Mechanochemical Synthesis of a TiO₂-Containing Biogenic Hydroxyapatite Ceramic Composite: Balancing Antibiofilm...

Implant-associated infections remain a major challenge in bone-related biomedical applications, where bacterial colonization and biofilm formation can compromise tissue integration and clinical performance. This study reports the mechanochemical synthesis, physicochemical characterization, antimicrobial activity, antibiofilm performance, and short-term fibroblast cytocompatibility of a TiO₂-containing bovine-derived biogenic hydroxyapatite ceramic composite (BHAp-TiO₂). The composite was prepared by high-energy mechanical milling using 10 wt% TiO₂ and characterized by X-ray diffraction, Rietveld refinement, Raman spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. XRD/Rietveld analysis identified a multiphase ceramic composite composed of hydroxyapatite, whitlockite, and rutile TiO₂, with no evidence of Ti⁴⁺ substitution into the hydroxyapatite lattice or detectable anatase within the XRD/Rietveld detection limit. SEM-EDS confirmed the granular agglomerated morphology of the powders and the elemental presence of Ti in BHAp-TiO₂. Compared with pristine BHAp, BHAp-TiO₂ produced a concentration-dependent reduction in AlamarBlue^®-derived bacterial metabolic activity against five clinically relevant planktonic strains. At 200 µg/mL, residual metabolic activity decreased to 10.90-32.90%, depending on the bacterial species, with the strongest response observed for Escherichia coli. In crystal violet assays, BHAp-TiO₂ markedly inhibited Pseudomonas aeruginosa biofilm biomass, reaching 91.9 ± 3.4% inhibition at 200 µg/mL. In NIH/3T3 fibroblasts, BHAp-TiO₂ preserved short-term cytocompatibility after 24 h of direct exposure within the 0.1-100 µg/mL range, with MTT- and AlamarBlue^®-derived responses remaining close to or above the 80% cytotoxicity limit. Overall, BHAp-TiO₂ is best interpreted as a rutile TiO₂-containing biogenic calcium phosphate ceramic composite with enhanced antimicrobial and antibiofilm performance while maintaining short-term fibroblast cytocompatibility under the evaluated conditions.