Optimized protocol for collecting root canal biofilms for in vitro studies

Abstract

Abstract Aim The goal of this study was to standardize a new protocol for collecting biofilm from the interior of the root canal system (RCS) for in vivo testing. Methodology: In this study, 44 bovine incisors were used. The samples were divided into three experimental groups: 14 teeth, 12 for counting colony-forming units (CFU), and two samples for scanning electron microscopy (SEM). The first group was used for the biofilm collection protocol proposed here, the second group for the 2nd Biofilm Collection Protocol collection, and the third group for biofilm collection with an absorbent paper tip. Two additional teeth were used as sterilization controls to ensure that the experiments were free of contamination. The coronal region was removed and standardized at 15 mm. They were fitted with a Protaper up to the F5 insert, and the apical foramen was sealed with composite resin. The roots were stabilized with acrylic resin in a 1.5 mL Eppendorf tube. The specimens were sterilized and then inoculated with Enterococcus faecalis NTCT 775 every 24 h for 21 days. Following the period, each group underwent biofilm collection protocols, and CFU and scanning electron microscopy (SEM) data were analyzed. Shapiro–Wilk and one-way ANOVA tests were used to determine statistically significant differences between groups. Results The biofilm collection protocol group had the most CFUs, with extremely high values when compared with the other groups when converted to Log10. The results of the One-Way ANOVA test revealed that the 2nd collection protocol and absorbent paper tip collection groups were statistically similar (p > 0.05), whereas the biofilm collection protocol group was not. Conclusion The biofilm collection protocol proposed in this study was effective at collecting microorganisms from within the RCS. Compared to the biofilm collection protocol with paper cones, the in vivo collection protocol from bovine teeth yielded significantly more CFUs. Thus, the proposed protocol significantly increases the bacterial load of biofilms collected from the RCS sample, bringing the experiments closer to the reality of endodontic infections.