Research Projects -
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Development of biodevices for mechanical stimulation of cells in living bone and comprehensive analysis of their mechanotransduction mechanism
Grant number：23KK0163 2023.09 - 2026.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (International Collaborative Research)
Grant amount：\21060000 （ Direct expense: \16200000 、 Indirect expense：\4860000 ）
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Elucidation of early bone tissue architecture by in vivo volumetric image analysis-from both sides of cells and bone matrix
Grant number：23H03114 2023.04 - 2027.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
上岡 寛, 原 徹, 中條 真奈, 伊豆 弥生
Grant amount：\18720000 （ Direct expense: \14400000 、 Indirect expense：\4320000 ）
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Elucidation of early bone tissue construction through biological volume image analysis - from both cell and bone matrix perspectives -
Grant number：23K27804 2023.04 - 2027.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
上岡 寛, 原 徹, 中條 真奈, 伊豆 弥生, 王 紫儀
Grant amount：\18720000 （ Direct expense: \14400000 、 Indirect expense：\4320000 ）
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Challenge to elucidate the mechanism of cleft palate development from gene expression analysis linked to spatial information
Grant number：22K19624 2022.06 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
上岡 寛, 早野 暁, 大野 充昭
Grant amount：\6500000 （ Direct expense: \5000000 、 Indirect expense：\1500000 ）
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Systemic understanding about epigenetic regulatory mechanism focused on bone remodeling
Grant number：22H03297 2022.04 - 2026.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
井澤 俊, 加治屋 幹人, 早野 暁, 上岡 寛
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
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後天的な顎骨形態決定の鍵となる遺伝子の同定とそれに基づく不正咬合抑制への新戦略
Grant number：22K10271 2022.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
河野 加奈, 早野 暁, 山城 隆, 上岡 寛
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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多元歯形状データベースに基づくAIベース歯科治療支援システムの開発
Grant number：22H03615 2022.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(B)
諸岡 健一, 上岡 寛, 宮内 翔子, 河野 加奈
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
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マウス骨細管超可視化による骨細管ネットワーク構築・維持のメカニズムの解明
Grant number：22K10244 2022.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
村田 有香, 犬伏 俊博, 佐々木 淳一, 山城 隆, 上岡 寛
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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Analysis of dysregulated bone healing mechanisms via aryl hydrocarbon receptor by smoking and its regulation
Grant number：2022G023 2022.04 - 2025.03
公益財団法人 喫煙科学研究財団 一般研究（喫煙と内分泌・代謝） 一般研究
研究代表者, 井澤 俊, 共同研究者, 上岡 寛, 早野 暁
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Coevolution of facial formation due to changes in food and environment after the bottleneck of ethnic migration
Grant number：20H05133 2020.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area) Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
上岡 寛
Grant amount：\5460000 （ Direct expense: \4200000 、 Indirect expense：\1260000 ）
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HLA genome editing of dental pulp cells for iPS cell stock.
Grant number：19K22707 2019.06 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
手塚 建一, 上岡 寛
Grant amount：\6500000 （ Direct expense: \5000000 、 Indirect expense：\1500000 ）
【乳歯の採取と細胞単離】コロナ感染症拡大下で、新しい乳歯の獲得が困難な状況が続いた。しかし、岡山大学とは継続して、オンラインで連絡を取り合っており、再開可能になったらすぐに単離と送付を再開する予定である。
【歯髄細胞へのゲノム編集】ゲノム編集のために用いたdCas9/BE3用いた実験を繰り返しているが、目的の変異を持った細胞が得られていない。実験系の検証のために、Zinc Finger Nuclease (ZFN)を代わりに用いいたところ、再現性よくIn-Del変異によるHLA-A2の不活化が確認されたため、歯髄細胞においてはdCas9/BE3の変異導入効率がZFNと比較して著しく低いという結論に達した。
【トレーサビリティシステム開発】歯髄細胞の輸送や製造工程を記録するShizuiNetの開発は順調に進み、株式会社しずい細胞研究所を設立することができた。スイスのヘルスケア関連ブロックチェーンとパートナーシップを結び、ブロックチェーン技術開発企業との共同研究契約締結や、地元企業と協力して、クリーンルームで使用可能なバーコード読み取りデバイスのプロトタイプ作製にも成功した。これにより、実動機は、これまでに作製したものと合わせて5台になり、実際に実験室やクリーンルームに設置してのPoC実験を開始することができる。
【エクソソームを用いた歯周病治療の可能性】HLAハプロタイプホモ歯髄細胞が分泌するエクソソームによる歯周病予防効果を確認した論文がJournal of Periodontal Researchに掲載され、今後はゲノム編集を施した歯髄細胞からもエクソソームを単離して、評価することができるようになると思われる。 -
Elucidation of mechanosensor function acquisition process of osteocytes using 3D-volumetric image data
Grant number：19H03859 2019.04 - 2023.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
上岡 寛, 植田 紘貴, 早野 暁, 亀尾 佳貴, 原 徹
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
本年度は、骨細管内の構造解析から得られた流体-構造連成解析を進めて流体応力が骨細胞の表面にどのように変形を与えているかを、共同研究者である亀尾佳貴先生を中心としたグループが国際誌Biomechanics and Modeling in Mechanobiologyに発表した。本研究では、骨細管壁と骨細胞突起を結ぶテザリングエレメントが骨細管内を流れる体液の移動で引っ張られ、細胞突起に付着する膜部位で10％を超える細胞膜変形を生じることが確認できた。この変形はメカニカルストレスにより細胞内カルシウム上昇などを生じるには十分な細胞膜変形量であることから、これら細胞突起周辺の微細構造が骨細胞の細胞突起をメカノセンサーとして重要な役割を果たしていることが示唆された。また、新たにFIB-SEM(Focused Ion Beam-Scanning Electron Microscopy)で取得した高解像度連続断層写真を用いて、これまでより広域の細胞突起の3次元形態解析を行った。得られた断層画像から細胞突起、骨細管壁を自動抽出するために機械学習を初めて応用することができた。結果、DSC(dice similarity coefficient)は、83%となり、人が手動で抽出する同等の程度まで精度を上げることができた。これにより抽出した3次元画像から形態計測を行い骨細管の微細構造を数値化することにも成功した。以上の結果をJournal of Bone and Mineral Metabolismに報告した。また、これまでの研究の成果を第80回日本矯正歯科学会学術大会で行われた日本学術会議歯学委員会臨床系歯学分科会主催の公開シンポジウム「進化・発生・メカニカルストレスから探る顎顔面形成・維持機構最先端」でシンポジストとして発表した。
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Elucidation of the complex promoting mechanism of bone formation and cognitive function using bio-multimodal analysis
Grant number：19K10405 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Ueda Hirotaka
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
To develop and apply the findings that vagus nerve stimulation promotes the secretory function of salivary glands and to clarify the effects of parasympathetic electrical stimulation of the autonomic nervous system on tissue blood flow in the salivary glands, masseter muscle, and mandibular periosteum in the oral and maxillary region, we performed vagus nerve stimulation in rodents. The results showed a close relationship between salivary gland tissue blood flow and salivation induced by vagus nerve stimulation, suggesting the existence of a muscarinic receptor-mediated control mechanism of tissue blood flow. Furthermore, the results suggest that vagus nerve stimulation affects tissue blood flow in the periosteum. Further studies are needed to elucidate the mechanism by which vagus nerve stimulation produces ischemia in the masseter muscle and its physiological significance in influencing tissue blood flow in the mandibular periosteum.
