脱细胞真皮复合骨髓基质干细胞构建组织工程前交叉韧带的实验研究

发布时间：2018-06-02 14:28

本文选题：前交叉韧带 + 脱细胞真皮基质　；参考：《第四军医大学》2013年硕士论文

【摘要】：目的：随着对组织工程ACL构建方法研究的不断深入，发现多种细胞生长因子在ACL愈合过程中起着关键的调控作用。其中TGF-β1和bFGF已成为研究ACL愈合的外源性生长因子的热点。ADM作为多种组织修复支架材料，其在临床中修复组织缺损已得到广泛应用。本实验的目的在于探讨特定浓度TGF-β1和bFGF对BMSCs在ADM支架上粘附，增值和分化的影响。并通过不同编织ADM重建兔ACL，比较重建ACL腱骨愈合，胶原含量分布和生物力学强度，从而为构建组织工程ACL提供实验依据。方法：1、将培养至第三代的兔BMSCs接种于ADM材料上，分别用普通培养液（对照组）及TGF-β1(浓度10ng/ml)和bFGF(浓度25ng/ml)培养液（实验组）培养，通过分别绘制第1、3、5、7、9、11天细胞增殖MTT曲线，第7天测定ALP含量，荧光显微镜及激光共聚焦扫描显微镜观察，分析编织ADM支架基本力学性能，以及两组培养液对细胞在ADM支架材料中的生长增殖，分化和粘附的影响；2、通过编织4股束形支架（对照组）和4股柱形支架（实验组）重建兔ACL，分别取材第6、12周重建ACL，通过大体观察关节功能和软骨退变、腱-骨HE染色、重建ACL天狼猩红染色及生物力学测试，分析两组编织方法ADM支架的力学性能，及细胞在支架上生长增值，，分泌I，III型胶原以及腱-骨愈合的差异。3、应用SPSS12.0统计软件对数据进行统计学分析，P0.05为统计学差异标准。结果：1、MTT曲线及荧光染色结果表明实验组培养液BMSCs在ADM支架上的粘附数量更多（P0.05），ALP染色证明TGF-β1(浓度10ng/ml)和bFGF(浓度25ng/ml)能够促进BMSCs细胞分泌更多的ALP(P0.05)，激光共聚焦扫描显微镜显示实验组细胞粘附数量多，细胞形态更优良。2、重建兔ACL术后6、12周腱-骨及ACL染色表明，实验组较对照组腱-骨愈合速度更快，两组ACL分泌I、 III型胶原情况基本相当。术后12周，生物力学测试结果实验组ACL的生物力学性能明显优于对照组(P0.05)。结论：1、TGF-β1（浓度10ng/ml）及bFGF(浓度25ng/ml)培养液对BMSCs在ADM上的生长，粘附及分化有明显的刺激增强效果。2、编织ADM支架具备一定的生物力学强度，其中4股编织柱形ADM支架在兔体内能够促进细胞分泌I,III型胶原，并提供较好的腱-骨愈合速度及生物力学强度。
[Abstract]:Aim: with the further study of ACL construction methods in tissue engineering, it was found that many cell growth factors play a key role in the healing of ACL. Among them, TGF- 尾 1 and bFGF have become the focus of exogenous growth factor in the study of ACL healing. ADM is used as a variety of scaffolds for tissue repair, and it has been widely used in clinical repair of tissue defects. The purpose of this study was to investigate the effects of specific concentrations of TGF- 尾 1 and bFGF on the adhesion, proliferation and differentiation of BMSCs on ADM scaffolds. The bone healing, collagen content distribution and biomechanical strength of reconstructed ACL tendon were compared by different braided ADM to provide experimental basis for the construction of tissue engineering ACL. Methods the third generation rabbit BMSCs was inoculated on the ADM material and cultured with normal culture medium (control group), TGF- 尾 1 (10 ng / ml) and bFGF- 尾 1 (25 ng / ml) culture medium (experimental group). The MTT curves of cell proliferation were plotted on the 7911 days of the first day, respectively. On the 7th day, the content of ALP was measured, the basic mechanical properties of braided ADM scaffolds were analyzed by fluorescence microscope and laser confocal scanning microscope, and the growth and proliferation of cells in ADM scaffold were also analyzed. ACLs were reconstructed by weaving 4 strands (control group) and 4 strands scaffolds (experimental group). ACLs were reconstructed at week 612, respectively. The articular function and cartilage degeneration were observed, and tendon-bone HE staining was performed. ACL sirius red staining and biomechanical test were performed to analyze the mechanical properties of ADM scaffolds and the growth of cells on the scaffolds. The secretion of type III collagen and tendon-bone union was different. The statistical analysis of the data by SPSS12.0 software was used as the standard of statistical difference (P0.05). Results the results of BMSCs and fluorescence staining showed that the adherent quantity of BMSCs on ADM scaffold was more than that of TGF- 尾 1 (10 ng / ml) and bFGF- 尾 1 (25 ng / ml). Laser confocal scanning microscope (confocal scanning microscope) showed that TGF- 尾 1 (10 ng / ml) and bFGF (25 ng / ml) could promote the secretion of ALP P0.05 by BMSCs cells. The results showed that the number of cell adhesion in experimental group was high, The morphology of the cells was better. The results of tendon bone and ACL staining at 612 weeks after ACL reconstruction showed that the healing rate of tendon-bone in the experimental group was faster than that in the control group. The level of I and III collagen secreted by ACL in the two groups was almost the same. 12 weeks after operation, the biomechanical properties of ACL in the experimental group were significantly better than those in the control group (P 0.05). Conclusion: TGF- 尾 _ 1 (10 ng / ml) and bFGF (25 ng / ml) can stimulate the growth, adhesion and differentiation of BMSCs on ADM significantly. The braided ADM scaffolds have a certain biomechanical strength. Among them, 4 strands of columnar ADM scaffolds could promote the secretion of type III collagen in rabbit cells, and provide a better rate of tendon-bone healing and biomechanical strength.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R686.5

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