Effects of in-office bleaching agent combined with different desensitizing agents on enamel

Abstract Objective: To analyze color change, microhardness and chemical composition of enamel bleached with in-office bleaching agent with different desensitizing application protocols. Materials and Methods: One hundred and seventeen polished anterior human enamel surfaces were obtained and randomly divided into nine groups (n = 13). After recording initial color, microhardness and chemical composition, the bleaching treatments were performed as G1: Signal Professional White Now POWDER&LIQUID FAST 38% Hydrogen peroxide(S); G2: S+Flor Opal/0.5% fluoride ion(F); G3: S+GC Tooth Mousse/Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) paste(TM); G4: S+UltraEZ/3% potassium nitrate&0.11% fluoride(U); G5: S+Signal Professional SENSITIVE PHASE 1/30% Nano-Hydroxyapatite (n-HAP) suspension(SP); G6: S-F mixture; G7: S-TM mixture; G8: S-U mixture; G9: S-SP mixture. Color, microhardness and chemical composition measurements were repeated after 1 and 14 days. The percentage of microhardness loss (PML) was calculated 1 and 14 days after bleaching. Data were analyzed with ANOVA, Welch ANOVA, Tukey and Dunnett T3 tests (p<0.05). Results: Color change was observed in all groups. The highest ΔE was observed at G7 after 1 day, and ΔE at G8 was the highest after 14 days (p<0.05). A decrease in microhardness was observed in all groups except G6 and G7 after 1 day. The microhardness of all groups increased after 14 days in comparison with 1 day after bleaching (p>0.05). PML was observed in all groups except G6 and G7 after bleaching and none of the groups showed PML after 14 days. No significant changes were observed after bleaching at Ca and P levels and Ca/P ratios at 1 or 14 days after bleaching (p>0.05). F mass increased only in G2 and G6, 1 day after bleaching (p<0.05). Conclusions: The use of desensitizing agents containing fluoride, CPP-ACP, potassium nitrate or n-HAP after in-office bleaching or mixed in bleaching agent did not inhibit the bleaching effect. However, they all recovered microhardness of enamel 14 days after in-office bleaching.


Introduction
Esthetics is a major concern in dentistry today.
Changes in the smile has surprising effects on the self-esteem of an individual, especially in a society that places too much emphasis on physical appearance. 1 Tooth bleaching is one of the most requested esthetic procedures asked for by patients who want an attractive smile. Today, many different bleaching systems have been introduced to meet this demand. 2 Although the at-home bleaching system using 10% carbamide peroxide was considered the standard treatment for vital teeth in the past, in-office technique has become more popular than at-home bleaching, as highly concentrated (30%-35% hydrogen peroxide) products promote faster tooth whitening. 3 Regardless of the technique or product used, the mechanism of action of bleaching agents is based on a complex oxidation process with release of reactive oxygen species, which penetrate through the pores of enamel rods and reach the dentin, breaking down organic molecules and producing lighter, smaller, and clearer compounds. 4 In-office bleaching has been validated as effective for lightening tooth color, most clinical studies have shown that more than 70% of the patients who undergo in-office bleaching complain of tooth sensitivity and this is the main deterrent for patients to successfully complete their bleaching treatment. 5 Some remineralizing components, such as fluoride, calcium, amorphous calcium phosphate, and hydroxyapatite are used to minimize adverse effects of bleaching treatments on the enamel. 6 In an attempt to decrease or limit dental sensitivity during bleaching, a number of different desensitizing agents have been introduced for use before or after bleaching or in association with bleaching gels. 7 These components are added in the bleaching gel to prevent demineralization of the enamel during bleaching and the decrease in dental sensitivity reported by many patients during and after bleaching treatment. 8 However, there are conflicting reports about the effects of bleaching agents on alterations of the surface morphology and chemical properties of dental tissues. 9 Studies comparing the effect of desensitizing agents on the potential of bleaching gels and on the enamel structure are limited. 6,10,11 Therefore, this study aimed to evaluate the effects of different desensitizing protocols on the efficacy of in-office bleaching agent and microhardness and chemical composition of human enamel in vitro. The null hypothesis tested was that the use of desensitizing agents with the bleaching system would alter the bleaching efficacy and would have no beneficial effect on enamel.

Materials and Methods
This study was conducted in accordance with all the provisions of the local human subjects oversight committee, with the Declaration of Helsinki (1964) and with the policies of Hacettepe University. The approval code for this study is FON 12/19.

Sample preparation
Recently extracted intact human incisors were stored at room temperature in distilled water until testing. The roots of the teeth were removed 2 mm apically to the cementoenamel junction using diamond discs, and the crowns were embedded in colorless

Materials Composition
Signal self-cure acrylic-liquid mixture (Panacryl, Rubydent, Istanbul, Turkey). The enamel surfaces were ground flat using 400-grit silicon carbide abrasive paper and polished with 600 and 1200-grit aluminum oxide papers on a polishing machine (Mecapol P230, Presi, France), until a circular area of 10 mm diameter was exposed. The specimens were then subjected to color, surface microhardness and chemical composition analyses. For the standardization of the specimens, 170 teeth with the similar initial Vickers hardness values were selected for the study.

