Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol.

DENTAL-3024; No. of Pages 14

ARTICLE IN PRESS d e n t a l m a t e r i a l s x x x ( 2 0 1 7 ) xxx–xxx

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Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol Paulo Henrique Perlatti D’Alpino a,b,∗ , Gioconda Emanuella Diniz de Dantas Moura c , Silvana Coelho de Arruda Barbosa b , Lygia de Azevedo Marques d , Marcos Nogueira Eberlin d , Fábio Dupart Nascimento e , Ivarne Luis dos Santos Tersariol c,e a

Biomaterials in Dentistry Program, Universidade Anhanguera de São Paulo (UNIAN-SP), São Paulo, Brazil Biotechnology and Innovation in Health Program, Universidade Anhanguera de São Paulo (UNIAN-SP), São Paulo, SP, Brazil c Department of Biochemistry, Molecular Biology Division, Federal University of São Paulo, São Paulo, Brazil d ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas (UNICAMP), Campinas, SP, Brazil e Interdisciplinary Center of Biochemistry Investigation (CIIB), University of Mogi das Cruzes, Mogi das Cruzes, SP, Brazil b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objectives. To evaluate the cytotoxic effects of exposing odontoblast cells to a variety of

Received 22 June 2017

commercial self-adhesive cements polymerized using different activation modes.

Received in revised form

Methods. Five cements: MaxCem Elite (MAX), Bifix SE (BSE), G-Cem LinkAce (GCE), Clearfil SA

19 July 2017

Luting (CAS), and RelyX U200 (U200) were mixed, dispensed into molds, and distributed in

Accepted 20 September 2017

groups, according to polymerization protocols: immediate photoactivation; delayed pho-

Available online xxx

toactivation (10 min self-curing plus light-activation); and chemical activation (no light

Keywords:

assessed by Trypan Blue staining and total cell death was assessed by annexin V-APC/7-AAD

exposure). Immortalized rat odontoblast cells (MDPC-23) were cultured. Cell viability was Self-adhesive resin cements

double staining and flow cytometry. Volatilized compounds from polymerized specimens

Odontoblastic cells

of cements were evaluated by gas chromatography/mass spectrometry (GC–MS). Data was

Cytotoxicity

analyzed with 2-way ANOVA/Tukey tests (˛ = 0.05).

Total death cell

Results. Exposure to all of the cements tested significantly reduced the cell viability, irre-

Gas Chromatography

spective of the activation protocol (p < 0.05). The least harmful cements were CSA and U200.

Mass spectroscopy

Total death of cells significantly increased when exposed to BSE, GCE, and MAX, especially when chemically activated (p < 0.05). Characteristic apoptotic cells increased after exposure to cements, mainly for MAX, regardless of the activation mode. Chemical activation of MAX also induced necrosis. Moreover, GCE and MAX exhibited higher percentages of late apoptotic/dead cells. Chromatograms revealed 28 compounds released from the cements tested,

∗ Corresponding author at: Universidade Anhanguera de São Paulo – UNIAN SP, Programa de Pós-Graduac¸ão em Biotecnologia e Inovac¸ão em Saúde, Av. Raimundo Pereira de Magalhães, 3.305, São Paulo, SP, CEP: 05145-200, Brazil. E-mail addresses: [email protected] (P.H.P. D’Alpino), gioconda [email protected] (G.E.D.d.D. Moura), [email protected] (S.C.d.A. Barbosa), [email protected] (L.d.A. Marques), [email protected] (M.N. Eberlin), [email protected] (F.D. Nascimento), [email protected] (I.L.d.S. Tersariol). https://doi.org/10.1016/j.dental.2017.09.011 0109-5641/© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: D’Alpino PHP, et al. Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol. Dent Mater (2017), https://doi.org/10.1016/j.dental.2017.09.011


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some of them with known carcinogenic effects. Selection of self-adhesive cements and polymerization protocols affect the cytotoxicity and cell viability of odontoblastic cells. Clinical significance. Despite the simplified cementation protocol, care is needed when cementing indirect restorations with self-adhesive cements, especially on recently exposed dentin. This category of material may cause differential cytotoxic effects and should be considered when selecting a cement. This is particularly true in clinical cases of light attenuation, where the polymerization depends on chemical activation, inducing higher cytotoxic damages when using some of the cements tested. © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Self-adhesive resin cements were launched considering the possibility to overcome the drawbacks of other types of materials used to cement indirect restorations, allowing for a less critical cementation procedure [1]. In addition to methacrylate monomers and low molecular weight resins found in the composition of resin-based restoratives, self-etching functionalized monomers were also added to the composition to significantly reduce the pH and to demineralize the tooth structure, promoting micromechanical adhesion [2]. Common methacrylate monomers modified with carboxylic or phosphoric acid groups include 4 META (4-methacryloyloxyethy trimellitate anhydride), 10-MDP (10-methacryloyloxi-decyl-dihydrogen-phosphate), GPDM (glyceroldimethacrylate dihydrogen phosphate), and Phenyl P (2-methacryloyloxyethyl phenyl phosphoric acid), among others [3]. As a result of these monomers, secondary reactions (ionic and covalent interactions) between the cement and enamel/dentin are formed, which convert the substances into a salt complex formed by calcium and the acid-modified monomers, thereby establishing chemical bonds with the tooth structure [2,4]. In this way, these materials dispense technique-sensitive steps, such as acid etching, priming, or bonding [4]. This unique bonding mechanism represents an important feature compared to other categories of resin cements, which are essentially micromechanical in nature [5]. The chemical composition of dental materials and their application protocols to the dentinal tissue also play a central role in defining their compatibility with the pulp–dentin complex [6,7]. (Co)monomers and other substances can be eluted from polymerized dental methacrylate-based materials [8]. These byproducts eluted from dental materials after manipulation and setting have been regarded as potentially affecting both the biocompatibility and the structural stability of the restoration [9]. Considering the fact that the self-adhesive resin cements are dual-cured materials [10], the different activation protocols may also affect the biocompatibility of these resin cements in different ways [11,12]. The degree of conversion of (co)monomers to polymers is claimed to influence the material properties and biocompatibility of a given material. This is because an insufficiently dense network arising from decreased conversion of double carbon bonds results in monomer leaching and the release of such substances as plasticizers, polymerization initiators, and inhibitors [13]. Thus, the lower the degree of conversion, the higher the amount

