Effect of pigmenting agents on tooth enamel staining during immediate tooth whitening: an in vitro study estudo in vitro GDS, Barbosa IF. pigmenting tooth enamel staining during immediate

de hidrogênio; corantes; pigmentação. Abstract Introduction: Considering the increased search for esthetic procedures, questions emerge about the maintenance and regression of color, especially regarding the need to restrict dark foods during bleaching procedures or their influence on the of the treatment. Objective: To assess the influence agents immediate The data obtained were subjected to one-way ANOVA and heteroscedasticity analysis by the Welch and Brown-Forsythe tests. The Tamhane test was used for group comparison. Result: The color change was visible to the naked eye, considering the mean ΔE in all groups tested was higher than 3.7. However, there was no statistical difference between the control group and the groups assessed. Conclusion: The contact of staining agents on bovine tooth enamel did not affect the final result of the immediate tooth bleaching. Descriptors:


INTRODUCTION
Smile harmony has a great influence on the life of patients, especially in the social aspect. The esthetic appearance of teeth is extremely important because white teeth are associated with both beauty and health 1 . Considering the increased search for cosmetic procedures, questions emerge about the maintenance and regression of color, especially regarding tooth bleaching 2,3 .
The difference between the types of staining is well established in the literature and divided into two main groups: intrinsic and extrinsic 4,5 ; The first has pigments integrated to the enamel and/or dentin structure and it is harder to remove, while the latter occurs due to diet habits, with the deposition of chromogenic substances, and it is easier to treat 6,7 . The treatment for both is also extensively established in the literature and divided into three techniques: mediate, immediate, and combined [8][9][10] . Its action mechanism has not been fully described but the more accepted theories are that bleaching agents, through oxidation, cleave the extensive conjugated chains that form the pigments, thus reducing the number of staining molecules 11 .
Dentists also often recommend that patients control the ingestion of darker-colored products such as coffee, teas, and wine during bleaching procedures, thus performing a white diet. This mostly appears in the written instructions of the leaflets of products in this field or it is recommended because of the lack of information from previous generations that believed in internal staining caused by external agents. Considering that several authors have reported tooth discoloration and staining by agents such as wine and coffee [12][13][14] , the need to restrict dark foods during the bleaching procedure has not been confirmed, as well as their influence on the stability and effectiveness of the treatment 15 .
Decalcification, porosities, and topographic changes in tooth enamel are promoted 16,17 , favoring discoloration and pigment build-up in teeth. However, the mineral loss is compensated by the remineralization properties of saliva 18 , which contains calcium and phosphate ions. Moreover, the carbamide peroxide, when dissociating into ammonia and carbon dioxide, increases the pH of the bleaching agent for 15 minutes and makes the oral cavity environment more basic, decreasing this demineralization even more 19,20 .
In this sense, as an impact on the questions that emerge for the type of diet after tooth bleaching, especially regarding industrialized beverages, this study aimed to assess in vitro the influence of different staining agents on tooth enamel during the immediate bleaching treatment with 35% hydrogen peroxide. The null hypothesis is that staining agents do not affect the result during tooth bleaching performed with 35% hydrogen peroxide when applied to bovine tooth enamel.

Obtaining, Cleaning, and Storing the Teeth
Sixty healthy bovine teeth were used in this study (Frigorífico Mondelli Indústria de Alimentos S/A, Bauru, SP, Brazil). The teeth were stored for one month in an 8% thymol solution after their extractions. For specimen preparation, the enamel surfaces were polished with #600 silicon carbide sandpapers (Carborundum Abrasivos, São Paulo, SP, Brazil) in a polisher (Instituto Alberto Luiz Coimbra de Pós-Graduação e Pesquisa em Engenharia, COPPE, Cidade Universitária, UFRJ, Rio de Janeiro, Brazil) for a standardized time of 30 seconds. For the first part of the study, a condensation silicone mold (heavy base) was produced in the buccal aspect of one single specimen to standardize the positioning during the spectrophotometer reading.

