Efficacy of new natural agents from Anacardiaceae extracts on dentin collagen crosslinking

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j ou rn a l h o m epa ge :w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / d e m a

Efficacy

of

new

natural

biomodification

agents

from

Anacardiaceae

extracts

on

dentin

collagen

cross-linking

M.A.

Moreira

_,

_N.O.

_Souza

_,

_R.S.

_Sousa

_,

_D.Q.

_Freitas

_,

_M.V.

_Lemos

_,

D.M.

De

Paula

_,

_F.J.N.

_Maia

_,

_D.

_Lomonaco

_,

_S.E.

_Mazzetto

_,

V.P.

Feitosa

a_{DentalSchool,FederalUniversityofCeara,CampusofSobral,Sobral,Brazil} b_{DentalSchool,PPGO-UFC,FederalUniversityofCeara,Fortaleza,Brazil}

c_{DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeara,Fortaleza,Brazil} d_{PauloPicanc¸oSchoolofDentistry,Fortaleza,Brazil}

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Articlehistory:

Received28March2017

Receivedinrevisedform7June2017 Accepted8July2017

Keywords:

Dentin Collagen Biomaterials Restorativedentistry Biomechanics

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Objectives.Severalpolyphenolsfromrenewablesourcesweresurveyedfordentin biomodi-fication.However,phenolsfromcashewnutshellliquid(CNSL,Anacardiumoccidentale)and fromAroeira(Myracrodruonurundeuva)extracthaveneverbeenevaluated.Thepresent inves-tigationaimedtocomparethedentincollagencrosslinking(biomodification)effectiveness ofpolyphenolsfromAroeirastembarkextract,proanthocyanidins(PACs)fromgrape-seed extract(Vitisvinifera),cardolandcardanolfromCNSLafterclinicallyrelevanttreatmentfor oneminute.

Methods.Three-pointbendingtestwasusedtoobtaintheelasticmodulusoffully dem-ineralizeddentinbeamsbeforeandafterbiomodification,whilstcolorchangeandmass variationwereevaluatedafterfourweekswaterbiodegradation.Coloraspectwasassessed byopticalimagesafterbiodegradationwhereascollagencross-linkingwasinvestigatedby micro-Ramanspectroscopy.Statisticalanalysiswasperformedwithrepeated-measurestwo wayANOVAandTukey’stest(p<0.05).

Results. The increase in elastic modulus after biomodification was in the order car-dol>cardanol>aroeira=PACswithcardolsolutionachievingmean338.2% increase.The massincreaseafterbiomodificationfollowedthesameorderaforementioned.Nevertheless, afterfourweeksaging,morehydrophobicagent(cardanol)inducedthehighestresistance againstwaterbiodegradation.Aroeiraandcardolattainedintermediateoutcomeswhereas PACsprovidedthelowerresistance.Tannin-basedagents(AroeiraandPACs)stainedthe specimens indarkbrowncolor. Nocolor alterationwasobservedwithcardol and car-danoltreatments.Allfouragentsachievedcrosslinkinginmicro-Ramanafteroneminute application.

Significance.Inconclusion,majorcomponentsofCNSLyieldoverallbestdentin biomodifi-cationoutcomeswhenappliedforoneminutewithoutstainingthedentincollagen.

©2017TheAcademyofDentalMaterials.PublishedbyElsevierLtd.Allrightsreserved.

∗ _{Correspondingauthorat:LabPPGO-UFC,DentalSchool,FederalUniversityofCeará,RuaMonsenhorFurtadoS/N,Fortaleza,CE60430−350,} Brazil.

E-mailaddresses:victorpfeitosa@hotmail.com,victor.feitosa@facpp.edu.br(V.P.Feitosa).

http://dx.doi.org/10.1016/j.dental.2017.07.003

1.