of uncured monomers and additives [14]. The consequences induced by the degradation of materials over time has also been highlighted, with extended effects on oral tissues and systemic effects from the ingestion of eluted components [15]. Considering the similarity of resin cements in terms of chemical composition with resin composites and adhesive systems, previous studies may provide important guidance in terms of the biocompatibility of this category of material [16]. Concerns have been expressed about the chemical composition of self-adhesive resin cements, specifically regarding the need for a balanced formulae due to the fact that the polymerization reaction occurs in an acidic environment [17,18]. Cytotoxicity may be induced not only due to chemical irritation from the materials but also due to pH changes occurring in the vicinity of the materials during setting [15]. In this way, the neutralization of the pH of the self-adhesive resin cements is important to avoid impacting the end conversion, especially considering the effect of both the formulation of new methacrylate monomers and the technology to initiate the polymerization process [17]. Consequently, varying biological responses with different cement types would be expected [15]. On the other hand, it has been claimed that a glass ionomer concept was added to the formulation to neutralize the initial low pH [1]. Thus, comprehension of the dynamic process in which the demineralization/monomer permeation process and the polymerization kinetics coexist in this category of material is of paramount importance [18]. The elution of matrix monomers and photoinitiators has been correlated with cytotoxic effects, because an inverse correlation has been found between the release of these components and cell survival [19]. Leaching of unreacted free monomers from resin-based materials in wet environments is regarded as being capable of inducing oxidative-stress-mediated pulp cell death, inflammatory mediator over-expression, and depletion of glutathione peroxidase and superoxide dismutase enzymes [20–23]. It has also been claimed that free monomers can alter the phenotypic characteristics of dental pulp stem cells, impacting the regenerative potential of the pulp tissue [24–26]. Depletion of glutathione, production of reactive oxygen species, and a few other molecular mechanisms were also identified as determining factors leading to apoptosis and/or pulp necrosis [27]. Studies in animals and humans have shown mild to severe inflammatory pulp reactions, leading in many cases to cell apoptosis, followed by severe pulp alteration [28].



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Table 1 – Self-adhesive resin cements characterized in the present study.a Material

Lot n◦ /Exp. date

Composition

Working time (min.)

Setting time (min.)

Curing time (s)

Filler content Wt. (%) Vol. (%)

RelyX U200 3M ESPE, St. Paul, MN, USA

1609600582 2017-04

2

6

20

72.0 43.0

Clearfil SA Luting Kuraray, Tokyo, Japan

4E0078 2017-03

1

2–4

20

44.0 (Vol.%)

MaxCem Elite Kerr Corporation, Orange, CA, USA

6088085 2018-02

1

4

10–20

69.9 46.0

Bifix SE Voco GmbH, Cuxhaven. Germany G-Cem LinkAce GC Corporation, Tokyo, Japan

1621122 2017-11

Base: Methacrylate monomers containing phosphoric acid groups, methacrylate monomers, initiators, stabilizers, rheological additives. Catalyst: Methacrylate monomers, alkaline fillers, silanated fillers, initiator components, stabilizers, pigments, rheological additives. Zirconia/silica fillers. Clicker delivery system. Paste A: Bis-GMA, TEGDMA, 10-MDP, DMA, hydrophobic aromatic dimethacrylate, silanated barium glass filler, silanated colloidal silica. Paste B: Bis-GMA, DMA, hydrophobic aromatic dimethacrylate, silanated barium glass filler, silanated colloidal silica, surface treated sodium fluoride. Hand Mixing. GPDM, co-monomers (mono-, di-, and tri-functional), proprietary self-curing redox activator, methacrylate monomers, water, acetone, and ethanol. Inert minerals and ytterbium fluoride. Automix system. Bis-GMA, UDMA, Gly-DMA, phosphate monomers, initiators, stabilizers. Glass. Automix system. Paste A: Fluoroalumino silicate glass, Initiator, UDMA, dimethacrylate, pigment, silicon dioxide, Inhibitor Paste B: Silicon dioxide, UDMA, Dimethacrylate, Initiator, Inhibitor. Automix system.

2

4

10–20

70.0 45.0

2 45

4

20

71.0 56.6

1607041 2018-07

Abbreviations: Bis-GMA: bisphenol A diglycidyl ether dimethacrylate; TEGDMA: Triethyleneglycol dimethacrylate; UDMA: Urethane dimethacrylate; 10-MDP: 10- methacryloxydecyl dihydrogen phosphate, DMA: Dimethylamine; Gly-DMA: glycerol dimethacrylate; GPDM: glycero-phosphate dimethacrylate. a Manufacturers’ information.

Considering the fact that self-adhesive resin cements are frequently applied to newly exposed dentin, especially in total crown preparations, and the fact that the polymerization reaction in methacrylate-based resins is radical-mediated [29], the evaluation of the biological effects resulting from the application of this class of material is of clinical relevance [30]. Drawing upon two hypotheses, this study attempts to investigate the effects of activation protocols on the cell viability and cell death of odontoblastic cells exposed to different commercial self-adhesive resin cements. Commercial cements were selected, manipulated, and polymerized using different activation protocols. The following research hypotheses were tested: (1) the brand of self-adhesive cement tested will significantly influence the cell viability and overall percentage of cell death; (2) the polymerization activation mode will signif-

icantly influence cell viability and overall percentage of cell death.

2.

Materials and methods

2.1.

Experimental design

In this in vitro study, the cytotoxicity according to the percentage of cell viability, and the % of total cell death were evaluated in resin cement, according to the following factors: (1) selfadhesive resin cements at five levels: MaxCem Elite (Kerr), Bifix SE (Voco), G-Cem LinkAce (GC), Clearfil SA Luting (Kuraray), and RelyX U200 (3M ESPE); and (2) activation mode at three levels: immediate photoactivation, delayed photoactivation, and



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chemical activation. The characteristics of the resin cements selected are described in Table 1. Fifteen groups were categorized and treated according to the various polymerization protocols.

2.2.

Specimen preparation

The self-adhesive resin cements were mixed according to the manufacturers’ instructions in terms of working and setting times. The unpolymerized resin cements were then placed into a Teflon mold (2 mm thick, 6 mm in diameter) and positioned over a polyester strip. After filling the mold to excess, the material surface was covered with a Mylar strip and a glass slide and was compressed to extrude excess material. The glass slide was then removed, leaving the Mylar strip. The distal end of the curing light guide was placed within 0.1 mm of the top of the Mylar surface. Specimens were then photoactivated after the working time (Table 1). Curing time followed the manufacturers’ recommendations as depicted in Table 1. The light-curing procedure was performed using a poly-wave LED light-curing unit (Bluephase, Ivoclar Vivadent, Schaan, Liechtenstein) with an irradiance of 1200 mW/cm2 . Prior to testing, and throughout the experiment, the light output was monitored using a handheld radiometer (Model 100, Demetron Research Corp., Danbury, CT, USA). After the photoactivation procedures (immediate and delayed photoactivation groups), the specimens were removed from the molds and were then stored in lightproof recipients for 24 h at room temperature (22 ◦ C). Specimens from the chemically activated groups were kept in the molds for 24 h at room temperature in the dark prior to their removal.

