s41467-024-47696-5 (1) by Biogenomics

Article

https://doi.org/10.1038/s41467-024-47696-5

Exosome-coatedoxygennanobubble-laden hydrogelaugmentsintracellulardeliveryof exosomesforenhancedwoundhealing

Received:8October2023

Accepted:9April2024

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XiaoxueHan 1,2,3,4,ChaimongkolSaengow3,5,LeahJu1,2,WenRen1,2, RandyH.Ewoldt 3,5 &JosephIrudayaraj 1,2,3,4,6

Woundhealingisanobviousclinicalconcernthatcanbehinderedbyinadequateangiogenesis,inflammation,andchronichypoxia.Whileexosomes derivedfromadiposetissue-derivedstemcellshaveshownpromiseinacceleratinghealingbycarryingtherapeuticgrowthfactorsandmicroRNAs,intracellularcargodeliveryiscompromisedinhypoxictissuesduetoactivated hypoxia-inducedendocyticrecycling.Toaddressthischallenge,wehave developedastrategytocoatoxygennanobubbleswithexosomesandincorporatethemintoapolyvinylalcohol/gelatinhybridhydrogel.Thisapproach notonlyalleviateswoundhypoxiabutalsooffersanefficientmeansofdeliveringexosome-coatednanoparticlesinhypoxicconditions.Theself-healing propertiesofthehydrogel,alongwithitscomponent,gelatin,aidsinhemostasis,whileitscrosslinkingbondsfacilitatehydrogenperoxidedecomposition, toamelioratewoundinflammation.Here,weshowthepotentialofthismultifunctionalhydrogelforenhancedhealing,promotingangiogenesis,facilitatingexosomedelivery,mitigatinghypoxia,andinhibitinginflammationina maleratfull-thicknesswoundmodel.

Poorwoundhealingfollowingtraumaandsurgicalproceduresconstitutesapressingglobalmedicalissue,impactingmillionsofindividualsannuallyandposingsubstantialchallengestohealthcare professionals1,2.Thisconcernarisesduetotheadverseeffectson patients’ qualityoflife,heightenedpsychosocialstress,andtheconsiderable financialburdenassociatedwithprolongedclinicalwound management.Thestatisticsrevealthatinadequatewoundhealing affects6.5millionpatientsintheUS,andamongallwoundtypes, surgicalwounds,beingthecostliest,significantlycontributetototal Medicarespending3.Themaingoalsinmanagingwoundsencompass expeditiouswoundclosureandtheattainmentofascarthatisboth functionalandaestheticallypleasing4–6.However,anomalouswound healingcanbetriggeredbythedevianceofinflammationandhypoxia

fromtheirtypicalpatternsduringthenormalhealingprocess,thereby inducingdelayedwoundclosure,keloidformation,andthedevelopmentofhypertrophicscars7 8.Therefore,effectivemanagementof hypoxiaandinﬂammationisofparamountimportanceinthedevelopmentofnextgenerationadvancedwounddressings.

Adipose-derivedstemcell(ADSC)-derivedexosomeshave emergedaspromisingtherapeuticagentsintissueregeneration.This isprimarilyattributedtotheircapabilitytofunctionasintercellular communicatorsandharborarichcargoofbioactivemolecules, includingproteins,nucleicacids,andlipids.Thesemoleculescontributetowoundhealingbyexertinganti-inﬂammatoryeffects,inhibitingapoptosis,promotingangiogenesis,andfacilitatingenhanced cellmigrationandproliferation9,10.Recentstudiesemphasizethe

1DepartmentofBioengineering,1102EverittLab,1406W.GreenSt.,UniversityofIllinoisatUrbana-Champaign,Urbana,IL61801,USA. 2BiomedicalResearch Center,MillsBreastCancerInstitute,CarleFoundationHospital,Urbana,IL61801,USA. 3CancerCenteratIllinois,BeckmanInstitute,Urbana,IL61801,USA. 4HolonyakMicroandNanotechnologyLaboratory,CarleR.WoeseInstituteforGenomicBiology,Urbana,IL61801,USA. 5DepartmentofMechanicalScience andEngineering,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,USA. 6CarleIllinoisCollegeofMedicine,UniversityofIllinoisatUrbana-Champaign, Urbana,IL61801,USA. e-mail: jirudaya@illinois.edu

potentialofexosome-loadedhydrogelsasmultifunctionaldressings forbothacuteandchronicwoundhealing11,12.Thedatashowcasestheir capabilitytomitigateoxidativestress,stimulateangiogenesis,and enhance fibroblastmigration,contributingpositivelytoallphasesof thewoundhealingprocess13–15.Notably,exosomesofferseveral advantagesoverstemcells,astheyareconsideredsafer,lacktumorigenicpotential,andposeaminimalriskofembolism16 17.Nevertheless, recentinvestigationshaverevealedthattheintracellularcargodelivery efficiencyofexosomesiscompromisedunderwoundhypoxicconditionsduetoactivatedhypoxia-inducedendocyticrecycling18,19.This limitationsignificantlyimpedestheirtherapeuticefficacyandnecessitatesapproachestoenhanceexosomedeliveryinhypoxicwound tissues.

Toaddressthischallengeaswellaswoundinﬂammationand hypoxia,weproposeastrategyutilizingADSC-derivedexosome coatedBSA-basedoxygennanobubbles(EBO)embeddedwithinaselfhealinghydrogelmatrixasamultifunctionalwounddressing.The oxygensupplycomponent,oxygennanobubbles(ONB),isformedby encapsulatingnanoscaleoxygenbubbleswithinaglycosylatedprotein conjugatecomposedofdextran-conjugatedbovineserumalbumin (BSA).Theutilizationofglycosylatedproteinsoffersenhancedprotein characteristics,includingimprovedthermalstabilityandself-assembly properties20.Additionally,theglycosylatedproteinconjugateshave beenestablishedtohavefreeradicalscavengingfeaturesandthe capacitytoimpedeoxidativedeterioration,renderingthemextensivelyusefulinbiomedicalapplicationssuchastumortherapyand woundhealing21.Simultaneously,theglycosylatedproteinconjugate encapsulatesthebulknanoscaleoxygenbubblesproducedthrough

ultrasoniccavitation,resultingintheformationofONB.Exosomes derivedfromhumanadiposetissuestemcellsarefurthercoatedonto thesurfaceoftheONB,resultinginthe ﬁnaloxygen-carryingnanosystem,whichexhibitsoxygenreleasepropertiesandfacilitatesthe intracellulardeliveryofexosomes.

Tofurtheroptimizethehemostaticandantioxidantpropertiesof wounddressings,ahybridhydrogelisdevelopedbycombiningpolyvinylalcohol(PVA),gelatin(GA),andborax.Thehydrogelpossesses dynamiccrosslinkingthroughchemicalborateesterbonds,imparting themwithexceptionaltissueadhesion,self-healingcapabilities,and shapeadaptability22.ThemechanismthatisresponsiblefortheselfhealingabilityofthePVA/GAhydrogelistheboroniccrosslinks.Two possiblerouteswerepresumedthatdepictsintraspecies(PVA-PVA) andinterspecies(PVA-GA)crosslink:(i)boronic-esters23;and(ii)ionic crosslinks24 (Fig. 1a).Consequently,itiswell-suitedforwoundsthat requirefrequentstretchingandforuseinpartsofthebodythatarein constantmotionandcanprovideprotectiontowoundsbyeffectively sealingthewoundsite,reducingtheriskoffurtherinjuryor contamination25.Moreover,theboratebondspresentinthehydrogel exhibittheuniqueabilitytoreactwithhydrogenperoxide(H2O2), therebymitigatingexcessiveinﬂammation26.Additionally,theincorporationofgelatin,knownforitshemostaticproperties,further enhancesthedressings’ abilitytopromotebloodclotting(Fig. 1b), facilitatingtheacceleratedhealingoftraumaticandsurgicalwounds27

Inthiswork,weshowthefeasibilityofintegratinganoxygencarryingnanobubble,EBO,intoawounddressingtofacilitate therapeuticexosomedeliverybyincorporatinganantioxidative andhemostaticself-healingPVA/GAgelasascaffold.Intherat

Fig.1|SchematicillustrationofEBO-Gel-mediatedwoundhealing. a CrosslinkingmechanismsandstructureofEBO-Gel. b Enhancedwoundhealing programmedbyhemostasis,promotedexosomedelivery,oxygensupply,

angiogenesis,andantioxidantpropertiesofferedbyEBO-Gel.PVApolyvinylalcohol,GAgelatin,EBOADSC-derivedexosomecoatedBSA-basedoxygennanobubbles,EBO-GelEBOnanoparticles-embeddedhydrogel.

full-thicknesswoundmodel,thehydrogeldemonstratesaccelerated woundhealingandreducedinﬂammation.Thisadvanceddressing synergisticallycombinesmultiplefunctionalities,includinghemostasis,antioxidantactivity,oxygendelivery,andenhancedexosome transport.

Results

SynthesisandcharacterizationofEBO

Adiposetissueservesasanindispensablereservoirofmesenchymal stemcellsforapplicationsintissueengineeringandstemcelltherapy duetoitsconvenientaccessibilitythroughminimallyinvasiveproceduresinhumansubjects28,29.Inthisstudy,weisolatedADSCsfrom humanadiposetissueandproceededtoextractandcharacterize exosomesderivedfromtheseADSCs.TheisolatedADSCsexhibited thecharacteristicspindleshapeand ﬁbroblast-likegrowthpattern commonlyassociatedwithmesenchymalstemcells(Supplementary Figs.1and2).Furthermore,thesecellsdemonstratedpositive expressionofapanelofmesenchymalstemcellmarker(CD90, CD105,andCD44),whiledisplayingnegativeexpressionofCD106, CD45,andCD19(SupplementaryFig.3).Theisolatedexosomesdisplayedauniformdistributionofparticlesizes,withanaveragediameterof125.2nmandaconcentrationof7.22×108 particles/mL measuredbynanoparticletrackinganalysis(NTA)(Fig. 2b).The proteinconcentrationofexosomewasquantiﬁedbybicinchoninic acid(BCA)proteinassay.Transmissionelectronmicroscopy(TEM) imagingdepictedexosomeswithatypicalcircularorcup-shaped morphology,withadiameterofapproximately100nm(Fig. 2eand SupplementaryFig.4).

