effect of exposure of glass ionomer cements to acid
TRANSCRIPT
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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
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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.
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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
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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
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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
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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
6.6Conclusion..........................................................................................................................1456.7References.........................................................................................................................146
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
7.6Conclusion..........................................................................................................................1647.7References.........................................................................................................................165
CHAPTER8.......................................................................................................................167
Discussion........................................................................................................................167
8.1Pilotstudyoutcomes......................................................................................................167
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8.2Acidchallengeoutcomes...............................................................................................1688.3Topicaltreatmentoutcomes.......................................................................................1698.4Importanceandroleofaluminiumions..................................................................1708.5References.........................................................................................................................174
CHAPTER9.......................................................................................................................176
ConclusionsandAreasforFurtherResearch......................................................176
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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
Figure4.7.F7EPblockstreatedwithTM+analysedusingEDXAatvarioussubsurface
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
solutionsandthreetreatmentgroups...............................................................132
Figure6.7.Cumulativethree-dayphosphateionreleasefromF7blocksacrossfour
solutionsandthreetreatmentgroups...............................................................133
Figure6.8.Cumulativethree-dayphosphateionreleasefromF7EPblocksacrossfour
solutionsandthreetreatmentgroups...............................................................134
Figure7.1.ComparisonofFluorideIonReleaseinFujiVIIEPandShofuBeautifilF03.
............................................................................................................................159
Figure7.2.Relationshipbetweendailyaluminiumandfluorideionsreleasedfrom
SBF03..................................................................................................................160
Figure7.3.Relationshipbetweendailyaluminiumandphosphateionsreleasedfrom
F7EP....................................................................................................................160
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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
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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,
PhosphateandFluorideionrelease.*-nostatisticalsignificance)...................138
Table6.10.Statisticalsignificanceincitricacid.(Hardness.Massloss.Calcium,
PhosphateandFluorideionrelease.*-nostatisticalsignificance)...................139
Table7.1.GeneralcompositionofGICs.SiO2makesuptherestupto100%...........153
Table7.2.Surfacehardnesscomparison(VH1.0).Absolutevaluesinbold,%dropsin
secondrow.........................................................................................................157
Table7.3.DailymasslossofSBF03infourstoragesolutions.Meanvaluesingrams.
............................................................................................................................158
Table8.1.GeneralcompositionofGICs.....................................................................172
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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.
5. Crisp S, Lewis BG, Wilson AD (1979). Characterization of glass-ionomer
cements.5.Effectofthetartaricacidconcentrationintheliquidcomponent.
JournalofDentistry7(4):304-312.
6. KentBE,LewisBG,WilsonAD (1979).Glass ionomercement formulations: I.
Thepreparationofnovelfluoroaluminosilicateglasseshighinfluorine.Journal
ofDentalResearch58(6):1607-1619.
7. Crisp S, Lewis BG, Wilson AD (1977). Characterization of glass-ionomer
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.
10. McLeanJW,WilsonAD(1977).Theclinicaldevelopmentoftheglass-ionomer
cement.III.Theerosionlesion.AustralianDentalJournal22(3):190-195.
11. McLeanJW,WilsonAD(1977).Theclinicaldevelopmentoftheglass-ionomer
cements. I. Formulations andproperties.AustralianDental Journal22(1):31-
36.
12. Crisp S, Lewis BG, Wilson AD (1976). Characterization of glass-ionomer
cements. 2. Effect of the powder: liquid ratio on the physical properties.
JournalofDentistry4(6):287-290.
13. Crisp S, Lewis BG, Wilson AD (1976). Characterization of glass-ionomer
cements. 1. Long term hardness and compressive strength. Journal of
Dentistry4(4):162-166.
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.
21
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
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157. Asmussen E, Peutzfeldt A (2002). Long-term fluoride release from a glass
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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.
71
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
4.3.1MaterialsandMethods
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
4.4.1MaterialsandMethods
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
Figure4.7.F7EPblockstreatedwithTM+analysedusingEDXAatvarioussubsurface
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.
95
96
4.6References
1. Margolis HC, Moreno EC (1994). Composition and cariogenic potential of
dentalplaquefluid.CriticalReviewsinOralBiology&Medicine5(1):1-25.
2. ZoergiebelJ,IlieN(2013).Evaluationofaconventionalglassionomercement
withnewzincformulation:effectofcoating,agingandstorageagents.Clinical
OralInvestigations17(2):619-626.
97
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
simulate a gastric erosive challenge (50mM NaCl adjusted to pH 2.0 with HCl), a
dietaryerosivechallenge(50mMcitricacidatpH5.0)andacariogenicacidchallenge
(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.
Themassofeachblockwasmeasuredevery24hoursbeforesurfacehardness
measurements were performed. Blocks were taken out of solution, pat dried and
thenweighedusingamicrobalance(PrecisaXT120A,Dietikon,Switzerland).
