Browning Of Adipocytes In Fat Grafts Associated With Higher Level Of Necrosis And Type 2 Macrophages Recruitment In Humans And Mice

Background:Fat transfer is an important treatment method for repairing soft tissue defects in plastic and reconstructive surgeries.However,complications such as necrotic cysts and nodules after fat grafting and low volume retention are the key issues that limit the application of this technology.Necrosis of fat grafts is a very complicated process,accompanied by changes in the metabolic function and morphology of adipocytes.The mechanism behind it has not yet been clear.Understanding the development of fat graft necrosis is of great significance for clinical intervention and reducing the occurrence of fat grafts necrosis,and improving the survival of fat grafts.In the pathological observation of the necrotic tissue of fat grafts,we accidentally discovered that there is a large number of browning adipocytes in the area of necrotic adipose tissue.Under normal circumstances,brown adipocytes only exist in specific parts of humans in a small number.Brown adipocytes can specifically express uncoupling protein(UCP-1),which is responsible for its high metabolism.Therefore,we speculate that browning of adipocytes after fat grafting may be highly related to fat necrosis,suggesting the occurrence of fat necrosis.To confirm this hypothesis,we conducted a clinical study on the browning of adipocytes in necrotic tissue after fat transfer.In recent years,studies have found that in animal models fat transfer can induce the browning of white adipocytes in the graft,forming beige adipocytes which are similar to brown adipocytes,but the mechanism and influencing factors behind this phenomenon are still unclear.The origin and biological properties of the cells are unclear.Based on previous studies on the biological characteristics of brown adipocytes,brown adipocytes are characteristic of high metablism level and they are more prone to necrosis;beige adipocytes appearing in wounds and chronic wound tissues have been confirmed to be less viable and more prone to necrosis than white adipocytes.Combined with the concomitant relationship between fat necrosis and browning of adipocytes after fat grafting that we found in clinical studies,we speculate that beige adipocytes that appeared in animal experiments and browning of adipocytes we found in humans after fat grafting share same origins and biological characteristics.It is a sign of poor adipocytes survival after fat grafting.To verify this hypothesis,we conducted in vivo experiments on the relationship between browning of adipocytes and fat grafts necrosis after fat grafting.Confirming the clinical findings,the source and biological nature of beige adipocytes were to be revealed.At present,there is no research reporting the mechanism and regulation mechanism of browning of adipocytes during fat grafting.Existing studies have confirmed that the inflammatory micro-environment has a very important influence on the survival of fat grafts.Among them,the most important factor is macrophages,and its two subtypes,M1 and M2 macrophages,play important roles in the inflammatory response.Since browning of adipocytes after fat grafting is closely related to fat necrosis,its regulation is regulated by macrophages in the inflammatory microenvironment.Therefore,we believe that the regulation mechanism of the browning of adipocytes is inseparable from the functioning of macrophages,and the effects of M1 and M2 macrophages are different.In order to further clarify the mechanism of fat browning,we conducted a study on the effect of M1/M2 macrophages on browning of adipocytes.Objectives:1.To clarify the relationship between browning of adipocytes and fat necrosis after fat grafting in humans;2.To clarify the relationship between browning of adipocytes and fat necrosis in animal experiments,and to reveal the origin and biological properties of browning adipocytes;3.To explore the regulatory mechanism of browning of adipocytes after fat grafting,and to clarify the regulatory role of M1 and M2 macrophages in browning of adipocytes.Methods:(1)Clinical trials1.Clinical tissue samples:1)Experimental group(necrotic nodules group):6 cases of fat necrotic nodules,obtained from necrosis remove surgeries after autologous fat transfer for breast augmentation;2)Control group 1(necrotic flaps group):5 cases of necrotic adipose tissue after grafting were obtained from the necrotic area of the flap after breast reconstruction with abdominal flaps;3)Control group 2(survived adipose group):5 cases of normal adipose tissue from the repair operation after breast reconstruction with abdominal flaps.2.Detection and identification of brown adipocytes in samples:1)Immunohistochemistry staining with specific browning marker UCP-1 antibody;2)transmission electron microscope:to observe the morphology of adipocytes and the amount of mitochondria;3)Real-time PCR:to detect the expression level of UCP-1 gene;4)Western Blot:to detect the expression level of UCP-1 protein.