Background: Fracture healing is strongly influenced by mechanical loading conditions, whereas hindlimb unloading induces bone loss and may further compromise skeletal repair. However, the metabolic remodeling of bone tissue under fracture, unloading, and their combination remains insufficiently characterized. This study aimed to investigate metabolic alterations in mouse bone tissue under fracture (Fx), tail-suspension hindlimb unloading group (HU), and fracture combined with tail-suspension hindlimb unloading group (Fx + HU) using untargeted metabolomics.

Methods: Male mice were randomly assigned to four groups: sham control (Sham), Fx, HU, and Fx + HU. Mechanical unloading was induced using a tail-suspension hindlimb unloading model, and fracture was established using a standardized murine fracture procedure. After 3 weeks of intervention, proximal tibial bone tissues were collected for micro-computed tomography (micro-CT) and untargeted metabolomics analysis. Multivariate statistical analyses, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA (OPLS-DA), were performed to identify metabolic differences among groups. Differential metabolites and enriched pathways were further analyzed.

Results: Micro-CT analysis showed significant deterioration of trabecular bone mass and microarchitecture in the Fx, HU, and Fx + HU groups compared with the Sham group (all p < 0.001), with the Fx + HU group showing the most pronounced impairment. Untargeted metabolomics demonstrated robust analytical reproducibility in both positive and negative ion modes. PCA and supervised multivariate analyses showed clear separation among the four groups, and the Fx + HU group exhibited the greatest metabolic divergence from the others. Differential metabolite screening and pathway enrichment analysis revealed marked alterations in metabolic pathways associated with amino acid metabolism, lipid metabolism, energy metabolism, and oxidative stress-related processes.

Conclusions: Fracture and hindlimb unloading each induced substantial metabolic remodeling in mouse bone tissue, while their combination produced more profound and distinct metabolic perturbations. These findings suggest that mechanical unloading may aggravate bone metabolic dysregulation after fracture and provide exploratory metabolomic evidence for understanding skeletal deterioration under combined injury and unloading conditions.