A Novel Dehydrated Human Umbilical Cord Particulate Medical Device: Matrix Characterization, Performance, and Biocompatibility for the Management of Acute and Chronic Wounds - FlowPaper FlipBook

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Citation:Croteau,D.;Buckley,M.; Mantay,M.;Brannan,C.;Roy,A.; Barbaro,B.;Grifﬁths,S.ANovel DehydratedHumanUmbilicalCord ParticulateMedicalDevice:Matrix Characterization,Performance,and BiocompatibilityfortheManagement ofAcuteandChronicWounds. Bioengineering2024,11,588. https://doi.org/10.3390/ bioengineering11060588 AcademicEditor:JaroslavaHalper Received:29April2024 Revised:28May2024 Accepted:4June2024 Published:8June2024 Copyright:©2024bytheauthors. LicenseeMDPI,Basel,Switzerland. Thisarticleisanopenaccessarticle distributedunderthetermsand conditionsoftheCreativeCommons Attribution(CCBY)license(https:// creativecommons.org/licenses/by/ 4.0/). bioengineering Article ANovelDehydratedHumanUmbilicalCordParticulate MedicalDevice:MatrixCharacterization,Performance,and BiocompatibilityfortheManagementofAcuteand ChronicWounds DominiqueCroteau,MollyBuckley,MorganMantay,CourtneyBrannan,AnneliseRoy,BarbaraBarbaro andSarahGrifﬁths* ResearchandDevelopment,StimlabsLLC,1225NorthmeadowParkway,Suite104,Roswell,GA30076,USA *Correspondence:sarah@stimlabs.com Abstract:Chronicwoundspresentasigniﬁcantsocioeconomicburdenforecastedtoincreasein prevalenceandcost.Minimallymanipulatedhumanplacentaltissueshavebeenincreasinglyem- ployedandproventobeadvantageousinthetreatmentofchronicwounds,showingimproved clinicaloutcomesandcost-effectiveness.However,technologicaladvanceshavebeenconstrainedby minimalmanipulationandhomologoususecriteria.Thisstudyfocusesonthecharacterizationofa noveldehydratedhumanumbilicalcordparticulate(dHUCP)medicaldevice,whichoffersaunique allogeneictechnologicaladvancementandtheﬁrsthumanbirthtissuedeviceforwoundmanagement. Characterizationanalysesillustratedacomplexextracellularmatrixcompositionconservedinthe dHUCPdevicecomparedtonativeumbilicalcord,withabundantcollagensandglycosaminoglycans imbibinganintricateporousscaffold.Dermalﬁbroblastsreadilyattachedtotheintactscaffoldofthe dHUCPdevice.Furthermore,thedHUCPdeviceelicitedasigniﬁcantparacrineproliferativeresponse indermalﬁbroblasts,incontrasttoﬁbrillarcollagen,aprevalentwounddevice.Biocompatibility testinginaporcinefull-thicknesswoundmodelshowedresorptionofthedHUCPdeviceandnormal granulationtissuematurationduringhealing.ThedHUCPdeviceisapromisingadvancementin woundmanagementbiomaterials,offeringauniquecombinationofstructuralcomplexityadeptfor challengingwoundtopographiesandamicroenvironmentsupportiveoftissueregeneration. Keywords:umbilicalcord;chronicandacutewounds;cellular,acellular,andmatrix-likeproduct (CAMP);skinsubstitute;cellular-and/ortissue-basedproduct(CTP);woundhealing;placental tissue;particulatewounddevice;woundanimalmodel;cellproliferation 1.Introduction Chronicwoundsbroadlydescribetissuedefectsthatfailtosuccessfullyprogress throughthenormalhealingcascadeinatimelyandorganizedmanner.Approximately 1–2%oftheglobalpopulationwillexperienceachronicwoundintheirlifetime,whichis showntodirectlycontributetoadecliningqualityoflife[1,2].Thesenonhealing,orhard-to- heal,woundsstemfromdiverseetiologiesandpatientcomorbiditiesandcreateanimmense publichealthburden[1,3].Thereexistsanunmetmedicalneedforcontinuedwoundcare innovationtoimproveoutcomesforchronicwounds.Medicaldevicewounddressings havebeenmarkedbytechnologicalandmaterialadvancementsinthepastcentury.While initiallygauzewasoncethetraditionalchoiceforawoundcovering,dressingshave