ToproduceEBO,theinitialstepinvolvedthepreparationofONB, whichwassubsequentlycoatedwiththeexosomemembrane.The synthesisofONBcommencedwiththeconjugationofBSAtodextran sulfatethroughamixturefollowedbyultrasonication.Meanwhile, oxygenwasintroducedintothesystemthroughouttheultrasonication process.ThesynthesisofEBOinvolvedsubjectingtheexosomeand ONBtoultrasonication(Fig. 2a).Themechanicalforcesinducedby ultrasonicationresultedinthetransientdisruptionoftheexosome structure,allowingforthereassemblyofexosomesaroundtheONB, formingacore-shellstructure.TEM(Fig. 2e)andscanningelectron microscopy(SEM)(SupplementaryFig.5)imagingconﬁrmedthe presenceofthiscore-shellstructure,withEBOexhibitingabilayer core-shellmorphologyandpossessingadiameterrangingfrom approximately150to200nm.TheconcentrationofEBOwastestedby NTAasshowninSupplementaryFig.6.Dynamiclightscattering(DLS) analysisrevealedthatONBpossessedanaveragehydrodynamicdiameterof122.50nmwithameanzetapotentialof 40.73mV,whileEBO exhibitedanaveragehydrodynamicdiameterof192.02nmwitha meanzetapotentialof 23.30mV(Fig. 2c,d).Thenegativechargeshad beenestablishedtostabilizethenanobubblesgeneratedviaultrasonic cavitation30.Consequently,thenegativelychargedEBOcaneffectively shieldtheoxygennanobubblesfromOstwaldripeningandcoalescence,therebyenhancingtheirstabilityduringthelong-termstorage, particularlyinclinicalapplications.

TheinternalizationofDio-labeledEBOintoHDF-a(humandermal fibroblasts,adult)cellswasdemonstratedthroughco-incubationfor 6h,asdepictedinFig. 2f.TheorthogonalXZandYZsectionsofa Z-stackconfirmthepresenceofEBOsinthecytoplasm(Fig. 2f(ii)and SupplementaryVideo1).Theeffectivenessoftheconjugationbetween dextransulfateandBSAwasevaluatedbymeasuringtheUV-vis absorbanceat294nmand420nm,whichcorrespondtotheintensity ofbrowning,indicativeofearlyandlateMaillardreactionproducts (MRP),respectively31.Theresultsindicatedasignificantincreasein absorbanceatboth294nmand420nmafterovernightmixing,comparedtonon-ultrasonicatedBSA(Fig. 2g).Furthermore,ultrasonicationleadstoafurtherincreaseinabsorbancecomparedtothemixture alone,confirmingtheformationofglycosylatedproteinconjugates.To

furthervalidatetheconjugation,sodiumdodecylsulfate

polyacrylamidegelelectrophoresis(SDS-PAGE)wasconducted (Fig. 2i).ThemonomerofBSAexhibitedamolecularweightof approximately66kDa.Afterultrasonication,asimilarproteinbandat approximately66kDawasobservedintheshell,ONB,andEBOsamples,indicatingthepresenceofBSAintheseformulations.However, theproteinbandappearedtobeslightlyshiftedcomparedtonative BSA.Thisshiftcanbeattributedtotheincreasedmolecularmass resultingfromtheconjugationbetweenproteinandpolysaccharides inducedbyultrasonication,indicatingthesuccessfulconjugation.FTIR analysiswasperformedtoidentifyfunctionalgroupsofONB(Fig. 2h). Theabsorptionbandsat1650cm 1 and1542cm 1 wereattributedto distinctiveamide І andamideIIbandsfromproteins32.Thespectral featuresofdextranappearedatapproximately1200–1000cm 1 region.Theintensitychangeobservedinthespectralregionsaround 1650cm 1 and1542cm 1 wereascribedtothemodiﬁcationsofC=O andC–NstretchingvibrationsoriginatingfromamideIandII, respectively,duetotheMaillardreaction.Anincreaseinbandintensity wasobservedintheregionof3700–3000cm 1,correspondingtothe vibrationsassociatedwithN-HandO-Hgroups33.Thisenhancement canbeattributedtothehigherabsorbanceofMRPthatwasgenerated. Theextendeddurationofoxygennanobubblepresenceduringstorageiscrucialforefﬁcientoxygenrelease.Toevaluatethestability, particularlythelongevityofthecore-shellstructureofEBO,TEM imageswereobtainedunderdifferentstorageconditions(2daysat 37°Cand1monthat4°C).AsillustratedinSupplementaryFig.7, despitethedissociatedproteinobservedinthebackgroundafter 1monthat4°C(possiblyexosomefragmentswithnegativestaining), EBOsuccessfullymaintainsitscore-shellstructureinbothconditions. Thisresilienceisadvantageousforensuringsustainedoxygensupply uponsubsequentuse.

CharacteristicsofEBO-Gel

TheaqueousmixtureofPVA,gelatinandEBOisalowviscosityliquid (SupplementaryVideo2),upontheadditionof2wt%ofborax,transientcross-linksrapidlyformboronicesterbonds,creatingaviscoelastichydrogelwithinseconds.Figure 3a,b,andSupplementaryFig.10 showevidenceofthesekeyrheologicalfeatures.Knownasprotorheology,theseimagescanbeusedforbothqualitativeandquantitativeinference(detailsinMethods).Forexample,thetiltedvialof EBO-GelinFig. 3aisevidenceofasufficientlyhighviscositytoinhibit flowontheobservationtimescaleof5min,withanimpliedviscosityon theorderof η ~104 Pa·satthisgravitationalstressof~100Pa.Athigher appliedstress,theEBO-Gelreadily flows,asevidencedinFig. 3bwith thematerialextrudedbyhandfromasyringewithcharacteristic flow stress~100,000Pa.Thisisevidencefordramaticshearthinning,since thematerialreadily flowsathighstress,butretainsitsshapeatlower stress(intheformofUIUCinFig. 3b).Thisinjectabledesignfurther facilitatesconformitywiththeshapeofadefect,enablingpreciseand tailoredapplication.

SEMimagesrevealtheporousstructureandthepresenceof nanoscaleparticleinEBO-Gel(Fig. 3i).IncontrasttothesmoothsurfaceobservedinBlank-Gel(SupplementaryFig.8),EBO-Gelexhibitsa roughersurfacewithnanoscaleparticlesadheringtothegelskeleton, indicatingtheexistenceofEBO.TheEBOreleaseprofilewasshownin SupplementaryFig.9,indicatingthatover80%ofEBOisreleasedfrom EBO-Gelwithin48h.Thiscorrespondswiththeintervalsforrefreshing wounddressings,ensuringtheefficientreleaseofnanoparticlestothe woundbedbeforethenextdressing.Toascertaintheshapeadaptability,thehydrogelunderwentiterativeremodeling,whereinitwas repeatedlyreconfiguredintovariousmorphologiesincludingstar shape,crescentmoonshape,round,andcrossshape,whichdemonstratesthefavorablecapacityofEBO-Geltoconformeffectivelyto irregularwoundshapes,thushighlightingitsadaptabilityinaclinical setting(Fig. 3c).

Fig.2|SynthesisandcharacterizationofEBO.a Schematicofthepreparationof ONBandEBO. b Concentrationdistributionandscatteredimages(background)of exosomes.Diameterdistribution(c)andzetapotentialvalues(d)ofONBandEBO (n =3independentsamples). e TEMimagesof(i)exosomes;(ii)ONB;(iii)EBO. Scalebar:50nm. f (i)EBOuptakeand(ii)Z-stackslicewithorthogonalviewsofEBO (Green)internalizationinHDF-acellsafter6hofincubation.Scalebar:10 μm. g The browningintensityofearly(A294)andlate(A420)MRP(n =3independentsamples). h InfraredspectraofBSAandONB.Pinkareasindicatethespectralregionsofgroup vibrations(Left)anddextran(Right).(i)SDS-PAGEofdifferentformulations.Lanes:

Self-healingabilityisanadditionalcrucialfactorthatinﬂuences thewoundadaptabilityofhydrogeldressings34–36.Themechanismthat isresponsiblefortheself-healingabilityofourPVA-GAhydrogelsisthe transientboroniccrosslinks.Twopossiblerouteswerepresumed, intra-(PVA-PVA)andinterspecies(PVA-GA)crosslinks:(i)boronicesters;and(ii)ioniccrosslinks.Weexaminedself-healingpropertiesat bothmacroandmicrolevels.Atthemacroscopiclevel,twoEBO-Gel

1.NaturalBSA;2.MixtureofBSAanddextransulfate;3.UltrasonicatedBSA;4.Shell; 5.ONB;6.Exosomes;7.EBO.Dataarepresentedasmean±SD(b, d, g).Statistical analysiswasperformedbytwo-wayANOVAwithDunnett’smultiplecomparisons (g).Representativeimagesareshownfromtwoindependentexperimentswith similarresults(e, f, i).SourcedataareprovidedasaSourceData ﬁle.BSAbovine serumalbumin,ONBoxygennanobubble,Exoadipose-derivedstemcell(ADSC)derivedexosomes,EBOADSC-derivedexosomecoatedBSA-basedoxygennanobubbles,Dexdextransulfate,MixmixtureofBSAanddextransulfate.

specimens,eachstainedwithadistinctcolor(purpleandyellow),were broughttogether.Inthisprotorheologydemonstration,thereassembledgelwassubjectedtostretchingafteraminutetoevaluateits healingcapacity.Notably,thedyesexhibitedrapidmigrationalongthe damagedinterface,whichcouldbevisuallyobservedfrom3to 100min(Fig. 3d).Toexploretheself-healingcharacteristic,anevaluationofthegellinearviscoelasticitywasassessedfurthercarefully

