Effect of exposure of glass ionomer cements to acid ...

1

Effectofexposureofglassionomercementstoacidenvironments

By

IlyaZalizniak

Submittedintotalfulfilmentoftherequirementsofthedegreeof

MasterofPhilosophy

October2016

MelbourneDentalSchool

FacultyofMedicine,DentistryandHealthScience

TheUniversityofMelbourne

2

ABSTRACT

This research focused on evaluating the effect of casein phosphopeptide-

amorphouscalciumphosphate(CPP-ACP) incorporated intoaglass ionomercement

(GIC).TwoconventionalGICmaterialswerethemainfocusofthestudy,FujiVIIand

FujiVIIEP(containing3%w/wCPP-ACP),thesematerialsweremadeintorectangular

blocks and subjected to various acidic media (lactic, citric and hydrochloric acids),

whiledailyobservationsofsurfacehardness,masslossandionreleasewerecarried

out over a three day period. Acid storage solutions were specifically chosen to

approximateconditions thematerialswouldbeexposed to inanoralenvironment;

bacterialacidchallenge,foodacidchallengeandstomachacidreflux.Laterstagesof

thestudyalsoincludedtopicalCPP-ACFP(caseinphosphopeptide-amorphouscalcium

fluoridephosphate)treatmentofGICsurface,aswellasamoredetailedanalysisof

ionreleaseofFujiVIIEPcomparedtoothermaterials(ShofuBeautifil).

BlocksofGICmaterialwerepreparedandallowedtosetinanincubator(37C,

95%+relativehumidity)for24hours.Surfacehardnesswasdeterminedusingmicro-

indentationusingamicroscope-aligned indenter.Acid storagemediawere changed

every24hoursandallsolutionswerekeptforfurtheranalysisofionrelease.Fluoride

ion concentrationsweremeasured using an ion selective electrode, phosphate ion

concentrations were determined using a UV spectrophotometry assay and calcium

ionconcentrationsweremeasuredusingatomicabsorptionspectroscopy(AAS).Mass

losswasdeterminedusingananalyticalmicrobalance.Laterstagesofthestudyalso

measuredaluminiumionconcentrations,whichwereacquiredusingAAS.Datafrom

samplegroupswasfoundtofollowthenormaldistribution,χ2testwasusedtotest

normality. Single factor ANOVA was used to analyse the results using Bonferroni-

Holmmultiplecomparison.Two-wayANOVAwasusedtodeterminethe interaction

3

between incorporation of CPP-ACP and exposure to different acids. Level of

significancewassetatα=0.05.

Over the course of three days of storage the surface hardness of both

materialsreducedsignificantlyinallacidsolutionsaswellasacontrolwatersolution.

Resultsshowedsignificant increases incalciumandphosphateionreleaseinFujiVII

EPgroupscomparedtoFujiVIIinthevastmajorityofacidsolutions.Fluoriderelease

remainedstableforbothmaterialswithnosignificantdifferencesmeasuredbetween

them. Further topical treatment provided additional increases in calcium and

phosphate ion release for bothmaterials. Exception to this were groups stored in

citricacid,whichexhibitedfargreatermasslosscomparedtoothergroups,veryhigh

phosphateionreleaseandanincreaseinsurfacehardness.Analysisofaluminiumion

release to indicated thataluminium ionsarepossiblychelated fromtheGICmatrix,

leadingtoadditionalreleaseofphosphateionsandincreasedmassloss.Formationof

acrustsurfacelayerseemstobethecauseofincreasedsurfacehardnessintopically

treatedcitricacidgroups.

Comparisonoffluoride,phosphateandaluminiumionreleasebetweenFujiVII

EP and Shofu Beautifil showed that ion release profiles are significantly and

fundamentallydifferentbetweenthetwomaterials,whichmaybeduetothelevelof

maturation(age)oftheglassandstructuredifferencesbetweenthematerials.

4

DECLARATION

This is to certify that this thesis is the original work of the author except where

indicated;dueacknowledgementhasbeenmade inthetext.This thesis is lessthan

40,000wordsinlengthexclusiveoffigures,tablesandbibliography.

IlyaZalizniak

October2016

5

ACKNOWLEDGEMENTS

Iwouldliketothankmysupervisors,withoutwhomthisresearchwouldnot

bepossible,ProfessorMichaelBurrows,AssociateProfessorJosephPalamaraandDr

RebeccaWongfortheirguidance,continuedsupportandadvice.

IwouldalsoliketothankProfessorEricReynoldsforhisadviceaswellasCRC

forOralHealthfortheirsupportthroughoutmydegreeandGCCorporation(Tokyo,

Japan)fortheirfinancialandmaterialsupport.

Iwouldliketoexpressmygratitudetothefollowingpeopleandorganisationsfor

theirsupportandadviceduringmyresearch:

• Dr.NathanCochrane

• DavidStanton

• Bio21

• MelbourneDentalSchool

• UniversityofMelbourne

• YiYuan

• Dr.FanChai

• MarioSmith

• DanielStalder

6

LISTOFABBREVIATIONS

GIC :Glassionomercement

CPP-ACP :Caseinphosphopeptide-amorphouscalciumphosphate

CPP-ACFP :Caseinphosphopeptide-amorphouscalciumfluoridephosphate

F7 :FujiVII

F7EP :FujiVIIEP

TM+ :ToothMoussePlus

SBF03 :ShofuBeautifilF03

AAS :Atomicabsorptionspectrcopy

EDXA :Electrondispersivex-rayanalysis

SEM :ScanningelectronMicroscopy

MPa :Megapascal

VH :Vickershardness

RH :Relativehumidity

N :Newton

w :width

l :length

t :thickness

TISAB :Totalionicstrengthadjustablebuffer

HCL :Hydrochloricacid

7

TABLEOFCONTENTS

CHAPTER1..........................................................................................................................15

Introduction.......................................................................................................................15

1.1References............................................................................................................................17

CHAPTER2..........................................................................................................................22

LiteratureReview.............................................................................................................222.1DentalCaries........................................................................................................................222.1.1Aetiology..........................................................................................................................................232.1.2Immunisation................................................................................................................................242.1.3FluorideandCariesprevention.............................................................................................252.1.4Fluoridecontainingdentalrestorativematerials..........................................................28

2.2GlassIonomerCements....................................................................................................292.2.1Origin.................................................................................................................................................292.2.2ChemistryoftheSettingReaction........................................................................................292.2.3PhysicalProperties.....................................................................................................................322.2.4FluorideReleasefromGICs.....................................................................................................332.2.5ClinicalApplicationandComparison..................................................................................342.2.6Resin-ModifiedGICs....................................................................................................................372.2.7ProximalandSecondaryCaries.............................................................................................40

2.3CaseinPhosphopeptide-AmorphousCalciumPhosphate....................................412.3.1ChemistryandRoleinOralEnvironment.........................................................................422.3.2InteractionbetweenCPP-ACPandFluoride....................................................................43

2.4AnalyticalMethods............................................................................................................442.5References............................................................................................................................46

CHAPTER3..........................................................................................................................69

AimsandObjectives........................................................................................................69

CHAPTER4..........................................................................................................................72

Preliminarystudies.........................................................................................................72

4.1Introduction.........................................................................................................................724.2Specimenshape...................................................................................................................74

8

4.2.1MaterialsandMethods..............................................................................................................744.2.2Discussion.......................................................................................................................................75

4.3Ionrelease............................................................................................................................764.3.1MaterialsandMethods..............................................................................................................764.3.2Results..............................................................................................................................................794.3.3Discussion.......................................................................................................................................86

4.4Hardness................................................................................................................................874.4.1MaterialsandMethods..............................................................................................................874.4.2Results..............................................................................................................................................904.4.3Discussion.......................................................................................................................................93

4.5Conclusion.............................................................................................................................944.6References............................................................................................................................96

CHAPTER5..........................................................................................................................98

IonreleaseandphysicalpropertiesofCPP-ACPmodifiedGICinacid

solutions..............................................................................................................................98

5.1Abstract(249words):.......................................................................................................985.2Introduction.........................................................................................................................995.3MaterialsandMethods..................................................................................................1015.4Results.................................................................................................................................1035.5Discussion..........................................................................................................................1085.5.1Surfacehardness.......................................................................................................................1085.5.2Changeinmass...........................................................................................................................1095.5.3IonRelease...................................................................................................................................110

5.6Conclusion..........................................................................................................................1115.7References.........................................................................................................................112

CHAPTER6.......................................................................................................................116

RechargeandionreleasefollowingtopicalToothMoussetreatmentofCPP-

ACPmodifiedGICinacidsolutions..........................................................................116

6.1Abstract(235words):....................................................................................................1166.2Introduction......................................................................................................................1176.3MaterialsandMethods..................................................................................................1196.4Results.................................................................................................................................123

9

6.4.1SurfaceHardness......................................................................................................................1236.4.2MassLoss......................................................................................................................................1246.4.3IonRelease...................................................................................................................................128

6.5Discussion..........................................................................................................................1356.5.1Watercontrol..............................................................................................................................1366.5.2Lacticacid.....................................................................................................................................1376.5.3Hydrochloricacid......................................................................................................................1386.5.4Citricacid......................................................................................................................................1396.5.5Surfacehardness.......................................................................................................................1406.5.6Changeinmass...........................................................................................................................1416.5.7IonRelease...................................................................................................................................1426.5.8GeneralDiscussion...................................................................................................................143


CHAPTER7.......................................................................................................................150

AluminiumionreleaseofconventionalGICsandgiomermaterialsinacid

solutions...........................................................................................................................1507.1Abstract(232words):....................................................................................................1507.2Introduction......................................................................................................................1517.3MaterialsandMethods..................................................................................................1547.4Results.................................................................................................................................1567.4.1SurfaceHardness......................................................................................................................1567.4.2MassLoss......................................................................................................................................1577.4.3IonRelease...................................................................................................................................158

7.5Discussion..........................................................................................................................1617.5.1SurfaceHardness......................................................................................................................1617.5.2Changeinmass...........................................................................................................................1617.5.3IonRelease...................................................................................................................................162


CHAPTER8.......................................................................................................................167

Discussion........................................................................................................................167

8.1Pilotstudyoutcomes......................................................................................................167

10

8.2Acidchallengeoutcomes...............................................................................................1688.3Topicaltreatmentoutcomes.......................................................................................1698.4Importanceandroleofaluminiumions..................................................................1708.5References.........................................................................................................................174

CHAPTER9.......................................................................................................................176

ConclusionsandAreasforFurtherResearch......................................................176

11

LISTOFFIGURES

Figure2.1.Stagesofcarieslesioninitiationandprogression.Dentalcare.com...........27

Figure4.1.MasslossofF7andF7EPintartaricacidoverathree-dayperiod.n=5.....84

Figure4.2.Calcium,aluminiumandphosphateionreleasefromF7inthreedifferent

acids.n=5.Al3+....................................................................................................85

Figure4.3.Calcium,aluminiumandphosphateionreleasefromF7EPinthree

differentacids.n=5..............................................................................................86

Figure4.4.DepthprofilecomparisonofTM+treatedF7EPfollowingthree-day

exposuretofoursolutions.InitialsurfaceVHshownasablackline...................88

Figure4.5.SurveyregionsforEDXA.............................................................................89

Figure4.6.F7EPblockstreatedwithTM+analysedusingEDXAatvarioussubsurface

regions..................................................................................................................92


regions..................................................................................................................93

Figure5.1.Measuredcalcium(a),phosphate(b)andfluoride(c)ionreleasefromF7

andF7EPwhenstoredinthefourdifferentsolutionsoverathreedayperiod.

Significantincrease(p<0.05)incalciumionswasmeasuredforallgroups(except

water)betweenF7EPandF7.Significantincrease(p<0.05)inphosphateion

releasewasmeasuredforcitricandhydrochloricacidscomparedtowater.

SignificantlymorephosphatewasreleasedfromF7EPcomparedtoF7incitric

andhydrochloricacids.Fluorideionreleasewassignificantlyhigher(p<0.005)in

allacidscomparedtowater.Groupsmarkedwithsamelowercaseindicateno

significantdifferenceinfluorideionreleasebetweenF7andF7EPwhenexposed

tothesamesolution..........................................................................................106

Figure5.2.Differenceincalciumandphosphateionreleaseasafunctionofchangein

massbetweenF7andF7EPincitricacid(a)andhydrochloricacid(b)..............107

12

Figure6.1.DailymasslossofF7blockstreatedwithplacebomousseandstoredin

fourdifferentsolutions......................................................................................125

Figure6.2.DailymasslossofF7EPblockstreatedwithplacebomousseandstoredin

fourdifferentsolutions......................................................................................126

Figure6.3.DailymasslossofF7blockstreatedwithTM+andstoredinfourdifferent

solutions.............................................................................................................127

Figure6.4.DailymasslossofF7EPblockstreatedwithTM+andstoredinfour

differentsolutions..............................................................................................128

Figure6.5.Cumulativethree-daycalciumionreleasefromF7blocksacrossfour

solutionsandthreetreatmentgroups...............................................................131

Figure6.6.Cumulativethree-daycalciumionreleasefromF7EPblocksacrossfour


Figure6.7.Cumulativethree-dayphosphateionreleasefromF7blocksacrossfour


Figure6.8.Cumulativethree-dayphosphateionreleasefromF7EPblocksacrossfour


Figure7.1.ComparisonofFluorideIonReleaseinFujiVIIEPandShofuBeautifilF03.

............................................................................................................................159

Figure7.2.Relationshipbetweendailyaluminiumandfluorideionsreleasedfrom

SBF03..................................................................................................................160

Figure7.3.Relationshipbetweendailyaluminiumandphosphateionsreleasedfrom

F7EP....................................................................................................................160

13

LISTOFTABLES

Table4.1.Materialsusedinpilotstudies.CPP-ACP(caseinphosphopeptide-

amorphouscalciumphosphate)...........................................................................74

Table4.2.Comparisonofmasslossbetweenonehoursettingtimeand24hour

settingtimefollowedbycitricacidexposureofF7.Percentagechangeinbold

numbers,allothernumbersingrams.n=5..........................................................80

Table4.3.Comparisonofmasslossbetweenonehoursettingtimeand24hour

settingtimefollowedbycitricacidexposureofF7EP.Percentagechangeinbold

numbers,allothernumbersingrams.n=5..........................................................81

Table4.4.DailyIonreleaseofF7,F7EP,RivaProtectandChemFilMolarusingliquid

chromatography.AllconcentrationvaluesareinmM.........................................82

Table4.5.MasslosscomparisonbetweenCFR,F7andF7EPwhenstoredincitricacid

andwateroverathree-dayperiod.Allvaluesareingrams................................83

Table4.6.SurfaceVHofF7,F7EP,RivaProtectandChemFilMolarinlacticacidover

28days.Storagevolume15ml.n=5.....................................................................90

Table4.7.SurfaceVHofChemfilRockcomparedtoF7andF7EPstoredincitricacid

overathreedayperiod.Standarderrorshowninsecondarycolumns.Allvalues

inMPaVH1.0........................................................................................................91

Table5.1.MeansurfacehardnessofF7andF7EPinacidicandneutralsolutions

measuredoverthreedays.Valuesmarkedwiththesamelowercasesubscript

indicatesignificantdifferencebetweenGICmaterialswithinthesamesolution

forthesametimeperiod...................................................................................104

Table5.2.MeanrelativemassofF7andF7EPwhenstoredinthefourdifferent

solutionsoverthreedays.Valuesmarkedwithuppercasesuperscriptindicate

significantdifferencebetweendifferentsolutionswithinthesamematerialfor

thesametimeperiod.Eachgroupisscaledaccordingtoitsinitialweight(100%).

............................................................................................................................104

14

Table6.1.Treatmentsandcontrolgroups.................................................................120

Table6.2.SurfaceHardness(%changerelativetofreshblocks)...............................123

Table6.3.MassLoss(%changerelativetofreshblocks)..........................................125

Table6.4.Calciumionrelease(allunitsinμM),n=10...............................................129

Table6.5.Phosphateionrelease(allunitsinμM),n=10...........................................130

Table6.6.Fluorideionrelease,n=10.........................................................................135

Table6.7.Statisticalsignificanceinwater.(Hardness.Massloss.Calcium,Phosphate

andFluorideionrelease.*-nostatisticalsignificance)......................................136

Table6.8.Statisticalsignificanceinlacticacid.(Hardness.Massloss.Calcium,

PhosphateandFluorideionrelease.*-nostatisticalsignificance)...................137

Table6.9.Statisticalsignificanceinhydrochloricacid.(Hardness.Massloss.Calcium,


Table6.10.Statisticalsignificanceincitricacid.(Hardness.Massloss.Calcium,


Table7.1.GeneralcompositionofGICs.SiO2makesuptherestupto100%...........153

Table7.2.Surfacehardnesscomparison(VH1.0).Absolutevaluesinbold,%dropsin

secondrow.........................................................................................................157

Table7.3.DailymasslossofSBF03infourstoragesolutions.Meanvaluesingrams.

............................................................................................................................158

Table8.1.GeneralcompositionofGICs.....................................................................172

15

CHAPTER1

Introduction

Originally introduced by Smith in 19681, glass ionomer cements (GIC)

underwentaperiodofrapiddevelopmentinthelate1970sandearly1980s2-21and

havebeenthesubjectofcontinuingsteadyimprovementeversince.Valuedfortheir

chemical adhesion to tooth structure and potential for long term fluoride release,

GICs have established a unique role as a restorative material with anti-cariogenic

propertiesabletopromoteremineralisationofcaries-affectedenamelanddentine22.

Overtheyears,numerouseffortstoimproveGICshaveyieldedmixedresults.

Incorporation of anti-bacterial agents, bio-active glass particles and silica fillers

greatlyreducedtheworkabilityofthematerial23-27.However,furtherdevelopments

of the GIC formula have provided fast setting times, enhanced radio-opacity and

efforts toprovide the releaseofcalciumandphosphate ions inaddition to fluoride

ionstofurtherenhanceGIC’sbiomimeticqualities28,29.

Ongoing research has increased our understanding of the chemical and

physicalpropertiesofGICsand theirbroaderapplicationswithindentistry.Through

thisresearchtheGICshavefoundwideclinicalapplicationaspitandfissuresealants,

bonding agents and ‘low’ load-bearing restorative materials30. Lower mechanical

16

strength compared to other materials as well as lower retention rates and initial

watersensitivityhavelimitedtheuseofGICstoseveralspecificapplications30,31.

TobetterunderstandmorerecentimprovementstoGICmaterialsaseriesof

laboratorystudieswereundertakenandarepresentedinthefollowingpagesofthis

thesis. Physical and chemical properties of GIC materials were investigated in

combination with a variety of environmental conditions and topical treatment

protocols.Additionalchemicalanalysiswasthenconductedbasedonobtainedresults

tofurtherunderstandtheunderlyingstructureofGICmaterials.

A comparison of conventionalGICs, amodified conventionalGIC and a pre-

reacted glass ionomer particle filled resin composite were investigated. The

experimentalworkintroducesanewapproachtotestingtheGIC-basedmaterialsand

howtheymightreactunderdifferentenvironmentalconditions.Thisworkhasshed

lightintotheirstructureandfinallysuggestssomepossiblewaystheGICformulacan

befurtherimprovedtoamelioratetheeffectsthedifferentenvironmentalconditions.

Thefirstpartoftheexperimentalworkwascentredonaseriesofpilotstudies

todevelopanoptimumspecimenshape,thebestmethodstodetermineionrelease

and hardness of testmaterials. The following chapter (Chapter 5) investigated the

changes in physical properties and ion release of a CPP-ACP-modified conventional

GICwhenitwassubjectedtoanacidicenvironment.Chapter6describestheeffects

of the potential to recharge the GICs from the aspect of fluoride, calcium and

phosphateions.ThecementswererechargedwithaCPP-ACPcontainingcrèmeand

then exposed to the acidic environment once more to determine changes in ion

release. The outcomes showed some unexpected results that are believed to be

relatedtothealuminiumionsintheGICs.ThisformedthebasisofChapter7.Finally,

Chapter8drawstogetheranddiscussesalloftheaspectsofthisresearchprojectand

briefly considers where GICs could bemodified to overcome some of the changes

thatmayoccurwhenexposedtodifferentacids.

17

1.1References

1. SmithDC(1968).Anewdentalcement.BritishDentalJournal124(9):381-384.

2. Prosser HJ, Powis DR, Brant P,Wilson AD (1984). Characterization of glass-

ionomercements .7.Thephysicalpropertiesof currentmaterials. Journalof

Dentistry12(3):231-240.

3. Crisp S, Lewis BG, Wilson AD (1980). Characterization of glass-ionomer

cements. 6. A Study of Erosion and Water-Absorption in Both Neutral and

AcidicMedia.JournalofDentistry8(1):68-74.

4. CrispS,KentBE,LewisBG,FernerAJ,WilsonAD(1980).Glass-ionomercement

formulations.II.Thesynthesisofnovelpolycarobxylicacids.JournalofDental

Research59(6):1055-1063.


cements.5.Effectofthetartaricacidconcentrationintheliquidcomponent.

JournalofDentistry7(4):304-312.

6. KentBE,LewisBG,WilsonAD (1979).Glass ionomercement formulations: I.

Thepreparationofnovelfluoroaluminosilicateglasseshighinfluorine.Journal

ofDentalResearch58(6):1607-1619.


cements.3.Effectofpolyacidconcentrationonphysicalproperties.Journalof

Dentistry5(1):51-56.

18

8. WilsonAD,CrispS,AbelG(1977).Characterizationofglass-ionomercements

.4. Effect of molecular-weight on physical properties. Journal of Dentistry

5(2):117-120.

9. McLeanJW,WilsonAD(1977).Theclinicaldevelopmentoftheglass-ionomer

cement.II.Someclinicalapplications.AustralianDentalJournal22(2):120-127.


cement.III.Theerosionlesion.AustralianDentalJournal22(3):190-195.


cements. I. Formulations andproperties.AustralianDental Journal22(1):31-

36.


cements. 2. Effect of the powder: liquid ratio on the physical properties.



cements. 1. Long term hardness and compressive strength. Journal of


14. Crisp S,Wilson AD (1976). Reactions in glass ionomer cements: V. Effect of

incorporating tartaric acid in the cement liquid. Journal of Dental Research

55(6):1023-1031.

15. WilsonAD,CrispS,FernerAJ(1976).Reactionsinglass-ionomercements:IV.

Effect of chelating comonomers on setting behavior. Journal of Dental

Research55(3):489-495.

19

16. Crisp S, Prosser H, Wilson A (1976). An infra-red spectroscopic study of

cement formation between metal oxides and aqueous solutions of poly

(acrylicacid).JournalofMaterialsScience11(1):36-48.

17. Crisp S, Wilson AD (1974). Reactions in glass ionomer cements: III. The

precipitationreaction.JournalofDentalResearch53(6):1420-1424.

18. Crisp S, Wilson AD (1974). Reactions in glass ionomer cements: I.

Decompositionofthepowder.JournalofDentalResearch53(6):1408-1413.

19. Crisp S, PringuerMA,Wardleworth D,Wilson AD (1974). Reactions in glass

ionomer cements: II. An infrared spectroscopic study. Journal of Dental

Research53(6):1414-1419.

20. CrispS,WilsonAD(1973).Formationofaglass-ionomercementbasedonan

lon-leachable glass and polyacrylic acid. Journal of Applied Chemistry and

Biotechnology23(11):811-815.

21. WilsonAD,KentBE(1972).Anewtranslucentcementfordentistry.Theglass

ionomercement.BritishDentalJournal132(4):133-135.

22. Tyas MJ, Burrow MF (2004). Adhesive restorative materials: a review.

AustralianDentalJournal49(3):112-121;quiz154.

23. TjandrawinataR,IrieM,SuzukiK(2005).Effectof10wt%sphericalsilicafiller

additionon the variouspropertiesof conventional and resin-modified glass-

ionomercements.ActaOdontologicaScandinavica63(6):371-375.

20

24. Yli-UrpoH, Lassila LV,Narhi T, Vallittu PK (2005). Compressive strength and

surface characterization of glass ionomer cements modified by particles of

bioactiveglass.DentalMaterials21(3):201-209.

25. Yli-Urpo H, Narhi M, Narhi T (2005). Compound changes and tooth

mineralization effects of glass ionomer cements containing bioactive glass

(S53P4),aninvivostudy.Biomaterials26(30):5934-5941.

26. SandersBJ,GregoryRL,MooreK,AveryDR(2002).Antibacterialandphysical

properties of resin modified glass-ionomers combined with chlorhexidine.

JournalofOralRehabilitation29(6):553-558.

27. Botelho MG (2004). Compressive strength of glass ionomer cements with

dental antibacterial agents. Journal of the South African Dental Association

59(2):51-53.

28. MazzaouiSA,BurrowMF,TyasMJ,DashperSG,EakinsD,ReynoldsEC(2003).

Incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa

glass-ionomercement.JournalofDentalResearch82(11):914-918.

29. Al Zraikat H, Palamara JE,Messer HH, BurrowMF, Reynolds EC (2011). The

incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa

glassionomercement.DentalMaterials27(3):235-243.

30. Tyas MJ (2006). Clinical evaluation of glass-ionomer cement restorations.

JournalofAppliedOralScience14Suppl(10-13.

31. Mount GJ (1998). Clinical performance of glass-ionomers. Biomaterials

19(6):573-579.

22

CHAPTER2

LiteratureReview

2.1DentalCaries

In today’sworld,despitemanyadvancedpharmaceuticalsandouradvanced

understandingofmedicine,dentalcariesremainsamajorhealthproblemworldwide1.Inmanycountriesitisthesinglemostcostlydiseasetotreat2,3.Manyeffortshave

beenmadetobetterunderstandandtreattheproblem.Ithaslongbeencommonly

acceptedthattheprevalenceofrefinedsugarsinthedietareamajorcausativefactor

in dental caries. The effect of sugars on dental caries has been the subject of

discussion for over 60 years despite the best efforts of the dental profession and

otherstocurtailtheproblem4,5.Raisingpublicawarenesshasbeenattheforefront

ofeffortstocombatdentalcaries6.

23

2.1.1Aetiology

In an attempt to describe the aetiology of caries lesion formation, several

studieshaveattempted to correlate theonsetof caries to traceelements found in

caries-affectedenamel7.Itwasestablishedthatfluoridemustpossessanti-cariogenic

propertiesduetotheconsistentobservationofitspresenceinthesurfacelayerfound

above the carious lesionand its absence in thede-mineralisedenamel surrounding

the lesion 8. For a long time therewas a significant amount of empirical evidence

linking caries to dietary habits, but the formation of carious lesions has been best

described by Featherstone et al., in 19799. Their study investigated the chemical

processesthatoccurredduringtheformationofcariouslesions.

Beginning in the 1890s, studies focused on studying the bacteria present

during the onset of dental caries10, 11. Replicating the formation of lesions in the

laboratorywasthefirststeptounderstandingtherelationshipbetweenbacteriaand

dental caries. It was found that certain strains of bacteria, Streptococcus mutans,

which is present in healthy saliva, consumes glucose. Fermentation of glucose

increases the growth of the bacterial colony and the metabolism of the bacteria

whichinturnproduceslacticacid12.ThelowerpHcausedbyincreasedconcentrations

of lactic acid results in tooth mineral dissolution and consequently dental caries.

Other Streptococcal strains and Lactobacilli, both found in healthy dental plaque,

were found to have similar characteristics as S. mutans. It is unclear what exactly

causessomespeciesofbacteriatoincreaseinpopulationinpreferencetoothers,but

inalmostallcasesofdentalcariesadominantglucoseconsumingstrainemergesand

asa result thepHof theoral environmentdropsasmore lactic acid isproduced12.

Thus the onset of dental caries depends on a number of factors; amature biofilm

containing the right bacterial strains, the supply of fermentable sugars and a

substrate forbacterial colonies togrowon,namelya toothsurface (enameland/or

dentine).Alltheseconditionsneedtobesatisfiedforcariestoinitiateandprogress.

Fermentable sugars remaining after food consumption in the oral

environment produce lactic acid over time when the bacterial biofilm is mature.

24

Lacticacid,initsun-ionizedform,penetratestheenamel,dissociatesandonceahigh

enoughconcentration isreachedbeginsattackingandremovingcalcium,phosphate

and fluoride ions from the tooth. The largely intact surface layer is formed as

subsurfaceionsdiffusetothesurfacewheretheyareadsorbedbackintothelattice

oftheenamel.Theratesoflacticacidpenetration,minerallossandadsorptionatthe

surfacealldependonconcentrationgradientsoflacticacidwithrespecttotheintra-

enamelfluid.Asthelesionprogressesfurtherintosub-surfacelayersgreateramounts

oftoothmineralaredisplaced,which inturn leadstothedropintheconcentration

gradient at the surface creating an equilibrium between mineral loss and ion

adsorption. From the outside this creates an intact surface layer while the lesion

continuestoprogressfurtherintothesub-surface9.Hence,thepresenceoffluoride

in the surface layer has been shown to be the result of carious lesion progression

rather thanhindering it.Despite thisevidence, thecariostaticabilityof fluoridehas

beenpromotedinfavourofotheragents(suchascalciumandphosphate),thatalso

havebeenfoundtoslowtheprogressionofdentalcaries.

2.1.2Immunisation

Early efforts to characterize dental caries anti-bodies found that such

antibodies either exist in very low concentrations in saliva or do not exist at all 13.

More recentattemptsat immunizationagainstdental caries foundsomesuccess in

animalstudies,howevertheefficacyofsuchvaccinesonhumansremainsunknown

asfurtherresearchisneeded14.Althoughultimatelytheonsetofdentalcariescanbe

traced to various strains of bacteria, the underlying cause is driven by de-

mineralisation of tooth structure caused by a falling pH in the oral environment.

Clinical observations suggest that patients on broad-spectrum antibiotics exhibit

reduced symptoms of dental caries; this highlights the problem surrounding

immunisation. Any effective vaccinewould need to be broad enough to immunise

againstallknowncariescausingbacteria12.

25

2.1.3FluorideandCariesprevention

Sincetheintroductionoffluorideintotheoralenvironment,eitherthrougha

fluoridatedwatersupply,toothpasteortopicaltreatments,theprevalenceofdental

carieshasfallen15.Theroleoffluorideincariespreventionhaslongbeenestablished7. Investigationshaveconcludedthat lowconcentrationsoffluoridehaveatwo-fold

effectininhibitingformationofcaries-likelesionsinhumanenamel16.Theinteraction

of fluoride with enamel mineral leads to an increase in re-mineralisation during

periods of super-saturation with respect to hydroxyapatite and also resulted in

reduced de-mineralisation during periods of under-saturation with respect to

fluorapatite. The same study also determined that even low concentrations of

fluorideledtoreducedbacterialacidproductionresultinginsmallerpHfallsindental

plaque16.Itisnowcommonlyacceptedthatfluorideisabletosubstituteforhydroxyl

ionsinhydroxyapatitefoundinenameltoformfluorapatite(Equation1),whichisless

solubletherebyprovidingincreasedprotectionagainstacidattack17.

(Equation1) Ca10(PO4)6(OH)2+2F-→Ca10(PO4)6F2+2OH-

Little research has been done to exactly determine the concentrations of

fluoride required to achieve an optimal cariostatic effect. In contrast, numerous

studies have investigated the effect of fluoride on the clinically observed levels of

fluorosis,especiallyinchildren18.

