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　　　　　　　　　　　　　　　
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　　　　　　　　　　　　　　　
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
網塚 憲生, 日本国

2003年, 日本骨粗鬆症学会研究奨励賞　　　　　　　　　　　　　　　
日本国

2000年, 歯科基礎医学会学会賞　　　　　　　　　　　　　　　
日本国

2000年, 日本骨代謝学会学術賞　　　　　　　　　　　　　　　
日本国

1996年, 日本骨代謝学会奨励賞　　　　　　　　　　　　　　　
日本国

1996年, William Geiz Award　　　　　　　　　　　　　　　

1993年, 若手研究者賞(Young Investigator Award)　　　　　　　　　　　　　　　

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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
日本語

DAP12/TREM2シグナルは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, 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
日本語

EBAG9-deficient mice display decreased bone mineral density with suppressed autophagy.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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
日本語

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
日本語

Histochemical assessment of osteoclast-like giant cells in Rankl-/- mice.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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, 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
日本語

Phosphorylated pullulan promotes calcification during bone regeneration in the bone defects of rat tibiae.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical assessment of accelerated bone remodeling and reduced mineralization in Il-6 deficient mice.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Morphological variety of capillary ends invading the epiphyseal plate in rat femora using scanning electron microscopy with osmium maceration.
Tsuneyuki Yamamoto, Shigeru Takahashi, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
Journal of oral biosciences, 64, 3, 346, 351, ELSEVIER, 2022年09月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Matrix Vesicle-Mediated Mineralization and Osteocytic Regulation of Bone Mineralization.
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, MDPI, 2022年09月01日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Deletion of Tfam in Prx1-Cre expressing limb mesenchyme results in spontaneous bone fractures.
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, SPRINGER JAPAN KK, 2022年08月10日, ［国内誌］
英語, 研究論文（学術雑誌）, 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
日本語

Tmem174, a regulator of phosphate transporter prevents hyperphosphatemia
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, Springer Science and Business Media LLC, 2022年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Bone biopsy findings in patients receiving long-term bisphosphonate therapy for glucocorticoid-induced osteoporosis.
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, SPRINGER JAPAN KK, 2022年03月25日, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Author Correction: Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-κB, ERK and JNK signaling pathways.
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, NATURE PORTFOLIO, 2022年03月02日, ［国際誌］
英語

Oral biosciences: The annual review 2021.
Hayato Ohshima, Kenji Mishima, Norio Amizuka
Journal of oral biosciences, 64, 1, 1, 7, ELSEVIER, 2022年03月, ［査読有り］, ［招待有り］, ［国際誌］
英語, 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.

Deterioration of Bone Quality in Rats with Glucocorticoid-Induced Osteoporosis Results in a Reduction in Cortical Bone Strength
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, WILEY, 2022年02月
英語

Characterization, pharmacokinetics, and pharmacodynamics of anti-Siglec-15 antibody and its potency for treating osteoporosis and as follow-up treatment after parathyroid hormone use.
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, ELSEVIER SCIENCE INC, 2022年02月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Morphological Assessment of the Biological Effects of Eldecalcitol
Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Norio Amizuka
Osteoporotic Fracture and Systemic Skeletal Disorders, 413, 430, Springer Singapore, 2021年12月
論文集(書籍)内論文

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
日本語

骨補填材としてのリン酸化プルラン/β-TCP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
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
日本語

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
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
日本語

Comparative immunolocalization of tissue nonspecific alkaline phosphatase and ectonucleotide pyrophosphatase/phosphodiesterase 1 in murine bone.
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］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Altered immunolocalization of FGF23 in murine femora metastasized with human breast carcinoma MDA-MB-231 cells.
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, SPRINGER JAPAN KK, 2021年09月, ［査読有り］, ［国際共著］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Three-dimensional reconstruction of the Golgi apparatus in osteoclasts by a combination of NADPase cytochemistry and serial section scanning electron microscopy.
Tsuneyuki Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
Histochemistry and cell biology, 156, 5, 503, 508, SPRINGER, 2021年08月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Involvement of distant octacalcium phosphate scaffolds in enhancing early differentiation of osteocytes during bone regeneration.
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, ELSEVIER SCI LTD, 2021年07月15日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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
日本語

Vitamin K-Dependent γ-Glutamyl Carboxylase in Sertoli Cells Is Essential for Male Fertility in Mice.
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, AMER SOC MICROBIOLOGY, 2021年03月24日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Oral biosciences: The annual review 2020.
Hayato Ohshima, Kenji Mishima, Norio Amizuka
Journal of oral biosciences, 63, 1, 1, 7, ELSEVIER, 2021年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological observation on the initial stage of vascular invasion into the secondary ossification of murine femoral epiphyseal cartilage.
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, BIOMEDICAL RESEARCH PRESS LTD, 2021年, ［査読有り］, ［国際共著］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical characteristics on minimodeling-based bone formation induced by anabolic drugs for osteoporotic treatment.
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, BIOMEDICAL RESEARCH PRESS LTD, 2021年, ［査読有り］, ［国際共著］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Bone histomorphometric and immunohistological analysis for hyperostosis in a patient with SAPHO syndrome: A case report.
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, ELSEVIER, 2020年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Calcinosis cutis in self-healing dominant dystrophic epidermolysis bullosa.
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, WILEY, 2020年12月, ［査読有り］, ［国際誌］
英語

Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone.
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, SPRINGER, 2020年11月11日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Intermittent PTH Administration Increases Bone-Specific Blood Vessels and Surrounding Stromal Cells in Murine Long Bones.
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, SPRINGER, 2020年11月10日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Abaloparatide Promotes Bone Repair of Vertebral Defects in an Ovariectomized Rat Model of Osteoporosis
Akito Makino, Tomoka Hasegawa, Hideko Takagi, Yoshimasa Takahashi, Naoki Hase, Norio Amizuka
JOURNAL OF BONE AND MINERAL RESEARCH, 35, 236, 236, WILEY, 2020年11月, ［査読有り］
英語

Osteocytic Osteolysis in PTH-treated Wild-type and Rankl-/- Mice Examined by Transmission Electron Microscopy, Atomic Force Microscopy, and Isotope Microscopy.
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, SAGE PUBLICATIONS LTD, 2020年10月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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).

Frequent administration of abaloparatide shows greater gains in bone anabolic window and bone mineral density in mice: A comparison with teriparatide.
Akito Makino, Tomoka Hasegawa, Hideko Takagi, Yoshimasa Takahashi, Naoki Hase, Norio Amizuka
Bone, 142, 115651, 115651, ELSEVIER SCIENCE INC, 2020年09月18日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical assessment on the cellular interplay of vascular endothelial cells and septoclasts during endochondral ossification in mice.
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, OXFORD UNIV PRESS, 2020年08月20日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Siglec-15-targeting therapy protects against glucocorticoid-induced osteoporosis of growing skeleton in juvenile rats.
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, ELSEVIER SCIENCE INC, 2020年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Osteocalcin is necessary for the alignment of apatite crystallites, but not glucose metabolism, testosterone synthesis, or muscle mass.
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, PUBLIC LIBRARY SCIENCE, 2020年05月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Oral biosciences: The annual review 2019
Hayato Ohshima, Norio Amizuka
JOURNAL OF ORAL BIOSCIENCES, 62, 1, 1, 8, ELSEVIER, 2020年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Role of sodium-dependent Pi transporter/Npt2c on Pi homeostasis in klotho knockout mice different properties between juvenile and adult stages.
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, WILEY, 2020年02月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical Assessment Of Abnormal Mineralization In Bone And Aorta Induced By Disrupted FGF23/alpha klotho
Tomoka Hasegawa, Yukina Miyamoto, Zixuan Qiu, Hiromi Hongo, Norio Amizuka, Tomomaya Yamamoto
JOURNAL OF BONE AND MINERAL RESEARCH, 34, 142, 142, WILEY, 2019年12月, ［査読有り］
英語

Imaging modalities for drug-related osteonecrosis of the jaw (3), Positron emission tomography imaging for the diagnosis of medication-related osteonecrosis of the jaw
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, ELSEVIER SCI LTD, 2019年11月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 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.

Histochemical examination on principal collagen fibers in periodontal ligaments of ascorbic acid-deficient ODS-od/od rats
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, OXFORD UNIV PRESS, 2019年10月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Brown tumor diagnosed three years after parathyroidectomy in a patient with nail-patella syndrome: A case report.
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, ELSEVIER, 2019年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Three-dimensional morphology of the Golgi apparatus in osteoclasts: NADPase and arylsulfatase cytochemistry, and scanning electron microscopy using osmium maceration
Tsuneyuki Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
MICROSCOPY, 68, 3, 243, 253, OXFORD UNIV PRESS, 2019年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Alternating lamellar structure in human cellular cementum and rat compact bone: Its structure and formation
Tsuneyuki Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Norio Amizuka
JOURNAL OF ORAL BIOSCIENCES, 61, 2, 105, 114, ELSEVIER SCIENCE BV, 2019年06月, ［査読有り］, ［国際誌］
英語, 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.

Immunohistochemical analysis of new bone formation in a patient with retroperitoneal liposarcoma; a case report
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, CIC EDIZIONI INT, 2019年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Sequential Treatment with Eldecalcitol After PTH Improves Bone Mechanical Properties of Lumbar Spine and Femur in Aged Ovariectomized Rats
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, SPRINGER, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Oral biosciences: The annual review 2018
Hayato Ohshima, Norio Amizuka
JOURNAL OF ORAL BIOSCIENCES, 61, 1, 1, 4, ELSEVIER SCIENCE BV, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Optimal administration frequency and dose of teriparatide for acceleration of biomechanical healing of long-bone fracture in a mouse model
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, SPRINGER JAPAN KK, 2019年03月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histological Effects of the Combined Administration of Eldecalcitol and a Parathyroid Hormone in the Metaphyseal Trabeculae of Ovariectomized Rats
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, SAGE PUBLICATIONS LTD, 2019年03月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Anabolic action of teriparatide to osteoporotic patients].
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］
日本語,

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．

Immunolocalization of podoplanin/E11/gp38, CD44, and endomucin in the odontoblastic cell layer of murine tooth germs
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, BIOMEDICAL RESEARCH PRESS LTD, 2019年, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Type 1 diabetes mellitus induced low bone turnover in ovariectomized rats
Bo Liu, Wei Feng, Tomoka Hasegawa, Norio Amizuka, Minqi Li
HISTOLOGY AND HISTOPATHOLOGY, 34, 1, 57, 67, F HERNANDEZ, 2019年01月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Aspirin promotes apoptosis and inhibits proliferation by blocking G0/G1 into S phase in rheumatoid arthritis fibroblast-like synoviocytes via downregulation of JAK/STAT3 and NF-κB signaling pathway.
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, SPANDIDOS PUBL LTD, 2018年12月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Siglec-15-Targeting Therapy Increases Bone Mass in Rats and Is a Potential Therapeutic Strategy for Juvenile Osteoporosis
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, WILEY, 2018年11月, ［査読有り］, ［国際誌］
英語

Biological Effects of Abaloparatide on Bone Mass and Bone Turnover in Mice, a Comparison with Teriparatide.
Akito Makino, Tomoka Hasegawa, Norio Amizuka
JOURNAL OF BONE AND MINERAL RESEARCH, 33, 295, 295, WILEY, 2018年11月, ［査読有り］, ［国際誌］
英語

Siglec-15-targeting therapy increases bone mass in rats without impairing skeletal growth
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, ELSEVIER SCIENCE INC, 2018年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Annexin A5 Involvement in Bone Overgrowth at the Enthesis
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, WILEY, 2018年08月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Eldecalcitol Causes FGF23 Resistance for Pi Reabsorption and Improves Rachitic Bone Phenotypes in the Male Hyp Mouse
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, ENDOCRINE SOC, 2018年07月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Identification of Differentially Expressed Genes Induced by Aberrant Methylation in Oral Squamous Cell Carcinomas Using Integrated Bioinformatic Analysis
Xiaoqi Zhang, Hao Feng, Dongfang Li, Shanshan Liu, Norio Amizuka, Minqi Li
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 19, 6, MDPI, 2018年06月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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］, ［国際共著］, ［国内誌］
英語, 研究論文（学術雑誌）

Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM)
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, SPRINGER, 2018年04月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

In focus in HCB: Hard Tissue Biology
Norio Amizuka, Sohei Kitazawa
HISTOCHEMISTRY AND CELL BIOLOGY, 149, 4, 287, 288, SPRINGER, 2018年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Dual function of peroxiredoxin I in lipopolysaccharide-induced osteoblast apoptosis via reactive oxygen species and the apoptosis signal-regulating kinase 1 signaling pathway
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, NATURE PUBLISHING GROUP, 2018年04月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The effect of switching from teriparatide to anti-RANKL antibody on cancellous and cortical bone in ovariectomized mice
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, ELSEVIER SCIENCE INC, 2018年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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］, ［国際共著］, ［国内誌］
英語, 研究論文（学術雑誌）

Zoledronate promotes bone formation by blocking osteocyte-osteoblast communication during bone defect healing
Pingping Cui, Hongrui Liu, Jing Sun, Norio Amizuka, Qinfeng Sun, Minqi Li
HISTOLOGY AND HISTOPATHOLOGY, 33, 1, 89, 99, F HERNANDEZ, 2018年01月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Anti-Siglec-15 antibody reduces bone resorption while maintaining bone formation in ovariectomized (OVX) rats and monkeys.
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, WILEY, 2017年12月, ［査読有り］, ［国際誌］
英語

Dose effects of beta-tricalcium phosphate nanoparticles on biocompatibility and bone conductive ability of three-dimensional collagen scaffolds
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, JAPANESE SOC DENTAL MATERIALS DEVICES, 2017年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Cellular function of osteocytes in normal and klotho-deficient mice.
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］, ［国際共著］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

An ENU-induced splice site mutation of mouse Col1a1 causing recessive osteogenesis imperfecta and revealing a novel splicing rescue
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, NATURE PUBLISHING GROUP, 2017年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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."

