Exosomes From Adipose-Derived STEM CELLS by Biogenomics

StemCells:TheEmergingRolesand

ApplicationsinTissueRegeneration ofPlasticandCosmeticSurgery

MingchenXiong † ,QiZhang † ,WeijieHu,ChongruZhao,WenchangLv,YiYi,YipingWu* andMinWu*

1 DepartmentofPlasticSurgery,TongjiHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology, Wuhan,China

Adipose-derivedstemcells(ASCs)areanimportantstemcelltypeseparatedfrom adiposetissue,withthepropertiesofmultilineagedifferentiation,easyavailability,high proliferationpotential,andself-renewal.Exosomesarenovelfrontiersofintercellular communicationregulatingthebiologicalbehaviorsofcells,suchasangiogenesis, immunemodulation,proliferation,andmigration.ASC-derivedexosomes(ASC-exos) areimportantcomponentsreleasedbyASCsparacrine,possessingmultiplebiological activities.Tissueregenerationrequirescoordinated“vitalnetworks”ofmultiplegrowth factors,proteases,progenitors,andimmunecellsproducinginflammatorycytokines. Recently,ascell-to-cellmessengers,ASC-exoshavereceivedmuchattentionfor thefactthattheyareimportantparacrinemediatorscontributingtotheirsuitability fortissueregeneration.ASC-exos,withdistinctpropertiesbyencapsulatingvarious typesofbioactivecargoes,areendowedwithgreatapplicationpotentialintissue regeneration,mechanicallyviathemigrationandproliferationofrepaircells,facilitation oftheneovascularization,andotherspecificfunctionsindifferenttissues.Here,this articleelucidatedtheresearchprogressofASC-exosabouttissueregenerationin plasticandcosmeticsurgery,includingskinanti-agingtherapy,dermatitisimprovement, woundhealing,scarremoval,flaptransplantation,bonetissuerepairandregeneration, obesityprevention,fatgrafting,breastcancer,andbreastreconstruction.Deciphering thebiologicalpropertiesofASC-exoswillprovidefurtherinsightsforexploringnovel therapeuticstrategiesoftissueregenerationinplasticandcosmeticsurgery.

Keywords:adipose-derivedstemcells,exosomes,tissueregeneration,biologicalactivity,function

INTRODUCTION

Adiposetissueprovidesamajorenergystoragedepotforthebody.Overthepastyears,adipose tissuehasbeenconsideredasamultifunctionalorganthatcontrolsmetabolichomeostasis, immunity,andsatiety(Yangetal., 2018).Adiposetissuecanbebroadlyseparatedintofat-storing adipocytesandtheadiposetissuestromalvascularfraction(SVF).SVFisaheterogeneouscellgroup thatistraditionallyisolatedusingenzymessuchascollagenase.SVFisaneasilyaccessiblesystem comprisedofvariousimmunecells,erythrocytes,endothelialcells,andadipose-derivedstemcells (ASCs),withtheexceptionofadipocytes(KimandLee, 2020).ASCsaredeﬁnedasasubsetof mesenchymalstemcells(MSCs)isolatedfromtheSVFwithinadiposetissuebyenzymaticdigestion

(Gentileetal., 2019b).ASCsarespecificallyvaluablebecause theycanbeeasilyharvestedwiththepropertiesofabundance andconvenientseparation.Intermsofcellidentification,ASCs exhibitamesenchymal-likemorphologyandtheexpression profileofCD34+ ,CD44+ ,CD31 ,andCD45 cellsurface markers(Shuklaetal., 2020).ASCsarenotonlyprecursorsto adipocytesbutalsomultipotentprogenitorstoavarietyofcells includingosteoblasts,chondrocytes,myocytes,epithelialcells, andneuronalcells.Furthermore,ASCspossessseveralunique characteristics,includingeasyavailability,highproliferation potential,self-renewal,andsecretionoftrophicfactorsand extracellularvesicles(EVs),thusoeringafeasibleandvalid alternativetoothersourcesofMSCs,suchasbonemarrow-MSCs (BMSCs)(Mazinietal., 2019).Thesecretedfactorsderivedfrom ASCs,suchasgrowthfactorsandcytokines,areknowntoexert paracrinesignalsresponsibleforchemoattractant,angiogenic, andprosurvivaleectsrequiredfortissueregeneration(Bajek etal., 2016).ASCsareparticularlyusefulastheycanbe easilyharvestedwithminimaldonorsitemorbidityandhave adierentiationpotentialsimilartootherMSCs.Thus,ASCs havebeensuccessfullyproposedasaprominentcandidateinthe developmentoftissueengineeringproducts.Aswell,plasticand cosmeticsurgeryisagrowingfielduniquelypositionedforthe applicationofASCs(Zuketal., 2001).

Theterm“exosome”specificallydefinesasmallsubsetof extracellularvesiclesthatrangefrom50nmto200nm, whichhavebeenfoundinnumerousbodyfluids,including blood,urine,cerebrospinalfluid,breastmilk,saliva,lymph, andbile,underbothhealthyandpathologicalconditions (Wangetal., 2018).Exosomesareformedwhentheendosome membraneinvaginatestoproduceamultivesicularbody whichuponfusingwiththecellmembranetoreleasethe vesicleswithinasexosomesintotheextracellularspace(Toh etal., 2018).Consequently,thekeyexpressionbiomarkersof exosomes,whicharegenerallyrecognizedasexosome-associated characteristics,areproteinsassociatedwithendocytosisand endosomaltrackingssuchastetraspanins(CD81,CD63,CD9), ALIX,TSG101,caveolins,clathrin,andtransferrinreceptors, duetothedierentmechanismsofsecretion(HongP.etal.,

Abbreviations: gH2AX,PhosphorylatedHistoneH2AX;AKT,ProteinKinase B;AP-1,ActivatorProtein1;Arg-1,Arginase1;ASC-CM,ASC-Conditioned Medium;ASC-exos,ASC-derivedexosomes;ASCs,Adipose-derivedStemCells; BAT,BrownAdiposeTissue;Bax,Bcl-2-associatedXProtein;Bcl-2,B-cell lymphoma/leukemia2;CAL,Cell-AssistedLipotransfer;Caspase-3/9,Aspartate ProteolyticEnzyme3/9;ECM,ExtracellularMatrix;EVs,ExtracellularVesicles; H2 O2 ,HydrogenPeroxide;HaCaTs,HumanKeratinocytes;HDFs,Human DermalFibroblasts;I/R,Ischemia-Reperfusion;IFN-g,InterferonGamma;IL4/5/6/13,Interleukin4/5/6/13;iNOS,InducibleNitricOxideSynthase;MAPKs, Mitogen-ActivatedProteinKinases;MMP-1/9,MatrixMetalloproteinase1/9; MSCs,MesenchymalStemCells;NF-kB,NuclearFactorKappaB;NOX-1/4, NADPHOxidase1/4;Nrf2,NF-E2-relatedfactor2;PDGF-AA,Platelet-Derived GrowthFactor-AA;PGE2,ProstaglandinE2;PI3K,Phosphatidylinositol-4,5Bisphosphate3-Kinase;RANKL,ReceptorActivatorofNuclearFactorKappa BLigand;ROS,ReactiveOxygenSpecies;SA-b-gal,Senescence-Associated b-galactosidase;SMP30,SenescenceMarkerProtein30;SVF,StromalVascular Fraction;TGF-b,TransformingGrowthFactorBeta;TIMP-1,TissueInhibitorof Metalloproteinases1;TNF-a,TumorNecrosisFactorAlpha;TSG-6,TNF-AlphaStimulatedGene/Protein6;UCP-1,UncouplingProtein1;UVB,UltravioletB; VECs,VascularEndothelialCells;VEGF,VascularEndothelialGrowthFactor; WAT,WhiteAdiposeTissue.

2019).Exosomesarepackedwithcell-type-speciﬁccombinations ofproteins(cytoskeletalproteins,transmembraneproteins,and heatshockproteins),nucleicacids(DNA,mRNA,miRNA,long andshortnon-codingRNA),lipids,andenzymes(GAPDH, ATPase,pgk1),shuttlingtheseactivecargoesbetweendierent cellsinvolvinginacomplexintercellularcommunicationsystem. Thesecargoesarewrappedinthemembranetoprotectfrom degradationandtransporttothesurroundingcells(Kalluriand LeBleu, 2020).Thus,exosomeshavesomespecialbiological characteristicsandprocessesandarecapableofpotentially modulatingthespeciﬁcactivityoftherecipienttargetcells.

