Cytotoxicity Analysis of Apatite Samples

For cytotoxic analysis, the powder of the apatite samples was formed into round pellets (12 mm diameter and 1 mm thickness) using unixial pressing (130 mg/pellet) and the pellets were sterilized using gamma irradiation (BBF GmbH, Kernen-Rommelshausen, Germany). For the apatite pellets, 160 μL of an isotonic NaCl solution (0.9% NaCl) were added for 30 min. After that, the system was incubated for 24 h at 37 °C and 5% CO₂ with 500 μL cell culture media (DMEM, 4.5 g/L D-glucose, + L-glutamine, Gibco by Life Technologies). This was supplemented with 2 mM glutamine (Stock 200 mM), 100 U/mL penicillin + 100 μg/mL streptomycin (Stock: 10,000 U/mL Pen, 10,000 μg/mL Strep), 1% insulin, transferrin and selenium (Stock: 100x ITS-G), and 10% fetal calf serum. All the supplements were also provided by Gibco by Life Technologies.