骨髓间充质干细胞和软骨细胞共培养种子细胞特征及体内成软骨活性  

Characteristics of co-culture of autogenic bone marrow mesenchymal stem cells with chondrocytes as seed cells and in vivo chondrogenic activity

作　　者：冯万文[1] 莱浙军[1] 李小民 常伶文[1] 徐毓林[1] 张丽[1] 王晓红[1] 何向红 夏亚一[2] 党跃修[2] 吴萌[2] 孙正义[2] 靳白玉[2] 李向东[1] 

机构地区：[1]连云港市第一医院东方医院暨连云港市涉外医院骨科,江苏省连云港市222042 [2]兰州大学第二医院骨科研究所,甘肃省兰州市730030

出　　处：《中国组织工程研究与临床康复》2008年第3期442-446,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:组织工程技术为解决关节软骨缺损修复这一难题提供了新的思路,但至今尚无一种细胞能完全满足软骨组织工程对种子细胞的要求。探讨以自体骨髓间充质干细胞与软骨细胞共培养为种子细胞修复关节软骨缺损的可行性,并评价修复效果。方法:实验于2005-03/2006-02在兰州大学骨科研究所组织工程实验室和连云港市东方医院骨科实验室完成。①取浓度为3×108L-1的第2代骨髓间充质干细胞和软骨细胞,按2:1比例混匀共培养作为种子细胞,观察细胞的增殖和基质合成,绘制细胞生长曲线。②将细胞终浓度为3×108L-1的共培养细胞加入含有同种异体脱钙骨基质的24孔培养板中进行接种培养2,4,6d,计算共培养细胞与脱钙骨基质的黏附率。③取54只青紫兰兔制备全层关节软骨缺损模型,随机分为实验组、脱钙骨基质对照组、空白对照组,每组18只。实验组于软骨缺损处植入同种异体脱钙骨基质与共培养细胞;脱钙骨基质对照组植入脱钙骨基质;空白对照组不作任何植入。移植术后6,12周取出膝关节对修复组织进行大体、组织学评分和免疫组织化学观测。结果:54只青紫兰兔均进入结果分析。①共培养的软骨细胞基质合成丰富,细胞增殖加快。脱钙骨基质与骨髓间充质干细胞和软骨细胞共培养第2,4,6天的黏附率分别为(46.50±1.40)%,(93.25±2.89)%和(88.34±0.76)%。②大体观察结果:实验组缺损修复组织呈软骨样,表面光滑平坦,与周围软骨整合的软骨细胞更为成熟,修复组织与软骨下骨结合牢固。脱钙骨基质对照组、空白对照组的修复组织呈纤维组织和无修复。③组织学评分:实验组优于脱钙骨基质对照组、空白对照组,差异具有显著性意义(P﹤0.01),脱钙骨基质对照组与空白对照组比较差异无显著性意义(P﹥0.05)。④免疫组织化学染色显示实验组修复组织的细胞为透明软骨样细胞,柱�AIM：Tissue engineering technique brings a new approach to repair articular cartilage defects. There was no ideal seed cell for cartilage tissue engineering. We investigate the feasibility of articular cartilage defects repaired with co-cultures of autogenic bone marrow mesenchymal stem cells with chondrocytes as seed cells, and evaluate repaired outcomes. METHODS Experiments were performed at the Laboratory of Tissue Engineering and Institute of Orthopaedics of Lanzhou University and Laboratory of Orthopaedics of Lianyungang Dongfang Hospital from March 2005 to February 2006. （1） Two-passaged bone marrow mesenchymal stem cells and chondrocytes （3×10^8 L^-1） were collected and then co-cultured at a ratio of 2 to 1 as seed cells. Extracellular matrix and proliferation of co-cultures were observed. Proliferative curve of the seed cells was drawn. （2）3×10^8 L^-1 co-cultured cells were incubated with allogenic demineralized bone matrix in 24-well plate for 2, 4 and 6 days. Adhesive rate of co-cultures into allogenic demineralized bone matrix were detected. （3）Totally 54 pigmented rabbits were made into models of articular cartilage defects in the knee joints. They were divided into experimental, demineralized bone matrix control and blank control groups, with 18 in each group. Rabbits in the experimental group were implanted with allogenic demineralized bone matrix and co-cultured cells; rabbits in the demineralized bone matrix control group were implanted with demineralized bone matrix only; rabbits in the blank control group did not receive any intervention Repaired tissues were evaluated with macroscopic views, histological scores and immunohistochemical stains at weeks 6 and 12 after transplantation. RESULTS： Fifty-four pigmented rabbits were involved in the result analysis. （1）Co-cultured chondrocytes was rich in extracellular matrix and proliferated promptly. Adhesive rate of co-cultures into allogenic demineralized bone matrix were （46.50±1.40） %, （93.25±2.89） % and （88.34

关 键 词：骨髓间充质干细胞 软骨细胞 共培养 种子细胞 脱钙骨基质 关节软骨修复 

分 类 号：R394.2[医药卫生—医学遗传学]