doi:10.1111/iej.12479

Cytocompatibility of HEMA–free resin–based luting cements according to application protocols on dentine surfaces

D. G. Soares1, C. A. Brito2, R. H. B. Tavares da Silva3, A. P. D. Ribeiro4, J. Hebling5 & C. A. de Souza Costa1 Department of Physiology and Pathology, Araraquara School of Dentistry, Universidade Estadual Paulista – UNESP, ania, Goi as; 3Department of Dental Materials and Araraquara, S~ ao Paulo; 2Department of Dentistry, Paulista University, Goi^ Prosthodontics, Araraquara School of Dentistry, Universidade Estadual Paulista – UNESP, Araraquara, S~ ao Paulo; 4Department 5 of Dentistry, Federal University of Brasilia, Brasilia, Campus Universitario Darcy Ribeiro, Brazilia; and Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, Universidade Estadual Paulista – UNESP, Araraquara, S~ao Paulo, Brazil 1

Abstract Soares DG, Brito CA, Tavares da Silva RHB, Ribeiro APD, Hebling J, de Souza Costa CA. Cytocompatibility of HEMA–free resin–based luting cements according to application

protocols

on

dentine

surfaces.

International

Endodontic Journal, 49, 551–560, 2016.

Aim To evaluate the transdentinal cytotoxicity of resin–based luting cements (RBLCs), with no HEMA in their composition, to odontoblast–like cells. Methodology Human dentine discs 0.3 mm thick were adapted to artificial pulp chambers (APCs) and placed in wells of 24–well plates containing 1 mL of culture medium (DMEM). Two categories of HEMA–free RBLCs were evaluated: group 1, self–adhesive Rely X Unicem (RU; 3M ESPE), applied directly to the dentine substrate; and group 2, Rely X ARC (RARC; 3M ESPE), applied to dentine previously acid–etched and treated with a bonding agent. In group 3 (control), considered as representing 100% cell metabolic activity, no treatment was performed on dentine. The APC/disc sets were incubated for 24 h or 7 days at 37 °C and 5% CO2. Then, the extracts (DMEM + dental materials components that diffused through dentine) were applied to cultured odontoblast–like MDPC–23 cells for 24 h. After that, the cell

Correspondence: Carlos Alberto de Souza Costa, Department of Physiology and Pathology, Univ Estadual Paulista – UNESP, Araraquara School of Dentistry, Humait a Street, 1680, Araraquara, SP 14801–903, Brazil (Tel: +55 (16) 3301–6477; Fax: +55 (16) 3301–6488; e–mail: [email protected]).

© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

viability (MTT assay), cell morphology (SEM), total protein production (TP) and alkaline phosphatase (ALP) activity were assessed. Data from MTT assay and TP production were analysed by Kruskal–Wallis and Mann– Whitney tests (a = 5%). Data from ALP activity were analysed by one–way ANOVA and Tukey’s test (a = 5%). Results In group 1, a slight reduction in cell viability (11.6% and 16.8% for 24–h and 7–day periods, respectively) and ALP activity (13.5% and 17.9% for 24–h and 7–day periods, respectively) was observed, with no significant difference from group 3 (control) (P > 0.05). In group 2, a significant reduction in cell viability, TP production and ALP activity compared with group 3 (control) occurred (P < 0.05), regardless of incubation time. Alteration in MDPC–23 cell morphology was observed only in group 2. Conclusions HEMA–free Rely X ARC cement caused greater toxicity to odontoblast–like MDPC–23 cells than did Rely X Unicem cement when both resin–based luting materials were applied to dentine as recommended by the manufacturer. Keywords: cell culture, resin–based cements.

cytotoxicity,

dentine,

Received 1 March 2015; accepted 5 June 2015

Introduction Resin–based materials, such as bonding agents, composite resins and luting cements, have been widely used in operative dentistry, as these dental materials

