纳米修饰可降解双层支架修复兔膝关节骨软骨缺损的研究

Repair of osteochondral defects is one of challenging problems in orthopedics. There is no ideal method to repair it in clinical treatment. The studies show it is significantly promising to engineering osteochondral graft using biological scaffolds for repairing osteochondral defects in animal test. However, it has not developed the ideal biological scaffold materials, yet. In our previous study, bilayered scaffold was successfully fabricated to mimic osteochondral matrix with degradable poly(lactide-co-glycolide) (PLGA). Stem cells-seeded bilayered PLGA scaffold repaired the osteochondral defect well. But similar to most studies, biomechanical strength of neotissues was not excellent. Fortunately, Nano scaffolds materials could improve the interaction between cells and scaffold and provide more beneficial microenvironment to facilitate tissue regeneration. In the present study, bilayered PLGA scaffolds would be modified by using collagen nanofibers and nano-sized β-tricalcium phosphate fabricated successfully in previous study, promoting the biocompatibility and mechanical properties of bilayered scaffolds and better mimicking extracellular matrix. And the nano-modified bilayered PLGA scaffolds, seeded with stem cells from bone marrow of rabbit, would be researched in vitro and would be implanted the osteochondral defects in the knee joint of rabbit. The impact of the nano-modified scaffolds on prolification and differentiation of stem cells and the effect of repairing osteochondral defects would be evaluated by cytology, histology, molecular biology and biomechanical techniques. According to these, we would discuss the significance of nano-modification for PLGA scaffolds and analyze the mechanism of osteochondral repair. It would lay the foundation to build the ideal osteochondral tissue engineering and provide new strategies for repairing osteochondral defects in clinical treatment.

骨软骨缺损修复是骨科最具挑战性问题之一，临床治疗尚无理想方法。实验表明，生物支架构建组织工程骨软骨展示良好应用前景，但迄今尚未找到理想生物支架材料。我们前期研究利用可降解聚乙交酯-丙交酯(PLGA)制备双层支架仿生骨软骨基质，修复骨软骨缺损获得满意效果；但同大多研究结果类似，新生组织生物力学强度不理想。而纳米支架材料能促进细胞-支架相互作用，提供更有利微环境促进组织再生。因此，本项目拟将胶原纳米纤维及前期研究制备的纳米β-TCP修饰PLGA双层支架，以提高其生物相容性及力学性能，更好仿生细胞外基质；纳米修饰双层支架通过负载干细胞行体外实验，并植入兔膝关节骨软骨缺损模型，利用细胞学、分子生物学及生物力学等技术评价纳米修饰双层支架对干细胞增殖、分化影响及修复骨软骨缺损效果，探讨双层支架表面纳米修饰意义，分析骨软骨修复机制，为构建理想组织工程骨软骨奠定实验基础，为临床治疗骨软骨缺损提供新策略。

骨软骨缺损修复是骨科最具挑战性问题之一，临床治疗尚无理想方法。实验表明，生物支架构建组织工程骨软骨展示良好应用前景，但迄今尚未找到理想生物支架材料。本项目前期研究利用可降解聚乙交酯-丙交酯(PLGA)制备双层支架仿生骨软骨基质，修复骨软骨缺损获得满意效果；但同大多研究结果类似，新生组织生物力学强度不理想。而纳米支架材料能促进细胞-支架相互作用，提供更有利微环境促进组织再生。因此，本项目利用胶原纳米纤维及纳米HA修饰PLGA双层支架表面，成功制备纳米表面修饰PLGA双层支架，即nCol-PLGA/nHA-PLGA。通过对纳米修饰支架表征进行检测及负载BMSCs的体外实验，结果显示纳米修饰双层支架有利于细胞黏附、增殖，具有良好生物相容性,纳米表面修饰未对PLGA双层支架的孔隙率产生明显影响，具有更好的压缩模量等力学性能；纳米修饰双层支架通过负载BMSCs植入兔膝关节骨软骨缺损模型，16周后取出新生骨软骨组织，利用组织学、分子生物学、蛋白印迹以及micro-CT等技术评价纳米修饰双层支架负载BMSCs修复骨软骨缺损效果，结果显示纳米修饰双层支架对骨软骨缺损具有一定的修复作用，而支架负载BMSCs具有更好的修复效果。组织学染色可见透明软骨组织形成及软骨下小梁骨形成，支架逐渐降解，micro-CT可见新生骨软骨表面光整，软骨下骨局部少量缺损；通过Western Blot检测新生组织蛋白表达，发现Collagen type I和type II以及P-smad 1和Smad 2四个蛋白的表达增高与新生骨软骨形成一致，提示TGF-β1/Smad信号通路参与了骨软骨组织的再生修复。总之，本项目通过体外及动物体内实验，阐明了PLGA双层支架纳米表面修饰的意义及纳米修饰PLGA双层支架构建组织工程骨软骨具有良好修复作用，揭示了其修复骨软骨缺损部分机制，为构建理想组织工程骨软骨奠定了实验基础，为临床治疗骨软骨缺损提供了新策略。