AimTo investigate the dynamic recovery of biofilms within dentinal tubules after primary irrigation with different protocols, and to evaluate the efficacy of various re‐irrigation protocols on recovered biofilm, considering factors such as smear layer, nutrient conditions, and primary irrigants.MethodologyA total of 416 mono or multi‐species biofilms samples were prepared from human teeth and incubated for 3 weeks. After inducing a smear layer on half of the samples, all specimens were irrigated with one of the following irrigant sequences: (1) 6% NaOCl +17% EDTA, (2) 6% NaOCl +8.5% EDTA, (3) 6% NaOCl and (8.5% EDTA +10 μg/mL DJK‐5 antimicrobial peptide), or (4) sterile water. Thirty‐two samples were used to assess immediate effect, whilst the rest were re‐incubated to assess biofilms recovery. Nutrient conditions were defined based on whether culture media were changed (nutrient‐rich) or not (nutrient‐poor) during re‐incubation. After 16 weeks, recovered biofilms underwent re‐irrigation using four additional protocols, with or without DJK‐5 peptide, based on primary irrigants. Confocal laser scanning microscopy was employed to evaluate immediate irrigant effects, biofilms recovery intervals (1, 3, 5, 8, 12, and 16 weeks after primary irrigation), and re‐irrigation effects at the 16‐week. Statistical analysis included one‐way anova and two‐way mixed anova tests.ResultsThe DJK‐5 peptide irrigation protocols demonstrated the highest killing rates during primary irrigation and resulted in a longer biofilms recovery time of 16 weeks compared to non‐peptide protocols (p < .001). Both primary irrigation type and smear layer presence significantly influenced biofilms recovery (p < .001). In the absence of smear layer, re‐irrigation efficacy didn't significantly differ from primary irrigation, regardless of primary irrigation type or nutrient conditions. However, with a smear layer present, re‐irrigation led to significantly higher proportion of dead bacteria compared to primary irrigation (p < .05). Inclusion of the DJK‐5 peptide into the re‐irrigation protocol displayed superior killing rate compared to other protocols (p < .001).ConclusionsBiofilms exhibited susceptibility to both peptide and non‐peptide protocols during re‐irrigation, irrespective of nutrient conditions or primary irrigation protocols. The DJK‐5 peptide irrigation protocols consistently displayed superior effectiveness compared to non‐peptide protocols.
