Effects of Bleaching Agents Combined with Regular and Whitening Toothpastes on Surface Roughness and Mineral Content of Enamel

Abstract

Objective: The purpose of this study was to evaluate surface roughness and changes in the composition of enamel submitted to different bleaching protocols and toothbrushing with regular and whitening toothpastes. Background data: Bleaching treatment could promote morphological and chemical changes in enamel surface. Methods: Enamel blocks were randomized into nine groups (n=10) according to the bleaching treatment (no bleaching, control group; 6% hydrogen peroxide, HP; or 10% carbamide peroxide, CP) and toothpaste used (placebo, PL; regular, R; or whitening dentifrice, W). Bleaching was performed according to manufacturers' instructions and all groups were submitted to 30,000 cycles of simulated toothbrushing with toothpaste (PL, R, or W). Mineral content evaluation and enamel roughness were evaluated initially (T₁), after bleaching (T₂), and after toothbrushing (T₃), using an energy-dispersive micro X-ray fluorescence spectrometer and profilometry, respectively. Data were statistically analyzed with two way ANOVA, Tukey, and Dunnett tests (5%). Results: Enamel surface roughness was influenced by bleaching and toothbrushing. Surface roughness increased for the groups that brushed with the placebo dentifrice (CP+PL, HP+PL, C+PL) and for the control group that brushed with whitening dentifrice (C+W). Enamel Ca/P ratio decreased after bleaching, but toothbrushing, regardless of the dentifrice used, did not reduce the enamel mineral content. Conclusions: The bleaching treatment resulted in a decrease of enamel mineral content, but the studied dentifrices did not contribute to surface mineral loss.

Introduction

Home-applied bleaching agents, typically containing carbamide peroxide (CP) as the active compound, have been extensively used to whiten stained teeth through oxidation of the pigments found in the dental structure.^1
–3 Currently, low-hydrogen peroxide (HP) concentrations (6–15%) are available for whitening, and the bleaching technique is similar to that of CP. The basic difference relies on the mode of application, as the manufacturers' instructions call for the use of 6% HP for 1–2 h/day instead of the conventional overnight protocol indicated for 10% CP agents.

In general, patients undergoing bleaching brush their teeth three or four times a day to maintain good oral health,⁴ and they frequently perceive the use of whitening toothpastes as a way to hasten the bleaching process. Dentifrices with whitening properties have emerged as an option to achieve and enhance whitening of the tooth structure.^5,6

Whitening dentifrices attempt to provide both therapeutic and aesthetic outcomes to patients.² Therapeutically, these pastes contain fluoride, an effective anticaries agent that also prevents and controls the demineralization of dental hard tissues.⁷ These dentifrices also contain additives, such as calculus-inhibiting, antiplaque, tooth-desensitizing, breath-freshening, stain-controlling and remineralizing agents. The dentifrice abrasives (including calcium carbonate, dicalcium phosphate dihydrate, hydrated silica, alumina, perlite, and sodium bicarbonate) play a cosmetic role because of their ability to remove extrinsic stains, therefore contributing to the tooth whitening aspect.^2,6,8,9 Whitening dentifrices may contain chemicals (enzymes, detergents, and oxygenation agents) that are able to inhibit or directly remove stains from the dental surface without the physical action promoted by the abrasives.^5,10

The combination bleaching treatment/whitening dentifrice is somehow disconcerting, as most whitening dentifrices contain a high amount of abrasives in different sizes and shapes to improve extrinsic stain removal.⁸ Furthermore, the exposure of hard dental tissues to bleaching agents can result in microstructural changes in the enamel surface, such as enamel defects and degradation.¹¹ Chemical evaluations have also detected mineral loss and decreases in Ca/P concentration in bleached enamel.^12,13

In previous investigations, CP agents were not able to increase enamel surface roughness, but a significant increase in roughness was observed when bleaching was associated with brushing and regular abrasive dentifrices.^4,14 The effects of the association of HP, acidic soft drinks, and toothbrushing with whitening dentifrices on enamel mineral content have been previously evaluated by FT-Raman and energy-dispersive X-ray (EDX) analyses, which demonstrated that this combination decreases enamel mineral content.¹²

Considering that limited data are available regarding the efficiency of peroxide agents in low concentration combined with whitening and regular dentifrices and their possible combined effect on the mineral content of the enamel structure, this study tested the alternative hypotheses that bleaching agents associated with whitening or low abrasive toothpastes would: (1) increase surface roughness and (2) decrease the Ca/P ratio of the enamel surface.

