Effects of zoledronic acid on odontoblast-like cells

Effects

of

zoledronic

acid

on

odontoblast-like

cells

F.G.

Basso

,

A.P.S.

Turrioni

,

J.

Hebling

,

C.A.

de

Souza

Costa

*

1.

Introduction

Bisphosphonatesareaclassofsyntheticanalogsof pyrophos-phate,whichhavebeenwidelyusedintreatmentofdiseases withintenseboneactivityresorption,suchasosteoporosis, Paget’s disease and some bone tumours,such as multiple myeloma, and bone metastases of breast and prostate tumours.1,2

Thesedrugsarephysiologicalmodulatorsofbone resorp-tion and calcification with high affinity for hydroxyapatite crystals,thusremainingadheredtothemineralizedtissuesof body.3,4 _{In the same way as the bone tissue, dentin is}

characterized as a partially mineralized connective tissue withgreathydroxyapatitecontent, andrecentstudieshave beensuggestedthatbisphosphonatescanalsoadheretothis

dental tissue.5_{However,todate,littleisknownabout how}

bisphosphonates adhere to the dental tissues, the mecha-nismsbywhichthisadherenceoccursortheconditionsunder whichthesedrugsarereleasedtothepulp.

In vivo studies have demonstrated that treatment with bisphosphonatesduringtheformationofteethwasassociated withtheoccurrenceofamelogenesisimperfectaand forma-tionofadisorganizeddentintissue.6,7 _Sakaietal.6_reported

that bisphosphonates can adhere to dentin, promoting a completeorintermittentinhibitionofdentinogenesis.

Ithasbeendescribedthatbisphosphonatescanbereleased frommineralizedtissuesduringboneresorptionor remodel-ling.8_{Itisknownthatremodellingprocessesofmineralized}

tissues do not occur in the adult dentition. However, pulpal injuries caused by events, such as trauma and chronic inflammatory processes,could activateodontoclast

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Articlehistory:

Accepted27September2012

Keywords: Bisphosphonates Cellculture Cytotoxicity

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b

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The aim of the studywas to evaluatethe effects ofa highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5104cells/cm2)wereseededfor48hinwellsof24-welldished.Then,theplainculture

medium(DMEM)wasreplacedbyfreshmediumwithoutfetalbovineserum.After24h,ZOL (1or5mM)wasaddedtothemediumandmaintainedincontactwiththecellsfor24h.After thisperiod, thesuccinicdehydrogenase(SDH) enzymeproduction(cellviability –MTT assay),totalprotein(TP)production,alkalinephosphatase(ALP)activity,andgene expres-sion(qPCR)ofcollagentypeI(Col-I)andALPwereevaluated.Cellmorphologywasassessed bySEM.FivemMZOLcausedasignificantdecreaseinSDHproduction.BothZOL concentra-tionscausedadose-dependentsignificantdecreaseinTPproductionandALPactivity.ZOL also produced discret morphological alterations inthe MDPC-23 cells. Regarding gene expression,1mM ZOLcaused asignificant increasein Col-Iexpression. Although5mM ZOLdidnot affectCol-Iexpression,itcaused asignificantalterationinALPexpression (ANOVAandTukey’stest,p< 0.05).ZOLpresentedadose-dependentcytotoxiceffectonthe

odontoblast-likecells,suggestingthatunderclinicalconditionsthereleaseofthisdrugfrom dentincouldcausedamagetothepulpo-dentincomplex.

#2012ElsevierLtd.

*Correspondingauthorat:DepartamentodeFisiologiaandPatologia,FaculdadedeOdontologiadeAraraquara,UniversidadeEstadual Paulista,RuaHumaita´,1680.Centro,CaixaPostal:331,Cep:14801903Araraquara,SP,Brazil.Tel.:+551633016477;fax:+551633016488.

E-mailaddress:casouzac@foar.unesp.br(C.A.deSouzaCosta).

