Single-Cell Mapping Reveals Age-Related Alterations in Periosteal Progenitor Cells and Immune Microenvironment Impairing Bone Regeneration

Aging profoundly impacts bone homeostasis and regeneration, yet the cellular and molecular mechanisms underlying periosteal aging remain poorly understood. Using single-cell RNA sequencing, we profiled the periosteum of 3-, 9-, and 18-month-old mice, revealing age-related shifts in progenitor, neutrophil, and macrophage subpopulations. Aging reduced mesenchymal cell populations and impaired osteogenic potential, disrupting periosteal homeostasis. Periosteal progenitor subsets exhibited distinct aging trajectories: Dpt? fibrous-layer cells undergoing early senescence, while Postn? progenitors showed osteogenic decline. Aging also shifted immune profiles, increasing inflammatory Cd38hi macrophages and dysfunctional Nlrp3hi neutrophils, further disrupting bone homeostasis. Notably, aged progenitor cells upregulated CSF1 and CXCL signaling, driving macrophage and neutrophil infiltration, exacerbating bone loss. Our findings provide a comprehensive periosteal aging atlas, highlighting disrupted progenitor-immune crosstalk as a key driver of bone homeostasis imbalance and impaired regeneration, offering potential therapeutic targets for age-related bone disorders. Overall design: Long bones were harvested from wild-type C57BL/6 J mice across three age groups: 3 months, 9 months, and 18 months, with six males per group (Aniphe Biolaboratory Inc.). The acquisition protocol was as follows: following cervical dislocation for euthanasia, the animals were sterilized by external application of 75% ethanol. The lower limbs were then aseptically dissected, and the overlying skin, muscles, fat, and other soft tissues were meticulously removed. The intact femur and tibia were subsequently isolated for further processing.