Furthermore,earlyevidenceshowedthattopicalfluorideapplicationsprevent

bacterial growth on the tooth surface19. Reduced levels of S. mutans have been

reported under stannous fluoride (SnF2) treatments in particular, more so than

sodiumfluoride(NaF2)20.Later,studiesinvestigatingcalciumfluoridehaveshownno

changeinS.mutanslevelsundertopicalfluoridetreatments21.Ithasbeenproposed

that in high enough concentrations of fluoride, topical treatments are able to

substitute fluoroapatite into demineralised enamel thereby arresting the onset of

cariouslesions22.Byprovidingaphysicalbarrierbetweentoothmineralandbacteria,

26

fluoridevarnishesareabletocreateafavourableconcentrationgradientfor inward

diffusionoffluoride,thuspromotingremineralisation23.Sugarsremainingafterfood

consumption,which aremetabolised into acid, aremore likely to be found on the

surface of teeth. Therefore, specifically protecting the surface of the tooth against

acid attack further adds to the cariostatic ability of fluoride. Could deep caries-like

artificial lesions also be re-mineralised using conventional calcium, phosphate and

fluoride topical treatments? It was found that topical treatments could only re-

mineralise the surface layer of enamel and that the ion penetration of the surface

wasnothighenoughtoreachdemineraliseddentine24.Thisissupportedbyprevious

theoriesaboutsubsurfacedissolutionofmineralsandsubsequentadsorptiononthe

surface layer of enamel. Further evidence in support of fluoride exposure is often

reported25,26, butother factors in theoral environment, suchasdietaryhabits and

theroleofhealthysalivaflowaregainingmoresignificanceandattentionwithregard

tocariesactivityandrisk27.Thecompositionofdentalplaque,orbiofilmasitisnow

called,hasbeenanongoingtopicof interest.Theanti-cariogenicpotentialofdental

plaquehasalwaysbeenseenasanimportantfactorinthedevelopmentofcaries,the

biofilm is supersaturated (with calcium and phosphate ions) with respect to tooth

mineralandconsequentlyhasthepotentialtore-mineralisecariouslesions.However,

fermentable carbohydrates will lower the pH of the biofilm and the degree of

saturation will therefore decrease rapidly28. A cycle of remineralisation and

demineralisation at the tooth surface involving calcium and phosphate ions is

currently the acceptedmechanism of a carious lesion formation and repair (Figure

2.1)29.

27

Figure2.1.Stagesofcarieslesioninitiationandprogression.(http://se.dentalcare.com/en-US/dental-education/continuing-

education/ce377/ce377.aspx?ModuleName=coursecontent&PartID=7&SectionID=-1)

2.1.3aMinimalInterventionDentistry

Bornoutof the ideaof trying to re-mineraliseapatitedepleteddentineand

enamel, minimal intervention dentistry is a concept that is now supported and

promulgated by many individuals30-32. The traditional approach of treating caries

involves removing all ‘infected’ and ‘affected’ dentine from the tooth for the

preparationofadefinedcavitythatwillbefilledwithamaterialofsomekind,either

dentalamalgamora resin-basedmaterial.Caries-affecteddentine isdefinedasde-

mineralised dentine that is ‘free’ from bacterial contamination and still retains its

collagenmatrixstructure33.Caries-infecteddentinecontainsahighconcentrationof

bacteriaandthecarieshasprogressedtothepointwherethecollagenfibrestructure

has broken down into an amorphous mass, consequently no re-mineralisation is

28

possible.Minimal intervention treatment is a conceptwhere traditional restorative

approachesofremovingallcaries-infectedand-affecteddentineisquestionedanda

method of removing only infected dentine and leaving affected dentine to be re-

mineralised is advocated 31, 34. The role of fluoride is a major one as fluoride

incorporatedintodentalrestorativematerialsmayhavethepotentialtore-mineralise

surroundingtissueandtissueatthebaseofacavity.Earlystudiesreportedonhow

fluoride release fromdentalmaterialswould affect enamel; oneof the conclusions

made from these studies was that materials containing no fluoride reduce the

fluoridecontentofenamel 35.Thisseemsto indicate that fluoridesaturationof the

oral environment has a strong influence on fluoride content of enamel; lack of

fluorideinplaquecanleadtolowfluoridelevelsinenamel.Thistiesinwellwithour

understandingofcariouslesionformationandtheeffectoffluoridesaturationlevels

onarrestingcariesprogression, low levelsof fluoride ionscan leadtoan imbalance

betweenplaqueandenamel,whichinturncanpromotedemineralisation.

2.1.4Fluoridecontainingdentalrestorativematerials

Historically,somedentalmaterialshaveproventobebetterfluoridereleasing

agentsthanothers.Onesuchtypeofmaterial isaglass ionomercement(GIC), first

proposed in the late1960s36andactivelydeveloped fromtheearly1970s37-54.GICs

haveestablishedanicheamongstotherdentalrestorativematerialsfornotonlytheir

high fluoride release but reliable chemical adhesion to tooth structure as well as

simpleuse55.

29

2.2GlassIonomerCements

2.2.1Origin

In1972aglass ionomercement (GIC)systemwas introducedbyWilsonand

Kentasanewtypeofdentalmaterial,describedbythereactionbetweenacrylicacid

andfluoroaluminosilicateglasspowder37.Theideaofadhesionandtheformationof

anionexchangelayertotoothstructureusingapoly-alkenoicacidwasfirstsuggested

bySmithafewyearsearlier36.Thebondiscreatedwhenacidiccarboxylgroupsattack

toothstructureliberatingcalciumandphosphateions,inreturnaluminium,fluoride,

silicaandstrontiumionsareliberatedfromtheglasspowderandpenetratethetooth

structure,resultinginalayerofexchangedionsbetweentheGICandtoothtissue56.

ThistypeofchemicaladhesiontotoothstructuremadeGICsuniqueamongstdental

restorativematerials,manystudiesfollowedattemptingto improveandunderstand

thebenefitsofGICs.

2.2.2ChemistryoftheSettingReaction

Research into possible alternatives to poly-alkenoic acids, which tend to

thickenandgelattherequiredconcentrationof50%,haveledtotartaricacidbeing

proposedasapossibleacceleranttobeusedinGICs43,50.Itwasalsodeterminedthat

certain copolymers of polyacrylic acid showed goodmobility and low viscosity at a

concentration of 50%. Of these, acrylic acid produced cements of higher tensile

strength 53. In subsequent studies the relationships between the glass powder and

acid liquid (P:L) ratio and its effect on setting time, compressive strength and

hardness were explored. It was claimed that higher P:L ratio leads to increased

compressivestrength,settingrate,surfacehardnessandmadethemixlesssensitive

tomoisture 41. The optimal powder:liquid ratio, which will depend on the specific

clinicalapplication, shouldbe tailored toaccommodateworking timeandmaximize

strengthanddurabilityofthemix41.Somestudiesfoundthatthemolecularweightof

30

thepolyacidusedinthereactionhadasignificanteffectonthestrengthoftheGIC46.

PolyacidsofhighermolecularweightproducedGICswithenhancedstrength,butthe

workingtimeofthemixwasreducedlimitingtheGICsuse.Ifalowerconcentrationof

the polyacid, with the same P:L ratio, was used to increase theworking time, the

strengthoftheGICwassubsequentlyreduced46.Investigationoftheeffectoftartaric

acidonthepropertiesofGICsdiscoveredthattartaricacidwasabletoacceleratethe

setting reaction, providing a “snap set,” as well as increase the strength of the

cement.However,whenusedinexcessitslowedthesettingreactionandweakened

thecement50.AchelatingeffectoftartaricacidonthesettingpropertiesofGICswas

also observed, it was speculated that the tartaric acid extracted metal ions (e.g.

aluminium)fromtheglassandwasabletoholdtheionsinsolutionpreventingearly

binding and thus extending the “working time.” The setting characteristics were

furtherimprovedasthiseffectalsoincreasedtherateofhardeningofthecementmix44.

A separate study focused on investigating the ability of polyacrylic acid to

extract fluoride ions from the glass powder during the setting reaction57. It was

concludedthattheextractionoffluorideionswasaidedbytheformationofcalcium

and aluminium salt complexes. Understanding of the extraction mechanism is

important in being able to control the reaction, if the fluoride is not extracted

efficiently the pH of the setting cement rises too quickly producing an unworkable

mix 39. Further research into the effect of pH on fluoride release and mechanical

properties concluded that while fluoride ion release was beneficial, more work is

neededtobedoneto improvethemechanicalpropertiesofmanymaterials if they

aretobeusedforloadbearingrestorations58.

2.2.2aRoleofAluminium

Aluminiumplaysanimportantroleintheacid-basesettingreactioninGICs.It

ispredominantlypresentintheformofaluminiumoxide.Althoughtheinitialsetting

reaction in theGIC involvescalciumorstrontiumsalts,asecondarysettingreaction

31

relies heavily on aluminium salts. The presence of aluminium adds basicity and

ensuresthatacompleteacid-basereactioncantakeplace,allowingthemixtofully

set.Exposure toanacidicchallengemay leadtoaluminiumbeing leached fromthe

GICrestoration;thebiologicaleffectsandstructuralsignificancearediscussedinthe

paperbyNicholsonetal59.

It is estimated that if a GIC restorationwere to dissolve completely and be

absorbed by the human body via the gastro-intestinal tract over a 5-year period it

wouldonlycontribute0.5%ofthemaximumallowablealuminiumintake.Attemptsto

makeaGICcontaininghighlevelsoffluorideprovedsuccessful,ofparticularinterest

wastherelativeratiobetweenaluminiumoxideandsilicondioxideanditseffecton

the stability of the cementmix51. It was also discovered that the Al2O3: SiO2 ratio

controlled the cements susceptibility to acid dissolution. The presence of Al3+ at

networkformingsitesmadethecementvulnerabletoacidattack,butaboveacertain

Al:Si ratio the inclusion of Al3+ ions made the GIC more resilient against an acid

challenge 51. The attenuated total reflectance spectroscopy techniquewas used to

investigatethevariousspeciespresentinthecementmixduringthesettingreaction

ofaGIC60. Itwasdiscoveredthatbothcalciumandaluminiumsaltswerepresent in

thefinalmixinequalamounts.Calciumsaltswereformedfirstandwereresponsible

forgelationandthe initial set,aluminiumsaltswere formed laterwhichresulted in

the hardening of the cement mix 40. In a follow-up study, it was discovered that

calciumsaltswere fully formedwithin the first3hoursof setting,whilealuminium

saltsonlystartedformingafteranhourandthereactioncontinuedforupto48hours

after the initial setting. The initial stages of setting consisted of a precipitation

reaction as fluoride and phosphate ions were extracted from the glass powder to

form insoluble salts and complexes. The structure of the final matrix was

characterizedbychainsofcovalentbonds,whichwerefurtherstrengthenedbyionic

bridgescross-linkingthechains,withglassparticlesembeddedinthehardmatrix38.

32

2.2.3PhysicalProperties

InvestigationofwearandmicrohardnessofGICsshowedthathydrationand

exposuretolacticacidplayasignificantroleinthehardnessandwearpropertiesof

GICs61. Characterization studies ofGICs found that surface hardness predominantly

developsduringthefirst24hoursaftermixing,withslight increasesbeingrecorded

uptooneyearfollowingtheinitialset42.Thecompressivestrengthoftheinvestigated

GICscontinued to increaseaftera24hourperiod,and theauthors speculated that

themaximumcompressivestrengthwouldbeachievedafteroneyear42.Astudyof

surface roughness and hardness of various types of GICs determined that the

compositionoftheGIChasasignificanteffectonitsroughnessandhardness62.GICs

werefoundtohavethehighestsurfaceroughness.AsilverreinforcedGICwasfound

tohavethehighestmicrohardness,howevernocorrelationbetweenroughnessand

hardness could be determined 62. Elsewhere it was reported that major chemical

changesinsomeconventionalGICsoccurredwhenexposedtoartificialsaliva,which

pointed to an increase in surfacemicrohardness after a period of storage63. In an

attempt to find an appropriate laboratory test to predict the resistance to

degradationofrestorativematerialsthreedifferentmethodswerecompared–mass

loss, degradation of the area of thin cement films, and reduction in transverse

strength64. Degradation of the area of thin films made from cements showed the

greatest correlation with in vivo results64. Research into incorporation of

hydroxyapatite(HA) intoGICsfoundthatthefracturetoughnesswasimprovedwith

incorporationofHA,whileat the same time leavingbondingand fluoride releasing

benefitsofthematerialunaffected65.

ItwasshownthatGIChadthelargestinitialwateruptakeinthefirst24hours

compared to other water-based dental cements (dental silicate cements and zinc

polycarboxylatecements),althougherosionoverasevendayobservationperiodwas

similar52.Whenexposedtolacticacidmedia,GICsdemonstratedtheleastamountof

erosion compared to other water-based cements52. While GICs have several

advantagesoverothermaterials,suchasstrongchemicaladhesiontotoothstructure,

33

they do have their shortcomings. Initial water sensitivity, setting time and other

physicalpropertieshavebeeninvestigatedandcomparedtomanyotherrestorative

materials.

2.2.4FluorideReleasefromGICs

GIC’s ability to deliver a sustained release of fluoride ions provides unique

benefitsincombatingcaries.StudiesoffluoridereleaseandabsorptionfromGICsat

differentpHlevelsshowthatfluoridereleaseisdependentonsurfacedegradationof

theGIC,whichiscausedbyvaryingthepHofthesolution66.However,itisimportant

to note that Kim et al.67, in their study of GIC’s ability to re-mineralise apatite

depleteddentine,claimthatmineralconcentrationaloneisnotasufficientendpoint

for assessing the successof contemporary remineralisation strategies 67. It isnoted

that GICs may be able to re-mineralise partially de-mineralised dentine, where

remaining apatite crystalsmay serve as centres for nucleation and subsequent re-

mineralisation.Inasplit-mouthstudy,proximalcarieslesionstreatedwithGICswere

foundtobemorelikelytoremaininorregresstotheouterhalfofenamel68.Astudy

ofminerallossonenameladjacenttoGICrestorationsconcludedthatGICscanexert

aprotectiveeffectonlyunderacariogenicchallengeandnotanerosivechallenge69,it

is speculated that fluoride ion releasemaynotbehighenough topreventa strong

erosive challenge. However, thismay also indicateGIC vulnerability against a citric

acid attack. Fluoride ion release comparison between fluoride varnish (Duraphat,

Colgate-PalmolivePtyLtd,USA)andGIC,foundthatbothproductspromotemorere-

mineralization of artificial lesions on proximal surfaces,with fluoride varnish (52%)

providing higher reduction in carious surface area than GIC (33%)70. Other studies

alsoclaimtohavefoundapositivecorrelationbetweenthedosageoffluorideandits

ability to inhibit demineralization 71. De-mineralisation and re-mineralisation of

enamelwasfoundtohaveastrongdependenceontheconcentrationoffluorideions,

itisclaimedthatfluoridehasasignificanteffectoninhibitingdemineralisationatthe

34

tooth surface, and that fluoride contributes to sub-surface remineralisation

potentiallyreversingtheeffectofcaries72.

A study by Williams et al 73 investigated the relationship between fluoride

release from GICs, volume of cement samples and their surface area. Several

different sample shapes were tested (discs, cylinders and bars); a strong positive

correlation was found between long-term fluoride release and surface area, as

opposedtothesamplevolume73.Ashort-termstudyproposedthatfluoridereleasing

gelsmayprolong the fluoride releaseofGICsby replenishing fluoride lost fromthe

GICsurface74.However,ithasbeenpointedoutthattheoverwhelmingevidenceof

superiorfluoridereleasebyGICsisnotconclusiveevidenceoftheircariostaticability,

whichcanonlybeascertainedthroughcarefullydesignedclinicaltrials75.

2.2.5ClinicalApplicationandComparison

In a seriesof articles,McLeanandWilsondiscussed the clinical implications

andusesofGICs,chemicaladhesiontodentineandenamelwashighlightedasoneof

themajorbenefitsofthematerial,desirableaestheticqualitieswerealsomentioned.

The authors suggested that themost promising use of GICwould be in preventive

dentistry,sealingerosionlesionsandfissures.Inaddition,GIC’scariostaticproperties

couldplayan important role in inhibitingocclusalcaries.Followingaclinical trialof

GICsasarestorativematerialforcervicallesionsanumberofrecommendationswere

madeandthelimitationsofGICapplicabilitywerehighlighted47-49.

Sincethenalotofprogresshasbeenmade,bothindevelopingGICmaterials

and tailoring them for specific applications. Further research has highlighted the

effectivenessofGICscomparedtoresin-basedfissuresealants,andconcludedthatno

materialhadanadvantageovertheotherandbothwereeffectiveasfissuresealant

materials76. Additional evidence comparing the effect of resin-based infiltrants and

sealants on the progression of caries lesions on proximal surfaces has shown

significant benefit to using either approach. Using resin infiltrants and sealants

promisestoalleviatetheissueofsecondarycaries,whichnon-fluoridereleasingresin

35

restorations are susceptible to77. In a review study of clinical trials pit and fissure

sealants were found to have a significant effect on prevention of dental caries,

howeverwhencomparingvarioussealanttypesamongstthemselvestheresultswere

conflicting78.Studiesshowingresin-basedsealantsasbeingsuperiortoGICsealants

reportedhighretentionrates,asopposedtoverylowretentionratesforbothsealant

typesinstudiesthatshowedGICstobesuperior.Long-term(7years)studies,which

showednodifferencebetweeneithersealanttypereportedhighretentionratesfor

resin-based sealants and poor retention rates for GIC-based sealants 79. Fluoride

varnisheswerecomparedwithsealantsintheirabilitytopreventdentalcariesandit

was found that sealantswere superior in their ability to prevent occlusal caries 79.

Some studies have claimed that resin-based sealants performed better in terms of

sealingabilitythanGICs80.However,comparisonofretentionandcariesprevention

ofaresincompositesealantandaGIChasshownthatalthoughtheretentionofGIC

waslower, itscariespreventativeeffectwashigherthantheresin-basedmaterial in

permanentfirstmolars81.Insupportofthisevidenceothershavefoundthatfissures

sealed with GIC continued to have a caries inhibiting benefit even after the

restoration was clinically deemed to have been totally lost. Only after careful

examinationoftoothreplicaswas itrevealedthatsomeoftheGICstill remained in

thefissurebase82.ItwouldseemthatcompleteretentionisnotnecessaryforGICsto

retaintheircariesinhibitingproperties.ThisisfurthersupportedbyTorppa-Saarinen

andSeppa83,whosuggestedthataslongasasmallpartoftheGICwasstillpresentin

the fissure itmay stillprovideananti-cariogeniceffect.Examining secondary caries

around margins restored with amalgam, resins and GICs revealed that GICs were

superior in inhibitingsecondarycariesoccurrence,althoughthemarginsthemselves

werenotas“tight”as theamalgamandresinmaterials tested84.A fiveyearclinical

evaluationofGICsusedaslutingcementsonfullcastrestorationsreportedlowrates

of secondary caries and excellent retention85. A survey of GIC use by Australian

dentists found that GICs were mainly used for tunnel restorations, approximal

restorationsinprimarymolarsaswellas“sandwich”restorations,whereaGICisused

for its chemical adhesion to dentine as an intermediate layer beneath a resin

36

restoration86. Very few biological complicationswere reported, but themajority of

surveyeddentistsreportedmorecomplicationswithGICsthanamalgamrestorations,

withfractureofthemarginalridgeandwearbeingthetwomostcitedcomplications

withGICs.Onlyafewdentistsreportedoccurrencesofsecondarycariesandgingival

inflammationwhenusing aGIC comparedwith threequartersof surveyeddentists

whenusingresincomposites86.

GICs are classified into several types depending on application, chemical

formulationofeachtypeareverysimilarandonlyvaryinpower:liquidratioandglass

particlesize55,87.Abriefsummaryofthesetypesandthespecificdifferencesbetween

themisoutlinedbelow.

TypeI–lutingcements

Fallingunderamoregeneralcategoryoflutingagents,lutingcementsfillthe

role of fluoride releasing adhesives used for dental prostheses, implants, crowns,

bridges, veneers and orthodontic appliances. A strong chemical bond to tooth

structureandtootherrestorativematerialsisthemajoradvantageofusingGICsasa

lutingagent88.

TypeII–Aesthetic(II-1)andReinforced(II-2)restorativecements

GICsinthiscategoryarecommonlyusedascavityrestorations.Dependingon

the cavity location and size, all types of GICs (conventional, resin-modified, fast

setting, highly flowable) find use as restorative cements. The clinical evidence

suggests thatconventionalGICsareonlysuitable in lowstress restorations, suchas

anteriorapproximalandcervicalrestorations87.Highfailurerateshavebeenreported

forconventionalGICsusedinposteriorapproximalrestorations89,90.

TypeIII–linersandbases

Commonlyusedin“closedsandwich”orlaminaterestorations,whereaGICis

placed at the base of the cavity and covered by another restorativematerial. This

37

method provides a superior chemical bond between the dentine and restoration,

whilemaintainingadurable,aestheticrestorationontheexposedsurface55,91.

TypeIV–fissureandsealant

ModernGICshavedevelopedspecificformulationsforuseasfissuresealants

on theocclusal surface, the glass particles are finer than in otherGIC formulations

andthepowder:liquidratioismodifiedtobeamoreflowablemix92.

2.2.6Resin-ModifiedGICs

Resin-modifiedGICs (RM-GICs)were introduced relatively recently andhave

found applications as fluoride releasing restorativematerials that could be used in

load-bearing restorations, as well as broadening the range of fluoride delivery

methodsingeneral.Hydroxyethylmethacrylate(HEMA)istheresinincludedintothe

GIC,whichintroducesanadditionalpolymerizationreaction,eitherlightactivatedor

self-cured.IntroductionofresinintotheGICreducestheinitialwatersensitivitythat

conventionalGICssufferfromduringtheearlystagesofsetting.Resin-modifiedGICs

undergo an acid-base setting reaction like conventionalGICs,with a separate resin

polymerization reaction occurring. Some reports claim that like conventional GICs

resin-modifiedGICsareable tobondviachemicaladhesiontodentineandenamel,

although studies on this topic are divided. Parallel efforts were also made to

introducefluoridereleasingsilicateglassesintoresincomposites.Poly-acidmodified

resincomposites (PAMRC)as theyarecalled, sharesomeof thepropertiesof resin

compositesandGICs, suchas fluoride release.However,PAMRCs lack thechemical

adhesiontotoothstructureandfluoriderechargepropertiesofGICs.

The study of fluoride release and recharge fromdifferentmaterials used as

fissure sealants determined that GICs showed the highest initial and sustained

fluorideionreleaseincomparisontoRM-GICsandresincomposites93.Anoverviewof

thetherapeuticeffectsofGICssurmisedthatGICsexhibitanti-cariogenicproperties

throughtheiruptakeandreleaseof fluoride ions 94.Fluoriderelease fromGICsand

resin composites shows that composites releasemuch lower levelsof fluoride than

38

GICs inthefirstyear,butfollowingthat,thereducedreleasefrombothmaterials is

verysimilar95.ThelowerhydrophilicityofresincompositesincomparisontoGICsisa

significantfactorinthelargedifferenceinfluoridereleasebetweenthetwomaterials

duringtheearlystagesofrestoration.IthasalsobeenreportedthatGICsshowbetter

fluoride recharge, responding to NaF treatments better than comparable resins 96.

Some studies claim that RM-GICs have similar levels of fluoride ion release as

conventionalGICs,andthatthecompressivestrengthofRM-GICsdidnotchangeover

time compared to a conventional GIC97. Further evidence of equivalent fluoride

release fromRM-GICs compared to conventionalGICshasbeen reported98. In vitro

comparisonbetweenRM-GICsandconventionalGICsusedtorestorecervicalcavities

showed no significant difference between the two materials, however, RM-GIC

provedtobemoreacidresistant99.Alaboratorystudyoffluoridereleasefromseveral

GICs, RM-GICs and PAMRCs demonstrated that the amount of fluoride released is

dependentonthestoragemediumperhapsmoresothanthetypeofmaterial100.A

coatofdentineadhesiveoverconventionalGICandRM-GICrestorationswasfound

to significantly reduce fluoride release from these materials 101. Under in situ

conditionsRM-GICsshowsignificantremineralisationabilitiescomparedtoamalgam,

which exhibited further demineralisation in the specimens 102. A meta-analysis by

Mickenautschetal concluded thatRM-GICsandcomposites containing fluorideare

equallyeffectiveatreducingdemineralisationinadjacenthardtoothtissueandthat

botharemoreeffectivethancompositescontainingnofluoride103.Alaboratorystudy

of orthodontic brackets bonded with either RM-GIC or fluoride containing resin

compositesshowednodifferenceindemineralisationbetweenthetwomaterials104.

Afouryearclinicalstudycomparingamalgam,resincompositesandRM-GICsdidnot

find any significant difference between the materials when used to restore

overdentureabutments105.

IthasbeenfoundthatRM-GICsareweakerindiametraltensilestrengththan

resin composites but stronger than conventional GICs. RM-GICs also showed less

susceptibilitytowaterlossoruptakeovera6monthstorageperiod106.LoadandpH

havebeenfoundtohaveasignificanteffectonthewearrateofdentineandenamel,

39

thesamestudyalsodemonstratedthatresincompositesaremoreresistanttowear

than conventional and RM-GICs 107. Research into erosion of dental restorative

materials compared with dental tissue at low pH showed that resin composites

exhibited no change when exposed to citric acid (pH 3.0 to 7.0) for seven days,

conventionalGICshowedincreasingsignsoferosionwithdecreasingpH,althoughit

wasreportedthattherateoferosionfortheGICwas lessthanthatofdentineand

enamel108.Anothercomparisonofwearratesunderacidicchallengebetweenresin

composite,RM-GICandaconventionalGIC reported that resincompositeexhibited

thelowestsusceptibilitytoacidchallenge,followedbyaRM-GIC,withaconventional

GIC having the lowest resistance to acid attack 109. A three-year clinical study by

Ostlund et al. focused on retention of posterior approximal restorations using

amalgam, resin composite and GIC materials. Restorations that used a GIC were

found to have much higher failure rates than resins and amalgam 110. Surface

propertiesofGICswere found tobegreatly affectedbyacidic challenge (pH4.3 to

6.2), which was not the case for resin composites. Additionally the pH of the

demineralising solution was found to be strongly correlated with the release of

fluorideions111.FurtherevidenceofhigherfluoridereleaseforaRM-GICcomparedto

aresincompositehasbeenreported,alsoofnoteistheresin’sslowerpolymerization

rateduetointerferencebyoxygenduringthepolymerizationprocess112.

2.2.6aAtraumaticRestorativeTreatment

Atraumatic Restorative Treatment (ART) technique was first proposed by

Frencken et al. as a means to provide oral healthcare to a wider population

throughouttheworld,especiallyinthoseareasthatareeconomicallylessdeveloped113. The need for such a treatment was first highlighted by Thorpe et al. 114. The

techniqueisanextensionofMinimalInterventionDentistry,andreliesonlyonhand

instruments and requires no electricity. Caries-infected tissue is removed and the

cavityisrestoredusinganadhesivefillingmaterial,usuallyaconventionalGIC.Atwo

yearstudyusingaGICandaresincompositeforocclusalpitandfissuresealingand

40

proximalrestorations(classIandIIrespectively)usingtheARTapproachdetermined

that neither material was significantly better than the other115. However, a

comparison between high viscosity GICs and resin composites according to ART

concluded that GICs were more effective under field conditions due to being less

techniquesensitive thanresincomposites116. Inaonce-offapplicationofcomposite

andGIC using the ART approach, GICwas found to have a 3.1 to 4.5 times higher

cariespreventiveeffect117.AreviewofGICsusedassealantsforARTclaimedthatthe

treatmentswereeffectiveandnewerARTtailoredGICshaveshownanimprovement

overpreviousversionsofGICs118.BasedaroundtheARTapproach,astudyrevealed

that retention rates of occlusal and posterior approximal restorations using resin

composite andGICmaterialsweremore affected by the type of restoration rather

than the typeofmaterialused,withocclusal restorations showinghigher retention

ratesforbothmaterials115.

2.2.7ProximalandSecondaryCaries

Anextensivesurveyshowedthatthemainreasonforrestorationreplacement

wassecondarycaries119.Astudyofproximalcariesprogressionhighlightedtheneed

to develop better preventive measures for proximal surfaces in caries affected

patients120. The effect of restorative materials on secondary caries prevention

indicates that conventionalGICsexhibit amorepronounced inhibition zonearound

therestorationthanaRM-GIC,andafluoridereleasingresincompositeexhibitedno

inhibition zone at all121. Consequently, a conventional GIC seems to provide more

protectionagainstsecondarycariesthanaRM-GIC.Otherstudiesreportmuchhigher

numbers of Streptococcusmutans when sampled frommargins of resin composite

restorationscomparedwiththemarginsofGICrestorations122.Furthermore,overa

5-year period retention rates in occlusal resin composite restorations were much

higherthanGICrestorations,buttheresincompositegroupshowedahigherrateof

recurrenceofcaries123.AdditionalclinicalevidenceofpoorretentionratesforRM-GIC

compared to other sealants has been reported, studies also highlight significantly

41

lowerStreptococcusmutanscountsforRM-GICrestorationswhichisattributedtothe

presenceoffluoriderelease124.Incervicalcariouslesionsahigherrateofrecurrence

of caries is claimed in resin composite restorations than GIC restorations125.

Consequently, several comparisons involving RM-GICs and fluoride containing resin

compositesshowedthat,althoughtheinclusionoffluorideintotheresincomposite

has a positive anti-cariogenic effect that prevents demineralisation in surrounding

toothtissue,RM-GICsprovideafargreaterbenefit126,127.Aproblemcanarisewhen

interpreting clinical trial results. It has been shown that fluoride releasing RM-GICs

can provide an anti-cariogenic benefit in bovine enamel some distance from the

restoration site, this can thereforemake evaluating splitmouth study designs very

complicated, ifnot invalid128.Atwo-yearclinicaltrial foundthatretentionratesfor

RM-GICs are much lower than for resin composites, consequently a higher caries

recurrenceratewasreportedfortheRM-GIC129.Athree-yearclinicalstudyevaluating

afluoridecontainingresincompositeandaRM-GICfoundthatbothmaterialswere

suitableforposteriorapproximalrestorations,buttheresinhadahigherfailurerate

duetosecondarycariesthantheRM-GIC89.WearratesofseveralRM-GICsrevealeda

cyclicbehaviour inwearpatternsof thematerials testedovera two-yearperiod130.

Although intermediateresultsat6,9and18monthsshowedgreatvariability, long-

term wear rates appeared to converge and the difference between the materials

after two years was insignificant. This may explain some conflicting reports in

comparisonofresincompositesandRM-GICswithregardstorecurrenceofcaries.

2.3CaseinPhosphopeptide-AmorphousCalciumPhosphate

First demonstrated to have potential anticariogenic properties in the

laboratory, animal and human in situ studies, casein phosphopeptide amorphous

calcium phosphate (CPP-ACP) has since been incorporated into several dietary

productsaswellasdentalmaterials131-134.AccordingtoReynoldsetal.theproposed

anticariogenic mechanism of CPP-ACP is that it incorporates nanocomplexes into

42

plaqueandontothetoothsurface,thelocalizedCPP-ACPnanocomplexesthenactto

buffer the freecalciumandphosphate ionactivities, therebymaintaininga stateof

supersaturation with respect to tooth enamel, preventing demineralisation and

promoting remineralisation 135.ThebondbetweenCPPandACP isdependentupon

thepHofthesurroundingenvironment,asthepHdropsfollowinganacidchallenge,

CPP-ACPdissociatesreleasingcalciumandphosphatewhichcounteractsthedropin

pHandleadstoaninhibitionofdemineralisationbymaintainingasupersaturationof

calciumandphosphateionswithrespecttotoothenamel136,137.