Fig.3|CharacterizationsofEBO-Gel.a FormationofEBO-Gel. b Injectabilityof EBO-Gelthroughthesyringe. c ShaperemodelingandadaptabilityofEBO-Gel. d Macroscopicself-healingpropertyofEBO-Gelovertime.Scalebar(inthe2nd row): 2mm. e Storagemodulus(G’)andlossmodulus(G’’)ofEBO-Gelaftercycliccutting andreassemblingprocess. f Adhesivecapacityondifferentsubstrates. g Adhesion to ﬁngerwithdifferentbendingangles. h LinearviscoelasticrheologicalcharacterizationofBlank-GelandEBO-Gel.Thetransitionfromtheblueregiontothe yellowregionsigniﬁestheshiftfromliquid-liketosolid-likebehaviorduringthe

followingacyclicprocessofcuttingandreassembling(Fig. 3e).The ﬁndingsrevealedthatevenafterundergoingthreecyclesofcutting andreassembling,thehydrogelscouldconsistentlymaintaintheir linearviscoelasticproperties,i.e., G’ and G’’.Wefurtherconﬁrmthe recoverabilitywiththealternatingstrainamplitudetest,i.e.,applicationofsmall-large-smallstrainamplitudeduringoscillatoryshear.The resultsshowalmostfullrecovery,wheretheunrecoverablepartis attributabletogelatinbundleat T < T g ≈ 60 C(SupplementaryFig.13). Theresultsfromtheserheologicalexperimentssuggestthatboth blank-andEBO-Gelscanself-healandretaintheirrheologicalpropertiesevenafterbeingstrained,whichisfavorableforclinical woundcare.

Adhesionpropertieswereinvestigatedonmaterialsandtissues. Theadhesivepropertiesofthehydrogelondiversesubstratesare showninFig. 3f,includingplastic,glass,rubber,andsteel.Linearviscoelasticcharacterizationwithoscillatoryshearfrequencysweep

gelationprocess. i SEMimagesofBlank-GelandEBO-Gel.Theredareaindicatesthe magniﬁedregion.Scalebarsareshownoneachimagerespectively. j Adhesionto ratorgans(fromlefttoright:heart,liver,spleen,lung,andkidney). k Adhesionto skintissuefromdifferentspecies(fromlefttoright:rat,porcine,mouse,and human).Dataarepresentedasmean±SD(h).Representativeimagesareshown fromtwoindependentexperimentswithsimilarresults(i).SourcedataareprovidedasaSourceData ﬁle.PVApolyvinylalcohol,GAgelatin,Blank-Gelhydrogel scaffoldwithoutnanoparticles,EBO-GelEBOnanoparticles-embeddedhydrogel.

experiments,Fig. 3h,showthatourhydrogelsareviscoelastic fluids witharelaxationtimebasedoncrossoverfrequency λ =1=ωc ≈1 5s,a largezeroshearviscosity~2kPa·s,andaplateauelasticmodulus~5kPa. Thislinearviscoelasticbehaviorrationalizeswhythematerialsappear solid-likewithminimal flow,butthegelscanalsoremodel,making themsuitableforirregularlyshapedandbleedingwoundsandsuitable forgluing.Wealsoshowthatthehydrogelexhibitedremarkable fixationonthe finger,retainingitsadhesionwhenthe fingerswere flexed withinarangeof0°to90°,providingfurtherevidenceofexceptional adaptabilityofEBO-Gel,particularlyonbodypartsthatexhibitawide rangeofmotion(Fig. 3g).Additionally,theEBO-Gelwasabletotightly adheretothesurfacesofratorgansandeffectivelysupportedtheselfweightoftheseorgans(Fig. 3j).Italsoshowedstrongadhesiononskin surfacesfromvariousspecies(Fig. 3k).Theadhesioncanbeattributed totheformationofstronghydrogenbondsbetweentheskintissue andEBO-Gel.ThisrobustadhesivefeatureestablishestheEBO-Gelasa

Fig.4|OxygensupplyandantioxidantpropertiesofEBO-Gel.a Illustrationof oxygensupplyandanti-inflammationmechanismsofferedbyEBO-Gel. b Oxygen releasecurvemonitoredwithin10h. c Intracellularhypoxiaconditionsofdifferent treatments.Scalebar:50 μm. d H2O2 consumptioncapacityofEBO-Gel(n =3 independentsamples). e ROS/SODdetectioninHDF-acells.Scalebar:100 μm. f QuantificationofROS/SOD fluorescence(n =3biologicallyindependentsamples). g EvaluationofH2DCFDAsignalaftertreatedwithBlank-Gel,ONB-Gel,ExoGel,andEBO-Gel.Dataarepresentedasmean±SD(d, f).Statisticalanalysiswas

reliablephysicalsealingagentforhemostasispurposes.Effective degradationiscrucialtopreventtheretentionofresidualhydrogelin deepwounds,thatcouldimpedethehealingduetoitsstrongadhesion andtissueadaptability.InvitrodegradationanalysisrevealedthatEBOGelcandegradeby80%within3daysundersimulatedphysiological conditions(SupplementaryFig.14),ensuringcompatibilitywiththe subsequenthealingprocessandalignmentwiththedressingchange intervalsininvivostudies.

OxygensupplyandROSeliminationassessment

Toverifytheoxygenreleaseproperties,oxygenconcentrationwas monitoredwithin10h.Figure 4bdemonstratestheoxygen-supplying capabilitiesofbothEBO-GelandONB-Gelinhypoxicconditions,in comparisontoExo-GelandBlank-Gel.ExtendedoxygenreleaseproﬁleswerecomparedwithaControl-Gelformedwithoutultrasonication.Despiteadeclineafter12h,thedissolvedoxygeninthehypoxic environmentexceededthatinControl-Gelby40h(Supplementary Fig.15).Thissupportsourtreatmentofchangingdressingsevery 2daysinsubsequentinvivoexperimentstosustainoptimaloxygen levelsinthewoundbed.Subsequently,hypoxiainhumandermal ﬁbroblast(HDF-a)cellswasinvestigatedbyemploying[Ru(dpp)3]Cl2 (RDPP)asacellularhypoxiaindicator(Fig. 4c).Remarkably,cells

performedbyone-wayANOVAwithTukey’smultiplecomparisons(f).Representativeimagesareshownfromthreeindependentexperimentswithsimilar results(c, e).SourcedataareprovidedasaSourceData ﬁle.Exoadipose-derived stemcell(ADSC)-derivedexosomes,ONBoxygennanobubble,NTnotreatments, Blank-Gelhydrogelscaffoldwithoutnanoparticles,Exo-Geladipose-derivedstem cell(ADSC)-derivedexosomes-embeddedhydrogel,ONB-Geloxygennanobubbleembeddedhydrogel,EBO-GelEBOnanoparticles-embeddedhydrogel.

incubatedwithEBO-GelandONB-GelexhibitedthelowRDPP ﬂuorescenceintensity,signifyingeffectivehypoxiamitigation,whichserves tovalidatetheexceptionalcellularoxygensupplycapabilityof EBO-Gel.

ROSscavengingpropertiescanbeascribedtotwoprimaryfactors:(1)thepresenceofboronicesterbondsinthegelenablesitto respondtoH2O2 andfacilitateitsdecomposition;(2)theexosomes derivedfromADSCshavebeendemonstratedtopossessantioxidant properties37 (Fig. 4a).Figure 4dshowedarapidreductioninH2O2 concentrationwithinthe first30minofincubationwiththegel,witha continueddecreaseobservedoverthecourseof240min,indicating theabilityofthegeltoeffectivelyscavengeH2O2.ROS/SOD fluorescencedetectiononHDF-acellsfurtherdemonstratedtheROSeliminationcapacitiesofEBO-Gel(Fig. 4e,f).Furthermore, flowcytometry analysisshowedEBO-Gel-treatedcellsexhibitedweakH2DCFDA fluorescence(Fig. 4g).

Exosomedeliveryenhancementstudy

Oxygensensingpathwaywasfoundtoregulateendocytosis38.Previous studieshavealsodemonstratedthatintracellularcargodeliveryof exosomesisunderminedinhypoxictissues18.Therefore,EBOis expectedtorestoreexosomedeliveryefﬁciencyinhypoxic

Fig.5|Enhancedintracellularexosomedelivery.a RepresentativeimmunofluorescentimagesofCFSE-ExoandLamp2underdifferenttreatments.Scalebar: 10 μm. b Fluorescenceintensityandcolocalizationefficiencyofdifferenttreatments(n =3biologicallyindependentsamples). c Illustrationsoftheprocedures fortheevaluationofexosomerecycling. d Quantificationofexosomerecyclingin theculturemedium(n =3biologicallyindependentsamples).Dataarepresented

microenvironmentsbysupplyingoxygen.Toexploretheexosome deliveryefﬁciency,HDF-acellswereco-culturedwithCFSE-labeled exosomesforadurationof8h.Immunoﬂuorescencewasperformed toassessthecolocalizationbetweenexosomesandLamp2,amarkerof endolysosomes(Fig. 5a).Remarkably,underhypoxicconditions,a notabledecreaseinthecolocalizationofexosomesandLamp2was

asmean±SD(b, d).Statisticalanalysiswasperformedbyone-wayANOVAwith Tukey’smultiplecomparisons(b)ortwo-wayANOVAwithDunnett’smultiple comparisons(d).Representativeimagesareshownfromtwoindependent experimentswithsimilarresults(a).SourcedataareprovidedasaSourceData ﬁle. EBO-GelEBOnanoparticles-embeddedhydrogel.

observed,indicatingadiminishedtransferofexosomestotheendolysosomes,whichtypicallyserveassitesforthereleaseofprotein cargointothecytoplasm(Fig. 5b).

