聚磷酸钙纤维和左旋聚乳酸软骨支架复合自体骨髓间充质干细胞构建组织工程化人工关节软骨

Construction of tissue engineering cartilage using bone marrow mesenchymal stem cells seeded onto CPP/PLLA

目的应用组织工程学技术,体外初步构建组织工程化人工关节软骨。方法制备三维多孔软骨支架材料CPP/PLLA,体外诱导兔MSCs向软骨细胞表型分化,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达,将诱导细胞与软骨支架材料CPP/PLLA复合,体外培养构建人工关节软骨,1周后终止培养,扫描电镜观察组织工程化人工软骨的微观结构;同时将构建人工软骨移植于兔大腿皮下,3周后处死动物,甲苯胺蓝染色观察。结果扫描电镜观察可见该复合材料CPP/PLLA为高孔隙率的网状、连通、微孔结构,微孔分布均匀,孔径大小为300～400μm之间;兔MSCs经体外软骨表型定向诱导后,Ⅱ型胶原免疫组化染色阳性。诱导后的MSCs可在支架材料内良好贴附生长,细胞被分泌的胶原基质包裹;从体内获取的培养物组织切片观察可见大量的软骨细胞生成,甲苯胺蓝染色阳性。结论经软骨起源诱导后的MSCs与CPP/PLLA复合培养可以构建自体软骨移植的替代物,为应用软骨组织工程方法修复关节软骨缺损和功能重建提供一种新材料,具有较大的潜在应用价值。

Objective To construct artificial cartilage with tissue-engineering techniques. Methods The CPP/PLLA composites of the scaffold for cartilage tissue engineering were fabricated with a solvent-casting, particulate-leaching method; Rabbit MSCs were isolated and induced to differentiate into chondrogenic phenotype in a defined medium in vitro, then immunohistochemical staining was applied to detect the expression of collagen type Ⅱ. The differentiated cells were implanted on the CPP/PLLA composites. After the cell-scaffold complex was cultured in vitro for one week, the ultrastructure of the scaffold was observed with scanning electron microscopy. At the same time, the cell-scaffold complex was transplanted subcutaneously adjacent to the knee joint for 3 weeks in vivo, stained with toluidine blue. Results The scaffold composites CPP/PLLA had micropores with the diameter of 300 -400μm, microporous sponge structure to fibrous net work in microstructure of cross sections. Immunohistochemical staining of collagen Ⅱ were positive for the MSCs after that was cultured under the defined medium. Induced MSCs were well adherent to the scaffold composites and the cell were embedded by the cell-matrix; The grafts developed into matured cartilage tissue assessed by toluidine blue staining after 3 weeks of transplantation in vivo. Conclusions The induced MSCs and CPP/PLLA composite cultured together could construct autograft substitute for cartilage defect. This experiment provides new biosynthetic materials and methods for repairing cartilage defect.