Construct Tissue-engineered Capillary Networks By Endothelial Progenitor Cells Cultured And Differentiated From Peripheral Blood Mononuclear Cells In Vitro

Object To investigate how to obtain endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMCs) cultured and differentiated in vitro. To investigate methods of verifying EPCs. To analysis the reproductive and differentiated difference of EPCs caused by different culture conditions; to research how to construct tissue-engineered capillary networks. To analysis the effects of the EPCs' growing status on scaffolds by using different methods of transferring seed cells.MethodsCells culture: Draw 24 portions of 15ml venous blood from volunteers: Experiment Group (EG) 14, Control Group 1 (CG1) 5, Control Group 2 (CG2) 5. Extract PBMCs by gradient-density centrifugation from vein blood. EG: Transplant PBMCs into flasks coated by human fibronectin (HFN). And PBMCs were affected by VFGF and bFGF; CG1: Culture PBMCs without VEGF and bFGF; CG2: Culture PBMCs without HFN coating. Passage primary generation cells (P1) in the 12th culture day. Observe the diversity of cell morphology. Defect the reproductive ability of each group by counting cell clones, counting cells per clone and MTT chromatometry. The P1 of each group were analysised by CD34, CD133, Flk-1 immunohistochemistry (IHC) staining; the P3 of each group were stained by CD31 staining. The PBMCs and P1, P2, P3 of EG were compared by CD34,CD133 flow cytometry (FCM); the P1, P3 of each group were compared by CD31 FCM. The P3 cells of EG group were stained by DiI-AcLDL, FITC-UEA-1.Construction of cell-scaffold complex and animal experiment: 8 nude mice: 2 mice in EG1, EG2, EG3, and EG4. EG1 is for scaffolds' absorption time experiment (control group); EG2 is for transferring full dose of seed cells; EG3 is for transferring half dose of seed cells; EG4 is for transferring half dose of seed cells twice. Construct PLGA scaffolds coated HFN by the particles leaching method. Defect scaffolds' absorption time by using EG1. Transplant P3 cells of EG into scaffolds and then culture the cell-scaffold complex for 4 weeks in vitro. Then examine the growth of EPCs on scaffolds by SEM. Insert cell-scaffold complex under EG2, EG3, EG4 nude mice's back skin, and then harvest skin and subcutaneous tissue which included cell-scaffold complex after scaffold absorbed. Analysis the tissue by HE, CD31 IHC staining and SEM. Count the ventages in which endothelial cells (ECs) grew and of integrated endo- membrane in which red blood cells existed.Results The reproductive and differentiated ability of EPCs cultured by EG was better than control groups. The CD34~+CD133~+ cells in P3 cells of EG were as 79 times as in PBMCs just centrifugated. The P3 cells of EG could intake DiI-AcLDL and bind FITC-UEA-1 what was the functions of ECs-like cells. Bioactive scaffolds were absorbed completely after having been implanted under nude mice's back skin 11 weeks ago. The P3 EPCs of EG could stick and grow on scaffolds. The ventages in which ECs grew and of integrated endomembrane in which red blood cells existed could be found in bioactive scaffolds by staining methods. These two effective kinds of ventages were mostly likely found on EG4 cell-scaffold complex.Conclusions It was successful to obtain proliferating and differentiating EPCs from PBMCs cultured in vitro, the quantity and the quality of EPCs could satisfy requests for constructing tissue-engineered capillary networks. The ability, velocity of proliferation and the cell clonality of EPCs were correlated with growth factors (GFs) and HFN coating. HFN coating didn't correlated with EPCs' differentiation, but GFs could promote EPCs differentiate into ECs; the micro-structure, physico-chemical property and absorption time of PLGA scaffolds coated by HFN could satisfy requests for constructing tissue-engineered capillary networks. Increase the transferring quantity of seed cells in the certain extent and transferring same quantity of EPCs by several times could promote EPCs' grow on scaffolds and ECs differentiated from EPCs form the integrated endomembrane.