ORIGINALARTICLEAnInvitroAnalysisofElementalReleaseandCytotoxicityofRecastNickelChromiumDentalCastingAlloysNagamRajaReddyAnandapandianPonsekarAbrahamKrishnanMurugesanVasanthakumarMatsaReceived:30December2010/Accepted:22May2011/Publishedonline:3June2011C211IndianProsthodonticSociety2011AbstractRecastingofthecastingalloysaffectsthecompositionandelementalreleasewhichmayhavecyto-toxiceffectdifferentfromthepurealloyinthesurroundingtissues.AnInvitrostudywasconductedtoinvestigatetheelementalreleaseandtheircytotoxiceffectsfromcom-merciallyavailableNiCrdentalcastingalloys,commonlyusedforfabricatingfixedpartialdentures.ThreeNiCralloysWiron99(A),Ceramet(B),andHiNickelCB(C)weretested.Alloyspecimens(disks395mm)werecastedandgroupedasfollows:GroupI(A1/B1/C1):100%purealloy；GroupII(A2/B2/C2):50%newwith50%recast；andGroupIII(A3/B3/C3):100%recast.DisksofeachalloytypefromeachgroupweretransferredtoDulbeccosmodifiedeaglemediumandleftfor3daysat37C176Cinanatmosphereof5%CO2.Ni,Cr,Co,CuandMoelementalreleasefrommetalalloysintoculturemediumwasinves-tigatedusingInductivelyCoupledPlasmaMassSpec-trometry.CytotoxicitywastestedusingmousefibroblastcellsandMTTAssay.Controlsconsistedof6wellscon-tainingcellswithnoalloyspecimens.Datawereanalyzedbytwo-wayanalysisofvariancefollowedbyt-test.ThetotalamountofelementsreleasedinpartsperbillionforvariouscastinggroupswereGroupI,A1-6.572,B1-6.732,C1-8.407；GroupII,A2-22.046,B2-26.450,C2-29.189；GroupIII,A3-84.554,B3-88.359,C3-92.264.MoreamountsofelementswerereleasedinHiNickelCBthanCerametandWiron99inallthethreetestgroups.Per-centageofviablecellsfromMTTanalysiswereGroupI,A1-62.342,B1-61.322C1-60.593,GroupII,A2-58.699,B2-56.494,C2-52.688,GroupIII,A3-53.101,B3-52.195,C3-47.586.TheviablecellspresentintheculturemediaweremoreinWiron99thanCerametandHiNickelCB.Elementalreleaseincreasedwithamountofrecastalloy.AmongstthethreealloystestedHiNickelCBhadsignif-icantlyhigherelementsreleasedcomparedtoCerametandWiron99in100%purealloys,50%recastand100%recastalloys.Wiron99showedleastelementreleasein100%purealloy,50%recastand100%recastspecimens.100%purealloysofallthreealloysarelesscytotoxic,buttheircytotoxicityismoreon50%and100%re-castedalloys.OutofallthreevariationsofcastingWiron99wasleastcytotoxic,followedbyCerametandHiNickelCB.Recastingofalloyssignificantlyincreasedtheelementsreleasedandtheircytotoxicity.KeywordsRecastingC1CytotoxicityC1ElementalreleaseC1DentalcastingalloysC1BasemetalIntroductionAdvancesinscienceandtechnologyinthefieldofdentalmaterialsciencehaveleadtotheinventionofbasemetalalloysforapplicationindentistry1.Introductionofchromealloyshasprovidedanalternativetogold,withtheseresultedinNiCrandCoCrbasedalloyswhichareClinicalImplicationsCastrestorationsplacedinclosecontactwithoraltissuesforvariousperiodsoftimemayelicitlocaladversetissuereactionssuchasgingivitisandperiodontitisadjacenttothem.Degreeofcytotoxicityandbiocompatibilityofdentalcastingalloyshasbeenrelatedtoalloycompositionandelementsreleasedfromalloysintosurroundingmedium.RecastingofNiCralloysincreasestheamountofelementsreleasedandhencethecytotoxicpotential.N.R.ReddyCKSTejaInstituteofDentalSciences,Tirupathi,AndhraPradesh,IndiaA.P.Abraham(&)C1K.MurugesanC1V.MatsaDepartmentofProsthodonticsandCrown&Bridge,SRMDentalCollege,SRMUniversity,Ramapuram,Chennai,Indiae-mail:123JIndianProsthodontSoc(Apr-June2011)11(2):106112DOI10.1007/consideredaseconomicallylessexpensiveandalsohavetherequiredqualitiesandbiocompatibilityforusageindentalrestorativework2,3.Basemetalalloysarepronetovarioustypesofcorrosiondependingonitscompositionandoralenvironment1.Thereleaseofmetallicelementsfromdentalalloysisapotentialhealthproblemtodentalpatient4.Metalsareknowntocausetoxicinflammatoryallergicormutagenicreactions.Importantconsequenceofelementalreleaseiscytotoxicityofadjacenttissuesincellcultures5,6.Castrestorationsbeingplacedinclosecontactwithoraltissuesforvariousperiodsoftimemayelicitlocaladversetissuereactionssuchasgingivitisandperiodontitisadjacenttothem.Degreeofcytotoxicityandbiocompatibilityofdentalcastingalloyshasbeenrelatedtoalloycompositionandelementsreleasedfromalloysintosurroundingmedium7.Recastingofusedalloysintheformofspruesordefectivecastinghasbeenpracticedtopreventwastageofmaterialaftercasting3.Identificationoftheelementsandtheirconcentrationsresponsibleforcytotoxiceffectsisimportantbecauseitwillhelpinimprovinganddesigningneweralloystoavoidthereleaseofelementswhichareharmfultooralcavity.Hypothesisofthestudywasthatrecastingofbasemetalalloyswouldchangethechemicalpropertiesofthealloysandthusaffecttheirelementalreleaseandsubsequentlyelicitcytotoxiceffects.TheaimofthisinvitrostudywastoinvestigatetheeffectsofrecastingofNiCrbasemetalalloysandtheirpotentialcytotoxiceffectsandelementalreleaseintoculturemedia.ObjectivesofstudyweretoassesstheelementalreleaseandcytotoxicityofNiCralloysandcorrelateelementalreleasewithcytotoxicityofpurealloy(GroupI),50%pureand50%re-casted(GroupII)and100%re-casted(GroupIII)alloy.MaterialsandMethodsTheinvitrostudywasundertakenwithNiCralloyformetalceramicfromthreedifferentmanufacturers(Fig.1；Table1).Alloyshavebeencodedforsimplicityandref-erencepurposes.PreparationofAlloySpecimensAnaluminiummetaldiewithacircularspaceof5mmradiusand3mmdepthwasmilledinthecentreportionof1cmthickaluminiumblockusingcomputerizedmillingmachine(Fig.2).Waxpatterns10mmindiameter93mmthicknesswerepreparedwithgreeninlaycastingwax(Fig.3)(Bego-Germany)usingthemetaldie.Atotalof108waxpatternsweremadeforthreealloysforGroupI:100%purealloy(A1/B1/C1),GroupII:50%pure50%recastalloy(A2/B2/C2),andGroupIII:100%re-castedalloy(A3/B3/C3).CastingofthePatternThewaxpatternsofthethreegroupsweresegregatedasgiveninTable2.Thespruedwaxpatternswereinvestedinphosphatebondedinvestmentinmetalcastingringwithdrycellulosepaperringliner.Followingthistheywerecastedbylostwaxtechniqueinaninductioncastingmachine(Fig.4)(Bego-Germany)withtherespectivealloys.MetalsusedforrecastingwasobtainedfromcleanedleftoverspruesandbuttonsofthealloysthathadbeenpreviouslycastingroupI.Afterbenchcoolingthecastingringwasdivestedandsandblastedusing250lmaluminiumoxidetoremovetheremnantinvestmentmaterial.Sprueswerecutoffandspecimenswerefinishedandpolishedusingcarborundumdiscs,metaltrimmers,rubberwheels,sandpapersandpolishingcakeusinghandmotorinstruments.Thepolisheddisksweresoakedinadetergentsolutionfor5minandthenscrubbedusingasoftbristlebrushandrinsedundertapwaterfor5min.Speci-menswerethenreplacedinsteriledistilledwaterandcleanedbysonificationandautoclavedatatemperatureof150C176Cfor60min.ElementalReleaseSixdisksofeachalloytypefromeachgroupweretrans-ferredto6mlofDulbeccosmodifiedeaglemedium(DMEM)andincubatedfor3daysat37C176Cinanatmo-sphereof5%CO2.DMEMispreparedbysupplementingwith5%newborncalfserum,100U/mlofPenicillin,Streptomycinand0.25lgm/lAmphotericinB(Fig.5).ThemediawasanalyzedforpresenceofNi,Cr,Mo,Co,andCuusinganInductivelyCoupledPlasmaMassSpec-trometry(ICPMS).MediumwasdilutedwithdeionizedwaterandkeptinICPMSandthereadingswererecorded.Triplicateabsorbancewasusedtodeterminethemeanconcentrationofdifferentelementsinpartsperbillion(ppb)releasedfromalloys.Fig.1NiCralloysformetalceramicJIndianProsthodontSoc(Apr-June2011)11(2):106112107123CellCultureandCytotoxicityTestForCytotoxicitytest,theremaining6disksfromvariouscastingalloysinthreegroupswereplacedincenterofa24welltissuecultureplateand0.5mlofmousefibroblastcellsuspensionwasaddedtocellwell(Fig.6)andwereleftfor2h.Followingthis0.5mlofDMEMwasaddedandcellswereincubatedfor3daysat37C176Cinanatmosphereof5%CO2.Thecontrolsconsistedof6wellscontainingcellswithnoalloyspecimens.AftertheincubationperiodtheplateswereremovedfromincubatorandcytotoxicityofTable1DescriptionofNiCrdentalcastalloys*CompositionasprovidedbymanufacturerSerialNo.TradenameManufacturerComposition(wt%)*Code1Wiron99Bego(Germany)Nickel-65AChromium-22.5Molybdenum-9.5Niobium-1Silica-1Iron-0.5Cesium-0.52CerametLaboline(Europe)Nickel-62BChromium-26Molybdenum-10Silica-1.5Others-0.53HiNickelCBHikari(Japan)Nickel-60CChromium-10Copper-15Manganese-7.9Others-7.1Fig.2MilleddieFig.3WaxpatternsFig.4CastspecimensTable2DistributionofspecimensGroupTypeNo.ofalloyspecimen(A)Wiron99(B)Ceramet(C)HiNickelCBI100%New12A112B112C1II50%New50%recast12A212B212C2III100%Recast12A312B312C3108JIndianProsthodontSoc(Apr-June2011)11(2):106112123materialswasassessedbymeasuringSuccinicDehydro-genaseactivity(SDH)ofthecellsbyusingMTTassay.MTTreagentisaddedtothecultureplatewithalloyspecimensanditistransferredto96wellplatesforread-ingsinEnzymeLinkedImmunosorbentAssay(ELISA)reader,theprotocolfollowedfortheMTTAssayissum-marizedinTable3.Thereadingswerecalculatedbytheformula:%CellViabilityMeanabsorbanceofsampleC2100Meanabsorbanceofcontrol:StatisticalAnalysisTheresultsobtainedfromtheMTTanalysisandICPMSanalysiswassubjectedtostatisticalanalysis.OnewayANOVAwasusedtocalculatetheP-value.MultiplerangetestsbyTukeyHSDprocedurewereemployedtoidentifythesignificantgroupsat5%level.ResultsElementalReleaseElementalreleasewasseeninallthreecastingalloygroups.ElementsreleasedwereNi,Cr,Co,CuandMo.Tables4,and5summarizetheelementalreleasebydif-ferentcastedalloysinthreecastingprocedures.HiNickelCBhadthehighestamountofelementalreleaseamongthethreecastingalloysfollowedbyCerametandWiron99.Elementalreleasesignificantlyincreasedwiththeper-centageofrecastmaterialusedinforallelementsanalyzedinthestudywithP0.05(Graph1).CytotoxicityResultsThepercentageofviablecellsinthreegroupsistabu-latedinTable6.MeanpercentagesofcellactivityofthreebasemetalalloysforthreecastingconditionswereanalyzedandcytotoxicitywasobservedtobemoreinHiNickelCB,followedbyCerametandWiron99.Resultsshowedthat100%recastalloyshad