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ReviewArticle PlacentalTissuesasBiomaterialsinRegenerativeMedicine AnneliseRoy,MorganMantay,CourtneyBrannan,andSarahGriffiths ResearchandDevelopment,StimLabsLLC,1225NorthmeadowParkway,Suite104,Roswell,GA30076,USA CorrespondenceshouldbeaddressedtoSarahGri ﬃ ths;sarah@stimlabs.com Received 27 August 2021; Accepted 19 March 2022; Published 21 April 2022 AcademicEditor:Fu-MingTsai Copyright©2022AnneliseRoyetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense, whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. Placentaltissuesencompassallthetissueswhichsupportfetaldevelopment,includingtheplacenta,placentalmembrane, umbilicalcord,andamniotic ﬂ uid.Sincethe1990stherehasbeenrenewedinterestintheuseofthesetissuesasarawmaterial forregenerativemedicineapplications.Placentaltissueshavebeenextensivelystudiedfortheirpotentialcontributiontotissue repairapplications.Studieshaveattributedtheire ﬃ cacyinaugmentingthehealingprocesstotheextracellularmatrixsca ﬀ olds richincollagens,glycosaminoglycans,andproteoglycans,aswellasthepresenceofcytokineswithinthetissuesthathavebeen showntostimulatere-epithelialization,promoteangiogenesis,andaidinthereductionofin ﬂ ammationandscarring.The compositionsandpropertiesofallbirthtissuesgivethemthepotentialtobevaluablebiomaterialsforthedevelopmentofnew regenerativetherapies.Herein,thedevelopmentandcompositionsofeachofthesetissuesarereviewed,withfocusonthe structuralandsignalingcomponentsthatarerelevanttomedicalapplications.Thisreviewalsoexplorescurrentcon ﬁ gurations andrecentinnovationsintheuseofplacentaltissuesasbiomaterialsinregenerativemedicine. 1.Introduction Placentaltissues,orextra-embryonictissues,alsocommonly referredtoasbirthtissues,supportandprotectfetaldevelop- mentduringgestation.Theyincludetheplacentaldisc,the umbilicalcord,placentalmembrane,andamniotic ﬂ uid (Figure1A).Thesetissuesaretransient,astheyonlyexist tosupportthefetusuntilbirth.Placentaltissuesareofgreat interestinmedicine,speci ﬁ callyasabiomaterialbecauseof theexceptionalpropertiesoffetaltissuesandtheeaseof accesstothemasrawmaterials[1,2].Unlikecadaverictis- sue,placentaltissuesareofaconsistentage,donatedfrom ascreenedpopulationofhealthydonors,donotrequire extensiveprocedurestocollect,andwouldotherwisebedis- cardedasmedicalwaste[3].Commontissuecollectionprac- ticesinvolvepackagingfortransportfollowingroutine cesariansectiondeliveries,andvaginaldeliveriesundercer- taincircumstances,withsterilematerialsandtransportsolu- tions.Sterilecontainersarecommonlyusedtotransportthe tissuetoavoidcontamination,andstoragetimeiskeptas minimalaspossibleunderrefrigeratedconditionstoensure materialstability[3,4]. Inthe16 th century,medicaluseofthehumanplacenta wasdescribedinChina[5].The ﬁ rstdocumenteduseofpla- centaltissuesinmodernmedicinedatesbacktothebegin- ningofthe20 th centurywhereapplicationofplacental membraneinskingraftingwas ﬁ rstpublishedshowingsupe- riore ﬃ cacyovercadaverictissuebyDavisin1910[6].The ﬁ rststudiesshowingtherapeutice ﬃ cacyofplacentaltissues appearbetweenthe1930sand1980s[2].Severalreports werepublishedinthe1930-1940sontheuseofplacental membraneinsurgicalandwoundapplicationsfollowedby clinicalreportsandtrialsthroughthe1970sand1980s.As contemporarystandardsofcleanlinessandsterilitydevel- opedthroughthe ‘ 80sand ‘ 90s,scientistsbegantoexperi- mentwithdehydrationandsterilizationofthetissue.This ledtotheincreasedsafety,usability,andaccessoftoday ’ s shelf-stableproducts. Giventheclinicalbene ﬁ tsofplacentalmembranegrafts inwoundandsurgicalapplications,researchhasfurther focusedondevelopingandtestingnewtissue-andcell- basedtherapiesderivedfromplacentaltissues[4].Since theturnofthecentury,applicationsofthesetissueshave includedusesincelltherapy,bioengineering,and Hindawi BioMed Research International Volume 2022, Article ID 6751456, 26 pages https://doi.org/10.1155/2022/6751456regenerativemedicine[3].Themajorityofclinicalstudies haveutilizedplacentalmembrane,umbilicalcordveins, andamniotic ﬂ uid[3].However,extensivepreclinicalexper- imentationhasbeenperformedontheplacentaldisc,Whar- ton ’ sJelly,andumbilicalarteries[3].Thewidearrayof applicationsstemsfromthevariedfunctionsandformsof theplacentaltissuesandtheirrichcompositionofextracel- lularmatrix(ECM)components,signalingmolecules,and growthfactors[3]. Overthelast30yearstherehasbeenvastinterestinthe useofimplanted,injected,orculturedstemcellsasatherapy tohelpregeneratedamagedtissues[7].Placentaltissuesare arichsourceofstemcellsthathavebeenextensively researched.Theuseofmultipotentmesenchymalstromal cells(MSCs)fromumbilicalcordblood,Wharton ’ sJelly, andplacenta,forinstance,havebeenthoroughlydescribed [8,9]andarenotasubjectofthisreview.Recentresearch