Available

online

at

www.sciencedirect.com

journalhomepage:http://www.elsevier.com/locate/aob

0003–9969#2012ElsevierLtd.

http://dx.doi.org/10.1016/j.archoralbio.2012.09.016

Open access under the Elsevier OA license.

differentiation and induce a resorptive process in dentin, ultimatelycausingthereleaseofthesedrugstointeractwith thepulptissue.5_{Anotherhypothesisoftheactionof}

bispho-sphonatesonthepulptissuewouldbetheircytotoxicityatthe momentofinfusion.However,itissuggestedthat,thelimited drugconcentrationatthemomentofinfusionwouldnotbe sufficienttoproduceacytotoxiceffectstothepulpcells.5

Recent studies have shown that bisphosphonates are cytotoxic todifferent cell types.5,9–11Therefore,the release ofthisdrugtothepulptissuecouldpromotecytotoxiceffects to the pulp cells, reducing the reparative capacity of this tissue.Inmammalianteeth,odontoblastsareorganizedina monolayerthat underliesthe coronaland rootdentin,and thustheseperipheralpulpcells wouldbethe firsttogetin contact with bisphosphonates released from dentin.12,13

Therefore,theaimofthisstudywastoevaluatetheeffects ofzoledronicacid(ZOL),ahighlypotent,heterocyclic nitro-gen-containingbisphosphonate,onodontoblast-likeMDPC-23 cells by evaluating succinic dehydrogenase (SDH) enzyme production (cell viability – MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, reverse transcriptasepolymerasechainreaction(qPCR)forcollagen typeI (Col-I) and ALP, and morphology (scanning electron microscopy–SEM).

2.

Material

and

methods

2.1. MDPC-23cellculture

Theodontoblast-likecellsMDPC-23usedinthisstudywere cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM; SigmaAldrichCorp.,St.Louis,MO,USA)supplementedwith 10%fetalbovineserum(FBS;Gibco,GrandIsland,NY,USA) andcontaining100IU/mLpenicillin,100mg/mLstreptomycin and2mmol/Lglutamine(Gibco)inanhumidifiedincubator with5%CO2and95%airat378C(IsotempFisherScientific,

Pittsburgh,PA,USA).TheMDPC-23cellsinDMEMcontaining 10%FBSweresub-culturedatevery2daysuntilanadequate numberofcellswereobtainedforthestudy,andthenplated (1.5104cells/cm2)ontosterile24-wellplates(CostarCorp.,

Cambridge, MA, USA), which were maintained in the humidifiedincubator with5%CO2and 95%air at378C for

48h.Threegroupswerethenestablished:onecontrolgroup, whichreceivednotreatment,andtwoexperimentalgroups, whichweretreatedwithZOLatconcentrationsof1mMand 5mM.

2.2. ZOLonMDPC-23cellculture

Zoledronicacid (ZOL) was selected forinvestigation inthe presentstudybecauseitisahighpotentbisphosphonateand, accordingtorecentstudies,14,15_{isoneofthemostfrequently}

prescribed bisphosphonates. In addition, several workers reportedthatthis nitrogencontainingbisphosphonatemay cause intense cytotoxic effects in different types of cell cultures,includingpulpcells.10,11,16

Afterthe48-incubationperiod,the culturemediumwas aspirated and replacedby freshFBS-freeDMEM containing ZOL(Zometa4mg;NovartisBiocieˆnciasS.A.,Sa˜oPaulo,SP,

Brazil)atconcentrationsofeither1mMor5mM,accordingto thegroup.Theseconcentrationswerechosenbasedonastudy byScheperetal.17_{whoshowedthatZOLcanbefoundatthese}

concentrations in the alveolarbone and saliva ofpatients undertreatmentwiththisdrug.Theculturemediumwiththe drugremainedincontactwiththecellsintheincubatorwith 5%CO2and95%airat378Cfor24h.