Hypoxiahasbeenknowntoimpactendocyticrecyclingviathe modulationofRab1119 39 40.Consequently,therecyclingofexosomes throughexocytosismayhindertheefﬁcientreleaseoftheircargointo

Fig.6|Biocompatibilityandhemostaticpropertiesassessment.a Cellviability ofHDF-acellsincubatedwithdifferentconcentrationsofExo-Gel,ONB-Gel,and EBO-Gel(n =5biologicallyindependentsamples). b Bloodcompatibilityevaluated byhemolysisassay(n =3biologicallyindependentsamples).Insertedimage:+: Positivecontrol(Triton);1:Blank-Gel;2:Exo-Gel;3:ONB-Gel;4:EBO-Gel. c Mechanismsofhemostasiscapacity:(i)Embolizationhemostasisofferedby remodeling,adhesive,andself-healingpropertiesofEBO-Gel;(ii)ActivatedplateletmediatedhemostasisofferedbyGA. d InvitroprocoagulanteffectsofEBO-Gel. e Illustration(Left)andDigitalphotos(Right)ofhemostasisevaluationonratliver

thecellularcytoplasm,thuscompromisingthedeliveryoftherapeutic cargo.Toprovethishypothesis,aperiodof24-hofinternalizationof CFSE-labeledADSC-derivedexosomewasallowed,followedbythe assessmentofCFSEsignalsthatweresubsequentlyre-secretedintothe culturemediumovera12-hintervalunderdistinctexperimentalconditions,includinghypoxia,normoxia,andtreatmentwithanoxygenenhancingagent(EBO)(Fig. 5c).Theobtainedresultsrevealeda heightenedpresenceofexosomeproteinsrecycledtotheculture mediumduringhypoxicincubationincontrastwithbothEBO-treated andnormoxicgroups(Fig. 5d).Thisobservationsigniﬁesthepotential ofEBOtreatmenttoaugmenttheevasionofexosomesfromenhanced endocyticrecyclingcausedbyhypoxia,therebyfacilitatingthetherapeuticcargoreleasewithinthecellularcytoplasm.

Evaluationofbiocompatibilityandhemostaticefﬁcacy

ToexaminethebiocompatibilityofEBO-Gel,cytotoxicitytestand hemolysisassaywasconducted.Forcytotoxicityexperiments,HDF-a cellswereincubatedwithfourdifferentconcentrationsofExo-Gel, ONB-Gel,andEBO-Gelfor24h.AsshowninFig. 6a,allgroupsperformedhighcellviability,surpassing80%,whichindicatestheir excellentbiocompatibility.Forhemolysisassay,allhydrogelgroups demonstratedaremarkablebloodcompatibility,asevidencedbya hemolysisratiooflessthan2%(Fig. 6b).

hemorrhagemodel. f Quantitationofbloodlossin e (n =3biologicallyindependentexperiments).Dataarepresentedasmean±SD(a, b, f).Statisticalanalysiswas performedbytwo-wayANOVAwithDunnett’smultiplecomparisons(a)ortwotailedStudent’s t test(f).Representativeimagesareshownfromthreeindependent experimentswithsimilarresults(b, d, e).SourcedataareprovidedasaSourceData ﬁle.GAgelatin,Exo-Geladipose-derivedstemcell(ADSC)-derivedexosomesembeddedhydrogel,ONB-Geloxygennanobubble-embeddedhydrogel,EBO-Gel EBOnanoparticles-embeddedhydrogel.

Hemostasisplaysapivotalroleintheinitialphaseofwoundhealing,servingasacriticalstepthatestablishesatemporarybarrierto safeguardtheunderlyingtissuesagainstadditionalharmandpotential infection8.Giventhefrequentoccurrenceofbleedinginbothsurgical woundsandtraumaticinjuries,advancedwounddressingsshould possesstheabilitytoexpeditethehemostasisprocess.Gelatinhas gainedsigniﬁcantattentiontobeapromisinghemostasismaterialdue toitssimilarityincompositiontotheextracellularmatrix,enabling gelatintoeffectivelytriggerplateletaggregationandfacilitate hemostasis41,42.Inaddition,PVAhydrogelasaself-healingbioadhesive hydrogeloffersapromisingapplicationasanembolichemostatic agent,allowingittoseamlesslyadheretothesiteofbleeding,forminga physicalsealthateffectivelycontrolsandmanagesthebleedingprocess (Fig. 6c).Therefore,thehemostaticfunctionofEBO-Gelwasassessed bothinvitroandinvivo.EBO-Gel,whenmixedwithratwholebloodand bloodcellsatavolumeratioof2:1for1min,showedcoagulation.This underscoresitshemostaticcapability,similartocommercialproducts includingCURAD®BloodStop®HemostaticGauzeandBleedStop™,in comparisontoboththecontrolgroupandanon-hemostaticgel(Carbopolhydrogelgroup)(Fig. 6dandSupplementaryFig.16).Intherat liverhemorrhagemodel,thebleedingsitewaspromptlysealedupon applicationofEBO-Gel,leadingtoasubstantialreductioninbloodloss, alignedwiththeinvitro ﬁndings(Fig. 6e,f,SupplementaryFigs.17,18).

Fig.7|Evaluationofenhancedcellproliferation,migration,andangiogenesis.

a ImmunoﬂuorescenceimagesofBrdUstaininginHDF-acells.Scalebar:50 μm.

b CellproliferationassayofHDF-acellswithdifferenttreatments(n =3biologicallyindependentsamples). c CalceinAM/PI ﬂuorescencestainingtoexaminelive/ deadcells.Scalebar:50 μm. d ScratchwoundhealingassayconductedonHDF-a cellswithdifferenttreatmentssubjectedto0h,12h,or24hofhypoxia.Scalebar: 200 μm. e Invitrowoundclosureratefollowedbyscratchingassayin d (n =3 biologicallyindependentsamples). f, g Brightﬁeldimagesandquantitativeresultof transwellmigrationofHDF-acells(n =3biologicallyindependentsamples).Scale bar:200 μm. h TubeformationabilityofHUVECswithdifferenttreatments.Scale

OurresultshighlighttheeffectivehemostaticperformanceofEBO-Gel, inadditiontowoundhealingcharacteristics.

Invitrofacilitationofproliferation,migration,andangiogenesis

CellproliferationwasassessedusingHDF-acells,whichwereexposed tohypoxicconditionsfor24and48haftertreatmentwithhydrogels. Notably,theEBO-Gelgroupexhibitedahighernumberofproliferating cellsincomparisontotheothergroups,asdemonstratedinFig. 7b,c.

bar:200 μm.Quantitativeresultsof(i)numberofbranchesand(j)totalbranches length(n =3biologicallyindependentsamplesin i and j. k Cellcycleanalysisof HDF-acellswithdifferenttreatments.Dataarepresentedasmean±SD(b, e, g, I, j). Statisticalanalysiswasperformedbytwo-wayANOVAwithDunnett’smultiple comparisons(b, e),one-wayANOVAwithTukey’smultiplecomparisons(g, i, j). Representativeimagesareshownfromtwo(a, c)orthree(d, f, h)independent experimentswithsimilarresults.SourcedataareprovidedasaSourceData ﬁle.NT notreatments,Blank-Gelhydrogelscaffoldwithoutnanoparticles,Exo-Geladiposederivedstemcell(ADSC)-derivedexosomes-embeddedhydrogel,ONB-Geloxygen nanobubble-embeddedhydrogel,EBO-GelEBOnanoparticles-embeddedhydrogel.

Furthermore,real-timemonitoringofcellproliferationwasconducted usingaBrdUincorporationassayandcellcycleanalysis.TheEBO-Gel groupdisplayedasignificantlyhigherpositivesignalofBrdU(Fig. 7a) andmorecellsinS/(G2/M)phasescomparedtotheothergroups (Fig. 7k),indicatinganincreaseinthenewlysynthesizedDNA,which corroboratestheabilityofEBO-Gelinpromotingcellproliferation. ADSC-Exoplaysacrucialroleinpromotingdermal fibroblast migrationandwoundhealingbyreleasingMALAT1,aspecificlong

non-codingRNA43.Additionally,themodulationofApoptosisPeptidaseActivatingFactor1(APAF1)throughmiR-93-3pinADSC-Exos contributestoimprovedcellularviabilityandmigration,particularlyin hypoxicconditions44.ThemigrationabilityofHDF-acellsunder hypoxicconditionswasassessedusingbothscratchingassayand transwellmigrationtest.Inthescratchingassay,cellmigrationwas visuallymonitoredat0,12,and24h.Remarkably,theEBO-Geltreatmentgroupdisplayedahigherwoundclosurerateincomparisonto theothergroups,indicatingenhancedcellmigration(Fig. 7d,e). Similarly,theEBO-Gelgroupexhibitedahighernumberofmigrated cellsinthetranswellafter24hofco-incubation(Fig. 7f,g).These resultscollectivelydemonstratethecapacityoftheEBO-Geltopromote ﬁbroblastmigration.

Angiogenesis,apivotalprocessinwoundhealing,entailsthe formationoftube-likestructuresbyendothelialcells,whichprogressivelyextend,branch,andestablishinterconnectednetworks45.The establishmentofanextracellularmatrix(ECM)isvitalforneovascularization,withcollagendepositionplayingafundamentalrolein aorticendothelialcellmigration,whichisanoxygen-dependent process46.Hence,oxygensupplymightbeapotentialstrategyfor enhancingbloodvesselformation.Moreover,EBOaidsangiogenesis throughexosomescontainingregulatorssuchasNrf2andFGF2,with exosomespromoting β-cateninactivationandharboringmicroRNAs (miR-31,miR-125a)thatmodulateangiogenicpathways,collectively enhancingproangiogeniceffects47,48.Invitrotubeformationcapacity wasevaluatedusingHUVECs(Fig. 7h).After6hofco-incubation,the EBO-Gel-treatedgroupdevelopedahighernumberofbranches (Fig. 7i)andthemaximumtotalbranchlength(Fig. 7j),indicatingthe potentialofEBO-Geltofacilitateangiogenesis.

