Synthesis and characterization of a newmethacrylate monomer derived from the cashewnut shell liquid (CNSL) and its effect on dentinaltubular occlusion

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Synthesis

and

characterization

of

a

new

methacrylate

monomer

derived

from

the

cashew

nut

shell

liquid

(CNSL)

and

its

effect

on

dentinal

tubular

occlusion

Madiana

Magalhães

Moreira

_,

_Lucas

_Renan

_Rocha

_da

_Silva

_,

Talita

Arrais

Daniel

Mendes

_,

_Sérgio

_Lima

_Santiago

_,

Selma

Elaine

Mazzetto

_,

_Diego

_Lomonaco

_,

_Victor

_Pinheiro

_Feitosa

a_{PostgraduatePrograminDentistry,FederalUniversityofCeará,Fortaleza,Brazil}

b_{DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeará,Fortaleza,Brazil}

c_{PauloPicanc¸oSchoolofDentistry,Fortaleza,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2February2018 Receivedinrevisedform 11April2018 Accepted30April2018 Keywords: Anacardiumoccidentale Cardanol Organicsynthesis Methacrylates Epoxyresins

Dentindesensitizingagents

a

b

s

t

r

a

c

t

Objective.Theaimofthisstudywastosynthesize,tocharacterizeandtoevaluatetheeffects

ontubularocclusionofnewmonomerderivedfromcashewnutshellliquid(CNSL),also studyingtheeffectsofacidchallenge(AC)ondentinsurfacestreatedwithdesensitizers.

Methods.Theintermediarycardanol-epoxy(CNE)wassynthesizedthroughepoxidationof

CNSL,followedbysynthesisofcardanol-methacrylate-epoxy(CNME)through methacry-loylchlorideesterification.Productswerepurifiedthroughchromatographycolumnand characterizedbyFouriertransforminfraredspectrometryandnuclearmagneticresonance. Resinousdentindesensitizers were formulatedcontainingeitherunsaturated cardanol (CNU),CNEorCNME.Dentindisksweredividedintosevengroups:SL—Smear-layer,EDTA —EDTA-treatedonly,GLUMA—GlumaDesensitizer,OCB—OneCoatBond,CNU—CNU desensitizer,CNE—CNEdesensitizerandCNME—CNMEdesensitizer.Dentinalfluidrate (DFF)wasobtainedusingaFlodecequipmentandtubularocclusionemployingascanning electronmicroscope(SEM),beforeandafterAC.DataofDFFweresubmittedtotwo-way ANOVAandTukey’stest(p<0.05).

Results.GLUMAshowedthelowerreductioninDFFwhencomparedtotheotherproducts,

whichwerestatistically similar.EvenafterAC,CNMEpresentedthe mosthomogenous andoccludedsurface,whileCNEandCNUwerepartiallyremoved,GLUMAwascompletely removedandOCBkeepanoccluded,butirregularsurface.

Significance.CNMEshowedagreatreductionofDFFandahomogenousoccludedsurface,

suggestingthatitmaybeasuitableandacid-resistanttreatmentoptionfordentine hyper-sensitivity.

©2018TheAcademyofDentalMaterials.PublishedbyElsevierInc.Allrightsreserved.

∗ _{Correspondingauthorat:LaboratoryPPGO-UFC,MonsenhorFurtadoSt.,RodolfoTeófilo,Fortaleza,Ceará60.430-350,Brazil.}

E-mailaddress:victor.feitosa@facpp.edu.br(V.P.Feitosa). https://doi.org/10.1016/j.dental.2018.04.011

1.

Introduction

Hypersensitive dentin(DH) is a common oralhealth issue in the adult population, associated with exposed cervical dentinandcharacterizedbyshortandsharppaininresponse to several external stimuli [1,2]. This significant increase in the dentin sensitivity is explained by the most widely accepted “hydrodynamic theory” proposed by Brännström etal.[3].Suchexplanationadvocatesthatmovementsoffluid withinthedentinaltubulesstimulatepulpal mechanorecep-tors, whichare interpretedas pain. Thisclinical condition affectsdirectlythequalityoflife,therebymotivatingthe inves-tigationsofmechanismsleadingtoDHandthedevelopment ofawiderangeoftreatmentmethodstoprevent,reduceor eliminatethiscondition[4].

