- Email: [email protected]
Responses of human dental pulp cells after application of a low-concentration bleaching gel to enamel
archives of oral biology 60 (2015) 1428–1436
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Responses of human dental pulp cells after application of a low-concentration bleaching gel to enamel Diana G. Soares a, Fernanda G. Basso b, De´bora Salles Scheffel b, Josimeri Hebling b, Carlos A. de Souza Costa a,* a
Department of Physiology and Pathology, Univ. Estadual Paulista – UNESP, Araraquara School of Dentistry, Humaita´ Street 1680, Araraquara, SP, Brazil b Department of Orthodontics and Pediatric Dentistry, Univ. Estadual Paulista – UNESP, Araraquara School of Dentistry, Humaita´ Street 1680, Araraquara, SP, Brazil
article info
abstract
Article history:
Objective: To evaluate the effect of a 17.5% H2O2 gel on the odontoblastic differentiation
Accepted 14 June 2015
capability of human dental pulp cells (HDPCs). Design: The bleaching gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to
Keywords:
transwells, positioned over previously cultured HDPCs. In the control group, no treatment
Odontoblasts
was performed on the discs. Immediately after samples were bleached, the cell viability
Dental pulp
(MTT assay) and death (Live/Dead assay) as well as the mRNA gene expression of inflam-
Tooth bleaching
matory mediators (TNFa, IL-1b, IL-6, and COX-2; real-time PCR) were evaluated. The mRNA
Hydrogen peroxide
gene expression of odontoblastic markers (DMP-1, DSPP, and ALP) and mineralized nodule
Differentiation
deposition (alizarin red) were assessed at 7, 14 and 21 days post-bleaching. The amount of H2O2 in contact with cells was quantified. Data were evaluated by Kruskal–Wallis and Mann– Whitney tests (a = 5%). Results: Significant cell viability reduction and cell death were observed for bleached groups relative to control in a time-dependent fashion. Also, significant overexpression of all inflammatory mediators tested occurred in the 45- and 15-min groups. In the bleached groups, the expression of ALP, DMP-1, and DSPP and the deposition of mineralized nodules were reduced in comparison with those in the control group, at the initial periods (7 and 14 days). However, the 15- and 5-min groups reached values similar to those in the control group at the 21-day period. Conclusions: The 17.5% H2O2 gel was cytotoxic to pulp cells; however, cells subjected to short-term bleaching are capable of expressing the odontoblastic phenotype over time. # 2015 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Physiology and Pathology, Univ. Estadual Paulista – UNESP, Araraquara School of Dentistry, Humaita´ Street 1680, Araraquara, SP 14801-903, Brazil. Tel.: +55 1633016477. E-mail address: [email protected] (C.A. de Souza Costa). http://dx.doi.org/10.1016/j.archoralbio.2015.06.014 0003–9969/# 2015 Elsevier Ltd. All rights reserved.
archives of oral biology 60 (2015) 1428–1436
1.
Introduction
It is well known that tooth-bleaching therapy can promote odontoblast death due to oxidative damage mediated by hydrogen peroxide and its by-products, capable of diffusing through enamel and dentine at toxic concentrations.1,2 These cells are essential for the homeostasis of the dentine-pulp complex, since they are responsible for the deposition and mineralization of dentine matrix throughout the life of the tooth, as well as for the orchestration of the inflammatory and immune responses of pulp tissue, since the noxious signal acts on the tooth surface.3,4 Odontoblasts are highly differentiated terminal cells. Therefore, when they are lost, reparative dentinogenesis takes place, which involves mesenchymal stem cell (MSC) recruitment, proliferation, and differentiation into odontoblast-like cells, resulting in the deposition of mineralized matrix and restoration of the homeostasis of pulp tissue.5–7 It was previously demonstrated that very low concentrations of H2O2 induce odontoblastic differentiation and mineralized matrix deposition8,9; conversely, toxic concentrations of H2O2 enhance inflammatory mediator expression associated with the impairment of odontoblastic differentiation capability.10–14 Recently, it was demonstrated that application of a lowconcentration H2O2 gel (17.5%) from 5 to 45 min onto dental structure was able to reduce in about 11.3–4.5 times, respectively, the indirect toxicity of in-office tooth-bleaching to human dental pulp cells (HDPCs), when compared with traditional therapy (35% H2O2; 3 15 min). The cells were able to proliferate significantly 3 days after samples were bleached, demonstrating that pulp cells were able to overcome the initial oxidative stress.2 However, the effect of this initial H2O2 toxicity on the odontoblastic differentiation capability of HDPCs as well as mineralized matrix deposition was not addressed. Therefore, the present study investigated the effect of a 17.5% H2O2 gel, applied to enamel surfaces for different periods, on the immediate viability of HDPCs and late odontoblastic differentiation and mineralized matrix deposition capability.
2.
Materials and methods
2.1.
Cell culture
The human dental pulp cell (HDPC) primary culture was obtained by enzymatic digestion of pulp tissue from one freshly extracted third molar, obtained from one donor (24 years of age) (Proc. n8 13/11; Ethics Committee of Araraquara School of Dentistry, SP, Brazil) after sign the informed consent according to the code of ethics of the world medical association (Declaration of Helsinki). The pulp tissue was removed aseptically, cut into small pieces (1 mm), and then incubated for 24 h in complete Dulbecco’s Modified Eagle Medium (DMEM; supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, 2 mmol/L glutamine; Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; Gibco) and 200 units/mL of type II collagenase (Worthington
1429
Biochemical Corporation, Lakewood, NJ, USA). After this period, the released cells were subcultured in complete DMEM with 10% FBS. Cells from the 4th to 6th passages were used.
2.2.
Enamel/dentine specimens
Bovine incisors, from 24- to 30-month-old bullocks, were cross-sectioned in its buccal surface with a diamond trephine bur (Dinser brocas diamantadas LTDA, Sa˜o Paulo, SP, Brazil) coupled to bench drilling machine (FSB 16 Pratika; Schultz, Joinville, SC, Brazil) to obtain 5.6 mm-diameter discs containing both enamel and dentine. To obtain the standardized thicknesses similar to those of central incisors (3.5 mm),15 we polished the dentine surfaces with 400- and 600-grit abrasive papers, followed by EDTA 0.5 N treatment for 30 s for smear layer removal. The enamel surfaces of the discs were cleaned with a solution of pumice stone and water by means of a low-speed handpiece. The discs were adapted to acrylic transwells (polycarbonate membrane/8 mm pore size; Corning Inc., Corning, NY, USA) by means of a fluid light-cured resin (TopDam, FGM, Joinville, SC, Brazil), which promoted a lateral seal, and the set was then sterilized with ethylene oxide.
2.3.
Experimental design
The HDPCs were seeded into wells of 24-well plates (6 104 cells/well) for 24 h in DMEM supplemented with 10% FBS, reaching 80% confluence. The culture medium was then replaced by 300 mL of DMEM with no FBS, and the disc/ transwell set was positioned onto the previously cultured cells, in such a way that only the dentine was in direct contact with the culture medium, and the enamel surface remained exposed to receive the bleaching procedure. The bleaching gel containing 17.5% of H2O2 was obtained by dilution of a 35% H2O2 gel (Whiteness HP; FGM) in distilled water immediately before the bleaching procedure (1:1).2 The gel (30 mg) was applied to the enamel surface for different periods according to the experimental groups: 45-min group – 3 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. Immediately after the bleaching treatment, the transwell/disc set was removed, the culture medium in contact with the cells was collected, and the cells were washed with 1 mL of phosphate-buffered saline (PBS) solution.
2.4.
Trans-enamel and trans-dentinal cytotoxicity
2.4.1.
Cell viability analysis (MTT assay)
The cells were incubated for 4 h at 37 8C and 5% CO2 with the MTT solution (Sigma–Aldrich Corp., St. Louis, MO, USA) diluted in DMEM (1:10). The formazan crystals formed in viable cells were then dissolved in acidified isopropanol, and the absorbance of the resulting solution was read at 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).
1430
2.4.2.
archives of oral biology 60 (2015) 1428–1436
Cell death analysis (Live/Dead assay)
For this assay, the cells were seeded onto glass slides (13 mm diameter) positioned at the bottoms of the 24-well plates. Immediately after the bleaching procedure, the cells were incubated for 45 min with the fluorescence probes Ethyl homodimer-1 (Eth-1; 4 mM; Invitrogen, San Francisco, CA, USA), which binds to DNA bands only in cells experiencing cell membrane rupture (undergoing necrosis), and Calcein AM (CA; 1 mM; Invitrogen), which is hydrolyzed by cytoplasmic esterases in viable cells. The glass slides were evaluated by fluorescence microscopy (20; Leica DM 5500B, Nussloch GmbH, Nussloch, Germany), and 6 photographs were taken for each sample. The numbers of viable and dead cells were counted by means of Image J software, and the percentage of dead cells from the total number of cells was calculated. The average value of dead cells per well was used for statistical analysis (n = 6).
