In vitro cytotoxicity of a composite resin and compomer

Introduction
Many new dental restorative materials have entered the market in recent years. It is claimed these products have many advantages, such as fluoride release and improved adhesion to enamel and dentine. However, in many cases, the product is released onto the market without prior independent evaluation. In this context, cytotoxicity testing is an assessment of the probability that a material will be hazardous to the body, particularly with respect to the material’s potential to cause pulpal problems (Wataha et al . 1994).
In the past, many varieties of restorative materials have been tested and found to be hazardous. Kawahara et al . (1968) found that silicate and zinc phosphate cements were toxic to cultured cells, particularly early in the setting process. Meryon & Riches (1982) subsequently reported that composite resin materials were cytotoxic to fibroblasts and macrophages and Muller et al . (1990) found that some glass ionomer cements were toxic to primary rabbit pulpal fibroblasts.
In addition to problems arising from leakage and bacterial contamination that particularly affected silicate cements (Powis et al . 1988), the direct toxicity of restorative materials largely results from the elution of compounds from the material. For example, Rathbun et al . (1991) studied the elution of the components of composite-resin restorative materials into various organic solvents. They found that the monomer Bis-GMA (bisglycidyldimethacrylate) was the main material eluted and they also identified a benzophenone UV stabiliser that did not participate in the photo-activated polymerization reaction. Furthermore, Geurtsen et al . (1998) showed that the elution of TEGDMA (triethylene glycol dimethacrylate) was one of the main causes for the cytotoxic reactions evoked by the light-cured glassionomer cements and compomers that they investigated. In fact, the National Institute of Occupational Safety and Health has classified TEGDMA as being irritating to various tissues (RTECS 1995).
It has been found that the greater the extent of the photo-initiated polymerization reaction, the fewer the monomers available to be leached (Ferracane 1994). This relationship was demonstrated by Rueggeberg & Craig (1988) for a light-cured composite consisting of the monomers Bis-GMA and TEGDMA, who showed that the degree of light-curing of this monomer mixture and the degree of conversion of carbon-carbon double bonds, to form the hardened material, was reduced by curing the composite through varying thickness of previously cured composite which reduced the light intensity.
Another significant factor to consider is whether or not the dentine provides protection for the pulp. Several features of dentine may influence the potential toxicity of a restorative material. The limited wetability of dentine limits the dissolution of the applied material, and the buffering capacity of the dentine hydroxyapaptite allows for the use of acid-base reaction materials and liquid acids (Pashley 1996). Furthermore, the presence of a smear layer may further reduce the permeability of dentine by acting as a diffusion barrier (Pashley et al . 1981). However, it can also be argued that the smear layer could harbour bacteria, which would then multiply beneath the applied restoration (Brannstrom 1996). Indeed Hanks et al . (1994), using a ‘split chamber device’, found that the diffusion of biological and synthetic materials through dentine was indirectly proportional to the dentine thickness. Similarly, Gerzina & Hume (1994) compared the diffusion of TEGDMA through dentine to the pulp space with its release directly into aqueous solution in vitro and reported that dentine provided an effective method of retarding the release of TEGDMA.
However, it is important to note that a restorative material which is hazardous in vitro may not necessarily be toxic in vivo , possibly due to biological barriers or insufficient time of contact between the offending restorations and susceptible tissues (Wataha et al . 1994). Therefore, in order to obtain an accurate risk assessment, the in vitro testing model must reflect the clinical situation as closely as possible. Two main strategies have been employed to date. The first is testing of the components of materials to cells in monolayer culture, constructing dose–response curves and then using this to estimate the cytotoxic potential of these components in vivo ; secondly, the use of barriers between the material and the cells to mimic barriers which might exist in vivo (Wataha et al . 1994). In the present study the second strategy was used, in which a dentine barrier was placed between cultured cells and the materials to be tested in vitro in order to simulate possible pulpal effects in the clinical situation.
All cells have the ability to die through activation of an internally encoded suicide programme. Once activated, this programme initiates a characteristic form of cell death called apoptosis (Thompson 1995). One of the essential features of apoptosis is that, although the membrane is thrown into massive convolutions (blebbing), it remains intact. The dead cells are rapidly removed through engulfment by macrophages and any leakage of their noxious contents and consequent inflammatory response is avoided (Cotter & Martin 1995). In contrast, necrosis is a pathological form of cell death that results from an overwhelming cellular injury. It is associated with an early loss of cell membrane integrity, resulting in leakage of cytoplasmic contents and the induction of an inflammatory response. Unlike apoptosis, where there is controlled auto-digestion of the cell, necrotic cells spill their contents out over the surrounding cells, thus spreading any toxic or infectious agent and resulting in a spreading wave of necrosis (Thompson 1995).
There have been few studies of the mode of cell death induced by dental restorative materials. In view of this, morphological and enzymatic assessments of the mode of cell death were performed after light curing samples of restorative materials for different time periods.