FT-Raman and Energy Dispersive X-Ray Fluorescence Spectrometric Analyses of Enamel Submitted to 38% Hydrogen Peroxide Bleaching,an Acidic Beverage,and Simulated Brushing

Abstract

Objective: This study aimed to investigate the effects on enamel surface treated with hydrogen peroxide bleaching and acidic soft drink immersion and/or brushing with whitening dentifrices. Materials and Methods: Fifty-six standardized enamel slabs obtained from labial surfaces of bovine incisors were used. Enamel slabs were ground flat, polished, and randomly assigned to one of seven treatment groups: (1) control, in which no treatment was performed, (2) soft drink immersion, (3) 38% hydrogen peroxide bleaching, (4) simulated toothbrushing with whitening dentifrice, (5) soft drink immersion and bleaching, (6) soft drink immersion, bleaching, and toothbrushing, and (7) bleaching and toothbrushing. The mineral concentration of enamel surfaces was determined before and after treatments by means of Fourier transform (FT)-Raman spectroscopy and energy dispersive x-ray fluorescence spectrometry (EDXRF). Data were statistically analyzed by ANOVA and Tukey test (p < 0.05). Results: Raman spectroscopy results indicated that enamel mineral content decreased after all treatments except in group 1, whereas EDXRF results exhibited mineral decrease in groups 3, 4, 5, and 7. Conclusion: Bleaching alone or in combination with soft drink immersion and brushing decreases enamel mineral content.

Introduction

H ydrogen peroxide has been largely accepted as the most popular active compound of dental bleaching agents for nearly a century.¹ Its acceptance is related to successful clinical performance at both low and high concentrations.^2,3 An in-office bleaching procedure is a practical alternative to in-home bleaching treatments, especially with severe discoloration, poor patient compliance, or the need for rapid results. High concentrations of peroxides are also recommended as an initial bleaching therapy that can be extended to or combined with home-bleaching procedures.^1,4

The widespread use of highly concentrated peroxides is a concern because it has been shown that bleaching may cause morphological alterations that compromise the enamel surface integrity,⁵ increase enamel roughness,^6,7 change the enamel inorganic composition,^8,9 decrease enamel microhardness,^3,7,10,11 and decrease enamel mineral content.¹²

In order to overcome hydrogen peroxide side effects, fluoride therapy is strongly recommended after bleaching.^13

–16 Among the existing fluoride treatments, dentifrices have been widely adopted as the main method of delivering topical fluoride and providing caries preventive benefits.¹⁷ Patients performing bleaching are more inclined to use dentifrices with whitening properties, but these dentifrices can be associated with wear due to their high abrasiveness.^18,19 It is assumed that patients undergoing bleaching treatment should use only low-abrasive toothpastes since bleaching itself can lead to enamel mineral loss.²⁰ In addition, mineral loss can be intensified by the consumption of acidic food or beverages, leading to enamel erosion described as enamel dissolution as a result of direct acid attack.^21,22 An in vitro evaluation of bovine enamel immersed in different acidic beverages (soft drinks and orange juice) showed a significant enamel mineral loss especially after exposure to those containing citric acid.²¹ Regarding this, it is speculated that bleaching agents and whitening toothpastes are harmful due to enamel loss, especially after the consumption of acidic drinks.

Enamel composition after bleaching, brushing, and contact with an acidic solution can be evaluated through sensitive analysis of the enamel's chemical aspects to determine inorganic modifications. In this regard, Fourier transform (FT)-Raman spectroscopy and energy dispersive x-ray fluorescence spectrometry (EDXRF) are established methods for simple and nondestructive analyses for obtaining information with respect to molecular composition and structure of substrates. These analyses permit identifying and quantifying enamel mineral compounds and, because they are nondestructive, enamel can be observed before and after treatments.^23

–26

Due to the described changes caused by bleaching on an enamel surface, the aim of the present study is to evaluate the effects of 38% hydrogen peroxide on enamel mineral content when abrasive dentifrices are used for brushing with and exposure to an acidic beverage occurs.

