透明质酸钠结合尿源性干细胞修复创伤性软骨缺损的实验研究

Sodium hyaluronate combined with human urine-derived stem cells in repairing traumatic cartilage defect：an experimental study

摘要:

目的：透明质酸钠复合尿源性干细胞复合溶液体系对比格犬膝关节软骨缺损动物模型进行关节腔注射治疗，观察其关节软骨修复效果。方法：应用离心贴壁法从健康成人尿液中分离提取出尿源性干细胞，进行原代及传代细胞培养，并行表面标志物及多向分化潜能鉴定。选用透明质酸钠作为细胞载体材料，将其与第4代尿源性干细胞混合制成细胞混悬液。将12只比格犬建立膝关节股骨内、外髁直径5 mm的软骨全层缺损动物模型，造模成功3周后随机平均分为3组，分别为透明质酸钠细胞悬液治疗组、透明质酸钠治疗组和生理盐水空白对照组，按分组相应行膝关节注射。注射后12周处死取材，取大体标本观察软骨缺损处形态变化，标本切片后进行HE染色和甲苯胺蓝染色评价膝关节软骨修复效果。结果：治疗后12周，透明质酸钠细胞悬液治疗组的膝关节软骨缺损修复效果明显优于透明质酸钠治疗组及生理盐水空白对照组，大体标本可见透明质酸钠细胞悬液组软骨缺损区基本被软骨样组织填满，具有光滑的表面。切片HE染色和甲苯胺蓝染色显示相比较于其他两组，其缺损部位软骨细胞呈柱状排列，透明样软骨组织覆盖良好，与相邻边缘软骨组织完全整合，但软骨厚度较周围正常软骨组织稍薄。结论：透明质酸钠复合尿源性干细胞溶液体系对关节软骨缺损具有明显的修复作用，为一种新的软骨缺损治疗方案提供实验学依据。

Abstract:

Objective：To investigate the effect of a complex solution system of sodium hyaluronate and human urine-derived stem cells （hUSCs） in repairing articular cartilage by intra-articular injection in beagles with knee cartilage defect. Methods：The centrifugation and adherence method was used to isolate hUSCs from fresh urine of healthy adults，and primary cell culture and subculture were performed. Surface markers and multipotential differentiation were identified. Sodium hyaluronate was used as a cell carrier and mixed with the fourth-generation hUSCs to prepare cell suspension. A total of 12 beagles were used to establish a model of full-thick－ness cartilage defect with a diameter of 5 mm at the medial and lateral epicondyles of the femur in the knee joint. At 3 weeks after the model being established，the beagles were equally and randomly divided into sodium hyaluronate cell suspension treatment group，sodium hyaluronate treatment group，and normal saline blank control group，and injection into the knee joint was performed accord－ingly. The beagles were sacrificed at 12 weeks after injection，and related samples were collected. Gross specimens were used to ob－serve the morphological changes of cartilage defect，and HE staining and toluidine blue staining were performed for sections to evaluate the repair of knee cartilage. Results：At 12 weeks after treatment，the sodium hyaluronate cell suspension treatment group had a sig－nificantly better improvement in knee cartilage defect than the sodium hyaluronate treatment group and the normal saline blank control group，and gross evaluation showed that in the sodium hyaluronate cell suspension treatment group，the area of cartilage defect was almost filled with newly formed cartilage-like tissue and had a smooth surface. HE staining and toluidine blue staining of sections showed that compared with the sodium hyaluronate treatment group and the normal saline blank control group，the sodium hyaluronate cell suspension treatment group had the columnar arrangement of cartilage cells and good coverage of hyaline cartilage tissue in the area of defect，which was fully inte－grated with the adjacent cartilage tissue and was slightly thinner than the adjacent normal cartilage tissue. Conclusion：The complex solution system of sodium hyaluronate and hUSCs has a marked effect in repairing articular cartilage defect，which provides an ex－perimental basis for a new treatment regimen for cartilage defect.