Theimportantaimofplasticandcosmeticfieldisclosely associatedwithtissueregeneration,andtorepairthemorphology andfunctionofcongenitaloracquireddefectsthroughmany treatmentmethods,includingmedicalimaging,microsurgery, compositetissueallotransplantation,nanotechnology,cell biology,andbiomaterials(Naderietal., 2017).Adiposetissue possessesimportantfunctionsinimmunemodulation,wound healing,andtissueregeneration.Nowadays,autologousadipose tissueapplicationisexploredtobeappliedinimprovingskin quality,contourirregularities,woundrepairandsofttissue regenerationinplasticandcosmeticsurgery(Strongetal., 2019). Itsecacyandsafetyarewidelyaccepted,butthereisalack ofuniversallyrecognizedmechanisms.However,giventhatthe characteristicsofadiposetissuevarydramaticallydependingon thedonorstatus,theeectisofindividualdierence(Wang etal., 2013).Besides,significantlyalteringthecomponents orbiomechanicalpropertiesoftheadiposetissue,suchasby removingstromalcellsfromtheadiposetissue,willsubjectto morecomplexapplications.Therefore,autologousadiposetissue cannotbeeasilyusedasadrug.Thus,ASCsandASC-exosare veryimportantderivativesfromfattissue,capturingintensive attention.ExosomescarryspecificcontentsoftheparentalASCs, includingDNAs,RNAs,lipids,cytokines,enzymes.Exosomes arecapableofprotectingtheircargoesfromdegradationand arehighlystableinserumandblood,thusecientlydelivering cargoestotargetcells.ASC-exosandASCsperformtheir functionsviadierentmechanisms.TheASC-exosareusedas toolsforrepairingandregeneratedactivationofdamagedcells, andarenowconsideredtoorchestratetheeventsrequiredfor tissueregeneration,immunefunction,tissuehomeostasisand developmentofcellfate.Hence,althoughwithoutdierentiation ability,ASC-exoscanmimicthecapacityofASCsforinnovative cell-freetherapy,suchastissueregenerationandrepair,reduction ofinjuries,andanti-inflammation.Intermsofstorageand delivery,unlikeASCs,exosomesaresmallnon-livingsubstances thatcanbesterilefilteredandfrozenwithoutcryo-preservatives. Frommanufacturingandstoragetodelivery,thereisnoneed tomaintaincellviabilityandfunction.ASC-exoshavecertain advantagesoverASCsinproduction,storage,shelflife,delivery, andpotentiallyready-to-usebiologicalproducts(Vizosoetal., 2017).Moreover,ASC-exosarepotentiallysafertherapeutic agentsthanASCs.ComparedwithASCs,ASC-exosmight avoidcelltherapy-associatedproblems,includinglimitedcell survival,immunerejectionecacy,senescence-inducedgenetic instability,inactivatefunction,andthepossibilityofunfavorable dierentiation(Figueroaetal., 2014).Owingtotheirmultiple

features,ASC-exoshaveshowntherapeuticpotentialinmany clinicaldiseases,especiallyintissueregeneration,suchasskin repairing,fatgrafting,andvariousreconstructionoperation.In addition,exosomeshavebeeninnovativelyutilizedfortargeted drugdeliveryandasgenecarriersforregenerativemedicine (Mehryabetal., 2020).Butactually,ASC-exoslackenough clinicaltrialstoconﬁrmthesafetyandeectiveness(Table1).

Inthisreview,wemainlysummarizethelatestresearchabout thefunctionsandinvestigationsofASC-exosconcernedwith tissueregenerationinplasticandcosmeticsurgery,including skinanti-agingtherapy,dermatitisimprovement,woundhealing, scarremoval,ﬂaptransplantation,bonetissuerepairand regeneration,obesityprevention,fatgrafting,breastcancer,and breastreconstruction.Wehopethiswillprovidefurtherinsights intothepivotalrolesandapplicationsofASC-exosintissue engineeringandregenerativetherapies.Forthesegoals,we searchedtheadipose-derivedstemcells/ASCs,exosomesandthe

ASC-exosASCs

SourceacquisitionfromASCswith exosomeseparationmethods, existinadiposetissueand stableinserumandblood

easyacquisitionandhighyield fromadipocytetissues, especiallyfromwhiteadipose tissue

Morphologysmalllipidbilayervesiclesmesenchymal-likecells

Managementcouldbesterileﬁlteredand frozenwithout cryo-preservatives,easily long-termstorageanddelivery, easilykeepbiologicalactivity

shouldpreservecellviability andfunctionfrommanufacture tostorageanddelivery,high storagerequirements,complex cultivation

Biological properties protectcargoesfrom degradation,targetspeciﬁcity, goodtissuepermeability, intercellularcommunication, immunefunction,tissue homeostasisanddevelopment ofcellfate multi-lineagedifferentiation, secret greatkindsofgrowthfactors byparacrinefunction, prosurvivaleffects,regulation ofimmunefunction, angiogenesis

SecretomeDNAs,RNAs,lipids,cytokines, enzymesfromtheparentcell exosomes,cytokines,DNAs, RNAs,lipids,enzymes

Biosafetylimitedimmunogenicity,high biosafety immunogenicity,biosafety

Applicationsconsideredasmultiple bioactivesubstancesfortissue regeneration,couldbegene modiﬁcation,uploaddrugsas carriers,uploadinothercarriers suchasnanomaterials

Clinicaltrialslackenoughclinicaltrialsto conﬁrmthesafetyand effectiveness

consideredasidealstemcell sourceforcellandtissue regeneration;couldbegene modiﬁcation,uploadincarriers suchasnanomaterials

securityandeffectivenessare veriﬁedinmanydiseases

Application disadvantages relativelowpurityandyield, complicatedcomponents, substantialdegreeof heterogeneityindosing regimensinthereportedcases, lacking invivo clinicaltrials limitedcellsurvival,immune rejectionefﬁcacy, senescence-inducedgenetic instability,inactivatefunction, andthepossibilityof unfavorabledifferentiation, individualdifferences

Abbreviations:ASCs,Adipose-derivedstemcells;ASC-exos, ASC-derivedexosomes.

above8relatedﬁeldsinplasticandcosmeticsurgeryaskeywords onPubmedintherecent5years.Thesesearchedstudiesinvolved incell,animalexperiments,orclinicaltrials,especiallythe originalarticleswereincludedaccordingtorelevancetothetopic.

ASC-EXOSINSKINAGING

Theskinissubjecttoanunpreventableintrinsicaging process,alongwiththeexogenousfactors-inducedagingstate. Particularly,ultravioletradiationresultsinprematureskinaging, alsoknownasextrinsicskinagingorphotoaging.Themost typicalfeaturesofskinagingarethelossofelasticityandthe generationofwrinkles,whichareattributedtothestructural andfunctionalchangesinskincellsandtissues(Lietal., 2019).Amongskincells,humandermalﬁbroblasts(HDFs)and keratinocytes(HaCaTs)areregardedasbarrierstopreventskin fromtimeagingandultravioletB(UVB)photoaging.Various improvementstrategies,suchasantioxidants,retinoids,peptides, growthfactors,andautologouspatientfatorcollagengraft,have beenusedtoﬁghtagainstskinaging(Kimetal., 2019).Because ofthepoorpenetrationthroughthestratumandshort-term maintenanceforseveralmonths,theseabovestrategiesarenow notideal.ItishopefultoreduceskinagingpinnedonASCs andASC-derivatives,whichcouldregulateHDFsproliferation, migration,andcollagenexpression.

ASC-conditionedmedium(ASC-CM)andASC-exos,both containingkeycytokinesandgrowthfactorssecretedbythe ASCs,couldfacilitatetheregenerationandrepairofvarious tissuesandorganstoexertinﬂuencesonanti-oxidation,antiwrinkle,andwhiteningskin.ASC-CMhasbeenprovedto protectHDFsfromoxidativestress invitro (Kimetal., 2008). Lietal. (2019) foundthatinUVBirradiation invitro model,ASC-CMcouldeectivelydown-regulatetheactivation andtranscriptionofUVB-inducedsignalingpathwayssuchas mitogen-activatedproteinkinases(MAPKs),activatorprotein 1(AP-1),andnuclearfactorkappaB(NF-kB),andupregulatetheexpressionofantioxidantresponseelementssuch asphaseIIgeneHO-1andtransforminggrowthfactor-beta (TGF-b),whilereducinginterleukin6(IL-6)secretion.Thereby ASC-CMshowedapositiveeectonprotectingHDFsand HaCaTsfromUVB-inducedphotoagingdamage.TheplateletderivedgrowthfactorAA(PDGF-AA)containedinASC-CM alsocouldactivatethephosphatidylinositol-4,5-bisphosphate3kinase(PI3K)/proteinkinaseB(AKT)signalpathway,and mediatephotoaging-inducedHDFsproliferation,extracellular matrix(ECM)depositionandremodelinginthe invitro experiment,whichwasreportedby Guoetal. (2020) group. Itdemonstratedthatthewell-preparedASC-CMplayeda positiveroleinpreventingHDFsfromintrinsicandextrinsic agingdamagestoacertaindegree.Meanwhile,theresult alsoclariﬁedthatthePDGF-AAmightcontributetobetter outcomeswithsomeothercomponentsofASC-CM.However, theingredientsinASC-CMarerathercomplextosynergistically achievetheanti-aginggoal.Theexosomesareimportant componentsinASC-CM,mightpossessapositivelyindependent orsynergisticroles. Huetal. (2019) showedthatexosomesfrom

TABLE1| CharacteristiccomparisonofASC-exosandASCs.

three-dimensionalculturedHDFspheroids(3D-HDF-exos)and BMSC-exoscouldbothdown-regulatetumornecrosisfactor alpha(TNF-a)andup-regulatedTGF-b expression,resultingin decreasedmatrixmetalloproteinase1(MMP-1)andincreased typeIprocollagen invitro andanudemousephotoagingmodel. Theseresultsindicatedthattheexosome-containing3D-HDFexosandBMSC-exosbothhadanti-skin-agingpropertiesandthe potentialtopreventandtreatcutaneousaging(Figure1A).

Atpresent,theexistingresearchofASC-exosonskinagingis limited.Astheepidermislayeris50–120 µmandtheepidermisdermisthicknessis2–5mminhumans,thelocaltreatmentwith exosomescanarriveattheepidermisandbeabsorbedonhuman skin(Xuetal., 2018).Exosomesderivedfromhumanstemcells, suchasASCs,areofmultiplebioactivefunctionsforskinaging treatment,deservingfurtherresearch.

