合成微小環境を用いた軟骨組織のｉｎ　ｖｉｔｒｏ構築

使用合成微环境体外构建软骨组织

• 批准号: 14F04106
• 负责人: 松本 卓也
• 金额: $ 1.54万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2014
• 资助国家: 日本
• 起止时间: 2014-04-25 至 2016-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14F04106/
• 关键词: 軟骨; 合成微小環境; DNAメチル化; 石灰化

The purpose of this study was to identify new methods and culture conditions to enhance chondrogenic differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (hBMSCs) by applying chemical, physical and biological cues, including overexpression of DNA methyltransferases (DNMTs) in cells. We attempted to fabricate a chemically- and physically-controlled microenvironment using materials (e.g., hydrogels) that recapitulate native cell niche characteristics to optimally modulate the chondrogenic differentiation of BMSCs. Besides the physical and chemical stimulations, we also evaluated the chondrogenic differentiation of hBMSCs after overexpression of DNMTs that eventually induce DNA methylation at CPG rich regions of key genes. To study the effect of DNMTs on chondrogenesis of hBMSCs, we transfected overexpression vectors of DNMT3A, DNMT3B and the pcDNA control in hBMSCs, and culture the cells in a 3D micromass culture system with growth factors and glucocorticoids.Additionally, since in vitro synthesized cartilage tissue generally undergoes through mineralization, we also attempted to understand the mechanisms involved in the chondrocyte death associated with initial mineralization process by using the secondary ossification center of mouse femur as in vivo model.

本研究的目的是通过应用化学、物理和生物线索，包括细胞中 DNA 甲基转移酶 (DNMT) 的过度表达，确定新的方法和培养条件，以增强人骨髓来源的间充质干细胞/祖细胞 (hBMSC) 的软骨分化。 。我们尝试使用能够重现天然细胞生态位特征的材料（例如水凝胶）构建化学和物理控制的微环境，以最佳地调节 BMSC 的软骨形成分化。除了物理和化学刺激之外，我们还评估了 DNMT 过表达后 hBMSC 的软骨分化，最终在关键基因的 CPG 丰富区域诱导 DNA 甲基化。为了研究DNMT对hBMSCs软骨形成的影响，我们将DNMT3A、DNMT3B和pcDNA对照的过表达载体转染到hBMSCs中，并在含有生长因子和糖皮质激素的3D微团培养系统中培养细胞。此外，由于体外合成的软骨组织通常通过矿化进行，我们还试图了解与初始矿化相关的软骨细胞死亡的机制采用小鼠股骨二次骨化中心作为体内模型进行过程。

项目成果

Comparison of platelet serotonin transporter activity in subjects with severe sleep bruxism and control

严重睡眠磨牙症受试者与对照组血小板血清素转运蛋白活性的比较

• DOI: 10.1016/j.jpor.2014.06.003
• 发表时间: 2014
• 影响因子: 3.6
• 作者: Hajime Minakuchi; Chiharu Sogawa C; Emilio Satoshi Hara; Haruna Miki; Kenji Maekawa; Norio Sogawa; Shigeo Kitayama; Yoshizo Matsuka; Glenn T Clark; Takuo Kuboki
• 通讯作者: Takuo Kuboki

Fluocinolone acetonide is a potent synergistic factor of TGF-β3-associated chondrogenesis of bone marrow-derived mesenchymal stem cells for articular surface regeneration.

Fluocinolone acetonide 是用于关节面再生的骨髓间充质干细胞的 TGF-β3 相关软骨形成的有效协同因子。

• 发表时间: 2016
• 作者: Hara ES; Ono M; Pham HT; Sonoyama W; Kubota S; Takigawa M; Matsumoto T; Young MF; Olsen BR; Kuboki T.
• 通讯作者: Kuboki T.

Efficient bone formation in a swine socket lift model using Escherichia coli-derived recombinant human bone morphogenetic protein-2 adsorbed in β-tricalcium phosphate.

使用吸附在 β-磷酸三钙中的大肠杆菌衍生重组人骨形态发生蛋白 2 在猪窝提升模型中实现有效骨形成。

• DOI: 10.1159/000369061
• 发表时间: 2014
• 作者: Mitsuaki Ono; Wataru Sonoyama; Katushi Yamamoto; Yasutaka Oida; Kentaro Akiyama; Shigehiko Shinkawa; Ryu Nakajima; Hai Than Pham; Emilio Satoshi Hara; Takuo Kuboki
• 通讯作者: Takuo Kuboki

A high-throughput screening identified fluocinolone acetonide as a potent synergistic factor of TGF-β3-mediated chondrogenesis of BMSCs for articular surface repair.

一项高通量筛选发现氟轻松是 TGF-β3 介导的 BMSC 软骨形成的有效协同因子，用于关节面修复。

• 发表时间: 2015
• 作者: Hara ES; Ono M; Pham HT; Sonoyama W; Kubota S; Takigawa M; Young MF; Olsen BR; Matsumoto T; Kuboki T.
• 通讯作者: Kuboki T.

Qualitative analysis of tissue mineralization during secondary ossification of mouse femur

小鼠股骨二次骨化过程中组织矿化的定性分析

• 发表时间: 2014
• 作者: Emilio Satoshi Hara; Masahiro Okada; Takako Hattori; Satoshi Kubota; Takuo Kuboki; TakuyaMatsumoto
• 通讯作者: TakuyaMatsumoto