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研究者情報

マスター

アカウント（マスター）

  • 氏名

    網塚　憲生(アミヅカ　ノリオ), アミヅカ　ノリオ

所属（マスター）

  • 歯学研究院　口腔医学部門　口腔健康科学分野

所属（マスター）

  • 歯学研究院　口腔医学部門　口腔健康科学分野

独自項目

syllabus

  • 2021, ソフトマター医工学特論, Soft Matter Medical Engineering, 修士課程, 生命科学院, 基礎医学、再生医学、バイオマテリアル、がん生物学
  • 2021, 発表・論文執筆法演習Ⅰ, Seminar Ⅰ for oral presentation and writing scientific papers, 博士後期課程, 歯学院, 研究計画，文献検索，研究のまとめ，口頭発表，論文作製
  • 2021, 発表・論文執筆法演習Ⅱ, Seminar Ⅱ for oral presentation and writing scientific papers, 博士後期課程, 歯学院, 研究計画，文献検索，研究のまとめ，口頭発表，論文作製
  • 2021, 発表・論文執筆法演習Ⅲ, Seminar Ⅲ for oral presentation and writing scientific papers, 博士後期課程, 歯学院, 研究計画，文献検索，研究のまとめ，口頭発表，論文作製
  • 2021, 硬組織機構解析学, Morphological Analysis of Hard Tissue, 博士後期課程, 歯学院, 硬組織，形態，発生，分子生物学，比較解剖学，系統発生学
  • 2021, 硬組織機構解析学研究, Morphological Research in Hard Tissue, 博士後期課程, 歯学院, 骨代謝、硬組織、微細構造、組織化学、電子顕微鏡
  • 2021, 硬組織発生生物学, Developmental Biology of Hard Tissue, 博士後期課程, 歯学院, 硬組織，形態形成，分子生物学
  • 2021, 硬組織発生生物学, Developmental Biology of Hard Tissue, 博士後期課程, 歯学院, 骨、軟骨、歯、硬組織、発生、石灰化、電子顕微鏡
  • 2021, 硬組織発生生物学研究, Research for Developmental Biology of Hard Tissue, 博士後期課程, 歯学院, 硬組織，歯，発生，形態学，顕微鏡
  • 2021, 口腔生物学と医学, Oral Biology and Medicine, 博士後期課程, 歯学院, 骨, 歯, 組織構造, 細胞外基質, 再生, 骨芽細胞, 破骨細胞, リモデリング, ホスファターゼ, ATPアーゼ, 材料, 歯科矯正学, 歯周病学
  • 2021, 歯学研究概論, Research Basics in Dental Sciences, 博士後期課程, 歯学院, 歯学研究，基本的研究技法，基礎知識
  • 2021, 基礎発生学, 学士課程, 歯学部, 発生学総論、発生学各論
  • 2021, 組織学, 学士課程, 歯学部, 細胞学、組織学、器官構築
  • 2021, 組織学実習, 学士課程, 歯学部, 組織学、細胞学、器官構築
  • 2021, 口腔組織学, 学士課程, 歯学部, 組織学、細胞学、器官構築
  • 2021, 口腔組織学実習, 学士課程, 歯学部, 組織学、細胞学、器官構築、顕微鏡観察
  • 2021, 関連臨床医学, 学士課程, 歯学部, 関連臨床医学、耳鼻咽喉科学、精神医学、臨床心理学、眼科、皮膚科、産婦人科
  • 2021, 歯学英語Ⅰ, 学士課程, 歯学部, professional English, practical English, English conversation

PositionHistory

  • 教育研究評議会評議員, 2018年4月1日, 2020年3月31日
  • 教育研究評議会評議員, 2020年4月1日, 2022年3月31日
  • 教育研究評議会評議員, 2022年4月1日, 2024年3月31日
  • 大学院歯学研究院副研究院長, 2018年4月1日, 2020年3月31日
  • 大学院歯学研究院副研究院長, 2020年4月1日, 2022年3月31日
  • 歯学部長, 2022年4月1日, 2024年3月31日
  • 大学院歯学研究院長, 2022年4月1日, 2024年3月31日
  • 大学院歯学院長, 2022年4月1日, 2024年3月31日

researchmap

プロフィール情報

学位

  • 歯学博士（新潟大学）
  • 歯科学修士（新潟大学）

プロフィール情報

  • 姓

    網塚, アミヅカ

  • 名

    憲生, ノリオ

  • ID各種

    200901033797255010

対象リソース

業績リスト

研究キーワード

  • 形態系基礎歯科学   Oral Anatomy and Morphological Basic Dentistry   

研究分野

  • ライフサイエンス / 常態系口腔科学

経歴

  • 2022年04月 - 現在 北海道大学大学院歯学研究院 歯学研究院長 歯学院長 歯学部長 兼任
  • 2017年04月 - 現在 北海道大学大学院歯学研究院 教授
  • 2018年04月 - 2022年03月 北海道大学大学院歯学研究院 副歯学研究院長 副歯学院長 副歯学部長 兼任
  • 2009年05月 - 2017年03月 北海道大学大学院歯学研究科 教授
  • 2005年09月 - 2009年04月 新潟大学 超域研究機構 教授
  • 2003年05月 - 2005年08月 新潟大学 超域研究機構 プロジェクトリーダー兼任
  • 2002年01月 - 2003年04月 新潟大学大学院医歯学総合研究科 助教授
  • 1992年04月 - 2001年12月 新潟大学歯学部 助手

学歴

  •         - 1992年   新潟大学   歯学研究科   歯科基礎系(口腔解剖学)
  •         - 1992年   新潟大学
  •         - 1988年   新潟大学   歯学部   歯学科(歯学専門課程)
  •         - 1988年   新潟大学

委員歴

  • 2018年05月 - 現在   歯科基礎医学会   理事
  • 2016年 - 現在   日本骨粗鬆症学会   評議員
  • 2002年 - 現在   日本骨形態計測学会   評議員   日本骨形態計測学会
  • 2002年 - 現在   日本解剖学会   評議員   日本解剖学会
  • 2002年 - 現在   日本骨代謝学会   評議員   日本骨代謝学会
  • 2002年 - 現在   歯科基礎医学会   評議員   歯科基礎医学会

受賞

  • 2017年01月 北海道大学 平成28年度北海道大学教育総長賞 奨励賞
     
    受賞者: 網塚 憲生
  • 2016年10月 日本学術振興会 科学研究費補助金・審査員賞
     
    受賞者: 網塚 憲生
  • 2005年 Best paper award of 2004, the 2nd joint meeting of the Europeans Calcified Tissue Society and International Bone and Mineral Society, Geneva, Switzerland.
  • 2003年 歯科基礎医学会 第３回歯科基礎医学会ライオン学術賞
     骨代謝調節因子における形態学的解析 
    受賞者: 網塚 憲生
  • 2003年 日本骨粗鬆症学会研究奨励賞
  • 2000年 歯科基礎医学会学会賞
  • 2000年 日本骨代謝学会学術賞
  • 1996年 日本骨代謝学会奨励賞
  • 1996年 William Geiz Award
  • 1993年 若手研究者賞(Young Investigator Award)

論文

  • Hideyuki Kobayashi, M Alaa Terkawi, Masahiro Ota, Tomoka Hasegawa, Tomomaya Yamamoto, Tomohiro Shimizu, Dai Sato, Ryo Fujita, Toshifumi Murakami, Norio Amizuka, Norimasa Iwasaki, Masahiko Takahata
    Bone research 12 1 35 - 35 2024年06月07日 
    DNAX-associated protein 12 kD size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5 A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2, Clec5a, Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec-15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Furthermore, biochemical analysis revealed that Sigelc-15 activates macrophage colony-stimulating factor (M-CSF)-induced Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.
  • DAP12/TREM2 signalはRANKL中和抗体中止後の過剰な骨吸収に影響を及ぼす
    石津 帆高, 清水 智弘, 長谷川 智香, 網塚 憲生, 岩崎 倫政
    日本骨形態計測学会雑誌 34 1 117 - 117 日本骨形態計測学会 2024年05月
  • 石津 帆高, 清水 智弘, 山本 知真也, 長谷川 智香, 網塚 憲生, 岩崎 倫政
    日本整形外科学会雑誌 98 3 S840 - S840 (公社)日本整形外科学会 2024年03月
  • Kotaro Azuma, Kazuhiro Ikeda, Sachiko Shiba, Wataru Sato, Kuniko Horie, Tomoka Hasegawa, Norio Amizuka, Shinya Tanaka, Satoshi Inoue
    iScience 27 2 108871 - 108871 2024年02月16日 
    Estrogen receptor-binding fragment associated antigen 9 (EBAG9) exerts tumor-promoting effects by inducing immune escape. We focused on the physiological functions of EBAG9 by investigating the bone phenotypes of Ebag9-knockout mice. Female Ebag9-knockout mice have fragile bones with lower bone mineral density (BMD) compared with wild-type mice. Histomorphometric analyses demonstrated that lower BMD was mainly caused by decreased bone formation. Serum bone turnover markers showed that enhanced bone resorption also contributed to this phenotype. We revealed that EBAG9 promoted autophagy in both osteoblastic and osteoclastic lineages. In addition, the knockdown of Tm9sf1, a gene encoding a protein that functionally interacts with EBAG9, suppressed autophagy and osteoblastic differentiation of the murine preosteoblastic cell line MC3T3-E1. Finally, overexpression of TM9SF1 rescued the suppression of autophagy caused by the silencing of Ebag9. These results suggest that EBAG9 plays a physiological role in bone maintenance by promoting autophagy together with its interactor TM9SF1.
  • 石津 帆高, 清水 智弘, 長谷川 智香, 網塚 憲生, 岩崎 倫政
    日本整形外科学会雑誌 97 8 S1739 - S1739 (公社)日本整形外科学会 2023年08月
  • RANKL中和抗体中止後の骨吸収過剰亢進におけるメカニズム解明アプローチ
    石津 帆高, 清水 智弘, 長谷川 智香, 網塚 憲生, 岩崎 倫政
    日本骨代謝学会学術集会プログラム抄録集 41回 141 - 141 (一社)日本骨代謝学会 2023年07月
  • Yukina Miyamoto, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Mai Haraguchi-Kitakamae, Miki Abe, Haruhi Maruoka, Hotaka Ishizu, Tomohiro Shimizu, Yasuyuki Sasano, Nobuyuki Udagawa, Minqi Li, Norio Amizuka
    Journal of oral biosciences 65 2 175 - 185 2023年04月21日 
    OBJECTIVES: We examined mice with gene deletion of Receptor activator of nuclear factor-κB (Rank) ligand (Rankl) to histologically clarify whether they contained progenitor cells committed to osteoclastic differentiation up to the stage requiring RANK/RANKL signaling. METHODS: The tibiae and femora of ten-week-old male wild-type, c-fos-/-, and Rankl-/- mice were used for immunohistochemistry and transmission electron microscopy (TEM). RESULTS: In Rankl-/- mice, we observed osteoclast-like giant cells, albeit in low numbers, with single or two nuclei, engulfing the mineralized extracellular matrix. TEM revealed that these giant cells contained large numbers of mitochondria, vesicles/vacuoles, and clear zone-like structures but no ruffled borders. They often engulfed fragmented bony/cartilaginous components of the extracellular matrix that had been degraded. Additionally, osteoclast-like giant cells exhibited immunoreactivity for vacuolar H+-ATPase, galectin-3, and siglec-15 but not for tartrate-resistant acid phosphatase, cathepsin K, or MMP-9, all of which are classical hallmarks of osteoclasts. Furthermore, osteoclast-like giant cells were ephrinB2-positive as they were near EphB4-positive osteoblasts that are also positive for alkaline phosphatase and Runx2 in Rankl-/- mice. Unlike Rankl-/- mice, c-fos-/- mice lacking osteoclast progenitors and mature osteoclasts had no ephrinB2-positive osteoclast-like cells or alkaline phosphatase-positive/Runx2-reactive osteoblasts. This suggests that similar to authentic osteoclasts, osteoclast-like giant cells might have the potential to activate osteoblasts in Rankl-/- mice. CONCLUSIONS: It seems plausible that osteoclast-like giant cells may have acquired some osteoclastic traits and the ability to resorb mineralized matrices even when the absence of RANK/RANKL signaling halted the osteoclastic differentiation cascade.
  • Takafumi Muneyama, Tomoka Hasegawa, Yimin, Tomomaya Yamamoto, Hiromi Hongo, Mai Haraguchi-Kitakamae, Miki Abe, Haruhi Maruoka, Hotaka Ishizu, Tomohiro Shimizu, Yasuyuki Sasano, Minqi Li, Norio Amizuka
    Journal of oral biosciences 65 2 163 - 174 2023年04月21日 
    OBJECTIVE: Toll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2-/-) mice to gain insight into TLR2 function in biological circumstances without microbial infection. METHODS: Eight-week-old male wild-type and TLR2-/- mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. RESULTS: TLR2-/- tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2-/- osteoclasts reduced cell fusion and bone resorption activity. CONCLUSION: It seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.
  • 石津 帆高, 清水 智弘, 岩崎 倫政, 長谷川 智香, 網塚 憲生
    北海道整形災害外科学会雑誌 65 142nd suppl 44 - 44 北海道整形災害外科学会 2023年
  • Yasuhito Morimoto, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Haruhi Maruoka, Mai Haraguchi-Kitakamae, Ko Nakanishi, Tsuneyuki Yamamoto, Hotaka Ishizu, Tomohiro Shimizu, Kumiko Yoshihara, Yasuhiro Yoshida, Tsutomu Sugaya, Norio Amizuka
    Frontiers in bioengineering and biotechnology 11 1243951 - 1243951 2023年 
    The current study aimed to evaluate bone tissue regeneration using a combination of β-tricalcium phosphate (βTCP) and phosphorylated pullulan (PPL, a phosphate-rich polysaccharide polymer consisting of maltotriose units). Round defects of 2 mm diameter were created in the arterial center of rat tibiae, which were further treated with vehicle (control group), βTCP (βTCP group), or βTCP + PPL (βTCP + PPL group) grafts. The control specimens without bone grafts exhibited rapid bone formation after 1 week; however, the regenerated bone was not resorbed until 4 weeks. In contrast, βTCP-grafted specimens exhibited fewer but thicker trabeculae, whereas the βTCP + PPL group displayed many fine trabeculae at 4 weeks. In the βTCP + PPL group, new bone was associated with the βTCP granules and PPL. Similarly, PHOSPHO1-positive osteoblasts were localized on the βTCP granules as well as the PPL. On the other hand, TRAP-reactive osteoclasts predominantly localized on newly-formed bone and βTCP granules rather than on the PPL. No significant differences were observed in the expression of Alp, Integrin αv, Osteopontin, Osteocalcin, and Dmp-1 in PPL-treated MC3T3-E1 osteoblastic cells, suggesting that PPL did not facilitate osteoblastic differentiation. However, von Kossa staining identified abundant needle-like calcified structures extending inside the PPL. Furthermore, transmission electron microscopy (TEM) revealed many globular structures identical to calcified nodules. In addition, calcified collagen fibrils were observed in the superficial layer of the PPL. Thus, PPL may serve as a scaffold for osteoblastic bone formation and promotes calcification on its surface. In conclusion, we speculated that βTCP and PPL might promote bone regeneration and could be integrated into promising osteoconductive materials.
  • Yasuhito Moritani, Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Yimin, Miki Abe, Hirona Yoshino, Ko Nakanishi, Haruhi Maruoka, Hotaka Ishizu, Tomohiro Shimizu, Masahiko Takahata, Norimasa Iwasaki, Minqi Li, Kanchu Tei, Yoichi Ohiro, Norio Amizuka
    Journal of oral biosciences 64 4 410 - 421 2022年10月11日 
    OBJECTIVES: Interleukin-6 (IL-6) contributes to the regulation of functions in various tissues and organs. Even though IL-6 has been reported to modulate bone metabolism in previous studies, this finding is controversial. This study aims to evaluate the possible involvement of IL-6 in bone metabolism by examining the histological activity of osteoblasts and osteoclasts in the femora of Il-6 deficient (Il-6-/-) mice. METHODS: Eight-week-old male Il-6-/- mice and their wild-type littermates were fixed with a paraformaldehyde solution, and their femora were extracted for micro-CT analysis, immunohistochemistry, and real-time PCR analysis. RESULTS: Il-6-/- femora showed an increased bone volume/tissue volume (TV) but a reduced bone mineral density compared with the wild-type. Furthermore, the tissue-nonspecific alkaline phosphatase positive area/TV ratio, the expression of Runx2, Osterix, and Rankl, and the number of tartrate-resistant acid phosphatase-positive osteoclasts were all increased in the Il-6-/- mice. A considerable number of unmineralized areas within the bone matrix and abundant sclerostin-reactive osteocytes were observed in Il-6-/- femoral metaphyses but not in the wild-type. Interestingly, the gene expression of Cd206 was elevated in Il-6-/- femora, and many F4/80-positive macrophages/monocytes and CD206-immunoreactive macrophages in the primary trabeculae had migrated closer to the growth plate, where intense RANKL immunoreactivity was detected. These results suggest that, in an IL-6-deficient state, CD206-positive macrophages may differentiate into osteoclasts when in contact with RANKL-reactive osteoblastic cells. CONCLUSION: In a state of IL-6 deficiency, the population and cell activities of osteoblast, osteoclasts, and macrophages seemed to be facilitated, except for the reduced mineralization in bone.
  • Tsuneyuki Yamamoto, Shigeru Takahashi, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
    Journal of oral biosciences 64 3 346 - 351 2022年09月 
    OBJECTIVES: The function of capillary ends at the epiphyseal plate has been actively investigated. However, their morphology is still poorly understood. This study was designed to examine the capillary ends invading the epiphyseal plate three-dimensionally by scanning electron microscopy and discuss the relationship between their morphology and function. METHODS: Distal halves of the femora of eight-week-old male Wistar rats were used. The specimens were divided into two groups for transmission and scanning electron microscopy. For transmission electron microscopy, sagittal ultrathin sections were routinely prepared after the demineralization of the specimens, and the chondro-osseous junction was examined at the epiphyseal plate. For scanning electron microscopy, the specimens were sagittally freeze-cracked, osmium-macerated, and routinely processed. RESULTS: Endothelial cells of capillary ends had fine fenestrations, and hence they were distinguishable from perivascular cells (also known as septoclasts). Based on the outline and the presence or absence of pores, the capillary ends were divided into four types: closed dome, closed spire, porous dome, and porous spire. The two dome types generally occupied more than half of a lacuna, whereas the two spire types generally occupied only a small part of a lacuna. The porous types engulfed cellular remnants, indicative of degraded chondrocytes, via their pores. Some of the spire types penetrated the transverse septum. CONCLUSIONS: The morphological variety of capillary ends reflected their functional variety. Observations suggest that the capillary ends change their morphology dynamically in response to various functions, including the removal of degraded chondrocytes and perforation of transverse septa.
  • Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Miki Abe, Hirona Yoshino, Mai Haraguchi-Kitakamae, Hotaka Ishizu, Tomohiro Shimizu, Norimasa Iwasaki, Norio Amizuka
    International journal of molecular sciences 23 17 2022年09月01日 
    Bone mineralization entails two mineralization phases: primary and secondary mineralization. Primary mineralization is achieved when matrix vesicles are secreted by osteoblasts, and thereafter, bone mineral density gradually increases during secondary mineralization. Nearby extracellular phosphate ions (PO43-) flow into the vesicles via membrane transporters and enzymes located on the vesicles' membranes, while calcium ions (Ca2+), abundant in the tissue fluid, are also transported into the vesicles. The accumulation of Ca2+ and PO43- in the matrix vesicles induces crystal nucleation and growth. The calcium phosphate crystals grow radially within the vesicle, penetrate the vesicle's membrane, and continue to grow outside the vesicle, ultimately forming mineralized nodules. The mineralized nodules then attach to collagen fibrils, mineralizing them from the contact sites (i.e., collagen mineralization). Afterward, the bone mineral density gradually increases during the secondary mineralization process. The mechanisms of this phenomenon remain unclear, but osteocytes may play a key role; it is assumed that osteocytes enable the transport of Ca2+ and PO43- through the canaliculi of the osteocyte network, as well as regulate the mineralization of the surrounding bone matrix via the Phex/SIBLINGs axis. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.
  • Hiroki Yoshioka, Shingo Komura, Norishige Kuramitsu, Atsushi Goto, Tomoka Hasegawa, Norio Amizuka, Takuya Ishimoto, Ryosuke Ozasa, Takayoshi Nakano, Yuuki Imai, Haruhiko Akiyama
    Journal of bone and mineral metabolism 40 5 839 - 852 2022年08月10日 
    INTRODUCTION: Osteoblasts require substantial amounts of energy to synthesize the bone matrix and coordinate skeleton mineralization. This study analyzed the effects of mitochondrial dysfunction on bone formation, nano-organization of collagen and apatite, and the resultant mechanical function in mouse limbs. MATERIALS AND METHODS: Limb mesenchyme-specific Tfam knockout (Tfamf/f;Prx1-Cre: Tfam-cKO) mice were analyzed morphologically and histologically, and gene expressions in the limb bones were assessed by in situ hybridization, qPCR, and RNA sequencing (RNA-seq). Moreover, we analyzed the mitochondrial function of osteoblasts in Tfam-cKO mice using mitochondrial membrane potential assay and transmission electron microscopy (TEM). We investigated the pathogenesis of spontaneous bone fractures using immunohistochemical analysis, TEM, birefringence analyzer, microbeam X-ray diffractometer and nanoindentation. RESULTS: Forelimbs in Tfam-cKO mice were significantly shortened from birth, and spontaneous fractures occurred after birth, resulting in severe limb deformities. Histological and RNA-seq analyses showed that bone hypoplasia with a decrease in matrix mineralization was apparent, and the expression of type I collagen and osteocalcin was decreased in osteoblasts of Tfam-cKO mice, although Runx2 expression was unchanged. Decreased type I collagen deposition and mineralization in the matrix of limb bones in Tfam-cKO mice were associated with marked mitochondrial dysfunction. Tfam-cKO mice bone showed a significantly lower Young's modulus and hardness due to poor apatite orientation which is resulted from decreased osteocalcin expression. CONCLUSION: Mice with limb mesenchyme-specific Tfam deletions exhibited spontaneous limb bone fractures, resulting in severe limb deformities. Bone fragility was caused by poor apatite orientation owing to impaired osteoblast differentiation and maturation.
  • X連鎖低リン血症性くる病モデルマウスは血管侵入抑制を伴う軟骨内骨化異常を示す
    大巻 真幸, 山本 知真也, 金子 一郎, 瀬川 博子, 網塚 憲生, 長谷川 智香
    日本骨粗鬆症学会雑誌 8 Suppl.1 94 - 94 (一社)日本骨粗鬆症学会 2022年08月
  • Sumire Sasaki, Yuji Shiozaki, Ai Hanazaki, Megumi Koike, Kazuya Tanifuji, Minori Uga, Kota Kawahara, Ichiro Kaneko, Yasuharu Kawamoto, Pattama Wiriyasermkul, Tomoka Hasegawa, Norio Amizuka, Ken-ichi Miyamoto, Shushi Nagamori, Yoshikatsu Kanai, Hiroko Segawa
    Scientific Reports 12 1 6353 - 6353 2022年05月 [査読有り]
     
    Abstract Renal type II sodium-dependent inorganic phosphate (Pi) transporters NaPi2a and NaPi2c cooperate with other organs to strictly regulate the plasma Pi concentration. A high Pi load induces expression and secretion of the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) that enhance urinary Pi excretion and prevent the onset of hyperphosphatemia. How FGF23 secretion from bone is increased by a high Pi load and the setpoint of the plasma Pi concentration, however, are unclear. Here, we investigated the role of Transmembrane protein 174 (Tmem174) and observed evidence for gene co-expression networks in NaPi2a and NaPi2c function. Tmem174 is localized in the renal proximal tubules and interacts with NaPi2a, but not NaPi2c. In Tmem174-knockout (KO) mice, the serum FGF23 concentration was markedly increased but increased Pi excretion and hypophosphatemia were not observed. In addition, Tmem174-KO mice exhibit reduced NaPi2a responsiveness to FGF23 and PTH administration. Furthermore, a dietary Pi load causes marked hyperphosphatemia and abnormal NaPi2a regulation in Tmem174-KO mice. Thus, Tmem174 is thought to be associated with FGF23 induction in bones and the regulation of NaPi2a to prevent an increase in the plasma Pi concentration due to a high Pi load and kidney injury.
  • Masahiko Takahata, Tomohiro Shimizu, Satoshi Yamada, Tomomaya Yamamoto, Tomoka Hasegawa, Ryo Fujita, Hideyuki Kobayashi, Tsutomu Endo, Yoshinao Koike, Norio Amizuka, Masahiro Todoh, Jun-Ichiro Okumura, Tomomichi Kajino, Norimasa Iwasaki
    Journal of bone and mineral metabolism 40 4 613 - 622 2022年03月25日 
    INTRODUCTION: Bisphosphonates (BPs) have been shown to reduce the incidence of vertebral fractures during the first year or two of glucocorticoid (GC) treatments and are therefore recommended as a first-line treatment for GC-induced osteoporosis (GIO). However, there are theoretical concerns about the long-term use of BPs in low-turnover osteoporosis caused by chronic GC therapy. MATERIALS AND METHODS: We analyzed the trabecular microarchitecture, bone metabolism, and material strength of iliac crest bone biopsy samples from 10 female patients with rheumatoid arthritis who received an average of 6.7 years of BP therapy for GIO (GIOBP group), compared with those of 10 age- and bone mineral density (BMD)-matched non-rheumatoid arthritis postmenopausal women (reference group). RESULTS: Patients in the GIOBP group had a significantly greater fracture severity index, as calculated from the number and the extent of vertebral fractures compared with the reference patients. Micro-computed tomography analysis showed that the degree of mineralization and trabecular microarchitecture were significantly lower in the GIOBP group than in the reference patients. Patients in the GIOBP group exhibited lower bone contact stiffness, determined by micro-indentation testing, than in the reference group. The contact stiffness of the bone was negatively correlated with the fracture severity index and the daily prednisolone dosage. Immunohistochemistry and serum bone turnover markers showed decreased osteoclastic activity, impaired mineralization, and an increased fraction of empty lacunae in the GIOBP group. CONCLUSION: Our findings indicate that patients receiving long-term BP for GIO are still at high risk for fragility fractures because of poor bone quality.
  • Wei Feng, Hongrui Liu, Tingting Luo, Di Liu, Juan Du, Jing Sun, Wei Wang, Xiuchun Han, Kaiyun Yang, Jie Guo, Norio Amizuka, Minqi Li
    Scientific reports 12 1 3746 - 3746 2022年03月02日
  • Hayato Ohshima, Kenji Mishima, Norio Amizuka
    Journal of oral biosciences 64 1 1 - 7 2022年03月 [査読有り][招待有り]
     
    BACKGROUND: The Journal of Oral Biosciences is devoted to advancing and disseminating fundamental knowledge concerning every aspect of oral biosciences. HIGHLIGHT: This review features review articles in the fields of "Extracellular Vesicles," "Propolis," "Odontogenic Tumors," "Periodontitis," "Periodontium," "Flavonoids," "Lactoferrin," "Dental Plaque," "Anatomy," "Induced Pluripotent Stem Cells," "Bone Cell Biology," "Dysgeusia," "Dental Caries," and "Dental Pulp Cavity," in addition to the review article by the winners of the "Lion Award" ("Sox9 function in salivary gland development") presented by the Japanese Association for Oral Biology. CONCLUSION: These reviews in the Journal of Oral Biosciences have inspired its readers to broaden their knowledge regarding various aspects of oral biosciences. The current editorial review introduces these exciting review articles.
  • Fumiya Nakamura, Yuya Kanehira, Dai Sato, Ryo Fujita, Tomoka Hasegawa, Hideyo Horiuchi, Tomomi Masuya, Masahiro Ota, Norio Amizuka, Masahiko Takahata, Hiromi Kimura-Suda
    JOURNAL OF BONE AND MINERAL RESEARCH 37 175 - 175 2022年02月
  • Eisuke Tsuda, Chie Fukuda, Akiko Okada, Tsuyoshi Karibe, Yoshiharu Hiruma, Nana Takagi, Yoshitaka Isumi, Tomomaya Yamamoto, Tomoka Hasegawa, Shunsuke Uehara, Masanori Koide, Nobuyuki Udagawa, Norio Amizuka, Seiichiro Kumakura
    Bone 155 116241 - 116241 2022年02月 
    Recent studies have established the idea that Siglec-15 is involved in osteoclast differentiation and/or function, and it is anticipated that therapies suppressing Siglec-15 function can be used to treat bone diseases such as osteoporosis. We have produced rat monoclonal anti-Siglec-15 antibody (32A1) and successively generated humanized monoclonal anti-Siglec-15 antibody (DS-1501a) from 32A1. Studies on the biological properties of DS-1501a showed its specific binding affinity to Siglec-15 and strong activity to inhibit osteoclastogenesis. 32A1 inhibited multinucleation of osteoclasts and bone resorption (pit formation) in cultured mouse bone marrow cells. 32A1 also inhibited pit formation in cultured human osteoclast precursor cells. Maximum serum concentration and serum exposure of DS-1501a in rats were increased in a dose-dependent manner after single subcutaneous or intravenous administration. Furthermore, single administration of DS-1501a significantly suppressed bone resorption markers with minimal effects on bone formation markers and suppressed the decrease in bone mineral density (BMD) of the lumbar vertebrae in ovariectomized (OVX) rats. In histological analysis, the osteoclasts distant from the chondro-osseous junction of the tibia tended to be flattened, shrunken, and functionally impaired in 32A1-treated rats, while alkaline phosphatase-positive osteoblasts were observed throughout the metaphyseal trabeculae. In addition, we compared the efficacy of 32A1 with that of alendronate (ALN) as follow-up medicine after treatment with parathyroid hormone (PTH) using mature established osteoporosis rats. The beneficial effect of PTH on bone turnover disappeared 8 weeks after discontinuing the treatment. The administration of 32A1 once every 4 weeks for 8 weeks suppressed bone resorption and bone formation when the treatment was switched from PTH to 32A1, leading to the maintenance of BMD and bone strength. Unlike with ALN, the onset of suppression of bone resorption with 32A1 was rapid, while the suppression of bone formation was mild. The improvement of bone mass, beneficial bone turnover balance, and suppression of osteoclast differentiation/multinucleation achieved by 32A1 were supported by histomorphometry. Notably, the effects of 32A1 on bone strength, not only structural (extrinsic) but also material (intrinsic) properties, were significantly greater than those of ALN. Since the effect of 32A1 on BMD was moderate, its effect on bone strength could not be fully explained by the increase in BMD. The beneficial balance of bone turnover caused by 32A1 might, at least in part, be responsible for the improvement in bone quality. This is the first report describing the effects of anti-Siglec-15 antibody in OVX rats; the findings suggest that this antibody could be an excellent candidate for treating osteoporosis, especially in continuation therapy after PTH treatment, due to its rapid action and unprecedented beneficial effects on bone quality.
  • Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Norio Amizuka
    Osteoporotic Fracture and Systemic Skeletal Disorders 413 - 430 2021年12月
  • 骨再生におけるバイオマテリアル開発と骨質評価アップデート 新規リン酸化多糖体"リン酸化プルラン"を用いた骨再生(Bone regeneration by using the novel phosphorylated polysaccharide "Phosphorylated pullulan")
    長谷川 智香, 森本 康仁, 久保田 恵亮, 本郷 裕美, 吉田 靖弘, 網塚 憲生
    Journal of Oral Biosciences Supplement 2021 90 - 90 (一社)歯科基礎医学会 2021年10月
  • 森本 康仁, 久保田 恵亮, 阿部 未来, 丸岡 春日, 長谷川 智香, 本郷 裕美, 吉田 靖弘, 網塚 憲生, 菅谷 勉
    日本歯周病学会会誌 63 秋季特別 120 - 120 (NPO)日本歯周病学会 2021年10月
  • 小林 英之, 太田 昌博, 長谷川 智香, 山本 知真也, 清水 智弘, 藤田 諒, 網塚 憲生, アラー・テルカウイ, 岩崎 倫政, 高畑 雅彦
    日本骨代謝学会学術集会プログラム抄録集 39回 128 - 128 (一社)日本骨代謝学会 2021年10月
  • Yamamoto T, Mae T, Hongo H, Yamamoto T, Abe M, Nasoori A, Morimoto Y, Maruoka H, Kubota K, Haraguchi M, Li M, Hasegawa T
    Journal of Oral Biosciences. 63 3 259 - 264 2021年09月 [査読有り]
     
    OBJECTIVE: This study aimed to demonstrate the immunolocalization and gene expression of tissue nonspecific alkaline phosphatase (TNALP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in osteoblasts, preosteoblasts, and osteocytes of murine bone to provide clues for a better understanding of the supply of phosphate ions (Pi) during bone mineralization. METHODS: Six-week-old male C57BL/6J mice (n = 6) were fixed with a paraformaldehyde solution, and the right femora were extracted for immunodetection of TNALP and ENPP1, while the left tibiae were used for reverse transcription polymerase chain reaction to evaluate Tnalp and Enpp1 gene expression. RESULTS: TNALP was intensely localized on the basolateral cell membranes of mature osteoblasts and preosteoblastic cells. There was little immunoreactivity of TNALP on the secretory surface of the osteoblasts and no TNALP reactivity in the osteocytes. In contrast, ENPP1 was observed throughout the cytoplasm of mature osteoblasts and osteocytes embedded in bone but was not observed in preosteoblasts. Together, despite the fact that the osteoid is a site of matrix vesicle-mediated mineralization, ENPP1, which inhibits mineralization by providing pyrophosphates, was localized in close proximity of the osteoid, whereas TNALP, which facilitates mineralization by providing Pi, was relatively distant from the osteoid. CONCLUSION: It seems likely that the differential localization of TNALP and ENPP1 around the osteoid observed at the microscopic level may provide preferential micro-circumstance for a balanced concentration of Pi and pyrophosphate for bone mineralization.
  • Ayako Yokoyama, Tomoka Hasegawa, Toru Hiraga, Tamaki Yamada, Yimin, Hiromi Hongo, Tomomaya Yamamoto, Miki Abe, Taiji Yoshida, Yasuo Imanishi, Shinichiro Kuroshima, Muneteru Sasaki, Paulo Henrique Luiz de Fraitas, Minqi Li, Norio Amizuka, Yutaka Yamazaki
    Journal of bone and mineral metabolism 39 5 810 - 823 2021年09月 [査読有り]
     
    INTRODUCTION: After the onset of bone metastasis, tumor cells appear to modify surrounding microenvironments for their benefit, and particularly, the levels of circulating fibroblast growth factor (FGF) 23 in patients with tumors have been highlighted. MATERIALS AND METHODS: We have attempted to verify if human breast carcinoma MDA-MB-231 cells metastasized in the long bone of nu/nu mice would synthesize FGF23. Serum concentrations of calcium, phosphate (Pi) and FGF23 were measured in control nu/nu mice, bone-metastasized mice, and mice with mammary gland injected with MDA-MB-231 cells mimicking primary mammary tumors. RESULTS AND CONCLUSIONS: MDA-MB-231 cells revealed intense FGF23 reactivity in metastasized lesions, whereas MDA-MB-231 cells cultured in vitro or when injected into the mammary glands (without bone metastasis) showed weak FGF23 immunoreactivity. Although the bone-metastasized MDA-MB-231 cells abundantly synthesized FGF23, osteocytes adjacent to the FGF23-immunopositive tumors, unlike intact osteocytes, showed no FGF23. Despite significantly elevated serum FGF23 levels in bone-metastasized mice, there was no significant decrease in the serum Pi concentration when compared with the intact mice and mice with a mass of MDA-MB-231 cells in mammary glands. The metastasized femora showed increased expression and FGFR1 immunoreactivity in fibroblastic stromal cells, whereas femora of control mice showed no obvious FGFR1 immunoreactivity. Taken together, it seems likely that MDA-MB-231 cells synthesize FGF23 when metastasized to a bone, and thus affect FGFR1-positive stromal cells in the metastasized tumor nest in a paracrine manner.
  • Tsuneyuki Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
    Histochemistry and cell biology 156 5 503 - 508 2021年08月26日 [査読有り]
     
    The three-dimensional morphology of the Golgi apparatus in osteoclasts was investigated by computer-aided reconstruction. Rat femora were treated for nicotinamide adenine dinucleotide phosphatase (NADPase) cytochemistry, and light microscopy was used to select several osteoclasts in serial semi-thin sections to investigate the Golgi apparatus by backscattered electron-mode scanning electron microscopy. Lace-like structures with strong backscattered electron signals were observed around the nuclei. These structures, observed within the Golgi apparatus, were attributed to the reaction products (i.e., lead precipitates) of NADPase cytochemistry. Features on the images corresponding to the Golgi apparatus, nuclei, and ruffled border were manually traced and three-dimensionally reconstructed using ImageJ/Fiji (an open-source image processing package). In the reconstructed model, the Golgi apparatus formed an almost-continuous structure with a basket-like configuration, which surrounded all the nuclei and also partitioned them. This peculiar three-dimensional morphology of the Golgi apparatus was discovered for the first time in this study. On the basis of the location of the cis- and trans-sides of the Golgi apparatus and the reported results of previous studies, we postulated that the nuclear membrane synthesized specific proteins in the osteoclasts and, accordingly, the Golgi apparatus accumulated around the nuclei as a receptacle.
  • Shizu Saito, Ryo Hamai, Yukari Shiwaku, Tomoka Hasegawa, Susumu Sakai, Kaori Tsuchiya, Yuko Sai, Ryosuke Iwama, Norio Amizuka, Tetsu Takahashi, Osamu Suzuki
    Acta biomaterialia 129 309 - 322 2021年07月15日 [査読有り]
     
    This study hypothesized that distant octacalcium phosphate (OCP) scaffolds may enhance osteocyte differentiation in newly formed bone matrices. The results obtained were compared with those of Ca-deficient hydroxyapatite (OCP hydrolyzate, referred to as HL hereafter). Granular OCP and HL, 300-500 µm in diameter, were implanted in critical-sized rat calvarial defects for eight weeks and subjected to histology, immunohistochemistry, histomorphometry, and transmission electron microscopy (TEM). Early osteocyte differentiation from an osteoblastic cell line (IDG-SW3) was examined using materials without contacting the surfaces for 10 days. The material properties and the medium composition were analyzed through selected area electron diffraction (SAED) using TEM observation and curve fitting of Fourier transform infrared (FT-IR) spectroscopy. The number of positive cells of an osteocyte earlier differentiation marker podoplanin (PDPN) in bone matrices, along the direction of bone formation, was significantly higher in OCP than that in HL. The ultrastructure around the OCP surfaces observed by TEM showed the infiltration of some cells, including osteocytes adjacent to the OCP surface layers. The OCP structure remained unchanged by SAED analysis. Nanoparticle deposition and hydrolysis on OCP surfaces were detected by TEM and FT-IR, respectively, during early osteocyte differentiation in vitro. The medium saturation degree varied in accord with ionic dissolution, resulting in possible hydroxyapatite formation on OCP but not on HL. These results suggested that OCP stimulates early osteocyte differentiation in the bone matrix from a distance through its metastable chemical properties. STATEMENT OF SIGNIFICANCE: This study demonstrated that octacalcium phosphate (OCP) implanted in critical-sized rat calvaria bone defects is capable of enhancing the early differentiation of osteocytes embedded in newly formed bone matrices, even when the surface OCP is separated from the osteocytes. This prominent bioactive property of OCP was demonstrated by comparing the in vivo and in vitro performances with a control material, Ca-deficient hydroxyapatite (OCP hydrolyzate). The findings were elucidated by histomorphometry, which analyzed the differentiation of osteocytes along the parallel direction of new bone growth by osteoblasts. Therefore, OCP should stimulate osteocyte differentiation through ionic dissolution even in vivo owing to its metastable chemical properties, as previously reported in an in vitro study (Acta Biomater 69:362, 2018).
  • リン酸化プルラン/β-TCPコンビネーションマテリアルにおける新規骨再生誘導
    森本 康仁, 久保田 恵亮, 阿部 未来, 丸岡 春日, 本郷 裕美, 吉田 靖弘, 菅谷 勉, 網塚 憲生, 長谷川 智香
    日本骨形態計測学会雑誌 31 2 S135 - S135 日本骨形態計測学会 2021年06月
  • Sachiko Shiba, Kazuhiro Ikeda, Kuniko Horie-Inoue, Kotaro Azuma, Tomoka Hasegawa, Norio Amizuka, Tomoaki Tanaka, Toshihiko Takeiwa, Yasuaki Shibata, Takehiko Koji, Satoshi Inoue
    Molecular and cellular biology 41 4 2021年03月24日 [査読有り]
     
    γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.
  • Hayato Ohshima, Kenji Mishima, Norio Amizuka
    Journal of oral biosciences 63 1 1 - 7 2021年03月 [査読有り]
     
    BACKGROUND: The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences. HIGHLIGHT: This review featured the review articles in the fields of "Microbiology," "Palate," "Stem Cells," "Mucosal Diseases," "Bone Cell Biology," "MicroRNAs," "TRPV1 Cation Channels," and "Interleukins" in addition to the review article by prize-winners of the "Rising Members Award" ("DKK3 expression and function in head and neck squamous cell carcinoma and other cancers"), presented by the Japanese Association for Oral Biology. CONCLUSION: These reviews in the Journal of Oral Biosciences have inspired the readers of the journal to broaden their knowledge regarding the various aspects of oral biosciences. The current editorial review introduces these exciting review articles.
  • Keiji Hashimoto, Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Y Imin, Miki Abe, Alireza Nasoori, Ko Nakanishi, Haruhi Maruoka, Yasuhito Morimoto, Keisuke Kubota, Tomohiro Shimizu, Mai Haraguchi, Masahiko Takahata, Norimasa Iwasaki, Minqi Li, Toshiaki Fujisawa, Norio Amizuka
    Biomedical research (Tokyo, Japan) 42 4 139 - 151 2021年 [査読有り]
     
    It remains unknown whether the histology of vascular invasion during secondary ossification of epiphyseal cartilage is the same as that seen in primary ossification; we examined the initial processes of vascular invasion of secondary ossification in the murine femora. Many endomucin-immunoreactive blood vessels gathered at the central region of the articular surface, and buds of soft tissue, including glomerular loops of endomucin-immunoreactive blood vessels and TNALPase- immunopositive osteoblastic cells accompanied by TRAP-positive osteoclasts, had begun to invade the epiphyseal cartilage. The invading soft tissues formed cartilage canals displaying MMP9 immunoreactivity in the tip region, and cartilaginous collagen fibrils were not visible in the vicinity of the vascular wall of the blood vessels. Thus, the histological profile marked by invading glomerular vasculature and the erosion of the cartilage matrix near the vascular walls during secondary ossification differs from that seen during primary ossification.
  • Tomomaya Yamamoto, Tomoka Hasegawa, Paulo Henrique Luiz de Fraitas, Hiromi Hongo, Shen Zhao, Tsuneyuki Yamamoto, Alireza Nasoori, Miki Abe, Haruhi Maruoka, Keisuke Kubota, Yasuhito Morimoto, Mai Haraguchi, Tomohiro Shimizu, Masahiko Takahata, Norimasa Iwasaki, Minqi Li, Norio Amizuka
    Biomedical research (Tokyo, Japan) 42 5 161 - 171 2021年 [査読有り]
     
    Modeling, the changes of bone size and shape, often takes place at the developmental stages, whereas bone remodeling-replacing old bone with new bone-predominantly occurs in adults. Unlike bone remodeling, bone formation induced by modeling i.e., minimodeling (microscopic modeling in cancellous bone) is independent of osteoclastic bone resorption. Although recently-developed drugs for osteoporotic treatment could induce minimodeling-based bone formation in addition to remodeling-based bone formation, few reports have demonstrated the histological aspects of minimodeling-based bone formation. After administration of eldecalcitol or romosozumab, unlike teriparatide treatment, mature osteoblasts formed new bone by minimodeling, without developing thick preosteoblastic layers. The histological characteristics of minimodeling-based bone formation is quite different from remodeling, as it is not related to osteoclastic bone resorption, resulting in convex-shaped new bone and smooth cement lines called arrest lines. In this review, we will show histological properties of minimodeling-based bone formation by osteoporotic drugs.
  • Shun Watanabe, Naoki Sawa, Hiroki Mizuno, Rikako Hiramatsu, Noriko Hayami, Masayuki Yamanouchi, Tatsuya Suwabe, Junichi Hoshino, Takeshi Fujii, Toshihide Hirai, Tomoka Hasegawa, Norio Amizuka, Yoshifumi Ubara
    Bone reports 13 100296 - 100296 2020年12月 [査読有り]
     
    A 56-year-old Japanese woman with a history of palmoplantar pustulosis was admitted for examination due to left femur pain. Radiography and computed tomography showed thickening of the bone on the outer portion of the left femur. Bone scintigraphy of the left femur showed intense radioactive uptake. Consequently, the patient was diagnosed with SAPHO syndrome. Bone histomorphometric analysis of the left femur showed cancellous bone with thickened cortical bone. Whilst normal bone shows cancellous bone with double labeling (normal turn over), and cortical bone with no labeling (low turn over, adynamic state), this case presented with both cancellous and cortical bone with marked double labeling (indicating high turn over), abundant osteoid and woven bone. Immunohistological analysis showed that cells lining the bone surface consisted of osteoblasts and were positive for alkaline phosphatase (ALP). Few to little of these cells were positive for tartrate-resistant acid phosphatase (TRAP)-5B, cathepsin K and matrix metallopeptidase 9 (MMP-9). These results indicate that, in this case study, excessive production of osteoblasts contributed to hyperostosis of the left femur, with abundant osteoid and woven bone. This type of bone formation in SAPHO syndrome is not lamellar bone seen in normal bone, but rather fragile and mechanically weak bone, resulting in bone pain. Doxycycline may be a therapeutic option for bone pain in this patient.
  • Shota Takashima, Yasuyuki Fujita, Satoru Shinkuma, Satoko Shimizu, Tomoka Hasegawa, Norio Amizuka, Hiroshi Shimizu, Ken Natsuga
    The Journal of dermatology 47 12 e457-e458 - E458 2020年12月 [査読有り]
  • Tomoya Nagai, Tomoka Hasegawa, Yimin, Tomomaya Yamamoto, Hiromi Hongo, Miki Abe, Taiji Yoshida, Ayako Yokoyama, Paulo Henrique Luiz de Freitas, Minqi Li, Atsuro Yokoyama, Norio Amizuka
    Histochemistry and cell biology 155 3 369 - 380 2020年11月11日 [査読有り]
     
    In this study, we examined the immunolocalization of podoplanin/E11, CD44, actin filaments, and phosphorylated ezrin in the osteoblasts on the verge of differentiating into osteocytes in murine femora and tibiae. When observing under stimulated emission depletion microscopy, unlike podoplanin-negative osteoblasts, podoplanin-positive osteoblasts showed a rearranged assembly of actin filaments along the cell membranes which resembled that of embedded osteocytes. In the metaphysis, i.e., the bone remodeling site, CD44-bearing osteoclasts were either proximal to or in contact with podoplanin-positive osteoblasts, but the podoplanin-positive osteoblasts also localized CD44 on their own cell surface. These podoplanin-positive osteoblasts, which either possessed CD44 on their cell surface or were close to CD44-bearing osteoclasts, showed phosphorylated ezrin-positivity on the cell membranes. Therefore, the CD44/podoplanin interaction on the cell surface may be involved in the osteoblastic differentiation into osteocytes in the metaphyses, via the mediation of podoplanin-driven ezrin phosphorylation and the subsequent reorganized assembly of actin filaments. Consistently, the protein expression of phosphorylated ezrin was increased after CD44 administration in calvarial culture. Conversely, in modeling sites such as the cortical bones, podoplanin-positive osteoblasts were uniformly localized at certain intervals even without contact with CD44-positive bone marrow cells; furthermore, they also exhibited phosphorylated ezrin immunoreactivity along their cell membranes. Taken together, it seems likely that the CD44/podoplanin interaction is involved in osteoblastic differentiation into osteocytes in the bone remodeling area but not in modeling sites.
  • Shen Zhao, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Miki Abe, Taiji Yoshida, Mai Haraguchi, Paulo Henrique Luiz de Freitas, Minqi Li, Kanchu Tei, Norio Amizuka
    Calcified tissue international 108 3 391 - 406 2020年11月10日 [査読有り]
     
    To verify whether PTH acts on bone-specific blood vessels and on cells surrounding these blood vessels, 6-week-old male mice were subjected to vehicle (control group) or hPTH [1-34] (20 µg/kg/day, PTH group) injections for 2 weeks. Femoral metaphyses were used for histochemical and immunohistochemical studies. In control metaphyses, endomucin-positive blood vessels were abundant, but αSMA-reactive blood vessels were scarce. In the PTH-administered mice, the lumen of endomucin-positive blood vessels was markedly enlarged. Moreover, many αSMA-positive cells were evident near the blood vessels, and seemed to derive from those vessels. These αSMA-positive cells neighboring the blood vessels showed features of mesenchymal stromal cells, such as immunopositivity for c-kit and tissue nonspecific alkaline phosphatase (TNALP). Thus, PTH administration increased the population of perivascular/stromal cells positive for αSMA and c-kit, which were likely committed to the osteoblastic lineage. To understand the cellular events that led to increased numbers and size of bone-specific blood vessels, we performed immunohistochemical studies for PTH/PTHrP receptor and VEGF. After PTH administration, PTH/PTHrP receptor, VEGF and its receptor flk-1 were consistently identified in both osteoblasts and blood vessels (endothelial cells and surrounding perivascular cells). Our findings suggest that exogenous PTH increases the number and size of bone-specific blood vessels while fostering perivascular/stromal cells positive for αSMA/TNALP/c-kit.
  • Akito Makino, Tomoka Hasegawa, Hideko Takagi, Yoshimasa Takahashi, Naoki Hase, Norio Amizuka
    JOURNAL OF BONE AND MINERAL RESEARCH 35 236 - 236 2020年11月 [査読有り]
  • Hiromi Hongo, Tomoka Hasegawa, Masami Saito, Kanako Tsuboi, Tomomaya Yamamoto, Muneteru Sasaki, Miki Abe, Paulo Henrique Luiz de Freitas, Hisayoshi Yurimoto, Nobuyuki Udagawa, Minqi Li, Norio Amizuka
    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 68 10 651 - 668 2020年10月 [査読有り]
     
    To demonstrate the ultrastructure of osteocytic osteolysis and clarify whether osteocytic osteolysis occurs independently of osteoclastic activities, we examined osteocytes and their lacunae in the femora and tibiae of 11-week-old male wild-type and Rankl-/- mice after injection of human parathyroid hormone (PTH) [1-34] (80 µg/kg/dose). Serum calcium concentration rose temporarily 1 hr after PTH administration in wild-type and Rankl-/- mice, when renal arteries and veins were ligated. After 6 hr, enlargement of osteocytic lacunae was evident in the cortical bones of wild-type and Rankl-/- mice, but not so in their metaphyses. Von Kossa staining and transmission electron microscopy showed broadly demineralized bone matrix peripheral to enlarged osteocytic lacunae, which contained fragmented collagen fibrils and islets of mineralized matrices. Nano-indentation by atomic force microscopy revealed the reduced elastic modulus of the PTH-treated osteocytic perilacunar matrix, despite the microscopic verification of mineralized matrix in that region. In addition, 44Ca deposition was detected by isotope microscopy and calcein labeling in the eroded osteocytic lacunae of wild-type and Rankl-/- mice. Taken together, our findings suggest that osteocytes can erode the bone matrix around them and deposit minerals on their lacunar walls independently of osteoclastic activity, at least in the murine cortical bone. (J Histochem Cytochem 68: -XXX, 2020).
  • Akito Makino, Tomoka Hasegawa, Hideko Takagi, Yoshimasa Takahashi, Naoki Hase, Norio Amizuka
    Bone 142 115651 - 115651 2020年09月18日 [査読有り]
     
    Abaloparatide (ABL) is a novel 34-amino acid peptide analog of parathyroid hormone-related protein. In clinical studies, although ABL showed a greater bone mineral density (BMD) increase than teriparatide (TPTD, human parathyroid hormone 1-34), the responses of ABL to bone formation and resorption markers were weaker, making it difficult to understand the relationship between the bone anabolic window (increase in bone formation versus resorption) and bone mass. In the present study, the effects of ABL and TPTD were compared in mice. Given that the rate of bone turnover is higher in rodents than in humans, the comparison was made with several administration regimens providing equivalent daily dosages: once daily (QD, 30 μg/kg every 24 h), twice daily (BID, 15 μg/kg every 12 h), or three times a day (TID, 10 μg/kg every 8 h). Frequent administration of ABL showed higher BMD with enhancement of trabecular and cortical bone mass and structures than that of TPTD, consistent with the clinical results seen with once daily administration. ABL increased bone formation marker levels more than TPTD with more frequent regimens, while bone resorption marker levels were not different between ABL and TPTD in all regimens. Analysis of bone histomorphometry and gene expression also suggested that ABL increased bone formation more than TPTD, while the effect on bone resorption was almost comparable between ABL and TPTD. The bone anabolic windows calculated from bone turnover markers indicated that ABL enhanced the anabolic windows more than TPTD, leading to a robust increase in BMD. The mechanism by which ABL showed a better balance of bone turnover was suggested to be partly due to the enhanced remodeling-based bone formation involved in Ephb4. Taken together, our findings would help elucidate the mechanism by which ABL shows excellent BMD gain and reduction of fractures in patients with osteoporosis.
  • Erika Tsuchiya, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Miki Abe, Taiji Yoshida, Shen Zhao, Kanako Tsuboi, Nobuyuki Udagawa, Paulo Henrique Luiz de Freitas, Minqi Li, Yoshimasa Kitagawa, Norio Amizuka
    Microscopy (Oxford, England) 70 2 201 - 214 2020年08月20日 [査読有り]
     
    This study was aimed to verify the cellular interplay between vascular endothelial cells and surrounding cells in the chondro-osseous junction of murine tibiae. Many CD31-positive endothelial cells accompanied with Dolichos Biflorus Agglutinin lectin-positive septoclasts invaded into the hypertrophic zone of the tibial epiphyseal cartilage. MMP9 immunoreactive cytoplasmic processes of vascular endothelial cells extended into the transverse partitions of cartilage columns. In contrast, septoclasts included several large lysosomes which indicate the incorporation of extracellular matrices despite no immunopositivity for F4/80 －a hallmark of macrophage/monocyte lineage. In addition, septoclasts were observed in c-fos-/- mice but not in Rankl-/- mice. Unlike c-fos-/- mice, Rankl-/- mice showed markedly-expanded hypertrophic zone and the irregular shape of the chondro-osseous junction. Immunoreactivity of PDGF-bb, which involved in angiogenic roles in the bone, was detected in not only osteoclasts but also septoclasts at the chondro-osseous junction. Therefore, septoclasts appear to assist the synchronous vascular invasion of endothelial cells at the chondro-osseous junction. Vascular endothelial cells adjacent to the chondro-osseous junction possesses endomucin but not EphB4, whereas those slightly distant from the chondro-osseous junction were intensely positive for both endomucin and EphB4, while being accompanied with ephrinB2-positive osteoblasts. Taken together, it is likely that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2.
  • Dai Sato, Masahiko Takahata, Masahiro Ota, Chie Fukuda, Tomoka Hasegawa, Tomomaya Yamamoto, Norio Amizuka, Eisuke Tsuda, Akiko Okada, Yoshiharu Hiruma, Ryo Fujita, Norimasa Iwasaki
    Bone 135 115331 - 115331 2020年06月 [査読有り]
     
    Effective treatment of juvenile osteoporosis, which is frequently caused by glucocorticoid (GC) therapy, has not been established due to limited data regarding the efficacy and adverse effects of antiresorptive therapies on the growing skeleton. We previously demonstrated that sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) targeting therapy, which interferes with osteoclast terminal differentiation in the secondary, but not primary, spongiosa, increased bone mass without adverse effects on skeletal growth, whereas bisphosphonate, a first-line treatment for osteoporosis, increased bone mass but impaired long bone growth in healthy growing rats. In the present study, we investigated the efficacy of anti-Siglec-15 neutralizing antibody (Ab) therapy against GC-induced osteoporosis in a growing rat model. GC decreased bone mass and deteriorated mechanical properties of bone, due to a disproportionate increase in bone resorption. Both anti-Siglec-15 Ab and alendronate (ALN) showed protective effects against GC-induced bone loss by suppressing bone resorption, which was more pronounced with anti-Siglec-15 Ab treatment, possibly due to a reduced negative impact on bone formation. ALN induced histological abnormalities in the growth plate and morphological abnormalities in the long bone metaphysis but did not cause significant growth retardation. Conversely, anti-Siglec-15 Ab did not show any negative impact on the growth plate and preserved normal osteoclast and chondroclast function at the primary spongiosa. Taken together, these results suggest that anti-Siglec-15 targeting therapy could be a safe and efficacious prophylactic therapy for GC-induced osteoporosis in juvenile patients.
  • Takeshi Moriishi, Ryosuke Ozasa, Takuya Ishimoto, Takayoshi Nakano, Tomoka Hasegawa, Toshihiro Miyazaki, Wenguang Liu, Ryo Fukuyama, Yuying Wang, Hisato Komori, Xin Qin, Norio Amizuka, Toshihisa Komori
    PLoS genetics 16 5 e1008586  2020年05月 [査読有り][通常論文]
     
    The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.
  • Hayato Ohshima, Norio Amizuka
    JOURNAL OF ORAL BIOSCIENCES 62 1 1 - 8 2020年03月 [査読有り][通常論文]
     
    Background: Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences.Highlight: This review features review articles in the fields of "Bone Cell Biology," "Microbiology," "Oral Heath," "Biocompatible Materials," "Mouth Neoplasm," and "Biological Evolution" in addition to the review articles by winners of the Lion Dental Research Award ("Role of nicotinic acetylcholine receptors for modulation of microcircuits in the agranular insular cortex" and "Phospholipase C-related catalytically inactive protein: A novel signaling molecule for modulating fat metabolism and energy expenditure") and the Rising Members Award ("Pain mechanism of oral ulcerative mucositis and the therapeutic traditional herbal medicine hangeshashinto," "Mechanisms underlying the induction of regulatory T cells by sublingual immunotherapy," and "Regulation of osteoclast function via Rho-Pkn3-c-Src pathways"), presented by the Japanese Association for Oral Biology.Conclusion: These reviews in the Journal of Oral Biosciences have inspired the readers of the journal to broaden their knowledge regarding various aspects of oral biosciences. The current editorial review introduces these exciting review articles. (C) 2020 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
  • Ai Hanazaki, Kayo Ikuta, Shohei Sasaki, Sumire Sasaki, Megumi Koike, Kazuya Tanifuji, Yuki Arima, Ichiro Kaneko, Yuji Shiozaki, Sawako Tatsumi, Tomoka Hasegawa, Norio Amizuka, Ken-Ichi Miyamoto, Hiroko Segawa
    Physiological reports 8 3 e14324  2020年02月 [査読有り][通常論文]
     
    SLC34A3/NPT2c/NaPi-2c/Npt2c is a growth-related NaPi cotransporter that mediates the uptake of renal sodium-dependent phosphate (Pi). Mutation of human NPT2c causes hereditary hypophosphatemic rickets with hypercalciuria. Mice with Npt2c knockout, however, exhibit normal Pi metabolism. To investigate the role of Npt2c in Pi homeostasis, we generated α-klotho-/- /Npt2c-/- (KL2cDKO) mice and analyzed Pi homeostasis. α-Klotho-/- (KLKO) mice exhibit hyperphosphatemia and markedly increased kidney Npt2c protein levels. Genetic disruption of Npt2c extended the lifespan of KLKO mice similar to that of α-Klotho-/- /Npt2a-/- mice. Adult KL2cDKO mice had hyperphosphatemia, but analysis of Pi metabolism revealed significantly decreased intestinal and renal Pi (re)absorption compared with KLKO mice. The 1,25-dihydroxy vitamin D3 concentration was not reduced in KL2cDKO mice compared with that in KLKO mice. The KL2cDKO mice had less severe soft tissue and vascular calcification compared with KLKO mice. Juvenile KL2cDKO mice had significantly reduced plasma Pi levels, but Pi metabolism was not changed. In Npt2cKO mice, plasma Pi levels began to decrease around the age of 15 days and significant hypophosphatemia developed within 21 days. The findings of the present study suggest that Npt2c contributes to regulating plasma Pi levels in the juvenile stage and affects Pi retention in the soft and vascular tissues in KLKO mice.
  • Tomoka Hasegawa, Yukina Miyamoto, Zixuan Qiu, Hiromi Hongo, Norio Amizuka, Tomomaya Yamamoto
    JOURNAL OF BONE AND MINERAL RESEARCH 34 142 - 142 2019年12月 [査読有り]
  • Yoshimasa Kitagawa, Noritaka Ohga, Takuya Asaka, Jun Sato, Hironobu Hata, Joseph Heiman, Kanako Tsuboi, Norio Amizuka, Yuji Kuge, Tohru Shiga
    JAPANESE DENTAL SCIENCE REVIEW 55 1 65 - 70 2019年11月 [査読有り][通常論文]
     
    Medication-related osteonecrosis of jaws (MRONJ) is one of the most complicated inflammatory conditions in oral and maxillofacial region. It is very difficult to correctly evaluate the degree and extent of necrosis and infection. This refractory osteonecrosis often needs extended surgery, leading to impaired quality-of-life. We have performed hyperbaric oxygen therapy (HBO) combined with conservative surgery for advanced cases. We have appraised the value of FDG-PET and 3-phase bone scintigraphy in the diagnosis and management of this condition. MRONJ showed significantly higher SUVmax on FDG-PET than the others. Although the 3 phase pool bone images did not change significantly, perfusion and static bone image as well as PET showed remarkable response to HBO for MRONJ. SUVmax after HBO was significantly lower than those of before HBO. These preliminary results indicate that FDG-PET is useful for monitoring the effect of HBO for MRONJ. (C) 2019 Published by Elsevier Ltd on behalf of The Japanese Association for Dental Science.
  • Tomoka Hasegawa, Yukina Miyamoto-Takasaki, Miki Abe, Zixuan Qiu, Tomomaya Yamamoto, Yimin, Taiji Yoshida, Hirona Yoshino, Hiromi Hongo, Ayako Yokoyama, Muneteru Sasaki, Shinichiro Kuroshima, Kuniko Hara, Masatoshi Kobayashi, Yasuhiro Akiyama, Takeyasu Maeda, Paulo Henrique Luiz de Freitas, Minqi Li, Norio Amizuka
    MICROSCOPY 68 5 349 - 358 2019年10月 [査読有り][通常論文]
     
    In this study, we aimed to clarify the role of ascorbic acid in collagen synthesis in periodontal ligaments using osteogenic disorder Shionogi (ODS)/ShiJcl-od/od rats lacking L-gulonolactone oxidase. These rats cannot synthesize ascorbic acid in vivo. Eight-week-old ODS/ShiJcl-od/od male rats were administered ascorbic acid solution at a concentration of 200 mg/dL (control group, n = 6) or ascorbic acid solution at concentration of 0.3 mg/dL (insufficient group, n = 12). Six rats of the insufficient group were then given with ascorbic acid solution at concentration of 200 mg/dL for additional 3 weeks (rescued group, n = 6), and then, their mandibles were histochemically examined. Consequently, the insufficient group specimens were seen to possess fewer collagen fibers, and silver impregnation revealed numerous fine, reticular fiber-like fibrils branching off from collagen in the periodontal ligaments. In control group, faint immunoreactivities for matrix metalloproteinase (MMP)2 and cathepsin H were seen in the periphery of blood vessels and throughout the ligament, respectively. In contrast, in the insufficient group, intense MMP2-immunoreactivity was observed to be associated with collagen fibrils in the periodontal ligaments, and cathepsin H-immunopositivity was seen in ligamentous cells. The rescued group showed abundant collagen fibers filling the periodontal ligament space. Under transmission electron microscopy, ligamentous fibroblasts incorporated collagen fibrils into tubular endosomes/lysosomes while simultaneously synthesizing collagen fibril bundles. Thus, ascorbic acid insufficiency affected the immunolocalization of cathepsin H and MMP2; however, ligamentous fibroblasts appear to possess the potential to synthesize collagen fibers when supplied with ascorbic acid.
  • Naoya Toriu, Toshiharu Ueno, Hiroki Mizuno, Akinari Sekine, Noriko Hayami, Rikako Hiramatsu, Keiichi Sumida, Masayuki Yamanouchi, Eiko Hasegawa, Tatsuya Suwabe, Junichi Hoshino, Naoki Sawa, Kenmei Takaichi, Takeshi Fujii, Tomoka Hasegawa, Norio Amizuka, Motoko Yanagita, Yoshifumi Ubara
    Bone reports 10 100187 - 100187 2019年06月 [査読有り][通常論文]
     
    We report a 48-year-old Japanese man with a brown tumor of the right distal tibia. At the age of 25 years, hemodialysis was initiated due to nail-patella syndrome. Severe secondary hyperparathyroidism and osteoporosis progressed over time, so parathyroidectomy was performed at age 45. Spontaneous fracture of the right distal tibia occurred suddenly at age 48. Imaging studies revealed a bone tumor-like lesion and surgery was performed. The resected specimen was a brown mass consisting of multinucleated giant cells on a fibrous tissue background, and these findings were consistent with a diagnosis of brown tumor. Immunohistochemistry revealed that multinucleated giant cells near areas of bone matrix were positive for tartrate-resistant acid phosphatase and cathepsin K, but the majority of the giant cells in the lesion were negative for these markers. Even after parathyroidectomy, brown tumor should be considered in the differential diagnosis of bone tumor-like lesions in patients on long-term dialysis. This case suggests that osteoclast activation may not contribute to development of brown tumors, although these lesions are generally considered to arise from subperiosteal bone resorption related to osteoclast overactivity in patients with hyperparathyroidism.
  • Tsuneyuki Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
    MICROSCOPY 68 3 243 - 253 2019年06月 [査読有り][通常論文]
     
    In this study the following were suggested: (1) in osteoclasts the nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum; (2) consequently, the Golgi apparatus accumulates and wraps all nuclei together in a multi-spherical configuration with the cis-side facing the nuclear membrane.Abstract This study was designed to observe osteoclasts in the rat femora by light and electron microscopic cytochemistry for nicotinamide adenine dinucleotide phosphatase (NADPase) and arylsulfatase, and scanning electron microscopy using osmium maceration to assess the three-dimensional morphology of the Golgi apparatus in osteoclasts. The Golgi apparatus showed strong NADPase activity and surrounded each nucleus with the cis-side facing the nucleus. The Golgi apparatus could be often traced for a length of 20 m or longer. Observations of serial semi-thin sections confirmed that a single line of reaction products (=lead precipitates) intervened somewhere between any two neighboring nuclei. The nuclear membrane showed strong arylsulfatase activity as well as rough endoplasmic reticulum and lysosomes. Scanning electron microscopy showed that the Golgi apparatus covered the nucleus in a porous sheet-like configuration. Under magnification, the cis-most saccule showed a sieve-like configuration with fine fenestrations. The saccules decreased fenestration numbers toward the trans-side and displayed a more plate-like appearance. The above findings indicate the following. (1) The Golgi saccules of osteoclasts have a three-dimensional structure comparable with that generally seen in other cell types. (2) The Golgi apparatus forms a porous multi-spherical structure around nuclei. Within the structure, in most cases a Golgi stack partitions the room into several compartments in each of which a nucleus fits. (3) The nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum. Consequently, the Golgi apparatus accumulates around nuclei with the cis-side facing the nucleus.
  • Tsuneyuki Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
    JOURNAL OF ORAL BIOSCIENCES 61 2 105 - 114 2019年06月 [査読有り][通常論文]
     
    Background: Human cellular cementum and compact bone exhibit an alternating lamellar structure, in which intensely and faintly stainable lamellae are stratified in an alternating manner. Many investigators, including our group, have accumulated considerable data regarding lamellar structure. In this review, we summarize the alternating lamellar structure, based on available data, and introduce our hypothesis regarding its formation.Highlight: We implemented 10% and 24% NaOH maceration methods for scanning electron microscopy. The 10% NaOH maceration method was used for detailed examination of the collagen fibril arrangement, whereas the 24% NaOH maceration method was used for examination of cell morphology in the absence of collagen fibrils. The following findings were obtained: (1) sections of cementum and bone showed two types of alternating lamellae-those comprising longitudinally and nearly longitudinally arranged fibril arrays, and those comprising transversely and nearly transversely arranged fibril arrays; (2) the fibril arrays appeared to shift arrangement in a regular and periodic manner, such that the alternating lamellar structure appeared in sections; (3) where the alternating lamellar structure was being formed, osteoblasts and cementoblasts extended slender processes alongside newly deposited fibrils.Conclusion: Our data showed that the alternating lamellar structure was consistent with the twisted plywood model previously proposed for osteonal lamellae. For the formation of this structure, there have been two major hypotheses: a self-assembly hypothesis and a cellular control hypothesis. Our data support the latter; osteoblasts and cementoblasts move their processes synchronously and periodically to control fibril arrangement, thereby forming the alternating lamellar structure. (C) 2019 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
  • Junichi Hoshino, Tomoka Hasegawa, Rikako Hiramatsu, Hiroki Mizuno, Akinari Sekine, Masahiro Kawada, Keiichi Sumida, Noriko Hayami, Masayuki Yamanouchi, Eiko Hasegawa, Tatsuya Suwabe, Naoki Sawa, Ken Mei Takaichi, Norio Amizuka, Masaji Hashimoto, Yoshifumi Ubara
    CLINICAL CASES IN MINERAL AND BONE METABOLISM 16 2 178 - 180 2019年05月 [査読有り]
     
    In April 2013, a 73-years-old Japanese man was admitted to our institute for the evaluation of metastatic liposarcoma. Soft tissue adjacent to the bone tissue was investigated immunohistochemically to evaluate the mechanism of new bone formation. Multinucleated giant cells accumulating on the bone tissue surface were strongly positive for tartrate-resistant acid phosphatase (TRAP) and cathepsin K, satisfying the histochemical criteria for identification of osteoclasts. In contrast, mononuclear cells on the bone tissue surface demonstrated immunopositivity for alkaline phosphatase (ALP) and were associated with osteocalcin staining of the bone surface and interior, indicating that these cells were probably osteoblasts.
  • Sadaoki Sakai, Hiromi Hongo, Tomomaya Yamamoto, Tomoka Hasegawa, Satoshi Takeda, Hitoshi Saito, Koichi Endo, Kenji Yogo, Norio Amizuka
    CALCIFIED TISSUE INTERNATIONAL 104 3 251 - 261 2019年03月 [査読有り][通常論文]
     
    Parathyroid hormone (PTH) analogs have a powerful anabolic effect on bone and are used in the treatment of patients with severe osteoporosis. However, there are limitations to how long they can be safely administered. Withdrawal of PTH results in the cancelation of its effects, necessitating subsequent treatment to maintain the bone quantity and quality. This study assessed the effects of Eldecalcitol (ELD), an active vitamin D3 derivative, after PTH in estrogen-deficient osteoporotic rats. Six-month-old female rats were ovariectomized, and PTH administration was started 7weeks later. After 4weeks of PTH treatment, the animals were divided into three groups and either continued to receive PTH (PTH-PTH), or were switched to ELD (PTH-ELD) or vehicle (PTH-Veh) for an additional 4weeks. In the femur, increased BMD by 4weeks treatment of PTH was significantly reduced in PTH-Veh but not in PTH-PTH and PTH-ELD. The same tendency was observed in the lumbar vertebrae. MicroCT imaging and histomorphometry analysis revealed that the favorable bone structure changes by PTH administration were also maintained in the femurs and tibias of the PTH-PTH and PTH-ELD groups. Increased bone strength by 4-week treatment of PTH in lumber also maintained in PTH-ELD. Furthermore, minimodeling was observed in the PTH-ELD group. These results demonstrate that treatment with ELD sequentially following PTH prevented the bone quantity and strength reduction that accompanies PTH withdrawal in estrogen-deficient rats.
  • Hayato Ohshima, Norio Amizuka
    JOURNAL OF ORAL BIOSCIENCES 61 1 1 - 4 2019年03月 [査読有り][通常論文]
     
    Background: The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge regarding every aspect of oral biosciences.Highlight: This editorial review features summaries of review articles in the fields of "Bone Biology," "Epigenomics," "Periodontium," and "Amelogenesis" in addition to review articles by winners of the Lion Dental Research Award ("Role of non-canonical Wnt signaling pathways in bone resorption," "Mechanisms of orofacial sensory processing in the rat insular cortex," and "Analysis of the mechanism in salivary gland development using gene database") and the Rising Members Award ("Synergistic findings from microbiological and evolutional analyses of virulence factors among pathogenic streptococcal species" and "Free fatty acids may be involved in the pathogenesis of oral-related and cardiovascular diseases"), presented by the Japanese Association for Oral Biology.Conclusion: These reviews published in the Journal of Oral Biosciences have inspired the readers of the Journal to broaden their knowledge of various aspects in the oral biosciences. This editorial review summarizes these exciting articles. (C) 2019 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
  • Masahiro Ota, Masahiko Takahata, Tomohiro Shimizu, Daisuke Momma, Hiroki Hamano, Shigeto Hiratsuka, Norio Amizuka, Tomoka Hasegawa, Norimasa Iwasaki
    JOURNAL OF BONE AND MINERAL METABOLISM 37 2 256 - 263 2019年03月 [査読有り][通常論文]
     
    Despite preclinical studies demonstrating the effectiveness of teriparatide for skeletal repair in small animals, inconclusive data from clinical trials have raised questions regarding the optimal teriparatide dosing regimen for bone repair. To address this, we assessed the effect of teriparatide frequency and dose on long-bone healing using a mouse femur osteotomy/fracture model. Eight-week-old male ICR mice were subjected to open femur osteotomies, then randomized into following five groups (n=8 per group): vehicle; low dose/high frequency: 3g/kg/dose, 3 times/day; low dose/low frequency: 9g/kg/dose, 1 time/day; high dose/high frequency: 9g/kg/dose, 3 times/day; high dose/low frequency: 27g/kg/dose, 1 time/day. Skeletal repair was assessed by microcomputed tomography, mechanical testing, and histology 4weeks after surgery. High-dose and/or high-frequency teriparatide treatment increased callus bone volume but failed to have a significant impact on the biomechanical recovery of fractured femurs, possibly because of impaired cortical shell formation in fracture calluses. Meanwhile, low-dose/low-frequency teriparatide therapy enhanced callus bone formation without interfering with cortical shell formation despite a lesser increase in callus bone volume, leading to significant two and fourfold increases in ultimate load and stiffness, respectively. Our findings demonstrate that administering teriparatide at higher doses and/or higher frequencies raises fracture callus volume but does not always accelerate the biomechanical recovery of fractured bone, which points to the importance of finding the optimal teriparatide dosing regimen for accelerating skeletal repair.
  • Tomoka Hasegawa, Tomomaya Yamamoto, Sadaoki Sakai, Yukina Miyamoto, Hiromi Hongo, Zixuan Qiu, Miki Abe, Satoshi Takeda, Kimimitsu Oda, Paulo Henrique Luiz de Freitas, Minqi Li, Koichi Endo, Norio Amizuka
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 67 3 169 - 184 2019年03月 [査読有り][通常論文]
     
    Intermittent administration of human parathyroid hormone (1-34) (hPTH(1-34)) promotes anabolic action in bone by stimulating bone remodeling, while eldecalcitol, an analog of active vitamin D3, suppresses osteoclastic bone resorption, and forms new bone by minimodeling. We have examined the biological effects of combined administration of eldecalcitol and hPTH(1-34) on 9-week-old Wistar rats that underwent an ovariectomy (OVX) or Sham operation. They were divided into a Sham group, OVX with vehicle (OVX group), OVX with 10 mu g/kg/day of hPTH(1-34) (PTH group), OVX with 20 ng/kg/day of eldecalcitol (eldecalcitol group) or OVX with 10 g/kg/day of hPTH(1-34), and 20 ng/kg/day of eldecalcitol (combined group) for 4 or 8 weeks. As a consequence, the combined group showed a marked increase in bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) than OVX and had the highest bone mineral density (BMD) compared with other groups. OVX and PTH groups exhibited a high osteoblastic surface/bone surface (Ob.S/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS) indices and many TRAP-reactive osteoclasts. Contrastingly, eldecalcitol and combined groups tended to attenuate the indices of osteoclastic surface/bone surface (Oc.S/BS) and Ob.S/BS than that the other groups. The combined group revealed histological profiles of minimodeling- and remodeling-based bone formation. Thus, the combined administration of eldecalcitol and hPTH(1-34) augments their anabolic effects by means of minimodeling and remodeling.
  • Hasegawa T, Miyamoto Y, Yamamoto T, Amizuka N
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 153 1 16 - 21 公益社団法人 日本薬理学会 2019年 [査読有り][通常論文]
     

    骨粗鬆症治療薬の副甲状腺ホルモン（PTH）製剤として，遺伝子組み換え型ヒトPTH［1-34］であるテリパラチドが用いられている．我が国においては，連日製剤と週1回製剤の2種類が存在しており，そのどちらも骨量を増加させ，また，骨折率を低下させることが報告されている．一般的にPTHを持続投与すると骨吸収を誘導するのに対して，PTH間歇投与では骨量増加につながることが知られている．その細胞学的メカニズムとして，PTHは骨芽細胞の前駆細胞である前骨芽細胞に対して細胞増殖を亢進する一方，成熟型骨芽細胞に対しては破骨細胞とのカップリングに依存して，骨形成を促進することが報告されている．さらに，我々は，PTH間歇投与の頻度（投与間隔）の違いにより，骨の細胞群の挙動が異なることを明らかにした．PTH高頻度投与では，成熟型骨芽細胞が活発に骨基質合成を行うだけでなく，前骨芽細胞の増殖が亢進して厚い細胞性ネットワークを形成し，その中で多数の破骨細胞が誘導されていた．その結果，高骨代謝回転の骨リモデリングにより骨形成が誘導されていた．しかし，PTH低頻度投与では，成熟型骨芽細胞による骨形成が亢進するが，前骨芽細胞はあまり増加せず，よって破骨細胞形成の誘導も上昇しなかった．この場合，骨リモデリングとミニモデリングの両方によって骨形成が誘導されることが明らかにされた．このように，PTH製剤の投与頻度が異なると，骨の形態や形成のされ方が違うことが強く示唆された．以上より，PTH製剤の投与頻度によって骨形成促進における作用機序が異なると思われる．

  • Naznin Khadiza, Tomoka Hasegawa, Tomoya Nagai, Tomomaya Yamamoto, Yukina Miyamoto-Takasaki, Hiromi Hongo, Miki Abe, Mai Haraguchi, Tsuneyuki Yamamoto, Yimin, Zixuan Qiu, Muneteru Sasaki, Shinichiro Kuroshima, Hayato Ohshima, Paulo Henrique Luiz de Freitas, Minqi Li, Yasutaka Yawaka, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 40 4 133 - 143 2019年 [査読有り][通常論文]
     
    In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.
  • Bo Liu, Wei Feng, Tomoka Hasegawa, Norio Amizuka, Minqi Li
    HISTOLOGY AND HISTOPATHOLOGY 34 1 57 - 67 2019年01月 [査読有り][通常論文]
     
    To investigate type 1 diabetes mellitus (T1DM) affecting bone remodeling in the context of menopause in female rats. The animals were subjected to either ovariectomy (OVX) which was performed to mimic postmenopausal estrogen deficiency, and/or type 1 diabetes mellitus which was established by the intra-abdominal administration of 50 mg/kg streptozotocin (STZ). Single-loaded groups were the OVX group and the STZ group, while the combined group was the OVX+STZ group. Bone histomorphometry was performed on the tibial metaphysis by Micro-CT scanning. Immunohistochemistry was used to assess the activity of osteoblast and osteoclast by counteracting with antibodies against their respective specific marker enzymes. The gene expression of key molecules involved in osteoblastic and osteoclastic signaling pathways were analyzed by RT-qPCR. The results showed a significant bone volume decrease in both single-loaded groups and combined group with the combined group suffering greatest bone loss and bone structural deterioration (p<0.001). Immunohistochetnical staining and RT-qPCR revealed an increase in osteoblastic (p<0.001) and osteoclastic (p<0.01) activities in OVX rats while there was a decrease (p<0.05) in those of STZ rats. When OVX and STZ were combined, the rats exhibited a further decrease in osteoblastic activity (p<0.001) and a similar level of osteoclastic activity (p>0.05) compared to their STZ counterparts. These results demonstrated that STZ-induced T1DM reverses OVX-associated high bone turnover osteoporosis to the type of low bone turnover, leading to greater bone loss and structural defect.
  • Zhang X, Feng H, Du J, Sun J, Li D, Hasegawa T, Amizuka N, Li M
    International journal of molecular medicine 42 6 3135 - 3148 2018年12月 [査読有り][通常論文]
     
    Rheumatoid arthritis (RA) is a commonly occurring autoimmune disease. Its defining pathological characteristic is the excessive proliferation of fibroblast-like synoviocytes (FLS), which is similar to tumor cells and results in a range of clinical problems. As a commonly used antipyretic, analgesic and anti-inflammatory drug, aspirin is the first-line treatment for RA. However, its mechanism of action has not been well explained. The goal is to investigate the biological effects of aspirin on primary RA-FLS and its underlying mechanisms. In this experiment we treated cells with various concentrations of aspirin (0, DMSO, 1, 2, 5, 10 mM). Cell proliferation activity was detected with CCK-8 assays. Apoptosis and cell cycle distribution were detected via flow cytometry. Apoptosis and cell cycle-associated proteins (Bcl-2, Bax, PRAP1, Cyclin D1, P21), as well as the key proteins and their phosphorylation levels of the NF-kappa B and JAK/STAT3 signaling pathways, were detected via western blot analysis. Bioinformatics prediction revealed that aspirin was closely associated with cell proliferation and apoptosis, including the p53 and NF-kappa B signaling pathways. By stimulating with aspirin, cell viability decreased, while the proportion of apoptotic cells increased, and the number of cells arrested in the G(0)/G(1) phase increased in a dose-dependent manner. The expression of Bax increased with aspirin stimulation, while the levels of Bcl-2, PRAP1, Cyclin D1 and P21 decreased; p-STAT3, p-P65 and p-50 levels also decreased while STAT3, P65, P50, p-P105 and P105 remained unchanged. From our data, it can be concluded that aspirin is able to promote apoptosis and inhibit the proliferation of RA-FLS through blocking the JAK/STAT3 and NF-kappa B signaling pathways.
  • Dai Sato, Masahiko Takahata, Masahiro Ota, Chie Fukuda, Eisuke Tsuda, Tomohiro Shimizu, Hiroki Hamano, Sigeto Hiratsuka, Akiko Okada, Ryo Fujita, Norio Amizuka, Tomoka Hasegawa, Nrimasa Iwasaki
    JOURNAL OF BONE AND MINERAL RESEARCH 33 34 - 34 2018年11月 [査読有り][通常論文]
  • Akito Makino, Tomoka Hasegawa, Norio Amizuka
    JOURNAL OF BONE AND MINERAL RESEARCH 33 295 - 295 2018年11月 [査読有り][通常論文]
  • Dai Sato, Masahiko Takahata, Masahiro Ota, Chie Fukuda, Eisuke Tsuda, Tomohiro Shimizu, Akiko Okada, Yoshiharu Hiruma, Hiroki Hamano, Shigeto Hiratsuka, Ryo Fujita, Norio Amizuka, Tomoka Hasegawa, Norimasa Iwasaki
    BONE 116 172 - 180 2018年11月 [査読有り][通常論文]
     
    The treatment of juvenile osteoporosis has not been established due to a lack of data regarding the efficacy and adverse effects of therapeutic agents. The possible adverse effects of the long-term use of antiresorptive therapies on skeletal growth in children is of particular concern. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec15) is an immunoreceptor that regulates osteoclast development and bone resorption, and its deficiency suppresses bone remodeling in the secondary spongiosa, but not in the primary spongiosa, due to a compensatory mechanism of osteoclastogenesis. This prompted us to develop an anti-Siglec-15 therapy for juvenile osteoporosis because most anti-resorptive drugs have potential adverse effects on skeletal growth. Using growing rats, we investigated the effects of an anti-Siglec-15 neutralizing antibody (Ab) on systemic bone metabolism and skeletal growth, comparing this drug to bisphosphonate, a first-line treatment for osteoporosis. Male 6-week-old F344/Jcl rats were randomized into six groups: control (PBS twice per week), anti-Siglec-15 Ab (0.25, 1, or 4 mg/kg every 3 weeks), and alendronate (ALN) (0.028 or 0.14 mg/kg twice per week). Treatment commenced at 6 weeks of age and continued for the next 6 weeks, Changes in bone mass, bone metabolism, bone strength, and skeletal growth during treatment were analyzed. Both anti-Siglec-15 therapy and ALN increased bone mass and the mechanical strength of both the femora and lumbar spines in a dose-dependent manner. Anti-Siglec-15 therapy did not have a significant effect on skeletal growth as evidenced by micro-CT-based measurements of femoral length and histology, whereas high-dose ALN resulted in growth retardation with histological abnormalities in the growth plates of femurs. This unique property of the anti-Siglec-15 Ab can probably be attributed to compensatory signaling for Siglec-15 inhibition in the primary spongiosa, but not in the secondary spongiosa. Thus, anti-Siglec-15 therapy could be a safe and effective for juvenile osteoporosis.
  • Akemi Shimada, Hisashi Ideno, Yoshinori Arai, Koichiro Komatsu, Satoshi Wada, Teruhito Yamashita, Norio Amizuka, Ernst Poschl, Bent Brachvogel, Yoshiki Nakamura, Kazuhisa Nakashima, Hiroaki Mizukami, Yoichi Ezura, Akira Nifuji
    JOURNAL OF BONE AND MINERAL RESEARCH 33 8 1532 - 1543 2018年08月 [査読有り][通常論文]
     
    Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5(-/-)) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5(-/-) mice than in wildtype (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5(-/-) mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5(-/-) mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5(-/-) and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5(-/-) mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. (c) 2018 American Society for Bone and Mineral Research.
  • Ichiro Kaneko, Hiroko Segawa, Kayo Ikuta, Ai Hanazaki, Toru Fujii, Sawako Tatsumi, Shinsuke Kido, Tomoka Hasegawa, Norio Amizuka, Hitoshi Saito, Ken-ichi Miyamoto
    ENDOCRINOLOGY 159 7 2741 - 2758 2018年07月 [査読有り][通常論文]
     
    X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D-3 analog, eldecalcitol [1 alpha,25-dihydroxy-2 beta-(3-hydroxypropyloxy) vitamin D-3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.
  • Xiaoqi Zhang, Hao Feng, Dongfang Li, Shanshan Liu, Norio Amizuka, Minqi Li
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 19 6 2018年06月 [査読有り][通常論文]
     
    Oral squamous cell carcinoma (OSCC) is a malignant disease. Methylation plays a key role in the etiology and pathogenesis of OSCC. The goal of this study was to identify aberrantly methylated differentially expressed genes (DEGs) in OSCCs, and to explore the underlying mechanisms of tumorigenesis by using integrated bioinformatic analysis. Gene expression profiles (GSE30784 and GSE38532) were analyzed using the R software to obtain aberrantly methylated DEGs. Functional enrichment analysis of screened genes was performed using the DAVID software. Protein-protein interaction (PPI) networks were constructed using the STRING database. The cBioPortal software was used to exhibit the alterations of genes. Lastly, we validated the results with the Cancer Genome Atlas (TCGA) data. Twenty-eight upregulated hypomethylated genes and 24 downregulated hypermethylated genes were identified. These genes were enriched in the biological process of regulation in immune response, and were mainly involved in the PI3K-AKT and EMT pathways. Additionally, three upregulated hypomethylated oncogenes and four downregulated hypermethylated tumor suppressor genes (TSGs) were identified. In conclusion, our study indicated possible aberrantly methylated DEGs and pathways in OSCCs, which could improve the understanding of the underlying molecular mechanisms. Aberrantly methylated oncogenes and TSGs may also serve as biomarkers and therapeutic targets for the precise diagnosis and treatment of OSCCs in the future.
  • Isotope microscopic assessment for localization of 15N-minodeonate in bone.
    Hongo H, Sasaki M, Hasegawa T, Tsuboi K, Qiu Z, Amizuka N
    Hokkaido Journal of Dental Science. 38 2 1 - 8 2018年04月 [査読有り][通常論文]
  • Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Zixuan Qiu, Miki Abe, Takuma Kanesaki, Kawori Tanaka, Takashi Endo, Paulo Henrique Luiz de Freitas, Minqi Li, Norio Amizuka
    HISTOCHEMISTRY AND CELL BIOLOGY 149 4 423 - 432 2018年04月 [査読有り][通常論文]
     
    The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes' cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.
  • Norio Amizuka, Sohei Kitazawa
    HISTOCHEMISTRY AND CELL BIOLOGY 149 4 287 - 288 2018年04月 [査読有り][通常論文]
  • Hao Feng, Ziyu Li, Juan Du, Jing Sun, Wei Feng, Dongfang Li, Shanshan Lui, Wei Wang, Hongrui Liu, Norio Amizuka, Minqi Li
    CELL DEATH DISCOVERY 4 1 47 - 47 2018年04月 [査読有り][通常論文]
     
    Lipopolysaccharide (LPS)-induced osteoblast apoptosis is a prominent factor to the defect in periodontal tissue repair in periodontal disease. LPS challenge contributes to the production of reactive oxygen species (ROS) in periodontitis, and peroxiredoxin 1 (Prx1) is an antioxidant protein that protect cells against oxidative damage from ROS. Without LPS stimulation, apoptotic rates were higher in both Prx1 knockout (Prx1(KO)) and Prx1 overexpression (Prx1(OE)) cells compared with wild type. After LPS stimulation, intracellular ROS in Prx1(KO) cells showed the highest level and Prx1(OE) cells showed the least. Treatment with LPS significantly elevated the expression of Bax, Cyto-c, and caspase 3 in Prx1(KO) cells compared with wild type, although this could be completely abolished by NAC. In Prx1(OE) cells, the expression and activation of ASK1 were significantly increased, and this was slightly reduced by LPS stimulation. NQDI-1 completely abolished the increased phosphorylation of JNK and p38 and the expression of caspase 3 in LPS-stimulated cells. These results indicate that Prx1 eliminates intracellular ROS and exhibits a cytoprotective role in LPS-induced apoptosis. However, under physiological conditions, Prx1 overexpression acts as a H2O2 messenger, triggering the expression of ASK1 and its downstream cascades.
  • Toshinobu Omiya, Jun Hirose, Tomoka Hasegawa, Norio Amizuka, Yasunori Omata, Naohiro Izawa, Hisataka Yasuda, Yuho Kadono, Morio Matsumoto, Masaya Nakamura, Takeshi Miyamoto, Sakae Tanaka
    BONE 107 18 - 26 2018年02月 [査読有り][通常論文]
     
    We examined the effect of teriparatide, and switching from teriparatide to anti-RANKL (receptor activator of nuclear factor kappa B ligand) monoclonal antibody, in ovariectomized mice. Twelve-week-old female C57BL/6 mice were ovariectomized or sham operated. Four weeks after surgery, ovariectomized mice were subjected to one of the following four treatments: phosphate-buffered saline (PBS) for 8 weeks; teriparatide for 4 weeks followed by PBS for 4 weeks (PTH4W group); teriparatide for 8 weeks (PTH8W group); or teriparatide for 4 weeks followed by anti-RANKL antibody (single subcutaneous injection of 5 mg/kg) (SWITCH group). Twelve weeks after the operation, bone mineral density was increased in PTH8W and SWITCH groups to broadly comparable levels, but these were significantly decreased in the PTH4W group after discontinuation of teriparatide. Histomorphometric analysis demonstrated that cancellous bone formation and resorption were profoundly suppressed in the SWITCH group. Bone formation was also suppressed on the endocortical surface of cortical bone but was maintained on the periosteal surface. Anti-RANKL antibody suppressed osteoclast activity immediately after treatment, while bone formation was only gradually decreased. These results suggest that anti-RANKL antibody maybe a therapeutic option after discontinuation of teriparatide therapy. (C) 2017 Elsevier Inc. All rights reserved.
  • The diversity of preosteoblastic morphology -Preosteoblastic response to parathyroid hormone-.
    Qiu Z, Miyamoto Y, Yamamoto T, Hongo H, Abe M, Yoshida T, Yoshino H, Sasaki M, Nagai T, Khadiza N, Yokoyama A, Zhao S, Mae T, Kirikoshi S, Moritani Y, Haraguchi M, Freitas PHL, Li M, Amizuka N, Hasegawa T
    Hokkaido Journal of Dental Science. 39 1 2 - 10 2018年 [査読有り][通常論文]
  • Pingping Cui, Hongrui Liu, Jing Sun, Norio Amizuka, Qinfeng Sun, Minqi Li
    HISTOLOGY AND HISTOPATHOLOGY 33 1 89 - 99 2018年01月 [査読有り][通常論文]
     
    Nitrogen-containing bisphosphonates (N-BPs) are potent antiresorptive drugs and their actions on osteoclasts have been studied extensively. Recent studies have suggested that N-BPs also target bone-forming cells. However, the precise mechanism of N-BPs in osteoblasts is paradoxical, and the specific role of osteocytes is worthy of in-depth study. Here, we investigated the cellular mechanisms of N-BPs regulating bone defect healing by zoledronate (ZA). Bone histomorphometry confirmed an increase in new bone formation by systemic ZA administration. ZA induced more alkaline phosphatase-positive osteoblasts and tartrate-resistant acid phosphatase-positive osteoclasts residing on the bone surface. Inexplicably, ZA increased SOST expression in osteocytes embedded in the bone matrix, which was not compatible with the intense osteoblast activity on the bone surface. ZA induced heterogeneous osteocytes and disturbed the distribution of the osteocytic-canalicular system (OLCS). Furthermore, according to the degree of OLCS regularity, dentin matrix protein 1 reactivity had accumulated around osteocytes in the ZA group, but it was distributed evenly in the OLCS of the control group. The control group showed a dense array of the gap junction protein connexin 43. However, connexin 43 was extremely sparse after ZA administration. In summary, ZA treatment reduces gap junction connections and blocks cellular communication between osteocytes and osteoblasts. Retaining SOST expression in osteocytes leads to activation of the Wnt signaling pathway and subsequent bone formation.
  • Chie Fukuda, Eisuke Tsuda, Akiko Okada, Norio Amizuka, Tomoka Hasegawa, Tsuyoshi Karibe, Yoshiharu Hiruma, Nana Takagi, Seiichiro Kumakura
    JOURNAL OF BONE AND MINERAL RESEARCH 32 S112 - S113 2017年12月 [査読有り][通常論文]
  • Shusuke Murakami, Hirofumi Miyaji, Erika Nishida, Kohei Kawamoto, Saori Miyata, Hiroko Takita, Tsukasa Akasaka, Bunshi Fugetsu, Toshihiko Iwanaga, Hiromi Hongo, Norio Amizuka, Tsutomu Sugaya, Masamitsu Kawanami
    DENTAL MATERIALS JOURNAL 36 5 573 - 583 2017年10月 [査読有り][通常論文]
     
    Three-dimensional collagen scaffolds coated with beta-tricalcium phosphate (beta-TCP) nanoparticles reportedly exhibit good bioactivity and biodegradability. Dose effects of (beta-TCP nanoparticles on biocompatibility and bone forming ability were then examined. Collagen scaffold was applied with 1, 5, 10, and 25 wt% beta-TCP nanoparticle dispersion and designated TCP1, TCP5, TCP10, and TCP25, respectively. Compressive strength, calcium ion release and enzyme resistance of scaffolds with (beta-TCP nanoparticles applied increased with beta-TCP dose. TCP5 showed excellent cell-ingrowth behavior in rat subcutaneous tissue. When TCP10 was applied, osteoblastic cell proliferation and rat cranial bone augmentation were greater than for any other scaffold. The bone area of TCP10 was 7.7-fold greater than that of non-treated scaffold. In contrast, TCP25 consistently exhibited adverse biological effects. These results suggest that the application dose of (beta-TCP nanoparticles affects the scaffold bioproperties; consequently, the bone conductive ability of TCP10 was remarkable.
  • Amizuka N, Hasegawa T, Yamamoto T, Mae T, Qiu Z, Abe M, Zhao S, Nagai T, Yokoyama A, Khadiza N, Haraguchi M, Yamamoto T, Freitas PHL, Li M
    Hokkaido Journal of Dental Science. 38 56 - 62 北海道歯学会 2017年09月 [査読有り][通常論文]
     
    During the last decade, osteocyte-derived factors i.e., sclerostin, dentin matrix protein-1, fibroblast growth factor 23 (FGF23) that reduces serum phosphate concentration by mediating FGF receptor 1c/αklotho in the kidney, have been highlighted for osteocytes' fine-turned regulation on bone remodeling and phosphate homeostasis. Osteocytes are interconnected through gap junctions between their cytoplasmic processes, and thereby, build upon the functional syncytia, referred to as the osteocytic lacunar-canalicular system (OLCS). Osteocytes appear to communicate surrounding osteocytes and osteoblasts by means of two possible pathways of molecular transport throughout the OLCS : One is a passageway of their cytoplasmic processes, and the other is a pericellular space in the osteocytic canaliculi. The regularly-oriented OLCS in mature compact bone appears to efficiently serve for molecular transport, mechanosensing and targeted bone remodeling that would erase microdamages in bone. In a disrupted signaling state of FGF23/αklotho, serum concentration of phosphate would be markedly-elevated. Despite highly-elevated serum phosphate, αklotho -deficient mice revealed defective mineralization in bone matrix. OLCS in αklotho -deficient mice were irregularly-distributed and the connectivity of cytoplasmic processes of osteocytes was very poor, so that osteocytes did not seem to form functional syncytia. Therefore, osteocytes'function cooperated with other bone cells, rather than serum concentration of calcium/phosphate, and this seems to play a central role in maintaining bone mineralization. In this review, the biological function of the regularly-arranged OLCS in a normal state will be introduced, as well as dysfunctional osteocytes in αklotho-deficient state, using animal models.
  • Koichi Tabeta, Xin Du, Kei Arimatsu, Mai Yokoji, Naoki Takahashi, Norio Amizuka, Tomoka Hasegawa, Karine Crozat, Tomoki Maekawa, Sayuri Miyauchi, Yumi Matsuda, Takako Ida, Masaru Kaku, Kasper Hoebe, Kinji Ohno, Hiromasa Yoshie, Kazuhisa Yamazaki, Eva Marie Y. Moresco, Bruce Beutler
    SCIENTIFIC REPORTS 7 1 11717 - 11717 2017年09月 [査読有り][通常論文]
     
    GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T. A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, alpha 1 chain, was responsible for the phenotype. Col1a1seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing "rescue."
  • Tingting Luo, Hongrui Liu, Wei Feng, Di Liu, Juan Du, Jing Sun, Wei Wang, Xiuchun Han, Jie Guo, Norio Amizuka, Xianqi Li, Minqi Li
    CELL PROLIFERATION 50 3 2017年06月 [査読有り][通常論文]
     
