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May2008,Volume2,No.5(SerialNo.6)JournalofLifeSciences,ISSN1934-7391,USA

    ;Applicationofmodifiedpolyethyleneglycolhydrogelsin ;theconstructionoftissueengineeredheartvalve ;0UYANGHuil.

    ;ZHA0RongI.ZHANGJin.bao.LIUYanglZHENGQi-jun1.YANGJianl. ;GUChun.hu1WEIXu-fenglCHENChangsheng2YIDing.hualLIUWei-yongl ;(i.DepartmentCardiovascularSurgery,X

    ingHospital,FourthMilitaryMedicalUniversity,Xi’an710032,China;

    ;2.DepartmentofHealthStatistics,FourthMilitaryMedicalUniversity,Xi’an710032,Chi

    na

    ;Abstract:Toenhancetheadhesionofseedingcellstothe

    ;biomaterialscaffolds.thePEGhydrogelsweremodified.

    ;PorcineaorticvalvesweredecellularizedwithTritonX100and

    ;trypsin.ThecellswereencapsulatedintothePEGhydrogelsto

    ;completetheprocessofthecellsattachingtotheacellular ;porcineaorticvalyes.Herein,theautologousmesenchymalstem ;cells(MSCs)ofgoatswereselectedastheseedingcellsandthe

    ;tendencyofMSCstowarddifferentiationwasobservedwhen ;thesinglesemilunarTEHVhadbeenimplantedintotheir ;abdominalaortas.Furthermore,VEGF,TGFB1,andtheeell

    ;adhesivepeptidemotifRGDwereincorporated.Lightand ;electronmicroscopyobservationswereperformed.Analysisof ;modifiedPEG—hydrogelsTeHV’s(PEG—TEHV)tensilestrength,

    ;andtheratioofreendothelialandmuralthrombosisrevealed ;muchbetterimprovementthanthenakedacellularporcineaortic ;valve(NAPAV).Thedataillustratedthecriticalimportanceof ;MSCdifferentiationintoendothelialandmyofibroblastfor ;remodelingintonativetissue.Ourresultsindicatethatjtis ;feasibletoreconstructTEHVefficientlybycombiningmodified ;PEGhydrogelswithacellularbiomaterialseaffoldand ;autologousMSCscells.

    ;Keywords:tissueengineering;biomaterials;decellularization; ;polyethyleneglycolhydrogel;heartvalves

    ;1.Introduction

    ;Tissueengineeringheartvalves(TEHV)may

    ;havethepotentialtoovercomeshortcomingsofthe ;prostheticcardiacvalvethatiscurrentlyused.The ;seedingcells,scaffoldbiomaterials,andmethodof

    ;Acknowledgments:ThisresearchwassupportedbytheState

    ;HighTech863Funding(No.2006AA02A138)andtheNatural ;SciencesFoundationofChina(No.30672086). ;OUYANGHui(1972),male,Ph.D.;researchfield:tissue ;engineeringheartvalve.

    ;constructivearethethreeimportantfactorsinthe ;constIuctionDrocessofTEHV[1-2].AduItbone ;marrowderivedstemcellshavetraditionallybeen ;consideredtobetissue?specificcellswithlimited ;capacityfordifferentiation.TheuseofboneMSCs ;providesalessinvasivesourceforcellsapplicableto ;tissueengineering,includingcardiovasculartissues ;suchasheartvalves.AsthescaffoldoftheTEHV.

    ;naturallyderivedmaterialscanoffermanymechanical, ;chemical,andbiologicaladvantagesoversynthetic ;materials;itthusholdstremendouspotentialforusein ;tissueengineeringtherapies4J.Decellularization

    ;approachesmayreducehostimmuneresponseto ;bioprostheticsandgeneratenaturalbiomaterialsforuse ;incellseedingandtissueengineeringapplications. ;Bioactivehydrogelsweredevelopedtofacilitate

    .Theywereproventotemporarily ;arterialhealing【引

    ;protectthearterialinjuryfrombloodcontact. ;Furthermore,vascularendothelialgrowthfactor ;(VEGF),transforminggrowthfactor-beta1(TGF-[31), ;andRGDcouldbeincorporatedinPEGhydrogels.

    ;Theydemonstratedefficientendothelialcellanchorage ;andactivationonPEGhydrogelmatrices.

