Comparative Study Of Exosome-mimetic Nanovesicles And Exosomes In Improving Survival Rate Of Fat Grafting

Objective: At present,autologous fat transplantation is playing an increasingly important role in plastic and cosmetic surgery.Not only is it widely used in cosmetic surgery such as facial rejuvenation,but also because of its simple operation,small trauma,fast recovery,easy promotion,good biocompatibility,rich sources,and good filling effects,it has also been gradually applied to fill tissue depressions and other tissue and organ repair and reconstruction areas.However,traditional fat collection and injection techniques have shortcomings such as high absorption rate and low survival rate.Especially when a large amount of fat transplantation is required for reconstruction of large-scale body surface tissue defects,the shortcomings of autologous fat transplantation technology limit its further application and promotion.In order to better promote the application of autologous fat transplantation technology in the field of plastic surgery,many methods have been proposed to improve the survival rate of fat grafts.Among them,the regeneration effect of stem cells in the field of fat transplantation has been verified by many studies.However,the regulation mechanism of stem cell proliferation and differentiation is not yet fully clear,and there is a risk of transforming into tumor cells.And recent studies have shown that adipose-derived stem cells(ADSCs)play a role mainly through the paracrine pathway.ADSCs can carry soluble factors by secreting exosomes(EXOs)and perform biological functions.However,the amount of EXOs released by cells is very small,and the acquisition process is cumbersome.One strategy to overcome this shortcoming is to prepare exosome-mimetic nanovesicles(NVs)from cell sources on a large scale.This study aims to compare the effects of artificially prepared nano-scale NVs and EXOs in improving the survival rate of fat transplantation.Methods: 1.Use the hydrodynamic liposuction method to obtain human adipose tissue,and extract the P4 generation hADSCs by enzymatic hydrolysis.Carry out adipogenic osteogenic differentiation induction and flow cytometric identification.2.Extract the NVs and EXOs derived from the P4 generation hADSCs.The morphology,particle size,surface protein label and zeta potential of NVs and EXOs were identified and compared using electron microscopy,NTA,nanoflow and zeta.3.Vitro cell experiment: Fluorescently label NVs and EXOs,and dynamically track the internalization of NVs and EXOs into human umbilical vein endothelial cells(HUVECs).Set up different concentration gradients of NVs and EXOs,and study their optimal concentration to promote HUVECs proliferation,migration,and angiogenesis.4.Animal experiment: Create nude mouse fat transplantation model.The optimal concentrations of NVs and EXOs obtained from the in vitro experiment results were mixed with the adipose tissue obtained by hydrodynamic liposuction,respectively,and transplanted under the skin of nude mice.After observation for 1,2,and 3 months,the nude mice were sacrificed to take out the transplanted tissue.Compare the volume and weight between groups.After sectioning,H&E staining was performed to observe the cell morphological integrity,vacuole formation and fibrosis in the transplanted tissue.Immunohistochemical staining(CD34,VEGF,Ki67)was used to observe neovascularization and proliferation.Results: 1.It can be seen from the optical microscope that hADSCs were slender and spindle-shaped.Oil red O staining results showed that there were red fat droplets with different sizes in the cells.Alizarin red staining showed a characteristic large number of red colored areas.The surface markers of hADSCs expressed CD29+/ CD34+/CD90+/ CD31-/CD45-.2.The electron microscope results showed that NVs and EXOs were both spherical double-layer membrane structures.NTA particle size range was about 50-150 nm.Nanoflow analysis showed positive expression of CD9 and CD81.The zeta potential was negative.3.In vitro experiments we found that when NVs and EXOs were at low concentrations,as the concentration increased,their ability to promote the proliferation,migration and angiogenesis of HUVECs was more significant.When the optimum concentration was reached,the effect of increasing the concentration will decrease instead.The optimal concentration of NVs was 60ug/m L,and the optimal concentration of EXOs was 40ug/m L.4.In vivo experiments we have verified that NVs and EXOs can effectively improve the survival rate of fat transplantation.Moreover,the mechanism of NVs and EXOs improving the survival of fat transplantation was mainly related to the promotion of neovascularization.Conclusions: The shape,size,and membrane structure of NVs were similar to EXOs.In the field of fat transplantation,it can achieve the same effect as EXOs.At the same time,the output of NVs was much higher than that of EXOs.Therefore,it is expected to replace EXOs in the field of fat transplantation and provide new ideas for other regenerative medicine fields.