带界面结构的一体化仿生骨软骨支架的研制及修复骨软骨缺损的实验研究

Articular osteochondral injury is common, while it is diffcult to repair. Osteochondral tissue engineering is a newly but promising treatment opition. In view of the important role of the "bone-cartilage interface" structure to isolate the microenvironment of bone and cartilage, fabrication of integrated biomimetic osteochondral scaffold with bone-cartilage interface structure is currently a hot and difficult research. In this project, cartilage portion of osteochondral scaffold with oriented microporous structure would be fabricated with silk fibroin (SF)/cartilage extracellular matrix (CECM) composite materials by TIPS technique.Bone portion with high perforation and porosity would be prepared with SF/nanometer-hydroxyapatite (n-HA) material by "phase separation/leaching" technique, and compact CECM/SF/n-HA layer would be used to integrate cartilage and bone portion of the scaffold. In this study, the integrated osteochondral scaffolds combined with chondrogenic and osteogenic induced adipose-derived stem cells were cultured in vitro and implanated into osteochondral defects of high-load-bearing area of rabbit femoral condyle(the scaffold without interface structure for control). A morphologic, histological,biochemical,and biomechanical assessment of repair effects of osteochondral defects would be conducted. The quality of bone-cartialge interface healing would be observed with high focus.The effects of bone-cartilge interface structure to tissue engineered osteochondral tissue repair would be investigated. Findings from these studies will enable improvement of biomimetic osteochondral construction with newly scaffold design and construction strategies.

关节骨-软骨联合损伤常见且修复困难，构建组织工程骨软骨复合体是目前非常有前景的治疗方法。鉴于骨软骨界面结构对隔离成骨和成软骨微环境的重要作用，制造带界面结构的一体化仿生骨软骨支架是目前研究的热点与难点。本项目拟采用丝素蛋白（SF）/软骨细胞外基质（CECM）材料,利用TIPS技术制备具有取向微孔结构的软骨支架部分；采用SF/纳米羟基磷灰石（n-HA）材料，利用"相分离/沥滤技术"制备具有高贯通性和孔隙率的骨支架部分；二者之间以致密的CECM/SF/n-HA层连接。将脂肪基质干细胞向成软骨、骨诱导分化后分别接种到一体化骨软骨支架相应部位，在体外培养后植入兔股骨髁负重区骨软骨联合缺损（以无界面结构支架为对照），通过组织学、生化成分及生物力学等指标检测骨软骨修复效果，重点观察骨软骨界面愈合情况，探讨界面结构对组织工程骨软骨组织修复的影响。本项目可为仿生构建理想的骨软骨复合体提供新材料和新策略。

关节骨-软骨联合损伤常见且修复困难，构建组织工程骨-软骨复合体是目前非常有前景的治疗方法。鉴于骨软骨界面结构对隔离成骨和成软骨微环境的重要作用，制造带界面结构的一体化仿生骨软骨支架是目前研究的热点与难点。.本项目首先采用丝素蛋白（SF）和软骨细胞外基质（CECM）材料,利用TIPS技术制备具有取向微孔结构的软骨支架，并在在体外评测了软骨支架的理化性能；后利用缓释释放系统和浸泡结合冷冻干燥技术，将软骨支架与TGF-β3结合，诱导脂肪干细胞在SF/CECM支架上的增值和分化,通过组织学、生化成分及生物力学等指标检测证明了支架，细胞因子，细胞一体化结构的成软骨能力。.基于以上基础，采用SF/纳米羟基磷灰石（n-HA）材料构建了带界面结构的骨软骨一体化支架。利用TIPS和“相分离/沥滤技术”制备具有取向微孔结构的软骨上层结构和具有高贯通性和孔隙率的骨支架下层部分；二者之间以致密的SF/n-HA层连接，体外实验通过组织学、生化成分及生物力学等指标评测脂肪基质干细胞在软骨上层和骨下层的诱导效果。体内实验将脂肪基质干细胞与骨软骨结构复合支架结合，植入兔股骨髁间负重区骨软骨联合缺损处（以无界面结构支架为对照），通过组织学、生化成分及生物力学等指标检测骨软骨修复效果，重点观察骨软骨界面愈合情况，探讨界面结构对组织工程骨软骨组织修复的影响，并发现界面结构对组织工程骨软骨组织修复尤其是软骨GAG生成有促进作用，对于软骨下骨无明显促进作用。