Experimental Study On The Role Of The Early Macrophage Infiltration In The Free Fat Grafting

Background and ObjectiveFat transplantation is the most popular method for tissue augmentation and repair of large volume soft tissue in plastic surgery.However,there is still a lack of complete understanding of the survival/regeneration mechanism of after free fat transplantation.At present,most studies have focused on the role of adipose derived mesenchymal stem cells(ASCs)in fat transplantation.Few studies have involved the role of inflammatory cells in fat transplantation.Many studies have observed that there are lots of "crown-like structures" formed by mononuclear macrophages in fat grafts.They are believed to be mainly related to the elimination of necrotic adipocytes.However,the specific role of macrophages have not been further studied.Macrophages are one of the in inflammatory cells that are closely related to tissue regeneration.Macrophages can be activated to different types responding to the changes in the environment.In general,M1 macrophages can secrete a large number of proi-nflammatory cytokines,mediating inflammatory responses.M2 macrophages regulate inflammatory response and promote tissue regeneration by secreting anti-inflammatory cytokines.Then,is macrophage involved in tissue regeneration after free fat transplantation?By which way can macrophages regulate this' regeneration process?Can we manipulate the macrophage-mediated inflammation to promote the retention rate of fat transplantation?Around these scientific problems,this study used a mouse-based free fat transplantation model to explore the infiltration mode of macrophages in the graft,to compare the retention rate of fat transplantation after up-regulating and down-regulating the macrophages by evaluating the angiogenesis,stem cell recruitment and extracellular matrix(ECM)reconstruction in fat grafts.Method1.observation of macrophage infiltration mode in free fat graftingThe inguinal fat flap from C57 mouse was obtained and cut into small pieces,and was grafts in the back of the mice.The grafts were harvested at different time points.Immunofluorescence staining,flow cytometry and qRT-PCR was applied to assess the inflammatory cell infiltration level and inflammatory cytokine expression level.2.Effects of up-regulationa and down-regulation of the macrophages on free fat transplantationUsing the mouse autologous fat transplantation model mentioned above,liposomes-encapsulated chloride phosphonates were used to deplete macrophages within one week after transplantation,monocyte activation stimulating factor(M-CSF)was applied to upregulate macrophage level and PBS were used as control.The retention rate between three groups of grafts at different time points was compared.The levels of inflammation,angiogenesis and stem cell recruitment in each group were evaluated by whole-mount staining,immunofluorescence staining,flow cytometry and qRT-PCR technology.The role of macrophages in free fat transplantation was also discussed.3.Effects of up-regulationa and down-regulation of the macrophages on ECM remodeling after free fat transplantation The mouse autologous fat transplantation model was used.The liposomes-encapsulated chloride phosphonates was used in the early and late stage after transplantation,respectively,to eliminate macrophages and liposome encapsulated PBS was used as control.The levels of inflammation,ECM collagen deposition,fibrosis and capsule thickness were evaluated by scanning electron microscopy,immunofluorescence staining,flow cytometry and qRT-PCR.The effects of macrophages on ECM reconstruction in transplanted fat were also discussed.4.Manipulation of the level of macrophages and stem cells by using granulocyte colony stimulating factors to promote the retention of fat transplantationA mouse model of autologous fat transplantation was used.The granulocyte colony-stimulating factor(G-CSF)of 10 ?g/kg body weight,G-CSF of 100 ?g/kg body weight and PBS were used in the mice receiving fat grafting in one week after transplantation.The difference of retention rate between three groups of grafts at different time points was observed.And the level of inflammation,angiogenesis,stem cell recruitment were assessed by histological observation,immunofluorescence staining,flow cytometry and qRT-PCR to investigate the efficacy of the strategy of manipulation of the macrophages and stem cell level to improve the fat graft retention rate.Results1.At one week after the free fat transplantation,macrophage began to become the dominating population of inflammatory cells,At this time,they are M1-polarized with high level expression of pro-inflammatory factors,e.g.,TNF-a,as well as VEGF and other angiogenic factors.Fat grafts are experiencing“inflammation period".After fourth weeks,the M2 macrophages become the leading inflammatory cells in the transplanted fat and highly expressed TGF-? and other anti-inflammatory factors.2.in the early stage of free fat transplantation,the angiogenic factor expression was significantly up-regulated in the macrophage up-regulation group,more neovascularization and blood stem cells were observed in the fat grafts,these are also involved in tissue fat transplantation of adipose in regeneration,so its long-term retention rate was significantly better than the control group.After depleting macrophages,the expression of angiogenic factors in transplanted fat was decreased significantly,and the number of new blood vessels was significantly less.Moreover,the number of blood derived stem cells collected from transplanted fat decreased and the long-term retention rate was not as good as that in the control group.3.in the early stage of free fat transplantation,the elimination of macrophages could lead to a decrease in the collagen secretion in the transplanted fat,and the ECM remodeling was delayed.At the late stage of transplantation,the elimination of macrophages can relieve the degree of fibrosis in the transplanted fat and reduce the thickness of the capsule.4.High dose of G-CSF can enhance the blood stem cell recruitment,improve the degree of vascularization,but also cause a prolonged inflammation,fat graft fibrosis,inhibition of adipogenesis,and lead to decreased long-term retention of fat transplantation.Low dose of G-CSF can increase the recruitment level of blood derived stem cells and increase the degree of early vascularization in fat transplantation,but it will not cause prolonged high level of inflammation and fibrosis,and can improve the long-term retention rate of transplanted fat successfully.ConclusionMacrophages play an important role in free fat transplantation.Early macrophage infiltration can promote angiogenesis in graft,promote recruitment of hematogenous stem cells,and stimulate the remodeling of ECM.Macrophage infiltration in later stage can cause fibrosis of adipose tissue and inhibit adipogenesis.The low dose of G-CSF strengthens the recruitment of sterm cells and promotes the degree of vascularization,and improves the long-term retention of transplanted fat.High dose of G-CSF can cause high levels of high inflammation,and cause fibrosis of the transplanted fat,which can not improve the long-term retention of the transplanted fat.Therefore,macrophages have a complex role in the regulation of fat transplantation.