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 the 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 were untreated (control). The rats were killed after 2 or 30 days, and the maxillae were examined by light microscopy. Inflammation was evaluated through histomorphometric analysis with inflammatory cell count in the coronal and radicular thirds of the pulp. Fibroblasts were also counted. Scores were attributed to odontoblastic layer and vascular changes. Tertiary dentin area and pulp chamber central area were measured histomorphometrically. Data were compared by analysis of variance and Kruskal-Wallis test (p<0.05). Results After 2 days, the amount of inflammatory cells increased in the coronal pulp occlusal third up to the 15-min application groups of each bleaching gel. In the groups exposed to each concentration for 30 and 45 min, the number of inflammatory cells decreased along with the appearance of necrotic areas. After 30 days, reduction on the pulp chamber central area and enlargement of the tertiary dentin area were observed, without the detection of inflammation areas. Conclusion The rat model of extracoronal bleaching showed to be adequate for studies of bleaching protocols, as it was possible to observe alterations in the pulp tissues and tooth structure caused by different concentrations and application periods of bleaching agents.

INTRODUCTION 2 O 2 gel is considered to be a conservative and affordable aesthetic treatment 18 . Its effectiveness is attributable 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 . ROS-induced oxidative stress can cause mutation, enzyme inactivation, protein degradation, and fragmentation in pulp cells, which might manifest as pulpitis and tooth sensitivity 3 . The severity bleaching protocol used, and this procedure has been increasingly questioned 2,6,8 .
An increase in vascular permeability depending on the duration of the bleaching procedures 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 increased deposition of reparative dentin, thinning of the odontoblastic layer, inflammatory infiltration, and internal root resorption. Some of the changes, such as inflammation and bleeding, reversed after 60 days 25 incisors by using the abovementioned protocol caused partial necrosis in the coronal pulp and a 8 . Moreover, 45-min bleaching with 35% H 2 O 2 gel resulted in necrosis near to 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 anatomical characteristics of the teeth and 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 greater penetration of H 2 O 2 , and consequently more damage to pulp tissues 8 . Therefore, it is essential to characterize appropriate protocol to be applied in this model and to allow the conduction 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, in addition to the evaluation of potential therapeutic agents that may be used to minimize the damage caused by H 2 O 2 to the pulp tissue, in different application protocols 6,9 . The choice of rats was due to the ease of standardization and control of these animals, and the possibility of performing other tests 7,9 . Thus, it is possible to study different variables in order to, in a second stage, with results already standardized and evaluated in animals, propose the validation of these results in humans, with smaller groups, following ethical principles 9 . Researches involving both dog and human teeth to study bleaching protocols are the required sample as well as ethical principles. Furthermore, Cintra, et al. 6 (2013), when analyzing on 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. It was hypothesized that: (I) the H 2 O 2 in bleaching gel is capable of penetrating pulp tissue and causing greater damages with increasing time of application and H 2 O 2 concentration; (II) pulp tissue is capable of recuperating from the damages caused by H 2 O 2 after long periods of time.

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

Histology
Animals were killed with an overdose of the anesthetic solution 2 or 30 days after the bleaching sessions. Their bilateral maxillae were separated, in a 10% ethylenediaminetetraacetic acid (EDTA) solution for three months, and then dehydrated in hematoxylin and eosin (H&E).
The serial histological sections of each specimen were selected from the point where the mesial root 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 . Inflammation was evaluated through count in the coronal and radicular thirds of the pulp. Fibroblasts were also counted. The cell count 2 the pulp tissue of each specimen, examined under 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 for vascular changes were also assigned as follows: 1-normality; 2-increase in the number of blood vessels; or 3-necrosis.
The mean central area of the pulp chamber was measured by image processing software (Leica QWin V3, Leica Microsystems, Wetzlar, Hesse, was possible to calculate the percentage reduction 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 counts of to two-way analysis of variance and Tukey's test level of 5% (p<0.05). The scores obtained in the analysis of odontoblastic layer and vascular changes were submitted to Kruskal-Wallis and Dunn's tests (p<0.05). The values obtained in the mean central area of the pulp chamber were submitted to Kolmogorov-Smirnov test of normality and one-way analysis of variance (p<0.05).

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

20%-5 min group
The dental pulp appeared similar to that of the control group. The odontoblastic layer was intact and the blood vessels showed normal characteristics. The cementum, periodontal ligament, alveolar bone, and other supporting structures also seemed 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). there was an increased amount of blood vessels. The odontoblastic layer was partially disorganized in the occlusal third ( Figure 3C).

20%-30 min group
found in the middle third of the coronal pulp in this group. There was a large reduction in the amount 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 seemed normal in all cases.

20%-45 min group
This group showed an 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
There was an increase in the number of of the coronal pulp in this group. The amount odontoblastic layer was absent in the occlusal third, and partly disorganized in the middle third of the coronal pulp. There was an increase in the number of blood vessels throughout the coronal

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).

35%-30 min group
reduced in the occlusal third, where necrotic areas were present. There was an increase in the cervical thirds of the coronary pulp. The amount of coronary pulp. The odontoblastic layer was absent in the occlusal and middle thirds of the crown. There was an increase in the number of blood vessels in the middle and cervical thirds of the coronal pulp.
The occlusal third was characterized as necrotic. A the cervical third of the radicular pulp ( Figure 3I).

35%-45 min group
There was necrosis in the occlusal third of this 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. The odontoblastic layer was absent in the occlusal and middle thirds of the crown, and partially disorganized in the cervical third. The number of blood vessels increased in the cervical third of the coronary and radicular pulp. The remaining thirds seemed normal ( Figure 3J).

