Matrix vesicle isolation from a three-dimensional in vitro bone model

Extracellular vesicles (EV) are nano-sized bilayer vesicles that are involved in biological functions and secreted by a wide variety of cells. Osteoblasts, the bone forming cells, can release a subset of EVs known as matrix vesicles (MtVs) which are believed to be involved in matrix mineralization and feature bone forming properties. Osteoblast-derived EVs or MtVs have been mostly isolated from conditions which are still far from nature, i.e. mesenchymal stromal cells (MSCs), or osteoblast cell lines cultured in two-dimensional (2D) tissue culture flasks. In our study, we aimed at investigating whether MtVs could also be isolated from an environment which better resembles the complex in vivo situation. This study investigated the EVs secretion during osteogenic differentiation of human bone marrow MSCs (hBMSCs) in the most advanced human three-dimensional (3D) in vitro woven bone constructs previously developed by our group. hBMSCs were cultured in spinner flask bioreactors which induced wall shear stress on cells and directed the cells to differentiate towards osteoblasts and osteocytes. The EVs secreted into the culture medium were isolated and characterized based on their morphological, biological, and functional properties. The characteristics of a part of isolated EVs shared similarities with MtVs. These vesicles were electron-dense and electron-lucent, showed alkaline phosphatase (ALP) activity, increased the amount of released free phosphate into the culture medium, and increased the amount of deposited phosphate within the ECM. The results indicate that a complex 3D environment mimicking bone development is favorable to stimulate MtV-producing cells to produce targeted MtVs in vitro. These MtVs potentially could be used as a biological agent for bone regeneration and fracture healing through, for instance, integration with biomaterials to target bone formation locally.