Research Projects -
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Constructing an in vivo High-Resolution Cell Lineage Analysis System Utilizing Cellular Barcoding Technology
Grant number：24K21302 2024.06 - 2027.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Pioneering)
大野 充昭, 窪木 拓男, 王 紫儀
Grant amount：\26000000 （ Direct expense: \20000000 、 Indirect expense：\6000000 ）
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Understanding the Mechanisms of Tooth Germ Development Using Spatial Single-Cell Epigenome Analysis
Grant number：24K22188 2024.06 - 2027.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
窪木 拓男, 大野 充昭, 王 紫儀
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
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1細胞解析で解き明かすMSCsの免疫調節メカニズムとオートファジー
Grant number：24K02633 2024.04 - 2027.03
日本学術振興会 科学研究費助成事業 基盤研究(B)
秋山 謙太郎, 窪木 拓男, 大野 充昭
Grant amount：\18460000 （ Direct expense: \14200000 、 Indirect expense：\4260000 ）
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エピゲノムに着目した変形性関節症関連因子WISP1の発現制御機構の解明
Grant number：24K13044 2024.04 - 2027.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
前田 あずさ, 窪木 拓男
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
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新規センシング技術と機械学習による歯への機械的負荷を定量化する評価システムの構築
Grant number：23K09294 2023.04 - 2027.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
水口 一, 窪木 拓男, 水口 真実, 三木 春奈
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
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MR画像による長期予後調査より関節円板後部結合組織の偽円板化と予後との関連を探る
Grant number：23K09295 2023.04 - 2026.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
三木 春奈, 水口 一, 窪木 拓男
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
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MSC/2型Mφ1細胞間連携による炎症・再生連関促進の分子メカニズム解明
Grant number：22K19627 2022.06 - 2025.03
日本学術振興会 科学研究費助成事業 挑戦的研究(萌芽) 挑戦的研究(萌芽)
秋山 謙太郎, 窪木 拓男, 大野 充昭
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
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BMP-2誘導性骨髄から紐解く骨髄ニッチ細胞・CAR細胞の起源と発生メカニズム
Grant number：22K19625 2022.06 - 2024.03
日本学術振興会 科学研究費助成事業 挑戦的研究(萌芽) 挑戦的研究(萌芽)
大野 充昭, 窪木 拓男
Grant amount：\6500000 （ Direct expense: \5000000 、 Indirect expense：\1500000 ）
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咀嚼が唾液中BDNFならびに認知症発症に与える影響-ヒト高齢者を対象とした研究-
Grant number：22K10101 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) 基盤研究(C)
三野 卓哉, 窪木 拓男, 大野 彩, 大野 充昭, 山下 徹, 黒崎 陽子
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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時空間的トランスクリプトーム解析を応用した歯肉角化制御メカニズムの解明
Grant number：22K10100 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) 基盤研究(C)
縄稚 久美子, 納所 秋二, 大野 充昭, 窪木 拓男, 大野 彩
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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高齢者の低栄養における腸内細菌叢の役割解明と新規シンバイオティクス療法の開発
Grant number：22K10057 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) 基盤研究(C)
小山 絵理, 後藤 和義, 大野 彩, 窪木 拓男, 大森 江, 大野 充昭
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
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IoTセンサーを利用した高齢者の口腔関連日常生活動作の測定と要介護後期介入転換
Grant number：22K10076 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) 基盤研究(C)
藤原 彩, 窪木 拓男, 大野 彩, 水口 一
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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高齢者の固定性インプラントを活用したシステム非依存的補綴治療介入の有効性の検討
Grant number：22K10058 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) 基盤研究(C)
黒崎 陽子, 窪木 拓男, 三野 卓哉, 大野 彩
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
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Elucidation of the regulatory mechmisum of odontoblast differentiation by singl cell analysis and its application to therapeutics
Grant number：22H03280 2022.04 - 2025.03
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)
大野 充昭, 窪木 拓男
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
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Elucidation of the regulatory mechmisum of odontoblast differentiation by singl cell analysis and its application to therapeutics
Grant number：23K24538 2022.04 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
大野 充昭, 窪木 拓男, 王 紫儀
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
本研究では，歯科界において必須の情報でありながら未解決である象牙芽細胞マスター遺伝子の同定，さらにはダイレクトリプログラミングによる象牙芽細胞分化誘導法を確立する．具体的には，位置情報を付加した1細胞レベルでの解析に加えて，細胞分化の時間軸を加味した分化経 路推定解析を駆使し，候補転写因子群の抽出を行う．そして，iPS細胞樹立技術を逆手に取ったマスター遺伝子同定法を駆使して象牙芽細胞分化のマスター遺伝子を同定し，同定したマスター遺伝子や誘導した象牙芽細胞を利用して，象牙質再生療法の基盤技術を確立する事を目的としている．
2022年度に，生後5~7日齢のCol1a1-GFPマウスの歯胚を摘出後，酵素処理により細胞の単一化を行った.GFP陽性象牙芽細胞およびGFP陽性骨芽 細胞が含まれる CD45 (白血球)，Ter119 (赤血球)，CD31 (血管内皮細胞)陰性分画に存在する細胞をセルソーターにて分離し，約5000個の単一細胞を得て，シングル解析システム (10x chromium)にてscRNA-seq解析を実施した.そして，細胞のアノテーションを行い，間葉系幹細胞が象牙芽細胞へと分化する過程を，velocity解析およびtrajectory解析を駆使し，解析した．その結果，歯胚に存在するMki67陽性の細胞増殖能が高い間葉系幹細胞を同定することができた．また，この細胞が，全象牙細胞から象牙芽細胞へと分化する過程を明らかにすることができた．
今後，本結果から抽出された遺伝子に対し，機能解析を行う予定である． -
Development of tooth regenerative technology based on the spatiotemporal transcriptome analysis and iPS interference
Grant number：21H04842 2021.04 - 2025.03
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)
窪木 拓男, 大野 充昭, 辻 孝, 渡辺 亮, 宝田 剛志
Grant amount：\42510000 （ Direct expense: \32700000 、 Indirect expense：\9810000 ）
本申請研究では，「臓器としての歯の再生」を最終目的に，1細胞レベルでのRNA発現解析に加えて，時空間情報を加味した遺伝子発現解析法を駆使し，歯胚発生における1細胞レベル時空間的トランスクリプトームMapを構築し，これらのデータベースをもとに，iPS干渉法を応用し，歯原性上皮・間葉細胞の誘導方法を開発する.そして，器官原基法により，非歯原性細胞から，生理的機能を有した臓器としての歯を世界で初めて再生することを目的としている．
本年度は，歯胚発生における1細胞レベル時空間特異的トランスクリプトームMapを構築した．
具体的には，マウスE10.5，E11.5，E12.5，E14.5，E18.5の歯胚および非歯原性口腔粘膜組織を摘出し，酵素処理にて約1万細胞の単一細胞を得て，Single cell RNA-Seq (scRNA-Seq)解析し，どの細胞が，どの遺伝子を，どの程度発現しているか1細胞レベルで解析を行った．また，メッシュ状に位置情報となるインデックス配列が付加されたスライドガラスに，E10.5，E11.5，E12.5，E14.5，E16.5の歯胚を含むマウス頭部前頭断の凍結切片を貼り付け，HE染色を行い，組織学的情報を取得した．次に，スライド上でmRNAを単離，位置情報のインデックス配列が付加されたcDNAを合成し，ライブラリー作製後にシークエンスを行い，インデックス情報から，二次元空間での遺伝子発現情報を構築し，遺伝子発現Mapを構築した． -
変形性関節症におけるエピジェネティクスを介したWISP1遺伝子発現制御機構の解明
Grant number：21K10019 2021.04 - 2024.03
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)
前田 あずさ, 窪木 拓男, 大野 充昭
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
世界でも群を抜いた高齢化率を示している我が国において，医療費や介護給付費による財源圧迫を回避すべく健康寿命の延伸は喫緊の課題であり，国民が要支援となる最大の原因である変形性関節症 (OA) の発症原因や予防法の解明は，臨床的・医療経済学的に大変意義深い．これまでに変形性関節症の原因遺伝子のひとつとされているWISP1遺伝子に着目し，WISP1遺伝子の発現抑制下では変形性関節症の進行が抑制されることを証明してきたが，本研究では，変形性関節症の予防法の確立を視野に入れ，変形性関節症を発症した関節軟骨でWISP1遺伝子がどのように発現制御されているのか解明することを目的としている．
ヒト間葉系幹細胞 (hBMSCs) の軟骨分化過程ではDNAメチル基転移酵素 (DNMT3A) が高発現しており，DNMT3Aを強制発現させたhBMSCsでは軟骨分化が促進されることがわかっているが，軟骨細胞分化に伴い発現することが確認されているWISP1遺伝子が，軟骨細胞分化過程においてどのようにDNAメチル化修飾の影響を受けるのかを検討した．DNAメチル化阻害薬である5-aza-2-deoxycytidine (5-aza) で24時間処理したhBMSCsをマイクロマス培養法にて21日間培養し，軟骨細胞分化過程におけるWISP1遺伝子の発現パターンを定量性RT-PCR法にて評価したところ，コントロール群と比較して5-aza処理群ではWISP1遺伝子の発現は低下しており，脱メチル化によりWISP1遺伝子の発現が低下することが確認された． -
機械学習を応用した咀嚼機能低下を精度高く検出する新規検査方法，評価基準の開発
Grant number：21K09977 2021.04 - 2024.03
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)
水口 真実, 窪木 拓男, 水口 一, 三木 春奈, 小山 絵理
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
要介護高齢者の増大に対して，要介護状態の発症を遅らせ，健康寿命を延伸することが強く望まれている。近年，フレイルが高齢者の自立喪失の有意なリスク因子であると報告された。フレイルサイクルの一端に口腔機能，特に咀嚼嚥下機能の低下による低栄養がある。そこで，口腔機能低下に関するリスク因子を早期に発見し，早期に口腔機能，栄養状態の改善を図ることができれば，高齢者の要介護状態への転落を遅延できると考えている。
実際の食事時の筋電図を音声波形解析技術の応用と機械学習により，早期の咀嚼機能低下を類推する試みを開始したが，教師データ獲得が困難であったため，他のリスク因子を検討する中でACTN3遺伝子多型が抽出された。そこで遺伝子多型との関連を検討することとした。
咀嚼嚥下機能の維持，賦活を目的とし行われる筋機能訓練により，オーラルディアドコキネシスに加え，舌圧も改善することが知られている。この舌圧は骨格筋量よりも体幹筋量の影響を受け，舌機能や舌骨上筋群は速筋繊維が優位な筋と言われている。一方，サルコペニア発症時には速筋線維優位な筋線維の萎縮が生じ，速筋はサルコペニアの影響を大きく受ける。このサルコペニア発症のリスク因子の一つに，ACTN3遺伝子R577X多型による筋線維の萎縮が明らかとなっているが，これらと舌機能低下並びに嚥下障害を呈するリスクについての関連は明らかでない。
そこで本年度は，ACTN3遺伝子R577X多型を評価するための予備的検討を行った。特に，高齢者を対象にDNAを採取する必要があることから，被検者負担の少ない採取方法並びにその結果の妥当性について検討した。その結果，唾液中，頬粘膜の擦過（10回，20回）を行い，R577X多型を評価したところ，同一の結果が得られた。これより，頬粘膜の10回擦過によってR577X多型を評価するのに十分なDNA採取ができることが明らかとなった。 -
New treatment strategies for periodontal disease and peri-implantitis from the point of Macrophage autophagy abnormality
Grant number：21H03131 2021.04 - 2024.03
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)
秋山 謙太郎, 窪木 拓男, 大野 充昭
Grant amount：\17290000 （ Direct expense: \13300000 、 Indirect expense：\3990000 ）
本研究は，歯周病やインプラント周囲炎における病態形成メカニズムを，歯槽骨破壊局所に集積するMφの活性化に注目し，オートファジー異常の観点から解明するとともに，炎症性サイトカインの産生経路を特定し，免疫トレランス獲得につながる新たな検査技術や 新規組織再生療 法開発につなげることを目的としている. 本年度の研究実績の概要を以下に示す．
１）実験的マウス周囲炎モデルにおけるマクロファージの分布
週齢の異なるマウス(C57BL/6, 5週齢および50週齢)の下顎第一臼歯に5-0絹糸を結紮した結果，5週齢と比較して50週齢で明らかな歯槽骨破壊が観察された．また，蛍光免疫染色によるマクロファージの分布を確認したところ，50週齢で炎症巣周囲に多くのマクロファージが分布していた．特に，炎症性マクロファージであるCD80陽性M1の分布が観察され，抗炎症性であるCD206陽性M2の分布は５週齢と比べて少ないことがわかった．また，組織の免疫トレランス維持に重要な役割を果たすと考えられている間葉系幹細胞の分布を検討したところ，50週齢ではPDGFra陽性間葉系幹細胞の分布は少ないことがわかった．
２）週齢の違いによる間葉系幹細胞とマクロファージの相互作用
５週齢，50週齢それぞれから単離・培養したマクロファージと間葉系幹細胞をカルチャーインサートを用いて共培養したところ，５週齢間葉系幹細胞はM1からM2へのマクロファージの極性変化を強く誘導したのに対して，50週齢間葉系幹細胞ではあまり誘導されないことがわかった． -
Clarification of the mechanism of mesenchymal stem cell stemness using iPS interference method
Grant number：20K21679 2020.07 - 2023.03
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)
窪木 拓男, 渡辺 亮, 大野 充昭, 秋山 謙太郎
Grant amount：\6500000 （ Direct expense: \5000000 、 Indirect expense：\1500000 ）
近年，加齢変化が引き起こす疾患群に共通するホストの本質的変化として，ステムセルエイジングが注目されている。我々は加齢に伴い間葉系幹細胞(MSCs)の免疫調節能が著明に低下すること，また，骨芽細胞分化能の低下と脂肪細胞分化傾向への転換により，脂肪髄を呈することも明らかにしてきた。その結果，傷害組織で休眠から覚める，もしくは新たに動員されるMSCsの機能が低下し，局所の組織修復能や免疫調節能が低下，加齢性疾患の罹患感受性が上昇すると考えられる。したがって，骨髄MSCsの老化を防ぎ，幹細胞性が高いフラクションをいかに保つかが，これらの加齢性疾患におけるホストの病因の理解，さらには予防と治療に寄与するものと考えられる。そこで本研究は，骨髄MSCsの幹細胞性維持機構を解明することを目的に以下の計画を立てた。1)MSCs幹細胞性維持に関わる因子の探索:ヒト骨髄MSCsとヒト成人皮膚線維芽細胞 (hADFs)から，RNAを抽出し，RNA-Seqにて網羅的に比較検討を行い，転写因子に焦点を絞り，データベースを構築し，関連遺伝子を抽出する。2) 1)にて抽出した関連遺伝子から，iPS干渉法を駆使し，骨髄MSCsの幹細胞性維持に関わるマスター遺伝子の同定を試みる。また，このマスター遺伝子を利用して，hADFsからMSCsを作成する技術を確立する。今年度は，MSCs幹細胞性維持に関わる因子の探索を目的に，ヒト骨髄MSCsとhADFsから，RNAを抽出し，RNA-Seqにて網羅的に比較検討を行い，転写因子に焦点を絞り，データベースを構築し，関連遺伝子を抽出した。そして，抽出した転写因子の強制発現ベクターを作製し，iPS干渉法にて，さらなる絞り込みに成功した。
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人工知能による深層学習を応用した運動障害性咀嚼障害の多軸診断支援システムの開発
Grant number：20K10071 2020.04 - 2023.03
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)
大野 彩, 窪木 拓男, 森田 瑞樹, 菊谷 武, 百田 龍輔
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
本申請研究では、①咀嚼運動を撮影した動画から評価できる「咀嚼機能評価プロトコール」の診断結果が、十分な信頼性・妥当性を有するかどうかを、臼歯部移送試験や専門医の診断等と比較して評価する。また②そのプロトコールを用いて、患者および正常咀嚼者の動画撮影および診断を行い、教師データを収集する。そして、③教師データを人工知能（AI)深層学習に供し、AIによる運動障害性咀嚼障害診断システムを開発することを目的としている。
本年度は、人を対象とする医学系研究に関する倫理指針に則り、本研究計画について倫理審査委員会の承認を得た。そして、動画を用いた運動性咀嚼機能評価法の信頼性・妥当性の確認のため、まずは健常者にてプロトコールおよび咀嚼解析システムの精度確認を行った。その結果、撮影角度、影や眼鏡等の影響による運動検出精度の低下が明らかとなった。そのため、システムの改良を行い、運動検出精度を向上させることに成功した。 -
下顎骨後方移動術に伴う睡眠呼吸障害の発症リスクおよび施術基準の確立
Grant number：19K10382 2019.04 - 2023.03
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)
中村 政裕, 川邉 紀章, 片岡 伴記, 鬼頭 慎司, 森本 泰宏, 水口 一, 窪木 拓男, 宮脇 卓也
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
骨格性下顎前突患者に対し安全に下顎骨後方移動術を施行するためには、術後の上気道の狭窄やそれに伴う睡眠呼吸状態への影響に配慮しなければならない。過去の研究においても、下顎骨後方移動術を行った患者の上気道形態の変化は調べられているが、これらは覚醒時に撮影したセファログラムやコンピューター断層撮影画像を解析した静態評価であった。そのため、本研究は、上気道の動態および三次元解析が可能であるMRI movieおよびvolumetric MRIを用いて下顎骨後方移動術前後の睡眠時における上気道形態の変化を観察し、下顎骨の後方移動量と睡眠時の上気道の容量との関連を解明し、睡眠時の上気道形態変化と睡眠時呼吸状態との関連を調べることで睡眠呼吸障害の発症リスクを明らかにすることを目的としている。
本年度の研究実績として、研究を開始するにあたり、臨床研究実施計画書および研究説明書、同意書等を作成し、岡山大学臨床研究審査専門委員会への申請を行った。臨床研究審査専門委員会の承認が得られたため、患者への同意説明を開始した。 -
基底膜構成分子の誘導制御による低侵襲角化歯肉獲得療法の確立
Grant number：19H03841 2019.04 - 2023.03
日本学術振興会 科学研究費助成事業 基盤研究(B) 基盤研究(B)
前川 賢治, 窪木 拓男, 冨田 秀太, ハラ エミリオ・サトシ, 大橋 俊孝, 大野 充昭
Grant amount：\17030000 （ Direct expense: \13100000 、 Indirect expense：\3930000 ）
基底膜直下に存在する角化歯肉由来間葉細胞と非角化歯肉由来間葉細胞の遺伝子発現の相違を明らかにすることで，上皮細胞の角化に関わっている間葉細胞からのシグナル分子を抽出することが可能であると考える。そこで，令和元年度は，レーザーマイクロダイセクション法とRNA-Seq を組み合わせた候補因子の抽出を目的に，以下の実験を実施してきた。
間葉組織は，筋肉や脂肪など様々な組織を含むことから，マクロレベルで口蓋粘膜から間葉組織を採取すると，様々な組織を含んでしまう。基底膜直下の間葉組織の遺伝子発現解析を正確にするには，特異的に組織を採取することが可能なレーザーマイクロダイセクション法を用いる必要がある。そこで，マウスの口蓋粘膜（角化粘膜）と頬粘膜（非角化粘膜）の凍結組織切片を作製し，サンプルの厚み，固定方法，染色方法，レーザーの強度の調整等，様々な条件検討を行い，本組織において最適な条件を見出した。そして，これらのサンプルからRNAを抽出し，RNA-seqが可能な質の高いRNAが回収できていることを，TapStation (アジレント)にて確認した。
また、in vitroにおいて，角化粘膜の間葉組織に高発現している遺伝子をRNA-seq解析から抽出後，さらなる候補因子の絞り込みをin vitroにて行う予定である。そこで，令和元年度は，ヒト口腔扁平上皮癌由来の口腔粘膜上皮細胞であるTR146と，ヒト口腔粘膜細胞由来線維芽細胞を用いた三次元共培養実験モデルを構築すべく，コラーゲンゲルの種類，濃度や細胞の濃度などの条件検討を行い，正常な口腔粘膜組織に類似したin vitro モデル構築に適正な条件の絞り込みを終えた。 -
オーラルフレイル回避を目指した筋機能訓練の有効性検討と新規個別スキームの開発
Grant number：19K10225 2019.04 - 2023.03
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)
水口 一, 三木 春奈, 水口 真実, 窪木 拓男
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
摂食機能の低下の抑制が可能となれば，健常高齢者の要介護状態への転落を遅延できるかもしれなという研究仮設のもと，高齢者の筋機能維持を筋機能訓練の観点から検証することを当初の目的としていた。その一方で，睡眠時の口腔周囲筋の機能亢進が筋機能訓練の一助となりうる可能性にも着目した。その結果，高齢者の就寝時の筋活動量と筋機能との関連についても検討を行うこととなった。
第一に，就寝中の咬筋の筋活動を簡便かつ適正に定量化することを目的に，音声動画撮影を追加したpolysomnography（PSG）検査をもとに，筋電図による睡眠時筋活動検査システムについて検討を行った。しかしながら従来の睡眠時の口腔周囲筋の活動評価は，筋電図をもとに一定の閾値を超えた筋活動の頻度を基準としてきたため，掻痒や体動による筋電図波形の亢進も筋活動の亢進として誤認されてしまう場合があり，その信頼性に問題があることが明らかとなった。すなわち，我々が行った動画記録を伴うポリソムノグラフ（PSG）検査において，咬筋の筋活動が亢進したイベントのうち，80.7±16.0%が体動や掻痒等による非特異的な筋活動やアーチファクトに起因したものであった事が示された。
本研究結果は，筋電計単体による口腔周囲筋の評価には多くの偽陽性イベントが含まれていることを示すものであり，従来の検査方法，評価方法の妥当性を高く評価できないとう結果となった。事実，閉塞性睡眠時無呼吸（OSAS）症状が出現する際には低酸素状態となることから，交感神経活動の亢進，微小／睡眠覚醒，筋活動の亢進が出現し，その結果，筋活動の亢進が認められることが知られている。
そこで，検査が容易な筋電図検査において，真の口腔周囲筋の活動と偽の筋電図の亢進現象（OSAS関連性，嚥下関連性，動作関連性，その他）を識別する手法の確立が急務と考えられた。 -
The clarification of bone formation and absorption mechanism in the bone marrow microenvironment
Grant number：19H03842 2019.04 - 2022.03
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)
Ono Mitsuaki
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
BMP-2 has been widely studied for its potent ability to induce osteoblast differentiation and ectopic bone formation, and is already in clinical use worldwide. On the other hand, we have shown that BMP-2 inhibits bone formation in the bone marrow. However, the mechanism by which BMP-2 suppresses bone formation remains unclear. Therefore, we analyzed the detail of BMP-2 induced bone. Single cell RNA-seq analysis revealed that BMP-2-induced bone formed bone marrow with hematopoietic function. These results suggest that BMP-2 has the ability to regenerate bone marrow as an organ, and may suppress bone formation in the bone marrow in order to reserve space for hematopoiesis.