    ;AcombinationthePEGhydrogelsandthe

    ;acellularizedporcineaorticvalveswasinvestigatedas ;themethodforreconstructionoftheTEHVinthis ;study.ThetendencyoftheMSCsfordifferentiation ;wasobservedaftertheTEHVswereimplantedin ;abdominalaortaingoatmodelsatthesametime. ;Applicationofmodifiedpolyethyleneglycolhydrogelsintheconstructionoftissueengine

    eredheartvalve

    ;2.Materialandmethods

    ;2.1Cellisolation,expansionandidentification ;Bonemarrowfromthegreatertrochanterof

    ;femurofadultgoatwasaspiratedintoaheparinized ;syringeusingastandard14gaugebonemarrow

    ;aspirationneedle.About10mlofbonemarrowwas ;aspiratedfromeachgoat.Itwasaddedto20mlof ;phosphate-bufferedsaline(PBS),andcentrifugedat ;900xgatroomtemperature(RT)for10minutes.

    ;Then,thesupernatantwasdiscarded,10ml ;Dulbecco’smodifiedEagle’smediumlow—glucose

    ;(DMEML,Gibco,USA)wasaddedandthesoJution ;wastransferredtoa20mlconicalflittubecontaining ;aPercolldensitygradient(p=1.073)andcentrifuged ;at900×gatRTfor30minutes.Themononuclear ;celllayerwassubsequentlyisolatedandwashed ;twicewithPBS,andthenplatedintissueculture ;plates(Falcon,BD,USA).Cellswereculturedin ;DMEMLwith10%fetalbovineserum(FBS,Gibco, ;USA),1%Lglutamine,penicillin,andstreptomycin ;(Sigma,USA).Cellswereexpandedina37.C,5% ;CO2airhumidifiedincubatorandpassagedat80% ;confluence.Cellswerecharacterizedonthebasisof ;indirectimmunohistochemistrystainingforSH2 ;(CD105)(Sigma,USA),andpotentialtodifferentiate ;alongaspecificcelllineageadiposecel1.TheMSCs

    ;weretreatedinanadipogenicinductionmedia ;containing0.5mmol/LmethylisObuty1xanthine,1 ;~tmol/Ldexamethasone,10gg/mlinsulin,and100 ;p.mol/LindomethacininDMEMwith10%FBSfor ;20daysJoJ.Maturefibroblastcellsderivedfromthe ;wallofagoat’sveinsunderwentthesameprotocolas

    ;acontro1.

    ;2.2Preparationofaceilularizedvalves

    ;Theaorticvalves0f18weekoldChinalandrace

    ;pigs,withweightsrangingfrom40to50kg,were ;obtainedfromalocalabattoir(Fangxinslaughterhouse, ;Xi’an,Shaanxi,China).TheywerestoredinHanks

    ;balancedsaltsolution(HBSS)(Hyclone,USA)at4.C ;2

    ;immediatelytoshortenwarmischemiatime.After ;arrivingatthelaboratory(within30minutes),the ;aorticvalveswereexcisedandfreedfromadherentfat ;andmostofthemyocardium.Theaorticvalveconduits ;wereplacedinPBScontaining1%trypsin.0.05% ;ethylenediaminetetra-aceticacid(EDTA)for18

    ;hoursat37.Cundercontinuousshakingaspreviously ;describedbyGU,etalIJ.Themediawasreplacedafter

    ;18hours.Then.thevalveswereputinto1%Triton ;X100hypotonicandhypertonicsolutioninsuccession. ;toremovethecells,undercontinuousshakingfor24h ;at33.C.Thevalveswerethanflushedfor24bat4.C ;inHankssolution,undercontinuousshakingtoremove

    ;residua1substances.andstoredinHBSSat4.C.Prior ;tofurtherprocessing,theyweresterilizedbyirradiation ;underCo6ogamma(25kGy).Samplesoftheaortic ;valvesweretakenbeforeandaftertreatment.Main ;extracellularmatrixrECM)componentswere

    pentachromestain. ;demonstratedbyMovat

    ;2.3PEG-hydrogelpreparationandmodified ;PEG.TEHVconstruction

    ;BranchedPEGhadalreadybeenfunctionalized ;withvinylsulfoneattheOHtermini(4?armPEGVS,