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Regulation of tooth movement-initiated mechanotransduction via osteocytic bone remodeling
Grant number：17H04413 2017.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
ISHIHARA Yoshihito
Grant amount：\16770000 （ Direct expense: \12900000 、 Indirect expense：\3870000 ）
Orthodontic tooth movement (OTM) is achieved by the remodeling of the periodontal ligament (PDL) and alveolar bone in response to balanced mechanical loading. We investigated the regulatory dynamics of the SOST/Scl expression generated by orthodontic tooth movement (OTM) in vivo and in vitro. Our results provide evidence to support that factors secreted by the PDL, including SOST/Sclerostin, control alveolar bone remodeling through osteocytic SOST/Sclerostin in OTM. In addition, our findings suggest that augmented mechanical stress-mediated Ca2+ oscillations in PDL enhance the production and release of bone regulatory signals.
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A Novel Method to Detect 3D Mandibular Changes Related to Soft-Diet Feeding
Grant number：17K17325 2017.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
KONO Kana, TANIKAWA Chihiro, YAMASHIRO Takashi, KAMIOKA Hiroshi
Grant amount：\4030000 （ Direct expense: \3100000 、 Indirect expense：\930000 ）
Soft-diet feeding is a widely-used experimental model for studying the association between the skeletal morphology and muscle-related loading on the bone. Traditionally, these studies have been based on two-dimensional (2D) radiographs in the lateral view. However, 2D observation cannot detect three-dimensional (3D) changes in detail. Therefore, we have newly developed and reported a modified surface-based analysis using micro-3D computed tomography (CT) to examine and detect 3D changes of the mandible associated with soft diet feeding.
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Grant number：17K17323 2017.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
Hayano Satoru, SHIMADA Akira, SAITO Megumu, KAMIOKA Hiroshi, KAWANABE Noriaki, Fukui Yuko
Grant amount：\4030000 （ Direct expense: \3100000 、 Indirect expense：\930000 ）
In this study, we focused on Diamond-Blackfan anemia (DBA), which is characterized by red blood cell aplasia and craniofacial defects. iPS cells derived from DBA patient was used in this study. Our current study indicates that craniofacial defect is caused by activation of p53-mediated cell death pathway which is induced by interaction between ribosomal proteins and MDM2.
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口蓋裂患者における鼻咽腔閉鎖率分析-VPIに対する治療基準の確立を目指して-
Grant number：17K11937 2017.04 - 2018.03
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
村上 隆, 飯田 征二, 片岡 伴記, 上岡 寛
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
期間中に来院した口蓋裂患者の中から本研究の目的を理解し賛同する者を被験者とするため、被験者のリストの作成を図った（但し、未成年で保護者の同意が得られない者、発達障害を有する者、多動性障害を有する者、MRI 撮像により障害を被る可能性のある者、言語評価が得られない者、従来の鼻咽腔閉鎖機能の評価が行えない者は除外する）。
データ採得を行う時期として、口唇裂・口蓋裂総合治療センターで通常診療時、音声言語評
価および鼻咽腔閉鎖機能を評価する時期と設定した（不必要な検査を行うことはない）。すなわち、データ採得時期は初診時、治療前、（軟口蓋挙上装置の使用の場合治療中も）、および治療後とした。鼻咽腔形態の撮像に際しては、被験者に仰臥位で、設定した専用プロトコルに従い発声を指示し、60 秒間MRI movie を撮像するため、撮像装置としてMRI（3.0T Magnetom Verio, Siemens, Erlangen, Germany）を用い、Gradient Echo sequence により撮影を行うよう研究プロトコルを検討した。音声言語の評価としては、現在日本で広く実施されている2 種類の聴覚判定法（遠城寺式・乳幼児分析発達検査表（九州大学小児歯科改訂版）、新版発音検査（千葉テストセンター））を用いて岡山大学病院口唇裂・口蓋裂総合治療センター検査室にて評価した。そして、鼻咽腔閉鎖機能の評価には、従来より定量可能な検査法であるセファログラムとナゾメータ検査を行うこととした。そのため、ナゾメータを新たに購入し、検査体制を整えた。 -
Roles of CCN2 and Rab14 in the formation of extracellular matrix
Grant number：16K11786 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Hoshijima Mitsuhiro
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
CCN2 and Rab14 strongly expressed in bone, cartilage and lung. To elucidate the roles of CCN2 and Rab14 in osteocyte maturation, we investigated the different expression of osteocyte-related genes between young osteocytes and developmentally mature osteocytes using 3D-cultured MLO-Y4 cells. As the results, in the mature MLO-Y4 cells, rab14, ccn2, col1a1, OCN, c-Fos, Cx43 and Panx3 mRNA expression were significantly increased in comparison with young cells. Furthermore, in the present study, we showed for the first time that intracellular Ca2+ levels were significantly increased in developmentally mature osteocytes in comparison with young osteocytes by flow-induced mechanical stress.
These findings show that the CCN2 and Rab14 plays an important role in the formation of extracellular matrix, and the intracellular Ca2+ responses in a 3D cellular network in osteocyte. -
Analysis of the new role of a collagen network for the morphogenesis during the bone modeling
Grant number：16H05549 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KAMIOKA HIROSHI, ADACHI Taiji, JAGER Edwin
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
We performed three-dimensional construction of collagen fibers in the osteogenic phase by using FIB-SEM. Furthermore, since this analysis extends to a cubic region of 25 micrometers per side, it was possible to capture a cellular network containing multiple osteocytes simultaneously. From these observations, processes extending from the osteoblasts to the matrix side have a certain regularity so as to avoid bundled collagen fibers. Furthermore, the bones pretreated with BAPN, which inhibits collagen fiber aggregation, cell processes extending from osteoblasts have no specific orientation. Thus, it has been revealed that changing the nature of bone matrix affects osteocyte network formation.
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The involvement of cell polarity in trabecular formation
Grant number：16K15837 2016.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
KAMIOKA HIROSHI
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
A knockdown experiment of Nesprin - 1 using siRNA for osteoblast cell line Saos 2 was performed. By introducing Si - Nesprin 1, it was confirmed by immunofluorescence that nuclear localization of Nesprin decreased. Furthermore, it was confirmed that si-Nesprin 1 suppresses expression at the protein level by the Western blotting method. Therefore, it was confirmed that si-Nesprin 1 was introduced into Saos 2 cells.
