INTRODUCTION

Tooth color is a result of the penetration into the enamel and dentin of light, which is scattered, partially absorbed, and reflected to observers. The unabsorbed wavelengths leave the tissue creating the color sensation; this depends on the interaction of light with certain molecules inside the mineralized tissues, which act as pigments or dyes.¹ Parts of these molecules (atom or group of atoms), known as chromophores, are responsible for its light absorption.² The small molecules of hydrogen peroxide, the most commonly used bleaching agent in dentistry, easily penetrate the tooth structure. Because of the inherent instability of hydrogen peroxide, it undergoes O-O bond homolysis, creating reactive oxygen species (ROS), also known as free radicals.^2,3 The ROS promote the oxidation of chromophore groups in the colored molecules inside enamel and dentin, reducing light absorption, and producing a chromatic change and whitening effect.^1,4–6

Bleaching treatment can be performed at home by using whitening agents with low peroxide concentrations for longer periods or in the dental office by using higher concentrations for shorter periods.⁶ Regardless of the technique and peroxide concentration, to allow a controlled application onto the tooth structure, the bleaching agent must contain a thickening agent, creating an appropriate gel consistency and allowing for topical application.⁷ A gel is a semisolid, containing small, colloidal inorganic particles or organic molecules (thickeners) dispersed in an interpenetrated liquid.⁸ These particles may attract and bond to neighboring ones, creating a three-dimensional network that immobilizes the liquid, in this case, the hydrogen peroxide and water, preventing flow when in a steady-state and increasing the viscosity of the system.⁹ Viscosity is a physical property of fluids related to their resistance to deformation because of internal frictional forces when in relative motion. The amount of thickener and the strength of the molecular interaction will determine the viscosity of the product.

The gel is a semisolid “dosage form” used to facilitate the administration and delivery of a medicament (active ingredient or drug) to a patient for different applications,⁸ such as skin care, dermatology, gynecology, and oral care. Internal diffusivity of the active ingredient inside the product has been reported to be inversely proportional to the viscosity of the medium^10,11 and directly proportional to the size of the penetrant molecule, as predicted with the Stokes–Einstein equation.^10,12 The composition of the thickener and its different electrical charge has been reported to result in different diffusivity of the drug, even in similar concentrations, because of the interaction between the electrical charge of the thickener and the drug.¹² Additionally, in different areas of medicine, studies have reported the negative impact of the higher viscosity of semisolid vehicles caused by the increase of thickener concentration on the drug release and the permeation into the tissues.^10,13–20 In addition, because of large differences among the thickener molecules, each specific agent produces a different kind of three-dimensional network responsible for the gel formation, which also can interfere with drug delivery. Therefore, to optimize formulation, the physicochemical factors that influence the release properties must be understood. Different kinds of thickener, as well as viscosity, may affect the interaction of peroxide-based bleaching gels with the tooth structure.

Once the peroxide has been delivered by the gel, it penetrates the intercrystalline spaces of the enamel and into the dentin tubules, reaching the pulp chamber. On its way, it interacts with tooth chromophores, creating the bleaching effect, and also with organic and inorganic substances of the dental tissues, producing side effects that should be minimized. The first is the demineralization of enamel, caused by the undersaturation of bleaching gels with respect to Ca²⁺ and PO₄³⁻ ions,²¹ low pH,²² or oxidation of enamel proteins.²³ In addition, some anionic thickeners have been reported to promote the demineralization of enamel by chelation,²⁴ reducing microhardness, and possibly increasing tooth wear²⁵ and staining.²⁶ When they reach the pulp, the ROS produce cell damage, interfering with the cellular metabolism and vascular permeability, resulting in an inflammatory response at various levels²⁷ and pain.²⁸ Therefore, to improve bleaching therapy, the aim is to reduce the side effects while maintaining or improving the whitening efficacy.

