Modification With Methoxy Polyethylene Glycol Promotes In Vivo Adipogenesis Of Acellular Adipose Matrix Xenotransplantation

Background and ObjectiveAutologous fat transplantation is the first choice for the treatment of large soft tissue defects,but there are some problems such as graft calcification.The use of acellular adipose matrix(AAM)is a new way to realize tissue regeneration through adipogenesis.The balance between the regenerative activity and immunogenicity of AAM needs to be considered in the preparation method.Our groups previously developed a physically prepared AAM,to retain more active components through a simplified preparation method,but there is no futher discussion on its possible residual immunogenicity.The purpose of our research is to explore the effect of possible immune response on adipogenesis of physical AAM in vivo.Immune modification of grafts to suppress immune response has a long history in the field of transplant medicine.In the initial chemical cross-linking method,glutaraldehyde(GA)is the most widely used,but its ability to modify antigenic epitopes is limited and its cytotoxicity is high.Immune camouflage is another means of immune modification,and methoxy polyethylene glycol(mPEG)is the most widely used.The immune modification effect of mPEG is remarkable,and it is relatively non-toxic,widely used in commercial,and has been gradually deeply studied in the field of blood transfusion and organ transplantation.The purpose of our research is to adopt fat transplantation model to compare the immune modification ability and cytotoxicity of GA and mPEG,and to select a better immune modifier.Acellular matrix removes most of the cellular components,but there is still residual antigen,which can't be definitely quantitied and can cause host immune response.However,compared with fresh tissue transplantation,acellular matrix induces less rejection-promoting Type 1 helper T cells(Th1)immune response and obtains better results in vivo.The purpose of our research is to study the effect of mPEG modification on the adipogenic ability of physical AAM in vivo,and to find its possible immunological mechanism.Methods1.HE staining for histological observation;Masson's Trichrome staining for fibrosis detection;Immunofluorescence for evaluation of adipogenesis and infiltration of Thl cells.2.qRT-PCR for detection of IL-2,IFN-?,IL-4,PPAR-? and CEBP-? expression in the grafts;ELISA for IgM and IgG detection in the hosts.3.Live-dead cell staining and CCK-8 for evaluation of hASCs viability;Flow cytometry for detection of apoptotic cell propotion and Thl cells propotion.Results1.The adipose regeneration of AAM grafted to nude mice was better than that of BALB/c mice.2.mPEG-modified fat grafts had lower degree of fibrosis,less inflammatory cells infiltration and lower level of immune rejection than GA group.3.The viability of hASCs treated with mPEG was higher than that of GA group,and the proportion of apoptotic cells was lower.4.The adipose regeneration of mPEG-modified AAM grafts was better than that of unmodified group,and the level of immune rejection was lower.5.AAM modified by mPEG has less promoting effect on the differentiation of Naive CD4+T cells into Th1 cells.Conclusions1.The immune response induced by physical AAM inhibits the adipogenic results of xenotransplantation in vivo.2.mPEG reduces the inflammation and immune rejection of xenogeneic fat transplantation significantly more than GA.3.The cytotoxicity of mPEG is significantly lower than that of GA.4.mPEG inhibits Th1 immune rejection of physical AAM xenotransplantation and promotes adipogenesis in vivo.SignificanceIn this study,mPEG,which is approved by FDA and widely used,is applied to the field of AAM for the first time.Through its immune camouflage,we find another way to solve the problem of immunogenic residue of AAM,which has high clinical application value and great commercial potential.