Osteoblast differentiation and mineralization assays

hFOB1.19 cells were plated in 12-well plates (2 × 10⁵ cells/well) and 2 h later treated with EVs from the indicated groups (100 µg). Culture media were replaced every 3 days and EVs were replenished with every media change. Osteoblast differentiation at the indicated times was assessed by measuring the levels of ALP activity; RUNX2, COL1, and OPN expression by Western blot (see list of antibodies in Supplementary Table ⁴); and mineralization by alizarin red staining as explained below.

ALP activity in cell culture supernatants was determined using an Alkaline Phosphatase Diethanolamine Activity Kit (Sigma). The supernatants of cultured cells (100 µL) were incubated with 400 µL of reaction buffer (1.0-M diethanolamine and 0.5-mM MgCl₂, pH 9.8) in the presence of 50 µL of 0.67 M p-nitrophenyl phosphate (ALP substrate) for 30 min at 37 °C. The reaction was stopped with 50 µL of 1-N NaOH, and the hydrolyzed substrate detected by absorbance at 405 nm using a plate reader (Spectra max 340PC, Molecular Devices), with a SoftMax Pro 6.2 software. ALP activity was calculated in units. One unit was defined as the amount of ALP that hydrolyzes 1 µmol of substrate per min at 37 °C. The activity of ALP found in the culture media was corrected by the amount of cellular protein in each well. Lysates were obtained by solubilization of cells in lysis buffer (150-mM NaCl, 10-mM Tris-HCl (pH 8.0), 1-mM EDTA, 1-mM Na₃VO₄, 0.5-mM PMSF, 5-mg/mL aprotinin, 5-mg/mL leupeptin, complete protease inhibitor cocktail (Roche) and 1% NP-40) for 15 min and protein content was determined with a Bradford Assay (Bio-Rad).

Maturation of hFOB1.19 into bone-producing osteoblasts was confirmed by alizarin red staining. At the indicated times, culture media were removed, and cells were fixed in 4% paraformaldehyde for 20 min, washed two times with PBS, and then stained with 1% alizarin red solution (Sigma-Aldrich) for 30 min. After washing five times with PBS to eliminate excess dye in the wells, 500 µl of PBS was added and cells examined for the presence of calcium deposits. The red dye staining the calcium deposits was then extracted by shaking the plates for 60 min at room temperature in PBS. The supernatants were then transferred to clean dishes and the absorbance of the extracted dye was measured at 562 nm using a plate reader (Spectra max 340PC).