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Bone marrow microenvironment of MRONJ and application of Escherichia coli derived BMP-2
Grant number：19K10246 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Nawachi Kumiko
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
The pathogenesis of medication - related osteonecrosis of the jaws (MRONJ) remains largely unknown. Therefore, it is essential to elucidate the pathogenesis of MRONJ and to develop a treatment for this disease. We hypothesized that BMP-2, which has strong osteogenic potential, might be useful in the treatment of MRONJ. In fact, a mouse MRONJ model was created and rhBMP-2 was administered into the extraction socket. The results showed that bone formation in the extraction socket was suppressed in the non-transplanted group, whereas bone formation was significantly enhanced by rhBMP-2 transplantation. These results suggest that BMP-2 may be a new treatment for MRONJ.
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Examination for the effect of chewing on cognitive function and comprehensive search for biomarkers for early diagnosis of dementia
Grant number：19K10205 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Mino Takuya
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
Whether chewing stimulation increase BDNF consistency or not has not been revealed on humans. We conducted 60 minutes chewing stimulation experiments with gum base on healthy male adults to investigate BDNF consistency in saliva and plasma using the BDNF ELISA kit. As a result, there were no increase of BDNF consistency in saliva and plasma during 60 minutes chewing stimulation experiments.
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Grant number：18K19646 2018.06 - 2020.03
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)
Kuboki Takuo
Grant amount：\6240000 （ Direct expense: \4800000 、 Indirect expense：\1440000 ）
Stem cells are required for lifelong homeostasis and regeneration of organs and tissues in mammals. However, aging of stem cells reduces cellular function and results in dysfunctional organs and tissues. Therefore, maintenance of the stemness of stem cells is of crucial importance, although its mechanisms are still unclear. The aim of this study was to identify the master regulator for the maintenance of stemness of bone marrow mesenchymal stem cells (BMSCs). First, we compared the RNA expression patterns between BMSCs derived from young and old mice, as well as between human BMSCs and human dermal fibroblasts using RNA-seq, and found that 30 transcription factors were highly expressed in BMSCs derived from young mice and in human BMSCs. Next, we found that several transcription factors could inhibit the induction to pluripotent stem (iPS) cells using iPS interfering method. The detailed function of these transcription factors in BMSCs are now being investigated.
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Understanding of molecular mechanisms of tooth development using iPS interference and application to tooth regeneration techniques
Grant number：18H02991 2018.04 - 2021.03
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)
Kuboki Takuo
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
Whole-tooth regeneration is ultimate goal in dental field and the goal of this study is to identify the master transcription factors that characterize tooth germ derived epithelial and mesenchymal cells and develop method to generate those cells. First, we analyzed the mouse embryonic tooth germ and human stem cell from the apical papilla (hSCAP) by RNA-Seq and performed iPS interference to identify the potential transcription factors. Finally, candidate transcription factors were overexpressed in human adult dermal fibroblast (hADF), resulting that hADF was induced into the cells similar to tooth germ derived mesenchymal cells.
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The functional analysis of WISP1 gene aimed at the prevention of osteoarthritis
Grant number：18K09682 2018.04 - 2021.03
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)
Maeda Azusa
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
The purpose of this study was to elucidate the relationship between WISP1 gene and osteoarthritis (OA). In this study, we found that severity of OA mouse models in the Wisp1 deficient (Wisp1-KO) mouse was milder compared to wild type mouse. We also found that expression level of genes related to the extracellular matrix degrading proteases in the Wisp1-KO-OA knee joint tissue was lower compared to wildtype, it is suggesting that WISP1 is involved in pathogenic mechanism and progress of OA by promoting the secretion of ECM-degrading proteases.
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Molecular understanding of the accumulation of mesenchymal stem cells in vivo by photo-manipulation techniques and its application to bone-related diseases
Grant number：17H04399 2017.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
Takarada Takeshi
Grant amount：\16640000 （ Direct expense: \12800000 、 Indirect expense：\3840000 ）
In this study, we generated a tTA-dependent photoactivatable Cre-loxP recombinase knock-in mouse model (TRE-PA-Cre mice) using a CRISPR/Cas9 system. These mice were crossed with ROSA26-tdTomato mice (Cre reporter mouse) to visualize DNA recombination as marked by tdTomato expression. We demonstrated that external noninvasive LED blue light illumination allows efficient DNA recombination in the liver of TRE-PA-Cre:ROSA26-tdTomato mice transfected with tTA expression vectors using hydrodynamic tail vein injection. The TRE-PA-Cre mouse established here promises to be useful for optogenetic genome engineering in a noninvasive, spatiotemporal, and cell-type specific manner in vivo.
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Understanding of periodontal disease and peri-implantitis with stem cell disfunction
Grant number：17H04392 2017.04 - 2020.03
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)
AKIYAMA Kentaro
Grant amount：\16640000 （ Direct expense: \12800000 、 Indirect expense：\3840000 ）
In this study, we investigated how the bone marrow-derived mesenchymal stem cells (MSCs) function is affected by host aging. And then, how the suppressed MSCs function contributes to the development of inflammatory diseases in periodontal tissues such as periodontal disease and peri-implantitis. As a result, in the experimentally induced periodontal disease model mice, increased bone resorption was observed in aged mice. Moreover, the function of MSCs derived from 50-week-old mice showed lower cell proliferation and osteoblast differentiation ability with higher adipocyte differentiation ability. More interestingly, MSCs from aged mice showed decreased immunoregulatory capacity.
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Grant number：17K11750 2017.04 - 2020.03
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)
Kimura-Ono Aya
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
Recently, we successfully regenerated the cortical bone-like bone, which is important to maintain the long-term stability of regenerated bone, using Escherichia coli-derived rhBMP-2 (E-rhBMP-2) adsorbed in PLGA membrane in rat model. The purpose of this study was to evaluate the E-rhBMP-2/PLGA membrane using peri-implantitis canine model. First, we generated the peri-implantitis model using beagle dogs and transplanted the autogenous bone to evaluate this animal model. Interestingly, autogenous bone graft could not recover the bone defect caused by peri-implantitis. Next, we transplanted the β-TCP containing E-rhBMP-2 in bone defects and covered with PLGA membrane containing E-rhBMP-2. Only β-TCP containing E-rhBMP-2 was transplanted as control group. As a result, PLGA membrane containing E-rhBMP-2 induced bone formation compared with control group. In conclusion, the PLGA membrane containing E-rhBMP-2 was efficient in bone formation in a peri-implantitis model in beagle dogs.
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必須アミノ酸トリプトファンによる幹細胞老化制御機構の解明・骨質改善治療への応用
Grant number：17K11751 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)
笈田 育尚, 窪木 拓男, 大野 彩, 宝田 剛志, 大野 充昭
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
口腔インプラント治療は，人工歯根が歯槽骨や顎骨と結合することにより強固な骨支持を得るため，骨量と骨質が重要な因子となる．しかし，日本人は欧米人と比べ歯槽骨が解剖学的に菲薄で，インプラント体埋入のために骨造成が必要な場合も少なくない．また，高齢化の進む日本で増加傾向にある骨粗鬆症患者へ口腔インプラント治療がなされる場合も多く，多くの研究者が骨造成や骨質改善に関する研究を進めてきた．
我々は，これまでの研究から骨髄由来間葉系幹細胞の幹細胞性維持という観点からスクリーニングし，同定したトリプトファンが，骨質改善や骨の創傷治癒を促進することが明らかにした．この結果は，トリプトファンの投与が口腔インプラントの骨結合促進においても有用である可能性を強く示唆するものである．しかし，口腔インプラントの埋入に伴うトリプトファンの投与が，①骨のリモデリングを担う骨芽細胞，破骨細胞や間葉系幹細胞にどのような影響を与えるのか，また，②インプラント体の初期固定や長期予後に有意に働くのか，また，トリプトファンによる幹細胞の活性化が骨粗鬆症をはじめとする老化疾患に対して有効なのか，未だその詳細は明らかでない．
そこで，本研究ではトリプトファンの骨代謝関連細胞に与える効果の検討を行うこととした．トリプトファンと間葉系幹細胞の骨芽細胞分化との関係性は明らかにしてきたが，破骨細胞との関係性は未だ不明である．はじめに，トリプトファンが破骨細胞分化に与える影響を検討した．すなわちトリプトファンを投与したマウスの大腿骨を経時的 (0, 3, 7, 14日)にサンプリングし，組織学的，分子生物学的に検討する計画をした．しかし本研究では研究期間が短く解析，評価するまでには至らなかった．
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Grant number：16H06990 2016.08 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
Hara Emilio Satoshi, Matsumoto Takuya, Okada Masahiro, Kuboki Takuo, Nagaoka Noriyuki, Hattori Takako
Grant amount：\2860000 （ Direct expense: \2200000 、 Indirect expense：\660000 ）
We investigated the initial stages of endochondral ossification during secondary ossification of mouse femur epiphysis from the Life Science and Material Science viewpoints. We found that the very initial starting point of mineral formation in the epiphysis occurs at post-natal day 6 (P6).
Electron microscopy-based ultrastructural analysis showed that cell-secreted matrix vesicles were absent in the early steps of osteoblast-independent endochondral ossification. Instead, chondrocyte membrane fragments were found in the fibrous matrix surrounding the hypertrophic chondrocytes. EDS analysis and electron diffraction study indicated that cell membrane fragments acted as nuclei for newly formed calcospherites which initially showed an amorphous calcium phosphate phase, and then transformed into apatite crystal phase. These findings would be valuable to develop novel organic-inorganic hybrid materials for manipulation of biomineralization. -
Examine the effect of HMGB1 on bone marrow derived mesenchymal stem cell
Grant number：16H06991 2016.08 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
Komi Keiko, KUBOKI Takuo, AKIYAMA Kentaro, ONO Mitsuaki, MARUHAMA Kotaro, YOSHIOKA Yuya, KUNITOMO Masayoshi
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
In recent years High mobility group box 1 (HMGB 1) is known as a factor promoting tissue regeneration. It is also reported that HMGB1 induces mesenchymal stem cells in the marrow to the damage skin. In this study, we investigated the influence of HMGB1 on bone marrow derived mesenchymal stem cells. In the mouse wound healing model, host mesenchymal stem cell accumulation was detected in 1 day after surgery, and HMGB1 was expressed around the stem cell accumulation site in same day. We examined the effect of HMGB1 on mesenchymal stem cells, but the effect on stemness, stem cell proliferation and migration was not clear. Further studies on multiple differentiation and immunomodulate function that may be affected in the presence of HMGB1 will be necessary in the future.
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Identification of risk factor of CKD and pathogenic bacteria of infectious complications in dialysis
Grant number：16H06989 2016.08 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
KOYAMA Eri, KUBOKI Takuo, ONO Aya, ONO Mitsuaki, AKIYAMA Kentaro
Grant amount：\2860000 （ Direct expense: \2200000 、 Indirect expense：\660000 ）
In our country, the chronic kidney disease (CKD) patients increase year by year, and it is reported that oral health is related to life prognosis and the onset of complications in CKD patients, and the relationship between oral and kidney is attracting a great deal of attention.
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Therefore, the goal of this study is to identify the bacterial flora in shunt infection and clarify the relationship between oral flora and shunt infection. From the results of 16S rRNA analysis, the ratio of staphylococcus in saliva of several patients was abnormally high and oral flora of saliva was similar to that of infected shunt．And we also found that the oral disease-related bacteria existed in the infected shunt. -
Evaluation of upper airway size using Magnet Resonance Imaging
Grant number：16K11787 2016.04 - 2020.03
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)
KATAOKA Tomoki
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
MRI movie and volumetric MRI, that could evaluate a soft tissue kinetically and three-dimensionally, were used to evaluate upper airway size. While the original aim to make a treatment standard of patients with obstructive sleep apnea syndrome was not accomplished, we established the evaluation method of the upper airway using MRI movie and volumetric MRI, and clarified the usefulness of MRI movie that could evaluate a state of upper airway kinetically. As a result, mandibular body length and anteroposterior position were correlated with upper airway size, although the anteroposterior length and position of maxillary bone, and the vertical position and length of mandible were not correlated.
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Grant number：16K11590 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)
Minakuchi Mami
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
The results of comprehensive analyses of the intestinal flora and metabolites in the elderly requiring long-term care showed that the ratio of bacterial flora X and Y, at the division level, were significantly higher in overweight and underweight individuals, respectively. At the genus level, the bacterial flora Z and A were identified, respectively, as a bacterium positively and negatively correlated with BMI. Moreover, it became clear that some samples of underweight individuals could be distributed in the same cluster.
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Grant number：16H05524 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)
Ono Mitsuaki
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, in this experiment, we revealed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application.
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Bone regenerative therapy using Escherichia coli-derived rhBMP-2 adsorbed in beta-TCP.
Grant number：16K11624 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)
NAWACHI Kumiko
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
Bone regeneration using BMP-2 is expected to have the highest therapeutic effect, but there is no BMP-2 product approved in Japan. We succeeded to develop the BMP-2 using E. coli system and it was clarified that the combination of E. coli-BMP-2 and the already approved biodegradable artificial bone, β-tricalcium phosphate (β-TCP), strongly induces bone formation in socket preservation model in dog.
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Search of master regulator of odontoblastogenesis using iPS interference
Grant number：16K15802 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
Ono Mitsuaki
Grant amount：\3380000 （ Direct expense: \2600000 、 Indirect expense：\780000 ）
It is still unknown not only the mechanism but also the master regulator of odontoblastogenesis. Therefore, the final goal of our study is to identify the mater regulator of odontoblastogenesis. We performed the comprehensive analysis from the histological and developmental point of view using RNA-Seq, and could narrow down the several candidate odontoblastogenesis related genes. In the future, we are planing to perform the functional analysis of these candidate genes.
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Chronic muscle pain pathophysiology from the viewpoint of mitochondria dysfunction
Grant number：16K15801 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
MAEKAWA KENJI
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
We made an attempt to elucidate the relationship between vitamin D function and muscle pain. This hypothesis was established based on the several previous reports, which describe the positive relationship between taking antilipemic and muscle pain onset, especially seen in hyperlipidemia patients with vitamin D deficiency.
As the results, gene expression levels of vitamin D receptor was significantly higher in trapezius and masseter muscles, which are easily affected by muscle pain than limb muscles, which are not very sensitive to muscle pain. These results suggest the possible relationship between vitamin D function and muscle pain. In addition, expression levels of positive cell number of pain sensitivity marker (c-fos) in central nervous system under noxious stimulus application in masseter muscle tended to be higher in vitamin D deficiency rats than those in rats without vitamin D deficiency. -
Grant number：15H05026 2015.04 - 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)
Maekawa Kenji
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
Keratinized gingiva around the tooth and dental implant is critical to maintain a healthy condition of periodontal and peri-implant tissues, however the mechanisms regulating keratinization of gingiva still remain unclear. In this study, we hypothesized that the basement membrane is a critical regulator of keratinization of the oral mucosal epithelium. From our results, we found out that the difference in keratinization of oral mucosa is associated with the composition of the basement membrane. In addition to this, the composition which highly expresses in keratinized gingiva induced human gingival epithelial cells to express keratinized marker in vitro.
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Grant number：15K20439 2015.04 - 2018.03
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)
Mino Takuya
Grant amount：\3900000 （ Direct expense: \3000000 、 Indirect expense：\900000 ）
It is well known that peri-implantitis is caused by bacterial infection but the constitution of bacteria causing peri-implantitis is still unclear. We did cluster analysis of NGS to investigate the bacterial flora of peri-implant exudate which was extracted from 6 patients suffering from peri-implantitis. As a result, we found five kinds of bacteria in peri-implantitis site which were not found in periodontitis teeth, and another five kinds of bacteria which were not found in normal implants.
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Identification of master genes for regulating developmental time in regenerated organs
Grant number：15K15707 2015.04 - 2017.03
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
OSHIMA Masamitsu
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
We aimed to study the identification of master genes for regulating developmental time and morphogenesis in regenerated organs. We hypothesized that essential regulatory factors of developmental time and morphogenesis in tooth development could be identified by comparing the deciduous and permanent tooth germs harvested from an identical beagle dog. In this study, we identified the FGF14 and FEZF2 genes as candidates of the regulating factors by the cDNA microarray analysis. Furthermore, we analyzed the effects of these molecules on the murine tooth germ development. We have indicated that FGF14 promoted substantial elongation of the incisor tooth germ by acceleration of ameloblast proliferation. Further study to elucidate the molecular mechanism of FGF14 and FEZF2 on tooth development is necessary for the identification of the regulatory factors of developmental time and morphogenesis in tooth organogenesis.
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Grant number：15K20480 2015.04 - 2017.03
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)
OIDA Yasutaka, KUBOKI Takuo, ONO Mitsuaki, Emilio Satoshi Hara
Grant amount：\4030000 （ Direct expense: \3100000 、 Indirect expense：\930000 ）
Osteoarthritis is the most common chronic condition of the joints, affecting approximately ten million Japanese and articular cartilage repair remains a challenging problem. Based on a high-throughput screening and functional analysis, we found that fluocinolone acetonide (FA) strongly potentiated chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF-b3 -treated hBMSCs could completely repair the articular surface.
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Grant number：15K15708 2015.04 - 2017.03
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
Kuboki Takuo
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
Amino acids are essential for life and cell metabolism. We hypothesized that amino acids can regulate the stem cell phenotype and we performed a screening and found tryptophan as stemness regulator of BMSCs. Indeed, migration and colony forming ability of BMSCs was enhanced by tryptophan treatment. In vivo mice bone defect model, Tryptophan accelerated bone healing, and increased bone volume and trabecular number compared to PBS-injected group. In summary, L-tryptophan enhances the stemness and osteoblastic differentiation of BMSCs, and may be used as an essential factor to accelerate bone healing and/or prevent bone loss, such as in the case of ageing and osteoporosis.
