Experimental Study Of Angiogenesis In The ADM Area By G-CSF Mobilizating Endothelial Progenitor Cells

Objective:Acellular dermal matrix(ADM) is an ideal material of tissue engineered skin,to study the ADM to vascularize rapidly for tissue engineered skin surviving are important,the experiment research that the granulocyte colony-stimulating factor(G-CSF) mobilizate endothelial progenitor cells(EPCs) to promote angiogenesis in the graft ADM area.To observe the effects of ADM transplantated and G-CSF mobilizating on the number of EPCs,which come from peripheral blood and endothelial progenitor cells homing and differentiating to the ADM,to observe the number of endothelial progenitor cells in peripheral blood at different time points,to compare the cell-specific surface markers and the number of wound angiogenesis.To provide some new ideas and simple method for ADM transplantated and other artificial stent to vascularize rapidly,as well as provide a reference for the tissue-engineering skin to vascularize.Methods:To choose granulocyte colony-stimulating factor that has been used in clinical,to mobilizate by the dose in accordance with the literature.Selected healthy mouse(to avoid the effects of estrogen on EPCs) ICR mouse 120,randomly divided into 4 groups:A group (simply transplanting ADM),B group(transplanting + G-CSF mobilizating),C group (G-CSF mobilizating),D group(negative control group),30 in each group.B group: grafting ADM and G-CSF mobilizating,A group:grafting ADM and normal saline injecting,C group:G-CSF mobilizating,D group:injection of normal saline alone as a negative control,A,B group were transplanted skin after they were injected for five days,and to mobilized sequentially in each group for 7 days.To select six mouse randomly in each group,before mobilization and in 48h,72h,1w,2w after injection (other groups in accordance with operated group),To take blood for Flow cytometry CD34,Flk-1,CD133-positive labeled cells.To cut of the grafted ADM and some surrounding tissue,which some tissues were fixated in 4%formaldehyde,and chiped by conventional pathology,using HE staining,immunohistochemical staining and other methods to observe the pathological changes and the angiogenesis.The results were used(?)±s to express,were analyzed by statistics.Results:Every group EPCs is very low before they were injected,there was no significant difference in the control group at 5 time point.After operation 48h,72h,1w and 2w,the proportion of dual-fluorescence-positive cells in G-CSF mobilizating group and the skin grafting group are increased than those in the control group(p＜0.01);the proportion of dual-fluorescence-positive cells in skin grafting+ G-CSF mobilizating group are increased more significantly than in the skin grating group and G-CSF mobilizating group(p＜0.05),the proportion of dual-fluorescence-positive cells in G-CSF mobilizating group are increased more significantly than in the skin grating group(p＜0.01).Capillary density in grafting ADM area in Skin grafting+ G-CSF mobilizating group are higher than the skin grafting group,there is significant difference statistically(p＜0.05).Conclusion:the quantity of EPCs in peripheral blood can be increased by G-CSF mobilizating;skin grafts,ischemia,trauma can mobilizate EPCs,so that the quantity of EPCs in peripheral blood can be increased; EPCs have peaked in peripheral blood by G-CSF mobilizated about seven days,the timing of transplantation choose the fifth day after mobilization,that is,before the arrival of peak of EPCs is more appropriate;EPCs can home and differentiate to ADM zone so that the number of blood vessels increase around the wound,which is beneficial to angiogenesis of AND and wound healing.