自体骨负载软骨细胞修复兔关节骨软骨缺损

Autologous bone loaded chondrocytes for repairing osteochondral defects in rabbitsAbstract

目的:观察原代单层培养的自体软骨细胞包埋于骨与骨膜之间修复关节骨软骨缺损的可行性,探索一种关节内骨软骨缺损或单纯软骨缺损的修复方法。方法:实验于2003-05/2006-10在安徽医科大学完成。实验分组:取4周龄健康新西兰大白兔27只,体质量460～520g,随机摸球法分为自体骨+软骨细胞+骨膜移植组、自体骨+骨膜移植组、骨膜移植组,每组9只。实验方法:取自体骨+软骨细胞+骨膜移植组兔子膝关节的软骨消化成软骨细胞并在体外原代单层培养近2周;每只兔子的双侧膝关节股骨滑车部造成3mm×4mm×4mm的骨软骨缺损,在自体骨+软骨细胞+骨膜移植组各取两块3mm×4mm的髂骨洗去血细胞,充填软骨下骨缺损,松质面向关节腔,骨膜覆盖,软骨细胞与纤维蛋白凝胶混合物注入腔隙内;自体骨+骨膜移植组不注射软骨细胞;骨膜移植组用骨膜修复。实验评估:分别在4,8,12周进行大体和苏木精-伊红染色、蕃红花”O“染色、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学进行形态学以及透射电镜超微结构观察软骨的生长情况,并按照O’Driscoll,Keeley and Salter评分标准,对各组进行半定量评分,组间比较采用q检验。结果:纳入新西兰大白兔27只,均进入结果分析。①3组软骨的生长情况:自体骨+软骨细胞+骨膜移植组软骨缺损被修复,外观与周围正常软骨没有明显区别,组织学检测为透明软骨或类透明软骨,软骨下骨修复完善;自体骨+骨膜移植组缺损表面被纤维软骨修复,12周出现明显退变;骨膜移植组缺损被纤维组织及骨样组织修复,对应的髌骨软骨出现磨损的创痕。②3组不同时间点组织学评分:自体骨+软骨细胞+骨膜移植组、自体骨+骨膜移植组、骨膜移植组4周分别是(10.83±4.40),(9.33±3.93),(5.83±1.94)分;8周:(15.17±4.71),(10.83±3.06),(6.67±1.51)分;12周:(15.83±4.58),(8.17±2.79),(3.83±0.75)分。在第8,12周,3组之间差异有显著性意义(P<0.01)。结论:自体骨复合纤维蛋白凝胶负载软骨细胞修复关节骨软骨缺损,可在重建软骨下骨的同时形成透明或类透明软骨表面。

AIM： To observe the possibility of primary monolayer culture chondrocytes embedded in autologous bone and periosteum for osteochondral defects, and to explore, a repair method of osteochondral defects or simple chondral defects. METHODS： The experiment was performed at the Anhui Medical University from May 2003 to October 2006. Grouping： A total of 27 4-week-old healthy New Zealand white rabbits with the body mass of 460-520 g were randomly divided into autologous bone plus chondrocytes plus periosteum transplantation group, autologous bone plus periosteum transplantation group and periosteum transplantation group, with 9 in each group. Experimental methods：Articular chondrocytes from the knee joints of autologous bone plus chondrocytes plus periosteum transplantation group were digested, and cultured in vitro for 2 weeks. 3 mm×4 mm×4 mm osteochondral defect was established at femoral trochlea of knee joint of each rabbit. Two 3 mm×4 mm iliac bones were obtained in autologous bone plus chondrocytes plus periosteum transplantation group and blood cells were washed-out. Osteochondral defects were filled; spongiosa faced to articular cavity; covered by periosteum; compounds of chondrocytes and brinolase gel were injected in lacouna. The rabbits in the autologous bone plus periosteum transplantation group did not receive chondrocytes. The rabbits in the periosteum transplantation group were treated with periosteum repair. Experimental evaluation： At weeks 4, 8 and 12 general and haematoxylin-eosin （HE） staining, Safranin-O, toluidine blue staining, Ⅱ type collagen immunohistochemical method were performed and transmission electron microscope were used to observed the cartilaginous growth. According to O＇Driscoll,Keeley and Salter, semi-quantitative score was conducted and q, test was done among groups. RESULTS： Totally 27 New Zealand white rabbits were involved in the result analysis. ①Cartilaginous growth in the 3 groups： osteochondral defects were repaired in the autologous bone plus chondrocytes plus periosteum transplantation group, and there was no significant difference with the normal cartilages. Histology showed that it was hyaline cartilage or hyaline-like cartilage and subchondral bone was repaired consummately. The defects were repaired by fibrocartilage and marked cataplasia appeared at week 12 in the autologous bone plus periosteum transplantation group. The defects were repaired by fibrous tissue and osteoid tissue and the wornout scar appeared in corresponding whirbone in the pedosteum transplantation group. ②Histology score at different time points in the 3 groups： It was （10.83±4.40）, （9.33± 3.93）, （5.83±1.94） points;at week 8：（15.17±4.71 ）, （10.83±3.06）, （6.67±1.51） points;at week 12： （15.83±4.58）, （8.17± 2.79）,（3.83±0.75） points in the autologous bone plus chondrocytes plus periosteum transplantation group, autologous bone plus periosteum transplantation group and periosteum transplantation group, respectively. There were significant differences among the 3 groups at weeks 8 and 12, （P〈 0.01 ）. CONCLUSION： Autologous bone combined with fibrin gel loading chondrocytes in the repair of osteochondral defects can form hyaline or hyaline-like cartilage when reestablishing subchondral bone.