Osteocytes extracellular vesicles cargos: a new approach for regenerative medicine of bone tissue – OVERbone

There are increasing clinical situations in which bone regeneration is required for skeletal reconstruction of large bone defects. A therapeutic issue is to restore bone mass by stimulating osteogenic activity. OVERbone aims to investigate a novel mechanism of bone formation based on extracellular vesicles secreted by bone cells, that could be used as therapy for bone regeneration.
Osteocytes, highly specialized cells that are embedded in the mineralized bone matrix, play an important role in bone homeostasis and in the maintenance of bone integrity (bone tissue repair) and are intimately linked to the process of bone aging. Many EVs are present in the bone matrix and are produced throughout life in response to many stimuli. EVs contain molecules (proteins, nucleic acids, etc.) which are characteristic of their cell of origin and guarantee their functionality. Osteocytes have been shown in vitro to produce EVs that could be a sophisticated mechanism of intercellular communication in bone. Thus, the decrease in renewal and integrity of bone tissue (appearance of micro-fractures) during aging could not only participate in compromising the mechanical properties of bone, but also in altering this communication network between bone cells.
Our hypotheses are that EVs are released by osteocytes in vivo, that this release is a key event in the dialogue between osteocytes and other bone cells and that EVs released by osteocytes are essential for their function in bone. In addition, we believe that these EVs are altered during aging either in quantity or in quality and that this alteration contributes to bone aging.
The main objective of the OVERbone project will be therefore to establish that these EVs: 1) are present in bone tissue using a nanometric imaging approach, 2) can control osteoformation and improve bone repair, 3) contain molecules responsible for their regenerative properties that we will try to identify, and 4) that these EVs (or certain molecules they contain) can be manipulated to stimulate bone formation and improve bone regeneration in vivo.
The OVERbone proposal main novelties lie on the fact that these VEs and their associated molecules have never been identified as players in physiological bone repair which is impaired in the elderly patient. In addition, information on the location/environment of EVs in bone tissue as well as on their ultrastructure observed in situ will help to better understand how bone cells communicate.
During the OVERbone project, EVs will be produced in vivo from the bones of young, adult and old mice. Two vesicular subpopulations (exosome-type vesicles (<100 nm) and larger vesicles (up to 1 micron)) will be extracted and characterized. This characterization will be carried out using biophysical measurements, biochemical analyses in order to confirm the nature of these EVs, their cellular origin as well as their content, and finally a functional analysis carried out in vitro using conventional cellular and molecular approaches to assess their effects on the differentiation and activity of osteoforming cells. The quantitative and qualitative modifications of the EVs isolated from mice bones of different ages will also be sought. The challenge of visualizing EVs in bone tissue will be met through the use of new high-resolution electronic imaging techniques. Finally, the osteogenic activity and the regenerative properties of these EVs will be demonstrated in vivo in association with biomaterials using tissue engineering approaches.
The success of OVERbone is based on the complementary expertise of its partners in the fields of purification and characterization of EVs, bone tissue physiopathology, high-resolution imaging and therapeutic regeneration and biomaterial grafts.