    Objectives: The purpose of this study was to investigate effects of adipocytes on osteoclast adhesion-related molecules.Materials and methods: ST2 cells, a cloned stromal cell line from mouse bone marrow, able to differentiate into adipocytes, were cultured in serum-free alpha-MEM which was then collected to be used as adipocyte-conditioned medium (ADIPO CM). RAW264.7 cells were cultured in ADIPO CM in the presence of RANKL, and bone marrow-derived macrophages were cultured in ADIPO CM in the presence of RANKL and macrophage-colony stimulating factor to induce osteoclast differentiation. TRAP staining, resorption pit assay, qRT-PCR and western blotting assays were performed.Results: ELISAs revealed that CXCL12 was abundant in ADIPO CM and CCK-8 assay revealed no proliferation of RAW264.7 cells after exogenous CXCL12 treatment. ADIPO CM enhanced osteoclast formation and resorption, both by RAW264.7 cells and BMMs. In addition, exogenous CXCL12 efficiently potentiated formation of TRAP-positive osteoclast and resorption by RAW264.7 cells. Western blotting and qRT-PCR suggested that ADIPO CM or combined treatment with exogenous CXCL12 caused significant increase in expression of NFAT2, src and osteoclast adhesion-related molecules, including beta 3 integrin, CD44 and osteopontin. However, these promotional effects were largely abrogated on treatment of AMD3100, a CXCR4 antagonist.Conclusions: Adipocytes promoted osteoclast differentiation, function and expression of adhesion-related molecules through the CXCL12/CXCR4 signalling pathway.
  • Tomoka Hasegawa, Tomomaya Yamamoto, Erika Tsuchiya, Hiromi Hongo, Kanako Tsuboi, Ai Kudo, Miki Abe, Taiji Yoshida, Tomoya Nagai, Naznin Khadiza, Ayako Yokoyama, Kimimitsu Oda, Hidehiro Ozawa, Paulo Henrique Luiz de Freitas, Minqi Li, Norio Amizuka
    The Japanese dental science review 53 2 34 - 45 2017年05月 [査読有り][通常論文]
     
    Matrix vesicle-mediated mineralization is an orchestrated sequence of ultrastructural and biochemical events that lead to crystal nucleation and growth. The influx of phosphate ions into the matrix vesicle is mediated by several proteins such as TNAP, ENPP1, Pit1, annexin and so forth. The catalytic activity of ENPP1 generates pyrophosphate (PPi) using extracellular ATPs as a substrate, and the resultant PPi prevents crystal overgrowth. However, TNAP hydrolyzes PPi into phosphate ion monomers, which are then transported into the matrix vesicle through Pit1. Accumulation of Ca2+ and PO43- inside matrix vesicles then induces crystalline nucleation, with calcium phosphate crystals budding off radially, puncturing the matrix vesicle's membrane and finally growing out of it to form mineralized nodules.
  • Michihito Kono, Tomoka Hasegawa, So Nagai, Toshio Odani, Kazumasa Akikawa, Yukiko Nomura, Hidetsugu Sato, Keisuke Kikuchi, Norio Amizuka, Hideaki Kikuchi
    MEDICINE 96 21 e6968  2017年05月 [査読有り][通常論文]
     
    Rationale: Palisaded neutrophilic and granulomatous dermatitis (PNGD) is a benign, inflammatory dermatosis with distinct histopathological features often observed in patients with systemic diseases. There were no reports of PNGD without underlying systemic diseases as an underlying cause of hypercalcemia. Herein, we report a case of a 62-year-old man with hypercalcemia due to PNGD, but with no underlying systemic diseases, including tuberculosis, sarcoidosis, or vasculitis.Patient concerns: Laboratory tests showed an elevated C-reactive protein level, an elevated corrected calcium level, a normal 25-hydroxyvitamin D level, and an elevated 1,25-dihydroxyvitamin D level. There were no other abnormalities to explain the hypercalcemia. Positron emission tomography-computed tomography showed abnormal uptake in his skin. Histopathological examination of the skin showed palisaded granulomatous infiltrate in the dermis. Neutrophils, degenerated collagen, and fibrin were present in the centers of the palisades without prominent mucin. There were no eosinophils, central necrosis, or necrotizing vasculitides. These features were consistent with PNGD.Diagnoses: A diagnosis of PNGD with hypercalcemia was established.Interventions: Oral prednisolone was administered to the patient.Outcomes: After treatment, his symptoms resolved, and his calcium, 1,25-dihydroxyvitamin D and CRP levels returned to normal. Skin specimens before and after treatment were assessed using immunohistochemistry for 1a-hydroxylase. Granuloma and epidermal cells were 1a-hydroxylase- positive before treatment. After treatment, the granuloma diminished in size and the 1ahydroxylase-positive areas of the epidermal cells decreased.Lessons: This case was particularly unique because increased 1a-hydroxylase expression in the granuloma and epidermal cells seemed to result in hypercalcemia due to excessive transformation of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Physicians should consider PNGD as an underlying cause of hypercalcemia.
  • Junko Shimomura-Kuroki, Muhammad Farooq, Tsuneo Sekimoto, Norio Amizuka, Yutaka Shimomura
    ODONTOLOGY 105 2 150 - 154 2017年04月 [査読有り][通常論文]
     
    Parathyroid hormone and parathyroid hormonerelated peptide (PTHrP), and its receptor (PTH1R) play an important role in differentiation of bone and cartilage in the developing stages. Constitutive dimers of PTH1R are believed to be dissociated by ligand binding, and monomeric PTH1R is capable of activating G protein. Jansen type metaphyseal chondrodysplasia is caused by missense mutations in PTH1R, which are constitutively active even without the presence of the ligands. However, the underlying pathomechanisms remained largely unknown. In this study, we have attempted to further characterize a PTH1R missense mutation H223R responsible for Jansen type metaphyseal chondrodysplasia. cDNAs encoding wild-type (Wt)- and H223R mutant (Mut)-PTH1R were transfected into HEK293T cells, and as a consequence of western blots, both the Wt- and Mut-PTH1R proteins showed several fragments between 55 and 65 kDa in size, while the patterns of N-glycosylation were distinct between them. Then we hypothesized that the Mut-PTH1R might physically interact with the Wt-PTH1R, leading to affect the downstream cAMP accumulation. Co-immunoprecipitation assays clearly showed that interaction occurred not only between the Wt-PTH1R themselves, but also between the Wt- and Mut-PTH1R. Furthermore, we performed CRE reporter assays to investigate cAMP accumulation. Constitutive, ligand-independent cAMP accumulation was observed in HEK293T cells expressing the Mut-PTH1R. Interestingly, there was a statistically lower constitutive activity in HEK293T cells co-expressing the Wt- and Mut-PTH1R proteins. Summarizing, it seems likely that Mut-PTH1R may be, at least in part, co-localized with Wt-PTH1R by forming a heterodimer, leading to affect the function to each other.
  • Jing Sun, Juan Du, Wei Feng, Boyao Lu, Hongrui Liu, Jie Guo, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 48 2 73 - 81 2017年04月 [査読有り][通常論文]
     
    This study evaluated the effects of metformin on orthodontic tooth movement in a rat model of type 2 diabetes mellitus. Rats were fed a high-fat diet for 4 weeks to induce fat accumulation and insulin resistance, and then injected with a low dose of streptozotocin (35 mg/kg) intraperitoneally to induce type 2 diabetes. An orthodontic appliance was placed in normoglycemic, type 2 diabetes, and type 2 diabetes with metformin-administrated rats. After 14 days, type 2 diabetes rats exhibited greater orthodontic tooth movement and had a higher number of tartrate-resistant acid phosphatase-positive osteoclasts, stronger cathepsin K expression, and weaker alkaline phosphatase immunostaining than normoglycemic rats. Metformin administration resulted in normalization of osteoclast numbers, cathepsin K immunostaining, and of tooth movement as well as partly recovery of alkaline phosphatase expression in diabetic rats. Metformin also reduced sclerostin expression and improved the immunolocalization of dentin matrix protein 1 in osteocytes of type 2 diabetes rats. These results suggest that metformin administration reversed the adverse effects of diabetes on orthodontic tooth movement.
  • Kumiko Hikone, Tomoka Hasegawa, Erika Tsuchiya, Hiromi Hongo, Muneteru Sasaki, Tomomaya Yamamoto, Ai Kudo, Kimimitsu Oda, Mai Haraguchi, Paulo Henrique Luiz de Freitas, Minqi Li, Junichiro Iida, Norio Amizuka
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 65 4 207 - 221 2017年04月 [査読有り][通常論文]
     
    To elucidate which of elevated serum concentration of inorganic phosphate (Pi) or disrupted signaling linked to alpha klotho/fibroblast growth factor 23 (FGF23) is a predominant regulator for senescence-related degeneration seen in alpha Klotho-deficient mice, we have examined histological alteration of the periodontal tissues in the mandibular interalveolar septum of alpha Klotho-deficient mice fed with Pi-insufficient diet. We prepared six groups of mice: wild-type, kl/kl, and alpha Klotho(-/-) mice with normal diet or low-Pi diet. As a consequence, kl/kl(norPi) and alpha Klotho(-/-norPi) mice showed the same abnormalities in periodontal tissues: intensely stained areas with hematoxylin in the interalveolar septum, dispersed localization of alkaline phosphatase-positive osteoblasts and tartrate-resistant acid phosphatase-reactive osteoclasts, and accumulation of dentin matrix protein 1 in the osteocytic lacunae. Although kl/kl(lowPi) mice improved these histological abnormalities, alpha Klotho(-/-lowPi) mice failed to normalize those. Gene expression of alpha Klotho was shown to be increased in kl/kl(lowPi) specimens. It seems likely that histological abnormalities of kl/kl mice have been improved by the rescued expression of alpha Klotho, rather than low concentration of serum Pi. Thus, the histological malformation in periodontal tissues in alpha Klotho-deficient mice appears to be due to not only increased concentration of Pi but also disrupted alpha klotho/FGF23 signaling.
  • Issei Takayama, Hideyuki Tanabe, Takashi Nishiyama, Harumi Ito, Norio Amizuka, Minqi Li, Ken-ichi Katsube, Isao Kii, Akira Kudo
    JOURNAL OF CELL COMMUNICATION AND SIGNALING 11 1 5 - 13 2017年03月 [査読有り][通常論文]
     
    CCN3 is a matricellular protein that belongs to the CCN family. CCN3 consists of 4 domains: insulin-like growth factor-binding protein-like domain (IGFBP), von Willebrand type C-like domain (VWC), thrombospondin type 1-like domain (TSP1), and the C-terminal domain (CT) having a cysteine knot motif. Periostin is a secretory protein that binds to extracellular matrix proteins such as fibronectin and collagen. In this study, we found that CCN3 interacted with periostin. Immunoprecipitation analysis revealed that the TSP1-CT interacted with the 4 repeats of the Fas 1 domain of periostin. Immunofluorescence analysis showed co-localization of CCN3 and periostin in the periodontal ligament of mice. In addition, targeted disruption of the periostin gene in mice decreased the matricellular localization of CCN3 in the periodontal ligament. Thus, these results indicate that periostin was required for the matricellular localization of CCN3 in the periodontal ligament, suggesting that periostin mediated an interaction between CCN3 and the extracellular matrix.
  • Tomoka Hasegawa, Takashi Endo, Erika Tsuchiya, Ai Kudo, Zhao Shen, Yasuhito Moritani, Miki Abe, Tomomaya Yamamoto, Hiromi Hongo, Kanako Tsuboi, Taiji Yoshida, Tomoya Nagai, Naznin Khadiza, Ayako Yokoyama, Paulo Henrique Luiz de Freitas, Minqi Li, Norio Amizuka
    Journal of Oral Biosciences 59 1 55 - 62 2017年02月01日 [査読有り][通常論文]
     
    Objectives The aim of this study was the biological application of focused ion beam-scanning electron microscopy (FIB-SEM) to obtain serial sectional images of skeletal tissues that showed the ultrastructure of 1) cartilaginous extracellular fibrils and 2) osteoblastic cytoplasmic processes. Methods Seven-week-old female wild-type mice were fixed with half-Karnovsky solution and then OsO4, and tibiae were extracted for block staining prior to observation under transmission electron microscope (TEM) and FIB-SEM. Results TEM showed the fine fibrillar but somewhat amorphous ultrastructure of the intercolumnar septa in the growth plate cartilage. In contrast, FIB-SEM revealed bundles of stout fibrils at regular intervals paralleling the septa's longitudinal axis, as well as vesicular structures embedded in the cartilaginous matrix of the proliferative zone. In the primary trabeculae, both TEM and FIB-SEM showed several osteoblastic cytoplasmic processes on the osteoid, in greater numbers than those seen in the bone matrix. FIB-SEM revealed the agglomeration of cytoplasmic processes beneath osteoblasts that formed a tubular continuum extending from those cells. Based on these findings, we postulated that osteoblasts not only extend their cytoplasmic processes to the bone matrix, but also stack these cell processes on the osteoid of the primary trabeculae. Conclusion Taken together, these data suggest that FIB-SEM imaging of serial bone sections may facilitate new insights on the ultrastructure of cartilage and bone tissues.
  • Xiuchun Han, Juan Du, Di Liu, Hongrui Liu, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 48 1 41 - 51 2017年02月 [査読有り][通常論文]
     
    The aim of this experiment was to elucidate the histological alterations after systemic administration of eldecalcitol (ELD) combined with guided bone regeneration during the restoration of bone defect healing in rats. The femurs of 8-week-old Wister rats were used to generate bone defect models. The defect was covered with a collagen membrane, and ELD group was administrated with eldecalcitol (50 ng/kg body weight) intragastrically once every other day. Femora were harvested at 1, 2, 4 and 8 weeks post-surgery. Decalcify tissue slices were made and used for histological and immunohistochemical examination. Bone biomarkers of RANKL, OPG and osteocalcin (OCN) were detected by western blot. The results revealed that the system administration of ELD could improve new bone formation demonstrated by the increased bone volume/tissue volume ratio and accelerated mineralization. ELD suppressed osteoclastic bone resorption by reducing the number of osteoclasts, decreasing the expression of cathepsin-K and the ratio of RANKL/OPG at the early stage of bone defect restoration (1 and 2 weeks) and upregulating OCN expression at the later stage of bone defect healing (4 and 8 weeks). These data suggested that systemic administration of eldecalcitol accelerated bone formation and promoted bone maturation by decreasing bone resorption and promoting bone mineralization during bone defect restoration.
  • Tomoka Hasegawa, Takashi Endo, Erika Tsuchiya, Ai Kudo, Zhao Shen, Yasuhito Moritani, Miki Abe, Tomomaya Yamamoto, Hiromi Hongo, Kanako Tsuboi, Taiji Yoshida, Tomoya Nagai, Naznin Khadiza, Ayako Yokoyama, Paulo Henrique Luiz de Freitas, Minqi Li, Norio Amizuka
    JOURNAL OF ORAL BIOSCIENCES 59 1 55 - 62 2017年02月 [査読有り][通常論文]
     
    Objectives: The aim of this study was the biological application of focused ion beam-scanning electron microscopy (FIB-SEM) to obtain serial sectional images of skeletal tissues that showed the ultrastructure of 1) cartilaginous extracellular fibrils and 2) osteoblastic cytoplasmic processes.Methods: Seven-week-old female wild-type mice were fixed with half-Karnovsky solution and then OsO4, and tibiae were extracted for block staining prior to observation under transmission electron microscope (TEM) and FIB-SEM.Results: TEM showed the fine fibrillar but somewhat amorphous ultrastructure of the intercolumnar septa in the growth plate cartilage. In contrast, FIB-SEM revealed bundles of stout fibrils at regular intervals paralleling the septa's longitudinal axis, as well as vesicular structures embedded in the cartilaginous matrix of the proliferative zone. In the primary trabeculae, both TEM and FIB-SEM showed several osteoblastic cytoplasmic processes on the osteoid, in greater numbers than those seen in the bone matrix. FIB-SEM revealed the agglomeration of cytoplasmic processes beneath osteoblasts that formed a tubular continuum extending from those cells. Based on these findings, we postulated that osteoblasts not only extend their cytoplasmic processes to the bone matrix, but also stack these cell processes on the osteoid of the primary trabeculae.Conclusion: Taken together, these data suggest that FIB-SEM imaging of serial bone sections may facilitate new insights on the ultrastructure of cartilage and bone tissues. (C) 2016 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
  • Wei Feng, Hongrui Liu, Tingting Luo, Di Liu, Juan Du, Jing Sun, Xiuchun Han, Kaiyun Yang, Jie Guo, Norio Amizuka, Minqi Li
    SCIENTIFIC REPORTS 7 2017年01月 [査読有り][通常論文]
     
    Interleukin (IL)-6 is known to indirectly enhance osteoclast formation by promoting receptor activator of nuclear factor kappa-B ligand (RANKL) production by osteoblastic/stromal cells. However, little is known about the direct effect of IL-6 on osteoclastogenesis. Here, we determined the direct effects of IL-6 and its soluble receptor (sIL-6R) on RANKL-induced osteoclast formation by osteoclast precursors in vitro. We found IL-6/sIL-6R significantly promoted and suppressed osteoclast differentiation induced by low-(10 ng/ml) and high-level (50 ng/ml) RANKL, respectively. Using a bone resorption pit formation assay, expression of osteoclastic marker genes and transcription factors confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R. Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-kappa B (nuclear factor kappa-light-chain-enhancer of activated B cells), ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) induced by low-and high level RANKL, respectively. Taken together, our findings demonstrate that IL-6/sIL-6R differentially regulate RANKL-induced osteoclast differentiation and activity through modulation of NF-kappa B, ERK and JNK signaling pathways. Thus, IL-6 likely plays a dual role in osteoclastogenesis either as a pro-resorption factor or as a protector of bone, depending on the level of RANKL within the local microenvironment.
  • Feng W, Liu H, Luo T, Liu D, Du J, Sun J, Wang W, Han X, Yang K, Guo J, Amizuka N, Li M
    Scientific reports 7 41411 - 41411 2017年01月 [査読有り][通常論文]
     
    Interleukin (IL)-6 is known to indirectly enhance osteoclast formation by promoting receptor activator of nuclear factor kappa-B ligand (RANKL) production by osteoblastic/stromal cells. However, little is known about the direct effect of IL-6 on osteoclastogenesis. Here, we determined the direct effects of IL-6 and its soluble receptor (sIL-6R) on RANKL-induced osteoclast formation by osteoclast precursors in vitro. We found IL-6/sIL-6R significantly promoted and suppressed osteoclast differentiation induced by low-(10 ng/ml) and high-level (50 ng/ml) RANKL, respectively. Using a bone resorption pit formation assay, expression of osteoclastic marker genes and transcription factors confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R. Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-kappa B (nuclear factor kappa-light-chain-enhancer of activated B cells), ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) induced by low-and high level RANKL, respectively. Taken together, our findings demonstrate that IL-6/sIL-6R differentially regulate RANKL-induced osteoclast differentiation and activity through modulation of NF-kappa B, ERK and JNK signaling pathways. Thus, IL-6 likely plays a dual role in osteoclastogenesis either as a pro-resorption factor or as a protector of bone, depending on the level of RANKL within the local microenvironment.
  • Atsunaka Sakurai, Tomoka Hasegawa, Ai Kudo, Zhao Shen, Tomoya Nagai, Miki Abe, Taiji Yoshida, Hiromi Hongo, Tomomaya Yamamoto, Tsuneyuki Yamamoto, Kimimitsu Oda, Paulo Henrique Luiz de Freitas, Minqi Li, Hidehiko Sano, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 38 4 257 - 267 2017年 [査読有り][通常論文]
     
    To assess the chronological participation of sclerostin and FGF23 in bone metabolism, this study tracked the immunolocalization of sclerostin and FGF23 in the metaphyses of murine long bones from embryonic day 18 (E18) through 1 day after birth, 1 week, 2 weeks, 4 weeks, 8 weeks, and 20 weeks of age. We have selected two regions in the metaphyseal trabeculae for assessing sclerostin and FGF23 localization: close to the chondro-osseous junction, i.e., bone modeling site even in the adult animals, and the trabecular region distant from the growth plate, where bone remodeling takes place. As a consequence, sclerostin-immunopositive osteocytes could not be observed in both close and distant trabecular regions early at the embryonic and young adult stages. However, osteocytes gradually started to express sclerostin in the distant region earlier than in the close region of the trabeculae. Immunoreactivity for FGF23 was observed mainly in osteoblasts in the early stages, but detectable in osteocytes in the later stages of growth in trabecular and cortical bones. Fgf23 was weakly expressed in the embryonic and neonatal stages, while the receptors, Fgfr1c and alpha Klotho were strongly expressed in femora. At the adult stages, Fgf23 expression became more intense while Fgfr1c and alpha Klotho were weakly expressed. These findings suggest that sclerostin is secreted by osteocytes in mature bone undergoing remodeling while FGF23 is synthesized by osteoblasts and osteocytes depending on the developmental/growth stage. In addition, it appears that FGF23 acts in an autocrine and paracrine fashion in fetal and neonatal bones.
  • Hasegawa T, Amizuka N
    Clinical calcium 27 12 1713 - 1722 2017年 [査読有り][通常論文]
  • Hisashi Toraya, Tomoka Hasegawa, Naoko Sakagami, Erika Tsuchiya, Ai Kudo, Shen Zhao, Yasuhito Moritani, Miki Abe, Taiji Yoshida, Tomomaya Yamamoto, Tsuneyuki Yamamoto, Kimimitsu Oda, Nobuyuki Udagawa, Paulo Henrique Luiz de Freitas, Minqi Li, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 38 2 123 - 134 2017年 [査読有り][通常論文]
     
    Since osteoblastic activities are believed to be coupled with osteoclasts, we have attempted to his-tologically verify which of the distinct cellular circumstances, the presence of osteoclasts themselves or bone resorption by osteoclasts, is essential for coupled osteoblastic activity, by examining c-fos(-/-) or c-src(-/-) mice. Osteopetrotic c-fos deficient (c-fos(-/-)) mice have no osteoclasts, while c-src deficient (c-src(-/-)) mice, another osteopetrotic model, develop dysfunctional osteoclasts due to a lack of ruffled borders. c-fos(-/-) mice possessed no tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts, and showed very weak tissue nonspecific alkaline phosphatase (TNALPase)-reactive mature osteoblasts. In contrast, c-src(-/-) mice had many TNALPase-positive osteoblasts and TRAPase-reactive osteoclasts. Interestingly, the parallel layers of TRAPase-reactive/osteopontin- positive cement lines were observed in the superficial region of c-src(-/-) bone matrix. This indicates the possibility that in c-src(-/-) mice, osteoblasts were activated to deposit new bone matrices on the surfaces that osteoclasts previously passed along, even without bone resorption. Transmission electron microscopy demonstrated cell-to-cell contacts between mature osteoblasts and neighboring ruffled border-less osteoclasts, and osteoid including many mineralized nodules in c-src(-/-) mice. Thus, it seems likely that osteoblastic activities would be maintained in the presence of osteoclasts, even if they are dysfunctional.
  • Yusuke Tanaka, Tomoka Hasegawa, Tamaki Yamada, Tomomaya Yamamoto, Muneteru Sasaki, Hiromi Hongo, Kanako Tsuboi, Mai Haraguchi, Paulo Henrique Luiz de Freitas, Minqi Li, Kimimitsu Oda, Yasunori Totsuka, Kanchu Tei, Norio Amizuka
    HISTOLOGY AND HISTOPATHOLOGY 31 12 1315 - 1326 2016年12月 [査読有り][通常論文]
     
    In order to provide a clue to understand the interplay between leptin and estrogen, we have examined femoral metaphyses of ovariectomized db/db mice carrying a mutated leptin receptor. We performed ovariectomy (OVX) or sham-operation (sham) on 12week old female wild-type and db/db mice, and then, after 8 weeks, divided the animals into four groups: wild-type sham, wild-type OVX, db/db sham and db/db OVX. Samples from all groups were prepared for histochemical and ultrastructural examinations. As a result, db/db sham mice showed a reduced number and thickness of metaphyseal trabeculae and excessive adipose tissue when compared to wild-type sham mice. The wild-type OVX group exhibited markedly diminished trabecular number, as well as lower populations of osteoblasts and osteoclasts in comparison to wild-type sham group. On the other hand, trabecular numbers were similar for the two db/db groups, suggesting that the effect of the ovariectomy, i.e., estrogen deficiency may be lessened in this animal model. Leptin receptor was mainly found in osteoblasts and in bone marrow stromal cells including adipocytes. In addition, the expression of estrogen receptor did not seem to change after OVX in wild-type mice and in db/db mice. Both db/db sham and OVX mice featured many adipocytes close to the metaphyseal chondro-osseous junction, while osteoblasts accumulated glycogen granules and lipid droplets. Therefore, it seems likely that the disruption of leptin signaling in db/db mice shifts the cell differentiation cascade towards the adipocyte lineage, resulting in an osteoporotic bone independently of estrogen deficiency.
  • Anne Roscher, Tomoka Hasegawa, Sebastian Dohnke, Carlos Ocaña-Morgner, Norio Amizuka, Rolf Jessberger, Annette I Garbe
    Bone reports 5 214 - 221 2016年12月 [査読有り][通常論文]
     
    Osteoclasts are bone resorbing cells acting as key mediators of bone disorders. Upon adhesion to bone, osteoclasts polarize and reorganize their cytoskeleton to generate a ring-like F-actin-rich structure, the sealing zone, wherein the osteoclast's resorptive organelle, the ruffled border, is formed. The dynamic self-organization of actin-rich adhesive structures, the podosomes, from clusters to belts is crucial for osteoclast-mediated bone degradation. Mice lacking the protein SWAP-70 display an osteopetrotic phenotype due to defective bone resorption caused by impaired actin ring formation in Swap-70-/- osteoclasts. To further elucidate the mechanisms underlying this defect, we investigated the specific function of SWAP-70 in the organization and dynamics of podosomes. These detailed studies show that the transition from podosome clusters to rings is impaired in Swap-70-/- osteoclasts. Live cell imaging of dynamic F-actin turnover and SWAP-70 localization during podosome patterning indicate that SWAP-70 is dispensable for cluster formation but plays a key role in F-actin ring generation. Our data provide insights in the role of SWAP-70's F-actin binding domain and pleckstrin homology (PH) domain in the proper localization of SWAP-70 and formation of a peripheral podosome belt, respectively. Ex vivo bone analyses revealed that SWAP-70-deficient osteoclasts exhibit defective ruffled border formation and V-ATPase expression. Our findings suggest an important role of membrane binding of SWAP-70 for the regulation of actin dynamics, which is essential for podosome patterning, and thus for the resorptive activity of osteoclasts.
  • Tomohiro Hikata, Tomoka Hasegawa, Keisuke Horiuchi, Nobuyuki Fujita, Akio Iwanami, Kota Watanabe, Ken Ishii, Masaya Nakamura, Norio Amizuka, Morio Matsumoto
    Bone reports 5 286 - 291 2016年12月 [査読有り][通常論文]
     
    Minimodeling is a type of focal bone formation that is characterized by the lack of precedent bone erosion by osteoclasts. Although this form of bone formation has been described for more than a decade, how anti-osteoporotic agents that are currently used in clinical practice affect the kinetics of minimodeling is not fully understood. We performed a bone morphometric analysis using human vertebral specimens collected from postmenopausal patients who underwent spinal surgery. Patients were divided into three groups according to osteoporosis medication; non-treated, Eldecalcitol (ELD, a vitamin D derivative that has recently been approved to treat patients with osteoporosis in Japan)-treated, and bisphosphonate-treated groups. Five to six patients were enrolled in each group. There was a trend toward enhanced minimodeling in ELD-treated patients and suppressed of it in bisphosphonate-treated patients compared with untreated patients. The differences of minimodeling activity between ELD-treated and bisphosphonate-treated patients were statistically significant. The present study suggests that ELD and bisphosphonates have opposite effects on minimodeling from one another, and show that minimodeling also takes place in vertebrae as has been described for the ilium and femoral head in humans.
  • Hiromi Hongo, Muneteru Sasaki, Sachio Kobayashi, Tomoka Hasegawa, Tomomaya Yamamoto, Kanako Tsuboi, Erika Tsuchiya, Tomoya Nagai, Naznin Khadiza, Miki Abe, Ai Kudo, Kimimitsu Oda, Paulo Henrique Luiz de Freitas, Minqi Li, Hisayoshi Yurimoto, Norio Amizuka
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 64 10 601 - 622 2016年11月 [査読有り][通常論文]
     
    Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate's effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([N-15]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [N-15]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [N-15]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase-positive osteoclasts and alkaline phosphatase-reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.
  • Juan Du, Wei Feng, Jing Sun, Cuijie Kang, Norio Amizuka, Minqi Li
    SCIENTIFIC REPORTS 6 35995 - 35995 2016年10月 [査読有り][通常論文]
     
    Peroxiredoxin (PRX), a family of peroxidases, is associated with various biological processes such as the detoxification of oxidants and cell apoptosis. Besides, the anti-apoptosis effect of estrogen results partially from its anti-oxidant function. The purpose of this study was to investigate the expression of PRXs in ovariectomy (OVX) mice and the related anti-oxidative mechanism of estrogen. Eight-weekold mice were subjected to ovariectomy. MC3T3-E1 cells were pretreatment with 17b-estradiol and N-acetyl cysteine followed by oxidative injury induced with H2O2. Western blot and real time-PCR were applied to clarify the expressions of PRX1 and caspase-3, with both wild-type and PRX1 knockout MC3T3-E1 cells generated by CRISPR/Cas9 technology. The results showed PRX1 and PRX5 were upregulated in osteoblasts in the proximal tibial metaphysis of ovariectomy mice. Interestingly, PRX1 and PRX5 showed different distribution patterns, with PRX1 mainly accumulated in cell nuclei and PRX5 in the cytoplasm. Gene expression analysis showed significantly reduced expressions of PRX1 and caspase-3 in the pretreatment groups when compared with cells treated with H2O2 alone. Also, a decrease of caspase-3 expressions was observed in PRX1 knockout MC3T3-E1 cells with or without H2O2 in comparison to wild-type cells. These findings suggested that PRX may play important roles in estrogen-deficient osteoporosis.
  • Kanako Tsuboi, Tomoka Hasegawa, Tomomaya Yamamoto, Muneteru Sasaki, Hiromi Hongo, Paulo Henrique Luiz de Freitas, Tomohiro Shimizu, Masahiko Takahata, Kimimitsu Oda, Toshimi Michigami, Minqi Li, Yoshimasa Kitagawa, Norio Amizuka
    HISTOCHEMISTRY AND CELL BIOLOGY 146 3 337 - 350 2016年09月 [査読有り][通常論文]
     
    In order to determine whether osteoclastic bone resorption is restarted after withdrawn of bisphosphonates, we conducted histological examinations on murine osteoclasts, osteoblasts and osteocytes after discontinuation of a daily regimen of alendronate (ALN) with a dosage of 1 mg/kg/day for 10 days. After drug discontinuation, metaphyseal trabecular number and bone volume remained unaltered for the first 4 days. Osteoclast number did not increase, while the number of apoptotic osteoclasts was elevated. On the other hand, tissue non-specific alkaline phosphatase-immunoreactive area was markedly reduced after ALN discontinuation. In addition, osteocytes showed an atrophic profile with empty lacunar areas during and after ALN treatment. Interestingly, as early as 36 h after a single ALN injection, osteocytes show signs of atrophy despite the presence of active osteoblasts. Structured illumination microscopy system showed shortening of osteocytic cytoplasmic processes after drug cessation, suggesting a possible morphological and functional disconnection between osteocytes and osteoblasts. Taken together, it appears that osteoclastic bone resorption is not resumed after ALN discontinuation; also, osteoblasts and osteocytes hardly seem to recover once they are inactivated and atrophied by ALN. In summary, it seems that one must pay more attention to the responses of osteoblasts and osteocytes, rather focusing on the resuming of osteoclastic bone resorption after the ALN discontinuation.
  • Jing Sun, Bao Sun, Wei Wang, Xiuchun Han, Hongrui Liu, Juan Du, Wei Feng, Bo Liu, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 47 4 389 - 399 2016年08月 [査読有り][通常論文]
     
    Vitamin D has an anabolic effect on bone developmental processes and is involved in maintaining skeletal integrity. In recent years, pediatric cases of vitamin D intoxication have attracted attention. Therefore, the aim of this study was to investigate the influence of long-term administration of physiologically-high-dose calcitriol (1,25(OH)(2)D-3) on bone remodeling in young developing rats. Neonatal rats received once-daily subcutaneous injection of calcitriol (250 ng/kg body weight), or PBS only as a control, for 3 weeks. At 1, 2 and 4 weeks' post-administration, rats were sacrificed and fixed by transcardial perfusion with 4 % paraformaldehyde, following which tibiae were extracted for histochemical analysis. Compared with the control group, the number of tartrate-resistant acid phosphatase- and Cathepsin K-positive osteoclasts were significantly increased, and the expression of alkaline phosphatase in osteoblasts was decreased in trabecular bone of rats administered high-dose 1,25(OH)(2)D-3, leading to decreased trabecular bone volume. In addition, the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) was increased, while that of osteoprotegerin was weaker in osteoblasts in the experimental group compared with the control group. Moreover, there was weaker immunoreactivity for EphrinB2 in osteoclasts and EphB4 in osteoblasts of trabecular bone in the experimental group compared with the control group. These findings suggest that long-term use of physiologically-high dose calcitriol may result in bone loss through RANKL/RANK/osteoprotegerin and EphrinB2-EphB4 signaling pathways, and that these negative effects could continue after drug withdrawal. Therefore, optimal limits for vitamin D administration need to be established for children and adolescents.
  • Bo Liu, Jian Cui, Jing Sun, Juan Li, Xiuchun Han, Jie Guo, Min Yi, Norio Amizuka, Xin Xu, Minqi Li
    MOLECULAR MEDICINE REPORTS 14 2 1099 - 1106 2016年08月 [査読有り][通常論文]
     
    The aim of the present study was to investigate the expression of matrix metalloproteinase (MMP) 9 and MMP2, and their potential roles in bone metastasis nests using a well-standardized model of breast cancer bone metastasis in nude mice. BALB/c nu/nu mice (5-week-old; n=10) were subjected to intracardiac injection of MDA-MB-231 human breast cancer cells. After 4 weeks, the mice exhibiting radiolucent lesions in tibiae were sacrificed, and the tibiae were removed for histochemical analysis. The gene expression of MMP2 and MMP9 in the tumor cells, metaphysis and diaphysis of normal BALB/c nu/nu mice were determined using reverse transcription-polymerase chain reaction analysis. The metastatic tumor tissue occupied almost the entire bone marrow cavity. Numerous tartrate-resistant acid phosphatase-positive osteoclasts were found in the metastasized lesions. The invaded tumor cells positive for mammaglobin 1 exhibited different proliferation activities and apoptosis between the metaphysis and diaphysis. Proliferating cell nuclear antigen was expressed at high levels in the metaphyseal area, whereas TdT-mediated dUTP nick-end labeling (TUNEL) -positive cells were more evident in the diaphysis area. Of note, MMP9 was expressed predominantly in the proliferating cell nuclear antigen-positive area, whereas the expression of MMP2 was observed predominantly in the diaphysis, which had more TUNEL-positive cells. Taken together, the results suggested that MMP9 and MMP2 may have their own importance in extracellular matrix degradation and trabecular bone damage in different zones of bone metastasis, including the metaphysis and diaphysis.
  • Norio Amizuka
    Journal of orthopaedic trauma 30 8 S2  2016年08月 [査読有り][通常論文]
     
    In bone, there are 3 distinct cell types: an osteoblast, a bone forming cell; an osteocyte embedded in bone matrix as a consequence of being differentiated from an osteoblast; and an osteoclast, a multinucleated giant cell responsible for bone resorption. Bone is always remodeled by replacing old bone with new bone (bone remodeling), by which bone can maintain its stiffness and flexibility. However, in an osteoporotic state, the disrupted balance between bone resorption and formation results in not only markedly reduced bone mass, but also in disorganized geometry of trabecules, which can often give rise to a bone fracture. Osteocytes located in their lacunae insert their fine cytoplasmic processes into narrow passageways referred to as osteocytic canaliculi. Neighboring osteocytes connect to each other by means of a gap junction in their cytoplasmic processes. Therefore, osteocytes and their lacunae/canaliculi appear to form functional syncytium called osteocytic lacunar canalicular system (OLCS). The geometrical distribution of OLCS is poorly arranged in immature bone, while it appears well-arranged distribution in mature bone (cortical bone), in which molecular transports and sensing mechanical stress seems to be efficient, and therefore, may be able to respond to mechanical stress. In this seminar, I will introduce our recent findings on the morphology and function of OLCS which may respond to mechanical stress.
  • Tsuneyuki Yamamoto, Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Norio Amizuka
    JAPANESE DENTAL SCIENCE REVIEW 52 3 63 - 74 2016年08月 [査読有り][通常論文]
     
    Cementum was first demonstrated by microscopy, about 180 years ago. Since then the biology of cementum has been investigated by the most advanced techniques and equipment at that time in various fields of dental sciences. A great deal of data on cementum histology have been accumulated. These data have been obtained from not only human, but also non-human animals, in particular, rodents such as the mouse and rat. Although many dental histologists have reviewed histology of human cementum, some descriptions are questionable, probably due to incorrect comparison of human and rodent cementum. This review was designed to introduce current histology of human cementum, i.e. its structure, function, and development and to re-examine the most questionable and controversial conclusions made in previous reports. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of Japanese Association for Dental Science.
  • Tomomaya Yamamoto, Tomoka Hasegawa, Muneteru Sasaki, Hiromi Hongo, Kanako Tsuboi, Tomohiro Shimizu, Masahiro Ota, Mai Haraguchi, Masahiko Takahata, Kimimitsu Oda, Paulo Henrique Luiz de Freitas, Aya Takakura, Ryoko Takao-Kawabata, Yukihiro Isogai, Norio Amizuka
    ENDOCRINOLOGY 157 7 2604 - 2620 2016年07月 [査読有り][通常論文]
     
    Evidence supports that daily and once-weekly administration of teriparatide, human (h) PTH(1-34), enhance bone mass in osteoporotic patients. However, it is uncertain whether different frequencies of hPTH(1-34) administration would induce bone formation similarly in terms of quantity and quality. To investigate that issue, mice were subjected to different frequencies of PTH administration, and their bones were histologically examined. Frequencies of administration were 1 time/2 days, 1 time a day, and 2 and 4 times a day. Mice were allocated to either to control or to 3 different dosing regimens: 80 mu g/kg of hPTH(1-34) per injection (80 mu g/kg per dose), 80 mu g/kg of hPTH(1-34) per day (80 mu g/kg.d), or 20 mu g/kg of hPTH(1-34) per day (20 mu g/kg.d). With the regimens of 80 mu g/kg per dose and 80 mu g/kg.d, high-frequency hPTH(1-34) administration increased metaphyseal trabecular number. However, 4 doses per day induced the formation of thin trabeculae, whereas the daily PTH regimen resulted in thicker trabeculae. A similar pattern was observed with the lower daily hPTH(1-34) dose (20 mu g/kg.d): more frequent PTH administration led to the formation of thin trabeculae, showing a thick preosteoblastic cell layer, several osteoclasts, and scalloped cement lines that indicated accelerated bone remodeling. On the other hand, low-frequency PTH administration induced new bone with mature osteoblasts lying on mildly convex surfaces representative of arrest lines, which suggests mini-modeling-based bone formation. Thus, high-frequency PTH administration seems to increase bone mass rapidly by forming thin trabeculae through accelerated bone remodeling. Alternatively, low-frequency PTH administration leads to the formation of thicker trabeculae through bone remodeling and minimodeling.
  • Bao Sun, Jing Sun, Xiuchun Han, Hongrui Liu, Juan Li, Juan Du, Wei Feng, Bo Liu, Jian Cui, Jie Guo, Norio Amizuka, Minqi Li
    HISTOLOGY AND HISTOPATHOLOGY 31 6 647 - 656 2016年06月 [査読有り][通常論文]
     
    Long-term use of glucocorticoids (GC) causes rapid bone loss and increases the risk of osteoporotic fractures. Matrix metalloproteinase (MMPs), the most prominent kind of proteases implicated in the proteolytic degradation of the extracellular matrix (ECM), have been reported to be involved in pathological process of GC induced osteoporosis. However, the underlining mechanisms are still unclear. The aim of this study was to investigate the spatial expression and the potential function of MMP 2, 9 and 13 in osteoporosis induced by prednisolone in the tibiae of mice. In this experiment, mice were given prednisolone (15 mg/kg body weight) in PBS intragastrically every other day, or only PBS as control. Two weeks later, mice were fixed with transcardial perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and tibiae were extracted for histochemical analysis. Compared with control group, the number of TRAP-positive osteoclasts and the immunoreactivity of MMP 2, 9 and 13 were significantly increased in the trabecular bone of mice administered with prednisolone, leading to the decrease of trabecular bone volume. On the other hand, lighter eosin staining areas containing numerous empty lacunae of osteocytes and crevices were seen in the narrowing cortical bone. Furthermore, intense immunoreaction of MMP 2 and MMP 13 were found in the enlarged lacunae and the crevices, respectively. Taken together, we concluded that prednisolone administration induced the increase of MMP 2, 9 and 13 expressions, while MMP 2 and MMP 13 played essential roles in the osteocytic osteolysis and the early impaired areas in the cortical bone. Therefore, MMPs might be new potential therapeutic targets for prevention and treatment of glucocorticoid induced osteoporosis, especially osteocytic osteolysis.
  • Bo Liu, Xiuchun Han, Wei Feng, Jian Cui, Tomoka Hasegawa, Norio Amizuka, Xin Xu, Minqi Li
    ARCHIVES OF ORAL BIOLOGY 65 82 - 86 2016年05月 [査読有り][通常論文]
     
    Objective: Ghrelin, an appetite-stimulating hormone, plays diverse regulatory functions in cell growth, proliferation, differentiation and apoptosis during mammalian development. There is limited information currently available regarding Ghrelin expression during mammalian tooth development, thus we aimed to establish the spatiotemporal expression of Ghrelin during murine molar odontogenesis.Design: Immunohistochemistry was performed to detect the expression pattern of Ghrelin in mandible molar from E15.5 to PN7 during murine tooth development.Results: The results showed that Ghrelin initially expressed in the inner enamel epithelium and the adjacent mesenchymal cells below, further with persistent expression in the ameloblasts and odontoblasts throughout the following developmental stages. In addition, Ghrelin was also present in Hertwig's epithelial root sheath at the beginning of tooth root formation.Conclusions: These results suggest that Ghrelin was present in tooth organs throughout the stages of tooth development, especially in ameloblasts and odontoblasts with little spatiotemporal expression differences. However, the potential regulatory roles of this hormone in tooth development still need to be validated by functional studies. (C) 2016 Elsevier Ltd. All rights reserved.
  • Tomoka Hasegawa, Erika Tsuchiya, Miki Abe, Norio Amizuka
    Clinical calcium 26 5 677 - 82 2016年05月 [査読有り][通常論文]
     
    During endochondral bone development, the longitudinal vascular invasion into cartilage primordium initially takes place, by which mineralized cartilage matrix would be exposed into bone. Thereafter, osteogenic cells differentiate into mature osteoblasts to deposit new bone onto the exposed mineralized cartilage. New bone formation at the chondro-osseous junction appears to be achieved by the process of modeling, but not by bone remodeling based on cellular coupling between osteoclasts and osteoblasts. Recently, a specific vessel subtype in bone was reported:Vascular endothelial cells close to the chondro-oseous junction showed intense CD31/Endomucin(CD31(hi)Emcn(hi), type H), while the endothelial cells of sinusoidal vessels in diaphysis revealed only weak CD31/Endomucin(CD31(lo)Emcnlo, type L). It is suggested crucial roles of endothelial HIF in controlling bone angiogenesis, type H vessel abundance, endothelial growth factor expression and osteogenesis.
  • Jian Cui, Juan Li, Wei Wang, Xiuchun Han, Juan Du, Jing Sun, Wei Feng, Bo Liu, Hongrui Liu, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 47 2 221 - 228 2016年04月 [査読有り][通常論文]
     
    High mobility group box 1 (HMGB1) is a late inflammatory cytokine that plays an important role in periodontal tissue remodeling during orthodontic tooth movement. Calcitriol (1,25-dihydroxyvitamin D-3 [1 alpha,25 (OH)(2)D-3]) is a systemic calcium-regulating hormone shown to downregulate expression of multiple proinflammatory cytokines in human periodontal ligament cells in response to orthodontic force. The purpose of this study was to investigate the effect of 1 alpha,25(OH)(2)D-3 on the expression of HMGB1 in periodontal ligament (PDL) cells during orthodontic tooth movement. Seven-week-old male Wistar rats were used for experimentation. Tooth movement was assessed using a nickel-titanium coil spring to apply mechanical loading to the tooth for 5 days. This was followed by administration of either 1 alpha,25(OH)(2)D-3 or normal saline by gavage every other day for up to 28 days. Immunohistochemistry was used to analyze the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and HMGB1. After discontinuation of orthodontic force, expression of the early inflammatory cytokines IL-6 and TNF-alpha were time-dependently reduced in the 1 alpha,25(OH)(2)D-3 group compared with the control group at each time point. Similarly, expression of HMGB1 was decreased over time in both the 1 alpha,25(OH)(2)D-3 and normal saline groups, and 1 alpha,25(OH)(2)D-3 administration enhanced this decline. These findings indicate that administration of 1 alpha,25(OH)(2)D-3 might provide a favorable microenvironment for orthodontic tooth movement by downregulating expression of HMGB1 in PDL cells.
  • Wei Feng, Bo Liu, Di Liu, Tomoka Hasegawa, Wei Wang, Xiuchun Han, Jian Cui, Yimin, Kimimitsu Oda, Norio Amizuka, Minqi Li
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 64 1 42 - 53 2016年01月 [査読有り][通常論文]
     
    In this study, we aimed to evaluate the influence of diet-induced obesity on IL-6 deficiency-induced bone remodeling abnormality. Seven-week-old IL-6(-/-) mice and their wild type (WT) littermates were fed a standard diet (SD) or high-fat diet (HFD) for 25 weeks. Lipid formation and bone metabolism in mice tibiae were investigated by histochemical analysis. Both IL-6(-/-) and WT mice fed the HFD showed notable body weight gain, thickened cortical bones, and adipose accumulation in the bone marrow. Notably, the HFD normalized the bone phenotype of IL-6(-/-) mice to that of their WT counterpart, as characterized by a decrease in bone mass and the presence of an obliquely arranged, plate-like morphology in the trabecular bone. Alkaline phosphatase and osteocalcin expressions were attenuated in both genotypes after HFD feeding, especially for the IL-6(-/-) mice. Meanwhile, tartrate-resistant acid phosphatase staining was inhibited, osteoclast apoptosis rate down-regulated (revealed by TUNEL assay), and the proportion of cathepsin K (CK)-positive osteoclasts significantly increased in IL-6(-/-) mice on a HFD as compared with IL-6(-/-) mice on standard chow. Our results demonstrate that HFD-induced obesity reverses IL-6 deficiency-associated bone metabolic disorders by suppressing osteoblast activity, upregulating osteoclastic activity, and inhibiting osteoclast apoptosis.
  • Makoto Tanaka, Hiroyuki Yamada, Satoshi Nishikawa, Hiroshi Mori, Yasuo Ochi, Naoto Horai, Minqi Li, Norio Amizuka
    INTERNATIONAL JOURNAL OF RHEUMATOLOGY 2016 8938916 - 8938916 2016年 [査読有り][通常論文]
     
    The role of cathepsin K in joint degradation in a model of collagen-induced arthritis (CIA) in cynomolgus monkey was examined using biochemical markers and histology. Joint swelling, urinary C-telopeptide of type II collagen (CTX-II), deoxypyridinoline (DPD), and N-and C-telopeptides of type I collagen (NTX and CTX-I, resp.) were analyzed. Immunohistochemistry of type II collagen, cathepsin K, and CTX-II were performed using joints. Joint swelling reached peak on day 42 and continued at this level. The CTX-II level peaked on day 28 and declined thereafter, while CTX-I, NTX, and DPD reached plateau on day 43. Joint swelling was positively correlated with CTX-II increases on days 20 and 42/43, with increases in CTX-I and NTX/Cr on days 42/43 and 84, and with DPD increases throughout the study period. Intense cathepsin K staining was observed in osteoclasts and in articular cartilage and synovial tissue in arthritic joints. CTX-II was present in the superficial layer of articular cartilage in CIA monkeys. Evidence from biochemical markers suggests that matrix degradation in the CIA model starts with degradation of cartilage, rather than bone resorption. Cathepsin K expressed in osteoclasts, articular cartilage, and synovial tissue may contribute to degradation of cartilage.
  • Zhusheng Liu, Tomomaya Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Kanako Tsuboi, Erika Tsuchiya, Mai Haraguchi, Miki Abe, Paulo Henrique Luiz de Freitas, Akira Kudo, Kimimitsu Oda, Minqi Li, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 37 2 141 - 151 2016年 [査読有り][通常論文]
     
    We employed a well-standardized murine rib fracture model to assess the distribution, in the cortical bone, of three important osteocyte-derived molecules-dentine matrix protein 1 (DMP1), sclerostin and fibroblast growth factor 23 (FGF 23). Two days after the fracture, the periosteum thickened, and up to the seventh day post-fracture, the cortical surfaces were promoting neoformation of two tissue types depending on the distance from the fracture site: chondrogenesis was taking place near the fracture, and osteogenesis distant from it. The cortical bones supporting chondrogenesis featured several empty lacunae, while in the ones underlying newly-formed woven bone, empty lacunae were hardly seen. DMP1-immunopositive osteocytic lacunae and canaliculi were seen both close and away from the fracture. In contrast, the region close to the fracture had only few sclerostin-and FGF23-immunoreactive osteocytes, whereas the distant region revealed many osteocytes immunopositive for these markers. Mature cortical bone encompassing the native cortical bone was observed at two-, three-and four-weeks post-fracture, and the distribution of DMP1, sclerostin and FGF23 appeared to have returned to normal. In summary, early stages of fracture healing seem to be important for triggering chondrogenesis and osteogenesis that may be regulated by osteocytes via their secretory molecules.
  • 長谷川智香, 網塚憲生
    Pain Research 31 4 210 - 219 2016年 [査読有り][通常論文]
     
    Bone is a supporting tissue consisting of a variety of bone cells (osteo blasts, osteoclasts, osteocytes), calcified bone matrix, blood vessels, nerves and so forth. Bone diseases-osteoporosis, bone fracture, arthritis, bone metas tasis -often induce bone pain, presumably due to various kinds of nociceptors linked to the signaling in sensory nerves. Nociceptive pain in bone is caused by several stimuli of acid secreted by osteoclasts, physiochemical damaging on peripheral nerves, ATPs, prostaglandins, and so forth. However, precious knowledge on the biological function of bone cells may provide a clue for better understanding on the cellular mechanism of bone pain. In a physio logical state, normal bone is always remodeled by balanced osteoclastic bone resorption and subsequent osteoblastic bone formation. Osteocytes connect to neighboring osteocytes and to osteoblasts on the bone surface by means of thin cytoplasmic processes that go through narrow passageways. i.e., osteo cytic canaliculi, and thereby building functional syncytia referred to as osteocytic lacunar-canalicular system. Thus, osteocytes are at the center of bone turnover's mainframe. In this review, we will introduce histological and ultrastructural aspects of bone cells, and give a rough outline of bone pain.
  • Bingzi Dong, Itsuro Endo, Yukiyo Ohnishi, Takeshi Kondo, Tomoka Hasegawa, Norio Amizuka, Hiroshi Kiyonari, Go Shioi, Masahiro Abe, Seiji Fukumoto, Toshio Matsumoto
    JOURNAL OF BONE AND MINERAL RESEARCH 30 11 1980 - 1993 2015年11月 [査読有り][通常論文]
     
    Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D-3 or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT-305/MK-5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT-305/MK-5442 suppressed the hypersensitivity to extracellular Ca2+ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock-in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT-305/MK-5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1-34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock-in mice exhibited low bone turnover due to the deficiency of PTH, and JTT-305/MK-5442 as well as PTH(1-34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. (c) 2015 American Society for Bone and Mineral Research.
  • Feng W, Lv S, Cui J, Han X, Du J, Sun J, Wang K, Wang Z, Lu X, Guo J, Oda K, Amizuka N, Xu X, Li M
    Materials science & engineering. C, Materials for biological applications 54 133 - 141 2015年09月 [査読有り][通常論文]
     
    The purpose of this study was to evaluate the effects of osteogenic differentiated adipose-derived stem cell (ADSC) loaded beta-tricalcium phosphate (beta-TCP) in the restoration of bone defects under intraperitoneal administration of 1 alpha,25-dihydroxyvitamin D3(1 alpha,25 (OH)(2)D-3). ADSCs were isolated from the fat tissue of 8 week old Wister rats and co-cultured with beta-TCP for 21 days under osteogenic induction. Then the ADSC-13-TCP complexes were implanted into bone defects in the femora of rats. 1 alpha,25(OH)(2)D-3 (VD) or normal saline (NS) was administrated intraperitoneally every other day after the surgery. Femora were harvested at day 7, day 14 and day 28 post-surgery. There were 4 groups for all specimens: beta-TCP-NS group; beta-TCP-ADSC-NS group; beta-TCP-VD group and beta-TCP-ADSC-VD group. Alkaline phosphatase (ALP) was up-regulated obviously in ADSC groups compared with non-ADSC groups at day 7, day 14 and day 28, although high expression of runt-related transcription factor 2 (RUNX2) was only seen at day 7. Furthermore, the number of TRAP-positive osteoclasts and the expression of cathepsin K (CK) were significantly decreased in VD groups compared with non-VD groups at day 7 and day 14. As a most significant finding, the beta-TCP-ADSC-VD group showed the highest BV/TV ratio compared with the other three groups at day 28. Taken together, ADSC-loaded beta-TCP under the administration of 1 alpha,25(OH)(2)D-3 made a promising therapy for bone defects restoration. (C) 2015 Elsevier B.V. All rights reserved.
  • Azuma K, Shiba S, Hasegawa T, Ikeda K, Urano T, Horie-Inoue K, Ouchi Y, Amizuka N, Inoue S
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 30 7 1245 - 1254 2015年07月 [査読有り][通常論文]
     
    Vitamin K is a fat-soluble vitamin that is necessary for blood coagulation. In addition, it has bone-protective effects. Vitamin K functions as a cofactor of -glutamyl carboxylase (GGCX), which activates its substrates by carboxylation. These substrates are found throughout the body and examples include hepatic blood coagulation factors. Furthermore, vitamin K functions as a ligand of the nuclear receptor known as steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR). We have previously reported on the bone-protective role of SXR/PXR signaling by demonstrating that systemic Pxr-knockout mice displayed osteopenia. Because systemic Ggcx-knockout mice die shortly after birth from severe hemorrhage, the GGCX-mediated effect of vitamin K on bone metabolism has been difficult to evaluate. In this work, we utilized Ggcx-floxed mice to generate osteoblast-specific GGCX-deficient (Ggcx(obl/obl)) mice by crossing them with Col1-Cre mice. The bone mineral density (BMD) of Ggcx(obl/obl) mice was significantly higher than that of control Col1-Cre (Ggcx(+/+)) mice. Histomorphometrical analysis of trabecular bones in the proximal tibia showed increased osteoid volume and a higher rate of bone formation in Ggcx(obl/obl) mice. Histomorphometrical analysis of cortical bones revealed a thicker cortical width and a higher rate of bone formation in Ggcx(obl/obl) mice. Electron microscopic examination revealed disassembly of mineralized nodules and aberrant calcification of collagen fibers in Ggcx(obl/obl) mice. The mechanical properties of bones from Ggcx(obl/obl) mice tended to be stronger than those from control Ggcx(+/+) mice. These results suggest that GGCX in osteoblasts functions to prevent abnormal mineralization in bone formation, although this function may not be a prerequisite for the bone-protective effect of vitamin K. (c) 2015 American Society for Bone and Mineral Research. (c) 2015 American Society for Bone and Mineral Research.
  • Wu Hao, Syoichi Tashiro, Tomoka Hasegawa, Yuiko Sato, Tami Kobayashi, Toshimi Tando, Eri Katsuyama, Atsuhiro Fujie, Ryuichi Watanabe, Mayu Morita, Kana Miyamoto, Hideo Morioka, Masaya Nakamura, Morio Matsumoto, Norio Amizuka, Yoshiaki Toyama, Takeshi Miyamoto
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 28 17106 - 17115 2015年07月 [査読有り][通常論文]
     
    Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that dedifferentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D-3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheralnervede-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D-3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition.
  • Juan Li, Wei Feng, Bo Liu, Bao Sun, Xiuchun Han, Juan Du, Jing Sun, Yimin, Jian Cui, Jie Guo, Akira Kudo, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 46 3 303 - 311 2015年06月 [査読有り][通常論文]
     
    Periostin is essential for the integrity and function of the periodontal ligament (PDL), and periostin knockout is related to an enhanced inflammatory status in PDL. High mobility group box 1 (HMGB1), a late inflammatory cytokine, is up-regulated in PDL cells in response to mechanical stress. This study aimed to investigate the effect of periostin deficiency (Pn-/-) on HMGB1 expression in PDL during orthodontic tooth movement. We used 8-week-old male mice homozygous for the disrupted periostin gene and their wild-type (WT) littermates. Tooth movement was performed according to Waldo's method, in which 0.5-mm-thick elastic bands were inserted between the first and second upper molars of anesthetized mice. After 3 days of mechanical loading, mice were fixed by transcardial perfusion of 4 % paraformaldehyde in phosphate buffer, and the maxilla was extracted for histochemical analyses. Compared with the WT group, Pn-/- mice showed higher basal expression of HMGB1 in the absence of mechanical loading. Following 3 days of orthodontic tooth movement, the PDL in the compression side of both groups was almost replaced by cell-free hyaline zones, and Pn-/- mice showed a much wider residual PDL than WT mice. In the tension side, the number of HMGB1-positive cells in PDL in both Pn-/- and WT groups increased remarkably without a significant difference between the two groups. Our findings suggest an inhibitory effect of periostin on HMGB1 production by PDL and confirmed the critical role of periostin in integrity of PDL collagen fibrils during orthodontic tooth movement, although mechanical loading is the predominant stimulant of HMGB1 expression relative to periostin deficiency.
  • Hongrui Liu, Jian Cui, Jing Sun, Juan Du, Wei Feng, Bao Sun, Juan Li, Xiuchun Han, Bo Liu, Yimin, Kimimitsu Oda, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 46 3 313 - 323 2015年06月 [査読有り][通常論文]
     
    The purpose of this study was to investigate the effect of zoledronate (ZA) on osteoclast functions and viability in the tibiae of 8-week-old male mice. After weekly intravenous administration of ZA (125 mu g/kg body weight) for 8 weeks, the mice were fixed by transcardial perfusion of 4 % paraformaldehyde under anesthesia, and their tibiae were extracted for histochemical analysis. Compared with the control group, many tartrate-resistant acidic phosphatase-positive osteoclasts were found on the surface of the trabecular bone, but cartilage cores were obviously increased in the metaphysis of the ZA group. Osteoclasts of both groups showed similar expression of cathepsin K and matrix metalloproteinase-9. However, hardly any expression of c-src, a gene necessary for ruffled border formation and bone resorption, was found in osteoclasts of the ZA group. Moreover, no expression of CD44 or osteopontin (OPN) was observed in osteoclasts of the ZA group. Taken together, our findings suggest that ZA administration decreases the bone resorption ability of osteoclasts by inhibiting c-src expression and suppressing osteoclast adhesion by interfering with CD44/OPN binding.
  • Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Kanako Tsuboi, Norio Amizuka
    Clinical calcium 25 5 693 - 9 2015年05月 [査読有り][通常論文]
     
    Klotho deficient (kl/kl) mice exhibit Möncheberg's vascular calcification in the tunica media due to hyperphosphatemia and hypercalcemia by mediating the disrupted signaling of FGF23/klotho axis. Recent studies have hypothesized the mechanism of medial vascular calcification : Vascular smooth muscle cells acquired excessive intake of phosphate ions undergo a phenotypic differentiation into osteoblasts and induce biological calcification in the tunica media. It is useful to clarify the underlying cellular mechanism of vascular calcification for the development of the treatment and preventive medicine. This review will introduce the histological and ultrastructual findings on medial vascular calcification in kl/kl mice.
  • Mitsuru Saito, Marc D. Grynpas, David B. Burr, Matthew R. Allen, Susan Y. Smith, Nancy Doyle, Norio Amizuka, Tomoka Hasegawa, Yoshikuni Kida, Keishi Marumo, Hitoshi Saito
    BONE 73 8 - 15 2015年04月 [査読有り][通常論文]
     
    Eldecalcitol (ELD), an active form of vitamin D analog approved for the treatment of osteoporosis in Japan, increases lumbar spine bone mineral density (BMD), suppresses bone turnover markers, and reduces fracture risk in patients with osteoporosis. We have previously reported that treatment with ELD for 6 months improved the mechanical properties of the lumbar spine in ovariectomized (OVX) cynomolgus monkeys. ELD treatment increased lumbar BMD, suppressed bone turnover markers, and reduced histomorphometric parameters of both bone formation and resorption in vertebral trabecular bone. In this study, we elucidated the effects of ELD on bone quality (namely, mineralization, microarchitecture, microdamage, and bone collagen crosslinks) in OVX cynomolgus monkeys in comparison with OVX-vehicle control monkeys. Density fractionation of bone powder prepared from lumbar vertebrae revealed that ELD treatment shifted the distribution profile of bone mineralization to a higher density, and backscattered electron microscopic imaging showed improved trabecular bone connectivity in the ELD-treated groups. Higher doses of ELD more significantly reduced the amount of microdamage compared to OVX-vehicle controls. The fractionated bone powder samples were divided according to their density, and analyzed for collagen crosslinks. Enzymatic crosslinks were higher in both the high-density (>= 2.0 mg/mL) and low-density (<2.0 mg/mL) fractions from the ELD-treated groups than in the corresponding fractions in the OVX-vehicle control groups. On the other hand, non-enzymatic crosslinks were lower in both the high- and low-density fractions. These observations indicated that ELD treatment stimulated the enzymatic reaction of collagen crosslinks and bone mineralization, but prevented non-enzymatic reaction of collagen crosslinks and accumulation of bone microdamage. Bone anti-resorptive agents such as bisphosphonates slow down bone remodeling so that bone mineralization, bone microdamage, and non-enzymatic collagen crosslinks all increase. Bone anabolic agents such as parathyroid hormone decrease bone mineralization and bone microdamage by stimulating bone remodeling. ELD did not fit into either category. Histological analysis indicated that the ELD treatment strongly suppressed bone resorption by reducing the number of osteoclasts, while also stimulating focal bone formation without prior bone resorption (bone minimodeling). These bidirectional activities of ELD may account for its unique effects on bone quality. (C) 2014 The Authors. Published by Elsevier Inc.
  • Hongrui Liu, Jian Cui, Wei Feng, Shengyu Lv, Juan Du, Jing Sun, Xiuchun Han, Zhenming Wang, Xiong Lu, Yimin, Kimimitsu Oda, Norio Amizuka, Minqi Li
    MATERIALS SCIENCE & ENGINEERING C-MATERIALS FOR BIOLOGICAL APPLICATIONS 49 14 - 24 2015年04月 [査読有り][通常論文]
     
    The aim of this study was to investigate the influence of calcitriol on osteoinduction following local administration into mandibular bone defects. Calcitriol-loaded absorbable collagen membrane scaffolds were prepared using the polydopamine coating method and characterized by scanning electron microscopy. Composite scaffolds were implanted into rat mandibular bone defects in the following groups: no graft material (control), bare collagen membrane (CM group), collagen membrane bearing polydopamine coating (DOP/CM group), and collagen membrane bearing polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and 8 weeks post-surgery, the osteogenic potential of calcitriol was examined by histological and immunohistochemical methods. Following in vivo implantation, calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic differentiation at weeks 1, and 2. Furthermore, statistical analysis revealed that the newly formed bone volume in the CAL/DOP/CM group was significantly higher than other groups at weeks 1, and 2. At weeks 4, and 8, the CAL/DOP/CM group showed more mineralized bone and uniform collagen structure. These data suggest that local administration of calcitriol is promising in promoting osteogenesis and mineralization for restoration of mandibular bone defects. (C) 2014 Elsevier B.V. All rights reserved.
  • Yasuhisa Abe, Kousuke Iba, Koichi Sasaki, Hironori Chiba, Kumiko Kanaya, Tomoyuki Kawamata, Kimimitsu Oda, Norio Amizuka, Muneteru Sasaki, Toshihiko Yamashita
    JOURNAL OF BONE AND MINERAL METABOLISM 33 2 125 - 134 2015年03月 [査読有り][通常論文]
     
    The aim of this study was to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) on pain in an ovariectomized (OVX) mouse model. We evaluated skeletal pain in OVX mice through an examination of pain-like behavior as well as immunohistochemical findings. In addition, we assessed the effects of alendronate (ALN), a potent osteoclast inhibitor, on those parameters. The OVX mice showed a decrease in the pain threshold value, and an increase in the number of c-Fos immunoreactive neurons in laminae I-II of the dorsal horn of the spinal cord. Alendronate caused an increase in the pain threshold value and inhibited c-Fos expression. The serum level of tartrate-resistant acid phosphatase 5b, a marker of osteoclast activity, was significantly negatively correlated with the pain threshold value. Furthermore, we found that an antagonist of the transient receptor potential channel vanilloid subfamily member 1, which is an acid-sensing nociceptor, improved pain-like behavior in OVX mice. These results indicated that the inhibitory effect of BP on osteoclast function might contribute to an improvement in skeletal pain in osteoporosis patients.
  • Shengyu Lv, Juan Li, Wei Feng, Hongrui Liu, Juan Du, Jing Sun, Jian Cui, Bao Sun, Xiuchun Han, Kimimitsu Oda, Norio Amizuka, Xin Xu, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 46 1 107 - 114 2015年02月 [査読有り][通常論文]
     
    Recent studies indicate that high mobility group box protein 1 (HMGB1) originating from periodontal ligament (PDL) cells can be a potential regulator in the process of orthodontic tooth movement and periodontal tissue remodeling. The aim of this study is to investigate HMGB1 expression in periodontal tissue during orthodontic tooth movement in mice according to Waldo's method. Six 7-week-old C57BL6 mice were used in these experiments. The elastic band was inserted into the teeth space between the right first and second maxillary molars. After 3 days of mechanical loading, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the maxillary was extracted for histochemical analyses. The histological examination revealed local PDL tear at the tension side and the formation of extensive cell-free hyaline zones at the compression side. The immunolocalization of HMGB1 was significantly presented at tension side of PDL, apical area and dental pulp, whereas at the compression side of PDL, the labeling of HMGB1 was almost undetectable as the presence of hyaline zone. Taken together, we concluded that the orthodontic tooth movement by Waldo's method leads to histological changes and HMGB1 expression pattern that differ from those of coil spring method, including PDL tear and extensive hyaline zone which may severely destroy periodontal tissue and in turn impede tooth movement.
  • Hongrui Liu, Jie Guo, Shanliang Wei, Shengyu Lv, Wei Feng, Jian Cui, Tomoka Hasegawa, Hiromi Hongo, Yang Yang, Xiangzhi Li, Kimimitsu Oda, Norio Amizuka, Minqi Li
    HISTOLOGY AND HISTOPATHOLOGY 30 1 77 - 85 2015年01月 [査読有り][通常論文]
     
    This study aimed to investigate the expression of matrix Gla protein (MGP) and osteocalcin (OCN) in the tibial epiphysis of developing mice. At 1, 2, 3, and 4 weeks after birth, tibiae were removed and processed for histochemical observations and western blot analyses under anesthesia. To evaluate bone volume, the specimens were scanned with Micro CT Scanner from the articular cartilage through the growth plate, along the long axis of tibia. At 1 week after birth, OCN reactivity was faint in the region of vascular invasion, while hardly any MGP reactivity was discernible. Subsequently, MGP reactivity was seen on the cartilaginous lacunar walls of hypertrophic chondrocytes, while OCN reactivity was evenly found not only in the bone matrix, but also in the cartilaginous lacunar walls and on the bone surfaces. Furthermore, double-immunostaining clearly showed that MGP reactivity appeared closer to the cartilage matrix than OCN reactivity until postnatal week 3. Interestingly, the immunoreactivities for MGP and OCN both showed tidemarks in the articular cartilage at postnatal week 4, and MGP reactivity was more intense than OCN reactivity. Statistical analyses showed an overall upward trend in MGP and OCN expression levels during tibial epiphysis development, even though OCN was more abundant than MGP at every time-point. Taken together, our findings suggest that the expression of MGP and OCN increased gradually in the murine developing tibial epiphysis, and the two mineral-associated proteins may occur at the same location during a particular period, but at different levels.
  • Tsuneyuki Yamamoto, Tamaki Yamada, Tomomaya Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Kimimitsu Oda, Norio Amizuka
    ACTA HISTOCHEMICA ET CYTOCHEMICA 48 3 95 - 101 2015年 [査読有り][通常論文]
     
    To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.
  • Tsuneyuki Yamamoto, Tomomaya Yamamoto, Tamaki Yamada, Tomoka Hasegawa, Hiromi Hongo, Kimimitsu Oda, Norio Amizuka
    HISTOCHEMISTRY AND CELL BIOLOGY 142 5 489 - 496 2014年11月 [査読有り][通常論文]
     
    This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig's epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial-mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle.
  • Shiba S, Ikeda K, Azuma K, Hasegawa T, Amizuka N, Horie-Inoue K, Inoue S
    Biochemical and biophysical research communications 453 3 350 - 355 2014年10月 [査読有り][通常論文]
     
    Vitamin K-dependent gamma-glutamyl carboxylase (GGCX) is an enzyme that catalyzes the conversion of glutamic acid to gamma-carboxyglutamic acid in substrate proteins. Among GGCX target proteins, recent evidence indicates that osteocalcin regulates insulin sensitivity and secretion. However, the precise contribution of GGCX to glucose metabolism remains to be clarified. To address this question, we generated osteoblast-specific Ggcx-deficient (i.e., conditional knockout [cKO]) mice using collagen type 1 alpha 1 (Col1)-Cre mice. Ggcx cKO mice exhibited altered metabolism compared with, their controls; serum glucose levels could be maintained with low amounts of insulin, and the weight of white adipose tissue (WAT) significantly decreased in Ggcx cKO mice. Our findings suggest that GGCX expressed in osteoblasts is critical for the maintenance of blood glucose and WAT. (C) 2014 Elsevier Inc. All rights reserved.
  • Hongrui Liu, Wei Feng, Yimin, Jian Cui, Shengyu Lv, Tomoka Hasegawa, Bao Sun, Juan Li, Kimimitsu Oda, Norio Amizuka, Minqi Li
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 62 8 556 - 564 2014年08月 [査読有り][通常論文]
     
    Interleukin-6 (IL-6) is a multifunctional cytokine considered to modulate bone homeostasis. Based on previous contradictory studies, we aimed to verify the influence of IL-6 deficiency on bone remodeling using an IL-6 knockout (IL-6-/-) murine model. Eight-month-old male mice, homozygous for the disrupted IL-6 gene, and their wild type (WT) littermates (control), were used. After transcardiac perfusion, tibiae were removed for histochemical analysis. Compared with the control group, IL-6 deficiency increased tartrate resistant acid phosphatase (TRAP)-positive osteoclast numbers and up-regulated the alkaline phosphatase (ALP) activity of osteoblasts in the metaphysis of the tibia. However, further analysis of serial histological sections from IL-6-/- ice found a significant discrepancy in osteoclast number, with the higher number of TRAP-positive osteoclasts conflicting with the lower number of cathepsin K-positive osteoclasts. Moreover, TUNEL staining identified a significantly higher rate of osteoclast apoptosis in IL-6-/- ice as compared with their WT controls. IL-6 deficiency induced abundant TRAP-positive osteoclasts but delayed bone remodeling by significantly inhibiting the bone resorption activity of osteoclasts and promoting osteoclast apoptosis.
  • Kimie Nakagawa, Natsumi Sawada, Yoshihisa Hirota, Yuri Uchino, Yoshitomo Suhara, Tomoka Hasegawa, Norio Amizuka, Tadashi Okamoto, Naoko Tsugawa, Maya Kamao, Nobuaki Funahashi, Toshio Okano
    PLOS ONE 9 8 e104078  2014年08月 [査読有り][通常論文]
     
    UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K-2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K-2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K-2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K-2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wildtype. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K-2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K-2, but may have additional functions beyond the biosynthesis of vitamin K-2.
  • Liu Hongrui, Guo Jie, Wei Shanliang, Lv Shengyu, Feng Wei, Cui Jian, Hasegawa Tomoka, Hongo Hiromi, Yang Yang, Li Xiangzhi, Oda Kimimitsu, Amizuka Norio, Li Minqi
    Histol Histopathol 2014年06月23日 [査読有り][通常論文]
     
    This study aimed to investigate the expression of matrix Gla protein (MGP) and osteocalcin (OCN) in the tibial epiphysis of developing mice. At 1, 2, 3, and 4 weeks after birth, tibiae were removed and processed for histochemical observations and western blot analyses under anesthesia. To evaluate bone volume, the specimens were scanned with Micro CT Scanner from the articular cartilage through the growth plate, along the long axis of tibia. At 1 week after birth, OCN reactivity was faint in the region of vascular invasion, while hardly any MGP reactivity was discernible. Subsequently, MGP reactivity was seen on the cartilaginous lacunar walls of hypertrophic chondrocytes, while OCN reactivity was evenly found not only in the bone matrix, but also in the cartilaginous lacunar walls and on the bone surfaces. Furthermore, double-immunostaining clearly showed that MGP reactivity appeared closer to the cartilage matrix than OCN reactivity until postnatal week 3. Interestingly, the immunoreactivities for MGP and OCN both showed tidemarks in the articular cartilage at postnatal week 4, and MGP reactivity was more intense than OCN reactivity. Statistical analyses showed an overall upward
  • Tabata Chihiro, Hongo Hiromi, Sasaki Muneteru, Hasegawa Tomoka, de Freitas Paulo, Henrique Luiz, Yamada Tamaki, Yamamoto Tomomaya, Suzuki Reiko, Yamamoto Tsuneyuki, Oda Kimimitsu, Li Minqi, Kudo Akira, Iida Junichiro, Amizuka Norio
    Histol Histopathol 29 6 731 - 742 2014年06月 [査読無し][通常論文]
     
    Verifying whether periostin affects the distribution of type I collagen, fibronectin and tenascin C in the periodontal ligament (PDL) is important to contribute to a more thorough understanding of that protein's functions. In this study, we have histologically examined incisor PDL of mandibles in 20 week-old male wild-type and periostin-deficient (periostin-/-) mice, by means of type I collagen, fibronectin, tenascin C, proliferating cell nuclear antigen, matrix metallo-proteinase (MMP)-1 and F4/80-positive monocyte/macrophage immunostaining, transmission electron microscopy and quantitative analysis of cell proliferation. Wild-type PDL featured well-arranged layers of collagen bundles intertwined with PDL cells, whose longitudinal axis ran parallel to the collagen fibers. However, cells in the periostin-/- PDL were irregularly distributed among collagen fibrils, which were also haphazardly arranged. Type I collagen and fibronectin reactivity was seen throughout the wild-type PDL, while in the periostin-/- PDL, only focal, uneven staining for these proteins could be seen. Similarly, tenascin C staining was evenly distributed in the wild-type PDL, but hardly seen in the periosti
  • Shengyu Lv, Hongrui Liu, Jian Cui, Tomoka Hasegawa, Hiromi Hongo, Wei Feng, Juan Li, Bao Sun, Akira Kudo, Norio Amizuka, Minqi Li
    JOURNAL OF MOLECULAR HISTOLOGY 45 3 303 - 309 2014年06月 [査読有り][通常論文]
     
    The purpose of this study was to investigate immunolocalization of collagenolytic enzymes including cathepsin K, matrix metalloproteinase (MMP) 1 and 2 in the compressed periodontal ligament (PDL) during orthodontic tooth movement using a periostin deficient (Pn-/-) mouse model. Twelve-week-old male mice homozygous for the disrupted periostin gene and their wild type (WT) littermates were used in these experiments. The tooth movement was performed according to Waldo's method, in which elastic bands of 0.5 mm thickness were inserted between the first and second upper molars of mice under anesthesia. At 1 and 3 days after orthodontic force application, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the first molars and peripheral alveolar bones were extracted for histochemical analyses. Compared with WT mice, immunolocalization of cathepsin K, MMP1 and MMP2 was significantly decreased at 1 and 3 days after orthodontic tooth movement in the compressed PDL of Pn-/- mice, although MMP1-reactivity and MMP2-reactivity decreased at different amounts. Very little cathepsin K-immunoreactivity was observed in the assessed regions of Pn-/- mice, both before and after orthodontic force application. Furthermore, Pn-/- mice showed a much wider residual PDL than WT mice. Taken together, we concluded that periostin plays an essential role in the function of collagenolytic enzymes like cathepsin K, MMP1 and MMP2 in the compressed PDL after orthodontic force application.
  • Chihiro Tabata, Hiromi Hongo, Muneteru Sasaki, Tomoka Hasegawa, Paulo Henrique Luiz de Freitas, Tamaki Yamada, Tomomaya Yamamoto, Reiko Suzuki, Tsuneyuki Yamamoto, Kimimitsu Oda, Minqi Li, Akira Kudo, Junichiro Iida, Norio Amizuka
    HISTOLOGY AND HISTOPATHOLOGY 29 6 731 - 742 2014年06月 [査読有り][通常論文]
     
    Verifying whether periostin affects the distribution of type I collagen, fibronectin and tenascin C in the periodontal ligament (PDL) is important to contribute to a more thorough understanding of that protein's functions. In this study, we have histologically examined incisor PDL of mandibles in 20 week-old male wild-type and periostin-deficient (periostin(-/-)) mice, by means of type I collagen, fibronectin, tenascin C, proliferating cell nuclear antigen, matrix metalloproteinase (MMP)-1 and F4/80-positive monocyte/ macrophage immunostaining, transmission electron microscopy and quantitative analysis of cell proliferation. Wild-type PDL featured well-arranged layers of collagen bundles intertwined with PDL cells, whose longitudinal axis ran parallel to the collagen fibers. However, cells in the periostin(-/-) PDL were irregularly distributed among collagen fibrils, which were also haphazardly arranged. Type I collagen and fibronectin reactivity was seen throughout the wild-type PDL, while in the periostin(-/-) PDL, only focal, uneven staining for these proteins could be seen. Similarly, tenascin C staining was evenly distributed in the wildtype PDL, but hardly seen in the periostin(-/-) PDL. MMP1 immunoreactivity was uniformly distributed in the wild-type PDL, but only dotted staining could be discerned in the periostin(-/-) PDL. F4/80-positive monocyte/macrophages were found midway between tooth-and bone-related regions in the wild-type PDL, a pattern that could not be observed in the periostin(-/-) PDL. In summary, periostin deficiency may not only cause PDL collagen fibril disorganization, but could also affect the distribution of other major extracellular matrix proteins such as fibronectin and tenascin C.
  • Liu Hongrui, Feng Wei, Yimin, Cui Jian, Lv Shengyu, Hasegawa Tomoka, Sun Bao, Li Juan, Oda Kimimitsu, Amizuka Norio, Li Minqi
    J Histochem Cytochem 62 8 556 - 64 2014年05月14日 [査読無し][通常論文]
     
    Interleukin-6 (IL-6) is a multifunctional cytokine considered to modulate bone homeostasis. Based on previous contradictory studies, we aimed to verify the influence of IL-6 deficiency on bone remodeling using an IL-6 knockout (IL-6-/-) murine model. Eight-month-old male mice, homozygous for the disrupted IL-6 gene, and their wild type (WT) littermates (control), were used. After transcardiac perfusion, tibiae were removed for histochemical analysis. Compared with the control group, IL-6 deficiency increased tartrate resistant acid phosphatase (TRAP)-positive osteoclast numbers and up-regulated the alkaline phosphatase (ALP) activity of osteoblasts in the metaphysis of the tibia. However, further analysis of serial histological sections from IL-6-/- mice found a significant discrepancy in osteoclast number, with the higher number of TRAP-positive osteoclasts conflicting with the lower number of cathepsin K-positive osteoclasts. Moreover, TUNEL staining identified a significantly higher rate of osteoclast apoptosis in IL-6-/- mice as compared with their WT controls. IL-6 deficiency induced abundant TRAP-positive osteoclasts but delayed bone remodeling by significantly inhibiting t
  • Norio Amizuka, Tomomaya Yamamoto, Tomoka Hasegawa
    Clinical calcium 24 4 475 - 85 2014年04月 [査読有り][通常論文]
     
    Bone micro-environment appears to reflect bone turnover, i.e., frequency of bone remodeling. There are many bone-synthesizing mature osteoblasts, bone-resorbing osteoclasts, and a thick cell layer of preosteoblasts overlying mature osteoblasts in the region which shows active bone remodeling. Bone lining cells, - flattened, resting form of osteoblasts cover the quiescent bone surface, in which, however, osteocyte-lacunar canalicular system tend to be geometrically well-arranged. Thus, bone micro-environment seems to be regulated by preosteoblasts, bone marrow stromal cells and vascular endothelial cells, as well as osteoblasts and osteoclasts. But, precious biological function of preosteoblasts and bone marrow stromal cells are still under the investigation, e.g., due to many phenotypes of preosteoblasts. In this review, we will introduce histological and ultrastructural aspects on cellular involvement in bone micro-environment.
  • Abe Yasuhisa, Iba Kousuke, Sasaki Koichi, Chiba Hironori, Kanaya Kumiko, Kawamata Tomoyuki, Oda Kimimitsu, Amizuka Norio, Sasaki Muneteru, Yamashita Toshihiko
    J Bone Miner Metab 2014年03月16日 [査読無し][通常論文]
     
    The aim of this study was to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) on pain in an ovariectomized (OVX) mouse model. We evaluated skeletal pain in OVX mice through an examination of pain-like behavior as well as immunohistochemical findings. In addition, we assessed the effects of alendronate (ALN), a potent osteoclast inhibitor, on those parameters. The OVX mice showed a decrease in the pain threshold value, and an increase in the number of c-Fos immunoreactive neurons in laminae I-II of the dorsal horn of the spinal cord. Alendronate caused an increase in the pain threshold value and inhibited c-Fos expression. The serum level of tartrate-resistant acid phosphatase 5b, a marker of osteoclast activity, was significantly negatively correlated with the pain threshold value. Furthermore, we found that an antagonist of the transient receptor potential channel vanilloid subfamily member 1, which is an acid-sensing nociceptor, improved pain-like behavior in OVX mice. These results indicated that the inhibitory effect of BP on osteoclast function might contribute to an improvement in skeletal pain in osteoporosis
  • Norio Amizuka, Tomoka Hasegawa, Tomomaya Yamamoto, Kimimitsu Oda
    Clinical calcium 24 2 203 - 14 2014年02月 [査読有り][通常論文]
     
    In bone, biomineralization induced by osteoblasts is known to be initiated by small extracellular vesicles referred to as "matrix vesicles". Matrix vesicles possess many enzymes and transporters, which synthesize and incorporate Ca²⁺ and PO4⁻ into the vesicles. Calcification initiates when crystalline calcium phosphates are nucleated inside these matrix vesicles, and calcium phosphates, i.e., hydroxyapatite crystals, grow and eventually break through the membrane to get out of the matrix vesicles. Exposed calcium phosphates featuring "ribbon-like" appearance assemble radially, forming spherical mineralized structure, referred to as "mineralized nodule" or "calcifying globule". This process is called "matrix vesicle mineralization". Thereafter, the mineralized nodules make contacts with surrounding collagen fibrils, extending mineralization along with their longitudinal axis from the contact points of collagen fibrils - collagen mineralization. Matrix vesicle mineralization and subsequent collagen mineralization are classified as primary mineralization associated with osteoblastic bone formation. After primary mineralization, secondary mineralization takes place, gradually increasing mineral density of bone matrix. This review will introduce the microscopic findings on matrix vesicle mineralization and subsequent collagen mineralization.
  • Kimimitsu Oda, Natsuko Numa Kinjoh, Miwa Sohda, Keiichi Komaru, Norio Amizuka
    Clinical calcium 24 2 233 - 9 2014年02月 [査読有り][通常論文]
     
    There are four isozymes for human alkaline phosphatase (ALP) : tissue-nonspecific ALP (TNSALP), intestinal ALP, placental ALP and germ cell ALP. We present a brief history of TNSALP and review progress in research on it and a rare inborn error of metabolism called hypophosphatasia (HPP), which is caused by various loss-of-function mutations in the ALPL gene encoding TNSALP. HPP is characterized by decreased levels of serum ALP activity and defect in mineralization of bone and teeth, thus establishing the direct link between TNSALP and biomineralization. In addition to its 3D structure, studies on TNSALP mutants expressed in mammalian cells have revealed how each mutation affects the structure and function of TNSALP at the molecular level, which contributes to our understanding of the molecular basis of HPP.
  • Bingzi Dong, Itsuro Endo, Takeshi Kondo, Yukiyo Ohnishi, Masahiro Abe, Seiji Fukumoto, Tomoka Hasegawa, Norio Amizuka, Shin-ichi Aizawa, Toshio Matsumoto
    JOURNAL OF BONE AND MINERAL RESEARCH 29 S75 - S75 2014年02月 [査読有り][通常論文]
  • Tomoka Hasegawa, Saito Mitsuru, Doyle Nancy, Chouinard Luc, Smith Susan, Yamamoto Tomomaya, Oda Kimimitsu, Saito Hitoshi, Amizuka Norio
    JOURNAL OF BONE AND MINERAL RESEARCH 29 S106 - S106 2014年02月 [査読有り][通常論文]
  • Aiji Yajima, Ken Tsuchiya, Kosaku Nitta, Masaaki Inaba, Yoshihiro Tominaga, Norio Amizuka, Akemi Ito, Hironari Shindo
    JOURNAL OF BONE AND MINERAL RESEARCH 29 S173 - S173 2014年02月 [査読有り][通常論文]
  • Sadaoki Sakai, Satoshi Takeda, Tomomaya Yamamoto, Tomoka Hasegawa, Norio Amizuka, Koichi Endo
    JOURNAL OF BONE AND MINERAL RESEARCH 29 S263 - S264 2014年02月 [査読有り][通常論文]
  • Tomomaya Yamamoto, Tomoka Hasegawa, Sadaoki Sakai, Satoshi Takeda, Kimimitsu Oda, Minqi Li, Koichi Endo, Norio Amizuka
    JOURNAL OF BONE AND MINERAL RESEARCH 29 S314 - S315 2014年02月 [査読有り][通常論文]
  • Muneteru Sasaki, Tomoka Hasegawa, Tamaki Yamada, Hiromi Hongo, Paulo Henrique Luiz de Freitas, Reiko Suzuki, Tomomaya Yamamoto, Chihiro Tabata, Satoru Toyosawa, Tsuneyuki Yamamoto, Kimimitsu Oda, Minqi Li, Nobuo Inoue, Norio Amizuka
    Bone 57 1 206 - 19 2013年11月 [査読無し][通常論文]
     
    In an attempt to identify the histological properties of the klotho-deficient (kl/kl) bone matrix, bone mineralization and the localization of Ca(2+)-binding bone matrix proteins - osteocalcin, dentin matrix protein-1 (DMP-1) and matrix Gla protein (MGP) - were examined in kl/kl tibiae. While a widespread osteocalcin staining could be verified in the wild-type bone matrix, localization of the same protein in the kl/kl tibiae seemed rather restricted to osteocytes with only a faint staining of the whole bone matrix. In wild-type mice, MGP immunoreactivity was present at the junction between the epiphyseal bone and cartilage, and at the insertion of the cruciate ligaments. In kl/kl mice, however, MGP was seen around the cartilaginous cores of the metaphyseal trabeculae and in the periphery of some cells of the bone surface. DMP-1 was identified in the osteocytic canalicular system of wild-type tibiae, but in the kl/kl tibiae this protein was mostly found in the osteocytic lacunae and in the periphery of some cells of the bone surface. Mineralization of the kl/kl bone seemed somewhat defective, with broad unmineralized areas within its matrix. In these areas, mineralized osteocytes along with their lacunae and osteocytic cytoplasmic processes were found to have intense osteocalcin and DMP-1 staining. Taken together, it might be that the excessive production of Ca(2+)-binding molecules such as osteocalcin and DMP-1 by osteocytes concentrates mineralization around such cells, disturbing the completeness of mineralization in the kl/kl bone matrix.
  • Muneteru Sasaki, Tomoka Hasegawa, Tamaki Yamada, Hiromi Hongo, Paulo Henrique Luiz de Freitas, Reiko Suzuki, Tomomaya Yamamoto, Chihiro Tabata, Satoru Toyosawa, Tsuneyuki Yamamoto, Kimimitsu Oda, Minqi Li, Nobuo Inoue, Norio Amizuka
    BONE 57 1 206 - 219 2013年11月 [査読有り][通常論文]
     
    In an attempt to identify the histological properties of the klotho-deficient (kl/kl) bone matrix, bone mineralization and the localization of Ca2+-binding bone matrix proteins - osteocalcin, dentin matrix protein-1 (DMP-1) and matrix Gla protein (MGP) - were examined in kl/kl tibiae. While a widespread osteocalcin staining could be verified in the wild-type bone matrix, localization of the same protein in the kl/kl tibiae seemed rather restricted to osteocytes with only a faint staining of the whole bone matrix. In wild-type mice, MGP immunoreactivity was present at the junction between the epiphyseal bone and cartilage, and at the insertion of the cruciate ligaments. In kl/kl mice, however, MGP was seen around the cartilaginous cores of the metaphyseal trabeculae and in the periphery of some cells of the bone surface. DMP-1 was identified in the osteocytic canalicular system of wild-type tibiae, but in the kl/kl tibiae this protein was mostly found in the osteocytic lacunae and in the periphery of some cells of the bone surface. Mineralization of the kl/kl bone seemed somewhat defective, with broad unmineralized areas within its matrix. In these areas, mineralized osteocytes along with their lacunae and osteocytic cytoplasmic processes were found to have intense osteocalcin and DMP-1 staining. Taken together, it might be that the excessive production of Ca2+-binding molecules such as osteocalcin and DMP-1 by osteocytes concentrates mineralization around such cells, disturbing the completeness of mineralization in the kl/kl bone matrix. (C) 2013 Elsevier Inc. All rights reserved.
  • Koichiro Komatsu, Akemi Shimada, Tatsuya Shibata, Satoshi Wada, Hisashi Ideno, Kazuhisa Nakashima, Norio Amizuka, Masaki Noda, Akira Nifuji
    JOURNAL OF ENDOCRINOLOGY 219 2 145 - 158 2013年11月 [査読有り][通常論文]
     
    Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.
  • Norio Amizuka, Tomoka Hasegawa, Muneteru Sasaki, Tomomaya Yamamoto, Tsuneyuki Yamamoto
    Clinical calcium 23 10 1453 - 61 2013年10月 [査読有り][通常論文]
     
    Osteocytes are known to synthesize FGF23 which would bind to FGFR1c/klotho complex in proximal renal tubules in kidney, thereby, reducing serum concentration of Pi and the activity of 1α-hydroxylase. Meanwhile, recent studies suggest the possibility that osteocytes might induce osteolysis of lacuna walls. Compact, cortical bone develops well-organized distribution of osteocyte-lacunar canalicular system (OLCS) , which appears to be efficient for osteocytic function. There seems some relation between the geometrical regularity of OLCS and osteocytic regulation of systemic and local mineral balance.
  • Sultana Sara, Al-Shawafi Hiba A, Makita Saori, Sohda Miwa, Amizuka Norio, Takagi Ritsuo, Oda Kimimitsu
    Mol Genet Metab 109 3 282 - 288 2013年07月 [査読無し][通常論文]
     
    Various loss-of function mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene cause a rare genetic disorder called hypophosphatasia (HPP), which is characterized by defective mineralization in the bones and teeth and a deficiency in serum alkaline phosphatase. A point mutation (c.1250A>G), which leads to replacement of an asparagine at position 417 of TNSALP with serine [TNSALP (N417S)], has been reported in a patient diagnosed with perinatal HPP (Sergi C. et al. Am, J. Med. Genet. 103, 235-240, 2001). In order to characterize the molecular properties of TNSALP (N417S), we expressed and analyzed TNSALP (N417S) both in COS-1 cells (transient expression) and CHO K1 Tet-On cells (inducible cell system). In contrast to wild-type TNSALP [TNSALP (W)], cells expressing TNSALP (N417S) lacked its alkaline phosphatase activity. However, this mutant underwent N-linked oligosaccharide processing and appeared on the cell surface similar to TNSALP (W). Importantly, this mutant failed to assemble into a dimer structure, which is needed for the catalytic function of TNSALP, as evidenced by newly developed SDS-PAGE as well as sucrose-density-gradient centrifugation. Substitution
  • Hitoshi Saito, Satoshi Takeda, Norio Amizuka
    JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 136 1 178 - 182 2013年07月 [査読有り][通常論文]
     
    Vitamin D is known as a potent stimulator of bone resorption. The active form of vitamin D-3, calcitriol (1 alpha,25-dihydroxyvitamin D-3), stimulates release of calcium (Ca) from bone in ex vivo organ culture, and treatment with large amounts of an active Vitamin D-3 analog induces hypercalcemia and bone resorption in mice in vivo. Calcitriol strongly induces both receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in osteoblasts in vitro. On the other hand, it has been reported that active vitamin D-3 inhibits bone resorption in Various experimental animal Models.We previously Showed that eldecalcitol [1 alpha,25-dihydroxy-2 beta-(3-hydrOXy-prOpylOXy)vitamin D-3; ED-71] suppresses bone resorption and increases bone mineral density (BMD) to a greater extent than alfacalcidol (la-hydroxyvitamin D-3) in ovariectomized (OVX) rats in vivo.To elucidate the histological events that follow administration of eldecalcitol compared to calcitriol, OVX rats were given either vehicle, eldecalcitol (10, 30, or 90 ng/kg), or calcitriol (33.3, 100, 300, or 900 ng/kg), and sham-operated control animals were given vehicle, 5-times per week for 12 weeks. The lumbar spine and femur were removed and processed for bone mineral density (BMD) assessments and the femur for histomorphometrical analyzes.Both eldecalcitol and calcitriol increased the lumbar and femoral BMD in a dose dependent manner. Bone histomorphometry revealed that osteoclast surface (Oc.S/BS) and eroded surface (ES/BS) were dose-dependently suppressed in the trabecular region of the femur. Both calcitriol and eldecalcitol dose-dependently stimulated focal bone formation that started without prior bone resorption, a process known as bone minimodeling. Both reduction of bone resorption and stimulation of focal bone formation were more clearly observed in the eldecalcitol-treated rats than in the calcithol-treated rats.Taken together, these findings suggest that eldecalcitol is a more potent vitamin D-3 analog that stimulates focal bone formation (minimodeling) and suppresses bone resorption more strongly than does calcitriol. This article is part of a Special Issue entitled 'Vitamin D Workshop'. (C) 2012 Elsevier Ltd. All rights reserved.
  • Sara Sultana, Hiba A. Al-Shawafi, Saori Makita, Miwa Sohda, Norio Amizuka, Ritsuo Takagi, Kimimitsu Oda
    MOLECULAR GENETICS AND METABOLISM 109 3 282 - 288 2013年07月 [査読有り][通常論文]
     
    Various loss-of function mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene cause a rare genetic disorder called hypophosphatasia (HPP), which is characterized by defective mineralization in the bones and teeth and a deficiency in serum alkaline phosphatase. A point mutation (c.1250A>G), which leads to replacement of an asparagine at position 417 of TNSALP with serine [TNSALP (N417S)], has been reported in a patient diagnosed with perinatal HPP (Sergi C. et al. Am, J. Med. Genet. 103, 235-240, 2001). In order to characterize the molecular properties of TNSALP (N417S), we expressed and analyzed TNSALP (N417S) both in COS-1 cells (transient expression) and CHO K1 Tet-On cells (inducible cell system). In contrast to wild-type TNSALP [TNSALP (W)], cells expressing TNSALP (N417S) lacked its alkaline phosphatase activity. However, this mutant underwent N-linked oligosaccharide processing and appeared on the cell surface similar to TNSALP (W). Importantly, this mutant failed to assemble into a dimer structure, which is needed for the catalytic function of TNSALP, as evidenced by newly developed SDS-PAGE as well as sucrose-density-gradient centrifugation. Substitution of the asparagine at position 417 with structurally related amino acids such as an aspartate and a glutamine also abolished the dimerization of TNSALP without perturbing its cell surface localization. Taken together, the asparagine at position 417 is crucial for the assembly and function of TNSALP, which may explain the severity of the N417S mutation. (C) 2013 Elsevier Inc. All rights reserved.
  • Taku Kojima, Tomoka Hasegawa, Paulo Henrique Luiz de Freitas, Tomomaya Yamamoto, Muneteru Sasaki, Keisuke Horiuchi, Hiromi Hongo, Tamaki Yamada, Naoko Sakagami, Naoaki Saito, Michiko Yoshizawa, Tadaharu Kobayashi, Takeyasu Maeda, Chikara Saito, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 34 3 119 - 128 2013年06月 [査読有り][通常論文]
     
    We have histologically examined vascular invasion and calcification of the hypertrophic zone during endochondral ossification in matrix metalloproteinase (MMP)-9 deficient (MMP-9(-/-)) mice and in their littermates at 3 days, 3 weeks and 6 weeks after birth. Capillaries and osteoclasts at the chondro-osseous junction showed an intense MMP-9 immunopositivity, suggesting that they recognize chemical properties of cartilaginous matrices, and then release MMP-9 for cartilage degradation. CD31-positive capillaries and tartrate-resistant acid phosphatase-reactive osteoclasts could be found in the close proximity in the region of chondro-osseous junction in MMP-9(-/-) mice, while in wild-type mice, vascular invasion preceded osteoclastic migration into the epiphyseal cartilage. Although MNP-9(-/-) mice revealed larger hypertrophic zones, the index of calcified area was significantly smaller in MMP-9(-/-) mice. Interestingly, the lower layer of the MMP-9(-/-) hypertrophic zone showed intense MMP-13 staining, which could not be observed in wild-type mice. This indicates that MMP-13 may compensate for MMP-9 deficiency at that specific region, but not to a point at which the deficiency could be completely rescued. In conclusion, it seems that MMP-9 is the optimal enzyme for cartilage degradation during endochondral ossification by controlling vascular invasion and subsequent osteoclastic migration.
  • Tomoka Hasegawa, Norio Amizuka, Tamaki Yamada, Zhusheng Liu, Yukina Miyamoto, Tomomaya Yamamoto, Muneteru Sasaki, Hiromi Hongo, Reiko Suzuki, Paulo Henrique Luiz de Freitas, Tsuneyuki Yamamoto, Kimimitsu Oda, Minqi Li
    BIOMEDICAL RESEARCH-TOKYO 34 3 153 - 159 2013年06月 [査読有り][通常論文]
     