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feature interesting properties such as enhanced aesthetics and adhesion to mineralized dental tissues (Radovic et al. 2008, Makkar & Malhotra 2013). However, a concern regarding these dental materials is related to their biocompatibility with the pulp–dentine complex (de Souza Costa et al. 2014). It was previously demonstrated that unreacted free monomers leached from resin–based materials in wet environments are capable of inducing oxidative stress–mediated pulp cell death, inflammatory mediator over– expression and cause depletion of glutathione peroxidase and superoxide dismutase enzymes (Hanks et al. 1991, Stanislawski et al. 2003, Lefeuvre et al. 2005, Reichl et al. 2008, Chang et al. 2009, 2010, Drozdz et al. 2011, Wisniewska–Jarosinska et al. 2011, Chang et al. 2012, Krifka et al. 2012, Batarseh et al. 2014, Botsali et al. 2014). Also, free toxic monomers can alter the phenotypic characteristics of dental pulp stem cells, interfering with the regenerative potential of pulp tissue (Bakopoulou et al. 2011, Galler et al. 2011, Martins et al. 2012, Paschalidis et al. 2014). In clinical situations, the ability of residual monomers to elicit toxic reactions in pulp tissue is also directly related to dentine permeability (Perdig~ ao 2002). Next to the pulp chamber, the diameter and number of dentinal tubules per area increase, resulting in high permeability (de Souza Costa et al. 2014). This permeable substrate features a large quantity of dentinal fluid, which may interfere with the polymerization of resin materials enhancing the local presence of toxic unreacted free monomers (Abedin et al. 2014, Tj€ aderhane 2015). However, it is known that the chemical composition of dental materials as well as the protocols of application to dentine play a central role in defining their compatibility with the pulp–dentine complex (de Souza Costa et al. 2002, 2007). Smear layer removal prior to the application of resin–based materials to dentine significantly increases the toxic potential of restorative procedures, as it enhances dentine permeability and local humidity (de Souza Costa et al. 2002, 2007, Lanza et al. 2009). Also, materials with a high content of 2–hydroxyethyl methacrylate (HEMA) cause high transdentinal toxicity to pulp cells (de Souza Costa et al. 2006, Barbosa et al. 2015), as this resin monomer has low molecular weight and hydrophilicity, which favour its transdentinal diffusion (Gerzina & Hume 1995, 1996, Hamid & Hume 1997, Ceting€ ußc et al. 2007). Therefore, it may be suggested that HEMA–free dental materials that do not require dentine conditioning prior to their clinical use would be

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considered a more biocompatible option to be applied on deep dentine. Therefore, the aim of the present study was to assess the indirect cytocompatibility of resin–based luting cements without HEMA in their composition according to their application protocols to dentine surface, following the manufacturer’s recommendations. The null hypothesis is that the tested cements have no transdentinal cytotoxic effects to odontoblast–like cells, regardless of the application protocol on dentine surfaces.

Materials and methods Dentine discs After approval by the Research Ethics Committee of the Araraquara School of Dentistry – UNESP (Proc.11/10), S~ ao Paulo, Brazil, 50 dentine discs, approximately 0.5 mm thick, were obtained from healthy human third molars. For this purpose, transverse cuts were made in the coronal portion of the teeth, above the projection of the pulp horns, and below the dentine–enamel junction, by means of a diamond disc (11–4254, 4″ 9 0.012″/series 15LC, Diamond Blade; Buehler Ltd., Lake Bluff, IL, USA) mounted in a metallographic cutter (ISOMET 1000, Buehler Ltd.). The discs were meticulously inspected under a stereoscopic loupe (Model SZX7; Olympus, S~ ao Paulo, Brazil), and those with pulp horns and/or enamel islands were excluded. Disc thickness was then reduced to 0.3 mm by means of 400– and 600– grit water–abrasive papers (T469–SF – Norton; Saint– Gobam Abrasivos Ltda., Jundiai, SP, Brazil). The smear layer formed by wear was removed by the application of EDTA (0.5 mol L–1; pH 7.4) for 60 s to each surface of the discs, followed by washing with deionized water. The dentine discs with final thickness standardized at 0.3 mm were mounted in a filtering chamber connected to a water column of 180 cm, and the hydraulic conductance (Lp; lL/cm2/min/cm H2O) was calculated for each disc (Lanza et al. 2009). After that, the occlusal surface of each disc was ground for 10 s with abrasive papers (T469–SF – Norton, Saint–Gobam Abrasivos Ltda.) to obtain a smear layer similar to that created when a thin diamond bur is used on the dentine surface (Reis et al. 2005). Finally, the dentine discs were distributed into three groups in such way that the mean values of dentine permeability showed no significant difference amongst them (Kruskal–Wallis, P > 0.05).

© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

Soares et al. Transdentinal toxicity of resin cements

MDPC–23 cell culture Odontoblast–like MDPC–23 cells (rat dental papillae cells) (Hanks et al. 1998) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Chemical Co., St. Louis, MO, USA) containing 10% heat–inactivated foetal bovine serum (FBS – GIBCO, Grand Island, NY, USA), 100 IU mL–1 and 100 lg mL–1, respectively, of penicillin and streptomycin and 2 mmol L–1 of glutamine (GIBCO) in a humidified atmosphere containing 5% CO2 at 37 °C. For the experiment, 10 000 cells per cm–2 were cultivated in each compartment of 24–well acrylic plates (Costar Corp., Cambridge, MA, USA) and kept in an incubator with 5% CO2 at 37 °C for 6 days.

Experimental design The dentine discs were individually placed in the artificial pulp chambers (APCs) between two silicone rings (Rodimar Rolamentos Ltda, Araraquara, SP, Brazil), with the occlusal surface facing up. After being sterilized by ethylene oxide, the APC/disc sets were placed into wells of 24–well plates containing 1 mL of DMEM with no FBS, in such a way that only the pulpal surface of the disc was in direct contact with DMEM. Each resin–based luting cement selected for this study was applied to the occlusal surface of each disc. After the predetermined incubation time, two aliquots of 500 lL of the culture medium in contact with dentine were collected from each APC/disc. Figure 1 illustrates the experimental design used in the present investigation. The materials were manipulated and applied to the occlusal surfaces of the discs in accordance with the manufacturer’s recommendations. The application protocol and composition of each resin–based luting cement are shown in Table 1.

Immediately after incubation periods, the 500–lL aliquots of each APC/disc set were collected and then applied to the MDPC–23 previously cultured into 24–well plates. The extract remained in contact with the cells for 24 h in an incubator at 37 °C and 5% CO2. After this period, cell viability, cell morphology and alkaline phosphatase (ALP) activity/total protein (TP) production were evaluated. In total, 10 APC/disc sets were used for each experimental group. Aliquots of 8 APC/disc sets were used for cell viability and ALP activity/TP production, and aliquots of 2 APC/ disc sets were used for cell morphology.

Cell viability assay Cell viability was measured by the MTT assay (n = 8). After the 24–h incubation, the extract was aspirated and the cells were incubated for 4 h at 37 °C and 5% CO2 with the MTT solution (5 mg mL–1; Sigma Chemical Co.) diluted in DMEM (1 : 10). Then, the culture medium/MTT solution was aspirated and replaced by 600 lL of acidified isopropanol solution (0.04 N HCl) to dissolve the formazan crystals. Three aliquots of 100 lL were transferred to 96–well plates (Costar Corp.), and cell viability was evaluated proportionally to the absorbance determined at 570 nm in an ELISA microplate reader (Tp Reader; Thermoplate, Nanshan District, Shenzhen, China).

ALP activity/TP production Analysis of ALP activity (n = 8) was performed by a colorimetric end–point assay (Labtest Diagn ostico S.A., Lagoa Santa, MG, Brazil) with a thymolphthalein monophosphate substrate. After the incubation period in contact with the extract, the cells underwent lysis

Transdentinal cytotoxicity After application of the materials to the occlusal dentine surface (one increment, 1 mm thick), the APC/ disc sets were incubated for a 24–h or 7–day periods at 37 °C and 5% CO2 to obtain the extracts (culture medium + components from the cements that diffused through the dentine). Therefore, the following groups were established: group 1 – Rely X Unicem (RU) (3M ESPE; St. Paul, MN, USA); and group 2 – Rely X ARC (RARC) (3M ESPE). In group 3 (control), no treatment was performed on dentine surfaces (APC/disc sets were incubated for 7 days in serum–free DMEM).

© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

Figure 1 Schematic representation of the artificial pulp chamber (APC)/disc set positioned in a well of 24–well plates to obtain the extracts.

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Table 1 Application protocols and chemical compositions of luting cements Groups

Application protocols

Compositions

Group 1

1. RelyX Unicem application (3M ESPE)Dosage of equal quantities of pastes A and B; Mixture for 20 s; Application of self–adhesive cement to dentine (1 mm thick); Light polymerization for 20 s (450 mW cm2, XL 300, 3M ESPE)

Group 2

1. Acid–etching (37% phosphoric acid; Scotchbond etchant; 3M ESPE) Application of 37% phosphoric acid to dentine for 15 s; The acidic agent was rinsed out with sterilized distilled water for 30 s, and the tooth structure was dried with sterilized cotton 2. Adhesive System application (Single Bond 2; 3M ESPE) One layer of bonding agent was applied, then let stand for 30 s, and gently air–dried for 10 s. A second application was performed. The product was light–cured for 20 s under a halogen lamp (450 mW cm2, XL 300, 3M ESPE)

