β-TCP三维灌注生物反应器用于骨髓间充质干细胞扩增及成骨分化研究

发布时间：2018-05-24 16:56

本文选题：间充质干细胞 + β-磷酸三钙　；参考：《上海交通大学》2008年硕士论文

【摘要】： 目的:建立一套小规模MSC高效扩增反应器系统,在实验室水平快速扩增骨髓间充质干细胞,并同期建立成骨分化诱导及动力学研究方法,为临床骨损伤修复在短期获得定量组织、细胞数提供实验室向生产过渡。材料方法:(1)采用平行实验探索最佳的β-TCP支架制备处方和工艺,获得脆碎度、硬度、外观规则度、细胞悬液和培养基吸收效率最理想的片状支架。(2)在静态培养测试中,用廉价易贴壁的Vero进行接种,观察与2D平板培养相比较的扩增速率优势。(3)在实验室利用可观测双联小室设计并搭建体外灌注生物反应器。(4)将提取的2代大鼠骨髓间充质干细胞接种于平置支架材料上,先于二氧化碳培养箱中静置,完全贴附后开始连续灌注培养,于定期分别取样,与二维平板对照比较,CCK-8染色检测细胞生长趋势、迁徙率变化、ALP活性染色测试有无成骨诱导剂下的成骨诱导水平。干燥细胞-支架混合体,在扫描电镜下观察细胞生长状态、迁移能力和支架亲和水平。结果:(1)制备出平滑均匀、有一定强度和较低脆碎度、细胞悬液和培养基吸收高效、孔隙率达78%的β-TCP支架。(2)在3D静态培养比较2D培养中显示出后期更大的承载量和扩增速率。(3)反应器在流速15.7 ml/h左右能优效扩增间充质干细胞。(4)提取的大鼠MSCs在三维支架的灌注培养中,显示了空白支架独特的成骨诱导能力,并且相对于平板培养,CCK-8染色检测证明细胞扩增速率提高了约6-10倍,总体碱性磷酸酶活性大大提升,存在成骨诱导剂环境下,比ALP活性基本保持不变。此外,三维灌注条件下早期呈现整体迁徙率为主,晚期则以局部迁徙为主,其迁徙能力可满足骨科修复要求。电镜扫描显示出胞外基质的分泌使细胞集落与支架很好的亲和,并在层面结构显示出独特的生长适应性。结论:用优化的工艺和处方,可获得外观规则光滑、质地均匀坚韧、脆碎度低、耐浸泡、高生物亲和性的β-TCP三维支架,可供贴壁细胞贴附。采用双联小室配合β-TCP三维支架,可实现在实验室搭建三维连续灌注生物反应器,具备接种、取样、观察、考察迁徙率生长速率等功能,相对于二维平板,β-TCP三维支架可显著提高间充质干细胞扩增速度,材料本身可在接近饱和诱导的水平促进间充质干细胞成骨分化。单个细胞MSC存在成骨诱导剂时的分化能力与细胞比生长速率无直接关联。干细胞能在三维支架上分泌胞外基质,借以相互接触并融合在支架表面。三维支架层面结构相对于条状和柱状结构更有利于细胞的贴附与生长。
[Abstract]:Objective: to establish a small scale MSC high efficiency amplification reactor system, to rapidly expand bone marrow mesenchymal stem cells (BMSCs) at laboratory level, and to establish a method of osteogenic differentiation induction and kinetics study at the same time. For clinical bone injury repair in a short period of time to obtain quantitative tissue, the number of cells to provide laboratory transition to production. Materials: (1) using parallel experiments to explore the best preparation prescription and process of 尾 -TCP scaffolds, the flake scaffolds with the best degree of brittleness, hardness, regularity of appearance, cell suspension and absorption efficiency of culture medium were obtained in the static culture test. Inoculated with cheap, easily adherent Vero, To observe the advantage of amplification rate compared with 2D flat plate culture, we designed and built an in vitro perfusion bioreactor in the laboratory with observable double chamber, and inoculated the 2 generation rat bone marrow mesenchymal stem cells on the flat scaffold material. The cell growth trend was detected by CCK-8 staining in comparison with two-dimensional plate control after continuous perfusion culture was carried out after complete attachment in a carbon dioxide incubator, and then samples were taken separately at regular intervals, and compared with two-dimensional plate control, CCK-8 staining was used to detect the growth trend of the cells. The change of migration rate and ALP activity staining were used to test the osteogenic induction level with or without osteogenic inducer. The dry cell-scaffold mixture was used to observe cell growth, migration ability and scaffold affinity under scanning electron microscope. Results 1) smooth and uniform, with a certain strength and low brittle degree, cell suspension and medium absorption efficiency, 尾 -TCP scaffold with a porosity of 78%.) compared with 2D culture in 3D static culture, the 尾 -TCP scaffold showed higher loading capacity and amplification rate in 3D static culture than 2D culture.) the MSCs extracted from rat MSCs in 3D static culture could be effectively amplified by expanding mesenchymal stem cells (MSCs) at a flow rate of about 15.7 ml/h. In the perfusion culture of scaffolds, The results showed that the blank scaffold had unique osteoblast induction ability, and compared with plate culture, CCK-8 staining showed that the rate of cell expansion was increased by 6-10 times, the activity of alkaline phosphatase was greatly increased, and the osteoblast inducer was found in the presence of osteogenic inducer. The activity of specific ALP remained basically unchanged. In addition, the whole migration rate was dominant in the early stage and the local migration in the late stage under the condition of 3D perfusion, and its migration capacity could meet the requirements of orthopedic repair. Scanning electron microscopy showed that the secretion of extracellular matrix (ECM) made the cell colony compatible with the scaffold and showed unique growth adaptability in the layer structure. Conclusion: 尾 -TCP scaffolds with smooth appearance, uniform and tough texture, low embrittlement, high biocompatibility and immersion resistance can be obtained by optimizing the process and prescription, and can be attached to adherent cells. Using the double chamber and 尾 -TCP three-dimensional scaffold, the three-dimensional continuous perfusion bioreactor can be built in the laboratory, which has the functions of inoculation, sampling, observation, investigation of the growth rate of migration rate and so on. Compared with two-dimensional plate, 尾 -TCP three-dimensional scaffold can significantly improve the expansion rate of mesenchymal stem cells, and the material itself can promote the osteogenic differentiation of mesenchymal stem cells near saturation level. There was no direct correlation between the differentiation ability of single cell MSC and the specific growth rate of cells in the presence of osteogenic inducer. Stem cells can secrete extracellular matrix on three-dimensional scaffolds to contact each other and fuse on the scaffold surface. Three-dimensional scaffold structure is more favorable for cell adhesion and growth than stripe and columnar structure.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【参考文献】