Hgf 1

Human gingival fibroblasts HGF-1 (CRL-2014, ATCC, Manassas, VA, USA) are normal adherent cells and were isolated from the gingiva of a 28-year-old male. Cells were cultured in a humidified incubator at 37 °C, 95% air, and 5% CO₂, using Dulbecco’s Modified Eagle’s Medium (DMEM, 31600-083) supplemented with 1.5 g/L NaHCO₃, 3.5 g/L glucose, 1% antibiotic-antimycotic mix (10,000 units penicillin, 10 mg streptomycin, and 25 μg amphotericin B per mL; A5955, Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum (10270-106, origin of South America, Gibco, Life Technologies, Carlsbad, CA, USA). Passage cell culture was performed using a 0.25% trypsin–0.53 mM EDTA solution and the culture medium was completely replaced every two days. For the determination of the total cell number from cultures, a hemocytometer was used.

Commercially available HGFs (HGF-1, CRL-2014; ATCC, Manassas, VA, USA) were first cultured in 10 ml Minimum Essential Medium α (α-MEM; Thermo Fisher Scientific, USA) containing 10 vol % foetal bovine serum (FBS; Thermo Fisher Scientific, USA) and 1 vol % antibiotics (penicillin/streptomycin, Thermo Fisher Scientific, USA) in the cell culture dish with a diameter of 10 cm at 37 °C under a 5% CO₂ atmosphere in the CO₂ incubator (BB150, Thermo Fisher Scientific, USA). We changed the medium every 3 days and passaged the cells by trypsin digestion when they grew 80% of the bottom of the dish. HGFs at passages 3–5 were used for the experiments.

MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)-assay was performed to evaluate the cytotoxicity of the orthodontic bonding agents containing fGtBAG. The orthodontic brace was placed on the tooth surface excessive bonding agent often flows on the gingiva. So human gingival fibroblasts-1 (HGF-1; ATCC, Rockville, MD, USA), the most abundant cell in periodontal connective tissue, used for cell viability test. The resin sample disks were inserted into 96 well plates and sterilized with low-temperature plasma (LOWTEM Crystal 50, Gunpo-si, Korea). HGF-1were cultured in Dulbecco’s modified Eagle’s medium (Hylone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 100 IU/mL penicillin/streptomycin (Hyclone, Logan, UT, USA). The HGF-1 cells were injected into 96 well plates containing the samples and cultured in a 5% CO₂ incubator at 37 °C for 24 h. Then, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) was added at a concentration of 5 mg/mL and reacted for 4 h in a dark room. The supernatant was removed and the samples were melted with MTT crystal dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA, 150 µl/well) formed in the cells. The absorbance was measured at 620 nm wavelength (Sunrise^TM, TECAN, Männedorf, Switzerland).

A primary normal human gingival fibroblast-1 (HGF-1) cells (CRL-2014, ATCC, VA, USA) was used as a normal oral cell line at 5–10 passages [42 (link)]. HSC-2 (JCRB0622, JCRB Cell Bank, Japan) and SCC-15 (CRL-1623, American type culture collection) were chosen as oral squamous cell carcinoma cell lines. The cells (1 × 10⁴ cells/100 μl) were prepared from 90% confluent cultures biological experiments and seeded in each well of 96 wells (SPL, Pocheon, Gyeonggi-do, Korea). All biological investigations used 96 wells and employed the appropriate cell culture media (DMEM for HGF-1, DMEM/F-12(3:1) for HSC-2 and DMEM/F-12(1:1) for SCC-15) supplemented with 10% fetal bovine serum (Gibco, NY, USA) and 1% antibiotics (penicillin/streptomycin, Gibco). The cells were grown in an incubator at 37°C in a humidified atmosphere containing 5% CO₂.

HGF-1 (ATCC CRL-2014, Virginia, USA) in the third to sixth passage were cultured in completed Dulbecco’s modified Eagle medium (DMEM) (Sigma Chemical Co., St. Louis, Missouri, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, California, USA) and 100 UT/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine (Gibco, Grand Island, New York, USA). The cell lines were maintained under the condition of a humidified atmosphere (5%) of CO₂ at 37 °C until the cells achieve a confluence of 90%. 10,000 cells were cultured on each material placed in 24-well plate and incubated for 24 h. On the day of imaging, 2 µM Calcein-AM (Molecular Probes, Eugene, OR, USA) was added to the cells and incubated for 60 min. Then, the dye was removed and washed the stained HGF cells by incubation with pH 7.2 phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific, USA) for 15 min to allow for AM ester removal. Live cells growing on the materials was analyzed by confocal microscopy (Stellaris 5, Leica Microsystems, Wetzlar, Germany) to determined mean intensity of the green fluorescent.