Indirect cytocompatibility of a low-concentration hydrogen peroxide bleaching gel to odontoblast-like cells.

doi:10.1111/iej.12426

Indirect cytocompatibility of a low-concentration hydrogen peroxide bleaching gel to odontoblastlike cells

D. G. Soares1, N. Marcomini1, F. G. Basso2, T. N. Pansani3, J. Hebling2 & C. A. de Souza Costa1 1

Department of Physiology and Pathology, Araraquara School of Dentistry, University of Estadual Paulista (UNESP), Araraquara; 2Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, University of Estadual Paulista (UNESP), Araraquara; and 3Department of Dental Materials and Prosthodontics, Araraquara School of Dentistry, University of Estadual Paulista (UNESP), Araraquara, Brazil

Abstract Soares DG, Marcomini N, Basso FG, Pansani TN, Hebling J, de Souza Costa CA. Indirect cytocompatibility of a low-concentration hydrogen peroxide bleaching gel to odontoblast-like cells. International Endodontic Journal, 49, 26–36, 2016.

Aim To assess the initial cytotoxicity and the late phenotype marker expression of odontoblast-like cells (MDPC-23) subjected to less aggressive in-office bleaching therapies. Methodology A 17.5% hydrogen peroxide (H2O2) gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to trans-wells positioned over cultured MDPC-23 cells. No treatment was performed on the negative control. Immediately after bleaching, the cell viability, gene expression of inflammatory mediators and quantification of H2O2 diffusion were evaluated. The ALP activity, DSPP and DMP-1 gene expression and mineralized nodule deposition (MND) were assessed at 7, 14 or 21 days post-bleaching and analysed statistically with Mann–Whitney U-tests (a = 5%). Results H2O2 diffusion, proportional to treatment time, was observed in all bleached groups. Reductions

of approximately 31%, 21% and 13% in cell viability were observed for the 45-, 15- and 5-min groups, respectively. This reduction was significant (P < 0.05) for the 45- and 15-min groups, which also presented significant (P < 0.05) over-expression of inflammatory mediators. The 45-min group was associated with significant (P < 0.05) reductions in DMP-1/ DSPP expression at all periods, relative to control. The ALP activity and MND were reduced only in initial periods. The 15-min group had less intense reduction of all markers, with no difference to control at 21 days. Conclusions The 17.5% H2O2 applied to tooth specimens for 5 min caused no alteration in the odontoblast-like cells. When this gel was applied for 45 or 15 min, a slight cytotoxicity, associated with alterations in phenotypic markers, was observed. However, cells were able to recover their functions up to 21 days post-bleaching. Keywords: cell differentiation, cytotoxicity, odontoblasts, tooth bleaching. Received 19 September 2014; accepted 23 December 2014

Introduction Correspondence: Carlos A. de Souza Costa, Department of Physiology and Pathology, University of Estadual Paulista – UNESP, Araraquara School of Dentistry, Humait a Street, 1680, Araraquara, SP 14801-903, Brazil (e-mail: [email protected]).

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International Endodontic Journal, 49, 26–36, 2016

Odontoblasts below dentine at the periphery of the pulps have their protracted processes anchored inside dentinal tubules, forming the pulp–dentine complex (Couve et al. 2013). Thus, these cells are the first to

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd

Soares et al. Tooth bleaching cytocompatibility

receive toxic components from dental materials capable of diffusing through enamel and/or dentine (de Souza Costa et al. 2014). The main function of odontoblasts is to produce dentine throughout the lifespan of the tooth, physiologically or in response to injury. Following relatively mild tooth injury, odontoblasts are upregulated to secrete and mineralize a tubular reactionary dentine to keep the pulp tissue away from noxious stimuli and reduce the diffusion of toxic components released from dental materials or microorganisms into the pulp space, thus protecting the underlying cells and maintaining pulp health (Goldberg & Smith 2004, Schmalz & Smith 2014). Odontoblasts are also involved in the initiation, development and maintenance of the pulp inflammatory/immune response (Cooper et al. 2010, 2014). Therefore, these cells are essential for the homeostasis and function of the pulp–dentine complex, representing the first line of pulp tissue defence for the host (Farges et al. 2009). Laboratory and histopathological studies have demonstrated that in-office bleaching therapy performed with 35 and 38% hydrogen peroxide (H2O2)-based gels applied for three times of 10–15 min to enamel causes intense oxidative stress to odontoblast-like cells, associated with the disruption of cell membranes and cell death by necrosis (de Souza Costa et al. 2010, Cintra et al. 2013, Soares et al. 2014a). These toxic effects have been correlated to the slight to intense tooth sensitivity claimed by a number patients subjected to this kind of aesthetic therapy (Reis et al. 2011, de Almeida et al. 2012, Tay et al. 2012, Bonafe et al. 2013). Therefore, alternative in-office bleaching protocols using reduced bleaching time and different H2O2 concentrations have been employed in the clinical situation with the advantage of minimizing the adverse events to the pulp–dentin complex (Martin et al. 2013, Moncada et al. 2013, Reis et al. 2013, Bortolatto et al. 2014, Ozcan et al. 2014). It was recently demonstrated that a 17.5% H2O2 gel promoted significant colour enhancement, associated with a reduction of around 60–90% in H2O2 diffusion through enamel and dentine, depending on the application regimen, in comparison with traditional protocols (Soares et al. 2014b). In addition, significant minimization in the trans-enamel and trans-dentinal toxicity to odontoblast-like cells was observed, and around 50% of the damaged cells demonstrated improved viability 3 days after being bleached (Soares et al. 2014a). However, the effect of this initial toxicity on odontoblastic marker regulation