2.3.1ChemistryandRoleinOralEnvironment

An in vitro study concluded that CPP-ACP complexes on dentine surface

provokelowerdemineralizationandhigherremineralizationcomparedtothedentine

surfaceswithouttheagent138.An insitutrialofdentalproductscontainingcalcium,

phosphate and fluoride, Tooth Mousse Plus (GC Corporation, Tokyo, Japan) and

Clinpro ToothCrème (3MESPE,MN,USA) had significantly higher levels of enamel

lesion remineralization than products containing only fluoride (1000ppm fluoride,

5000ppm fluoride)139. Cochrane et al. have demonstrated subsurface lesion

remineralization with CPP-ACP and CPP-ACFP with greatest effect recorded at pH

5.5140.

IncorporationofCPP-ACPintoaGIChasshownthatadditionof3%w/wCPP-

ACP has the potential to improve the GIC’s anti-cariogenic properties without

adverselyaffectingitsphysicalproperties141.Furtherstudieslookingatincorporation

of CPP-ACP intoGICs claim that incorporation of 1.56%w/w of CPP-ACP increased

microtensile bond and compressive strengths, as well as increased the release of

calciumandphosphateionsfromtheGIC.TheauthorsalsostatedthattheCPP-ACP

containingGICwasmoreresistanttoacidchallengeinvitro142.

PriorapplicationofCPP-ACPcontainingpaste(ToothMousse,GCCorporation,

Tokyo, Japan) has been shown to reduce the fall of plaque pH following a sucrose

challenge143.ContinuouslubricationwithToothMousse(TM)hasalsobeenshownto

43

reduceerosivedentinewear,whileatthesametimepromotingremineralisation144.

FurtherstudiesofenamelerosioninvitroconcludedthatProNamel(GlaxoSmithKline

plc.,Brentford,Middlesex,UK)andToothMoussemayofferadegreeofprotection

fromerosion of permanent enamel145. CPP-ACP’s ability to bind to plaque has also

beeninvestigatedandariseoffreecalciuminplaquebiofilmasaresultofapplication

ofCPP-ACPwasdemonstrated.ThispropertyofCPP-ACPmayaidinremineralisation,

additionally the ability to provide a large reservoir of calcium may also restrict

minerallossduringacariogenicepisode146.

Severalattemptshavebeenmadeto incorporateCPP-ACPintocommercially

availableproducts.Atrialofsugar-freechewinggumcontainingCPP-ACPpresented

evidenceofsubsurfacelesionremineralisationfollowinganacidattack,thestudyalso

claimed that the re-mineralised mineral is more resistant to further acid

challenges147.FurtherevidenceoutliningCPP-ACP’sabilitytoremineralisesubsurface

lesionswhencomparedtoconventionalsourcesofcalciumshowedthatmouthrinses

andchewinggumscontainingCPP-ACPprovidedhigher levelsofremineralisation135,

148,149.

Asignificantcorrelationbetweentoothwearandanumberofacidicdietary

productsanddrinkinghabitshasbeenreportedinadultsbetween18-30yearsofage150. The addition of CPP-ACP into sports drinks in vitro demonstrated that

concentrations of 0.063% w/w of CPP-ACP and higher prevented erosive lesions

occurringintestspecimens.However,surfaceirregularitieswereobservedwhichmay

beattributedtore-depositedcalciumandphosphatefromCPP-ACP151.

2.3.2InteractionbetweenCPP-ACPandFluoride

Saturationofplaquewithcalciumandphosphateionsisanimportantaspect

of preventing demineralisation and promoting remineralisation, however fluoride

ions continue to play an important role in caries prevention. At the core of caries

preventionisfluoride’sabilitytosubstituteintohydroxyapatitetocreatefluorapatite,

which is more resistant to acid attack152. It has been shown that the presence of

44

amorphous calcium and phosphate ions can help localise fluoride ions in plaque

thereby creating ideal conditions for fluorapatite formation153. Several studieshave

investigated themechanism by which calcium and phosphate ions help to localise

fluoride ions on the tooth surface140,154-156, investigators conclude that amorphous

calcium, phosphate and fluoride nano-complexes are formed in plaque in the

presenceofCPPcreatingabioavailablestabilisedreservoiroftheseion.

2.4AnalyticalMethods

The overview of dental caries and GICs has identified several important

parameters of interest such as concentrations of beneficial ions in the oral

environment(calcium,phosphateandfluoride)andphysicalpropertiesofrestorative

materials(compressiveandtensilestrength,hardness,andsolubility).

A number of laboratory methods have been used to measure the

concentration of ions released from dental materials. Atomic absorption

spectroscopy has been used in a number of studies to detect concentration of

calcium ions in solution released fromGICs followingaperiodof storageanywhere

between24hoursandafewweeks141,142.Thesamestudiesalongwithseveralothers

have used an ion selective electrode to measure the release of fluoride ions into

storage solutions58, 66, 93, 141, 142, 157-159. Phosphate ion release has been measured

colorimetrically using a UV-visible spectrophotometer141, 142, 160. Some studies have

used ion chromatography tomeasure fluoride, calcium and phosphate ions at the

sametime139,161.Oneofthecommonfactorsinmanystudiesistheuseoflacticacid

asan‘acidchallenge’solution,thepHofthesolution isoftensetto5.0asacritical

levelfordissolutionoftoothmineral.Theseparametersarespecificallychosentobe

asclosetoclinicalconditions.Thelevelsofionconcentrationmeasurementsdepend

on the volume of the storage solution as well as the period of storage; sufficient

accuracy is achievedby fine tuning these twoparameters at the pilot stage of any

experimentalprotocol.

45

Clinical evidence shows that GICs perform differently to other restorative

materials when it comes to physical properties, especially at the early stages of

polymerisation before a ‘complete set’ is achieved. Due to self-curing nature of

conventionalGICs theseearly stages aremore critical to long-termperformanceof

GICscomparedtootherrestorativematerialssuchasresincompositesandRM-GICs.

To better understand how GIC behaves many physical characteristics of these

materialshavebeenstudiedusingavarietyoftechniques.Roughnessofthesurface

can indicateamaterial’sability to trapplaqueandcanhaveanundesiredeffecton

the aesthetics of the material, which can lead to discoloration of the surface. In

Dental Research, surface roughness has been measured using atomic force

microscopy (AFM)162 and surface profilometry62. AFM is able to achieve a higher

resolutionsoitisoftenusedformeasuringsurfaceroughnessofenamel.Restorative

materials can undergo higher levels of degradation, especially when subjected to

abrasivelaboratorytesting,intheseinstancessurfaceprofilometryhasproventobe

more suitable. In conjunction with surface roughness, surface hardness is also a

property of interest, especially when looking at occlusal surfaces of restorations,

where restorative materials are subjected to masticatory forces. Either Knoop or

Vickers hardness tests are used to determine the surface hardness of materials,

enamelanddentine.KnoophardnessisoftenchosenoverVickerswhensmallsample

sizeposesarestrictionontheavailablesurfacearea.Micro-indentation62andnano-

indentation163,164arewellestablishedmethodsformeasuringsurfacehardness.

GICs are very sensitive to the levels of moisture in the surrounding

environmentduringtheearlystagesofthesettingreactionandcansufferfromeither

wateruptakeorwaterloss.Analyticalbalancesareoftenusedtomeasurethemass

ofGICsamples161asawayofmonitoringthemoisture levelofthematerials.When

investigating ion release,massmeasurements can also supplement the ion release

data.

46

2.5References

1. Griffin SO, JonesK, TomarSL (2001).Aneconomicevaluationof community

waterfluoridation.JournalofPublicHealthDentistry61(2):78-86.

2. Widström E, Eaton KA (2004). Oral healthcare systems in the extended

Europeanunion.OralHealth&PreventiveDentistry2(3):155.

3. MarinoRJ,KhanAR,MorganM (2013).Systematicreviewofpublicationson

economic evaluations of caries prevention programs. Caries Research

47(4):265-272.

4. Tanz RD (1949). The etiology of dental caries: A correlation of reports.Oral

Surgery,OralMedicine,OralPathology2(8):1080-1089.

5. GooseD(1965).Dietanddentalcaries.PublicHealth79(3):131-135.

6. Bibby BG (1961). Prevention of dental caries. The Journal of Prosthetic

Dentistry11(6):1124-1130.

7. MurrayJJ(1976).Fluoridesincariesprevention:JohnWright&SonsLtd.

8. CurzonM,CrockerD(1978).Relationshipsoftraceelementsinhumantooth

enameltodentalcaries.ArchivesofOralBiology23(8):647-653.

9. FeatherstoneJD,DuncanJF,CutressTW(1979).Amechanismfordentalcaries

basedonchemicalprocessesanddiffusionphenomenaduring in-vitrocaries

simulationonhumantoothenamel.ArchivesofOralBiology24(2):101-112.

47

10. Miller WD (1891). The human mouth as a focus of infection. The Lancet

138(3546):340-342.

11. Clarke JK (1924). On the bacterial factor in the aetiology of dental caries.

BritishJournalofExperimentalPathology5(3):141.

12. Fejerskov O, Kidd EAM (2008). Dental caries : the disease and its clinical

management.2nded.Oxford;Ames,Iowa:BlackwellMunksgaard.

13. SimsW(1972).Theconceptofimmunityindentalcaries:II.Specificimmune

responses.OralSurgery,OralMedicine,OralPathology34(1):69-86.

14. Koga T, Oho T, Shimazaki Y, Nakano Y (2002). Immunization against dental

caries.Vaccine20(16):2027-2044.

15. WinterG(1990).Epidemiologyofdentalcaries.ArchivesofOralBiology35(S1-

S7.

16. Wahab FK, Shellis RP, Elderton RJ (1993). Effects of low fluoride

concentrations on formation of caries-like lesions in human enamel in a

sequential-transferbacterialsystem.ArchivesofOralBiology38(11):985-995.

17. Moreno EC, Kresak M, Zahradnik RT (1974). Fluoridated hydroxyapatite

solubilityandcariesformation.Nature247(5435):64-65.

18. Carey CM (2014). Focus on fluorides: update on the use of fluoride for the

preventionofdentalcaries.TheJournalofEvidence-BasedDentalPractice14

Suppl:95-102.

48

19. LoescheWJ,Murray RJ,Mellberg JR (1973). The effect of topical acidulated

fluorideonpercentageofStreptococcusmutansandStreptococcussanguisin

interproximalplaquesamples.CariesResearch7(4):283-296.

20. Zickert I, Emilson CG (1982). Effect of a fluoride-containing varnish on

Streptococcus mutans in plaque and saliva. Scandinavian Journal of Dental

Research90(6):423-428.

21. Beltran-AguilarED,Goldstein JW, LockwoodSA (2000). Fluoridevarnishes.A

reviewoftheirclinicaluse,cariostaticmechanism,efficacyandsafety.Journal

oftheAmericanDentalAssociation131(5):589-596.

22. Ogaard B, Rolla G, Helgeland K (1984). Fluoride retention in sound and

demineralized enamel in vivo after treatment with a fluoride varnish

(Duraphat).ScandinavianJournalofDentalResearch92(3):190-197.

23. Holmen L, Ogaard B, Rolla G, Thylstrup A (1986). A polarized light and

scanningelectronmicroscopestudyoftheeffectofDuraphattreatmentonin

vivocaries.ScandinavianJournalofDentalResearch94(6):521-529.

24. ten Cate J (2008). Remineralization of deep enamel dentine caries lesions.

AustralianDentalJournal53(3):281-285.

25. ForstenL (1998). Fluoride releaseanduptakebyglass-ionomersand related

materialsanditsclinicaleffect.Biomaterials19(6):503-508.

26. Burke FM, Ray NJ, McConnell RJ (2006). Fluoride-containing restorative

materials.InternationalDentalJournal56(1):33-43.

49

27. SelwitzRH,IsmailAI,PittsNB(2007).Dentalcaries.TheLancet369(9555):51-

59.

28. Margolis HC, Moreno EC (1994). Composition and cariogenic potential of

dentalplaquefluid.CriticalReviewsinOralBiology&Medicine5(1):1-25.

29. Silverstone LM (1981). Dental caries: aetiology, pathology and prevention:

Macmillan.

30. AnusaviceKJ(1995).Preservativedentistry:thestandardofcareforthe21st

century.JournalofPublicHealthDentistry55(2):67-68.

31. TyasMJ, Anusavice KJ, Frencken JE,Mount GJ (2000).Minimal intervention

dentistry--areview.FDICommissionProject1-97.InternationalDentalJournal

50(1):1-12.

32. Anderson MH (1992). Changing paradigms in caries management. Current

OpinioninDentistry2(157-162.

33. Fusayama T, Terachima S (1972). Differentiation of two layers of carious

dentinbystaining.JournalofDentalResearch51(3):866.

34. Mount GJ (1998). Clinical performance of glass-ionomers. Biomaterials

19(6):573-579.

35. NormanRD,PhillipsRW,SwartzML (1960).Fluorideuptakebyenamel from

certaindentalmaterials.JournalofDentalResearch39(1):11-16.

36. SmithDC(1968).Anewdentalcement.BritishDentalJournal124(9):381-384.

50

37. WilsonAD,KentBE(1972).Anewtranslucentcementfordentistry.Theglass

ionomercement.BritishDentalJournal132(4):133-135.







Research53(6):1414-1419.





cements. 1. Long term hardness and compressive strength. Journal of


43. Crisp S,Wilson AD (1976). Reactions in glass ionomer cements: V. Effect of

incorporating tartaric acid in the cement liquid. Journal of Dental Research

55(6):1023-1031.



Research55(3):489-495.

51


cements.3.Effectofpolyacidconcentrationonphysicalproperties.Journalof


46. WilsonAD,CrispS,AbelG(1977).Characterizationofglass-ionomercements

.4. Effect of molecular-weight on physical properties. Journal of Dentistry

5(2):117-120.


cement.II.Someclinicalapplications.AustralianDentalJournal22(2):120-127.


cement.III.Theerosionlesion.AustralianDentalJournal22(3):190-195.


cements. I. Formulations andproperties.AustralianDental Journal22(1):31-

36.


cements.5.Effectofthetartaricacidconcentrationintheliquidcomponent.


51. KentBE,LewisBG,WilsonAD (1979).Glass ionomercement formulations: I.

Thepreparationofnovelfluoroaluminosilicateglasseshighinfluorine.Journal



cements. 6. A Study of Erosion and Water-Absorption in Both Neutral and

AcidicMedia.JournalofDentistry8(1):68-74.

52

53. CrispS,KentBE,LewisBG,FernerAJ,WilsonAD(1980).Glass-ionomercement

formulations.II.Thesynthesisofnovelpolycarobxylicacids.JournalofDental

Research59(6):1055-1063.

54. Prosser HJ, Powis DR, Brant P,Wilson AD (1984). Characterization of glass-

ionomercements .7.Thephysicalpropertiesof currentmaterials. Journalof

Dentistry12(3):231-240.

55. Tyas MJ, Burrow MF (2004). Adhesive restorative materials: a review.

AustralianDentalJournal49(3):112-121;quiz154.

56. Sennou H, Lebugle A, Gregoire G (1999). X-ray photoelectron spectroscopy

study of the dentin–glass ionomer cement interface. Dental Materials

15(4):229-237.

57. CrispS,WilsonAD(1973).Formationofaglass-ionomercementbasedonan

lon-leachable glass and polyacrylic acid. Journal of Applied Chemistry and

Biotechnology23(11):811-815.

58. MoreauJL,XuHHK(2010).Fluoridereleasingrestorativematerials:Effectsof

pHonmechanicalproperties and ion release.DentalMaterials26(11):e227-

235.

59. NicholsonJW,CzarneckaB (2009).Reviewpaper:Roleofaluminuminglass-

ionomer dental cements and its biological effects. Journal of Biomaterials

Applications24(4):293-308.

60. Crisp S, Prosser H, Wilson A (1976). An infra-red spectroscopic study of

cement formation between metal oxides and aqueous solutions of poly

(acrylicacid).JournalofMaterialsScience11(1):36-48.

53

61. Mckinney JE, Antonucci JM, Rupp NW (1987). Wear and microhardness of

glass-ionomercements.JournalofDentalResearch66(6):1134-1139.

62. Bala O, Arisu HD, Yikilgan I, Arslan S, Gullu A (2012). Evaluation of surface

roughnessandhardnessofdifferentglassionomercements.EuropeanJournal

ofDentistry6(1):79-86.

63. Yip H, To W (2005). An FTIR study of the effects of artificial saliva on the

physical characteristics of the glass ionomer cements used for art. Dental

Materials21(8):695-703.

64. Swartz M, Phillips R, Pareja C, Moore B (1989). In vitro degradation of

cements: a comparison of three test methods. The Journal of Prosthetic


65. LucasME,AritaK,NishinoM(2003).Toughness,bondingandfluoride-release

properties of hydroxyapatite-added glass ionomer cement. Biomaterials

24(21):3787-3794.

66. GandolfiMG,ChersoniS,AcquavivaGL,PianaG,PratiC,MongiorgiR(2006).

FluoridereleaseandabsorptionatdifferentpHfromglass-ionomercements.

DentalMaterials22(5):441-449.

67. KimYK,YiuCK,KimJR,GuL,KimSK,WellerRNetal.(2010).Failureofaglass

ionomer to remineralizeapatite-depleteddentin. JournalofDentalResearch

89(3):230-235.

54

68. Trairatvorakul C, Itsaraviriyakul S, Wiboonchan W (2011). Effect of glass-

ionomer cement on the progression of proximal caries. Journal of Dental

Research90(1):99-103.

69. Salas CFC, Guglielmi CAB, Raggio DP, Mendes FM (2011). Mineral loss on

adjacent enamel glass ionomer cements restorations after cariogenic and

erosivechallenges.ArchivesofOralBiology56(10):1014-1019.

70. Trairatvorakul C, Techalertpaisarn P, Siwawut S, Ingprapankorn A (2009).

Effectofglassionomercementandfluoridevarnishontheremineralizationof

artificial proximal caries in situ. The Journal of Clinical Pediatric Dentistry

34(2):131-134.

71. RodriguesE,DelbemACB,PedriniD,OliveiraMSR(2008).pH-cyclingmodelto

verifytheefficacyof fluoride-releasingmaterials inenameldemineralization.

OperativeDentistry33(6):658-665.

72. Featherstone JD, Glena R, Shariati M, Shields CP (1990). Dependence of in

vitro demineralization of apatite and remineralization of dental enamel on

fluoride concentration. Journal of Dental Research 69 Spec No(620-625;

discussion634-626.

73. WilliamsJ,BillingtonR,PearsonG(1999).Theinfluenceofsampledimensions

onfluorideionreleasefromaglassionomerrestorativecement.Biomaterials

20(14):1327-1337.

74. Diaz-Arnold AM, Holmes DC, Wistrom DW, Swift Jr EJ (1995). Short-term

fluoride release/uptake of glass ionomer restoratives. Dental Materials

11(2):96-101.

55

75. Hörsted-Bindslev P (1994). Fluoride release from alternative restorative

materials.JournalofDentistry22(S17-S20.

76. Yengopal V, Mickenautsch S, Bezerra AC, Leal SC (2009). Caries-preventive

effectofglassionomerandresin-basedfissuresealantsonpermanentteeth:a

metaanalysis.JournalofOralScience51(3):373-382.

77. Martignon S, Ekstrand KR, Gomez J, Lara JS, Cortes A (2012).

Infiltrating/sealingproximal caries lesions:A 3-year randomized clinical trial.

JournalofDentalResearch91(3):288-292.

78. Ahovuo-SalorantaA,HiiriA,NordbladA,MakelaM,WorthingtonHV (2008).

Pitandfissuresealantsforpreventingdentaldecayinthepermanentteethof

childrenandadolescents.CochraneDatabaseofSystematicReviews4).

79. Hiiri A, Ahovuo-Saloranta A, Nordblad A, Makela M (2010). Pit and fissure

sealantsversusfluoridevarnishesforpreventingdentaldecayinchildrenand

adolescents.CochraneDatabaseofSystematicReviews3).

80. GaneshM,ShobhaT (2007).Comparativeevaluationof themarginal sealing

ability of Fuji VII and Concise as pit and fissure sealants. Journal of

ContemporaryDentalPractice8(4):10-18.

81. ArrowP,RiordanPJ (1995).RetentionandcariespreventiveeffectsofaGIG

andaresin-basedfissuresealant.CommunityDentistryandOralEpidemiology

23(5):282-285.

82. Mjör IA (1990). Glass ionomer and resin-based fissure sealants: a clinical

study.EuropeanJournalofOralSciences98(4):345-350.

56

83. Torppa-Saarinen E, Seppä L (1990). Short-term retention of glass-ionomer

fissuresealants.ProceedingsoftheFinnishDentalSociety86(2):83.

84. Hotz PR (1979). Experimental secondary caries around amalgam, composite

and glass ionomer cement fillings in human teeth. Schweizerische

Monatsschrift für Zahnheilkunde Revue mensuelle suisse d'odonto-

stomatologie/SSO89(9):965.

85. BrackettWW,MetzJE(1992).Performanceofaglassionomerlutingcement

over5yearsinageneralpractice.TheJournalofProstheticDentistry67(1):59-

61.

86. ForstenL,MountGJ,KnightG(1994).ObservationsinAustraliaoftheuseof

glass ionomer cement restorative material. Australian Dental Journal

39(6):339-343.

87. NagarajaUpadhyaP,KishoreG (2005).Glass ionomercement:Thedifferent

generations.TrendsinBiomaterialsandArtificialOrgans18(2):158-165.

88. RosenstielSF,LandMF,CrispinBJ(1998).Dentallutingagents:Areviewofthe

currentliterature.JournalofProstheticDentistry80(3):280-301.

89. MjörI,JokstadA(1993).Five-yearstudyofClassIIrestorationsinpermanent

teethusingamalgam,glasspolyalkenoate (ionomer) cermetand resin-based

compositematerials.JournalofDentistry21(6):338-343.

90. Ostlund J, Moller K, Koch G (1992). Amalgam, composite resin and glass

ionomercementinClassIIrestorationsinprimarymolars--athreeyearclinical

evaluation.SwedishDentalJournal16(3):81-86.

57

91. Tyas MJ (2006). Clinical evaluation of glass-ionomer cement restorations.

JournalofAppliedOralScience14Suppl(10-13.

92. SmalesRJ,GaoW,HoFT(1997).Invitroevaluationofsealingpitsandfissures

withnewerglass-ionomercementsdevelopedfortheARTtechnique.Journal

ofClinicalPediatricDentistry21(4):321-323.

93. BayrakS,TuncES,AksoyA,ErtasE,GuvencD,OzerS(2010).Fluoriderelease

and recharge from different materials used as fissure sealants. European


94. Mickenautsch S, Mount G, Yengopal V (2011). Therapeutic effect of glass-

ionomers:anoverviewofevidence.AustralianDentalJournal56(1):10-15.

95. Asmussen E, Peutzfeldt A (2002). Long-term fluoride release from a glass

ionomercement,acompomer,andfromexperimentalresincomposites.Acta

Odontologica60(2):93-97.

96. Takahashi K, Emilson C, Birkhed D (1993). Fluoride release in vitro from

various glass ionomer cements and resin composites after exposure to NaF

solutions.DentalMaterials9(6):350-354.

97. Musa A, Pearson G, Gelbier M (1996). In vitro investigation of fluoride ion

release from four resin-modified glass polyalkenoate cements. Biomaterials

17(10):1019-1023.

98. Momoi Y, McCabe J (1993). Fluoride release from light-activated glass

ionomerrestorativecements.DentalMaterials9(3):151-154.

58

99. Dunne S, Goolnik J,Millar B, Seddon R (1996). Caries inhibition by a resin-

modified and a conventional glass ionomer cement, in vitro. Journal of


100. Carvalho A, Cury J (1999). Fluoride release from some dental materials in

differentsolutions.OperativeDentistry24(1):14.

101. Mazzaoui S, BurrowM, TyasM (2000). Fluoride release from glass ionomer

cements and resin composites coated with a dentin adhesive. Dental

Materials16(3):166-171.

102. ten Cate JM, vanDuinen RN (1995). Hypermineralization of dentinal lesions

adjacent to glass-ionomer cement restorations. Journal of Dental Research

74(6):1266-1271.

103. Mickenautsch S, Yengopal V (2010). Demineralization of hard tooth tissue

adjacent to resin-modified glass-ionomers and composite resins: a

quantitativesystematicreview.JournalofOralScience52(3):347-357.

104. Wilson R, Donly K (2001). Demineralization around orthodontic brackets

bondedwithresin-modifiedglassionomercementandfluoride-releasingresin

composite.PediatricDentistry23(3):255.

105. KeltjensH,CreugersT,Van'tHofM,CreugersN(1999).A4-yearclinicalstudy

on amalgam, resin composite and resin-modified glass ionomer cement

restorationsinoverdentureabutments.JournalofDentistry27(8):551-555.

106. Uno S, Finger WJ, Fritz U (1996). Long-term mechanical characteristics of

resin-modifiedglassionomerrestorativematerials.DentalMaterials12(1):64-

69.

59

107. RichardsLC,Kaidonis JA,TownsendGC (2010).New insights into toothwear

based on in vitro studies. Proceedings of the Institution of Mechanical

EngineersPartJ-JournalofEngineeringTribology224(J6):551-558.

108. EisenburgerM,AddyM,RossbachA(2003).Acidicsolubilityoflutingcements.


109. Shabanian M, Richards LC (2002). In vitro wear rates of materials under

different loadsandvaryingpH.TheJournalofProstheticDentistry87(6):650-

656.

110. Ostlund J, Möller K, Koch G (1992). Amalgam, composite resin and glass

ionomercementinClassIIrestorationsinprimarymolars--athreeyearclinical

evaluation.SwedishDentalJournal16(3):81.

111. SilvaKG,PedriniD,DelbemAC,CannonM(2007).EffectofpHvariationsina

cyclingmodelon thepropertiesof restorativematerials.OperativeDentistry

32(4):328-335.

112. Cattani-LorenteM,DupuisV,MoyaF,PayanJ,MeyerJ-M(1999).Comparative

studyofthephysicalpropertiesofapolyacid-modifiedcompositeresinanda

resin-modifiedglassionomercement.DentalMaterials15(1):21-32.

113. Frencken JE, Pilot T, Songpaisan Y, Phantumvanit P (1996). Atraumatic

restorative treatment (ART): rationale, technique, and development. Journal

ofPublicHealthDentistry56(3SpecNo):135-140;discussion161-133.

60

114. ThorpeS(1992)Oralhealth,reachingouttotheyear2000.Keynoteaddress

at the 7th IADRmeeting of the East and Southern African section. Harare,

Zimbabwe.

115. Ersin NK, Candan U, Aykut A, Oncag O, Eronat C, Kose T (2006). A clinical

evaluation of resin-based composite and glass ionomer cement restorations

placedinprimaryteethusingtheARTapproach:resultsat24months.Journal

oftheAmericanDentalAssociation(1939)137(11):1529-1536.

116. Oba AA, Dulgergil T, Sonmez IS, Dogan S (2009). Comparison of caries

preventionwithglassionomerandcompositeresinfissuresealants.Journalof

FormosanMedicalAssociation108(11):844-848.

117. Beiruti N, Frencken JE, van't Hof MA, Taifour D, Helderman WHV (2006).

Caries-preventiveeffectofaone-timeapplicationofcompositeresinandglass

ionomersealantsafter5years.CariesResearch40(1):52-59.

118. YipHK,SmalesRJ(2002).Glassionomercementsusedasfissuresealantswith

the atraumatic restorative treatment (ART) approach: review of literature.

InternationalDentalJournal52(2):67-70.

119. BurkeF,CheungS,MjörI,WilsonN(1999).Restorationlongevityandanalysis

of reasons for the placement and replacement of restorations provided by

vocational dental practitioners and their trainers in the United Kingdom.

QuintessenceInternational(Berlin)30(4):234.

120. Martignon S, Chavarria N, Ekstrand KR (2010). Caries status and proximal

lesion behaviour during a 6-year period in young adult Danes: an

epidemiologicalinvestigation.ClinicalOralInvestigations14(4):383-390.

61

121. Pereira PN, Inokoshi S, Tagami J (1998). In vitro secondary caries inhibition

aroundfluoridereleasingmaterials.JournalofDentistry26(5-6):505-510.

122. SvanbergM,Mjör I, Ørstavik D (1990).Mutans streptococci in plaque from

margins of amalgam, composite, and glass-ionomer restorations. Journal of

DentalResearch69(3):861-864.

123. Mejàre I, LingströmP,PeterssonLG,HolmA-K,TwetmanS,KällestålCetal.

(2003).Caries-preventiveeffectoffissuresealants:asystematicreview.Acta

Odontologica61(6):321-330.

124. Amin HE (2008). Clinical and antibacterial effectiveness of three different

sealantmaterials.AmericanDentalHygienistsAssociation82(5):45-45.

125. TyasMJ(1991).Cariostaticeffectofglassionomercement:afive-yearclinical

study.AustralianDentalJournal36(3):236-239.

126. Park SH, Kim KY (1997). The anticariogenic effect of fluoride in primer,

bondingagent,andcompositeresininthecavosurfaceenamelarea.Operative

Dentistry22(3):115-120.

127. ToriiY,ItotaT,OkamotoM,NakaboS,NagamineM,InoueK(2001).Inhibition

ofartificialsecondarycariesinrootbyfluoride-releasingrestorativematerials.

OperativeDentistry26(1):36.

128. Tantbirojn D, Douglas W, Versluis A (1997). Inhibitive effect of a resin-

modified glass ionomer cement on remote enamel artificial caries. Caries

Research31(4):275-280.

62

129. Baseggio W, Scarparo Naufel F, de Oliveira Davidoff DC, Salata Nahsan FP,

FluryS,AlmeidaRodrigues J (2010).Caries-preventiveefficacyand retention

ofaresin-modifiedglassionomercementandaresin-basedfissuresealant:a

3-yearsplit-mouthrandomisedclinicaltrial.OralHealth&PreventiveDentistry

8(3):261.

130. SidhuSK,SherriffM,WatsonTF(1997).Invivochangesinroughnessofresin-

modifiedglassionomermaterials.DentalMaterials13(3):208-213.

131. ReynoldsE,JohnsonI(1981).Effectofmilkoncaries incidenceandbacterial

composition of dental plaque in the rat.Archives ofOral Biology26(5):445-

451.

132. Reynolds E, Riley P, Storey E (1982). Phosphoprotein inhibition of

hydroxyapatitedissolution.CalcifiedTissueInternational34(S52.

133. Reynolds EC,Wong A (1983). Effect of adsorbed protein on hydroxyapatite

zetapotentialandStreptococcusmutansadherence. Infectionand Immunity

39(3):1285-1290.

134. ReynoldsE (1987).Thepreventionofsub-surfacedemineralizationofbovine

enamel and change in plaque composition by casein in an intra-oralmodel.

JournalofDentalResearch66(6):1120-1127.

135. Reynolds E, Cai F, Shen P, Walker G (2003). Retention in plaque and

remineralization of enamel lesions by various forms of calcium in a

mouthrinseorsugar-freechewinggum.JournalofDentalResearch82(3):206-

211.

63

136. Reynolds E (1997). Remineralization of enamel subsurface lesions by casein

phosphopeptide-stabilized calcium phosphate solutions. Journal of Dental

Research76(9):1587-1595.

137. Reynolds EC (1998). Anticariogenic complexes of amorphous calcium

phosphate stabilized by casein phosphopeptides: a review. Special Care in


138. Rahiotis C, Vougiouklakis G (2007). Effect of a CPP-ACP agent on the

demineralizationandremineralizationofdentineinvitro.JournalofDentistry

35(8):695-698.

