ABSTRACT Bone injuries are a major health problem. There are 7.9 million bone fractures sustained annually in the U.S. Healing is impaired in about 10% of these fractures with seriously delayed union or non-union, causing morbidity for patients and enormous healthcare costs. While strategies such as bone grafting, synthetic polymers, low intensity pulsed ultrasound and electromagnetic fields, growth factors and cell therapy are currently being used or investigated to promote bone healing, each of these therapies have their own advantages and disadvantages in terms of cost, effectiveness and safety. Thus, there is a compelling need to find novel effective therapies that promote fracture healing. Vitamin C and thyroid hormone (TH) are known to play key roles in endochondral bone formation (EBF). Our recent studies on the molecular pathways for TH and vitamin C actions revealed evidence that sequential activation of hedgehog and hypoxia signaling pathways contribute to key steps involved in EBF. Our focus in this project is on the therapeutic utility and mechanisms of action of two small molecules, SAG 21k and IOX2, that activate hedgehog and hypoxia signaling pathways to promote EBF at the fracture site. In this proof of concept study, we propose to deliver SAG21K and IOX2 locally using 3D printed fibrin gel/β-tricalcium phosphate (βTCP) scaffolds at the defect site to provide mechanical strength and minimize unwanted side effects on other tissues. A clinically relevant segmental defect model in the femoral midshaft in which a 2.5-mm defect is stabilized by an intramedullary threaded rod with attached plastic spacers that does not heal over a prolonged period will be used. Three aims are proposed. In aim 1, we will 3D print fibrin gel/β-tricalcium phosphate (βTCP) scaffold preparations containing SAG21k and IOX2 and evaluate the suitability of these preparations for delivery of effective concentrations of SAG21k and IOX2 at the optimal therapeutic time window for activation of hedgehog and hypoxia signaling at the fracture site by measurement of downstream signaling targets of these signaling pathways by immunohistochemistry (IHC) and real time PCR in the fracture callus of mice at different times. In aim 2, we will test the hypothesis that sequential activation of hedgehog followed by hypoxia signaling will be effective in promoting healing of femoral segmental defects. We will compare the efficacy of bone healing with SAG21k and IOX2 with that of autografts, a gold standard used for healing of nonunion defects. We will use validated microCT, bone strength and histological measurements to evaluate the fracture healing phenotype. Therapeutic effectiveness of SAG21k/IOX2 combination therapy will be studied using aged and diabetic mice with impaired fracture healing. In aim 3, we will test the hypothesis that sequential activation of sonic hedgehog and hypoxia signaling induces bone healing by promoting direct conversion of chondrocytes-to-osteoblas...