以软骨细胞外基质与脱细胞骨基质构建组织工程骨软骨双层支架及其性能的研究

A novel bilayered scaffold constructed with cartilage extracellular matrix /acellular bone matrix for osteochondral tissue engineering

目的 探讨采用软骨细胞外基质（CECM）与脱细胞骨基质（ACBM）为材料制作新型组织工程骨软骨双层支架的可行性,并检测其性能.方法 双层支架的骨部分以犬松质骨制备的ACBM为原料,软骨部分以人CECM为材料,采用冷冻冻干法制备CECM/ACBM双层支架并交联.测定支架孔隙率,采用四甲基偶氮唑盐（MTT）法分析支架浸提液毒性.分离培养犬骨髓基质干细胞（BMSCs）,成软骨诱导后种植到支架上,倒置显微镜、电镜、Dead/Live荧光染色观察细胞在支架的生长、分化情况.结果 扫描电镜及Micro-CT观察显示支架内孔洞相互贯通呈海绵状,CECM部分孔径（155±34）μm,孔隙率为91.3%±2.0%;ACBM部分具有大然骨的孔径和空隙率,骨软骨部分结合良好.培养1～6 d不同浓度支架浸提液与对照培养液吸光度值比较羌异均无统计学意义（P＞0.05）.倒置显微镜、电镜检查结果表明BMSCs在支架上黏附良好,细胞基质分泌增加,Dead/Live荧光染色表明双层支架内细胞均呈绿色.结论 CECM/ACBM骨软骨双层支架具备良好的孔径和孔隙率,骨、软骨两层间结合良好,无毒,生物相容性良好,可作为支架载体用于组织工程骨软骨复合体的构建.

Objective To fabricate a novel bilayered scaffold constructed with cartilage extracellular matrix （CECM） and acellular bone matrix （ACBM） for osteochondral tissue engineering. Methods The bone layer of the osteochondral scaffold was prepared using canine bone cancellous bone columns, and the cartilage layer was fabricated using CECM. After CECM microfilaments were decellularized, the biphasic scaffolds were fabricated by soaking the ACBM columns into cylindrical silicon moulds with a 30 g/L CECM suspension using simple freeze-drying method. After the scaffolds were cross-linked, the porosity was measureed. MTT test was also done to assess cytotoxicity of the scaffolds. Canine bone marrow-derived mesenehymal stem cells （BMSCs） were induced by chondrogenic medium and seeded into novel scaffold. Cell proliferation and differentiation were analyzed using inverted microscopy, scanning electron microscopy （SEM） and Dead/Live staining method. Results SEM and Micro-CT revealed a 3-D interconnected porous structure, with the CECM pore diameter of 155±34μm and the porosity of 91.3%± 2.0%. Cytotoxicity testing with MTT revealed no significant difference in absorbanee among different extracts, showing no cytotoxic effect of the scaffold on BMSCs. Inverted microscopy and SEM showed that the novel scaffold could provide a suitable 3-D environment to support the adhesion, proliferation and differentiation of BMSCs to chondrocytes in culture with chondrogenic medium. Confocal microscopy of cell-scaffold constructs revealed cells with green fluorescence. Conclusion Since the novel CECM/ACBM bilayered integrated osteochondral scaffold has good mircostructure, non-toxicity and good biocompatibility, it may be a suitable candidate as an alternative cell-carrier for osteochondral tissue engineering.