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This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/iej.12891

This article is protected by copyright. All rights reserved.

DR. FRANCINE BENETTI (Orcid ID : 0000-0002-5459-353X)

PROF. JOÃO EDUARDO GOMES FILHO (Orcid ID : 0000-0001-5994-2287)

PROF. LUCIANO TAVARES ANGELO CINTRA (Orcid ID : 0000-0003-2348-7846)

Article type : Original Scientific Article

Concentration-dependent effect of bleaching agents on the immunolabeling of interleukin-6,

interleukin-17, and CD5-positive cells in the dental pulp

F Benetti1, JE Gomes-Filho1, LL Ferreira1, G Sivieri-Araújo1, E Ervolino2, ALF Briso3, LTA Cintra1

Department of 1Endodontics, 2Basic Science, 3Restorative Dentistry, São Paulo State University

(UNESP), School of Dentistry, SP, Brazil.

Running Head: Dental bleaching and the dental pulp

Keywords CD5, dental pulp, hydrogen peroxide, interleukin-17, interleukin-6, tooth bleaching.

Name and address of the corresponding author:

Dr. Luciano Tavares Angelo Cintra

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Department of Restorative Dentistry, Araçatuba Dental School, UNESP-Univ Estadual Paulista, R:

José Bonifácio, 1193. Vila Mendonça. Araçatuba - São Paulo, Brazil

Tel: (0055) 18 36362867

Fax: (0055) 18 36363253

e-mail: [email protected]

Abstract

Aim To evaluate lymphocyte-like cell activation (CD5-positive cells) and the expression of interleukin

(IL)-6 and IL-17 in the pulp after dental bleaching with two concentrations of hydrogen peroxide

(H2O2).

Methodology The right and left maxillary molars from 40 rats were treated randomly with bleaching

gel with 20% H2O2 (BLUE group, 1 application of 50 min), 35% H2O2 (MAXX group, 3 applications of 15

min), or placebo gel (Control). After 2 and 30 days, the rats were killed (n=10), and the jaws were

processed for histological and immunohistochemistry analysis of the pulp tissue. The scores of

inflammation and immunolabeling (IL-6/IL-17) were submitted to Mann-Whitney and Kruskal-Wallis

followed Dunn tests, respectively; ANOVA tests were used for comparisons of number of CD5-

positive cells and pulp chamber area values (P<0.05).

Results At 2 days, 60% of specimens of the BLUE group were associated with moderate

inflammation in pulp horns, and in the MAXX group with necrosis (P<0.05). At 30 days, the pulp was

organized, and tertiary dentine was formed. The MAXX group had superior immunolabeling of IL-17

at 2 days differing significantly from other groups (P<0.05). At 2 days, 90% of the specimens of the

BLUE group had moderate immunolabeling of IL-6, and 50% of the MAXX group had severe

immunolabeling, both significantly different from the control (P<0.05). There was no significant

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difference between the groups at 30 days (P>0.05). CD5-positive cells were present at 2 and 30 days,

particularly in the bleached groups (P<0.05), without significant difference between time periods

(P>0.05).

Conclusions IL-6 and IL-17 participated in inflammation in the pulp tissue of rats after dental

bleaching, particularly at 2 days. The immunolabeling was greater with increasing H2O2

concentration. This process was accompanied by the prolonged activation of CD5-positive cells.

Introduction

The hydrogen peroxide (H2O2) within bleaching gels dissociates, giving rise to reactive oxygen species

(ROS), responsible for the bleaching process (Kawamoto & Tsujimoto 2004). The ROS are unstable

and react with other substances to achieve molecular stability (Kawamoto & Tsujimoto 2004).

Studies have reported diffusion of H2O2 in to enamel and dentine (Cintra et al. 2016a), reaching the

dentine–pulp complex and reacting with pulp cells (Costa et al. 2010, Cintra et al. 2013, 2016b,

Benetti et al. 2017a). This would explain why most patients who undergo this treatment have tooth

sensitivity (Charakorn et al. 2009).

