Evaluation of redox dynamics in callus-forming skeletal progenitor cells during fracture healing using in vivo DNP-MRI

Fracture healing is a dynamic biological process involving inflammation, activation of callus-forming skeletal progenitor cells (CaSPCs), and tissue remodeling, in which redox regulation plays a critical role. However, there is no established imaging technique that evaluates CaSPC-related redox activity at fracture sites in vivo, as conventional imaging modalities reflect structural changes rather than cellular activity. In vivo dynamic nuclear polarization magnetic resonance imaging (DNP-MRI) enables visualization of tissue redox status. In this study, we investigated the utility of DNP-MRI to visualize spatiotemporal redox changes at fracture sites using a mouse tibial fracture model. In vivo DNP-MRI with the nitroxyl radical probe carbamoyl-PROXYL (CmP) revealed stage-dependent, accelerated CmP signal decay within the fracture callus during early- and mid-phase healing. Complementary in vitro redox analyses demonstrated that CaSPCs exhibit significantly higher CmP decay rates than intact bone-derived skeletal progenitor cells (IB-SPCs). Ex vivo analyses revealed that CaSPCs exhibited higher mitochondrial ROS production than IB-SPCs. These findings indicate that redox dynamics within the fracture callus are stage-dependent and reflect intrinsic metabolic features associated with CaSPCs. In vivo DNP-MRI represents a functional imaging approach for visualizing redox metabolism during fracture healing, providing metabolic information beyond structural imaging and enabling earlier evaluation of bone regeneration.