I nvest i gat i onofi nhi bi t or yef f ect sof
semi conduct ort oot hbr ushonbi of i l m f or mat i on
Depar t men tofBehavi or alPedi at r i cDen t i s t r y
Ok ayamaUni ver si t yGr aduat eSchoolofMedi ci ne.Dent i s t r yand Phar maceu t i cal Sci ences
Kazi Ani surRahman
December1 2,201 2
1.Introduction
Streptococcusmutansisimplicatedasapnmarycausativeagentofdental cariesinhumans1‑2)IOneofitsimportantvirulencepropertiesistheabilityto form biofilms,termed dentalplaque,on tooth surfaces,after which accumulationofacidsindentalplaqueleadstolocalizeddecalcificationofthe enamelsurface.Therefore,removalofcanogenicdentalplaquefrom tooth surfacesisconsideredtobeafundamentaltechniqueforpreventionofdental Ca｢leS.
Semi‑conductorpowdershavebeenshowntohaveabactericidaleffectfor photocatalyzed disinfectionofwater,while photosensitive titanium dioxide (TiO2)suspended inwaterwasdemonstrated to reduce the time oflight exposure requlred to induce disinfection 3) Furthermore,TiO2 has a bactericidaleffectonseveralspeciesofmicroorganisms,suchasEscherichI'a coII･ 4)and Streptococcus sobrI･nusAHT5) ltwas also reported thatSI sobrinusceHsweredamagedduetoslgnificantdisruptionofceHmembranes andce"wallsafterphotocatalyticactionfor60‑120min6)･
A newlyintroducedtoothbrushwithTiO2embeddedjnasemiconductorin thebodyofthebrush,namedthe(Soladeyl2),wasrepo什edtohavebetter plaql｣e ｢emOVlng POtentialwhen compared to conventionaltoothbrushes, Suggestingthatthephotocatalyticpropertiesofthesemiconductormayhave bactericidalactivitiesandreducedentalplaqueontoothsurfaces7).However, themechanism causlngthereducedplaqueremainstobeelucidated.The (Soladey‑3)containsasemiconductorwithaTiO2rodandsolarpanel,as TiO2WaSreportedtobemoresensitiveunderUVlightinwater8).lnthisstudy, wefoundreducedbiofHm formationbyS.mutansfoIIowlnguseOfSoladey13,
anewlyintroducedtoothbrushthatcontainsasemiconductoralongwitha TiO2rodandsolarpanel･lnaddition,wevisuallyobservedthemorphologyof thesuげaceofhydroxyapatite(HA)pelletsa冊erbrushingwiththeSoladey‑3 andconsideredthepossl‑blemechanismsrelatedtoitse斤ects.
2.Materialsandmethods
2･1,HApelletsandtoothbrushes
HA pellets (10xlOx2 mm,APP‑100,PENTAX,Tokyo,Japan)were employedinthisstudy.Forthetoothbrushes,weusedaSoladey‑3toothbrush (ShikenLtd.C0.,Osaka,Japan)ContainingaTiO2rodembeddedintheneck and a sola｢panelonthe topofthe hand一e ofthe body,and a regular toothbrushwithastainlesssteelrodintheneckasaplacebo.
2.2.Bacteriaandculturemedia
S.mutansMT8148R,whichisresistanttostreptomycln,wasusedinthis study.BacteriaweregrowninToddHewitt(TH)Broth(BectonDickinsonCo., Maryland,USA)includingstreptomycin(500トtg/ml)overnightat370Cpriorto theexpe｢imentalprocedure.
2.3.Sa/ivasample
Unstimulated whole saliva was collected from a healthy subjectinto a beakerplacedoniceandclarifiedbycentrifugationfor30minat12,000g, afte｢whichthesupematantwascollectedandkeptaト200Cuntiluse.
