INTRODUCTION

Carbamide peroxide applied at home is the technique most employed by clinicians for bleaching vital teeth,^1,2 and it provides a high satisfaction rate among patients.³ However, some patients have indicated that they could not adapt to the use of whitening trays or disliked waiting 2 to 3 weeks for the bleaching results.^4,5 In these cases, an in-office bleaching technique is indicated because it is possible to obtain faster bleaching results compared with the at-home treatment,^4,6 with the advantage of exclusive in-office application and full control of the whole procedure, which prevents the intake of product.^4,7,8

Unfortunately, due to the higher concentration of hydrogen Peroxide (HP) used, in-office bleaching is related to a higher percentage of patients with tooth sensitivity (TS).^4-8 Bleaching-induced TS is associated with an inflammatory response from the pulp cells due to the diffusion of HP,⁹ which may alter the dental pulp.^10,11 This is the main reason for several changes to the in-office bleaching gels recently launched by manufacturers.

In the past, in-office bleaching gels were delivered with a low pH (around 2.0)^12,13 to increase the product's shelf life.¹⁴ However, this leads to several modifications in the chemical composition, morphology, and mechanical properties of the tooth structure,^15,16 which can increase the passage of HP and, consequently, increase TS.^17-20 These enamel alterations are more aggressive if the bleaching gels are in contact with the enamel surface for a long period of time. This occurs due to the prolonged contact of enamel with low pH in-office gels^21,22 and is one of the reasons manufacturers have indicated replenishing the gel during in-office bleaching. The HP is typically applied and left undisturbed on tooth surfaces for 10-20 minutes during in-office bleaching, and this procedure is repeated two to five times at each clinical appointment, depending on the bleaching gel brand.^23,24

Recently, in-office bleaching gels began presenting with neutral to alkaline pH, which are less aggressive to the tooth structure.^21,22,25,26 Some clinical studies have shown that neutral/alkaline pH bleaching gels reduced TS compared with acidic pH bleaching gels.^17,19,20 Due to this, manufacturers have suggested that these new brands could be applied without replenishing the gel during in-office bleaching. This simplifies the clinical procedure, reduces the risk of occasional soft tissue burns, and lowers the cost because less material is used per patient.

Unfortunately, the pH of different available commercial products is not clear to clinicians, nor is the best application mode made known to clinicians. Therefore, the aim of the present study was to compare the amount of HP that reaches the pulp chamber using in-office bleaching gels with different pH applied using two application modes. Also, the pH of these in-office bleaching gels was evaluated during application, and color change was evaluated immediately and 7 days after bleaching. The null hypotheses tested were that the pH and application mode of in-office bleaching gels does not affect (1) the amount of HP that reaches the pulp chamber and (2) color change after bleaching.

METHODS AND MATERIALS

This study was reviewed and approved by the Ethics Committee of the local university, under protocol No. 1355037. A total of 96 sound premolar human teeth, free of fracture lines, enamel defects, fissures, or any pathologic lesions, were divided according to a combination of the following main factors: 1) bleaching agents (Opalescence Boost PF 38% [Ultradent, South Jordan, UT, USA]; Whiteness HP Blue 35% [FGM, Joinville, SC, Brazil]; Whiteness HP Maxx 35% [FGM]; Lase Peroxide Sensy 35% [DMC, São Carlos, SP, Brazil]; or Total Blanc Office 35% [Nova DFL, Estrada do Guerengué, RJ, Brazil]) (Table 1); and 2) application technique (three 15-minute applications [3×15] or a single 45-minute [1×45] application). An additional, unbleached control group was added to the experimental design.

Table 1 Material, Batch Number, Composition, pH, and Manipulation for In-office Bleaching Gels

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Quantification of HP Penetration

The roots of all teeth (n=6 for each group) were sectioned using a diamond disc (KG Sorensen, Barueri, SP, Brazil, double-sided segmented, No. 7011) under constant water irrigation 3 mm apical to the cementoenamel junction. The pulp tissue was then carefully removed, and the pulp chamber washed with distilled water. The pulp chamber was carefully widened with a round bur (#1014, KG Sorensen). The entrance to the pulp chamber was widened using a round bur (No. 1014, KG Sorensen) to accommodate a micropipette (LABMATE Soft, 50 μL of capacity, HTL Lab Solutions, Warsaw, Poland), while avoiding touching the pulp chamber walls.

All teeth were vertically fixed to a wax plaque. Adhesive tape (Missner, Missner & Missner Ltda, Blumenau, SC, Brazil) was placed before nail polish application to limit the exposure area to 6 mm (radius) (Colorama, L'Oreal Brasil, Rio de Janeiro, RJ, Brazil), standardizing the area for bleaching gel application and color measurement.

Before applying the bleaching gels, an analytic curve was prepared with a standard HP solution at 30% concentration (Labsynth, Diadema, SP, Brazil) to obtain the relation between the light absorbance and the HP concentration. For this purpose, an acetate buffer solution (pH 4.5) was used to attract and stabilize the HP that might penetrate the pulp chamber. The solution was titrated with a potassium permanganate standard solution (Figure 1).²⁹ Next, a 25-μL aliquot of acetate buffer (pH 4.5) was placed into the pulp chamber of each tooth to absorb and stabilize any HP that might penetrate the pulp chamber.