During the bleaching procedure, the pH of gels applied over the tooth surface reduces at a variable rate according to the specific formulation.²⁹ This occurs simultaneously with a reduction in peroxide concentration³⁰ because of decomposition and permeation toward the tooth. However, the number of free radicals inside the peroxide solution is directly proportional to the pH,³¹ and it has been reported that bleaching efficacy is associated with a higher pH.^32,33 Therefore, pH and peroxide concentration should be kept at a stable level while applied to the tooth to reduce the demineralization of enamel and maintain the whitening effect. The kind of the thickener and the level of viscosity might affect peroxide penetration and the presence of ROS in the pulp chamber.

Although extensively studied in other medical specialties,^10,34–38 the authors are aware of only one study that evaluated the effect of the vehicle’s viscosity on the bleaching efficacy.³⁹ Products from each manufacturer have different formulations and contain different excipients in different amounts, which results in quite different rheological properties. Knowledge of the effect of viscosity on a product’s efficacy is typically proprietary, and under the control of the dental company’s research and development department. That the full composition of each product is generally not released hampers an intensive and specific analysis of the topic.

Considering the paucity of scientific information available, the aim of this study was to investigate the influence of the viscosity and kind of thickener in 35% hydrogen peroxide bleaching gels on the tooth structure (color change, demineralization of enamel, and permeation) and inside the gel (ROS, pH, and peroxide concentration) during the application. The null hypotheses tested were that the viscosity and the kind of thickener in the formulation would not interfere with the effect of the bleaching treatment and the reactions inside the gel.

METHODS AND MATERIALS

Specimen Preparation

Two hundred forty cylindrical specimens (6 mm in diameter) were obtained from the labial surface of bovine incisors with a diamond trephine mill in a circular cutting machine under water cooling (Figure 1A). Enamel and dentin surfaces were flattened to create a disk shape, and the final thickness standardized at 2 mm (1 mm of enamel and 1 mm of dentin) (Figure 1B). The enamel surface was polished by using an automatic polishing machine (Panambra, Sao Bernardo do Campo, Brazil) and SiC abrasive paper grits P1200, P2400, and P4000 (Extec Corp, Enfield, USA) for 30, 60, and 120 s, respectively.⁴⁰ The disks were ultrasonically cleaned after the use of each abrasive paper to remove residue. The dentin side of the specimens was etched for 15 s using a 35% phosphoric acid gel (Ultra-Etch, Ultradent Products, Inc, South Jordan, UT) to remove the smear layer created by the grinding procedure, opening the dentin tubules to simulate the clinical condition.^41,42 The specimens were then immersed in individual Eppendorf tubes containing 2 mm of ultrapure water for rehydration. Each tube was numbered to identify the specimen.

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Formulation of Gels

Experimental bleaching gels were prepared using five different thickeners, as shown in Table 1. Each thickener was combined with 60% hydrogen peroxide solution (Kolovec, Diadema, SP, Brazil), ultrapure water, and pure triethanolamine (TEA) to adjust the pH, creating 35% hydrogen peroxide gels (w/w) with a pH of 6.5. The amount of TEA used in each formulation (w/w) for low, medium, and high viscosity, respectively, for the different thickeners were as follows: Carbopol (2.92, 3.53, 3.93), Salcare (3.66, 4.86, 7.96), Aristoflex (0.50, 0.62, 0.87), Aerosil (1.33, 0.90, 0.36), and CMC (0.59, 4.86, 7.04). For the semisynthetic polysaccharide (SSP) thickener, a small amount of propylene glycol was used to prehydrate the powder, and to allow water hydration and gel formation.⁹ The gels were mixed with a dual asymmetric centrifugal laboratory mixer (model DAC 150.1 FVZ, SpeedMixer, FlackTek, Landrum, USA).

Table 1: Information about the Thickeners Tested

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The amount of each thickener was adjusted to obtain different levels of viscosity and measured with a Brookfield DV2T Viscometer (Brookfield Engineering Laboratories, Middleboro, MA, USA), as previously described,^{14–19,35,36,43} and associated with the Helipath stand and a T-Bar Spindle (#96),⁴⁴ inside a 10-mL Becker flask at 25°C. Three different viscosities were created, named low (50,000 cP), medium (250,000 cP), and high (1,000,000 cP).

