Enhanced fracture repair in p21^-/- mice is mediated through increased callus mineralization

Fracture healing is a complex, multi-phase process involving mesenchymal progenitor cell (MPC) recruitment, chondrogenesis, osteogenesis and bone remodelling. The cyclin-dependent kinase inhibitor p21 is known to regulate cell cycle progression and has been implicated in tissue regeneration. Here, we investigated the role of p21 in endochondral fracture repair using a transverse tibial fracture model in physiologically normal mice. Prx1CreERT2-GFP;R26RTdtomato mice on C57BL/6 and p21^-/- backgrounds enabled lineage tracing of MPCs. Longitudinal in vivo micro-CT, histological analysis, tissue cytometry and mechanical testing were used to assess callus formation, cellular composition, and mechanical integrity. Our results demonstrate that p21^-/- mice exhibit enhanced bone regeneration, with significantly higher bone mineral density (BMD) and bone volume fraction (BV/TV) in the fracture callus at 2 and 4 weeks post-fracture (wpf). Histology revealed increased Prx1⁺ cell recruitment, along with greater expression of chondrogenic (Sox9) and osteogenic (BSP) markers in p21^-/- mice at 2wpf. Biomechanical testing showed that despite similar strength, p21^-/- calluses had reduced toughness, suggesting altered matrix remodelling. Collectively, our findings highlight p21 as a negative regulator of bone regeneration, likely through modulation of MPC recruitment and differentiation. Together, these data suggest therapeutic targeting of p21 may enhance fracture healing and counteract osteoporosis in terms of bone remodelling and repair.