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Enamelsurfaceremineralization:Usingsyntheticnanohydroxyapatite:JShantiSwarup,ArathiRao,ContemporaryClinicalDentistry

ORIGINALARTICLE
Year:2012|Volume:3|Issue:4|Page:433436

Enamelsurfaceremineralization:Usingsyntheticnanohydroxyapatite
JShantiSwarup,ArathiRao
DepartmentofPaedodonticsandPreventiveDentistry,ManipalCollegeofDentalSciences,ManipalUniversity,Mangalore,India
CorrespondenceAddress:
ArathiRao
DepartmentofPaedodonticsandPreventiveDentistry,ManipalCollegeofDentalSciences,ManipalUniversity,LightHouseHillRoad,Mangalore.575001,KarnatakaState
India

Abstract
Objective:Thepurposeofthisstudywastoevaluatetheeffectsofsyntheticallyprocessedhydroxyapatiteparticlesinremineralizationoftheearlyenamellesionsincomparisonwith
2% sodium fluoride. MaterialsandMethods: Thirty sound human premolars were divided into nanohydroxyapatite group (n0 = 15) and the sodium fluoride group (n = 15). The
specimensweresubjectedtodemineralizationbeforebeingcoatedwith10%aqueousslurryof20nmnanohydroxyapatiteor2%sodiumfluoride.Theremineralizingefficacyofthe
materialswasevaluatedusingsurfacemicrohardness(SMH)measurements,scanningmicroscopicanalysisandanalysisoftheCa/Pratioofthesurfaceenamel.Dataanalysiswas
carriedoutusingpairedttestandindependentttest.Results:Theresultsshowedthatthenanohydroxyapatitegroupproducedasurfacemorphologyclosetothebiologicenamel,the
increase in mineral content (Ca/P ratio) was more significant in the nanohydroxyapatite group ( P < 0.05) and the SMH recovery was closer to the baseline level in the
nanohydroxyapatitegroup(P<0.05).Boththegroupsdidnotshowanysignificantdifferenceinthickness(P>0.05).Conclusion:Theuseofbiomimeticnanohydroxyapatiteasa
remineralizingagentholdspromiseasanewsyntheticenamelbiocompatiblematerialtorepairearlycariouslesions.

Howtocitethisarticle:
SwarupJS,RaoA.Enamelsurfaceremineralization:Usingsyntheticnanohydroxyapatite.ContempClinDent20123:433436

HowtocitethisURL:
SwarupJS,RaoA.Enamelsurfaceremineralization:Usingsyntheticnanohydroxyapatite.ContempClinDent[serialonline]2012[cited2016Oct2]3:433436
Availablefrom:http://www.contempclindent.org/text.asp?2012/3/4/433/107434

FullText
Introduction

Prevention of dental caries has always been difficult to tackle. Dental professionals have actively participated in caries prevention through plaque removal and dental hygiene
techniquesreinforcingtheneedforreductionincariogenicrefinedcarbohydratesingestiontopicalapplicationoffluoridateddentrifices,rinsesandgelssystemicwaterfluoridation
placementofpitandfissuresealants.[1]
Cariesisadynamicprocesswithinterspersedperiodsofdemineralizationandremineralization.Thephenomenonofreversalofincipientorearlyenamelcariesformsanimportant
partofpreventionleadingtoapparentrepairofthelesion.[2]Severalinvestigatorshaveworkedtowardsdevelopingtheidealremineralizingagent,whichdiffusesintothesubsurface
ordeliverscalciumandphosphateintothesubsurface.[3]
Fluorides,overthepast25years,havebeeninstrumentalincausingthedeclineofdentalcariesexperienceinmostindustrializedcountries.Topicalfluoridespromotetheformationof
flourapatiteinthepresenceofcalciumandphosphateionsproducedduringenameldemineralization.However,forevery2fluorideions,10calciumions,and6phosphateionsare
requiredtoformoneunitcelloffluorapatite[Ca10(PO4)6F2].[4]Hence,theavailabilityofcalciumandphosphateionscanbethelimitingfactorfornetremineralizationtooccur.
Also given the mechanism of remineralization by topical fluorides they attempt only to reduce apatite dissolution rather than aiming to promote mineralization of apatite crystal or
replacementofthelostminerals.[5]
Thestructureofenamelistoocomplextoberemodeledandthebasicenamelbuildingblocksaregenerally2040nmhydroxyapatite(HA[Ca10(PO4)6(OH)2)nanoparticles.
Therefore,theremineralizationofenamelmineralsbyusingsyntheticapatiteorHA,thatresemblesenamelHAmaybebeneficial.[5],[6]Theaimofthisstudythuswastoexplorethe
effectsofsyntheticallyprocessednanosizedbiomimeticHAparticlesincausingremineralizationoftheearlyenamellesionsincomparisonwith2%sodiumfluoride.