was not assessed. Therefore, in this study, the immediate cytotoxic effects of several bleaching protocols with a 17.5% H2O2 gel on odontoblast-like cells were assessed, and the regulation of phenotypic markers by this pulp cell line was analysed up to 21 days post-bleaching. The null hypothesis was that the 17.5% H2O2 gel has no toxic effect on the MDPC-23 independent of the contact time with dental structure.

Materials and methods Cell culture The odontoblast-like cell MDPC-23, kindly donated by Dr. Carl Hanks, from the University of Michigan, USA, was used. The cells were cultured and subcultured into cell culture flasks with complete DMEM (Dulbecco’s Modified Eagle medium; supplemented with 100 IU mL1 penicillin, 100 lg mL1 streptomycin, 2 mmol L1 glutamine; Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; Gibco) at 37 °C and 5% CO2 until adequate numbers of cells were obtained. Cells from the 56th to 60th passages were used.

Experimental procedure A total of 120 standardized enamel/dentine discs (from 24- to 30-month-old bullocks), 5.6 mm in diameter and 3.5 mm in thickness, were obtained and adapted to acrylic trans-wells (polycarbonate membrane per 8 lm pore size; Corning Inc., Corning, NY, USA) by means of a fluid light-cured resin (TopDam, FGM, Joinville, SC, Brazil) and sterilized by ethylene oxide. The standardized thickness was obtained after polishing the dentine surface with wet 400- and 600-grit silicon carbide paper (T469SF- Norton, Saint-Gobain Abrasivos Ltda., Jundiaı, SP, Brazil) until 3.5-mm-thick enamel/dentine discs were obtained. Three measurements were performed in three different areas of the discs with a digital calliper (Mitutoyo 500-144B; Mitutoyo Sul America Ltda., S~ ao Paulo, SP, Brazil). The cells were seeded into 24-well plates at 80% confluence (6 9 104 cells per well; 24 h). Thereafter, the culture medium was replaced by 300 lL of complete DMEM with no FSB, and the disc/trans-well set was placed over previously cultured cells in such a way that only the dentine was in direct contact with the culture medium, and the enamel surface remained


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Tooth bleaching cytocompatibility Soares et al.

exposed to receive the bleaching procedure (Fig. 1). A bleaching gel with 17.5% H2O2 was freshly prepared by dilution of the liquid phase of a commercial 35%-H2O2 gel (Whiteness HP; FGM, Joinville, SC, Brazil) in distilled water. The gel was applied to dental enamel (30 mg) for different times, giving rise to the following groups: 45-min group – three applications of 15 min each; 15-min group – one application of 15 min; 5-min group – one application of 5 min; and control group – no treatment. For the 45-min group, after each 15-min application, the gel was aspirated with a sterilized Pasteur pipette coupled to a vacuum bomb. Immediately after the last application, the bleaching gel was aspirated, the disc/trans-well set was removed and the cells assessment was performed.

MTT assay For cell viability, immediately after being bleached, the cells were incubated for 4 h with MTT solution (Sigma-Aldrich Corp., St. Louis, MO, USA) at 37 °C and 5% CO2, after which the absorbance of formazan crystals in the viable cells was read (570 nm; ELISA microplate reader, Tp Reader, Thermoplate, Nanshan District, Shenzhen, China). The mean absorbance of the control group was considered as 100% of cell viability, and the percentage values for experimental groups were calculated based on this parameter (n = 6).

