IntroductionExtracellular vesicles (EV) have long been recognized as an important means of cell to cell communication, but current metrics to delineate various subpopulations of EV are limited. Recently, a distinctive subpopulation of EV embedded within the extracellular matrix of soft tissues, termed matrix-bound nanovesicles (MBV), has been described. Although the lipid membrane composition and intravesicular cargo of MBV clearly differ from liquid phase EV (i.e. exosomes), a more comprehensive characterization of the physical and biologic properties of MBV vs. exosomes and those of a separate subpopulation of EV, specifically bone matrix vesicles, would contribute to our understanding of the biogenesis and physiologic role of these three EV subpopulations. ResultsThe physical characteristics, protein and miRNA cargo profiling, vesicle membrane lipidomics, and immunomodulatory activity were used to compare skeletal muscle-derived MBV, liquid phase plasma exosomes, and mineralization-competent matrix vesicles of provisional bone matrix. We show that despite similar physical characteristics, these three preparations of EV represent distinct entities with different biologic activity. ConclusionThese results inform metrics for the categorization of EV and provide tools for the isolation of EV for potential diagnostic and therapeutic applications.