目的:研究骨软骨镶嵌成形术与联合基因增强组织工程方法治疗急性骨软骨缺损,并观察在不同比例下的组织修复情况,以期发现两者间的最佳组合.方法:对携带hTGF-β1的重组腺病毒转染BMSCs(hTGF-β1转染组),与采用Adv-βgal转染BMSCs(Adv-βgal转染组)及未转染BMSCs(未转染空白对照组)进行Western Blot检测hTGF-β1、Ⅱ型胶原及Aggrecan表达.雄性6月龄崇明山羊18只,体重22～25 kg,取自体骨髓进行BMSCs分离及培养,传至第3代.每只动物双膝股骨内髁进行实验,分为AR、AL、BR、BL、CR、CL 6组.AR为骨软骨移植覆盖面积为44.44%单纯组,AL为44.44%联合组,BR为33.33%单纯组,BL为33.33%联合组,CR为22.22%单纯组,CL为22.22%联合组.于双膝股骨内髁负重区采用骨钻制备直径为9 mm,深为3 mm的骨软骨缺损后,单纯组采用骨软骨镶嵌成形术的自体骨软骨柱修复,联合组同时将转染hTGF-β1的BMSCs藻酸钠混合液注入空隙,加入氯化钙产生凝胶.术后24周取材,行大体及组织学观察,并参照O'Driscoll,Keeley and Salter组织形态学评分标准进行评分,行免疫组织化学及透射电镜观察.结果:hTGF-β1转染组细胞hTGF-β1、Ⅱ型胶原及Aggrecan表达均强于Adv-βgal转染组及未转染空白对照组.大体观察,组织学染色以及透射电镜检查显示各组的缺损有不同程度的修复.33.33%联合组、44.44%单纯组、44.44%联合组的整体修复效果差别无统计学意义,33.33%单纯组、22.22%单纯组、22.22%联合组的整体修复效果差于前3组.结论:骨软骨镶嵌成形术联合基因增强组织工程的方法可以有效修复骨软骨缺损.随着自体骨软骨移植覆盖缺损面积的减少,骨软骨镶嵌成形术联合基因增强组织工程方法的修复优越性可以得到更好的体现.%Objective:To investigate the optimum proportion of Mosaicplasty and genes-enhanced tissue engineering for the repair of acute osteochondral defects. Methods: Western blot test was conducted to detect the expression of Htgf-betal, Col II and Aggrecan in 3 groups,including Htgf-betal, transduction group, Adv-betagal transduction group and control group without transduction. Eighteen 6-month-old Shanghai male goats (weight :22 to 25 kg) were used. BMSCs were isolated from the autologous bone marrow,and were subcultured to get the cells at passage 3. Thirty-six medial femoral condyles were used and divided into 6 groups named AR,AL,BR,BL,CR,and CL. Acute cylindrical defects (9 mm in diameter and 3 mm in depth)were created in the weight bearing area of the medial femoral condyle of hind limbs. In the single group, the autologous osteochondral mosaicplasty was performed to repair the defect;in the combination group,besides the mosaicplasty,the dead space between the cylindrical grafts and the host cartilage were injected with the suspension of Htgf-betal ,gene enhanced autogenous BMSCs in sodium alginate.and CaCl2 was dropped into it to form calcium alginate gels. The autologous osteochondral transplantation cover rates of group AR was 44.44% single group, AL was 44.44% combination group, BR was 33.33% single group,BL was 33.33% combination group,CR was 22.22% single group,and CL was 22.22% combination group. The goats were killed 24 weeks after operation to receive gross and histology observation,which was evaluated by the histological grading scale of O'Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were also performed. Results: Western blot test showed the expression of the Htgf-betal ,Col II and the Aggrecan in the Htgf-betal transduction group were significantly higher than that of the Adv-betagal transduction and the blank control groups. The gross and histology observation revealed that each defects of six groups had different degrees of repairing. There was no significantly difference among the BL, AR, and AL groups. But the scores of the other three groups (BR, CR, and CL) were significantly poorer than the former three groups. Conclusion: Mosaicplasty associated with genes enhanced tissue engineering could repair the osteochondral defects effectively. With the autologous osteochondral transplantation coverage reducing,the advantage of the combinationcould have a better representation.
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