Quantification of H2O2 diffusion The amount of H2O2 capable of diffusing through enamel and dentine was quantified by the leuco-crystal violet/peroxidase reaction, as previously described (Soares et al. 2013, 2014a). A 100-lL aliquot of the extract from each group of MTT assay (n = 6) was transferred to tubes containing 900 lL of acetate buf-

Figure 1 Schematic representation procedure.

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of


the

experimental

fer solution (2 mol L1, pH 4.5), to avoid H2O2 degradation. Then, a 500 lL quantity of buffer solution plus extract was transferred to experimental tubes to react with leuco-crystal violet (0.5 mg mL1; Sigma-Aldrich Corp.) and horseradish peroxidase enzyme (1 mg mL1; Sigma-Aldrich Corp.). The final volume of reaction was adjusted to 3 mL with distilled water, and the optical density of the solutions was measured at 600-nm wavelength in an ELISA microplate reader. A standard curve of known H2O2 concentrations was used for conversion of the optical density obtained in the samples into lg mL1 of H2O2, and the data were related to lg mL1 of culture medium.

Gene expression of inflammatory mediators The gene expression of tumour necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), interleukin 6 (IL-6) and cyclooxygenase 2 (COX-2) was performed by the real-time PCR technique. Immediately after bleaching, the culture medium was replaced by fresh DMEM with no SFB, and cells were incubated for 6 h at 37 °C and 5% CO2 (n = 6). Total RNA was then extracted with an RNAqueousâ-micro kit (Ambion, Austin, TX, USA) by following the manufacturer’s protocols. One microgram of total RNA, following DNase I treatment, was reverse-transcribed into single-stranded cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), according to the recommended protocol [25 °C (10 min), 37 °C (120 min), 85 °C (5 s), 4 °C]. For relative quantification of mRNA levels, SYBRâ Green primers (Sigma-Aldrich Corp.) were used, as follows: TNF-a (forward) 50 -CCCTCCTGGCC AACGGCA-30 and (reverse) 50 -TCGGGGCAGCCTTGTC CC-30 ; IL-1b (forward) 50 -AAAGCCTCGTCGTGTCGG -30 and (reverse) 50 -CCTTTGAGGCCCAAGGGC-30 ; IL-6, (forward) 50 -GAGGATACCACTCCCAACAGACC-30 and (reverse) 50 -AAGTGCATCATCGTTGTTCATACA-30 ; COX2, (forward) 50 -ACCCTGCCTACGAAGGAACT-30 and (reverse) 50 -ACCACGGTTTTGACATGGGT-30 ; and b-actin, (forward) 50 -GGACCTGACGGACTACCTCATG-30 and (reverse) 50 -TCTTTGATGTCACGCACGATTT-30 . Amplification assays were performed with SYBRâ Green Master Mix (Applied Biosystems), and fluorescence was determined with StepOne Plus equipment (Applied Biosystems). The CT values for each sample were normalized by an endogenous control gene (b-actin). Thereafter, the mean CT value of the con-



trol group was used to normalize the CT values of both control and experimental groups.

Alkaline phosphatase (ALP) activity assay For this analysis, immediately after being bleached, the cells were cultured in osteogenic medium (DMEM plus 10% SFB, supplemented with 10 nmol L1 b-glycerophosphate and 50 lg mL1 sodium ascorbate; SigmaAldrich Corp.), for periods of 7, 14 and 21 days (the medium was changed every day) (n = 6). After each time-point, cell lysis was performed with 0.1% sodium lauryl sulphate (Sigma-Aldrich Corp.). ALP activity was assessed with a kit (End-point assay; Labtest, Lagoa Santa, MG, Brazil) as previously described (Soares et al. 2013). This assay is based on the reactivity of ALP with the thymolphthalein monophosphate substrate. Released thymolphthalein becomes purple in the presence of sodium carbonate and sodium hydroxide. Absorbance was then read at a 590-nm wavelength with an ELISA microplate reader and converted into U L1 by means of a standard curve with known amounts of ALP. Total protein dose was performed for normalization of ALP according to the Read & Northcote (1981) protocol, as previously described (Soares et al. 2013). The absorbance of the test and blank tubes was measured at a 655-nm wavelength with the ELISA microplate reader. The absorbance value obtained was converted into mg L1 by a standard protein curve. The final value of ALP was normalized by total protein obtained from each well, with the value of ALP activity as U mg1 of protein.

tation to solubilize the nodules. The absorbance of the resulting solution was measured at 570 nm by means of an ELISA microplate reader. The percentage of calcium deposition for each experimental group was calculated based on the mean value of the control group at 7 days as 100% of staining.