139. ShenP,MantonDJ,CochraneNJ,WalkerGD,YuanY,ReynoldsCetal.(2011).

Effectofaddedcalciumphosphateonenamelremineralizationbyfluorideina

randomizedcontrolledinsitutrial.JournalofDentistry39(7):518-525.

140. Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC (2008). Enamel

subsurface lesion remineralisation with casein phosphopeptide stabilised

solutionsofcalcium,phosphateandfluoride.CariesResearch42(2):88-97.







64

143. CaruanaPC,MulaifySA,MoazzezR,BartlettD(2009).Theeffectofcaseinand

calciumcontainingpasteonplaquepHfollowingasubsequentcarbohydrate

challenge.JournalofDentistry37(7):522-526.

144. Ranjitkar S, Narayana T, Kaidonis JA, Hughes TE, Richards LC, TownsendGC

(2009). The effect of casein phosphopeptide-amorphous calcium phosphate

onerosivedentinewear.AustralianDentalJournal54(2):101-107.

145. Rees J, Loyn T, Chadwick B (2007). Pronamel and tooth mousse: an initial

assessmentoferosionpreventioninvitro.JournalofDentistry35(4):355-357.

146. RoseR(2000).Bindingcharacteristicsofstreptococcusmutansforcalciumand

caseinphosphopeptide.CariesResearch34(5):427-431.

147. Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds E (2004). Acid

resistanceofenamelsubsurfacelesionsremineralizedbyasugar-freechewing

gumcontainingcaseinphosphopeptide-amorphouscalciumphosphate.Caries

Research38(6):551-556.

148. Shen P, Cai F, Nowicki A, Vincent J, Reynolds E (2001). Remineralization of

enamel subsurface lesions by sugar-free chewing gum containing casein

phosphopeptide-amorphous calcium phosphate. Journal of Dental Research

80(12):2066-2070.

149. MorganM, Adams G, Bailey D, Tsao C, Fischman S, Reynolds E (2008). The

anticariogeniceffectofsugar-freegumcontainingCPP-ACPnanocomplexeson

approximal caries determined using digital bitewing radiography. Caries

Research42(3):171-184.

65

150. Bartlett DW, Fares J, Shirodaria S, Chiu K, AhmadN, SherriffM (2011). The

associationof toothwear,dietanddietaryhabits inadultsaged18-30years

old.JournalofDentistry39(12):811-816.

151. RamalingamL,Messer L, Reynolds E (2005).Adding caseinphosphopeptide-

amorphouscalciumphosphate to sportsdrinks toeliminate invitroerosion.

PediatricDentistry27(1):61-67.

152. Moreno EC, Kresak M, Zahradnik RT (1974). Fluoridated hydroxyapatite

solubilityandcariesformation.Nature247(5435):64-65.

153. CochraneNJ,CaiF,HuqNL,BurrowMF,ReynoldsEC(2010).Newapproaches

to enhanced remineralization of tooth enamel. Journal of Dental Research

89(11):1187-1197.

154. Heshmat H, Banava S, Abdian H, Fard MJK (2014). Effects of casein

phosphopeptide amorphous calcium phosphate and casein phosphopeptide

amorphous calcium phosphate fluoride on alterations of dental plaque pH

following sucrose consumption. Journal of IslamicDentalAssociationof Iran

26(4):4.

155. HambaH,NikaidoT,InoueG,SadrA,TagamiJ(2011).EffectsofCPP-ACPwith

sodium fluoride on inhibition of bovine enamel demineralization: a

quantitative assessment using micro-computed tomography. Journal of

Dentistry39(6):405-413.

156. ReynoldsEC,CaiF,CochraneNJ,ShenP,WalkerGD,MorganMVetal.(2008).

Fluoride and casein phosphopeptide-amorphous calcium phosphate. Journal


66

157. Asmussen E, Peutzfeldt A (2002). Long-term fluoride release from a glass

ionomercement,acompomer,andfromexperimentalresincomposites.Acta

odontologicaScandinavica60(2):93-97.

158. CarvalhoAS,Cury JA (1999). Fluoride release from somedentalmaterials in

differentsolutions.OperativeDentistry24(1):14-19.

159. MazzaouiSA,BurrowMF,TyasMJ(2000).Fluoridereleasefromglassionomer

cements and resin composites coated with a dentin adhesive. Dental

Materials16(3):166-171.

160. Xu HH, Weir MD, Sun L (2009). Calcium and phosphate ion releasing

composite: effect of pH on release and mechanical properties. Dental

Materials25(4):535-542.

161. PrabhakarRL,Brocchini S,Knowles JC (2005). Effectofglass compositionon

thedegradationpropertiesandionreleasecharacteristicsofphosphateglass--

polycaprolactonecomposites.Biomaterials26(15):2209-2218.

162. PoggioC,CeciM,BeltramiR,LombardiniM,ColomboM(2014).Atomicforce

microscopystudyofenamelremineralization.AnnalidiStomatologia5(3):98-

102.

163. Towler MR, Bushby AJ, Billington RW, Hill RG (2001). A preliminary

comparison of the mechanical properties of chemically cured and

ultrasonically cured glass ionomer cements, using nano-indentation

techniques.Biomaterials22(11):1401-1406.

67

164. EllakuriaJ,TrianaR,MinguezN,SolerI,IbasetaG,MazaJetal.(2003).Effect

of one-year water storage on the surface microhardness of resin-modified

versusconventionalglass-ionomercements.DentalMaterials19(4):286-290.

69

CHAPTER3

AimsandObjectives

Review of published literature highlighted the lack of laboratory evidence

exploringtheeffectofvariousacidsonGICs.Anecdotalclinicalevidenceisabundant

that points to acid exposure causing increased fluoride release fromGICs but poor

performancewhen it comes to theeffectsonphysicalproperties.By restricting the

number of variables, a laboratory study can help understand the specific factors

responsible for influencing ion release and physical properties of GICs undergoing

acidchallenge.

Theaimsofthisresearchwereto:

a) Investigate the effects of various acids on selected physical properties of

conventional GICs. Determine if surface hardness and mass loss vary

dependingonwhichacidtheGICisexposedto;

b)Investigatetheeffectsofvariousacidsonfluoride,phosphateandcalcium

ionreleasefromGICs;

70

c) Evaluate the ion release and physical characteristics of GICs after topical

applicationsofacalciumandphosphatereleasingcream;

d)InvestigaterechargepotentialoftopicallydeliveredCPP-ACFPwhenapplied

tothesurfaceofconventionalGICswithandwithoutincorporatedCPP-ACP.

e) To characterise the role of aluminium in the structure of GICs and the

impactofchelatingagentsonthephysicalpropertiesofGICs.

72

CHAPTER4

Preliminarystudies

4.1Introduction

A series of pilot studies were developed to determine the most suitable

methodsformeasuringtheremineralisationanddemineralisationpropertiesofglass

ionomer cements (GICs) and other GIC-likematerials.Measurements ofmass loss,

surface hardness and ion concentrations were determined to be the most

fundamental parameters related to remineralisation and demineralisation of tooth

mineral. Several different materials were investigated, including conventional GICs

and resin-modifiedGICs. In parallel, several acid solutionswere evaluated as acidic

challengemediatoreplicatevariousclinicalscenarios.

Several measuring techniques were found to be incompatible with the

selected materials and acid solutions. Difficulties with measuring fluoride ion

concentration when using lactic acid required either the use of other methods to

measure fluorideorusingadifferentacid toprovideanacid challenge for selected

materials. Liquid chromatography, previously used tomeasure ion release in acidic

73

solutionswas unable tomeasure fluoride ions in lactic acid. Results indicated that

spectralpeaksforfluorideandlacticacidoverlappedtocauseaccuracyproblemsfor

determiningfluoriderelease.Atthisstageapilotstudywasruntoinvestigatetheuse

of other acids. Citric acid was chosen due to its presence in the diet and oral

environment,withaspecificacidconcentrationandpHvaluebeingchosentoretain

relevance to clinical conditions. Although citric acid presented itself as a suitable

replacement for lactic acid for the purpose of measuring fluoride ions, it was

discovered that it posed a considerable threat of contamination to liquid

chromatographyequipment.CitricacidwasnotedtocauselargemasslossintheGICs

whichwasbelievedtobecausedbychelationofaluminiumions,consequentlyarisk

of aluminium contamination of the liquid chromatography column led to its

abandonmentasananalyticaltool.Alternativemethodswereidentifiedandvalidated

foraccuracyandconsistencyofmeasurement.The ionselectiveelectrodewasused

to obtain fluoride ion concentrations in solution. Atomic absorption spectroscopy

(AAS) was used to conduct calcium assays and colorimetric assays were used to

measure phosphate ion concentrations. AAS was also later used to conduct

aluminiumassays.

Pilot studieswith citric acid did show that theGICs responded to citric acid

verydifferentlytolacticacid,soexposuretocitricacidbecameanadditionalfocusof

further study and following several pilots, citric acid was included in the study

protocol. Results from pilot citric acid studies also highlighted the importance of

investigatingotherlowpHmediathatmaybeencounteredintheoralenvironment,

consequently hydrochloric acid was also included. The ionic concentration of each

solutionwascarefullyselectedbasedonvaluesfoundpreviouslyinplaquefluid.1

As several acid solutions were trialled during the early stages of the pilot

studiestheprotocolsforsurfacehardnessandionreleasehadtobere-evaluatedand

anewsetofparameterstested.

Theaimsofthepilotstudieswereto:

74

a)investigatesuitablematerialsandmethodstofurthertheunderstandingof

demineralization and remineralisation processes in dental restorative

materials;and

b) establish protocols that provide consistent outcomes formeasuringmass

loss, surface hardness and ion release for dental restorative materials

followinganacidchallenge.

4.2Specimenshape

4.2.1MaterialsandMethods

Various glass ionomer cement (GIC) restorativematerialswere used in pilot

studies todetermine suitableparameters for themain investigationsundertaken in

this research (Table 4.1). The GICs used were ChemFilMolar (CFM), ChemFil Rock

(CFR)(DentsplyDeTreyGmbH,Germany),RivaProtect(SDILtd.,Australia),whichisa

resin-modifiedGIC (RM-GIC),and thehigh fluoride releasingconventionalGICs,Fuji

VII(F7)(GCCorporation,Tokyo,Japan)andFujiVIIEP(F7EP)(GCCorporation,Tokyo,

Japan).Materialsweretestedformassloss,ionreleaseandsurfacehardness.

Material Notes

ChemFilMolar

ChemfilRock ReplacedChemFilMolar

RivaProtect

FujiVII Highfluoriderelease

FujiVIIEP Fuji VII that contain 3% (w/w) CPP-

ACP

Table4.1.Materialsusedinpilotstudies.CPP-ACP(caseinphosphopeptide-amorphouscalciumphosphate).

75

Polyvinyl siloxane impression material (eliteHD+ light body, Zhermack SpA,

Badia Polesine, Italy) moulds were used to create standardised discs of the

restorativematerials thatmeasured1mmx10mm (thickness x diameter). Thiswas

laterchangedtorectangularblocksmeasuring3mmx6mmx6mm(thicknessxwidth

x length).Discsorblocksofeachrestorativematerialwerepreparedbyplacing the

materials in themouldwith the top andbottom surfaces coveredby plastic strips,

held between two glass slides. The glass slides were pressed together to extrude

excessmaterial.Thespecimenswereallowedtosetinsidethemouldsfor24hoursin

anincubator(37°C,~100%relativehumidity).Aftercoolingtoroomtemperaturethe

blocks were removed from the moulds, and lapped with 600-grit paper (Norton

Tufbak,Saint-GobainAbrasivesLtd.,Auckland,NZ)underrunningdistilledwater.

4.2.2Discussion

Specimen shape was changed to accommodate subsurface analysis of the

restorative materials. The thickness of discs was insufficient for this purpose;

additionally the disc shape lent itself to only two surfaces being suitable for

subsurfaceanalysisasopposedtosixsurfacesofathickerblock.Rectangularblocks

insteadofdiscswereproven tobemoresuitable for investigatingdepthprofilesof

tested materials following acid challenge. A pilot study was run to determine the

feasibility of using blocks instead of discs. Later pilot studies, using blocks, were

performedtoinvestigatehardnessasafunctionofdepthaswellasEnergyDispersive

X-Ray Analysis (EDXA) assays of subsurface regions of dental materials, something

whichwouldnotbepossibleusingdiscsduetoathicknessofonly1mm.

76

4.3Ionrelease


Acidsolutions,namelytartaricacid,citricacid,lacticacidandhydrochloricacid

were trialled in conjunction with measuring equipment to be used to determine

calcium,phosphateandfluorideionconcentrationstoensurecompatibility.

FourdifferentsolutionswerepreparedtoexposetheblocksofGICtoavariety

of acidic and neutral environments. The three acidic solutions were formulated to

simulate a gastric erosive challenge (50mM NaCl adjusted to pH 2.0 with HCl), a

dietaryerosivechallenge(50mMcitricacidatpH5.0)andacariogenicacidchallenge

(50mMlacticacidatpH5.0).Afurtherpilotstudywasconductedusingtartaricacid

(pH 5.0, 50mM), which was used to investigate the effect of the disassociation

strengthofacids.Disassociationstrengthofacidsvariesdependingonthemolecular

structure, specifically thenumberofH+ ionsanacid candonate. Lactic and tartaric

acidareable todonateamaximumof twoH+ ions.Citricacid candonate threeH+

ions;thismeansthatcitricacidisabletochelatealuminiumions(Al3+),whereaslactic

acidandtartaricacidcannot.Consequently,anotherimportantgoalofthepilotwork

was to establish a protocol formeasuring aluminium ion concentration in aqueous

solutionusingAAS. Inadditiontomass lossandaluminiumionrelease,calciumand

phosphate ion releasewere alsomeasured. The control solution in all studieswas

distilled deionised water at pH 6.9 (MilliQ water, Millipore Corporation, Victoria,

Australia).

Initialpilotstudiesuseda28daydurationformeasurementsthatwerecarried

outafter1,2,3,7,14,21and28days.Thestoragevolumewasinitially15mL(plastic

containers), however this protocol was quickly replaced with a three-day duration

setupin5mLofsolution.Twotosixblockspergroupofeachmaterialwereexposed

to5mLofoneof the four solutionsabove. Solutionswere changedevery24hours

and the samples were measured for change in mass and surface hardness. The

solutionswere analysed to determine calcium, phosphate and fluoride ion release.

77

Later experiments also focused on aluminium ion release, the protocol used for

aluminiumassayswassimilar tocalciumassaysas itusedthesameequipment,but

wasdevelopedatalaterstage.

Themassofeachblockwasmeasuredevery24hoursbeforesurfacehardness

measurementswereperformed.Blocksweretakenoutofthesolutionpatdriedusing

Kimwipes (Kimtech Science, Kimberly-Clarke Professional, Australia) and then

weighed using a microbalance (Precisa XT 120A, Dietikon, Switzerland). Readings

wereobtainedtowithin±0.0001ofagram.

Theionreleaseofcalcium,aluminium,phosphateandfluorideaftereach24-

hourperiodof storageweredeterminedusingatomicabsorption spectroscopy (for

calciumandaluminium), colourimetry (for inorganic phosphate) andan ion-specific

electrode (for fluoride). To determine the calcium concentration, sample solutions

(0.5mL)weredilutedwithwater(0.5mL,MilliQ),acidifiedwith1MHCl(0.5mL)and

dilutedwith2%lanthanumchloride(0.5mL)andanalysedonaVarianAA240atomic

absorption spectroscope (AAS, Varian Australia Pty. Ltd.) against a set of seven

standardsrangingfrom0to250µMcalcium.TheVarianAA240AASwasalsousedto

determine the concentration of aluminium ions. Sample solutions (0.5 mL) were

dilutedwithpotassiumchloride(0.5mL,8mg/L)andlanthanumchloride(0.5mL,2%

w/w) and then acidified with 1M HCl (0.5 mL) and compared against a set of

standards ranging from 0 to 125 µM aluminium. Inorganic phosphate ion

concentrations were determined colourimetrically using a spectrophotometer (UV-

visiblespectrophotometer,VarianAustralia,Pty.Ltd).Thesampleswerepreparedby

taking100µLofsolution,dilutingitwith500µLof4.2%ammoniummolybdateand

adding 20 µL 1.5% of Tween ® 20. (Sigma-Aldrich, St. Louis, MO). The phosphate

concentrationwasdeterminedbycomparingthespectrophotometerreadingsofthe

samplesagainstasetofsevenstandardsranginginphosphateconcentrationsfrom0

to100µM.Theconcentrationoffluorideionswasdeterminedusinganion-selective

electrode (Radiometer analytical, ISE C301F, Lyon, France) connected to an ion

analyser(Radiometeranalytical,IonCheck45,Lyon,France).Samplesolutions(1mL)

weredilutedwith1mLtotalionicstrengthadjustmentbuffer(MerckPtyLtd,Kilsyth,

78

VIC,Australia)andmeasuredagainstasetofeightfluoridestandardsrangingfrom0

to 1000 µM. Liquid Chromatography (ICS-3000, Dionex Corporation, CA, USA)

machinewasusedat the initial stage tomeasure the concentrationof various ions

(seetable).

As the main body of experimental work progressed additional pilots were

conducted.Oneof theparameters testedwas theeffectof topical treatmentusing

CPP-ACFPcontainingcream(ToothMoussePlus,GCCorporation,Tokyo,Japan).The

pilotestablishedsuitableparametersfordilutionofthecreamandstandardisedthe

duration of treatment duration. This additional step was integrated into already

establishedprotocoloftestingsurfacehardness,masslossandcollectingsolutionsfor

laterionconcentrationassays.

Later stages of themain experimentalwork focusedon aluminium ions and

the effect on the structure of GICs under acid challenge. In order to rule out the

bufferingeffectoftheCPP-ACPcontainingcreamontheinitialsettingreactionapilot

studywasconducted.Somegroupswereallowedtosetfor24hoursinanincubator

(37C,95%relativehumidity),whileothergroupsonlysetforonehourinanincubator

(37C,95%relativehumidity).BlocksofF7andF7EPwerestoredfor24hoursineither

water or citric acid solution. F7 and F7EP groups were subjected to either Tooth

Mousse Plus (TM+, GC Corporation, Tokyo, Japan) treatment or a placebomousse

treatment,whileacontrolgrouphadnotopicaltreatment.After24hours,themass

loss of each block was measured, however no surface hardness or ion release

measurementsweremade.

Datafromsamplegroupswerefoundtofollowanormaldistributionafteruse

ofχ2test.SinglefactorANOVAwasusedtoanalysetheresultsusingBonferroni-Holm

multiple comparison. Two-way ANOVA was used to determine the interaction

between application of CPP-ACFP and exposure to different acids. The level of

significancewassetatα=0.05.

79

4.3.2Results

F7 results showedno significantdifferencesbetweenone-hour andone-day

groups when stored in water (Table 4.2). When stored in citric acid, however,

significantdifferences inmass losswereobserved inboth theplacebomousse and

TM+ groups when one-day results were compared to one-hour results (p<0.05).

However, when blocks were only allowed to set for one hour, the placebo group

showedadecreaseinmasslosscomparedtotheone-daygroups,whereastheTM+

groupshowedanincreaseinmassloss.F7EPresultsshowedthatallowingtheblocks

toonlysetforonehourbeforeplacingtheminsolutionhadasignificantimpactonall

one-day/one-hourmass loss comparisons (Table 4.3). In bothwater and citric acid,

the one-hour groups showed significantly (p<0.05) higher mass gain (water) or

significantly(p<0.05)lowermassloss(citricacid).

80

Table4.2.Comparisonofmasslossbetweenonehoursettingtimeand24hoursettingtimefollowedbycitricacidexposureofF7.Percentagechangeinbold

numbers,allothernumbersingrams.n=5.

81

Table4.3.Comparisonofmasslossbetweenonehoursettingtimeand24hoursettingtimefollowedbycitricacidexposureofF7EP.Percentagechangeinbold

numbers,allothernumbersingrams.n=5.

Theevaluationofionreleaseoftheinitialfourrestorativematerialsovera28-

day period inmany cases showed very low concentrations for daily ion release, in

someinstancesthelowreleaselevelswerebelowthedetectionlimit(phosphateion

releaseinwater)oftheinstrumentation(Table4.4).Theseresultsalsohighlightedthe

inabilityof liquidchromatographytomeasurefluoride ions in lacticacid,something

ofgreatinteresttothisstudy.

82

Table4.4.DailyIonreleaseofF7,F7EP,RivaProtectandChemFilMolarusingliquidchromatography.AllconcentrationvaluesareinmM.

Inadditiontotheabovefourmaterials,ChemFilRock(CFR)was investigated

separately. Compared to F7 and F7EP, Chemfil Rock exhibited less mass loss over

threedays,thisdifferencewasfoundtobestatisticallysignificant(p<0.05)(Table4.5).

F7EPwasalso found tohave lostmoremass thanF7 (p<0.05).Massgains found in

water storage were also found to be statistically significant among all groups

(p<0.05).Precisionandconsistencyof themassmeasurementsproducedverysmall

errors.

83

Table4.5.MasslosscomparisonbetweenCFR,F7andF7EPwhenstoredincitricacid

andwateroverathree-dayperiod.Allvaluesareingrams.

F7EPshowedagainofmasswhenstored intartaricacid,F7showedamass

loss, thedifferencebetweenthetwomaterialswassignificant (p<0.05) (Figure4.1).

Citricacidshowedamuchhigheraluminiumionreleasethaneitherlacticortartaric

acids(p<0.05).TherewasnosignificantdifferencebetweenF7andF7EPintermsof

aluminium ion release for all solutions (Figure 4.2 and 4.3). Significantly higher

phosphate ion release (p<0.05)wasmeasured in tartaricacid forbothF7andF7EP

compared to phosphate ion release in lactic acid. Phosphate ion release was

significantlylowerforbothmaterialsintartaricacidcomparedtocitricacid(p<0.05).

84

Calciumionreleasewassignificantlyhigherintartaricacidcomparedtolacticacidin

F7EPonly(p<0.05).

Figure4.1.MasslossofF7andF7EPintartaricacidoverathree-dayperiod.n=5

85

Figure4.2.Calcium,aluminiumandphosphateionreleasefromF7inthreedifferentacids.n=5.Al3+

86

Figure 4.3. Calcium, aluminium and phosphate ion release from F7EP in threedifferentacids.n=5.

4.3.3Discussion

Someof theacidparameterswere investigatedcoincidentlyaspartofother

pilotstudies,suchasthetartaricacidandaluminiumAASstudy.Thestudiesinvolving

RivaProtect,ChemFilMolarandF7andF7EPusedstoragevolumesof15mLforeach

block.Thisvolumewasdeemedtoolarge,asionconcentrationsweretoodilutedand

inmanycaseswerefallingbelowthedetectionlimitofequipment.Surfacehardness,

whichwasmeasuredupto28days,showedthatpasttheseven-daypointstandard

deviations became too large and therefore statistical comparisons became

meaningless. The protocol was therefore changed to a three-day duration and a

storagevolumeof5mL forall furtherpilotwork.Thenewprotocolproducedmuch

greater precision and consistency when comparing ion release profiles between

groups due to a higher concentration of ions, while still being able to detect

87

differences between materials and solutions in terms of mass loss and surface

hardness.

Foreachmaterial, twotosixdiscsorblocksweredivided intoequalgroups.

Most groups were not subjected to topical treatments, however some later pilot

work included an additional topical treatment step. Topical treatment pilot studies

(ToothMoussePlus,placebomousse) followed largelythesameprotocolas for the

other ion release andmass/hardness change studies. The equipment usedwas the

sameasforthestudieswithouttopicaltreatment(massloss,ionreleaseandsurface

hardness). MilliQ water was used to rinse the blocks following topical treatment.

Kimwipeswereusedtopatdrytheblocksbeforetheywereplacedinacidsolution(or

water control). Topical treatmentswerealways carriedout immediatelybefore the

blockswereplacedback intoanacidsolution,aftermass lossandsurfacehardness

measurements.

4.4Hardness


Vickersmicrohardnessmeasurementsweredeterminedfromindentationson

the lapped GIC surface using aMicrohardness tester (MHT-10, Anton Paar GmbH,

Graz, Austria) attached to a microscope (Leica DMPL, Leica Microsystems Wetzlar

GmbH,Germany).Indentationsweremadeusingarangeofforces(0.5,1.0,1.5,2.0

and2.5N)on freshandacidexposedblocksof F7andF7EP.Dwell timewasvaried

between6and20secondsatafixedforceof1.0N.Rateofforceapplicationwasfixed

at0.99N/minforallmeasurements.Theindentationswereseparatedbyadistance

ofatleastthreetimestheindentationsize.Imagesoftheindentationswereacquired

throughacalibrateddigitalcamera(LeicaDFC320)mountedonthemicroscopeand

distancemeasurementsmade using Image Tool software (Version 3.0, UTHSC, San

88

Antonio, TX)whichwere then converted into Vickers hardness values. Blockswere

thenplacedintofreshsolutionandreturnedtotheincubator.

During investigations of surface hardness of F7 and F7EP high surface

hardness was measured in some groups. This prompted a further pilot study to

determinewhetheracrust layerwasformedonthesurfaceofthematerials. Inthis

pilot, surface hardness was measured in layers as a function of depth. The depth

profileofsurfacehardnessinvolvedlappingthematerialsurfaceinstagesof100μm

andmeasuring the hardness of the exposed surface at each depth, up to 500 μm

(Figure 4.4).Microhardnesswasmeasured using the same equipment as described

above (Vickershardness). Blockswere lappedusingwet600-grit lappingpaper and

rinsedwithMilliQwater.

Figure4.4.DepthprofilecomparisonofTM+treatedF7EPfollowingthree-dayexposuretofoursolutions.InitialsurfaceVHshownasablackline.

89

Figure4.5.SurveyregionsforEDXA.

Tosupplementthedepthprofilehardnessstudyanotherpilotwasconducted

using electron dispersion X-ray analysis (EDXA). Samples for EDXA using scanning

electronmicroscope(FeiQuantaCryoSEM,FeiCorporation,ON,USA)wereselected

fromgroupsalreadyexposedtothethree-dayacidchallenge.Blockswerefractured

in two (usinga sharpblade) inorder toexpose thecentral and subsurface regions.

Samples were air dried and secured to stubs using two sided carbon tape. Ion

concentrationswere sampledand comparedbetween the surface layer, subsurface

regionand‘bulk’material,onlyrelativeconcentrationsofionscouldbemeasured,no

absolutevalueswereavailable(Figure4.5).

90

4.4.2Results

Surfacehardnesswasmeasuredovera28-dayperiod,statisticallysignificant

(p<0.05) differences between materials appeared by the third day (Table 4.6). In

somecasesstatisticallysignificantdifferencescouldonlybedetectedbytheseventh

day,butbythe14thdaythestandarddeviationsbecametoolargeandnosignificant

differencesbetweenmaterialscouldbemeasured.

ChemFilMolarwas initiallysignificantlyharder(p<0.05)thantheotherthree

materials and Riva protect was significantly (p<0.05) softer than F7 and F7EP.

Considerablesofteningwasobservedinthefirst24hours;atthisstagenosignificant

differencesbetweenanyofthematerialscouldbemeasured.

Table4.6.SurfaceVHofF7,F7EP,RivaProtectandChemFilMolarinlacticacidover28days.Storagevolume15ml.n=5

Inadditiontotheabovefourmaterials,ChemFilRock(CFR)was investigated

separately.Onlymass lossandsurfacehardnessweremeasured.ChemFilRockwas

initiallyofverysimilarsurfacehardnesstoF7andF7EP(p>0.05)(Table4.7).Atthe48-

hourmark significant differences could be detected between CFR, F7 and F7EP. In

fact, inthecitricacidgroups,followingthesofteningobservedinthefirst24hours,

CFRdidnotrecordanyfurthersofteningatthesurface(p>0.05betweendays1,2and

3).F7andF7EPcontinuedtograduallysoftenwitheachdaywhenstoredincitricacid.

Blocksstoredinwatershowedsignificantdifferencesamongallmaterials,withF7EP

recordingtheleastreductioninsurfacehardness,followedbyCFRandF7.

91

Table4.7.SurfaceVHofChemfilRockcomparedtoF7andF7EPstoredincitricacidoverathreedayperiod.Standarderrorshowninsecondarycolumns.Allvaluesin

MPaVH1.0

Fourgroupswere investigated fordepthprofilehardness,F7EP treatedwith

TM+exposedtocitric,lacticandhydrochloricacidsaswellasawatercontrolgroup.

Resultsshowedastatisticallysignificant(p<0.05)decreaseinsubsurfaceVH(around

100 μmbelow the surface) in the citric acid group compared to ‘bulk’ VH (i.e. the

hardness of the material in a region not directly exposed to the solutions). This

decreasewasalsosignificantwhencomparedtomeasurementstakenatadepthof

100μm in groupsplaced in theother solutions.No statistical significance couldbe

detected when lactic acid, hydrochloric acid and water groups were compared

amongsteachotheratthe100μmdepth(p>0.05).

EDXAof surface and subsurface regionsof F7EPblocks showed therewas a

significantdifferencebetweenblocksstoredinwaterandthosestoredincitricacid.

Theconcentrationofionsinthe‘bulk’ofthematerialinbothgroupswasverysimilar.

However, insomecasesthewaterstoragegroupsshowedlower ionconcentrations

on the surface and in subsurface regions compared to the citric acid groups and

comparedtothematerialbulk(aluminiumandstrontiumions)(Figure4.6).Thereare

also cases where citric acid exposed groups showed higher concentrations of ions

(sodiumandcarbon)onthesurfaceandinsubsurfaceregionscomparedto‘bulk’of

thematerial(sodiumandcarbon)(Figure4.7).

92

Figure4.6.F7EPblockstreatedwithTM+analysedusingEDXAatvarioussubsurfaceregions.

93


regions.

4.4.3Discussion

Microhardnessmeasurements showedanexpected spreadof results,higher

load values produced larger indentations. When the surface of the material was

softened followinga three-dayexposure toacid the indentationswereeven larger.

Variationsindwelltimeproducednoperceptiblechangeinindentationsize.

Themicrohardness pilot studywas conducted to determinewhich range of

indentationsizeswouldprovidethemostaccurateresultsforallcases(softandhard

restorativematerialsurfaces).Thevaluesobtainedatthisstagewerenotimportant,

the goal was to establish a set of parameters (dwell time and applied force) that

wouldworkforallmaterialsandconditionswithoutchangingthemagnificationofthe

microscope during post indentation image capture. In some cases very large

94

indentationswerestillobservedandthemagnificationneededtobechangedinorder

to obtain precise measurements. This was not ideal, however using lower applied

forcewouldhave resulted in reducedprecision for small indentations.Apragmatic

set of parameters (1.0N, 6 sec dwell time) was chosen to minimise the impact of

havingtochangemagnificationsduringindentationmeasurements.

InclusionofChemFilRockwasonlyintendedforpilottests,CFRisadifferent

type of GIC compared to F7/F7EP and Riva. Most GICs are based on aluminium,

however some new materials include zinc in their formulation; CFR is one such

material. The addition of zinc improves the flexural strength of material and

potentially improves resistance to dissolution2. The intended application of CFR is

different to that of the other tested materials, nevertheless including CFR in pilot

workformsabaselinemeasurement.

Changes with depth were marginal and there were many difficulties in

determiningtheoptimaldepth interval. Ideally, itwasnotsocompellingtofindout

whatthehardnessdropwasbutratheratwhichdepthitoccurred,ifatall.Evenusing

averycoarseresolutionof100μmthesignificantdropandthenriseinhardnessfor

the citric acid group could still be measured. Additionally, EDXA surveys showed

evidenceof ionmigrationfromthe‘bulk’andsub-surfaceregionsofthematerialto

thesurfacelayer.