    Sclerostin, an osteocyte-derived molecule, has been reported to serve as a negative regulator of osteoblastic activity as well as bone remodeling. However, there is no report that verified the regional difference for sclerostin synthesis, and in this study we have investigated immunolocalization of sclerostin by comparing dentin matrix protein (DMP) 1, an osteocyte-derived factor broadly expressed in tibial metaphyses and cortical bone. In metaphyseal primary trabecules, a site of bone modeling, strong DMP1-reactivity was observed in osteocytic lacunar-canalicular system (OLCS), while faint staining for sclerostin was visible only in a few osteocytes. In secondary trabecules, in which bone remodeling begins, some osteocytes showed intense sclerostin-immunopositivity, though there were many DMP1-positive osteocytes. In cortical bone, there were more osteocytes reactive for sclerostin, when compared with those in the secondary trabecules. Silver impregnation verified that immature, primary trabecules contained randomly-oriented OLCS, while mature, cortical bone showed geometrically well-arrangement of OLCS. Taken together, though DMP1 is broadly synthesized in bone, sclerostin appears to be abundantly synthesized in regular OLCS of cortical bone, but less produced in irregular OLCS as seen in primary trabecules, indicating the regional difference for sclerostin synthesis.
  • Minqi Li, Tomoka Hasegawa, Hiromi Hogo, Sawako Tatsumi, Zhusheng Liu, Ying Guo, Muneteru Sasaki, Chihiro Tabata, Tsuneyuki Yamamoto, Kyoji Ikeda, Norio Amizuka
    HISTOLOGY AND HISTOPATHOLOGY 28 3 327 - 335 2013年03月 [査読有り][通常論文]
     
    The purpose of this study was to examine histological alterations on osteoblasts from the alveolar bone of transgenic mice with targeted ablation of osteoctyes. Eighteen weeks-old transgenic mice based on the diphtheria toxin (DT) receptor-mediated cell knockout (TRECK) system were used in these experiments. Mice were injected intraperitoneally with 50 mu g/kg of DT in PBS, or only PBS as control. Two weeks after injections, mice were subjected to transcardiac perfusion with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4), and the available alveolar bone was removed for histochemical analyses. Approximately 75% of osteocytes from alveolar bones became apoptotic after DT administration, and most osteocytic lacunae became empty. Osteoblastic numbers and alkaline phosphatase (ALP) activity were markedly reduced at the endosteum of alveolar bone after DT administration compared with the control. Osteoblastic ALP activity in the periodontal ligament region, on the other hand, hardly showed any differences between the two groups even though numbers were reduced in the experiment group. Silver impregnation showed a difference in the distribution of bone canaliculi between the portions near the endosteum and the periodontal ligament: the former appeared regularly arranged in contrast to the latter's irregular distribution. Under transmission electron microscopy (TEM), the osteoblasts in the periodontal ligament showed direct contact with the Sharpey's fibers. Thus, osteoblastic activity was affected by osteocyte ablation in general, but osteoblasts in contact with the periodontal ligament were less affected than endosteal osteoblasts.
  • Muneteru Sasaki, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Norio Amizuka
    Clinical calcium 23 3 347 - 53 2013年03月 [査読有り][通常論文]
     
    Intermittent administration of parathyroid hormone, PTH, appears to promote preosteoblastic proliferation and osteoblastic bone formation, giving rise to anabolic effect in bone. We investigated the behavior of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. As a consequence, bone formation was increased in PTH-treated wild-type mice, whereas in the osteoclast-deficient c-fos - / - mice, there was no significant increase in bone formation, despite the highly-increased population of preosteoblasts. It seems likely that the absence of osteoclasts might hinder PTH-driven bone anabolism, and also that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration. We will also discuss the pivotal role of osteocytes in PTH-mediated anabolic effect.
  • Rikako Hiramatsu, Yoshifumi Ubara, Noriko Hayami, Masayuki Yamanouchi, Eiko Hasegawa, Keiichi Sumida, Tatsuya Suwabe, Junichi Hoshino, Naoki Sawa, Norio Amizuka, Kenmei Takaichi
    BONE 52 2 684 - 688 2013年02月 [査読有り][通常論文]
     
    A 55-year-old woman who had been on hemodialysis for 5 years was admitted for evaluation of a hard mass in the right hip region. Her serum calcium (Ca)-phosphate (P) product was elevated. Radiographs showed periarticular calcified masses in the soft tissues around both hips and shoulders, which were characteristic of uremic tumoral calcinosis (UTC). Biopsy specimens were obtained from both right hip mass and the right iliac crest. Histological examination of hip mass revealed bone-like tissue with marrow, as well as calcified material. The bone-like tissue was categorized as heterotopic ossification (HO), because it had been formed inside soft tissue where bone-like tissue does not normally exist. Histological analysis of HO showed the formation of cancellous bone-like tissue. Woven mineralized bone-like tissue was predominant over lamellar bone-like tissue. High bone turnover combined with osteitis fibrosa-like lesion was diagnosed because of an increase of the fibrous volume, as well as clear double tetracycline labeling. Near a site of HO, numerous ALP- and Runx2-positive cuboidal osteoblast-like cells and TRAP- and cathepsin K-positive multinucleated osteoclast-like cells were noted. Histomorphometric analysis of the right iliac crest revealed osteitis fibrosa. This is the first report of HO in a patient with UTC. After parathyroidectomy, the patient's Ca-P imbalance was corrected and UTC subsided. Although the mechanism by which new bone-like tissue formation arises in the soft tissues has not yet been determined, secondary hyperparathyroidism may have contributed to the progression of UTC in this patient. (C) 2012 Elsevier Inc. All rights reserved.
  • Hasegawa T, Sasaki M, Liu Z, Yamada T, Yamamoto T, Hongo H, Suzuki R, Miyamoto Y, Yamamoto T, Freitas PHL, Li M, Amizuka N
    Hokkaido Journal of Dental Science. 34 1 2 - 10 北海道歯学会 2013年 [査読有り][通常論文]
     
    Klotho deficient （kl/kl） mice are well known to develop hyperphosphatemia and resultant Möncheberg's vascular sclerosis, which consists of elongated or fragmented elastic lamellae and abundant collagen fibrils inside the vessels. Instead of normal vascular smooth muscle cells （VSMCs）, the tunica media of the kl/kl aorta has cells rich with abundant endoplasmic reticulum and Golgi apparatus, somewhat resembling osteoblasts. There were many matrix vesicle-like structures and calcifying nodules in the vicinity of these osteoblast-like cells in kl/kl aorta. The calcifying nodules seem to trigger calcification in the elastic lamellae, without promoting it in the collagen fibrils inside the kl/kl aorta. Also, mineral deposition was observed within the intravascular amorphous organic component, suggesting dystrophic calcification. Thus, two possible pathways for vascular calcification exist: one mediated by matrix vesicle-like structures, and another taking place after the deposition of calcium phosphates in the amorphous organic component. Compared to the latter, which consists of the classical view of intravascular calcification, the former appears to mimic osteoblastic mineralization in bone, and could be the result of trans-differentiation of VSMCs into osteoblastic cells. In this work, we will review our current findings on the process of medial vascular calcification found in kl/kl mice.
  • Hasegawa T, Sasaki M, Yamada T, Ookido I, Yamamoto T, Hongo H, Yamamoto T, Oda K, Yokoyama K, Amizuka N
    J Oral Biosci. 55 1 10 - 15 2013年 [査読有り][通常論文]
     
    Examination of klotho-deficient (kl/kl) mice showed that they had developed vascular medial calcification. Von Kossa and van Gieson staining showed calcification of the straight elongated elastic lamellae. Interestingly, elastic fibers, but not the abundant collagen fibrils surrounding them, were preferentially calcified in the kl/kl aorta. Unlike normal vascular smooth muscle cells (VSMCs), tunica media cells of kl/kl mice had developed cisterns of rough endoplasmic reticulum and Golgi apparatus; thus, they resembled matrix-synthesizing cells rather than authentic VSMCs. Additionally, many matrix vesicle-like structures and calcifying nodules were observed in the vicinity of these VSMC-like cells in kl/kl tunica media. Several calcifying nodules made contact with the amorphous organic materials, allowing the extension of calcification into the amorphous materials from these contact points. In contrast, mineral deposition within the amorphous organic materials was also observed, indicating dystrophic calcification. These observations suggest 2 possible pathways of vascular calcification: one involving matrix vesicle-like structures that could include mineral crystals and the other involving the deposition of calcium phosphates within the amorphous materials. The former pathway suggests the possibility of trans-differentiation of VSMCs into the osteoblastic lineage that could induce biological calcification of vascular tissues, in addition to the classical hypothesis involving dystrophic calcification. Here, we review our current findings on the process of vascular medial calcification of the aorta in klotho-deficient mice. © 2013 Japanese Association for Oral Biology.
  • Ying Guo, Minqi Li, Liu Zhusheng, Tamaki Yamada, Muneteru Sasaki, Tomoka Hasegawa, Hiromi Hongo, Chihiro Tabata, Reiko Suzuki, Kimimitsu Oda, Tsuneyuki Yamamoto, Masamitsu Kawanami, Norio Amizuka
    JOURNAL OF ELECTRON MICROSCOPY 61 5 309 - 320 2012年10月 [査読有り][通常論文]
     
    This study aimed at elucidating whether estrogen deficiency would affect the synthesis of an osteocyte-derived factor, sclerostin, in the mesial region of alveolar bone. Eight 9-week-old Wistar female rats were ovariectomized (OVX) and eight other rats were Sham-operated (Sham). After 4 weeks, the interradicular septa of mandibular first molar were embedded in paraffin and then histochemically examined. Sclerostin-positive osteocytes were located in the superficial layer of the mesial region of Sham bones, whereas the OVX mesial region showed less sclerostin-reactive osteocytes. There was no significant difference in the distribution of estrogen receptor alpha and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling -positive cells in the groups studied. The Sham mesial region featured many osteoclasts, and OVX specimens showed numerous osteoclasts in association with intense immunolabeling of the receptor activator of the nuclear factor kB ligand. Contrary to the observations in Sham specimens, a complex meshwork of cement lines was seen in the OVX mesial region, accompanied by an irregularly distributed osteocytic lacunar-canalicular system. In conclusion, estrogen deficiency appears to inhibit osteocyte-derived sclerostin synthesis in the mesial region of the interradicular septum, in a process that seems to be mediated by accelerated bone remodeling rather than by direct effects on osteocytes.
  • Tsuneyuki Yamamoto, Tomoka Hasegawa, Muneteru Sasaki, Hiromi Hongo, Chihiro Tabata, Zhusheng Liu, Minqi Li, Norio Amizuka
    JOURNAL OF ELECTRON MICROSCOPY 61 2 113 - 121 2012年04月 [査読有り][通常論文]
     
    This study was designed to elucidate details of the structure and formation process of the alternate lamellar pattern known to exist in lamellar bone. For this purpose, we examined basic internal lamellae in femurs of young rats by transmission and scanning electron microscopy, the latter employing two different macerations with NaOH at concentrations of 10 and 24%. Observations after the maceration with 10% NaOH showed that the regular and periodic rotation of collagen fibrils caused an alternation between two types of lamellae: one consisting of transversely and nearly transversely cut fibrils, and the other consisting of longitudinally and nearly longitudinally cut fibrils. This finding confirms the consistency of the twisted plywood model. The maceration method with 24% NaOH removed bone components other than cells, thus allowing for three-dimensional observations of osteoblast morphology. Osteoblasts extended finger-like processes paralleling the inner bone surface, and grouped in such a way that, within a group, the processes arranged in a similar direction. Transmission electron microscopy showed that newly deposited fibrils were arranged alongside these processes. For the formation of the alternating pattern, our findings suggest that: (1) osteoblasts control the collagen fibril arrangement through their finger-like process position; (2) osteoblasts behave similarly within a group; (3) osteoblasts move their processes synchronously and periodically to promote alternating different fibril orientation; and (4) this dynamic sequential deposition of fibrils results in the alternate lamellar (or twisted plywood) pattern.
  • Tomoka Hasegawa, Hiromi Hongo, Muneteru Sasaki, Tamaki Yamada, Norio Amizuka
    Clinical calcium 22 3 373 - 9 2012年03月 [査読有り][通常論文]
     
    Parathyroid hormone (PTH) -driven anabolism in bone appears to involve the osteoblastic activation and the increased population of preosteoblastic lineages. Given that the activities of osteoclasts and osteoblasts are intertwined during normal bone remodeling, it is plausible that the anabolic action of PTH is either directly or indirectly related to the osteoclast. We have recently reported that the absence of osteoclasts in c-fos( - / - ) mice might hinder PTH-driven bone anabolism, and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration. Alternatively, it was suggested that PTH administration inhibits sclerostin synthesis by osteocytes, thereby allowing for active bone formation. Taken together, PTH affects bone cells in a dual pathway - mediating osteoblastic (preosteoblastic) activities or osteocytic synthesis of sclerostin -.
  • Nagata M, Hoshina H, Li M, Arasawa M, Uematsu K, Ogawa S, Yamada K, Kawase T, Suzuki K, Ogose A, Amizuka N, Fuse I, Okuda K, Uoshima K, Nakata K, Yoshie H, Takagi R
    Bone 50 5 1123 - 1129 2012年 [査読有り][通常論文]
  • Norio Amizuka, Tomoka Hasegawa, Kimimitsu Oda, Paulo Henrique Luiz de Freitas, Kazuto Hoshi, Minqi Li, Hidehiro Ozawa
    Frontiers in bioscience (Elite edition) 4 2085 - 100 2012年01月01日 [査読無し][通常論文]
     
    Cartilage calcification is carried out by chondrocytes as they hypertrophy and begin to secrete matrix vesicles. Calcification initiates when calcium phosphates appear inside these matrix vesicles, forming hydroxyapatite crystals that eventually break through the membrane to form calcifying globules, as in bone calcification. However, the extracellular environment in cartilage is different from that in bone: cartilage is abundant in proteoglycans but contains a small amount of osteopontin. Hypertrophic chondrocytes secrete vesicles in the cartilaginous matrix of intercolumnar septae only, forming well-calcified longitudinal septae and poorly-calcified transverse partitions. Such pattern of vesicle deposition permits the invasion of endothelial cells, which infiltrate into cartilage and induce migration of osteogenic and osteoclastic cells. Osteoclasts resorb the excess of calcified globules in the partitions, shaping calcified cartilage cores paralleling the longitudinal axis of long bones. After the formation of these calcified cartilage cores, endochondral ossification involves a series of well-defined events in which osteogenic cells deposit new bone onto the cartilage core and form primary trabecules. This review presents the histology of epiphyseal cartilage calcification and endochondral ossification.
  • Guo Ying, Li Minqi, Zhusheng Liu, Yamada Tamaki, Sasaki Muneteru, Hasegawa Tomoka, Hongo Hiromi, Tabata Chihiro, Suzuki Reiko, Oda Kimimitsu, Yamamoto Tsuneyuki, Kawanami Masamitsu, Amizuka Norio
    J Electron Microsc (Tokyo) 61 5 309 - 320 2012年 [査読無し][通常論文]
     
    This study aimed at elucidating whether estrogen deficiency would affect the synthesis of an osteocyte-derived factor, sclerostin, in the mesial region of alveolar bone. Eight 9-week-old Wistar female rats were ovariectomized (OVX) and eight other rats were Sham-operated (Sham). After 4 weeks, the interradicular septa of mandibular first molar were embedded in paraffin and then histochemically examined. Sclerostin-positive osteocytes were located in the superficial layer of the mesial region of Sham bones, whereas the OVX mesial region showed less sclerostin-reactive osteocytes. There was no significant difference in the distribution of estrogen receptor alpha and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling -positive cells in the groups studied. The Sham mesial region featured many osteoclasts, and OVX specimens showed numerous osteoclasts in association with intense immunolabeling of the receptor activator of the nuclear factor kB ligand. Contrary to the observations in Sham specimens, a complex meshwork of cement lines was seen in the OVX mesial region, accompanied by an irregularly distributed osteocytic lacunar-canalicular system.
  • Amizuka N, Hongo H, Sasaki M, Hasegawa T, Suzuki R, Tabata C, Ubaidus S, Masuki H, Guo Y, Freitas PHL, Oda K, Li M
    J Oral Biosci. 54 1 37 - 42 2012年 [査読有り][通常論文]
     
    Osteocytes build up functional syncytia, i.e., the osteocytic lacunar-canalicular system (OLCS). The sites of active bone remodeling revealed the irregularly arranged OLCS, while those of slow remodeling featured well arranged OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of OLCS. Recently, osteocytes were shown to regulate phosphorus serum levels and osteoblastic activity through the expression of fibroblast growth factor (FGF) 23 and sclerostin. In our observations, FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS: both proteins were consistently expressed by osteocytes in epiphyses and cortical bones showing regularly arranged OLCS. In contrast, osteoprotegerin-deficient (OPG-/-) mice revealed rapid bone remodeling with irregular OLCS. Sclerostin-immunoreactivity was markedly diminished in OPG-/- bones. However, sclerostin expression seemed to be a function of osteoclastic and osteoblastic activities rather than being influenced by the regularity of OLCS. This review will introduce our recent studies on the regularity of OLCS and the synthesis of osteocyte-derived FGF23 and sclerostin. © 2012 Japanese Association for Oral Biology.
  • Sasaki M, Hongo H, Hasegawa T, Suzuki R, Liu Z, Freitas PHL, Yamada T, Oda K, Yamamoto T, Li M, Totsuka Y, Amizuka N
    Oral Science International. 9 1 1 - 8 2012年 [査読有り][通常論文]
     
    Osteocytes are organized in functional syncytia collectively referred to as the osteocytic lacunar-canalicular system (OLCS). The osteocytes are interconnected through gap junctions between their cytoplasmic processes, which pass through narrow passageways referred to as osteocytic canaliculi. There are two possible ways molecules can be transported throughout the OLCS: via the cytoplasmic processes and their gap junctions, and via the pericellular space in the osteocytic canaliculi. Transport of minerals and small molecules through a spatially well-organized OLCS is vital for bone mineral homeostasis, mechanosensing, and bone remodeling control. Recently, osteocyte-derived molecules - sclerostin, dentin matrix protein-1, fibroblast growth factor 23 (FGF23) - have been put in evidence as they may be related to osteocytic functions such as mechanosensing, regulation of bone remodeling, and so forth. FGF23 regulates serum phosphate concentration by affecting renal function, while sclerostin can inhibit osteoblastic activities. In our observations, FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS. This review will introduce our recent morphological studies on the regularity of OLCS and the synthesis of osteocyte-derived FGF23 and sclerostin. © 2012 Japanese Stomatological Society.
  • Norio Amizuka, Tomoka Hasegawa, Kimimitsu Oda, Paulo Henrique Luiz Freitas De, Kazuto Hoshi, Minqi Li, Hidehiro Ozawa
    Frontiers in Bioscience - Elite 4 6 2085 - 2100 2012年01月01日 [査読有り][通常論文]
     
    Cartilage calcification is carried out by chondrocytes as they hypertrophy and begin to secrete matrix vesicles. Calcification initiates when calcium phosphates appear inside these matrix vesicles, forming hydroxyapatite crystals that eventually break through the membrane to form calcifying globules, as in bone calcification. However, the extracellular environment in cartilage is different from that in bone: cartilage is abundant in proteoglycans but contains a small amount of osteopontin. Hypertrophic chondrocytes secrete vesicles in the cartilaginous matrix of intercolumnar septae only, forming well-calcified longitudinal septae and poorlycalcified transverse partitions. Such pattern of vesicle deposition permits the invasion of endothelial cells, which infiltrate into cartilage and induce migration of osteogenic and osteoclastic cells. Osteoclasts resorb the excess of calcified globules in the partitions, shaping calcified cartilage cores paralleling the longitudinal axis of long bones. After the formation of these calcified cartilage cores, endochondral ossification involves a series of welldefined events in which osteogenic cells deposit new bone onto the cartilage core and form primary trabecules. This review presents the histology of epiphyseal cartilage calcification and endochondral ossification.
  • Norio Amizuka, Hiromi Hongo, Muneteru Sasaki, Tomoka Hasegawa, Paulo Henrique Luiz de Freitas, Hiroshi Mori, Minqi Li
    ARTHRITIS RESEARCH & THERAPY 14 2012年 [査読有り][通常論文]
  • Hiromi Hongo, Muneteru Sasaki, Tomoka Hasegawa, Norio Amizuka
    Clinical calcium 21 11 63 - 70 2011年11月 [査読有り][通常論文]
     
    This review will show the histological findings in femora of ovariectomized (OVX) rats administered with or without eldecalcitol, a second-generation of vitamin D analog, which were published in our recent article. The OVX group showed markedly-reduced bone mineral density, and the decreased trabecular number and thickness, which was consistent to increased osteoclastic number and bone resorption parameters. After eldecalcitol administration, the number of osteoclasts was diminished, accompanied with elevated bone mineral density. Interestingly, eldecalcitol did promote a type of focal bone formation that is independent of bone resorption, a process known as bone mini-modeling. Taken together, our findings suggest that eldecalcitol mainly inhibits osteoclastic bone resorption, but, in part, stimulates focal bone formation in the OVX bone.
  • de Freitas Paulo, Henrique Luiz, Hasegawa Tomoka, Takeda Satoshi, Sasaki Muneteru, Tabata Chihiro, Oda Kimimitsu, Li Minqi, Saito Hitoshi, Amizuka Norio
    Bone 49 3 335 - 342 2011年09月 [査読無し][通常論文]
     
    To elucidate the histological events that follow administration of eldecalcitol, a second-generation of vitamin D analog currently awaiting approval as a drug for treatment of osteoporosis, we employed the ovariectomy (OVX) rat model. OVX rats received vehicle or 30ng/kg of eldecalcitol, and sham-operated animals received vehicle only. Rats were sacrificed after 12weeks and had their femora and tibiae removed and processed for histochemical and histomorphometrical analyses. When compared with OVX group, osteoclastic number and bone resorption parameters were significantly reduced in eldecalcitol-treated rats, accompanied by decreased bone formation parameters. The preosteoblastic layer, with which osteoclastic precursors interact for mutual differentiation, was poorly developed in the eldecalcitol group, indicating less cell-to-cell contact between preosteoblasts and osteoclast precursors. Interestingly, eldecalcitol did promote a type of focal bone formation that is independent of bone resorption, a process known as bone minimodeling. While the number of ED-1-positive macrophages was higher in the bone marrow of treated rats, though osteoclastic number was deceased. Taken toge
  • Paulo Henrique Luiz de Freitas, Tomoka Hasegawa, Satoshi Takeda, Muneteru Sasaki, Chihiro Tabata, Kimimitsu Oda, Minqi Li, Hitoshi Saito, Norio Amizuka
    BONE 49 3 335 - 342 2011年09月 [査読有り][通常論文]
     
    To elucidate the histological events that follow administration of eldecalcitol, a second-generation of vitamin D analog currently awaiting approval as a drug for treatment of osteoporosis, we employed the ovariectomy (OVX) rat model. OVX rats received vehicle or 30 ng/kg of eldecalcitol, and sham-operated animals received vehicle only. Rats were sacrificed after 12 weeks and had their femora and tibiae removed and processed for histochemical and histomorphometrical analyses. When compared with OVX group, osteoclastic number and bone resorption parameters were significantly reduced in eldecalcitol-treated rats, accompanied by decreased bone formation parameters. The preosteoblastic layer, with which osteoclastic precursors interact for mutual differentiation, was poorly developed in the eldecalcitol group, indicating less cell-to-cell contact between preosteoblasts and osteoclast precursors. Interestingly, eldecalcitol did promote a type of focal bone formation that is independent of bone resorption, a process known as bone minimodeling. While the number of ED-1-positive macrophages was higher in the bone marrow of treated rats, though osteoclastic number was deceased. Taken together, our findings suggest that eldecalcitol stimulates preosteoblastic differentiation rather than their proliferation, which in turn may prevent or diminish cell-to-cell contact between preosteoblasts and osteoclastic precursors, and therefore, lead to lower osteoclast numbers and decreased bone resorption. (C) 2011 Elsevier Inc. All rights reserved.
  • Hasegawa Tomoka, Li Minqi, Hara Kuniko, Sasaki Muneteru, Tabata Chihiro, de Freitas Paulo, Henrique Luiz, Hongo Hiromi, Suzuki Reiko, Kobayashi Masatoshi, Inoue Kiichiro, Yamamoto Tsuneyuki, Oohata Noboru, Oda Kimimitsu, Akiyama Yasuhiro, Amizuka Norio
    Biomed Res 32 4 259 - 269 2011年08月 [査読無し][通常論文]
     
    Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous alpha-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodul
  • Tomoka Hasegawa, Minqi Li, Kuniko Hara, Muneteru Sasaki, Chihiro Tabata, Paulo Henrique Luiz de Freitas, Hiromi Hongo, Reiko Suzuki, Masatoshi Kobayashi, Kiichiro Inoue, Tsuneyuki Yamamoto, Noboru Oohata, Kimimitsu Oda, Yasuhiro Akiyama, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 32 4 259 - 269 2011年08月 [査読有り][通常論文]
     
    Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous a-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model.
  • Maiko Haga, Kayoko Nozawa-Inoue, Minqi Li, Kimimitsu Oda, Sumio Yoshie, Norio Amizuka, Takeyasu Maeda
    Anatomical record (Hoboken, N.J. : 2007) 294 6 1074 - 82 2011年06月 [査読無し][通常論文]
     
    Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian's staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23--a regulator for the serum concentration of phosphorus--and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone.
  • Maiko Haga, Kayoko Nozawa-Inoue, Minqi Li, Kimimitsu Oda, Sumio Yoshie, Norio Amizuka, Takeyasu Maeda
    ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY 294 6 1074 - 1082 2011年06月 [査読有り][通常論文]
     
    Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian's staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23-a regulator for the serum concentration of phosphorus-and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone. Anat Rec, 294:1074-1082, 2011. (C) 2011 Wiley-Liss, Inc.
  • Hitoshi Saito, Satoshi Takeda, Fumiaki Takahashi, Paulo Luiz de Freitas, Norio Amizuka
    OSTEOPOROSIS INTERNATIONAL 22 62 - 63 2011年03月 [査読有り][通常論文]
  • Hitoshi Saito, Satoshi Takeda, Fumiaki Takahashi, Paulo Luiz de Freitas, Norio Amizuka
    OSTEOPOROSIS INTERNATIONAL 22 206 - 207 2011年03月 [査読有り][通常論文]
  • Tomoka Hasegawa, Muneteru Sasaki, Chihiro Tabata, Hideo Masuki, Minqi Li, Norio Amizuka
    Clinical calcium 21 2 190 - 6 2011年02月 [査読有り][通常論文]
     
    Osteoprotegerin (OPG) acts as a decoy receptor for the receptor activator of the nuclear factor κ B (RANK) ligand (RANKL) , preventing its association with RANK and inhibiting osteoclastogenesis. Therefore, mice homozygous for targeted disruption of the OPG gene reveal stimulated bone resorption and bone formation, resulting in enhanced bone remodeling. The OPG deficient (OPG( - / - )) mouse showed the diturbed distribution of collagen fibers and complex meshwork of cement lines, which implies weakened strength of OPG( - / - ) bone against mechanical stress. In addition, the abnormally promoted remodeling of the OPG( - / - ) bone caused the disorganized distribution of osteocyte lacunar canalicular system (OLCS) . Histochemical assessment revealed the markedly reduced synthesis of sclerostin in the OPG( - / - ) OLCS while the synthesis of dentin matrix protein-1 was not extremely affected by the OPG deficiency. Taken together, OPG deficient mouse appears to be a valid model for extremely-stimulated bone remodeling, and would provided important clues for better understanding for activities of bone cells in a pathological state in bone.
  • Yumi Watanabe, Kosei Takeuchi, Susumu Higa Onaga, Michiko Sato, Mika Tsujita, Manabu Abe, Rie Natsume, Minqi Li, Tatsuya Furuichi, Mika Saeki, Tomomi Izumikawa, Ayumi Hasegawa, Minesuke Yokoyama, Shiro Ikegawa, Kenji Sakimura, Norio Amizuka, Hiroshi Kitagawa, Michihiro Igarashi
    BIOCHEMICAL JOURNAL 432 1 47 - 55 2010年11月 [査読有り][通常論文]
     
    CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.
  • Masuki Hideo, Li Minqi, Hasegawa Tomoka, Suzuki Reiko, Ying Guo, Zhusheng Liu, Oda Kimimitsu, Yamamoto Tsuneyuki, Kawanami Masamitsu, Amizuka Norio
    Biomed Res 31 5 307 - 318 2010年10月 [査読無し][通常論文]
     
    In order to define the osteocytic function in accelerated bone remodeling, we examined the distribution of the osteocytic lacunar-canalicular system (OLCS) and osteocyte-secreting molecules--dentin matrix protein (DMP) 1 and sclerostin--in the epiphyses and cortical bones of osteoprotegerin deficient (OPG(-/-)) mice. Silver impregnation visualized a well-arranged OLCS in the wild-type epiphyses and cortical bone, whereas OPG(-/-) mice had an irregular OLCS in the epiphyses, but well-arranged canaliculi in the cortical bone. DMP1-positive osteocytes were evenly distributed throughout the wild-type epiphyses and cortical bone, as well as the OPG(-/-) cortical bone. However, OPG(-/-) epiphyses revealed weak DMP1-immunoreactivity. Thus, osteocytes appear to synthesize more DMP1 as the OLCS becomes regular. In contrast, sclerostin-immunoreactivity was significantly diminished in the OPG(-/-) epiphyses and cortical bone. In OPG(-/-) epiphyses and cortical bone, triple staining demonstrated few sclerostin-positive osteocytes in the periphery of a thick cell layer of alkaline phosphatase-positive osteoblasts and many tartrate resistant acid phosphatase-positive osteoclasts. Summarizin
  • Kaya Narimatsu, Minqi Li, Paulo Henrique Luiz de Freitas, Sara Sultana, Sobhan Ubaidus, Taku Kojima, Liu Zhucheng, Guo Ying, Reiko Suzuki, Tsuneyuki Yamamoto, Kimimitsu Oda, Norio Amizuka
    JOURNAL OF ELECTRON MICROSCOPY 59 5 427 - 436 2010年10月 [査読有り][通常論文]
     
    Preosteoblasts are currently defined as the precursors of mature osteoblasts. These cells are morphologically diverse and may represent a continuum during osteoblast differentiation. We have attempted to categorize the different preosteoblastic phenotypes in vivo by examining bone cells expressing the runt-related transcription factor 2, alkaline phosphatase and BrdU incorporation - histological traits of a preosteoblast - under transmission electron microscopy (TEM). TEM observations demonstrated, at least, in part two preosteoblastic subtypes: (i) a cell rich in cisterns of rough endoplasmic reticulum (rER) with vesicles and vacuoles and (ii) a subtype featuring extended cytoplasmic processes that connect with distant cells, with a small amount of scattered cisterns of rER and with many vesicles and vacuoles. ER-rich cells, whose cellular machinery is similar to that of an osteoblast, were often seen adjacent to mature osteoblasts, and therefore, may be ready for terminal differentiation. In contrast, ER-poor and vesicle-rich cells extended their cytoplasmic processes to mature osteoblasts and, frequently, to bone-resorbing osteoclasts. The abundant vesicles and vacuoles identified in this cell type indicate that this cell is involved in vesicular transport rather than matrix synthesis activity. In summary, our study verified the morphological diversity and the ultrastructural properties of osteoblastic cells in vivo.
  • Hideo Masuki, Minqi Li, Tomoka Hasegawa, Reiko Suzuki, Guo Ying, Liu Zhusheng, Kimimitsu Oda, Tsuneyuki Yamamoto, Masamitsu Kawanami, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 31 5 307 - 318 2010年10月 [査読有り][通常論文]
     
    In order to define the osteocytic function in accelerated bone remodeling, we examined the distribution of the osteocytic lacunar-canalicular system (OLCS) and osteocyte-secreting molecules dentin matrix protein (DMP) 1 and sclerostin-in the epiphyses and cortical bones of osteoprotegerin deficient (OPG(-/-)) mice. Silver impregnation visualized a well-arranged OLCS in the wild-type epiphyses and cortical bone, whereas OPG(-/-) mice had an irregular OLCS in the epiphyses, but well-arranged canaliculi in the cortical bone. DMP1-positive osteocytes were evenly distributed throughout the wild-type epiphyses and cortical bone, as well as the OPG(-/-) cortical bone. However, OPG(-/-) epiphyses revealed weak DMP1-immunoreactivity. Thus, osteocytes appear to synthesize more DMP1 as the OLCS becomes regular. In contrast, sclerostin-immunoreactivity was significantly diminished in the OPG(-/-) epiphyses and cortical bone. In OPG(-/-) epiphyses and cortical bone, triple staining demonstrated few sclerostin-positive osteocytes in the periphery of a thick cell layer of alkaline phosphatase-positive osteoblasts and many tartrate resistant acid phosphatase-positive osteoclasts. Summarizing, the regular distribution of OLCS may affect DMP1 synthesis, while the cellular activities of osteoclasts and osteoblasts rather than the regularity of OLCS may ultimately influence sclerostin synthesis.
  • Hideyuki Tanabe, Issei Takayama, Takashi Nishiyama, Masashi Shimazaki, Isao Kii, Minqi Li, Norio Amizuka, Ken-ichi Katsube, Akira Kudo
    PLOS ONE 5 8 e12234  2010年08月 [査読有り][通常論文]
     
    Background: Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells.Methods and Findings: In this study, however, we reported that periostin physically associates with the Notch1 precursor at its EGF repeats in the inside of cells. Moreover, by using the periodontal ligament of molar from periostin-deficient adult mice (Pn(-/-) molar PDL), which is a constitutively mechanically stressed tissue, we found that periostin maintained the site-1 cleaved 120-kDa transmembrane domain of Notch1 (N1 (TM)) level without regulating Notch1 mRNA expression. N1 (TM) maintenance in vitro was also observed under such a stress condition as heat and H(2)O(2) treatment in periostin overexpressed cells. Furthermore, we found that the expression of a downstream effector of Notch signaling, Bcl-xL was decreased in the Pn(-)(-/) molar PDL, and in the molar movement, cell death was enhanced in the pressure side of Pn(-/-) molar PDL.Conclusion: These results suggest the possibility that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition, which is caused by its physical association with the Notch1 precursor.
  • YAMAMOTO Tsuneyuki, LI Minqi, LIU Zhucheng, GUO Ying, HASEGAWA Tomoka, MASUKI Hideo, SUZUKI Reiko, AMIZUKA Norio
    Odontology : Official journal of the Society of the Nippon Dental University = 歯学 98 2 102 - 109 2010年07月 [査読有り][通常論文]
     
    Cementum is mineralized tissue with collagen fibrils as its major organic component, and it can be roughly classified into acellular and cellular cementum. The latter generally consists of a stack of cellular intrinsic fiber cementum layers, in which intensely and weakly stained lamellae (each about 2.5 mu m thick) alternate in light microscopic observations. It has been suggested that the alternate lamellar pattern results from periodic changes of the intrinsic fiber arrangement, but owing to the difficulty of observing the fibril arrangement three dimensionally, details were not understood until recently. The NaOH-maceration method has been developed to overcome this difficulty. For the past two decades, we have studied the structure and development of cementum by scanning electron microscopy using NaOH-maceration, as well as by light and transmission electron microscopy, and have accumulated a significant amount of data with regard to the structure and formation of cementum. In light of these data, we have arrived at the following conclusions: (1) The alternate lamellar pattern conforms to the twisted plywood model, in which collagen fibrils rotate regularly in the same direction to form two alternating types of lamellae; one type consists of transversely and almost transversely cut fibrils and the other consists of longitudinally and almost longitudinally cut fibrils. (2) The development of the intrinsic fiber arrangement may be controlled by cementoblasts; the cementoblasts move finger-like processes synchronously and periodically to create alternate changes in the intrinsic fiber arrangement, and this dynamic sequence results in the alternate lamellar pattern.
  • Li Minqi, Seki Yukie, Freitas Paulo H. L., Nagata Masaki, Kojima Taku, Sultana Sara, Ubaidus Sobhan, Maeda Takeyasu, Shimomura Junko, Henderson Janet E., Tamura Masato, Oda Kimimitsu, Liu Zhusheng, Guo Ying, Suzuki Reiko, Yamamoto Tsuneyuki, Takagi Ritsuo, Amizuka Norio
    Journal of Electron Microscopy 59 3 227 - 236 Oxford University Press 2010年06月 [査読有り][通常論文]
     
    The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.
  • Norio Amizuka, Minqi Li, Paulo H. L. D. Freitas, Zhucheng Liu, Ying Guo, Reiko Suzuki, Tsuneyuki Yamamoto
    ENDOCRINE JOURNAL 57 S244 - S245 2010年03月 [査読有り][通常論文]
  • Li Minqi, Seki Yukie, Freitas Paulo H L, Nagata Masaki, Kojima Taku, Sultana Sara, Ubaidus Sobhan, Maeda Takeyasu, Shimomura Junko, Henderson Janet E, Tamura Masato, Oda Kimimitsu, Liu Zhusheng, Guo Ying, Suzuki Reiko, Yamamoto Tsuneyuki, Takagi Ritsuo, Amizuka Norio
    J Electron Microsc (Tokyo) 59 3 227 - 236 2010年 [査読無し][通常論文]
     
    The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but
  • Isao Kii, Takashi Nishiyama, Minqi Li, Ken-ichi Matsumoto, Mitsuru Saito, Norio Amizuka, Akira Kudo
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 3 2028 - 2039 2010年01月 [査読有り][通常論文]
     
    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.
  • Ubaidus Sobhan, Li Minqi, Sultana Sara, de Freitas Paulo, Henrique Luiz, Oda Kimimitsu, Maeda Takeyasu, Takagi Ritsuo, Amizuka Norio
    J Electron Microsc (Tokyo) 58 6 381 - 392 2009年12月 [査読無し][通常論文]
     
    This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was
  • Sobhan Ubaidus, Minqi Li, Sara Sultana, Paulo Henrique Luiz de Freitas, Kimimitsu Oda, Takeyasu Maeda, Ritsuo Takagi, Norio Amizuka
    JOURNAL OF ELECTRON MICROSCOPY 58 6 381 - 392 2009年12月 [査読有り][通常論文]
     
    This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.
  • Norio Amizuka, Minqi Li, Ying Guo, Zhucheng Liu, Reiko Suzuki, Tsuneyuki Yamamoto
    Clinical calcium 19 12 1788 - 96 2009年12月 [査読有り][通常論文]
     
    Post-transcriptional maturation with the presence of vitamin K(2) promotes gamma-carboxylation of osteocalcin, enabling further binding to hydroxyapatite, from which one could infer that vitamin K(2) increased the quality of bone matrix. For instance, vitamin K(2) rescued the impaired collagen mineralization caused by Mg insufficiency, by promoting a re-association of the process of collagen mineralization with mineralized nodules. Sodium warfarin, which antagonizes the function of vitamin K(2), reduced the binding of osteocalcin to bone matrices, and consequently resulted in crystalline particles being dispersed throughout the osteoid without forming mineralized nodules. Therefore, gamma-carboxylated Gla proteins mediated by vitamin K(2) appear to play a pivotal role in normal mineralization in bone.
  • Hiroko Segawa, Akemi Onitsuka, Junya Furutani, Ichiro Kaneko, Fumito Aranami, Natsuki Matsumoto, Yuka Tomoe, Masashi Kuwahata, Mikiko Ito, Mitsuru Matsumoto, Minqi Li, Norio Amizuka, Ken-ichi Miyamoto
    American journal of physiology. Renal physiology 297 3 F671-8 - F678 2009年09月 [査読有り][通常論文]
     
    Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodium-dependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (P(i)) reabsorption in adult mice. In fact, mice that are null for Npt2c (Npt2c(-/-)) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insights and the relative importance of Npt2a and Npt2c, we now studied Npt2a(-/-)Npt2c(+/+), Npt2a(+/-)Npt2c(-/-), and Npt2a(-/-)Npt2c(-/-) double-knockout (DKO). DKO mice exhibited severe hypophosphatemia, hypercalciuria, and rickets. These findings are different from those in Npt2a KO mice that show only a mild phosphate and bone phenotype that improve over time and from the findings in Npt2c KO mice that show no apparent abnormality in the regulation of phosphate homeostasis. Because of the nonredundant roles of Npt2a and Npt2c, DKO animals showed a more pronounced reduction in P(i) transport activity in the brush-border membrane of renal tubular cells than that in the mice with the single-gene ablations. A high-P(i) diet after weaning rescued plasma phosphate levels and the bone phenotype in DKO mice. Our findings thus showed in mice that Npt2a and Npt2c have independent roles in the regulation of plasma P(i) and bone mineralization.
  • Paulo Henrique Luiz de Freitas, Minqi Li, Tadashi Ninomiya, Midori Nakamura, Sobhan Ubaidus, Kimimitsu Oda, Nobuyuki Udagawa, Takeyasu Maeda, Ritsuo Takagi, Norio Amizuka
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 24 9 1586 - 97 2009年09月 [査読有り][通常論文]
     
    This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. After administering PTH intermittently to wildtype and c-fos(-/-) mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH-treated wildtype mice, whereas in the osteoclast-deficient c-fos(-/-) mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double-positive for alkaline phosphatase and BrdU, suggesting active pre-osteoblastic proliferation. Ultrastructural examinations showed two major pre-osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c-fos(-/-) mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH-administered wildtype mice. We concluded that the absence of osteoclasts in c-fos(-/-) mice might hinder PTH-driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.
  • Hiroko Segawa, Akemi Onitsuka, Junya Furutani, Ichiro Kaneko, Fumito Aranami, Natsuki Matsumoto, Yuka Tomoe, Masashi Kuwahata, Mikiko Ito, Mitsuru Matsumoto, Minqi Li, Norio Amizuka, Ken-ichi Miyamoto
    AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 297 3 F671 - F678 2009年09月 [査読有り][通常論文]
     
    Segawa H, Onitsuka A, Furutani J, Kaneko I, Aranami F, Matsumoto N, Tomoe Y, Kuwahata M, Ito M, Matsumoto M, Li M, Amizuka N, Miyamoto K. Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development. Am J Physiol Renal Physiol 297: F671-F678,2009. First published July 1, 2009; doi:10.1152/ajprenal.00156.2009.-Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodium-dependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (P-i) reabsorption in adult mice. In fact, mice that are null for Npt2c (Npt2c(-/-)) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insights and the relative importance of Npt2a and Npt2c, we now studied Npt2a(+/+) Npt2c(+/+), Npt2a(+/-) Npt2c(-/-), and Npt2a(-/-) Npt2c(-/-) double-knockout (DKO). DKO mice exhibited severe hypophosphatemia, hypercalciuria, and rickets. These findings are different from those in Npt2a KO mice that show only a mild phosphate and bone phenotype that improve over time and from the findings in Npt2c KO mice that show no apparent abnormality in the regulation of phosphate homeostasis. Because of the nonreddundant roles of Npt2a and Npt2c, DKO animals showed a more pronounced reduction in P-i transport activity in the brush-border membrane of renal tubular cells than that in the mice with the single-gene ablations. A high-P-i diet after weaning rescued plasma phosphate levels and the bone phenotype in DKO mice. Our findings thus showed in mice that Npt2a and Npt2c have independent roles in the regulation of plasma P-i and bone mineralization.
  • Paulo Henrique Luiz de Freitas, Minqi Li, Tadashi Ninomiya, Midori Nakamura, Sobhan Ubaidus, Kimimitsu Oda, Nobuyuki Udagawa, Takeyasu Maeda, Ritsuo Takagi, Norio Amizuka
    JOURNAL OF BONE AND MINERAL RESEARCH 24 9 1586 - 1597 2009年09月 [査読有り][通常論文]
     
    This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. After administering PTH intermittently to wildtype and c-fos(-/-) mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH-treated wildtype mice, whereas in the osteoclast-deficient c-fos(-/-) mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double-positive for alkaline phosphatase and BrdU, suggesting active pre-osteoblastic proliferation. Ultrastructural examinations showed two major pre-osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c-fos(-/-) mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH-administered wildtype mice. We concluded that the absence of osteoclasts in c-fos(-/-) mice might hinder PTH-driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration. J Bone Miner Res 2009;24:1586-1597. Published online on April 27, 2009; doi: 10.1359/JBMR.090413
  • Norio Amizuka, Minqi Li, Masato Tamura, Kimimitsu Oda
    Clinical calcium 19 7 935 - 43 2009年07月 [査読有り][通常論文]
     
    Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) , which were shown to bind the common receptor, PTH/PTHrP receptor, affect fetal chondrogenesis and adult bone turnover, respectively. PTH appears to predominantly stimulate proliferation of osteoblastic precursors, and consequently affect osteoblastic differentiation mediating coupling with osteoclasts. A recent study has reported the biological function of C-terminal region of PTHrP including nucleolar targeting signal. We will review recent reports for the action of PTH and PTHrP on bone and cartilage.
  • 網塚憲生, UBAIDUS Sobhan, FREITAS Paulo, SULTANA Sara, LI Minqi
    日本臨床 67 5 868 - 71 2009年05月 [査読有り][通常論文]
  • Norio Amizuka, Minqi Li, Kuniko Hara, Masatoshi Kobayashi, Paulo H. L. de Freitas, Sobhan Ubaidus, Kimimitsu Oda, Yasuhiro Akiyama
    JOURNAL OF ELECTRON MICROSCOPY 58 2 55 - 65 2009年04月 [査読有り][通常論文]
     
    This study aimed to elucidate the ultrastructural role of Gla proteins in bone mineralization by means of a warfarin-administration model. Thirty-six 4-week-old male F344 rats received warfarin (warfarin group) or distilled water (control group), and were fixed after 4, 8 and 12 weeks with an aldehyde solution. Tibiae and femora were employed for histochemical analyses of alkaline phosphatase, osteocalcin and tartrate-resistant acid phosphatase, and for bone histomorphometry and electron microscopy. After 4, 8 and 12 weeks, there were no marked histochemical and histomorphometrical differences between control and warfarin groups. However, osteocalcin immunoreactivity was markedly reduced in the warfarin-administered bone. Mineralized nodules and globular assembly of crystalline particles were seen in the control osteoid. Alternatively, warfarin administration resulted in crystalline particles being dispersed throughout the osteoid without forming mineralized nodules. Immunoelectron microscopy unveiled lower osteocalcin content in the warfarin-administered osteoid, which featured scattered crystalline particles, whereas osteocalcin was abundant on the normally mineralized nodules in the control osteoid. In summary, Gla proteins appear to play a pivotal role in the assembly of mineralized nodules.
  • Hiroko Segawa, Akemi Onitsuka, Masashi Kuwahata, Etsuyo Hanabusa, Junya Furutani, Ichiro Kaneko, Yuka Tomoe, Fumito Aranami, Natsuki Matsumoto, Mikiko Ito, Mitsuru Matsumoto, Minqi Li, Norio Amizuka, Ken-ichi Miyamoto
    JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 20 1 104 - 113 2009年01月 [査読有り][通常論文]
     
    Primary renal inorganic phosphate (Pi) wasting leads to hypophosphatemia, which is associated with skeletal mineralization defects. In humans, mutations in the gene encoding the type IIc sodium-dependent phosphate transporter lead to hereditary hypophophatemic rickets with hypercalciuria, but whether Pi wasting directly causes the bone disorder is unknown. Here, we generated Npt2c-null mice to define the contribution of Npt2c to Pi homeostasis and to bone abnormalities. Homozygous mutants (Npt2c(-/-)) exhibited hypercalcemia, hypercalciuria, and elevated plasma 1,25-dihydroxyvitamin D-3 levels, but they did not develop hypophosphatemia, hyperphosphaturia, renal calcification, rickets, or osteomalacia. The increased levels of 1,25-dihydroxyvitamin D, in Npt2c(-/-) mice compared with age-matched Npt2c(+/+) mice may be the result of reduced catabolism, because we observed significantly reduced expression of renal 25-hydroxyvitamin D-24-hydroxylase mRNA but no change in 1 alpha-hydroxylase mRNA levels. Enhanced intestinal absorption of calcium (Ca) contributed to the hypercalcemia and increased urinary Ca excretion. Furthermore, plasma levels of the phosphaturic protein fibroblast growth factor 23 were significantly decreased in Npt2c(-/-) mice. Sodium-dependent Pi co-transport at the renal brush border membrane, however, was not different among Npt2c(+/+), Npt2c(+/-) and Npt2c/ mice. In summary, these data suggest that Npt2c maintains normal Ca metabolism, in part by modulating the vitamin D/fibroblast growth factor 23 axis.
  • Norio Amizuka, Minqi Li, Masatoshi Kobayashi, Kuniko Hara, Shoji Akahane, Kiichi Takeuchi, Paulo H. L. Freitas, Hidehiro Ozawa, Takeyasu Maeda, Yasuhiro Akiyama
    HISTOLOGY AND HISTOPATHOLOGY 23 11 1353 - 1366 2008年11月 [査読有り][通常論文]
     
    It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.
  • Junko Shimomura-Kuroki, Sobhan Ubaidus, Paulo H. L. Freitas, Minqi Li, Yoko Ishida, Naoaki Saito, Kimimitsu Oda, Shohachi Shimooka, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 29 2 61 - 69 2008年04月 [査読有り][通常論文]
     
    A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PTH)/parathyrold hormone-related peptide (PTHrP), i.e., PTH-R, has been discovered in human Blomstrand's lethal chondrodysplasia. As skeletal deformities in this type of chondrodysplasia appear to compromise the receptor binding to its ligands, we examined the possibility that rat PTH-R carrying P132L mutation (PTH-R-P132L) would result in abnormal intracellullar localization. Osteoblastic MC3T3-E1 cells were transfected with expression vectors containing cDNAs encoding either wild-type PTH-R or mutant PTH-R-P132L. The cells expressing the wild-type PTH-R produced a receptor protein with a molecular mass of 66.3 kDa, which localized its immunoreactivity mainly on the cell surfaces. In contrast, the PTH-R-P132L was hardly detected on the cell surfaces, but accumulated within the rough-surfaced endoplasmic reticulum.. Consistent with this localization, the cells expressing the mutant receptor failed to generate cyclic AMP in response to PTH. Furthermore, a remarkably weaker intensity of the 66.3 KDa band compared with the wild-type counterpart suggests that PTH-R-P132L is prone to degradation in the transfected cells. In summary, these findings indicate that defective transport of PTH-R-P132L to the cell surface Would be a molecular basis for Blomstrand's chondrodysplasia.
  • Hironobu Suzuki, Norio Amizuka, Kimimitsu Oda, Masaki Noda, Hayato Ohshima, Takeyasu Maeda
    JOURNAL OF ANATOMY 212 3 275 - 285 2008年03月 [査読有り][通常論文]
     
    The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho(-/-) mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization - according to von Kossa's staining method - and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho(-/-) metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho(-/-) mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho(-/-) mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho(-/-) mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.
  • Minqi Li, Tomonao Saito, Norio Amizuka
    Clinical calcium 18 3 328 - 35 2008年03月 [査読有り][通常論文]
     
    Steroid-induced osteoporosis has been reported to show reduced osteoblastic bone formation and accelerated osteoclastic bone resorption. However, a fracture risk of patients with steroid-induced osteoporosis is higher than an assumed risk merely estimated by the bone mineral density, and therefore, bone quality appears to be reduced in the steroid-induced osteoporosis. Precious mechanism how steroid can reduce the quality of bone is still veiled, but one possible explanation may be osteocytes' apoptosis by steroid, probably resulting in the failure of their maintaining of bone minerals and mechanosensing in bone.
  • Masayoshi Nakadate, Norio Amizuka, Minqi Li, Paulo H. L. Freitas, Kimimitsu Oda, Shuichi Nomura, Katsumi Uoshima, Takeyasu Maeda
    MICROSCOPY RESEARCH AND TECHNIQUE 71 2 93 - 104 2008年02月 [査読有り][通常論文]
     
    This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R (R)). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R (R). Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R (R)-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R (R) surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R (R) are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology.
  • Hironobu Suzuki, Norio Amizuka, Kimimitsu Oda, Masaki Noda, Hayato Ohshima, Takeyasu Maeda
    ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY 291 2 183 - 190 2008年02月 [査読有り][通常論文]
     
    Klotho-deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho-deficient mice-comparing the findings with those from their wild-type litter-mates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization.
  • Masashi Shimazaki, Kazuto Nakamura, Isao Kii, Takeshi Kashima, Norio Amizuka, Minqi Li, Mitsuru Saito, Keiichi Fukuda, Takashi Nishiyama, Satoshi Kitajima, Yumiko Saga, Masashi Fukayama, Masataka Sata, Akira Kudo
    JOURNAL OF EXPERIMENTAL MEDICINE 205 2 295 - 303 2008年02月 [査読有り][通常論文]
     
    Acute myocardial infarction (AMI) is a common and lethal heart disease, and the recruitment of fibroblastic cells to the infarct region is essential for the cardiac healing process. Although stiffness of the extracellular matrix in the infarct myocardium is associated with cardiac healing, the molecular mechanism of cardiac healing is not fully understood. We show that periostin, which is a matricellular protein, is important for the cardiac healing process after AMI. The expression of periostin protein was abundant in the infarct border of human and mouse hearts with AMI. We generated periostin(-/-) mice and found no morphologically abnormal cardiomyocyte phenotypes; however, after AMI, cardiac healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness caused by a reduced number of alpha smooth muscle actin-positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes were rescued by gene transfer of a spliced form of periostin. Moreover, the inhibition of FAK or alpha v-integrin, which blocked the periostin-promoted cell migration, revealed that alpha v-integrin, FAK, and Akt are involved in periostin signaling. Our novel findings show the effects of periostin on recruitment of activated fibroblasts through FAK-integrin signaling and on their collagen fibril formation specific to healing after AMI.
  • Hidehiro Ozawa, Kazuto Hoshi, Norio Amizuka
    JOURNAL OF ORAL BIOSCIENCES 50 1 1 - 14 2008年 
    This review aims to propose a more consistent interpretation of the concept of matrix vesicle mineralization, and includes current views on biomineralization in general. We first focused on the ultrastructural, cytochemical and biophysical characteristics of matrix vesicles which confirm the assertion that matrix vesicles are the initial sites of bone biomineralization. We offer a scheme describing how cells regulate biomineralization by means of matrix vesicles during bone formation and remodeling and then discuss the subsequential biomineralization, the growth of hydroxyapatite crystals by direct extension, as well as secondary nucleation from previously formed seams of bone and collagen mineralization.
  • Morphological Aspects on Osteocytic Function on Bone Mineralization.
    Sasaki M, Hongo H, Hasegawa T, Suzuki R, Liu Z, Freitas PHL, Yamada T, Oda K, Yamamoto T, Li M, Totsuka Y, Amizuka N
    Oral Science International. 9 1 1 - 8 2008年 [査読有り][通常論文]
  • Tatsuya Ohashi, Masahiko Sunaga, Toshihide Miura, Kumiko Sasagawa, Yasuhito Sato, Wataru Ohashi, Ken Katagiri, Yoshinori Katano, Iwao Kiyokawa, Ryo Kojima, Takeshi Tomonaga, Fumio Nomura, Norio Amizuka, Kimimitsu Oda, Toyoji Sato, Katsuhiro Katayama
    HYBRIDOMA 26 6 401 - 406 2007年12月 [査読有り][通常論文]
     
    Tissue-nonspecific alkaline phosphatase (TNSALP) in serum comprises liver alkaline phosphatase (liver-ALP) and bone alkaline phosphatase (bone-ALP). Liver-ALP is a marker of liver disease; thus a specific method for its measurement would be useful. Measurement of ALP by electrophoresis is difficult, although all of the isozymes can be assessed simultaneously. Total ALP can also be measured by automated analyzer, but it is difficult to determine the cause of a high ALP value because bone-, intestine-, placenta-, and tumor-ALP are measured together. Thus, anti-TNSALP monoclonal antibodies that can resolve these problems are needed. Here we have generated an anti-TNSALP monoclonal antibody, 3-29-3R. This clone has specificity to liver-ALP rather than to bone-ALP. In electrophoresis, 3-29-3R reacted with TNSALP and shifted the bands. The use of 3-29-3R enabled easy interpretation of the results. Furthermore, we tested 3-29-3R by developing an immunocapture enzymatic assay (IEA). Preliminary results of the IEA show that this method is effective for measurement of liver-ALP. Thus, the monoclonal antibody that we have established may be a useful tool for clinical diagnosis.
  • N. Yoshiba, K. Yoshiba, A. Hosoya, M. Saito, T. Yokoi, T. Okiji, N. Amizuka, H. Ozawa
    European Cells and Materials 14 SUPPL.2 142  2007年11月
  • KOJIMA Taku, AMIZUKA Norio, SUZUKI Akiko, DE FREITAS Paulo Henrique Luiz, YOSHIZAWA Michiko, KUDO Akira, SAITO Chikara, MAEDA Takeyasu
    Journal of bone and mineral metabolism 25 6 361 - 373 2007年11月 [査読有り][通常論文]
     
    In this study, we present a novel guided bone regeneration (GBR) concept that consists of combining Boneject, a bone substitute containing atelocollagen and bovine hydroxyapatite particles, with thermoplastic, bioresorbable plates (DeltaSystem) known to resist mechanical loading. In rat calvariae, standardized bone defects were filled with Boneject and covered with a convex DeltaSystem plate. Tissue from rats at 1, 2, 4, 8, and 12 weeks postoperation were fixed with an aldehyde solution, and the new bone formed at the defects was histologically assessed. At 1 week, alkaline phosphatase (ALP)-negative cells deriving from the bottom region of the defect could be found up to half the height of the cavity. Boneject particle surfaces in the bottom region revealed an intense osteopontin immunopositivity whereas those in the upper region did not. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts accumulated on the surfaces of osteopontin-coated particles. A newly formed, woven-like bone featuring ALP-positive osteoblasts extended from the native bone. At the second week, the newly formed woven bone had surrounded the Boneject particles. Cement lines, which indicate active bone remodeling, could be observed in the new bone despite its immaturity. Four weeks after surgery, the new bone had reached the height of the DeltaSystem plate, and just beneath it a periostin-positive fibrous layer covered the mix of new bone and Boneject particles. By then, despite having acceptable histological features, electron probe microanalyzer (EPMA) and transmission electron microscope (TEM) analyses revealed that the new bone could not be regarded as compact bone. At 8 and 12 weeks, the new bone showed compact bone-like features according to TEM and EPMA assessments. Summarizing, the combination of a bone substitute such as Boneject and a rigid, bioresorbable plate appears to be osteoconductive and to promote bone remodeling, leading to the genesis of a tissue similar to the one that is regarded as the "gold standard" for bone regeneration: the compact bone.
  • HIROSE Satoshi, LI Minqi, KOJIMA Taku, DE FREITAS Paulo Henrique Luiz, UBAIDUS Sobhan, ODA Kimimitsu, SAITO Chikara, AMIZUKA Norio
    Journal of bone and mineral metabolism 25 6 374 - 382 2007年11月 [査読有り][通常論文]
     
    Giving the complexity that characterizes the mechanisms of bone remodeling and the number of events that have to be in absolute harmony for it to occur flawlessly, the postulation that temporospatial distribution of osteocytes and their lacunar canalicular system might influence and be influenced by bone remodeling can be regarded, at least, as feasible. In this study, using Schoen's silver staining, we have examined the distribution of the osteocytic lacunar canalicular system (OLCS) in bones of developing mice. Trabecular bones of 3-day-old, 2-week-old, and 3-week-old mice displayed osteocytic cytoplasmic processes without any perceptible alignment. Also, many plump osteocytes were embedded in the mineralized bone matrix in a disorderly manner. At 4 weeks of age, however, mice bones showed some osteocytic processes that reached the bone surface on a right angle, while other osteocytes displayed the same features seen on 3-week specimens. Samples at 8 weeks of age featured osteocytes with their usual spindle shape, organized so as to parallel the longitudinal axis of trabecular bone. They also extended their cytoplasmic processes perpendicularly to the bone surface. However, several osteocytes immersed in older bone, i.e., a residual mix of cartilage and bone matrices, still showed a random pattern of distribution of their cytoplasmic processes. Up to 12 weeks of age, the majority of the osteocytes became flattened and were shown to be aligned with their long axis paralleling the bone surface. This tendency for such a gradual arrangement was also observed in cortical bones. We have further demonstrated that 8-week-old osteoprotegerin-deficient mice, which demonstrated histological evidence of higher than average bone turnover, revealed a disorganized OLCS. Given the data gathered in this work, the OLCS appears to assume an organized, probably function-related spatial distribution as normal bone remodeling goes on.
  • Nagako Yoshiba, Kunihiko Yoshiba, Akihiro Hosoya, Masahiro Saito, Takamasa Yokoi, Takashi Okiji, Norio Amizuka, Hidehiro Ozawa
    CELL AND TISSUE RESEARCH 330 1 133 - 145 2007年10月 [査読有り][通常論文]
     
    Matrix remodeling is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Periostin, originally identified in a mouse osteoblastic library, plays a role in cell adhesion and migration and in mechanical stress-induced matrix remodeling. In this study, we analyzed and compared the distribution patterns of TIMP-2 and periostin during mouse mandible development. Immunohistochemical staining for TIMP-2 and periostin was carried out on serial cryosections obtained from mice at embryonic days 13-16, postnatal day 2 (P2), P35, and 12 weeks of age. TIMP-2 and periostin exhibited a strikingly similar protein distribution during mandible development. From bud to early bell stages of molars, TIMP-2 and periostin were highly expressed on the lingual and anterior sides of the basement membrane and on the adjacent jaw mesenchyme. In pre- and postnatal incisors, the basement membrane of the apical loop and dental follicle was immunostained for TIMP-2 and periostin. At postnatal stages, TIMP-2 and periostin were prominently confined to the extracellular matrix (ECM) of gingival tissues, periodontal ligaments, and tendons (all recipients of mechanical strain). However, periostin was solely detected in the lower portion of the inner root sheath of hair follicles. Gingiva of P2 cultured in anti-TIMP-2 antibody-conditioned medium showed markedly reduced staining of periostin. We suggest that TIMP-2 and periostin are co-distributed on ECM exposed to mechanical forces and coordinately function as ECM modulators.
  • Paulo Henrique Luiz de Freitas, Taku Kojima, Soblian Ubaidus, Minqi Li, Guangwei Shang, Ritsuo Takagi, Takeyasu Maeda, Kimimitsu Oda, Hidehiro Ozawa, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 28 4 191 - 203 2007年08月 [査読有り][通常論文]
     
    We have examined the morphological changes in chondrocytes after exposure to experimental hypergravity. Tibial epiphyseal cartilages of 17-days-old mouse fetuses were exposed to centrifugation at 3G for 16 It mimicking hypergravitational environment (experimental group), or subjected to stationary cultures (control group). Centrifugation did not affect the sizes of epiphyseal cartilage, chondrocyte proliferation, type X collagen-positive hypertrophic zone, and the mRNA expressions of parathyroid hormone-related peptide and fibroblast growth factor receptor Ill. However, centrifuged chondrocytes showed abnormal morphology and aberrant spatial arrangements, resulting in disrupted chondrocytic columns. Through histochemical assessments, actin filaments were shown to distribute evenly along cell membranes of control proliferative chondrocytes, while chondrocytes subjected to centrifugal force developed a thicker layer of actin filaments. Transmission electron microscopic observations revealed spotty electron-dense materials underlying control chondrocytes' cell membranes, while experimental chondrocytes showed their thick layer. In the intracolumnar regions of the control cartilage, longitudinal electron-dense fibrils were associated with short cytoplasmic processes of normal chondrocytes, indicating assumed cell-to-matrix interactions. These extracellular fibrils were disrupted in the centrifuged samples. Summarizing, altered actin filaments associated with cell membranes, irregular cell shape and disappearance of intracolumnar extracellular fibrils suggest that hypergravity disturbs cell-to-matrix interactions in our cartilage model.
  • Taku Kojima, Paulo H. L. Freitas, Sobhan Ubaidus, Akiko Suzuki, Minqi Li, Michiko Yoshizawai, Kimimitsu Oda, Takeyasu Maeda, Akira Kudo, Chikara Saito, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 28 4 219 - 229 2007年08月 [査読有り][通常論文]
     
    We aimed to histologically elucidate whether bioresorbable plates (DeltaSystem (R)) can induce cortical bone formation, which is essential for long-lasting bone augmentation. Standardized bone defects in rat calvariae were covered with a convexly-shaped DeltaSystem (R) plate, and then processed for histological observations. At 1 week, alkaline phosphatase-positive osteoblasts were seen in the newly-formed bone extending from the cavity's bottom, indicating accelerated osteogenesis. A thick layer of soft connective tissue positive for periostin, a hallmark of periosteum, covered this new bone. At 2 weeks, a spongy bone had filled the cavity up to half its height. The inner layer of the soft tissue facing the spongy bone revealed abundant periostin and osteopontin, and had many tartrate-resistant acid phosphatase-positive osteoclasts. At 4 weeks, this layer had given rise to thin new bony matrices without relation to the spongy bone arising from the cavity. These bone matrices had been thickened by 8 weeks, and turned into a thick cortical bone outlining the regenerated bone at 12 weeks. Thus, our study has provided histological evidences of cortical osteogenesis when DeltaSystem (R) plates are used for bone augmentation procedures.
  • Naoki Sawada, Yutaka Taketani, Norio Amizuka, Masako Ichikawa, Chiharu Ogawa, Kaori Nomoto, Kunitaka Nashiki, Tadatoshi Sato, Hidekazu Arai, Masashi Isshiki, Hiroko Segawa, Hironori Yamamoto, Ken-ichi Miyamoto, Eiji Takeda
    BONE 41 1 52 - 58 2007年07月 [査読有り][通常論文]
     
    Caveolin-1 is an essential and signature protein of caveolae, which are small invaginations of the plasma membrane enriched in cholesterol and sphingolipids. Although high levels of expression of caveolin-1 have been demonstrated in osteoblasts as welt as endothelial cells, fibroblasts, and muscular cells, the role of caveolin-1 in osteoblasts has not been clarified. Here, we show that caveolin-1 is secreted from osteoblasts in the form of matrix vesicles; extracellular vesicles released from the plasma membrane of osteoblasts. In this study, caveolae and matrix vesicles were similarly enriched in cholesterol and sphingomyelin in fractions isolated from mineralizing MC3T3-E1 cells. Interestingly, in the MC3T3-E1 cells caveolin-1 was enriched in the matrix vesicle fraction as well as the caveolar membrane fraction, and the amount of caveolin-1 in the matrix vesicle fraction increased as differentiation progressed. Localization of caveolin-1 in matrix vesicles was also confirmed in murine tibia. Furthermore, overexpression of caveolin-1 enhanced matrix calcification in MC3T3-E1 cells, whereas knockdown of caveolin-1 diminished it. These results suggest that secreted caveolin-1 as a component of matrix vesicles may play an important role in osteoblast calcification. (c) 2007 Elsevier Inc. All rights reserved.
  • Zenjiro Maruyama, Carolina A. Yoshida, Tatsuya Furuichi, Norio Amizuka, Masako Ito, Ryo Fukuyama, Toshihiro Miyazaki, Hideki Kitaura, Kouhei Nakamura, Takashi Fujita, Naoko Kanatani, Takeshi Moriishi, Kei Yamana, Wenguang Liu, Hiroshi Kawaguchi, Kozo Nakamura, Toshihisa Komori
    DEVELOPMENTAL DYNAMICS 236 7 1876 - 1890 2007年07月 [査読有り][通常論文]
     
    Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenie mice that expressed dn-Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild-type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that Runx2 is not essential for maintenance of the expression of major bone matrix protein genes in postnatal bone development and maintenance. Developmental Dynamics 236:1876-1890, 2007. (c) 2007 Wiley-Liss, Inc.
  • Sawako Tatsumi, Kiyoaki Ishii, Norio Amizuka, Minqi Li, Toshihiro Kobayashi, Kenji Kohno, Masako Ito, Sunao Takeshita, Kyoji Ikeda
    CELL METABOLISM 5 6 464 - 475 2007年06月 [査読有り][通常論文]
     
    Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function hasremained anenigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%-80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteolblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these "osteocyte-less" mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechano-transduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.
  • Minqi Li, Tomoyo Sasaki, Katsuhiro Ono, Paulo Henrique Luiz de Freitas, Ubaidus Sobhan, Taku Kojima, Junko Shimomura, Kimimitsu Oda, Norio Amizuka
    BIOMEDICAL RESEARCH-TOKYO 28 3 127 - 137 2007年06月 [査読有り][通常論文]
     
    The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c truce. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive rnacrophages/rnonocytes were localized adjacent to, or invading the metastatic tissue. In addition, son-ie F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone mat-row. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.
  • Naoto Kosaki, Hironari Takaishi, Satoru Kamekura, Tokuhiro Kimura, Yasunori Okada, Li Minqi, Norio Amizuka, Ung-il Chung, Kozo Nakamura, Hiroshi Kawaguchi, Yoshiaki Toyama, Jeanine D'Armiento
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 354 4 846 - 851 2007年03月 [査読有り][通常論文]
     
    Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloprotemase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process. (c) 2007 Elsevier Inc. All rights reserved.
  • Junichiro James Kazama, Norio Amizuka, Masafumi Fukagawa
    THERAPEUTIC APHERESIS AND DIALYSIS 10 SUPPL. 1 S34 - S38 2006年12月 [査読有り][通常論文]
     
    Vascular calcification is observed frequently in hemodialysis patients. The guidelines for kidney disease outcomes quality initiative recommend a strict control of serum calcium and phosphorus concentrations. Calcium-phosphorus product in extracellular fluids is almost at an oversaturation level in dialysis patients and in healthy individuals as well, but crystallization of hydroxyapatite does not occur in healthy individuals. Presumably, some systemic mechanism that has yet to be defined works to block the formation of hydroxyapatite crystals in healthy individuals, whereas in dialysis patients this defense mechanism is disrupted in hard tissues, leading to progressive biomineralization. Matrix vesicles released from specialized mesenchymal cells such as osteoblasts, play a central role in disrupting the putative defense mechanism against calcification. Matrix vesicles are internally in a highly favorable environment for progressive calcification, and essentially a nidus for hydroxyapatite crystal nucleation and its external growth through disruption of the defense mechanism. Osteoblastic cells release matrix vesicles that form initial crystal hydroxyapatite by condensation of phosphate and calcium, referred to as matrix vesicle calcification. Hydroxyapatite crystals break through vesicular membranes to reach a nearby collagen fiber network and form a continuous layer of calcified bone matrix. Thereafter follows the formation of additive bone matrix by osteoblasts and the advance of the calcification front. Therefore, ectopic calcification arises mechanistically from: (i) disruption of a systemic defense mechanism against calcification; and (ii) appearance of osteoblast-like cells in hard tissues that normally are localized in soft tissues. Abnormal accumulation of calcium/inorganic phosphate in dialysis patients is accounted for by the former disruption of systemic defense mechanism against calcification, and arterial calcification in dialysis patients by the latter osteoblast-like cells transformed from tunica media or vascular smooth muscle cells.
  • Oshima Shigeki, Onodera Shin, Amizuka Norio, Li Minqi, Irie Kazuharu, Watanabe Satoshi, Koyama Yoshikazu, Nishihira Jun, Yasuda Kazunori, Minami Akio
    FEBS LETTERS 580 27 6519  2006年11月27日 [査読有り][通常論文]
  • Naoki Kondo, Kunihiko Tokunaga, Tomoyuki Ito, Katsumitsu Arai, Norio Amizuka, Li Minqi, Hiroshi Kitahara, Masayuki Ito, Makoto Naito, Jiang Shu-Ying, Kimimitsu Oda, Takehiro Murai, Reiko Takano, Akira Ogose, Naoto Endo
    MICROSCOPY RESEARCH AND TECHNIQUE 69 10 839 - 846 2006年10月 [査読有り][通常論文]
     
    We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.
  • Katsuhiro Ono, Sadahiro Kamiya, Takuhiko Akatsu, Chika Nakamura, Minqi Li, Norio Amizuka, Kunio Matsumoto, Toshikazu Nakamura, Nobuo Kugai, Seiki Wada
    BONE 39 1 27 - 34 2006年07月 [査読有り][通常論文]
     
    Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13 +/- 0.25 ng/ml), whereas HGF was undetectable (< 0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALE/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling. (c) 2005 Elsevier Inc. All rights reserved.
  • Katsushi Tsukiyama, Yuichiro Yamada, Chizumi Yamada, Norio Harada, Yukiko Kawasaki, Masahito Ogura, Kazuhisa Bessho, Minqi Li, Norio Amizuka, Masahiro Sato, Nobuyuki Udagawa, Naoyuki Takahashi, Kiyoshi Tanaka, Yutaka Oiso, Yutaka Seino
    MOLECULAR ENDOCRINOLOGY 20 7 1644 - 1651 2006年07月 [査読有り][通常論文]
     
    Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone.
  • Minqi Li, Norio Amizuka
    Clinical calcium 16 7 1221 - 27 2006年07月 [査読有り][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) is known as an important local factor for chondrogenesis, promoting chondrocyte proliferation and inhibiting their differentiation into the hypertrophic phenotype. Signaling transduction through the PTH/PTHrP receptor has two possible pathways: the activation of adenylate cyclase and subsequent protein kinase A (PKA), and the activation of phospholipase C (PLC). Recent studies with mice carrying PTH/PTHrP receptor inactivated for PLC and chondrocyte-specific deletion of the G (s) gene have shown that cAMP/PKA signaling appears to stimulate chondrocyte proliferation and inhibit their differentiation, whereas PLC signaling enhanced chondrocyte differentiation and inhibited their proliferation. In a physiological state, cAMP/PKA signaling may predominate over PLC pathway. Also, Na(+)/H(+)exchanger regulatory factor 2 (NHERF2) has been reported to down-regulate adenylate cyclase activity, in a switch mechanism that results in signal transduction through the PLC pathway.
  • Akiko Suzuki, Kayoko Nozawa-Inoue, Norio Amizuka, Kazuhiro Ono, Takeyasu Maeba
    Anatomical Record - Part A Discoveries in Molecular, Cellular, and Evolutionary Biology 288 6 646 - 652 2006年06月 [査読有り][通常論文]
     
    Previous studies have pointed out a lack of adhesion structures in the synovial lining layer of the rat temporomandibular joint (TMJ) despite showing an epithelial arrangement. CD44, a major cell adhesion molecule, plays crucial roles as an anchor between cells and extracellular matrices by binding hyaluronan (HA) for the development of organs or the metastasis of tumors. The present study examined the localization of CD44 in the synovial membrane of the rat TMJ by immunocytochemistry for OX50, ED1, and Hsp25, which are markers for the rat CD44, macrophage-like type A, and fibroblast-like type B synoviocytes, respectively. Histochemistry for HA-binding protein (HABP) was also employed for the detection of HA. OX50 immunoreactions were found along the cell surface and, in particular, accumulated along the surface of the articular cavity. Observations by a double immunostaining and immunoelectron microscopy revealed that all the OX50-immunopositive cells were categorized as fibroblastic type B cells, which had many caveolae and a few vesicles reactive to intense OX50. However, the macrophage-like type A cells did not have any OX50 immunoreaction in the synovial lining layer. A strong HABP reaction was discernable in the extracellular matrix surrounding both OX50-positive and -negative cells in the synovial lining layers, exhibiting a meshwork distribution, but weak in its sublining layer. This localization pattern of CD44 and HABP might be involved in the formation of the epithelial arrangement of the synovial lining layer. Furthermore, OX50 immunonegativity in the type A cells suggests their low phagocytotic activity in the rat TMJ under normal conditions. © 2006 Wiley-Liss, Inc.
  • Akiko Suzuki, Kayoko Nozawa-Inoue, Norio Amizuka, Kazuhiro Ono, Takeyasu Maeda
    ANATOMICAL RECORD PART A-DISCOVERIES IN MOLECULAR CELLULAR AND EVOLUTIONARY BIOLOGY 288A 6 646 - 652 2006年06月 [査読有り][通常論文]
     
    Previous studies have pointed out a lack of adhesion structures in the synovial lining layer of the rat temporomandibular joint (TMJ) despite showing an epithelial arrangement. CD44, a major cell adhesion molecule, plays crucial roles as an anchor between cells and extracellular matrices by binding hyaluronan (HA) for the development of organs or the metastasis of tumors. The present study examined the localization of CD44 in the synovial membrane of the rat TMJ by immunocytochemistry for OX50, ED1, and Hsp25, which are markers for the rat CD44, macrophage-like type A, and fibroblast-like type B synoviocytes, respectively. Histochemistry for HA-binding protein (HABP) was also employed for the detection of HA. OX50 immunoreactions were found along the cell surface and, in particular, accumulated along the surface of the articular cavity. Observations by a double immunostaining and immunoelectron microscopy revealed that all the OX50-immunopositive cells were categorized as fibroblastic type B cells, which had many caveolae and a few vesicles reactive to intense OX50. However, the macrophage-like type A cells did not have any OX50 immunoreaction in the synovial lining layer. A strong HABP reaction was discernable in the extracellular matrix surrounding both OX50-positive and -negative cells in the synovial lining layers, exhibiting a meshwork distribution, but weak in its sublining layer. This localization pattern of CD44 and HABP might be involved in the formation of the epithelial arrangement of the synovial lining layer. Furthermore, OX50 immunonegativity in the type A cells suggests their low phagocytotic activity in the rat TMJ under normal conditions.
  • Shin Onodera, Satoshi Sasaki, Shigeki Ohshima, Norio Amizuka, Minqi Li, Nobuyuki Udagawa, Kazuharu Irie, Jun Nishihira, Yoshikazu Koyama, Ayako Shiraishi, Harukazu Tohyama, Kazunori Yasuda
    JOURNAL OF BONE AND MINERAL RESEARCH 21 6 876 - 885 2006年06月 [査読有り][通常論文]
     
    The bone phenotype of mice overexpressing MIF was studies. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP-3, -9, and -13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo.
  • SUZUKI Hironobu, AMIZUKA Norio, NODA Masaki, AMANO Osamu, MAEDA Takeyasu
    Archives of histology and cytology 69 2 119 - 128 2006年06月 [査読有り][通常論文]
     
    The submandibular gland (SMG) has been regarded as an age-stable organ in spite of reports on its structural changes with aging. Although the klotho gene is involved in aging, little information is available regarding its effects on morphological changes of SMGs. The present study examined the histological and immunohistochemical features of SMGs in klotho-deficient mice - which are well-established aging models - by immunohistochemical and histochemical techniques. Five kinds of cellular markers - against NGF, EGF, Mn- and Cu/Zn-SOD, and RITC-conjugated phalloidin were used for the identification of cell types. In klotho-deficient mice, the SMGs lost their granular ducts and each lobe diminished. The granular duct showed strong immunoreactivities for NGF and EGF in the wild-type mice, but the NGF- and EGF-immunopositive ducts decreased in number remarkably in klotho-deficient mice. Interestingly, instead of a loss of the granular duct, the striated duct located on the distal portion in the homozygous mice came to show NGF- and EGF-immunoreactions. Neither Mn- and Cu/Zn-SOD immunoreactivities in the duct system nor the phalloidin-reaction in the myoepithelial cells differed between the wild-type and klotho-deficient mice. Our findings suggest that the klotho gene inhibited the differentiation of the granular duct from the striated duct due to the repression and/or down-regulation of sexual and growth hormones.
  • Isao Kii, Norio Amizuka, Li Minqi, Satoshi Kitajima, Yumiko Saga, Akira Kudo
    Biochemical and biophysical research communications 342 3 766 - 72 2006年04月14日 [査読有り][通常論文]
     
    A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin-/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin-/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone.
  • Minqi Li, Norio Amizuka
    Clinical calcium 16 4 591- 97 - 597 2006年04月 [査読有り][通常論文]
     
    Three types of bone metastasis can be identified : the osteolytic, osteoblastic and the intertrabecular metastasis. Bone resorption is believed to be responsible for osteolytic metastasis. The early stage of metastatic lesion showed tumor cells loosely intermingled with osteoblasts, fibroblastic stromal cells, osteoclasts and endothelial cells. In the metastatic nest, many tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts accumulated in direct contact with alkaline phosphatase (ALP)--or receptor activator of NF-kappaB ligand (RANKL)-positive osteoblastic cells, indicating the osteoclastogenesis. On the other hand, osteoclasts express CD44 while osteopontin was abundant in the stromal tissue of tumor nests. Therefore, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration. We review the histopathological microenvironment featuring osteoclastic bone resorption, angiogenesis and matrix degradation, which appears to facilitate proliferation of tumor cells after the onset of bone metastasis.
  • Shigeki Oshima, Shin Onodera, Norio Amizuka, Minqi Li, Kazuharu Irie, Satoshi Watanabe, Yoshikazu Koyama, Jun Nishihira, Kazunori Yasuda, Akio Minami
    FEBS letters 580 5 1251 - 6 2006年02月20日 [査読有り][通常論文]
     
    A link between macrophage migration inhibitory factor (MIF) and estrogen has recently emerged. We examined the involvement of MIF in osteoporotic changes in bone after ovariectomy (OVX), and revealed that MIF-deficient mice (MIF-KO) were completely protected from this phenomenon. The increase in osteoclast number per bone surface and serum IL-1beta levels, which were observed in wild-type mice after OVX, did not occur in MIF KO. Our data suggest that MIF plays an important role in the pathogenesis of postmenopausal osteoporosis, and could be a novel target for the treatment of this disease.
  • Minqi Li, Norio Amizuka, Kiichi Takeuchi, Paulo H L Freitas, Yoshiro Kawano, Masaaki Hoshino, Kimimitsu Oda, Kayoko Nozawa-Inoue, Takeyasu Maeda
    Microscopy research and technique 69 2 73 - 83 2006年02月 [査読有り][通常論文]
     
    The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.
  • Nozawa-Inoue Kayoko, Akiko Suzuki, Norio Amizuka, Takeyasu Maeda
    Anatomical Record - Part A Discoveries in Molecular, Cellular, and Evolutionary Biology 288 1 8 - 12 2006年01月 [査読有り][通常論文]
     
    This immunocytochemical study revealed the expression of caveolin-1, a major protein of caveolae, in the rat temporomandibular joint. In the synovial lining layer, immunoreactive products for caveolin-1 were detected on the cell membrane of the fibroblast-like type B cells, as confirmed by immunocytochemistry for heat shock protein 25. The cells in the articular disk, the articular layer, and zone of proliferation of the mandibular condyle also showed intense immunoreactions for caveolin-1. © 2005 Wiley-Liss, Inc.
  • Kayoko Nozawa-Inoue, Akiko Suzuki, Norio Amizuka, Takeyasu Maeda
    The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 288 1 8 - 12 2006年01月 [査読有り][通常論文]
     
    This immunocytochemical study revealed the expression of caveolin-1, a major protein of caveolae, in the rat temporomandibular joint. In the synovial lining layer, immunoreactive products for caveolin-1 were detected on the cell membrane of the fibroblast-like type B cells, as confirmed by immunocytochemistry for heat shock protein 25. The cells in the articular disk, the articular layer, and zone of proliferation of the mandibular condyle also showed intense immunoreactions for caveolin-1.
  • Hironobu Suzuki, Norio Amizuka, Kimimitsu Oda, Minqi Li, Hiromasa Yoshie, Hayato Ohshima, Masaki Noda, Takeyasu Maeda
    Archives of histology and cytology 68 5 371 - 81 2005年12月 [査読有り][通常論文]
     
    Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.
  • Yuya Taguchi, Norio Amizuka, Masayoshi Nakadate, Hideo Ohnishi, Noritaka Fujii, Kimimitsu Oda, Shuichi Nomura, Takeyasu Maeda
    Biomaterials 26 31 6158 - 66 2005年11月 [査読有り][通常論文]
     
    This study was designed to evaluate the histological changes during ossification and cellular events including osteogenic differentiation responding to collagenous bioresorbable membranes utilized for GBR. Standardized artificial bony defects were prepared at rat maxillae, and covered with a collagenous bioresorbable membrane. These animals were sacrificed at 1, 2, 3 and 4 weeks after the GBR-operation. The paraffin sections were subject to tartrate resistant acid phosphatase (TRAP) enzyme histochemistry and immunohistochemistry for alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC). In the first week of the experimental group, woven bone with ALP-positive osteoblasts occupied the lower half of the cavity. The collagenous membrane included numerous ALP-negative cells and OP-immunoreactive extracellular matrices. At 2 weeks, the ALP-, OP- and OC-immunoreactivity came to be recognizable in the region of collagenous membrane. Since ALP-negative soft tissue separated the collagenous membrane and the new bone originating from the cavity bottom, the collagenous membrane appeared to induce osteogenesis in situ. At 3 weeks, numerous collagen fibers of the membrane were embedded in the adjacent bone matrix. At 4 weeks, the membrane-associated and the cavity-derived bones had completely integrated, showing the same height of the periosteal ridge as the surrounding alveolar bones. The collagen fibers of a GBR-membrane appear to participate in osteogenic differentiation.
  • Akiko Suzuki, Kayoko Nozawa-Inoue, Nobuyuki Ikeda, Norio Amizuka, Kazuhiro Ono, Ritsuo Takagi, Takeyasu Maeda
    Anatomical Record - Part A Discoveries in Molecular, Cellular, and Evolutionary Biology 286 2 908 - 916 2005年10月 [査読有り][通常論文]
     
    Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively. © 2005 Wiley-Liss, Inc.
  • Hironobu Suzuki, Norio Amizuka, Takeyasu Maeda
    Nihon rinsho. Japanese journal of clinical medicine 63 Suppl 10 188 - 93 2005年10月 [査読有り][通常論文]
  • Norio Amizuka, Minqi Li
    Nihon rinsho. Japanese journal of clinical medicine 63 Suppl 10 278 - 83 2005年10月 [査読有り][通常論文]
  • Norio Amizuka, Minqi Li
    Nihon rinsho. Japanese journal of clinical medicine 63 Suppl 10 300 - 5 2005年10月 [査読有り][通常論文]
  • Norio Amizuka, Minqi Li
    Nihon rinsho. Japanese journal of clinical medicine 63 Suppl 10 382 - 6 2005年10月 [査読有り][通常論文]
  • Norio Amizuka, Minqi Li, Takeyasu Maeda
    Clinical calcium 15 7 57 - 61 2005年07月 [査読有り][通常論文]
     
    Magnesium (Mg) is most likely restored in bone matrix, implicating a pivotal role in bone mineralization. Mg-insufficient bone reveals fragility to mechanical loading despite normal or higher levels of bone mineral content, permitting stimulated osteoclastic bone resorption. In contrast, vitamin K(2) (MK-4:menatetrenone) inhibited osteoclastic bone resorption stimulated by the Mg-insufficiency, thereby normalizing bone remodeling. The Mg-insufficiency caused an increased concentration of calcium, which resulted in an extremely-high purity of hydroxyapatite (HA) crystal [Ca(10)(PO(4))(6)(OH)(2)] and accelerated mineralization in bone. In contrast, MK-4 did not affect the calcium-concentration nor HA-purity, but repressed mineralization accelerated by Mg-insufficiency. Thus, MK-4 appears to recover the "bone quality" lessened by the Mg-insufficiency by two mechanisms:controlling bone turnover and mineralization.
  • Kayoko Nozawa-Inoue, Norio Amizuka, Akiko Suzuki, Takeyasu Maeda
    The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 284 2 522 - 8 2005年06月 [査読有り][通常論文]
     
    One series of our research has shown an intense expression of immunoreaction for heat shock protein 25 (Hsp25) in various cellular elements in the rat temporomandibular joint (TMJ). This protein is the major substrate of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), which mediates an intracellular stress-activated signaling pathway to stimulate cytosolic actin reorganization under various stresses. The present study was undertaken to examine the localization of MAPKAPK-2 in the rat TMJ by immunocytochemical techniques. Furthermore, confocal microscopy with double staining was employed to demonstrate the colocalization of MAPKAPK-2 and Hsp25. Immunocytochemistry for MAPKAPK-2 showed an intense immunoreaction in the cytoplasm of the synovial lining cells, the endothelial cells, and the fibroblasts in the synovial membrane of the rat TMJ. Double immunostaining under a confocal microscope succeeded in demonstrating the colocalization of MAPKAPK-2 and Hsp25 immunoreactions in the cytoplasm of fibroblastic type B synoviocytes in the TMJ. On the other hand, the macrophage-like type A-cells expressed MAPKAPK-2 immunoreactions but lacked Hsp25 immunoreactivity. The cells in the articular disk and the chondrocytes in the maturative and hypertrophic layer of the mandibular cartilage also showed intense immunoreactions for MAPKAPK-2 and Hsp25. In addition to cytoplasmic localization, MAPKAPK-2 immunoreactions were found in the nucleus of some synovial lining cells, cells in the articular disk, and chondrocytes. Current observations imply the presence of the phosphorylation of Hsp25 via activated MAPKAPK-2 in the cytoplasm. MAPKAPK-2 and Hsp25 possibly participate in the induction of cytoskeletal changes to the various cellular elements in rat TMJ under normal conditions.
  • Naoko Sakagami, Norio Amizuka, Minqi Li, Kiichi Takeuchi, Masaaki Hoshino, Midori Nakamura, Kayoko Nozawa-Inoue, Nobuyuki Udagawa, Takeyasu Maeda
    Micron (Oxford, England : 1993) 36 7-8 688 - 95 2005年 [査読有り][通常論文]
     
    Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization.
  • Hironobu Suzuki, Norio Amizuka, Isao Kii, Yoshiro Kawano, Kayoko Nozawa-Inoue, Akiko Suzuki, Hiromasa Yoshie, Akira Kudo, Takeyasu Maeda
    The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 281 2 1264 - 75 2004年12月 
    Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption.
  • Isao Kii, Norio Amizuka, Junko Shimomura, Yumiko Saga, Akira Kudo
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 19 11 1840 - 9 2004年11月 [査読有り][通常論文]
     
    UNLABELLED: We studied cadherin-11 function in the differentiation of mesenchymal cells. Teratomas harboring the cadherin-11 gene generated bone and cartilage preferentially. Cadherin-11 transfectants of C2C12 cells and cadherin-11 and/or N-cadherin transfectants of L cells showed that cadherin-11 together with N-cadherin-induced expression of ALP and FGF receptor 2. These results suggest that cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage in a different manner from N-cadherin. INTRODUCTION: Cell-cell interaction is an essential event for tissue formation; however, the role of cell-cell adhesion in mesenchymal tissue formation as well as in cell differentiation in this tissue remains unclear. cadherins, which are calcium-dependent cell adhesion receptors, form adherence junctions after adherence and aggregation of cells. Because cadherin-11 as well as N-cadherin has been reported to be a mesenchyme-related cadherin, we examined the cadherin-11 action in teratomas and in the cell lines C2C12 and L cell. Herein, we show that cell-cell interaction mediated by cadherin-11 is responsible for bone and cartilage formation. MATERIALS AND METHODS: It has been previously reported that N-cadherin-expressing E-cadherin-/- ES transfectants formed neuroepithelium and cartilage in teratomas. Thus, we transfected the E-cadherin-/- ES cell line with the cadherin-11 gene. Moreover, we also transfected C2C12 cells and L cells with the cadherin-11 gene for morphological analysis and study of the induced differentiation at the molecular level. RESULTS AND CONCLUSION: Teratomas derived from embryonic stem cells in which the cadherin-11 gene had been expressed exogenously contained bone and cartilage preferentially, showing that cadherin-11 is involved in mesenchymal tissue formation, specifically in controlling the differentiation of these cells into osteoblasts and chondrocytes. Therefore, we further examined the functional difference between cadherin-11 and N-cadherin. The expression patterns of cadherin-11 and N-cadherin in cells of the mouse osteoblastic cell line MC3T3-E1 showed that each cadherin was located independently of the cell-cell adhesion site and acted individually. In hanging drop cultures, cadherin-11 L cell transfectants aggregated in a sheet-like structure, whereas N-cadherin transfectants aggregated in a spherical form, indicating that each cadherin confers a different 3D architecture because of its individual adhesive property. To investigate the molecular mechanism of cadherin-11 action in cell differentiation, we analyzed cadherin-11 transfectants of C2C12 cells and cadherin-11 and/or N-cadherin transfectants of L cells and showed that cadherin-11, together with N-cadherin, induced expression of alkaline phosphatase (ALP) and fibroblast growth factor receptor 2. These results suggest that cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage in a different manner from N-cadherin.
  • Fabricio I Tapety, Norio Amizuka, Katsumi Uoshima, Shuichi Nomura, Takeyasu Maeda
    Clinical oral implants research 15 3 315 - 24 2004年06月 [査読有り][通常論文]
     
    This study was designed to investigate the responses of bone cells to a deproteinized bovine bone material, Bio-Oss (Geistlich-Pharma, Wolhunsen, Switzerland), which was grafted in artificial bone defects of rat femurs. Standardized bone defects in the cortical bone of the right femurs were grafted with Bio-Oss particles. Narrow penetrations were prepared on the bottom of the cavity, enabling osteogenic cells to migrate from the bone marrow. A defect in the left femur without Bio-Oss was used as a control. The treated femurs were histochemically examined at 1, 3, 5, 7, and 14 days after the operation. At day 1, no osteogenic migration into the cavities occurred in either the control or experimental groups. At day 3, alkaline phosphatase (ALPase) immunohistochemistry showed a migration of the positive cells at the bottom of the cavities of the experimental groups, but not in the control ones. At day 5, new bone formation was recognized at the bottom of the cavity of both groups. In the experimental group, ALPase-positive cells were localized on Bio-Oss and/or on the thin bone matrix that covered this material. The superficial layer of Bio-Oss underlying the newly formed bone exhibited osteocalcin immunoreactivity. Transmission electron microscopy revealed osteoblasts depositing bone matrices--including collagen fibers--on the surface of Bio-Oss. At days 7 and 14, woven bone occupied the previous cavities of both control and experimental groups, accompanied by osteoclasts. Thus, Bio-Oss appears to serve as a scaffold for osteogenic cells as well as to promote osteoblastic differentiation and matrix synthesis.
  • Norio Amizuka
    Clinical calcium 14 4 589 - 93 2004年04月 [査読有り][通常論文]
     
    Bone is abundant in extracellular matrices, and therefore,"bone quality" appears to reflect the property of the bone matrix. The bone matrix is composed of minerals and organic materials. The volume of collagen fiber is approximately 90% of the whole organic materials of the bone matrix. Since collagen fibers could resist tension, the elasticity of the bone seems to come from the property of the collagen fibers. The mineralization of the bone matrix is achieved by the formation of hydroxyapatite crystals. The non-collagenous proteins and proteoglycans may regulate the growth of the mineralized crystal.
  • Hisaaki Kudo, Norio Amizuka, Kazuo Araki, Keiji Inohaya, Akira Kudo
    Developmental biology 267 2 473 - 87 2004年03月15日 [査読有り][通常論文]
     
    The myoseptum of fishes, composed of dense collagen, is a connective tissue layer that forms in the embryo, dividing somites from the trunk, and its structure and function are similar to those of the mammalian tendon. Both the myoseptum and tendon serve as the transmitter of muscular contractility to bones and adjoining muscles, and their structure is indispensable for movement of vertebrate animals. We cloned the zebrafish periostin gene and examined its expression and function in the myoseptum. The expression in embryos started in the rostral part of each segmented somite in the early segmentation stage; and consequently, metameric stripes were observed. At the end of segmentation, the expression region shifted to the transverse myoseptum and the myotome-epidermis boundary, and each myotome was surrounded by periostin. Using a polyclonal antibody, we found that the periostin protein was localized to the transverse myoseptum. Consistently, periostin morpholino antisense oligonucleotide led to defects in myoseptum formation, a delay in the differentiation of myofibers, and disorder of connection between myofibrils and myoseptum. We demonstrated here that periostin is the first molecule involved in myoseptum formation and propose that periostin secretion on the surface of the myoseptum is required for the adhesion of muscle fiber bundles to the myoseptum and the differentiation of muscle fibers.
  • Hirotsugu Hao, Norio Amizuka, Kimimitsu Oda, Noritaka Fujii, Hideo Ohnishi, Atsushi Okada, Shuichi Nomura, Takeyasu Maeda
    Biomaterials 25 3 431 - 42 2004年02月 [査読有り][通常論文]
     
    This study aimed to elucidate the biological effects of a self-setting tricalcium phosphate bone substitute (BIOPEX) applied in rat femoral cortical bone cavities. Narrow penetrations through the cavity and bone marrow were prepared to obtain cellular sources. In the experimental group at day 1, a thin cell layer intruded into a narrow space between the grafted BIOPEX and the bottom of the cavity. From days 5 to 10, a range of tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts accumulated on the surface of the BIOPEX facing the bottom of the cavity, whilst many alkaline phosphatase (ALPase)-positive osteoblasts were localized on the bone surface opposing the BIOPEX. However, at day 20, osteoblasts were localized neighboring the osteoclasts on the BIOPEX, and deposited bone matrices onto this material, implying a coupling between osteoclasts and osteoblasts. At days 30 and 40 post-operation, small remnants of BIOPEX particles were present in the new bone with a profile of compact bone. Thus, BIOPEX is resorbed by osteoclasts, and succeeded by osteoblastic bone apposition with a coupling of osteoclasts and osteoblasts at the later stage. In conclusion, the use of BIOPEX provides adequate bone regeneration with the profile of compact bone.
  • Yamada Kazuho, Nozawa-Inoue Kayoko, Kawano Yoshiro, Kohno Shoji, Amizuka Norio, Iwanaga Toshihiko, Maeda Takeyasu, 野澤-井上 佳世子
    The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 274 2 934 - 941 John Wily & Sons Ltd 2003年10月 
    Numerous epidemiological studies have pointed out a higher frequency of temporomandibular disorder (TMD) in women than in men, suggesting the involvement of a sex hormone such as estrogen in the pathogenesis of TMD. Although estrogen is known to play pivotal roles in osteoarthrosis or rheumatoid arthritis in systemic joints, there have been few reports about the role of estrogen in the temporomandibular joint (TMJ). Its effect is generally mediated by estrogen receptors (ERs), ERα and ERβ, the former which is a predominant type. In this study, we examined the expression of ERα protein and mRNA in the TMJ of adult male rats by immunocytochemistry and in situ hybridization histochemistry. Intense ERα-immunoreactivity was localized in the synovial lining cells, stromal cells in the articular disc, and chondrocytes in the TMJ. These ERα-immunopositive synovial lining cells were characteristic of cytoplasmic processes identified with confocal and immuno-electron microscopy, suggesting that they were synovial type B cells. In situ hybridization histochemistry confirmed intense signals for ERα in the synovial lining cells and the sublining fibroblasts at mRNA levels. The nuclei of chondrocytes showed an intense immunoreaction for ERα in the area of the maturative and hypertrophic layer of the articular cartilage. In addition to the nuclear localization of ERα, a weak immunoreaction appeared in the cytoplasm of some ERα-positive cells. These findings supported the hypothesis that the TMJ tissue --at least in the male rat-- has the potential to be an estrogen target tissue.
  • A Suzuki, K Nozawa-Inoue, Y Kawano, N Amizuka, T Maeda
    BIOMEDICAL RESEARCH-TOKYO 24 5 249 - 260 2003年10月 [査読有り][通常論文]
     
    The distribution and type of immunocompetent cells were investigated in rat oral mucosae using immunocytochemistry and enzyme histochemistry, focusing on histological structures. We used two antibodies, OX6 and ED1, which recognize the rat la-antigen and macrophage/monocyte lineage, respectively. Enzymatic histochemistry for acid phosphatase (ACPase) activity and adenosine triphosphatase (ATPase) activity was also employed to identify macrophages and Langerhans cells, respectively. Many OX6-immunopositive cells, dendritic or irregular in shape, were recognizable in the lamina propria of oral mucosae: some cells extended their dendritic processes into the epithelial layer of the buccal and sublingual mucosae. Dendritic cells within the epithelium showed intense ATPase reaction, indicating they could be categorized as Langerhans cells. A small number of ED1-positive cells existed in the lamina propria, but none were present in the epithelial cell layer. Double staining either with OX6 and ED1 or OX6 and ACPase made it possible to divide the immunocompetent cells in the lamina propria into three types: the OX6-positive cells without ED1 or ACPase-reaction, the OX6-negative cells with ED1 and ACPase-reactions, and the OX6-ED1/ACPase-co-expressing cells, each of which possessed characteristic ultrastructural features demonstrated by immunoelectron microscopy. Taking these findings together with previous reports, these three types of cells were regarded as a dendritic-like cell, a macrophage without antigen-presentation ability, and a macrophage with antigen-presentation ability, respectively. There were regional differences in the distribution and density of these immunocompetent cells; they were densely distributed in order of the buccal and sublingual mucosae, the palatal mucosa, and the dorsal surface of tongue. The region-specific distribution and density of the immunocompetent cells might be due to the histological. structure of each oral mucosa, suggesting the presence of different immune-defense systems among each portion of the oral mucosae.
  • Kazuho Yamada, Kayoko Nozawa-Inoue, Yoshiro Kawano, Shoji Kohno, Norio Amizuka, Toshihiko Iwanaga, Takeyasu Maeda
    The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 274 2 934 - 41 2003年10月 [査読有り][通常論文]
     
