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Evaluation of an experimental rat model for comparative studies of bleaching agents

ABSTRACT Dental materials, in general, are tested in different animal models prior to their clinical use in humans, except for bleaching agents. Objectives To evaluate an experimental rat model for comparative studies of bleaching agents by investigating the influence of different concentrations and application times of H2O2 gel in the pulp tissue during in-office bleaching of rats’ vital teeth. Material and methods The right and left maxillary molars of 50 Wistar rats were bleached with 20% and 35% H2O2 gels, respectively, for 5, 10, 15, 30, or 45 min (n=10 rats/group). Ten animals (control) were untreated. The rats were killed after 2 or 30 days, and the maxillae were examined by light microscopy. Inflammation was evaluated by histomorphometric analysis with inflammatory cell counting in the coronal and radicular thirds of the pulp. The counting of fibroblasts was also performed. Scores were attributed to the odontoblastic layer and to vascular changes. The tertiary dentin area and the pulp chamber central area were histomorphometrically measured. Data were compared by the analysis of variance and the Kruskal-Wallis test (p<0.05). Results After 2 days, the amount of inflammatory cells increased in the occlusal third of the coronal pulp until the time of 15 min for both concentrations of bleaching gels. In 30 and 45 min groups of each concentration, the number of inflammatory cells decreased along with the appearance of necrotic areas. After 30 days, a reduction in the pulp chamber central area and an enlargement of tertiary dentin area were observed without the detection of inflammation areas. Conclusion The rat model of extra coronal bleaching showed to be adequate for bleaching protocols studies, as it was possible to observe alterations in the pulp tissues and in the tooth structure caused by different concentrations and periods of application of bleaching agents.

INTRODUCTION 2 O 2 gel is considered a conservative and affordable aesthetic treatment 18 . Its effectiveness is due to the low molecular mass of the main active compound: H 2 O 2 , which easily diffuses through enamel and dentin and releases reactive oxygen species (ROS), thus oxidizing organic structures 2 .
Importantly, H 2 O 2 and its by-products have varying biological effects on human oral tissues 30 . The ROS-induced oxidative stress can cause mutation, enzyme inactivation, protein degradation, and fragmentation of pulp cells that might manifest as pulpitis and tooth sensitivity 3 . The severity bleaching protocols, and this procedure has been increasingly questioned 2, 6,8 .
An increase in vascular permeability that is dependent of the bleaching procedures duration has been observed in rats' incisors 12 . A 30 min bleaching session using 35% H 2 O 2 gel, with or without heat, caused a severe inflammatory reaction in the dental pulp of dogs, including an increased deposition of reparative dentin, the reversed after 60 days 25 bleaching of mandibular incisors using the abovementioned protocol caused partial necrosis in the the radicular pulp 8 . Moreover, a 45 min bleaching with 35% H 2 O 2 gel resulted in necrosis near the pulp horns in rats 6 . On the other hand, the application of 38% H 2 O 2 gel on human premolars did not cause pathological changes in the dental pulp 17 . Therefore, it is evident that the anatomical characteristics of the teeth and of the in vivo model analyzed, as well as the bleaching protocols employed, determined different results.
Thus, the lesser thickness of enamel and dentin in teeth of rats might allow a greater penetration of H 2 O 2 , consequently causing more damages to pulp tissues 8 . Therefore, it is essential to characterize the protocol to be applied in this model and to allow the performance of further studies on H 2 O 2 damage to pulp tissues. This model will enable the evaluation of new dosages, formulations, and concentrations of bleaching agents that arise in the market, and also of the potential therapeutic agents that may be used to minimize the damage caused by H 2 O 2 in different application protocols 6,9 . The choice by the rats in this animal model was due to the easiness in standardizing and controlling these animals, and the possibility of performing other tests 7,9 . Thus, it is possible for us to study different variables and to be able to propose, in a second stage and with the results already standardized and evaluated for animals, the validation of the same results in smaller groups of humans by following the ethical principles 9 . Research involving both dogs' and human's teeth to study bleaching protocols are impractical because and also because of ethical principles. Furthermore, Cintra, et al. 6 of the number of bleaching sessions in pulp tissues, indicated the possibility of using teeth of rats for the study of bleaching protocols. Using the rat model for studying bleaching agents is relatively simple and easy to reproduce. Therefore, the purpose of this study was to characterize an experimental animal model for comparative studies of bleaching agents by and application times of H 2 O 2 bleaching of rats' vital teeth. We hypothesized that: (I) the H 2 O 2 bleaching gel is capable of penetrating the pulp tissue and causing greater damages when the time of application and the concentration of H 2 O 2 are increased; (II) the pulp tissue is capable of recovering from the damages caused by H 2 O 2 after long periods of time.