Severaldesensitizing agentshavebeen assessedforthe treatmentofDH,relyingontwoapproaches:nerve desensi-tizationorocclusionofpatentdentinaltubules[5].However, inagreementwiththehydrodynamictheory[3],mostofthe currentlyemployedagentsareusedtooccludethedentinal tubules,oncethisstrategymaysuppressdentinalfluid move-mentregardlessthestimulievoked,therebydecreasingDH. Althoughmanystudieshavereportedthe initialefficacy of these agents, most of them have their therapeuticeffects readilyarrestedordiminishedover timeduetodailytooth brushingorintermittentconsumingofacidicbeverages[6].

GlumaDesensitizer(HereausKulzer,Hanau,Germany)is oneofthedesensitizingagentswiththe longesthistory of useindentalclinics.Itsmechanismofactionrelieson pre-cipitationofplasmaproteinswithindentinaltubulesthanks to the high capacity of glutaraldehyde in promoting pro-tein crosslinking. Nevertheless, such compound is highly detrimentaltothemetabolismofodontoblast-likepulpcells (MDPC-23) [7] and possesses high dissolution capacity in acidicsolutions[8],whatmightcauseseveralcytotoxicissues. Recently,severalmorebiocompatibleplant-derivedproducts wereinvestigatedinordertoreplaceglutaraldehydeasdentin collagencrosslinkingagent[9].Inthisregard,newmonomers couldbecreatedfromtheseplant-derivedcompounds yield-ingimproveddesensitizingagents.

Cashewnut shellliquid(CNSL)emergesasanabundant renewableresourceofnaturallyoccurringphenols,also high-lightingaffinitywithdentincollagenand potentialtoform crosslinking[10].Aftertheindustrialthermaltreatment,CNSL ismainlycomposedbycardanol(approximately70%),along carbonchainphenol(Fig.1),whichgrantsthiscompoundhigh hydrophobicityandmanyreactivesitesfororganicsynthesis [11].Besides,itdoesnotdepictcytotoxicormutageniceffects [12].Advancesinthedevelopmentoflong-lastingdentin seal-ingtechnologiesshouldachieveacid-resistantdesensitizing agents. Therefore, the synthesis of a methacrylate epoxy cardanolmonomercouldobtainbiocompatible,hydrophobic andacid-resistantagent,oncetheoxiraneringcanopenin acidenvironments[13,14]andactasalatentpolymerization improvingthematerialovertime.

Theaimofthismanuscriptwastosynthesize,to charac-terizeandtoevaluatetheeffectsondentindesensitization (tubular occlusion)of new methacrylate monomer derived from the CNSL, also studying the effects of acid

chal-Fig.1–Syntheticroute:unsaturatedcardanol(CNU)was epoxidizedwithhydrogenperoxide,usingformicacidasa catalyst,inordertoincorporateanoxiraneringwherethe unsaturationwaslocatedandformcardanol-epoxy(CNE). CNEwasesterifiedwithmethacryloylchloride,inthe presenceoftriethylamineasacatalyst,toincorporateone methacrylatefunctionalitywherethehydroxylwaslocated andtoobtainthefinalmonomer

cardanol-methacrylate-epoxy(CNME).

lenge on dentin surfaces treated with desensitizers. The studyhypothesesunderinvestigationwerethat(1)thereare differencesinthedentinaltubuleocclusionamongthe desen-sitizingagents,and(2)thenewmonomercanpromoteacid resistance.

2.

Materials

and

methods

2.1. Reagents

CNSL(technicalgrade)waskindlysuppliedbyAmêndoasdo BrasilLTDA(Fortaleza,Brazil).Formicacid,hydrogen perox-ide, triethylamine, 3,5-Di-tert-4-butylhydroxytoluene (BHT), methacryloylchloride,anhydroussodiumsulfateandsolvents werepurchasedfromSigma-Aldrich(St.Louis,USA)andused asreceived.Silicagel(Merck)wasemployedinthe chromato-graphicseparations.

2.2. Synthesisofcardanol-epoxy(CNE)

Briefly,thecardanol-epoxy(CNE)synthesiswasaccomplished withepoxidationreactionofunsaturationsoftheCNSL,using hydrogenperoxideandformicacidwithamolarratioof1:3:1,

respectively.First,CNSLwasplacedinaroundbottomflask andthenformicacidwasadded,followedbytheslow addi-tion ofhydrogen peroxideunder magneticstirring atroom temperature (25◦C). The resulting solution was vigorously stirredat65◦Cfor2hand30minandmonitoredbythinlayer chromatography.Next, theproduct wasisolated bysolvent extractionfromtheaqueoussolutionandtheorganicphase wasdriedusinganhydroussodiumsulfate.Finally,the prod-uctwasfilteredfollowedbytheevaporationofthesolventina rotaryevaporator,andthecrudeproductwaspassedthrough asilica chromatographycolumntoafford the purifiedCNE monomer(Fig.1).

