不同细胞接种密度体外构建“自组装”工程化软骨的实验研究  

Effect of cell seeding concentration on the quality of in vitro generated tissue-engineered cartilage constructed by the technique of self-assembly

作　　者：贾杰[1] 杨述华[1] 张宇坤[1] 孙志博[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中国矫形外科杂志》2012年第11期1030-1033,共4页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(编号:30800654)

摘　　要：[目的]探究以人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hMSCs)为种子细胞体外构建"自组装"工程化软骨对应的合理细胞接种密度。[方法]体外分离与培养hMSCs。用含100 ng/ml生长分化因子5(growth differentiation factor 5,GDF-5)的软骨诱导液(chondrogenic medium,CM)定向诱导培养第3代hM-SCs,诱导3周后重悬细胞,分别以A组2.5×106/ml,B组5×106/ml,C组1×107/ml,D组2×107/ml四种细胞密度接种于2%琼脂糖包被的24孔板,每组设5个复孔。自组装培养3周后,对标本进行大体观察、组织学及免疫组化检测并进行生化分析,比较不同组标本的软骨生物学特性的差异。[结果]"自组装"培养3周后,各组都形成了软骨样组织团块,团块直径与湿重随细胞接种密度而增加。Bern评分结果显示C、D两组明显高于A、B两组(P<0.05),且C组与D组间差异无统计学意义,Ⅱ型胶原免疫组化染色检测到C组与D组细胞外基质内有较强的阳性信号弥漫分布,蛋白多糖(GAG)含量C、D两组亦明显高于A、B两组(P<0.05),且C组与D组间差异无统计学意义。[结论]在一定细胞接种密度范围内,"自组装"工程化软骨的生物学特性呈密度依赖性增加。以1×107/ml接种时,可以获得具有良好生物学特性的"自组装"工程化软骨。[Objective]To explore the effect of varying cell seeding concentration on the quality of in vitro generated tissue-engineered cartilage constructed by the technique of self-assembly using human bone marrow mesenchymal stem cells（hMSCs） as seed cells.[Methods]hMSCs were isolated and cultured in vitro.The cells from passage 3 were induced by chondrogenic medium（CM） supplemented with 100 ng/ml growth differentiation factor 5（GDF-5）.After 3 weeks of induction,cells were re-suspended and were inoculated on 2% agarose-coated 24-well plates according to different concentrations.The 4 groups were divided according to this cell seeding concentration difference： group A 2.5×10^6/ml,group B 5×10^6/ml,group C 1×10^7/ml,group D 2×10^7/ml,with five multiple wells for each group.After continuously induced with CM for 3 weeks,grossly observation,histological and immunohistochemistry staining,biochemical analysis were performed.[Results]After 3 weeks of self-assembly culture,tissue-engineered cartilage were formed in all groups.The diameter and wet weight of the tissue-engineered cartilage increased,varying the cell seeding concentration.Bern score in group C and D are obviously higher than in group A and B,but there was no statistical difference between group C and D.Type II collagen expression and GAG content in group C and D were also greater than in group A and B.[Conclusion]Within a certain range,the biological characteristics of the tissue-engineered cartilage was increased in cell seeding concentration-dependent manner.The cell seeding concentration 1×10^7/ml is suitable for in vitro generated tissue-engineered cartilage constructed by the technique of self-assembly.

关 键 词：成人骨髓间充质干细胞 生长分化因子5 “自组装”培养 软骨分化 

分 类 号：R318[医药卫生—生物医学工程]