A new role for peroxidases in bone repair

Abstract

When bone undergoes trauma or the architecture deteriorates, due to disease and is neglected or misdiagnosed, non-unions can occur, whereby bone does not heal correctly. As a consequence, patients experience pain, stiffness, loss of mobility and disability. In many cases this can result in an inability to perform normal duties in employment, which causes significant financial burden to the patient and economy. The repair of these large bone defects remains a significant challenge for orthopaedic surgeons. Bone grafting strategies have been developed to repair and restore bone function, however the demand for functional bone grafts is extremely high, with an estimated 2.2 million patients worldwide undergoing bone grafting procedures annually. Due to an aging population these numbers are expected to double by 2020, which will put further burden on health care costs worldwide. Autologous bone grafting remains the current standard to repair bone defects and fractures, however, this method of treatment has numerous surgical-associated morbidities and complication rates of up to 30%. Therefore, researchers are attempting to identify substitute grafting materials which possess the critical bone reparative characteristics required for successful healing. To date, a bone graft material which is comparable to autologous bone, with fewer associated morbidities is yet to be identified, thus, continued research is required to identify and develop new agents to promote and accelerate bone repair. Agents which have been thoroughly investigated to enhance the bone repair process in combination with bone graft substitutes include the use of BMP-2. BMP-2 has proven to be successful due to its pro-osteogenic role whereby it promotes osteoblast functionality through the regulation of genes necessary for collagen biosynthesis and mineralisation of the extracellular matrix (ECM). Osteoblasts are one of the main cell types responsible for bone formation and bone repair. These cells are derived from the mesenchymal progenitor cell population, along with endothelial cells and fibroblasts. Work published by our laboratory provides evidence that a group of enzymes with peroxidase activity, namely mammalian-derived myeloperoxidase (MPO) and eosinophil peroxidase (EPO) as well as plant-derived soybean peroxidase (SBP) stimulate the migration of fibroblastic cells and promote their ability to generate a functional ECM. In addition, we have presented evidence demonstrating the ability of these peroxidases, in promoting endothelial cell function and inhibiting osteoclastogenesis, suggesting a potential role for these enzymes in bone repair. The work described in this thesis aims to provide evidence that mammalian and plant derived peroxidase enzymes including, MPO, EPO and SBP possess pro-osteogenic activities by influencing osteoblast functionality. Using physiologically relevant concentrations of peroxidases, this study showed that the enzymatic catalytic activities and substrate specificities of each of these enzymes which were shown to be different, resulted in differential responses in the context of osteoblast function. EPO and SBP demonstrated a well-conserved pro-osteogenic capacity to stimulate the biosynthesis of collagen I by primary human osteoblasts and promote mineralisation of the deposited ECM. In contrast, MPO, while it was able to promote ECM deposition, it failed to promote mineralisation and therefore unlikely to contribute to bone formation. The ability of EPO and SBP to stimulate mineralisation by osteoblasts suggests that these enzymes may possess key properties for promoting bone repair. Of the two tested peroxidases however, SBP is more readily available and significantly cheaper than EPO, making it an attractive and realistic candidate for further pre-clinical assessment. Data presented in this thesis demonstrate for the first time the pro-osteogenic ability of SBP, in combination with a commercially available scaffold to significantly accelerate bone repair in an ovine critical-sized defect model. This was confirmed by quantitative micro-CT analysis. Histological assessment showed evidence of intramembranous bone formation and viable osteoblast and osteocyte cell populations, indicative of bone repair and maturation. These results suggest that SBP may be beneficial as a therapeutic agent to accelerate localised repair of damaged bone. The use of rodents over larger animals for different models of bone repair allows for high throughput analyses of multiple variables, such as dose and time. Using wildtype mice, we established a critical size defect model to validate SBP in this species, prior to investigating other models of bone repair. The doses of SBP investigated in this study demonstrated significant inhibition of bone formation with increased fibrous tissue present and an absence of bone remodelling indicators. The results presented in this thesis highlight the importance of further mechanistic investigation, to determine how SBP regulates the remodelling process and the necessity for optimisation before assessing the role of SBP in fracture healing. In conclusion, our findings demonstrate for the first time that peroxidase enzymes likely regulate multiple cellular processes involved in new bone formation, including collagen I biosynthesis, bone matrix mineralisation and osteogenic regulation. Specifically, the plant derived peroxidase, SBP, displays significant pro-osteogenic potential by promoting intramembranous ossification. The studies presented in this thesis provide the first in vivo evidence for peroxidase enzymes as therapeutic agents with the potential to enhance bone repair and, identifies peroxidase inhibitors as a preventative target of pathological ossification