人转化生长因子β1基因转染骨髓基质干细胞复合藻酸钙修复骨软骨缺损

Repair of osteochondral defects using human transforming growth factor beta 1 gene transfected bone marrow stem cells combined with calcium alginate

目的:拟利用人转化生长因子β1基因转染骨髓基质干细胞,再与可降解藻酸钙材料复合构建具有较好生物学活性的组织工程化人工骨软骨,以期获得良好的软骨间整合。方法:雄性12个月龄的崇明山羊18只,由上海交通大学附属第九人民医院动物实验中心提供。携带人转化生长因子β1基因的重组腺病毒由上海交通大学附属第九人民医院骨科研究室提供。制备藻酸钙凝胶的氯化钙、藻酸钠为Gibco公司产品。从羊髂嵴抽取8mL骨髓,贴壁法分离培养骨髓基质干细胞,传至第3代以携带人转化生长因子β1基因的重组腺病毒转染过夜。18只山羊均于双膝股骨内髁负重区建立骨软骨缺损模型,造模后分为3组,6只/组:转染细胞-藻酸钙组将转染人转化生长因子β1基因的骨髓基质干细胞悬于1.2%藻酸钠中,调整细胞密度为5×1010L-1,吸取5mL注入骨软骨缺损处,随后缓慢滴入过量的2.5%氯化钙,使氯化钙与藻酸钠发生交联反应形成藻酸钙凝胶。复合对照组同法注入未转染人转化生长因子β1基因的骨髓基质干细胞-藻酸钙凝胶,模型组不给予任何干预。转染后Westernblot法检测细胞外源基因的表达。移植后组织形态学检查及缺损评分。结果:人转化生长因子β1基因转染后,骨髓基质干细胞表达人转化生长因子β1,并分泌细胞外基质Ⅱ型胶原及Aggrecan。移植后24周,转染细胞-藻酸钙组缺损获得类透明样软骨修复,复合对照组及模型组均为纤维组织或纤维样软骨修复。与模型组比较,移植后第12,24周复合对照组、转染细胞-藻酸钙组骨软骨缺损组织形态学评分均明显升高(P<0.05),且转染细胞-藻酸钙组升高幅度显著大于复合对照组(P<0.05);转染细胞-藻酸钙组缺损评分随着修复时间的延长而明显升高(P<0.05)。结论:实验成功将组织工程学与分子生物学有机结合,经基因修饰的骨髓基质干细胞可使人转化生长因子β1持续高效发挥作用,并逐步转化为成熟的软骨细胞且分泌软骨基质,与藻酸钙复合修复骨软骨缺损效果满意。

AIM： To transduce the human transforming growth factor- β1 （bTGF- β1） gene to the bone marrow stem calls （BMSCS） and then combine with the degradable alginate to compose the tissue engineering artificial cartilage with biological activity, expecting to get a better integration of the cartilages. METHODS： Eighteen masculine goats of 12 months old were provided by the animal experiment center of the Ninth Hospital Affiliated to Shanghai Jiao Tong University. The recombinant adenoviroses carrying hTGF- β1 were provided by the Orthopaedic Laboratory of the Ninth Hospital Affiliated to Shanghai Jiao Tong University. Calcium chloride and sodium alginate were the products of Gibeo Company. Eight milliliters of bone marrow were obtained from the iliac crest of the goats. The adherent method was used to separate and culture the BMSCs. The third passage cells were transduced with bTGF- β1 and incubated ovemighL An acute osteocbondral defect model was created in the weight-bearing area of the medial femoral condyle on the both knees of eighteen goats. The goats were randomized into three groups, with six in each group. In the group of transduced cells with calcium alginate, the hTGF- β1 gene transduced BMSCs were suspended in the 1.2% sodium alginate. The cell density was adjusted to 5×10^10 cells L^-1. Five milliliters of the suspension were injected into the osteochondral defects, and excessive 2.5% calcium chloride was dropped to make the calcium chloride and sodium alginate cross-link to form calcium alginate gels. In the compound control group, the same method was used to inject the gels of non-gene transduced BMSCs into the calcium alginate gels. In the model group, none of the intervention was given. Western blot was employed to detect the expression of the exogenous gene. The reparative tissues were determined for the morphology observation and the defect scoring. RESULTS： After the transduction of the hTGF- β1 gene, the BMSCs expressed hTGF- β1, synthesized the extracellular matrix of type U collagen and excreted aggrecan. At 24 weeks after the transplantation, the group of transduced cells with calcium alginate got a hyaline cartilage-like restoration. The compound control group and the model group got a fibrous tissue or fibrocartilage-like repair. Comparing the reparative tissues of 12 weeks and 24 weeks, the scores of the group of transduced cells with calcium alginate and the compound control group were significantly higher than those in the model group （P 〈 0.05）. The scores of the reparative tissue in the group of transduced cells with calcium alginate were significantly higher than those in the compound control group （P 〈 0.05）. The scores in the group of transduced cells were significantly increased as the prolonging duration （P 〈 0.05）. CONCLUSION： The experiments combine the tissue engineering and molecular biology with a success. The gene-transduced BMSCs can make the hTGF- β1 bring into effect continuously and effectively, and gradually transform into mature chondrocytes to excrete the matrix. The hTGF- β1 gene transduced BMSCs combined with calcium alginate could repair the osteochondral defects with satisfactory results.