In a previous study, an increase in the number of bleaching sessions led to greater damage in

the pulp (Cintra et al. 2013). In vitro and in vivo studies have demonstrated the cytotoxicity

generated by bleaching gels in pulp cells in the early post-operative period (Costa et al. 2010, Soares

et al. 2014a, Cintra et al. 2016a, Benetti et al. 2017a, 2017b). Also, cytokines have been shown to be

involved in acute inflammation six hours after dental bleaching (Soares et al. 2015). However, little is

known about the inflammatory response generated in the pulp some time after the bleaching

procedure, and the possible presence of inflammatory mediators involved in chronic inflammatory

response after this procedure remains unknown.

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The cytokines, secreted by leukocytes and other cells, are excellent markers of inflammation,

acting as modulators of inflammatory responses (Ashida et al. 2011). Interleukin (IL)-6 was observed

soon after dental bleaching in human dental pulp cells (Soares et al. 2015), but this has not been

investigated over time. This interleukin can promote the attraction of neutrophils and increase

vascular permeability in the pulp chamber (Elsalhy et al. 2013, Xiong et al. 2015), consequently

generating increased oedema (Elsalhy et al. 2013), which contributes to inflammation of the pulp

tissue (Azuma et al. 2015).

IL-17, produced by T-helper-17 cells (Xiong et al. 2015), acts in the inflammatory response,

inducing the expression of other proinflammatory cytokines, such as IL-6 (Xiong et al. 2015), and

neutrophil recruitment (Tong et al. 2014). IL-17 can stimulate the production of T cells (Chen et al.

2011), associated with an exacerbation of inflammatory response (Xiong et al. 2009). Considering

that IL-17 has potent effects on numerous cells, it is likely to stimulate pulp cells to secrete other

cytokines after a dental bleaching procedure and may mediate the inflammatory process.

CD5 is a receptor present in mature lymphocytes and is an important regulator of the

activation of these cells (de Wit et al. 2011). The lymphocyte is a defence cell responsible for specific

responses, and the balance between proliferation and cell death is responsible for the homeostasis

of these cells. Disruption of this balance may cause disease progression (Gillet et al. 2007).

Cintra et al. (2016b) observed that alterations in the pulp tissue increase with greater

concentrations of bleaching gel, and after a certain period, the pulp is organized and partially

occupied by tertiary dentine. Maturation of collagen fibres in the pulp was also observed (Cintra et

al. 2016c). However, it is not known which factors mediate the inflammatory process by the

oxidative stress generated by bleaching gel over time. This present study investigated in vivo the

lymphocyte-like cell activation and expression of IL-17 and IL-6 proinflammatory cytokines in pulp

tissue after dental bleaching with two concentrations of H2O2 both in the initial period and over

time.

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Immunolabeling provides information on whether the inflammation in the pulp tissue

caused by dental bleaching results in chronic inflammation or can be repaired. It is hypothesized that

IL-6 and IL-17 have a greater immunolabeling, as well as greater amounts of CD5-positive cells, in the

presence of a higher H2O2 concentration. The presence of these markers in the inflammatory process

generated after dental bleaching could indicate them as potential targets for further therapeutic

agents. The null hypothesis of this study was that the bleaching gel concentration does not influence

the immunolabeling of these cytokines in the pulp tissue.

Materials and Methods

A total of 40, 2 month-old male Wistar albino rats (250 g) were used. The sample size was

established on the basis of a previous studies (Cintra et al. 2013, 2016a, 2016b). The rats were

housed in a temperature-controlled environment (22ºC±1ºC, 70% humidity) on a standard light/dark

schedule with access to food and water ad libitum. The experimental protocol was approved by the

local Ethics Committee (CEUA 2014-00591) and conducted in accordance with the Guide for the Care

and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD, USA).