Invivowoundhealinganalysis

EBO-Gelshowedrobusttissueadhesion,oxygenrelease,antioxidant, andenhancedexosomedeliveryproperties,allofwhichareexpected tobepromisingfornoninvasivewoundclosure.Therefore,thewound healingperformanceoftheEBO-Gelwasassessedinaratfull-thickness defectmodel.Figure 8adepictsthesurgicalprocedure,dressing application,andhealingtimeline.ResultsshowthattheEBO-Geltreatedgroupexhibitedanotablyacceleratedhealingratecompared totheothergroups,particularlyduringtheproliferationstagefrom day2today10(Fig. 8b–e).Remarkably,theTegadermgroupexhibited signsofinfectionandinﬂammationondays2and4,whilethegroups treatedwithhydrogeldressingsshowednosigniﬁcantinfection (Fig. 8b).Throughoutthetreatmentduration,areductioninbody weightwasobservedduringtheinitialtwodayspost-surgery,which canbeattributedtotherecoveryfromanesthesia.Subsequently,the animalsexhibitedagradualweightgain,withanincreaseinweight observedbyday14(Fig. 8f).Additionally,after14daysoftreatment, thewoundstreatedwiththeEBO-Geldisplayedexcellentrecovery quality,characterizedbytheabsenceofhypertrophicandkeloidscars.

Tocomprehensivelyassessthetherapeuticeffectivenessofthe differenttreatments,tissuesamplescollectedonDay4andDay14 weresubjectedtohistopathological evaluationusinghematoxylinand eosin(H&E)stainingandMasson’strichromestaining,focusingonskin repairparametersincludingscarindex,dermisthickness,epidermis thickness,andcollagenfraction49 50.Day4representsacrucialtime windowfortheinflammationandproliferationstagesofthehealing process,whileDay14canreflecttheregenerationstageandhealing efficacy.AsillustratedinSupplementaryFigs.19and20,theEBO-Gel groupexhibitedaconsistentlyregeneratedepidermis,increased fibroblastproliferation,enhancedangiogenesis,andcollagen fiber formation.Incontrast,woundsintheTegadermandBlank-Gelgroups showedincompleteclosureandhigherareasofinflammationinfiltration.ThishighlightstheroleofEBO-Gelininfluencingtheinflammation,proliferation,andearlyregenerationstagesofhealing.The stainingresultsindicatedvisiblewoundclosureandthedevelopment

ofnewlyformedepidermisacrossalltreatmentgroupsonDay14 (Fig. 9a,b).However,theEBO-Geltreatedgroupexhibiteddistinct characteristicswith flatterwoundsurfaces,demonstratingacontinuousandcoherentepidermis,highlightingitssignificantpotential inpreventingkeloidformationandachievingscarlesswoundhealing. Furthermore,theEBO-Gelgroupdisplayedahigherdensityofnewly formedbloodvesselsandreducedinflammationareas,signifyingits capabilitytoenhanceangiogenesisandpossessantioxidantproperties.Theincreasedcountofnewlygeneratedhairfolliclesandglands (SupplementaryFigs.21and22),andtheorganizedformationof granulationtissuefurtherunderscoredthesuperiorqualityofhealing achievedthroughEBO-Geltreatment.

TheScarIndex(SI),representingscarsize,isdeterminedby dividingthescararea(mm²)bythecorrespondingaveragedermal thickness(mm).Thecalculatedresultsshowedthatthegrouptreated withEBO-Gelobtainedthelowestscarindex,indicatingamore favorablehealingoutcome(Fig. 9c).Completewoundhealingisalso markedbytherepairofdermis.Dermalthicknessmeasurements revealedthatthegrouptreatedwithEBO-Gelexhibitedathickerdermallayer,indicatingamorerobusthealingprocessandwell-repaired tissue(Fig. 9d).Conversely,thethicknessofthenewlyformedepidermiswasobservedtobeclosertothatofnormalskintissue,indicatingamorebalancedhealingresponse(Fig. 9f).Thisthinner epidermisisadvantageousinpreventingtheformationofthickscabs orkeloids,whichcanotherwiseimpairthenormalfunctioningofthe regeneratedtissue.Furthermore,Masson’strichromestaining demonstratedcollagendepositionandorganizationinvarioustreatments.TheEBO-Geltreatedgroupshowedanabundanceofdensely packedcollagen fibersinmagnifiedregions.Notably,these fibers demonstratedamorematurephenotypecharacterizedbywellorganizedalignmentandanintricatecollagennetworktopology, especiallyincomparisontotheTegadermandBlank-Gelgroup (Fig. 9e).Thequantitativeassessmentofcollagenvolumefraction alignedwiththeobserved findings,whichverifiedtheimprovedcollagendepositionabilityofEBO-Gel.

ToassessthemultifacetedimpactofEBO-Gelonangiogenesisand anti-inflammatoryresponsesinvivo,weperformed fluorescentstainingofwoundtissuesonDay14post-treatment.TheimmunofluorescentstainingforCD31,amarkerindicatingangiogenesis, exhibitedheightenedexpressionand fluorescenceintensityinthe EBO-Geltreatedgroup,showcasingitspotentialinpromotingangiogenesis(Fig. 9gandSupplementaryFig.23).ByincorporatingADSCexosomes,EBO-Gelhasthepotentialtomitigateexcessiveinflammation,forestallingthedevelopmentofdysfunctionalscarsthroughout thehealingprocess.OurexaminationencompassedROSlevels, inflammatorycytokineexpression,andmacrophagephenotypes. Dihydroethidium(DHE)stainingunveiledsignificantlylowerROS levelsinwoundstreatedwithEBO-GelincomparisontotheTegaderm controlandBlank-Gel(Fig. 9handSupplementaryFig.23).Notably,the heightenedDHEintensityintheTegadermcontrolandBlank-Gel groupssuggestsprolongedelevatedROSlevelsinhealedtissueeven after14daysoftreatment,possiblycontributingtohypertrophicscar formation.IntheevaluationofimmunecellsthroughimmunofluorescentstainingforCD86andCD206withF4/80 markersforM1 inflammatoryandM2anti-inflammatorymacrophages,respectively EBO-GeldemonstratedlowerCD86andhigherCD206expression comparedtocontrolgroups(Fig. 9i,SupplementaryFigs.23–25). Furthermore,theimmunofluorescentstainingofinterleukin-6(IL-6), aninflammatorymarker,exhibitedthelowestexpressionintheEBOGelgroup,solidifyingitsefficaciousanti-inflammatoryeffects(SupplementaryFigs.26and27).

Effectivewoundclosurecouldbepotentiallyhinderedifthe adaptivehydrogelfailstodegradeadequatelyindeepwounds, underscoringthecrucialimportanceofitsdegradability51.Forinvivo degradationassessment,thevisualobservationofEBO-Gelrevealsa

Fig.8|InvivoefﬁcacyofEBO-Gelinaratfull-thicknesswoundmodel. a Illustrationofwoundcreationandtreatmenttimeline. b Representativedigital photosofwoundswithvariedtreatmentsatdifferenttimepoints.Scalebar:7mm. c Tracesofwoundclosurefromday0today14.Quantitativeanalysisof(d)wound area(cm2)and(e)woundclosurerate(n =6biologicallyindependentwounds) overtime. f Relativebodyweightofratsaftervariedtreatments(n =4animalsper group).Dataarepresentedasmean±SD(e, f).Statisticalanalysiswasperformedby two-wayANOVAwithDunnett’smultiplecomparisons(e).Sourcedataareprovided asaSourceData ﬁle.Tegadermnotreatments,Blank-Gelhydrogelscaffoldwithout nanoparticles,Exo-Geladipose-derivedstemcell(ADSC)-derivedexosomesembeddedhydrogel,ONB-Geloxygennanobubble-embeddedhydrogel,EBO-Gel EBOnanoparticles-embeddedhydrogel,D0-D14dayspost-surgery.

Fig.9|histologicalanalysisofwoundsthatunderwenttreatmentswithdifferenthydrogels. Representativeimages(top)andmagnifiedimages(bottom)of (a)H&Eand(b)Masson’strichromestainingofthewoundtissueonDay14.Red arrows:newbloodvesselformation;Blackarrows:inflammationarea;Yellow arrows:newlygenerated-hairfollicles.Scalebar:500 μm(a)and1mm(b)forthe normal-sizedimage;250 μm(a)and500 μm(b)forthemagnifiedimage.Various parametersforwoundhealingevaluation,including:(c)Scarindex;(d)Dermis thickness;(e)Collagenvolumefraction;and(f)Epidermisthickness(n =3biologicallyindependentsamplesin c–f).Representative fluorescenceimagesof(g) CD31immunostaining,(h)DHE(red)staining,and(i)CD86andF4/80

progressivereductioninsizeandcompletedegradationwithina3-day period(SupplementaryFig.28).H&Estainingoftissuessurrounding theinjectionsitesonDay3post-injectionrevealednodiscernible inﬂammationinducedbytheinjectioncomparedtonormalskinand subcutistissues(SupplementaryFig.29).Thissuggeststheinvivo biocompatibilityofEBO-Gel.Autolysischaracterizesthedegradation ofEBO-Gel,wherewatermolecules,withunsharedelectronpairs, initiatethehydrolysisofboronatoms51,52.Thishydrolysiscausesthe gradualbreakdownoftheboraxbondcrosslinkednetwork,weakening bothboraxandhydrogenbondcrosslinks.Consequently,thehydrogel networkprogressivelyloosenswhenexposedtoawaterenvironment

immunostainingofwoundtissuesonDay14post-treatment.Cellnucleiwere stainedwithDAPI(blue).Scalebar:100 μm(CD31)and50 μm(DHEandCD86). Datainarepresentedasmean±SD(c–f).StatisticalanalysiswasperformedbyonewayANOVAwithTukey’smultiplecomparisons(c–f).Representativeimagesare shownfromthreeindependentexperimentswithsimilarresults(a, b, g, h, i). SourcedataareprovidedasaSourceData ﬁle.Tegadermnotreatments,Blank-Gel hydrogelscaffoldwithoutnanoparticles,Exo-Geladipose-derivedstemcell(ADSC)derivedexosomes-embeddedhydrogel,ONB-Geloxygennanobubble-embedded hydrogel,EBO-GelEBOnanoparticles-embeddedhydrogel.

overtime,ultimatelyresultinginthecompletedissolutionofthe hydrogel.TocomprehensivelyassessthebiosafetyofEBO-Gel,we conductedH&Estainingonvisceralslicesobtainedfromanimals treatedfora14-dayperiod(SupplementaryFig.30).Theresults revealednoobvioussystemictoxicityinanimalstreatedwithEBO-Gel comparedtountreatedanimals,validatingthebiosafetyofEBO-Gel.