Next, culture experiments of knockdown cells using uneven surface structures were performed. As a result, there was no significant difference between the two in the arrangement of the cells. Next, in order to examine the change of Saos 2 in cell polarity in mechanical stress, cell extension experiment with flexer cell unit was performed. However, in Saos 2, no change in polarity to mechanical stress was observed, so Nesprin's involvement was not considered. -
後天的に顔面骨格形態を決定する新たな分子メカニズムの解明
Grant number：26463093 2014.04 - 2016.03
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
柳田 剛志, 上岡 寛, 久保田 聡
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
現代社会では食生活の変化に伴い咀嚼機能が低下し、顎骨が退化縮小した結果、不正咬合が増加したと言われている。しかし、食生活などの後天的な影響がどのようにして顎態を変化させるのか、その分子機構は未だ不明である。今回我々はその分子機構を調査するため以下の実験を開始した。
生後3週のICRマウスを2群に分け、粉餌、固形餌を与え5週齢まで飼育した。粉餌飼育群をさらに2群に分け、粉餌、固形餌を与え7週齢まで飼育した。その後同様に2群に分け9週齢まで飼育した。生後3週、5週、7週、9週の各時点においてサンプルを回収し実験を行った。小動物用X線CT装置にて撮影したマウス頭部を立体構築し、セファログラム分析法を応用してサンプル間の詳細な形態比較を行った。また各サンプルの咬筋からtotal RNAを回収し、筋機能を反映すると言われているMyh遺伝子群の発現をRealtime RT-PCR法により比較した。また、咬筋の筋線維特性を速筋と遅筋を分けることのできるNADH-TR染色を用いて観察し、顎骨の形態変化と遺伝子発現の変化及び筋線維特性の関連性を考察した。Micro-3DCTを用いてマウスの顎骨の形態を解析した結果、食餌の硬さの違いで下顎頭の高さ、歯槽骨の高さが変化した。また、Myh遺伝子群が食餌の硬さを変更させた後に応答し、発現を変化させていた。さらに、組織染色から筋線維の特性が変化した。マウスの顎骨の形態を詳細に解析した結果、粉末食を与えたマウスは骨格性ハイアングルの形態的特徴を有していた。特に生後3から7週の期間に与えた食餌の硬さが最終的な骨格形態に影響を与えることが示された。また、粉餌を与え続けたマウスでは硬餌を与えたマウスに比較してMyh4遺伝子の発現量が増加、Myh2遺伝子の発現量が減少し、これに伴い組織染色では速筋の比率が減少することが分かった。 -
Search for new mechanism of bone remodeling by the collagen nano-model analysis
Grant number：25293419 2013.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KAMIOKA HIROSHI, KAMEO YOSHITAKA, SUMIYOSHI KUMI, HARA TORU, ADACHI TAIJI
Grant amount：\17290000 （ Direct expense: \13300000 、 Indirect expense：\3990000 ）
Osteocytes form the cellular network by their long processes and act as mechanosensory cells in bone. However, it is unknown how this cellular network is formed and what kind of factors influence the network formation. Focused ion beam-scanning electron microscopy (FIB-SEM) has increasingly found use in biological research. The main application is in the acquisition of three-dimensional data by tomography. We therefore reconstructed three-dimensional images of the osteocyte network as well as the collagen fibrils during bone modeling. Based on the positional relationships of osteocytes and collagen fibrils, we analyzed the influence of the collagen bundle formation on the osteocyte network formation.
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Novel functions of Runx genes as causal genes for ectodermal dysplasia
Grant number：24249093 2012.04 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
Yamashiro Takashi, NAKAGAWA ICHIRO
Grant amount：\45240000 （ Direct expense: \34800000 、 Indirect expense：\10440000 ）
In this study, we investigated the possible roles of epithelial Runx1/Cbfb signaling in the organogenesis and craniofacial development. Using the epithelial-specific Runx1 knock-out and epithelial-specific Cfbf knock-out mice, we provided evidence that Runx1/Cbfb has indispensable roles in the development of the teeth, the salivary gland, and the palate. In salivary gland, Runx1 is involved in sexual dimorphism in the induction of the granular convoluted tubules of the submandibular gland in the presence of androgen. In the incisors, Runx1 is involved in the regulation of Lgr5-positive epithelial stem cells that differentiate into ameloblasts, through regulation of phosphorylation of Stat3 in the cervical loop of the growing incisors. In the palate, Runx1 epithelial-specific deletion led to the failed disintegration of the contacting palatal epithelium and that Tgfb3 is a critical downstream target in the pathogenesis of anterior cleft palate in the mutants.
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THE EFFECT OF MYOSTATIN ON BONE REMODELING
Grant number：24659909 2012.04 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
KAMIOKA HIROSHI, YANAGITA Takeshi
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
In order to examine the participation of muscle to the bone via humoral factor, we focused on the myostatin, which inhibits muscle growth. Firstly, we tried to examine the effect of myostatin on the bone formation. In the process of examining the bone formation, we could establish realtime calcium imaging of bone cells as well as 3D analysis of microstructure of bone.
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The possible role of endoplasmic reticulum stress in orthodontic tooth movement
Grant number：24792283 2012.04 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
MURAKAMI Takashi, YANAGITA Takeshi, KAMIOKA Hiroshi
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
The detailed mechanisms of orthodontic tooth movement have not been revealed. On the other hand, it was revealed that endoplasmic reticulum stress induced that cell survival and cell necrosis. The aim of this study was to examine the participation of the endoplasmic reticulum stress in periodontal membrane and the alveolar bone using an experimental tooth movement. Because the endoplasmic reticulum stress marker expressed in bone addition and the hypoxia situation, it was suggested that endoplasmic reticulum stress participated in the orthodontic tooth movement.
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The elucidation of molecular mechanisms of amelogenesis by epitherial-specific Runx knockout mouse
Grant number：22390390 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
HONJO Tadashi, YAMASHIRO Takashi, KAMIOKA Hiroshi
Grant amount：\19370000 （ Direct expense: \14900000 、 Indirect expense：\4470000 ）
Targeted deletion of Runx genes has revealed distinct roles for these proteins in development. Here, we report that epithelial Runx genes are involved in regulation of ameloblastdifferentiation. We analyzed the functional role of Cbfb and Runx1 in the enamel formation invivo, using a conditional genetic approach in which the Cbfb and Runx1 gene was inactivated in mouse epithelial cells(K14-Cre/Cbfbfl/fl, K14-Cre/Runx1 fl/fl). We found that mice deficient in epithelial Cbfb and Runx1 resulted in enamel aplasia in the incisors and in hypoplasia in the molars. The present results provide the genetic evidence that Runx1 and Cbfb genes function in the enamel formation in developing molar and incisors.
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Mechanical response in artificially controlled osteocyte network
Grant number：21592594 2009 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
KAMIOKA Hiroshi, SUGAWARA Yasuyo, KATANOSAKA Yuki
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
Osteocyte in bone are thought to be mechanosensory cells. They form cellular network. However, the role of osteocyte network is not well known. We employed the nano-patterning method to form artificially-formed osteocyte network and applied mechanical stress with fluid shear stress in micro-fluid flow chamber.
