Bacterial surface colonisation has significant implications for the spread of infections in healthcare, industrial, and agricultural settings, often causing serious health problems and economic losses. Despite the widespread use of chemical-based antimicrobial surface coatings, such as antibiotics, metal derivatives, and polyammonium salts, bacterial infections continue to rise. These chemistry-based strategies face significant limitations, particularly the escalating problem of antimicrobial resistance and the ability of bacteria to form resilient biofilms that diminish the effectiveness of chemical agents. Concerns about potential cytotoxicity and environmental leaching of chemicals further challenge the long-term viability of such approaches. Consequently, there is an urgent need for alternative, chemistry-independent antibacterial strategies. Structural features found on the surfaces of certain living organisms, such as shark skin, plant leaves, and insect wings, exhibit antibiofouling, self-cleaning, and bactericidal activities due to the presence of micro- and nanostructured topographies, which provide mechano-antibacterial action through the physical deterrence or damage of surface-adherent bacterial cells. However, the specific contribution of individual geometrical factors, including height, shape, spacing, aspect ratio, and pattern arrangement, to mechano-antibacterial activity remains unclear, as natural surfaces exhibit inherent structural variability that prevents independent control of these parameters. Similarly, the biomimetic surfaces produced using conventional fabrication techniques lack the precision, uniformity and dimensional control needed to isolate and systematically investigate how these geometrical parameters influence antibacterial activity. Additive manufacturing has made significant progress in recent years by enabling the precise and efficient production of intricate geometries that are difficult to achieve with conventional manufacturing methods. Specifically, the two-photon polymerisation (2PP) additive manufacturing technique stands out for its ability to fabricate arbitrarily complex three-dimensional structures with sub-micrometre resolution. In this work, the 2PP technique was optimised through systematic tuning of key printing parameters, including the laser power, exposure time, hatching and slicing distances, point distances, and interface settings, to precisely produce hexagonally arranged micro- and nanostructures with controlled variations in height, spacing, and wall surface texture. These refinements ensured consistent feature fidelity across all printed designs, enabling systematic investigation of the influence of structural geometry on antibacterial responses. The bactericidal properties of the nanopillars and the antibiofouling properties of the micropillars were evaluated against pathogenic Pseudomonas aeruginosa. The fabricated nanopillars exhibited spacing-dependent bactericidal activity, causing bacterial membrane deformation and rupture. Those with 800 nm centre-to-centre spacing exhibited the highest percentage (57.5 ± 8.5%) of surface-adherent dead cell count. Whole transcriptome analysis showed activation of stress-related signalling PA3305.1 pathways in bacteria exposed to the nanopillared surface. Despite showing pronounced bactericidal activity, the total number of adherent cells (both live and dead) was also noticeably high on this surface, suggesting that dead cell remnants may facilitate the attachment of incoming cells, promote biofilm development and compromise the nanopillars’ long-term bactericidal efficacy under dense bacterial colonisation. Micropillars (with 5 µm centre-to-centre spacings), in contrast, significantly reduced biofilm formation by up to 61.2 ± 6.86 % in comparison to the flat surface. However, some cells managed to evade the micropillar barrier and settled around the bases of the pillars, where they remained intact with no visible cell deformation as observed in the scanning electron micrograph. These hexagonally arranged nano- and micropillars primarily induced a spacing-dependent response in pathogenic Pseudomonas aeruginosa, affecting cell viability, adhesion and biofilm formation. However, each surface scale poses its own limitations. Therefore, the nanopillars were integrated into a micropillar design to create dual-scale topographies, aiming to achieve a complementary combination of bactericidal and antibiofouling properties that reduce surface-adherent cells and prevent colonised cells from proliferating and maturing into biofilms. Biological analysis through crystal violet biofilm assay, morphological, and cell viability analysis of the dual-scale topographies demonstrated notable improvements in antibacterial performance, with a significantly higher reduction (73.5 ± 3.24%) in biofilm and 4.5-fold lower cell density on the surface compared to the single-scale microtopographies. The proportion of membrane-damaged cells was substantially higher on the dual-scale surface compared to the flat surface. The observed phenomenon is likely due to the complementary effect of the underlying nanopillars, which damage cells that manage to evade the initial micropillar barrier and hinder the proliferation of adherent cells. Overall, this work establishes 2PP as a versatile and precise fabrication technique for creating next-generation antibacterial surfaces with tunable micro- and nanoscale geometries that inhibit bacterial proliferation and subsequent biofilm formation without relying on chemical agents. These findings contribute to the ongoing efforts in combating antimicrobial resistance and promote global health, offering a promising strategy for implementation in biomedical devices and other high-touch surfaces to impede bacterial colonisation and minimise the risk of bacterial infections.
Tan Ning (Sat,) studied this question.