Discussion

Thehealingoftraumaticandsurgicalwoundsrepresentsasigniﬁcant challengethataffectsmillionsofindividualsworldwide,placing immensestrainonhealthcaresystemsandpatientsalike.Amongthe

mostcommoncomplicationsencounteredinsuchwoundsare bleeding,poorhealing,andthedevelopmentofhypertrophicscarring. Adipose-derivedstemcell-derivedexosomeshaveemergedaspromisingtherapeuticagents,boastingantioxidantandpro-angiogenic propertiesthatpromotetissueregeneration.However,thehypoxic microenvironmentofpoorlyhealingwoundscancompromisetheir deliveryefﬁcacy.

Althoughacutehypoxiacanstimulateinitialtissueregeneration byincreasingvascularendothelialgrowthfactor(VEGF),prolonged hypoxiahindersoptimalrecovery53.Whilehypoxiainitiatesneovascularization,itcannotsustaintheprocess54,emphasizingthe importanceofrestoringoxygenlevelsaftertheinitialacutehypoxia phaseinwoundhealing.Moreover,loweroxygentensionduring hypertrophicscarprogressionsuggestsoxygensupplyasapotential strategytoexpeditewoundhealingandpreventhypertrophicscar formation55.Addressingthesemultifacetedchallenges,ourstudy introducesatissueadhesivePVA/GAhybridhydrogelthatexhibitsa remarkablehemostaticsynergisticeffectbyincorporatingthetherapeuticpropertiesofgelatin,specificallytargetingirregularlyshaped andbleedingwounds.Moreover,wehavetakenapioneeringstepby embeddingexosome-coatedoxygennanobubbleswithinthehydrogel,therebyelevatingthedeliveryefficiencyofexosomesand addressinghypoxia.Thiscombinationnotonlyacceleratesthewound healingprocessbutalsoeffectivelyremovesexcessROSfromthe woundtissue,alleviatinghypoxiaandmitigatingtheriskofhypertrophicscars.ThescarlesshealingpotentialofEBO-Gelisattributedto themultifacetedfunctionsofADSC-Exos,enrichedwithmicroRNAs suchasmiR-192-5pandmiR-29athatregulate fibroticresponsesand keloid fibroblastmigration56.Moreover,EBO-Gelshowspromisein addressingabnormalmetabolicconditionsandoxygentensionin hypoxichypertrophicscars57 58,offeringapromisingavenueforfurther investigationinscarmanagement.

Insummary,ourresearchhasculminatedinthedevelopmentofa hybridhydrogelthatpossessesremarkablecapabilitiesinhemostasis, anti-inﬂammation,hypoxiamitigation,andenhancedexosomedelivery,which,whenappliedtofull-thicknesswounds,demonstratedan acceleratedhealingspeedandsigniﬁcantlyimprovedhealingquality. Notably,EBO-Gelcouldalsobepotentiallyusedfordiabeticchronic woundtreatment,leveragingitsoxygen-supplyingcapability,antioxidantproperties,promotionofcellmigration,andenhancementof vascularization.Futureresearchcantargetotherischemicconditions, includingchronicwoundsandpotentiallycancer.

Methods

Ethicalstatement

Thisstudycomplieswithallrelevantethicalregulations.Allanimalrelatedprocedureswereconductedinaccordancewiththeguidelines oftheInstitutionalAnimalCareandUseCommitteeandtheDivisionof AnimalResourcesattheUniversityofIllinoisundertheprotocol approvedbytheInstitutionalAnimalCareandUseCommitteeatthe UniversityofIllinois(IACUCProtocol#:23012).

Materials

Bovineserumalbumin,dextransulfatesodiumsalt(Mw200kDa), poly(vinylalcohol)(Mw89–98kDa),sodiumtetraboratedecahydrate, collagenasetypeI,5-Bromo-2′-deoxyuridinewerepurchasedfrom Sigma-Aldrich.MTT(Catalog#M6494),CFSE(Catalog#C34554),Dio (Catalog#V22886),LAMP2monoclonalantibody(Catalog#MA1-205), H2DCFDA(Catalog#D399),propidiumiodide,BrDUprimaryantibody (Catalog#B35128),AlexaFluor™ Plus555Phalloidin(Catalog #A30106),CD86primaryantibody(Catalog#PA5-114995),CD206 primaryantibody(Catalog#MA5-44409),F4/80monoclonalantibody (Catalog#14-4801-82),CD31primaryantibody(Catalog#MA1-26196), IL-6primaryantibody(Catalog#MA545069),Dihydroethidium(Catalog#D1168)wereobtainedfromThermoFisherScientiﬁc.Secondary

antibodiesusedinthisstudywereobtainedfromThermoFisherScientiﬁc,includingGoatanti-MouseIgG(H+L)Cross-AdsorbedSecondaryAntibody,FITC(Catalog#F-2761),Goatanti-RabbitIgG(H+L) SecondaryAntibody,FITC(Catalog#65-6111),andGoatanti-RatIgG (H+L)Cross-AdsorbedSecondaryAntibody,AlexaFluor™ 555(Catalog#A-21434).Allantibodieswerediluted100timesexceptGoatantiRabbitIgG(H+L)SecondaryAntibody(1:50).ROS/Superoxidedetectionassaykit(ab139476),calceinAM(ab141420),DAPI(ab228549) werepurchasedfromAbcam.Humanmesenchymalstemcellmarker antibodypanelwaspurchasedfromR&Dsystems(Catalog#SC017).

Celllinesandanimals

TheHDFacelllinewaspurchasedfromAmericanTypeCultureCollection(PCS-201-012,ATCC)andcellswereculturedinFibroblastBasal Medium(PCS-201-013,ATCC)supplementedwiththeFibroblastGrowth Kit-LowSerum(PCS-201-041,ATCC),1×Antimycotic–Antibiotic (15240096,ThermoFisher)at37°C,5%CO2.HUVECs(LonzaC2517A, usedexperimentallybeforepassage6)weremaintainedinEndothelial CellGrowthMedium2(EGM)EBMTM-2BasalMedium(Catalog#CC3156,Lonza)supplementedwithEGMTM-2SingleQuotsTMSupplements(Catalog#CC-4176,Lonza),1×Antimycotic–Antibiotic(15240096, ThermoFisher)at37°C,5%CO2.PrimaryADSCswerederivedfrom discardeddonoradiposetissue,as describedbelow.MaleSpragueDawleyratswerepurchasedfromEnvigoLaboratory(Indianapolis,IN, USA).Allratswerehousedataconstanttemperatureandhumidityina roomwithanartiﬁcial12hrslight/darkcycleandallowedfreeaccessto foodandwater.Allanimalstudieswereperformedinaccordancewith theguidelinesoftheInstitutionalAnimalCareandUseCommitteeand theDivisionofAnimalResourcesattheUniversityofIllinois.

ADSCsisolation,culture,andcharacterization

ADSCswereisolatedfromhumanadiposetissueobtainedfromThe SpecimenProcurementServiceCenter,CarleHealth,CarleFoundation Hospital.Thetissuesutilizedinthisstudyarediscarded “de-identified tissues” withnopersonalinformationandareexemptfromtheneed forformalInstitutionalReviewBoard(IRB)approvalandinformed consent.TheydonotfallunderHumanSubjects.Briefly,tissuewas digestedin0.075%collagenasetype1preparedinPBScontaining2% penicillin/streptomycinfor30minat37°C,5%CO2,andthemixture wascentrifugedat2000rpmfor5min.Then,thecellpelletwas resuspendedin3mLofDMEMsupplementedwith20%FBS,1%Lglutamine,and1%penicillin/streptomycin,andthecellsuspensionwas then filteredthrough70 μmcellstrainer.Thecellswereplatedina lysinecoatedtissuecultureplateandadheredfor48h.Afterthe confluencyreached80%,theADSCsweresubcultured.

TheisolatedADSCswerecharacterizedby flowcytometry.The cellswereincubatedwithhumanmesenchymalstemcellmarker antibodypanelfor30minatroomtemperature,followedbycentrifugationat300× g for5min,andthesamplewashedtwicein2mLof PBSwith3%BSA.Then,thecellswereincubatedwithsecondaryantibodyfor30mininthedarkandwerewashedandresuspendedin flow cytometrystainingbufferfor flowcytometricanalysisusingBD FACSDiva(v9.0).

Exosomeisolationandcharacterization

Atthe3rd 8th passage,ADSCswereculturedinDMEMwith5% exosome-depletedFBS,andthespentmediawascollectedafter48h andcentrifugedat2000× g for10minat4°Ctoremovecelldebris. Thesupernatantwasthenultraﬁlteredwitha100kDa ﬁlter(Amicon 15),andtheresultingsolutionwasultracentrifugedat120,000× g for 90minat4°Ctoobtainexosomepellets.TheexosomewasresuspendedincoldPBSandstoredat 80°Candusedwithinonemonth.

Tocharacterizetheisolatedexosome,nanoparticletracking analysis(NTA)NTA(NanoSightNS300,MalvernPanalytical)wasused tomeasuretheconcentrationandsizeofexosome.TEMimageswere

obtainedbyScanningTransmissionElectronMicroscope(S)TEM (ThermoFisherFEI,TecnaiG2F20S-TWIN).Sampleswerenegatively stainedwithuranylacetate(1w/w%).

