2.11. Resorption assay using calcium phosphate‐coated plates

Resorption assays were performed as previously described (Song et al., 2015). To obtain COCs, BMMs (2 × 10⁴/well) were plated on calcium phosphate‐coated 48‐well plates and cultured with M‐CSF (30 ng/ml) and RANKL (60 ng/ml) for 3 days. Then, the COCs were stimulated with F. alocis EVs (1, 3, or 10 μg/ml of protein; 0.6 ×, 1.8 ×, or 6 × 10⁶ of EV particles/cell) in the presence of M‐CSF (30 ng/ml) for 10 days. The plates were washed with distilled water to remove cells and subjected to von Kossa staining. For the staining, the plates were incubated with 5% silver nitrate solution for 30–60 min in the dark. The plates were then washed with distilled water and incubated in carbonate‐formaldehyde solution (5% Na₂CO₃, 9.25% formaldehyde) for 2 min before washing and drying. Representative images of the resorption area from triplicate samples were obtained by digital inverted microscopy (DS‐Ri2). The resorption area was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA) from representative images of triplicate samples.