Vickersmicrohardnessmeasurementsweredeterminedfromindentationson
the lapped GIC surface using aMicrohardness tester (MHT-10, Anton Paar GmbH,
5
Graz, Austria) attached to a microscope (Leica DMPL, Leica Microsystems Wetzlar
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
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
1. ForstenL,MountGJ,KnightG(1994).ObservationsinAustraliaoftheuseof
glass ionomer cement restorative material. Australian Dental Journal
39(6):339-343.
2. GaneshM,ShobhaT (2007).Comparativeevaluationof themarginal sealing
ability of Fuji VII and Concise as pit and fissure sealants. Journal of
ContemporaryDentalPractice8(4):10-18.
3. 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.
4. MazzaouiSA,BurrowMF,TyasMJ,DashperSG,EakinsD,ReynoldsEC(2003).
Incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa
glass-ionomercement.JournalofDentalResearch82(11):914-918.
5. Al Zraikat H, Palamara JE,Messer HH, BurrowMF, Reynolds EC (2011). The
incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa
glassionomercement.DentalMaterials27(3):235-243.
6. Skrtic DA, J. M. Eanes, E. D. (2003). Amorphous calcium phosphate-based
bioactivepolymericcompositesformineralizedtissueregenration.Journalof
Research108(3):167-182.
7. 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.
5
8. ShenP,MantonDJ,CochraneNJ,WalkerGD,YuanY,ReynoldsCetal.(2011).
Effectofaddedcalciumphosphateonenamelremineralizationbyfluorideina
randomizedcontrolledinsitutrial.JournalofDentistry39(7):518-525.
9. Bartlett DW, Fares J, Shirodaria S, Chiu K, AhmadN, SherriffM (2011). The
associationof toothwear,dietanddietaryhabits inadultsaged18-30years
old.JournalofDentistry39(12):811-816.
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.
11. Margolis HC, Moreno EC (1994). Composition and cariogenic potential of
dentalplaquefluid.CriticalReviewsinOralBiology&Medicine5(1):1-25.
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.
15. NicholsonJW,CzarneckaB (2009).Reviewpaper:Roleofaluminuminglass-
ionomer dental cements and its biological effects. Journal of Biomaterials
Applications24(4):293-308.
5
16. CochraneNJ,ReynoldsEC(2009).Caseinphosphopeptidesinoralhealth.Food
ConstituentsandOralHealth172):185-224.
115
116
CHAPTER6
Rechargeandionreleasefollowing
topicalToothMoussetreatmentof
CPP-ACPmodifiedGICinacid
solutions
6.1Abstract(235words):
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.
6.3MaterialsandMethods
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.
Themassofeachblockwasmeasuredevery24hoursbeforesurfacehardness
measurementswereperformed.Blocksweretakenoutofsolution,gentlydriedusing
KimwipesDelicateTaskWipers (KimtechScience,Kimberly-ClarkProfessional,NSW,
Australia) and then weighed using a microbalance (Precisa XT 120A, Dietikon,
Switzerland).Onlysinglemeasurementswerecarriedouttominimisedehydrationof
theGICsurfaceandmaintainconsistencyamongstallsamples.
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).Twoindentationsweremadeoneachblock(Force,1.0N;Dwell,6
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
1. ForstenL,MountGJ,KnightG(1994).ObservationsinAustraliaoftheuseof
glass ionomer cement restorative material. Australian Dental Journal
39(6):339-343.
2. GaneshM,ShobhaT (2007).Comparativeevaluationof themarginal sealing
ability of Fuji VII and Concise as pit and fissure sealants. Journal of
ContemporaryDentalPractice8(4):10-18.
3. 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.
4. MazzaouiSA,BurrowMF,TyasMJ,DashperSG,EakinsD,ReynoldsEC(2003).
Incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa
glass-ionomercement.JournalofDentalResearch82(11):914-918.
5. Al Zraikat H, Palamara JE,Messer HH, BurrowMF, Reynolds EC (2011). The
incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa
glassionomercement.DentalMaterials27(3):235-243.
6. Skrtic DA, J. M. Eanes, E. D. (2003). Amorphous calcium phosphate-based
bioactivepolymericcompositesformineralizedtissueregenration.Journalof
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
8. 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.
9. ShenP,MantonDJ,CochraneNJ,WalkerGD,YuanY,ReynoldsCetal.(2011).
Effectofaddedcalciumphosphateonenamelremineralizationbyfluorideina
randomizedcontrolledinsitutrial.JournalofDentistry39(7):518-525.
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.
11. Bartlett DW, Fares J, Shirodaria S, Chiu K, AhmadN, SherriffM (2011). The
associationof toothwear,dietanddietaryhabits inadultsaged18-30years
old.JournalofDentistry39(12):811-816.
12. 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.