Experimental Groups
Sixty bovine teeth were used, distributed into six experimental groups (n=10): Group 01 -Immediate bleaching with 35% hydrogen peroxide, once a week, three applications of 15 minutes, according to the manufacturer's instructions, resulting in 45 minutes per session. After each weekly session, the bovine teeth were stored in artificial saliva for 24 hours and then immersed in distilled water for 15 minutes daily. Next, the bovine teeth were again stored in artificial saliva for the remaining time (positive control group).
Group 02 -Immediate bleaching with 35% hydrogen peroxide, once a week, three applications of 15 minutes, according to the manufacturer's instructions, resulting in 45 minutes per session. After each weekly session, the bovine teeth were stored in artificial saliva for 24 hours and then immersed in coffee for 15 minutes daily. Next, the bovine teeth were again stored in artificial saliva for the remaining time.
Group 03 -Immediate bleaching with 35% hydrogen peroxide, once a week, three applications of 15 minutes, according to the manufacturer's instructions, resulting in 45 minutes per session. After each weekly session, the bovine teeth were stored in artificial saliva for 24 hours and then immersed in a cola soft drink for 15 minutes daily. Next, the bovine teeth were again stored in artificial saliva for the remaining time.
Group 04 -Immediate bleaching with 35% hydrogen peroxide, once a week, three applications of 15 minutes, according to the manufacturer's instructions, resulting in 45 minutes per session. After each weekly session, the bovine teeth were stored in artificial saliva for 24 hours and then immersed in red wine for 15 minutes daily. Next, the bovine teeth were again stored in artificial saliva for the remaining time.
Group 05 -Immediate bleaching with 35% hydrogen peroxide, once a week, three applications of 15 minutes, according to the manufacturer's instructions, resulting in 45 minutes per session. After each weekly session, the bovine teeth were stored in artificial saliva for 24 hours and then immersed in mate tea for 15 minutes daily. Next, the bovine teeth were again stored in artificial saliva for the remaining time.
Group 06 -Immediate bleaching with 35% hydrogen peroxide, once a week, three applications of 15 minutes, according to the manufacturer's instructions, resulting in 45 minutes per session. After each weekly session, the bovine teeth were stored in artificial saliva for 24 hours and then immersed in industrialized açaí for 15 minutes daily. Next, the bovine teeth were again stored in artificial saliva for the remaining time.

Tooth Bleaching
For the immediate bleaching of bovine teeth, the Whiteness HP 35% hydrogen peroxide bleaching gel was used. The hydrogen peroxide gel was handled according to the manufacturer's recommendations, which consists of mixing three drops of 35% hydrogen peroxide to one drop of thickener. The gel was applied directly on the buccal surfaces in the enamel of bovine teeth, which were partially immersed in artificial saliva, leaving the buccal surfaces exposed, and the Whiteness HP bleaching gel filled the entire bleaching area. Bleaching was performed in vitro and, according to the manufacturer's instruction, the gel remained in contact with the enamel for 45 minutes in three applications of 15 minutes, resulting in three treatment sessions with a 7-day interval between each session. During the sessions, the specimens remained exposed to room temperature. The gel was removed with flexible plastic rods with cotton on the ends (Cotonetes -Johnson & Johnson, Brazil) and then washed abundantly in running water. Later, the bovine teeth were again stored in artificial saliva at room temperature, with the crown completely immersed. The artificial saliva was replaced once a week after each bleaching session to maintain its properties.

Staining Protocol
The samples from group 02 were stained with coffee (Nescafé Tradição, Nestlé™, Switzerland) containing 5 g of sugar (União Cristalçúcar) mixed with a teaspoon equivalent to 3 g, which was obtained with a digital scale. Then, the portion was dissolved in 50 ml of boiling water, as suggested by the manufacturer, and agitated for about three minutes. Staining was performed by immersing the specimens in coffee for 15 minutes, considering this time was determined by the manufacturers who established it as the average time to drink a cup of coffee, and among coffee drinkers, to whom the average intake is 3.2 cups a day 21 .
The samples from the other groups (03 -cola soft drink; 04 -red wine; 05 -mate tea; and 06açaí) were stained by immersing the specimens in their respective staining agents without changes in preparation for the standardized time of 15 minutes daily, once a day. Next, the samples were washed in running water and returned to storage in artificial saliva until the next staining session.

Color Analysis
The color was analyzed on enamel at two different moments. The initial moment occurred before starting bleaching (t0) with the bovine teeth having been stored for 24 hours in artificial saliva. The second moment occurred after three weeks, at the end of bleaching/staining (tf). For the color readings, the bovine teeth were placed in the condensation silicone mold (heavy base) for standardization. A Vita Easyshade™ Advance 4.0 spectrophotometer (VITA Zahnfabrik H. RauterGmbH & Co, BadSäckingen, Germany) was used, previously calibrated according to the manufacturer's recommendations. The color was determined using the parameters quantified in the CIE Lab system with three coordinates: L* (a*) and (b*), where L* represents the tooth value in a scale from 0 (black) to 100 (white) and a* and b* represent the shadow, where (a*) is the measure along the red axis (positive a*) -green axis (negative a*) and (b*) is the measure along the yellow axis (positive b*) -blue axis (negative b*). The color comparison before and after bleaching will be determined by the difference between both colors (ΔE), which will be calculated with the formula: ΔE = [(ΔL)2 +(Δa)2 +(Δb)2]1/2. When ΔE is higher than 3.7, an easily visible difference is considered; when it is between 3.7 and 1, a clinically acceptable difference is considered; and when ΔE is lower than 1, a clinically non-perceptible difference is considered 22 .

Statistical Analysis
The data obtained were analyzed with the SPSS software, version 13.0. All the data obtained were subjected to one-way analysis of variance (one-way ANOVA) and, due to heteroscedasticity conditions, to the Welch and Brown-Forsythe tests. The Tamhane test was used for group comparison (5% significance level). Table 1 presents the ΔE, mean, and standard deviation values obtained in the experimental conditions established.

