Effects of black tea tooth staining previously to 35% hydrogen peroxide bleaching

Abstract

Aim

To determine if the artificial staining with black tea (BT) influences the enamel microhardness before in-office bleaching and if BT staining is necessary to evaluate the efficacy of bleaching with 35% hydrogen peroxide

Methods

Enamel/dentin blocks were randomized into groups according to the staining protocol (n=5/group): (CO) control – maintained in artificial saliva solution (AS); (BT₄) immersed in black tea solution for 4 h; (BT₂₄) immersed in black tea solution for 24 h. After the staining protocols, all specimens were kept in AS for one week, followed by bleaching (three sessions of HP application for 40 min). Knoop surface microhardness (kgF/mm²) was determined at baseline (T₀), after staining (T₁), after 7 days of storage in AS (T₂), and after bleaching (T₃). The color (∆E₀₀) and coordinate changes (∆L, ∆a, ∆b) were measured using a digital spectrophotometer at T₀ and T₃. Data were submitted to one-way (∆E₀₀, ∆L, ∆a, ∆b) or two-way ANOVA repeated measures (kgF/mm²) and Tukey’s test (a=5%).

Results

The staining protocols (BT₄ and BT₂₄) promoted significantly lower microhardness (T₁ and T_2, p<0.05) than CO, whereas CO was the only group to maintain microhardness values over time. Bleaching promoted perceptible ∆E₀₀ without a significant difference among the groups regardless of the staining protocol (p=0.122). CO and BT₄ showed no differences in terms of ∆L and ∆a (p>0.05), but BT₄ displayed a higher ∆b than CO.

Conclusion

The artificial staining with BT negatively affected the enamel surface microhardness and was not essential to evaluate the efficacy of 35% hydrogen peroxide bleaching.

Bleaching agents; Hydrogen peroxide; Staining and labeling

Introduction

Tooth discoloration is classified based upon its location¹. Intrinsic color is associated with structural and thickness alterations to dentin during tooth formation or after its eruption², while extrinsic discoloration is determined by the adsorption of polyphenolic compounds onto the surface of dental enamel and their interaction with pellicle proteins³. Most of these organic chromogens are present in the daily habits of the patients, i.e., intake of foods and smoking⁴. Additionally, enamel defects, dental caries, or restorative materials may facilitate the incorporation of such compounds within the tooth structure⁵. However, it should be noted that the extrinsic staining deposited on the enamel is not resistant to removal with either regular toothbrushing or professional prophylaxis⁴,⁶.

Tooth bleaching remains the most ultraconservative and efficient approach to treat dental discoloration even after prophylaxis⁷. The technique consists of applying carbamide or hydrogen peroxide gels on the enamel surface⁸ in concentrations that vary according to the bleaching regimen. The chemical byproducts of the reaction, the reactive oxygen species (ROS) resulting from peroxide decomposition, diffuse through the enamel into the dentin⁹, supposedly breaking down the intrinsic staining into smaller molecules. Consequently, teeth look whiter⁸.

Several in vitro studies evaluated tooth bleaching protocols’ efficacy and adverse effects by using artificial staining on enamel before HP bleaching. One of the reasons for that approach is the necessity of the standardization of tooth color among groups before the bleaching treatments¹⁰,¹¹. Previous studies reported tooth staining with soft drinks¹², tobacco smoke, coffee¹³, red wine¹⁴, and black tea¹¹. There is evidence that black tea exhibits a higher staining effect than cola-based soft drinks and coffee¹⁵. In this regard, Sulieman et al.³ (2003) validated an artificial staining protocol with black tea specifically for a dental bleaching evaluation. According to the authors, the 24-hour immersion of enamel/dentin specimens in black tea solution did not differ from a 6-day immersion³. In other words, an overnight staining protocol would suffice to assess the bleaching efficacy of peroxides.

