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Year : 2020  |  Volume : 31  |  Issue : 6  |  Page : 924-929
Effect of different home-bleaching techniques for a regular or an extended time on enamel properties

1 Department of Restorative Dentistry, Faculty of Dentistry, University of Rio de Janeiro State, Rio de Janeiro, RJ, Brazil
2 Metallurgical and Materials Engineering Program, COPPE, Rio de Janeiro, RJ, Brazil
3 Nanotechnology Engineering Program, COPPE, Rio de Janeiro, RJ, Brazil
4 Biophysics, Physics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil
5 Prosthesis Department, School of Dentistry of State University of Rio de Janeiro (UERJ), Rio de Janeiro, RJ, Brazil

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Date of Submission10-Oct-2019
Date of Decision13-Mar-2020
Date of Acceptance05-Aug-2020
Date of Web Publication22-Mar-2021


Context: The side effects of bleaching products are still incompletely known. Aims: This work aims to evaluate the effects of bleaching regimens on colour variation, microstructure, roughness, composition and nanohardness of human dental enamel until 8 weeks. Settings and Design: Twenty teeth were cross-sectioned to obtain eighty enamel fragments (50 × 50 mm) divided into four groups: CN (control Negative-artificial saliva), CP10 (10% carbamide peroxide), HP10 (10% hydrogen peroxide), and WS (whitening strips-10% hydrogen peroxide). Methods and Material: Roughness (atomic force microscopy–AFM and 3-D surface scanning), morphology (confocal laser scanning microscopy–CLSM and AFM), hardness and elastic modulus (nanoindentation), and composition (Raman microspectroscopy) were analysed before the therapy and after 4 and 8 weeks. Colour measures were performed weekly. Statistical Analysis Used: Two-way ANOVA with repeated measures (P < 0.05). Results: Bleaching stabilizes after 3 weeks for HP10 and after 4 weeks for CP10 and WS. Roughness evaluation showed statistical difference for HP10 after 8 weeks for Sa and Sq, for HP10 and WS after 4 weeks and for CP10 after 8 weeks. The same occurred for hardness and elastic modulus. The morphological evaluation demonstrated the most significant effects after 8 weeks of treatment for HP10 and WS. Composition analysis revealed modifications in peaks related to the organic content spectra (protein) with an increase in detection after 4 weeks, followed by a decrease after 8 weeks. Conclusions: H2O2-based products caused morphological and compositional alterations on enamel.

Keywords: Atomic force microscopy, dental bleaching, human enamel, hydrogen peroxide, Raman spectroscopy

How to cite this article:
De Miranda MS, Eltom AE, Souza Camargo Sd, Rocha GM, Reis Perez Cd. Effect of different home-bleaching techniques for a regular or an extended time on enamel properties. Indian J Dent Res 2020;31:924-9

How to cite this URL:
De Miranda MS, Eltom AE, Souza Camargo Sd, Rocha GM, Reis Perez Cd. Effect of different home-bleaching techniques for a regular or an extended time on enamel properties. Indian J Dent Res [serial online] 2020 [cited 2022 Jun 27];31:924-9. Available from:

   Introduction Top

Bleaching agents decompose, causing oxidation of coloured inorganic and organic compounds.[1],[2],[3],[4],[5] Excessive exposure during application and longer treatments than usually proposed can exaggerate the possible adverse effects of the products.[6],[7],[8],[9],[10],[11] The sophisticated high inorganic and small organic structure of enamel are responsible for mechanical properties.[12],[13]

This study aims to evaluate the effects of applications' regimens for up to 8 weeks on the microstructure, roughness, composition and nanohardness of human dental enamel associated with three different home-bleaching agents. Confocal laser scanning microscopy (CSLM), atomic force microscopy (AFM), micro-spectroscopy Raman and nanoindentation measurements were performed.

   Methods and Materials Top

The ethics committee on human research from Pedro Ernesto University Hospital (CEP-HUPE) approved the protocols. Twenty sound human third molars were donated by the Bank of Human Teeth of UERJ Dentistry School and examined under a stereomicroscope loupe (Lumagny® 6X, n° 7547, Hong Kong, China) to eliminate the ones with defects. They had their roots removed and were cut in a cross-shape section on the occlusal surface with a water-cooled, low-speed diamond saw to obtain four fragments (buccal, lingual, mesial, and distal). The sections were divided into four groups (CN, CP10, HP10, WS) and treated following the manufacturer's instructions [Table 1].
Table 1: Bleaching agents and protocol

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The full treatment period was 8 weeks. Samples were analysed before the therapy and after 4 and 8 weeks of bleaching. Colour measurements were made weekly to observe the approximate time of whitening saturation, from which no gain in the process was observed.