MaterialsandMethods

Thisisatripleblindstudycarriedoutafterobtainingapprovalfromtheinstitutionalethicscommittee.
Specimenpreparation
Thirtyhumanpremolarsextractedfororthodonticreasonswereselectedforthestudy.Soundnoncariousteethwithoutanyrestorationswereincludedandteethwithdeformities,
defects,fractureswereexcludedfromthestudy.Theteeththusselectedwerethoroughlycleanedofdebrisandstoredinsaline(0.9%sodiumchloridesolution)untilrequired.The
crownwasseparatedfromtherootandsectionedintotwohalvesusingahighspeeddiamonddisc.Theabradedsurfaceswerepolishedusingpumicepolishingpaste.Thespecimens
wererandomlydividedintoacontrolgroupandatreatmentgroup,eachconsistingof15specimens.
Lesionformation
Eachspecimenwereplacedin15mlofdemineralizingsolutionofthecomposition2.2mMCaCl2,0.05Mlacticacid,and0.5ppmFadjustedtopH4.5with50%NaOHfor48h.At

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theendof48hchalkywhiteincipientcarieslikelesionsdevelopedonthesurfaceofthespecimens.
Remineralizationregimen
Nanohydroxyapatitegroup
NanohydroxyapatitepowderwasprocuredfromM/s.DynamicOrthopedics(P)Ltd.,Cochin,India.Thenanohydroxyapatitepowderhadacrystaldimensionof50100nminlengthand
2040nminwidth.ItwasensuredthatthesenanohydroxyapatitecrystalsaresimilartoapatitecrystalsinhumanenamelbyXraydiffractionanalysistoguaranteetheirbiomimetic
property.[7]Thedemineralizedspecimenswerecoatedwith10%aqueousslurryofnanohydroxyapatiteandimmersedinartificialsaliva[8](FusayamaMeyer'sartificialsalivaofthe
compositionKCl0.4g/l,NaCl0.4g/l,CaCl22H2O0.906g/l,NaH2PO42H2O0.690g/l,Na2S9H2O0.005g/l,Urea1g/lpH6.5)for10days.
Sodiumfluoridegroup
2%neutralsodiumfluoridesolutionwaspreparedbydissolving20gofsodiumfluoridepowderin1lofdistilledwater.Fifteendemineralizedspecimenswereimmersedinthefreshly
prepared2%sodiumfluoridesolutionfollowedbyimmersioninartificialsalivafor10days.
Scanningelectronmicroscopicanalysis
Environmental scanning electron microscope (SEM) (Carl Zeiss, Oxford Instrument, INCA Oxford Software package) was used for analyzing the surface morphology and enamel
thickness of the specimens at the baseline level, after demineralization and remineralization treatments. The procedures were carried out under extended pressure (100 Pa air
pressure)withLaB6filament.Enamelthicknesswasmeasuredatthreepointsfromthedentinoenameljunctiontotheenamelsurfacebydrawingtangentsandanaveragewastaken
outofthethreereadings.
EnergydispersiveXrayanalysis
ThecalciumandphosphatecontentinthesurfaceenamelwasmeasuredwiththeaidofSEMfittedwithenergydispersiveXrayanalysis.ThereadingswerethenconvertedtoCa/P
ratio(wt%).Ca/Pratiowasassessedbeforeandafterdemineralizationandafterremineralizationtoevaluatethechangeinthemineraldensity.
Surfacemicrohardnessanalysis
The surface microhardness (SMH) of the specimens were measured as Vikers hardness number (VHN) during three stages of this study: Before demineralization, after
demineralization, and after remineralization using an automatic digital microhardness tester (Micro Vickers Hardness Tester, Omni Tech). Three indentations were made on each
sample during each stage of the study using a single load of 200gf (gram force) with a holding period of 15 s and the SMH data obtained were the average values of three
measurements.Aminimumdistanceof150mwasensuredbetweenadjacentindentationsinordertoavoidmeasurementerrors.
Statisticalanalysis
ThedatawerecomputerizedandanalyzedusingSPSS(SoftwarePackageusedforStatisticalAnalysis)version11.5.Theresultsobtainedonenamelthickness,mineralcontent,and
microhardnessdeterminationbetweeneachstepinthesamegroupwasanalyzedusingpairedttestfollowedbyanalysisusingindependentsample'sttesttocomparetheresults
betweenboththegroups.