4.5Conclusion

TheprotocolformeasuringsurfacehardnesswasestablishedusingtheAnton

Paar microhardness testing machine and a Leica microscope using parameters of

forceof1.0N,dwelltimeof6secondsandrateof0.99N/min.Protocolsformeasuring

calciumandaluminiumionswereestablishedusingatomicabsorptionspectroscopy

(AAS). An Ion selective electrode was determined to be the only feasible way to

measurefluorideionconcentrationsforthetestedcombinationofmaterialsandacid

storagemedia.Colourimetrywasusedtomeasurephosphateionconcentrations.

96

4.6References



2. ZoergiebelJ,IlieN(2013).Evaluationofaconventionalglassionomercement

withnewzincformulation:effectofcoating,agingandstorageagents.Clinical

OralInvestigations17(2):619-626.

5

CHAPTER5

Ionreleaseandphysical

propertiesofCPP-ACPmodified

GICinacidsolutions

(ThefollowingchapterhasbeenpublishedinJournalofDentalResearchMonthYear,

seeappendixforfullpublication)

5.1Abstract(249words):

Recentcommercializationofanewglass-ionomercement(GIC)(FujiVIITMEP)claims

enhanced tooth protection due to the incorporation of 3% (w/w) casein

phosphopeptide amorphous calciumphosphate (CPP-ACP).Objectives: Aims of this

studyweretoassessthisnewGICcomparedwithaGICwithoutCPP-ACP(FujiVII™)in

regards to ion release, changes in surfacehardness and inmass under a variety of

acidicandneutralconditions.Methods:EightyblocksofFujiVII™(F7)andFujiVII™EP

5

(F7EP) were subjected to three acidic solutions (lactic and citric acids pH 5.0,

hydrochloric acid pH 2.0) andwater (pH 6.9) over a three day period. Ion release,

surfacehardnessandweightmeasurementswerecarriedoutevery24hours.Results:

Higher calcium ion release from F7EP was observed under all acidic conditions.

IncreasedinorganicphosphateionreleasewasobservedforF7EPinhydrochloricand

citricacids.FluorideionreleasewassimilarbetweenF7andF7EPunderallconditions

butwassignificantlyhigherinacidscomparedtowater.Afterthreedaystherewasno

significantdifferenceinsurfacehardness(p>0.05)betweenthetwomaterialsunder

allconditionsexcepthydrochloricacid.MinimalchangeinmasswasobservedforF7

and F7EP in water, lactic and hydrochloric acids, however citric acid caused

significantly more mass loss compared with water (p<0.001). Conclusion:

Incorporationof3%w/wCPP-ACPintoF7hasenhancedcalciumandphosphate ion

release,withnosignificantchange influoride ionreleaseandnoadverseeffectson

surfacehardnessorchangeinmass.

Keywords:GIC,CPP-ACP,hardness,ionrelease

5.2Introduction

Glass ionomercements(GICs)arewidelyusedforavarietyofpurposessuch

as for intermediate restorations, caries stabilization,definitive restorationofmicro-

cavities or non-carious cervical lesions, adhering orthodontic brackets and bands,

fissure sealing erupting molars and as a surface protecting material for high risk

surfacessuchasrootsurfaces1,2.ThemainadvantagesofGICsaretheirstrongability

tochemicallybind todentineand theirability to release fluoride ions.This fluoride

ionreleasehasbeenshowntoslowtheprogressionandaid theregressionofearly

cariouslesions3.Thetoothsurfacewillonlydemineralisewhenthefluidbathingthe

tooth is undersaturated with respect to the tooth mineral which is composed of

5

hydroxyapatitehencethecalciumandphosphateionactivityatthetoothsurfacewill

alsobeimportant.

Anumberofinvestigatorshaveexploredthemodificationofdentalmaterials

inattempttohavethemreleasecalcium,phosphateandfluorideions4-6.Skrticetal.

(2003)exploredmodifyingdentalcompositeswithbioactiveglasses.Mazzaouietal.

(2003) and Al Zraikat et al. (2011) assessed the addition of casein phosphopeptide

amorphous calcium phosphate (CPP-ACP) to GICs. The casein phosphopeptides

stabilisecalciumandphosphateionsinabioavailableformthatallowthemtoinhibit

demineralisation and promote the remineralisation of early lesions 7. CPP-ACP is

stable in thepresenceof fluoride andhas been shown towork synergisticallywith

fluoride 8. This makes CPP-ACP a promising additive to dental products and

restorativematerials. The study byMazzaoui et al. (2003) was a proof of concept

studythatshowedthatCPP-ACPcouldbeaddedtoGIC.AlZraikatetal.(2011)later

explored the effect of CPP-ACP concentration on GIC mechanical properties.

Additionally,bothoftheseofstudiesshowedthattheadditionofCPP-ACPimproved

calciumandphosphateionreleaseinlacticacid.

Thetoothsurfacecanbeexposedtoavarietyofdemineralizationchallenges

including: acids formed from metabolic processes of oral bacteria (predominantly

lactic acid); foodacids suchas citric andphosphoric acid that are commonly found

softdrinks9;andhydrochloricacidfromtheregurgitationofstomachacids. Ifthese

acidsoverwhelmsaliva’sprotectivefunctionsmineralmaybelostfromtheteeth10.

Therefore, the overall aim of this study was to determine how GIC with CPP-ACP

performedunderavarietyofacidicconditionscomparedwithGICwithoutCPP-ACPin

termsofionrelease,changeinhardnessandchangeinmass.Theresearchquestions

were:what effectwould the addition of CPP-ACP have on; (1) surface hardness of

F7EP compared to F7; (2) change in mass between F7EP and F7 under neutral or

acidic environments and (3) change in calcium, phosphate or fluoride ion release

betweenF7EPandF7underneutralordifferentacidicenvironments.

5

5.3MaterialsandMethods

F7andF7EPcontaining3%w/wCPP-ACPfromthesamebatchwereprovided

by GC Corporation (Japan) in capsule form. Polyvinyl siloxane impression material

(eliteHD+lightbody,ZhermackSpA,BadiaPolesine,Italy)mouldswereusedtocreate

standardisedGICblocksmeasuring3mmx6mmx6mm(thicknessxwidthxlength).

40blocksofeachGICwerepreparedbyplacingthematerialsinthemouldwiththe

topandbottomsurfacescoveredbyplasticstrips,whichwasheldbetweentwoglass

slides.Theglassslidesweregentlypressedtogethertoextrudeanyexcessmaterial.

The specimenswereallowed to set inside themoulds for 24hours in an incubator

(37°C,~100%relativehumidity).Aftercoolingtoroomtemperaturetheblockswere

removed from themoulds, and the twomajor parallel surfaces of the blockswere

lapped with 600 grit paper (Norton Tufbak, Saint-Gobain Abrasives Ltd., Auckland,

NZ).

Four different solutionswere prepared to expose the blocks to a variety of

acidic and neutral environments. The three acidic solutions were formulated to



(50mMlacticacidatpH5.0)(thisconcentrationwasselectedbasedonvaluesfound

previouslyinplaquefluid11).TheneutralsolutionwasdistilleddeionisedwateratpH

6.9(MilliporeCorporation,Victoria,Australia).

Ten blocks of each type of GIC were exposed to 5mL of one of the four

solutions (stored inplastic containers). Solutionswere changedevery24hoursand

thesamplesweremeasuredforchange inmass,surfacehardnessandthesolutions

wereanalysedtodetermineionreleaseofcalcium,phosphateandfluoride.


measurements were performed. Blocks were taken out of solution, pat dried and

thenweighedusingamicrobalance(PrecisaXT120A,Dietikon,Switzerland).



5


GmbH,Germany).Twoindentationsweremadeoneachblock(Force,1.0N;Dwell,6

s;Rate,0.99N/min).Theindentationswereseparatedbyadistanceofatleastthree

times the indentation size. Images of the indentations were acquired through a

calibrated digital camera (Leica DFC320) mounted on the microcope (Leica DMLP,

Leica Microsystems Wetzlar GmbH, Germany) and distance measurements made

using Image Tool software (Version 3.0, UTHSC, San Antonio, TX)whichwere then

converted intoVickershardnessvalues.Blockswerethenplaced intofreshbatchof

solutionandreturnedtotheincubator.

Theionreleaseofcalcium,phosphateandfluorideaftereach24hourperiod

of storageweredeterminedusingatomicabsorptionspectroscopy, colorimetryand

an ion-specific electrode respectively. To determine the calcium concentration

sample solutions (1mL) were acidified with 1M HCl (0.5mL) and diluted with 2%

lanthanum chloride (0.5 mL) and analysed on a Varian AA240 atomic absorption

spectroscope(VarianAustraliaPty.Ltd.)againstasetofsevenstandardsrangingfrom

0 to 250 µM calcium. Inorganic phosphate ion concentrations were determined

colorimetrically using a spectrophotometer (UV-visible spectrophotometer, Varian

Australia,Pty.Ltd).Thesamplesthatwereanalysedwerepreparedbytaking100µL

of solution, diluting with 500µL of 4.2% ammoniummolybdate and adding 20µL

1.5%ofTween®20.(Sigma-Aldrich,St.Louis,MO).Thephosphateconcentrationwas

determinedbycomparingthespectrophotometerreadingsofthesamplesagainsta

setofsevenstandardsranginginphosphateconcentrationsfrom0and100µM.The

concentration of fluoride ions was determined using an ion-selective electrode

(Radiometeranalytical,ISEC301F,France)connectedtoanionanalyzer(Radiometer

analytical,IonCheck45,France).Samplesolutions(1mL)weredilutedwith1mLtotal

ionicstrengthadjustmentbuffer(MerckPtyLtd,Kilsyth,VIC,Australia)andmeasured

againstasetofeightfluoridestandardsrangingfrom0to1000µM.

Datafromsamplegroupswasfoundtofollowthenormaldistribution,χ2test

wasusedtotestnormality.SinglefactorANOVAwasusedtoanalysetheresultsusing

Bonferroni-Holmmultiplecomparison.Two-wayANOVAwasused todetermine the

5

interactionbetweenincorporationofCPP-ACPandexposuretodifferentacids.Level

ofsignificancewassetatα=0.05.

5.4Results

Table 5.1 shows surface hardness of F7EP and F7 in three different acidic

solutions and distilled deionised water measured over three days. A significant

decreaseinsurfacehardnesswasmeasuredfromdaytodayinallsolutionsexceptin

thefollowingcases.F7EPincitricandhydrochloricacidsbetweendaytwoandthree

didnotregisterasignificantchangeinsurfacehardness.F7inwaterdidnotregistera

significant change between days one and two, and two and three. The surface

hardnessofF7EPinwaterwasnotsignificantlydifferentbetweenitsinitialhardness

anddayoneaswellasbetweendaystwoandthree.TheinitialhardnessofF7EPwas

significantly (p<0.001) lower than thatof F7.However, after24hoursof storage in

lactic and citric acid F7EPPhada similar surfacehardness to F7andwasno longer

significantly weaker for citric, lactic acids and water (p>0.05), but it was still

significantly (p<0.01) weaker in hydrochloric acid. After three days of storage no

significant difference in surfacehardness couldbemeasuredbetween F7EP and F7

(p>0.05)forcitric,lacticacidsandwater,butF7EPstillremainedsignificantly(p<0.05)

weakerinhydrochloricacid.

5

Table5.1.MeansurfacehardnessofF7andF7EPinacidicandneutralsolutionsmeasuredoverthreedays.Valuesmarkedwiththesamelowercasesubscript

indicatesignificantdifferencebetweenGICmaterialswithinthesamesolutionforthesametimeperiod.

Table5.2.MeanrelativemassofF7andF7EPwhenstoredinthefourdifferentsolutionsoverthreedays.Valuesmarkedwithuppercasesuperscriptindicate

significantdifferencebetweendifferentsolutionswithinthesamematerialforthesametimeperiod.Eachgroupisscaledaccordingtoitsinitialweight(100%).

Table5.2showsthemeanrelativemasschangeofF7andF7EPblockswhen

storedinthefourdifferentsolutionsoverthreedays.Eachgroupisscaledaccording

toitsinitialweight(100%).ThegreatestmasslossatalltimepointswasfromF7and

F7EPthatwereexposedtocitricacid.

Figures 5.1a-c show themeasured concentration of calcium, phosphate and

fluoride ionsreleasedfromF7andF7EPwhenstored in the fourdifferentsolutions

overathreedayperiod.TherateofcalciumandphosphateionreleaseforF7EPwas

significantlygreaterthanthatofF7forbothcitricandhydrochloricacids(Figures5.1a

and5.1b),andtherewasalsogreatercalciumionreleaseinlacticacid(Figure5.1a).

The fluoride ion release in all solutionswas generally similar for both F7 and F7EP

(Figure5.1c).

5

Figure5.1.Measuredcalcium(a),phosphate(b)andfluoride(c)ionreleasefromF7andF7EPwhenstoredinthefourdifferentsolutionsoverathreedayperiod.

Significantincrease(p<0.05)incalciumionswasmeasuredforallgroups(exceptwater)betweenF7EPandF7.Significantincrease(p<0.05)inphosphateionreleasewasmeasuredforcitricandhydrochloricacidscomparedtowater.SignificantlymorephosphatewasreleasedfromF7EPcomparedtoF7incitricandhydrochloricacids.

Fluorideionreleasewassignificantlyhigher(p<0.005)inallacidscomparedtowater.Groupsmarkedwithsamelowercaseindicatenosignificantdifferenceinfluorideion

releasebetweenF7andF7EPwhenexposedtothesamesolution.

Wherea change inmasswasdetected foranexposure toanacidic solution

overthethreedays(Table5.2)thecalciumandphosphateionreleasewasfoundto

begreaterforF7EP(Figures5.1aand5.1b).Thedifferenceincalciumandphosphate

ionreleaseasafunctionofchangeinmassforF7andF7EPisshowninFigures5.2a

and5.2bforcitricandhydrochloricacidsrespectively(lacticacidandwatercouldnot

beplottedastherewasnomeasurablemassloss).Thesefiguresshowthatperunitof

mass loss therewas a greater releaseof calciumandphosphate ions from theGIC

containingCPP-ACP.

5

Figure5.2.Differenceincalciumandphosphateionreleaseasafunctionofchangein

massbetweenF7andF7EPincitricacid(a)andhydrochloricacid(b).

5

5.5Discussion

InallthetestsconductedonF7andF7EP,thematerialsbehaveddifferentlyin

water and acidic environments. The surface hardness of the GIC's significantly

decreased intheacidicenvironmentsalthoughthisdidnotcorrelatetoachangeof

mass.Thereleaseofionswasgreaterinacidicconditionscomparedwithwater.The

releaseofcalciumandphosphateionsfromthematerialonlyunderacidicconditions

isidealasthisiswhentheywouldbeneededtoprotecttheadjacenttoothstructure

fromdemineralization.ThehigherreleaseofcalciumandphosphateionsfromF7EP

under acidic conditions may saturate the fluid in the oral environment with

appropriateionsthusactingasa"smartmaterial"fortheprotectionofatrisktooth

surfaces.

Previousstudieshaveinvestigatedionreleaseandsurfacehardnessofvarious

GICs inacidicandneutralenvironments4,5,12.Pastresultshaveshownthatfluoride

release fromF7 isdecreasedwith incorporationofCPP-ACPwhenstored inneutral

solution(water)orlacticacid5.IthasalsobeenreportedthatlowerpHenvironments

negatively affect surfacehardnessofGICs 13.However, therehavebeenno studies

lookingationreleaseorsurfacehardnessofGICscoveringawidevarietyofacidsthat

canbeencounteredintheoralenvironment.

5.5.1Surfacehardness

TheresultsindicatethatthedegradationofsurfacehardnessispHdependant

ratherthansolutiondependant.Lacticandcitricacids(bothpH5)producedasimilar

reduction inmicrohardness over three days,whileHCl (pH 2) showed the greatest

andwater (pH6.9) the least. Even though initially F7EPwas significantlyweaker in

surface hardness than F7, a converging pattern occurred across all solutions such

that, except for HCl, the difference after three dayswas no longer significant. It is

possible that F7EP has a greater buffering capacity than F7 alone as CPP-ACP has

previouslybeenshowntohavebufferingpotential14.

5

5.5.2Changeinmass

Aswithsurfacehardness,therewasaninitialdifferenceinthechangeinmass

betweenthesetwoGICmaterials.ThiscanbepartlyattributedtoF7EPbeingslightly

lessdensethanF7.Significantmasslosswasobservedforspecimensplacedincitric

acid for bothmaterials.While other solutionsproduced small changes fromday to

daythedifferencesbetweenthemwerenotsignificant.Thedifferenceinthechange

in mass between F7EP and F7, in citric acid, was not significant when taking into

account the difference in initial weights. Although surface hardness showed very

similar trend in a decrease in hardness for all solutions this was not repeated for

change in mass. For citric and lactic acids the mass loss shows a large difference

betweentwoacidsolutionssuchthat, in lacticacidaslightmassgainwasobserved

forF7EPandaslightlossforF7.Incitricacidthelargestmasslosswasobservedfor

bothmaterials,whileamassgainwasobservedinwater.Thechelatingeffectofthe

citricacidmaycontributetothe largemass loss.Citricacid isatriproticacid,which

can chelate transition metals, in this case aluminium. Aluminium is an initial

componentoftheGICmatrixintheformofaluminiumphosphateandthealuminium

ion is involved in the acid-base setting reaction. The cross-linking between the

polyalkenoic acid chains is formed by a slow final maturation reaction involving

aluminium ions,which increases the strength of theGIC 15. This hardening process

maytakedaystocomplete.WhenexposedtocitricacidAl3+ ionsarecomplexedby

citrate removing them from theGICmatrix leading to the greatermass loss of the

material.Furtherevidence tosupport this theorycanbeseen in thephosphate ion

releasedatashown inFigure5.1b.TheGICwithoutCPP-ACPreleasedhigh levelsof

phosphatealthoughitcontainednoCPP-ACPsupportingthechelationofaluminium

andthedissolutionofthealuminiumphosphatepresentwithinallGICs.

5

5.5.3IonRelease

When subjected to aggressive environments such as citric, lactic and

hydrochloric acids a substantial release of ions occurred from both GIC materials.

Calciumreleaseinthethreeacidsrangedfrom0.07to0.20µmol/mm2forF7butwas

higher for F7EP, ranging from 0.36 to 1.34 µmol/mm2, equating to an increase of

564%, 405%, 462% for citric, lactic and hydrochloric acids respectively. This can be

explainedbytheCPP-ACPactingasasourceofadditionalcalciumions.TheCPP-ACP

complexesmay release calcium and phosphate ions by four proposedmechanisms

withonebeingpHdependentrelease16.An increase incalciumionswouldworkto

maintainthedegreeofsaturationwithrespecttotoothmineralandworktoprevent

demineralization.Phosphate ionrelease in lacticacidandwaterwasnotdetectable

foreitherGICmaterial.Dailyphosphateionreleaseforcitricacidrangedfrom9.8to

13.9nmol/mm2forF7andwashigherforF7EP,rangingfrom14.5to17.6nmol/mm2,

anaverageincreaseof39%.Dailyphosphateionreleaseforhydrochloricacidranged

from0.42to0.51nmol/mm2forF7andwashigherforF7EP,rangingfrom0.7to1.07

nmol/mm2, an average increaseof 85%. This implies that somephosphate is being

suppliedbyCPP-ACP,butunlikethecalciumionreleasealotofthephosphateisalso

beingsuppliedbytheGICmatrixmostlikelyfromdissolutionofAlPO4atthesurface

of the GIC. Dissolution of the AlPO4 by citric and hydrochloric acids releases PO43-

fromtheGICmatrix.Dailyaveragefluoridereleaseforlacticacidandwaterdropped

with addition of CPP-ACP, from 63.79 to 54.38 µmol/mm2 in lactic acid and from

11.47 to 6.02 µmol/mm2 in water (Figure 5.1b). This effect has been observed in

previous studies conducted on F7 with CPP-ACP 5. Daily average fluoride release

remained steady forbothGICmaterialswhenexposed to citric (103.53and108.46

µmol/mm2)andhydrochloric acids (107.40and109.38µmol/mm2) for F7andF7EP

respectively(Figure5.1b).

5

5.6Conclusion

Ithasbeenshownthat,althoughinitialsurfacehardnessofF7EPislowerthan

F7, after three days of storage in three of the four types of solution therewas no

significantdifferencebetweenthetwoGICs.AdditionofCPP-ACPtoF7increasedthe

releaseofcalciumionsbyanaverageof477%whenexposedtoacidicsolutionswith

very little or no change in fluoride ion release. However, phosphate ion release

rangedgreatly(belowdetectionlimitto17.5nmol/mm2daily)dependingonthetype

ofacidexposuretotheGICmaterialsandwhetherCPP-ACPhasbeenincorporated.

5

5.7References



39(6):339-343.






Research90(1):99-103.







6. Skrtic DA, J. M. Eanes, E. D. (2003). Amorphous calcium phosphate-based

bioactivepolymericcompositesformineralizedtissueregenration.Journalof

Research108(3):167-182.




5







10. Roberts MW, Li SH (1987). Oral findings in anorexia nervosa and bulimia

nervosa: a study of 47 cases. Journal of the American Dental Association

115(3):407-410.



12. Bala O, Arisu HD, Yikilgan I, Arslan S, Gullu A (2012). Evaluation of surface

roughnessandhardnessofdifferentglassionomercements.EuropeanJournal

ofDentistry6(1):79-86.

13. SilvaKG,PedriniD,DelbemAC,CannonM(2007).EffectofpHvariationsina

cyclingmodelon thepropertiesof restorativematerials.OperativeDentistry

32(4):328-335.

14. CaruanaPC,MulaifySA,MoazzezR,BartlettD(2009).Theeffectofcaseinand

calciumcontainingpasteonplaquepHfollowingasubsequentcarbohydrate

challenge.JournalofDentistry37(7):522-526.




5

16. CochraneNJ,ReynoldsEC(2009).Caseinphosphopeptidesinoralhealth.Food

ConstituentsandOralHealth172):185-224.

116

CHAPTER6

Rechargeandionreleasefollowing

topicalToothMoussetreatmentof

CPP-ACPmodifiedGICinacid

solutions


Recent commercialization of a glass-ionomer cement (GIC) (Fuji VIITM EP)

claims enhanced tooth protection due to the incorporation of 3% (w/w) casein

phosphopeptide amorphous calcium phosphate (CPP-ACP).Objectives: The aims of

this studywere toassess theability for calciumandphosphate ion rechargeof the

modifiedGICusingatopicaltreatmentcontainingCPP-ACFPandcomparingitwitha

GIC without CPP-ACP under a variety of acidic and neutral conditions.Methods:

EightyblocksofGICFujiVII™andFujiVII™EPweresubjectedtothreeacidicsolutions

117

(lacticandcitricacidsatpH5.0,hydrochloricacidatpH2.0)andwater(pH6.9)overa

three-day period. Ion release, surface hardness and weight measurements were

carriedoutevery24hours.Every12hourshalfoftheblocksweretreatedwithTooth

Mousse Plus (TM+), the remainder received a placebo treatment. Results:

Significantly higher calcium and phosphate ion releasewere recorded for bothGIC

materials treated with either placebo or TM+ when exposed to citric acid,

accompaniedby increasedmass lossand increase insurfacehardness.Themajority

ofothercaseswithTM+treatmentresultedinlowermasslossandincreasedrelease

of calcium and phosphate ions compared to groups treated with placebomousse.

Conclusion: Topical treatment affects the GIC in a way that is poorly understood.

TopicalToothMoussePlustreatment increasedsurfacehardnessand ionreleaseof

bothGICsincitricandhydrochloricacids.

Keywords:GIC,CPP-ACP,surfacehardness,ionrelease,recharge,toothmousse

6.2Introduction

In restorative dentistry glass ionomer cements (GICs) are widely used for a

variety of purposes such as restorations, caries stabilization aswell as adhesion of

orthodontic brackets1, 2. GICs bind chemically to dentine and enamel and release

fluoride ionsover time,whichhasbeenshowntoslowtheprogressionandaid the

regression of early carious lesions 3. In an effort to further improve the caries

inhibition and repair potential of GICs, a number of attempts have beenmade to

modifythemwiththeaimtoallowreleaseofcalciumandphosphateionsinaddition

to fluoride4-6. One such attempt has incorporated the compound, casein

phosphopeptide amorphous calcium phosphate (CPP-ACP) into the GIC. Previous

studies assessed the potential of incorporating CPP-ACP intoGICs and have shown

verypromisingresults4,5,7.HighercalciumandphosphateionreleasefromtheCPP-

ACPmodifiedGICwhen exposed to lactic acidwere demonstrated4,5. CPP-ACPhas

118

beenshowntoworksynergisticallywithfluorideandthebioavailablephosphateand

calcium ions from CPP-ACP, and demonstrated to inhibit demineralisation and

promotetheremineralisationofearlycariouslesions8,9.Incorporationoffluorideinto

the ACP complex to form amorphous calcium fluoride phosphate (ACFP) has been

characterised and demonstrated to be more effective than ACP at remineralising

enamelsubsurfacelesions8,10.

A variety of demineralization challenges including acids formed from

metabolic processes, food acids and gastric acids can leave the surface of teeth

exposed to loss of surface mineral11. If these acids overwhelm the protective

functionsofsaliva,toothmineralwillbelost12.Lossoftoothmineralhasbeenstudied

extensively,however lossofdental restorativematerialshas largelybeenexamined

fromtheclinicalpointofviewonly,andmuchofthemechanismshowthelossoccurs

arenotwellunderstood.Recently,aninvestigationoftheeffectsofvariousacidson

thephysicalpropertiesofGICsrevealedthatthecommonfoodacid,citricacid,hada

farmorepronounced impactonGICsthan lacticacid7,which is theacidassociated

withtheformationofcariouslesions.AsignificantmasslossofGICblocksexposedto

citricacid,comparedtolacticacid,wasaccompaniedbyanincreaseinthereleaseof

calciumandphosphateions.

EarlyindicationssuggestthatanincreasedreleaseofphosphateionsfromGIC

incitricacidmaybetheresultofchelationofaluminiumions.Aluminiumiscontained

in four components of GICs - aluminium fluoride, cryolyte, aluminium oxide and

aluminiumphosphate.Duringthe initialmixingbetweentheglasspowderandacid,

aluminium ions are leached from the glass and replace hydrogen ions in carboxyl

groups in theacid, forming salts13.Manyof thealuminium ions remain in theglass

particles and do not initially participate in the setting reaction. The initial ratio of

aluminiumandsilicacontrolstherateoftheGICsettingreactionthroughthegelation

and cross-linking phase, so presence of extra aluminium ions is an important

consideration for theworkabilityof themix 14. This studywill attempt toprovidea

furtherunderstandingof themechanisms thatproduce such anunexpectedly large

releaseofionsandmasslossinconventionalGICsandcomparethesefindingsagainst

119

resin-modified GICs, which theoretically are not as susceptible to chelation by

exposure tocitricacid.Early indications fromaprevious studysuggest thatamuch

higher release of phosphate ions in citric acid may be the result of chelation of

aluminium ions within the GIC7. The aims of this study were to build on the data

collectedinthepreviousstudy7andfurtherinvestigatetheeffectofaddingCPP-ACP

intheformofacrèmewithrespecttoionreleaseandphysicalpropertiesofFujiVII

(F7)andFujiVIIEP(F7EP)whensubjectedtoanacidicchallenge.TheeffectofCPP-

ACFPwillbe investigatedaftera topical treatmentusingToothMoussePlus (TM+),

which contains 10% w/w CPP-ACFP compared to a placebo mousse without CPP-

ACFP.

The null hypotheses to be tested are; 1) topical treatment using placebo

moussedoesnotaltersurfacehardness,masslossprofileandprovidesnoadditional

ion release, under acid challenge compared to “no treatment” groups; 2) topical

treatmentusingToothMoussePlusdoesnotaltersurfacehardness,masslossprofile

and provides no additional ion release under acid challenge compared to (a) “no

treatment”groupsand(b)groupstreatedwithplacebomousse.


EncapsulatedF7andF7EPcontaining3%w/wCPP-ACPfromthesamebatch

ofglasspowderwereprovidedbyGCCorporation(Tokyo,Japan).Polyvinylsiloxane

impressionmaterial(eliteHD+lightbody,ZhermackSpA,BadiaPolesine,Italy)moulds

were used to create standardised GIC blocks measuring 3mm x 6mm x 6mm

(thicknessxwidthx length).EightyblocksofeachGICwerepreparedbyplacingthe

materials in themouldwith the top andbottom surfaces coveredby plastic strips,

held between two glass slides. The glass slides were pressed together to extrude

excessmaterial.Thespecimenswereallowedtosetinsidethemouldsfor24hoursin

anincubatorat37°Cand100%relativehumidity.Aftercoolingtoroomtemperature

the blocks were removed from the moulds, and lapped with wet 600-grit paper

120

(NortonTufbak,Saint-GobainAbrasivesLtd.,Auckland,NZ)toprovideastandardised

surfacefinish.

For each material, the 80 blocks were divided into two groups of 40. One

group received topical treatmentofToothMoussePlus (TM+,GCCorporation), the

otherreceivedaplacebomoussetreatment(moussebasewithnoactiveingredient,

provided by GC Corporation, Japan) (Table 6.1). TM+ (10% w/w CPP-ACFP) was

dilutedwithwater(MilliQ,MilliporeCorporation,Victoria,Australia)to2%w/wCPP-

ACFP,placebomoussewasdilutedusingthesameratios.Eachblockwasplacedinto

2mLofdilutedmoussesolution,eitherTM+orplacebo,for2minutes,followedbya

gentlerinseusingdistilledwaterafterwhichtheblockwasplacedbackintoanacidor

control solution (water). Topical treatmentsweredeliveredevery 12hours starting

with a pre-treatment dose applied after the blocks were lapped but before being

placedintothestoragesolutionforthefirsttime.

Controlsolution -Water(pH6.7)controlforAcidsolutions

Controltreatment-PlacebotreatmentcontrolforTM+

-“Notreatment”controlforPlaceboandTM+

Controlmaterial -FujiVIIcontrolforFujiVIIEP

Table6.1.Treatmentsandcontrolgroups.

Four different solutions were prepared using three acidic and one neutral

environment.Thethreeacidicsolutionswereformulatedtosimulateagastricerosive

challenge (50mM NaCl adjusted to pH 2.0 with HCl), a dietary erosive challenge

(50mMcitricacidatpH5.0)andacariogenicacidchallenge(50mMlacticacidatpH

5.0). The control solution was distilled deionised water at pH 6.9 (Millipore

Corporation, Victoria, Australia). In the absence of saliva as a storagemedium and

taking into account the highly controlled nature of a laboratory study, the ionic

concentrationsofacidicsolutionswereselectedbasedonvaluespreviouslyreported

inplaquefluid15.

