There are approximately 15 million bone fractures annually and the mandible sustains the majority of fractures of the craniofacial skeleton. Importantly, prevalence of impaired healing is significant and remains an unmet clinical need. Understanding the mechanisms that direct fracture healing is imperative to the development of improved therapies. The mandible heals through the process of endochondral ossification, in which a cartilage intermediate forms and is later replaced by bone. Recent work has revealed a new model of endochondral ossification in which chondrocytes of the cartilage intermediate transdifferentiate into osteoblasts that form the new bone at a region adjacent to the invading vasculature. The mechanisms underlying chondrocyte transdifferentiation have not been explored, but my preliminary data, along with previously published work, indicate that canonical Wnt signaling may be a central mediator of chondrocyte transdifferentiation. For this project I aim to understand the role of canonical Wnt signaling during endochondral bone repair, and then test the therapeutic effect of a novel, water-soluble molecule that strongly activates Wnt signaling. The central hypothesis for this project is that canonical Wnt signaling regulates chondrocyte transdifferentiation by inducing the osteogenic program and that activation of the Wnt pathway through administration of Wnt-Surrogate accelerates mandible fracture healing by increasing the rate of conversion of chondrocytes to osteoblasts. To determine the role of canonical Wnt signaling during endochondral fracture repair, in my first Aim, I will use transgenic mouse strains to conditionally inhibit or activate canonical Wnt signaling in chondrocytes comprising the fracture callus. I will assess the effects of Wnt signaling on chondrocyte transdifferentiation by measuring the rate of cartilage to bone conversion. The effect of Wnt signaling on cellular re-programming will be determined by measuring the expression levels and patters of chondrogenic and osteogenic genes in chondrocytes using qPCR, in situ hybridization, immunohistochemistry, and stereology. In the second Aim, I will test the therapeutic effect of a novel surrogate Wnt ligand to promote fracture repair. The Garcia Laboratory (Stanford) has kindly provided us with the Wnt-Surrogate that strongly activates Wnt signaling in vitro. To determine the osteogenic effects of Wnt-surrogate, cartilage explants will be assessed for matrix mineralization and alkaline phosphatase activity in vitro. Additionally, mandible fracture models will be assessed for bone mineral density and rate of healing. Design of Experiments (DOE) methodologies will be used to optimize the dose and timing of Wnt-Surrogate administration, which will be applied to further in vivo analysis of the effect of Wnt-Surrogate on biomechanical strength and rate of bone formation. Taken together this study will provide improtant information regarding the role of canonical Wnt signaling in chondrocyte transdifferentiation and pre-clinical evidence for Wnt-Surrogate as a novel approach to fracture healing.
The mandible heals through endochondral ossification, a process in which a cartilage intermediate forms and is later replaced by bone. A new model of endochondral ossification has recently emerged in which chondrocytes from the cartilage intermediate transdifferentiate into osteoblasts that form the new bone; however the mechanisms that govern this transdifferentiation are unknown. The experiments outlined in this grant proposal will define the role of canonical Wnt signaling in the regulation of chondrocyte transdifferentiation and will determine the effect of a novel Wnt surrogate on the rate of mandible fracture repair.