Nevertheless, staining agents may influence composition and structure other than tooth optical properties. For instance, it has been reported that teeth stained with black tea exhibited lower ROS penetration after highly concentrated HP bleaching than unstained teeth¹⁶. In addition, long-lasting enamel exposure to solutions or products with a pH lower than 5.5 can trigger enamel demineralization¹⁷. Since evidence shows that black tea’s pH can range from 3.3 to 6.5 in commercial ready-to-drink beverages¹⁸, artificial staining before bleaching may compromise the enamel mineral composition and surface morphology. Moreover, tooth bleaching may modify the enamel composition and structure by decreasing the enamel mineral content¹⁹ and surface microhardness²⁰, increasing the surface roughness¹⁴, and changing the enamel morphology¹¹.

Given these facts, artificial black tea staining may interfere with the outcomes and decrease the reliability of the results related to tooth bleaching itself. Although several studies have already investigated the effects of tooth bleaching on enamel surface properties¹¹,¹⁴,¹⁹,²⁰, there is no evidence showing if the step of the artificial tooth staining isolated would negatively affect the enamel microhardness and become a confounding variable. Additionally, studies have not indicated whether the specimens’ darkening in different exposure times is necessary to determine enamel color change compatible with efficient tooth bleaching. Hence, this study determined the effect of artificial staining with black tea, in short (4 h) or long (24 h) exposures, on enamel surface microhardness and the efficacy of tooth bleaching with 35% hydrogen peroxide (HP) on stained and nonstained teeth. The null hypotheses were that (a) artificial staining would not decrease the enamel surface microhardness and that (b) the staining protocols tested would not affect the color change after bleaching.

Materials and Methods

Experiment Design

Dental blocks were submitted to artificial staining protocols with or without black tea solution (BT):

• CO: Control - without black tea and stored in artificial saliva (AS)

• BT₄: black tea immersion for 4 h (short exposure)

• BT₂₄: black tea immersion for 24 h (long exposure)

After staining, all specimens were stored in AS for 7 d and submitted to an in-office bleaching protocol (35% HP). The color and the surface microhardness of the specimens were assessed before (T₀), after staining (T₁), after storage in AS (T₂), and after bleaching treatment (T₃).

Specimen preparation

Bovine incisors were extracted, cleaned with periodontal scrapers, and stored in 0.1% thymol solution at 4°C for no longer than 30 days. Fifteen teeth without enamel cracks or defects were selected, and the roots were separated from the crowns using a low-speed diamond saw (Isomet, Buehler; Lake Bluff, Illinois, USA) under refrigeration, 2 mm below the cementoenamel junction. Blocks (6 mm × 6 mm and 3 mm thick) were obtained from the central portion of the crown. The dentin was flattened using a rotary polisher (Arotec Ind. Com., São Paulo, SP, Brazil) with silicon carbide paper #600 under water-cooling. The enamel surface was finished with aluminum oxide grit #600 and #1200 and polished using diamond aqueous suspensions (6, 3, 1, and 0.25 μm, Metaldi Supreme, Buehler, Lake Bluff, Illinois, USA). The blocks were ultrasonically cleaned with distilled water for 5 min among each progression in grits.

Artificial Staining and Groups Distribution

The dentin of specimens was isolated using wax to allow the exposure of the enamel surface only. The blocks were randomly assigned to groups according to the black tea protocol (n=5/group): (CO) without BT and stored in AS [1,5 mM Ca; 0,9 mM P; 150 mM KCl e 0,1 M Tris; pH 7,0]²¹, (BT₄) immersion in BT for 4 h, (BT₂₄) immersion for 24 h. The sample size was calculated using the software G*Power. The mean and standard deviation values of the enamel surface microhardness reported by Costa et al.²² (2021) were used to detect the effect size since these authors also used artificially stained enamel to evaluate the action of hydrogen peroxide bleaching. The detected effect size (f = 1.5) was applied on G*Power with a predetermined significance level of 0.05, and a power test of 80%. As a result, five specimens were required for each group.