Adipocytes enhance expression of osteoclast adhesion-related molecules through the CXCL12/CXCR4 signalling pathway
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, WILEY, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization.
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, Elsevier Ltd, 2017年05月, ［査読有り］, ［国際誌］
英語, 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.

Palisaded neutrophilic and granulomatous dermatitis as a novel cause of hypercalcemia A case report
Michihito Kono, Tomoka Hasegawa, So Nagai, Toshio Odani, Kazumasa Akikawa, Yukiko Nomura, Hidetsugu Sato, Keisuke Kikuchi, Norio Amizuka, Hideaki Kikuchi
MEDICINE, 96, 21, e6968, LIPPINCOTT WILLIAMS & WILKINS, 2017年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Characterization of a PTH1R missense mutation responsible for Jansen type metaphyseal chondrodysplasia
Junko Shimomura-Kuroki, Muhammad Farooq, Tsuneo Sekimoto, Norio Amizuka, Yutaka Shimomura
ODONTOLOGY, 105, 2, 150, 154, SPRINGER, 2017年04月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histological evidence that metformin reverses the adverse effects of diabetes on orthodontic tooth movement in rats
Jing Sun, Juan Du, Wei Feng, Boyao Lu, Hongrui Liu, Jie Guo, Norio Amizuka, Minqi Li
JOURNAL OF MOLECULAR HISTOLOGY, 48, 2, 73, 81, SPRINGER, 2017年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical Examination on Periodontal Tissues of Klotho-Deficient Mice Fed With Phosphate-Insufficient Diet
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, SAGE PUBLICATIONS LTD, 2017年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Periostin is required for matricellular localization of CCN3 in periodontal ligament of mice
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, SPRINGER, 2017年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes
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, Japanese Association for Oral Biology, 2017年02月01日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Histochemical examination of systemic administration of eldecalcitol combined with guided bone regeneration for bone defect restoration in rats
Xiuchun Han, Juan Du, Di Liu, Hongrui Liu, Norio Amizuka, Minqi Li
JOURNAL OF MOLECULAR HISTOLOGY, 48, 1, 41, 51, SPRINGER, 2017年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes
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, ELSEVIER SCIENCE BV, 2017年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-kappa B, ERK and JNK signaling pathways
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, NATURE PUBLISHING GROUP, 2017年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-κB, ERK and JNK signaling pathways.
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, NATURE PUBLISHING GROUP, 2017年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Chronological immunolocalization of sclerostin and FGF23 in the mouse metaphyseal trabecular and cortical bone
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, BIOMEDICAL RESEARCH PRESS LTD, 2017年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

[Bone remodeling and modeling/mini-modeling.]
Hasegawa T, Amizuka N
Clinical calcium, 27, 12, 1713, 1722, 2017年, ［査読有り］

Histochemical assessment for osteoblastic activity coupled with dysfunctional osteoclasts in c-src deficient mice
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, BIOMEDICAL RESEARCH PRESS LTD, 2017年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Histological assessment for femora of ovariectomized obesity (db/db) mice carrying mutated leptin receptor
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, F HERNANDEZ, 2016年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The F-actin modulator SWAP-70 controls podosome patterning in osteoclasts.
Anne Roscher, Tomoka Hasegawa, Sebastian Dohnke, Carlos Ocaña-Morgner, Norio Amizuka, Rolf Jessberger, Annette I Garbe
Bone reports, 5, 214, 221, Elsevier Inc, 2016年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histomorphometric analysis of minimodeling in the vertebrae in postmenopausal patients treated with anti-osteoporotic agents.
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, Elsevier Inc, 2016年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Localization of Minodronate in Mouse Femora Through Isotope Microscopy
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, SAGE PUBLICATIONS LTD, 2016年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ovariectomy upregulated the expression of Peroxiredoxin 1 & 5 in osteoblasts of mice
Juan Du, Wei Feng, Jing Sun, Cuijie Kang, Norio Amizuka, Minqi Li
SCIENTIFIC REPORTS, 6, 35995, 35995, NATURE PUBLISHING GROUP, 2016年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Effects of drug discontinuation after short-term daily alendronate administration on osteoblasts and osteocytes in mice
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, SPRINGER, 2016年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical examination of the effects of high-dose 1,25(OH)(2)D-3 on bone remodeling in young growing rats
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, SPRINGER, 2016年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of MMP9 and MMP2 in osteolytic metastasis originating from MDA-MB-231 human breast cancer cells
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, SPANDIDOS PUBL LTD, 2016年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

1. Morphological Implication on Cellular Response to Mechanical Stress in Bone.
Norio Amizuka
Journal of orthopaedic trauma, 30, 8, S2, 2016年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histology of human cementum: Its structure, function, and development
Tsuneyuki Yamamoto, Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Norio Amizuka
JAPANESE DENTAL SCIENCE REVIEW, 52, 3, 63, 74, ELSEVIER SCI LTD, 2016年08月, ［査読有り］, ［国際誌］
英語, 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.

Frequency of Teriparatide Administration Affects the Histological Pattern of Bone Formation in Young Adult Male Mice
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, ENDOCRINE SOC, 2016年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of MMP 2, 9 and 13 in prednisolone induced osteoporosis in mice
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, F HERNANDEZ, 2016年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Altered distribution of Ghrelin protein in mice molar development
Bo Liu, Xiuchun Han, Wei Feng, Jian Cui, Tomoka Hasegawa, Norio Amizuka, Xin Xu, Minqi Li
ARCHIVES OF ORAL BIOLOGY, 65, 82, 86, PERGAMON-ELSEVIER SCIENCE LTD, 2016年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Cellular interplay of bone cells and vascular endothelial cells in bone].
Tomoka Hasegawa, Erika Tsuchiya, Miki Abe, Norio Amizuka
Clinical calcium, 26, 5, 677, 82, 2016年05月, ［査読有り］, ［国内誌］
日本語, 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.

The effect of calcitriol on high mobility group box 1 expression in periodontal ligament cells during orthodontic tooth movement in rats
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, SPRINGER, 2016年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Long-Term Administration of High-Fat Diet Corrects Abnormal Bone Remodeling in the Tibiae of Interleukin-6-Deficient Mice
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, SAGE PUBLICATIONS LTD, 2016年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Joint Degradation in a Monkey Model of Collagen-Induced Arthritis: Role of Cathepsin K Based on Biochemical Markers and Histological Evaluation
Makoto Tanaka, Hiroyuki Yamada, Satoshi Nishikawa, Hiroshi Mori, Yasuo Ochi, Naoto Horai, Minqi Li, Norio Amizuka
INTERNATIONAL JOURNAL OF RHEUMATOLOGY, 2016, 8938916, 8938916, HINDAWI LTD, 2016年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of osteocyte-derived molecules during bone fracture healing of mouse ribs
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, BIOMEDICAL RESEARCH PRESS LTD, 2016年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

骨の組織・微細構造 --顕微解剖学的知見--
長谷川智香, 網塚憲生
Pain Research, 31, 4, 210, 219, JAPANESE ASSOC STUDY PAIN, 2016年, ［査読有り］
日本語, 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.

Calcilytic Ameliorates Abnormalities of Mutant Calcium-Sensing Receptor (CaSR) Knock-In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)
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, WILEY, 2015年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical examination of adipose derived stem cells combined with β-TCP for bone defects restoration under systemic administration of 1α,25(OH)2D3.
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, ELSEVIER SCIENCE BV, 2015年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Osteoblast-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Enhanced Bone Formation With Aberrant Mineralization.
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, WILEY-BLACKWELL, 2015年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation
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, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Altered distribution of HMGB1 in the periodontal ligament of periostin-deficient mice subjected to Waldo's orthodontic tooth movement
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, SPRINGER, 2015年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical evidence of zoledronate inhibiting c-src expression and interfering with CD44/OPN-mediated osteoclast adhesion in the tibiae of mice
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, SPRINGER, 2015年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Vascular Calcification - Pathological Mechanism and Clinical Application - . Vascular calcification in klotho deficient environment].
Tomoka Hasegawa, Tomomaya Yamamoto, Hiromi Hongo, Kanako Tsuboi, Norio Amizuka
Clinical calcium, 25, 5, 693, 9, 2015年05月, ［査読有り］, ［国内誌］
日本語, 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.

Treatment with eldecalcitol positively affects mineralization, microdamage, and collagen crosslinks in primate bone
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, ELSEVIER SCIENCE INC, 2015年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Local administration of calcitriol positively influences bone remodeling and maturation during restoration of mandibular bone defects in rats
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, ELSEVIER SCIENCE BV, 2015年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Inhibitory effect of bisphosphonate on osteoclast function contributes to improved skeletal pain in ovariectomized mice
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, SPRINGER JAPAN KK, 2015年03月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Expression of HMGB1 in the periodontal tissue subjected to orthodontic force application by Waldo's method in mice
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, SPRINGER, 2015年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of matrix Gla protein and osteocalcin in the developing tibial epiphysis of mice
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, F HERNANDEZ, 2015年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Hertwig's Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars
Tsuneyuki Yamamoto, Tamaki Yamada, Tomomaya Yamamoto, Tomoka Hasegawa, Hiromi Hongo, Kimimitsu Oda, Norio Amizuka
ACTA HISTOCHEMICA ET CYTOCHEMICA, 48, 3, 95, 101, JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY, 2015年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Hertwig's epithelial root sheath cell behavior during initial acellular cementogenesis in rat molars
Tsuneyuki Yamamoto, Tomomaya Yamamoto, Tamaki Yamada, Tomoka Hasegawa, Hiromi Hongo, Kimimitsu Oda, Norio Amizuka
HISTOCHEMISTRY AND CELL BIOLOGY, 142, 5, 489, 496, SPRINGER, 2014年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

γ-Glutamyl carboxylase in osteoblasts regulates glucose metabolism in mice.
Shiba S, Ikeda K, Azuma K, Hasegawa T, Amizuka N, Horie-Inoue K, Inoue S
Biochemical and biophysical research communications, 453, 3, 350, 355, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological Evidence of Increased Osteoclast Cell Number and Asymmetric Bone Resorption Activity in the Tibiae of Interleukin-6-Deficient Mice
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, SAGE PUBLICATIONS LTD, 2014年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Vitamin K-2 Biosynthetic Enzyme, UBIAD1 Is Essential for Embryonic Development of Mice
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, PUBLIC LIBRARY SCIENCE, 2014年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of matrix Gla protein and osteocalcin in the developing tibial epiphysis of mice.
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］
英語, 研究論文（学術雑誌）, 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

Altered distribution of extracellular matrix proteins in the periodontal ligament of periostin-deficient mice.
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
英語, 研究論文（学術雑誌）, 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

Histochemical examination of cathepsin K, MMP1 and MMP2 in compressed periodontal ligament during orthodontic tooth movement in periostin deficient mice
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, SPRINGER, 2014年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Altered distribution of extracellular matrix proteins in the periodontal ligament of periostin-deficient mice
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, F HERNANDEZ, 2014年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological Evidence of Increased Osteoclast Cell Number and Asymmetric Bone Resorption Activity in the Tibiae of Interleukin-6-Deficient Mice.
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, SAGE PUBLICATIONS LTD, 2014年05月14日, ［国際誌］
英語, 研究論文（学術雑誌）, 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

[Bone and Stem Cells. Cellular network in bone micro-environment - histological and ultrastructural aspects -].
Norio Amizuka, Tomomaya Yamamoto, Tomoka Hasegawa
Clinical calcium, 24, 4, 475, 85, 2014年04月, ［査読有り］, ［国内誌］
日本語, 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.

Inhibitory effect of bisphosphonate on osteoclast function contributes to improved skeletal pain in ovariectomized mice.
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
英語, 研究論文（学術雑誌）, 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

[Microscopic aspects on biomineralization in bone].
Norio Amizuka, Tomoka Hasegawa, Tomomaya Yamamoto, Kimimitsu Oda
Clinical calcium, 24, 2, 203, 14, 2014年02月, ［査読有り］, ［国内誌］
日本語, 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.

[Tissue-nonspecific alkaline phosphatase and hypophosphatasia].
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］, ［国内誌］
日本語, 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.

