Human bone marrow stem/stromal cell osteogenesis is regulated via mechanically activated osteocyte- 1 derived extracellular vesicles

39 Bone formation or regeneration requires the recruitment, proliferation and osteogenic differentiation of 40 stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are 41 mechanosensitive cells which play fundamental roles in coordinating loading-induced bone formation via 42 the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical 43 signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the 44 potency of the mechanically stimulated osteocyte secretome in driving human MSC recruitment and 45 differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and 46 bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection 47 of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of 48 extracellular vesicles (EVs) in driving these effects. This demonstrates the pro-osteogenic potential of 49 osteocyte-derived mechanically activated extracellular vesicles (MA-EVs), which have great potential as a 50 cell-free therapy to enhance bone regeneration and repair in diseases such as osteoporosis. 51