2.3. Analysisofcellviability(SDHproduction–MTT assay)

Cell viability was evaluated using the methyltetrazolium (MTT) assay.18–20 _{This method determines the activity of}

SDHenzyme,whichisameasureofcellular(mitochondrial) respiration,andcanbeconsideredasthemetabolicrate of cells.After24hofincubationofthecellsincontactwithDMEM alone (controlgroup)orcontainingthetwoZOL concentra-tions(experimentalgroups),theculturemediumwas aspirat-edandreplacedby900mLoffreshDMEMplus100mLofMTT solution (5mg/mLsterile PBS). Thecells wereincubated at 378Cfor4h. Thereafter,theculturemediumwiththeMTT solution was aspiratedand replaced by 700mL of acidified isopropanolsolution(0.04NHCl)ineachwelltodissolvethe violet formazancrystalsresulting fromthecleavage ofthe MTTsaltringbytheSDHenzymepresentinthemitochondria ofviablecells,producingahomogenousbluishsolution.After agitation and confirmation of the homogeneity of the solutions,three100mLaliquotsofeachwellweretransferred toa96-wellplate(CostarCorp.).Cellviabilitywasevaluatedby spectrophotometry asbeingproportionaltotheabsorbance measuredat570nmwavelengthwithanELISAplatereader (ThermoPlate,NanshanDistrict,Shenzhen,Gandong,China). Thevaluesobtainedfromthethreealiquotswereaveragedto provide a single value. The absorbance was expressed in numericalvalues,whichweresubjectedtostatisticalanalysis todeterminetheeffectofZOLonthemitochondrialactivityof thecells.

2.4. Totalproteinexpression

at655nmwavelengthwiththeELISAplatereader(Thermo Plate). Total protein production was calculated from a standardcurvecreatedusingknownproteinconcentrations.

2.5. ALPactivity

AnalysisofALPactivitywasperformedusingthe colorimet-ricendpointassay(ALPKit;LabtestDiagno´sticoS.A.,Lagoa Santa,MG,Brazil)employedinpreviousstudies.20Thistest usesathymolphthaleinmonophosphatesubstrate,whichis a phosphoric acid ester substrate. ALP hydrolyzes the thymolphthaleinmonophosphatesubstrate, releasing thy-molphthalein.Therefore,itispossibletomeasuredirectly the productofhydrolysis,altering the pH.Thealtered pH interruptstheenzymaticactivityandprovidesbluishcolour tothesolution,whichischaracteristicofthereaction.The intensityofthe resultingcolourisdirectly proportionalto theenzymatic activityandisanalyzed spectrophotometri-cally. After 24h incubation of the cells in contact with DMEM alone (control group) or containing the two ZOL concentrations(experimentalgroups),the culturemedium with ZOLwas aspirated andthe cells were washed three times with1mLPBS at378C.An amountof1mLof 0.1% sodiumlaurylsulphate(SigmaAldrichCorp.)wereaddedto eachwellandmaintainedfor40minatroomtemperatureto producecelllysis.Thetestwasperformedaccordingtothe instructions ofthe Kit’s manufacturer. Theabsorbance of the samples was measured at 590nm wavelength with a spectrophotometer(ThermoPlate).ALPactivity was calcu-latedbyastandardcurveusingknownconcentrationsofthe enzyme.

2.6. AnalysisofcellmorphologybySEM

SEM analysiswas usedto identify possiblemorphological alterations caused by the additionof different concentra-tionsofZOLtoDMEMculturemediuminwhichtheMDPC-23 cellswerecultured.Thefollowingprotocolusedinprevious studies was employed.20,21 _{Sterile 13-mm-diameter cover}

glasses(FisherScientific,Pittsburgh,PA,USA)weresterilized in70%ethanolfor24handplacedonthebottomofthewells immediatelybeforeseedingthecells.After24hof incuba-tionofthecellsincontactwithDMEMalone(controlgroup) or containing the two ZOL concentrations (experimental groups),theculturemediumwithZOLwasaspiratedandthe cellswerefixedin1mLof2.5%glutaraldehydeinPBSfor1h. Then,the glutaraldehydewas removedandthecellswere washedwithPBSandpost-fixedwith1%osmiumtetroxide for 1h at room temperature. The cells that remained adheredtothe glasssubstratewere washedwithPBSand distilledwatertwoconsecutivetimes(5mineach)andthen dehydratedinaseriesofincreasingethanolconcentrations (30,50and70%,onetimefor30mineach;95and100%,two timesfor60mineach)andcoveredthreetimeswith200mL of 1,1,1,3,3,3-hexamethyldisilazane (HMDS; Sigma Aldrich Corp.) at 20min intervals. The cells were mounted on metallic stubs, stored in a desiccator overnight, sputter-coatedwithgold,andtheirmorphologywasexaminedwith ascanningelectronmicroscope(JMS-T33Ascanning micro-scope,JEOL,Tokyo,Japan).