Effect of a Calcilytic Compound in Autosomal Dominant Hypocalcemia Model Mice.
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, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語

Eldecalcitol, a new-generation vitamin D3 analog, increases trabecular bone via "minimodeling'' in ovariectomized cynomolgus monkeys.
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, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語

The Importance of Activated Vitamin D for the Mineralization by the Osteocyte in Patients with Renal Hyperparathyroidism.
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, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語

Combination Treatment of 1-34 PTH and Eldecalcitol Showed Synergistic Effect on BMD without Severe Hypercalcemia.
Sadaoki Sakai, Satoshi Takeda, Tomomaya Yamamoto, Tomoka Hasegawa, Norio Amizuka, Koichi Endo
JOURNAL OF BONE AND MINERAL RESEARCH, 29, S263, S264, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語

Effects of the Combination of Eldecalcitol, an Analog of Active Vitamin D-3, and Parathyroid Hormone in Ovariectomized Rat Bones
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, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語

Altered distribution of bone matrix proteins and defective bone mineralization in klotho-deficient mice.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Altered distribution of bone matrix proteins and defective bone mineralization in klotho-deficient mice
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, ELSEVIER SCIENCE INC, 2013年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Alendronate promotes bone formation by inhibiting protein prenylation in osteoblasts in rat tooth replantation model
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, BIOSCIENTIFICA LTD, 2013年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Updates on rickets and osteomalacia: biological function of osteocyte and FGF23/klotho system].
Norio Amizuka, Tomoka Hasegawa, Muneteru Sasaki, Tomomaya Yamamoto, Tsuneyuki Yamamoto
Clinical calcium, 23, 10, 1453, 61, 2013年10月, ［査読有り］, ［国内誌］
日本語, 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.

An asparagine at position 417 of tissue-nonspecific alkaline phosphatase is essential for its structure and function as revealed by analysis of the N417S mutation associated with severe hypophosphatasia.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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

Eldecalcitol and calcitriol stimulates 'bone minimodeling,' focal bone formation without prior bone resorption, in rat trabecular bone
Hitoshi Saito, Satoshi Takeda, Norio Amizuka
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY, 136, 1, 178, 182, PERGAMON-ELSEVIER SCIENCE LTD, 2013年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

An asparagine at position 417 of tissue-nonspecific alkaline phosphatase is essential for its structure and function as revealed by analysis of the N417S mutation associated with severe hypophosphatasia
Sara Sultana, Hiba A. Al-Shawafi, Saori Makita, Miwa Sohda, Norio Amizuka, Ritsuo Takagi, Kimimitsu Oda
MOLECULAR GENETICS AND METABOLISM, 109, 3, 282, 288, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2013年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical aspects of the vascular invasion at the erosion zone of the epiphyseal cartilage in MMP-9-deficient mice
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, BIOMEDICAL RESEARCH PRESS LTD, 2013年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Sclerostin is differently immunolocalized in metaphyseal trabecules and cortical bones of mouse tibiae
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, BIOMEDICAL RESEARCH PRESS LTD, 2013年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histological examination on osteoblastic activities in the alveolar bone of transgenic mice with induced ablation of osteocytes
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, F HERNANDEZ, 2013年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Bone histology after intermittent PTH treatment - animal models -].
Muneteru Sasaki, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto, Norio Amizuka
Clinical calcium, 23, 3, 347, 53, 2013年03月, ［査読有り］, ［国内誌］
日本語, 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.

Occurrence of new bone-like tissue formation in uremic tumoral calcinosis
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, ELSEVIER SCIENCE INC, 2013年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Medial vascular calcification: a new concept challenging the classical paradigm of dystrophic calcification.
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］
英語, 研究論文（学術雑誌）, 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.

Histochemical examination of vascular medial calcification of aorta in klotho-deficient 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, ELSEVIER SCIENCE BV, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of sclerostin synthesized by osteocytes in relation to bone remodeling in the interradicular septa of ovariectomized rats
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, OXFORD UNIV PRESS, 2012年10月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Structure and formation of the twisted plywood pattern of collagen fibrils in rat lamellar bone
Tsuneyuki Yamamoto, Tomoka Hasegawa, Muneteru Sasaki, Hiromi Hongo, Chihiro Tabata, Zhusheng Liu, Minqi Li, Norio Amizuka
JOURNAL OF ELECTRON MICROSCOPY, 61, 2, 113, 121, OXFORD UNIV PRESS, 2012年04月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

[Biological function of bone cells on the PTH-driven anabolic effect].
Tomoka Hasegawa, Hiromi Hongo, Muneteru Sasaki, Tamaki Yamada, Norio Amizuka
Clinical calcium, 22, 3, 373, 9, 2012年03月, ［査読有り］, ［国内誌］
日本語, 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 -.

A clinical study of the alveolar bone tissue engineering with cultured autogenous periosteal cells: coordinated activation of bone formation and resorption.
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］
英語, 研究論文（学術雑誌）

Histology of epiphyseal cartilage calcification and endochondral ossification.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of sclerostin synthesized by osteocytes in relation to bone remodeling in the interradicular septa of ovariectomized rats.
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, OXFORD UNIV PRESS, 2012年, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

The distribution of osteocytic lacunar-canalicular system, and immunolocalization of FGF23 and sclerostin in osteocytes.
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, ELSEVIER SCIENCE BV, 2012年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Morphological aspects of the biological function of the osteocytic lacunar canalicular system and of osteocyte-derived factors.
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, WILEY, 2012年, ［査読有り］
英語, 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.

Histology of epiphyseal cartilage calcification and endochondral ossification
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］
英語, 研究論文（学術雑誌）, 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.

Assessment of histological alterations in cartilage and extracellular matrix driven by collagen-induced arthritis in Macaca fascicularis
Norio Amizuka, Hiromi Hongo, Muneteru Sasaki, Tomoka Hasegawa, Paulo Henrique Luiz de Freitas, Hiroshi Mori, Minqi Li
ARTHRITIS RESEARCH & THERAPY, 14, BIOMED CENTRAL LTD, 2012年, ［査読有り］
英語

[Frontiers in vitamin D; basic research and clinical application. A review on histological findings in bones administered with eldecalcitol].
Hiromi Hongo, Muneteru Sasaki, Tomoka Hasegawa, Norio Amizuka
Clinical calcium, 21, 11, 63, 70, 2011年11月, ［査読有り］, ［国内誌］
日本語, 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.

Eldecalcitol, a second-generation vitamin D analog, drives bone minimodeling and reduces osteoclastic number in trabecular bone of ovariectomized rats.
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
英語, 研究論文（学術雑誌）, 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

Eldecalcitol, a second-generation vitamin D analog, drives bone minimodeling and reduces osteoclastic number in trabecular bone of ovariectomized rats
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, ELSEVIER SCIENCE INC, 2011年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats.
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
英語, 研究論文（学術雑誌）, 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

Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats
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, BIOMEDICAL RESEARCH PRESS LTD, 2011年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

A morphological analysis on the osteocytic lacunar canalicular system in bone surrounding dental implants.
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, WILEY-BLACKWELL, 2011年06月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A Morphological Analysis on the Osteocytic Lacunar Canalicular System in Bone Surrounding Dental Implants
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, WILEY-BLACKWELL, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

ELDECALCITOL SUPRESSES BONE RESORPTION INDEPENDENT OF ITS CALCEMIC EFFECT BY DIMINISHING OSTEOCLAST ON BONE SURFACE
Hitoshi Saito, Satoshi Takeda, Fumiaki Takahashi, Paulo Luiz de Freitas, Norio Amizuka
OSTEOPOROSIS INTERNATIONAL, 22, 62, 63, SPRINGER LONDON LTD, 2011年03月, ［査読有り］
英語

ELDECALCITOL SUPRESSES BONE RESORPTION INDEPENDENT OF ITS CALCEMIC EFFECT BY DIMINISHING OSTEOCLAST ON BONE SURFACE
Hitoshi Saito, Satoshi Takeda, Fumiaki Takahashi, Paulo Luiz de Freitas, Norio Amizuka
OSTEOPOROSIS INTERNATIONAL, 22, 206, 207, SPRINGER LONDON LTD, 2011年03月, ［査読有り］
英語

[Animal models for bone and joint disease. Bone disease of osteoprotegerin deficient mouse].
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］, ［国内誌］
日本語, 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.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 is required for normal cartilage development
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, PORTLAND PRESS LTD, 2010年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of DMP1 and sclerostin in the epiphyseal trabecule and diaphyseal cortical bone of osteoprotegerin deficient mice.
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
英語, 研究論文（学術雑誌）, 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

Ultrastructural observation on cells meeting the histological criteria for preosteoblasts - a study in the mouse tibial metaphysis
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, OXFORD UNIV PRESS, 2010年10月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of DMP1 and sclerostin in the epiphyseal trabecule and diaphyseal cortical bone of osteoprotegerin deficient mice
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, BIOMEDICAL RESEARCH PRESS LTD, 2010年10月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Periostin Associates with Notch1 Precursor to Maintain Notch1 Expression under a Stress Condition in Mouse Cells
Hideyuki Tanabe, Issei Takayama, Takashi Nishiyama, Masashi Shimazaki, Isao Kii, Minqi Li, Norio Amizuka, Ken-ichi Katsube, Akira Kudo
PLOS ONE, 5, 8, e12234, PUBLIC LIBRARY SCIENCE, 2010年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological review of the human cellular cementum with special reference to an alternating lamellar pattern
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, SPRINGER, 2010年07月, ［査読有り］, ［国内誌］
英語, 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.

FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line
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］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histological assessment for PTH/PTHrP signaling on osteoblasts
Norio Amizuka, Minqi Li, Paulo H. L. D. Freitas, Zhucheng Liu, Ying Guo, Reiko Suzuki, Tsuneyuki Yamamoto
ENDOCRINE JOURNAL, 57, S244, S245, JAPAN ENDOCRINE SOC, 2010年03月, ［査読有り］
英語

FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line.
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
英語, 研究論文（学術雑誌）, 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

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture
Isao Kii, Takashi Nishiyama, Minqi Li, Ken-ichi Matsumoto, Mitsuru Saito, Norio Amizuka, Akira Kudo
JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 3, 2028, 2039, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling.
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, ［国内誌］
英語, 研究論文（学術雑誌）, 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

FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling
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, OXFORD UNIV PRESS, 2009年12月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

[Biological effects of vitamin K2 on bone quality].
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］, ［国内誌］
日本語, 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.

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development.
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］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Intermittent PTH administration stimulates pre-osteoblastic proliferation without leading to enhanced bone formation in osteoclast-less c-fos(-/-) mice.
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, WILEY-BLACKWELL, 2009年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development
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, AMER PHYSIOLOGICAL SOC, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Intermittent PTH Administration Stimulates Pre-Osteoblastic Proliferation Without Leading to Enhanced Bone Formation in Osteoclast-Less c-fos(-/-) Mice
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, WILEY-BLACKWELL, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

[Hormones and osteoporosis update. Histological aspects on the action of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) on bone and cartilage].
Norio Amizuka, Minqi Li, Masato Tamura, Kimimitsu Oda
Clinical calcium, 19, 7, 935, 43, 2009年07月, ［査読有り］, ［国内誌］
日本語, 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.

骨細胞の微細構造と機能
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］, ［国内誌］
日本語

Warfarin administration disrupts the assembly of mineralized nodules in the osteoid
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, OXFORD UNIV PRESS, 2009年04月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Type IIc Sodium-Dependent Phosphate Transporter Regulates Calcium Metabolism
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, AMER SOC NEPHROLOGY, 2009年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Vitamin k2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency
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, F HERNANDEZ, 2008年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Rat wild-type parathyroid hormone receptor (PTH-R) and mutant PTH-R-P132L show the different intracellular localization in vitro
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, BIOMEDICAL RESEARCH PRESS LTD, 2008年04月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histological and elemental analyses of impaired bone mineralization in klotho-deficient mice
Hironobu Suzuki, Norio Amizuka, Kimimitsu Oda, Masaki Noda, Hayato Ohshima, Takeyasu Maeda
JOURNAL OF ANATOMY, 212, 3, 275, 285, WILEY, 2008年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Assessment of bone quality. Bone quality of steroid-induced osteoporosis].
Minqi Li, Tomonao Saito, Norio Amizuka
Clinical calcium, 18, 3, 328, 35, 2008年03月, ［査読有り］, ［国内誌］
日本語, 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.

Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement
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, WILEY-LISS, 2008年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Involvement of the klotho protein in dentin formation and mineralization
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, WILEY, 2008年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Periostin is essential for cardiac healing after acute myocardial infarction
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, ROCKEFELLER UNIV PRESS, 2008年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Current Concepts of Bone Biomineralization
Hidehiro Ozawa, Kazuto Hoshi, Norio Amizuka
JOURNAL OF ORAL BIOSCIENCES, 50, 1, 1, 14, ELSEVIER SCIENCE BV, 2008年
英語, 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］

Generation of a monoclonal antibody specific for tissue-nonspecific alkaline phosphatase and its use in a clinical diagnostic study
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, MARY ANN LIEBERT INC, 2007年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Association of TIMP-2 with extracellular matrix exposed to mechanical stress and its co-distribution with periostin during mouse tooth development
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
研究論文（国際会議プロシーディングス）

Histological examination of bone regeneration achieved by combining grafting with hydroxyapatite and thermoplastic bioresorbable plates
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, SPRINGER JAPAN KK, 2007年11月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining
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, SPRINGER JAPAN KK, 2007年11月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Association of TIMP-2 with extracellular matrix exposed to mechanical stress and its co-distribution with periostin during mouse mandible development
Nagako Yoshiba, Kunihiko Yoshiba, Akihiro Hosoya, Masahiro Saito, Takamasa Yokoi, Takashi Okiji, Norio Amizuka, Hidehiro Ozawa
CELL AND TISSUE RESEARCH, 330, 1, 133, 145, SPRINGER, 2007年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological assessments on the abnormalities of mouse epiphyseal chondrocytes with short term centrifugal loading
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, BIOMEDICAL RESEARCH PRESS LTD, 2007年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical examinations on cortical bone regeneration induced by thermoplastic bioresorbable plates applied to bone defects of rat calvariae
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, BIOMEDICAL RESEARCH PRESS LTD, 2007年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Caveolin-1 in extracellular matrix vesicles secreted from osteoblasts
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, ELSEVIER SCIENCE INC, 2007年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Runx2 determines bone maturity and turnover rate in postnatal bone loss in estrogen deficiency
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, WILEY-LISS, 2007年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Targeted ablation of Osteocytes induces osteoporosis with defective mechanotransduction
Sawako Tatsumi, Kiyoaki Ishii, Norio Amizuka, Minqi Li, Toshihiro Kobayashi, Kenji Kohno, Masako Ito, Sunao Takeshita, Kyoji Ikeda
CELL METABOLISM, 5, 6, 464, 475, CELL PRESS, 2007年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Distribution of macrophages, osteoclasts and the B-lymphocyte lineage in osteolytic metastasis of mouse mammary carcinoma
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, BIOMEDICAL RESEARCH PRESS LTD, 2007年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice
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, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2007年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ectopic calcification as abnormal biomineralization
Junichiro James Kazama, Norio Amizuka, Masafumi Fukagawa
THERAPEUTIC APHERESIS AND DIALYSIS, 10, SUPPL. 1, S34, S38, WILEY, 2006年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Macrophage migration inhibitory factor-deficient mice are resistant to ovariectomy-induced bone loss (vol 580, pg 1251, 2006)
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］

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat
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, WILEY, 2006年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Involvement of hepatocyte growth factor in the development of bone metastasis of a mouse mammary cancer cell line, BALB/c-MC
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, ELSEVIER SCIENCE INC, 2006年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion
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, OXFORD UNIV PRESS INC, 2006年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Histological function of PTHrP in cartilage].
Minqi Li, Norio Amizuka
Clinical calcium, 16, 7, 1221, 27, 2006年07月, ［査読有り］, ［国内誌］
日本語, 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.

