Responses of Dental Pulp Cells to a Less Invasive Bleaching Technique Applied to Adhesively Restored Teeth Diana Gabriela Soaresa / Nancy Tomoko Saconob / Ana Paula Dias Ribeiroc / Fernanda Gonçalves Bassod / Débora Sales Scheffele / Josimeri Heblingf / Carlos Alberto de Souza Costag Purpose: To assess the cytotoxicity of 35% hydrogen peroxide (HP) bleaching gel applied for 15 min to sound or restored teeth with two-step self-etching adhesive systems and composite resin. Materials and Methods: Sound and restored enamel/dentin disks were stored in water for 24 h or 6 months + thermocycling. The disks were adapted to artificial pulp chambers and placed in compartments containing culture medium. Immediately after bleaching, the culture medium in contact with dentin was applied for 1 h to previously cultured odontoblast-like MDPC-23 cells. Thereafter, cell viability (MTT assay) and morphology (SEM) were assessed. Data were analyzed by two-way ANOVA and Tukey’s test (_ = 5%). Results: In comparison to the negative control group (no treatment), no significant cell viability reduction occurred in those groups in which sound teeth were bleached. However, a significant decrease in cell viability was observed in the adhesive-restored bleached groups compared to negative control. No significant difference among bleached groups was observed with respect to the presence of restoration and storage time. Conclusion: The application of 35% HP bleaching gel to sound teeth for 15 min does not cause toxic effects in pulp cells. When this bleaching protocol was performed in adhesive-restored teeth, a significant toxic effect occurred. Keywords: laboratory research, toxicity, odontoblasts, tooth bleaching, adhesives. J Adhes Dent 2015; 17: 155–161. doi: 10.3290/j.jad.a33892
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lternative in-office bleaching strategies have been the focus of several studies on preservation of the pulpdentin complex.19,35-39 It has been well demonstrated in the literature that traditional in-office protocols cause
a
Postdoctoral Student, Department of Physiology and Pathology, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Experimental procedure, wrote manuscript.
b
Postdoctoral Student, Department of Stomatology and Oral Pathology, Federal University of Goiás, Goiás, Brazil. Idea, hypothesis, experimental procedure.
c
Professor, Department of Dentistry, Federal University of Brasilia, and Campus Universitario Darcy Ribeiro, Brasilia, DF, Brazil. Experimental procedure.
d
Postdoctoral Student, Department of Physiology and Pathology, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Experimental procedure, contributed to discussion.
e
Postdoctoral Student, Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Experimental procedure, proofread manuscript.
f
Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Statistical analysis, contributed to discussion.
g
Professor, Department of Physiology and Pathology, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Hypothesis, idea, proofread manuscript, co-wrote manuscript.
Correspondence: Professor Carlos Alberto de Souza Costa, Department of Physiology and Pathology, University Estadual Paulista, UNESP, Araraquara School of Dentistry, Humaitá Street 1680, Araraquara, SP, Brazil 14801-903. Tel: +55-163301-6477. Fax: +55-16-3301-6488. e-mail: [email protected]
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Submitted for publication: 12.03.14; accepted for publication: 23.02.15
intense tooth sensitivity10,18,25,28,29,32,42 as well as damage to pulp cells in vitro19,35-39 and in vivo,15,20 which indicates that there should be concern about the safety of such procedures.25 This kind of professional bleaching protocol is based on 35% to 40% hydrogen peroxide (HP) gels applied to enamel for 30 to 45 min at each clinical appointment.10,18,28,32,42 Recently, Soares et al36 demonstrated that reducing contact time of the 35% HP gel to 15 min caused the same bleaching effect as the traditional protocol after five sessions. Additionally, about 60% of HP diffusion was observed,36 accomplished by minimizing the toxicity to cultured pulp cells.35,39 According to the authors, a high quantity of free non-reacted HP molecules diffuses through the enamel/dentin substrate to reach the pulp chamber after the application of highly concentrated bleaching gels to enamel for long periods, such as 45 min. Conversely, when bleaching time is reduced, the reaction of HP with dentin chromophores becomes more specific, reducing the amount of free HP capable of reaching the pulp space.35,36,39 A few studies have demonstrated that the presence of restorations on tooth surfaces scheduled for bleaching treatment enhances HP diffusion into the pulp chamber.9,11 A recent clinical investigation showed a higher prevalence of tooth sensitivity in patients with adhesive restorations in incisors subjected to a traditional in-office 155
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SILICON RING
WAX SEAL
HP GEL 1.6 mm ENAMEL 2.5 mm DENTIN
COMPOSITE RESIN
SILICON RING
IMMEDIATELY AFTER BLEACHING
EXTRACT 1 HOUR CONTACT TIME
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Fig 1 Schematic representation of the artificial pulp chamber (APC) and experimental protocols. a: APC/disk set positioned in a 24-well plate compartment. b: MDPC-23 cells previously cultured in wells of 24-well plates in contact with the extracts.
tooth-bleaching protocol.10 In contrast, Soares et al37 recently demonstrated that a 45-min treatment with highly concentrated bleaching gels performed on enamel/dentin disks containing an etch-and-rise adhesive/composite resin restoration had no toxic effect on odontoblast-like cells. According to Bonafé et al,10 the dental material and technique used to perform the cavity restoration, as well as the quality of restoration margins, may interfere with HP diffusion and consequently may affect tooth sensitivity. Therefore, as several restorative materials and bleaching protocols are available in clinical practice, the question related to the safety of bleaching performed on restored teeth remains. Much has been written about the effects of bleaching on composite resin materials;8,12,17,30,44,46 however, there is a dearth of studies evaluating the effect of the adhesive interface on the pathway of HP diffusion to the pulp chamber. Therefore, the aim of the present study was to assess the toxic effects on pulp cells caused by a less invasive in-office tooth-bleaching technique performed on enamel/dentin disks restored with a self-etching adhesive and composite resin, subjected or not to the aging process.
