Saos 2 cells

For cell culture experiments, samples were sterilized under UV light, inserted into 12-well polystyrene cell culture plates (TPP, Switzerland; internal well diameter 21.4 mm) and seeded with: i) human osteoblast-like MG-63 cells (European Collection of Cell Cultures, Salisbury, UK; Cat. No. 86051601) suspended in Dulbecco's modified Eagle's Minimum Essential Medium (DMEM; Sigma, USA, Cat. No. D5648), ii) Saos-2 cells (European Collection of Cell Cultures, Salisbury, UK, Cat. No. 89050205) suspended in McCoy medium (Sigma, Cat. No. M4892), or iii) murine macrophage-like RAW 264.7 cells (ATCC, TIB-71, USA) suspended in RPMI-1640 medium (Sigma, Cat. No. R8758). All culture media were supplemented with 10% fetal bovine serum (Sebak GmbH, Aidenbach), and gentamicin (40 µg/mL, LEK). Each well contained i) 36,000 MG-63 cells (i.e., approximately 10,000 cells/cm²), ii) 20,000 Saos-2 cells (5,600 cells/cm²) or iii) 50,000 RAW 264.7 cells (14,000 cells/cm²), and 2 mL of the medium. The cells were cultured for 1 and 3 days (to evaluate cell numbers) or for 7 days (for PCR detection of markers of osteoblast differentiation, and to measure the potential immune activation of RAW cells) at 37°C in a humidified air atmosphere containing 5% CO₂. Two samples were used for each experimental group and time interval.