Initial and pulp chamber concentration of hydrogen peroxide using different bleaching products

Objective: This study’s aim was to quantify the hydrogen peroxide (HP) penetration into the pulp chamber of teeth submitted to different protocols of bleaching. Material and Methods: Ninety premolars were randomly divided into nine groups according to the bleaching agent protocol (n = 10): control (no bleaching), carbamide peroxide 10% [10% CP], carbamide peroxide 16% [16% CP], carbamide peroxide 22% [22% CP], hydrogen peroxide 4% [4% HP], hydrogen peroxide 6% [6% HP], hydrogen peroxide 7.5% [7.5% HP], hydrogen peroxide 10% [10% HP] and hydrogen peroxide 35% [35% HP]. The penetration of HP was measured via spectrophotometric analysis of the acetate buffer solution from the pulp chamber. The absorbance of the resulting solution was determined in a spectrophotometer and converted into equivalent concentration of HP (μg/ mL). To analyze the concentration of HP, the titration of bleaching agents with potassium permanganate was used. Data were subjected to ANOVA and Tukey’s test for pairwise comparison (α = 0.05). Results: Higher concentration of HP in the pulp chamber was found in the HP 35% group (p < 0.0001). No significant difference between at-home protocols were observed (p = 0.64). Titration values showed that the concentration of the products was similar to that claimed by the manufacturer. Conclusion: It follows that the amount of HP that reaches the pulp chamber is not proportional to the concentration of whitening gels, but depends on the application time recommended by the manufacturers. RESUMO


O R I G I N A L A R T I C L E
Initial and pulp chamber concentration of hydrogen peroxide using different bleaching products INTRODUCTION T he demand for dental bleaching products has increased significantly over the past year. In some countries, 85.9% of the surveyed adult population have the desire to perform tooth bleaching [1]. Hydrogen peroxide and carbamide peroxide are the most common dentist-supervised products used in dental clinics for bleaching [2]. For at-home bleaching, the products employed different concentrations of carbamide peroxide (5% to 22%) or hydrogen peroxide ranging from 3% to 10%. On the other hand, in-office bleaching employs highly concentrated hydrogen peroxide gels -typically in the range of 20 to 38% [3][4][5].
All these bleaching products have shown effective color changes both at the immediate and in middle-term follow-ups [6,7]. However, more than 50% of the patients submitted to at-home and in-office bleaching experience bleaching-induced tooth sensitivity. The risk and severity of tooth sensitivity depends on: 1) the bleaching technique [8,9], which are directly related to the hydrogen peroxide concentration and usage time [10][11][12] and 2) patient-related factors, such as baseline tooth color and age [8]. Unfortunatelly, cell viability reduction, H 2 O 2 diffusion, cell morphology alteration, oxidative stress, and cell membrane damage of pulp cells depend on the concentration of hydrogen peroxide and contact time of this products [13,14] In regard to the bleaching technique, Rezende et al. (2016) [8] reported that athome bleaching protocols have lower tooth sensitivity than in-office bleaching. In a 0 to 4 pain scale, the mean tooth sensitivity of at-home bleaching was 0.5 ± 0.9, while inoffice bleaching was 2.8 ± 2.9. Perhaps, such differences may be related to the amount of hydrogen peroxide that reaches the pulp chamber. Hydrogen peroxide from carbamide peroxide products, for instance, appears to penetrate less than the hydrogen peroxide of equivalent concentration [15]. Tooth exposure with 15% carbamide peroxide gel (equivalent to 5.3% hydrogen peroxide) resulted in a mean pulp chamber concentration of peroxide that was less than half caused by exposure to pure 5% hydrogen peroxide [15].
The penetration of hydrogen peroxide within the dental structures, that occurs in only 15 minutes [16], seems to be dose dependent [16]. In a clinical study using similar protocols, a higher risk of tooth sensitivity was experienced by the patients who used the 20% CP (71.4%) than the ones who used 10% CP (36.8%) [10]. Variations in the amount of the hydrogen peroxide that reaches the pulp chamber using different bleaching protocols are of clinical interest such it may allow clinicians to choose the best protocol for highly sensitive patients. In face of that the aim of this study was to quantify and correlate the initial amount of hydrogen peroxide of bleaching products with the amount of hydrogen peroxide that reaches the pulp chamber using different bleaching protocols.

