Evaluation of a 6% hydrogen peroxide toothwhitening gel on enamel and dentinemicrohardness in vitro
Andrew Joinera,*, Gopal Thakkera, Yvonne Cooperb
aUnilever Oral Care, Quarry Road East, Bebington, Wirral CH63 3JW, UKb4-Front Research, Unit 6, Capenhurst Technology Park, Capenhurst, Cheshire CH1 6ER, UK
Received 4 September 2003; revised 14 October 2003; accepted 15 October 2003
KEYWORDSIn vitro model; Enamel;
Dentine; Bleaching;
Tooth whitening;
Microhardness; Colour
Summary Objectives. The aims of this study were to evaluate the effects of a novel 6%hydrogen peroxide containing tooth whitener, Xtra White (XW), on enamel and dentinemicrohardness in vitro.Methods. Polished human enamel and dentine specimens were prepared and
baseline microhardness determined. In study 1, enamel specimens were exposed to20 min cycles of either water, XW or Sprite Light for up to 28 cycles. In studies 2 and 3,enamel specimens were treated with 20 min cycles of either XW or water and exposedto whole saliva at all other times. In study 3, an additional exposure to a fluoridecontaining toothpaste was conducted. In total, 28 treatments were conducted in orderto simulate a 2 weeks product use. In study 4, dentine specimens were treated as perstudy 3. Final microhardness measurements were taken and for studies 3 and 4 colourmeasurements were additionally taken.Results. XW and water gave no statistically significant ðp . 0:05Þ changes in enamel
and dentine microhardness after 28 treatments. Sprite Light gave a significantðp , 0:00002Þ reduction in enamel microhardness after one 20 min treatment. XWshowed significant bleaching of enamel and dentine specimens as compared to thewater control.Conclusions. XW does not have any significant effect on enamel and dentine
microhardness.q 2003 Elsevier Ltd. All rights reserved.
Introduction
Aesthetic dentistry, particularly tooth whitening,has increased dramatically in recent years since theintroduction of nightguard vital bleaching in 1989
by Haywood and Heymann1 followed by the launchof a myriad of professional and mass market toothwhitening products.2 –5 These products often con-tain hydrogen peroxide or carbamide peroxide asthe active agent. Carbamide peroxide is an alterna-tive source of hydrogen peroxide since it is readilydissociated into urea and hydrogen peroxide oncontact with water. The tooth whitening mode ofaction involves diffusion of the hydrogen
0300-5712/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.jdent.2003.10.010
Journal of Dentistry (2004) 32, 27–34
www.elsevier.com/locate/jdent
*Corresponding author. Tel.: þ44-151-641-3000; fax: þ44-151-641-1806.
E-mail address: [emailprotected]
peroxide through enamel to the enamel–dentinejunction and dentine where it oxidises colouredpigments to a lighter colour.6,7
As the whitening process often involves thedirect contact of the whitening product on thesurface of the teeth for an extended period oftime, many studies have evaluated the effects ofperoxide-based products on the physical andchemical properties of tooth enamel. In somecases, in vitro evaluations have reported alterationsin microhardness and micromorphology afterexposure to peroxide based formulations8 – 10
whereas others have found no changes.11 –13 Furtherstudies have evaluated effects of peroxide ondentine microhardness and micromorphology andhave also found conflicting results. Changes wereobserved for some studies14 – 16 whilst othersobserved none.17,18 Therefore, it is important forany new peroxide-based formulation to beextensively evaluated for its potential effects ondental hard tissues.
The aims of the current study were to evaluatethe effects of a new tooth whitening productcontaining 6% hydrogen peroxide on themicrohardness of enamel and dentine. Theapproach described by Lussi et al.19 was used, asthis in vitro model has been successfully applied tothe determination of changes in enamel microhard-ness and is particularly sensitive for determiningthe erosive potential of foods and beverages.19,20
This model applies the test material directly ontofreshly polished enamel surfaces. In addition, theeffects on enamel and dentine microhardness wasevaluated in simulated use experiments in vitro,where the enamel and dentine samples wereexposed to the hydrogen peroxide gel and eithersaliva or saliva and a fluoride containingtoothpaste.