Introduction

Majordrawbacksindentinbondarerelatedtoenzymaticand hydrolyticdegradationofhybridlayerandresin-sparse colla-genmatrix[1].Itishypothesizedthatthequalityandlongevity ofthedentinadhesionmaybeimprovedbyincreasingthe dentin collagen mechanical properties [2]. In this regard, dentinbiomodificationimprovesthestiffnessoftheadhesive interfaceandprotectsthecollagenfibrilsfrombiodegradation bypromotingcollagencrosslinking[3].

A variety of crosslinking (biomodification) agents have provedtoeffectivelyincreasemechanicalpropertiesofdentin organic matrix [4]. Nevertheless, condensed tannins, also knownasproanthocyanidins(PACs),fromgrapeseedextract (Vitisvinifera)areagroupofplant-derivedpolyphenolswith highlight and high crosslink potential to collagen thereby providingbiostability[3,5–7].Thepositiveinfluenceofthese substancesonbondstrength[8]anddentinpropertiessuch asultimatetensile strength[4], resistanceto demineraliza-tion/biodegradation [9,10] and elastic modulus [11,12] was describedinseveralinvestigations.Duetothedarkcolorof PACssolution,themainshortcomingforitsclinicalusestillis therisktostaintoothsubstrates[13].

Further plant extracts from Anacardiaceae such as

Anacardiumoccidentale (cashew) andMyracrodruonurundeuva

(Aroeira) [14] have potential significant capabilities for application indentin biomodification.Cardol and cardanol are long carbon-chain phenols (Fig. 1) obtained from the

industrial extractionofcashew nutshell liquid (CNSL)[15]

during productionofnuts. They haveantioxidant capacity

[16,17],enzymeinhibitorypotential[18,19]andtwo hydrox-ylsinp-position(inthecaseofcardol)similartothoseofPACs (Fig.1).ExtractsofAroeirashowedanti-inflammatory prop-erties[20,21]andantimicrobialactivity[22].Moreover,major polyphenols of Aroeira are characterized by hydrolysable tannins [23] with high chemical interaction with proteins producingtannin-proteincomplexesrelativelyinsolubleand resistanttodegradation[24].

Although several plant-derived polyphenols were tested fordentincollagencrosslinking[25,26],cardol/cardanolfrom cashewnutshellliquidandAroeiraextracthaveneverbeen evaluated. Therefore, the aim ofthe present study was to determinetheeffectsofcardol,cardanolandAroeiraextract ascrosslinkersonmechanicalpropertiesanddegradationof demineralizeddentinincomparisontoPACswithinclinically relevantapplicationtime.Thehypothesistestedwasthat car-dol,cardanol,PACsandAroeiraprovidesimilarincreaseon elasticmodulusandmassvariationofdemineralizeddentin beams.

2.

Materials

and

methods

2.1. Preparationofbiomodificationsolutions

Cardolandcardanolwereobtainedfromindustrialcashewnut shellliquid(CNSL)suppliedbyAmendoasdoBrasilLTDA (For-taleza,Brazil)separatedemployingamethodologydescribed

Fig.1–Chemicalstructuresofbiomodificationagentssurveyed.(a)Mainpolyphenolfromaroeiraextract.(b)Major structureofproanthocyanidinsfromgrapeseedextract.(c)ChemicalstructureandNMRgraphofpurifiedcardanol.(d) ChemicalstructureofcardolwithitsrespectiveNMRgraph.*Descriptionofnuclearmagneticresonancegraphsand outcomesofgaschromatography/massspectroscopyanalyses:cardanol—Lightbrownoil,1_HNMR(CDCl

3,ı):1,03(t);1,06;