MATERIALS AND METHODS Cell Culture An immortalized odontoblast-like cell culture (MDPC-23) was used. The cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Gibco; Grand Island, NY, USA), supplemented with antibiotics (100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l glutamine; Gibco) and 10% fetal bovine serum (FBS; Gibco). To carry out the experimental procedure, the cells were seeded in 24-well plates at a density of 2 × 104 cells/cm2 for 48 h (80% confluence) at 37°C and 5% CO2 (Isotemp, Fisher Scientific; Pittsburgh, PA, USA). Enamel/Dentin Disks Enamel/dentin disks (5.6 mm diameter) were obtained from intact bovine incisors by the same method de156
scribed by Soares et al.37 The thickness of the disks was set at 3.5 mm by polishing the dentin surfaces with 400- and 600-grit abrasive papers. Enamel surfaces were cleaned with pumice stone and water solution, and standardized round cavities (1.6 mm diameter × 2.5 mm depth) were prepared in the experimental disks with a high-speed water-cooled cylindrical diamond bur (#1095, KG Sorensen; Barueri, SP, Brazil). Therefore, the remaining dentin thickness between the cavity floor and the pulp space was set at 1.0 mm.37 The cavities were restored with a two-step self-etching adhesive system (Adper SE Plus, 3M ESPE; St Paul, MN, USA) and a nanofilled composite resin (Filtek Z350; 3M ESPE) as follows: One layer of liquid A (primer) of Adper SE Plus was applied to the dentin surface and left untouched for 20 s. A first layer of liquid B (adhesive) of Adper SE Plus was applied under friction for 20 s, followed by 10 s of gentle air drying. Then, a second layer was applied and light cured for 20 s under a halogen lamp (450 mW/cm2 Curing Light XL 300, 3M ESPE). The cavity was restored by the application of two increments of a nanofilled composite resin (Filtek Z350; 3M ESPE), individually light cured for 20 s. Twentyfour hours after cavity restoration, the resin surface was polished with sequential Soflex disks (3M ESPE) at low speed. In the non-restored control disks, only a thin layer of composite resin was applied to the non-cavitated enamel surface, as described in detail by Soares et al.31 This procedure allowed us to evaluate the influence of the tooth/restoration interface (aged or not) on the diffusion of H2O2 and its cytotoxicity to cultured pulp cells. Thereafter, the disks (sound and restored) were stored for either 24 h or 6 months in water at 37°C, which was replaced every day until the moment of evaluation. The disks stored for 6 months were subjected to a thermocycling regimen in a thermal cycler (MSCT-3 plus, Marcelo Nucci-ME; São Carlos, SP, Brazil) for a total of 20,000 cycles at 5°C and 55°C, with a 30-s dwell time in each bath. After the storage periods, the dentin surfaces of disks were treated with EDTA 0.5 N for 30 s for smear layer removal. The disks were individually adapted to artificial The Journal of Adhesive Dentistry
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Experimental Procedure The disk/APC sets were positioned in 24-well plates containing 1 ml of DMEM with no FBS. The bleaching gel with 35% HP (Whiteness HP, FGM Produtos Odontológicos; Joinville, SC, Brazil) was applied to the enamel surfaces of the disks for 15 min. The following groups were studied (n = 13): G1 – negative control: sound disk + 24 h of water storage/non-bleached; G2 – sound disk + 24 h of water storage/bleached; G3 – restored disk + 24 h of water storage/non-bleached; G4 – restored disk + 24 h of water storage/bleached; G5 – sound disk + 6 months of water storage/non-bleached; G6 – sound disk + 6 months of water storage/bleached; G7 – restored disk + 6 months of water storage/non-bleached; G8 – restored disk + 6 months of water storage/bleached. Immediately after bleaching, the culture medium in contact with dentin was applied to previously cultured odontoblast-like MDPC-23 cells in 24-well plates and left for 1 h at 37°C and 5% CO2 (Isotemp; Fisher Scientific). A schematic representation of the APC/disk set and experimental protocols is shown in Fig 1. Cell Viability For this analysis, 11 APC/disk sets were used for each experimental group. After the contact time with the extracts, the cells were incubated for 4 h with MTT solution (5 mg/ml) diluted in DMEM without FBS (1:10), and absorbance of formazan crystals on viable cells was read in an ELISA microplate reader (Tp Reader, Thermoplate; Shenzhen, China) at 570 nm wavelength. The numeric values obtained from the MTT assay were converted into percentages according to mean absorbance observed in the control group (G1), which was considered as presenting 100% cell viability. Cell Morphology For morphological evaluation of the MDPC-23 cells, two APC/disk sets were used. Sterile 12-mm-diameter glass disks (Fisher Scientific) were placed at the bottom of the 24-well dishes before the cells were seeded. The same experimental procedure was performed as described above. After the extracts were removed, the cells attached to the glass substrate were washed with PBS, fixed in 1 ml buffered 2.5% glutaraldehyde for 60 min, and post-fixed for an additional 60 min in 1% osmium tetroxide, dehydrated with ethanol solutions (30%, 50%, 70%, 90%, and 100%), and chemically dried with HMDS (1,1,1,3,3,3-hexamethyldisilazane). The glass disks were mounted on metallic stubs, kept in a desiccator for 72 h, coated with gold, and analyzed by scanning electron microscopy (JEOL-JMS-T33A Scanning Microscope, JEOL; Tokyo, Japan). Vol 17, No 2, 2015
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A,a
A,a
100
AB,a
A,a
A,a AB,a
B,a B,a
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pulp chambers (APCs) as described by Soares et al.37 Two silicon rings (Rodimar Rolamentos; Araraquara, SP, Brazil) were used to promote the lateral seal between the upper and lower compartments of the APC, and an additional seal was performed with wax on the edges of the disks. The APCs with the enamel/dentin disks in position were sterilized in ethylene oxide gas.
80
60
40
20
0 24 hours
6 months
Water storage periods Error bars ± 1,00 SD Sound disk/ Non bleached Sound disk/ Bleached Restored disk/ Non bleached Restored disk/ Bleached
Fig 2 Bar graph of cell viability percentages according to treatment and water storage times. Different uppercase letters indicate statistically significant differences among groups (treatments) for each water storage period. Identical lowercase letters indicate no statistically significant differences for each treatment according to the water storage periods (Tukey’s test, p < 0.05). Values are mean ± SD (n = 11). G1 negative control: sound disk + 24 h water storage/non-bleached; G2 sound disk + 24 h water storage/bleached; G3 restored disk + 24 h water storage/non-bleached; G4 restored disk + 24 h water storage/ bleached; G5 sound disk + 6 months water storage/nonbleached; G6 sound disk + 6 months water storage/bleached; G7 restored disk + 6 months water storage/non-bleached; G8 restored disk + 6 months water storage/bleached.
Statistical Analysis Three independent experiments were performed in this investigation to assess the reproducibility of the data, which were then compiled and subjected to Levene’s test to verify homoscedasticity. Thereafter, the percentage of cell viability was analyzed by two-way (treatment x storage time) ANOVA and Tukey’s test. The significance level was set at 5% (_ = 0.05).
RESULTS The MTT assay results of cell viability are presented in Fig 2. No significant reductions in cell viability were observed in G2 (9.4%) and G6 (7.9%) (sound bleached teeth) compared with the control group (G1). Also, the restored and non-bleached groups (G3 and G7) did not differ from the negative control, demonstrating that the adhesive restoration had no cytotoxic potential. In contrast, the restored and bleached groups presented significant reductions in cell viability (G4 – 17.4%; 157
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GS
a
b
GS c
d
GS
e
f
GS
g 158
h
Fig 3 Representative scanning electron micrographs of cell morphology (original magnification 500X). a, c, e, g: Non-bleached groups: non-restored/24 h, non-restored/6 months, restored/24 h, and restored/6 months, respectively. A large number of MDPC-23 cells close to confluence can be observed adhering to the glass substrate. b, d: Non-restored/ bleached/24 h and non-restored/ bleached/6 months, respectively. The cells that remained adhering to the glass substrate exhibited an altered morphology, which caused some reduction in cell size, leaving small cell-free areas on the cover glass surface (GS). f, h: Restored/ bleached/24 h and restored/ bleached/6 months, respectively. Reduced cell size was observed, resulting in round cells and leaving large cell-free areas on the cover glass surface (GS).
