The Study Of The Effect Of Washing On Fat Graft Survival And Associated Mechanisms

BackgroundAs an ideal soft tissue grafting material,autologous fat has been widely performed in cosmetic and reconstructive surgery.However,the unpredictability of fat graft survival limits its wider applications.In fat grafting surgery,fat processing plays a key role in fat graft survival.Although plastic surgeons have made great efforts to optimize’ the processing procedure,a standardized method remains undetermined.According to our long-term clinical practice and observation,we found that the outcome of fat grafting was markedly improved after adding washing to fat processing procedures.Although some researchers were also in favor of lipoaspirate washing,in fact,it was only adopted by a small part of plastic surgeons,which is still a debated issue.The damaging effect of blood on fat grafts has been demonstrated in our previously published study.Therefore,it is of great significance to remove blood component by washing.However,it is unclear whether washing to remove other contaminants is still necessary in the absence of significant bleeding.Thus,this study investigated the effect of lipoaspirate washing on fat graft outcome after excluding the influence of blood through animal experiments,and then explored the underlying mechanism.Objectives1.To clarify the effect of washing on fat graft survival by comparing the retention rate and survival quality of the fat particles obtained by sedimentation and washing after removing blood cells;2.To explore the relevant mechanisms of washing on fat grafts from four aspects:inflammatory response,oxidative stress,apoptosis and adipose-derived stem cell(ADSC).MethodsPart Ⅰ:Comparative study on the effect of lipoaspirate washing on fat graft survival 1.Fat harvesting and processing:Young healthy females who underwent abdominal or thigh liposuction were selected as donors.Lipoaspirates naturally separated into three layers from top to bottom after decantation.The infranatant of unwashed and twice-washed lipoaspirate was processed by lysing red blood cells to obtain de-Erythrocyte Infranatant(dEI),and the purified fat was obtained by cotton pad absorption method.2.Animal experiment of fat grafting:dEI or PBS and purified fat were mixed at a ratio of 1:3 and divided into 3 groups:washed group,unwashed group and control group.The grafts were injected to the back of BALB/c nude mice.Fat specimens were harvested at 1 day and 1,3,8 weeks after grafting.Gross observation,Volume and weight retention rates were compared.Hematoxylin-eosin(HE)staining,immunostaining of perilipin-1,and CD31 were conducted.And expression levels of adipogenic associated genes PPAR-y and C/EBPα,angiogenic associated genes VEGF and bFGF were detected by qRT-PCR.Graft survival was comprehensively assessed by the above methods.Part Ⅱ:Investigation of mechanisms of washing to promote fat graft survival1.Detection of inflammatory levels:CD45 and Ly6g immunofluorescent staining was used to respectively detect the number of inflammatory cells and neutrophils in the graft.ELISA and qRT-PCR were used to detect protein and gene expression levels of inflammatory factors IL-6,IL-10,and TNF-α,TGF-β to assess the alteration of inflammatory state.2.Detection of oxidative stress levels:The content of 8-iso-PGF2α protein at day 1 and week 1 was detected to evaluate the level of oxidative stress injury.3.Detection of apoptosis levels:qRT-PCR was used to detect the expression of Bax and Bcl-2 genes at each time point,and the Bcl-2 to Bax ratio was calculated to compare and analyze the trend of apoptosis.4.The effect of dEI on biological characteristics of ADSCs in vitro:ADSCs were extracted from the lipoaspirate and underwent routine culture to the second passage.Immunophenotype of ADSCs were detected by flow cytometry,direct toxicity and proliferation ability of dEI to ADSCs was detected by CCK-8 assay.The adipogenic ability of ADSCs pretreated by washed and unwashed dEIs and PBS was compared.Scratch assay was used to compare the cell migration ability.ResultsPart Ⅰ:At week 3 and 8 after transplantation,volume and weight retention rates,histological scores,number of viable adipocytes and new blood vessels of washed group is higher than unwashed group with statistical significance.No obvious difference was found between washed and control group.The expression levels of related genes such as PPAR-γ,C/EBPα,VEGF and bFGF showed the same trend.Part Ⅱ:1.Inflammatory response:The number of neutrophils at day 1 and overall inflammatory cells at each time point except week 8 of washed group is obviously lower than unwashed group with statistical significance.No obvious difference was found between washed and control group.At the early stage of transplantation,the expressions of pro-inflammatory factors TNF-α and IL-6 of washed group is obviously lower than unwashed group.And at the late stage of transplantation,expressions of anti-inflammatory factors IL-10 and TGFβ of washed group is higher than unwashed group with obvious significance.2.Oxidative stress:At day 1 after transplantation,the expression of 8-iso-PGF2α of unwashed group is higher than washed group,the difference was statistically significant.3.Apoptosis level:After transplantation,Bcl-2 to Bax ratio declined slowly after grafting,reached to a minimum at week 3,and sharply increased thereafter,which indicated a transition from pro-apoptosis to anti-apoptosis.Washed group exhibited higher ratios than unwashed group at each experimental time point.And no statistical difference was noted between washed group and control group.4.The effect of dEI on the biological characteristics of ADSCs:dEI has direct cytotoxicity to ADSCs,but does not affect the proliferation,adipogenic differentiation and migration abilities.Conclusions1.Removal of mixed components except blood cells in lipoaspirate by washing is beneficial to improve the survival rate and quality of fat grafts.2.Relieving inflammation,reducing oxidative stress and inhibiting apoptosis may be the potential mechanisms of washing to promote fat graft survival.