Effects of aging on the immune and periosteal response to fracture injury.

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, subsequently, complete bone healing. Irrespective of age, how periosteal mesenchymal and immune cells interact during early fracture healing is incompletely understood, limiting our ability to modulate this process. 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. Comprehensive analysis, corroborated by bulk RNA-sequencing, flow cytometry, and histology, demonstrated that aging decreased pSSPC proliferation, markedly reduced expression of genes required for callus formation, and increased senescence signature. During the regeneration phase, at 14 days post injury, aged mice demonstrated reduced mineralization of the callus, accompanied by elevated Sox9 expression and increased cartilage content, suggesting delayed repair. We also found that the chemokine Cxcl9 was highly upregulated in aged intact Prrx1+ pSSPCs, which has the potential to directly regulate other pSSPCs, and was associated with increased recruitment of CD8+ T cells at the fracture site. Cell-to-cell communication analysis provided further appreciation of the complex interactions among the many mesenchymal and hematopoietic cell types regulating fracture healing and highlighted the impact of aging on these interactions. Together, these results provide insight into age-induced alterations in early fracture healing, which could facilitate the development of improved therapeutic approaches for fracture repair in the elderly.