2.3.

Seeding MDPC-23

MDPC-23 is an immortalized cell line from fetal rat molar dental papillae capable of expressing dentin sialoprotein and other proteins expressed by odontoblasts [31,32]. The cells were subcultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich Corp., St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Cultilab, Campinas, SP, Brazil), 100 IU/mL penicillin, 100 mg/mL streptomycin and 2 mmol/L glutamine (Gibco, Grand Island, NY, USA) in a humidified incubator with 5% CO2 and 95% air at 37 ◦ C (Isotemp Fisher Scientific, Pittsburgh, PA, USA) for 3 days until the number of cells necessary to perform the study was reached. The cells (1 × 105 ) were seeded in the lower compartment of 24-well plates (HTS Transwell, Ref. #3374, Corning, NY, USA) and maintained in an incubator with 5% CO2 and 95% air at 37 ◦ C. After 48 h, individual resin cement discs, produced according to the experimental groups, were placed onto a Transwell insert that comprised a permeable support device with a microporous membrane with pores 8 ␮m in diameter. The discs remained in incubation with the cells for 24 h. This allowed for the elution of unreacted monomers from polymerized resin and low molecular weight material with harmful potential in relation to the viability of odontoblastic cells.

2.4. Quantification of apoptotic vs. necrotic cells after exposure to the self-adhesive cements (annexin V-APC/7-AAD staining) Cell death was investigated using annexin V-APC/7-AAD double staining kit (BD Pharmingen, BD Biosciences, Franklin Lakes, NJ, EUA) and was analyzed through flow cytometry. APC Annexin V staining was used in conjunction with the vital dye 7-Amino-Actinomycin (7-AAD) allowing identification of early apoptotic cells (7-AAD negative, APC Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. Cells that are considered viable are both APC Annexin V and 7-AAD negative; cells that are in early apoptosis are APC Annexin V positive and 7-AAD negative; cells that are in late apoptosis or already dead are both APC Annexin V and 7-AAD positive [33,34]. The cells were seeded and incubated with different resins, as previously mentioned, followed by harvesting with 10 mM EDTA in phosphate-buffered saline (PBS) and resuspending in binding buffer (0.01 M HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2 ). The suspensions were transferred to tubes, centrifuged, and resuspended with annexin V-APC and 7-AAD according to the manufacturer’s instructions. The cells were incubated at room temperature for 30 min and the viable, apoptotic or necrotic cell populations were evaluated through flow cytometry. The data were collected using a FACSCalibur flow cytometer (Becton–Dickinson, CA, USA) and CellQuest software (Becton–Dickinson), followed by analysis using FlowJo software (Tree Star, CA, USA). A total of 10,000 events were collected for each sample. The results were calculated as the percentage of cell death compared to the control (unexposed cells). The experiment ran in triplicate and was expressed as the mean ± standard error of the mean (SEM).

2.5. Evaluation of % cell death in cultures exposed to the self-adhesive cements (Trypan Blue exclusion) Trypan blue solution was used for cell viability assay. To determine total cell count and cell viability, 20 ␮L of cell suspension was mixed with 20 ␮L of 0.05% trypan blue solution. Viable cells and number of total cells were counted by hemocytometer.

2.6.

Statistical analysis

Statistical significance among the groups was assessed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Student’s t test was also used to analyze the data when pertinent. Differences were considered significant when the P-values were less than 0.05 (p < 0.05). All statistical analyses were performed using GraphPad Prism software (GraphPad, CA, USA).

2.7. Gas-chromatography/mass spectrometry (GC–MS) analysis GC–MS was performed to complement the results, and to establish possible correlations between the released compounds and their potential cytotoxic effects on odontoblastic




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Fig. 1 – Quantification of total cell death (%) by apoptosis and/or necrosis in odontoblastic cell line exposed to self-adhesive resin cements as a function of the activation mode (immediately photoactivated, photoactivated after 10 min, and chemically activated). Data are expressed as mean + standard error. * significant difference from the respective control (p < 0.05).

cells. For this analysis, the specimens of cements were obtained as previously described. In this case, the specimens were photoactivated following the manufacturer’s instructions (immediately photoactivated). Analyses were carried out on a GC–MS system from Agilent Technologies consisting of a gas chromatograph (7890A) coupled to a quadrupole mass spectrometer (MSD 5975C) operating in the electron ionization (EI) mode (70 eV), coupled to a Gerstel MPS2-twister as headspace autosampler. The chromatographic separations were performed on a J&W DB-WAX fused silica capillary column (30 m × 250 mm i.d., 0.25 ␮m film thickness) from Agilent Technologies using helium as carrier gas (1 mL min−1 ). The injector, GC–MS interface and MSD source temperature were 250, 310, and 230 ◦ C, respectively. The following oven temperature program was used: initial temperature 40 ◦ C, held for 2 min, ramped at 5 ◦ C min−1 to 240 ◦ C and held for 10 min. A solvent delay time of 5 min was used to protect the ion multiplier of the MS instrument from saturation. The MS was operated in full scan mode being the m/z range from 30 to 500. Each cement was introduced to a headspace vial, which was tightly closed. The vial was kept for 30 min at 37 ◦ C before injection. A volume of 1 mL of the gas phase was injected using the split injection mode with a split ratio 20:1. The volatile components were identified by comparing the mass spectrum with those available in the Nist08 spectra library. Chemical composition was reported as the percentage of the relative area after obtaining the sum of all peak areas in the chromatograms. Results were also expressed as retention time (tR ), quoted in units of minutes.

3.