Materials and Methods

This study had the approval of the Ethical Committee Guidelines of the University of Taubaté in accordance with the National Health Council (n°0004/07).

Experimental design

Experimental units consisted of 56 bovine enamel slabs. The factor under study was enamel treatment (seven levels) according to the following conditions: (1) control group, with no bleaching, no simulated brushing, and no acidic soft drink immersion; (2) soft drink immersion (Coca-Cola^® Light Lemon, Coke Co-FEMSA, Jundiaí, SP, Brazil); (3) 38% hydrogen peroxide bleaching (Opalescence Xtra Boost-Ultradent Products, South Jordan, UT, USA); (4) simulated toothbrushing with whitening dentifrice (MaxWhite; Colgate-Palmolive, Co. Osasco, SP, Brazil); (5) soft drink immersion and bleaching; (6) soft drink immersion, bleaching, and toothbrushing; (7) bleaching and toothbrushing.

Changes in inorganic content were measured by means of FT-Raman spectroscopy and by EDXRF before and after the treatments. The variable responses were statistically analyzed.

Specimen preparation

Twenty-eight sound, extracted bovine incisors, stored in 0.1% thymol solution at 5°C for no longer than 2 weeks after extraction, were used in this study. The teeth were cleaned of gross debris and placed in deionized water for 24 h before the experiment. The root was sectioned from the coronary portion with a double-faced diamond disk (No. 7020; KG Sorensen, Barueri, SP, Brazil) on a low-speed handpiece (Intramatic 2068, Kavo, Joinville, SC, Brazil) and the crown was sectioned to obtain dental slabs (4.0 × 4.0 × 4.0 mm). The slabs were leveled using a water-cooled mechanical grinder (Aropol 2V; Arotec, Cotia, SP, Brazil), ground flat with water-cooled aluminum oxide grit papers (no. 600, 800, 1000, and 1200; 3M ESPE, St. Paul, MN, USA), and polished with 6.0, 3.0, 1.0, and 0.25 μm diamond pastes (Arotec, Cotia, SP, Brazil). After polishing, enamel slabs were randomly divided into one of seven groups (n = 8):

Control, stored in remineralizing solution (0.1 M Tris buffer, 1.5 mM calcium, 0.9 mM phosphate, 150 mM KCl [pH 7.0]²⁷) through the experiment

Soft drink immersion (pH 2.0; Coca-Cola Light Lemon)

Bleaching (38% hydrogen peroxide, Opalescence Xtra Boost)

Toothbrushing (Tek; Johnson and Johnson, São José dos Campos, SP, Brazil) with whitening dentifrice (pH 6.7–7.7; MaxWhite)

Soft drink immersion and bleaching

Soft drink immersion, bleaching, and toothbrushing

Bleaching and toothbrushing

Treatment protocols

Samples were subjected (according to the previous group distribution) to one of three treatments: immersion in acidic beverage, bleaching procedure, or simulated brushing. These events were not performed simultaneously due to the limitations of each treatment. Therefore, when the group was not designed to join a specific treatment, it remained immersed in the remineralizing solution at 37°C. Group 1 was not treated and remained stored in artificial saliva through the experimental period.

The total amount of time of the experimental phase was 14 d: the immersion in acidic beverage was performed in 3 d, bleaching was performed in two sessions during 7 d, and the 30,000 simulated brushing cycles were performed during 4 d. Throughout the experimental period the remineralizing solution was replaced every day. Before and after treatments, samples of all groups were submitted to FT-Raman and EDXRF analyses.

Immersion in an acidic beverage

Each specimen from Groups 2, 5, and 6 was immersed in 2 mL of an acidic soft drink (Coca-Cola Light Lemon, replaced daily) in individually capped vials for 72 h. Specimens were not agitated and were kept at 37°C. Afterward, specimens were rinsed thoroughly with distilled and deionized water and stored in a second vial containing 2 mL of remineralizing solution at 37°C. The remineralizing solution was replaced every day.

