Engineered Nanovesicles Derived From Stromal Vascular Fraction For Adipose Regeneration In Dermis Scaffold And Its Mechanism Study

Objective:Insufficient volume retention is the primary problem that needs to be solved in autologous fat transplantation.It is difficult to avoid oil droplet implantation and secondary inflammatory reaction in the transplantation process,which is an important cause for the above problem.Stromal vascular fraction(SVF)based transplantation technology removed part of the oil droplets in adipose tissue and coagulated the active components,and achieved good clinical effects.However,short in vitro preservation time and residual oil droplets still affected the stability of SVF transplantation effect.In situ tissue engineering techniques that induce cells to regenerate fat in the transplanted recipient may break through the volume retention bottleneck.In this paper,adipose tissue was prepared into nanovesicles without oil droplets,which were transplanted together with dermal microparticles with mechanical stress close to adipose tissue to explore the effect and possible mechanism of fat regeneration in situ of the graft.Methods:(1)The nanovesicles(SVF-EVs)were prepared from adipose tissue by mechanical emulsification and multiple filtration membrane extrusion,and the morphology of the vesicles was observed by transmission electron microscopy.The particle size distribution of vesicles was analyzed by a nanoparticle tracker.Fluorescence staining was used to evaluate the effect of vesicle uptake.The protein content in vesicles was determined by the BCA method.One week after the injection of engineered SVF vesicles through the tail vein,the biological application safety of the vesicles was evaluated by the results of HE staining of the mouse organs.Adipose tissue-derived stem cells(ADSCs)were extracted,and the growth of ADSCs was interfered with in vitro vesicles.Alizarin red staining was used to evaluate whether the vesicles promoted adipogenic differentiation.(2)Dermal tissue was prepared into dermal microparticles by mechanical grinding,and the surface morphology of the microgranular scaffold was observed by scanning electron microscopy(SEM).Histological HE and Masson staining were used to evaluate the internal structure of dermal micro granular stents.Cell adhesion and proliferation in the scaffold were evaluated by cell inoculation rate and cell death staining.The dermal micro particles were transplanted subcutaneously into nude mice.After 4 weeks,the mechanical stress of the dermal microparticles scaffold,dermal tissue,and orbital septum fat were compared using a universal tensile tester.(3)Respectively,the preparation of pig source and important engineering SVF vesicles,mixed with corresponding dermal microparticles after subcutaneous transplanted into animals and control group for dermal micro particle mixing normal saline.After 4 weeks and 6 weeks after materials,compare graft volume and quality of change,through histological HE,oil red O,perilipin,CD31,CD206 dyeing evaluation in situ fat regenerative effects,and resistance to human by weeks of immunofluorescence staining nuclear membrane protein renewable source of fat cells.Results:(1)After a series of treatments,the adipose tissue was prepared into engineered vesicles,which showed obvious membrane structure under a transmission electron microscope.Most of the vesicles were distributed between 100 nm and 200 nm in diameter and could be phagocytized into the intracellular cells by endothelial cells.The total protein content of engineered vesicles was positively correlated with the number of vesicles,and the average protein content in a single vesicle was about 4.423×10^-11mg by the BCA method.After the engineered vesicles were injected into the caudal vein,no abnormality was found in the histological HE staining of the nude mice.Engineered vesicles can promote the formation of more intracellular lipid droplets in ADSCs in vitro,and the effect of lipid formation becomes more obvious with the increase of vesicle concentration and the prolongation of intervention time.(2)Injectable particles were formed after dermal grinding.Scanning electron microscopy and histological HE and Masson staining results showed that there were pores between the particles.The inoculation rate of dermal micro granular scaffold cells was 80.27±6.01%,significantly higher than that of dermal mass(50.59±15.29%).A large number of cells were observed in the dermal micro particle at 1,4 and 7 days after implantation.The results of the universal tensile machine showed that the Young's modulus of the micro granular scaffold was 0.368±0.221 MPa,which was consistent with that of the natural adipose tissue(0.114±0.048 MPa),and decreased 88 times compared with that of the dermal tissue(32.378±9.037MPa).(3)After mixed transplantation of porcine-engineered vesicles and porcine dermal microparticles,the graft volume in the experimental group(152.00±24.9mm³)was larger than that in the control group(111.75±26.25mm³)4 weeks later,and there was no significant difference in the mass between the two groups.The oil red O results showed that the staining area of the experimental group was larger than that of the control group,and the central area was more stained.After the mixed transplantation of human-engineered vesicles and human dermal microparticles,the volume of grafts in the experimental group(262.67±33.87mm³)was larger than that in the control group(212.67±40.95mm³)after 4 weeks,but there was no significant difference in the mass between the two groups.After 6 weeks,the volume of the grafts in the experimental group(228.01±24.71 mm³)was still larger than that in the control group(192.83±16.13 mm³),and the mass of the grafts in the control group(244.88±28.09 mg)was lighter than that in the control group(280.08±15.12 mg).Histological perilipin staining showed that there were more regenerated adipocytes in the experimental group than in the control group,and they were more widely distributed.CD206immunohistochemical staining showed that there were fewer macrophages in the experimental group than in the control group.Immunofluorescence staining of the regenerated adipose cells showed that there were no human cells labeled with green fluorescence in the adipose cell band.Conclusion:(1)Adipose tissue can be prepared into 100 nm-200 nm diameter vesicles(engineered SVF vesicles),containing protein components,which can be engulfed by endothelial cells,and has good safety in a biological application and lipogenic ability in vitro.(2)Dermal microparticles have good cytocompatibility.The interspaces between the microparticles are conducive to cell migration and nutrient exchange,and the mechanical stress is similar to that of natural adipose tissue.(3)The engineered SVF vesicles could promote the regeneration of adipocytes in dermal micro granules,which was consistent with the cytological results in vitro,and the volume retention rate was higher than that of the control group.At the same time,it can reduce the infiltration of inflammatory cells,reduce the inflammatory response in the tissue,and facilitate the in-situ regeneration of fat.The regenerated adipocytes in the tissue are most likely to be lipogenic differentiation from the surrounding incoming cells.