2212-8271 - PDF document

2212-8271© 2013 The Authors. Published b
2212-8271© 2013 The Authors. Published by Elsevier B.V.Selection and/or peer-review under responsibility of Professor Mamoru Mitsuishi and Professor Paulo Bartolodoi: 10.1016/j.procir.2013.01.044 Procedia CIRP 5 ( 2013 ) 222 – 225 TheFirstCIRPConferenceonBiomanufacturingTheeffectofatypeIphotoinitiatoroncurekineticsandcelltoxicityinprojection-microstereolithographyR.Baila*,A.Patelb,H.Yanga,C.M.Rogersb,F.R.A.J.Roseb,J.I.Segala,S.M.RatchevaaManufacturingResearchDivision,FacultyofEngineering,UniversityofNottingham,UniversityPark,NottinghamNG72RD,U.K.bWolfsonCentreforStemCells,TissueEngineeringandModelling(STEM),CentreforBiomolecularSciences,SchoolofPharmacy,UniversityPark,NottinghamNG72RD,U.K.*Correspondingauthor.Tel.:+44-(0)115-956-6083;fax:+44-(0)115-951-3800;E-mailaddress:epxrb2@nottingham.ac.ukAbstractProjection-microstereolithographyisanadditivemanufacturingtechniquebasedonthespatiallycontrolledsolidificationofaliquidphotopolymeronexposuretodigitallymanipulatedlightpatterns.Thisstudypresentsamethodologytoevaluatetheeffectofatype-Iphotoinitiatoronthereactionkineticsintheprocessandthecytocompatibilityoftheproducedcomponents.Whilethereactionspeedanddegreeofconversionwereheavilydependentontheappliedamountoftheinitiator,acleartoxiceffectwasobservedwithalltestedconcentrations,andapost-processingstepof7dayswasrequiredtoleachouttheinitiatorresidues.©2012TheAuthors.PublishedbyElsevierB.V.Selectionand/orpeer-reviewunderresponsibilityofProfessorMamoruMitsuishiandProfessorPauloBartoloKeywords:Microstereolithography;photopolymerization;photoinitiator;BAPO;FTIR;AlamarBlueassay1.IntroductionStereolithographyisaprocessinitiallyappliedinrapidprototypingandbasedonthespatiallycontrolledsolidificationofaliquidandlight-sensitivepolymer(resin)uponinteractionwithalightsource(photo-polymerization).Projection-microstereolithography(PMSL)appliesapatterngenerator,usuallyanLCDoraDigitalMicromirrordevice(DMD),todigitallymodulatealaserorlightbeamaccordingtolayerprofilesdeterminedfromanSTLfile[1].Onceanarrayoflightspots(pixels)isprojectedontotheresinsurface,thelightpropagateswithineachilluminatedvoxelinthedirectionofthebeamwhereitprovidestheenergythatleadstosimultaneoussolidificationintheirradiatedpixels.Thiscuringprocessisrepeatedlayerbylayer.Thesolidificationofastereolithographicresininahighlycrosslinked3Dpolymernetworkresultsfromadistinctivecuringmechanismthatinvolvesthesuccessivegelationandvitrificationofthephoto-polymer.Photopolymerizationreactionscanbeinitiatedbyfreeradicals,inwhichcaseoligomerswithphoto-crosslinkablegroupsarerequiredaswellasfreeradicalsthataregeneratedbyaphotoinitiator(PI)whenirradiatedwithlight.Bisacylphosphineoxide(BAPO)derivativesbelongtotheNorrishtypeIinitiatorsthatundergoalpha-cleavageofacarbonylorcarbonyl-phosphinoylbondwhenirradiatedwithlightandthatexhibitabsorbancemaximabetween365and416nm,dependingontheirexactchemicalcomposition[2].characterizethecrosslinkingofmonomersinathechemicalstructureandchainlengthofthemonomer[3],itisalsoinfluencedbytheappliedPIandlightalmost80%canbeachievedinsetupsusingaquartz-tungstenhalogenlamp(380-500nm)andaviolet-LEDlightsource(390-430nm)[4].Anotherrequirementwithbiomedicalcomponentsisthebiocompatibilityofthephotoinitiatorattheappliedconcentration.BAPOistoleratedinconcentrationsofupto0.5wt%byosteoblast-likecells[5],andupto1.0Available online at www.sciencedirect.com© 2013 The Authors. Published by Elsevier B.V.Selection and/or peer-review under responsibility of Professor Mamoru Mitsuishi

and Professor Paulo BartoloOpen access