ASC-EXOSINATOPICDERMATITIS

Atopicdermatitis(AD)isachronicinﬂammatoryskindisease accompaniedwithpruritus,erythema,edema,excoriation, andthickeningoftheskin,leadingtodecreasedunaesthetic

appearanceofskin(Leeetal., 2018).Bothdefectiveskinbarrier andabnormalimmuneresponsesarecrucialfactorsinAD development.ThereforeADwithimmunologicabnormalities couldbetreatedbydierentormulti-prongedapproaches focusedonreducingtheseverityandfrequencyofsymptoms, suchasdietarymanagement,drugtherapy,andultraviolet assistedtherapy(Parketal., 2019).Severalstudieshave demonstratedthatallergicprogressinADcouldbesuppressed byBMSCsandASCswhilemodulatingmultipletargets(Shin etal., 2017).Forinstance, Sahetal. (2018) foundthatsuperoxide dismutase3-transducedMSCsamelioratedADpathologyand enhancedtheecacyofMSCtherapybycontrollingactivated immunecells,reducingexpressionlevelsofpro-inﬂammatory mediatorsintheskinofADmice.

Nevertheless,giventhatADchronicandrecurrent characteristics,thetherapeuticutilizationofMSCshasseveral drawbacks,suchaspoorengraftmenteciency,undesired immuneresponses,shorthalf-life,anddicultiesinquality control(Louetal., 2017).Exosomesareinvolvedinthe developmentandprognosisofADskindiseases,including repairingleakyskinbarriersaswellassuppressingskin inﬂammation(Alvesetal., 2016). Choetal. (2018) established

FIGURE1| ASC-exos function invariousskinassociatedapplications. (A) ASC-CMandBMSC-exoscouldproduceROSatalowlevel,downregulateTNF-a, upregulateTGF-b toincreaseMMP-1andprocollagentypeIexpressionforcollagensynthesis,thusenhancingtheskinelasticityandeasethewrinklesfor anti-aging. (B) ASC-exoswascapabletoenhancestratumcorneumhydration,reducethesecretionofinflammatorycytokinessuchasIL-4,IL-5,IL-13,IFN-g,and TNF-a,andalleviatetheinfiltrationofmastcells,dendriticepidermalcells(DECs)inskinlesionsandeosinophilsintheblood,andproduceceramidestorestorethe epidermalbarrier,thusrelievingthedermatitisofskin. (C) ASC-exosreducedtheproductionofROS,decreasetheexpressionofIL-6,IL-1b,TNF-a,andthe oxidativestress-relatedproteinssuchasNADPHoxidase1/4(NOX1/4),increaseMMP-9andVEGFtoameliorateECMreconstruction,thusfosteringHDFs proliferationandmigrationtoreinforcethere-epithelialization. (D) ASC-exoswasconducivetopromotetubeformationofVECs,increasetissuethickness,and reducetheinfiltrationofinflammatorycellstorelievetheinflammationandapoptosisforthehighsurvivalrateoftheskinflap.ASCs,Adipose-derivedstemcells; ASC-exos,ASC-derivedexosomes;HDFs,HumanDermalFibroblasts;HaCaTs,HumanKeratinocytes;ECM,ExtracellularMatrix;ROS,ReactiveOxygenSpecies; MMP-1/9,MatrixMetalloproteinase1/9;IFN-g,InterferonGamma;TNF-a,TumorNecrosisFactorAlpha;TGF-b,TransformingGrowthFactorBeta;IL-4/5/6/13, Interleukin4/5/6/13;NOX-1/4,NADPHOxidase1/4;VEGF,VascularEndothelialGrowthFactor;VECs,VascularEndothelialCells,VECs.

anADmodelofNC/NGAmicetreatedwithhousedust miteantigens.Inthismousemodel,ASC-exoswerefoundto amelioratepathologicalsymptomssuchasthelevelsofserum IgE,thenumberofeosinophilsintheblood,andtheinfiltration ofmastcells,dendriticepidermalcellsinskinlesions.Thestudy suggestedtheimmuneregulationroleofASC-exosinAD.Inthe latterstudy,thesamegroupalsofoundthatthesubcutaneous injectionofASC-exosinanoxazolone-induceddermatitismodel remarkablyreducedtrans-epidermalwaterloss,andenhanced stratumcorneumhydrationandmarkedlydecreasedthelevels ofinflammatorycytokinessuchasIL-4,IL-5,IL-13,TNF-a, interferongamma(IFN-g),IL-17andTSLP,allinadosedependentmanner(Shinetal., 2020).Interestingly,ASC-exos alsoinducedtheproductionofceramidesanddihydroceramides topromoteskinbarrierrestoration(Shinetal., 2020).These studiessuggestedthatthesystemicadministrationofASC-exos amelioratedAD-likesymptomsthroughtheregulationof inflammatoryresponsesandthepotentialofeectivelyrestoring epidermalbarrierfunctionsinAD(Figure1B).ASC-exos couldbeapromisingcell-freecandidatetocurrentlylimited treatmentoptionsforAD.

ASC-EXOSINWOUNDANDSCAR

Manyexposed,unsightly,orchronicwounds,suchasdiabetic ulcers,arediculttoheal,notonlycausingfunctional disabilitiesbutalsoaectingmentalhealth.Poorwoundhealing eventuallyleadstohypertrophicscarsorkeloidformation, pigmentation,prolongedhealing,andulcerativeskindefects (Emingetal., 2014).Therearemanytraditionaltreatment methodsforskinandsofttissuetrauma,suchaslow-intensity lasers,advancedtreatmentdressings,negativepressurewound treatment,hyperbaricoxygen,andskintransplantation(Bellei etal., 2018).However,aswoundhealingisacomplicatedprocess referringtomultiplecelltypes,growthfactors,andextracellular matrix,sometraditionaltreatmentsjustplayanauxiliaryrole accompaniedbyundesirablehealing.

ASCsandSVFcanproduceabundantsecretomegroups, leadingtocellproliferationanddierentiation,migration,and healingmicroenvironment.ThemigrationandfunctionsofASCs couldbeenhancedviaPI3K/AKTpathwayactivatedbyintegrin b1,resultedintheimprovedchronicrefractorywound(WangJ. etal., 2020).Interestingly,inthewoundmousemodel, Bi etal. (2019) foundthatbothSVFandhumanASCsimproved thefunctionofendotheliocytesandfibroblasts,regulatedgene expression,andjointlypromotedskinhealing.Thisstudyshowed thatSVFcouldreplaceASCsforwoundhealing,duetothe convenienceofSVFapplications.Intheburnwoundmodel, onlyautologousASCs,butnotallogeneicASCs,significantly improvedhealinginacuteburnwoundsoftherat(Changetal., 2018). Fujiwaraetal. (2020) alsoconstructedanovineburn modelandprovedthatASCsimprovedgraftedburnwound healingbypromotingbloodflowandvascularendothelialgrowth factor(VEGF)expression.Besides,thelocalizedinjectionof ASCscouldaccelerateandenhancetheclosureofpressureulcers (Xiaoetal., 2019).Likewise, Bukowskaetal. (2020) systematically

ensuredthesafetyofhumanSVFwheninjectedintoamurine pressureulcerinjurymodel.Thishealingfunctionusually dependsonthetrophicfactorsofASCsandSVF,including cytokines,growthfactors,andchemokines.Notably,exosomes containingsecretomefromASCshaveopenedthewaytoanewly emergingcell-freetherapy.

StudieshaveshownthatASC-exosplayedapositiverolein cutaneouswoundhealingbymeansofactingonHDFsand HaCaTsandothermaintargetcellsthroughvarioussignal channels(Figure1C; Qiuetal., 2020). Maetal. (2019) exposedHaCaTstohydrogenperoxide(H2 O2 )forestablishing askinlesionmodel,discoveringthatASC-exoscouldfoster HaCaTsproliferation,migration,andinhibitapoptosisthrough Wnt/b-cateninsignalingpathway.Likewise, Heetal. (2020) also confirmedthatMALAT1-containingASC-exosimprovedwound healingbytargetingmiR-124andactivatingWnt/b-catenin pathway.Inaddition,ASC-exosmightalsopromoteand optimizecollagensynthesisviaupregulatingPI3K/Aktpathway duringcutaneouswoundhealing(Zhangetal., 2018). LiX. etal. (2018) foundthatexosomesfromNF-E2-relatedfactor 2(Nrf2)-overexpressingASCssignificantlyreducedtheulcer areainthefeetofdiabeticrats,bypromotingtheproliferation andangiogenesisofendothelialcells,improvinglevelsof senescencemarkerprotein30(SMP30)andVEGFandvascular endothelialgrowthfactorreceptor2(VEGFR2)phosphorylation toacceleratethewoundhealing,aswellasinhibitingreactive oxygenspecies(ROS)productionandinflammatorycytokine expressions,suchasIL-1b,IL-6,andTNF-a.Notwithstanding thissuperiority,exosome-basedtherapyofwoundhealingstill facesthechallengesofrapidclearancerateandrelativelyshort half-life invivo (Liuetal., 2017).Thesustainedreleaseand retentionofexosomesinthetargetareaisanimportantfactor forhealing.Liuetal.showedhyaluronicacid(HA)mightserveas exosomesimmobilizerandwounddressingfordurableexosomes retentionatwoundsitestoeectivelyreparativeeect.ASCexoscombinedwithHAwasabletoactivatetheHDFsactivity ofthewoundsurfaceandreinforcedthere-epithelializationand vascularizationofthewoundsurface(Liuetal., 2019).Moreover, severalstudieshaveconfirmedtheroleofASC-exosmiRNAsin skinhealing.Forinstance, Yangetal. (2020) foundthathighly expressedmiRNA-21derivedfromASC-exoscouldenhancethe migrationandproliferationoftheHaCaTs,byincreasingthe matrixmetalloproteinase9(MMP-9)expressionthroughthe PI3K/AKTpathway.TheoverexpressingmiRNA-21couldalso enhancecollagensynthesisandoptimizecollagendeposition, significantlyimprovethehealingeectoffull-thicknessskin woundsinmice(Yangetal., 2020). Shietal. (2020) verifiedthat mmu_circ_0000250-modifiedASCsderivedexosomespromoted woundhealingindiabeticmicebyinducingmiR-128-3p/SIRT1mediatedautophagy.