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G8 – 22.2%) compared with the negative control. No difference among the bleached groups was observed with respect to storage time and the presence of restorations. SEM analysis showed alterations in cell morphology after application of the bleaching gel to enamel. The MDPC-23 cells exhibited a rounder morphology compared with cells in the non-bleached control group. There was a decrease in the number of cells attached to the glass substrate, indicating the occurrence of cell damage or death (Fig 3). These alterations were more evident in those groups restored with composite resin, especially in the disks subjected to aging by 6-month water storage and thermocycling (Figs 3F, 3H). In the non-bleached groups, numerous cells were observed to be near confluence. These cells exhibited a wide cytoplasm and were spread over the entire surface of the coverglass.
DISCUSSION It has been reported that the presence of restored cavities in teeth selected to be bleached may influence HP penetration into the pulp chamber, potentially subjecting the pulp-dentin complex to its toxic effects.9,11 Bonafé et al10 observed more intense tooth sensitivity immediately after applying a 35% HP bleaching gel for 45 min to anterior adhesively restored vital teeth compared with non-restored teeth. Therefore, in the present study, the influence of adhesive restorations on trans-enamel and trans-dentinal cytotoxicity was evaluated. It was shown that restoration contributed to enhanced cytotoxicity caused by bleaching therapy with a 35% HP gel applied to enamel for 15 min. Additionally, the alterations in cell morphology after the bleaching procedure were more accentuated in the restored groups, regardless of storage time (Fig 3). A recent study using a similar experimental protocol demonstrated the mechanisms of MDPC-23 viability reduction mediated by 35% HP bleaching gel applied to enamel/ dentin disks for 15 min.35 Those authors showed that the cell viability was reduced by 28.3% immediately after the bleaching procedure. Cell death by necrosis was determined by using the ethyl homodimer-1 probe that only binds to DNA bands of cells with membrane rupture, which consequently undergoe necrosis. It was also shown that 22.8% of cells exposed to this specific protocol were necrotic, which was proportional to the percentage of cell viability reduction, and around of 70% of remaining cells were under oxidative stress.35 According to the literature, this kind of cell death may occur because of direct contact of ROS from HP degradation with cell membranes, as well as the diffusion of HP through membranes, leading cells to an oxidative stress condition associated with lipid peroxidation.14,40 In that same study, other bleaching protocols were evaluated, showing that these adverse effects were proportional to the amount of HP able to diffuse through enamel and dentin.35 In this way, based on the results of the present study, it can be speculated that the greater cell viability decrease observed in restored/bleached groups occurred at least in part due to necrosis, followed by intense oxidative stress generation resulting from the higher diffusion of HP through Vol 17, No 2, 2015
the disks. Although the data obtained from laboratory investigations cannot be extrapolated to clinical conditions, an analysis of the results of the present study may partially explain the high tooth sensitivity described by Bonafé et al10 in their clinical study, as the amount of HP that reaches the pulp chamber has been directly related to the extent of oxidative damage to pulp cells.35 Tooth sensitivity has been associated with inflammatory pulp reactions due to the chemical irritation of pulp cells by oxidizing compounds from bleaching gels that reached the pulp chamber.20 This histopathological condition was previously demonstrated by in vivo studies in which human and animal teeth were bleached; its intensity was dependent on the bleaching protocol performed.15,20 If tooth-bleaching procedures cause pulp damage, then the release of cell-derived factors such as prostaglandins would excite or sensitize pulpal nociceptors.4,16,24,41 In addition, increased pulpal expression of substance P (a vasoactive neuropeptide) was observed in human premolars subjected to an in-office bleaching technique, indicating that neurogenic inflammation plays a role in bleaching-mediated tooth sensitivity.13 Moreover, it is known that during pulpal inflammation, the fluid shifts that occur in dentinal tubules due to vasodilation and increased pulp pressure trigger impulses in the intradentinal pulpal nerve fiber endings.5 Therefore, the higher the HP penetration into the pulp chamber, the higher the oxidative damage to the pulp cells and tooth sensitivity. According to the data presented in the present investigation, it seems that the adhesive interface may play an important role in this process. It has been shown that different adhesive systems present various levels of sensitivity to bleaching gels.21,22 In a recent study, Soares et al37 observed no significant difference concerning cytotoxicity and trans-enamel and trans-dentinal diffusion of HP when adhesive-restored and non-restored teeth were bleached with a 20% or 35% HP gel for 45 min. In that study, the authors used an etchand-rinse bonding agent (Single Bond, 3M ESPE), while a two-step self-etching adhesive (Adper SE, 3M ESPE) was used in the present investigation. Alves et al2 reported that Adper SE showed poor microtensile and nanoleakage results compared with Single Bond 2 (3M ESPE) 24 h after the restorative procedure. Additionally, Teixeira and Chain43 observed that Adper SE presented the lowest bond strength value compared with that of other two-step self-etching adhesive systems (Clearfil SE Bond, Kuraray; Optibond Solo Plus SE, Kerr; Tyrian SPE, Bisco). According to Van Landuyt et al,45 two-step self-adhesive systems have better mechanical performance than other self-etching categories. However, Roubickova et al33 observed that a two-step self-etching adhesive (Clearfil SE Bond) allowed a slight increase in microleakage at the enamel margins of resin composite restorations after bleaching treatment, indicating its sensitivity to peroxide. The authors also reported that the microleakage was not significantly affected by the bleaching procedure when an etch-and-rinse adhesive system (Gluma Comfort Bond, Heraeus Kulzer) was used. Dudek et al22 observed that tooth-bleaching procedures may significantly decrease bond strength among single-step self-etching adhesives and enamel/ 159
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dentin substrates. Conversely, these authors reported that tooth bleaching does not interfere with bond strength when etch-and-rinse systems are used. These data were corroborated by Didier et al,21 who observed that short treatment times with highly concentrated HP gels do not significantly influence the bond strength of etch-and-rinse restorations to mineralized dental tissues. The negative effects promoted by HP on self-etching adhesive interfaces have been correlated with the following: (1) the compromised bonding performance of self-etching adhesives to enamel and dentin creates a more permeable tooth/restoration interface, facilitating HP diffusion;33,45 (2) once at the interface, HP promotes oxidative degradation of adhesive components;33 (3) HP may denature dentin proteins at the interface, resulting in morphological changes that could reduce the bond between resin restorations and dentin.31 All of these factors may increase the ability of HP to diffuse through the interface to reach the pulp chamber. These mechanisms are believed to occur simultaneously during the application of bleaching gels to adhesive-restored teeth, with the adhesive systems showing different degrees of susceptibility, as follows: one-step self-etching > two-step selfetching > etch-and-rinse systems.22,33 In the present investigation, the aging process performed in half of the restored samples did not significantly influence the cytotoxicity caused by the bleaching procedure on the cultured cells. According to the literature, water storage is the most commonly used material aging process,3 and thermocycling simulates the thermal changes that occur in the oral cavity by repeated thermal cycles that induce contraction/expansion stress.27 This kind of tooth/restoration stress may create