    Numerous epidemiological studies have pointed out a higher frequency of temporomandibular disorder (TMD) in women than in men, which indicates the involvement of a sex hormone, such as estrogen, in the pathogenesis of TMD. Although estrogen is known to play pivotal roles in osteoarthrosis or rheumatoid arthritis in systemic joints, there have been few reports about the role of estrogen in the temporomandibular joint (TMJ). The effect of estrogen is generally mediated by the estrogen receptors (ERs) ER alpha (the predominant type) and ER beta. In this study we examined the expression of ER alpha protein and mRNA in the TMJ of adult male rats by immunocytochemistry and in situ hybridization histochemistry. Intense ER alpha immunoreactivity was localized in the synovial lining cells, stromal cells in the articular disc, and chondrocytes in the TMJ. These ER alpha-immunopositive synovial lining cells are characteristic of cytoplasmic processes identified with confocal and immunoelectron microscopy, which indicates that they are synovial type B cells. In situ hybridization histochemistry confirmed intense signals for ER alpha in the synovial lining cells and the sublining fibroblasts at mRNA levels. The nuclei of chondrocytes showed an intense immunoreaction for ER alpha in the maturative and hypertrophic layers of the articular cartilage. In addition to the nuclear localization of ER alpha, a weak immunoreaction appeared in the cytoplasm of some ER alpha-positive cells. These findings support the hypothesis that TMJ tissue-at least in the male rat-has the potential to be an estrogen target tissue.
  • NOZAWA-INOUE Kayoko, AMIZUKA Norio, IKEDA Nobuyuki, SUZUKI Akiko, KAWANO Yoshiro, MAEDA Takeyasu
    Archives of histology and cytology 66 4 289 - 306 2003年10月 [査読有り][通常論文]
     
    This paper reviews recent findings of the synovial membrane, in particular the morphology, function and development of synovial lining cells, in the temporomandibular joint (TMJ). Electron microscopic studies have confirmed the synovial membrane in TMJ consists of macrophage-like type A cells and fibroblast-like type B cells identical to those in other systematic joints. The macrophage-like type A cells react with anti-macrophage and macrophage-derived substances including the major histocompatibility class II molecule, and show a drastic increase in their number in the inflamed synovial membrane. In addition, they have the ability to produce substances involved in the progression of TMJ inflammation such as nitric oxide and inducible nitric oxide synthase. Observation of osteopetrotic mice revealed that macrophage-like type A cells in TMJ are derived from monocyte lineage. Immunocytochemistry for 25kDa heat shock protein was able to depict the entire shape of fibroblast-like type B cells including their unique processes. The expression of an estrogen receptor alpha-immunoreaction in the fibroblast-like type B cells may explain the etiology of temporomandibular disorders at a higher frequency in females than in males, suggesting that TMJ is a target tissue for estrogen. Furthermore, fibroblast-like type B cells are equipped with a basement membrane to serve as an adhesion molecule for the fibroblast-like type B cells to keep their epithelial arrangement. A clear understanding of the morphology of the intact synovial membrane will serve to clarify the etiology and development of temporomandibular disorders.
  • Norio Amizuka, Junko Shimomura, Takeyasu Maeda, Hidehiro Ozawa
    Clinical calcium 13 4 405 - 12 2003年04月 [査読有り][通常論文]
     
    The epiphyseal cartilage is composed of the distinct zones of resting, proliferative and hypertrophic chondrocytes. The intercolumnar cartilage matrix of the hypertrophic zone is subjected to mineralization whereas the transverse partitions of the cartilage column are poorly mineralized. Therefore, mineralized cartilage matrices are formed parallel to the longitudinal axis of the epiphyseal cartilage. Vascular endothelial cells invade the cartilage by penetrating the poorly mineralized transverse partition at the chondro-osseous junction, resulting in the exposure of mineralized intercolumnar matrix to bony tissue. The exposed mineralized cartilage matrices appear to serve as scaffolds for osteoblastic attachment. These osteoblasts deposit bone matrices onto the cartilage cores, forming primary trabecular bones. Vascular endothelial growth factor, VEGF, is a strong angiogenic factor, and play a pivotal role in vascular invasion into cartilage. The invading endothelial cells possess the receptor for VEGF, and secret matrix metallo protatenase to digest the cartilage matrices of the unmineralized transverse partition of the column.
  • Masahiro Ito, Norio Amizuka, Shohei Tanaka, Yukiko Funatsu-Ozawa, Shin-ichi Kenmotsu, Kimimitsu Oda, Tamio Nakajima, Hidehiro Ozawa
    Journal of bone and mineral metabolism 21 6 353 - 62 2003年 [査読有り][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) induces pathological bone resorption in an endocrine manner, resulting in hypercalcemia of malignancy. However, the histopathological aspect of the action of PTHrP secreted by tumor cells on bone resorption has not well been documented. Therefore, we studied cell-cell interactions between bone cells, stromal cells, and PTHrP-secreting tumor cells (EC-GI-10) morphologically. Tumor cells injected subcutaneously into the parietal region formed a tumor mass, invading the bone marrow. The tumor mass was surrounded by a membrane structure consisting of stromal cells. These stromal cells were positive for alkaline phosphatase (ALPase). Tartrate-resistant acid phosphatase (TRAPase)-positive osteoclasts were localized close to the ALPase-positive cells, and numerous osteoclasts were observed on the neighboring bone surfaces. PTHrP, vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-9 were detected in the tumor cells. Using RT-PCR, expression of interleukin (IL)-1Alpha, IL-1Beta, and PTHrP, which are strong bone resorption factors, was detected in the tumor cells. Some ALPase-positive cells localizing on the neighboring bone surfaces and endothelial cells revealed PTH/PTHrP receptor immunoreactivity. Ultrastructurally, numerous blood vessels were observed between the tumor nests and the stromal cells. The nests were surrounded by a basement membrane, but it was discontinuous, therefore permitting direct contact between the tumor cells and the stromal cells. These results indicate that PTHrP secreted by tumor cells appears to stimulate osteoclast differentiation and bone resorption in a paracrine manner through PTH/PTHrP receptor-immunopositive cells. IL-1Alpha, IL-1Beta, VEGF, and MMP-9 may also be involved in facilitating osteoclast formation and the subsequent bone resorption.
  • Norio Amizuka, Junko Shimomura, Minqi Li, Yukie Seki, Kimimitsu Oda, Janet E Henderson, Atsuko Mizuno, Hidehiro Ozawa, Takeyasu Maeda
    Journal of electron microscopy 52 6 503 - 13 2003年 [査読有り][通常論文]
     
    Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG-/- mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG-/- epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG-/- mice appear to cause bone fragility.
  • AMIZUKA Norio, ODA Kimimitsu, SHIMOMURA Junko, MAEDA Takeyasu
    Anatomical science international 77 4 225 - 36 2002年12月 [査読有り][通常論文]
     
    Parathyroid hormone (PTH)-related peptide (PTHrP) has been believed to act by binding the common receptor to PTH (PTH/PTHrP receptor). However, PTHrP is localized not only in the secretory pathway, but also in nucleoli by virtue of its nucleolar targeting signal (NTS). This review demonstrates the bipartite action of PTHrP on chondrocytes, the receptor-mediated and -independent signaling pathway. Mice with deletion of the PTHrP gene were characterized by a chondrodysplasia due to markedly reduced proliferation of epiphyseal chondrocytes. The PTH/PTHrP receptor was localized mainly in proliferative chondrocytes in the epiphyseal cartilage, indicating that PTHrP modulates normal proliferation via the receptor. In contrast to the receptor-mediated action, the mid-region of the amino acid sequence of PTHrP contains an NTS. The PTHrP-translation was found to initiate from both methionine-coding AUG and downstream leucine-coding CUGs in its signal sequence. When translated from CUGs, PTHrP accumulated in the nucleoli, and the translation from AUG localized PTHrP in both the Golgi apparatus and nucleoli. Therefore, nucleolar PTHrP appears to be derived from the translation initiating from both AUG and CUGs. A chondrocytic cell line expressing a full-length PTHrP, but not PTHrP lacking NTS, were resistant to apoptosis caused by serum depletion, suggesting that the nucleolar PTHrP in chondrocytes serves as a survival factor against apoptosis. Thus, PTHrP regulates chondrocyte proliferation, differentiation and apoptosis by mediating its receptor or acting directly on the nucleolus.
  • Roberto Lin, Norio Amizuka, Tomoyo Sasaki, Michelle M Aarts, Hideharo Ozawa, David Goltzman, Janet E Henderson, John H White
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 17 9 1604 - 12 2002年09月 [査読有り][通常論文]
     
    Vitamin D deficiency results in defects in endochondral bone development characteristic of rickets, which include elongation of the cartilaginous growth plates and disorganization of the primary spongiosa. These defects are caused in part by impaired cartilage mineralization and vascularization of the chondro-osseous junction. Blood vessel invasion of mineralized cartilage is an essential step in endochondral ossification, providing access for cells that degrade cartilage as well as those that form bone. Vascular endothelial growth factor (VEGF) was shown to be a key regulator of this process when infusion of a dominant negative VEGF receptor effectively blocked vascular invasion and endochondral ossification in the growth plates of juvenile mice. Here, we show that the active metabolite of vitamin D 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] directly stimulates transcription of mRNAs encoding VEGF121 and -165 isoforms in the CFK2 chondrogenic cell line. Enhanced VEGF expression also was evident in growth plate chondrocytes and osteoblasts in the tibia of juvenile mice treated systemically with 1alpha,25(OH)2D3. This was seen in conjunction with enhanced expression of matrix metalloproteinase (MMP) 9, which activates VEGF stored in the cartilage matrix, in osteoclastic cells adjacent to the chondro-osseous junction. The alterations in VEGF and MMP-9 expression were accompanied by enhanced vascular invasion of mineralized cartilage, as assessed by CD31 immunoreactivity. These results provide evidence that 1alpha,25(OH)2D3 signaling stimulates VEGF and MMP-9 gene expression and promotes neovascularization of the epiphyseal growth plate in vivo through increased availability of active growth factor.
  • Norio Amizuka, Yukie Seki, Takeyasu Maeda
    Clinical calcium 12 6 835 - 43 2002年06月 [査読有り][通常論文]
  • Katsuhiro Ono, Takuhiko Akatsu, Takehiko Murakami, Ryuichi Kitamura, Michiko Yamamoto, Nariyoshi Shinomiya, Makoto Rokutanda, Tomoyo Sasaki, Norio Amizuka, Hidehiro Ozawa, Naokazu Nagata, Nobuo Kugai
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 17 5 774 - 81 2002年05月 [査読有り][通常論文]
     
    We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.
  • Norio Amizuka, Tomoyo Sasaki, Takeyasu Maeda
    Clinical calcium 12 3 327 - 36 2002年03月 [査読有り][通常論文]
     
    Endothelial cells play an important role in endochondral bone formation. In the chondro-osseous junction, endothelial cells appear to invade into cartilage by the cellular mechanism of angiogenesis evidenced by cell duplication, disappearance of basement membranes and activated migration. The endothelial cells penetrate the unmineralized transverse partition of the cartilage columns.
  • K Hoshi, N Amizuka, T Kurokawa, K Nakamura, R Shiro, H Ozawa
    ORTHOPEDICS 24 3 273 - 277 2001年03月 [査読有り][通常論文]
     
    Melorheostotic bone was examined histopathologically. In the severely affected areas, an abundance of osteoid and increased angiogenesis was observed. Increased osteoid without mineralization indicated the overproduction of bone matrix. Bone resorption also appeared to increase because osteoclasts were numerous in melorheostotic bone, thus suggesting a high rate of bone turnover. In addition, transforming growth factor-beta was immunolocalized in the periosteal fibroblasts, mesenchymal cells surrounding vessels, endothelial cells, and osteoblasts, while basic fibroblast growth factor was found in endothelial cells and mast cells near vessels. These cytokines may have some association with the exuberant bone matrix production and angiogenesis in melorheostosis.
  • K Horiuchi, N Amizuka, S Takeshita, H Takamatsu, M Katsuura, H Ozawa, Y Toyama, LF Bonewald, A Kudo
    JOURNAL OF BONE AND MINERAL RESEARCH 14 7 1239 - 1249 1999年07月 [査読有り][通常論文]
     
    We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure, Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed "periostin," Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription- polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3-E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in tell adhesion. The protein is highly homologous to beta ig-h3, a molecule induced by transforming growth factor beta (TGF-beta) that promotes the adhesion and spreading of fibroblasts, Because TGF-beta has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression, By Western blot analysis, TGF-beta increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.
  • Kazuyoshi Wakida, Chikage Murakami, Takahiro Satoda, Takashi Uchida, Norio Amizuka, Hidehiro Ozawa, Makoto Fukae, James P. Simmer
    Histochemistry and Cell Biology 111 4 297 - 303 1999年 [査読有り][通常論文]
     
    Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage.
  • AC Karaplis, B He, MTA Nguyen, ID Young, D Semeraro, H Ozawa, N Amizuka
    ENDOCRINOLOGY 139 12 5255 - 5258 1998年12月 [査読有り][通常論文]
     
    ii single homozygous nucleotide exchange in exon E3 of the gene encoding the parathyroid hormone receptor type 1 (PTHR1) was identified in an infant with Blomstrand chondrodysplasia born to consanguineous parents. This alteration changes a strictly conserved proline residue at position 132 in the receptor's amino terminal extracellular domain to leucine. COS-1 cells expressing the mutant receptor did not accumulate cyclic adenosine 3',5'-monophosphate in response to PTH or PTH-related peptide (PTHrP) and did not bind the radiolabeled ligand. Expression of the mutant protein on the cell surface of transiently transfected COS-I cells and in growth plate chondrocytes derived from the affected infant suggests that proline 132 is critical for the receptor's intrinsic binding activity. These findings suggest that the Blomstrand form of human short-limbed dwarfism arises from defective PTHR1 signaling in the developing cartilaginous skeleton.
  • Kazuto Hoshi, Norio Amizuka, Kimimitsu Oda, Yukio Ikehara, Hidehiro Ozawa
    Histochemistry and Cell Biology 107 3 183 - 191 1997年 [査読有り][通常論文]
     
    Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-μm tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes.
  • JE HENDERSON, N AMIZUKA, H WARSHAWSKY, D BIASOTTO, BMK LANSKE, D GOLTZMAN, AC KARAPLIS
    MOLECULAR AND CELLULAR BIOLOGY 15 8 4064 - 4075 1995年08月 [査読有り][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) is a mediator of cellular growth and differentiation as well as a cause of malignancy-induced hypercalcemia. Most of the actions of PTHrP have been attributed to its interaction with a specific cell surface receptor that binds the N-terminal domain of the protein. Here we present evidence that PTHrP promotes some of its cellular effects by translocating to the nucleolus. Localization of transiently expressed PTHrP to the nucleolus was dependent on the presence of a highly basic region at the carboxyl terminus of the molecule that bears homology to nucleolar targeting sequences identified within human retroviral (human immunodeficiency virus type 1 and human T-cell leukemia virus type 1) regulatory proteins. Endogenous PTHrP also localized to the nucleolus in osseous cells in vitro and in vivo. Moreover, expression of PTHrP in chondrocytic cells (CFK2) delayed apoptosis induced by serum deprivation, and this effect depended on the presence of an intact nucleolar targeting signal. The present findings demonstrate a unique intracellular mode of PTHrP action and a novel mechanism by which this peptide growth factor may modulate programmed cell death.

MISC

  • 網塚憲生, 阿部未来, 槙野彰人, 長谷川智香 日本整形外科学会雑誌 96 (3) 2022年
  • 網塚 憲生, 長谷川 智香, 本郷 裕美, 山本 知真也 日本骨形態計測学会雑誌 31 (2) S101 -S101 2021年06月
  • 森本 康仁, 久保田 恵亮, 阿部 未来, 丸岡 春日, 本郷 裕美, 吉田 靖弘, 菅谷 勉, 網塚 憲生, 長谷川 智香 日本骨形態計測学会雑誌 31 (2) S135 -S135 2021年06月
  • 北川善政, 浅香卓哉, 佐藤淳, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2021 2021年
  • 森本康仁, 久保田恵亮, 阿部未来, 丸岡春日, 長谷川智香, 本郷裕美, 吉田靖弘, 網塚憲生, 菅谷勉 日本歯周病学会会誌(Web) 63 2021年
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  • 久保田恵亮, 久保田恵亮, 横山敦郎, 網塚憲生, 長谷川智香 Journal of Oral Biosciences Supplement (Web) 2021 2021年
  • 阿部未来, 山本知真也, 山本知真也, 本郷裕美, ALIREZA Nasoori, 網塚憲生, 長谷川智香 Journal of Oral Biosciences Supplement (Web) 2021 2021年
  • 長谷川智香, 森本康仁, 森本康仁, 久保田恵亮, 久保田恵亮, 本郷裕美, 吉田靖弘, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2021 2021年
  • 中村郁哉, 兼平裕也, 佐藤大, 藤田諒, 長谷川智香, 堀内秀与, 枡谷朋美, 太田昌博, 高畑雅彦, 網塚憲生, 木村(須田)廣美 日本骨代謝学会学術集会プログラム抄録集(CD-ROM) 39th 2021年
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  • 丸岡春日, 丸岡春日, 長谷川智香, 中西康, 中西康, 網塚憲生, 佐藤嘉晃 日本矯正歯科学会大会プログラム・抄録集 80th 2021年
  • 塩飽由香利, 塩飽由香利, 齊藤志都, 濱井瞭, 長谷川智香, 網塚憲生, 蔡優広, 高橋哲, 鈴木治 日本バイオマテリアル学会大会予稿集(Web) 43rd 2021年
  • 丸岡春日, 長谷川智香, 中西康, 佐藤嘉晃, 網塚憲生 日本再生医療学会総会(Web) 20th 2021年
  • 森本康仁, 長谷川智香, 本郷裕美, 久保田恵亮, 阿部未来, 丸岡春日, 吉田靖弘, 菅谷勉, 網塚憲生 日本再生医療学会総会(Web) 20th 2021年
  • 齊藤志都, 齊藤志都, 塩飽由香利, 濱井瞭, 長谷川智香, 網塚憲生, 高橋哲, 鈴木治 日本再生医療学会総会(Web) 20th 2021年
  • 久保田恵亮, 長谷川智香, 本郷裕美, 森本康仁, 阿部未来, 丸岡春日, 吉田靖弘, 横山敦郎, 網塚憲生 日本再生医療学会総会(Web) 20th 2021年
  • 小林英之, 高畑雅彦, 太田昌博, 清水智弘, 佐藤大, 藤田諒, 岩崎倫政, 長谷川智香, 網塚憲生 北海道整形災害外科学会 140th 2021年
  • 阿部 未来, 長谷川 智香, 宇田川 信之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 38回 132 -132 2020年10月
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 阿部 未来, 小守 壽文, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 38回 133 -133 2020年10月
  • 槙野 彰人, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 38回 145 -145 2020年10月
  • 中村 郁哉, 兼平 裕也, 佐藤 大, 藤田 諒, 長谷川 智香, 堀内 秀与, 枡谷 朋美, 太田 昌博, 高畑 雅彦, 居城 邦治, 網塚 憲生, 木村 廣美[須田] 日本骨代謝学会学術集会プログラム抄録集 38回 145 -145 2020年10月
  • 中嶋 悠斐, 山本 知真也, 本郷 裕美, 網塚 憲生, 長谷川 智香 日本骨代謝学会学術集会プログラム抄録集 38回 159 -159 2020年10月
  • RANKL-RANK-OPGシグナル研究の最前線 骨粗鬆症治療薬候補としてのSiglec-15抗体の研究
    津田 英資, 福田 千恵, 宇田川 信之, 高橋 直之, 長谷川 智香, 網塚 憲生, 佐藤 大, 高畑 雅彦 Journal of Oral Biosciences Supplement 2020 101 -101 2020年09月
  • 副甲状腺ホルモン間歇投与による皮質骨多孔化の組織化学的検索
    阿部 未来, 山本 知真也, 本郷 裕美, 網塚 憲生, 長谷川 智香 Journal of Oral Biosciences Supplement 2020 176 -176 2020年09月
  • メッケル軟骨前方部に形成される膜性骨の形態学的特徴と役割
    井上 貴一朗, 丸岡 春日, 長谷川 智香, 山本 恒之, 八若 保孝, 網塚 憲生 Journal of Oral Biosciences Supplement 2020 193 -193 2020年09月
  • 反射電子像を利用した走査型電子顕微鏡による破骨細胞のゴルジ装置の立体復構
    山本 恒之, 長谷川 智香, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2020 204 -204 2020年09月
  • マウス下顎骨切歯エナメル器におけるCD44とendomucin陽性血管の局在について
    越石 麟, 本郷 裕美, 網塚 憲生, 長谷川 智香 Journal of Oral Biosciences Supplement 2020 392 -392 2020年09月
  • 網塚 憲生, 長谷川 智香, 本郷 裕美, 山本 知真也 日本骨形態計測学会雑誌 30 (1) S84 -S84 2020年05月
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 山本 恒之, 小守 壽文, 網塚 憲生 日本骨形態計測学会雑誌 30 (1) S127 -S127 2020年05月
  • 中村 郁哉, 佐藤 大, 藤田 諒, 長谷川 智香, 堀内 秀与, 桝谷 朋美, 太田 昌博, 高畑 雅彦, 居城 邦治, 網塚 憲生, 木村 廣美[須田] 日本骨形態計測学会雑誌 30 (1) S134 -S134 2020年05月
  • 山本 恒之, 本郷 裕美, 長谷川 智香, 網塚 憲生 THE BONE 33 (3) 259 -263 2020年03月
  • 網塚憲生, 長谷川智香 日本セラミックス協会秋季シンポジウム講演予稿集(CD-ROM) 33rd 2020年
  • 齊藤志都, 塩飽由香利, 濱井瞭, 高橋哲, 鈴木治, 長谷川智香, 網塚憲生 日本セラミックス協会年会講演予稿集(CD-ROM) 2020 2020年
  • 本郷裕美, 森本康仁, 森本康仁, 長谷川智香, 菅谷勉, 吉田靖弘, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 125th 2020年
  • 山本知真也, 山本知真也, 本郷裕美, 井上貴一朗, 長谷川智香, 山本恒之, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 125th 2020年
  • 網塚憲生, 本郷裕美, 長谷川智香 日本再生医療学会総会(Web) 19th 2020年
  • 本郷裕美, 長谷川智香, 森本康仁, 森本康仁, 菅谷勉, 吉田靖弘, 網塚憲生 日本再生医療学会総会(Web) 19th 2020年
  • 長谷川智香, 本郷裕美, 山本知真也, 山本恒之, 井上貴一朗, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 125th 2020年
  • 井上貴一朗, 丸岡春日, 長谷川智香, 山本恒之, 八若保孝, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2020 2020年
  • 津田英資, 福田千恵, 宇田川信之, 高橋直之, 長谷川智香, 網塚憲生, 佐藤大, 高畑雅彦 Journal of Oral Biosciences Supplement (Web) 2020 2020年
  • 阿部未来, 山本知真也, 本郷裕美, 網塚憲生, 長谷川智香 Journal of Oral Biosciences Supplement (Web) 2020 2020年
  • 山本恒之, 長谷川智香, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2020 2020年
  • 越石麟, 本郷裕美, 網塚憲生, 長谷川智香 Journal of Oral Biosciences Supplement (Web) 2020 2020年
  • 中村郁哉, 佐藤大, 藤田諒, 長谷川智香, 堀内秀与, 枡谷朋美, 太田昌博, 高畑雅彦, 三友秀之, 居城邦治, 網塚憲生, 木村(須田)廣美, 木村(須田)廣美 分析化学討論会講演要旨集(Web) 80th 2020年
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 網塚 憲生 THE BONE 33 (2) 129 -133 2020年01月
  • 森本 康仁, 長谷川 智香, 本郷 裕美, 吉田 靖弘, 網塚 憲生, 菅谷 勉 日本歯周病学会会誌 61 (秋季特別) 142 -142 2019年10月
  • RANKL-RANK-OPGシグナル研究の最前線 骨粗鬆症治療薬候補としてのSiglec-15抗体の研究
    津田 英資, 福田 千恵, 宇田川 信之, 高橋 直之, 長谷川 智香, 網塚 憲生, 佐藤 大, 高畑 雅彦 Journal of Oral Biosciences Supplement 2019 43 -43 2019年10月
  • 骨粗鬆症モデルラットにおけるエルデカルシトール誘導性ミニモデリングの組織化学的解析
    長谷川 智香, 山本 知真也, 本郷 裕美, 坂井 貞興, 與語 健二, 網塚 憲生 Journal of Oral Biosciences Supplement 2019 227 -227 2019年10月
  • 酵素細胞化学と準超薄連続切片を用いた破骨細胞のゴルジ装置の立体復構
    山本 恒之, 長谷川 智香, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2019 338 -338 2019年10月
  • アレンドロネート投与が骨特異的血管に与える影響について
    吉野 弘菜, 吉田 泰士, 宮本 幸奈, 邱 紫旋, 網塚 憲生, 長谷川 智香 Journal of Oral Biosciences Supplement 2019 417 -417 2019年10月
  • Jansen型PTH/PTHrP受容体変異トランスジェニックマウスの形態および機能異常解析
    下村 淳子[黒木], 梨田 智子, 森田 貴雄, 大島 勇人, 網塚 憲生, 下村 裕 Journal of Oral Biosciences Supplement 2019 337 -337 2019年10月 [査読有り][通常論文]
  • 森本康仁, 長谷川智香, 本郷裕美, 吉田靖弘, 網塚憲生, 菅谷勉 日本歯周病学会会誌(Web) 61 142 2019年10月01日 [査読無し][通常論文]
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 宮本 幸奈, 網塚 憲生 腎と骨代謝 32 (4) 275 -283 2019年10月 [査読無し][通常論文]
     
    ＜文献概要＞FGF23は,おもに骨細胞から産生・分泌されて腎臓の近位尿細管に存在するFGFR1c/αklotho複合受容体に作用することで,血中リン濃度を調節している.したがって,kl/klマウスにおいてFGF23/αklotho軸が破綻すると,高リン・高カルシウム血症を呈し,動脈中膜にメンケベルグ型中膜石灰化が誘導される.その組織病理機序として,血管平滑筋細胞が骨芽細胞様細胞へとtrans-differentiationすることで,骨芽細胞と同様に生物学的石灰化が生じる可能性が示唆されている.電顕観察では,骨芽細胞が分泌するような基質小胞様構造物が観察されたが,必ずしも,骨基質石灰化のようにI型コラーゲン線維に基質小胞性石灰化が波及しているのではなく,弾性線維が優先的に石灰化しているように観察された.また,低リン餌をkl/klマウスに与えると血管石灰化が低下したが,αklotho遺伝子の発現が若干回復していることから,血中リン濃度の低下,および,αklotho遺伝子回復の両方の可能性が考察された.
  • 佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 藤田 諒, 小林 英之, 福田 知恵, 津田 英資, 長谷川 智香, 網塚 憲生, 岩崎 倫政 日本骨代謝学会学術集会プログラム抄録集 37回 184 -184 2019年09月
  • 佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 藤田 諒, 小林 英之, 岩崎 倫政, 福田 千恵, 津田 英資, 網塚 憲生, 長谷川 智香 日本整形外科学会雑誌 93 (8) S1810 -S1810 2019年09月 [査読無し][通常論文]
  • 佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 藤田 諒, 小林 英之, 岩崎 倫政, 福田 千恵, 津田 英資, 網塚 憲生, 長谷川 智香 日本整形外科学会雑誌 93 (8) S1810 -S1810 2019年09月 [査読無し][通常論文]
  • 吉野弘菜, 山本知真也, 宮本幸奈, 邱紫せん, 網塚憲生, 長谷川智香 日本骨形態計測学会雑誌 29 (1) S141 -S141 2019年05月29日 [査読無し][通常論文]
  • 渡邉駿, 平松里佳子, 山内真之, 諏訪部達也, 澤直樹, 平井利英, 森川鉄平, 長谷川智香, 網塚憲生, 乳原善文 日本骨形態計測学会雑誌 29 (1) S134 -S134 2019年05月29日 [査読無し][通常論文]
  • 長谷川智香, 山本知真也, 宮本幸奈, 邱紫せん, 網塚憲生 Bone 33 (1) 3‐8 2019年05月20日 [査読無し][通常論文]
  • 渡邉 駿, 平松 里佳子, 山内 真之, 諏訪部 達也, 澤 直樹, 平井 利英, 森川 鉄平, 長谷川 智香, 網塚 憲生, 乳原 善文 日本骨形態計測学会雑誌 29 (1) S134 -S134 2019年05月
  • 吉野 弘菜, 山本 知真也, 宮本 幸奈, 邱 紫せん, 網塚 憲生, 長谷川 智香 日本骨形態計測学会雑誌 29 (1) S141 -S141 2019年05月
  • 下村 淳子[黒木], 林 幸子[坂井], 梨田 智子, 森田 貴雄, 網塚 憲生, 下村 裕 小児歯科学雑誌 57 (2) 245 -245 2019年05月 [査読無し][通常論文]
  • 長谷川 智香, 山本 知真也, 宮本 幸奈, 邱 紫せん, 網塚 憲生 THE BONE 33 (1) 3 -8 2019年05月 [査読無し][通常論文]
  • 『骨形態計測データから骨粗鬆症治療を吟味する』骨粗鬆症治療薬における細胞組織・微細構造in vivo解析
    網塚 憲生, 長谷川 智香, 槙野 彰人, 本郷 裕美, 山本 知真也 日本骨形態計測学会雑誌 29 (1) S61 -S61 2019年05月 [査読無し][通常論文]
  • 『画像解析技術による骨組織評価』骨特異的血管と骨細胞における組織化学・電顕イメージング
    長谷川 智香, 邱 紫せん, 宮本 幸奈, 山本 知真也, 網塚 憲生 日本骨形態計測学会雑誌 29 (1) S65 -S65 2019年05月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 槙野 彰人, 本郷 裕美, 山本 知真也 日本骨形態計測学会雑誌 29 (1) S61 -S61 2019年05月 [査読無し][通常論文]
  • 長谷川 智香, 邱 紫せん, 宮本 幸奈, 山本 知真也, 網塚 憲生 日本骨形態計測学会雑誌 29 (1) S65 -S65 2019年05月 [査読無し][通常論文]
  • Podoplanin陽性骨芽細胞および骨細胞における組織化学的検索
    永井 伯弥, 長谷川 智香, 網塚 憲生, 横山 敦郎 北海道歯学雑誌 39 (2) 163 -163 2019年03月
  • 本郷裕美, 長谷川智香, 井上貴一朗, 山本恒之, 網塚憲生 日本顕微鏡学会北海道支部学術講演会講演要旨集 2019 2019年
  • 長谷川 智香, 宮本 幸奈, 山本 知真也, 網塚 憲生 日本薬理学雑誌 153 (1) 16 -21 2019年01月 [査読無し][通常論文]
     
    骨粗鬆症治療薬の副甲状腺ホルモン(PTH)製剤として、遺伝子組み換え型ヒトPTH[1-34]であるテリパラチドが用いられている。我が国においては、連日製剤と週1回製剤の2種類が存在しており、そのどちらも骨量を増加させ、また、骨折率を低下させることが報告されている。一般的にPTHを持続投与すると骨吸収を誘導するのに対して、PTH間歇投与では骨量増加につながることが知られている。その細胞学的メカニズムとして、PTHは骨芽細胞の前駆細胞である前骨芽細胞に対して細胞増殖を亢進する一方、成熟型骨芽細胞に対しては破骨細胞とのカップリングに依存して、骨形成を促進することが報告されている。さらに、我々は、PTH間歇投与の頻度(投与間隔)の違いにより、骨の細胞群の挙動が異なることを明らかにした。PTH高頻度投与では、成熟型骨芽細胞が活発に骨基質合成を行うだけでなく、前骨芽細胞の増殖が亢進して厚い細胞性ネットワークを形成し、その中で多数の破骨細胞が誘導されていた。その結果、高骨代謝回転の骨リモデリングにより骨形成が誘導されていた。しかし、PTH低頻度投与では、成熟型骨芽細胞による骨形成が亢進するが、前骨芽細胞はあまり増加せず、よって破骨細胞形成の誘導も上昇しなかった。この場合、骨リモデリングとミニモデリングの両方によって骨形成が誘導されることが明らかにされた。このように、PTH製剤の投与頻度が異なると、骨の形態や形成のされ方が違うことが強く示唆された。以上より、PTH製剤の投与頻度によって骨形成促進における作用機序が異なると思われる。(著者抄録)
  • 津田英資, 福田千恵, 宇田川信之, 高橋直之, 長谷川智香, 網塚憲生, 佐藤大, 高畑雅彦 Journal of Oral Biosciences Supplement (Web) 2019 43 (WEB ONLY) 2019年 [査読無し][通常論文]
  • 山本知真也, 山本知真也, 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 163 2019年 [査読無し][通常論文]
  • 松本愛子, 松本愛子, NAZNIN Khadiza, 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 218 2019年 [査読無し][通常論文]
  • 邱紫せん, 伊敏, 山本知真也, 李敏啓, 森本景之, 網塚憲生, 長谷川智香 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 142 2019年 [査読無し][通常論文]
  • 下村(黒木)淳子, 梨田智子, 森田貴雄, 大島勇人, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2019 337 (WEB ONLY) 2019年 [査読無し][通常論文]
  • 森本康仁, 長谷川智香, 邱紫せん, 松本愛子, 桐越晶子, 森谷康人, 橋本圭司, 趙申, 前壮功仁, 本郷裕美, 菅谷勉, 吉田靖弘, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 162 2019年 [査読無し][通常論文]
  • 宮本幸奈, 宮本幸奈, 阿部未来, 邱紫せん, 宇田川信之, 網塚憲生, 長谷川智香 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 142 2019年 [査読無し][通常論文]
  • 山本恒之, 長谷川智香, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2019 338 (WEB ONLY) 2019年 [査読無し][通常論文]
  • 橋本圭司, 橋本圭司, 長谷川智香, 藤澤俊明, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 163 2019年 [査読無し][通常論文]
  • 吉野弘菜, 吉田泰士, 宮本幸奈, QIU Z, 網塚憲生, 長谷川智香 Journal of Oral Biosciences Supplement (Web) 2019 417 (WEB ONLY) 2019年 [査読無し][通常論文]
  • 長谷川智香, 山本知真也, 本郷裕美, 坂井貞興, 與語健二, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2019 227 (WEB ONLY) 2019年 [査読無し][通常論文]
  • 吉田泰士, 吉田泰士, 本郷裕美, 阿部未来, 阿部未来, 吉野弘菜, 吉野弘菜, 邱紫せん, 網塚憲生, 長谷川智香 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 125 2019年 [査読無し][通常論文]
  • 吉野弘菜, 吉野弘菜, 宮本幸奈, 邱紫せん, 阿部未来, 阿部未来, 吉田泰士, 吉田泰士, 網塚憲生, 長谷川智香 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 125 2019年 [査読無し][通常論文]
  • 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 99 2019年 [査読無し][通常論文]
  • 森谷康人, 森谷康人, 伊敏, 邱紫せん, 山本知真也, 鄭漢忠, 網塚憲生, 長谷川智香 日本解剖学会総会・全国学術集会講演プログラム・抄録集 124th 162 2019年 [査読無し][通常論文]
  • 北川 善政, 浅香 卓哉, 佐藤 淳, 秦 浩信, 坪井 香奈子, 網塚 憲生, 久下 裕司, 志賀 哲 臨床放射線 63 (10) 1071 -1081 2018年10月 [査読無し][通常論文]
  • 長谷川智香, 宮本幸奈, 本郷裕美, 阿部未来, 阿部未来, 網塚憲生 日本組織細胞化学会総会・学術集会講演プログラム・予稿集 59th 43 -43 2018年09月29日 [査読無し][通常論文]
  • 小澤英浩, 長谷川智香, 網塚憲生 Bone 32 (2) 139‐144 -144 2018年09月20日 [査読無し][通常論文]
  • FGF23/klothoシグナルによる基質石灰化制御機構
    長谷川 智香, 宮本 幸奈, 本郷 裕美, 山本 知真也, 網塚 憲生 Journal of Oral Biosciences Supplement 2018 270 -270 2018年09月
  • 新規肥満2型糖尿病モデルでSDT fattyラットの骨組織解析について
    本郷 裕美, 坪井 香奈子, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2018 271 -271 2018年09月
  • 破骨細胞のゴルジ装置の立体形態 酵素細胞化学を用いた光学顕微鏡およびオスミウム浸軟法を用いた走査型電子顕微鏡による研究
    山本 恒之, 長谷川 智香, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2018 369 -369 2018年09月
  • 骨系細胞におけるJansen型PTH/PTHrP受容体の機能異常の解析
    下村 淳子[黒木], 梨田 智子, 森田 貴雄, 網塚 憲生, 下村 裕 Journal of Oral Biosciences Supplement 2018 446 -446 2018年09月 [査読無し][通常論文]
  • 小澤 英浩, 長谷川 智香, 網塚 憲生 THE BONE 32 (2) 139 -144 2018年09月 [査読無し][通常論文]
  • 組織化学イメージングで探る硬組織の細胞機能 電子顕微鏡を駆使した骨の細胞組織学的アプローチ
    長谷川 智香, 宮本 幸奈, 本郷 裕美, 阿部 未来, 網塚 憲生 日本組織細胞化学会総会・学術集会講演プログラム・予稿集 59回 43 -43 2018年09月 [査読無し][通常論文]
  • 骨改造制御の新局面:骨吸収から骨形成・骨再生への橋渡し機構を探る 組織学的知見からみる骨改造
    長谷川 智香, 宮本 幸奈, 網塚 憲生 Journal of Oral Biosciences Supplement 2018 122 -122 2018年09月 [査読無し][通常論文]
  • 長谷川智香, 宮本幸奈, 本郷裕美, 山本知真也, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2018 ROMBUNNO.P1‐50 (WEB ONLY) -270 2018年09月 [査読無し][通常論文]
  • 山本恒之, 長谷川智香, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2018 ROMBUNNO.P2‐13 (WEB ONLY) -369 2018年09月 [査読無し][通常論文]
  • 下村(黒木)淳子, 梨田智子, 森田貴雄, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2018 ROMBUNNO.P3‐13 (WEB ONLY) -446 2018年09月 [査読無し][通常論文]
  • 本郷裕美, 坪井香奈子, 長谷川智香, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2018 ROMBUNNO.P1‐51 (WEB ONLY) -271 2018年09月 [査読無し][通常論文]
  • 邱 紫せん, 長谷川 智香, 本郷 裕美, 山本 知真也, 李 敏啓, 伊 敏, 網塚 憲生 日本骨粗鬆症学会雑誌 4 (Suppl.1) 294 -294 2018年09月 [査読無し][通常論文]
  • 槙野 彰人, 長谷川 智香, 網塚 憲生 日本骨粗鬆症学会雑誌 4 (Suppl.1) 293 -293 2018年09月 [査読無し][通常論文]
  • 長谷川 智香, 宮本 幸奈, 邱 紫セン, 山本 知真也, 本郷 裕美, 松井 功, 網塚 憲生 日本骨粗鬆症学会雑誌 4 (Suppl.1) 294 -294 2018年09月 [査読無し][通常論文]
  • 本郷 裕美, 坪井 香奈子, 長谷川 智香, 網塚 憲生 日本骨粗鬆症学会雑誌 4 (Suppl.1) 294 -294 2018年09月 [査読無し][通常論文]
  • 長谷川 智香, 宮本 幸奈, 邱 紫せん, 阿部 未来, 本郷 裕美, 網塚 憲生 日本骨粗鬆症学会雑誌 4 (3) 394 -396 2018年08月 [査読無し][通常論文]
     
    生後7週齢aklotho遺伝子変異(kl/kl)マウス、aklotho遺伝子欠損マウスの大腿骨・脛骨及び大動脈を用い、FGF23/klothoシグナルが与える基質石灰化への影響について検討した。大腿骨、脛骨、大動脈は組織化学的、微細構造学的な解析を行った。また、2種類のマウスの基質石灰化異常を引き起こすのが血中リン・カルシウム濃度異常おるいはFGF23/klothoシグナルの局所作用であるかを明らかにする目的で、血中リン濃度離乳後3週間低リン食で飼育したkl/klマウス、aklotho遺伝子欠損マウスについても同様に解析した。その結果、FGF23/klothoシグナルの破綻は、骨組織では骨細胞および骨芽細胞の機能異常を生じて骨基質の石灰化異常を誘導し、大動脈では血管平滑筋細胞が骨芽細胞様細胞に変化し血管石灰化及び血管骨化を誘導することを示唆した。これらの異常は血中リン濃度の是正で回復しないことより、FGF23/klothoシグナル自体が影響していると考えられた。
  • 佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 福田 千恵, 岡田 顕子, 津田 英資, 濱野 博基, 長谷川 智香, 網塚 憲生, 岩崎 倫政 日本整形外科学会雑誌 92 (8) S1704 -S1704 2018年08月 [査読無し][通常論文]
  • イメージング最前線 骨における組織化学・電顕イメージング
    網塚 憲生, 長谷川 智香 日本骨代謝学会学術集会プログラム抄録集 36回 119 -119 2018年07月 [査読無し][通常論文]
  • 小児骨粗鬆症に対するSiglec-15分子標的治療の有用性 成長骨格発達に影響の生じない骨吸収抑制薬の開発
    佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 福田 千恵, 津田 英資, 濱野 博基, 平塚 重人, 藤田 諒, 網塚 憲生, 長谷川 智香, 岡田 顕子, 岩崎 倫政 日本骨代謝学会学術集会プログラム抄録集 36回 160 -160 2018年07月 [査読無し][通常論文]
  • Fgf23遺伝子欠損マウスの骨基質石灰化異常におけるSIBLING familyの局所作用
    長谷川 智香, 邱 紫セン, 山本 知真也, 本郷 裕美, 松井 功, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 36回 170 -170 2018年07月 [査読無し][通常論文]
  • マウスにabaloparatideまたはteriparatideを皮下投与した時の骨量及び骨代謝に及ぼす作用の比較
    槙野 彰人, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 36回 184 -184 2018年07月 [査読無し][通常論文]
  • イメージング最前線 骨における組織化学・電顕イメージング
    網塚 憲生, 長谷川 智香 日本骨代謝学会学術集会プログラム抄録集 36回 119 -119 2018年07月 [査読無し][通常論文]
  • 槙野 彰人, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 36回 184 -184 2018年07月 [査読無し][通常論文]
  • 長谷川 智香, 邱 紫セン, 山本 知真也, 本郷 裕美, 松井 功, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 36回 170 -170 2018年07月 [査読無し][通常論文]
  • 佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 福田 千恵, 津田 英資, 濱野 博基, 平塚 重人, 藤田 諒, 網塚 憲生, 長谷川 智香, 岡田 顕子, 岩崎 倫政 日本骨代謝学会学術集会プログラム抄録集 36回 160 -160 2018年07月 [査読無し][通常論文]
  • 補綴治療に求められる骨質を科学する 骨質に関する骨の微細構造と細胞群の役割
    網塚 憲生, 長谷川 智香 日本補綴歯科学会誌 10 (特別号) 85 -85 2018年06月 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香, 邱紫旋, 本郷裕美 日本透析医学会雑誌 51 (Supplement 1) 387 2018年05月28日 [査読無し][通常論文]
  • リン-FGF23-Klotho axis:臨床から基礎へ 骨基質石灰化におけるリン-FGF23-Klotho axis
    網塚 憲生, 長谷川 智香, 邱 紫旋, 本郷 裕美 日本透析医学会雑誌 51 (Suppl.1) 387 -387 2018年05月 [査読無し][通常論文]
  • 副甲状腺摘除後に顕在化した長期透析患者のBrown tumorの一例
    鳥生 直哉, 上野 智敏, 水野 裕樹, 渡邉 駿, 小黒 昌彦, 大島 洋一, 井熊 大輔, 関根 章成, 早見 典子, 住田 圭一, 山内 真之, 長谷川 詠子, 星野 純一, 澤 直樹, 乳原 善文, 高市 憲明, 藤井 丈士, 長谷川 智香, 網塚 憲生 日本透析医学会雑誌 51 (Suppl.1) 543 -543 2018年05月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 山本 知真也, 本郷 裕美 日本骨形態計測学会雑誌 28 (2) S100 -S100 2018年05月 [査読無し][通常論文]
  • 長谷川 智香, 邱 紫せん, 山本 知真也, 本郷 裕美, 松井 功, 網塚 憲生 日本骨形態計測学会雑誌 28 (2) S155 -S155 2018年05月 [査読無し][通常論文]
  • 阿部 未来, 長谷川 智香, 網塚 憲生 日本骨形態計測学会雑誌 28 (2) S164 -S164 2018年05月 [査読無し][通常論文]
  • 佐藤 大, 高畑 雅彦, 太田 昌博, 清水 智弘, 福田 千恵, 岡田 顕子, 津田 英資, 長谷川 智香, 網塚 憲生, 岩崎 倫政 日本骨形態計測学会雑誌 28 (2) S145 -S145 2018年05月 [査読無し][通常論文]
  • 長谷川 智香, 本郷 裕美, 邱 紫せん, 網塚 憲生, 阿部 未来 THE BONE 32 (1) 3 -8 2018年05月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 邱 紫旋, 本郷 裕美 日本透析医学会雑誌 51 (Suppl.1) 387 -387 2018年05月 [査読無し][通常論文]
  • 副甲状腺摘除後に顕在化した長期透析患者のBrown tumorの一例
    鳥生 直哉, 上野 智敏, 水野 裕樹, 渡邉 駿, 小黒 昌彦, 大島 洋一, 井熊 大輔, 関根 章成, 早見 典子, 住田 圭一, 山内 真之, 長谷川 詠子, 星野 純一, 澤 直樹, 乳原 善文, 高市 憲明, 藤井 丈士, 長谷川 智香, 網塚 憲生 日本透析医学会雑誌 51 (Suppl.1) 543 -543 2018年05月 [査読無し][通常論文]
  • 網塚 憲生, 山本 知真也, 本郷 裕美, 邱 紫せん, 長谷川 智香 日本骨形態計測学会雑誌 28 (2) S89 -S89 2018年05月 [査読無し][通常論文]
  • 網塚 憲生, 邱 紫璇, 山本 知真也, 長谷川 智香 バイオマテリアル = Journal of Japanese Society for Biomaterials : 生体材料 36 (1) 34 -37 2018年01月 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香 日本補綴歯科学会誌(Web) 10 85 (WEB ONLY) 2018年 [査読無し][通常論文]
  • 長谷川智香, 宮本幸奈, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2018 ROMBUNNO.UDS09‐1 (WEB ONLY) 2018年 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香 日本骨代謝学会学術集会プログラム抄録集 36th 119 2018年 [査読無し][通常論文]
  • 前壮功仁, 前壮功仁, 山本知真也, 趙申, 邱紫せん, 長谷川智香, 山崎裕, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 123rd 178 2018年 [査読無し][通常論文]
  • 吉野弘菜, 吉野弘菜, 長谷川智香, 邱紫せん, 坪井香奈子, 本郷裕美, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 123rd 179 2018年 [査読無し][通常論文]
  • 佐藤大, 高畑雅彦, 太田昌博, 清水智弘, 福田千恵, 津田英資, 濱野博基, 平塚重人, 藤田諒, 網塚憲生, 長谷川智香, 岡田顕子, 岩崎倫政 北海道整形災害外科学会 135th (135th suppl) 21 -21 2018年 [査読無し][通常論文]
  • 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 123rd 98 2018年 [査読無し][通常論文]
  • 網塚 憲生, 邱 紫せん, 山本 知真也, 長谷川 智香 バイオマテリアル-生体材料- 36 (1) 34 -37 2018年01月 [査読無し][通常論文]
     
    骨再生を誘導するバイオマテリアルには骨誘導能あるいは骨伝導能を有するものがあり、骨欠損の大きさや形状、また、欠損部の微細環境に適応した様々なバイオマテリアルが開発されている。一方で、骨形成には、骨の形づくりであるモデリングと骨の置換えである骨リモデリングがある。骨を再生する場合、破骨細胞と骨芽細胞との細胞学的カップリングを基盤とした骨リモデリングによって、幼弱骨から緻密骨へと成熟させてゆく必要がある。再生骨の最終的なゴールは、力学負荷の感知や骨強度を備えた成熟骨の形成にあると思われる。(著者抄録)
  • 坪井 香奈子, 長谷川 智香, 邱 紫せん, 網塚 憲生 THE BONE 31 (4) 345 -350 2018年01月 [査読無し][通常論文]
  • 長谷川 智香, 宮本 幸奈, 邱 紫璇, 阿部 未来, 本郷 裕美, 網塚 憲生 日本骨粗鬆症学会雑誌 = The journal of Japan Osteoporosis Society 4 (3) 394 -396 2018年 [査読無し][通常論文]
  • 網塚 憲生 日本骨粗鬆症学会雑誌 3 (4) 427 -430 2017年11月 [査読無し][通常論文]
     
    骨粗鬆症と関連する歯科領域の最新の研究を取り上げた。本稿では以下の項目で述べた。1)歯周組織の再生、2)歯科領域の細胞生物学、3)薬剤関連顎骨壊死。歯科領域における骨再生は、複雑な形状を示す歯槽骨が足場となること、その一方で上皮組織の侵入や口腔内細菌からの感染を防ぐ必要があることから、さまざまな組織再生技術が開発されてきた。近年では、患者の自家血からplatelet-rich plasma(PRP)を作成し、それを抜歯窩に充填することで抜歯窩の治癒促進および良好な骨再生が可能となっている。また、2017年には、歯のエナメル質形成や象牙質形成にかかわる新しい因子や既知の因子の新規作用が発見されている。
  • 長谷川 智香, 網塚 憲生 Clinical Calcium 27 (12) 1713 -1722 2017年11月 [査読無し][通常論文]
     
    ヒトの発生期や成長期では骨の形づくりであるモデリングが主に認められるが,成人では骨の置き換えである骨リモデリングが優位を占める。また,モデリングは骨全体の形づくりであるマクロモデリングと顕微レベルでの形づくりであるミニモデリングに分けて考えることができる。ミニモデリングとは,破骨細胞の骨吸収に依存せず,休止期骨芽細胞が活性化し活性型骨芽細胞となって,既存骨の上に新しい骨を添加してゆく現象である。骨粗鬆症治療薬であるエルデカルシトール(ビタミンDアナログ製剤)とテリパラチド(遺伝子組換えヒト副甲状腺ホルモン1-34)はミニモデリングを誘導することが知られている。ミニモデリングを組織学的に観察すると,基質合成を盛んに行う活性型骨芽細胞が新生骨の表面に局在するが,その周囲では,前骨芽細胞の厚い細胞性ネットワークの発達が悪く,また,破骨細胞もあまり誘導されない。ここでは,骨リモデリングとモデリング・ミニモデリングにおける組織学的特徴を述べたい。(著者抄録)
  • 長谷川 智香, 網塚 憲生 THE BONE 31 (3) 239 -244 2017年10月 [査読無し][通常論文]
  • 土屋恵李佳, 土屋恵李佳, 長谷川智香, 北川善政, 網塚憲生 北海道歯学雑誌 38 (1) 59‐60 2017年09月15日 [査読無し][通常論文]
  • 八木沼 洋行, 松村 讓兒, 藤山 文乃, 中村 桂一郎, 網塚 憲生, 一條 裕之, 瀬藤 光利, 柴田 昌宏, 渡辺 雅彦 解剖学雑誌 92 2 -8 2017年09月 [査読無し][通常論文]
     
    日本解剖学会教育・若手育成委員会は、研究医の養成に関する取組の実施状況と、若手研究者のキャリアアップを解剖学会としてどのように支援すべきかについての会員の意向を知ることを目的としてアンケート調査を行った。研究医の養成に関する取組で、この5年間の変化で特筆すべきことは、希望する学生を対象とした研究医養成コースの実施が、医学部では半数以上、歯学部でも4分の1程度までに増えており、履修者も多い状況となっていることである。多くの場合、このコースの履修によって大学院の履修期間の短縮、入学金や授業料の減免、奨学会や学会参加費の支援が行われ、さらに、大学間の連携で研修やリトリートも行われている。分野別認証評価導入の影響については、過度の臨床中心のカリキュラムを懸念する回答が多かったが、良い結果となったとする回答もあった。新専門医制度の導入については、回答者の約6割が、研究医を目指す人材がさらに少なくなることを懸念していた。今後の若手育成における学会の役割については、以下のような意見が寄せられた。(1)これまでの取組の維持・発展、(2)表彰制度の拡充、(3)経済的支援や負担軽減、(4)若手間や他分野の研究者との交流促進(若手の会の結成)、(5)研究情報交流の促進、(6)教育能力向上のための各種セミナーや合宿の実施、(7)教育能力に関する認定制度の導入、(8)キャリアアップ支援の強化、(9)その他。(著者抄録)
  • 土屋 恵李佳, 長谷川 智香, 北川 善政, 網塚 憲生 北海道歯学雑誌 38 (1) 59 -60 2017年09月 [査読無し][通常論文]
     
    骨形成の機序は，骨の発生・成長の場で生じるモデリングと古い骨を新しい骨に置き換えるリモデリング（骨改造）に大別される． 成長期の長骨（長管骨）における軟骨内骨化では，血管の軟骨侵入により石灰化軟骨基質が露出し，そこに骨芽細胞が骨形成を行うといったモデリングが認められる．本稿では，軟骨内骨化のモデリングにより骨形成が誘導される領域，すなわち，骨幹端領域において，近年，注目を集めている骨特異性血管の存在や，septoclast（別名：perivascular cell）について紹介する．
  • 土屋 恵李佳, 長谷川 智香, 北川 善政, 網塚 憲生 北海道歯学雑誌 38 (1) 59 -60 2017年09月 [査読無し][通常論文]
  • Osteocyte Biologyアップデート 骨芽細胞から骨細胞へ 微細構造学的知見
    長谷川 智香, 永井 伯弥, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2017 147 -147 2017年09月 [査読無し][通常論文]
  • 骨代謝調節機構における新しい研究展開 骨基質石灰化における局所リン酸供与とFGF23/klothoシグナル
    長谷川 智香, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2017 170 -170 2017年09月 [査読無し][通常論文]
  • 骨端板に侵入する毛細血管の形態に関する走査型電子顕微鏡観察
    山本 恒之, 長谷川 智香, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2017 419 -419 2017年09月 [査読無し][通常論文]
  • 網塚 憲生 日本骨粗鬆症学会雑誌 3 (Suppl.1) 220 -220 2017年09月 [査読無し][通常論文]
  • 大宮 俊宣, 廣瀬 旬, 宮本 健史, 長谷川 智香, 網塚 憲生, 田中 栄 日本整形外科学会雑誌 91 (8) S1648 -S1648 2017年08月 [査読無し][通常論文]
  • 網塚憲生 CLINICAL CALCIUM 27 (8) 124 -125 2017年07月28日
  • 長谷川 智香, 山本 知真也, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 35回 168 -168 2017年07月 [査読無し][通常論文]
  • 島田 明美, 新井 嘉則, 小松 浩一郎, 和田 悟史, 出野 尚, 中島 和久, 山下 照仁, 江面 陽一, 網塚 憲生, 中村 芳樹, 二藤 彰 日本骨代謝学会学術集会プログラム抄録集 35回 170 -170 2017年07月 [査読無し][通常論文]
  • 山本 知真也, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 35回 175 -175 2017年07月 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香 日本透析医学会雑誌 50 (Supplement 1) 385 2017年05月28日 [査読無し][通常論文]
  • 長谷川智香, 網塚憲生 日本骨形態計測学会雑誌 27 (1) S57 -S57 2017年05月22日 [査読無し][通常論文]
  • H. Segawa, A. Onitsuka, J. Furutani, Kaneko, I, F. Aranami, N. Matsumoto, Y. Tomoe, M. Kuwahata, M. Ito, M. Matsumoto, M. Li, N. Amizuka, K. Miyamoto AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 312 (5) F848 -F848 2017年05月 [査読無し][通常論文]
  • 電子顕微鏡・光学顕微鏡による最先端・骨イメージング FIB-SEMを用いた骨の細胞群の三次元微細構造イメージング
    長谷川 智香, 網塚 憲生 日本骨形態計測学会雑誌 27 (1) S57 -S57 2017年05月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香 日本透析医学会雑誌 50 (Suppl.1) 385 -385 2017年05月 [査読無し][通常論文]
  • 工藤 愛, 長谷川 智香, 網塚 憲生 日本骨形態計測学会雑誌 27 (1) S114 -S114 2017年05月 [査読無し][通常論文]
  • 長谷川 智香, 網塚 憲生 日本骨形態計測学会雑誌 27 (1) S109 -S109 2017年05月 [査読無し][通常論文]
  • 山本 知真也, 長谷川 智香, 網塚 憲生 日本骨形態計測学会雑誌 27 (1) S99 -S99 2017年05月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 山本 知真也 日本骨形態計測学会雑誌 27 (1) S88 -S88 2017年05月 [査読無し][通常論文]
  • 長谷川 智香, 彦根 久美子, 土屋 恵李佳, 工藤 愛, 山本 恒之, 網塚 憲生 THE BONE 31 (1) 3 -8 2017年04月 [査読無し][通常論文]
  • 堀内 圭輔, 日方 智宏, 長谷川 智香, 藤田 順之, 岩波 明生, 渡邉 航太, 石井 賢, 中村 雅也, 網塚 憲生, 松本 守雄 日本整形外科学会雑誌 91 (2) S640 -S640 2017年03月 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香 日本解剖学会総会・全国学術集会講演プログラム・抄録集 122nd 2017年
  • 吉田泰士, 吉田泰士, 坪井香奈子, 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 122nd 2017年
  • 長谷川智香, 坪井香奈子, 坪井香奈子, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 122nd 2017年
  • 山本恒之, 長谷川智香, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2017 2017年
  • 工藤愛, 工藤愛, 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 122nd 2017年
  • 長谷川智香, 永井伯弥, 永井伯弥, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2017 2017年
  • 長谷川智香, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2017 2017年
  • 網塚憲生, 長谷川智香 再生医療 16 2017年
  • 永井伯弥, 永井伯弥, 長谷川智香, 横山敦郎, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 122nd 2017年
  • 横山亜矢子, 横山亜矢子, 山田珠希, 平賀徹, 長谷川智香, 山崎裕, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 122nd 162 2017年 [査読無し][通常論文]
  • 本郷裕美, 佐々木宗輝, 長谷川智香, 坪井香奈子, 網塚憲生 THE BONE 30 (4) 3 -7 2017年 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 坪井 香奈子, 網塚 憲生 THE BONE 30 (4) 303 -307 2017年01月 [査読無し][通常論文]
  • 高橋 直之, 西村 理行, 竹田 秀, 網塚 憲生, 高柳 広 THE BONE 30 (4) 389 -397 2017年01月 [査読無し][通常論文]
  • 長谷川 智香, 網塚 憲生 PAIN RESEARCH 31 (4) 210 -219 2016年12月 [査読無し][通常論文]
     
    骨組織は、骨基質や血管、神経に加えて、骨芽細胞、破骨細胞、骨細胞といった様々な細胞から構成される複雑な組織である。近年、骨組織に存在する神経が、これら骨の細胞群の機能を調節する可能性が示唆されており、骨に分布する神経は多彩な機能を有していると考えられる。骨に存在する細胞群の構造と役割を中心に概説すると共に、現在考えられている骨痛に対する考察、そして、骨に分布する感覚神経が骨の細胞群に与える影響について概説した。
  • 戸井田 侑, 川野 晋平, 丁 世俊, 長谷川 智香, チョウドリ・アルマス, サイケオ・ピポップ, アハメッド・ズバエル, 成徳 英理, 勝俣 愛一郎, 福澤 尚幸, 松本 真理子, 角田 晋一, 星加 修平, 池田 考績, 田中 享, 島田 康史, 田上 順次, 網塚 憲生, 吉田 靖弘, 佐野 英彦 特定非営利活動法人日本歯科保存学会学術大会プログラムおよび講演抄録集 145回 24 -24 2016年10月 [査読無し][通常論文]
  • 八木沼 洋行, 松村 讓兒, 佐藤 洋一, 飯野 哲, 鶴尾 吉宏, 大和田 祐二, 藤山 文乃, 網塚 憲生, 日本解剖学会教育・若手育成委員会 解剖学雑誌 91 (4) 33 -40 2016年09月 [査読無し][通常論文]
     
    日本解剖学会教育・若手育成委員会は、(1)肉眼解剖学教育の現状と課題の把握、(2)肉眼解剖学教育における教育効果を高めるための取組に関する情報交換の促進を目的として、平成25年9月にアンケート調査を行った。肉眼解剖学の授業と実習は、多くの大学で第2学年に行われており、肉眼解剖学実習の平均回数は医学部で34.6回、歯学部で27.4回、正規の平均総時間数は125時間と97時間であった。ただし、8割の大学で正規時間だけでは足らず、実習時間の相当の延長を行っていた。教育認証制度の導入に対応するためのカリキュラム改訂が進んでいるが、今のところ、実習時期が早くなる以外の影響は少ないという結果であった。医学部の入学定員増の影響として、教員増なしの教育負担増による研究等への影響を心配する声が多かった。また、設備や献体数の制約から一体あたりの学生数を増やさざるを得なかったという回答もあった。教育上有効と思われる取組として、教材や剖出手順の工夫、臨床医による関連の講義や実習指導の導入、高学年での2回目の解剖学実習の採用など、他の参考となる多数の実践例が寄せられた。自由意見として、解剖学教育を担う若手人材の育成システムの必要性、教育に関する情報交換の機会の重要性を訴える声が多かった。(著者抄録)
  • II型糖尿病モデルSDT fattyラットにおける下顎骨歯周組織の組織化学的検索
    吉田 泰士, 本郷 裕美, 坪井 香奈子, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2016 418 -418 2016年09月 [査読無し][通常論文]
  • c-fos遺伝子欠損マウスの骨細胞における微細構造学的検索
    阿部 未来, 長谷川 智香, 山本 知真也, 土屋 恵李佳, 宇田川 信之, 網塚 憲生 Journal of Oral Biosciences Supplement 2016 421 -421 2016年09月 [査読無し][通常論文]
  • 骨組織におけるFGF23産生細胞の経時的局在変化について
    櫻井 敦中, 長谷川 智香, 山本 知真也, 佐野 英彦, 網塚 憲生 Journal of Oral Biosciences Supplement 2016 480 -480 2016年09月 [査読無し][通常論文]
  • オスミウム浸軟法による破骨細胞のゴルジ装置の立体微細構造および分布に関する走査型電子顕微鏡観察
    山本 恒之, 坪井 香奈子, 長谷川 智香, 本郷 裕美, 網塚 憲生 Journal of Oral Biosciences Supplement 2016 492 -492 2016年09月 [査読無し][通常論文]
  • 八木沼 洋行, 松村 讓兒, 佐藤 洋一, 飯野 哲, 鶴尾 吉宏, 大和田 祐二, 藤山 文乃, 網塚 憲生 解剖学雑誌 = Acta anatomica Nipponica 91 (4) 33 -40 2016年09月 [査読無し][通常論文]
  • 網塚 憲生 日本整形外科学会雑誌 90 (8) S1439 -S1439 2016年08月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香 日本整形外科学会雑誌 90 (8) S1653 -S1653 2016年08月 [査読無し][通常論文]
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 坪井 香奈子, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 34回 184 -184 2016年07月 [査読無し][通常論文]
  • 土屋 恵李佳, 本郷 裕美, 山本 知真也, 長谷川 智香, 宇田川 信之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 34回 186 -186 2016年07月 [査読無し][通常論文]
  • 清水 智弘, 高畑 雅彦, 山田 悟史, 山本 知真也, 長谷川 智香, 濱野 博基, 太田 昌博, 平塚 重人, 亀田 裕亮, 但野 茂, 網塚 憲生, 岩崎 倫政 日本骨代謝学会学術集会プログラム抄録集 34回 208 -208 2016年07月 [査読無し][通常論文]
  • 島田 明美, 新井 嘉則, 和田 悟史, 出野 尚, 中島 和久, 小松 浩一郎, 山下 照仁, 江面 陽一, 網塚 憲生, 中村 芳樹, 二藤 彰 日本骨代謝学会学術集会プログラム抄録集 34回 214 -214 2016年07月 [査読無し][通常論文]
  • 山本 知真也, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 34回 216 -216 2016年07月 [査読無し][通常論文]
  • 坪井 香奈子, 本郷 裕美, 山本 知真也, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 34回 218 -218 2016年07月 [査読無し][通常論文]
  • 吉野 寧維, 半田 佐保子, 四馬田 恵, 鈴木 敦詞, 長谷川 智香, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 34回 219 -219 2016年07月 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 斎藤 雅美, 宇田川 信之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 34回 228 -228 2016年07月 [査読無し][通常論文]
  • 長谷川 智香, 網塚 憲生 PAIN RESEARCH 31 (2) 52 -52 2016年06月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香 日本骨形態計測学会雑誌 26 (1) S71 -S71 2016年06月 [査読無し][通常論文]
  • 山本 知真也, 長谷川 智香, 小守 壽文, 網塚 憲生 日本骨形態計測学会雑誌 26 (1) S115 -S115 2016年06月 [査読無し][通常論文]
  • 土屋 恵李佳, 本郷 裕美, 山本 知真也, 長谷川 智香, 北川 善政, 網塚 憲生 日本骨形態計測学会雑誌 26 (1) S123 -S123 2016年06月 [査読無し][通常論文]
  • 清水 智弘, 高畑 雅彦, 山田 悟史, 山本 知真也, 長谷川 智香, 亀田 裕亮, 太田 昌博, 但野 茂, 網塚 憲生, 岩崎 倫政 日本骨形態計測学会雑誌 26 (1) S157 -S157 2016年06月 [査読無し][通常論文]
  • 長谷川智香, 土屋恵李佳, 阿部未来, 網塚憲生 CLINICAL CALCIUM 26 (5) 25 -30 2016年04月28日
  • 網塚憲生 CLINICAL CALCIUM 26 (5) 763 -763 2016年04月28日
  • 網塚憲生 CLINICAL CALCIUM 26 (5) 111 -111 2016年04月28日
  • 長谷川 智香, 土屋 恵李佳, 阿部 未来, 網塚 憲生 Clinical Calcium 26 (5) 677 -682 2016年04月 [査読無し][通常論文]
     
    骨の長軸方向の成長は軟骨内骨化によって成し遂げられ、そこには、血管内皮細胞を中心とした複雑な細胞連関や微細環境が形成されている。血管内皮細胞は石灰化軟骨基質の間に存在する未石灰化横隔壁を壊しながら進むことで、石灰化軟骨基質が露出し、その表面に骨芽細胞が骨基質合成を行う。骨・軟骨移行部では、マクロファージではなくperivascular cellが存在し、細胞・基質の残渣を処理すると考えられている。さらに、この部位では破骨細胞から骨芽細胞へのカップリングを基盤とした骨リモデリングではなく、モデリングによって骨芽細胞が新生骨を形成している。近年、骨に特異的な血管のサブタイプが報告されている。それは、CD31hi Emcnhiを示すtype H血管であり、その周囲にosterix陽性の骨原性細胞やPDGFRβ(platelet-derived growth factor receptor β)陽性間葉系細胞が近接しており、type H血管は血管新生能力を有しているという。このtype H血管は、Hif1αがその形成に重要な因子であることを推察している。ここでは、特に軟骨内骨化における血管内皮細胞の組織学的・微細構造学的特徴と、近年の報告をご紹介する。(著者抄録)
  • 宮治裕史, 古月文志, 加藤昭人, 小川幸佑, 村上秀輔, 西田絵利香, 宮田さほり, 川本康平, 滝田裕子, 岩永敏彦, 本郷裕美, 網塚憲生, 川浪雅光 再生医療 15 297 2016年02月01日 [査読無し][通常論文]
  • 網塚憲生 超音波骨折治療研究会プログラム・抄録集 19th 2016年
  • 網塚憲生, 長谷川智香, 本郷裕美 日本解剖学会総会・全国学術集会講演プログラム・抄録集 121st 2016年
  • 長谷川智香, 佐々木宗輝, 山本知真也, 本郷裕美, 坪井香奈子, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 121st 2016年
  • 阿部未来, 本郷裕美, 山本知真也, 長谷川智香, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 121st 2016年
  • 山本恒之, 坪井香奈子, 長谷川智香, 本郷裕美, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2016 2016年
  • 櫻井敦中, 櫻井敦中, 長谷川智香, 山本知真也, 佐野英彦, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2016 2016年
  • 吉田泰士, 本郷裕美, 坪井香奈子, 長谷川智香, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2016 2016年
  • 阿部未来, 長谷川智香, 山本知真也, 土屋恵李佳, 宇田川信之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2016 2016年
  • 網塚憲生 日本腎性骨症研究会プログラム抄録集 2016 2016年
  • 長谷川 智香, 網塚 憲生 PAIN RESEARCH 31 (4) 210 -219 2016年 [査読無し][通常論文]
     
    <p>Bone is a supporting tissue consisting of a variety of bone cells (osteo blasts,osteoclasts, osteocytes), calcified bone matrix, blood vessels, nerves and so forth.<b> </b>Bone diseases — osteoporosis, bone fracture, arthritis, bone metastasis — often induce bone pain, presumably due to various kinds of nociceptors linked to the signaling in sensory nerves.<b> </b>Nociceptive pain in bone is caused by several stimuli of acid secreted by osteoclasts, physiochemical damaging on peripheral nerves, ATPs, prostaglandins, and so forth.<b> </b>However, precious knowledge on the biological function of bone cells may provide a clue for better understanding on the cellular mechanism of bone pain.<b> </b>In a physiological state, normal bone is always remodeled by balanced osteoclastic bone resorption and subsequent osteoblastic bone formation.<b> </b>Osteocytes connect to neighboring osteocytes and to osteoblasts on the bone surface by means of thin cytoplasmic processes that go through narrow passageways. <i>i.e., </i>osteocytic canaliculi, and thereby building functional syncytia referred to as osteocytic lacunar–canalicular system.<b> </b>Thus, osteocytes are at the center of bone turnover's mainframe.<b> </b>In this review, we will introduce histological and ultrastructural aspects of bone cells, and give a rough outline of bone pain.</p>
  • 網塚 憲生, 本郷 裕美, 坪井 香奈子, 長谷川 智香, 山本 知真也, 原口 真衣 顕微鏡 50 (3) 191 -196 2015年12月 [査読無し][通常論文]
     
    骨組織に存在する骨芽細胞はコラーゲン分泌とリン酸カルシウム結晶の沈着を行うことで石灰化骨基質合成を行う細胞である。しかし、骨芽細胞は基質合成を行うとともに、自らは骨細胞に分化して骨基質の中に埋め込まれてゆく。骨芽細胞と骨細胞は互いに細胞突起を介した細胞性ネットワークを形成し、グループとして機能すると考えられている。破骨細胞は骨吸収を行う多核巨細胞であり、波状縁から酸と基質分解酵素を分泌して骨基質を吸収している。破骨細胞は古い骨あるいは脆弱な骨を認識して吸収すると考えられており、その後、骨芽細胞が新しい骨を添加させてゆく。このように、骨は絶えず、破骨細胞により吸収され、また、骨芽細胞により新しく形成されているが、この新旧の骨基質の置換を骨リモデリング(骨改造)という。骨リモデリングは、破骨細胞と骨芽細胞系細胞との細胞学的カップリングを基盤としている。(著者抄録)
  • 網塚憲生 CLINICAL CALCIUM 25 (10) 21 -21 2015年09月28日
  • 骨の細胞イメージング-a new horizon-骨のメカニズムをどこまで観ることができるか オーバービュー 骨を観る 組織化学・微細構造学からバイオイメージングへの展開
    網塚 憲生, 長谷川 智香, 坪井 香奈子, 本郷 裕美, 原口 真衣, 山本 知真也 Journal of Oral Biosciences Supplement 2015 98 -98 2015年09月 [査読無し][通常論文]
  • 骨の細胞イメージング-a new horizon-骨のメカニズムをどこまで観ることができるか SIM・FIB-SEM・原子間力顕微鏡・同位体顕微鏡を用いた骨組織解析への応用
    長谷川 智香, 坪井 香奈子, 本郷 裕美, 山本 知真也, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 99 -99 2015年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン投与ならびに授乳期にカルシウム欠乏食投与によって誘導される骨細胞周囲の骨基質変化について
    本郷 裕美, 佐々木 宗輝, 斎藤 雅美, 宇田川 信之, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 171 -171 2015年09月 [査読無し][通常論文]
  • 軟骨内骨化における血管内皮細胞の微細構造学的検索
    土屋 恵李佳, 本郷 裕美, 山本 知真也, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 172 -172 2015年09月 [査読無し][通常論文]
  • 糖尿病が骨細胞に与える組織化学的影響について SDT fattyラットを用いた解析
    坪井 香奈子, 本郷 裕美, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 173 -173 2015年09月 [査読無し][通常論文]
  • PTHの投与頻度の違いによるリモデリング・ミニモデリングを基盤とした骨形成
    山本 知真也, 長谷川 智香, 本郷 裕美, 原口 真衣, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 199 -199 2015年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン投与ならびに授乳期にカルシウム欠乏食投与によって誘導される骨細胞周囲の骨基質変化について
    本郷 裕美, 佐々木 宗輝, 斎藤 雅美, 宇田川 信之, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 307 -307 2015年09月 [査読無し][通常論文]
  • 糖尿病が骨細胞に与える組織化学的影響について SDT fattyラットを用いた解析
    坪井 香奈子, 本郷 裕美, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 308 -308 2015年09月 [査読無し][通常論文]
  • 土屋 恵李佳, 本郷 裕美, 山本 知真也, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 311 -311 2015年09月 [査読無し][通常論文]
  • 骨芽細胞特異的副甲状腺ホルモン関連ペプチド(PTHrP)トランスジェニックマウスにおける歯胚および歯槽骨の組織化学的検索
    山崎 七愛, 山本 知真也, 長谷川 智香, 本郷 裕美, 原口 真衣, 織田 公光, 小守 壽文, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 538 -538 2015年09月 [査読無し][通常論文]
  • c-fos遺伝子欠損マウスにおける骨芽細胞・骨細胞の微細構造学的検索
    阿部 未来, 本郷 裕美, 山本 知真也, 長谷川 智香, 網塚 憲生 Journal of Oral Biosciences Supplement 2015 542 -542 2015年09月 [査読無し][通常論文]
  • 本郷 裕美, 坂井 貞興, 山本 知真也, 長谷川 智香, 武田 聡, 遠藤 弘一, 斎藤 一史, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 33回 177 -177 2015年07月 [査読無し][通常論文]
  • 本郷 裕美, 斎藤 雅美, 宇田川 信之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 33回 167 -167 2015年07月 [査読無し][通常論文]
  • 島田 明美, 新井 嘉則, 和田 悟史, 出野 尚, 上運天 太一, 中島 和久, 小松 浩一郎, 山下 照仁, 江面 陽一, 網塚 憲生, 中村 芳樹, 二藤 彰 日本骨代謝学会学術集会プログラム抄録集 33回 168 -168 2015年07月 [査読無し][通常論文]
  • 山本 知真也, 長谷川 智香, 山崎 七愛, 織田 公光, 小守 壽文, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 33回 174 -174 2015年07月 [査読無し][通常論文]
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 坪井 香奈子, 山本 恒之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 33回 212 -212 2015年07月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香 日本医師会雑誌 144 (特別1) S50 -S52 2015年06月 [査読無し][通常論文]
  • 網塚 憲生, 山本 知真也, 本郷 裕美, 長谷川 智香 日本骨形態計測学会雑誌 25 (2) S47 -S47 2015年05月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 25 (2) S65 -S65 2015年05月 [査読無し][通常論文]
  • 土屋 恵李佳, 本郷 裕美, 山本 知真也, 長谷川 智香, 原口 真衣, 北川 善政, 網塚 憲生 日本骨形態計測学会雑誌 25 (2) S72 -S72 2015年05月 [査読無し][通常論文]
  • 坂井 貞興, 本郷 裕美, 山本 知真也, 長谷川 智香, 武田 聡, 松本 義弘, 斎藤 一史, 網塚 憲生, 遠藤 弘一 日本骨形態計測学会雑誌 25 (2) S81 -S81 2015年05月 [査読無し][通常論文]
  • ラット上顎の抜歯即時埋入後早期荷重を行ったチタンインプラント周囲骨の組織学的検索
    池田 欣希, 長谷川 智香, 山内 貴紀子, 網塚 憲生, 横山 敦郎 日本補綴歯科学会誌 7 (特別号) 229 -229 2015年05月 [査読無し][通常論文]
  • 長谷川智香, 山本知真也, 本郷裕美, 坪井香奈子, 網塚憲生 CLINICAL CALCIUM 25 (5) 69 -75 2015年04月28日
  • 長谷川 智香, 山本 知真也, 本郷 裕美, 坪井 香奈子, 網塚 憲生 Clinical Calcium 25 (5) 693 -699 2015年04月 [査読無し][通常論文]
     
    klotho遺伝子欠損(kl/kl)マウスは、FGF23/klotho軸の破綻により著明な高リン・高カルシウム血症を呈し、動脈中膜にメンケベルグ型血管石灰化が生じるモデル動物である。近年、中膜石灰化の病理機序として、血管平滑筋細胞内にリンが流入することにより、血管平滑筋細胞が骨芽細胞の様々な表現型を獲得し、生物学的に石灰化が生じる可能性が示唆されている。血管石灰化の病理機序の詳細を明らかにすることは、本疾患の治療・予防法の開発に広く応用できると考えられる。本稿では、kl/klマウスの大動脈でみられる血管石灰化について組織学的・微細構造学的な知見を紹介する。(著者抄録)
  • 小島拓, 芳澤享子, 小野由起子, 坂上直子, 齋藤直朗, 長谷川智香, 網塚憲生, 前田健康, 小林正治 日本歯科医学会誌 34 105 2015年03月31日 [査読無し][通常論文]
  • 網塚 憲生 日本歯科理工学会誌 34 (2) 64 -64 2015年03月25日 [査読無し][通常論文]
  • 山本 恒之, 山本 知真也, 本郷 裕美, 長谷川 智香, 網塚 憲生 THE BONE 29 (1) 3 -7 2015年03月 [査読無し][通常論文]
  • 網塚 憲生 日本歯科理工学会誌 34 (2) 64 -64 2015年03月 [査読無し][通常論文]
  • 坪井 香奈子, 北川 善政, 網塚 憲生 北海道歯学雑誌 35 (2) 176 -178 2015年03月 [査読無し][通常論文]
  • 本郷裕美, 佐々木宗輝, 斎藤雅美, 宇田川信之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 阿部未来, 本郷裕美, 山本知真也, 長谷川智香, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 長谷川智香, 坪井香奈子, 本郷裕美, 山本知真也, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 土屋恵李佳, 土屋恵李佳, 本郷裕美, 山本知真也, 長谷川智香, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 坪井香奈子, 本郷裕美, 長谷川智香, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 山本知真也, 長谷川智香, 本郷裕美, 原口真衣, 織田公光, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 池田欣希, 長谷川智香, 山内貴紀子, 網塚憲生, 横山敦郎 日本補綴歯科学会誌(Web) 7 2015年
  • 山崎七愛, 山本知真也, 長谷川智香, 本郷裕美, 原口真衣, 織田公光, 小守壽文, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2015 2015年
  • 骨微細構造解析に骨組織検査は必要かHistomorphometryの利点(vs Imaging)．
    網塚憲生, 李 敏啓, 長谷川智香, 山本恒之 Calcium Pros and Cons～カルシウム代謝研究・議論の変遷～ 35 -39 2015年 [査読無し][通常論文]
  • 矢島 愛治, 土谷 健, 稲葉 雅章, 冨永 芳博, 荒井 純子, 浅宮 由香理, 小俣 百世, 松上 桂子, 網塚 憲生, 伊藤 明美, 進藤 廣成, 新田 孝作 腎と骨代謝 28 (1) 63 -67 2015年01月 [査読無し][通常論文]
     
    慢性腎臓病では,40年前から骨小腔の著明な形態学的変化が論文化されたが,その後さらなる検討は成されていなかった.2000年頃より骨細胞の詳細な研究はProfessor Lynda F. Bonewaldにより成され,この10年間における骨細胞研究はめざましいものがある.しかしながら,慢性腎臓病領域ではいまだにSherrardの分類が使用され骨細胞そのものの研究は進んでおらず,ヒト慢性腎臓病の英論文はMEDLINE上で検索したかぎりわずかに2編のみである.一方,骨細胞が発現するFGF-23, sclerostin, RANKLの研究はすさまじいものがあり,今後の慢性腎臓病領域の骨細胞研究において心強いかぎりである.本論文では,論文化された慢性腎臓病領域の骨細胞変化につき最近のtopicを交えて述べる.(著者抄録)
  • 田幡 千尋, 佐々木 宗輝, 工藤 明, 本郷 裕美, 長谷川 智香, 山本 知真也, 網塚 憲生 THE BONE 28 (3) 237 -243 2014年09月 [査読無し][通常論文]
  • アレンドロネート連日投与中・投与中止後における骨の細胞群における組織化学的変化
    坪井 香奈子, 佐々木 宗輝, 長谷川 智香, 虎谷 彌, 織田 公光, 北川 善政, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 106 -106 2014年09月 [査読無し][通常論文]
  • ミノドロン酸の同位体顕微鏡を用いた骨組織分布の観察と骨の細胞群に対する影響(Localization of 15N-minodronate by isotope microscopy and histochemical assessment for the biological effects of minodronate in mice)
    佐々木 宗輝, 本郷 裕美, 小林 幸雄, 圦本 尚義, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 106 -106 2014年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン(PTH)間歇投与の投与量・投与頻度の違いによる骨の細胞群の変化について
    山本 知真也, 長谷川 智香, 佐々木 宗輝, 虎谷 彌, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 108 -108 2014年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン投与ならびに授乳期・カルシウム欠乏食で誘導される骨細胞周囲の骨基質解析について
    本郷 裕美, 佐々木 宗輝, 斎藤 雅美, 虎谷 彌, 宇田川 信之, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 108 -108 2014年09月 [査読無し][通常論文]
  • 抜歯即時埋入後早期咬合負荷を与えたチタンインプラント周囲骨組織の組織学的検索(Histological examinations on bone tissue around titanium implants bearing early occlusal loading after their implantation)
    池田 欣希, 長谷川 智香, 織田 公光, 網塚 憲生, 横山 敦郎 Journal of Oral Biosciences Supplement 2014 122 -122 2014年09月 [査読無し][通常論文]
  • αklotho-/-マウスおよびkl/klマウスの下顎臼歯部歯周組織における組織化学的解析
    彦根 久美子, 長谷川 智香, 佐々木 宗輝, 本郷 裕美, 土屋 恵李佳, 虎谷 彌, 織田 公光, 飯田 順一郎, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 131 -131 2014年09月 [査読無し][通常論文]
  • 骨組織における組織非特異型アルカリフォスファターゼ(TNALP)とENPP1の免疫組織化学的解析
    小林 博和, 本郷 裕美, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 138 -138 2014年09月 [査読無し][通常論文]
  • アレンドロネート連日投与中・投与中止後における骨の細胞群における組織化学的変化
    坪井 香奈子, 佐々木 宗輝, 長谷川 智香, 虎谷 彌, 織田 公光, 北川 善政, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 139 -139 2014年09月 [査読無し][通常論文]
  • FIB-SEMを用いた骨細胞・骨細管系の3次元微細構造解析(The analysis of three-dimensional ultrastructure of osteocyticlacunar-canalicular system(OLCS) by using FIB-SEM)
    長谷川 智香, 山本 知真也, 虎谷 彌, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 141 -141 2014年09月 [査読無し][通常論文]
  • ミノドロン酸の同位体顕微鏡を用いた骨組織分布の観察と骨の細胞群に対する影響(Localization of 15N-minodronate by isotope microscopy and histochemical assessment for the biological effects of minodronate in mice)
    佐々木 宗輝, 本郷 裕美, 小林 幸雄, 圦本 尚義, 網塚 憲生 Journal of Oral Biosciences Supplement 2014 144 -144 2014年09月 [査読無し][通常論文]
  • 矢島 愛治, 土谷 健, 伊藤 明美, 網塚 憲生, 新田 孝作, 進藤 廣成 Osteoporosis Japan 22 (Suppl.1) 159 -159 2014年09月 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 斎藤 雅美, 虎谷 彌, 宇田川 信之, 網塚 憲生 Osteoporosis Japan 22 (Suppl.1) 230 -230 2014年09月 [査読無し][通常論文]
  • 坂井 貞興, 武田 聡, 位高 美香, 山本 知真也, 長谷川 智香, 網塚 憲生, 遠藤 弘一 Osteoporosis Japan 22 (Suppl.1) 337 -337 2014年09月 [査読無し][通常論文]
  • 山本 知真也, 長谷川 智香, 坂井 貞興, 武田 聡, 本郷 裕美, 織田 公光, 李 敏啓, 遠藤 弘一, 網塚 憲生 Osteoporosis Japan 22 (Suppl.1) 337 -337 2014年09月 [査読無し][通常論文]
  • レプチン受容体遺伝子変異(db/db)マウスの卵巣摘出(OVX)後における骨組織の組織化学的解析
    田中 祐介, 鄭 漢忠, 網塚 憲生 日本口腔科学会雑誌 63 (4) 353 -353 2014年09月 [査読無し][通常論文]
  • 矢島 愛治, 網塚 憲生, 土谷 健, 新田 孝作, 進藤 廣成, 伊藤 明美 透析ケア 20 (7) 626 -628 2014年07月 [査読無し][通常論文]
  • 矢島 愛治, 網塚 憲生, 土谷 健, 新田 孝作, 進藤 廣成, 伊藤 明美 透析ケア 20 (7) 629 -631 2014年07月 [査読無し][通常論文]
  • 網塚 憲生, 本郷 裕美, 坪井 香奈子, 山本 知真也, 長谷川 智香 日本骨代謝学会学術集会プログラム抄録集 32回 163 -163 2014年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 32回 193 -193 2014年07月 [査読無し][通常論文]
  • 長谷川 智香, 山本 知真也, 織田 公光, 斎藤 一史, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 32回 221 -221 2014年07月 [査読無し][通常論文]
  • 柴 祥子, 東 浩太郎, 長谷川 智香, 池田 和博, 堀江 公仁子, 網塚 憲生, 井上 聡 日本骨代謝学会学術集会プログラム抄録集 32回 222 -222 2014年07月 [査読無し][通常論文]
  • 董 冰子, 遠藤 逸朗, 大西 幸代, 近藤 剛史, 安倍 正博, 福本 誠二, 長谷川 智香, 網塚 憲生, 相澤 慎一, 松本 俊夫 日本骨代謝学会学術集会プログラム抄録集 32回 223 -223 2014年07月 [査読無し][通常論文]
  • 山本 知真也, 田中 祐介, 長谷川 智香, 坂井 貞興, 武田 聡, 本郷 裕美, 佐々木 宗輝, 織田 公光, 李 敏啓, 遠藤 弘一, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 32回 264 -264 2014年07月 [査読無し][通常論文]
  • 坪井 香奈子, 佐々木 宗輝, 織田 公光, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 32回 293 -293 2014年07月 [査読無し][通常論文]
  • 網塚 憲生, 山本 知真也, 長谷川 智香, 本郷 裕美 THE BONE 28 (2) 143 -152 2014年06月 [査読無し][通常論文]
     
    電子顕微鏡は真空中の電子線を利用する顕微鏡であり、最も分解能が優れた観察機器である。電子顕微鏡から得られる情報は、微細構造、元素分布、特定の蛋白や酵素活性の微細局在、さらには細胞機能にまで及ぶ。光学顕微鏡により、組織切片のさまざまなものを可視化する方法を組織化学と呼び、酵素抗体法、免疫組織化学、in situハイブリダイゼーション、オートラジオグラフィなどがあげられる。このような各種の組織化学は電子顕微鏡にも応用することができる。本稿では、光学顕微鏡の組織化学を概説した後、電子顕微鏡の種類と用途、特に一般の微細構造だけでなく免疫電顕や電顕オートラジオグラフィといった応用手法について、筆者らの所見をご紹介する。(著者抄録)
  • 矢島 愛治, 進藤 廣成, 長谷川 智香, 稲葉 雅章, 冨永 芳博, 池田 恭治, 西澤 良記, 土谷 健, 新田 孝作, 網塚 憲生, 伊藤 明美 日本骨形態計測学会雑誌 24 (2) S125 -S125 2014年05月 [査読無し][通常論文]
  • 網塚 憲生, 山本 知真也, 長谷川 智香, 佐々木 宗輝 実験医学 32 (7) 1044 -1053 2014年05月 [査読無し][通常論文]
     
    前骨芽細胞、骨芽細胞、骨細胞といった骨形成系細胞は1つの分化の流れに存在しているが、それぞれ特有の機能をもって骨代謝に参画している。前骨芽細胞は骨芽細胞の前駆細胞である一方、破骨細胞の分化形成に関与しており、副甲状腺ホルモン(PTH)の間歇投与により細胞増殖が亢進するが、PTH間歇投与の方法によっては、前骨芽細胞の増加や骨基質のでき方が異なる。また、骨芽細胞は破骨細胞とのカップリングを基盤にした骨リモデリングを行っているが、生体内ではミニモデリングで誘導される骨形成もあり、骨粗鬆症治療薬であるエルデカルシトールは骨芽細胞に対してミニモデリングを効率よく誘導させると考えられる。骨芽細胞から分化した骨細胞は、骨芽細胞が形成した石灰化骨基質を維持したり、また、微細環境によっては溶解する機能が備わっていると推察される。(著者抄録)
  • 坂井 貞興, 武田 聡, 山本 知真也, 長谷川 智香, 網塚 憲生, 遠藤 弘一 日本骨形態計測学会雑誌 24 (2) S87 -S87 2014年05月 [査読無し][通常論文]
  • 齋藤 直朗, 李 敏啓, 辰巳 佐和子, 池田 恭治, 網塚 憲生, 小林 正治 日本骨形態計測学会雑誌 24 (2) S100 -S100 2014年05月 [査読無し][通常論文]
  • 山本 恒之, 長谷川 智香, 山本 知真也, 本郷 裕美, 網塚 憲生 日本骨形態計測学会雑誌 24 (2) S102 -S102 2014年05月 [査読無し][通常論文]
  • 長谷川 智香, 山本 知真也, 織田 公光, 齋藤 一史, 網塚 憲生 日本骨形態計測学会雑誌 24 (2) S108 -S108 2014年05月 [査読無し][通常論文]
  • 山本 知真也, 田中 祐介, 長谷川 智香, 坂井 貞興, 武田 聡, 本郷 裕美, 佐々木 宗輝, 織田 公光, 遠藤 弘一, 網塚 憲生 日本骨形態計測学会雑誌 24 (2) S109 -S109 2014年05月 [査読無し][通常論文]
  • 佐々木 宗輝, 本郷 裕美, 小林 幸雄, 織田 公光, 圦本 尚義, 網塚 憲生 日本骨形態計測学会雑誌 24 (2) S112 -S112 2014年05月 [査読無し][通常論文]
  • 坪井 香奈子, 佐々木 宗輝, 織田 公光, 北川 善政, 網塚 憲生 日本骨形態計測学会雑誌 24 (2) S114 -S114 2014年05月 [査読無し][通常論文]
  • 山本 知真也, 佐々木 宗輝, 長谷川 智香, 織田 公光, 網塚 憲生 日本骨形態計測学会雑誌 24 (2) S115 -S115 2014年05月 [査読無し][通常論文]
  • 道家 孝幸, 阿部 恭久, 金谷 久美子, 佐々木 宗輝, 網塚 憲生, 山下 敏彦 日本骨形態計測学会雑誌 24 (2) S118 -S118 2014年05月 [査読無し][通常論文]
  • 遠藤 逸朗, 董 冰子, 長谷川 智香, 網塚 憲生, 福本 誠二, 松本 俊夫 日本内分泌学会雑誌 90 (1) 219 -219 2014年04月 [査読無し][通常論文]
  • 網塚憲生, 山本知真也, 長谷川智香 CLINICAL CALCIUM 24 (4) 13 -23 2014年03月28日
  • 網塚 憲生, 山本 知真也, 長谷川 智香 Clinical Calcium 24 (4) 475 -485 2014年03月 [査読無し][通常論文]
     
    骨の微細環境は、骨リモデリングの代謝回転の程度によって異なる組織像を示す。骨リモデリングが活発に行われている部位では、活性型骨芽細胞、活発に骨吸収を行う破骨細胞、厚い前骨芽細胞層が発達するが、骨リモデリングがあまり行われない部位では、骨芽細胞は扁平化し休止期骨芽細胞として骨表面を覆っている。そこでは前骨芽細胞の細胞層はあまり発達せず、破骨細胞前駆細胞の分布も少ないが、骨基質内部には骨細胞が幾何学的に整然と配列する傾向が認められる。このような骨の微細環境は、骨芽細胞や破骨細胞ばかりでなく、前骨芽細胞、骨髄間質細胞、血管内皮細胞などによっても調節されている。しかし、前骨芽細胞は様々な表現型が報告されており、多くの機能を有すると思われる。ここでは、骨の微細環境を形成する細胞群について組織学的・微細構造学的な知見を紹介する。(著者抄録)
  • 佐々木 宗輝, 本郷 裕美, 山本 知真也, 長谷川 智香, 網塚 憲生 THE BONE 28 (1) 7 -12 2014年02月 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香, 山本知真也, 織田公光 CLINICAL CALCIUM 24 (2) 47 -58 2014年01月28日
  • 織田公光, 沼-金城奈津子, 相田美和, 小丸圭一, 網塚憲生 CLINICAL CALCIUM 24 (2) 77 -83 2014年01月28日
  • 小林博和, 本郷裕美, 織田公光, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2014 2014年
  • 彦根久美子, 彦根久美子, 山本知真也, 織田公光, 工藤明, 飯田順一郎, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 119th 2014年
  • 虎谷彌, 長谷川智香, 山本知真也, 織田公光, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 119th 2014年
  • 本郷裕美, 山田珠希, 柳沼祐美子, 斎藤雅美, 中野貴由, 宇田川信之, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 119th 111 2014年 [査読無し][通常論文]
  • 坪井香奈子, 佐々木宗輝, 北川善政, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 119th 109 2014年 [査読無し][通常論文]
  • 長谷川智香, 山田珠希, 佐々木宗輝, 笹野泰之, 網塚憲生 日本解剖学会総会・全国学術集会講演プログラム・抄録集 119th 111 2014年 [査読無し][通常論文]
  • 坪井香奈子, 佐々木宗輝, 坪井香奈子, 佐々木宗輝, 長谷川智香, 虎谷彌, 織田公光, 北川善政, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2014 ROMBUNNO.O1‐C6 (WEB ONLY) 2014年 [査読無し][通常論文]
  • 本郷裕美, 佐々木宗輝, 斎藤雅美, 虎谷彌, 宇田川信之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2014 ROMBUNNO.O1‐C15 (WEB ONLY) 2014年 [査読無し][通常論文]
  • 山本知真也, 長谷川智香, 佐々木宗輝, 虎谷彌, 織田公光, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2014 ROMBUNNO.O1‐C14 (WEB ONLY) 2014年 [査読無し][通常論文]
  • 彦根久美子, 彦根久美子, 長谷川智香, 佐々木宗輝, 本郷裕美, 土屋恵李佳, 土屋恵李佳, 虎谷彌, 織田公光, 飯田順一郎, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2014 ROMBUNNO.O2‐F8 (WEB ONLY) 2014年 [査読無し][通常論文]
  • ナノテクノロジーEXPRESS 第29回 FIB-SEMを用いた骨組織の3次元微細構造解析．
    長谷川智香, 山本知真也, 網塚憲生, 小澤英浩, 遠堂敬史 NanotechJapan Bulletin 7 (5) 190 -194 2014年 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 山本 知真也, 織田 公光 Clinical Calcium 24 (2) 203 -214 2014年01月 [査読無し][通常論文]
     
    骨のバイオミネラリゼーションは、骨芽細胞が分泌する基質小胞に始まる。基質小胞には様々な酵素や膜輸送体が存在しており、Ca2+やPO4-の生成やそれらを基質小胞内部に輸送する仕組みが備わっている。基質小胞内でリン酸カルシウムの核形成が誘導されると、リン酸カルシウムの結晶が放射状に成長し、基質小胞の単位膜を破り外界へ露出するようになる。その結果、リボン状・短冊状を示すリン酸カルシウム結晶塊が放射状に集積した球状の石灰化構造物、すなわち、石灰化球が形成されることになる。ここまでのプロセスは骨芽細胞が分泌した基質小胞に依存するものであることから、基質小胞性石灰化という。その後、石灰化球が、骨芽細胞が分泌したコラーゲン線維に接すると、その接触部位からコラーゲン線維に石灰化が波及してゆく。この過程をコラーゲン性石灰化という。骨芽細胞による基質小胞性石灰化とコラーゲン性石灰化は、骨形成に伴って行われる石灰化現象であり一次石灰化と呼ぶ。一方、石灰化骨基質が形成された後も、徐々に石灰化度が上昇してゆくが、この過程を二次石灰化という。本稿では、骨基質の一次石灰化におけるバイオミネラリゼーションについて、我々の顕微解剖学的な知見をもとに概説する。(著者抄録)
  • 織田 公光, 沼 奈津子[金城], 相田 美和, 小丸 圭一, 網塚 憲生 Clinical Calcium 24 (2) 233 -239 2014年01月 [査読無し][通常論文]
     
    ヒトアルカリフォスファターゼには4種類のアイソザイムが存在する:組織非特異型(TNSALP)、小腸型、胎盤型、生殖細胞型酵素。この総説では歴史的な事項を踏まえて、TNSALPに関する基礎研究と低フォスファターゼ症(HPP)の研究の進展についてレビューする。HPPは、TNSALP遺伝子上の突然変異に起因する骨や歯などの硬組織の低石灰化を主症状とする先天性遺伝疾患で、TNSALPと石灰化を結ぶ直接の証拠である。3次元モデルに加えて、細胞に発現させた個々のTNSALPの変異型酵素を解析することは、特定の変異がTNSALPの構造や機能に与える影響を分子レベルで明らかにし、HPPの分子基盤の理解に役立つ。(著者抄録)
  • 田幡 千尋, 工藤 明, 網塚 憲生, 飯田 順一郎 日本矯正歯科学会大会プログラム・抄録集 72回 179 -179 2013年10月 [査読無し][通常論文]
  • 網塚憲生, 長谷川智香, 佐々木宗輝, 山本知真也, 山本恒之 CLINICAL CALCIUM 23 (10) 61 -69 2013年09月28日
  • 山本恒之, 長谷川智香, 山本知真也, 本郷裕美, 山田珠希, 織田公光, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.P2‐50 (WEB ONLY) -196 2013年09月 [査読無し][通常論文]
  • 田中祐介, 田中祐介, 長谷川智香, 山田珠希, 織田公光, 鄭漢忠, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐2 (WEB ONLY) -141 2013年09月 [査読無し][通常論文]
  • 山田珠希, 坪井香奈子, 平賀徹, 山本知真也, 田中祐介, 長谷川智香, 織田公光, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐75 (WEB ONLY) -140 2013年09月 [査読無し][通常論文]
  • 本郷裕美, 山田珠希, 宇田川信之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐48 (WEB ONLY) -141 2013年09月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 佐々木 宗輝, 山本 知真也, 山本 恒之 Clinical Calcium 23 (10) 1453 -1461 2013年09月 [査読無し][通常論文]
     