regardingtheregenerativemechanismofactionofMSCs hasshownthattheyprovidetherapeuticbene ﬁ tthrough thereleaseofcytokines[10].This,alongwiththeclinical successofnon-viableplacentaltissueallografts,furthersug- gestsatherapeuticpotentialforplacentaltissuesasbiomate- rials[2].Thisreviewwilldiscusstherawmaterialpotential ofplacentaltissuesassourcesofECMcomponents,scaf- folds,signalingmoleculesandgrowthfactorsforusein regenerativetherapies.Wewillemphasizethestructural Maternal vessels Intervillas Spaces Fetal vessels Chorionic Plate Cytotrophoblasts Syncytiotrophoblasts Decidua Amniotic ﬂuid Umbilical cord Placental membrane Placental disc Umbilical arteries Umbilical vein Umbilical cord cross section Fetal-facing side Maternal-facing side SA EL WJ UA UA UV Epithelium Basement membrane Compact layer Fibroblast layer Intermediate layer Reticular layer Basement membrane Trophoblast layer Amnion Chorion Intermediate layer (a)(b) (c) D Figure 1:Theanatomyofplacentaltissues.(a)Theanatomyofthefetalcompartment,(b)theanatomyoftheplacentalmembranelayers, (c)theanatomyandcrosssectionoftheumbilicalcord,includingtheepitheliallayer(EL),subamnion(SA),Wharton ’ sJelly(WJ),umbilical arteries(UA),andumbilicalvein(UV),and(d)theanatomyoftheplacentaldisc. 2BioMedResearchInternationalandsolublebiochemicalcompositionofthesetissuesand discusscurrentinnovationsusingthesetissuesasbiomate- rialsinregenerativemedicine. 2.PlacentalMembrane 2.1.AnatomyandDevelopment.Theplacentalmembrane (PM)surroundsthedevelopingfetusandcreatestheamni- otic ﬂ uid- ﬁ lledfetalcompartmentduringgestation (Figure1A).ThePMisstructuredinthreemajorlayers: thefetal-facingamnion,thematernal-facingchorion,and theintermediatelayerinbetween,asshowninFigure1B [11,12].Theamnion,whichiscomposedofepithelium, basementmembrane,compactlayer,and ﬁ broblastlayer, enclosesthefetusandtheamniotic ﬂ uid,whichcushions andprotectsthefetusduringgestation[13].Thechorion, thematernal-facinglayer,incontactwiththematernal decidua,iscomprisedofthreelayers:thereticularlayer,a basementmembraneandatrophoblastlayer[11,14,15]. Thechorionformsabarrierbetweenthefetalenvironment andthematernalimmunesystem[16,17].Theintermediate layerisaspongeynetworkofcollagensandproteoglycans thatfunctionsasaphysicalbarrierbetweentheamnion andchorion.Thestructureofthislayeraidsinthemove- mentoftheamnionalongthechorion,supportedbythe highconcentrationofhyaluronicacid(HA)whichhydrates andlubricatesthetissue[11,18 – 20]. Duringgestation,theamnionandchorionformsepa- ratelyafterblastocystimplantationaroundday14ofgesta- tion,alongsidethedevelopmentoftheplacentaldisc[21, 22].Thewalloftheblastocystbecomesthechorion.By day10post-conception,theamnionisvisible,formingfrom theamniogeniccellsthatmakeuptheinnertrophoblast layerofthecellularmassoftheembryo[21].ThePMcon- tinuouslygrowsthroughoutgestationtoaccommodatethe growingfetus.Followingtheendofmonth3ofgestation, theamnionisvisiblyseparatedfromthechorionleave,the precursortothechorion,bythechorioniccavity.Asthe amnioticsac ﬁ llswith ﬂ uidandgraduallyexpands,the amnionadherestotheinnermostsurfaceofthechorion andthechorioniccavitydisappears.Althoughnowclosely adhered,amnioticandchorioniclayersofthePMdonot fuseandremainhistologicallyseparate[21].Atterm,thelit- eraturereportsnativePMthicknesscanrangefrom0.02mm to0.6mmandtheaveragesurfaceareaofthePMis1600 cm 2 [1,23]. 2.2.Physiology.ThePMactstoprovideabarrierandprotect thefetusfromthematernalenvironmentandwithstandthe stretchingandpressurecreatedbythefetusasitdevelops andmoves[14,24].Inaddition,thePMismetabolically activeandprovidesanimmunebarriertoinfectionanda barriertoimmunerejectionbythemother[25].Overthe courseofgestation,thePMconstantlyundergoesmatrix remodelingtomaintainitsbarrierintegrityandaccommo- dateagrowingfetus[26].Thecompositionofeachlayerof thePMvariesslightlytosupporttheoverallfunctionof themembrane.Thebasementmembraneoftheamnionis adensetissuelayerthatactsasapermeablebarrierforthe exchangeofnutrients[11,27].Thecompactlayerprovides tensilestrengthtothemembraneandisthemost ﬁ brous oftheamnionlayersduetothehighcontentofcollagens secretedfromthemesenchymalcellsinthe ﬁ broblastlayer [11,12,28].The ﬁ broblastlayercontributestothetensile strengthofthemembraneandprovidesasca ﬀ oldforcell- cellinteractions[29,30].Theintermediatelayerfunctions asabarrierbetweentheamnionandthechorion,providing mechanicalsupportintheformofanon ﬁ brillarnetwork madeupmostlyofproteoglycans,glycosaminoglycans (GAGs)andtypeIIIcollagen[11,18,20].Thereticularlayer providesasca ﬀ oldforthechorionwithanetworkof ﬁ bers andisembeddedwithmesenchymalcellssimilartothe ﬁ broblastlayerintheamnion[31].Thebasementmem- branebetweenthereticularlayerandthetrophoblasts increasesthestructuralintegrityofthechorionbyproviding