Groups
Main components Batch number

Quantification of the hydrogen peroxide concentration in the pulp chamber
The roots of all teeth were cut approximately 3 mm apical to the cementoenamel junction. The entrance to the pulp cavities was widened carefully, with a round bur (#1014, KG Sorensen, Barueri, SP, Brazil) to allow the introduction of a micropipette (LABMATE Soft, HTL Lab Solutions, Warsaw, Poland) inside the pulp chamber. This procedure was done carefully to not touch the internal walls of the pulp chamber. The pulp tissue was removed, and pulp chamber washed with distilled water.
Throughout this study, analytical-grade chemicals without previous purification were used; they were prepared with deionized water from a Millipore Milli-Q system (MS2000, Gehaka, São Paulo, SP, Brazil). Hydrogen peroxide was purchased from LABSYNTH (LABSYNTH, Diadema, SP, Brazil) (34%-36%). A 5000 µg/mL stock solution was prepared in acetate buffer solution (pH = 4) and standardized by conventional methods. The solution was titrated with potassium permanganate standard solution [17]. Aliquots of the stock solution of HP were diluted volumetrically to obtain working standard solutions of 0.032 -0.397 µg/mL [18] (Figure 1).
The occlusal surface of all teeth was fixed vertically to a wax plaque and the labial surface area of each tooth where the bleaching agent were applied was isolated by applying a light-cured resin dam (KG Sorensen, Barueri, SP, Brazil). A 25 µL aliquot of acetate buffer (pH 4.5) was placed into the pulp chamber of each tooth to absorb and stabilize any peroxide that might penetrate into the pulp chamber.
The bleaching gels were applied over the enamel surface according to the manufacturers' recommendations (Table I) to reproduce the clinical condition. A single-day session was performed for each material (Table I). After the exposure period (Table I), the acetate buffer solution in the pulp chamber of each tooth were removed by means of a mechanical micropipette and transferred to a glass tube. This procedure was done by rinsing the pulp chamber of each tooth four times with 25 µL of acetate buffer, and transferring this solution to the same glass tube. Next, more deionized water (2.725 µL) was added to the glass tube along with 100 µL of 0.5 mg/mL of leucocrystal violet (Sigma Chemical Co, St Louis, MO, USA) and 50 µL of 1 mg/mL enzyme horseradish peroxidase (Peroxidase Type VI-A, Sigma Chemical Co, St Louis, MO, USA). This procedure was repeated separately for each tooth.
The absorbance at 596 nm of the resultant violet color in the tubes was measured in a Cary 50 UV-Vis spectrophotometer (Varian, Palo Alto, CA, USA). According to Beer's Law, absorbance is directly proportional to the concentration. Therefore, the concentration of hydrogen peroxide (µg/mL) was determined by comparing it to the calibration curve previously obtained (Figure 1). By knowing the concentration (µg/mL) and volume of the solution, the hydrogen peroxide mass (µg) can be calculated [18].
We also calculated the theoretical availability of hydrogen peroxide during each application session by multiplying the titrated concentration of the product (% HP) by the number of hours a day recommended by manufacturers.

Statistical analysis
The data related to the concentration of hydrogen peroxide that reached the pulp chamber were subjected to one-way analysis of variance (ANOVA) and Tukey's tests for pairwise comparisons (alpha = 0.05). The Pearson correlation between the means of the hydrogen peroxide concentration that reached the pulp and the initial hydrogen peroxide concentration in each product was calculated.

RESULTS
Different amounts of hydrogen peroxide were found in the pulp chamber of the different bleaching protocols (Table II). The amount of hydrogen peroxide from the 35% HP group was significantly higher than all athome protocols evaluated (p < 0.0001). No statistically significant differences between athome protocols were observed (p = 0.649).

Quantification of the initial hydrogen peroxide concentration in the bleaching products
Potassium permanganate was used as an oxidizing agent to describe the initial concentration of hydrogen peroxide in each bleaching product. Hydrogen peroxide reduces the permanganate to a colorless product based on a reduction-oxidation reaction, according to the following formula [17,19] instructions and were analytically weighted. A sample of bleaching product (approximately 0.2 g) was collected and diluted in 50 mL of distilled water and 10 mL of 1 mol.L -1 sulfuric acid. Then, the potassium permanganate solution (0.06 mol.L -1 ) was added to this solution [17,19], resulting in a violet color solution. This color change indicated the equivalence point, i.e., the moment when all of the H 2 O 2 have been consumed. Three titrations for each bleaching gel system were performed.
The hydrogen peroxide mean concentration was calculated by the following formula: Braz Dent Sci 2020 Apr/Jun;23 (2) 5 The HP concentrations claimed by the manufacturer and the values obtained from permanganate potassium titration are shown in Table II. Titration values showed that the concentration of the products was similar to that claimed by the manufacturer.
A strong and positive correlation (r = 0.98; p = 0.0026) was found between the titrated concentration of all products and the concentration of peroxide in the pulp chamber ( Figure 4). However, when the same correlation was run only for titrated concentration of the at-home products (excluding the in-office bleaching product), a non-significant correlation was observed (r = 0.52; p = 0.22; Figure 2). The theoretical availability of peroxide per application time varied per product and it was not proportional to the concentration of the hydrogen peroxide of the products.

