Alteration and Biological Response of Two In-office Bleaching Protocols

but also alterations in pulpal tissues. Hard-tissue penetration, color alteration and the pulp response alterations were evaluated for two in-office bleaching protocols with H2O2. For trans-enamel/dentin penetration and color alteration, discs of bovine teeth were attached to an artificial pulp chamber and bleached according to the groups: BLU (20% H2O2 1x50 min, Whiteness HP Blue); MAX (35% H2O2 3x15 min, Whiteness HP Maxx); Control (1x50 min, placebo). Trans-enamel/dentin penetration was quantified based on the reaction of H2O2 with leucocrystal violet and the color analyzed by CIELab System. Twenty Wistar rats were divided into two groups (BLU and MAX) and their maxillary right molars were treated according to the same protocols of the in vitro study; the maxillary left molars were used as controls. After 2 days, the animals were killed and their maxillae were examined by light microscopy. The inflammation of pulp tissue was scored according to the inflammatory infiltrate (1, absent; 2, mild; 3, moderate; 4, severe/necrosis). Data were analyzed by statistical tests (a=0.05). MAX showed higher trans-enamel/dentinal penetration of H2O2 (p<0.05). The color alteration was similar for both groups (p>0.05), and different when compared to Control group (p<0.05). MAX showed severe inflammation in the upper thirds of the coronal pulp, and BLU showed moderate inflammation (p<0.05). In-office bleaching protocols using lower concentrations of hydrogen peroxide should be preferred due to their reduced trans-enamel/dentinal penetration since they cause less pulp damage and provide same bleaching efficiency. Pene t r a t i on Capa c i t y, Co l o r Alteration and Biological Response of Two In-office Bleaching Protocols


Introduction
In-office bleaching is a popular dental aesthetic treatment option because of its high success rate (1).Several methods and approaches have been described in the literature for bleaching vital teeth, such as different bleaching agents, concentrations, time of application and product presentation (2).Hydrogen peroxide (H 2 O 2 ) is the main active compound of most commercially available bleaching agents (3), whitens teeth by oxidizing organic structures through reactive oxygen species (ROS) (4).
The use of H 2 O 2 at high concentrations (25-40%) leads to tooth color enhancement and reduced shade guide units.Of note, higher the concentration and application time of H 2 O 2 , greater is the ROS production (5), such as superoxide anions (O 2-), H 2 O 2 , hydroxyl radical (-OH) and O 2 (5).The low molecular weight of H 2 O 2 and its sub products favors the rapid diffusion of these ROS through mineralized dental tissues (4).Here, ROS induce oxidative stress in a time/concentration-dependent fashion (6), at least in part, because of imbalance between the amount of ROS and that of endogenous or exogenous antioxidants (7).This stress, in turn, causes mutation, enzyme inactivation, protein fragmentation and degradation of pulp cells (8), resulting in pain or necrosis (5).
The success of in-office bleaching protocols is directly related to the diffusion capacity of H 2 O 2 through enamel and dentin (9).However, deeper penetration increases the risk of pulp damage (9).New products based on 20% H 2 O 2 were placed on the market, but there are few studies comparing the efficacy and the effects on pulp of this concentration to traditional products (2).It is important to maintain the effectiveness of dental bleaching, however the indicated posology must be evaluated due its biologic safety.Changes in in-office dental bleaching technique may provide alternatives for maintaining its effectiveness and prevent or at least reduce the harmful effects of this therapy on pulp tissue (7).
Considering the great demand for dental bleaching procedures and the aesthetic benefits obtained due this treatment, it is required deeper studies for different H 2 O 2 protocols considering its final results and their consequences.Therefore, the aim of this study was to evaluate trans-enamel/dentinal penetration of H 2 O 2 , color alteration, and the influence on pulp damage of two inoffice bleaching protocols with H 2 O 2 .The null hypotheses tested were: the concentration of the H 2 O 2 does not L.T.A. Cintra et al. influence the trans-enamel/dentinal penetration, the bleaching effectiveness and the pulp damage.