microgaps that allow the passage of oral fluids, favoring the hydrolytic degradation of the adhesive interface.3 In the present study, the storage time of 6 months was used with daily changes of water, which accelerates the degradation of the adhesive interface.27 In addition, the thermal regimen of 20,000 cycles was carried out to simulate approximately 2 years of clinical wear.23 However, Soares et al37 previously showed that this laboratory protocol does not significantly influence the indirect toxic effects caused by bleaching therapy on cultured MDPC-23 cells. This may be partially explained by the fact that the margins of the restored, thermocycled enamel/dentin samples were in enamel. In this specific condition, the adhesive/dentin bond is protected against degradation.1,3 It is known that when exposed adhesive/dentin-bonded surfaces are subjected to aging methods similar to those performed in this study, significant decreases in resin-bond strength occur after 6 months.6,7,26 Therefore, in addition to the adhesive system used for the restoration of dental cavities, the restoration margins may also play a role in the diffusion of HP through enamel and dentin to cause toxic effects on pulp cells. The cytotoxicity data obtained in this study for the sound teeth subjected to the alternative in-office protocol agree with those from previous investigations, as no difference in cell viability was found between sound and sound/bleached groups.35 Similar results were found by 160
Soares et al,39 where application of the 35% HP gel onto sound enamel/dentin disks for 15 min promoted only 1% viability reduction in MDPC-23 cells, after 1-h exposition time to the bleaching components able to diffuse through the disks. However, alterations in cell morphology and reductions in total protein production (34.5%) and alkaline phosphatase activity (50.9%) were also observed.39 Another recent study demonstrated that MDPC-23 cells exposed to this bleaching protocol experienced oxidative stress associated with cell membrane damage.35 Analysis of these data demonstrated that even the reduced amount of HP diffusing through enamel and dentin according to this bleaching technique35,39 is capable of causing alterations in pulp cells. However, it was also shown that the remaining pulp cells were able to proliferate significantly in a short time after bleaching (72 h) and to recover their odontoblastic phenotype.35 Therefore, this tooth-bleaching technique appears to be an interesting alternative for anterior teeth, minimizing pulpal damage and post-operative sensitivity. Nevertheless, further studies are needed to evaluate the effectiveness and safety of this technique in vivo. According to the data obtained in the present investigation, the presence of adhesive restorations performed with a self-etching adhesive system and composite resin significantly enhanced the toxicity of a 35% HP gel to odontoblast-like cells. Despite the limitations of this in vitro study, such as the absence of outward dentin fluid movement that may prevent the inward diffusion of dental materials components to the pulp chamber,34 clinicians should be aware that the esthetic procedure of bleaching in an adhesive-restored tooth may increase the possibility of pulp damage. The diffusion of toxic products across the tooth/restoration bond interface may be influenced by the adhesive system used as well as by the location and quality of restoration margins. Therefore, an accurate evaluation of restoration margins must be performed before adhesively restored teeth are subjected to bleaching. In addition, alternatives for the bleaching of restored teeth should be the focus of future studies, to enable clinicians to apply safe, painless bleaching protocols in treating their patients.
CONCLUSION It can be concluded that the application of a 35% HP bleaching gel to enamel for 15 min is not toxic to cultured odontoblast-like MDPC-23 cells. However, the presence of adhesive restorations performed with a twostep self-etching adhesive system enhanced the cytotoxicity of this tooth-bleaching protocol.
ACKNOWLEDGMENTS This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant # 2013/23520-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (grant # 301291/2010-1), and FUNDUNESP (grant # 0024/021/13-PROPE-CDC).
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Abdalla AI, Feilzer AJ. Four-year water degradation of a total-etch and two self-etching adhesives bonded to dentin. J Dent 2008;36:611-617. Alves FB, Lenzi TL, Reis A, Loguercio AD, Carvalho TS, Raggio DP. Bonding of simplified adhesive systems to caries-affected dentin of primary teeth. J Adhes Dent 2013;15:439-445. Amaral FB, Colluci V, Palma-Dib RG, Corona SAM. Assessment of in vitro methods used to promote adhesive interface degradation: a critical review. J Esthet Restor Dent 2007;19:340-353. Andersson DA, Gentry C, Moss S, Bevan S. Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J Neurosci 2008;28:2485-2494. Andrew D, Matthews B. Displacement of the contents of dentinal tubules and sensory transduction in intradental nerves of the cat. J Physiol 2000;529:791-802. Armstrong SR, Keller JC, Boyer DB. The influence of water storage and C-factor on the dentin-resin composite microtensile bond strength and debond pathway utilizing a filled and unfilled adhesive resin. Dent Mater 2001;17:268-276. Armstrong SR, Vargas MA, Fang Q, Laffoon JE. Microtensile bond strength of a total-etch 3-step, total-etch 2-step, self-etch 2-step, and a self-etch 1-step dentin bonding system through 15-month water storage. J Adhes Dent 2003;5:47-56. Barcellos DC, Benetti P, Fernandes Junior VVB, Valera MC. Effect of carbamide peroxide bleaching gel concentration on the bond strength of dental substrates and resin composite. Oper Dent 2010;35:463-469. Benetti AR, Valera MC, Mancini MNG, Miranda CB, Baldicci I. In vitro penetration of bleaching agents into the pulp chamber. Int Endod J 2004;37:120-124. Bonafé E, Bacovis CL, Iensen S, Loguercio AD, Reis A, Kossatz S. Tooth sensitivity and efficacy of in-office bleaching in restored teeth. J Dent 2013;41:363-369. Camargo SEA, Valera MC, Camargo CHR, Mancini MNG, Menezes MM. Penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique. J Endod 2007;33:1074-1077. Canay S1, Cehreli MC. The effect of current bleaching agents on the color of light-polymerized composites in vitro. J Prosthet Dent 2003; 89:474-478. Caviedes-Bucheli J, Ariza-Garcia G, Restrepo-Mendez S, Rios-Osorio N, Lombana N, Munoz HR. The effect of tooth bleaching on substance P expression in human dental pulp. J Endod 2008;34:1462–1465. Cecarini V, Gee J, Fioretti E, Amici M, Angeletti M, Eleuteri AMet al Protein oxidation and cellular homeostasis: emphasis on metabolism. Biochim Biophys Acta 2007;1773:93-104. Cintra LT, Benetti F, da Silva Facundo AC, Ferreira LL, Gomes-Filho JE, Ervolino E, Rahal V, Briso AL. The number of bleaching sessions influences pulp tissue damage in rat teeth. J Endod 2013;39:1576-1580. Cook SP, McCleskey EW. Cell damage excites nociceptors through release of cytosolic ATP. Pain 2002;95:41–47. Cullen DR, Nelson JA, Sandrik JL. Peroxide bleaches: effect on tensile strength of composite resins. J Prosthet Dent 1993;69:247-249. de Almeida LCAG, de Souza Costa CA, Riehl H, dos Santos PH, Sundfeld RH, Briso ALF. Occurrence of sensitivity during at-home and in-office tooth bleaching therapies with or without use of light sources. Acta Odontol Latinoam 2012;25:3-8. de Oliveira Duque CC, Soares DG, Basso FG, Hebling J, de Souza Costa CA. Bleaching effectiveness, hydrogen peroxide diffusion, and cytotoxicity of a chemically activated bleaching gel. Clin Oral Investig 2014;18:1631-1637. 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;109:59-64. Didier VF, Batista AUD, Montenegro RV, Fonseca RB, Carvalho FG, Barros S, Carlo HL. Influence of hydrogen peroxide-based bleaching agents on the bond strength of resin–enamel/dentin interfaces. Int J Adhes Adhesives 2013;47:141-145. Dudek M, Roubickova A, Comba L, Housova D, Bradna P. Effect of postoperative peroxide bleaching on the stability of composite to enamel and dentin bonds. Oper Dent 2013;38:394-407. Gale MS, Darvell BW. Thermal cycling procedures for laboratory testing of dental materials restorations. J Dent 1999;27:89-99. Goodis HE, Bowles WR, Hargreaves KM. Prostaglandin E2 enhances bradykinin-evoked iCGRP release in bovine dental pulp. J Dent Res 2000;79:1604–1607.
