Research Projects -
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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.