Tooth Bleaching

All rats were anaesthetised by intramuscular injections of ketamine (80 mg/kg, Ketamina Agener

10%, União Química Farmacêutica Nacional S/A, Embu-Guaçu, São Paulo, Brazil) and xylazine (10

mg/kg, Xilazin, Syntec do Brazil LTDA, Cotia, São Paulo, Brazil). After the application and

photoactivation of the resinous gingival barrier (Top Dam; FGM Dental Products, Joinville, SC, Brazil),

the maxillary molars received, randomly, 0.01mL of bleaching or placebo gels (Cintra et al. 2013), as

follow (Table 1): the BLUE group: 1 application of 50 min, 20% H2O2, (Whiteness HP Blue; FGM

Dental Products, Joinville, SC, Brazil); the MAXX group: 3 applications of 15 min, 35% H2O2

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(Whiteness HP Maxx; FGM Dental Products); and the Control: 3 applications of 15 min or 1

application of 50 min (in according with bleaching gel respective), placebo gel - thickener of the

bleaching gel. Syringes of 1.0 mL were used to standardise the volume of the applied bleaching gel

or placebo gel in molar rats.

Laboratory procedures

At 2 and 30 days, 20 rats were killed with an overdose of anaesthetic solution (Thipentax, Cristália—

Produtos Químicos Farmacêuticos LTDA, Itapira, São Paulo, Brazil), resulting in ten hemimaxillae per

bleaching group in each period and ten more hemimaxillae of the contra-lateral side were randomly

selected for control group (n=10). Then, the right and left hemimaxillae were separated, dissected,

and fixed in a solution of 4% buffered formaldehyde for 24 hours. The specimens were decalcified in

a 10% ethylenediaminetetraacetic acid (EDTA) solution for three months and then dehydrated,

clarified, and embedded in paraffin.

Serial histological sections of each specimen were selected from the point where the mesial

root of the first molar was seen in all its longitudinal extension. Five-micron sections were cut in the

buccal–lingual plane and stained with haematoxylin–eosin or submitted to immunohistochemistry

using an indirect immunoperoxidase technique (Garcia et al. 2013) for IL-6, IL-17 and CD5. The first

slide with histological sections obtained was selected for staining in H.E., and the next two for

immunohistochemistry. This sequence was repeated, obtaining two slides with histological sections

for each staining.

For immunohistochemical reactions, the histological sections were deparaffinised in xylene

and hydrated in a decreasing ethanol series. Antigen retrieval was achieved by immersing the

histological slides in buffer citrate solution (Antigen Retrieval Buffer, Spring Bioscience, Pleasanton,

CA, USA) in a pressurised chamber (Decloaking Chamber; Biocare Medical, Concord, CA, USA) at 95°C

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for 10 minutes. The slides were rinsed with phosphate-buffered saline (PBS) at the end of each stage

of the immunohistochemical reaction. The histological sections were immersed in 3% H2O2 solution

for 1 h and 20 minutes and in 1% bovine serum albumin for 12 h to block endogenous peroxidase

activity and the nonspecific sites, respectively. The histological slides were divided and incubated

with one of the following primary antibodies: anti-IL-6 (primary antibody rabbit, SC-1265, Santa Cruz

Biotechnology, CA, USA), anti-IL-17 (primary antibody rabbit, SC-7927, Santa Cruz Biotechnology), or

anti-CD5 (primary antibody goat, SC-6986, Santa Cruz Biotechnology). The primary antibodies were

diluted (Antibody Diluent with Background Reducing Components, Dako Laboratories, Carpinteria,

CA, USA), and placed in a moist chamber for 24 h. The histological sections were incubated with a

biotinylated secondary antibody for 1 h and 30 min and were subsequently treated with

streptavidin–horseradish peroxidase conjugate for 1 h and 30 min (Universal Dako Labelled

Streptavidin-Biotin kit; Dako Laboratories). The slides were rinsed with PBS and the reaction was

developed using the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB Chromogen kit;

Dako Laboratories) and was counterstained with Harris’s Haematoxylin. The negative controls

consisted of specimens submitted to the procedures previously mentioned but without the primary

antibodies.

