Evaluation of biofilm disruption on root dentin following conventional and nanoparticle-enhanced photodynamic therapies using scanning electron microscopy and FIJI-based image analysis

Enterococcus faecalis biofilm is a major contributor to persistent root canal infections due to its high resistance to conventional irrigants. This study aimed to evaluate and compare different disinfection protocols, including sodium hypochlorite (NaOCl), photodynamic therapy (PDT) with methylene blue (MB), and MB functionalized with reduced graphene oxide (MB-rGO) nanoparticles with or without sonic activation, in disrupting E. faecalis biofilm on root canal dentin using SEM and quantitative FIJI-based image analysis. Eighty root dentin specimens were obtained from forty extracted single-rooted human teeth. Following canal preparation and instrumentation, thirty-five teeth were inoculated with Enterococcus faecalis and incubated for 21 days to develop mature biofilms, while five teeth served as negative controls. Each tooth was longitudinally sectioned to yield two standardized dentin specimens, which were randomly allocated to six experimental groups (n = 10 specimens per group) according to the disinfection protocol. Groups 1–2 received 5% sodium hypochlorite (NaOCl), Groups 3–4 underwent photodynamic therapy (PDT) using methylene blue (MB), and Groups 5–6 received PDT with methylene blue functionalized with reduced graphene oxide (MB-rGO). Sonic activation was applied in Groups 2, 4, and 6. Biofilm removal was assessed using scanning electron microscopy (SEM) and quantified with FIJI software employing the Trainable Weka Segmentation plugin. A 6-point visual scoring scale was additionally applied. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test and the Wilcoxon signed-rank test (p < 0.05). The positive control showed extensive biofilm coverage (73.36 ± 6.36%). NaOCl-treated groups showed moderate reduction (Group 1: 35.57 ± 3.11%; Group 2: 31.67 ± 2.09%). PDT with MB exhibited greater reduction (Group 3: 21.68 ± 2.65%; Group 4: 19.11 ± 1.78%). The MB-rGO groups presented the highest efficacy (Group 5: 17.71 ± 2.66%; Group 6: 10.46 ± 1.92%), with Group 6 significantly outperforming all others (p < 0.001). Semi-quantitative scores reflected similar trends, with Group 6 showing the lowest score (1.20 ± 0.63, p < 0.001). MB-rGO combined with sonic and laser activation demonstrated the highest efficacy against E. faecalis biofilm, indicating a promising approach for enhanced endodontic disinfection.