Therefore, 15 experimental gels were created from the combination of the five thickeners (CAR, MSA, ASE, SSP, and PAC) and three viscosities (low, medium, and high). In addition, a negative control group (NC) was treated with ultrapure water adjusted to pH 6.5 with TEA.

The baseline hydrogen peroxide concentration (w/w) in each bleaching gel was checked by titration with potassium permanganate by using a potentiometric titrator (HI902C1-02, Hanna Instruments, Woonsocket, USA) with an ORP electrode (HI3618D, Hanna Instruments).³⁰ The pH was measured with a pH meter (Digimed, Sao Paulo, Brazil) and a microbulb electrode (HI1083B, Hanna Instruments). The ROS quantification in the bleaching gel was performed with aminophenyl fluorescein (A36003, Invitrogen, Paisley, UK), which has a high fluorescence response to the hydroxyl free radical (•OH). The fluorescence was measured by wavelength excitation and detection of 485 nm/528 nm, respectively, with a multimode microplate reader (Synergy HTX, Biotek, Vermont, USA). The results were obtained in relative fluorescent units (RFU).

RESULTS

One-way ANOVA showed no significant differences among the groups for the baseline values of microhardness (p=0.99), L^* (p=0.4487), and b^* (p=0.4327) color coordinates, showing that all groups had similar conditions before the treatment began.

Two-way ANOVA showed significant differences for viscosity and the kind of thickener for all analysis performed. The results of ANOVA and the Tukey tests for the factor “viscosity” are shown in Table 2. The high viscosity reduced the bleaching effect, peroxide penetration, softening of enamel, and ROS quantity in the gels in relation to the low viscosity. However, it increased the drop in pH and peroxide content in the bleaching gels.

Table 2: Means (SD) and Results of the Two-way ANOVA and Tukey Test for the Factor Viscosity ^a

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The results of ANOVA and the Tukey tests for the factor “kind of thickener” are shown in Table 3. Gels produced with MSA showed higher color change in comparison with SSP and ASE, while the others showed no significant differences among them. The peroxide penetration was higher for PAC, and smaller for SSP and CAR. The microhardness reduction was higher for CAR and smaller for PAC. The higher pH reduction was seen for ASE and CAR, and the smaller for SSP. The higher peroxide concentration reduction was for SSP and the smaller was for CAR. The higher amount of ROS was for MSA and the smaller was for ASE.

Table 3: Means (SD) and Results ANOVA and Tukey Test for the Factor “Kind of Thickener” ^a

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In relation to the factor “time point” of measurement for ROS, a statistically significant reduction (p=0.0001) was observed from the beginning (17.08±8.02) to the end of the treatment (10.48±6.48).

The Dunnett test showed that the negative control group (water) had significantly smaller color change (ΔEab = 0.52±0.12) and percentage of microhardness reduction (%KHN reduction = −0.02%±0.006) in relation to the experimental groups. Also, there was a significant increase in pH (11.83%±2.39) in the negative control, while the experimental groups showed a reduction.

DISCUSSION

In this study, the viscosity of the bleaching gels and the kind of thickener significantly influenced all parameters analyzed, thus rejecting the null hypothesis. The higher the viscosity, the smaller the bleaching effect, the lower the peroxide penetration, the smaller reduction in microhardness and the number of free radicals in the gel. However, the pH and peroxide concentration reductions were higher for the more viscous gels (Table 2). A previous study reported the effect of the viscosity level (low, medium, and high) on the bleaching effect and pulpal penetration of a 10% hydrogen peroxide bleaching gel.³⁹ Although nonsignificant differences were observed among the groups in relation to bleaching, a higher penetration was observed for the low and medium viscosity (0.48 μg/mL and 0.44 μg/mL, respectively) in relation to the high viscosity (0.35 μg/mL),³⁹ consistent with the results of our study. Another study evaluated the effect of the viscosity of desensitizing gels on the pulpal penetration of potassium nitrate, also reporting a strong inverse correlation.⁵⁰ Of interest is why the increase in viscosity influenced the pulpal penetration of the active principles, either the peroxide³⁹ or the potassium nitrate50 reported in those studies, or the hydrogen peroxide in the current study.