Materials and Methods

This study was reviewed and approved by the Ethical Research Committee of the Taubaté Dental School, UNITAU, Brazil (protocol #0041/07). Forty-five bovine incisors were extracted and stored in 0.1% thymol solution at 5^°C for 1 week. The teeth were cleaned and placed in artificial saliva for 24 h. Then, the root was cut from the coronary portion and four dental slabs (4×4×3 mm) were obtained from each incisor and leveled using a water-cooled mechanical grinder (Aropol 2V, Arotec, Cotia, SP, Brazil). The enamel surface was flattened with water-cooled aluminum oxide grit papers (Paper Sheets # 600, 800, 1000, and 1200, 3M ESPE, St. Paul, MN) and polished with 6, 3, 1, and 0.25 μm diamond pastes (Diamond Paste, Arotec, Cotia, SP, Brazil).

Enamel slabs were randomly divided into nine groups (n=20), according to bleaching treatment (no bleaching, control, C; 10% carbamide peroxide, CP; or 6% hydrogen peroxide, HP) and toothpaste used (regular, R; whitening, W; or placebo dentifrice, PL) as follows: CP+R; CP+W; CP+PL; HP+R; HP+W; HP+PL; C+R; C+W; and C+PL.

Bleaching treatments were performed according to the regimen proposed by the manufacturer, followed by simulated toothbrushing. Mineral content and enamel roughness were evaluated initially (T₁), after bleaching (T₂) and after simulated toothbrushing (T₃) using an energy-dispersive micro X-ray fluorescence spectrometer (μEDX) and a profilometer, respectively. Before treatments (bleaching/toothbrushing) and analysis (μEDX/ profilometry), samples were kept in a remineralizing solution (artificial saliva: 1.45 mM calcium, 5.4 mM phosphate, 0.1 M Tris buffering solution) at 37°C.¹⁵

Treatments

Bleaching

The HP bleaching agent (White Class, FGM, SC, Brazil), pH 6.0-7.0, was applied for 1 h/day for 15 days while CP (NiteWhite ACP, Discus Dental Inc., Culver City, CA, USA), pH 6.5, was applied 6 h/day for 30 days, according to manufacturer instruction. In each bleaching agent application, a 1 mm thick pellicle of the gel was applied on the enamel surface. Afterward, samples were rinsed with distilled water and stored in the remineralizing solution at 37°C until the next application. Samples not submitted to bleaching (C+R, C+W, and C+PL) remained stored in the remineralizing solution at 37°C. After the bleaching regimen, samples were stored for 12 h prior to analysis (T₂).

Simulated toothbrushing

Samples were subjected to 30,000 cycles (corresponding to ∼1.7 year of normal brushing)¹⁶ in a simulated toothbrush machine (Equilabor, Piracicaba, SP, Brazil), with the cycle's speed set at 4.5 strokes/sec and 200 g of load/weight. Soft nylon toothbrush heads (Oral B Indicator, Procter & Gamble, SP, Brazil) were adjusted to the brushing machine and replaced at 15,000 cycles.¹² Toothbrushing cycles were performed using three dentifrices: Colgate Total 12 (R) (Colgate-Palmolive Industries Ltd., São Bernardo do Campo, SP, Brazil), a low abrasive dentifrice containing hydrated silica (15–21%) and sodium fluoride (1450 ppm), with pH 7.0–8.0, according to recommended dietary allowance (RDA)=68; Crest Multicare Whitening (W) (Procter & Gamble, Mason, OH), a highly abrasive dentifrice containing tetrasodium pyrophosphate (3–7%) and hydrated silica (15–40%), and sodium fluoride (1100 ppm), with pH 7, according to RDA=128; and a manipulated dentifrice (PL) (Byofórmula, São José dos Campos, SP, Brazil), a highly abrasive, with calcium carbonate (40%), sodium bicarbonate (4%), and sodium fluoride (1000 ppm), with pH 5.3, according to RDA=135. The manipulated dentifrice was used to compare its efficacy with the manufactured whitening dentifrice, because both were considered highly abrasive toothpastes. A slurry was prepared with each dentifrice (PL, R, and W) and distilled water (1 g:1 mL w/v).¹² After toothbrushing, samples were ultrasonically cleaned for 10 min and immersed in the remineralizing solution for 12 h prior to the analyses.

Analyses

The analyses were performed at baseline (T₁), after bleaching treatment (T₂), and after simulated toothbrushing (T₃), as follows.