    骨細胞はfibroblast growth factor 23(FGF23)を分泌し、腎臓の近位尿細管に発現するFGFR1c/klotho複合体に結合することで、リン再吸収と1α-hydroxylaseを抑制し血中リン濃度を低下させる。一方、骨細胞が骨小腔や骨細管周囲の骨基質を溶解・添加する可能性も指摘されている。緻密骨において、骨細胞は規則的な骨細管系を発達させており、その規則性は骨細胞機能に対して効率的に作用すると考えられる。つまり、骨細胞の全身的なリン濃度調節および局所的な骨基質石灰化の調節と骨細胞の規則的配列性とは、何らかの関連性があると思われる。(著者抄録)
  • 骨細胞のバイオロジー オーバービュー 骨の司令塔 骨細胞
    網塚 憲生, 宮本 幸奈, 本郷 裕美, 佐々木 宗輝, 長谷川 智香 Journal of Oral Biosciences Supplement 2013 102 -102 2013年09月 [査読無し][通常論文]
  • 卵巣摘出レプチン受容体遺伝子変異(db/db)マウスの骨組織における組織化学的検索
    田中 祐介, 長谷川 智香, 山田 珠希, 織田 公光, 鄭 漢忠, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 107 -107 2013年09月 [査読無し][通常論文]
  • RANKL遺伝子欠損マウスにおける破骨細胞様細胞の微細構造学的解析
    宮本 幸奈, 長谷川 智香, 佐々木 宗輝, 織田 公光, 宇田川 信之, 山本 恒之, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 109 -109 2013年09月 [査読無し][通常論文]
  • 咬合負荷が抜歯即時埋入チタンインプラント周囲の骨組織に与える影響について
    池田 欣希, 長谷川 智香, 網塚 憲生, 横山 敦郎 Journal of Oral Biosciences Supplement 2013 111 -111 2013年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン投与による骨細胞周囲の骨基質改変について
    本郷 裕美, 山田 珠希, 宇田川 信之, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 119 -119 2013年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン間歇投与の頻度が骨の細胞動態に及ぼす影響について
    山本 知真也, 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 山田 珠希, 山本 恒之, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 119 -119 2013年09月 [査読無し][通常論文]
  • 乳癌骨転移巣における骨細胞産生因子の組織化学的解析
    山田 珠希, 坪井 香奈子, 平賀 徹, 山本 知真也, 田中 祐介, 長谷川 智香, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 125 -125 2013年09月 [査読無し][通常論文]
  • FGF23/klotho軸の破綻は血管骨化を誘導する klotho遺伝子変異マウスを用いた組織学的検索
    長谷川 智香, 山田 珠希, 佐々木 宗輝, 笹野 泰之, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 126 -126 2013年09月 [査読無し][通常論文]
  • bisphosphonate投与中止後の骨の細胞群における組織化学的検索
    坪井 香奈子, 佐々木 宗輝, 長谷川 智香, 北川 善政, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 134 -134 2013年09月 [査読無し][通常論文]
  • ミノドロン酸の同位体顕微鏡を用いた骨組織分布と破骨細胞に対する影響
    佐々木 宗輝, 本郷 裕美, 小林 幸雄, 圦本 尚義, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 135 -135 2013年09月 [査読無し][通常論文]
  • アレンドロネート直接作用による骨芽細胞分化の制御
    小松 浩一郎, 島田 明美, 柴田 達也, 中島 和久, 網塚 憲生, 二藤 彰 Journal of Oral Biosciences Supplement 2013 135 -135 2013年09月 [査読無し][通常論文]
  • 乳癌骨転移巣における骨細胞産生因子の組織化学的解析
    山田 珠希, 坪井 香奈子, 平賀 徹, 山本 知真也, 田中 祐介, 長谷川 智香, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 140 -140 2013年09月 [査読無し][通常論文]
  • FGF23/klotho軸の破綻は血管骨化を誘導する klotho遺伝子変異マウスを用いた組織学的検索
    長谷川 智香, 山田 珠希, 佐々木 宗輝, 笹野 泰之, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 140 -140 2013年09月 [査読無し][通常論文]
  • bisphosphonate投与中止後の骨の細胞群における組織化学的検索
    坪井 香奈子, 佐々木 宗輝, 長谷川 智香, 北川 善政, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 140 -140 2013年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン投与による骨細胞周囲の骨基質改変について
    本郷 裕美, 山田 珠希, 宇田川 信之, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 141 -141 2013年09月 [査読無し][通常論文]
  • 卵巣摘出レプチン受容体遺伝子変異(db/db)マウスの骨組織における組織化学的検索
    田中 祐介, 長谷川 智香, 山田 珠希, 織田 公光, 鄭 漢忠, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 141 -141 2013年09月 [査読無し][通常論文]
  • 咬合負荷が抜歯即時埋入チタンインプラント周囲の骨組織に与える影響について
    池田 欣希, 長谷川 智香, 網塚 憲生, 横山 敦郎 Journal of Oral Biosciences Supplement 2013 143 -143 2013年09月 [査読無し][通常論文]
  • ミノドロン酸の同位体顕微鏡を用いた骨組織分布と破骨細胞に対する影響
    佐々木 宗輝, 本郷 裕美, 小林 幸雄, 圦本 尚義, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 146 -146 2013年09月 [査読無し][通常論文]
  • セメント質初期形成における上皮鞘細胞と歯小嚢細胞の動態について
    山本 恒之, 長谷川 智香, 山本 知真也, 本郷 裕美, 山田 珠希, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2013 196 -196 2013年09月 [査読無し][通常論文]
  • 山本 知真也, 佐々木 宗輝, 長谷川 智香, 網塚 憲生 Osteoporosis Japan 21 (Suppl.1) 255 -255 2013年09月 [査読無し][通常論文]
  • 田中 祐介, 長谷川 智香, 本郷 裕美, 佐々木 宗輝, 山田 珠希, 戸塚 靖則, 鄭 漢忠, 網塚 憲生 Osteoporosis Japan 21 (Suppl.1) 265 -265 2013年09月 [査読無し][通常論文]
  • 長谷川 智香, 山田 珠希, 佐々木 宗輝, 網塚 憲生 Osteoporosis Japan 21 (Suppl.1) 266 -266 2013年09月 [査読無し][通常論文]
  • 網塚 憲生, 山本 知真也, 佐々木 宗輝, 長谷川 智香 THE BONE 27 (3) 263 -269 2013年08月 [査読無し][通常論文]
     
    骨細胞は細胞突起を張り巡らせた骨細管系というグループで機能している。それは幼弱骨や緻密な成熟骨では異なる分布パターンを示し、骨基質の石灰化度やコラーゲン線維の走行を含めた組織構築に対応した配列を示していると考えられる。さらに、骨細胞だけがネットワークを形成しているのではなく、骨基質上の骨芽細胞や前骨芽細胞とも細胞間相互作用を行うと考えられ、骨細胞は骨基質の情報を周囲に伝え、また、周囲の情報を骨基質に反映させるといった重要な役割を担っていると考えられる。(著者抄録)
  • 道家 孝幸, 射場 浩介, 金谷 久美子, 阿部 恭久, 佐々木 宗輝, 網塚 憲生, 山下 敏彦 日本整形外科学会雑誌 87 (8) S1383 -S1383 2013年08月 [査読無し][通常論文]
  • 佐々木 宗輝, 本郷 裕美, 小林 幸雄, 圦本 尚義, 網塚 憲生 日本骨形態計測学会雑誌 23 (1) S81 -S81 2013年06月 [査読無し][通常論文]
  • 山本 知真也, 佐々木 宗輝, 本郷 裕美, 田中 祐介, 長谷川 智香, 山田 珠希, 山本 恒之, 網塚 憲生 日本骨形態計測学会雑誌 23 (1) S83 -S83 2013年06月 [査読無し][通常論文]
  • 本郷 裕美, 山田 珠希, 山本 知真也, 中野 貴由, 宇田川 信之, 網塚 憲生 日本骨形態計測学会雑誌 23 (1) S84 -S84 2013年06月 [査読無し][通常論文]
  • 山本 恒之, 長谷川 智香, 本郷 裕美, 山本 知真也, 網塚 憲生 日本骨形態計測学会雑誌 23 (1) S97 -S97 2013年06月 [査読無し][通常論文]
  • 長谷川 智香, 大城戸 一郎, 山田 珠希, 横山 啓太郎, 網塚 憲生 日本骨形態計測学会雑誌 23 (1) S99 -S99 2013年06月 [査読無し][通常論文]
  • 網塚 憲生, 山本 知真也, 長谷川 智香, 佐々木 宗輝 日本骨代謝学会学術集会プログラム抄録集 31回 63 -63 2013年05月 [査読無し][通常論文]
  • 長谷川 智香, 大城戸 一郎, 庄司 繁市, 山田 珠希, 織田 公光, 横山 啓太郎, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 31回 79 -79 2013年05月 [査読無し][通常論文]
  • 鈴木 敦詞, 長谷川 智香, 植田 佐保子, 平井 博之, 四馬田 恵, 網塚 憲生, 伊藤 光泰 日本骨代謝学会学術集会プログラム抄録集 31回 81 -81 2013年05月 [査読無し][通常論文]
  • 田中 祐介, 長谷川 智香, 本郷 裕美, 佐々木 宗輝, 山田 珠希, 柳 鋳晟, 山本 恒之, 織田 公光, 戸塚 靖則, 鄭 漢忠, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 31回 93 -93 2013年05月 [査読無し][通常論文]
  • 亀田 啓, 永井 聡, 三好 秀明, 渥美 達也, 千田 大, 岩倉 洋一郎, 網塚 憲生, 清水 力 日本骨代謝学会学術集会プログラム抄録集 31回 94 -94 2013年05月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 佐々木 宗輝, 山田 珠希, 山本 恒之 日本臨床 71 (増刊2 最新の骨粗鬆症学) 31 -38 2013年04月 [査読無し][通常論文]
  • 網塚 憲生, 本郷 裕美, 宮本 幸奈, 長谷川 智香, 佐々木 宗輝, 山田 珠希, 山本 恒之 解剖学雑誌 88 (1-2) 25 -28 2013年03月 [査読無し][通常論文]
     
    私たち歯学部・歯科大学の役割は歯科治療における充分な技術と知識、深い洞察を備えた歯科医師を養成することである。しかし、その一方で、多くの歯学部・歯科大学はリサーチマインドを持った歯学部学生の育成を目指して、歯科医療だけでなく基礎研究を激励するとともに、研究、あるいは、それに準じた機会を与えることが多いと思われる。北海道大学歯学部では、5年から6年生の学部学生を対象に教室配属を行っており、数名の学生が各基礎教室に配属されている。そのような基礎配属をとおして、何人かの学生が新しい発見をみいだし、また、それを学会発表する機会を得ている。その一方で、日本の多くの学会や研究会は学生セッションなどの場を設け、そこでは学部生が指導教官ともに行った研究成果を発表している。近年における歯学部・歯科大学や学会の若手研究者育成に対する努力は近い将来実を結ぶものと期待している。(著者抄録)
  • 網塚憲生 CLINICAL CALCIUM 23 (3) 112 -112 2013年02月28日
  • 佐々木宗輝, 長谷川智香, 本郷裕美, 山本知真也, 網塚憲生 CLINICAL CALCIUM 23 (3) 41 -47 2013年02月28日
  • 佐々木 宗輝, 長谷川 智香, 本郷 裕美, 山本 知真也, 網塚 憲生 Clinical Calcium 23 (3) 347 -353 2013年02月 [査読無し][通常論文]
     
    副甲状腺ホルモン(parathyroid hormone:PTH)の間歇投与によって、骨芽細胞の骨形成および前骨芽細胞の増殖が亢進し骨形成優位となる。我々は、PTHの間歇投与による骨芽細胞系細胞の動向およびPTHの骨形成に対して破骨細胞がどのように関与するのか検索した。その結果、野生型マウスにPTHを投与すると骨形成の亢進が認められたが、破骨細胞が存在しないc-fos欠損マウスにPTHを投与しても、前骨芽細胞数が増加したにも関わらず、骨芽細胞による骨形成は誘導されなかった。したがって、破骨細胞が存在しない状況では、PTHの骨形成作用が誘導されないこと、すなわち、破骨細胞は前骨芽細胞から骨芽細胞への分化、そしてその後の骨形成に必要な存在であることが推測された。また、本稿では、PTH間歇投与における骨細胞の関与についても概説したい。(著者抄録)
  • 池田欣希, 長谷川智香, 網塚憲生, 横山敦郎 Journal of Oral Biosciences Supplement (Web) 2013 2013年
  • 小松浩一郎, 島田明美, 柴田達也, 中島和久, 網塚憲生, 二藤彰 Journal of Oral Biosciences Supplement (Web) 2013 2013年
  • 島田明美, 新井嘉則, 出野尚, 上運天太一, 中島和久, 小松浩一郎, 山下照仁, 江面陽一, 網塚憲生, 高橋直之, POESCHL Ernst, 二藤彰 日本分子生物学会年会プログラム・要旨集(Web) 36th 1P-0587 (WEB ONLY) 2013年 [査読無し][通常論文]
  • 坪井香奈子, 坪井香奈子, 佐々木宗輝, 長谷川智香, 北川善政, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐110 (WEB ONLY) 2013年 [査読無し][通常論文]
  • 長谷川智香, 山田珠希, 佐々木宗輝, 笹野泰之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐79 (WEB ONLY) 2013年 [査読無し][通常論文]
  • 宮本幸奈, 宮本幸奈, 長谷川智香, 佐々木宗輝, 織田公光, 宇田川信之, 山本恒之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐11 (WEB ONLY) 2013年 [査読無し][通常論文]
  • 山本知真也, 佐々木宗輝, 本郷裕美, 長谷川智香, 山田珠希, 山本恒之, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐49 (WEB ONLY) 2013年 [査読無し][通常論文]
  • 血管石灰化における病理機序の解明－Klotho欠損マウスを用いた微細構造学的解析－.
    長谷川智香, 本郷裕美, 佐々木宗輝, 大城戸一郎, 横山啓太郎, 網塚憲生 第15回Vitamin K & Aging研究会 記録集 55 -58 2013年 [査読無し][通常論文]
  • 佐々木宗輝, 本郷裕美, 小林幸雄, 圦本尚義, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2013 ROMBUNNO.O‐112 (WEB ONLY) 2013年 [査読無し][通常論文]
  • 佐々木 宗輝, 長谷川 智香, 山本 知真也, 本郷 裕美, 虎谷 彌, 網塚 憲生 THE BONE 26 (4) 365 -370 2012年12月 [査読無し][通常論文]
  • Histochemical examination on osteocytes and their lacunae after administration of parathyroid hormone in mice
    Hongo H, Sasaki M, Yamada T, Hasegawa T, Yamamoto T, Nakano T, Shimoji S, Amizuka N 22nd ANZBMS Annual scientific meeting with 1st Asia-Pacific Bone & Mineral Research Meeting 2012年09月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 赤堀 永倫香, 山田 珠希 日本生理学雑誌 74 (5) 235 -236 2012年09月 [査読無し][通常論文]
  • レプチン受容体遺伝子変異(db/db)マウスにおける骨芽細胞・骨細胞および骨基質の形態学的解析
    田中 祐介, 長谷川 智香, 本郷 裕美, 佐々木 宗輝, 山田 珠希, 山本 恒之, 戸塚 靖則, 鄭 漢忠, 網塚 憲生 北海道歯学雑誌 33 (1) 34 -34 2012年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン投与による骨細胞周囲の骨基質に関する組織化学的検索
    本郷 裕美, 佐々木 宗輝, 長谷川 智香, 田幡 千尋, 川浪 雅光, 山本 恒之, 下地 伸司, 網塚 憲生 北海道歯学雑誌 33 (1) 34 -34 2012年09月 [査読無し][通常論文]
  • インプラント周囲骨細胞の免疫組織化学的検索
    羽下 麻衣子[辻村], 網塚 憲生, 前田 健康, 吉江 紀夫 Journal of Oral Biosciences Supplement 2012 119 -119 2012年09月 [査読無し][通常論文]
  • Jausen型PTH/PTHrP受容体の機能異常の解析
    下村 淳子[黒木], 竜 佑宗, 松田 貴絵, 田中 聖至, 織田 公光, 網塚 憲生 Journal of Oral Biosciences Supplement 2012 165 -165 2012年09月 [査読無し][通常論文]
  • 山田 珠希, 佐々木 宗輝, 長谷川 智香, 本郷 裕美, 網塚 憲生 O.li.v.e. 2 (3) 214 -218 2012年08月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 佐々木 宗輝, 本郷 裕美, 田幡 千尋, 山田 珠希, 赤堀 永倫香, 山本 恒之 日本整形外科学会雑誌 86 (8) S1210 -S1210 2012年08月 [査読無し][通常論文]
  • 網塚 憲生 日本整形外科学会雑誌 86 (8) S1212 -S1212 2012年08月 [査読無し][通常論文]
  • 山田 珠希, 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 赤堀 永倫香, 網塚 憲生 腎と骨代謝 25 (3) 215 -224 2012年07月 [査読無し][通常論文]
     
    新規活性ビタミンD製剤であるエルデカルシトールは,アルファカルシトールに比べて,骨折抑制および骨密度上昇作用を示す.エルデカルシトールの作用としては,破骨細胞性骨吸収の抑制にあると考えられてきたが,エルデカルシトール投与後の骨型アルカリフォスファターゼの上昇および骨吸収に依存しない骨形成(ミニモデリング)が観察されており,エルデカルシトールの骨形成作用が注目を浴びている.エルデカルシトールは,前骨芽細胞を細胞増殖ではなく活性型骨芽細胞へ分化させることで,ミニモデリングという局所的な骨形成を誘導すると推測される.(著者抄録)
  • 網塚 憲生, 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 山本 知真也, 山田 珠希, 山本 恒之 日本骨代謝学会学術集会プログラム抄録集 30回 108 -108 2012年07月 [査読無し][通常論文]
  • 長谷川 智香, 大城戸 一郎, 山田 珠希, 横山 啓太郎, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 30回 115 -115 2012年07月 [査読無し][通常論文]
  • 網塚 憲生, 長谷川 智香, 佐々木 宗輝, 本郷 裕美, 田幡 千尋, 山田 珠希, 山本 恒之 日本骨代謝学会学術集会プログラム抄録集 30回 118 -118 2012年07月 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 山田 珠希, 田幡 千尋, 山本 恒之, 中野 貴由, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 30回 215 -215 2012年07月 [査読無し][通常論文]
  • 山本 恒之, 長谷川 智香, 佐々木 宗輝, 田幡 千尋, 本郷 裕美, 山田 珠希, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 30回 239 -239 2012年07月 [査読無し][通常論文]
  • 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 山田 珠希, 網塚 憲生 O.li.v.e. 2 (2) 154 -158 2012年05月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 柳 鋳晟, 山本 知真也, 山田 珠希, 山本 恒之 日本骨形態計測学会雑誌 22 (1) S37 -S37 2012年05月 [査読無し][通常論文]
  • 田中祐介, 長谷川智香, 本郷裕美, 佐々木宗輝, 山田珠希, 山本恒之, 戸塚靖則, 鄭漢忠, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2012 8 2012年04月 [査読無し][通常論文]
  • 本郷裕美, 佐々木宗輝, 長谷川智香, 田幡千尋, 川浪雅光, 山本恒之, 下地伸司, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2012 9 2012年04月 [査読無し][通常論文]
  • 佐々木 宗輝, 長谷川 智香, 本郷 裕美, 山田 珠希, 山本 知真也, 山本 恒之, 網塚 憲生, 小澤 英浩 THE BONE 26 (2) 125 -129 2012年04月 [査読無し][通常論文]
  • 大城戸 一郎, 横山 啓太郎, 長谷川 智香, 網塚 憲生, 細谷 龍男 日本腎臓学会誌 54 (3) 324 -324 2012年04月 [査読無し][通常論文]
  • 長谷川 智香, 山田 珠希, 佐々木 宗輝, 李 敏啓, 大城戸 一郎, 横山 啓太郎, 網塚 憲生 Osteoporosis Japan 20 (2) 279 -281 2012年04月 [査読無し][通常論文]
     
    血管石灰化の病理機序を解明するため、klotho欠損マウスの大動脈を形態学的な手法を用いて検索した。Klotho欠損マウスの大動脈中膜では、血管平滑筋細胞が骨芽細胞のいくつかの性質を獲得する細胞へとtrans-differentiationし、石灰化を誘導することが示唆された。石灰化領域の血管平滑筋細胞は、基質小胞性様構造物を分泌し、骨基質にみられるような、生物学的な基質小胞性石灰化を誘導する可能性が示唆された。さらに、klotho欠損環境では、血中に豊富に存在するリンやカルシウムが、中膜に存在する有機性の不定形構造物内部へと物理的に沈着する、病理学的な石灰化も起こることが示唆された。また、matrix Gla protein(MGP)陽性反応は石灰化基質を取り囲むように局在していた。中膜石灰化によってMGPの発現が誘導され、MGPは石灰化抑制に作用する可能性が推測された。
  • HONGO Hiromi, HASEGAWA Tomoka, SASAKI Muneteru, SUZUKI Reiko, MASUKI Hideo, YAMADA Tamaki, SHIMOJI Shinji, KAWANAMI Masamitsu, YAMAMOTO Tsuneyuki, AMIZUKA Norio 北海道歯学雑誌 32 (2) 93 -103 2012年03月15日 [査読無し][通常論文]
     
    Osteocytes build up functional syncytia, i.e., the osteocytic lacunar-canalicular system（OLCS）. The osteocytes are interconnected through gap junctions between their cytoplasmic processes, which pass through narrow passageways referred to as osteocytic canaliculi. There are two possible ways, in which molecules can be transported throughout the OLCS: via the cytoplasmic processes and their gap junctions, and via the pericellular space in the osteocytic canaliculi. Transport of minerals and small molecules through a spatially well-organized OLCS appears to be pivotal for bone mineral homeostasis and bone remodeling control. Recently, osteocyte-derived molecules -- sclerostin, dentin matrix protein-1, fibroblast growth factor 23（FGF23）-- have been put in evidence as they may be related to osteocytic functions such as regulation of bone remodeling and so forth. Osteocytes were shown to regulate phosphorus serum levels and osteoblastic activity through the expression of FGF23 and sclerostin. In our observations, FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS: both proteins were consistently expressed by osteocytes in epiphyses and cortical bones showing regularly arranged OLCS. In contrast, mice bearing high bone turnover, e.g., osteoprotegerin deficient mice, revealed markedly-diminished sclerostin. This review will introduce our recent studies on the regularity of OLCS and the osteocytic function.
  • 副甲状腺ホルモン投与による骨細胞性骨融解に関する組織化学的検索
    本郷裕美, 佐々木宗輝, 長谷川智香, 鈴木礼子, 下地伸司, 網塚憲生 第117回日本解剖学会総会・全国学術大会 2012年03月 [査読無し][通常論文]
  • 小島 拓, 芳澤 享子, 小野 由起子, 鈴木 晶子, 坂上 直子, 長谷川 智香, 網塚 憲生, 織田 公光, 前田 健康, 齊藤 力 日本歯科医学会誌 31 (31) 34 -38 2012年03月 [査読無し][通常論文]
     
    腫瘍、嚢胞、外傷、炎症などの疾患によって広範囲で複雑な形態の顎骨欠損が生じた場合、形態を考慮した顎骨再建が必要になる。われわれのこれまでの基礎的研究から、熱可塑性吸収性プレートと骨補填材を用いることで三次元形態を有する骨再生が可能であることが示唆されたが、さらなる骨再生の向上を目指すには、間葉系幹細胞とバイオマテリアルを組み合わせる培養技術を用いた骨再生が有用ではないかと考えた。そこで本研究では、骨髄由来間葉系幹細胞・多孔性β-TCPブロック複合体をラット頭蓋骨骨膜下に移植する動物実験モデルを用いて組織学的検討を行った。生後4週齢の雄性Fischer系ラット大腿骨から骨髄細胞を採取し、間葉系幹細胞を分離培養後、多孔性β-TCPブロックに播種して骨芽細胞様細胞への分化を誘導した。この間葉系幹細胞・多孔性β-TCPブロック複合体を生後12週齢の同系ラット頭蓋骨骨膜下に移植したものを培養群とし、β-TCPブロック単体を移植したものを対照群とした。術後2、8週目に灌流固定を行い、移植部位を組織化学的に解析した。培養群の術後2週目ではβ-TCPブロック辺縁部に新生骨を認めた。新生骨表層には多数の骨芽細胞が配列し、周囲のβ-TCP上には破骨細胞が局在していた。術後8週目ではブロック中央部にも新生骨を認め、その骨梁は太く層板様を呈していた。一方、対照群ではブロック内部は線維性結合組織で占められており、術後8週目においても新生骨はわずかしか認められなかった。以上の結果から、骨髄由来間葉系幹細胞を骨芽細胞様細胞に分化させ多孔性β-TCPブロックに播種することで、β-TCPブロック単体よりも広範囲に骨が新生されることが明らかとなった。したがって骨再生バイオマテリアルに間葉系幹細胞を供給することの有用性が示唆された。(著者抄録)
  • 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 山田 珠希, 網塚 憲生 O.li.v.e. 2 (1) 76 -80 2012年02月 [査読無し][通常論文]
  • 長谷川 智香, 本郷 裕美, 佐々木 宗輝, 山田 珠希, 網塚 憲生 Clinical Calcium 22 (3) 373 -379 2012年02月 [査読無し][通常論文]
     
    PTH間歇投与により骨形成が誘導される。その考え方として、骨芽細胞に作用して骨形成を亢進させるだけでなく、前骨芽細胞の細胞数を増加させることも重要であると思われる。増加した前骨芽細胞から活性型骨芽細胞への分化には、破骨細胞とのカップリングが必要と考えられ、PTHの骨形成促進作用に破骨細胞は無関係でないことが示唆される。一方、PTHは骨細胞に直接作用してスクレロスチン産生を抑制させることで、骨芽細胞を活性化し、骨形成を促進する作用も提唱されている。おそらく、骨芽細胞はPTHによる二極性の調節を受けて、骨形成促進に誘導されると推測される。(著者抄録)
  • 下村(黒木)淳子, 竜佑宗, 松田貴絵, 田中聖至, 織田公光, 網塚憲生 Journal of Oral Biosciences Supplement (Web) 2012 2012年
  • The biological function of parathyroid hormone and parathyroid hormone-related peptide in osteoblastic cells.
    Amizuka N, Sasaki M, Hongo H, Hasegawa T, Yamada T, Yamamoto T, Freitas PHL, Li M Progres in Photonics: Materials, Nano-and Bio-Imaging, and Communications. 65 -69 2012年 [査読無し][通常論文]
  • 羽下(辻村, 麻衣子, 網塚憲生, 前田健康, 吉江紀夫 Journal of Oral Biosciences Supplement (Web) 2012 ROMBUNNO.P1‐49 (WEB ONLY) 2012年 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 網塚 憲生 腎と骨代謝 25 (1) 7 -16 2012年01月 [査読無し][通常論文]
     
    骨細胞は,骨細胞・骨細管系を発達させており,FGF23,sclerostin,DMP-1といった因子を分泌することで,血中リン濃度,骨芽細胞抑制,骨基質石灰化を調節している.klothoはFGF23の受容体を形成する因子であり,そのklotho遺伝子が欠損したマウスでは,骨細胞はDMP-1過剰産生とFGF23の産生低下を示すほか,一次骨梁では過剰な石灰化を,しかし,二次骨梁では広範囲な未石灰化領域を呈した.klotho遺伝子欠損マウスでは,高リン血症を発症するため骨形成時の石灰化は亢進するが,その後,骨細胞による骨基質ミネラル維持は破綻している可能性が考えられた.(著者抄録)
  • 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 山本 恒之, 網塚 憲生 O.li.v.e. 1 (1) 6 -9 2011年11月 [査読無し][通常論文]
  • 長谷川 智香, 李 敏啓, 佐々木 宗輝, 大城戸 一郎, 横山 啓太郎, 網塚 憲生 Osteoporosis Japan 19 (Suppl.1) 299 -299 2011年11月 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 李 敏啓, 網塚 憲生, 小澤 英浩 THE BONE 25 (4) 351 -355 2011年10月 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 網塚 憲生 Clinical Calcium 21 (11) 1647 -1654 2011年10月 [査読無し][通常論文]
     
    エルデカルシトールを卵巣摘出(OVX)ラットに投与すると、破骨細胞数の減少と破骨細胞性骨吸収の低下だけではなく、骨吸収に依存しない骨形成(ミニモデリング)が誘導される。組織学的には、破骨細胞数の減少および破骨細胞の骨吸収抑制、活性型マクロファージ数の増加、骨密度の上昇を認めた。ミニモデリングは破骨細胞による骨吸収に依存しない骨形成であり、その骨表面上には活性型骨芽細胞が観察された。しかし、破骨細胞分化を支持する前骨芽細胞はその数が減少しており、発達した前骨芽細胞のネットワークが観察されなかった。また、エルデカルシトールは、前骨芽細胞を、細胞増殖ではなく活性型骨芽細胞へ誘導させることでミニモデリングという局所的な骨形成を可能にすると推測された。一方、エルデカルシトールによって減少した前骨芽細胞のため、破骨細胞・マクロファージ前駆細胞との細胞間接触が低下し、破骨細胞形成が抑制されると推察された。(著者抄録)
  • T. Hasegawa, M. Li, M. Sasaki, T. Chihiro, I. Ookido, Z. Liu, T. Yamamoto, K. Yokoyama, N. Amizuka OSTEOPOROSIS INTERNATIONAL 22 S568 -S569 2011年09月 [査読無し][通常論文]
  • T. Hasegawa, K. Oda, M. Sasaki, C. Tabata, Z. Liu, Y. Guo, K. Inoue, M. Li, T. Komori, T. Yamamoto, N. Amizuka OSTEOPOROSIS INTERNATIONAL 22 S578 -S579 2011年09月 [査読無し][通常論文]
  • 佐々木 宗輝, 田幡 千尋, 長谷川 智香, 郭 頴, 李 敏啓, 山本 恒之, 井上 農夫男, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 113 -113 2011年09月 [査読無し][通常論文]
  • 長谷川 智香, 本郷 裕美, 佐々木 宗輝, 田幡 千尋, 柳 鋳晟, 山本 恒之, 李 敏啓, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 114 -114 2011年09月 [査読無し][通常論文]
  • 田中 祐介, 長谷川 智香, 佐々木 宗輝, 柳 鋳晟, 織田 公光, 韋 山良, 李 敏啓, 戸塚 靖則, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 117 -117 2011年09月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 宗輝, 本郷 裕美, 長谷川 智香 Journal of Oral Biosciences 53 (Suppl.) 84 -84 2011年09月 [査読無し][通常論文]
  • 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 郭 頴, 虎谷 彌, 田幡 千尋, 鈴木 礼子, 李 敏啓, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 131 -131 2011年09月 [査読無し][通常論文]
  • 田幡 千尋, 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 李 敏啓, 山本 恒之, 飯田 順一郎, 工藤 明, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 134 -134 2011年09月 [査読無し][通常論文]
  • 郭 頴, 柳 鋳晟, 長谷川 智香, 織田 公光, 山本 恒之, 網塚 憲生, 李 敏啓 Journal of Oral Biosciences 53 (Suppl.) 148 -148 2011年09月 [査読無し][通常論文]
  • 柳 鋳晟, 郭 頴, 長谷川 智香, 織田 公光, 山本 恒之, 網塚 憲生, 李 敏啓 Journal of Oral Biosciences 53 (Suppl.) 148 -148 2011年09月 [査読無し][通常論文]
  • 山本 知真也, 佐々木 宗輝, 本郷 裕美, 長谷川 智香, 虎谷 彌, 柳 鋳晟, 李 敏啓, 森 裕史, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 151 -151 2011年09月 [査読無し][通常論文]
  • 李 敏啓, 長谷川 智香, 佐々木 宗輝, 田幡 千尋, 織田 公光, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 162 -162 2011年09月 [査読無し][通常論文]
  • 山本 恒之, 長谷川 智香, 佐々木 宗輝, 郭 頴, 田幡 千尋, 柳 鋳晟, 李 敏啓, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 162 -162 2011年09月 [査読無し][通常論文]
  • 吉沢 早織, 本郷 裕美, 佐々木 宗輝, 長谷川 智香, 李 敏啓, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 167 -167 2011年09月 [査読無し][通常論文]
  • 宮本 幸奈, 佐々木 宗輝, 長谷川 智香, 織田 公光, 宇田川 信之, 山本 恒之, 李 敏啓, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 167 -167 2011年09月 [査読無し][通常論文]
  • 韋 山良, 佐々木 宗輝, 柳 鋳晟, 郭 穎, 田幡 千尋, 織田 公光, 田中 祐介, 李 敏啓, 戸塚 靖則, 網塚 憲生 Journal of Oral Biosciences 53 (Suppl.) 192 -192 2011年09月 [査読無し][通常論文]
  • ラット臼歯歯根膜コラーゲン線維とシャーピー線維の構造について
    田幡 千尋, 李 敏啓, 長谷川 智香, 佐々木 宗輝, 山本 恒之, 飯田 順一郎, 網塚 憲生 北海道歯学雑誌 32 (1) 76 -76 2011年09月 [査読無し][通常論文]
  • Klotho遺伝子欠損マウスにおける骨細胞の組織学的所見
    佐々木 宗輝, 田幡 千尋, 長谷川 智香, 郭 頴, 李 敏啓, 山本 恒之, 井上 農夫男, 網塚 憲生 北海道歯学雑誌 32 (1) 76 -76 2011年09月 [査読無し][通常論文]
  • Klotho遺伝子欠損環境における骨基質石灰化について
    佐々木 宗輝, 田幡 千尋, 長谷川 智香, 郭 頴, 李 敏啓, 山本 恒之, 井上 農夫男, 網塚 憲生 北海道歯学雑誌 32 (1) 77 -77 2011年09月 [査読無し][通常論文]
  • アスコルビン酸合成能欠如ラットの歯根膜異常における形態学的検索
    長谷川 智香, 和田 悟史, 佐々木 宗輝, 柳 鋳晟, 山本 恒之, 大畑 昇, 李 敏啓, 網塚 憲生 北海道歯学雑誌 32 (1) 77 -77 2011年09月 [査読無し][通常論文]
  • アスコルビン酸合成能欠如ラットの骨基質石灰化における組織学的検索
    長谷川 智香, 李 敏啓, 田幡 千尋, 和田 悟史, 郭 頴, 大畑 昇, 網塚 憲生 北海道歯学雑誌 32 (1) 78 -78 2011年09月 [査読無し][通常論文]
  • klotho欠損マウス血管石灰化における病理機序の微細構造学的解明
    長谷川 智香, 李 敏啓, 佐々木 宗輝, 田幡 千尋, 郭 頴, 柳 鋳晟, 山本 恒之, 大畑 昇, 網塚 憲生 北海道歯学雑誌 32 (1) 78 -78 2011年09月 [査読無し][通常論文]
  • 網塚 憲生 Dental medicine research 31 (2) 209 -209 2011年07月31日 [査読無し][通常論文]
  • 李 敏啓, 長谷川 智香, 武田 聡, 佐々木 宗輝, 田幡 千尋, 織田 公光, 斎藤 一史, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 29回 188 -188 2011年07月 [査読無し][通常論文]
  • 長谷川 智香, 織田 公光, 佐々木 宗輝, 田幡 千尋, 柳 鋳晟, 郭 穎, 井上 貴一朗, 李 敏啓, 小守 壽文, 山本 恒之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 29回 192 -192 2011年07月 [査読無し][通常論文]
  • 佐々木 宗輝, 田幡 千尋, 長谷川 智香, 郭 頴, 柳 鋳晟, 李 敏啓, 山本 恒之, 井上 農夫男, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 29回 195 -195 2011年07月 [査読無し][通常論文]
  • 長谷川 智香, 李 敏啓, 佐々木 宗輝, 田幡 千尋, 大城戸 一郎, 郭 頴, 柳 鋳晟, 山本 恒之, 横山 啓太郎, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 29回 196 -196 2011年07月 [査読無し][通常論文]
  • 田幡 千尋, 佐々木 宗輝, 長谷川 智香, 李 敏啓, 山本 恒之, 飯田 順一郎, 工藤 明, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 29回 204 -204 2011年07月 [査読無し][通常論文]
  • 山本 恒之, 長谷川 智香, 佐々木 宗輝, 郭 頴, 田幡 千尋, 柳 鋳晟, 李 敏啓, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 29回 235 -235 2011年07月 [査読無し][通常論文]
  • 柳 鋳晟, 郭 穎, 長谷川 智香, 織田 公光, 山本 恒之, 網塚 憲生, 李 敏啓 日本骨代謝学会学術集会プログラム抄録集 29回 250 -250 2011年07月 [査読無し][通常論文]
  • 島田 明美, 小松 浩一郎, 中島 和久, 網塚 憲生, ブラッハフォーゲル・ベント, 二藤 彰 日本骨代謝学会学術集会プログラム抄録集 29回 252 -252 2011年07月 [査読無し][通常論文]
  • 郭 穎, 柳 鋳晟, 長谷川 智香, 山本 恒之, 網塚 憲生, 李 敏啓 日本骨代謝学会学術集会プログラム抄録集 29回 283 -283 2011年07月 [査読無し][通常論文]
  • 長谷川 智香, 増木 英郎, 佐々木 宗輝, 田幡 千尋, 李 敏啓, 山本 恒之, 網塚 憲生, 小澤 英浩 THE BONE 25 (2) 95 -100 2011年05月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 21 (2) S53 -S53 2011年05月 [査読無し][通常論文]
  • 佐々木宗輝, 田幡千尋, 長谷川智香, 郭頴, 柳鋳晟, 李敏啓, 山本恒之, 井上農夫男, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2011 8 2011年04月 [査読無し][通常論文]
  • 佐々木宗輝, 田幡千尋, 長谷川智香, 郭頴, 柳鋳晟, 李敏啓, 山本恒之, 井上農夫男, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2011 9 2011年04月 [査読無し][通常論文]
  • 長谷川智香, 和田悟史, 佐々木宗輝, 柳鋳晟, 山本恒之, 大畑昇, 李敏啓, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2011 10 2011年04月 [査読無し][通常論文]
  • 田幡千尋, 李敏啓, 長谷川智香, 佐々木宗輝, 山本恒之, 飯田順一郎, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2011 7 2011年04月 [査読無し][通常論文]
  • 長谷川智香, 李敏啓, 佐々木宗輝, 田幡千尋, 郭頴, 柳鋳晟, 山本恒之, 大畑昇, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2011 12 2011年04月 [査読無し][通常論文]
  • 網塚 憲生 医科学応用研究財団研究報告 28 355 -359 2011年02月 [査読無し][通常論文]
  • 長谷川智香, 李敏啓, 田幡千尋, 田幡千尋, 和田悟史, 郭頴, 大畑昇, 網塚憲生 北海道歯学会学術大会プログラム講演内容抄録 2011 2011年
  • Histochemical examination on the periodontal ligaments in the ascorbic acid-insufficient microenviroment.
    Hasegawa T, Inoue K, Wada S, Miyamoto Y, Sasaki M, Tabata C, Yamamoto T, Oohata N, Li M, Amizuka N J Physiol Sci. 61 (suppl) S185 -S185 2011年 [査読無し][通常論文]
  • Morphological aspects of biological function of osteocytes.
    Amizuka N, Hasegawa T, Sasaki M, Tabata C, Yamamoto T, Li M, Wada S J Physiol Sci. 61 (suppl) S83 -S83 2011年 [査読無し][通常論文]
  • 長谷川 智香, 佐々木 宗輝, 田幡 千尋, 増木 英郎, 李 敏啓, 網塚 憲生 Clinical Calcium 21 (2) 190 -196 2011年01月 [査読無し][通常論文]
     
    オステオプロテジェリン(OPG)欠損マウスでは、破骨細胞形成が促進されるだけでなく、骨芽細胞による骨形成も亢進される。そのため、骨改造も亢進するため骨基質には複雑なセメントラインが形成されるが、このことは骨細胞・骨細管系の配列を著しく不規則にしてしまい、骨細胞によるsclerostin産生が低下していることが明らかとなった。OPG欠損マウスは、破骨細胞形成系に関与するモデルマウスだけでなく、骨代謝研究に広く応用できると考えられる。(著者抄録)
  • 下村 淳子[黒木], 松田 貴絵, 網塚 憲生, 下岡 正八 小児歯科学雑誌 48 (5) 569 -569 2010年11月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 山本 恒之 Journal of Oral Biosciences 52 (Suppl) 88 -88 2010年09月 [査読無し][通常論文]
  • 増木 英郎, 李 敏啓, 郭 頴, 長谷川 智香, 柳 鋳晟, 鈴木 礼子, 織田 公光, 山本 恒之, 川浪 雅光, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 102 -102 2010年09月 [査読無し][通常論文]
  • 李 敏啓, 長谷川 智香, 柳 鋳晟, 増木 英郎, 佐々木 宗輝, 田幡 千尋, 山本 恒之, 小守 壽文, 織田 公光, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 102 -102 2010年09月 [査読無し][通常論文]
  • 佐々木 宗輝, 李 敏啓, 増木 英郎, 長谷川 智香, 田幡 千尋, 織田 公光, 山本 恒之, 井上 農夫男, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 104 -104 2010年09月 [査読無し][通常論文]
  • 郭 頴, 李 敏啓, 柳 鋳晟, 増木 英郎, 織田 公光, 山本 恒之, 川浪 雅光, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 104 -104 2010年09月 [査読無し][通常論文]
  • 長谷川 智香, 李 敏啓, 井上 貴一朗, 田幡 千尋, 佐々木 宗輝, 鈴木 礼子, 織田 公光, 山本 恒之, 大畑 昇, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 124 -124 2010年09月 [査読無し][通常論文]
  • 田幡 千尋, 李 敏啓, 長谷川 智香, 佐々木 宗輝, 織田 公光, 山本 恒之, 工藤 明, 飯田 順一郎, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 124 -124 2010年09月 [査読無し][通常論文]
  • 羽下 麻衣子, 野澤 佳世子[井上], 李 敏啓, 吉江 紀夫, 網塚 憲生, 前田 健康 Journal of Oral Biosciences 52 (Suppl) 128 -128 2010年09月 [査読無し][通常論文]
  • 山本 恒之, 李 敏啓, 郭 頴, 柳 鋳晟, 増木 英郎, 長谷川 智香, 鈴木 礼子, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 147 -147 2010年09月 [査読無し][通常論文]
  • 宮本 幸奈, 李 敏啓, 長谷川 智香, 柳 鋳晟, 郭 頴, 増木 英郎, 織田 公光, 宇田川 信之, 山本 恒之, 網塚 憲生 Journal of Oral Biosciences 52 (Suppl) 189 -189 2010年09月 [査読無し][通常論文]
  • 網塚 憲生, 郭 頴, 長谷川 智香, 柳 鋳晟, 佐々木 宗輝, 沢村 祥子, 鈴木 礼子, 山本 恒之, 李 敏啓 THE BONE 24 (3) 229 -233 2010年08月 [査読無し][通常論文]
     
    骨細胞・骨細管系は骨基質内に細胞突起を張り巡らせることにより細胞性ネットワークを形成している。骨細胞の機能としては、力学的負荷の感知、骨基質ミネラルなどの物質輸送、骨改造調節などがあげられ、特に、規則的配列を示す骨細管系は、力学的負荷の感知や物質輸送にも都合がよい構造を示すと思われる。また、骨細胞から産生されるFGF23、sclerostin、DMP-1は骨基質の性状、すなわち骨質にも影響を与えると考えられる。(著者抄録)
  • 網塚 憲生, 李 敏啓, 長谷川 智香, 山本 恒之 Clinical Calcium 20 (8) 1272 -1275 2010年07月 [査読無し][通常論文]
  • 田辺 英幸, 高山 一成, 西山 尚志, 島崎 雅司, 喜井 勲, 李 敏啓, 網塚 憲生, 勝部 憲一, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 28回 221 -221 2010年07月 [査読無し][通常論文]
  • 郭 穎, 李 敏啓, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 28回 243 -243 2010年07月 [査読無し][通常論文]
  • 李 敏啓, 郭 頴, 宇田川 信之, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 28回 245 -245 2010年07月 [査読無し][通常論文]
  • 李 敏啓, 郭 穎, 小守 壽文, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 28回 253 -253 2010年07月 [査読無し][通常論文]
  • 多部田 康一, 高橋 直紀, 前川 知樹, 山崎 和久, 網塚 憲生 新潟歯学会雑誌 40 (1) 79 -81 2010年06月 [査読無し][通常論文]
  • 卵巣摘出ラットの下顎歯槽骨における骨細胞再生蛋白の局在について
    郭 頴, 李 敏啓, 柳 鋳晟, 山本 恒之, 川浪 雅光, 網塚 憲生 北海道歯学雑誌 31 (1) 20 -20 2010年06月 [査読無し][通常論文]
  • 李 敏啓, 原 久仁子, 秋山 康博, 織田 公光, 網塚 憲生 解剖学雑誌 85 (2) 56 -56 2010年06月 [査読無し][通常論文]
  • 宮本 幸奈, 李 敏啓, 長谷川 智香, 柳 鋳晟, 郭 頴, 鈴木 礼子, 沢村 祥子, 宇田川 信之, 山本 恒之, 網塚 憲生 日本骨形態計測学会雑誌 20 (1) S73 -S73 2010年05月 [査読無し][通常論文]
  • 李 敏啓, 長谷川 智香, 柳 鋳晟, 郭 頴, 鈴木 礼子, 沢村 祥子, 山本 恒之, 網塚 憲生 日本骨形態計測学会雑誌 20 (1) S74 -S74 2010年05月 [査読無し][通常論文]
  • 郭 穎, 李 敏啓, 柳 鋳晟, 山本 恒之, 川浪 雅光, 網塚 憲生 日本骨形態計測学会雑誌 20 (1) S75 -S75 2010年05月 [査読無し][通常論文]
  • 山本 恒之, 李 敏啓, 網塚 憲生 日本骨形態計測学会雑誌 20 (1) S80 -S80 2010年05月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 鈴木 晶子, 芳澤 享子, 齊藤 力, 前田 健康 日本歯科医学会誌 29 118 -118 2010年03月 [査読無し][通常論文]
  • 佐々木 理絵, 李 敏啓, 齋藤 直朗, Freitas Paulo, 織田 公光, 網塚 憲生 解剖学雑誌 85 (Suppl.) 201 -201 2010年03月 [査読無し][通常論文]
  • 郭穎, 李敏啓, 網塚憲生 日本骨代謝学会学術集会プログラム抄録集 28th 2010年
  • LI Minqi, HASEGAWA Tomoka, MASUKI Hideo, LIU Zhusheng, GUO Ying, SUZUKI Reiko, YAMAMOTO Tsuneyuki, FREITAS Paulo HL, AMIZUKA Norio Journal of oral biosciences 52 (2) 94 -99 2010年 [査読無し][通常論文]
     
    Mineralization of bone matrix starts with the formation of matrix vesicles secreted by osteoblasts, which subsequently grow into mineralized nodules. These nodules are globular structures formed by mineral crystals assembled in a radial fashion, and appear to be the final inducers of collagen mineralization. Despite its apparent simplicity, the course of bone mineralization is tightly controlled by many factors in the microenvironment. In this review, we will present our recent experiments on the general processes of bone mineralization. First, we will discuss the ultrastructural alteration of mineral crystals in the context of magnesium (Mg) insufficiency, which causes an elevation of calcium (Ca) concentrations in bone and of the pure form of hydroxyapatite. Such elevated Ca concentrations also induced premature collagen mineralization without the mediation of mineralized nodules. This could mean that the availability of elements, such as Mg, in the bone microenvironment is important for the in vivo generation of the pure form of hydroxyapatite. Next, we will examine collagen mineralization in the context of ascorbic acid insufficiency. Ascorbic acid is essential for collagen formation and, when it is not available, the typical, stout collagen microfibrils with striations cannot be identified. In their place, filamentous fibrils of collagen without striations, i. e., immature collagen microfibrils, are observed. In the osteoid, however, many mineralized nodules feature some sort of “mineral extensions” towards the immature microfibrils. It seems that collagen microfibrils, even if immature, serve as a scaffold for the mineralization of collagen fibrils. These findings reaffirm that a sophisticated set of regulatory steps is necessary for proper mineralization in bone.
  • 李 敏啓, 郭 頴, 柳 鋳晟, 鈴木 礼子, 山本 恒之, 網塚 憲生 北海道歯学雑誌 30 (2) 119 -121 2009年12月15日 [査読無し][通常論文]
  • 李 敏啓, 郭 頴, 柳 鋳晟, 鈴木 礼子, 山本 恒之, 網塚 憲生 北海道歯学雑誌 30 (2) 119 -121 2009年12月 [査読無し][通常論文]
  • 網塚 憲生 北海道歯学雑誌 30 (2) 131 -131 2009年12月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 鈴木 晶子, 芳澤 享子, 齊藤 力 日本再生歯科医学会誌 7 (1) 90 -90 2009年12月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 郭 頴, 柳 鋳晟, 鈴木 礼子, 山本 恒之 Clinical Calcium 19 (12) 1788 -1796 2009年11月 [査読無し][通常論文]
     
    ビタミンK2によりカルボキシル化されたオステオカルシンは、ハイドロキシアパタイト結晶のカルシウム(Ca)との結合性が上昇し、骨基質の性状が向上するものと考えられる。例えば、低マグネシウム飼育ラットにおいてコラーゲン性石灰化は異常を示すが、低マグネシウムラットにビタミンK2を投与すると、石灰化球とコラーゲン線維の会合を保つことで正常な石灰化を回復させることができた。一方、ビタミンK2の結抗剤であるワーファリンを投与すると、骨基質におけるオステオカルシンの局在性が低下するばかりでなく、本来、石灰化球を構成するはずの石灰化結晶塊がほぐれて散在してしまう。従って、ビタミンK2によってカルボキシル化されたオステオカルシンなどのグラタンパクは、骨における正常な石灰化に重要な役割を演ずると考えられる。(著者抄録)
  • Ubaidus Sobhan, Sultana Sara, 織田 公光, 小島 拓, 李 敏啓, 鈴木 礼子, 柳 鋳晟, Freitas Paulo, 網塚 憲生, 小澤 英浩, 下村 淳子[黒木] THE BONE 23 (4) 361 -366 2009年10月 [査読無し][通常論文]
  • 李 敏啓, 長谷川 智香, 郭 頴, 柳 鋳晟, 鈴木 礼子, 山本 恒之, 網塚 憲生 骨粗鬆症治療 8 (4) 267 -270 2009年10月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 芳澤 享子, 齊藤 力 日本口腔科学会雑誌 58 (4) 274 -275 2009年09月 [査読無し][通常論文]
  • 網塚 憲生, Ubaidus Sobhan, Sultana Sara, Freitas Paulo, 李 敏啓 腎と骨代謝 22 (3) 185 -194 2009年07月 [査読無し][通常論文]
     
    骨基質には,リン酸カルシウムといった石灰化結晶だけでなく多くの有機成分が存在するが,そのほとんどがコラーゲン線維である.幼若な骨基質ではさまざまな方向を示すコラーゲン線維が観察されるが,緻密骨である皮質骨などを観察すると,コラーゲン線維が束状となり規則的な走行を示す.また,電顕観察を行うと,コラーゲン細線維の横紋構造に一致してリボン状の石灰化結晶塊が配列しており,コラーゲン線維の内部構造と石灰化結晶の配列は無関係ではない.しかしながら,未だにコラーゲン線維の石灰化過程についてはさまざまな説が提唱されており,今後の詳細な研究が期待される.(著者抄録)
  • 武田 聡, 斎藤 一史, 田村 達也, 高橋 文明, 李 敏啓, Luiz de Freitas Paulo Henrique, 網塚 憲生, 尾形 悦郎 日本骨代謝学会学術集会プログラム抄録集 27回 214 -214 2009年07月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 田村 正人, 織田 公光 Clinical Calcium 19 (7) 935 -943 2009年06月 [査読無し][通常論文]
     
    副甲状腺ホルモン(parathyroid hormone:PTH)および副甲状腺ホルモン関連ペプチド(parathyroid hormone-related peptide:PTHrP)は同一の受容体であるPTH/PTHrP受容体に作用することが知られており、PTHは生後の骨代謝に、PTHrPは胎生期の軟骨に対して主に影響を及ぼす。PTHは骨芽細胞前駆細胞の増殖促進に作用し、その後の細胞分化・機能には破骨細胞とのカップリングが必要になることが示唆されている。また、軟骨形成におけるPTHrPのC末端領域の作用も明らかにされつつある。ここでは、近年報告されたPTHおよびPTHrPの骨・軟骨における作用について総説する。(著者抄録)
  • 網塚 憲生, Ubaidus Sobhan, Freitas Paulo, Sultana Sara, 李 敏啓 日本臨床 67 (5) 868 -871 2009年05月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 19 (1) S31 -S31 2009年05月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本骨形態計測学会雑誌 19 (1) S54 -S54 2009年05月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 解剖学雑誌 84 (Suppl.) 71 -71 2009年03月 [査読無し][通常論文]
  • 成松 花弥, 李 敏啓, 織田 公光, 網塚 憲生 解剖学雑誌 84 (Suppl.) 199 -199 2009年03月 [査読無し][通常論文]
  • 網塚 憲生 新潟歯学会雑誌 38 (2) 129 -129 2008年12月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, Freitas Paulo HL, Ubaidus Sobhan, Sultana Sara, 小林 正敏, 原 久仁子, 秋山 康博, 赤羽 章司, 小澤 英浩, 前田 健康 THE BONE 22 (6) 687 -692 2008年11月 [査読無し][通常論文]
  • 網塚 憲生, Ubaidus Sobhan, Freitas Paulo, Sultana Sara, 李 敏啓 THE BONE 22 (6) 729 -735 2008年11月 [査読無し][通常論文]
     
    骨細胞は、骨基質内で骨基質ミネラルを調節するために都合のよい骨細管系を発達させており、FGF23、sclerostin、DMP-1といった因子を分泌することによって、全身性ミネラル、骨改造、骨基質石灰化を調節している。しかし一方で、骨細胞・骨細管系がどのような微細環境を形成し、また、どのようにミネラルの流出・流入を可能にするかについては今後の解明に期待がもたれる。(著者抄録)
  • 喜井 勲, 西山 尚志, 原田 伊知郎, 李 敏啓, 松本 健一, 斉藤 充, 松本 裕子, 市川 知宏, 高山 一成, 田辺 英幸, 高久田 和夫, 網塚 憲生, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 26回 172 -172 2008年10月 [査読無し][通常論文]
  • H. Segawa, A. Onitsuka, Y. Tomoe, J. Furutani, I. Kaneko, F. Aranami, S. Kuwahara, M. Li, N. Amizuka, M. Matsumoto, M. Ito, K. Miaymoto JOURNAL OF BONE AND MINERAL RESEARCH 23 S127 -S127 2008年09月 [査読無し][通常論文]
  • 網塚 憲生 The Quintessence 27 (9) 46 -47 2008年09月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 芳澤 享子, 齊藤 力 日本口腔科学会雑誌 57 (4) 553 -553 2008年09月 [査読無し][通常論文]
  • 網塚 憲生, Ubaidus Sobhan, 小島 拓, Freitas Paulo HL, 李 敏啓, 下村 淳子 腎と骨代謝 21 (3) 183 -190 2008年07月 [査読無し][通常論文]
     
    骨細胞は骨基質に埋め込まれた細胞であり,無数に張り巡らされた細胞突起で細胞性ネットワークを形成している.この細胞性ネットワークは,グループとして機能する可能性から骨細胞・骨細管系と呼ばれ,おもに,物質輸送,力学負荷感知,骨代謝回転の調節に影響を与えると考えられている.骨細胞・骨細管系は発生段階でみられる線維性骨などでは不規則な構造を示し,骨基質がより成熟・綴密化した骨では規則的な配列を示すようになる.このような骨細胞・骨細管系の再配列を可能にするのは骨改造である.成熟した骨の皮質骨では骨単位において,また,骨梁ではBSU(basic structural unit)において,機能的な骨細胞・骨細管系が発達しているとみなされる.ここでは,骨細胞・骨細管系における顕微解剖学的なわれわれの知見について述べたい.(著者抄録)
  • 李 敏啓, Ubaidus Sobhan, Freitas Paulo HL, 小澤 英浩, 網塚 憲生 THE BONE 22 (4) 451 -455 2008年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 18 (2) S37 -S37 2008年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 18 (2) S47 -S47 2008年07月 [査読無し][通常論文]
  • 李 敏啓, 原 久仁子, 秋山 康博, 網塚 憲生 日本骨形態計測学会雑誌 18 (2) S88 -S88 2008年07月 [査読無し][通常論文]
  • OVXラットの柵状織においてED-71は骨微小モデリングを促進し、破骨細胞数を減少させる(ED-71 drives bone minimodeling and reduces osteoclastic number in trabeculae of OVX rats)
    Henrique Luiz de, Freitas Paulo, 李 敏啓, 齋藤 直朗, 高木 律男, 網塚 憲生 日本骨形態計測学会雑誌 18 (2) S90 -S90 2008年07月 [査読無し][通常論文]
  • Freitas Paulo HL, 小島 拓, Ubaidus Sobhan, 李 敏啓, 高木 律男, 前田 健康, 織田 公光, 小澤 英浩, 網塚 憲生 THE BONE 22 (3) 247 -251 2008年06月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, Freitas Paulo HL, Ubaidus Sobhan THE BONE 22 (3) 267 -274 2008年06月 [査読無し][通常論文]
     
    骨芽細胞は骨基質蛋白合成とその石灰化を行う一方、自ら産生した骨基質に埋まり骨細胞へと分化してゆく。骨芽細胞と骨細胞は互いに細胞突起を介した細胞性ネットワークを形成し、機能的なグループを作ると考えられる。前骨芽細胞は骨芽細胞の前駆細胞として存在するだけでなく、積極的に骨代謝に関与する細胞であり、in vivoではいくつかの表現型が存在する。また、破骨細胞は骨吸収を営む細胞であり、酸と基質蛋白分解酵素を分泌して骨基質を吸収する。破骨細胞は骨芽細胞系細胞の支持によって形成されるが、一方で、破骨細胞は骨芽細胞の活性や機能に対して影響を与えると推測される。ここでは、骨の細胞形態学を概説した。(著者抄録)
  • 李 敏啓, 網塚 憲生 Clinical Calcium 18 (7) 1029 -1030 2008年06月 [査読無し][通常論文]
  • 瀬川 博子, 鬼塚 朱美, 伊藤 美紀子, 松本 満, 網塚 憲生, 宮本 賢一 日本腎臓学会誌 50 (3) 307 -307 2008年04月 [査読無し][通常論文]
  • 池田 恭治, 辰巳 佐和子, 石井 清朗, 小林 利寛, 竹下 淳, 網塚 憲生, Li Minqi, 河野 憲二, 伊東 昌子, Ikeda Kyoji, Tatsumi Sawako, Ishii Kiyoaki, Kobayashi Toshihiro, Takeshita Sunao, Amizuka Norio, Li Minqi, Kono Kenji, Ito Masako 宇宙利用シンポジウム 第24回 平成19年度 = Space Utilization Research: Proceedings of the Twenty-fourth Space Utilization Symposium 24th 234 -235 2008年03月 
    資料番号: AA0063706060
  • 齋藤 直朗, 李 敏啓, 辰巳 佐和子, 池田 恭治, 網塚 憲生 解剖学雑誌 83 (Suppl.) 160 -160 2008年03月 [査読無し][通常論文]
  • 李 敏啓, 原 久仁子, 秋山 康博, 網塚 憲生 解剖学雑誌 83 (Suppl.) 160 -160 2008年03月 [査読無し][通常論文]
  • 李 敏啓, 齋藤 直朗, 網塚 憲生 Clinical Calcium 18 (3) 328 -335 2008年02月 [査読無し][通常論文]
     
    ステロイド性骨粗鬆症は、従来、骨組織における骨芽細胞の骨形成低下、破骨細胞の骨吸収亢進を示すと考えられてきた。しかし、骨密度(bone mineral density:BMD)が減少していなくとも骨折頻度が高いことから、ステロイド性骨粗鬆症は骨質が低下していることが指摘されてきた。ステロイド性骨粗鬆症の骨質低下のメカニズムはいまだに明らかにされていないが、近年、ステロイドによる骨細胞のアポトーシスやそれに伴う骨基質ミネラル維持・力学感知の阻害が、骨質低下の原因の一つであることが推測されている。(著者抄録)
  • Hidehiro Ozawa, Kazuto Hoshi, Norio Amizuka Journal of Oral Biosciences 50 (1) 1 -14 2008年 [査読無し][通常論文]
     
    This review aims to propose a more consistent interpretation of the concept of matrix vesicle mineralization, and includes current views on biomineralization in general. We first focused on the ultra-structural, cytochemical and biophysical characteristics of matrix vesicles which confirm the assertion that matrix vesicles are the initial sites of bone biomineralization. We offer a scheme describing how cells regulate biomineralization by means of matrix vesicles during bone formation and remodeling and then discuss the subsequential biomineralization, the growth of hydroxyapatite crystals by direct extension, as well as secondary nucleation from previously formed seams of bone and collagen mineralization.
  • 小島 拓, 網塚 憲生, 芳澤 享子, 齊藤 力 日本口腔科学会雑誌 57 (1) 180 -180 2008年01月 [査読無し][通常論文]
  • 網塚 憲生, Freitas Paulo H.L., 小島 拓, Ubaidus Sobhan, 尚 光偉 日本臨床 65 (増刊9 新時代の骨粗鬆症学) 194 -201 2007年11月 [査読無し][通常論文]
  • 網塚 憲生, Freitas Paulo HL, Ubaidus Sobhan, 齋藤 直朗, 李 敏啓 THE BONE 21 (6) 723 -729 2007年11月 [査読無し][通常論文]
     
    前骨芽細胞は骨芽細胞の前駆細胞であるとともに、それ自体、副甲状腺ホルモン(PTH)などに対して反応性を示す細胞である。また、前骨芽細胞の形態学的なphenotypeは1種類ではなく、ER-rich cell,endocytic cell,undifferentiated cell,PT-cellなどが報告されている。これら前骨芽細胞の微細構造と想定される役割について解説する。(著者抄録)
  • N. Amizuka, T. Komori, K. Oda, D. Goldtzman, A. Karaplis, M. Li JOURNAL OF BONE AND MINERAL RESEARCH 22 S156 -S156 2007年09月 [査読無し][通常論文]
  • H. Segawa, A. Onitsuka, M. Kuwahata, F. Aranami, J. Furutani, I. Kaneko, Y. Tomoe, S. Kuwahara, N. Amizuka, M. Matsumoto, K. Miyamoto JOURNAL OF BONE AND MINERAL RESEARCH 22 S34 -S34 2007年09月 [査読無し][通常論文]
  • P. H. Luiz de Freitas, M. Li, T. Ninomiya, M. Nakamura, K. Oda, R. Takagi, N. Udagawa, T. Maeda, N. Amizuka JOURNAL OF BONE AND MINERAL RESEARCH 22 S107 -S107 2007年09月 [査読無し][通常論文]
  • U. Sobhan, M. Li, K. Oda, T. Maeda, R. Takagi, N. Amizuka JOURNAL OF BONE AND MINERAL RESEARCH 22 S377 -S377 2007年09月 [査読無し][通常論文]
  • M. Li, K. Hara, Y. Akiyama, N. Amizuka JOURNAL OF BONE AND MINERAL RESEARCH 22 S478 -S478 2007年09月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生 Journal of Oral Biosciences 49 (Suppl.) 97 -97 2007年08月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 天野 修, 前田 健康 Journal of Oral Biosciences 49 (Suppl.) 110 -110 2007年08月 [査読無し][通常論文]
  • 網塚 憲生, 小守 壽文, 李 敏啓 Journal of Oral Biosciences 49 (Suppl.) 151 -151 2007年08月 [査読無し][通常論文]
  • 網塚 憲生 Osteoporosis Japan 15 (3) 563 -563 2007年07月 [査読無し][通常論文]
  • 李 敏啓, 原 久仁子, 秋山 康博, 網塚 憲生 日本骨形態計測学会雑誌 = Journal of Japanese Society of Bone Morphometry 17 (1) S66 2007年06月30日 [査読無し][通常論文]
  • 李 敏啓, 小守 壽文, 網塚 憲生 日本骨形態計測学会雑誌 = Journal of Japanese Society of Bone Morphometry 17 (1) S74 2007年06月30日 [査読無し][通常論文]
  • 網塚 憲生, Freitas Paulo H.L., Ubaidus Sobhan 日本骨代謝学会学術集会プログラム抄録集 25回 105 -105 2007年06月 [査読無し][通常論文]
  • 網塚 憲生, Ubaidus Sobhan, Freitas Paulo H.L. 日本骨代謝学会学術集会プログラム抄録集 25回 151 -151 2007年06月 [査読無し][通常論文]
  • 網塚 憲生, 小守 壽文, 李 敏啓 日本骨代謝学会学術集会プログラム抄録集 25回 180 -180 2007年06月 [査読無し][通常論文]
  • 骨細胞の分布/陰窩-細管系に関する組織学的評価(Histochemical assessments on the distribution of osteocytes / lacunar canalicular system)
    Ubaidus Sobhan, 李 敏啓, 小島 拓, 織田 公光, 前田 健康, 高木 律男, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 25回 203 -203 2007年06月 [査読無し][通常論文]
  • 喜井 勲, 網塚 憲生, 李 敏啓, 中川 一樹, 高山 一成, 島崎 雅司, 田辺 英幸, 西山 尚志, 松本 裕子, 高久田 和夫, 市川 知広, 原田 伊知郎, 赤池 敏宏, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 25回 238 -238 2007年06月 [査読無し][通常論文]
  • 李 敏啓, 原 久仁子, 秋山 康博, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 25回 249 -249 2007年06月 [査読無し][通常論文]
  • 骨芽細胞系細胞のPTHによる間歇的刺激は骨芽細胞の存在に非依存性である(Intermittent PTH stimulates cells of the osteoblastic lineage independently of osteoblastic presence)
    Freitas Paulo, 李 敏啓, 二宮 禎, 中村 美どり, 織田 公光, 高木 律男, 宇田川 信之, 前田 健康, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 25回 289 -289 2007年06月 [査読無し][通常論文]
  • 【ビタミンKの今日的意義と役割】基礎編 ビタミンK2の骨基質に対する微細構造学的メカニズム
    網塚 憲生, 李 敏啓 Pharma Medica 25 (Suppl.) 12 -18 2007年06月 [査読無し][通常論文]
  • 細胞接着・細胞外基質・細胞間相互作用 骨芽細胞の石灰化におけるcaveolin-1の重要な役割(Caveolin-1 plays an important role in the calcification of osteoblasts)
    澤田 直樹, 竹谷 豊, 網塚 憲生, 山本 浩範, 武田 英二 日本発生生物学会・日本細胞生物学会合同大会要旨集 40回・59回 51 -51 2007年05月 [査読無し][通常論文]
  • マウスの頭蓋顔面骨格形成時におけるFGF受容体1のカドヘリン-11による安定化(Cadherin-11 stabilizes FGF Receptor 1 during craniofacial skeletal development in mice)
    喜井 勲, 徐 方, 下村 淳子, 網塚 憲生, 常 智杰, 工藤 明 日本発生生物学会・日本細胞生物学会合同大会要旨集 40回・59回 169 -169 2007年05月 [査読無し][通常論文]
  • 李 敏啓, 原 久仁子, 秋山 康博, 網塚 憲生 日本骨形態計測学会雑誌 17 (1) S66 -S66 2007年05月 [査読無し][通常論文]
  • 李 敏啓, 小守 壽文, 網塚 憲生 日本骨形態計測学会雑誌 17 (1) S74 -S74 2007年05月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 下村 淳子 医学のあゆみ 221 (1) 5 -13 2007年04月07日 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 下村 淳子 医学のあゆみ 221 (1) 5 -13 2007年04月 [査読無し][通常論文]
     
    骨の構造は身体の支柱として、また血清カルシウムなどのミネラル調節に対応した機能を果たす。骨組織に存在する骨芽細胞、破骨細胞、骨細胞は、それらの分化・形成あるいは細胞活性にたがいに影響を及ぼしあいながら機能している。骨芽細胞は骨形成を担う細胞であり、骨基質蛋白合成と基質小胞を介した石灰化を誘導するが、自ら産生した骨基質に埋まり骨細胞へと分化をなし遂げる。骨芽細胞と骨細胞はたがいに細胞突起を介した細胞性ネットワークを形成し、機能的なグループをつくっていると考えられる。一方、破骨細胞は骨吸収を営む細胞であり、酸と基質蛋白分解酵素を分泌して骨基質を吸収する。破骨細胞は骨芽細胞系細胞の支持によって形成されるが、一方で、破骨細胞は骨芽細胞の活性や機能に対して影響を与えると推測される。このような破骨細胞と骨芽細胞は古い骨基質と新しい骨基質へと改変する骨改造を営むが、それは単に基質だけではなく、骨細胞の分布を規則的に構築する現象でもある。ここでは骨の構造と機能を概説した。(著者抄録)
  • 瀬川 博子, 鬼塚 朱美, 塩沢 和代, 巴 由佳, 桑波田 雅士, 伊藤 美紀子, 松本 満, 網塚 憲生, 宮本 賢一 日本腎臓学会誌 49 (3) 236 -236 2007年04月 [査読無し][通常論文]
  • 小島 拓, 齊藤 力, 網塚 憲生 Clinical Calcium 17 (5) 795 -796 2007年04月 [査読無し][通常論文]
  • 中川一樹, 喜井勲, 松本裕子, 西山尚志, LI Minqi, 網塚憲生, 高久田和夫, 工藤明 生化学 2007年
  • 網塚 憲生, Ubaidus Sobhan, 李 敏啓 骨粗鬆症治療 6 (1) 33 -40 2007年01月 [査読無し][通常論文]
     
    骨基質の石灰化は「骨密度」だけでは評価しきれない点が多く、骨基質の有機成分や細胞との関連性を考える必要がある。骨芽細胞がおこなう初期石灰化は、基質小胞性石灰化とコラーゲン性石灰化の過程に分けられるが、ともにカルシウムやリン酸を供給する酵素やプロテオグリカンなどの有機成分などによる調節を受ける。皮質骨においては、コラーゲン線維は幾何学的に規則的な配列を示し、また、骨細胞の骨細管もコラーゲン線維と関連して規則的な走行を示す。この形態学的特徴は骨細胞・骨細管系のmechanosensor,chemotransducerとして効率的に機能するために都合の良い構造と考えられる。(著者抄録)
  • 李 敏啓, 網塚 憲生, Henrique Paulo, Ubaidus Sobhan, 小島 拓, 小澤 英浩 THE BONE 21 (1) 3 -7 2007年01月 [査読無し][通常論文]
  • 小澤 英浩, 中村 浩彰, 李 敏啓, 網塚 憲生 THE BONE 21 (1) 21,13 -34,13 2007年01月 [査読無し][通常論文]
     
    骨は生体の中で最も細胞外基質が豊富な組織であり、そこにヒドロキシアパタイト結晶が特異的に沈着することによって特徴付けられている。したがって、骨質の要因の多くは「骨基質」に由来すると考えられるが、その調節は「骨の細胞群」が行っている。骨基質における石灰化は、基質小胞性石灰化とコラーゲン性石灰化の過程に分けられるが、それらは非コラーゲン性蛋白質やプロテオグリカンなどの有機成分による調節を受ける。一方、骨基質に多量に含まれるコラーゲン線維は、皮質骨では規則的な配列を示すことでヒドロキシアパタイト結晶の沈着・配向とともに力学的負荷に対する抵抗を示す。(著者抄録)
  • 李 敏啓, Ubaidus Sobhan, 網塚 憲生 THE BONE 21 (1) 59 -65 2007年01月 [査読無し][通常論文]
     
    骨は生体の中で最も細胞外基質が豊富な組織である。したがって、骨質の要因の多くは「骨基質」に由来すると考えられる。骨基質における石灰化は、基質小胞性石灰化とコラーゲン性石灰化の過程に分けられるが、非コラーゲン性蛋白やプロテオグリカンなどの有機成分による調節を受ける。一方、骨基質に多量に含まれるコラーゲン線維は、皮質骨では規則的な配列を示すことで力学的負荷に対する抵抗を示す。また、石灰化骨基質に張り巡らされた骨細胞・骨細管系は内部歪みの感知や物質輸送に関与することで骨質に寄与すると考えられる。(著者抄録)
  • 網塚 憲生, Sobhan Ubaidus, 李 敏啓 骨粗鬆症治療 6 (1) 33 -40 2007年01月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生 新潟歯学会雑誌 36 (2) 317 -317 2006年12月 [査読無し][通常論文]
  • 青木 由香莉, 李 敏啓, Sobhan Ubaidus, 小澤 英浩, 前田 健康, 吉江 弘正, 山崎 和久, 網塚 憲生 新潟歯学会雑誌 36 (2) 317 -318 2006年12月 [査読無し][通常論文]
  • 小島 拓, 李 敏啓, 齊藤 力, 前田 健康, 網塚 憲生 新潟歯学会雑誌 36 (2) 318 -318 2006年12月 [査読無し][通常論文]
  • 広瀬 聡, 李 敏啓, 坂上 直子, 小島 拓, 織田 公光, 網塚 憲生, 齊藤 力 新潟歯学会雑誌 36 (2) 318 -319 2006年12月 [査読無し][通常論文]
  • 坂上 直子, 李 敏啓, Sobhan Ubaidus, Henrique Paulo, 小島 拓, 織田 公光, 小澤 英浩, 網塚 憲生 新潟歯学会雑誌 36 (2) 319 -319 2006年12月 [査読無し][通常論文]
  • Shigeki Oshima, Shin Onodera, Norio Amizuka, Minqi Li, Kazuharu Irie, Satoshi Watanabe, Yoshikazu Koyama, Jun Nishihira, Kazunori Yasuda, Akio Minami FEBS LETTERS 580 (27) 6519 -6519 2006年11月 [査読無し][通常論文]
  • S. Tatsumi, N. Amizuka, M. Li, K. Ishii, K. Kohno, M. Ito, S. Takeshita, K. Ikeda JOURNAL OF BONE AND MINERAL RESEARCH 21 S18 -S18 2006年09月 [査読無し][通常論文]
  • P. H. L. Freitas, N. Amizuka, M. Li, R. Takagi, T. Maeda JOURNAL OF BONE AND MINERAL RESEARCH 21 S321 -S321 2006年09月 [査読無し][通常論文]
  • T. Kojima, N. Amizuka, M. Li, N. Kosaki, C. Saito, H. Takaishi, T. Maeda JOURNAL OF BONE AND MINERAL RESEARCH 21 S137 -S137 2006年09月 [査読無し][通常論文]
  • N. Kosaki, S. Kamekura, H. Kawaguchi, T. Kimura, Y. Okada, L. Minqi, N. Amizuka, Y. Toyama, J. D'Armiento, H. Takaishi JOURNAL OF BONE AND MINERAL RESEARCH 21 S82 -S82 2006年09月 [査読無し][通常論文]
  • N. Sawada, Y. Taketani, N. Amizuka, M. Isshiki, M. Ichikawa, C. Ogawa, K. Nashiki, E. Shuto, T. Sato, H. Yamamoto, H. Arai, E. Takeda JOURNAL OF BONE AND MINERAL RESEARCH 21 S334 -S334 2006年09月 [査読無し][通常論文]
  • 小島 拓, 李 敏啓, 齊藤 力, 前田 健康, 網塚 憲生 Journal of Oral Biosciences 48 (Suppl.) 126 -126 2006年09月 [査読無し][通常論文]
  • 吉羽 永子, 吉羽 邦彦, 興地 隆史, 斎藤 正寛, 横井 隆政, 細矢 明宏, 網塚 憲生, 小澤 英浩 Journal of Oral Biosciences 48 (Suppl.) 153 -153 2006年09月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 THE BONE 20 (5) 599 -604 2006年09月 [査読無し][通常論文]
     
    副甲状腺ホルモン関連ペプチド(PTHrP)は、1940年代に副甲状腺ホルモン(PTH)様の物質としてその存在が示唆され、1980年代にクローニングされている。PTHrPは、当初、原癌遺伝子として認識されていたが、その後に軟骨形成に関与すること、核小体移行を行うこと、骨芽細胞のsurvival因子として作用することなどが明らかにされてきた。ここでは軟骨に関与するPTHrP研究の20年を振り返ってみたい。(著者抄録)
  • ポリ乳酸プレートとハイドロキシアパタイト補填材併用による骨再生の組織化学的検索
    小島 拓, 網塚 憲生, 芳澤 享子, 齊藤 力 日本口腔外科学会雑誌 52 (Suppl.) 212 -212 2006年09月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 腎と骨代謝 19 (3) 199 -205 2006年07月 [査読無し][通常論文]
     
    1991年にクローニングされたPTH受容体(PTH-R1)はおもに骨芽細胞,軟骨細胞,腎臓の尿細管細胞などに存在し,PKA/cAMP経路とPLC経路によってそのシグナルを伝える.PTH-R1シグナルは骨・軟骨形成に重要であることが明らかにされているが,とくにPKA/cAMP経路を介して軟骨細胞の分化増殖を調節すると考えられる.PTHrPの軟骨細胞の増殖に対する作用はp57Kip2を介して,また増殖層から肥大化細胞層への分化に対してはIndian hedgehogを介して間接的に作用する.一方で,PTHrPは骨芽細胞から産生されautocrine/paracrine的に作用することで,骨芽細胞の増殖やアポトーシスの抑制に作用することが推測されている(著者抄録)
  • 鈴木 啓展, 大島 勇人, 織田 公光, 李 敏啓, 網塚 憲生, 吉江 弘正, 野田 政樹, 前田 健康, 小澤 英浩 THE BONE 20 (4) 395 -399 2006年07月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本骨形態計測学会雑誌 16 (2) S56 -S56 2006年07月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本骨代謝学会学術集会プログラム抄録集 24回 92 -92 2006年07月 [査読無し][通常論文]
  • 喜井 勲, 松本 裕子, Li Minqi, 田辺 英幸, 中川 一樹, 高山 一成, 東 由明, 高久田 和夫, 網塚 憲生, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 24回 170 -170 2006年07月 [査読無し][通常論文]
  • 田辺 英幸, 喜井 勲, 森山 明美, 李 敏啓, 網塚 憲生, 勝部 憲一, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 24回 171 -171 2006年07月 [査読無し][通常論文]
  • 李 敏啓, 石井 清朗, 辰巳 佐和子, 池田 恭治, 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 24回 185 -185 2006年07月 [査読無し][通常論文]
  • 澤田 直樹, 竹谷 豊, 網塚 憲生, 山本 浩範, 武田 英二 日本骨代謝学会学術集会プログラム抄録集 24回 185 -185 2006年07月 [査読無し][通常論文]
  • 瀬川 博子, 鬼塚 朱美, 塩沢 和代, 荒波 史, 古谷 順也, 伊藤 美紀子, 桑波田 雅士, 松本 満, 網塚 憲生, 宮本 賢一 日本骨代謝学会学術集会プログラム抄録集 24回 186 -186 2006年07月 [査読無し][通常論文]
  • 小崎 直人, 亀倉 暁, 川口 浩, 李 敏啓, 網塚 憲生, 戸山 芳昭, 高石 官成 日本骨代謝学会学術集会プログラム抄録集 24回 207 -207 2006年07月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 李 敏啓, 小崎 直人, 齊藤 力, 高石 官成, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 24回 219 -219 2006年07月 [査読無し][通常論文]
  • マウス乳癌細胞株4Tlの骨転移における骨破壊メカニズムの解析
    中村 智香, 小野 加津広, 神谷 貞浩, 赤津 拓彦, 久貝 信夫, 李 敏啓, 網塚 憲生, 大湾 一郎, 二村 典行, 和田 誠基 日本骨代謝学会学術集会プログラム抄録集 24回 254 -254 2006年07月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本骨形態計測学会雑誌 = Journal of Japanese Society of Bone Morphometry 16 (2) S56 2006年07月01日 [査読無し][通常論文]
  • 青木 由香莉, 李 敏啓, 網塚 憲生 日本骨形態計測学会雑誌 16 (2) S73 -S73 2006年07月 [査読無し][通常論文]
  • 李 敏啓, 石井 清朗, 辰巳 佐和子, 池田 恭治, 網塚 憲生 日本骨形態計測学会雑誌 16 (2) S69 -S69 2006年07月 [査読無し][通常論文]
  • DE FREITAS PAULO, HENRIQUE Luiz, AMIZUKA Norio, LI Minqi, TAKAGI Ritsuo, MAEDA Takeyasu 日本骨形態計測学会雑誌 = Journal of Japanese Society of Bone Morphometry 16 (2) S70 2006年07月01日 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 李 敏啓, 小崎 直人, 齊藤 力, 高石 官成, 前田 健康 日本骨形態計測学会雑誌 16 (2) S88 -S88 2006年07月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生 Clinical Calcium 16 (7) 1221 -1227 2006年06月 [査読無し][通常論文]
     
    副甲状腺ホルモン関連ペプチド(parathyroid hormone-related peptide:PTHrP)は軟骨細胞の分化増殖に重要な因子として明らかにされてきた.その作用は,軟骨細胞の増殖亢進と肥大化軟骨細胞への分化抑制によるものと考えられる.一方で,シグナル伝達の経路としては,GタンパクG(s)を介してアデニレートシクラーゼが活性化され,環状アデノシン-リン酸(cAMP)産生に伴うプロテインキナーゼA(PKA)の活性化シグナルと,Gタンパク質G(q)を介してホスホリパーゼC(PLC)が活性化され,イノシトールトリスリン酸(IP3)とジアシルグリセロール(DAG)の産生に伴うPKCの活性シグナルが大まかに考えられる.近年の遺伝子組み換えマウスの技術により,PLC活性を欠落した副甲状腺ホルモン(PTH)/PTHrP受容体を発現するマウス,および軟骨細胞特異的にG(s)αを欠損するマウスによって,cAMP/PKAは軟骨細胞の増殖亢進・分化抑制に,また,PLCは軟骨細胞の増殖抑制・分化促進に作用していることが明らかにされた.しかしながら,生理的にはcAMP/PKA活性がPLPC活性を上回ると考えられるが,N+/H+交換体調節因子(NHERF2)がPTH/PTHrP受容体のC末端に結合し,cAMP/PKA活性を抑制する機構も明らかにされている(著者抄録)
  • 坂上 直子, 李 敏啓, 網塚 憲生, 宇田川 信之, 小澤 英浩 THE BONE 20 (3) 263 -267 2006年05月 [査読無し][通常論文]
  • 池田 順行, Hossain Kazi Sazzad, 井上 佳世子, 鈴木 晶子, 高木 律男, 前田 健康, 網塚 憲生 日本顎関節学会雑誌 18 (1) 42 -42 2006年04月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生 Clinical Calcium 16 (4) 591 -597 2006年03月 [査読無し][通常論文]
     
    骨転移は骨吸収型,骨形成型,骨梁間型に大きく分けられるが,骨吸収型の骨転移は破骨細胞による骨吸収によるところが大きい.初期の骨転移巣では,腫瘍細胞が骨芽細胞,間質細胞,破骨細胞,血管内皮細胞などと混在するとともに,活発な細胞増殖を示す.このような初期転移巣では,破骨細胞前駆細胞がアルカリホスファターゼ/receptor activator of NF-κB ligand(RANKL)陽性骨芽細胞と細胞接触を示すことから,破骨細胞形成が既に始まっていることが推測された.一方で,一度形成された破骨細胞は,その細胞膜に発現するCD44と細胞外基質のオステオポンチンとの結合を介して,腫瘍巣の間質内を自由に移動できるものと考えられる.本稿では,腫瘍巣の増大を可能にすると思われる破骨細胞性骨吸収,血管新生,基質分解など組織病理学的な微細環境について述べたい(著者抄録)
  • 中村智香, 小野加津広, 神谷貞浩, 赤津拓彦, 久貝信夫, 李敏啓, 網塚憲生, 大湾一郎, 二村典行, 和田誠基 日本骨代謝学会学術集会プログラム抄録集 24th 2006年
  • 山田千積, 山田祐一郎, 月山克史, 原田範雄, 李敏啓, 網塚憲生, 高橋直之, 宇田川信之, 田中清, 稲垣暢也, 清野裕, 清野裕 日本骨代謝学会学術集会プログラム抄録集 24th 2006年
  • LI Minqi, 網塚憲生 Clinical Calcium 16 (7) 2006年
  • LI Minqi, LI Minqi, 網塚憲生 Clinical Calcium 16 (4) 2006年
  • 李 敏啓, 網塚 憲生, 前田 健康, 島村 拓也, 小澤 英浩 THE BONE 20 (1) 3 -7 2006年01月 [査読無し][通常論文]
  • 顎関節関節腔形成における血管内皮細胞とマクロファージの動態
    鈴木 晶子, 野澤 佳世子[井上], 網塚 憲生, 前田 健康 新潟歯学会雑誌 35 (2) 246 -246 2006年01月 [査読無し][通常論文]
  • 骨芽細胞の活性に及ぼす破骨細胞の影響(第一報) op/opマウスを用いた組織化学的検討
    坂上 直子, 網塚 憲生, 李 敏啓, 前田 健康 新潟歯学会雑誌 35 (2) 257 -257 2006年01月 [査読無し][通常論文]
  • 骨芽細胞の活性に及ぼす破骨細胞の影響(第二報) c-src遺伝子欠損マウスを用いた組織化学的検討
    坂上 直子, 網塚 憲生, 李 敏啓, Freitas Paulo H.L., 前田 健康 新潟歯学会雑誌 35 (2) 257 -257 2006年01月 [査読無し][通常論文]
  • ポリ乳酸プレートおよびhydroxyapatite補填材併用による骨再生の組織化学的検索
    小島 拓, 網塚 憲生, 鈴木 晶子, 芳澤 享子, 齊藤 力, 前田 健康 新潟歯学会雑誌 35 (2) 264 -264 2006年01月 [査読無し][通常論文]
  • チタンインプラント植立時に併用したα-TCP系自己硬化型補填材が骨再生に及ぼす影響
    中舘 正芳, 網塚 憲生, 野村 修一, 前田 健康 新潟歯学会雑誌 35 (2) 265 -265 2006年01月 [査読無し][通常論文]
  • Y Taguchi, N Amizuka, M Nakadate, H Ohnishi, N Fujii, K Oda, S Nomura, T Maeda BIOMATERIALS 26 (31) 6158 -6166 2005年11月 [査読無し][通常論文]
     
    This study was designed to evaluate the histological changes during ossification and cellular events including osteogenic differentiation responding to collagenous bioresorbable membranes utilized for GBR. Standardized artificial bony defects were prepared at rat maxillae, and covered with a collagenous bioresorbable membrane. These animals were sacrificed at 1, 2, 3 and 4 weeks after the GBR-operation. The paraffin sections were subject to tartrate resistant acid phosphatase (TRAP) enzyme histochemistry and immunohistochemistry for alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC). In the first week of the experimental group, woven bone with ALP-positive osteoblasts occupied the lower half of the cavity. The collagenous membrane included numerous ALP-negative cells and OP-immunoreactive extracellular matrices. At 2 weeks, the ALP-, OP- and OC-immunoreactivity came to be recognizable in the region of collagenous membrane. Since ALP-negative soft tissue separated the collagenous membrane and the new bone originating from the cavity bottom, the collagenous membrane appeared to induce osteogenesis in situ. At 3 weeks, numerous collagen fibers of the membrane were embedded in the adjacent bone matrix. At 4 weeks, the membrane-associated and the cavity-derived bones had completely integrated, showing the same height of the periosteal ridge as the surrounding alveolar bones. The collagen fibers of a GBR-membrane appear to participate in osteogenic differentiation. (c) 2005 Elsevier Ltd. All rights reserved.
  • Akiko Suzuki, Kayoko Nozawa-Inoue, Nobuyuki Ikeda, Norio Amizuka, Kazuhiro Ono, Ritsuo Takagi, Takeyasu Maeda The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 286 (2) 908 -16 2005年10月 [査読無し][通常論文]
     
    Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation; the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively.
  • 鈴木 啓展, 網塚 憲生, 前田 健康 日本臨床 63 (増刊10 臨床分子内分泌学(3)) 188 -193 2005年10月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本臨床 63 (増刊10 臨床分子内分泌学(3)) 278 -283 2005年10月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本臨床 63 (増刊10 臨床分子内分泌学(3)) 300 -305 2005年10月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓 日本臨床 63 (増刊10 臨床分子内分泌学(3)) 382 -386 2005年10月 [査読無し][通常論文]
  • 近藤 直樹, 徳永 邦彦, 伊藤 知之, 荒井 勝光, 伊藤 雅之, 北原 洋, 網塚 憲生, 李 敏啓, 高野 玲子, 遠藤 直人 別冊整形外科 1 (48) 35 -40 2005年10月 [査読無し][通常論文]
     
    8週齢雌ラットの右脛骨骨髄を穿孔して作成した骨髄損傷モデルに,100mg/kg/日のステロイドを3日間臀筋内に投与し,骨再生過程におけるステロイドの影響を組織学的・分子生物学的に検討した.ステロイド投与群(M群)は組織学的に投与7日で新生骨と線維芽細胞様細胞が混在し,10,14日では対照群と比較してより多くの新生骨が残存していた.しかし,新生骨単位面積あたりの破骨細胞数は両群間で有意差を認めず,Cathepsin Kの免疫染色でも染色性に差を認めなかった.また,M群の最大骨量は対照群より有意に小さかったが,骨芽細胞分化マーカー(アルカリホスファターゼ,I型プロコラーゲンα1鎖,オステオポンチン,オステオカルシン)は両群間で差を認めず,ステロイドは出現した骨芽細胞の分化には影響を与えないと考えられた
  • Shumpei Niida, Takako Kondo, Sachie Hiratsuka, Shin-Ichi Hayashi, Norio Amizuka, Tetsuo Noda, Kyoji Ikeda, Masabumi Shibuya Proceedings of the National Academy of Sciences of the United States of America 102 (39) 14016 -21 2005年09月27日 [査読無し][通常論文]
     
    VEGF receptor 1 (VEGFR-1/Flt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte/macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic Csf1op/Csf1op (op/op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colony-stimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency (Flt1(TK)-/-) into op/op mice. The original Flt1(TK)-/- mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op/opFlt1(TK)-/-) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op/opFlt1(TK)-/- mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op/opFlt1(TK)-/- mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions.
  • Dengshun Miao, Bin He, Yebin Jiang, Tatsuya Kobayashi, Maria A Sorocéanu, Jenny Zhao, Hanyi Su, Xinkang Tong, Norio Amizuka, Ajay Gupta, Harry K Genant, Henry M Kronenberg, David Goltzman, Andrew C Karaplis The Journal of clinical investigation 115 (9) 2402 -11 2005年09月 [査読無し][通常論文]
     
    Mice heterozygous for targeted disruption of Pthrp exhibit, by 3 months of age, diminished bone volume and skeletal microarchitectural changes indicative of advanced osteoporosis. Impaired bone formation arising from decreased BM precursor cell recruitment and increased apoptotic death of osteoblastic cells was identified as the underlying mechanism for low bone mass. The osteoporotic phenotype was recapitulated in mice with osteoblast-specific targeted disruption of Pthrp, generated using Cre-LoxP technology, and defective bone formation was reaffirmed as the underlying etiology. Daily administration of the 1-34 amino-terminal fragment of parathyroid hormone (PTH 1-34) to Pthrp+/- mice resulted in profound improvement in all parameters of skeletal microarchitecture, surpassing the improvement observed in treated WT littermates. These findings establish a pivotal role for osteoblast-derived PTH-related protein (PTHrP) as a potent endogenous bone anabolic factor that potentiates bone formation by altering osteoblast recruitment and survival and whose level of expression in the bone microenvironment influences the therapeutic efficacy of exogenous PTH 1-34.
  • N Amizuka, M Li, K Hara, M Kobayashi, Y Akiyama, T Maeda JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S194 -S195 2005年09月 [査読無し][通常論文]
  • T Kojima, N Amizuka, C Saito, T Maeda JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S205 -S205 2005年09月 [査読無し][通常論文]
  • M Nakadate, N Amizuka, S Nomura, T Maeda JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S202 -S202 2005年09月 [査読無し][通常論文]
  • J Shimomura, N Amizuka, T Kojima, T Maeda, D Goltzman, A Karaplis, S Shimooka JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S183 -S183 2005年09月 [査読無し][通常論文]
  • H Suzuki, N Amizuka, M Noda, H Ohshima, T Maeda JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S195 -S195 2005年09月 [査読無し][通常論文]
  • M Li, N Amizuka, K Takeuchi, T Maeda JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S252 -S252 2005年09月 [査読無し][通常論文]
  • K Ono, S Kamiya, T Akatsu, N Kugai, M Li, N Amizuka, K Matsumoto, T Nakamura, S Wada JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S214 -S214 2005年09月 [査読無し][通常論文]
  • A Mizuno, N Amizuka, M Li, M Suzuki JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S198 -S198 2005年09月 [査読無し][通常論文]
  • 中舘 正芳, 網塚 憲生, 野村 修一, 前田 健康 Journal of Oral Biosciences 47 (Suppl.) 100 -100 2005年09月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 鈴木 晶子, 芳澤 享子, 齊藤 力, 前田 健康 Journal of Oral Biosciences 47 (Suppl.) 100 -100 2005年09月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 野田 政樹, 大島 勇人, 前田 健康 Journal of Oral Biosciences 47 (Suppl.) 100 -100 2005年09月 [査読無し][通常論文]
  • 下村 淳子, 網塚 憲生, 小島 拓, 前田 健康, 下岡 正八 Journal of Oral Biosciences 47 (Suppl.) 101 -101 2005年09月 [査読無し][通常論文]
  • 野澤 佳世子[井上], 鈴木 晶子, 網塚 憲生, 前田 健康 Journal of Oral Biosciences 47 (Suppl.) 102 -102 2005年09月 [査読無し][通常論文]
  • 鈴木 晶子, 野澤 佳世子[井上], 網塚 憲生, 前田 健康 Journal of Oral Biosciences 47 (Suppl.) 102 -102 2005年09月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, パウロエンリケ・ルイズデフレイタズ, 前田 健康 Journal of Oral Biosciences 47 (Suppl.) 105 -105 2005年09月 [査読無し][通常論文]
  • 網塚 憲生 Osteoporosis Japan 13 (Suppl.1) 71 -71 2005年09月 [査読無し][通常論文]
  • 網塚 憲生 Osteoporosis Japan 13 (Suppl.1) 121 -121 2005年09月 [査読無し][通常論文]
  • 大嶋 茂樹, 小野寺 伸, 伊東 学, 西平 順, 網塚 憲生, 三浪 明男 Osteoporosis Japan 13 (Suppl.1) 211 -211 2005年09月 [査読無し][通常論文]
  • Kazi Sazzad Hossain, Norio Amizuka, Nobuyki Ikeda, Kayoko Nozawa-Inoue, Akiko Suzuki, Minqi Li, Kiichi Takeuchi, Megumi Aita, Yoshiro Kawano, Masaaki Hoshino, Kimimitsu Oda, Ritsuo Takagi, Takeyasu Maeda Microscopy research and technique 67 (6) 325 -35 2005年08月15日 [査読無し][通常論文]
     
    The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the "articular zone," "articular layer," and "resting zone" from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles.
  • Takuya Shimamura, Norio Amizuka, Minqi Li, Paulo H. L. Freitas, John H. White, Janet E. Henderson, Susumu Shingaki, Tamio Nakajima, Hidehiro Ozawa BIOMEDICAL RESEARCH-TOKYO 26 (4) 159 -172 2005年08月 [査読無し][通常論文]
     
    Bone tissue, with its dynamic microenvironment featuring osteoclastic bone resorption, angiogenesis and matrix degradation, appears to facilitate proliferation of tumor cells after the onset of bone metastasis. In this study, we examined metastatic lesions in the femora of BALB/c nu/nu mice two weeks after intracardiac injection with human breast carcinoma MDA-231 cells. Histopathological observations showed the metastatic lesions close to the chondro-osseous junction, and revealed MDA-231 cells loosely intermingled with different cell types such as osteoblasts, fibroblastic stromal cells, osteoclasts and endothelial cells. In the metastatic nest, many tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts accumulated in direct contact with or were close to alkaline phosphatase (ALPase)- or receptor activator of NF-kappa B ligand (RANKL)positive osteoblastic cells. It seems likely that osteoclastogenesis is mediated through cell-to-cell contacts with ALPase- and RANKL-expressing osteoblastic cells. Formation of many capillaries lacking complete basal membranes and pericytes ratified the results of in situ hybridization, which revealed intense expression of VEGF in tumor nests, and therefore, indicated ongoing tumor-induced angiogenesis. The tumor cells possessed matrix metallo-proteinases (MMPs)-1 and -9, and frequently extended their stout cytoplasmic processes into fragmented fibrillar components of the growth plate cartilage, implicating degradation of cartilaginous matrix. Thus, osteolytic bone metastasis has demonstrated pathological features as tumor-induced angiogenesis and degradation of extracellular matrix, in addition to osteoclastogenesis. This complex interplay between tumor cells and host tissues may enable and nourish the establishment of a microenvironment that facilitates tumor progression.
  • 網塚 憲生, 李 敏啓, 前田 健康 Clinical Calcium 15 (7) 1155 -1159 2005年07月 [査読無し][通常論文]
     
