人异体骨软骨移植物保存方法研究

Six kinds of preservation of human osteochondral allografts:a comparative study

[目的]探讨六种保存方法对人关节软骨组织结构和细胞活性的影响,寻求一种保存效果较好的方法,为临床提供一种有活性的异体骨软骨移植物。[方法]自捐献新鲜尸体膝关节利用专用手术器械获取4.5 mm×4.5 mm大小的人骨软骨块,分别采用梯度降温法、Co60射线照射+梯度降温法、玻璃化法、连续降温法、直接液氮法和酒精浸泡法对软骨块进行保存处理,分别于保存第8、15、30、60 d时,采用蕃红-O组织染色、扫描电镜、软骨细胞胎盼蓝染色、MTT法等,观察并比较以上6种方法保存后关节软骨细胞存活率及其代谢功能变化。[结果]除了酒精保存方法外,其余保存方法随着时间延长软骨组织的细胞成活率和细胞代谢活性逐渐降低;保存60 d时,采用玻璃化法保存软骨组织的细胞存活率为62.47%,软骨基质成分丢失较少,胶原纤维断裂不明显;采用慢速梯度降温法保存软骨组织的细胞存活率为59.75%,软骨基质成分丢失较多,胶原纤维断裂明显;其他保存方法软骨细胞存活率不足40%,软骨基质成分大量丢失,胶原纤维杂乱。[结论]六种保存方法中,玻璃化保存法能够较好的保存关节软骨,活性最好,其次是梯度降温保存法,两者均具有一定临床价值。Co60射线照射对软骨细胞有一定的损伤作用;酒精浸泡不能保存软骨细胞活性。

[ Objective] To evaluate the effect of six kinds of osteochondral allograft storage in vitro. L MethodsJ The col- umned osteochondral allografts was harvested from the articular surface of fresh human cadaver femoral condyle, which was 4. 5 millimeter in diameter and 4. 5 millimeter in length. The allografts were preserved by cooling cryopreservation, step cooling cro preservation with cobalt 60 radiation, continued cooling eryoperservation, vitrification cropreservation, liquid nitrogen storage, and alcohol immersing. Their effects were compared with fresh human osteochondral allografts. The viability and metabolism ofarticular cartilage allografts were detected with MTT method, histology, inmmunohistology and ultrostmcture obeservaiton. [ Re- stilts ] Except the alcohol immersing, the viability and metabolism of chondroycyte was gradually decreased with time. At sixty days of allograft storage, the chondrocyte viability was 62. 5 percent with slight proteoglycan loss in vitrification cryopreservation, fifty -nine point five percent with more proteoglycan loss and collagen fiber disorder in step cooling cropreservation, and less than 40 percent in other preservation group with matrix dramatic disorder. [ Conclusion] The best preservation of osteochondral allografts was vitrification cryopreservation. The next best was step cooling cryopreservation. The cobalt 60 radiation had slight injury to articular cartilage. The alcohol cold not preserve the viability of articular cartilage.