Experimental gel containing bioactive glass-ceramic to minimize the pulp damage caused by dental bleaching in rats

Abstract Objectives This study evaluated if the use of a bioactive glass-ceramic-based gel, named Biosilicate (BS), before, after or mixed with bleaching gel, could influence the inflammation of the dental pulp tissue of rats’ molars undergoing dental bleaching with hydrogen peroxide (H2O2). Methodology The upper molars of Wistar rats (Rattus norvegicus, albinus) were divided into Ble: bleached (35% H2O2, 30-min); Ble-BS: bleached and followed by BS-based gel application (20 min); BS-Ble: BS-based gel application and then bleaching; BS/7d-Ble: BS-based gel applications for 7 days and then bleaching; Ble+BS: blend of H2O2 with BS-based gel (1:1, 30-min); and control: placebo gel. After 2 and 30 days (n=10), the rats were euthanized for histological evaluation. The Kruskal-Wallis and Dunn statistical tests were performed (P<0.05). Results At 2 days, the Ble and Ble-BS groups had significant alterations in the pulp tissue, with an area of necrosis. The groups with the application of BS-based gel before H2O2 had moderate inflammation and partial disorganization in the occlusal third of the coronary pulp and were significantly different from the Ble in the middle and cervical thirds (P<0.05). The most favorable results were observed in the Ble+BS, which was similar to the control in all thirds of the coronary pulp (P>0.05). At 30 days, the pulp tissue was organized and the bleached groups presented tertiary dentin deposition. The Ble group had the highest deposition of tertiary dentin, followed by the Ble-BS, and both were different from control (P<0.05). Conclusion A single BS-based gel application beforehand or BS-based gel blended with a bleaching gel minimize the pulp damage induced by dental bleaching.


Introduction
Studies on the bleaching gel containing hydrogen peroxide (H 2 O 2 ) have indicated some adverse effects caused by this product. [1][2][3][4] For example, there are reports on the morphological changes in the structure of dental enamel after contact with H 2 O 2 , such as increase in roughness and decrease in hardness. 2,3,5,6 Histochemical changes in dental tissues are observed as a loss of calcium and phosphorus ions in enamel and dentin. 2,5,6 Decrease in the flexural strength of the teeth was also observed. 3 These damages occur due to H 2 O 2 releasing reactive oxygen species (ROS), which react with organic and inorganic molecules. 2,3,5 ROS can reach the dental pulp due to their low molecular weight 7 and cytopathological effects are mutation, enzymatic inactivation, protein degradation, and cellular apoptosis or tissue necrosis. 1,4,[8][9][10] As a consequence, most patients who undergo dental bleaching have postoperative sensitivity characterized by tingling or needles. 11 This sensitivity can also affect healthy teeth, unlike common dental sensitivity which occurs only in the presence of exposed dentin or cracks in the enamel. 11 Many therapeutic agents have been tested in an attempt to minimize pulp damage and teeth sensitivity, such as anti-inflammatory substances, 12,13 antioxidants, 14 desensitizing 15 or remineralizing agents. 2,5 The use of remineralizing agents, such as bioactive glasses and glass-ceramics, is common for these materials present remarkable positive interaction with hard (bone and teeth) and soft tissues. [16][17][18][19] The ability of these materials to form a hydroxycarbonate apatite (HCA) layer on their surfaces in vivo, promoting an interface and strong bonds between bone and teeth, can be used to prevent enamel demineralization after dental bleaching. 20 Biosilicate ® (BS), a bioactive glass-ceramic, has been successfully tested in several medical and dental applications. 18,19 This material can induce the deposition of HCA in dentin tubules 21 , reducing tooth hypersensitivity. 22 Other studies demonstrated that BS was able to increase microhardness of the enamel after dental bleaching, 23 in addition to minimizing the demineralization of dental structures caused by bleaching gel. 5 Furthermore, it showed the ability to reduce the dental sensitivity caused after this aesthetic procedure. 11 However, the effects on the pulp tissue generated by the use of BS in dental bleaching have not yet been investigated.
The model of rat molars for pulp tissue analysis after dental bleaching was proposed by Cintra, et al. 24 (2016). This model is easy to standardize specimens, given the difficulty of obtaining a sufficient number of samples for studies in humans. 1,4 It is thus possible to study different variables in rats, validating the results in humans later, in accordance with ethical principles.  Afterwards, the product was removed with a cotton ball and the molars were rinsed thoroughly with water.
The experimental BS-based gel (glass-ceramic) with ~500 nm granulometry was applied using a microapplicator (Microbrush ® , Kage Sorensen, Cotia, SP, Brazil) over the occlusal surface of upper molars. The material was rubbed for 30 sec and then remained active for 20 min, similarly to other studies. 2,22 For the BS-based gel preparation, the BS powder was mixed with distilled water immediately before application, using a ratio of powder weight (mg) to water volume (