Bleaching procedure

Group 1 was not treated. Group 2 specimens were immersed in soft drink as described for specimens of Groups 2, 5, and 6. Group 3 specimens were bleached with highly concentrated hydrogen peroxide (pH = 7.0, 38% Opalescence Xtra Boost). The bleaching activator was mixed with the bleaching agent and a 1 mm pellicle of the gel was applied to the exposed enamel surface and light activated for 1 min (light emitting diode [Ultraled]; Heatless Curing Light, Dabi Atlante, Ribeirão Preto SP, Brazil), left undisturbed for 8 min, and agitated for 5 min. The gel was again left undisturbed for 1 min with a total time of 15 min of bleaching. Afterwards, samples were rinsed with distilled and deionized water and a second bleaching application was performed after a 3 min interval, as described before. After bleaching, samples were stored in 2 mL of remineralizing solution for 7 d. After this period of storage, a second bleaching session (with two applications of the gel) was performed.

Group 4 samples were brushed according to the simulated brushing protocol, Group 5 samples were immersed in the acidic soft drink for 72 h and then bleached, and Group 6 samples were first immersed in the acidic soft drink for 72 h and then bleached. At the end, samples were cycled 30,000 times in the simulated brushing machine. G7 samples were bleached and then submitted to the simulated brushing.

Simulated brushing

The simulated brushing procedure was performed 7 d after the bleaching. Group 4, Group 6, and Group 7 specimens were subjected to 30,000 cycles in a simulated toothbrushing machine (Equilabor; Piracicaba, SP, Brazil) with the cycle speed set at 4.5 strokes/sec and 200 g of load/weight. Soft nylon toothbrushes heads (Tek) were adjusted to the brushing machine and replaced at 15,000 cycles. Slurry was prepared with whitening dentifrice and distilled water (1 g/1 mL) and then placed on the enamel surface slabs at 0.4 mL/min. The simulated tooth brushing was done during four consecutive days. Afterward, specimens were removed from the simulated brushing machine, ultrasonically cleaned with water for 10 min, and immersed in remineralizing solution for 24 h. After this period, samples of all groups were submitted to FT-Raman and EDXRF analyses.

FT-Raman spectroscopy

The enamel surface was analyzed by FT-Raman spectroscopy to evaluate changes in enamel components. There was no need for specific sample preparation prior to Raman and EDXRF analyses. Both analytical methods are nondestructive and the data collection was based on laser or x-ray interactions with the sample without contact. Spectra of the samples before and after enamel bleaching were obtained using an FT-Raman Spectrometer (RFS 100/S^®; Bruker Inc., Karlsruhe, Germany). To excite the spectra, the defocused 1064.1 nm line of a Nd:YAG laser source was used. Maximum incident laser power on the sample surface was about 100 mW and spectrum resolution was 4 cm⁻¹.

The samples were positioned over a glass slide in the sample holder compartment and an IR354 lens collected radiation scattered over 90° on the enamel surface. For each sample, one spectrum was collected at a central point on the enamel surface. In order to obtain a good signal to noise ratio, 100 scans were co-added for each spectra. Altogether, 112 spectra were obtained.

Data analysis

The spectra in the region of interest, from 300 to 4000 cm⁻¹, were analyzed using a curve-fitting routine, which allowed the subtraction of background scattering and reduction of noise level by digital filtering. All spectra were processed by fitting six Raman vibrational stretching modes: 430 cm⁻¹ (p1), 449 cm⁻¹ (p2), 587 cm⁻¹ (p3), 610 cm⁻¹ (p4), 1044 cm⁻¹ (p5), and 1070 cm⁻¹ (p6). For the qualitative and semiquantitative spectral analysis, the spectra were baseline corrected and then normalized to the 960 cm⁻¹ peak. In order to obtain the area of each band, band decomposition was performed by Gaussian shapes. The integrated areas of the peaks were calculated by the Microcal Origin 5.0^® software (Microcal Software, Inc., Northampton, MA, USA) for the control and treated samples. The averages of integrated areas of the evaluated Raman peaks (p1–p6) were calculated for the control and bleached data.