and Professor Paulo BartoloOpen access under CC BY-NC-ND license.Open access under CC BY-NC-ND license. R. Bail et al. / Procedia CIRP 5 ( 2013 ) 222 – 225 wt%byMC3T3pre-osteoblastsonaphotopolymerscaffold[6].Withothercelllineshowever,thetolerancelevelforBAPOcanbemuchlower[7].Projection-microstereolithographyisapowerfultechniquetomanufacturemicrostructureswithcomplexgeometriesforbiomedicalapplications.Akeychallenge,however,istoidentifyPIcompositionsandtheoptimumconcentrationswhereasufficientcuringspeedcanbeachievedwithoutcompromisingbiocompatibility.Thisstudysuggestsasystematicmethodologytospecificallyevaluatetheconcentration-dependentperformanceofatypeIphotoinitiatorinaPMSLprocessregardingthecuringspeed,thedegreeofconversionandthetoxiceffectswithinacell-containingenvironment.2.MaterialsandMethods2.1.MaterialpreparationPhenylbis(2,4,6-trimethylbenzoyl)phosphineoxideservedasphotoinitiatordissolvedinPoly(ethyleneglycol)-dimethacrylate(PEGDMA),M=750g/mol,atroomtemperatureandintermediatestirringratefor5hours.Fourdifferentphotopolymermixturescontaining0.1,0.25,0.5and1.0wt%initiatorwerepreparedinseparateunitsandusedinthekineticsandtoxicityassessments.PEGDMAandinitiatorwerepurchasedfromSIGMAAldrichandusedasreceived.Cell-culturemediawasmadeusingDulbecco’smodifiedeagle’smedium(DMEM)andfetalcalfserum(FCS)at10%v/v.Atransformedcelllineofgreenfluorescentprotein(GFP)MouseEmbryonicFibroblastNIH3T3cells(ECACC)wereculturedinDMEMmedia.When80-90%confluent,cellsweredetachedfromtheculturesurfacewith0.25%()trypsin0.02%)EDTAinPBS.Cellswerecentrifuged(250)for5minutes,resuspendedinDMEMmediumandsplit1in5.Theculturemediawaschangedevery3daysandkeptina37C,5%COhumidifiedincubator.AnAlamarBlue®assaysolutionwasproducedbydilutinginHanksBalancedSaltsSolution(HBSS)inaoftheassaysolutionwaspipettedintoeachwellandcontrolplatethatwerelefttoincubate(37°C,5%CO90min).Then100lofthesolutionwastakenfromeachwellintriplicateandplacedintoa96wellplatetomeasurefluorescenceat570nminaTecanmicroplatereader.Thesolutionwasreplacedwith1mlDMEM,andtheassaywasrepeatedatday1,2,3,6and7.2.2.KineticassessmentTheinfraredabsorbancespectraintheuncuredandcuredresinweremonitoredusingaBrukerTensor27®FourierTransformInfrared(FTIR)spectrophotometer.Athinfilmcellwasmadebysandwichingtworound25mmsodiumchloride(NaCl)crystalwindowsinaPresslok®cellholderlockedinpositionwithalid.Absorptionspectrawererecordedfrom2,000towavenumberataresolutionof4cm,andchangeswereassessedinthe1,700to1,550cmregion.AnEnvisiontecPerfactory®Mini-MultiLensprojection-microstereolithographymachineemittingvisiblebluelightof380-480nmwavelengthservedastheirradiationsource.TheprojectorbrightnessofthePMSLsystemwasadjustedtoalightintensityof600mW/dm,whichcorrespondedto500mW/dmwhenprojectedthroughoneNaCldiskhorizontallyplacedonthecalibrationplateofthemachine.Thelightbeamwasrecalledviathe‘Showcompensationmask’functionandexposuretimes(t)of5,10,20,30and60secondsweremanuallycontrolledwithastopwatch.Thefinalexposurewascarriedoutwith2000flashesinaG171Otoflash®UV-flashpostcuringdevice.AninfraredabsorbancereadingforasingleandcleanNaCldiscwasusedasthebackgroundreading,A.ThethinfilmcellwasloadedwiththephotopolymertogeneratetheIRreadingsfortheuncuredmaterial,andafterexposureinthePMSLmachineforthe(partly)calculatedassignedtotheC=Cstretchingvibrationsat1637cmforeachexposureaccordingtoequation(1):)([])([1)(000AtAAtAtCCiCCiCCWhere,AC=C)quantifiestheIRabsorptionpeakat1637cmintheuncuredmaterial,AC=C)istheabsorptioninthecuredmaterialafteranexposuretimetandAisthebackgroundnoise.2.3.BiocompatibilityassessmentSquarechipsmeasurin

g10x10x2mmweremodeledinCADandmadeinsetso