Surface roughness

A profilometer (Surf test-211, Mitutoyo, Tokyo, Japan) was used to perform three measurements in different directions on the enamel surface using the parameter Ra (μm) and a cut-off of 0.25 mm to determine the average surface roughness (Ra) of each sample.

μEDX

A μEDX fluorescence spectrometer (1300, Shimadzu, Kyoto, Japan), equipped with a rhodium X-ray tube and a Si (Li) detector cooled by liquid nitrogen, was coupled to a computer data system and used to determine the Ca/P ratio and analyze the Ca P elements on the enamel surface. The experimental measure of the proportion of Ca/P standard used was 1.68 (0.05). The energy electronic transition experimental (Kα) was 3.689 keV for the Ca element and 2.010 keV for the P element. The parameters used for the μEDX and subsequent data analysis were developed according to the method described by Paula and co-authors.¹²

Statistical analysis

The normal distribution of values was analyzed by the Shapiro–Wilk test. The exploratory data analysis was performed with SAS 9.1 software (SAS Institute, Cary, NC) to determine the normal distribution of the results of the μEDX and profilometer analysis for the ANOVA parameters. The factors “groups” and “time” and their levels were considered for statistical analysis. The Tukey test was used to perform multiple comparisons of mean values whenever significant interactions between “groups” and “time” were observed (p<0.05). An additional data analysis (ANOVA/Dunnett) was performed to compare the group results obtained after bleaching (T₂) with the baseline (T₁) and final evaluations (T₃). In this analysis, T₁ was considered a negative control and T₃ was considered a positive control. The control groups C+R, C+W, and C+PL were not included in this analysis because they received no bleaching. All analyses were conducted using the SAS 9.1 software with the level of significance (α) fixed at 5%. Surface roughness data were converted into base 10 log for statistical analysis. The ANOVA test with repeated measures in time was applied for all response variables. For the surface roughness analysis, a significant interaction was detected by ANOVA between the factors “group” and “time” (p=0.0006) and the Tukey test identified the differences among the levels of the factors.

Results

Surface roughness

The results (Table 1) indicate that surface roughness significantly increased after toothbrushing for groups CP+PL, HP+PL, C+W, and C+PL. In group C+R, the inverse occurred, and a surface roughness decrease was observed. Groups CP+R, CP+W, HP+R, and HP+W presented similar results at T₁ and T₃. At baseline (T₁), all groups presented similar surface roughness. After toothbrushing (T₃), the highest roughness values were observed in groups CP+R, CP+PL, HP+W, HP+PL, and C+PL whereas the lowest values were observed for groups HP+R and C+R. Group CP+W presented intermediate values that were statistically similar to all studied groups. Group C+W also displayed intermediate surface values that were similar to groups CP+R, CP+PL, HP+R, HP+W, HP+PL, and C+PL. Group C+W, however, presented a higher value than group C+R.

Table 1.

Mean Values (Standard Deviation) of Surface Roughness of the Groups at Baseline (T₁), After Bleaching (T₂), and After Toothbrushing (T₃)

	Time
Groups	Baseline (T₁)	After bleaching (T₂)	After toothbrushing (T₃)	Roughness increase (%)
CP+R	5.1 (1.4) Aa^*	3.7 (0.8)	5.8 (1.3) Aa^*	14
CP+W	4.4 (0.5) Aa^*	3.5 (1.2)	5.0 (1.2) ABCa^*	14
CP+PL	4.7 (1.1) Aa	3.7 (1.4)	5.7 (1.6) Ab^*	21
HP+R	3.7 (1.1) Aa	3.5 (1.1)	3.9 (1.4) BCa	5
HP+W	5.2 (1.6) Aa	4.6 (1.5)	6.0 (1.2) Aa^*	15
HP+PL	4.8 (1.5) Aa	4.3 (1.0)	5.9 (1.5) Ab^*	23
C+R	4.6 (1.3) Aa		3.7 (1.3) Cb	−20
C+W	4.5 (1.1) Aa		5.6 (1.2) ABb	24
C+PL	4.4 (0.8) Aa		6.1 (1.1) Ab	39

Mean followed by different letters indicate statistical differences according to ANOVA and Tukey tests, α=5%. Capital letters compare groups (columns) and lower case letters compare time (lines).

Statistical differences of those of T₂ (after bleaching) according to Anova and Dunnet tests, α=5%.

C, control; CP, carbamide peroxide; HP, hydrogen peroxide; PL, placebo (toothpaste); R, regular (toothpaste); W, whitening (toothpaste).