Results

Surfacemorphologicanalysis
ObservablesurfacechangeswereseenintheSEManalysisoftheenamel.[Figure1]adepictsthenormalsmoothandintactenamelsurfacebeforedemineralizationwiththetypical
keyholepatternsofenamelprisms.Thesurfacestructurechangedtoirregularwithvoidsandnumerousmicroporesafterdemineralization[Figure1]b.Thespecimenstreatedwith2%
sodiumfluorideshoweddepositionofcalciumfluorideglobulesonthedemineralizedenamelsurfaces[Figure2].Thesurfacesoftheteethtreatedwithnanohydroxyapatitedisplayed
nucleationofapatiticcrystalsintheporescreatedbydemineralization[Figure3].Theporousinterprismaticandprismaticenamelstructureswerecompletelyhiddenbyathick,uniform
apatiticlayerwithtypicalfastenedneedlelikeHAcrystallites[Figure4]incontrasttothespecimenstreatedby2%sodiumfluoridewhichexhibitedevidenceofuncoveredporous
surfaces.{Figure1}{Figure2}{Figure3}{Figure4}
Surfacemineralcontentanalysis
TheCa/Pratiofelltoaconsiderablelevelinboththegroupsafterdemineralization.ThoughboththegroupsshowedasignificantincreaseinCa/Pratio(P<0.05),itwasfoundthatthe
recoveryofmineralcontentwasmoreinHAgroupthanin2%sodiumfluoridegroupandthedifferencewasstatisticallysignificant(P<0.05)[Table1].{Table1}
Enamelthicknessanalysis
Therewasaconsiderablereductioninthethicknessofthesurfaceenamelafterdemineralization[Table2].Treatmentwithboth2%sodiumfluorideandnanohydroxyapatiteresultedin
aslightincreaseinthicknessofthesurfaceenamel.Nanohydroxyapatitegrouphadabetterincreaseinenamelthickness,butthedifferencewasnotstatisticallysignificant(P>0.05).
{Table2}
Surfacemicrohardnessanalysis
The results of enamel SMH analysis of the specimens are shown in [Table 3]. The specimens in both the treatment groups had experienced a decrease in microhardness after
demineralizationandhadrehardenedafterremineralization.TheincreaseinSMHwasstatisticallysignificantinboththegroups(P<0.05).SMHrecoverywasmorepronouncedinHA
groupthaninsodiumfluoridegroupandwasstatisticallysignificant(P<0.05).{Table3}

Discussion

Fluorides have been the most favored remineralizing agent. Topically applied sodium fluoride solution has been known to cause remineralization, mainly by reducing apatite
dissolutionbyforminglesssolublefluorapatiteandincreasingSMH,butareunabletoreconstructthelostmineralstructure.[9],[10]NanostructuredHAcrystalsexhibithighlevelsof
biomimeticpropertiesduetotheircomposition,structure,morphology,bulkandsurfacephysicalchemicalproperties.20nmHAusedinthisstudyarebioinspiredmoleculesthathasa
surface area of 100 m 2 /g, which makes them possess strong affinity to the demineralized surfaces. [6] The SEM analysis of the surface after remineralization induced by
nanohydroxyapatite reflects this observation where HA nanocrystals were found to adhere to the pores created by demineralization. These adherent nanocrystals were found to
aggregate and grow into microclusters and form a uniform apatite layer on the demineralized surface. The surface also revealed the newly formed apatite layer to be completely
coveringtheprismaticandinterprismaticenamelstructures.Thesameobservationwasnoticedinanearlierstudywhere100nmcarbonatedHAparticleswerefoundtocoverthe

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demineralizedenamelsurfacesmoreeffectivelywhencomparedwithfluoridatedtoothpaste.[11]
The fall in the surface Ca/P ratio following demineralization did not practically increase after remineralizing with 2% sodium fluoride application. This finding suggests that only
structuralmodificationofapatiteoccursandisrestrictedtoapartialhydroxylgroupreplacementbyfluorideionswithoutinfluencingtheCaandphosphatecontent.Thespecimens
treatedwithnanohydroxyapatiteexhibitedsurfaceCa/Pratioclosetothatofthebiologicalenamelandthesyntheticnanohydroxyapatiteusedindicatinganapatitecoatingdeposition
onthedemineralizedenamelsurface.Anearlierstudy[11]doneonremineralizationbycarbonatednanohydroxyapatitecrystalsexpressedthattheenamelslabsbrushedwiththe
materialshowedanincreaseinCa/Pratio,thoughnotclosertothebiologicalenamelwhichmightbeattributedtotheabsenceofsalivaryeffectontheremineralization.Thestudyalso
depictstheCa/PratioofthespecimenstreatedwithfluoridedidnotresembletheCa/Pratioofbiologicenamel.
The significant increase in SMH in the nanohydroxyapatite group establishes that this new material rehardens the softened enamel by gradual deposition of the mineral that
precipitatesandnucleatesinthedarkzoneofdemineralizationtherebyofferingcompletebiomimeticregenerationofthelostenamelcrystallites.A48hremineralizationstudy[12]
usingnanohydroxyapatiterevealedasimilarincreaseinSMHaftertreatmentwiththematerial.TheearlierstudyalsopicturesthattheincreaseinSMHdidnotreachcloseenoughto
thebiologicenamelsurfacehardnesswhichmightbebecauseofthedecreasedcontacttimewiththetestmaterialandalsotheblockadeofthesurfacelayer.[13]

Conclusion

10%biomimeticnanohydroxyapatiteoftheparticlesize20nmhasthepotentialtoremineralizeinitialenamelcariesunderinvitroconditionswhencomparedwith2%sodiumfluoride.
This documented biomimetic apatite coating on the demineralized enamel suffices the need for a synthetic enamel biocompatible material able to repair early enamel lesions.
Nanohydroxyapatitewouldthereforebebeneficialinpromotingremineralizationwithregulardailyusage.

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