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RESEARCHARTICLE Intermediatelayercontributioninplacentalmembrane allografts AnneliseRoy|SarahGriffiths ResearchandDevelopmentDepartment, StimLabs,LLC,Roswell,GA,USA Correspondence SarahGriffiths,StimLabs,LLC,1225 NorthmeadowParkway,Suite104,Roswell, GA30076,USA. Email:sarah@stimlabs.com Fundinginformation StimLabs,LLC Abstract Placentalmembrane(PM)allograftsarecommonlyusedtotreatchronicwounds.Native PMiscomposedofanamnion,chorion,andintermediatelayer(IL)thatcontainmatrix structuresandregulatorycomponentsbeneficialinwoundhealing.Historically, commerciallyavailableallograftswerecomposedofonlyoneortwolayersofthe PM.TomaximizetheconservedmaterialinPMallografts,adehydratedcomplete humanplacentalmembrane(dCHPM)allograftprocessedusingtheClearify ™ process wasdeveloped.HistologicalandproteomiccharacterizationcomparingdCHPM allograftswithnativePMdemonstratedthatthemajorityofmatrixstructuresand regulatoryproteinsareretainedindCHPMallograftsthroughprocessing.Toevaluate theimportanceofmaintainingtheentireintactPMandthecontributionoftheIL,the structuralandproteomicmakeupoftheILwascomparedwiththatofdCHPM allografts.Thisisthefirstknowncharacterizationofregulatoryproteinsinthe IL.ResultsdemonstratethattheILcontainsover900regulatoryandsignaling components,includingchemokines,growthfactors,interleukins,andproteaseinhibitors. Thesecomponentsarekeyregulatorsofangiogenesis,neurogenesis,osteogenesis, inflammation,tissueremodeling,andhostdefense.Theresultsshowthattheproteomic compositionoftheILisconsistentwiththatoftheentiredCHPMallograft.Although furtherinvestigationisrequiredtofullyunderstandthecontributionoftheILinPM allografts,theseresultsdemonstratethattheILcontainsstructuralandregulatory proteinsthatcanenhancethebarrierandwoundhealingpropertiesofPMallografts. KEYWORDS allograft,amnion,chorion,extracellularmatrix,placentalmembrane,woundhealing 1|INTRODUCTION TherecentincreaseduseofPMallograftsinwoundhealing applicationsstemsfromtheirprovenefficacy,uniquebioscaffold, andtherapeuticcomponents,aswellastheimmunologicalprivilegeof fetaltissue(Castellanos,Bernabé-García,Moraleda,&Nicolás,2017; Parolini,Solomon,Evangelista,&Soncini,2010).Studiesperformed oncompositeorsinglelayerallograftsconfirmthepresenceof growthfactors,cytokines,andtissueinhibitorsofmetalloproteinases (TIMPs;Koob,Lim,Massee,Zabek,&Denozière,2014;Mrugala etal.,2016).Additionally,compositeandsinglelayerPMallografts havedemonstratedsuccessinwoundhealingapplications.Since themid-1990s,singlelayerplacentalmembraneshavebeenpopular inophthalmologyasatreatmentincornealsurgeries(J.C.Kim& Tseng,1995;Solomonetal.,2002).Acrossarangeofapplications, thesegraftshavebeenshowntoacceleratewoundclosureand successfullyhealchronicwounds(Castellanosetal.,2017;Mrugala etal.,2016). Anatomically,thehumanPMsurroundsthefetusandseparatesit frommaternaltissue.ThePMisametabolicallyactivetissuethat continuallydevelops,expands,andremodelsduringgestation.The membraneprovideselasticityandstrengthtohold,cushion,and Received:18November2019Revised:8May2020Accepted:2June2020 DOI:10.1002/term.3086 JTissueEngRegenMed.2020;1 – 10.wileyonlinelibrary.com/journal/term©2020JohnWiley&Sons,Ltd.1protectadevelopingfetusandactsasacriticalbarrier,preventinga mixingofmaternalandfetalbloodandthetransferofpathogens, whileallowingkeynutrientstopasstothefetus(Gude,Roberts, Kalionis,&King,2004;Watson&Burton,1998).Malfunctionsofthe PMareassociatedwithahostofcomplications,suchaspremature ruptureoffetalmembranes,underliningtheimportanceofPMs (Janzenetal.,2017;Kumaretal.,2016;Watson&Burton,1998).As donoreligibilityfortransplantablematerialwouldexcludethese donorsforacceptanceinmanufacturing,thepotentialimpactson efficacyinwoundrepairarenotknown. NativePMhaseightidentifiablelayersthateachcontributeto theoverallstrength,elasticity,andbarrierpropertiesofthetissue,as summarizedinTable1(Bryant-Greenwood,1998).Commonly,these