3 mm thick disks were obtained at the root cervical third from 9 maxillary human molars and a standardized smear layer was produced Cosite images of the dentin surface were obtained after several cumu lative demineralization times Sixteen images wer ID: 75736
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DentinDemineralizationWhenSubjectedtoBioPureMTAD:ALongitudinalandQuantitativeAssessmentGustavoDe-Deus,MD*,ClaudiaReis,MS,SandraFidel,PhD,RivailFidel,PhD,andSidneiPaciornik,PhDInthepresentstudy,thedemineralizingabilityofBioPureMTAD(Dentsply/Tulsa,Tulsa,OK),17%EDTA,and5%citricacidonradiculardentinwasquantitativelyandlon-gitudinallyanalyzed.3mmthickdiskswereobtainedattherootcervicalthirdfrom9maxillaryhumanmolars,andastandardizedsmearlayerwasproduced.Cositeimagesofthedentinsurfacewereobtainedafterseveralcumu- Fromthe*VeigadeAlmeidaUniversityand BasicResearch—Technology De-Deusetal.—Volume33,Number11,November2007 selectedbytheSEMoperator.ItisacommonfindingthatmostSEMoperatorstendtoselectcleancanalareaswithopendentinaltubulesratherthanareaswithlargebulkofdebrisorsmearlayer(15,16Moreover,SEMinvestigationsdonotallowlongitudinalobserva-tionsofthedentinalmorphology.Inthiskindofstudy,thestatusofthedentinalsurfacebeforetheapplicationofthechelatorisunknownbe-causetheevaluationisnotperformedinthesamesample.Inanimpor-tantarticle,Petersetal(),usingahigh-qualitymicrocomputedto-mographymethod,showedthattheamountofuninstrumentedcanalareasafterpreparationis35%ormoreoftheirtotal.So,itispossibletoconcludethatuninstrumenteddentinalwallsaresmearfree,andthisfacthighlightstheimportanceofalongitudinalcharacterinsmear-removalinvestigations,inwhichthestatusofthedentinalsurfaceiswellknownbeforetheinitialapplicationofthechelator.Itisworthmentioningthatatomicforcemicroscopy(AFM)hasbeenusedforlongitudinalevaluationofthedentindemineralizationprocess(14,19).Thisapproachshowedrelevantadvantagessuchastheobservationoftheprocessinnearrealtimeasthesampleswereimmersedinthechelatingsubstanceduringobservation.However,lim-itationscausedbyspecificcharacteristicsofAFMprecludedobtainingaccuratequantitativeresults.Watari()usedAFMtoobtainquantita-tiveresultsregardingreliefmeasurementssuchasroughness.Cositeopticalmicroscopy(CSOM)wasrecentlyintroduced(anditrepresentsanefficientmethodfordirectcomparisonofthedemi-neralizingpowerofthesolutionsusedinEndodontics.TheaccuracyandreproducibilityofCSOMhavebeenverifiedpreviously(),anditprovedtobefastandrobust.Moreover,themethodprovidesquantita-tivedatalinkedtothelongitudinalobservationofthedentinalsubstrateHence,thepresentworkaimedtoassess,bothlongitudinallyandquantitatively,thedemineralizingabilityofBioPureMTADthroughCSOManddigitalimageanalysis.Both17%EDTAand5%citricacidwereusedasreferencesolutionstocomparetheresults.Thenullhy-pothesistestedwasthatthereisnodifferenceindemineralizationofdentinirrigatedwitheitherBioPureMTADorEDTAor5%citricacid.MaterialsandMethodsSpecimenSelectionandPreparationThisstudywasrevisedandapprovedbytheEthicsCommittee,NucleusofCollectiveHealthStudies,RiodeJaneiroStateUniversity,RiodeJaneiro,Brazil.NinemaxillaryhumanmolarswereselectedfromthetoothbankofRiodeJaneiroStateUniversity.Eachspecimenwasmountedinanepoxyresincylinder(Arazyn1.0;AraQuímica,SaoPaulo,Brazil)tofacilitatemanipulationandimprovethemetallographicpreparation.Followingaprotocoldescribedpreviously(),dentindiscsapproximately30.3mmthickwerecutatthemiddlethirdlevelofthecrownsbutabovetherootcanalusingalow-speedsaw(Isomet;Buhler,Ltd,LakeBluff,NY)withadiamonddisc(125mm12.7mm,33°C),withcontinuouswaterirrigationinordertopreventoverheating.Thedentinsurfaceswerecarefullyinspectedtoensurethattheywerefreeofcoronalenamelorpulpalexposures.Thedecisiontoperformthepresentevaluationoncrowndentininsteadofonintraradiculardentinwasbasedonapilotstudyinwhichthedifficultytoobtainusefuldisksfromintraradiculardentinwasde-termined.Inaddition,crowndentincanprovidemorehomogeneousthanintraradiculardentin(),andthisrepresentsadesirablefeatureincomparativeassessments.Astandardmetallographicprocedure(grindingwithSiCpaper[200,300,400,and600]gritsand3mdiamondpaste)wasusedonthepulpalsurfaceofthetoothtopreparethemfortheexperimentalprocessandtoproduceastandardizedsmearlayer(14,17,19,23Atthispoint,thesampleswererandomlydividedinto3groupsaccordingtothechelatingagentusedasfollows(n3pergroup):(1)G1:BioPureMTAD,(2)G2:17%EDTA(pH7.7),and(3)G3:5%citricacid(pH2.0).EDTAandcitricacidwerefreshlypreparedbytheman-ufacturer(Formula&AçãoLtda.,SaoPaulo,Brazil).ExperimentalProcedure(CositeMicroscopy)TheexperimentsweredevelopedinanAxioplan2Imagingmotor-izedmicroscope(CarlZeissVisionGmbh,Hallbergmoos,Germany).AnEpiplan100HDobjectivelenswasusedcoupledtoa1300pixelsAxiocamHRdigitalcamera(CarlZeiss),leadingtoatotalmag-nificationofapproximately1000X,andaresolutionof0.1Inthecositemicroscopyexperiment,aspecialholderallowedapplicationofthechelatingsolutionswithoutremovingthedentinsam-plefromthemicroscope.Amotorizedspecimenstagewasusedtoautomaticallyacquire16imagefieldsatspecificx-ypositionsofagivensampleforseveralcumulativedemineralizationtimes(0,15,30,60,180,and300seconds).Thus,itwaspossibletofollowthesamefieldswithhighreproducibilityofthex-ypositionsandautofocus,allowingtheobservationoftheeffectofdemineralizationintheverysameregions.Apilottestwasperformedforthefirstmethodologicarticle()inwhichanumberof16imagefieldspertoothwasdefined.Thisdecisionwasbasedonthefollowingcriteria:()theavailabledentinareaand()theamountofusefuldataobtainedperimage.ThedetailsoftheprocedureweredescribedbyDe-Deusetal().ThecompleteimageacquisitionsequencewascontrolledbyaspecialroutineimplementedundertheAxioVision4.5software(CarlZeissVision).ImageAnalysisApreviouslydevelopedimageanalysisroutine()wasusedtoenhanceimagecontrast,discriminate(17,19,24),andmeasureopendentintubulesineachacquiredimage.Then,theratiobetweenthetotalareaofopentubulesandtheareaofthefullimagefield,theso-calledareafraction(AF),wasmeasured.ThechangeofAFoverdemineraliza-tiontimewasusedtoquantifytheprocess.Theroutinewasappliedwithoutoperatorinfluencetothevastmajorityoftheimagesacquiredfordifferentsamplesatdifferenttimes.AllstepswereimplementedasamacroroutineundertheKS4003