The analyses were performed by a single calibrated operator in a blinded manner under light

microscopy (400X, DM 4000 B; Leica, Wetzlar, Germany). The pulp chamber was divided into thirds

(occlusal, middle, and cervical) (Cintra et al. 2013), and the inflammation was scored in each third as

follow: 0, inflammatory cells absent or negligible in number; 1, mild inflammatory infiltrate (<25 cells

per field); 2, moderate inflammatory infiltrate (between 25 and 125 cells per field); 3, severe

inflammatory infiltrate (>125 cells per field); and 4, tissue necrosis (adapted from Cintra et al. 2013).

The immunolabeling for IL-6 and IL-17 was defined as a brownish colour in the cytoplasm of

the cells and extracellular matrix. Because immunolabeling of both the cells and the extracellular

matrix is of great importance, semiquantitative analysis, which provides information on the numbers

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of immunolabeling cells and immunolabeling intensity of the extracellular matrix was performed

(Cintra et al. 2016d). The scores were assigned as follows (adapted from Cintra et al. 2016d): 0,

immunolabeling missing; 1, low standard of immunolabeling; 2, moderate standard of

immunolabeling; 3, severe standard of immunolabeling; and 4, very severe standard of

immunolabeling.

To analyse CD5, cells that had brown cytoplasm were counted (Benetti et al. 2017a), and the

area of each third of pulp chamber was measured in each specimen by image processing software

(Leica QWin V3, Leica Microsystems), to subsequently to express the number of cells per mm2

(Cintra et al. 2016b, Benetti et al. 2017a). The values of total central area of the pulp chamber was

also obtained, and by taking into account the values obtained it was possible to calculate the

reduction percentage in the central area of the pulp chamber in the treated groups in comparison to

the central area of the control group.

Statistical analysis

Mann-Whitney tests were used for statistical comparisons of inflammatory response. Kruskal-Wallis

and Dunn tests were used for comparisons of immunolabeling of IL-6 and IL-17. Two-way ANOVA

test was used for comparisons of number of immunolabeling CD5 cells, and one-way ANOVA

statistical test for comparisons of pulp chamber area values (P<.05).

Results

Histological analysis

The representative images of the inflammatory response can be visualized in Figure 1.

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At 2 days, the Control group exhibited intact pulp tissue and absence of inflammation (Figure

1; A,a). The BLUE group had cellular disorganization, especially in the pulp horns; most specimens

had moderate inflammation in this region, whereas in the middle and cervical third mild

inflammation was observed (Figure 1; B,b). The MAXX group had necrosis in the occlusal third,

moderate inflammation in the middle third, and mild inflammation in the cervical third (Figure 2;

C,c).

At 30 days, all specimens had cellular organization (Figure 1; D-F), with organized

odontoblastic layer (Figure 1; d1,d2-f1,f2). There was a large formation of tertiary dentine in the

bleached groups (Figure 1; E,e1,e2;F,f1,f2), occupying part of the pulp chamber.

Analysis of inflammatory mediators

The representative images of immunolabeling for IL-17 and IL-6 can be visualized in Figure 2.

At 2 days, IL-17 analysis revealed that the control group had low immunolabeling (Figure 2;

A); the BLUE group exhibited low and moderate immunolabeling (Figure 2; B); and the MAXX group

had a moderate immunolabeling (Figure 2; C). At 30 days (Figure 2; D-F), all groups had lower

immunolabeling for IL-17.

In the IL-6 analysis at 2 days, the control group had low immunolabeling (Figure 2; G); the

BLUE group had moderate immunolabeling (Figure 2; H); and the MAXX had moderate and high

immunolabeling (Figure 2; I). At 30 days, the control group had low immunolabeling (Figure 2; J);

BLUE group, moderate immunolabeling (Figure 2; K); and the MAXX group moderate

immunolabeling (Figure 2; L).

