One out of every 200 people in the U.S. will suffer an amputation, and this number is expected to double in the next 40 years. Amputation has protracted effects on vocational outcome, individual productivity, and personal independence far beyond the initial trauma. While complete limb regeneration in humans may someday become the ultimate therapy, the ability to coordinately control bone regeneration would reduce pain from ectopic bone spurs, and provide a better fit for prosthetics, resulting in a better quality of life in the near term. Amputation of the digit tip within the terminal phalangeal bone of rodents, monkeys, and humans results in near-perfect regeneration from the blastema, which has been well characterized in both adult and neonatal mice. However, it remains unclear why more proximal digit amputations or amputations within limbs, fail to produce the same regenerative result. The long term goal of this work is to understand the influence of oxygen on regeneration, so that these mechanisms can be exploited to confer regenerative capacity where it is lacking. The objective of this study is to gain insight into the role of oxygen in the regenerative process by manipulating oxygen within the digit tip to alter regeneration. Our central hypothesis is that a biphasic oxygen response facilitates the regenerative process. Our hypothesis stems from the biphasic nature of the regenerative models, and oxygen's known influence in the process of wound healing. The highly proliferative, undifferentiated blastema forms in a hypoxic environment. Exposure to increased oxygen tension initiates the differentiation of the blastema into bone, supporting the idea of a biphasic oxygen response that drives the regenerative process. The rationale for this study is that understanding the oxygen gradients that promote bone regeneration will not only provide a better understanding of the regenerative process, but will also provides opportunities for therapeutic intervention. The central hypothesis will be teste by two specific aims: 1) Determine the influence of oxygen on the formation and maintenance of the blastema. Based on evidence of an avascular blastema, and that hypoxia maintains the multipotency and proliferation rate of osteoblast precursor cells, our working hypothesis is that an initial hypoxic event is critical for promoting the blastema structure 2) Establish to what extet oxygen modifies the rate of regeneration. We hypothesize that stimulating hypoxic cascades when the regenerating digit is already hypoxic, and increasing oxygen tension when the digit is actively mineralizing bone will have a net additive effect on the rate of the regenerative process. Completion of these aims will provide predictive capacity for the regenerative process to form new bone in the context of oxygen, and provide defined points of intervention to manipulate regenerative outcome. Research enabled by this work will lead to a defined mechanistic understanding of oxygen's role in bone regeneration and identify therapeutic targets to enhance regenerative capacity. PUBLIC HEALTH RELEVANCE: Limb amputation has a profound impact on a person's vocational options, individual productivity, and independence. One out of every 200 people in the United States is an amputee, and this number is expected to double in the next 40 years due to an increase in amputations secondary to vascular disease. Wound healing is thought to be contingent upon appropriate oxygen levels at the wound site, and our long-term goal is to understand oxygen's role in limb regeneration in order to apply the findings as a critical step towards optimizing treatment for amputees.