PBMSCs凝胶对兔肌腱移植物重建ACL腱骨愈合的影响

发布时间：2018-06-27 13:36

本文选题：前交叉韧带 + 腱骨愈合　；参考：《安徽医科大学》2015年硕士论文

【摘要】：目的探讨在兔自体肌腱移植物重建前交叉韧带(anterior cruciate ligament,ACL)模型股骨道内注入自体外周血间充质干细胞(peripheral blood mesenchymal stem cells,PBMSCs)凝胶对早期腱骨愈合的影响。方法健康3至4月龄新西兰大白兔32只,随机分为实验组与对照组,每组16只,实验组予以皮下注射粒细胞集落刺激因子(G-CSF)动员6天后于耳缘静脉采集外周血,密度梯度离心法分离纯化PBMSCs,细胞培养箱内采用贴壁培养法培养并传代至第三代,光镜下观察细胞形态后行流式细胞检测鉴定所培养细胞表面特定抗原(CD11b、CD29、CD34、CD90)的表达。对全部32只新西兰大白兔采用抛硬币法随机选取一侧膝关节,建立兔自体肌腱移植物重建前交叉韧带模型,实验组于动物模型股骨道内注入PBMSCs凝胶,对照组仅注入凝胶。分别于术后第2、4、8、12周每组随机取4只实验动物处死取出膝关节,其中1只做Masson三色染色观察移植物在股骨道中组织愈合的病理表现,3只做股骨道移植物抗牵拉试验观察其愈合强度,记录移植物从骨道内脱出时的拉力值,实验组与对照组所得数据采用两样本t检验,比较两组数值有无显著性差异(选取a=0.01为检验水准)。结果光镜下观察待测细胞以集落方式贴壁生长,呈长梭型或多角形。流式细胞学检测得出被检测细胞表面CD29、CD90、CD11b、CD34阳性表达率分别为97.3%、98.4%、1.3%、1.3%,提示被检测细胞表面表达CD29、CD90,不表达CD11b、CD34。Masson三色染色结果提示:术后第2周:两组在腱骨交界处均有大量坏死组织形成,纤维排列紊乱,成纤维细胞与胶原纤维均无明显增生;术后第4周:对照组仍有大量坏死组织,纤维排列不齐,有少量成纤维细胞增生,但未见明显胶原纤维。PBMSCs组坏死组织较空白组减少,组织排列欠规则,有少量成纤维细胞及胶原纤维长入;术后第8周:对照组有大量成纤维细胞长入,纤维排列欠规则,腱骨愈合界面有少量胶原纤维长入。PBMSCs组有大量成纤维细胞长入,部分纤维排列整齐,部分欠规则,腱骨愈合界面有大量胶原纤维长入;术后第12周:对照组成纤维细胞数量减少,胶原纤维生成增多,腱骨愈合交界处成纤维细胞与胶原纤维数量比约为1:1。PBMSCs组仅残余少量成纤维细胞,大量胶原纤维规则排列在腱骨愈合交界处。在实验组与对照组中,股骨道移植物抗牵拉力强度均随时间延长呈上升趋势,在术后第2周、第4周,对两组的抗牵拉强度进行比较,无明显统计学意义(P0.01)。在术后第8周及第12周,对两组的抗牵拉强度进行比较,PBMSCs组抗牵拉力强度大于对照组,差异有统计学意义(P0.01)结论PBMSCs凝胶对兔自体肌腱移植物重建ACL的早期腱骨愈合有促进作用。
[Abstract]:Objective to investigate the effect of autologous peripheral blood mesenchymal stem cells (peripheral blood mesenchymal stem cells, PBMSCs) gel on early tendon bone healing in the femoral canal of anterior cruciate ligament (ACL) model of autogenous tendon graft in rabbits. Methods 32 rabbits were randomly divided into 3 to 4 month old New Zealand white rabbits. Group and control group, with 16 rats in each group, the experimental group was subcutaneously injected with granulocyte colony stimulating factor (G-CSF) to collect peripheral blood in the ear vein for 6 days, and the density gradient centrifugation was used to separate and purify PBMSCs. The cell culture box was cultured and passed to the third generation in the cell culture box, and the cell morphology was observed by the flow cytometry. The expression of the specific antigen (CD11b, CD29, CD34, CD90) on the surface of the cultured cells. All 32 New Zealand white rabbits were randomly selected to select one side of the knee joint with a coin toss method. The rabbit autologous tendon graft was established to reconstruct the anterior cruciate ligament. In the experimental group, the PBMSCs gel was injected into the femoral canal of the animal model and the control group was injected only with the gel. The knee joints were taken out of 4 experimental animals in each group at 2,4,8,12 weeks. 1 of them were stained with Masson Tri Color staining to observe the pathological manifestation of tissue healing in the femoral canal. 3 of the femur graft resistance test was performed to observe the healing strength of the graft, and the tension values of the graft from the bone canal were recorded. The data obtained from the experimental group and the control group were recorded. Two samples t test was used to compare the significant differences between the two groups (select a=0.01 as the test level). Results the cells under the microscope were observed under the light microscope, and the cell surface was adhered to the wall. The flow cytometry showed that the positive expression rates of CD29, CD90, CD11b and CD34 were 97.3%, 98.4%, 1.3%, 1.3%, respectively. The expression of CD29, CD90, and non expression of CD11b on the surface of the cells showed that second weeks after the operation: second weeks after the operation, there were a large number of necrotic tissue in the two groups at the junction of the tendon and bone, the fibrous arrangement was disorderly, and the fibroblasts and the collagen fibers had no obvious hyperplasia; fourth weeks after the operation, there were still a large number of necrotic tissue in the control group. There was no obvious collagen fibrous fibroblast proliferation, but no obvious collagen fibrous.PBMSCs group was less than the blank group, the tissue arrangement was less regular, and a small amount of fibroblasts and collagen fibers grew. Eighth weeks after the operation, there were a large number of fibroblasts in the control group, the fiber arrangement was not regular, and the tendon bone healing interface with a small amount of collagen fibers grew into the.PBMSCs group. For Twelfth weeks after the operation, the number of fibrous cells in the control group decreased, the collagen fibers increased, and the number of fibroblasts and collagen fibers at the junction of tendon bone healing was only a small amount of fibroblasts in group 1:1.PBMSCs, and the number of fibroblasts and collagen fibers at the juncture of tendon bone healing were only a small amount of fibroblasts. A large number of collagen fibers were arranged at the juncture of tendon bone healing. In the experimental group and the control group, the tensile strength of the femur transplants increased with time. In the second week and fourth weeks after the operation, the tensile strength of the two groups was compared. There was no significant statistical significance (P0.01). The anti traction of the two groups at eighth and 12 weeks after the operation. The strength of the PBMSCs group was higher than that of the control group, and the difference was statistically significant (P0.01). Conclusion PBMSCs gel could promote the early tendon bone healing of the rabbit autologous tendon graft for the reconstruction of ACL.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R687

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