evolvedovertimetoincludethoseincorporatedwithantimicrobials,anti-inﬂammatory properties,andotherelements[4].Morerecently,biomaterial-basedoptions,collectively referredtoasCellular,Acellular,andMatrix-likeProducts(CAMPs;formerlyknownas skinsubstitutesandCellular-and/orTissue-basedProducts(CTPs))havebeendeveloped andnowexistinanarrayofofferings[5,6].Scientiﬁcadvancesinrecentdecadeshave Bioengineering2024,11,588.https://doi.org/10.3390/bioengineering11060588https://www.mdpi.com/journal/bioengineeringBioengineering2024,11,588 2of16 enabledtheprogressionfromsingle-componentcollagendressingstotheemergenceof intactextracellularmatrixdeviceswiththeultimategoalofcloselymimickingcomplex nativehumantissuecompositionandstructure[4,5,7,8].Whilethisevolutionofdressings derivedfromvariousbiomaterialsourcesbringsimmensevaluetotheﬁeldofwound treatment,immunologicalbarrierstoproductdevelopmentoftenpersist,requiringhighly involved,andfrequentlyharsh,tissuepreparationmethodstoensurexenoantigenicityis adequatelyaddressed[9,10]. Theregenerativepotentialofhumanconnectivetissueproductsprovidesanappealing allogeneicapproachtoaddressthischallenge,butuntilnowhasbeenrestrictedduetothe existingregulatoryframeworkforhumantissueproductswhichlimitsinnovationpoten- tial[11].Humanumbilicalcordtissueisadistinctiveallogeneicbiomaterialsourcethathas shownpromiseforusesinacuteandchronicwoundsduetotheregenerativepotentialofa complexconnectivetissueextracellularmatrix[12].Theumbilicalcordlinkstheplacenta andfetustoprovidenutrientsandoxygentothegrowingfetus[13].Amucoidconnective tissuematrix—originallydescribedbyThomasWhartonin1656—ensurestheunrestricted ﬂowofnutrients,impartingdistinctivestructuralpropertiestoresistexternalforces[14,15]. Theintricatestructuralattributesofthisconnectivetissuematrixthatistypicallyconsidered biologicalwastemakesforauniqueopportunityinregenerativemedicineandwoundcare applications.Herein,weprovideacomprehensivematrixcompositioncharacterization andinvitroperformanceevaluation,aswellasinvivobiocompatibilityandperformance, ofanewlyUnitedStatesFoodandDrugAdministration(FDA)-clearednoveldehydrated humanumbilicalcordparticulate(dHUCP)medicaldevice.Thisstudyhighlightstheﬁrst humanumbilicalcord-derivedmedicaldeviceandtheﬁrsthumanbirthtissueintendedfor woundmanagementthathasreceived510(k)clearancefromtheU.S.FDA[16,17]. 2.MaterialsandMethods 2.1.BiomaterialSourcingandProcessing Humanbirthtissues,consistingoftheplacentaandumbilicalcord,wereobtainedvia donationfromhealthy,full-termcesariansectionbirthsandwerescreenedandtestedfor riskofcommunicablediseasesinaccordancewithFoodandDrugAdministration(FDA) regulations[18]andAmericanAssociationofTissueBanks(AATB)standardscurrentat thetimeofmanufacturing.Umbilicalcordtissueswereseparatedfromtheplacentaand dissectedtoremovetheumbilicalarteriesandvein.Tissueswerethensubjectedtopropri- etaryprocessing,inclusiveofaseriesofrinsesdesignedtoremovebloodremnantsand unwantedconstituentswhileretainingcriticaldesiredextracellularmatrixcomponentsand structuralcharacteristics.Theprocessingstepsrenderalyophilized,terminallysterilized (SAL10 − 6 viaelectronbeamirradiation)dehydratedhumanumbilicalcordparticulate (dHUCP)medicaldevice(StimlabsLLC,Roswell,GA,USA).Forhistologicalcomparisons torawsourcematerial,freshumbilicalcord(f-UC)tissuewasasepticallydissectedfrom thematernaldiscandbrieﬂyrinsedwithpuriﬁedwatertoremoveexcesssurfaceblood content.Further,f-UCtissuewascutlaterallythroughtheveinwalltoopenthematerial forcross-sectionalanalysis. 