动态压力促进骨基质支架中微血管形成的实验研究  

Study of dynamic pressure to promote the angiogenesis of bone matrix

作　　者：杨军[1] 周振东[2] 李建军[1] 张钦明[1] 

机构地区：[1]中国医科大学附属盛京医院脊柱创伤骨科,110004 [2]沈阳市第四人民医院骨科

出　　处：《中华骨科杂志》2011年第4期372-378,共7页Chinese Journal of Orthopaedics

摘　　要：目的 将血管内皮祖细胞（endothelial progenitor cells,EPCs）种植于体外制备的猪脱钙骨基质支架中,予适当压力干预,观察动态压力对EPCs形成血管能力的影响.方法 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EPCs,并对诱导细胞进行免疫荧光鉴定.取猪脊柱椎体松质骨,通过组织固定、脱脂、脱钙等步骤制备脱钙骨基质支架.取第三代EPCs种植在脱钙骨基质支架,将细胞一支架复合物分为两组,压力组给予动态压力,对照组直接培养,观察两组支架上的细胞血管形成能力.RT-PCR检测EPCs分化相关因子vWF和Flk-1的mRNA表达.结果 两组HE染色和荧光染色可见血管束形成,且压力组细胞血管束形成明显多于对照组.RT-PCR检测vWF和Flk-1 mRNA的表达情况,压力组表达明显高于对照组.结论 给予适当压力干预后脱钙骨基质与EPCs复合构建组织工程骨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.Objective To observe the effects of dynamic pressure for the ability of endothelial progenitor cells （EPCs） to form blood vessels, when EPCs seeded into DBM with load. Methods Use the Ficoll density gradient centrifuge combined with difference-speed adherence screening method to separate MNCs from rat bone marrow. Identify the induced EPCs by means of immunohistochemistry and immunofluorescence. Through the organization of fixed, defatted, decalcified and other steps use of spine vertebral body,demineralized bone matrix （DBM） samples of pig were prepared in vitro. Divided scaffolds into two groups A and groups B. Induced EPCs were seeded into DBM. The cell-seeded scaffolds of groups A were dynamically loaded in compression using a sine wave at 1 Hz, 5% strain in the media-filled chamber for 4 h on days 5 of culture. and cell-seeded scaffolds of groups B were cultured directly without any load. Both of two groups were cultured two weeks. Then the ability of EPCs to form blood vessels was observed. Primer desig;Extract total RNA from cells with Trlzol; Reverse transcription reaction; PCR. Results Two groups of cells in HE staining and fluorescent staining showed the formation of vascular bundles. There were formation of blood vessels. It was obvious that the formation in group A was more than that in group B. Test the mRNA expression of vWF and Flk-1 during the EPCs differentiationby RT-PCR. Group A was significantly stronger than that of group B. Conclusion When DBM combines together with EPC, it has become organization engineering bone, then with press on it, the bone graft has been vascularized, so it has clinical application on the direction of repair bone defect.

关 键 词：压力 新生血管化 生理 组织工程 

分 类 号：R687.3[医药卫生—骨科学]