2.3. Synthesisofcardanol-methacrylate-epoxy(CNME)

Inagenericway,cardanol-methacrylate-epoxy(CNME) syn-thesiswasaccomplishedwithanesterificationreactionofthe aromatichydroxylofCNE,usingtriethylamineand methacry-loylchloridewiththemolarratioof1:1:1,respectively.Overall, CNEwasplacedintoaroundbottomflaskanddissolvedin tolueneatroomtemperature(25◦C)undervigorousstirring. Triethylaminewasaddedascatalysttothemixture,followed byBHT,aspolymerizationinhibitor.Thereafter,methacryloyl chloridewasslowlyaddedandtheslurrywasstirredat60◦C for4h,beingmonitoredbythinlayerchromatography.Then, theproductwasisolatedbysimplesolventextractionfrom theaqueoussolutionandtheorganicphasewasdriedusing anhydroussodiumsulfate.Finally,theproductwasfiltered fol-lowedbytheevaporationofthesolventinarotaryevaporator, andthecrudeproductwaspassedthroughasilica chromatog-raphycolumntoaffordthepurifiedCNMEmonomer(Fig.1). Duetothepresenceofthemethacrylategroup,theproduct waskeptinafridge(4◦C)andprotectedbyaluminumfoilafter theadditionofBHTtoavoidspontaneouspolymerization.

2.4. Fouriertransforminfraredspectroscopy(FTIR)

Samples of the synthesized and isolated products were characterized by a Fourier transform infrared spectropho-tometer (Spectrum Frontier, Perkin-Elmer Corp., Norwalk, USA) equipped with a crystal to perform attenuated total reflectance (ATR-FTIR) analysis. Samples were individually dispensedontothe crystal and spectrawere obtainedin a spectral range of 4000–550cm−1 with 4cm−1 resolution in transmittancemode.FTIRspectrawereobtainedintriplicate foreachproduct,usingtheunsaturatedcardanolasreference, andthenprocessedforbaselinecorrectionandnormalization.

2.5. Nuclearmagneticresonance(NMR)

Thenuclearmagneticresonance(NMR)spectraofcarbonand hydrogen were obtained in aBrucker spectrometer instru-ment,modelDPX 300(Rheinstetten,Germany) operatingat 75MHzfor NMR13C and 300MHzfor NMR1H. Deuterated chloroformwasusedasasolventatroomtemperature.

2.6. Resin-baseddentindesensitizerformulation

A resinous dentin desensitizer was formulated contain-ing 20wt% CNME, 50wt% triethyleneglycoldimethacrylate

(TEGDMA), 28.5wt% urethane dimethacrylate (UDMA), 0.5wt% camphorquinone (CQ) and 1wt% ethyl-4-dimethylamine benzoate (EDAB). Similar resin-based desensitizers were also formulated with 20wt% of unsat-urated cardanol (CNU)or 20wt% CNEinstead ofCNME.As commercial controls, Gluma Desensitizer (Heraeus Kulzer, Hanau,Germany)wasused,whichisbasedonglutaraldehyde andhydroxyethylmethacrylate(HEMA),andOneCoatBond SL(Coltène/WhaledentInc.,Mahwah,USA)wasemployedas resin-basedadhesivefordesensitization.

2.7. Preparationofspecimens

Forty nineextracted soundhumanthirdmolarswere used after approval by the Research Ethics Committee of Fed-eralUniversityofCeará(protocol1482602).Theywerestored in 0.1% thymol solution at 4◦C for no longer than one month. Occlusal enamel and roots were removed using a slow-speedwater-cooleddiamondsaw(Isomet4000;Buehler, Lake Bluff, USA) in order to obtain a disk (0.9±0.2mm in thickness) from middle coronal dentin from each tooth as described by Sauro et al. [15]. These specimens were wet-abraded using a 600-grit silicon carbide paper (30s) to create a standard smear layer, that was subsequently removedusing17%EDTA(pH7.4)for2minutesfollowedby anultrasonicbathcontainingdistilled waterfor5min.The dentindisks wererandomlydivided into thegroups(n=7). Group SL comprised the reference specimens, which were untreatedwithretainedsmear-layer.GroupEDTAcomprised controlEDTA-treatedspecimenswithopeneddentintubules (100%permeability)andthatnodesensitizingtreatmentwas applied. Specimensin further groups were stored individ-ually withmoist paperto keepthem hydrated. They were hydratedindistilledwaterfor30min[16]beforetreatment asfollows:groupGLUMAwastreatedwithGluma Desensi-tizer;groupOCBwastreatedwithOneCoatBondSL;group CNUwastreatedwiththeresin-baseddesensitizercontaining unsaturatedcardanol;groupCNEwastreatedwiththe resin-based desensitizer containing cardanol-epoxy and group CNMEwastreatedwiththeresin-baseddesensitizer contain-ingcardanol-methacrylate-epoxy.Aftertreatmentandinitial investigation, two specimens of each group were exposed to acidchallenge with6%citricacid forfurtherevaluation [15].