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Grant number：26253088 2014.04 - 2018.03
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)
Kuboki Takuo
Grant amount：\41600000 （ Direct expense: \32000000 、 Indirect expense：\9600000 ）
Whole-tooth regeneration therapy has great potential for the replacement of lost teeth．Recently, our group have been reported an in vitro three-dimensional cell manipulation method called the bioengineered organ germ method. However, it is unclear the mechanism of tooth development. Therefore, we performed the comprehensive analysis and could narrow down the several candidate genes. In the future, we are planing to perform the functional analysis of these candidate genes.
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Development of tissue regeneration therapy using host stem cell homing
Grant number：26713053 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A) Grant-in-Aid for Young Scientists (A)
Akiyama Kentaro, KUBOKI TAKUO, ONO MITSUAKI, OSHIMA MASAMITSU
Grant amount：\22490000 （ Direct expense: \17300000 、 Indirect expense：\5190000 ）
In this study, we investigated the mechanism of stem cell accumulation in regeneration site during wound healing process. In the mouse wound healing model, host mesenchymal stem cell accumulation was detected in 1 day after surgery. By using cDNA micro-array analysis, TNFa was detected as one of the stem cell accumulation factor. Thus, we confirmed the effect of TNFa on stem cell function including, cell proliferation, mobilization, immune-modulatory property. While TNFa could inhibit the cell proliferation, cell mobilization was strongly up-regulated. Furthermore, immune-modulatory property was, which was evaluated FASL expression, totally up-regulated. These results indicated that TNFa could stimulate host stem cell function and cause immune tolerance in wound healing site.
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Grant number：26861637 2014.04 - 2017.03
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)
Ono Aya
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
It is still unclear the constitution of bacteria in peri-implantitis. We analyzed the bacterial flora of the exudate around dental implant body suffered from peri-implantitis by NGS. The experimental results were revealed that there are differences between individuals in the bacterial flora, and the peri-implantitis and the periodontal disease were similar bacterial flora and the healthy dental implants and the healthy tooth were also similar in the bacterial flora.
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Therapeutic effect of human dental pulp stem cell on Experimental autoimmune encephalomyelitis.
Grant number：26670837 2014.04 - 2016.03
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
Akiyama Kentaro, KUBOKI TAKUO, ONO MITSUAKI, OSHIMA MASAMITSU
Grant amount：\3250000 （ Direct expense: \2500000 、 Indirect expense：\750000 ）
In this study，we evaluated therapeutic effect of human dental pulp stem cells (DPSCs) against experimental autoimmune encephalomyelitis (EAE) model mice. In comparison with human bone marrow derived mesenchymal stem cells (BMSCs) injection, DPSCs ameliorate clinical symptom including hind leg paralysis and immunological status (up-regulation of regulatory T cells and down-regulation of inflammatory Th 17 cells). However, the accumulation of injected cells into injured nerve system wasn't detected. Thus nerve regeneration mechanism still unclear.
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The identification and functional analysis of attached gingiva related gene and protein
Grant number：25893138 2013.08 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
KUMAZAKI Asuka, KUBOKI Takuo, ONO Mitsuaki, UEDA Junji
Grant amount：\2730000 （ Direct expense: \2100000 、 Indirect expense：\630000 ）
The purpose of this study was to identify the specific gene of attached gingiva. First, to compare the ability of mesenchymal cells derived from attached gingiva and free gingiva, we collected them using out growth method. Mesenchymal cell derived from free gingiva had high ability in cell adhesion, proliferation, and migration, compared with attached gingiva. These data indicated mesenchymal cells derived from attached gingiva and free gingiva were completely different and we hypothesized that the mesenchymal cells were involved in the difference of attached gingiva and free gingiva. Therefore, next we performed the co-culture assay using human squamous cell carcinoma cells and confirmed that cell adhesion and extra cell matrix molecules are important for keratinization of epithelial cells. And from cDNA maicroassay data using mesenchymal tissue and epithelial tissue, several genes were selected as candidate factors of attached gingiva related gene, especially, keratinization.
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The mechanism identification of environmental selective bone induction/suppression of BMP-2
Grant number：25463050 2013.04 - 2016.03
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)
Nawachi Kumiko, KUBOKI TAKUO, SONOYAMA WATARU, ONO MITSUAKI, AKIYAMA KENTARO, SHINKAWA SHIGEHIKO
Grant amount：\5070000 （ Direct expense: \3900000 、 Indirect expense：\1170000 ）
Recombinant human BMP-2 (rhBMP-2) has been widely used to treat bone defects or bone-associated diseases in human. However, recently our research group found that rhBMP-2 inhibited bone formation in in the bone marrow space. The purpose of this research was to elucidate the mechanism of environmental selective bone induction/suppression of BMP-2. In the mice model, BMP-2 could not induce the bone formation in the bone marrow rich environment. Moreover, large animal model experiments revealed that rhBMP-2 were suitable for operative procedure in the bone marrow poor environment, such as socket lift and socket preservation.
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Do severe sleep bruxism subjects have the cardiovascular risk due to sleep hypertension?
Grant number：25670819 2013.04 - 2016.03
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
Minakuchi Hajime, KUBOKI Takuo, MAEKAWA Kenji
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
Validation of the miniature sleep bruxism (SB) detection/analyzing device has not been evaluated. This study determined the validity of device compared to polysomnogram puls AV recording (PSG). Sensitivity, specificity and accuracy between device and PSG assessment were 1.00，0.88 and 0.93 in case cut-off criteria was set at score 1 to 2. These results suggested that this modified miniature detection/analyzing device would obtain the moderate to high validity in clinical use.
Association between SB and simultaneous comorbid motor activites is unclear. This study aimed to compare the the incidences of body motions between sleep arousal (SA) with SB (SAwSB) and SA without SB (SAw/oSB). Motor activities in SAwSB was significantly higher than that in SAw/oSB (t-test, p<0.01, 95.1+/-6.6 %, 69.2+/-19.5 %). Leg movement and swallowing were significantly frequent among the comorbidities. This suggested that SB can be cocomitantly activated with leg movemnet and swallowing in relation to SA. -
Development of technologies for next-generation regenerative therapies
Grant number：25242041 2013.04 - 2016.03
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)
Tsuji Takashi, KUBOKI Takuo, OSHIMA Masamitsu, EGUSA Hiroshi, TSUBOTA Kazuo, KAGIHARA Yasuhiro, FUJITA Satoshi, KISHIDA Akio, SATO Akio, TAKEDA Akira, TOYOSHIMA Ko-ei
Grant amount：\47190000 （ Direct expense: \36300000 、 Indirect expense：\10890000 ）
In this project, we aimed at the development of fundamental technology for the next generation of organ regenerative medicine. 1) We successfully demonstrated to regenerate of secretory glands including salivary and lacrimal glands by the transplantation of their bioengineered germs, which were reconstituted by using "Organ Germ Methods". 2) To identify the cell seeds for organ regeneration, we demonstrated the possibility for the development of the functional bioengineered 3D integumentary organ system and the realization of organ replacement therapy by using iPS cells. 3) We further achieved the fundamental technology developments of new functional gel materials using the functional sugar chain and self-assembling peptides, for the application to clinical application for organ regenerative medicine. These results indicated that we successfully developed the fundamental technology for clinical application of the wide variety of organ regenerative therapies.
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Development of production technique of tissue derived stem cell using novel reprogramming system.
Grant number：25670818 2013.04 - 2015.03
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
KUBOKI Takuo, ONO Mitsuaki, AKIYAMA Kentaro
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
Small non-coding microRNAs (miRNAs) have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. We herein sorted stem-cell-enriched side population (SP) cells from human DPCs and PDLCs, and performed a miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/ progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
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Study of the effect in perioperative oral management
Grant number：24890136 2012.08 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
MINAKUCHI Mami, SOGA Yoshihiko, KUBOKI Takuo
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
The clinical survey of intraoral condition has been performed for the patients who received the surgical therapy of malignant tumors of the gastrointestinal region in order to promote the intraoral management in perioperative periods effectively. And, case report article was published regarding to the relationship between postoperative recovery of the esophageal cancer patient and orally nutrition approaches. Furthermore, 2nd Advanced perioperative dental approach symposium was held at January 26th, 2014. In this meeting, discussions with the nationwide practitioners were performed to exchange the opinion of the newest finding of oral functional management in perioperative periods, develop the novel approach in perioperative oral management and verify these efficacies in order to future applications.
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Human dental pulp stem cell ameliorate Experimental autoimmune encephalomyelitis.
Grant number：24890135 2012.08 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
AKIYAMA Kentaro, KUBOKI Takuo, ONO Mitsuaki, OSHIMA Masamitsu
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
In this study, we evaluated the therapeutic effect of bone marrow derived mesenchymal stem cells (BMMSCs) or dental pulp stem cells (DPSCs) systemic injection on experimental autoimmune encephalomyelitis (EAE) model mice. In the clinical evaluation, both BMMSCs and DPSCs showed significant therapeutic effect including hind leg paralysis. However, BMMSCs injected group showed, in terms of immunological evaluation, higher up regulation of regulatory T cells (Tregs) in peripheral blood after 40days from EAE induction compare to DPSCs injected group.
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Grant number：24792083 2012.04 - 2015.03
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)
MINO Takuya, KUBOKI Takuo, SONOYAMA Wataru, ONO Mitsuaki, ONO Aya
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
The purpose of this study was to identify the biological markers that are involved in the onset and progression of peri-implantitis. First, we developed a clinical examination protocol by referring to the previous reports and investigated the prospective study for the initial peri-implantitis patient using the developed protocol. The subject of this study was five and, basic information of subjects was shown below, average age: 71.0 ± 4.6 years, average number of implants per patient : 4.8 ± 2.4, average number of implant with peri-implantitis：1.4 ± 0.5) . To identify the biological markers, gingival crevicular fluids around the oral implant and natural tooth were collected by using the paper points. ,The adequate amount of bacterial DNA could be collected for 16S rRNA sequencing using this protocol.
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Grant number：24659875 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
SONOYAMA Wataru, KUBOKI Takuo, ONO Mitsuaki
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
It is well known that adult tissue stem cells, which exit in the niche, are involved in the maintenance or restoration of homeostasis in various tissue. In this research, we focused Ccn4 gene and that revealed that Ccn4 gene could be involved in tooth development and wound healing. In future, we will clarify the relationship between Ccn4 gene and maintenance mechanism of niche.
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Grant number：24792142 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)
OIDA Yasutaka, KUBOKI Takuo, SONOYAMA Wataru, ONO Mitsuaki, SHINKAWA Shigehiko, NAKAJIMA Ryu
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
CCN2 / connective tissue growth factor (CTGF) has been reported to have essential role in cartilage development, chondrocyte proliferation and differentiation as well as regulation of the extracellular matrix metabolism. This study screened a compound library and identified harmine as a novel inducer of CCN2. This finding indicates harmine as a potential drug for prevention and / or repair of cartilage degradation.
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Grant number：23792227 2011.04 - 2015.03
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)
MINE Atsushi, YATANI Hirofumi, KUBOKI Takuo, YOSHIDA Toshimasa, KUROSAKI Youko, MINO Takuya
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
Three-unit resin-bonded fixed partial prosthesis (Rb-FPP, n = 86) and Three-unit conventional fixed partial prosthesis (Co-FPP, n = 100) installed at the Fixed Prosthodontic Clinic of Okayama University Dental Hospital between April 1989 and March 1992 were clinically evaluated. The corresponding survival rate for Rb-FPP and Co-FPP were 69 % and 72 % after 10 years and 50 % and 46 % after 25 years respectively. There was no significant difference between the two groups (p = 0.88).
Regarding to the incidence of complications, “removal of FPP” was significantly higher for Co-FPP (p = 0.01). Cox proportional hazards test revealed that “FPP type”, “sex”, “age”, “number of remaining teeth”, “insertion position” and “abutment teeth condition” were not significant predictors of FPP failure (p = 0.82). -
Grant number：23390442 2011.04 - 2015.03
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)
MINAKUCHI Hajime, KUBOKI Takuo, SOGAWA Chiharu, MAEKAWA Kenji, SOGAWA Norio, KITAYAMA Shigeo
Grant amount：\18200000 （ Direct expense: \14000000 、 Indirect expense：\4200000 ）
This aimed to evaluate the correlation between SERT uptake ability in human peripheral platelets and SB frequency. Subjects were consecutively recruited from sixth-year students. SB frequency was determined as the summary score of 3-consecutive night assessments using a self-contained EMG detector/analyzer in their home environment. Fasting peripheral venous blood samples were collected in the morning following the final SB assessment. Amount of SERTs and platelets were quantified by ELISA assay. Functional characterization of SERT, 5-HT uptake, maximum velocity (Vmax) and affinity constant (Km) were assessed by [3H] 5-HT uptake assay. The correlation between these aforementioned variables and SB level was evaluated.
A small but significant negative correlation between SB level and [3H] 5-HT uptake was observed (p<0.05, R2=0.063, Spearman correlation). Platelet serotonin uptake is significantly correlated to SB frequency, although it only explains a small amount of SB variability. -
Functional regeneration of the oral cavity marginal muscles by cybernics approach
Grant number：23659898 2011 - 2013
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
MAEKAWA KENJI, OKA Hisao, KUBOKI Takuo
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
While attempts were made to establish the electrical stimulation methods to induce the reactive muscle activity for the patients with muscle dysfunction in orofacial regions by systemic disease such as a stroke, it was difficult to achieve it. Therefore, we next conceived to evaluate the degree of burden for care-givers to nursing to the persons requiring care with masticatory disturbance. By carrying out this investigation, we thought that we could appeal the importance of the rehabilitation of oral function and could indirectly lead to the development of novel rehabilitation methods. The results of the investigation to evaluate the degree of burden for the care-givers to persons requiring care suggested that time necessary to feeding and oral care, and the number of remaining teeth significantly associated with the degree of burden.
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Grant number：23890123 2011 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
NAWACHI Kumiko, OIDA Yasutaka, SONOYAMA Wataru, KUBOKI Takuo
Grant amount：\3250000 （ Direct expense: \2500000 、 Indirect expense：\750000 ）
BMP-2 is widely known as an osteogenic inducer, but conflicting results are related to its function inside the bone marrow space. This study aimed to clarify the molecular mechanisms and impact of BMP-2 on bone metabolism environment of the bone marrow space. Of great interest, we found that BMP-2 inhibits bone formation in bone marrow space. To date, in an attempt to elucidate this mechanism, we are focusing on BMP-2 negative feedback regulation as well as on cellular and molecular interactions from an immunological perspective.
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Development of a technic to generate somatic stem cells using a newly reprogramming method.
Grant number：23659899 2011 - 2012
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
KUBOKI Takuo, SONOYAMA Wataru, UCHIBE Kenta, ONO Mitsuaki
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
We performed a screening for factors involved in the regulation of maintenance of stemness in dental pulp cells (DPCs). As a result, we found that TNF-αwas involved in maintenance of stemness, and increased the number of cells positive for stem cell surface markers CD146 and SSEA4. Moreover, we also identified some microRNAs that regulate stemness, proliferation and differentiation of DPCs.
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Development of neuro-regeneration with Botulinum toxin
Grant number：23659897 2011 - 2012
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
MATSUKA Yoshizo, KUBOKI Takuo, MATSUO Ryuji, OGUMA Keiji, YAMAMOTO Yumiko, KUMADA Ai
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
Dissociated rat trigeminal ganglion somata were incubated with matrigel in 37℃ CO_2condition. Commercially available hippocampus neuron was used in this study. Neuronal change was observed and the neuron that incubated with type A Botulinum toxin was survived longer than control. Also, dendrite of the neuron with Rotulinum toxin was longer than control neuron.
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低分子化合物ライブラリーを用いた骨形成過程における新規BMP2活性制御因子の探索
Grant number：23592844 2011
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)
藤澤 拓生, 園山 亘, 窪木 拓男, 服部 高子
Grant amount：\5070000 （ Direct expense: \3900000 、 Indirect expense：\1170000 ）
骨欠損部に対して骨形成タンパク(BMP)を用いて骨造を図る方法は次世代の骨造法の最も有望な方法と考えられているが,ターゲットとする細胞の応答性の低さから大量のタンパクが必要となり高コストとなること,および大量のタンパク投与による副作用のリスクが高まる危険があり,より低用量で高効果の得られる投与方法の開発が望まれている。そこで本研究はBMPの生理活性を増強する低分子化合物を同定し,その機能を解明することを目的に以下の実験を行った。
まず初めに一次スクリーニングとして低分子化合物(FDA approved Drug Library)の細胞増殖能,細胞障害度ならびにBMP-2の生物学的活性に与える影響について検討した。細胞増殖能についてはMTS assayで,細胞障害度に関してはLDH assayでそれぞれ評価した。BMP-2の生物学的活性に関してはBMP-2シグナルの増強の有無をBMP-2にのみ特異的な反応を示すId-1プロモーター領域を有するレポーター遺伝子を導入したC2C12細胞を用いたルシフェラーゼアッセイで評価した。その結果,640個の低分子化合物ライブラリーから細胞に障害を与えることなくBMP-2の生物学的活性を相乗的に増強する,あるいは化合物単体でBMP-2様の生物学的活性を示す可能性のある化合物を40個抽出した。さらに二次スクリーニングとしてin vitroでアルカリホスファターゼ活性の測定とアリザリンレッド染色による石灰化能の検討を行い40個の候補化合物からBMP-2の骨形成能を増強している可能性のある化合物を7個抽出した。 -
Grant number：22249064 2010.04 - 2014.03
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)
KUBOKI Takuo, TAKIGAWA Masaharu, SONOYAMA Wataru, TSUJI Takashi, ASAHARA Hiroshi
Grant amount：\46670000 （ Direct expense: \35900000 、 Indirect expense：\10770000 ）
In the dental field, the regeneration of complete tooth is ultimate goal. First, we examined whether fully functional bioengineered tooth could regenerate utilizing an organ germ method with the permanent tooth bud tissue of the post-natal canine, we succeeded in it, for the first time in the world. Next, in order to understand the mechanism of tooth development, we performed the screening and clarified that several Hox gene specifically expressed in the development of tooth.
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Development of a new treatment for trigeminal neuron sensitization
Grant number：22390365 2010.04 - 2014.03
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)
MATSUKA Yoshizo, KUBOKI Takuo, YAMAMOTO Yumiko, KUMADA Ai, OGUMA Keiji
Grant amount：\18720000 （ Direct expense: \14400000 、 Indirect expense：\4320000 ）
Fluorescein labeled botulinum toxin heavy chain which works for endocytosis was injected into rat face skin and found in ipsilateral trigeminal ganglion. The labeled botulinum toxin heavy chain was not found on the contralateral side or in the only fluorescein injection. The uptake was inhibited by colchicine treatment, which blocks axonal transport. Intradermal injection of botulinum toxin alleviates infraorbital nerve constriction induced thermal hyperalgesia in an operant assay. Direct application of botulinum toxin to dorsal root ganglia reversed the sciatic nerve entrapment induced decreases in withdrawal thresholds.
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A multifunctional implant which can achieve the site-specific tissue regeneration
Grant number：22390366 2010.04 - 2014.03
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)
MAEKAWA Kenji, HAYAKAWA Satoshi, SONOYAMA Wataru, KUBOKI Takuo, ITOH Yoshihiro
Grant amount：\19240000 （ Direct expense: \14800000 、 Indirect expense：\4440000 ）
Our previous technique for apatite deposition onto the titanium surface after insertion in human body, can be only applied to the pure titanium surface. However, in order to apply this surface modification technique to clinically used implants, improvement of the technique is necessary, because all the commercially available titanium implants are made from titanium alloy. For this purpose, we developed liquid phase deposition (LPD) technique, which can induce the apatite formation onto the titanium alloy surface after insertion in human body. We found that rat bone marrow stromal cells (rBMSC) can be attached to this modified titanium alloy surface (treated by LPD technique) and the osseogenesis of rBMSC tended to be accelerated. Recently, the investigation of the bioactivity of this surface modification technique using rat model is still ongoing.