    ;MW20KDa,Sunbio,SouthKorea).VEGFandTGF-131 ;werepurchasedfromPeprotechCompanyofEngland. ;Afunctionalcellaaachmentoligopeptide ;(AcGCGYGRGDSPGNH2),containingthecell

    ;adhesionmotifRGDanda16aminoacidoligopeptide,

    ;containingamatrixmetalloproteinase(MMP)cleavage ;substratebetweencysteineresiduesatoppositeends

    GCRDGPQGIWGQDRCG-NH2)werepurchased ;(Ac

    ;fromSangonbioengineeringCo.,LtdofShanghai, ;China.Thepurifiedpeptideswereanalyzedusinga ;VoyagerEliteMALDITOFmassspectrometer

    ;(PerSeptiveBiosystems,UK).Inaccordancewith ;Hubbell,etalI]J,thepreparationofPEGhydrogels ;involveddissolving4--arm-PEG-?VSin

    ;triethanolaminebufferedsaline(TEAbuffer,0.3M, ;pH8.0,Sigma)togivea1O%(w/v)solution.The ;Applicationofmodifiedpolyethyleneglycolhydrogelsintheconstructionoftissueengine

    eredheartvalve

    ;specimenswerecoatedwithgoldpalladiumand

    ;imageswereobtainedwithaHITACHIS-3400N ;scanningelectronmicroscopeat5kV.SEMwasused ;tofurtherassessendothelialmorphology.Foranalysis, ;boththesurfacesoftheleaflets’ventricularisand

    ;spongiosalayerwereevaluated.Ninerandomvisual ;fieldsfromeachsideofspecimenwereinspectedto ;calculatetheECs’percentageofcoverageprecisely.

    ;Onlycelllayerswiththetypicalcobblestone ;characteristicswereacceptedasEClayers.Thearea ;occupiedbyECswasmeasuredandcomparedtothe ;totalimageareatoobtainanaccuratecellcoverage ;measurement.Thereportedcoveragewastheaverage ;oftheseanalyzedimagesforeachotherfromtheImage ;Pro3DS6.0software(MediaCyberneticsInc.,Sliver ;Spring,MD,USA).

    ;2.8EnergydispersiveX-rayspectrometry ;quantifiedcalciumdetermination

    ;ThecalciumcontentofPEG-TEHVandNAPAV

    ;inthegoatmodelat16weeksafterimplantationwas

    ray ;measuredandanalyzedbyenergydispersiveX

    ;spectrometer(INCAEnergysoftware1.80,EMAX, ;OxfordInstrumentsCorp.,UK),andexpressedasa ;percentageofatomicweight.

    ;2.9Mechanicalstrengthtests

    ;Themechanicalstrengthofthegroups

    ;PEGTEHVofstaticculturedfor7daysbefore ;implantation(PEG7D),PEGTEHV,andnativegoat

    ;aoaicvalve(NGAV)wasmeasured(each,n=8)atRT. ;Atensiletester(Instron4302mechanicaltester,Instron ;Corp.,USA)wasusedtoperformuniaxialstretching ;onspecimensatastrainrateof10mm/min.Tissue ;specimensofleafletswereattachedatbothendstoan ;atraumaticclampandsubjectedtounaxialtensile ;loadingtofailure(radialandcircumferential ;directions).Themaximumpointcompleteruptureof ;thespecimenswasdetermined.

    ;2.10Statistics

    ;4

    ;Datawerepresentedasmean?standarddeviation ;(SD).Statisticalcomparisonofdatawasmadeusing ;SPSSsoftware(Version13.0,Chicago,IL,USA)for ;bothaone.samplet-testandTukey’shonestly

    ;significantdifferencetest.AvalueofP<0.05was ;consideredstatisticallysignificant.

    ;3.Results

    ;3.1Cellisolation,expansionand

    ;characterization.

    ;Tento15millioncellswereobrainedfromeach ;adultgoat.Cellsattachedtocultureplatesoverafour ;tofivedayperiod,andproliferatedtoconfluence ;within10days.Cellswerepassagedat80%

    ;confluence,andexpansioncontinuedsuccessivelyin ;largercellcultureplates.Approximately25(+3)days ;(passage46)wereneededtoobtainanadequate

    ;numberofMSCs(approximately3-5xl0.cells).Early ;passagecellsstaineduniformlypositiveforSH2and ;cellsstaineduniformlynegativeforCD31.After ;initialtreatmentinadipogenicinductionmedia, ;intracellularlipidvacuolesbecameevidentbyOilRed

    ;Ostaining.Maturefibroblastcellsunderwentthesame ;protocol,butdidnotdevelopintracellularlipid ;vacuoles(Fig.1).