13. NicholsonJW,CzarneckaB (2009).Reviewpaper:Roleofaluminuminglass-
ionomer dental cements and its biological effects. Journal of Biomaterials
Applications24(4):293-308.
14. Crisp S, Lewis BG, Wilson AD (1976). Characterization of glass-ionomer
cements. 2. Effect of the powder: liquid ratio on the physical properties.
JournalofDentistry4(6):287-290.
148
15. Margolis HC, Moreno EC (1994). Composition and cariogenic potential of
dentalplaquefluid.CriticalReviewsinOralBiology&Medicine5(1):1-25.
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.
149
150
CHAPTER7
Aluminiumionreleaseof
conventionalGICsandGiomer
materialsinacidsolutions
7.1Abstract(232words):
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
7.3MaterialsandMethods
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
simulate a gastric erosive challenge (50mM NaCl adjusted to pH 2.0 with HCl), a
dietaryerosivechallenge(50mMcitricacidatpH5.0)andacariogenicacidchallenge
(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
Themassofeachblockwasmeasuredevery24hoursbeforesurfacehardness
measurementswereperformed.Blocksweretakenoutofthesolutionpatdriedand
then weighed using an analytical microbalance (Precisa XT 120A, Dietikon,
Switzerland).Onemeasurementpersamplewasperformedwithanerrorof±0.1mg.
Vickersmicrohardnessmeasurementsweredeterminedfromindentationson
the lapped GIC surface using a microhardness tester (MHT-10, Anton Paar GmbH,
Graz, Austria) attached to a microscope (Leica DMPL, Leica Microsystems Wetzlar
GmbH,Germany).Twoindentationsweremadeoneachblock(Force,1.0N;Dwell,6
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
7.4.1SurfaceHardness
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
7.5.1SurfaceHardness
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
1. ForstenL,MountGJ,KnightG(1994).ObservationsinAustraliaoftheuseof
glass ionomer cement restorative material. Australian Dental Journal
39(6):339-343.
2. GaneshM,ShobhaT (2007).Comparativeevaluationof themarginal sealing
ability of Fuji VII and Concise as pit and fissure sealants. Journal of
ContemporaryDentalPractice8(4):10-18.
3. 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.
4. MazzaouiSA,BurrowMF,TyasMJ,DashperSG,EakinsD,ReynoldsEC(2003).
Incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa
glass-ionomercement.JournalofDentalResearch82(11):914-918.
5. Al Zraikat H, Palamara JE,Messer HH, BurrowMF, Reynolds EC (2011). The
incorporationofcaseinphosphopeptide-amorphouscalciumphosphateintoa
glassionomercement.DentalMaterials27(3):235-243.
6. Skrtic DA, J. M. Eanes, E. D. (2003). Amorphous calcium phosphate-based
bioactivepolymericcompositesformineralizedtissueregenration.Journalof
Research108(3):167-182.
7. 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.
166
8. ShenP,MantonDJ,CochraneNJ,WalkerGD,YuanY,ReynoldsCetal.(2011).
Effectofaddedcalciumphosphateonenamelremineralizationbyfluorideina
randomizedcontrolledinsitutrial.JournalofDentistry39(7):518-525.
9. Bartlett DW, Fares J, Shirodaria S, Chiu K, AhmadN, SherriffM (2011). The
associationof toothwear,dietanddietaryhabits inadultsaged18-30years
old.JournalofDentistry39(12):811-816.
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.
11. Zalizniak I, Palamara JEA,WongRHK,CochraneNJ,BurrowMF,ReynoldsEC
(2013). Ion releaseandphysical propertiesofCPP–ACPmodifiedGIC in acid
solutions.JournalofDentistry41(5):449-454.
12. NicholsonJW,CzarneckaB (2009).Reviewpaper:Roleofaluminuminglass-
ionomer dental cements and its biological effects. Journal of Biomaterials
Applications24(4):293-308.
13. Margolis HC, Moreno EC (1994). Composition and cariogenic potential of
dentalplaquefluid.CriticalReviewsinOralBiology&Medicine5(1):1-25.
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.
2. Margolis HC, Moreno EC (1994). Composition and cariogenic potential of
dentalplaquefluid.CriticalReviewsinOralBiology&Medicine5(1):1-25.
3. 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.
4. Crisp S, Wilson AD (1974). Reactions in glass ionomer cements: III. The
precipitationreaction.JournalofDentalResearch53(6):1420-1424.
5. Crisp S, Wilson AD (1974). Reactions in glass ionomer cements: I.
Decompositionofthepowder.JournalofDentalResearch53(6):1408-1413.
6. WilsonAD,CrispS,FernerAJ(1976).Reactionsinglass-ionomercements:IV.
Effect of chelating comonomers on setting behavior. Journal of Dental
Research55(3):489-495.
175
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).
.
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 4450
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%).
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 4452
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.
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 4454
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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
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