Investigation of bacterial attachment patterns on micro-and nano-restricted surface topographies

The use of nanotechnology in the design and fabrication of nanomaterials is rapidly increasing, particularly in commercial applications that span electronics, renewable energy, cosmetics, automotive and medical products. The use of metallic implants is increasing and diversifying; as is research into the use of titanium that has undergone bioactive surface modification to increase biocompatibility and eliminate bacterial biofilm formation. Biofilm formation by human pathogenic bacteria on medical implants can be problematic, most often leading to failure of the device and requiring its surgical removal from the patient. Biofilms can be associated with systemic infection, loss of limb or organ function, amputation or death. Therefore it is critical to identify ways by which implant surfaces can be improved so that they can modulate the degree of bacterial attachment that takes place on their surfaces. In this study, the modification of titanium surfaces was achieved using two different approaches, namely the alteration of the surface architecture of the bulk titanium, and by coating substrate surfaces with thin films of titanium. For bulk titanium materials, equal channel angular pressing (ECAP) and femtosecond laser ablation were employed to alter the surface micro-and nanoscopically topographic parameters. A magnetron physical vapour deposition system was employed to fabricate titanium thin films containing particular sub-nanometric surface features. Radio-frequency plasma enhanced chemical vapour deposition (RF PECVD) was also investigated as a method by which titanium surfaces could be improved by coating the titanium surface with thin films of terpinen-4-ol, a constituent of tea-tree oil that has been shown to be anti-microbial. An optimised experimental procedure allowed the application of RF PECVD of terpinen-4-ol such that the resulting film retained the chemical integrity and functional properties of the original terpinen-4-ol. The physical and chemical properties of all types of fabricated or/and modified titanium surfaces were thoroughly characterised using contact angle goniometry, atomic force microscopy (AFM), optical interferometry, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared and Raman spectroscopy. Two medically important bacteria, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus CIP 65.8T, were used to study their attachment and biofilm formation on the fabricated and/or modified surfaces. The results obtained in this study suggest that micro-to sub-nanoscale surface topography plays an important role in modulating the degree of bacterial attachment. On superhydrophobic titanium surfaces fabricated using femtosecond laser ablation, the spherical S. aureus appeared to be able to successfully colonise the surface, whilst the rod-shaped P. aeruginosa cells were not able to do so. It was found that whilst the total surface area of the surface increased as a result of the laser processing, it appeared that not all of this increased area was available for bacterial attachment; in fact it is likely that the increased resistance to bacterial colonisation by P. aeruginosa cells arose from a greatly diminished surface area that was available for cell attachment. The spherical bacteria, on the other hand, appeared to require a much lower degree of surface contact to allow successful attachment. It is proposed that in order to sustain their attachment on nano-structured titanium surfaces, bacteria employ a few different attachment mechanisms which are dependent on the nanoscale of the surface topography. Cell membrane deformability, as a result of the Helfrich’s repulsive force of different cell morphologies, may play a major role during the process of bacterial attachment onto a molecularly smooth surface, where previously proposed mechanisms of attachment, such as through interactions with the flagella and fimbriae, or by the production of extracellular polymeric substances (EPS) appeared not to promote bacterial adhesion.