The Effect Of The Structure And Viability Of Adipocytes On Fat Grafts Survival

BackgroundAutologous fat transplantation(AFT)has become a well established method of soft tissue augmentation for both reconstructive and cosmetic indications.However,the clinical outcome following fat grafting remains unpredictable and technique-dependent.The mechanisms underlying fat graft survival are not fully understood,particularly regarding the contributions of different cell types.The leading theory of graft survival suggests that tissue remodeling is fueled by the Adipose-derived stem cells(ADSCs).Thus the current studies focus on ADSCs and pay little attention to adipocytes,and lack the understanding of the structure and viability of adipocytes on fat grafts survival.In this study,we first triedtoobtaine fat particles which are different in structure and viabilityofadipocytes—but not in other variables(thesizeoffat particles,the fat components,the number and viability of thestromal vascular fraction cells(SVFs),the number and function of ADSCs,etc)in in vitro experiment,then further studied the fat survival after transplantation in vivo.Lastly,patients who underwentbreast aesthetic and reconstructive surgerieswith autologous fat graftingwere retrospectively recruited,to evaluate the clinical significance of protecting the structure and activity of adipocytes in large volume fat grafting.Objective1.To obtain fat particleswhich are different inadipocytes structure and viability,but are the same in other variables.2.To investigate the fat survival after transplantation of differentfat particles in nude mice.3.To evaluate the clinical effect of the adipocytes structure and viability protecting method in breast augmentation and reconstruction with autologous fat grafting.Methods1.Construct fat particles differing in adipocytesstructure and viability—but not in other variablesHuman lipoaspirate was harvested using conventional liposuction and processed using three separate methods,namely,cotton-pad filtration,soft centrifugation(400 × g,1 min),and Coleman centrifugation(1,200×g,3 min).The three sample types were then subjected to secondary concentration for 15min.The following experiments were used to evaluate whether the fat particles differing in adipocytes structure and viability—but not in other variableswere successfully constructed:the scanning electron microscopy(SEM)was used to study the structure of adipose tissue,and theglucose transport testwas used to measure the viability of adipose tissue.The stromal vascular fractions(SVF)among thethree groups was quntified and chracterized by Muse cell analyzer and flow cytometry.The SVF cells were cultured adherently to the third generation,then Cell proliferation was quantified using Cell Counting Kit-8,and the Multilineage differentiation was initiated using adipogenic,osteogenic,and chondrogenic media.2.Compare fat survival after transplantation of differentfat particles in nude miceThe animal model where granular fat was transplanted was constructed with immunodeficient miceand the transplanted fat was taken out 2 weeks and 8 weeks after the injection.The general feature and volume retention rate of the grafts were compared.The hematoxylin-eosin staining and immunohistochemical staining(perilipin,CD31,vascular endothelial growth factor)were used to compare the quality of grafts in terms of fat survival,angiogenesis and cytokine secretion.3.To evaluate the clinical effect of the adipocytes structure and viability protecting method in breast augmentation and reconstruction with autologous fat graftingPatients who underwentbreast augmentation and reconstructive surgerieswith autologous fat graftingwere retrospectively recruited.The incidence of complications,patient satisfaction,and physician satisfaction assessed by independent surgeons were detected,to evaluate the clinical significance of protecting the structure and activity of adipocytes in high-volume fat transplantation.ResultsPart oneAfter a 15 min secondary concentration,the adipocytes struture and viability was different among the three groups(P<0.05),while the fat components,the number and viability of SVFs,the number of live ADSCs and their proliferation and multilineage differentiation abilities all showed no significant differences(P>0.05),indicating that the fat particleswhich are different in structure and viabilityofadipocytes—but not in other variables was successfully constructed.Part two(1)Volume retention rate:Both at weeks 2 and 8,there were no significant differences in the volume retention rates among the three groups(P>0.05).(2)Hematoxylin-Eosin staining:The better the structure and viability of adipocytes,the better the quality of grafts,with the highest graft viability and the lowest vacuole numbers,necrotic areas,fibrosis,and inflammation.Conversely,the worse the structure and viability of adipocytes,the worse the quality of grafts.(3)Immunohistochemical staining for perilipin:The better the structure and viability of adipocytes,the more active adipocytes after transplantation,and the higher the effective volume retention rate.There were significant differences among the three groups(P<0.05).(4)Vessel counting(CD31+):Both at weeks 2 and 8,the better the structure and viability of adipocytes,the more vascularity was found in the grafts.There were significant differences among the three groups(P<0.05).(5)Vascular endothelial growth factor(VEGF)secretion:At week 2,the better the structure and viability of adipocytes,the more VEGF secrection was found in the grafts.There were significant differences among the three groups(P<0.05).The change of VEGF was parallel to the trend of angiogenesis.Partthree(1)For patients underwentsimultaneous implant exchange with fat,protecting the structure and viabilityof adipocytesas much as possible during the wholeprocessofsurgery could achieve good clinicalresults.The results showed that the breast was natural and soft,the satisfaction of patients and the third independent surgeons were high.Compared with the previous studies,the number of treatments needed to achievesatisfactionwas less,and the incidence of complications was lower.(2)For patients underwent breast reconstruction with autologous fat grafting,protecting the structure and viabilityof adipocytesas much as possible during the wholeprocessofsurgery could achieve good clinicalresults.The results showed that the breast deformity was correctedeffectively,the satisfaction of patients and the third independent surgeons were high.Compared with the previous studies,the number of treatments needed to achievesatisfactionwas less,and the incidence of complications was lower.Conclusion:1.In the first part of the study,through the secondary concentration of fat obtained by different fat purification methods,the differences of fat components,number and viability of SVF cells,number and function of ADSCsamongthethree groups were successfully eliminated,while the differences of structure and viability of adipocytes were retained.In brief,the fat particleswhich are different in structure and viability ofadipocytes-but not in other variables was successfully constructed,laying the foundations for further studies on the effect of the structure and viability of adipocytes on fat grafts survival.2.Better adipocytes structure and viability provided higher-quality fat grafts after transplantation,with higher integrity of adipose tissue,more living adipocytes and vessel density,while with less vacuole,necrosis,fibrosis and inflammation.3.Better adipocytes structure andviability was associated with morefunctional adipocytes,which have better angiogenesis-related cytokines secreting abilityto promote angiogenesis and fat survival.4.For patients underwentbreast augmentation and reconstructive surgeries,protecting the structure and viability of adipocytes as much as possible during the wholeprocessof surgery couldobtain satisfactory clinical effects.