Comparison of the antimicrobial efficacy of contemporary intracanal medicaments and hydrogels used in regenerative endodontic treatment against Enterococcus faecalis and Candida albicans

The aim of this in vitro study was to compare the antimicrobial efficacy of various intracanal medicaments against dual-species biofilms formed by Enterococcus faecalis and Candida albicans in immature teeth within the context of regenerative endodontic treatment (RET). For this purpose, the following medicaments were evaluated: calcium hydroxide (Ca(OH)₂), 2% chlorhexidine (CHX) gel, double antibiotic paste (DAP), chitosan hydrogel, chitosan-diclofenac hydrogel, CASA hydrogel, and chitosan-chlorhexidine hydrogel. Using infected root canal samples, the XTT assay was performed to comprehensively assess the antimicrobial effects of these medicaments both on microbial growth capacity and metabolic activity, thereby enabling simultaneous evaluation of both microorganisms. Additionally, another aim of the study was to investigate potential differences between the paper point method and the dentin chip method, which are commonly used sampling techniques during antimicrobial efficacy assessment. A total of 270 single-rooted human teeth were used in this study. Following root canal preparation, the teeth were infected with standardized suspensions of E. faecalis and C. albicans for three weeks. At the end of the infection period, the intracanal medicaments assigned to the experimental groups were applied, and the teeth were incubated for 7 days. Microbial sampling was performed using two different methods, with 15 teeth from each group. In the paper point method, sterile #25 paper points were inserted into the root canals and then transferred into tubes containing phosphate-buffered saline (PBS). In the dentin chip method, dentin shavings were collected with Gates Glidden burs and transferred into PBS-containing tubes. In both methods, the samples were vortexed, and 100 µl aliquots were serially diluted and plated on bile esculin agar for E. faecalis and Sabouraud dextrose agar for C. albicans. The plates were incubated for 48 hours, after which colony-forming units were counted and converted to log₁₀ values. To provide a more comprehensive evaluation of antimicrobial activity, the XTT assay was also performed on root canal samples, and absorbance was measured at 450 nm. Since normality could not be achieved, non parametric tests (Mann-Whitney U, Kruskal-Wallis H, Dwass-Steel-Critchlow-Fligner) were applied. According to the findings, significant differences in antimicrobial efficacy were observed among the medicaments. CASA hydrogel and chitosan chlorhexidine hydrogel completely eradicated the biofilms of both E. faecalis and C. albicans. Chitosandiclofenac hydrogel showed high efficacy against E. faecalis, but relatively lower efficacy against C. albicans. Chlorhexidine gel reduced the biofilm biomass, while calcium hydroxide exhibited only limited antimicrobial activity. Double antibiotic paste demonstrated pronounced effects on E. faecalis, but weaker effects on C. albicans. Chitosan hydrogel showed generally low antimicrobial efficacy. No significant differences were found between the sampling methods Keywords: regenerative endodontics, biofilm, intracanal medicaments, hydrogel, XTT