Mineralizing cells release a special class of extracellular vesicles known as matrix vesicles (MV), crucial for bone mineralization. Following their release, MV anchor to the extracellular matrix (ECM), where their highly specialized enzymatic machinery facilitates the formation of seed mineral within the MV’s lumen, subsequently releasing it onto the ECM. However, how MV propagate mineral onto the collagenous ECM remains unclear. In this study, we address these questions by exploring the “protein corona” paradigm whereby nanoparticles entering a biological milieu become cloaked by a corona of soluble proteins modifying their biological functions. We isolated native MV from the growth plates of chicken embryos. After removing the protein corona from the native MV using high ionic strength buffer, we obtained shaved MV. Reconstituted MVs were produced by incubating shaved MV with the removed protein corona constituents. Our results show that both the removal and reconstitution of protein corona significantly affect the biochemical and physicochemical properties of MV, resulting in 3 well-defined groups. Shaved MV exhibited an increase in tissue nonspecific alkaline phosphatase (TNAP) activity and a decrease in mineral deposition compared to native MV. Reconstituted MV partially recovered these functions, showing a reduction of TNAP activity and mineral deposition compared to native MV. Furthermore, changes in the protein corona affect the MV ability to anchor to the collagenous ECM, which is crucial for initiating the propagation of the mineral phase within this organic matrix. Proteomic analyses revealed changes in the protein profile of the MV resulting from the removal of the protein corona, indicating that shaved proteins were primarily related to external structural and ECM organization and catabolism. These findings underscore the role of the protein corona in modulating the mineralization capabilities of MV. Understanding these interactions could lead to new therapeutic strategies for enhancing bone repair and regeneration.

Pathological calcification of soft tissue, particularly in vascular structures, is a hallmark of several cardiovascular diseases and significantly contributes to vascular stiffening and dysfunction. Despite sharing similarities with physiological ossification, the mechanisms driving the transdifferentiation of mouse vascular smooth muscle cells (MOVAS) into osteochondroblast-like phenotypes remain poorly understood. This transdifferentiation plays a critical role in the initiation and progression of pathological calcification. In this study, we developed a bioinspired 3D scaffold, composed of type I collagen (Col) and κ-carrageenan (κ-Carr), designed to mimic key aspects of the vascular extracellular matrix (ECM). This novel scaffold provides a physiologically relevant platform to study soft tissue calcification under osteogenic conditions. We demonstrated that this 3D system supports MOVAS cell adhesion, spreading, and transdifferentiation into a mineralizing phenotype in a controlled manner, as evidenced by the overexpression of osteogenic markers (TNAP and RUNX2), increased alkaline phosphatase activity, and controlled calcium phosphate deposition. Spectroscopic and thermogravimetric analyses revealed the formation of carbonated apatite minerals and a calcium-deficient apatite structure, indicative of controlled mineral deposition within the organic matrix. The incorporation of κ-carrageenan enhanced the calcification process, underscoring the importance of biochemical cues in directing the MOVAS phenotype changes. This scaffold system effectively replicates the spatial organization and physicochemical cues of the vascular ECM, providing a unique and innovative model to study pathological calcification processes. Moreover, this approach holds significant potential for developing regenerative biomaterials and therapeutic strategies aimed at preventing vascular calcification, opening new avenues for clinical applications and therapeutic interventions in cardiovascular disease.