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New approach to the mechanism of the pain by orthodontic tooth movement
Grant number：21592596 2009 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MURAKAMI Takashi, KAMIOKA Hiroshi, YAMASHIRO Takashi
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
Orthodontic tooth movement evokes a pain. As for the past research to the mechanism of the pain, the focus has been placed on neural cells. This research newly focused on glial cells surrounding neurons, and searched how glial cells involved in the development of the pain by orthodontic tooth movement. This research revealed not only temporal distribution of activated glial cells but also the involvement of CGRP and VR1 in central nervous system during orthodontic tooth movement.
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歯の移動時における一酸化窒素(NO)の機能解明―NOSノックアウトマウスを用いて
Grant number：20592402 2008 - 2010
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
湊 雅直, 山城 隆, 本城 正, 上岡 寛, 黒坂 寛, 藤井 昭仁
Grant amount：\3250000 （ Direct expense: \2500000 、 Indirect expense：\750000 ）
歯に矯正力が加わると、歯根膜組織で炎症様反応を含む初期反応が生じる。歯の移動の痛みや歯根吸収といった矯正治療に伴う副作用の多くは、この初期反応に関連することが示唆されるものの、その発生の詳細は不明である。歯根膜には間葉組織、血管、神経組織といった異なる組織が混在し、これらの相互作用を含む反応を理解することは容易ではない。そこで本研究では、歯の移動によるこの初期炎症反応を強力に誘導する一酸化窒素(NO)の役割の詳細を検討することを目的とした。本年度においては、NOSノックアウトマウスの表現型を検討するための予備実験としてC57BLマウスを用いて実験条件の検討を行った。これまでの歯の移動実験では、ほとんどの実験動物にラットが用いて行われており、本年度の研究の目的として、遺伝子改変マウスにおいて骨や歯槽骨での表現型を検討するための、実験条件の詳細な設定を試みた。その結果、歯の移動方法として、これまでラットの歯を移動させるために用いられたエラスティックと比べて、弾力が高く、非常に細い500μmの弾性スレッドを用いる方法を考案した。組織像から、歯に適切な矯正力が加わり、過度な骨や歯の吸収が生じることなく歯が移動することが確認された。この実験モデルを用いて、TRAP染色をおこなった結果、歯の移動開始、6時間後、12時間後、24時間後、72時間後に、TRAP陽性細胞数を確認し、骨吸収の動態を確認するのが適切であることが明らかとなった。特に、ラットにおける歯の移動モデルと異なり、歯の移動開始6時間後にはTRAP陽性細胞数が増加することを見出した。また、in situ hybridizationによって骨代謝マーカー遺伝子のmRNAの分布を検討した。これらの研究結果を元に来年度はNOSノックアウトマウスを用いて、TRAP細胞の出現と骨代謝マーカーmRNAの発現動態を元に表現型の解析を行い、歯の移動においてNOSが果たす役割を検討する予定である。
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Possible involvement of sulfation of heparan sulfate proteoglycan in tooth regeneration.
Grant number：19390529 2007 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
YAMASHIRO Takashi, SAITO Masahiro, KAMIOKA Hiroshi, HARADA Hidemitsu
Grant amount：\19630000 （ Direct expense: \15100000 、 Indirect expense：\4530000 ）
糖鎖は生体のシグナルとしてはたらくタンパク質を多彩に修飾し、その生理的機能および伝達を制御する。本研究の目的は、歯や硬組織における形態形成と分化における糖鎖の役割を明らかにすることである。ヘパラン硫酸プロテオグリカン硫酸基分解酵素であるSulf1とSulf2遺伝子に注目し、その発現部位、ノックアウトマウスの表現型、またin vitroの解析の結果から、糖鎖の修飾が少なくともWntシグナルの制御を介して、歯の形態形成と分化に関与していることが明らかとなった。
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矯正的歯の移動における結合組織成長因子の役割-アポトーシスによる歯槽骨の改造-
Grant number：18659614 2006 - 2007
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
山本 照子, 上岡 寛, 黒田 晋吾, 湊 雅直
Grant amount：\3300000 （ Direct expense: \3300000 ）
結合組織成長因子(CTGF)は、in vivoにおける機械的刺激において、骨細胞、骨芽細胞で遺伝子発現することが知られているが、その役割は明らかではない。一方、in vitroにおける機械的刺激によって、CTGFがヒト腎線維芽細胞で発現し、アポトーシスを誘導したとの報告がされている。本研究は、マウス実験的歯の移動モデルを作成し、in situハイブリダイゼーション法を用いて歯周組織におけるCTGF mRNAの発現を検討した。また、TUNEL染色及びISOL染色にてDNAの断片化を経時的に検索し、歯周組織に発現するCTGFが歯槽骨リモデリング時の細胞のアポトーシスに関与している可能性を検討した。
機械的刺激による骨リモデリング時に、CTGF mRNAは骨細胞、骨芽細胞、破骨細胞に発現しており、CTGFが骨のリモデリングに関与していることが強く示唆された。CTGF mRNAを発現する骨細胞は、歯の移動開始から12時間後まで増加し、その後1日から21日まで経時的に減少した。すなわち、CTGF mRNAは歯の移動の機械的刺激が加わった初期において、歯槽骨圧迫側の骨細胞で一過性に多く発現することが明らかになった。また、歯槽骨圧迫側におけるTUNEL陽性骨細胞は、歯の移動開始から12時間後まで増加し、その後1日から21日まで経時的に減少した。この変化は骨細胞におけるCTGF mRNAの発現様相とほぼ一致していた。さらに、歯の移動12時間後では、歯槽骨圧迫側にISOL染色陽性を示す骨細胞が多く認められることから、骨細胞の細胞死はアポトーシスによると考えられる。以上のことから、実験的歯の移動時の骨細胞において、CTGFが発現しアポトーシスを誘導することによって、骨リモデリングに関与している可能性が示唆された。これらの結果は国際誌に投稿中である。 -
Virtual bone reflecting microstructure ofreal bone
Grant number：18592234 2006 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
KAMIOKA Hiroshi, YAMAMOTO Teruko
Grant amount：\3880000 （ Direct expense: \3400000 、 Indirect expense：\480000 ）
Aim: We applied electron tomography to the osteocytes in bone in order to make virtual bone.
Materials & Methods: Chick calvariae were desected from 16-day old chick embryo. The calvariae were treated by 1 mM collagenase and 5 mM EDTA and trimmed in 3x3-mm for further use. These sections were fixed with 3% paraformaldehyde for 24h, stained with 2% protargol solution containing copper beads for 48h as silver-staining, and embedded in paraffin. After these treatment, 2 μm sections were placed on fromvar coated cupper grids. For tomographic data collection, sections were dipped in a solution of colloidal gold for 1-2 min. Electron tomographic data collection was carried out with H-3000 ultra-high voltage EM (Hitachi) at 2 MV equipped with a 360 °-tilt specimen holder. Images were recorded on a Tietz Tem-Cam 4k x 4k-pixel F415S slow scan CCD camera (TVIPS) binned to 2k x 2k pixels. Alignment of the tilt series and reconstruction of 3D volumes from aligned images was performed by the weighted back-projection method by using a tomography software package IMOD.
