F.-M. Huang & Y.-C. Chang
Department of Prosthodontics, Chung Shan Medical University Hospital, Taichung, Taiwan.
School of Dentistry, College of Oral Medicine, Chung Shan Medical University, Taichung, Taiwan.

Introduction.
Dentine-bonding agents are recommended in the placement of resin-based restorative materials. The agents are used to improve the contact between the restorative material and the walls of the prepared cavity of the tooth. As dentine-bonding agents come into close and prolonged contact with vital dentine, their influence on pulp tissue is of great interest. Therefore, dentine-bonding agents should have good biocompatibility.
In general, the biocompatibility of dentine-bonding agents is assessed by a three-step approach. The first step is to screen a candidate material using a series of in vitro cytotoxicity assays. Second, if the material tested is not a cytotoxic agent in vitro, it can be implanted subcutaneously and the local tissue reaction evaluated. Finally, the in vivo effects of the material on target tissue cells must be evaluated in animals or human beings.
In vitro cytotoxic screening as a primary factor of biocompatibility is determined by cell culture. Experimentation in vitro has the advantage of easily controlled experimental factors that are often a problem when performing experiments in vivo. In vitro methods are simple, reproducible, cost-effective, relevant, and suitable for the evaluation of basic biological properties of dental materials (Mjor 1978).
The biocompatibility of dentine-bonding agents has been extensively studied using murine fibroblast cell line L929 (Schedle et al. 1998, Hashieh et al.1999, Kaga et al. 2001), mouse odontoblast-like cell line MDPC-23 (de Souza Costa et al. 1999) and human gingival fibroblasts (Szep et al. 2002, Huang et al. 2002a). Although culture systems vary considerably in the way cytotoxicity is measured, most employ cells that are transformed or established cell lines as the model for cell response. However, normal diploid cells differ from established, or transformed cells in many ways (Holley 1975, Lechner & Kaighn1979, Feigal et al.1985). In vitro cytotoxicity tests should be performed with cells homologous to the human tissue of ultimate concern (Haustveit et al.1984, Yesilsoy & Feigal1985, Chang et al.1998; 2000).
To date, the sensitivity of cultured human pulp cells to dentine-bonding agents has not been adequately studied. It is important to clarify the effects of dentine-bonding agents on cells derived from oral tissues, such as pulp cells, because bonding agents come into close contact with pulp tissues. The aim of this study was to evaluate the cytotoxicity of five dentine-bonding agents on cultured human pulp cells.

Materials and methods.

Dentine-bonding agents.
Five dentine-bonding agents were evaluated: Clearfil SE Bond (CB), Heliobond (HB) Prime & Bond NT (PB NT), Single Bond (SB), and Syntac Single Component (SSC) (Table 1).

Table 1. Principal components of the dentine-bonding agents tested.

Sample fabrication.
In order to evaluate the effects of the photo-polymerized dentine-bonding agents, 60 mm _10 mm cellulose strips were exposed to UV light for 30 min to prevent bacterial contamination. On the strips, 10 mL of each dentine- bonding agent was applied and light-cured for 20 s. After polymerization, specimens were placed in polyethylene vials directly after mixing.

Elute preparation.
Each specimen was placed in 8 mL of fresh Dulbecco’s modified Eagle’s medium (DMEM) and transferred into fresh media after 48 h and 5 days. All samples were extracted twice consecutively in culture medium. After each elution period, the extracts were removed, and the vials were filled again with fresh medium. The extraction media were then collected into sterile syringes at the end of this period and passed through a 0.22-mm filter. Subsequently, these extraction media were prepared to be used in this study.

Cell culture.
Human dental pulp cells were cultured using an explant technique as described previously (Chang et al. 1998; 2000).The tooth root was removed by horizontal section below the cementoenamel junction with a no. 330 bur in high-speed handpiece with water spray. The pulp tissue was removed aseptically, rinsed with Dulbecco’s modified Eagle’s medium (DMEM), and placed in a 35- mm Petri dish. Pulp tissue was cut into small fragments with a no.15 blade and grown in DMEM supplemented with10% foetal calf serum and antibiotics (100 U mL_1 of penicillin, 100 mg mL_1 of streptomycin, and 0.25 mg mL_1 of fungizone), that is, complete medium. Cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. Confluent cells were detached with 0.25% trypsin and 0.05% EDTA for 5 min, and aliquots of separated cells were subcultured. Cell cultures between the third and eighth passages were used in this study.