Localization of CD44 and hyaluronan in the synovial membrane of the rat temporomandibular joint
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］
英語, 研究論文（学術雑誌）, 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.

Localization of CD44 and hyaluronan in the synovial membrane of the rat temporomandibular joint
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, WILEY-LISS, 2006年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Transgenic mice overexpressing macrophage migration inhibitory factor (MIF) exhibit high-turnover osteoporosis
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, WILEY, 2006年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological and immunohistochemical changes in the submandibular gland in klotho-deficient mice
SUZUKI Hironobu, AMIZUKA Norio, NODA Masaki, AMANO Osamu, MAEDA Takeyasu
Archives of histology and cytology, 69, 2, 119, 128, INT SOC HISTOLOGY & CYTOLOGY, 2006年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Periostin is an extracellular matrix protein required for eruption of incisors in mice.
Isao Kii, Norio Amizuka, Li Minqi, Satoshi Kitajima, Yumiko Saga, Akira Kudo
Biochemical and biophysical research communications, 342, 3, 766, 72, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2006年04月14日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Histopathological observations on osteolytic bone metastasis].
Minqi Li, Norio Amizuka
Clinical calcium, 16, 4, 591- 97, 597, 2006年04月, ［査読有り］, ［国内誌］
日本語, 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.

Macrophage migration inhibitory factor-deficient mice are resistant to ovariectomy-induced bone loss.
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, ELSEVIER SCIENCE BV, 2006年02月20日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC-5) cells.
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, WILEY-LISS, 2006年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of caveolin-1 in the rat temporomandibular joint
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］
英語, 研究論文（学術雑誌）, 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.

Expression of caveolin-1 in the rat temporomandibular joint.
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, WILEY-LISS, 2006年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histological evidence of the altered distribution of osteocytes and bone matrix synthesis in klotho-deficient mice.
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, INT SOC HISTOLOGY & CYTOLOGY, 2005年12月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

A histological evaluation for guided bone regeneration induced by a collagenous membrane.
Yuya Taguchi, Norio Amizuka, Masayoshi Nakadate, Hideo Ohnishi, Noritaka Fujii, Kimimitsu Oda, Shuichi Nomura, Takeyasu Maeda
Biomaterials, 26, 31, 6158, 66, ELSEVIER SCI LTD, 2005年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Development of the articular cavity in the rat temporomandibular joint with special reference to the behavior of endothelial cells and macrophages
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］
英語, 研究論文（学術雑誌）, 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.

[Calcitonin receptor gene recombinant animals].
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］, ［国内誌］
日本語

[Parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor and the signal transduction].
Norio Amizuka, Minqi Li
Nihon rinsho. Japanese journal of clinical medicine, 63 Suppl 10, 278, 83, 2005年10月, ［査読有り］, ［国内誌］
日本語

[Parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor gene recombinant mice].
Norio Amizuka, Minqi Li
Nihon rinsho. Japanese journal of clinical medicine, 63 Suppl 10, 300, 5, 2005年10月, ［査読有り］, ［国内誌］
日本語

[Parathyroid hormone-related peptide (PTHrP) gene recombinant mice].
Norio Amizuka, Minqi Li
Nihon rinsho. Japanese journal of clinical medicine, 63 Suppl 10, 382, 6, 2005年10月, ［査読有り］, ［国内誌］
日本語

[The interplay of magnesium and vitamin K2 on bone mineralization].
Norio Amizuka, Minqi Li, Takeyasu Maeda
Clinical calcium, 15, 7, 57, 61, 2005年07月, ［査読有り］, ［国内誌］
日本語, 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.

Immunocytochemical localization of MAPKAPK-2 and Hsp25 in the rat temporomandibular joint.
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, WILEY-LISS, 2005年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Reduced osteoblastic population and defective mineralization in osteopetrotic (op/op) mice.
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, PERGAMON-ELSEVIER SCIENCE LTD, 2005年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunohistochemical localization of periostin in tooth and its surrounding tissues in mouse mandibles during development.
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, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Cell-cell interaction mediated by cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage.
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, AMER SOC BONE & MINERAL RES, 2004年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A histological evaluation of the involvement of Bio-Oss in osteoblastic differentiation and matrix synthesis.
Fabricio I Tapety, Norio Amizuka, Katsumi Uoshima, Shuichi Nomura, Takeyasu Maeda
Clinical oral implants research, 15, 3, 315, 24, BLACKWELL MUNKSGAARD, 2004年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Bone quality in the respect of bone matrix].
Norio Amizuka
Clinical calcium, 14, 4, 589, 93, 2004年04月, ［査読有り］, ［国内誌］
日本語, 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.

Zebrafish periostin is required for the adhesion of muscle fiber bundles to the myoseptum and for the differentiation of muscle fibers.
Hisaaki Kudo, Norio Amizuka, Kazuo Araki, Keiji Inohaya, Akira Kudo
Developmental biology, 267, 2, 473, 87, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2004年03月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A histological evaluation on self-setting alpha-tricalcium phosphate applied in the rat bone cavity.
Hirotsugu Hao, Norio Amizuka, Kimimitsu Oda, Noritaka Fujii, Hideo Ohnishi, Atsushi Okada, Shuichi Nomura, Takeyasu Maeda
Biomaterials, 25, 3, 431, 42, ELSEVIER SCI LTD, 2004年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The Expression of Estrogen Receptor α (Er α) in the rat Temporomandibular Joint
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
英語, 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.

Regional differences in the distribution and type of immunocompetent cells in the rat oral mucosae
A Suzuki, K Nozawa-Inoue, Y Kawano, N Amizuka, T Maeda
BIOMEDICAL RESEARCH-TOKYO, 24, 5, 249, 260, BIOMEDICAL RESEARCH PRESS LTD, 2003年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Expression of estrogen receptor alpha (ER alpha) in the rat temporomandibular joint.
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］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Synovial membrane in the temporomandibular joint-Its morphology, function and development
NOZAWA-INOUE Kayoko, AMIZUKA Norio, IKEDA Nobuyuki, SUZUKI Akiko, KAWANO Yoshiro, MAEDA Takeyasu
Archives of histology and cytology, 66, 4, 289, 306, INT SOC HISTOLOGY & CYTOLOGY, 2003年10月, ［査読有り］, ［国内誌］
英語, 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.

[Mineralization and vascular invasion during endochondral bone formation].
Norio Amizuka, Junko Shimomura, Takeyasu Maeda, Hidehiro Ozawa
Clinical calcium, 13, 4, 405, 12, 2003年04月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, 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.

Ultrastructural and cytobiological studies on possible interactions between PTHrP-secreting tumor cells, stromal cells, and bone cells.
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, SPRINGER-VERLAG TOKYO, 2003年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Defective bone remodelling in osteoprotegerin-deficient mice.
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, OXFORD UNIV PRESS, 2003年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Biological action of parathyroid hormone (PTH)-related peptide (PTHrP) mediated either by the PTH/PTHrP receptor or the nucleolar translocation in chondrocytes
AMIZUKA Norio, ODA Kimimitsu, SHIMOMURA Junko, MAEDA Takeyasu
Anatomical science international, 77, 4, 225, 36, 2002年12月, ［査読有り］, ［国内誌］
英語, 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.

1Alpha,25-dihydroxyvitamin D3 promotes vascularization of the chondro-osseous junction by stimulating expression of vascular endothelial growth factor and matrix metalloproteinase 9.
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, AMER SOC BONE & MINERAL RES, 2002年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[Not Available].
Norio Amizuka, Yukie Seki, Takeyasu Maeda
Clinical calcium, 12, 6, 835, 43, 2002年06月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）

Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines.
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, AMER SOC BONE & MINERAL RES, 2002年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

[The biological role of blood vessels during endochondral bone formation].
Norio Amizuka, Tomoyo Sasaki, Takeyasu Maeda
Clinical calcium, 12, 3, 327, 36, 2002年03月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, 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.

Histopathological characterization of melorheostosis
K Hoshi, N Amizuka, T Kurokawa, K Nakamura, R Shiro, H Ozawa
ORTHOPEDICS, 24, 3, 273, 277, SLACK INC, 2001年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta
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, AMER SOC BONE & MINERAL RES, 1999年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Maturation ameloblasts of the porcine tooth germ do not express amelogenin
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］
英語, 研究論文（学術雑誌）, 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.

Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia
AC Karaplis, B He, MTA Nguyen, ID Young, D Semeraro, H Ozawa, N Amizuka
ENDOCRINOLOGY, 139, 12, 5255, 5258, ENDOCRINE SOC, 1998年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Immunolocalization of tissue non-specific alkaline phosphatase in mice
Kazuto Hoshi, Norio Amizuka, Kimimitsu Oda, Yukio Ikehara, Hidehiro Ozawa
Histochemistry and Cell Biology, 107, 3, 183, 191, 1997年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

NUCLEOLAR LOCALIZATION OF PARATHYROID HORMONE-RELATED PEPTIDE ENHANCES SURVIVAL OF CHONDROCYTES UNDER CONDITIONS THAT PROMOTE APOPTOTIC CELL-DEATH
JE HENDERSON, N AMIZUKA, H WARSHAWSKY, D BIASOTTO, BMK LANSKE, D GOLTZMAN, AC KARAPLIS
MOLECULAR AND CELLULAR BIOLOGY, 15, 8, 4064, 4075, AMER SOC MICROBIOLOGY, 1995年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

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, 日本整形外科学会雑誌, 96, 3, 2022年

骨形成促進剤で誘導されるmodeling-based/remodeling-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, 日本骨形態計測学会雑誌, 31, 2, S101, S101, 2021年06月
日本骨形態計測学会, 日本語

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, 2021年06月
日本骨形態計測学会, 日本語

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, Journal of Oral Biosciences Supplement (Web), 2021, 2021年

骨補填材としてのリン酸化プルラン/β-TCP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, 日本歯周病学会会誌(Web), 63, 2021年

高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, 中嶋悠斐, 山本知真也, 山本知真也, 本郷裕美, NASOORI Alireza, 網塚憲生, 長谷川智香, Journal of Oral Biosciences Supplement (Web), 2021, 2021年

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, 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

副甲状腺ホルモン製剤投与による皮質骨多孔化の細胞学的メカニズムについて
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

新規リン酸化多糖体“リン酸化プルラン”を用いた骨再生
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

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, 日本骨代謝学会学術集会プログラム抄録集(CD-ROM), 39th, 2021年

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, 大巻真幸, 長谷川智香, 山本知真也, 山本知真也, 金子一郎, 瀬川博子, 網塚憲生, 日本骨代謝学会学術集会プログラム抄録集(CD-ROM), 39th, 2021年

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, 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

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, 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

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, 日本再生医療学会総会(Web), 20th, 2021年

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, 日本再生医療学会総会(Web), 20th, 2021年

リン酸八カルシウム(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, 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

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

免疫受容体Siglec-15は生理的骨吸収制御する主要調節因子である
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

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
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
(一社)日本骨代謝学会, 日本語

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
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
(一社)日本骨代謝学会, 日本語

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
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
(一社)日本骨代謝学会, 日本語

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
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
(一社)日本骨代謝学会, 日本語

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
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
(一社)日本骨代謝学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

Remodeling-based/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, 日本骨形態計測学会雑誌, 30, 1, S84, S84, 2020年05月
日本骨形態計測学会, 日本語

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
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
日本骨形態計測学会, 日本語

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
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
日本骨形態計測学会, 日本語

骨層板の微細構造と形成について 水酸化ナトリウム浸軟法を用いた走査型電子顕微鏡観察
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
(株)メディカルレビュー社, 日本語

動物モデルを用いた骨組織再生の顕微学的検索
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

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, 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

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
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

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
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

