应用骨髓基质干细胞体外构建组织工程化管状软骨的实验研究  

In vitro engineering tubular cartilage using polyglycolic acid and bone marrow stroma cells

作　　者：夏万尧[1] 刘伟[1] 刘天一[1] 陈付国[1] 崔磊[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学附属第九人民医院整形外科上海第二医科大学组织工程研究中心,200011

出　　处：《中华显微外科杂志》2005年第4期328-330,i0004,共4页Chinese Journal of Microsurgery

基　　金：国家高技术研究发展计划资助(863计划;2002AA205021)

摘　　要：目的以骨髓基质干细胞(bone marrow stroma cells,BMSCs)为种子细胞,接种于管状聚羟基乙酸(PGA)支架上,在体外构建组织工程化管状软骨. 方法取长枫杂交仔猪髂骨骨髓,在低糖DMEM完全培养液培养2周,传代后以浓度为5×107/ml细胞悬液均匀接种于管状PGA支架上,以高糖DMEM低血清特定培养液诱导(含胰岛素2 mg/L、转铁蛋白3 mg/L、丙酮酸100mg/L、地塞米松10-7mol/L、TGF-β 10ng/L、葡萄糖4.5 mg/ml、2%胎牛血清),连续诱导培养10周,从大体、组织学和Ⅱ型胶原免疫组化对再生组织进行评价. 结果 6周时染色见BMSCs-PGA复合物表层为2～4层成纤维样细胞组成的软骨膜,下层为较成熟的软骨组织,软骨细胞包埋在软骨陷窝内,有很多散在PGA纤维,而在中间部分,组织量较少,结构较构散.10周时BMSCs-PGA复合物外观呈乳白色软骨样,管壁较厚,有一定的弹性,但中间部管腔塌陷较明显,苏木素-伊红染色见实验组管状BMSCs-PGA和6周时相似,但结构更致密和规则,细胞数量较正常软骨组织少,还可见少量的未降解的PGA纤维.免疫组化证实形成的组织有Ⅱ型胶原分布. 结论管状BMSCs-PGA复合物在特定培养液诱导下,在体外能形成管状软骨,这为将来临床应用BMSCs作为种子细胞,修复软骨缺损或构建复合组织气管提供了实验基础和技术参数.Objective To investigate the feasibility of in vitro engineered tube-shaped cartilage using bone marrow stroma cells （BMSCs） seeding on tube-shaped polyglyeolie acid （PGA）. Methods Bone marrow was aspirated from swine iliac crest and cultured in a complete medium of low glucose of DMEM for two weeks. Then BMSCs suspension with a density of 50 million cells/ml in DMEM-LG medium was seeded onto the tube-shaped PGA scaffold at a volume of 0. 5 ml for each construct. The BMSCs-PGA constructs were then incubated constituitivel in a defined medium （DMEM containing insulin 2mg/ml, transferrin 3mg/ml,dexamethasone 10.7 mol/L, pyruvate 100mg/L,TGF-β1 10ng/L,glucose 4. 5mg/ml,2% fetal bovine serum） in an incubator at 37℃ for ten weeks. Constructs were harvested and analyzed by gross observation, histology and immunohistochemical localization of type IT collagen after 6 and 10 weeks of culture in vitro respectively. Resuits HE staining showed elongated lining cells appeared as perichondriumo-like cells at the surface,and at the substrate of the neocartilage some mature ehondrocytes were embeded in the lacuna and undegraded PGA fibers were observed after 6 weeks in vitro. When harvested at 10 weeks,the BMSCs-PGA constructs exhibited a pearly opalescence and was resistant to external compression except the sink in the middle of tube-shaped cartilage. HE staining showed more mature and homogeneous cartilages than that of harvested at 6 weeks and the PGA fibers were almost completely degraded. Immunohistochemical staining revealed the presence of type IT collagen in the ECM of tissue engineered cartilage. Conclusion The experiments demonstrated that a tubular cartilage could be engineered in vitro using PGA and BMSCs,suggesting its potential to serve as an optimal autologous cell source to repair cartilage defects and to reconstruct composite tissue trachea.

关 键 词：骨髓基质干细胞 生物医学工程 分化 组织工程化 体外构建 软骨膜 实验研 管状 苏木素-伊红染色 PGA支架 

分 类 号：R318.0[医药卫生—生物医学工程]