负载TGF-β_1微球的壳聚糖-丝素支架复合骨髓间充质干细胞修复兔关节软骨缺损的实验研究  

Experimental Study on the Reparative Effects on Rabbit Articular Cartilage Defects by Using Bone Marrow-derived Mesenchymal Stem Cells Compounded Chitosan-silk Fibroin Scaffold Load with TGF-β_1 Microspheres.

作　　者：杨亚冬[1] 张文元[1] 房国坚[1] 

机构地区：[1]浙江省医学科学院,杭州310013

出　　处：《医学研究杂志》2011年第6期70-76,共7页Journal of Medical Research

基　　金：浙江省科技计划项目(2008C33025)

摘　　要：目的研制负载转化生长因子β1(transforming growth factor-β1,TGF-β1)壳聚糖微球的壳聚糖-丝素支架复合骨髓间充质干细胞(mesenchymal stem cells,MSCs)体外构建组织工程软骨,观察将其移植修复兔膝关节软骨缺损的效果。方法通过密度梯度离心法及贴壁培养法分离纯化MSCs,以抗兔CD14、CD44等单抗采用流式细胞仪进行MSCs表面抗原鉴定,获得纯化的MSCs。采用乳化交联法获得包裹TGF-β1壳聚糖缓释微球,并将该微球负载在冷冻干燥获得的壳聚糖-丝素支架上。将培养的MSCs种植到支架上,体外构建组织工程软骨,移植到兔关节软骨缺损处。移植空白支架的为实验对照组,移植体外构建的组织工程软骨为实验组。主要观察指标,流式检测的MSCs细胞表面标记情况,微球的形态,支架与细胞共培养后电镜下情况,支架移植后的兔关节修复情况及组织学检查。结果流式检测MSCs第4代及第9代细胞的CD14及CD44表面抗原的表达情况发现两代MSCs CD14表达基本呈阴性,CD44表达呈阳性,符合间充质干细胞的特性。扫描电镜观察所制备的微球基本呈球形,表面光滑,直径约为1μm,大小较均一,分散度好。细胞在壳聚糖-丝素三维支架上生长状态良好,电镜观察,可见支架孔隙内有细胞黏附生长。移植治疗后发现实验组修复明显好于对照组,实验组术后3个月实验组已经有较硬的类软骨组织填充修复,切片显示有软骨细胞规则排列,甲苯胺蓝染色为阳性,而对照组仅为纤维组织修复,切片显示为纤维组织或纤维软骨组织修复,软骨细胞排列紊乱,甲苯胺蓝染色阴性或弱阳性。结论负载TGF-β1壳聚糖微球的壳聚糖-丝素支架复合骨髓间充质干细胞体外复合培养后移植治疗修复兔膝关节软骨缺损,具有较好的疗效。Objective To explore the reparative effects of complex of bone marrow - derived mesenchymal stem cell and chitosan - silk fibroin scaffold load with TGF -β1 microspheres on rabbit articular cartilage defects. Methods Bone marrow - derived mesenchymal stem cells （MSCs） were isolated and purified by a combination of density gradient centrifugation and adhesive culture in vitro, and surface antigens CD14 and CD44 of MSCs were detected by flow cytometre. Chitosan - silk fibroin scaffolds and chitosan microspheres loaded with TGF- β1 were prepared by the freeze- drying and the emulsion- crosslinking method respectively,then the microspheres were loaded on the scaffolds. Tissue engineering cartilage constructed by seeding MSCs on the scaffolds in vitro was transplanted to the defects of rabbit ar- ticular cartilage. The experimental group and the control group transplanted tissue engineering cartilage and chitosan - silk fibroin scaffoldonly, respectively. Results Negative surface antigen CD14 and positive CD44 of purified MSCs were detected. The microspheres were spherical in shape, and had a smooth surface. The diameter was approximately 1 ~m. Chitosan - silk fibroin scaffolds had favorable struc- tural features for MSCs attachmen and proliferation. In experimental group, defect area was replaced by hyaline - like cartilages. Most of the cartilage cells were arranged regularly and styloidly. Result of toluidine blue staining was positive. In control group, there were fibrous - like tissue and fibrocartilages. Cartilaginous cells were disarranged. The result of toluidine blue staining was negative or weakly positive. Conclusion The complex of bone marrow - derived mesenchymal stem cell and chitosan - silk fibroin scaffold load with TGF - β1 microspheres had obvious therapeutic effect on rabbit articular cartilage defects.

关 键 词：壳聚糖-丝素 支架 骨髓间充质干细胞 软骨 TGF—β1 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]