The representative images of the immunolabeling for CD5 can be visualized in Figure 3. The

immunolabeling for CD5 was observed in diffusely distributed cells over the pulp. At 2 days, greater

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immunolabeling was observed in occlusal third of the BLUE group, and in the middle and cervical

thirds of both bleached groups. At 30 days, the number of immunolabeling cells was determined

only in the cervical third of the crown because the occlusal and middle thirds were occupied by

tertiary dentine. The greater immunolabeling was observed in bleached groups. The CD5 cell

counts/mm2 is shown in Figure 3; G.

Comparison among the Groups

The statistical analysis of inflammatory responses can be visualized in Table 2. The bleached groups

had significant differences in the occlusal and middle thirds of the coronal pulp at 2 days after dental

bleaching, with more damage in the MAXX group (P<.05). At 30 days, there was no significant

difference between the groups (P>0.05).

Immunohistochemical analysis for IL-17 and IL-6 (Table 3), the MAXX group revealed

significant immunolabeling for IL-17 compared with other groups, 2 days after bleaching (P<0.05);

the bleached groups had significant immunolabeling for IL-6 compared with the control group, at 2

days after bleaching (P<0.05). At 30 days, there was no significant difference between the groups

(P>0.05).

In the immunohistochemical analysis for CD5 (Figure 3; G), after 2 days, there was a

significant difference between the BLUE group and the other groups in the occlusal third (P<0.05); in

the middle and the cervical third, both bleached groups had a significant difference from the control

group (P<0.05). The immunolabeling was present at 30 days, with no significant difference compared

to 2 days (P>0.05).

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Discussion

This study investigated lymphocyte-like cell activation and immunolabeling of IL-6 and IL-17

proinflammatory cytokines in pulp tissue after tooth bleaching with two concentrations of H2O2. The

null hypothesis was rejected, since greater immunolabeling, as well as a larger number of CD5-

positive cells, were observed with greater H2O2 concentrations.

A study using Wistar rat molars reported that one bleaching session with 35% H2O2 was

capable of causing necrosis in the occlusal third of the coronal pulp (Cintra et al. 2013). A similar

study in human mandibular incisors revealed similar pulp reactions (Costa et al. 2010), suggesting

that this experimental model may be useful to predict outcomes of bleaching procedures performed

in human mandibular incisors (Cintra et al. 2013).

The concentration of bleaching gels can influence the damage to pulpal tissue, due to the

greater penetration of H2O2 (Cintra et al. 2016b). In this study, approximately 60% of the specimens

of the group that received the bleaching gel with 20% H2O2 exhibited moderate inflammatory

infiltrate in the occlusal third of the coronal pulp, whereas 60% of the specimens of the group that

received bleaching gel with 35% H2O2 had necrosis in this area. The pulp tissue was able to recover

its organization, forming an odontoblastic layer around the entire pulp. However, a large amount of

tertiary dentine was produced, which reduced the volume of the pulp chamber.

After an injury to tooth tissue, pulp progenitor cells are recruited for the repair process and

differentiate into second-generation odontoblasts to produce reparative dentine (Kitamura et al.

1999). Odontoblasts that persist in the tissue also participate in the repair process, secreting

reactionary dentine (Smith et al. 1995). Thus, the dentine gradually increases in thickness over the

space formerly occupied by pulp tissue (Goldberg & Smith 2004), as occurred in the present study.

Several studies have shown that H2O2 is capable of inducing mineralization of pulp tissue

(Matsui et al. 2009, Soares et al. 2014). Lower H2O2 concentrations strongly induced the expression

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of proteins associated with the mineralization process, whereas these proteins were adjusted

according to the concentration of H2O2. This study showed that the MAXX group was associated with

a greater decrease in the pulp chamber area. This indicates that the highest concentration of H2O2

can stimulate more odontoblasts to produce tertiary dentine as a form of protection against ROS.

