摘要
目的:观察聚己内酯-聚乙二醇共聚物与兔关节软骨细胞在体外复合培养以及体内植入后异位成软骨情况。方法:实验于2006-01/06在北京积水潭医院骨科研究所完成。①实验材料:3周龄新西兰白兔1只,SPF级4～6周龄BALB/C-nu/nu裸鼠7只,雌雄不限,聚己内酯-聚乙二醇嵌段共聚物(由清华大学机械系提供)。②实验干预:取兔双膝及股骨头关节软骨,通过酶消化法获取软骨细胞,进行原代和传代培养;将聚己内酯-聚乙二醇载体材料切成小块,乙醇浸泡并三蒸水冲洗后晾干,60Co辐照灭菌;收集第3代软骨细胞,以3×10^(10)L-1浓度接种于6片材料上,孵箱中培养1周。③实验分组:将7只裸鼠随机分为材料细胞复合组5只,背部植入培养1周的材料细胞复合物,单纯材料组2只,植入单纯材料。④实验评估:对第3代软骨细胞通过甲苯胺蓝染色进行鉴定;通过倒置显微镜和扫描电镜观察软骨细胞在载体材料上的生长增殖情况;材料种植于体内6周后取材,进行大体观察和组织学观察。结果:①关节软骨细胞形态学观察和鉴定:1～3代软骨细胞形态基本一致,大多呈多角形,少数为梭形,核圆且清晰可见,3代以后软骨细胞形态变宽变扁。甲苯胺蓝染色证实第3代软骨细胞胞浆内呈蓝紫色异染颗粒。②软骨细胞在载体材料上的生长增殖情况:细胞接种于材料1d后,倒置显微镜下观察,可见细胞以单个、多层或团状形式吸附在材料表面。电镜观察:细胞形状为圆球形,贴附在材料表面。③体内植入物大体和组织学观察:体内植入6周后,材料细胞复合组材料孔隙间形成透明软骨组织,单纯材料组材料孔隙间为纤维组织充填。结论:聚己内酯-聚乙二醇共聚物可以作为软骨细胞生长的支架载体,可以异位构建组织工程化软骨,材料的降解性还需要继续观察。
AIM： To observe the growth of rabbit chondrocytes incubated in the polycaprolactone-polyethylene glycol block copolymer （PCE） in vitro, and investigate the ectopic chondrogenesis by implantation of chodrocyte-PCE composite. METHODS： The research had been completed in the Institute of Orthopaedics, Beijing Jishuitan Hospital from January to June in 2006. ①Materials： one New Zealand rabbit of 3 weeks old; seven BALB/C-nu/nu nude mice of SPF grade, either gender, aging 4-6 weeks; PCE was offered by Department of Mechanics, Tsinghua University②The chondrocytes were obtained by enzyme digestion from articular cartilage of rabbit＇s knees and femoral heads, and then the primary cultivation and serial subcultivation were done; The PCE carrier was chipped into pieces, soaked in alcohol, flushed with water, and then sterilized with ^60Co; Chondrocytes at the third passage were collected and seeded into six pieces of carder, then cultivated for one week in the incubator. ③Seven athymic mice were separated into two groups at random. Five of them in experiment group, the composites of chondrocyte-carder were planted into their backs; two of them in control group, the pure carriers were planted into their backs. ④The chondrocytes at the third passage were verified by toluidine blue dye. The growth and proliferation of chondrocytes in the carriers were observed by inverted microscope and scanning electron microscope. Six weeks after implantation, the gross and histological observations were performed to evaluate the turnover of the chondrocyte-carriers and pure carders. RESULTS： ①Morphology of chondrocytes： The appearance of the chondrocytes at 1-3 passages was the same, most of them were polygon and minor of them were fusiform. The nuclear was round and clear, after the third generation, they were broadened and flattened. The result of toluidine blue dye confirmed that the intracytoplasm of chondrocytes of the third generation was amethyst metachromatic granule②The growth and proliferation of chondrocytes in the carriers： One day after seeding into the carrier, the chondrocytes under inverted microscope were sticking on the carrier surface in single, multiplayer, or group. Those under electron microscope were round shaped and sticking on the surface of the carrier. ③Gross and histological observation of implants： Six weeks after implantation, hyaline cartilage formed in the porus of carrier in experiment group, and in control group, the porus of carder were filled with fibrous tissue. CONCLUSION： PCE can act as the carrier of chondrocytes, construct tissue-engineered cartilage in ectopia, its degradation performance needs future observation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第26期5066-5069,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
第二炮兵总医院青年科研基金(715-009-0160)~~