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
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

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索
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

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, 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

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 (Web), 2020, 2020年

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 (Web), 2020, 2020年

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 (Web), 2020, 2020年

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 (Web), 2020, 2020年

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 (Web), 2020, 2020年

赤外イメージングによるステロイド性骨粗鬆症モデルラット腰椎の骨質解析
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

アスコルビン酸合成能欠如(ODS/ShiJcl-od/od)ラットにおける歯根膜異常
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
(株)メディカルレビュー社, 日本語

リン酸三カルシウム(TCP)およびリン酸化プルラン(PPL)含有新規骨補填材の開発
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
(NPO)日本歯周病学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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］
(一社)歯科基礎医学会, 日本語

リン酸三カルシウム(TCP)およびリン酸化プルラン(PPL)含有新規骨補填材の開発
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
日本語

【軟部組織の石灰化-血管と心臓弁】血管石灰化における組織学的・微細構造学的知見 FGF23/αklotho軸破綻によるマウス大動脈石灰化
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
＜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., (株)日本メディカルセンター, 日本語

小児ステロイド性骨粗鬆症に対するSiglec-15分子標的治療の有用性
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
(一社)日本骨代謝学会, 日本語

骨代謝研究による骨粗鬆症治療の改革 小児ステロイド性骨粗鬆症に対するSiglec-15分子標的治療の有用性
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
(公社)日本整形外科学会, 日本語

骨代謝研究による骨粗鬆症治療の改革 小児ステロイド性骨粗鬆症に対するSiglec-15分子標的治療の有用性
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
(公社)日本整形外科学会, 日本語

アレンドロネート投与による骨芽細胞・破骨細胞と骨特異的血管の組織学的変化について
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
日本骨形態計測学会, 日本語

骨生検で骨形成亢進を捉えたSAPHO症候群の一例
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
日本骨形態計測学会, 日本語

副甲状腺ホルモン(PTH)製剤と活性型ビタミンD₃製剤(エルデカルシトール)の併用投与による骨形成作用
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
日本語

骨生検で骨形成亢進を捉えたSAPHO症候群の一例
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
日本骨形態計測学会, 日本語

アレンドロネート投与による骨芽細胞・破骨細胞と骨特異的血管の組織学的変化について
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
日本骨形態計測学会, 日本語

Jansen型PTH/PTHrP受容体の骨系細胞における機能異常解析
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
(公社)日本小児歯科学会, 日本語

副甲状腺ホルモン(PTH)製剤と活性型ビタミンD3製剤(エルデカルシトール)の併用投与による骨形成作用
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
(株)メディカルレビュー社, 日本語

『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
日本骨形態計測学会, 日本語

『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
日本骨形態計測学会, 日本語

『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
日本骨形態計測学会, 日本語

『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
日本骨形態計測学会, 日本語

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
北海道歯学会, 日本語

副甲状腺ホルモン投与および授乳期カルシウム欠乏食給餌によって誘導される骨細胞周囲骨基質の微細構造学的変化について
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

【進化する骨粗鬆症治療薬 国内患者1,300万人のQOL向上へ】副甲状腺ホルモン製剤の骨形成作用について
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
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), (公社)日本薬理学会, 日本語

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 (Web), 2019, 43 (WEB ONLY), 2019年
日本語

副甲状腺ホルモン(PTH)単回投与後における骨の組織化学的変化・遺伝子発現の変化について
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
日本語

マウス歯根膜の発生過程におけるendomucin陽性血管の局在変化について
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
日本語

Toll like receptor(TLR)2遺伝子欠損マウスの破骨細胞における組織学的検索
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
日本語

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(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
日本語

リン酸三カルシウム(TCP)およびリン酸化プルラン(PPL)含有新規骨補填剤による新生骨誘導における組織学的解析
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
日本語

Rankl遺伝子欠損マウスにおける破骨細胞様大型細胞の組織学的・微細構造学的検索
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
日本語

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 (Web), 2019, 338 (WEB ONLY), 2019年
日本語

マウス大腿骨二次骨化における骨特異的血管と骨芽細胞・破骨細胞の局在変化
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
日本語

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, QIU Z, 網塚憲生, 長谷川智香, Journal of Oral Biosciences Supplement (Web), 2019, 417 (WEB ONLY), 2019年
日本語

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 (Web), 2019, 227 (WEB ONLY), 2019年
日本語

II型糖尿病モデルSDT Fattyラットで誘発された糖尿病性骨粗鬆症および歯周病変の組織化学的解析
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
日本語

アレンドロネート外頸静脈投与後における骨芽細胞・破骨細胞と骨特異性血管の経時的局在変化
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
日本語

骨組織における骨特異的血管と骨細胞分化の微細構造学的解析
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
日本語

IL‐6遺伝子欠損マウスの骨組織における組織化学的解析
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
日本語

【薬剤関連顎骨壊死の画像診断up to date】薬剤関連顎骨壊死のPET検査
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
金原出版(株), 日本語

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, 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
日本組織細胞化学会, 日本語

骨基質石灰化の微細構造学
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
(株)メディカルレビュー社, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

骨基質石灰化の微細構造学
小澤 英浩, 長谷川 智香, 網塚 憲生, THE BONE, 32, 2, 139, 144, 2018年09月
(株)メディカルレビュー社, 日本語

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
日本組織細胞化学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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 (Web), 2018, ROMBUNNO.P1‐50 (WEB ONLY), 270, 2018年09月
(一社)歯科基礎医学会, 日本語

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 (Web), 2018, ROMBUNNO.P2‐13 (WEB ONLY), 369, 2018年09月
(一社)歯科基礎医学会, 日本語

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(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
(一社)歯科基礎医学会, 日本語

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 (Web), 2018, ROMBUNNO.P1‐51 (WEB ONLY), 271, 2018年09月
(一社)歯科基礎医学会, 日本語

Toll like receptor(TLR)2遺伝子欠損マウスの破骨細胞における組織学的検索
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
(一社)日本骨粗鬆症学会, 日本語

PTHrPアナログであるabaloparatideをマウスに皮下投与した時の骨量及び骨代謝に及ぼす作用について teriparatideとの比較
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
(一社)日本骨粗鬆症学会, 日本語

骨基質石灰化におけるFGF23/klothoとSIBLING familyの連関 Fgf23遺伝子欠損マウスを用いた組織化学・微細構造解析
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
(一社)日本骨粗鬆症学会, 日本語

新規肥満2型糖尿病モデルSDT fattyラットにおける骨組織異常について
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
(一社)日本骨粗鬆症学会, 日本語

FGF23/klothoシグナルによる基質石灰化制御機構の解明
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
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。, (一社)日本骨粗鬆症学会, 日本語

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革新的な小児骨粗鬆症治療薬の開発
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
(公社)日本整形外科学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(一社)日本骨代謝学会, 日本語

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
(公社)日本補綴歯科学会, 日本語

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, Supplement 1, 387, 2018年05月28日
日本語

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
(一社)日本透析医学会, 日本語

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
(一社)日本透析医学会, 日本語

テリパラチドの骨形成作用における細胞組織学的検索
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
日本骨形態計測学会, 日本語

Fgf23遺伝子欠損マウスの骨基質石灰化異常におけるSIBLING familyの関与
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
日本骨形態計測学会, 日本語

骨リモデリングとモデリングにおける活性型骨芽細胞と周囲の細胞群の組織学的検索
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
日本骨形態計測学会, 日本語

成長期小児モデル動物に対するビスフォスフォネート製剤投与の影響 骨格成長発育障害への影響
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
日本骨形態計測学会, 日本語

FIB-SEMを用いた骨芽細胞・骨細胞の三次元微細構造観察
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
(株)メディカルレビュー社, 日本語

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
(一社)日本透析医学会, 日本語

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
(一社)日本透析医学会, 日本語

細胞組織学・微細構造学からみた骨形成
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
日本骨形態計測学会, 日本語

骨再生における骨の細胞群の役割 (特集 ロコモティブシンドロームのためのバイオマテリアル)
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
日本バイオマテリアル学会 ; 2002-, 日本語

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(Web), 10, 85 (WEB ONLY), 2018年
日本語

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 (Web), 2018, ROMBUNNO.UDS09‐1 (WEB ONLY), 2018年
日本語

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, 36th, 119, 2018年
日本語

PTH間歇投与マウスの骨組織におけるリン酸イオン供与膜輸送体・酵素群の局在
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
日本語

アレンドロネートが骨芽細胞系細胞と骨特異性血管に及ぼす組織学的影響
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
日本語

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革新的な小児骨粗鬆症治療薬の開発―
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, 北海道整形災害外科学会, 135th, 135th suppl, 21, 21, 2018年
北海道整形災害外科学会, 日本語

骨の細胞・組織における微細構造解析~FIB‐SEM・SIM・STEDによる新規イメージング~
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
日本語

【ロコモティブシンドロームのためのバイオマテリアル】骨再生における骨の細胞群の役割
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
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), 日本バイオマテリアル学会, 日本語

ビスホスホネート投与および投与中止後の骨芽細胞・骨細胞の組織学的変化
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
(株)メディカルレビュー社, 日本語

FGF23/klothoシグナルによる基質石灰化制御機構の解明 (2017年度 日本骨粗鬆症学会 研究奨励賞)
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
日本骨粗鬆症学会, 日本語

歯科の分野でトピックとなっている論文のレビュー
網塚 憲生, 日本骨粗鬆症学会雑誌, 3, 4, 427, 430, 2017年11月
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。, (一社)日本骨粗鬆症学会, 日本語

【骨リモデリング制御と疾患】骨リモデリングとモデリング・ミニモデリング
長谷川 智香, 網塚 憲生, Clinical Calcium, 27, 12, 1713, 1722, 2017年11月
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), (株)医薬ジャーナル社, 日本語

FIB-SEMを用いた軟骨細胞および骨芽細胞の微細構造観察
長谷川 智香, 網塚 憲生, THE BONE, 31, 3, 239, 244, 2017年10月
(株)メディカルレビュー社, 日本語

軟骨内骨化における骨特異的血管とseptoclastの組織学的知見
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
日本語

若手育成に関するアンケート調査の結果報告(2017年3月)
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
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), (一社)日本解剖学会, 日本語

軟骨内骨化における骨特異的血管とseptoclastの組織学的知見
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
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．, 北海道歯学会, 日本語

最新の歯学 軟骨内骨化における骨特異的血管とseptoclastの組織学的知見
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
北海道歯学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

顕微鏡解析からみたビスホスホネート製剤の作用機序
網塚 憲生, 日本骨粗鬆症学会雑誌, 3, Suppl.1, 220, 220, 2017年09月
(一社)日本骨粗鬆症学会, 日本語

デノスマブの骨吸収、骨形成への早期効果
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
(公社)日本整形外科学会, 日本語

連載 注目の海外文献(64) １.血流は骨の血管機能と骨形成を調節する ２.骨芽細胞はCxcl9を分泌し骨の血管新生を抑制する
網塚憲生, CLINICAL CALCIUM, 27, 8, 124, 125, 2017年07月28日
医薬ジャーナル社

kl/klマウスにおける基質小胞性石灰化の抑制メカニズムについて
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
(一社)日本骨代謝学会, 日本語

腱・靱帯付着部(enthesis)においてAnnexin A5は線維軟骨の分化を負に制御する
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
(一社)日本骨代謝学会, 日本語

骨芽細胞特異的PTHrP過剰発現マウスの骨における組織化学・微細構造学的解析
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
(一社)日本骨代謝学会, 日本語

骨の細胞に対するPTH作用の組織学的知見
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
日本語

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22日
日本骨形態計測学会, 日本語

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development (vol 297, pg F671, 2009)
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
AMER PHYSIOLOGICAL SOC, 英語, その他

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
日本骨形態計測学会, 日本語

二次性副甲状腺機能亢進症治療の新たな知見 骨の細胞に対するPTH作用の組織学的知見
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
(一社)日本透析医学会, 日本語

マウス長管骨におけるsclerostinとFGF23産生細胞の経時的局在変化
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
日本骨形態計測学会, 日本語

αklotho/FGF23破綻による骨組織石灰化異常は低リン食給餌により回復する
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
日本骨形態計測学会, 日本語

副甲状腺ホルモン(PTH)単回投与後における大腿骨の組織化学的変化について
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
日本骨形態計測学会, 日本語

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
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
日本骨形態計測学会, 日本語

αklotho遺伝子変異マウスの低リン餌飼育による歯槽骨の組織異常回復
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
(株)メディカルレビュー社, 日本語

エルデカルシトールは閉経後骨粗鬆症患者においてミニモデリング骨形成を誘導する
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
(公社)日本整形外科学会, 日本語

骨ネットワークと他臓器連関:オーバービュー
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

II型糖尿病モデルSDT fattyラットにおける歯周組織の組織化学的解析
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

超解像共焦点レーザー顕微鏡とFIB-SEMでみる骨の世界
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

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 (Web), 2017, 2017年

マウス骨組織におけるsclerostinとFGF23産生細胞の経時的局在変化
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

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, 永井伯弥, 本郷裕美, 網塚憲生, Journal of Oral Biosciences Supplement (Web), 2017, 2017年

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 (Web), 2017, 2017年

骨質に影響を及ぼす骨の微細構造と細胞群の役割
網塚憲生, 長谷川智香, 再生医療, 16, 2017年

骨細胞分化におけるE11/gp38 glycoproteinの組織化学的局在について
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

