Experimental Study Ischemic Flap Survival Improvement By Composition-selective Fat Grafting With Novel Adipose Tissue Derived Product-Stromal Vascular Fraction Gel

BackgroundFlap necrosis due to insufficient blood supply is a common postoperative complication in random pattern flaps.Stem cell therapies have emerged as promising biologics for tissue ischemia.A novel fat derived product,stromal vascular fraction gel(SVF-gel),can be prepared with lipoaspirate through simple mechanical processing,removing only the lipid content.SVF-gel enriches adipose-derived stem cells and potentially beneficial for flap necrosis.Methods and resultsNude mice ischemic flaps were treated with human SVF-gel,stromal vascular fraction(SVF)cell suspension or saline(n=10).They were injected to the flap recipient beds,and necrosis and vascularization was assessed on postoperative day 14.We harvested the necrosis-free distal to evaluated skin healthiness and neovasculogenesis by Masson's trichrome stain and immunofluorescence,etc.Pro-angiogenic factors were assessed with tissue qRT-PCR.Finally,we traced the grafted human tissue with immunofluorescenceSVF-gel-treated flaps have the smallest necrotic zones(22.05%±0.0438)compared with the saline controls(53.78%±0.1412)or SVF-treated ones(35.54%±0.0850,p=0.039).Numerous functional musculocutaneous perforators were developed around SVF-gel grafts.The SVF-gel-treated skin had the best fat restoration(231.3±48.1?m)among three groups(F=10.83,p=0.0102)while saline-treated flap distal appeared fibrotic.SVF-gel-treated flaps also had-43%more CD31+ capillaries(p=0.0152)with-3 folds more gene expression of angiogenic cytokines of VEGF and bFGF(p=0.0310 and 0.0303,respectively)than salinetreated controls.Furthermore,we found hSVF-gel cells(hGolgi+)had directly engrafted as vessel component(a-smooth muscle actin,a-SMA+)to the flap.ConclusionsAdipose cellular matrix enhanced flap neovascularization partly by direct incorporation,improved flap survival and fat restoration.The composition-selective fat grafting with SVF-gel demonstrated efficacy comparable with stem cell therapy and is especially valuable for clinical translationDiscussionBrowsing literature published in the decade,researchers have been working on improving flaps survival via various approaches but end up focusing on one topic:neovascularization.VEGF is a fundamental GF that required in the angiogenic process.Researchers applied VEGF by direct subdermal injection to minimize necrosis.However,recombinant GF therapies raise safety and efficacy concerns regarding their non-physiology level and short half-life.Then biological vectors(e.g.adeno-associated virus)were adopted to deliver GFs genes like VEGF,basic fibroblast growth factor(b-FGF),and hepatocyte growth factor(HGF),etc.Kuo et al.used extracorporeal shock wave(ESW)to stimulate innate VEGF-dependent angiogenesis.In the decade,scientists have started to use viable cells/stem cells for sophisticated trophic effect and cell replenishment.In reality,even with autologous adult stem cell,engraftment yield is extremely low(<3%)nullifying their curative intent.This means that most cell therapies depend on the temporary paracrine effect.Current experimental solutions raise safety concerns(recombinant GF or biological drugs),require particular instruments(ESW treatment),or meet challenges complying regulations/guidelines(stem cell therapies).Translating them into routine therapies is a highly-complicated process.SVF-gel is a potent and convenient alternative injectable tool containing SVF/ASCs,and the 'minimal manipulation' preparation procedure can be performed in the same flap surgery and shows comparable efficacy with cell therapy.Different from most cell therapies,SVF-gel was delivered on the recipient bed instead ofdiffusing the graft into the flap dermis/subdermal layer.Based on our unsuccessful preliminary data with intra-flap injection,we hypothesized that SVF-gel,as composite tissue,itself needs sufficient oxygen/nutrient from blood supply to survive;thus,this administration route would increase the hypoxic/ischemic burden of the flap.Both SVF suspension and SVF-gel can improve the flap survival;moreover,larger survival area and better angiogenesis were observed in the SVF group.Cells in suspension are easily diffused or absorbed;thus,they are hardly localized in the target region.Furthermore,they lost the intrinsic cell-ECM adhesion.Then diverse biological processes got influenced including cell behavior,metabolism,fate and secreted factors,etc.Stem cells with their niche(e.g.ASCs in SVF-gel)is likely to work differently with cell suspension upon delivery,thus result in variable efficacy.Little h-Golgi-positive cells were found in the SVF group,suggesting SVF suspension improved the flap survival by paracrine effect,instead of direct participation in the tissue differentiation and regeneration.With comparable number of cells of the same population,as we demonstrated before,SVF-gel not only keeps ASCs' cell-ECM interaction but also provide physical protection(adipose ECM niche)and impose better therapeutic effect.It was reported that ASCs delivered in a 3D construct could differentiated into capillary component in vivo.In our flap tissue,we were encouraged to see that SVFgel facilitated angiogenesis partly by directly joining the neovasculature as a-SMA(+)cells on the capillary walls.It is possible that because of the transplanted a-SMA(+)human cells migrated or hASC migrated and differentiated into pericytes.However,this was seldom found in SVF treated flap.Our in vitro also study suggested that cells can migrate from the SVF-gel and have the ability to differentiate into multiple lineage.In this study,we focused on the therapeutic effect of SVF-gel as the first step to develop a cellular/tissue product to enhance flap survival.Herein SVF-gel grafts not only remained,they also demonstrated pro-angiogenic and pro-adipogenesis capacity in non-fibrotic tissue restoration.After free transplantation,SVF-gel's survival is necessary for deliberate trophic factors secretion.This is a possible explanation why flap still exhibited higher levels of angiogenic factors(VEGF and bFGF)expression.Regarding the local retention,SVF-gel can stay at injection site and survive,while most SVF in single-cell suspension were found disappear in 1 week after soft tissue injection.Low cellular retention would inevitably shorten its working period and limit the efficacy.Other than the above possible mechanisms,another study is needed to assess the trophic effect and microenvironmental improvement with SVF/ASC in suspensions and SVF-gel.