Materials and methods
Preparation of enamel and dentinespecimens
The roots of extracted human incisors wereremoved by using a diamond abrasion wheel. Thelingual part of the tooth was then flattened using ahigh abrasive disc (Tycet Ltd. Hemel Hempstead,Herts, UK), turned over and the facial surface thenpolished with the abrasive disc to flatten thesurface. The polished sections were cut on a TwoWell Model 3242 Wire Cutter (Ebner, Le Locle,Switzerland) into approximately 3 £ 3 £ 2 mm3
blocks, containing both enamel and dentine
surfaces. The enamel or dentine surfaces of theblocks were polished successively with 1200 gritsilicon carbide paper (3M United Kingdom plc,Berks, UK), followed by 3, 1 and 0.25 mm diamondpolish (Kemet International Ltd, Maidstone UK).
Microhardness measurement
Hardness measurements of enamel were performedwith a Knoop diamond under a load of 50 g on aMicromet 2100 Microhardness Tester (Buehler, LakeBluff, IL, USA). The indents were made at intervalsof approximately 50 mm in a parallel fashion. Thelengths of the indents were measured with anoptical analysis system. Five indents were made atbaseline and at all time points and the mean indentlength calculated.
Hardness measurements of dentine were per-formed with a Vickers diamond under a load of 500 gon a Micromet 2100 Microhardness Tester (Buehler,Lake Bluff, IL, USA). Five indents were made atbaseline and at the end point, and the mean Vickershardness (VH) calculated.
Study 1: enamel only
Five baseline hardness indents per specimen weremade and measured. The average indent length ofeach specimen was calculated and the blocks wererandomly assigned to one of three treatment groupsðn ¼ 5Þ of either 6% hydrogen peroxide gel (XtraWhite1, Unilever Oral Care), deionised water (Milli-Q Plus, Millipore, USA) or a carbonated beverage(Sprite Light, The Coca-Cola Co., Uxbridge, UK).The specimens were exposed to water (3 ml), SpriteLight (3 ml), or the hydrogen peroxide gel (0.01 ml)for 20 min and then rinsed well with water toremove any residual treatment solution or gel. Fivefurther indents were taken near to the baselineindents, and the mean indent length of the sampledetermined. The specimens were subjected to atotal of 28 £ 20 min treatments in total. The meanindent length was determined after 14 and 28treatments for the water and Xtra White groups.The Sprite Light specimens had the indent lengthmeasured after one and two treatments onlybecause of the excessive changes in the enamelsurface.
The surface morphology of the specimens wasanalysed using light microscopy (Olympus AX70Provis, Olympus UK Ltd, London, UK) and SEM(Cambridge S360, Cambridge Scientific InstrumentsLtd, Ely, Cambs, UK) techniques.
1 Marketed as Signal/Mentadent/Pepsodent/Aim Xtra White.
A. Joiner et al.28
Study 2: enamel plus saliva
The enamel specimens were prepared and indentedas per study design 1. The specimens were randomlyassigned to the treatment groups ðn ¼ 6Þ of eitherthe 6% hydrogen peroxide gel (Xtra White) ordeionised water. They were then treated withsterile (gamma-irradiated) whole stimulated salivaovernight at 37 8C in order to form a salivarypellicle, rinsed with water, dried with a tissue andtreated with either the hydrogen peroxide gel(0.01 ml) or deionised water (3 ml) for 20 min at37 8C. The specimens were thoroughly rinsed withwater to remove any residual product and returnedto the saliva at 37 8C. The treatments wererepeated twice per day until 28 treatments intotal were completed, thus simulating a 2-week’sproduct use. The specimens were finally rinsed withwater, indented with the Knoop diamond and thefinal mean indent length determined.