乳がん骨転移巣における骨細胞の組織化学的検索
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
日本語

ミノドロネートの骨組織分布および破骨細胞に対する作用．
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
日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

ミノドロネートの骨組織分布および破骨細胞に対する作用
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
(株)メディカルレビュー社, 日本語

骨・運動器領域の基礎研究の国内外の動向
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
(株)メディカルレビュー社, 日本語

骨の組織・微細構造 顕微解剖学的知見
長谷川 智香, 網塚 憲生, PAIN RESEARCH, 31, 4, 210, 219, 2016年12月
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。, 日本疼痛学会, 日本語

接着性を有するMTA含有直接覆髄材の開発
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
(NPO)日本歯科保存学会, 日本語

内眼解剖学教育に関するアンケート調査結果(平成26年3月)
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
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), (一社)日本解剖学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

肉眼解剖学教育に関するアンケート調査結果(平成26年3月)
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
日本解剖学会, 日本語

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
網塚 憲生, 日本整形外科学会雑誌, 90, 8, S1439, S1439, 2016年08月
(公社)日本整形外科学会, 日本語

骨の細胞における微細構造・組織化学的知見
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
(公社)日本整形外科学会, 日本語

低リン食給餌がkl/klマウスおよびa klotho-/-マウスの骨・血管石灰化に及ぼす組織学的影響
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
(一社)日本骨代謝学会, 日本語

軟骨内骨化における血管内皮細胞とperivascular cellの細胞間連携に関する組織化学的検索
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
(一社)日本骨代謝学会, 日本語

ステロイド長期使用の関節リウマチ患者は骨粗鬆症治療に関わらず骨質異常が生じ骨脆弱性を引き起こす
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
(一社)日本骨代謝学会, 日本語

Annexin a5による腱・靱帯付着部(enthesis)における軟骨層の肥大化と石灰化の調節
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
(一社)日本骨代謝学会, 日本語

副甲状腺ホルモン(PTH)単回投与後における骨の細胞群の組織化学的変化について
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
(一社)日本骨代謝学会, 日本語

新規肥満2型糖尿病モデルSDT fattyラットの骨組織の組織化学的変化について
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
(一社)日本骨代謝学会, 日本語

III型Na依存性リン酸輸送担体による過剰なリン酸負荷は大動脈壁における弾性線維の構造を劣化させる
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
(一社)日本骨代謝学会, 日本語

授乳期のカルシウム欠乏食給餌マウスにおける骨細胞周囲の骨基質の微細構造学的変化について
本郷 裕美, 佐々木 宗輝, 斎藤 雅美, 宇田川 信之, 網塚 憲生, 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
(一社)日本骨代謝学会, 日本語

骨の痛み 骨の組織・微細構造 顕微解剖学的知見
長谷川 智香, 網塚 憲生, PAIN RESEARCH, 31, 2, 52, 52, 2016年06月
日本疼痛学会, 日本語

クル病、骨軟化症を考える 骨基質石灰化異常における微細構造学的知見
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
日本骨形態計測学会, 日本語

骨芽細胞特異的PTHrP過剰発現マウスの下顎骨・長管骨における組織化学・微細構造学的解析
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
日本骨形態計測学会, 日本語

軟骨内骨化における血管内皮細胞とperivascular cell(septoclast)の組織化学・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, 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
日本骨形態計測学会, 日本語

ステロイド長期使用のRA患者は骨粗鬆症治療に関わらず骨質異常が生じ骨脆弱性を引き起こす
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
日本骨形態計測学会, 日本語

特集 骨免疫学 ～境界を越えて!～ Review 骨組織を構成する細胞群と血管系の連関
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
医薬ジャーナル社

理解を助けるトレーニング問題 軟骨内骨化における骨の細胞群の役割について
網塚憲生, CLINICAL CALCIUM, 26, 5, 763, 763, 2016年04月28日
医薬ジャーナル社

理解を助けるトレーニング問題 軟骨内骨化における骨の細胞群の役割について
網塚憲生, CLINICAL CALCIUM, 26, 5, 111, 111, 2016年04月28日
医薬ジャーナル社

【骨免疫学〜境界を越えて!〜】骨組織を構成する細胞群と血管系の連関
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
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), (株)医薬ジャーナル社, 日本語

TCPナノ粒子を用いた組織再生用ナノ材料の創製と応用
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
日本語

骨の微細環境における細胞性ネットワーク-メカニカルストレスとの関連性-
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

Overview:骨基質石灰化における微細構造学
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

FGF23/klotho軸破綻による骨・血管の石灰化異常
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

c-fos遺伝子欠損マウスのモデリング・リモデリング領域における骨芽細胞の組織化学的・微細構造学的相違について
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

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 (Web), 2016, 2016年

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, 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

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 (Web), 2016, 2016年

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 (Web), 2016, 2016年

「オステオサイトの顕微形態学~入門編~」
網塚憲生, 日本腎性骨症研究会プログラム抄録集, 2016, 2016年

骨の組織・微細構造 ──顕微解剖学的知見──
長谷川 智香, 網塚 憲生, PAIN RESEARCH, 31, 4, 210, 219, 2016年
<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>, 日本疼痛学会, 日本語

骨の細胞における組織学的・微細構造学的知見
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
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), (公社)日本顕微鏡学会, 日本語

特集 顕微学的アプローチによる骨の細胞機能の解明 Preface 巻頭言～骨の顕微学的アプローチ～
網塚憲生, CLINICAL CALCIUM, 25, 10, 21, 21, 2015年09月28日
医薬ジャーナル社

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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, 311, 311, 2015年09月
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

PTHからELDへ切替投与した閉経性骨粗鬆症モデルラットにおける骨組織検索
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
(一社)日本骨代謝学会, 日本語

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小腔周囲の骨基質の微細構造学的検索
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
(一社)日本骨代謝学会, 日本語

Annexin a5による腱・靱帯付着部(enthesis)における骨形成の調節
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
(一社)日本骨代謝学会, 日本語

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と顎骨における組織化学的解析
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
(一社)日本骨代謝学会, 日本語

各種電子顕微鏡を用いた骨細胞間ネットワークの微細構造解析
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
(一社)日本骨代謝学会, 日本語

【ロコモティブシンドロームのすべて】ロコモティブシンドロームの基礎 運動器を構成する組織 骨
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
(公社)日本医師会, 日本語

骨粗鬆症治療による骨の形態と質の変化 細胞組織学からみた骨粗鬆症治療薬の作用機序
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
日本骨形態計測学会, 日本語

骨粗鬆症治療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
網塚 憲生, 日本骨形態計測学会雑誌, 25, 2, S65, S65, 2015年05月
日本骨形態計測学会, 日本語

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, 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
日本骨形態計測学会, 日本語

OVXラットにおけるPTHからELDへの切替投与の効果の検討
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
日本骨形態計測学会, 日本語

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
(公社)日本補綴歯科学会, 日本語

特集 血管石灰化の基礎と臨床 Topics klotho欠損環境における血管石灰化
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
医薬ジャーナル社

【血管石灰化の基礎と臨床】Klotho欠損環境における血管石灰化
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
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), (株)医薬ジャーナル社, 日本語

バイオマテリアルと間葉系幹細胞による顎骨再生を目指した三次元的骨再生法の開発―その骨質と機能の評価―
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
日本語

骨の細胞・石灰化基質および骨再生における微細構造学的知見(特別講演1,第65回日本歯科理工学会学術講演会)
網塚 憲生, 日本歯科理工学会誌, 34, 2, 64, 64, 2015年03月25日
一般社団法人日本歯科理工学会, 日本語

骨の成熟度と骨細胞形態との関連性 浸軟法を用いた走査型電子顕微鏡観察
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
(株)メディカルレビュー社, 日本語

骨の細胞・石灰化基質および骨再生における微細構造学的知見
網塚 憲生, 日本歯科理工学会誌, 34, 2, 64, 64, 2015年03月
(一社)日本歯科理工学会, 日本語

ビスフォスフォネート製剤に関する新たな知見
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
北海道歯学会, 日本語

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 (Web), 2015, 2015年

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 (Web), 2015, 2015年

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 (Web), 2015, 2015年

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, 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

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 (Web), 2015, 2015年

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 (Web), 2015, 2015年

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(Web), 7, 2015年

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 (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
日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

【慢性腎臓病と老化-Phosphate connection】慢性腎臓病における骨細胞の変化
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
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), (株)日本メディカルセンター, 日本語

ペリオスチン欠損マウスの切歯歯根膜における組織学的検索
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
(株)メディカルレビュー社, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 英語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 英語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 英語

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
(一社)歯科基礎医学会, 英語

CKDに併発する骨粗鬆症(透析骨症含む)の病態・診断 CKDでの皮質骨骨粗鬆症・骨折リスク(PTH上昇との関連で)
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
ライフサイエンス出版(株), 日本語

骨細胞性骨溶解における微細構造学的・組織化学的解析
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
ライフサイエンス出版(株), 日本語

PTH(1-34)とエルデカルシトールの海綿骨に対する異なる作用 卵巣摘除ラットを用いたμCTによる骨構造解析
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
ライフサイエンス出版(株), 日本語

副甲状腺ホルモン(PTH)とエルデカルシトール(ELD)を併用投与した骨粗鬆症モデルラットにおける骨組織化学
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
ライフサイエンス出版(株), 日本語

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
(NPO)日本口腔科学会, 日本語

【骨から全身へ、いま学びなおしたい よくわかるCKD-MBD】(Q9)CKD-MBDによる異所性石灰化ってどういうもの?
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
(株)メディカ出版, 日本語

【骨から全身へ、いま学びなおしたい よくわかるCKD-MBD】(Q10)CKD-MBDだと骨折しやすいの?
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
(株)メディカ出版, 日本語

骨代謝制御における骨細胞の役割 骨細胞・骨細管系における形態学的アプローチ
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
(一社)日本骨代謝学会, 日本語

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
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
(一社)日本骨代謝学会, 日本語

Eldecalcitolは卵巣摘出カニクイザルにおいてミニモデリングによる骨形成を誘導する
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
(一社)日本骨代謝学会, 日本語

骨芽細胞におけるビタミンK依存性γ-グルタミルカルボキシラーゼは骨代謝と糖代謝を制御する
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
(一社)日本骨代謝学会, 日本語

常染色体優性低Ca血症(ADH)モデルマウスに対するcalcilyticsの効果
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
(一社)日本骨代謝学会, 日本語

副甲状腺ホルモン(PTH)とエルデカルシトール(ELD)を併用投与した骨粗鬆症モデルラットにおける骨組織解析
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
(一社)日本骨代謝学会, 日本語

骨芽細胞および骨細胞に対するビスフォスフォネート連日投与の組織化学的作用
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
(一社)日本骨代謝学会, 日本語

【骨バイオイメージング】静的イメージング 光顕と電顕による骨組織の画像イメージング
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
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), (株)メディカルレビュー社, 日本語

腎性副甲状腺機能亢進症に対する副甲状腺全摘除術後の骨細胞数、骨細胞-骨細管系の石灰化について
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
日本骨形態計測学会, 日本語

【骨代謝 つくり、壊し、変える-そのメカニズムと最新治療 分子機構から骨粗鬆症・リウマチなど骨疾患への応用まで】(第I部) 骨代謝制御機構を知る (第3章)骨をみる イメージングの現状と展望 骨形成系にかかわる細胞群の微細構造学的知見
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
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), (株)羊土社, 日本語

テリパラチドとエルデカルシトールの併用投与が卵巣摘除ラットにおよぼす影響
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
日本骨形態計測学会, 日本語

骨細胞特異的死滅マウスの骨小腔基質溶解における微細構造学的検索
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
日本骨形態計測学会, 日本語

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形態について
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
日本骨形態計測学会, 日本語

卵巣摘出カニクイザルにおけるエルデカルシトールの効果 組織化学的検索
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
日本骨形態計測学会, 日本語

骨粗鬆症モデルラットを用いた副甲状腺ホルモン(PTH)とエルデカルシトール併用投与の効果
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
日本骨形態計測学会, 日本語

同位体顕微鏡を用いたミノドロン酸の局在と骨の細胞に対する組織化学的検討
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
日本骨形態計測学会, 日本語

ビスフォスフォネート連日投与による骨芽細胞・骨細胞への影響について
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
日本骨形態計測学会, 日本語

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微細構造の変化
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
日本骨形態計測学会, 日本語

骨粗鬆症に伴う疼痛と骨代謝回転状態との関係
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
日本骨形態計測学会, 日本語

骨・ミネラル代謝異常症診療の進歩 Ca感知受容体関連疾患の最新の知見とその治療
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
(一社)日本内分泌学会, 日本語

特集 骨と幹細胞 Review 骨の微細環境における細胞性ネットワーク -組織学･微細構造学的見解から-
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
医薬ジャーナル社

【骨と幹細胞】骨の微細環境における細胞性ネットワーク 組織学・微細構造学的見解から
網塚 憲生, 山本 知真也, 長谷川 智香, Clinical Calcium, 24, 4, 475, 485, 2014年03月
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), (株)医薬ジャーナル社, 日本語

Klotho欠損環境における骨細胞と基質石灰化
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
(株)メディカルレビュー社, 日本語

特集 バイオミネラリゼーション ～結晶学から臨床まで～ Seminar 骨基質バイオミネラリゼーションの顕微解剖学
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
医薬ジャーナル社

特集 バイオミネラリゼーション ～結晶学から臨床まで～ Topics アルカリフォスファターゼと低フォスファターゼ症
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
医薬ジャーナル社