    骨基質の石灰化ミネラルにはさまざまな元素が存在するが,特に体内のマグネシウムは半分以上が骨基質に存在しており,カルシウム,リンとともに重要な骨基質ミネラルを構成している.低マグネシウム環境では,破骨細胞の吸収活性が上昇するとともに骨芽細胞も活性化され,骨代謝回転が亢進する.一方,ビタミンK2(MK-4:menatetrenone)は破骨細胞の活性を抑制するために骨代謝回転の過剰な亢進を是正する.一方で,低マグネシウム環境では,骨基質内のカルシウム濃度が上昇するために,純度の高いハイドロキシアパタイト結晶を形成し,石灰化の過剰成長までも誘導してしまう.ビタミンK2はハイドロキシアパタイト結晶の純度には影響を与えないが,石灰化の過剰成長を抑制することが明らかとなった(著者抄録)
  • 李 敏啓, 網塚 憲生, 前田 健康, 小澤 英浩 THE BONE 19 (4) 355 -359 2005年07月 [査読無し][通常論文]
  • M Li, N Amizuka, K Oda, Y Kawano, K Takeuchi, T Maeda BONE 36 S206 -S206 2005年06月 [査読無し][通常論文]
  • M Nasu, N Amizuka, M Li, S Nomura, JE Henderson, T Maeda BONE 36 S175 -S175 2005年06月 [査読無し][通常論文]
  • N Amizuka, M Li, M Kobayashi, S Akahane, K Hara, K Takeuchi, M Nasu, H Ozawa, Y Akiyama, T Maeda BONE 36 S168 -S168 2005年06月 [査読無し][通常論文]
  • T Kojima, N Amizuka, A Suzuki, M Yoshizawa, C Saito, T Maeda BONE 36 S159 -S160 2005年06月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会学術集会プログラム抄録集 23回 138 -138 2005年06月 [査読無し][通常論文]
  • 高石 官成, 小崎 直人, 川口 浩, 鄭 雄一, 宇田川 信之, 網塚 憲生, 戸山 芳昭 日本骨代謝学会学術集会プログラム抄録集 23回 161 -161 2005年06月 [査読無し][通常論文]
  • 小野 加津広, 赤津 拓彦, 神谷 貞浩, 網塚 憲生, 李 敏啓, 大湾 一郎, 久貝 信夫, 和田 誠基 日本骨代謝学会学術集会プログラム抄録集 23回 174 -174 2005年06月 [査読無し][通常論文]
  • 下村 淳子, 網塚 憲生, 小島 拓, 前田 健康, 下岡 正八 日本骨代謝学会学術集会プログラム抄録集 23回 174 -174 2005年06月 [査読無し][通常論文]
  • 小野 加津広, 神谷 貞浩, 網塚 憲生, 李 敏啓, 赤津 拓彦, 久貝 信夫, 和田 誠基 日本骨代謝学会学術集会プログラム抄録集 23回 175 -175 2005年06月 [査読無し][通常論文]
  • 辰巳 佐和子, 網塚 憲生, 伊東 昌子, 河野 憲二, 池田 恭治 日本骨代謝学会学術集会プログラム抄録集 23回 206 -206 2005年06月 [査読無し][通常論文]
  • 中舘 正芳, 網塚 憲生, 野村 修一, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 23回 220 -220 2005年06月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 野田 政樹, 大島 勇人, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 23回 228 -228 2005年06月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, 喜井 勲, 工藤 明, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 23回 229 -229 2005年06月 [査読無し][通常論文]
  • 小島 拓, 網塚 憲生, 鈴木 晶子, 芳澤 享子, 齊藤 力, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 23回 236 -236 2005年06月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 原 久仁子, 小林 正敏, 竹内 亀一, 秋山 康博, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 23回 274 -274 2005年06月 [査読無し][通常論文]
  • 低マグネシウム飼料飼育により低下した「骨質」に対するビタミンK2(MK-4)の効果
    網塚 憲生, 李 敏啓, 小林 正敏, 赤羽 章司, 原 久仁子, 竹内 亀一, 小澤 英浩, 秋山 康博, 前田 健康 マグネシウム 24 (1) 3 -11 2005年06月 [査読無し][通常論文]
     
    生後4週雄性Wistar系ラットを用いて,コントロール群,低Mg飼育群(低Mg群),低Mg+ビタミンK2(MK4:メナテトレノン)(MK4群)を作製し,4週および8週後の大腿骨,脛骨の骨改造ならびに石灰化ミネラルについて検索した.低Mg群の骨幹端骨梁には波状縁を発達させた多数の破骨細胞を観察し,亢進した骨吸収と骨改造が示唆された.MK4群では,波状縁の発達が悪い破骨細胞を認め,MK4は低Mgで亢進した破骨細胞性骨吸収に対して抑制的に作用すると考えられた.MK4は,骨代謝回転と骨ミネラルをコントロールすることで,低Mgで生じた骨質の低下を改善すると考えられた
  • 癌の骨転移シンポジウム 骨転移巣の微細環境における組織学的知見
    網塚 憲生 日本骨形態計測学会雑誌 15 (2) 65 -65 2005年06月 [査読無し][通常論文]
  • 肺癌骨転移巣における破骨細胞の骨吸収及び基質分解の形態学的観察
    李 敏啓, 網塚 憲生, 前田 健康 日本骨形態計測学会雑誌 15 (2) 77 -77 2005年06月 [査読無し][通常論文]
  • ポリ乳酸プレートとハイドロキシアパタイト骨補填材を併用した骨増生の試み
    小島 拓, 網塚 憲生, 鈴木 晶子, 芳澤 享子, 齊藤 力, 前田 健康 日本骨形態計測学会雑誌 15 (2) 86 -86 2005年06月 [査読無し][通常論文]
  • 歯科インプラント周囲に植立と同時に充填されたα-TCP系自己硬化型補填材が誘導する新生骨について
    中舘 正芳, 網塚 憲生, 野村 修一, 前田 健康 日本骨形態計測学会雑誌 15 (2) 87 -87 2005年06月 [査読無し][通常論文]
  • T Kawase, K Okuda, Y Saito, N Amizuka, H Suzuki, M Yoshie IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL 41 (5-6) 171 -176 2005年05月 [査読無し][通常論文]
     
    Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor beta 1 (TGF-beta 1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous Studies, we demonstrated that PRP mimics TGF-beta 1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type 1 collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult Volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells IRA failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.
  • 中舘正芳, 魚島勝美, 網塚憲生, 織田公光, 前田健康, 野村修一 日本補綴歯科学会学術大会プログラム・抄録集 113th 74 2005年05月 [査読無し][通常論文]
  • インプラント周囲に充填した自己硬化型α-TCP系骨補填材に対する組織反応
    中舘 正芳, 魚島 勝美, 網塚 憲生, 織田 公光, 前田 健康, 野村 修一 日本補綴歯科学会雑誌 49 (113回特別) 74 -74 2005年05月 [査読無し][通常論文]
  • 下村 淳子, 網塚 憲生, 小島 拓, 下岡 正八 小児歯科学雑誌 43 (2) 205 -205 2005年04月 [査読無し][通常論文]
  • op/opマウスにおける骨基質石灰化と骨芽細胞の局在について
    坂上 直子, 網塚 憲生, 李 敏啓, 宇田川 信之, 前田 健康 解剖学雑誌 80 (Suppl.) 143 -143 2005年03月 [査読無し][通常論文]
  • 骨吸収型骨転移病巣で破骨細胞が産生するcathepsin KとMMP-9の免疫局在
    李 敏啓, 網塚 憲生, 井上 佳世子, 河野 芳郎, 竹内 亀一, 前田 健康 解剖学雑誌 80 (Suppl.) 161 -161 2005年03月 [査読無し][通常論文]
  • 致死型軟骨無形成症の組織異常と細胞内シグナリング
    新潟歯学会雑誌 印刷中 2005年 [査読無し][通常論文]
  • N Sakagami, N Amizuka, MQ Li, K Takeuchi, M Hoshino, M Nakamura, K Nozawa-Inoue, N Udagawa, T Maeda MICRON 36 (7-8) 688 -695 2005年 [査読無し][通常論文]
     
    Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization. (c) 2005 Elsevier Ltd. All rights reserved.
  • Norio Amizuka, Junko Shimomura, Minqi Li, Makiko Nasu, Takeyasu Maeda Journal of bone and mineral metabolism 23 Suppl (SUPPL. 1) 43 -7 2005年 [査読無し][通常論文]
  • 網塚 憲生, UCHIDA Takashi, NOZAWA INOUE Kayoko, KAWANO Yoshiro, SUZUKI Akiko, LI Minqi, NASU Makiko, KOJIMA Taku, SAKAGAMI Naoko, OZAWA Hidehiro, MAEDA Takeyasu Journal of oral biosciences 47 (1) 33 -41 2005年 [査読無し][通常論文]
     
    Human enamel tufts appeared as corrugated ribbon-like structures located on the dentin parallel to the tooth axis when observed under the binocular microscope and scanning electron microscope (SEM). SEM observation disclosed enamel tufts as bundles of well-extended tubular structures attributable to hypomineralized enamel sheaths. Plate-like structures, previously referred to as “tuft-root” ran in the center of the enamel tufts, connecting the dentin surface. When observing under the transmission electron microscope, the plates of tufts extended from the superficial layer of the dentin, penetrating the hypermineralized zone adjacent to the dentin-enamel (D-E) junction, and then, reaching the tuft region. In the tuft region, the plates of tufts ran mainly along the enamel sheaths and partially across the enamel prisms. The immuno-gold technique verified an intense immunoreactivity for amelogenin in the superficial layer of the dentin as well as the enamel prisms in the tufts, although no reaction was found over the “plates of tufts”. The immunoreactivity for 13–17 kd sheath proteins, also denoted as sheathlin, ameloblastin or amelin, was detected over the filamentous structures closely associated with the enamel sheaths in the enamel tuft. Thus, our study demonstrated that enamel tufts consist of both well extended hypomineralized enamel prisms and “plates of tufts”. The major organic substance of the enamel tufts is suggested to be 13–17 kd sheath proteins rather than amelogenin. © 2005, Japanese Association for Oral Biology. All rights reserved.
  • Anat. Rec. 284A (2) : 522-528 2005年 [査読無し][通常論文]
  • 近藤直樹, 徳永邦彦, 伊藤知之, 網塚憲生, 李敏啓, 荒井勝光, 伊藤雅之, 高野玲子, 遠藤直人 再生医療 4 2005年
  • C Yamada, Y Yamada, K Tsukiyama, N Harada, H Fujiwara, K Miyawaki, K Tanaka, N Amizuka, N Inagaki, Y Seino DIABETES 54 A364 -A365 2005年 [査読無し][通常論文]
  • C Yamada, Y Yamada, K Tsukiyama, N Harada, H Fujiwara, K Miyawaki, K Tanaka, N Amizuka, N Inagaki, Y Seino DIABETOLOGIA 48 A203 -A204 2005年 [査読無し][通常論文]
  • 那須 真樹子, 網塚 憲生, 李 敏啓, 野村 修一, 前田 健康 新潟歯学会雑誌 34 (2) 243 -246 2005年01月 [査読無し][通常論文]
  • 変異型線維芽細胞増殖因子受容体III型による軟骨細胞のアポトーシス
    那須 真樹子, 網塚 憲生, 李 敏啓, 野村 修一, 前田 健康 新潟歯学会雑誌 34 (2) 286 -286 2005年01月 [査読無し][通常論文]
  • 致死型軟骨無形成症II型における骨端軟骨への血管侵入異常
    那須 真樹子, 網塚 憲生, 李 敏啓, 野村 修一, 前田 健康 新潟歯学会雑誌 34 (2) 287 -287 2005年01月 [査読無し][通常論文]
  • 肺癌骨転移病巣において破骨細胞が産生するcathepsin KとMMP-9の局在
    李 敏啓, 網塚 憲生, 織田 公光, 河野 芳郎, 竹内 亀一, 前田 健康 新潟歯学会雑誌 34 (2) 287 -287 2005年01月 [査読無し][通常論文]
  • 低マグネシウムラットにおける骨代謝および石灰化に関する微細構造学的検討
    李 敏啓, 網塚 憲生, 竹内 亀一, 前田 健康 新潟歯学会雑誌 34 (2) 288 -288 2005年01月 [査読無し][通常論文]
  • 下村 淳子, 網塚 憲生, 小島 拓, 下岡 正八 小児歯科学雑誌 43 (2) 205 -205 2005年 [査読無し][通常論文]
  • 網塚 憲生 助成研究報告集 2005 35 -42 2005年 [査読無し][通常論文]
  • SUZUKI Hironobu, AMIZUKA Norio, ODA Kimimitsu, LI Minqi, YOSHIE Hiromasa, OHSHIMA Hayato, NODA Masaki, MAEDA Takeyasu Archives of histology and cytology 68 (5) 371 -381 2005年 [査読無し][通常論文]
     
    Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.
  • H Suzuki, N Amizuka, Kii, I, Y Kawano, K Nozawa-Inoue, A Suzuki, H Yoshie, A Kudo, T Maeda ANATOMICAL RECORD PART A-DISCOVERIES IN MOLECULAR CELLULAR AND EVOLUTIONARY BIOLOGY 281A (2) 1264 -1275 2004年12月 [査読無し][通常論文]
     
    Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption. (C) 2004 Wiley-Liss, Inc.
  • 那須 真樹子, 網塚 憲生, 李 敏啓〔他 新潟歯学会雑誌 = 新潟歯学会雑誌 34 (2) 79 -82 2004年12月 [査読無し][通常論文]
  • 網塚 憲生, 前田 健康, 小澤 英浩 THE BONE 18 (6) 661 -665 2004年11月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 那須 真樹子 モレキュラ-メディシン 41 (11) 1387 -1393 2004年11月 [査読無し][通常論文]
  • 網塚 憲生, 下村 淳子, 李 敏啓, 那須 真樹子, 坂上 直子, 野澤 佳世子[井上], 前田 健康 Osteoporosis Japan 12 (4) 522 -526 2004年10月 [査読無し][通常論文]
     
    高代謝回転モデル動物であるオステオプロテジェリン(OPG)欠損マウスの骨基質を微細構造学的に検索した.骨基質は不規則な走行のコラーゲン線維で構成されていた.セメントラインを電子顕微鏡観察すると電子密度が低いことから骨基質間の密着性は低いと考えられた.生後10〜15週にはOPG欠損によって破骨細胞と骨芽細胞の数が増加し活発な骨改造が認められたが,17週になると成長板軟骨のほとんどが吸収されてしまい,破骨細胞と骨芽細胞の数は野生型マウスと大差がなかった.骨髄組織における脂肪細胞数は生後15週,17週とも増加していた
  • Y Asawa, N Amizuka, K Hara, M Kobayashi, M Aita, M Li, S Kenmotsu, K Oda, Y Akiyama, H Ozawa BONE 35 (4) 870 -880 2004年10月 [査読無し][通常論文]
     
    To evaluate the biological effects of vitamin K2 (menatetrenone, MK-4) on ovariectomy (OVX)-induced bone loss, we have examined histological alterations of femoral metaphyses of sham-operated (sham group), ovariectomized (OVX group), and MK-4 dietary-supplemented OVX (MK-4 group; 50 mg/kg per day) female Fischer rats 1, 2, 5, and 8 weeks after OVX. In the first week, rats of the OVX and MK-4 groups showed discontinuous trabeculae compared with sham-operated rats. At 2 weeks after OVX, the OVX rats revealed many large tartrate resistant acid phosphatase (TRAP)-positive osteoclasts, while osteoclasts in the MK-4-treated rats were similar in size to those of the sham group. At 5 weeks, the OVX and MK-4 groups revealed fragmented trabeculae in femoral metaphyses. The cartilage matrix was partially exposed due to stimulated bone resorption in the OVX group, but not in the MK-4 group. After 8 weeks, the OVX rats had little metaphyseal trabeculae, whereas the MK-4-treated rats had maintained short trabeculae. Despite the presence of intense alkaline phosphatase-positive osteoblasts on trabeculae in the MK-4 group, TRAP-positive osteoclasts were flattened without developing ruffled borders. Therefore, MK-4 appeared to lessen the increase in osteoclastic bone resorption induced by OVX, as well as to maintain the accelerated osteoblastic activity. It is of importance to identify the target cells for MK-4 in bone. Autoradiography localized [H-3]-labeled MK-4 mainly in osteoblasts and adjacent bone matrices, but not in osteoclasts, indicating that MK-4 targets osteoblasts. Thus, MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy. (C) 2004 Elsevier Inc. All rights reserved.
  • H Tanabe, Kii, I, N Amizuka, K Katsube, A Kudo JOURNAL OF BONE AND MINERAL RESEARCH 19 S275 -S275 2004年10月 [査読無し][通常論文]
  • Kii, I, N Amizuka, S Kitajima, M Li, K Takeuchi, T Maeda, J Kanno, T Inoue, Y Saga, A Kudo JOURNAL OF BONE AND MINERAL RESEARCH 19 S21 -S21 2004年10月 [査読無し][通常論文]
  • MA Soroceanu, DS Miao, Y Jiang, JJ Zhao, XY Bai, H Su, HK Genant, N Amizuka, D Goltzman, AC Karaplis JOURNAL OF BONE AND MINERAL RESEARCH 19 S97 -S97 2004年10月 [査読無し][通常論文]
  • 低マグネシウムラットの骨組織学的検討
    網塚 憲生 マグネシウム 23 (2) 129 -129 2004年10月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 那須 真樹子 Molecular Medicine 41 (11) 1387 -1393 2004年10月 [査読無し][通常論文]
     
    骨転移を示す腫瘍細胞の多くが副甲状腺ホルモン関連ペプチド(PTHrP)を産生していることからPTHrPの重要性が推察されてきたが,1994年にPTHrP欠損マウスが作製されると,PTHrPは腫瘍ばかりでなく軟骨形成に重要な役割を演ずることが知られるようになった.また,PTH/PTHrPレセプターの点突然変異で,Jansen型よびBlomstrand型の軟骨異形成症が発症することが発見されている.そこで,こうしたPTHrPの骨転移における作用と,正常組織における作用について,それぞれ概説した
  • 発生期マウス下顎関節突起における肥大層の組織化学的変化(Histochemical changes of the hypertrophic zone in developing mouse mandibular condyle)
    Hossain Kazi Sazzad, 池田 順行, 野澤 佳世子, 網塚 憲生, 織田 公光, 高木 律男, 前田 健康 Journal of Oral Biosciences 46 (5) 396 -396 2004年09月 [査読無し][通常論文]
  • 中舘 正芳, 網塚 憲生, 魚島 勝美, 田口 裕哉, 那須 真樹子, 前田 健康, 野村 修一 Journal of Oral Biosciences 46 (5) 385 -385 2004年09月 [査読無し][通常論文]
  • SAZZAD HOSSAIN KAZI, 池田 順行, 野澤-井上, 佳世子, 網塚 憲生, 織田 公光, 高木 律男, 前田 健康 Journal of oral biosciences 46 (5) 396 -396 2004年09月01日 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, 織田 公光, 河野 芳郎, 竹内 亀一, 那須 真樹子, 小島 拓, 高木 律男, 前田 健康 Journal of Oral Biosciences 46 (5) 405 -405 2004年09月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 織田 公光, 野田 政樹, 大島 勇人, 前田 健康 Journal of Oral Biosciences 46 (5) 405 -405 2004年09月 [査読無し][通常論文]
  • 那須 真樹子, 網塚 憲生, 李 敏啓, 野村 修一, 前田 健康 Journal of Oral Biosciences 46 (5) 406 -406 2004年09月 [査読無し][通常論文]
  • 鈴木 晶子, 野澤 佳世子[井上], 池田 順行, 網塚 憲生, 前田 健康 Journal of Oral Biosciences 46 (5) 406 -406 2004年09月 [査読無し][通常論文]
  • 喜井 勲, 網塚 憲生, 李 敏啓, 竹内 亀一, 前田 健康, 北嶋 聡, 菅野 純, 井上 達, 相賀 裕美子, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 22回 98 -98 2004年08月 [査読無し][通常論文]
  • 水野 敦子, 網塚 憲生, 李 敏啓, 鈴木 誠 日本骨代謝学会学術集会プログラム抄録集 22回 121 -121 2004年08月 [査読無し][通常論文]
  • 大嶋 茂樹, 小野寺 伸, 西平 順, 網塚 憲生, 三浪 明男 日本整形外科学会雑誌 78 (8) S1061 -S1061 2004年08月 [査読無し][通常論文]
  • 小野寺 伸, 大嶋 茂樹, 西平 順, 網塚 憲生, 三浪 明男 日本整形外科学会雑誌 78 (8) S1064 -S1064 2004年08月 [査読無し][通常論文]
  • 小野 加津広, 赤津 拓彦, 網塚 憲生, 前原 博樹, 大湾 一郎, 金谷 文則, 和田 誠基, 久貝 信夫 日本骨代謝学会学術集会プログラム抄録集 22回 146 -146 2004年08月 [査読無し][通常論文]
  • 田辺 英幸, 喜井 勲, 網塚 憲生, 勝部 憲一, 工藤 明 日本骨代謝学会学術集会プログラム抄録集 22回 163 -163 2004年08月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, 織田 公光, 河野 芳朗, 竹内 亀一, 高木 律男, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 22回 201 -201 2004年08月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 小林 正敏, 赤羽 章司, 原 久仁子, 竹内 亀一, 小澤 英浩, 秋山 康博, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 22回 202 -202 2004年08月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 織田 公光, 野田 政樹, 吉江 弘正, 前田 健康 日本骨代謝学会学術集会プログラム抄録集 22回 202 -202 2004年08月 [査読無し][通常論文]
  • Minqi Li, Norio Amizuka, Kimimitsu Oda, Kunihiko Tokunaga, Tomoyuki Ito, Kiichi Takeuchi, Ritsuo Takagi, Takeyasu Maeda Microscopy research and technique 64 (4) 330 -42 2004年07月01日 [査読無し][通常論文]
     
    We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.
  • FI Tapety, N Amizuka, K Uoshima, S Nomura, T Maeda CLINICAL ORAL IMPLANTS RESEARCH 15 (3) 315 -324 2004年06月 [査読無し][通常論文]
     
    This study was designed to investigate the responses of bone cells to a deproteinized bovine bone material, Bio-Oss(R) (Geistlich-Pharma, Wolhunsen, Switzerland), which was grafted in artificial bone defects of rat femurs. Standardized bone defects in the cortical bone of the right femurs were grafted with Bio-Oss(R) particles. Narrow penetrations were prepared on the bottom of the cavity, enabling osteogenic cells to migrate from the bone marrow. A defect in the left femur without Bio-Oss(R) was used as a control. The treated femurs were histochemically examined at 1, 3, 5, 7, and 14 days after the operation. At day 1, no osteogenic migration into the cavities occurred in either the control or experimental groups. At day 3, alkaline phosphatase (ALPase) immunohistochemistry showed a migration of the positive cells at the bottom of the cavities of the experimental groups, but not in the control ones. At day 5, new bone formation was recognized at the bottom of the cavity of both groups. In the experimental group, ALPase-positive cells were localized on Bio-Oss(R) and/or on the thin bone matrix that covered this material. The superficial layer of Bio-Oss(R) underlying the newly formed bone exhibited osteocalcin immunoreactivity. Transmission electron microscopy revealed osteoblasts depositing bone matrices - including collagen fibers - on the surface of Bio-Oss(R). At days 7 and 14, woven bone occupied the previous cavities of both control and experimental groups, accompanied by osteoclasts. Thus, Bio-Oss(R) appears to serve as a scaffold for osteogenic cells as well as to promote osteoblastic differentiation and matrix synthesis.
  • Tetsuro Nakazawa, Arata Nakajima, Naohiko Seki, Akihiko Okawa, Masaki Kato, Hidesige Moriya, Norio Amizuka, Thomas A Einhorn, Masashi Yamazaki Journal of orthopaedic research : official publication of the Orthopaedic Research Society 22 (3) 520 -5 2004年05月 [査読無し][通常論文]
     
    To comprehensively evaluate gene expression in the early stage of fracture healing, we used a cDNA microarray with 2304 cDNA clones derived from an oligo-capped mouse embryo library. Closed mid-diaphyseal fractures were created in mouse tibiae and expression profiles were analyzed 3 days after fracture. Six genes were up-regulated in comparison to those in unfractured bones and these included three genes previously identified but never shown to be present in fractures, periostin, calumenin, and FHL-1. Cloning of these genes has been completed but their expression pattern and function during fracture healing and bone formation remain to be elucidated. Up-regulation of the six genes was reconfirmed by semi-quantitative RT-PCR analysis. Spatial and temporal expression of one of the newly identified fracture-induced genes, periostin, was analyzed using in situ hybridization, because it displayed the highest up-regulation ratio. A signal for periostin was detected in undifferentiated mesenchymal cells and immature preosteoblastic cells in the periosteal tissues between days 3 and 14 after fracture. Northern analysis showed that periostin gene expression rapidly increased by day 3, reached a peak on day 7, and declined by day 14. These findings suggest that periostin is a specific marker for preosteoblasts and may play an important role in periosteal callus formation during the early stage of fracture healing.
  • 野澤 佳世子[井上], 網塚 憲生, 前田 健康, 山田 一穂, 小澤 英浩 THE BONE 18 (3) 251 -255 2004年05月 [査読無し][通常論文]
  • 小野 加津広, 網塚 憲生, 赤津 拓彦, 角 誠二郎, 久貝 信夫 日本内分泌学会雑誌 80 (1) 162 -162 2004年04月 [査読無し][通常論文]
  • 骨質とビタミンK 組織学的知見
    網塚 憲生 応用薬理 66 (1〜2) 104 -104 2004年04月 [査読無し][通常論文]
  • H Kudo, N Amizuka, K Araki, K Inohaya, A Kudo DEVELOPMENTAL BIOLOGY 267 (2) 473 -487 2004年03月 [査読無し][通常論文]
     
    The myoseptum of fishes, composed of dense collagen, is a connective tissue layer that forms in the embryo, dividing somites from the trunk, and its structure and function are similar to those of the mammalian tendon. Both the myoseptum and tendon serve as the transmitter of muscular contractility to bones and adjoining muscles, and their structure is indispensable for movement of vertebrate animals. We cloned the zebrafish periostin gene and examined its expression and function in the myoseptum. The expression in embryos started in the rostral part of each segmented somite in the early segmentation stage; and consequently, metameric stripes were observed. At the end of segmentation, the expression region shifted to the transverse myoseptum and the myotome-epidermis boundary, and each myotome was surrounded by periostin. Using a polyclonal antibody, we found that the periostin protein was localized to the transverse myoseptum. Consistently, periostin morpholino antisense oligonucleotide led to defects in myoseptum formation, a delay in the differentiation of myofibers, and disorder of connection between myofibrils and myoseptum. We demonstrated here that periostin is the first molecule involved in myoseptum formation and propose that periostin secretion on the surface of the myoseptum is required for the adhesion of muscle fiber bundles to the myoseptum and the differentiation of muscle fibers. (C) 2004 Elsevier Inc. All rights reserved.
  • 網塚 憲生 Clinical Calcium 14 (4) 589 -593 2004年03月 [査読無し][通常論文]
     
    骨は細胞外基質に最も富む組織であり,「骨質」は骨基質の性状によるものと思われる.骨基質は石灰化成分と有機質成分に分けて考えることができるが,有機質の中でもコラーゲン線維は90%を占めている.コラーゲン線維は引っ張り強さを有することから,骨の弾性に影響を及ぼすと考えられる.一方,骨基質の石灰化成分の多くはハイドロキシアパタイト結晶で構成されているが,周囲の非コラーゲン性蛋白質やプロテオグリカンにより成長が調節されている
  • H Hao, N Amizuka, K Oda, N Fujii, H Ohnishi, A Okada, S Nomura, T Maeda BIOMATERIALS 25 (3) 431 -442 2004年02月 [査読無し][通常論文]
     
    This study aimed to elucidate the biological effects of a self-setting tricalcium phosphate bone substitute (BIOPEX(R)) applied in rat femoral cortical bone cavities. Narrow penetrations through the cavity and bone marrow were prepared to obtain cellular sources. In the experimental group at day 1, a thin cell layer intruded into a narrow space between the grafted BIOPEX(R) and the bottom of the cavity. From days 5 to 10, a range of tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts accumulated on the surface of the BIOPEX(R) facing the bottom of the cavity, whilst many alkaline phosphatase (ALPase)-positive osteoblasts were localized on the bone surface opposing the BIOPEX(R). However, at day 20, osteoblasts were localized neighboring the osteoclasts on the BIOPEX(R), and deposited bone matrices onto this material, implying a coupling between osteoclasts and osteoblasts. At days 30 and 40 post-operation, small remnants of BIOPEX(R) particles were present in the new bone with a profile of compact bone. Thus, BIOPEX(R) is resorbed by osteoclasts, and succeeded by osteoblastic bone apposition with a coupling of osteoclasts and osteoblasts at the later stage. In conclusion, the use of BIOPEX(R) provides adequate bone regeneration with the profile of compact bone. (C) 2003 Elsevier Ltd. All rights reserved.
  • Kayoko Nozawa-Inoue, Nobuyuki Ikeda, Akiko Suzuki, Norio Amizuka, Takeyasu Maeda Journal of Oral Biosciences 46 (6) 519 -522 2004年 [査読無し][通常論文]
     
    The synovial lining layer in the temporomandibular joint (TMJ) consists of macrophage-like type A cells and fibroblast-like type B cells. Their identification, however, has been difficult because of the lack of a specific cell marker. This review summarizes the characteristic configuration and development of the type B cell in the murine TMJ. Immunocytochemistry for 25kDa-heat shock protein (Hsp25) revealed two profiles of the fibroblast-like type B cells with cytoplasmic processes in the adult rat TMJ: one projected horizontally slender processes which covered the synovial membrane, and the other extended a thick, long process towards the articular cavity. The former appeared earlier than the latter during the development of synovial membrane, in close relation with the formation of the articular cavity and the commencement of active jaw movement. Since immature type B cells with Hsp25-immunoreactivity were found in the mesenchymal tissue which corresponded to the future articular cavity, type B cells may differentiate directly from mesenchymal cells. © 2004, Japanese Association for Oral Biology. All rights reserved.
  • J. Bone Miner. Res. 19(11) : 1840-1849 2004年 [査読無し][通常論文]
  • AMIZUKA Norio, HENDERSON Janet E., WHITE John H., ODA Kimimitsu, LI Miniqi, NOZAWA (INOUE) Kayoko, KAWANO Yoshiro, SUZUKI Akiko, KARAPLIS Andrew C., GOLTZMAN David, MAEDA Takeyasu Journal of oral biosciences 46 (2) 79 -96 2004年 [査読無し][通常論文]
     
    Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and precocious maturation of chondrocytes. The hypertrophic zone of the mutant epiphyseal cartilage revealed aberrant heterogeneous populations of chondrocytes, i.e., non-hypertrophic cells at different stages of differentiation. Therefore, PTHrP appears to play a central role in modulating cell proliferation and differentiation. The biological function of PTHrP is mediated by signaling pathway linked to the PTH/PTHrP receptor. However, the amino acids at 87-107 positions of PTHrP show a putative nucleolar targeting signal, and thought to involve in the resistance to apoptosis. Chondrocytic cell line, CFK2, transfected with truncated forms of PTHrP cDNA showed this peptide in the nucleoli mediated by translation initiating from AUG-codon and alternatively initiating from CUG codons. Thereby, PTHrP appears to act as a bipartite modulator of chondrocyte proliferation/differentiation, both through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus. Meanwhile, PTH/PTHrP receptor expression is controlled by two promoters in mouse, and the downstream promoter acts predominantly in bone and cartilage. We found 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3] downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes. This indicates that the interplay between PTH and 1,25 (OH)2D3 is specific to their overlapping roles of serum calcium regulation in bone, and not to their complementary effects on proliferation/differentiation of chondrocytes. Thus, we will review our recent examinations on cartilage development in conjunction with the biological role in PTHrP, PTH/PTHrP receptor and 1,25 (OH)2D3.
  • Norio Amizuka, David Davidson, Hanlong Liu, Gladys Valverde-Franco, Sen Chai, Takeyasu Maeda, Hidehiro Ozawa, Vicki Hammond, David M Ornitz, David Goltzman, Janet E Henderson Bone 34 (1) 13 -25 2004年01月 [査読無し][通常論文]
     
    Bone development is regulated by conserved signalling pathways that are linked to multifunctional growth factors and their high affinity receptors. Parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) have been shown to play pivotal, and sometimes complementary, roles in the replication, maturation and death of chondrocytes during endochondral bone formation. To gain further insight into how these pathways coordinate cartilage and bone development, we generated mice lacking expression of both PTHrP and FGFR3. The phenotype of compound mutant mice resembled that of their PTHrP-deficient littermates with respect to neonatal lethality, facial dysmorphism and foreshortening of the limbs. The absence of PTHrP in the developing epiphyseal cartilage of PTHrP-/- and PTHrP-/-/FGFR3-/- mice resulted in a dominant hypo-proliferative phenotype. However, abnormalities such as the presence of nonhypertrophic cells among hypertrophic chondrocytes and excessive apoptosis seen in the hypertrophic zone of PTHrP-/- mice were absent in the PTHrP-/-/FGFR3-/- mice. Furthermore, the absence of FGFR3 in single and compound mutant mice led to decreased expression of vascular endothelial growth factor (VEGF) and an increase in depth of hypertrophic chondrocytes. These observations indicate that FGFR3 deficiency can rescue some of the defects seen in PTHrP-deficient mice and that it plays an important role in the regulation of chondrocyte differentiation and hypertrophy. These studies support a dominant role for PTHrP in regulating the pool of proliferating cells during limb development and suggest that signalling by FGFR3 plays a more prominent role in cartilage maturation and vascular invasion at the chondro-osseous junction.
  • 網塚 憲生, 李 敏啓, 前田 健康 THE BONE 18 (1) 39 -44 2004年01月 [査読無し][通常論文]
     
    副甲状腺ホルモン関連ペプチド(PTHrP)は軟骨細胞などの分化増殖に重要な役割を果たすことが,PTHrP遺伝子欠損マウスをはじめとして一連の遺伝子組み換えマウスによって示されてきた.又,線維芽細胞増殖因子受容体III型(FGFR3)とPTHrPの二重遺伝子欠損マウスではPTHrP遺伝子欠損マウスの異常に類似していた.更に,PTH/PTHrP受容体の遺伝子変異により,Jansen型及びBlomstrand型骨幹端軟骨異形成症や内軟骨腫が生じることが明らかにされている
  • 軟骨細胞においてFGFR3はJAK/STAT系を介してPTH/PTHrP受容体発現抑制を行う
    関 雪絵, 網塚 憲生, 永田 昌毅, 織田 公光, 高木 律男, 前田 健康 新潟歯学会雑誌 33 (2) 289 -290 2004年01月 [査読無し][通常論文]
  • 骨折の初期治癒過程における組織化学的検討
    李 敏啓, 網塚 憲生, 竹内 亀一, 高木 律男, 前田 健康 新潟歯学会雑誌 33 (2) 290 -290 2004年01月 [査読無し][通常論文]
  • ラット上顎骨における吸収性膜を用いたGBR法の組織学的観察
    田口 裕哉, 網塚 憲生, 中舘 正芳, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 新潟歯学会雑誌 33 (2) 290 -291 2004年01月 [査読無し][通常論文]
  • 網塚 憲生, 下村 淳子, 李 敏啓 オステオポローシスジャパン 12 (4) 522 -526 2004年 [査読無し][通常論文]
  • NOZAWA INOUE Kayoko, IKEDA Nobuyuki, SUZUKI Akiko, AMIZUKA Norio, MAEDA Takeyasu Journal of oral biosciences 46 (6) 519 -522 2004年 [査読無し][通常論文]
     
    The synovial lining layer in the temporomandibular joint (TMJ) consists of macrophage-like type A cells and fibroblast-like type B cells. Their identification, however, has been difficult because of the lack of a specific cell marker. This review summarizes the characteristic configuration and development of the type B cell in the murine TMJ. Immunocytochemistry for 25kDa-heat shock protein (Hsp25) revealed two profiles of the fibroblast-like type B cells with cytoplasmic processes in the adult rat TMJ: one projected horizontally slender processes which covered the synovial membrane, and the other extended a thick, long process towards the articular cavity. The former appeared earlier than the latter during the development of synovial membrane, in close relation with the formation of the articular cavity and the commencement of active jaw movement. Since immature type B cells with Hsp25-immunoreactivity were found in the mesenchymal tissue which corresponded to the future articular cavity, type B cells may differentiate directly from mesenchymal cells. © 2004, Japanese Association for Oral Biology. All rights reserved.
  • 網塚 憲生 日本骨形態計測学会雑誌 13 (3) 1 -7 2003年12月 [査読無し][通常論文]
     
    培養軟骨細胞に様々なPTHrP cDNAをtransfectionしたところ,PTHrPの翻訳は開始メチオニンをコードするAUGばかりでなく,下流に存在するロイシンをコードするCUGからのalternative translationが可能であることが明らかにされた.これらのPTHrPはAUG或いはCUGコドンから開始された場合でも,内在する核小体移行シグナルによって核に局在することが示された.以上のことから,PTHrPは受容体に結合するか,或いは核小体局在の二方向性の経路で軟骨細胞の分化増殖を調整すると考えられた
  • FI Tapety, N Amizuka, S Nomura, T Maeda JOURNAL OF DENTAL RESEARCH 82 398 -398 2003年12月 [査読無し][通常論文]
  • 網塚 憲生, 前田 健康, 小澤 英浩 THE BONE 17 (6) 543 -547 2003年11月 [査読無し][通常論文]
  • Reiko Mochizuki, Minori Dateki, Kazuyuki Yanai, Yasuyuki Ishizuka, Norio Amizuka, Hiroyuki Kawashima, Yoshihiko Koga, Hidehiro Ozawa, Akiyoshi Fukamizu Biochemical and biophysical research communications 310 (4) 1219 -26 2003年10月31日 [査読無し][通常論文]
     
    Neurochondrin/norbin is a cytoplasmic protein involved in dendrite outgrowth. The expression of the gene has been restricted to neural, bone, and chondral tissues. To identify the functions of the gene in vivo, we have generated mice with a disrupted mutation in the neurochondrin/norbin gene. Histological analysis of heterozygous mutant mice indicates the possibility of specific functions of neurochondrin/norbin in chondrocyte differentiation. We defined the expression patterns of neurochondrin/norbin-lacZ fusion protein in the central nervous system. In the developing olfactory bulb, beta-galactosidase activity was detected in the mantle layer at 12.5 dpc and the strongest activity was detected in the presumptive mitral or tufted cell layer at 15.5 dpc. beta-Galactosidase activity was also detected in the lateral choroid plexus. In homozygous (-/-) mutant mice, the disruption of the neurochondrin/norbin gene leads to early embryonic death between 3.5 and 6.5 dpc. This result indicates that neurochondrin/norbin gene function is essential for the early embryogenesis.
  • 田口 裕哉, 網塚 憲生, 中舘 正芳, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 日本補綴歯科學會雜誌 = The journal of the Japan Prosthodontic Society 47 71 -71 2003年10月24日 [査読無し][通常論文]
  • 田口裕哉, 網塚憲生, 中舘正芳, 大西英夫, 藤井規孝, 野村修一, 前田健康 日本補綴歯科学会学術大会プログラム・抄録集 110th 71 2003年10月 [査読無し][通常論文]
  • 田口 裕哉, 網塚 憲生, 中舘 正芳, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 日本補綴歯科学会雑誌 47 (110回特別) 71 -71 2003年10月 [査読無し][通常論文]
  • 永田 昌毅, 関 雪絵, 高木 律男, 網塚 憲生, Nuckolls Glen, Slavkin Harold 新潟医学会雑誌 117 (10) 601 -602 2003年10月 [査読無し][通常論文]
  • 永田 昌毅, 関 雪絵, 高木 律男, 網塚 憲生, Glen Nuckolls, Harold Slavkin 新潟医学会雑誌 = 新潟医学会雑誌 117 (10) 601 -602 2003年10月 [査読無し][通常論文]
  • 野澤-井上, 佳世子, 河野 芳朗, 網塚 憲生, 前田 健康 歯科基礎医学会雑誌 45 (5) 303 -303 2003年09月01日 [査読無し][通常論文]
  • H Kitahara, K Tokunaga, T Ito, M Ito, N Amizuka, K Oda, N Endo JOURNAL OF BONE AND MINERAL RESEARCH 18 S281 -S281 2003年09月 [査読無し][通常論文]
  • 関 雪絵, 永田 昌毅, 網塚 憲生, 前田 健康, 高木 律男 歯科基礎医学会雑誌 45 (5) 266 -266 2003年09月 [査読無し][通常論文]
  • 野澤 佳世子[井上], 河野 芳朗, 網塚 憲生, 前田 健康 歯科基礎医学会雑誌 45 (5) 303 -303 2003年09月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, 織田 公光, 河野 芳朗, 竹内 亀一, 高木 律男, 前田 健康 歯科基礎医学会雑誌 45 (5) 266 -266 2003年09月 [査読無し][通常論文]
  • 関 雪絵, 永田 昌毅, 網塚 憲生, 前田 健康, 高木 律男 歯科基礎医学会雑誌 45 (5) 266 -266 2003年09月01日 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 織田 公光, 野田 政樹, 吉江 弘正, 前田 健康 歯科基礎医学会雑誌 45 (5) 270 -270 2003年09月 [査読無し][通常論文]
  • 田口 裕哉, 網塚 憲生, 関 雪絵, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 歯科基礎医学会雑誌 45 (5) 297 -297 2003年09月 [査読無し][通常論文]
  • Hossain Kazi Sazzad, 池田 順行, 井上 佳世子, 網塚 憲生, 織田 公光, 高木 律男, 前田 健康 歯科基礎医学会雑誌 45 (5) 304 -304 2003年09月 [査読無し][通常論文]
  • 北原 洋, 徳永 邦彦, 伊藤 知之, 伊藤 雅之, 網塚 憲生, 織田 公光, 遠藤 直人 日本整形外科学会雑誌 77 (8) S1089 -S1089 2003年08月 [査読無し][通常論文]
  • 網塚 憲生, 下村 淳子, 関 雪絵, 前田 健康 腎と骨代謝 16 (3) 217 -227 2003年07月 [査読無し][通常論文]
     
    副甲状腺ホルモン関連ペプチド(PTHrP)シグナルが軟骨形成に重要な役割を演ずることが明らかにされ,特にPTHrPとIndian hedgehogのnegative feedbackによって軟骨分化が調節されることが明らかとなっている.又,PTH/PTHrP受容体のpoint mutationでJansen型及びBlomstrand型の軟骨異形成症が発症すること,更に内軟骨腫が発症することも明らかにされている.近年の報告では,PTH/PTHrP受容体にNa+/H+交換体調節因子が結合することにより,PTH/PTHrP受容体からのホスフォリパーゼCのシグナル伝達が活性化することが発見された.これら軟骨細胞の分化制御機構について解説した
  • N Amizuka, Y Seki, J Shimomura, S Kenmotsu, A Karaplis, T Maeda BONE 32 (5) S109 -S110 2003年05月 [査読無し][通常論文]
  • T Nakazawa, A Nakajima, A Okawa, H Moriya, N Amizuka, N Seki, M Yamazaki BONE 32 (5) S101 -S101 2003年05月 [査読無し][通常論文]
  • S Arai, N Amizuka, Y Azuma, A Kudo BONE 32 (5) S153 -S153 2003年05月 [査読無し][通常論文]
  • Y Taketani, N Amizuka, C Ogawa, N Sawada, K Nomoto, H Arai, E Takeda BONE 32 (5) S137 -S137 2003年05月 [査読無し][通常論文]
  • Y Seki, N Amizuka, J Henderson, M Nagata, K Oda, R Takagi, T Maeda BONE 32 (5) S100 -S100 2003年05月 [査読無し][通常論文]
  • H Kitahara, K Takunaga, T Ito, M Ito, N Amizuka, K Oda, N Endo BONE 32 (5) S133 -S133 2003年05月 [査読無し][通常論文]
  • J Shimomura, N Amizuka, Y Seki, T Noda, T Maeda BONE 32 (5) S142 -S142 2003年05月 [査読無し][通常論文]
  • 竹谷 豊, 網塚 憲生, 小川 千春, 澤田 直樹, 野本 香, 武田 英二 日本細胞生物学会大会講演要旨集 56回 28 -28 2003年05月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 吉江 弘正 日本歯科保存学雑誌 46 (春季特別) 52 -52 2003年05月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 13 (2) S17 -S17 2003年05月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, 竹内 亀一, 高木 律男, 前田 健康 日本骨形態計測学会雑誌 13 (2) S37 -S37 2003年05月 [査読無し][通常論文]
  • 網塚 憲生, 李 敏啓, 小林 正敏, 原 久仁子, 竹内 亀一, 下村 淳子, 秋山 康博, 前田 健康 日本骨形態計測学会雑誌 13 (2) S49 -S49 2003年05月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 13 (1) 5 -9 2003年04月 [査読無し][通常論文]
     
    近年,いかに強固かつ弾力に富む丈夫な骨を増やしてゆくかといった「骨質」が注目を浴びてきている.骨は人体の中でも細胞外基質が最も発達した組織であり,骨基質の性状・量・立体的構築が「骨質」を規定すると考えて良い.しかし,長管骨の皮質骨と海面骨の構造は異なり,それらを覆う骨芽細胞の活性,類骨層の広さ,骨代謝回転などは同じではない.したがって,「骨質」を単純に石灰化度のみで評価しきれない面がある.そこで,動物モデルを用いて,正常の骨組織における骨基質,骨代謝回転と骨基質,ビタミンKと骨基質に分け,骨基質から読みとれる「骨質」を組織学的な見解を述べた
  • Satoshi Arai, Norio Amizuka, Yoshiaki Azuma, Sunao Takeshita, Akira Kudo Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 18 (4) 686 -95 2003年04月 [査読無し][通常論文]
     
    Osteoclastogenesis is regulated by RANKL expressed on stromal cells. In this study, we sought to isolate a new surface molecule regulating osteoclastogenesis on stromal cells by generating monoclonal antibodies. A rat was immunized with the mouse stromal cell line, TSB13, which can support osteoclastogenesis, and a monoclonal antibody, A15-1, was obtained. A15-1 bound to a surface antigen on TSB13 cells, termed osteoclastogenesis-related antigen (OCRA), and immunoprecipitation with this antibody revealed that OCRA was a 220-kDa molecule. By means of flow cytometry, the A15-1 antigen (OCRA) was found to be expressed on various mesenchymal cell lines but not on hematopoietic cell lines, and the expression level of OCRA on the TSB13 cells was slightly increased by treatment with 1alpha,25(OH)2D3. When osteoclast progenitors and TSB13 cells were co-cultured in the presence of 1alpha,25(OH)2D3, the addition of A15-1 inhibited osteoclast differentiation in a dose-dependent manner; however, no significant inhibition of soluble RANKL-induced osteoclastogenesis was observed, suggesting that A15-1 inhibited only stromal cell-dependent osteoclastogenesis. The same inhibitory effect of A15-1 was also observed when primary bone marrow-derived stromal cells were used. The osteoclastogenesis-promoting effects of other osteotropic factors, such as parathyroid hormone (PTH) and interleukin (IL)-1beta, were also inhibited by A15-1. Time-course analysis of osteoclast differentiation in vitro indicated that the initial 2 days of treatment with A15-1 was sufficient for inhibition, suggesting that A15-1 inhibits the early stages of osteoclast differentiation. Finally, we investigated the in vivo effects of A15-1 on PTH-induced hypercalcemia in mice. Treatment with A15-1 significantly decreased the osteoclast surface in the PTH-administered mice. Taken together, our data indicate that OCRA, a novel A15-1-detected antigen, regulates stromal cell-dependent osteoclastogenesis.
  • 網塚 憲生, 関 雪絵, 下村 淳子, 監物 新一, 前田 健康 解剖学雑誌 78 (Suppl.) 190 -190 2003年04月 [査読無し][通常論文]
  • 下村 淳子, 網塚 憲生, 関 雪絵, 野田 忠, 前田 健康 解剖学雑誌 78 (Suppl.) 191 -191 2003年04月 [査読無し][通常論文]
  • 網塚 憲生 昭和歯学会雑誌 23 (1) 81 -81 2003年03月31日 [査読無し][通常論文]
  • 【骨の細胞と形態機能】軟骨内骨化における石灰化と血管侵入
    網塚 憲生, 下村 淳子, 前田 健康, 小澤 英浩 Clinical Calcium 13 (4) 405 -412 2003年03月 [査読無し][通常論文]
     
    骨端軟骨は大きく,静止層,増殖層,肥大化層に分けられる.肥大化層のカラム間基質は石灰化を受けるが,カラム内の横隔壁は石灰化をあまり受けない.そのために,石灰化軟骨基質は軟骨の長軸方向に形成される.血管内皮細胞は軟骨・骨境界部であまり石灰化を受けていない横隔壁を貫通することで,軟骨に侵入してゆき,その結果,石灰化したカラム間基質が骨組織へと露出する.この露出した軟骨基質は骨芽細胞が接着する足がかりとして役立つと考えられる.又,これらの骨芽細胞は骨基質を軟骨基質上へ分泌することにより,一次骨梁を形成する
  • 大田 秀隆, 東 浩太郎, 堀内 敏行, 風間 広仁, 荒木 厚, 細井 孝之, 沢辺 元司, 網塚 憲生, 折茂 肇 日本老年医学会雑誌 40 (2) 167 -171 2003年03月 [査読無し][通常論文]
     
    93歳女.食欲低下と意識障害(JCS20)が出現した.表在リンパ節は蝕知せず,腹部は平坦,かつ柔軟で,腫瘤は触れなかった.生化学検査では血清補正カルシウム値16.6mg/dl,iP2.3mg/dlであり,内分泌検査では血漿副甲状腺ホルモン5ng/ml未満,副甲状腺ホルモン関連タンパク(PTHrP)5.0pmol/Lと高値であった.頭部CTでは新規脳血管障害の所見はなかった.生理食塩水と利尿剤,カルシトニンで高カルシウム血症の治療を行ったが効果はみられず,播種性血管内凝固症候群と腎不全のため死亡した.その後の病理解剖で胃原発の非ホジキンリンパ腫と診断された.リンパ腫が浸潤した脾臓を用いてRT-PCRを行ったところ,PTHrPの発現が見られ,免疫染色でもリンパ腫細胞にPTHrP陽性像が認められた
  • Y Hirose, H Suzuki, N Amizuka, J Shimomura, Y Kawano, K Nozawa-Inoue, A Kudo, T Maeda BIOMEDICAL RESEARCH-TOKYO 24 (1) 31 -37 2003年02月 [査読無し][通常論文]
     
    The immunolocalization of periostin, previously termed osteoblast-specific factor 2, was investigated in developing long bones of 17-day-old fetal mice and of 1-, 2-, 3- and 8-week-old mice at the light and electron microscopic levels. Fetal femurs showed immunoreactions for periostin in the periosteum, perichondrium, articular surface of the epiphyseal cartilage, joint ligaments, and fascias of surrounding muscles. In particular, intense immunoreactivity for periostin was found in the fibrous layer of the periosteum and perichondrium. At postnatal 1-and 2-weeks, in contrast, the immunoreactivity was restricted to the periosteum and thick fascias of surrounding muscles when compared with the fetal bones. Immunoelectron microscopic observation of the periosteum demonstrated immunoreaction products for periostin at the junction of periosteal fibroblasts and collagen bundles, suggesting its competence in the cell-to-matrix interaction. Mice at 3 and 8 weeks, unlike 2-week-old mice, showed a periostin-immunoreaction dominantly in the osteoblastic layer but not in the fibroblastic layer of the periosteum. Furthermore, the perichondrium and fascias of surrounding muscles were devoid of immunoreaction. Thus, periostin was confirmed to be widely distributed in bone and concomitant tissues at the fetal stage. As mice grew, however, its immunoreactivity gradually came to be restricted to the osteoblastic layer of the periosteum. Our findings suggest that periostin acts at the site of the cell-to-matrix interaction in periosteum, fascias, and joint ligament during morphogenesis of these tissues.
  • 網塚憲生, 下村淳子, 前田健康, 小沢英浩 Clinical Calcium 13 (4): 15-22 (4) 2003年 [査読無し][通常論文]
  • PTHrPと軟骨細胞
    腎と骨代謝 16(3): 217-228 2003年 [査読無し][通常論文]
  • 軟骨内骨化における血管の役割
    Clinical Calcium 12(3):15-24 2003年 [査読無し][通常論文]
  • 乳癌骨転移モデルを使った組織化学的研究
    新潟歯学会雑誌 32(1):111-112 2003年 [査読無し][通常論文]
  • AMIZUKA Norio, SHIMOMURA Junko, LI Minqi, SEKI Yukie, ODA Kimimitsu, HENDERSON Janet E., MIZUNO Atsuko, OZAWA Hidehiro, MAEDA Takeyasu Journal of electron microscopy 52 (6) 503 -513 2003年 [査読無し][通常論文]
     
    Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG-/- mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG-/- epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG-/- mice appear to cause bone fragility.
  • ITO MASAHIRO, AMIZUKA NORIO, TANAKA SHOHEI, FUNATSU OZAWA YUKIKO, KENMOTSU SHIN-ICHI, ODA KIMIMITSU, NAKAJIMA TAMIO, OZAWA HIDEHIRO Journal of bone and mineral metabolism 21 (6) 353 -362 2003年 [査読無し][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) induces pathological bone resorption in an endocrine manner, resulting in hypercalcemia of malignancy. However, the histopathological aspect of the action of PTHrP secreted by tumor cells on bone resorption has not well been documented. Therefore, we studied cell-cell interactions between bone cells, stromal cells, and PTHrP-secreting tumor cells (EC-GI-10) morphologically. Tumor cells injected subcutaneously into the parietal region formed a tumor mass, invading the bone marrow. The tumor mass was surrounded by a membrane structure consisting of stromal cells. These stromal cells were positive for alkaline phosphatase (ALPase). Tartrate-resistant acid phosphatase (TRAPase)-positive osteoclasts were localized close to the ALPase-positive cells, and numerous osteoclasts were observed on the neighboring bone surfaces. PTHrP, vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-9 were detected in the tumor cells. Using RT-PCR, expression of interleukin (IL)-1alpha, IL-1beta, and PTHrP, which are strong bone resorption factors, was detected in the tumor cells. Some ALPase-positive cells localizing on the neighboring bone surfaces and endothelial cells revealed PTH/PTHrP receptor immunoreactivity. Ultrastructurally, numerous blood vessels were observed between the tumor nests and the stromal cells. The nests were surrounded by a basement membrane, but it was discontinuous, therefore permitting direct contact between the tumor cells and the stromal cells. These results indicate that PTHrP secreted by tumor cells appears to stimulate osteoclast differentiation and bone resorption in a paracrine manner through PTH/PTHrP receptor-immunopositive cells. IL-1alpha, IL-1beta, VEGF, and MMP-9 may also be involved in facilitating osteoclast formation and the subsequent bone resorption.
  • Expression of estrogen receptor  (ER ) in the rat temporomandibular joint.
    Anat. Rec. 274 (2):934-941 2003年 [査読無し][通常論文]
  • 工藤久智, 荒木和男, 網塚憲生, 工藤明 日本発生生物学会大会発表要旨集 36th 2003年
  • 自己硬化型骨補填材(バイオペックス)に対する骨組織の反応
    羽尾 博嗣, 網塚 憲生, 野村 修一, 前田 健康 新潟歯学会雑誌 32 (2) 342 -342 2002年12月 [査読無し][通常論文]
  • ラット顎関節におけるエストロゲンレセプターαの免疫組織化学的研究
    山田 一穂, 河野 正司, 野澤 佳世子[井上], 網塚 憲生, 前田 健康 新潟歯学会雑誌 32 (2) 353 -353 2002年12月 [査読無し][通常論文]
  • マウス骨格組織におけるperiostinの局在について
    広瀬 泰史, 鈴木 啓展, 網塚 憲生, 前田 健康 新潟歯学会雑誌 32 (2) 355 -355 2002年12月 [査読無し][通常論文]
  • 田口 裕哉, 網塚 憲生, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 日本補綴歯科学会雑誌. 特別号, 日本補綴歯科学会学術大会抄録集 = Proceedings of the ... conference, the Japan Prosthodontic Society 46 (108) 150 -150 2002年10月11日 [査読無し][通常論文]
  • 羽尾博嗣, 網塚憲生, 藤井規孝, 織田公光, 野村修一, 前田健康 日本補綴歯科学会学術大会プログラム・抄録集 108th 189 2002年10月 [査読無し][通常論文]
  • 田口 裕哉, 網塚 憲生, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 日本補綴歯科学会雑誌 46 (108回特別) 150 -150 2002年10月 [査読無し][通常論文]
  • 羽尾 博嗣, 網塚 憲生, 藤井 規孝, 織田 公光, 野村 修一, 前田 健康 日本補綴歯科学会雑誌 46 (108回特別) 189 -189 2002年10月 [査読無し][通常論文]
  • 網塚 憲生, 監物 新一, 天谷 吉宏, 織田 公光, 前田 健康 歯科基礎医学会雑誌 44 (5) 387 -387 2002年09月20日 [査読無し][通常論文]
  • R Lin, N Amizuka, T Sasaki, MM Aarts, H Ozawa, D Goltzman, JE Henderson, JH White JOURNAL OF BONE AND MINERAL RESEARCH 17 (9) 1604 -1612 2002年09月 [査読無し][通常論文]
     
    Vitamin D deficiency results in defects in endochondral bone development characteristic of rickets, which include elongation of the cartilaginous growth plates and disorganization of the primary spongiosa. These defects are caused in part by impaired cartilage mineralization and vascularization of the chondro-osseous junction. Blood vessel invasion of mineralized cartilage is an essential step in endochondral ossification, providing access for cells that degrade cartilage as well as those that form bone. Vascular endothelial growth factor (VEGF) was shown to be a key regulator of this process when infusion of a dominant negative VEGF receptor effectively blocked vascular invasion and endochondral ossification in the growth plates of juvenile mice. Here, we show that the active metabolite of vitamin D 1alpha,25-dihydroxyvitamin D-3 [1alpha,25(OH)(2)D-3] directly stimulates transcription of mRNAs encoding VEGF121 and -165 isoforms in the CFK2 chondrogenic cell line. Enhanced VEGF expression also was evident in growth plate chondrocytes and osteoblasts in the tibia of juvenile mice treated systemically with 1alpha,25(OH)(2)D-3. This was seen in conjunction with enhanced expression of matrix metalloproteinase (MMP) 9, which activates VEGF stored in the cartilage matrix, in osteoclastic cells adjacent to the chondro-osseous junction. The alterations in VEGF and MMP-9 expression were accompanied by enhanced vascular invasion,of mineralized cartilage, as assessed by CD31 immunoreactivity. These results provide evidence that 1alpha,25(OH)(2)D-3 signaling stimulates VEGF and MMP-9 gene expression and promotes neovascularization of the epiphyseal growth plate in vivo through increased availability of active growth factor.
  • N Amizuka, Y Seki, S Kenmotsu, T Sasaki, T Maeda JOURNAL OF BONE AND MINERAL RESEARCH 17 S306 -S306 2002年09月 [査読無し][通常論文]
  • T Sasaki, K Ono, T Akatsu, N Kugai, T Maeda, N Amizuka JOURNAL OF BONE AND MINERAL RESEARCH 17 S311 -S311 2002年09月 [査読無し][通常論文]
  • 網塚 憲生, 監物 新一, 天谷 吉宏, 織田 公光, 前田 健康 歯科基礎医学会雑誌 44 (5) 387 -387 2002年09月 [査読無し][通常論文]
  • 関 雪絵, 網塚 憲生, 織田 公光, 高木 律男, 前田 健康 歯科基礎医学会雑誌 44 (5) 387 -387 2002年09月 [査読無し][通常論文]
  • 鈴木 啓展, 網塚 憲生, 河野 芳朗, 吉江 弘正, 工藤 明, 前田 健康 歯科基礎医学会雑誌 44 (5) 407 -407 2002年09月 [査読無し][通常論文]
  • 山田 一穂, 河野 正司, 井上 佳世子[野澤], 網塚 憲生, 前田 健康 歯科基礎医学会雑誌 44 (5) 408 -408 2002年09月 [査読無し][通常論文]
  • 李 敏啓, 網塚 憲生, 竹内 亀一, 高木 律男, 前田 健康 歯科基礎医学会雑誌 44 (5) 438 -438 2002年09月 [査読無し][通常論文]
  • 羽尾 博嗣, 網塚 憲生, 織田 公光, 藤井 規孝, 野村 修一, 前田 健康 歯科基礎医学会雑誌 44 (5) 441 -441 2002年09月 [査読無し][通常論文]
  • 田口 裕哉, 網塚 憲生, 大西 英夫, 藤井 規孝, 野村 修一, 前田 健康 歯科基礎医学会雑誌 44 (5) 443 -443 2002年09月 [査読無し][通常論文]
  • 網塚 憲生 日本整形外科学会雑誌 76 (8) S1085 -S1085 2002年08月 [査読無し][通常論文]
  • 島村 拓也, 網塚 憲生, 小澤 英浩 新潟歯学会雑誌 = 新潟歯学会雑誌 32 (1) 111 -112 2002年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 = Journal of Japanese Society of Bone Morphometry 12 (2) S10 2002年05月15日 [査読無し][通常論文]
  • 網塚 憲生, 関 雪絵, 前田 健康 Clinical Calcium 12 (6) 835 -843 2002年05月 [査読無し][通常論文]
  • 網塚 憲生 日本骨形態計測学会雑誌 12 (2) S10 -S10 2002年05月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小野 加津広, 赤津 拓彦, 久貝 信夫, 前田 健康 日本骨形態計測学会雑誌 12 (2) S21 -S21 2002年05月 [査読無し][通常論文]
  • 新井 智, 網塚 憲生, 東 由明, 関口 清俊, 工藤 明 Connective Tissue 34 (1) 62 -62 2002年04月 [査読無し][通常論文]
     
    <目的>RANKLはpreB細胞上に発現が認められるが、破骨前駆細胞との共存培養で破骨細胞は形成されない。このことは、破骨細胞の分化はRANK-RANKLによりてのみ制御されるのでなく、RANKL以外のストローマ細胞上の因子によりて、調節を受けている可能性がある。本研究では、破骨細胞の分化を制御するストローマ細胞表面分子を検出することを目的とした。<方法と結果>破骨細胞形成を支持するストローマ細胞株TSB13を抗原とし、モノクローナル抗体を作成した。スクリーニングの結果、TSB13上の表面分子に結合する抗体A15-1を得た。免疫組織染色で抗原は骨表面のストローマ細胞に発現が認められ、活性型ビタミンD_3の投与を続けたマウスの骨髄で抗原の発現上昇が見られた。抗原は、間葉系細胞株のみに広く発現ていた。A15-1を破骨前駆細胞とストローマ細胞の共存培養系に加えたところ、破骨前駆細胞から単核破骨細胞への分化がおこる最初の2日間のみ加えたとき、破骨細胞分化阻害が認められた。しかし、ストローマ細胞を用いない可溶型RANKLによる破骨細胞分化は阻害しなかった。高カルシウム血症モデルマウスの腹腔内にA15-1を投与したところ、破骨細胞数、骨吸収活性ともに抑制傾向が認められた。大量に蛋白を集め、免疫沈降後CBB染色でone-bandを切り出し、アミノ酸配列を決定したところ、この抗原はフィブロネクチンであった。フィブロネクチンのantisense-oligopeptideをTSB13にtransfectionし、細胞表面での発現を低下させたところ、共存培養系における破骨細胞形成活性は低下した。この結果はフィブロネクチンが破骨細胞分化を制御することを抗体のみならず、遺伝子レベルでも証明できたことを示す。<考察>A15-1の処理により、in vivo、in vitroで破骨細胞形成が阻害されたことから、細胞表面のフィブロネクチンがストローマ細胞依存性の破骨細胞分化に必須である可能性が示唆された。フィブロネクチンと破骨前駆細胞上のインテグリンと相互作用により生じたシグナルがRANK-RANKLによる破骨細胞形成シグナルを補助したものと考えられる。
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 解剖学雑誌 77 (Suppl.) J.A.A.34 -J.A.A.34 2002年03月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 浅輪 幸世, 小澤 英浩 解剖学雑誌 77 (Suppl.) J.A.A.34 -J.A.A.34 2002年03月 [査読無し][通常論文]
  • Masahiro Ito, Norio Amizuka, Hidehiro Ozawa, Kimimitsu Oda The Biochemical journal 361 (Pt 3) 473 -80 2002年02月01日 [査読無し][通常論文]
     
    Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn(153)-->Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308-314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse-chase experiments showed that newly synthesized TNSALP (N153D) remained endo-beta-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface.
  • 網塚 憲生, 佐々木 朝代, 前田 健康 Clinical Calcium 12 (3) 327 -336 2002年02月 [査読無し][通常論文]
     
    軟骨内骨化を担う細胞群の主役の1つとして血管内皮細胞があげられるが,血管内皮細胞の軟骨侵入に至る迄の軟骨細胞の整然とした細胞増殖・分化は重要なプロセスである.石灰化軟骨基質は軟骨カラム間に形成されており,血管内皮細胞はカラム中の石灰化されていない薄い横隔壁を貫通することで軟骨に侵入すると考えられる.軟骨内骨化における軟骨細胞と血管内皮細胞に焦点をあてて解説した
  • マウス尾椎の軟骨内一次骨化におけるアルカリ性ホスファターゼとオステオポンチンの局在
    新潟歯学会雑誌 31(2): 83-84 2002年 [査読無し][通常論文]
  • 癌の骨転移の微細構造
    Clinical Calcium 12(6): 137-145 2002年 [査読無し][通常論文]
  • Involvement of cyclooxygenase 2 in osteoclast formation and bone destruction in bone metastasis of mouse breast cancer cell lines.
    J Bone Miner. Res. 17: 774-781 2002年 [査読無し][通常論文]
  • 伊藤 将広, 織田 公光, 網塚 憲生, 小澤 英浩 THE BONE 16 (1) 3 -7 2002年01月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 新潟歯学会雑誌 = 新潟歯学会雑誌 31 (2) 83 -84 2001年12月 [査読無し][通常論文]
  • 泉 直也, 網塚 憲生, 小澤 英浩 THE BONE 15 (6) 619 -622 2001年11月 [査読無し][通常論文]
  • 骨転移障害部位における破骨性骨溶解とマクロファージ遊走(Osteoclastic Osteolysis and Macrophage Migration in Bone Metastastic Lesions)
    Amizuka Norio, Sasaki Tomoyo, Ono Kazuhiro, Ito Masahiro, Kenmotsu Shinichi, Oda Kimimitsu, Akatsu Takuhiko, Ejiri Sadakazu, Kugai Nobuo, Nagata Naokazu Journal of Bone and Mineral Metabolism 19 (Suppl.) 58 -58 2001年10月 [査読無し][通常論文]
  • 外島 正樹, 横手 耐治, 大月 哲也, 小松 則夫, 小澤 敬也, 網塚 憲生 臨床血液 42 (10) 1027 -1027 2001年10月 [査読無し][通常論文]
  • S Arai, N Amizuka, Y Azuma, A Kudo JOURNAL OF BONE AND MINERAL RESEARCH 16 S209 -S209 2001年09月 [査読無し][通常論文]
  • K Shibasaki, M Ito, N Amizuka, S Tanaka, H Yuyama, U Matsukawa, H Asano, K Miyata, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 16 S269 -S269 2001年09月 [査読無し][通常論文]
  • N Amizuka, M Chen, T Sasaki, Y Asawa, H Ozawa, JE Henderson JOURNAL OF BONE AND MINERAL RESEARCH 16 S187 -S187 2001年09月 [査読無し][通常論文]
  • Y Asou, N Amizuka, K Kashimada, T Yamashita, Y Nabeshima, H Ozawa, M Noda JOURNAL OF BONE AND MINERAL RESEARCH 16 S449 -S449 2001年09月 [査読無し][通常論文]
  • 西野 幾子, 網塚 憲生, 小澤 英浩 THE BONE 15 (5) 413 -417 2001年09月 [査読無し][通常論文]
  • 網塚 憲生 歯科基礎医学会雑誌 43 (4) 360 -369 2001年08月 [査読無し][通常論文]
     
    PTHrPはシグナル配列を有する分泌型蛋白質であるにも拘わらず,87位から107位迄のアミノ酸配列の両端には塩基性アミノ酸が存在し,HIV-1やHTLV-1に認められる核小体内局在様配列を有する.もしPTHrPが細胞外へ分泌されPTHと共有のレセプターに結合する一方,細胞内の核小体に局在し,何らかの機能を営むとすれば,PTHrPは粗面小胞体・ゴルジ系を経て細胞外分泌される経路と,細胞内で合成され核小体へと移行する経路の双極性を示すことになる.PTHrPの作用について,PTHrP遺伝子欠損マウスの異常,PTHrP及びPTH/PTHrPレセプターの局在を紹介し,PTHrPの核小体移行とそのメカニズムについて解説した
  • 渡邊 淳一, 網塚 憲生, 小澤 英浩 歯科基礎医学会雑誌 43 (5) 572 -572 2001年08月 [査読無し][通常論文]
  • 伊藤 将広, 網塚 憲生, 小澤 英浩, 織田 公光 歯科基礎医学会雑誌 43 (5) 533 -533 2001年08月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 歯科基礎医学会雑誌 43 (5) 557 -557 2001年08月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 浅輪 幸世, 伊藤 将広, 織田 公光, 小澤 英浩 歯科基礎医学会雑誌 43 (5) 558 -558 2001年08月 [査読無し][通常論文]
  • 西野 幾子, 網塚 憲生, 小澤 英浩 新潟歯学会雑誌 31 (1) 49 -50 2001年07月 [査読無し][通常論文]
  • 浅輪 幸世, 網塚 憲生, 佐々木 朝代, 入江 一元, 監物 新一, 原 久仁子, 秋山 康博, 江尻 貞一, 小澤 英浩 日本骨代謝学会雑誌 19 (2) 34 -34 2001年07月 [査読無し][通常論文]
  • 新井 智, 網塚 憲生, 東 由明, 工藤 明 日本骨代謝学会雑誌 19 (2) 50 -50 2001年07月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 浅輪 幸世, 伊藤 将広, Henderson Janet, 織田 公光, 小澤 英浩 日本骨代謝学会雑誌 19 (2) 5 -5 2001年07月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 19 (2) 143 -143 2001年07月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 浅輪 幸世, 小澤 英浩 整形・災害外科 44 (8) 894 -895 2001年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会雑誌 19 (1) 5 -12 2001年06月 [査読無し][通常論文]
  • M Ito, N Amizuka, T Nakajima, H Ozawa BONE 28 (6) 609 -616 2001年06月 [査読無し][通常論文]
     
    We examined the effects of a third generation bisphosphonate [YM-175; disodium dihydrogen (cycloheptylamino)-methylene-1,1-bisphosphonate] on osteoclasts in osteosclerotic (oc/oc) mice to elucidate the cellular mechanism for incorporation of the bisphosphonate, Osteoclasts of oc/oc mice were in direct contact with bone matrix but devoid of ruffled borders. Tartrate-resistant acid phosphatase (TRA-Pase) showed spotty localization intercellularly, whereas vacuolar H+-ATPase (V-ATPase) immunoreactivity was observed homogeneously in the cytoplasm, Upon injection of bisphosphonate, most osteoclasts lost cell polarity and were detached from bone surfaces. The detached osteoclasts underwent apoptosis as characterized by condensation of chromatin, absence of Golgi apparatus, and formation of many vesicles in the cytoplasm, Both TRAPase and V-ATPase were evenly distributed in the cytoplasm, The pyknotic nuclei of osteoclasts revealed DNA fragments as evidenced by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The results indicate that osteoclasts lacking ruffled borders in oc/oc mice incorporated the bisphosphonate from a site different from ruffled borders and that bisphosphonate may directly affect osteoclasts without mediating its deposition to the bone matrix. (C) 2001 by Elsevier Science Inc. All rights reserved.
  • 網塚 憲生, 佐々木 朝代, 小澤 英浩 整形・災害外科 44 (7) 802 -803 2001年06月 [査読無し][通常論文]
  • 浅輪 幸世, 網塚 憲生, 佐々木 朝代, 入江 一元, 監物 新一, 原 久仁子, 秋山 康博, 江尻 貞一, 小澤 英浩 日本骨形態計測学会雑誌 11 (1) S53 -S53 2001年06月 [査読無し][通常論文]
  • 渡邊 淳一, 野田 忠, 網塚 憲生, 小澤 英浩 THE BONE 15 (3) 199 -202 2001年05月 [査読無し][通常論文]
  • 上宮 文, 松本 美環, 風間 広仁, 荒木 厚, 細井 孝之, 小野内 常子, 網塚 憲生, 沢辺 元司, 堀内 敏行 日本内分泌学会雑誌 77 (1) 164 -164 2001年04月 [査読無し][通常論文]
  • 網塚 憲生 解剖学雑誌 76 (1) 43 -43 2001年02月 [査読無し][通常論文]
  • 網塚憲生 整形・災害外科 44 802 -803 2001年 [査読無し][通常論文]
  • 多指症とGli遺伝子
    整形・災害外科 44 849 -895 2001年 [査読無し][通常論文]
  • 骨の構造と細胞群、骨の知識と骨粗鬆症
    医薬情報センター 1 10 -13 2001年 [査読無し][通常論文]
  • Kawabata Shinichi, Amizuka Norio, Hanada Kooji, Ozawa Hidehiro Orthodontic waves : journal of the Japanese Orthodontic Society : 日本矯正歯科学会雑誌 60 (6) 354 -361 2001年 [査読無し][通常論文]
     
    The upper-left first molar of 16-month-old Fischer rats was moved mesially by 20 g of orthodontic force, in order to examine the histological alteration of the interradicular septum in the aged rats. The tooth was subjected to inclination force as well as mesial shift, showing the degenerated distal ligament around the area of bifurcation and compression of the central region of the mesial ligament. Therefore, we have observed the central region of mesial interradicular septum. In the control, a few tartrate resistant acid phosphatase (TRAP)-positive flattened osteoclasts were present on the mesial interradicular septum. Parallel osteopontin (OPN)-immunopositive lines, identical to the cement lines, were seen in the alveolar bone, due to slowly-advancing bone remodeling. On the first day of the experiment, TRAP-and ED1-positive osteoclasts were resorbing the mesial surface of the interradicular septum, and ALP-positivity was seen in cells in the mesial periodontal ligament and on the bone surface. On day 3, active osteoclastic bone resorption was still observable. ALP-positive osteoblasts were localized on the alveolar bone, interspersing among the active osteoclasts. Consistently, an intense OPN-immunoreactivity was found beneath osteoblasts, indicating deposition of bone matrix. This study demonstrated that mechanical stress could cause an immediate rise of activation of osteoclasts as early as on the first day, and induced osteoblastic activity including bone formation until the third day in the aged rats. Thus, aged rats seem to be promptly subject to osteoclastic bone resorption and subsequent osteoblastic activation under tooth movement.
  • Naoya Izumi, Norio Amizuka, Yasunori Sakakura, Kazuharu Irie, Toshihiko Yajima, Hidehiro Ozawa Acta Histochem Cytochem 34 (5) 363 -369 2001年 [査読無し][通常論文]
     
    We have raised specific antisera against the extracellular domain of the rat/mouse calcitonin receptor (CTR), consequently immunolocalizing the CTR-positive cells in mouse tibiae. As expected, the immunoreactivity for the CTR was intensely detected in cells identical to tartrate-resistant acid phosphatase (TRAP) positive multinucleated osteoclasts and mononuclear cells, whereas no immunoreactivity was detected in osteoblasts and bone marrow cells. Most osteoclasts on the bone surface possessed the CTR, therefore indicating that bone resorbing osteoclasts could be prompted to respond to endogenous calcitonin. Immuno-electron microscopy revealed CTR-immunoreactivity mainly on the plasma membranes including the pits associated with the cell membranes, and sometimes on intracellular translucent vacuoles and in the vesicles in the vicinity of the Golgi apparatus in the osteoclasts. These results lead to the postulation that CTR is chiefly localized to the cell surface of osteoclast, but is subjected to continuous internalization followed by the receptor-transport to the Golgi apparatus.
  • High bone turnover associated with increased angiogenesis in melorheostosis : Histopathological studies.
    Orthopedics. 24 273 -277 2001年 [査読無し][通常論文]
  • Nishino, I, N Amizuka, H Ozawa JOURNAL OF BONE AND MINERAL METABOLISM 19 (5) 267 -276 2001年 [査読無し][通常論文]
     
    Osteopetrotic (op/op) mice do not exhibit bone remodeling because of defective osteoclast formation caused by the depletion of macrophage colony-stimulating factor (M-CSF). In the present study, we investigated tibial bones of op/op mice with or without prior injections of M-CSF to determine whether osteoclast formation and subsequent bone resorption could activate osteoblasts, which is known as a "coupling" phenomenon. In op/op mice, no osteoclasts were present, but the metaphyseal osteoblasts adjacent to the growth plate cartilage seemed to be active, revealing an intense alkaline phosphatase (ALPase) immunoreactivity. Consequently, primary trabecular bones were extended continuously to the diaphysis, indicating that bone modeling is well achieved in op/op mice. In contrast with the metaphysis, most of the diaphyseal osteoblasts were flattened and showed weak ALPase activity, and, as a result, they seemed to be less active. Osteopontin (OPN) was localized slightly at the interface between bone and cartilage matrices of the primary trabeculae. In contrast, in op/op mice injected with M-CSF, tartrate-resistant acid phosphatase-positive osteoclasts appeared, resorbing trabecular bones of the diaphyseal region. The diaphyseal osteoblasts in the vicinity of the active osteoclasts were cuboidal and exhibited strong ALPase immunoreactivity. OPN was observed not only at the bone-cartilage interface, but also significantly on the resorption lacunae beneath the bone-resorbing osteoclasts. These observations indicate that the activation of diaphyseal osteoblasts appears to be coupled with osteoclast formation and subsequent osteoelastic bone resorption. Alternatively, the metaphyseal osteoblasts at the chondro-osseous junction seemed to be less affected by osteoclastic activity.
  • 網塚 憲生 歯科基礎医学会雑誌 43 (4) 360 -369 2001年 [査読無し][通常論文]
  • 島村 拓也, 網塚 憲生, 新垣 晋, 中島 民雄, 小澤 英浩 新潟歯学会雑誌 30 (2) 258 -258 2000年12月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 新潟歯学会雑誌 30 (2) 257 -258 2000年12月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 入江 一元, 江尻 貞一, 小澤 英浩 新潟歯学会雑誌 30 (2) 257 -257 2000年12月 [査読無し][通常論文]
  • J Watanabe, N Amizuka, T Noda, H Ozawa CELL AND TISSUE RESEARCH 301 (3) 375 -387 2000年09月 [査読無し][通常論文]
     
    Since odontoclasts share similar characteristics with osteoclasts, this study has examined whether odontoclasts exhibit cytological alteration after treatment with bisphosphonate, which induces apoptosis of osteoclasts. After the administration of bisphosphonate to 6-day-old rabbits, many odontoclasts detached from the dentine surface of the deciduous teeth, resulting in the reduction of tartrate-resistant acid phosphatase (TRAPase) and immunoreactivity for cathepsin K. Transmission electron microscopy revealed a number of odontoclasts showing poorly developed or a lack of ruffled borders, a Golgi apparatus markedly reduced in size, and numerous cytoplasmic vesicles. The bisphosphonate-treated odontoclasts displayed fragmented DNA in the pyknotic nuclei evidenced by terminal deoxynucleotidyl-transferasemediated dUTP-biotin nick-end labeling, indicating that bisphosphonate can induce apoptosis of the odontoclasts. Ultrastructural observations of the apoptotic odontoclasts revealed condensed heterochromatin at the margin of the nuclear envelope, assembled arrays of rough endoplasmic reticulum, and many vacuoles and vesicles. Some apoptotic odontoclasts showed ladder-like structures between the adjacent nuclear envelopes, enlargement of the nuclear envelopes, and the formation of a ribosome-like granular structure in the nuclei. Thus, odontoclasts are able to undergo apoptosis after bisphosphonate treatment; this results in cytological alterations, including reduced resorption activity and the inhibition of protein synthesis/transport as indicated by the diminished TRAPase and cathepsin K and the poorly developed Golgi apparatus, respectively. Nuclear alteration as evidenced by the appearance of ladder-like and ribosome-like structures was characteristic of apoptotic odontoclasts.
  • T Sasaki, N Amizuka, K Oda, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 15 S243 -S243 2000年09月 [査読無し][通常論文]
  • R Lin, N Amizuka, D Wang, H Ozawa, D Goltzman, JE Henderson, JH White JOURNAL OF BONE AND MINERAL RESEARCH 15 S342 -S342 2000年09月 [査読無し][通常論文]
  • Y Asou, N Amizuka, T Yamashita, Y Nabeshima, H Ozawa, M Noda JOURNAL OF BONE AND MINERAL RESEARCH 15 S468 -S468 2000年09月 [査読無し][通常論文]
  • N Amizuka, H Liu, D Wang, H Ozawa, D Goltzman, JE Henderson JOURNAL OF BONE AND MINERAL RESEARCH 15 S157 -S157 2000年09月 [査読無し][通常論文]
  • T Shimamura, N Amizuka, N Izumi, T Nakajima, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 15 S350 -S350 2000年09月 [査読無し][通常論文]
  • N Amizuka, T Sasaki, R Lin, D Goltzman, JE Henderson, H Ozawa, JH White JOURNAL OF BONE AND MINERAL RESEARCH 15 S206 -S206 2000年09月 [査読無し][通常論文]
  • マウス乳癌細胞株の骨破壊メカニズムの検討
    小野 加津広, 赤津 拓彦, 村上 健彦, 山本 通子, 四ノ宮 成祥, 六反田 亮, 網塚 憲生, 小澤 英浩, 永田 直一, 久貝 信夫 Osteoporosis Japan 8 (Suppl.1) 107 -107 2000年09月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 THE BONE 14 (4) 399 -402 2000年08月 [査読無し][通常論文]
  • 麻生 義則, 網塚 憲生, 山下 照仁, 二藤 彰, 四宮 謙一, 鍋島 陽一, 小澤 英浩, 野田 政樹 日本整形外科学会雑誌 74 (8) S1825 -S1825 2000年08月 [査読無し][通常論文]
  • 網塚 憲生 歯科基礎医学会雑誌 42 (5抄録集) 395 -395 2000年08月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 歯科基礎医学会雑誌 42 (5抄録集) 402 -402 2000年08月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 小澤 英浩 歯科基礎医学会雑誌 42 (5抄録集) 402 -402 2000年08月 [査読無し][通常論文]
  • 福士 真理子[入江], 伊藤 将広, 天谷 吉宏, 網塚 憲生, 佐々木 朝代, 小澤 英浩, 織田 公光, 大村 智, 池原 征夫 歯科基礎医学会雑誌 42 (5抄録集) 446 -446 2000年08月 [査読無し][通常論文]
  • 渡邊 淳一, 網塚 憲生, 小澤 英浩 歯科基礎医学会雑誌 42 (5抄録集) 448 -448 2000年08月 [査読無し][通常論文]
  • N Amizuka, JE Henderson, JH White, AC Karaplis, D Goltzman, T Sasaki, H Ozawa HISTOLOGY AND HISTOPATHOLOGY 15 (3) 957 -970 2000年07月 [査読無し][通常論文]
     
    Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and premature maturation of chondrocytes. Abnormal, heterogeneous populations of chondrocytes at different stages of differentiation were seen in the hypertrophic zone of the mutant growth plate. Although the homozygous null animals die within several hours of birth, mice heterozygous for PTHrP gene deletion reach adulthood, at which time they show evidence of osteopenia. Therefore, PTHrP appears to modulate cell proliferation and differentiation in both the pre and post natal period. PTH/PTHrP receptor expression in the mouse is controlled by two promoters. We recently found that, while the downstream promoter controls PTH/PTHrP receptor gene expression in bone and cartilage, it is differentially regulated in the two tissues. 1 alpha,25-dihydroxyvitamin D3 downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes, both in vivo and in vitro. Most of the biological activity of PTHrP is thought to be mediated by binding of its amino terminus to the PTH/PTHrP receptor. However, recent evidence suggests that amino acids 87-107, outside of the amino terminal binding domain, act as a nucleolar targeting signal. Chondrocytic cell line, CFK2, transfected with wild-type PTHrP cDNA showed PTHrP in the nucleoli as well as in the secretory pathway. Therefore, PTHrP appears to act as a bifunctional modulator of both chondrocyte proliferation and differentiation, through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus.
  • T Sasaki, N Amizuka, K Irie, S Ejiri, H Ozawa ARCHIVES OF HISTOLOGY AND CYTOLOGY 63 (3) 271 -284 2000年07月 [査読無し][通常論文]
     
    We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent, In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immuorecactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules, When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization, Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.
  • N Amizuka, M Fukushi-Irie, T Sasaki, K Oda, H Ozawa BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 273 (2) 621 -629 2000年07月 [査読無し][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) is not only secreted out of cells, but also targeted to the nucleoli due to a nucleolar targeting signal (NTS). We assessed the molecular mechanism underlying the dual targeting of PTHrP by constructing a series of truncated forms of rat PTHrP cDNA and expressing them in CHO cells. Immunostaining was observed in both the Golgi apparatus and nucleoli in the same cell expressing PTHrP with the N-terminal full-length signal sequence. When PTHrP molecules were translated from CUGs downstream of the AUG-initiator codon in the signal sequences, potential alternative initiators of the translation, they were exclusively localized in the nucleoli. In contrast, when a construct containing only the ATG-initiator codon was expressed, PTHrP was found to localize in both the nucleolus and the Golgi apparatus. No nucleolar staining of PTHrP was observed in the CHO cells transfected with PTH/ PTHrP receptors even after incubating with a conditioned medium containing PTHrP, ruling out a possibility that PTHrP is, once secreted, internalized via receptor-mediated endocytosis and subsequently conveyed to nucleoli. Compatible with these morphological observations, a preproform of PTHrP was found in the cells expressing PTHrP in addition to proPTHrP, indicative of molecules along the secretory pathway. These results strongly indicate that the signal sequence of PTHrP is not sufficient to direct all the newly synthesized molecules across the endoplasmic reticulum, resulting in part of it being delivered to the nucleoli due to the NTS. (C) 2000 Academic Press.
  • 尾椎の発達している軟骨における基質鉱化中アルカリホスファターゼ及びオステオポンチンの局在
    Sasaki Tomoyo, Amizuka Norio, Irie Kazuharu, Ejiri Sadakazu, Ozawa Hidehiro Archives of Histology and Cytology 63 (3) 271 -284 2000年07月 [査読無し][通常論文]
     
    軟骨原基におけるアルカリホスファターゼ,鉱化及びオステオポンチンの場所的及び時間的局在を示した.アルカリホスファターゼ及び早期鉱化は増殖性及び肥大性帯の間の境界に沿って検出されたのに対し,オステオポンチンは殆ど独占的に肥大性帯に局在した.選択的に,一旦血管侵入が起こると,オステオポンチンは肥大性帯の下部領域に限られた.オステオポンチンは示唆されたように鉱化に対する規定者として作用するが,血管侵入,ならびに遊走する骨芽細胞及び破骨細胞の続いて起こる接着の誘導者として機能し得ると考えられた
  • 網塚 憲生, 島村 拓也, 伊藤 将広, 佐々木 朝代, 小澤 英浩 ホルモンと臨床 48 (7) 585 -594 2000年07月 [査読無し][通常論文]
  • M Fukushi-Irie, M Ito, Y Amaya, N Amizuka, H Ozawa, S Omura, Y Ikehara, K Oda BIOCHEMICAL JOURNAL 348 (3) 633 -642 2000年06月 [査読無し][通常論文]
     
    Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54), CYS (R54C) or an Asp(277)--> Ala (D277A) substitution was found in a patient with hypophosphatasia [Henthorn, Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9924-9928]. To examine effects of these missense mutations on properties of TNSALP, the TNSALP mutants were expressed ectopically in COS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP (R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP (D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A) was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP (R54C) interferes with folding and assembly of TNSALP (D277A) in trans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.
  • 泉 直也, 網塚 憲生, 小澤 英浩 THE BONE 14 (3) 273 -276 2000年06月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, Henderson Janet, 小澤 英浩 日本骨代謝学会雑誌 18 (2) 9 -9 2000年06月 [査読無し][通常論文]
  • 小澤 英浩, 網塚 憲生, 伊藤 将広, 島村 拓也 日本骨代謝学会雑誌 18 (2) (79) -(79) 2000年06月 [査読無し][通常論文]
  • 島村 拓也, 網塚 憲生, 中島 民雄, 小澤 英浩 日本骨代謝学会雑誌 18 (2) 157 -157 2000年06月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 織田 公光, 小澤 英浩 日本骨代謝学会雑誌 18 (2) 11 -11 2000年06月 [査読無し][通常論文]
  • 小野 加津広, 赤津 拓彦, 村上 健彦, 北村 竜一, 山本 通子, 網塚 憲生, 小澤 英浩, 永田 直一, 久貝 信夫 日本骨代謝学会雑誌 18 (2) 158 -158 2000年06月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 日本骨形態計測学会雑誌 10 (1) S34 -S34 2000年06月 [査読無し][通常論文]
     
    血管内皮細胞増殖因子は肥大化軟骨細胞より産生され,HS-GAG等の細胞外基質に結合し,細胞・基質間相互作用にて効率よく血管内皮細胞に受容される機構が推測された
  • 佐々木 朝代, 網塚 憲生, 小澤 英浩 日本骨形態計測学会雑誌 = Journal of Japanese Society of Bone Morphometry 10 (1) S34 2000年06月01日 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 小澤 英浩 医学のあゆみ 192 (6) 740 -746 2000年02月05日 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 小澤 英浩 医学のあゆみ 192 (6) 740 -746 2000年02月 [査読無し][通常論文]
  • 島村 拓也, 網塚 憲生, 中島 民雄, 小澤 英浩 解剖学雑誌 75 (1) 147 -147 2000年02月 [査読無し][通常論文]
  • 網塚 憲生, 佐々木 朝代, 小澤 英浩 解剖学雑誌 75 (1) 147 -147 2000年02月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 入江 一元, 江尻 貞一, 小澤 英浩 解剖学雑誌 75 (1) 147 -147 2000年02月 [査読無し][通常論文]
  • Norio Amizuka, Hidehiro Ozawa, Tomoyo Sasaki Acta Anatomica Nipponica 75 (5) 415 -425 2000年 [査読無し][通常論文]
     
    Parathyroid hormone (PTH)-related peptide (PTHrP) was determined to be a factor inducing malignancy-associated hypercalcemia by activating a common receptor (PTH/PTHrP receptor) with PTH. PTHrP gene "knock out" mice showed a form of dyschondroplasia due to reduced proliferation of chondrocytes. In addition, heterogenous populations of variously-differentiated chondrocytes were present in the hypertrophie zone of the mutant epiphyseal plate. Although the homozygotes die within several hours after birth, the adult mice, heterozygous for PTHrP gene deletion, showed a delayed skeletal abnormality at 3 month old, with a reduced amount of PTHrP transcript, therefore, PTHrP appears to modulate cell proliferation and differentiation at both fetal and adult stages. The co-localization of PTHrP and its receptor in osteoblastic cells and chondrocytes suggested a paracrine/autocine mode of action manner of these molecules. Recently, fibroblast growth factor receptor 3 (FGFR3) deficient mice demonstrated skeletal defects including kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae, which are caused by an increase in proliferation. Therefore, it seems that PTHrP and FGFR3 serve as positive and negative regulators on the chondrocyte proliferation, respectively. In this paper, we review our recent studies on the histological abnormality of long bone seen in PTHrP gene deficient- and FGFR3 gene deficient-mice.
  • 網塚憲生 総合健康推進財団研究報告書 1997 2000年
  • 網塚憲生, 佐々木朝代, 小沢英浩, 織田公光, 入江真理子 Bone 14 (5) 2000年
  • M Ito, N Amizuka, T Nakajima, H Ozawa BONE 25 (4) 447 -452 1999年10月 [査読無し][通常論文]
     
    The process of apoptosis and fate of osteoclasts are not well elucidated because dying osteoclasts are rarely seen in normal bone. Histological, cytochemical, and ultrastructural features of osteoclasts undergoing apoptosis were studied in the femur and tibia of rats treated with a third-generation bisphosphonate (disodium dihydrogen (cycloheptylalmino)methylene-l, l-bisphosphonate), After the bisphosphonate administration, osteoclasts decreased significantly in number. Initially, they became devoid of ruffled borders and detached from the bone surface. In such osteoclasts, the Golgi apparatus was degraded, or dispersed in the cytoplasm, Later, osteoclasts revealed typical features of apoptosis, with pyknotic nuclei showing condensation and margination of heterochromatins and DNA fragmentation. They were often convoluted to give rise to apoptotic bodies. In addition, enlargement and fusion of nuclear envelopes and subsequent disruption leading to leakage of nuclear contents into the cytoplasm were observed in osteoclasts in the late stage of apoptosis, These osteoclasts as well as apoptotic bodies were surrounded by cytoplasmic processes of macrophages, which often contained degenerated cytoplasmic fragments of osteoclasts, Apoptotic osteoclasts migrating into or present in capillaries were also observed in some areas. In conclusion, bisphosphonate induces apoptosis of osteoclasts, which was characterized by ultrastructural changes of the nucleus typical of apoptosis accompanied by degradation of cell organelles. The majority of them are eliminated by macrophages, but there are some that escape into blood vessels. (C) 1999 by Elsevier Science Inc. All rights reserved.
  • N Amizuka, MF Chen, C Goodyer, H Ozawa, JE Henderson JOURNAL OF BONE AND MINERAL RESEARCH 14 S187 -S187 1999年09月 [査読無し][通常論文]
  • N Amizuka, D Wang, J Colvin, H Ozawa, D Goltzman, D Ornitz, JE Henderson JOURNAL OF BONE AND MINERAL RESEARCH 14 S435 -S435 1999年09月 [査読無し][通常論文]
  • 堀内 圭輔, 網塚 憲生, 竹下 淳, 小澤 英浩, 戸山 芳昭, 工藤 明 日本細胞生物学会大会講演要旨集 52回 69 -69 1999年08月 [査読無し][通常論文]
  • 島村 拓也, 網塚 憲生, 中島 民雄, 小澤 英浩 歯科基礎医学会雑誌 41 (5) 425 -425 1999年08月 [査読無し][通常論文]
  • 佐々木 朝代, 網塚 憲生, 入江 一元, 小澤 英浩 歯科基礎医学会雑誌 41 (5) 427 -427 1999年08月 [査読無し][通常論文]
  • 網塚 憲生, 小澤 英浩 歯科基礎医学会雑誌 41 (5) 429 -429 1999年08月 [査読無し][通常論文]
  • 堀内 圭輔, 網塚 憲生, 竹下 淳, 小澤 英浩, 工藤 明, 戸山 芳昭 日本整形外科学会雑誌 73 (8) S1921 -S1921 1999年08月 [査読無し][通常論文]
  • 網塚 憲生 新潟歯学会雑誌 29 (1) 33 -34 1999年07月 [査読無し][通常論文]
  • 網塚 憲生 腎と骨代謝 12 (3) 253 -266 1999年07月 [査読無し][通常論文]
  • 網塚 憲生, 小澤 英浩 THE BONE 13 (2) 3 -6 1999年06月 [査読無し][通常論文]
  • 小澤 英浩, 網塚 憲生 THE BONE 13 (2) 47 -56 1999年06月 [査読無し][通常論文]
  • 網塚 憲生 THE BONE 13 (2) 73 -79 1999年06月 [査読無し][通常論文]
  • 網塚 憲生, Goltzman David, Ornitz David, Henderson Janet, 小澤 英浩 日本骨代謝学会雑誌 17 (2) 7 -7 1999年06月 [査読無し][通常論文]
  • 堀内 圭輔, 網塚 憲生, 竹下 淳, 小澤 英浩, 戸山 芳昭, 工藤 明 日本骨代謝学会雑誌 17 (2) 18 -18 1999年06月 [査読無し][通常論文]
  • K Wakida, N Amizuka, C Murakami, T Satoda, M Fukae, JP Simmer, H Ozawa, T Uchida HISTOCHEMISTRY AND CELL BIOLOGY 111 (4) 297 -303 1999年04月 [査読無し][通常論文]
     
    Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage.
  • 歯科臨床のための骨の科学 軟骨発生におけるPTHrPの役割とその欠損による異常について
    網塚 憲生 The Quintessence 18 (3) 674 -681 1999年03月 [査読無し][通常論文]
  • K Hoshi, S Kemmotsu, Y Takeuchi, N Amizuka, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 14 (2) 273 -280 1999年02月 [査読無し][通常論文]
     
    To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19-day post-coitum embryonic rat calvariae, Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts, In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 mn in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils, In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side, Where calcification was progressed, this: fusion was even more so, Some very large fribrils exhibited complicated contours, 400 nm or more in diameter, Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification, The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.
  • N Amizuka, MY Kwan, D Goltzman, H Ozawa, JH White JOURNAL OF CLINICAL INVESTIGATION 103 (3) 373 -381 1999年02月 [査読無し][通常論文]
     
    Transcription of the mouse parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR) gene is controlled by promoters P1 and P2. We performed transcript-specific in situ hybridization and found that P2 is the predominant promoter controlling PTHR gene expression in bone and cartilage. Treatment with 1 alpha,25-dihydroxyvitamin D3 (D3) in vivo specifically downregulated P2-specific transcripts in osteoblasts, but not in chondrocytes, under conditions where it enhanced bone resorption. Treatment of the osteoblastic cell Line MC3T3-E1 with D3 in vitro reduced expression of both P2-specific transcripts and PTHR protein. This effect was not blocked by cycloheximide, indicating that D3 inhibits PTHR expression by downregulating transcription of the P2 promoter. A similar inhibitory effect of D3 was not observed in the chondrocytic cell line CFK2. Gene-transfer experiments showed that P2, but not P1, is active in both MC3T3-E1 and CFK2 cells, and that D3 specifically inhibited P2 promoter activity in MC3T3-E1, but not in CFK2 cells. Inhibition of P2 activity by D3 required promoter sequences lying more that 1.6 kb upstream of the P2 transcription start site. Thus, the P2 promoter controls PTHR gene expression in both osteoblasts and chondrocytes. D3 downregulates PTHR gene transcription in a cell-specific manner by inhibiting P2 promoter activity in osteoblasts, but not in chondrocytes.
  • 骨組織の再建 微細形態学的・細胞化学的特徴
    小澤 英浩, 星 和人, 入江 一元, 中村 浩彰, 網塚 憲生, 江尻 貞一 解剖学雑誌 74 (1) 12 -12 1999年02月 [査読無し][通常論文]
  • 変異型組織非特異的アルカリホスファターゼの細胞内局在と酵素活性
    福士 真理子, 網塚 憲生, 織田 公光, 小澤 英浩 解剖学雑誌 74 (1) 125 -125 1999年02月 [査読無し][通常論文]
  • 軟骨・骨の分化形成におけるFGFレセプター3とPTHrPの作用 FGFR3/PTHrP遺伝子二重欠損マウスを用いた形態学的解析
    網塚 憲生, 小澤 英浩 解剖学雑誌 74 (1) 137 -137 1999年02月 [査読無し][通常論文]
  • 網塚憲生 メディカルレビュー社、東京 13 (2) 73 -79 1999年 [査読無し][通常論文]
  • 小沢英浩, 網塚憲生 メディカルレビュー社 13 (2) 47 -56 1999年 [査読無し][通常論文]
  • 軟骨におけるPTHrPの作用 腎と骨代謝
    骨の分子発生学 12 (3) 253 -266 1999年 [査読無し][通常論文]
  • 網塚憲生 クインテッセンス出版 18 (3) 174 -180 1999年 [査読無し][通常論文]
  • カルシウム その基礎・臨床・栄養 骨とカルシウム -骨の細胞とカルシウム-
    社団法人 全国牛乳普及協会 1999年 [査読無し][通常論文]
  • THE BONE
    13 (2) 73 -79 1999年 [査読無し][通常論文]
  • THE BONE
    13 (2) 47 -56 1999年 [査読無し][通常論文]
  • Keisuke Horiuchi, Norio Amizuka, Sunao Takeshita, Hiroyuki Takamatsu, Mieko Katsuura, Hidehiro Ozawa, Yoshiaki Toyama, Lynda F. Bonewald, Akira Kudo Journal of Bone and Mineral Research 14 (7) 1239 -1249 1999年 [査読無し][通常論文]
     
