## 3 Results
### Antibacterial Activity  
The antibacterial performance of the different surfaces was assessed against the Gram-positive bacterium _Staphylococcus aureus_, using a combination of qualitative and quantitative methods. Initially, antibacterial effects were assessed qualitatively through a halo inhibition assay and SEM in SE and BSE modes after 24 h of incubation (Figure 14). _S. aureus_ was selected for this proof-of-concept study due to its clinical relevance as one of the primary pathogens associated with implant-related infections [56]. Its strong ability to adhere to biomaterial surfaces and form biofilms makes it a representative and challenging model organism for evaluating antibacterial performance in titanium-based implant applications. Figure 13: Representative EDS spectra of the HAp coatings after soaking for 15 days in SBF. Although no visible inhibition zones were detected in the halo assay, SEM analysis revealed distinct differences in bacterial colonization across the tested surfaces. The uncoated titanium substrate exhibited extensive bacterial adhesion, indicating no inherent antibacterial activity. The TiO\({}_{2}\) pre-coated surface exhibited a moderate reduction in bacterial density, possibly explained by its photocatalytic activity [57] and the higher surface energy of titanium oxide [58]. According to [PERSON] et al., TiO\({}_{2}\) surfaces exhibit higher surface energy than uncoated titanium controls, which may promote the adsorption of proteins. These adsorbed proteins could subsequently interact with bacterial cell membranes, preventing bacterial attachment to the surface, thereby reducing adhesion and inhibiting biofilm formation [58]. Notably, the HAp-coated surface exhibited a dramatic reduction in bacterial presence, with no _S. aureus_ cells detected, suggesting that the surface possesses significant antibacterial activity. BSE imaging was instrumental in distinguishing bacterial cells from HAp particles. Due to the higher atomic numbers of calcium and phosphorus, HAp regions appeared brighter in BSE mode, whereas bacterial cells--mainly composed of lighter elements such as carbon and nitrogen--appeared darker. This contrast facilitated the accurate interpretation of surface bacterial adhesion and/or colonization. To quantitatively validate these observations, colony-forming unit (CFU) assays were conducted after 24 h of incubation (Figure 15). Figure 14: Top-view SEM micrographs acquired in secondary electron (SE) and backscattered electron (BSE) modes, showing _Staphylococcus aureus_ populations after 24 h of incubation on different surfaces: (**a**,**b**) uncoated substrate, (**c**,**d**) TiO\({}_{2}\) pre-coated surface, and (**e**,**f**) HAp-coated surface. The uncoated substrate exhibited the highest bacterial load, confirming its lack of antibacterial activity. In contrast, the TiO\({}_{2}\) pre-coated surface demonstrated substantial antibacterial efficacy. Although SEM images revealed the presence of bacterial cells on the TiO\({}_{2}\)-coated surface, this observation does not necessarily contradict the high antibacterial rate, as SEM cannot differentiate between metabolically active and inactive bacteria. Remarkably, the HAp-coated surface not only achieved the highest antibacterial efficacy (-98.8% CFU reduction) but also showed no detectable bacterial cells in SEM micrographs, indicating a strong correlation between the structural evidence and the viability-based quantitative data. As discussed above, the antibacterial effect of the TiO\({}_{2}\) coating is primarily attributed to its photocatalytic activity and increased surface energy. However, the superior and potentially synergistic antibacterial performance of the HAp-coated surface suggests the involvement of additional mechanisms. In this study, HAp was synthesized using a green, bio-inspired method with _Hylocreus undatus_ (dragon fruit) extract as the natural reaction medium.