单纯骨软骨镶嵌成形术与联合组织工程方法治疗急性骨软骨缺损的对比研究

COMPARATIVE RESEARCH ON REPAIRING ACUTE OSTEOCHONDRAL DEFECT BY MOSAICPLASTY AND THE COMBINATION OF MOSAICPLASTY WITH TISSUE ENGINEERING METHODS

目的对比研究单纯骨软骨镶嵌成形术与联合组织工程方法及联合未转染基因的BMSCs-藻酸钙修复急性骨软骨缺损的效果。方法对携带hTGF-β1的重组腺病毒转染BMSCs(hTGF-β1转染组)、采用携带Lac-Z报告基因的重组腺病毒(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、ColⅡ及Aggrecan表达。将雄性6月龄崇明山羊18只,体重22～25kg,随机分为A、B、C组(n=6)。取B、C组自体骨髓进行BMSCs分离、培养,传至第3代。B组细胞行hTGF-β1重组腺病毒转染。3组动物于双后肢股骨内髁负重区采用骨钻制备直径5mm、长3mm的缺损,A组采用自体骨软骨柱修复;B组修复材料同A组,并同时将5mL转染hTGF-β1的BMSCs藻酸钠混合液注入空隙,加入CaCl2产生凝胶;C组:修复方法同B组,采用未转染hTGF-β1的自体BMSCs藻酸钠混合液。术后观察山羊一般情况,于术后12周及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;术后24周行免疫组织化学及透射电镜观察。结果转染后5d,hTGF-β1转染组细胞hTGF-β1、ColⅡ及Aggrecan表达均显著强于Adv-βgal转染组及未转染空白对照组。术后动物均存活至实验完成,伤口Ⅰ期愈合。大体观察B组修复组织边界模糊,移植修复区域表面光滑;A、C组软骨间空隙有裂隙存在。组织学观察A组软骨间空隙区纤维软骨样组织修复、纤维组织填充或相邻软骨增生;B组修复软骨细胞排列规律,交界区整合良好;C组软骨间的空隙区见纤维软骨样组织,存在裂隙。各时间点B组组织学评分均高于A、C组,C组高于A组,差异均有统计学意义(P<0.05);B组12周与24周差异有统计学意义(P<0.05)。术后24周免疫组织化学示B组软骨间修复组织染色以软骨细胞和陷窝周围明显,A、C组呈淡染。透射电镜观察示B组修复组织可见典型软骨细胞,A、C组见平行或交错排列的胶原纤维束。结论骨软骨镶嵌成形术联合组织工程方法可解决单纯骨软骨镶嵌成形术残留缺损愈合不良及软骨间整合不良的问题。

Objective To compare the effect of mosaicplasty, mosaicplasty with gene enhanced tissue engineering and mosaicplasty with the gels of non-gene transduced BMSCs in alginate on the treatment of acute osteochondral defects. Methods Western blot test was conducted to detect the expression of hTGF-β1, Col II and Aggrecan in 3 groups, namely hTGF-β1 transduction group, Adv-βgal transduction group and blank control group without transduction. Eighteen 6-month-old Shanghai mascul ine goats weighing 22-25 kg were randomized into groups A, B and C （n=6）. BMSCs were isolated from the autologous bone marrow of groups B and C, and were subcultured to get the cells at passage 3. In group B, the BMSCs were transduced with hTGF-β1. For the animals of 3 groups, acute cyl indrical defects 5 mm in diameter and 3 mm in depth were created in the weight bearing area of the medial femoral condyle of hind l imbs. In group A, the autologous osteochondral mosaicplasty was performed to repair the defect; in group B, besides the mosaicplasty, the dead space between the cyl indrical grafts and the host cartilage were injected with the suspension of hTGF-β1 gene transduced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels; in group C, the method was the same as the group B, but the BMSCs were not transduced. General condition of the goats after operation was observed, the goats were killed 12 and 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 performed 24 weeks after operation. Results Western blot test showed the expression of the hTGF-β1, Col II and the Aggrecan in the hTGF-β1 transduction group were significantly higher than that of the Adv-βgal transduction and the blank control groups. All the goats survived until the end of experiment and all the wounds healed by first intention. Gross observation revealed the boundaries of the reparative tissue in group B were indistinct, with smooth and continuous surfaces of the whole repaired area; while there were gaps in the cartilage spaces of groups A and C. Histology observation showed the dead space between the cyl indrical grafts in group A had fibrocartilage-l ike repair tissue, fill ing of fibrous tissue or overgrowth of the adjacent cartilage; the chondrocytes in group B had regular arrangements, with favorable integrations; while the dead space between the cyl indrical grafts in group C had fibrocartilage-l ike repair tissue, with the existence of gaps. The histology scores of group B at different time points were significantly higher than that of groups A and C, and group C was better than group A （P 〈 0.05）; for group B, significant difference was detected between 12 weeks and 24 weeks in the histology score （P 〈 0.05）. Immunohistochemistry staining for Col II 24 weeks after operation showed the chondrocytes and lacuna of the reparative tissue in group B was obviously stained, while groups A and C presented l ight staining. TEM observation showed there were typical chondrocytes in the reparative tissue in group B, while parallel or interlaced arrangement collagen fiber existed in groups A and C. Conclusion Combining mosaicplasty with tissue engineering methods can solve the problem caused by single use of mosaicplasty, including the poor concrescence of the remnant defect and poor integration with host cartilages.