Results

The results for the total cell death are depicted in Figs. 1 and 2. Fig. 1 displays the percentage of total cell death by apoptosis and/or necrosis in cell lines exposed to self-adhesive resin cements as a function of the activation mode. Flow cytometric analysis showed that the cell death varied as a function of the exposure to the cements and different activation modes. Compared to the control group (unexposed cells), significant increase in the total cell death occurred when exposed to

cement MaxCem Elite, irrespective of the activation mode (p < 0.05). In addition, cements Bifix SE and G-Cem LinkAce also induced a significant increase in the total cell death when photoactivated after 10 min or chemically activated (p < 0.05). Although the exposure to RelyX U200 and to Clearfil SA Luting seemed to increase the total cell death, no significance was observed in comparison to that of the control group (p > 0.05). Fig. 2 depicts the results of flow cytometry assay using annexin V-APC/7-AAD double staining to distinguish living cells (7-AAD−/Annexin V−), dead cells (7-AAD+/AnnexinV−), early apoptotic cells (7-AAD−/Annexin V+), and late apoptotic/dead cells (7-AAD+/Annexin V+). Regarding the fraction of early apoptotic cells, the results varied from 10% (RelyX U200, when immediately photoactivated) to 70% (MaxCem Elite, when chemically activated), compared to 6.15% for the control, unexposed group. The highest percentages of apoptosis were observed for MaxCem Elite, irrespective of the activation mode (Fig. 2). Clearfil SA Luting also exhibited increased % of apoptosis when immediately photoactivated and chemically activated (28.8% and 24.8%, respectively), and Bifix SE when photoactivated after 10 min or when chemically activated (25.8% and 30.6%, respectively). G-Cem LinkAce and MaxCem Elite also exhibited higher percentages of late apoptotic/dead cells (9.76 and 24.2%, respectively) compared to the control, unexposed cells (0.25%). Chemically activated specimens of MaxCem Elite also induced increased % of dead cell (3.86%) compared to the control (0.15%). Viability of MDPC-23 cells after contact with specimens of self-adhesive resin cements are graphically represented in Fig. 3. Statistical analysis showed that there was a significant reduction in cell viability in all of the experimental groups, irrespective of the activation mode (p < 0.05). The descending order in the reduction of cell viability was as follows: MAX > GCE > BSE > CSA > U200. When G-Cem LinkAce and MaxCem Elite were chemically activated, the percentages of viable cells were even lower (31.9% and 1.7%, respectively) compared to other activation modes. Fig. S4 in the online version at DOI:10.1016/j.dental.2017.09.011 displays the test chroof the self-adhesive cements tested. matograms Chromatographic analysis of volatile organic compounds



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Fig. 2 – Flow cytometry assessment of apoptosis and necrosis of MDPC-23 cells after exposure to specimens of self-adhesive resin cements, activated with different polymerization protocols (immediately photoactivated, photoactivated after 10 min, and chemically activated). Treated cells were stained with annexin V-APC/7-AAD double staining, with the experiment containing a technical triplicate. Four quadrants (Q) are representing: Q1, dead cells (AnnexinV−/7-AAD+); Q2, late apoptotic/dead cells (AnnexinV+/7-AAD+); Q3, early apoptotic cells (AnnexinV+/7-AAD−); Q4, living cells (AnnexinV−/7-AAD−). Values in the quadrants represent percent positive cells.

Table 2 – Results of most probable compounds found in cements samples detected by GC–MS, using NIST library as database. Cements

U200

CSA

BSE

GCE

MAX

Chemical compounds (NIST library by GC–MS)

tR

Area

tR

Area

tR

Area

tR

Area

tR

Cyclopentasiloxane, decamethyl- (silane) 2-Hexanol, methyl ether Acetic acid l-Menthone Cyclohexanone Propylene Glycol dl-Menthol Levomenthol Acetophenone 1,2-Ethanediol (ethylene glycol) 2-Propenoic acid, 2-methyl- (methacrylic acid, MAA) Methyl methacrylate (MMA) n-Butyl methacrylate (n-BMA) (acrylic acid) Benzenemethanol or Benzyl alcohol (BA) 2,4-Dinitrophenyl crotonate (2,4-DNP) 2-Hydroxyethyl methacrylate (HEMA) 2-Butenoic acid, butyl ester or Butyl crotonate Cyclopropanecarboxylic acid Butylated Hydroxytoluene (BHT) Crotonic anhydride dl-Camphorquinone Biphenyl Hexanoic acid Phenol 4-Isopropoxy-2-butanone Pentanoic acid 1,2-Propanediol, 3-chloro- (3-MCPD) Phenol, 2,4-bis(1,1-dimethylethyl)- (PD) Total

6.6 – – – – – – – – – 16.4 – – – – 18.3 – – – – 21.5 22.0 22.2 22.4 23.5 24.5 – 28.0

1.0 – – – – – – – – – 52.5 – – – – 5.8 – – – – 12.1 0.7 2.3 1.1 1.1 1.2 – 2.1

– – – – – – – – – – 16.3 16.9 – – – 18.3 – 20.0 20.5 21.1 21.5 – – 22.4 – 23.5 – –

– – – – – – – – – – 19.2 6.7 – – – 5.3 – 2.9 1.0 57.4 0.5 – – 1.1 – 1.1 – –

– – – – – 14.2 – – – 14.9 16.2 16.9

– – – – – 2.5 – – – 1.0 20.1 30.3 – – 2,63 16.0 – – 0.9 – 0.9 1.4 – 4.6 1.0 – – –

– – 11.6 – – – – – – – 16.1 – 17.4 – – 18.3 – – 20.5 – – – – – – – 23.6 –

– – 0.7 – – – – – – – 76.9 – 1.1 – – 14.1 – – 0.35 – – – – – – – 1.3 –

– 7.2 – 11.8 12.3 – 14.3 15.2 15.4 – 16.2 – – 17.6 – – 18.3 – – – 21.5 – – 22.4 – – – –

10

9

– 18.2 18.3 – – 20.6 – 21.5 22.0 – 22.4 23.5 – – – 11

6

Area – 0.6 – 3.3 1.7 – 2.4 14.1 1.7 – 15.7 – – 4.3 – – 33.0 – – – 0.9 – – 6.5 – – – – 11

tR = retention time in minutes; area in %.




released by the cements revealed the presence of at least 28 major common compounds (Table 2). The released organic compounds were material-dependent as only one of the substances was commonly found in all of the cements tested (Table 2). This byproduct is methacrylic acid (MAA), derived from TEGDMA (triethylene glycol dimethacrylate) and HEMA (2-hydroxyethyl methacrylate) [35]. MaxCem Elite and Bifix SE were the cements from which more organic substances were volatilized (11 compounds), while G-Cem LinkAce was the cement from which less compounds were volatilized (6 compounds). Eight out of the 11 substances released from MaxCem Elite were exclusively observed in this cement (Table 2). CSA was the cement from which the released byproducts were most commonly found in most of the cements (7 compounds). Except for G-Cem LinkAce, all cements present the camphorquinone (CQ)-amine complex initiation system (Table 2). G-Cem LinkAce contains butyl methacrylate and the presence of volatilized acetic acid seems to be related to the polymerization initiator system [36]. HEMA was also found in most of the cements (exception for MAX) (Table 2). Possible reasons for the higher death cell and lower cell viability mostly relate to the release of certain compounds and their byproducts, such as 2,4-Dinitrophenyl crotonate (2,4-DNP) [37], 1,2-Propanediol, 3-chloro- (3-MCPD) [38], and Benzenemethanol (Benzyl alcohol) [39], respectively from BSE, GCE, and MAX (Table 2).

