Defying the ‘wettability determinism’: dynamic interfacial evolution directs cell fate on femtosecond laser-textured Ti6Al4V

Highly hydrophobic surfaces, with contact angles approaching or exceeding 150° (the superhydrophobic criterion), are often considered poor at supporting cell adhesion, a view largely derived from wettability theories that did not account for proteins. Although protein adsorption in serum-containing media has been shown to mitigate the adverse effects of high hydrophobicity, it remains unclear whether, among surfaces with comparably high contact angles, the specific microscale topography governs cellular behavior and how the wettability of the cell-conditioned surface evolves with culture time. To address these questions, three femtosecond laser-induced micro-/nanostructures (concentric rings HL1, vertical grooves HL2, and rhombic textures HL3) were fabricated on Ti6Al4V and heat-treated to comparably high contact angles of 142.3° ± 2.5°, 148.7° ± 3.1°, and 141.3° ± 6.1°, respectively. HL2 and HL3 significantly promoted the adhesion, spreading, viability, and proliferation of MC3T3-E1 cells despite the comparably high contact angles, whereas HL1 and the ground control performed poorly. These results indicate that high hydrophobicity per se does not necessarily inhibit cell adhesion and that the microscale topographical feature is a major determinant of cellular behavior under comparable high-contact-angle conditions. Droplet-impact experiments suggested a plausible mechanism: from day 1 to day 7, the cell-conditioned HL2 surface showed markedly increased droplet spreading and reduced rebound, likely attributable to progressive cell adhesion, spreading, and cytoskeletal network maturation. Atomic force microscopy revealed that HL2 and HL3 guided cells into flattened morphologies (thickness 757 ± 491 nm and 591 ± 429 nm) with moderate cytoskeletal stiffness (3.54 ± 3.36 kPa and 3.53 ± 2.26 kPa), whereas HL1 induced excessive polarization and high stiffness (4.39 ± 3.55 kPa), reflecting a distinct mechanical phenotype associated with its elongated, polarized morphology.