微环境基质对骨髓间充质干细胞凝聚行为及成软骨分化的影响

Cartilage regeneration has been a major focus of tissue engineering, both because of the constantly growing need for more effective options for joint repair and the expectation that this apparently simple tissue will be easy to engineer. After several decades, however, cartilage regeneration has proven to be anything but easy. Although there are good pre-clinical results of "material-induced" cartilage regeneration without the use of growth factors, its mechanism is poorly understood. The development of native cartilage during embryonic development can provide valuable insights on cartilage regeneration and important guidelines for developing more effective cell therapies and tissue engineering products. Mesenchymal cell condensation, or precartilage condensation, is regarded as the major event of the mesenchymal stem cell commitment to the chondrogenic lineage, after which cartilage-tissue-specific transcription factors and structural proteins begin to accumulate. In this study, we propose to investigate how microenvironment matrix affects mesenchymal cell condensation in vitro and how it further affects chondrogenic differentiation and cartilage tissue formation. Type I/II composite collagen hydrogel with varied final collagen concentration and type I/II ratio will be used to encapsulate bone-marrow-derived mesenchymal stem cells. Cell migration during cultivation will be monitored by time-lapse imaging and chondrogenic differentiation will be characterized by gene expression and protein markers. Then microenvironment matrix turnover will be investigated by characterizing degradation and remodeling of the pre-existing type I and type II collagen, and synthesis of new cartilage-tissue-specific matrix proteins including type X collagen and lubricin. Finally, the functionality of the engineered cartilage tissue will be characterized by its mechanical properties such as compression modulus and friction coefficient. This study would help us understand the dynamic regulation and interactions between microenvironment matrix and mesenchymal stem cells during cartilage tissue formation, and will lay the foundation for matrix design in cartilage tissue engineering.

受损软骨组织的功能性修复一直是组织工程面临的挑战之一，虽然不依赖生长因子的“材料诱导”软骨再生已得到较好的临床前实验结果，其作用机理仍有待进一步探索。胚胎发育时的组织形成过程一直是相关组织工程借鉴模仿的主要对象。本课题拟将从胚胎发育过程中间充质凝聚的角度出发，分析胶原微环境基质对骨髓间充质干细胞体外凝聚行为的影响，进而探索间充质干细胞所在微环境基质对其成软骨分化及形成软骨组织的调控作用。研究将使用不同浓度、不同比例的I/II复合型胶原水凝胶包埋不同细胞密度的骨髓间充质干细胞，观察细胞迁移及成软骨凝聚情况，在细胞和分子水平衡量其成软骨分化程度，研究间充质干细胞成软骨分化后对微环境基质的重建，并对最终形成的软骨组织进行功能性评价。本课题特色在于系统的研究微环境基质与骨髓间充质干细胞的动态调节和相互作用，将为软骨组织工程基质材料的制备与设计提供理论的支持和指导。

胚胎发育时的软骨组织形成过程一直是软骨组织工程借鉴模仿的主要对象。本项目从胚胎发育过程中间充质凝聚的动态发展过程出发，分析了细胞间相互作用和胶原微环境基质对骨髓间充质干细胞体外凝聚行为的影响。前期研究表明胶原水凝胶能诱导间充质干细胞（MSCs）成软骨分化，但其构成的基质微环境对MSCs成软骨分化过程的调控作用和机制仍不清楚。本项目主要研究内容与结果如下：1、研究了胶原浓度及细胞密度对MSCs体外凝聚行为的影响，成功构建了模拟成软骨凝聚初期及后期不同阶段微环境的培养体系，筛选出最有利于MSCs凝聚及启动成软骨分化的关键因子条件；2、在细胞和分子学水平研究了钙黏蛋白N-cadherin对MSCs凝聚行为及启动成软骨分化的影响和规律，并探索前期N-cadherin与后期胶原基质微环境对MSCs成软骨分化的协同调控作用，明确成软骨分化不同阶段微环境中的关键因子；3、探索MSCs成软骨分化后对基质微环境的重建作用，阐明微环境基质与MSCs的动态调控机制和相互作用，为临床软骨损伤再生修复使用的软骨诱导胶原水凝胶的优化制备提供理论基础；4、在不添加化学交联剂的条件下，探究胶原成胶关键条件对胶原水凝胶的纤维结构及黏弹性的影响，在三维培养中初步考察胶原水凝胶的黏弹性对MSCs成软骨分化不同阶段的调控作用。本项目成功建立了构建模拟MSCs成软骨分化不同阶段的三维培养模型，明确了MSCs启动成软骨分化前期关键信号蛋白和维持软骨分化后期微环境因素，研究了胶原水凝胶动态力学性能对MSCs成软骨分化的调控作用，为临床软骨损伤再生修复使用的软骨诱导胶原水凝胶的优化制备提供了理论基础。