2.7. AnalysisofCol-IandALPexpression

2.7.1. RNAextractionandcDNAsynthesis

TheeffectsofZOLonCol-IandALPexpressionwasevaluated after48hofcontactofthedrugwiththecellsbytwo-stepreal timepolymerasechainreaction(qPCR),whichisasensitive andfastmethodtoevaluategeneexpression.Unlike conven-tionalPCR,thistechniqueneedsasmallnumberofsamples, less methodological standardization and no contaminant reagents. Another advantage is that the amplification can beobservedatanycycle,andnopostprocessingofsamplesis required.22

Forthistest,thecellweretransferredtomicrocentrifuge tubestowhich1mLoftrizol(Invitrogen,Carlsbad,CA,USA) wasaddedtoinhibittheactionofRNAasesandthecellswere incubated for 5min at room temperature. Next, 0.2mL of chloroformwasaddedforeach1.0mLdetrizol(SigmaAldrich Corp., St. Louis, MO, USA) to promote release of the cytoplasmicproteins.Thetubeswereagitatedmanuallyfor 15s,leftrestfor2–3minatroomtemperature,andcentrifuged at 1200rcf(MicrocentrifugeEppendorf model5415R, Eppen-dorf,Hamburg,Germany)for15minat48C.After centrifuga-tion, the samples presented three phases: a precipitated phase,correspondingtotheorganicportion(phenol, chloro-form, DNA), an intermediate phase (proteins) and a more aqueoussupernatantphase,correspondingtoRNA(RNAand buffer).

Theaqueousphasewasaliquotedtoanewtube,inwhich 0.5mLofisopropanol (Sigma–AldrichCorp.) was addedfor each 1.0mL of trizol to promote precipitation of RNA in solution.Thesamplesweremaintainedatroomtemperature for10minandthencentrifugedat12,000rcffor10minat48C. Afterthisstage,formationofaprecipitatedfraction(pellet) was observed at the bottom of the tube. Thesupernatant fractionwasdiscardedandtheprecipitatedphasewasdried by inverting the tubes onto a blotting paper sheet during 10min.Afterdrying, 1.0mLof75% ethanol(Sigma–Aldrich Corp.)was addedforeach1.0mLoftrizolandthe samples wereagitatedandcentrifugedat7500rcffor5minat48C.The supernatant fraction was discarded and the RNA was subjected to the same drying procedure for 30min. Next, the RNA was resuspended in 10mL of ultrapure water (Invitrogen) and the resulting solution was incubated at 558Cfor10min.PartoftheobtainedRNA(1.0mL)wasdiluted in ultrapure water at 1:50 forquantification ofRNA in an Eppendorfbiophotometer(modelEppendorfRS-232C, Eppen-dorf,Hamburg,Germany).

cDNAwas synthesizedfromeachRNAsampleforqPCR using the High Capacity cDNA Reverse Transcriptions Kit (AppliedBiosystems,FosterCity,CA,USA),accordingtothe followingprotocol.Inamicrocentrifugetubewereadded10

RTBuffer,10RTRandomPrimers,25dNTPMix,reverse transcriptase and 0.5mg of the RNA of each sample. The sampleswerethensubjectedtothefollowingamplification cycling conditions:258C (10min),378C(120min),858C(5s) and48Cthereafter.

2.7.2. qPCR

specificprimersweresynthesizedfromthemRNAsequence (Table1).Thereactionswerepreparedwithstandardreagents forqPCR(SyberGreenPCRMasterMix;AppliedBiosystems) together with the primer/probesets specific foreach gene (Table1).Thefluorescencereadingswereperformedusingthe StepOnePlusSystem(AppliedBiosystems)ateach amplifica-tioncycle,andwereanalyzedsubsequentlyusingtheStepOne Software 2.1 (Applied Biosystems). All reactions were sub-jectedtothesameanalyticalconditionsandwerenormalized by the ROXTM _{passive reference dye signal to correct}

fluctuationsonreadingresultingfromvariationsofvolume andevaporationduringthereaction.Theresult,expressedin CTvalues,referstothe numberofcyclesnecessaryforthe fluorescent signal to reach the detection threshold. The individualresults expressedinCT valueswererecordedin worksheets,groupedaccordingtothegroupsandnormalized according to the expression of the selected endogenous referencegene(b-actin).Then,theRNAmconcentrationsof eachtargetgenewereanalyzedstatistically.