(2)Animal study:1.Animal models:1)We symmetrically inject different volumes of human adipose tissue on the back of Balb/c nude mice subcutaneously;2)Experimental group(necrosis group):the injection volume of adipose tissue is 100?l per site;3)Control group(survival group):the injection volume of adipose tissue is 500?l per site;4)Control group(Sham):fresh adipose tissue from liposuction without further grafting.2.Harvest time:2 weeks,4 weeks,8 weeks and 12 weeks after grafting.3.Detection and identification of browning adipocytes:1)Immunohistochemistry staining with specific browning marker UCP-1 antibody;2)transmission electron microscope:to observe the morphology of adipocytes and the amount of mitochondria;3)Real-time PCR and Western blot:to detect UCP-1 gene and protein expression levels;4)Western blot:to detect the level of necrosis factor cleaved caspase-3;5)Western blot and ELISA:to detect CD206 and other M1/M2 cell surface markers;6)Western blot and ELISA:to detect IL-6,TNF?,TGF?,IL-10,IL1?,MCP-1 and other M1/M2 inflammatory factors.Results:1.UCP-1 antibody-specific staining showed that the fat graft tissue samples with necrosis showed positive staining of adipocytes in humans,which contained multilocular lipid droplets and were small in shape.The results of transmission electron microscopy further confirmed that the cells have abundant mitochondria,which is consistent with the characteristics of browning adipocytes.However,this type of adipocytes is not observed in normal fat grafts.2.Real-time fluorescent quantitative PCR results showed that the UCP-1 gene expression level in the fat graft tissue with necrosis in humans was significantly higher than that in the normal fat graft group(>5 times,**p<0.01).3.Western Blot semi-quantitative analysis indicates that in animal studies,necrosis group expresses a higher level of cell necrosis marker cleaved caspase-3 protein than the survival group and the sham group,indicating that the level of tissue necrosis is higher.4.UCP-1 antibody specific staining showed that necrosis group in mice contained stained-positive adipocytes,which contained multilocular lipid droplets,and were small in shape.The results of transmission electron microscopy further confirmed that the cells are rich in mitochondria,which is consistent with the characteristics of browning adipocytes.However,this type of adipocyte was not observed in the samples of the survival group and the sham group.5.Western Blot semi-quantitative analysis further clarified that the animal experimental necrosis group expressed a higher level of beige adipocyte marker UCP-1 protein than the survival group and the sham group,indicating that the tissue browning level was higher.6.ELISA analysis revealed that there was continuous activation and local infiltration of M2 macrophages(>2 folds)in fat grafts in the necrotic group,accompanied by an increase in the level of browning marker UCP1 in the grafts.At the same time,it was accompanied by a significant increase of IL-6 which could promote browning development(749.0±134.1 pg/ml).Conclusions:1.Browning of adipocytes is clearly found in the necrotic tissues after fat grafting in humans.It has been further verified in the necrotic tissues of animal models,but this phenomenon is not found in the fat grafts that survive well,indicating that browning of adipocytes after fat grafting is related to fat necrosis.A large number of browning of adipocytes indicates that fat necrosis has occurred.2.Brown adipocytes appearing in necrotic adipose tissue after grafting are manifested as small cells,containing multilocular lipid droplets,rich in mitochondria,and expressing high levels of UCP-1 at the gene and protein level.Browning/beige/brite adipocytes share the same biological characteristics as brown adipocytes,which is a sign of poor adipocyte survival after fat grafting.Browning biogenesis of adipocytes after fat grafting is regulated by macrophages.The type and infiltration of macrophages can affect the level of browning.The recruitment and infiltration of M2 macrophages is positively correlated with browning.Among them,IL-6 is secreted by M1 type macrophages,but it has the effect to promote the polarization of M2 type macrophages and then accelarate browning development.Its level in tissues is positively correlated with the level of browning of adipocytes after fat grafting.