Effects of Aging on the Immune and Periosteal Response to Fracture in Mice

Abstract Aging predisposes individuals to reduced bone mass and fragility fractures, which are costly and linked to high mortality. Understanding how aging affects fracture healing is essential for developing therapies to enhance bone regeneration in older adults. During the inflammatory phase of fracture healing, immune cells are recruited to the injury site as periosteal skeletal stem/progenitor cells (pSSPCs) rapidly proliferate and differentiate into osteochondral lineages, allowing for fibrocartilaginous callus formation and complete bone healing. Irrespective of age, how periosteal mesenchymal and immune cells interact during early fracture healing is incompletely understood, limiting our ability to potentially modulate these processes. To address this, we directly analyzed, in parallel, at a single-cell level, isolated murine CD45(+) and CD45(-) periosteal cells dissected from intact and fractured bones, collected three days after injury. Through comprehensive analysis, corroborated by bulk RNA-sequencing, flow cytometry, and histology, we found aging decreases pSSPCs proliferative, marked by a reduced expression of genes required for callus formation and an increased senescence signature. We found that the chemokine Cxcl9 was highly upregulated in aged intact Prrx1+ pSSPCs, predicted to interact with other pSSPCs directly, and associated with increased recruitment of CD8+ T cells at the fracture site three days after injury. Cell-to-cell communication analysis provided insight into the complexity of interactions among the many cell types regulating fracture healing and the impact of aging on these processes. Together, these results provide insight into age-induced alterations in fracture healing, informing the development of improved therapeutic approaches for fragility fractures.