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 »  Home  »  Endodontic Articles 2  »  Cytotoxicity of resin-, zinc oxide–eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells
Cytotoxicity of resin-, zinc oxide–eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells
Introduction - Materials and methods.



Introduction.
Teeth with severe pulpal or periapical inflammation can be successfully treated with the established techniques of cleaning and shaping the root canals, followed by obturation of the root canal system. A large variety of root canal sealers have been advocated for use, in conjunction with solid or semisolid filling materials. Currently, root canal sealers are available based on various formulas such as expoxy resin, calcium hydroxide and zinc oxide–eugenol. Ideally, root canal sealers should be biocompatible and have satisfactory physico-chemical properties. They should also be well tolerated by the periradicular tissues. Indeed, since these materials will be in direct contact with periapical tissues for prolonged periods of time, their biocompatibility is of primary importance. A biocompatible sealer should neither prevent nor hinder tissue repair, but should aid or stimulate the reorganization of injured structures. One method of testing the biological compatibility of root canal sealers is to use an in vitro model to determine the cellular response. This has the advantage that many factors and variables can be controlled (Arenholt-Bindslev & Bleeg 1990, Barbosa et al . 1993) and the cytotoxicity can be determined with reliability and reproducibility (Arenholt-Bindslev & Horsted-Bindslev 1989, Beltes et al . 1995). Numerous permanent cell lines and oral primary fibroblasts derived from humans or animals have been used to evaluate cytotoxic effects of extracts, as well as solid specimens of root canal sealers (Arenholt-Bindslev & Horsted-Bindslev 1989, Matsumoto et al . 1989, Briseno & Willershausen 1991, Gerosa et al . 1995, Beltes et al . 1995, Vajrabhaya et al . 1997, Osorio et al . 1998, Koulaouzidou et al . 1998, Geurtsen et al . 1998, Guigand et al . 1999, Cohen et al . 2000, Leonardo et al . 2000). Although test systems vary considerably in the way cytotoxicity is measured, most employ cells that are transformed or of tumour origin as the model for cell response. However, normal diploid cells differ from established or transformed cells in many ways such as: mitotic rate, density-dependent regulation of growth, mitochondrial function, and media selection (Holley 1975, Lechner & Kaighn 1979, Feigal et al . 1985). To date, there has been very little data on the cytotoxicity of various types of root canal sealer in different culture systems. The purposes of this investigation were to study the cytotoxic effects and the long-term biocompatibility of elutes of three types of root canal sealer (resin-based, zinc oxide–eugenol-based, and calcium hydroxide-based) on human primary periodontal ligament (PDL) cells and a permanent hamster cell line (V79 cells).

Materials and methods.

Sealers.
Six root canal sealers were evaluated: N2, Endomethansone, AH26, AHPlus, Canals and Sealapex. The materials tested were resin-based (Table 1), zinc oxide–eugenol-based- (Table 2), or calcium hydroxide-based (Table 3).

Sample fabrication.
The cements were mixed according to the manufacturers’ instructions. Triplicate sample disks of the root canal sealers were fabricated in sterile cylindrical glass moulds 10 mm in height and 3 mm in diameter. Excess flash was removed with a sterile scalpel. The specimens were placed in polyethylene vials directly after mixing.

Elute preparation.
Immediately after the initial setting period, each specimen was placed in 10 mL of fresh culture medium and then transferred into fresh media after 24 h, 48 h, 72 h and 7 days. After each elution period, the medium was removed and cytotoxicity determined after the cells and elutes were incubated for 24 h. Cells without addition of elutes acted as untreated control.

 Resin-based root canal sealers tested
Table 1. Resin-based root canal sealers tested.

Zinc oxide-eugenol-based root canal sealers tested
Table 2. Zinc oxide-eugenol-based root canal sealers tested.

Calcium hydroxide-based root canal sealers tested
Table 3. Calcium hydroxide-based root canal sealers tested.

Cell cultures.
The PDL cells were cultured from healthy premolars (Chang et al . 1999, Tai & Chang 2000). Explants were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% foetal calf serum and antibiotics (100 units mL 1 penicillin, 100 g mL 1 streptomycin, and 0.25 g mL 1 of fungizone). To avoid contamination from gingival tissue, the periodontal ligament was carefully removed from the middle third of the root with a scalpel. The fragments were grown in DMEM supplemented with 10% foetal calf serum and antibiotics. Cells from passages 3–8 were used in this study. A permanent cell line derived from Chinese hamster lung fibroblasts (V79) was cultivated in minimal essential medium, supplemented with 10% foetal calf serum and 1% penicillin, streptomycin, and neomycin. Subcultivation was performed on confluent cultures.

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. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) solution was prepared as 1 mg mL –1 in complete medium just before use. Cells were diluted in fresh complete medium and seeded in 96-well plates (V79: 1 10 4 cells well 1 , PDL: 2 10 4 cells well 1 ). After overnight attachment, cells were treated with various extracts of sealers (200 L well 1 ) for 20 h, then 50 L MTT dye was added to each well. Plates were incubated in a CO 2 incubator for 4 h. Optical density was determined by eluting the dye with dimethyl sulfoxide (Sigma, St. Louis, MO, USA), and the spectrophotometric absorbance was 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.