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

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 T2, 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.


Introduction
Tooth discoloration is classified based upon its location 1 . Intrinsic color is associated with structural and thickness alterations to dentin during tooth formation or after its eruption 2 , while extrinsic discoloration is determined by the adsorption of polyphenolic compounds onto the surface of dental enamel and their interaction with pellicle proteins 3 . Most of these organic chromogens are present in the daily habits of the patients, i.e., intake of foods and smoking 4 . Additionally, enamel defects, dental caries, or restorative materials may facilitate the incorporation of such compounds within the tooth structure 5 . However, it should be noted that the extrinsic staining deposited on the enamel is not resistant to removal with either regular toothbrushing or professional prophylaxis 4,6 .
Tooth bleaching remains the most ultraconservative and efficient approach to treat dental discoloration even after prophylaxis 7 . The technique consists of applying carbamide or hydrogen peroxide gels on the enamel surface 8 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 dentin 9 , supposedly breaking down the intrinsic staining into smaller molecules. Consequently, teeth look whiter 8 .
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 treatments 10,11 . Previous studies reported tooth staining with soft drinks 12 , tobacco smoke, coffee 13 , red wine 14 , and black tea 11 . There is evidence that black tea exhibits a higher staining effect than cola-based soft drinks and coffee 15 . 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 immersion 3 . 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 teeth 16 . In addition, long-lasting enamel exposure to solutions or products with a pH lower than 5.5 can trigger enamel demineralization 17 . Since evidence shows that black tea's pH can range from 3.3 to 6.5 in commercial ready-to-drink beverages 18 , 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 content 19 and surface microhardness 20 , increasing the surface roughness 14 , and changing the enamel morphology 11 .
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 sur-face properties 11,14,19,20 , 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.

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) • BT 4 : black tea immersion for 4 h (short exposure) • BT 24 : 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 (T 0 ), after staining (T 1 ), after storage in AS (T 2 ), and after bleaching treatment (T 3 ).

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 , (BT 4 ) immersion in BT for 4 h, (BT 24 ) 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/mm 2 among groups were found at baseline (T 0 ). The microhardness was evaluated immediately after staining (T 1 ), after stabilization in AS (T 2 ), and seven days after bleaching (T 3 ).

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 (T 2 ) and 7 days after the last bleaching session (T 3 ). The color variation was calculated according to CIEDE2000 (∆E 00 ), with the color difference between T 3 and T 2 . The 50:50% perceptibility threshold adopted was 1.2 ∆E 00 units 23 . 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 (∆E 00 , ∆L, ∆a, ∆b) or two-way repeated-measures ANOVA (microhardness), and Tukey's test detected differences among groups at a significance level of 5%.

Results
Microhardness evaluation. At baseline (T 0 ), no differences in microhardness were detected among groups (Table 1), but both staining protocols (BT 4 and BT 24 ) decreased enamel microhardness (T 1 ) in comparison with CO (p<0.001). At this time-point (T 1 ), BT 24 exhibited lower microhardness than BT 4 (p<0.001). After storage in AS for 7 d (T 2 ), the results remained unaltered.
Seven days after bleaching (T 3 ), CO and BT 4 displayed no differences (p=0.36) but exhibited higher Knoop values than BT 24 (p<0.05). CO microhardness remained unaltered over time, while microhardness recovery was detected for BT 24 at T 3. Color evaluation. No differences in ∆E 00 among groups were detected (p=0.122) after bleaching (T 3 -T 2 ). The mean color differences promoted by bleaching were above the perception threshold (∆E 00 >0.8), regardless of the staining protocol (Table 2). BT 24 showed higher ∆L, ∆a, and ∆b values than CO but no difference from BT 4 in ∆L and ∆b. BT 4 and CO displayed no differences in ∆L and ∆a (>0.05) but higher ∆b than CO.

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 lifetime 3 . The protocol described in 2003 3 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 groups [24][25][26] . Nevertheless, that study 3 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 surface 22,27 . 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 5.5 [28][29][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 tea 31 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 drinks 32 . 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 BT 4 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 BT 24 , 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 ∆E 00 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 [∆E 00 = 9.0 (1.1)]. Additionally, in vitro studies using artificial staining may exhibit exacerbated ∆E 00 results. For instance, different studies evaluating the same bleaching protocols presented higher ∆E 00 values when teeth were artificially stained 11,13 compared to nonstained teeth 13,33,34 .
Differences in the L* and a* coordinate changes among the groups could be explained by the absence (CO) or lower amount (BT 4 ) 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 CIEDE2000 35, could overcome the drawbacks of previous calculations (CIELAB or visual shade guides). Moreover, ∆E 00 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 indexes 7 .
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