Results: Stereo-pair image of osteocyte was taken by ultrahigh-voltage electron microscopy with tilt +/-7 °. Osteocyte clearly appeared with numerous processes and brunches. The tomograms were generated from two +1-60 ° tilt series of a 2.0 μm section of chick calvaria and the microgram were taken 2 ° intervals. We could observe the series with 0.3 μm interval in depth of osteocyte tomography. The final pixel size on the specimen was 8 nm. We could distinguish the osteocyte cell body and their processes with high-density dots of silver on the surface of cell membrane.
Conclusions: It is possible to observe silver-stained osteocytes in thick section (2 μm) by ultrahigh-voltage electron microscopy. In addition, high-resolution (8 nm) electron tomography was performed to the osteocytes in chick calvariae. -
Systematic study on the osteocyte network formation and their mechanical responsibility
Grant number：17209064 2005 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
YAMAMOTO Teruko, KAMIOKA Hiroshi, DEGUCHI Toru, SUGAWARA Yasuyo
Grant amount：\39780000 （ Direct expense: \30600000 、 Indirect expense：\9180000 ）
Aim: We examined mechanical property to analyze the change from osteoblasts to osteocytes. Furthermore, gap junctional intercellular communication among osteocytes in chick calvaria was examined using FRAP analysis. We established 3D cultures of osteoblast and osteocytes.
Materials & Methods: Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody. The elastic modulus of living cells was analyzed with Atomic Force Microscopy. Using Fluorescence Replacement After Photobleaching (FRAP) analysis we examined the effect of changes in pH_0, [Ca^<2+>]_e, and addition of PTH on GJIC in osteocytes in chick calvaria. Anti-Connexin43 (Cx43) immunolabelling was used to localize gap junctions.
Results: The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053±934 Pa, that of osteoid osteocytes was 7971±422 Pa and that of mature osteocytes was 4471±198 Pa. Cx43 immunoreactivity was detected in most of the osteocyte processes. FRAP analysis showed dye-coupling among osteocytes. In untreated osteocytes, fluorescence intensity recovered 43.7±2.2% within 5 min after photobleaching. Pretreatment of osteocytes with 18 α-GA, significantly decreased. When pH_0 was decreased, fluorescence recovery significantly decreased. Conversely, when pH_0 was increased, fluorescence recovery was significantly increased. When [Ca^<2+>]_e was increased from 1 to 25 mM, fluorescence recovery was significantly decreased. In bone fragments exposed to 1.0 to 10 nM rPTH for 3 h, replacement of fluorescence was significantly increased. Chelating intracellular calcium ions affected GJIC regulation by [Ca^<2+>]_e and PTH. There were significant differences between actin and microtubule cytoskeletons in the process of MC3T3-E1 cells and primary osteocytes.
Conclusions: There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells. Furthermore, our study showed for the first time that GJIC among osteocytes is regulated by the extracellular environment and by hormonal stimulation during bone remodeling. This method may be more biologically relevant to living bone than current methods. Osteoblasts processes may have a different functional role than the osteocyte dendritic network. -
Visualization of osteocyte differentiation in living bone
Grant number：16390606 2004 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KAMIOKA Hiroshi, TAKANO Teruko, FUKUNAGA Tomohiro
Grant amount：\14600000 （ Direct expense: \14600000 ）
First, we could succeeded to reconstruct osteocyte network in bone using IMARIS, NEURON TRACER, SURPASS software. We informed the results in international journal "BONE" in 2005. Precise information is in the paper. Briefly, we measured osteocyte morphometry in chick calvaria and also showed morphological differences between osteocytes and osteoblasts. This technique was applied to visualize living osteocytes in bone by using OB7.3 chick osteocyte specific antibody. Now, we are in the way to visualize osteoblasts and osteocytes in living bone.
Besides the morphological differences between osteoblasts and osteocytes, we for the first time demonstrated significant difference between the cells in response to mechanical stress, especially in fluid shear stress. In the study, osteocytes were less respositive to fluid shear stress than osteoblasts. It was believed that osteocytes are the chief mechanosensory cells in bone.
However, in our knowledge based on the present study, osteocyte showed less calcium response than osteoblasts. In addition, we elucidated the implication of different focal adhesion formation between the cells.
Therefore, we now showed morphological and functional differences in osteocytes by the grant this time. -
生骨組織中での骨細胞の機械的刺激応答能の検討
Grant number：14704052 2002 - 2003
日本学術振興会 科学研究費助成事業 若手研究(A) 若手研究(A)
上岡 寛
Grant amount：\21190000 （ Direct expense: \16300000 、 Indirect expense：\4890000 ）
平成14年度の研究により共焦点レーザー顕微鏡を用いて骨組織中の生きた骨細胞を蛍光的に可視化できることを確認した。また、同様の方法を用いれば細胞内カルシウムインディケーターであるFluo3を骨組織中の骨細胞に取り込まし、細胞内カルシウムの動態を観察することが可能であると思われた。平成15年度は、まず、骨系細胞の力学的負荷による細胞内カルシウムの動態を検討するために、骨細胞、骨芽細胞、ライニング細胞を骨組織より取り出し培養し、流体剪断応力を負荷してこれらの細胞の細胞内カルシウムの変化を追跡した。これにより、骨系細胞の細胞カルシウムの濃度、オシレーションの頻度などの基礎的な細胞内カルシウムの動態を得た。その結果、骨細胞、骨芽細胞、ライニング細胞は、ともに流体剪断応力に応答することが明らかになったが、予想に反して骨細胞のカルシウム応答能は、他の細胞に比べて非常に低いことがわかった。細胞内カルシウムの変動は各群で有意差を生じなかった。これらの結果から、in vitroでの骨細胞は機械的応答能が低いことが予測された。また、生骨への機械的負荷は現在負荷装置を開発中であり、今後詳細な情報は得られるものと考えられている。得られた結果と今回のin vitroの結果を比較することにより、骨細胞のまわりの環境が細胞の機械的応答能に影響をあたえるかどうかを検討することができる。
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CTGF gene delivery for enhancement the healing of mandibular ramus fracture
Grant number：13672152 2001 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
KURODA Shingo, KURATANI Tsuyoshi, KAMIOKA Hiroshi, TAKANO Teruko
Grant amount：\3900000 （ Direct expense: \3900000 ）
The purpose of the present study was to investigate the CTGF gene delivery for enhancing the repair of fractured condylar and mandibular ramus. Using six-week-old wistar rat, 12 hours, 1, 3, 5, 7, and 14 days after the fracture, mandible including fractured condylar and ramus were dissected and decalcified with EDTA. After embedded in paraffin, 5 μ, β horizontal and sagittal sections were made. RNA probes of CTGF, collagen type I, II, III, X, Cbfal and TGF 0 were used for in situ hybridization to examine the expression of these factors during fracture healing. CTGF were expressed not only hypertrophic but hyperplastic chondrocyte in the healing site and this expression was changed in the time course. The present findings suggest that CTGF may play an important roll for repair and healing in condylar fracture and this model is useful as a model of fracture healing.