    ;Fig.1

    ;Notes:A:Goatbo~owMSCsconfluenceat10dafter ;primaryculture.20×:B:Immunohistochemistrystainingof ;MSCsstaineduniformlypositiveforSH2,20×;C:Afterinitial ;treatmentinadipogenicinductionmedia,intracellularlipid ;

    ;Applicationofmodifiedpolyethyleneglycolhydrogelsintheconstructionoftissueen#uee

    redheartvalve

    ;E.F:DopplersignalsindicatedthattheTEHVleafletflapped ;withthepulseofthebloodflow,andthebloodinflowand ;outflowfromthesinusofgroupPEGTEHV;G.H:Ongross

    ;examination,nomurMthrombusexistedonthesurfaceofthe ;groupPEG.TEHV(whitearrowhead)andtheleafletwasintact ;andverysmooth.

    ;InNAPAV.asparsemonolayeroffactorVIII

    ;positivecellswaspresentonthesurfaceofthegrafted ;tissue.InPEGTEHV,moreabundantcellrepopulation

    VIIIpositiveandmorphologic ;wasseen.Factor

    ;findingssupportedthatthosecellswereECs.Many ;ocSMApositivecellswerespreadthroughoutthetissue. ;Inaddition,vasculogenesis,indicatingaproper ;remodeling,wasobservedinthemiddleofthegraftin ;PEG-TEHV(Fig.4).

    ;

    ;Fig.4

    ;Notes:A:AsparingmonolayeroffactorVIIIpositive

    ;cellswaspresentonthesurfaceofthegraftedtissueinthe ;groupNAPAV(blackarrowhead),40×;B:FactorVIII ;stainingdemonstratedacontinuousmonolayerofendothelial ;cellsliningonthesurfaceofthevalveofgroupPEGTHV

    ;(blackarrowhead),40×;C:Vasculogenesiswasobservedin ;themiddleofthegraftofthegroupPEGTEHV(white

    ;arrowhead),40×;D:o【一SMApositivecellswerespread

    ;throughoutthetissueofgroupPEGTEHV,20~.

    ;3I3.3SEMexaminationandratioofreendothelia1 ;calculation

    ;SEMshowedthedifferencebetweenbothofthe

    ;groups.Fig.5Ashowedsmoothandconfluence

    ;endotheliallayerOfPEGTEHVandFig.5Bshowed

    ;theintercellularconnectivegapclearly.Fig.5Cand5D ;showedtherewerealotofcollagensdisplayedunder

;6

    ;thefragmentedmuralthrombusandafewof

    ;endothelia1cellsexistedinNAPAV.Theratioof ;reendothelialindicatedthatNAPAVwassignificantly

    TEHVandNGAVafter16 ;lowerthanthatofPEG

    ;weeks(<0.05),andtherewasasignificantdifference ;betweenthePEG-TEHVandNGAV(P<0.05)(Fig. ;6A).

    ;3.3.4Transmissionelectronmicroscope(TEM) ;examination

    ;Thecollagenwaspreservedperfectlythroughthe ;processofdecellularizationbytheexaminationof ;TEM.After16weeksimplantedinthegoatabdomen, ;TEMdemonstratedthatconsecutiveEClayersexisted ;onthesurfaceofthevalve,andthatthefibroblast ;existedundertheEClayersinPEGTEHV(Fig.5E,

    ;5F).