Gene expression of odontoblastic markers The gene expression of the odontoblastic markers dentine matrix phosphoprotein 1 (DMP-1) and dentine sialophosphoprotein (DSPP) was assessed by real-time PCR, as described above (n = 6). The endogenous control gene used was b-actin. Taqman assays were used for relative quantification of mRNA levels, as follows: DMP-1 – Rn01450122_m1, DSPP – Rn02132391_s1 and b-actin – Rn00667869_m1. The CT values for each sample were normalized by the endogenous control gene. Thereafter, the mean CT value of the control group at 7 days was used to normalize the CT values of both control and experimental groups at all periods.

Statistical analysis To verify the reproducibility of data, two independent experiments were performed for all protocols. Thereafter, data were compiled and subjected to Levene’s test to verify homoscedasticity. Cell viability, H2O2 diffusion, mRNA gene expression, ALP activity and percentages of Alizarin Red staining were subjected to the Kruskal–Wallis and Mann–Whitney U-tests at a significance level of 5%. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used to run the statistical analyses.

Mineralized nodule deposition (MND) For assessment of the quantity of mineralized nodules deposited, the cells were cultured for 7, 14 and 21 days in osteogenic medium after being bleached (n = 6). At each time-point, the cells were washed twice with PBS, fixed with cold 70% ethanol for 1 h, washed one time with deionized water and then stained with Alizarin Red dye (40 mmol L1, pH 4.2; Sigma-Aldrich Corp.) for 20 min, under gentle shaking (VDR Shaker, Biomixer, Ribeir~ ao Preto, SP, Brazil). After aspiration of unincorporated dye, the cells were washed twice with deionized water for the removal of excess stain, and representative photographs from each group were taken by light microscopy (Olympus BX51, Olympus, Miami, FL, USA). The cells were then incubated with 10% cetylpyridinium chloride (Sigma-Aldrich Corp.) for 15 min under agi-


Results Cell viability/H2O2 diffusion Data on H2O2 concentrations in the extracts and percentages of cell viability are shown in Table 1. H2O2 diffusion was observed for all bleached groups and was proportional to the bleaching time. Significant reductions in cell viability relative to the control group were observed for the 45- and 15-min groups (P < 0.05). Cell viability was reduced by 31%, 21.4% and 12.9% for the 45-, 15- and 5-min groups, respectively.

Inflammatory mediator gene expression The highest inflammatory mediator mRNA expression was observed for the 45-min group compared with


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Table 1 Percentage of cell viability and quantification of H2O2 diffusion (lg mL1) for each experimental group

min group was not significantly different when compared with the negative control at all periods of analysis (Table 2; Fig. 3).

Analysis Groups

H2O2 (lg mL1)

% cell viability

Negative control 45 min 15 min 5 min

99.8 69.0 78.6 87.1

(99.1–100.5)a (64.9–69.7)c (71.9–78.8)b (81.9–87.4)ab

Odontoblastic marker gene expression

n.d. 0.71 (0.59–0.81)b 0.25 (0.18–0.27)c 0.10 (0.09–0.15)c

For the negative control group, mRNA expression of DMP-1 and DSPP increased significantly (P < 0.05) over time, reaching a peak at 21 days. The bleached groups showed a similar pattern, with increasing gene expression throughout periods of analysis, reaching a peak at 21 days. For DMP-1, the 45-min group showed gene expression over time similar to that of the negative control at 7 and 14 days; however, this group did not reach a peak at 21 days as in the control group, showing a significant (P < 0.05) difference compared with this group at this time-point. When compared with the negative control, the 15- and 5-min groups were not significantly different at 7 days, a significant (P < 0.05) increase at 14 days and no difference at the 21-day period. For DSPP, the 45-min group was associated with significantly (P < 0.05) lower mRNA expression at all periods of analysis when compared with the control group. The 15-min group was significantly (P < 0.05) different from control only at 21 days, whereas the 5-min group showed no significant difference at all periods of analysis. These data are demonstrated in Fig. 4.

Numbers are medians (25th to 75th percentiles), n = 6. Groups identified by the same letter do not differ significantly (Mann– Whitney, P > 0.05). n.d. = not detected.

the negative control group, which was significant (P < 0.05) for all genes assessed. The 15-min group had significant (P < 0.05) increases in TNF-a, IL-1b and COX-2 expression when compared with the control group. No significant increase in inflammatory mediator mRNA expression was observed for the 5-min group (Fig. 2).