Paste A: silanized glass powder 85–95%, silane– treated silica 5–10%, calcium hydroxide 1–5%, substituted pyrimidine 1–5%, sodium persulfate 1%. Paste B: methacrylated phosphoric acid esters 40– 50%, triethylene glycol dimethacrylate, bisphenol–A –diglycidyl ether methacrylate 25–35%, substituted dimethacrylate 22–34% 37% phosphoric acid, thickener

TM

3. RelyXTM ARC application (3M ESPE) Equal parts of paste A and paste B were hand–mixed for 10 s. The material was applied to dentine (1 mm thick) and light–polymerized for 40 s (450 mW cm2, XL 300, 3M ESPE)

with 1.050 mL of 0.1% sodium lauryl sulphate (SLS) (Sigma Chemical Co.) for 40 min at room temperature. Thereafter, a 50–lL quantity of each well was transferred to test tubes containing 50 lL of substrate (thymolphthalein monophosphate 22 mmol L–1 – reagent No. 1 of the Kit) and 500 lL of buffer (300 mmol L–1, pH 10.1 – reagent No. 2 of the Kit) at 37 °C. After 10 min of incubation, a 2–mL quantity of Color Reagent (94 mmol L1 sodium carbonate and 250 mmol L1 sodium hydroxide – reagent No. 3 of the Kit) was added to each tube. The absorbance was determined in an ELISA reader, at the wavelength of 590 nm. TP production was assessed in the remaining samples of each well (1 mL) used for ALP activity (n = 8). A 1–mL quantity of Lowry reagent solution (Sigma Chemical Co.) was added and remained in contact for 20 min, at room temperature. Then, a 500–lL quantity of Folin solution (20 min; Sigma Chemical Co.) and Ciocalteu0 s phenol reagent (30 min; Sigma – Aldrich) was added. Three aliquots of 100 lL from each tube were then transferred to a 96–well plate, and the absorbance of the test and blank tubes was

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Ethyl alcohol 25–35%, methylethylidene bis methacrylate, bis[4,1–phenyleneoxy(2–hydroxy–3,1– propanediol)] 10–20%, Silica treated with silicon 10 –20%, 2–hydroxyethyl methacrylate 5–15%, 1,3– glycerol dimethacrylate 5–10%, Copolymer of acrylic and itaconic acid 5–10%, dimethacrylate diurethane 1–5%, water < 5% Paste A: Ceramic treated with 60–70% silicone, triethylene glycol dimethacrylate 10–20%, bisphenol –A–diglycidyl ether methacrylate 10%–20%, silica treated with silicon 1–10%, functionalized dimethacrylate polymer 1–10%. Paste B: ceramic treated with silicon 55–65%, triethylene glycol dimethacrylate 10–20%, bisphenol–A–diglycidyl ether methacrylate 10–20%, silica treated with silicon 1–10%, functionalized dimethacrylate polymer 1–10%

read at the wavelength of 655 nm in an ELISA microplate reader. The ALP activity (U mL1) and TP production (mg mL1) were calculated from a standard curve. The ALP activity values were then normalized by the TP value.

Cell morphology analysis Two wells of each group (n = 2) were selected for cell morphology analysis by scanning electron microscopy (SEM). For this purpose, glass slides (Fisher Scientific, Pittsburgh, PA, USA), previously sterilized in 70% ethanol, were placed at the bottom of each well immediately before the MDPC–23 cells were seeded. The extracts were applied to the cells as previously described. After the 24–h incubation period, the cells that still adhered to the glass slides were fixed in 2.5% buffered glutaraldehyde. Then, the slides were washed with PBS, post–fixed in 1% osmium tetroxide, dehydrated in solutions with increasing concentrations of ethanol and processed for evaluation by SEM (DSM 960, Carl Zeiss Inc., Oberk€ ochen, Germany).

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Soares et al. Transdentinal toxicity of resin cements

pared to the negative control when the cells were exposed to 24–h or 7–day extracts, respectively (P > 0.05). Concerning TP production, no significant difference related to the negative control was also observed for group 1 (P > 0.05). However, in group 2 (Rely X ARC) a significant reduction related to the negative control was observed for TP production (12.9% and 22% for the 24–h and 7–day extracts, respectively) and ALP activity (25.4% and 34.6% for the 24–h and 7–day extracts, respectively) (P < 0.05). Regarding TP production and ALP activity, no significant difference was observed between groups 1 and 2 when the MDPC–23 cells were exposed to 24–h and 7–day extracts (P > 0.05).

