Date of Award
Doctor of Philosophy (PhD)
In most instances, the skeleton has a remarkable capacity for repair following injury. However, in 5 to 10% of patients, fractures fail to properly heal resulting in non-union. A need exists for a more comprehensive understanding of the complex biology of fracture repair, which involves the coordinated work of many cell types including osteoblasts, osteoclasts, and immune cells. Depending on the extent of injury, fractures will heal through either intramembranous bone formation, involving the direct formation of bone callus, or endochondral bone formation, featuring a cartilage intermediary prior to bone callus formation. Both processes begin with inflammation, which sets the stage for a successful repair cascade. However, the production of inflammatory cytokines and recruitment of immune cells in the early stages of fracture repair, and the implications for these processes on effective fracture repair remain incompletely understood.For the first aim of the dissertation, we examined gene expression in response to injury across a broad range of time points for both intramembranous and endochondral bone formation. We used RNA sequencing to observe gene expression and immunohistochemistry to observe select protein expression throughout the early repair process. While some degree of overlap existed, we observed divergent gene expression between intramembranous and endochondral bone formation, with each possessing a distinct transcriptome contributing to repair. An inflammatory reaction was present for both bone formation types, however endochondral repair had expression of inflammatory genes and recruitment of immune cells that were orders of magnitude greater than observed in intramembranous repair. Intramembranous repair was more enriched for genes associated with osteoblasts and bone formation and was uniquely enriched for PI3K-Akt signaling. This is likely reflective of the more simplified nature of bone formation in intramembranous repair Рwhich does not require production of a cartilage intermediary callus. We also observed a dramatic downregulation of voltage gated ion channels in endochondral repair, which was not present in intramembranous repair.For the second aim of this dissertation, we tested the involvement of interleukin-6 (IL-6) in fracture repair. IL-6 is a component of the inflammatory reaction to skeletal injury and has high upregulation in early callus tissue. We used an IL-6 global knockout mouse (IL-6 KO) with two models of skeletal injury, full fracture and stress fracture, which heal through endochondral and intramembranous bone formation respectively. Full fracture repair was not altered in IL-6 KO mice. However, repair of stress fracture was enhanced in IL-6 KO mice, which had larger calluses and more bone formation than control animals. IL-6 KO did not alter osteoclast numbers or recruitment of the innate immune system in stress fracture callus. IL-6 KO mice had slight reductions in inflammatory signaling and higher expression of the bone anabolic protein Wnt1 after injury. Furthermore, IL-6 KO animals displayed an increased ability to form woven bone when challenged with a non-injurious model of loading induced bone formation. Thus IL-6 KO directly affects the bone building potential of osteoblasts, allowing for the increased formation of bone.For the third aim of this dissertation, we examined the CXC family of chemokines in skeletal repair through deletion of CXC family receptor 2 (Cxcr2). Several members of the CXC chemokine family are among the most highly upregulated genes following skeletal injury. Despite this, their impact of fracture repair has not been reported. We used a global knockout mouse for Cxcr2 (Cxcr2 KO) to study bone formation after full and stress fracture. The ability of Cxcr2 KO mice to form bone was unchanged following full fracture. However, after stress fracture Cxcr2 KO mice had a diminished capacity for repair, producing smaller calluses with less bone formation than control animals. Cxcr2 KO mice had blunted recruitment of neutrophils, the earliest responding immune cell to injury, within stress fracture callus. Thus, we concluded that the blunted immune response in Cxcr2 KO mice leads to the reduced capacity for bone formation.This dissertation work provides insight into the transcriptional profile and inflammatory response of fracture repair following skeletal injury. We are hopeful that this work will benefit the planning of future research on fracture repair biology and provide insight for potential fracture therapeutics to aid in repair and prevent nonunion.
Matthew J. Silva
Roberta . Faccio, Anthony . French, Daniel . Link, Lori . Setton,