2.4.3. PCR)
Gene expression of inflammatory mediators (real-time
The gene expressions of tumour necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), interleukin 6 (IL-6), and cyclooxygenase 2 (COX-2) were performed 6 h after the bleaching procedure (the cells were incubated in fresh DMEM with no FBS) by the real-time PCR technique (n = 6). This period (6 h after bleaching) was used based on the fact that the highest values of mRNA expression for all inflammatory mediators assessed in this in vitro study were previously observed after HDPCs exposition to H2O2 (data not shown) at a concentration capable to cause alteration on the odontoblastic differentiation capability.16 Total RNA was extracted with an RNAqueous1micro kit (Ambion1, Austin, TX, USA), and 1 mg was then reverse-transcribed into single-stranded cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), in accordance with the manufacturer’s protocols. Taqman Assays (Invitrogen) for human genes were used, as follows: IL-1b, Hs01555410_m1; IL-6, Hs00985639_m1; TNF-a, Hs00174128_m1; COX-2, Hs00153133_m1; and b-actin, 4333762T. The CT values for each sample were normalized by the endogenous control gene b-actin. Thereafter, the mean CT value of the control group was used to normalize the CT values of both control and experimental groups.
2.5.
Odontoblastic differentiation capability
For this analysis, immediately after being washed in PBS solution, the cells were incubated in osteogenic medium (DMEM plus 10% SFB, supplemented with 10 nmol/L b-glycerophosphate and 50 mg/mL sodium ascorbate; Sigma–Aldrich Corp.) for 7, 14 and 21 days, with medium being changed daily. The expression of odontoblastic markers and the deposition of mineralized matrix were then assessed at each time-point.
2.5.1. PCR)
gene expression of odontoblastic markers (real-time
The gene expression of dentine matrix phosphoprotein 1 (DMP-1), dentine sialophosphoprotein (DSPP), and alkaline phosphatase (ALP) was assessed by real-time PCR, as described above (n = 6). Taqman assays (Invitrogen) were used for relative quantification of mRNA levels, as follows: DMP-1,
Hs01009391_g1; DSPP, Hs00171962_m1; ALP, Hs01029144_m1; and b-actin, 4333762T. The CT values for each sample were normalized by the endogenous control gene b-actin. 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.
2.5.2.
Deposition of mineralized matrix (Alizarin red staining)
At each time-point, the cells were fixed with cold 70% ethanol, washed with deionized water, and then stained with alizarin red dye (40 mM, pH 4.2; Sigma–Aldrich Corp.) under gentle shaking (VDR Shaker, Biomixer, Ribeira˜o Preto, SP, Brazil). The cells were than 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.), and the absorbance of the resulting solution was measured at 570 nm (ELISA microplate reader). The percentage of alizarin red staining, which represents the calcium deposition, for each experimental group was calculated based on the mean value of the control group at 7 days as representing 100% of staining.
2.6.
Quantification of H2O2 diffusion
Immediately after the bleaching procedure, one aliquot of DMEM in direct contact with cells was transferred to tubes containing acetate buffer solution (2 mol/L, pH 4.5). This solution was then transferred to tubes containing leucocrystal violet (0.5 mg/mL; Sigma–Aldrich Corp.) and horseradish peroxidase enzyme (1 mg/mL; Sigma–Aldrich Corp.), and the final volume of reaction was adjusted to 3 mL with distilled water. The absorbance of the resulting solution was read 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 mg/mL of H2O2, and the data were related to mg/mL of culture medium.
2.7.
Statistical analysis
Two independent experiments were performed for all protocols in this study. Data were compiled and subjected to Levene’s test to verify homoscedasticity. Cell viability, cell death, H2O2 diffusion, mRNA gene expression, and percentages of alizarin red staining were subjected to the Kruskal– Wallis and Mann–Whitney tests at a significance level of 5%. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used to run the statistical analyses.
3.
Results
3.1.
Cytotoxicity/H2O2 diffusion
Data on cell viability, cell death, and H2O2 quantification are given in Table 1. With the control group considered as presenting 100% of cell viability, reductions of around 80.1%, 74.6%, and 63.6% of HDPC viability were observed for the 45-, 15-, and 5-min groups, respectively. Similar results were
1431
archives of oral biology 60 (2015) 1428–1436
Table 1 – Data on cell viability, cell death, and H2O2 diffusion for the control and experimental groups. Group Control 45-min 15-min 5-min
% Cell viability (MTT assay)
% Cell death (Eth-1-positive cells)
a
100 (90.8–105.3) 19.9 (11.1–20.1) c 25.4 (13.3–28.9) bc 36.4 (33.1–37.9) b
2.9 64.9 58.1 34.5
d
(2.3–3.1) (63.1–67.8) ab (53.4–67.7) b (33.8–45.3) c
H2O2 diffusion (mg/mL) n.d.* 0.80 (0.59–0.85) b 0.25 (0.20–0.31) c 0.11 (0.09–0.15) c
Values are medians (from 25th to 75th percentiles), n = 6. In each column, groups identified with the same letter do not differ statistically (Mann–Whitney, p > 0.05). * not detected.
observed for the cell death assay, in which 64.9%, 58.1%, and 34.5% of Eth-1-positive cells were observed for the 45-, 15-, and 5-min groups, respectively. All of these values were significantly different from those observed in the control group. The 5-min group featured the lowest values of cell viability reduction and cell death, being significantly different from the other bleached groups. Representative images of live (CApositive) and dead cells (Eth-1-positive) are demonstrated in Fig. 1. The H2O2 diffusion values also followed the timedependent pattern, with the 45-min group featuring the highest values, which were significantly different from those of the other bleached groups.
3.2.
Inflammatory mediator gene expression
The results for inflammatory mediator mRNA expression are presented in Fig. 2. IL-6, TNF-a, COX-2, and IL-1b mRNA expression was significantly enhanced for the 45- and 15-min groups compared with the control. These groups showed a similar behaviour for inflammatory mediators’ gene expression, and no significant difference among them was observed
for the IL-6, COX-2, and IL-1b mRNA expression. A discrete increase in the expression of these genes was observed for the 5-min group; however, a significant difference from the control was observed only for IL-6.
3.3.
Odontoblastic marker gene expression
The mRNA gene expression of odontoblastic markers is presented in Fig. 3. The gene expression of ALP in the control group presented a peak at 14 days. This peak was not observed in bleached groups, which resulted in a significant difference from the control at this time-point; however, the 15- and 5min groups featured significantly higher mRNA ALP expression at 21 days relative to control. The mRNA gene expression of DMP-1 was significantly reduced at 7, 14 and 21 days for all bleached groups compared with control, except for the 5-min group at 7 days. Regarding DSPP, the 45-min group showed the lowest values of mRNA expression, with a significant difference relative to control at all time-points; conversely, the 15and 5-min groups featured no significant difference from the control at 21 days.
Fig. 1 – Representative images of live (green) and dead (red) cells for each group, obtained by fluorescence microscopy (20T). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
1432
archives of oral biology 60 (2015) 1428–1436
Fig. 2 – Box-Whisker plot of inflammatory mediator mRNA expression. Vertical axis represents relative gene expression normalized by the negative control group, and horizontal axis represents the experimental groups. Different letters indicate a statistically significant difference among groups (n = 6) (Mann–Whitney, p > 0.05).
3.4.
Mineralized matrix deposition
The mineralized matrix deposition in bleached groups was reduced, relative to control, at the 7- and 14-day periods, being more intense for the 45-min group; however, at the 21-day period, only the 45-min group presented a significantly lower percentage than the control (Table 2). Fig. 4 reveals a remarkable increase in mineralized matrix deposition in the control and 5-min groups at 21 days. The 45-min group presented no mineralized nodule deposition at any period of analysis.
4.
Discussion
In the present investigation, it was demonstrated that the 17.5% H2O2 bleaching gel was cytotoxic to the HDPCs since the cell viability reduction, as well as the cell death percentage,
was above 30%.17 However, the cytotoxicity was dependent on the contact time of the gel with the enamel surface. Soares et al.2 demonstrated that pulp cells exposed to these experimental bleaching protocols were under oxidative stress, in which the intensity was proportional to the contact time of the gel with enamel; however, the cells were able to proliferate significantly over time, recovering their viability at around 3–4 times three days after the bleaching procedure. In the present study, cell viability reduction occurred partly as a consequence of cell death by necrosis, since the cells were positively stained for the Eth-1 probe, which binds to the DNA of only cells with disrupted membranes. The percentage of positive Eth-1 cells was also proportional to the treatment time and the amount of H2O2 capable of diffusing through enamel and dentine to reach the underlying cells. This demonstrates that the higher the H2O2 concentration in contact with pulp cells, the higher the cell viability reduction mediated by necrosis. Wu et al.18 demonstrated that, at toxic concentrations, H2O2
1433
archives of oral biology 60 (2015) 1428–1436
Fig. 3 – Box-Whisker plot of odontoblastic marker mRNA expression. 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 timepoints for each group. Different letters indicate a statistically significant difference among groups (n = 6) (Mann–Whitney, p > 0.05).
Table 2 – Alizarin red staining percentage for HDPCs. Groups
Periods of analysis 7 days
Alizarin stain (%)
Control 45-min 15-min 5-min
100.4 9.7 40.7 57.6
(100.0–100.7) cA (9.1–11.2) bC (37.9–44.5) cB (54.9–62.7) bB
14 days 392.5 54.5 77.3 120.5
(382.7–414.6) bA (51.0–57.1) aC (70.8–87.2) bC (104.3–133.2) bB
21 days 557.1 71.0 327.7 570.9
(557.0–557.9) aA (70.4–71.1) aB (317.7–512.2) aA (554.5–574.9) aA
Numbers are medians (from 25th to 75th percentiles), n = 6. Lowercase letters allow for comparisons in rows and uppercase letters comparisons in columns. Groups identified by the same letter do not differ statistically significantly (Mann–Whitney, p > 0.05).