Comparedtothesinglefactortherapy,MSCsapplicationis alsosuperiorinscarremovalduetotheMSCs-secretedvarious inflammatorymodulators.Intherabbitscarmodel, Liuetal. (2014) locallyinjectedBMSCstoregulateinflammationand preventedhypertrophicscarformation,attributingtoBMSCs secretionofananti-inflammatoryprotein,TNF-a-stimulated gene/protein6(TSG-6). Wuetal. (2015) showedthat invitro

assay,theMSC-CMdecreasedviability, a-SMAexpression, andcollagensecretionofhumankeloidfibroblasts.Besides, inamousedermalfibrosismodel,MSC-CMinfusioninduced asignificantdecreaseinskinfibrosisduetotheTGF-b3 inCM-mediatedtherapeuticeectsonpreventingcollagen accumulation(Wuetal., 2015).TheapplicationofASCs andASC-derivativesmightalsoprovidenovelscarlessrepair methods.Intheearlystageofwoundhealing,exogenousASCexospromotedtheexpressionoftypeIandtypeIIIcollagento shortenthehealingtime,andmightinhibitcollagensynthesis tominimizescarformationinthelaterperiod(Huetal., 2016). Thistendencyfollowedthehistologicalchangesobservedduring thenaturalhealingofsofttissuewounds.Thatis,collagen depositionwasmoreimportantintheearlyphaseofhealing, whileinthelatephaseofhealing,matrixreconstructionwas morecritical.ASC-exosamelioratedECMreconstructionand reducedthescarformationbyregulatingtheratiosoftypeIII collagen/typeIcollagen,TGF-b3/TGF-b1,andMMP-3/tissue inhibitorofmetalloproteinases1(TIMP-1),aswellasfacilitating HDFsdierentiation(Wangetal., 2017).TheteamofWang etal.firstlydevelopedtheFHEhydrogelandFEPhydrogel scaoldbothwithstimuli-responsiveASC-exos.TheASC-exos releasedbythesetwocarriermaterialssignificantlyincreasedthe regenerationofskinappendagesandreducedtheformationof scartissue(WangC.etal., 2019)(WangM.etal., 2019).The sustainedreleaseofbioactiveexosomeshelpstoachievebetter woundhealingandscarremoval.

ASC-EXOSINSKINFLAPSINJURY

Flaptransplantationisanessentialmethodtorepairrefractory traumaandorganreconstruction,includingthealarrim,external ear,andfingertipdefects.Theinsucientneovascularizationand ischemia-reperfusion(I/R)injuryareresponsibleforpoorflap healingoutcomes(Sorkinetal., 2020).ASC-basedtherapyhas becomeanapplicablemethodtopreventI/Rinjuryforassisting flaptransplantation.Forinstance,ASCsareabletoenhance angiogenesistoincreasetheviabilityofchondrocutaneous compositegrafts,fortheapplicationofdefectsinthenose,ear scales,andskin(Yuceletal., 2016).

RecentstudieshaveshownthatASC-exoswerehopefulto improvethesurvivalstatusofskinflaps.Bybioinformatics analysis,Xiongetal.pointedoutthatthemiRNA-760 upregulationandmiRNA-423-3pdownregulationinASC-exos couldregulatetheexpressionofITGA5andHDAC5genes, respectively,consequentlypromotedthevascularizationofthe skinflap.Inalegwoundmodelofrat,themicrovascular angiographyof28dayspost-flaptransplantationrevealedthatthe ASC-exostreatedgroupsexhibitedbettervascularizationdegrees oftheartificialdermisprefabricatedflapsovercontrolgroups (Xiongetal., 2020).Undeniably,manydierentiallyexpressed miRNAsinASC-exosareassociatedwiththevascularization offlaps.Thestudyby Puetal. (2017) showedthatIL-6richhumanASC-exospromotedflapangiogenesisandflap repairafterI/Rinjuryinmice.Inthiscase,theemployment ofASC-exostodeliverIL-6isbeneficialforpatientsafety

becauseitdoesnotrequiretheuseofviralvectors. Bai etal. (2018) alsoshowedthatlow-doseH2 O2 -stimulatedASCexoscouldincreasetheneovascularizationoftheflapand relievetheinflammationandapoptosisafterI/Rinjury,thus increasingthesurvivalrateoftheflap invivo.Insummary, ASC-exosplayanimportantroleinthevascularizationof skinflaps,andtherebyresolvetheproblemofinsucient neovascularizationoftheflaps,thusexpandingtheapplicationof flaptransplantation(Figure1D).

ASC-EXOSINBONETISSUEDAMAGE

Fractures,tumorbonesurgery,deformity,revisionofthe prosthesis,andosteomyelitiscanbefullyidentifiedassegmental lossofbonestructure.Particularly,boneregenerationisthemain emphasisinvolvingbothsurgeryandaestheticsincraniofacial surgery.Exosomes,asnanoscaleextracellularvesicleswith anintercellularcommunicationfunction,provideanexcellent mediumforthepackagingandtransportationofRNAsand proteins,benefitingforbroadapplicationinbonetissue engineering(Paduanoetal., 2017).Besides,ASC-exos,with significantosteogenicinductionability,caneectivelyregulate themicroenvironmentofbonetissuebytransportingavarietyof bioactivemolecules.

Theboneremodelingcycleiscomposedofconsequential phases:resorption,reversal,andformation.Studieshavefound thatosteocytesinbonetissuesequipwithmanyfunctions ofcoordinatingtheboneremodelingofosteoclastsand osteoblasts,whichmaintainthebonehomeostasis(Borciani etal., 2020).An invitro studyconfirmedthatASC-exos couldantagonizeosteocyteapoptosistriggeredbyischemiaand hypoxia,anddecreaseosteocyte-mediatedosteoclastogenesis, whichwasattributedtothedecreaseinreceptoractivatorof nuclearfactorkappabligand(RANKL)expression(Renetal., 2019).RANKLinteractswithitsreceptorRANK,whichishighly expressedbyosteoclastsortheirprecursorsandisessential forosteoclastactivation(Figure2A).ASC-exoscanbeused intissueengineeringcombinedwithecientlybiocompatible ecientcarrierstoimproveosteogenesiseciency.According toarecentstudy,ASC-exoscouldbeimmobilizedonthe polydopamine-coatingPLGAscaolds.Thiscell-freenano-sized carrierenhancedboneregenerationsignificantly,atleastpartially throughitsosteoinductiveeectsandcapacitiesofpromoting MSCsmigrationandhominginthenewlyformedbonetissue (LiW.etal., 2018).Itwasdefinitelyestablishedthatan idealscaoldforexosomesloadingwouldbebiocompatible, biodegradable,andcapableofcontrolledreleasingexosomes. ChenS.etal. (2019) showedthattheexosomesderivedfrommiR375-overexpressingASCsincorporatedwithhydrogelpossessed theabilitytoenhanceboneregenerationinaratmodelof calvarialdefect.Moreeectiveandconvenientloadingstrategies shouldbedeveloped.Furthermore,appropriatechangesin thecultureconditionsofASCswillfacilitatetheproduction ofcustomizedASC-exos.Byusinghypoxia/serumdeprivation (H/SD)inducedosteocyteapoptosismodelwithmurinelong boneosteocyteY4(MLO-Y4), Zhuetal. (2017) demonstrated

FIGURE2| ASC-exosfunctionintheboneremodelingcycle. (A) Inboneresorptionandreversal,ASC-exoscoulddecreasetheexpressionofRANKL,whichwas highlyexpressedbyosteoclastsortheirprecursorsforosteoclastactivation,toantagonizeosteocyte-mediatedosteoclastogenesis. (B) Inboneformation, ASC-exospossessedtheabilityofloweringtheproductionofIL-6andPGE2,downregulatingSA-b-galactivityandreducingtheaccumulationof gH2AXin osteoblasts.Additionally,ASC-exoscouldupregulatetheradioofBcl-2/Bax,diminishtheproductionofROSandcytochromec,andsubsequentactivationof caspase-3/9inosteocytes.ASCs,Adipose-derivedstemcells;ASC-exos,ASC-derivedexosomes;RANKL,ReceptorActivatorofNuclearFactorKappaBLigand; IL-6,Interleukin6;PGE2,ProstaglandinE2;SA-b-gal,Senescence-Associated b-galactosidase; gH2AX,PhosphorylatedHistoneH2AX;Bcl-2,B-cell lymphoma/leukemia2;Bax,Bcl-2-associatedXprotein;Caspase-3/9,AspartateProteolyticEnzyme3/9.

ASC-exoscouldecientlyantagonizeosteocyteapoptosisand osteocyte-mediatedosteoclastogenesis,viaupregulatedradio ofB-celllymphoma2(Bcl-2)/Bcl-2-associatedXprotein (Bax),diminishedproductionofROSandcytochromec,and subsequentactivationofaspartateproteolyticenzyme9(caspase9)andcaspase-3.Thisresultalsoprovidedthe invitro evidence ofASC-exosapplicationinage-relatedbonedisease. Luetal. (2017) demonstratedthatASC-exos,especiallyprimedbyTNFa pre-conditionedASCs,couldpromotetheproliferationand dierentiationofhumanosteoblaststhroughWntsignaling pathway.Therefore,themethodsforproducingspeciﬁcASCexos,oerapromisingapproachtoreplacedirectstemcell transplantation,furtherwideningtheapplicationofexosomesin boneregeneration(Figure2B).