In vitro Experiment
Collection and Standardization of Specimens Forty-five bovine incisors (n=45) were obtained from animals aged 24-30 months (9,10).To perform the enamel surface standardization, teeth were analyzed under a stereomicroscope, and those with spots, disparate morphological changes, and excessive wear of the incisor and the presence of cracks were excluded.The selected teeth were cleaned with periodontal curettes, pumice and water.They were fixed on a device attached to a drill platform bench (FGC-16; Ferrari, São Paulo, SP, Brazil) and cut with a diamond tip (0.8 mm in diameter; Dinser Ferramentas Diamantadas Ltda, São Paulo, SP, Brazil) under constant irrigation.Cylinders (5.7 mm in diameter) were created from the middle third of the buccal surface.
The dentinal surface was manually smoothened with 600-grit aluminum oxide sandpaper (T469-SF-Noton, Saint Gobam Abrasives Ltda, Jundiai, SP, Brazil) until 3.5-mmthick discs (~1.3 and 2.2 mm of enamel and dentin, respectively), measured by a digital caliper (500 to 144 B, Mitutoyo South America Ltda, Suzano, SP, Brazil), were obtained.The smear layer was removed by applying EDTA solution for 30 s, followed by a rinsed with deionized water.

Artificial Pulp Chambers (APCs)
Each disc was adapted to an APC (9)(10)(11).Each APC had upper and lower compartments with 8-and 6-mmdiameter openings, respectively, to allow appropriate positioning and lateral sealing of the specimen.The lower compartment also had lateral perforations for circulation of the bleaching agent and was used to quantify H 2 O 2 penetration in the specimen.The specimens were attached in the upper compartment of the APCs between two silicone rings (5.60-mm inner diameter, 1.78-mm thickness; Ref. OR 008; Rodimar Rolamentos Ltda, Araraquara, SP, Brazil), which maintained fixation of the disks and sealed with pink wax, restricting lateral penetration of the bleaching agent.The lower compartment side had holes to allow circulation of the solutions that remained in contact with the dentin surface (acetate buffer solution).

In Vitro Bleaching Procedure
The APCs were individually placed in wells of acrylic cell culture plates filled with 1-mL acetate buffer solution.
The dentinal surface remained in contact with the acetate solution.The specimens were then treated with either 0.04 mL of 20% H 2 O 2 (Whiteness HP Blue) once for 50 min (BLU group), or 0.04 mL of 35% H 2 O 2 (Whiteness HP Maxx) in three times for 15 min each (MAX group) with a 2-min interval between sessions, or 0.04 mL of placebo gel (Control group) for 50 min.All procedures were performed in accordance with the manufacturer's instructions (FGM Produtos Odontologicos Ltda, Joinville, SC, Brazil).

Quantification of H 2 O 2 Penetration
After the bleaching procedure, 25 µL of the acetate buffer solution was removed from each well and mixed with 2,750 µL of distilled water, 100 mL of leucocrystal violet (0.5 mg/mL; Sigma Chemical Co, St. Louis, MO, USA), and 50 µL of peroxidase (1 mg/mL, Sigma Chemical Co).The solution was diluted to a final volume of 3 mL with distilled water.
Penetration was evaluated on the basis of the reaction of H 2 O 2 with leucocrystal violet, catalyzed by peroxidase enzyme (9).The color of the mixture changes according to the amount of H 2 O 2 .As the signal is proportional to the absorbance of H 2 O 2 , the amount of H 2 O 2 penetrating the tooth surface and solution in the wells can be indirectly assessed.An ultraviolet visible reflectance spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) was used for measurements 30 min after each bleaching session.The calibration factor (CF) was calculated as the ratio of the concentration of the standard solution of H 2 O 2 to its absorbance.The average CF was used to calculate the H 2 O 2 concentration in each specimen.