121

Ten blocks per group of each GICwere exposed to 5mL of one of the four

solutions (stored inplastic containers). Solutionswere changedevery24hoursand

thesamplesweremeasuredforchangeinmassandsurfacehardness.Thesolutions

wereanalysedtodetermine ionreleaseofcalcium,phosphateand fluoride.Topical

treatmentswerealwayscarriedoutimmediatelybeforetheblockswereplacedback

intothesolution,aftermasslossandsurfacehardnessmeasurements.


measurementswereperformed.Blocksweretakenoutofsolution,gentlydriedusing

KimwipesDelicateTaskWipers (KimtechScience,Kimberly-ClarkProfessional,NSW,

Australia) and then weighed using a microbalance (Precisa XT 120A, Dietikon,

Switzerland).Onlysinglemeasurementswerecarriedouttominimisedehydrationof

theGICsurfaceandmaintainconsistencyamongstallsamples.





s;Rate,0.99N/min).Theindentationswereseparatedbyadistanceofatleastthree

times the indentation size. Images of the indentations were acquired using a

calibrated digital camera (Leica DFC320)mounted on themicroscope (Leica DMLP,

Leica Microsystems Wetzlar GmbH, Germany) and distance measurements were

madeusingImageToolsoftware(Version3.0,UTHSC,SanAntonio,TX),whichwere

converted intoVickershardnessvalues.Blockswere thenplaced into freshsolution

and returned to the incubator. At no time were the blocks allowed to become

dehydrated.

Theionreleaseofcalcium,aluminium,phosphateandfluorideaftereach24-

hourperiodof storageweredeterminedusingatomicabsorption spectroscopy (for

calciumandaluminium), colourimetry (for inorganic phosphate) andan ion-specific

electrode (for fluoride). To determine the calcium concentration, sample solutions

(0.5mL)weredilutedwithwater(0.5mL,MilliQ),thenacidifiedwith1MHCl(0.5mL)

and dilutedwith 2% lanthanum chloride (0.5mL) and analysed on a Varian AA240

122

atomicabsorptionspectroscope(AAS,VarianAustraliaPty.Ltd.)againstasetofseven

standardsrangingfrom0to250µMcalcium.TheVarianAA240AASwasalsousedto

determine the concentration of aluminium ions. Sample solutions (0.5 mL) were

dilutedwithpotassiumchloride(0.5mL,8mg/L)andlanthanumchloride(0.5mL,2%

w/w) and then acidified with 1M HCl (0.5 mL) and compared against a set of

standards ranging from 0 to 125 µM aluminium. Inorganic phosphate ion

concentrations were determined colourimetrically using a spectrophotometer (UV-

visible spectrophotometer, Varian Australia, Pty. Ltd). Samples were prepared by

taking100µLofsolution,dilutingthemwith500µLof4.2%ammoniummolybdate

andadding20µL1.5%ofTween®20.(Sigma-Aldrich,St.Louis,MO).Thephosphate

concentrationwasdeterminedbycomparingthespectrophotometerreadingsofthe

samplesagainstasetofsevenstandardsranginginphosphateconcentrationsfrom0

and 100 µM. The concentration of fluoride ions was determined using an ion-

selective electrode (Radiometer analytical, ISE C301F, France) connected to an ion

analyser(Radiometeranalytical,IonCheck45,France).Samplesolutions(1mL)were

dilutedwith1mLtotal ionicstrengthadjustmentbuffer (MerckPtyLtd,Kilsyth,VIC,

Australia)andmeasuredagainsta setofeight fluoride standards ranging from0 to

1000µM.

ControlgroupsconsistedofF7andF7EPblocksthatunderwentacidchallenge

only(notopicaltreatments).Datafromsamplegroupswerefoundtofollowanormal

distribution, χ2 test was used to test normality. Single factor ANOVA was used to

analyse the results using Bonferroni-Holm multiple comparison (Microsoft Excel

2011). Two-way ANOVA was used to determine the interaction between

incorporationofCPP-ACFPandexposuretodifferentacids.Levelofsignificancewas

setatα=0.05.

123

6.4Results

6.4.1SurfaceHardness

.

Topical treatment with TM+ of F7 surface in control solution (water)

preventednearlyallof thesofteningcompared tono treatment (p<0.05),TM+was

also significantly better than the placebo treatment (p<0.05) at reducing the

softeningofthesurface(Table6.2).PlacebotreatmentoftheF7surfaceshowedno

statisticallysignificantimprovementcomparedtothecontrol(p>0.28).F7EPshowed

noimprovementwheneitherofthetopicaltreatmentswereapplied(p>0.42inboth

cases)comparedtonotreatment.

Table6.2.SurfaceHardness(%changerelativetofreshblocks).

n=10.Standarderrorinbrackets

GICblocksexposedtocitricandlacticacidswithnotopicaltreatmentshowed

considerable softening on the surface, with an average a drop of 51% and 52% in

Vickers hardness respectively (Table 6.2)7. When the topical treatment cycle

occurred, blocks exposed to citric acid not only showed much less softening, but

actually demonstrated an increase in surface hardness. This effect was more

124

pronounced in groups treated with TM+, however blocks treated with placebo

mousse also showed an increase in surface hardness (Table 6.2). The increasewas

statistically significant for F7 and F7EP with the placebo and TM+ treatments

comparedtothecontrol(p<0.001inallcases);nostatisticaldifferencewasmeasured

betweenplaceboandTM+(p>0.05).

In lactic acid, only the placebo treatment of F7 showed any significant

difference (p<0.05),where the surfacehardnesswas lower than the control group,

little or no change was measured for TM+ application for either F7 or F7EP. TM+

treatment showed a statistically significant reduction in softening of F7 treated

groups in hydrochloric acid compared with the control and placebo treatments

(p<0.05bothcases).Verysimilarresultswerealsoobservedinblocksstoredinwater,

where a significant improvement in surface hardness was measured in F7 treated

withTM+comparedtothecontrolandplacebo(p<0.001andp<0.01respectively).No

statistically significant differences in hardness change were measured for F7EP

exposed to lactic and hydrochloric acids and water for both placebo and TM+

treatments.

StatisticallysignificantdifferencesbetweenF7andF7EPwereobservedinonly

two cases, placebo treatment in lactic acid (p<0.001) and TM+ treatment inwater

(p<0.05).

6.4.2MassLoss

Bothmaterialsthatunderwenttopicaltreatmentsandwereexposedtocitric

acidshowedasignificantincreaseinlossofmasscomparedtothecontrols(p<0.001

in all cases (Table 6.3). The loss inmass increased dramatically (an increase in the

range of 4.8% to 8.7%) in all four groups. Placebo treatment showed significantly

higher mass loss compared to TM+ treatment in citric acid for both F7 and F7EP

(p<0.05). F7 and F7EP responded differently to placebo and TM+ treatments, F7EP

wasmoreresistanttomasslossunderbothplaceboandTM+treatments(p<0.05and

p<0.001 respectively). The largest daily loss of mass occurred on the last day of

125

measurement for all four groups (F7, F7EP in citric acid with TM+ and placebo

treatments,Figures6.1a-d).

Table6.3.MassLoss(%changerelativetofreshblocks).

n=10.Standarderrorinbrackets.

Figure6.1.DailymasslossofF7blockstreatedwithplacebomousseandstoredinfourdifferentsolutions.

126

Thelargestmeasureddifferenceoccurredinthecitricacidchallenge,however

statisticallysignificantdifferenceswerealsodetectedinotherstoragemedia.Placebo

treatmentinlacticacidshowedahighermasslosscomparedtotheacidcontroland

TM+ treatment for both F7 and F7EP groups (p<0.001 in all four cases), TM+

treatmentwasnodifferenttotheacidcontrol(p>0.11forbothmaterials).F7EPalso

showedreducedmass losscomparedwithF7forbothplaceboandTM+treatments

whenstoredinlacticacid(p<0.001inbothcases).

Figure6.2.DailymasslossofF7EPblockstreatedwithplacebomousseandstoredinfourdifferentsolutions.

127

Figure6.3.DailymasslossofF7blockstreatedwithTM+andstoredinfourdifferentsolutions.

Inhydrochloricacid,theTM+treatedgroupsshowedastatisticallysignificant

reduction in mass loss compared to the control and placebo groups for both F7

(p<0.01andp<0.05respectively)andF7EP(p<0.001 inbothcases).Aswasthecase

forcitricandlacticacids,F7EPshowedlessmasslossinhydrochloricacidcompared

withF7forbothplaceboandTM+(p<0.001forbothcases).

TopicaltreatmentsoftheF7EPblocksstoredinwaterforthreedaysshoweda

significant difference in the measured mass compared to the control groups (no

topical treatment).BothplaceboandTM+showedasignificantly reducedmass loss

compared with the control untreated blocks (p<0.05 both cases). No statistical

difference between the two topical treatments was detected for F7EP. Only TM+

treatmentshowedasignificantdifferenceinmasslosswhenusedtotreatF7stored

inwatercomparedtocontrolandplacebotreatment(p<0.05inbothcases).

128

Figure6.4.DailymasslossofF7EPblockstreatedwithTM+andstoredinfourdifferentsolutions.

6.4.3IonRelease

Overall,F7EPshowedconsistentlyhighercalciumionreleaseacrossallgroups

comparedwithF7 (p<0.001),withtheexceptionofwaterwherethedetection limit

wasunabletomeasuretheionlevels(Table6.4).Calciumionreleaseforspecimens

thatwere exposed to lactic and hydrochloric acids showed an increased release of

ionswhentreatedwithTM+comparedtothecontrolandplacebotreatmentgroups

(p<0.05inallcases),thiswastrueforbothF7andF7EP.Theincreasewasverysimilar

inmagnitudeforbothmaterials(around130µMforHCland20-50µMforlacticacid).

PlacebomoussetreatmentofF7showedsignificantly lesscalcium ionreleasewhen

storedinhydrochloricacid(p<0.05,30.3μMvs16.6μM),thedifferenceintheF7EP

groupwassimilar inmagnitude (173.0μMvs156.6μM),but itwasnotstatistically

significant(Figures6.5and6.6).F7EPblocksexposedtocitricacidshowednochange

in calcium ion releasewhen treatedwith TM+, but showed a large increasewhen

treated with the placebo (p<0.01). F7 blocks treated with placebo also showed a

129

highercalcium ionrelease thanthose treatedwithTM+(p<0.05). Inmostcases ion

releaseduringthefirst24hourswaslargerthanonsubsequentdays.Theexception

tothiswastheTM+treatedgroupsthatwereexposedtohydrochloricacid; inboth

casesthedailyreleaseremainedsteady.

Table6.4.Calciumionrelease(allunitsinμM),n=10

130

Table6.5.Phosphateionrelease(allunitsinμM),n=10

F7EPconsistentlydisplayedaslightlyhigherreleaseofphosphateionsthanF7

(Table 6.5). Lactic acid groups treated with placebo mousse showed higher ion

releasecomparedtotherespectivecontrolgroups(p<0.05forbothmaterials),TM+

treated groups produced significantly higher ion release compared to the “no

treatment” control (p<0.05 for both F7 and F7EP) and placebo treatment groups

(p<0.05forbothF7andF7EP).

131

Figure6.5.Cumulativethree-daycalciumionreleasefromF7blocksacrossfoursolutionsandthreetreatmentgroups.

Dataforthecontrolgroupswhenblockswereexposedtocitricacidshowed

very high phosphate ion release compared to other solutions (Figure 6.3a,b). F7

treated with placebo mousse showed a slightly higher amount of phosphate ion

releasecomparedwiththecontrolgroup(p<0.05),eventhoughthereadingforF7EP

compared to the control was also higher, no significant difference was detected

(p>0.25). However, F7 and F7EP treatedwith TM+ exposed to citric acid showed a

significantly lower release of phosphate ions compared with the control groups

(p<0.05), the TM+ treated groups also showed significantly lower phosphate ion

releasethanplacebotreatedgroupsforbothmaterials(p<0.05).

132

Figure6.6.Cumulativethree-daycalciumionreleasefromF7EPblocksacrossfoursolutionsandthreetreatmentgroups.

In hydrochloric acid exposed groups, the placebo treated blocks released a

statistically lower amount of phosphate ions compared to “no treatment” control

groups, the magnitude of difference was similar in both materials although only

significant for F7 (p<0.05). Blocks treated with TM+ showed significantly higher

phosphateionreleasethancontrolandplacebotreatedgroups(p<0.05inallcases).

Waterstoredblocksdisplayedasimilarpatterntothehydrochloricacidgroups,many

results fell below the detection limit, thus making statistical comparisons difficult.

TM+treatedblocksreleasedsignificantlymorephosphateionsthanbothcontroland

placebogroups(p<0.05inallcases).

133

Figure6.7.Cumulativethree-dayphosphateionreleasefromF7blocksacrossfoursolutionsandthreetreatmentgroups.

F7EPconsistentlyproducedhigherphosphateionreleasethanF7inallthree

acids;theonlystatisticallynon-significantresultwasforTM+treatedblocksinlactic

acid(p<0.05inallothercases),althoughtheamountswerehigherforF7EPtheywere

also highly variable fromday to day. In several groups, the largest daily release of

phosphatewasobservedonthethirddayofmeasurement.

134

Figure6.8.Cumulativethree-dayphosphateionreleasefromF7EPblocksacrossfoursolutionsandthreetreatmentgroups.

Topical treatment with TM+ had a similar effect in groups exposed to acid

solutions, amodest increase in the amountof fluoride ionswere released into the

solution(Table6.6).Blocksexposedtocitricacidshowedmuchhigherreleaseofions

than blocks stored in other solutions. Placebo mousse treatment produced an

increaseinfluorideionreleasecomparabletoTM+treatment,howeverthiswasonly

observed in citric acid exposed groups. TM+ treated group produced significantly

higher fluoride ion release than the “no treatment” control inF7 (p<0.05),butwas

notsignificantlydifferent fromtheplacebotreatedgroup;nosignificantdifferences

weremeasuredbetween control andboth treatments for F7EP. In all other groups

placebotreatmentshowednosignificantdifference in ion releasecomparedto“no

treatment”controlgroups.

0

200

400

600

800

1000

1200

1400

1600

HCl Citric Lactic Water

IonRelease(μM)

Solution

FujiVIIEP(Phosphate)

Not-ment

Placebo

TM+

135

Table6.6.Fluorideionrelease,n=10

6.5Discussion

Althoughnodensitometrymeasurementsweremadeduringtheexperiment,

therewasnovisiblechange involumeoftheblocks inallgroups.Measurementsof

volumechangemayshedmorelightonwherethemasslossiscomingfrom.Itmaybe

possibletocorrelatealuminiumionreleasewithvarioussourcesofaluminiuminGIC

composition.Cross-linkingaluminiumionsinthematrixifchelatedmaycauseachain

reactionleadingtogreatermassloss,whereasfilleramorphoussourcesofaluminium

ionsmayhaveafarlesspronouncedeffect.

More calcium and phosphate ions are expected to be released from F7EP

(comparedtoF7),whereadditional ionsareprovidedbyCPP-ACPincorporated into

theGIC.Similarly,TM+isasourceofadditionalcalcium,phosphateandfluorideions

due to the presence of CPP-ACFP in the mousse; these additional ions can also

contribute to higher calcium, phosphate and fluoride ion release. Ion release from

TM+treatmentofF7EPshouldbeclosetocombinedplacebotreatmentofF7EPand

TM+treatmentofF7.Thisstraightforward,additiveionreleasepatternisobservedin

lacticandhydrochloricacidgroupsforcalciumandphosphateionrelease.

136

6.5.1Watercontrol

Table6.7.Statisticalsignificanceinwater.(Hardness.Massloss.Calcium,PhosphateandFluorideionrelease.*-nostatisticalsignificance)

The control solution, water provides a baseline for the rest of the results.

ThereisaconsiderablesofteningofthesurfaceoftheGICwhenstoredinwaterfor

threedays,thesofteningismorepronouncedinF7thaninF7EP(Table6.7).Topical

treatmentwithTM+doesnotseemtohaveanyeffect(positiveornegative)onF7EP

blocks,howeverTM+treatmentofF7blocksmakesabigdifferenceandisthehighest

surfacehardnessvalueafterthreedaysofstorageoutofallwatergroups(albeitstill

3.7% lower than freshGICblocks).Placebomoussedidnot registerany statistically

significantresultssuggestingthatitisinfactCPP-ACFPspecificallythatisresponsible

forimprovedsurfacehardnessinF7andF7EPblocks,asevidencedbyhighersurface

hardnessinF7EPandimprovedsurfacehardnessinF7onlywhentreatedwithTM+.

Allgroupsstored inwatershowedaslightmass increase,somecomparisons

werestatisticallysignificanteventhoughthemagnitudeofthedifferenceswassmall,

thisispossiblyduetotheaccuracyofmassmeasurementsleadingtoverytighterror

margins.Theslight increaseinmasscouldbeduetonaturalvariancebutcouldalso

beduetotheGIC’swateruptake,especiallyintheearlystagesofmaturation16.Since

thesampleswerenotdesiccatedprior toweightmeasurements it stands to reason

that somemoisturewould remain in theGICblocks.DetailedanalysisofGICwater

uptakeisbeyondthescopeofthisstudy,thesenumbersformedthebasisascontrol

groupsfortherestofthestudy.

137

CalciumandPhosphateionreleaseinwatergroupswasverylow,oftenbelow

the detection limits of our techniques, the exceptions were groups where TM+

treatmentswereusedandthehighestreleaseofionsoccurredinthefirst24hours,

droppingoffsignificantlyinthefollowingdays.

6.5.2Lacticacid

Table6.8.Statisticalsignificanceinlacticacid.(Hardness.Massloss.Calcium,

PhosphateandFluorideionrelease.*-nostatisticalsignificance)

Hardness remained largely unchanged under the topical treatments, both

placeboandTM+,theexceptionwasplacebotreatmentforF7,whichshowedmore

softeningofthesurface,whichwassignificant(Table6.8).Thereisnoclearpatternto

explain this particular result, the difference is not great (although statistically

significant)andmaybeduetonaturalvariation.

Several groups showed statistical significance when it came to mass loss,

howevermeasurementsfellbetween0.5%and1.1%ofeachotherbetweengroups,

thissuggeststhatthedifferenceinmasslossisnotsubstantialandthesignificanceis

almostentirelyaresultofhighprecisioninabsolutemeasurements.Inmanycasesan

increaseinmasswasobserved,whichindicatesthatGIC’searlystagewateruptakeis

having a more pronounced effect than acid erosion. Competing factors of water

uptake and acid erosion make it difficult to assign significance to these results,

especiallywhenthedifferencesbetweengroupsaresosmall.

138

Therewassignificantlymorecalciumandphosphate inTM+groups forboth

F7 an F7EP compared to placebo and control treatments, presence of CPP-ACFP in

TM+ is most likely responsible for this increase, extra ions were being released

directly from topically applied TM+ as it is exposed to acidmedia and the peptide

bond is broken. Fluoride levels were affected very little, this is not only true for

topical treatments but also between different solutions tested. There was some

variationbetweengroups,butnofirmpatterncouldbeobserved.

6.5.3Hydrochloricacid

Table6.9.Statisticalsignificanceinhydrochloricacid.(Hardness.Massloss.Calcium,PhosphateandFluorideionrelease.*-nostatisticalsignificance)

The only significant result in surface hardness was detected in F7 when

treatedwithTM+,no significantdifferencewasobserved inF7EP, thismay suggest

thatCPP-ACP/ACFPishelpingtoprotectthesurfaceandthealreadypresentCPP-ACP

in F7EP is the reason no statistical significance was observed in the F7EP groups

(Table6.9).

Aswiththewaterandlacticacidgroupsthedifferencesinmasslossbetween

groups were minor, however there was a strong pattern in the HCl results that

suggestedTM+treatmentishavingaprotectiveeffect.Thisresultwasevenobserved

intheF7EPgroups,whereGICalreadycontainsCPP-ACP.

More calcium and phosphate ions were being released when F7 and F7EP

were treated with TM+. In the case of phosphate ion release from F7EP the daily

139

releasewas increasingeachday suggesting thatperhaps thepeak releasepotential

maynotbereacheduntilsometimeafterthreedays.Thiseffectwasnotobservedin

F7groupstreatedwithTM+.Fewercalciumandphosphateionswerereleasedunder

the placebo treatment for both F7 and F7EP suggesting that TM+ was having an

additiveeffectonionrelease.

6.5.4Citricacid

Table6.10.Statisticalsignificanceincitricacid.(Hardness.Massloss.Calcium,

PhosphateandFluorideionrelease.*-nostatisticalsignificance)

Surfacehardness improvedgreatlyundertopical treatments forbothF7and

F7EP. These results showed lot of variability and even though TM+ numbers were

muchhigherthantheplacebonosignificantdifferencebetweenthetwotreatments

could bemeasured (Table 6.10). The large standard error present in placebo and

TM+ results may suggest they are unreliable, however the increase in surface

hardnesswassolargethattheeffectwasnotindoubt.Sinceplacebogroupsfollowed

thesamepatternasTM+itisclearthattopicallyappliedCPP-ACFPwasnotthemain

causeofthesubstantialincreaseinsurfacehardness,butsomethingcommontoboth

topicaltreatmentsmustbethereasonfortheincrease.

Unlike theother solutions,which showed veryminor variation inmass loss,

thecitricacidtreatedGICblockssufferedfromsignificantlossofmass.Undertopical

treatments this mass loss increased. Placebo and TM+ treatments were both

significantly different from the control (untreated) groups and were very close to

140

eachother.StatisticalsignificancewasalsodetectedbetweentheplaceboandTM+

treatments.

More calcium and phosphate ions were released by the F7EP groups

comparedtoF7,whichcanbeattributedtoCPP-ACPpresentinF7EP.However,TM+

treated groups released significantly less calcium and phosphate than the placebo

treated groups for all groups (F7 and F7EP). In other acid groups TM+ application

showedthatCPP-ACP/ACFPhasanadditiveeffecton ionrelease,morecalciumand

phosphateionswerereleasedfromF7EPthanF7andthiswasfurtherelevatedunder

TM+ treatment. Furthermore, placebo treatment in the other acids did not

demonstrateaconsistentincreaseinionrelease,thiswascompletelytheoppositeof

the citric acid measurements. It stands to reason that an ingredient, possibly

glycerine,commontoboththeplaceboandTM+wasinteractingwiththecitricacid

toproducehighionrelease,increasedmasslossandhardeningofthesurface.Slightly

lowermasslossandlower(althoughstillincreasedrelativetothecontrol)ionrelease

of TM+ compared to placebo may indicate that CPP-ACFP was inhibiting the

interactionbetweenthecitricacidandglycerine.

6.5.5Surfacehardness

Exposure to any liquid storage medium has a softening effect on the GIC

surface,asdemonstratedbythereductionofVickershardnessinwaterforallgroups.

TheprotocolfollowedwasexactlythesameasinthatdeterminedinChapter5.The

results of repeated measurements were in good agreement with the previously

publisheddata7.

Resultsfromthecontrolgroupindicatedthatreductioninsurfacehardnessis

stronglyassociatedwiththepHoftheacidsolution.However,thecitricacidgroups

treatedwith TM+ and placebomousse behaved very differently. Topical treatment

witheitherTM+ortheplacebomoussenotonlyshowedreductioninthesofteningof

thesurface,butalsoanincreaseinhardnesscomparedtoinitialmeasurementsprior

to topical treatmentor acid challenge. The increase inmass loss forblocks treated

141

with TM+ and placebo suggests that more ions were displaced as a result of the

topicaltreatments.Someofthesedisplacedionsmayhavemigratedtothesurfaceof

thespecimenwherethey formedazoneofsaturationandthusprecipitatedonthe

surfacetoformacrust.Inaddition,thetopicaltreatmentitselfmostlikelyleftafilm

onthesurface,whichcouldhaveformedanidus forother ionstoprecipitateonto.

CPP-ACPhasademonstratedabilityof localisingcalciumandphosphateionsonthe

tooth surface 8, 9, consequently formation of a surface crust-like layer seems quite

possible andwould explain the dramatic increase in surface hardness amongst the

citricacidexposedgroups.

Onlyinafewcasesforthelacticandhydrochloricacidexposedgroupswere

thereanystatisticallysignificantchangesinsurfacehardness.InoneF7group(lactic

acid) the placebo treatment was associated with softening of the surface, also in

lactic acid, but in the F7EP blocks treated with TM+ showed a slight reversal in

softening.

6.5.6Changeinmass

Baselinemeasurements from the groups stored inwater indicated that the

GICsused in this experiment tended to swell and retain aportionof liquid storage

media. This early stage water uptake was the most likely reason of the modest

softening of the surface ofmanyGICs andhas been reported in the past 13. Tooth

moussetreatedGICblocksstoredinlacticandhydrochloricacidsweremoreresistant

to dissolution and exhibited lowermass loss compared to the control and placebo

treatments,F7EPalsoperformedbetterthanF7inthisregard.Althoughstatistically

significant,thesedifferenceswereverysmall.

Significant loss of mass in the citric acid exposed blocks with no topical

treatment has already been reported7, and interestingly the mass loss was even

higherforthetopicallytreatedgroups.Thepresenceofahardsurfacelayersuggests

that any loss of ions due to erosion would be inhibited, it would seem that the

mechanism for increased mass loss was slightly different in the topically treated

142

groups.F7showedaslightlygreatermasslossthanF7EP,whichmaysuggestthatthe

CPP-ACPinthecementactsasabufferingagent.However,thegreatestdailylossof

massalmostalwaysoccurredonthethird(last)dayofmeasurement,indicatingthat

theprocessseemedtobeaccelerating.ThissuggeststhatthetopicallyappliedCPP-

ACPwasnothavingthesamebufferingeffectastheCPP-ACPalreadypresentwithin

theGIC(F7EP).

6.5.7IonRelease

In TM+ treated groups exposed to citric acid both F7 and F7EP showed a

reduction in phosphate ion release, a steady calcium ion release and an increased

fluorideionrelease.Atthesametimeanincreasedlossofmasswasalsoobserved.In

thecaseof theplacebotreatedgroupsexposedtocitricacid, increasedphosphate,

calciumandfluoridereleasewasmeasuredforbothGICs,accompaniedbythelargest

measuredlossofmassamongstallgroups.Asdemonstratedbytheplacebomousse

results,applicationofthetopicaltreatmentitselfintroducedaninteractionbetween

theacidstoragemediaandtheGIC. Insomecasestheionreleasewasgreaterthan

thatwhichwouldbeexpectedfromtheTM+alone,whichitselfhasadditionalionsin

theformofCPP-ACFP.Understandingofadditionalmechanisms,suchasformationof

acrust surface layerandchelationofaluminium ionsmayhelp toexplain the large

differencesobservedbetweenthecontrol,placeboandTM+groups.Thepresenceof

CPP-ACFPinTM+seemstoprovidesomemitigationagainstmasslosscomparedwith

theplacebotreatment.Additionalcalcium,phosphateandfluoride ionsprovidedby

the TM+ itself were hard to distinguish from those released from the GIC as the

additional buffering capacity produced by TM+ ions could potentially inhibit these

sameionsbeingreleasedfromtheGIC,especiallyinthecaseofF7EP.

143

6.5.8GeneralDiscussion

Itwouldseemalotof ionicmovementisoccurringasevidencedbythehigh

mass loss and ion release,more so after the topical treatments, wheremore ions

werenotonlybeingreleasedintostoragesolutionsbutalsobeingdepositedonthe

GIC surface. This indicates that surface erosion of GIC may not be the main

mechanismcontributing to increased ion release. It ismore likely that the inherent

porosity of the GIC plays a key role in facilitating high ion release. Previous

speculationsuggestedthatchelationofaluminiumionsincitricacidexposedgroups

may be responsible for the large phosphate ion release observed in the untreated

control F7andF7EPblocks. Inmany cases, thehighestdailyphosphate ion release

and mass loss were observed on the last day of measurements, which not only

suggests that the processmay not have reached its peak, but also theremaybe a

stronglinkbetweenphosphateionreleaseandmassloss.Apossibleexplanationfor

increasedlossofmassandincreasedsurfacehardnesscouldbethatcitricacidhasan

inherent ability to chelate aluminium from the GIC matrix producing an ion rich

environment that when coupledwith topical treatments of either TM+ or placebo

mousseresultedintheformationofacrust-likesurfacelayer.Inturn,thepresenceof

precipitation in the surface crust layer would produce a solubility concentration

gradient that increases from the subsurface inwardly towards the bulk of the GIC,

allowing citric acid to penetrate deeper into the GIC thus chelating additional

aluminium as the acid continues to diffuse through the cement. Extra ions are

believed to then increase the thickness and strength of the crust-like surface layer

that in turn improves the resistance to dissolution, but results in deeper acid

penetration intotheGIC.Thedeeper ingressof theacidcanthen leadto increased

lossofmasswithinthebulkofthecementbutisassociatedwithanincreasedsurface

hardnessthatexceedsthehardnessof freshGICs.Thisprocess is thoughttobenot

unliketheprocessofanactivecariouslesioninteeth,wherecalcium,phosphateand

fluoride ionsaredisplaceddue to solubilityof fluorapatite in thepresenceof lactic

acid creating a concentration gradient, which promotes the inward progression of

144

carious lesions17, but is also associatedwith the initialmaintenance of the surface

integrity of the enamel. It would be interesting to perhaps extend the length of

exposureoftheGICinacidtonotewhetherthesurfaceeventuallydissolvestoforma

pitorcavity.

Clinical evidence suggests that conventional GIC restorations have poor

retention rates and suffer from high wear rates when placed on load bearing

surfaces18. A lot of these drawbacks have been addressedwith the introduction of

resin-modifiedGICs,howeversustainedhighfluoridereleaseissomethingthatresin-

modified GICs are not currently able to achieve. High mass loss is certainly an

undesirable property to have in a restorative material, but in this case it is also

accompaniedbyincreasedcalciumandphosphateionrelease.Thesecombinationsof

drawbacksandbenefitshavecreatedaspecial role forGICswithin thespectrumof

restorativematerials,particularly intheareasofcariesprevention.However, itmay

be possible to reduce some of these limitations if interactions between citric acid,

topicaltreatmentsandGICmaterialsarebetterunderstood.Greatercontroloverion

release and potentially higher “on-demand” ion releasemay be possible through a

betterunderstandingofthemechanismsthatcause lossofmassandaccompanying

ion release in the cements. This may provide great benefit in cases where high

erosionoftoothmineralispresentandmaybecounteractedbyincreasedionrelease

fromtheGIC,whichmaybepreferentiallylostratherthantoothstructure.

The null hypotheses tested, it was found that; 1) topical treatment using

placebomousse increasedsurfacehardness, causedhighermass lossandproduced

increasedcalciumandphosphate ionrelease forF7andF7EPblocksstored incitric

acidcomparedtountreatedblocks,noconsistentsignificantdifferencewasobserved

inotherstoragemedia;2) topical treatmentusingTM+ increasedsurfacehardness,

causedhighermasslossandproducedincreasedcalciumandphosphateionrelease

forF7andF7EPblocksstoredincitricacidcomparedto(a)untreatedblocksand(b)

showedsignificantdifferenceswhencomparedtoblocksalsostoredincitricacidbut

treated with placebo mousse. A significant increase in calcium and phosphate ion

145

releasewasalsoobservedinTM+treatedgroupsinothersolutionswhencompared

tountreatedgroups.

6.6Conclusion

ToothMoussePlusandplacebomoussetopical treatmentshaveshownthat

the topical treatment itself has a very pronounced effect on the GICs tested (F7,

F7EP).Inthecaseofplacebomousse,additionalphosphate,calciumandfluorideions

werereleased incitricacid fromtheGICat theexpenseofsignificantmass loss,no

additionalionswerereleasedinthelacticandhydrochloricacidchallengeshowever,

nosignificantmass losswasobservedeither.ToothMoussePlus treatedGICblocks

showed increased calcium, phosphate and fluoride ion release in lactic and

hydrochloricacids,aswellas increasedcalciumandphosphate ionrelease inwater

withnoadditionaladverseeffects. Incitricacid,ToothMoussePlus showedsimilar

resultstotheplacebomousse,withCPP-ACPprovidingsomemitigationagainstmass

loss and increased ion release compared to the “no treatment” control. Further

research into the importance of aluminium compounds present in the GICs may

explain the vulnerability of fluoroaluminosilicate glassmaterials to chelating agents

suchascitricacid.