RESULT
The result shows a significant difference between the mean values obtained for ΔE (p<0.05%; one-way ANOVA) ( Table 2). The graphic analysis of the groups clearly shows the irregular behavior of the treatment given to group 04 (higher variability). Group 03 had more homogeneous behavior in the set of samples (Figure 1).
Thus, considering heteroscedasticity conditions, the Welch and Brown-Forsythe tests were applied, again showing a significant difference between the means of the groups (p<0.05%).
Lastly, the intergroup comparative analysis showed a significant difference only between groups 02 and 03 (p=0.01%, Tamhane test), while the comparisons between the other groups had no significant differences (p>0.05%, Tamhane test) ( Table 2).

DISCUSSION
The study assessed the influence of different staining agents on the color change of tooth enamel, which may occur during the immediate bleaching treatment. Bovine teeth were used because of the morphological and histological similarity to human teeth 23 , the uniform composition, and the ease of obtaining and standardizing them 24 . The color was analyzed with an objective method that promotes more reliable and accurate results, named spectrophotometry 22 , when compared to the results obtained with the visual method, thus preventing the interference of personal factors. Moreover, the bovine teeth used in the study were stored in artificial saliva, simulating the remineralization in the oral cavity 15,21 , which may reverse the erosion process and porosities caused by bleaching agents with low pH and oxidative ability.
The results in this study showed a color change visible to the naked eye, considering the mean ΔE in all the groups tested was higher than 3.7 ( Table 1). As shown by De Geus et al. 22 , a ΔE mean higher than 3.7 is considered a clinically acceptable difference in comparison with measures lower than 3.7, and it is clinically visible.
Moreover, the result of the present study did not show a significant difference between the control group and the other groups assessed (Table 2). Thus, the null hypothesis was accepted. A study by Côrtes et al. 10 , 2013 showed that during tooth bleaching, the staining agents cannot change treatment effectiveness but using the same pigments caused losses of the results obtained in the post-treatment phase, specifically during the ingestion of wine and coffee. It also reported that even if the enamel is susceptible to extrinsic staining when immersed in different fluids, the susceptibility of additional staining does not increase.
Another study by Matis et al. 15 , 2015 showed that individuals that consumed red wine or dark fruits did not present changes in enamel color when compared with individuals who did not consume such staining agents, which concludes that adhering to a white diet during the tooth bleaching process did not improve the esthetic result of tooth bleaching. However, the study by Berger et al. 4 , 2008 indicates that beverages containing dyes with an acidic pH may affect negatively the effectiveness of tooth bleaching, changing the topography of enamel and allowing staining after consuming staining agents. Nonetheless, the authors tested the staining immediately after bleaching and for a long exposure time (1 hour and 48 hours), which may show results that do not reflect the clinical reality of patients.
It is believed that the extrinsic staining of teeth by dying agents such as tea, red wine, cocoa, coffee, grape, pear, apple, passion fruit, pomegranate, açaí, nuts, almond, clove, cinnamon, red beans, among others results from the deposition of tannins on the tooth surface. The connection of tannins or other polyphenols to the hydroxyapatite component of the enamel may be exacerbated by salivary proteins. The enamel permeability, which allows ion exchanges between tooth and saliva 6 facilitates the entry of ions and substances with a low molecular weight in the dental structure, such as bleaching agents and staining substances. These pigments in the oral cavity may penetrate the enamel and reach the dentin, causing dental staining, which cannot be removed with prophylaxis.
Another important aspect to highlight is that these substances consist of macromolecular chains and, therefore, are hardly able to permeate through human enamel, which allows only the passage of molecules with low molecular weight. Tooth enamel works as a semipermeable membrane that allows the passage of ions and small molecules, therefore, the bleaching process does not occur in the structure of the mineralized enamel but possibly as a result of the oxidation of organic tissues in the human dentin.
Previous studies showed that bleaching may increase enamel roughness 10,17 . However, the present study corroborates recent ones that do not show changes in topography or staining after the bleaching process, which is likely explained by the differences in the methodologies used. Moreover, the addition of calcium and fluoride to the bleaching gel and the presence of artificial saliva decreased the loss in microhardness and increased enamel surface smoothness in the bleaching process, making the teeth less susceptible to staining. The in vitro trial is not a full representation of the clinical response but it takes on importance in the control and development of a base for the next research stage. Bovine teeth are often used due to their availability and surface size and for presenting morphological characteristics and physical properties similar to human teeth 25 , however, further studies are required to investigate the relationship between diet and tooth bleaching, advancing to in situ and in vivo models.

CONCLUSION
Based on the experimental conditions of this study and within the limitations of an in vitro research, it may be concluded that: • Bleaching occurred with color changes visible to the naked eye because the mean ΔE in all the groups tested was higher than 3.7; • The contact of staining agents on bovine tooth enamel did not affect the final result of immediate tooth bleaching with 35% hydrogen peroxide.