Surface Functionalization of Ti6Al4V-ELI Alloy with Antimicrobial Peptide Nisin

Abstract

Implant-associated infections are a severe global concern, especially in the case of orthopedic implants intended for long-term or permanent use. The traditional treatment through systemic antibiotic administration is often inefficient due to biofilm formation, and concerns regarding the development of highly resistant bacteria. Therefore, there is an unfulfilled need for antibiotic-free alternatives that could simultaneously support bone regeneration and prevent bacterial infection. This study aimed to perform, optimize, and characterize the surface functionalization of Ti6Al4V-ELI discs by an FDA-approved antimicrobial peptide, nisin, known to hold a broad antibacterial spectrum. Accordingly, nisin bioactivity was also evaluated by in vitro release tests both in physiological and inflammatory pH conditions. Several methods, such as X-ray photoelectron spectroscopy (XPS), and Kelvin Probe atomic force microscopy confirmed the presence of a physisorbed nisin layer on the alloy surface. The functionalization performed at pH 6–7 was found to be especially effective due to the nisin configuration exposing its hydrophobic tail outwards, which is also responsible for its antimicrobial action. In addition, the first evidence of gradual nisin release both in physiological and inflammatory conditions was obtained: the static contact angle becomes half of the starting one after 7 days of soaking on the functionalized sample, while it becomes 0° on the control samples. Finally, the evaluation of the antibacterial performance toward the pathogen Staphylococcus aureus after 24 h of inoculation showed the ability of nisin adsorbed at pH 6 to prevent bacterial microfouling into biofilm-like aggregates in comparison with the uncoated specimens: viable bacterial colonies showed a reduction of about 40% with respect to the un-functionalized surface and the formation of (microcolonies (biofilm-like aggregates) is strongly affected.

Functionalization of a chemically treated Ti6Al4V-ELI alloy with nisin for antibacterial purposes

“…It can be assumed that the release is almost complete at this stage. This behavior is significantly different from that of polished and functionalized Ti6Al4V-ELI alloy as reported in [28] where the change in contact angle after the release test, compared to the functionalized surface, was lower in all cases. It can be concluded that the CT surface is able to release a larger amount of grafted nisin than the polished one; a role of physisorption instead of chemisorption in the different release can be supposed.…”

“…As last, the presence of calcium ions on the functionalized surfaces is in line with previous observations [31] and suggests that it has a role in the functionalization mechanism as a linker between the negatively charged functional groups of CT and nisin. This is different from what observed after functionalization with nisin of mechanical polished Ti6Al4V-ELI where the substrate has not such a high density of deprotonated functional groups and physisorption instead of chemisorption occurred [28].…”

“…MP is reported as a control. compared to the CT Nisin6 specimens' results to confirm that the grafted nisin did not reduce its bioactivity due to the pH variation as we previously demonstrated by comparing MP and MP nisin-doped specimens where the latter showed a promising antifouling activity by preventing the formation of 3D biofilm-like structures [28].…”

“…The authors have previously investigated nisin physisorption on the polished titanium alloy Ti6Al4V with Extra Low Interstitial content (Ti64ELI) in [28], as it was considered an interesting substrate for musculoskeletal application. The low content of interstitials (0.12 wt% of oxygen content against 0.20 wt% characterizing the more common Ti6Al4V) increases toughness, resistance to corrosion, and crack propagation [29].…”

“…The low content of interstitials (0.12 wt% of oxygen content against 0.20 wt% characterizing the more common Ti6Al4V) increases toughness, resistance to corrosion, and crack propagation [29]. Nisin was physisorbed on the polished substrate and exercised an activity of inhibition of the formation of bacteria microcolonies (biofilm-like aggregates) [28]. However, because of inert surface, no bioactivity was observed inducing any osteoconductive effect.…”