PreparationofONBandEBO

ONBwaspreparedthroughanultrasonicationmethod.Briefly,40mg ofBSAand80mgofdextransulfateweredissolvedin10mLsterile 1×PBSbuffer(pH=7.4),andthenfullymixedusingamagneticstirrer overnight.Further,ultrasonicationwasconductedinicebathwithan ultrasoniccelldisruptor(SFX250,Branson,USA)with3sonand3soff cycle,at50%ofamplitudefor7minwhileoxygenwascontinuously introducedinthissystem.Theresultingsolutionwas filteredbya 0.22 μmcelluloseacetate filter.Finally,ONBwascollectedthrough ultrafiltrationwithAmicon15 filter(100kDa).

EBOwasgeneratedbyutilizingultrasonicationtocoattheexosomemembraneontotheONB.Typically,exosomesandONBswere thoroughlymixedattheratioof1:2,followedbyultrasonication.The ultrasonicationprocesswascarriedoutinanicebath,withacycleof 10sonand10soff,atanamplitudeof50%,foradurationof5min.This cyclewasrepeatedthreetimes.

CharacterizationofONBandEBO

ThehydrodynamicsizedistributionandzetapotentialofONBandEBO wasobtainedbytheLitesizer™ 500(AntonPaar).(S)TEMwas employedtoacquireTEMimages,withthesamplesbeingnegatively stainedwith1w/w%uranylacetate.TheNanoDrop™ OneMicrovolume UV-VisSpectrophotometer(Catalognumber:ND-ONE-W,ThermoFisherScientiﬁc)wasutilizedtomeasuretheUV-visabsorbance.The infraredspectraofBSAandONBwereobtainedbyaFT-IRspectrometer(Thermo-NicoletIs-50FTIR).Oxygenreleasepropertywas monitoredbyOrion™ VersaStarPro™ DissolvedOxygenElectrochemistryBenchtopMeter(ThermoFisherScientiﬁc)every0.5hinthe durationof10h.

EBO-Gelsynthesisandcharacterization

PVAandgelatinwerecompletelydissolvedindistilledwater,resulting inthe ﬁnalconcentrationsof8wt%and2wt%,respectively.Subsequently,a2wt%solutionofboraxwasaddedtothemixture,facilitatingtheformationoftheblankPVA/GAgel.TopreparetheExo-Gel, ONB-Gel,andEBO-Gelformulations,exosomes,ONBs,andEBOwere evenlydistributedwithinthePVA/GAmixture(10wt%PVAand2.5wt% GA)andwerefurthercrosslinkedbyanequivalentvolumeofa2wt% boraxsolution.

AllrheologicaltestsweredoneonanARES-G2rotationalrheometer(TAInstruments,DE)usinga25-mmparallel-plategeometryat 1mmgapwithsandpapersattachedtobothplatestopreventslippage. Thegelswereheatedwhilebeingstirredat T = T g ,GA ∼ 60 Cfor10min andthesampleswereallowedtorestfor5minbeforeany rheologicaltests.

Oscillatorystrain-amplitudesweepexperimentwasusedto identifythecriticalyield-strainamplitude, γ y ,thatapproximatesthe boundarybetweenlinearandnonlinearregimesofbothblankand EMO-ladenPVA/GAgels.Thesetestswereperformedat ω =1rad=s,and sweptupfrom γ 0 =0 1%to500%.BothblankandEMO-ladengels exhibit γ y ∼ 100%,seeSupplementaryFig.11.Toassesstheinjectability, thecomplexviscosity, jη * j ω 1 G0 2 + G’’2 p ,isnextextractedfrom thisstrain-amplitudesweepexperiment,andplottedagainstapplied stressamplitude, σ 0 ,forbothblankandEBO-hydrogels,SupplementaryFig.12.Bothhydrogelsexhibityieldstress, σ y ,ofabout2kPa. Dramaticshearthinningoccurswhentheappliedstressexceeds σ y .To furtherassesstheinjectability,thestainedhydrogelwasloadedintoa syringewithaninnerdiameterof4.7mmandsubsequentlyextruded toconformtotheshapeof “UIUC”,seeFig. 3b.Thissubﬁgureshows thevisualconﬁrmationofshearthinningwherethehydrogelswere

extrudableyetbeabletoretaintheirshapeforatleast60minonthe substrate.Aquantitativeestimateofforcerequiredtoextrudecomes fromabalanceofappliedforcegeneratingapressurethatmustexceed theyieldstressatthewallsincircularpipe ﬂow, F =2σ y L R A =0 8N. Invertingthisequation,wecanestimatetheappliedwallshearstress during ﬂowinthesyringe,e.g.foraforceontheorderofafewNewtons,withL/R~10,thewallshearstressis~100,000Pa.Ourhydrogels exhibitgoodshaperetentionandshearthinningandthussuitablefor invivoapplication.

Linearviscoelasticity(LVE)wasassessedusingoscillatoryshearfrequencysweepexperimentsatsmall-strainamplitude(γ 0 =5%).TheLVE resultsarereportedinFig. 3h,andrelaxationtimeisextractedfromthe crossoverfrequency, ωc : λ =1=ωc ≈1 5s.Thissignifiesthesolid-likebehavioratatimescaleshorterthan λ,andthegelscan flowat η * ≈2,000Pa sfromnetworkrearrangingatalongertimescale.Visualevidenceconfirmsthisbehavior.ThetiltedvialinFig. 3a,wasusedtomakeanestimate ofthe flowviscosity.Usinggravitational flowdownanincline,anupper boundviscosityisestimatedtobe η = ρgh2 sin θ=2vs =1 9×104 Pa s basedonanassumed fluidthickness h =10mm,tiltangle θ =40 ,and maximumvelocitybasedonadisplacementof5mmoverthe5minof observation59.Thisviscosityisobservedatthecharacteristicdriving stressfromgravityof σ = ρgh =98Pa.

Twomethodswereusedtodemonstratetheself-healingproperties. The firstmethodwastofragmentizethePVA-GAsamplesandreprocess them.Toreprocessthesamples,theywerecutupintosmallpieces (about5×5mmsquares)beforeblobbingandsandwichingthem betweentwoglassplatesat1.2mmgap.Foreachreprocessing,about 10wt%offreshsamplewasaddedtocompensateforsmallmasslost fromthefragmentation.Linearviscoelasticity(videsupra)ofthegels weremeasuredasaprobingsurrogatemetricforbothvirgin(anewly crosslinkedgel)andreprocessedsamples.Resultsrevealnosignificant differenceuptothethirdreprocessing,withonlyaslightincreaseinLVE attributabletoevaporation.Thesecondmethodwastoprobetherheologicalrecoveryfromlargedeformation.Forthisexperiment,asmallamplitudeoscillatoryshear(SAOS)atthestrainamplitudeof5%was first appliedfor60s,andthenlarge-amplitudeoscillatoryshear(LAOS)at 200%for60s,andlastlybacktoSAOSat5%toprobetherecoveryof linearviscoelasticity.Therecoverywasmonitoredfor30minuntilthe PVA-GAgelsreacha finallogarithmicagingregime.

Collagenprecursorsolutionsaremadeandkeptoniceuntilthe testtime.Approximately55 μLofthecollagenprecursorsolutionswas depositedonthebottomplateandtheupperplatewasthenlowered intoposition.Athinlayerofheavymineraloil(FCC/USP,FisherChemical)wasappliedtothesamplefreesurfacetopreventevaporation. Thetemperaturewasthenraisedto37°Ctoinitiategelation.

ESEM(FEIQuantaFEG450ESEM)wasemployedtovisualize microstructuresofhydrogels.Macroscopicself-healingpropertywas evaluatedasfollows:TwoEBO-Gelspecimens,eachwithadiameterof 2.2cm,werefabricatedandsubsequentlysubjectedtostainingusing crystalvioletandreddyes,respectively.Thehydrogelswerethen halved,andonehalffromeachtypeofthehydrogelwasjuxtaposed. After1min,thereassembledgelwassubjectedtostretchingtoassess itshealingcapacity.Thediffusionofdyewasmonitoredfor100min. Theassessmentofshapeadaptabilityinvolvedplacingthehydrogel withinvarioussiliconemoldstoevaluateitsabilitytoconformtodifferentshapes.Todemonstratetheinjectabilityofthehydrogel,the stainedhydrogelwasloadedintoasyringewithaninnerdiameterof 4.7mmandsubsequentlyextrudedtoformtheshapeof “UIUC” . Oxygenreleasewithin48hwasmonitoredbyOrion™ VersaStarPro™ DissolvedOxygenElectrochemistryBenchtopMeter(ThermoFisher Scientiﬁc)usingapreviouslydevelopedmethodology60

Invitrodegradation

Forinvitrodegradationanalysis,EBO-Gelsweresubmergedina5mL PBSsolution(pH=7.4)withlysozyme(1000U/mL).Theincubationwas

inaconstant-temperatureincubatorsetat37°C.Thehydrogelswere takenoutforlyophilizationandweighedatintervalsof0,1,2,and3days. Tomaintainenzymefunctionality,thelysozymesolutionwasrefreshed daily.Theinvitrodegradationratewasdeterminedusingtheformula:

Degradationrate= W 0 W t =W 0 ×100%

where W 0 and W t representedtheweightsoftheoriginaland remaininghydrogels,respectively.

Hemolysisassay

FreshratbloodwasobtainedviacardiacpuncturefollowingeuthanasiaandanticoagulatedusingHeparin.Subsequently,erythrocytes wereisolatedthroughcentrifugationat10,000× g for5minand washedusingD-PBS.Next,0.8mLofdilutederythrocyteswere exposedto200 μLofTritonX-100,PBS,anddifferenthydrogels. Followinga4-hincubationatroomtemperature,themixtureswere centrifugedat10,016× g for5min,andtheabsorbanceofthesupernatantwasmeasuredat540nmusingamicroplatereader.The hemolyticratiowascalculatedusingthefollowingformula:Hemolysis ratio=ODt ODn ðÞ=ðODp ODnÞ ×100%.ODt:ODvalueoftested samples,ODn:ODvalueofnegativecontrol,ODp:ODvalueofpositive control.