aredescribedastwomajorlayers:theamnionandthechorion.On thefetalsideofthePM,theamnioniscomposedofanepithelium, basementmembrane,compactlayer,andfibroblastlayer.Theamnion istheinnermostmembranetothefetus,providingtensilestrength andactingasafibrousskeleton(Niknejadetal.,2008).Towardthe maternalsideisthechorion,whichincludesthereticularlayer,a pseudobasementmembrane,andtrophoblastlayerthatcontactsthe maternaldecidua(Niknejadetal.,2008).Thechorioncontributes toelasticityandstabilityandprovidesascaffoldfornativecells (Bryant-Greenwood,1998;Hieberetal.,1997;Mamedeetal.,2012; Niknejadetal.,2008).Theamnionandchorionattachandinteract throughathird,dynamicmiddlelayercalledtheintermediatelayer (IL),alsoreferredtoasthespongylayerorzonaspongiosa. Eachlayerhasacharacteristicextracellularmatrix(ECM)makeup thatisinstrumentaltothestructuralintegrityandbarrierpropertiesof thePM.Theepitheliallayerismadeupofathinlayeroftightly packedepithelialcellsthatsitdirectlyonabasementmembrane. Theamnioticbasementmembraneiscomposedofdenselypacked collagens,laminin,nidogen,fibronectin,andproteoglycanslike heparansulfate(Keene,Sakai,Lunstrum,Morris,&Burgeson,1987; Malaketal.,1993;Mamedeetal.,2012;Niknejadetal.,2008; TABLE1 Extracellularmatrixcomponentsandfunctionsofthenativeplacentalmembranelayers LayerECMcomponentsFunctionSources AmnionEpithelium • Epithelialcells • Containsgrowthfactorsthatpromote epithelialization Parolinietal.(2010);Mamedeetal.(2012); Malaketal.(1993);Keeneetal.(1987); Rousselleetal.(1997);Smithetal. (1994);Niknejadetal.(2008) Basement membrane • Collagens • Laminin • Nidogen • Fibronectin • Heparansulfate • Actsaspermeablebarrier,allowing transportofnutrientsandbuilding blocks • Hemostaticpropertiespreventhematoma andreducemicrobialaccumulation • Increasesbarrierintegrity,stabilizing membraneandcells • Providesscaffoldingforother extracellularcomponents Compactlayer • CollagentypesI,III, V,andVI • Fibronectin • Mainfibrousskeletonofnativescaffold • Increasestensilestrength Fibroblast layer • CollagentypesI,III, andIV • Proteoglycans • Anchorscellstoscaffolding • Increasestensilestrength Intermediate layer Intermediate layer • CollagentypesI,III, andIV • Heparansulfate • Hyaluronicacid • Createsanacellularbarrierbetween amnionandchorion • Nonfibrillarmeshworkstructurethat providesscaffoldandcushioning • Tissuehydrationandlubrication • Mechanicalsupporttomembranes • Provideselasticityandtractional resistancetomembrane Meinertetal.(2001);Mamedeetal. (2012);Bryant-Greenwood(1998) ChorionReticularlayer • CollagentypesI – VI • Elastin • Proteoglycans • Contributestomembraneintegrity • Providesascaffoldforotherlayersof membraneandcellgrowth • Contributestoelasticityofthemembrane Malaketal.(1993);Hieberetal.(1997); Malaketal.(1993);Niknejadet al.(2008);Keeneetal.(1987);Rousselle etal.(1997);Smith&Ockleford(1994); Bryant-Greenwood(1998) Basement membrane • CollagentypeIV • Fibronectin • Laminin • Increasesintegrityofmembranebarrier • Providesscaffoldingforother extracellularcomponents • Stabilizesmembranesandhelpsstabilize cells Trophoblasts • Trophoblasts • Collagen • Chemicalbarriertomaternalhormones andlocalmaternalsignals Abbreviation:ECM,extracellularmatrix. 2ROY AND GRIFFITHSRousselleetal.,1997;Smith&Ockleford,1994).Thecompactlayer andfibroblastlayerarealsomadeupofcollagentypeI,typeIII, typeV,andtypeVI,alongwithlaminins,nidogens,andfibronectins, creatingthefibrousstructureoftheamnion(Keeneetal.,1987; Mamedeetal.,2012;Niknejadetal.,2008;Rousselleetal.,1997; Smith&Ockleford,1994). Thechorionlayersprovidestructuralsupportandanchoringfor cellsacrossthePM(Bryant-Greenwood,1998;Malaketal.,1993). ThereticularlayeriscomposedofcollagentypesI – VIandanelastin networkcontributingaddedelasticity(Hieberetal.,1997;Malak etal.,1993;Niknejadetal.,2008).Thetrophoblastsandcollagen inthetrophoblastlayerfacilitatesattachmentofthePMstothe maternaldecidua(Bryant-Greenwood,1998). TheILactsasaninterfacebetweenthematernal-facingand