Isolation of Extracellular Vesicles from hBMSCs

Isolation of EVs from naïve hBMSCs CM and osteogenic CM was performed by ultracentrifugation as described previously [24 (link),26 (link),27 (link)]. In brief, the CM was centrifuged at 300× g (Sigma, Osterode a. Harz, Germany) and 4 °C for 10 min to remove cells, and subjected to centrifugation again at 2000× g to remove dead cells. Afterwards, centrifugation for 30 min at 10,000× g was applied to remove cell debris, then the supernatant was passed through 0.22 μm filters (Sarstedt, Nümbrecht, Germany), and was subjected to ultracentrifugation once at 120,000× g for 70 min at 4 °C. After careful removal of the supernatant, the pellet was resuspended with PBS and centrifuged again at 120,000× g for 70 min at 4 °C. The hereof resulting EV pellets were resuspended in the presence of 25 mM Trehalose (Carl Roth, Karlsruhe, Germany) in PBS. The protein concentration of EVs was measured using BCA (bicinchoninic acid) Protein Assay Kit Pierce (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s instructions.