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25. Goldstein GR1, Kiremidjian-Schumacher L. Bleaching: is it safe and effective? J Prosthet Dent 1993;69:325-328. 26. Jacques P, Hebling J. Effect of dentin conditioners on the microtensile bond strength of a conventional and a self-etching primer adhesive system. Dent Mater 2005;21:103-109. 27. Kitasako Y, Burrow MF, Nikaido T, Tagami J. The influence of storage solution on dentin bond durability of resin cement. Dent Mater 2000;16:1-6. 28. Kossatz S, Dalanhol AP, Cunha T, Loguercio A, Reis A. Effect of light activation on tooth sensitivity after in-office bleaching. Oper Dent 2011;36:251-257. 29. Lee DH, Lim BS, Lee YK, Yang HC. Effects of hydrogen peroxide (H2O2) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines. Cell Biol Toxicol 2006;22:39-46. 30. Mujdeci A, Gokay O. Effect of bleaching agents on the microhardness of tooth-colored restorative materials. J Prosthet Dent 2006;95:286-289. 31. Perdigao J, Francci C, Swift EJ Jr, Ambrose WW, Lopes M. Ultra-morphological study of the interaction of dental adhesives with carbamide peroxide-bleached enamel. Am J Dent 1998;11:291–301. 32. Reis A, Dalanhol AP, Cunha TS, Kossatz S, Loguercio AD. Clinical effects of prolonged application time of an in-office bleaching gel. Oper Dent 2011;36:590-596. 33. Roubickova A, Dudek M, Comba L, Housova D, Bradna P. Effect of postoperative peroxide bleaching on the marginal seal of composite restorations bonded with self-etch adhesives. Oper Dent 2013;38:644-654. 34. Sauro S, Pashley DH, Montanari M, Chersoni S, Carvalho RM, Toledano M, Osorio R, Tay FR, Prati C. Effect of simulated pulpal pressure on dentin permeability and adhesion of self-etch adhesives. Dent Mater 2007;23:705-713. 35. 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;42:185-198. 36. Soares DG, Basso FG, Pontes EC, Garcia LD, Hebling J, de Souza Costa CA. Effective tooth-bleaching protocols capable of reducing H2O2 diffusion through enamel and dentine. J Dent 2014;42:351-358. 37. Soares DG, Pastana JV, Duque CC, Ribeiro AP, Basso FG, Hebling J, de Souza Costa CA. Influence of adhesive restorations on diffusion of H2O2 released from a bleaching agent and its toxic effects on pulp cells. J Adhes Dent 2014;16:123-128. 38. Soares DG, Pontes ECV, Ribeiro APD, Basso FG, Hebling J, de Souza Costa CA. Low toxic effects of a whitening strip to cultured pulp cells. Am J Dent 2013;26:283-285. 39. 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;17:1901-1909. 40. Squier TC. Oxidative stress and protein aggregation during biological aging. Exp Gerontol 2001;36:1539-1550. 41. Sawada Y, Hosokawa H, Matsumura K, Kobayashi S. Activation of transient receptor potential ankyrin 1 by hydrogen peroxide. Eur J Neurosci 2008;27:1131–1142. 42. Tay LY, Kose C, Herrera DR, Reis A, Loguercio AD. Long-term efficacy of in-office and at-home bleaching: a 2-year double-blind randomized clinical trial. Am J Dent 2012;25:199-204. 43. Teixeira CS, Chain MC. Evaluation of shear bond strength between selfetching adhesive systems and dentin and analysis of the resin-dentin interface. Gen Dent 2010;58:e52-e61. 44. Turker SB, Biskin T. Effect of three bleaching agents on the surface properties of three different esthetic restorative materials. J Prosthet Dent 2003;89:466-473. 45. Van Landuyt KL, Mine A, De Munck J, Jaecques S, Peumans M, Lambrechts P, Van Meerbeek B. Are one-step adhesives easier to use and better performing? Multifactorial assessment of contemporary onestep self-etching adhesives. J Adhes Dent 2009;11:175-190. 46. Yu H, Li Q, Cheng H, Wang Y. The effects of temperature and bleaching gels on the properties of tooth-colored restorative materials. J Prosthet Dent 2011;105:100-107.
Clinical relevance: This study demonstrated that a less invasive bleaching technique might become more toxic to the pulp-dentin complex when applied to teeth containing an adhesive interface. Therefore, a detailed analysis of adhesive interfaces must be performed to verify the safety of performing bleaching on restored teeth.
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lternative in-office bleaching strategies have been the focus of several studies on preservation of the pulpdentin complex.19,35-39 It has been well demonstrated in the literature that traditional in-office protocols cause
a
Postdoctoral Student, Department of Physiology and Pathology, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Experimental procedure, wrote manuscript.
b
Postdoctoral Student, Department of Stomatology and Oral Pathology, Federal University of Goiás, Goiás, Brazil. Idea, hypothesis, experimental procedure.
c
Professor, Department of Dentistry, Federal University of Brasilia, and Campus Universitario Darcy Ribeiro, Brasilia, DF, Brazil. Experimental procedure.
d
Postdoctoral Student, Department of Physiology and Pathology, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Experimental procedure, contributed to discussion.
e
Postdoctoral Student, Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Experimental procedure, proofread manuscript.
f
Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Statistical analysis, contributed to discussion.
g
Professor, Department of Physiology and Pathology, Araraquara School of Dentistry, UNESP, University Estadual Paulista, Araraquara, São Paulo, Brazil. Hypothesis, idea, proofread manuscript, co-wrote manuscript.