Bleaching simulation was performed following Sorozini et al., (2017)[8] methodologies. A 0.05 mL layer of each bleaching gel was applied using a calibrated micropipette. During the therapy, the samples were stored in 100% humidity at 37°C. After the established time, the gel was it held under running tap water for 30 seconds. Subsequently, the specimens were placed in an individual receptacle containing 13.5 mL of artificial saliva solution and stored at 37°C. These solutions were changed daily. The specimens used in CP10 and HP10 were covered by a custom-made tray of silicon during the application. A compatible piece of the strip was used to cover each fragment of WS.

As part of the experiments, all specimens were submitted to simulated tooth brushing with an electric brush (Oral-B Vitality, Model 3709, Procter & Gamble Oral Care, Kronberg, Germany). Only one operator performed the brushing for 5 s twice a day, before the application of the bleaching gel, and after 15 min of storage in saliva after the gel's removal. The technique was performed with 90 g of pressure with distilled water.[8] Samples were evaluated for topographic changes with confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM), and roughness, composition, nanohardness, and elastic modulus changes. Colour measurements were performed weekly to observe the bleaching saturation time of each material.

The specimens were marked with two lines forming a dagger before the beginning of the analysis to define the analysis region. For this, a scratch test was performed on the nanoindenter G200 Agilent (MTS Systems Corporation, Santa Clara, CA, USA). Flatter areas of the sample were selected. The test was programmed to perform two crossed lines (200 μm each) with a 50 mN force.

Weekly, each specimen was analysed using digital spectrophotometer Vita Easy Shade (Vita Zahnfabrik, Bad Säckingen, Germany), which calculates CIEL *a*b* values. L* evaluates the brightness of the sample. At the same time, a * and b * are related to the saturation of the colour, following the methodology presented by Sagel and Gerlach, 2007.[14] The differences for the values were calculated from ΔL*, Δa* e Δb*. Changes of less than two units of ΔL* or ΔE are considered imperceptible, while values above three are considered as evidence of a clinically visible change.

CLSM measurements: The samples were analysed at three periods, by Zeiss apparatus, model LSM 510 META. This analysis was chosen as it allows a bigger view of the sample (300 μm × 300 μm) without destruction and allowing it to be its control. The objective was to verify structural changes and was performed according to Sorozini et al. (2018).[15]

AFM measurements: surfaces were imaged by Dimension Icon Scanning Probe Microscopy (Bruker–Santa Barbara, CA, USA) using Peak Force Tapping. Image processing (line-wise flatten only) was performed using NanoScope Analysis software (Bruker, Santa Barbara, CA, USA); according to Lippert et al. (2004)[12] and Sorozini et al., (2018),[15] selections of 30 μm × 30 μm were performed in all images to evaluate roughness and profile analysis, and the values of Sq and Sa were recorded for statistical analysis.

Raman microspectroscopy: A Raman spectrophotometer (inVia Qontor, Renishaw, New Mills, Gloucestershire, UK) equipped with a 785 nm diode laser coupled with an optical microscope and operating in the StreamLine HR™ mode was used in line with the postulates of Rae et al. (2014).[16] Three different equipment's settings were used to scan the same area.: 1-Range from 390 to 1551 cm−1 (centre 1000 cm−1) with 10% of the laser potency, one integration time of 5 s, 2-Range from 1130 to 2550 cm−1 (centre 1665 cm−1) with 10% of the laser potency, one integration time of 10 s, and 3-Range from 2494 to 3271 cm−1 (centre 2900 cm−1) with 100% of the laser potency, one integration time of 10 s. The collected spectra were normalised according to the absorbance of the phosphate band (n1PO4) at 960 cm−1 using the Raman spectrophotometer's software (Wire, Renishaw).

Nanoindentation: Tests were performed using Agilent G200 Nanoindenter (Santa Clara, CA, USA), which has a displacement resolution of <0.01 nm and a force resolution of 50 nN with a loading cycle in 15 s and a peak hold for 2 s. This methodology was based on the works of Oyen (2006)[17] and Ge et al. (2005).[18]

Statistical analysis: The sample size calculation was performed with a power of 0.8 and a significance level of α < 0.05 (Axum 7, Mathsoft, Cambridge, Massachusetts, USA), and twenty teeth were selected. Statistical analysis was performed by SPSS 20.0 for Windows. Two-way repeated-measures ANOVA and Bonferroni's adjustment for multiple comparisons were used.