2.2.HistologyandImmunohistochemistry Histologicalanalysisofnativef-UCandthedHUCPdevicewasperformedbyPremier Laboratory,LLC(Longmont,CO,USA),accordingtotheirstandardprocedures.Samples wereﬁxedin4%paraformaldehydeandprocessedforparafﬁnembedding,thensubse- quentlysectionedandmountedontochargedslides.Glycosaminoglycans(GAGs)and glycoproteinswerestainedwithAlcianBlue(pH2.5).SlidesforAlcianBluestainingwere incubatedin3%aceticacidfor2minpriortobeingsubmergedinAlcianBlue2.5for45min (AnatechLTD,BattleCreek,MI,USA),re-incubatedin3%aceticacid,rinsedintapwater, andplacedinNuclearFastRedfor10min.Slidesforimmunohistochemicalstainingwere incubatedin3.0%hydrogenperoxidefollowedbyepitoperetrievalviaProteinaseK(Agi- lentTechnologiesInc.,SantaClara,CA,USA),withtheexceptionofslidesforHyaluronicBioengineering2024,11,588 3of16 AcidBindingProtein(HABP)staining,whichwereincubatedinAvidinsolution(Agilent TechnologiesInc.,SantaClara,CA,USA)andBiotinsolution(AgilentTechnologiesInc., SantaClara,CA,USA).Slideswerethenblockedwithaserum-freeproteinsolution(Agilent TechnologiesInc.,SantaClara,CA,USA)priortoantibodyincubation.Primaryantibodies againstanti-CollagenI(Abcam,Waltham,MA,USA),anti-CollagenIII(ThermoFisher Scientiﬁc,Waltham,MA,USA),anti-laminin(EnCorBiotechnology,Gainesville,FL,USA), anti-ﬁbronectin(Abcam,Waltham,MA,USA),andbiotinylatedanti-HyaluronicAcidBind- ingProtein(MilliporeSigma,Burlington,MA,USA)wereusedtostaintheslidesatroom temperaturefor30min(60minforHABP).Uponconclusionofprimaryantibodyincuba- tion,appropriateHRP-conjugatedsecondaryantibodies(EnVision+;AgilentTechnologies Inc.,SantaClara,CA,USA)wereapplied,followedbyapplicationofDAB+Chromogen solution(AgilentTechnologiesInc.,SantaClara,CA,USA).Slideswerethenrinsedand counterstainedinamodiﬁedHarrishematoxylinsolution(AgilentTechnologiesInc.,Santa Clara,CA,USA).StainedsectionswerescannedusingtheAperioScanScopeXTimaging system(LeicaBiosystems,DeerPark,IL,USA)andImageScopesoftwareversion12.4.6. ScalebarswereformattedusingImageJversion1.54f. 2.3.TotalCollagenQuantiﬁcation TotalcollagenconcentrationswerequantiﬁedusingaTotalCollagenKit(Perchlorate- Free)(Abcam,Waltham,MA,USA)accordingtomanufacturer’sinstructions,withsome modiﬁcations.ThedHUCPdevicematerialwassubjectedtoalkalinehydrolysisat120 ◦ C for1hwithoutdilutioninwaterorhomogenization.Sampleswerechilled,neutralized withconcentratedhydrochloricacid,mixed,andcentrifuged.Theextractedsamples wereheatedat65 ◦ CuntildriedandthenreconstitutedwiththeOxidationReagentMix (chloramineTconcentrate,oxidationbuffer).Sampleswereincubatedfor20minatroom temperature,mixedwithDevelopersolutionfor5minat37 ◦ C,thenfurthermixedwith DMABConcentratefor45minat65 ◦ C.Sampleabsorbancewasmeasuredat560nm.Total collagenconcentrationwasquantiﬁedfromastandardcurveofCollagenIandconverted intomgoftotalcollagenpergramofthedHUCPdevice’sinitialdryweight. 2.4.TotalSulfatedGlycosaminoglycan(sGAG)Quantiﬁcation TotalsulfatedGAGconcentrationswerequantiﬁedusingtheBlyscanSulfatedGly- cosaminoglycanAssayKit(BioColorLtd,Carrickfergus,CoAntrim,UK)accordingtothe manufacturer’sinstructions,withsomemodiﬁcations.Allreagentsnotprovidedwith theassaykitweresourcedfromSigma-Aldrich(St.Lous,MO,USA)unlessotherwise stated.ThedHUCPdevicematerialwasdigested(5mg/mL)inpapainextractionsolution (0.098Msodiumacetate,0.014MEDTA,4.55mMcysteineHCl(ThermoFisherScientiﬁc, Waltham,MA,USA),0.44mMpapain,0.2Msodiumphosphatebuffer,pH6.4)at65 ◦ C for3h,andlysatesweredilutedwithdeionizedwater.Blyscandyereagentwasaddedto dilutedsamplesthatwerethengentlyagitatedfor30mintoprecipitateoutdyedsGAG material.Sampleswerethencentrifuged,dissociationreagentwasaddedtoeachsample pellet,andsamplesweretransferredtoaclear-bottommicrotiterplateandreadat656nm absorbance.TheconcentrationoftotalsGAGwasquantiﬁedfromastandardcurveof chondroitinsulfateandconvertedintomgoftotalsGAGpergramofthedHUCPdevice’s initialdryweight. 