SDF-1/CXCR4介导生物活性骨软骨仿生支架促MSCs归巢及修复骨软骨缺损的实验研究

Osteochondral injuries are common and difficult to treat. Stromal cell-derived factor-1(SDF-1) could promote the homing of stem cells. It is still unclear whether biomimetic and bioactive osteochondral scaffold promotes osteochondral regeneration through stromal cell-derived factor-1/CXCR4-mediated MSCs homing. Recent studies show that small molecule Kartogenin (KGN) could induce mesenchymal stem cells into chondrocytes.The project intends to build a bilayered osteochondral scaffold composed of silk fibroin, cartilage ECM and nano hydroxyapatite (Nano-HA), in which the cartilage layer controlled release of small molecules Kartogenin and SDF-1, and the bone layer controlled release SDF-1. The release kinetic of SDF-1 and the effect of SDF-1 on the raising MSCs in vitro and in vivo would be examined. The chondrogenic effect of the cartilage layer scaffold with slow release KGN would also be researched. Using tissue-engineered method, we would evaluate the effect of this acellular osteochondral scaffold on repairing the osteochondral defect in animal model through mobilization, recruitment and migration of MSCs, and make clear the role and mechanism of SDF-1/CXCR4 axis during the process. Through this study, new chemotactic and chondrogenic osteochondral scaffold would be developed, which will provide the experimental basis for clinical treatment for osteochondral injuries.

骨软骨损伤常见且治疗困难。SDF-1能促进干细胞的归巢，而SDF-1/CXCR4 轴能否在介导生物活性骨软骨支架促进 MSCs 归巢及修复骨软骨缺损过程中发挥重要作用，目前尚不清楚。最近研究表明小分子物质Kartogenin（KGN）能诱导间充质干细胞转变为软骨细胞。本项目拟以丝素蛋白、软骨ECM和纳米羟基磷灰石（Nano-HA）为材料，构建软骨层缓释（SDF-1+KGN）、骨层缓释SDF-1的骨软骨仿生支架，研究新型支架SDF-1的释放动力学及其体内、外募集MSCs的作用；研究缓释KGN的软骨层支架促进MSCs成软骨作用及生物学特性；运用组织工程手段评价此无细胞骨软骨支架动员、募集和迁移MSCs修复骨软骨缺损动物模型的效果，明确SDF-1/CXCR4轴发挥的作用及效能。通过此课题，将获得兼具有“趋化引导”及“成软骨诱导”作用的新型骨软骨支架，并为骨软骨的临床治疗新手段开发提供实验基础。

骨软骨损伤是临床上常见的运动损伤性疾病且治疗困难。组织工程为骨软骨损伤修复带来新的希望。本项目拟以丝素蛋白、软骨ECM和纳米羟基磷灰石（nano-HA）为材料，构建软骨层缓释（SDF-1α+KGN）、骨层缓释SDF-1α的骨软骨支架，研究新型支架SDF-1的释放动力学及其体内、外募集BMSCs的作用；研究缓释KGN的软骨层支架促进MSCs成软骨作用及生物学特性；运用组织工程手段评价此无细胞骨软骨支架动员、募集和迁移MSCs修复骨软骨缺损动物模型的效果，明确SDF-1α/CXCR4轴发挥的作用及效能。研究结果发现运用乳化溶剂挥发法制备的载SDF-1α以及KGN的PLGA微球平均直径在50μm左右，表面光滑，可以缓慢释放SDF-1α和KGN至30天左右。同时Transwell迁移实验发现SDF-1α可以促进BMSCs的迁移，并且呈剂量依赖性，以100ng/mL最为显著。SDF-1α促迁移能力被SDF-1α的受体CXCR-4拮抗剂AMD3100部分削弱。骨软骨支架由层次鲜明的“透明软骨-软骨下骨”2部分组成，2层之间连接紧密。软骨层有明显取向结构，软骨下骨层具有良好的三维孔状结构，骨层具有良好的三维大孔结构，连通性好，电镜显示微球均匀分布在支架内。BMSCs体外分离培养并接种至支架上，死活染色以及鬼笔环肽染色显示软骨层和骨层具有良好的组织相容性。载SDF-1α以及KGN微球的骨软骨支架软骨再生情况较空白支架组和物理吸附SDF-1α以及KGN组更好，主要体现在软骨表面平滑度和完整度，且SDF-1α以及KGN组新生软骨组织中GAG和II型胶原含量高于空白支架组和物理吸附SDF-1α以及KGN组。Micro-CT结果显示载SDF-1α以及KGN微球的骨软骨支架软骨下骨再生要优于空白支架组和物理吸附SDF-1α以及KGN组。因此，我们获得兼具有“趋化引导”及“成软骨诱导”作用的新型无细胞骨软骨支架，该支架具有良好的生物相容性，通过募集自体干细胞并诱导分化为软骨细胞，从而有利于兔膝关节骨软骨缺损的修复，并为骨软骨的临床治疗新手段开发提供实验基础。