Color Evaluation
Color assessment of the same specimens was performed by using an ultraviolet visible spectrophotometer, Model UV-2450 (Shimadzu, Kyoto, Japan), which uses the color model CIE L* a* b* established by the Commission Internacionale I'Éclairage-CIE.For this purpose, we made a black silicone mold containing a central hole, which allowed standard positioning of the specimen during the readings.Three measurements were obtained for each specimen, and the average between them was calculated.The analyses were performed before the initiation of treatment and 24 h after the bleaching session.The CIE L* a* b* color calculates the distance between two points by using the following formula:

In Vivo Experiment
Animals Twenty male Wistar rats (180-200 g) were used in this study.The animals were housed in a temperature-controlled environment (22 °C±1 °C, 70% humidity and 12 h light-

In vivo and in vitro study of bleaching protocols
dark cycle) and received water and food ad libitum.All the animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD, USA).The experimental protocol (CEUA-2012-00846) was approved by the Ethics Committee of Universidade Estadual Paulista (UNESP; Araçatuba, SP, Brazil).

In Vivo Bleaching Procedure
The rats were anesthetized by intramuscular injections of ketamine (87 mg/kg; Francotar, Virbac do Brasil Ind. e Com.Ltda; Roseira, SP, Brazil) and xylazine (13 mg/kg; Rompum, Bayer SA, São Paulo, SP, Brazil) and assigned to two equal groups (12).The BLU group (n=10) underwent one bleaching session with 20% H 2 O 2 (Whiteness HP Blue) for 50 min.The MAX group (n=10) underwent three 15-min sessions with 35% H 2 O 2 (Whiteness HP Maxx) with a 2-min interval between sessions.Both the protocols were performed according to the manufacturer's recommendations (FGM).The products were applied to three maxillary right molars in both the groups; the contralateral molars served as control group.

Histology
Two days after the bleaching procedure, the animals were killed with an overdose of the anesthetic solution.The bilateral maxillae were separated, dissected and fixed in 10% buffered formalin solution for 24 h.The tissues were then dehydrated through a graded series of ethanol, embedded in paraffin, cut into 5-μm sagittal sections, and stained with hematoxylin and eosin (H&E).

Statistical Analysis
Data were collected and analyzed by a single calibrated operator in a blinded manner.Normality and homoscedasticity were verified.Analysis of variance (ANOVA) and Student t-test were used to analyze hard-tissue penetration and color alteration, while one-way ANOVA and Mann-Whitney U-test were used for statistical comparisons of pulp damage at the significance level of 5% (a=0.05).

Trans-Enamel/Dentinal Penetration of H 2 O 2 and Color Alteration
BLU, MAX and control group had mean penetration values of 4.42±0.46,6.14±0.67 and 0.04±0.03µg/mL, respectively.Student t-test revealed significant differences for trans-enamel/dentinal penetration of hydrogen peroxide between the groups (p<0.05),showing that higher the concentration, higher the penetration through hard dental tissue (Table 1).
The analysis of Δa, Δb, ΔL and ΔE showed significant differences for color alteration between Control group as compared with BLU and MAX (p<0.05).However, the BLU and MAX groups showed similar results to all analysis (p>0.05) (Table 1).

Inflammatory Response
The control group exhibited well-defined acellular and cell-rich layers under an intact odontoblast layer and even distribution of cell components, blood vessels, and extracellular matrix structures in the dental pulp (Fig. 1A-C).
All the BLU specimens showed various changes in the coronal pulp.Seven and three specimens showed moderate and severe inflammation or necrosis, respectively, in the occlusal third, with absence of cells and the odontoblast layer as well as tissue disorganization in some pulp horns (Fig. 1D).The middle third showed moderate to mild inflammation (Fig. 1E).The cervical thirds of coronal pulp had mild inflammation, narrowed odontoblast layer and congested blood vessels (Fig. 1F).Two specimens showed mild inflammation in the cervical third of the radicular pulp.
In the MAX group, eight specimens exhibited necrosis in the pulp horns (Fig. 1G) and severe or moderate inflammation in the middle third, with an absent odontoblast layer and congested blood vessels (Fig. 1H).Three and five specimens showed moderate and mild inflammation, respectively, in the cervical third of the coronal pulp (Fig. 1I).Three specimens showed mild inflammation in the cervical third of the radicular pulp.
In the upper two-thirds of the coronal pulp, a significant difference in inflammatory response was observed between  BLU and MAX groups (p<0.05).The intensity decreased apically (Table 2).Overall, BLU and MAX had median scores of 3 and 4, respectively in the occlusal third and, 2 and 3, in middle third of the coronal pulp (p<0.05).