Color measurements
The color distribution (L*, a* and b*) of each specimen was measured with a spectrophotometer (VITA Easy Shade, Vident, Brea, CA, USA).
Measurements were taken at the middle third region of the specimens, which were repeated three times at each evaluation, and then their averages were calculated. The spectrophotometer was calibrated with a white reflectance standard according to the manufacturer's protocol before each measurement.
The overall color difference of specimen in each group was calculated by the following expressions 12,13 :

Microhardness evaluation
The microhardness values of the enamel surfaces were obtained with a microhardness tester (Shimadzu

Results
Sample characteristics were similar for each group before test procedures for all the tested parameters.          a protocol, which may promote remineralization after bleaching and recover the microhardness loss and surface deterioration of enamel caused by bleaching.
The mixture of remineralizing and bleaching agents was capable of reducing sensitivity and recover or at least avoid alterations in the surface morphology of enamel. 17 Fluoride, potassium nitrate, ACP or n-HAP have been introduced in recent bleaching products to prevent either hypersensitivity or demineralization effect. [18][19][20] Moreover, these novel techniques did not decrease the bleaching potential of the peroxide. 21 In this study, the color change effect of the bleaching agent, both alone or combined with desensitizing agents (after bleaching or mixed with the bleaching gel), significantly decreased 14 days after the bleaching treatment when compared to 1 day after bleaching. This excludes any possible dehydration effect and represents the real effect in color change.
Similarly, Zekonis, et al. 22 (2003) reported that the greatest values of color change were observed after bleaching, followed by a relapse 7 days after bleaching.  Laboratory studies have shown microscopic surface enamel defects, associated with bleaching, could be repaired using a paste containing n-HAP crystals. 25 Because of its nano metric size, n-HAP can easily penetrate inside the dentinal tubules and enamel microcracks; thereby it promotes a reliable sealing for the tubules and microcracks, and it restores the microstructure and chemical composition of the tooth tissues. 18,26,27 The n-HAP crystals are also very resistant to acidic challenges that routinely take place in the oral environment. 28 In this study, only 1 day after bleaching, 2 desensitizing agent groups showed an increase in initial microhardness values, all the groups were In the daily routine of oral environment, acidic challenges lead to conditions for enamel remineralization, and previously demineralized enamel is known to be more susceptible to further remineralization. Bleaching agents may cause demineralization on the enamel, by which ionic changes are induced and mineral uptake is increased to replace the mineral loss during treatment.
Therefore, the specimens were stored in artificial saliva to mimic the oral conditions. On the other hand, some studies suggest that saliva may partially lead to the replacement of the mineral loss caused by the bleaching treatment. 29,30 Basting, et al. 31 (2005) reported that immersion in a solution similar to human saliva for two weeks after bleaching may increase microhardness of bleached enamel. Similar to previous findings, saliva could present a reformative effect on the microhardness loss in this study.
Although the saliva is expected to remineralize the bleached enamel, some in situ studies have reported a decrease in microhardness on enamel immediately after bleaching treatments. 15 The microhardness loss could be related to mineral content loss caused by demineralization. The effects of bleaching agents on mineral loss of enamel and dentin are usually tested by microhardness studies for being directly related to the mineral content of the tooth. 15,32 For this reason, microhardness tests can be used both as a comparative measure of hardness changes and as a direct measure of mineral loss or gain as a consequence of demineralization and remineralization processes.
On the other hand, EDS determines the mineral content of dental hard tissues. The main advantage of this system is its capability to provide an accurate and non-destructive analysis of the specimens. 33,34 As a consequence, this method was used to evaluate the changes on mineral content of enamel in this study. The findings revealed that bleaching or desensitizing agents used after bleaching or as a mixture in bleaching agent did not affect Ca and P levels, but F levels increased in the groups treated with F-containing desensitizers after bleaching. The possible explanation for this increase may be the use of F-containing bleaching agent to prevent either sensitivity or demineralization during the bleaching treatment. However, controversial results exist on the topic, since no supporting evidence regarding the influence of F-containing bleaching gels on the demineralization has been documented. In an in vitro study, the bleaching agent either combined with F or Ca was insufficient to prevent the reduction in the surface microhardness of enamel. 35 Conversely, Lee, et al. 36 (2006) reported a decrease in the Ca/P ratio of bleached bovine enamel after application of 30% HP. In contrast to their study, although 38% HP was used, the Ca/P ratios were not changed in this study.
With the limitation of this study, a specimen was examined per group under SEM and revealed no deleterious effects on enamel. The majority of the previous SEM studies that investigated the surface morphology following bleaching were in the same line with his study, reporting no significant changes. 33,37 On the other hand, some other studies have reported slight alterations on the enamel morphology with an increased number of pits, pores and erosion areas, which may also indicate demineralization and dissolution. 15,38,39 Therefore, modifications of the bleaching gel did not influence the color change in this study.
The desensitizing agents used after bleaching or as a mixture in the bleaching gel were capable of increasing the microhardness of enamel. Thus, the null hypothesis is accepted. However, more studies are needed to evaluate the effect of desensitizers used after bleaching or used in different modifications on dental hard tissues after extended periods of time.

Conclusions
Within the experimental limitations of this in vitro study, the following conclusions could be drawn: 1. The use of desensitizing agents containing fluoride, CPP-ACP, potassium nitrate or n-HAP either after in-office bleaching or added to bleaching agent did not affect the color change.
2. Microhardness of enamel increased 14 days after in-office bleaching used with desensitizing agents.
3. Ca, P contents and Ca/P ratios did not change 14 days after bleaching. The F content increased in groups that contained only fluoride [G2 (S&F) and G6 (S-F mixture)] one day after bleaching.