The black tea solution was prepared according to an adaptation of the protocol described by Sulieman et al.³ (2003). Two grams of black tea (Dr. Oetker, São Paulo, SP, Brazil) was diluted in 100 mL of boiling distilled water for 5 min, filtered, and cooled. The specimens were immersed in BT (20 mL) at a constant temperature of 37°C. The pH of the solution was checked before and after immersion (4 h: 4.7 – 4.9 and 24 h: 4.6 – 4.9). After staining, the specimens were washed in distilled water and stored in AS for 7 d to allow color stabilization of the specimens.

Bleaching Procedures

In-office bleaching (35% HP, Total Blanc Office H35, Nova DFL, Rio de Janeiro, RJ, Brazil) was performed according to the manufacturer’s recommendation: three 40-min sessions at 72-h intervals. The specimens were stored in AS at intervals for seven consecutive days after bleaching.

Surface Microhardness

The mean enamel surface microhardness was obtained by three impressions in the central area of the block, with a Knoop diamond penetrator (Future Tech-FM-1e, Tokyo, Japan), under a static charge of 25 grams for 5 seconds and 100 µm of distance from each other. No differences in kgF/mm²among groups were found at baseline (T₀). The microhardness was evaluated immediately after staining (T₁), after stabilization in AS (T₂), and seven days after bleaching (T₃).

Colorimetric Evaluation

The colorimetric measurements on the enamel surface were performed using a digital spectrophotometer (EasyShade, Vita Zahnfabrik, Bad Säckingen, Germany). The equipment was fixed on a platform to allow the tip to face enamel as parallel as possible. The background in which the dental blocks were placed was standardized with an opaque tile. This apparatus was positioned inside a light-controlled chamber. The L*, C*, h*, a*, and b* coordinates were obtained from the average of three measurements. L* represents the luminosity of an object (black to white), while a* and b* represent + red/-green and + yellow/-blue, respectively. The C* parameter determines the chroma of the substrate, and h*, the tooth hue. Because this study aimed to evaluate the effect of bleaching in stained teeth, colorimetric data were collected 7 days after staining and storage in AS (T₂) and 7 days after the last bleaching session (T₃). The color variation was calculated according to CIEDE2000 (ΔE₀₀), with the color difference between T₃ and T₂. The 50:50% perceptibility threshold adopted was 1.2 ΔE₀₀ units²³. The variation in the L*, a*, and b* coordinates were also individually calculated, taking into consideration the same time points mentioned above. The groups exhibited no significant differences in the mean baseline L*, a*, and b* values.

Statistical analysis

The normal distribution (Shapiro–Wilk, p>0.05) and homoscedasticity (Levene, p>0.05) of the data were confirmed. Data were statistically analyzed by one-way (ΔE₀₀, ΔL, Δa, Δb) or two-way repeated-measures ANOVA (microhardness), and Tukey’s test detected differences among groups at a significance level of 5%. All analyses were performed by SPSS version 23 (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp.).

Results

Microhardness evaluation. At baseline (T₀), no differences in microhardness were detected among groups (Table 1), but both staining protocols (BT₄ and BT₂₄) decreased enamel microhardness (T₁) in comparison with CO (p<0.001). At this time-point (T₁), BT₂₄ exhibited lower microhardness than BT₄ (p<0.001). After storage in AS for 7 d (T₂), the results remained unaltered.

Table 1
Mean values and standard deviation of microhardness values (kgF/mm2).

Groups	T0	T1	T2	T3
CO	307.1 (20.5) Aa	288.5 (14.5) Aa	286.5 (6.2) Aa	285.89 (10.74) Aa
BT4	307.0 (16.3) Aa	260.1 (16.9) Bb	259.5 (17.7) Bb	267.82 (25.35) Ab
BT24	310.3 (16.2) Aa	189.0 (24.4) Cc	204.6 (25.0) Cc	224.60 (11.97) Bb

• Means followed by distinct letters are different according to two-way ANOVA repeated measurements and Tukey’s test at a significance level of 5%. Capital letters compare staining protocols (columns), and lowercase letters compare time points (lines). T₀: baseline; T₁: after staining; T₂: after 7 days of storage in AS; T₃: after bleaching. CO: control; BT₄: black tea immersion for 4 h; BT₂₄: black tea immersion for 24 h.