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高脂血症治療薬；スタチンを応用した象牙質形成促進作用を持つ新規覆髄材の開発
Grant number：22592150 2010.04 - 2013.03
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)
岡本 洋介, 窪木 拓男, 松香 芳三, 園山 亘, 大野 充昭
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
1.in vitroでのスタチンの歯髄幹綱胞における作用機序の検討
スタチンはメバロン酸経路の上流にあるHMG-CoA還元酵素を抑制しコレステロール産生の抑制することが報告されている。歯髄幹細胞(Dental Pulp Stem Cell : DPSC)でも同様の作用機序であるか検討するため,HMG-CoAの下流に位置するメバロン酸を用いた。培地にスタチン1μMとメバロン酸1mMの濃度で同時に添加し,MTS法で細胞増殖に与える影響を検討した。その結果,5日目にスタチン単独での細胞増殖抑制効果が消失していることを確認した。
またスタチンはメバロン酸経路の中間産物の抑制により,Rho経路を介し細胞周期をG1/S期で停止させることが報告されている。そこで細胞周期に与える影響を検討するため,スタチン1μM添加し3日間培養したDPSCをPIにて染色後,FACSを用い解析を行った。その結果,G0/G1期への集積像およびG2/M期ピークの減弱が観察された。以上の結果よりDPSCにおけるスタチンの作用はメバロン酸-Rho経路を介していることが示唆された。
2,イヌを用いた覆髄モデルの作製
本研究の臨床モデルは,歯髄に近接したカリエスが考えられる。そこで本研究ではビーグル犬(1歳齢)の犬歯を用い覆髄モデルを作製した。歯の遠心面がら近心に向け歯科用5倍速エンジンで窩洞の形成を行い,通常の歯科用覆髄剤を用い覆髄処置を行い,歯科用セメントを用い窩洞の充填を行った。1か月後に組織を回収し,通法に従い組織標本を作製した。その結果,安定して歯髄に近接した窩洞を形成できていることが確認された。
次に,このイヌ覆髄モデルを用い,スタチンの象牙質形成効果を検討した。つまり,スタチン2mMならびにPBSを各10μlを含むコラーゲンスポンジを覆髄剤として窩洞内に設置し,歯科用セメントにて充填した。1か月後に組織を回収し,組織標本を作製しHE染色を行なった結果,2mMのスタチンに有意な象牙質形成促進作用は認められなかった。 -
分子イメージングとバイオマーカー探索による慢性筋痛の局所病態解析
Grant number：22592151 2010 - 2012
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)
小野 剛, 前川 賢治, 水口 一, 松香 芳三, 窪木 拓男
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
我々は,以前,近赤外線分光法を用いて慢性筋痛を訴える被験者の有痛筋組織内では,筋作業や交感神経活動亢進時の筋組織内の血管拡張機能が低下している所見を得た.さらに分子イメージング技術であるPositron emission tomography (PET)を用いて,筋組織のエネルギー源であるグルコースの取り込み量を僧帽筋に慢性筋痛を訴える被験者の僧帽筋と,非筋痛者の僧帽筋内とで比較した結果,筋痛者の僧帽筋組織内のグルコース取り込み量が非筋痛者のそれに比較して有意に抑制されることを明らかとした.これらの知見から更に慢性筋痛の病態を明らかとすることを目的に,筋組織内代謝と組織内血流の相互関係に着目した.相互関係を明らかとするためには,グルコース代謝の指標となる18F-FDGと,血流の指標となる150の筋組織内取り込み量をPETでダイレクトに測定することが有効と考え,150ガスをプローブとして実験に用いる手法の確立を目指して予備的検討を行っている.その一方で,150ガスを用いた血流動態評価が技術的な問題等で困難な場合に備え,血流の絶対量が測定可能な近赤外線分光計(オメガモニターBOM-L1 TR W)を用いて測定する血流動態測定の予備的検討も同時進行させている.加えて,最近,PETを用いた実験的研究で虚血のマーカーとして最近着目されている64Cu-ATSMを研究に用いることが可能かどうかも含めて検討中である.さらに,動物を対象としてPETを用いた基礎研究を開始することも考慮にいれ,理研分子イメージングセンターの研究者にコンタクトを取り準備を開始した.採択決定が11月となり,今年度の実質的な研究期間が3ヶ月と短かったため,実際の研究データの採取は来年度行う予定である
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インプラント周囲炎の早期診断ならびに新規治療法の開発
Grant number：21592451 2009 - 2011
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)
山崎 聖也, 窪木 拓男, 荒川 光, 小野 剛
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
本研究の目的はインプラント周囲の炎症を未然に察知するためにインプラント周囲の炎症性歯肉を採取し特異的な生物学的マーカーを同定することである.昨年度に引き続き,当科で口腔インプラント治療を行った患者の中でどれだけの患者がインプラント周囲炎によりインプラントを喪失しているのかを把握する目的で,これまでに当院で口腔インプラント治療を行った患者のデータベースの整理を行った.過去15年間に当科にて口腔インプラント治療を行った全患者は453名であった,これらの患者に埋入されているインプラント体1本1本について追跡調査をおこないインプラント周囲炎の発症頻度ならびにインプラント周囲に炎症を引き起こすリスクファクターについて調査した.また,インプラント周囲に特異的に炎症が起こるのか(隣在歯から炎症が波及しているのではないか)を確認するためにインプラントの隣在歯に起こるトラブルの詳細を同時に調査した.
《方法》
1.1990年2月から2007年3月までの間に当科にて口腔インプラント治療を受けた全患者393人1062本の中でオッセオインテグレーションが獲得されたものが721本であった.これらのインプラント体を対象に多変量解析による生存分析をおこなった
2.多数歯の遊離端欠損患者に対して,インプラント群と可撤性部分床義歯群を選別し,欠損の隣在歯におこる炎症に起因するトラブルの発生を生存分析を用いて比較した
《結果》
1.インプラント体の10年累積生存率は94%であり.10本のインプラント体が除去に至っていた.インプラント体が除去に至るリスク要因は上部構造が術者可撤式である事と喫煙習慣である事が明らかとなった
2.インプラントと可撤性部分床義歯では欠損部の隣在歯におけるトラブルの発生率は有意に可撤性部分床義歯の方が高かった
今後はこれらの結果をふまえてインプラント体を除去するに至ったインプラント周囲炎をもった患者のインプラント体周囲歯肉からインプラント周囲に起こる炎症の特異的なマーカーを具体的に調査していく予定である -
VALIDITY OF PROSTHETIC CLINICAL GUIDELINE DEVELOPED ON THE BASIS OF TREATMENT DIFFICULTY INDICES
Grant number：21249092 2009 - 2011
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)
HIRAI Toshihiro, SASAKI Keiichi, SATO Yuji, ISHIBASHI Kanji, KUBOKI Takuo, BABA Kazuyoshi, HIDESHIMA Masayuki, KOBAYASHI Hiroshi, SAKURAI Kaoru, MASUMI Shinichi, KOSHINO Hisashi, AITA Hideki, KIMURA Aya, KONO Mai, KOYAMA Shigeto, KITAGAWA Noboru, TANABE Norimasa, TUKASAKI Hiroaki, WAKABAYASHI Noriyuki, RYU Masahiro, KAWANO Toshihiro
Grant amount：\46150000 （ Direct expense: \35500000 、 Indirect expense：\10650000 ）
The purpose of this study was to develop the prosthodontic clinical guideline based on treatment difficulty indices. A multi-centered epidemiological study showed the newly developed multi-axis assessment protocol was useful and reliable to estimate the level of difficulty in treating patients who needed prosthodontic care. The results suggest that the established evidence-based clinical guideline is expected to have a significant contribution to the practice of qualified prosthodontic treatment with sufficient safety and efficacy.
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Grant number：20592267 2008 - 2010
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)
KANYAMA Manabu, KUBOKI Takuo, SONOYAMA Wataru, KATAOKA Ken
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
In this study, we isolated human dental follicle epithelial (DFE) cells that were different from gingival epithelial (GE) cells in terms of their gene expression profiles and proliferative capacity. Furthermore, DFE cells were revealed to be putative amelogenic cells based on their gene expression of an ameloblast marker, amelogenin. We found that a direct contact between DFE and dental papilla mesenchymal (DPM) cells increased an amelogenin expression in DFE cells, suggesting transmembrane signaling from the DPM cells played some pivotal roles for DFE cells to enter into the ameloblast lineage. More importantly, this increase was remarkable when DFE cells were cultured with DPM cells in comparison to DFM cells. We confirmed that DPM cells expressed dentin sialophosphoprotein (DSPP), one of odontoblast markers, but DFM cells did not. Therefore, this DPM cells-driven DFE cells differentiation might be a reproduction of epithelial - mesenchymal interaction, found in tooth generation. However, the underlying mechanisms of this induced differentiation of DFE cells were not cleared. Much more signaling works are required to elucidate epithelial - mesenchymal interaction in human tooth generation. Novel human amelogenic cells, DFE cells, might be useful tool for such kind of signaling works.
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Grant number：20592264 2008 - 2010
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)
ARAKAWA Hikaru, KUBOKI Takuo, MATSUKA Yoshizo, KANYAMA Manabu, YAMAZAKI Seiya, KIMURA Aya, NODA Kinji, YAMAMOTO Michiyo
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
The realities that it becomes impossible for the elderly patients with dental implants to respond to the recall because of the change in the general status, especially the dementia and the circulatory system disease were clarified. Moreover, healthy elderly patients also are holding uneasiness in the maintenance method and the prognosis of the dental implants. We dentists should do the information presentation concerning the prognosis, and the reconsideration of a long-term caring plan to stare at the patient's aging is requested
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A multi-center study on prosthetic rehabilitation for Shortened Dental Arch
Grant number：20249077 2008 - 2010
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)
IGARASHI Yoshimasa, SASAKI Keiich, KOYANO Kiyoshi, KUBOKI Takuo, MAEDA Yoshinobu, BABA Kazuyoshi, AKAGAWA Yasumasa, KASUGAI Shouhei, FUEKI Kenji, KOYAMA Shigeto, TUKASAKI Hiroaki, IKEBE Kazunori, OGINO Youichirou, KORETAKE Katsunori, YOSHIDA Eiko, KONDO Hisatomo, KURODA Shinji, GARRETT Neal R
Grant amount：\34060000 （ Direct expense: \26200000 、 Indirect expense：\7860000 ）
The aim of this study was to investigate efficacy of prosthetic rehabilitation for shortened dental arch. Oral health-related quality of life and masticatory function were evaluated in patients with shortened dental arch before and after prosthetic treatments and in patients without treatment. Oral health-related quality of life and masticatory function were impaired with increase of missing posterior teeth, and they were improved after prosthetic treatment, suggesting that prosthetic rehabilitation for shortened dental arch is effective.
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Development of the basic technology for tooth regenerative therapy system
Grant number：20249078 2008 - 2010
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)
TSUJI Takashi, KUBOLKI Takuo, KASUGAI Shohei, TOMOOKA Yasuhiro, YAMAMOTO Teruko, SONOYAMA Wataru, SAITO Masahiro
Grant amount：\48620000 （ Direct expense: \37400000 、 Indirect expense：\11220000 ）
In current research on whole-tooth regenerative therapy, a basic strategy is being pursued in which a bioengineered tooth germ is induced to develop into a fully functional tooth. We developed a three-dimensional organ-germ culture method for the reconstitution a bioengineered organ germ. We successfully demonstrated that our bioengineered tooth germ could develop a fully functioning tooth, which has hardness for masticatory potential, the functional responsibility against a mechanical stress, and perceptive potentials of neural fibers. Furthermore, we demonstrated a further development in this regard in which a bioengineered tooth unit comprising mature tooth, periodontal ligament and alveolar bone, was successfully transplanted into a properly-sized bony hole in the alveolar bone through bone integration by recipient bone remodeling in a murine transplantation model system. These results showed that the bioengineered tooth germ and mature tooth unit could regenerate a fully functioning tooth and an organ replacement regenerative therapy might be feasible.
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第二世代BMP-2を応用した口腔インプラントの骨結合促進と歯槽骨再生の実用化
Grant number：20592268 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)
縄稚 久美子, 窪木 拓男, 園山 亘, 完山 学, 山崎 聖也
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
1. 大腸菌由来BMP-2によるインプラント体周囲の骨再生の検討
大腸菌由来BMP-2を用いてインプラント体周囲への骨再生が可能かをラット背部皮下での異所性骨再生モデルで検討した.濃度は,0,1,5,10ug/ulのBMP-2水溶液を調整し,インプラント体を3分間浸漬したうえで移植した.移植から,2・3週間後にレントゲンで骨形成状態を確認したところ,5,10ug/ulのBMP-2溶液を用いたものでは明らかな骨形成が確認された.3週後に,インプラント体を回収し,非脱灰切片を作成し顕微鏡的に評価したところ,インプラント体と骨の直接的な接触が得られていることが確認された.
2. 大腸菌由来BMP-2を骨補填材と併用した際の骨再生の検討
大腸菌由来BMP-2と骨補填材とを併用した際の骨再生をラット背部皮下での異所性骨再生モデルで検討した.骨補填材は炭酸機含有アパタイトとβTCPを用いた.事前に両骨補填材の吸水量を検討し,一移植体当たり0,1.25,2.5,12.5,25,125ugのBMP-2を含有するように調整した上で移植した.レントゲン的に骨形成を確認したうえで,移植から3週後に移植体を回収し,組織学的に評価した.その結果,12.5,25,125ugのBMP-2を用いた群では両方の骨補填材を用いた群で明らかな骨新生を認めた.0.25,2.5ugでは3週の時点では骨形成は認めなかった.また,高濃度のBMP-2を用いた場合,移植体のボリュームは約10倍以上となるが,その中心部には赤血球を主体とする血液が停滞している部分が広く認められることが確認できた. -
Grant number：20592265 2008 - 2010
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)
MINAKUCHI Hajime, KUBOKI Takuo, MATSUKA Yoshizo, SOGAWA Norio, KITAYAMA Shigeo, UEHARA Junji
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
(1) This project evaluated the night-to-night variability and examine for a systematic alteration on the first night in EMG levels. The results suggested that recordings should be at least 5-6 nights duration to establish a reasonable measure of an individual's average nightly masseter EMG level.
(2) The aim of this study was to compare the uptake ability of serotonin transporter (SERT) from peripheral platelets between severe and non-bruxism human subjects. In response, the SERT ability would be related to sleep bruxism frequency. -
Grant number：19592237 2007 - 2008
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)
YAMAZAKI Seiya, KUBOKI Takuo, MATSUKA Yoshizou, SONOYAMA Wataru
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
本研究では, 変形性顎関節症に対する新規関節腔内注入薬として可溶型腫瘍壊死因子受容体(sTNFR-II)を対象に考えており, 既に関節リウマチの治療薬として使用されているエタネルセプトにより, 関節軟骨破壊に関与する一部のマトリックスメタロプロテアーゼの関節軟骨細胞からの産生を抑制する傾向がみられた. また, 併せて変形性顎関節症に特異的なタンパク質を同定することで, 本治療の効果判定の指標としたり、新規治療に応用するため, 顎関節滑液中のタンパク質に関してもサイトカインアレイ解析や網羅的解析を行った.
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咬合感覚異常症患者における末梢および中枢知覚神経活動の亢進と治療法開発
Grant number：19659506 2007 - 2008
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
松香 芳三, 窪木 拓男, 福島 俊士, 小川 匠, 坂口 千代美, 小野 剛
Grant amount：\3200000 （ Direct expense: \3200000 ）
1.健常者における周囲歯肉への局所麻酔薬塗布後の歯の知覚閾値の低下
昨年に引き続き、咬頭嵌合位の咬合接触異常を訴えない健常者に局所麻酔薬(ハリケインゲル)を歯周歯肉へ塗布前後の歯の知覚閾値を計測したところ、閾値の低下が観察された。さらに、部位により知覚閾値の低下に差があることが理解できた。以上から、咬合感覚異常症患者の歯肉への局所麻酔薬の塗布により、歯の知覚を減少させることができる可能性、歯種により、反応が異なる可能性が示唆された。
2.咬合感覚異常症患者に対する局所麻酔薬の効果
昨年に引き続き、咬合感覚異常症患者に研究目的・内容を説明し、研究への参加を依頼した。包含基準は、1.咬頭嵌合位での咬合接触の異常を6か月以上にわたり訴える、2.患者は問題歯を特定可能であるとした。除外基準は、1.歯髄・歯周病変・顎関節症が存在する、2.多数の欠損歯のために、咬頭嵌合位が不安定である、3.歯科用局所麻酔薬に対するアレルギーがあるとした。それらの患者に対し、局所麻酔薬を塗布したところ、症状の軽快が観察された。
3.咬合感覚異常症患者と健常者の脳磁図計測
東京歯科大学の機器を使用し、脳磁図計測を行うため、プロトコールに関して東京歯科大学の担当研究者と検討した。被験者に能動的に咬合接触してもらうと、脳内の活動が生じるため、受動的に当該歯を押すこと装置の開発が必要であることが理解できた。研究費補助期間終了後になるが、今後、計測を継続していく予定である。
4.三叉神経刺激動物モデルにおける三叉神経節でのDNA変化の網羅的解析
ラット眼窩下神経をゆるく結紮することにより、刺激したモデルにおいて、三叉神経節内のDNA変化を網羅的に解析した。その結果、伝達物質遊離に関連するGRP75が増加していることが理解できた。 -
付着歯肉の分化に関連した特異的遺伝子・蛋白の同定とその機能解析
Grant number：19659507 2007 - 2008
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
窪木 拓男, 高柴 正悟, 園山 亘, 滝川 正春
Grant amount：\3200000 （ Direct expense: \3200000 ）
1.付着歯肉組織と遊離歯肉組織の遺伝子発現解析
前年度のマウスならびにラット歯肉組織の組織学的検討をもとにして,成体マウスの口腔内から実態顕微鏡下で付着歯肉と遊離歯肉をそれぞれ採取した、この組織からmRNAを抽出し,cDNAマイクロアレイを行い,両者の遺伝子発現を比較・検討した.
その結果,付着歯肉で発現量の高い遺伝子として,mmp12, integrin(alpha6), laminin(beta3), HIF-1a, VEGF, tenomodulin, collagen(typeV, alpha2), integrin(beta4)などが抽出された.一方,遊離歯肉で発現量の高い遺伝子としてIGFBP2, RABL3(member of RAS oncogene family-like3), RASA3(RAS p21 protein activator3), elastinなどが抽出された.既知の発現パターンと機能から考察するといくつかの遺伝子はたいへん興味深い研究対象と考えられた.
2,ヒト歯肉上皮細胞の培養
倫理委員会の許可を得て,抜歯時に得られたヒト歯肉サンプルから歯肉上皮細胞を分離し,同時に得た歯原性上皮細胞とその差異を比較,検討した.
その結果,歯肉上皮細胞は培養条件下では寿命が短く,cumulative population doubling(cPD)は平均8であった。一方,歯原性上皮細胞は平均16のcPDを示した.また,両者ともに上皮細胞のマーカーであるサイトケラチン14とE-cadherinを遺伝子レベルで発現していたが,amelogeninの発現は歯肉上皮細胞では認めなかった.すなわち,歯肉上皮細胞は歯原性上皮細胞と比較して,明らかに異なるフェノタイプを有していることを明らかにした. -
Grant number：18390512 2006 - 2009
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)
MATSUKA Yoshizo, MAEKAWA Kenji, KUBOKI Takuo, KANYAMA Manabu, SUGIMOTO Tomosada, TAKEI Koji, ONO Tsuyoshi, OGUMA Keiji, YAMAMOTO Yomiko, KITAMURA Yoichi, KUMADA Ai, SPIGELMAN Igor, NEUBERT John k.
Grant amount：\16850000 （ Direct expense: \14300000 、 Indirect expense：\2550000 ）
We found that trigeminal nerve constriction induced facial pain and increased neurotransmitter release from trigeminal ganglion cell body. Also, facial injection of highly purified Botulinum toxin type A 150 kDa (BoNT/A/A) decreased the pain level and the neurotransmitter release.
