Effects of TiO₂ Nano Glass Ionomer Cements Against Normal and Cancer Oral Cells

Materials and Methods

Materials. Three commercially available glass ionomer cements (GICs) were used: Base cement, Core shade cement and FX-II (Shofu Dental Corp. Kyoto, Japan). Chemical compositions of the cements are listed in Table I. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM): GIBCO BRL, Grand Island, NY, USA; fetal bovine serum (FBS): JRH Bioscience, Lenexa, KS, USA; Carisolv gel, single mix: MediTeam, Göteborg, Sweden; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): Aldrich, St. Louis, MO, USA; PGE₂ assay kit: Cayman Chemical Co., Ann Arbor, MI, USA); IL-1β: R&D Systemes Mineeapolis, MN, USA; culture plastic dishes: 6-well and 96-microwell plates: Becton Dickinson, Franklin Lakes, NJ, USA; dimethyl sulfoxide (DMSO): Wako Pure Chem Ind., Osaka, Japan.

Preparation of fine powder of cements with TiO₂ nanoparticles. Each GIC was blended with 3% or 5% (w/w) TiO₂ nanoparticles, anatase phase, particle size <25 nm (Sigma-Aldrich, St. Louis, MO, USA) (Table I). The cement powder was mixed with the cement liquid under the manufacturer's recommended conditions. The cement mixture was placed into the metallic mold, and then standardized GIC blocks (4×4×1 mm) of cements were prepared in a metal mold. The GICs/TiO₂ nanoparticle samples were ground up with a mortar and pestle to yield a fine powder of cements, and then subjected to the sterilization by autoclaving (20 min, 120°C, 2 atm).

Cell culture. Human OSCC (HSC-2, HSC-3, HSC-4) and gingival carcinoma cell lines (Ca9-22) were obtained from (Riken Cell Bank, Tsukuba, Japan). Normal human oral cells (HGF, HPC, HPLF) were prepared from periodontal tissues, as previously reported (17). Cells were cultured at 37°C with in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified atmosphere with 5% CO₂. Cells were then harvested by treatment with 0.25% trypsin-0.025% EDTA-2Na in PBS(−) and either subcultured or used for experiments.

Assay for cytotoxic activity. Cells (3×10⁴ cells/0.1 ml) were inoculated into each well of 96-microwell plates and incubated for 48 h to achieve the complete cell adherence. The cements were suspended in DMEM culture medium and were added at 0, 0.62, 1.25, 2.5, 5, 10, 20 and 40 mg/ml. The cements were incubated for further 48 h. The relative viable cell number was then determined by the MTT method. In brief, the culture medium was replaced with MTT (0.2 mg/ml) dissolved in DMEM, and cells were incubated for 4 h at 37°C. After replacing the medium, the formazan product was dissolved with DMSO, and the absorbance at 540 nm of the lysate was determined by using a microplate reader (Multiskan; Biochromatic Labsystem, Osaka, Japan). The 50% cytotoxic concentration (CC₅₀) was determined from the dose–response curve and the mean CC₅₀ (±S.D.) value of each cement was calculated in triplicate of three independent experiments.

PGE₂ production. Near-confluent HPC and HGF cells were treated with different concentrations (0, 1.25, 2.5 and 5 mg/ml) of FX-II:TiO₂ nanoparticles (100:0) and FX-II:TiO₂ nanoparticles (97:3) for 30 min in the fresh culture medium. Cells were induced to produce PGE₂ with IL1β (3 ng/ml) for a further 24 h, respectively. The concentration of PGE₂ released into the culture supernatant was then determined by ELISA with PGE₂ assay kit, according to manufacturer's recommended procedures (16).

Fine cell structure. HPC, HPLF and HGF cells were treated with 0, 2.5, and 5 mg/ml of FX-II:TiO₂ nanoparticles (100:0) and FX-II:TiO₂ nanoparticles (97:3) for 3 h. After washing three times with 5 ml of cold PBS(−), the cells were fixed for 1 h with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C. The cells were scraped with a rubber policemen, post-fixed for 90 min with 1% osmium tetraoxide-0.1 M cacodylate buffer (pH 7.4), dehydrated and embedded in Araldite M (CIBA-GEIGY Swiss; NISSHIN EN Co., Ltd., Tokyo, Japan). Thin sections were stained with uranyl acetate and lead citrate, and were then observed under a JEM-1210 transmission electron microscope, Japan Electron Optics Laboratory (JEOL, Co., Ltd Akishima, Tokyo, Japan) (magnification: ×5,000) at an accelerating voltage of 100 kV (18).

Statistical analysis. Data are expressed as the mean±standard deviation (SD). Statistical analysis was performed by paired t-test and ANOVA post-hoc Tukey test using SPSS (Statistical Package for the Social Sciences, Chicago, IL, USA). Differences were considered significant at p<0.05.