Correspondence: Professor Carlos Alberto de Souza Costa, Department of Physiology and Pathology, University Estadual Paulista, UNESP, Araraquara School of Dentistry, Humaitá Street 1680, Araraquara, SP, Brazil 14801-903. Tel: +55-163301-6477. Fax: +55-16-3301-6488. e-mail: [email protected]
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Submitted for publication: 12.03.14; accepted for publication: 23.02.15
intense tooth sensitivity10,18,25,28,29,32,42 as well as damage to pulp cells in vitro19,35-39 and in vivo,15,20 which indicates that there should be concern about the safety of such procedures.25 This kind of professional bleaching protocol is based on 35% to 40% hydrogen peroxide (HP) gels applied to enamel for 30 to 45 min at each clinical appointment.10,18,28,32,42 Recently, Soares et al36 demonstrated that reducing contact time of the 35% HP gel to 15 min caused the same bleaching effect as the traditional protocol after five sessions. Additionally, about 60% of HP diffusion was observed,36 accomplished by minimizing the toxicity to cultured pulp cells.35,39 According to the authors, a high quantity of free non-reacted HP molecules diffuses through the enamel/dentin substrate to reach the pulp chamber after the application of highly concentrated bleaching gels to enamel for long periods, such as 45 min. Conversely, when bleaching time is reduced, the reaction of HP with dentin chromophores becomes more specific, reducing the amount of free HP capable of reaching the pulp space.35,36,39 A few studies have demonstrated that the presence of restorations on tooth surfaces scheduled for bleaching treatment enhances HP diffusion into the pulp chamber.9,11 A recent clinical investigation showed a higher prevalence of tooth sensitivity in patients with adhesive restorations in incisors subjected to a traditional in-office 155
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SILICON RING
WAX SEAL
HP GEL 1.6 mm ENAMEL 2.5 mm DENTIN
COMPOSITE RESIN
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IMMEDIATELY AFTER BLEACHING
EXTRACT 1 HOUR CONTACT TIME
EXTRACT
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Fig 1 Schematic representation of the artificial pulp chamber (APC) and experimental protocols. a: APC/disk set positioned in a 24-well plate compartment. b: MDPC-23 cells previously cultured in wells of 24-well plates in contact with the extracts.
tooth-bleaching protocol.10 In contrast, Soares et al37 recently demonstrated that a 45-min treatment with highly concentrated bleaching gels performed on enamel/dentin disks containing an etch-and-rise adhesive/composite resin restoration had no toxic effect on odontoblast-like cells. According to Bonafé et al,10 the dental material and technique used to perform the cavity restoration, as well as the quality of restoration margins, may interfere with HP diffusion and consequently may affect tooth sensitivity. Therefore, as several restorative materials and bleaching protocols are available in clinical practice, the question related to the safety of bleaching performed on restored teeth remains. Much has been written about the effects of bleaching on composite resin materials;8,12,17,30,44,46 however, there is a dearth of studies evaluating the effect of the adhesive interface on the pathway of HP diffusion to the pulp chamber. Therefore, the aim of the present study was to assess the toxic effects on pulp cells caused by a less invasive in-office tooth-bleaching technique performed on enamel/dentin disks restored with a self-etching adhesive and composite resin, subjected or not to the aging process.
MATERIALS AND METHODS Cell Culture An immortalized odontoblast-like cell culture (MDPC-23) was used. The cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Gibco; Grand Island, NY, USA), supplemented with antibiotics (100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l glutamine; Gibco) and 10% fetal bovine serum (FBS; Gibco). To carry out the experimental procedure, the cells were seeded in 24-well plates at a density of 2 × 104 cells/cm2 for 48 h (80% confluence) at 37°C and 5% CO2 (Isotemp, Fisher Scientific; Pittsburgh, PA, USA). Enamel/Dentin Disks Enamel/dentin disks (5.6 mm diameter) were obtained from intact bovine incisors by the same method de156
scribed by Soares et al.37 The thickness of the disks was set at 3.5 mm by polishing the dentin surfaces with 400- and 600-grit abrasive papers. Enamel surfaces were cleaned with pumice stone and water solution, and standardized round cavities (1.6 mm diameter × 2.5 mm depth) were prepared in the experimental disks with a high-speed water-cooled cylindrical diamond bur (#1095, KG Sorensen; Barueri, SP, Brazil). Therefore, the remaining dentin thickness between the cavity floor and the pulp space was set at 1.0 mm.37 The cavities were restored with a two-step self-etching adhesive system (Adper SE Plus, 3M ESPE; St Paul, MN, USA) and a nanofilled composite resin (Filtek Z350; 3M ESPE) as follows: One layer of liquid A (primer) of Adper SE Plus was applied to the dentin surface and left untouched for 20 s. A first layer of liquid B (adhesive) of Adper SE Plus was applied under friction for 20 s, followed by 10 s of gentle air drying. Then, a second layer was applied and light cured for 20 s under a halogen lamp (450 mW/cm2 Curing Light XL 300, 3M ESPE). The cavity was restored by the application of two increments of a nanofilled composite resin (Filtek Z350; 3M ESPE), individually light cured for 20 s. Twentyfour hours after cavity restoration, the resin surface was polished with sequential Soflex disks (3M ESPE) at low speed. In the non-restored control disks, only a thin layer of composite resin was applied to the non-cavitated enamel surface, as described in detail by Soares et al.31 This procedure allowed us to evaluate the influence of the tooth/restoration interface (aged or not) on the diffusion of H2O2 and its cytotoxicity to cultured pulp cells. Thereafter, the disks (sound and restored) were stored for either 24 h or 6 months in water at 37°C, which was replaced every day until the moment of evaluation. The disks stored for 6 months were subjected to a thermocycling regimen in a thermal cycler (MSCT-3 plus, Marcelo Nucci-ME; São Carlos, SP, Brazil) for a total of 20,000 cycles at 5°C and 55°C, with a 30-s dwell time in each bath. After the storage periods, the dentin surfaces of disks were treated with EDTA 0.5 N for 30 s for smear layer removal. The disks were individually adapted to artificial The Journal of Adhesive Dentistry
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Experimental Procedure The disk/APC sets were positioned in 24-well plates containing 1 ml of DMEM with no FBS. The bleaching gel with 35% HP (Whiteness HP, FGM Produtos Odontológicos; Joinville, SC, Brazil) was applied to the enamel surfaces of the disks for 15 min. The following groups were studied (n = 13): G1 – negative control: sound disk + 24 h of water storage/non-bleached; G2 – sound disk + 24 h of water storage/bleached; G3 – restored disk + 24 h of water storage/non-bleached; G4 – restored disk + 24 h of water storage/bleached; G5 – sound disk + 6 months of water storage/non-bleached; G6 – sound disk + 6 months of water storage/bleached; G7 – restored disk + 6 months of water storage/non-bleached; G8 – restored disk + 6 months of water storage/bleached. Immediately after bleaching, the culture medium in contact with dentin was applied to previously cultured odontoblast-like MDPC-23 cells in 24-well plates and left for 1 h at 37°C and 5% CO2 (Isotemp; Fisher Scientific). A schematic representation of the APC/disk set and experimental protocols is shown in Fig 1. Cell Viability For this analysis, 11 APC/disk sets were used for each experimental group. After the contact time with the extracts, the cells were incubated for 4 h with MTT solution (5 mg/ml) diluted in DMEM without FBS (1:10), and absorbance of formazan crystals on viable cells was read in an ELISA microplate reader (Tp Reader, Thermoplate; Shenzhen, China) at 570 nm wavelength. The numeric values obtained from the MTT assay were converted into percentages according to mean absorbance observed in the control group (G1), which was considered as presenting 100% cell viability. Cell Morphology For morphological evaluation of the MDPC-23 cells, two APC/disk sets were used. Sterile 12-mm-diameter glass disks (Fisher Scientific) were placed at the bottom of the 24-well dishes before the cells were seeded. The same experimental procedure was performed as described above. After the extracts were removed, the cells attached to the glass substrate were washed with PBS, fixed in 1 ml buffered 2.5% glutaraldehyde for 60 min, and post-fixed for an additional 60 min in 1% osmium tetroxide, dehydrated with ethanol solutions (30%, 50%, 70%, 90%, and 100%), and chemically dried with HMDS (1,1,1,3,3,3-hexamethyldisilazane). The glass disks were mounted on metallic stubs, kept in a desiccator for 72 h, coated with gold, and analyzed by scanning electron microscopy (JEOL-JMS-T33A Scanning Microscope, JEOL; Tokyo, Japan). Vol 17, No 2, 2015
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A,a
A,a
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AB,a
A,a
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pulp chambers (APCs) as described by Soares et al.37 Two silicon rings (Rodimar Rolamentos; Araraquara, SP, Brazil) were used to promote the lateral seal between the upper and lower compartments of the APC, and an additional seal was performed with wax on the edges of the disks. The APCs with the enamel/dentin disks in position were sterilized in ethylene oxide gas.