The Dunnett test was additionally applied to compare groups after bleaching (T₂). Groups CP+R and CP+W presented lower roughness values at T₂ than at T₁. The roughness values of groups CP+R, CP+W, CP+PL, HP+W, and HP+PL significantly increased at T₃.

μEDX

Table 2 presents data obtained from μEDX analysis in which the Ca/P ratio was considered for statistical analysis. The experimental measure of the proportion of Ca/P standard used was 1.68 (0.05). The interaction between the factors “group” and “time” was not significant (p=0.23); therefore, the main factors were analyzed separately. No differences were observed among the levels of the factor “group” (p=0.18); however, differences were observed among “time” (p<0.0001), and an increase in the Ca/P ratio was observed after toothbrushing (T₃).

Table 2.

Mean Values (Standard Deviation) of Ca/P Ratio of the Groups at Baseline (T₁), After Bleaching (T₂), and After Toothbrushing (T₃)

	Time
Groups	Baseline (T₁)	After bleaching (T₂)	After toothbrushing (T₃)
CP+R	2.0 (0.1)^* Aa	3.6 (1.0)	3.7 (0.9) Ab
CP+W	1.9 (0.2)^* Aa	3.7 (0.7)	3.0 (0.4)^* Ab
CP+PL	2.0 (0.2)^* Aa	3.5 (1.4)	3.5 (1.0) Ab
HP+R	2.0 (0.1)^* Aa	2.7 (0.4)	3.0 (0.6) Ab
HP+W	2.2 (0.3)^* Aa	3.7 (0.9)	3.5 (0.4) Ab
HP +PL	1.9 (0.2)^* Aa	3.0 (0.9)	3.3 (1.1) Ab
C+R	1.9 (0.1) Aa		3.2 (0.7) Ab
C+W	2.0 (0.9) Aa		2.9 (0.7) Ab
C+PL	2.0 (0.1) Aa		3.2 (0.7) Ab

Mean followed by different letters indicate statistical differences according to ANOVA and Tukey tests, α=5%. Capital letters compare groups (columns) and lower case letters compare time (lines).

Statistical differences of those of T₂ (after bleaching) according to ANOVA and Dunnet tests, α=5%.

C, control; CP, carbamide peroxide; HP, hydrogen peroxide; PL, placebo (toothpaste); R, regular (toothpaste); W, whitening (toothpaste).

The Dunnett test presents the comparisons among the Ca/P ratio obtained after bleaching (T₂) with the other phases (T₁ and T₃). The values obtained for the six bleached groups at baseline (T₁) were significantly lower than after bleaching (T₂). Group CP+W also presented higher Ca/P ratio values after bleaching (T2) than after the final evaluation (T₃).

Discussion

In recent years, effects of home-applied bleaching agents on enamel morphology have been reported, such as surface roughness and microhardness, enamel mineral loss potential, and decrease in adhesive resistance after bleaching.^{8,17

–25} In addition, it has been demonstrated that surface changes on bleached enamel can be intensified by toothbrushing procedures, especially those that use abrasive dentifrices.^4,6,19,26,27 Whitening dentifrices have the potential to reduce and remove extrinsic stains because of the presence of specific chemicals that decrease or inhibit stain either by lightening the stain or by physical desorption of adherent stains (surfactants, enzyme systems, calcium-chelating builders, and calcium phosphate absorbents).² Additionally, dentifrice abrasives incorporated into the paste eliminate the pellicle and the extrinsic pigment itself.^5,10 However, whitening dentifrices, which possibly contain more abrasive particles than conventional toothpastes, may be more likely to increase enamel surface roughness when combined with bleaching.

The bleaching treatment showed no differences in surface roughness when compared with baseline for most studied groups (CP+PL, HP+R, HP+W, and HP+PL), and groups CP+R and CP+W showed a decrease in surface roughness when compared with baseline means (Table 1, T₁ T₂). Previous results also showed that surface roughness was not changed,^16,28

–31 or showed a slight decrease³² after bleaching treatment. Navimipour et al.³² demonstrated that the enamel surface bleached with 15% CP presented a slight decrease in surface roughness. The authors related that enamel surface roughness only increased when submitted to immediate brushing after bleaching treatment. Pachaly and Pozzobon¹⁶ also reported increased roughness after bleaching treatment associated with brushing with a whitening dentifrice. In the present study, the association of bleaching and toothbrushing was able to increase surface roughness, regardless of the toothpaste (R, W, or PL) or the bleaching agent used (CP or HP) (Table 1, T₂ T₃). These results are in accordance with previous reports that attested that bleaching agents alone were unable to increase surface roughness,^16,28