Following experiments were performed in vitro and in vivo, to examine the optimum conditions of HVJ liposome method for the condylar fracture model. (1) Luciferase gene was delivered to mesenchymal cell, isolated from the bone marrow in mouse femur, by using HVJ-liposome method in vitro. Luciferase reporter assay was performed to evaluate delivery rate. (2) After the fracture, β-galactosidase gene was delivered by using HVJ-liposome method. Three days after gene introduction, X-gal staining was performed to evaluate the delivery rate. These pilot studies indicated that the gene delivery by using HVJ-liposome was efficient to the condylar fracture model.
CTGF gene delivered by using HVJ-liposome in rat is performed and both in situ hybridization and immuno-histochemistry were used to analyze the expression of CTGF during fracture healing. We are preparing the paper about these observations to submit to the international journals, and some of them have been accepted. -
Study on the mechanism for the periodontal ligament tissue regeneration using human periodontal ligament tissues derived from complex odontoma
Grant number：13672151 2001 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
TSUBOI Yoshiko, MIYAMOTO Manabu, KAMIOKA Hiroshi, TAKANO Teruko
Grant amount：\3900000 （ Direct expense: \3900000 ）
Orthodontic tooth movement depends on the active periodontal ligament remodeling and regeneration. To analyze this mechanism, we utilized odontoma PDL and tried to find the genes specific in odontoma PDL. Human PDL tissue derived from a premolar complex odotoma was excised, which was extracted from orthodontic reasons, and placed onto flexible bottom plate. One month later, the heterogeneous cells reached confluence and they were cloned. Each clone was collected at 6-8 passages and stocked. Cell morphology and growth and differentiation ability of each clone is analyzing.
Total RNA was extracted from the heterogeneous primary culture cells from the odontoma PDL and from normal tooth PDL. To investigate the difference in the expression of Msx-1, Msx-2, which has been reported to control the tooth development, in odontoma PDL and normal tooth PDL, total RNA was reverse transcribed into cDNA, followed by quantitative real-tune PCR, using specific primers. As a result, all RNAs expressed the Msx-1 and Msx-2 mRNAs, though the expression level varied with the cells. The morphology of the cells also differed from each other.
Furthermore, another periodontal ligament tissues from another complex odontoma were used and analyzed the expression of Msx-1 and Msx-2 and the same tendency was observed. These results suggested that the process for the formation of odontoma involves at least Msx-1 and Msx-2, but also other transcriptional factors. We are analyzing cDNA array method and differential display PCR method, to identity the genes that is expressed specific in odotoma PDL. We are also preparing to immortalize the cloned cells, and to identify the clone specific genes. -
Studies for linage of periodontal ligament cells by gene transfection technique.
Grant number：12671997 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MIYAMOTO Manabu, TAKANO Teruko, KAMIOKA Hiroshi
Grant amount：\3000000 （ Direct expense: \3000000 ）
To clarify a lineage of cells isolated from periodontal ligament (PDL), the following studies were undertaken. First, we constructed (he experimental protocol for introducing and expression of the foreign genes into primary culture of human PDL cells. We constructed the plasmid vector which have human telomerase gene at downstream for cytomegarovirus immediate early enhancer and promoter, and introduced it to the primary cell culture which were established by the outgrowth method from human PDL. The expression of telomerase gene in cloned cells selected by an antibiotic marker was confirmed by RT-PCR. Secondly, expression of alkaline phosphatase (ALP) which were known to be a classical marker for cells in osteoblastic lineage were analyzed in single cell derived-clones form human PDL cells. The cloned cells showed same morphology, however expressed different levels of ALP activities and showed different responses against dexamethasone. The RNA expression levels for ALP gene were almost identical among these clones, suggesting that the different levels for ALP activities were derived from translational and/or transmembrane process of ALP protein. Thirdly, we investigated the gene-expression profiles in different clones for human PDL cells. Total RNAs expressed in two different PDL clones, one is high ALP clone and other is low ALP clone, were analyzed by a microarray assay, and compared with each other in over 1,500 different genes. These PDL clones showed almost same RNA expression profiles and showed slight difference in several specific genes. Finally, we also analyzed the roles of nerve growth factors in mouse PDL clones.
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The role of cytoskeleton on the transaction of mechanical stress to biological response in osteocytes -Transfection of GFP gene to cytoskeleton
Grant number：12671998 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
KAMIOKA Hiroshi, MIYAMOTO Manabu, TAKANO Teruko
Grant amount：\600000 （ Direct expense: \600000 ）
Morphological changes of cytoskeleton in osteoblasts and osteocytes were investigated after subjection to fluid shear stress. It was observed that osteoblastic cell line MC3T3-E1 react to fluid shear stress and changes its cytoskeleton (especially, actin) after application of fluid shear stress of 12 dynes/cm2 for 1 hour. Hence, other cytoskeletal protein, such as vimentin and tubulin did not - change their morphology after adaptation of fluid shear stress. When the cells were microinjected with fluoresncence-labeled vinculin and subjected to fluid shear stress, it was confirmed that the assembly of vinculin was performed within 10 minutes after application of fluid shear stress. So, it was suggested that morphological changes after fluid shear stress happens very rapidly. However, when osteocytes were subjected to fluid shear stress, the cytoskeletal changes was not observed. Based on these results, the cytoskeleton in osteocytes was very stable comparing to osteoblasts. Next, we focused on the stability of osteocyte morphology and observed the three dimensional distribution of osteocyte processes in bone using confocal microscopy and differential interference contrast microscopy.
Furthermore, we found that connective tissue growth factor is a candidate of marker of cytoskeletal changes in bone cells. -
Analysis of genes induced in human periodontal ligament cells respond to the mechanical stress
Grant number：12470460 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
TAKANO Teruko, TSUBOI Yoshiko, MIYAMOTO Manabu, KAMIOKA Hiroshi
Grant amount：\13800000 （ Direct expense: \13800000 ）
Human PDL tissue was excised from a premolar or a third molar, which were extracted from orthodontic reasons, and placed onto flexible bottom plate with Dalbecco's modified eagle medium (DMEM) containing 10 % fetal bovine serum. After 10 to 14 days fibroblastic cells were extended and after 1 or 3 months later, tension force was applied on the primary culture cells for 24h. The cells derived from the PDL tissue from the same patient without stress were used for control. Total RNA was extracted from both cells and reverse transcribed into cDNA, followed by PCR. The difference of the expression level of 1176 kinds of genes was analyzed by Micro Array method. Same experiments were done with other 3 patient's periodontal tissue and mRNA was collected at some points from early stage to late stage after mechanical stress. The genes reinforced by the mechanical stress, and those suppressed were identified. Some genes expressed strongly in early stage transiently, that decreased with time, and some showed reverse pattern. On the other hand, the mechanical tension force was applied using the cells that were subcultured to 3 passages. The results were almost the same to the experiments using the primary culture cells, although the number of genes responded to the stress were less and the amount of the expression change by stress was smaller. These results suggested that the aging accompanied with the long culture days or subculture lowed down the sensitivity for the mechanical stress or caused the selection for the cells that cannnot respond to the stress.