Before penetrating the tooth, the peroxide molecules in the gel layer applied on the enamel must leave the three-dimensional network formed by the thickener and permeate toward the gel–enamel interface. Those located at the boundary will leave the gel first, reducing the gradient of concentration in that area. The other peroxide molecules in the gel must diffuse from the more concentrated areas toward the less, as described in Fick’s first law, passing through the maze formed by the thickener’s chains until they reach the boundary, as they try to reestablish a concentration equilibrium. However, they are delivered rapidly, creating a flux of the active ingredients toward the tooth. As the peroxide concentration in the enamel is smaller than in the gel, the permeation is maintained during the whole treatment period. Therefore, the phenomena of internal diffusion of the drug, its release, and the penetration into the target tissue are all essential to the effect of the treatment.^11,16,51,52

Hydrogen peroxide release by a bleaching gel has been reported to be higher for those with lower viscosity, although those with higher viscosity sustained release over longer periods.²⁰ Another study reported that drug release was inversely proportional to the concentration of the active ingredient and gel viscosity.¹⁴ According to the authors, the differences may be related to the three-dimensional structure of the thickener network, where the liquid phase is maintained by capillary, adsorption, and molecular interaction.¹⁴ Gels with thicker and more compact structure have a smaller network mesh that hinders free diffusion of drug molecules and consequently the release rate.¹⁴ Other studies agree that the drug release of a gel is controlled by the complexity of the network formed by the thickener, which creates a long diffusion path for the active ingredient.^51,52 The solvent may be trapped in smaller thickener cells, acting as a diffusion barrier.¹⁰

The aim during the development of any formulation for topical application is to obtain the maximum flux of the active ingredient to the tissue to be treated instead of retaining the drug in the vehicle.¹⁶ Among the critical factors affecting the flux is the concentration of the drug in the vehicle.¹⁶ A higher peroxide concentration in the bleaching gel has been associated with higher pulpal penetration.^53,54 However, in the present study, although the peroxide concentration in the experimental gels was the same, permeation was reduced at higher viscosities, showing the importance of the vehicle in the flux and delivery of the active ingredient. It is important that the excipients (nontherapeutic ingredients) help obtain the target flux¹⁶ instead of being an obstacle.

Although permeation of peroxide into the tooth structure is necessary for the bleaching effect, whether a higher penetration can increase the bleaching effect is still unclear.⁴ Higher pulpal penetration has been reported to be unrelated to bleaching efficacy.⁵⁵ However, in our study, the less viscous gels showed higher bleaching efficacy and higher peroxide penetration. Additional studies on this topic should be performed to better understand this relationship. From a biological point of view, the increase in viscosity seems to be a good option to protect the pulp, while other methods such as chemical activation can improve the bleaching efficacy.^42,45,49,56 However, the present study did not simulate a positive outward pulpal pressure, which can strongly interfere with in vivo penetration.

Although studies evaluating low concentration peroxide gels for home bleaching concluded that the higher the viscosity, the slower the degradation of the peroxide remaining in the gel^57,58; our study showed the opposite. The more viscous gels had increased reduction of peroxide concentration and of the ROS, which may be related to the higher amount of the base required to achieve the pH of the formulation. This may have destabilized the peroxide molecules, resulting in oxygen release instead of free radical formation,⁵⁹ which also may be associated with the higher pH drop in those groups (Table 2). The solvent evaporation rate and the consequent increase of the viscosity during the treatment can also interfere with the effect of the treatment^18,60,61 and may help explain the differences in peroxide concentration reduction among the groups. Additional studies are necessary to evaluate this effect during bleaching procedures, mainly for products where a single application is recommended by the manufacturer, and the gel is not reapplied.