cellsca ﬀ oldingforthetrophoblastlayerandcontributesto theimmuneprivilegeofthetissue[16,32,33].Thetropho- blastlayer,theoutermostlayerofthechorion,consistsof trophoblasts,myo ﬁ broblastsandmacrophages[16]. TheoverallPMstructuresupportsresidentcellswhile maintainingtensilestrengthandelasticityduringtheregular turnoveroftheECMthatisfacilitatedbyendogenouscyto- kinesinthePM[34].ThePMisanimmuneprivilegedtissue contributingtoitsfunctionasanimmunebarrierbetween themotherandfetus,preventingmaternalrejectionofthe fetus.Studiesintheearly1980sfoundnohumanleukocyte antigen(HLA)-A,-B,-C,or-DRmoleculesinhuman amnion,butmorerecentstudieshavedetectedtheseclassI andIImoleculesinvariouscellsofthetissue[35].However, whilealmostallcellsofthePMhavebeenshowntoexpress classIHLAmolecules,thetissuehasbeenfoundtobe immunologicallyinertwithalmostnoreportsofimmune rejection[35,36]andthereislittletonomajorhistocompat- ibilitycomplex(MHC)reactivityinunmatchedadultrecip- ients[36].Thislimitedreactivityisreportedlyduetothe activesuppressionofBcells,Tcells,anddendriticcells,from interactionssuchasthebindingofCD8+TcellstoHLA-G molecules[35,36].FurtherFasligandexpressiononamnion epithelialcellsissuggestedtosignalapoptosisofhostlym- phocytes,helpingtopreventmaternalrejectionofthefetus, alongwiththeimmunosuppressiveactionsofcellsurface andsolubleHLA-G[35]. 2.3.StructuralComposition.ThePMiscomposedofanet- workofcollagens(typesI,III,IV,VandVI),laminin, ﬁ bro- nectin,HA,vitronectin,elastin,andproteoglycans,which providemechanicalstrength,elasticity, ﬂ exibility,and appropriatesti ﬀ nesstosupportthebarrierintegrityinthe uterineenvironment(Table1)[12,18,34,37,38].Eachlayer ofthePMhasadi ﬀ erentcompositiontoserveitsindividual function[20].TheECMoftheamnioncontainscollagens typesI,III,IV,VandVIsecretedbythemesenchymalcells inthe ﬁ broblastlayer,aswellaselastin, ﬁ bronectin,laminin, HA,andsulfatedproteoglycans[12,19,34].Thechorion ECMconsistsofcollagenstypesI,III,IV,V,andVI,elastin, ﬁ bronectin,lamininandHA[34].Theintermediatelayeris composedofcollagentypesI,III,andIV,sulfatedproteogly- cansandglycoproteins.Duetothehighconcentrationof 3BioMedResearchInternationalproteoglycansandglycoproteins,theintermediatelayer allowstheamnionandchoriontoglideagainsteachother [14,18,28]. ThePMcontainsdi ﬀ erenttypesofcollagen(typesI,III, IV,V,andVI)secretedby ﬁ broblastswhichservedi ﬀ erent purposesdependingonwhichlayertheyarefoundin[28]. CollagentypesIandIIIformparallelbundlesthroughout allPMlayers,exceptthetrophoblastlayer,tosupportthe mechanicalintegrityofthetissueduringgestation[20,28]. CollagenstypeIVandVformconnectionstothebasement membranesofboththeamnionandthechorion[39].The ﬁ brousstructureoftheamnionisattributedtothecollagens typeI,III,VandVIpresent[20].Thechorion,thethickest ofthelayers,containslargeamountsofcollagenstypeI andIV,whichformsca ﬀ oldingforresidentcellsinthePM [20,28].Additionally,thepresenceofcollageninthetro- phoblastlayerofthechorionassistsintheanchoringof thePMtothematernaldecidua[14,20].Theintermediate layeriscomprisedofcollagentypeI,IIIandIVandcontains ahigherdensityofproteoglycansandmucopolysaccharides [20].PMisattractivetoresearchersinterestedincollagen- richsourcesforwoundhealingapplicationsbecauseofthe roleofcollageninreducingin ﬂ ammationinwoundheal- ing[40]. Glycoproteins,suchas ﬁ bronectinandlaminin,areECM componentsthatcontributetothestructuralintegrityofthe Table 1:Extracellularmatrixcomponentsofplacentalmembrane,amniotic ﬂ uid,umbilicalcordWharton ’ sJellyandplacentaldisc. ExtracellularMatrixComponentsofPlacentalTissues ECMComponentPlacentalMembrane Amniotic Fluid Umbilical Cord PlacentalDisc CollagenI[26,28] [109, 127] ∗∗ [188][232,233,235,254] CollagenII[28]NR[205][235] CollagenIII[28] [109, 127] ∗∗ [188][232,233,254] CollagenIV[28] [108, 109] ∗∗ [188][3,232,254] CollagenV[28]NR[188][232,235] CollagenVI[28]NR[205][235] CollagenXII[28]NR[205][254] CollagenXIII[28]NRNR[254] CollagenXIV[28]NR[258][235,254] CollagenXXNRNRNR[254] CollagenXXVIIINRNRNR[254] Aggrecan[28]NRNR[254] Agrin[259]NRNR[254] Chondroitinsulfateproteoglycans[260] [135] ∗∗ [191][254] Elastin[28]NRNR[254] FibrinogenNRNRNR[254] Fibrillin[28]NR[261][3] Fibronectin[28] [127] ∗∗ [262] [3,232,235,236,238, 240,254] Ficolin-2NRNRNR[254] Heparinsulfateproteoglycan[12] [135] ∗∗ [188][3,254] Hyaluronicacid[12] [135] ∗∗ [194][241] Dermatansulfate[260] [135] ∗∗ [263][241] Chondroitin6-sulfate[264] [135] ∗∗ [188][241] Chondroitin4-sulfate[264] [135] ∗∗ [188][241] Lamininsalpha1,2,3,4;beta1,2; gamma1,3 [43]Laminina2,a3,a5[43]a1,a4:ND[43]b1- 3,gamma1-2 [43]gamma3:ND NRNR[3,218,232,235,254] Nidogen[265]NRND[254] Vitronectin[28]NRNR[254] OsteopontinNRNRNR[254] ∗∗ LevelsarefromsecondtrimesterAFcollectionandmeasurements;NR=notreported;ND=notdetected. 