fetal-facingsideofthePMandvariesinthickness(Baergen,2011).It iscomposedofcollagentypeI,typeIII,andtypeIVandahighdensity ofproteoglycansandglycoproteins,includinghyaluronicacidand heparansulfate(Bryant-Greenwood,1998;Mamedeetal.,2012; Meinertetal.,2001;Niknejadetal.,2008).TheseECMproteins existinaloosematrixthatformsaspongy,acellularnetwork (Niknejadetal.,2008).Itactsasabarrierbetweentheamnionand chorionlayersandallowstheamnionlayertoglidealongthe chorion(Bryant-Greenwood,1998).Althoughthemechanical characteristicsareimperativetoPMintegrity,littleelsehasbeen studiedabouttheIL. CompositePMallograftshavebeenshowntobeefficaciousin woundhealing,especiallyinchronicwounds,cornealwounds,and burns(Castellanosetal.,2017;Mermetetal.,2007;Solomon etal.,2002).Typically,followingdebridement,PMallograftsare appliedtothewoundsurfacetostimulatehealthywoundclosure, followedbyapplyingasecondarydressing(Ganatra,2003).The allograftsarereapplieduntilresolutionofthewound,similarto methodsdescribedbyGanatra(2003)andMermetetal.(2007). AnimalandclinicalresearchhasshownthatPMisanti-inflammatory, antimicrobial,analgesic,pro-angiogenic,immunologicallyprivileged, andhaspropertiesthatreducescarringandenhancecellular proliferationandmigration,manyofwhichcanbeattributedtothe reciprocityoftheECMstructureandtheregulatoryproteinspresent inPM(Castellanosetal.,2017;Malhotra&Jain,2014;Mermet etal.,2007;Schultz&Wysocki,2009).Sincethecommercialsuccess oftheseallografts,alargebodyofresearchhasfocusedonidentifying thespecificcytokinesintheamnionorchorionlayersofPMsthat helptoregulatetissuerepair(Castellanosetal.,2017;Fetterolf& Snyder,2012;Tsengetal.,2004).Componentsthatregulate fibroblastmigrationandproliferation,includingbasicfibroblast growthfactor(bFGF),transforminggrowthfactor-beta(TGF- β 1),and platelet-derivedgrowthfactors(PDGF-BBandPDGF-AA),andthose thatregulatere-epithelialization,likeepithelialgrowthfactor(EGF), havebeenidentifiedinPM(Barrientos,Stojadinovic,Golinko,Brem,& Tomic-Canic,2008;Koob,Lim,Zabek,&Massee,2015;Werner& Grose,2003).TIMPsareregulatorsofECMturnoverthatarecritical tomediatingfibrosisandhealthyECMdepositionandarefoundin PMs(Arpino,Brock,&Gill,2015;Visse&Nagase,2003).Stimulators ofangiogenesis,includingvascularendothelialgrowthfactor(VEGF), havealsobeenstudiedinPM(Werner&Grose,2003).Focus hasbeengiventoidentifyingcomponentsthatreduceinflammation, likespecificinterleukinsandhyaluronicacid(Necas,Bartosikova, Brauner,&Kolar,2008;Werner&Grose,2003).Hyaluronicacidisa componentofinterestinPMbecauseofitsroleinanti-inflammatory, analgesic,andantifibroticsignalingpathways(Gupta,Lall,Srivastava,& Sinha,2019;Mohan,Bajaj,&Gundappa,2017;Mohseni,Saem, Sekhavati,Molaszem,&Tabrizi,2018;Necasetal.,2008).These biochemicalpropertiesarecomplementedbytheinherentphysical, mechanical,andvaporbarrierprovidedbythelayersofPMallografts (Ganatra,2003). ThemanufacturingofPMallograftsnecessitateswashingthe tissuetoremoveresidualmaternalblood.Popularprocessing techniquesprescribetheseparationoftheamnionandchorionto facilitatecompletewashingofthemembranesbeforerelamination (Daniel,Tofe,Spencer,&Russo,2012;Koobetal.,2015).These techniquesexposethecenterofthePMtoagitationandmanipulation resultinginthelossofsubstantialECMcontent,includingthe majorityofthehydrophilicIL.Commonly,thetissueistheneither cryopreserved,dehydrated,orlyophilized(freeze-dried)topreserve thetissue.Commerciallyavailableshelf-stablePMallograftsaresold asamnionandamnion/chorioncompositegrafts(Koobetal.,2015). TheseprocessessubstantiallyremovetheIL,removingkeynutrients andstructuresfromtheallograft.Furthermore,theseparationand ovendehydrationdisruptsthenaturalarchitectureofthegrafts (Johnson,Gyurdieva,Dhall,Danilkovitch,&Duan-Arnold,2017). Aproprietaryprocessingtechnique(Clearifyprocess)was developedbyStimLabs,LLC(Roswell,GA),toproduceadehydrated completehumanplacentalmembrane(dCHPM)allograft(Revita ™ ), designedtoconservetheintactarchitectureandoptimizethe retentionofkeycomponentsthroughprocessing.ThedCHPMretains thefullyintactstructureoftheamnion,IL,andchorionstructures throughprocessing. ThepurposeofthisstudywastocomparedCHPMallograftswith nativePMtodemonstratethatthecompletenativebarrierstructures andcytokinecompositionareconservedthroughprocessing. Additionally,dataarepresentedontheregulatorycomponentcontent oftheILanditspotentialcontributiontotheoverallbarrierand woundhealingpropertiesofdCHPMallografts. 