   Results Top

Color measurements: For HP10 the colour remained stable from the 3rd week of treatment on when the colour achieved a level 1 on Classic Vita Shade Colour. For the CP10 and the WS, the color stabilisation occurred from the 4th week until 8 weeks.

CLSM measurements: The three most significant images of the same sample are presented in [Figure 1] that shows the evolution of the different therapies through time. In CN and CP10, there were no significant changes in the images obtained. The depth of the cross increased after 8 weeks of treatment for HP10, and the presence of risks from the brushing was evident. Also, it was no longer possible to observe the presence of prisms in [Figure 1]I. These events were also observed through AFM. Likewise, WS presented an increase in depth of the cross after 4 weeks and, after 8 weeks, loss of prisms in some of the areas, probably related to the disorganisation of the prismatic pattern by the combination of bleaching strips and brushing in the proposed regimen.
Figure 1: Evolution of CSLM measurements, (initial, 4 weeks, and 8 weeks): CN – a, b, c; CP10 — d, e, f; HP10 —g, h, i; WS — j, k, l

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AFM measurements: [Figure 2] shows the characteristic keyhole-like prisms, which are arranged compactly and separated with each other by their organic sheaths. The WS did not show any changes with the application of brushing and storage in artificial saliva. The prismatic structure can be well observed and easily identified. In CP10, after 8 weeks, it was possible to identify a more granular structure with small points, meaning more exposure to the nanoparticles of hydroxyapatite crystals. This feature was found in HP10 after 4 and 8 weeks. [Figure 2]G, [Figure 2]H and [Figure 2]I, which represents the specimens bleached with HP10, show significant changes in the time intervals tested. After 4 weeks, the prismatic structure was less defined, with a degradation of the original pattern. The most significant effects on morphology, however, were observed after 8 weeks of treatment: the prismatic pattern was wholly lost, and there was a smoother surface. Besides, scratches could be observed in the specimens, a contribution of the wear promoted by the brushing. It was also observed in WS. After 4 weeks of bleaching strips, the prisms became more evident, and a loss of substance is suggested mainly in the interprismatic region, which seems to have portions torn out. After 8 weeks, the prismatic pattern became less evident and scratches were observed on the surface with few remaining prisms. The evolution of roughness parameters Sq and Sa is shown in [Table 2]. There was a statistically significant difference for HP10 and WS after 4 weeks and CP10 after 8 weeks of treatment (P < 0.05).
Figure 2: Evolution of AFM measurements, (initial, 4 weeks and 8 weeks): CN – a, b, c; CP10 - d, e, f; HP10 - g, h, i; WS - j, k, l

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Table 2: Mean and standard deviation of Sq and Sa values for each group (nm). Groups with statistical difference (P<0.05) are highlighted in bold

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Raman microspectroscopy results: The mean spectra obtained for the previously described ranges were analysed and can be seen in [Figure 3]. For HP10, after 4 weeks, there was an increased detection of the 1450 cm−1 peak relative to the C-H bending of protein molecules [Figure 3]B. Likewise, peak 1243 cm −1 corresponding to amide I showed an increase of count in that range. After 8 weeks, HP10 and WS had a decrease in the detection of protein molecules. For WS, the spectra returned to be like the control. For the third group of spectra [Figure 3]C, the same pattern previously described was found for these bands.
Figure 3: (a) Characteristic band of the v1 po4 at 960 cm-1 - hydroxyapatite; (b) Detection of the 1450 cm-1 peak relative to the c-h bending from protein molecules; (c) Peaks located at 2881 cm-1 and 2950 cm-1 related to the C-H stretching of organic molecules

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Nanoindentation measurements: The mean and standard deviation for nanohardness and elasticity is shown in [Table 3]. Specimens of CN kept their values of elasticity. On the other hand, HP10 and WS presented a decreasing tendency for the values of elasticity after 4 weeks. Qualitative image analysis and roughness values showed an increase in roughness after 4 weeks. In CP10, a decrease was observed after 4 weeks (P < 0.05). The behaviour of nanohardness for the groups can be observed in [Table 3].
Table 3: Mean and standard deviation of the values of nanohardness and elasticity (GPa) for each group. Groups with statistical difference (P<0.05) are highlighted in bold

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   Discussion Top

In this study, the bleaching gels were compared concerning their influence on the surface, composition and physical properties of enamel specimens. The absence of planing and polishing allows a better comparison to clinical reality and more reliable results despite the challenge for the execution of evaluation techniques.[2],[15],[19],[20],[21] Another strategy was to use the sample as its control once only non-destructive techniques were performed.[22] It was also decided to use artificial saliva with daily changes.[2],[8],[20],[23],[24],[25],[26] There are few articles in the literature with the use of bleaching gels for more than four weeks.[5],[6],[7],[8],[9] It was observed that the colour stabilisation occurred from the 3rd week on for HP10 and the 4th week on for CP10 and WS.