4BioMedResearchInternationalPM.Fibronectininteractswithcollagensandproteoglycans, andin ﬂ uencescellmorphology,adhesion,migration,prolif- erationanddi ﬀ erentiationbybindingtothecellsinboththe amnionandchorion[28,41]Lamininsprovideaconnection betweenneighboringmembranelayersbybindingtoother proteins,likecollagens[28,42],[43].Proteoglycanscontrib- utetothetensilestrengthandintegrityofthePM,aswellas tocellproliferationanddi ﬀ erentiation,andassistinbinding ofgrowthfactorsintheotherlayersofthePM[14,18]. Whenutilizedasabiomaterialinwoundmanagement,this naturallyoccurringECMsca ﬀ oldcanprovidestructural supportandfacilitateintercellularandintracellularsignaling betweencells,cellattachmentandcellularmigrationessen- tialtoproperwoundremodeling[44,45].PMECMcompo- nentshavebeenshowntocontributetodecreased in ﬂ ammation,preventionofinfection,andreductionin scarringandpainatthesiteofinjury[44,46]. TheECMalsocontainscomponentsthatgivethePM ﬂ exibilityandcushioningfromthepressuresofgestation. Elastin ﬁ berscontributetotheintegrityandtensilestrength ofthePMasitstretchesduringgestation[15,20].GAGs, suchasHA,areanothercomponentthatprovidesthe ECMwithelasticityandlubrication.Studiesonthedistribu- tionofHAinPMhaveobserveditspresenceincollagen-rich (speci ﬁ callytypesIVandV)areasofthetissue,forexample, thebasementmembraneorreticularlayerofPM,aswellas intermediatelayer,butnotintheepitheliumoftheamnion. Thechorionandintermediatelayerhaveagreaterpresence ofHAthantheamnion[34,47,48].TheGAGcontentof theintermediatelayer,speci ﬁ callytheHA,contributesto thehydrophilicnatureofthelayer[20].HAhasalsobeen showntoplayaroleinthereductionofin ﬂ ammationand readilyaccessiblebiomaterialsthatprovideHAareofgrow- inginterestintheregenerativemedicine ﬁ eld[20,38]. 2.4.Cytokines&AntimicrobialComponents.Extensive researchintothecytokinecontentofbothnativePMand processedPMallograftshasbeendonetohelpunderstand theroleofPMingestationandpotentialclinicalapplica- tions.ThelayersofthePMareareservoirofcytokinesand growthfactorswhichregulatethepathwaysthatmaintain theECMstructuralintegrityduringgestation(Table2). Evaluatingthecytokineandgrowthfactorcontentinthe amnion,theintermediatelayer,andchorionisofinterest forvaryingapplicationsofthetissue[20,49 – 51].Speci ﬁ c focushasbeengiventogrowthfactorssuchasepithelial growthfactor(EGF), ﬁ broblastgrowthfactors(FGFs),vas- cularendothelialgrowthfactor(VEGF),transforming growthfactoralpha(TGF α ),transforminggrowthfactor beta(TGF β ),keratinocytegrowthfactor(KGF),hepatocyte growthfactor(HGF),andplatelet-derivedgrowthfactors (PDGFs),interleukins(ILs)suchasIL-4,IL-6,IL-8,and IL-10,interferons(IFNs),andproteaseinhibitorsincluding tissueinhibitorsofmetalloproteinases(TIMPs)TIMP-1, TIMP-2,andTIMP-4becauseoftheirapplicabilitytothera- peuticapplications[49,50,52 – 54].Thesesignalingcompo- nentshelpregulatemultipleaspectsofthehealingcascade, includingangiogenesis,in ﬂ ammation,andtissueremodeling [20,51].BasicFGF(bFGF),endocrine-derivedvascular endothelialgrowthfactor(EG-VEGF),VEGFandPDGF- AA,andPDGF-BBstimulatetheactivityandmigrationof matrixmetalloproteinases(MMPs),whichareresponsible forthebreakdownofexistingECMinpreparationfor remodelingofthePM[34,55].TIMPsareimportantinhib- itorsthatregulatetheactivityofMMPstopreventexcessive breakdownoftheECManddepositECMcomponents[56, 57].InadditiontotheactiveroleMMPsplayintheremod- elingoftheplacentalmembraneduringgestation,theyare anactivecomponentintheonsetoflabor,wherehighlevels ofMMPsandreducedlevelsofTIMPsinducethedegrada- tionofthePMECMresultinginmembranerupture[55]. Speci ﬁ cally,attheonsetoflabor,MMP-9concentrations aremainlylocalizednearthecervixandtheconcentrations risethroughoutthePMaslaborprogresses[55].Thetensile strengthofhealthytermPMhasbeencorrelatedtothedis- tributionofMMP-9withtheweakestareashavingthehigh- estconcentration[31]. Theamnionandchorionarecomparableintermsofthe typesofcytokinespresentbutdi ﬀ erintheirdistributionand concentration[51].Theconcentrationsrangefromlessthan 1pg/mgtoover6,000pg/mg,normalizedbydryweight, dependingonthethicknessandlocation[33,38,50,58]. Multiplecytokineshavebeenfoundinsigni ﬁ cantlyhigher concentrationsinfreshchorioncomparedtootherlayers, includingadiponectin(APN),angiogenin(ANG),ANG-2, bFGF,EG-VEGF,HGF,insulin-likegrowthfactor1(IGF- 1),PDGF-AA,PDGF-BB,TIMP-2andTIMP-4[51].The greaterconcentrationspersurfaceareafoundinthechorion areattributedtothechorionbeingthickerthantheamnion [51].Theamnionlayer,however,doescontainhigherlevels ofseveralcomponents,includinggalectin-7,IL-1F5and TGF- β 1[51].Theproteomicpro ﬁ leoftheintermediate layerhasbeenrecentlyinvestigated,demonstratingthatthe layernotonlycontributestotheoverallstructuralintegrity