2|MATERIALSANDMETHODS 2.1|Tissueprocessing dCHPMallograftswereprocessedfromdonatedhumanplacentas (FigureS1).Placentaswererecoveredfromfull-term,healthybirths underfullconsentofthemothers.Thedonationprocessfollowedthe regulationsoftheFoodandDrugAdministration(FDA)andthe AmericanAssociationofTissueBanks(AATB).Patientscreeningwas performedtotestforhumanimmunodeficiencyvirustype1andtype 2antibody,humanT-lymphotropicvirustype1andtype2antibody, ROY AND GRIFFITHS3hepatitisCantibody,hepatitisBsurfaceantigen,hepatitisBcore totalantibody,rapidplasmaregain(RPR)forsyphilisorserologic testforsyphilis(SRS),humanimmunodeficiencyvirustype1nucleic acid,hepatitisCvirusnucleicacid,hepatitisBvirusnucleicacid, cytomegalovirusantibody,andWestNilevirusnucleicacid. IntactPMswereremovedfromtheplacentaldiskbyblunt dissection.Samplesoffresh,unprocessedtissuewerecutanddigested foranalysis.Membraneswerethenfurtherprocessedthroughthe Clearifyprocess.Afterwashing,smallsectionsofmembranewerecut tocollectILsamples.Usinggentlemassage,thegelatinousILwas pushedoutfrombetweentheamnionandchorionlayers.Processed membranesandisolatedILwerethenlyophilizedbeforeanalysis. 2.2|Histology Histologicalanalysisoffresh,unprocessedPMandthedCHPM sampleswasperformedbyPremierLaboratory,LLC(Longmont,CO), accordingtotheirstandardprocedures.Stainswereselectedto highlightthemorphologyandprinciplestructuralproteinsinthePM: cellnuclei(hematoxylinandeosin[H&E]),collagen(Masson's trichromeandVerhoeff'sstainwithvanGiesoncounterstain),elastic fibers(Verhoeff'sstainwithvanGiesoncounterstain;EVG),and glycosaminoglycans(GAGs)andproteoglycans(Alcianblue). 2.3 | PMlayerthicknessanalysis Scannedhistologicalimagesoffresh,unprocessedPMsamples(n=3 donors)wereanalyzedinImageScopev12.2.2.5015.Multiple(n>10) measurementsoftheamnion,IL,andchorionthicknessweretakenat consistentintervalsacrosseachsample.Thethicknessmeasurements wereaveragedtoobtainameanlayerthicknessforeachlayerin eachgivensample.Themeanlayerthicknessisreportedasamean acrossdonors. 2.4|Samplepreparationforproteomicanalysis Tissuesampleswereweighed,andsurfaceareameasurementswere taken,whereapplicable.Sampleswerethenmincedandplacedincell lysisbufferwith1%v/vproteaseinhibitor.Tissuesweredigested overnightat4 Candthenhomogenized.Thehomogenatewas centrifugedandthesupernatantcollectedforanalysis. 3|ENZYME-LINKEDIMMUNOSORBENT ASSAY Specificcomponentsrelevanttowoundhealingwereidentified andconcentrationsofthesecomponentsweremeasuredusing enzyme-linkedimmunosorbentassay(ELISA)ineachsampletype. PDGF-BB,PDGF-AA,TIMP-1,TIMP-2,TIMP-4,TGF- β 1,TGF- α , bFGF,andEGFELISAkitswereobtainedfromRayBiotech,Inc (Norcross,GA).Hyaluronan(Hyaluronicacid[HA])andVEGFELISA kitswereobtainedfromR&DSystems(Bio-TechneCorporation,Min- neapolis,MN).LactoferrinELISAkitswereobtainedfromAssayPro, LLC(St.Charles,MO).Assayswereperformedaccordingtoeachkit manufacturer'sinstructions.ComponentconcentrationsintheIL arereported.Tocomparecomponentconcentrations,dCHPM componentconcentrationswerenormalizedtothecomponent concentrationsoffresh,unprocessedPM. 3.1|KiloplexELISAarray Concentrationsof1,000cytokines,growthfactors,andregulatory proteinsindCHPMandILsamplesweremeasuredwithkiloplex ELISAarray(Raybiotech,Inc).TestingwasperformedbyRayBiotech, Inc(Norcross,GA).Concentrationswerenormalizedtodryweight forcomparison. 