3.1. Characterization of stem cell-derived EVs
EVs were isolated from both BMSCs and PSCs cultured in growth media (D0) as well as osteogenic media at 7 days (D7) and 21 days (D21) of culture. The yield of the BMSC-derived EVs was 71.0±38.3, 149.3±22.9, and 123.6±16.9 μg/mL for D0, D7, and D21, respectively. BMSCs exhibited similar yields to the values reported in the literature [44], but PSCs consistently yielded higher EV protein quantities in comparison to BMSCs. The PSC-derived EVs had yields of 394.6±38.2, 959.3±59.7, and 695.7±90.8 μg/mL at D0, D7, and D21, respectively (Figure 2A ). The lipid concentration of the EVs was also quantified so that a comparison could be made between the protein to lipid ratios of each EV group as is standard according to ISEV to ensure EVs are pure and the isolation method does not co-isolate unbound proteins. The BMSC-derived EVs had lipid concentrations of 185.2±17.2, 495.2±32.9, and 1339.37±74.2 μg/mL at D0, D7, and D21 respectively. The PSC-derived EVs had lipid concentrations of 3486.5±227.1, 818.1±85.8, and 247.7±8.2 μg/mL at D0, D7, and D21 respectively (Figure 2B ). This means that for the protein to lipid ratios, BMSC-derived EVs had a ratio of 0.383, 0.302, and 0.092 at D0, D7, and D21 respectively, while the PSC-derived EVs were 0.113, 1.172, and 2.808 at D0, D7, and D21, respectively.
EVs were characterized by TEM and DLS to confirm that the size and morphology were consistent with the values and shape reported by other groups isolating EVs. The TEM images showed that all six groups of examined EVs had a cup-shaped morphology and an average hydrodynamic diameter between 120-150 nm (Figure 2C) . The TEM images revealed that the different groups of isolated EVs featured similar shape and size ranges, confirming that the techniques for isolation yielded consistent batches of EVs.
DLS was also performed to quantitatively confirm the EV size(Figure 2D) . DLS is able to calculate the hydrodynamic diameter of EVs from the Stokes-Einstein Equation, which is used to characterize how large particles move slower due to Brownian motion. From this experiment, we found that the BMSC-derived EVs had diameters of 137.4±11.97, 158.7±29.41, and 218.3±20.3 nm on D0, D7, and D21, respectively. The PSC-derived EVs had diameters of 135.5±1.17, 129.3±25.22, and 197.5±34.85 nm on D0, D7, and D21, respectively.