2.4.Biofilm formationassay
HApeHetsweredividedinto3groups,theneitherbrushedwiththeSoladey13 toothbrushastheexperimental(EX group)o｢the placebotoothbrush(PL group),whilethethirdgroupwasnotbrushedandusedasacontrol･Brushing wasdoneinajarcontainingdistilledwaterandunderaHghtsource(40W, 750LUX)bythesameindividuals,whomaintainedthesamelevelofconstant
forceandusedatechniquesimilartonormaltoothbrushing.Toestimate biofilm formation,HApelletswithorwithoutbrushingwereexposedtohuman salivafor2hoursat37oC.ThreepeHetseachfrom theEXgroup,PLgroup, and controlgroups were placed into 6‑we" culture plates (CELLSTAR, Greinerbiol0ne,Frickenhausen.Germany)with5mlofTH brothcontaining lx106colony‑formingunits(CFU)/mlofS.mutansineachweH.Thepellets were then incubated at370C on a shakerat60 rpm underanaerobic conditions(80% N2,10% H2,10% CO2).FoHowing incubation,thepe‖ets wererinsed3timeswithphosphatebu斤eredsaline(PBS)toremoveJoosely boundcells.Next,theywereplacedintofreshPBS(5ml),chiHedonice,and theattachedbacteriawereremovedbyultrasonicvibrationfor1min.P巨s containlngbacteriawereseriallydilutedwithnormalsalinetoprepare102‑,
104‑,and106‑folddilutionsample･Next,100LLloforlglnalandeachofthe d仙tedsampleswereplatedonMitis‑Saliva｢山S(MS)a9ar(Becton,Dickinson) for48hoursat370C underananaerobiccondition,FinaHy,thenumbersof bacterialcolonieswerecountedandtheresultsweresubjectedtostatistical analysIS.
2.5.X‑rayphotoelectronspectroscopy(XPS)
ThechemicalcompositionsoftheHA pelletswithandwithoutbrushingwas analyzed using anXPS device (AXISINOVA,Shimadzu Co.,Ltd.Kyoto, Japan)equippedwithanAIKα photonwith1486･6eVofenergy･Allbinding ener9‑eSWerereferredtoastheCIspeakofthealiphaticsurfacecarbonat 276‑288eV,andthehigh‑resolutionXPSspectraoftheCa2p(338‑356 eV),P2p(125‑137eV),andO ls(525‑537eV)photoelect｢onpeakswere analyzed.
2･6･MorphologicalanalysisofHApellets
PelletsintheEXandPLgroupswerebrushed10,20,30,40,or50times, thenexposedtohumansalivafor2hoursat37oC,fotfowedbylnCubationin dist川edwaterfor24 hoursat37oC,Toevaluatethe recoverytimeofthe perimeterand area ofthe HA crystals,pellets brushed 50 times were incubatedindistilledwaterfor12.24,48,72,96,or120hoursat37oC.To reducetechnicalerror,aHbrushingprocedureswereperformed3timesbythe same individuals.To evaluate the HA pelletsurfacesafterbrushing and bacteria一adherence,they were obseⅣed uslng a scann∫ng e一ectron microscope (SEM;HitachiS‑4800,Tokyo,Japan)atlow (x800)and high (xlOO.000) magnifications.Measurements ofthe length ofthe crystal perimeterandsurfaceareaweredoneatx100,000magni¶CationuslngZeiss LSM Image Browserso斤ware,version 3･1･0199 (www･zeiss･de)I Data obtainedwerethensubjectedtostatisticalanaJysIS.