2.5.HyaluronanEnzyme-LinkedImmunosorbentAssay Theconcentrationofhyaluronicacid(HA)wasmeasuredusinganenzyme-linked immunosorbentassay(ELISA)forHyaluronan(R&DSystems,Minneapolis,MN,USA). ThedHUCPdevicematerialwasdigestedinpapainextractionsolution(0.098Msodium acetate,0.014MEDTA,4.55mMcysteineHCl,0.44mMpapain,0.2Msodiumphosphate buffer,pH6.4)at65 ◦ Cfor3h.Thehomogenateswerecentrifuged,andthelysates werecollectedanddilutedwithassaydiluent.AssayswereperformedaccordingtotheBioengineering2024,11,588 4of16 manufacturer’sinstructions.ThedHUCPHAconcentrationswerenormalizedtosample dryweights. 2.6.dHUCPDeviceAbsorbanceCapacity ThecontentsofavialofthedHUCPdevice(1cc)wereweighedpre-andpost- hydrationwith0.9%normalsaline(McKesson,Irving,TX,USA).Theabsorbancecapacity ofthedHUCPdevicewasassessedasthechangeintotalmassfromitsdrystatetoits hydratedstate. 2.7.ScanningElectronMicroscopy(SEM) SEMimagingwasusedtoelucidatetheultrastructureofthedHUCPdevice.Samples weremountedusingdouble-sidedcarbontapeandimagedwithaHitachiS3700-Nscanning electronmicroscope(HitachiHigh-TechnologiesCorporation,Tokyo,Japan)byApplied TechnicalServices(ATS;Marietta,GA,USA)accordingtotheirstandardprocedures.Images weretakenusingthebackscatterdetectorinvariablepressureat60Pawithanoperating voltageof20kV.HitachiS-3700NSEMsoftware(OperationVer8.1;EvacuationVer2.8) wasusedforimageacquisition.ScalebarswereformattedusingImageJversion1.54f. 2.8.InVitroCellularPerformanceMeasurements Normaldermalﬁbroblasts(WS1cellline,ATCC,Manassas,VA,USA)werecul- turedinEagle’sMinimalEssentialMedium(EMEM;ATCC,Manassas,VA,USA)sup- plementedwith10%fetalbovineserum(FBS;ATCC,Manassas,VA,USA)and1%peni- cillin/streptomycin(ATCC,Manassas,VA,USA),accordingtothemanufacturer’srecom- mendations.Forcellularattachmentstudies,250,000cellswereseededontothedHUCP device.After6h,themediumwasremovedandtheseededdevicewasincubatedwiththe livecellstainCalceinAM(MolecularProbes,Eugene,OR,USA)diluted1:4inDulbecco’s phosphate-bufferedsaline(DPBS;ATCC,Manassas,VA,USA)atroomtemperaturefor 15min.ThestainsolutionwasremovedandreplacedwithfreshDPBS.PiecesofthedHUCP devicewereplacedintonewwellsafterrinsingandwereimagedunderGFPﬂuorescence tocaptureadheredcellswhilethedHUCPdevicetopographywassimultaneouslycap- turedviatransilluminationusinganEVOSM5000imagingsystem(Invitrogen,Waltham, MA,USA).Asanegativecontrol,piecesofthedHUCPdevicenotseededwithcellswere subjectedtothesameincubationandstainingprocedure.Scalebarswereformattedusing ImageJversion1.54f. Forcellularproliferationstudies,thedHUCPdeviceandatypeIbovineﬁbrillar collagenpowderdevicewereextractedinEMEMsupplementedwith2%fetalbovine serumat37 ◦ Cfor6hwithgentleagitation.Thesolublefractionwascollectedandused forproliferationtreatmentconditions.Cellswereseededatadensityof6250cells/cm 2 understandardcultureconditions.After24h,mediumwasremovedandreplacedwitha starvationmedium(EMEMsupplementedwith2%serum)for6h—exceptforthepositive controlwhichwasmaintainedinEMEMsupplementedwith10%serumfortheentirestudy duration—followedbytheapplicationoftheprepareddHUCPdeviceandcollagenpowder extractspreparedasdescribedabove,ataﬁnalconcentrationof2mg/mL.Intandem,asep- arateplatesubjectedtothe6hstarvationwasthenremovedofmediaandfrozenat − 80 ◦ C, servingasa‘Day0’referencecontrol.Cellswereincubatedinthetreatmentconditionsfor 72h,themediumwasremoved,andtheplatewasfrozenat − 80 ◦ C.Uponthawingtoroom temperature,theproliferationofcellswasquantiﬁedwithCyQUANTCellProliferationAs- saykit(MolecularProbes,Eugene,OR,USA),accordingtothemanufacturer’sinstructions, andreadonaplatereaderat480nmexcitation/520nmemission.Cell-containingwells werebackground-ﬂuorescence-correctedformedia-matchedcontrolwellsthatdidnot containcells.Therelativeproliferationofthe72htreatmentwascalculatedandreportedas thepercentchangeinﬂuorescencewithrespectto,orfrom,the‘Day0’plateﬂuorescence valuesforeachtreatmentcondition.Bioengineering2024,11,588 5of16 2.9.BiocompatibilityTesting Aporcinefull-thicknesswoundimplantstudywasutilizedtoassessthebiocompati- bilityofthedHUCPdevice.ThestudywasconductedbyBridgePTS,Inc.(SanAntonio, TX,USA),approvedbytheirInstitutionalAnimalCareandUseCommittee,andperformed perU.S.FoodandDrugAdministrationGoodLaboratoryPractice(GLP)regulationsset forthin21CFRPart58andInternationalOrganizationforStandardization(ISO)10993- 6:2016testsforlocaleffectsafterimplantation[19].Inpreparationforwoundcreation, three(3)femaleYorkshire-crosspigs(35 ± 10kg)wereprovidedintramuscularinjection ofAtropine(0.02–0.05mg/kg),thensedatedwithTiletamine–Zolazepam(4.0–6.0mg/kg) andisoﬂurane(0.5–5%)mixedwithoxygen.Theskinwaspreparedusingachlorhexidine scrubandisopropylalcoholinalternatingfashionthreetimestomimictheskinpreparation inhumans. Full-thicknesssquare(2cm × 2cm)woundswerecreatedusingasterilescalpelblade andscissorsonthedorsalthoraxparalleltoeachsideofthespine,spaced2–3cmapart. Hemostasiswasconﬁrmed,andwoundswerewipedcleantoensuretheperiwoundarea wasdrypriortotreatment.ThedHUCPdevice(2cc)wasappliedtowoundsonDay0. Perthedevice’sinstructionsforuse,thedHUCPdevicewaspre-hydratedwithsaline, thenappliedtothebaseofthewoundbed,gentlyspreadevenlyacrossthewoundsurface uptothewoundmargins.Eachwoundwasthendressedandmonitoredregularly.The dHUCPdevicewasnotremovednorreappliedforthedurationofthisstudy.Pigswere assesseddailyforanysignsofpainandprovidedappropriatepainmanagementmedication asnecessary. Ondays7,14,and35,asubsetofwounds(10pertimepoint,sampledacrossall pigs)wassampledforhistopathologicalanalysesbyexcisingastripthroughthecenter ofthewoundtoincludemarginsofhealthyskin.Thesetimepointswereselectedbased onguidanceinISO10993-6:2016tocapturetheestimatedearlyandmid-degradation proﬁletimeframeoftheproductataclinicallyrelevantimplantationsiteandextendup toorbeyondtheestimatedpointofcompletedegradation.Sampleswereinitiallyﬁxed in10%neutralbufferedformalinforatleast24h,thensubsequentlyﬁxedinafresh aliquotofformalinforanother48htoensurefullformalinpenetration.Sampleswere parafﬁn-embedded,sectioned,mountedonslides,andstainedbyStageBio(Frederick, MD,USA)pertheirstandardprocedures.Masson’sTrichromestainwasusedtoevaluate thegranulationtissue,namely,toidentifyregionsofresidualimplantmaterialwithin thewoundsitesaswellastocharacterizethegranulationtissueingrowth,ﬁlling,and maturationovertime.Thepreparedstainedslidesweresenttoanindependentthirdparty, Inotiv(FortCollins,CO,USA),forhistopathologicalassessmentvialightmicroscopy.Scale barsonrepresentativeimageswereformattedusingImageJversion1.54f. Scoringofstainedslideswasperformedaccordingtothreeparameters:thedegreeof granulationtissueingrowthintothedHUCPdevicematerial,thedegreeofgranulation tissuethatﬁlledthewoundbedasthewoundhealed,andthematurationstageofthe granulationtissueinboththesuperﬁcialandthedeepwoundbed.Tissueingrowth intothedHUCPdevicematerialwasscoredasfollows:(0)absent;(1)minimal;(2)mild; (3)moderate;(4)marked,asinthedegreeofchangeisascompleteaspossible(i.e.,devices