In vitro interaction of MC3T3-derived extracellular vesicles with collagen-based scaffolds

Mineralization is a complex, dynamic process crucial for bone formation, studied extensively from structural, chemical, and biological perspectives. Governed by mineralization-competent cells within an extracellular matrix (ECM) primarily composed of collagen and sulfated glycosaminoglycans (GAGs), this process is essential. Osteoblasts, key players in bone formation, drive mineralization by secreting ECM components and releasing heterogeneous extracellular vesicles (EVs). Among EVs, matrix vesicles (MVs) are unique membrane-bound vesicles involved in mineralization, displaying distinct ECM interactions, unlike medium extracellular vesicles (mEVs). However, the mechanisms remain unclear. This study investigated the interaction between MVs and mEVs with organic matrix components, using an ECM bioinspired by bone tissue to replicate MV-mediated mineralization in vitro. Collagen-based scaffolds [S(Col)] mimicking the native tissue microenvironment were prepared through slow evaporation of concentrated type I collagen solutions followed by NH_3(g) exposure to induce fibrillogenesis and stabilize supramolecular order. To mimic the role of GAGs, 5 wt% κ-Carrageenan (κ-Carr) was incorporated into the scaffolds. MC3T3-E1 cell maturation into a mineralizing phenotype was promoted through 14 days of osteogenic supplementation, followed by isolation of MVs and mEVs secreted by mature osteoblasts. The mineralizing capabilities and roles of MVs and mEVs in ECM mineralization in vitro were assessed. Findings revealed a distinct biochemical profile, highlighting MVs' specialized function in vesicle anchoring and ECM mineralization induction. Microscopic and spectroscopic analyses demonstrated that incorporating κ-Carr into the scaffolds enhanced MVs anchoring, promoting biomimetic ECM mineralization. This study explored the interplay between MVs and κ-Carr in tissue mineralization, providing insights into tissue homeostasis and advancing diagnostic and therapeutic applications in tissue engineering and regenerative medicine.

This work was supported by FAPESP (2018/25871-8 and 2019/25054-2).