Statistical analysis Two independent experiments were performed. The numerical data obtained by cell viability, ALP activity and TP production were compiled and evaluated regarding their distribution and homoscedasticity. As there was no adherence to the normal curve for the variables cell viability and TP production, the nonparametric Kruskal–Wallis and Mann–Whitney tests were selected. For the variable ALP activity, the one– way analysis of variance (ANOVA) and Tukey’s test were applied. All the statistical tests were considered at the pre–established level of significance of 5%.

Results Cell morphology

The results of cell viability, ALP activity and TP production are presented in Table 2.

The SEM assessment of cell morphology demonstrated in group 3 (negative control) numerous MDPC–23 cells exhibiting large cytoplasm organized in dense nodules attached to the glass substrate. Similar cell morphology was observed in group 1, in which the RU cement was applied to dentine for a 24–h or 7– day periods. However, an alteration in cell morphology was observed in group 2, in which most of the MDPC–23 cells were associated with a size reduction with some exhibiting membrane disruption. This cell morphology was observed in both 24–h and 7–day periods. Representative images of control and experimental groups are shown in Fig. 2.

Cell viability In group 1 (Rely X Unicem), cell viability reductions of 11.6% and 16.8% occurred when the MDPC–23 cells were exposed to the 24–h and 7–day extracts, respectively. No significant difference was observed when compared with negative control (group 3) in which the cell viability was regarded as 100% (P > 0.05). In group 2 (Rely X ARC), a significant cell viability reduction of 29.5% and 30.5% was observed after exposing the cells to the 24–h and 7–day extracts, respectively (P < 0.05). No significant difference was observed when each group was evaluated separately regarding the extract time (24 h versus 7 days) (P > 0.05).

Discussion Many self–adhesive resin–based luting cements have been introduced with the aim of reducing the number of clinical steps as well as enhancing biocompatibility with the pulp–dentine complex (Radovic et al. 2008, Makkar & Malhotra 2013). A histopathological study

ALP activity/TP production A slight reduction of 13.5% and 17.9% on ALP activity was observed in group 1 (Rely X Unicem) com-

Table 2 Numeric values and percentages of cell viability, ALP activity and TP production for each experimental group Cell viability Groups Negative control Rely X Unicem –24 h Rely X Unicem –7d Rely X ARC –24 h Rely X ARC –7 d

OD (nm) 0.95 0.84 0.79 0.67 0.66

(0.51–1.42)*a (0.47–1.24)a (0.38–1.30)a,b (0.41–0.99)b (0.42–0.99)b

TP production %† 100 88.4 83.2 70.5 69.5

mg L 167.86 181.46 166.18 146.20 130.93

1

ALP activity U mg1

%†

(139.41–212.21)*a (155.27–222.24)a (125.44–215.44)a (110.95–195.64)b (98.03–178.66)b

100 108.1 99.0 87.1 78.0

5.20 4.50 4.27 3.88 3.40

(1.19)**a (0.80)a,b (0.65)a,b (0.60)b (0.77)b

%† 100 86.5 82.1 74.6 65.4

*Values are median (25th–75th percentile), n = 8 (Mann–Whitney). **Values are mean (standard deviation), n = 8 (Tukey’s test). †Percentages were calculated based on the negative control value as 100%. Different lowercase letters in columns indicate statistically significant differences among groups (P < 0.05).

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(a)

(b)

(c)

Figure 2 Representative images of cell morphology – SEM, 500x. (a) – Group 3 (control): Large numbers of MDPC–23 cells can be observed. MDPC–23 cells with large cytoplasm are covering the entire glass substrate on which they were cultivated. SEM, 500x. (b) – Group 1 (RU – 7 days): As observed in group 3 (control), the MDPC–23 cells exposed to RU extract do not present morphological alterations. SEM, 500x. (c) – Group 2 (RARC – 7days): Despite the large number of MDPC–23 cells remaining on the glass substrate, cell size reduction occurred, and some cells exhibited membrane disruption.