2.8. Statisticalanalysis

Afteranalysisofdatadistribution(Shapiro-Wilk,p> 0.05)and

homogeneityofvariances(Levene,p> 0.05),cellviability(SDH

production), TP production, ALP activity and Col-I and ALP expression data were independently subjected to one-way analysisofvariance(treatment:control,1mMor5mMZOL).Once rejectedthenullhypothesisofabsenceofdifferencesamongthe groups,additionalTukey’stestswerealsoappliedforpairwise comparison.Asignificancelevelof5%wassetforallanalyses.

3.

Results

DatafromSDHproduction,TPproductionandALPactivityare presentedinTable 2. Theuse of1mMZOL didnotcause a significant(p> 0.05)reductioninSDHproductioncompared

withthecontrolgroup.However,SDHproductiondecreased significantlycomparedwiththecontrolgroup(p< 0.05)when

ZOLconcentrationincreasedto5mM.Nostatistically signifi-cant difference was found between the 1 and 5mM ZOL concentrations(Table2).

ApplicationofZOLontheodontoblast-likecellscauseda significant (p< 0.05) decrease in TP production and ALP

activity (Table 2) compared with the control group. No statistically significant difference (p> 0.05) was found

be-tweenthe1and5mMZOLconcentrations(Table2).

Col-IandALPexpressiondetectedbyqPCRarepresentedin

Fig.1.WhentheMDPC-23cellswereexposedtoZOLat5mM concentration, Col-I expression did not differ significantly

(p> 0.05)fromthecontrolgroupinwhichthedrugwasnot

used.Ontheotherhand,exposureto1mMZOLstimulatedthe odontoblast-likecellstoproduceCol-I,leadingtoastatistically significantincrease(p< 0.05)initsexpressioncomparedto

theothergroups.Astatisticallysignificantdecrease(p< 0.05)

inALPexpressionwasobservedwhenthecellswereexposed to5mMZOLcomparedwiththeexpressionofthisproteinin theothergroups(controland1mMZOL).

The SEManalysis ofthe odontoblast-like cells MDPC-23 incubatedincontactwithZOLrevealedthatboth concentra-tionsofthedruginducedmorphologicalalterations,especially reductionofcellsize,whichcreatedlargeintercellularspaces andexposedthecoverglassthatservedassubstrateforcell culture.Ontheotherhand,inthecontrolgroup,theMDPC-23 cellswerenearconfluenceandhadawidecytoplasmcovering theentiresurfaceoftheglasssubstrate(Fig.2).

Table1–Primersequencesusedforanalysisofgene expressionoftheodontoblast-likecellsMDPC-23(Mus musculus)subjectedtotreatmentwithzoledronicacid.

Gene Sequencesofprimers

Col-I Forward–50_ACGTCCTGGTGA AGTTGGTC30

Reverse–50_CAGGGAAGCCTC TTTCTCCT30

ALP Forward–50_GCTGATCATTCC CACGTTTT30

Reverse–50_CTGGGCCTGGTA GTTGTTGT30

b-actin Forward–50_AGCCATGTACGT

AGCCATCC30

Reverse–50_{CTCTCAGCTGTGGT} GGTGAA30

Table2–DatafromSDHproduction,TPproductionand ALPactivityofMDPC-23cellsexposedtozoledronicacid (ZOL)atdifferentconcentrations.

Group SDH

productiona _productionTP b _activityALP b

Controlgroup 0.659(0.094)a _{284.47(27.81)}a _{155.99(45.11)}a 1mMZOL 0.567(0.204)ab _{190.05(17.73)}b _86.60(39.59)b 5mMZOL 0.450(0.117)b _{171.86(30.03)}b _87.02(40.01)b

Allvaluesaregivenasmean(standarddeviation),n=8. a _{Valuesexpressedinabsorbance(470nm).}

b_{Values expressedin U/L.Means followed by sameletters in}

columnsdonotdiffersignificantly(Tukey’stest,p>0.05).

