Objective:To investigate the feasibility of construction of tissue engineered cartilage by co-culture of bone marrow mesenchymal stem cells (BMSCs) and costal chondrocytes (CCs),and to provide theoretical basis and experimental basis for clinical repair of articular cartilage defects by Wuzhishan miniature pig knee cartilage defects with co-cultured cells.Methods:Density gradient centrifugation method was used to isolate BMSCs from Wuzhishan miniature pig.The double enzyme digestion method was used to isolate CCs.The passage 3 generation of BMSCs and passage 2 generation of CCs were randomly divided into 3 groups:a co-culture group of BMSCs∶CCs for 1∶2 (Group A),a simple CCs (Group B),and a simple BMSCs (Group C).The cell growth curve was drawn,and the content of glycosaminoglycan (GAG) of external separation in chondrocytes was determined.The 12 Wuzhishan miniature pigs were randomly divided into a co-culture cells/collagen membrane experimental group,a collagen membrane control group and the blank group.In the co-culture cells/collagen membrane experimental group,the co-cultured cells/collagen membrane were implanted into the cartilage defects of the mandibular condyle;in the collagen membrane control group,only collagen membrane was implanted;while in the blank group,nothing was implanted.Six animals were sacrificed at 8 and 16 weeks after surgery respectively (2 animals in each group).General observation,cartilage histological score and histopathological examination were carried out.Results:The BMSCs and co-culture cells grew well.The biological activity of CCs was good.After 16 weeks of operation,the repair tissues in the co-cultured cells/collagen membrane experimental group showed hyaline cartilage features:smooth,flat,and integrated well with the surrounding cartilage and subchondral bone.The collagen membrane in the collagen membrane control group was fibrously repaired.Repair tissue gross score in the co-culture cells/collagen membrane experimental group was significantly better than that in the collagen membrane control group and the blank group (both P<0.05),but there was no significant difference between the collagen membrane control group and the blank group (P>0.05).Conclusion:BMSCs,CCs and co-cultured cells can function as the seed cells for cartilage tissue engineering,and the co-culture cells (BMSCs∶CCs=1∶2) possess more advantages;the short-term effect of co-culture cells with collagen membrane on repairing cartilage defects is satisfied.%目的:探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)和肋软骨细胞(costal chondrocytes,CCs)共培养构建组织工程软骨及共培养细胞修复五指山小型猪膝关节软骨缺损的可行性,为临床修复关节软骨缺损提供理论依据和奠定实验基础.方法:密度梯度离心法分离五指山小型猪BMSCs,双酶消化法分离CCs.取第3代BMSCs和第2代CCs,随机分为3组:BMSCs:CCs为1:2的共培养组为A组,单纯CCs为B组,单纯BMSCs为C组.绘制细胞生长曲线图,测定软骨细胞外分泌基质糖胺多糖(glycosaminoglycan,GAG)的能力.将12只五指山小型猪随机分为共培养细胞/胶原膜实验组、胶原膜对照组和空白组.共培养细胞/胶原膜实验组髁间窝软骨缺损处植入共培养细胞/胶原膜,胶原膜对照组单纯植入胶原膜,空白组不做任何植入,于术后第8和16周分别处死6只动物,每组2只.取材行大体观察、软骨组织学评分及病理组织学检测.结果:密度梯度离心法分离的BMSCs生长良好,双酶消化法分离的CCs生物活性良好,共培养细胞生长良好.术后16周共培养细胞/胶原膜实验组修复组织呈透明软骨样,表面光滑平坦,周围软骨及软骨下骨整合良好,而胶原膜对照组和空白组为纤维性修复和无修复.共培养细胞/胶原膜实验组修复组织大体评分明显优于胶原膜对照组和空白组(均P<0.05),胶原膜对照组和空白组之间差异无统计学意义(P>0.05).结论:BMSCs,CCs和共培养细胞均适合作为软骨组织工程的种子细胞,其中共培养细胞(BMSCs∶CCs为1∶2)更具优势;共培养细胞复合胶原膜修复软骨缺损近期效果满意.
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