Hydrogen reshapes the senescent microenvironment of callus to enhance the healing of anti-osteoporotic-drug-induced atypical femoral fracture

Long-term bisphosphonates (BPs) are widely used to treat osteoporosis, however, they are paradoxically associated with the development of atypical femoral fractures (AFFs), which often characterized by impaired healing. In this study, we induced an AFF model using zoledronate (ZOL) administration in ovariectomized (OVX) osteoporotic rats, following a unilateral femoral fracture. Here we identified that a local pro-senescent microenvironment causes persistent inflammation and impairs effective regeneration in rat AFFs. Molecular hydrogen has demonstrated anti-senescence and anti-inflammatory properties, yet its effects on AFF healing remain unexplored. Therefore, we treated the AFF rats with hydrogen rich water (HRW). The outcomes were assessed by radiographs, histology, micro-CT, and biomechanical tests. The fracture microenvironment was analyzed based on the indicators of senescence, fibrosis, macrophage polarization, cytokine expression, and angiogenesis. We found that HRW significantly promoted callus bridging and resolved the non-union gap in ZOL-induced AFFs, whereas the ZOL group exhibited persistent fibrous tissue. Micro-CT and biomechanical tests confirmed that HRW did not compromise the mechanical strength of the bone mass elevated by BPs. Instead, HRW specifically attenuated the local senescent microenvironment, with a reduction in both SA-β-gal activity and the expression of p16, p21 within the fracture gap. This was accompanied by clearance of pathological fibroblasts, a shift from pro-inflammatory M1 to anti-inflammatory M2 macrophages, a rebalanced cytokine profile, and restored formation of osteogenesis-coupled type-H vessels. Our findings confirm that molecular hydrogen facilitates AFFs healing by locally reversing the senescent microenvironment, rather than boosting systemic bone formation. This creates a favorable niche for callus bridging, representing a novel therapeutic strategy for fracture delayed union or non-union.