in human teeth demonstrated that when the self– adhesive HEMA–free luting cement Rely X Unicem (RU) was applied in deep cavities, neither transdentinal displacement of dental material components nor resin tag formation within dentinal tubules was observed (de Souza Costa et al. 2006). However, in teeth with remaining dentine thickness (RDT) lower than 0.3 mm (very deep cavities), disruption of the odontoblastic layer associated with a discrete inflammatory response in the pulp zone related to the cavity floor occurred 7 days after cavity restoration. No pulp damage took place when the cavity floor was lined with hard–setting calcium hydroxide cement. When the conventional cementation protocol characterized by dentine conditioning and application of bonding agent prior to resin–luting cement placement on the cavity floor was performed, intense pulpal damage and irreversible alteration of pulp tissue were observed (de Souza Costa et al. 2006). Therefore, in the present investigation, as the composition and application protocol of resin–based materials plays a role in their biocompatibility with the pulp–dentine complex, two HEMA–free resin–based luting cements, used as recommended by the manufacturer, were applied to very thin dentine discs (0.3 mm). This methodology constitutes a great challenge for both materials assessed in this study. The cell viability was measured by MTT assay, complemented by cell morphology analysis. Reduction on cell viability and alteration on cell morphology have been directly associated with the toxic potential of dental materials to pulp cells (de Souza Costa et al. 2014). In addition, the phenotype characteristic of MDPC–23 cells related to dentine matrix deposition was assessed by

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measurement of TP production and ALP activity. As one of the main functions of odontoblasts is to deposit dentine matrix and then mineralize it throughout the tooths’ lifespan, any clinical procedure that can disturb this odontoblast activity may cause severe alteration to pulp tissue homeostasis (Goldberg & Smith 2004, de Souza Costa et al. 2014). Amongst several markers for odontoblastic differentiation/maturation, ALP activity has been widely assessed (Goldberg & Smith 2004). This protein is responsible by dephosphorylation of extracellular matrix proteins, providing inorganic phosphate during the dentine matrix mineralization process. It is also known that increased protein expression has a direct relationship with cell differentiation (Goldberg & Smith 2004). Despite this unfavourable experimental condition, a slight reduction in cell viability (from 11.6 to 16.8%) was observed in group 1 (RU), with no significant difference compared with the control group. This result corroborates those obtained by SEM analysis, which demonstrated no alteration in MDPC–23 cell morphology after exposure to the RU extracts. In addition, nonsignificant reductions in TP production and ALP activity occurred in group 1, regardless of the contact time with dentine discs (24 h or 7 days). These results demonstrate that RU features low toxic potential to MDPC–23 cells when applied to nonconditioned thin dentine surfaces. In contrast, significant reductions in cell viability, TP production and ALP activity, associated with alterations in MDPC–23 cell morphology, occurred in group 2, in which the resin– based luting cement RARC was applied to dentine substrates previously acid–etched and subjected to bonding agent application.

© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

Soares et al. Transdentinal toxicity of resin cements

In the last few years, it has been demonstrated that discrete reductions in cell viability occur when cultured pulp cells were exposed to extracts obtained from RU, whereas extracts from HEMA–containing luting cements caused intense cytotoxic effects (de € Mendoncßa et al. 2007). However, Ulker et al. (2012) observed that cells exposed to RU extracts were under oxidative stress, demonstrating that components leached from this HEMA–free cement affected the homeostasis of human dental pulp cells. In the present study, slight reductions in cell viability and ALP activity were observed in the MDPC–23 cells exposed to RU extracts. This cell activity inhibition may be caused by other toxic components present in the RU composition, such as triethylene glycol dimethacrylate (TEGDMA) and bisphenol–A–diglycidyl ether methacrylate (BisGMA). It was previously demonstrated that exposure of pulp cells to these monomers causes depletion of glutathione activity and increases reactive oxygen species (ROS) production, inflammatory mediator expression, apoptosis and/or necrosis in a dose– dependent fashion (Hanks et al. 1991, Stanislawski et al. 2003, Lefeuvre et al. 2005, Reichl et al. 2008, Chang et al. 2009, 2010, 2012, Drozdz et al. 2011, Wisniewska–Jarosinska et al. 2011, Krifka et al. 2012, Batarseh et al. 2014, Botsali et al. 2014). However, the size and hydrophilicity of penetrating monomers have been considered as determining factors in the resin infiltration process on dentine (Eliades et al. 2001, Abedin et al. 2014). Researchers have reported that bonding agents suffer a separation phase when applied to humid dentine, with large and hydrophobic monomers, such as BisGMA, being preferentially retained at the superficial region of the hybrid layer, and small and hydrophilic monomers, such as HEMA, promoting resin infiltration in demineralized dentine (Eliades et al. 2001, Cadenaro et al. 2009, Ye et al. 2012, Abedin et al. 2014). Despite presenting a diffusion rate lower than that of HEMA, it has already been demonstrated that TEGDMA is able to diffuse through dentine discs up to 0.6 mm thick in vitro, with or without pulp pressure simulation (Gerzina & Hume 1995, 1996, Hamid & Hume 1997). Therefore, one may suggest that TEGDMA is the main component of RU responsible for the slight cell viability reduction observed in this study. The transdentinal diffusion of TEGDMA may also be related to the reduction on ALP activity observed in the cells exposed to RU extracts. Galler et al. (2011) demonstrated that subtoxic concentrations of TEGDMA (0.3 mmol L–1) in direct contact with

© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

human dental pulp cells were capable of reducing by 5–20% the expression of the mineralization–related genes collagen I, ALP, bone sialoprotein, osteocalcin, Runx2 and dentine sialophosphoprotein, after 4 h, and by 50% after 12–h exposure. This concentration was also able to reduce ALP activity and mineralized nodule deposition significantly after 14 days. In the present investigation, ALP activity was reduced by 13.5% to 17.9% when the MDPC–23 cells were exposed to RU extracts, but no significant difference was observed relative to negative controls. Also, no reduction on TP production was observed in this group, demonstrating only slight alteration on cell functions. Therefore, one may speculate that only a small amount of TEGDMA and/or other toxic components of RU were able to diffuse through dentine discs to cause a slight toxic effect. However, the long–term effects on ALP activity reduction mediated by RU should be further considered. The positive results observed in the present study for group 1 (RU) may be attributed to the self–adhesive characteristics of this luting cement as well as the absence of HEMA in its composition. The presence of the methacrylate monomers with acidic groups in RU cement eliminates the necessity for prior acid– etching and bonding agent application to dentine. Therefore, the protocol recommending clinical use of the resin–based RU cement does not significantly increase dentine permeability, which may prevent or at least reduce the transdentinal diffusion of high numbers of unreacted free monomers (Radovic et al. 2008, Makkar & Malhotra 2013). It was previously demonstrated that the application of RU to coronal dentine does not result in the formation of a hybrid layer and resin tags (De Munck et al. 2004, Pisani– Proencßa et al. 2011). The histopathological study performed by de Souza Costa et al. (2006) showed that the pulp–dentine complex was able to recover normal characteristics 60 days after the slight initial pulp response promoted by RU applied to very deep cavities (RDT < 0.3 mm) prepared in human premolars. However, the authors observed intense and irreversible pulp inflammation associated with inner dentine resorption in the group subjected to dentine acid– etching followed by the application of a HEMA–containing adhesive system. The transdentinal cytocompatibility of RU may also be a consequence of high monomer/polymer conversion and low degradation rates (Araujo et al. 2002). Franz et al. (2009) stated that the degree of conversion of resin–based materials has a direct relationship

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with its cytotoxic potential. In a previous in vitro study, de Mendoncßa et al. (2011) observed that RU subjected only to chemical cure caused significant reductions in MDPC–23 viability and ALP activity; however, when RU was subjected to dual cure, no significant cytotoxic effect occurred. The monomer/ polymer conversion of RU is chemically initiated by the presence of potassium persulfate. This chemical activator is able to reduce the number of toxic residual monomers (Bourke et al. 1992, Kakaboura et al. 1996, Araujo et al. 2002). Also, the resin matrix of RU after setting features a high degree of cross–linking between and amongst monomers, due to reactions amongst double carbon bonds of acidic methacrylate with methacrylate monomers. The RU pH is increased during the setting process, and the material achieves a neutral level after polymerization reaction (Radovic et al. 2008, Makkar & Malhotra 2013). The high number of fillers in the RU cement also minimizes its degradation rate (Araujo et al. 2002), and the silanated fillers become chemically embedded in cement matrix after setting (Radovic et al. 2008, Makkar & Malhotra 2013). All of these characteristics confer long–term stability on the RU cement and reduce the number of free toxic monomers (Araujo et al. 2002). In group 2 (RARC), the conventional cementation technique reduced MDPC–23 viability by 29.5% and 30.5% after exposure of cultured cells to 24–h and 7–day extracts, respectively. Morphological alterations in the odontoblast–like cells, as well as reductions in TP (12.9–22%%) and ALP activity (25.4–34.6%), also occurred in this experimental group. Pontes et al. (2014) reported no cytotoxic effects when round RARC specimens were immersed in culture medium for 24 h and the extracts were applied to MDPC–23 cells for an additional 24–h period. This cytocompatibility was probably because of a lack of HEMA in RARC composition and the high content of fillers and cross–linking bonds after setting, as well as the presence of sodium persulfate as a chemical initiator (Araujo et al. 2002, Radovic et al. 2008, Makkar & Malhotra 2013). Therefore, the cytotoxicity observed in this study for this luting cement may be related to the cementation protocol performed rather than to the chemical composition of RARC. As RARC does not contain acidic monomers in its composition, this material requires dentine conditioning and bonding agent application before its placement on dentine substrate (Radovic et al. 2008, Makkar & Malhotra 2013). Therefore, one may suggest that acid–etching, followed by the application of a bonding agent with a