This leads to aging of the pulp tissue and impairs its ability to defend against new aggressors (Cintra

et al. 2016c).

IL-6 was present most strongly in the tissue, both in the BLUE group and the MAXX group.

Significant levels of this interleukin were identified in the inflamed dental pulp (Barkhordar et al.

1999). This IL-6 production can have a beneficial or deleterious effect, depending on the quantity

produced and the time of its action (Balto et al. 2001). Elevated levels of IL-6 are associated with

increased inflammation and clinical symptoms (Prso et al. 2007), and IL-6 is present just after the

bleaching procedure in cell cultures (Soares et al. 2015). In this study, the immunolabeling of IL-6

was more intense at two days than at 30 days, suggesting that inflammation and symptoms

decrease after some time.

Greater immunolabeling found for IL-17 in the MAXX group may indicate that this cytokine

acts in the pulp inflammatory process during oxidative stress caused by H2O2. As the labelling was

present in various types of pulp cells, it is likely that it stimulated pulp tissue cells to secrete other

cytokines. The treatment of human dental pulp cells with IL-17 showed that it is able to induce IL-6

in a dose-dependent manner. Thus, it has been suggested that IL-17 can exacerbate the

inflammatory response in pulpits (Xiong et al. 2015). Studies have also shown that IL-17 promotes

bone remodelling, as well as increased osteogenic differentiation markers on human mesenchymal

stem cells (Huang et al. 2009).

CD5-positive cells were present in both the specimens analysed two days after the bleaching

procedure and those analysed after 30 days, with no significant difference between them. CD5 is a

receptor present on T and B cells, related to the progression of diseases that involve pulp tissue

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(Cooper et al. 2010). However, this immunolabeling was also found in other cells. These data are

consistent with studies showing that H2O2 bleaching gel, when used in low concentrations, can

promote the expression of inflammatory mediators by odontoblasts, which are the first defence cells

of the pulp tissue due to peripheral location and intimate contact with dentine (Cooper et al. 2010).

Thus, the odontoblasts are immunocompetent cells, capable of activating an immune response

(Cooper et al. 2010).

With the increase of the pathogenic agent, the expression of inflammatory markers by other

cells occurs. This fact can be observed during infection, where the progression of disease involves

the release of cytokines and chemokines by fibroblasts, stem cells, endothelial cells, and, finally, cells

of the immune system (Cooper et al. 2014). In the present study, the effects of H2O2 were intense, so

there was a release of inflammatory mediators by other cells of the pulp tissue, and this caused the

attraction of immune cells.

Positive immunolabeling for CD5 at 30 days suggests that the pulp tissue remains in an

inflammatory state, even though organized. This finding can be confirmed in future studies, with

markers showing the permanence of oxidative stress in the pulp after bleaching procedures.

A greater number of CD5-positive cells was observed in the occlusal third of the BLUE group,

which received a lower concentration of H2O2. This result can be explained by the fact that the MAXX

group had necrosis in this region, which decreased tissue cellularity. Similar results were obtained

previously with lower H2O2 concentration that resulted in greater presence of apoptotic cells than

higher concentration that resulted in the presence of necrosis (Benetti et al. 2017a). However, these

results disagree with those observed with the immunolabeling of IL-6 and IL-17, where the intensity

was proportional to H2O2 concentration in bleaching gels. This disagreement can be attributed to the

different ways interleukins were analysed: in the present study, interleukins were analysed in the

entire coronal pulp instead of an analysis of coronal pulp thirds, according to a previous study

(Ferreira et al. 2017).

