Military personnel are at a substantially increased risk of bone fracture during combat. A major complication of fracture, especially in cases of high-energy trauma, is delayed union or non-union, meaning that the bone does not heal in a timely manner or does not heal at all. Remodeling of skeletal bone requires the recruitment and proliferation of stem cells with the capacity to differentiate to functional osteoblasts that deposit and mineralize extracellular bone matrix. Given the close association of bone and bone marrow (BM), it has been suggested that BM may serve as a source of these progenitors. Using mice whose bone marrow was reconstituted by a clonal population of cells derived from a single enhanced green fluorescent protein positive (EGFP+) hematopoietic stem cell (HSC), our data shows that the HSC gives rise to osteoblasts, osteocytes and chondrocytes during non-stabilized fracture repair. These findings are paradigm shifting in that most studies focus on the use of the mesenchymal stem cell for repair of musculoskeletal injuries and disease. Based on this novel source for osteo-chondrogenic cells, we hypothesize that HSC-derived osteo- chondrogenic cells may be exploited to enhance fracture repair. The proposed studies will elucidate mechanisms regulating differentiation and maturation of osteoblasts, osteocytes and chondrocytes from the HSC. Based on these mechanistic studies, HSC-derived osteo-chondrogenic progenitors will be manipulated in vivo to demonstrate their ability to effect healing of fracture. The hypothesis of this study will be tested using our novel clonal HSC cell transplantation method in conjunction with non-stabilized and non-union fracture models through two Specific Aims. Aim (1) is to define the molecular mechanisms regulating HSC differentiation and maturation to osteo-chondrogenic lineages in vitro. This Aim seeks to determine the mechanisms governing the commitment of HSCs to the osteo- chondrogenic lineage and identify factors regulating the differentiation and maturation of HSC-derived osteo- chondrogenic progenitors. In vitro experiments based on sorted HSCs (Lin-sca1+ckithiCD34-) cells will profile the temporal expression of osteo-chondrogenic lineage-specific genes. Genes associated with HSC-derived cell differentiation will also be profiled to identify genes involved in differentiation/maturation from this unique source. Complimentary inhibition and stimulation studies will be used to demonstrate the functional importance of identified genes. Aim (2) is to enhance fracture repair by modulation of HSC-derived osteo-chondrogenic precursors in vivo in clonally engrafted animals in which HSC-derived cells can be traced back to a single sorted EGFP+ HSC. The effects of HSC mobilization on fracture repair will be examined alone or in combination with administration of exogenous differentiation factors at the fracture site. The contribution of HSC-derived cells to fracture healing will be examined histochemically, biochemically and quantitatively using micro-computed tomography (micro-CT), static and dynamic histomorphometry. These studies are significant in that they challenge existing dogma by suggesting a novel HSC origin for bone and cartilage cells that may be exploited to enhance healing in cases of fracture and non-union. Methods to enhance and accelerate the fracture healing process based on this novel osteo-chondrogenic stem cell source would have far-reaching benefits for military personnel. Given that high-impact, high-velocity trauma such as those seen in combat, have an increased risk of resulting in non-union, the findings from this study have great relevance to the VA mission. Findings from the proposed studies have the potential to impact Veterans Health Care by identifying unique stem cell-based therapies for improving recovery from fracture.