In relation to the comparison among the thickeners, significant differences were observed for all the analyses. The synthetic polymeric thickeners carbomer (CAR) (Carbopol 980), alkali swellable emulsion (ASE) (Salcare SC 81), and modified sulfonic acid (MSA) polymer (Aristoflex AVC), as well the semisynthetic polymer (CMC) are polyelectrolytes, being similar to electrolytes (salts) and polymers (high-molecular-weight molecules), also known as polysalts.⁹ They are available as particles formed by tightly coiled state molecules. For crosslinked thickeners, each particle can be considered as a network structure formed by many tightly coiled linear polymer chains interconnected by crosslinks, forming what can be considered a single, giant molecule. Without the crosslinks, the particles would be only a group of intertwined linear polymer chains but not chemically bonded.⁶² After dissociation in water and neutralization, the acid groups become electrically charged. The repulsion among the similar charges modifies the three-dimensional configuration of the polymer chain to an uncoiled state, increasing the hydrodynamic volume. The entanglement and the weak bonding among some areas of the chains creates a network that immobilizes the solvent and increases the viscosity.

Aristoflex is an ammonium acryloyl dimethyl taurate/vp copolymer,⁶³ while Salcare and Carbopol are polyacrylates, which are copolymers containing a combination of acrylic acid, methacrylic acid, or its simple esters. CMC is produced from cellulose gum modified with carboxymethyl groups. They also have quite different molecular weight (Table 1). Therefore, the configuration of the network created is completely different, which may influence the internal diffusion of peroxide and its permeation into the tooth. Salcare and Carbopol have carboxyl groups (-COOH) that are protonated, creating an acidic medium after hydration, but remaining in a coiled state.^63,64 To produce the ionization of the carboxyl groups and thickening effect, it is necessary to add base to increase the pH of the medium,⁹ resulting in ionization and negative charges. As CMC and Aristoflex are salts, the acidic groups were previously neutralized by the manufacturer with sodium (−CH₂CO₂Na) and ammonia (−CH₂SO₃NH4), respectively. Undergoing ionization just after hydration, they release Na⁺ or NH₄⁺, respectively,^63,65,66 which can have some influence on the effect. Carbopol also showed the smaller reduction in peroxide concentration, which was consistent with previous studies.^57,58

Although the thickening mechanism is similar, the Aristoflex AVC group showed increased bleaching compared to CMC or Salcare SC 81, which was also associated with more free radicals (Table 3). This can be related to the characteristics of the polymer network, since it has a higher molecular weight of around 10,000 g/mol, while the other two are smaller (262.19 and 314.37 g/mol) (Table 1). Another reason could be the release of NHNH₄⁺ by the polymer, which may have interfered with the formation of free radicals. Additionally, Aristoflex required much less base (TEA) than all the other thickeners to reach the required pH, indicating the efficacy of the original neutralization. However, it produced one the highest microhardness reduction values, just below Carbopol, probably because of its high molecular weight and the great number of acidic groups in the molecule that have affinity with the calcium in the tooth structure.

The highest pH reductions during the period of treatment were observed for Salcare and Carbopol, which also required much more TEA than all the other thickeners because OH⁻ must be released from the base to induce ionization of the carboxyl groups. TEA (C₆H₁₅NO₃) is a tertiary amine and weak base of low toxicity that is frequently used to adjust the pH of gel formulations. After ionization, the anion OH⁻ will increase the pH, but the cation release will also be part of the gel and may interfere with the ionic interactions during the many reactions taking place. Both the cations and anions have been reported to be released by the bases used as pH conditioner in dental bleaching gels and interfere with the bleaching effect.⁵⁹ Another difference is that the Aristoflex and Carbopol are highly crosslinked and that, after hydration, they remain as a spherical particle of microgel, not as stretched molecules as for Salcare and CMC.^63,67,68 The expanded particles approximate each other, creating spherical globules and producing a hierarchical structure.⁶⁹ The higher the molecular weight and the crosslinking, the higher is the thickening effect.⁶³