Animals
Sixty male Wistar rats (180-200 g) were used in this study. The animals were housed in a temperature-controlled environment (22°C ± 1°C) in a standard light-dark schedule with unrestricted access to food and water. The experimental protocol was approved by the Ethics Committee (CEUA 2013-01253) and conducted according to Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, Maryland, USA).

Histology
Animals were killed by the anesthetic solution overdose 2 or 30 days after the bleaching sessions. Their bilateral maxillae were separated, dissected, solution for three months and then dehydrated in hematoxylin and eosin (H&E). The serial histological sections of each specimen were selected from the seen in all its longitudinal extension.
The coronal pulp was divided into occlusal, middle, and cervical thirds and the radicular pulp was divided into cervical, middle, and apical thirds 6 .
coronal and radicular thirds of the pulp. The 2 third of the pulp tissue of each specimen, examined under light microscopy (×1000 magnification; DM4000 B, Leica Microsystems, Wetzlar, Germany).
Scores were attributed to the odontoblastic layer in each third of the pulp tissue as follows: 1-intact odontoblastic layer; 2-disorganized odontoblastic layer; or 3-disruption of the odontoblastic layer. Scores to vascular changes were also assigned as follows: 1-normality; 2-increase in the number of blood vessels; or 3-necrosis.
The mean of central area of the pulp chamber was measured by image processing software (Leica QWin V3, Leica Microsystems, Wetzlar, Germany) ( Figure 1). By taking into account the obtained values it was possible to calculate the reduction percentage in the central area of the pulp chamber in the treated groups considering the central area of the control group.
After the application of the Kolmogorov-Smirnov test of normality, the data obtained in the counting to the Two-way analysis of variance and Tukey tests 5% (p<0.05). The scores obtained in the analysis of the odontoblastic layer and of vascular changes were submitted to the Kruskal-Wallis and Dunn tests (p<0.05). The values obtained in the mean of central area of the pulp chamber were submitted to Kolmogorov-Smirnov test of normality and One-way analysis of variance test (p<0.05).

Control group
The dental pulp of control animals exhibited wellodontoblastic layer and even distribution of cells, blood vessels, and extracellular matrix structures ( Figure 2).

20%-5 min group
The dental pulp appeared to be similar to that in the control group. The odontoblastic layer was intact and blood vessels showed normal characteristics. The cementum, periodontal ligament, alveolar bone, and other supporting structures also appeared to be normal ( Figure 3A).

20%-10 min group
This group did not exhibit a considerable amount thirds of the coronal pulp. The odontoblastic layer was partially disorganized in the occlusal third, and there was an increase in the number of blood vessels in the occlusal and middle thirds of the coronal pulp ( Figure 3B).

20%-15 min group
There was an increase in the amount of of the coronal pulp, a reduction in the amount blood vessels. The odontoblastic layer was partially disorganized in the occlusal third ( Figure 3C).

20%-30 min group
cells was found in the middle third of the coronal pulp. There was a large reduction in the amount of disruption of the odontoblastic layer. The amount of blood vessels increased in the occlusal and middle thirds of the coronal pulp ( Figure 3D). The radicular pulp appeared to be normal in all cases.

20%-45 min group
This group showed increased number of of the coronal pulp. The occlusal third showed necrotic areas. A reduction in the number of crown. The odontoblastic layer was absent in the occlusal third and partly disorganized in the middle of the radicular pulp ( Figure 3E).

