Study On The Vacularization Of Tissue Engineering Dermal Substitutes With Human Umbilical Vein Endothelial Cells

The standard method of healing a severe wound by surgical repair is by split thickness skin autografting. However, there is a lack of donor in patients with a high percentage of skin defects sites, in addition to this autografting, Many skin substitutes are in development or have been introduced to the market in USA admitted by FAD.In November,2007,the first tissue engineering skin-An ti fu developed by Tissue Engineering Center of the Forth Military Midical University are registed by FAD, and applied in clinic in China,Which are another source of skin substitutes for reparing skin defect. But they are not the same as the anatomy and physiology of normal skin, such as lack of caplliary-like structure. The tissue engineering skin can not supply plenty of nutrition and o2 after transplantation, that limited the use of tissue engineering skin in large and deep skin lesions.Vascularization of tissue engineering skin is only way to resolve this problem.This study try to vascularize the tissue engineering skin in different method,such as interact of seed cells,graft material and control release system.1.Culture and identification of seed cells (human umbilical vein endothelial cell, HUVEC) for vascularization used in tissue engineering skin.Aim: To obtain purified HUVECs with good proliferation ability.The HUVECs were obtain from new born human umbilical coreby enzyme digestion employed in our lab.The HUVECs were identified by cell morphology(inverted phase contrast microscop,SEM), immunofluorescent histochemical staining for VIII factor-related antigen, and transmission electron microscopy for Weibel-Palade bodies. Result: The HUVECs showed very good proliferation ability and with 100% of HUVEC.2. Reconstruction of a dermal-like tissue provided with a capillary-like structure with HUVECs, Fbs and collagen.Aim: To study the interaction of HUVECs and Fbs in forming capillary-like structure in 2 dimension and 3 dimensional tissue engineering dermal substitude.The passage 3 HUVECs were no-contact cocultured with Fbs in snap-on dish. Meanwhile the HUVECs marked by Hochost-33342 were cocultured with Fbs in dish.The interaction of two kinds of cells were observed by contrast phase microscope and fluorescence microscope.The passage 3 HUVECs were cocultured with Fb (1:1 in density of 106/ml )in collagen. The culture of passage 3 HUVECs in collagen with density of 106/ml as control group.Result:The Fbs can improve ability of the forming capllary-like structure of HUVECs in 2 dimension and 3 dimension;The dermal-like tissue provided with a capillary-like structure were constructed in this study.3. Reconstruction of a dermal-like tissue provided with a capillary-like structure with HUVECs, acellular dermal matrixAim:To inspect the role of acellular dermal matrix (ADM) in Reconstruction of a dermal-like tissue provided with a capillary-like structure with HUVECs.ADM were obtain by novel trypsogen-collagenase double digestion method in this study ,The ADM were observed by HE,SEM and its strength were tested. The result showed that The ADM proved high porosity and breaking strength.To test the biocompatibility of ADM, coculture of ADM with HUVECs and Fbs were carried out, observation of HE and SEM showed that the ADM applied in this study have good biocompatibility and could form capillary-like structure in case of coculture of ADM with HUVECs.The ADM without cells were transplanted subcutaneouly in nude mouse as control group.The ADM seeded with HUVEC were transplanted subcutaneouly in nude mouse as experiment group.The result showed that both transplant had no capillary-like structure formed at 2 week after transplantation. The plenty of capillary-like structure were observed in transplant at 3 week after transplantation in experiment group . However, no capillary-like structure were observed in control group. Immunochemical staining of monoclone antibody CD31 and VIII Factor-related antigen indicated positive expression on the capillary-like structure cells. The result show that the ADM seeded with HUVECs can shorten the time of forming capillary-like structure after transplantation in vivo, farther improve the achievement ratio of transplantation4. Study of control release of growth factor with Gel microspheres in reconstruction of a dermal-like tissue provided with a capillary-like structure Cytokine bFGF play an important role in angiogenesis and vasculogenesis, It can promote vasculogenesis in a reasonable concentration. However interval feeded with medium in reconstruction of a dermal-like tissue in vitro can not stabilize the concentration of Cytokine in deepth of tissue engineering derma.To resolve this problem , The control release growth factor with gel microspheres were applied in reconstruction of a dermal-like tissue provided with a capillary-like structure. To test the biocompatibility of gel microspheres, coculture of gel microspheres with HUVECs carried out. The gel microspheres combined with bFGF were mixtured with HUVECs to reconstruct the TE derma. The gel microspheres uncombined bFGF were mixtured with HUVECs to reconstruct the TE derma as control group.The study indicated that control release gel microspheres is valid method in forming capllary of HUVEC in TE derma.