Fig. 4 – Panel of mineralized nodule deposition through periods of analysis. For each group and period, the left image represents a digital photograph of the well, and at right is a light-microscopic (20T) image from a delimited area of the well. Absence of nodule deposition was observed in all groups at 7 days. Some nodules were observed in the control group and the 15- and 5-min groups at 14 days. At the 21-day period, the control and 5-min groups presented an intense deposition of mineralized nodules, and increased numbers of nodules were observed in the 15-min group. Absence of nodule deposition was observed for the 45-min group at all periods of analysis.
1434
archives of oral biology 60 (2015) 1428–1436
causes cell viability reduction by necrosis and apoptosis, with higher concentrations leading to a higher percentage of necrotic cells. This specific type of cell death causes intense damage in vivo, since high quantities of intracellular components are released, including lysosomal enzymes, causing damage to the neighbouring cells and triggering an inflammatory tissue reaction.19,20 Pulpal inflammation associated with local tissue necrosis was previously demonstrated by in vivo studies in which high-concentration H2O2 bleaching gels (35–38%) were applied to rat or human teeth.20–23 In the present investigation, the remaining HDPCs showed increased expression of IL-6, TNF-a, COX-2, and IL-1b in comparison to the control group. Key and well-characterized cytokines, including IL-1a, IL-1b, TNF-a, IL-4, IL-6, IL-8, and IL-10, are highly expressed by structural cells from pulp tissue, such as odontoblasts, fibroblasts and mesenchymal stem cells, along with immune cells, when they are exposed to bacterial and dental materials components.24–28 These molecules play regulatory functions for lymphocytes, macrophages, and neutrophils, activating the inflammatory cascade,29,30 which may result in release of proteolytic enzymes, such as metalloproteinases, with the consequent disruption of extracellular matrix components.31,32 Prostaglandin E2, synthesized by COX-2 gene is also a major pro-inflammatory mediator, which is known to accelerate inflammation and injury in several tissues, including the dental pulp.33–35 The inflammatory mediators IL-6, TNF-a, COX-2, and IL-1b were assessed in the present investigation since they are strongly expressed in inflamed pulp tissue.24,28,34,35 Also, previous studies demonstrated that depending on the concentration of these inflammatory mediators in the pulp they may play either regenerative or degenerative tissue effects.10–13,18,28 Therefore, these mediators have been used as inflammatory markers by in vitro studies performed with pulp cells such as this one.25–27,33 The effect of the immediate cytotoxicity of the 17.5% H2O2 gel on the odontoblastic differentiation capability of HDPCs was also evaluated in this study. It was observed that the cells exposed to the bleaching protocols showed mRNA expression of DSPP, DMP-1, and ALP at all periods of analysis. However, alteration in the gene expression pattern relative to the control group was observed, mainly at the initial periods. Interestingly, the cells subjected to the less aggressive protocols (5 and 15 min of bleaching) were able to increase gene expressions over time, reaching a pattern similar to that of the control group. The proteins encoded by these genes are directly related to the odontoblastic differentiation process, by providing inorganic phosphate, binding calcium to the collagenous matrix and promoting the nucleation and growth of the mineral phase.4,36 Regarding the mineralized matrix deposition, instead of the reduction in the percentage of alizarin red staining that was observed at 7 and 14 days for all bleached groups, an increase in this percentage over time was observed, with the 15- and 5-min groups presenting no significant difference from control at 21 days. Therefore, one may conclude that cells of these groups were able to overcome the initial oxidative damage and proliferate and differentiate into cells with an odontoblastic phenotype able to deposit mineralized matrix in vitro.
Some molecules are directly related to the cytoprotection of pulp cells against oxidative stress mediated by H2O2. Min et al.8 demonstrated that overexpression of the heat-shock protein heme oxygenase-1 (HO-1) in HDPCs significantly improved their viability against H2O2-induced cytotoxicity. The authors also observed that the overexpression of DSPP on H2O2-treated cells was via an OH-1-dependent pathway. Another molecule related to the cytoprotection of HDPCs against H2O2 was recently described, the peroxisome proliferator-activated receptor gamma (PPARg), a transcriptional factor belonging to the ligand-activated nuclear receptor superfamily.14 This molecule plays an important role in LPS-mediated pulp inflammation, by removing ROS from cells and tissues.37,38 PPARg overexpressed HDPCs increased their viability after exposure to toxic concentrations of H2O2, associated with increased activity of the anti-oxidant enzymes Cu/ZnSOD and MnSOD, and up-regulation of DSPP and DMP-1 proteins.14 Therefore, both OH-1 and PPARg plays a protective role in the induction of dentinogenesis under oxidative stress in human pulp cells. The 45-min group featured the most intense alteration in the odontoblastic differentiation ability of HDPCs. The remaining cells were unable to deposit mineralized matrix in all periods of analysis, which may have been caused, at least in part, by the intense alterations in DSPP and DMP-1 mRNA expression. According to Ye et al.,39 down-regulation of DSPP and DMP-1 leads to failure of the maturation of predentin into dentine, with reduced dentine in postnatal tooth development. This fact may also be a consequence of the more intense expression of the inflammatory mediators observed for this group. According to Cooper et al.,19 there is a fine balance between inflammatory mediator dosage/contact time and odontoblastic differentiation, as demonstrated by Paula-Silva et al.,12 who applied low-dose TNF-a to HDPCs and observed notable overexpression of DPP, DSP, and DMP-1 after a short treatment time (6 h). Min et al.11 observed increased ALP activity and dentine matrix non-collagenous protein overexpression after 72-hour cultivation of HDPCs with proinflammatory 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.10 showed increased ALP activity in reversible pulpitis in comparison with healthy or irreversible pulpitis samples. Analysis of these data corroborates those reported by Alongi et al.,13 who verified that MSCs from teeth diagnosed with irreversible pulpitis presented lower expression of odontoblastic markers (DSPP, ALP, and osteocalcin) than did normal pulp tissue MSC-derived cells. A second explanation for the more intense alteration of the odontoblastic differentiation capability observed for the 45min group may be related to the intensity of oxidative stress on remaining cells. Soares et al.2 showed that the oxidative stress on HDPCs exposed to the 17.5% H2O2 for 45 min was significantly higher in comparison with that for the 15- and 5min treatment times. Lee et al.14 demonstrated that HPDCs under intense oxidative stress by long-term exposure to H2O2 showed down-regulation of DSPP and DMP-1 mRNA associated with impaired mineralized matrix deposition in vitro. Conversely, the treatment of HDPCs with non-toxic concentrations of H2O2 enhanced ALP activity, up-regulated DSPP,
archives of oral biology 60 (2015) 1428–1436
OPN (osteopontin), and OCN (osteocalcin) expression, and increased calcified nodule deposition in vitro.1,8,9 Therefore, the lower the concentration of H2O2 in contact with pulp cells after being bleached, the higher the regenerative capability of human pulp tissue to overcome the oxidative damage. One can conclude that low-concentration bleaching gels applied for reduced contact times to dental structure may be an interesting alternative for low-thickness teeth, i.e., human incisors, which are more susceptible to pulp-dentine complex oxidative damage, since they allow greater H2O2 diffusion into the pulp chamber.21–24 Previous studies demonstrated that bleaching gels with 15–17.5% H2O2 caused significant tooth colour improvement by increasing the number of sessions.40,41 Nevertheless, the data obtained in the present in vitro study should be considered with caution, because the toxicity observed in laboratory tests (in vitro studies) may be minimized in clinical conditions, since, in vital teeth, there is outward dentinal fluid movement as well as the presence of collagen and odontoblast cytoplasmic processes inside dentinal tubules. All these factors interfere with the inward displacement of toxic components released from dental products, such as bleaching agents.36 In addition, the presence of extracellular matrix, anti-oxidant, and lymphatic systems in pulp tissue protects pulp cells against external aggression.42–44
5.
Conclusions
According to the methodology carried out in this in vitro study, one may conclude that bleaching gel with 17.5% H2O2 applied to enamel for 15 or 5 min promotes cytotoxicity to pulp cells associated with the overexpression of inflammatory mediators; however, the surviving cells were able to overcome the initial aggression and differentiate into cells with an odontoblastic phenotype able to deposit mineralized matrix.
Funding This study was supported by the Sa˜o Paulo Research Foundation – FAPESP (grants # 2013/23520-0 and 2013/058790) and by the National Council of Technological and Scientific Development – CNPq (grant # 301029/2010-1).
Competing interests The authors have no conflict of interest.
Ethical approval Proc. n8 13/11; Ethics Committee of Araraquara School of Dentistry, SP, Brazil.
