合成微小環境を用いた軟骨組織のｉｎ　ｖｉｔｒｏ構築

本研究的目的是通过化学、物理和生物线索，包括细胞中DNA甲基转移酶（DNMTs）的过表达，确定新的方法和培养条件来增强人骨髓间充质干细胞（hBMSCs）的软骨分化。我们尝试使用材料（如水凝胶）制造化学和物理控制的微环境，再现天然细胞生态位特征，以最佳地调节骨髓间充质干细胞的软骨分化。除了物理和化学刺激外，我们还评估了dnmt过表达后hBMSCs的软骨分化，最终诱导关键基因富含CPG区域的DNA甲基化。为了研究DNMTs对hBMSCs软骨形成的影响，我们在hBMSCs中转染DNMT3A、DNMT3B过表达载体和pcDNA对照，并在生长因子和糖皮质激素的三维微质量培养系统中培养细胞。此外，由于体外合成的软骨组织通常会经历矿化过程，我们也试图通过小鼠股骨继发性骨化中心作为体内模型来了解初始矿化过程中软骨细胞死亡的机制。

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.