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

Abstract

Objectives

Polymerized dental resin materials release residual monomers that may interact with pulp tissues. We hypothesized that dental adhesives might cause cytotoxicity in pulp cells via the generation of reactive oxygen species (ROS), which may also contribute to genotoxic effects in vitro.

Methods

For cytotoxicity testing, transformed human pulp-derived cells were exposed to extracts of primers and bonding agents of Clearfil SE bond, Clearfil Protect bond, AdheSE, Prompt L-Pop, and Excite for 24 h. The cytotoxicity of the same materials was also analyzed in a dentin barrier test device using three-dimensional pulp cell cultures. The generation of ROS in monolayer cultures was measured after a 1 h exposure period by flow cytometry (FACS), and genotoxicity as indicated by the formation of micronuclei was determined in V79 cells after a 24 h exposure period.

Results

The dentin primers and bonding agents decrease cell survival in a dose-related manner. Cytotoxicity of bonding agents based on concentrations which caused 50% cell death (EC50) were ranked as follows: Excite (0.16 mg/ml) > AdheSE bond (0.30 mg/ml) > Clearfil Protect bond (0.35 mg/ml) > Clearfil SE bond (0.37 mg/ml), and Prompt L-Pop bond (0.68 mg/ml). Dentin primers were about 10-fold less effective. In contrast, no cytotoxic effects of the dental adhesives were observed in a dentin barrier test device. Yet, all dental adhesives increased the amounts of ROS about fivefold in pulp cells in a dose-related manner, and, again, the bonding agents were more efficient than the dentin primers. Finally, the number of micronuclei was increased about sixfold by extracts of the AdheSE primer.

Significance

Our results suggest that the cytotoxic potencies demonstrated by these materials might be of clinical relevance, since all dental adhesives disturbed the cellular redox state of pulp cells in monolayer cultures. As a result, the concentrations of biologically active ingredients of some of the agents may be high enough to modify pulp cell metabolism when the materials are used in deep cavities or directly contact pulp tissue.

Introduction

Modern dental adhesive systems are used to improve the contact between the restorative material and the walls of the prepared cavity of the tooth. As these materials come in close and prolonged contact with vital dentin, their influence on pulp tissue is critical. Thus, the biocompatibility of dentin bonding agents is a relevant aspect of the clinical success of these materials [1], [2], [3], [4]. Dentin bonding agents alone proved to be cytotoxic, and it has been found that the type and quantity of leachable components significantly influence the biological behavior of resin restorations [5], [6], [7], [8], [9], [10]. Cytotoxicity of dentin bonding agents has been examined using a variety of cell lines including primary human pulp and pulp-derived cells [5], [6], [7], [8], [11], [12], [13], [14]. Yet, the isolation of primary cells from target tissues is labor intensive and time consuming, and the resulting cell numbers are often very low compared to the almost unlimited number of cells obtained from continuous cell lines. Most important, primary cells have a limited life span. These shortcomings might be overcome by the construction of transformed primary cells with an expanded life span retaining properties of the original cells. Recently, stable cell lines were generated after transfection of primary bovine and human pulp-derived cells with coding sequences of transforming oncogenes [15], [16], [17]. Moreover, in vitro pulp chambers have been designed, introducing dentin as a barrier between test material and target cells. Transformed pulp-derived cells were grown as three-dimensional cultures and included in a dentin barrier test device to mimic the interactions between a target tissue, matrix proteins and filling materials that occur in vivo [18], [19].

As a consequence of aerobic metabolism, small amounts of reactive oxygen species (ROS), are constantly generated in cells and tissues. Cellular antioxidants like glutathion act in unison to detoxify these reactive molecules, but when the balance between oxidants and antioxidants is disrupted, a condition referred to as oxidative stress exists. If oxidative stress persists, oxidative damage to lipids, proteins and nucleic acids accumulates and eventually results in biological effects ranging from the alteration of signal transduction pathways and gene expression levels to cell transformation, mutagenesis and cell death [4]. Leachables from resin-based materials such as HEMA and TEGDMA are a likely cause of cellular stress via the formation of ROS. Recently, it was shown that there was a possible link between ROS production and cytotoxic activity [20], [21], [22], [23], [24]. Moreover, the induction of genotoxic effects of TEGDMA and HEMA has been demonstrated in vitro as well, indicating DNA reactivity of the compounds. In addition, cytotoxic resin materials were shown to cause cytotoxicity and elevated numbers of micronuclei [25], [26]. In the present study, we combined and analyzed various aspects of cell responses towards dental adhesives. First, we hypothesized that biologically active monomers or additives may be released from dentin bonding agents to cause cytotoxicity in pulp cell monolayers. Furthermore, the production of ROS may indicate an early onset of processes leading to cell death via apoptosis, or the generation of DNA damage as indicated by elevated numbers of micronuclei. In addition, the cytotoxicity of dentin bonding agents was also evaluated in a dentin barrier test device using three-dimensional cultures of transformed pulp-derived cells to mimic an in vivo situation.