Co-transplantation Of Exosomes Derived From Hypoxia-preconditioned Adipose Mesenchymal Stem Cells Promotes Neovascularization And Graft Survival In Fat Grafting

Background Autologous fat transplantation is one of the most important techniques for repair and reconstruction in plastic surgery.It is widely used in facial deformities caused by various congenital diseases and acquired wounds,breast reconstruction and breast augmentation after breast cancer surgery,and cosmetic surgery.However,the post-operative graft reabsorption and the unpredictability of fat volume retention have been the bottleneck preventing the development of this technology.The study of the mechanism of survival of transplanted tissue indicates that the establishment of early blood supply plays a key role in the survival of fat granules.The stem cell technology that has emerged in the last decade has provided new ideas for this,and surgeons have applied the stromal vascular fraction(SVF),which is rich in stem cells,to clinics and achieved good results,but the underlying mechanism is not clear,moreover,what role stem cells play in this process is also unknown.Exosomes is a new hotspot in stem cell research in recent years.They are nanoscale biological vesicles secreted by cells and serve as a "communication"vector between cells.Previous studies have shown that exosomes participate in a variety of pathophysiological processes in the body and are effective in repairing tissue damage and promoting angiogenesis.It is one of the bearers of the powerful paracrine function of stem cells.Therefore,this study aims to investigate whether stem cell exosomes can promote the survival of free grafted adipose tissue by stimulating the angiogenesis of the graft,and provide a new idea for clinical improvement of the success rate of autologous fat filling surgery.Methods 1.Purification and identification of exosomes.(1)Human primary adipose-derived mesenchymal stem cells(hADSCs)were cultured and induced to differentiate.Specific differentiation was used to identify their multi-differentiation capabilities.Surface markers of mesenchymal stem cells were identified by flow cytometry.(2)The exosomes in the supernatants of hADSCs cultured in vitro were extracted by ultracentrifugation,and the morphological and diameter parameters of the exosomes were measured using transmission electron microscope and Nanosight.Western blotting and flow cytometry were used to detect specific marker proteins on the surface of exosomal membranes.(3)Using Nanosight to examine the morphology of exosomes produced by hADSCs in a hypoxic microenvironment and a normoxic environment,respectively.2.Exosomes play a role in animal models of fat free grafting.(1)Human subcutaneous liposuction fat was transplanted to the back of a nude mouse to establish an animal model.Two kinds of exosomes,hypoxic hADSC-Exo and hADSC-Exo,were injected into the surrounding tissue to establish two experimental groups.(2)Animals were sacrificed at 2 weeks,4 weeks,6 weeks and 8 weeks to obtain specimens and weighed.Laser Doppler flowmetry was used to detect subcutaneous skin mass around the back of cellulite in nude mice before animals were sacrificed.Immunofluorescence staining was used to detect the number of vascular endothelial cells in the specimen.HE staining and immunohistochemical staining were used to examine the pathology of the specimens and the activity of adipocytes.(3)Use angiogenesis-related factor-specific protein microarray to detect the expression and differences of factors in hypoxic hADSC-Exo and hADSC-Exo two kinds of exosomes,as well as the expression and differences of factors in adipose tissue specimens.3.Effects of exosomes on HUVECs in vitro angiogenesis model.(1)Human umbilical vein endothelial cells(HUVECs)were cultured in vitro.The surface markers of endothelial cells were identified by immunofluorescence staining and flow cytometry.(2)With different colors of fluorescence on exosomes and cell membranes,dynamic observation of whether the exosomes can enter the interior of the cell was perforrmed.(2)Using CCK-8,scratch healing test and matrix-filler cavity formation test,the effects of co-culture of exosomes and cells on the proliferation,migration,and lumen formation of HUVECs were examined.Results 1.hADSCs have been successfully cultured,have three lines of differentiation ability,and express mesenchymal stem cell specific marker proteins.The exosomes were successfully extracted from the stem cell culture supernatant,with a diameter of about 100 nm and a "cup" shape under an electron microscope,expressing CD9,CD63 and other exosome membrane-specific proteins.The exosomes produced under hypoxic microenvironment have a larger average diameter and a wider range of diameter distribution than those produced under normoxic conditions.2.Human fat particles co-transplanted with exosomes compared to the control group:(1)The rate of reabsorption decreased,the number of active fat cells increased,and complications such as inflammatory cell infiltration,fibrosis,and formation of oil capsules decreased;2)The recovery of early blood circulation around the graft is accelerated and capillary angiogenesis is increased in the specimen.(3)Compared with hADSC-Exo,vascular endothelial growth factor-related factors increased in hypoxic hADSC-Exo,especially VEGF/VEGF-R family.The trend of expression of relevant tissue specimens is basically consistent with this trend.3.Exosomes can be phagocytosed by HUVECs and can increase HUVECs proliferation,migration,and tube formation.Conclusion 1.Adipose-derived stem cell exosomes can increase the survival rate of free-grafted adipose tissue by promoting angiogenesis and reduce resorption.2.The hypoxic microenvironment can further strengthen this effect by stimulating the adipose stem cells to secrete exosomes containing more growth factors.