| BackgroundIn the field of plastic surgery,Autologous fat grafting is a common method ofaugmentation and reconstruction of soft tissue defects.With the advancement of fat graft assist technologies,the long-term retention rate of fat graft has been relatively controllable,but still not ideal.How to obtain higher and more stable fat retention has been the focus of research.Research to date have concentrated on the harvesting,processing,and injection techniques of fat grafts,but little attention has been paid to the effect of the donor site on fat survival after grafting..In autologous fat grafting,both the donor and recipient sites require restoration.Both of them require early infiltration of macrophages to initiate repair.However,the numbers of macrophages that body can recruit within certain periods of time is limited.Therefore,there may be a competition between the restorations at the donor and recipient sites.The current animal models simply evaluate the histological changes of grafted fat without considering the effect of the donor site,and cannot simulate the actual situation well.Therefore,based on the traditional free fat grafting model,this study created donor site damage to simulate liposuction,and evaluated its effect on the inflammatory response and regeneration process in the recipient site.Also,macrophages were used along with fat grafting in this model,trying to provide a new idea for improving the long-term retention of grafts.Methods1.The effect of donor site injury on the repair process and outcome of grafted fat.C57BL/6 mice were randomly divided into two groups.The experimental group(Pro-Grafting group)underwent dorsal fat grafting immediately after simulated liposuction in the inguinal fat flap.The control group(Grafting Only group)performed dorsal fat grafting immediately after sham operation.On day 3,7,14,30,60 and 90 after grafting,samples were harvested and evaluated:the gross morphology of grafts was observed,the volume of the grafts were measured by drainage method,serum CRP levels were measured by ELISA,infiltration of inflammatory cells and tissue structure of grafts were assessed by hematoxylin-eosin(H&E)staining,and the infiltration and shifting of macrophages were evaluated by immunofluorescence staining.2.The effect of delayed fat grating on grafts outcome.After simulating liposuction in mice,fat grafting was underwent on the 3rd,7th,and 14th day after surgery,named Delay 3d group,Delay 7d group,Delay 14d group.Pro-Grafting(Delay 0d)group and Grafting Only(Delay ?)group were used as positive and negative controls respectively.The grafts were harvested on day 14,30,and 90 after grafting and relevant evaluations were performed:the gross morphology of grafts was observed,the volume of the grafts were measured by drainage method,infiltration of inflammatory cells and tissue structure of grafts were assessed by H&E staining,the infiltration of CD34-positive cells,the infiltration and shifting of macrophages were evaluated by immunofluorescence staining,macrophage-related inflammatory factors and adipogenesis proteins were detected by RT-PCR and WB respectively.3.The role of macrophages in delayed repair of grafts at recipient site.The donor and recipient fats in the Pro-Grafting group were harvested at different time points,the infiltration of inflammatory cells and tissue structure were observed by H&E staining,the infiltration and shifting of macrophages were evaluated by immunofluorescence staining,and the difference of inflammatory factors between donor and recipient sites was evaluated by RT-PCR.Based on the Pro-Grafting group,1 × 106 GFP mouse-derived M0 macrophages were injected into the tail vein for tracing,and the fats of recipient and donor sites were harvested on day 7,14,and 30 after grafting,and the infiltration and shifting of macrophages were evaluated by immunofluorescence staining.4.Macrophage assisted fat grafting reverses delayed fat repair in recipient site.In the Pro-Grafting+M2 group,1 × 106 GFP mouse-derived M0 macrophages were mixed with fat after simulated liposuction of the inguinal fat flap,and IL-4 was injected into the grafts on the 3rd day after grafting to promote the shifting of macrophages into M2 type,and the Pro-Grafting group was used as the control group.The grafts were harvested on day 3,7,14,30,60,and 90 after grafting,and the gross morphology of the grafts were observed.The volume of grafts was measured by drainage method.H&E staining was used to observe the long-term tissue structure.Immunofluorescence staining was performed to observe the infiltration of macrophages and the adipogenesis of grafts.WB was used to detect the levels of adipogenesis-related proteins.Results1.The damage in the donor sites led to the reduction of the long-term retention rate,as well as the delay of the repair process.In the short-term after the damage to the donor site,CRP level increased rapidly and back to normal in about 7 days.Histological observations showed that the grafts in the Grafting Only group were in the initial stage of inflammation on day 3,in the peak stage of inflammation on day 7,and in the declining stage of inflammation on day 30,while in the Pro-Grafting group,the initial stage of inflammation was delayed to the 7th day after grafting.The peak stage of inflammation was on day 30 and the declining stage of inflammation was on day 60.Immunofluorescence results showed that the infiltration and shifting of macrophages were also delayed.2.Compared with the Pro-Grafting group,the fat grafting performed on the 7th and 14th days after the donor site injured,the infiltration and shifting of macrophages were advanced,and the infiltration of CD34-positive precursor cells was advanced.Also,there was no statistics difference with the Grafting Only group,which indicated that the delayed repair phenomenon disappeared.The disappearance of the delayed repair phenomenon is accompanied by a stronger ability to regenerate fat,a more complete tissue structure,and a higher retention rate.3.In the Pro-Grafting group,early inflammation in donor site was higher than it in the recipient site,enabling rapid priority repair.Traces of macrophages showed that the macrophages preferentially infiltrated the donor site,and the recipient site largely infiltrated until the inflammation in the donor site subsided.4.Compared with the Pro-Grafting group,the Pro-Grafting+M2 group has faster fat regeneration and leads to a higher graft retention rate.ConclusionsIn the fat grafting model with donor site damage,the donor site inflammation level first increased,that is,macrophages preferentially infiltrated the donor site to initiate repair,and the repair process in the recipient site was delayed,which led to a reduction in graft retention.Promoting infiltration and shifting of macrophages in the grafts can reverse the delayed repair phenomenon.Accelerating macrophage infiltration and transformation is a new way toinitiate fat regeneration and increasing retentionin the future. |
PDF Full Text Request