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 (Web), 2014, 2014年

αKlotho遺伝子欠損マウスの下顎臼歯部歯槽骨における組織化学的解析
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

c-src遺伝子欠損マウスにおける破骨細胞と骨芽細胞のカップリング現象
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

副甲状腺ホルモン投与で誘導される骨細胞周囲の骨基質変化について
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
日本語

ビスフォスフォネート投与中止後における骨の細胞群の組織化学的検索
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
日本語

klotho遺伝子変異マウスの血管石灰化・血管骨化における組織学的検索
長谷川智香, 山田珠希, 佐々木宗輝, 笹野泰之, 網塚憲生, 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
日本語

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, 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
日本語

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 (Web), 2014, ROMBUNNO.O1‐C15 (WEB ONLY), 2014年
日本語

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 (Web), 2014, ROMBUNNO.O1‐C14 (WEB ONLY), 2014年
日本語

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, 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
日本語

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
日本語, 機関テクニカルレポート，技術報告書，プレプリント等

【バイオミネラリゼーション〜結晶学から臨床まで〜】骨基質バイオミネラリゼーションの顕微解剖学
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
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), (株)医薬ジャーナル社, 日本語

【バイオミネラリゼーション〜結晶学から臨床まで〜】アルカリフォスファターゼと低フォスファターゼ症
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
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), (株)医薬ジャーナル社, 日本語

マウス切歯歯根膜におけるペリオスチンの役割 細胞外マトリックスとの組織学的関連性
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
(公社)日本矯正歯科学会, 日本語

特集 くる病･骨軟化症 UPDATE Seminar 骨細胞の機能とFGF23/klothoシステム
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
医薬ジャーナル社

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 (Web), 2013, ROMBUNNO.P2‐50 (WEB ONLY), 196, 2013年09月
(一社)歯科基礎医学会, 日本語

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, 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
(一社)歯科基礎医学会, 日本語

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 (Web), 2013, ROMBUNNO.O‐75 (WEB ONLY), 140, 2013年09月
(一社)歯科基礎医学会, 日本語

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 (Web), 2013, ROMBUNNO.O‐48 (WEB ONLY), 141, 2013年09月
(一社)歯科基礎医学会, 日本語

【くる病・骨軟化症UPDATE】骨細胞の機能とFGF23/klothoシステム
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
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), (株)医薬ジャーナル社, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

副甲状腺ホルモン(PTH)間歇投与における投与量・投与頻度が骨形成に及ぼす影響について
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
ライフサイエンス出版(株), 日本語

レプチン受容体遺伝子変異(db/db)マウスの卵巣摘出後における骨組織の変化について
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
ライフサイエンス出版(株), 日本語

Klotho遺伝子変異マウスで生じた血管骨化における組織学的検索
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
ライフサイエンス出版(株), 日本語

【骨細胞:骨を制御する司令塔】骨の微細環境における骨細胞
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
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), (株)メディカルレビュー社, 日本語

骨粗鬆症に伴う疼痛と骨組織の酸性環境変化
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
(公社)日本整形外科学会, 日本語

ミノドロン酸の同位体顕微鏡による骨組織分布と破骨細胞に及ぼす影響
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
日本骨形態計測学会, 日本語

副甲状腺ホルモンの間歇投与頻度が骨の細胞群に及ぼす作用 モデル動物を用いた形態学的検索
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
日本骨形態計測学会, 日本語

副甲状腺ホルモン投与による骨細胞性骨溶解に関する微細構造学的検索
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
日本骨形態計測学会, 日本語

オスミウム浸軟法による破骨細胞波状縁の立体微細形態について
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
日本骨形態計測学会, 日本語

kl/klマウスは血管石灰化だけでなく血管骨化を示す 微細構造元素分析および組織形態解析
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
日本骨形態計測学会, 日本語

骨粗鬆症治療の新展開 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
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
(一社)日本骨代謝学会, 日本語

FGF23/klotho軸の破綻は血管石灰化だけでなく血管骨化を誘導する
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
(一社)日本骨代謝学会, 日本語

III型Na輸送担体過剰発現ラットにおける大動脈壁の微細構造の変化について
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
(一社)日本骨代謝学会, 日本語

卵巣摘出を行った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, 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
(一社)日本骨代謝学会, 日本語

メラノコルチン2受容体ノックアウトマウスにおける骨遺伝子発現解析
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
(一社)日本骨代謝学会, 日本語

【最新の骨粗鬆症学-骨粗鬆症の最新知見】骨の基礎研究 骨の構造と機能 骨の細胞と組織
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
(株)日本臨床社, 日本語

【研究医養成】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
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
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), (一社)日本解剖学会, 日本語

理解を助けるトレーニング問題 テリパラチドの間歇投与が骨の細胞や基質に及ぼす作用について
網塚憲生, CLINICAL CALCIUM, 23, 3, 112, 112, 2013年02月28日
医薬ジャーナル社

特集 骨評価法の進歩 Seminar PTH治療による骨組織変化-動物実験モデル-
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
医薬ジャーナル社

【骨評価法の進歩】PTH治療による骨組織変化 動物実験モデル
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
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), (株)医薬ジャーナル社, 日本語

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 (Web), 2013, 2013年

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 (Web), 2013, 2013年

Annexin a5による骨と腱・靭帯付着部(enthesis)形成の調節
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
日本語

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, 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
日本語

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 (Web), 2013, ROMBUNNO.O‐79 (WEB ONLY), 2013年
日本語

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, 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
日本語

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 (Web), 2013, ROMBUNNO.O‐49 (WEB ONLY), 2013年
日本語

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
日本語, 研究発表ペーパー・要旨（全国大会，その他学術会議）

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 (Web), 2013, ROMBUNNO.O‐112 (WEB ONLY), 2013年
日本語

卵巣摘出ラットの根間中隔歯槽骨における骨改造とスクレロスチンの局在
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
(株)メディカルレビュー社, 日本語

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

骨形成と骨吸収の先端研究から探る、骨修復促進のストラテジー 骨細胞の細胞生理における形態学的アプローチ 骨細胞による石灰化骨基質の調節
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
(一社)日本生理学会, 日本語

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
北海道歯学会, 日本語

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
北海道歯学会, 日本語

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
(一社)歯科基礎医学会, 日本語

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
(一社)歯科基礎医学会, 日本語

骨の微細構造学(第4回) 破骨細胞 骨を食べる巨人
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
(株)メディカルレビュー社, 日本語

骨粗鬆症の動物モデル 骨粗鬆症のモデル動物における骨の細胞群
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
(公社)日本整形外科学会, 日本語

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
網塚 憲生, 日本整形外科学会雑誌, 86, 8, S1212, S1212, 2012年08月
(公社)日本整形外科学会, 日本語

【ビタミンDの新たな展開】骨に対する活性型ビタミンDの直接作用 新規ビタミンD製剤の骨吸収抑制と骨形成促進
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
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), (株)日本メディカルセンター, 日本語

骨粗鬆症治療薬によるリモデリング調整を介した骨質への効果 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
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
(一社)日本骨代謝学会, 日本語

慢性腎臓病と骨・ミネラル代謝 klotho欠損マウスにおける血管石灰化の組織化学的・微細構造学的検索
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
(一社)日本骨代謝学会, 日本語

ビタミンD最前線 エルデカルシトールがin vivoの骨に及ぼす細胞学的作用について
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
(一社)日本骨代謝学会, 日本語

副甲状腺ホルモン投与で誘導される骨小腔周囲の骨基質溶解について
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
(一社)日本骨代謝学会, 日本語

長骨の部位による骨細胞ネットワークの立体微細形態の違いについて
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
(一社)日本骨代謝学会, 日本語

Appeal to eye 骨の微細構造学(第3回) 前骨芽細胞 その多様性と機能
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
(株)メディカルレビュー社, 日本語

PTH治療と骨形態 PTH投与における骨の細胞群の動向について
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
日本骨形態計測学会, 日本語

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, 北海道歯学会学術大会プログラム講演内容抄録, 2012, 8, 2012年04月
日本語

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, 北海道歯学会学術大会プログラム講演内容抄録, 2012, 9, 2012年04月
日本語

PTH間歇投与後の前骨芽細胞における組織化学的検索
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
(株)メディカルレビュー社, 日本語

塩酸セベラマー投与による血管石灰化抑制効果の検討
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
(一社)日本腎臓学会, 日本語

klotho遺伝子欠損マウスの血管石灰化における形態学的検索
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
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。, ライフサイエンス出版(株), 日本語

Bone-Orchestrating Cells, Osteocytes
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
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., 北海道歯学会, 英語

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

骨再生バイオマテリアルと間葉系幹細胞併用による骨再生向上の解析 培養技術を応用した新しい骨再生法の展開
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
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), 日本歯科医学会, 日本語

Appeal to eye 骨の微細構造学(第2回) 卵巣摘出ラット骨粗鬆症に対する新規ビタミンD製剤の効果
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
(株)メディカルレビュー社, 日本語

【骨形成促進薬としてのPTH】PTHの骨形成促進作用における骨の細胞群の役割
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
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), (株)医薬ジャーナル社, 日本語

Jausen型PTH/PTHrP受容体の機能異常の解析
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
英語, 記事・総説・解説・論説等（国際会議プロシーディングズ）

インプラント周囲骨細胞の免疫組織化学的検索
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
日本語

【FGF23/Klotho研究の進歩】骨細胞の機能とFGF23 Klotho遺伝子欠損マウスと野生型マウスとの比較
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
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), (株)日本メディカルセンター, 日本語

Appeal to eye 骨の微細構造学(第1回) 骨を支える細胞 骨細胞
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
(株)メディカルレビュー社, 日本語

klotho遺伝子欠損マウスの血管石灰化における形態学的検索
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
ライフサイエンス出版(株), 日本語

卵巣摘出ラットにおけるエルデカルシトールの効果
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
(株)メディカルレビュー社, 日本語

【ビタミンDの新たな展開】エルデカルシトールによるラット骨組織微細形態の変化 エルデカルシトールの骨吸収抑制作用と骨形成作用
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
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), (株)医薬ジャーナル社, 日本語

ULTRASTRUCTURAL ASSESSMENT FOR VASCULAR CALCIFICATION IN THE AORTA OF KLOTHO-/- MICE
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
SPRINGER LONDON LTD, 英語, 研究発表ペーパー・要旨（国際会議）

ENDOCHONDRAL OSSIFICATION IN MICE OVEREXPRESSING TISSUE NONSPECIFIC ALKALINE PHOSPHATASE DRIVEN BY TYPE II COLLAGEN PROMOTER
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
SPRINGER LONDON LTD, 英語, 研究発表ペーパー・要旨（国際会議）

klotho-/-マウスにおける骨細胞産生蛋白の局在と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, Journal of Oral Biosciences, 53, Suppl., 113, 113, 2011年09月
(一社)歯科基礎医学会, 日本語

klotho遺伝子欠損マウスの血管石灰化における組織化学的検索
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
(一社)歯科基礎医学会, 日本語

レプチン受容体遺伝子変異マウス(db/dbマウス)における骨基質ミネラルと骨細胞産生因子の形態学的解析
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
(一社)歯科基礎医学会, 日本語

骨代謝における新領域研究の挑戦 血管石灰化の機序における微細構造学的アプローチ
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
(一社)歯科基礎医学会, 日本語

副甲状腺ホルモン投与による骨細胞性骨溶解の可能性について
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
(一社)歯科基礎医学会, 日本語

ペリオスチン欠損がマウス切歯歯根膜の基質線維改変に与える組織学的影響について
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
(一社)歯科基礎医学会, 日本語

卵巣摘出ラットの下顎歯槽骨におけるsclerostinの組織学的観察
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
(一社)歯科基礎医学会, 日本語

マウス肋骨骨折の治癒過程におけるFGF23の組織学的検討
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
(一社)歯科基礎医学会, 日本語

カニクイザル関節炎モデルの第二指近位関節における組織化学的解析
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
(一社)歯科基礎医学会, 日本語

ビタミンDアナログ(Eldecalcitol)が卵巣摘出ラットの脛骨に及ぼす効果について
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
(一社)歯科基礎医学会, 日本語

層板骨基質線維の立体構築に関わる骨芽細胞の役割について
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
(一社)歯科基礎医学会, 日本語

MMP-13遺伝子欠損マウスの軟骨内骨化における組織化学的解析
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
(一社)歯科基礎医学会, 日本語

RANKL遺伝子欠損マウスの破骨細胞様大型細胞における組織化学的検索
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
(一社)歯科基礎医学会, 日本語

骨組織におけるオステオカルシンと基質グラ蛋白の局在について
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
(一社)歯科基礎医学会, 日本語

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
北海道歯学会, 日本語

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
北海道歯学会, 日本語

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
北海道歯学会, 日本語

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
北海道歯学会, 日本語

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
北海道歯学会, 日本語

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
北海道歯学会, 日本語

骨細胞の形態と機能における最近の知見
網塚 憲生, Dental medicine research, 31, 2, 209, 209, 2011年07月31日
日本語

卵巣摘出ラット脛骨の海綿骨におけるビタミンDアナログ-Eldecalcitol-の投与効果について
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
(一社)日本骨代謝学会, 日本語

アルカリフォスファターゼトランスジェニックマウスにおける基質石灰化機構の解明
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
(一社)日本骨代謝学会, 日本語

klotho-/-環境における骨細胞は基質ミネラル維持不能を示す
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
(一社)日本骨代謝学会, 日本語