ALP activity/MND The negative control group was associated with peak ALP activity at 7 days, which was significantly decreased at 14 and 21 days. The bleached groups showed significant (P < 0.05) reductions in ALP activity at 7 days compared with negative control; however, all bleached groups reached a peak at 21 days, with significant (P < 0.05) differences relative to negative control and with the 5-min group featuring the highest values (Table 2). MND was significantly (P < 0.05) reduced at 7 and 14 days for the 45-min group and at 7 days for the 15-min group, compared with the negative control; however, these groups reached the same pattern as the negative control at 21 and 14 days, respectively. The 5-

Discussion Biocompatible in-office bleaching therapies have been the focus of several studies aimed at the preservation of the pulp–dentine complex (Soares et al. 2013, 2014a,b). In the present investigation, the cytocompatibility of an experimental low-concentration

Table 2 ALP activity (U mg1) and Alizarin Red staining (%) for the experimental groups at each period of analysis Periods of analysis Groups 1

ALP (U mg )

Alizarin stain (%)

Control 45 min 15 min 5 min Control 45 min 15 min 5 min

7 days 3.28 1.62 2.22 2.57 100.0 51.6 77.2 95.5

14 days a A

(3.17–3.64) (1.62–2.09)b C (2.21–2.34)b CB (2.57–2.81)b AB (100.0–100.1)c A (50.9–51.8)c C (76.3–77.4)c BC (95.4–95.9)c AB

1.15 1.68 2.34 2.28 735.1 142.1 755.3 710.4

21 days b C

(1.03–1.44) (1.62–2.15)b BC (2.33–2.43)b A (2.20–2.55)b AB (706.5–735.8)b A (142.0–148.9)b B (755.1–757.3)b A (710.3–726.3)b A

1.46 3.03 3.46 5.57 1246.3 1163.1 1160.8 1198.4

(1.45–1.88)b C (3.02–3.97)a B (3.38–3.69)a B (5.39–5.97)a A (1212.6–1246.8)a (1161.3–1182.8)a (1145.4–1161.4)a (1195.9–1198.8)a

A B B AB

Numbers are medians (25th–75th percentiles), n = 6. Lower case letters allow for comparisons in rows and upper case letters comparisons in columns. Comparisons are possible for each cellular product. Groups identified by the same letter do not differ significantly (Mann–Whitney, P > 0.05).

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(a)

(b)

(c)

(d)

Figure 2 Box–whisker plot of mRNA gene expression of IL-6 (a), COX-2 (b), TNF-a (c) and IL-1b (d). Vertical axis represents

relative gene expression normalized by the negative control group, and horizontal axis represents the experimental groups. Different letters indicate a significant difference amongst groups (Mann–Whitney, P > 0.05).

bleaching gel, containing 17.5% H2O2, was assessed. In the 45-min group, the bleaching gel was applied three times of 15 min because a previous study showed that repeated short applications of H2O2based gels on enamel are more effective than one prolonged application time (e.g. 1 9 45 min) and cause less intense post-bleaching tooth sensitivity (Reis et al. 2011). The bleaching time of 15 and 5 min was also evaluated to determine the effect of the contact time on the cellular parameters assessed in this in vitro study. Application of this gel to enamel/dentine discs simulating the thickness of human central incisors (3.5 mm) (Harris & Hicks 1998) caused a slight reduction in cell viability, which was directly related to the contact time of gel. This cell viability reduction was significant only when the gel was applied for 45 min (31%) or


15 min (21.4%), associated with over-expression of IL-6, TNF-a, IL-1b and COX-2 mRNA, key inflammatory mediators of pulp tissue (Cooper et al. 2010, 2014, Horst et al. 2011). These results corroborate those found in the literature, which demonstrates a time-dependent bleaching toxicity to pulp cells in vitro (Coldebella et al. 2009, Trindade et al. 2009, Soares et al. 2013, 2014a,b). As shown in the present study, this side effect, which has been correlated to the amount of H2O2 able to diffuse through enamel and dentine to come into contact with pulp cells, seems to be proportional to bleaching time (Soares et al. 2013, 2014a,b). In clinical situations, bleaching gels containing 15–20% of H2O2 in their composition are capable of promoting significant colour change when applied for 45–60 min on tooth surface, reaching a similar


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Figure 3 Panel of mineralized nodule deposition through periods of analysis for MDPC-23 cells. For each group and period, the left image represents a digital photograph of the well, and at right is a light microscopic (209) image from the delimited area of the well. The mineralized nodule is demarcated with asterisks (*). A peak of mineralized nodule deposition was observed at 14 days for the control group and for the 15- and 5-min groups. The 45-min group showed no mineralization nodule deposition at 7 days and a reduced number at 14 days; however, this group reached a pattern similar to that of the negative control at 21 days. The other bleached groups showed mineralized nodules from the 7-day period.

