Stage-Dependent Antibiofilm Effects of UVA Combined with Cinnamaldehyde Against Staphylococcus aureus Biofilms on Titanium Surfaces

Staphylococcus aureus biofilms formed on titanium surfaces are highly relevant to orthopedic implant-associated infection and remain difficult to control after maturation. This study aimed to evaluate whether ultraviolet A (UVA, 365 nm) combined with cinnamaldehyde (CA) could improve antibiofilm activity against titanium-associated S. aureus biofilms in a stage-resolved in vitro model and to examine whether the observed responses were associated with reactive oxygen species (ROS). Early stage (8 h) and 24 h biofilm models were established on total hip arthroplasty (THA)-derived titanium discs. After condition screening, 0.5 mM CA combined with 5 min UVA exposure was selected for subsequent experiments. Biofilm biomass was assessed by crystal violet staining, bacterial viability by live/dead staining and colony-forming unit (CFU) enumeration, ROS-associated fluorescence by dihydroethidium (DHE) imaging, and biofilm-associated gene expression by quantitative real-time PCR (qRT-PCR). Chondrocyte viability was also evaluated under the selected antibiofilm-effective conditions. The combined treatment showed stage-dependent antibiofilm effects, with greater biomass reduction in the 8 h biofilm model and marked impairment of bacterial viability and culturability in both models. ROS-associated fluorescence increased under combined exposure and was partially attenuated by N-acetyl-L-cysteine (NAC) in the 24 h biofilm model. In parallel, CA + UVA was associated with lower expression levels of clfA, icaA, and icaD in the 8 h biofilm model and of icaA, icaB, and icaD in the 24 h biofilm model, with partial NAC attenuation in the latter. Chondrocyte viability was lower in all treatment groups than in the untreated control, although the combined treatment did not show an obvious additional decrease compared with the single-treatment groups. These findings indicate that UVA combined with CA exerts stage-dependent antibiofilm effects in an in vitro titanium-associated S. aureus biofilm model. The observed ROS-associated responses were consistent with, but do not establish, mechanistic involvement. The current treatment setting also requires further optimization before translational applicability can be more confidently considered.