Osteoarthritis(OA)isacommondegenerativejointdisease characterizedbycartilagedegeneration,synovitis,subchondral bonesclerosis,andosteophyteformation(Henrotinetal., 2012). Currenttreatmentsarebasicallysymptomatictohandlepainand swelling,andmainlyrelyonantalgicsandanti-inﬂammatory drugs.Articularcartilagehasalimitedpotentialtorepair, withprogressivelymorecliniciansemphasizingcellulartherapy. Multipleadiposetissue-associatedcomponentsandextractions arepromisingtobeappliedinOAtherapy,includingSVF, ASCs,ASCs-exos,ASC-CM,andmicrofragmentedadiposetissue

(MFAT).Inaclinicaltrial,SpasovskipointedoutthattheOA patientstreatedwithsingleinjectionofASCsshowedsignificant cartilagerestoration(Spasovskietal., 2018).Similarly,inanother trialofOApatientsreceivedwithASCs,theASCsmightcause animmediatelocalresponseduetoreleasedparacrinefactors andcytokinesforOAamelioration(Persetal., 2018). Tranetal. (2019) employedSVFtoregeneratedamagedkneecartilageof OApatients,revealingatrendtowardabetterecacyofSVF withthemicrofracturemethodforOAtreatmentoveraperiodof twoyears. HongZ.etal. (2019) alsosuggestedthatintra-articular SVFinjectionwasasafetreatmentofOA,andcouldeectively relievepain,improvefunction,andrepaircartilagedefectsin patientswithbilateralsymptomatickneeosteoarthritis.MFAT couldreducethephaseofcellmanipulationwithoutexpansion orenzymatictreatmentinashortperiod(Paolellaetal., 2019).In aninflammatorycellmodelofOAsynoviocytes,MFATreduced typicalmacrophagesmarkersanditspotentialitytoinducean anti inflammatoryeecttoaddressOA(Mautneretal., 2019). Asthecytoprotectiveandanti-inflammatorypropertiesof ASCsinhumanchondrocytesandexperimentalOAmaybe mediatedbyparacrineeects,theparacrinemediatorsASC-exos areattractiveforalternativetherapiesofOA.ASC-exosmightbe safer,cheaper,andmoreeectiveOAtherapy(Persetal., 2015). ASC-exosareabletodownregulateinflammationandoxidative

stress,whichmightsuccessfullymediateantisenescenceinOA (WangQ.etal., 2020).Theintra-articularinjectionofASCexoscouldinhibitcartilageandsubchondralbonedegradation, decreaseosteophyteformation,andanti-synovialinflammation, thusslowingtheprogressionofOA(ZhangR.etal., 2019). Tofiño-Vianetal. (2017) showedthatbothASC-CMandASCexosloweredtheproductionofIL-1b-stimulatedinflammatory mediatorsIL-6andprostaglandinE2(PGE2),down-regulated senescence-associated b-galactosidase(SA-b-gal)activity,and reducedtheaccumulationofphosphorylatedhistoneH2AX (gH2AX),inOAosteoblasts.Furthermore,inthenextstudy, theyconfirmedthatmicrovesiclesandexosomessecretedfrom ASCscouldaectthemetabolismofOAchondrocytesby modulatinginflammatoryanddegradativepathwaysassociated withjointdestruction(Tofiño-Vianetal., 2018). Zhaoetal. (2020) separatedthepatientASC-exos,whichexertedastrong stimulatoryeectonchondrocytemigrationandproliferation withtheupregulationofmiR-145andmiR-221inthe modeloftheinflammation-inflictedoxidativestress.Woo etal.alsoconfirmedthathumanASC-EVscouldpotentially protectcartilagefromdegenerationandcoulddelaycartilage degenerationinOAratandmousemodels.Themechanism wasprobablythathumanASC-EVssuppressedIL-1b upregulatedcatabolicmoleculesandenhancedtypeIIcollagen expressioninhumanOAchondrocyte(Wooetal., 2020). Ragniestablishedan invitro modelofhumanfibroblast-like synoviocytes(FLSs)fromOApatients,showingthatASC-EVs possessedtheimmunoregulatorypropertiesforOAregulation andthathyaluronanwasinvolvedinASC-EVsinternalizationin FLSs(Ragnietal., 2019).

Thesepioneeringresultsreinforcedthegreatprospectsfor ASC-exosandASC-EVsasanoveltherapeuticoptionfor OA.However,thenumberofstudiesissmall.Inthecontext ofOA,althoughtheASCsandSVFhavebeenconﬁrmed theirclinicallytherapeuticecacyandsafety,theASC-exos therapieshavenotyetbeenusedinclinicaltrials.Therestill needstoexecuteadetailedexplorationinlargecohortsto investigatethatthefunctionsandmechanismsofASC-exos arenecessary.Inpre-clinicalstudies,theoptimizedconditions andobtainmentsforASC-exos invitro andthemechanismsof ASC-exos invivo requirefurtherstudies.Inclinicaltrials,the ASC-exosbasedtherapyshouldsetoptimalcriteria,including exosomeconcentrationanddose,injectiontimesandintervals. Inaddition,thecomprehensiveimmuneimpactfollowingthe ASC-exosadministrationshouldbeperformedtodetermine theimmuneresponseoftherecipient.Totally,ASC-exosmay representtheeectiveclinicalstrategyofOAoncetrials havebeenfullycontrolledandtheirbeneﬁtsandsafetyhave beenfullyassessed.

ASC-EXOSINOBESITY

Obesityisagrowinghealthpandemicwhoseglobalprevalence hasincreaseddramaticallyoverthelastfewdecades.Inaddition tobringingthephysicalchanges,obesityalsocausesconsiderable obesity-relatedinﬂammationandmetabolicdisorders,including

dysfunctionofadiposetissueandinsulinresistanceinkey metabolicorgansandinsucientsecretionofinsulinbythe pancreas(ZhangB.etal., 2019).Apartfromtheenergystorage function,theadiposetissueisalsoanimportantendocrine tissueandagreatsourceofASC-exos.ASCsplaycriticalroles incontrollingobesity-associatedinflammationandmetabolic disorders.Thus,thesecretionquantityandfunctionofASCexosarehypotheticallyshapedbyobesity. Zhaoetal. (2018) foundthatASC-exoscouldtransferintomacrophagestoinduce anti-inflammatoryM2phenotypesthroughthetransactivation ofarginase1(Arg-1)andIL-10byexosome-carriedactive STAT3,andincreasetheexpressionofuncouplingprotein 1(UCP-1)topromotewhiteadiposetissue(WAT)beiging, therebyimprovingobesity-relatedinflammationandmetabolism (Figure3).Mechanistically,thisstudydelineatedanovel exosome-mediatedASC-macrophagecross-talkthatfacilitated immuneandmetabolichomeostasisinWAT,thusprovidinga potentialtherapyforobesityanddiabetes.

Extracellularvesiclesbelongtoaheterogeneitysystem, includingexosomes,apoptoticbodies,microvesicles(PerezHernandezetal., 2017).ItisworthnoticingonASC-EVs peculiarityindierentphysiologicalorpathologicalcontexts. TheconcreteuseofASC-EVsincell-basedtherapyintheobese settingshouldbetakenintoaccount.Somestudiessuggested thatobesityordiabetescouldimpairthecapacityofASCsfor anti-inﬂammationandwoundhealing,aswellasinﬂuencethe productionandbioactivitiesofASCs-exos,thusincreasingthe riskforimmuneormetabolicdisorders(Strongetal., 2016). Togliattoetal. (2016) investigatedthetherapeuticimpactofASCEVsrecoveredfromobesesubjectsvisceralandsubcutaneous tissues.ComparedwithASC-EVsfromnon-obesesubjects,ASCEVsfromobesesubjectsshowedimpairedangiogenicpotential invitro becauseofthedecreaseofEVscargoesincludingVEGF, MMP-2,andmiRNA-126(Togliattoetal., 2016).Obesityimpacts onASC-EVsandASC-exospro-angiogenicpotentialandmay raisemoreconcernsaboutthesecrucialtissuerepairmediators.

ASC-EXOSINFATGRAFTING

Fatgraftinghasbeengaininglargeattentionintissue augmentationoverthepastdecadesforhemifacialatrophy, lipodystrophy,andbreastreconstruction(TanandLoh, 2017). BothASCsandfatgraftcanexertawrinkle-reducingeectand synergisticallyaectcollagensynthesisandneovascularization (Kimetal., 2019).However,thesurvivalrateoffatgrafts remainsunsatisﬁedduetothedevascularizationandischemic injuryofadiposetissuesmadebyliposuction,injection,and long-termfatabsorption.Severaltechnicalmodiﬁcationshave beendescribedtoenhancefatgraftsurvivalwithmorecomplete adiposetissuestructure.Cell-assistedlipotransfer(CAL)isan ecienttechniquethatmixesASCs-richSVFwithlipoaspirate, forreinforcingadipogenesisandangiogenesistoaugmentfat graftreliability(Yoshimuraetal., 2008).Importantly, Kølle etal. (2020) conductedarandomizedcontrolledclinicaltrial comparingfatgraftsenrichedwith exvivo-expandedautologous ASCstonon-enrichedfatgraftsinbreastaugmentation,

FIGURE3| ASC-exoscouldimproveobesity-relatedinflammationandmetabolism.Intheobesitymicroenvironmentofmice,ASC-exoswasabletoreducethe secretionofiNOS,TNF-a,andIL-12,andincreasethesecretionofArg-1andIL-10toactivateM2macrophagepolarization,thusamelioratingWATinflammation. ASC-exosalsoincreasedtheexpressionofUCP-1topromoteWATbeiging,therebyimprovingobesity-relatedinflammationandmetabolism.ASCs, Adipose-derivedstemcells;ASC-exos,ASC-derivedexosomes;WAT,WhiteAdiposeTissue;BAT,BrownAdiposeTissue;iNOS,InducibleNitricOxideSynthase; TNF-a,TumorNecrosisFactorAlpha;IL-12,Interleukin12;Arg-1,Arginase1;IL-10,Interleukin10;UCP-1,UncouplingProtein1.

demonstratingthatASCssignificantlyimprovedthevolume retentionoffatgraftscomparedwithconventionalfatgrafting andnoadverseeectswereobserved.Thisresultfurther confirmedthesignificanceofCALinbothreconstructiveand cosmeticvolumerestoration.However,limitationsofcell-based therapieshaveconstrainedtheiruse,includinguncommitted dierentiation,unwantedsideeects,immunerejection, andregulatoryhurdles(O’Halloranetal., 2018).ASC-exos havebeenidentifiedtomotivatefunctionalrecoveryinfat graftingandfilling.