    We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed 'periostin.' Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription- polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3- E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to βig-h3, a molecule induced by transforming growth factor β (TGF-β) that promotes the adhesion and spreading of fibroblasts. Because TGF-β has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF-β increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.
  • Blomstrand chondrodisplasia caused by a missense mutation in the human parathyroid hormone receptor-1. (共著)
    Endocrinology. 139 (12) 5255 -5258 1999年 [査読無し][通常論文]
  • IZUMI Naoya, AMIZUKA Norio, ODA Kimimitsu, MISUMI Yoshio, IKEHARA Yukio, OZAWA Hidehiro Acta histochemica et cytochemica 32 (5) 393 -405 1999年 [査読無し][通常論文]
     
    Osteoclasts secrete protons and lysosomal enzymes through ruffled borders, resulting in demineralization and degradation of bone matrix. Golgi apparatus is thought to play a key role in this process. In order to elucidate the specific functions of Golgi apparatus in osteoclasts, we observed the ultrastructural and cytochemical changes resulting from the administration of brefeldin A (BFA), an inhibitor of Golgi structures, and wortmannin (WT), an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). When treated with BFA, osteoclasts showed a dissociated Golgi apparatus, while the peanut agglutinin (PNA)-positive ruffled borders were poorly developed or disappeared. Moreover, the acid phosphatase (ACPase) activity was somewhat reduced. WT-treated osteoclasts, on the other hand, exhibited no ultrastructural alteration of the Golgi apparatus, but were marked by the disappearance of PNA-positive ruffled borders, and a consequent increase in the number of ACPase-positive vesicles and vacuoles in the cytoplasm. These results indicate that the Golgi apparatus of osteoclasts not only plays an important role in the formation of lysosomal enzymes but also the supply of ruffled border membranes. Furthermore, PI 3-kinase appears to be involved in the transport of lysosomal enzymes and the maintenance of ruffled borders.
  • 網塚 憲生, 小澤 英浩 THE BONE 12 (4) 3 -6 1998年12月 [査読無し][通常論文]
  • Bisphosphonateにより誘導される破歯細胞のアポトーシスに関する細胞化学的検索
    渡邊 淳一, 網塚 憲生, 野田 忠, 小澤 英浩 新潟歯学会雑誌 28 (2) 93 -93 1998年12月 [査読無し][通常論文]
  • op/opマウスへのM-CSF投与による骨基質改変について
    西野 幾子, 網塚 憲生, 小澤 英浩 新潟歯学会雑誌 28 (2) 98 -98 1998年12月 [査読無し][通常論文]
  • 致死型低ホスファターゼ症例 突然変異型組織非特異的アルカリホスファターゼ(Gly317→Asp)の細胞内における保持と分解
    福士 真理子, 網塚 憲生, 小澤 英浩, 織田 公光 新潟歯学会雑誌 28 (2) 98 -99 1998年12月 [査読無し][通常論文]
  • 望月 礼子, 柳内 和幸, 石塚 保行, 谷口 圭子, 高塚 美紀, 網塚 憲生, 川島 博行, 小澤 英浩, 深水 昭吉 日本分子生物学会年会プログラム・講演要旨集 21 607 -607 1998年12月01日 [査読無し][通常論文]
  • 泉 直也, 網塚 憲生, 小澤 英浩 電子顕微鏡 33 (3) 202 -204 1998年11月30日 [査読無し][通常論文]
  • 泉 直也, 網塚 憲生, 小澤 英浩 電子顕微鏡 33 (3) 202 -204 1998年11月 [査読無し][通常論文]
  • 網塚 憲生, 入江 一元, 小澤 英浩, 水野 敦子, 東尾 侃二 THE BONE 12 (3) 3 -6 1998年09月 [査読無し][通常論文]
  • 組織非特異的アルカリホスファターゼの変異による酵素活性及び細胞内局在の変化について
    福士 真理子, 網塚 憲生, 小澤 英浩, 織田 公光 歯科基礎医学会雑誌 40 (抄録) 448 -448 1998年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン関連ペプチド(PTHrP)の核小体移行はalternative translationに起因する
    網塚 憲生, 福士 真理子, 織田 公光, 小澤 英浩 歯科基礎医学会雑誌 40 (抄録) 387 -387 1998年09月 [査読無し][通常論文]
  • 細胞分化 骨・脳特異的新規因子 C16Nのノックアウトマウス作製とその解析
    望月 礼子, 柳内 和幸, 石塚 保行, 谷口 圭子, 漆崎 美紀, 網塚 憲生, 川島 博行, 小澤 英浩, 深水 昭吉 生化学 70 (8) 755 -755 1998年08月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会雑誌 16 (2) 74 -74 1998年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 16 (1) 40 -41 1998年06月30日 [査読無し][通常論文]
  • A Mizuno, N Amizuka, K Irie, A Murakami, N Fujise, T Kanno, Y Sato, N Nakagawa, H Yasuda, S Mochizuki, T Gomibuchi, K Yano, N Shima, N Washida, E Tsuda, T Morinaga, K Higashio, H Ozawa BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 247 (3) 610 -615 1998年06月 [査読無し][通常論文]
     
    Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG; is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities. (C) 1998 Academic Press.
  • A Mizuno, N Amizuka, K Irie, A Murakami, N Fujise, T Kanno, Y Sato, N Nakagawa, H Yasuda, S Mochizuki, T Gomibuchi, K Yano, N Shima, N Washida, E Tsuda, T Morinaga, K Higashio, H Ozawa BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 247 (3) 610 -615 1998年06月 [査読無し][通常論文]
     
    Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG; is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities. (C) 1998 Academic Press.
  • 網塚 憲生, 小澤 英浩 THE BONE 12 (2) 3 -6 1998年06月 [査読無し][通常論文]
  • M Fukushi, N Amizuka, K Hoshi, H Ozawa, H Kumagai, S Omura, Y Misumi, Y Ikehara, K Oda BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 246 (3) 613 -618 1998年05月 [査読無し][通常論文]
     
    One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C.R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, me have examined the biosynthesis of TNSALP with a Gly(317)-->Asp substitution, When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP. (C) 1998 Academic Press.
  • N Amizuka, M Yamada, JI Watanabe, K Hoshi, M Fukushi, K Oda, Y Ikehara, H Ozawa MICROSCOPY RESEARCH AND TECHNIQUE 41 (4) 313 -322 1998年05月 [査読無し][通常論文]
     
    Woven bone induced by direct injection of basic fibroblast growth factor (bFGF) into rat bone marrow was examined. On the first day after injection, fibrous tissues formed in the treated region of the bone marrow. Tissue-nonspecific alkaline phosphatase (TNAPase)-immunopositive osteoblastic cells and osteopontin immunopositive-extracellular matrices were observed in the fibrous tissues, indicating bone induction. On the fifth day, the bFGF-induced bone was found broadly in the bone marrow. In the originally existing bone, osteopontin-immunoreactivity was observed at cement Lines, but not in the fully calcified matrix, whereas the woven bone displayed immunoreactivity throughout the matrix. Numerous TRAPase-positive osteoclasts were present on the surfaces of the woven bone, but no obvious cement Line was observed. Therefore, both bone formation and resorption appeared highly active, without normal cellular coupling equilibrated between bone formation and resorption performed by osteoblasts and osteoclasts. On the tenth day, the bFGF-induced bone was almost replaced by bone marrow. Thus, bone formation actively occurred in the first half of the experimental period, whereas bone resorption came to be predominant thereafter. This study demonstrated that bFGF stimulates bone formation, which, however, is subject to subsequent resorption, probably due in part to the absence of coordinated cellular coupling between osteoclasts and osteoblasts.
  • M Fukushi, N Amizuka, K Hoshi, H Ozawa, H Kumagai, S Omura, Y Misumi, Y Ikehara, K Oda BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 246 (3) 613 -618 1998年05月 [査読無し][通常論文]
     
    One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C.R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, me have examined the biosynthesis of TNSALP with a Gly(317)-->Asp substitution, When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP. (C) 1998 Academic Press.
  • N Amizuka, M Yamada, JI Watanabe, K Hoshi, M Fukushi, K Oda, Y Ikehara, H Ozawa MICROSCOPY RESEARCH AND TECHNIQUE 41 (4) 313 -322 1998年05月 [査読無し][通常論文]
     
    Woven bone induced by direct injection of basic fibroblast growth factor (bFGF) into rat bone marrow was examined. On the first day after injection, fibrous tissues formed in the treated region of the bone marrow. Tissue-nonspecific alkaline phosphatase (TNAPase)-immunopositive osteoblastic cells and osteopontin immunopositive-extracellular matrices were observed in the fibrous tissues, indicating bone induction. On the fifth day, the bFGF-induced bone was found broadly in the bone marrow. In the originally existing bone, osteopontin-immunoreactivity was observed at cement Lines, but not in the fully calcified matrix, whereas the woven bone displayed immunoreactivity throughout the matrix. Numerous TRAPase-positive osteoclasts were present on the surfaces of the woven bone, but no obvious cement Line was observed. Therefore, both bone formation and resorption appeared highly active, without normal cellular coupling equilibrated between bone formation and resorption performed by osteoblasts and osteoclasts. On the tenth day, the bFGF-induced bone was almost replaced by bone marrow. Thus, bone formation actively occurred in the first half of the experimental period, whereas bone resorption came to be predominant thereafter. This study demonstrated that bFGF stimulates bone formation, which, however, is subject to subsequent resorption, probably due in part to the absence of coordinated cellular coupling between osteoclasts and osteoblasts.
  • L Beck, AC Karaplis, N Amizuka, AS Hewson, H Ozawa, HS Tenenhouse PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95 (9) 5372 -5377 1998年04月 [査読無し][通常論文]
     
    Npt2 encodes a renal-specific, brush-border membrane Na+-phosphate (P-i) cotransporter that is expressed in the proximal tubule where the bulk of filtered P-i is reabsorbed. Mice deficient in the Npt2 gene mere generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of P-i homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal P-i reabsorption in humans. Homozygous mutants (Npt2(-/-)) exhibit increased urinary P-i excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal P-i reabsorption. However; unlike HHRH patients, Npt2(-/-) mice do not have rickets or osteomalacia, At weaning, Npt2(-/-) mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype, Our findings demonstrate that Npt2 is a major regulator of P-i homeostasis and necessary for normal skeletal development.
  • T Kameda, H Mano, Y Yamada, H Takai, N Amizuka, M Kobori, N Izumi, H Kawashima, H Ozawa, K Ikeda, A Kameda, Y Hakeda, M Kumegawa BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 245 (2) 419 -422 1998年04月 [査読無し][通常論文]
     
    Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption. (C) 1998 Academic Press.
  • L Beck, AC Karaplis, N Amizuka, AS Hewson, H Ozawa, HS Tenenhouse PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95 (9) 5372 -5377 1998年04月 [査読無し][通常論文]
     
    Npt2 encodes a renal-specific, brush-border membrane Na+-phosphate (P-i) cotransporter that is expressed in the proximal tubule where the bulk of filtered P-i is reabsorbed. Mice deficient in the Npt2 gene mere generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of P-i homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal P-i reabsorption in humans. Homozygous mutants (Npt2(-/-)) exhibit increased urinary P-i excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal P-i reabsorption. However; unlike HHRH patients, Npt2(-/-) mice do not have rickets or osteomalacia, At weaning, Npt2(-/-) mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype, Our findings demonstrate that Npt2 is a major regulator of P-i homeostasis and necessary for normal skeletal development.
  • T Kameda, H Mano, Y Yamada, H Takai, N Amizuka, M Kobori, N Izumi, H Kawashima, H Ozawa, K Ikeda, A Kameda, Y Hakeda, M Kumegawa BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 245 (2) 419 -422 1998年04月 [査読無し][通常論文]
     
    Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption. (C) 1998 Academic Press.
  • カルシトニン受容体と副甲状腺ホルモン受容体との関連・異同について
    ライフサイエンス出版 1998年 [査読無し][通常論文]
  • マイクロウェーブを用いた骨組織の迅速固定、迅速脱灰 マイクロウェーブ照射による生物試料の固定・染色法の基本とその応用(共著)
    学際企画 1998年 [査読無し][通常論文]
  • イヌの歯槽上部インプラント周囲骨欠損の骨再生法において脱灰凍結乾燥を施した同種骨移植の影響
    クインテッセンスDental Implantology 5 (2) 100 -109 1998年 [査読無し][通常論文]
  • Possible involvement of stromal cells in the differentiation and activation of osteoclasts at the site of bone metastasis. International Bone Forum
    1998年 [査読無し][通常論文]
  • 網塚憲生 上原記念生命科学財団研究報告集 12 1998年
  • 網塚 憲生, 小澤 英浩, 田中 瑞栄 THE BONE 11 (4) 3 -6 1997年12月 [査読無し][通常論文]
  • 副甲状腺ホルモン関連ペプチドの細胞内局在について
    網塚 憲生 歯科基礎医学会雑誌 39 (抄録) 456 -456 1997年09月 [査読無し][通常論文]
  • 網塚 憲生, 小澤 英浩 THE BONE 11 (3) 39 -52 1997年09月 [査読無し][通常論文]
  • 星 和人, 網塚 憲生, 監物 新一, 小澤 英浩 日本整形外科學會雜誌 = The Journal of the Japanese Orthopaedic Association 71 (8) S1516 1997年08月25日 [査読無し][通常論文]
  • K Hoshi, S Kenmotsu, Y Takeuchi, N Amizuka, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 12 F301 -F301 1997年08月 [査読無し][通常論文]
  • N Amizuka, M Kwan, D Goltzman, H Ozawa, JH White JOURNAL OF BONE AND MINERAL RESEARCH 12 S372 -S372 1997年08月 [査読無し][通常論文]
  • M Ikegame, O Ishibashi, N Amizuka, H Ozawa, H Kawashima JOURNAL OF BONE AND MINERAL RESEARCH 12 F227 -F227 1997年08月 [査読無し][通常論文]
  • K Hoshi, N Amizuka, T Sakou, T Kurokawa, H Ozawa BONE 21 (2) 155 -162 1997年08月 [査読無し][通常論文]
     
    To elucidate the process of ossification in spinal ligaments, an aqueous solution containing recombinant human bone morphogenetic protein (BMP)-2 (40 mu g/100 mu L) was injected into murine ligamenta flava, and the ossification process was analyzed morphologically. In the control group, the solution administered lacked the protein; these flattened ligamentous fibroblasts possessing BMP receptors type IA and type II existed among type I collagen bundles, In the week immediately following the injection of BMP-2, ligamentous fibroblasts began to proliferate, differentiating into alkaline phosphatase-positive chondrocytes surrounded by an extracellular matrix composed of type I and II collagen, By the second week, differentiated chondrocytes of various stages were observed in type II collagen-rich matrix, These chondrocytes showed an abundance of BMP receptors type IA and II, The pathologically induced cartilage was resorbed by chondroclasts, permitting migration of blood vessels and osteogenic cells, as well as providing a site for endochondral ossification, By the third week, BMP-induced ossification had compressed the spinal cord, and by the sixth week, the ligamentous tissue had been almost completely replaced by bone, Ligamentous fibroblasts appeared to possess BMP receptors, as well as the potentiality to differentiate into chondrocytes, BMP receptors were upregulated during chondrification of ligamentous fibroblasts induced by exogenous BMP-2, suggesting that BMPs may play an important role in ossification of spinal ligaments. (C) 1997 by Elsevier Science Inc. All rights reserved.
  • 網塚 憲生, 小澤 英浩 新潟歯学会雑誌 27 (1) 61 -62 1997年07月 [査読無し][通常論文]
  • 網塚 憲生, 池亀 美華, 小澤 英浩 日本骨代謝学会雑誌 15 (2) 234 -234 1997年06月20日 [査読無し][通常論文]
  • 星 和人, 監物 新一, 竹内 靖博, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 亀田 剛, 真野 博, 山田 芳司, 網塚 憲生, 泉 直也, 小堀 正人, 宮澤 浩史, 森 芳久, 川島 博行, 小澤 英浩, 池田 恭治, 亀田 晃, 羽毛田 慈之, 久米川 正好 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 61 -61 1997年06月20日 [査読無し][通常論文]
  • 池亀 美華, 石橋 宰, 網塚 憲生, 小澤 英浩, 川島 博行 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 138 -138 1997年06月20日 [査読無し][通常論文]
  • 伊藤 将広, 網塚 憲生, 中島 民雄, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 西野 幾子, 網塚 憲生, 山田 まりえ, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 泉 直也, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 渡邊 淳一, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 近藤 尚知, 春日井 昇平, 網塚 憲生, 向山 仁, 尾澤 昌悟, 菊川 郁雄, 黒田 真司, 大谷 啓一, 小澤 英浩, 大山 喬史 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 15 (1) 1 -7 1997年06月 [査読無し][通常論文]
  • R Kremer, D Goltzman, N Amizuka, MM Webber, JS Rhim CLINICAL CANCER RESEARCH 3 (6) 855 -859 1997年06月 [査読無し][通常論文]
     
    Immortalized adult and fetal prostate cell lines grown in serum-free conditions produce low levels of parathyroid hormone-related peptide (PTHRP) in the presence of growth factors as assessed by mRNA analysis, PTHRP immunoreactivity, and immunohistochemistry. Subsequent infection of these cells with Kirsten murine sarcoma virus containing an activated Ki-ms oncogene induces at least a 10-20-fold increase in PTHRP expression and production of both adult and fetal immortalized cell lines in the presence of the same growth factors. These results provide the first evidence of direct activation of PTHRP by the ras oncogene in human prostate cells and suggest its potential usefulness as a tumor marker in prostate malignancies.
  • 網塚 憲生 日本骨代謝学会雑誌 15 (2) 234 -234 1997年06月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会雑誌 15 (2) 312 -312 1997年06月 [査読無し][通常論文]
  • Y Matsuki, M Nakashima, N Amizuka, H Warshawsky, D Goltzman, K Yamada, P Krebsbach, SK Lee, C Kozak, Y Yamada JOURNAL OF DENTAL RESEARCH 76 (5) 1179 -1179 1997年05月 [査読無し][通常論文]
  • K Hoshi, N Amizuka, K Oda, Y Ikehara, H Ozawa HISTOCHEMISTRY AND CELL BIOLOGY 107 (3) 183 -191 1997年03月 [査読無し][通常論文]
     
    Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-mu m tissue slices: and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes.
  • ハイドロキシアパタイト・コーテッドMicro-Ventインプラントとチタン素材Swede-Ventインプラントの比較
    クインテッセンスDental Implantology 4 (1) 111 -113 1997年 [査読無し][通常論文]
  • 歯周疾患モデルにおける抜歯後即時埋入インプラントとポリラクテックメンブレンの併用による垂直的骨高径の変化-イヌにおける動物実験モデル(共著)
    クインテッセンス・Dental Implantology 4 (5) 82 -83 1997年 [査読無し][通常論文]
  • インプラント形成における熱発生.in vitroにおける研究
    クインテッセンスDental Implantology 4 (4) 83 -86 1997年 [査読無し][通常論文]
  • インプラント周囲の深い欠損における微生物に関する知見.(共著)
    クインテッセンスDental Implantology 4 (3) 83 -85 1997年 [査読無し][通常論文]
  • 突然変異型組織非特異的アルカリホスファターゼの生合成と分解(共著)
    生化学会雑誌 69 849 1997年 [査読無し][通常論文]
  • 生物学的石灰化における骨芽細胞分化マーカーの免疫細胞化学的局在(共著)
    新潟日整会誌 71 (8) S1516 1997年 [査読無し][通常論文]
  • 組織非特異型アルカリホスファターゼの免疫局在(共著)
    日本解剖学会雑誌 152 1997年 [査読無し][通常論文]
  • 破骨細胞に対するbisphosphonateの作用機序(共著)
    日本解剖学会雑誌 76 1997年 [査読無し][通常論文]
  • 破歯細胞と破骨細胞のbisphosphonateおよびcalcitoninに対する形態変化の比較について(共著)
    日本解剖学会雑誌 76 1997年 [査読無し][通常論文]
  • PI3-kinaseの阻害剤であるwortmannin投与後のラット破骨細胞の形態変化(共著)
    日本解剖学会雑誌 73 1997年 [査読無し][通常論文]
  • ラット頸骨骨髄内bFGF投与による異所性骨の形態学的検索
    日本解剖学会雑誌 73 1997年 [査読無し][通常論文]
  • 矯正力に対する老齢ラットの歯槽骨改造現象(共著)
    歯科基礎医学会雑誌 39 111 1997年 [査読無し][通常論文]
  • 網塚憲生, 小沢英浩 Molecular Medicine 34 (Dec) 422 -423 1997年 [査読無し][通常論文]
  • 網塚憲生, 小沢英浩 日本骨代謝学会雑誌 15 (2) 312 1997年 [査読無し][通常論文]
  • 近藤 尚知, 春日井 昇平, 網塚 憲生, 向山 仁, 尾澤 昌悟, 菊川 郁雄, 黒田 真司, 大谷 啓一, 小澤 英浩, 大山 喬史 日本骨代謝学会雑誌 15 (2) 311 -311 1997年 [査読無し][通常論文]
  • 渡邊 淳一, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 15 (2) 310 -310 1997年 [査読無し][通常論文]
  • 泉 直也, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 15 (2) 309 -309 1997年 [査読無し][通常論文]
  • 西野 幾子, 網塚 憲生, 山田 まりえ, 小澤 英浩 日本骨代謝学会雑誌 15 (2) 230 -230 1997年 [査読無し][通常論文]
  • 伊藤 将広, 網塚 憲生, 中島 民雄, 小澤 英浩 日本骨代謝学会雑誌 15 (2) 166 -166 1997年 [査読無し][通常論文]
  • 池亀美華, 石橋宰, 網塚憲生, 小沢英浩, 川島博行 日本骨代謝学会雑誌 15 (2) 138 1997年 [査読無し][通常論文]
  • 亀田剛, 真野博, 山田芳司, 網塚憲生, 泉直也, 小堀正人, 宮沢浩史, 森芳久, 久米川正好 日本骨代謝学会雑誌 15 (2) 61 1997年 [査読無し][通常論文]
  • 星 和人, 監物 新一, 竹内 靖博, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 15 (2) 39 -39 1997年 [査読無し][通常論文]
  • N Amizuka, N Takahashi, N Udagawa, T Suda, H Ozawa ACTA HISTOCHEMICA ET CYTOCHEMICA 30 (4) 351 -362 1997年 [査読無し][通常論文]
     
    We have previously established a mouse coculture system of osteoblastic cells and spleen cells for examining osteoclast differentiation. In the present study, we examined the morphological features of the cell-cell contact between mouse spleen cells and osteoblastic cells in the co-cultures. Light microscopic investigations revealed that tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated spleen cells appeared in the vicinity of alkaline phosphatase (ALP)-positive osteoblastic cells. Ultrastructurally, spleen cells extended long cytoplasmic filopodia in all directions, by which spleen cells touched adjacent osteoblastic cells. The adjacent osteoblastic cells and spleen cells adhered to each other by forming electron-dense cytoplasmic materials on their inner leaflets of plasma membranes at cell-cell contact sites. Some adjacent osteoblastic and spleen cells made contact on the plasma membranes, forming an extracellular microenvironment. Coated pits were also formed on the plasma membranes facing this microenvironment. These morphological features of the cell-cell contact between osteoblastic cells and spleen cells indicate that there is internalization of organic components, i.e., receptor-mediated endocytosis in the contact sites between the two types of cells.
  • Mizue Tanaka, Norio Amizuka, Kang Jung Kim, Tatsuo Itoh, Hidehiro Ozawa Journal of Bone and Mineral Metabolism 15 (2) 67 -76 1997年 [査読無し][通常論文]
     
    We examined the rapid formation and subsequent resorption of woven bone induced by partial ablation of rat bone marrow. On the 1st day after ablation, masses of clots occupied the region from which marrow was eliminated. On the 3rd day, alkaline phosphatase- (ALPase-) positive osteoblastic cells appeared in the vicinity of the marrow-eliminated region, forming woven bone. Other ectopic woven bone extended from the endosteal surface toward the bone marrow. Therefore, the newly formed bone originated in two different sites, the endosteal bone surface and the marrow tissues near the marrow-eliminated region. On the 7th day, numerous tartrate-resistant acid phosphatase- (TRAPase-) positive osteoclasts and ALPase-positive osteoblasts expressing the osteonectin gene indicated high activity in both formation and resorption of ectopic woven bone. On the 10th day, the ectopic bone had been markedly resorbed and replaced by bone marrow tissue as the ectopically formed woven bone had not been dynamically maintained, probably because of reduced bone formation activity. Immunoreactivity for basic fibroblast growth factor (bFGF) was indistinctly observed on osteoblastic and preosteoblastic cells on the 1st day after ablation. The fibroblastic cells in the marrow-eliminated region on the 3rd day, and both osteoblasts and preosteoblasts in the woven bone on the 7th day, showed strong immunoreactivity for bFGF. Unlike fractured cortical bone, no chondrogenesis was observed. This model appears to provide convenient material and an important clue for investigation of imbalanced bone formation and subsequent resorption.
  • N Amizuka, HS Lee, MY Kwan, A Arazani, H Warshawsky, GN Hendy, H Ozawa, JH White, D Goltzman ENDOCRINOLOGY 138 (1) 469 -481 1997年01月 [査読無し][通常論文]
     
    The kidney is the major site of expression of the PTH/PTH-related peptide receptor (PTHR) gene. Previously we have shown that the PTHR gene is expressed from two promoters in kidney, an upstream kidney-specific promoter (P1) and a downstream promoter (P2) that is active in a wide variety of tissues. Here, we have used immunohistochemical and transcript-specific in situ hybridization techniques to map the expression of the PTHR gene and protein and to determine the distribution of P1- and PB-driven messenger RNAs in renal tissue. Immunohistochemical and immunoelectron microscopic analysis showed that PTHR protein is expressed on both basolateral and luminal membranes of proximal tubular epithelial cells, strongly suggesting a bipolar mode of action of PTH. Receptor protein also was detected on the surface of glomerular podocytes. Strikingly, immunoelectron microscopic analysis showed that endothelial cells of the peritubular vasculature, but not the glomerular vasculature, contain high levels of PTHR protein. We found that both P1 and P2 are expressed at moderate levels in both cortical and medullary epithelial cells of nephrons, correlating well with the immunohistochemical localization of PTHR protein. However, although abundant transcripts were detected in peritubular endothelial cells with P1-specific and coding sequence probes, P2-specific expression was not observed in these cells. These results provide evidence that the physiological effects of PTH- and/or PTH-related peptide on renal tubular function may be mediated not only through direct effects on epithelial cells but also indirectly through endothelial cell-based signaling. In addition to expression in vascular endothelial cells, high levels of P1-specific, but not P2-specific, PTHR messenger RNA were detected in vascular smooth muscle. Taken together, these experiments provide evidence for strong PTHR gene expression in renal vascular tissues. Moreover, given that previous studies have shown that P2, but not P1, is active in other tissues with an abundant vasculature, our results suggest that regulation of PTHR gene expression in renal vascular tissue is distinct from that of other organs.
  • K Hoshi, N Amizuka, T Kurokawa, H Ozawa ACTA HISTOCHEMICA ET CYTOCHEMICA 30 (4) 371 -379 1997年 [査読無し][通常論文]
     
    In order to elucidate the causes of ossification in spinal ligaments, ultrastructural alteration and immunolocalization of transforming growth factor-beta (TGF-beta), as well as that of the TGF-beta receptor were examined during the chondrification of ligamentous fibroblasts, by inducing ossification in ligamenta flava of murine lumbar spines, employing recombinant human bone morphogenetic protein-2 (rhBMP-2). Normal ligamenta flava, consisting of flattened fibroblasts, showed no immunolocalization of TGF-B isoforms. From the second week following BMP-administration, cartilage gradually replaced the ligaments. Cells in the central portion of the ligament contained abundant Golgi apparatus as well as enlarged endoplasmic reticula. Proliferative or hypertrophic chondrocytes in this area showed immunoreactivity to TGF-beta(3) latency-associated peptide, while calcified hypertrophic chondrocytes accompanied by both matrix vesicle-and collagen-calcification were noticed at the insertion site to spinal lamina, where there was no immunolocalization of this peptide. In addition, the TGF-beta type I receptor was localized on the proliferative and hypertrophic cells of the central portion. Therefore, as regards the pathological chondrification of ligamentous fibroblasts induced by exogenous BMP-2, TGF-beta is speculated to promote matrix-formation and chondrocytic maturation, under the autocrine/paracrine system, in concert with exogenous BMF-2.
  • Norio Amizuka, Naoyuki Takahashi, Nobuyuki Udagawa, Tatsuo Suda, Hidehiro Ozawa Acta Histochemica et Cytochemica 30 (4) 351 -362 1997年 [査読無し][通常論文]
     
    We have previously established a mouse co-culture system of osteoblastic cells and spleen cells for examining osteoclast differentiation. In the present study, we examined the morphological features of the cell-cell contact between mouse spleen cells and osteoblastic cells in the co-cultures. Light microscopic investigations revealed that tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated spleen cells appeared in the vicinity of alkaline phosphatase (ALP)-positive osteoblastic cells. Ultrastructurally, spleen cells extended long cytoplasmic filopodia in all directions, by which spleen cells touched adjacent osteoblastic cells. The adjacent osteoblastic cells and spleen cells adhered to each other by forming electron-dense cytoplasmic materials on their inner leaflets of plasma membranes at cell-cell contact sites. Some adjacent osteoblastic and spleen cells made contact on the plasma membranes, forming an extracellular microenvironment. Coated pits were also formed on the plasma membranes facing this microenvironment. These morphological features of the cell-cell contact between osteoblastic cells and spleen cells indicate that there is internalization of organic components, i.e., receptor-mediated endocytosis in the contact sites between the two types of cells.
  • 網塚 憲生, 伊藤 将広, 入江 一元 現代医療 29 (1) 171 -180 1997年01月 [査読無し][通常論文]
  • 副甲状腺ホルモンセプターの骨,軟骨,腎臓における局在について
    網塚 憲生, 入江 一元, 小沢 英浩 THE BONE 10 (4) 3 -6 1996年12月 [査読無し][通常論文]
  • N Amizuka, JE Henderson, K Hoshi, H Warshawsky, H Ozawa, D Goltzman, AC Karaplis ENDOCRINOLOGY 137 (11) 5055 -5067 1996年11月 [査読無し][通常論文]
     
    In previous work we showed that the chondrodysplastic phenotype of mice homozygous for a null mutation of the PTH-related peptide (PTHrP) gene was due in part to reduced proliferation and aberrant differentiation of growth plate chondrocytes. In the present study we have extended those observations by examining chondrocytes for evidence of PTH/PTHrP receptor expression, proliferation, and programmed cell death. Receptor messenger RNA and protein were expressed in chondrocytes in the resting and proliferative zones of both wild-type and mutant mice. In normal animals, expression was abundant in the area of transition between proliferative and hypertrophic chondrocytes and absent from cells in the lower hypertrophic region. On the other hand, the hypertrophic zone in mutant mice contained nonhypertrophic chondrocytes, which exhibited characteristics of proliferating cells, including PTH/PTHrP receptor expression, [H-3]thymidine incorporation, and expression of proliferating cell nuclear antigen. In contrast to the situation in normal animals, some cells adjacent to the zone of vascular invasion in mutant growth plates showed biochemical and morphological evidence of programmed cell death. In addition to these alterations in the maturation of growth plate chondrocytes, homozygous mutants demonstrated signs of aberrant differentiation of periosteal precursor cells. In some specimens, clusters of chondrocytes embedded in a cartilaginous matrix were observed between the layers of periosteal osteoblasts and the bony collar in the sterna and tibiae of mice homozygous for a null mutation of the PTHrP gene. Taken together, these observations indicate that PTHrP plays a pivotal role in the orderly progression of chondrocytes through stages of proliferation, differentiation, and programmed cell death in the epiphyseal growth plate and may also facilitate the commitment of precursors to cells of the chondrocytic or osteoblastic lineages.
  • SA Kawas, N Amizuka, JJM Bergeron, H Warshawsky CALCIFIED TISSUE INTERNATIONAL 59 (3) 192 -199 1996年09月 [査読無し][通常論文]
     
    In order to examine our hypothesis that maturation ameloblasts could degrade the enamel matrix in a manner analogous to bone resorption mediated by osteoclasts, we have assessed the distribution of lysosomal enzymes in the enamel organ by immunolocalizing the cation-in-independent mannose 6-phosphate receptor (MPR) and the lysosomal enzyme cathepsin B at all stages of amelogenesis. Secretory ameloblasts showed strong immunoreactivity for MPR in the supranuclear Golgi region and in the cytoplasm between the Golgi region and the distal junctional complexes. However, cathepsin B immunoreactivity was mainly seen in the distal portion of Tomes' process, which was unreactive for MPR immunogenicity. In maturation ameloblasts, the MPR was observed on the ruffled border of the ruffle-ended ameloblast (SA) but not on the distal cell membrane of the smooth-ended ameloblast (SA), although both cell types demonstrated strong immunoreactivity for MPR in the Golgi region. Immunoreactive cathepsin B was seen at the distal ends of both RA and SA. It is postulated that the nascent lysosomal enzymes bind to the mannose 6-phosphate receptors which target them not only to intracellular lysosomes, but also to the ruffled border of maturation ameloblasts where these enzymes are secreted into the enamel. Since MPR and lysosomal enzymes were also detected on the ruffled border of osteoclasts (Ocl) adjacent to alveolar bone, our immunocytochemical approach provides strong evidence for a similarity between the maturation process in enamel, as mediated by the ruffle-ended maturation ameloblasts, and bone resorption mediated by osteoclasts. This study has established that a common mechanism, based on MPR-targeted lysosomal secretion and matrix degradation, is basic to the maturation process involved in calcified tissues as different as bone and enamel.
  • 骨と腎臓における副甲状腺ホルモンレセプターの局在とその上流と下流のプロモーターの機能に関する研究
    網塚 憲生 歯科基礎医学会雑誌 38 (抄録) 455 -455 1996年08月 [査読無し][通常論文]
  • B Sidler, L Alpert, JE Henderson, R Deckelbaum, N Amizuka, JE Silva, D Goltzman, AC Karaplis JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM 81 (8) 2841 -2847 1996年08月 [査読無し][通常論文]
     
    PTH-related peptide (PTHrP) is the major factor responsible for humoral hypercalcemia of malignancy. This paraneoplastic syndrome has been described in association with a number of malignancies, but rarely with carcinoma of the colon. Moreover, little is known about the molecular mechanisms that underlie PTHrP overexpression in tumors. Here we report a patient who presented with hypercalcemia 6 months after resection of a neuroendocrine colonic carcinoma (tumor I). At the time of admission, intact PTH was decreased, circulating PTHrP levels were elevated, and there was tumor recurrence (tumor II). Immunohistochemical staining of paraffin-embedded sections from tumor I did not stain for PTHrP, whereas cells from tumor II stained intensely positive. Southern blot analysis and differential PCR of genomic DNAs from tumor specimens and the patient's leukocytes demonstrated amplification of the PTHrP gene in tumor II. Moreover, staining for p53 protein was evident in tumor II, but not in tumor I, consistent with the presence of a mutant form of p53 and associated loss of tumor suppressor function in the recurrent tumor. PTHrP gene amplification was also detected in one of five other tumors associated with humoral hypercalcemia of malignancy. These findings suggest that a potential mechanism contributing to PTHrP overexpression in malignancies is gene amplification, which could arise from increased genomic instability associated with the progressive stages of neoplasia.
  • N Amizuka, HS Lee, GN Hendy, D Goltzman, JH White JOURNAL OF BONE AND MINERAL RESEARCH 11 M484 -M484 1996年08月 [査読無し][通常論文]
  • M Itoh, N Amizuka, T Nakajima, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 11 S401 -S401 1996年08月 [査読無し][通常論文]
  • K Hoshi, N Amizuka, T Sako, T Kurokawa, H Ozawa JOURNAL OF BONE AND MINERAL RESEARCH 11 51 -51 1996年08月 [査読無し][通常論文]
  • Z Pausova, E Soliman, N Amizuka, N Janicic, EM Konrad, A Arnold, D Goltzman, GN Hendy JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM 81 (7) 2711 -2718 1996年07月 [査読無し][通常論文]
     
    Parathyroid tumors occur either sporadically or as part of inherited syndromes such as multiple endocrine neoplasia (MEN) types 2A and 2B. The development of both of these familial syndromes has been related to specific germline gain-of-function mutations predominantly in exons 10 and 11 (MEN 2A) and 16 (MEN 2B) of the RET proto-oncogene. The same mutations have also been implicated in the pathogenesis of sporadic medullary thyroid carcinoma and sporadic pheochromocytoma. The RET mutations are thought to have a transforming effect only in cells of neural crest origin such as thyroid parafollicular (C-cells) and adrenal chromaffin cells, which normally express the RET proto-oncogene. Expression of RET messenger RNA has not yet been studied in the parathyroid, however, we demonstrate in this study by a sensitive, semiquantitative RT-PCR technique and in situ hybridization, that RET is expressed in MEN 2A parathyroid tumors and in sporadic adenomas. Although DNA from a parathyroid tumor of a MEN 2A patient displayed an expected mutation, none of the previously described MEN 2A or 2B mutations were found in DNA of 34 sporadic adenomas. Our data suggest that parathyroid disease is an integral part of the MEN 2A syndrome, but that MEN 2 mutations in RET rarely play a part in the pathogenesis of sporadic parathyroid tumors.
  • 網塚 憲生, 小澤 英浩 実験医学 14 (10) 1492 -1498 1996年07月 [査読無し][通常論文]
  • 網塚 憲生 日本骨代謝学会雑誌 14 (2) 115 -115 1996年06月 [査読無し][通常論文]
  • 星 和人, 網塚 憲生, 酒匂 崇, 黒川 高秀, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 14 (2) 139 -139 1996年06月01日 [査読無し][通常論文]
  • 伊藤 将広, 網塚 憲生, 中島 民雄, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 14 (2) 304 -304 1996年06月01日 [査読無し][通常論文]
  • 泉 直也, 網塚 憲生, 小澤 英浩 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 14 (2) 305 -305 1996年06月01日 [査読無し][通常論文]
  • N Amizuka, AC Karaplis, JE Henderson, H Warshawsky, ML Lipman, Y Matsuki, S Ejiri, M Tanaka, N Izumi, H Ozawa, D Goltzman DEVELOPMENTAL BIOLOGY 175 (1) 166 -176 1996年04月 [査読無し][通常論文]
     
    Although apparently phenotypically normal at birth, mice heterozygous for inactivation of the gene encoding parathyroid hormone-related peptide (PTHrP) develop haplotype insufficiency by 3 months of age. In addition to histologic and morphologic abnormalities similar to those seen in homozygous mutants, heterozygous animals demonstrated alterations in trabecular bone and bone marrow These included metaphyseal bone spicules which were diminished in volume, irregularly distributed, and less well developed than those seen in age-matched controls as well as bone marrow, which contained an inordinate number of adipocytes. A substantial reduction in PTHrP mRNA was detected in heterozygous tissue, while circulating parathyroid hormone (PTH) and calcium concentrations were normal. Thus, while a physiologic concentration of PTH was capable of maintaining calcium homeostasis, it was incapable of compensating for PTHrP haploinsufficiency in developing bone. In normal animals, both PTHrP and the PTH/PTHrP receptor were expressed predominantly in chondrocytes situated throughout the proliferative zone of the tibial growth plate. In the metaphysis, the PTH/PTHrP receptor was identified on osteoblasts and preosteoblastic cells situated in the bone marrow, while PTHrP was expressed only by osteoblasts. These observations indicate that postnatal bone development involves susceptible pathways that display exquisite sensitivity to critical levels of PTHrP and imply that the skeletal effects of PTH are influenced by locally produced PTHrP. Moreover, identification of both the ligand and its N-terminal receptor in metaphyseal osteoblasts and their progenitors suggests an autocrine/paracrine role for the protein in osteoblast differentiation and/or function. Impairment in this function as a consequence of PTHrP haploinsufficiency may critically influence the course of bone formation, resulting in altered trabecular architecture and perhaps low bone mass and increased bone fragility. (C) 1996 Academic Press, Inc.
  • 網塚 憲生 日本内分泌学会雑誌 72 (2) 176 -176 1996年04月 [査読無し][通常論文]
  • 網塚 憲生, 小澤 英浩 日本内分泌学会雑誌 72 (2) 176 -176 1996年03月20日 [査読無し][通常論文]
  • チタン表面に形成された実験的唾液ペリクルはレンサ球菌の接着を仲介する(共著)
    クインテッセンスDental Implantology 3 (6) 105 -106 1996年 [査読無し][通常論文]
  • ウシ胎仔下顎骨由来の骨芽細胞による石灰化初代培養を用いたチタン合金と骨芽細胞および基質との境界面に関する微細形態学的研究(共著)
    クインテッセンスDental Implantology 3 (3) 107 -118 1996年 [査読無し][通常論文]
  • ラット脛骨モデルを用いたチタニンプラントにおける骨形成の形態学的、ラジトオートグラフィー的研究
    クインテッセンスDental Implantology 3 (1) 115 -127 1996年 [査読無し][通常論文]
  • rh BMP-2による脊椎靭帯骨化症モデルの形態学的観察(共著)
    日本パラプレジア医学会誌 9 (1) 210 -211 1996年 [査読無し][通常論文]
  • 骨、腎臓における副甲状腺ホルモンレセプターの局在
    解剖誌 71 (4) 332 1996年 [査読無し][通常論文]
  • 破骨細胞のゴルジ体からの膜輸送課程にはPI3-kinaseが関与する(共著)
    日骨代謝誌 14 (2) 305 1996年 [査読無し][通常論文]
  • 破骨細胞は細胞死の後、マクロファージに貧食されるか血管内に入り込んで他の器官で処理される(共著)
    日骨代謝誌 14 (2) 304 1996年 [査読無し][通常論文]
  • Bisphosphonate投与における破歯細胞の細胞死について(共著)
    歯基礎誌 38(補冊) 102 1996年 [査読無し][通常論文]
  • 軟骨と骨組織におけるアポトーシス、特集:アポトーシスと疾患
    現代医療 29 (1) 181 -186 1996年 [査読無し][通常論文]
  • 「骨代謝研究の最前線」骨、軟骨組織における副甲状腺ホルモン関連ペプチドの生物学的役割
    日本内分泌学会雑誌 72 176 1996年 [査読無し][通常論文]
  • 副甲状腺ホルモン関連ペプチド(PHTRP)と骨、軟骨の成長-PTHRP遺伝子欠損ヘテロマウスに認められた骨、軟骨の異常について-
    10 (2) 1996年 [査読無し][通常論文]
  • 網塚憲生、小澤英浩:実験動物の骨モデル9 骨端軟骨異常と副甲状腺ホルモン関連ペプチド
    骨と関節 9 (5) 1996年 [査読無し][通常論文]
  • 軟骨と骨組織におけるアポトーシス現代医療
    特集アポトーシスと疾患 29 (1) 18 1996年 [査読無し][通常論文]
  • 副甲状腺ホルモン関連ペプチド遺伝子ノックアウトマウスとレセプターの新しい研究展開
    実験医学増刊号「骨形成・骨吸収のシグナリング」 14 (10) 190 1996年 [査読無し][通常論文]
  • PTHrPの生理学的役割
    内分泌,糖尿病 科学評論社 1 139 1996年 [査読無し][通常論文]
  • ウシ胎仔下顎骨由来の骨芽細胞による石灰化初代培養を用いたチタン合金と骨芽細胞および基質との境界面に関する微細形態学的研究(共著)
    クインテッセンス社 3 115 1996年 [査読無し][通常論文]
  • Ossification of spinal ligaments induced by rhBMP-2. The international conference on hormone and cytokine in bone. (共著)
    J. Bone Miner. Metab. 14 (2) 108 1996年 [査読無し][通常論文]
  • Norio Amizuka, David Goltzman, Hidehiro Ozawa Tissue Engineering 2 (4) 277 -287 1996年 [査読無し][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) was originally identified as a factor inducing malignancy-associated hypercalcemia by activating a common receptor (PTH/PTHrP receptor) with PTH. Recently, PTHrP gene 'knock- out' mice showed a form of dyschondroplasia due to reduced proliferation of chondrocytes. In addition, heterogenous populations of variously differentiated chondrocytes were seen in the hypertrophic zone of the mutant epiphyseal plate. Although the homozygotes die within several hours after birth, the adult mice heterozygous for PTHRP gene deletion display a delayed skeletal abnormality that is apparent at 3 months of age, which expresses a reduced amount of PTHrP transcript. Therefore PTHrP appears to modulate cell proliferation and differentiation at both fetal and adult stages. The colocalization of PTHrP and its receptor in osteoblastic cells and chondrocytes suggested a paracrine/autocrine mode of action. More recently, region 87-107 of the amino acid sequence of PTHrP was reported to contain a putative nucleolar localization sequence. Although the presence of the leader sequence of PTHrP preferentially targets this protein to the secretory pathway, COS-7 cells and chondrocytic CFK-2 cells, which were transfected with plasmids expressing PTHrP with or without the leader sequence, both showed nucleolar localization of both forms of PTHrP. Thus PTHrP appears to regulate normal proliferation and differentiation of bone and cartilage cells by activating the PTH/PTHrP receptor, and possibly by directly acting on the nucleus of target cells.
  • N Amizuka, AC Karaplis, JE Henderson, H Warshawsky, D Goltzman, H Ozawa JOURNAL OF DENTAL RESEARCH 75 2034 -2034 1996年 [査読無し][通常論文]
  • Immunolocalization of the bone morphogenetic protein receptor and transforming growth factor-β during ectopic osteo-chondrogenesis induced by exogenous bone morphogenetic protein-2. (共著)
    Histochemistry and Cytochemistry, Proceedings of the Xth International Congress 461 1996年 [査読無し][通常論文]
  • F HaraIrie, N Amizuka, H Ozawa BONE 18 (1) 29 -39 1996年01月 [査読無し][通常論文]
     
    We performed immunohistochemical and ultrastructural studies to disclose a possible relationship between nerve fibers and bone metabolism. Immunohistochemical distribution of calcitonin gene-related peptide (CGRP)-positive nerve fibers during bone development was assessed in the femurs of rats, CGRP-positive nerve fibers were denser in the epiphysis than in the metaphysis. These nerve fibers particularly ran along the epiphyseal trabecules facing the growth plate and came in contact with osteoclasts. Many osteoclasts at the epiphyseal trabecules facing the growth plate contained abundant toluidine blue and periodic acid-Scfiiff-positive granules. Electron microscopy revealed that these osteoclasts have many membrane-bound, electron-dense granular structures and dilated cisterns of rough endoplasmic reticulum containing electron-dense material. They were often surrounded by clear cells displaying features of nerve fiber and had no ruffled border. Furthermore, ultrastructural observations revealed electron-dense structures coating the cytoplasmic side of plasma membranes of the nerve fibers. We also observed coated pits in the cytoplasm of the osteoclasts: facing the nerve fibers. To further clarify the role of innervation, we compared trabecules of rats undergoing denervation of the sciatic nerve with those from unoperated rats. Denervation resulted in a significant increase in the number of cement lines on the epiphyseal trabecules facing the growth plate. These results suggest that the osteoclasts at the epiphyseal trabecules facing the growth plate are in part regulated by CGRP-positive nerve fibers. Thus, CGRP-positive nerve fibers could be a crucial element in bone metabolism during bone growth and development.
  • Norio Amizuka, David Goltzman, Hidehiro Ozawa Tissue Engineering 2 (4) 277 -287 1996年 [査読無し][通常論文]
     
    Parathyroid hormone-related peptide (PTHrP) was originally identified as a factor inducing malignancy-associated hypercalcemia by activating a common receptor (PTH/PTHrP receptor) with PTH. Recently, PTHrP gene 'knock- out' mice showed a form of dyschondroplasia due to reduced proliferation of chondrocytes. In addition, heterogenous populations of variously differentiated chondrocytes were seen in the hypertrophic zone of the mutant epiphyseal plate. Although the homozygotes die within several hours after birth, the adult mice heterozygous for PTHRP gene deletion display a delayed skeletal abnormality that is apparent at 3 months of age, which expresses a reduced amount of PTHrP transcript. Therefore PTHrP appears to modulate cell proliferation and differentiation at both fetal and adult stages. The colocalization of PTHrP and its receptor in osteoblastic cells and chondrocytes suggested a paracrine/autocrine mode of action. More recently, region 87-107 of the amino acid sequence of PTHrP was reported to contain a putative nucleolar localization sequence. Although the presence of the leader sequence of PTHrP preferentially targets this protein to the secretory pathway, COS-7 cells and chondrocytic CFK-2 cells, which were transfected with plasmids expressing PTHrP with or without the leader sequence, both showed nucleolar localization of both forms of PTHrP. Thus PTHrP appears to regulate normal proliferation and differentiation of bone and cartilage cells by activating the PTH/PTHrP receptor, and possibly by directly acting on the nucleus of target cells.
  • PTHrP, a mediator of cell proliferation and differentiation of both chondrocytes and osteoblasts : The biological action of PTHrP on skeletal cells.
    International Bone Forum 4 18 -21 1996年 [査読無し][通常論文]
  • Phosphatidylinositol 3-kinase is involved in membrane transport between Golgi apparatus and ruffled border in osteoclast. (共著)
    J. Bone Miner. Res. 11 S184 1996年 [査読無し][通常論文]
  • N Amizuka, AC Karaplis, JE Henderson, H Warshawsky, D Goltzman, H Ozawa JOURNAL OF DENTAL RESEARCH 75 2034 -2034 1996年 [査読無し][通常論文]
  • F. Hara-Irie, N. Amizuka, H. Ozawa Bone 18 (1) 29 -39 1996年 [査読無し][通常論文]
     
    We performed immunohistochemical and ultrastructural studies to disclose a possible relationship between nerve fibers and bone metabolism. Immunohistochemical distribution of calcitonin gene-related peptide (CGRP)-positive nerve fibers during bone development was assessed in the femurs of rats. CGRP-positive nerve fibers were denser in the epiphysis than in the metaphysis. These nerve fibers particularly ran along the epiphyseal trabecules facing the growth plate and came in contact with osteoclasts. Many osteoclasts at the epiphyseal trabecules facing the growth plate contained abundant toluidine blue and periodic acid-Schiff-positive granules. Electron microscopy revealed that these osteoclasts have many membrane-bound, electron dense granular structures and dilated cisterns of rough endoplasmic reticulum containing electron-dense material. They were often surrounded by clear cells displaying features of nerve fiber and had no ruffled border. Furthermore, ultrastructural observations revealed electron dense structures coating the cytoplasmic side of plasma membranes of the nerve fibers. We also observed coated pits in the cytoplasm of the osteoclasts facing the nerve fibers. To further clarify the role of innervation, we compared trabecules of rats undergoing denervation of the sciatic nerve with those from unoperated rats. Denervation resulted in a significant increase in the number of cement lines on the epiphyseal trabecules facing the growth plate. These results suggest that the osteoclasts at the epiphyseal trabecules facing the growth plate are in part regulated by CGRP-positive nerve fibers. Thus, CGRP-positive nerve fibers could be a crucial element in bone metabolism during bone growth and development.
  • 入江 史子, 網塚 憲生, 小澤 英浩 THE BONE 9 (4) 1 -4 1995年12月 [査読無し][通常論文]
  • 副甲状腺ホルモン関連ペプチド(PTHRP)遺伝子ノックアウトマウスにおける骨組織異常
    網塚 憲生, 小澤 英浩 THE BONE 9 (3) 巻頭 -巻頭 1995年09月 [査読無し][通常論文]
  • 網塚 憲生, 小澤 英浩 THE BONE 9 (3) 83 -92 1995年09月 [査読無し][通常論文]
  • 副甲状腺ホルモン関連ペプチド欠損マウスにおける軟骨細胞の形態学的検索
    網塚 憲生 歯科基礎医学会雑誌 37 (抄録) 136 -136 1995年08月 [査読無し][通常論文]
  • Z PAUSOVA, E SOLIMAN, N AMIZUKA, N JANICIC, EM KONRAD, A ARNOLD, D GOLTZMAN, GN HENDY JOURNAL OF BONE AND MINERAL RESEARCH 10 S191 -S191 1995年08月 [査読無し][通常論文]
  • N AMIZUKA, H LEE, WARSHAW, H WARSHAWSKY, H OZAWA, D GOLTZMAN, JH WHITE JOURNAL OF BONE AND MINERAL RESEARCH 10 S281 -S281 1995年08月 [査読無し][通常論文]
  • N AMIZUKA, JE HENDERSON, H WARSHAWSKY, H OZAWA, D GOLTZMAN, AC KARAPLIS JOURNAL OF BONE AND MINERAL RESEARCH 10 S285 -S285 1995年08月 [査読無し][通常論文]
  • N AMIZUKA, H LEE, WARSHAW, H WARSHAWSKY, H OZAWA, D GOLTZMAN, JH WHITE JOURNAL OF BONE AND MINERAL RESEARCH 10 S281 -S281 1995年08月 [査読無し][通常論文]
  • PTHrPの生理学的役割
    網塚 憲生, 小澤 英浩 内分泌・糖尿病科 1 (2) 139 -145 1995年08月 [査読無し][通常論文]
  • 網塚 憲生, Goltzman David, 小澤 英浩 日本骨代謝学会雑誌 13 (2) 28 -28 1995年07月 [査読無し][通常論文]
  • A UTANI, JB KOPP, CA KOZAK, Y MATSUKI, N AMIZUKA, S SUGIYAMA, Y YAMADA LABORATORY INVESTIGATION 72 (3) 300 -310 1995年03月 [査読無し][通常論文]
     
    BACKGROUND: Kalinin, an anchoring filament-associated protein, is a member of the laminin family which is believed to play a role in the attachment of particular epithelial cells to the basement membrane. EXPERIMENTAL DESIGN: A newborn mouse lung cDNA library was screened under low stringency conditions using as a probe a murine laminin pi chain cDNA, an approach designed to identify novel laminin pi chain homologs. RESULTS: We have identified a novel cDNA sequence comprising 4049 base pahs, with an open reading frame that potentially codes for 1168 amino acids, including a presumptive signal peptide. The gene was mapped to mouse chromosome 1. The amino acid sequence is homologous to laminin beta 1 and beta 2 chains, but is distinguished from these molecules by having a truncated short arm region. The sequence has considerable similarity to the human kalinin B1 chain (laminin beta 3), suggesting that it is murine beta 3 chain. Northern blot analysis demonstrated a single 4.0-kilobase species in the mouse newborn skin, lung, and Pam 212 cells. Immunolocalization studies using the 14-day embryo and adult mouse tissues demonstrated that the murine beta 3 chain is located at the dermal-epidermal junction of the skin and the basement membrane of the oral mucosa, oropharynx, and upper portions of the tracheobronchial tree. In situ hybridization studies of the mammary gland showed that the beta 3 mRNA chain was produced by the epithelial cells of the gland but not by the mesenchymal cells. Western blotting under nonreducing conditions using the antibody to beta 3 chain detected molecules of 400 and 440 kilodaltons (kDa) in the conditioned medium of human foreskin keratinocytes, A431 cells, and Pam 212 cells. Western blotting under reducing conditions detected only a single 140-kDa species, suggesting that beta 3 chain is present within disulfide-linked complexes. This 140-kDa species was contained in the kalinin complex immunoprecipitated by anti-kalinin beta 2 (laminin gamma 2) antibody from the conditioned medium of HT1080 cells, suggesting that both the beta 3 and gamma 2 chains are present in the multimeric complex. CONCLUSIONS: Murine laminin beta 3 chain shows considerable homology to the human homolog and has a tightly restricted tissue distribution, confined to the basement membrane of cells in contact with the external environment. In vitro studies indicate that the beta 3 chain participates in trimer formation with the gamma 2 chain and possibly with other laminin chains as well.
  • Y MATSUKI, N AMIZUKA, H WARSHAWSKY, D GOLTZMAN, Y YAMADA ARCHIVES OF ORAL BIOLOGY 40 (2) 161 -164 1995年02月 [査読無し][通常論文]
     
    Epimorphin has been recently identified as an important factor in the morphogenesis of epithelial cells. A cDNA encoding epimorphin from skin of newborn mice was cloned by the polymerase chain-reaction technique before the preparation of digoxigenin-labelled cRNA probes. In situ hybridization of longitudinal sections of rat incisors revealed a distinct pattern of expression of epimorphin mRNA in the ameloblast layer. Epimorphin mRNA was detected from the presecretory stage up to the beginning of the maturation stage of amelogenesis. With the identification of this expression by epithelial-derived cells, i.e. ameloblasts, it is thought likely that epimorphin is one of the factors that modulate the differentiation cascade of ameloblasts in the course of amelogenesis.
  • ヘルトビッヒ上皮鞘由来エナメル上皮細胞によるアメロゲニン産生について(共著)
    歯科基礎医学会誌 37 191 1995年 [査読無し][通常論文]
  • ラット頚骨モデルを用いたチタンプラントにおける骨形成の形態学的, ラジオオートグラフィー的研究
    クインテッセンス社 2 155 1995年 [査読無し][通常論文]
  • Paratyroid hormone-related peptide (PTHRP) modnlates rormal differentiation of osteoprogenitor ceiis : Morphological evidence of alnormality in mutantmice homozygous and heterozygous for PTHRP gene deficiency.
    Bone 16 206 1995年 [査読無し][通常論文]
  • Bin Liu, Norio Amizuka, David Goltzman, Shafaat A. Rabbani International Journal of Cancer 63 (2) 276 -281 1995年 [査読無し][通常論文]
     
    To attain full biological activity, the precursor pro‐parathyroid hormone‐related peptide (ProPTHRP) must be converted to the mature peptide PTHRP. We have examined the effect of inhibiting expression of the pro‐hormone convertase furin in H‐500 rat Leydig tumor cells on PTHRP production and action in vitro and in vivo. H‐500 Leydig tumor cells were stably transfected with a mammalian expression plasmid containing furin cDNA in an anti‐sense orientation. This resulted in inhibition of endogenous furin mRNA expression and of protein production as assessed by immunocytochemistry. These experimental cells secreted extended NH2‐terminal PTHRP forms with reduced adenylate cyclase‐stimulating activity. This was associated with a marked decrease in the proliferation of these tumor cells in vitro. Transfected and control cells were then implanted into male Fischer rats. Animals implanted with control cells became hypercalcemic. In contrast, animals implanted with experimental cells maintained near normal levels of plasma calcium. Experimental cells inoculated in vivo developed into tumors of significantly decreased volume compared to control cells and animal survival time was prolonged. Our results indicate that alteration of the processing of PTHRP can diminish the hypercalcemic endocrine actions of PTHRP and can reduce autocrine/paracrine effects of PTHRP on tumor cell growth both in vitro and in vivo. Furin may also exert a broader role in processing other factors required for tumor proliferation. Consequently, anti‐sense modulation of furin activity may be a potential modality for understanding the mechanism of neoplastic growth and progression. Copyright © 1995 Wiley‐Liss, Inc., A Wiley Company
  • HENDERSON J. E. Mol. Cell Biol. 15 4064 -4064 1995年 [査読無し][通常論文]
  • Y MATSUKI, M NAKASHIMA, N AMIZUKA, H WARSHAWSKY, D GOLTZMAN, KM YAMADA, Y YAMADA JOURNAL OF DENTAL RESEARCH 74 (1) 307 -312 1995年01月 [査読無し][通常論文]
     
    The formation of tooth organs is regulated by a series of developmental programs. We have initiated a genome project with the ultimate goal of identifying novel genes important for tooth development. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3- to 4-week-old rats, sequenced cDNA clones, and classified their sequences by homology search through the GenBank data base and the PIR protein data base. Here, we report partial DNA sequences obtained by automated DNA sequencing on 400 cDNA clones randomly selected from the library. Of the sequences determined, 51% represented sequences of new genes that were not related to any previously reported gene. Twenty-six percent of the clones strongly matched genes and proteins in the data bases, including amelogenin, alpha 1(I) and alpha 2(I) collagen chains, osteonectin, and decorin. Nine percent of clones revealed partial sequence homology to known genes such as transcription factors and cell surface receptors. A significant number of the previously identified genes were expressed redundantly and were found to encode extracellular matrix proteins. Identification and cataloging of cDNA clones in these tissues are the first step toward identification of markers expressed in a tissue- or stage-specific manner, as well as the genetic linkage study of tooth anomalies. Further characterization of the clones described in this paper should lead to the discovery of novel genes important for tooth development.
  • 網塚 憲生 日本骨代謝学会雑誌 12 (3) 136 -147 1994年11月 [査読無し][通常論文]
  • N AMIZUKA, H WARSHAWSKY, JE HENDERSON, D GOLTZMAN, AC KARAPLIS JOURNAL OF CELL BIOLOGY 126 (6) 1611 -1623 1994年09月 [査読無し][通常論文]
     
    To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [H-3]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these non-hypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.
  • 小沢 英浩, 網塚 憲生 日本臨床 52 (9) 2246 -2254 1994年09月 [査読無し][通常論文]
     
    Bone modeling and remodeling as well as systemic calcium homeostasis are modulated predominantly by bone cells such as mesencymal-derived osteoblasts and osteocytes and haematopoietic-derived osteoclasts. Osteoblasts are classified into preosteoblasts, mature osteoblasts and bone lining cells (BLC) based on their cell structures and biological functions. Preosteoblasts appear to regulate the activities of mature osteoblasts and osteoclasts as well as to differentiate into mature osteoblasts. Cuboidal mature osteoblasts show well-developed Golgi apparatus and rough endoplasmic reticulum, and synthesize actively major bone matrix proteins including type I collagen, osteopontin, osteonectin and osteocalcin. Unlike mature osteoblasts, BLCs display flattened cell bodies and few cell organelle, therefore, indicating less activities. These osteoblasts communicate through gap junctions and adheres junctions with each other or with osteocytes, consequently forming three-dimensional cellular network in the bone tissues. In contrast, osteocytes, compacted cell type embedded in the bone matrix, provide a cellular environment in the bone matrix by means of their numerous cytoplasmic processes. Moreover, it is likely that they participate in calcium transport from bone matrix to tissue fluid, that is "osteocytic osteolysis". Osteoclasts, multinucleated giant cells with ruffled borders and clear zones, are responsible for bone resorption, although probably they are controlled by surrounding osteoblast phenotype. Active osteoclasts secrete H+ and proteolytic enzyme such as cathepsin and ACPase towards resorption pits through ruffled borders. These bone cells play an important role in bone modeling and remodeling.
  • S NIIDA, N AMIZUKA, F HARA, H OZAWA, H KODAMA JOURNAL OF BONE AND MINERAL RESEARCH 9 (6) 873 -881 1994年06月 [査読無し][通常論文]
     
    Osteoclast deficiency in op/op mice is cured by a single injection of 5 mu g recombinant human macrophage colony-stimulating factor (rhM-CSF). In this study, we found that mouse osteoclasts are positive for Mac-2 antigen, but not for F4/80, MOMA-2, Mac-1, or BM8 antigen. By using F4/80 and MOMA-2 monoclonal antibodies, we confirmed the absence of mature macrophages in the femora of op/op mice and found that multiple injections of rhM-CSF are required for the recruitment of macrophages in the bones. After a single rhM-CSF injection, we found Mac-2 positive mononuclear cells in the femora of op/op mice. The time course of the appearance of Mac-2-positive cells was very similar to that of tartrate-resistant acid phosphatase (TRAP)-positive cells. In bone sections prepared from the mutant mice that received rhM-CSF 3 days earlier, 91% of the TRAP-positive mononuclear cells were also positive for Mac-2 antigen. These results demonstrate the expression of Mac-2 antigen in preosteoclasts. The antigen was detected on the plasma membrane of preosteoclasts, as well as in their cytoplasm and nucleus, and in the extracellular matrix in the space between the cells and bone. Since Mac-2 is a galactose-specific lectin, a potential role of the lectin in cell-cell and cell-matrix adhesion during osteoclast differentiation is suggested.
  • 小沢英浩, 網塚憲生 日本臨床 52 (9) 1994年 [査読無し][通常論文]
  • 網塚憲生, 小沢英浩, WARSHAWSKY H, GOLTZMAN D 日本骨代謝誌 12 (2) 181 1994年 [査読無し][通常論文]
  • Y MATSUKI, N AMIZUKA, H WARSHAWSKY, D GOLTZMAN, M NAKASHIMA, KM YAMADA, Y YAMADA JOURNAL OF DENTAL RESEARCH 73 146 -146 1994年 [査読無し][通常論文]
  • Localization of gene expression of PTHRP and of the PTH/PTHRP receptor in normal adult bone tissues : consequences of bone formation in PTHRP-deficient animals. (共著)
    J. Bone Min. Res. 9 S128 1994年 [査読無し][通常論文]
  • Localization of gene expression of PTHRP and of the PTH/PTHRP receptor in normal adult bone tissues : Consequence for bone formation in PTHRP-deficient animals
    Journal of Bone & Mineral Research 9 S128 1994年 [査読無し][通常論文]
  • Identification of a functional nucleolar localization signal in parathyroid hormone-related peptide. (共著)
    Journal of Bone & Mineral Research 9 S121 1994年 [査読無し][通常論文]
  • 網塚憲生 日骨代謝誌 12 118 1994年
  • 石井 ヒロ子, 石井 史郎, 角田 俊彦, 野田 忠, 鈴木 誠, 網塚 憲生, 小澤 英浩 小児歯科学雑誌 32 (2) 350 -350 1994年 [査読無し][通常論文]
  • N AMIZUKA, H WARSHAWSKY, D GOLTZMAN, AC KARAPLIS JOURNAL OF BONE AND MINERAL RESEARCH 8 S148 -S148 1993年08月 [査読無し][通常論文]
  • N AMIZUKA, H WARSHAWSKY, D GOLTZMAN, AC KARAPLIS JOURNAL OF BONE AND MINERAL RESEARCH 8 S148 -S148 1993年08月 [査読無し][通常論文]
  • T SATO, E ABE, HJ CHENG, HH MEI, T KATAGIRI, T KINOSHITA, N AMIZUKA, H OZAWA, T SUDA ENDOCRINOLOGY 133 (1) 397 -404 1993年07月 [査読無し][通常論文]
     
    We previously reported that 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] tissue-specifically stimulated the production of the third component of complement (C3) in bone in vitro and in vivo. In the present study, we examined the possible roles of C3 in bone using bone marrow cultures with antibodies against C3 and C3 receptors (Mac 1, 8C12, and 7G6) and the purified mouse C3 protein. The C3 protein produced preferentially by stromal cells in response to 1alpha,25-(OH)2D3 was distributed in macrophage-like mononuclear cells and small cells with few nuclei. Adding anti-C3 antibody together with 1alpha,25-(OH)2D3 to bone marrow cultures greatly inhibited not only the appearance of tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells, but also the growth of macrophage-like mononuclear cells and stromal cells. The inhibitory effect of anti-C3 antibody on osteoclast-like cell formation was most prominent when it was added between days 2-4 of the 6-day culture period, which corresponded to the late proliferative phase and the early differentiation phase of osteoclast development. Adding anti-C3 receptor antibodies also inhibited osteoclast-like cell formation induced by 1alpha,25-(OH)2D3. When C3 receptors were detected by the binding of C3-coated sheep red blood cells or immunostaining, the localization of C3 receptor-positive cells coincided exactly with that of C3 protein-positive cells. C3 receptors were expressed mainly in macrophage-like mononuclear cells, TRAP-positive mononuclear cells, and TRAP-positive small cells with few nuclei. TRAP-positive large cells with many nuclei were totally negative for C3 receptors. When macrophage-colony-stimulating factor (M-CSF), the purified C3 protein, and 1alpha,25-(OH)2D3 were added to bone marrow methylcellulose cultures, separately or in combination, M-CSF-dependent colony formation was strikingly inhibited by 1alpha,25-(OH)2D3, but the inhibition was prevented by simultaneously adding C3. These results provide additional evidence that osteoclast progenitors are indeed cells of the monocyte-macrophage lineage. It is likely that the C3 produced by stromal cells in response to 1alpha,25-(OH)2D3 is somehow involved in osteoclast development by potentiating M-CSF-dependent proliferation of bone marrow cells and induction of osteoclast differentiation.
  • マイクロウェーブを用いた骨組織の迅速固定、迅速脱灰:マイクロウェーブ照射による生物試料の固定・染色法の基本とその応用
    1993年 [査読無し][通常論文]
  • OP/OPマウス大腿骨における遺伝子組換型ヒトM-CSF投与後の破骨細胞の分化とMac-2抗原の発現について(共著)
    歯科基礎医学会雑誌 1993年 [査読無し][通常論文]
  • The localization of H+-ATPase/Carbonic anhydrase in ameloblasts at the maturation stage. (共著)
    J. Dent. Res. 72 204 1993年 [査読無し][通常論文]
  • The localization of H+ ATPase/Carbonic Anhydrase in ameloblasts at the maturation (共著)
    J. Dental. Res. 72 204 1993年 [査読無し][通常論文]
  • 骨組織内のM-CSFの局在に関する免疫組織化学的研究
    網塚 憲生 歯科基礎医学会雑誌 34 (抄録) 189 -189 1992年09月 [査読無し][通常論文]
  • E ABE, T SATO, CH JIN, T KINOSHITA, N AMIZUKA, H OZAWA, T SUDA JOURNAL OF BONE AND MINERAL RESEARCH 7 S304 -S304 1992年08月 [査読無し][通常論文]
  • 骨芽細胞における1α,25(OH)2D3レセプターの細胞内局在について
    網塚 憲生 解剖学雑誌 67 (4) 556 -556 1992年08月 [査読無し][通常論文]
  • 網塚 憲生, 小沢 英浩 THE BONE 6 (2) 1 -5 1992年06月 [査読無し][通常論文]
  • 網塚 憲生, UCHIDA Takashi, FUKAE Makoto, YAMADA Marie, OZAWA Hidehiro Ａｒｃｈｉｖｅｓ ｏｆ Ｈｉｓｔｏｌｏｇｙ ａｎｄ Ｃｙｔｏｌｏｇｙ 55 (2) 179 -190 1992年05月 [査読無し][通常論文]
     
    Enamel tufts were exposed after decalcification of the enamel matrix and their fine structures and immunocytochemical characteristics were examined. Under the binocular microscope and the scanning electron microscope (SEM), enamel tufts appeared as corrugated ribbon-like structures located on the dentine parallel to the tooth axis. SEM observation disclosed enamel tufts as bundles of well extended tubular structures with cross striations attributable to hypocalcified enamel sheaths. Plate-like structures were observed at the center of enamel tufts, where they ran parallel to the enamel tufts. Under the transmission electron microscope (TEM), the plates of tufts revealed their origin in the superficial layer of the dentine, penetrating the hypercalcified zone adjacent to the dentineenamel (D-E) junction, and then reaching the tuft region. The plates of tufts ran mainly along the enamel sheaths and partially across the prisms in the tuft region. The protein-A-gold technique revealed an intense immunoreactivity for amelogenin over the superficial layer of the dentine, but over the enamel prisms in the tufts nor over the plates of tufts. The immunoreactivity for 13-17kd protein was detected over the filamentous structures closely associated with the enamel sheaths in the enamel tuft. Thus our study disclosed that enamel tufts consist of both well extended hypocalcified enamel prisms and plates of tufts. The major organic component of the enamel tufts is suggested to be 13-17kd protein rather than amelogenin.
  • N AMIZUKA, T UCHIDA, M FUKAE, M YAMADA, H OZAWA ARCHIVES OF HISTOLOGY AND CYTOLOGY 55 (2) 179 -190 1992年05月 [査読無し][通常論文]
     
    Enamel tufts were exposed after decalcification of the enamel matrix and their fine structures and immunocytochemical characteristics were examined. Under the binocular microscope and the scanning electron microscope (SEM), enamel tufts appeared as corrugated ribbon-like structures located on the dentine parallel to the tooth axis. SEM observation disclosed enamel tufts as bundles of well extended tubular structures with cross striations attributable to hypocalcified enamel sheaths. Plate-like structures were observed at the center of enamel tufts, where they ran parallel to the enamel tufts. Under the transmission electron microscope (TEM), the plates of tufts revealed their origin in the superficial layer of the dentine, penetrating the hypercalcified zone adjacent to the dentine-enamel (D-E) junction, and then reaching the tuft region. The plates of tufts ran mainly along the enamel sheaths and partially across the prisms in the tuft region. The protein-A-gold technique revealed an intense immunoreactivity for amelogenin over the superficial layer of the dentine, but over the enamel prisms in the tufts nor over the plates of tufts. The immunoreactivity for 13-17kd protein was detected over the filamentous structures closely associated with the enamel sheaths in the enamel tuft. Thus our study disclosed that enamel tufts consist of both well extended hypocalcified enamel prisms and plates of tufts. The major organic component of the enamel tufts is suggested to be 13-17 kd protein rather than amelogenin.
  • N AMIZUKA, H OZAWA ARCHIVES OF HISTOLOGY AND CYTOLOGY 55 (1) 77 -88 1992年03月 [査読無し][通常論文]
     
    The intracellular localization and translocation of the 1-alpha,25-dihydroxyvitamin D3 receptor (1-alpha,25(OH)2D3 receptor) in osteoblasts of mouse parietal bone and MC3T3-E1 cells were examined immunocytochemically using a rat monoclonal antibody to 1-alpha,25(OH)2D3 receptor. In osteoblasts of parietal bones in vivo, immunoreactivity for 1-alpha,25(OH)2D3 receptor was detected not only in the nuclei but also in lysosomal structures, and also sparsely in the cytoplasmic matrix. The transport of 1-alpha,25(OH)2D3 receptor was investigated immunocytochemically after incubation with 1-alpha,25(OH)2D3. In osteoblasts of parietal bones, after 1 min incubation with 10(-8)M 1-alpha,25(OH)2D3, the perinuclear cytoplasm showed intense immunoreactivity for 1-alpha,25(OH)2D3 receptor. After 10 min incubation, immunoreactivity was intense in the nuclei, especially in the heterochromatin. In MC3T3.E1 cells, after 1 min incubation with 1-alpha,25(OH)2D3, immunoreactivity for 1-alpha,25(OH)2D3 receptor was found in the form of a fibrillar structure extending radially to the periphery of the cells. The immunostaining pattern of anti-1-alpha,25(OH)2D3 receptor was similar to the distribution of microtubules stained with anti-/3-tubulin antibody. After 10 min incubation, the nuclei showed intense immunoreactivity for 1-alpha,25(OH)2D3 receptor. Incubation with colchicine dissociated the fibrillar structures and inhibited the intranuclear localization of the 1-alpha,25(OH)2D3 receptor. These findings suggest that the 1-alpha,25(OH)2D3 receptor is located in the nuclei, in lysosomal structures and also sparsely in the cytoplasmic matrix of osteoblasts in vivo, and that the receptor is transported to the perinuclear cytoplasm via microtubules to be then translocated into the nucleus, especially into the heterochromatin.
  • 阿部悦子, 佐藤利之, 金成河, 洪梅花, 木下タロウ, 網塚憲生, 小沢英浩, 須田立雄 日本骨代謝誌 10 (3) 320 1992年 [査読無し][通常論文]
  • 活性型ビタミンDにより骨組織で産生される補体第三成分(C3)とその生理学的役割(共著)
    歯科基礎医学会雑誌 34 133 1992年 [査読無し][通常論文]
  • 骨芽細胞における1α,25(OH)2D3レセプターの細胞内局在について
    解剖誌 67 556 1992年 [査読無し][通常論文]
  • ラット切歯エナメル芽細胞におけるH+-ATPaseと炭酸脱水素酵素の局在について、-免疫組織細胞化学的研究- I:成熟期エナメル芽細胞 (共著)
    歯科基礎医学会雑誌 34 195 1992年 [査読無し][通常論文]
  • 骨組織に分布するCGRP陽性神経の免疫組織化学的研究(共著)
    歯科基礎医学会雑誌 34 188 1992年 [査読無し][通常論文]
  • E. Satoh, C. H. Wu, T. Suzuki, K. Hara, N. Amizuka, H. Ozawa Periodontal Clinical Investigation 14 (1) 7 -13 1992年 [査読無し][通常論文]
     
    The efficacy of the air-powder abrasive device (APAD) in root planing during flap surgery was assessed in vitro and in vivo. Two teeth on which full-thickness flaps were raised underwent root planing by hand-scaling alone and hand-scaling combined with APAD. One tooth extracted prior to surgery underwent unrestricted hand-scaling under a binocular microscope (control). The two teeth root planed in situ and the tooth root planed in vitro were observed by scanning electron microscopy (SEM). Then, 10 patients with periodontitis requiring flap surgery were selected for a clinical study. After raising full-thickness flaps, 29 sites from five patients underwent root planing using hand-scaling combined with APAD, whereas 22 sites from six patients underwent root planing by hand scalers only. SEM revealed that root surfaces planed in situ by hand-scaling combined with APAD were smoother than those root-planed using hand scalers alone. The control tooth surface was as smooth as those submitted to combined root planing. The clinical study showed that sites submitted to combined root planing displayed enhanced attachment level gain and pocket reduction. These results, associated with the shortened instrumentation time, suggest APAD as a useful instrument for root planing during flap surgery.
  • 網塚 憲生 歯科基礎医学会雑誌 33 (抄録) 93 -93 1991年09月 [査読無し][通常論文]
  • 骨芽細胞における1α,25(OH)2D3レセプターの細胞内局在について
    新潟歯学会誌 21 204 1991年 [査読無し][通常論文]
  • 骨芽細胞における1α,25(OH)2D3レセプターの局在について
    歯科基礎医学会雑誌 33 93 1991年 [査読無し][通常論文]
  • Intracellular localization of 1α, 25(OH)2D3 receptor in osteoblasts.
    Jpa. J. Oral. Biol. 33 93 1991年 [査読無し][通常論文]
  • 網塚 憲生 歯科基礎医学会雑誌 32 (抄録) 61 -61 1990年09月 [査読無し][通常論文]
  • ヒト永久歯エナメル叢の微細構造学的および免疫細胞化学的研究
    網塚 憲生 解剖学雑誌 65 (4) 325 -325 1990年08月 [査読無し][通常論文]
  • 破骨細胞の分化と骨芽細胞系細胞の相互作用に関する微細構造学的研究 マウス頭蓋骨由来の骨芽細胞様細胞と脾細胞の共存培養により形成されるTRAP陽性多核細胞について
    網塚 憲生 日本骨代謝学会雑誌 8 (3) 315 -315 1990年06月 [査読無し][通常論文]
  • ラット大腿骨におけるED1陽性マクロファージと破骨細胞の免疫細胞化学的研究
    歯科基礎医学会雑誌 3 61 1990年 [査読無し][通常論文]
  • ヒト永久歯エナメル叢の微細構造学的研究
    解剖誌 65 325 1990年 [査読無し][通常論文]
  • Ultrastructural observation of TRAP-positive osteoclastic cells differentiated by co-culture with mouse spleen cells and calvaria stronal cells
    J. Bone & Mineral Metab. 8 213 1990年 [査読無し][通常論文]
  • Immunocytochemical observation of ED1-positive macrophage and osteoclast in rat femur.
    Jap. J. Oral Biol. 3 61 1990年 [査読無し][通常論文]
  • Ultrastructural study of Enamel tufts in human permanent teeth.
    65 325 1990年 [査読無し][通常論文]
  • ヒト永久歯におけるエナメル叢の微細構造学的観察
    網塚 憲生 新潟歯学会雑誌 19 (1) 89 -90 1989年06月 [査読無し][通常論文]
  • N AMIZUKA, H OZAWA JOURNAL OF DENTAL RESEARCH 68 (4) 667 -667 1989年04月 [査読無し][通常論文]
  • ヒト永久歯エナメル叢における微細構造学的観察
    新潟歯学会雑誌 19 90 1989年 [査読無し][通常論文]
  • Ultrastructural and cytochemical studies on the coupling hpenomena between osteoclasts and osteoblastic cells.
    J. Bone Min. Res. 4 212 1989年 [査読無し][通常論文]
  • 低アルカリホスファターゼ症における突然変異型組織非特異型アルカリホスファターゼの生合成について(共著)
    第39回歯科基礎医学会 39 111 [査読無し][通常論文]
  • 骨の細胞におけるゴルジ装置の構造と機能-BrefeldinA投与による骨芽細胞と破骨細胞のゴルジ装置の形態変化について-(共著)
    歯科基礎医学会誌 37 1 [査読無し][通常論文]
  • 破骨細胞の細胞死に関する微細構造学的, 細胞化学的研究(共著)
    歯科基礎医学会誌 37 2 [査読無し][通常論文]
  • The biological action of parathyroid hormone (PTH)-related peptide (PTHrP) mediated either by the PTH/PTHrP receptor or the mucleolar translocation in chondrocytes
    Anat Sci Int 771, 225-236 [査読無し][通常論文]
  • Ultrastructural and cytochemical observation of apoptotic osteoclasts induced by bisphosphonate. (共著)
    Bone [査読無し][通常論文]
  • High bone turnover associated with increased angiogenesis in melorheostosis : Histopathological studies. (共著)
    Orthopedics. [査読無し][通常論文]
  • Blomstrand chondrodisplasia caused by a missense mutation in the human parathyroid hormone receptor-1. (共著)
    Endocrinology in press [査読無し][通常論文]

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    医歯薬出版 2016年05月 (ISBN: 9784263457955) 376p
  • ファーマナビゲーター抗RANKL抗体編
    網塚憲生, 長谷川智香, 本郷裕美, 山本知真也, 原口真衣 (範囲:RANKL欠損状態における骨組織異常)
    メディカルレビュー社 2016年
  • 骨・臓器ネットワークとオステオサイト
    網塚憲生, 原口真衣, 本郷裕美, 坪井香奈子, 山本知真也, 長谷川智香 (範囲:Overview 骨細胞の形態学的知見)
    メディカルビュー社 2016年
  • 脇田, 稔, 前田, 健康, 中村, 浩彰, 網塚, 憲生 
    医歯薬出版 2015年02月 (ISBN: 9784263457849) xvii, 363p
  • 脇田 稔, 前田 健康, 中村 浩彰, 網塚 憲生 (担当:共編者(共編著者))
    医歯薬出版 2015年02月 (ISBN: 4263457846) 384
  • 「骨」を知る53の質問～ウェルエイジングをサポートするために～
    網塚憲生, 長谷川智香, 原口真衣, 山本知真也, 本郷裕美 (範囲:モデリングとリモデリングの違いは何ですか)
    医薬ジャーナル社 2015年
  • 骨ペディア 骨疾患・骨代謝キーワード事典
    網塚憲生, 長谷川智香 (範囲:モデリング、リモデリングとミニモデリング・軟骨内骨化と膜性骨化・骨・硬組織の染色法・微細構造解析)
    羊土社 2015年
  • 網塚, 憲生, 中村, 浩彰, 星, 和人, 小澤, 英浩 
    メディカルレビュー社 2014年02月 (ISBN: 9784779211829) 438p
  • 新しい骨形態計測～ Modern Bone Histomorphometry ～
    長谷川智香, 網塚憲生 (範囲:電子顕微鏡 Electron Microscopy)
    ウィネット出版 2014年
  • 口腔科学
    網塚 憲生 (範囲:骨の解剖生理)
    朝倉書院 2014年
  • ビタミンDと疾患 改訂版 －基礎の理解と臨床への応用－
    網塚憲生, 長谷川智香, 山本知真也, 宮本幸奈, 佐々木宗輝, 本郷裕美 (範囲:Ⅲ．ビタミンDの作用 活性型ビタミンＤ3製剤（エルデカルシトール）による骨リモデリング制御)
    医薬ジャーナル社 2014年
  • CKD-MBDハンドブック2nd Edition
    網塚憲生, 長谷川智香, 佐々木宗輝 (範囲:第1章 ミネラル代謝の生理 6．骨代謝回転と石灰化)
    日本メディカルセンター 2013年
  • ガイドラインサポートハンドブック 慢性腎臓病に伴う骨ミネラル代謝異常（CKD-MBD）
    長谷川智香, 佐々木宗輝, 山田珠希, 網塚憲生 (範囲:CKD-MBDと骨細胞)
    医薬ジャーナル社 2013年
  • 最新の骨粗鬆症学－骨粗鬆症の最新知見
    網塚憲生, 佐々木宗輝, 山田珠希, 山本恒之 (範囲:骨の細胞と組織)
    日本臨牀社 2013年
  • がん骨転移のバイオオロジーとマネージメント
    長谷川智香, 佐々木宗輝, 本郷裕美, 山田珠希, 山本恒之, 網塚憲生 (範囲:転移標的臓器としての骨の解剖学・組織学)
    医薬ジャーナル社 2012年
  • トートラ解剖学 第2版
    網塚 憲生 (範囲:骨組織)
    丸善株式会社 2010年
  • 網塚 憲生 
    [網塚憲生] 2008年03月 332p
  • 網塚 憲生 
    [網塚憲生] 2006年03月 274p
  • 副甲状腺ホルモン/副甲状腺ホルモン関連ペプチド受容体とシグナル伝達
    臨床内分泌学３－甲状腺・副甲状腺・骨内分泌代謝系－ 2005年
  • 副甲状腺ホルモン/副甲状腺ホルモン関連ペプチド受容体遺伝子改変マウス
    臨床内分泌学３－甲状腺・副甲状腺・骨内分泌代謝系－ 2005年
  • 副甲状腺ホルモン関連ペプチド遺伝子改変動物
    臨床内分泌学３－甲状腺・副甲状腺・骨内分泌代謝系－ 2005年
  • 網塚, 憲生 
    [網塚憲生] 2004年03月 1冊
  • 骨の細胞の形態と機能
    新しい透析骨症 日本メデイカルセンター 2003年
  • 軟骨の形態学
    骨・関節疾患 朝倉書店 2003年
  • カルシトニンの標的組織と作用「カルシウムと骨」
    朝倉書店 2001年
  • 骨原生細胞「カルシウムと骨」
    朝倉書店 2001年
  • 副甲状腺ホルモン関連ペプチド遺伝子組み替えマウスの軟骨・骨の異常
    医学のあゆみ 2000年
  • 副甲状腺ホルモン/副甲状腺ホルモン関連タンパク受容体の機能「新・分子骨代謝学と骨粗鬆症」
    メディカルレビュー社 2000年
  • The Bipartite action of parathyroid hormone(PTH)-related peptide(PTHrP)mediating by binding PTH/PTHrP receptor and translocation to nucleolus. In Recent Research Development of Endocrinology.
    Transworld Research Network. India. 2000年
  • 骨端軟骨異常と副甲状腺ホルモン関連ペプチド
    骨と関節 協和企画通信 1996年
  • 副甲状腺ホルモン関連ペプチド(PTHRP)と骨, 軟骨の異常について
    メディカルレビュー社 1996年
  • 骨組織におけるオステオポンティンの局在について
    骨と関節 協和企画通信 1995年
  • カルシトニン遺伝子関連ペプチド(CGRP)陽性神経の分布と破骨細胞(共著)
    メディカルレビュー社 1995年
  • マイクロウェーブを用いた骨組織の迅速固定, 迅速脱灰(共著)
    マイクロウェーブ照射による生物試料の固定・染色法の基本とその応用 1993年
  • 骨芽細胞におけるビタミンDレセプターの細胞内局在と細胞内移動
    THE BONE メディカルレビュー社 1992年
  • 破骨細胞と骨芽細胞の分化・活性化における細胞間,細胞・基質間相互作用に関する形態学的見解
    骨代謝調節因子-最近の進歩羊土社 1991年
  • Ultrastructural Observation of Enamel Tufts in Human Permanent Teeth
    Proceedings of Tooth Enamel V Florence Publishers 1989年
  • 口腔組織発生学 第3版
    網塚 憲生 (担当:単著範囲:第11章 歯の研究法)
    医歯薬出版株式会社