ofthemembranebutalsofunctionsasasourceofsignaling componentsimportanttowoundhealingapplications,such asPDGF-AA,PDGF-BB,TIMP-1,TIMP-2,TIMP-4,TGF- β 1,bFGF,EGF,andVEGF[20]. ThePMalsoservesasabarriertobacterialinfectiondur- inggestation,containinglayersofantimicrobialpeptides, suchashumandefensins,ela ﬁ n,secretoryleukocyteprotease inhibitor(SLPI),andhistonesH2AandH2B[59,60]. Human β -defensin(HBD)isproducedbyepithelialcellsin thePM[61 – 63].Defensinsareantimicrobialpeptidesthat preventthegrowthofbacteria[61].ThePMalsocontains cystatinEwhichhasanti-viralproperties[64].Thereare greaterlevelsofbacterialinhibitionfromthematernal- facing chorionthanthefetal-facingamnion,whichpoints totheroleofthechorionasakeybarrierlayerinpreventing infectionandrejection[63]. 2.5.ClinicalApplicationsandUsesinRegenerativeMedicine. ThePMhasthelongesthistoryofuseinmodernmedicine ofallplacentaltissueswiththe ﬁ rstdocumentedcasein 1910whereitwasusedinskingrafting[6].Thismembrane hasbeenextensivelyresearchedforuseinwoundapplica- tions,wherethesheetcon ﬁ gurationeasilytranslatestoa woundcovering.Apartfromitsregenerativeproperties,the 5BioMedResearchInternationalTable 2:Signalingcomponentsofplacentalmembrane,amniotic ﬂ uid,umbilicalcordWharton ’ sjellyandplacenta. SignalingComponentsofPlacentalTissues Functional rolein regenerative medicine CytokinePlacentalmembraneAmniotic ﬂ uidUmbilicalcordPlacentaldisc Angiogenic signal TGF- β 2[266] [267]**;[268]** (TGF- β 1and- β 2) [269] [3,217,243,245,252,254, 270] TGF- α [266][271][272][273] Angiogenin[51][274]**NR[252] VEGF [26]VEGF-A;[26, 275 – 277] ND(VEGF-D) [142]**;[278] [194](VEGF-A);NR (VEGF-B);NR(VEGF- C);NR(VEGF-D) [3,244,245,252,254,270] PlGF[51,58][279]**NR[245,246,252] FGF-1(aFGF)[51]NR[198][252,254] FGF-6NRNRNR[252,254] FGF-2(bFGF)[266][271][198][252,254] HGF[51][271][194][252,254] PDGF-AA,-BB [26]PDGF-AA;[51] PDGF-BB [280]**(PDGF-BB) [194]PDGF-AA;NR (PDGF-BB) [244,252,254] ANG[51][281]NR[3] Anti in ﬂ ammatory signal IL-4[26][142]**;[280]**NR[243,245,282,283] IL-10[26][142]**;[267]**NR[243,245,282,283] IL-13NR[142]**NR[243] IL-1ra[51][142]**[194][243] IL-6[58] [142]**; [267]**;[284] [194][217,243,244,252,283] IL-2[51] [285][142]**; [280]**;[284] NR[243,283] Adiponectin[286][287]NR[244,288] Protease inhibitor TIMP-1[26][289],[290][194][252] TIMP-2[26][289 – 291][194][252] TIMP-4[26][290][292][293] Antimicrobial IFN γ NR[142]**;[280]**[194][243,245,283] Lactoferrin[20][144]NR[294] HBD-1[20,60][153]NR[60] HBD-2[295][145][296][60,248] HBD-3[295]NRNR[60] HNP-3[297][298]NR[297] Ela ﬁ n/Trappin-2[297][297]NR[60,248] SLPI[297][200]NRND[60] Tissue remodeling TGF- β 1,2,3[26] [268]**(TGF- β 1 and β 2) [186] [3,17,243,245,252,254, 270] TGF- α [51][271][194][273] Activin[38][299]NR[245] VEGF [26]VEGF-A;[26, 275 – 277] ND(VEGF-D) [142]**,[278] [194](VEGF-A);NR (VEGF-B);NR(VEGF- C);NR(VEGF-D) [3,244,245,252,254,270] PlGF[33,51][279]**NR[245,246,252] FGF-1,-2,-3,-4, -5,-6,-7 FGF-1:[51];FGF-2: [266];FGF-3:NR;FGF-4: [33];FGF-5:[20];FGF-6: [20];FGF-7:[33] NR FGF-1:[198];FGF-2: [198];FGF-3:NR;FGF-4: [300];FGF-5:NR;FGF-6: NR;FGF-7:[194] [252,254] 6BioMedResearchInternationaltissueisattractiveinwoundhealingapplicationsbecauseitis asustainablematerial,typicallyclassi ﬁ edasmedicalwaste, thatisimmuneprivileged[36,65].Whileacuteandchronic woundsandburnsarestillthemostprevalentapplication forPM,thereisawidevarietyofsurgicalandsportsmedi- cineapplicationswherePMisbeinginvestigatedasatreat- ment,includingocularreconstruction,spinalsurgery, nervereconstruction,dermatologicalcases,andasabarrier membraneforadhesionorscarringprevention[4,11,12, 36,66]. Thewidearrayofapplicationsandadvancesintissue processingtechnologyhasledtotheemergenceandinvesti- gationofnumerousPMcon ﬁ gurations,includingassingle- layer,multi-layer,full-thicknessandcompositegrafts,asa micronizedpowder,andasaninjectablematrix[11,34, 67 – 70].Processingandpreservationtechniquesalsovary. Dependingupontheapplication,thePMmaybeleft untreatedordecellularizedtoremovedonorcellsand DNAfromthetissue,forexampleusingdispase,EDTA, etc.