Periapical lesions and endodontic treatment failures are frequently attributed to persistent microbial infections, with Enterococcus faecalis emerging as a predominant pathogen due to its ability to colonize dentinal tubules and form resilient biofilms.^[1] Conventional disinfection relies on sodium hypochlorite (NaOCl), yet its cytotoxicity, limited penetration into anatomical complexities, and incomplete biofilm eradication underscore the need for safer, more effective adjunctive therapies.^[2]

Photodynamic therapy (PDT) presents a promising solution by combining light-sensitive agents (photosensitizers; PS) with laser irradiation to generate reactive oxygen species (ROS), selectively targeting pathogens without thermal damage.^[3-5] Unlike traditional methods, PDT’s mechanism_oxidative destruction of bacterial membranes, proteins, and DNA_addresses biofilm resistance while preserving host tissues, making it ideal for intricate root canal systems.^[6,7]

Recent advancements highlight near-infrared (NIR) lasers (e.g. 810 nm) for deeper tissue penetration and reduced scattering. Indocyanine green (ICG), an FDA-approved NIR-absorbing dye, excels in ROS generation and biocompatibility, whereas methylene blue (MB), a phenothiazine dye, offers broad-spectrum antimicrobial activity.^[8,9] Cetrimide (CT), a quaternary ammonium compound, enhances PDT efficacy through surfactant-mediated biofilm disruption and intrinsic antibacterial properties.^[10]

Despite promising findings, comparative studies on PS efficacy in endodontic PDT remain limited. Existing research predominantly focuses on visible-light-activated agents, with sparse data on NIR-compatible PS like ICG. Furthermore, the synergistic potential of antimicrobial surfactants like CT in PDT protocols warrants exploration to optimize bacterial eradication.

This in vitro study evaluates the antimicrobial efficacy of PDT using ICG, MB, and CT activated by an 810 nm diode laser against E. faecalis in infected root canals. By addressing gaps in PS selection and laser parameters, this work aims to advance PDT’s clinical translation as a standardized adjunct in endodontic disinfection.

All experimental groups showed significant reductions in bacterial load compared to the untreated control group (P < 0.05) [Figure 1]. In untreated canals (control), a robust biofilm was established, with a mean bacterial load of 6.59 ± 0.08 log₁₀ CFU/mL, confirming the persistence of infection without intervention. In contrast, irradiation with the 810 nm diode laser alone reduced the CFU count to 5.94 ± 0.07 log₁₀ CFU/mL, demonstrating the intrinsic antibacterial activity of the laser.

PDT further enhanced bacterial eradication. Canals treated with CT-mediated PDT exhibited a reduction to 5.55 ± 0.10 log₁₀ CFU/mL, significantly lower than both the control and laser-alone groups. MB-PDT achieved an even more marked reduction, with bacterial counts dropping to 5.08 ± 0.20 log₁₀ CFU/mL, thereby outperforming CT-mediated PDT. Remarkably, ICG-activated PDT achieved complete eradication of E. faecalis, with no detectable CFUs observed posttreatment, highlighting its superior efficacy.

Statistical analysis supported these observations. Shapiro–Wilk testing confirmed the normal distribution of the data (P > 0.05), and one-way ANOVA revealed significant differences among the groups (F = 1204.6, P < 0.001). Tukey’s post hoc analysis further delineated these differences, showing that all treatment groups differed significantly from the control (P < 0.001). Comparisons between the laser-alone group and the PDT groups yielded statistically significant differences, with PDT + CT (P = 0.003), PDT + MB (P < 0.001), and PDT + ICG (P < 0.001) all showing superior performance. In addition, the PDT + ICG group differed significantly from both the PDT + MB and PDT + CT groups (P < 0.001), underscoring the enhanced bactericidal potential of ICG-activated PDT.

The findings of this study underscore the exceptional efficacy of ICG-mediated PDT in eradicating E. faecalis from infected root canals, achieving complete bacterial elimination (0 CFU/mL). This aligns with its recognized role as a resilient pathogen in chronic endodontic infections, often evading conventional disinfection due to its biofilm-forming ability and dentinal tubule colonization.^[11] The superiority of ICG-PDT over the MB and CT groups highlights the critical influence of PS properties and laser parameters. ICG’s NIR absorption (810 nm) enables deeper tissue penetration (4–6 mm) compared to MB’s visible-light activation (665 nm), effectively targeting bacteria in anatomically complex regions, such as isthmuses and apical deltas.^[8,12]

CT’s dual role as a surfactant and antimicrobial agent likely enhanced PDT efficacy by disrupting biofilm matrices and increasing bacterial membrane permeability, consistent with prior studies demonstrating its substantivity and biofilm clearance.^[13] However, its lower reduction (5.55 ± 0.10 log~10~CFU/mL) compared to ICG emphasizes the need for PS-specific optimization. Importantly, PDT’s safety advantage over traditional laser protocols _avoiding thermal risks such as dentin carbonization or root resorption_ was evident. While diode lasers alone (5.94 ± 0.07 log~10~CFU/mL) can generate cytotoxic heat, PDT’s oscillatory fiber motion and intermittent irradiation (three 20-s cycles) mitigated temperature rises, as validated by Alfredo et al.^[14] Furthermore, cytotoxicity studies corroborate PDT’s biocompatibility: Kashef et al. reported no fibroblast toxicity, whereas George and Kishen noted 97.7% bacterial kill versus only 30% fibroblast damage, contrasting sharply with NaOCl’s neurotoxic risks.^[15,16]

Despite robust outcomes, discrepancies with earlier studies warrant consideration. For instance, Souza et al. observed statistically insignificant PDT effects, potentially due to inadequate oxygen levels in root canals or suboptimal PS diffusion.^[17] This study’s rigorous PS incubation times (15 min for ICG) and standardized laser parameters (0.5 W, 143 J/cm²) may explain the superior results, aligning with protocols by Garcez et al. and Foschi et al.^[6,18]

Future research should prioritize clinical validation through long-term trials to assess PDT’s durability in vivo, such as the 6-month follow-up protocol by Garcez et al.,^[18] which could confirm therapeutic consistency in dynamic clinical environments. Combination therapies integrating ICG-PDT with conventional agents, such as EDTA or NaOCl, warrant exploration to dismantle residual biofilm matrices and enhance synergistic antimicrobial outcomes. Finally, cost-effectiveness analyses are essential to evaluate PDT’s economic viability, particularly in resource-limited settings, ensuring equitable access to this advanced disinfection modality without compromising clinical standards. These steps will bridge translational gaps, optimizing PDT for widespread adoption in endodontic practice.

While promising, this in vitro model simplifies clinical realities. Natural infections involve polymicrobial consortia, and variations in root canal anatomy (e.g. curvature, accessory canals) may alter PDT efficacy.^[19] In addition, the single-strain focus on E. faecalis excludes interactions with fungi or resistant Gram-negative species.

ICG-PDT emerges as a paradigm shift in endodontic disinfection, combining unparalleled antimicrobial efficacy with minimal cytotoxicity. By addressing anatomical and microbial complexities in future studies, this modality could bridge the gap between laboratory success and clinical adoption, revolutionizing standards in root canal therapy.