(a)

(b)

Figure 4 Box–whisker plot of mRNA gene expression of DMP-1 (a) and DSPP (b). Vertical axis represents relative gene expression normalized by the negative control group, and horizontal axis represents the experimental groups. Lower case letters allow for comparisons within the groups at each time-point; upper case letters allow for comparisons within the time-points for each group. Different letters indicate a significant difference amongst groups (Mann–Whitney, P > 0.05).

outcome than those observed for the 35%-H2O2 gel throughout the clinical appointments (Martin et al. 2013, Moncada et al. 2013, Reis et al. 2013, Bortolatto et al. 2014, Ozcan et al. 2014). In these studies,

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the tooth sensitivity rate was reduced in around three times when the low-concentrated gels were used in comparison with the high-concentrated ones. Also, the intensity and duration of tooth sensitivity were



reduced by the use of low-concentrated gels (Martin et al. 2013, Moncada et al. 2013, Reis et al. 2013, Bortolatto et al. 2014, Ozcan et al. 2014). Similar effect was also observed by reducing the contact time of the bleaching gel with tooth structure (Cardoso et al. 2011); however, the application of low-concentrated gels for reduced time on enamel interfered with the bleaching effectiveness in such way that additional sessions were needed to achieve significant colour improvement (Soares et al. 2014b). In this way, it seems that these bleaching protocols are interesting alternatives for low-thickness teeth, such as incisors, which features the highest rates of tooth sensitivity and are more easily bleached (de Almeida et al. 2012, Bonafe et al. 2013). The pathway for H2O2 mediation of MDPC-23 cell viability reduction involves the generation of an oxidative stress condition, followed by cell membrane damage and cell death by necrosis (Soares et al. 2014a). This oxidative condition has been correlated with the inflammatory reaction in pulp tissue observed in histological studies after bleaching (Seale et al. 1985, Kina et al. 2010, de Souza Costa et al. 2010, Cintra et al. 2013). As odontoblasts are the first cells to come into contact with toxic components capable of diffusing through dentine, they initiate the immune/inflammatory reaction in pulp tissue to protect underlying cells against noxious signals by expressing inflammatory mediators, such as proinflammatory cytokines and chemokines, promoting regulatory functions for lymphocytes, macrophages and neutrophils (Farges et al. 2009, Cooper et al. 2010, 2014, Horst et al. 2011). This effect was observed in the present investigation, in which odontoblast-like cells expressed mRNA of IL-1b, TNF-a, IL6 and COX-2 after being exposed to bleaching gel components at toxic concentrations. Depending on the amount of immune cell recruitment, intense tissue damage may take place, as defence cells release proteolytic enzymes as metalloproteinases, which break down the extracellular matrix components and cellular contacts (Sato et al. 2013, Fouad & Verma 2014). However, it has been shown that inflammation is essential for pulp tissue healing after it is challenged or damaged, which depends on the period and intensity of tissue damage after injury (Paula-Silva et al. 2009, Fouad & Verma 2014). It has been demonstrated that, after teeth with healthy pulps are exposed to mild injury, the pulp healing process takes place by the activation of reactionary dentinogenesis. This phenomenon results in


deposition of an organic matrix by odontoblasts, followed by its biomineralization (Goldberg & Smith 2004, Schmalz & Smith 2014). Several noncollagenous proteins and enzymes secreted by odontoblasts are directly related to this process. Alkaline phosphatase (ALP) is involved with the initial phase of dentine matrix biomineralization, as it promotes dephosphorylation of extracellular matrix proteins, providing inorganic phosphate (Goldberg et al. 2011). Other proteins, such as DMP-1, DSP (dentine sialoprotein) and DPP (dentine phosphoprotein), are implicated in the nucleation and growth of the mineral phase, being expressed in mature odontoblasts and upregulated during the active mineralization phase (D’Souza et al. 1997, Simon et al. 2009, Goldberg et al. 2011, Schmalz & Smith 2014). DSP and DPP are encoded by the DSPP gene, which is cleaved immediately after secretion (Goldberg et al. 2011). These proteins are rapidly discharged in the odontoblastic process on the mineralization front after the deposition of dentine matrix (Weinstock & Leblond 1974, Couve et al. 2013), having direct participation in calcium binding to previously synthesized collagenous matrix, initiating the formation of hydroxyapatite crystals within collagen fibres and controlling the rate of crystal growth (Goldberg et al. 2011, Couve et al. 2013). It was previously demonstrated that MDPC-23 cells indirectly exposed to the same bleaching protocols as tested in the present investigation were able to overcome oxidative stress and proliferate significantly 72 h thereafter, increasing their viability 1.5, 2.2 and 2.6 times, for the 45-, 15- and 5-min protocols, respectively (Soares et al. 2014a). However, the effect of this initial damage on odontoblast phenotype regulation related to the regenerative process of pulp tissue was no longer demonstrated. In the present investigation, it was demonstrated that the cells exposed to the 17.5% H2O2 gel showed DMP-1 and DSPP mRNA gene expression, ALP activity and MND at all periods of analysis. Therefore, the MDPC-23 cells maintained their odontoblastic phenotype after contact with the components released from the bleaching agent capable of diffusing across enamel and dentine. However, alteration of these odontoblastic marker expression patterns in comparison with those observed in the negative control group occurred for the 45- and 15-min groups. As observed for the cell viability assay, the higher the contact time, the higher the alteration of odontoblastic marker expression. However, over-expression of these markers over