2.8. Dentindesensitizingtreatment

GLUMA and OCBwereappliedaccording tomanufacturer’s instructionsasdescribedinTable1.Specimenstreatedwith CNSLcompoundswerepretreatedwithabsoluteethanolfor 30sandtheexperimentalresin-baseddesensitizerswithCNU, CNE or CNMEwere applied withmicrobrush for20s, gen-tly air-dried, appliedagain for20s and light-curedfor 40s using theLEDBiolux(BioArt,SãoCarlos,São Paulo,Brazil). Afterwards,specimensofeachgroupwereimmersed individ-uallyfor1minina12-wellplate,with6%citricacid.After theimmersion,theywerethoroughlyrinsedwithdeionized water.

Table1–Commercialdesensitizingagentsandapplicationprocedures.

Desensitizingagent Components Procedure Manufacturer

GlumaDesensitizer (GLUMA)

Aqueoussolutioncontaining5% glutaraldehydeand35%HEMA

Applyandleavefor60s,dry,rinse HereausKulzer,Hanau,Germany OneCoatBondSL(OCB) Methacrylatea_{,polyalkenoat}

methacrylierta_,watera

Apply(dwellfor20s),gentleair, light-cureb_(30s)

Coltène/WhaledentInc.,Mahwah, USA

HEMA:2-hyroxyethylmethacrylate. a _{Notprovidedbythemanufacturer.}

b _{LEDBiolux(BioArt,SãoCarlos,SãoPaulo,Brazil).}

2.9. Dentinepermeabilityevaluation

The rate of fluid flow was obtained using Flodec equip-ment(Flodec-System,DemarcoEngineering,Switzerland)by recordingmeasurementsofhydraulicconductance(Lp). Spec-imenswereattachedtoaplexiglassplate,penetratedwitha stainlesssteeltube,withanexposedstandarddentinsurface, whichwereindividuallyconnectedtothehydraulicpressure device.Fluidpassedunderaconstantdeionizedwater pres-sureof15cmH2O(1.47kPa).Themovementofanairbubble insideacapillarytubeinthehydraulicsystemwasrecorded during5min;thelast3minrecordswereusedtoobtaintheLp foreachexperimentalcondition[17].

AftertheimmersionofallspecimensintotheEDTA solu-tion,theinitialLp ofeach specimenwascalculated,which weresubmittedtostatisticalanalysis.Following,thedentin disks were grouped into the experimentalgroups (n=3) in ordertoobtainnormaldata(p>0.05)andnostatistical differ-enceamongbaselinepermeabilityvalues.Subsequently,the specimenswerewet-abradedontheocclusalsidewith 600-gritSiCpaperfor30stocreatestandardizedsmearlayer,then Lpwasmeasured toevaluatetheminimumpermeability.To obtainthemaximumpermeability(Lpmax=100%was arbi-trarilyassigned),thatallowsanevaluationofthechangesin dentinpermeability afterthe applicationoftreatments, all specimenswereetchedfor15swith37%phosphoricacid solu-tion,followedbyrinsingwithdeionizedwater.

Moreover,theapplicationofexperimentalmaterialsonthe dentinspecimenswascarriedout asaforementioned.After treatments,hydraulicconductancewasmeasuredagainand treatmentpermeabilitywasexpressedasapercentage(Lp%) ofthemaximumLpvalue(100%).Finally,eachtreateddentin diskwasimmersedin6%citricacidsolutionfor60sinorderto evaluatetheacidresistanceofdesensitizingtreatmentsand thelastLpwasperformedafterwashingtheacid.Themeans (%reduction)and standard deviationsforeach groupwere calculated and the statisticalanalysis was performed with two-wayANOVAandTukey’stest(p<0.05).

