新型脱细胞软骨基质三维多孔支架的制备  

FABRICATION OF A NOVEL CARTILAGE ACELLULAR MATRIX SCAFFOLD FOR CARTILAGE TISSUE ENGI- NEERING

作　　者：杨强[1] 彭江[1] 卢世璧[1] 孙明学[1] 黄靖香[1] 张莉[1] 许文静[1] 赵斌[1] 眭祥[1] 姚军[1] 袁玫[1] 

机构地区：[1]解放军总医院全军骨科研究所,北京100853

出　　处：《中国修复重建外科杂志》2008年第3期359-363,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30330570);北京市科委任务课题资助项目(H060920050630);全军医药卫生科研基金资助项目(06Z057)~~

摘　　要：目的探讨脱细胞软骨基质三维多孔支架的制备方法以及将其应用于关节软骨组织工程的可行性。方法取天然人软骨粉碎后,采用梯度离心法取100 nm～5μm软骨微丝,脱细胞处理后制备为质量体积比为3%的悬液,采用冷冻冻干法制备脱细胞软骨基质三维多孔支架。254 nm紫外线和碳化二亚胺/ N-羟基琥珀酰亚胺对支架进行交联。冷冻冻干后,对支架材料进行组织学及扫描电镜观察,测定支架孔径和孔隙率、吸水率,并采用MTT法分析支架浸提液毒性。分离培养犬BMSCs,用TGF-β1成软骨诱导后种植至支架,倒置显微镜、电镜观察细胞在支架上的生长、分化情况。结果组织学观察显示,三维多孔支架中无软骨细胞碎片残留,甲苯胺蓝染色、番红O染色、Ⅱ型胶原免疫组织化学染色均呈阳性。扫描电镜显示支架内孔洞相互连通,孔径为(155 ± 34)μm,孔隙率为91.3% ± 2.0%,吸水率为2 451% ± 155%。MTT 法显示不同浓度支架浸提液与对照DMEM培养液吸光度值比较,差异无统计学意义(P > 0.05),支架无细胞毒性。倒置显微镜观察,细胞在支架上黏附良好;扫描电镜下细胞在支架上均匀分布,细胞呈圆形或椭圆形,并有基质分泌。结论制备的脱细胞软骨基质三维多孔支架去细胞彻底,保留了软骨ECM主要成分,无毒,具备合适的孔径和孔隙率,生物相容性良好,是软骨组织工程良好的支架载体。Objective To develop a novel cartilage acellular matrix （CACM） scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-（3-dimethylaminopropyl） carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell proliferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocyte fragments in the scaffolds and that toluidine blue, safranin O and anti-collagen Ⅱ immunohistochemistry staining were positive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter （155 ± 34） Ima, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or elliptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative cell-carrier for cartilage ti

关 键 词：组织工程软骨 ECM 脱细胞 支架 冷冻冻干法 

分 类 号：R318.08[医药卫生—生物医学工程]