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All bleaching gels were manipulated according to the description in Table 1 and placed over the enamel surface. In the 1×45 group, the product was placed on the enamel surface and remained untouched for the entire time (45 minutes). In contrast, the product on the tooth surface in the 3×15 group was removed using an aspirating tip, and the product reapplied two additional times, until 45 minutes had elapsed (Table 1).

After the treatment period, a micropipette was used to transfer the acetate buffer solution from the pulp chamber of each tooth to a glass tube. The pulp chamber of each tooth was then rinsed four times with 25 μL of acetate buffer, which was placed into the same glass tube. Then, deionized water (2.725 μL) was added to the glass tube along with 4 mmol/L of 4–amino-2,3-dimethyl-1-phenyl–3-pyrazolyl–5, 24 mol/L of phenol, 0.4 U/mL of peroxidase dissolved in phosphate buffer, 0.1 M pH = 7.0 (Glucose pp, Gold Análise Diagnóstica Ltda, Belo Horizonte, MG, Brazil). When stored at 4°C, the peroxidase catalyzes the degradation of HP in the presence of aminophenazone 4-aminoantipyrine with phenol. This reaction releases oxygen that oxidizes the chromogenic hydrogen donor, making the originally transparent solution turn to a shade of pink.

The amount of HP in the solution was then measured by evaluating the color absorbance using an ultraviolet-Vis spectrophotometer (Shimadzu UV 1601, Kyoto, Japan) at a wavelength of 510 nm to obtain the optical density.³⁰ According to Beer's Law, absorbance is directly proportional to the concentration; therefore, the concentration of HP (μg/mL) was determined by comparing it to the calibration curve obtained previously (Figure 1).^29,30 Once the concentration (μg/mL) and volume of the solution were known, the HP mass (l g) was calculated using the following equation: m = C * MM * V, where m represents mass, C is the concentration, MM is the HP molar mass (34.158), and V is the volume (3×10⁻³ L).³⁰ This procedure was repeated separately for each tooth.

RESULTS

Quantification of HP Penetration

The HP concentration that reached the pulp chamber in each group is depicted in Table 2. The two-way ANOVA revealed a statistically significant effect for the cross-product interaction (Table 2; p=0.001). The amount of HP was lower in the pulp chamber of the control group than the bleached groups (Table 2; p<0.05). In general, there was no difference between the in-office bleaching agents tested when they were used in the 3×15 technique (Table 2; p>0.41). However, there was a significant difference when the gels were applied using the 1×45 technique (Table 2; p=0.001). In the 1×45 technique, the neutral/alkaline in-office bleaching gels Opalescence Boost PF 38% and Whiteness HP Blue 35% showed lower mean HP penetration than the acidic bleaching gels (Table 2; p=0.001).

Table 2 Means and Standard Deviations of the Hydrogen Peroxide (HP) Concentration (μg/mL) Detected Inside the Pulp Chamber for the Treatment Groups and the Statistical Comparison

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Color Change Evaluation

The ΔE in each group is shown in Table 3. Two-way ANOVA revealed no statistically significant effect for the cross-product interaction and the main factors (Table 3; p>0.54). These results indicate that no significant difference in color change was observed between groups. However, a significant whitening effect was detected by all groups, regardless of the pH and application mode of the evaluated in-office bleaching gels (Table 3).

Table 3 Means and Standard Deviations of the ΔE for the Treatment Groups and the Statistical Comparison

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Quantification of pH in Contact With Enamel Surface

The quantification of pH in contact with the enamel surface in each group is shown in Table 4. The two-way ANOVA revealed a statistically significant effect for the cross-product interaction (Table 4; p=0.01). The baseline results confirmed that Opalescence Boost PF 38% and Whiteness HP Blue 35% are neutral/alkaline in-office bleaching gels. On the other hand, Whiteness HP Maxx 35%, Lase Peroxide Sensy 35%, and Total Blanc Office 35% were confirmed to be acidic in-office bleaching gels (Table 4). The pH remained stable during the 3×15 application technique for all bleaching gels evaluated (Table 4; p>0.05). However, when applied in the 1×45 technique, there was a significant difference between the in-office bleaching agents tested (Table 4; p=0.01). When the neutral/alkaline in-office bleaching gels were applied, no significant change in pH was observed during the 1×45 application (Table 4; p>0.61). On the other hand, all acidic bleaching gels significantly decreased in pH during the 1×45 technique, mainly after 30 minutes (Table 4; p<0.01).