Study 3: enamel plus saliva and fluoride
The enamel specimens were prepared and indentedas per study design 1. The specimens were randomlyassigned to the treatment groups ðn ¼ 5Þ of eitherthe 6% hydrogen peroxide gel (Xtra White) ordeionised water. They were treated with sterile(gamma-irradiated) whole stimulated saliva over-night at 37 8C, rinsed with water, dried with a tissueand the baseline colour measured with a MinoltaCR-241 Chroma meter (Minolta Camera Co. Ltd,Osaka, Japan) in the Lpapbp mode. The Lp; ap and bp
values form a three-dimensional colour space suchthat Lp represents light-to-dark, ap green-to-redand bp blue-to-yellow.21 The specimens weretreated with a water:toothpaste slurry (2:1, Signal,Lever-Faberge, London, UK; 1450 ppm F as NaF) for30 s and then rinsed with water. The specimenswere dried with a tissue and then treated witheither the hydrogen peroxide gel (0.01 ml) ordeionised water (3 ml) for 20 min at 37 8C. Thespecimens were thoroughly rinsed with water toremove any residual product and returned to thesaliva at 37 8C. The fluoride toothpaste and whiten-ing gel or water treatments were repeated twiceper day until 28 in total were completed, thussimulating a 2-week’s fluoride toothpaste andwhitening product use. The specimens were finallyrinsed with water and the final colour and meanindent length determined. Colour differences werecalculated from
DLp ¼ LpðpostÞ2 Lpð0Þ
Dap ¼ apðpostÞ2 apð0Þ
Dbp ¼ bpðpostÞ2 bpð0Þ
where LpðpostÞ is Lp value post-treatment and Lpð0Þis Lp value at baseline; apðpostÞ is ap valuepost-treatment and apð0Þ is ap value at baseline,and bpðpostÞ is bp value post-treatment and bpð0Þ isbp value at baseline.Total colour change ðDEÞ wascalculated from
DE ¼ ½ðDLpÞ2 þ ðDapÞ2 þ ðDbpÞ2�1=2
Study 4: dentine plus saliva and fluoride
The dentine specimens were prepared and indentedfive times with the Vickers diamond in order to givebaseline microhardness. The specimens were ran-domly assigned to the treatment groups ðn ¼ 7Þ ofeither the 6% hydrogen peroxide gel (Xtra White) ordeionised water. The method of study 3 wasfollowed.
Results
Study 1: enamel only
The mean Knoop indent lengths are shown inTable 1 for all three treatments. For water andthe Xtra White treatments, the differences inmean indent length between baseline and 1, 14and 28 treatments were not of statistical signifi-cance (p . 0:05; t-test, two sample two tailed).However, for Sprite Light there was an increasein indent length after only one 20 min treatmentand this was of high statistical significanceðp , 0:00002Þ compared to baseline. The indentlength increased further for Sprite Light after asecond 20 min treatment. Subsequent treatmentswere discontinued for this group due to the largechanges in the enamel surface roughness increas-ing the surface light scattering and thus makingfurther measurements difficult.
Table 1 Mean Knoop indent lengths for enamel treated witha carbonated beverage, water or Xtra White.
Number oftreatments
Mean Knoop indent length (mm, s.d.)
Carbonatedbeverage
Water Xtra White
Baseline 47.77 (1.25) 49.53 (1.29) 47.65 (0.71)1 61.28 (3.15) 48.60 (1.29) 48.06 (2.25)2 73.31 (6.49) n.d. n.d.14 n.d. 49.88 (1.06) 48.13 (0.79)28 n.d. 49.28 (1.05) 47.69 (0.88)
n.d., not determined.
Evaluation of a 6% hydrogen peroxide tooth whitening gel on enamel and dentinemicrohardness in vitro
29
Typical light microscopy images of enamel after28 treatments with water and Xtra White are shownin Figures 1 and 2, respectively. Qualitatively, therewere no differences between treatments in termsof visual inspection. For the carbonated beveragetreated enamel surfaces, these were too rough toobtain useful images. Therefore, samples weresubmitted for SEM imaging and typical images areshown in Figures 3–5 for the water, Xtra White andcarbonated beverage treated specimens respect-ively. Significant enamel surface degradation hasoccurred with the carbonated beverage treatedspecimens compared to the water and Xtra Whitetreated specimens.
Study 2: enamel plus saliva
The mean Knoop indent lengths for the water andXtra White groups are shown in Table 2. For bothtreatment groups, the differences in mean indentlength between pre- and post treatment were not
Fig. 1 Light microscopy image of enamel after28 £ 20 min water treatments.
Fig. 4 SEM image of enamel after 28 £ 20 min XtraWhite treatments.
Fig. 5 SEM image of enamel after 2 £ 20 min carbo-nated beverage treatments.
Fig. 3 SEM image of enamel after 28 £ 20 min watertreatments.
Fig. 2 Light microscopy image of enamel after28 £ 20 min Xtra White treatments.
A. Joiner et al.30
of statistical significance (p . 0:05; t-test, twosample two tailed). Thus, the microhardness ofenamel had not significantly changed followingeither of these treatments.
