Hepatocyte growth factor (hgf)

Human squamous cell carcinoma cell line FaDu (referred to as FaDu; ATCC®HTB-43^TM, USA) was cultured in RPMI1640 medium supplemented with 10% low endotoxin fetal bovine serum FBS and 1% Penicillin/Streptomycin (GIBCO). Human Gingival Fibroblasts (referred to as hGF; 300703, CLS Cell Lines Service, Germany) were cultured in DMEM:Ham’s F12 medium (GIBCO), supplemented with 10% FBS (GIBCO) and 1% Penicillin/Streptomycin (GIBCO). Both cell types were held under standard conditions, i.e., at $37{}^{\circ }$ C and a humidified atmosphere containing 5% CO ${}_2$ . In all experiments, asynchronously and exponentially growing cells were used.

Human gingival fibroblasts (HGF, CLS Cell Lines Service GmbH, Eppelheim, Germany) were seeded into 96-well tissue, culture-treated plates at a density of 0.5 × 10⁵ cells/mL and allowed to adhere overnight in complete cell culture medium: MEM α medium with 10% fetal bovine serum (FBS, both from Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland). Extracts were done for every investigated sample in complete cell culture medium at 10 mg/mL, for 24 h, at 37 °C. The CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS, Promega, Madison, WI, USA) was used to assess the biocompatibility of samples’ extracts, according to the manufacturer instructions and ISO 10993-5:2009(E). Cells were incubated for 24 h with fresh, complete medium (Control) or different concentrations of samples’ extracts (2.5 mg/mL, 5 mg/mL, 7.5 mg/mL, and 10 mg/mL). After incubation with MTS reagent (3 h), absorbance readings were done at 490 nm on a FLUOstar^® Omega microplate reader (BMG LABTECH, Ortenberg, Germany). Experiments were done in triplicate, and treated cell viability was expressed as a percentage of the Control cells’ viability (means ± standard deviation). Data were statistically analyzed by the independent two-tailed (Student’s) t-test, considering p < 0.05 to be statistically significant.

Normal human gingival fibroblasts (HGF) (CLS Cell Lines Service, Eppelheim, Germany) were maintained in DMEM supplemented with 10% FBS. Cells were cultured in flasks at 37 °C in a humidified incubator with 5% CO₂ supplementation and the medium was changed every 2–3 days. The cells were used for experiments or subcultured once they reached 70–80% confluence.