These results made clear the mechanisms of trigeminal neuralgia and peripheral BoNT/A injection may be an important treatment in the near future. -
Dentin regeneration by dental pulp stem cell, dental epithelium and dental mesenchyme
Grant number：18390514 2006 - 2008
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)
KANYAMA Manabu, KUBOKI Takuo, MATSUKA Yoshizou, UEHARA Jyunji, SONOYAMA Wataru, TSUCHIMOTO Yohei
Grant amount：\15200000 （ Direct expense: \12800000 、 Indirect expense：\2400000 ）
歯髄幹細胞, 歯胚上皮細胞, 歯胚間葉細胞を用いて, 歯胚発生段階で生じている上皮間葉相互作用を再現することで象牙質を再生しようと試みた.その結果, 上皮間葉相互作用に関連するSonic Hedgehog(Shh)と呼ばれる成長因子が象牙質を産生する象牙芽細胞の増殖や分化を促進することが明らかとなり, このShhが象牙質再生のキーファクターであることが示唆された.また, ヒトの智歯から採取した上皮細胞と間葉細胞を用い上皮間葉相互作用が試験管内で再現できる可能性が見いだされた.
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結合組織成長因子(CCN2/CTGF)を用いた顎顔面領域の三次元軟骨再生
Grant number：18592121 2006 - 2007
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)
藤澤 拓生, 服部 高子, 滝川 正春, 窪木 拓男, 上原 淳二
Grant amount：\3950000 （ Direct expense: \3500000 、 Indirect expense：\450000 ）
本研究では,ドナーサイトから採取した自己軟骨細胞をCCN2/CTGFとともに培養・増幅し、付形した3次元スキャフォードに播種後に移植する新しい顎顔面再生療法を開発するための基礎研究を行い,以下の知見を得た.
細胞実験にはすべて4周齢の日本白色ウサギの耳介より採取した初代耳介軟骨細胞を用いた.
1.CTGFの細胞増殖に対する効果をMTS assayで評価したところ,50ng/mlのCTGF添加により耳介軟骨細胞の細胞増殖は,細胞播種後5日目と7日目にコントロール群と比較すると有意に促進された.
2.DNA合成に対するCTGFの効果を[^3H]thymidineの取り込みを指標に検討したところ,CTGFは濃度依存性に耳介軟骨細胞のDNA合成を上昇させ,50ng/mlでピークに達した(コントロールの約1.5倍).
3.プロテオグリカン合成に対するCTGFの効果は[^<35>S]sulfateの細胞内への取り込みを指標に検討した.プロテオグリカン合成もDNA合成と同様に添加したCTGFの濃度依存性に上昇し,50ng/mlでピークに達した(コントロール群の約1.4倍).
4.軟骨細胞の分化関連マーカー遺伝子の遺伝子発現に対する効果はリアルタイムPCRにて検討した.50ng/mlのCTGFでコンフルエントに達した耳介軟骨細胞を48時間刺激することにより,CTGFの遺伝子発現は1.9倍,エラスチンの遺伝子発現は5倍,2型コラーゲンの遺伝子発現は1.5倍上昇したが,10型コラーゲンの遺伝子発現には有意な発現上昇は認められなかった.またエラスチンのタンパク産生をビクトリアブルー染色で確認したところ,CTGF添加によりビクトリアブルーの染色性は亢進していた.すなわち,エラスチンのタンパク産生はCTGF添加によりコントロール群と比べると亢進していた.一方,アリザリンレッド染色ではその染色性にコントロール群との差は認められなかった.すなわち,CTGF添加により耳介軟骨細胞の石灰化は誘導されなかった.
5.In vivoにおける軟骨再生に対するCTGFの効果は,細胞ペレットをヌードマウスの背部皮下に移植することで検討した.移植後4週に移植片を取り出したところ,CTGF処理群はコントロール群と比べると移植片の大きさが明らかに大きくなっていた.移植片をサフラニン染色したところ,CTGF群,コントロール群ともにサフラニン染色陽性であったが,CTGF群のほうがその染色性は亢進していた.
これらの結果から,CTGFには耳介軟骨細胞においてそのphenotypeを増強する働きがあると考えら,CTGFを弾性軟骨の修復・再生にも応用できる可能性が示唆された。 -
アドレナリンレプターの遺伝子多型からみた慢性筋痛の病態解明と症型分類
Grant number：18659572 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
前川 賢治, 窪木 拓男, 水口 一, 松香 芳三, 小野 剛
Grant amount：\3200000 （ Direct expense: \3200000 ）
1) 頭頸部筋症状とβ2アドレナリン受容体多型の関連性の検討
岡山大学歯学部のヒトゲノム倫理委員会から承認を受け,本学附属病院の顎関節症・口腔顔面痛み専門外来を受診した患者の中で,研究参加に同意の得られた被験者より採血を行った.各被験者のDNAサンプルを取得開始し,現在,約200名のサンプルを保有している.また,切断酵素を用いたβ2アドレナリン受容体の遺伝子多型解析手法を確立した.今後は,β2受容体の遺伝子多型や筋症状に関する因子を予測因子として,多変量解析を用いて統計解析を進めていく予定である.
2) 頚部慢性筋痛者における筋組織内代謝特性に関する疫学的検討
岡山画像センターをがん検診を目的としてPET(Positron Emission Tomography)検査を受診した患者の中で,研究参加に同意の得られたものを対象とした.各被験者からはPET撮像前に頭頸部の慢性筋痛に関するアンケートに回答してもらい,筋痛に有無や場所,疼痛の強さに関するデータを得た.PET画像は,両側僧帽筋上部の規格化された部位にROI (region of interest)を設定し,その部位におけるFDG(F-18フルオロデオキシグルコース)の集積値を求めた.各被験者の基礎データ(年齢,性別,頭頸部慢性筋痛の有無,疼痛強度等)と僧帽筋部のFDGの集積値を予測因子とした多変量解析を行った.その結果,慢性筋痛を訴える被験者ではFDGの集積は有意に低下している結果が得られた.さらに,疼痛強度が強いほど,FDGの集積値は低い傾向にあった.これらの結果は,慢性筋痛者においては筋組織内の代謝が抑制されて可能性を示すものと思われた. -
Bone regeneration with degradable hydrogels and genetically modified, recombinant human BMP-2 with two additional repeats of the heparin-binding domain.
Grant number：18592125 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)
NAWACHI Kumiko, KUBOKI Takuo, KANYAMA Manabu
Grant amount：\3790000 （ Direct expense: \3400000 、 Indirect expense：\390000 ）
A genetically modified, recombinant human bone morphogenetic proteirr2 with two additional repeats of the heparin-binding domain, derived from E coli (rhBMP-2 T4) has shown a stronger osteoinductivity than wild-type rhBMP-2. The aim of this study was to find a suitable carrier fix rhBMP-2 T4 in order to enhance bone regeneration. The in vitro and in vivo release profiles of^<125>I-labelled rhBMP-2 T4 incorporated into 6 kinds of gelatin hydrogels with different isoelectric point (IEP) were investigated. As a result, the higher the IEP was, the longer the rhBMP2 T4 radioactivity was preserved. To elucidate the osteoinductivity difference of the rhBMP2 T4/hydrogels composites, two high IEP gelatin hydrogels: a basic gelatin hydrogel (PI9) and a modified PI9 with spermine (SM), were implanted at critical-sized defects in the rat calvaria. The mean normalized radio-opacity against the surrounding intact bone in the PI9 group was significantly higher than those in the SM (p<0.001) and the control (without carrier) groups (p<0.001). The observed radio-opacity increase in the PI9 group was clearly related to the bone formation at the defect. Therefore, we concluded that PI9 is a suitable carrier for the rhBMP-2 T4 to enhance bone regeneration.
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Genetic analysis of monoamine transporter and relation to the frequency of the nocturnal bruxism.
Grant number：18592122 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)
MINAKUCHI Hajime, MAEKAWA Kenji, KUBOKI Takuo, KITAYAMA Shigeo
Grant amount：\3920000 （ Direct expense: \3500000 、 Indirect expense：\420000 ）
RESEARCH #1
Purpose: The relationship between sleep bruxism (SB) and temporomandibular disorders (TMDs) is unclear. This study aimed to estimate SB prevalence in an adolescent population and investigate the relationship between SB frequency and prevalence of the TMD signs/symptoms. Materials and Methods: First year high school students were recruited, and 195 subjects responded in this study. The subjects were divided into severe and non-severe SB groups with SB frequency and were received clinical examinations and interview. The odd-ratio (OR) were calculated to test the relationship between SB frequency gender difference and presence of the TMD signs/symptoms by multiple regression analysis. Results: Severe SB was significantly related to the presence of TMJ click (OR: 3.74, P= 02), while gender(male) was not related to that In contrast, severe SB was not related to headache, while gender (male)was significantly related to headache(OR: 2.52, P= 04). Conclusion: These results suggested that the presence of a TMJ click was closely related to the severe SB in an adolescent population.
RESEARCH #2
Objective: Novel miniature EMG analyzer (BiteStripR, S.L.P. Israel) occasionally showed error message when applied in Asian subjects. In response, the version-up model has been developed, and this study verified this modification. Methods: Six severe bruxism volunteers who had been diagnosed by portable electromyography and video recording participated this continuous 5-nights BiteStrip recording. The incidence rates of error message were compared. Results: The incidence of error message in the version-up systems (1/30, 3.3%) was significantly lower than that in the conventional system (14/30, 46.7%, p<0.01, Chi-square test). Conclusion: This modification was able to reduce the incidence of the error message.
RESEARCH #3
Objective: No research has been achieved regarding to the validity of bruxism assessment considering the daily fluctuation. This study aimed to verify the 1-night, 2-night and 3-night assessment of bruxism compared to the result of 10-straght night assessment. Methods: Fifteen TMD clinic patients were asked to measure individual bruxism level by means of using BiteStrip for 10 straight nights. The mode value of the result of 10-straight night was set as gold standard. The validity of 1-night test, 2-night test and 3-night test was calculated compared to prior gold standard. Result and summary: Totally 8 patients participated this study. The sensitivity, specificity and validity of 1-night test were 0.87-0.90, 0.74-0.80 and 0.82-0.88, respectively. This indicated the 1-night test could demonstrate the moderated diagnostic accuracy. -
オゾン照射によるエルビウムヤグレーザー照射象牙質の脆弱層強化
Grant number：18592123 2006
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)
峯 篤史, 窪木 拓男, 松香 芳三, 鈴木 一臣, 土本 洋平
Grant amount：\2600000 （ Direct expense: \2600000 ）
牛切歯掛冠部唇側象牙質を用い,試料の半分はう蝕試料として人工脱灰溶液に5日間象牙質を浸漬した.それぞれの試料は3等分し.2試料にレーザー(Er : YAG, CO2)照射を行った.Scanning Electron Microscopy (SEM) (DS-720,TOPCON)を使用し,加速電圧20kVの条件で観察し,Energy Dispersive X-ray Spectroscopy (EDS) (Voyager III M3100. Noran Instrument Inc.)を用いてビーム電流50〜100pA,測定時間150秒の条件で測定した.測定結果からO/PおよびCa/Pを算出した.
その後オゾン照射(ヒールオゾン,Kavo)し同様に形態観察および元素測定を行った.
統計解析はまず歯質(健全,う蝕),処理(無処理,レーザー照射),オゾン照射(有,無)という三条件の影響を三元配置分散分析にて確認した後. Sheffe法を用いて有意水準5%で有意差検定した.
形態観察では各処理による著明な変化は認められないものの,CO2レーザー照射およびオゾン照射により閉じた象牙細管がより観察される傾向があった.
三元配置分散分析の結果,O/Caにおいては歯質(F=1.173,P=.281),処理(F=2.443,P=.0955),オゾン照射(F=2.320,P=.1330)のいずれも有意な影響を受けさなかった.一方,Ca/Pにおいては,オゾン照射の有無に有意な影響を受けなかったが(F=3.330,P=.0730),歯質(F=7.097,P=.0099,)および処理(F=7.846,P=.0009)は有意な影響を及ぼした.表面処理においてSheffe法によりCO2レーザー,Er : YAGレーザー間に有意差が認められた.
以上により,象牙質に対するオゾン照射により形態的ならびに元素的には大きな変化が認められないが明らかになった. -
Newly developed bio-active implant by minute control of apatite coating layer.
Grant number：17390516 2005 - 2008
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)
MAEKAWA Kenji, YOSHIDA Yasuhiro, KUBOKI Takuo
Grant amount：\17020000 （ Direct expense: \15400000 、 Indirect expense：\1620000 ）
失われた歯を補う目的で顎の骨に埋入されるチタンインプラントが,早期に確実に骨と結合して治療期間が短縮できるよう,チタン表面に生物学的な表面の改質を施した.具体的には,生体内に埋入された時に骨と親和性の高いアパタイトがチタン表面に自己形成される酸化膜を施し,骨の形成能が亢進される表面を開発した。また,ポリリン酸をチタン表面に吸着させることにより,高い生体活性を有するチタンインプラントの開発に繋がる可能性を示した。
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Establishment of Information Basis for Tooth Regeneration
Grant number：17209062 2005 - 2008
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)
KUBOKI Takuo, UEDA Minoru, KANYAMA Manabu, TAKASHIBA Syogo, TSUJI Takashi, TAKIGAWA Masaharu, ASAHAR Hiroshi, TUCHIMOTO Youhei, SONOYAMA Wataru, TAGAWA Youichi
Grant amount：\48880000 （ Direct expense: \37600000 、 Indirect expense：\11280000 ）
マウスの歯の発生時に認められる遺伝子を検索し、従来報告のなかった28個の遺伝子を同定した。エナメル質形成細胞の成熟は、周囲に存在する細胞が制御していることを証明した。高脂血症治療薬(スタチン)は、象牙質の形成を促進し、歯科治療薬として応用しうることを示した。顎骨に存在する細胞は、手足の骨の細胞とは異なる性質を有していること、また、顎骨の再生促進に成長因子(結合組織成長因子、塩基性線維芽細胞増殖因子)が応用可能であることを確認した。
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Synthesis of a novel functional monomer and development of an adhesive system appropriate to laser-irradiated dental tissues
Grant number：17390517 2005 - 2007
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)
SUZUKI Kazuomi, YOSHIDA Yasuhiro, IRIE Masao, TANAKA Jiro, NISHIYAMA Norihiro, KUBOKI Takuo
Grant amount：\16390000 （ Direct expense: \15700000 、 Indirect expense：\690000 ）
Introduction: The use of Er:YAG laser for catty preparations during treatment of dental caries has received attention because there is no unpleasantness of noise and vibration as with air turbine and micromotor, and there is no need of anesthesia for treatment. However, we found that conventional dental adhesive systems do not perform sufficiently well on Er:YAG laser irradiated dental surfaces, and moreover, one of its reasons is the weakness of dental surfaces after laser irradiation. So, due to its importance and clinical significance, we aimed to clarify the dental structure after Er:YAG laser irradiation and its quality as an adhesive surface, along with the development of a new adhesive system appropriate to this surface.
Materials and Methods: 1) Adhesive surfaces preparation: Er:YAG laser (Erwin, Morita Co.) was irradiated with 127mJ, 10pps for 2 sec for enamel and with 69mJ, 10pps for 2 sec for dentin. 2) The primers of the adhesive systems were prepared with triazine-, aminoacid-, and epoxy-methacrylate/HEMA and alcohol. 3) The adhesive surfaces above were treated with each type of primer, phosphoric acid esther bonding agent and filled with composite resin (Clearfil APX, Kuraray Co.), then immersed in water at 37□for 24 hours before bond strength measurements.
Results and Discussion: The bond strength of epoxy type primer system to Er:YAG laser irradiated dental adhesive surfaces was 18.6MPa for enamel (control: Megabond/Kuraray Co.; 12MPa), and 17.2MPa for dentin (control: 9MPa). The authors say that these results may be due to oxygen that exists in the space created by microcracks that occurred during laser irradiation of dental adhesive surfaces. As conventional dental adhesives, used in the control group, are of radical polymerization type, the polymerization reaction decrease remarkably in the presence of oxygen. On the other hand, the ring opening polymerization type resin tested in this study may be not much influenced by the presence of oxygen during reaction. -
Development of chair-side assay system for ongoing bone loss with peri-implant sulcus fluids
Grant number：17592025 2005 - 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)
ARAKAWA Hikaru, KUBOKI Takuo, KANYAMA Manabu, UEHARA Junji, NAWACHI Kumiko, SEIYA Yamasaki
Grant amount：\3440000 （ Direct expense: \3200000 、 Indirect expense：\240000 ）
The first aim of this study was to detect MMP-1, MMP-8 and MMP-13 in the pen-implant sulcular fluid (PISF) collected from peri-implantitis sites and to determine any correlations between the MMPs presence/absence and the ongoing or future bone loss. Four peri-implantitis and four matched control patients were included in this study. Annually adjusted vertical bone loss (AVBL) was calculated to determine the annual velocity of the bone loss. PISF was analyzed by Western blotting utilizing anti-human monoclonal antibodies specific for MMP-1, -8 and -13. In the group of peri-implantitis patients, MMP-8 was detected in the PISF from three active peri-implantitis sites whose AVBL were more than 0.8 mm per year. Meanwhile, MMP-8 was not detected in the PISF from the remaining six implants whose AVBL at PISF sampling was less than 0.6 mm per year. In the control patients, MMP-8 was not detected in any PISF. MMP-1 and -13 could not be found in any PISF samples. According to the findings, it was clarified that MMP-8 could be a good marker for predicting ongoing bone loss around dental implants. The next aim was to identify the risk factors in clinical success rates of osseointegration (SRO) using multivariate analysis. Subjects were consecutive patients who had been installed dental implants with rough surface in our hospital from February 1990 to March 2007. Logistic regression analysis showed that no predictor variables were risk factors for SRO with recent rough surface dental implants. The third aim was to assess oral-health-related QoL (OHQoL) levels before and after treatment in partial edentulous patients with implant-supported dentures, fixed and removable partial dentures and to compare OHQoL-based treatment effectiveness among those treatment modalities. This indicates that implant-supported dentures can improve the patients OHQoL level much higher than the fixed and removable restorations.
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The practice of anti-cytokine treatment for temporomandibular joint osteoarthritis
Grant number：17592024 2005 - 2006
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)
UEHARA Junji, KUBOKI Takuo, FUJISAWA Takuo, MINAKUCHI Hajime
Grant amount：\3400000 （ Direct expense: \3400000 ）
We examined in vitro and in vivo for achievement of "The practice of anti-cytokine treatment for temporomandibular joint osteoarthritis".
Confluent cultured cells of rat primary articular chondrocytes were stimulated with TNFα, IL-1β or both cytokines for 24 hours. The anti-TNFα biological drug (etanercept) was added after stimulation with cytokines 6 and18 hours later. The gene expressions of mmp3, 9 and 13 in chondrocytes were evaluated by real-time RT-PCR.
1) Compared with control condition, cytokine stimulation significantly up-regulate the gene expression of each mmps at 24h. Under both cytokines stimulation condition, the gene expression of each mmps was more up-regulation than under TNFα or IL-1β stimulation condition.
2) Etanercept addition inhibited the up-regulation of mmp3, 9 and 13 gene expressions under TNFα or both cytokines stimulation condition. But each mmps gene expressions were not inhibited by etanercept at any time points under IL-1β stimulation condition.
3) The up-regulation of the gene expression of mmp3 or mmp9 stimulated by TNFα was significantly inhibited by etanercept at 24 h, at 12 and 24 h, respectively.
Etanercept was injected into the knee joint of the monoiodoacetic acid (MIA) induced experimental rat osteoarthritis model to histologically evaluate the effect on inhibition of damaged cartilage.
1) All of rats injected etanercept were not inhibited their articular cartilage damage.
These results indicated that etanercept had the inhibitory effect on the expression of MMP3, 9 and 13 induced TNFα in OA-like condition. Though mmp3, 9 and 13 gene expressions were not inhibited in the IL-1β addition, each mmps gene expressions were inhibited by etanercept in the both TNFα and IL-1β addition. Therefore it may be hopeful that etanercept is effective on the treatment of osteoarthritis with severe inflammation such as synovial tissue inflammation. Unfortunately the effectiveness of etanercept was not positive in vivo. We should evaluate the validity of this investigation in the future study.
In this investigation, we could not achieve the final objective of "The practice of anti-cytokine treatment for temporomandibular joint osteoarthritis". But present results are needful data for us to accomplish the purpose of this study. -
Assessment of gene transfer method for induction of osseo integration on dental implant
Grant number：16591948 2004 - 2006
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)
SUZUKI Koji, KUBOKI Takuo, KANYAMA Manabu
Grant amount：\3500000 （ Direct expense: \3500000 ）
CTGF gene transfer to the osteoblasts could be a promising strategy to accelerate bone formation. In this study, we investigated the possibility of recombinant adenovirus to transfer CTGF genes to a mouse osteoblastic (MC3T3-E1) cell line in vitro, and bone regeneration process after tooth extraction.
