构建组织工程软骨中机械刺激的作用研究

发布时间：2018-01-05 15:35

本文关键词：构建组织工程软骨中机械刺激的作用研究　出处：《浙江大学》2010年博士论文　论文类型：学位论文

【摘要】：机械刺激是软骨组织工程中非常重要的调控因子。本文以软骨细胞和骨髓间充质干细胞为种子细胞,研究了机械刺激在关节软骨组织工程中的作用。首先研究了机械刺激对牛膝关节不同软骨细胞亚群在PRG4、HAS1、HAS2mRNA表达和PRG4、透明质酸蛋白质合成方面的影响。发现机械刺激可以诱导软骨细胞亚群发生显型的改变以适应其环境。软骨细胞各个亚群之间的相互作用对透明质酸的合成有促进作用。使用混合亚群的软骨细胞,在最佳的机械刺激条件和生物化学条件下,可以诱导复合物表层的细胞重新分化具有浅表区细胞显型,从而构建出较完整的关节软骨层次结构和功能性的软骨滑液界面。这对于通常以来自整个关节的各软骨细胞亚群混合细胞作为种子细胞的组织工程有重要的意义。由于软骨细胞作为种子细胞存在来源不足,获取手段复杂且对患者损伤较大的缺点,继而研究了以hBMSCs为种子细胞,体外构建软骨组织过程中机械刺激的作用。为了建立体外hBMSCs成软骨细胞培养体系,研究了纤维蛋白凝胶——聚氨酯支架体系是否支持体外hBMSCs向软骨细胞的诱导分化。通过测量细胞的GAG/DNA值,以Real-time PCR测量细胞的mRNA水平,和组织学及免疫生物化学染色方法,对支架培养和微团培养进行了比较。实验结果表明与微团培养体系相比,在最佳接种密度(5x106个细胞／支架)下,细胞支架复合物具有相似的软骨细胞标志性蛋白合成量GAG/DNA,较高的软骨细胞标志性基因COL2、AGG mRNA水平,和较低的成骨细胞标志性基因Sp7、ALP、BSP2 mRNA水平,同时还具有能够为新生组织的积聚提供空间的优点,有利于进一步使用生物力学的手段优化体外干细胞向软骨细胞的诱导分化。在不同的外源TGF-βl浓度下,研究了机械刺激对hBMSCs分化过程的影响,并使用TGF-βⅠ型受体抑制剂LY364947阻断TGF-β信号转导通路,研究了TGF-β信号转导通路在该过程中的作用。实验结果表明机械剌激可以诱导纤维蛋白凝胶——聚氨酯支架培养的hBMSCs向软骨细胞分化,前提条件是外源TGF-β浓度较低且培养液中含有地塞米松。由于TGF-β本身可诱导hBMSCs成软骨细胞,过高的外源TGF-β浓度下机械刺激的作用会被覆盖。机械刺激通过TGF-β信号转导通路诱导hBMSCs向软骨细胞分化,TGF-β受体抑制剂可以阻断TGF-β信号转导通路从而抑制机械刺激诱导干细胞向软骨细胞分化的作用。对hBMSCs生物支架复合物施加不同强度和频率的循环动压力和表面剪切摩擦力,以观察不同力学环境下hBMSCs的反应。发现机械刺激的频率和幅度可以调控hBMSCs向软骨细胞分化的过程。在本文所采用的机械刺激范围内,较高的机械刺激频率和较高的动压力幅度更有利于hBMSCs向软骨细胞分化,更有利于抑制软骨细胞肥大化。由此说明在以BMSCs为种子细胞的软骨组织工程研究中,对细胞支架复合物施加适当强度的机械刺激非常重要。将视黄酸β受体抑制剂LE135与机械刺激相结合,研究其对hBMSCs分化过程的影响。发现在微团培养和支架培养体系中,视黄酸β受体抑制剂LE135对外源TGF-β和机械刺激诱导的hBMSCs向软骨细胞分化的过程有抑制作用。同样的条件下,LE135对hBMSCs向成骨细胞分化的过程没有影响。
[Abstract]:Mechanical stimulation is a very important regulatory factor in cartilage tissue engineering. In this paper, chondrocytes and bone marrow mesenchymal stem cells were used as seed cells, and the role of mechanical stimulation in articular cartilage tissue engineering was studied.
The first study of mechanical stimulation on knee joints of different cartilage cell subsets in PRG4, HAS1, HAS2mRNA and PRG4 expression, affect the synthesis of hyaluronic acid protein. It is found that mechanical stimulation can induce changes of chondrocyte subpopulations phenotype to adapt to its environment. The cartilage cell subpopulations interaction has a promoting effect on the synthesis of hyaluronic acid. The use of mixed cartilage cell subsets, in the best mechanical and chemical conditions, can induce the complex surface of the cell differentiation with superficial cell phenotype, in order to build a more complete articular cartilage structure and function of cartilage synovial fluid interface. This usually takes for the cartilage cell subsets of mixed cells from the entire joint as seed cells in tissue engineering has important significance.
