This study examines bacterial adhesion on titanium-substrates used for bone implants. Adhesion is the most critical phase of bacterial colonization on medical devices. The surface of titanium was modified by hydrothermal treatment (HT) to synthesize nanostructured TiO2-anatase coatings, which were previously proven to improve corrosion resistance, affect the plasma protein adsorption, and enhance osteogenesis. The affinity of the anatase coatings toward bacterial attachment was studied by using a green fluorescent protein-expressing Escherichia coli (gfp-E. coli) strain in connection with surface photoactivation by UV irradiation. We also analyzed the effects of surface topography, roughness, charge, and wettability. The results suggested the dominant effects of the macroscopic surface topography, as well as microasperity at the surface roughness scale, which were produced during titanium machining, HT treatment, or both. Macroscopic grooves provided a preferential site for bacteria deposit within the valleys, while the microscopic roughness of the valleys determined the actual interaction surface between bacterium and substrate, resulting in an "interlocking" effect and undesired high bacterial adhesion on nontreated titanium. In the case of TiO2-coated samples, the nanocrystals reduced the width between the microasperities and thus added nanoroughness features. These factors decreased the contact area between the bacterium and the coating, with consequent lower bacterial adhesion (up to 50% less) in comparison to the nontreated titanium. On the other hand, the pronounced hydrophilicity of one of the HT-coated discs after pre-irradiation seemed to enhance the attachment of bacteria, although the increase was not statistically significant (p > 0.05). This observation may be explained by the acquired similar degree of wetting between gfp-E. coli and the coating. No correlation was found between the bacterial adhesion and the ζ-values of the samples in PBS, so the effect of surface charge was considered negligible in this study.

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Surface charge is one of the most significant properties for the characterisation of a biomaterial, being a key parameter in the interaction of the body implant with the surrounding living tissues. The present study concerns the systematic assessment of the surface charge of electrochemically anodized TiO2 nanotubular surfaces, proposed as coating material for Ti body implants. Biologically relevant electrolytes (NaCl, PBS, cell medium) were chosen to simulate the physiological conditions. The measurements were accomplished as titration curves at low electrolytic concentration (10−3 M) and as single points at fixed pH but at various electrolytic concentrations (up to 0.1 M). The results showed that all the surfaces were negatively charged at physiological pH. However, the zeta potential values were dependent on the electrolytic conditions (electrolyte ion concentration, multivalence of the electrolyte ions, etc.) and on the surface characteristics (nanotubes top diameter, average porosity, exposed surface area, wettability, affinity to specific ions, etc.). Accordingly, various explanations were proposed to support the different experimental data among the surfaces. Theoretical model of electric double layer which takes into account the asymmetric finite size of ions in electrolyte and orientational ordering of water dipoles was modified according to our specific system in order to interpret the experimental data. Experimental results were in agreement with the theoretical predictions. Overall, our results contribute to enrich the state-of-art on the characterisation of nanostructured implant surfaces at the bio-interface, especially in case of topographically porous and rough surfaces.

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