应用生物蛋白胶与胚胎软骨细胞混合移植修复兔膝关节实验性软骨缺损区(英文)

Repair of experimental defects of articular cartilage in rabbits with homografts of fibrin sealant and embryonic chondrocytes

背景:应用异体软骨细胞移植修复关节软骨缺损的研究较多,但其可引起免疫反应。由于胚胎细胞具有较低的抗原性和较强的增殖能力,期望其能为组织工程研究与应用开辟新的载体材料。目的:采用兔胚胎软骨细胞培养移植修复关节软骨缺损,并对其组织形态学进行观察。设计:随机分组观察对比实验。单位:广西医科大学组织胚胎学试验室。材料:孕4周新西兰大白兔1只;成年新西兰大白兔24只,雌雄不限,体质量2～2.5kg。方法:实验于2000-12/2002-06在广西医科大学组织胚胎学试验室完成。在成兔股骨内侧髁作关节软骨缺损模型,实验组采用生物蛋白胶与胚胎软骨细胞相混后,植入兔膝关节实验性软骨缺损区,对照组不做任何处理或单纯用生物蛋白胶移植修补,分别于术后4,8,12周观察关节软骨缺损修复的情况,并按改良Pineda方法对其进行组织学评分,标准为细胞形态、基质染色、表面平整、软骨厚度、宿主结合5项,0分表示正常,分值越高表示改变越严重。主要观察指标:①兔膝关节标本大体观察结果。②兔膝关节标本组织学观察结果。③关节软骨组织学半定量计分结果。④关节软骨缺损修复区进行疗效评价结果。结果:24只兔均进入结果分析。①兔膝关节标本大体观察结果:胚胎软骨细胞+纤维蛋白封闭剂组缺损区颜色接近正常软骨,质韧弹性好,与周围软骨界限消失;纤维蛋白封闭剂组与未处理组始终未愈合,但创面略小,有白色纤维组织充填。②兔膝关节标本组织学观察结果:胚胎软骨细胞+纤维蛋白封闭剂组优势组织为透明样软骨,深方为骨组织,表面略凸或平整,基质着色正常,与周围软骨完全结合分界不清,组织中未发现淋巴细胞浸润;纤维蛋白封闭剂组与未处理组为纤维组织,部分有残留明显的凹陷性瘢痕,与周围组织接合或部分接合。③关节软骨组织学半定量计分结果:按改良自Pineda方法,12周胚胎软骨细胞+纤维蛋白封闭剂组评分显著低于纤维蛋白封闭剂组和未处理组[(0.50±0.76),(7.88±1.13),(8.13±1.36)分,P<0.05],而单纯用纤维蛋白封闭剂修补组在4周与未处理组比较有显著性差异(P<0.05),在8,12周则无差异。④关节软骨缺损修复区进行疗效评价结果:12周时胚胎软骨细胞+纤维蛋白封闭剂组完全修复8膝,纤维蛋白封闭剂组不完全修复1膝,未修复7膝,未处理组未修复8膝。结论:胚胎软骨细胞移植组所产生的修复组织接近正常软骨组织,明显优于纤维蛋白封闭剂组和未处理组,该方法作为关节软骨缺损修复是可行的。

BACKGROUND：Research about the repair of articular cartilage with heterograft chondrocytes is frequently reported, but the method may cause immune rejection. Since the embryonic cells possess lower antigenicity and stronger proliferation capability, it is hoped that they can be used as a novel carrier substitute in tissue engineering research.DESIGN： A randomized grouping observation and comparative experiment.SETTING： Histological Embryonic Laboratory in Guangxi Medical University.MATERIALS： A big white adult New Zealand rabbit pregnant for 4 weeks was adopted; and another 24 big white adult New Zealand rabbits were selected, with no limitationin whether they were female or male and with a body mass of 2 to 2.5 kg.METHODS： This experiment was carried out at the Histological Embryonic Laboratory in Guangxi Medical University between December 2000and June 2002. The models of defects in articular cartilage were made artificially in femur medial malleolus of the mature rabbits. In the experimental group, defects were repaired by the implantation of Fibrin Sealant and embryonic chondrocytes mixture, but for the control group, only Fibrin Sealant was implanted or nothing was done about the defect. The restoration of articular cartilage defect was then observed 4,8 and 12 weeks after the operation, and was scored according to modified Pineda＇s method. The standard consists of 5 items, I.e., cellular morphology, matrix staining, surfacing smoothness, cartilage thickness and host union. 0 refers to normal and the higher the score is, the more serious the pathological changes are.MAIN OUTCOME MEASURES： ①The general observation of rabbit knee joint; ② Histological observation of rabbit knee joints; ③ Histological semi-quantitative score of articular cartilage; ④ Appraisal of the curative effect of articular cartilage defects.RESULTS： Totally 24 rabbits were enrolled in this experiment and all entered the stage of result analysis. ① The general observation of rabbit knee joint： In embryonic chondrocytes plus fibrin sealant group, the color in defect area was basically the same as that of the normal cartilage, showing a strong quality and better elasticity with the boundaries from the surrounding cartilage approximately vanishing. In Fibrin sealant group and the control group, the defect did not heal completely, but the defect area became small and filled with white fibrous tissues. ② Histological observation of rabbit knee joints： In embryonic chondrocytes plus fibrin sealant group,tissues were predominated by hyaline cartilage, with bone tissues appearing in deeper position, and the surface was slightly raised or smooth, and the matrix showed normal staining, and were completely united with the surrounding cartilage and the division line was not clear. There was no lymphocyte infiltration in the tissues. In Fibrin sealant group and the control group, tissues were predominantly fibrous tissues, and part of them showed obvious residual hollow scar, connecting or partly connecting with the surrounding tissues. ③ Histological semi-quantitative score of articular cartilage： According to modified Pineda＇s method, the scores of embryonic chondrocytes plus fibrin sealant group after 12 weeks were obviously lower than those of the fibrin sealant group and the control group [（0.50±0.76） vs （7.88±1.13）, （8.13±1.36）, P ＜ 0.05]; moreover, the difference between pure fibrin sealant group and the control group was of statistical significance 4weeks after the operation （P ＜ 0.05）, but there was no obvious difference 8and 12 weeks after the operation. ④Appraisal of the curative effect of articular cartilage defects： 12 weeks after the operation, in the embryonic chondrocytes plus fibrin sealant group, the defect of 8 cases healed completely. In fibrin sealant group 1 case healed but not completely, and 7cases did not get repaired for their defect. In the control group, 8 cases failed to get their defect repaired.CONCLUSION： The repaired tissues in embryonic chondrocytes trans plantation group were basically the same as normal cartilage, obviously superior to the fibrin sealant group and the control group, suggesting that such method is feasible in the repair of articular cartilage defects.