Mechanical Cues and Lineage Commitment Govern the Angiogenic Potential of Mesenchymal Cell‐Derived Extracellular Vesicles

Bone regeneration requires a finely tuned interplay between osteogenesis and angiogenesis. While current treatments, such as auto/allografts, provide support, they often fail to promote adequate vascularization necessary for complete repair. Extracellular vesicles (EVs), as mediators of intercellular communication, have emerged as promising acellular nanotechnologies for tissue regeneration due to their bioactive cargo and low immunogenicity. Mechanical stimulation, a known enhancer of bone cell function, can modulate EV cargo and potentially improve regenerative efficacy. In this study, we investigated how mechanical stimulation and the stage of mesenchymal lineage commitment influence the angiogenic potential of secretomes and EVs derived from mesenchymal stromal/stem cells, osteoblasts, and osteocytes. Our findings reveal that both cell mechanical stimulation and their differentiation stage significantly modulate the angiogenic properties of the resulting EVs. Among the tested conditions, mechanically-stimulated osteocyte-derived EVs demonstrate superior angiogenesis, promoting endothelial cell migration, tube formation, and CD31 expression. These effects were further validated in a pre-clinical ex ovo chick chorioallantoic membrane assay, where robust neovascularization was observed. This work highlights the critical role of both mechanical cues and cell differentiation stage in regulating the angiogenic capacity of EVs and proposes mechanically activated osteocyte-derived EVs as a novel pro-angiogenic nanotherapeutic for bone repair.