| The blood supply is a fundamental requisition in skin transplantation. Vascularization of transplanted tissue can achieved through the blood vessels contained in flap pedical or anastomosed during transplant ion, besides which another important way is the new vessels from recipient site grow into transplanted tissue. In tissue engineering skin transplantation, the prominent drawbacks are the poor survival and the unpredictable viability after grafting. The proposed main reasons for these, include poor diffusion of nutrients, lack of ready-formed capillary network, and slow neovascularization. All these lead to the graft slough and dead. Therefore, vascularization of dermal substitutes is one of the important aspects in skin tissue engineering study.The skin flap transplantation is one of the most familiar treatment in plastic surgery. Too big flap often causes its distal part ischemic and necrotic, so how to promote the skin flap blood vessel, break the limitation of length to breadth ratio, and obtain the larger and super-routine flap, is an important research project in plastic surgery field.Recent study had discovered EPC (endothelial progenitor cells) existing in adult marrow, peripheral blood, cord blood and fetal liver, experiment of EPC transplanting show this kind of cell participated various physiologic and pathologic process of blood vessel reconstruction. As a new kind of cell, whichcan be use to establish a treatment method, the EPC provides a new clinic strategy for ischemic diseases.[Objectives] To establish the methods of seeding EPC in porous biodegradable scaffold PGA (the polyglycolic acid) by leading EPC into tissue engineering skin and ischemic skin flap. To study EPC biological characteristics and its neovascularization in tissue engineering skin and ischemia skin flap transplantation by constructing tissue engineering skin with EPC and fibroblast, and injecting the EPC into corresponding skin flap, further, evaluate its effect and investigate the related mechanism.[Methods] CD133~+cells were enriched from human umbilical cord blood by immunomagnetic sorting, and cultured with EGM-2MV media. The percentage of CD 13 3+cells in cord blood monocytes and sorting efficiency were measured by fluorescence activate cell sorting (FACS). The cells cultured for different times were identified by immunochemical staining with anti- CD 133, CD34, vWF antibodies and observed with transmission electron microscope. The mixture of EPCs and human stomach cell GC7901 were injected into athymic mice to observe the tumor growth and vascularization in tumor. After labeled with PKH26 (Fluorescent Cell Linker), the EPC were injected into the over-length flap models made on athymic mice. Observing the EPCs trace and their participating in the flap vascularization were done with fluorescent microscope. The potential of EPC neovascularization in ischemic tissue of skin flap was evaluated through measuring the necrotic area and vessel diameter and quantity in the skin flap. On these foundations, EPCs and fibroblasts were seeded into dermal substitute constructed by mixing PGA with fibronogen. When EPCs and fibroblasts in PGA-collagen gel showed better morphological character and a network form, the surface of the substitute was covered with a thin layer keratinocyte sheet to construct tissue engineering skin. This compound skin was cultured in vitro, then transplanted to repair the athymicmouse full-thickness wound, at last biopsied to be observed for its structure and vascularization. After transplantation, the fates of EPCs and keratinocys were traced by immunohistochemical staining of anti-human vWF MAb and anti-human keratin 14 MAb. In the control group, PGA alone covered with keratinocytes sheet as compound skin was transplanted to athymic mice. [Results] (1) The percentage of CD133+ cells in cord blood monocytes was 0.91%, and after sorting, the percentage of CD133+ cells became 85.52%. The culture cells showed a typical spindle-shaped morphology at 1 week and cluster areas of cobblestone-like cells after 2 weeks. The expression of CD 133 decreased, which vWF increased on the cell surface during the culture time. The cells after 10 days culture have typical Weibel-Palade body observed under transmission electron microscope. (2) Tumor forming experiment in athymic mice showed that with smaller necrosis area, much more and larger blood vessel, the tumor size of EPC group was larger than that of control (p<0.05). Immunohistological analysis many human vWF antigen-positive endothelial cells involved in the tumor vascularization. (3) In EPC promoting skin flap experiment, the skin flap necrosis area of EPC group is significantly smaller than that of control (p<0.05), the dermal and hypodermal blood perfusion of EPC group significantly more than that of control (p<0.05). Immunohistological and label fluorescent analyses showed vWF antigen-positive cells and labeled cells constructing blood vessels of flap. (4) In tissue engineering skin transplantation experiment, the EPCs and fibroblasts could evenly distribute the dermal scaffold, meaning while attaching to the PGA and stretching into the spindle or multiangular shap gradually, then there were large amounts of them growing in the compound skin. Keratinocyte sheet could closely adhere to dermal substitute and form multiplayer. After athymic mice transplanting by 21 days, histological section showed epidermis proliferating obviously, forming multiplayer and cuticular layer, and immunohistological analysis showed... |
PDF Full Text Request