Reparative dentin area
Thirty days after the bleaching sessions, all the specimens showed normal dental pulp. However, the central area of the pulp chamber reduced, and the tertiary dentin area increased (Figure 4). groups and the control group in the occlusal third (p<0.05), except for the 35%-45 min group, which in the middle third of the coronal pulp were noted between the control group and the 20%-10 to 45 min and 35%-5 to 45 min groups (p<0.05). In the cervical third, the difference from the control group was also present in the 20%-15 to 45 min and 35%-10 to 45 min groups (p<0.05). In the cervical  were noted between the 20%-45 min and 35%-30 min groups from the other groups, and between differences were not observed in the other radicular thirds (p>0.05).

Intergroup comparisons
the experimental groups. The 20%-10 to 45 min compared with the control group (p<0.05). This decrease was also present 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 were not observed in the radicular thirds of any group (p>0.05). Table 3 shows the scores assigned to the odontoblast layer of each experimental group. In the occlusal third, the 20%-45 min and 35%-30 control and 20%-5 min groups (p<0.05). In the middle third of the coronal pulp, the 35%-45 min and 20%-5 to 30 min groups (p<0.05). There were radicular thirds (p>0.05). from the control and 20%-5 min groups in the occlusal third (p<0.05). In the middle third of the coronal pulp, the 35%-45 min group also differed cervical third and radicular thirds (p>0.05). At 30 days, the specimens showed a gradual differences were observed between the 35%-45 min group and the other groups, except for the 35%-30 min group (p<0.05). The 20%-5 min, 20%-10 min, 20%-15 min, and 35%-5 min groups (p>0.05) ( Table 5).

DISCUSSION
Tooth bleaching is an aesthetic alternative for discolored teeth, but it has potential adverse effects that are not yet completely understood 28 . A single results, but longer application time 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 that ROS generated by the H 2 O 2 of bleaching gels are capable of causing histochemical and morphological changes in enamel and dentin 4,5 .
In vivo as mild to severe. These include studies performed in dog teeth 25,26 , human mandibular incisors 8,19 , rat  incisors 12,13 , and rat molars 6. Cell culture studies also demonstrated cellular damage as apoptosis 14 , 3 , cytotoxicity 30 , damage to the DNA 23 , cell viability reduction 27 , or ageing of the dental pulp 1,28 . The cytotoxicity of bleaching gel to pulp tissue was also observed in this study.
Studies . Therefore, in vivo experiments are the ones that best represent the reality of bleaching effects.
Application of 38% H 2 O 2 gel on human premolars does not cause pathological changes in the dental pulp 17 . However, application of the same concentration on human mandibular incisors causes necrosis in the coronal pulp, similar to what was observed in rat molars 6 , possibly because of the thinner enamel and dentin 8 that morphological characteristics of different tooth upper anterior human teeth should be used to exactly determine pulp changes. Even under these such as age, 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 easy reproduction and standardization for the study of new bleaching agents, posology, concentrations, and application time.
In dog teeth, dental bleaching using 35% H 2 O 2 showed greater changes immediately beneath the region where the gel was applied 25 , similar 2 O 2 gel applied for 30 or 45 min and 20% H 2 O 2 gel for 45 min. Severe pulp damage may occur when bleaching agents are applied on the buccal surface of teeth with thin enamel and dentin 8,25,26 . Dog Furthermore, studies in dogs have been avoided nowadays for ethical reasons.
The use of rats as the experimental model presents advantages such as ease of handling, reproduction, control, predictability 22 , and standardization 6 . Moreover, this model further presents better acceptance regarding ethical and economic concerns 9 .
Despite the difference in enamel and dentin thickness between humans and rat teeth (2.5 mm vs. 100 μm, respectively), they both show the same proportion of these structures 6,9 . In addition, rat 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 pulp tissues, where the essential biological reactions and the wound healing of rat molar teeth are comparable to that of other mammals 9 . Conversely, rat incisors are typical of rodents, of 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 to 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 to 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 in this concentration and application time 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, as well as 20% and 35% H 2 O 2 gel for 45 min, was related to changes in vascular permeability 12 . Therefore, our study was conducted with several application times and two concentrations, one of which more commonly employed in clinical dentistry (35% H 2 O 2 ) 16,29 , and a lower one (20% H 2 O 2 ). Our results allow choosing a concentration and time of application for comparative analysis in the initial the subsequent reparative process (at 30 days).
In our evaluation at 30 days after bleaching, we observed that all the groups showed signs of repair. Tertiary dentin was formed to protect the dental pulp, reducing the pulp chamber central area, and differences from those of high concentration/ application time. 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 10,30 . H 2 O 2 can penetrate the cell membrane, increase alkaline phosphatase activity, and 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 the dentin production 28 . The model in rats can also be used in long-term analysis to determine clinical protocols of application that The characterization of this experimental model does not replace human trials, but allows the knowledge of new bleaching agents mechanism of action; the comparison between protocols of bleaching; and the study of desensitizing and remineralizing agents used before and after bleaching to minimize effects on pulp tissues.

CONCLUSION
In conclusion, the rat model of extracoronal bleaching showed to be adequate for studies of bleaching protocols, as it was possible to observe alterations in pulp tissues and tooth structure caused by different concentrations and application times of bleaching agents. In-office bleaching with H 2 O 2 accelerated aging of the dental pulp by inducing deposition of tertiary dentin, and the degree of damage increased with increasing concentration and application time of the bleaching agent.