Recently, Hanetal. (2018) discoveredthatco-transplantation ofASC-exosandhypoxia-treatedASC-exosinnudemouse modelsofsubcutaneousfatgraftingbothcouldparticipatein neovascularizationandattenuateinflammationinthegrafts.In thesubsequentstudy,theyfurtherinvestigatedthemolecular mechanismofhypoxia-enhancingpromotingtheeectofASCexosinfatgrafting,raisingthatthehypoxia-treatedASC-exos significantlyenhancetheangiogenesisoftheischemicadipose tissuebyregulatingVEGF/VEGF-Rsignalingpathway(Han etal., 2019).AsthefunctionalnanovesiclessecretedbyASCs, ASC-exospossessmoreadvantagesinimprovingthevolume retentionrateoffatgraftsthanASCs. ChenB.etal. (2019) have shownthatASCs-exoswereequivalenttoASCsinimproving thesurvivaloffatgraftsbyup-regulatingearlyinflammation andenhancingangiogenesisinmice.Whereasduringthemid tolatestagesoffatgrafting,ASC-exosexertedapro-adipogenic eectandalsoincreasedcollagensynthesislevel,similarly,to theirsourceofASCs(ChenB.etal., 2019). Zhuetal. (2020) foundthatinan invivo mousemodelofautologousfatgrafting,

graftstreatedbyASC-EVssigniﬁcantlyexhibitedsurvivalwith moreneovascularization,increasedfatretention,anddecreased ﬁbrosisandnecrosis.TheASC-EVsuptakebymacrophages promotedM2typepolarizationandcatecholaminesecretion, thustheM2macrophages-CMcouldenhancebrowningadipose dierentiationwithenhancedenergyexpenditure(Zhuetal., 2020).Theseresultssuggestthat,asacell-freestrategy,ASCexoscouldbeaneectiveandappealingpathtoheightengraft survivalinlipotransfer(Figure4).However,amajordrawbackof fatgraftingistheunpredictabilityoftheclinicaloutcomesince highvolumeabsorptionratesarecommon.Itisnotyetclear howlongthiseectlastsorwhetherASC-exosfrombothhealthy individualsanddiseasedpatientsareequallyeective.Further experimentalandclinicalstudiesarerequiredtodetermine theoptimalconcentrationandsourceofASC-exosenrichment requiredtoimprovefatgraftwithoutsideeects.

ASC-EXOSINBREASTCANCERAND BREASTRECONSTRUCTION

Adipocytesaretheabundantcellularcomponentsinthebreast cancermicroenvironment.Theinvasionofbreastcancercells leadstotheproximityofcancercellsandadipocytes,whichhave beenreferredtoascancer-associatedadipocytes(CAAs)(Choi etal., 2018; DeLopeetal., 2018).CAAsexhibitaﬁbroblasticlikemorphologyandoverexpressedECMproteins.CAAscan interactwithcancercellsviaseveralwaystotriggerbreastcancer initiation,metastasis,angiogenesis,andcachexia.Studieshave

FIGURE4| Thesequenceflowdiagramoffatgrafting.Intheclinicalapplicationoffatgraftingforfacialfillerandbreastaugmentation,sterileadiposetissueis collectedthroughliposuction.Afterenzymedigestionandcentrifugationofthecollectedadiposetissue,theobtainedheterogeneousmixtureofendothelialcells, smoothmusclecells,fibroblast,pericytes,mastcells,andpreadipocytesisnamedSVF.InCAL,halfthevolumeoftheaspiratedfatisprocessedforisolationofthe SVFcontainingASCs,andtheotherhalfoftheaspiratedfatispreparedforgrafting.Finally,theSVF-supplementedfatisinjectedintothetargetsitesofgrafting.In animalstudiesofthesubcutaneousfatgrafting,co-transplantationofASC-exoswithadipocytescaneffectivelypromotetheneovascularizationtoenhancesurvival inthefatgrafts.ASCs,Adipose-derivedstemcells;ASC-exos,ASC-derivedexosomes;SVF,StromalVascularFraction;CAL,Cell-AssistedLipotransfer,CAL.

demonstratedthesurvivalrate,growth,andinvasivenessof tumorsafterinteractingwithASCs,whichareCAAscomponents. Byexcretingavarietyofbioactivefactorsincludingvisfatin, adipsin,CCL5,IGF,HGF,VEGF,IL-8,andTGF-b,ASCscanexert biologicalinﬂuencesonproliferation,epithelial-mesenchymal transition,andmetastasis(Huangetal., 2019; Gotoetal., 2019). ASCscoulddierentiateintocancer-associatedﬁbroblastsin breastcancerenvironment,especiallywithamorepronounced eectonobesepatients(Choetal., 2012; Eckeretal., 2019).

Theexosomes,releasedfromtheadipocytes andASCs,are emergingasanewtypeinthecrosstalkbetweenbreasttumors andadipocytes(Sauteretal., 2019; Teufelsbaueretal., 2019). Previously, Linetal. (2013) investigatedtheeectofASC-exos onbreastcancerMCF7cells,showingthatASC-exosactivated theWntandHhsignalingpathwaystostrengthentumor cellmigration.Ontheotherhand,thebreastcancer-derived exosomescouldreciprocallyshapethefunctionofadipocytes. ArecentstudyconﬁrmedthatmiRNA-144andmiRNA126-secretingexosomesfrombreastcancercellsco-cultured withadipocytescouldpromotecancermetastasisbyinducing beige/browndierentiationandreprogrammingthemetabolism insurroundingadipocytes(Wuetal., 2019).Interestingly, Lee etal. (2019) emphasized theeectofcancerstemcells(CSCs) treatedwithexosomesderivedfromosteogenicdierentiating humanASCs.Theresultsshowedthattheexpressionofdrugresistancegenes(ATPbindingcassettetransporter),thebreast cancergenefamily(BCRA1andBCRA2),andtheErbBgene familywereallsigniﬁcantlydecreasedinCSCs(Leeetal., 2019).

TheosteoinductiveASCs-exoscouldbeabiochemicalcuefor CSCsreprogrammingintonon-tumorigeniccellsandcontribute toovercomingtherapeuticresistance. Heetal. (2018) noted thatMSC-CMcouldalsosuppressthegrowthofbreastcancer cellsbyinhibitingtheSTAT3signalingpathwayandMSCCMcombinedwithradiotherapysigniﬁcantlydelayedxenograft tumorgrowthinmice.

Forpatientswithbreastcancer,breastreconstructionrequires theunificationoftumorsafetyandaesthetics.Fatgrafting usingmammaplastymightcomplicatebreastimagingand breastcancersurveillanceduetothevaryinglevelsofnodule formationandcalcificationsinbreasttissue(Bayrametal., 2019). TheASCsassistedfattransplantationisacommontechnique forCALwhichmakesgreatimprovementsinthesurvival rateoftransplantedfatwithlessfatresorptionandnecrosis forthefavorableaestheticoutcomeofbreastaugmentation. Nevertheless,tumorsafetyinbreastreconstructionisthe primaryconsideration(O’Halloranetal., 2017). Silvaetal. (2019) suggestedthatfattransplantationdidnotpromote tumorproliferationandmetastasisinmousemodelsof residualbreastcancer.Krastevetal.carriedoutalongtermfollow-uptrailof587breastcancerpatientswho underwentautologousfattransplantationandtraditionalbreast reconstruction,respectively.Theresultsshowedthatafter 5yearsthelocalrecurrencerateofbreastcancerhadno significantdierenceinthesetwomethods(Krastevetal., 2019).Similarly, Gentileetal. (2019a) alsoconducteda3-year follow-upof121patientswithbreastcancerwhounderwent

adiposestromalcell-enhancedengineeringfattransplantation, whichalsoshowedsatisfactorysafetyandeectiveness.Thus, theseclinicalstudiesusingASCsreportsafetydatain breastreconstruction.

Tosomeextent,exosomesoriginatefromthetumoradipocytesinteractioninacomplexmetabolicnetworkfavoring malignantprogression.However,atthisstage,therearelimited studiesonASC-exosusedforbreastreconstruction.Inclinical trialsandbasicexperiments,itisworthnotingthattheroleof ASC-exosinbreastreconstructionafterbreastcancersurgery isnotentirelyclear.Toavoidthetumorigenicpotentialrisk, themechanismsofASCsandASCs-exosinbreastcancer andbreastconstructionremaintobecarefullyelucidated. Additionally,itisimportanttoconductrandomizedtrials forilluminatingthesafetyandecacyoftransplantedASCs orASC-exos,incomparisontocommonlyapplyingwith conventionaltechniques.

