Osteocyte transition induced by quiescent vascular smooth muscle cells through paracrine signaling is independent of shear stress

We investigated how vascular smooth muscle cells (VSMCs) shape osteolineage fate under mechanosignaling, with emphasis on validating effects in mesenchymal stromal cells (MSCs), a more primitive stage than differentiated osteoblasts. Conditioned media from shear-stressed and non-stressed VSMCs challenged primary human osteoblasts and human MSCs for up to 28 days. Across both cell types, VSMCs robustly promoted osteoblast-to-osteocyte plasticity, evidenced by morphological remodeling and increased expression of osteocyte markers (GP38, SOST, DMP1, miR-23a, FGF23). Notably, non-stressed VSMC–conditioned medium elicited stronger osteocyte-function signatures, including a > 10-fold rise in RANKL transcripts, and these responses were recapitulated in MSCs, demonstrating that VSMC cues instruct osteogenic commitment at an earlier lineage stage and converge on an osteocyte-like phenotype. Mechanistically, small extracellular vesicles (sEVs) emerged as key mediators of VSMC–bone crosstalk: sEV cargo from non-stressed VSMCs displayed higher SOST and DMP1 transcripts, along with phosphor - β-catenin, phospho-connexin-43, and ATP, suggesting pathways that support osteocyte survival and function. Collectively, our data position VSMCs as pivotal instructors of osteolineage progression - from MSC commitment to osteocyte specification - via sEV-dependent communication, with non-stressed VSMCs exerting the strongest effect, particularly on functional readouts such as RANKL. Further, these findings support VSMC-sEV–inspired, cell-free approaches to modulate osteocytogenesis and regulate bone remodeling, while proposing SOST/DMP1/miR-23a as candidate circulating markers of osteocyte function and treatment response.