3.2|Statisticalanalyses AllstatisticalcomparisonswereperformedusingunpairedStudent ttestswitha95%confidenceinterval.Concentrationsofcomponents werecomparedbetweendCHPMandfresh,nativePMtissue. pvalues<0.05wereconsideredtobestatisticallysignificant. 4|RESULTS 4.1|HistologyofdCHPMandnativePM HistologicalanalysisofdCHPMandfreshPM(Figure1)showsthat dCHPMretainstheeightindividuallayersandmembranestructureof freshPMthroughtheClearifyprocess.Thisisevidentinthecellnuclei oftheepitheliallayerandtrophoblastlayerclearlyindarkpurple,and thediscernable,densepinkofthebasementmembranes,compact layer,andreticularlayerthatarevisibleinbothtissuetypes. Importantly,thelightlystained,wavy,andsubstantiallyacellularILis identifiableinbothsamples.EVGstainingofdCHPMshowsthe definedcollagencontentofthefibroblastandILandbasement membranesinred,aswellastheelastincontentofthereticularlayer inblack(Figure2a).ThecollagencontentofdCHPM(stainedblue, Figure2b)appearstobemostheavilystainedintheamnionand IL.ThedenseAlcianbluestainingofdCHPMshowstherichGAG contentoftheIL,thefibroblastlayer,andthereticularlayer(Figure2c). 4.2 | NativePMlayerthickness Thethicknessesweremeasuredforeachofthethreemajor membranelayers(amnion,IL,andchorion)infreshPMsamples.The meanamnionlayerthicknesswas69.43 μ m±22.34 μ m(n=3donors, 4ROY AND GRIFFITHSp=0.05).ThemeanILthicknesswas257.97 μ m±83.45 μ m(n=3 donors),whichis3.72timesthickerthanthemeanamnionlayer thickness.Themeanchorionthickness(291.04 μ m±62.79 μ m,n=3 donors)was1.1-foldthickerthantheIL(p=0.61)andwas significantlythickerthanthemeanamnionthickness(p=0.02).The meanILthicknessaccountsforover40%ofthemeanoverall thicknessofthenativePM. 4.3|ProteomicanalysisofdCHPM,nativePM, andIL Theconcentrationsofapanelofcomponentsthatareknowntobe relevanttowoundhealingweremeasuredintheIL(Table2). ThepercentoffreshPMproteincontentretainedfollowing processinganddehydrationwascalculatedfordCHPMsamplesand FIGURE1 Histologicalsectionsofacomplete,freshplacentalmembrane(a)anddehydratedcompletehumanplacentalmembrane(dCHPM) (b)stainedwithhematoxylinandeosin(H&E).Theindividuallayersandthelayergroupingsmakingupthecompleteplacentalmembranearelisted [Colourfigurecanbeviewedatwileyonlinelibrary.com] FIGURE2 Histological sectionsofdehydratedcomplete humanplacentalmembrane (dCHPM)allograftsstainedwith (a)Verhoeff'sstainwithVan Giesoncounterstainshowing collageninredandelasticfibers inblack,(b)Masson'strichrome showingcollageninblue,and (c)Alcianblueshowing glycosaminoglycansinlightblue [Colourfigurecanbeviewedat wileyonlinelibrary.com] TABLE2 Meancomponentconcentrationsinintermediatelayer Component Intermediatelayer Mean(pg/mg)SD n PDGF-BB5.304.775 PDGF-AA255.47322.815 TIMP-11,051.26500.303 TIMP-2877.69413.653 TIMP-40.540.475 TGF- β 122.798.053 bFGF177.9155.194 EGF0.050.098 Hyaluronicacid5,283,723.821,830,219.426 Lactoferrin39,906.3822,609.993 VEGF0.390.363 ROY AND GRIFFITHS5representedinFigure3.Therewasnosignificantdifferencein concentrationsofPDGF-BB,PDGF-AA,TIMP-1,TIMP-2,TIMP-4, bFGF,EGF,HBD-1,lactoferrin,orTGF- α betweenfreshPMand dCHPM.TherewasasignificantlygreaterconcentrationofHA measuredindCHPMallografts(43.56 μ g/cm 2 ±14.89 μ g/cm 2 ,n=6) comparedwithfreshPM(25.00 μ g/cm 2 ±6.76 μ g/cm 2 ,n=3,p=0.04). Anarrayof1,000proteintargetswasperformedonILanddCHPM samplestocomparetheproteincategoriesdetectableinthetissues. Detectedproteinswerecategorizedintogroupsbythetypeofprotein andthefunctionalroletheyperform.Acomparisonofthenumberof proteinsineachcategorydetectedinILanddCHPMsamples,aswell asspecificsignalingfactorsmeasuredineachsampletype,isdetailedin Figure4.Functionalroleswereassignedbaseduponactivitiesrelevant totissuerepairandremodeling,asreportedbythehttp://www. uniport.orgdatabase.Proteinsthatwerenotassociatedwiththese functionalroleswerenotincludedinthisanalysis. 