1,08;1,36;1,45;1,50;1,71;2,19(t,2H);2,65;2,94;2,95;5,13(m)5,21(m);5,53(m);6,63(m,1H);6,65(m,1H);6,73(d,2H);7,13 (t,1H)ppm.GC/MS(EI):m/z=302.Cardol—Darkbrownoil,1_{HNMR(acetone-d6,ı):0,80(t);1,26(m);1,51(m);2,05(m);2,39}

byLomonacoetal.[27].Theproductswerealsocharacterized bygaschromatography/mass spectrometry and 1_{H nuclear}

magneticresonance (NMR) toensure their purity[27]. The CNSLcomponentsweredilutedinEtOH/H2O(1:1volumeratio)

at2wt% concentration. Aroeira extractwas obtained from commercial mastic stem bark (M&A Natural Products, For-taleza,Brazil) of M. urundeuva species through infusion in EtOH/H2O(1:1volumeratio)with15%bark(15gbarkin100mL

hydroethanolicsolution).After5minagitation in25◦_C,the

mixturewasfilteredtwicetoobtain thesolutionofAroeira polyphenols with approximately 2% concentration, as the totalamountofpolyphenolsinAroeirastembarkis approx-imately 13% [23]. Proanthocyanidins (PACs) solution was preparedafterdissolving6.5%grapeseedextract(V.vinifera, MeganaturalGold,Madera,CA,USA)inEtOH/H2O(1:1volume

ratio)with5minagitationat25◦_{Candfinaldoublefiltering.}

ThesolutionswerebufferedtopH7.2.Ethanol/watersolution wasemployedtostandardizethedissolutionofagents,once cardanolhasverylowsolubilityinonlydistilledwater. Chem-icalstructuresofmajorpolyphenolfromAroeira,monomeric unitofproanthocyanidinsfromgrape-seedextract,and struc-turesofcardolandcardanolaredepictedinFig.1.

2.2. Structuralcharacterizationofcardanolandcardol

NMR spectra were recorded on Avance DRX-500 (500MHz for 1_{H, Bruker, Bremen, Germany) using CDCl}

3 as solvent

for cardanol, and acetone-d6 for cardol. The gas chro-matography/massspectrometryanalyseswereperformedon GC/MS QP-2010 Ultra (Shimadzu, Tokyo, Japan), equipped witha(5%-phenyl)-methylpolysiloxane(DB-5) capillary col-umn(30m×0.25mm),usinghelium(He)ascarriergasand

aflowrateof1mL/mininasplitlessmode.

2.3. Samplepreparation

Twentyfiveextractedcaries-free humanthirdmolarswere used in this study after approval by the Research Ethics CommitteeofFederalUniversityofCeará(protocol1482602). Teethwerecutwithslow-speedwater-cooleddiamondsaw (Isomet 4000; Buehler, Lake Bluff, USA). One 0.5mm-thick disk from middle coronal dentin was obtained from each tooth [12]. Disks were then sectioned into dentin beams (1.7mm×0.5mm×6mm).Atotalof75beamswereobtained

andwerecompletelydemineralizedin10%H3PO4solutionfor

5hat20◦_{C[12].Demineralizationwasconfirmedwithdigital}

radiography.

2.4. Dentinbiomodification

Demineralized dentin beams (n=15/group) were randomly dividedinfivegroupsandtreatedwithdistilledwater(control) oroneofthefourbiomodificationsolutionstested(PACsfrom grapeseedextract,cardol,cardanolandAroeirabarkextract). Beforethetreatment,theinitialdryweightandinitialflexural modulus(three-pointbendingtest)wereassessedas previ-ouslydescribed[12].Forbiomodification,thedemineralized beamswereindividuallyimmersedin1mLofeachsolution for1min,tosimulateaclinicallyrelevanttime.Afterwards, beamswerevigorouslyrinsedwithdistilledwaterfor30sto

removeunboundcrosslinkers.Theflexuralmoduluswas re-assessed immediately afterimmersion,and treatedbeams were individually storedin 1mL artificial salivacontaining 5mMHEPES,2.5mMCaCl2,0.05mMZnCl2and120mMNaCl

(pH7.4)at37◦_{Cforfourweekstoundertakecollagen}

degrada-tion[28].