4.

Discussion

The results of the present study indicated that the exposure of odontoblastic cells to different self-adhesive resin cements induced injuries to the odontoblastic cells. This chemically-induced damage may be due to the release of cytotoxic substances that seems to activate the apoptotic response, causing a material-dependent, wide-ranging cell death as functions of the chemical composition of the cements and their activation modes. In addition, the exposure of these cells to two of the self-adhesive cements tested also led to a significant increase in the late apoptotic/dead cells when

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chemically activated (GCE and MAX). Thus, the first research hypothesis, which anticipated that the brand of self-adhesive cement tested will significantly influence the cell viability and overall percentage of cell death, was accepted by the experimental data. Two types of cell death have generally been demonstrated: apoptosis and necrosis [40]. Apoptosis represents a controlled physiological process intended to eliminate a specific group of cells at a given time or in response to a particular signal to preserve the overall well-being of the organism [41]. The predominant phenotypic features of apoptosis include DNA fragmentation, loss of cell volume or cell shrinkage, chromatin condensation (pyknosis), fragmentation of the nucleus (karyorrhexis), and apoptotic body formation [41]. It is an energy-demanding process with wide-ranging implications in tissue kinetics. Despite these processes, there is no release of cellular contents. Finally, dead cells are readily phagocytosed by macrophages, thus avoiding an acute inflammatory reaction [42]. By contrast, necrosis is a passive, toxicity-induced cell death, a no energy-demanding process that follows the release of cellular contents, which may generate an inflammatory reaction [43]. Cells swell and the chromatin and nucleus are cleaved by DNase (karyolysis), disrupting the cellular homeostasis [41]. Possible reasons for these results include residues of the unreacted cross-linking agents, which have been claimed to be bioactive in relation to the pulp tissue [44]. In this way, these compounds generated by the incomplete monomer conversion during polymerization of the cements can be regarded as potential sources of cytotoxicity [45]. In the present study, a significant reduction in the percentage of viable cells was observed in all of the experimental groups (p < 0.05). The reduction in cell viability followed the descending order: MAX > GCE > BSE > CSA > U200. Chemical activation of GCE and MAX induced the highest reduction in cell viability (31.9% and 1.7%, respectively) compared with other activation modes. In this way, the second hypothesis, which speculated that the polymerization activation mode will significantly influence cell viability and overall percentage of cell death, was also proven valid. In a previous study [46], mild to no toxic effects were observed when an odontoblastic cell line (MDPC-23) was exposed to an experimental

Fig. 3 – Mean percentage of viable odontoblastic cells exposed to self-adhesive resin cements activated with different polymerization protocols (immediately photoactivated, photoactivated after 10 min, and chemically activated). Data are expressed as mean + standard error. * significant difference from the respective control (p < 0.05). Please cite this article in press as: D’Alpino PHP, et al. Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol. Dent Mater (2017), https://doi.org/10.1016/j.dental.2017.09.011


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resin-based material, irrespective of the light-activation protocols. Conversely, intense cytotoxic effects were observed when the resin-based material was not light-cured. The crucial problems relating to the ability of the dual cements to effectively polymerize after immediate exposure to light, and the effectiveness of the chemical-cure component, have previously been discussed [47]. However, no consensus has been obtained: on the one hand, it has been pointed out that the chemical-cure component may provide an improved network structure and polymer properties, and, on the other hand, others believe that this activation mode would not allow for maximum cement hardening [11]. Some dual cements were regarded as having a limited self-curing mechanism when immediately light-activated in the dual-cure mode, thereby restricting the materials from achieving their maximum mechanical properties [48]. Concerns have been previously raised as to whether self-adhesive resin cements can be successfully used in clinical applications because lower-end conversions are obtained in a self-curing mode in the case of light attenuation [49]. In a previous study, self-cured specimens were found to present significantly higher cytotoxicity than dual-cured specimens of adhesive and self-adhesive resin cements [50]. This is clinically relevant, because manufacturers usually recommend immediate photoactivation of the cement. In this way, the authors recommended a clinically convenient time delay to photoactivate the dual-cured cements, thereby avoiding the interference of the activating light in the kinetics of the polymerization of the self-curing component [48]. The chemical curing mechanism proceeds slowly and is expected to ensure polymerization especially in those areas where the curing-light is unable to reach [11,18]. The light irradiation after the initial “dark period” in the 10-min delay groups may help the polymerizing chains to build a packaged network, especially at the cement surface, thereby acting as an isolating layer and impeding the diffusion of unreacted monomers and byproducts from the deeper areas [51]. This may be particularly true for GCE and MAX which exhibited better results for the group in which the photoactivation was delayed for 10 min. Despite the recommended delay, the ideal time lapse needed to photoactivate the cement line after mixing has yet to be determined, but a delay time from 5 to 10 min has been suggested to avoid interference in the final cure and in its properties [47]. According to previous publications and the results of the present study, it seems that different activation modes induce the formation of different three-dimensional polymer networks, thereby allowing different amounts of released substances within one material, some of them rather cytotoxic. Conventionally, polymerization initiators used in light-cured restorative materials usually consist of a photosensitizer, mainly CQ and a reducing agent, frequently a tertiary amine, such as dimethyl-para-toluidine (DMPT) or dimethylaminoethyl methacrylate (DMAEMA) [52]. In a similar way, in the polymerization process in self-curing materials the amine reacts with the peroxide in the monomer medium, resulting in the formation of free radical intermediates that initiate the polymerization. In this case, polymerization is usually induced by a benzoyl peroxide (BPO)/amine redox or DMPT/amine redox couples [53]. At modulated irradiation

