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Date of Award
Doctor of Philosophy (PhD)
Bone is a dynamic organ that readily undergoes bone formation and remodeling in response to its mechanical environment. This response is accelerated during injury such as full fracture and or stress fracture, where woven bone must rapidly form for successful repair and restoration of mechanical function. However, in 5-10% of all clinical fracture cases, bone formation is impeded and the fracture fails to heal leading to non-union. Furthermore, widely used bone active anti-resorptive pharmaceuticals such as bisphosphonates have been linked to nonhealing stress fractures called Atypical Femoral Fractures (AFFS). Animal models of full fracture and stress fracture have demonstrated that angiogenesis is critically important for osteogenesis and subsequent bone repair. In addition, it has been shown that cytokines driving these angiogenic and osteogenic processes, like vascular endothelial growth factor (VEGFA), are critical for bone repair. However, the role that bisphosphonates and osteoblast lineage cells play in modulating these indispensable angiogenic and osteogenic factors in vivo remains undetermined.
For the first aim of the dissertation we tested the role that bisphosphonates play in regulating angiogenesis during stress fracture repair. We determined this by dosing rats (Female, 5 month, Fisher 344) with the bisphosphonate alendronate (5 mg/kg) following forelimb cyclic compression to create a stress fracture. Early gene expression and histological results demonstrated that short-term alendronate administration improved early angiogenesis and osteogenesis following stress fracture.
For the second aim of the dissertation we used lineage tracing and the inducible diphtheria toxin (iDTR) and 3.6Collagen type 1 thymidine kinase (3.6Col1 tk) cell specific ablation models to test the contribution of various osteoblast lineage cell subsets (Osterix (Osx), Dentin Matrix Protein-1 (Dmp1), 3.6Col1) to osteogenesis and angiogenesis necessary for bone repair. From these studies, we determined that pre-existing osteoprogenitor (Osx+ lineage) cells but not more mature osteoblasts (Dmp1+ lineage) cells readily contribute to woven bone forming osteoblasts during fracture. Although depletion of the Osx+ and Dmp1+ cell lineages during stress fracture repair did reduce injury induced osteoegensis, nonspecific effects of the DT toxin on mouse vitality greatly confounded these effects. Finally, ablating replicating osteoblast lineage cells during full fracture and stress fracture repair using the 3.6Col1 tk model, severely diminished post fracture callus and bone formation.
For the third aim of the dissertation, we sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. To test this, Ubiquitin C (UBC), Osx, or Dmp1 Cre-ERT2 mice containing floxed VEGFA alleles (VEGFAfl/fl) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect. UBC CreERT2 VEGFAfl/fl (UBC cKO) mice, which were used to mimic non-specific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture but not stress fracture repair. Osx CreERT2 VEGFAfl/fl (Osx cKO) mice, but not Dmp1 CreERT2 VEGFAfl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+) but not mature osteoblasts/osteocytes (Dmp1+) is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair.
The work presented in this dissertation, by improving our understanding of the molecular and cellular mechanisms regulating angiogenesis and osteogenesis during fracture repair, will hopefully aid in the development of future fracture therapeutics and intervention strategies to prevent nonunion.
Matthew J. Silva
Roberto Civitelli, David Ornitz, Lori Setton, Simon Tang,
Available for download on Friday, January 24, 2020