Department of Micro Orthopaedics, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
Exp Biol Med (Maywood). 2013 Jan;238(1):23-30. doi: 10.1258/ebm.2012.012223.
The aim of this work is to explore the feasibility and therapeutic effect of repairing rabbit articular cartilage defects using thermo-sensitive chitosan/poly (vinyl alcohol) composite hydrogel engineered Ad-hTGF-β1-transfected bone marrow mesenchymal stem cells. Rabbit's bone marrow stromal cells (BMSCs) were obtained and cultured in vitro and transfected with a well-constructed Ad-hTGF-β1 vector, the cartilage phenotype of the transfected cells was tested by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Twenty-four New Zealand white rabbits with articular cartilage defects were randomly divided into four groups: group A was treated with CS/PVA gel and transfected BMSCs; group B received CS/PVA gel and un-transfected BMSCs; group C was treated with CS/PVA gel alone and group D was the untreated control group. Experimental animals of each group were killed at 16 weeks after operation. General observation, Masson's trichrome staining and collagen II immunohistological staining of the specimens were performed to evaluate the repair effect. The Wakitani scoring method was used to evaluate the repair effect. RT-PCR and Western blot confirmed that the hTGF-β1 gene was expressed in BMSCs and triggered the expression of specific markers of cartilage differentiation such as aggrecan mRNA and Collagen II in BMSCs after transfection with Ad-hTGF-β1. Sixteen weeks after operation, the defects in group A had smooth and flat surfaces, and the defects appeared to have completely healed, exhibiting almost the same color and texture as the surrounding cartilage. Masson's trichrome staining showed that the cell arrangement and density of regenerated cartilage tissue in group A was not significantly different from that of normal cartilage tissue. The immunohistochemical staining of Col II showed a strong expression in group A and weak expression in group B, but no expression in groups C and D. According to the Wakitani score, the difference between experimental group A and all of the other groups was statistically significant (P < 0.01). To conclude, as a thermosensitive and injectable scaffold material, CS/PVA gel engineered with BMSCs transfected with hTGF-β1 can effectively repair rabbit articular cartilage defects.
本研究旨在探讨温敏性壳聚糖/聚乙烯醇复合水凝胶复合转染人转化生长因子-β1（hTGF-β1）基因的骨髓间充质干细胞（BMSCs）修复兔关节软骨缺损的可行性及疗效。体外分离培养兔骨髓基质细胞（BMSCs），构建高效表达载体 Ad-hTGF-β1，转染 BMSCs，采用逆转录聚合酶链反应（RT-PCR）和 Western blot 检测转染后细胞软骨表型。将 24 只新西兰大白兔关节软骨缺损模型随机分为 4 组：A 组为壳聚糖/聚乙烯醇复合水凝胶联合转染 BMSCs 组；B 组为壳聚糖/聚乙烯醇复合水凝胶联合未转染 BMSCs 组；C 组为单纯壳聚糖/聚乙烯醇复合水凝胶组；D 组为空白对照组。术后 16 周处死实验动物，大体观察、Masson 三色染色及Ⅱ型胶原免疫组织化学染色观察标本修复效果，采用 Wakitani 评分标准评价修复效果。RT-PCR 和 Western blot 证实，Ad-hTGF-β1 转染 BMSCs 后，hTGF-β1 基因在 BMSCs 中表达，并触发 BMSCs 中软骨分化特异性标志物，如聚集蛋白聚糖 mRNA 和 Collagen II 的表达。术后 16 周时，A 组的软骨缺损部位表面光滑、平整，缺损几乎完全愈合，颜色和质地与周围软骨相似。Masson 三色染色显示，A 组再生软骨组织的细胞排列和密度与正常软骨组织无明显差异。Col II 免疫组化染色显示 A 组表达较强，B 组表达较弱，C、D 组无表达。根据 Wakitani 评分，实验组 A 与其他组之间的差异均有统计学意义（P < 0.01）。结论：作为一种温敏性、可注射的支架材料，复合转染 hTGF-β1 的 BMSCs 的壳聚糖/聚乙烯醇复合水凝胶可有效修复兔关节软骨缺损。