Acknowledgments This study was supported by the Sa˜o Paulo Research Foundation–FAPESP (grants # 2013/23520-0 and 2013/05879-0) and by
1435
the National Council of Technological and Scientific Development–CNPq (grant # 303599/2014-6). The authors deny any conflicts of interest related to this study within three years of beginning the work.
references
1. Soares DG, Ribeiro AP, da Silveira Vargas F, Hebling J, de Souza Costa CA. Efficacy and cytotoxicity of a bleaching gel after short application times on dental enamel. Clin Oral Investig 2013;17(8):1901–9. 2. Soares DG, Basso FG, Hebling J, de Souza Costa CA. Concentrations of and application protocols for hydrogen peroxide bleaching gels: effects on pulp cell viability and whitening efficacy. J Dent 2014;42(2):185–98. 3. Couve E, Osorio R, Schmachtenberg O. The amazing odontoblast: activity, autophagy, and aging. J Dent Res 2013;92(9):765–72. 4. Schmalz G, Smith AJ. Pulp development, repair, and regeneration: challenges of the transition from traditional dentistry to biologically based therapies. J Endod 2014;40(Suppl 4):S2–5. 5. Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, et al. Comparative analysis of in vitro osteo/ odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Arch Oral Biol 2011;56(7):709–21. 6. Morad G, Kheiri L, Khojasteh A. Dental pulp stem cells for in vivo bone regeneration: a systematic review of literature. Arch Oral Biol 2013;58(12):1818–27. 7. Dimitrova-Nakov S, Baudry A, Harichane Y, Kellermann O, Goldberg M. Pulp stem cells: implication in reparative dentin formation. J Endod 2014;40(Suppl 4):S13–8. 8. Min KS, Lee HJ, Kim SH, Lee SK, Kim HR, Pae HO, et al. Hydrogen peroxide induces heme oxygenase-1 and dentin sialophosphoprotein mRNA in human pulp cells. J Endod 2008;34(8):983–9. 9. Matsui S, Takahashi C, Tsujimoto Y, Matsushima K. Stimulatory effects of low-concentration reactive oxygen species on calcification ability of human dental pulp cells. J Endod 2009;35(1):67–72. 10. Spoto G, Fioroni M, Rubini C, Tripodi D, Di Stilio M, Piattelli A. Alkaline phosphatase activity in normal and inflamed dental pulps. J Endod 2001;27(3):180–2. 11. Min KS, Kwon YY, Lee HJ, Lee SK, Kang KH, Lee SK, et al. Effects of proinflammatory cytokines on the expression of mineralization markers and heme oxygenase-1 in human pulp cells. J Endod 2006;32(1):39–43. 12. Paula-Silva FW, Ghosh A, Silva LA, Kapila YL. TNF-alpha promotes an odontoblastic phenotype in dental pulp cells. J Dent Res 2009;88(4):339–44. 13. Alongi DJ, Yamaza T, Song Y, Fouad AF, Romberg EE, Shi S, et al. Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential. Reg Med 2010;5(4):617–31. 14. Lee YH, Kang YM, Heo MJ, Kim GE, Bhattarai G, Lee NH, et al. The survival role of peroxisome proliferator-activated receptor gamma induces odontoblast differentiation against oxidative stress in human dental pulp cells. J Endod 2013;39(2):236–41. 15. Harris EF, Hicks JD. A radiographic assessment of enamel thickness in human maxillary incisors. Arch Oral Biol 1998;43(10):825–31. 16. Soares DG, Basso FG, Hebling J, de Souza Costa CA. Effect of hydrogen-peroxide-mediated oxidative stress on human dental pulp cells. J Dent 2014. http://dx.doi.org/10.1016/ j.jdent.2014.12.006. [Epub ahead of print].
1436
archives of oral biology 60 (2015) 1428–1436
17. ISO 10993-5:2009(E). Biological evaluation of medical devices – part 5: tests for cytotoxicity in vitro. Geneva, Switzerland. 2009. 18. Wu TT, Li LF, Du R, Jiang L, Zhu YQ. Hydrogen peroxide induces apoptosis in human dental pulp cells via caspase-9 dependent pathway. J Endod 2013;39(9):1151–5. 19. Cooper PR, Holder MJ, Smith AJ. Inflammation and regeneration in the dentin-pulp complex: a double-edged sword. J Endod 2014;40(Suppl 4):S46–51. 20. Fouad AF, Verma P. Healing after regenerative procedures with and without pulpal infection. J Endod 2014;40(Suppl 4):S58–64. 21. de Souza Costa CA, Riehl H, Kina JF, Sacono NT, Hebling J. Human pulp responses to in-office tooth bleaching. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109(4):59–64. 22. Cintra LT, Benetti F, da Silva Facundo AC, Ferreira LL, Gomes-Filho JE, Ervolino E, et al. The number of bleaching sessions influences pulp tissue damage in rat teeth. J Endod 2014;39(12):1576–80. 23. Ferreira VG, Nabeshima CK, Marques MM, Paris AF, Gioso MA, dos Reis RS, et al. Tooth bleaching induces changes in the vascular permeability of rat incisor pulps. Am J Dent 2013;26(5):298–300. 24. Roderjan DA, Stanislawczuk R, Hebling J, de Souza Costa CA, Soares DG, Reis A, et al. Histopathological features of dental pulp tissue from bleached mandibular incisors. J Mater Sci Eng B 2014;4(6):178–85. 25. Horst OV, Horst JA, Samudrala R, Dale BA. Caries induced cytokine network in the odontoblast layer of human teeth. BMC Immunol 2011;12:9. 26. Kim RH, Williams DW, Bae S, Lee RS, Oh JE, Mehrazarin S, et al. Camphorquinone inhibits odontogenic differentiation of dental pulp cells and triggers release of inflammatory cytokines. J Endod 2013;39(1):57–61. 27. Asgary S, Nazarian H, Khojasteh A, Shokouhinejad N. Gene expression and cytokine release during odontogenic differentiation of human dental pulp stem cells induced by 2 endodontic biomaterials. J Endod 2014;40(3):387–92. 28. Diomede F, Caputi S, Merciaro I, Frisone S, D’Arcangelo C, Piattelli A, et al. Pro-inflammatory cytokine release and cell growth inhibition in primary human oral cells after exposure to endodontic sealer. Int Endod J 2014;47(9):864–72. 29. Yazid FB, Gnanasegaran N, Kunasekaran W, Govindasamy V, Musa S. Comparison of immunodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth. Clin Oral Investig 2014;18(9):2103–12. 30. Durutu¨rk L, Sarˇˇi S, Sengu¨l A. Immunocompetent cell level as a diagnostic reference for pulpal pathosis of primary teeth. Arch Oral Biol 2013;58(10):1517–22. 31. Cooper PR, Takahashi Y, Graham LW, Simon S, Imazato S, Smith AJ. Inflammation-regeneration interplay in the dentine-pulp complex. J Dent 2010;38(9):687–97.
