“三维功能性同轴电纺丝支架”的构建及其募集骨髓间充质干细胞修复关节软骨的疗效和生物学机制的研究

Currently,lots of patients were suffered from cartilage injury, but the capacity of cartilage self-repair is poor. As a result,the cartilage injury brings heavy burden to patients and society.Currently, scientists attempt to repair cartilage injury with the method of tissue engineering. However,there are still lots of challenges to be overcomed and no specific and useful cartilage repair system has been developed. The appropriate seed cells,growth factors and scaffolds are three basic elements of tissue engineering. In cartilage tissue engineering, bone mesenchymal stem cells (BMSCs) are the comparatively ideal seed cells; and TGF-β1 is the growth factor commonly used, which could promote the chondrogenic differentiation of BMSCs.In the previous study, we have developed the “film-like functional CBrhTE scaffold”,which could synchronously recruit BMSCs, support BMSCs, and release TGF-β1 in a controlled manner to promote the chondrogenic differentiation of BMSCs. However,the shortcoming of the “film-like functional CBrhTE scaffold” is the low porosity, and the BMSCs can not easily immigrate into the inside of the scaffold,which prevent the cartilage regeneration. Therefore,in this subject, we attempt to enhance the porosity of the “film-like functional CBrhTE scaffold” to construct the “3D functional CBrhTE scaffold”, furtherly explore the physical and chemical properties of the “3D functional CBrhTE scaffold”,and determine the sustained release of TGF-β1 by the scaffold. And meanwhile, in vitro experiments, we attempt to explore the capacity of recruiting BMSCs and promoting BMSCs chondrogenic differentiation by the “3D functional CBrhTE scaffold”; in vivo experiments, we attempt to explore the effect of cartilage regeneration by the “3D functional CBrhTE scaffold”. Finally, we attempt to explore the biological mechanism of cartilage regeneration by the BMSCs recruited onto the “3D functional CBrhTE scaffold”. Consequently, the completion of this subject could obtain the “3D functional CBrhTE scaffold”,and explicit the mechanism of cartilage regeneration by this scaffold, which could provide the theory and experimental evidence for cartilage repair.

随着全民运动水平提高，关节软骨损伤患者逐年增多，严重的是：关节软骨自我修复能力差。组织工程技术修复软骨损伤较有应用前景，但目前尚未发现一种成熟的修复方法。在软骨组织工程技术中，骨髓间充质干细胞（BMSCs）是理想的种子细胞；TGF-β1是常用的生物因子，能促进BMSCs成软骨分化。在以前的研究中，我们用同轴电纺丝技术构建了“膜状功能性CBrhTE支架”，其能同时募集BMSCs、缓释TGF-β1促进BMSCs成软骨分化。不足之处在于，此支架孔隙率偏低，无法形成内外均充满BMSCs的3D支架，从而影响软骨再生效率。因此，在本课题中，为提高软骨再生效率，我们拟提高“膜状功能性CBrhTE支架”的孔隙率，构建“三维功能性CBrhTE支架”，并行体内外实验探索此支架募集BMSCs修复关节软骨的疗效；最终，探索此支架利用BMSCs修复软骨的生物学机制。总之，本课题的完成，可为软骨损伤的修复提供新思路。

随着全民运动水平提高及人口老龄化的进展，关节软骨损伤和退变的患者逐年增多，严重的是：关节软骨自我修复能力差。组织工程技术修复软骨损伤较有应用前景，但目前尚未发现一种成熟的修复方法。在软骨组织工程技术中，支架材料、种子细胞和细胞因子是三大要素。其中，骨髓间充质干细胞（bone marrow mesenchyml stem cell，BMSCs）是理想的种子细胞；TGF-β1是常用的生物因子，能促进BMSCs成软骨分化。在以前的研究中，我们用同轴电纺丝技术构建了“膜状功能性CBrhTE支架”，其能同时募集BMSCs、缓释TGF-β1促进BMSCs成软骨分化。不足之处在于，此支架孔隙率偏低，无法形成内外均充满BMSCs的3D支架，从而影响软骨再生效率。因此，在本课题中，为提高软骨再生效率，我们进一步改良了支架制作工艺，联合使用“同轴电纺丝技术”和“淋盐技术”，以改善“膜状功能性CBrhTE支架”的孔隙率，构建“三维功能性CBrhTE支架”。使用扫描电镜和透射电镜检测“三维功能性CBrhTE支架”的纤维结构，结果显示“三维功能性CBrhTE支架”是由“外壳—夹芯”双层结构的纤维组成，E7多肽均匀的耦连到支架的表面，耦连E7肽后，支架的亲水性升高、力学强度无显著性改变。“三维功能性CBrhTE支架”能够缓释TGF-β1，并且缓释的TGF-β1仍然保持生物学活性。体外实验方面，使用“三维功能性CBrhTE支架”培养BMSCs，结果显示，“三维功能性CBrhTE支架”能够促进BMSCs粘附和成软骨分化。体内实验方面，构建了新西兰大白兔软骨损伤模型，并用“三维功能性CBrhTE支架”、“膜状功能性CBrhTE支架”和“单纯微骨折”方法修复软骨损伤，结果显示，修复术后24周，“三维功能性CBrhTE支架”的软骨修复能力优于“膜状功能性CBrhTE支架”和“单纯微骨折组”；最终，经生物信息学分析，发现“三维功能性CBrhTE支架”利用BMSCs修复关节软骨的生物学机制可能与Indian hedgehog信号通路有关。总之，本课题的完成，可为关节软骨损伤的修复提供新的思路。