CONCLUSIONANDPERSPECTIVE

Adipose-derivedstemcellsarestemcellpopulationswithinthe adiposestromalcompartmentthathavemultipledierentiation potentials,easyacquisition,andthehighyield,makingASCs attractivefortissueengineeringandcelltherapyasanideal stemcellsource.ASC-exos,containingimportantparacrine mediators,havereceivedmuchattentionrecentlyforfunctioning inintercellularcommunication.Ascell-to-cellmessengers, ASC-exosarevaluablesupplementwiththeregenerativeand reconstructivestrategies,andisparticularlysuccessfuland safelyappliedtochronicwounds,scars,boneinjuries,and cosmeticsurgery.BothASCsandASC-exospossesshuge applicationpotentialonthetissueregenerativeﬁeldinplasticand cosmeticsurgery.

Intermsofsourceandproduction,ASCsboastthebenefits thatthestemcellyieldfromfatismuchgreaterthanthatobtained fromothersources.ASC-exospossessadvantagesofhugesources andhighavailability,indicatingthatASC-exoscanbean alternativewhenexosomesfromothersourceshavediculties toextractorarenotsuitablefortherapy(Haetal., 2020). ASC-exosandotherMSC-exoshavesimilarcharacteristics, suchasmorphologyandcellsurfacemarkers,butsome importantbiologicaldierenceshavebeenfoundinproliferation, geneexpression,dierentiationability,andimmunosuppressive pathways.ForexosomesfromdierentsourcesofMSCs,they partlycontainsimilarproteins.Interestingly, Villatoroetal. (2019) showedthatcanineASCshadadvantagesofproliferative capacity,whereascanineBMSCsshowedasignificantlyhigher secretoryproductionofsomesolublefactors.Therefore,when selectingthesourceofMSCs,thosebiologicaldierencesshould beconsideredforcellimplantationorthesecretomeisdirectly usedforspecificclinicalapplications.Inaddition,itisobvious thatthoseofMSC-exosvaryaccordingtotheoriginofMSCs. However,comparativestudiesofMSC-exosbytheirtissueorigin arestilllimited,andonlyafewreportshavecompareddierent exosomeswithinthesamestudy.Forexample,comparedwith humanBMSC-exos,ASC-exosexhibitedahigheractivityof

neprilysin,whichwasanamyloid b peptidedegradingenzyme, suggestingthetherapeuticrelevanceofASC-exosinAlzheimer disease(Katsudaetal., 2013).ASCsmightbesaferandmore eectivethanthatfromBMSCs,includinglackofmajor histocompatibilitycomplex(MHC)classIIonASCs,induction ofhigherlevelsofanti-inﬂammatorymacrophagesandprolymphangiogenicactivity(Maguire, 2019).

Furthermore,comparedwithASCs,ASC-exosoerdistinct advantagesthatuniquelypositionthemashighlyeective bioactiveconstituents.Theproposedseveralmainreasonsare asfollowsbasedonpreviousreports:(1)Biosafety:exosomes, includingASCs-exos,arenaturallyoccurringsecretedmembrane vesiclesfromthereleasingcellswithlowerimmunogenicity, posingthefavorablebiosafetyofASC-exos.(2)Biological activity:althoughwithoutsimilarself-dierentiationfunction asASCs,ASCs-exoscontainabroadrepertoireofcargoes, includingnucleicacid,proteins,andenzymesformodulating multiplecellularactivities,actinginbothimmediateandremote areasinaparacrinemanner.(3)Stability:theASC-exosare comprisedofnaturallybimolecularphospholipidstructure, providingsucientstabilitytoavoidbiodegradation.Thus,ASCexosareverywelltoleratedinbiologicalfluidsalongwiththe ubiquitouspresence.(4)Carrierfeatures:ASCs-exosfunctionas acarrierforitself,alsocanbeusedasacomponentuploadedin well-designedbiomedicalmaterials.Duetotheintrinsichoming capacityorartificiallymodifiedtargetingability,ASC-exoscan serveasstableandeectivecarrierstoloadspecificproteins, lipids,andgeneticmaterial,andpreferentiallytransportitto targetedtissuesororgans.Exosome-baseddeliverysystemsmay beofprecedenceinthetreatmentofdiseasesattributingto theirendogenousorigin,whichminimizestheimmunogenicity andtoxicityandexertstheoptimaleect.Thedevelopment ofmultifunctionalbioactivebiomaterialswithlong-termASCexosomesreleaseisalsoveryimportanttosynergisticallyenhance tissueregenerationandtherapy.Withtheabovedesirable properties,ASC-exosholdclinicallypromisingpotentialinthe novelcell-freetherapeuticstrategies.Therefore,asmentioned inthisreview,ASC-exosareexpectantlyrecognizedasnew candidatesfortheskinanti-agingtherapy,skininflammation treatment,woundandscarrepair,flapgrafting,bonetissuerepair, obesityprevention,fattransplantation,breastcancer,andbreast reconstruction(Table2).Collectively,thesefindingsreinforce thesignificanceofASC-exos-participatedcellcommunication andapplicationsinplasticandcosmeticsurgery.Thoughlacking adequateapplicationinclinicalpractice,ASC-exosareplayingan increasinglygreaterroleespeciallyinmaximizingthetherapeutic eectofdermopathicfeaturesandtissuereconstruction.

Nevertheless,therearestillsomechallengesinthe developmentofASC-exosapplication.Firstly,obtaining ASCscontinuestobeaninconvenience.ThesourcesofASCs, aswellastheseparationandcultivationmethods,medium compositionanddosage,cellpassage,cellfusionandviability, mycoplasma,andothermicrobialcontamination,allshould betightlycontrolledtomaintainreliablebiologicalecacy andASC-exoswithhighquality.Secondly,theextractedASCexosmighthavelowpurityandyieldinthelab.Nowadays ASC-exosseparationmethodsincludeultracentrifugation,

TABLE2| ThemechanismsandfunctionsofASC-exosintissueregeneration.

DiseaseSourceModelFunctionMechanismReferences

SkinagingHuman

ASC-CM

Human ASC-CM

Human BMSC-exos

Atopic dermatitis

PhotoaginginducedHDFsand HaCaTs

Photoaginginduced HDFs

PhotoaginginducedHDFsand mice

HumanASC-exosADmodelof NC/NGAmice

HumanASC-exosADmodelof SKH-1mice

SkinwoundHumanASC-exosSkinlesionmodel ofHaCaTs

HumanASC-exosSkinlesionmodel ofHaCaTsand HDFs

HumanASC-exosfull-thicknessskin woundofmice

HumanASC-exosDiabeticfootulcer ofrat

HumanASC-exosFulllayerskin woundofmice

ASC-exosSkinwoundof diabeticmice

ScarformationHumanASC-exosSkinwoundof mice

HumanASC-exosSkinwoundof mice

SkinflapinjuryHumanASC-exosArtificialdermis prefabricatedflap andlegwoundof rat

SkinﬂapI/R injury

HumanASC-exosSkinﬂapI/Rinjury ofmice

BonedefectHumanASC-exosHypoxic-ischemic osteocyte

HumanASC-exosCalvarialdefectsof rats

HumanASC-exosHumanprimary osteoblasticcells

OsteoarthritisHumanASC-exosOAmodelof osteoblasts

HumanASC-exosChondrocytes stimulatedwith H2 O2

Photoaging prevention

Dermatitis improvement

EpidermalBarrier Repair

HaCaTsviability enhancement

HaCaTsandHDFs viability enhancement

DownregulatetheactivationandtranscriptionofUVB-induced signalingpathwaysandupregulatetheexpressionof antioxidantresponseelements

PDGF-AAinASC-CMpromotedHDFsproliferationand activatedPI3K/AktsignalpathwaytofacilitateECMdeposition andremodeling

ProduceROSatalowlevel,downregulateTNF-a,upregulate TGF-b toincreaseMMP-1andprocollagentypeIexpressionfor collagensynthesis

Decreasethelevelsofinﬂammatorycytokinesandreducethe numberofeosinophilsintheblood,andtheinﬁltrationofmast cells,dendriticepidermalcells

Reducetrans-epidermalwaterlossandenhanceepidermal lamellarbodiesandformlamellarlayerattheinterfaceoftheSC andstratumgranulosum.

FosterHaCaTsproliferation,migration,andinhibitapoptosis throughWnt/b-cateninsignalingpathway

ASC-exoscontainingMALAT1couldmediateH2O2-induced woundhealingviatargetingmiR-124throughactivatingthe Wnt/b-cateninpathway

WoundhealingPromoteﬁbroblastsproliferationandmigrationandoptimize collagendepositionviathePI3K/Aktsignalingpathwayto acceleratewoundhealing.

WoundhealingASC-exosoverexpressing-Nrf2promotedtheproliferationand angiogenesisofendothelialcells,andincreasedtheexpression ofwoundgrowthfactor,decreasedthelevelsofinﬂammation andoxidativestress-relatedproteins.