講演・口頭発表等

  • Chronological distribution and gene expression of osteoclasts and their precursors after the single administration of anti-RANKL antibody in mice.
    Ishizu H, Shimizu T, Hasegawa T, Amizuka N, Iwasaki N
    ORS(Orthopaedic Research Society) 2023 Annual Meeting
  • 破骨細胞の分化・機能に対するRANKL/Siglec-15の作用について
    長谷川智香, 山本知真也, 本郷裕美, 宮本幸奈, 宗山昂史, 福田千恵, 津田英資, 網塚憲生
    第64回歯科基礎医学会学術大会
  • アレンドロネート投与後における骨特異的血管の組織化学的変化
    吉野弘菜, 長谷川智香, 網塚憲生
    日本解剖学会第68回東北・北海道連合支部学術集会
  • 副甲状腺ホルモン製剤投与による皮質骨多孔化の微細構造学的解明
    阿部未来, 山本知真也, 本郷裕美, 吉野弘菜, 網塚憲生, 長谷川智香
    日本解剖学会第68回東北・北海道連合支部学術集会
  • 新規肥満2型糖尿病モデルSDT fatty ラットにおける骨組織の解析
    本郷裕美, 姚 斉, 長谷川智香, 網塚憲生
    日本解剖学会第68回東北・北海道連合支部学術集会
  • エストロゲン応答遺伝子Ebag9欠損マウスにおける骨量低下とオートファジー制御
    東 浩太郎, 池田和博, 柴 祥子, 佐藤 航, 堀江 公仁子, 長谷川智香, 網塚憲生, 田中伸哉, 井上 聡
    第24回日本骨粗鬆症学会
  • X連鎖低リン血症性くる病モデルマウスは血管侵入抑制を伴う軟骨内骨化異常を示す
    大巻真幸, 山本知真也, 金子一郎, 瀬川博子, 網塚憲生, 長谷川智香
    第24回日本骨粗鬆症学会
  • エボカルセトは骨細胞異常で誘導される皮質骨多孔化を抑制する: 二次性副甲状腺機能亢進症を伴う慢性腎臓病モデルラットを用いた動物実験
    長谷川智香, 徳永 紳, 山本知真也, 本郷裕美, 酒井まり子, 川田剛央, 網塚憲生
    第24回日本骨粗鬆症学会
  • テリパラチド/アバロパラチドで誘導されるリモデリングとモデリングの微細構造 - 動物モデル実験による組織解析
    長谷川智香, 山本知真也, 阿部未来, 槙野彰人, 網塚憲生
    第24回日本骨粗鬆症学会
  • Histological alteration of bone-specific blood vessels by alendronate administration
    Yoshino H, Hasegawa T, Amizuka N
    The 99th General Session & Exhibition of The IADR/ The 50th Annual Meeting of The AADR/ The 45th Annual Meeting of The CADR
  • 肢間葉系特異的Tfam ノックアウトマウスは四肢の自然骨折をきたす
    吉岡大輝, 河村真吾, 蔵満紀成, 後藤篤史, 長谷川智香, 網塚憲生, 石本卓也, 小笹良輔, 中野貴由, 今井祐記, 秋山治彦
    第40回日本骨代謝学会学術集会
  • PTH 製剤投与の皮質骨多孔化を誘導する破骨細胞・血管連関の微細構造学的解明
    阿部未来, 山本知真也, 本郷裕美, 吉野弘菜, 網塚憲生, 長谷川智香
    第40回日本骨代謝学会学術集会
  • 骨芽細胞特異的に副甲状腺ホルモン関連ペプチドを過剰発現させたマウスの長管骨の組織化学的解析及び遺伝子解析
    山本知真也, 阿部未来, 吉野弘菜, 原口真衣, 網塚憲生, 長谷川智香
    第40回日本骨代謝学会学術集会
  • 二次性副甲状腺機能亢進症の皮質骨多孔化はPTH-Pi ループで誘導される骨細胞性骨溶解と骨小腔石灰化抑制により発症する－エボカルセトによる皮質骨多孔化抑制の検討
    長谷川智香, 徳永 紳, 山本知真也, 本郷裕美, 酒井まり子, 網塚憲生
    第40回日本骨代謝学会学術集会
  • アバロパラチドまたはテリパラチドを投与した動物モデルの組織学的知見
    網塚憲生, 槙野彰人, 山本知真也, 長谷川智香
    第40回日本骨代謝学会学術集会
  • 骨芽細胞における形態学知見 －過去から近年までの研究
    網塚憲生, 長谷川智香
    第40回日本骨代謝学会学術集会
  • 部分腎摘ラットにおける骨病変解析～エボカルセトの作用検討～
    徳永 紳, 長谷川智香, 酒井まり子, 川田剛央, 網塚憲生
    第67回日本透析医学会学術集会・総会
  • PTH/PTHrP 製剤の骨形成における組織学的作用機序 ̶動物モデルを用いた基礎研究―
    網塚憲生, 阿部未来, 槙野彰人, 長谷川智香
    第95回日本整形外科学会学術総会
  • リン酸八カルシウム（OCP）による骨再生と骨細胞分化
    塩飽由香利, 齊藤志都, 濱井 瞭, 長谷川智香, 網塚憲生, 蔡 優広, 髙橋 哲, 鈴木 治
    第43回日本バイオマテリアル学会大会
  • 歯根形成期のマウス臼歯歯根膜におけるαSMA陽性/c-kit陽性細胞の局在変化について
    丸岡春日, 長谷川智香, 中西康, 佐藤嘉晃, 網塚憲生
    第80回日本矯正歯科学会＆第5回国際学術大会
  • 骨代謝回転状態によるpodoplanin/PHOSPHO1 陽性骨芽細胞の局在変化
    中嶋悠斐, 山本知真也, 本郷裕美, Nasoori Alireza, 網塚憲生, 長谷川智香
    第63回歯科基礎医学会学術大会
  • リン酸化多糖体/βTCP 混合新規骨補填材によるインプラント周囲骨再生について
    久保田恵亮, 横山敦郎, 網塚憲生, 長谷川智香
    第63回歯科基礎医学会学術大会
  • 副甲状腺ホルモン製剤投与による皮質骨多孔化のメカニズムについて
    阿部未来, 山本知真也, 本郷裕美, Nasoori Alireza, 網塚憲生, 長谷川智香
    第63回歯科基礎医学会学術大会
  • 骨補填材としてのリン酸化プルラン/βTCPコンビネーションマテリアルの応用
    森本康仁, 久保田恵亮, 阿部未来, 丸岡春日, 長谷川智香, 本郷裕美, 吉田靖弘, 網塚憲生, 菅谷 勉
    第64回 秋季日本歯周病学会学術大会
  • 顎骨壊死の病態と評価
    北川善政, 浅香卓哉, 佐藤 淳, 網塚憲生
    第63回歯科基礎医学会学術大会
  • 新規リン酸化多糖体“リン酸化プルラン”を用いた骨再生  [招待講演]
    長谷川智香, 森本康仁, 久保田恵亮, 本郷裕美, 吉田靖弘, 網塚憲生
    第63回歯科基礎医学会学術大会
  • 骨の恒常性維持および骨成長における免疫受容体群を介した骨吸収制御機構
    小林英之, 太田昌博, 長谷川智香, 山本知真也, 清水智弘, 藤田 諒, 網塚憲生, アラー テルカウイ, 岩崎倫政, 高畑雅彦
    第39回日本骨代謝学会学術集会
  • ステロイド性骨粗鬆症モデルラット大腿骨皮質骨におけるコラーゲン線維配向性は上昇する
    中村郁哉, 兼平裕也, 佐藤 大, 藤田 諒, 長谷川智香, 堀内秀与, 枡谷朋美, 太田昌博, 高畑雅彦, 網塚憲生, 木村-須田廣美
    第39回日本骨代謝学会学術集会
  • 低リン血症性くる病モデルマウス（Hypマウス）における軟骨内骨化異常の組織化学的検索
    大巻真幸, 長谷川智香, 山本知真也, 金子一郎, 瀬川博子, 網塚憲生
    第39回日本骨代謝学会学術集会
  • 骨基質石灰化の組織学・微細構造学  [招待講演]
    長谷川智香, 網塚憲生
    2021年度東北大学金属材料研究所共同研究ワークショップ・日本バイオマテリアル学会東北ブロック講演会
  • 副甲状腺ホルモン投与で誘導される皮質骨多孔化について
    阿部未来, 山本知真也, 本郷裕美, 網塚憲生, 長谷川智香
    日本解剖学会第67回東北・北海道連合支部学術集会
  • Histochemical assessment of perivascular αSMA/c-kit immunoreactive cells in periodontal ligaments.
    Maruoka H, Hasegawa T, Nakanishi K, Inoue K, Yamamoto T, Sato Y, Amizuka N
    The 99th General Session & Exhibition of The IADR/ The 50th Annual Meeting of The AADR/ The 45th Annual Meeting of The CADR
  • 骨形成促進剤で誘導されるmodeling-based/remodeling-based bone formationの組織学  [招待講演]
    網塚憲生, 長谷川智香, 本郷裕美, 山本知真也
    第41回日本骨形態計測学会 2021年07月
  • リン酸化プルラン/β-TCPコンビネーションマテリアルにおける新規骨再生誘導
    森本康仁, 長谷川智香, 本郷裕美, 久保田恵亮, 阿部未来, 丸岡春日, 吉田靖弘, 菅谷 勉, 網塚憲生
    第41回日本骨形態計測学会
  • マウス上顎臼歯歯根膜におけるαSMA陽性間葉系細胞の組織化学的検索
    丸岡春日, 長谷川智香, 中西 康, 井上貴一朗, 山本知真也, 佐藤嘉晃, 網塚憲生
    第126回日本解剖学会総会・全国学術集会/第98回日本生理学会大会合同大会
  • リン酸化多糖体とTCP混合補填材を用いたインプラント周囲骨再生における組織学的検索
    久保田恵亮, 長谷川智香, 山本知真也, 本郷裕美, 森本康仁, 阿部未来, 丸岡春日, 吉田靖弘, 横山敦郎, 網塚憲生
    第126回日本解剖学会総会・全国学術集会/第98回日本生理学会大会合同大会
  • リン酸三カルシウム(TCP)およびリン酸化プルラン(PPL)含有新規骨補填材を用いた骨再生
    森本康仁, 長谷川智香, 山本知真也, 本郷裕美, 久保田恵亮, 吉田靖弘, 菅谷 勉, 網塚憲生
    第126回日本解剖学会総会・全国学術集会/第98回日本生理学会大会合同大会
  • PTH間歇投与マウス大腿骨におけるpodoplanin/PHOSPHO1陽性骨芽細胞の局在
    中嶋悠斐, 山本知真也, 本郷裕美, 網塚憲生, 長谷川智香
    第126回日本解剖学会総会・全国学術集会/第98回日本生理学会大会合同大会
  • Ⅱ型コラーゲンプロモーターを用いた組織非特異的アルカリフォスファターゼ過剰発現マウス大腿骨骨端軟骨における石灰化抑制について
    長谷川智香, 山本知真也, 本郷裕美, 小守壽文, 網塚憲生
    第126回日本解剖学会総会・全国学術集会/第98回日本生理学会大会合同大会
  • マウス臼歯歯根膜における未分化間葉系細胞と周囲組織の組織化学的検索
    丸岡春日, 長谷川智香, 中西 康, 佐藤嘉晃, 網塚憲生
    第20回日本再生医療学会総会
  • リン酸化多糖体とTCP混合補填材を用いたインプラント周囲骨再生における組織学的検索
    久保田恵亮, 長谷川智香, 本郷裕美, 森本康仁, 阿部末来, 丸岡春日, 吉田靖弘, 横山敦郎, 網塚憲生
    第20回日本再生医療学会総会
  • リン酸三カルシウム（TCP）およびリン酸化プルラン（PPL）含有新規骨補填材を用いた骨再生
    森本康仁, 長谷川智香, 本郷裕美, 久保田恵亮, 阿部未来, 丸岡春日, 吉田靖弘, 菅谷勉, 網塚憲生
    第20回日本再生医療学会総会
  • ン酸八カルシウム（OCP）が新生骨基質の骨細胞分化に及ぼす影響の検討
    齊藤志都, 塩飽由香利, 濱井 瞭, 長谷川智香, 網塚憲生, 髙橋 哲, 鈴木 治
    第20回日本再生医療学会総会
  • Bone regeneration by novel combination materials using phosphorylated pullulan and triphosphate calcium.
    Morimoto Y, Hasegawa T, Hongo H, Sugaya T, Yoshida Y, Amizuka N
    American Academy of Periodontology 106th Annual Meeting
  • ステロイド性骨粗鬆症ラットの腰椎の骨質は，大腿骨の骨質に比べて緩やかに変化する  [通常講演]
    中村郁哉, 兼平裕也, 佐藤 大, 藤田 諒, 長谷川智香, 堀内秀与, 枡谷朋美, 太田昌博, 高畑雅彦, 居城邦治, 網塚憲生, 木村-須田廣美
    第38回日本骨代謝学会学術集会
  • 椎体を欠損させた卵巣摘除ラットにおけるアバロパラチドの骨欠損修復促進作用  [通常講演]
    槙野彰人, 長谷川智香, 網塚憲生
    第38回日本骨代謝学会学術集会
  • PTH投与による高骨代謝回転状態でのpodoplanin/PHOSPHO1陽性骨芽細胞の局在  [通常講演]
    中嶋悠斐, 山本知真也, 本郷裕美, 網塚憲生, 長谷川智香
    第38回日本骨代謝学会学術集会
  • モデリングおよび骨リモデリング領域の骨芽細胞の活性化における組織細胞学的検索  [通常講演]
    阿部未来, 長谷川智香, 宇田川信之, 網塚憲生
    第38回日本骨代謝学会学術集会
  • 軟骨細胞特異的アルカリホスファターゼ過剰発現マウスの骨端軟骨における組織学的・微細構造学的解析  [通常講演]
    長谷川智香, 山本知真也, 本郷裕美, 阿部未来, 小守壽文, 網塚憲生
    第38回日本骨代謝学会学術集会
  • The distribution of vascular endothelial cells and surrounding alphaSMA-/c-kit-immunoreactive mesenchymal cells in periodintal ligaments of murine developing morars.
    Maruoka H, Inoue K, Sato Y, Amizuka N
    The 9th International Orthodontic Congress
  • Abaloparatide promotes bone repair of vertebral defects in an overiectomized rat model of osteoporosis.
    Makino A, Hasegawa T, Takagi H, Takahashi Y, Hase N, Amizuka N
    The American Society for Bone and Mineral Research (ASBMR) 2020 Annual Meeting
  • マウス下顎骨切歯エナメル器におけるCD44とendomucin陽性血管の局在について  [通常講演]
    越石 麟, 本郷裕美, 網塚憲生, 長谷川智香
    第62回歯科基礎医学会学術大会
  • 副甲状腺ホルモン間歇投与による皮質骨多孔化の組織化学的検索  [通常講演]
    阿部未来, 山本知真也, 本郷裕美, 網塚憲生, 長谷川智香
    第62回歯科基礎医学会学術大会
  • メッケル軟骨前方部に形成される膜性骨の形態学的特徴と役割  [通常講演]
    井上貴一朗, 丸岡春日, 長谷川智香, 山本恒之, 八若保孝, 網塚憲生
    第62回歯科基礎医学会学術大会
  • 反射電子像を利用した走査型電子顕微鏡による破骨細胞のゴルジ装置の立体復構  [通常講演]
    山本恒之, 長谷川智香, 本郷裕美, 網塚憲生
    第62 回歯科基礎医学会学術大会
  • 骨粗鬆症治療薬候補としてのSiglec-15 抗体の研究
    津田英資, 福田千恵, 宇田川信之, 高橋直之, 長谷川智香, 網塚憲生, 佐藤 大, 高畑雅彦
    第62回歯科基礎医学会学術大会 シンポジウム・ワークショップパネル（指名）
  • 動物モデルを用いた骨組織再生の顕微学的検索  [通常講演]
    網塚憲生, 長谷川智香
    公益社団法人日本セラミックス協会第33回秋季シンポジウム シンポジウム・ワークショップパネル（指名）
  • Abaloparatide promotes bone repair of vertebral defects in an overiectomized rat model of osteoporosis.
    Makino A, Hasegawa T, Takagi H, Takahashi Y, Hase N, Amizuka N
    WCO-IOF-ESCEO 2020
  • ステロイド性骨粗鬆症モデルラットにおける骨強度と骨密度および骨質の関係  [通常講演]
    中村郁哉, 佐藤 大, 藤田 諒, 長谷川智香, 堀内秀与, 桝谷朋美, 太田昌博, 高畑雅彦, 居城邦治, 網塚憲生, 木村-須田廣美
    第40回日本骨形態計測学会
  • 軟骨細胞特異的組織非特異型アルカリフォスファターゼ過剰発現マウスの骨端軟骨における組織化学・微細構造学的解析  [通常講演]
    長谷川智香, 山本知真也, 本郷裕美, 山本恒之, 小守壽文, 網塚憲生
    第40回日本骨形態計測学会
  • Remodeling/Modeling-based bone formationの組織学
    網塚憲生, 長谷川智香, 本郷裕美, 山本知真也
    第40回日本骨形態計測学会 公開講演，セミナー，チュートリアル，講習，講義等
  • マウス下顎骨切歯エナメル器におけるpodoplanin、CD44およびendomucinの局在について  [通常講演]
    越石 麟, 本郷裕美, 井上貴一朗, 網塚憲生, 長谷川智香
    第125回日本解剖学会総会・全国学術集会
  • 副甲状腺ホルモン間歇投与マウスの長管骨におけるpodoplaninの免疫局在について  [通常講演]
    中嶋悠斐, 本郷裕美, 井上貴一朗, 網塚憲生, 長谷川智香
    第125回日本解剖学会総会・全国学術集会
  • RANKL中和抗体を単回投与したマウスの大腿骨の組織学的変化について  [通常講演]
    山本知真也, 本郷裕美, 井上貴一朗, 長谷川智香, 山本恒之, 網塚憲生
    第125回日本解剖学会総会・全国学術集会
  • リン酸三カルシウム（TCP）/リン酸化プルラン（PPL）含有新規骨補填材による新生骨形成誘導について  [通常講演]
    本郷裕美, 森本康仁, 長谷川智香, 菅谷 勉, 吉田靖弘, 網塚憲生
    第125回日本解剖学会総会・全国学術集会
  • 骨血管連関における細胞間コミュニケーション
    長谷川智香, 本郷裕美, 山本知真也, 網塚憲生
    第125回日本解剖学会総会・全国学術集会 シンポジウム・ワークショップパネル（指名）
  • リン酸八カルシウム（OCP）による骨形成進展における骨細胞分化の観察
    齊藤志都, 塩飽由香利, 濱井 瞭, 長谷川智香, 網塚憲生, 高橋 哲, 鈴木 治
    日本セラミックス協会 2020 年年会
  • 歯科領域の骨再生における細胞学的・微細構造学的検索 －動物モデルの顕微解析．  [通常講演]
    網塚憲生, 本郷裕美, 長谷川智香
    第19回日本再生医療学会総会 2020年03月 シンポジウム・ワークショップパネル（指名）
  • リン酸三カルシウム（TCP）/リン酸化プルラン（PPL） 含有新規骨補填材によって形成された新生骨の組織学的検  [通常講演]
    本郷裕美, 長谷川智香, 森本康仁, 菅谷勉, 吉田靖弘, 網塚憲生
    第19回日本再生医療学会総会
  • Siglec-15-targeting Therapy Protects Against Glucocorticoid-induced Osteoporosis With A Suitable Profile For Growing Skeleton In Rats.
    Sato D, Takahata M, Ota M, Fukuda C, Tsuda E, Shimizu T, Fujita R, Kobayashi H, Hiratsuka S, Amizuka N, Hasegawa T, Iwasaki N
    The OrgtoPaedic Research Society 2020 Annual Meeting
  • 骨粗鬆症治療薬候補としてのSiglec-15抗体の研究
    津田英資, 福田千恵, 宇田川信之, 高橋直之, 長谷川智香, 網塚憲生, 佐藤 大, 高畑雅彦
    第61回歯科基礎医学会学術大会 シンポジウム・ワークショップパネル（指名）
  • Microscopic imaging of osteoblasts/osteocytes in bone.  [通常講演]
    網塚 憲生, 長谷川智香
    第60回日本組織細胞化学会総会・学術集会/第13回日中合同組織細胞化学セミナー 2019年09月 シンポジウム・ワークショップパネル（指名）
  • 骨特異的血管と骨細胞における組織化学・電顕イメージング．
    長谷川智香, 邱 紫璇, 宮本幸奈, 山本知真也, 網塚憲生
    第39回日本骨形態計測学会 シンポジウム・ワークショップパネル（指名）
  • 骨粗鬆症治療薬における細胞組織・微細構造in vivo解析  [通常講演]
    網塚憲生, 長谷川智香, 槙野彰人, 本郷裕美, 山本知真也
    第39回日本骨形態計測学会 2019年07月 シンポジウム・ワークショップパネル（指名）
  • Histological Assessment for Osteocyte Function. Special Lecture.  [通常講演]
    網塚 憲生
    International Collaborative Symposium on Development of Human Resources in Practical Oral Health and Treatment 2019年02月 シンポジウム・ワークショップパネル（指名）
  • 顕微鏡の世界に魅せられて  [通常講演]
    網塚 憲生
    第20回日本骨粗鬆症学会 2018年10月 公開講演，セミナー，チュートリアル，講習，講義等
  • 骨における組織化学・電顕イメージング  [通常講演]
    網塚 憲生
    第36回日本骨代謝学会学術集会 2018年07月 公開講演，セミナー，チュートリアル，講習，講義等
  • 骨基質石灰化におけるリン-FGF23-Klotho axis  [通常講演]
    網塚 憲生
    第63回日本透析医学会学術集会・総会 2018年06月 シンポジウム・ワークショップパネル（指名）
  • 細胞組織学・微細構造学からみた骨形成  [通常講演]
    網塚 憲生
    第38回日本骨形態計測学会 2018年06月 シンポジウム・ワークショップパネル（指名）
  • テリパラチドの骨形成作用における細胞組織学的検索  [通常講演]
    網塚 憲生
    第38回日本骨形態計測学会 2018年06月 シンポジウム・ワークショップパネル（指名）
  • 骨質に関する骨の微細構造と細胞群の役割  [通常講演]
    網塚 憲生
    公益社団法人日本補綴歯科学会第127回学術大会 2018年06月 シンポジウム・ワークショップパネル（指名）
  • Morphological assessment for osteocyte function in bone.  [通常講演]
    網塚 憲生
    KSBMR-JSBMR joint symposium The 6th Seoul Symposium on Bone Health 2018年05月 シンポジウム・ワークショップパネル（指名）
  • 顕微鏡解析からみたビスホスホネート製剤の作用機序  [通常講演]
    網塚 憲生
    第19回日本骨粗鬆症学会 2017年10月 シンポジウム・ワークショップパネル（指名）
  • Osteocyte Biologyオーバービュー  [通常講演]
    網塚 憲生
    第59回歯科基礎医学会学術大会 2017年09月 シンポジウム・ワークショップパネル（指名）
  • ミニモデリングと骨リモデリングにおける骨形成－組織化学・微細構造学的知見－  [通常講演]
    網塚 憲生
    第37回日本骨形態計測学会 2017年06月 シンポジウム・ワークショップパネル（指名）
  • 骨の細胞に対するPTH作用の組織学的知見  [通常講演]
    網塚 憲生
    第62回日本透析医学会学術集会・総会 2017年06月 シンポジウム・ワークショップパネル（指名）
  • 骨ネットワークと他臓器連関：オーバービュー  [通常講演]
    網塚 憲生
    第122回日本解剖学会総会・全国学術集会 2017年03月 シンポジウム・ワークショップパネル（指名）
  • 骨質に影響を及ぼす骨の微細構造と細胞群の役割  [通常講演]
    網塚 憲生
    第16回日本再生医療学会総会 2017年03月 シンポジウム・ワークショップパネル（指名）
  • 骨粗鬆症治療薬における骨の細胞・組織学的知見について－動物モデルを用いた基礎研究－  [通常講演]
    網塚 憲生
    第31回日本整形外科学会基礎学術集会 2016年10月 シンポジウム・ワークショップパネル（指名）
  • 骨の細胞における微細構造・組織化学的知見  [通常講演]
    網塚 憲生
    第31回日本整形外科学会基礎学術集会 2016年10月 公開講演，セミナー，チュートリアル，講習，講義等
  • 骨基質石灰化異常における微細構造学的知見  [通常講演]
    網塚 憲生
    第36回日本骨形態計測学会 2016年06月 シンポジウム・ワークショップパネル（指名）
  • Overview：骨基質石灰化における微細構造学  [通常講演]
    網塚 憲生
    第121回日本解剖学会総会全国学術集会 2016年03月 シンポジウム・ワークショップパネル（指名）
  • オーバービュー：骨を観る －組織化学・微細構造学からバイオイメージングへの展開－  [通常講演]
    網塚 憲生
    第57回歯科基礎医学会学術大会 2015年09月 シンポジウム・ワークショップパネル（指名）
  • 骨粗鬆症治療薬における基礎から得られた新たな知見－ビスホスホネート製剤を中心として－  [通常講演]
    網塚 憲生
    第35回日本骨形態計測学会 2015年06月 シンポジウム・ワークショップパネル（指名）
  • 細胞組織学からみた骨粗鬆症治療薬の作用機序  [通常講演]
    網塚 憲生
    第35回日本骨形態計測学会 2015年06月 シンポジウム・ワークショップパネル（指名）
  • FIB-SEM を用いた骨組織の微細構造解析  [通常講演]
    網塚 憲生
    日本顕微鏡学会第71回学術講演会 2015年05月 シンポジウム・ワークショップパネル（指名）
  • Morphological examination on anabolic effects of intermittent administration of PTH in bone.  [通常講演]
    網塚 憲生
    13th Congress of the International Societyof Bone Morphometry 2015年04月 シンポジウム・ワークショップパネル（指名）
  • Histological aspects on the biological function of osteocytes.  [通常講演]
    網塚 憲生
    13th Congress of the International Society of Bone Morphometry 2015年04月 シンポジウム・ワークショップパネル（指名）
  • 副甲状腺ホルモン製剤に対する骨の細胞群の反応 －組織学的知見－  [通常講演]
    網塚 憲生
    第32回日本骨代謝学会学術集会 2014年07月 シンポジウム・ワークショップパネル（指名）
  • 骨細胞・ 骨細管系における形態学的アプローチ  [通常講演]
    網塚 憲生
    第32回日本骨代謝学会学術集会 2014年07月 シンポジウム・ワークショップパネル（指名）
  • Histopathological examination on bisphosphonate-related osteonecrosis of the jaw (BRONJ).  [通常講演]
    網塚 憲生
    The 9th Scientific Meeting of the Asian Academy of Osseointegration 2014年07月 シンポジウム・ワークショップパネル（指名）
  • Localization of 15N-minodronate by isotope microscopy and histochemical assessment for the biological effects of minodronate to bone cells in mice.  [通常講演]
    網塚 憲生
    SISS-16 2014年06月 シンポジウム・ワークショップパネル（指名）
  • 骨基質石灰化の形態学  [通常講演]
    網塚 憲生
    第34回日本骨形態計測学会 2014年06月 シンポジウム・ワークショップパネル（指名）
  • オーバービュー： 骨の司令塔－骨細胞－  [通常講演]
    網塚 憲生
    第55回歯科基礎医学会学術大会・総会 2013年09月 シンポジウム・ワークショップパネル（指名）
  • リモデリング・ミニモデリングの観点からみたエルデカルシトールとテリパラチドの骨形成作用  [通常講演]
    網塚 憲生
    第31回日本骨代謝学会学術集会／国際骨代謝学会・日本骨代謝学会第２回合同国際会議 2013年05月 シンポジウム・ワークショップパネル（指名）
  • マグネシウムは骨組織に必要か  [通常講演]
    網塚 憲生
    第32回日本マグネシウム学会学術集会 2012年11月 シンポジウム・ワークショップパネル（指名）
  • 新規活性型ビタミンD₃製剤エルデカルシトールの作用機序－動物実験モデルにおける組織学的解析－  [通常講演]
    網塚 憲生
    第27回日本整形外科学会基礎学術集会 2012年10月 シンポジウム・ワークショップパネル（指名）
  • 骨粗鬆症のモデル動物における骨の細胞群  [通常講演]
    網塚 憲生
    第27回日本整形外科学会基礎学術集会 2012年10月 シンポジウム・ワークショップパネル（指名）
  • Bisphosphonate投与およびPTH投与モデル動物の組織学的解析－骨質変化に付随する現象－  [通常講演]
    網塚 憲生
    第30回日本骨代謝学会学術集会 2012年07月 シンポジウム・ワークショップパネル（指名）
  • エルデカルシトールがin vivoの骨に及ぼす組織細胞学的作用について  [通常講演]
    網塚 憲生
    第30回日本骨代謝学会学術集会 2012年07月 シンポジウム・ワークショップパネル（指名）
  • PTH投与における骨の細胞群の動向について  [通常講演]
    網塚 憲生
    第32回日本骨形態計測学会 2012年06月 シンポジウム・ワークショップパネル（指名）
  • klotho欠損マウスにおける骨・血管石灰化のカルシウムパラドックス  [通常講演]
    網塚 憲生
    日本顕微鏡学会第68回学術講演会 2012年05月 シンポジウム・ワークショップパネル（指名）
  • 骨細胞の細胞生理学における形態学的アプローチ  [通常講演]
    網塚 憲生
    第89回日本生理学会大会 2012年03月 シンポジウム・ワークショップパネル（指名）
  • 将来の解剖学・組織学を担う歯学部学生に対する期待と取り組み  [通常講演]
    網塚 憲生
    第117回日本解剖学会総会・全国学術集会 2012年03月 シンポジウム・ワークショップパネル（指名）
  • Assessment of histological alterations in cartilage and extracellular matrix driven by collagen-induced arthritis in Macaca fascicularis.  [通常講演]
    網塚 憲生
    The 1st Bio-Rheumatology International Congress (BRIC2011) 2011年11月 シンポジウム・ワークショップパネル（指名）
  • Histochemical imaging of bone forming cells.  [通常講演]
    網塚 憲生
    12th Chitose International Forum on Photonics Science & Technology 2011年10月 シンポジウム・ワークショップパネル（指名）
  • 血管石灰化の機序における微細構造学的アプローチ  [通常講演]
    網塚 憲生
    第53回歯科基礎医学会学術大会 2011年09月 シンポジウム・ワークショップパネル（指名）
  • 骨細胞機能における形態学的アプローチ  [通常講演]
    網塚 憲生
    第29回日本骨代謝学会学術集会 2011年07月 シンポジウム・ワークショップパネル（指名）
  • Histological assessment for the biological functions of PTH and PTHrP in skeletal tissue.  [通常講演]
    網塚 憲生
    Research Unit of Metabolic bone disease in Korean Endocrine Society 2011年07月 シンポジウム・ワークショップパネル（指名）
  • 骨形成におけるPTHと活性型ビタミンDの作用－形態学的知見－  [通常講演]
    網塚 憲生
    第31回日本骨形態計測学会 2011年05月 シンポジウム・ワークショップパネル（指名）
  • 骨細胞の機能における形態学的知見  [通常講演]
    網塚 憲生
    第88回日本生理学会大会第116回日本解剖学会総会・全国学術集会合同大会 2011年03月 シンポジウム・ワークショップパネル（指名）
  • 石灰化基質に対する骨細胞の作用  [通常講演]
    網塚 憲生
    第52回歯科基礎医学会学術大会 2010年09月 シンポジウム・ワークショップパネル（指名）
  • ビスフォスフォネート関連顎骨壊死（BRONJ）に対する形態学的考察  [通常講演]
    網塚 憲生
    日本学術会議会・第64回日本口腔科学会学術大会共催市民公開シンポジウム 2010年06月 シンポジウム・ワークショップパネル（指名）
  • Histological assessment for PTH/PTHrP signaling on osteoblasts.  [通常講演]
    網塚 憲生
    14th international congress of endocrinology (ICE 2010) 2010年03月 シンポジウム・ワークショップパネル（指名）
  • Histological assessment for biological function of osteocytic lacunar-canalicular system.  [通常講演]
    網塚 憲生
    連携機能を活用した口腔からQOL向上を目指す連携研究・国際シンポジウム 2010年02月 シンポジウム・ワークショップパネル（指名）
  • 骨組織における組織化学の実際  [通常講演]
    網塚 憲生
    第27回日本骨代謝学会 2009年07月 シンポジウム・ワークショップパネル（指名）
  • 骨・軟骨組織における免疫組織化学  [通常講演]
    網塚 憲生
    第29回日本骨形態計測学会 2009年05月 シンポジウム・ワークショップパネル（指名）
  • 骨質を石灰化基質の微細構造から考える  [通常講演]
    網塚 憲生
    第29回日本骨形態計測学会 2009年05月 シンポジウム・ワークショップパネル（指名）

所属学協会

  • 国際骨代謝学会   日本骨粗鬆症学会   日本顕微鏡学会   日本骨形態計測学会   アメリカ骨代謝学会   国際歯科研究学会   日本解剖学会   日本骨代謝学会   歯科基礎医学会   日本再生医療学会   

Works（作品等）

  • FGFR3-/-/PTHrP-/-マウスの骨格異常
    2001年
  • Skeletal Abnormalities of FGFR3-/-/PTHrP-/- mouse
    2001年

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2024年04月 -2027年03月 
    代表者 : YAMAUTI MONICA, 友清 淳, 戸井田 侑, 網塚 憲生, 星加 修平, 川本 千春
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2022年06月 -2024年03月 
    代表者 : 北川 善政, 平田 健司, 渡邊 史郎, 佐藤 淳, 浅香 卓哉, 竹内 康人, 犬伏 正幸, 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 網塚 憲生, 長谷川 智香
     
    本研究は、副甲状腺ホルモン(PTH)、または、副甲状腺ホルモン関連ペプチド(PTHrP)が、骨芽細胞系細胞、あるいは、血管内皮細胞や血管平滑筋細胞への作用の解明を目標としている。PTH/PTHrPが骨形成促進作用を有する一方、研究代表者らはPTH/PTHrPが骨の血管にも作用し、血管周囲にalpha smooth muscle actin(αSMA)陽性・alkaline phosphatase(ALP)陽性・c-kit陽性が増加することを報告してきた。このことは、PTH/PTHrPは骨芽細胞と血管への両極性の作用を有する可能性を示唆する。一方で、Gli1が未分化間葉系細胞のマーカーとなり得ることを利用して、PTH/PTHrPで増加する血管周囲細胞ならびに骨芽細胞系細胞は、Gli1陽性未分化間葉系細胞由来なのか、それとも、異なる由来なのか検索した。そのモデル動物として、Gli1-CreErt2マウスとRosa26-loxP-stop-loxPtdTomatoマウスを交配してタモキシフェン投与依存的にGli1陽性細胞がtdTomato蛍光蛋白質を発現する成獣期の遺伝子改変マウス(Gli1-CreErt2:Rosa26-tdTomatoマウス)を作成した。その結果、正常状態では、Gli1/tdTomato陽性未分化間葉系細胞は成長板直下の骨幹端部にわずかに観察され、特に、成長板直下の骨梁間に存在することが明らかとなった。一方、PTH投与（２週間）タモキシフェン投与（２日間）すると、tdTomato陽性・αSMA陽性細胞（血管周囲細胞）、および、tdTomato陽性・ALP陽性細胞（骨芽細胞系細胞）はその数を増加させること、また、分布の範囲が血管側および骨芽細胞側と広がる傾向が認められた。このことは、PTH/PTHrPはGli1陽性を示す未分化間葉系細胞に作用し、細胞増殖を亢進させるとともに、血管周囲細胞ならびに骨芽細胞系細胞の両方への分化経路を促すことが推察された。
  • 日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2021年07月 -2023年03月 
    代表者 : 網塚 憲生
     
    ミニモデリングは、破骨細胞と骨芽細胞との共役（カップリング）で誘導される骨リモデリングとは異なり、破骨細胞の骨吸収に依存せずに、骨芽細胞を活性化させて骨基質合成を誘導する様式であり、成人においても、ある種の骨粗鬆症治療薬で誘導されることが知られている。現在のところ、ミニモデリングは、主に抗スクレロスチン抗体と活性型ビタミンＤアナログで誘導されるが、その機序については不明な点が多い。本研究では、ミニモデリングの発生機序を解明することで、それを誘導する “ミニモデリングファクター”が存在するか明らかにすることを目標とした。令和３年度では、活性型ビタミンＤアナログ（エルデカルシトール）によるミニモデリング誘導を骨細胞ネットワークとスクレロスチン抑制に焦点を当ててラットモデル実験を実施した。その際、骨の部位によって骨リモデリングが活発に行われている箇所が異なるため、ラットにエルデカルシトールを投与した大腿骨・脛骨を骨端部と骨幹端部に分けてサンプル採取し、ミニモデリングで誘導された新生骨の内部におけるスクレロスチン陽性骨細胞の割合を統計学的に検索した。その結果、骨幹端部では、通常、骨リモデリングが優位に誘導されており、一方、骨端部では骨リモデリング活性が低いことが示された。さらに、エルデカルシトール投与後におけるミニモデリングの発生箇所・頻度は、骨幹端部よりも骨端部の骨梁で多いことが示された。また、骨幹端部・骨端部の骨梁において、ミニモデリングで生じた骨基質ではスクレロスチン陽性骨細胞の割合が著しく低下していた。よって、エルデカルシトールのミニモデリング誘導作用は、スクレロスチンの作用をブロックすることで生じる可能性が高いことが強く示唆された。現在、これらの所見に基づき、論文作成中である。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 網塚 憲生
     
    本研究の目的は、副甲状腺ホルモン(PTH）シグナルが前骨芽細胞（骨芽細胞前駆細胞）の細胞増殖および成熟骨芽細胞の基質合成を亢進するだけでなく、石灰化基質の構成要素であるリン酸イオンの生成・供与に関する膜輸送体・酵素群に直接作用する可能性を明らかにすることである。 平成30年度では、PTH投与においてリン酸イオン供与膜輸送体・酵素の発現・局在が、基質合成を行っている成熟骨芽細胞、また、それ以外の前骨芽細胞・骨細胞においてどのように変化するか解析した。具体的には、申請者が2016年にEndocrinologyに報告した方法に準じてPTH高頻度投与マウスを作成し、成熟骨芽細胞が活発に基質小胞・石灰化球を産生しながら基質合成を行うこと、また、骨形成速度が亢進することを確認した。ところが、PTH高頻度投与では主要なリン酸イオン供与膜輸送体・酵素である組織非特異型アルカリホスファターゼ(TNAP),ピロリン酸合成酵素(ENPP1),基質小胞内でリン酸を生成するPHOSPHO1の発現が上昇したが、ピロリン酸を輸送するANKの発現に変化は認められなかった。これら膜輸送体や酵素を免疫組織化学的に解析したところ、コントロール群（PTH非投与群）ではTNAPとPHOSPHO1は成熟骨芽細胞と前骨芽細胞に、一方、ピロリン酸合成を行うENPP1は主に骨芽細胞に教材した。しかし、PTH高頻度投与では、骨芽細胞ばかりでなく前骨芽細胞や骨細胞にもENPP1の強陽性反応を認めた。real time PCRで解析すると、PTH投与群におけるTNAP発現の約2倍のENPP1発現上昇が確認された。 以上から、PTHシグナルによる骨基質石灰化の低下は、単に骨基質合成が上昇しているからだけではなく、石灰化抑制に作用するENPP1発現が亢進することに一因していることが示唆された。
  • 日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2018年06月 -2020年03月 
    代表者 : 網塚 憲生
     
    本研究では、骨芽細胞から骨細胞として骨基質に埋め込まれる時空的タイミングがどのような因子・環境によって制御されるのか、その細胞マーカーは存在するのか、また、骨細胞へのスイッチングが決定している骨芽細胞では細胞極性や細胞骨格といった細胞内小器官にダイナミックな変化が生じるのかといった、基質内に埋め込まれる前に特定の骨芽細胞に「分化指令」がなされるか否か解明することを目的としている。 平成30年度の研究において、申請者はpodoplaninに注目し、podoplaninを介した骨芽細胞から骨細胞へのスイッチング誘導の可能性、podoplaninとCD44との結合を介したEMR familyのリン酸化ならびにアクチンフィラメントの再構築を誘導することで、骨芽細胞から骨細胞への分化の可能性を検索した。その結果、骨リモデリング部（骨幹端）ではCD44陽性破骨細胞がpodoplanin陽性骨芽細胞に接触すること、また、その骨芽細胞は細胞膜にリン酸化ezrin陽性反応を示し、さらに、その骨芽細胞のアクチン線維は他の骨芽細胞よりも骨細胞に類似した細胞内分布を示すことが明らかとなった。一方、モデリング部位である皮質骨骨髄側では、podoplanin陽性骨芽細胞が骨表面に一定間隔で局在していたが、podoplanin陽性骨芽細胞とCD44陽性細胞との細胞間接触像は観察されなかったにも関わらず、podoplanin陽性骨芽細胞の細胞膜周囲にはリン酸化ezrinの局在が認められた。 以上から、モデリング部位では、骨細胞への分化におけるpodoplaninシグナルはCD44陽性破骨細胞に依存しないのに対して、骨リモデリング部位では、podoplanin陽性骨芽細胞が、CD44陽性破骨細胞と接触することで、骨細胞への分化が増強される可能性が推察された。
  • 台湾陽明大学・北海道大学主催 国際カンファレンス
    公益財団法人伊藤医薬学術交流財団：平成31年度(第25回)助成
    研究期間 : 2019年04月 -2020年03月 
    代表者 : 網塚 憲生
  • 骨芽細胞から骨細胞へのスイッチングにおけるON-OFF機構の新規解明
    日本学術振興会：学術研究助成基金助成金 挑戦的研究（萌芽）
    研究期間 : 2018年04月 -2020年03月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 下村 淳子, 梨田 智子, 中原 賢, 下村 裕, 網塚 憲生
     
    副甲状腺ホルモン・副甲状腺ホルモン関連ペプチドの受容体(以下PTH1R)は骨代謝だけではなく、歯の萌出に重要な役割を持つ。本研究では、Jansen型PTH1Rの分子病理について解明し、歯槽骨におけるPTH1Rの作用を解明するとともに、PTH1Rの関与する歯の萌出機構を明らかにすることを目的とした。本研究の結果、変異型PTH1Rの機能異常は、野生型PTH1Rと構造上の違い、およびその細胞内局在が異なることに起因し、タンパク質の糖鎖修飾の相違がその原因であると考えられた。さらに、この機能異常により、in vivoにおいて軟骨内及び膜内骨化異常が生じる可能性が示唆された。
  • 二国間交流事業共同研究・セミナー（日本学術振興会・国際交流事業）
    公益財団法人伊藤医薬学術交流財団：平成30年度（第24回）助成
    研究期間 : 2018年04月 -2019年03月 
    代表者 : 網塚 憲生
  • ミニモデリングにおける骨細胞ネットワークの新規調節機構
    日本学術振興会：二国間交流事業共同研究
    研究期間 : 2016年04月 -2018年12月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 網塚 憲生, 長谷川 智香, 坪井 香奈子, 本郷 裕美
     
    骨細胞ネットワークは、骨の成熟に伴って幾何学的に規則性を示していた。また、骨形成機序が個体成長初期ではミニモデリング（モデリング）であるのに対して、成長後期では骨リモデリングへと移行していた。骨細胞ネットワークは、胎生期・生後ではスクレロスチンをあまり産生せず、成獣期で骨リモデリングが行わる時期になると骨細胞から産生されることが観察された。一方、成獣期でも力学負荷やビタミンＤアナログによってミニモデリングが誘導されることが知られている。今回の研究では、骨細胞ネットワークが局所的にスクレロスチン産生を抑制することで、ミニモデリングを誘導する可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 網塚 憲生, 織田 公光, 長谷川 智香, 本郷 裕美, 山本 知真也
     
    αklotho-/-マウスおよびkl/klマウスに低リン食飼育を行うと、両マウスとも血中リン濃度の上昇が抑えられ、kl/klマウスの骨の石灰化異常は回復したが、αklotho-/-マウスの骨異常は回復しなかったことから、骨基質石灰化は血中リン濃度にだけ依存するのではないと考えられた。また、骨芽細胞から骨細胞に分化しつつある細胞はpodoplaninとリン酸化erzrinを細胞膜周囲に局在させていたが、ENPP1-/-マウスとkl/klマウスでは、早期にpodoplanin陽性骨芽細胞が誘導されたことから、血中リン濃度またはリン酸イオン供与体が骨細胞分化を促進させる可能性が推察された。
  • 二国間交流事業共同研究・セミナー（日本学術振興会・国際交流事業）
    公益財団法人伊藤医薬学術交流財団：平成29年度（第23回）助成
    研究期間 : 2017年04月 -2018年03月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 若手研究(B)
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 佐々木 宗輝, 黒嶋 伸一郎, 網塚 憲生
     
    本研究の目的は骨細胞と血中リン濃度の関係を解明することを目的とした。Kl/klマウスは、低リン食によって表現型が改善され、αklotho-/-マウスでは、表現型の改善が認められなかった。従って、αklotho-/-マウスの表現型の発現には血中リン濃度以外の因子の存在が考えられた。次に、野生型マウスの頭蓋骨から骨細胞を単離し、様々なリン濃度で培養を行い、各濃度における骨細胞産生蛋白の発現を調べた。その結果、リン濃度の変化に応じて発現する蛋白質の変化を認めた。従って、骨細胞はリン濃度を直接感知すると考えられ、そのためにはαklotho蛋白の存在が必要な可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 小島 拓, 網塚 憲生, 芳澤 享子, 小林 正治
     
    骨再生向上のために培養技術が有用ではないかと考え、骨髄または脂肪組織由来間葉系幹幹細・多孔性β-TCPブロック複合体を用いた骨再生について組織学的に検討を行った。4週齢GFPラットの大腿骨と脂肪組織から間葉系幹細胞を分離培養後、多孔性β-TCPブロックに播種して骨芽細胞様細胞へと分化誘導した。この複合体を10週齢のヌードラット頭蓋骨に移植し、移植部位を組織化学的に解析した。その結果、分化誘導された骨髄細胞と脂肪組織由来細胞は高い骨再生能を有することが明らかとなった。これらの結果から骨髄細胞だけでなく脂肪組織由来の幹細胞も骨再生における有用な細胞源になることが示唆された。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 網塚 憲生
     
    本研究は、骨における副甲状腺ホルモン（PTH）の二つの作用について検索を行った。第一点は、PTHの高頻度投与では多数の骨梁が誘導されるが、PTHの低頻度投与では骨梁数が増加しないものの、骨梁幅が増加することを明らかにした。前者は活発な骨リモデリングに依存する一方、後者はミニモデリング（破骨細胞の骨吸収に依存しない骨形成）と骨リモデリングの両方に依存することが推察された。第二点は、PTH投与後短時間において、骨細胞はその周囲の骨基質を溶解（骨細胞性骨溶解）し、また、再石灰化する可能性を明らかにした。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 高畑 雅彦, 須田 廣美, 東藤 正浩, 網塚 憲生
     
    関節リウマチ（RA）患者の骨脆弱性と骨質異常の関連を手術時に採取した腸骨生検サンプルを用いて調査した. 慢性的にステロイド剤を投与されているRA長期罹患患者では閉経後骨粗鬆症患者と比較して有意に力学的強度が低下しており，脆弱性骨折の既往も多かった. 骨密度は二群間で有意差はなかったことから，対象症例における骨強度と靱性の低下は，骨微細構造の劣化や石灰化障害による骨質異常が原因であると考えられた．副次的な知見として，臨床骨折評価ツールであるFRAXスコアが，ステロイド剤を使用しているRA患者の骨折リスクの評価に有用であることが示された．
  • 骨粗鬆症治療を見据えた新規骨形成メカニズム「ミニモデリング」の解明
    日本学術振興会：二国間交流事業共同研究
    研究期間 : 2013年04月 -2015年12月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 網塚 憲生
     
    FGF23/klotho軸が破綻し血中リン濃度が上昇したkl/klマウスおよびαklotho-/-マウスを解析したところ、骨基質石灰化が亢進するのではなく、骨細胞周囲の骨基質ミネラルが流失していた。低リン食飼育したkl/klマウスでは、骨組織異常が改善されたが、αklotho-/-マウスでは骨組織の改善は認められなかった。さらに、低リン食を与えたkl/klマウスでは、腎臓のαklothoの発現がある程度、回復していた。よって、FGF23/klotho軸の破綻で生じる骨細胞周囲の基質石灰化異常は、血中リン濃度異常によるものではなく、別の要因で生じる可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2011年04月 -2015年03月 
    代表者 : 横山 敦郎, 安田 元昭, 山本 悟, 網塚 憲生
     
    咬合に起因する微小動揺がオッセオインテグレーションに与える影響を明らかにすることを目的に、ラット上顎臼歯を抜歯後、チタンスクリューを即時埋入し、1週間後に咬合接触を付与し、咬合接触を与えないラットを対照群とし、組織化学的検索を行うとともに、マイクロアレイを用いて遺伝子発現の比較を行った。咬合接触を付与したラットにおいてはスクリュースレッド間の骨梁幅は有意に太くなり、スクレロスチン陽性骨細胞率は低下していた。遺伝子発現については、骨形成やBMP調整に関与する遺伝子が発現していた。これらの結果から、抜歯後即時埋入早期に与える適度の咬合負荷は、オッセオインテグレーションを早めることが示唆された。
  • klothoシグナル破綻による血管石灰化および血管骨化 －マウスおよびヒトの血管における組織学的検索－
    日本腎臓財団：腎不全病態研究助成
    研究期間 : 2014年04月 -2015年03月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 網塚 憲生
     
    副甲状腺ホルモン(PTH) の間歇投与頻度が高い場合、前骨芽細胞の細胞増殖亢進および多数の破骨細胞形成を伴った活発な骨リモデリングが誘導され、また、投与頻度を下げると、前骨芽細胞の細胞増殖よりも骨芽細胞の骨基質合成が優位になることが明らかとなった。一方、同じ受容体に結合する副甲状腺ホルモン関連ペプチド(PTHrP)は骨髄ストローマ細胞や前骨芽細胞の増殖促進に作用するが、破骨細胞形成には大きく作用しない可能性が示唆された。
  • klotho-FGF23作用破綻における血管石灰化の細胞生物
    日本腎臓財団：腎不全病態研究助成
    研究期間 : 2012年04月 -2013年03月 
    代表者 : 網塚 憲生
  • Klotho欠損状態における骨代謝異常・血管石灰化の微細構造学的検索
    日本腎臓財団：腎不全病態研究助成
    研究期間 : 2011年04月 -2012年03月 
    代表者 : 網塚 憲生
  • 骨細胞・骨細管系による骨基質ミネラル維持機構の解明
    日本学術振興会：科学研究費補助金 基盤研究（B）
    研究期間 : 2009年04月 -2012年03月 
    代表者 : 網塚 憲生
  • 変異型副甲状腺ホルモン受容体の細胞内輸送と組織異常における新たな展開
    日本学術振興会：科学研究費補助金 挑戦的萌芽研究
    研究期間 : 2009年04月 -2011年03月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2009年 -2011年 
    代表者 : 網塚 憲生, 李 敏啓, 織田 公光, 宇田川 信之
     
    本研究の目的は、骨に無数に張りめぐらされている骨細胞ネットワーク(骨細胞・骨細管系)が骨基質ミネラルの調節・維持および骨リモデリングに対してどのような細胞学的作用を示すのかin vivoで解明することである。本研究によって明らかにされた点は以下である。1)骨細胞・骨細管系の配列における幾何学的規則性は、成熟骨である皮質骨で発達しており、そのような規則的に配列した骨細胞からはスクレロスチンやFGF23といった因子が多量に産生されること、2)オステオプロテジェリン遺伝子欠損マウスやklotho遺伝子欠損マウスといった骨代謝回転が異常を示す状態では、骨細胞・骨細管系の配列が不規則となり、DMP-1やスクレロスチンなどの産生が影響を受けることも明らかにしている。3)さらに、規則的な骨細管系に存在する骨細胞は、副甲状腺ホルモンに反応して、周囲の骨基質ミネラルを溶出する可能性も得られた。
  • 骨芽細胞分化誘導蛋白を保持する新しいリン酸カルシウム材料の骨誘導
    ノーステック財団：「研究開発事業」スタートアップ補助金
    研究期間 : 2009年04月 -2010年03月 
    代表者 : 網塚 憲生
  • 骨・軟骨細胞における副甲状腺ホルモン受容体シグナルの分子細胞学的解析
    日本学術振興会：科学研究費補助金・特別研究費
    研究期間 : 2009年04月 -2010年03月 
    代表者 : 網塚 憲生
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2009年 -2010年 
    代表者 : 網塚 憲生
     
    副甲状腺ホルモン(PTH)および副甲状腺ホルモン関連ペプチド(PTHrP)の変異型受容体による組織異常を主にトランスジェニックマウスを用いて解析することが本研究の目的である。前年度では、ヒトBlomstrand型軟骨異形成症の組織異常を明らかにするため、type II collagen promoterに組み込んだBlomstrand型PTH-R^のcDNAを作製し、その組織異常を解析した(下村、網塚他日本小児歯科学会2010年)。本年度は、PTH-Rシグナルを過剰発現するトランスジェニックマウスの構築であったことから、申請者は胎生18日齢の胎仔において骨芽細胞特異的にPTHrPを過剰発現させることで、骨芽細胞や軟骨細胞の組織異常および細胞増殖・分化の解析を行った。その結果、PTHrPは骨芽細胞特異的に過剰産生されていたが、Runx2/ALP陽性骨芽細胞の数も低下しており、破骨細胞数も低下していた。一方、ALP陰性を示す多くの紡錘形細胞がPCNA陽性を呈したことから、胎生期におけるPTHrPの役割として、骨芽細胞だけでなく他の細胞の増殖を亢進させる可能性が強く示唆された。さらに、このTgマウスでは軟骨が増大しており、肥大化層の形成が認められないことから、分泌型のPTHrPが軟骨細胞の増殖抑制・肥大化促進を誘導していることも示唆された。以上、PTHrP過剰産生モデルの結果から、PTH-Rシグナルは軟骨・骨組織の形態形成に重要であると推測された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2007年 -2008年 
    代表者 : 伊東 昌子, 上谷 雅孝, 池田 恭治, 森諭 史, 網塚 憲生, 中野 貴由, 真柴 賛, 池田 恭治, 森 諭史, 真柴 賛, 網塚 憲生, 中野 貴由
     
    グルチコルチコイド(GC)は、骨密度に対して以上に、骨質を悪化させて骨脆弱化を高めると考えられるが、その実態は明らかにされていない。骨細胞群、骨微細構造、コラーゲン架橋に対してのGCの作用を、同じ症例の同一骨から得られた標本で解析した。結果として、1) 骨細胞アポトーシスを示唆するempty lacunaeの増加を認めた。破骨細胞の増加は認めなかった2) コラーゲン生理的架橋の減少・AGEs架橋の軽度の減少が見られた3) 骨梁構造悪化以上に、最大強度の減少が認められた
  • 日本学術振興会：科学研究費助成事業 萌芽研究
    研究期間 : 2007年 -2008年 
    代表者 : 網塚 憲生, 小澤 英浩, 小守 壽文
     
    平成20年度は核小体型PTHrP cDNAを組み込んだtype I collagen promoter enhancer cassetteを受精卵にmicroinjectionしてトランスジェニックマウスを作製した。さらに、核小体型PTHrPに対して分泌型の正常PTHrPを過剰産生するトランスジェニックマウスもコントロール実験として同時に作製している。その結果、我々の予想に反して、核小体型PTHrPトランスジェニックマウスではダイナミックな組織異常を観察することができなかったことから、核小体型PTHrPは軟骨細胞(前々年度に報告)と骨芽細胞に対する作用が異なる可能性が示唆された。一方、分泌型PTHrPを骨芽細胞に過剰発現させた場合は新生仔は死産となり、組織解析を行うと軟骨が伸張する一方で軟骨内骨化が阻害されていること、さらには一部の軟骨原器が異常拡大していることが明らかとなった。原因として皮質骨や僅かに形成される骨梁の骨芽細胞から過剰量のPTHrPが産生されて、軟骨細胞の受容体に作用したと考えられる。この表現系についても我々の予想を超えており、従来、パラクライン的に作用すると考えられてきたPTHrPが、比較的離れた部位に影響を及ぼすことが明らかとなった。現在、核小体型PTHrPトランスジェニックマウスでの微細構造学的異常、および骨芽細胞系細胞の分化異常がないか検索していると共に、副産物的に得られた分泌型PTHrPトランスジェニックマウスにおける軟骨伸張の分子細胞メカニズムを引き続き解析している。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2006年 -2007年 
    代表者 : 網塚 憲生, 前田 健康, 小澤 英浩
     
    本研究の解析目標は(1)骨細胞のミネラル調節の微細メカニズム、(2)骨細胞・骨細管系が骨質に与える影響、(3)PTHが骨細胞および骨基質ミネラルの流出・流入に与える影響、である。 (1)骨細胞特異的に死滅させるTgマウスの解析を前年度から引き続き行ったところ、骨細胞の死滅により骨基質ミネラルの流出を確認した。骨細胞は酸ホスファターゼやMMP-1ではなく、DMP-1やFGF23などのミネラル調節因子を過剰産生することで骨基質ミネラルの流出・流入を調節すると推測された。本研究の一部は論文掲載(次頁)であり、一部は現在、作成中。 (2)鍍銀染色にて骨細胞・骨細管系の分布を検索すると、骨代謝回転が高い部位ではその分布が乱れており、骨代謝回転が低い部位では規則的な幾何学配列を示した。後者ではリン調節に関するFGF23の発現が高く機能的な骨細管系を構築していると考えられた。論文掲載済み(次頁) (3)PTHによってosteocytic osteolysis(骨細胞性骨溶解)が報告されていることから(Belanger,1967)、PTHを野生型マウス(骨改造あり)、また破骨細胞の存在しないc-fos^<-/->マウス(骨改造なし)に投与し、骨細胞骨溶解の有無とその微細メカニズムを解析した。その結果、野生型マウスでは骨小腔の拡大を示したが、c-fos^<-/->マウスでは骨小腔の拡大を示さなかった。また、野生型マウスでは骨細胞におけるsclerostinの産生が低下した。従って、PTH投与における骨細胞性骨溶解は、骨細胞のみで調節されているのではなく、sclerostinなどを介して骨芽細胞に作用し、骨芽細胞・骨細胞系として機能することが示唆された。本研究は新知見が多く、引き続き、検索を行っている。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2006年 -2007年 
    代表者 : 前田 健康, 網塚 憲生, 井上 佳世子, 河野 芳朗
     
    我々はこれまで歯根膜ルフィニ神経終末がTrkBが発現し、そのリガンドであるBDNFならびにNT-4/5がこの機械受容器の発生、成長、再生に深く関与していることを明らかにしてきた。しかしながら、ルフィニ神経終末の成熟過程に関する分子については明らかにされていない。そこで神経栄養因子の一つであるGDNFの発現変化ならびに動物性レクチンの一つであるgalectinの局在について検討した。 1.三叉神経節ではGDNF、GFRα1、RetのmRNAの発現が確認された。免疫染色の結果、約30%の神経細胞がGFRα1およびRET陽性を示し、画像解析法により、それらは中型の神経細胞であることが明らかになった。しかしながら、GDNFは一部の衛星細胞のみに認められるのみで、神経細胞はGDNF陰性であった。 2.歯根膜ではこれら3つの分子が終末シュワン細胞に発現していたが、ルフィニ神経終末の軸索ではGDNF陰性であった。またルフィニ神経終末はgalectinの反応を欠いていた。 3.生後3日ではこれら3つの分子の発現は認められなかったが、生後1週でGFRα1陽性を示すようになり、生後2週になると一部の軸索がRet陽性となるとともにGDNF陽性終末シュワン細胞が出現し、ルフィニ神経終末が急激に形成される生後3週ではGDNF陽性終末シュワン細胞の数が増加した。 4.GDNF陽性シュワン細胞は下歯槽神経切断後、2週後から出現したが、その形態は紡錘形であり、成熟組織で見られる円形を呈する終末シュワン細胞は術後3週以降に観察された。 これらの時期特異的なGDNFの発現はGDNFがルフィニ神経終末の成熟・維持過程に深く関与していることを示している。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2005年 -2006年 
    代表者 : 河野 芳朗, 前田 健康, 網塚 憲生, 井上 佳世子
     
    歯根上皮鞘は歯根象牙質形成及び歯周組織の発生に大きく関与していることが知られている。この歯根上皮鞘の運命については2つの考え方が提唱され、1つは、アポトーシスによって消滅するという考え方であり、もう1つは、上皮-間葉形質変換によって、セメント芽細胞に変異するという考え方である。上皮細胞の間葉系細胞への形質転換は発生段階のミュラー管、二次口蓋の正中縫合部において報告され、また病的環境ではガン細胞の侵襲性転移性細胞への変換において認められる。 これまでの歯根上皮鞘に関する報告では、(1)上皮細胞の細胞内小器官が変化すること、(2)セメント芽細胞に象牙芽細胞のような段階的な分化途上の細胞が認められない。また、近年、培養ヘルトビッヒ上皮鞘の上皮細胞が表現型を変化させ、間葉系細胞特有の遺伝子発現をするように形質転換することを報告されている。これらの所見は、いずれも歯根上皮鞘細胞がセメント芽細胞に分化するという上皮-間葉形質転換説を支持する。しかしながら、上皮鞘細胞の時空間的挙動については全く理解されておらず、現在のところ、上皮-間葉形質転換説は仮説にすぎず、そのメカニズムも全く不明である。 これに関連して、様々なタンパクについて切歯歯根膜舌側上皮鞘とセメント芽細胞での発現を免疫組織化学的に検索した結果、ある種のヒートショックタンパク質が歯根象牙質に接触する、上皮細胞からセメント芽細胞まで連続的に発現する像が観察され、また、ラット切歯舌側セメント芽細胞に、これまで報告されていない、アクアポリン、S100タンパクが発現していることを明らかにした。この免疫染色結果は歯根上皮鞘上皮がセメント芽細胞に直接分化することを示唆するとともに、これらの新しく発現が明らかにされたタンパク質がセメント質の形成に重要な働きをすることが明らかにされた。
  • 日本学術振興会：科学研究費助成事業 萌芽研究
    研究期間 : 2005年 -2006年 
    代表者 : 網塚 憲生, 前田 健康, 井上 佳世子, 河野 芳朗, 織田 公光
     
    平成18年度では、17年度で作製した核小体型PTHrP(副甲状腺ホルモン関連ペプチド)トランスジェニックマウス(Tgマウス)の組織解析を行った。 胎生18日齢のTgマウスのgenotypingを挿入DNA特異的なprimerを用いて、核小体型PTHrP cDNAが過剰発現されているマウス個体を確認した。それらの大腿骨において、type II collagenを発現する骨端軟骨の静止層・増殖層では、PTHrPおよびmyc-tagは軟骨細胞の核および核小体に局在することが免疫組織学的に明らかにされた。このような、Tgマウス骨端軟骨を野生型マウスと比較すると、Tgマウスの静止層、増殖層、肥大化層の大きさは変わらず、骨端軟骨におけるPCNA陽性(増殖)軟骨細胞の比率、およびtype X collagen陽性肥大化層の領域も野生型マウスと比較しても違いが認められなかった。従って、核小体型PTHrPは、PTH/PTHrP受容体を介した作用とは異なり、軟骨細胞の分化増殖に著しい影響を及ぼさないと推測された。ところが、野生型マウスでは骨の長軸方向に平行な軟骨カラムが形成されるのに対して、Tgマウスの増殖層では、大きさおよび形状の異なる軟骨細胞が不規則に局在した。以前に報告したPTHrP欠損マウスでは軟骨細胞の形状や配列が不規則であったことを考え合わせると、軟骨細胞の形や配列性には核小体型PTHrPが関与する可能性が結論づけられた。本研究成果は平成18年度新潟歯学会第2回例会(平成18年11月)で報告するとともに論文作成中であり、平成19年度の日本骨形態計測学会、日本骨代謝学会にて発表予定(抄録提出済み)である。
  • 日本学術振興会：科学研究費助成事業 特別研究員奨励費
    研究期間 : 2004年 -2006年 
    代表者 : 網塚 憲生, LI MINQI, LI MinQi
     
    四肢短縮型小人症の一つである致死型軟骨無形成症(TD:thanatophoric dysplasia)の発症原因は変異型線維芽細胞増殖因子受容体III型(FGFR3)で誘導される骨・軟骨異常に起因することが知られている。これまでに、我々は軟骨細胞の分化段階に対するFGFR3の作用機序を副甲状腺ホルモン関連ペプチド(PTHrP)との比較で検討を行うと共に、ヒトTDサンプルの組織病理解析を行ってきた。しかし、これらは全て胎生期におけるFGFR3シグナルが軟骨・骨に及ぼす影響を検討したものであり、成人(成獣)を念頭に置いた解析が必要と考えた。そこで、我々はアメリカ・ワシントン大学・David Ornitz博士とカナダ・マッギル大学・Janet Henderson博士の協力を得て、成獣のFGFR3遺伝子欠損マウスの試料を関節軟骨および骨組織の異常を解析した。成獣期(生後4ヶ月)FGFR3遺伝子欠損マウスでは、関節軟骨における軟骨細胞の肥大化が若干亢進し、また、野生型マウスと比較すると骨量がやや増加するものの、骨基質の石灰化は低下傾向を示した。このことは、FGFR3シグナルの欠乏は、成獣期においても胎生期同様に軟骨細胞の肥大化への分化亢進、さらには骨芽細胞の増殖を促進するが、一方で、その成熟化を抑制するために骨基質石灰化が低下する可能性が推測された。さらに、FGFR3欠損状態では無秩序に増大した肥大化軟骨細胞の石灰化誘導が長軸方向に行われないため、軟骨内骨化部位における血管内皮細胞の軟骨侵入も抑制されていた。このように、FGFR3シグナルはヒト・マウス間、また、胎生期・成獣期ともに同じ作用を示すことが確認された。現在、本年度の成獣FGFR3マウスの所見結果をさらに発展させ、学会発表・論文化を進めている。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2004年 -2006年 
    代表者 : 山崎 和久, 網塚 憲生, 中島 貴子
     
    歯周炎は臨床的には結合組織破壊と歯槽骨の吸収で、免疫組織学的には多数のB細胞・形質細胞の浸潤で特徴づけられるが、多数のT細胞も認められる。我々はそれらの一部は自己抗原を認識し、自己反応性の応答が歯周炎の病態形成に重要な役割を演じていることを明らかにした。近年、形態的・機能的に異なる様々なT細胞サブセットが免疫応答の調節に複雑に関与していることが明らかになってきた。そこで、歯周疾患感受性の決定に深く関わっていると考えられる歯周炎局所のT細胞に関して免疫学的・分子生物学的手法を用いて網羅的に解析した。 その結果、免疫組織学的検索から歯周炎局所に浸潤しているCD4陽性のT細胞の一部はCTLA-4とCD25を発現していることが明らかになった。このCD4^+CD25^+CTLA-4^+T細胞はregulatory T細胞のphenotypeであり、B細胞の浸潤が強くなるのに伴って比率の上昇が見られた。また、遺伝子レベルではnaturalregulatory T細胞マーカーとされるFOXP3、1L-10,TGF-βの発現が歯周炎組織で上昇していた。さらに、別の制御性T細胞集団であるNKT細胞も歯周炎における免疫制御に関与し、その機能はB細胞上に発現するCDld分子によって制御されていることが示唆された。 さらに、局所に浸潤している細胞の機能を詳細に検討するため、歯周炎組織から多数のT細胞クローンを樹立し、それらの遺伝子発現とCD4^+CD25^-T細胞の増殖に及ぼす影響についても検索した。ほとんどのT細胞クローンはTh1,Th2いずれのサイトカインおよびマウスにおけるnatural regulatory T細胞のマーカー分子であるFOXP3遺伝子を発現していたが、骨吸収に関わるIL-17,RANKLについては患者間で発現しているT細胞クローンの頻度に差が見られた。 これまでの歯周炎局所のT細胞が発現する受容体遺伝子の解析からは歯周ポケット内細菌叢の複雑さを考慮すると比較的限られた種類の抗原を認識することがわかっていたが、同じ受容体レパートリーを持つT細胞が胸部・腹部動脈瘤の組織からも検出され、歯周炎と動脈硬化症の関連についても示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2005年 -2005年 
    代表者 : 小野 加津広, 神谷 貞浩, 網塚 憲生, 善本 隆之, 和田 誠基
     
    【目的】肝細胞増殖因子(HGF)は癌の増殖・浸潤・血管新生を促す。また骨芽細胞はHGFを産生するが、骨転移とHGFの関連は明らかではない。そこで我々はマウス乳癌細胞株、BALB/c-MC(BALB)を用いて骨転移へのHGF関与を検討した。 【方法および成績】癌細胞のマウスにおける骨転移組織(左心室腔への癌細胞投与による転移系)を評価すると腫瘍周囲基質にHGF発現がみられ、その部位に一致して血管新生の指標となるCD31陽性血管が認められた。詳細を検討するためにBALB、骨髄細胞および骨芽細胞の培養上清中のHGF濃度を測定するとBALB、および骨髄細胞では感度(0.4ng/ml)以下であったが、骨芽細胞では3.13±0.25ng/mlであり、この濃度は既報より血管新生を促進しうる濃度であった。以前の検討ではBALBを骨髄細胞と共存培養したとき、培養上清中のPGE_2濃度が上昇したので、培養系にPGE_2を添加すると骨芽細胞からのHGF産生は増加した。それに対してBALBおよび骨髄細胞においては依然として測定感度以下であった。5ng/mlのHGFを添加するとBALBの増殖は亢進し、HGF受容体であるc-MetのBALBにおける発現もWestern blotで確認された。浸潤能を評価するためBALBを基底膜モデルであるマトリゲルコートインサート上に播種し、下層にHGFを添加してインサート通過数を計測するとHGF添加はBALBの通過を3倍に増加させた。また下層に骨芽細胞を培養すると、BALBの通過は増加し、その効果はHGF阻害剤NK4で抑制され、さらにc-Met中和抗体添加でも抑制された。 【結論】骨芽細胞の産生するHGFはマウス乳癌細胞株、BALBの骨・骨髄への浸潤を促進し、さらに増殖を刺激すると考えられた。またHGFは転移巣での血管新生を誘導し癌の増殖・進展を促進すると考えられた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2004年 -2005年 
    代表者 : 前田 健康, 網塚 憲生, 河野 芳朗, 井上 佳世子
     
    我々は先に歯根膜機械受容器であるルフィニ神経終末の発生・再生過程に脳由来神経栄養因子(brain derived neurotrophic factor ; BDNF)が深く関与していることを明らかにした。BDNFの受容体であるTrkBはさらにneurotrophin-4/5(NT-4/5)と結合する。本研究ではNT-4/5遺伝子欠損マウス(ホモ型)と野生型マウスにおける歯根膜ルフィニ神経終末の発生・再生過程を免疫組織学的に検討した。 1.歯根膜ルフィニ神経終末における免疫組織化学と定量分析 ルフィニ神経終末の分布密度の経日変化は、両遺伝子型ともに経日的に神経分布密度は増加し、生後8週でほぼプラトーに達した。また野生型と比較してホモ型の神経分布密度は生後8週まで有意に低く、さらにホモ型マウスでは生後2週と3週間、生後3週と8週間で有意差があることを明らかにした。 2.TrkB, p75-NGFRのタンパクレベル,遺伝子レベルでの発現の変化 歯根膜,三叉神経節共に発現量の変化はなかった. 3.終末シュワン細胞の動態 両遺伝子型ともに非特異的コリンエステラーゼ染色パターンおよび細胞形に相違はなく,歯根膜単位面積に対する陽性細胞数の経時変化にも相違は認められなかった. 4.下歯槽神経切断におけるNT-4/5欠損マウス歯根膜ルフィニ神経終末の再生過程 観察期間を通してホモ型マウスが有意に低い値を示し,特に,術後3〜10日にかけて有意に低く,NT-4/5は再生過程の初期段階において重要な役割を果たしていることが示唆された. 以上のことより,NT-4/5は歯根膜ルフィニ神経終末の発生.再生過程の早期に関与することが明らかとなった.また,NT-4/5やBDNFのようなさまざまな神経栄養因子が時期依存的に歯根膜ルフィニ神経終末の発生・再生過程に関与していることが示唆された.
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2004年 -2005年 
    代表者 : 網塚 憲生, 前田 健康, 小澤 英浩
     
    骨・軟骨形成において重要な遺伝子である線維芽細胞増殖因子受容体III型(FGFR3)および副甲状腺ホルモン関連ペプチド(PTHrP)について、遺伝子欠損マウス、変異型PTH/PTHrP受容体蛋白、PTHrP産生腫瘍細胞の骨転移のモデル動物を用いて統合的な検索を行った。平成16年度には、FGFR3/PTHrP遺伝子二重欠損マウスに認められる軟骨・骨組織の異常について解析を行い、軟骨細胞の増殖に関してはPTHrPがFGFR3よりも優位に機能すること、しかしながら肥大化層への分化後は、アポトーシスならびに血管内皮増殖因子(VEGF)産生においてはFGFR3優位であることを明らかにした(Amizuka et al., Bone 2004、Best Paper Award、2005年国際骨代謝学会)。また、平成17年度では、骨組織への異常を解析し、骨芽細胞から分泌されるPTHrPがautocrine的に作用し骨芽細胞の増殖を正に調節する機構の解析を行った(Miao, Amizuka et al., J Clin Invest, 2005)。さらに、Jansen型およびBlomstrand型PTH/PTHrP受容体(PTH-R)といった変異型PTH-R蛋白の骨芽細胞における細胞内プロセッシングを解析した結果、変異型蛋白の小胞体への蓄積と細胞質内へのleakageならびにその後の分解を明らかにしている(下村、網塚ら:日本骨代謝学会2005年、論文作成中)。さらに、PTHrPは高カルシウム血症誘導物質であることから、PTHrP産生腫瘍細胞が骨転移を生じた場合の局所的な病理組織学的な知見についても検索している(Li, Amizuka et al., Micro Res.Tech,2005)。以上、FGFR3/PTHrP遺伝子の変異における軟骨・骨病変の統合的解析を行った。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2004年 -2005年 
    代表者 : 織田 公光, 天谷 吉宏, 網塚 憲生
     
    低ホスファターゼ症はヒト組織非特異型アルカリホスファターゼ(tissue-nonspecific alkaline phosphatase, TNSALP)遺伝子上の変異に起因する先天性骨代謝異常の疾患であり、多くは劣性形式で、まれに優性形式で遺伝することが知られている。2005年11月現在全世界で178の変異が報告されているが、これまで本疾患の分子病態機序に関する研究は数例ついて報告されただけである。筆者の研究室ではこの数年ミスセンス変異を中心に突然変異がTNSALP分子の生合成や触媒活性に与える影響を知る目的で、一連の細胞生物学的な解析を試みて来ている。本研究では(1)日本人患者にのみ高頻度で出現するcDNAの1559番目のチミンが欠損したフレームシフト変異(1559delT)と優性遺伝することが報告されている(2)TNSALP分子の99番目のアラニンがトレオニンに置換されたミスセンス変異例(A99T)について主に研究を行った。野生型及び変異を導入したcDNAを哺乳動物細胞に導入した一過性の発現系での解析に加えて、Tet-On細胞を用いた条件発現細胞系の樹立に成功し、より正常に近い発現レベルで変異型酵素分子と野生型分子との比較検討を行った。 (1)1559delT:in vitro翻訳系の結果と合わせて1559delTは読み枠の変更によりC末端に野生型よりも80アミノ酸残基延長を持つ大きな分子として合成されることが確認された。また、野生型酵素がグリコシルホスファチジルイノシトール(GPI)によって修飾されるのに対して1559delTはフレームシフトによりGPIで修飾されない活性を保持する可溶性のタンパク質として合成されるが、延長ペプチド上の3つのシステイン間のジスルフィド結合により高分子凝集体を形成することが原因で新生タンパク質のごく一部が分泌されずにすぎず、大部分は小胞体に蓄積後、ポリユビキチン化されて最終的にプロテアソームで分解されることが明らかになった。 (2)(A99T):野生型と同じく80kDaの成熟型分子としてGPIを介して細胞表面に発現するが、最大の特徴は活性を失っている点で、99番目のアラニンが触媒活性に重要な寄与をしていることが示唆された。また、野生型のポリペプチドとヘテロ2量体を形成することがわかり、この性質が優性遺伝する原因と考えられた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2003年 -2004年 
    代表者 : 河野 芳朗, 前田 健康, 網塚 憲生, 井上 佳世子
     
    アミロライド感受性上皮ナトリウムチャンネルはα、β、γの3つのサブユニットからなるアピカルメンブレンタンパクで、セルボリュームやメカノトランスダクションに関与していることが知られている。これらのサブユニットは腎臓の集合管や、大腸上皮に均等に共発現している。そこで、我々は、ラット切歯の3つのステージ毎の、歯性上皮での発現をmRNAレベルで検索した。RT-PCRによる分析では、前期成熟期、後期成熟期、基質分泌期のエナメル器、に3つのサブユニットの発現がみられた。さらに、インサイチューハイブリダイセイションと免疫組織学的検索により、ラット切歯エナメル芽細胞層に特異的な発現がみられた。 熱ショックタンパクはストレス状態のみならず生理的状態においても、シャペロンとして発現している。Hsp90は正常状態において多くのトランスデゥーサーや、細胞周期、発生調節因子の経路において作用すると考えられている。我々は、ラット切歯を免疫組織学的手法により、Hsp90が歯性上皮に広く発現し、また、中間層細胞に強く発現していることを示した。さらに、新しく形成された象牙質上のセメント芽細胞にも、発現がみられた。 ソニックヘッジホッグは哺乳類ヘッジホッグファミリーの1つで、胎生発生や、器官発生に重要な役割をしている。Shhは、その拡散する範囲内に存在する細胞の行動や運命に影響を与える。そこで、我々は、ラット切歯におけるShhタンパクの局在を免疫組織学的に検索した。強いShh免疫陽性反応が重層化した中間層細胞と、そのすぐ下層の内エナメル上皮に観察された。またその免疫陽性反応は、エナメル芽細胞が分化するにつれて減弱した。さらに、Shh免疫反応は、内エナメル上皮下層から間葉に向かって段階的に局在していた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2002年 -2003年 
    代表者 : 前田 健康, 河野 芳朗, 網塚 憲生, 山田 好秋, 山村 健介, 井上 佳世子
     
    BDNF遺伝子欠損(ホモ型、ヘテロ型)マウスおよび野生型マウスを用い、歯根膜ルフィニ神経終末の終末形成状態、発達・再生過程について免疫組織化学的に検討した。得られた成果は以下の通りである。 1.野生型マウス歯根膜ではルフィニ神経終末が歯槽骨寄りの歯根膜に密に分布しており、これら神経終末は外形が不規則なI型神経終末と外形が円滑なII型神経終末の2種に分けられた。一方、BDNFノックアウトマウス(ヘテロ型)ではI型ルフィニ神経終末の低形成ならびに形成不全が認められた。このルフィニ神経終末は分岐状態が野生型のものより悪く、また軸索終末の膨隆も劣っていた。画像解析法による神経分布密度の検討ではヘテロ型の神経分布密度は野生型のものより18%低いことが明らかになった。 2.生後1から3週間までのホモ型、ヘテロ型、野生型マウスの切歯歯根膜を検討したところ、野生型とヘテロ型における神経密度に差は認められなかったが、ホモ型の神経密度は低い値を示した。生後3週のホモ型マウスではルフィニ神経終末をほとんど観察することはできなかった。 3.下歯槽神経切断モデルを用い、BDNF遺伝子欠損マウスヘテロ型(+/-)の歯根膜ルフィニ神経終末の再生過程を免疫細胞化学的に検討したところ、BDNFの減少により歯根膜ルフィニ神経終末の再生遅延が引き起こされることが明らかとなり、歯根膜ルフィニ神経柊末の再生過程においてBDNFが重要な役割を果たしていることが示唆された。 本研究の結果から、歯根膜ルフィニ神経終末の再生過程ならびに生後発達期、特に成熟期、にBDNFが関与している可能性が示された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2002年 -2003年 
    代表者 : 永田 昌毅, 網塚 憲生
     
    私たちは変異Fgfr2が影響する主要な組織のひとつとして軟骨を仮説した。私たちはApert症候群型変異Fgfr2遺伝子(Apert-Fgfr2)を軟骨細胞に発現させたトランスジェニックマウスを用いた。それらのマウスは頭蓋底の成長点である軟骨縫合の早期癒合をきたし、頭蓋に限局した奇形を結果した。この奇形は軟骨縫合における軟骨細胞の肥大化の亢進、頭蓋底を形成する蝶形骨の前後的縮小と頭蓋底軟骨の厚みの減少を伴っていた。頭蓋に特徴的に現出する軟骨の異常の原因としてFGFファミリー分子の発現の特異性を仮定した。私たちは頭蓋の凍結切片からレーザーマイクロダイセクション(LMD)によって軟骨細胞を選択的に採取し、その遺伝子発現を検討した。その結果、Fgf2とFgf10が頭蓋の軟骨において特徴的に発現が見られた。 Apert-Fgfr2はこれらのFGF2とFGF1Oに対し親和性の増強が報告されており、トランスジェニックマウスでみられた頭蓋特異的奇形にFGFライガンドの分布が起因する可能性が示唆された。頭蓋底軟骨内におけるFGFR2シグナリング異常活性化がもたらす結果を分子レベルで解明することを目的にLMDとリアルタイムPCRを用いた遺伝子発現解析を進めている。これまでに肥大化軟骨におけるCbfa1、Ihh、MMP-13の亢進が示唆されており、今後これらの結果をより詳細に検証することが必要である。以上より頭蓋の形態形成と発育においてFGFR2シグナリングが重要な役割を果たすと考えられる。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2001年 -2003年 
    代表者 : 江尻 貞一, 池亀 美華, 網塚 憲生, 河野 正司, 田中 みか子
     
    ヒト顎骨の骨粗髭化の病態を把握し、治療・予防法を確立することを目的として、卵巣を摘出したサルの顎骨についてpQCT及びマイクロCTを用いて骨量変化と骨構造変化を解析しhPTH(1-34)の間欠投与効果も検索した。 【平成13-14年度】雌カニクイザル12匹を2群に分け、卵巣摘出術(OVX)または偽手術(Sham)を施し、術後78週目に屠殺した。下顎骨を摘出後、pQCT及びマイクロCTにて、右下顎第1大臼歯遠心根を通る前頭断面を観察し、画像解析装置を用い海綿骨および皮質骨の2次元的構造解析を行った。また顎骨の3次元構築像を作成し、骨構造変化と皮質骨中の管腔構造の解析も試みた。 【平成14-15年度】雌カニクイザル12匹を用いた。動物を3匹ずつ以下の4群に分け実験した。1群:Sham手術+溶媒週1回投与群,2群:OVX+溶媒週1回投与群,3群:OVX+hPTH(1-34)1.26μg/kg週1回投与群,4群:OVX+hPTH(1-34)6μg/kg週1回投与群とした。術後52週目に屠殺し、下顎骨を摘出。マイクロCTと画像解析装置を用いて解析し、Sham群、OVX群間で比較検討した。 【結果と考察】卵巣摘出したサル顎骨では、エストロゲン欠乏により、海綿骨量が減少するとともに皮質骨中を近遠心方向に走る管腔が拡大する事によって粗髭化が生じる事が明らかとなった。hPTH(1-34)1.26μg/kg週1回投与あるいはhPTH(1-34)6μg/kg週1回投与処置で、骨梁は増加する事が示された。その効果は1.26μg/kg週1回投与より6μg/kg週1回投与がより有効であった。しかしながら皮質骨中の血管腔の増大を阻止する効果は認められず、低濃度ではエストロゲン欠乏によって生じる皮質骨の粗髭化を逆に助長してしまう可能性も示唆された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2001年 -2003年 
    代表者 : 網塚 憲生
     
    先天性骨格異常である軟骨無形成症と軟骨異形成症は、線維芽細胞増殖因子受容体III型(FGFR3)あるいは副甲状腺ホルモン関連ペプチド(PTHrP)の恒常的なシグナル伝達で発症することが明らかにされている。そこで、軟骨細胞の増殖分化に対するFGFR3とPTHrPの作用を解析した。一方で、PTHrPには核小体移行シグナルが存在し、受容体を介さず細胞の核に移行する可能性も考えられる。以上、軟骨内骨化に関与する因子であるPTHrP、FGFR3を中心に組織学的な解析を行った。 (1)胎生18日齢におけるFGFR3^<-/->/PTHrP^<-/->マウスは四肢の発達が悪く、PTHrP^<-/->マウスの骨格異常に類似した像を示した。特に、骨端軟骨の低形成、ならびに軟骨細胞の増殖抑制を示したことから、軟骨細胞の増殖に対してPTHrPはFGFR3よりも優位に機能するか、機能的に下流に位置することが考えられた。しかしながら、PTHrP^<-/->マウスで観察された軟骨細胞のアポトーシスは、FGFR3^<-/->/PTHrP^<-/->マウスでは僅かにしか認められず、また、VEGFの発現もPTHrP^<-/->マウスでは肥大化軟骨細胞に一致してその発現が認められたのに対して、FGFR3^<-/->マウスおよびFGFR3^<-/->/PTHrP^<-/->マウスではVEGFの発現が低下していた。このことから、FGFR3は軟骨細胞の増殖に対してはPTHrPの上流で機能するが、肥大化期以降の軟骨細胞に対してはFGFR3独自の作用を有することが示唆された。 (2)PTHrPの多くはPTHと共通の受容体を介して作用すると考えられるが、一方でPTHrPは核小体移行シグナルを有している。PTHrPの核小体移行のメカニズムは開始メチオニンをコードするAUGと下流に存在するロイシンをコードするCUGからの翻訳によるものであることが明らかにされた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2000年 -2002年 
    代表者 : 前田 健康, 藤井 規孝, 網塚 憲生, 山本 仁, 大島 勇人, 田口 直幸
     
    1.チタン薄膜上での神経細胞の生存に関する研究 チタン薄膜上で、PC12細胞を培養した。培養液中にNGFを添加すると、PC12細胞はチタン薄膜上で神経突起を伸展させたが、これらの細胞はチタン表面から容易に剥離した。このことはチタン薄膜とPC12細胞は接着する可能性が低いことを示唆した。 2.チタンインプラント植立に対するラット上顎骨組織の組織反応 ラット上顎骨インプラント植立モデルを用いて、チタンインプラント周囲における新生骨形成過程をインプラント骨窩洞界面の間隙の差異について、微細構造学的、酵素組織化学的に検討した。顎骨におけるインプラント植立時の間隙の違いによる詳細な新生骨形成過程が明らかとなった。新生骨による骨性結合が達成されるためには、適度な間隙が必要であることが示唆された。 3.インプラント上皮の再生 インプラント周囲上皮の再生過程を組織学的に観察したところ、インプラント窩洞形成により破壊された口腔粘膜上皮は、植立後15日までに正常ラット接合上皮と同じような細胞形態、角化様式を示した。 4.インプラント周囲上皮内神経線維の再生 植立5日まではインプラント周囲上皮に神経線維は全く観察されなかった。植立後10日になると、上皮基底部付近にPGP9.5およびCGRP陽性神経線維が集積はじめ、植立後15日で正常接合上皮同様に神経線維が上皮層内に進入していた。骨性結合が獲得された20-30日では神経線維の再生は終了したように思えたが、その分布密度は正常のものより低いことが明らかとなった。 5.表面性状の相違が骨性結合獲得に及ぼす影響 インプラント体表面の形態は骨形成過程に影響を及ぼさないことが明らかとなった。一方、表面性状により、骨形成速度の著名な変化は認められなかったことから、表面の材質の違いにより骨形成の方向が異なることが明らかとなった。
  • 日本学術振興会：科学研究費助成事業 基盤研究(A)
    研究期間 : 1999年 -2000年 
    代表者 : 小澤 英浩, 星 和人, 網塚 憲生, 江尻 貞一, 池亀 美華, 中村 浩彰
     
    骨の形態は、構成細胞である骨芽細胞、骨細胞と破骨細胞のバランスや、軟骨、血管、神経、腱などの周囲組織との協調関係によって維持されると推測されているものの、年齢や環境、あるいは種差によって骨組織の形態が多様化する機構は未知である。本研究では、骨形態の制御機構を明らかにする事を目的として、(1)石灰化骨基質の形成・維持機構を微細形態学的、組織細胞化学的に分子レベルで解析し、さらに(2)加齢現象、内外の環境変化、種差による形態変化あるいは分子細胞学的変化を検索した。 (1)に関しては、類骨においてコラーゲン細線維の間隙にプロテオグリカンの網目構造が観察され、カルシウムはプロテオグリカンの領域に、リンはコラーゲン細線維の領域に、相対的に集積していることが明かとなり、それぞれの構造がカルシウムおよびリンを保持し、カルシウム・リン酸イオン積の上昇を抑制する事により、未石灰化状態を維持しているものと思われた。一方、石灰化球ではカルシウムとリンの両者が共在化しており、カルシウム・リンの共在化機構が石灰化の進行に重要な役割を果たしていることが明らかとなった。 (2)に関しては、骨は、エストロゲンなどの性ホルモン、副甲状腺ホルモンなどのカルシウム調節ホルモン、骨形成因子などの増殖因子、あるいは張力刺激などの力学的因子など、様々な因子による形成性あるいは吸収性の作用を受け、合目的に骨形態変化させて内外の環境に対応することが明らかとなった。とくに張力刺激に関しては、骨芽細胞分化促進作用の機序として、同部位の骨膜細胞および前骨芽細胞におけるBMP4遺伝子発現の増加が関与していることが示唆された。また、種差に伴う骨形態変化に関しては、魚類であるキンギョの咽頭歯および咽頭骨をμCTで評価し、その咽頭歯および咽頭骨の特異性を明らかにすることができた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(A)
    研究期間 : 1999年 -2000年 
    代表者 : 小澤 英浩, 網塚 憲生, 入江 一元, 江尻 貞一, 小石川 篤, 菊池 一夫
     
    生体の同一被検体における骨密度の定量解析と3次元的骨形態の解析を経時的に可能とする高分解能マイクロフォーカスX線CTの開発・改良を目的とし次の様な研究を行った。【撮影条件と被爆線量と画質限界のトレード・オフの見極め】基準画像撮影時の被爆線量は従来比1/16〜1/50に低減したが、被爆線量目標値の110μSVとは12〜35倍の乖離があった。被爆線量をさらに軽減するため高感度型検出器を再検討した結果、高解像度(640×480ピクセル→2048×2048ピクセル)高濃度階調(10ビット→12ビット)検出器(デジタルフラットパネル)の採用を試み、撮影条件と撮影時間のトレードオフのテストを実施した。【ワーク固定型(X線管と検出器回転型)のX線CT装置の開発】被検体搭載台サイズ600mm×1000mmで、イヌ、サル、ウサギ等の生体を対象とする大型動物生体用X線マイクロCT装置の基本設計を完了した。【高分解能マイクロフォーカスX線CT用の3次元骨形態計測画像解析システムの構築】撮影時間の短縮化と3次元CT画像再構成の高速化を実現する為に、新たに高速CT3次元再構成方式(コーンビーム方式)を開発し、それを3/1.8インチ高感度検出器を搭載した骨組織無観血的高分解能形態解析システムに取り入れた。【骨体積密度(皮質骨&海綿骨)の測定と評価システムの確立】皮質骨等価物質よりなる体積密度既知のファントムと海綿骨等価物質よりなる体積密度既知のファントムを複数組み合わせることにより、皮質骨が海綿骨CT値に与える影響を定量的に数式化することが可能となり、海綿骨部の絶対体積密度を正確に得ることができた。【新規開発した高分解能マイクロフォーカスX線CT装置の応用】卵巣摘出によって生じるサルの顎骨の変化、ビタミンD投与したマウスの骨格の変化、Gli遺伝子欠損マウスの骨格異常などを明らかにし、日本骨代謝学会、アメリカ骨代謝学会などにおいて報告した。
  • 日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1999年 -2000年 
    代表者 : 網塚 憲生
     
    平成12年度の目的として、線維芽細胞増殖因子レセプターtype III(FGFR3)遺伝子欠損マウス、ならびにGli-Krupper familyを構成する転写調節因子であるGli遺伝子の欠損モデルマウスを用いて骨・軟骨形成を検索した【平成12年研究計画1】。また、軟骨の増殖分化に関連する副甲状腺ホルモン関連ペプチド(PTHrP)の核小体移行に関する細胞生物学的検討を加えた【平成12年研究計画2】。その結果、FGFR3マウスでは、軟骨細胞の増殖亢進と軟骨・骨移行部における血管侵入の抑制が認められ、特に、後者は血管内皮増殖因子(VEGF)を介していることを報告した(第22回アメリカ骨代謝学会;Acta,Anat.Nipponica,Amizuka,et al.,2000;Histol.Histopath,Amizuka et al.,2000.)。さらに、Gli3遺伝子欠損マウスでは多趾症・多指症とともに、発達の悪い脛骨が観察された。組織学的に、軟骨内骨化に携わる骨原生細胞が過剰なアポトーシスを生じており、アポトーシス誘導因子として作用するBMP-2を介することが明らかとなった(第22回アメリカ骨代謝学会、第17回日本骨代謝学会、網塚ら)。さらに、PTHrPの核小体移行については、開始メチオニンをコードするAUGコドンばかりでなく下流のCUGコドンからのalternative translationが認められること、またその際に、PTHrPタンパクは核小体に移行することを明らかにした(Biochem.Biophys.Res.Commun.,Amizuka et al.,2000)。以上の研究成果は、本来計画した内容を充分満足しており、そのほとんどが論文に受理中である。また、本研究によって日本骨代謝学会学術賞、歯科基礎医学会学会賞を受賞した。
  • 日本学術振興会：科学研究費助成事業 萌芽的研究
    研究期間 : 1999年 -1999年 
    代表者 : 小澤 英浩, 網塚 憲生, 江尻 貞一, 中村 浩彰, 入江 一元
     
    本年度は、電子エネルギー損失分光電子顕微鏡(EELS)の運用に改良を加え、今までなし得なかったÅレベルの元素の局在解析法と高分解免疫電顕法を開発し、生物学的石灰化におけるコラーゲン性石灰化の機序を解明することを目的として、EELSとイメージングプレートとを組み合わせた分析電顕システムを用いてコラーゲン性石灰化機構を解析した。 実験試料には、胎生期ラット頭頂骨を液体ヘリウムで急速凍結し、さらに凍結置換を行ったものを用いた。この試料を超薄切して、EELSで観察し、微細形態の検索や、元素分析、オステオネクチンの免疫電顕等を行った。 胎生期頭蓋骨では、基質小胞内での結晶析出から、石灰化の形成、コラーゲン性石灰化による広範な石灰化基質の確立にいたる一連の過程が観察できる。石灰化球では石灰化結晶の集積が観察されるが、結晶表面には、酢酸ウラン・クエン酸鉛の電子染色で染色される幅約1nmの結晶鞘が存在しており、その部位には、Nの明らかな集積は見られなかったことから、結晶鞘は比蛋白性成分から構成されていることが示唆された。このように、免疫電顕では物質の局在同定は困難である微細な構造においても、この電顕を用いた分析では、組成に関する情報がえられることが明らかとなった。 さらに、この電顕を用いた高分解能免疫電顕では、石灰化球とコラーゲン細線維の境界部にオステオネクチンが集積することが明らかとなり、オステオネクチンがコラーゲン性石灰化に重要な役割を果たすことが示唆された。
  • 日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1997年 -1998年 
    代表者 : 網塚 憲生
     
    平成10年度では、骨代謝および骨形態形成に重要な副甲状腺ホルモン関連ペプチド(PTHRP)および線維芽細胞増殖因子(FGF)とそのレセプターに対する微細構造学的、分子細胞生物学的検索を行った。今回の研究成果では、PTH/PTHRPレセプター遺伝子には上流と下流のプロモーターが存在し骨・軟骨組織では下流のプロモーターが機能すること、さらには骨芽細胞の下流プロモーターは活性型ビタミンDにより抑制されるが、軟骨細胞では制御されないことを、in situ hybridizationやPTH/PTHRPレセプター抗体を用いた免疫組織化学、さらにはRT-PCRやWestern blottingを用いて明らかにした。また、PTH/PTHRPレセプターの132番目のプロリンがリジンに変異したレセプター遺伝子配列を明らかにし、それはPTH/PTHRPなどのリガンドが存在してもsignal transductionが伝わらないことを報告した。一方、FGFR3レセプターも重要な軟骨・骨の分化増殖因子であることから、FGFR3(-/-)/PTHRP(-/-)二重遺伝子欠損マウスを作製して、PTHRP(-/-)欠損マウスとFGFR3(-/-)欠損マウスとの組織学的異常を検索した。その結果、FGFR3(-/-)/PTHRP(-/-)マウスは、PTHRP(-/-)マウスと同様に軟骨細胞の増殖抑制を示していることから、軟骨の分化増殖に関してはFGFR3よりもPTHRPが機能的に上位であることが強く示唆された。さらに、骨組織におけるFGFの作用を明らかにするために、ラット骨髄内にbFGFを投与し、組織変化を経時的に観察したところ、すでに1日目でアルカリホスファターゼ活性を示す骨芽細胞系細胞の増殖とそれに付随した骨芽細胞への分化や骨形成が認められた。以上から、PTHRP,FGFおよびそれらのレセプターは軟骨・骨に重要な分化調節因子であることが明らかにされた。
  • 日本学術振興会：科学研究費助成事業 基盤研究(A)
    研究期間 : 1996年 -1998年 
    代表者 : 小澤 英浩, 網塚 憲生, 中村 浩彰, 入江 一元, 江尻 貞一
     
    【新しい骨量計測法と骨組織形態計測法による骨組織の加齢変化の検索】レーザー顕微鏡用の骨形態計測解析ソフトを開発するとともに、μCTを骨形態計測に応用する事で、顎骨の骨構造解析とその骨量測定に成功し、顎骨加齢現象に関する基礎的研究システムを確立した。その解析結果から、顎骨においてもエストロジェン欠乏による骨粗髭化が生じることや、加齢に伴う全身性ホルモンの変化と咬合によるメカニカルストレスによって顎骨の骨構造維持改変が行われていることを明らかにした。【組織細胞化学的検索】ラット下顎頭ではII型コラーゲンを産生していた軟骨細胞が加齢とともに肥大化細胞が消失し、I型コラーゲンを産生する線維軟骨様組織に変化した。また加齢ラット、卵巣摘出(OVX)ラットの骨髄組織では、脂肪細胞が顕著に認められた。このような下顎頭軟骨の変化や骨髄間質細胞の変化から、骨組織の加齢現象は軟骨細胞、骨芽細胞細、破骨細胞の増殖・分化制御機構を介して発現している可能性が示唆された。【分子細胞生物学的検索】加齢、老化に伴う骨の細胞の遺伝子発現をin situ hybridizationにて検索した。OVXラットやPTHRP遺伝子欠損マウスではosteonectinやALPaseあるいはPTHRPやFGFなどとそれらのレセプターの遺伝子発現が抑制傾向を示すとともに、細胞分化の制御に伴いこれらの遺伝子発現にも変化が認められた。【骨誘導・骨形成促進実験】加齢に伴って生じる骨量減少を予防・改善することを目的として、PTHの間欠投与あるいはBMP-2を用いて骨の再建法を検討した。PTHを間欠投与すると、OVXラット下顎頭骨量の減少が予防されるとともに、減少した骨量をも回復させた。BMP-2を抜歯窩に投与すると、間葉系細胞の増殖亢進および骨芽細胞への分化が促進し多量の新生骨が形成された。またBMP-2とe-PTFE膜を併用すると、骨形成の困難な部位に骨形成が誘導され、その骨量が維持されるため、骨形態付与が可能である事が示された。
  • 日本学術振興会：科学研究費助成事業 萌芽的研究
    研究期間 : 1996年 -1997年 
    代表者 : 小澤 英浩, 網塚 憲生, 入江 一元, 中村 浩彰, 江尻 貞一
     
    骨形成蛋白(BMP-2)の生涯的役割を包括的に解明するために、昨年度はマウス成獣の腰椎脊柱靱帯にBMP-2を用いて異所性骨化を誘導する研究や、成獣ラット上顎の抜歯窩にBMP-2を投与する実験を行い、靱帯線維芽細胞がBMP-2の投与により線維軟骨様軟骨細胞を経て硝子軟骨様軟骨細胞へ分化し内軟骨骨化により骨へ置換される現象や、抜歯窩の骨形成が促進される事を明らかにした。 本年度は、BMP-2による骨形成現象に関わるサイトカインネットワークを解明する事が目標であったため、これらの実験系を用いてBMP-2やTGF-βなどのリガンドやレセプターを免疫組織細胞化学的に検索した。その結果、BMP-2により誘導される骨化現象において、BMP標的細胞におけるBMP receptorのup-regulationや、TGF-βのauto crine/paracrineによる分化誘導が起こる事が明らかとなった。 骨形成蛋白が肢芽発生にも重要な役割を果たすという従来の報告も考えあわせると、骨形成蛋白は他のサイトカインとも密接な連係を保ちながら生涯にわたり骨の形成や再生、組織の形態維持に重要な役割を担っていると思われた。 これらの結果および関連所見は、国際雑誌Bone Acta Hist ochemi ca et Cytochemica Histochemistry and Cell Biology等で出版された他、第102回日本解剖学会、第15回日本骨代謝学会、第18回米国骨代謝学会、第12回日本整形外科基礎学術集会、平成9年度新潟歯学会第1回定例会等で公表し、高い評価を受けた。
  • 日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1996年 -1996年 
    代表者 : 網塚 憲生
     
    我々は、副甲状腺ホルモン関連ペプチド(PTHRP)およびそのレセプターに対する一連の分子細胞生物学的検索を行い、平成8年度中に関連論文で国際雑誌に約10編を報告するとともに、国際および国内学会において活発に活動している。従来我々は腫瘍関連タンパクとして発見されたPTHRPが、正常組織の発生に重要な役割を果たすタンパクであることを明らかにし、特に軟骨や骨組織の形態形成および細胞の分化増殖におよぼす作用を分子細胞生物学的に検索してきた。前年度の主だった研究を挙げると、遺伝子組み替えにより、PTHRPの相同遺伝子を一部削除したHeterozygousミュータントマウスを検索し、個体成長に伴いその異常が蓄積し著明になること、さらにPTHRPやレセプターの局在や遺伝子発現を明らかにしている(Amizuka et al.Deve.Biol.)。またPTHRP欠損Homozygousマウスでは軟骨の発達が著しく阻害されるばかりでなく、軟骨細胞のアポトーシス(細胞死)が促進され、軟骨から骨への置換部位で軟骨内骨化が抑制されていることを報告している(Amizuka et al.,Endocrinology)。PTHRPに対するレセプターであるPTH/PTHRPレセプターでについても検索しており、軟骨細胞と骨芽細胞および腎臓の細胞を用いて、PTH/PTHRPレセプターの遺伝子発現における上流と下流のプロモーターの機能についてin situ hybridizationを用いて解明している(Amizuka et al.,Endocrinology)。また、PTHRPは本来、腫瘍にて発見されたことから、腫瘍細胞におけるPTHRPが細胞増殖にも関与するとともに、細胞内プロセッシングにも関与していることを明らかにしている(Bin et al,Int.J.Cancer,Sidler et al.,J.Clin.Endcorinol.Metab.)。
  • 日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1995年 -1995年 
    代表者 : 網塚 憲生
     
    副甲状腺ホルモン関連ペプチド(PTHRP)は悪性腫瘍に併発する高カルシウム血症の起因物質として発見された。PTHRPは病理的には腫瘍細胞から産生される副甲状腺ホルモン(PTH)様の物質であり、そのアミノ酸配列においてはN末端13個のアミノ酸がPTHと高い相同性を示している。1991年にクローニングされたPTHレセプターはPTHRPとも結合し(PTH/PTHRPレセプター)、PTH様の生物学的活性を示すことが明らかにされている。ところが、PTHRPは正常組織、特に胎生期に多く産生されていることから、PTHRPは単に血清カルシウム調節因子ではなく、個体発生に重要な役割を果たす因子であることが考えられる。近年、gene targeting法によりPTHRP遺伝子欠損マウスが作製された。我々はこれらPTHRP遺伝子欠損homozygousマウスの骨端軟骨を形態学的に検索してきた(Amizuka et al.,J.Cell Biol.,1994)。これらマウスでは軟骨細胞の増殖と分化の抑制が認められる他、早期に細胞死に移行する軟骨細胞が存在することが明らかにされた。このことから、軟骨細胞の細胞死とPTHRPとの関係を明らかにする目的で、培養軟骨細胞にPTHRPcDNAをtransfectionして無血清培地で培養したところ、正常細胞に比べてPTHRPを過剰発現する軟骨細胞が長期間生き延びることが明らかにされた(Henderson,Amizuka,et al.,Mol.Cell Biol.,1995)。さらに我々は、PTHRPが成獣期においても同様な生物学的作用を有するか否かを解明する目的で、single geneを有する3カ月齢のheterozygousマウス(+/-)を用いて検索した。heterozygousマウスは血清カルシウムやPTH濃度は正常であるが、骨端軟骨における軟骨細胞の細胞増殖とカラム形成が悪く、それに続く骨組織の骨梁の形成もわずかに抑制されていた(Amizuka et al.,Dev.Biol.,1996)。このことから、PTHRPは成獣期においても同様に軟骨細胞の増殖および分化を調節する因子であることが推測される。
  • 日本学術振興会：科学研究費助成事業 一般研究(A)
    研究期間 : 1994年 -1995年 
    代表者 : 小澤 英浩, 網塚 憲生, 中村 浩彰, 江尻 貞一, 入江 一元
     
    本研究では、まず非脱灰硬組織試料作製のための基本設備(薄切器、研磨器、ソフッテクス)と本研究の中核機種である共焦点レーザー顕微鏡に励起波長、マイクロボクセル、形態計測システムを追加することにより、3次元的組織・細胞学的形態計測法の開発と、個体レベルから細胞レベルに及ぶ硬組織代謝の形態変化を定量的に捉えることを目標に、以下の研究を行った。(1)顎骨の老化現象など骨粗鬆症を主体とした骨組織の生理的・病的組織変化を共焦点レーザー走査顕微鏡を用いて検索し、その画像データをミクロ画像情報処理システムによりディジタル化して集積した。(2)ファイリングされたディジタル画像データを用いて、顎骨形態計測定量化解析ソフトの開発と定量解析後のデータ処理システムの開発を試み、以下の解析項目についてLuzex-F(ニレコ社製)画像解析装置にて半自動解析を可能にした。:(1)全骨梁面(2)全形成面(3)活性形成面(4)全吸収面(5)活性吸収面(6)全骨量(7)全類骨量(8)全骨組織量(9)線維組織量(10)1重標識面(11)2重標識面(12)2重標識面(13)類骨層幅(14)均骨梁単位幅(15)休止面(16)。低石灰化骨量(3)レーザー顕微鏡から連続的に取り込んだテトラサイクリン・カルセイン標識未脱灰骨標本の光学的切片情報を、本システム7用い解析するとともに、その連続断面画像情報をmicrovoxel (INDEC Systems,Inc.)およびLuzex-III(ニレコ社製)にて立体構築を試みた。その結果、microvoxelを用いることにより、テトラサイクリン・カルセインで標識されている領域を研磨切片の表面から深さ方向100μまで立体的に再構築するとともに、その体積をもとめることが可能となった。(4)surface modelingソフトであるLuzex-III(ニレコ社製)を用いることにより、破骨細胞や骨芽細胞などの細胞成分と標識骨面などを同時に立体的に構築することが可能であることが示された。これらの方法を用い、(1)ラット下顎頭の加齢変化に関する形態学的、免疫組織化学的研究(2)ラット歯槽骨における加齢変化に関する組織学的、組織化学的研究(3)骨形態計測法による副甲状腺ホルモン(PTH)の骨代謝に対する作用の検討-ラットにおける骨の部位による効果の比較-(4)卵巣摘出ラットにおける骨梁減少様式に関する微細構造学的研究:等の骨代謝基礎的研究を行い学会論文等にて発表した。
  • 日本学術振興会：科学研究費助成事業 試験研究(B)
    研究期間 : 1994年 -1995年 
    代表者 : 小澤 英浩, 藤井 和博, 網塚 憲生, 中村 浩彰, 江尻 貞一, 入江 一元
     
    本研究は研究室で増大多用化するミクロ画像情報に対して、一時保存、永久保存、集中管理、報告書作成、画像解析処理を含めたミクロ画像情報処理システムを開発することにより、ミクロ画像情報の検索から画像解析処理、Desk Top Publishing (DTP)により報告書作成、高精細印字出力による電子写真化までを一元的に扱い、研究業務の能率化、研究画像データの共同利用とデータ蓄積の一元化を指向することを目的として計画された。さらに画像データをデジタル画像として格納することにより、画像劣化のない画像情報を保存でき、最新の情報処理技術と融合させ、保存されたデータを過去に遡って検索、印刷出力、利用することができるシステムの構築を試みた。実際に構築したシステムは、画像を含めた報告書を集中管理する画像ファイリング部を中心に、レーザー顕微鏡、光学顕微鏡、EM902電子顕微鏡の画像データを入力するインターフェース部と、画像編集機能と高精細印字機能による報告書作成、学会投稿原稿作成等をサポートするDTP部、および画像処理部から構成されている。画像データの入力方式としてはCCDカメラより光学顕微鏡画像および透過原稿あるいは反射原稿の画像情報をデジタル画像インターフェース部を介して入力し、またレーザー顕微鏡画像およびEM902電子顕微鏡画像は光磁気ディスクを媒体として本システムに入力する構成とした。画像データのファイリングは画像処理部内のハードディスクおよび画像処理部に接続された光磁気ディスクを使用することとした。またファイリングされた画像データの出力としては高精細プリンタおよび新規開発したRGB切り替え器を介して既設のFR-3000フィルムレコーダーに接続し使用することとした。さらに我々は、構築したミクロ画像処理システムを用い、1.共焦点レーザー走査顕微鏡を応用した歯牙組織の微細構造学的観察 2.ラット下顎頭の加齢変化に関する形態学的・免疫組織化学的研究 3.ラット歯槽骨の生理的加齢変化及び矯正力に対する組織反応の加齢変化に関する細胞学的、組織化学的研究 4.実験的歯周炎における歯槽骨吸収に関する細胞化学的研究 5.ラット切歯エナメル芽細胞におけるH^+-ATPaseと炭酸脱水酵素の局在性について-免疫組織、細胞化学的研究等の基礎研究を行い学会論文等にて発表した。
  • 骨組織と腎におけるPTH/PTHRPレセプターの局在
  • 骨芽細胞の分化過程におけるPTH/PTHRPレセプターとFGFレセプターの発現について
  • 骨組織におけるPTHrPの生物学的作用に対する形態学的検索
  • Localization of PTH/PTHRP receptor in bone and kidney
  • Gene expression of PTH/PTHRP receptor and FGF receptor during osteoblast differentiation
  • Morphological assessment for biological note of PTHRP in bone tissues

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