35%-5 min group
reduced in the occlusal third of the coronal pulp, where the odontoblastic layer was partially disorganized. An increase in the number of blood vessels was observed in all areas of the coronal pulp ( Figure 3F).

35%-10 min group
In this group, there was an increase in the middle thirds of the coronal pulp. The amount odontoblastic layer was absent in the occlusal third and was partly disorganized in the middle third of the coronal pulp. There was an increase in the number of blood vessels throughout the coronal pulp ( Figure 3G).

35%-15 min group
There was an increase in the number of reduced. The odontoblastic layer was absent in the occlusal third, and partly disorganized in the middle third of the coronary pulp. There was an increase in the number of blood vessels throughout the coronal pulp ( Figure 3H). the cervical third of the radicular pulp ( Figure 3I).

35%-45 min group
There was necrosis in the occlusal third of this group with absence of inflammatory cells and fibroblasts. The number of inflammatory cells increased in the cervical and middle thirds of the coronal pulp, and in the cervical third of the in these thirds. There was no odontoblastic layer in the occlusal and middle thirds of the crown, and this layer was partially disorganized in the cervical third. The number of blood vessels increased in the cervical third of coronary and radicular pulps. The remaining thirds seemed to be normal ( Figure 3J).

Reparative Dentin Area
Thirty days after the bleaching sessions, all specimens showed normal dental pulp. However, the central area of the pulp chamber reduced, and the tertiary dentin area increased (Figure 4).

Intergroup Comparisons
of all experimental groups. The most predominant inflammatory cells found were mononuclear cells, such as lymphocytes, macrophages, and gradually with the increasing concentrations and the application time of the bleaching gel, up to the 15 min application groups of each bleaching gel, in the occlusal third of the coronal pulp. The groups that received the application of 30 and 45 min of each bleaching agent showed areas of necrosis in the occlusal third with a decrease in the amount of inflammatory cells. Significant differences were observed between the bleached groups and   other radicular thirds (p>0.05).
experimental groups. The 20%-10 to 45 min, and to the control group (p<0.05). This decrease remained in the middle third of the coronal pulp in the 20%-15 to 45 min, and 35%-15 to 45 min groups (p<0.05). In the cervical third, only the groups that received the bleaching gels for 45 min in the radicular thirds of any group (p>0.05). Table 3 shows the scores assigned to the odontoblast layer of each experimental group. the 20%-45 min, 35%-30 and 45 min in the occlusal third compared to the control and the 20%-5 min groups (p<0.05). In the middle third of the coronal pulp, the difference remained between the 35%-45 min group with the control and 20%-5 to differences in the cervical third and in radicular thirds (p>0.05). Table 4 shows the scores assigned to the vascular changes of each experimental group.   20%-45 min and 35%-45 min in the occlusal third compared to the control and 20%-5 min groups (p<0.05). In the middle third of the coronal pulp, the difference remained between the 35%-45 min group with the control and 20%-5 groups (p<0.05).