5|DISCUSSION HistologicalanalysisshowsthattheClearifyprocesseddCHPM preservesthenativearchitectureofthePM,includingtheIL (Figure1).Thelayersareneverseparatedduringprocessingandso retaintheirnaturalborders,connections,andsubstance.Further,the freeze-dryingmethodpreservesthenaturalstructureofthetissue, unlikeheatdehydratedalternativesthatcancausethetissuelayersto compact,whichhasbeenshowntoalterbiomatrixfunction(Johnson etal.,2017;Koobetal.,2014).ThedCHPMstillretainstheopen, spongytissueoftheIL.Thisopenarchitectureandtheintact processingtechniquemakedCHPMmoreanalogoustothenative FIGURE3 Meantargetconcentrationsindehydratedcomplete humanplacentalmembrane(dCHPM)allograftsnormalizedtotarget contentinfreshPM.Proteinanalysisperformedusingenzyme-linked immunosorbentassay.Horizontallineindicatesconcentrationsof freshplacentalmembrane(PM). * Resultsofattestwithsignificant differencesbetweenthedCHPMconcentrationsandthefreshPM concentrations(p<0.05) FIGURE4 Proteincategoriesandfunctionalgroupsofproteinsdetectedindehydratedcompletehumanplacentalmembrane(dCHPM)and intermediatelayer.(a)ComparisonofthenumberofproteinsdetectedineachcategorybetweendCHPMandintermediatelayer.(b)Tableof signalingfactorsdetectedindCHPMand/orintermediatelayergroupedbyfunctionalroles.Thecomponentsincludedinthisfigurearea selectionofrelevantcomponentstestedinthekiloplexassayandarenotacompletelistofdetectedcomponents 6ROY AND GRIFFITHStissuecomparedwithsinglelayerorcompositePMallografts.Native tissuematrixstructureisakeycomponentofadvancedwound healing,actingasascaffoldformorefacilecellinfiltration(Schultz, Davidson,Kirsner,Bornstein,&Herman,2011).Theconservedelastin contentofthereticularandtrophoblastlayersishighlightedinthe EVGstainingofdCHPM(Figure2a).Thiscompositionaddstothe integrityofthemembrane,givingelasticityandscaffoldingtosupport connectionstootherlayers(Hieberetal.,1997;Malaketal.,1993). PreservingtheseECMstructureswithindCHPMallograftsallows themtoactasnaturalphysicalbarrierscapableofprotectingboth surfaceandsurgicalwoundsduringthehealingprocess. ThemajorityofthePMECMisinterstitialcollagen,which mechanicallystrengthensthetissue,andmakesitmoreresistantto proteolyticenzymes(Bryant-Greenwood,1998;Mamedeetal.,2012). Thisisadvantageousinchronicwoundapplicationsthat,bydefinition, donotproperlyregulatematrixdegradingenzymes,suchasmatrix metalloproteinases(MMPs;Caley,Martins,&O'Toole,2015).Based onMasson'strichromestaininginthisstudy,alargequantityofcollagen wasfoundtobepresentintheIL.Inaddition,itwasdemonstrated thatthemostheavilystainedregionsofmucopolysaccharidesand glycoproteins,includingGAGsandproteoglycans,arefoundintheIL andreticularlayer.ThehydrophilicnatureoftheGAGcontentofthe IL,specificallythehyaluronicacid,givesitlubricating,elastic,and cushioningproperties,whichmakeitanalmostincompressiblefluidor jelly(Goa&Benfield,1994;Meinertetal.,2001).Ourresultssupport studiesthatdemonstratethattheILispredominantlycomposedof collagen,proteoglycans,andGAGsthatexistinaloosematrixthat formsaspongy,acellularnetwork(Bryant-Greenwood,1998;Meinert etal.,2001;Niknejadetal.,2008). AlthoughtheILiscrucialtothemechanicalandbarrierproperties ofthePM,itiscommonlygroupedtogetheraspartoftheamnion. Becauseofthis,thereislittleresearchavailablecharacterizingtheIL specifically,eventhoughthelayerisvisiblydistinctinhistological stainingandcanaccountforover40%ofthefullthicknessofnative PM.Thisdesignationoverlooksitsuniquecontributiontothebarrier, mechanical,andmetabolicpropertiesofthePM(Bryant-Greenwood, 1998;Niknejadetal.,2008).Ourdatademonstratethatthemajority ofthethicknessofnativePMcomesfromthechorionand IL.AlthoughthethicknessoftheILisvariablebetweendonors,these datademonstratedthatonaverage,itis3.72timesasthickasthe amnionlayer(Baergen,2011). OptimalPMallograftsintendedforuseinwoundhealingshould maximizetheconservationofplacentalECMproteins.Structural ECM,suchascollagenandelastin,providestensilestrength.GAGs andproteoglycans,beingextremelyhydrophilicproteins,promote tissuehydration,flowresistance,andmolecularexclusion.Adhesive glycoproteinssuchasfibronectinandlamininprovidestructural integrity,amatrixforcellgrowth,andfacilitateinteractionsbetween