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In general, this study shows that higher concentrations of H2O2 cause greater damage to pulp

tissue: presence of areas of necrosis, decreased cellularity, and increased inflammation. However, it

should be noted that the bleaching gel of the BLUE group, in addition to the lower concentration of

H2O2, has in its composition calcium gluconate (Table 1), which may influence the results. The

presence of calcium gluconate in bleaching gel minimizes the necrosis observed in human teeth after

dental bleaching (Roderjan et al. 2015). This is because calcium can maintain a stable pH of the

bleaching gel, besides preventing demineralization of the enamel, which would consequently reduce

H2O2 penetration and tooth sensitivity (Kossatz et al. 2012, Roderjan et al. 2015).

Furthermore, a recent study revealed areas of necrosis after 6 hours of application of

bleaching gel (Lima et al. 2016). Thus, it may be that the pulp tissue observed in the present study at

2 days is in the process of regeneration. This fact may also have influenced the absence of necrosis in

the BLUE group, once its lower H2O2 concentration could have caused less damage to pulp tissue

allowing its rapid recovering after 2 days. Thus, further studies are needed in this experimental

model performing analyses in shorter periods after treatment.

Previous studies have reported different pulp tissue repair capacity linked to the animal

species, suggesting that each species should have detailed histological biocompatibility studies

(Watts & Patterson 1981, Hebling et al. 1999, Costa et al. 2003). In addition, rat molars have small

areas of exposed dentine on the occlusal surface, which received the bleaching gel (Hunt et al.

1970). However, studies have shown that the essential biological reactions and healing of pulp tissue

from rat molars are comparable to those of other mammals (Dammaschke 2010), and that rat

molars have histological and biological characteristics similar to human molars (Sasaki & Kawamata-

Kido 1995, Dammaschke 2010). Damage found in rat pulp tissue after bleaching with high H2O2

concentration (Cintra et al. 2013) were similar to those found in human maxillary incisors (Costa et

al. 2010). These results should not be directly extrapolated to human teeth but this study, as well as

preview study in molar rats (Benetti et al. 2017a), shows that the H2O2 concentration influences the

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cellular mechanisms that involve the response of the pulp tissue. To better evaluate the influence of

the concentration of the bleaching agent in pulp tissue, this study evaluated separately each third of

the coronal pulp, considering that deeper thirds would be affected by lower concentrations of

permeated H2O2.

Conclusion

IL-6 and IL-17 participated in the inflammatory process occurring in rat pulp tissue after tooth

bleaching, particularly in the early periods. The immunolabeling was greater with increasing H2O2

concentration, and was accompanied by the prolonged activation of CD5-positive cells.

Conflict of Interest

The authors have stated explicitly that there are no conflicts of interest in connection with this

article.

Acknowledgement

This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (n.

2013/25429-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (n.

455943/2014-1).

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Figure Legends

Figure 1 (A-C, a-c) Two days after bleaching: Representative photomicrographs of the Control group

(A, a1) show normal pulp tissue and intact odontoblastic layer (black arrows), and (a2) evidenced

tissue organization; the BLUE group (B, b1) presents cellular disorganization (black arrowheads) and

(b2) severe inflammatory infiltrate (yellow arrowheads) in the occlusal third; and the MAXX group (C,

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c1) shows areas of necrosis and a decrease in cellularity in the occlusal third, (c2) that were

evidenced in higher magnification. (D-F, d-f) Thirty days after bleaching: Representative

photomicrographs of Control (D, d1, d2), BLUE (E, e1, e2), and MAXX (F, f1, f2). In the comparative

panoramic photomicrographs (D-F), the pulp chamber as a whole can be observed, showing the

formation of tertiary dentine in the bleached groups (E, F); Black arrows indicate the intact

odontoblastic layer, asterisks show the predentine layer, yellow stars indicate the tertiary dentine,

red arrows show the blood vessels in the subjacent tissue [H&E staining, A-F: 100X; a1-f1, d2-f2:

400X; a2-c2: 1000X].