2.7.Statisticalanalysis
StatisticalanaJysJSWasPerformeduslngaOne‑WayANOVAtest,alongwith Fisher's protected leastsignificantdifference (PLSD)posthoc testand Student'st‑tes
t
.A p‑vaLue <0･05 was considered to be slgnificant
･All calculationswereperformedusingSPSS1610so冊ware(SPSS,lnc･,Chicago, IL).3.Results
lnthe biofilm formationassay,the proportion ofS･mutansorganismsin biofitmsformedinculturesofpelletswithoutsucroseandbrushedwiththe Soladey13(EX group)wassignificantly reduced after6 (p=0･006)and 12
(p=01004),(Fig.1A).ln the EX group,a few S.mutans colonies were observed,while biofHmsformed with the peHetsfrom the controland PL groupsconsistedofthickerandmoreuniform layersascomparedtotheEX group･When1% sucrosewasadded,thequantityofbiofilm intheEXgroup was21fold lessascomparedtothecontro一groupa什er12 hours.The cell countofS.mutansintheEXgroupdecreased(p=0.004)a什er24hoursand wassignificantlysmallerthanthatobservedincontrolgroup(Fig.1B).lnthe controlgroup,the biofilm wasdense and coveredthe HA peHetsurfaces.
TheseresultsindicatethatuseoftheSoJadey‑3toothbrushreducedbacterial colonizationunderthevarioustestconditions.
XPS high‑resolutionspectra forthe HA pelletswJLth orwithoutbrushing revealed a slgnificantchemjcalshin ofthe peak.ln comparisons among groups,the peak representing the binding energy ofCa 2P showed a significantincreaseof1.2eV intheEX groupascomparedtoPLgroup(Fig.
2A),SuggestingthatuseoftheSoladey‑3toothbrushproducedashin owards higherbindingenergy.lncontrast,theP2psurfaceconcentrationwas13110 eV inthisgroup,lowerthanthatinthePL(132.1eV)andcontrol(13117eV) groups.Thus,the binding energyforP 2pwasslgnificantly reduced a什er brushingwithSoladey‑3(1.1eV)(Figure2A).Interestingly,a什er120hoursof incubationbllowln9b｢ushin9WiththeSoladey‑3,allofthepeaks｢etumedto normallevels(Fig.2B).Thepeakrepresenting0IswasunchangedunderaH ofthetestedconditions(Fig.2A,B).
ThesurfacesofthepeHetsintheEX,PL andcontroJgroupswereobserved usln9 an SEM.Atleast10 areaswere selected foreach 9｢OuP,andthe surfaceareaandperimeterlengthofthecrystalsweremeasured.lntheEX
group,themeanvaluesforthesurfaceareaandperimeterlengthoftheHA crystalsweredecreased,andthesizedecreasedwithincreaslngnumbersof brushing.Somegapswereobservedbetweenthecrystalswithadecreasein thecrystalsize(Fig13A).ThesizeofpeHetssubjectedto50tinleSbrushing wassignificantlysmaHerthanthatofthosesubjectedtofewerbrushings(Fig.
3B,C).TherewasalsoasignificantdifferenceinmeancrystalsizeintheEX groupascomparedtothePLandcontrolgroups(Fig.3B,C).Toevaluate recovery,pelletsslJbjectedto50timesbrushingwereselectedfrom theEX groupand incubatedfor12,24,48,72,96,or120 hours,afterwhichthe surfaceareaandperimeterlengthofthecrystalsweremeasured.Thecrystals wererestoredtotheoriginalsizeafter120hours(Fig.4).
4.Discussion
TiO2isthoughttoenhancesolardisinfectionbyformlngOHlg,10),aradical consideredtohaveverydamaglngeffectsoni｢｢adiatedbiologlCalsystemsat diffusioncontro"edrates11112),asweHasceHwaHsandmembranes14).Thus, whenaTiO2equlPPedtoothbrushisused,biofilmslikelybecomesdisrupted byhydroxylradicals.Furthermore,theeffectivenessofthesemiconductorin thetoothbrushisdependentontheconcentrationofTiO2andlightintensity31 10)Alnaddition,OH･generatedinwaterdunnglightexposurejnthepresence ofTiO2WasShown to be responsible forbacterialdisinfection 10) ln the presentstudy,WeusedatoothbrushequlPPedwithasolarpanelandTiO2 Semiconductor to enhance the production of free radicals to prevent adherenceofS,mutansorganismstoHA.