Study 3: enamel plus saliva and fluoride
The mean Knoop indent lengths before and after 28treatments with a fluoride toothpaste and eitherwater or Xtra White are shown in Table 3. For bothtreatments, the mean post-treatment indent lengthwas numerically lower than the pre-treatmentindent length indicating an increase in enamelhardness. However, the differences between pre-and post treatments were not of statistical signifi-cance (p . 0:05; t-test, two sample two tailed).Colour change values (DLp;Dap;Dbp and DE) areshown in Table 4. The enamel specimens treatedwith Xtra White became whiter (increase in Lp) andsignificantly less yellow (decrease in bp) (p , 0:001;t-test, two sample two tailed) compared to base-line indicating that bleaching had occurred. Inaddition, compared to the water control, XtraWhite gave a significantly greater reduction in
yellow ðp , 0:05Þ and greater overall colour changeassociated with bleaching ðp , 0:02Þ:
Study 4: dentine plus saliva and fluoride
The mean Vickers Hardness Numbers (VHN) beforeand after 28 treatments with a fluoride toothpasteand either water or Xtra White are shown in Table 5.The differences between pre- and post treatmentsand the differences between water and Xtra Whitewere not of statistical significance (p . 0:05; t-test,two sample two tailed). Colour change values(DLp;Dap;Dbp and DE) are shown in Table 6. Thedentine specimens treated with Xtra White becamesignificantly whiter (p , 0:006; t-test, two sampletwo tailed), significantly less yellow ðp , 0:05Þ andgave a significantly greater total colour changeðp , 0:02Þ compared to the water control.
Discussion
The approach used by Lussi et al.19 was chosen forthe current studies because it offers a simple andeffective method for screening small changes toenamel surface microhardness.22 Indentations weremade at intervals of 50 mm in such a manner thatone indent at baseline was followed by thecorresponding indentation after one treatment.Subsequent indents after 2, 14 and 28 treatmentswere placed close to the line of the original indents.Thus, this overall indent design attempts todiminish the influence of possible inhom*ogeneitiesof the substrate surface.
Table 3 Mean Knoop indent lengths for human enamel pre-and post 28 treatments with a fluoride toothpaste and eitherwater or Xtra White.
Treatment Knoop length (mm, s.d.)
Pre Post
Water 51.6 (3.3) 48.7 (3.3)Xtra White 51.7 (1.8) 50.3 (2.9)
Table 6 Colour changes of dentune specimens: DLp;Dap;Dbp
and DE (s.d.).
Treatment DLp Da Dbp DE
Water 2.0 (3.1)a 0.0 (0.7) 20.2 (2.5)b 3.7 (2.7)c
Xtra White 7.2 (2.7)a 0.7 (0.5) 23.5 (2.8)b 8.0 (2.9)c
Statistical significant differences (t-test, two sample, twotailed). ap , 0:006; bp , 0:05; cp , 0:02:
Table 2 Mean Knoop indent lengths for human enamel pre-and post 28 treatments with water and Xtra White ðn ¼ 6Þ:
Treatment Knoop length (mm, s.d.)
Pre Post
Water 52.3 (4.3) 51.5 (5.1)Xtra White 52.6 (4.3) 49.1 (1.1)
Table 4 Colour changes of enamel specimens: DLp;Dap;Dbp
and DE (s.d.).
Treatment DLp Da Dbp DE
Water 20.4 (3.1) 21.1 (1.0) 22.0 (1.5)a 3.6 (1.8)b
Xtra White 2.0 (4.1) 21.1 (0.5) 24.6 (1.9)a 6.5 (1.1)b
Statistical significant differences (t-test, two sample, twotailed). ap , 0:05; bp , 0:02:
Table 5 Mean Vickers Hardness Numbers (VHN) for humandentine pre- and post 28 treatments with a fluoride tooth-paste and either water or Xtra White.
Treatment VHN (s.d.)
Pre Post
Water 50.5 (4.3) 46.8 (3.2)Xtra White 50.2 (5.9) 47.4 (4.2)
Evaluation of a 6% hydrogen peroxide tooth whitening gel on enamel and dentinemicrohardness in vitro
31
Microhardness changes are related to a loss orgain of mineral (demineralisation or remineralisa-tion) of the dental structure.23 It has been shownthat the microhardness test is suitable fordetermining small changes in surface microhard-ness that demonstrate the effects of acids,24 acidicbeverages19,20 and bleaching products12,25,26
on enamel. The suitability of the method wasagain confirmed in the current studies where nosignificant changes in enamel microhardness wereobserved after treatment with water or XtraWhite. Indeed, in the first study where no salivaand no protective effects of a salivary pelliclewere present,27 perhaps the most severe of thethree types of tests, there were no significantchanges in microhardness or differences in sur-face morphology for the water and Xtra Whitetreated enamel blocks. However for the acidiccarbonated beverage Sprite Light (pH ¼ 2.88)19
the enamel was significantly softened even afteronly one 20 min treatment as seen by thesignificant increase in Knoop indent length posttreatment. This confirms the previous findings ofLussi et al.19 who observed an approximately20 mm increase in Knoop indent length for enamelexposed to the same cabonated beverage after20 min. The SEM image of enamel after 2 £ 20 mintreatment of the carbonated beverage clearlyshows similarities to enamel that has been acidetched with 37% phosphoric acid for 60 s,11 thusconfirming the reason for the changes in surfacemicrohardness. The surface morphology ofenamel samples treated 28 £ 20 min with wateror Xtra White showed none of these etchedfeatures.
