摘要
目的观察异种异体脱细胞软骨支架材料（ACM）复合同种异体兔骨髓间充质干细胞（rBMSCs）修复兔股骨内髁关节软骨缺损的效果。方法（1）密度梯度离心和差速贴壁法获得原代兔BMSCs，选择第3代BMSCs作为种子细胞；（2）利用冷冻干燥、胰酶消化和化学去垢剂等方法制备脱细胞软骨支架材料；（3）3个月龄新西兰兔股骨内髁制备直径4mm，深3mm关节软骨缺损模型，24只新西兰兔以2个时间段随机分为5组，ⅠACM—BMSCs组：第3代BMSCs1×10^6个／ml与ACM于37℃5％CO2饱和湿度复合48h；ⅡACM组；Ⅲ空白对照组。（4）移植6、12周后大体及组织学观察，免疫组织化学染色观察修复组织Ⅱ型胶原，Wakitani评分评估修复效果。结果（1）大体观察及组织学观察：6和12周Ⅰ组再生组织与正常关节软骨面平齐，修复部位表面较平整，界限模糊，接近正常软骨。Ⅱ组修复组织表面不平整并有明显下陷，修复组织全层可见成纤维样细胞，深层可见极少数透明软骨样细胞。Ⅲ组未见明显修复，肉芽组织形成伴成纤维样细胞增生；（2）Wakitani组织学评分可见在不同的时间段Ⅰ组和Ⅱ组均低于Ⅲ组，差异有统计学意义（P〈0．05），Ⅰ组和Ⅱ组间组织学评分差异无统计学意义（P〉0．05）。（3）免疫组织化学：ACM—BMSCs组修复组织的细胞为软骨样细胞，可见柱状排列，周围软骨基质Ⅱ型胶原染色阳性。结论以ACM为支架材料，同种异体BMSCs为种子细胞制备的组织工程化软骨对兔股骨内髁关节软骨缺损有修复作用，形成的新生软骨为透明软骨样组织。
Objective To investigate the feasibility of repairing the whole layer cartilage defects of medial femoral condyles of adult rabbits with the acellular cartilage material （ACM）/boue marrow stem cells （BMSCs）. Methods （ 1 ） BMSCs of New Zealand rabbit were obtained, and purified by gradient centrifuge and adhesion culture in vitro. The passage 3 BMSCs were used for the seeding cells of cartilage tissue engineering. （2） The acellular pig articular cartilage was prepared by lyophilization, trypsinization and chemical subtraction. （3） Full thickness empty defects measuring 4 mm in diameter by 3 mm depth were prepared in the medial femoral condyle of 24 3-month-old New Zealand white rabbits, and the rabbits were randomized to two groups. Each group was randomized to receive transplantations with ACM-BMSCs （ 8 knees）, ACM （ 8 knees ）, or no grafts （ 8 knees ） into the cartilage defects. （4） The repairing effects of the condyles were macroseopically observed and the morphological changes of repaired defects were evaluated 6 and 12 weeks after the operation. The histological scores and the type-Ⅱ collagen immunohistochemistrical stains were analyzed. Results （1） At the 6th and 12th week after the operation ,the morphology, distribution and arrangement of the regenerated tissues were similar to normal cartilage in the knees with ACM-BMSCs transplantation, and the regenerated tissues grew to be integrated with the surrounding normal cartilage with obscure boundary between them. In the ACM group, the rough surface of regenerated tissue sunk obviously and the fibroblasts in all layer and the few chondrocytes in the deep layer were found. The thin reddish grey layer of soft granulation tissue formed in the defect and the fibroblasts were increased in non-transplantation group. （2） At every time interval, the histological scores of both groups Ⅰ and Ⅱbased on Wakitani scoring were lower than in group Ⅲ （ P 〈 0.05 ）, but the differences between groups Ⅰ and Ⅱ were not significant （ P 〉 0.05 ）. （ 3 ） Immunohistochemistrical stains showed that cells in the zones of repaired tissues were abundant, arranged columnedly and the type Ⅱ collagen staining was positive. Conclusion Tissue-engineering cartilage based on BMSCs seeded into ACM can repair the defects of the whole layer cartilage defects of medial femoral condyles of rabbits. The novel tissue was confirmed to be hyaline cartilage.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第3期342-345,F0004,共5页
Chinese Journal of Experimental Surgery
基金
基金项目：山西省国际技术合作项目（2007081037）
