动态压力促进骨基质支架中微血管形成的实验研究

发布时间：2018-11-13 09:23

【摘要】： 目的分离、获取大鼠骨髓单个核细胞,条件培养基诱导成血管内皮祖细胞,观察体外培养主要生物学特点,免疫组化及免疫荧光鉴定。在体外制备猪脱钙骨基质支架,电镜扫面观察组织形态。将血管内皮祖细胞种植在脱钙骨基质支架上,将细胞-支架复合体置于生物型压力反应器给予适当压力干预,观察其形成血管能力。方法取50克Wistar大鼠,采用密度梯度离心法结合差速贴壁筛选法分离出骨髓单个核细胞,用条件培养液从原代开始诱导培养,诱导其形成血管内皮祖细胞,并对诱导细胞进行免疫组化及免疫荧光鉴定。取适当大小猪脊柱椎体松质骨,通过组织固定、脱脂、脱钙等步骤制备出脱钙骨基质支架。取第三代血管内皮祖细胞种植在脱钙骨基质支架,将细胞-支架复合物随机分为A、B两组,A组置放在生物型压力反应器,给予1Hz频率,5%形变压力值,五天后将细胞-支架复合物取出,继续培养直至两周,B组直接培养两周,观察两组支架上的细胞血管形成能力。RT-PCR检测两组EPCs分化相关因子mRNA表达。结果新提取的细胞呈圆形,大小不一。24小时差速贴壁,36小时后可见部分细胞贴壁,并逐渐出现梭形、三角形、纺锤形或不规则形等形态。7天左右出现细胞集落,中央呈圆形,周围梭形细胞放射性生长。10～12天集落细胞呈单层铺路石样生长。细胞-支架复合物电镜下可见脱钙骨基质孔隙网架结构,骨小梁、小梁间隙和骨内管腔系统同时存在,细胞铺满材料表面,临近细胞相互融合。A、B两组HE染色和荧光染色可见血管束形成,且A组细胞明显多于B组。用RT-PCR法检测两组EPCs培养后vWF和Flk-1 mRNA的表达情况,A组表达明显强于B组。结论密度梯度离心法结合差速贴壁法提取血管内皮祖细胞简便、快捷,生长状态稳定,增殖能力强。可以认为血管内皮祖细胞是一种理想的骨组织工程种子细胞。脱钙骨基质是一种良好的载体支架材料,与血管内皮祖细胞复合构建组织工程骨给予适当压力干预后能明显促进移植骨的血管化,在修复骨缺损方面具有临床应用价值。
[Abstract]:Objective to isolate and obtain rat bone marrow mononuclear cells and induce vascular endothelial progenitor cells in conditioned medium, observe the main biological characteristics of culture in vitro, immunohistochemistry and immunofluorescence identification. Porcine decalcified bone matrix scaffolds were prepared in vitro. Vascular endothelial progenitor cells were implanted on the decalcified bone matrix scaffold and the cell-stent complex was placed in a biologic pressure reactor for appropriate pressure intervention to observe its vascular formation ability. Methods Bone marrow mononuclear cells were isolated from 50 g Wistar rats by density gradient centrifugation combined with differential adherent screening method. The mononuclear cells were cultured in conditioned medium from primary culture to induce endothelial progenitor cells (EPCs). The induced cells were identified by immunohistochemistry and immunofluorescence. The cancellous bone of the appropriate size pig spinal vertebrae was used to prepare the scaffold of decalcified bone by tissue fixation, degreasing and decalcification. The third generation vascular endothelial progenitor cells (VECs) were implanted in the decalcified bone matrix scaffolds. The cell-stent complexes were randomly divided into two groups: group A was placed in a biometric pressure reactor and given 1Hz frequency and 5% deformed pressure. After 5 days, the cell-stent complex was removed and cultured for two weeks. Group B was cultured directly for two weeks to observe the ability of cell angiogenesis. RT-PCR was used to detect the expression of EPCs differentiation related factor mRNA in the two groups. Results the newly extracted cells were round and varied in size. After 24 hours of differential adhesion, some cells could be seen sticking to the wall, and gradually appeared fusiform and triangular. After 7 days, the colony appeared round in the center, and the peripheral fusiform cells grew radially. At 10 ~ 12 days, the colony cells grew like monolayer paving stone. Electron microscope of cell-scaffold complex showed that the porous network structure of decalcified bone matrix, trabeculae, trabecular space and intraosseous lumen system existed simultaneously, the cells were covered with the surface of material, and the adjacent cells fused with each other. Blood bundle formation was observed in group B by HE staining and fluorescence staining, and the number of cells in group A was significantly higher than that in group B. The expression of vWF and Flk-1 mRNA in group A was significantly higher than that in group B after EPCs culture by RT-PCR method. Conclusion the method of density gradient centrifugation combined with differential adherent method is simple, rapid, stable and proliferative. Vascular endothelial progenitor cells are ideal seed cells for bone tissue engineering. Decalcified bone matrix is a good carrier scaffold material. Combined with vascular endothelial progenitor cells to construct tissue engineered bone can obviously promote the vascularization of bone graft after appropriate pressure intervention. It has clinical application value in repairing bone defect.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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