2.5. Elasticmodulus

Specimens were testedon three-pointbending set-up ina universaltestingmachine(Instron4484;InstronInc.,Canton, USA), witha 5Nload cell at0.5mm/mincrosshead speed. Load–displacement curves were converted to stress–strain curves.Widthandthicknessofthespecimensweremeasured andtheapparentelasticmoduluswascalculatedat3%strain aspreviouslydescribed[12].DatawereexpressedinMPa,and thepercentagevariationinelasticmoduluswascalculatedas theratioofthefinalvalue(afterbiomodification)totheinitial values(baseline).

2.6. Masschange

Demineralizeddentinbeamswereweighed before(M1)and

after(M2)dentinbiomodificationwithananalyticalbalance

(0.01mgprecision,AUX-220,Shimadzu,Tokyo,Japan).Further massassessmentwassurveyedafter4weeksdegradationin artificialsaliva(M3).Ineachperiod,specimenswereinitially

driedinavacuumdesiccatorcontainingsilicagelbeadsfor 72hatroomtemperature.Massvariation(Wmc%)was

deter-mined as the percentage ofgain or lossin mass foreach specimenaspreviouslydescribed[12]basedonthefollowing formulaforbiomodification:

Wmc(%)= [(M2×100)/M1]−100,

where M1 is the demineralized dentin beam mass before

dentin biomodification and M2 is the massof biomodified

dentinmatrix. Moreover,to assess the biodegradation per-centagemassvariation,thefollowingformulawasused:

Wde(%)= [(M3−M1)×100]/M1,

where M1 is the demineralized dentin beam mass before

dentinbiomodificationandM3isthemassofdentinmatrix

after four weeksartificial salivaimmersion. Afterthe final weighing,pictures ofeachspecimenwereobtainedusinga professional camera(Canon, Tokyo,Japan)withMacro lens (100mm,Canon)inordertoobservethefinalcolorandaspect ofbiomodifieddemineralizeddentinbeams.

2.7. Micro-Ramanspectroscopy

Vibrationalanalysisofthe demineralizeddentinspecimens beforeandafterbiomodificationtreatmentasaforementioned was assessed using the Micro-Raman spectrophotometer (Xplora,HoribaJobinYvon,Paris,France)calibratedinternally inzerousingthesiliconstandardsampleprovidedbythe man-ufacturer.TheconfigurationoftheequipmentwereHeNelaser with 3.2mW power, 633nmlaser wavelength, 10s acquisi-tiontime,5accumulations,1.5␮mspatialresolution,2.5cm−1

Table1–Mean(SD)ofelasticmodulus(MPa)ofdemineralizeddentinspecimens.

Initialmodulus(MPa) Modulusaftertreatment(MPa) Elasticmodulusvariation(%)

Control(H2O) 2.76(0.8)a 2.62(0.6)a -5.0(2.3)D

Grapeseedextract 2.45(0.7)a 3.83(0.9)b 56.3(5.0)C

Cardol 2,23(0.5)a 9.78(2.1)d 338.2(45.1)A

Cardanol 2.91(1.1)a 6.11(1.4)c 109.9(16.0)B

Aroeirabarkextract 2.92(0.9)a 4.66(1.2)b 59.5(7.9)C

Differentcapitallettersdepictsignificantdifferenceinpercentagevariation(p<0.05)whilstdifferenttinylettersindicatestatisticaldifference onelasticmodulus(p<0.05).

UK)and60×70␮mfieldarea.Forobservationofdentin

col-lagencross-linking,the rangewas700–1800cm−1 _tosurvey peaks/shouldersat1117cm−1 _and1235cm−1_accordingtoa previousinvestigation[29].Ramananalyseswereperformed intriplicatepergroup.