DISCUSSION
Tooth bleaching is an aesthetic alternative for discolored teeth, but it has potential adverse effects that are not completely understood yet 28 . A single results, but longer application times and multiple sessions may be required for optimal outcomes, increasing the risk of tooth sensitivity 20 and pulp damage 6 .
A large number of in vitro studies have shown the ROS generated by H 2 O 2 of bleaching gels is capable of causing histochemical and morphological changes in enamel and dentin 4,5 .
In vivo as mild to severe. These include studies performed in dogs 25,26 , human mandibular incisors 8,19 , and in   . Therefore, in vivo experiments are the ones that best represent the reality of bleaching effects.
The application of 38% H 2 O 2 gel in human premolars does not cause pathological changes in the dental pulp 17 . However, the application of the same concentration in human mandibular incisors causes necrosis in the coronal pulp in a similar way to that observed in rats' molars 6 , possibly because of the thinner enamel and dentin 8 . These pulp damage. Ideally, upper anterior human teeth should be used to determine the pulp changes accurately. Even under these conditions, other presence of restorations, previous trauma, among others.
Even though variations of the pulp response have been shown in human teeth, our study aimed to characterize an experimental animal model of ease reproduction and standardization to the study of new bleaching agents, posology, concentrations, and application time.
In dogs' teeth, dental bleaching using 35% H 2 O 2 showed greater changes immediately beneath the region where the gel was applied 27 , similarly to that observed in groups of 35% H 2 O 2 gel applied for 30 or 45 min, and 20% H 2 O 2 gel for 45 min in this study. Severe pulp damage may occur when bleaching agents are applied in the buccal surface of teeth with thin enamel and dentin 8,25,26 . Dog's teeth Furthermore, nowadays, studies in dogs have been avoided by ethical reasons.
The use of rats in the experimental model presents advantages such as straightforwardness in the handling and in the reproduction, and mainly, the easiness for controlling, predicting 22 , and standardizing 6 . Moreover, this model is better accepted regarding ethical and economic concerns 9 .
Despite the difference in enamel and dentin thickness between humans' and rats' teeth (2.5 mm vs the same proportion of these structures 6,9 . In addition, rats' molars have anatomical, histological, biological, and physiological features similar to human molars 9,24 . Also, rat molars exhibit the same structural characteristics of the pulp chamber and of the pulp tissues, where the essential biological reactions and the wound healing of rats' molar teeth are comparable to that of other mammals 9 .
Conversely, rats' incisors are typical of rodents, having permanent growth, with a wide-open apex, and cannot be compared to human teeth 9 .
In the present study, 35% H 2 O 2 gel applied for 30 response in the dental pulp, especially in the upper with high H 2 O 2 concentrations for 30-45 min in a single session is frequently associated with a high incidence of tooth sensitivity 8 . Considering the similarity of the results found in this study with the results of Costa, et al. 8 (2010), we suggested that rat molars can be targeted and improved as an experimental model to predict the results of procedures performed in human mandibular incisors with the same concentration and duration of application 6 . The amount of H 2 O 2 detected in the pulp chamber is related to the concentration and application time of the gel 2 . The use of 35% H 2 O 2 gel applied for 30 min, and 20% and 35% H 2 O 2 gel for 45 min was related to changes in the vascular permeability 12 . Therefore, our study was carried out with several application times and two concentrations, one of which more commonly employed at the clinic (H 2 O 2 35%) 16,29 , and another with lower concentration (H 2 O 2 20%). Our results allow us to choose the concentration and the time of application to analyze (after two days) as well as in the subsequent reparative process (after 30 days).
In our evaluation by 30 days after bleaching, we observed that all groups showed signs of repair. Tertiary dentin was formed to protect the dental pulp, reducing pulp chamber central concentration/application time groups showed concentrations/application time groups. Studies of the effects of high concentrations of bleaching gels on pulp cell cultures have shown that products released by 35% H 2 O 2 gel can diffuse through enamel and dentin and cause significant cell damage 10,30 .
Cell membrane can be penetrated by H 2 O 2 , increase alkaline phosphatase activity, induce apoptosis in the periodontal ligament and dental pulp 15 as well as stimulate mineralization 21 .
Increased alkaline phosphatase activity and extracellular matrix mineralization reveal dentin production 28 . The model in rats can also be used in long term analysis to determine clinical protocols of application that produce less pulp damages along time.
The characterization of this experimental model does not replace human trials, but allows us to know the action mechanisms of new bleaching agents; to compare bleaching protocols; and to study desensitizing and remineralizing agents used before and after bleaching to minimize its effects on pulp tissues.
Therefore, the rat model of extra coronal bleaching showed to be adequate to study bleaching protocols, as it was possible to observe alterations in the pulp tissues and in tooth structure caused by different concentrations and periods of application 2 O 2 the aging of the dental pulp by inducing the deposition of the tertiary dentin, and the degree of damage grew with the increasing concentrations and application times of the bleaching agent.