The Aerosil 200 thickener is basically composed of synthetic silicon dioxide particles of nanometric dimensions, also known as pyrogenic silica. It is insoluble in water or acid, and is not electrically charged.⁹ When dispersed in a liquid, the particles form a network structure through hydrogen bonding of their surface silanol groups,⁷⁰ creating chain-like aggregates and producing a loose tridimensional network that immobilizes the solvent and increases viscosity.⁹ Our results showed that the pulpal penetration was higher for Aerosil than for the other thickeners, probably because the peroxide released from this gel was also higher. An important problem with drug delivery is understanding the effect of the thickener matrix on the transport of low-molecular-weight active ingredients inside a gel. The transport conditions inside the network of uncharged thickener molecules, as in the case of silica particles, are expected to be similar to those inside the water.^12,71 However, drug and thickener chain interactions can occur if the gel is charged, giving rise to a non-Fickian transport.¹² In addition, the current study showed the smaller demineralizing effect for Aerosil, probably because of the absence of ionic interaction with the calcium in the tooth enamel, as seen for the other thickeners. The amount of TEA necessary to reach the required pH was smaller with higher gel viscosity, which was the opposite of the other thickeners. The pH reduction was smaller for this thickener than for Salcare and Carbopol, although the amount of ROS was higher.

In relation to the demineralization of enamel, the current study showed that the increase of the viscosity reduced its effects on the enamel, which may be related to the internal diffusion of the calcium captured by the thickener. In addition, all anionic thickeners tested showed a higher microhardness reduction than the nonionic one. The highest reduction was observed for Carbopol, followed by Aristoflex, which are both highly crosslinked and have higher molecular weights. Previous studies reported that Carbopol gels, even without peroxide, were able to reduce enamel microhardness^72–75 and increase surface roughness.²⁴ Calcium ions released by the enamel have been reported to permeate the unsaturated gels.^75,76 The demineralization of enamel and inhibition of hydroxyapatite crystal growth after exposure to Carbopol and CMC gels have been previously reported, even at neutral pH values, and were related to the negative charges of both the polyanions.⁷⁷ The polymers created complexes with the positively charged calcium ions and made the medium undersaturated.⁷⁷ The CMC also can strongly bond to multivalent ions such as Ca²⁺.^67,78

The negative control group showed significantly smaller values of color change and percentage of microhardness reduction in relation to the experimental groups, indicating that the method was sufficiently sensitive to detect even small color changes. The negative control also showed a significant increase in pH after application over the enamel (11.83±2.39%), while the experimental groups presented a reduction.

A bleaching effect from the thickener by itself was not expected, since its molecules cannot penetrate the tooth structure. Nevertheless, the absence of control groups evaluating the effect of peroxide free gels is a limitation of this study. Additional analysis is necessary to determine whether the best bleaching effect for some thickeners in the hydrogen peroxide gel formulation in this study may not be attributed to the thickener alone.

Based on our results, the effect of the viscosity and kind of thickener on the therapeutic and side effects of bleaching gels over the tooth structure was identified. The study of the vehicles of bleaching products is of high clinical significance, since most researchers are unaware that some aspects of the formulation may influence the results of the treatment more than the concentration of the active ingredient. Additional research is necessary to fully understand all aspects influencing the performance of the gels to create better and safer bleaching products.

CONCLUSIONS

The increase in bleaching gel viscosity reduced the color change, number of free radicals, peroxide permeation (PP), and demineralization of enamel, but increased peroxide decomposition and pH alteration during the treatment. The kind of thickener significantly interfered with the effect of the treatment on the tooth and chemical alterations in the gel during its application.