血管石灰化における微細構造学的機序の解明 klotho-/-マウス血管平滑筋細胞のtrans-differentiationと基質小胞形成
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
(一社)日本骨代謝学会, 日本語

ペリオスチン遺伝子欠損マウスの切歯歯根膜の基質線維改変における組織化学的検索
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
(一社)日本骨代謝学会, 日本語

層板骨における骨芽細胞突起とコラーゲン線維の立体構築形成との関係について
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
(一社)日本骨代謝学会, 日本語

マウス肋骨骨折の治癒過程におけるFGF23の組織学的観察
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
(一社)日本骨代謝学会, 日本語

歯周組織形成過程におけるAnnexin a5の発現と機能
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
(一社)日本骨代謝学会, 日本語

卵巣摘出ラットの下顎歯槽骨におけるsclerostinの局在
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
(一社)日本骨代謝学会, 日本語

高骨代謝回転モデル(OPG欠損)マウスにおけるDMP-1とsclerostinの産生
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
(株)メディカルレビュー社, 日本語

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
網塚 憲生, 日本骨形態計測学会雑誌, 21, 2, S53, S53, 2011年05月
日本骨形態計測学会, 日本語

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, 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
日本語

Klotho遺伝子欠損マウス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, 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
日本語

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, 北海道歯学会学術大会プログラム講演内容抄録, 2011, 10, 2011年04月
日本語

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, 北海道歯学会学術大会プログラム講演内容抄録, 2011, 7, 2011年04月
日本語

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, 北海道歯学会学術大会プログラム講演内容抄録, 2011, 12, 2011年04月
日本語

糖尿病性・ステロイド性骨粗鬆症に対する骨質を踏まえた病態解明アプローチ 骨細胞の障害機序および基質ミネラルの異常について
網塚 憲生, 医科学応用研究財団研究報告, 28, 355, 359, 2011年02月
(公財)鈴木謙三記念医科学応用研究財団, 日本語

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, 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
英語, 記事・総説・解説・論説等（国際会議プロシーディングズ）

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
英語, 記事・総説・解説・論説等（国際会議プロシーディングズ）

【骨・関節疾患の動物モデル】骨粗鬆症 OPG欠損マウスの骨病態
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
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), (株)医薬ジャーナル社, 日本語

PTH/PTHrP受容体トランスジェニックマウスにおける骨・軟骨異常
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
(公社)日本小児歯科学会, 日本語

基質からみた硬組織の調節機構 石灰化基質に対する骨細胞の作用
網塚 憲生, 李 敏啓, 山本 恒之, Journal of Oral Biosciences, 52, Suppl, 88, 88, 2010年09月
(一社)歯科基礎医学会, 日本語

オステオプロテジェリン遺伝子欠損マウスにおける骨細胞産生蛋白の局在
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
(一社)歯科基礎医学会, 日本語

骨芽細胞特異的PTHrPトランスジェニックマウスの骨組織における解析
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
(一社)歯科基礎医学会, 日本語

Klotho遺伝子欠損マウスの脛骨骨端部における骨細胞産生蛋白の局在について
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
(一社)歯科基礎医学会, 日本語

根間中隔歯槽骨の骨細胞は骨改造に応じてsclerostin産生を調節する
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
(一社)歯科基礎医学会, 日本語

アスコルビン酸欠乏ラットの歯根膜異常における組織化学的検索
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
(一社)歯科基礎医学会, 日本語

ラット臼歯の歯周組織におけるペリオスチンとファイブロネクチンの局在について
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
(一社)歯科基礎医学会, 日本語

歯科インプラント周囲における骨細胞・骨細管系の組織化学的変化
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
(一社)歯科基礎医学会, 日本語

有細胞セメント質層板構造に関する走査型および透過型電子顕微鏡検索
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
(一社)歯科基礎医学会, 日本語

RANKL遺伝子欠損マウスにおける骨・軟骨基質を取り込む細胞と活性型骨芽細胞の組織化学的検索
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
(一社)歯科基礎医学会, 日本語

【骨質】骨細胞による骨基質と骨質の調節機構
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
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), (株)メディカルレビュー社, 日本語

Calcium Pros and Cons 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, Clinical Calcium, 20, 8, 1272, 1275, 2010年07月
(株)医薬ジャーナル社, 日本語

periostinは歯根膜においてNotch1の安定性を調節することで細胞生存を制御している
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
(一社)日本骨代謝学会, 日本語

卵巣摘出ラットの下顎歯槽骨における骨細胞機能に関連する蛋白の局在について
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
(一社)日本骨代謝学会, 日本語

RANKL-/-マウスにおける骨芽細胞と骨・軟骨基質を取り込む細胞の組織学的解析
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
(一社)日本骨代謝学会, 日本語

骨芽細胞特異的PTHrP過剰発現マウスの骨組織における組織化学的解析
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
(一社)日本骨代謝学会, 日本語

新たな骨形成不全症モデルマウスに見られた選択的スプライシング sealにおけるGT-AGルールの例外
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
新潟歯学会, 日本語

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
北海道歯学会, 日本語

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微細構造学的観察
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
(一社)日本解剖学会, 日本語

RANKL遺伝子欠損マウスの骨芽細胞系細胞における組織学的解析
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
日本骨形態計測学会, 日本語

type I collagen promoterを用いたPTHrP Tgマウスの骨芽細胞における解析
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
日本骨形態計測学会, 日本語

卵巣摘出ラットの根間中隔歯槽骨におけるDMP-1およびsclerostinの局在
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
日本骨形態計測学会, 日本語

セメント質のコラーゲン線維層板構造における走査型電子顕微鏡観察
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
日本骨形態計測学会, 日本語

吸収性プレートによる顎骨の再生
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
日本歯科医学会, 日本語

c-fos遺伝子欠損マウスにおける骨細胞の組織化学的解析
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
(一社)日本解剖学会, 日本語

卵巣摘出ラットの下顎歯槽骨における骨細胞機能に関連する蛋白の局在について
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

Ultrastructural Assessment of Mineral Crystallization and Collagen Mineralization in Bone
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
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., 歯科基礎医学会, 英語, 書評論文,書評,文献紹介等

骨細胞・骨細管系の役割における新知見
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
日本語

最新の歯学 骨細胞・骨細管系の役割における新知見
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
北海道歯学会, 日本語

骨の形態学から学んだこと
網塚 憲生, 北海道歯学雑誌, 30, 2, 131, 131, 2009年12月
北海道歯学会, 日本語

熱可塑性吸収プレートとβ-TCP補填材併用による骨増生法の開発 ラット頭蓋骨を用いた実験モデルにおける組織化学的検討
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
日本再生歯科医学会, 日本語

【ビタミンKと骨UPDATE】基礎的事項 ビタミンK2と骨質 構造特性
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
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), (株)医薬ジャーナル社, 日本語

骨細胞・骨細管系におけるFGF23とDMP-1の免疫局在
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
(株)メディカルレビュー社, 日本語

目で見るBone Biology(第20回) 骨細胞の機能
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
(株)先端医学社, 日本語

β-TCP補填材を用いた骨再生におけるカップリング現象の組織化学的解明
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
(NPO)日本口腔科学会, 日本語

【骨質因子としてのコラーゲン研究の進歩】コラーゲン線維の石灰化とその異常 微細構造学的見地から
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
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), (株)日本メディカルセンター, 日本語

活性型ビタミンD3は高カルシウム血症を引き起こす投与量においても骨吸収を抑制する
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
(一社)日本骨代謝学会, 日本語

【ホルモンと骨粗鬆症UPDATE】カルシウム・リン調節系ホルモンと骨 骨・軟骨に対するPTH/PTHrP作用における形態学的知見
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
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), (株)医薬ジャーナル社, 日本語

【骨粗鬆症の薬物療法 薬効評価と臨床研究の進歩】骨細胞の微細構造と機能
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
(株)日本臨床社, 日本語

骨の形態的解析法の進歩 骨・軟骨組織における免疫組織化学
網塚 憲生, 日本骨形態計測学会雑誌, 19, 1, S31, S31, 2009年05月
日本骨形態計測学会, 日本語

骨質を石灰化基質の微細構造から考える
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
日本骨形態計測学会, 日本語

系統組織学から見た骨改造の顕微解剖学 オーバービュー 小型哺乳類の骨改造
網塚 憲生, 李 敏啓, 解剖学雑誌, 84, Suppl., 71, 71, 2009年03月
(一社)日本解剖学会, 日本語

前骨芽細胞として分類される細胞群における微細構造学的検索
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
(一社)日本解剖学会, 日本語

骨リモデリングを調節する細胞群の新しい展開
網塚 憲生, 新潟歯学会雑誌, 38, 2, 129, 129, 2008年12月
新潟歯学会, 日本語

ビタミンK2は骨基質石灰化の微細構造学的プロセスを正常化する
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
(株)メディカルレビュー社, 日本語

【リン代謝をめぐる諸問題 FGF23を中心として】カルシウム・リン代謝における骨細胞の役割
網塚 憲生, Ubaidus Sobhan, Freitas Paulo, Sultana Sara, 李 敏啓, THE BONE, 22, 6, 729, 735, 2008年11月
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), (株)メディカルレビュー社, 日本語

ペリオスチンは、ゴルジ体においてフィブロネクチンとテネイシン-Cの結合の足場として機能し、脛骨過労性骨膜炎の発症を抑制する
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
(一社)日本骨代謝学会, 日本語

The Synergistic Role of Npt2a and Npt2c in Inorganic Phosphate Metabolism of Mice
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
AMER SOC BONE & MINERAL RES, 英語, 研究発表ペーパー・要旨（国際会議）

【バイオサイエンスの現在 骨膜シートの臨床応用法を探る】骨膜のバイオロジー
網塚 憲生, The Quintessence, 27, 9, 46, 47, 2008年09月
クインテッセンス出版(株), 日本語

吸収性プレートを用いた骨増生モデルにおける新生骨の組織学的解析
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
(NPO)日本口腔科学会, 日本語

【骨細胞】骨細胞・骨細管系の顕微解剖学
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
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), (株)日本メディカルセンター, 日本語

骨リモデリングと骨細胞・骨細管系の構築に関する組織学的検索
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
(株)メディカルレビュー社, 日本語

骨の形と機能への疾患分子的アプローチ 骨形成系細胞におけるPTHとPTH/PTHrP受容体分子の画像イメージングと機能解析
網塚 憲生, 日本骨形態計測学会雑誌, 18, 2, S37, S37, 2008年07月
日本骨形態計測学会, 日本語

骨の組織化学・免疫電顕の実際と今後の展開
網塚 憲生, 日本骨形態計測学会雑誌, 18, 2, S47, S47, 2008年07月
日本骨形態計測学会, 日本語

アスコルビン酸合成能欠如ラットの骨組織異常における組織化学的検索
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
日本骨形態計測学会, 日本語

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
日本骨形態計測学会, 英語

力学負荷モデル実験における軟骨細胞の組織学的変化
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
(株)メディカルレビュー社, 日本語

【骨・軟骨研究の基礎と臨床】基礎編 骨の細胞形態学
網塚 憲生, 李 敏啓, Freitas Paulo HL, Ubaidus Sobhan, THE BONE, 22, 3, 267, 274, 2008年06月
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), (株)メディカルレビュー社, 日本語

歯槽骨における骨芽細胞と骨細管系の役割における検討 骨細胞特異的死滅マウスを用いた組織学的解析
李 敏啓, 網塚 憲生, Clinical Calcium, 18, 7, 1029, 1030, 2008年06月
(株)医薬ジャーナル社, 日本語

高Ca尿を伴う遺伝性低リン血症性くる病(HHRH;heredirary hypophosphatemic rickes with hypercalciuria)発症機構の解明
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
(一社)日本腎臓学会, 日本語

骨の機械応答とosteocyte
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, 宇宙航空研究開発機構宇宙科学研究本部, 日本語

骨細胞特異的死滅マウスの骨小腔・骨細管系における微細構造学的検索
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
(一社)日本解剖学会, 日本語

アスコルビン酸合成能欠如による骨組織異常の形態学的検索
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
(一社)日本解剖学会, 日本語

【骨質の評価】ステロイド性骨粗鬆症における骨質
李 敏啓, 齋藤 直朗, 網塚 憲生, Clinical Calcium, 18, 3, 328, 335, 2008年02月
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), (株)医薬ジャーナル社, 日本語

Current concepts of bone biomineralization
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 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., Japanese Association for Oral Biology, 英語, 書評論文,書評,文献紹介等

ポリ乳酸プレートをGBR法に応用した骨増生について
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
(NPO)日本口腔科学会, 日本語

【新時代の骨粗鬆症学 骨折予防を見据えて】骨質 構成要素・評価方法など 骨基質の石灰化
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
(株)日本臨床社, 日本語

【骨形成・骨吸収の最近のトピックス】骨形成のトピックス 骨形態からみた骨形成調節機構 前骨芽細胞の形態と骨形成に対する役割
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
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), (株)メディカルレビュー社, 日本語

Mice overexpressing nucleolar PTHrP driven by type II collagen promoter show disordered distribution of chondrocytes in the epiphyseal cartilage
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
AMER SOC BONE & MINERAL RES, 英語, 研究発表ペーパー・要旨（国際会議）

The role of the type IIc sodium-dependent phosphate transporter (Npt2c), which is involved in hereditary hypophosphatemic rickets with hypercalciuria (HHRH)
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
AMER SOC BONE & MINERAL RES, 英語, 研究発表ペーパー・要旨（国際会議）