Butyl toluidine blue as a photosensitizer for antimicrobial photodynamic therapy on titanium-associated biofilms

Abstract

This in vitro study aimed to determine a safe concentration of butyl toluidine blue (BuTB) and an appropriate pre-irradiation time for antimicrobial photodynamic therapy (aPDT) to reduce polymicrobial oral biofilm without causing cytotoxicity to human cells. Titanium (Ti) discs simulating implant abutment surfaces were inoculated with saliva from patients diagnosed with peri-implantitis to create a clinically relevant polymicrobial biofilm model. Antimicrobial PDT was performed using BuTB at 0.05 mg/mL and two pre-irradiation times (1 and 5 min), followed by diode laser irradiation (InGaAlP, 660 nm, 75 mW, 60 s, 4.5 J). The antimicrobial efficacy of each protocol was evaluated, and cytotoxicity was assessed on keratinocyte (HaCaT) and human gingival fibroblast (HGF) cell lines using both quantitative and qualitative approaches. Significant bacterial reduction was observed for both 1-min (8.91 ± 0.5 log₁₀ CFU/mL; p < 0.0001) and 5-min (7.75 ± 0.6 log₁₀ CFU/mL; p < 0.0001) pre-irradiation times, compared to untreated Ti controls (12.21 ± 0.1 log₁₀ CFU/mL). Although the 5-min protocol showed slightly greater antimicrobial activity (p = 0.0076), the 1-min protocol demonstrated favorable cell viability and mitochondrial activity in both HaCaT and HGF cells. These results suggest that BuTB-mediated aPDT, at a low concentration and short pre-irradiation time, can effectively reduce polymicrobial biofilm while preserving cell viability in the conditions studied. While further studies are needed, these in vitro findings provide a valuable foundation for developing safer and more effective aPDT protocols for potential future clinical applications.