146

6.7References



39(6):339-343.






Research90(1):99-103.









Research108(3):167-182.

7. Zalizniak I, Palamara JEA,WongRHK,CochraneNJ,BurrowMF,ReynoldsEC

(2013). Ion releaseandphysical propertiesofCPP–ACPmodifiedGIC in acid

solutions.JournalofDentistry41(5):449-454.

147







10. CrossKJ,HuqNL,StantonDP,SumM,ReynoldsEC(2004).NMRstudiesofa

novel calcium, phosphate and fluoride delivery vehicle-alpha(S1)-casein(59-

79) by stabilized amorphous calcium fluoride phosphate nanocomplexes.

Biomaterials25(20):5061-5069.






115(3):407-410.







148



16. Cattani-LorenteMA,Dupuis V, Payan J,Moya F,Meyer JM (1999). Effect of

water on the physical properties of resin-modified glass ionomer cements.

DentalMaterials15(1):71-78.

17. FeatherstoneJD,DuncanJF,CutressTW(1979).Amechanismfordentalcaries

basedonchemicalprocessesanddiffusionphenomenaduring in-vitrocaries

simulationonhumantoothenamel.ArchivesofOralBiology24(2):101-112.

18. YipHK,SmalesRJ(2002).Glassionomercementsusedasfissuresealantswith

the atraumatic restorative treatment (ART) approach: review of literature.

InternationalDentalJournal52(2):67-70.

150

CHAPTER7

Aluminiumionreleaseof

conventionalGICsandGiomer

materialsinacidsolutions


RecentadvancesintheunderstandingoftheinteractionbetweenGICmaterialsand

acidic environments have shed new light on the clinically encountered problem of

erosion of GIC restorations.Objectives: The aim of this studywas to evaluate the

importanceofaluminiumionsintheGICmatrixstructureanditsroleinbeneficialion

releasefromGICsandGiomermaterials.Methods:Fortyblocksof,FujiVII™(F7),Fuji

VII™EP (F7EP) and ShofuBeautifil (SBF03)were subjected to threeacidic solutions

(50mMlacticandcitricacidsatpH5.0,50mMhydrochloricacidatpH2.0)andwater

(pH 6.9) over a three day period. Ion release, surface hardness and weight

measurements were carried out every 24 hours. Results: A significantly higher

151

(p<0.05) mass loss and ion release were recorded for conventional GICs when

exposed to citric acid compared to theGiomermaterial. SBF03 showed substantial

fluorideionreleaseinthefirst24hourscomparedtofollowingdays(p<0.05).F7and

F7EP showed significantly higher daily fluoride ion release than SBF03 in all tested

acids (p<0.05). Conclusion: Results show that pre-reacted glass additives in SBF03

produce substantial fluoride release in the first24hoursonly.Whenexamining ion

release profiles from SBF03, F7 and F7EP each material has a different source of

fluoride ion release. Differences in ion release profiles are strongly linked to

aluminiumionsandaluminiumcompoundsfoundinglassparticles.

7.2Introduction

Glass ionomercements(GICs)arewidelyusedforavarietyofpurposessuch

asforinterimrestorations,cariesstabilization,definitiverestorationofmicro-cavities

or non-carious cervical lesions, bonding orthodontic brackets and bands, fissure

sealing eruptingmolars and as a surface protectingmaterial for high risk surfaces

such as root surfaces 1, 2. The main advantages of GICs are their strong ability to

chemicallybindtodentineandtheirabilitytoreleasefluorideions.Thisfluorideion

release is believed to slow the progression and aid the regression of early carious

lesions3.Thetoothsurfacewillonlydemineralisewhenthefluidbathingthetoothis

under-saturatedwithrespecttothetoothmineral (fluoride,calciumandphosphate

ions),whichiswhyionactivityatthetoothsurfaceisanimportanttopictostudy.

Anumberofinvestigatorshaveexploredthemodificationofdentalmaterials

inattempttohavethemreleasecalcium,phosphateandfluorideions4-6.Skrticetal6

exploredmodifyingdentalcompositeswithbioactiveglasses.Mazzaouietal4andAl

Zraikat et al5 assessed the addition of casein phosphopeptide amorphous calcium

phosphate (CPP-ACP) to GICs. The casein phosphopeptides stabilise calcium and

phosphateionsinabioavailableformthatallowthemtoinhibitdemineralisationand

promotetheremineralisationofearlylesions7.CPP-ACPisstableinthepresenceof

152

fluorideandhasbeenshowntoworksynergisticallywithfluoride8.ThismakesCPP-

ACPapromisingadditivetodentalproductsandrestorativematerials.Thestudyby

Mazzaouietal.(2003)wasaproofofconceptstudythatshowedthatCPP-ACPcould

be added to GIC. Al Zraikat et al. (2011) later explored the effect of CPP-ACP

concentration on GIC mechanical properties. Additionally, both of these studies

showedthattheadditionofCPP-ACPimprovedcalciumandphosphateionreleasein

lacticacid.

Thetoothsurfacecanbeexposedtoavarietyofdemineralizationchallenges

including: acids formed from metabolic processes of oral bacteria (predominantly

lacticacid);foodacidssuchascitricandphosphoricacidthatarecommonlyfoundin

softdrinks9;andhydrochloricacidfromtheregurgitationofstomachacids. Ifthese

acidsoverwhelm theprotective functionsof saliva,mineralmaybe lost from tooth

surfaces 10. Recently, a closer investigation of the effects of various acids on the

physicalpropertiesofGICshasrevealedthatcommonfoodacids,suchascitricacid,

havea farmorepronounced impactonGICs than lacticacid 11.A significant loss in

massofGICblocksexposed toa citricacid challengewas foundcompared to lactic

acid. Thiswasalsoaccompaniedbyan increased releaseof calciumandphosphate

ions.Early indicationssuggest thatanabnormallyhigh releaseofphosphate ions in

citricacidmaybetheresultofchelationofaluminiumions.

Aluminium is contained in GICs in four compounds - aluminium fluoride,

cryolyte (sodium aluminium fluoride), aluminium oxide, and aluminium phosphate

(Table 7.1). During the initial mixing between the glass powder and acid some

aluminium ions are leached from the glass and replace hydrogen ions in carboxyl

groups in the acid, forming salts12. A lot of the aluminium ions remain in the glass

particlesanddonotparticipateinthesettingreaction.Theinitialratioofaluminium

and silica controls the rate of the setting reaction through the gelation and cross-

linkingphase,sopresenceofextraaluminiumionsisanimportantconsiderationfor

the workability of the mix12. In this study it was aimed to better understand the

mechanismsthatproducesuchanunexpectedlylargereleaseofionsandlossofmass

inconventionalGICsandcomparethesefindingsagainstresincompositecontaining

153

pre-reactedglassionomerfillersreferredtoasaGiomer,whichtheoreticallyisnotas

susceptibletochelationbyexposuretocitricacid.

Powder weightpercent

Cryolite(Na3AlF6) 5%

AlF3 5%

SrF2orCaF2 22-34%

AlPO4 10%

Al2O3 16%

Table7.1.GeneralcompositionofGICs.SiO2makesuptherestupto100%.

AcomparisonbetweenaconventionalGICandaGiomerrestorativematerial

(Shofu Beautifil) is presented. Giomers are resin-based tooth coloured restorative

materialsthatcontainpre-reactedglassparticles.Theglassparticlesareincludedto

facilitate fluoride ion release, enhancing the materials ‘therapeutic’ benefits.

However, being a resin-based material the Giomer should be less susceptible to

chelationcausedbyleachingofaluminiumions.Thenullhypothesisis;a)Thereisno

difference in fluoride ion release between F7, F7EP (a conventional GIC containing

CPP-ACP) and SBF03 (a resin-based material containing pre-reacted, fluoride

containing, glass elements); and b) Aluminium ions are not related to fluoride ion

releaseineitherF7,F7EPorSBF03.

154


Two conventional glass ionomer cements, Fuji VII and Fuji VII EP, (GC

Corporation Tokyo, Japan, F7 and F7EP) in capsule form, and a pre-reacted glass

particle filled resin composite, Beautifil F03 (Shofu Inc., Kyoto, Japan, SBF03)were

usedinthisstudy.Mouldsmadeofapolyvinylsiloxaneimpressionmaterial(eliteHD+

lightbody,ZhermackSpA,BadiaPolesine,Italy)wereusedtocreatestandardisedGIC

andGiomerblocksmeasuring3mmx6mmx6mm(thicknessxwidthxlength).Forty

blocksofeachmaterialwerepreparedbyplacingtheGICorGiomerintothemould

withthetopandbottomsurfacescoveredbyplasticstrips,whichwasheldbetween

twoglassslides.Theglassslidesweregentlypressedtogethertoextrudeanyexcess

material, SBF03 samples were light-cured for 10 sec on twomajor surfaces of the

block using and LED light curing unit (bluephase C8, Ivoclar Vivadent AG,

Liechtenstein).Allspecimenswereallowedtosetinsidethemouldsfor24hoursinan

incubator (37°C, ~100% relative humidity). After cooling to room temperature the

blockswere removed from themoulds, and lappedwithwet 600-gritwet and dry

silicon carbide paper (Norton Tufbak, Saint-Gobain Abrasives Ltd., Auckland, NZ)

ensuringspecimensdidnotbecomedehydrated.

Four different solutionswere prepared to expose the blocks to a variety of

acidic and neutral environments. The three acidic solutions were formulated to



(50mMlacticacidatpH5.0)(thisconcentrationwasselectedbasedonvaluesfound

previouslyinplaquefluid13).TheneutralsolutionwasdistilleddeionisedwateratpH

6.9(MilliQ,MilliporeCorporation,Victoria,Australia).

Tenblocksofeacheachmaterial,namelyGIC,GICcontainingCPP-ACPandthe

Giomer were exposed to 5mL of each of the four solutions (stored in plastic

containers). The solutions were changed every 24 hours and the samples were

measuredforchange inmassandsurfacehardness.Thesolutionswereanalysedto

determinereleaseofaluminium,calcium,phosphateandfluorideions.

155


measurementswereperformed.Blocksweretakenoutofthesolutionpatdriedand

then weighed using an analytical microbalance (Precisa XT 120A, Dietikon,

Switzerland).Onemeasurementpersamplewasperformedwithanerrorof±0.1mg.


the lapped GIC surface using a microhardness tester (MHT-10, Anton Paar GmbH,



s; Rate, 0.99 N/min) at each time interval. A distance of least three times the

indentation size separated the indentations and both of themajor surfaces of the

blockwereusedforindentation.Imagesoftheindentationswereacquiredthrougha

calibrated digital camera (Leica DFC320)mounted on themicroscope. Diagonals of

the indent were measured using Image Tool software (Version 3.0, UTHSC, San

Antonio, TX) that were converted into Vickers hardness values. Blocks were then

placedintofreshbatchofsolutionandreturnedtotheincubator.

Ionreleaseofcalcium,aluminium,phosphateandfluorideaftereach24-hour

periodofstorageweredeterminedusingatomicabsorptionspectroscopy(forcalcium

and aluminium), colourimetry and an ion-specific electrode respectively. To

determine the calcium concentration sample solutions (0.5 mL) were diluted with

water(0.5mL,MilliQ),werethenacidifiedwith1MHCl(0.5mL)anddilutedwith2%

lanthanum chloride (0.5 mL) and analysed on a Varian AA240 atomic absorption

spectroscope(AAS,VarianAustraliaPty.Ltd.)againstasetofsevenstandardsranging

from0 to250µMcalcium. TheVarianAA240AASwas alsoused todetermine the

concentration of aluminium ions. Sample solutions (0.5 mL) were diluted with

potassium chloride (0.5mL, 8mg/L) and lanthanum chloride (0.5mL, 2%w/w) and

were then acidified by 1M HCl (0.5 mL) and compared against a set of standards

rangingfrom0to125µMaluminium. Inorganicphosphate ionconcentrationswere

determined colourimetrically using a spectrophotometer (UV-visible

spectrophotometer,VarianAustralia,Pty.Ltd).Thesamplesthatwereanalysedwere

prepared by taking 100 µL of solution, diluting with 500 µL of 4.2% ammonium

156

molybdateandadding20µL1.5%ofTween®20.(Sigma-Aldrich,St.Louis,MO).The

phosphate ionconcentrationwasdeterminedbycomparing thespectrophotometer

readings of the samples against a set of seven standards ranging in phosphate

concentrations from 0 and 100 µM. The concentration of fluoride ions was

determined using an ion-selective electrode (Radiometer analytical, ISE C301F,

France)connectedtoan ionanalyser (Radiometeranalytical, IonCheck45,France).

Samplesolutions(1mL)weredilutedwith1mLtotalionicstrengthadjustmentbuffer

(TISAB,Merck Pty Ltd, Kilsyth, VIC, Australia) andmeasured against a set of eight

fluoridestandardsrangingfrom0to1000µM.

Data fromsamplegroupswere foundto followanormaldistribution.Theχ2

testwasusedtotestnormalityofthedata.SinglefactorANOVAwasusedtoanalyse

theresultsusingBonferroni-Holmmultiplecomparison.Two-wayANOVAwasusedto

determine the interaction between incorporation of CPP-ACP and exposure to

differentacids.Thelevelofsignificancewassetatα=0.05.

7.4Results


Surface hardness for SBF03 samples was initially higher than F7 and F7EP,

however incitricacid,hydrochloricacidandwaterthepercentagedrop inhardness

washighercomparedtoF7andF7EP(Table7.2).Incitricacidtheabsolutehardness

remainedhigherthanF7andF7EP,butinwatertheabsolutehardnessofSBF03was

lowerthanF7EPandhigherthanF7.TheabsolutehardnessofSBF03inHClwaslower

thanbothF7andF7EPfollowingthethree-dayacidchallenge.Lacticacidwastheonly

group where SBF03 showed lower percentage drop and higher absolute hardness

afterthreedayscomparedtoF7andF7EP.

157

Table7.2.Surfacehardnesscomparison(VH1.0).Absolutevaluesinbold,%dropsinsecondrow.

7.4.2MassLoss

Shofu Beautifil F03 showed no significant mass loss in all solutions tested.

After three days there was some change (increase and decrease depending on

group),butnomorethan0.5%inallgroups(Table7.3).BeautifilF03blocksstoredin

watershowedsomevariabilityinresults,whichmayindicatesomewaterabsorption.

F7 and F7EP experienced significant mass loss over three days; depending on the

solutionthelossofmasswassubstantialandsignificantasshowninChapters5and6.

158

Table7.3.DailymasslossofSBF03infourstoragesolutions.Meanvaluesingrams.

7.4.3IonRelease

Fluoride ion release comparison between F7 and F7EP has been described

previouslyinChapter5,inmanygroupsnostatisticalsignificancecouldbemeasured,

forthepurposesoffurthercomparisononlyF7EPwillbeused.Fluorideionreleasein

F7EPwasmeasuredtobeconsistentoverthe3daysofthestudyforeachsolution.

Theacidicsolutionsshowedsignificantlyhigher(p<0.05)fluorideionreleasethanthe

control(water).FluorideionreleasefromSBF03wassignificantlyhigher(p<0.05)on

thefirstdaythanthefollowingdays,butwassignificantly lower(p<0.05)thanF7EP

foreachdayinallsolutions(Figure7.1).

159

Figure7.1.ComparisonofFluorideIonReleaseinFujiVIIEPandShofuBeautifilF03.

Asimilarpatternwasobservedforthedailyreleaseprofileofaluminiumions

and fluoride ions in SBF03 (Figure 7.2). A similar patternwas observed for F7 and

F7EP(Chapters5and6),howeverforF7/F7EPitwasaluminiumandphosphateions

thatsharedsimilardailyreleaseprofiles(Figure7.3).

160

Figure7.2.RelationshipbetweendailyaluminiumandfluorideionsreleasedfromSBF03.

Figure7.3.RelationshipbetweendailyaluminiumandphosphateionsreleasedfromF7EP.

161

7.5Discussion


InitialsurfacehardnessofF7andF7EPwasverysimilar,SBF03onaveragehad

a 28.7%higher initial surfacehardness than F7 and F7EP. Thehigher initial surface

hardness of SBF03 did not always translate into higher surface hardness after the

three-day acid challenge. Absolute values are ultimatelymore important, however

percentagedropsinhardnessindicatehowamaterialcanpotentiallyperformovera

longer period of time if the acid challenge was extended beyond the three-day

duration. SBF03 was especially vulnerable to hydrochloric acid, where the drop in

hardnesswaslargestoutofallstoragesolutions,converselyF7andF7EPperformed

best when exposed to hydrochloric acid compared to other acids tested. When

lookingatsurfacehardness,citricandlacticacidshadasimilareffectonallmaterials

tested.

7.5.2Changeinmass

The results fromChapters 5 and 6 showed F7/F7EP underwent a significant

mass loss,particularlywhenexposed to citric acid.Ahighphosphate ion release in

thehighmasslossgroupssuggestedthataluminiumchelationmightbethecausefor

the high loss of mass; this hypothesis is further supported in that aluminium ion

releasehasnowbeenmeasureddirectly.It isworthnotingthatalthoughthelossin

masswassignificanttherewasnoobviousvisiblereductioninthesizeoftheblocks.

This needs to be investigated further. No significant mass loss was measured in

Giomer groups. However, in the F7/F7EP groups the high mass loss was always

accompaniedbyhighionrelease.

162

7.5.3IonRelease

TheGiomermaterialshowedsignificantlylowerfluorideionreleasecompared

toF7EP in the first24hours,and fluoride ion release significantly reducedafter24

hours(p<0.05).AluminiumionreleaseinShofuBeautifilcloselyfollowedfluorideion

release, which is different to the pattern observed in F7EP. The GIC materials

demonstratedasteadyfluoride ionreleaseover3days,whichwasalsohigherthan

that of Beautifil, and aluminium ion release also closely followed phosphate ion

release (Chapters 5 and 6). This may indicate that, while Beautifil contains glass

particles, the source of fluoride ions may be different for eachmaterial. Table 7.I

shows thepossible sourcesofaluminium, fluorideandphosphate ionscontained in

theGICmaterials.InconventionalGICs,cross-linkingaluminiumionsarethesourceof

secondarysettingreactionfollowingthe initialsettingreaction involvingcalcium(or

strontium)ions.Thesecondarysettingreactioninvolvingaluminiumionsproceedsat

amuchslowerrateovermanydaysorevenweeks(dependingonmaterial)12.AsF7EP

issubjectedtoacidchallengethesettingreactionisstillongoingandaluminiumions

are not as tightly bound as in the matrix of a completely matured material. This

creates many potential sources of aluminium ions in F7EP, particularly under

chelation of citric acid,which results in higher phosphate and fluoride ion release.

Beautifil contains pre-reacted glass particles that containmostly bound aluminium,

phosphate and fluoride compounds. The most substantial release of fluoride and

aluminium ions observed in Beautifil occurred in the first 24 hours. However, the

greatest ion releasemayhavebeenduringa shorterperiodof timewithin the first

24-hourmeasurement period. There are twopossible reasons for this pattern. The

initial burst release of fluoride and aluminium ions is from the accessible surface

regionsof thematerial.Compared toGICs, resin-basedmaterialsarenotasporous

and permeable. GICs are able to sustain a higher fluoride release over time as

additional ions are released from sub-surface regions; this compromises the

structuralintegrityoftheGICmaterialsasevidencedbythemasslossdataandhigh

ionreleaseon the lastdayofmeasurement.Anotherpossibleexplanationcouldbe

163

thatthesealuminiumandfluorideions,containedinpre-reactedglassfillerparticles,

are excess ions that have not taken part in the initial setting reaction of the glass.

Theseionswouldnotbetightlyboundandconsequentlycouldbewashedoutofthe

materialmuchmoreeasily thanmore tightlybound ions.Thesedifferencesmaybe

the reason behind the different fluoride ion release profiles between F7EP and

Beautifiloverthe3-dayperiod.

The release of aluminium ions has emerged as an important piece in

understandinghowotherbeneficialionsarestoredandreleasedfromGICsandGIC-

basedmaterials.Whenweconsideracidchallengefromnotonlylacticacidbutalso

other acids encountered within the oral environment, such as citric acid the

mechanismsinvolvedinthe ionreleasecanbeestablished.Theevidence isbuilding

thatpoints to thechelationofaluminium ionsbycitricacidas themain reason for

highmasslossinF7andF7EP(Chapters5and6),aswellastheformationofa‘crust-

like’ surface layeronF7andF7EP samples following topical treatments.Aluminium

ions facilitate better cross-linking of theGICmatrix and take part in the long-term

maturingprocessoftheGIC,howevertheyarealsopresentinmanyfillercompounds

withintheGICstructure.GiventhehighmasslossandAL3+andPO4-ionreleasedata

the evidence suggests that it is not only the filler aluminium ions that are being

chelated but also the cross-linking ions. This is of great importance, however the

exactmechanisms that cause this are not clear, someparameters havebeen ruled

out as potentially contributing factors. For example, lowpH (HCl at pHof 2.0)was

found to not be a major factor in high dissolution of GIC structure. Also, topical

treatmentofGICsurfacedidnotalwaysprovideabenefit,andinsomespecificcases

reducedtheperformanceoftheGICmaterial.Theseunknowninteractionsaswellas

thephysicalevidencepresented in relation toaluminium ion releasemerits further

study.

164

7.6Conclusion

FluorideionreleasewasfoundtobesignificantlyhigherinF7EPcomparedto

Beautifil. Aluminium ions have a strong effect on ion release, especially when the

materialsareexposedtocitricacid.Aluminiumionreleaseholdsastrongcorrelation

withthereleaseofotherions,fluorideforBeautifilandphosphateforF7EP.Foreach

materialthesourceoffluorideionsisdifferent,whichcanbeattributedtoF7EPstill

being in a non-matured state andBeautifil containing elements frommaturedpre-

reactedglass.

165

7.7References



39(6):339-343.






Research90(1):99-103.









Research108(3):167-182.




166









115(3):407-410.

11. Zalizniak I, Palamara JEA,WongRHK,CochraneNJ,BurrowMF,ReynoldsEC

(2013). Ion releaseandphysical propertiesofCPP–ACPmodifiedGIC in acid

solutions.JournalofDentistry41(5):449-454.






167

CHAPTER8

Discussion

8.1Pilotstudyoutcomes

Fromtheverystarttheintentionwastoonlyuselacticacidasitisthemajor

acid associated with the formation of carious lesions1, however difficulties were

encountered with the measuring equipment, so alternative options needed to be

evaluated. Conflicts arose when using lactic acid andmeasurement of fluoride ion

release.Thepilotworkfocusedondeterminingifadifferentacidcouldbeused(citric

acidinsteadoflacticacid),howeveritwasfoundcitricacidcausedcontaminationof

theequipmentandhencewasalsodeemedincompatible.Thecitricacidpilotstudy

revealedsomeinterestingfindings(highphosphateionreleaseandhighermassloss

compared to lactic acid). A differentmeasuring setup needed to be used, but the

citricacidpilotresultswarrantedfurtherinvestigationduetothedifferentoutcomes,

thisledtocitricacidbeingincludedinthefinaltestingprotocol.Itbecameapparent

thatwhenimmersedindifferentacids,evenatthesamepHandionicstrength,the

GICsseemedtoreactinverydifferentways.Consequently,hydrochloricacidwasalso

168

addedtotheprotocol,eventhoughtherewerenoinitialdatatosuggestthatitwould

produce different results. After acid challenge solutions were established the

remainderofthepilotstudieswereabletoproceed.

Microhardnesspilotworkfocusedondeterminingthecorrectcombinationof

test parameters such that upper and lower hardness values could be measured

withoutadjustingtheequipment.Theuppervalueswereexpectedatthestartwhen

GIC blocks were fresh, shortly after the initial set and not yet exposed to acid

solution.Afterthreedaysofstoragehowever,itwasexpectedthatthesurfaceofthe

materialwoulddeteriorateandsoftenleadingtolowersurfacehardnessvalues.The

pilottestsfocusedonthetwoextremehardnessparameters.

Ionreleaseassayswereinitiallycarriedoutonsamplesstoredin15mLofacid

solution, this volume was found to be too large as some readings fell below the

detection limitsof theanalyticalapparatusused.Thepilotwas re-runusing5mLof

solution,which produced an improvement in accuracy for the low reading groups,

however, the high reading groups required dilution in some instances. This was

established at the pilot stage and carried throughout the remainder of the

experiments.Themicrohardnesspilotwasalso re-runwith5mLstoragevolumes to

determine if a smaller volume of acidwas adequate for consistentmeasurements.

Only in a few extreme cases did the magnification of the microscope need to be

altered(andthosereadingswerere-calibratedappropriately).

8.2Acidchallengeoutcomes

Themain focus of these studies beganwith testing the response of F7 and

F7EPGICstoa3-dayacidchallenge.Theresultsshowedthatsubstantially increased

calcium,phosphateand fluoride ion releasewasobserved inF7EPcompared toF7.

The increasewasattributed to thepresenceofCPP-ACP inF7EP.Statisticalanalysis

showedthatchangestophysicalproperties(surfacehardness,massloss)wereeither

notsignificantorshowedanimprovementforF7EPincomparisontoF7.Masslossin

169

citricacidwasunexpectedlyhighandwasobservedforbothF7andF7EP.Statistical

analysis could not attribute this effect to CPP-ACP, consequently no further

investigationwasconductedatthetimeasthefocusofthestudywasontheeffectof

CPP-ACP.However,thecitricacidgroupsalsoshowedamuchhigherphosphateion

releasethantheotheracidsolutions.

Itbecameevidentthat,atleast,conventionalGICsresponddifferentlytothe

differentacidsthatarereadilyfoundintheoralenvironment.Priortothisstudythe

mainfocusofresearchhasalwaysbeenonlacticacidasthechiefcausativeagentin

cariogenic activity1, 2. Lactic acid has proven to be an appropriate solution to use

when studying the effect of caries initiation in teeth under laboratory conditions.

However,when investigating its effect on restorativematerials itmayonly provide

partof thepicture.Citric acid ispresent inmanyprocessed foodsand itseffecton

GICsisthereforeofgreatinterest.

8.3Topicaltreatmentoutcomes

Building on the knowledge of the benefits of CPP-ACP a further study

investigating whether topically applied CPP-ACFP (using TM+) could impart its

benefits onto conventional GICs by enhancing calcium, phosphate and fluoride ion

storageandrelease, inamannersimilar toworkdone in thepast to ‘recharge’ the

fluoridecontentofGICbytopicalfluorideapplications.Thestudywasdesignedwith

several control groups; essentially, each variable had its own control material,

solutionortreatment.Placebomoussewasusedasthecontroltreatment,waterwas

usedasthecontrolsolutionandF7wasthecontrolmaterialforF7EP.Inadditionto

placebo mousse served as a control treatment, the previous study’s results (no

treatment)werealsousedasafurthercontrolgroup.

ThepresenceofCPP-ACPinTM+showedanincreaseinionrelease,reduction

inmasslossandincreaseinsurfacehardnesswhenusedtotreatF7blockscompared

to placebo mousse treatment. However, data also showed, with a high level of

170

statisticalsignificance,thatmasslosscomparedtothe‘no-treatment’controlgroups

wasmuchhigherandsurfacehardnessincreasedbeyonditsinitialvalues.Phosphate

ion release also increased from its already elevated levels observed in the ‘no-

treatment’ groups of the previous study. The hypothesis that aluminium chelation

wasthemaincauseoftheobservationshadgainedagreatersignificance.Theresults

werenotonlyunusual forexposure tocitricacidcomparedtootheracids,butalso

carriedimportantimplicationsofhowGICspotentiallyreacttovariousacidchallenges

inclinicalsituations.

Thefirststagewastoinvestigatetheincreasedsurfacehardness.Apilotstudy

was conducted to measure hardness throughout specimens and not just on the

surface. The surface was progressively lapped (in 100 μm increments) and the

hardness of the newly exposed surfacemeasured. The results revealed a crust-like

layer on the surface of the GIC blocks in citric acid with the highest values being

measuredatthe initialexposedsurface,whilethe lowestvalueswererecordedjust

belowthesurface layer (100μmbelowthesurface).Valuesof the lowesthardness

andthespecificdepthbelowthesurfaceatwhichitoccursareofgreatimportance,

howeverduetotheverycoarseresolutionofthepilotworkonlytheinitialevidence

ofthiseffecthasbeenobserved.Thisaspectoftheeffectoftheacidsneedsamore

comprehensive study. The presence of the crust-like layer indicates that there is a

migrationofionsfromthematerialbulkandsub-surfaceregionsoftheGICblocksto

the surface.Amoredirect investigationof aluminium ionsand their link to surface

hardness,masslossandgeneralionreleasewasthefocusofthefollow-upstudy.

8.4Importanceandroleofaluminiumions

Theearlystudies(Chapter5) foundanunexpectedcorrelationbetweenhigh

phosphatereleaseandmasslossforF7andF7EPstoredincitricacid.Atthatstageno

evidence was found that both of these measurements were caused by the same

mechanism, however aluminium chelation by citric acidwas a plausible reason for

171

both results. This outcome was interesting, but the focus of further investigations

remained with GICs and CPP-ACP. In the follow-up study (Chapter 6), topical CPP-

ACFPtreatmentintheformofToothMoussePlusonthesamesetofGICmaterials,

F7andF7EP,againshowedthesameeffect.Undertopicaltreatment,phosphateion

releaseandmasslosswerehighercomparedtonotreatment.Thiswastrueevenfor

the control groups, which used a placebomousse, where no additional phosphate

wasavailable.Whateverwas the causeof thehighphosphate ion releaseandhigh

mass loss itwas being further exacerbated by the topical treatment. An additional

unexpectedresultwastheincreasedsurfacehardnessbeyondthatoffreshGICblocks

(beforeanytreatmentoracidchallenge).Furtherinvestigationofthismechanismwas

warranted.

It was determined that the most simple and direct way to validate our

hypothesis that aluminium was somehow involved was to measure the release of

aluminium ions. A new protocol was developed to employ Atomic Absorption

Spectroscopy assays to measure aluminium ions released into solution. Results

showedthataluminiumionreleasecorrelatedcloselywithphosphateionreleaseand

massloss.Evenday-to-dayandsample-to-samplevariationsbetweenphosphateion

releaseandaluminiumionreleasefollowedeachotherclosely.

Examining the composition of GICs (Table 8.1) revealed that aluminium is

presentinanumberofcompounds,ofwhichaluminiumphosphateisone,butthere

aremanyothers.Chelationofaluminiumphosphatewouldaccountforthehigh ion

release and consequently high mass loss, however the relationship of other

aluminium-basedcompoundsandcitricacidremainsunclear.

Investigation of a resin composite containing so-called pre-reacted glass

ionomerfillerspresentedanopportunitytoinvestigatetheroleofaluminiuminGICs

fromadifferentperspective.ThematerialselectedwasShofuBeautifilF03(SBF03).

SBF03 is a ‘giomer restorative’material. It is resin-basedwith the inclusion of pre-

reacted glass particles that are claimed to allow fluoride ion release. The results

showed that SBF03 released a substantial amount of fluoride, however the daily

release fell sharply after the first 24 hours. Examination of aluminium ion release

172

showedthat,unlikeinF7andF7EP,SBF03sharedaclosecorrelationwithfluorideion

releasebutnotphosphate.Thismajordifferencemaybeexplainedbythedifferent

levelsofmaturationoftheglasspresentineachmaterial.Inordertounderstandthis

moreclearlyacloserexaminationofthepolymerisationprocessofGICsisneeded.