.0software(CarlZeissVision).Theinitialimages(0seconds)withthestandardizedsmearlayerwerenotanalyzedbecauseofthelowcontrastofthetubulescoveredbythesmearlayer,whichpreventedtheirdiscrimination.Asanovertimeevaluation,eachspecimenservedasitsowncontrol.DataPresentationandAnalysisDataarepresentedastubuleAFinpercentageofthewholedentinarea().Thepreliminaryanalysisofthepooleddatafromtheexper-imentalgroups(SPSSforWindows,Version8.0;SPSSInc,Chicago,IL)didnotshownormaldistribution(Kolmogorov-Smirnovtest).Furtherstatisticalanalysiswasperformedwithnonparametrictestmethodsus-ingtheKruskal-WallisH-testwithBonferronicorrectiontocomparetheAFvaluesbetweenthegroupsattherespectiveexperimentaltimes.TheAFandthetimewereusedasfactorsandthelevelofsignificancewassetat0.05.Origin6.0(MicrocalSoftware,Inc,Northampton,MA)wasalsousedasanalytictool.ThecolumnsoftheimagemontageinFigure1showtheevolutionofdemineralizationovertimefor17%EDTA,BiopureMTAD,and5%CA.Ineachcolumn,animagefieldataspecificx-ypositionofasampleisshownfor5cumulativedemineralizationtimes.Figure2showstheincreaseofAFofopentubulesagainsttimeforeachgroup.BasedonthepresentdatathefollowingstatementscanbeBasicResearch—Technology JOE—Volume33,Number11,November2007DentinDemineralizationandBioPureMTAD made:(1)generally,allimagesweresmearfreeafter15/30secondsofetchingandonecanobservetheenlargementofthedentinaltubules,asexpectedinthetypicalevolutionofdemineralization;(2)BioPureMTADand5%citricacidweremoreeffectivethan17%EDTAforallexperimentaltimes(p0.05);(3)BioPureMTADwasmoreeffectivethan5%citricacidatboth15secondsand30seconds(pwhereas5%citricacidwasmoreeffectivethanBioPureMTADatboth180secondsand300seconds;(4)thereisaclearsaturationonthedemineralizingabilityofBioPureMTADafter30seconds;(5)thereisatrendofsaturationonthedemineralizingabilityof5%citricacidafter30seconds;and(6)thedemineralizationkineticspromotedbyboth5%citricacidandBioPureMTADwasclearlyfasterthanfor17%EDTA. Figure1.Timeevolutionofagivendentinregionduringdemineralizationwitheachchelator.Thecolumnsshowtheevolutionofdemineralizationovertimefor17%EDTA,5%CA,andBioPureMTAD(fromleft).BasicResearch—Technology De-Deusetal.—Volume33,Number11,November2007 Basedonthepresentresults,thenullhypothesistestedwasplainlyrejected.ItcanbestraightforwardlyobservedinthegraphofFigure2thatunderthesameexperimentalconditions,bothBioPureMTADand5%CAweremoreeffectivethan17%EDTAatallexperimentaltimes.Regardingthedataobtained,twointerestingpointscanbedrawn:(1)thefastandsharpincreaseofthetubularareafractionpromotedbyBioPureMTADand5%CAand(2)thefullsaturationofthedeminer-alizingabilityofBioPureMTADafter30seconds.Thefirstpoint,thefasterdemineralizationkineticsofBioPureMTADand5%CA,canbeviewedaspositivefromaclinicalpointofviewbecausesmearremovalisfastandefficient.ThefastereffectofCAhasbeenreportedinafewearlierstudies.Recently,De-Deusetal(usingalongitudinalobservationmethod(AFM)foundthatCAhadthestrongesttime-effectrelationshipwhencomparedwithEDTAandEDTAC.However,tothebestoftheauthors’knowledge,thisfastereffecthasnotbeenreportedforBioPureMTADsofar.However,becauseofthecurrentconcernregardingerosiveeffectsstemmingfromtheasso-ciationofchelatorswithNaOCl,onecannotstatethatthefastestormostpowerfulsolutionisthebestindicatedforsmearlayerremoval.Thesecondpoint,theself-limitingeffectofBioPureMTAD,sug-gestsanoveltyinrelationtoitswell-knownproperties.Theself-limitingeffectofEDTAhasalreadybeenshown(13,14).Onceallthechelatingionshavereactedwiththecalciumionsofthedentin,equilibriumisestablishedanddemineralizationstops.However,thefewreportsaboutthesmear-removalabilityofBioPureMTADdonotpointoutaself-limitingeffect.Consideringtheconcernsmentionedearlierregardingerosiveeffects,asolutionthatpresentsaself-limitingeffectassociatedtoefficiencyisveryattractive.TheconsequencesofdentinmatrixdestructionremainsundefinedalthoughParketal()speculatedthatanincreasedcoronalleakageinsamplestreatedwithEDTAcomparedwiththosetreatedwithBioPureMTADmightbecausedbytheerosivepropertyofEDTAandthelengthofdentinexposuretothissolution.Futureresearchshouldtrytoanswerthefollowingquestion:whatisthebestclinicalindication?Eitheraverystrongchelatorthatremovescompletelythesmearlayerbutthatmightalsodestroythedentinmatrixorarelativelyweakerchelatorwiththeoppositebehavior?Theanswerrequiresabetterunderstandingofthemechanismofdentinmatrixdestructionanditseffectsontheadaptationofthefillingandachievedsealingaswellasapossibleinfluenceonrootstrength.TheerosiveeffectsofEDTAandcitricacidhavebeenreportedinseveralstudies(12,13,14,26,27).Ontheotherhand,themanufac-turer()andTorabinejadetal()reportedthat,unliketheuseofEDTAasthefinalrinse,minimalerosionofintraradiculardentinhasbeenreportedwhenNaOClandBioPureMTADwereusedinasimilarsequence.ThecitricacidanddoxycyclinepresentinBioPureMTADareresponsibleforitschelatingability.However,De-Deusetal()re-portedthatCAledtostrongerosiveeffects.Possibly,somekindofchemicalinteractionbetweenthecompo-nentsofBioPureMTADisresponsiblefortheself-limitingeffect.Tor-abinejadetal()reportedthatapparentlyBioPureMTADreactswiththedentinsurfacedifferentlywhencomparedwithCAorEDTA,andthesefindingsareinagreementwiththecurrentresults.TheuseofBioPureMTADwithdoxycyclineaffinitytodentinmayleadtoadifferenteffectonthedentinstructurewhencomparedwiththe17%EDTAand5%CA().Krauseetal()pointoutthatintheBioPureMTADpreparation,CAmayservetoremovethesmearlayer,thusallowingdoxycyclinetopenetratethedentinaltubulesandexertanantimicrobialeffect.However,somestudieshaveshownthatbothdoxy-cyclineandCAhaveacidetchingability(31,32Analyzingthepresentresults,itisapparentthat5%CAandBioPureMTADhavecomparableeffects.So,despiteitsself-limitingeffect,itisnotclearthatBioPureMTADcausedminimaldentinerosion.MoreinvestigationisnecessarytoclarifywhethertheclinicalprotocolfortheuseofBioPureMTADreallypreventsdentinerosion.Transmissionelec-tronmicroscopycanbeconsideredtheidealmethodtoassesstheeffectsoftheirrigationprotocolsonthedentinmatrix(Thisprojectisapartofalargerstudycomparingthedemineral-izationpowerofthedemineralizingagentsavailableincurrentend-odonticpractice.Inthepresentstudy,BioPureMTADwasevaluatedthroughCSOManddigitalimageanalysis.Themethodprovidesquan-titativedatalinkedtothelongitudinalobservationofthedentinalsub-stratechanges.BecausethegoalofthepresentworkwasrestrictedtoadirectlongitudinalandquantitativeassessmentofthechelatingabilityofBio-PureMTAD,theapplicationoftheseresultstotheclinicalsituationisnotstraightforward.Thelimitationsofthepresentresultaredirectlylinkedtotherequirementsofthelightopticalmicroscopytech-niques.Thechelatorsolutionwasappliedtoaflathorizontaldentinsurface,differentfromtheclinicalsituation,inwhichthecontactbetweenthechelatingsubstanceandthedentinsurfaceisaffectedbytheverticalpositionoftheteethandtheintrinsicanatomicalvari-abilityoftherootcanalsystem.Moreover,inthepresentmethod,arinsingprocedurewasnotperformedandthisisalsonotinlinewiththeclinicalsituation.However,foradirectcomparisonoftheche-latingabilityofthesolutionsthepresentmethodologicalmodelprovedtobefast,robust,reliableandstatisticallysound.Moreover,themethodprovideslongitudinalinformationaboutthedentinmor-phologyanddoesnotdependonthetraditionaldescriptivescoresofSEMstudies.Undertheconditionsofthepresentexvivoevaluation,thefollow-ingconclusionscanbedrawn:(1)thedemineralizationkineticspro-motedbyboth5%citricacidandBioPureMTADwasclearlyfasterthanfor17%EDTA,and(2)CSOMrepresentsapowerfulapproachtocom-paredirectlylongitudinallyandquantitativelytheabilityofthedeminer-alizingsolutions. Figure2.TimeevolutionoftheopentubuleAF.Datapointsaretheaverageof48measurementsfrom3differentsamples.Theframehighlightstheexperimentaltimesforwhichallthesolutionswerestatisticallydifferent.BasicResearch—Technology JOE—Volume33,Number11,November2007DentinDemineralizationandBioPureMTAD TheauthorswishtothankDr.CarlosCleberGarciaforhisessentialassistance.1.SchilderH.Fillingrootcanalsinthreedimensions.DentClinNorthAm1967;11:723–44.2.ShahravanA,HaghdoostA,AdlA,RahimiH,ShadifarF.Effectofsmearlayeronsealingabilityofcanalobturation:asystematicreviewandmeta-analysis.JEndod2007;33:96–105.3.GoldmanLB,GoldmanM,KronmanJH,LinPS.Theefficacyofseveralirrigatingsolutionsforendodontics:ascanningelectronmicroscopicstudy.OralSurgOralMedOralPatholEndod1981;52:197–204.4.YamadaRS,ArmasA,GoldmanM,LinOS.Ascanningelectronmicroscopiccom-parisonofahighvolumefinalflushwithseveralirrigatingsolutions:part3.JEndod1983;9:137–42.5.TorabinejadM,KhademiAA,BabagoliJ,etal.Anewsolutionfortheremovalofthesmearlayer.JEndod2003;29:170–5.6.ZhangW,TorabinejadM,LiY.EvaluationofcytotoxicityofMTADusingtheMTT-tetrazoliummethod.JEndod2003;29:654–7.7.TorabinejadM,ChoY,KhademiAA,BaklandLK,ShabahangS.TheeffectofvariousconcentrationsofsodiumhypochloriteontheabilityofBioPureMTADtoremovethesmearlayer.JEndod2003;29:233–9.8.BeltzRE,TorabinejadM,PouresmailM.QuantitativeanalysisofthesolubilizingactionofMTAD,sodiumhypochlorite,andEDTAonbovinepulpanddentin.JEndod2003;29:334–7.9.TorabinejadM,ShabahangS,AprecioRM,KetteringJD.TheantimicrobialeffectofMTAD:Aninvitroinvestigation.JEndod2003;29:400–3.10.BaumgartnerJC,JohalS,MarshallJG.Comparisonoftheantimicrobialefficacyof1.3%NaOCl/BioPureMTADto5.25%NaOCl/15%EDTAforrootcanalirrigation.JEndod2007;33:48–51.11.GhoddusiJ,RohaniA,RashedT,GhazianiP,AkbariM.AnevaluationofmicrobialleakageafterusingMTADasafinalirrigation.JEndod2007;33:173–6.12.TakedaFH,HarashimaT,KimuraY,MatsumotoK.Acomparativestudyoftheremovalofsmearlayerbythreeendodonticirrigantsandtwotypesoflaser.IntEndodJ13.