微骨折结合未培养的兔自体骨髓单个核细胞修复全层关节软骨缺损的实验观察

Experimental research on repairing full-thickness articular cartilage defects by transplantation of autologous uncultured bone-marrow-derived mononuclear cells in combination with micro-fracture

目的探讨微骨折结合未培养的自体骨髓单个核细胞对关节软骨缺损的修复效果。方法新西兰大白兔40只，随机分为A、B、C、D组，每组各10只。于兔右膝股骨滑车中部建立软骨缺损模型。A与C组均自兔左股骨远端髓腔抽取5ml骨髓，分离提取自体骨髓单个核细胞。A组：软骨缺损区造成微骨折后植入未培养的自体骨髓单个核细胞一自体纤维蛋白凝胶复合体；B组：软骨缺损区造成微骨折后植入自体纤维蛋白凝胶；C组：软骨缺损区植入未培养的自体骨髓单个核细胞一自体纤维蛋白凝胶复合体；D组：软骨缺损区植入自体纤维蛋白凝胶。术后8、12周各处死5只实验动物，取右侧股骨滑车部。行大体观察，然后对标本脱钙后行石蜡包埋，常规切片，在光镜下观察包括HE染色和甲苯胺蓝染色，根据wakitani评分标准评价标本的修复效果并进行统计学分析，以及Ⅱ型胶原的免疫检测。结果术后8、12周大体观察与织学观察结果，A组关节软骨缺损的再生修复均明显优于B、C与D组，差异具有统计学意义【（1．0±0．7）分比（8．2±0．8）分，P〈0．05]，修复组织为透明样软骨。结论微骨折和未培养的自体BM—MNCs复合纤维凝胶都有促进软骨修复的作用，两者结合起来对软骨再生的促进作用更明显。

Objective To examine the feasibility of autologous uncultured bone-marrow-derived mononuclear cells （BM-MNCs） in combination with microfracture in a full-thickness articular cartilage defect model so as to provide experimental rationales for clinical applications. Methods A total of 40 rabbits were divided randomly into groups A, B, C and D （n = 10 each）. In groups A and C, 5 ml marrow samples were harvested from left femur and then autologous BM-MNCs isolated. The full-thickness articular cartilage defects were made on femoral intercondylar fossa in fight knees of rabbits. Group A ： micro-fracture was made on cartilage defect and then autologous uncultured BM-MNCs-autologous fibrin gel complex implanted; Group B：the same micro-fracture was made on cartilage defect and autologous fibrin gel implanted; Group C：the cartilage defect was implanted with autologous uncultured BM-MNCs-autologous fibrin gel complex; Group D：the cartilage defect was implanted with autologous fibrin gel. Five rabbits were sacrificed at Weeks 8 and 12 post-transplantation in each group. And the reparative tissue samples evaluated grossly, histologically and immunohistochemically were graded according to the gross and histological scales. Results The statistical analyses of histological gradings at Weeks 8 and 12 showed that group A was significantly better than groups B, C and D （P 〈0. 05） , groups B and C were better than group D （P 〈0. 05） and each group at Week 12 was better than itself at Week 6 （ P 〈0. 05 ）. Conclusion Both of micro-fracture and transplantation of uncultured autologous BM-MNCs plus autologous fiber gel can promote the repair of cartilage defects. The combined use of micro-fracture and autologous uncultured BM-MNCs promotes the regeneration of articular cartilage so that it may provide theoretical rationales for clinical applications.