Fig.1–Col-IandALPexpression(qPCR)ofMDPC-23cells exposedtozoledronicacid(ZOL)atdifferent

concentrations.Columnsrepresentmeansanderrorbars representstandarddeviations,n=4.Columnsindicated

4.

Discussion

Bisphosphonates have been indicated for treatment of osteopenicandosteoporoticconditions.2_{Thehighaffinityof}

bisphosphonates forCa2+ _{ions and their strong binding to}

hydroxyapatitepromotesarapidincorporationofthesedrugs tothe tissues.4 _{ZOL is a highlypotent nitrogen-containing}

bisphosphonatethatpresentsaprolongedadhesiontobone surface and effect, and has been widely used for various clinicalconditions.14

Arecentstudy5_{demonstratedthatbisphosphonatesmay}

adheretodentinbecausethismineralizeddentaltissueisvery similartothoseofbonetissue.Thisadhesionprocess may occur duringodontogenesis,in childrentreatedwith these drugs during the formation and mineralization of dental tissues,aswellasduringphysiologicaldepositionof second-arydentin.15

Events that induce bone resorption or remodelling are capable of triggering the osteoclastic activity, resulting in adherenceoftheosteoclaststothebonesurfacesanddecrease oflocalpH.Theconsequentlossofaffinitybetween bispho-sphonatesand themineralized tissueleadstodrugrelease fromthetissue.23

Regardingtheoralcavity,somefactors,suchasprogression of caries lesions, dental trauma and toxicity of dental materialsmaydisorganizetheodontoblastlayeroreventhe pre-dentin,triggeringandactivatingtheactionoflocalclasts, whichstartsthedentinresorptionprocess.13,24,25The induc-tionoftheseeventsinpatientsunderbisphosphonatetherapy may result in release of the drug adhered to dentin hydroxyapatite,intensifyingthedamagestothe dentinopul-par complex. When bisphosphonates are released from dentin,the pulpodontoblastsarethefirstcelllineexposed to these drugs because they underlies the dentin and are responsibleforitsformationandmaintenance.12,13

Apreviousstudy26_{usingdentindiscsshowedthat}

bispho-sphonates are capable to adhere to dentin, inhibiting its resorption.Otherstudiesrevealedthatdailyadministrationof bisphosphonatesduringperiodsof7and14daysaffectedthe mineralizationofthedentinmatrix6_{andthattheinfusionof}

sometypes ofbisphosphonatesinratscausedenameland

dentin malformations.7 _{More recently, the findings of a}

bioassay showedthatthe ZOL concentrationsfoundinthe oralcavityofpatientsundertreatmentwiththisdrugranged from0.4 to5mM.17_{Thereafter, someauthorshave}

demon-stratedthatthisdrugcanbetoxictodifferentcelltypes,such as osteoblasts, endothelial cells and fibroblasts.10,11,16 _This

cytotoxiceffectcouldbeduetocontactofhighconcentrations ofbisphosphonatesreleasedfromthemineralizedtissuesto theadjacentcells.Arecentstudy5_{evaluatingthecytotoxicity}

ofZOLtopulpcellsinvitroshowedthatthisdrugcauseda significant decrease of the viability, proliferation and TP productionofthesecells.Thesedatawereconfirmedinthe presentstudyinwhichZOLconcentrations(1mMand5mM) simulatingthosefoundinthealveolarbonetissueofpatients under treatment withthis drug,17_{caused reduction of cell}

viability.