EDXRF measurements

Semiquantitative elemental analyses of calcium (Ca) and phosphorus (P) were carried out with an energy-dispersive micro X-ray fluorescence spectrometer (model μEDX 1300; Shimadzu, Kyoto, Japan) equipped with a rhodium X-ray tube and a Si(Li) detector cooled by liquid nitrogen (N₂) and coupled to a computer system for data processing. The voltage in the tube was set at 15 kV, with automatic adjustment of the current and beam diameter at 50 μm. Three spectra from each specimen were collected before and after the treatments. The measurements were performed with a count rate of 100 sec/point (live time) and a dead time of 25%. The energy range of scans was 0.0–40.0 eV. The equipment was adjusted using a certified (SIGMA, 2008) commercial reagent of stoichiometric hydroxyapatite [Sigma-Aldrich, (Poole, UK) synthetic Ca₁₀(PO₄)₆(OH)₂, grade 99.999%, lot 10818HA] as reference. The measurements were collected under fundamental parameters of characteristic X-ray emission of the elements Ca and P, and the elements O and H were used as chemical balance. The energy calibration was performed using internal standards of the equipment.

Statistical analysis

The measurements of the Ca/P ratio obtained by EDXRF and the area values of the Raman peaks were statistically analyzed using BioEstat 2.0 software (Mamirawá Beréon do Pará, PA, Brazil). Statistical analyses were performed using the Wilcoxon test to compare data at baseline and after treatment and the Kruskal–Wallis test to compare among treatments. For all analyses, 5% was considered the limit of significance. The linear Pearson correlation was used because the data were only quantitative.

Results

EDXRF results are shown on Table 1. At baseline, all groups presented similar Ca/P ratios on enamel surface (p > 0.05). After treatments, enamel mineral content of Group 1 remained unaltered. There was a significant decrease in the Ca/P ratio of Group 2–Group 7 after treatments (p < 0.05) and no differences were found among these groups after treatment (p > 0.05). A positive correlation was found between EDXRF and FT-Raman analyses with the value of r = 0.3532 (p = 0.0001).

Table 1.

Median and Range (Minimum and Maximum Data) of the Ca/P Ratio of Enamel at Baseline and After Treatment

Groups	Baseline	After treatment
1. Control	2.12 (1.8–2.4) Aa	2.15 (1.8–2.6) Aa
2. Soft drink immersion	2.19 (1.9–2.5) Aa	2.00 (1.8–2.2) Bb
3. Bleaching	2.17 (1.9–2.2) Aa	1.89 (1.8–2.0) Bb
4. Toothbrushing	2.11 (2.0–2.2) Aa	1.99 (1.9–2.2) Bb
5. Soft drink immersion + bleaching	2.34 (2.1–2.7) Aa	1.91 (1.7–2.1) Bb
6. Soft drink immersion + bleaching + toothbrushing	2.11 (1.9–2.3) Aa	1.87 (1.7–2.1) Ba
7. Bleaching + toothbrushing	2.23 (1.9–2.9) Aa	1.87 (1.5–2.1) Bb

Median followed by distinct letters differ statistically at 5% (Wilcoxon and Kruskal–Wallis). Uppercase letters show significant differences among groups (columns). Lowercase letters show significant differences between treatment times (lines).

Raman spectra of the six vibrational stretching modes corresponding to the phosphate and carbonate enamel concentrations were analyzed. The typical Raman spectra for the control and bleached bovine enamel were evaluated in the region of 300–1200 cm⁻¹. The spectra were vertically shifted for clarity and the peaks at 431 cm⁻¹ and 590 cm⁻¹ were related to PO₄ ³⁻ ν₂ and PO₄ ³⁻ ν₄ modes of phosphate, respectively. The peak at 1044 cm⁻¹ was attributed to PO₄ ³⁻ ν₃ mode of phosphate and the peak at 1071 cm⁻¹ was attributed to both carbonate (B type CO₃ ²⁻ ν₁) and phosphate (PO₄ ³⁻ ν₃) vibrations.