Cellviability

5×103 HDF-acellswerepre-seededinto96-wellplate.After24hof culture,themediumwasrefreshed,andtheHDF-acellswerecoincubatedwithhydrogels.MTTassaywasperformedtoevaluatecell viability.Inbrief,20 μLoftheprepared5mg/mLMTTsolutionwas addedtoeachwelloftheplateandincubatedat37°Cfor4h.Followingtheadditionof100 μLofDMSO,theplatewasincubatedforan additional4hat37°Cinthedark.Ultimately,theabsorbanceat 490nmwasmeasuredusingamicroplatereader(SynergyH1,BioTek Instruments).

Cellproliferation

2×103 HDF-acellswerepre-seededinto96-wellplate.After24hof normoxiaincubation,themediumwasrefreshed,andthehydrogels wereadded.Then,thewellplateswerekeptinhypoxiachamberwitha mixtureof3%O2,5%CO2,92%N2 at37°Cfor0h,24hand48h, respectively.Atdifferenttimepoint,WST-1reagentwasaddedandthe opticaldensityat440nmwasthenmeasuredafter4hofincubation accordingtomanufacturerdescription.

Cellmigration

Forscratchingassay,HDF-acellswerepre-seededina24-wellplate. Aftertheconﬂuencyreachedto80–90%,scratchingwoundswere createdby200 µLsterilepipettetips,andtheculturemediumwas replacedwithDMEMcontaining1%FBS.Then,hydrogelsweretreated intheupperchamberofcellcultureinserts(Millicell®,Sigma-Aldrich), andtheplateswerekeptinhypoxiachamberwiththehypoxicmixture asdescribed.Cellmigrationwasobservedbymicroscope(ZEISS)at differenttimepointsandwasquantiﬁedbyImageJsoftware(v1.53t).

Fortranswellmigrationassay,HDF-acellswereseededinthe upperchamberofcellcultureinsertswithadensityof1×104 cells/mL, whilethehydrogelsweretreatedintothelowerchamberwithculture medium.After24h,thecellsontheuppersideof ﬁlmweregently removedbyacottonswab,andthecellsthatmigratedtothelowerside werestainedwith0.5%crystalvioletfor1h.Thenumberofmigrated cellswerequantiﬁedbyImageJsoftware(v1.53t).

Exosomeuptake

ToprepareDio-labeledexosome,EBOwasincubatedwithDio(10 μM) for20min,followedbyultraﬁltrationusingAmicon15at1000× g for 10min.HDF-acellswerepre-seededin35mmcoverslipdishesand

culturedwithexosome-depletedmedium.Afterattachment,DiolabeledEBOwasaddedandincubatedfor6h.Thecellswere ﬁxedby 4%PFAafterco-incubation,andthenucleuswasstainedwith4′,6diamidino-2-phenylindole.Intracellularuptakewasobservedby Z-stack3Dconfocalimagingwithorthogonalviewsatthestepsizeof 0.3 µm(LeicaSP8).

Immunoﬂuorescenceanalysis

HDF-acellswerepre-seededin35mmcoverslipdishes.Afterattachment, hydrogelswereadded,andtheimagingdishesweretransferredto hypoxiachamberunderthesamehypoxicconditionsasdescribed.Followingthecompletionofhypoxiaincubation,thecellswerewashedwith PBSandsubsequently fixedwith4%paraformaldehyde.Thiswasfollowedbypermeabilizationusing0.3%TritonX-100foranadditional 10min.Then,thecellswereincubatedwithasolutionof3%BSAinPBS for1hat37°Ctopreventnonspecificbinding.Next,variousprimary antibodies(anti-BrdU,anti-Lamp2)wereappliedtothecellsandallowed toincubateovernightat4°C,andthecellswerethenincubatedwith secondaryantibodyfor1hat37°C.ForBrdUstaining,themediumwas replacedwith10 μMBrdUinculturemediumbeforehydrogelswere added.After fixation,thecellswereincubatedwith2MHClfor0.5hfor acidhydrolysis.CytoskeletonwasstainedbyAlexaFluor™ Plus555 Phalloidinfor20min.Imageswerecapturedbyaconfocallaserscanning microscope(LeicaSP8)andanalyzedbyImageJsoftware(v1.53t).

Tubeformationassay

HUVECsfortubeformationassaywereusedbeforepassage6.Typically,100 μLofMatrigel(Corning)wasaddedina96-wellplateand incubatedtogelat37°Cfor1h.10,000HUVECswereseededintoeach wellandhydrogelswerethenadded.Plateswerefurthertransferred intothehypoxiachamber,andsuppliedwithamixtureof3%O2,5% CO2,92%N2 at37°C.After6h,brightﬁeldimagesoftubeformation werecapturedbyLeicamicroscope.Thenumberofbranchesandtotal lengthofbrancheswasquantiﬁedbyImageJsoftware(v1.53t).

Invitrohemostasisassay

HeparinizedratbloodandhemocytesweremixedwithControl,Carbopolgel(1wt%),andEBO-Gel,respectively,atthevolumeratioof1:2.After 1min,thevialwasinversed,andthehemostasispropertywasobserved.

Invivowoundhealingstudy

MaleSprague-Dawleyratsof250–300gweight(8weeksold)were obtainedfromEnvigoLaboratory(Indianapolis,IN).Allanimalstudies wereperformedinaccordancewiththeguidelinesoftheInstitutional AnimalCareandUseCommitteeandtheDivisionofAnimalResourcesat theUniversityofIllinois(IACUCProtocol#:23012).Theratswererandomlysortedinto fivegroups:NT,Blank-Gel,Exo-Gel,ONB-Gel,andEBOGel.Theprecursorsolutionswerepreparedbythoroughlymixing0.4mL ofPVA/GA(10wt%PVAand2.5wt%GA)and0.1mLofnanoparticle solutions(Exo,ONB,andEBO)toachieve finalconcentrationsof8wt% PVAand2wt%GArespectively.Hydrogelswerecreatedbycombining theprecursorsolutionwithanequalvolumeof2wt%boraxsolution beforeapplication.Full-thicknesswoundswerethenmadeontheback usingasteriledisposable8mmdiameterdermalbiopsypunch(MEDLINE)toadepthof2mm.Thehydrogelswereappliedtothewounds aftersurgeryandrefreshedeverytwodays.IntheTegadermgroup,no hydrogelswereapplied.Thehealingprocesswasmonitoredbyadigital camera,andthewoundareawasquantifiedbyImageJsoftware.

Onday4andday14,animalswereeuthanized,andthewound tissueswerecollected.Then,thetissueswere ﬁxedin10%formalin neutralbufferedsolution(Sigma-Aldrich)andfurtherembeddedin parafﬁnandsectionedforH&E/MassonTrichromestainingaccording tothemanufacturermanual.Thehistologyimageswereacquiredby microscope.TheScarIndex(mm),denotedasScarArea(mm²)divided bytheAverageDermalThickness(mm).Dermalthickness,epidermal

thicknessandscarareawerequantiﬁedbyNDPView2software.The collagenvolumefractionwasquantiﬁedbyImageJsoftware(v1.53t).

Statisticsandreproducibility

StatisticalanalysiswasperformedusingGraphPadPrism(v9.0).The differencesbetweenanytwogroupswereanalyzedusingatwo-tailed, unpairedStudent’s t test.Forcomparisonsinvolvingmorethantwo groups,one-wayANOVAwithTukey’smultiplecomparisonswas employed.Groupeddatawereassessedusingtwo-wayANOVAwith Dunnett’smultiplecomparisons. P <0 05wasconsideredstatistically signiﬁcant.Thepresenteddataindicatethemean±SDfromatleast twoindependentexperiments.

Reportingsummary

FurtherinformationonresearchdesignisavailableintheNature PortfolioReportingSummarylinkedtothisarticle.

Dataavailability

Theauthorsdeclarethatallthedatasupportingthe ﬁndingsofthis studyareavailablewithinthearticleanditssupplementaryinformation.SourcedataareprovidedasaSourceData ﬁle.Sourcedataare providedwiththispaper.

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Acknowledgements

JIacknowledgesfundingfromtheHealthmakerlab(Award#HML0000) attheCarleIllinoisCollegeofMedicine.JIacknowledgesfundingfrom theNSF-SBIR(Award#2031313).XHacknowledgesthesupportfromthe CancerScholarsforTranslationalandAppliedResearch(C*STAR)ProgramsponsoredbytheCancerCenteratIllinoisandtheCarleCancer Center.CSacknowledgestheBeckmanInstituteforAdvancedScience andTechnologyattheUniversityof Illinois,Urbana-Champaignfora postdoctoralfellowship.ThankstotheTumorEngineeringandPhenotyping(TEP)SharedResourceatCancerCenteratIllinois(UIUC)for assistanceinbiologicalevaluation.OursincerethankstoKarenDotyat theHistologyfacilityintheDepartmentofComparativeBiosciences (UIUC)forassistanceintissueprocessingandimmunoﬂuorescent staining.ThankstoYunleiZhaoforassistanceindataanalysis.Figure 5c wascreatedwithBioRender.com(Agreementnumber:PQ26LNGIOK).

Authorcontributions

X.H.wasresponsiblefortheoverallmethodology,experiments,results anddiscussion,dataanalysis,andthe ﬁrstdraftofthemanuscriptand revision;J.I.conceivedtheidea,providedsupervisionandguidanceon experimentalplanningandfunding;W.R.assistedinanimalstudies;L.J. assistedinanimalexperimentsandbiologicalevaluation;C.S.andR.H.E. evaluatedtherheologicalcharacteristicsandrelateddiscussion.All authorscontributedtothewritingofthemanuscriptandhavereadand approvethemanuscript.

Competinginterests

J.I.andX.H. ﬁledaprovisionalpatentapplicationfortheexosomecoatedoxygennanobubble-ladenhydrogel(U.S.PatentApplicationNo.: 63/631,255).Theremainingauthorsdeclarenocompetinginterests.

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