Powder weight%

Cryolite(Na3AlF6) 5%

AlF3 5%

SrF2orCaF2 22-34%

AlPO4 10%

Al2O3 16%

Table8.1.GeneralcompositionofGICs

The initial setting reactionduring thepolymerisationprocess involveseither

calciumorstrontiumsaltsandwasdevelopedtogiveafastsettingpropertytoGICs3,

4. Historically, tartaric acid was used to provide this ‘snap-set’. Aluminium salts

participate in the polymerisation process at a much slower rate and the reaction

continuesformanyweeks4-6.Thismeansthattherelativecompositionofcompounds

present ina ‘fresh’GICwouldbeverydifferent to thosepresent ina ‘mature’GIC,

aluminium ionswouldbeboundtodifferentcompoundsandwithdifferentbinding

strengths. This may explain the differences observed between F7/F7EP and SBF03

whenitcomestofluoride,phosphateandaluminiumionrelease.

The studies examined two types of material, a conventional GIC that was

mixed fresh and immediately subjected to acid challenge and a giomer material,

173

which contains ‘matured’ glass particles. The results indicated that glass particles

might react differently to acid challenge depending on their stage of maturation.

These resultsmay carry implications formany conventionalGICs. If the functionof

aluminiumionscanbemorefullyunderstoodandtheirinteractionwithexternalacids

better traced over time the changes observed in the current work can be better

explained. This could then possibly allow modifications of the cements to be

introducedtoimprovetheirproperties.

174

8.5References

1. FeatherstoneJD,RodgersBE(1981).Effectofacetic, lacticandotherorganic

acidsontheformationofartificialcariouslesions.CariesResearch15(5):377-

385.





Research53(6):1414-1419.







Research55(3):489-495.

176

CHAPTER9

ConclusionsandAreasforFurther

Research

Conclusionsfromtheresearchundertakenaresummarisedbelow:

§ CPP-ACP, incorporated into Fuji VII EP, significantly increased the release of

calciumandphosphate ionscomparedtoFujiVII;nosignificantdifferencein

fluorideionreleasecouldbedetectedbetweenFujiVIIEPandFujiVII.

§ GICmaterials(FujiVIIEPandFujiVII)respondedtovariousacids indifferent

ways.Thetypeofacidwas foundtobeamoresignificant factor thanpHor

ionicstrengthoftheacid.

§ Topical CPP-ACFP treatment (ToothMousse Plus) of GIC surface (Fuji VII EP

andFujiVII)wasfoundtobemorebeneficialthanplacebocontroltreatment

(no active ingredient) and “no treatment” when considering calcium and

fluoride ion release. Topical treatment (Tooth Mousse Plus and placebo

177

moussecontrol)greatlyincreasedphosphateionreleaseandmasslossofGIC

blockscomparedto“notreatment”.ToothMoussePluswasfoundtoprovide

greaterprotectiontomasslosscomparedtoplacebotreatment.

§ Aluminiumchelationwas identifiedasa key factor inphosphate ion release

fromFujiVIIEPandFujiVIIwhenbothGICmaterialswereexposed tocitric

acid. Increased phosphate and aluminium ion release combined with

increasedsurfacehardnesssuggestsaformationofa“crust-like”layeronthe

surfacesofFujiVIIEPandFujiVII.

§ A comparison between Fuji VII EP and a glass-containing giomer material

(ShofuBeautifil F03) showeddifferent ion releaseprofilesdependingon the

maturationoftheglassineachmaterial.Aluminiumchelationwaspresentin

both cases, however aluminium ion release was closely related to fluoride

releaseinBeautifilandphosphatereleaseinFujiVIIEP.

Following the investigations covered in this thesis several areasof further research

havebeenidentified:

§ Furtherstudyintohowdentalrestorativematerials(notjustGICs)respondto

acid challenge, especially relating to ion release. Laboratory studies have

shownGICsresponddifferentlytoacidchallengedependingontheacidused.

Clinically the environment is more complex as different acids will exist

concurrentlyaswellasproteinadsorptiononthematerialsurface.Thismore

complex system may cause different interactions compared with the

outcomesfromindividualacidsusedinthecurrentresearch.

§ AfurtherinvestigationofrechargepotentialofCPP-ACFP(ToothMoussePlus)

onGICs andothermaterials is required. The results presented in this thesis

178

showed a significant improvement in ion release potential of conventional

GICswhentopically treatedwithToothMoussePlus.Thismaybe important

foruseinpatientswhohaveadrymouthandincreasedcariesrisk,aswellas

the appropriate selection ofGIC products depending on environmental acid

exposure.

§ TopicaltreatmentofGICsstoredincitricacidshowedtheformationofwhat

appeared to be a ‘crust-like’ surface layerwith an ion-depleted sub-surface

region.Mass loss, iondisplacementandmigrationtothesurface layer isnot

unlike the formation of an early enamel carious lesion in teeth, where a

fluoride-rich surface layer is located above a sub-surface region of

demineralised tooth structure. The effect of aluminium chelation in GICs

needsfurtherinvestigation.

§ AmoredetailedexaminationofaluminiumionswithinGICsandhowtheloss

ofaluminiumionscanbeaddressedtoimprovethelongevityanditsrelation

totheclinicalapplicationofGICs.Substitutionofaluminiumionswithanother

ion can potentially helpmitigate the chelation caused by citric acid in GICs

(andpossiblyothertriproticacidsencounteredintheoralenvironment).The

substitution can occur either via a topical treatment or ideally during the

manufacturing process. The manufacturing process of the glass of GICs to

substitutealuminiumcouldbeconsidered.

179

APPENDIX

Ion release and physical properties of CPP–ACP modified GICin acid solutions

I. Zalizniak a, J.E.A. Palamara a, R.H.K. Wong a, N.J. Cochrane a,M.F. Burrow b, E.C. Reynolds a,*aOral Health CRC, Melbourne Dental School, The University of Melbourne, Australiab Faculty of Dentistry, University of Hong Kong, Hong Kong

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 4 4 9 – 4 5 4

a r t i c l e i n f o

Article history:

Received 25 October 2012

Received in revised form

7 February 2013

Accepted 11 February 2013

Keywords:

GIC

CPP–ACP

Hardness

Ion release

a b s t r a c t

A new glass-ionomer cement (GIC) (Fuji VIITM EP) includes 3% (w/w) casein phosphopeptide–

amorphous calcium phosphate (CPP–ACP) to enhance ion release.

Objectives: To assess this new GIC compared with a GIC without CPP–ACP (Fuji VIITM) with

respect to ion release, changes in surface hardness and in mass under a variety of acidic and

neutral conditions.

Methods: Eighty blocks of Fuji VIITM (F7) and Fuji VIITM EP (F7EP) were subjected to three

acidic solutions (lactic and citric acids pH 5.0, hydrochloric acid pH 2.0) and water (pH 6.9)

over a three-day period. Ion release, surface hardness and weight measurements were

carried out every 24 h.

Results: Higher calcium ion release from F7EP was observed under all acidic conditions.

Increased inorganic phosphate ion release was observed for F7EP in hydrochloric and citric

acids. Fluoride ion release was similar between F7 and F7EP under all conditions but was

significantly higher in acids compared with water. After three days there was no significant

difference in surface hardness ( p > 0.05) between the two materials under all conditions

except hydrochloric acid. Minimal change in mass was observed for F7 and F7EP in water,

lactic and hydrochloric acids, however citric acid caused significantly more mass loss

compared with water ( p < 0.001).

Conclusion: Incorporation of 3% (w/w) CPP–ACP into F7 enhanced calcium and phosphate ion

release, with no significant change in fluoride ion release and no adverse effects on surface

hardness or change in mass.

Clinical significance statement: GICs have the potential to release fluoride ions particularly

under acidic conditions associated with dental caries and erosion. A new GIC containing

CPP–ACP and fluoride releases not only fluoride ions but also calcium and phosphate ions

under acidic conditions which should help to inhibit demineralisation associated with

caries and erosion.

# 2013 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

1. Introduction

Glass ionomer cements (GICs) are widely used for a variety of

purposes such as intermediate restorations, caries

* Corresponding author at: Melbourne Dental School, The University oTel.: +61 3 9351 1547; fax: +61 3 9341 1599.

E-mail addresses: [email protected], k.fletcher@unimelb

0300-5712/$ – see front matter # 2013 Elsevier Ltd. All rights reservedhttp://dx.doi.org/10.1016/j.jdent.2013.02.003

stabilisation, definitive restoration of micro-cavities or

non-carious cervical lesions, adhering orthodontic brackets

and bands, fissure sealing erupting molars and as a surface

protecting material for high risk surfaces such as root

surfaces.1,2 The main advantages of GICs are their strong

f Melbourne, 720 Swanston Street, Victoria 3010, Australia.

.edu.au (E.C. Reynolds).

.

http://dx.doi.org/10.1016/j.jdent.2013.02.003

mailto:[email protected]

mailto:[email protected]

http://www.sciencedirect.com/science/journal/03005712

http://dx.doi.org/10.1016/j.jdent.2013.02.003


ability to chemically bind to dentine and their ability to release

fluoride ions. This fluoride ion release has been shown to slow

the progression and aid the regression of early carious lesions.3

Tooth enamel will only demineralise when the fluid bathing the

enamel crystals is undersaturated with respect to the enamel

mineral (carbonated hydroxyapatite). Hence, calcium and

phosphate ion activity is an important component influencing

the level of enamel mineral saturation.

A number of investigators have explored the modification of

dental materials in attempt to have them release calcium,

phosphate and fluoride ions.4–6 Skrtic et al.6 explored modifying

dental composites with bioactive glasses. Mazzaoui et al.5 and

Al Zraikat et al.4 assessed the addition of casein phosphopep-

tide–amorphous calcium phosphate (CPP–ACP) to GICs. The

casein phosphopeptides stabilise calcium and phosphate ions

in a bioavailable form that allow them to inhibit demineralisa-

tion and promote the remineralisation of early lesions.7 CPP–

ACP is stable in the presence of fluoride and has been shown to

work synergistically with fluoride.8 This makes CPP–ACP a

promising additive to dental products and restorative materials.

The study by Mazzaoui et al.5 was a proof of concept study that

showed that CPP–ACP could be added to GIC. Al Zraikat et al.4

later explored the effect of CPP–ACP concentration on GIC

mechanical properties. Additionally, both of these of studies

showed that the addition of CPP–ACP improved calcium and

phosphate ion release in lactic acid.

The tooth surface can be exposed to a variety of

demineralisation challenges including: acids formed from

metabolic processes of oral bacteria (predominantly lactic

acid); food acids such as citric and phosphoric acid that are

commonly found in soft drinks9; and hydrochloric acid from

the regurgitation of stomach contents. If these acids over-

whelm saliva’s protective functions mineral may be lost from

the teeth.10 Therefore, the overall aim of this study was to

determine how GIC with CPP–ACP performed under a variety

of acidic conditions compared with GIC without CPP–ACP in

terms of ion release, change in hardness and change in mass.

Fuji VII (F7) is a low viscosity strontium glass based ionomer

cement and is commonly used as a surface protectant,

providing effective wetting and intimate adhesion to tooth

surfaces, as well as enhanced remineralisation capabilities. It

has higher fluoride release than most other GICs which makes

it a good candidate to establish if there is an additional benefit

through the incorporation of CPP–ACP as a source of calcium

and phosphate ions together with the fluoride ions. This study

will establish whether F7 plus CPP–ACP has the potential to

further protect surrounding hard tissue and enhance the

remineralisation of demineralised tissue by additional ion

release.

The research questions were: what effect on the GIC would

the addition of CPP–ACP have on; (1) surface hardness; (2)

change in mass under neutral or acidic environments and (3)

calcium, phosphate and fluoride ion release under neutral or

different acidic environments.

2. Materials and methods

Fuji VII GIC (F7) and F7 with added 3% (w/w) CPP–ACP (F7EP)

from the same batch were provided by GC Corporation (Japan)

in capsule form. Polyvinyl siloxane impression material

(eliteHD + light body, Zhermack SpA, Badia Polesine, Italy)

moulds were used to create standardised GIC blocks measur-

ing 3 mm � 6 mm � 6 mm (thickness � width � length). 40

blocks of each GIC were prepared by placing the materials

in the mould with the top and bottom surfaces covered by

plastic strips, which was held between two glass slides. The

glass slides were gently pressed together to extrude any excess

material. The specimens were allowed to set inside the

moulds for 24 h in an incubator (37 8C, �100% relative

humidity). After cooling to room temperature the blocks were

removed from the moulds, and the two major parallel surfaces

of the blocks were lapped with 600 grit paper (Norton Tufbak,

Saint-Gobain Abrasives Ltd., Auckland, NZ).

Four different solutions were prepared to expose the blocks

to a variety of acidic and neutral environments. The three

acidic solutions were formulated to simulate a gastric erosive

challenge (50 mM NaCl adjusted to pH 2.0 with HCl), a dietary

erosive challenge (50 mM citric acid at pH 5.0) and a cariogenic

acid challenge (50 mM lactic acid at pH 5.0) (this concentration

was selected based on values found previously in plaque

fluid).11 Ionic strength of all the acidic solutions was made up

to 50 mM including the HCl solution which was modified with

NaCl to approximate that found in stomach acid.12

The neutral solution was distilled deionised water at pH 6.9

(Millipore Corporation, Victoria, Australia).

Ten blocks of each type of GIC were exposed to 5 mL of one

of the four solutions. Solutions were changed every 24 h and

the samples were measured for change in mass, surface

hardness and the solutions were analysed to determine ion

release of calcium, phosphate and fluoride every 24 h over

three days.

The mass of each block was measured every 24 h before

surface hardness measurements were performed. Blocks were

taken out of solution, and then weighed using a microbalance

(Precisa XT 120A, Dietikon, Switzerland). Mass loss measure-

ment was performed under the same conditions for each

sample. Blocks were gently pat dried in the same manner and

weighed immediately to ensure consistent treatment before

testing. All mass loss measurements were obtained under

ambient conditions of 60 � 5% relative humidity and 23 � 2 8C.

The percentage change in mass was a combination of water

uptake (absorption) and dissolution (solubility) of the GIC.

Vickers microhardness measurements were determined

from indentations on the lapped GIC surface using a

Microhardness tester (MHT-10, Anton Paar GmbH, Graz,

Austria) attached to a microscope (Leica DMPL, Leica Micro-

systems Wetzlar GmbH, Germany). Two indentations were

made on each block (force, 1.0 N; dwell, 6 s; rate, 0.99 N/min).

The indentations were separated by a distance of at least three

times the indentation size. Images of the indentations were

acquired through a calibrated digital camera (Leica DFC320)

mounted on the microcope (Leica DMLP, Leica Microsystems

Wetzlar GmbH, Germany) and distance measurements made

using Image Tool software (Version 3.0, UTHSC, San Antonio,

TX) which were then converted into Vickers hardness values.

Blocks were then placed into fresh batch of solution and

returned to the incubator.

The ion release of calcium, phosphate and fluoride after

each 24 h period of storage was determined using atomic

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 4 4 9 – 4 5 4 451

absorption spectroscopy, colorimetry and an ion-specific

electrode respectively. To determine the calcium concentra-

tion sample solutions (1 mL) were acidified with 1 M HCl

(0.5 mL) and diluted with 2% lanthanum chloride (0.5 mL) and

analysed on a Varian AA240 atomic absorption spectroscope

(Varian Australia Pty. Ltd.) against a set of seven standards

ranging from 0 to 250 mM calcium. Inorganic phosphate ion

concentrations were determined colorimetrically using a

spectrophotometer (UV-visible spectrophotometer, Varian

Australia, Pty. Ltd.). The samples that were analysed were

prepared by taking 100 mL of solution, diluting with 500 mL of

4.2% ammonium molybdate and adding 20 mL 1.5% of Tween1

20 (Sigma–Aldrich, St. Louis, MO). The phosphate concentra-

tion was determined by comparing the spectrophotometer

readings of the samples against a set of seven standards

ranging in phosphate concentrations from 0 to 100 mM. The

concentration of fluoride ions was determined using an ion-

selective electrode (Radiometer analytical, ISE C301F, France)

connected to an ion analyser (Radiometer analytical, Ion

Check 45, France). Sample solutions (1 mL) were diluted with

1 mL total ionic strength adjustment buffer (Merck Pty. Ltd.,

Kilsyth, VIC, Australia) and measured against a set of eight

fluoride standards ranging from 0 to 1000 mM.

The x2 test was used to test normality and data were found

to be normally distributed. Single factor ANOVA was used to

analyse the results using Bonferroni–Holm multiple compari-

son. Two-way ANOVA was used to determine the interaction

between incorporation of CPP–ACP and exposure to different

acids. Level of significance was set at a = 0.05.

3. Results

Table 1 shows surface hardness of F7EP and F7 in three

different acidic solutions and distilled deionised water

measured over three days. A significant decrease in surface

hardness was measured from day to day in all solutions except

in the following cases. F7EP in citric and hydrochloric acids

between day two and three did not register a significant

change in surface hardness. F7 in water did not register a

significant change between days one and two, and two and

three. The surface hardness of F7EP in water was not

significantly different between its initial hardness and day

one as well as between days two and three. The initial

hardness of F7EP was significantly ( p < 0.001) lower than that

of F7. However, after 24 h of storage in lactic and citric acid

Table 1 – Mean surface hardness of F7 and F7EP in acidic and

HV (kg/mm2) n = 10 F7 (Std. Dev.)

Water Lactic acid Hydro-chloricacid

Ci

Initial 53.8a (5.3) 51.8b (5.5) 53.3c (7.6) 5

Day 1 48.5e (6.8) 32.6 (5.1) 28.1f (4.5) 3

Day 2 44.4 (4.2) 28.5 (3.1) 22.0g (3.9) 3

Day 3 42.0 (4.7) 24.8 (2.3) 15.4h (2.7) 2

Values marked with the same letter (a–h) indicate a significant differenc

period.

F7EPP had a similar surface hardness to F7 and was no longer

significantly weaker for citric, lactic acids and water ( p > 0.05),

but it was still significantly ( p < 0.01) weaker in hydrochloric

acid. After three days of storage no significant difference in

surface hardness could be measured between F7EP and F7

( p > 0.05) for citric, lactic acids and water, but F7EP still

remained significantly ( p < 0.05) weaker in hydrochloric acid.

Table 2 shows the mean relative mass change of F7 and

F7EP blocks when stored in the four different solutions over

three days. Each group is scaled according to its initial weight

(100%). The greatest mass loss at all time points was from F7

and F7EP that were exposed to citric acid.

Fig. 1a–c shows the measured concentration of calcium,

phosphate and fluoride ions released from F7 and F7EP when

stored in the four different solutions over a three-day period.

The rate of calcium and phosphate ion release for F7EP was

significantly greater than that of F7 for both citric and

hydrochloric acids (Fig. 1a and b), and there was also greater

calcium ion release in lactic acid (Fig. 1a). The fluoride ion

release in all solutions was similar for both F7 and F7EP

(Fig. 1c).

Where a change in mass was detected for an exposure to an

acidic solution over the three days (Table 2) the calcium and

phosphate ion release was found to be greater for F7EP (Fig. 1a

and b). The difference in calcium and phosphate ion release as

a function of change in mass for F7 and F7EP is shown in Fig. 2a

and b for citric and hydrochloric acids respectively (there was

no measurable mass loss in lactic acid and water). These

figures show that per unit of mass loss there was a greater

release of calcium and phosphate ions from the GIC contain-

ing CPP–ACP.

4. Discussion

In all the tests conducted on F7 and F7EP, the materials

behaved differently in water and acidic environments. The

surface hardness of the GIC’s significantly decreased in the

acidic environments although this did not correlate to a

change of mass. The release of ions was greater in acidic

conditions compared with water. The release of calcium and

phosphate ions from the material only under acidic conditions

is ideal as this is when they would be needed to protect the

adjacent tooth structure from demineralisation. The higher

release of calcium and phosphate ions from F7EP under acidic

conditions may saturate the fluid in the oral environment with

neutral solutions measured over three days.

F7EP (Std. Dev.)

tric acid Water Lactic acid Hydrochloricacid

Citric acid

3.2d (7.1) 47.1a (6.0) 46.2b (6.2) 46.6c (6.4) 46.7d (7.0)

9.5 (7.4) 45.6e (4.3) 34.0 (5.9) 22.6f (4.0) 38.1 (9.0)

2.9i (5.8) 42.2 (3.3) 28.3 (4.8) 15.5g (3.5) 28.5i (6.3)

5.8 (5.6) 41.5 (3.2) 24.0 (3.0) 13.3h (2.3) 25.6 (6.8)

e between GIC materials within the same solution for the same time

Table 2 – Mean relative mass of F7 and F7EP when stored in the four different solutions over three days.

(%) relative toinitial mass(100%) n = 10

F7 (Std. Dev.) F7EP (Std. Dev.)

Water Lacticacid

Hydrochloricacid

Citric acid Water Lactic acid Hydrochloricacid

Citric acid

Day 1 100.3a (5.7) 99.8 (3.8) 99.4 (4.5) 96.9a (4.2) 101.5d (5.1) 101.1 (5.5) 99.9 (4.3) 97.2d (4.4)

Day 2 100.5b (5.7) 99.7 (3.8) 98.6 (4.6) 92.8b (4.3) 102.0e (5.1) 101.3 (5.5) 99.5 (4.2) 93.2e (4.5)

Day 3 100.6c (5.6) 99.6 (3.8) 97.7 (4.6) 89.2c (4.4) 102.2f (5.2) 101.3 (5.4) 98.8 (4.4) 89.0f (4.6)

Values marked with the same letter (a–f) indicate significant difference between different solutions within the same material for the same

time period. Each group is scaled according to its initial weight (100%).


appropriate ions thus acting as a ‘‘smart material’’ for the

protection of at risk tooth surfaces. The three-day continuous

exposure in vitro would have been equivalent to a much longer

term periodic acid challenge in vivo. However, the in vitro

performance of these GIC materials should be confirmed

clinically.

Previous studies have investigated ion release and surface

hardness of various GICs in acidic and neutral environ-

ments.4,5,13 Past results have shown ion release from F7 with

Fig. 1 – Measured calcium (a), phosphate (b) and fluoride (c) ion

solutions over a three-day period. Significant increase ( p < 0.05)

between F7EP and F7. Significant increase ( p < 0.05) in phospha

acids compared to water. Significantly more phosphate was rel

acids. Fluoride ion release was significantly higher ( p < 0.005) i

letter indicate no significant difference in fluoride ion release b

incorporation of CPP–ACP when stored in neutral solution

(water) or lactic acid.4 It has also been reported that lower pH

environments negatively affect surface hardness of GICs.14

However, there have been no studies looking at ion release or

surface hardness of GICs covering a wide variety of acids that

can be encountered in the oral environment.

The results indicate that the degradation of surface

hardness was pH dependent rather than solution dependent.

Lactic and citric acids (both pH 5) produced a similar reduction

release from F7 and F7EP when stored in the four different

in calcium ions was measured for all groups (except water)

te ion release was measured for citric and hydrochloric

eased from F7EP compared to F7 in citric and hydrochloric

n all acids compared to water. Groups marked with same

etween F7 and F7EP when exposed to the same solution.

Fig. 2 – Difference in calcium and phosphate ion release as a function of change in mass between F7 and F7EP in citric acid (a)

and hydrochloric acid (b).

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 4 4 9 – 4 5 4 453

in microhardness over three days, while HCl (pH 2) showed the

greatest and water (pH 6.9) the least. Even though initially F7EP

was slightly softer in surface hardness than F7, a converging

pattern occurred across all solutions such that, except for HCl,

the difference after three days was no longer significant. It is

possible that F7EP has a greater buffering capacity than F7

alone as CPP–ACP has previously been shown to have

buffering potential.15,16

As with surface hardness, there was an initial difference in

the change in mass between these two GIC materials. This can

be partly attributed to F7EP being slightly less dense than F7.

Significant mass loss was observed for specimens placed in

citric acid for both materials. While other solutions produced

small changes from day to day the differences between them

were not significant. The difference in the change in mass

between F7EP and F7, in citric acid, was not significant when

taking into account the difference in initial weights. Although

surface hardness showed very similar trend in a decrease in

hardness for all solutions this was not repeated for change in

mass. For citric and lactic acids the mass loss showed a large

difference between the two acid solutions such that, in lactic

acid a slight mass gain was observed for F7EP and a slight loss

for F7. In citric acid a large mass loss was observed for both

materials. A mass gain was observed in water. The chelating

effect of the citric acid is likely to contribute to the large mass

loss. Citric acid is a triprotic acid, which can chelate transition

metals, in this case aluminium. Aluminium is an initial

component of the GIC matrix in the form of aluminium

phosphate and the aluminium ion is involved in the acid–base

setting reaction. The cross-linking between the polyalkenoic

acid chains is formed by a slow final maturation reaction

involving aluminium ions, which increases the strength of the

GIC.17 This hardening process may take days to complete.

When exposed to citric acid Al3+ ions are complexed by citrate

removing them from the GIC matrix leading to the greater

mass loss of the material. Further evidence to support this

theory can be seen in the phosphate ion release data shown in

Fig. 1b. The GIC without CPP–ACP released high levels of

phosphate although it contained no CPP–ACP supporting the

chelation of aluminium and the dissolution of the aluminium

phosphate present within all GICs.

When subjected to aggressive environments such as citric,

lactic and hydrochloric acids a substantial release of ions

occurred from both GIC materials. Calcium release in the three

acids ranged from 0.07 to 0.20 mmol/mm2 for F7 but was higher

for F7EP, ranging from 0.36 to 1.34 mmol/mm2, equating to an

increase of 564%, 405% and 462% for citric, lactic and

hydrochloric acids respectively. This can be explained by

the CPP–ACP acting as a source of bioavailable calcium ions.

The CPP–ACP complexes will release calcium and phosphate

ions in a pH dependent manner.16,18 An increase in calcium

ions would help to maintain the degree of saturation with

respect to tooth mineral which would prevent demineralisa-

tion. Phosphate ion release in lactic acid and water was not

detectable for either GIC material. Daily phosphate ion release

for citric acid ranged from 9.8 to 13.9 nmol/mm2 for F7 and was

higher for F7EP, ranging from 14.5 to 17.6 nmol/mm2, an

average increase of 39%. Daily phosphate ion release for

hydrochloric acid ranged from 0.42 to 0.51 nmol/mm2 for F7

and was higher for F7EP, ranging from 0.7 to 1.07 nmol/mm2,

an average increase of 85%. This implies that some phosphate

is being supplied by CPP–ACP, but unlike the calcium ion

release a lot of the phosphate is also being supplied by the GIC

matrix most likely from dissolution of AlPO4 at the surface of

the GIC. Dissolution of the AlPO4 by citric and hydrochloric

acids releases phosphate from the GIC matrix. Fluoride release

from both materials was similar and highest in citric (103.53

and 108.46 mmol/mm2) and hydrochloric acids (107.40 and

109.38 mmol/mm2) for F7 and F7EP respectively (Fig. 1b).

5. Conclusion

Although initial surface hardness of F7EP was slightly lower

than F7, after three days of storage in three of the four types of

solution there was no significant difference between the

surface hardness values of the two GICs. Addition of CPP–ACP

to F7 increased the release of calcium ions by an average of

477% when exposed to acidic solutions with no change in

fluoride ion release. However, phosphate ion release ranged

greatly depending on the type of acid exposure and whether

CPP–ACP had been incorporated.


r e f e r e n c e s

1. Ganesh M, Shobha T. Comparative evaluation of themarginal sealing ability of Fuji VII and concise as pit andfissure sealants. Journal of Contemporary Dental Practice2007;8:10–8.

2. Forsten L, Mount GJ, Knight G. Observations in Australia ofthe use of glass ionomer cement restorative material.Australian Dental Journal 1994;39:339–43.

3. Trairatvorakul C, Itsaraviriyakul S, Wiboonchan W. Effect ofglass-ionomer cement on the progression of proximalcaries. Journal of Dental Research 2011;90:99–103.

4. Al Zraikat H, Palamara JE, Messer HH, Burrow MF, ReynoldsEC. The incorporation of casein phosphopeptide–amorphous calcium phosphate into a glass ionomercement. Dental Materials 2011;27:235–43.

5. Mazzaoui SA, Burrow MF, Tyas MJ, Dashper SG, Eakins D,Reynolds EC. Incorporation of casein phosphopeptide–amorphous calcium phosphate into a glass-ionomercement. Journal of Dental Research 2003;82:914–8.

6. Skrtic DA, Eanes JMED. Amorphous calcium phosphate-based bioactive polymeric composites for mineralized tissueregenration. Journal of Research 2003;108:167–82.

7. Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC.Enamel subsurface lesion remineralisation with caseinphosphopeptide stabilised solutions of calcium, phosphateand fluoride. Caries Research 2008;42:88–97.

8. Shen P, Manton DJ, Cochrane NJ, Walker GD, Yuan Y,Reynolds C, et al. Effect of added calcium phosphate onenamel remineralization by fluoride in a randomizedcontrolled in situ trial. Journal of Dentistry 2011;39:518–25.

9. Bartlett DW, Fares J, Shirodaria S, Chiu K, Ahmad N, SherriffM. The association of tooth wear, diet and dietary habits inadults aged 18–30 years old. Journal of Dentistry 2011;39:811–6.

10. Roberts MW, Li SH. Oral findings in anorexia nervosa andbulimia nervosa: a study of 47 cases. Journal of the AmericanDental Association 1987;115:407–10.

11. Margolis HC, Moreno EC. Composition and cariogenicpotential of dental plaque fluid. Critical Reviews in Oral Biologyand Medicine 1994;5:1–25.

12. Lindahl A, Ungell AL, Knutson L, Lennernas H.Characterization of fluids from the stomach and proximaljejunum in men and women. Pharmaceutical Research1997;14:497–502.

13. Bala O, Arisu HD, Yikilgan I, Arslan S, Gullu A. Evaluation ofsurface roughness and hardness of different glass ionomercements. European Journal of Dentistry 2012;6:79–86.

14. Silva KG, Pedrini D, Delbem AC, Cannon M. Effect of pHvariations in a cycling model on the properties of restorativematerials. Operative Dentistry 2007;32:328–35.

15. Caruana PC, Mulaify SA, Moazzez R, Bartlett D. The effect ofcasein and calcium containing paste on plaque pH followinga subsequent carbohydrate challenge. Journal of Dentistry2009;37:522–6.

16. Cochrane NJ, Cai F, Huq NL, Burrow MF, Reynolds EC. Newapproaches to enhanced remineralization of tooth enamel.Journal of Dental Research 2010;89:1187–97.

17. Nicholson JW, Czarnecka B. Review paper: role of aluminumin glass-ionomer dental cements and its biological effects.Journal of Biomaterials Applications 2009;24:293–308.

18. Cochrane NJ, Reynolds EC. Casein phosphopeptides in oralhealth. Food Constituents and Oral Health 2009:185–224.

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

Zalizniak, Ilya

Title:

Effect of exposure of glass ionomer cements to acid environments

Date:

2016

Persistent Link:

http://hdl.handle.net/11343/124099

File Description:

Effect of exposure of glass ionomer cements to acid environments

Terms and Conditions:

Terms and Conditions: Copyright in works deposited in Minerva Access is retained by the

copyright owner. The work may not be altered without permission from the copyright owner.

Readers may only download, print and save electronic copies of whole works for their own

personal non-commercial use. Any use that exceeds these limits requires permission from

the copyright owner. Attribution is essential when quoting or paraphrasing from these works.

Effect of exposure of glass ionomer cements to acid ...

Documents

Transcript of Effect of exposure of glass ionomer cements to acid ...