times (i.e. 10-min photoactivation delay) or in chemical activations, a lower amount of activated initiator molecules may induce fewer polymerization nuclei, thus facilitating the building of longer polymer chains [54]. In a previous study, solutions containing diverse initiators induced necrosis in human gingival fibroblast cells [55]. Another concern has been raised regarding the concentration of the photoinitiation system present in the composition of dental materials. It has been advocated that there is a positive correlation between conversion rate and the concentration of this photoinitiating system until the point where it starts to decrease due to an excessive initiator concentration, called the inner shielding effect [56]. This would lead to unreacted photoinitiators in the polymerizing material [57]. It is generally known that CQ is not incorporated into the polymer network, and the fraction that is not consumed in the reaction may be mostly eluted after polymerization, irrespective of the concentration [58]. CQ has also been described as being poorly soluble in water [59]. These facts explain the varied relative concentrations of whole CQ molecules observed in the chromatograms of the GC–MS analysis. Except for GCE, all cements contained the initiator CQ in the composition, which may be mostly consumed during the polymerization reaction when exposed to light. Based on the results, U200 presents higher relative concentration of CQ compared to the other cements tested. Non-irradiated CQ has been correlated with induced oxidative stress, DNA damage, and cytotoxicity in primary human gingival fibroblasts [60]. In another study [61] evaluating the cytotoxic effects of monomers and additives of resin composites in permanent 3T3 cells and three primary human oral fibroblast cultures, CQ exhibited moderate cytotoxic effects. The authors found significant amounts of CQ released in aqueous extracts from the resin-based materials tested. In another study, the cytotoxic mechanisms of CQ was found to be unrelated to the formation of reactive oxygen species (ROS) as neither hydrogen peroxide nor singlet oxygen was produced [62]. In fact, CQ was regarded to induce cell death possibly due to the formation of free radicals [62]. According to the results of the present study, it seems that the concentration of this initiator system released from the cements was not the prevailing cytotoxic compound of the CQ-based cements tested. The methacrylate monomer HEMA was found in 4 out of 5 cements evaluated (excepted for MAX). In a previous study, no significant changes in cell viability were observed when human lymphocytes were exposed to methacrylate monomers, including HEMA, but these effects were linked with apoptosis and cell cycle changes [63]. This can be attributed to a HEMA-induced increased production of ROS and oxidative DNA damage through double-strand breaks [64]. As a consequence, the glutathione (GSH) detoxifying intracellular pool forms adducts with HEMA through its cysteine motif, starting an inflammatory reaction [64]. Oxidative stress caused by ROS in resin-exposed cells seems to activate the pathways that control the cell death and survival and the cell proliferation and differentiation [23]. The biochemical execution of apoptosis or the support of cell survival is finally determined by crosstalk between the regulators of distinct signaling pathways and the cellular redox status [65].




The monomer-induced disturbance of the intracellular redox homeostasis has been associated with the onset of apoptosis [66]. Moreover, resin monomers are claimed to down-regulate gene expression in pulp cells and to inhibit mineralization, thereby indicating a decrease in the regenerative potential of the dentin–pulp complex [67]. In a another study, the exposure of human bronchial epithelial cells (BEAS-2B) to HEMA led to an increased apoptosis, interruption of the cell cycle, and decreased cell proliferation, thereby reducing the viability [68]. Although a depletion of cellular glutathione and increased levels of ROS can be observed, more studies are necessary to understand the cytotoxic effect of this monomer, considering that HEMA-induced cell damage may not be solely caused by these mechanisms [68]. In addition, although increased ROS levels are generally correlated with genotoxicity [69], not all resin-based restorative material are genotoxic, inducing DNA damage or successive gene mutations [70,71]. In spite of the induction of possible cytotoxic and genotoxic effects of HEMA, these effects should be assigned more to its degradation products than to the monomer itself [72]. The main product of HEMA hydrolysis is methacrylic acid (MAA) which may undergo further degradation, producing several intermediates of potential biological activity [72]. In the present study, MAA was eluted by all of the cements evaluated. An increase of eluted MAA has been previously demonstrated, and the exposure time varied from 20 to 40 s in resin composites [14]. The authors speculated that MAA might be formed during the polymerization process or by photo-induced degradation during the elution process [14]. It has also been found that intra- and intermolecular reactions may occur during the polymerization process of resin-based materials, thereby influencing the polymer network formation, and forming byproducts such as MAA [73]. TEGDMA, a major monomer of resin matrix, especially in dentin bonding agents (30–50%), is also derived from MAA [74]. The metabolic pathway of TEGDMA may start with hydrolyzation by unspecific esterases to methacrylic acid and triethylene glycol [23]. TEGDMA was found to be a main leachable compound from the less polymerized resin [61], which caused public concern for the adverse biological effects of the material [71]. In the present study, based on the manufacturers’ information, the only TEGDMA-containing cement is Clearfil SA Luting. HEMA and TEGDMA-free methacrylate-based restorative materials seems to present advantages with respect to biocompatibility. Although most of the cements tested are either HEMA- and/or TEGDMA-based materials, their byproducts were found by GC–MS in all of the cements tested. The results of the present study do not agree with those obtained in a previous study [7], in which it was claimed that RelyX U200 contained no HEMA in the chemical composition. Based on the GC–MS results (Table 2), U200 released a relative percentage of HEMA (5.8%). The authors possibly referred to the absence of HEMA based on the manufacturer’s information, as the authors did not refer to any methods of evaluating its presence in the chemical formulation. In addition, the authors generally attributed to HEMA-free cements an absence of cell viability reduction when cultured pulp cells were exposed to extracts obtained from these cements, whereas extracts from HEMA-containing ones would cause