AFM worked complementary to CSLM as it represents a non-destructive technique that allows the observation of surfaces and their alterations on a nanometer scale, being one of the most powerful tools for evaluating the crystals of HAp and the organisation of the prisms in the enamel.[25],[27],[28] The parameters Sq and Sa revealed vertical variations of the sample during therapy.[29] The effects on microstructure observed by CLSM and AFM showed an initial loss of structural organisation for HP10 and WS after 4 weeks and for CP10 after 8 weeks. The behaviour of the enamel samples in these groups is related to the application of gels of higher concentration of H2O2, which have been shown to have a more deleterious effect on the surface. The presence of scratches shows that toothbrushing seems to have had an essential role in removing minerals depleted due to the oxidation of the organic matrix.[19],[24],[30],[31] Thus, a smoother surface, with possible exposure of subsurface layers beyond the markings from the brushing, was observed after 8 weeks for HP10 and WS. However, it should be emphasized that even in the groups that presented a tendency to increase roughness (CP10 after eight weeks and HP10 and WS after four weeks), Sa values were lower than the 0.2 μm, which is not enough to induce biofilm retention.[7],[19]

For the samples' composition analysis, Raman spectroscopy was chosen since it is a non-destructive method that evaluates changes in molecular composition.[22],[32],[33],[34] Some studies found differences in composition.[16],[22],[34],[35],[36] Changes occurred in the spectra readings with the centre at 1600 cm−1 (HP10 and WS). After 4 weeks of use, the counts increased for both groups, and the peaks related to the organic content, 1243 cm−1 (amide I) and 1450 cm−1 (CH bending), became more evident. After 8 weeks, the count decreased again, with the same peaks remaining more evident when compared to the control. In HP10, the change was more evident at both tested intervals. These changes suggest a possible effect of brushing on the specimens. Combining information on the composition and microstructural findings of disorganisation (observed by AFM and CLSM), it could be suggested that groups bleached with H2O2 presented removal of superficial layers with exposure of subsurface proteins, generating higher counts after 4 weeks of use.

The nanohardness and elastic modulus evaluations determine the response of the materials to plastic and elastic deformations.[17],[18],[24],[26],[35],[36],[37] The non-uniform arrangement of the HAp crystals can promote energy dissipation while maintaining its resistance. The crystals would be able to slide between them, mainly due to the presence of proteins, and, therefore, become responsible for the anisotropic and viscoelastic behaviour of the enamel.[16],[33],[34] Proteins in enamel have sacrificial bonds capable of unfolding against tensions and recovering their arrangement with the removal of forces.[17],[18],[24],[37],[38],[39],[40],[41] Therefore, the oxidation of these components seems to be decisive for altering the mechanical behaviour of the enamel.[22] In the present study, the cumulative morphological changes and storage in artificial saliva seem to have decreased the nanohardness and elasticity in CP10 after 8 weeks of treatment and HP10 and WS after 4 weeks, with an increase after 8 weeks. Probably, a smoother surface offers more resistance to the penetration of the indenter. For HP10 and WS, the decline with the subsequent increase of the values for this same parameter was statistically significant. These results can be related to the surface alteration with loss of the organisation of the structural arrangement, besides the possible influence of the remineralisation with artificial saliva as a function of the bleaching challenge. The results of the nanoindentation tests obtained are contrary to most findings in the literature,[14],[26],[39],[40],[41],[42] but this can be attributed to the employed method involving simulated brushing and ultrasound application, which could remove weak surface layers, like what happens clinically. Indeed, the use of artificial saliva (with daily changes) associated with simulated brushing distinguishes this work from others, where the cumulative effect of bleaching agents can produce different results.

It was possible to conclude that there were differences between the times of evaluation and between the home-bleaching agents. All bleaching methods tested induced changes, morphologically and ultra-structurally, in the nanohardness and the composition. Materials have shown to be safe while respecting the whitening effectiveness time.


The authors would like to thank CAPES for scientific support.

Financial support and sponsorship

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES)

Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Dr. Cesar dos Reis Perez
R. São Francisco Xavier, 524 . Maracana . Rio de Janeiro, RJ - Cep 20550-900
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdr.IJDR_791_19

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]


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