Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol
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.
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2021, Dental MaterialsCitation Excerpt :A few experiments have been conducted to evaluate the cytotoxicity of resinous cements, mostly focusing on the evaluation of a widely-since several years-used commercially available product of the self-adhesive resin cement, RelyX (3M, USA). Most studies utilized 2D in vitro experimental settings [15–18] or in vivo settings on teeth that were predetermined to be extracted [13]. Somewhere between the two settings (2D in vitro and in vivo), other examples of more sophisticated in vitro models can be found in the literature, such as the dentin barrier test (perfusion chamber), which was initially trialed with cells seeded in a 2D monolayer on dentin disks [19,20] or more recently within 3D surrogates [21–23], following the ISO 7405:2018 specifications [24].
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