Periodic Reporting for period 1 - HIPPOX (The mechanobiology of hypoxia during bone regeneration)
Periodo di rendicontazione: 2022-07-01 al 2023-08-31
Bone regeneration is a challenging clinical problem. Each year, millions of patients worldwide experience bone fractures and 10-15% of these fractures do not fully heal. Two critical events early in fracture repair determine the outcome of the healing process: changes in oxygen supply caused by blood vessel rupture and mechanical instability between the broken bone ends. Thus, cells that will eventually form cartilage and bone to heal the bone must simultaneously adapt to both different local oxygen levels and a mechanical microenvironment to ensure full tissue regeneration. The project aims to define new cellular and molecular mechanisms that mediate crosstalk between the local oxygen environment and mechanical signaling during fracture repair and target this crosstalk in an innovative regenerative therapy to accelerate fracture repair. Improvement in therapeutic strategies and rehabilitation will have a global impact and are already included in European health care efforts to ensure health throughout the life course, and to reduce hospitalization time and mortality in the (elderly) population.
The first part of the project and content of this report has been conducted at the University of Pennsylvania, United States (PENN) under the supervision of Prof. Joel D. Boerckel. Within this project part, we wanted to understand how intracellular oxygen levels vary between cells in the bone marrow and how tissue oxygen levels change during fracture repair. We established two methodologies to i) determine intracellular oxygen levels using EF5 staining and to ii) measure tissue oxygen levels using Oxyphor probes. Nitroimidazoles such as EF5 are selectively reduced by nitroreductase enzymes under hypoxic conditions, resulting in the formation of EF5 adducts that can be visualized with fluorophore-coupled antibody (collaboration partner: Cameron Koch, UPenn). Oxyphor probes enable the measurement of oxygen-dependent quenching of phosphorescence with a phosphoromoter under in vivo conditions in real time (collaboration partner: Sergei Vinogradov, UPenn). The precise definition of the local microenvironment during the initial phase of fracture healing allows to determine how intracellular and extracellular oxygen levels guide bone fracture repair and to define new therapeutic targets and avenues towards bone healing disorders.
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.
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.
Our data establish that the initial phase of fracture repair is not hypoxic, identify a putative cellular mechanism, and establish a novel intervention that improves bone repair. These findings contribute to new fundamental understanding in the field of bone regeneration and establishes a unique methodology to monitor cellular and tissue oxygen levels which can be transferred to various other fields. We are currently seeking IPR support to transfer the knowledge into a viable therapeutic strategy. In addition, further research is needed to validate the human relevance and provide evidence for a successful clinical translation.