Now, we are preparing the paper about these observations to submit to the international journals, and some of them have been accepted. -
THE ELUCIDATION OF THE FUNCTION OF THE OSTEOCYTES AS CALCIUM SENSORY CELLS
Grant number：10671937 1998 - 1999
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MIKI Yoshiki, KAMIOKA Hiroshi, HIURA Kenji, MORIYAMA Keiji
Grant amount：\2100000 （ Direct expense: \2100000 ）
Recently, a calcium-sensing receptor (CaSR) from bovine parathyroid tissue was cloned, and Northern blot analysis revealed expression of CaSR mRNA in several tissues other than the parathyroid gland. In the present study, we fractionated bone cells from 1-to 2-day-old rat calvaria by digestion with collagenase and EDTA and examined the expression of CaSR mRNA in cells of the various fractions by reverse transcription polymerase chain reaction (RT-PCR) analysis and in situ hybridization histochemistry. Fraction I cells had an elongate shape, and cells from fraction II through IV had a stellate and polygonal shape. Cells from fraction VI were small and stellate-shaped, and possessed a large number of long cytoplasmic processes. Fraction m cells expressed alkaline phosphatase (ALPase) activity and osteocalcin (Osc) mRNA, suggesting them to be osteoblasts. On the other hand, only fraction vl cells expressed CaSR mRNA ; and they displayed no ALPase activity or Osc mRNA. Furthermore, fraction VI cells responded to the elevated extracellular calcium with a rapid elevation of their cytosolic calcium. The phenomenon was independent of membrane voltage and insensitive to organic calcium channel modulators, such as BAY K 8644, nifedipine, and nicardipine. These findings strongly suggest that rat mature osteocytes express a CaSR that responds to elevated extracellular calcium.
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BIOLOGICAL CONTROL OF OSTEOCLASTS AND OSTEOBLASTS
Grant number：07557133 1995 - 1996
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
SUMITANI Koji, HIURA Kenji
Grant amount：\7500000 （ Direct expense: \7500000 ）
In this study, we examined the biological controls of osteoblasts to osteoclasts in the activation and formation. To examine them, we used a culture system as follows,
(1) unfractionated cells obtained from mouse long bones for osteoclastic formation
(2) stromal cell free hematopoietic blast cells (mouse) for multinucleated cell formation
(3) cloned mouse osteoblastic cells (MC3T3-E1)
The results were obtained as follows,
1) The conditioned medium (CM) of MC3T3-E1 cells precultured for 3days contained only the inhibitory factor of multinucleated cell formation. On the other hand, both inhibitory factor and stimulatory factor were found in the CM of MC3T3-E1 cells precultured for 60days. No such effectors were found in the CM of fibroblasts.
2) The production of inhibitory and stimulatory factors were depressed by indomethacin and cycloheximide, respectively.
3) The media of MC3T3-E1 cells precultured for 3 and 60days contained prostaglandin E2 which inhibited multinucleated cell formation.
4) 1alpha, 25 (OH)_2D_3 was essential for stimulation of MNC formation by both MC3T3-E1 cells derived stimulatory factor and GM-CSF.
These results suggest that the inhibitory factor is prostaglandin E2 and the stimulatory factor is GM-CSF like protein.
5) Although TGF-beta1 has potent inhibitory effect on osteoclastic bone resorption, in the presence of 1alpha, 25 (OH)_2D_3, TGF-beta1 stimulated the formation of osteoclast.
6) TGF-beta1 inhibited multinucleated cell formation induced by 1alpha, 25 (OH)_2D_3, but the conditioned medium of TGF-beta1-treated MC3T3-E1 cells stimulated such formation.
7) Both bafilomycin A_1 (H^+-ATPase inhibitor) and acetazolamide (carbonic anhydorase II inhibitor) inhibited osteoclastic bone resorption stimulated by parathyroid hormone.
On the other hand, bafilomycin A1 did not affect procathepsin L secretion while acetazolamide inhibited the secretion.
These findings suggest that osteoblasts have the effect on osteoclastic formation through calcium regulating factors such as prostaglandin E2, GM-CSF and TGF-beta1. -
THE EFFECTS OF BONE MATRIX PROTEINS ON OSTEOCLAST FORMATION
Grant number：06454584 1994 - 1995
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B) Grant-in-Aid for General Scientific Research (B)
HIURA Kenji, KONDO Kahori, KAMIOKA Hiroshi, SUMITANI Koji
Grant amount：\800000 （ Direct expense: \800000 ）
Osteoclasts are multinucleated cells derived from hematopoietic precursors that have the unique ability to excavate bone matrix. In cultured rat marrow cells with bone particles (100mug/cm^2 ; 20-53mum), osteoclast-like cell number and bone resorbing activity increased by 1.5- and 30-fold, respectively. Recent observations on mice engineered to have targeted knockout of the c-src gene resulted in a phenotype bearing a recessive form of osteopetrosis, a consequence of a decrease in osteoclast function. since osteoclasts were still present in the homozygous src-mutants, it was postulated that the defect could be with the osteoclast itself. Therefore, we have used Chicken osteoclasts as a model system to study of signal transduction. Activation of osteoclasts by bone particles results in elevated tyrosine phosphorylation of three proteins, p200, p130, and p85. By immunoprecipitation and western blotting technique, we demonstrated that p85 was identified with Cortactin. This tyrosine phosphoprotein is a previously characterized cytoskeletal substrate for c-src in transformed cells. The data suggested that Cortactin was one of the members for src dependent signal transduction in activated osteoclast.
Furthermore, we demonstrated that cytosolic calcium elevation that occurred in osteocytes on exposure to elevated extracellular calcium was independent of membrane voltage and was insensitive to modulation by organic calcium channel modulators, namely, BAY K 8644, nicardipine, and nifedipine. However, an intracellular calcium antagonist such as TMB-8 affected the cytosolic calcium level, suggesting that the cytosolic calcium elevation was due to mobilization of this cation from an intracellular store. -
PARTICIPATION OF CATHEPSIN L ON ORTHODONTIC TOOTH
Grant number：06672056 1994 - 1995
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (C) Grant-in-Aid for General Scientific Research (C)
SUMITANI Koji, TAGAMI Kahori, HIURA Kenji
Grant amount：\2200000 （ Direct expense: \2200000 ）
Osteoclasts are multinucleated giant cells responsible for the resorption of the calcified extracellular bone matrix. The degradation of the bone matrix protein including type I collagen is a key process for osteoclastic bone resorption. It has been suggested that lysosomal cysteine proteinase in osteoclasts is essential to degrade bone collagen, but the proteinase(s) for this collagenolysis have not been identified.
In this study, the participation of cathepsin L on osteoclastic bone resorption was investigated and the results obtained were as follows.
1) In cultured rat bone marrow cells, parathyroid hormone (PTH)-stimulated bone resorption was markedly suppressed by all of specific and non-specific inhibitors of cathepsin L tested but not by a specific inhibitor (CA-074) of cathepsin B.