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time took place in all bleached groups, as also observed in the negative control. Nevertheless, the 45-min group still had lower gene expression of DMP1/DSPP at 21 days relative to the control instead of similar MND, and increased ALP activity compared to the controls was observed at this time-point. The more intense alteration in odontoblastic marker expression observed for the 45- and 15-min groups was probably a consequence of the more intense reduction in cell viability associated with the overexpression of IL-1b, TNF-a, IL-6 and COX-2 in the remaining cells. According to Cooper et al. (2010), there is a fine balance between inflammatory mediator dose and pulp tissue regeneration. Alongi et al. (2010) verified that mesenchymal stem cells (MSCs) from teeth diagnosed with irreversible pulpitis presented lower expression of odontoblastic markers (DSPP, ALP and osteocalcin) than normal pulp tissue MSC-derived cells. Also, exposure of MSCs from normal tissue to high doses of TNF-a and IL1-b for 48 h reduced their odontoblastic differentiation capacity and mineralization rate at short-term periods. Conversely, when low doses of TNF-a were applied to human dental pulp cells (HDPCs), notable over-expression of DPP, DSP and DMP-1 was observed after a short treatment time (6 h) (Paula-Silva et al. 2009). Min et al. (2006) reported enhanced ALP activity and dentine matrix noncollagenous protein over-expression after 72-h incubation of pulp cells with low-dose pro-inflammatory cytokines (IL-1a and TNF-a). However, the contact of the cells with these cytokines for 14 days significantly decreased ALP activity and odontoblastic marker expression. Additionally, Spoto et al. (2001) reported increased ALP activity in reversible pulpitis in comparison with healthy or irreversible pulpitis samples. One can conclude that the exposure of pulp cells to low doses of inflammatory mediators may play an initial regulatory role in the pulp regeneration process, whereas high dosage may cause delayed regenerative capability. The contact of pulp cells with reactive oxygen species (ROS) and the intensity of oxidative stress in damaged cells have similar effects. Previous studies showed that low doses of H2O2 applied to MDPC-23 cells or HDPCs resulted in oxidative stress associated with over-expression of odontoblastic markers and increased mineralized nodule deposition in vitro (Lee et al. 2006, Min et al. 2008, Matsui et al. 2009). Matsui et al. (2009) observed that a short treatment (5–10 min) of HDPCs with low amounts of H2O2 (0.9 mmol L1) promoted odontoblastic differentiation

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and increased calcified nodule deposition in vitro. Similarly, a low H2O2 dose (0.2 mmol L1) applied for 24 h to cultured HDPCs strongly induced DSPP mRNA expression (Min et al. 2008). Lee et al. (2006) observed that 0.1–0.3 mmol L1 of H2O2 applied on alternate days for 4 days promoted oxidative stress associated with significantly increased ALP activity and mineralized nodule deposition by MDPC-23 cells. In the present study, when the 17.5%-H2O2 gel was applied for 5 min, the low amount of H2O2 able to diffuse through enamel and dentine (0.1 lg mL1) did not cause significant alterations in cell viability or over-expression of inflammatory mediators mRNA. Also, the highest values of ALP activity occurred at 21 days for this group, and no alteration in DMP-1/ DSPP gene expression and MND relative to control was observed. Nevertheless, high H2O2 doses released from bleaching gels in contact with pulp cells led to intense cell death and impairment of proliferative capability (Coldebella et al. 2009, Trindade et al. 2009, Soares et al. 2013, 2014a). A recent study also demonstrated that exposure of pulp cells to toxic concentrations of H2O2 for long periods (12 days) resulted in intense oxidative stress generation associated with low cell viability and down-expression of antioxidant molecules and odontogenic markers, impairing odontoblastic differentiation (Lee et al. 2013). Based upon the data obtained in the present investigation, it may be speculated that the odontoblastlike cells subjected to the bleaching procedures tested retained their phenotypic characteristics, being able to deposit and mineralize dentine matrix in vitro. The lower the contact time of gel with the enamel/dentine substrate, the lower the cytotoxicity and inflammatory mediator over-expression and the higher the odontoblastic phenotype regulation. However, not only the biocompatibility but also the bleaching effectiveness is crucial to selection of the ideal bleaching protocol to be performed in clinical situations. Soares et al. (2014b) observed that three 15-min applications of 17.5% H2O2 gel to enamel/dentine discs with the same thickness as used in the present study (3.5 mm) achieved the same colour alteration as the traditional protocol after four sessions. Analysis of these data, associated with those of the present investigation, demonstrates that this bleaching protocol may be an interesting alternative to be tested in the clinical situation. Conversely, it seems that a single application of this gel for 15 or 5 min caused a less intense colour alteration over time, and these protocols were