2.8. Statisticalanalysis

Analysisofelasticmodulusdatawascarriedoutviaone-way repeated-measuresANOVA(biomodificationagent),followed by Tukey’s post hoc test (p<0.05), after passing normality (p=0.657)andequalvariance(p=0.335)tests.Thedataof per-centagemodulusvariationresults,percentagemasschange after biomodification, and percentage mass variation after 4weeksdegradation were separately analyzed byone-way ANOVAandTukey’stest(˛=5%).

3.

Results

NMR analysis demonstrated 99.8% purityand detection of solelycardanol (Fig. 1c) andcardol (Fig.1d) structures sep-arately.Means ofelastic modulusand standard deviations are presented in Table 1. The statistical analysis depicted significantdifferencesbetweenbiomodificationgroupswhen compared to control group (p<0.05). Modulus was signifi-cantlyincreasedbybiomodificationregardlesstheagentused. Thehigherresultswereobservedwhenusingcardol(mean 338.2%increase)followedbycardanol(mean109.9%increase). ThetreatmentswithAroeira(mean59.5%increase)and proan-thocyanidinsfromgrape-seedextract(mean56.3%increase) werestatisticallysimilarandachievedhighermodulusthan control(distilledwater),butlowerthanCNSLreagents.

MasschangeoutcomesareshowninTable2.Cardol pro-vided the highestincrease ofweightafter biomodification, followed by cardanol. Aroeira (mean 8.6%) and PAC(mean 9.1%) induced similar mass increase (p=0.739). After four weekswaterdegradation,controldemonstratedmean39.5% massdecrease which wasstatistically similar (p=0.892) to PAC (mean 38.6% decrease). Cardol and Aroeira presented

Table2–Mean(SD)ofmassvariation(%)ofdentin specimens.

Massvariation(%) Afterbiomodification 4weeks degradation

Control(distilledwater) +0.8(0.3)d −39.5(6.9)C Grapeseedextract +9.1(3.0)c −38.6(4.3)C

Cardol +22.9(5.8)a −21.5(2.9)B

Cardanol +15.1(2.2)b −5.0(2.3)A

Aroeirabarkextract +8.6(1.8)c −19.8(7.6)B

Differentlettersindicatestatisticallysignificantdifference(p<0.05) withineachcolumn.

lowermassreduction(mean21.5%and19.8%respectively)in comparisontocontrolandPAC(p<0.05). Cardanolafforded thehighestresistanceagainstbiodegradationwithmean5% massdecrease.

Representativepicturesofspecimensafterbiodegradation arepresentedinFig.2.Darkcolorwasobservedinspecimens treatedwithPACandAroeirawhilstthosetreatedwith car-dolandcardanoldidnotdemonstratesignsofpigmentation. Controlspecimensdepictedaspectofhighlydegraded dem-ineralizeddentin.Ramanspectra(Fig.3)showed,forallfour biomodificationagents,emergenceofashoulderat approxi-mately1117cm−1_{andincreaseofapeakat1235cm}−1_which

representscollagencrosslinking.

4.

Discussion

Thepresentstudydeterminedtheeffectivenessofthreenew plant-derivedcompoundsasbiomodifiersinfully demineral-izeddentin.Itwasobserveddifferencesinspecimens’elastic moduli among experimentalsolutions, and biodegradation rateswerestrikinglydistinct.Therefore,thetestedhypothesis needstoberejected.

Crosslinkingofdentinmayaffordstabilization, biodegra-dationresistanceandstrengtheningtocollagenmatrix[26]. Morestableandresistantcollagennetworkishighly signifi-cantforitsfunctionasascaffoldsubstratefordentaladhesive infiltration [30]. The use of biomodification agents is

Fig.3–Vibrationalspectraofmicro-Ramananalysisfromsamespecimensbeforeandafter1minbiomodification treatment.Allreagentsinducedemergenceofshoulderat∼1117cm−1(blackarrow)andincreaseofAmideIIIpeakat

