The pace of bone repair slows with aging, increasing the chance of developing a delayed union or non-union. These complications are treated with surgical procedures causing significant morbidity and even mortality, especially in older adults. Here we will build on our previous work using heterochronic parabiosis (in which two mice of a different age share a blood supply) showing that exposure to a young circulation and young macrophage cells rejuvenates fracture repair in older mice. In our preliminary data we used cell lineage tracing analysis and parabiosis experiments to determine the developmental source of macrophage in fracture repair, and found these derived from a subpopulation of cells of yolk sac origin. Interestingly these cells reside in the spleen and are recruited through the circulation during bone repair. As mice age, this subpopulation of cells becomes depleted. In this proposal we study the role of this cell population and the factors they produce in the rejuvenation of fracture repair by undertaking the following aims: 1) Identify the role of macrophages derived from yolk sac progenitors in the rejuvenation of fracture repair. Heterochronic parabiosis in which these cells can be labeled or depleted will be investigated to define the contribution of young cells from this population of macrophage cells that can improve the quality of fracture repair in older animals. 2) Determine the function of genes expressed in unique macrophage subpopulations present in young mice in bone repair: We used single cell RNA sequencing and found a unique subpopulation of macrophages cells present in bone repair in only young animals. Mice lacking genes which encode for secreted proteins in various macrophage populations will be used in heterochronic parabiosis to determine their contribution to the rejuvenation of fracture repair. 3) Define how specific macrophage populations and the proteins they secrete alter mesenchymal differentiation in fracture repair. Our prior work showed an important role for beta-catenin in mesenchymal cell differentiation and in fracture repair rejuvenation. Here we will use in-vitro approaches to determine how specific subpopulations of macrophage cells and the proteins they secrete alter mesenchymal cell differentiation in cells from young and old animals. There will be an initial focus on beta-catenin, but an unbiased approach will be used as well. This proposed work builds on our prior studies of rejuvenation by heterochronic parabiosis in fracture repair. It will address critical gaps in our knowledge about the mechanism responsible for the rejuvenation phenotype driven by heterochronic parabiosis. Our work will also identify a novel therapeutic approach to address a critical clinical problem in older patients, delayed fracture healing.