not able to cause the same colour alteration as the traditional protocol, even after six sessions (Soares et al. 2014b). However, these more cytocompatible bleaching protocols should be an interesting alternative for teeth with little enamel and less dentine thickness, such as mandibular incisors, which are more susceptible to damage resulting from traditional therapy, as demonstrated by previous histopathological and clinical studies (de Souza Costa et al. 2010, de Almeida et al. 2012, Bonafe et al. 2013). Nevertheless, the scientific data obtained in this study must be carefully analysed, considering that in in vivo situations, several factors may interfere with H2O2 diffusion into pulp chambers, such as the presence of organic and inorganic components within the dentinal tubules, as well as the exudation of dentinal fluid (Sauro et al. 2007). Also, in the in vivo situation, the presence of extracellular matrix and the antioxidant system may reduce the initial cytotoxicity and increase the regenerative capacity of pulp tissue (Wataha 2012). Therefore, further in vivo studies are needed to evaluate the bleaching effectiveness and tooth sensitivity as well as the response of pulp tissue to different bleaching therapies, taking into account several clinical factors that can influence the results, such as the thickness of enamel and dentine as well as the age of the patients and the degree of dentine darkness.

Conclusion Application of a 17.5% H2O2 bleaching gel for 5 min can be considered cytocompatible with odontoblastlike cells. However, when this gel was applied for 45 or 15 min, a slight cytotoxicity associated with alterations in phenotypic marker expression was observed. These cells were able to recover their functions over time.

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Vital bleaching: a new light-activated hydrogen peroxide system.

Evaluation of Extraradicular Diffusion of Hydrogen Peroxide during Intracoronal Bleaching Using Different Bleaching Agents.

In-office bleaching gel with 35% hydrogen peroxide enhanced biofilm formation of early colonizing streptococci on human enamel.

Tooth bleaching with hydrogen peroxide and nano-hydroxyapatite: a 9-month follow-up randomized clinical trial.

Penetration of hydrogen peroxide and degradation rate of different bleaching products.

In vitro determination and quantification of 30% hydrogen peroxide penetration through dentin and cementum during bleaching.

Bleaching effect of activation of hydrogen peroxide using photon-initiated photoacoustic streaming technique.

Hydrogen peroxide signaling in vascular endothelial cells.

Bovine aortic endothelial cells release hydrogen peroxide.

Bactericidal properties of a titanium-peroxy gel obtained from metallic titanium and hydrogen peroxide.

Hydrogen peroxide formation by cells treated with a tumor promoter.

Effect of a New Bleaching Gel on Tooth Whitening.

The effect of the teeth bleaching with 35% hydrogen peroxide on the tensile bond strength of metal brackets.

Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

Regulation of hydrogen peroxide generation in cultured endothelial cells.

Method for detection of hydrogen peroxide in HT22 cells.

Cytolysis of corneal epithelial cells by hydrogen peroxide.

Effects of the concentration and composition of in-office bleaching gels on hydrogen peroxide penetration into the pulp chamber.

Penetration of the pulp chamber by carbamide peroxide bleaching agents.

Increased hydrogen peroxide concentration in human tumor cells due to a nitroxide free radical.

In vitro effects of hydrogen peroxide combined with different activators for the in-office bleaching technique on enamel.

Nox4: a hydrogen peroxide-generating oxygen sensor.

Effects on gastric mucosa induced by dental bleaching--an experimental study with 6% hydrogen peroxide in rats.

hydrogen peroxide--mediated activation of phenol and binding to protein.