Analysis at 2 days after the bleaching session
There was no loss of animals in this study. Rats were healthy on days when the hemimaxillae were removed for further processing and histological analysis. The histological aspect of the groups at 2 days can be observed in Figure 1, and scores for inflammation are presented in Table 1. The statistical analysis of scores attributed to the inflammatory infiltrate (see Table 1

Analysis at 30 days after the bleaching session
The histological aspect of the groups at 30 days in Figure 2 showed absence of inflammatory cells in their specimens, with organized pulp tissue and an intact odontoblastic layer throughout the pulp chamber.
However, the groups that received the bleaching gel presented the deposition of tertiary dentin in a large part of the pulp chamber.
The central area value of each group at 30 days is presented in Table 2. The deposition of tertiary dentin was higher in the Ble group compared to the control and Ble+BS groups (P<0.05). The Ble-BS group was Experimental gel containing bioactive glass-ceramic to minimize the pulp damage caused by dental bleaching in rats J Appl Oral Sci. 2020;28:e20190384 5/10    Therefore, the null hypothesis that BS-based gel does not minimize the damage that dental bleaching causes to the pulp tissue was rejected.
In this study, a high-concentration bleaching gel was used and it caused severe damage to the pulp tissue, such as necrosis. These results were observed in previous studies 4,7,28,29 and they were similar to those found in human mandibular incisors. 1 Severe toxic effects caused by bleaching gels have also been observed in pulp cell cultures, decreasing cellular metabolism and causing morphological changes. 9,10 The anti-inflammatory substances and low level laser therapy used have been tested and can promote reduction of pulp damage after dental bleaching, but the results are not conclusive. 9,14 The reduction of the bleaching gel concentration used in in-office bleaching is also an option to minimize the damage. 7,8,31 However, this product requires repeated sessions containing lower H 2 O 2 concentration, 6 a procedure which may also increase the damage to pulp tissue. 4 However, BS was effective in reducing the post-operative sensitivity reported by patients with bleached teeth, besides minimizing the effects of the bleaching gel on mineralized dental tissues. 11,23 Previous studies showed that formulations of 16% carbamide peroxide bleaching gel containing BS were relevant to improve the enamel microhardness after bleaching. 23 The BS powder, with granulometry inferior to 10μm, could induce the deposition of HCA in exposed dentinal tubules 21 and its use immediately after bleaching reduced the demineralization that bleaching products cause, 5 thus preventing the exposure of dentinal tubules. BS powder was also indicated for the treatment of dentin hypersensitivity, with highly positive results, 22 and its applications after dental bleaching significantly reduced post-operative sensitivity of patients with bleached teeth. 11,23 BS and other similar glass-ceramic materials also presented high positive-interaction with tissues, stimulating tissue regeneration. 17,18,19 Thus, this study investigated if the application of BS-based gel after bleaching gel could positively affect the pulp cells, stimulating tissue recovery. Our results indicate that BS was not able to induce the healing of pulp tissue after damage from the dental bleaching. It may be that BS did not have capacity to penetrate the pulp tissue to promote sufficient cellular stimuli, even with a sample produced via sol-gel that enables the creation of powder with finer particles and higher reactivity.
Thus, this result corroborates the idea that the reduction of sensitivity in bleached teeth after the use of BS originated from the sealing of the dental surface, which prevents or minimizes the external stimuli that could stimulate the inflamed pulp, increasing the sensation of pain. 22 The sol-gel system did not allow precise control of the particle sizes. It is not yet known if there is significant influence of the particle size and/or the synthesis route applied for BS in this particular application. Tirapelli, et al. 21 (2010) showed that the size of BS particles influences the penetration capacity in dentinal tubes and that these particles possibly decrease over time in contact with water. Our study did not identify whether the particles in BS-based gel were sufficient to penetrate the dentinal tubes of molar rats and reach the pulp. Moreover, this study applied BS-based gel in teeth immediately after its preparation, minimizing the reduction of particle size by contact with liquid.
The use of BS-based gel before bleaching had positive results in the inflammatory process. The BS is constituted by the Na 2 CaSi 2 O 6 crystalline phase, 18,19 which allows a higher local concentration of Na + and Ca 2+ released to the dental surface, maintaining a slightly alkaline medium. We observed that BS-based gel used in this study actually had alkaline pH. This may have contributed positively to our results by minimizing the acidification caused by the H 2 O 2 . 10 Furthermore, the alkaline medium is a favorable condition for the formation of an HCA layer 18,19,25,32 on the dental surface, reinforcing and protecting such surface. Moreover, the BS ability to improve the hardness and ionic concentration of dental enamel 5 can favor a reduction in the penetration of H 2 O 2 to the pulp tissue, which should be evaluated. applications had no significant effects. The difference in particle size should also be considered (~500 nm in this study versus 1 -20 μm in a previous study 22 ) and in the synthesis procedure to produce the BS (sol-gel in this study versus traditional glass-ceramics route in a previous study). 22 These parameters influence the reactivity of material. Moreover, the same authors also observed that 20 min of a single application of the product led to reduction in the pain response due to the stimulation of air from the triple syringe, indicating the quick action of BS.  [33][34][35] However, a previous study demonstrated that the use of bioglass was associated with bleaching gel maintains the ionic concentration in dental tissues, a fact that supports the integrity of the tissues during the bleaching. 2 The study also showed that the bleaching efficacy did not change when associating the bioglass with the bleaching gel. Thus, the benefits of blending the bleaching gel with the BS-based gel may be greater than just reducing its H 2 O 2 concentration.