9

intense cytotoxic effects [7]. In addition, it was speculated that HEMA-free dental materials not requiring a pre-conditioning clinical step would be considered a more biocompatible option to be applied to deep dentin [20]. In the present study, the only HEMA-free cement (MaxCem Elite) caused the highest reduction in the percentages of cell viability and significant increase in total cell death. Conversely, there was an agreement in terms of results that considered U200 to be less cytotoxic for inducing lower reduction in cell viability in both studies, with similar results compared to that of the control, unexposed cell groups. Other studies also demonstrated lower cytotoxicity when varied cell lines were exposed to a previous version of RelyX U200 (RelyX Unicem) [6,50]. In this way, previous concerns related to the presence of chromium ion in the formulation of U200 [75], known for its cytotoxicity and mutagenicity in certain species, and also to the presence of significantly higher free radical entrapment observed for this self-adhesive cement [18] were not proven valid considering the results of the present study for this material. Despite the lower cytotoxicity, mild inflammatory pulp response was found when inlays were cemented with RelyX Unicem in an in vivo study [76]. Clearfil SA Luting and Bifix SE cements, based on the manufacturers’ information, contain BisGMA (bisphenol A diglycidil dimethacrylate). Bifix SE also contains UDMA (urethane dimethacrylate). For methodical reasons, monomers with a higher molecular weight such as BisGMA or UDMA cannot be quantified with GC–MS, [51], which limits the comprehension of the results of the present study. On the other hand, this method allows the detection of low molecular weight compounds, which are claimed to be the most elutable substances from resin-based restorative materials [51]. Lee and coworkers [71] ranked the cytotoxicity of the most used resin monomers in RPC-C2A pulp cells as follows: Bis-GMA > UDMA > TEGDMA > HEMA > MMA. The same ranking order was observed by Gupta et al. (2012) in isolated human gingival fibroblasts [27]. Despite the cytotoxicity of BisGMA-based materials, Clearfil SA Luting was one of the least cytotoxic cements tested. Bifix SE, a BisGMA-, UDMA-based self-adhesive cement, was found to induce an increase in cell apoptosis when either photoactivated after 10 min or chemically activated (25.8% and 30.6%, respectively) (Fig. 2). Chemical activation of Bifix SE caused the highest reduction of viable cells (68.9%). Based on GC–MS results, 2,4-Dinitrophenyl crotonate was one of the substances released by this cement (Table 2). This compound is derivative of dinitrophenols (DNPs), a group of substances widely used as insecticides, acaricides, and fungicides in the agriculture or as raw materials in the dye industry [37]. DNPs are extremely noxious, producing cataracts, lowering leucocyte levels, disturbing the general metabolism, and producing carcinogenic effects [77]. Cases of death cases have also been reported [37]. It is true that the nature and amount of components released from the cements also depend on the molecular weight and hydrophilicity of the penetrating monomers and organic substances [78]. These chemical characteristics facilitate the ability of the lixiviating compounds to reach the pulp tissue, depending on the remaining dentin thickness [79]. Hydrophilic monomers, such as HEMA and TEGDMA, were



10


regarded as more easily diffusing through the dentin into the pulp. Conventional resin cements, associated with adhesive systems, exhibited less cytotoxicity compared to when a fibroblast cell line was exposed to self-adhesive cements [50]. Despite the tubule area opening promoted by etching, larger and hydrophobic monomers, such as BisGMA, may be retained by the interfacial hybrid layer, whereas smaller and hydrophilic monomers, such as HEMA, may infiltrate into the demineralized dentin [80]. Conversely, as self-adhesive resin cements require no acid etching, priming, or bonding [4], and considering that TEGDMA and Bis-GMA monomers have lower hydrophilicity and higher molecular weight, the absence of pre-treated dentin may hamper the diffusion of these high molecular weight monomers through the dentin at a toxic concentration [80]. GC–MS methods detected the presence of phenolic compounds in all the self-adhesive cements evaluated (Table 2). Phenolic compounds are considered cytotoxic agents but no genotoxic effects were observed in human pulp fibroblasts in vitro [81]. One of the phenolic components found in 3 out 5 cements tested (CSA, BSE, and GCE) is the inhibitor BHT, which presents severe cytotoxic effects [27]. This chemical derivative of phenol is generally used in the food industry, the cosmetics industry, and the therapeutic industry, and it has been claimed that it is highly volatile and unstable at elevated temperatures [82]. BHT has also been regarded as cytotoxic and as inducing apoptosis [83]. Conversely, according to the results of the present study, the phenolic compound phenol, 2,4-bis(1,1-dimethylethyl)- (PD), which is present in the cement U200, seems to present no cytotoxic effect. A previous study demonstrated that PD affected the regulated virulence factor production in a gram-negative bacterium (S. marcescens) and resulted in significant reduction in mediated biofilm formation and protease, among other developments, without hampering growth [84]. Considering that monomers may be a substrate for cariogenic bacterial strains, the addition of additives with intrinsic antibacterial properties would minimize the effects of the long-term degradation of the polymers, and consequently the failure in restorations [85]. Chemical activation of MaxCem Elite induced significantly higher % of total cell death (82.8%). In the present study, extracts of MAX reduced the number of MDPC-23 cells to 36.1% (when immediately photoactivated), 42.9% (when photoactivated after 10 min), and 1.7% (when chemically activated). Cell apoptosis and cell death increased when the odontoblastic cell line was exposed to the chemically activated specimens (50.8% and 3.86%, respectively). In this way, MAX was the material with the most cytotoxic material among the cements tested, irrespective of the activation mode. These results were in accordance with previous studies [30,50]. This cement contains, among other components, adhesive monomers including GPDM and an exclusive initiator system [30]. MAX released 11 substances, among them benzyl alcohol (BA). According to a previous study, the cytotoxic mechanisms of BA were found to induce an increasing apoptosis and necrosis levels in exposed human dermal fibroblasts [39]. BA also led to necrosis of human retinal pigment epithelial cells and triggered mitochondrial apoptosis [86]. Cyclohexanone was also another compound released by MAX. Chromosomal aberrations and aneuploidy have been described as being induced

by cyclohexanone in cultured human lymphocytes [87] and in the bone-marrow cells of rats treated in vivo [88]. An increased ROS production after exposure to MAX extracts has also been regarded as the main reason for the higher cytotoxicity [30]. Despite the correlation between the presence of ROS and cytotoxicity, this has not generally been associated with all resin cements, particularly for U200 [30]. G-Cem LinkAce cement was also found to have cytotoxic effects on the cell line tested. The results of cell viability assay demonstrated that the activation mode also influenced the results, mainly when chemically activated. In this case, only 31.9% of the cells were viable, compared to 63.0% when immediately photoactivated and 68.1% when activated after 10 min. According to the manufacturer, GCE is a BisGMA-, UDMA-based cement in which a proprietary acidic dimethacrylate was also added to the formulation. GCE is the cement from which less compounds were detected using GC–MS. Most of them are methacrylate derivatives (MMA, acrylic acid, and HEMA) and acetic acid. Among the released compounds, BHT was also found (Table 2), which is a synthetic antioxidant widely used to control free radicals, thereby preventing oxidation [89]. As previously described, BHT is considered an antioxidant with carcinogenic effects [90]. Another carcinogenic compound released by GCE is 3-MCPD (Table 2). This substance has been reported to be carcinogenic, nephrotoxic, and reproductively toxic in laboratory animal testing, especially in rodents [91]. Although it is difficult to correlate the cause and effect between cytotoxicity and a specific compound, the results of the present study suggest that the potential cytotoxicity demonstrated by these materials is of clinical relevance, as all self-adhesive cements evaluated induced varied cytotoxic effects to odontoblastic cells. Based on the results, it is valid to state that the cytotoxicity of luting cements is not only material dependent but also dependent on their activation mode. In this way, differential toxic effects of resin cements on the pulp cells should be considered during the selection of suitable resin cements for restoration [92]. Concentrations of biologically active components released by most of the self-adhesive resin cements may be high enough to modify the pulp cell metabolism [70], especially its regenerative and reparative capacities [30]. This is particularly true in clinical situations in which the materials are applied to newly exposed dentin, particularly in total crown preparations and deeper preparations, or when the materials are in direct contact with the pulp tissue [18,30,51]. In addition, specific techniques for inlay cementation using different luting cements may cause diverse toxic effects on pulp tissue [8]. The digital pressure required to seat the inlay restoration during cementation also seems to mechanically stimulate inward dentinal fluid movement, which would somehow lead to the diffusion of unreacted residual resin components toward the pulp tissue [8]. On the other hand, it has been claimed that cytoplasmic extensions of odontoblasts and collagen fibrils observed in vital teeth, which are associated with the presence of fluid within the dentinal tubules, seem to be the main mechanism for avoiding the inward diffusion of potential cytotoxic compounds released from dental materials, thereby minimizing the impact on the pulp cells [93].




