【作者】 杨金凤；

【导师】 王金福；

【作者基本信息】 浙江大学 ， 细胞生物学， 2008， 硕士

【摘要】 骨组织工程为骨缺损修复提供了一种新的疗法。人骨髓间充质干细胞(hMSCs)是一种理想的骨组织工程种子细胞,被广泛用于与支架材料复合构建组织工程骨。聚乳酸-乙醇酸(PLGA)支架在软骨和骨组织工程应用中显示出较好的性能而被广泛接受。灌流式生物反应器常用于培养骨骼组织。本文目的是评价灌流培养条件下人骨髓间充质干细胞在PLGA支架中的生长增殖及分化动态。实验所用支架孔隙率为94.5%-98.0%,孔径大小为280-450μm。首先通过对三个不同接种密度(1×10~6,2×10~6,4×10~6个/ml)接种效率的比较,确定了较为合适的接种密度(2×10~6个/ml)。通过对三个不同流速(0.2,0.4,0.8ml/min)培养效果的比较,确定了较为合适的灌流流速(0.2ml/min)。通过合适的接种密度和合适的灌流流速立体培养hMSCs,研究细胞在支架中的生长动态。MTT法检测细胞活力显示,在整个灌流培养期间,细胞活力持续显著增加,表明细胞增殖旺盛。DNA定量分析表明从接种到培养第6天细胞数量显著增加,之后增殖变缓。用扫描电镜(SEM)来观察细胞在支架中的分布以及细胞外基质的分泌情况,结果显示第5天细胞已经长入支架孔内部,并见有薄层基质合成;第11天,细胞外基质已形成厚厚的一层将支架基本完全覆盖。活细胞荧光染色和苏木素-伊红(H&E)染色可见细胞在支架中均匀分布。通过成骨细胞定向诱导分化实验,检测支架中hMSCs的成骨细胞分化效率。碱性磷酸酶染色和酶活性检测结果表明,成骨诱导第7天细胞已经开始进入早期分化,诱导期间持续分化;诱导组碱性磷酸酶活性显著高于对照组(未诱导组);未诱导组也检测到微量碱性磷酸酶存在,推测是灌流培养产生的剪切力所致。以上结果表明,用该灌流生物反应器系统来进行hMSCs的三维立体扩增和分化是可行的,可以用于骨组织工程研究。更多还原

【Abstract】 Bone tissue engineering beckons a new frontier for clinical treatment to bone defects. hMSCs, as an ideal cell source, are widely used in the treatment of bony defects. PLGA scaffold has exhibited good biocompatibility in bone tissue engineering. Perfusion bioreactor can provided favorable conditions for the three-dimensional cultivation of bone tissue. The objective of this study was to determine the viability and growth of hMSCs in a three-dimensional porous PLGA scaffold in combination with perfusion bioreactor culture system. The scaffold has a porosity of 94.5%-98.0% and pore size of 280-450μm. hMSCs were seeded at a density of 2×10~6cells/ml, and the perfusion rate was 0.2ml/min. Firstly, three different cell density(1×10~6, 2×10~6, 4×10~6个/ml) were used in the seeding process, and a cell density of 2×10~6个/ml was found to be the more proper seeding density by the comparison of seeding efficiency. Comparison of cell status after 3-days culture under three different perfusion rates (0.2, 0.4, 0.8ml/min) generated the more proper perfusion rate (0.2ml/min). Cell viability by analyzing with MTT colorimetry increased with time, indicating a significant proliferation. The cell quantity based on DNA assay increased in a fast proliferation at first 6 days, and then in a slower proliferation. SEM images showed, at day 5, hMSCs has migrated into the scaffold holes, and a dense layer of extracellular matrix has been formed; and the entire surface of the scaffold was covered by a dense layer of extracellular matrix at day 11. FDA staining and H&E staining showed that the cells spreaded uniformly through the whole scaffold. ALP staining and ALP activity assay showed that the early differentiation began at 7 days after osteogenic differentiation, followed by a continuing differentiation. Moreover, ALP activity of osteogenic medium induced group is much higher than that of the control group.These results demonstrate the feasibility of culturing cell/PLGA in a flow perfusion bioreactor for bone tissue engineering applications.更多还原