WoundhealingASC-exosoverexpressingmiRNA-21enhancedthemigration andproliferationoftheHaCaTsbyincreasingtheMMP-9 expressionthroughthePI3K/AKTpathway

Woundhealingmmu_circ_0000250enhancedthetherapeuticeffectof ASCs-exosomestopromotewoundhealingindiabetesby absorptionofmiR-128-3pandupregulationofSIRT1

ScarremovalInhibitcollagenexpressiontoreducescarformationinthelate stageofwoundhealing

ScarremovalRegulatetheratiosoftypeIIIcollagen/typeIcollagen, TGF-b3/TGF-b1,andMMP-3/TIMP-1,aswellasfacilitating HDFsdifferentiation

FlapvascularizationUpregulationofmiRNA-760anddownregulationof miRNA-423-3pinASC-exoscouldregulatetheexpressionof ITGA5andHDAC5genes,respectively,topromotethe vascularizationoftheskinﬂap

FlaprepairIL-6highlycontainedinASC-exoscouldenhanceskinﬂap recoveryandangiogenesisafterI/Rinjury

FlaprepairH2 O2 -treatedASC-exosincreasedtheneovascularizationand relievetheinﬂammationandapoptosisoftheﬂapafterI/Rinjury

OsteogenesisAmeliorateosteocyteapoptosisandosteocyte-mediated osteoclastogenesisbyloweringtheexpressionofRANKL

BoneformationASC-exosoverexpressingmiRNA-375wereabsorbedby hBMSCs,andinhibittheexpressionofIGFBP3toexert osteogeniceffects

BoneformationTNF-a-preconditionedASC-exospromotedtheproliferation anddifferentiationofhumanosteoblaststhroughtheWnt signalingpathway

Inﬂammation improvement

Lietal., 2019

2020

Huetal., 2019

Choetal., 2018

Shinetal., 2020

Maetal., 2019

2020

2018

LiX.etal., 2018

Shietal., 2020

2016

2020

Puetal., 2017

Baietal., 2018

Renetal., 2019

ChenS.etal., 2019

Luetal., 2017

DownregulateSA-b-galactivityandtheaccumulationof gH2AX Toﬁño-Vianetal., 2017

ChondrogenesisDownregulatedthepro-inflammatorymarkersIL-6,NF-kBand TNF-a,whiletheyupregulatedtheanti-inflammatorycytokine IL-10whenco-culturedwithactivatedsynovialfibroblasts, promotedchondrogenesisinperiostealcellsandincreased collagentypeIIand b-catenin

Zhaoetal., 2020

(Continued)

Guoetal.,

Heetal.,

Zhangetal.,

Yangetal., 2020

Huetal.,

Wangetal., 2017

Xiongetal.,

TABLE2| Continued

DiseaseSourceModelFunctionMechanismReferences

ObesityMouseASC-exosObesemiceObesitypreventionActivateM2-typemacrophagepolarization,improve inﬂammation,andpromotethebrowningofwhiteadipose tissue

FatgraftingHumanASC-exosMiceFatgrafts survivalpromotion

Mouse ASC-exos MiceFatgrafts survivalpromotion

BreastcancerHumanASC-exosBreastcancer MCF-7cells

Hypoxia-treatedASC-exosenhancedtheangiogenesisby regulatingtheVEGF/VEGF-Rsignalingpathway

Promoteangiogenesisandup-regulateearlyinﬂammation,exert proadipogeniceffectandincreasecollagensynthesisduringthe midtolatestages

TumorprogressionActivatetheWntandHhsignalingpathwaystostrengthenthe growthofMCF-7cells

Zhaoetal., 2018

Hanetal., 2019

ChenS.etal., 2019

Linetal., 2013

ASCs,Adipose-derivedstemcells;ASC-exos,ASC-derivedexosomes;HDFs,HumanDermalFibroblasts;HaCaTs,HumanKeratinocytes;UVB,UltravioletB;ASC-CM, ASC-ConditionedMedium;ECM,ExtracellularMatrix;H2 O2 ,HydrogenPeroxide;PDGF-AA,Platelet-DerivedGrowthFactor-AA;ROS,ReactiveOxygenSpecies;MMP1/9,MatrixMetalloproteinase1/9;TNF-a,TumorNecrosisFactorAlpha;TGF-b,TransformingGrowthFactorBeta;IL-4/5/6/13,Interleukin4/5/6/13;VEGF,Vascular EndothelialGrowthFactor;Nrf2,NF-E2-relatedfactor2;TIMP-1,TissueInhibitorofMetalloproteinases-1;I/R,Ischemia-Reperfusion;RANKL,ReceptorActivatorof NuclearFactorKappaBLigand;SA-b-gal,Senescence-Associated b-galactosidase; gH2AX,PhosphorylatedHistoneH2AX;Bcl-2,B-celllymphoma/leukemia2;Bax, Bcl-2-associatedXprotein.

exclusion,ultraﬁltration,two-aqueoussystem,immunoanity, andpolymerprecipitation.Althoughtheultracentrifugation isthemostcommonmethodforlargelyseparatingexosome, butaccompaniedbytheshortcomingsoftime-consuming, labor-intensive,costlyinstrumentation,andmultipleovernight centrifugationsteps.Asitisoverlyidealistictocompletelyisolate ASC-exosfromothercomponents,theecient,appropriate,and aordabletechniquesshouldbethoughtfultoacquireexosomes. Thesetechnologiesstillneedtoachieveequilibriuminimproving theyieldandpurityofASC-exos.

Thirdly,giventhatplasticsurgeryfrequentlyperforms liposuctionandautologousfattransplantation,plasticsurgeons andresearchershaveuniqueadvantagesinobtainingASCsand ASCs-exos.However,intheapplicationofASC-exosintissue regenerationofplasticandcosmeticsurgery,therearemany aspectsworthnoting.EmergingstudieshavereportedthatASCexoscanbeutilizedinplasticandcosmeticsurgery,butalmost allthesestudiesarelimitedincellularandanimalassays,without thelargescaleexplorationofclinicaltrialslikeASCs.Inaddition, exosomescouldbeabsorbedonhumanskin,butevenifinthe existingstudies,thereisstilllackofenoughreportsinskinaging, atopicdermatitisandskinﬂapsinjury,whichareimportantand intractableskindisease.Therefore,theactualclinicalprospects ofASC-exosarealmostablank,alsomeaningthatthereexist vastspacefordevelopment.Anotherimportantpointisthat auxiliarytherapyofASC-exos.Clinically,forplasticandcosmetic surgery-relateddiseases,lasertherapy,drugtherapy,tissueﬁlling andsurgicaloperationarecommontreatmentsandachievegood clinicaleects.Nevertheless,toacertainextent,ASC-exoscan onlybeusedasasupplementarytreatment,ratherthanasole therapyinthebeginning.Mostoftheexistingstudiesfocus onASC-exosasthemainorsoletreatment.Hence,whether ASC-exospossesssynergisticeectsorinhibitoryeectsonthe above-mentionedcommontreatmentsneedsfurtherresearch.

AccordingtotheaboveconsiderableinsightsintotheASCexosapplicationsandlimitations,itneedstocarryoutmore comprehensiveresearchesinthefollowingaspects.(1)Quality control.Thereexistsasubstantialdegreeofheterogeneityin

dosingregimensinthereportedcases.Forbetteroutcomes ofplasticandcosmeticsurgery,ASC-exosutilizationdetails, includingthestorageconditions,eectivedoses,concentration, andperiodoftreatment,arealltheimportantpoints.It isnecessarytofurtherexploresuitablemicroenvironmental conditionsorgeneticengineeringtechniquestoascertainthe eciencyofASC-exostreatment.(2)Componentsandfunctions. TheASC-exosarecomprisedofmultiplebioactivecomponents. Thecomplexmulti-componentsubstancesinASC-exosmay producediversebiologicalcharacteristicswhenfinallyusedin practice.ThepropensityforsomecontroversialeectsofASCexosiscontingentuponthetypeandstateofthehostcells, thetypeandstateoftherecipientcells,andtheinteracting microenvironments.ForpossiblyutilizingASC-exosinclinical application,adeepunderstandingofASC-exosandtheir componentsisthepriority.Identifyingthekeycomponentsand reformingASC-exostooverexpressthesecomponentsmight maximizethetherapeuticeectwhilereducingthesideor o-targeteects.Inthesubsequentstudies,ongoingadvances intheanalysisofthefunctionofASC-exoswillprobably unraveltheASC-exoscharacteristics,allowingdeepeningthe understandingoftheirroleinpathogenesisandregeneration properties.(3)Carrierpeculiarityexploration.ASC-exosisof carrierpeculiarityduetotheintricatestructureofexosomes.It meansthatexosomesaretheidealtherapeuticdeliverysystem. ASC-exosareeectivetoolsforcargotransportationofeective therapeuticagentswithlowerimmunogenicityandtoxicity. Ontheotherhand,ASC-exoscouldalsobeuploadedinthe specificnanomaterialsorhydrogelmaterialstopromoteskin repair.EngineeringASC-exostobeeectiveandsaferequires acomprehensiveunderstandingoftheirnecessarycomponents, includingbutnotlimitedtomembranestability,architecture, andpackagingoftheinteriorcomponents.(4)Largeclinical trials.Atpresent,excavationsonASC-exosstudiesbelongto basicresearchoranimallevel.Theseexperimentscannotclearly andactuallyreflecttheASC-exosusagesandtheirphysiological levels invivo.Infattransplantation,itisofgreatvalueto clinicallyexplorewhethertheexogenousASC-exoscouldbeused

forcelltransplantationwithsafetyandeectiveness.Especially, clinicalimplementationofanyoperationmustbebasedon safety.However,therolesofASC-exosinsomediseases remaincontroversial.TheoncologicalsafetyofASC-exosin breastcancerandbreastreconstructionisworthyofextreme attention.Moreover,theASC-exosimpactsonthecommon treatmentofplasticandcosmeticsurgeryareurgentclinical problems.Intheend,largerprospective,blinded,randomized clinicaltrialsareinurgentneedtofurtherestablishthelongtermeectiveness,safetyanddoseofASC-exosinhumans. Collectively,ASC-exosarepromisingcandidatesforcell-free therapystrategyanddeserveintenseinvestigationtoaccelerate ASC-exosapplicationsintissueregenerationofplasticand cosmeticsurgery.

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AUTHORCONTRIBUTIONS

MX andQZperformedtheliteraturesearchandwrotethe manuscript.MWandYWconceivedtheprojectandrevisedthe manuscript.WH,CZ,WL,andYYeditedthemanuscript.All authorsreviewedthemanuscriptandapprovedtheﬁnalversion.

ACKNOWLEDGMENTS

Thissectionacknowledgescontributionsfromthe ChinaGuangHuaScienceandTechnologyFoundation (No.2019JZXM001).

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