Figure 2 Immunohistochemical labelling of IL-17 and IL-6. Representative photomicrographs of the

immunolabeling for IL-17(A-F) in the Control (A) and BLUE (B) groups showing low immunolabeling

at 2 days, and MAXX group (C) showing moderate immunolabeling; and Control (D), BLUE (E) and

MAXX (F) groups with low immunolabeling at 30 days. Representative photomicrographs of the

immunolabeling for IL-6 (G-L) in the Control (G) group showing low immunolabeling at 2 days, and

BLUE (H) and MAXX (I) groups showing moderate immunolabeling; and Control (J) and BLUE (K)

groups showing low immunolabeling at 30 days, and MAXX (L) group with moderate

immunolabeling. [Immunolabeling for IL-17 and IL-6, 400X].

Figure 3 Immunohistochemical labelling of CD5. Representative photomicrographs of the cervical

third of Control (A, a, D, d), BLUE (B, b; E, e), and MAXX (C, c; F, f) groups at 2 (A–C, a-c) and 30 (D–F,

d-f) days. The black arrows in the largest magnification point to labelling cells. [Immunolabeling for

CD5, A-F: 400X; a-f: 1000X]. The graph (G) shows the cell counts for CD5, expressed in number of

cells per mm2, and statistical analysis. Different superscript lowercase letters indicate statistically

significant differences between groups at each analysis time, and same superscript uppercase letters

indicate statistically no significant differences between 2 and 30 days in the cervical third (P<0.05).

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Table 1 Composition of the bleaching gel and protocol of use according to manufacturer

Bleaching gel Composition Protocol of use

Whiteness HP

Blue

20% hydrogen peroxide, thickeners, blue

inert pigment, neutralizing agents, calcium

gluconate, glycol and deionized water

1 application of the 50 min

Whiteness HP

Maxx

35% hydrogen peroxide, thickeners,

coloring mixture, glycol, inorganic filler

and deionized water

3 applications of the 15 min each

Table 2 Scores for inflammatory infiltrate at 2 days and area of the pulp chamber at 30 days

Third of the

coronal pulp

Score 2

days

Group (n=10)

P value

Control BLUE MAXX

Occlusal

0 10 0 0

Mann-

Whitney

BLUE x

MAXX

P=0.007

1 0 2 0

2 0 6 2

3 0 1 2

4 0 1 6

Median* 1 3a 5b

Middle 0 10 0 0 Mann-

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1 0 6 2 Whitney

BLUE x

MAXX

P=0.049

2 0 4 6

3 0 0 1

4 0 0 1

Median* 1 2a 3b

Cervical

0 10 4 3

Mann-

Whitney

BLUE x

MAXX

P=0.372

1 0 6 5

2 0 0 2

3 0 0 0

4 0 0 0

Median* 1 2a 2a

Area of pulp

chamber at 30

days (µm²)

Mean (105)* 19.86a 9.25b 7.00c One-Way

ANOVA

P<0.001 SD (105) 2.35 0.59 0.25

*Different letters in the same line indicate statistically significant differences between groups.

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Table 3 Scores for immunohistochemical labeling of IL-6 and IL-17 at 2 and 30 days

Immunochemistry Score

Group

P value Control BLUE MAXX

2d 30d 2d 30d 2d 30d

IL-17

0 0 0 0 0 0 0

Kruskal-Wallis

2d: P<0.001

30d: P=1.000

1 9 10 5 10 0 10

2 1 0 5 0 6 0

3 0 0 0 0 4 0

4 0 0 0 0 0 0

Median* 1a 1A 1a 1A 2b 1A

IL-6

0 0 0 0 0 0 0

Kruskal-Wallis

2d: P<0.001

30d: P=0.014

1 10 10 1 5 0 4

2 0 0 9 5 5 6

3 0 0 0 0 5 0

4 0 0 0 0 0 0

Median* 1a 1A 2b 1A 2b 2A

*Different superscript lowercase letters in the same line indicate statistically significant

differences between groups at 2 days and superscript uppercase letters at 30 days.

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