Rrecombinant adenovirus encoding the LacZ gene (Ax1CALacZ) were applied to the MC3T3-E1 cells with 5, 10 and 50 multiplicity of infection (MOI). At one day after transfection, those cells were stained with X-gal. As the result, the MOI 50 transfection dosage produced almost 80% X-gal staining of the cells without any obvious cell damages. Recombinant adenovirus encoding the human CTGF gene with CAG promotor (Ax1CACTGF) were fabricated and applied to the MC3T3-E1 cells in the MOI 50 dose. CTGF mRNA translation and protein production by the transfected cells were assessed by RT-PCR and western blotting of the harvested cells using a prepared primer set and an anti-human-CTGF antibody at 1, 3 and 7 days after transfection. Ax1CACTGF transfection caused a marked up-regulation in CTGF mRNA expression and CTGF protein even 7 days after transfection.
The sequential phase appearances of extraction wound healing were clearly observed by histological examination from 2 to 14 days after extraction. At 7 days after extraction, infilling with woven bone was observed from the socket margin with trabecular bone radiating toward the center of the socket, and at 14 days after, the extraction socket was almost fully filled by newly generated bone that underwent remodeling During any healing processes observed in the extraction wound, no cartilage-like cells were evident in the socket as determined by S-0 staining -
Epidemiological and clinical research by a miniature bruxism detection device - Verify the prevalence, proportionate contribution for TMD and efficacy of oral appliance therapy -
Grant number：16591949 2004 - 2005
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)
MINAKUCHI Hajime, MAEKAWA Kenji, KUBOKI Takuo
Grant amount：\3600000 （ Direct expense: \3600000 ）
Objective : Sleep bruxism (SB) has been assessed by a self-estimated questionnaire and/or degree of tooth attrition. However these reliabilities were recognized insufficient to detect on-going SB events. One solution can be a miniature self-contained EMG detector-analyzer (BiteStrip○!R, S.L.P. Israel) that can be applied directly to the masseter muscle. Our previous study demonstrated this device has sufficient reliability to detect bruxism events with polysomnography as a golden standard (sensitivity : 0.92, specificity : 0.91); however, this device occasionally showed error message when applied in Asian subjects. This would be potentially due to skin resistance difference between Asian and Caucasians. In response, the version-up model has been developed recently to solve this problem. This study verified the improvement of this device. SB also has been hypothesized as a precipitating/perpetuating factor in TMD signs and symptoms. However, the direct relationship between the SB level and prevalence of the TMD signs and symptoms were still unclear, because of the difficulty in measuring the SB severity epidemiologically. Therefore, the aim of this study was to investigate the relationship in an adolescent population by using a miniature SB detection device (BiteStrip○!R, S.L.P. Israel).
Methods : Six healthy volunteers (1 female and 5 males, mean age 24.5+/-0.55) who had been diagnosed as bruxisers by means of portable electromyography assessment and concomitant video recording and participated continuous 10-nights BiteStrip recordings. Each subject was applied both conventional and version-up BiteStrip on their bilateral buccal skin surface individually during continuous 5-nights. The incidence rates of error message were compared. The 1st degree students of a high school were exhaustively recruited, and 195 students (male/female : 86/109, mean age 15.4±0.5 yr) among them participated in this survey. They received clinical examination and were explained the usage of BiteStrip. Bruxism episodes were detected at their 30% MVC threshold, and total number of SB episodes was classified into 0-3 (<40, 41-74, 75-124 and ≦ 125, respectively). Relationship between their SB levels and presence/absence of the TMD signs and symptoms (clicking, limitation of mouth opening (LMO), headache, shoulder stiffness) were calculated, and odd-ratio and confidence interval (CI) were estimated by a simple regression analysis.
Results : The incidence (rates) of error message in the conventional and version-up systems were 14/30 (46.7%) and 1/30 (3.3%) and the incidence in the version-up system was significantly lower than that in the conventional systems (p<0.01, Chi-square test). Of which 195 responders, 29 subjects didn't return BiteStrip and 40 BiteStrip showed error or no indication. Severe bruxier (≧score 3) had 3.58 times higher prevalence of TMJ clicking (95% CI : 1.21-10.39, P=0.02), whereas other signs and symptoms showed no significant relation.
Conclusion : This modification made in the version-up BiteStrip was able to reduce the incidence of the error message in this system. The device appears to be clinically applicable even for Asian population. These results supported TMJ clicking can be associated with severe bruxism. However this study measured SB in one night, multiple SB recordings should be included in a further research. -
チタン表面への生体分子および細胞接着挙動のナノレベル・2次元・リアルタイム測定
Grant number：16659533 2004 - 2005
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
吉田 靖弘, 鈴木 一臣, 窪木 拓男, 平田 伊左雄, 田川 陽一, 長岡 紀幸
Grant amount：\3300000 （ Direct expense: \3300000 ）
チタンは医学・歯学の分野において人工歯根・人工骨の材料として重要な位置を占めており,その用途・規模は年々拡大している。本研究はチタンを用いた人工歯根・人工骨の治療期間短縮ならびに適応範囲拡大を目指し,チタン表面での生体物質の相互作用をnmオーダーでかつ多点で解析するシステムとして,2次元イメージング表面プラズモン解析装置(SPR)に適したイメージングSPR用チタンセンサーの設計・チタンSPRセンサー作製を目指した。
本研究のチタンSPRセンサーは二酸化チタンを直接表面に被覆せず,金属チタン表面を大気中で酸化させた。測定結果より,本研究で開発したチタンSPRセンサーは,タンパク質吸着量をng/mm^2のオーダーで精密に測定できることが明らかとなった。また,このセンサーを用いることにより,タンパク質の吸着過程を経時的に測定することも可能となった。これより,チタンセンサーは大気中で酸化された金属チタン表面での生体分子の相互作用を求める上で極めて有用であることが示唆された。
また,今回作製した2次元イメージングSPRは,(1)表面に存在する極微量の物質をpg〜ng/cm^2のオーダーでリアルタイムに定量可能,(2)多数の生体分子間相互作用に関するデータを瞬時に取得可能,(3)2次元平板上で同時に起こる複数(〜数1,000種類)の反応をパラレル分析することが可能であった。さらにユニークな特徴として(4)イメージングSPRでありながら正確な入射光角度-反射光強度のSPRスペクトルを測定可能であり,他のイメージングSPRと比べてより正確な物質吸着量を求めることができるなどの優れた長所を有していることも明らかとなった。
以上より,2次元イメージングSPRとチタンSPRセンサーはチタン材料の生体適合性の研究に重要な役割を果たすことが期待できる。 -
自己骨髄由来間葉系幹細胞にタンパク質導入法を応用した歯槽骨再生技術の開発
Grant number：16659534 2004 - 2005
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
完山 学, 窪木 拓男, 荒川 光, 縄稚 久美子, 小島 俊司
Grant amount：\3200000 （ Direct expense: \3200000 ）
本研究は,タンパク質導入法を用いて,BMP,SHH,FGF,IGF,TGF-b,CTGFなど骨形成に関連する成長因子を自己骨髄由来間葉系幹細胞内に導入し,次にそれらをキャリアとともに抜歯窩や実験的な歯槽骨欠損部位に移植することで歯槽骨の再生を試みようとするものである。
昨年度は,FITCとCTGF,そのファミリーであるcyr61,novなどの骨形成関連遺伝子のクローニングを行った。今年度はBMP-2,-4,SHH,FGF-2,galectin-1,-3,-9,sod-1など骨形成に関連する成長因子をコードするcDNAのクローニングを行った。現在,これらの遺伝子にPTD配列を含んだ遺伝子を増幅しPETベクターにクローニングを行っている。
in vivo実験においては,昨年度行った抜歯窩モデルが実験群とコントロール群で差が認められなかったことから今年度は大きな骨欠損モデルで試みた。はじめに生後8週齢のWistar系雄性ラット頭蓋骨全層骨欠損モデル(critical size defect)を作製した。これはラット頭蓋骨に直径6mmの全層骨欠損を作成したもので,この欠損部にBMP-2,FGF-2を含浸させた直径6mmのゼラチンハイドロゲルシート,I型コラーゲンシートを移植したところ,術後4週でゼラチンハイドロゲルシートもしくはI型コラーゲンシートとBMP-2を用いたものは,BMP-2単体やbFGF単体と比較して著名な骨再生が認められた。しかし,再生部位を歯槽骨に近づけるべく3.0kg雄性日本白色ウサギ下顎骨骨欠損モデル(defect size : 6mm)を用いた場合,I型コラーゲンキャリアとbFGFを用いても術後4週でコントロールとの著名な差を見いだすことができなかった。
今後は,PTD配列を含んだ骨形成関連遺伝子とゼラチンハイドロゲルやI型コラーゲンキャリア,さらに骨髄由来間葉系幹細胞を組み合わせた実験系を構築する予定である。 -
Development of functional Ti implants with release of growth factors
Grant number：16390557 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)
YOSHIDA Yasuhiro, SUZUKI Kazuomi, KUBOKI Takuo, HIRATA Isao, TAGAWA Yoh-ichi, NAGAOKA Noriyuki
Grant amount：\13800000 （ Direct expense: \13800000 ）
In order to develop functional Ti implants with release of growth factors, biodegradable materials as a carrier have to adhere strongly to Ti surface. Lactide copolymers synthesized in this study hardly adhered to TI surface.
Ti plates were treated with 0.1 and 1 N hydrochloric acid, 37wt% phosphoric acid or kept untreated. The tensile bond strength of the composite cement to the untreated and pre-treated Ti plates was determined without and after 20,000 thermo-cycles. XPS was used to chemically analyze the effect of the three Ti pre-treatments, as well as the interaction of 10-methacryloxydecyl dihydrogen phosphate (10-MDP) with Ti treated with 1 N HCl. Although no significant difference in immediate tensile bond strength was measured, thermo-cycling significantly decreased the bond strength of all experimental groups except for Ti treated with 1 N HCl. XPS demonstrated that H_3PO_4 was strongly adsorbed on the Ti surface. These results clearly suggest that phosphorylation was effective to get strong attachment to the Ti surface. Ti pre-treated with 1 N HCl improved the adsorption of 10-MDP as compared to untreated Ti.
Cleaned Ti disks were immersed into three different concentrations of polyphosphoric acid solution (0.1,1 and 10 wt%) for 24 hours at 37℃. Ti immersed in distilled water for 24 hours at 37℃ served as control. Degrees of cell attachment (1,3,5 hours after cell seed) and proliferation (1,3,5 and 7 days after cell seed) on each treated Ti disk were evaluated by MTS assay. A significantly higher cell attachment was found on Ti treated with polyphosphoric acid in contrast to untreated Ti (control) for all three culture periods. MTS assay also revealed that cell proliferation levels significantly increased following a polyphosphoric-acid dose dependency. It was concluded that polyphosphoric-acid treatment of Ti enhanced the attachment and proliferation of hBMSCs. -
Establishment of an autologous cell transplantion method using mesenchymal stem cells for alveolus bone regeneration.
Grant number：16591947 2004 - 2005
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)
FUJISAWA Takuo, KUBOKI Takuo, TAKIGAWA Masaharu, UEHARA Junji
Grant amount：\3100000 （ Direct expense: \3100000 ）
The purposes of this research are to investigate the possibility of autologous mesenchymal stem cell transplantion method for alveolus bone regeneration using connective tissue growth factor (CTGF).
1. Isolation of human bone marrow cells and cell characterization.
Bone marrow cells were isolated from human iliac bone marrow of volunteer. Human bone marrow cells were plated and cultured in DMEM containing 10% FBS. Moreover, STRO-1 expressing cells were identified by immunostaining from culture of passage 9 cells, and these cells showed the multilineage differentiation (osteoblastic and adipogenic).
2. Effects of CTGF on stem cells behavior.
1) Cell attachment efficiency onto hydroxyapatite disks is enhanced by coating CCN2/CTGF dose-dependently, and CCN2/CTGF activated p38 MAPK signal pathway via αvβ3 integrin.
2) CCN2/CTGF significantly increased the cell proliferation dose dependently.
3) The migration assay using Chemotaxicell indicated that CCN2/CTGF significantly increased the cell migration.
4) CCN2/CTGF did not affect cell differentiation to the osteoblast.
5) CCN2/CTGF also enhanced the endothelial cell attachment and proliferation.
6) In vivo implantation study indicated that CCN2/CTGF enhance the stem cell survival and induce their migration into the porous hydroxyapatite scaffold. -
Cloning, Expression and Identification of Specific Genes related Osseointegration to Titanium.
Grant number：15390592 2003 - 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)
KANYAMA Manabu, KUBOKI Takuo, TAKIGAWA Masaharu, ARAKAWA Hikaru, SUZUKI Kouji, FUJISAWA Takuo
Grant amount：\14800000 （ Direct expense: \14800000 ）
The aim of this study is to clarify the cell behavior induced by titanium and to isolate specific genes that promote the titanium implant-bone integration. Especially, to identify the master key genes related osseointegration to titanium, the osteoblastic cell line, MC3T3-E1 cells were cultured on titanium coated glasses and the gene expression were analyzed.
1)Titanium and chrome coated glasses
Ti and Cr were coated to polished glass surfaces (33 mm in diameter by 1.5 mm in thickness) in a high vacuum condition. Ti and Cr coated glass disks were placed in a 6 well plate and fixed by silicone rings. Non-coated glass disks were served as controls.
2)Cell attachment, proliferation and differentiation assessments.
Initial cell attachment and proliferation of MC3T3-E1 cells were estimated by MTS-assay (CellTiter 96【○!R】 AQueous One Solution, Promega, USA), and cell differentiation was evaluated by alkaline phosphatase activity assay. The mean relative amount of the attached cells on the Ti-coated glass was significantly higher than those on non-coated and Cr-coated glass at 3 hours after seeding. On the same way the mean cell number on Ti-coated glass at 3 days after seeding was significantly higher than those of other conditions. The mean alkaline phosphatase(ALP) activity of the seeded osteoblasts also increased with time in these three conditions, while the ALP activity level on the Ti-coated glass at 14 days after seeding was significantly higher than those of other conditions. These results suggest that, Ti-coated glass plate accelerated the cell attachment, proliferation and differentiation of the MC3T3-E1 cells, compared to the non- and Cr-coated glass plates
3)Effect of gene expression of osteoblast on titanium
Osteoblasts were cultured on Ti coated, Cr coated and non-coated glass plates. And then differentially expressed genes were identified by cDNA subtractive hybridization. Twenty independent clones were isolated and by nucleotide sequencing of these clones, seven clones were identified including EST genes ; xab-2,sod-1,galectin-1,actin related protein 2/3 mRNA, RIKEN cDNA 2210013021 gene, EST 601086505F1, and EST 01439. And galectin-1,xab-2,sod-1 gene expression on Ti-coated glass were higher than non-coated, Cr-coated glass.
These results suggest that Ti-coating is more advantageous for osteoblastic cell attachment, proliferation and differentiation than Cr or non-coating and galectin-1,xab-2 and sod-1 up-regulation could be related to the Ti-bone integration. -
The trial of treatment for temporomandibular joint osteoarthritis with soluble tumor necrosis factor receptor
Grant number：15592050 2003 - 2004
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)
MAEKAWA Kenji, FUJISAWA Takuo, KUBOKI Takuo, UEHARA Junji
Grant amount：\3400000 （ Direct expense: \3400000 ）
In this study, we tried to confirm soluble tumor necrosis factor receptor(sTNFR)-I,II concentrations in synovial fluids of osteoarthritic temporomandibular joint (OA) and to investigate the expression changes of sTNFR-I,II in the articular chondrocytes under an osteoarthritis-mimicking condition.
1.sTNFR-I and -II concentrations were higher in the synovial fluids from OA patients than from asymptomatic controls.
2.sTNFR-I concentration was higher than sTNFR-II concentration in the synovial fluids from both of patients and asymptomatic controls.
3.sTNFR-I concentration in the synovial fluids was significantly related to the OA level.
4.Higher sTNFR-II concentration in the synovial fluids was associated with less pain and less restricted range of mouth opening in the osteoarthritic patients.
5.In the knee osteoarthritis rat model, both of TNFR-I and TNFR-II were found at the knee joint chondrocyte of arthritic side and control side, and in the arthritic side both of receptors were up-regulated.
6.IL-1β and/or TNFα addition to the culture medium up-regulated tnfr-I,-II gene expression levels of the cultured chondrocytes and specifically sTNFR-II protein concentration in the cultured medium.
Since IL-1β and/or TNFα addition to the culture medium up-regulated tnfr-II gene expression levels of the cultured chondrocytes and sTNFR-II protein concentration in the cultured medium, observed sTNFR-II up-regulation in the synovial fluids of the osteoarthritic patients could reflect the protective chondrocyte metabolism of the osteoarthritic joint. Even in the osteoarthritic joints, sTNFR-II could modulate inflammation and destruction of the temporomandibular articular tissues.
Therefore, novel therapies may include agents that specifically mimic the anti-inflammatory feedback mechanism of sTNFR-II in vivo. We speculate that injection into temporomandibular joint with Etanercept, a potent inhibitor of TNFα, will be effective treatment for OA. -
口腔インプラントの骨結合獲得難易度を予測する生物学的診断法の開発
Grant number：15659463 2003 - 2004
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
窪木 拓男, 高柴 正悟, 滝川 正春, 荒川 光, 藤沢 拓生
Grant amount：\3300000 （ Direct expense: \3300000 ）
1.チタンの細胞培養および遺伝子発現への影響
骨芽細胞様細胞株(MC3T3-E1細胞)の細胞培養培養および遺伝子発現に対するチタンの影響を検討した。
1)チタンプレート
ポリスチレン製の培養皿と表面粗さを同程度にするために,研磨ガラスにチタンを真空蒸着したものを使用した。
2)細胞接着への影響
通常の培養皿と比較してチタンは細胞接着を抑制する傾向にあった。
3)細胞増殖への影響
通常の培養皿と比較して,細胞播種後1,2日ではチタンでは増殖が抑制されるものの3日では両材料ともコンフルエントに達した。
4)細胞分化への影響
骨芽細胞の分化の指標のひとつであるアルカリホスファターゼ活性は,両材料ともに細胞がコンフルエントになった後5日目ごろより上昇し,14日目でピークを向え,21日目では低下した。チタンでは通常の培養皿と比べてアルカリホスファターゼ活性は抑制された。
5)遺伝子発現への影響
通常の培養皿と比較し,チタンの遺伝子発現への影響をサブトラクティブハイブリダイゼーション法にて検討したところ,両材料間で発現に差のあるsod-1,xab-2の遺伝子を検出した。
6)リアルタイムPCR法による遺伝子発現の変動
サブトラクティブハイブリダイゼーション法にて検出した発現に差のあるsod-1,xab-2の経時的な発現の変動を検討したところ,培養皿では細胞播種後5日目で発現のピークを向え,その後低下した。チタンでは発現のピークが10日目前後と培養皿より遅延し,発現も抑制されていた。 -
Development of chair-side assay system for ongoing bone loss with peri-implant sulcus fluids
Grant number：15592049 2003 - 2004
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)
ARAKAWA Hikaru, KUBOKI Takuo, KANYAMA Manabu, KOJIMA Shunji
Grant amount：\3300000 （ Direct expense: \3300000 ）
The first aim of this study was to correlate MMP-1,MMP-8 and MMP-13 in the PISF samples collected from ongoing bone loss sites around per-implants with the periodontal treatments using Western blotting and to determine expression of MMP-8 by immunohistochemistry. Four peri-implantitis patients and four healthy subjects (control group) were selected from a sample of 64 consecutive patients who had attended the Okayama University Hospital from 1990 to 2000. Initial positive test was observed only in three patients. Following the first sampling, two patients received peri-implant mechanical and anti-microbial treatment, while one patient received no active treatment because of his inability to attend the hospital regularly. At the second PISF sampling analysis, an 85 kDa band corresponding to pro-MMP-8 was observed only in one patient received no active periodontal treatment. Half a year following the second PISF sampling, the implant in this patient unfortunately was lost because of severity of peri-implantitis. And MMP-8-like immunoreactivity was clearly seen around inflammatory connective tissue cells in from active peri-implantitis patients.