notintendedtobefullyresorbable);or(N/A)materialabsent/resorbed.Granulation tissueﬁllingofthewoundbedwasscoredasfollows:(0)nogranulationtissueﬁlling wound;(1)~1–25%ofwoundbedﬁlled;(2)~26–50%ofwoundbedﬁlled;(3)~51–75% ofwoundbedﬁlled;(4)~76–100%ofwoundbedﬁlled;or(5)>100%ofwoundbed ﬁlled.Granulationtissuematurationwasscoredasfollows:(0)nocollagendeposition; (1)scantycollagendepositionasloose,poorlyorganizedstroma;(2)morenotablecollagen depositionthanScore1,majorityofstromaislooseandpoorlyorganizedwithcollagen ﬁberspredominantlyorientedparallelandperpendiculartotheskinsurface;(3)more notablecollagendepositionthanScore2,majorityofstromaisdenseandorganizedwith collagenﬁbersorientedparalleltotheskinsurface;or(4)morenotablecollagendeposition thanScore3,majorityofstromahastheappearanceofnativedermalcollagen.Bioengineering2024,11,588 6of16 2.10.StatisticalAnalysis StatisticalanalysiswasperformedusingGraphPadPrism10.1.0software(SanDiego, CA,USA).Speciﬁcstatisticaltestsareindicatedintheﬁgurelegends.A p <0.05was consideredstatisticallysigniﬁcant. 3.Results 3.1.HistologicalComparisonofthedHUCPDeviceandItsSourceBiomaterial Nativeumbilicalcordconnectivetissueextracellularmatrix(ECM)isknowntocon- sistofanarrayofcollagens,glycoproteins,andglycosaminoglycans,includingHA[12]. Histologicalandimmunohistochemicalstainingprocedureswereperformedonnative, freshumbilicalcord(f-UC)aswellastheumbilicalcord-deriveddevice,dHUCP,tocom- parerelativecompositionalandstructuralattributes.Bothf-UCandthedHUCPdevice samplescontainedabundantcomplexnetworksofCollagenI(Figure1A)andCollagenIII (Figure1B).Similarly,ﬁbronectinandlamininimmunohistochemicalexaminationofthe dHUCPdevice(Figure1C,D)showeddistributionsreﬂectiveofthestructuralorganization observedinnativef-UC.AlcianBluestain,performedwithpH2.5solution,wasused tocollectivelyidentifythepresenceofglycosaminoglycans(GAGs)andglycoproteins. Bothf-UCandthedHUCPdevicedisplayedpositivestainingviathismethod(Figure1E). Further,HA,evaluatedviaHABPimmunohistochemicalvisualization,wasubiquitousin f-UCandretainedinthedHUCPdevice(Figure1F). Figure1.ThedHUCPdevicepreservesinherentstructuralextracellularmatrixcomponentsand tissueorganizationmirroringnativehumanumbilicalcord.Upperrowshowsnative,freshumbilical cord(f-UC);lowerrowshowsthedHUCPdeviceforallpanels.Sectionsofeachtissue/material werestainedforthefollowingconnectivetissuematrixcomponents:(A)anti-CollagenI;(B)anti- CollagenIII;(C)anti-laminin;(D)anti-ﬁbronectin;(E)proteoglycansandglycosaminoglycansvia AlcianBlue;and(F)anti-HABP(HyaluronicAcidBindingProtein).Representativeimagesareshown. Scalebar=100microns. 3.2.ExtracellularMatrixProﬁleofthedHUCPDevice TofurtherunderstandthecompositionofthedHUCPdevice,ECMcomponentswere quantiﬁedrelativetothedryweightofthedevice(Figure2A).Thetotalcollagencontentof thedHUCPdevicewasexaminedandfoundtobe460.9 ± 57.5mg/g,orapproximately 46%ofthedeviceweightasmeasuredbytheTotalCollagenkit(Perchlorate-Free)(Abcam). Thepredominantnon-sulfatedGAG,hyaluronicacid,accountedfor14.87 ± 2.82mg/gvia ELISA(R&DSystems).Sulfatedglycosaminoglycans(GAGs)comprised5.40 ± 0.66mg/g ofthedHUCPdevicepertheBlyscanSulfatedGlycosaminoglycanAssaykit(BioColorLtd, Carrickfergus,CoAntrim,UK).Consideringtheexceptionallyhydrophilicnatureofthese matrixcomponents,themoistureabsorbancecapacityofthedHUCPdevicewasevaluated. Inresponsetosalinehydration,thedHUCPdevicemasssigniﬁcantlyincreasedmorethan 6-foldrelativetoitsdryweight(Figure2B).Bioengineering2024,11,588 7of16 Figure2.ThedHUCPdevicecontainshydrophilicmatrixcomponentsandreadilyabsorbsmoisture. ThedHUCPdevicewasassessedforquantiﬁedabundanceofextracellularmatrixproteinspreviously identiﬁedviahistologystudies.