4.2.1. MG-63 Cell Monocultures and Exposure to Mg(OH)2 NPs

MG-63 (ATCC^®CRL-1427™) cells were cultured in 96-well plates (2 × 10⁴ cells/cm²) in basal medium containing RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL amphotericin B (basal medium; all reagents from Gibco) at 37 °C, 95% humidity and 5% CO₂ atmosphere. After a 24 h incubation period for adherence, the medium was substituted for fresh basal medium or basal medium supplemented with 50 µg/mL ascorbic acid and 10 nM dexamethasone (osteogenic medium; all reagents from Sigma-Aldrich), used as controls, or exposed to Mg(OH)₂ NPs (1, 10 and 100 µg/mL). This concentration range was selected based on a previous work performed with similar NPs, showing its cytocompatibility in these levels [19]. Cultures were grown until day 6 and characterized for metabolic activity (MTT assay), alkaline phosphatase (ALP) activity and histochemical staining, and immunostaining of nucleus and osteopontin (SPP1).