| Objective The goal of this study was to isolate, culture and identify late outgrowth endothelial progenitor cells from bone marrow, observe the biological characteristics of late EPCs(L-EPCs). The purpose of the second part of research was to investigate the biocompatibility of SIS and L-EPCs by co-cultivating L-EPCs and SIS in vitro and observe L-EPCs growth on the SIS. To observe the properties of small-diameter vascular grafts composed of SIS tube seeded L-EPCs, and experimental studies of rabbit carotid artery repair defects with them.Methods Bone marrow-derived mononuclear cells were isolated and cultured in EGM-2MV medium. Related markers of CD34 CDl33, VEGFR-2, and v WF were detected by immunohistochemistry, immunofluorescence and flow cytometry. The uptake capacity of the Dil-ac LDL and FITC-UEA-1 was comfirmed by confocal laser. The L-EPCs growth curve was drew by MTT assay. The ability of forming vascular-like structure was measured on Matigel glue. Endothelial cells features were observed by transmission electron microscopy. The porcine SIS was prepared. L-EPCs were seeded onto the SIS and production of ANG-1 and VEGF by L-EPCs was examined by ELISA and immunoblotting assays. The SIS membrane bonded with collagen and chondroitin sulfate mixture and was stitched for tublar. Test burst pressure, hemolysis, the impact on clotting time in vitro.L-EPCs were seeded onto the SIS tublar and were observed by SEM. The patency of transplanted vessels were examined by Ultrasound and DSA. The remodel of SIS tublar were evaluated by histological examination. Endothelial cell layer were observed by SEM and fluorescence microscopy.Results The early EPCs(E-EPCs) was the colony like growth, L-EPCs exhibit cobblestone morphology, spindle-like shape. L-EPCs surface antigen markers of CD133,CD34, VEGF-2 v WF expressed as varying degrees of positive. Flow cytometry showedthat the levels of CD133 higher in E-EPCs, then decreased. VEGFR-2 rises in L-EPCs.The L-EPCs could form vascular-like structure, and had greater proliferation potential. The L-EPCs can be seen in the endothelial cells characteristic cell-Weible-Palade(WP) bodies.Light microscopy showed that the mechanically and chemically treated small intestinal submucosa was composed of cell-free extracellular matrix. SIS significantly enhanced the growth of L-EPCs in vitro. ELISA showed that ANG-1 levels increased after seeding on the surface of the SIS and peaked at day 7 post seeding, the level of VEGF was markedly higher in the endothelial progenitor cells starting 7 days. The immunoblotting assays further showed that apparently higher levels of both ANG-1 and VEGF upon seeding of the EPCs onto the surface of the SIS. SIS tublar had a good burst pressure, can prolong clotting time, hemolysis rate in line with medical materials testing requirements, will not cause damage to the red blood cells. Seeded cells can be grown on SIS tublar. There is a50% patency rate at 8w. Histological examination showed that SIS had good remodeling reaction, visible collagen fibers and smooth muscle cells, and could be seen intact endothelial cell layer by SEM, part of the endothelial cells derived from the marked L-EPCsConclusion There are different qualities of E-EPCs and L-EPCs in the bone marrow.The L-EPCs could form vascular-like structure, and had greater proliferation potential.Limited experimental results show that the L-EPCs may be an attractive candidate cell for angiogenesis. SIS has good biocompatibility with L-EPCs. SIS significantly enhanced the growth of L-EPCs in vitro. SIS pre-seeded with L-EPCs can be potentially applied as an alternative scaffold material in artificial blood vessel prosthesis. SIS tublar can be produced in line with the requirements of the graft vessel, can be integrated into the host,and form a complete layer of endothelial cells in vivo experiments. |
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