(A)Totalcollagen,totalhyaluronicacid(HA),andtotalsulfated glycosaminoglycan(s-GAG)contentsweredeterminedandnormalizedpermgofdHUCPdryweight. n=7pertest.(B)TheabilityofthedHUCPdevicetoreadilyabsorbmoisturewasexaminedby comparingthedevicemassfromdrytoasaline-hydratedstate.Pairedttest;****p<0.001.Data presentedasmean ± SEM,n=15. 3.3.UltrastructureAssessmentofthedHUCPDevice Incorroboratingthehistologicalstructuralandorganizationalcharacterization,Scan- ningElectronMicroscopy(SEM)imagingofthedHUCPdevicerevealedacomplex,porous connectivetissuematrix(Figure3).ThedHUCPdevicewasobservedtoconsistofaspongy- texturedarchitecturethroughout(Figure3A).Uponcloserexamination,itisevidentthat thisporouslatticeworkconﬁgurationpermeatesthroughoutthescaffold,indicativeof anintricate,multi-scaledstructure(Figure3B).Theoverallarchitectureintheseimages isconsistentwithpreviouslypublishedscanningelectronmicrographsofnativehuman umbilicalcord[20]. Figure3.TheultrastructureofthedHUCPdevicerevealsaporousconnectivetissuematrix.SEM imagesacquiredofarepresentativedHUCPparticleunder45 × magniﬁcation(A)and200 × magniﬁ- cation(B)presentedacomplex,openscaffoldstructureintheﬁnished,shelf-stabledevice.Scalebars asindicatedineachmicrograph.Scalebar=200micronsforbothpanels. 3.4.InVitroCellularPerformanceofthedHUCPDevice Animportantattributeofscaffoldsinwoundmanagementistosupportthemicroen- vironmentofdamagedanddeﬁcienttissuesbyprovidingastructureconduciveforhost cellularingrowthandproliferation.Dermalﬁbroblastcellswereseededdirectlyonto thedHUCPdeviceinvitro,andafter6h,thecellularattachmenttothedevice’sscaffoldBioengineering2024,11,588 8of16 architecturewasassessed.AsshowninFigures4AandA1,theseededcellsreadilyadhered tothedHUCPdevice,andtheﬂuorescencestainingwasconﬁrmedtobespeciﬁctothelive seededcells.Theﬁbroblastmorphologywasconsistentwithadhesiontoascaffold,dis- playingaﬂattenedmorphologywithspindleprojectionselongatingoutacrossthedHUCP device.Separately,theabilityofthedHUCPdevicetoaffectcellularproliferationwas assessedbymeasuringtheinductionofcellgrowthinresponsetoextractsofthedHUCP devicecomparedtountreatedmedia-matchedcontrols.Inaddition,abovineﬁbrillar collagenpowderwasassessedforproliferativeeffects,asarepresentativecollagen-based CAMPcomparator.After3days,untreatedcontrolﬁbroblastsdisplayedminimaltono proliferation(102.6 ± 13.0%)comparedtothecellquantityobservedattreatmentinitiation (Day0)(indicatedbythedashedlineatthe100%markonFigure4B).Asigniﬁcantprolifer- ativeeffectwasobservedinthedHUCPdeviceextract-treatedﬁbroblastsof172.1 ± 33.3% growthcomparedtotheuntreatedcontrols(102.6 ± 13.0%;p<0.05).Ontheotherhand, ﬁbrillarcollagenpowderextractsdidnotelicitanyproliferativephenotype(p>0.05vs. untreatedcontrol)(Figure4B). Figure4. ThedHUCPdevicesupportsscaffoldadherenceandinducesproliferationofdermal ﬁbroblasts.DermalﬁbroblastcellswereseededontothedHUCPdeviceandallowedtoadherefor6h. (A)LivecellswerestainedwithCalceinAMandvisualizedunderGFPﬂuorescencewithanoverlay imageofthedHUCPdevicetopographyvisualizedviatransillumination.Scalebar=200microns. (B)Dermalﬁbroblastsweretreatedwithdeviceextracts(2mg/mLof2%serummedia)preparedfrom eitherthedHUCPdeviceorbovinetypeIﬁbrillarcollagenpowder,andtherelativeproliferativeindex wascalculatedasthegrowthafter3daysrelativetobaselineDay0values(indicatedbythedashed line).ThedHUCPdeviceelicitedaparacrineproliferativeresponsecomparedtothe2%serumcontrol, whereasﬁbrillarcollagenwasnotstatisticallydifferentfromcontrolconditions.Datapresentedas mean ± SEM,n=4–6/group,witheachnassessedintriplicate.One-wayANOVAwithDunnett’s correctionformultiplecomparisons;*p<0.05,****p<0.0001,ns=notstatisticallysigniﬁcant. 