The effects of nanopattern surface technology and targeted metabolic therapies on orthopaedic implant related infections

Hansom, Donald (2017) The effects of nanopattern surface technology and targeted metabolic therapies on orthopaedic implant related infections. MD thesis, University of Glasgow.

Abstract

Bacterial biofilm infections cause significant morbidity in orthopaedic joint replacement. One of the most common bacteria in orthopaedic prosthetic infections is Staphylococcus aureus. Infection causes implant failure due to bacterial adherence and subsequent biofilm production. Nanotopography refers to the topography of a surface at the nanometre level and has major effects on cell behaviour. Studies suggest that surface nanotopography impacts the differential ability of staphylococci species to adhere, and may reduce orthopaedic implant infection rate. This research thesis focuses on bacterial adhesion on nanofabricated materials, and investigates the related metabolic changes and possible interventions.

Staphylococcus aureus growth and quantification methods were optimised, with regard to growth media, incubation time and lysozyme incubation time. Both polystyrene and titanium (Ti) nanosurfaces were studied. Adhesion analysis was performed using fluorescence imaging, quantitative PCR, and bacterial percentage coverage. Metabolomic analysis was conducted by substitution with ‘heavy’ labelled glucose into growth medium, thus allowing for bacterial metabolomic analysis and identification of up-regulated, labelled metabolites and pathways.

Bacterial growth was optimal using DMEM + supplement media, with adhesion occurring after 1hr bacterial incubation. Optimal lysozyme incubation for bacterial quantification using qPCR was 2hr. These parameters were used for all subsequent experimentation. Surface topography affects cell behaviour, bacterial adhesion and long term implant survival can be affected. This study found reduced bacterial adhesion on the SQ and HEX polystyrene patterns. While not found to be significant, this trend was supported by a lower average percentage bacterial coverage on both the SQ and HEX patterns (P=0.05 and P=0.01, respectively). It may be that the SQ and HEX nanopatterns are the optimal nanopit orientation required to prevent bacteria microcolony formation, keeping the bacteria in small, isolated clusters. In addition, this series of investigations showed an increase in bacterial concentrations on both the 2.5Hr and 3Hr treated Ti nanowire discs when compared to the polished Ti control disc, suggesting nanoroughness increases are associated with elevated bacterial adhesion. This theory was further supported by average percentage coverage, being significantly higher on the 2.5Hr and 3Hr treated discs. If, however, a disordered NC Ti nanopattern, hexagonal in nature, is used bacterial adhesion is significantly reduced when compared to a polished, control surface. The bacterial percentage coverage was also noted to be significantly lower on the NC surfaces, with over a 10-fold reduction when compared to the control surface. It is postulated that this reduction is through similar mechanisms to those described by Ivanova et al, and primarily related to altered surface interactions. Metabolomic analysis demonstrated increased intensity counts for key metabolites (pyruvate, aspartate, alanine and carbamoyl aspartate) involved in bacterial aggregation, proteoglycan and DNA synthesis. These pathways are also known to be important in bacterial biofilm production. Therapeutic targeting of these pathways was found to result in significantly reduced bacterial adhesion.

This study shows that by altering nanotopography bacterial adhesion, and therefore, biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. The identification of metabolic pathways involved in adhesion allows for a targeted approach to biofilm eradication in S. aureus. This is of significant benefit to the patient, the surgeon and the NHS, and may well extend far beyond the realms of orthopaedics.

• Item Type: Thesis (MD)
• Qualification Level: Doctoral
• Keywords: Orthopaedeics, total joint replacement infection, nanotopography, bacterial metabolomics, bacterial adhesion.
• Subjects: R Medicine > R Medicine (General); R Medicine > RD Surgery
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
• Supervisor's Name: Ramage, Prof. Gordon, Burgess, Dr. Karl and Millar, Mr. Neal
• Date of Award: 2017
• Depositing User: Mr Donald Hansom
• Unique ID: glathesis:2017-7917
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Feb 2017 08:33
• Last Modified: 23 Feb 2017 11:56
• URI: https://theses.gla.ac.uk/id/eprint/7917