骨髓间充质干细胞和软骨细胞共培养复合同种异体脱钙骨基质修复关节软骨全层缺损

Full-thickness articular cartilage defect repairment by coculture of autogenic bone marrow-derived mesenchymal stem cells with chondrocytes and implantation into allogenic demineralized bone matrix in rabbits

目的：探讨自体骨髓间充质干细胞（BMSCs）与软骨细胞共培养复合同种异体脱钙骨基质（DBM）修复关节软骨缺损的可行性，评价修复效果，为优化种子细胞源提供依据．方法：取浓度为5×10^9／L的第二代BMSCs和软骨细胞，按2：1比例混匀共培养作为种子细胞．DBM与共培养细胞复合植入修复为实验组（A组），单纯材料DBM组（B组）和不处理组（C组）作为实验对照组．移植8和16wk后经大体观察、组织学评分和免疫组化染色评价缺损修复．结果：共培养的软骨细胞基质合成丰富，细胞增殖快，共培养5—7d两种细胞比例达1：1以上．A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．B组和C组的修复组织呈纤维组织．组织学评分表明A组优于B，C两组，差异具有统计学意义（P〈0．01），B组与C组差异无统计学意义（P〉0．05）．免疫组化染色显示A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BMSCs与软骨细胞共培养，BMSCs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DBM复合后能有效修复关节软骨缺损．

AIM： To investigate the feasibility of articular cartilage defect repairment by coculture of autogenic bone marrowderived mesenchymal stem cells（BMSCs） with chondrocytetes and implantation into allogenic demineralized bone matrix （ DBM ）, evaluate the outcomes of repairment in order to provide a theoretical basis for optimizing cell resources for seeding. METHODS： Two-passaged BMSCs and chondrocytes were collected both at a concentration of 5× 10^9/L and then cocuhured at 2：1. Articular cartilage defects in the knee joints of rabbits repaired by the cocuhured cells seeded into allogenic DBM served as experimental group A, by simple DBM as control group B and by nothing as control group C. Repaired tissues were evaluated with macroscopic observation, histological scores and immunohistochemical staining at week 8 and 16 after transplantation. RESULTS： Chondrocytes cocuhured became rich in extracellular matrix and proliferated promptly. The ratio of chondrocytes to BMSCs achieved above 1：1 at 5 -7 d after cocuhure. In the experimental group A repaired tissues represented hyaline, smooth and flat. Chondrocytes in the zones of integrating with peripheral native cartilages were more mature. In the control group B and C, repaired tissues were fibers. Histological scores indicated that experimental group A excelled group B and C, and the differences were statistically significant（P〈0.01 ）; differences between group B and C were not significant （ P 〉 0.05 ）. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnnedly, rich in type-Ⅱ collagen matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in the experimental group A at week 16 postoperatively. CONCLUSION： Coculture of autogenic BMSCs with chondrocytes can promote the proliferation of chondrocytes and the production of chondral matrix. Cocultured cells for seeding can save a number of chondrocytes, shorten culture periods and reduce subculture times. Cocultured cells embedded into DBM can repair articular cartilage defects effectively.