Project Summary/Abstract There are approx. 15 million bone fractures annually and the mandible sustains the vast majority of craniofacial bone fractures. Currently clinical approaches, such as maxillofacial fixation, are exceedingly invasive and the prevalence of impaired healing remains. Therefore, the objective of this grant is to address the clinical need for a translational and clinically relevant approach to mandibular fractures. Accomplishing this goal requires cross- disciplinary methods that harness expertise in biomaterials and drug delivery coupled with an understanding of the mechanisms that drive functional bone repair. The mandible primarily heals through endochondral ossification, in which a cartilage intermediate forms and is later replaced by bone. In recent years, many groups, including ours, have published significant evidence to show that chondrocytes transdifferentiate into osteoblasts during bone development and fracture healing. The mechanisms underlying chondrocyte transdifferentiation have thus far not been thoroughly explored. However, my preliminary data, along with previously published work, indicate that β-catenin signaling is a critical mediator of chondrocyte-derived osteoblastogenesis. Activation of β-catenin by NGF/TrkA signaling has been observed in various cell types and interestingly; our preliminary data show an increase in NGF and TrkA expression in fracture calluses. Finally, our preliminary data show that NGF administration onto fractures during the cartilaginous phase accelerates bone repair. During this fellowship I aim to understand the role of NGF in chondrocyte transdifferentiation, and develop a therapeutic delivery system for local and sustained release of a “painless” NGF, NGFR100W. The central hypothesis for this project is that sustained release of NGFR100W via PEGDMA microparticles will accelerate endochondral fracture healing by activating β-catenin signaling in hypertrophic chondrocytes. In the first Aim I will build on our preliminary data of enhanced bone repair in NGF-treated mice by engineering NGFR100W-eluting PEGDMA microparticles to accelerate healing. NGFR100W-loaded PEGDMA microparticles will be injected percutaneously onto fracture calli followed by assessment of tissue composition, biomechanical strength, and rate of healing by using histology, microCT imaging, three-point bending tests, and stereology. In the second Aim, I will determine the mechanism by which NGF stimulates osteogenesis. I will use an ex vivo system of fracture callus-derived cartilage cultured with NGF to measure downstream markers of osteogenesis, angiogenesis, and candidate pathways including β-Catenin, Sox2, and hedgehog by RT-qPCR and western blot. In vivo I will conditionally delete TrkA from chondrocytes by crossing the TrkAfl/fl and aggrecan-CreER transgenic mice to test if NGF is required for chondrocyte transdifferentiation during fracture healing using the same functional outcome measures described in Aim...