Periodic Reporting for period 1 - HIPPOX (The mechanobiology of hypoxia during bone regeneration)
Période du rapport: 2022-07-01 au 2023-08-31
We performed in vitro studies by culturing mouse bone marrow cells under defined oxygen concentrations (10%, 2%, 0.5%, 0.1% pO2) and in vivo experiments investigating the cellular oxygen levels in bone marrow cells by systemic injection of EF5 in mice. Our data confirmed that the frequency of EF5-positive cells correlated positively with environmental hypoxia: less oxygen produces greater EF5 signal. Next, we analyzed EF5 in cells isolated from the fracture gap at 3, 5 or 7 days post-fracture (dpf). The frequency of EF5-positive cells at 3, 5 or 7 dpf was significantly lower than the EF5-positive frequency in the controls (adjacent bone marrow of the same limb or bone marrow of the contralateral bone). These findings suggest that the initial fracture healing is not hypoxic. To verify this unexpected result, we performed non-invasive oxygen measurements in live mice using Oxyphor PtG4 at 3, 7 and 14 dpf. These measurements confirmed that the fracture hematoma is not hypoxic, but rather exhibits oxygen levels which gradually decreases and approaches bone marrow pO2 levels by 14 dpf. Next, we sought to determine why the fracture gap exhibits such high oxygen levels. An inevitable consequence of bone fracture is the rupture of blood vessels, resulting in bleeding, release of red blood cells (erythrocytes) and invasion of progenitor cells. We performed single cell RNA sequencing and identified distinct cell progenitor subpopulations which eventually direct fracture gap oxygen levels and subsequent repair. Modulating these cell progenitors significantly enhanced fracture repair and therefore, opens a whole new field for musculoskeletal, regenerative therapies. Final data analysis, mathematical simulations and preparation of the manuscript are currently being conducted.