Elsevier

Dental Materials

Volume 33, Issue 12, December 2017, Pages 1402-1415
Dental Materials

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

https://doi.org/10.1016/j.dental.2017.09.011Get rights and content

Highlights

  • We evaluated the cytotoxicity of self-adhesive cements activated with different protocols.

  • Volatilized compounds from polymerized cements were evaluated by headspace GC–MS.

  • All of the cements tested significantly reduced the cell viability, irrespective of the activation protocol.

  • Total cell death increased, especially when exposed to chemically activated cements.

  • Chromatograms revealed 28 compounds released from the cements, some with carcinogenic effects.

Abstract

Objectives

To evaluate the cytotoxic effects of exposing odontoblast cells to a variety of commercial self-adhesive cements polymerized using different activation modes.

Methods

Five cements: MaxCem Elite (MAX), Bifix SE (BSE), G-Cem LinkAce (GCE), Clearfil SA Luting (CAS), and RelyX U200 (U200) were mixed, dispensed into molds, and distributed in groups, according to polymerization protocols: immediate photoactivation; delayed photoactivation (10 min self-curing plus light-activation); and chemical activation (no light exposure). Immortalized rat odontoblast cells (MDPC-23) were cultured. Cell viability was assessed by Trypan Blue staining and total cell death was assessed by annexin V-APC/7-AAD double staining and flow cytometry. Volatilized compounds from polymerized specimens of cements were evaluated by gas chromatography/mass spectrometry (GC–MS). Data was analyzed with 2-way ANOVA/Tukey tests (α = 0.05).

Results

Exposure to all of the cements tested significantly reduced the cell viability, irrespective of the activation protocol (p < 0.05). The least harmful cements were CSA and U200. 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, 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.

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], [21], [22], [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], [25], [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].

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 significantly influence cell viability and overall percentage of cell death.

Section snippets

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) self-adhesive 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 chemical activation. The characteristics of the resin

Results

The results for the total cell death are depicted in Fig. 1, Fig. 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

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

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).

  • 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

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.

References (97)

  • A.P. Manso et al.

    Cements and adhesives for all-ceramic restorations

    Dent Clin North Am

    (2011)
  • T.A. Pegoraro et al.

    Cements for use in esthetic dentistry

    Dent Clin North Am

    (2007)
  • A.D. Vrochari et al.

    Curing efficiency of four self-etching, self-adhesive resin cements

    Dent Mater

    (2009)
  • M. Schmid-Schwap et al.

    Cytotoxicity of four categories of dental cements

    Dent Mater

    (2009)
  • W. Spahl et al.

    Determination of leachable components from four commercial dental composites by gas and liquid chromatography/mass spectrometry

    J Dent

    (1998)
  • J. Volk et al.

    Non-irradiated campherquinone induces DNA damage in human gingival fibroblasts

    Dent Mater

    (2009)
  • T. Christensen et al.

    In vitro photosensitization initiated by camphorquinone and phenyl propanedione in dental polymeric materials

    J Photochem Photobiol B

    (2010)
  • E. Pawlowska et al.

    Genotoxicity and cytotoxicity of 2-hydroxyethyl methacrylate

    Mutat Res

    (2010)
  • G. Spagnuolo et al.

    Effect of 2-hydroxyethyl methacrylate on human pulp cell survival pathways ERK and AKT

    J Endod

    (2008)
  • M. Demirci et al.

    The induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives

    Dent Mater

    (2008)
  • D.H. Lee et al.

    Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures

    Dent Mater

    (2006)
  • H. He et al.

    Photopolymerization and structure formation of methacrylic acid based hydrogels: the effect of light intensity

    React Funct Polym

    (2008)
  • J. Durner et al.

    Metabolism of TEGDMA and HEMA in human cells

    Biomaterials

    (2010)
  • P.H. D’Alpino et al.

    Inorganic characterizations and filler particles morphology of self-adhesive cements

    Int J Adhes Adhes

    (2016)
  • C.A. de Souza Costa et al.

    Human pulp response to resin cements used to bond inlay restorations

    Dent Mater

    (2006)
  • K.L. Takahashi et al.

    Effects of dinoseb, 4,6-dinitro-o-cresol, and 2,4-dinitrophenol on rat Sertoli-germ cell co-cultures

    Reprod Toxicol

    (2003)
  • M. Cadenaro et al.

    Effect of adhesive hydrophilicity and curing time on the permeability of resins bonded to water vs: ethanol-saturated acid-etched dentin

    Dent Mater

    (2009)
  • Y.C. Chang et al.

    Cytotoxic and nongenotoxic effects of phenolic compounds in human pulp cell cultures

    J Endod

    (2000)
  • W.A. Yehye et al.

    Understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (BHT): a review

    Eur J Med Chem

    (2015)
  • A.M. Papas

    Diet and antioxidant status

    Food Chem Toxicol

    (1999)
  • C.A. de Souza Costa et al.

    Response of human pulps following acid conditioning and application of a bonding agent in deep cavities

    Dent Mater

    (2002)
  • V. Di Hipolito et al.

    Effectiveness of self-adhesive luting cements in bonding to chlorhexidine-treated dentin

    Dent Mater

    (2012)
  • J.L. Ferracane

    Resin composite-state of the art

    Dent Mater

    (2011)
  • N. Hiraishi et al.

    Effect of 2% chlorhexidine on dentin microtensile bond strengths and nanoleakage of luting cements

    J Dent

    (2009)
  • S. Ito et al.

    Effects of resin hydrophilicity on water sorption and changes in modulus of elasticity

    Biomaterials

    (2005)
  • I. Radovic et al.

    Self-adhesive resin cements: a literature review

    J Adhes Dent

    (2008)
  • W.M. Palin et al.

    Resin-based cements used in dentistry

  • J.L. Ferracane et al.

    Self-adhesive resin cements – chemistry, properties and clinical considerations

    J Oral Rehabil

    (2011)
  • B. Van Meerbeek et al.

    Comparative SEM and TEM examination of the ultrastructure of the resin-dentin interdiffusion zone

    J Dent Res

    (1993)
  • A.A. de Mendonca et al.

    Cytotoxic effects of hard-setting cements applied on the odontoblast cell line MDPC-23

    Oral Surg Oral Med Oral Pathol Oral Radiol Endod

    (2007)
  • L. da Fonseca Roberti Garcia et al.

    Transdentinal cytotoxicity of resin-based luting cements to pulp cells

    Clin Oral Investig

    (2016)
  • J.L. Ferracane

    Elution of leachable components from composites

    J Oral Rehabil

    (1994)
  • R. Svizero Nda et al.

    Effects of curing protocols on fluid kinetics and hardness of resin cements

    Dent Mater J

    (2013)
  • R. Trumpaite-Vanagiene et al.

    Cytotoxicity of commonly used luting cements — an in vitro study

    Dent Mater J

    (2015)
  • E.C. Pontes et al.

    Cytotoxicity of resin-based luting cements to pulp cells

    Am J Dent

    (2014)
  • R.R. Vaz et al.

    Bond strength and interfacial micromorphology of etch-and-rinse and self-adhesive resin cements to dentin

    J Prosthodont

    (2012)
  • A.V. Susila et al.

    Correlation of elution and sensitivity of cell lines to dental composites

    Dent Mater

    (2016)
  • D.G. Soares et al.

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

    Int Endod J

    (2016)
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