1235cm−1_{(dottedline)whichdemonstratescollagencrosslinkingaccordingtoLiuetal.[29].(A)Spectraoftreated(black)}

anduntreated(grey)specimenssubjectedtotreatmentwithproanthocyanidinsofgrapeseedextract.(B)Spectraoftreated (black)anduntreated(grey)specimenssubmittedtotreatmentwithAroeiraextract.(C)Spectraoftreated(black)and untreated(grey)specimenswhichundergonetreatmentwith2wt%cardolsolution.(D)Spectraoftreated(black)and untreated(grey)specimenssubjectedtotreatmentwith2wt%cardanolsolution.

posed to improve mechanical properties of the dentin, to increaseresin–dentinbondstrength,toreducebiodegradation ratesofdemineralizedcollagenand,consequently,toextend thelongevityofadhesiverestorations[2,4,31].However,one limitationofthe present investigation andresearch design comprises the usageof0.5mm-thick demineralized dentin specimens,whilstthephosphoricaciddemineralized thick-nessofdentinin the clinicalscenario representsonlyfew micrometers[10].Indeed,thecross-linkingpotentialoftested biomodificationagentsmightbeimprovedinathinnerzone forinteraction.

Tannins are naturaland biocompatible polyphenols [14]

withoutstandingpotentialasbiomodificationagents[26]due totheirhighmolecularweightandtendencytoformcomplex aggregateswithproteins.Inthisstudy,twomaincategories oftanninsweretested:thehydrolysabletannins,whichare presentinAroeirabarkextracts(M.urundeuva)[23]and con-densed tannins or proanthocyanidins (PACs), the principal polyphenolsofgrape-seedextract[26].Furthermore,wetested cardolandcardanolfromCNSL.Toourknowledge,thisisthe firststudytosurveycardanolandcardolfordentininteraction (anddentalresearch).

Biomodificationagentswithlowermolecularweight(such ascardolandcardanol)mayattaingreaterandfaster pene-trationindentincollagenmatrix[32],whichmayexplainthe highestincreaseinmassandinelasticmodulusafter1min applicationofcardolandcardanolsolutions(Tables2and1

and grape-seedextract areunlikely toinduce formationof hydrophobicinteractions.

Apartfromtheincreaseinelasticmodulus,the biodegrada-tionresistanceisrelatednotonlytotheformationofcollagen crosslinks,butalsowiththehydrophilicityofthe biomodifica-tionagent[7].Cardolandcardanolarerelativelyhydrophobic incomparisonwithtannins,buttheformerisslightlymore hydrophilic than the latterdue tothe additionalhydroxyl. Morehydrophobicfeatureofcardanoljeopardizesthedentin diffusionandincreaseofmassaftertreatment.However, col-lagenfibrilsconnectedwithcardanolmoleculesmighttendto becomemorehydrophobic,avoidingthewaterseepage dur-ing 4-week storageand consequently diminishing collagen degradation(Table2).

AlthoughPACspresentmultiplefreephenolfunctionalities (Fig.1b),theoligomericstructureswhichhavehigh biomod-ification potency [33], also have higher molecular weight, whatcouldjustifytheirslowerdiffusionincollagenmatrix, thereby explaining the reduced effectiveness in increasing elasticmodulusanddegradationresistancewhenappliedfor 1min.MostinvestigationsdepictedtheefficacyofPACsfrom grape-seedextractwhentheywereappliedfor1horlonger periods[4,11,12]. However,theefficacy ofPACsinclinically relevantapplicationtimehasalsobeendemonstratedinthin (fewmicrometers)demineralizeddentinlayer[10].

