Fibroblast basal medium

The cell lines used in the present study were: HGF—human primary gingival fibroblasts (ATCC^® PCS-201-018™), SCC-4—human squamous cell carcinoma cell line (ATCC^® CRL-1624™), HaCaT—immortalized human keratinocytes (CLS Cell Lines Service GmbH), and A375—human melanoma cells (ATCC^® CRL-1619™).
For cell culture and cell viability assay, the following reagents were needed: specific culture medium—Fibroblast Basal Medium (ATCC PCS-201-030) and Fibroblast growth kit—low serum (ATCC PCS-201-041) for HGF cells, DMEM:F12 Medium (ATCC^® 30-2006™)—for SCC-4 cells were acquired from ATCC, and Dulbecco’s Modified Eagle Medium (DMEM) high glucose −4.5 g/L for HaCaT and A375 cells, together with the other reagents used, as: trypsin—EDTA solution, PBS (phosphate saline buffer), fetal bovine serum (FCS), Trypan blue, Alamar blue (resazurin sodium salt) and hydrocortisone were purchased from Sigma Aldrich (Darmstadt, Germany) and Thermo Fisher Scientific (Waltham, MA, USA).

hGFs (PCS-201-018 ATCC, Manassas, VT, USA) were cultured in Fibroblast Basal Medium (ATCC PCS-201-030) in addition to Fibroblast Growth Kit-Low Serum (ATCC PCS-201-041), containing 5 ng/mL of rh FGF b, 7.5 mM of L-glutamine, 50 μg/mL Ascorbic acid, 1 μg/mL of Hydrocortisone Hemisuccinate, 5 μg/mL of rh Insulin and 2% Fetal Bovine Serum. The culture was maintained in an incubator at 37 °C in a humidified atmosphere with 5% CO₂ and 95% air, and when the cells reached 75–80% confluence, subcultures were produced.

The cell line used in the current study consisted of a primary human gingival fibroblast (HGF) monolayer. The cells were supplied by American Type Culture Collection (ATCC^® PCS-201-018™, Manassas, VA, USA), together with the culture medium—Fibroblast Basal Medium (ATCC^® PCS-201-030™)—and the required supplements—Fibroblast Growth Kit-Low Serum (ATCC^® PCS-201-041™) and 0.1% Penicillin-Streptomycin-Amphotericin B Solution (ATCC^® PCS-999-002™). The cells were cultured under sterile conditions and humidified atmosphere, enriched with 5% CO₂ (Steri-Cycle i160 incubator; Thermo Fisher Scientific, Waltham, MA, USA).
The method employed in the present study to assess the in vitro cytocompatibility profile of the Ti-6Al-V metal alloy used for the development of the custom-made orthodontic mini-implant was based on the extraction means, a technique suggested by ISO standard 10993-5:2009 [41 ] for medical devices. Thus, test mini-implant alloy was submerged into the culture medium and exposed to intermittent shaking for 24 h. Afterwards, the sample was removed and the resulting extraction medium was used to stimulate the HGF cells for different time intervals (24, 48, 72 h).