| BACKGROUND Chronic wound is defined as skin tissue defect which can not reach the state of the anatomical and functional integrity though normative and timely repair process. Clinically, it refers to the wounds that are still unhealed or showing no tendency of healing after more than 1 month`s treatment. The main reason include two aspects, the first is the local microcirculatory disturbances,which leads to the weakened cell self-repair, the second is the significantly reduced endogenous growth factors. Basic fibroblast growth factor(b FGF) and vascular endothelial growth factor(VEGF) play very important roles among all the growth factor in the process of wound healing. b FGF can promote fibroblast proliferation, collagen deposition, granulation tissue formation, thus accelerate wound healing. VEGF can increase capillary density locally, so as to improve the perfusion and metabolism, and eventually accelerate wound healing. It has been suggested keratinocytes can be accelerated for migrating and proliferating from low concentration to high concentration by epidermal growth factor concentration gradient. It has been reported rarely in recent years that growth factor concentration gradient induce cells accelerating migrating and proliferating eventually shortening the wound healing time. We aim to investigate whether the fibroblasts and vascular endothelial cells can be accelerated migrating and proliferating from low concentration to high concentration by growth factor concentration gradient to provides a theoretical basis for improving the chronic wound healing.METHODS 1.Cultured swine vascular endothelial cells and fibroblasts in vitro.2 Detected the effects on vascular endothelial cell proliferation and migration with different concentration of VEGF, and the effects on fibroblast proliferation and migration with different concentration of b FGF by MTT method and wound healing assay. 3.Binded b FGF and VEGF to the collagen scaffold by soaking method. Used ELISA method through standard curve to calculate the combination ratio and slow-releasing potential of VEGF and b FGF in collagen scaffold. 4.Screened out different concentrations of VEGF or b FGF with different proliferation activity and migrate activity in various time points, and binded them to the collagen scaffold to constitute a concentration gradient. 5.Cultured the vascular endothelial cell with different concentrations of VEGF binded to circular collagen scaffold, determined the proliferation and migration of vascular endothelial cell by MTT method and the measuring distance of the cell from the center of the collagen scaffold. 6.Cultured the fibroblasts with different concentrations of b FGF binded to circular collagen scaffold, determined the proliferation and migration of vascular endothelial cell by MTT method and the measuring distance of the cell from the center of the collagen scaffold.RESULTS 1.The proliferation and migration of vascular endothelial cell are affected in the different concentration of VEGF stimulation, the two sets of data were analysed by single factor analysis of variance, respectively. Selected three groups(5ng/ml, 25ng/ml, 100ng/ml), show statistical significant difference in every time points(P<0.05). 2.The proliferation and migration of fibroblasts are affected under the different concentration of b FGF stimulation, the two sets of data were analysed by single factor analysis of variance, respectively. Select three groups(10ng/ml, 50ng/ml, 100ng/ml), which show statistical significant difference in every time points(P<0.05). 3.The combination ratio of VEGF is 42.35%,while the combination ratio of b FGF is38.23%.Both of them show certain slow-releasing potential. 4.VEGF concentration gradient group(5ng/ml,25ng/ml, 100ng/ml) shows no significant impact on vascular endothelial cell proliferation activity, but has significant effects on vascular endothelial cell migration activity compared with single VEGF concentration group(P<0.05). 5.b FGF concentration gradient group(10ng/ml,50ng/ml,100ng/ml) shows no significant impact on vascular endothelial cell proliferation activity, but has significant effects on fibroblast migration activity compared with single b FGF concentration group(P<0.05).CONCLUSION A certain concentration of VEGF has a promoting effect on vascular endothelial cell proliferation and migration, and VEGF concentration gradient has a stronger role in promoting migration activity of vascular endothelial cell compared with single concentration of VEGF. Similarly, a certain concentration of b FGF has a promoting effect on fibroblast proliferation and migration, and b FGF concentration gradient has a stronger role in promoting migration activity of fibroblast compared with single concentration of b FGF. |
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