Restoration completion requires numerous steps before a restorative process is completed. The simplification of clinical steps in using restorative materials is critical to the success of a restorative procedure, because many aspects need to be considered [18]. Innovative research into acidic monomers in self-adhesive systems used for the development of self-adhesive cements has focused on increasing hydrolytic stability at the tooth-adhesive interface, especially under acidic conditions [51]. Clinically, self-adhesive cements are applied to the dentin substrate in relative humidity [94]. When in contact with hydrated dental substrates, the acid-functionalized monomers are ionized, acidifying the medium, which triggers an acid-based reaction between the glass fillers and the dental substrates (called glass ionomer concept) [1], which, in sequence, neutralizes the monomers’ ionization, and accordingly creates a polyacid matrix [95]. On the other hand, this humidity favors the solubility of the interfacial bonded area [96]. Accelerated degradation of the interfacial area formed by poorly polymerized acidic resin adhesives has also been reported as a result of the reaction between acidic monomers and the amine co-initiator that is commonly used in conventional camphorquinone/amine photoinitiator systems [97]. In this way, further studies are necessary for a better understanding of the curing mechanisms of self-adhesive cements in contact with the dentinal tissue. In the present study, it was demonstrated that the exposure of an odontoblastic cell line to different self-adhesive resin cements induced cytotoxicity in diverse magnitudes. In addition, the activation protocol is of clinical relevance, because the results were influenced by the mode in which the cements were polymerized. In this way, the concentrations of biologically active ingredients released by the self-adhesive cements, a function of the chemical formulation and activation mode, were high enough to modify the pulp metabolism of odontoblastic cells. In addition, it is important to highlight the problems related to the fact that the polymerization completion is compromised in areas in which the curing light energy does not reach, which has been found in various clinical cases in which the chemical reaction may occur [12]. Considering that only a relative percentage of the monomers are converted into polymers, and the fact that these materials are dual-cured restoratives, the amount of residual unreacted monomers may vary whether the cements are photoactivated or chemically activated. Biocompatibility and the understanding of cellular mechanisms are vital for managing and supporting dentin formation and pulp regeneration in dental therapy [23]. Further understanding of the cellular mechanisms involved in these processes, and clarification of the causal relationship between the released substances and compound-induced apoptosis, will certainly contribute to the development of newly bioactive dental restorative materials.

5.

Conclusions

Within the limitations of this study, it can be concluded that: 1. All of the self-adhesive cements tested induced a significant decrease in the viability of MDPC-23 cells (hypothesis rejected).

11

2. Choice of polymerization protocols in most of the cements tested affects the cytotoxicity, the total cell death, and the type of cell death in odontoblastic pulp cells exposed to different self-adhesive cements (hypothesis rejected). 3. Based on the parameters evaluated, one of the tested products induced higher cytotoxic effects than the others did: higher percentage of total death cell, increased characteristic apoptotic cells, higher percentages of necrosis and late apoptotic/dead cells, irrespective of the activation protocol. 4. The varied monomers, their byproducts, and other organic substances released from the cements seem to be highly detrimental to the metabolism of odontoblast pulp cells.

Acknowledgements This study was developed as partial fulfillment of the requirements of Dr. Barbosa’s PhD degree (UNIAN – SP). This study was partially supported by a grant from FAPESP (#13/05822-9). The authors are grateful to Universidade de Campinas (UNICAMP) for the technical support in the headspace gas chromatography/mass spectrometry analysis. The authors are also grateful to Universidade Federal de São Paulo (UNIFESP) for the technical support in the cell viability analysis.

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Microshear Bond Strength of Resin Cements to Lithium Disilicate Substrates as a Function of Surface Preparation.

Photo-activation of resin cements through porcelain veneers.

Effects of plasma-emulating light emitting diode (LED) versus conventional LED on cytotoxic effects of orthodontic cements as a function of polymerization capacity.

Influence of volume and activation mode on polymerization shrinkage forces of resin cements.

Cytotoxic effects of resin components on cultured mammalian fibroblasts.

Self-adhesive resin cements: a clinical review.

Effect of the Acidic Dental Resin Monomer 10-methacryloyloxydecyl Dihydrogen Phosphate on Odontoblastic Differentiation of Human Dental Pulp Cells.

Setting Time of Dual-cured Resin Cements.

Effects of barriers on chemical and biological properties of two dual resin cements.

Effects of different surface treatments on bond strength between resin cements and zirconia ceramics.

Effect of root canal rinsing protocol on dentin bond strength of two resin cements using three different method of test.

Resin composite characterizations following a simplified protocol of accelerated aging as a function of the expiration date.

Matrix metalloproteinase-3 in odontoblastic cells derived from ips cells: unique proliferation response as odontoblastic cells derived from ES cells.

Cytotoxicity evaluation of luting resin cements on bovine dental pulp-derived cells (bDPCs) by real-time cell analysis.

Macrophage activation induced by different carbon fiber-epoxy resin composites.

Polymerization kinetics of resin cements after light activation through fibre posts: an in vitro study.

The effect of resin cement type and cleaning method on the shear bond strength of resin cements for recementing restorations.

Comparison of the shear bond strength of self-adhesive resin cements to enamel and dentin with different protocol of application.

Self-adhesive resin cements: a new perspective in luting technology.

Differential cytotoxic activity of the petroleum ether extract and its furanosesquiterpenoid constituents from Commiphora molmol resin.

Effects of EDTA irrigation and water storage on the bonding durability of different adhesive resin cements to intra-radicular dentin.

Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells.

The effect of simplified adhesives on the bond strength to dentin of dual-cure resin cements.

The influence of resin cements on the final color of ceramic veneers.