[71,72].Additionalprocessingtechniquescaninclude dehydrationmethodssuchasovendehydrationandlyophi- lization,cryopreservation,andcryomilling[26,67,69,70]. Studieshaveshownthatlyophilizationpreventsdeteriora- tionatroomtemperatureforamorestableproductthatis comparabletofreshplacentaltissue[66,69,73].Character- izationofplacentalmembranecon ﬁ gurationshasdemon- stratedretentionoftheirregenerativepotentialafter processing.However,somestudieshavereportedreduced concentrationsofcytokines,aswellasalteredtissuestruc- turesandappearancecomparedtofreshPM[20,37,51,58]. Overthelasttwodecades,therehasbeenanincreased useofPMallograftsinthetreatmentofwoundsandburns asmoreevidencecontinuestoemergeregardingtheECM, therapeuticcomponents,andprovene ﬃ cacy[20,74,75]. ProcessedPMhasbeenasuccessfultreatmentforvenous legulcers(VLUs),diabeticfootulcers(DFUs),andchronic wounds,demonstratingdecreasedtimetowoundclosure [74],Onacellularlevel,thePMECMhassca ﬀ oldcharacter- isticsthatarebene ﬁ cialforanchorage-dependentcellattach- mentandendogenouscytokinesthatpromoteproliferation, angiogenesis,cellularmigration,tissueremodeling,andcre- ateanenvironmentfortheresolutionofthein ﬂ ammatory stageofhealingandimprovedremodeling[12,20,21,34, 37,38].ClinicalapplicationsofdehydratedPMasburn dressingshaveshownthatthePMencouragesre- epithelializationofdamagedtissue,stimulatedbyEGF, KGFandHGF,reducingin ﬂ ammation,decreasinghealing timeanddecreasingscartissuedevelopment[76].Implanted acellularPMgraftinasubcutaneousmousemodelencour- agedmigrationandin ﬁ ltrationofhostcellsduringtheheal- ingprocess,speci ﬁ callyof ﬁ broblast-likecells,macrophages, endothelialcells,andfewerT-cellsthanthecontrolgraft [77].PMhasbeensuccessfullyusedinophthalmologyfor almost30yearsforthetreatmentofcorneallesions,retinal detachment,andlimbalcellregeneration[78,79].Both intactanddenudedPMusedinocularreconstructionina rabbitmodelachievedre-epithelializationandintegration intotherabbittissueby3-weekspost-surgery,withlower in ﬂ ammatoryresponsesnotedindenudedPMtreatedeyes [80].Extractfromdigestedamnionusedineyedropsfor cornealtreatmenthasbeenshowntosuccessfullycultivate limbalstemcellsclinically[81]. Placentalmembrane-derivedmaterialshavebeen increasinglyusedsurgicallyinreconstructive,vascular,OB- GYN,abdominal,spinal,andneurologicalcases.Dehydrated PMusagehasbeenshowntoresultinsuperiorrecoveryand lowerratesofreherniationfollowingmicrodiscectomy[82]. Retrospectivecasestudieshaveshownthepotentialbene ﬁ ts ofPMtopreventretetheringofthespinalcordfollowing microsurgicalintradurallysisadhesion[83].Thepreventa- tiveuseofPMagainstfailedbacksurgerysyndrome(FBSS) causedbyepiduraladhesionsresultedinasigni ﬁ cant decreaseintheincidenceofepidural ﬁ brosisformationcom- paredtostandardclosureincanineandratmodels[84,85]. Table 2:Continued. SignalingComponentsofPlacentalTissues Functional rolein regenerative medicine CytokinePlacentalmembraneAmniotic ﬂ uidUmbilicalcordPlacentaldisc EGF[33][271,301][186][252,254,270] HGF[51][271][194][3,252,254] IGF-1[51][271,302]NR[3,252,254] PDGF-AA,-BB[26,51][280]**(PDGF-BB) [194]PDGF-AA;NR (PDGF-BB) [244,252,254] Adiponectin[286][279]**NR[244,288] IGFBP-1,-2,-3, -4 [303] ND(IGFBP-1),ND (IGFBP-2),IGFBP-3,ND (IGFBP-4) [304],[305]NR[252] MMP-2[55][55],[306][197][56] MMP-9[31],[55][55],[125],[306][197][56] ∗∗ LevelsarefromsecondtrimesterAFcollectionandmeasurements;NR=notreported;ND=notdetected. 7BioMedResearchInternationalThereisevidenceofPMuseinavarietyofreconstructive applicationsincludingvaginoplastyandvestibuloplasty[86, 87].TheclinicalusesofPMinthe ﬁ eldofurologyare numerous,includingpenilereconstruction,microsurgical corddenervations,posteriorurethroplasty,hypospadias repairs,andurogenitalindications,suchasvesicovaginal ﬁ s- tulas,asreviewedbyOotamasathienetal.(2017). Areviewoftheliteraturewillshowdozensofanimal studyinvestigationsofPMasanintraabdominaladhesion barrier,resultinginimprovedoutcomesinvariousanimal models.Aretrospectivecohortstudyof120patientswith 40patientsreceivingdehydratedPMbarriersusedfollowing intrauterineadhesiolysisand80patientsinthecontrol groupfoundPMtoreduceadhesionreformationand improvesubsequentmenstruationcomparedtothecontrol group[89].AstudyinvestigatingtheuseofPMasanadhe- sionbarrierfollowinglaparoscopicresectionofendometri- osiswithadhesiolysisfoundthatafter1-2weeksno adhesionswereobservedatpointofPMplacementin14 outof15cases[90]Inaratmodel,PMusedasanervewrap- pingresultedinadecreaseinscartissuedevelopmentandan increaseinsciaticfunction,suggestingthatinadditionto functioningasabarrier,thePMassistedinregenerating thenerve[91].Recently,areconstituted,injectablePMpow- derwasinvestigatedinapost-myocardialinfarctiontissue repairprocedureinaratmodel,resultinginadecreaseof myocardialinfarctionscarformationandimprovedleftven- tricularfunction[68]. Morerecently,useofPMinthe ﬁ eldofdermatologyhas beenexplored,includingapplicationsofPMinMohssur- gery,inthemanagementofchronicradiationnecrosis,and inthetreatmentofpyodermagangrenosumcases[92 – 94], AcaseseriesoftheuseofdehydratedPMMohsmicro- graphicsurgeryforlargefull-thickness(tothebone)scalp woundsfoundtreatmentwaswelltoleratedandresultedin thedevelopmentofgranulationtissue[95].Acaseofa77- yearoldfemalewithpyodermagangrenosumtreatedwith dehydratedPMasanadjuncttoimmunosuppressives reportedpainreductionfrom10/10to5/10withinhours ofapplicationthatdecreasedto0/10after1weekanda 56%reductioninwoundsizeafter3applications.