UCSF

The majority of fracture research has been conducted in long bones with significantly less work done in the craniofacial skeleton. Craniofacial bones differ from long bones in both their developmental mechanism and embryonic origin. Thus, it is possible that their healing mechanisms may differ as well. We sought to determine the regulatory mechanisms that govern fracture repair in both the craniofacial and appendicular skeletons using mandibular and tibial fracture models. Stabilized and unstabilized mandible fracture models were created which heal via intramembranous and endochondral ossification, respectively. Whereas bone forms directly via intramembranous ossification, endochondral ossification occurs through a cartilage intermediate that is gradually replaced with bone. Using our mandible fracture models, we confirmed that mechanical motion promotes robust cartilage formation whereas mechanical stabilization favors intramembranous ossification, a phenomenon previously demonstrated in long bone fractures. Due to the mobility experienced at the fracture site, the majority of clinical fractures heal through the process of endochondral ossification. Recent genetic studies conducted in the appendicular skeleton have demonstrated that during endochondral ossification a significant portion of callus chondrocytes transform into osteoblasts that derive the new bone. Using genetic lineage tracing and immunohistochemistry, we demonstrated that mandibular callus chondrocytes also undergo chondrocyte-to-osteoblast transformation, that they significantly contribute to new bone formation, and that they express pluripotent stem cell (Sox2) and osteogenic (OC) markers while undergoing their transition. Due to the important role of cartilage during fracture repair, we tested the efficacy of using cartilage grafts to heal critical-sized mandibular defects. Cartilage grafts not only produced complete bony-bridging of the defects, but the newly formed bone was also highly vascularized and primarily derived from donor / graft tissue. Using unstabilized tibial fracture models which heal via endochondral ossification, we sought to determine the molecular signals that regulate chondrocyte-to-osteoblast transformation. We hypothesized that canonical Wnt signaling plays a key role in this process and used loss-of-function and gain-of-function experiments to test our hypothesis. By inhibiting or over-activating the pathway through conditional expression in chondrocytes of a truncated or stabilized form of β-catenin, a key downstream co-factor required for canonical Wnt signaling, we demonstrated that pathway inhibition leads to impaired bone formation whereas pathway overstimulation results in osteopetrotic bone. Interestingly, inhibition of canonical Wnt signaling did not significantly increase chondrocyte cell death. Rather chondrocytes remained as detached cells in the marrow space. Additionally, global inhibition of Wnt ligand palmitoylation, a key step in ligand packaging and release, through conditional Porcupine deletion did not significantly inhibit chondrocyte-to-osteoblast transformation. Thus, although canonical Wnt signaling plays a key role in chondrocyte-to-osteoblast transformation during endochondral repair, this process appears to be independent of Wnt ligands modified via Porcupine-mediated palmitoylation. These data confirm that chondrocyte-to-osteoblast transformation occurs in both the craniofacial and appendicular skeletons, that canonical Wnt signaling plays a critical role in regulating chondrocyte transformation, and that this process is independent of Wnt ligands palmitoylated via Porcupine. Moreover, these data lend key insight into the development of novel and improved fracture therapies including the use of cartilage as a graft alternative for craniofacial fractures.