2.4. Cell Viability Evaluation of CNMs in OCs and OBs

OCs were prepared using the following procedure in all experiments: RAW264 cells were seeded first in a 96-well plate at a density of 3.1×10³ cells/cm² and cultured in αMEM supplemented with 10% FBS and penicillin-streptomycin-amphotericin B suspension. Twenty-four hours after incubation, a receptor activator of nuclear factor kappa-B ligand (RANKL; Oriental Yeast Co., Ltd., Tokyo, Japan) was added to the cultured medium at a concentration of 200 ng/mL and further incubated for 4 days.

OBs were seeded in a 96-well plate at a density of 3.1 × 10⁴ cells/cm². After 24 h, CNMs were added to the cultures and incubated for 24 or 48 h. The cell viability was analyzed using alamarBlue® reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Briefly, the alamarBlue® reagent was diluted in the medium (1:10) and added to the cultures. After 1 h of incubation, fluorescence intensity was measured using a PlateReader AF2200 (Eppendorf, Hamburg, Germany) with the excitation/emission wavelengths set at 535/590 nm. The group to which only the dispersant was added was designated as the control, and cell viability was calculated with the control set as 100%.