–31 but that the association with abrasive dentifrices promoted roughness on the enamel surface.^4,14,16,32 It has also been observed that the opposite situation occurs and that toothbrushing itself would not increase enamel surface roughness.²⁶

After toothbrushing, a significant increase in surface roughness, ranging from 21% to 39%, was observed in groups CP+PL, HP+PL, C+W, and C+PL (Table 1, T₁ T₃). Groups CP+PL, HP+PL, and C+PL underwent toothbrushing with calcium carbonate (40%) and sodium bicarbonate (4%) enhanced dentifrice (manipulated), and group C+W underwent toothbrushing with the whitening dentifrice containing hydrated silica (15–40%) and tetrasodium pyrophosphate (3–7%). In contrast, group C+R exhibited a decrease in surface roughness, possibly because no bleaching treatment was performed and the dentifrice used was of low abrasiveness (Colgate Total 12). Therefore, the selection of a toothpaste with low abrasivenes is important to maintain enamel roughness at a minimum, especially when bleaching is performed.

The abrasive cleaning process is influenced by some toothpaste factors, including particle hardness, shape, size, size distribution, concentration, applied load,³³ and even pH.³⁴ These factors are possibly responsible for the surface roughness increase or decrease promoted by the three dentifrices, as it has been observed that whitening toothpastes can produce greater surface roughness after bleaching becaue of the higher amounts and types of abrasives in their composition.³⁵

The regular toothpaste used (Colgate Total 12) contains hydrated silica and titanium dioxide as abrasives, whereas the whitening dentifrice (Crest Whitening Multicare) contains a combination of hydrated silica, pyrophosphate, sodium bicarbonate, and sodium carbonate. Dentifrices with a combination of abrasives such as hydrated silica, bicarbonate, and sodium bicarbonate plus calcium pyrophosphate, which are typically found in whitening toothpastes, are reported to be more abrasive than calcium carbonate and silica plus alumina.⁶ Although it would be expected that there is a higher abrasiveness in whitening toothpastes, the manipulated dentifrice was the one that was able to increase roughness, despite its association with the bleaching treatment. The manipulated dentifrice contained a high concentration of calcium carbonate (40%) and sodium bicarbonate, which could itself promote roughness. Additionally, the manipulated dentifrice had the lowest pH among the toothpastes used (5.3 against the neutral pH of the others), which could assist with stain removal and probably could also lead to enamel surface alterations.

The Ca/P ratio decreased after bleaching, and no differences between bleaching agents were observed (Table 2). The ratio between Ca and P elements (Ca/P) in the samples was calculated by the ratio of the relative weights determined by direct reading equipment, which provides software data from the conversion calculation of the relative intensity of the x-rays of the transition energies characteristic of the elements. We can then consider that values >2 indicate enamel mineral loss, which probably caused the decrease in the relative weight of the Ca element used to calculate the proportion Ca/P. Enamel mineral loss remains a controversial topic among researchers, because some microchemical studies have shown no mineral loss after tooth bleaching.^25,36 Tooth whitening occurs because of the dissociation of HP and CP into unstable free radicals that break down the stain molecules in dental hard tissues,¹³ and this redox reaction could affect the organic and inorganic enamel matrix.^12,13 In the current study, the mineral loss observed after bleaching is in accordance with previous EDX and FT-Raman evaluations, which demonstrated Ca/P loss of enamel bleached with different agents.^{12,13,22,37

–42}

Toothbrushing, regardless of the dentifrice used, did not decrease the enamel mineral concentration. On the contrary, it was observed that group CP+W (Table 2) presented higher Ca/P concentration after toothbrushing than after bleaching. This result was expected, because the presence of fluoride in the dentifrices and the storage media used (remineralizing solution) could possibly increase the enamel mineral content.⁴³

The hypotheses tested were partially accepted because (1) increased enamel surface roughness but no changes in enamel were observed when bleaching alone was performed, and, finally, (2) bleaching was able to increase mineral loss, but toothbrushing seemed to increase enamel mineral content.

Conclusions

Overall, the use of bleaching agents led to a decrease in enamel mineral content, and the toothpastes studied did not contribute to surface mineral loss.

Footnotes

Acknowledgments

We thank Prof. Dr. Lourenco Correr Sobrinho for the use of the Laboratory of Dental Materials at Piracicaba Dental School (University of Campinas) and Prof. Dr. Ana Paula R. Alves Claro for the use of the Materials Laboratory at Guaratinguetá (State University of São Paulo).

Author Disclosure Statement

No competing financial interests exist.

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