[93]. PMproductshavealsobeeninvestigatedandusedinthe pastdecadetotreatvariousmusculoskeletalandorthopedic conditions,includingligamentandtendonrepairandosteo- arthritis(OA)[79].Inacasestudyfocusedon ﬂ exortendon injuries,abluntdissectedamnionwrappedtendonresulted inahigherrangeofactivemotion,ahigherrankonthe StricklandandGlogovacscale,comparedtothecontrol group[96].Similarly,acombinationinjectablematrixcom- posedofPMandumbilicalcordhasbeenusedtotreatknee OAinhumans.Resultsfromthepilotstudyshowedan increaseincartilagethickness/volumeandadecreasein lesionscomparedtothecontrolgroup[97].Inaratmodel forAchillestendonrepairutilizingtypeIIdiabeticratsto impairhealing,dehydratedduallayeramnion/chorion showedimprovedcellmigration,lowerfailurerates,and morefavorablemechanicalstrengthofthetendoncompared tountreatedcontrols[98].MicronizedPMhasbeenutilized asaninjectabletreatmentforOA,andinratswithinduced OAhasshowntoresultinjointswithhigherproteoglycan contentthancontrols[99].Intra-articularinjectionoflyoph- ilizedPMinrabbitsforOAshowedsimilarimprovements comparedtoasalinecontrol[79].Acontrolled,multi- centertrialforPM-derivedbarrierstoencase ﬂ exortendons followingrepairresultedinsigni ﬁ cantlyincreasedrangeof motionandfunctionalscorescomparedtocontrol[79]. 3.AmnioticFluid 3.1.AnatomyandDevelopment.Amniotic ﬂ uid(AF)begins toformaroundweektwoofgestationandistheproductof wateraccumulatedfrommaternalcirculationthathas ﬁ lled theextracelomiccavity(Figure1A).Inadditiontowater accumulationviamaternalvesselsofthedeciduaandcho- rionlaeve,AFconstituentsarederivedfrom ﬁ ltrationfrom thefetalvesselsoftheumbilicalcordandchorionicplate, secretoryprocessesoftheamnioticepithelium,and ﬁ ltration fromintracorporealfetalvesselsviathefetalskin[100].As pregnancyprogressestoweek8,priortothekeratinization offetalskin,water,proteinsandothermetabolitesdi ﬀ use freelyandbidirectionallybetweenthefetusandAF [101 – 103].Duringthistime,thecompositionofAFresem- blesmaternalandfetalserum.Tomeettherequirements ofdevelopment,towardsthesecondhalfofgestation,the compositionofAFbeginstochangedueto ﬂ uidregulation andexchangepathways,suchasfetalswallowing,lung ﬂ uid excretion,fetalurination,andintramembranousabsorption [104].AFvolumeisprimarilyregulatedthroughfetalswal- lowingandintramembranousexchangeacrossthePM.This intramembranous ﬂ owtransferssolutesand ﬂ uidbetween thematernalcirculationandtheamnioticcavity.Mean- while,fetalswallowingallowsthesesubstancestobetrans- ferredfromtheamnioticcavityintofetalcirculation[104]. AlowdensitypopulationofheterogeneousAFcells calledamniocytesispresentinAFthatarederivedfrom thefetaltissue[105].Reportedcellconcentrationsinfreshly collectedAFsamplesareontheorderofmagnitudeof10 5 cells/mL[106,107].Amniocytescollectedfromroutine amniocentesisperformedduring14-16weeksgestationhave beendi ﬀ erentiatedandsubclassi ﬁ edintooneof3main groupsbasedontheirmorphologicalandgrowthcharacter- isticsinculture:60.8%amniotic ﬂ uidspeci ﬁ ctype(AF-type) cells,33.7%epithelioid(E-type),and5.5% ﬁ broblastictype (F-type)[108].Toidentifysubclassi ﬁ cationofamniocytes inAF,cellculturemustbeperformedtoobservethemor- phologicalcharacteristicsofthematuredcolonies[108]. Morphologically,matureE-typecellsarelargepolygonal cellswithsmoothmarginsthatgrowinclosecontactwith eachotherinculture.TheyresembleAF-typecellsbutcan bedistinguishedbytheirbroadergrowthmargin[108]. MatureAF-typecellcolonieshavepleomorphicmorphology andcanbedi ﬀ erentiatedafter12-14days.Theyaredistin- guishedbytheircharacteristic “ bulls-eye ” growthpattern thatisnotfoundinE-orF-typeAFcellcolonies[108, 109].IthasbeensuggestedthatAF-typecellsinAF ﬂ uid arederivedfromextraembryonic,fetaltrophoblastictissues, yettheirfunctionduringgestationisnotwellunderstood [110,111].F-typecellsoriginatefromfetal ﬁ brous 8BioMedResearchInternationalconnectivetissueanddermal ﬁ broblastsandgeneratea ribbedandstreamingpatternastheymatureinculture. Theyarespindle-shapedandoverlap,formingmulti- layeredsheetsinareasofgreaterdensitywhencultured [108,109].Averysmallsubpopulation,approximately 0.1-0.5%,ofAFcellsareCD117+andhavedi ﬀ erentiation potential.AFmesenchymalstemcells(AF-MSCs)have beenanareaoffocusforregenerativemedicineinrecent years[112 – 115].Asthevastmajorityofstudiesfocused onamniocyteshavebeenperformedinvitro,ourunder- standingoftheirproliferativepotentialandheterogenous phenotypeinvivoisstillevolving,especiallygiventhe processingofAF-derivedproductsthatwouldlikelyren- derMSCsnon-viable[112,116,117].A2019studyfound thatwhile3AF-derivedinjectableproductsallcontained signalingcomponentsthatmodulatehealing,therewere nocellswithknowncharacteristicsofMSCsintheprod- uct[117] 3.2.Physiology.OneofthemostimportantfunctionsofAF