单一生物材料聚乳酸/羟基乙酸共聚物构建组织工程骨软骨的可行性

Feasibility of constructing a scaffold for osteochondral tissue engineering using poly(lactide-co-glycolide) alone

背景:为仿生软骨和软骨下骨的生理和功能需求,近年来一些研究通过制备双层支架构建组织工程骨软骨来修复软骨缺损。但大多研究使用了不同的复合生物材料,很少文献报道使用单一生物材料制备双层多孔支架。目的:研究一体化双层聚乳酸/羟基乙酸共聚物(poly(lactic-co-glycolide),PLGA)多孔支架材料的生物相容性,探讨其构建组织工程骨软骨的可行性。方法:使用PLGA作为生物材料,采用室温模压/粒子浸出法制备一体化双层多孔支架。①体外实验:将第3代兔骨髓间充质干细胞接种于双层多孔支架上,接种1周后进行扫描电镜观察,接种48 h后进行LIVE/DEAD荧光染色;②体内实验:将骨髓间充质干细胞-双层多孔支架复合物、双层多孔支架分别植入裸鼠皮下,4,8周后取出植入物,进行苏木精-伊红染色和DAPI染色。结果与结论:①成功获得一体化双层多孔支架,支架上层(软骨层)孔径为100-200μm,下层(软骨下骨层)孔径为300-450μm,孔隙率为85%;②体外实验:LIVE/DEAD染色显示,骨髓间充质干细胞可在支架良好存活;扫描电镜可见细胞黏附在支架孔壁上,并可见大量沉积的细胞外基质;③体内实验:植入后8周苏木精-伊红染色显示,实验组支架上层纤维组织中夹杂有少量软骨细胞,下层可见大量骨小梁样组织形成,上下层组织紧密结合,支架部分降解,DAPI染色显示骨髓间充质干细胞可在裸鼠体内存活8周;对照组支架上下层可见少量纤维组织,未见软骨细胞及明显的小梁骨组织形成,支架部分降解;④结果表明:单一生物材料PLGA制备的一体化双层支架生物相容性良好,构建组织工程骨软骨具有可行性。

BACKGROUND： To mimic physiological and functional requirements of cartilage and subcondral bone, some recentstudies have fabricated bilayered scaffolds for osteochondral tissue engineering in cartilage repair. However, in cartilagerepair, and little is reported on the use of a single biomaterial instead of composite biomaterials to fabricate bilayeredporous scaffolds.OBJECTIVE： To investigate the biocompatibility of an integrated bilayered porous scaffold fabricated withpoly（lactic-co-glycolide） （PLGA）, and then to discuss its feasibility of constructing tissue-engineered osteochondral graft.METHODS： The integrated bilayered porous scaffolds were fabricated with PLGA by changing the ultrastructure ofscaffold. In vitro, bone marrow mesenchymal stem cells （BMSCs） isolated from rabbits were seeded into the bilayeredporous PLGA scaffold, co-cultured for 1 week and then observed under scanning electron microscope. After co-culturing48 hours, the cells were stained by LIVE/DEAD Kit. In vivo, the composite of DiI-labeled BMSCs and the scaffold or thescaffold alone were implanted subcutaneously into the skin of nude mice, and the implants were taken out and stained byhematoxylin-eosin and DAPI staining at 4 and 8 weeks after implantation.RESULTS AND CONCLUSION： （1） We successfully obtained the bilayered porous scaffolds with different pore sizes（the upper layer： 100-200 μm and the lower layer： 300-450 μm） and 85% porosity. （2） In the in vitro experiment, theLIVE/DEAD staining showed that BMSCs survived well in the scaffold, and the cells adhered well to the wall of pores inall the scaffolds and extracellular matrix deposition was found under the scanning electron microscope. （3） In the in vivoexperiment, shown by the hematoxylin-eosin staining, a small amount of chondrocytes formed in the fibrous tissue in theupper layer and a large amount of trabecular bones in the lower layer were found at 8 weeks after implantation in theexperimental group. The close integration between the upper layer and the lower layer appeared. BMSCs could survivefor 8 weeks in vivo shown by the DAPI staining. However, in the control group, a little fibrous tissue was found withoutchondrocytes and apparent trabecular bone, and the scaffold was partially degraded. To conclude, the integratedbilayered porous scaffold fabricated with PLGA alone has good biocompatibility, which is feasibly used in osteochondral tissue engineering.