2.10. Scanningelectronmicroscopy(SEM)

Fourspecimenspergroupwereanalyzedbyscanningelectron microscopy(SEM).TheywerepreparedascitedinSubsections 2.7and2.8andusedtwospecimensaftertretamentandtwo afteracidchallenge.Specimensweredehydratedinsilicagel, mountedonstubs,gold-sputtercoatedandobservedinSEM (InspectS50,FEICompany, Amsterdam,Netherlands) oper-atedat20kV.Representativescanningelectronmicrographs weretaken atdifferent magnificationsandwere chosenby

twoevaluatorsbasedonthefrequentlyobservedappearance ofthedentinsurface.

3.

Results

3.1. CharacterizationofCNU,CNEandCNME

Fig. 2 illustrates the infrared spectra (FTIR) of CNU, CNE andCNME.ThestretchoftheC O C(oxiranering)bondat 826cm−1 ispresentedinCNEand CNMEspectra.The char-acteristicsignalofthe vibrationalstretchofthe O Hbond ofphenolicringspresentinthespectraofCNUandCNEat 3388cm−1,disappearedinCNME,indicatingthesubstitution ofthisgroup.Absorptionsbandsat1740cm−1and1238cm−1 arereferredtothebondsC OandC CO O,respectively,are duethepresenceofthemethacrylicgroupofCNME.

The1Hand13C NMRspectraofthereactant(CNU) and products(CNEandCNME) aredisplayedinFig.3.Inthe1H NMRspectra(Fig.3a)wereobservedcommonsignalsforall thesecompoundsintheregionsbetween0.8to1.7ppmand 6.5to7.5ppm,attributedtothealiphatichydrogensformthe sidechainandthearomaticringhydrogens,respectively.The respectivecarbon atomsattachedtothese hydrogenswere observedinthe13CNMRspectra(Fig.3b),wheresignalsfrom 10 to 40ppm were attributed to aliphatic carbon atomsof theside-chain,whilethesignalsbetween110–160ppmwere originatedfromboththearomaticringasalsofromthe unsat-uratedcarbonsformthemethacrylicgroup.Specificchanges wereobservedinthespectraofCNEandCNMEwhen com-paredtothestartingmaterial(CNU).Signalsat␦2.75–3.00, atributedtotheoxiraneringofCNEandCNME(Fig.3a,3),was confirmedbythesignalattherangeof55–60ppminthe13C NMR(Fig.3b,5).Thesuccessfulesterificationofthearomatic hydroxylwasconfirmedbythesignalsat␦5.5–6.5,referredto thevinylichydrogens(Fig.3a,1)ofCNME,andthesharp sig-nalat2.2ppm(Fig.3a,4),attributedtotheterminalmethyl group.Inthe13CNMRspectra,wasalsoobservedasignalat 166ppm,characteristicofthecarbonyliccarbon(Fig.3b,4)of themethacrylicgroup.

3.2. Dentinpermeabilityevaluation

The results of percentage reduction in dentinal fluid flow achievedbythedesensitizingagentswhencomparedtothe baselineflowrateareillustratedinFig.4.CNMEshowedthe highest reductioninflow rate aftertreatment (87.1%).The applicationofOCB,CNU,CNEandCNMEontothedentindisks reduced dentine permeability withno statistical difference

Fig.2–FTIRspectraobtainedfromthemonomersunsaturatedcardanol(CNU—topspectrum),cardanol-epoxy(CNE— middlespectrum)andcardanol-methacrylate-epoxy(CNME—bottomspectrum).ThestretchoftheC Hsp2_{(aliphatic)bond}

at3009cm−1,isonlyobservedinCNU.ThestretchoftheC O C(oxiranering)bondat826cm−1ispresentedinCNEand CNMEspectra(firstdottedlineintheright),demonstratingthesuccessfulepoxidation.ThespectraofCNUandCNEshows theabsorptionbandat3388cm−1,characteristicofvibrationalstretchoftheO Hbondofthephenol,thathasdisappeared inCNME.Theabsorptionsbandsat1740cm−1andat1238cm−1referringtothebondsC O(carbonylfrommethacrylate) andC CO O,respectively,occurredonlyinCNME.

betweenthem,whilstGLUMAinducedthelowestreduction ofthe dentinal flow(p<0.05). There wasno statistical dif-ferencebetweenbeforeandafteracidchallengewithineach desensitizingtreatment.

