Research About Extracellular Vesicles Improve The Survival Of Transplanted Fat Grafts And The Related Mechanisms

Background:Free fat grafting is a promising surgical technique for soft-tissue augmentation,reconstruction,and rejuvenation.Despite these uses,transplanted fat often has a low survival rate,and adipose tissue can be quickly resorbed and replaced by fibrous tissue and oil cysts.Currently,the generally accepted explanation for the decreasing volume of fat is insufficient blood supply after transplantation.In addition to cell-based therapies for the treatment of ischemic diseases,interest in the vesicles released by these cultured cells has recently been increasing.A number of work showed that bone marrow mesenchymal stem cell-derived extracellular vesicles(BMSC-EVs)are pro-angiogenic.Based on this pro-angiogenic potential of BMSC-EVs,we hypothesized that co-transplantation of BMSC-EVs and fat during grafting would prevent fat cell death and stimulate neovascularization,preventing graft resorption.Objective:1)Our goal in this work was to explore hypoxia-Induced changes of the quantity of EVs secreted by BMSCs.2)To observe the effects of BMSC-EVs on the survival of transplanted fat grafts,and the possible mechanisms were also explored.Methods:1)EVs from BMSC culture supernatants were isolated through a series of ultracentrifugation steps.To ensure that the EVs were correctly identified,scanning electron microscopy,particle sizing analysis, and flow cytometric technique were used.2)Human fat tissue grafts with various concentrations of BMSC-EVs were subcutaneously injected into nude mice.Twelve weeks after transplantation,the animals were sacrificed and the grafts harvested.The harvested fat tissue grafts were weighed on a balance and their volumes were determined by the liquid overflow method.3)Human fat tissue grafts with BMSC-EVs were subcutaneously injected into nude mice.One week after transplantation,the animals were sacrificed and the grafts harvested.PDGFA,PDGFA-R,TGF-βand VEGF gene expressions were analyzed by qRT-PCR.Results:1)Scanning electron microscopy showed that the EVs were heterogeneous with a spheroid-shaped morphology,and particle sizing determined that the average EV diameter was 228.4±28.8 nm.Flow cytometry showed that the EVs expressed high levels of CD81,CD63,CD90,CD29,but not CD31 and CD45,confirming that they were of BMSC origin.2)The experiments showed that the total protein content was 2.455± 0.426 mg/10~6 BMSC in normoxia,significantly lower than that of cells in hypoxia(0.523±0.098 mg/10~6 BMSC,P<0.05).3)The grafts in the EVs-treated groups appeared larger than those in the non-treated group,and the tissue weight and volume analyses confirmed that the EV treatment increased the graft retention size.4)More CD31-positive capillaries were observed in the EVs-treated groups than in the control group.Further,qRT-PCR analysis of the grafts showed significantly higher expression levels of PDGF-A,PDGFR-A,VEGF-A and TGF-βin the EV-treated group compared with those in the control group.Conclusion:1)Hypoxia can increase the release of EVs from BMSC.2)Supplementation of fat grafts with BMSC-EVs improves the long-term retention of the transplanted fat in a mouse model.This beneficial effect is likely mediated by the pro-angiogenic effects of the BMSC-EVs.