Abstract
This study investigates a novel strategy combining ultrashort pulsed-direct laser interference patterning (USP-DLIP) and chemical etching to create hierarchically micro- and nanorough topographies on zirconia with improved cell-instructive and antibacterial properties. Linear (L3) and grid (G3) micropatterns of 3 μm periodicity were fabricated via USP-DLIP, and subsequently treated with hydrofluoric acid to introduce an additional homogeneous nanotopography across the patterns. The individual and combined effects of micropatterning and etching on biological responses were evaluated using human mesenchymal stem cells (hMSCs) and two bacterial strains (Pseudomonas aeruginosa and Staphylococcus aureus) in mono- and co-culture settings. Micropatterns primarily guided cell morphology, alignment, and migration, while the introduced nanotopography enhanced focal adhesion formation and modulated cell-surface interactions. Antibacterial effects were found to be topography- and species-specific: micropatterns restricted P. aeruginosa colonization through a bacterial confinement mechanism, whereas etching-induced nanotopography effectively reduced S. aureus adhesion by limiting contact points. In co-culture assays, hMSC survival depended on a complex interplay between cell spreading and bacterial retention, dictated by surface features. Notably, chemical etching improved the antibacterial potential of the patterns against S. aureus, but it did not result in a synergistic improvement on cellular responses. Indeed, among all tested surfaces, the non-etched linear pattern (L3) consistently exhibited the most favorable outcomes -enhancing hMSC adhesion, migration, and osteogenic differentiation and mineralization, while reducing bacterial colonization and supporting cell survival under infection-like co-culture conditions.