lnthepresentstudy,Calcium and/Orphosphoruswasdetectedjnthebinding
energyregl0nOfCa2pandP2pelectronsuslngXPSafter50timesbrushing with the Soladey‑3 toothbrush.Spontaneous re‑precIPitation has been reported to be dependent on environmental pH, temperature, ionic concentration,calcium/phosphorus(Ca/P)molarratio,andthepresenceof organicorinorganicelements14)IOrganicphosphatesandphosphonatesare weHknowntohavehighaffinitiesforadsorptiontoapatitesurfaces15116)･ln thehumanoralenvironment.S,mutansadhesionismodulatedbysalivary proteinadsorptionontotheenamelsurface17)AWe foundthatchangesin surfacecorTlPOSitionoftheHA peHetsoccurredasaresultofbrushingthe surfacesofthepe"ets.ThesesurfacechangesresultedintowerS,mutans aqinityascomparedtothenon‑brushedpellets.
lt is wellknown that organic phosphoric compounds are exceHent antibacterialagents18)･Therefore.itispossib一ethatthepresenceofcertain amounts ofphosphor‑containJng components on the surface may induce antibacterialactivitythatinhibitsthegrowthofbacterialce"S.Also,changesin surfaceroughnessandsurfacemorphologyareimportantfactorsthatimpact bacterialbehavior19)･ThecrystalsizeofpeHetsbrushed50timeswiththe Soladey‑3inthepresenceoflightandwatermayhavebecomesIgnificantly decreasedduetoshrinkage.whichcreatedsomegapspacesbetweenthe crystals.BrushingwithSoladey‑3producesnegativeenergyelectronsthat changethesurfacemorphologyofHA,thusgapsthatform asaresultmaybe dueto a hydrophobicaction ofthe crystals.These findingssuggestthat brushing50timeswiththistoothbrushcanmodifythe HA surface,which mightbeanimportantfactorforpreventionofbacterialadhesion.
lnsummary,theSoladey‑3TiO2SemiconductortoothbrushequlPPedwitha
Solarpanel,hasantibacteriaIeffectsandcancontrolbacterialadhesionbetter thancommoncommerciallyavailabletoothbrushes.Ourfindingssuggestthat thissemiconductortoothbrushcouldbeusefulforcontroHngofdentalcaries.
Acknowledgmen
t :
WethankAssociateProfessorHiroshiKominamiofKinkiUniversity,Japan, forperformlngtheXPSanalysIS.
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FigureLegend:
Figure1ViableceHcountsofS.mutansontheHA peHets,HA pelretswere treated50timeswithSoladey‑3(EXgroup)orcontroltoothbrush (controlgroup)andwereincubationwithS.mutansfor6,12or24 hours.Incubationwasperformedundertheconditionwith(B)or withoutsucrose(A).★P<0.05了★P<0.001.
Figure2XPSanalysISforbindingenergyontheSurfaceofHA treatedwith Soladey‑3 (experimentalgroup)ornormaltoothbrush (placebo group).HA peHetwithoutthebrushingtreatmentwasusedasa controL.(A)The peakpositionofCa,P and O inexperimental, placeboandcontrolgroupafter24hoursincubation.(B)After120 hoursincubation,thepeakpositionofCa,Pand0inexperimental, placeboandcont｢olgroupwasalmostinsameposition.Asterisks indicate'P<0.05.
Figure3SEM imageof(A)physicalchangeofHAmolecularsize(B)surface a｢eaofmoleculesand(C)pe｢imete｢ofmoleculesafterbrushing withsoladey13andplacebotoothbrush.Asterisksindicate'P<0.05,
★★P<0.001了 ★★P<0.0001
Figure4(A)lnHA peHetsexposedto50timesbrushingwiththeSoladey‑3, thecrystalsreturnedtonormalsizeafter120hoursofincubation (x100,000).Graphsdepictchangesinthe(B)surfaceareaand(C) perimete｢ofHA moleculesafter12.24,48,72,96,and120hours ofincubation,as weHas comparisons with the controlgroupI Asterisksindicate'P<0.05,''P<0.001,'''P<0.0001.
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