The chondrocytes as seed cell sources exist insufficient means of acquiring complex and larger defects injury patients, then studied with hBMSCs as seed cells, effects of mechanical stimulation of cartilage tissue in vitro. In the process of building a hBMSCs in order to establish in vitro chondrogenic culture system of fibrin gel - polyurethane stent system supports differentiation in vitro hBMSCs to chondrocytes. By measuring cellular GAG/DNA, Real-time PCR mRNA to measure the cell level, staining and histological and immunological biochemistry, to support training and micromass culture were compared. The experimental results show that compared with the micromass culture system, the optimum inoculation density (5x106 cells / scaffold), cell scaffold composite has the sign of similar GAG/DNA protein synthesis of cartilage cells, cartilage cell marker COL2 high, AGG MRNA level and low osteoblast marker genes Sp7, ALP, BSP2 mRNA level, at the same time, it also has the advantages of providing space for the accumulation of new tissue, which is conducive to further optimize the differentiation of stem cells into chondrocytes in vitro.
In different concentrations of exogenous TGF- beta L, studied the mechanical stimulation effect on the differentiation of hBMSCs, and use the TGF- receptor type inhibitor LY364947 beta TGF- signaling pathway, on the role of TGF- beta signaling pathway in this process. The experimental results show that the mechanical stimulating induced by fibrin gel - polyurethane scaffold hBMSCs chondrogenic differentiation, is a prerequisite for exogenous TGF- beta concentration was low and medium containing dexamethasone. Because TGF- beta itself can chondrogenic hBMSCs, effects of mechanical stimulation of high concentration of exogenous TGF- beta would be covered. HBMSCs were induced to chondrocytes by TGF- B signal transduction machinery stimulation of TGF- beta receptor inhibitor can inhibit the TGF- beta signaling pathways to inhibit the mechanical stimulation induced stem cells differentiating into chondrocytes.
Cyclic shear and friction surface pressure applied with different intensity and frequency on hBMSCs biological scaffold, to observe the different mechanical environment hBMSCs reaction. Found that the frequency and amplitude of mechanical stimulation can regulate the differentiation of hBMSCs into chondrocytes. This paper used by the mechanical stimulus range, high frequency and high mechanical stimulation the dynamic pressure amplitude is more conducive to the differentiation of hBMSCs into chondrocytes, more conducive to inhibit chondrocyte hypertrophy. Therefore in the BMSCs as the seed cells in cartilage tissue engineering, applying the appropriate strength on the cell scaffold composite mechanical stimulation is very important.
The retinoic acid receptor inhibitor LE135 and the combination of mechanical stimulation, to study its effect on the differentiation of hBMSCs. Found in micromass culture and culture support system, the process of retinoic acid receptor inhibitor LE135 on induced by exogenous TGF- beta and mechanical hBMSCs chondrogenic differentiation was inhibited. The same condition under the influence of LE135 on hBMSCs differentiation of osteoblasts from there.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【共引文献】