Seven days after bleaching (T₃), CO and BT₄ displayed no differences (p=0.36) but exhibited higher Knoop values than BT₂₄ (p<0.05). CO microhardness remained unaltered over time, while microhardness recovery was detected for BT₂₄ at T_3.

Color evaluation. No differences in ∆E₀₀ among groups were detected (p=0.122) after bleaching (T₃ -T₂). The mean color differences promoted by bleaching were above the perception threshold (∆E₀₀>0.8), regardless of the staining protocol (Table 2). BT₂₄ showed higher ∆L, ∆a, and ∆b values than CO but no difference from BT₄ in ∆L and ∆b. BT₄ and CO displayed no differences in ∆L and ∆a (>0.05) but higher ∆b than CO.

Table 2
Mean values and standard deviation of the alteration in Euclidian coordinates (∆L, ∆a, and ∆b) and color alteration between color stabilization in AS and bleaching.

Groups	∆L	∆a	∆b	∆E00
CO	-2.8 (3.7) B	-2.3 (1.4) A	-15.1 (1.8) B	9.0 (1.1) A
BT4	-1.0 (6.1) AB	-4.2 (1.6) A	-18.8 (2.1) A	10.7 (0.2) A
BT24	6.4 (5.9) A	-8.6 (1.4) B	-17.2 (1.0) A	11.5 (2.9) A

• Means followed by distinct letters are different according to one-way ANOVA and Tukey’s test at a significance level of 5%. Capital letters compare staining protocols (columns). CO: control; BT₄: black tea immersion for 4 h; BT₂₄: black tea immersion for 24 h.

Discussion

Black tea staining reduced the enamel microhardness before the bleaching procedures, regardless of immersion time in BT. This outcome remained unchanged even after storage in artificial saliva for 7 days elapsed from the last bleaching session. Therefore, the first null hypothesis was rejected because artificial staining with BT affected the enamel surface microhardness.

Artificial dental staining is a method developed to mimic the natural intrinsic staining that occurs with the deposition of secondary and tertiary dentin along the tooth´s lifetime³. The protocol described in 2003³ has been extensively used by researchers as an attempt to simulate an ideal substrate to be bleached and as an approach to standardize the teeth color of groups²⁴-²⁶. Nevertheless, that study³ failed to report the pH of black tea and its effects on enamel after 24 h of immersion.

To the best of our knowledge, studies investigating the bleaching effect on enamel have not reported the surface microhardness immediately after immersion in the staining solution. For instance, a previous report displayed the microhardness values at baseline and after bleaching while evaluating the effects of light-activated 6% HP on the enamel surface²²,²⁷. In that study, black tea with an unknown pH and manufacturer was used to immerse dental blocks for 18 h. Since a mean surface microhardness reduction was observed over time, the black tea immersion should also be considered in the interpretation of the results. Bearing this in mind, the time points of the present study allowed a more comprehensive understanding of the real impact of bleaching on the enamel surface. The results showed that 35% HP was not responsible for reducing the enamel surface microhardness, contrary to the black tea solution.

Similar to the tea used in our study, with a pH ranging from 4.6 to 4.9, others have reported the use of BT with a pH lower than 5.5²⁸-³⁰. The study of Farawati et al.³⁰ (2019) investigated the effect of black tea, wine, soda, coffee, and water on the surface properties of enamel submitted to bleaching with carbamide peroxide. The data from that study suggested that only black tea and wine changed the enamel mineral composition. One might say that the presence of fluoride in tea³¹ could uphold the level of enamel mineralization; nonetheless, it was demonstrated that the immersion of eroded enamel in a ready-to-drink black tea with 0.760 mg/L of fluoride, in a much lower immersion time than our study, decreases the enamel microhardness significantly more compared to other types of drinks³². Additionally, Jameel et al.²⁹ (2016) showed that a black tea solution (pH 4.9, 0.938 mg/L) negatively impacted not only the surface microhardness but also the enamel roughness. Hence, the existing evidence suggests that the pH of the solutions might play a more important role than the presence of fluoride in the maintenance of the surface properties.