Inadditiontotheanalysisofodontoblast-likecellviability, TP production and ALP activity, molecular biology experi-ments were also carried out in the present study, which indicatedthatCol-IandALPexpressioncanbeinhibitedina dose-dependentbytheactionofZOL.Thisinhibitoryeffectof ZOL could affect negatively the repair ofthe pulpo-dentin complexinvivo,asCol-Iisthemaincomponentofreactionary dentinmatrix,whichisproducedbyodontoblaststhatsuffer aggressions12,27_{andALPisdirectlyinvolvedinthe}

minerali-zationofthisnewlyformeddentinmatrix.28,29

ThepresentstudydemonstratedthatZOLatconcentration of 1mM increase Col-I expression. Similar result was also reportedinpreviousstudiesthatrevealedanincreaseinthe expression of this gene in vitro within the first days after contact of the drug with the cells.30,31 _{However, Col-I}

expressiondecreasedovertime,suggestingthattheinhibitory effectofZOLwasbothdose-andtime-dependent.31

The results of ZOL cytotoxicity to the odontoblast-like MDPC-23 cells demonstrated by the in vitro cellular and molecularbiologyprotocolsusedinthepresentstudywere confirmedintheanalysisofcellmorphologybySEM.Thecells incubatedincontactwithbothZOLconcentrationspresented sizereduction,probablyduetocytoskeletalshrinkage.This cellresponsepatternincontactwithlowtoxicagentshasbeen extensively described in the literature,19,21 _{which helps}

establishing theeffects ofthe tested drug.This isbecause

Fig.2–SEMmicrographsshowingthemorphologyofodontoblast-likecellsMDPC-23nottreated(controlgroup)and exposedtozoledronicacidat1mMor5mM.Inthecontrolgroup,cellsarenearconfluencecoveringtheentiresurfaceofthe glasssubstrate.Intheexperimentalgroups,thecellsexhibitedshrunkencytoplasmandlossoftheirnormalmorphology. Inaddition,itcanbenotedthatcellsdetachedfromtheglasssubstrate,leavingcell-freespaces.SEM,Original

thedecreaseofcellviabilityindicatedbytheMTTassaymight beduetoadirectinhibitoryeffectofthedrugoncellactivity, which resultsinreversible morphological alterations, or to necroticorapoptoticcelldeath,whichrepresentsan irrevers-ible condition. In both situations,the MTT assay provides valuesthatrepresentasmallernumberofformazancrystals formed. However, SEM analysis of the morphology and numberofcellsthatremainedadheredtotheglasssubstrate providescomplementarydatathatindicatetheactualeffectof thedrug.Thisway,themaintenanceofthenumberof MDPC-23 cells and the discrete alterations in their morphology observed in present study demonstrate that in spite of presenting cytotoxic effects, ZOL did not cause direct cell deathevenatthehigherconcentration(5mM).Perhaps,the sameZOL concentrationsevaluatedinthepresent study(1 and 5mM) could cause more intense cytopathic effects, if maintainedforalongertimeincontactwiththe odontoblast-likecellcultures,asdescribedbyKochetal.31

Theeffects of bisphosphonateson odontoblas-likecells couldberelatedtotheactivationofdifferentpathways,such asMitogen-activatedproteinkinase(MAPK),JunN-terminal kinase (JNK) as well as caspase pathways that regulate mitogenicactivity,geneexpressionandapoptosisofcells.17,32

Further in vitro and in vivo studies are necessary to characterize the relationshipbetween cytotoxicity and the concentrationandcontacttimeofZOLwithblastcells.

5.

Conclusions

Basedonthemethodologyusedinthepresentinvitrostudy and the obtainedresults, it maybe concluded thatZOL at concentrationsof1mMand5mMpresentedadose-dependent cytotoxic effectstothe odontoblast-like cells MDPC-23and decreasedtheexpressionoftypical dentinmatrix proteins, suggestingthatunderclinicalconditionstherelease ofthis drug from dentin may cause damage to the pulp–dentin complex.

Funding

Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP),Grant#2009/54722-1,BPDR2009/52326-1.

Competing

interests

Theauthorsdeclarenoconflictofinterests.

Ethical

approval

Notrequired.

Acknowledgements

TheauthorsacknowledgetheFundac¸a˜odeAmparoa` Pesquisa do Estado de Sa˜o Paulo-FAPESP (grants: 2009/54722-1 and

BP.DR: 2009/52326-1)and the Conselho Nacionalde Desen-volvimento Cientı´ficoand Tecnolo´gico-CNPq(grant:301291/ 2010-1)forthefinancialsupport.

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