3.3. SEMevaluationoftheocclusionofdentinal tubules

CharacteristicSEMimagesofthe dentinsurfacesonwhich thedesensitizingmaterialswereappliedareshowninFig.5. Differentchangesofthemorphologiesofthedentinsurfaces were observed according to the groups. GLUMA presented partiallyoccludedtubules(Fig. 5c), whichwere virtuallyall exposedafteracidchallenge(Fig.5d).OCBshowedoccluded

surfacewithsomewaterdroplets(Fig.5e),andsome porosi-tiesaftercitricacid(Fig.5f).CNUandCNEpresentedoccluded dentin tubulesaftertreatment (Fig. 5gand i, respectively), but they were partially removed after acid, showingsome patent tubules(Fig.5hand j,respectively).CNMEproduced anhomogenousandoccludedsurface(Fig.5k),evenafteracid challenge(Fig.5l).

4.

Discussion

The present study evaluated the effectiveness of dentinal tubuleocclusionamongdesensitizingagents,whichdepicted differentdegreesofocclusion.Therefore,hypothesis(1)needs tobeaccepted.Besides, theacidresistanceoftheseagents

Fig.3–1_HNMR(a)and13_{CNMR(b)spectraofthemonomerscardanolunsaturated(CNU—topspectrum),cardanol-epoxy}

(CNE—middlespectrum)andcardanol-methacrylate-epoxy(CNME—bottomspectrum).CNUshowedasignalattherange ofı5–6(Fig.3a—dottedrectangle2)relativetotheolefinicshydrogensofthelateralchain,thatwasabsentinCNEand CNME.Thesignalattherangeofı2.75–3(Fig.3a—dottedrectangle3)isattributedtotheoxiraneringofCNEandCNME, whichwasconfirmedbythesignalattherangeof55–60ppmin13_{CNMR(Fig.3b—dottedrectangle5).Betweenı5.5–6.5}

Fig.4–Meansandstandarddeviationsofthereductionindentinalfluidflow(%)valuesaftertreatmentandafteracid challenge.Differentcapitallettersindicatesignificantdifferencesbetweenthefivedesensitizingagents(p<0.05).No statisticaldifferencewasfoundaftertreatmentandafteracidchallengewithineachdesensitizer(p>0.05).Numerically, CNMEshowedthehighestreductionindentinalflowrateaftertreatment(87.1%).GLUMAinducedthelowestreductionof thedentinalflowwhencomparedtofurtherproducts(p<0.05),whichwerestatisticallysimilar.GLUMA—Gluma Desensitizer,OCB—OneCoatBondSL,CNU—unsaturatedcardanol,CNE—cardanol-epoxy,CNME—

cardanol-methacrylate-epoxy.

was evaluated, which fully accomplished only by the new monomer. Consequently, the hypothesis (2) also requires acceptance.

TheCNMEmonomerwassynthesizedviaatwo-step pro-cedure(Fig. 1). Inthe first step,the intermediaryCNEwas successfullyachieved,bymeans ofanadaptationofa pre-viously published protocol [18]. The second step was the esterificationofthephenolichydroxylinCNEwith methacry-loylchloride,inordertoaffordthefinaldesiredCNMEinhigh yields(>75%).Themonomerswereisolatedandcharacterized byFTIR,1_Hand13_{CNMR.Allspectraclearlyshowthe} func-tionalizationofunsaturationwithanoxiranering(CNEand CNME)andofthephenolmoietywithamethacrylatependant (CNME)(Figs.2and3).Bytheauthor’sknowledge,this two-stepreactiontosynthesizedentalmonomershasnotbeen reportedintheliteraturesofar.Furthermore,thisisthefirst studyinvestigatingadentalmonomersynthesizedfromCNSL compounds.

Theinfluenceofdesensitizing agentson dentin perme-abilitywastested withaFlodeccomputerizeddevice. After introducinganairbubbleinthesystem,thelinear displace-mentofthebubblewithintheglasscapillarywasmeasured. Thismovementoccursbywaterpressurethroughthediscs atsimulatedpulpalpressure,onceonlyalow,physiological

pressureissuggestedtobesuitableformeasuringhydraulic conductanceindentin[19].Thisinformationwastransmitted bymicroflowsensorstoacomputerconnectedtothe equip-ment,whichconvertsbysoftwarethisdisplacementtorateof fluidflow.