80
60
40
20
0 24 hours
6 months
Water storage periods Error bars ± 1,00 SD Sound disk/ Non bleached Sound disk/ Bleached Restored disk/ Non bleached Restored disk/ Bleached
Fig 2 Bar graph of cell viability percentages according to treatment and water storage times. Different uppercase letters indicate statistically significant differences among groups (treatments) for each water storage period. Identical lowercase letters indicate no statistically significant differences for each treatment according to the water storage periods (Tukey’s test, p < 0.05). Values are mean ± SD (n = 11). G1 negative control: sound disk + 24 h water storage/non-bleached; G2 sound disk + 24 h water storage/bleached; G3 restored disk + 24 h water storage/non-bleached; G4 restored disk + 24 h water storage/ bleached; G5 sound disk + 6 months water storage/nonbleached; G6 sound disk + 6 months water storage/bleached; G7 restored disk + 6 months water storage/non-bleached; G8 restored disk + 6 months water storage/bleached.
Statistical Analysis Three independent experiments were performed in this investigation to assess the reproducibility of the data, which were then compiled and subjected to Levene’s test to verify homoscedasticity. Thereafter, the percentage of cell viability was analyzed by two-way (treatment x storage time) ANOVA and Tukey’s test. The significance level was set at 5% (_ = 0.05).
RESULTS The MTT assay results of cell viability are presented in Fig 2. No significant reductions in cell viability were observed in G2 (9.4%) and G6 (7.9%) (sound bleached teeth) compared with the control group (G1). Also, the restored and non-bleached groups (G3 and G7) did not differ from the negative control, demonstrating that the adhesive restoration had no cytotoxic potential. In contrast, the restored and bleached groups presented significant reductions in cell viability (G4 – 17.4%; 157
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GS
a
b
GS c
d
GS
e
f
GS
g 158
h
Fig 3 Representative scanning electron micrographs of cell morphology (original magnification 500X). a, c, e, g: Non-bleached groups: non-restored/24 h, non-restored/6 months, restored/24 h, and restored/6 months, respectively. A large number of MDPC-23 cells close to confluence can be observed adhering to the glass substrate. b, d: Non-restored/ bleached/24 h and non-restored/ bleached/6 months, respectively. The cells that remained adhering to the glass substrate exhibited an altered morphology, which caused some reduction in cell size, leaving small cell-free areas on the cover glass surface (GS). f, h: Restored/ bleached/24 h and restored/ bleached/6 months, respectively. Reduced cell size was observed, resulting in round cells and leaving large cell-free areas on the cover glass surface (GS).
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G8 – 22.2%) compared with the negative control. No difference among the bleached groups was observed with respect to storage time and the presence of restorations. SEM analysis showed alterations in cell morphology after application of the bleaching gel to enamel. The MDPC-23 cells exhibited a rounder morphology compared with cells in the non-bleached control group. There was a decrease in the number of cells attached to the glass substrate, indicating the occurrence of cell damage or death (Fig 3). These alterations were more evident in those groups restored with composite resin, especially in the disks subjected to aging by 6-month water storage and thermocycling (Figs 3F, 3H). In the non-bleached groups, numerous cells were observed to be near confluence. These cells exhibited a wide cytoplasm and were spread over the entire surface of the coverglass.
DISCUSSION It has been reported that the presence of restored cavities in teeth selected to be bleached may influence HP penetration into the pulp chamber, potentially subjecting the pulp-dentin complex to its toxic effects.9,11 Bonafé et al10 observed more intense tooth sensitivity immediately after applying a 35% HP bleaching gel for 45 min to anterior adhesively restored vital teeth compared with non-restored teeth. Therefore, in the present study, the influence of adhesive restorations on trans-enamel and trans-dentinal cytotoxicity was evaluated. It was shown that restoration contributed to enhanced cytotoxicity caused by bleaching therapy with a 35% HP gel applied to enamel for 15 min. Additionally, the alterations in cell morphology after the bleaching procedure were more accentuated in the restored groups, regardless of storage time (Fig 3). A recent study using a similar experimental protocol demonstrated the mechanisms of MDPC-23 viability reduction mediated by 35% HP bleaching gel applied to enamel/ dentin disks for 15 min.35 Those authors showed that the cell viability was reduced by 28.3% immediately after the bleaching procedure. Cell death by necrosis was determined by using the ethyl homodimer-1 probe that only binds to DNA bands of cells with membrane rupture, which consequently undergoe necrosis. It was also shown that 22.8% of cells exposed to this specific protocol were necrotic, which was proportional to the percentage of cell viability reduction, and around of 70% of remaining cells were under oxidative stress.35 According to the literature, this kind of cell death may occur because of direct contact of ROS from HP degradation with cell membranes, as well as the diffusion of HP through membranes, leading cells to an oxidative stress condition associated with lipid peroxidation.14,40 In that same study, other bleaching protocols were evaluated, showing that these adverse effects were proportional to the amount of HP able to diffuse through enamel and dentin.35 In this way, based on the results of the present study, it can be speculated that the greater cell viability decrease observed in restored/bleached groups occurred at least in part due to necrosis, followed by intense oxidative stress generation resulting from the higher diffusion of HP through Vol 17, No 2, 2015
the disks. Although the data obtained from laboratory investigations cannot be extrapolated to clinical conditions, an analysis of the results of the present study may partially explain the high tooth sensitivity described by Bonafé et al10 in their clinical study, as the amount of HP that reaches the pulp chamber has been directly related to the extent of oxidative damage to pulp cells.35 Tooth sensitivity has been associated with inflammatory pulp reactions due to the chemical irritation of pulp cells by oxidizing compounds from bleaching gels that reached the pulp chamber.20 This histopathological condition was previously demonstrated by in vivo studies in which human and animal teeth were bleached; its intensity was dependent on the bleaching protocol performed.15,20 If tooth-bleaching procedures cause pulp damage, then the release of cell-derived factors such as prostaglandins would excite or sensitize pulpal nociceptors.4,16,24,41 In addition, increased pulpal expression of substance P (a vasoactive neuropeptide) was observed in human premolars subjected to an in-office bleaching technique, indicating that neurogenic inflammation plays a role in bleaching-mediated tooth sensitivity.13 Moreover, it is known that during pulpal inflammation, the fluid shifts that occur in dentinal tubules due to vasodilation and increased pulp pressure trigger impulses in the intradentinal pulpal nerve fiber endings.5 Therefore, the higher the HP penetration into the pulp chamber, the higher the oxidative damage to the pulp cells and tooth sensitivity. According to the data presented in the present investigation, it seems that the adhesive interface may play an important role in this process. It has been shown that different adhesive systems present various levels of sensitivity to bleaching gels.21,22 In a recent study, Soares et al37 observed no significant difference concerning cytotoxicity and trans-enamel and trans-dentinal diffusion of HP when adhesive-restored