2.3. EV characterization
The EV morphology was observed by transmission electron microscopy (TEM). EVs were fixed with 2% paraformaldehyde for 15 min; then, the mixture (about 8 μL) was dropped onto carbon-coated copper grids and air-dried for 10 min before staining with 1% uranyl acetate two times (6 min each). Images were obtained using an FEI Tecnai BioTwin TEM (Thermo Fisher Scientific). To determine the size distribution, dynamic light scattering (DLS), (Brookhaven Instruments Corporation; Holtsville, NY) was utilized for early (0 day; D0), middle (7 days; D7) and late (21-days; D21) time points of osteogenic differentiation.