Hydrolysable tannins were related tosome interactions withproteins[23].However,noneofthebiomodification stud-ieshavetestedtheireffectsonthedentincollagenmatrix[26]. Aroeirabarkextracthasrepresentativehydrolysabletannins withmanygalloylgroups(Fig.1a)whichareabletointeract through hydrogen bondswiththe side chainsofhydroxyl, carboxyl-amineor amidegroups ofthecollagen molecules

[5]andcapableofinducingmorecovalentandnon-covalent bonds[3,26]. The formation ofthese hydrogen bondsmay occurlately afterpartialhydrolysisofthepolyphenols dur-ingwaterstorage.Therefore,althoughthepositiveeffecton elasticmoduluswassimilar(Table1)betweenbothtannins (PACs and Aroeira), the degradation resistance after using hydrolysabletannins(Aroeirabarkextract)solutionwas aug-mentedincomparisonwithPACs(Table2),likelyduetolater formationofcollagencrosslinks.

One of the benefits of using biomodification agents to crosslink dentinal collagen is that during formation of crosslinker-protein bonds, they may remove proteoglycans fromdentinmatrix,replacewaterboundtofibrillarcollagen andreducehydrophilicity[7]whatmayalsoaffordhigher dif-fusionofresinmonomersinto thefully expandedcollagen network[32].Hence,theyare abletoreducebiodegradation rates[26,31].Indeed,suchmechanismofimprovingadhesive penetrationandincreasinglongevityofadhesiveinterfaces needstobeverifiedforthesenewbiomodificationagents sur-veyedinthisstudy.

DentinspecimenstreatedwithPACsandAroeirapresented brownishcolor (Fig.2).This colorchange outcome may be attributedtotheiroxidativeproperties,darkcolorofsolutions and content of polymeric high-molecular weight polyphe-nols[13,14,26].Althoughpurecardolandcardanolare dark oils,their solutionsare transparent andtreated specimens didnotdepictnoteworthycoloralteration(Fig.2).Thismay be explained by their lower molecular weight and lesser

capacitytoformpolymericstructures.Infact,thecolor sta-bilityofdentinafterbiomodificationwithcardanolandcardol mightbeconsideredanotheradvantagefortheirclinicaluse andadrawbackwhenusingPACsandAroeirasolutions.

Cardolandcardanolcorrespondtomorethan95%ofthe compositionoftechnicalcashewnutshellliquidproducedin theindustries[27].Suchliquidisaproductofdiscard (byprod-uct) resultedfromtheboiling ofnutsand itwasestimated thatonemilliontonsareproducedyearlyworldwide[34].This liquidcannotbereleasedintheenvironment[27]duetoits veryslownaturalbiodegradation.Therefore,dental applica-tionofcardolandcardanolmayrepresentasortof“recycling” processtherebyprovidingusefulutilizationinrestorative den-tistrywithsustainability.

Cardol and cardanol are non-cytotoxic [15] compounds in low concentrations possessing antioxidant effects [16]

and,similar toPACs[13], theypresent inhibitionofmatrix metalloproteinase-2 and matrix metalloproteinase-9 [18]. Bothcardolandcardanoldemonstratedremarkablepotential asdentinbiomodificationagentsinthepresentinvestigation asalsodemonstratedbymicro-Ramanspectroscopy(Fig.3), attaining thehighestincreaseinstiffnessofdemineralized dentin and resistance to degradation without specimens’ staining.

Furthermore,unlikepolyphenolsfromAroeiraandPACs, bothreagentsaresinglemolecules(Fig.1)pronetochemical synthesisofdentalmonomerswhichwouldaffordthe design-ingofcollagen-bindingandcollagen-crosslinkingmonomers withsubstantialperspectivefordentinbond.Indeed,further studiesareneededtounderstandlong-termbenefitsofthese

Anacardiaceae derived crosslinkers aswell astheir biocom-patibility with odontoblast-like cells. In conclusion, cardol demonstrated the highest potential to increase the elastic modulusofdemineralizeddentinwhilstcardanoltreatment attainedthelowestdegradation.Bothreagentsfromcashew nutshellliquidachievedoverallbestoutcomeswithout stain-ingdentinspecimens.

Acknowledgment

This research was supported by CNPq-Brazil (grant 457931/2014-0). The authors have no financial interest in anyoftheproductsusedinthisstudy.

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