3.5.BiocompatibilityofthedHUCPDeviceInVivo Consideringthefavorableinvitroﬁndingsofcellularattachmentandparacrineprolif- erationinductionindermalﬁbroblasts,wesubsequentlyinvestigatedtheinvivobiocom- patibilityofthedHUCPdeviceinaporcinefull-thicknessskinwoundingmodel.Residual dHUCPdevicematerialwasmicroscopicallydetectedinallstudywoundbedssampledat Day7andhalfofwoundssampledatDay14andwasabsentfromallwoundssampled onDay35,indicativeoffullresorption(Table1,representativeimagesinFigure5).The residualmaterialpresentedasdarkblueaggregatesontheMasson’sTrichromestainand wastypicallylocatedwithinthesuperﬁcialwoundbedbutwasalsooccasionallynoted inthemid-woundbedineightoftensamplesatDay7andrarelyobservedinthedeep woundbed(oneoftensamplesatDay14)(Table1;Figure5A,B).ByDay35,noresidual dHUCPdevicewasdetectableinanyofthetensampledwounds.TissueingrowthintoBioengineering2024,11,588 9of16 thedHUCPdevicematerial,asevaluatedviahistopathologicmicroscopicanalysis,was observedasminimaltomildatDay7(averagescore1.7 ± 0.5)andmoderateatDay14 (3.0 ± 0.0)inthisporcinemodel.Concordantly,sincenoresidualdHUCPdevicematerial wasmicroscopicallydetectedinanyoftheDay35samples,tissueingrowthwasunableto bescored(‘N/A’)(Table1;Figure5C). Table1.dHUCPdevicematerialtissueingrowthscores,devicepresence,anddevicedistribution withinwoundbedsovertimeinaporcinefull-thicknesswoundmodel. Parameter Day7Day14Day35 n Score Mean ± SD n Score Mean ± SD n Score Mean ± SD 01234N/A01234N/A01234N/A Tissueingrowth intodHUCPdevice (score) 10 037000 1.7 ± 0.5 10 000505 3.0 ± 0.0 10 0000010N/A ResidualdHUCP devicematerial presence P(10of10wounds)P(5of10wounds)A ResidualdHUCP devicematerial location S(10of10wounds) M(8of10wounds) D(0of10wounds) S(5of10wounds) M(0of10wounds) D(1of10wounds) N/A P=present;A=absent;S=superﬁcialwoundbed;M=mid-woundbed;D=deepwoundbed;SD=standard deviation;N/A=notapplicable;n=10woundspertimepointdistributedacross3pigs. Figure5.ThedHUCPdeviceisbiocompatibleandcompletelyresorbableinvivo.Full-thickness dermalwoundswerecreatedonpigs,andthedHUCPdevicewasappliedonDay0ofwounding. SamplewoundswereexcisedatDays7,14,and35atstudyconclusion.Masson’sTrichrome- stainedsectionswerescoredformicroscopicresidualdHUCPdevicematerialpresenceandtissue ingrowthintodHUCPdevice,aswellasgranulationtissueﬁllingandmaturation.Representative photomicrographsarepresented.(A)ByDay7,thedHUCPdevicewasnotedwithinthesuperﬁcial woundbed(openarrowheads),andathicklayerofresidualdHUCPdevicematerialwasalsolocated withintheserocellulardebrislayer(closedarrowheads).(B)ByDay14,onlyhalfoftheexcisedsample woundscontaineddetectableresidualdHUCPdevicematerial,andforthesamplesthatdidshow residualdHUCPdevice,itwasnotedonlynearthewoundsurfaceinthesuperﬁcialwoundbed(open arrowheads)aswellastheserocellulardebrislayer(closedarrowheads).(C)ByDay35,nodHUCP devicematerialinanyofthesampledwoundswasmicroscopicallyobserved.S=serocellulardebris liningthewoundsurface;G=granulationtissue;arrow=woundmargin;greendiamond=oneedge ofre-epithelialization.Scalebarsasindicatedineachmicrograph. GranulationtissueﬁlledmorethanhalfofeachsampledwoundbedbyDay7(with ascoreof3.3 ± 0.5)andappearedfullyﬁlledbyDay14(withascoreof4.3 ± 0.5),as evidencedbywoundssampledatthattimepoint(Table2).Asthewoundshealed,the tissuematurationprogressionwasobserved.Thedeepwoundbedexhibitedahigher tissuematurationscorerelativetothesuperﬁcialwoundbed(2.0 ± 0.0vs.1.0 ± 0.0atNext >