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large amount of HEMA to dentine, caused the toxic effects on the cultured pulp cells. Previous in vivo studies demonstrated that when a bonding agent is applied to dental cavities (RDT ≤ 0.5 mm), displacement of components leached from the resin–based material takes place, triggering persistent pulpal inflammation (de Souza Costa et al. 2002, 2006, 2007). This pulp damage is even more intense when the dentine is subjected to acid–etching, such as was performed in the present investigation (de Souza Costa et al. 2002, 2007). Rather than the cell viability/morphology alteration observed in the present investigation, the intense alteration on TP production and ALP activity after application of RARC onto thin dentine discs should be considered a concern. These results demonstrate that the phenotype characteristics of odontoblast–like cells can be intensely altered by the cementation procedure associated with this dental material, which may lead to impaired regeneration of pulp tissue through time. Histopathological studies demonstrated that application of adhesive systems onto deep dentine substrate previously subjected to acidic–etching lead to impaired capability of pre–dentine deposition by odontoblasts, resulting in internal dentine resorption mediated by immune system cells activation (de Souza Costa et al. 2002, 2007). These negative effects have been attributed to the transdentinal diffusion of a high amount of unreacted free HEMA leached from the bonding agent (Gerzina & Hume 1995, 1996, Hamid & Hume 1997, de Souza Costa et al. 2014). This resin monomer has recognized toxic potential, causing cell death associated with oxidative stress when in contact with pulp cells, and promotes reduction in the expression of type I collagen, osteonectin and dentine sialoprotein, proteins that play a fundamental role in the process of pulp healing and dentine matrix deposition/mineralization (Reichl et al. 2008, Bakopoulou et al. 2011, Botsali et al. 2014, Barbosa et al. 2015). Recently, Barbosa et al. (2015) demonstrated that removal of HEMA from bonding agents significantly reduced the cytotoxic potential of this kind of resin–based dental material. In a literature review performed by Radovic et al. (2008), the authors reported that RU has behaviour comparable with that of conventional cements, featuring bond strength similar to that of dentine/restorative materials, as well as similar microleakage and similar mechanical properties. According to Rodrigues et al. (2015), RU and RARC are equally effective alternatives for the bonding of indirect restorations to

© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

Soares et al. Transdentinal toxicity of resin cements

enamel and dentine. Therefore, according to the data from the present study, it may be suggested that HEMA–free self–adhesive resin cements, applied to dentine surfaces that have not been pre-treated, appears to be a less aggressive protocol for pulp cells in vitro. In this way, further studies are needed to evaluate the chemical compounds leached from these categories of luting cements that are capable of diffusing through dentine to reach the pulpal space in vivo and its effects to the pulp tissue.

Conclusion The self–adhesive HEMA–free resin–based luting cement Rely X Unicem featured indirect cytocompatibility when applied to dentine discs not subjected to pretreatment. However, acid–etching followed by bonding agent application to dentine prior to placement of the HEMA–free Rely X ARC luting cement resulted in significant cytotoxicity to the odontoblast– like MDPC–23 cells.

Acknowledgements This study was supported by the S~ ao Paulo Research Foundation – FAPESP (grants # 2013/23520–0 and 2013/05879–0) and by the National Council of Technological and Scientific Development – CNPq (grant # 303599/2014–6). The authors deny any conflict of interests related to this study.

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© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd

Cytocompatibility of HEMA-free resin-based luting cements according to application protocols on dentine surfaces.

To evaluate the transdentinal cytotoxicity of resin-based luting cements (RBLCs), with no HEMA in their composition, to odontoblast-like cells...
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