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ORIGINALARTICLEANINVITROANALYSISOFELEMENTALRELEASEANDCYTOTOXICITYOFRECASTNICKELCHROMIUMDENTALCASTINGALLOYSNAGAMRAJAREDDYANANDAPANDIANPONSEKARABRAHAMKRISHNANMURUGESANVASANTHAKUMARMATSARECEIVED30DECEMBER2010/ACCEPTED22MAY2011/PUBLISHEDONLINE3JUNE2011C211INDIANPROSTHODONTICSOCIETY2011ABSTRACTRECASTINGOFTHECASTINGALLOYSAFFECTSTHECOMPOSITIONANDELEMENTALRELEASEWHICHMAYHAVECYTOTOXICEFFECTDIFFERENTFROMTHEPUREALLOYINTHESURROUNDINGTISSUESANINVITROSTUDYWASCONDUCTEDTOINVESTIGATETHEELEMENTALRELEASEANDTHEIRCYTOTOXICEFFECTSFROMCOMMERCIALLYAVAILABLENICRDENTALCASTINGALLOYS,COMMONLYUSEDFORFABRICATINGFIXEDPARTIALDENTURESTHREENICRALLOYSWIRON99A,CERAMETB,ANDHINICKELCBCWERETESTEDALLOYSPECIMENSDISKS395MMWERECASTEDANDGROUPEDASFOLLOWSGROUPIA1/B1/C1100PUREALLOYGROUPIIA2/B2/C250NEWWITH50RECASTANDGROUPIIIA3/B3/C3100RECASTDISKSOFEACHALLOYTYPEFROMEACHGROUPWERETRANSFERREDTODULBECCOSMODIFIEDEAGLEMEDIUMANDLEFTFOR3DAYSAT37C176CINANATMOSPHEREOF5CO2NI,CR,CO,CUANDMOELEMENTALRELEASEFROMMETALALLOYSINTOCULTUREMEDIUMWASINVESTIGATEDUSINGINDUCTIVELYCOUPLEDPLASMAMASSSPECTROMETRYCYTOTOXICITYWASTESTEDUSINGMOUSEFIBROBLASTCELLSANDMTTASSAYCONTROLSCONSISTEDOF6WELLSCONTAININGCELLSWITHNOALLOYSPECIMENSDATAWEREANALYZEDBYTWOWAYANALYSISOFVARIANCEFOLLOWEDBYTTESTTHETOTALAMOUNTOFELEMENTSRELEASEDINPARTSPERBILLIONFORVARIOUSCASTINGGROUPSWEREGROUPI,A16572,B16732,C18407GROUPII,A222046,B226450,C229189GROUPIII,A384554,B388359,C392264MOREAMOUNTSOFELEMENTSWERERELEASEDINHINICKELCBTHANCERAMETANDWIRON99INALLTHETHREETESTGROUPSPERCENTAGEOFVIABLECELLSFROMMTTANALYSISWEREGROUPI,A162342,B161322C160593,GROUPII,A258699,B256494,C252688,GROUPIII,A353101,B352195,C347586THEVIABLECELLSPRESENTINTHECULTUREMEDIAWEREMOREINWIRON99THANCERAMETANDHINICKELCBELEMENTALRELEASEINCREASEDWITHAMOUNTOFRECASTALLOYAMONGSTTHETHREEALLOYSTESTEDHINICKELCBHADSIGNIFICANTLYHIGHERELEMENTSRELEASEDCOMPAREDTOCERAMETANDWIRON99IN100PUREALLOYS,50RECASTAND100RECASTALLOYSWIRON99SHOWEDLEASTELEMENTRELEASEIN100PUREALLOY,50RECASTAND100RECASTSPECIMENS100PUREALLOYSOFALLTHREEALLOYSARELESSCYTOTOXIC,BUTTHEIRCYTOTOXICITYISMOREON50AND100RECASTEDALLOYSOUTOFALLTHREEVARIATIONSOFCASTINGWIRON99WASLEASTCYTOTOXIC,FOLLOWEDBYCERAMETANDHINICKELCBRECASTINGOFALLOYSSIGNIFICANTLYINCREASEDTHEELEMENTSRELEASEDANDTHEIRCYTOTOXICITYKEYWORDSRECASTINGC1CYTOTOXICITYC1ELEMENTALRELEASEC1DENTALCASTINGALLOYSC1BASEMETALINTRODUCTIONADVANCESINSCIENCEANDTECHNOLOGYINTHEFIELDOFDENTALMATERIALSCIENCEHAVELEADTOTHEINVENTIONOFBASEMETALALLOYSFORAPPLICATIONINDENTISTRY1INTRODUCTIONOFCHROMEALLOYSHASPROVIDEDANALTERNATIVETOGOLD,WITHTHESERESULTEDINNICRANDCOCRBASEDALLOYSWHICHARECLINICALIMPLICATIONSCASTRESTORATIONSPLACEDINCLOSECONTACTWITHORALTISSUESFORVARIOUSPERIODSOFTIMEMAYELICITLOCALADVERSETISSUEREACTIONSSUCHASGINGIVITISANDPERIODONTITISADJACENTTOTHEMDEGREEOFCYTOTOXICITYANDBIOCOMPATIBILITYOFDENTALCASTINGALLOYSHASBEENRELATEDTOALLOYCOMPOSITIONANDELEMENTSRELEASEDFROMALLOYSINTOSURROUNDINGMEDIUMRECASTINGOFNICRALLOYSINCREASESTHEAMOUNTOFELEMENTSRELEASEDANDHENCETHECYTOTOXICPOTENTIALNRREDDYCKSTEJAINSTITUTEOFDENTALSCIENCES,TIRUPATHI,ANDHRAPRADESH,INDIAAPABRAHAMTABLE1ALLOYSHAVEBEENCODEDFORSIMPLICITYANDREFERENCEPURPOSESPREPARATIONOFALLOYSPECIMENSANALUMINIUMMETALDIEWITHACIRCULARSPACEOF5MMRADIUSAND3MMDEPTHWASMILLEDINTHECENTREPORTIONOF1CMTHICKALUMINIUMBLOCKUSINGCOMPUTERIZEDMILLINGMACHINEFIG2WAXPATTERNS10MMINDIAMETER93MMTHICKNESSWEREPREPAREDWITHGREENINLAYCASTINGWAXFIG3BEGOGERMANYUSINGTHEMETALDIEATOTALOF108WAXPATTERNSWEREMADEFORTHREEALLOYSFORGROUPI100PUREALLOYA1/B1/C1,GROUPII50PURE50RECASTALLOYA2/B2/C2,ANDGROUPIII100RECASTEDALLOYA3/B3/C3CASTINGOFTHEPATTERNTHEWAXPATTERNSOFTHETHREEGROUPSWERESEGREGATEDASGIVENINTABLE2THESPRUEDWAXPATTERNSWEREINVESTEDINPHOSPHATEBONDEDINVESTMENTINMETALCASTINGRINGWITHDRYCELLULOSEPAPERRINGLINERFOLLOWINGTHISTHEYWERECASTEDBYLOSTWAXTECHNIQUEINANINDUCTIONCASTINGMACHINEFIG4BEGOGERMANYWITHTHERESPECTIVEALLOYSMETALSUSEDFORRECASTINGWASOBTAINEDFROMCLEANEDLEFTOVERSPRUESANDBUTTONSOFTHEALLOYSTHATHADBEENPREVIOUSLYCASTINGROUPIAFTERBENCHCOOLINGTHECASTINGRINGWASDIVESTEDANDSANDBLASTEDUSING250LMALUMINIUMOXIDETOREMOVETHEREMNANTINVESTMENTMATERIALSPRUESWERECUTOFFANDSPECIMENSWEREFINISHEDANDPOLISHEDUSINGCARBORUNDUMDISCS,METALTRIMMERS,RUBBERWHEELS,SANDPAPERSANDPOLISHINGCAKEUSINGHANDMOTORINSTRUMENTSTHEPOLISHEDDISKSWERESOAKEDINADETERGENTSOLUTIONFOR5MINANDTHENSCRUBBEDUSINGASOFTBRISTLEBRUSHANDRINSEDUNDERTAPWATERFOR5MINSPECIMENSWERETHENREPLACEDINSTERILEDISTILLEDWATERANDCLEANEDBYSONIFICATIONANDAUTOCLAVEDATATEMPERATUREOF150C176CFOR60MINELEMENTALRELEASESIXDISKSOFEACHALLOYTYPEFROMEACHGROUPWERETRANSFERREDTO6MLOFDULBECCOSMODIFIEDEAGLEMEDIUMDMEMANDINCUBATEDFOR3DAYSAT37C176CINANATMOSPHEREOF5CO2DMEMISPREPAREDBYSUPPLEMENTINGWITH5NEWBORNCALFSERUM,100U/MLOFPENICILLIN,STREPTOMYCINAND025LGM/LAMPHOTERICINBFIG5THEMEDIAWASANALYZEDFORPRESENCEOFNI,CR,MO,CO,ANDCUUSINGANINDUCTIVELYCOUPLEDPLASMAMASSSPECTROMETRYICPMSMEDIUMWASDILUTEDWITHDEIONIZEDWATERANDKEPTINICPMSANDTHEREADINGSWERERECORDEDTRIPLICATEABSORBANCEWASUSEDTODETERMINETHEMEANCONCENTRATIONOFDIFFERENTELEMENTSINPARTSPERBILLIONPPBRELEASEDFROMALLOYSFIG1NICRALLOYSFORMETALCERAMICJINDIANPROSTHODONTSOCAPRJUNE2011112106112107123CELLCULTUREANDCYTOTOXICITYTESTFORCYTOTOXICITYTEST,THEREMAINING6DISKSFROMVARIOUSCASTINGALLOYSINTHREEGROUPSWEREPLACEDINCENTEROFA24WELLTISSUECULTUREPLATEAND05MLOFMOUSEFIBROBLASTCELLSUSPENSIONWASADDEDTOCELLWELLFIG6ANDWERELEFTFOR2HFOLLOWINGTHIS05MLOFDMEMWASADDEDANDCELLSWEREINCUBATEDFOR3DAYSAT37C176CINANATMOSPHEREOF5CO2THECONTROLSCONSISTEDOF6WELLSCONTAININGCELLSWITHNOALLOYSPECIMENSAFTERTHEINCUBATIONPERIODTHEPLATESWEREREMOVEDFROMINCUBATORANDCYTOTOXICITYOFTABLE1DESCRIPTIONOFNICRDENTALCASTALLOYSCOMPOSITIONASPROVIDEDBYMANUFACTURERSERIALNOTRADENAMEMANUFACTURERCOMPOSITIONWTCODE1WIRON99BEGOGERMANYNICKEL65ACHROMIUM225MOLYBDENUM95NIOBIUM1SILICA1IRON05CESIUM052CERAMETLABOLINEEUROPENICKEL62BCHROMIUM26MOLYBDENUM10SILICA15OTHERS053HINICKELCBHIKARIJAPANNICKEL60CCHROMIUM10COPPER15MANGANESE79OTHERS71FIG2MILLEDDIEFIG3WAXPATTERNSFIG4CASTSPECIMENSTABLE2DISTRIBUTIONOFSPECIMENSGROUPTYPENOOFALLOYSPECIMENAWIRON99BCERAMETCHINICKELCBI100NEW12A112B112C1II50NEW50RECAST12A212B212C2III100RECAST12A312B312C3108JINDIANPROSTHODONTSOCAPRJUNE2011112106112123MATERIALSWASASSESSEDBYMEASURINGSUCCINICDEHYDROGENASEACTIVITYSDHOFTHECELLSBYUSINGMTTASSAYMTTREAGENTISADDEDTOTHECULTUREPLATEWITHALLOYSPECIMENSANDITISTRANSFERREDTO96WELLPLATESFORREADINGSINENZYMELINKEDIMMUNOSORBENTASSAYELISAREADER,THEPROTOCOLFOLLOWEDFORTHEMTTASSAYISSUMMARIZEDINTABLE3THEREADINGSWERECALCULATEDBYTHEFORMULACELLVIABILITYMEANABSORBANCEOFSAMPLEC2100MEANABSORBANCEOFCONTROLSTATISTICALANALYSISTHERESULTSOBTAINEDFROMTHEMTTANALYSISANDICPMSANALYSISWASSUBJECTEDTOSTATISTICALANALYSISONEWAYANOVAWASUSEDTOCALCULATETHEPVALUEMULTIPLERANGETESTSBYTUKEYHSDPROCEDUREWEREEMPLOYEDTOIDENTIFYTHESIGNIFICANTGROUPSAT5LEVELRESULTSELEMENTALRELEASEELEMENTALRELEASEWASSEENINALLTHREECASTINGALLOYGROUPSELEMENTSRELEASEDWERENI,CR,CO,CUANDMOTABLES4,AND5SUMMARIZETHEELEMENTALRELEASEBYDIFFERENTCASTEDALLOYSINTHREECASTINGPROCEDURESHINICKELCBHADTHEHIGHESTAMOUNTOFELEMENTALRELEASEAMONGTHETHREECASTINGALLOYSFOLLOWEDBYCERAMETANDWIRON99ELEMENTALRELEASESIGNIFICANTLYINCREASEDWITHTHEPERCENTAGEOFRECASTMATERIALUSEDINFORALLELEMENTSANALYZEDINTHESTUDYWITHP005GRAPH1CYTOTOXICITYRESULTSTHEPERCENTAGEOFVIABLECELLSINTHREEGROUPSISTABULATEDINTABLE6MEANPERCENTAGESOFCELLACTIVITYOFTHREEBASEMETALALLOYSFORTHREECASTINGCONDITIONSWEREANALYZEDANDCYTOTOXICITYWASOBSERVEDTOBEMOREINHINICKELCB,FOLLOWEDBYCERAMETANDWIRON99RESULTSSHOWEDTHAT100RECASTALLOYSHADMORECYTOTOXICITYTHAN50PUREAND50RECASTEDAND50PUREAND50RECASTEDALLOYHADMORECYTOTOXICITYTHANPUREALLOYGRAPH2DISCUSSIONMETALSHAVEBEENANINTEGRALPARTOFDENTALRESTORATIVEPROCEDURESDENTALCASTINGALLOYSWHICHRESTORETHEFORMANDFUNCTIONOFMISSINGTOOTHSTRUCTURESHOULDBEBIOLOGICALLYINERTRELEASEOFMETALLICELEMENTSFROMDENTALCASTINGALLOYSISAPOTENTIALHEALTHPROBLEM2,8METALIONSLEACHEDFROMCASTINGALLOYSAREKNOWNTOCAUSETOXICINFLAMMATORY,ALLERGICORMUTAGENICREACTIONS9,10THESERELEASEDELEMENTSHAVEBEENDETECTEDBYINVITROANDINVIVOSTUDIESANIMPORTANTCONSEQUENCEOFELEMENTRELEASEISITSCYTOTOXICEFFECTONADJACENTTISSUEANDTHEAMOUNTOFTISSUEREACTIONDEPENDSUPONELEMENTRELEASED57,11,12FIG5DULBECCOSMODIFIEDEAGLEMEDIUMDMEMFIG6TISSUECULTUREPLATEWITH05MLOFMOUSEFIBROBLASTCELLSUSPENSIONTABLE3PROTOCOLFORPERFORMINGMTTASSAYSTEPACTION1PLATECELLSAT1,000100,000PERWELL2INCUBATEFOR624H3ADD10LLMTTREAGENT4INCUBATEFOR24HUNTILPURPLEPRECIPITATEISVISIBLE5ADD100LLDETERGENTREAGENT6LEAVEATROOMTEMPERATUREINTHEDARKFOR2H7RECORDABSORBANCEAT570NMJINDIANPROSTHODONTSOCAPRJUNE2011112106112109123THESELECTIONOFANALLOYFORSPECIFICCONDITIONSLIKEFIXEDPARTIALDENTURESAREINFLUENCEDBYTHREEFACTORSNAMELYPHYSICALPROPERTIES,BIOCOMPATIBILITYANDCOSTGOLDCONSIDEREDASTHEHIGHNOBLEMETALWASTHEALLOYOFCHOICEFORRESTORATIVEPROCEDURESANDITWASCONSIDEREDTHATNOIONSLEACHEDFROMITBUT,HISTOLOGICALSTUDIESDISPROVEDTHISMISCONCEPTION2HIGHCOSTOFGOLDHASLEADTOTHEINTRODUCTIONOFBASEMETALDENTALCASTINGALLOYS2,3NICKELCOMBINEDWITHCHROMIUMFORMSHIGHLYCORROSIONRESISTANTALLOY,THESEBASEMETALALLOYSWHICHAREPRIMARILYNICKELBASEDCOMPAREDWITHGOLDALLOYSARECHEAPER,HAVEHIGHELASTICMODULUSSTIFFER,HARDER,LESSDENSEANDPOSSESSCOMPARABLERESISTANCETOTARNISHANDCORROSION1DUETOCOMPARABLEPHYSICALPROPERTIESBASEMETALSHAVESLOWLYREPLACEDGOLDCASTINGALLOYS2,3,13,14COMMERCIALLYAVAILABLEBASEMETALALLOYSFORFIXEDPARTIALDENTURESAREUSUALLYNICRALLOYS1,2THECOMPOSITIONOFTHESEALLOYSMAYVARYACCORDINGTOMANUFACTURERBUTTHEBASICCONSTITUENTSARENI,CRANDMO1ADDITIONOFMINORCOMPONENTSSIGNIFICANTLYCHANGESTHEMICROSTRUCTUREANDPROPERTIESWHICHAFFECTTHEBONDSTRENGTHOFCERAMICTOMETALOXIDELAYERREQUIREDFORACHIEVINGBONDINGITISATTHEMANUFACTURERSDISCRETIONTOINCLUDEANYOFTHESEMINORCOMPONENTSTOPRODUCETHEDESIREDPROPERTIES1TABLE4COMPARISONOFMEANVALUESOFELEMENTALRELEASEBETWEENDIFFERENTRECASTEDALLOYSINTHREECASTINGGROUPSGROUPVARIABLEMATERIALMEANSDPVALUESIGNIFICANTGROUPSAT5LEVELI100PUREALLOYA11095005200001SIGC1VSA1ANDB1B111220081C114010082II50PURE50RECASTEDALLOYA23674028800001SIGC2VSA2ANDB2B244080231C248650296III100RECASTEDALLOYA314092073900001SIGC3VSA3ANDB3B3147272028C3149442301TABLE5THEAMOUNTOFVARIOUSELEMENTRELEASEDFROMCASTINGALLOYSFORTHREECASTINGGROUPSMEANSDELEMENTAL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