The next aim of this study was to identify the risk factors in acquisition and maintenance of osseointegration using multivariate analysis. Subjects were consecutive patients who had been installed and finished the secondary surgery osseointegrated dental implants in our hospital from February 1990 to March 2002. Based on the adjusted multivariate models, tobacco use, jaw and implant coating were statistically associated with acquisition of osseointegration. While the risk factors in maintenance of osseointegration were jaw and implant length. -
Gene Therapy for Periodontal Diseases -Regulation of host response by local gene delivery-
Grant number：14370710 2002 - 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)
TAKASHIBA Shogo, KUBOKI Takuo, NISHIMURA Fusanori, KUBOTA Satoshi, MYOKAI Fumio
Grant amount：\13500000 （ Direct expense: \13500000 ）
Because understanding the target factor was important, we decided to specify the target factor from both sides of non-specific host defense and tissue regeneration. Moreover, because it was also anxiety against clinical application of gene therapy because of its toxicity, the experiments are performed for testing it.
1.Target gene
In the rat, cytochrome c oxidase gene expressed strongly at 1 week and pro-α-2 type I collagen gene expressed strongly at 2.5 weeks after alveolar bone begun regeneration. Activation of these genes seem to be needed for alveolar bone regeneration. In dental pulp would, the homolong of human 12.7K-interacting protein 2 expressed strongly. We named it rat FIP-2 gene. It is under analyzing now for physiological meaning. In addition, inflammation, promoter region required for LPS-induced transcription of LITAF was revealed, which is new transcription factor for human tumor necrosis factor(TNF)-α.
2.Introduction of β-defensin by non-viral vector
Anti-bacterium peptide β-defensin gene was transferred to human epithelial cells and rat salivary glands to test its effect for reduce bacteria around cultured cells or rat oral cavity. Furthermore, its effect for local tissue inflammation was also examined. We found that bacterial numbers are reduced and that no obvious inflammation in rat salivary gland tissue. However, when electroporation was used for gene delivery, obvious inflammation was detected. Furthermore, It was revealed that β-defensin gene transcription requires 2 regions of NF-kB binding domain on its promoter, but that NF-IL6 biding domain on it acts to reduce its transcription. -
Establishment of a autologous cell transplantion method using mesenchymal stem cells for perio-dontal tissue and alveolus bone regeneration.
Grant number：14370632 2002 - 2004
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)
KUBOKI Takuo, UEDA Minoru, TAKIGAWA Masaharu, TAKASHIBA Shogo, MAEKAWA Kenji, YOSHIDA Yasuhiro
Grant amount：\14800000 （ Direct expense: \14800000 ）
1.Isolation human bone marrow cells and cell culture.
Bone marrow cells were isolated from human iliac bone marrow of volunteer. Human bone marrow cells were plated and cultured in DMEM containing 10% FBS. Culture medium was changed every 3 days, and their multipotent (osteoblastic and adipogenic) differentation abilities were confirmed
2.Connective tissue growth factor (CTGF/CCN2) enhanced hMSC attachment, migration and survival in a hydroxyapatite scaffold
Human bone marrow cells were incubated to attach onto porous HA blocks for a week. The porous HA/cells hybrids were implanted subcutaneously in nude mice (4 week-old) with CTGF (1ug) or distilled water (control).
The implants were harvested after 4 weeks for SEM observation. SEM observation supported that hBMSC-like cells migrated and survived inside of the porous HA scaffold with CTGF application, while without CTGF, no viable cells were observed inside of the scaffold.
3.hMSC initial attachment and proliferation enhanced on titanium
Adsorption of poly phosphoric acid to Ti disk surface was achieved by immersing Ti disk poly phosphoric acid solution (1 wt%) for 24 hours. Adsorption of polyphosphoric acid onto Ti disks enhanced attachment and proliferation of hMSC.
4.Effect of gene expression of osteoblast on titanium
Osteoblast was cultured on titanium dish and searched a titanium specific gene by cDNA subtractive hybridization. It became clear on titanium that sod-1, gene expression of ribosomal protein L19 were restrained significantly. -
Development of the methods for application of the factors that biologically accelerate reparative dentin formation
Grant number：14571844 2002 - 2003
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)
SONOYAMA Wataru, TAKIGAWA Masaharu, TAKASHIBA Shougo, KUBOKI Takuo
Grant amount：\3900000 （ Direct expense: \3900000 ）
1. Pulp cell isolation from human extracted tooth and confirmation of their phenotype
Under permission of ethical committee, pulp cells were isolated from human extracted tooth. RT-PCR was carried our to confirm their gene expression profile and phenotype. As a result, they expressed odont oblast-specific gene, dentin sialophosphoprotein (DSPP), and were suspected to be odont oblast lineage.
2. Effects of Growth factors on their attachment, proliferation, and differentiation
Effects of growth factors, e.g., transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), and connective tissue growth factor (CTGF), on their attachment to plastic dish were investigated. As a result, adsorption of these growth factors enhanced their attachment to plastic dish. Effects on their proliferation and alkaline phosphatase (ALPase) activity were also investigated. Concerning about proliferation, only TGF-beta1 enhanced their proliferation. While ALPase activity was downregulated by TGF-beta1 and bFGF.
3. Effects of hydroxyapatite (HAP) on their attachment
To estimate compatibility of HAP with pulp-derived cells, their attachment onto HAP was investigated. As a result, attachment onto HAP was significantly higher compared to plastic dish made of polystyrene. Adsorption of growth factors onto HAP tended to enhance attachment, but the difference was not significant.
4. Effect of HAP on their gene expression
To estimate that HAP have an effects on gene expression of pulp-derived cells or not, they were seeded onto HAP and their gene expression were investigated by RT-PCR. As a result, DSPP and type 1 collagen gene expression were significantly enhanced. -
Molecular mechanism of temporomandibular joint osteoarthritis and gene therapy for degraded, cartilage
Grant number：14571843 2002 - 2003
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)
FUJISAWA Takuo, TAKIGAWA Masaharu, NISHIDA Takashi, KUBOKI Takuo, MAEKAWA Kenji
Grant amount：\4000000 （ Direct expense: \4000000 ）
The purposes of this research are to clarify the mechanism of cartilage degradation in osteoarthritis (OA) and to investigate the possibility of gene therapy for articular cartlige restoration using connective tissue growth factor (CTGF).
First, we investigated the effects of CTGF/Hcs24 transduced by recombinant adenoviruses on the rabbit articular cartilage (RAC) cells in vitro. When RAC cells were infected with adenoviruses containing the CTGF/Hcs24 gene, RAC cells expressed CTGF/Hcs24 mRNA and produced CTGF/Hcs24 protein. RAC cells synthesized more proteoglycan than the control cells. These results suggest that CTGF is useful factor for cartilage repair.
Second, we establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive forced jaw opening (RFJO) 3 hours/day for 5 days was applied. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the RFJO period. Furthermore, apoptotic chondrocytes were observed in the cartilage degradation area at 7 days after the RFJO period. And nitrotyrosine, a marker of NO production, and MMP-3, a key factor of cartilage ECM degradation, were observed where chondrocyte apoptosis was evident. These results suggest the RFJO protocol without any surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and cartilage degradation in OA may be induced via chondrocytes apoptosis. This OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA. -
Responses of Hypothalamic-Pituitary-Adrenal axis to cold pressor stimulation
Grant number：13672032 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)
SUZUKI Koji, KASAI Teruo, MAEKAWA Kenji, KUBOKI Takuo
Grant amount：\3500000 （ Direct expense: \3500000 ）
Objective : Functional abnormalities of Hypothalamic-Pituitary-Adrenal (HPA) axis have been known implicated in chronic pain conditions (e.g., fibromyalgia). This study evaluated the changes in plasma endogenous opioid, HPA hormones concentration and trigeminal heat pain threshold upon cold pressor (CP) stimulation. Methods : In CP trials, ten healthy males (mean age : 25.3^^+__-1.3yrs) immersed their right hands for 2 minutes in 4℃ cold water. Mock trials were also done without the cold water. Whole blood samples were collected 5 minutes before, during, and 5, 15, 30, 45, 60 minutes after CP and mock stimulation for measuring Corticotropin-Releasing-Factor (CRF), Adrenocorticotropic hormone (ACTH), β-endorphine and cortisol. Whole blood was centrifuged for plasma separation, then frozen (-20℃) for later assay. Plasma CRF, ACTH, β-endorphine and cortisol concentrations were determined using radioimmunoassay techniques. Heat pain thresholds in subjects' right V2 region were simultaneously recorded 5 minutes before and 5, 30, 60 minutes after CPS using Thermal Sensory Analyzer (Medoc, Israel). Results : Mean plasma ACTH and β-endorphine levels significantly increased 5 minutes after cessation of CP stimulation and returned to the baseline in 60 minutes (p<0.01, One-way repeated measure ANOVA). Mean plasma cortisol levels also increased 30 minutes after cessation of CP stimulation (p<0.01), while no plasma CRF change was detectable (p=0.83). Mean heat pain thresholds did not show statistically significant changes during the experimental time course (p=0.41), since 4 of 10 subjects showed no obvious change in the heat pain thresholds even with the same CP stimulation schedule. Meanwhile, no variables observed in this study showed a significant change in the mock trial. Conclusions : These results suggest that CP stimulation induces progressive increase in plasma endogenous opioid and HPA hormones levels in healthy males, but this acute plasma endogenous opioid increase does not always associate to heat pain threshold responses in V2 skin region.
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MMP-3転写調節部位遺伝子多型からみた顎関節症の予後に関する分子遺伝学的研究
Grant number：13672033 2001 - 2002
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)
水口 一, 滝川 正春, 矢谷 博文, 窪木 拓男, 藤澤 拓生
Grant amount：\4000000 （ Direct expense: \4000000 ）
MMP-3遺伝子のプロモーター領域の多型(5A/6A)と関節疾患感受性との関連性を明らかにすることを目的として遺伝子多型解析を検討した.まず,本年度は遺伝子多型解析に先立ち,本研究計画に対して岡山大学歯学部倫理委員会の承認を得る必要があった.そのため,平成13年11月から岡山大学歯学部倫理委員会の承認を受けた後,岡山大学歯学部附属病院第一補綴科に顎関節部の疼痛,機能障害を主訴に来院し,本研究の趣旨を文書にて説明し自発的同意の得られた被検者から一律3mlの血液採取を行った.
平成14年1月30日現在での被検者数は,男性18名,女性26名の計44名であり,これら被検者の血液よりDNAの抽出を行った.このDNA抽出に関する手法は確立し得た.
現在,抽出されたDNAから、MMP-3プロモーター領域の対立遺伝子の多型(variable number of tandem repeat : VNTR)ならびにIX型コラーゲンα3鎖の遺伝子多型(SNPs)を検討すべく,Ye et al.(1995)の方法ならびにPaassiltaらの方法に従い、PCR産物に対してTthlllI酵素処理を応用し,被検者個々の対立遺伝子の多型を明らかにした.
その結果,現在集積された被検者数では,遺伝子多型と関節疾患感受性との間に統計学的に有為な関連性を見いだすことはできなかった.しかしながら,現段階では被検者数が少なくtype II errorが生じている可能性があり,今後も被検者数の継続的蓄積が必要となると考えられた. -
血管径調節関連遺伝子多型からみた慢性筋痛の分子遺伝学的検討
Grant number：13877327 2001 - 2002
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
窪木 拓男, 矢谷 博文, 鈴木 康司, 前川 賢治
Grant amount：\2000000 （ Direct expense: \2000000 ）
全身の多部位に慢性筋痛を訴える線維性筋痛症(FMS)患者のβ_2アドレナリン受容体(β_2AR)機能評価のため,末梢血由来単核球細胞上に存在するβ_2ARを用い,情報伝達の際に産生されるcAMPを機能性マーカーとして,ELISA法により測定し,年齢,性別をマッチングさせた正常被験者のそれと比較した.FMS群(女性8名,平均年齢44.3歳)と無症状群(女性9名,平均年齢35.8歳)から静脈血を採取し,単核球を分離した.分離した単核球は10^6個ずつ分注し,安静時ベースラインと10^<-3>,10^<-5>Mに希釈したβAR刺激薬(Isoproterenol, IP)を5分間作用させた低・高濃度IP刺激後の3条件のcAMP量を測定した。その結果,低濃度である10^<-5>M IP作用後のcAMP量は,無症状群では有意な増加を示したが,FMS群では変化がみられなかった(無症状群:p<0.001,FMS群:P=0.520).FMS群において単核球におけるβ_2ARの刺激に対する反応が抑制されているという事実は,FMSの病態に全身性のβ_2AR機能の脱感作が関与する可能性を示唆する重要な所見と思われた.一方で,FMSとの病態メカニズムの差異が論争されている慢性局所性筋痛症のβ_2AR機能評価を,慢性筋痛者11名,正常被験者21名を対象にして前述の手法に従って行った.その結果,単核球が産生するcAMP濃度はIP刺激濃度依存性に増大したが,その濃度変化量は両群間に有意な差が見られなかった.本結果は,局所性慢性筋痛症の病態には,全身性のβ_2AR機能異常の関与は認められないこと,換言すれば,線維性筋痛症と局所性慢性筋痛症の病態が異なることを示す重要な所見であると考えられた.遺伝子多型の解析については「岡山大学歯学部ヒトゲノム・遺伝子研究倫理審査委員会」より患者の遺伝子解析について承認を得た後,本学歯学部附属病院顎関節症・口腔顔面痛み外来を受診した患者のなかで,本研究計画の被験者選択基準に合致し,研究の参加に同意の得られた者より,末梢血3mlを供与してもらい,サンプル数を増加させ,解析を進めている.
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Risk assessment of peri-inplantitis patients with chair-side MMP-8 assay system
Grant number：13672031 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)
ARAKAWA Hikaru, KASAI Teruo, YATANI Hirofumi, KUBOKI Takuo
Grant amount：\4000000 （ Direct expense: \4000000 ）
Matrix metalloproteinases (MMPs) are known to play an important role in pathological tissue destruction in the periodontal tissues. Therefore, MMPs related to bone matrix degradation could be a possible marker for ongoing bone destruction around the peri-implantitis implants. In this study, four peri-implantitis patients (male/female : 2/2 ; mean age : 68.5^^+__-6.7 yrs ; 9 implants ) and age- and sex- matched four control patients (male/female : 2/2, mean age : 66.0^^+__-5.8 yrs, 8 implants) were selected from 64 consecutive patients who were installed implant-supported superstructures in our school from 1990 to 2000. Inclusion criteria for selection of the peri-implantitis and control patients in this study were having an implant "with accumulated vertical bone loss (ABL) of more than 0.6 mm per year" and "with ABL of less than 0.2 mm per year" after superstructure installation, respectively. Bone levels around implants were assessed by dental x-ray films taken annually and mean values of three calibrated observers readings were used as outcome. Ongoing vertical bone loss per year (OBL) after superstructure installation was also calculated. Gingival crevicular fluids (GCFs) were collected at the mesio-buccal comer of the gingival crevices by using absorbent paper points. Then the samples were dissolved in 0.1% phoshate buffered saline with Tween 20 (PBS-T) and stored at -20℃. MMPs in GCFs were detected by Western blotting using anti-human monoclonal antibodies specific for MMP-1, -8 and -13 (Fuji Chemical Industry, Japan) with positive controls. As the results, in the peri-implantitis patients, MMP-8 was detected in three actively bone-resorbing implants whose OBL at GCF sampling was more than 0.8 mm and not detected in the other six implants whose OBL at GCF sampling was less than 0.3 mm, while no MMP-8 was detected in the controls. MMP-1 and -13 could not be detected in any GCFs in this study. These results suggest that MMP-8 might be a marker for prediction of ongoing bone destruction around the peri-implantitis implants.
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Gene therapy for temporomandibular joint osteoarthritis
Grant number：12557169 2000 - 2002
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)
KUBOKI Takuo, NAKANISHI Tohru, TAKIGAWA Masaharu, FUJISAWA Takuo, KASAI Teruo, YATANI Hirofumi
Grant amount：\12000000 （ Direct expense: \12000000 ）
The purposes of this research are to clarify the mechanism of cartilage degradation in osteoarthritis (OA) and to investigate the possibility of gene therapy for articular cartlige restoration using connective tissue growth fector (CTGF).
First, we establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive forced jaw opening (RFJO) 3 hours/day for 5 days was applied. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the RFJO period. These results suggest the RFJO protocol without any surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and this OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA.
Second, we investigated the effects of CTGF/Hcs24 transduced by recombinant adenoviruses on the rabbit articular cartilage (RAC) cells in vitro. When RAC cells were infected with adenoviruses caontaining the CTGF/Hcs24 gene, RAC cells expressed CTGF/Hcs24 mRNA and produced CTGF/Hcs24 protein. RAC cells synthesized more proteoglycan than the control cells. -
Molecular cloning of the factors that biologically accelerate reparative dentin formation
Grant number：12470418 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)
KUBOKI Takuo, TAKIGAWA Masaharu, TAKASHIBA Shougo, SONOYAMA Wataru, KANYAMA Manabu, NAKANISHI Tohru
Grant amount：\13800000 （ Direct expense: \13800000 ）
1. Gene delivery to cultured cells
We prepared recombinant adenovirus vector carrying beta-galactosidase gene. Mouse osteoblast-like cells, MC-3T3 -E1, were cultured with this vector. As a result, almost all cells were transfected with beta-galactosidase gene.
2. Localization of known factors (TGF-beta 1 and CTGF) in vivo animal model
Localization of TGF-beta 1, that is supposed to be involved in reparative dentinogenesis, and CTGF were confirmed with immunohistochemical staining in animal (wister rat) experimental model. As a result, in 2 weeks from tooth reduction reparative dentin-like tissues were observed, and strong staining of TGF-beta 1 and CTGF were observed around these tissues.
3. Gene expression of TGF-beta 1 and CTGF in cultured cells stimulated with proinflammatory factors
MDPC-23, mouse-derived odontoblast-like cells were used in this study. The cells were stimulated with IL-1 beta and bacterial LPS. Changes in CTGF and TGF-b1 genes expression were examined by RT-PCR. As a result, MDPC-23 cells were expressing the CTGF and TGF-beta 1 genes coastitutively, and both factors increased CTGF gene expression and decreased TGF-b1 gene expression in the odontoblast-like cells (MDPC-23) within one-day period after stimulation.
4. Effect of TGF-beta 1 and CTGF to cultured cells
The effects of rCTGF and rTGF-beta 1 on cell proliferation were determined by the MTT assay. rTGF-beta 1 tended to decrease the proliferation dose-dependently, whereas effect of rCTGF was not evident. Next, to investigate the effects of rCTGF on calcification of MDPC-23 cells, cells were cultured with medium containing rCTGF. Sequential addition of AA and b-GP up-regulated the ALPase activity, while addition of rCTGF had no obvious effects. -
MRI brain mapping image analysis of oral and maxillofacial region by various sensory stimuli
Grant number：12671825 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)
KISHI Kanji, SHIGEHARU Hiroshi, WAKASA Toru, SUGIMOTO Tomosada, KUBOKI Takuo
Grant amount：\3400000 （ Direct expense: \3400000 ）
The aim of this study was to decide the adequate pulse sequence suitable for fMRI study of various sensory stimuli, and to establish the fixation of the head and image processing method, first of all. Secondly, we applied fMRI to the healthy volunteers on the tasks of gustatory stimulation and swallowing, and investigated the possibility of fMRI in the oral and maxillofacial region on clinical application.
In 2000, first of all, we investigated the scanning method. We performed fMRI study of hand-grasping task by two pulse sequences, gradient echo type EPI(GE-EPI), and spin echo type EPI(SE-EPI), and compared the fMRI images by these two sequences. As a result, the specificity and accuracy of the signal detectability by SE-EPI were higher than those by GE-EPI. Therefore, we decided the SE-EPI sequence on the following study.
The fixation of the head was performed by applying the hair-band and sponge to the head-coil and the head of the subjects. Visual stimulation and auditory stimulation were shut by using eye-mask and ear-plug.
Image processing was performed by the software (Numaris) included in Magnetom Vision, and fMRI images were produced by the statistical calculation using z-score.
Then, fMRI study by gustatory stimulation was performed. We used two tastants ; 1M NaCl and 3mM saccharin. These tastants were dropped into the oral cavity of the subjects through the tube, and fMRI scanning was done. As a result, pariental operculum, frontal operculum, and insula were mainly activated. There were no differences of activated areas between NaCl and saccharin.
