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

Abstract

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. ①Two-passaged bone marrow mesenchymal stem cells and chondrocytes (3×108 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. ②3×108 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. ③Totally 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. ①Co-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±0.76）% at 2, 4 and 6 days after co-culture respectively. ②In the experimental group, repaired tissues represented hyaline-like, smoothness and flat. Chondrocytes in the zones of integrating with peripheral native cartilages were more mature, repaired tissues integratinged with subchondral bones firmly. Repaired tissues were fibers and no repair in the demineralized bone matrix control group and the blank control group. ③Histological scores were higher in the experimental group than the demineralized bone matrix control group and the blank control group (P < 0.01). There was no significant difference between the demineralized bone matrix control group and the blank control group (P > 0.05). ④Immunohistochemical stains showed that cells repaired tissues were hyaline cartilage-like cells, arranged columnnedly，riched in type Ⅱcollagen matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in the experimental group.

CONCLUSION: Co-cultures of autogenic bone marrow mesenchymal stem cells with chondrocytes can promote proliferation of chondrocytes and production of chondral matrix. Co-cultures as seeding cells can save a number of chondrocytes, shorten culturing periods and reduce subcultured times. Co-cultures as seeding cells embedded into demineralized bone matrix can repair articular cartilage defects effectively.

Feng WW, Lai ZJ, Li XM, Chang LW, Xu YL, Zhang L, Wang XH, He XH, Xia YY, Dang YX, Wu M, Sun ZY, Jin BY, Li XD.Characteristics of co-culture of autogenic bone marrow mesenchymal stem cells with chondrocytes as seed cells and in vivo chondrogenic activity.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):442-446(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-442(ps).pdf]

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3×108 L-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3×108 L-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2，4，6 d，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，柱状排列，Ⅱ型胶原染色阳性，与周围软骨及软骨下骨整合良好。

结论：自体骨髓间充质干细胞与软骨细胞共培养作为种子细胞，骨髓间充质干细胞能增强软骨细胞的增殖，促进软骨细胞基质合成，缩短软骨细胞培养时间和减少传代次数，可节省大量的软骨细胞，与脱钙骨基质复合后能有效修复关节软骨缺损。
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

冯万文，莱浙军，李小民， 常伶文，徐毓林，张 丽，王晓红，何向红，夏亚一，党跃修，吴萌，孙正义，靳白玉，李向东.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[J].中国组织工程研究与临床康复，2008，12(3):442-446
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-442(ps).pdf]