32. Sato C, Rodrigues FA, Garcia DM, Vidal CM, Pashley DH, Tja¨derhane L, et al. Tooth bleaching increases dentinal protease activity. J Dent Res 2013;92(2):87–92. 33. Sousa NG, Cardoso CR, Silva JS, Kuga MC, Tanomaru-Filho M, Faria G. Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development. Arch Oral Biol 2014;59(9):944–53. 34. Jeong GS, Lee DS, Li B, Lee HJ, Kim EC, Kim YC. Effects of sappanchalcone on the cytoprotection and antiinflammation via heme oxygenase-1 in human pulp and periodontal ligament cells. Eur J Pharmacol 2010;644(1– 3):230–7. 35. Ohkura N, Shigetani Y, Yoshiba N, Yoshiba K, Okiji T. Prostaglandin transporting protein-mediated prostaglandin E2 transport in lipopolysaccharide-inflamed rat dental pulp. J Endod 2014;40(8):1112–7. 36. Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: structure, composition and mineralization. Front Biosci 2011;3(Elite Ed):711–35. 37. Yu MK, Lee JC, Kim JH, Lee YH, Jeon JG, Jhee EC, et al. Antiinflammatory effect of peroxisome proliferator activated receptor gamma on human dental pulp cells. J Endod 2009;35(4):524–8. 38. Kim JC, Lee YH, Yu MK, Lee NH, Park JD, Bhattarai G, et al. Anti-inflammatory mechanism of PPARg on LPS-induced pulp cells: role of the ROS removal activity. Arch Oral Biol 2012;57(4):392–400. 39. Ye L, MacDougall M, Zhang S, Xie Y, Zhang J, Li Z, et al. Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 2004;279(18):19141–8. 40. Sulieman M, Addy M, MacDonald E, Rees JS. The effect of hydrogen peroxide concentration on the outcome of tooth whitening: an in vitro study. J Dent 2004;32(4):295–9. 41. Soares DG, Basso FG, Pontes EC, Garcia LF, Hebling J, de Souza Costa CA. Effective tooth-bleaching protocols capable of reducing H2O2 diffusion through enamel and dentine. J Dent 2014;42(3):351–8. 42. Bowels WH, Burns H. Catalase/peroxidase activity in dental pulp. J Endod 1992;18(11):527–34. 43. Esposito P, Varvara G, Murmura G, Terlizzi A, Caputi S. Ability of healthy and inflamed human dental pulp to reduce hydrogen peroxide. Eur J Oral Sci 2003;111(5): 454–6. 44. Sauro S, Pashley DH, Montanari M, Chersoni S, Carvalho RM, Toledano M, et al. Effect of simulated pulpal pressure on dentin permeability and adhesion of self-etch adhesives. Dent Mater 2007;23(6):705–13.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Responses of human dental pulp cells after application of a low-concentration bleaching gel to enamel Diana G. Soares a, Fernanda G. Basso b, De´bora Salles Scheffel b, Josimeri Hebling b, Carlos A. de Souza Costa a,* a
Department of Physiology and Pathology, Univ. Estadual Paulista – UNESP, Araraquara School of Dentistry, Humaita´ Street 1680, Araraquara, SP, Brazil b Department of Orthodontics and Pediatric Dentistry, Univ. Estadual Paulista – UNESP, Araraquara School of Dentistry, Humaita´ Street 1680, Araraquara, SP, Brazil
article info
abstract
Article history:
Objective: To evaluate the effect of a 17.5% H2O2 gel on the odontoblastic differentiation
Accepted 14 June 2015
capability of human dental pulp cells (HDPCs). Design: The bleaching gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to
Keywords:
transwells, positioned over previously cultured HDPCs. In the control group, no treatment
Odontoblasts
was performed on the discs. Immediately after samples were bleached, the cell viability
Dental pulp
(MTT assay) and death (Live/Dead assay) as well as the mRNA gene expression of inflam-
Tooth bleaching
matory mediators (TNFa, IL-1b, IL-6, and COX-2; real-time PCR) were evaluated. The mRNA
Hydrogen peroxide
gene expression of odontoblastic markers (DMP-1, DSPP, and ALP) and mineralized nodule
Differentiation
deposition (alizarin red) were assessed at 7, 14 and 21 days post-bleaching. The amount of H2O2 in contact with cells was quantified. Data were evaluated by Kruskal–Wallis and Mann– Whitney tests (a = 5%). Results: Significant cell viability reduction and cell death were observed for bleached groups relative to control in a time-dependent fashion. Also, significant overexpression of all inflammatory mediators tested occurred in the 45- and 15-min groups. In the bleached groups, the expression of ALP, DMP-1, and DSPP and the deposition of mineralized nodules were reduced in comparison with those in the control group, at the initial periods (7 and 14 days). However, the 15- and 5-min groups reached values similar to those in the control group at the 21-day period. Conclusions: The 17.5% H2O2 gel was cytotoxic to pulp cells; however, cells subjected to short-term bleaching are capable of expressing the odontoblastic phenotype over time. # 2015 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Physiology and Pathology, Univ. Estadual Paulista – UNESP, Araraquara School of Dentistry, Humaita´ Street 1680, Araraquara, SP 14801-903, Brazil. Tel.: +55 1633016477. E-mail address: [email protected] (C.A. de Souza Costa). http://dx.doi.org/10.1016/j.archoralbio.2015.06.014 0003–9969/# 2015 Elsevier Ltd. All rights reserved.
archives of oral biology 60 (2015) 1428–1436
1.
Introduction
It is well known that tooth-bleaching therapy can promote odontoblast death due to oxidative damage mediated by hydrogen peroxide and its by-products, capable of diffusing through enamel and dentine at toxic concentrations.1,2 These cells are essential for the homeostasis of the dentine-pulp complex, since they are responsible for the deposition and mineralization of dentine matrix throughout the life of the tooth, as well as for the orchestration of the inflammatory and immune responses of pulp tissue, since the noxious signal acts on the tooth surface.3,4 Odontoblasts are highly differentiated terminal cells. Therefore, when they are lost, reparative dentinogenesis takes place, which involves mesenchymal stem cell (MSC) recruitment, proliferation, and differentiation into odontoblast-like cells, resulting in the deposition of mineralized matrix and restoration of the homeostasis of pulp tissue.5–7 It was previously demonstrated that very low concentrations of H2O2 induce odontoblastic differentiation and mineralized matrix deposition8,9; conversely, toxic concentrations of H2O2 enhance inflammatory mediator expression associated with the impairment of odontoblastic differentiation capability.10–14 Recently, it was demonstrated that application of a lowconcentration H2O2 gel (17.5%) from 5 to 45 min onto dental structure was able to reduce in about 11.3–4.5 times, respectively, the indirect toxicity of in-office tooth-bleaching to human dental pulp cells (HDPCs), when compared with traditional therapy (35% H2O2; 3 15 min). The cells were able to proliferate significantly 3 days after samples were bleached, demonstrating that pulp cells were able to overcome the initial oxidative stress.2 However, the effect of this initial H2O2 toxicity on the odontoblastic differentiation capability of HDPCs as well as mineralized matrix deposition was not addressed. Therefore, the present study investigated the effect of a 17.5% H2O2 gel, applied to enamel surfaces for different periods, on the immediate viability of HDPCs and late odontoblastic differentiation and mineralized matrix deposition capability.
2.
Materials and methods
2.1.
Cell culture
The human dental pulp cell (HDPC) primary culture was obtained by enzymatic digestion of pulp tissue from one freshly extracted third molar, obtained from one donor (24 years of age) (Proc. n8 13/11; Ethics Committee of Araraquara School of Dentistry, SP, Brazil) after sign the informed consent according to the code of ethics of the world medical association (Declaration of Helsinki). The pulp tissue was removed aseptically, cut into small pieces (1 mm), and then incubated for 24 h in complete Dulbecco’s Modified Eagle Medium (DMEM; supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, 2 mmol/L glutamine; Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; Gibco) and 200 units/mL of type II collagenase (Worthington
1429
Biochemical Corporation, Lakewood, NJ, USA). After this period, the released cells were subcultured in complete DMEM with 10% FBS. Cells from the 4th to 6th passages were used.
2.2.
Enamel/dentine specimens
Bovine incisors, from 24- to 30-month-old bullocks, were cross-sectioned in its buccal surface with a diamond trephine bur (Dinser brocas diamantadas LTDA, Sa˜o Paulo, SP, Brazil) coupled to bench drilling machine (FSB 16 Pratika; Schultz, Joinville, SC, Brazil) to obtain 5.6 mm-diameter discs containing both enamel and dentine. To obtain the standardized thicknesses similar to those of central incisors (3.5 mm),15 we polished the dentine surfaces with 400- and 600-grit abrasive papers, followed by EDTA 0.5 N treatment for 30 s for smear layer removal. The enamel surfaces of the discs were cleaned with a solution of pumice stone and water by means of a low-speed handpiece. The discs were adapted to acrylic transwells (polycarbonate membrane/8 mm pore size; Corning Inc., Corning, NY, USA) by means of a fluid light-cured resin (TopDam, FGM, Joinville, SC, Brazil), which promoted a lateral seal, and the set was then sterilized with ethylene oxide.
2.3.
Experimental design
The HDPCs were seeded into wells of 24-well plates (6 104 cells/well) for 24 h in DMEM supplemented with 10% FBS, reaching 80% confluence. The culture medium was then replaced by 300 mL of DMEM with no FBS, and the disc/ transwell set was positioned onto the previously cultured cells, in such a way that only the dentine was in direct contact with the culture medium, and the enamel surface remained exposed to receive the bleaching procedure. The bleaching gel containing 17.5% of H2O2 was obtained by dilution of a 35% H2O2 gel (Whiteness HP; FGM) in distilled water immediately before the bleaching procedure (1:1).2 The gel (30 mg) was applied to the enamel surface for different periods according to the experimental groups: 45-min group – 3 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. Immediately after the bleaching treatment, the transwell/disc set was removed, the culture medium in contact with the cells was collected, and the cells were washed with 1 mL of phosphate-buffered saline (PBS) solution.
2.4.
Trans-enamel and trans-dentinal cytotoxicity
2.4.1.
Cell viability analysis (MTT assay)
The cells were incubated for 4 h at 37 8C and 5% CO2 with the MTT solution (Sigma–Aldrich Corp., St. Louis, MO, USA) diluted in DMEM (1:10). The formazan crystals formed in viable cells were then dissolved in acidified isopropanol, and the absorbance of the resulting solution was read at 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).
1430
2.4.2.
archives of oral biology 60 (2015) 1428–1436
Cell death analysis (Live/Dead assay)
For this assay, the cells were seeded onto glass slides (13 mm diameter) positioned at the bottoms of the 24-well plates. Immediately after the bleaching procedure, the cells were incubated for 45 min with the fluorescence probes Ethyl homodimer-1 (Eth-1; 4 mM; Invitrogen, San Francisco, CA, USA), which binds to DNA bands only in cells experiencing cell membrane rupture (undergoing necrosis), and Calcein AM (CA; 1 mM; Invitrogen), which is hydrolyzed by cytoplasmic esterases in viable cells. The glass slides were evaluated by fluorescence microscopy (20; Leica DM 5500B, Nussloch GmbH, Nussloch, Germany), and 6 photographs were taken for each sample. The numbers of viable and dead cells were counted by means of Image J software, and the percentage of dead cells from the total number of cells was calculated. The average value of dead cells per well was used for statistical analysis (n = 6).