Discussion
The high concentration gel influence H 2 O 2 penetration and induced more severe alterations in the pulp tissues, however not obtained more bleaching effectiveness.In vivo and in vitro studies are among the experimental models used to analyze the alterations caused by H 2 O 2 in the pulp tissue and the level of penetration through enamel and dentin reaching the pulp chamber (9,13).Bovine teeth were used because human teeth are more susceptible to H 2 O 2 penetration than bovine (10), as well as rat teeth that allows the analysis on dental pulp (12).
The literature has shown that bleaching agents at high concentrations can produce injuries to the pulpal tissue (5,12,13), for this reason it was used two different bleaching agents, one containing 35% and the other containing 20% hydrogen peroxide.In this study, we found significant differences in bovine teeth hard-tissue penetration between MAX and BLU, which can be attributed to the different concentrations of the H 2 O 2 .The result showed that higher the H 2 O 2 concentration, deeper is the penetration through enamel and dentin, rejecting the first null hypothesis; the application time is also important ( 14), causing microporosities in the tooth structure (15), which was also found by previous research (9).Furthermore, the H 2 O 2 BLU has calcium in its composition, which tends to reduce possible structural alterations in the enamel after bleaching procedures by increasing the saturation of the gel and the enamel resistance to demineralization (16).
The data analysis showed similar bleaching effectiveness to both products, accepting the second null hypothesis.When the coordinates (Δa*, Δb* e ΔL*) are individually analyzed, it can be observed that the control group values remains unaltered, demonstrating the color maintenance during the experiment.In addition, the specimens submitted to dental bleaching procedure presented negative Δb values, showing that regardless the used product, the specimens became less yellowish (17).
Dental luminosity increase (ΔL) is the most expected and important optical effect in the effectiveness analysis of different dental bleaching therapy (18).In this context, the present study showed that both in-office bleaching protocols with H 2 O 2 presented similar behavior in the experimental conditions, suggesting that the product concentration can not be considered as a priority for the selection of dental bleaching technique in relationship to the color alteration.
On the other hand, the Δa change was very low.

