组织工程骨-软骨复合组织种子细胞的获取与培养(1)
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[摘要]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。
[关键词]组织工程；骨髓间充质干细胞；软骨细胞；种子细胞
[中图分类号]Q813.1 [文献标识码]A [文章编号]1008-6455(2012)05-0748-04
Obtainment and culture of seeding cells of tissue engineered osteochondral composites
LIU Peng1, FU Chong-jian1, DENG Tian-zheng2, JIANG Ming1, LI Ji-guo1, YU Shu-juan1, ZHU Guo-xiong1
(1.Department of Stomatology, Jinan General Military Hospital, Jinan 250031,Shangdong,China;2.Department of Stomatology, General Hospital of the Air Force of PLA)
Abstract: 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.
Key words: tissue engineering; bone marrow stem cells; chondrocytes; seeding cells
骨-软骨复合组织主要存在于关节、肋骨等区域，尤其是颞下颌关节、膝关节等运动关节，并承担着重要的功能。各种原因导致的关节软骨缺损是常见疾病，由于关节软骨自身修复能力有限，目前无特效治疗方法[1]。近年来组织工程的发展为治疗软骨缺损带来了良好的前景[2]。种子细胞、支架材料和生长因子是组织工程的三大要素，种子细胞的选择是组织工程化骨-软骨复合组织构建的前提。本研究选择了软骨及骨髓来源的细胞，通过对软骨细胞和骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)的分离方法、培养条件及生长测定 ......
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