cells(Schultz&Wysocki,2009).Woundhealingdependsonthe interactionofECM,cytokines,andcells,inaprocessknownas dynamicreciprocity(Schultzetal.,2011).AlthoughECMhelps managecytokineactivity,cellularattachmentstoECMstructuresare generallyrequiredforacellularresponsetocytokinessignaling (Schultzetal.,2011).ThepresenceofenoughintactECMcomponents andscaffoldingiscrucialtothehealingprocessandimportantto maintaininPMallograftsintendedtoaidintissueregeneration.The resultsdescribedaboveprovideevidencethatdCHPMallografts conserveamajorityoftheECMofnativePM,includingcollagens, elastins,andproteoglycans.Theseareimportantfortissueremodeling andfurtherassistinthefunctionofcytokinesknowntoimprovethe progressionoftissueremodeling(Schultzetal.,2011). ThePMisalsoaselectiveimmunologicalbarrier(H.S.Kim etal.,2002).Extensivestudieshavebeendoneontheantimicrobial propertiesofPM,itsroleinmodulatinginflammation,andthe interplayofinnateandadaptiveimmunityinthetissue(Frew& Stock,2011).ThePMcontainsawealthofantimicrobialpeptides (AMPs),including α -and β -defensinsandlactoferrin,whichisan iron-bindingpeptidethatinhibitsbacterialgrowthandisknownto playakeyroleinimmunologicaldefenseduringgestation(Frew& Stock,2011;Niemelä,Kulomaa,Vija,Tuohimaa,&Saarikoski,1989; Underwood,Gilbert,&Sherman,2005).invitroassayshaveshown PMtobebactericidaltoahostofbacteria,includingStaphylococcus aureus,Escherichiacoli,andPseudomonasaeruginosa,whichisa majorconcerninthetreatmentofburns,andindicatethatPMs haveanindependentmechanismofantimicrobialdecontamination (Kjaergaardetal.,2001;Zare-Bidaki,Sadrinia,Erfani,Afkar,& Ghanbarzade,2017).Previously,nostudyhasdescribedthe antimicrobialproteincontentoftheIL.Kiloplexarraydataofferthe firstinsightintotheadditionalbacteriostaticbarrierpropertiesofthe IL.Whereas17hostdefenseproteinsweredetectedinbothdCHPM andILsamples,includingIL-28A,PGRP-S,IL-17C,granulysin,IFNb, CL-P1,andReg3A,sevenspecificfactorswereonlydetectedintheIL, specificallyincludingCA15-3,S100A8,andTrappin-2(Figure4).Our resultsalsoshowthatlactoferrinispresentintheIL.Thisfurther suggestsanimmunologicalbarrierroleoftheILandunderlinesthe therapeuticimportanceofconservingthislayerinPMallografts.Our dataindicatethattheILcanenhancethehostdefensecontentof PMallograftsandpotentiallytheirantimicrobialproperties. Alongwiththepropertiesmentionedabove,PMallograftshave beenshowntohaveanti-inflammatory,analgesic,andpro-angiogenic properties,toreducescarringandenhancecellularproliferationand migrationduetothesignalingproteinandmatrixcontent(Castellanos etal.,2017;Malhotra&Jain,2014;Mermetetal.,2007;Schultz& Wysocki,2009).Here,wecomparedfresh,unprocessedPMto processeddCHPMtoinvestigatetheconservationofcomponents relevanttoapplicationsinwoundhealing.TheClearifyprocessdid notsignificantlydecreasetheamountofPDGF-AAorPDGF-BB, bFGF,EGF,TGF- α ,TIMP-1,TIMP-2,TIMP-4,hyaluronicacid,or lactoferrinfoundinPMtissue.Thesegrowthfactorsareimportantfor re-epithelializationandmatrixremodeling,whereasTIMPsarecrucial totheregulationofECMproteolysisandremodeling(Arpino etal.,2015);specifically,TIMPscanprovidelocalizedcontrolofECM turnoverandremodeling(Rileyetal.,1999).Thesedatademonstrate thattheClearifyprocessisabletoconservethesecriticalcomponents atthephysiologicallyrelevantconcentrationsfoundinfreshPM.It shouldbenotedthatseveralproteintargetsweremeasuredathigher ROY AND GRIFFITHS7concentrationsindCHPMthaninfreshPMwhennormalizedto surfacearea,includingHA.Itispossiblethatthereexistsgreater donorvariabilityintheconcentrationofthesetargetproteins,which wouldaccountfortheseapparentincreases.Hyaluronicacidcontent isreportedassignificantlyhigherindCHPMthaninfreshPM; however,HAinnativePMmaynotbeasreadilydigestedduetothe gelatinousnatureoffreshPMsampleswhencomparedwiththe dehydratedHAcontentindCHPM.TheseresultsdoconfirmthatHA ishighlyconservedindCHPMallograftsrelativetounprocessedPM, asisalsosuggestedbythehistologicalstaining.HAisabeneficial