2.4.3. PCR)
Gene expression of inflammatory mediators (real-time
The gene expressions of tumour necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), interleukin 6 (IL-6), and cyclooxygenase 2 (COX-2) were performed 6 h after the bleaching procedure (the cells were incubated in fresh DMEM with no FBS) by the real-time PCR technique (n = 6). This period (6 h after bleaching) was used based on the fact that the highest values of mRNA expression for all inflammatory mediators assessed in this in vitro study were previously observed after HDPCs exposition to H2O2 (data not shown) at a concentration capable to cause alteration on the odontoblastic differentiation capability.16 Total RNA was extracted with an RNAqueous1micro kit (Ambion1, Austin, TX, USA), and 1 mg was then reverse-transcribed into single-stranded cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), in accordance with the manufacturer’s protocols. Taqman Assays (Invitrogen) for human genes were used, as follows: IL-1b, Hs01555410_m1; IL-6, Hs00985639_m1; TNF-a, Hs00174128_m1; COX-2, Hs00153133_m1; and b-actin, 4333762T. The CT values for each sample were normalized by the endogenous control gene b-actin. Thereafter, the mean CT value of the control group was used to normalize the CT values of both control and experimental groups.
2.5.
Odontoblastic differentiation capability
For this analysis, immediately after being washed in PBS solution, the cells were incubated in osteogenic medium (DMEM plus 10% SFB, supplemented with 10 nmol/L b-glycerophosphate and 50 mg/mL sodium ascorbate; Sigma–Aldrich Corp.) for 7, 14 and 21 days, with medium being changed daily. The expression of odontoblastic markers and the deposition of mineralized matrix were then assessed at each time-point.
2.5.1. PCR)
gene expression of odontoblastic markers (real-time
The gene expression of dentine matrix phosphoprotein 1 (DMP-1), dentine sialophosphoprotein (DSPP), and alkaline phosphatase (ALP) was assessed by real-time PCR, as described above (n = 6). Taqman assays (Invitrogen) were used for relative quantification of mRNA levels, as follows: DMP-1,
Hs01009391_g1; DSPP, Hs00171962_m1; ALP, Hs01029144_m1; and b-actin, 4333762T. The CT values for each sample were normalized by the endogenous control gene b-actin. 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.
2.5.2.
Deposition of mineralized matrix (Alizarin red staining)
At each time-point, the cells were fixed with cold 70% ethanol, washed with deionized water, and then stained with alizarin red dye (40 mM, pH 4.2; Sigma–Aldrich Corp.) under gentle shaking (VDR Shaker, Biomixer, Ribeira˜o Preto, SP, Brazil). The cells were than 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.), and the absorbance of the resulting solution was measured at 570 nm (ELISA microplate reader). The percentage of alizarin red staining, which represents the calcium deposition, for each experimental group was calculated based on the mean value of the control group at 7 days as representing 100% of staining.
2.6.
Quantification of H2O2 diffusion
Immediately after the bleaching procedure, one aliquot of DMEM in direct contact with cells was transferred to tubes containing acetate buffer solution (2 mol/L, pH 4.5). This solution was then transferred to tubes containing leucocrystal violet (0.5 mg/mL; Sigma–Aldrich Corp.) and horseradish peroxidase enzyme (1 mg/mL; Sigma–Aldrich Corp.), and the final volume of reaction was adjusted to 3 mL with distilled water. The absorbance of the resulting solution was read 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 mg/mL of H2O2, and the data were related to mg/mL of culture medium.
2.7.
Statistical analysis
Two independent experiments were performed for all protocols in this study. Data were compiled and subjected to Levene’s test to verify homoscedasticity. Cell viability, cell death, H2O2 diffusion, mRNA gene expression, and percentages of alizarin red staining were subjected to the Kruskal– Wallis and Mann–Whitney tests at a significance level of 5%. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used to run the statistical analyses.
3.
Results
3.1.
Cytotoxicity/H2O2 diffusion
Data on cell viability, cell death, and H2O2 quantification are given in Table 1. With the control group considered as presenting 100% of cell viability, reductions of around 80.1%, 74.6%, and 63.6% of HDPC viability were observed for the 45-, 15-, and 5-min groups, respectively. Similar results were
1431
archives of oral biology 60 (2015) 1428–1436
Table 1 – Data on cell viability, cell death, and H2O2 diffusion for the control and experimental groups. Group Control 45-min 15-min 5-min
% Cell viability (MTT assay)
% Cell death (Eth-1-positive cells)
a
100 (90.8–105.3) 19.9 (11.1–20.1) c 25.4 (13.3–28.9) bc 36.4 (33.1–37.9) b
2.9 64.9 58.1 34.5
d
(2.3–3.1) (63.1–67.8) ab (53.4–67.7) b (33.8–45.3) c
H2O2 diffusion (mg/mL) n.d.* 0.80 (0.59–0.85) b 0.25 (0.20–0.31) c 0.11 (0.09–0.15) c
Values are medians (from 25th to 75th percentiles), n = 6. In each column, groups identified with the same letter do not differ statistically (Mann–Whitney, p > 0.05). * not detected.
observed for the cell death assay, in which 64.9%, 58.1%, and 34.5% of Eth-1-positive cells were observed for the 45-, 15-, and 5-min groups, respectively. All of these values were significantly different from those observed in the control group. The 5-min group featured the lowest values of cell viability reduction and cell death, being significantly different from the other bleached groups. Representative images of live (CApositive) and dead cells (Eth-1-positive) are demonstrated in Fig. 1. The H2O2 diffusion values also followed the timedependent pattern, with the 45-min group featuring the highest values, which were significantly different from those of the other bleached groups.
3.2.
Inflammatory mediator gene expression
The results for inflammatory mediator mRNA expression are presented in Fig. 2. IL-6, TNF-a, COX-2, and IL-1b mRNA expression was significantly enhanced for the 45- and 15-min groups compared with the control. These groups showed a similar behaviour for inflammatory mediators’ gene expression, and no significant difference among them was observed
for the IL-6, COX-2, and IL-1b mRNA expression. A discrete increase in the expression of these genes was observed for the 5-min group; however, a significant difference from the control was observed only for IL-6.
3.3.
Odontoblastic marker gene expression
The mRNA gene expression of odontoblastic markers is presented in Fig. 3. The gene expression of ALP in the control group presented a peak at 14 days. This peak was not observed in bleached groups, which resulted in a significant difference from the control at this time-point; however, the 15- and 5min groups featured significantly higher mRNA ALP expression at 21 days relative to control. The mRNA gene expression of DMP-1 was significantly reduced at 7, 14 and 21 days for all bleached groups compared with control, except for the 5-min group at 7 days. Regarding DSPP, the 45-min group showed the lowest values of mRNA expression, with a significant difference relative to control at all time-points; conversely, the 15and 5-min groups featured no significant difference from the control at 21 days.
Fig. 1 – Representative images of live (green) and dead (red) cells for each group, obtained by fluorescence microscopy (20T). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
1432
archives of oral biology 60 (2015) 1428–1436
Fig. 2 – Box-Whisker plot of inflammatory mediator mRNA expression. Vertical axis represents relative gene expression normalized by the negative control group, and horizontal axis represents the experimental groups. Different letters indicate a statistically significant difference among groups (n = 6) (Mann–Whitney, p > 0.05).
3.4.
Mineralized matrix deposition
The mineralized matrix deposition in bleached groups was reduced, relative to control, at the 7- and 14-day periods, being more intense for the 45-min group; however, at the 21-day period, only the 45-min group presented a significantly lower percentage than the control (Table 2). Fig. 4 reveals a remarkable increase in mineralized matrix deposition in the control and 5-min groups at 21 days. The 45-min group presented no mineralized nodule deposition at any period of analysis.
4.
Discussion
In the present investigation, it was demonstrated that the 17.5% H2O2 bleaching gel was cytotoxic to the HDPCs since the cell viability reduction, as well as the cell death percentage,
was above 30%.17 However, the cytotoxicity was dependent on the contact time of the gel with the enamel surface. Soares et al.2 demonstrated that pulp cells exposed to these experimental bleaching protocols were under oxidative stress, in which the intensity was proportional to the contact time of the gel with enamel; however, the cells were able to proliferate significantly over time, recovering their viability at around 3–4 times three days after the bleaching procedure. In the present study, cell viability reduction occurred partly as a consequence of cell death by necrosis, since the cells were positively stained for the Eth-1 probe, which binds to the DNA of only cells with disrupted membranes. The percentage of positive Eth-1 cells was also proportional to the treatment time and the amount of H2O2 capable of diffusing through enamel and dentine to reach the underlying cells. This demonstrates that the higher the H2O2 concentration in contact with pulp cells, the higher the cell viability reduction mediated by necrosis. Wu et al.18 demonstrated that, at toxic concentrations, H2O2
1433
archives of oral biology 60 (2015) 1428–1436
Fig. 3 – Box-Whisker plot of odontoblastic marker mRNA expression. 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 timepoints for each group. Different letters indicate a statistically significant difference among groups (n = 6) (Mann–Whitney, p > 0.05).
Table 2 – Alizarin red staining percentage for HDPCs. Groups
Periods of analysis 7 days
Alizarin stain (%)
Control 45-min 15-min 5-min
100.4 9.7 40.7 57.6
(100.0–100.7) cA (9.1–11.2) bC (37.9–44.5) cB (54.9–62.7) bB
14 days 392.5 54.5 77.3 120.5
(382.7–414.6) bA (51.0–57.1) aC (70.8–87.2) bC (104.3–133.2) bB
21 days 557.1 71.0 327.7 570.9
(557.0–557.9) aA (70.4–71.1) aB (317.7–512.2) aA (554.5–574.9) aA
Numbers are medians (from 25th to 75th percentiles), n = 6. Lowercase letters allow for comparisons in rows and uppercase letters comparisons in columns. Groups identified by the same letter do not differ statistically significantly (Mann–Whitney, p > 0.05).
