Generating ADSC-Derived Endothelial-Like Cells To Promote Angiogenesis And Performance Of Fat Grafting

Background:Fat grafting is increasingly used in the field of soft tissue regeneration and reconstruction,but retention rate of the graft is still uncontrollable.ASCs assist fat grafting can promote angiogenesis and increase retention rate of graft,but ASCs have limited differentiation ability after grafting and cannot directly differentiate into endothelial cells to participate in vascularization.Therefore,we induced ASCs to differentiate into E-ASCs in vitro for assisting fat grafting.Objective:To study whether ASCs can be induced to have a feature of endothelial cells in vitro and then transplant with fat graft to observe the vascularization and retention rate of the graft.Methods:ASCs were obtained by standardized process and their surface antigen and multidirectional differentiation ability were identified.Basic medium with VEGF-A and FGF-2 cultured ASCs in vitro for one week,and then identified the expression of CD31 and CD34.Using Matrigel to explore the vascularization ability of the E-ASCs.Nude mice as the research objects,the models were made by fat grafting,ASCs-assisted fat grafting,and E-ASCs-assisted fat grafting.The samples were obtained at 2,4,and 12 weeks after grafting and their appearance,quality,volume were compared.Collagen volume fraction(CVF),microvessel density(MVD)and adipocyte number were compared through HE staining,Masson staining and immunofluorescence staining.ASCs or E-ASCs labeled with CM-Dil were used for fat grafting.The fate of two type of cells were also compared.Result:The ASCs used in this study have multidirectional differentiation potential.The results of surface antigens confirmed that ASCs express CD29 and CD44,while don’t express CD31 and CD34.After being induced by VEGF-A and FGF-2,the expression of CD31 and CD34 turned positive and with the ability to form tubes in vitro.After assisted with fat grafting,partially E-ASCs can retain their endothelial characteristics and directly participate in the formation of the neovascularization,the others that are not involved in microvascular formation no longer expressed CD31.There was no significant difference in mass and volume between the E-ASCs group and the ASCs group at 12 weeks of grafting,but both were superior to the control group.Compared with ASCs group,E-ASCs group showed a significant increase in graft microvessel density as well as a decreases in collagen deposition.Immunofluorescence showed that the adipocytes in the E-ASCs group had the same size and higher density than those in the ASCs group.The outcome of grafts in the E-ASCs group was significantly improved.Conclusion:ASCs can obtain the characteristics of endothelial cells after being induced by VEGF-A and FGF-2 in vitro,which can directly participate in the formation of the neovascularization and improved survival of the graft.