Cytotoxicity assay.
A simple colorimetric assay developed by Mosmann (1983), as a test for cell proliferation and survival, has been adapted for the measurement of cytotoxicity. A 1mg mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) in complete medium was prepared just before use. Cells were diluted in fresh DMEM complete medium and seeded in 96-well plates (2 _104 cells/well). After overnight attachment, cells were treated with various extracts of dentine-bonding agents (200 mL/well) for 20 h, then 50 mL MTT dye was added to each well. Plates were incubated in a CO2 incubator for 4 h. Optical density was determined by eluting the dye with dimethyl sulfoxide (Sigma, St. Louis, MO, USA), and the spectrophotometric absorbance measured at 550 nm using a spectrophotometer (Hitachi, Tokyo, Japan).

Statistical analysis.
Five replicates of each concentration were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was conducted by one-way analysis of variance. Tests of differences of the treatments were analysed by Duncan’s test and a value of P < 0.05 was considered statistically significant.

Results.
The results showed that dentine-bonding agents were cytotoxic to primary human pulp cells by MTT assay. The sensitivity of cytotoxicity to human pulp cells depended on the materials tested.
As shown in Fig. 1, eluates from five dentine-bonding agents were cytotoxic to primary human pulp cells (P < 0.05). SB was significantly more cytotoxic than the other dentine-bonding agents tested. Day 2 eluates from all materials tested significantly inhibited growth of cells (P < 0.05), but the leaching of toxic substances diminished at Day 5 (Fig.1). In addition, the Day 5 eluates of CB and HB on pulp cells were not significantly different from control values (P > 0.05).
In general, the rank orders with respect to cytotoxicity were found to be as follows: SB > PB > SC > HB > CB.

Figure 1. Effect of the eluates from five dentine-bonding agents on human pulp cells by MTT assay. Percentage of absorbance from each material, compared with that of control was calculated.

Discussion.
In this study, cultured human pulp cells were used to evaluate the cytotoxicity of dentine-bonding agents. Primary pulp cells are more closely related to their original tissue and much easier to identify. In addition, pulp cells have a nearly unchanged metabolic state relative to their original tissue; the in vitro experiments thus approximate the in vivo situation. This is the reason why primary human pulp cells were chosen in this study.
Under normal conditions, few pulp cells proliferate in pulp tissue (Fitzgerald 1979). Pulp cells in the resting stage seem to reflect the in vivo condition more closely than do cells in the growing phase. Thus, in the present study, the cytotoxicity of dentine-bonding agents were examined on confluent cultures according to our recent studies (Chang & Chou 2001, Huang & Chang 2002b).
Several dentine-bonding agents have been evaluated for their cytotoxic effects (Hashieh et al. 1999, Schedle et al.1998, de SouzaCosta et al.1999, Kaga et al.2001, Szep et al. 2002, Huang & Chang 2002a). In accordance with this study, all dentine-bonding agents proved to be cytotoxic on different cell culture systems. Substances leached from dentine-bonding materials may be the reason why these materials exhibit cytotoxic effects. It has been demonstrated that unconverted monomers can be released from resin into an adjacent aqueous phase (Gerzina & Hume 1996) and can diffuse through dentine to the pulp space (Gerzina & Hume 1994). The toxic potential of components from dentine-bonding agents has been shown in vitro. Constituents of dentine- bonding agents, such as bisphenol-A-glycidyl methacrylate (Bis-GMA), triethyleneglycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA), were found to leach from dentine-bonding agents and may cause adverse effects (Geurtsen et al. 1998). It was found that hydrophilic monomers such as 2-hydroxyethyl methacrylate (HEMA) or TEGDMA were cytotoxic but to a lesser grade than the more hydrophobic monomers Bis-GMA or UDMA (Ratanasthien et al. 1995). In addition, HEMA or TEGDMA may also have an influence on the immune system (Rakich et al.1999).
In addition, dentine-bonding agents were found to release photoinitiator camphoroquinone (CQ) (Geursten et al. 1999). CQ is one of the photosensitizers, a widely used aliphatic type, which has been found attributed to free radicals including reactive oxygen generation (Atsumi et al. 1998). It also has been documented not only as acytotoxic agent (Atsumi et al.1998, Geursten et al. 1999) but also as a mutagen (Heil et al. 1996). Thus, CQ leached from dentine-bonding agents may partly explain why dentine-bonding agents are toxic agents.
From the results observed in the present study, it is concluded that dentine-bonding agents are cytotoxic to human pulp cells. However, the dentine might be important in protecting the dental pulp from the effects of substances leached from dentine-bonding agents. It is difficult to determine how much leach able substances actually act on pulp cells after diffusing through the dentine. Furthermore, the pulp circulation in vivo will wash out and reduce the concentrations of cytotoxic agents on pulp cells. Because dentine-bonding agents come into close and prolonged contact with vital dentine, comprehensive studies should be undertaken to clarify the substances leached from dentine-bonding agents effects pulp tissue both in vitro and in vivo.

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