ECMcomponentforwoundcareproductsasitcontributestocell proliferation,cellularmigration,tissuehydration,andlubrication,as wellassignalinganti-inflammatorypathways(Goa&Benfield,1994; Pienimakietal.,2001). Importantly,thisstudydemonstratesthatthesamecomponents foundindCHPMallograftsarealsofoundintheIL.Notably,thereare additionalproteinsrelevanttowoundhealingthatweredetectedin theILthatwerenotatdetectablelevelsindCHPMsamplesasa whole(Figure4).Acomparisonofproteincategoriesandfunctional groupsdetectedindCHPMandILalonefurtherhighlightsthe compositionalsimilaritybetweentheILandtheentiredCHPM. Proteomiccomparisonofthetwonotablyshowsthatagreater numberofproteinsineachcategorywereatdetectablelevelsinIL, includinggrowthfactors,interleukins,andcytokines.Althoughthe detectionlimitsofthekiloplexassayperformedresultedinthe detectionofmorecomponentsintheILsamples,whichmayhave beenmorediluteindCHPM,theresultshighlighttherichproteomic arraythatexistsintheIL.Thesesimilaritiesindetectablecomponents showthatthedevelopedprocessingmethodfordCHPMallografts conservesamajorityoftheuniqueproteinaceouscontentfoundin theIL.TheILcontributesadditionalconcentrationsofalmostall componentstestedinthePM.Of1,000testedproteins,834were detectedindCHPMand927weredetectedinIL.Thedetection overlapdemonstratesthatover98%ofthecomponentstestedinthe wholedCHPMallograftsarealsofoundinIL.Thesesimilaritieshold throughoutthefunctionalrolesofthedetectedproteins,includingthe numberofangiogenicfactorsdetected,aswellasproteinsinvolved intissueremodeling(Figure4b).Notably,anumberofadditional proteinsrelatingtoanti-inflammatoryandhostdefenseroleswere detectedinILthatwerenotdetectedindCHPMasawhole,including IL-4,IL-11,IL-13,CA15-3,S100A8,andTrappin-2.Thisextensive arrayshows,forthefirsttime,thecomplexproteomiccompositionof theILandthesignalingcontentthatitcontributestotheoverall nativePM.Theangiogenic,anti-inflammatory,hostdefense,and tissueremodelingcomponents,specifically,suggestarolefortheILin theuseofPMallograftsinwoundhealing.Thesefindingswarrant furtherstudyandclinicalevaluationstodeterminethepotential therapeuticbenefitoftheinclusionoftheILinPMallografts.Atthe timeofthisstudy,anongoingrandomizedclinicaltrialisevaluating theefficacyofdCHPMindiabeticfootulcerswhencomparedwith standardofcare(clinicaltrials.gov,NCT03708029). Previouslyreportedplacentalcharacterizationfocusesoncomposite orsinglelayerPMallograftsthatlackanILand/orachorion,asno previouslyavailableproductspreservedallthreemajorlayersofthe PM(Koobetal.,2014;Koobetal.,2015;McQuilling,Vines, Kimmerling,&Mowry,2017).Historically,ithasbeenassumedthat themajorityofcomponentsrelevanttowoundhealingarefoundin theamnionorchorionofPMallografts.Thisissupportedbythelack ofliteratureavailableontheproteomicmakeupoftheIL.However, althoughthesecomponentsdoexistintheamnionandchorion,these resultsshowthatthereisalsoadenseconcentrationofthesewound healingcomponentsintheIL,someofwhichmayaccountforthe majorityofthecomponentconcentrationinthecompletemembrane. InclusionoftheILintoPMallograftscanincreasetheconcentration ofproteinsshowntomodulatewoundhealing.Furthermore,the ILmaycontributeotherphysical,mechanical,andantimicrobial barriercharacteristicstoPMallograftsusedtotreatwounds.These resultsarethefirsttodescribetheprocessingofnativePMsto preservethecorematrixlayer.CharacterizationoftheILdistinguishes itasafunctionallydistinctandvitallayerofthePM,ratherthana subcomponentoftheamnionstroma. ACKNOWLEDGMENT ThisresearchwassupportedbyStimLabs,LLC. CONFLICTOFINTEREST Authorsdisclosethattheyareemployeesofandholdequityinterestin StimLabs,LLC.SGisaninventoronpatentapplicationsencompassing theintellectualpropertyoftheClearifyprocess. 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Howtocitethisarticle:RoyA,GriffithsS.Intermediatelayer contributioninplacentalmembraneallografts.JTissueEng RegenMed.2020;1 – 10.https://doi.org/10.1002/term.3086 10ROY AND GRIFFITHSNext >