isitsroleinfetalsupportandprotectionthroughoutgesta- tion[103].WhileAFprovidesauniqueenvironmentthat supportsfetalgrowthandmovement,itplaysalargerole incushioningandprotectingthefetusagainstthermaland mechanicalshock[103].AFalsoactstohelpprotectthe integrityoftheumbilicalcordbyprovidingamechanical cushionbetweenthefetusandtheuterinewall,preventing theumbilicalcordfrombeingcompressedduringgestation [101].Separatefrommechanicalprotection,AFcontributes totheinnateimmunityofthefetalsystem.Ahostofantimi- crobialandbacteriostaticpeptideshavebeenidenti ﬁ edin AF(Table2),whichhelpprotectthefetalenvironmentfrom infectionorcommonbacterialorfungalpathogens[103]. Additionally,AFactsasareservoirfornutrients,suchas essentialfreeaminoacids,proteins,lipids,electrolytes,and carbohydrates,asobservedinmid-trimesterAF[118].These nutrientsoriginatefrommaternalplasmaandtheplacenta andaretransportedtothefetalcavitythroughplacentalcir- culationor,lesscommonly,acrossthePMandintotheAF throughcarrier-mediatedtransportersandchannelslocated inthePM[104,119,120].Thecompositionofthesenutri- entsconstantlychangestomeettheneedsofthedeveloping fetus[118].Atfull-term,AFcompositionisabout98%water andelectrolytes.Peptides,carbohydrates,lipids,hormones, andsignalingmoleculescomprisetheother2%[101].These non-waterconstituentsaresolublecomponentsthataidin thestructure,protection,nutrition,andoverallwell-being offetaldevelopment.Glucoseistheprimaryenergysource forthefetusduringgestationandistransportedtothefetus viatheplacentaaswellasfromswallowingAF[121,122].In ananimalmodel,essentialaminoacidsinAF,suchasgluta- mine,arerequiredforgastrointestinaldevelopment,growth, andfunction,aswellasfetalnitrogenandcarbonmetabo- lism.Aminoacidconcentrationsnaturallydecreasetowards theendofgestationbutcouldbefurtherreducedbyalter- ationsinmaternaldiet[120]. 3.3.StructuralComposition.AFanalyzedmid-trimestercon- tainsECMcomponentsthataresecretedbyfetalcellssus- pendedintheAForshedfromplacentaltissuesand depositedintoAFbyfetalurine(Table1)[123,124].Colla- gentypesI,III,andIVaremajorcomponentsofthePM [125].AsthePMisweakenedinpreparationforspontane- ousruptureofthefetalmembranes,enzymaticconstituents ofAF,suchasMMP-9,degradeandmetabolizethecollagen presentinthePM,likelyleadingtocollagendepositioninto AFatterm[125,126]. IthasalsobeenproposedthatculturedfetalF-typecells inAFareadictatorofcollagendeposition,themaincom- ponentofthefetaldermalECM[108],andareresponsible fortheproductionoftypeIandtypeIIIcollagenand ﬁ bro- nectininfetalECMdevelopment[109,111,127].Incul- ture,F-typeandAF-typeamniocytesproduceaspectrum ofcollagenousproteins.InvitroF-typecellspredominantly synthesizetypeIandtypeIIIcollagen,butAF-typecells synthesizeconsiderablylesscollagen[128].Althoughthe importanceoftheseamniocytesduringgestationisnotwell studied,these ﬁ ndingssuggestaroleofamniocytesincol- lagensynthesis[127],whichmaycontributetosignaling fetalwoundrepairanddermalgrowth[109,129].Thefunc- tionofE-typecellsinAFisnotwidelyunderstoodbutthey havebeenshowntobeinvolvedinsecretionoftypeIVcol- lagenandcell-to-cellorganizationofbothkeratinand vimentin ﬁ bersinculture,furthersuggestingtheirrolein fetaldevelopment[108,130]. AFcontainsmultipleGAGsandnutrientsthatmayplay animportantroleinprovidingprotection,cushioning,and lubricationtothedevelopingfetus[131 – 133].Thecushion- ingandlubricationpropertiesofECMcomponentspresent inAF,suchasHA,havebeenatopicofinterestinregener- ativemedicinefortheirpotentialtherapeutice ﬀ ectsontreat- ingsynovialjointpainandpreventionofadhesion formation[47,134].HA(34%),chondroitin-6-sulfate (20%),non-sulfatedchondroitin(14%),chondroitin-4- sulfate(13%),heparansulfate(6%),anddermatansulfate (5%)makeupmostoftheGAGsfoundinAF[135]. TherelativelylowconcentrationsofhyaluronidaseinAF allowforanenvironmentrichinHA[136].HAservesto increaseAFviscosity,providessupportandlubricationin theuterineenvironment,andmayalsoplayanimportant roleinfetalwoundhealing[137].StudiesshowthatHApro- videsamesenchymalsignalforregenerativehealinginfetal wounds,contributingtothescarlesshealingobservedin fetuses,andpotentiallyprovidingintercommunication betweenthefetusandsurroundingtissuesthroughtheAF [132,138,139]. Fibronectinisaglycoproteinfoundinhighconcentra- tionsinAFcomparedtoplasma[140].Itissecretedby bothF-typeandAF-typecellsandisassociatedwiththe pericellularandPMECM[127].Thephysiologicalfunc- tionsof ﬁ bronectininAFarerelativelyunknown,but ﬁ bronectinpresentinAFcarriesdoublethecarbohydrates whencomparedtoplasma ﬁ bronectin,allowingforbetter protectionagainstproteolyticdigestion[140].Fibronectin mayberesponsiblefortherapid,scarlesswoundhealing observedinfetalwoundsasdepositionof ﬁ bronectin occursearlierinfetalwoundsthaninadultwounds [131,141]. 9BioMedResearchInternationalNext >