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

Alexandra Olsen; Priyatha Premnath; Scott Brunet; Leila Larijani; Sarah L Manske; Leah Ferrie; Neil A Duncan; Derrick E Rancourt; Ralph S Marcucio; Roman Krawetz

doi:10.1016/j.bone.2025.117732

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

Bone. 2026 Feb:203:117732. doi: 10.1016/j.bone.2025.117732. Epub 2025 Nov 18.

Authors

Affiliations

¹ McCaig Institute for Bone & Joint Health, University of Calgary, Calgary, AB, Canada; Department of Biomedical Engineering, University of Calgary, Calgary, AB, Canada.
² McCaig Institute for Bone & Joint Health, University of Calgary, Calgary, AB, Canada; Department of Biomedical Engineering, University of Wisconsin-Milwaukee, Milwaukee, WI, USA.
³ McCaig Institute for Bone & Joint Health, University of Calgary, Calgary, AB, Canada.
⁴ McCaig Institute for Bone & Joint Health, University of Calgary, Calgary, AB, Canada; Department of Biomedical Engineering, University of Calgary, Calgary, AB, Canada; Department of Radiology, University of Calgary, Calgary, AB, Canada.
⁵ McCaig Institute for Bone & Joint Health, University of Calgary, Calgary, AB, Canada; Department of Biomedical Engineering, University of Calgary, Calgary, AB, Canada; Department of Civil, University of Calgary, Calgary, AB, Canada.
⁶ Department of Orthopaedic Surgery, University of California at San Francisco, San Francisco, USA.
⁷ McCaig Institute for Bone & Joint Health, University of Calgary, Calgary, AB, Canada; Department of Biomedical Engineering, University of Calgary, Calgary, AB, Canada; University of Calgary, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada. Electronic address: rkrawetz@ucalgary.ca.

PMID: 41260533
DOI: 10.1016/j.bone.2025.117732

Abstract

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.

Keywords: Bone regeneration; Endochondral ossification; Fracture healing; Lineage tracing; Mesenchymal progenitor cells (MPCs); p21 knockout.

MeSH terms

Animals
Biomechanical Phenomena
Bone Density
Bony Callus* / diagnostic imaging
Bony Callus* / metabolism
Bony Callus* / pathology
Calcification, Physiologic*
Chondrogenesis
Cyclin-Dependent Kinase Inhibitor p21* / deficiency
Cyclin-Dependent Kinase Inhibitor p21* / genetics
Cyclin-Dependent Kinase Inhibitor p21* / metabolism
Fracture Healing* / physiology
Mesenchymal Stem Cells / metabolism
Mice
Mice, Inbred C57BL
Mice, Knockout
Osteogenesis
Tibial Fractures / pathology
X-Ray Microtomography

Substances

Cyclin-Dependent Kinase Inhibitor p21