自体骨髓间充质干细胞藻酸钙载体复合物对兔膝关节软骨缺损修复影响的实验研究

The experimental study of repairing effect after embedding compound including auto-bone marrow mesenchymal stem cells and calcium-algitate in rabbits articular genu defect

目的：软骨组织多处于人体骨骼的重要部位，其缺损修复一直为临床急待解决的难题，用组织工程方法修复关节软骨缺损是近年来正在研究的新途径。其中绝大多数研究几乎均着重体外培养条件的研究[1,2]，而忽略了对于改善局部微环境的探讨，为此，本实验试图在载体复合物植入软骨缺损微环境内时，增加能促进MSCs分裂、增殖、分化及血管新生的bFGF及参与和活跃成软骨细胞合成软骨基质及纤维的维生素C等，从而达到提高软骨缺损修复疗效的目的。方法从24只3月龄新西兰大耳白兔髂骨处抽取骨髓，以密度梯度离心法分离出骨髓间充质干细胞（MSCs）作为种子细胞进行扩增培养至第三代，制成细胞悬液，而后在自制模具中与藻酸钙制备成与兔膝关节软骨全层缺损（直径4mm、深度4mm）相一致的载体复合物同时加入bFGF和维生素C，将该载体复合物植入兔左膝关节软骨全层缺损处作为A组，而右膝关节的关节软骨全层缺损处则植入MSCs与藻酸钙的载体复合物作为B组。另取兔6只，左膝按上述方法作一缺损植入单纯藻酸钙载体作为C组；右膝缺损则作空白对照D组。待术后不同时间点（30 d，60 d及90 d）取材，常规石蜡包埋后制成切片，分别用于HE、Masson、番红O、透射电镜、免疫组化等指标以观察软骨缺损修复效果。结果 A，B，C三组软骨缺损均被透明软骨和纤维组织充填，只是A组的透明软骨组织较B组多，C组最少。随植入时间后移，A组几乎均被透明软骨样组织填平并且充填组织与周边正常关节软骨的交界逐渐分辨不出，并与深层软骨下骨相连接，软骨细胞间质的胶原纤维从表层向深层呈放射状排列于软骨细胞基质中。而B组的缺损处表面尚有较多的纤维组织且充填组织与周边正常关节软骨的分界较A组清晰，C组缺损处表面可见有较多纤维组织和未降解的藻酸钙，与周边正常关节软骨分界较A，B组清晰。A组填充的软骨细胞具有明显陷窝的，互相距离较远的3～5个细胞排列呈柱状的成熟软骨细胞明显较B组多，C组则很难观察到成行排列的软骨细胞。功能旺盛的软骨细胞（Ⅱ型胶原阳性细胞，胞质粗面内质网扩张等）则属A组最多，C组最少。D组术后90 d时缺损处为大量纤维组织填充且与周边正常关节软骨和软骨下骨整合较差。结论用自体MSC藻酸钙载体复合物植入膝关节软骨缺损同时向该缺损微环境内加bFGF和维生素C有利于软骨修复，能提高软骨缺损修复效果。

Objective Cartilage tissue is mostly located at the important site of bones in human body, and the repair of its defect remains in suspense in clinical. With the development of the tissue engineering, it brings new approach for the repair of the defect of the articular cartilage. The researchs emphases on the ex＆amp;nbsp;vivo condition. Accordingly, scholars ignore the effect of improving the local micro-circumstances. Consequ-ently we add the bFGF and VitC in the micro-circumstances when embedding the carrier compound in order to improve the effect of the repair. Methods Bone marrow is obtained from the iliac bone of 20 rabbits respectively. As the seed cells, Mesenchymal Stem Cells are purified with the density gradient centrifugation and ampilifid. The cell suspension is prepared with the 3rd generation and bFGF/VitC, then finishing the carrier compound which is coincident with the articular cartilage full-thickness defect （diameter 4mm, depth 4mm） and embedding. The left articular genu defects are embedded with carrier compound as the experimental group （A）, and the right embedded without bFGF/VitC as the group （B）. The left articular genu defects of another 6 rabbits are given the implantation of calcium alginate exclusively as group （C）, and the right defects remain blank as control group （D）. Sacrificing the responding rabbits in the different time after operation（30d,60d,90d）, and paraffin imbedding with routine methods and preparing the microtome section. Then observing the recovery result with the index of HE, Masson, Saffrine O, EM, Immunity-histchemistry. Results The chondro-defects in group A, B and C are filling with hyaline cartilage-similar tissue and fibre tissue. Hyaline cartilage-similar tissue in group A is much more than group B, and it is the lest in group C. With the time extending, the defects in group A are mostly fell with hyaline cartilage-similar. The boundary between normal circum-articular cartilage and filling tissue becomes illegible and the connecting with subcartilage-bone is visible. There are many collagen fibre presented emission-like in the extracellular matrix. On the surface of defects in group B, however, there are still plenty of fibre tissue and the boundary remains distinctive. 3-5 chondrocytes possessing obvious lacunes stand in line in the filling tissue of group A, and much more than that in group B. The phenomenon above can be scarcely observed in group C. The number of vigorous chondrocytes-collagen typeⅡ （＋）, rough endoplasmic reticulum in cytoplasm dialating etc. In group A is more than that of group B. It is the lest in group C. Considerable fibra tissue can be seen in the defect of group D after 90 days. The integration between filling tissue and subcartilage-bone or normal circum-articular cartilage is un-ideal. Conclusion Adding bFGF and VitC has positive function in improving the repairing effect of the defect location when embedding the compound composed of auto-MSCs and calcium alginate to the defect of knee joint.