Department of Orthopedics, Kunming Medical University, Kunming, China (mainland).
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China (mainland).
Med Sci Monit. 2016 Oct 28;22:4037-4045. doi: 10.12659/msm.897884.
BACKGROUND The aim of this study was to investigate the proliferation, differentiation, and tube formation of human outgrowth endothelial progenitor cells (OECs) cultured with porous demineralized bone matrix (DBM) under a dynamic perfusion system in vitro. MATERIAL AND METHODS OECs were isolated, expanded, characterized, eGFP-transfected and seeded on DBM scaffold and cultured under static or dynamic perfusion conditions, and continuously observed under fluorescence microscope. DBM scaffolds were harvested on day six for RT-PCR and western blot assay to analyze the mRNA and protein expression level of CD34, VE-cadherin, and VEGF. Scanning electron microscope (SEM) was used to observe the tube formation of OECs seeded on DBM scaffolds. RESULTS The results showed the cell density of OECs on DBM was higher when exposed to shear stress generated by a dynamic perfusion system. Shear stress also markedly increased the expression level of VE-cadherin and VEGF and decreased the expression of CD34, at both mRNA and protein levels. SEM showed that the shear-stressed OECs formed tube-like structures inside the pores of DBM scaffolds. CONCLUSIONS A dynamic perfusion system can be used as an innovative method for the rapid vascularization in tissue engineering, which can accelerate the proliferation and differentiation of OECs and the vascularization of implanted scaffolds.
本研究旨在探讨在体外动态灌注系统中，培养人血管生成内皮祖细胞（OECs）与多孔脱钙骨基质（DBM）的增殖、分化和管形成。
分离、扩增、鉴定、eGFP 转染 OECs 并接种于 DBM 支架上，在静态或动态灌注条件下培养，并在荧光显微镜下连续观察。在第 6 天收获 DBM 支架进行 RT-PCR 和 Western blot 分析以分析 CD34、VE-cadherin 和 VEGF 的 mRNA 和蛋白表达水平。扫描电子显微镜（SEM）用于观察接种于 DBM 支架上的 OECs 的管形成。
结果表明，与静态培养相比，在动态灌注系统产生的切应力作用下，OECs 在 DBM 上的细胞密度更高。切应力还明显增加了 VE-cadherin 和 VEGF 的表达水平，同时降低了 CD34 的表达水平，无论是在 mRNA 还是蛋白水平上。SEM 显示，受切应力的 OECs 在 DBM 支架的孔内形成管状结构。
动态灌注系统可作为组织工程中快速血管化的创新方法，可加速 OECs 的增殖和分化以及植入支架的血管化。