DISCUSSION
In the present study, a higher penetration of hydrogen peroxide in extracted human teeth were observed for the in-office tooth bleaching product than any of at-home bleaching agents. These results are in agreement with several previous studies that demonstrated that significant higher amounts of hydrogen peroxide can diffuse through dentin with inoffice protocols [15,16,20]. Additionally, some authors report that higher concentrate hydrogen peroxide products create micropores on enamel surface, which affects the enamel inner structure and enamel surface and may further make easy the passage of hydrogen peroxide through enamel [21].
In the present study, the concentration of hydrogen peroxide found in the pulp chamber for 35% HP group was higher than other published studies [18,22,23]. Amounts of HP ranging from 1.22 to 6.22 µg was observed in the pulp chamber of teeth bleached with 35% hydrogen peroxide in other studies [18,22,23], while in the present study an average of 28.5 µg was detected for 35% HP group. These differences may be attributed to variations in the experimental design such as the type of dental substrate used (human or bovine), composition of the gels, experimental protocol, hydrogen peroxide delivery method and differences of cavity preparation. However, this does not reduce the internal validity of all studies as the same conditions were applied for the different treatments within each one of these studies.
In regard to at-home bleaching protocols, the amount of hydrogen peroxide that reached the pulp chamber was similar for all groups. This was expected for products that have the same amount of active hydrogen peroxide. For instance, 10% carbamide peroxide gel, yields a maximum of 3.5% hydrogen peroxide, and therefore we anticipated the similar hydrogen peroxide diffusion of 10% carbamide peroxide and 4% hydrogen peroxide. The same phenomenon was observed to 22% carbamide peroxide and 10% hydrogen peroxide, which presents similar concentrations of active hydrogen peroxide.
However, the non-significant differences between systems with different initial hydrogen peroxide concentration was, at first glance, surprising, as previous studies suggested that the amount of hydrogen peroxide that penetrates the dental structure is influenced by its original concentration in the bleaching agent [15,16,20]. But this was also observed in several clinical trials.
In all clinical studies cited above, the products were employed according to the manufacturer's instructions, and this may be the key for understanding why similar hydrogen peroxide concentrations was observed for at-home products. In the present study, we also followed the manufacturer´s recommendations of all products in a way to simulate the clinical condition, as we wanted to compare bleaching protocols rather than bleaching product concentrations.
Bleaching protocols vary in the mode and duration of product application. Products with low active hydrogen peroxide were applied for longer periods of times than highly concentrated products. Therefore, the low concentration of hydrogen peroxide might have been compensated for the longer exposure time on the enamel surface (Table I). This means that not only concentration, but also application time is responsible for the amount of product that reaches the pulp chamber. This explains the lack of correlation between initial hydrogen peroxide concentration and the percentage of hydrogen peroxide that reached the pulp chamber for at-home bleaching products. Had the products been used for the same time, which is not representative of a clinical condition, a significant correlation might have been observed.
This correlation was, however, significant only when the 35% hydrogen peroxide; the product with the highest hydrogen peroxide concentration, was included in the data. The titrated concentration of hydrogen peroxide multiplied by the usage time (hour), according as manufacturers recommendations, give us the theoretical availability of the product on the enamel surface. This relation was highest for the 35% hydrogen peroxide product (23% per application session) in comparison with all other at-home products, which ranged from 5 to 19% per application session). The only product that showed a different behavior was 16% carbamide peroxide and deserves further investigations. It is worth pointing out that we have tested two material lots of 16% carbamide peroxide in the present study to confirm the results herein presented.
Another influential factor of hydrogen peroxide diffusion is the composition of the bleaching gel. The at-home groups present in their composition potassium nitrate and sodium fluoride. The sodium fluoride can form a calcium fluoride layer on the enamel surface which inhibits demineralization [31], which may contribute to a reduced penetration of hydrogen peroxide. Additionally, the at-home hydrogen peroxide gels wherein evaluated contain calcium in their composition. It is assumed that the presence of calcium is as the precursor of hydroxyapatite [32], and consequently, lower hydrogen peroxide can reach the pulp chamber when products with calcium phosphate are applied [18]. The pH of bleaching gels becomes an important factor when correlated with dental permeability, since most bleaching gels are acidic in order to stabilize and improve product life, they generate higher permeability [23,18]. According to other studies [33,34] either the pH of the bleaching gels used in this study are considered neutral, or what may have caused the lowest hydrogen concentration in hydrogen compared to the most commercially available acids [23,35].
Therefore, we can conclude from this in vitro study that the at-home products herein tested and used by the time recommended by the manufacturer led to similar hydrogen peroxide concentration in the pulp chamber of bleached teeth.

CONCLUSION
Bleaching agents used in office bleaching presented higher amounts of hydrogen peroxide in the pulp. While in home systems, the amount of HP that reaches the pulp chamber is not proportional to the concentration of whitening gels, but depends on the application time recommended by the manufacturers.