Date of Award

Winter 2018

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Steven Kerrigan

Second Supervisor

Professor Fergal O'Brien

Funder/Sponsor

Irish Research Council

Keywords

Osteomyelitis, Staphylococcus Aureus, Tissue Scaffolds, Bone Diseases, Infectious

Abstract

Osteomyelitis is an inflammatory bone disease that causes bone destruction and bone loss. It is a condition that affects both adults and children. There is an increasing incidence of osteomyelitis, which can be in part attributable with the increase of diabetes globally, as osteomyelitis is a common complication of diabetic foot ulcers/ foot infections. The most common microorganism to cause osteomyelitis is Staphylococcus aureus (S. aureus), with this species implicated in up to 80% of cases. Treatment of osteomyelitis heavily relies on systemic and local administration of antibiotics in conjunction with surgical intervention. However this dual treatment strategy is proving inadequate due to the increased resistance to antibiotics in the community and hospitals, and also the impairment to patients as a result of bone debridement. Therefore there is an unmet clinical need to successfully treat the infection and preserve the unaffected bone. Development of novel treatment strategies for osteomyelitis requires an indepth understanding of the disease, from its pathophysiology to the molecular interactions between bacteria and bone cells. Notably, the majority of the knowledge about bacterial and bone interactions was founded using 2 dimensional (2D) traditional tissue culture techniques. These culture conditions however are not very physiologically representative of in vivo, with a clear disconnect between in vitro and in vivo results. The use of animals provide a more physiologically representative environment, however there poses issues with ethics, cost, and translation into humans.

This research project developed a three dimensional (3D) collagen based model of staphylococcal infection that is used to evaluate the importance of the cell wall virulence factor Protein A (SpA) on S. aureus and how it is involved in the pathogenesis of osteomyelitis. This model may be used to study bone infection due to its biomimetic bone microenvironment, with particular focus on the molecular interactions between bacteria and bone cells. This can help advance our knowledge of the area, thus revealing potential novel drug targets in the treatment of osteomyelitis.

We describe for the first time a 3D model of staphylococcal bone infection using our collagen based scaffolds that have been previously used for tissue 19 engineering. We demonstrated that our scaffolds resisted collagen degradation in the presence of both osteoblasts and S. aureus, and retained a structured porous architecture that is necessary for supporting cellular survival and differentiation, similar to that of real bone. Using this model, we assessed the efficacy of two antibiotics commonly used to treat bone infection, gentamicin and vancomycin, against S. aureus in our 3D model compared to 2D. The S. aureus minimum inhibitory concentration (MIC) for vancomycin was higher than in 2D. This result corroborates the literature that vancomycin is a poor penetrator of bone (1), proving to be less effective against S. aureus within a collagen based matrix, resulting in a higher MIC than S. aureus tested using traditional antimicrobial susceptibility methods. Additionally, we have shown that when cultured in 3D, osteoblasts are more metabolically active in the presence of antibiotics compared to 2D, which may affect the pathophysiology of the disease. These results highlight usefulness of using a 3D scaffold for clinical applications.

Using this model, we functionally assessed the effect of S. aureus interactions with osteoblasts and in particular the role of previously identified SpA. The role of SpA in our 3D model was shown to affect osteoblast activity with osteoblasts in the presence of S. aureus expressing SpA on their surface, more metabolically active and producing more mineral than that uninfected osteoblasts. This was not previously seen in our previous 2D studies whereby S. aureus demonstrated a decrease in osteoblast metabolic activity and a decrease in mineral production. Moreover, this increase in mineral production with the onset of infection has been seen in patients with osteomyelitis, supporting our results. These differences between 2D and 3D settings demonstrates the importance of using 3D microenvironment to further represent the in vivo bone mileu.

Significant advances have been made recently identifying the underlying pathophysiology of osteomyelitis. In this body of work, we have demonstrated the potential of using a 3D bone microenvironment to assess the treatment of bone infection clinically and also study the interactions between bacteria and cells. Not only have we optimised a model that is biomimetic to mirror physiological bone ECM, the scaffold can also be used 20 to deliver novel therapeutics locally due to its biocompatible nature. Using these 3D in vitro surrogate models can provide further insight into the pathophysiology and progression of disease, contributing towards developing more tailored and innovative treatment strategies.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2018.

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