    ;Fig.5

    ;Notes:SEMexaminationA:Showedsmoothand

    ;confluenceendotheliallayerofgroupPEGTEHV(1,O00x);B:

    ;Showedtheintercellularconnectivegapclearlyinthegroup ;PEGTEHV(3,0o0×);c(1,000x)D(3,000×)showedthe ;collagenunderthefragmentedofmuralthrombusandthere ;wereafewofendothelialcellsexistedingroupNAPAV.TEM ;Applicationofmodifiedpolyethyleneglycolhydrogelsintheconstructionoftissueengine

    eredheartvalve

    ;examination(E,F)E:Wellarrangedcollagen

    ;acellularporcineaorticvalve(5,000x);F

    ;fiberofthe

    ;Blackarrow

    ;indicatedtheendothelialeellonthesurfaceofthevalve16 ;weeksafterimplantedingoatabdominalaortic;whitearrow ;indicatedthefibroblastcellundertheEC(5,000x). ;Fig.6

    ;Notes:A:Intheratioofreendothelia1.NAPAV ;f0.14_+-0.01)wassignificantlylowerthanthatofPEGTEHV

    ;(0.85?0.02)andNGAV(1.00)after16weeks(<O.05);there ;wasasignificantdifferencebetweenthePEGTEHVand

    ;NGAVf<0.05):B:Themaximalmechanicaltensilestrength ;wasmeasuredusinganInstron4302.PEG7Df8.77?0.39)

    ;versusPEGTEHVf12.87~1.34),P<0.001:PEG7Dversus

    ;NGAV(12.72?1.09).P<0.0ol:PEGTEHVversusNGAV,

    ;P=O.953>0.05(n=8);C:TheenergydispersiveXray

    ;spectrometershowedthattherewasasignificantdifference

    ;betweenthecalciumcontentofNAPAVrO.32~0.06)and ;NGAV(0.24_-~.05),=O.012<0.05.Therewerenosignificant ;differencebetweenthecalciumcontentofPEGTEHV

    ;(0I31?0.04)andNGAV(P=0.051>0.05)orbetween ;PEGTEHVandNAPAVrP=0.777>0.O5).

    ;3.3.5Mechanicalstrengthtest

    ;Themeanvaluesofthemaximumcomplete

    ;rupturepointwere8.77-+0-39MPainPEG7D.

    ;12.87+1.34MPainPEGTEHV.and12.72_+1.09MPa

    ;inNGAVrespectively.ThevalueofNAPAVafter16 ;weeksinvivowasuntestedbecausetheleafletshad ;beengraduallyruinedbythedepositedseveremural ;thrombus.Nosignificantdifferencewasfound ;betweenPEGTEHVandNGAVfp>0.05).Yetthere

    ;wasaverysignificantdifferenceinPEG7Dversusthe

    ;othersfP<0.OO1)(Fig.6B).

    ;3-3.6Ouantitativecalciumdetermination

    rayspectrometershowed ;TheenergydispersiveX

    ;thatthecalciumcontentofPEGTEHVafter16weeks

    ;waslowerthanthatofNAPAV.althoughtherewasno ;significantvariancebetweenthetwogroupsfP>0.05). ;However,therewasasignificantdifferencebetween ;NAPAVandNGAVfP<0.05)(Fig.6C).

    ;4.Discussion

    ;IntheTEHVresearch,itisveryimportantto

    ;understandtherecombinationoftheseeding-cellsand ;scaffoldmaterials.Theacellularscaffoldofnatural ;biomaterialscouldprovideahotbedforthe

    ;seedingcells.Thehydrogelusedinthisexperiment ;performedanimportantroleintheuniformdistribution ;ofseedingcells.TheHEstainingandSEM

    ;examinationconfirmedthisphenomenon.Thenature, ;shape,andsizeoftheporcineaorticvalvehad ;considerablecompatibilitywiththehumanheartvalve. ;Itsthreedimensional(3D)andcomplicated

    ;architecturewereperfect,andcannotbereplicatedby ;artificialmaterials.Furthermore,acquisitionsourcesof ;porcineaorticvalvesareextensive,cheap,andreadily ;7

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    ;Applicationofmodifiedpolyethyleneglycolhydrogelsintheconstructionoftissueengine

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    ;available.Thus,theseshouldbesuitableforuseasthe ;scaffoldofTEHV.Althoughtensiletesting

    ;demonstratedagradualweakeningofmechanical

    .the ;strengthwithincreaseddecellularizationtimef8-9

    ;acellularporcineaorticvalvemadeusingourmethod ;showedsimilarmechanicalstrengthtothatofthe ;nativegoataorticvalve.Inthisresearch,itiscriticalto ;notonlyknowhowtoenhancetheabilityofthe ;seedingcellstocombinetogetherwiththeacellular ;biomaterials,buttoalsotoleratetheimpulsionofthe ;systemiccirculationinvivo.Spec

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