The results of the integrated area of the vibrational stretching modes are presented in Table 2. At baseline, all groups had similar results (p > 0.05), and Group 2 through Group 7 presented similar phosphate concentration and were similar to Group 1 (p > 0.05). The same differences and similarities among groups were found after treatments. After treatment, a significant decrease of phosphate concentration was observed for Group 2 (acidic beverage), Group 3 (bleaching treatment), Group 4 (toothbrushing treatment), and Group 6 (acidic beverage, bleaching, and toothbrushing) compared to Group 5 and Group 7. Group 1, the control group, was similar to all groups after treatment (p < 0.05).

Table 2.

Median and Range (Minimum and Maximum) of the Integrated Area of Raman Picks (p1–p6) at Baseline and After Treatments

Groups	Baseline	After treatment
1. Control	2.92 (2.7–3.1) Aa	2.89 (2.7–3.0) ABa
2. Soft drink immersion	2.96 (2.8–3.2) Aa	2.79 (2.6–2.9) Bb
3. Bleaching	3.03 (2.9–3.2) Aa	2.84 (2.6–3.0) ABb
4. Toothbrushing	2.88 (2.8–3.0) Aa	2.77 (2.6–3.0) Bb
5. Soft drink immersion + bleaching	3.09 (2.6–3.0) Aa	2.93 (2.8–3.6) Ab
6. Soft drink immersion + bleaching + toothbrushing	2.99 (2.8–3.6) Aa	2.78 (2.8–3.1) Bb
7. Bleaching + toothbrushing	2.99 (2.9–3.2) Aa	2.92 (2.4–3.1) Ab

Median followed by distinct letters differ statistically at 5% (Wilcoxon and Kruskal–Wallis test). Uppercase letters show significant differences among groups (columns). Lowercase letters show significant differences between treatment times (lines).

Discussion

A number of studies have examined the influence of tooth bleaching on enamel microhardness, morphology, and enamel strength.^{3,5
–7,10,11,19,28
–30} Although controversial, it has been observed that tooth bleaching, even at low peroxide concentration, causes perceptible alterations on enamel prism peripheries and mild enamel prism core loss.²⁸ These mineral content changes could be aggravated by acidic soft drink consumption as well as common clinical procedures such as brushing with abrasive whitening dentifrices.^18

–21

This study evaluated enamel behavior before and after different surface treatments, and the enamel mineral content was determined by means of FT-Raman spectroscopy and EDXRF analysis. The EDXRF analysis provided information regarding Ca/P ratio whereas FT-Raman was able to detect differences in the phosphate and carbonate enamel peaks.^5,23

–26 After data collection, integrated areas of the evaluated Raman peaks (p1–p6) were averaged and a possible loss of the enamel's phosphate concentration was established, whereas EDXRF data showed enamel mineral loss related to the Ca/P ratio of enamel surface before and after treatments.

The control group, which was not treated and remained in remineralizing solution, presented the same Ca/P ratio and phosphate and carbonate concentrations at baseline and at the end of the experimental period as shown by the EDXRF and FT-Raman results. In a previous investigation, enamel micromorphological alterations were demonstrated after 10% carbamide peroxide bleaching, whereas no surface alterations were detected on in situ bleached enamel.³¹ This in situ–in vitro contrast may be explained by the remineralization effect of saliva under in situ conditions.^30,31

The soft drink used (Coca-Cola Light Lemon) was chosen due to its acidic pH of 2, which could promote enamel erosion.²² The EDXRF results showed a significant enamel mineral loss after immersion in the soft drink. A significant decrease of enamel mineral content after acidic beverage immersion and bleaching and after acidic beverage, bleaching, and toothbrushing was also observed.