Fig. 4 – Panel of mineralized nodule deposition through periods of analysis. For each group and period, the left image represents a digital photograph of the well, and at right is a light-microscopic (20T) image from a delimited area of the well. Absence of nodule deposition was observed in all groups at 7 days. Some nodules were observed in the control group and the 15- and 5-min groups at 14 days. At the 21-day period, the control and 5-min groups presented an intense deposition of mineralized nodules, and increased numbers of nodules were observed in the 15-min group. Absence of nodule deposition was observed for the 45-min group at all periods of analysis.
1434
archives of oral biology 60 (2015) 1428–1436
causes cell viability reduction by necrosis and apoptosis, with higher concentrations leading to a higher percentage of necrotic cells. This specific type of cell death causes intense damage in vivo, since high quantities of intracellular components are released, including lysosomal enzymes, causing damage to the neighbouring cells and triggering an inflammatory tissue reaction.19,20 Pulpal inflammation associated with local tissue necrosis was previously demonstrated by in vivo studies in which high-concentration H2O2 bleaching gels (35–38%) were applied to rat or human teeth.20–23 In the present investigation, the remaining HDPCs showed increased expression of IL-6, TNF-a, COX-2, and IL-1b in comparison to the control group. Key and well-characterized cytokines, including IL-1a, IL-1b, TNF-a, IL-4, IL-6, IL-8, and IL-10, are highly expressed by structural cells from pulp tissue, such as odontoblasts, fibroblasts and mesenchymal stem cells, along with immune cells, when they are exposed to bacterial and dental materials components.24–28 These molecules play regulatory functions for lymphocytes, macrophages, and neutrophils, activating the inflammatory cascade,29,30 which may result in release of proteolytic enzymes, such as metalloproteinases, with the consequent disruption of extracellular matrix components.31,32 Prostaglandin E2, synthesized by COX-2 gene is also a major pro-inflammatory mediator, which is known to accelerate inflammation and injury in several tissues, including the dental pulp.33–35 The inflammatory mediators IL-6, TNF-a, COX-2, and IL-1b were assessed in the present investigation since they are strongly expressed in inflamed pulp tissue.24,28,34,35 Also, previous studies demonstrated that depending on the concentration of these inflammatory mediators in the pulp they may play either regenerative or degenerative tissue effects.10–13,18,28 Therefore, these mediators have been used as inflammatory markers by in vitro studies performed with pulp cells such as this one.25–27,33 The effect of the immediate cytotoxicity of the 17.5% H2O2 gel on the odontoblastic differentiation capability of HDPCs was also evaluated in this study. It was observed that the cells exposed to the bleaching protocols showed mRNA expression of DSPP, DMP-1, and ALP at all periods of analysis. However, alteration in the gene expression pattern relative to the control group was observed, mainly at the initial periods. Interestingly, the cells subjected to the less aggressive protocols (5 and 15 min of bleaching) were able to increase gene expressions over time, reaching a pattern similar to that of the control group. The proteins encoded by these genes are directly related to the odontoblastic differentiation process, by providing inorganic phosphate, binding calcium to the collagenous matrix and promoting the nucleation and growth of the mineral phase.4,36 Regarding the mineralized matrix deposition, instead of the reduction in the percentage of alizarin red staining that was observed at 7 and 14 days for all bleached groups, an increase in this percentage over time was observed, with the 15- and 5-min groups presenting no significant difference from control at 21 days. Therefore, one may conclude that cells of these groups were able to overcome the initial oxidative damage and proliferate and differentiate into cells with an odontoblastic phenotype able to deposit mineralized matrix in vitro.
Some molecules are directly related to the cytoprotection of pulp cells against oxidative stress mediated by H2O2. Min et al.8 demonstrated that overexpression of the heat-shock protein heme oxygenase-1 (HO-1) in HDPCs significantly improved their viability against H2O2-induced cytotoxicity. The authors also observed that the overexpression of DSPP on H2O2-treated cells was via an OH-1-dependent pathway. Another molecule related to the cytoprotection of HDPCs against H2O2 was recently described, the peroxisome proliferator-activated receptor gamma (PPARg), a transcriptional factor belonging to the ligand-activated nuclear receptor superfamily.14 This molecule plays an important role in LPS-mediated pulp inflammation, by removing ROS from cells and tissues.37,38 PPARg overexpressed HDPCs increased their viability after exposure to toxic concentrations of H2O2, associated with increased activity of the anti-oxidant enzymes Cu/ZnSOD and MnSOD, and up-regulation of DSPP and DMP-1 proteins.14 Therefore, both OH-1 and PPARg plays a protective role in the induction of dentinogenesis under oxidative stress in human pulp cells. The 45-min group featured the most intense alteration in the odontoblastic differentiation ability of HDPCs. The remaining cells were unable to deposit mineralized matrix in all periods of analysis, which may have been caused, at least in part, by the intense alterations in DSPP and DMP-1 mRNA expression. According to Ye et al.,39 down-regulation of DSPP and DMP-1 leads to failure of the maturation of predentin into dentine, with reduced dentine in postnatal tooth development. This fact may also be a consequence of the more intense expression of the inflammatory mediators observed for this group. According to Cooper et al.,19 there is a fine balance between inflammatory mediator dosage/contact time and odontoblastic differentiation, as demonstrated by Paula-Silva et al.,12 who applied low-dose TNF-a to HDPCs and observed notable overexpression of DPP, DSP, and DMP-1 after a short treatment time (6 h). Min et al.11 observed increased ALP activity and dentine matrix non-collagenous protein overexpression after 72-hour cultivation of HDPCs with proinflammatory 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.10 showed increased ALP activity in reversible pulpitis in comparison with healthy or irreversible pulpitis samples. Analysis of these data corroborates those reported by Alongi et al.,13 who verified that MSCs from teeth diagnosed with irreversible pulpitis presented lower expression of odontoblastic markers (DSPP, ALP, and osteocalcin) than did normal pulp tissue MSC-derived cells. A second explanation for the more intense alteration of the odontoblastic differentiation capability observed for the 45min group may be related to the intensity of oxidative stress on remaining cells. Soares et al.2 showed that the oxidative stress on HDPCs exposed to the 17.5% H2O2 for 45 min was significantly higher in comparison with that for the 15- and 5min treatment times. Lee et al.14 demonstrated that HPDCs under intense oxidative stress by long-term exposure to H2O2 showed down-regulation of DSPP and DMP-1 mRNA associated with impaired mineralized matrix deposition in vitro. Conversely, the treatment of HDPCs with non-toxic concentrations of H2O2 enhanced ALP activity, up-regulated DSPP,
archives of oral biology 60 (2015) 1428–1436
OPN (osteopontin), and OCN (osteocalcin) expression, and increased calcified nodule deposition in vitro.1,8,9 Therefore, the lower the concentration of H2O2 in contact with pulp cells after being bleached, the higher the regenerative capability of human pulp tissue to overcome the oxidative damage. One can conclude that low-concentration bleaching gels applied for reduced contact times to dental structure may be an interesting alternative for low-thickness teeth, i.e., human incisors, which are more susceptible to pulp-dentine complex oxidative damage, since they allow greater H2O2 diffusion into the pulp chamber.21–24 Previous studies demonstrated that bleaching gels with 15–17.5% H2O2 caused significant tooth colour improvement by increasing the number of sessions.40,41 Nevertheless, the data obtained in the present in vitro study should be considered with caution, because the toxicity observed in laboratory tests (in vitro studies) may be minimized in clinical conditions, since, in vital teeth, there is outward dentinal fluid movement as well as the presence of collagen and odontoblast cytoplasmic processes inside dentinal tubules. All these factors interfere with the inward displacement of toxic components released from dental products, such as bleaching agents.36 In addition, the presence of extracellular matrix, anti-oxidant, and lymphatic systems in pulp tissue protects pulp cells against external aggression.42–44
5.
Conclusions
According to the methodology carried out in this in vitro study, one may conclude that bleaching gel with 17.5% H2O2 applied to enamel for 15 or 5 min promotes cytotoxicity to pulp cells associated with the overexpression of inflammatory mediators; however, the surviving cells were able to overcome the initial aggression and differentiate into cells with an odontoblastic phenotype able to deposit mineralized matrix.
Funding This study was supported by the Sa˜o Paulo Research Foundation – FAPESP (grants # 2013/23520-0 and 2013/058790) and by the National Council of Technological and Scientific Development – CNPq (grant # 301029/2010-1).
Competing interests The authors have no conflict of interest.
Ethical approval Proc. n8 13/11; Ethics Committee of Araraquara School of Dentistry, SP, Brazil.