2) In rats with bone resorption stimulated by a low calcium diet, serum calcium level was decreased by an intraperitoneal injection of cathepsin Linhibitors but not by CA-074.
3) Activities of cathepsin L and B were shown to be more than 80% of total proteinase activity in the culture medium of rat bone marrow cells.
4) In bone marrow cells cultured on bone slices, PTH increased both bone resorption and cathepsin L activity in the medium and these increases were suppressed by calcitonin.
5) The cultured bone marrow cells secreted cathepsin L as 39kD molecule and the secretion was suppressed by calcitonin.
6) The 39kD cathepsin L purified from rat long bones was converted to 25kD cathepsin L under acidic condition.
7) Rat long bone-derived 25kD cathepsin L as well as purified cathepsin L of rat liver degraded type I collagen at pH 4.05.0.
These results suggested that cathepsin L secreted from osteoclast plays a crucial role in the degradation of bone matrix proteins including type I collagen during bone resorption. -
骨細胞のカルシウムの取り込みについて
Grant number：06772009 1994
日本学術振興会 科学研究費助成事業 奨励研究(A) 奨励研究(A)
上岡 寛
Grant amount：\900000 （ Direct expense: \900000 ）
矯正的歯牙移動に伴う骨リモデリングは、破骨細胞による骨吸収および骨芽細胞による骨形成によってなされている。しかし、成熟した骨組織中には、これら細胞の約10倍もの骨細胞が占めている。しかし、骨細胞は回りを硬組織にとり囲まれているため単離ができず、その機能については、いまだ明らかにされていない。
最近、我々は、ニワトリ胚頭頂骨より非常に高純度で(95%)で、多量(1匹当たり10,000個)の骨細胞を単離することができた(Isolation Method of Osteoidosteocytes .J Bone Miner Res:S202.1993)。この骨細胞を用いて、骨リモデリングにおける骨細胞の役割を探っていくことは、意義のあることと思われる。
【結果】 骨リモデリング時に骨吸収側では、細胞外カルシウム濃度が生理的濃度の約20倍の40mMにも上昇することが報告されているが、この高濃度のカルシウムに骨細胞が応答し、骨細胞が骨代謝を調節するメカニズムついて検討を行うために以下の実験を行った。
1)本学にあるACAS570 WORK STATIONをもちいて、まず単離した骨細胞の細胞内カルシウムの変化を指標として、細胞外カルシウムに対する骨細胞の応答を観察した。その結果、骨細胞は細胞外液のカルシウムに濃度依存的に応答することがわかった。
2)骨細胞内のカルシウムの上昇がどのような機序によってなされているかを検討するために、膜電位依存性カルシウムチャンネルアゴニストおよびアンタゴニストを用いて細胞内カルシウムの変動を追った。アゴニストであるBAY K8644は細胞内カルシウムを上昇することはなかった。また、アンタゴニストであるnicardipine,nifedipineは細胞外カルシウムによって惹起される細胞内カルシウムの上昇を抑制することができなかった。
3)また、二価の陽イオンであるニッケルおよびカドミウムは、カルシウムによって惹起される骨細胞の反応と競合し、外液カルシウム非存在下でも単独に細胞内カルシウムを上昇することがわかった。
これらのことから骨細胞は細胞外液にカルシウムチャンネル以外の経路を介して応答していることが示唆された。 -
矯正力を作用した骨細胞が骨芽細胞の増殖および機能に及ぼす影響について
Grant number：05454556 1993
日本学術振興会 科学研究費助成事業 一般研究(B) 一般研究(B)
河田 照茂, 石川 啓詞, 上岡 寛, 住谷 光治
Grant amount：\6900000 （ Direct expense: \6900000 ）
矯正力の作用機序を解明する上で、骨細胞に対する矯正力の影響を検討することは有用であると思われる。そこで、培養した骨細胞を用いて以下の実験を行った。
実験方法
閉鎖培養皿(ローズチャンバー)上に骨細胞を培養し静水圧を負荷し、矯正力を加えた骨細胞のモデル型を想定した。静水圧の調整はローズチャンバーに連結した点滴の高さを調整することにより行った。0, 25,100g/cm^2の各圧で24時間0.1%BSA含有α-MEMで培養された培養上清を回収し骨芽細胞培養系(MC3T3-E1細胞)に添加した。
結果及び考察
1.矯正力を負荷した骨細胞の培養上清が骨芽細胞の増殖に与える影響
Meyer J.E.& Grundman H.の方法により、MC3T3-E1細胞中のDNA量を測定したところ、矯正力を負荷した骨細胞の培養上清は、骨芽細胞数の多少の増加を促すものの有意差は認められなかった。
2.矯正力を負荷した骨細胞の培養上清が骨芽細胞のALP活性に与える影響
ALP活性は、100g/cm^2の培養上清において未処理対照に比べ増加傾向がみられた
以上の結果より矯正力を受けた骨細胞からは骨芽細胞の分化を促すような液性因子が産生されている可能性が示唆された。 -
矯正力が骨の細胞の接着機構に与える影響について
Grant number：05671712 1993
日本学術振興会 科学研究費助成事業 一般研究(C) 一般研究(C)
住谷 光治, 石川 啓詞, 上岡 寛, 河田 照茂
Grant amount：\2000000 （ Direct expense: \2000000 ）
破骨細胞は骨基質に存在するArg-Gly-Aspの3つの連続するアミノ酸を認識して骨基質へ接着するインテグリンが存在することが知られている。我々の興味の対象はメカニカルストレスを負荷された骨の細胞が破骨細胞の基質への接着に与える影響である。本研究は、これによって歯科矯正学的な歯の移動のメカニズムの一端を知ろうというものである。
1. マウス骨芽細胞株MC3T3E-1細胞とニワトリ卵より採取した骨細胞を各々培養し、静水圧(10-20g/cm^2)および培養面が軟膜でできた培養皿を変形させて物理的圧刺激(メカニカルストレス)を加えた。
(1)24時間静水圧を負荷した場合には、MC3T3E-1細胞および骨細胞ともにおおきな形態変化は見られなかった。48時間負荷によってMC3T3E-1細胞はコントロール群と比較して星形から多少、紡錘形に変化した。
(2)圧刺激を加えたMC3T3E-1細胞培養上清中に明らかにプロスタグランディンE_2が産生されていた。プロスタグランディンE_2を外因性に骨細胞に作用させると形態が比較的速やかに変化した。
(3)(2)の培養上清を2次元電気泳動で展開すると明らかに静水圧、物理的圧刺激負荷群でコントロール群と比較して新しい蛋白性産生物が見られた。
2. 24時間静水圧を負荷したMC3T3E-1細胞の培養上清を濃縮し、プラスチックディッシュ上のラット由来単離破骨細胞に与えても形態や剥離に対する影響は見られなかった。
以上より、矯正力によって骨芽細胞や骨芽細胞影響を受けプロスタグランディンE_2以外の蛋白性物質を産生促進させるが、それが破骨細胞のインテグリンに直接は影響してはいないことが分かった。