In vivo and in vitro study of bleaching protocols
Although it is not a frequent coordinate influenced by the bleaching procedure, the statistical analysis detected that Δa values of bleached groups differed from the control group.It is believed that this reading may have detected any dyes in the bleaching gels used topically on the dental enamel, not influencing the aesthetic obtained with the treatment.
The similar results between MAX and BLU products, which might be explained by the diffusion coefficients, application time and bleaching agents concentration.Borges et al. (19) demonstrated that H 2 O 2 at the concentration of 35% has bleaching effectiveness greater than the concentration of 20% when both were applied for 30 min.However, de Almeida et al. (2), obtained similar results to our study observing the same bleaching effectiveness when H 2 O 2 35% and 20% was applied for 45 min.It means that a less concentrated product applied for a longer time can produce results as good as a higher concentrated product (2,18).Besides the concentration difference and the application time, the similar color alteration between both experimental groups might also be related to different viscosity of the hydrogen peroxide gels (20).
The oxidizing ability of H 2 O 2 may be responsible for the reduction in dentinal organic components when bleaching product is applied directly to dentin (8).Bleaching agent penetration occurs mainly because of the low molecular mass and ability to denature proteins, increasing ion movement through tooth structure (11), but it is also influenced by tooth density and dentinal tubule diameter (21).
After applying a bleaching agent, changes in the prismatic structure and biochemical properties, such as loss of carbonate and proteins, of enamel and dentin are notable.The loss becomes greater with enhanced proteolytic activity and reduced collagen, increasing the number of diffusion channels and cell permeability (22), and the pH of Whiteness HP Blue and HP Max with or without reapplications remains above the critical value for enamel (pH 5.5) even after the application time, influencing the penetration of H 2 O 2 .Therefore, possible pulp damage caused by ROS-promoted oxidation should be checked after bleaching (8).Despite the penetration power of H 2 O 2 , its diffusion through dentinal tubules of teeth with vital pulps may be prevented by dentinal fluid flow produced by intrapulpal pressure, cytoplasmic extensions of odontoblasts and other intratubular components (23).
In the present study, the histological findings showed that higher concentration of H 2 O 2 induced greater pulp changes, rejecting the third null hypothesis.The MAX group had disorganized odontoblast layer and pulp with areas of necrosis, liquefaction, absence of cells, intense inflammatory infiltrate, and dilated and congested vessels, which goes against to a previous research that shows pulp alterations until pulp necrosis as increasing the number of sessions (12).
In-office bleaching with 35% H 2 O 2 for 30 min causes severe pulp reactions such as dentin deposition, inflammation, and damage to odontoblast-like cells, and internal resorption in dogs (13).However, human premolars bleached with 38% H 2 O 2 mostly show normal dental pulp and no inflammation (23).Further, human incisors bleached with 38% H 2 O 2 become sensitive, but not treated human premolars (5).These differences may be related to the thickness of the hard tissues and their structural features.
Results obtained in animal studies cannot be extrapolated directly to human teeth (5,22,23), especially because of the difference in enamel thickness between humans and rats (2.5 mm vs. 100 µm) (24).Rat teeth are less dense than human teeth and extremely porous, allowing easy diffusion of fluids and dyes (24).However, in a previous study, human mandibular incisors subjected to repeated bleaching demonstrated large areas of necrosis in the coronal and radicular pulp, with mild inflammation (5).Considering the similarity of these results with our findings, rat molars may be a suitable model to predict the results of procedures performed in human mandibular incisors.
It was found that most of the treated specimens exhibited inflammatory infiltrate, congested blood vessels, disorganized pulp especially in the pulp horns of both the groups, and large areas of necrosis in the MAX group.This suggests that the intense and rapid penetration of dental hard tissue by H 2 O 2 may be beyond the capacity and speed of the pulp's protective mechanism, resulting in an immediate response to this highly aggressive compound.The problem lies in not knowing exactly the concentration of H 2 O 2 that the dental pulp can tolerate without damage (13).However, our results suggest that application of high concentrations of H 2 O 2 cause intense changes in dental tissues (25).
It was also shown that the concentrations of bleaching agents are more related to the inflammatory response intensity than to the bleaching efficacy.Thus, the results show that it is possible to have a variety of bleaching treatments and protocols, obtaining a similar aesthetic result with less damage to the dental tissues.
This study indicates that in-office bleaching protocols using lower concentrations of hydrogen peroxide should be preferred due to their reduced trans-enamel/dentinal penetration causing less pulp damage, and providing the same bleaching efficiency.

Figure 1 .
Figure 1.Representative images of H&E-stained sections 2 days after bleaching.The three right panels in each row are magnified views (×400) of the images (×100 magnification) from the CG (A-C), BLU (D-F) and MAX (G-I) groups.The asterisks indicate predentin, the black arrows depict the odontoblastic layer and the yellow arrows show pulp cells and blood vessels.

Table 1 .
Mean and standard deviation (SD) of H 2 O 2 penetration and color alteration

Table 2 .
Inflammation Intensity among the experimental groups aDifferent letters in the same row indicate statistically significant differences in median score (p<0.05).