Stage-Specific Proteomic Signatures of Matrix Vesicles Support Functional Transitions during Biomineralization

Matrix vesicles (MVs) are extracellular vesicles released by mineralizing-competent cells, including chondrocytes and osteoblasts. Although MVs are classically associated with biomineralization, whether distinct MV populations are produced throughout osteoblast differentiation remains poorly understood. Here, we investigated the biophysical, biochemical, and proteomic profiles of MVs isolated throughout osteogenic differentiation to evaluate the hypothesis of functional transitions during biomineralization progression. MVs were isolated from osteoblast cultures at three time points, representing the onset, establishment, and propagation stages of biomineralization. Biophysical and biochemical characterization revealed temporal differences in MV properties. Stage-specific proteomic signatures were identified among MV groups. Early stage MVs (7-14 days) exhibited enrichment of proteins associated with the extracellular matrix (ECM) organization, intracellular trafficking, cytoskeletal dynamics, and adhesion, suggesting a role in preparing the mineralizing microenvironment. In contrast, later-stage MVs (14-21 days) showed enrichment of proteins related to ECM interactions, calcium response, proteoglycan binding, metabolism, and regulatory signaling pathways, consistent with enhanced mineral propagation. Collectively, our findings demonstrate that distinct MVs are produced throughout osteoblast differentiation and support a functional transition model during biomineralization, in which MV cargo and properties are dynamically coordinated with the temporal demands of ECM mineralization.