组织工程骨-软骨复合组织种子细胞的获取与培养

目的:探索体外培养组织工程骨-软骨复合组织种子细胞的条件,并观察其部分生物学活性。方法:采用机械-酶消化法对3周龄新西兰大白兔耳软骨和关节软骨消化来获得软骨细胞,采用全骨髓贴壁法来获得骨髓间充质干细胞,对两种细胞进行原代和传代培养,通过倒置相差显微镜观察细胞形态、绘制生长曲线、免疫组化染色等对细胞进行生物学特性分析。结果:原代培养的软骨细胞以多角形或三角形为主,传代3次以后出现去分化。形态学和免疫组化显示细胞3代以内可以保持表型稳定,具有较强的增殖及分泌细胞外基质的能力,而且耳软骨及关节软骨细胞在实验中没有表现出明显的生物学差别。采用贴壁筛选法获得的BMSCs呈长梭形或多边形,生长曲线呈"S"形,Ⅱ型胶原免疫组化染色阳性,细胞生长增殖能力旺盛。结论:体外分离培养的软骨细胞和骨髓间充质干细胞符合组织工程要求,能够作为骨-软骨复合组织的种子细胞。

Obtainment and culture of seeding cells of tissue engineered osteochondral composites

Objective To explore the optimized conditions for isolation and culture of seeding cells of osteochondral composites and observe some biological features of the seeding cells.Methods 3-week-old New Zealand White rabbits were selected for BMSCs and chondrocytes isolation and culture.Articular cartilage cells were obtained by mechanical-collagenase digestion.BMSCs were saparated using whole marrow culture method and purified by attachment method.Morphology and growth characteristics were observed.Results The cultivation of cartilage cells presented with polygonal or triangle,morphology and immunohistochemistry showed that stable phenotype could be maintained within three generations,and the biological characters of chondrocytes from ears or joints were almost the same.BMSCs developed a spindle or polygonal appearance,growth curve was S-shaped,expression of type Ⅱcollagen was positive.Adhesive culture method was proved to be an effective method to provide a large number of BMSCs.Conclusion The chondrocytes and BMSCs are suitable for tissue engineering,and these can be used as seeding cells of osteochondral composites.