PROTEOMIC SHIFTS IN MATRIX VESICLES ACROSS OSTEOBLAST DIFFERENTIATION

Matrix vesicles (MV) are a group of extracellular vesicles released by mineralizing cells. Biomineralization is a complex, multi-step regulated process whose dynamics remain partially understood. Although previous studies have suggested a role for MV in the early stages of mineral nucleation and propagation into the extracellular matrix, it is unclear whether the properties of MV change throughout the mineralization process. To address this, we conducted proteomic and physicochemical characterizations of MV isolated at distinct time points during osteogenic differentiation. MV were obtained from the MC3T3-E1 osteoblast cell line at days 7 (MV-7), 14 (MV-14), and 21 (MV-21) using differential centrifugation. Their morphology, size, particle concentration, zeta potential, and tissue-nonspecific alkaline phosphatase (TNAP) activity were assessed. Proteomic profiling was performed using TMT10plex and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We observed significant temporal changes in MV features. MV-14 were smaller (163 nm) compared to MV-7 (178 nm) and MV-21 (188 nm), and exhibited a higher zeta potential. TNAP activity progressively increased over time, with the highest levels detected on day 21. Proteomic analysis identified 1,605 gene products commonly present in all samples. Distinct protein profiles were observed at each time point, although a core set of proteins—including key mineralization mediators such as TNAP—remained consistently present. Differential protein composition was also evident among groups: MV-7 and MV-14 shared 31 proteins, MV-7 and MV-21 shared 25, and MV-14 and MV-21 shared 27. Time-resolved clustering analysis (Mfuzz) identified five dynamic protein clusters, indicating coordinated shifts in MV protein content over the 21-day period. Our findings demonstrate that MVs undergo dynamic remodeling during osteoblast differentiation, resulting in MV with distinct characteristics and protein contents as the mineralization process progresses. These insights contribute to a deeper understanding of MV biology in bone formation and may support the development of novel regenerative medicine strategies.

This work was supported by FAPESP grants 19/08568-2, 22/04885-6, 24/12254-1, 24/17743-0, 24/04140-6, 21/13140-1, 25/06947-7; and CNPq 305426/2021-4.