BS
These data are favorable for the use of BS blended with bleaching gel.
After 30 days of the bleaching session, the pulp tissue of all groups was organized and a large amount of tertiary dentin was observed in the bleached groups.
Thus, it was possible to verify that the pulp tissue is able to recover from the damage caused by H 2 O 2 .
Other studies also demonstrated the reversal of such damage. 8,29,31 Notably, this tertiary dentin formation indicates the aging of the pulp tissue impairing its ability to defend against new aggressors. 28,31,36 In this study, the BS-based gel before or blended with bleaching gel enabled a significant reduction in deposition of tertiary dentin, and once again, showed the beneficial effect of using this blend. Thus, the remineralizing products previously used to recover lost ions from dental tissues after contact with H 2 O 2 can also be applied to prevent the pulp inflammation and to reduce the tertiary dentine production of this tissue. Consequently, postoperative sensitivity can be reduced. Moreover, when blended with bleaching gel, the results are better, besides reducing the clinical time.
A limitation of this study is the fact it has been performed on rat teeth, and thus, the results cannot be extrapolated directly to results in humans. However, rat molars have similar anatomical, histological, and physiological characteristics with human teeth. 24 Furthermore, standardized animal studies allow different groups to be compared without the influence of other variables, showing in this case the significant effects of mixing BS-based gel with bleaching gel. 24 However, this was the first study using a BSbased gel blended with a bleaching gel with high H 2 O 2 concentration, and further evaluation is required before its clinical use. Moreover, this study highlights that the effects in pulp tissue should be priority when performing procedures on the tooth surface, since some dental materials may cause severe alterations in this tissue, 4,31 as occurs with dental bleaching. 2,3,9,37 This damage alters the pulp tissue even after a long period. 28,36 Conclusions A single BS-based gel application beforehand or BS-based gel blended with bleaching gel can minimize the pulp damage induced by dental bleaching.