Toothbrushing alone or with whitening dentifrice or bleaching treatment promoted mineral loss after treatment. Although dentifrices may assist enamel uptake because a satisfactory fluoride concentration (1100 ppm) is available and delivered during brushing,¹⁷ most whitening dentifrices are abrasive¹⁸ and may affect enamel surface by removing mineral compounds, possibly resulting in enamel abrasion.¹⁹

Some authors reported that low hydrogen peroxide concentrations do not affect microhardness and enamel chemical composition.²⁵ However, it is possible that the high hydrogen peroxide concentration used in the present study leads to enamel mineral loss. Oltu and Gürgan⁵ observed a significant decrease of the Ca/P ratio after beaching with 35% carbamide peroxide compared with low carbamide peroxide concentrations by means of infrared absorption spectroscopy and X-ray diffraction analysis. A previous study⁸ demonstrated more Ca²⁺ loss from enamel surface after 35% and 38% hydrogen peroxide bleaching than after 10% carbamide peroxide treatment. The authors linked this significant enamel calcium loss to the potential of highly concentrated hydrogen peroxide to cause dental demineralization.⁸ In a previous Fourier-transformed infrared spectroscopy (FT-IR) evaluation, researchers observed enamel chemical changes after 10%, 20%, and 30% hydrogen peroxide treatment. The enamel alterations in the area of the biological PO₄ ν1 and PO₄ ν2 were directly proportional to the treatment time and peroxide concentration.⁹

Enamel mineral loss after bleaching may be explained by the whitening agent composition and its oxidation mechanism. Hydrogen peroxide breaks down into unspecific, unstable, and highly reactive free radicals that are believed to decompose the organic and inorganic enamel matrix.³² It is assumed that the effects of free radicals on the organic and inorganic enamel matrix may alter the chemical and morphological enamel structure.^{5
–7,12,16,28}

Highly concentrated hydrogen peroxide is an effective and widespread technique for tooth whitening. However, as observed, it may promote Ca/P enamel loss and being associated with acidic soft drinks and toothbrushing possibly increases enamel mineral injury.

Toothbrushing with whitening dentifrices may change the enamel surface due to abrasiveness, but in contrast, fluoride delivery to a bleached surface may prevent enamel inorganic damage. Although the results of both chemical analyses indicate that toothbrushing alone leads to enamel mineral loss, such loss is possibly related to enamel abrasion and wear²⁰ rather than demineralization. Conversely, the immersion of enamel in an acidic beverage (pH 2) for an extended time (72 h) may promote enamel dissolution or erosion,²¹ whereas enamel bleaching with 38% hydrogen peroxide (pH 7) may lead to enamel demineralization due the oxidation process and the effects of free radicals on the organic and inorganic matrix.^{5
–7,12,16,28} Since EDXRF and FT-Raman are not able to evaluate erosion or demineralization depth, these assumptions should be confirmed by a perfilometer test, which would determine enamel surface topography and/or polarized light microscopy and cross-sectional microhardness to evaluate enamel demineralization depth.

Although treatments (acidic beverage, bleaching, and toothbrushing) singly or collectively promoted a significant decrease of enamel Ca/P ratio compared to baseline values, FT-Raman results after treatment showed that the control group had similar results to all groups. The lowest phosphate/carbonate content was observed after acidic drink immersion (Group 2), toothbrushing (Group 4), and the treatments together (Group 6). One possibility for the differences presented by FT-Raman data of the enamel compared to EDXRF is that the first provides the concentration of carbonate, which is possibly the first element of enamel lost by erosive, abrasive challenges and bleaching procedures, since carbonate apatite is more soluble than hydroxyapatite.³³

The effects of acidic beverages, bleaching, and toothbrushing on enamel alone or together should be further investigated. Based on the results and considering the limitations of this in vitro study, care must be taken when performing in-office bleaching, specifically with frequency of application. Additionally, acidic soft drinks and toothbrushing with highly abrasive dentifrices should be avoided during bleaching treatment.

Conclusion

The application of an acidic beverage, bleaching treatment with 38% hydrogen peroxide, and toothbrushing whitening dentifrices to enamel surface were able to increase enamel mineral loss, leading to a decrease in the Ca/P ratio and phosphate/carbonate enamel content.

Footnotes

Acknowledgments

This work was supported by FAPESP (01/14384-8 and 05/50811-9).

Author Disclosure Statement

No competing financial interests exist.

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