and non-restored teeth were bleached with a 20% or 35% HP gel for 45 min. In that study, the authors used an etchand-rinse bonding agent (Single Bond, 3M ESPE), while a two-step self-etching adhesive (Adper SE, 3M ESPE) was used in the present investigation. Alves et al2 reported that Adper SE showed poor microtensile and nanoleakage results compared with Single Bond 2 (3M ESPE) 24 h after the restorative procedure. Additionally, Teixeira and Chain43 observed that Adper SE presented the lowest bond strength value compared with that of other two-step self-etching adhesive systems (Clearfil SE Bond, Kuraray; Optibond Solo Plus SE, Kerr; Tyrian SPE, Bisco). According to Van Landuyt et al,45 two-step self-adhesive systems have better mechanical performance than other self-etching categories. However, Roubickova et al33 observed that a two-step self-etching adhesive (Clearfil SE Bond) allowed a slight increase in microleakage at the enamel margins of resin composite restorations after bleaching treatment, indicating its sensitivity to peroxide. The authors also reported that the microleakage was not significantly affected by the bleaching procedure when an etch-and-rinse adhesive system (Gluma Comfort Bond, Heraeus Kulzer) was used. Dudek et al22 observed that tooth-bleaching procedures may significantly decrease bond strength among single-step self-etching adhesives and enamel/ 159
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dentin substrates. Conversely, these authors reported that tooth bleaching does not interfere with bond strength when etch-and-rinse systems are used. These data were corroborated by Didier et al,21 who observed that short treatment times with highly concentrated HP gels do not significantly influence the bond strength of etch-and-rinse restorations to mineralized dental tissues. The negative effects promoted by HP on self-etching adhesive interfaces have been correlated with the following: (1) the compromised bonding performance of self-etching adhesives to enamel and dentin creates a more permeable tooth/restoration interface, facilitating HP diffusion;33,45 (2) once at the interface, HP promotes oxidative degradation of adhesive components;33 (3) HP may denature dentin proteins at the interface, resulting in morphological changes that could reduce the bond between resin restorations and dentin.31 All of these factors may increase the ability of HP to diffuse through the interface to reach the pulp chamber. These mechanisms are believed to occur simultaneously during the application of bleaching gels to adhesive-restored teeth, with the adhesive systems showing different degrees of susceptibility, as follows: one-step self-etching > two-step selfetching > etch-and-rinse systems.22,33 In the present investigation, the aging process performed in half of the restored samples did not significantly influence the cytotoxicity caused by the bleaching procedure on the cultured cells. According to the literature, water storage is the most commonly used material aging process,3 and thermocycling simulates the thermal changes that occur in the oral cavity by repeated thermal cycles that induce contraction/expansion stress.27 This kind of tooth/restoration stress may create microgaps that allow the passage of oral fluids, favoring the hydrolytic degradation of the adhesive interface.3 In the present study, the storage time of 6 months was used with daily changes of water, which accelerates the degradation of the adhesive interface.27 In addition, the thermal regimen of 20,000 cycles was carried out to simulate approximately 2 years of clinical wear.23 However, Soares et al37 previously showed that this laboratory protocol does not significantly influence the indirect toxic effects caused by bleaching therapy on cultured MDPC-23 cells. This may be partially explained by the fact that the margins of the restored, thermocycled enamel/dentin samples were in enamel. In this specific condition, the adhesive/dentin bond is protected against degradation.1,3 It is known that when exposed adhesive/dentin-bonded surfaces are subjected to aging methods similar to those performed in this study, significant decreases in resin-bond strength occur after 6 months.6,7,26 Therefore, in addition to the adhesive system used for the restoration of dental cavities, the restoration margins may also play a role in the diffusion of HP through enamel and dentin to cause toxic effects on pulp cells. The cytotoxicity data obtained in this study for the sound teeth subjected to the alternative in-office protocol agree with those from previous investigations, as no difference in cell viability was found between sound and sound/bleached groups.35 Similar results were found by 160
Soares et al,39 where application of the 35% HP gel onto sound enamel/dentin disks for 15 min promoted only 1% viability reduction in MDPC-23 cells, after 1-h exposition time to the bleaching components able to diffuse through the disks. However, alterations in cell morphology and reductions in total protein production (34.5%) and alkaline phosphatase activity (50.9%) were also observed.39 Another recent study demonstrated that MDPC-23 cells exposed to this bleaching protocol experienced oxidative stress associated with cell membrane damage.35 Analysis of these data demonstrated that even the reduced amount of HP diffusing through enamel and dentin according to this bleaching technique35,39 is capable of causing alterations in pulp cells. However, it was also shown that the remaining pulp cells were able to proliferate significantly in a short time after bleaching (72 h) and to recover their odontoblastic phenotype.35 Therefore, this tooth-bleaching technique appears to be an interesting alternative for anterior teeth, minimizing pulpal damage and post-operative sensitivity. Nevertheless, further studies are needed to evaluate the effectiveness and safety of this technique in vivo. According to the data obtained in the present investigation, the presence of adhesive restorations performed with a self-etching adhesive system and composite resin significantly enhanced the toxicity of a 35% HP gel to odontoblast-like cells. Despite the limitations of this in vitro study, such as the absence of outward dentin fluid movement that may prevent the inward diffusion of dental materials components to the pulp chamber,34 clinicians should be aware that the esthetic procedure of bleaching in an adhesive-restored tooth may increase the possibility of pulp damage. The diffusion of toxic products across the tooth/restoration bond interface may be influenced by the adhesive system used as well as by the location and quality of restoration margins. Therefore, an accurate evaluation of restoration margins must be performed before adhesively restored teeth are subjected to bleaching. In addition, alternatives for the bleaching of restored teeth should be the focus of future studies, to enable clinicians to apply safe, painless bleaching protocols in treating their patients.
CONCLUSION It can be concluded that the application of a 35% HP bleaching gel to enamel for 15 min is not toxic to cultured odontoblast-like MDPC-23 cells. However, the presence of adhesive restorations performed with a twostep self-etching adhesive system enhanced the cytotoxicity of this tooth-bleaching protocol.
ACKNOWLEDGMENTS This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant # 2013/23520-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (grant # 301291/2010-1), and FUNDUNESP (grant # 0024/021/13-PROPE-CDC).
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Clinical relevance: This study demonstrated that a less invasive bleaching technique might become more toxic to the pulp-dentin complex when applied to teeth containing an adhesive interface. Therefore, a detailed analysis of adhesive interfaces must be performed to verify the safety of performing bleaching on restored teeth.
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