It is important to highlight that group BT₄ was set as an alternative to diminishing the necessary time of staining. Despite the lower immersion time, a significant decrease in KHN was also observed for this group. However, it is noteworthy that this reduction in the microhardness was significantly lower than that of BT₂₄, which may indicate a time-dependent effect of black tea on the enamel microhardness. Even though other protocols could adjust the pH of the staining solution as an attempt to overcome the low-pH value, raising the pH of this solution could impact the capability of the staining molecules to penetrate the dental structure. Since no studies were found attempting this approach, future studies could investigate its application.

The colorimetric data inferred that the staining protocols did not affect the color change promoted by bleaching. The group stored in artificial saliva showed a similar ∆E₀₀ to the BT groups. Thus, the second null hypothesis was accepted since the staining protocols did not affect the color change after bleaching.

According to Paravina et al.²³ (2019), a color change above 5.4 units is compatible with the excellent effectiveness of tooth bleaching, thereby indicating a very highly perceptible outcome. Therefore, the commercial bleaching gel used herein was very effective even without a previous staining step [∆E₀₀ = 9.0 (1.1)]. Additionally, in vitro studies using artificial staining may exhibit exacerbated ∆E₀₀ results. For instance, different studies evaluating the same bleaching protocols presented higher ∆E₀₀ values when teeth were artificially stained¹¹,¹³ compared to nonstained teeth¹³,³³,³⁴.

Differences in the L* and a* coordinate changes among the groups could be explained by the absence (CO) or lower amount (BT₄) of pigments, suggesting a minor challenge for the hydrogen peroxide gel. On the other hand, the high shift in the b* coordinate from b+ (yellow) to b- (blue), independent of the staining protocol, might suggest that the bleaching gel could breakdown the chromogens even without previous artificial staining. These results indicate that artificial staining before bleaching to mimic and standardize the specimens for color evaluation may be discarded since bleaching therapy was effective in nonstained teeth. In this regard, an interesting research approach would be avoiding dental blocks with a high initial luminosity (L*) and low values of the b* coordinate to observe the bleaching effects.

Updated colorimetric systems, such as the color change calculated by CIEDE2000³⁵, could overcome the drawbacks of previous calculations (CIELAB or visual shade guides). Moreover, ΔE₀₀ adjusts the low influence that the a* coordinate plays on the color of teeth. The Sulieman et al.³ (2003) study only applied a subjective color evaluation of the specimens while establishing the staining protocol. This fact emphasizes the need for further analysis using appropriate objective color measurements and corresponding updated color and whiteness indexes⁷.

A limitation of the present research is the absence of topography analysis and groups with additional BT immersion. However, the purpose of this investigation was to stress the importance of carefully employing artificial staining for bleaching evaluation. Artificial staining should be carefully designed since overexposure and pH might damage the enamel surface and lead to a mistaken interpretation of the HP bleaching effects and enamel mineral content. In addition, excluding artificial staining may not impact bleaching efficacy, and the results could be more reliable and translated to a clinical setting.

In conclusion, artificial staining with black tea showed detrimental effects on enamel surface microhardness, wherein the higher the time of exposure to tea was, the lower the hardness. In addition, bleaching with 35% HP did not decrease surface microhardness in the stained or nonstained specimens, and the staining protocols (4 h or 24 h immersion in black tea) were not essential to detect the bleaching effects. All groups displayed similar color changes after bleaching.

Acknowledgments

This study was supported by the São Paulo State Research Foundation (FAPESP #2020/08440-3 and #2020/06782-4). This study was also supported in part by the Coordenação de Apoio ao Pessoal de Nível Superior (CAPES – Financial Code 001).

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• Data Availability
  Datasets related to this article will be available upon request to the corresponding author.

Edited by

Editor: Dr. Altair A. Del Bel Cury

Publication Dates

• Publication in this collection
  17 Oct 2022
• Date of issue
  2023

History

• Received
  09 Jan 2022
• Accepted
  11 June 2022