ÇaltS,SerperA.Time-dependenteffectsofEDTAondentinstructures.JEndod14.De-DeusG,PaciornikS,PinhoMauricioM,PrioliR.Real-timeatomicforcemicros-copyofrootdentinduringdemineralizationwhensubjectedtochelatingagents.IntEndodJ2006;39:683–92.15.GulabivalaK,PatelaB,EvansG,YuanLingN.Effectsofthemechanicalandchemicalproceduresonrootcanalsurfaces.EndodTopics2005;10:103–22.16.HülsmannM,HeckendorffM,LennonA.Chelatingagentsinrootcanaltreatment:modeofactionandindicationsfortheiruse.IntEndodJ2005;36:810–30.17.De-DeusG,ReisCM,FidelRAS,FidelSR,PaciornikS.Co-sitedigitalopticalmicros-copyandimageanalysis:anapproachtoevaluatetheprocessofdentindemineral-ization.IntEndodJ2007;40:1–12.18.PetersOA,SchonenbergerK,LaibA.EffectsoffourNi-Tipreparationtechniquesonrootcanalgeometryassessedbymicrocomputedtomography.IntEndodJ19.PaciornikS,De-DeusG,ReisCM,PinhoMauricioMH,PrioliR.Insituatomicforcemicroscopyandimageanalysisofdentinsubmittedtoacidetching.JMicrosc2007;3:235–42.20.WatariF.Insituquantitativeanalysisofetchingprocessofhumanteethbyatomicforcemicroscopy.JElectronMicrosc2005;54:299–308.21.GrégoireG,JoniotS,GuignesP,MillasA.Dentinpermeability:self-etchingandone-bottledentinbondingsystems.JProsthetDent2003;90:42–9.22.MjörI,NordhalI.Thedensityandthebranchingofdentinaltubulesinhumanteeth.ArchOralBiol1996;41:401–12.23.De-DeusG,PaciornikS,MauricioMHP.EvaluationoftheeffectofEDTA,EDTACandcitricacidonthemicrohardnessofrootdentin.IntEndodJ2006;39:401–7.24.PaciornikS,MauricioMH.Digitalimaging.In:VanderVoortGF,ed.ASMHandbook:MetallographyandMicrostructures.MaterialsPark,OH:ASMInternational;2004:368–402.25.ParkDS,TorabinejadM,ShabahangS.TheeffectofMTADonthecoronalleakageofobturatedrootcanals.JEndod2004;30:890–2.26.CergneuxM,CiucchiB,DietschiJM,HolzJ.Theinfluenceofthesmearlayeronthesealingabilityofcanalobturation.IntEndodJ1987;20:228–32.27.MeryonSD,TobiasRS,JakemanKJ.Smearremovalagents:aquantitativestudyinvivoandinvitro.JProsthetDent1987;57:174–9.28.DentsplyTulsaDental.BioPureMTAD.Availableat:http://www.biopuremtad.com/.AccessedMarch24,2007.29.BakerPJ,EvansRT,SlotsJ,GencoRJ.Susceptibilityofhumanoralanaerobicbacteriatoantibioticssuitablefortopicaluse.JClinPeriodontol1985;12:201–8.30.KrauseTA,LiewehrFR,HahnCL.TheantimicrobialeffectofMTAD,sodiumhypo-chlorite,doxycycline,andcitricacidonEnterococcusfaecalis.JEndod2007;33:28–30.31.SmithJ,WaymanB.Anevaluationoftheantimicrobialeffectivenessofcitricacidasarootcanalirrigant.JEndod1986;12:54–8.32.DemirelK,BaerPN,McNamaraTF.Topicalapplicationofdoxycyclineonperiodon-tallyinvolvedrootsurfacesinvitro:Comparativeanalysisofsubstantivityoncemen-tumanddentin.JPeriodontol1991;62:312–6.33.TayFR,PashleyDH,LoushineRJ,etal.Ultrastructureofsmearlayer-coveredintrara-diculardentinafterirrigationwithBioPureMTAD.JEndod2006;32:218–21.BasicResearch—Technology De-Deusetal.—Volume33,Number11,November2007