Bone turns over continuously and is highly regenerative following injury. a means of investigating C75 the role of stem cell-intrinsic and extrinsic C75 molecular regulators for bone regeneration and repair. Imaging Lineage tracking Bone regeneration Fracture repair Mx1 Introduction Degenerative bone diseases and age-related bone loss leading to a high risk of osteoporotic fracture has become a major challenge in public health1. Bone maintenance is controlled by bone-forming osteoblasts and bone-resorbing osteoclasts. Defects of bone forming cells are a main cause of age-related bone loss and degenerative NOS2A C75 bone diseases2 3 While extensive research has focused on the improvement of fracture healing the discovery of reliable drugs to remedy degenerative bone diseases and to reverse the weakness of osteoporotic fractures remains an important issue. Thus studying C75 the source of bone forming cells and their control mechanisms in bone regeneration and repair provides a novel insight to enhance skeletal regeneration and reverse bone loss diseases. The presence of multipotent mesenchymal cells in bone marrow has been proposed based on the identification of clonogenic populations that could differentiate into osteogenic adipogenic and chondrogenic lineages identity of stem cells and how such cells react to fracture injury and supply bone-forming cells are unclear. Therefore it is important to develop a method that is able to analyze the migration proliferation and differentiation of endogenous SSCs/MSCs in under physiological circumstances. Fracture repair is usually a multi-cellular and dynamic process regulated by an array of complex cytokines and growth factors7. The most popular approach for fracture studies is to use an animal model with long-bone fracture and to analyze bones by bone sectioning and immunofluorescent techniques8-10. This repair process can be monitored by multiple imaging techniques including micro-CT11 near-infrared fluorescence12 and chemiluminescence imaging13. However each technique has certain limitations and there has been no effective way to monitor SSCs/MSC function at the cellular level kinetics of osteogenic stem/progenitor cells in fracture repair. This protocol offers sequential imaging to track the relocation of C75 osteogenic stem/progenitors into fracture sites and the quantitative measurement of osteoprogenitor growth in the early repair process. This approach may be useful in multiple contexts including the evaluation of therapeutic candidates to improve bone repair. Protocol 1 Mice and Preconditioning Note: All mice were maintained in pathogen-free conditions and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the Massachusetts General Hospital. All surgery should be performed under sterile condition using autoclaved sterile gear. (Rosa-YFP) and (Rosa-Tomato) were purchased from Jackson Laboratories. mice were provided by Dr. C75 Henry Kronenberg. For the quantitative analysis of OSPC migration and proliferation (single color-reporter) mice were used. For more detailed tracking of their differentiation into mature osteoblasts we used trigenic mice. To label cells reporter mouse intraperitoneally once every other day for 10 days. To eliminate resident hematopoietic cells irradiate mice with a single dose of 9.5 Gy. After 24 hr of irradiation transplant wild type bone marrow cells (1×106 cells/mouse) intravenously16. Note: Since hematopoietic cells improves the imaging quality and quantitation of osteogenic cells. After bone marrow transplantation monitor animals for four to six weeks in order to achieve a successful repopulation of donor marrow cells. 2 Mouse Preparation Anesthetize mouse by intraperitoneal injection of 50 μl of ketamine/xylazine (100 mg/kg xylazine 12 mg/kg body weight) using an IACUC-approved procedure. Note: Duration of the effect may be extended by an additional dose of ketamine/xylazine. Determine if the mouse is usually fully anesthetized by the lack of response to toe and/or tail pinches. (Optional) When the mouse is usually anesthetized secure an isoflurane gas mask over the nose by taping. Change the level of isoflurane to ensure the animal is usually fully sedated. Clip the scalp hair using an electric trimmer or small scissors. Remove the hair fragments and sterilize the uncovered skin with 70% alcohol swab. Apply tear gel to prevent corneal dehydration. 3 Microfracture Injury After.