Acknowledgments This study was supported by the Sa˜o Paulo Research Foundation–FAPESP (grants # 2013/23520-0 and 2013/05879-0) and by
1435
the National Council of Technological and Scientific Development–CNPq (grant # 303599/2014-6). The authors deny any conflicts of interest related to this study within three years of beginning the work.
references
1. Soares DG, Ribeiro AP, da Silveira Vargas F, Hebling J, de Souza Costa CA. Efficacy and cytotoxicity of a bleaching gel after short application times on dental enamel. Clin Oral Investig 2013;17(8):1901–9. 2. Soares DG, Basso FG, Hebling J, de Souza Costa CA. Concentrations of and application protocols for hydrogen peroxide bleaching gels: effects on pulp cell viability and whitening efficacy. J Dent 2014;42(2):185–98. 3. Couve E, Osorio R, Schmachtenberg O. The amazing odontoblast: activity, autophagy, and aging. J Dent Res 2013;92(9):765–72. 4. Schmalz G, Smith AJ. Pulp development, repair, and regeneration: challenges of the transition from traditional dentistry to biologically based therapies. J Endod 2014;40(Suppl 4):S2–5. 5. Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, et al. Comparative analysis of in vitro osteo/ odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Arch Oral Biol 2011;56(7):709–21. 6. Morad G, Kheiri L, Khojasteh A. Dental pulp stem cells for in vivo bone regeneration: a systematic review of literature. Arch Oral Biol 2013;58(12):1818–27. 7. Dimitrova-Nakov S, Baudry A, Harichane Y, Kellermann O, Goldberg M. Pulp stem cells: implication in reparative dentin formation. J Endod 2014;40(Suppl 4):S13–8. 8. Min KS, Lee HJ, Kim SH, Lee SK, Kim HR, Pae HO, et al. Hydrogen peroxide induces heme oxygenase-1 and dentin sialophosphoprotein mRNA in human pulp cells. J Endod 2008;34(8):983–9. 9. Matsui S, Takahashi C, Tsujimoto Y, Matsushima K. Stimulatory effects of low-concentration reactive oxygen species on calcification ability of human dental pulp cells. J Endod 2009;35(1):67–72. 10. Spoto G, Fioroni M, Rubini C, Tripodi D, Di Stilio M, Piattelli A. Alkaline phosphatase activity in normal and inflamed dental pulps. J Endod 2001;27(3):180–2. 11. Min KS, Kwon YY, Lee HJ, Lee SK, Kang KH, Lee SK, et al. Effects of proinflammatory cytokines on the expression of mineralization markers and heme oxygenase-1 in human pulp cells. J Endod 2006;32(1):39–43. 12. Paula-Silva FW, Ghosh A, Silva LA, Kapila YL. TNF-alpha promotes an odontoblastic phenotype in dental pulp cells. J Dent Res 2009;88(4):339–44. 13. Alongi DJ, Yamaza T, Song Y, Fouad AF, Romberg EE, Shi S, et al. Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential. Reg Med 2010;5(4):617–31. 14. Lee YH, Kang YM, Heo MJ, Kim GE, Bhattarai G, Lee NH, et al. The survival role of peroxisome proliferator-activated receptor gamma induces odontoblast differentiation against oxidative stress in human dental pulp cells. J Endod 2013;39(2):236–41. 15. Harris EF, Hicks JD. A radiographic assessment of enamel thickness in human maxillary incisors. Arch Oral Biol 1998;43(10):825–31. 16. Soares DG, Basso FG, Hebling J, de Souza Costa CA. Effect of hydrogen-peroxide-mediated oxidative stress on human dental pulp cells. J Dent 2014. http://dx.doi.org/10.1016/ j.jdent.2014.12.006. [Epub ahead of print].
1436
archives of oral biology 60 (2015) 1428–1436
17. ISO 10993-5:2009(E). Biological evaluation of medical devices – part 5: tests for cytotoxicity in vitro. Geneva, Switzerland. 2009. 18. Wu TT, Li LF, Du R, Jiang L, Zhu YQ. Hydrogen peroxide induces apoptosis in human dental pulp cells via caspase-9 dependent pathway. J Endod 2013;39(9):1151–5. 19. Cooper PR, Holder MJ, Smith AJ. Inflammation and regeneration in the dentin-pulp complex: a double-edged sword. J Endod 2014;40(Suppl 4):S46–51. 20. Fouad AF, Verma P. Healing after regenerative procedures with and without pulpal infection. J Endod 2014;40(Suppl 4):S58–64. 21. de Souza Costa CA, Riehl H, Kina JF, Sacono NT, Hebling J. Human pulp responses to in-office tooth bleaching. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109(4):59–64. 22. Cintra LT, Benetti F, da Silva Facundo AC, Ferreira LL, Gomes-Filho JE, Ervolino E, et al. The number of bleaching sessions influences pulp tissue damage in rat teeth. J Endod 2014;39(12):1576–80. 23. Ferreira VG, Nabeshima CK, Marques MM, Paris AF, Gioso MA, dos Reis RS, et al. Tooth bleaching induces changes in the vascular permeability of rat incisor pulps. Am J Dent 2013;26(5):298–300. 24. Roderjan DA, Stanislawczuk R, Hebling J, de Souza Costa CA, Soares DG, Reis A, et al. Histopathological features of dental pulp tissue from bleached mandibular incisors. J Mater Sci Eng B 2014;4(6):178–85. 25. Horst OV, Horst JA, Samudrala R, Dale BA. Caries induced cytokine network in the odontoblast layer of human teeth. BMC Immunol 2011;12:9. 26. Kim RH, Williams DW, Bae S, Lee RS, Oh JE, Mehrazarin S, et al. Camphorquinone inhibits odontogenic differentiation of dental pulp cells and triggers release of inflammatory cytokines. J Endod 2013;39(1):57–61. 27. Asgary S, Nazarian H, Khojasteh A, Shokouhinejad N. Gene expression and cytokine release during odontogenic differentiation of human dental pulp stem cells induced by 2 endodontic biomaterials. J Endod 2014;40(3):387–92. 28. Diomede F, Caputi S, Merciaro I, Frisone S, D’Arcangelo C, Piattelli A, et al. Pro-inflammatory cytokine release and cell growth inhibition in primary human oral cells after exposure to endodontic sealer. Int Endod J 2014;47(9):864–72. 29. Yazid FB, Gnanasegaran N, Kunasekaran W, Govindasamy V, Musa S. Comparison of immunodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth. Clin Oral Investig 2014;18(9):2103–12. 30. Durutu¨rk L, Sarˇˇi S, Sengu¨l A. Immunocompetent cell level as a diagnostic reference for pulpal pathosis of primary teeth. Arch Oral Biol 2013;58(10):1517–22. 31. Cooper PR, Takahashi Y, Graham LW, Simon S, Imazato S, Smith AJ. Inflammation-regeneration interplay in the dentine-pulp complex. J Dent 2010;38(9):687–97.
32. Sato C, Rodrigues FA, Garcia DM, Vidal CM, Pashley DH, Tja¨derhane L, et al. Tooth bleaching increases dentinal protease activity. J Dent Res 2013;92(2):87–92. 33. Sousa NG, Cardoso CR, Silva JS, Kuga MC, Tanomaru-Filho M, Faria G. Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development. Arch Oral Biol 2014;59(9):944–53. 34. Jeong GS, Lee DS, Li B, Lee HJ, Kim EC, Kim YC. Effects of sappanchalcone on the cytoprotection and antiinflammation via heme oxygenase-1 in human pulp and periodontal ligament cells. Eur J Pharmacol 2010;644(1– 3):230–7. 35. Ohkura N, Shigetani Y, Yoshiba N, Yoshiba K, Okiji T. Prostaglandin transporting protein-mediated prostaglandin E2 transport in lipopolysaccharide-inflamed rat dental pulp. J Endod 2014;40(8):1112–7. 36. Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: structure, composition and mineralization. Front Biosci 2011;3(Elite Ed):711–35. 37. Yu MK, Lee JC, Kim JH, Lee YH, Jeon JG, Jhee EC, et al. Antiinflammatory effect of peroxisome proliferator activated receptor gamma on human dental pulp cells. J Endod 2009;35(4):524–8. 38. Kim JC, Lee YH, Yu MK, Lee NH, Park JD, Bhattarai G, et al. Anti-inflammatory mechanism of PPARg on LPS-induced pulp cells: role of the ROS removal activity. Arch Oral Biol 2012;57(4):392–400. 39. Ye L, MacDougall M, Zhang S, Xie Y, Zhang J, Li Z, et al. Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 2004;279(18):19141–8. 40. Sulieman M, Addy M, MacDonald E, Rees JS. The effect of hydrogen peroxide concentration on the outcome of tooth whitening: an in vitro study. J Dent 2004;32(4):295–9. 41. Soares DG, Basso FG, Pontes EC, Garcia LF, Hebling J, de Souza Costa CA. Effective tooth-bleaching protocols capable of reducing H2O2 diffusion through enamel and dentine. J Dent 2014;42(3):351–8. 42. Bowels WH, Burns H. Catalase/peroxidase activity in dental pulp. J Endod 1992;18(11):527–34. 43. Esposito P, Varvara G, Murmura G, Terlizzi A, Caputi S. Ability of healthy and inflamed human dental pulp to reduce hydrogen peroxide. Eur J Oral Sci 2003;111(5): 454–6. 44. Sauro S, Pashley DH, Montanari M, Chersoni S, Carvalho RM, Toledano M, et al. Effect of simulated pulpal pressure on dentin permeability and adhesion of self-etch adhesives. Dent Mater 2007;23(6):705–13.