软骨诱导性天然高分子非匀质水凝胶的构建及诱导机制研究

Bone marrow mesenchymal stem cells(BMSCs), as one of the seeding cells sources in cartilage tissue engineering, is popularly studied in current research and is considered as one of the priorities in the future development. Not only will appropriate biological inducing factors affect the chrondrogenesis of BMSCs, but other physical factors would influence the progress obviously too, such as cell density, nutrient supply and mechanical stimulus. In order to establish a co-cultured system of inhomogeneous construct hydrogels which bears local high density BMSCs and has good performance of mass transfer and nutrient exchanging, the collagen microsphere loaded with high density BMSCs will be prepared and distributed in moderately modified hyaluronic acid and chondroitin sulfate derivatives embedded with chondrocytes, following by photopolymerization. The influence that material factors（such as composition, construction and chem-physical properties）exert on the inducing differentiation of BMSCs will be thoroughly studied by using methods like response surface methodology, computer aided quantification of imagines and mathematical models. By using the mechanical stimulus models in vitro experiments, the effect that chondrocytes micro-environment and mechanical stimulus have on BMSCs will be comprehensively evaluated, and the mechanism of inducing differentiation will be further discussed. Finally, the regeneration of cartilage tissue will be evaluated through subcutaneous or muscle implantation, and the repair of cartilage defect will be commented based on animal experiment. The successful implementation of this program will show potential of natural polymer based hydrogels in cartilage tissue engineering, equipping with appropriate BMSCs and chondrocytes co-culture system. The achievements of this program will accelerate the development of biomaterials for clinical applications and deepen the understanding of differentiation mechanism of BMSCs.

骨髓间充质干细胞（BMSCs）是目前软骨组织工程种子细胞的研究热点和未来发展重点之一，但BMSCs的软骨分化条件和诱导机制尚不十分明确。BMSCs不仅需要适当的生物学诱导因素实现软骨分化，而且细胞密度、营养供应、力学刺激等多种非生物学因素对其分化结果有显著影响。本项目拟将高密度BMSCs负载于胶原微球，并将其分布于包埋软骨细胞的改性透明质酸-硫酸软骨素水凝胶中，构建具有局部BMSCs高密度和良好传质与营养交换性能的非匀质水凝胶共培养体系。通过响应曲面设计、计算机图形量化处理、数学模型等方法，系统研究水凝胶的组成、结构、理化性能等材料学因素对BMSCs诱导分化的影响；结合水凝胶体外培养的力学刺激模型，综合评价软骨细胞微环境和力学刺激对BMSCs分化命运的作用，并进一步探讨BMSCs诱导分化机制；结合异位植入和软骨缺损修复动物实验，研究软骨再生和缺损修复的效果，为软骨再生修复提供新的途径。

利用骨髓间充质干细胞（BMSCs）的多能分化特性，通过营造适宜的细胞微环境调控干细胞分化命运并实现正常的功能表达，是目前生物医用材料研发与组织器官修复替代的核心和关键问题之一。软骨缺损难以自愈，通过干细胞成软骨分化是研究重点和趋势。. 本项目以天然软骨的主要细胞外基质成分，即透明质酸、硫酸软骨素和胶原为主要原料，通过适当的化学改性方法获得透明质酸和硫酸软骨素衍生物，使其能通过光交联的方法制备化学交联水凝胶；通过乳化法制备胶原微球并验证了细胞负载工艺，可以进一步用于构建非匀质复合三维网络水凝胶；通过制备光化学交联和物理交联共存的复合水凝胶材料，在不添加任何外源性生长因子的情况下进行体外细胞培养，研究了水凝胶组成与干细胞成软骨分化性能之间的关联，最终探讨通过材料学因素调控干细胞分化命运的方法与可行性，为进一步讨论软骨诱导机理提供条件并奠定基础。. 通过对制备的材料进行系统表征，以及体外细胞培养结果的分析，研究结果表明：（1）采用MA对透明质酸进行化学改性，可以获得储能模量接近300kPa且溶胀率低于100%的透明质酸水凝胶材料；与硫酸软骨素衍生物共混光交联后，获得的水凝胶储能模量可超过300kPa；（2）获得胶原微球制备及负载细胞的最佳条件，胶原微球具有良好的传质性能，有利于负载细胞的增殖与功能表达，有利于软骨细胞外基质的快速分泌与构建；（3）复合水凝胶中，化学交联使水凝胶具有良好的稳定性，胶原的物理交联网络改善了细胞的微环境，在细胞培养初期有效的促进了细胞的粘附与增殖，加快干细胞成软骨细胞分化进程。组织学、免疫组化以及基因和蛋白分析表明，在培养3周左右已完全分化，水凝胶内部有充分的基质分泌，良好的软骨细胞形态和大量的软骨陷窝。. 通过本项目的实施，制备了具有仿生组成与结构的复合水凝胶，有效的促进BMSCs的增殖、分化与功能表达，初步探讨和揭示了材料学因素对干细胞分化命运的影响和规律，为进一步丰富和完善软骨诱导机理提供基础。