GLUMArevealedsignificantlowerreductionofthedentinal flowwhencomparedtothefurtheragents(Fig.4).Thisresult was alsofoundin other study[20], which foundthat pho-tocurableresindesensitizers(Seal&Protect;DentsplyCaulk, Milford,DE,USA)demonstratesgreaterreductionon perme-abilitythanGlumaDesensitizer.TheinvestigationofCamps etal.[21]mayprovideanexplanationforthisresult,asthey foundthatwhentubuleswerefilledwithphosphate-buffered saline (PBS),it wasobservedan decreaseinhydraulic con-ductance indentindisks treatedwith Glumadesensitizing agent.Contrariwise,otherstudy[22],evenusingPBS,revealed thesameamountofreductioninpermeabilitybyGlumaand Seal &Protect, buttheyshowedthe highesthydraulic con-ductance(lessthan40%ofpermeabilityreduction)amongthe testedagents.ThepresentstudydidnotusePBSorsimulated bodyfluid.Therefore,themechanismofprecipitationof pro-teinsbyglutaraldehydemayhavebeennegativelyaffectedby theuseofdeionizedwater.Yet,itisimportanttoremember thatbesidesplasmaproteins,therearemanyothersourcesof

The3_{CNMRconfirmedthepresenceofthemethacrylategroupshowingthesignalattherangeof160–170ppm(Fig.3b—dottedrectangle4),} characteristicofthecarbonyl(C O).

Fig.5–SEMmicrographsdepictingthemostcommonfeaturesofdentinsurfacesaftertreatmentandafteracidchallenge with6%citricacid.Whitearrowsindicatepartiallyoccludeddentinaltubules,whileblackarrowsaredesignatedforwater droplets.Thepointerisindicatingphaseseparation.Thedifferentgroupsaredividedasfollows:(a)retainedsmear-layer,(b) EDTA-treatedonly,withopeneddentintubules,(c)dentinsurfacetreatedwithGlumaDesensitizer(GLUMA),(d)

GLUMA-treateddentinafteracidchallenge,(e)surfacetreatedwithOneCoatBondSL(OCB),(f)OCB-treateddentinafteracid, (g)dentinsurfacetreatedwithdesensitizercontainingunsaturatedcardanol(CNU),(h)CNU-treatedspecimensafteracid,(i) surfacetreatedwithdesensitizercontainingcardanol-epoxy(CNE),(j)CNE-treatedspecimensafteracid,(k)surfacetreated withdesensitizercontainingcardanol-methacrylate-epoxy(CNME)and(l)CNME-treatedspecimensafteracid.SEM demonstratedthatinitiallyGLUMAonlypartiallyoccludedthetubules(c)butshowedalmostcompletedentinexposure afteracidchallenge(d).OCBcreatedanoccludedsurface,butirregularduetoagreatamountofwaterdroplets(observedine andf).Besides,aninitialdegradationandexposureoftubuleswerenoted(f).CNUcoveredthedentinsurfacewithafrilled filmthankstoitsincompletepolymerization(g),thereasonwhyitwaspartiallyremovedafteracid(h).CNEshowedphase separationonoccludedsurface(pointerini),whichfacilitatedtheopeningoftubulesentranceafteracid(j).CNMEachieved acompleteobliterationofthedentinaltubules,evenafteracidchallenge,presentingahomogenousfilm(k,l).

proteinssuchascollagenandnon-collagenousproteinsfrom dentin.Nevertheless,theuseofPBSorsimulatedbodyfluidas adentinaltubularfluidcouldresultinsedimentationinthe glasscapillarywhatwouldimpairconsistencyoftheresults. Thisisthereasonwhydeionizedwaterwaschosenforthis study.

TheSEMimagesaftertheapplicationofGLUMA(Fig.5c) revealedonlyminorocclusionoftubulesdemonstratedbythe decreaseintubulediameter,whichbecamewide-openafter acidexposure(Fig.5d).GLUMApossesseslowacid dissolu-tionresistance[8],whichmaybeexplainedbyitshydrophilic componentsthatcanbeeasilyremovedordegradedinacidic environment.Itisproposedthatduringthereactionbetween aminogroup-containingsubstancesindentinand

glutaralde-hyde,thepolymerization ofHEMA takeplaceandstart the formationofaHEMApolymer[23].Nevertheless,the leacha-bilityofHEMAiswell-knownduetoitssmallmolecularweight andhydrophilicity,whichindeedmaycontributetohigher dis-solutioninacidsolutions.Clinicallythisimplicatesinmany returnsofpatientstore-applythedesensitizingagent,thereby increasingthecostsandreducingthesatisfactionwith treat-ment.