Lussi et al.19 describes the extrapolation of invitro results like those in a study similar to study1 to the in vivo situation as difficult, as there areother parameters involved such as bufferingcapacity and flow rate of saliva, and salivarypellicle formation. Thus, in order to simulate thein vivo situation more closely, the experimentaldesigns in studies 2–4 included, exposure tosaliva where the additional protective nature ofsaliva and salivary pellicle are involved,27,28 andfollowed the recommended in vivo product useinstructions. In studies 2 and 3, water and XtraWhite treated enamel again showed no significantchange in microhardness. The mean post-treat-ment indent lengths were numerically lower thanthe pre-treatment indent lengths indicating aslight increase in enamel hardness, althoughthese changes were not of statistical significance.This effect is due to the remineralisation beha-viour of saliva or saliva and fluoride on theenamel.29
In study 4, the microhardness values of thedentine specimens changed only slightly and thedifference between baseline and post treatmentwas not of statistical significance. In addition,differences between water and Xtra White werenot of statistical significance. Dentine is ananisotropic material in terms of its collagen fibrilorganisation and dentinal tubule orientation.30
However, it has been shown that dentine isisotropic with respect to Vickers microhardnessbut is anisotropic with respect to the Knoopmicrohardness measurement.30 Thus, the Vickersdiamond was chosen for the current dentinestudy, allowing for more consistent microhard-ness measurements irrespective of sampleorientation.
In the third and fourth study, the colour of theenamel and dentine specimens respectively weremeasured and for the Xtra White group it wasshown that they had increased in whiteness anddecreased in yellow. This shows that significantbleaching had occurred in contrast to the watercontrol group where there were no significantchanges in colour. This makes the subsequentmeasurements of the effects of peroxide on theenamel and dentine properties even more rel-evant since many literature studies of the effectsof bleach on hard tissues do not confirm orreport actual tooth bleaching.31
In considering the quantity of product applied inthese experiments, the amount can be consideredto be in excess of in vivo applications since therewas no elution of the gel from the enamel surface.This is in contrast to the in vivo situation where it isknown that peroxide levels within bleaching pro-ducts are depleted during use.32 Thus the exper-imental design in the current studies were in excessof what is anticipated under normal use, and can beused as further confirmation of the safety of thisproduct when used according to the manufacturer’sinstructions.
The microhardness evaluation of the currentstudy revealed no significant softening effects ofthe enamel and dentine by Xtra White. This is inagreement with many previous reportedstudies5,11 – 13,18,31,33 on the passivity of thebleaching process on dental hard tissues. It isnot fully understood why other studies havereported a decrease in hardness with peroxide-based products. This is probably due to differ-ences in experimental design, for example,Shannon et al.34 have suggested that some ofthe studies have not taken the remineralisationpotential of saliva into account because testingwas performed under conditions where possiblemineral loss could occur. Indeed, de Freitas
A. Joiner et al.32
et al.16 observed a slight decrease in dentinemicrohardness immediately post bleaching with a10% carbamide peroxide product which recoveredto its original baseline value after 14 days inartificial saliva. In addition, Price et al.35 reportslarge differences between 26 commercially avail-able tooth whitening products in terms of theirperoxide concentration (up to 35%) and pH range(3.67–11.13). These wide differences in formu-lation could well be a factor in the reporteddifferent product effects on hard tissues. Thus,the impact of any new tooth whitening producton tooth tissue integrity needs to be fullycharacterised prior to launch.
In conclusion, the data from a number ofstudies indicate that a new tooth whiteningproduct, Xtra White, containing 6% hydrogenperoxide does not have any detrimental effectson enamel and dentine microhardness ormorphology.
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A. Joiner et al.34