g10x10x2mmweremodeledinCADandmadeinsetsof3ontheabovePMSLmachine(500mW/dm,20sec)intheresinatthefourinitiatorconcentrations.Thepartswererinsedforthreeminutesinanultrasonicbathwithisopropylalcohol,airdried,sterilizedunderUVlight,andthentheresinchips(n=3)wereplacedintwo12wellplatesin1mlofDMEMandleftinthemediafor24hours.Cellswereseededataconcentrationof2.5x10cellsperwell,centrifuged,thesupernatantwasaspiratedoffandthecellpelletswereplacedina24wellplate.700ofthesupernatantinthechip-containingwell(n=2)wasaddedtothecorrespondingpellet-containingwell.Afurther700lfromthethirdchipwasaddedtoanemptywell.Fig.1showsthepositivecontrolcontaining2.5xcellsin1mlmedia(n=3)andthenegativecontrolcontaining1mlofpurecell-culturemedia(n=3).Eachresinchipwasthenre-suspendedin1mlmedia.Astandardcurveofcellcountversusfluorescenceat590nmwasproducedfordifferentcellcountsfrom30x R. Bail et al. / Procedia CIRP 5 ( 2013 ) 222 – 225 cellsto12.5x10cells(n=3).Toeachoftheinitiatorandthestandard-curvewells,1mlofAlamarBluesolutionwasadded.Thewellplateswerelefttoincubateandafluorescencereadingat590nmwasrun.Theremaining300loneachchipwascollectedandfrozenforlateruseinthereleasestudy.Fig.1.SchematicdiagramofthebiocompatibilityassessmentbasedonsupernatantcollectedfromresinchipscontainingdifferentBAPOconcentrations(0.1,0.25,0.5and1wt%)setupina24wellplate.ThewavelengthpeakandastandardcurveofabsorbanceatthepeakwavelengthversusBAPOconcentrationinDMEMwasgeneratedinanabsorbancescanat300-550nm.ToquantitativelyassessthePIreleasefromthechips,the300-lsamplestakenfromtheresinchipsatday1,2,3,6,7,8,9,10,13and14werethawed.100lofeachsamplewasplacedina96wellplate(n=3)foreachdayandconcentration.Anabsorbancescanwasperformedatthepeakwavelengthandagraphwasplottedtodeterminehowmuchofthephotoinitiatorhadbeenreleased.3.Results3.1.KineticcharacterizationTheinfraredabsorbancereadingsweretakenfortheuncuredmaterialandaftereachexposuretolight,andthiswasrepeatedlycarriedoutforthefourtestedinitiatorconcentrations.Alargenumberofpeakswasobservedinthe‘FingerprintRegion’between1,500and1,000cmthatcanbeassignedtovibrationsofthewholemolecularskeletonandtheP=Ostretchinthephotoinitiatormolecule.Irrespectiveofthat,thepeakofinterestat1,637cmwasclearlyvisibleandhighlyresponsivetoexposuretothelightbeamofthePMSLmachine.ThepeakheightsdecreasedwithincreasingexposuretimesasshowninFig.2,whiletheextendofthischangestronglydependedonthephotoinitiatorcontent.AlthoughtheinitialpeakheightslightlyvariedwiththematerialvolumeappliedbetweentheNaCldiscsforthedifferentmaterialsets,thisdidnotaffectthecalculatedinrelationtotheinitialpeakheight.Fig.2.FTIRdataforthePEG-basedphotoresincontaining0.5wt%BAPOphotoinitiator,revealingagradualchangeintheIR-absorbancepeakat1,637cmwithincreasingexposuretothelightsourceintheprojection-microstereolithographymachineusedascuringunit.increasingexposuretimesfordifferentBAPO-initiatorconcentrationsinthePEG-basedphotopolymermatrix.Withallfourinitiatorconcentrations,characteristickineticprofilesofaphoto-polymerizationreactionappearedalthoughthesensitivityofthecrosslinkingprocesswashighlydependentontheBAPOcontent.Largedifferenceswereobservedinthekineticratesofthechemicalcontrolledstage,thepositionoftheonsetpoints,andthemaximumconversion.ThemostBAPOconcentrationof1.0wt%,whilealethargicreactionwasrecordedforthelowestconcentrationof0.1wt%.Withalltestedmaterials,theonsetpointswherethechemically-controlledstagepassesintoamuchidentifiable.TheonsettimesincreasedwithincreasingBAPOcontentasshowninFig.3.Bycontrast,thedegreeofcrosslinkingintheonsetpointandthe R. Bail et al. / Procedia CI

RP 5 ( 2013 ) 222 – 225 maximumdegr
