Matrix Vesicle Nanostructures Guide Spatially Organized Bone-Like Mineralization in Biomimetic Collagen Scaffolds

Bone formation is tightly regulated by osteoblasts, which secrete extracellular matrix (ECM) components and heterogeneous populations of extracellular vesicles (EVs). Among EVs, matrix vesicles (MVs) are uniquely associated with mineralization and act as bioactive extracellular nanostructures that initiate and spatially direct mineral growth within the ECM. However, how MVs interact with the ECM and how their functions differ from the functions of medium-derived EVs (mEVs) remain unclear. To address this question, we developed a bioinspired collagen-based scaffold designed to mimic the bone ECM organic phase and to recreate MV-mediated mineralization in vitro. Type I collagen scaffolds were slowly concentrated and exposed to NH3(g) to induce fibrillogenesis and stabilize supramolecular organization, while 5 wt % κ-carrageenan, a sulfated polysaccharide that functionally emulates glycosaminoglycans, was incorporated to support mineral nucleation. MC3T3-E1 pre-osteoblasts were cultured under osteogenic conditions to induce differentiation and vesicle secretion. Biochemical, microscopic, and spectroscopic analyses revealed that MVs and mEVs exhibit distinct nano-biointerface behaviors, particularly regarding ECM anchoring and the ability to initiate mineral deposition. Furthermore, Raman chemical imaging and X-ray nanotomography demonstrated that MVs not only trigger mineral formation but also direct the spatial organization of phosphate deposition within the ECM at the micro/nanoscale. By distinguishing MV- from mEV-mediated mineralization in a biomimetic ECM system, this study provides new insights into vesicle-guided biomineralization and spatial matrix organization, with implications for skeletal development, pathological calcification, and vesicle-based regenerative strategies.