Biomineralization-inspired tuning of hydroxyapatite nanostructures: Decoupling topography and material effects on antimicrobial properties

Bacterial infections remain a major complication in orthopedic and maxillofacial surgery, particularly when associated with biomaterial surfaces, where they become exceedingly difficult to eradicate. Inspired by naturally occurring biocidal surface topographies, nanostructured surfaces have emerged as promising strategies for reducing the risk of infection. In this study, we investigated the potential for fine-tuning the nanostructures of calcium-deficient hydroxyapatite (CDHA) by adjusting the reaction conditions during the hydrolysis of a-tricalcium phosphate. By systematically varying synthesis parameters, including temperature, pH, duration, and nucleation seed, we generated a diverse set of nanostructured topographies. From these, three morphologies were selected: interconnected nanosheets, densely packed needles, and nest-like nanowire structures. Analogous nanotopographies were replicated in titanium oxide (TiO2) to isolate topographical from material-specific antimicrobial effects. All nanostructured surfaces exhibited significant bactericidal activity against P. aeruginosa. However, while needle- and nest-like topographies showed comparable efficacy across both materials, the lower elastic modulus of CaP pits resulted in reduced antibacterial performance compared to their Ti counterparts for both P. aeruginosa and S. aureus. Overall, the needle-like topographies demonstrated the highest antibacterial efficacy across all tested surfaces. Against S. aureus, no evidence of cell wall damage was found; however, metabolic assays revealed a significantly reduced activity, especially in needle-like topographies, where a reduction of Colony Forming Units (CFU) was also observed, suggesting a subtler, potentially indirect bactericidal mechanism. Our results demonstrate that a range of mechanobactericidal nanotopographies can be engineered on calcium phosphate surfaces via controlled processing, offering a platform for antibiotic-free infection control in bone grafting applications.