In 2001, fMRI by swallowing task was performed. Distilled water was injected into the oral cavity of the subjects at 3ml/sec. The subjects swallowed the water intermittently. As a result, primary motor cortex, and primary somatosensory cortex were mainly activated. -
Molecular biological study on pithophysiology of chronic muscle pain from the view of abnormal beta2-adrenergic receptor function.
Grant number：12671883 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)
MAEKAWA Kenji, MINAKUCHI Hajime, SUZUKI Kouji, KUBOKI Takuo, YATANI Hirofumi
Grant amount：\3400000 （ Direct expense: \3400000 ）
The purpose of this study was to investigate alteration of the beta-adrenergic receptor (BAR) in fibromyalgia and chronic localized myalgia patients. Since the BARs are present on circulating lymphocytes and activation of the G-protein coupled receptors like BAR leads to an increase in the intracellular level of cyclic adenosine monophosphate (cAMP), we measured cAMP levels to assess indirectly the functional status of the receptor. Thirty cc peripheral blood samples were drawn from subjects' anterocubital vein. Lymphocyte cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit (cAMP EIA system, Amersham, England). Aliquots of even amount of cells were incubated with or without stimulation of beta-agonist isoproterenol (ISO) for 5min. Results : Basal intracellular cAMP levels were not significantly different between FM patients and healthy controls and between localized myalgia and healthy controls. However, beta-agonist induced mean intracellular cAMP increase was significantly reduced in FM patients compared with controls. Regarding the localized myalgia patients, beta-agonist induced mean intracellular cAMP increase was almost identical with that of controls (p=0.806). These results suggest that diminished βAR function might be related to FM pathophysiology. In addition, these findings indirectly support the notion that the pathophysiology of fibromyalgia and localized myalgia are different.
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Effect of mastication on 3-dimensional brain hemodynamics measured by functional MRI
Grant number：12557170 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)
MAEKAWA Kenji, KISHI Kanji, SUZUKI Kouji, KUBOKI Takuo, WAKASA Touru
Grant amount：\6500000 （ Direct expense: \6500000 ）
The original purpose of this study was not achieved, because mastication provoked motion artifact to image brain hemodynamics using MRI. For this reason, we were forced to change the research protocol and investigated the cerebral cortical activation during volitional swallowing using functional MRI. The results showed that the swallowing task activated the inferior parts of the pre- and postcentral gyri. Additionally, we also investigated the correlation between T2 weighted MRI signal intensity (S1) and intramuscular blood volume in human muscle. First, we compared blood volume changes transcutaneously measured using near-infrared (NIR) spectroscopy against SI changes taken from a transverse T_2-weighted MR image of the masseter muscle in healthy human subjects before, during and after contraction. The data showed that both NIR-based total blood volume and T_2-weighted MRI-based SI levels clearly decreased during muscle contraction and a clear post-contraction rebound response was evident after the contraction. The NIR data were found to be highly correlated to MRI-based SI data (Pearson's r=0.945, p<0.0001). Next, we evaluated the ability of SI in T2-weighted trapezius muscle MRI to detect intramuscular hemodynamic changes upon cold pressor stimulation (sympathetic nerve activator). Cold pressor stimulation (4℃) was applied to each subject's right foot and ankle for 2 min. The SI changes were recorded continuously for 7 min before, 2 min during, and 6 min after withdrawal of cold pressor stimulation. The mean SI level in T2-weighted trapezius muscle MRI significantly increased during cold pressor stimulation (p<0001, one way repeated measure ANOVA and post hoc contrast analysis) and returned to the baseline level after cold pressor withdrawal. These findings are identical to the cold pressor-induced hemodynamic changes documented in the trapezius muscle by NIR spectroscopy evaluation (Acero et al., 1999). Both studies provided the evidence that SI measurement in T2-weighted muscle MRI is sufficiently sensitive to detect intramuscular hemodynamics in humans.
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Ex Vivo Gene Delivery Using an Adenovirus Vector in Treatment for Dental Implant
Grant number：12470419 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)
KANYAMA Manabu, NAKANISHI Tohru, TAKIGAWA Masaharu, KUBOKI Takuo, ARAKAWA Hikaru
Grant amount：\8200000 （ Direct expense: \8200000 ）
The purpose of this study was to contribute to shortening the healing period of the dental implant and expanding its application by ex vivo gene delivery using an adenovirus vector. It has been well-documented that connective tissue growth factor (CTGF) is up-regulated in the healing process at the bone fracture site in vivo and known as a potent stimulator for the proliferation and differentiation of osteoblasts in vitro. Therefore, CTGF gene transfer to the osteoblasts could be a promising strategy to accelerate bone formation. First, we investigated the possibility of recombinant adenovirus to transfer CTGF genes to a mouse osteoblastic (MC3T3-E1) cell line in vitro. Recombinant adenovirus encoding the LacZ gene and the human CTGF gene with CAG promotor (Ax1CACTGF) were applied to the MC3T3-E1 cells with 5, 10 and 50 multiplicity of infection (MOI). As the result, the MOI 50 transfection dosage produced almost 80% X-gal staining of the cells without any obvious cell damages and Ax1CACTGF transfection caused a marked up-regulation in CTGF mRNA expression and CTGF protein even 7 days after transfection. Second, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets to know the mechanisms of new alveolar bone formation. As the result, CTGF was expressed in the endothelial cells migrating into the granulation tissue at the sockets during 4 days after tooth extraction. Osteoblast-like cells proliferated in the sockets with CTGF expression at 4, 7, 10 and 14 days after extraction. Finally, we tried the ex vivo gene delivery, but we could not isolate osteoblasts from the defects of rat alveolar bone.
Based on these findings, adenovirus-mediated CTGF gene transduction to the cultured osteoblast-like cells was highly successful. However, in order to do the ex vivo gene transfer, it is necessary to develop the new methods of isolating osteoblasts. -
アデノウイルスベクター法を用いた早期osseointegrationの獲得
Grant number：11877338 1999 - 2001
日本学術振興会 科学研究費助成事業 萌芽的研究 萌芽的研究
李 起学, 滝川 正春, 中西 徹, 窪木 拓男
Grant amount：\2200000 （ Direct expense: \2200000 ）
休止期や分裂増殖の遅い細胞へも高い効率で遺伝子導入が可能であり,in vivo遺伝子導入が容易な,アデノウイルスベクター法を用い,アデノウイルスベクターに骨形成能が高いCTGF, TGF-βの遺伝子を組み込む方法を用い,続いて,ラット脛骨にアデノウイルスベクターを投与し,周囲組織(骨芽細胞,間葉系細胞)に感染,遺伝子導入させることで,持続的にそれらの遺伝子を発現させ,早期osseointegrationの獲得を試みることを目的とした実験を行ってきた。
前年度において,アテロコラーゲンとウイルスベクターの複合化と,複合化したウイルスベクターのin vivo LacZ遺伝子導入を行った。しかし導入効率が低く他のキャリアを用いた実験系が必要であると考えられた。そこで本年度はポリ乳酸や他の高分子生体材料をキャリアとして用いウイルスベクターと複合化を行いin vivo実験を行った。
1.ポリ乳酸とウイルスベクターの複合化
ポリ乳酸とウイルスベクター液を混ぜ凍結乾燥を行い,複合化させることに成功した。
2.複合化したウイルスベクターのin vivo LacZ遺伝子導入
ラット脛骨内に1で作製したLacZ遺伝子を発現するアデノウイルスベクターを注入し,経時的にラットを屠殺し,脛骨におけるLacZ遺伝子発現をX-gal染色にて観察したところ,ポリ乳酸を用いても導入効率の上昇にはつながらなかった。
3.ウイルスベクターの他臓器への感染の有無
2のラット屠殺時,固定直前に気管,肝臓,膵臓,腎臓,骨格筋を摘出し,total RNAを抽出し,LacZに対するprimerを用い,RT-PCRを行ったところ,他臓器にLacZ遺伝子の発現は認められなかったことから,ウイルスベクターの他臓器への影響がないことが確認できた。 -
The cloning and application of growth factors in restorative dentine
Grant number：10470417 1998 - 1999
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)
KANYAMA Manabu, SUZUKI Kouji, KUBOKI Takuo, TAKIGAWA Masaharu
Grant amount：\11500000 （ Direct expense: \11500000 ）
This study first sought to find unknown genes that expressed specifically to be made the restorative dentine by odontblast. Next, to confirm that foreign genes can be transferred to dental pulp of rats and to clarify the in vivo transfer availability of the adenovirus vectors.
When tooth was prepared the cavity by diamond points, we detected the specific 14 clones from dental pulp by subtractive hybridization.
Recombinant adenovirus harboring LacZ gene was injected into the prepared tooth cavity of 6-week-old Wistar rats. At 1week after injection, the tooth was dissected and X-gal staining examined the expression of delivered LacZ. To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart, and brain and expression of LacZ mRNA were analysed by RT-PCR. Expression of LacZ was observed a few odontblasts in the cavity. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the tooth cavity using adenovirus vector is feasible as an effective in vivo method. -
Effect of mechanical stress on chondrocytes metabolism
Grant number：10470415 1998 - 1999
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)
KUBOKI Takuo, HATTORI Takako, TAKIGAWA Masaharu
Grant amount：\11300000 （ Direct expense: \11300000 ）
To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes, The chondrocytes were exposed to CTF by using Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen and protein. Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro-and active-Mmp-9. the degradation of proteoglycan was inhibited by MMP Inhibitor, indicating that MMPs are involved in the degradation of proteoglycan induced by high frequency CTF.
Moreover reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis. These findings suggest that the CTF frequency is one of the key determinants of the chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this magnitude of CTF causes only minor change of cartilage matrix. High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -8 protein suggest that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation. -
主要組織適合性抗原からみた顎関節症の免疫遺伝学的要因の研究
Grant number：09877376 1997 - 1998
日本学術振興会 科学研究費助成事業 萌芽的研究 萌芽的研究
山下 敦, 窪木 拓男
Grant amount：\1500000 （ Direct expense: \1500000 ）
顎関節症の発症における遺伝的素因は,顎関節内障患者の家族歴からこの可能性が推測されているのみ(TaUentsら,1996)で,全く推測の域を出ていない.本申請は,当講座で行った双生児研究に引き統き,顎関節症に関連する遺伝的素因をヒトMHCすなわちHLAのタイプから免疫遺伝学的に解明しようとするものである。
現在まで、1)変形性顎関節症群16名:重度の顎関節の変形を伴うもの、2)顎関節リウマチ群5名:慢性関節リウマチが顎関節にも波及したものの2群に分けて継続して末梢血採取を行っている。サシブル数が少ないこともあり、はっきりした傾向は明言できないが、顎関節リウマチ群は、従来遺伝的なリスクが高いと報告された遺伝形質を持っていたが、変形性顎関節症の場合にはその傾向は曖昧であった。 -
顎関節潤滑因子産生機序に関する研究
Grant number：09671988 1997 - 1998
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)
篠田 一樹, 辻 清薫, 窪木 拓男
Grant amount：\2600000 （ Direct expense: \2600000 ）
1.関節円板前方転位患者と変形性顎関節症患者,正常者の顎関節滑液の採取ならびに滑液中のヒアルロン酸濃度の測定
顎関節潤滑因子であるヒアルロン酸,リウブリシンの濃度の低下が滑液中において生じると,関節潤滑が円滑に行われなくなることにより関節円板転位を引き起こし,また,関節軟骨への栄養供給が低下することによって軟骨の破壊が進行し,結果として変形性関節症を引き起こすことが考えられている(Kamelchuk et al,1995)。本研究では,まず,関節円板前方転位患者と変形性顎関節症患者の患側顎関節から治療目的として希釈回収法にて滑液を採取した。また,ボランティアの正常者の顎関節から同様に希釈回収法にて滑液を採取した。そして,タンパク量測定キットならびに吸光度測定機器を用いて総タンパク濃度を測定したところ関節円板前方転位患者と変形性顎関節症患者のタンパク濃度が正常者のそれと比較して有意に高いことが明かとなった。続いて,各サンプルをSDS-PAGEにて電気泳動し,それぞれのタンパクの構成に違いがあるかを一次元電気泳動解析装置にて検討したところ,SDS-PAGEレベルでは特に各群で有意差は認められなかった。現在,市販されている抗ヒトヒアルロン酸抗体を用いたwestern blottingにて各群のヒアルロン酸のバンドの強度を測定し,各群に有意差があるか検討中である。 -
Chronic skeletal muscle pain and regeonal hemodynamic abnormality
Grant number：08457528 1996 - 1997
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)
YAMASHITA Atsushi, AZUMA Yoshiharu, KUBOKI Takuo
Grant amount：\5800000 （ Direct expense: \5800000 ）
1) Effect of cold pressor test (CPT) on resting masseter muscle hemodynamics.
Since CPT was reported to evoke an increase of muscle sympathetic nerve activity in the human skeletal muscle (Victor et al.1987), CPT-induced hemodymanic change in the resting human masseter muscle was measured by using a near infrared spectroscopy device. Results : CPT induced strong increase in intramuscular blood volume and oxygen saturation levels. Moreover, after cessation of 4 degree C CPT task clear rebound Hb decrease was demonstrated. Therefore it can be speculated that increase in muscle sympathetic nerve activity induces hyperemia in normal subjects.
2) Effect of alpha and beta adrenergic blocking agents on intramuscular hemodynamic changes.
This study evaluated that the effect of intravenous infusion of non-selective alpha- and beta-adrenergic blocking agents on masseter muscle hemodynamics. Results : Intramuscular blood volume and oxygen saturation levels significantly increased by intravenous infusion of alpha blocking agent and trended to decrease by beta blocking agent.
3) Comparison of hemodynamic response to CPT between healthy subjects and chronic trapezius muscle pain subjects.
This study determined the difference in blood volume of the upper trapezius muscle area between normal and chronic shouder muscle pain subjects during CPT.Results : In both group of subjects the mean blood volume increased above the baseline livel during CPT,however, pain subjects exibited significantly lower mean blood volume during CPT.These results suggest that intramuscular hemodynamic abnormality might be related to the chronic muscle pain pathophysiology. -
Osteoarthritis of the TMJ and Mechanical Stress
Grant number：07672110 1995 - 1996
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)
KUBOKI Takuo, YAMASHITA Atsushi
Grant amount：\2300000 （ Direct expense: \2300000 ）
Excessive mechanical stress has been proposed as a cause breakdown of the articular cartilage. To investigate the effect of mechanical stress on chondrocyte metabolism, we examined the extracellular matrix synthesis and the gene expression of cartilage degradation factors and growth factors.
1.15 kPa cyclic mechanical stress (CTF) caused less increase in proteoglycan (PG) synthesis than 5 kPa (230.3 and 179.1% increase at 5 and 15 kPa)
2.5 kPa of CTF caused more decrease in DNA synthesis (50.0 and 30.1% decrease, at 5 and 15 kPa).
3.The mean normalized IL-1beta mRNA level against the corresponding control transiently increased during both level of CTF.
4.The mean normalized bFGF mRNA level against the corresponding control transiently increased during 15 kPa of CTF,while remarkable change was not observed at 5 kPa of CTF.
5.The mean normalized MMP-9 mRNA level against the corresponding control progressively increased during 15 kPa of CTF,while the transient increase was observed during 5 kPa of CTF.
6.Mean normalized MMP-2, TIMP-1, agrecan, collagen type II and type X mRNA level against the control did not show remarkable change at the both level of CTF.
7.When 15 kPa of CTF was applied, the production level of pro-MMP-9 did not change, but the level of active form MMP-9 was slightly increased, while 5 kPa of CTF was applied, the production level of pro-MMP-9 did not show remarkable change and it was not activated to active form MMP-9. Both level of CTF did not change the production of MMP-2. -
片側噛みしめ時におけるヒト顎関節関節円板動態
Grant number：05771684 1993
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)
窪木 拓男
Grant amount：\900000 （ Direct expense: \900000 ）
顎関節内障の発症には顎関節負荷の異常が密接に関連していると推測される。申請者は1986年より行ってきた顎関節部負荷のモデル解析に始まり、これまで顎関節部負荷が顎関節構造に与える影響について研究を行ってきた。その結果、持続片側噛みしめにより顎関節断層エックス線写真上において非噛みしめ側下顎頭の著しい後上方変位と前関節空隙の縮小が誘発されることを見出した。
しかし、関節硬組織のみを描出対象とするエックス線写真法では関節円板動態や筋活動状態を把握することはできない。したがって、本研究では関節円板の描出が可能なMRI法を用い、実験的前歯部噛みしめによる下顎頭-関節円板関係の変化を直接明らかとした。
本研究の結果、実験的前歯部噛みしめにより、T1強調MR画像上でも前関節空隙の縮小が生じ、滑液の層を描出していると思われる高信号域が移動、縮小することが明らかとなった。また、この層の移動を定量的に評価することを目的に、下顎頭-関節結節を結んだ直線上の信号強度の変化をプロファイル像として比較する方法を開発した。関節空隙の縮小は、関節負荷受圧複合体(関節結節上の関節軟骨、上関節腔の滑液、関節円板、下関節腔の滑液、下顎頭上の関節軟骨)の移動ならびに圧縮変形に起因することが明らかとなった。 -
成人顎関節の骨改造能に関する研究
Grant number：04671183 1992
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)
矢谷 博文, 窪木 拓男
Grant amount：\1900000 （ Direct expense: \1900000 ）
ヒト顎関節に生じる形態学的変化(リモデリング)については、下顎頭骨折や顎外科矯正処置後にみられたとする症例が数例報告されているだけで、ほとんど明らかにされていないといってよい。また、この形態学的変化は新しい下顎頭位に対する適応変化の一つの表われであると解釈されているが、その証拠はない。
そこで、関節円板が前方偏位した94名の顎関節内障患者の188顎関節(このうち61関節は復位性、58関節は非復位性関節円板前方転位と診断された)に、保存療法後にどのような形態的変化が現われるかを放射線学的に検討し、次のような結果を得た。
1.形態的変化のほとんどは下顎頭に生じ、側頭骨(下顎窩、関節結節)にはほとんど変化が生じなかった。すなわち、39の下顎頭に進行性リモデリング、15の下顎頭に退行性リモデリングが生じたのに対して、3関節結節に退行性変化が生じたのみであった。
2.進行性変化としては、下顎頭の二重輪郭像の出現および下顎頭全体が丸みを帯びるという変化が観察された。これに対して、退行性変化としては、下顎頭前関節面あるいは後関節面の扁平化、下顎頭の短縮化、あるいは関節結節の扁平化が観察された。
3.顎関節の形態学的変化を起こす頻度と患者の年齢には関連があり、高齢になるにつれて、進行性リモデリングの頻度は減少し、退行性リモデリングの頻度は増加した。
4.正常顎関節にはほとんど形態的変化が生じなかったのに対し、復位性関節円板前方転位では36%、非復位性関節円板前方転位では41%に形態変化が生じた。復位性関節円板前方転位ではそのほとんどが進行性リモデリングであった(34%)のに対し、非復位性関節円板前方転位は進行性変化と退行性変化の頻度がそれぞれ23%と18%であった。
5.円板の復位が得られた関節と得られなかった関節では、前者のほうが形態的変化を起こす頻度が高く、そのほとんどは進行性リモデリングであった。すなわち、円板復位に得られた関節全体の42%に進行性、3%に退行性リモデリングが生じたのに対し、復位の得られなかった関節全体の13%に進行性、17%に退行性リモデリングが生じた。
6.保存治療により下顎頭位が後方あるいは上方に変位した関節では形態的変化がまったくみられず、前方あるいは下方に変化した関節では47%、下顎頭位が変らなかった関節では33%に形態変化が観察された。前方あるいは下方に変位した場合のほとんどは進行性リモデリングであった(41%)のに対し、下顎頭位が変らなかった関節では、19%が進行性、14%が退行性変化であった。
以上より、ヒト顎関節は成人であっても本質的にリモデリング能を有しており、進行性および退行性リモデリングは保存治療により変えられた異なる関節内環境に対する異なる適応変化であると考えられた。また、形態変化を生じた患者の予後は良好であることから、これらの形態変化は関節内環境が正常化されたことの一つの指標となり得ることが示唆された。 -
顎関節円板,顎関節軟骨の耐圧縮特性に関する生物力学的ならびに組織学的研究
Grant number：03771429 1991
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)
窪木 拓男
Grant amount：\900000 （ Direct expense: \900000 ）