Table 4 Means and Standard Deviations of pH at the Different Assessment Points for the Treatment Groups and the Statistical Comparison

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DISCUSSION

The results of the present study showed that when the different application techniques were evaluated, a significant difference was observed for the acidic pH in-office gels compared with the other gels, leading us to reject the first null hypothesis. A higher amount of HP was usually found inside the pulp chamber for the 1×45 technique. According to the pH measurement, three products (Whiteness HP Maxx 35%, Lase Peroxide Sensy 35% and Total Blanc Office 35%) were considered acidic pH in-office gels in agreement with the measurements of several authors.^21,22,26 This is expected because acidic gel could cause more microhardness loss and morphologic change of enamel due to the enamel surface demineralization compared with the neutral/alkaline gels.^15,16

The results of the present study also showed a significant decrease in the pH of the acidic in-office gels with prolonged contact with enamel.^21,22 This may lead to further damage to the surface of the teeth, resulting in an increase in the superficial porosities of the enamel^15,16,31 and, consequently, greater passage from HP to the pulp chamber, in agreement with the results of HP penetration in the present study. This is the main reason the replenishing technique is indicated for the majority of in-office bleaching gels available on the market.^7,32 When the acidic gel is removed after 15-20 minutes of application, it prevents further changes on the enamel surface and, consequently, decreases the passage of HP, as observed in the results of the present study.

On the other hand, the results of the present study showed no significant difference for Opalescence Boost PF 38% and Whiteness HP Blue 35% with regards to the application technique. According to the pH measurement, these gels are considered neutral/alkaline pH gels. It has been described that a higher pH in bleaching gels leads to more dissociation of HP into free radicals. For instance, HP with a pH of 9 dissociated 2.7 times more than HP with a pH of 4.4.³³ Thus, if more HP dissociates into free radicals within the dental structure, less surplus of HP is available to travel within the dentin and reach the pulp chamber. This may explain the lower amount of diffused HP for the Opalescence Boost PF 38% and Whiteness HP Blue 35%.

When the 1×45 technique was evaluated, the neutral/alkaline gels typically showed a lower amount of HP inside the pulp chamber compared with the acidic gels, which was previously shown by Mena-Serrano and others.²⁷ This is in agreement with recent published clinical studies that showed a lower percentage of patients who reported TS when neutral/alkaline pH bleaching gels were compared with acidic pH bleaching gels.^17,19,20

The results of the present study and the study of Mena-Serrano and others²⁷ are not in consensus with the current literature. For instance, Pignoly and others³⁴ and Marson and others³⁵ showed that the amounts of HP found in the pulp chamber were similar, regardless of the pH of the gel used. However, methodologic differences may explain this controversy. While some studies used a small part of a bovine tooth, turning it into a simulated pulp chamber, the present study used human teeth without any preparation, which better simulates the clinical situation.^34,35 Also, Kwon,³⁶ who was the first to introduce the protocol of not replenishing the gel during in-office bleaching more than 10 years ago, showed that the amount of HP inside the pulp chamber was significantly lower when one prolonged application was evaluated. However, the application of the bleaching gel was evaluated using a different technique (sealed technique), so a clear comparison cannot be made between the results of the present study and Kwon's results.³⁶

It is worth mentioning that the results of the present study showed that these neutral/alkaline pH gels maintain their pH for the entire application duration, which was also previously observed by Trentino and others.²¹ This could be explained by the fact that these gels have a pH that is almost neutral; few ion exchange reactions occur with the surface of the enamel,^37,38 which enables pH to be maintained. This helps to explain why the application technique did not significantly change the amount of HP inside the pulp chamber when neutral/alkaline gels were applied.

Unfortunately, the products with the lower concentration of HP in the pulp chamber (alkaline/neutral gels) contain desensitizing agents. Opalescence Boost PF 38% contains potassium nitrate and sodium fluoride, and Whiteness HP Blue 35% contains calcium digluconate. These agents act in different ways; for instance, potassium nitrate reduces sensitivity by decreasing the capacity of the nerve fibers to propagate nerve impulses. On the other hand, sodium fluoride and calcium digluconate act by blocking the dentinal tubules, leading to a reduced flow of fluid into the pulp chamber.³⁹ A closer view of clinical trials that evaluated the TS of in-office bleaching gels containing desensitizer agents have shown controversial results.^17,19,20,40 Future clinical studies need to be done to evaluate the effect of neutral/alkaline gels with and without desensitizer on HP penetration.

It is also worth mentioning that it is not only the pH and the addition of desensitizing agents that could influence the amount of HP inside the pulp chamber. Other factors may affect the amount of HP that enters the pulp cavity, such as HP concentration and the viscosity of the bleaching gels.^27,41 Future studies need to be done to evaluate how these factors interact with the pH of different bleaching gels.

Finally, independent of the pH and application technique applied, a significant whitening was detected and no significant difference in color change was observed between groups, leading us to accept the second null hypothesis. This is in agreement with several clinical studies that have shown that all in-office bleaching materials evaluated in the present study are effective in terms of whitening.^17-19,20,40 However, according to the results of the present study, the pH and application technique of in-office bleaching gels could impair the amount of HP reaching the pulp chamber.

CONCLUSION

The amount of HP that reaches the pulp chamber was lower when neutral/alkaline pH gels were used, independent of the application technique. For acidic pH gels, it is preferable to use the 3×15 application technique, mainly because the 1×45 technique results in lower pH.