RP 5 ( 2013 ) 222 – 225 maximumdegreeofconversiondecreasedwithhigherinitiatorcontents,exceptforthelowestconcentration.mixcontaining0.25%initiator,whereastheresincompositionswiththehighest(1.0wt%)andthelowest(0.1wt%)initiatorcontentsyieldedvaluesbelow70%.3.2.ToxicityandreleaseWhilethecellcountonthetissuecultureplasticshowedasteadyincreaseoverthe7days,adecliningnumberoflivingcellsandlowcellviabilitywithculturetimewereobservedinthePI-containingwells(Fig.4).ThisindicatedaconsiderabletoxiceffectoftheBAPOinitiatoratalltestedconcentrationsontheusedcells.AlthoughtheviablecellcountincreasedoverthefirstthreedaysforthelowestBAPOconcentrationof0.1wt%andthiswasalsothemaximumpeakcomparedtothehigherconcentrations,itdeclinedbackatday7.Forthe0.25wt%and0.5wt%resinchips,thenumberofproliferatingcellspeakedatday2andthengraduallydecreased.TheAlamarBlueassayfurtherconfirmedthattherewasnoconsiderablenumberofviablecellsthroughouttheexperimentfortheresinchipscontaining1wt%initiator.Theopticalinspectionofthecellsattheendofday7showednocellsunderfluorescenceforallBAPOconcentrationsandonlycelldebrisremained.Fig.4.NumberofviablecellsontissuecultureplasticandforcomparisonatdifferentBAPOconcentrationsintheresinchipsoveraperiodof7days.Datapresentedasamean+/-SEM(n=6);absolutecellcountsindicatedforpurecellcultureand0.10wt%photoinitiator.TheweightreleaseofthePIfromtheswollenchipswasmeasuredviatheabsorbanceat400nminthesupernatant.Thedataindicatedahysteresiscurvewithlowreleaseratesoverthefirst4days,areleasepeakforallconcentrationsonday7,followedbylowreleaseratesafterthat(Fig.5).Thehighestreleasewasobservedfromthe1wt%resinchips,closelyfollowedbythe0.5and0.25wt%chips.Asub-peakintheleachingrateat1wt%BAPOoccurredonday2,whilethiswasnotobservedfortheotherchips.Fig.5.Cumulativephotoinitiatorrelease(weight%)fordifferentBAPOconcentrations,datapresentedasamean+/-SEM(n=6).4.ConclusionsTheresultsdemonstratetheefficiencyofBAPOphotoinitiatorsintheprocessbutatoxiceffectattherequiredconcentrationsonMouseEmbryonicFibroblastNIH3T3cells,suggestingthefurtherinvestigationoftypeIphotoinitiatorswithdifferentcelltypes.AcknowledgementsTheauthorsaregratefulforthetechnicalsupportprovidedbytheEPSRCCentreforInnovativeManufacturinginRegenerativeMedicineandthePrecisionManufacturingCentreinNottingham.[1]Sun,C.,Fang,N.,Wu,D.M.,Zhang,X.,2005.Projectionmicro-stereolithographyusingdigitalmicro-mirrordynamicmask,SensorsandActuators,A:Physical121,p.113.[2]Ikemura,K.,Ichizawa,K.,Yoshida,M.,Ito,S.,Endo,T.,2008.UV-VISspectraandphotoinitiationbehaviorsofacylphosphineoxideandbisacylphosphineoxidederivativesinunfilled,light-cureddentalresins,DentalMaterialsJournal27,p.765.[3]Sideridou,I.,Tserki,V.,Papanastasiou,G.,2002.Effectofchemicalstructureondegreeofconversioninlight-cureddimethacrylate-baseddentalresins,Biomaterials23,p.1819.[4]Arikawa,H.,Takahashi,H.,Kanie,T.,Ban,S.,2009.Effectofvariousvisiblelightphotoinitiatorsonthepolymerizationoflight-activatedresins,DentalMaterialsJournal28,p.454.[5]Schuster,M.,Turecek,C.,Kaiser,B.,Stampfl,J.,Liska,R.,Varga,F.,2007.EvaluationofbiocompatiblephotopolymersI:Photoreactivityandmechanicalpropertiesofreactivediluents.JournalofMacromolecularScience,PartA44,p.547.[6]Lee,J.W.,Ahn,G.,Kim,D.S.,Cho,D.W.,2009.Developmentofnano-andmicroscalecomposite3DscaffoldsusingPPF/DEF-HAandmicro-stereolithography,MicroelectronicEngineering86,p.1465.[7]Zhang,H.,Wang,L.,Song,L.,Niu,G.,Cao,H.,Wang,G.etal.,2011.Controllablepropertiesandmicrostructureofhydrogelsbasedoncrosslinkedpoly(ethyleneglycol)diacrylateswithdifferentmolecularweights.JournalofAppliedPolymerScience121,p.531.