The further desensitizers obtained statistically similar reductionondentinal fluidflow.However,CNMEmonomer achievedanumericallyhigherreductiononpermeabilitythan the others, especially when compared to OCB, which pre-sentedanintermediaryreduction(Fig.4).OCBisawater-based two-step etch-and-rinseadhesivechosenforthisstudydue

togreatbonding performanceand resistanceto thermocy-clingdegradation [24]. Moreover,OCBcontains HEMAinits composition(similartoGLUMA)andwaterasasolvent,thus, it isinterestingtoinvestigate the resistanceofarelatively hydrophilicadhesivetoacidchallenge.Thepresenceofthese hydrophiliccompoundsmayleadtohigherpermeabilitydue toleachingofHEMAorevenahydrolyticdegradationofthe polymer [25]. On the other hand, CNME is a hydrophobic monomerthatcancreatearegularlayertherebypreventing thesetypesofdegradation.

ThesurveyofBrunton et al.[8] revealed alevel ofacid resistance of OCB. On the other hand, in this study OCB exhibited greatamount ofwater droplets beforeand after acidchallenge(Fig.5eandf)onSEM,alsodepictingsignsof adhesivelayerdegradation(Fig.5f).Thewaterpermeabilityof simplifiedhydrophilicdentaladhesivesresemblespermeable membranes,whichallowshigherfluiddiffusion[26].Indeed, inaclinicalpointofview,thelongevityofdesensitizing treat-mentmaybejeopardizedbysuchimperfections[27].

The desensitizing containing unsaturated cardanol depictedacorrugatedlayer(Fig.5g)duetoanincomplete poly-merization.Itisexpectedthatonlymethacrylatemonomers have been light-cured and unsaturated cardanol remained unchangedwithinthepolymerformed.Theeffective antiox-idantrole ofphenols oftheCNSL has been evidenced [28] andmay playarole onsuchincompletepolymerization of desensitizer.Thisdefectiveandsuperficiallayerwasremoved afteracid,althoughsometubulesremainedpartiallyoccluded (Fig.5h).ThedesensitizingcontainingtheintermediaryCNE exhibiteda layerwithclearphaseseparation (Fig. 5i).This couldbeduetothepresenceofthehydroxylfunctionalityof cardanol-epoxypendant toaromatic ringthat canactas a hydrophilicsitepermeatingthehydrophobicUDMA-polymer. Afteracid challenge,the entrance ofthe tubules was also exposed, most likely caused by hydrolytic degradation of CNE (Fig. 5j), which does not participate on methacrylate co-polymerizationduetotheabsenceofsuchpolymerizable functionality. Nevertheless, CNE showed higher occlusion afteracidthanCNU,thankslikelytoanincreaseof crosslink-ing obtained by the acid-catalyzed hydrolysis of epoxides [13,14].

CNUandCNEshowedagreatreductionofpermeability, evenafteracidchallenge,whichseemscontradictorywiththe SEMresults.Thisdiscrepancybetweendentinalfluidflowand SEMobservationsmightbeexplainedbecauseSEM observa-tion isableto assessonlysuperficial characteristics whilst dentinalfluidflowmightbeaffectedbyinternaltubular occlu-sion. Kolker et al. also demonstrated that there is not a correlationbetweenthesetwoexperiments[22].

The desensitizing containing the final monomer CNME attained uniformand occludedlayer beforeand afteracid (Fig.5kandl).CNMEmethacrylatefunctionalityensuredthe regularphoto-polymerizationandformationofahomogenous polymer.Furthermore,methacrylateremovedthehydrophilic hydroxyland,consequently,generatedamorehydrophobic monomer.Thishydrophobicityyieldsapolymerwithlower wettability, water sorption, water droplets and hydrolytic degradationthan thehydrophilicones[25].Also,the open-ingoftheoxiraneringinacidenvironmentandtheresulting increased crosslinking might create a smart material [29],

whichbecomesmoreresistantincontactwithmanydifferent acidsfromthedietofpatientsovertheyears.

CNMEmonomerwassynthesizedthroughasimplified pro-cedure and emerges as a potential dental monomer that can add value to a natural by-product from the cashew nut industrial processing,which isproduced inlarge scale (1,000,000tonnes in 2013) [11]. Besides, it is a HEMA-free desensitizer, which might be a safer option to reduce dentin sensitivity [7]. Furthermore, CNME confirmed that itshydrophobicityandacid-resistancedevelopedapotential long-lastingdesensitizingagent,improvingthematerialover time.

5.

Conclusion

CNME showeda significant reductionofdentin permeabil-ityandauniformoccludedsurfaceevenafteracidexposure, suggestingthatitmaybeasuitableacid-resistanttreatment optionfordentinhypersensitivity.

Acknowledgments

This research was supported by CNPq-Brazil (grant 457931/2014-0) and Capes (grant 23038.006958/2014-96) (PIVPF).

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