Multispecies Biofilms Treated With Endodontic Sealers or Calcium Hydroxide: Antimicrobial Activity and Changes in Community Composition

Abstract

Introduction: To evaluate the antimicrobial activity and changes in community composition in biofilms treated with two root canal sealers or calcium hydroxide.

Material and methods: Forty-nine extracted bovine dentine cylinders were inoculated with subgingival dental plaque for 2 weeks in a CDC biofilm reactor. Three treatment groups were assigned: AH Plus, BC Sealer, and calcium hydroxide. Propylene glycol inert vehicle (PG) and untreated contaminated samples were used as controls. The infected root canal space was in direct contact with the materials for 7 days under anaerobic incubation. Anaerobic culture (colony forming units, CFU), quantification PCR (qPCR), and next generation sequencing were used to assess the effect of each material. Differences in the number of molecules (qPCR), CFU, and abundances of genera were evaluated using the Kruskal-Wallis test. The Shannon and Chao1 indices were used to measure alpha diversity. Differences in community composition were evaluated using non-parametric analysis of similarity (ANOSIM).

Results: The CFU, Shannon, and Chao1 indices revealed significant differences between BC Sealer, AH Plus, and calcium hydroxide groups versus the untreated control group and PG (p < 0.003). Calcium hydroxide and BC Sealer significantly reduced the qPCR values compared to the control group and PG. The biofilm composition (98.5%) was primarily composed of Peptostreptococcus, Streptococcus, Parvimonas, Fusobacterium, Veillonela, Mogibacterium, Lancefieldella, Eubacterium, Slackia, and Prevotella. Significant differences in overall community composition and beta diversity between untreated controls and AH Plus were observed (ANOSIM R = 0.674, p < 0.001). Parvimonas, Streptococcus, Eubacterium, and Lancefieldella were not affected by any of the materials tested.

Conclusion: Calcium hydroxide and BC Sealer significantly reduced the viability and the total number of DNA copies. AH Plus sealer reduced the bacterial viability but did not affect the DNA concentration. AH Plus significantly alters overall biofilm community composition compared to other groups. None of the materials tested eliminated the multispecies biofilm completely.

Keywords: endodontic sealers; intracanal medication; microbiome; next generation sequencing.

FIGURE 1

SEM images of biofilms of the immediate control. Images on top and centre were obtained at 3500 and 5000× magnification, and the image at the bottom is at 2000× magnification.

FIGURE 2

(A) Kruskal–Wallis analysis comparing culture and qPCR results among the treatment and control groups on a log₁₀ scale. Statistically significant differences are noted by the asterisks. PG (Inert vehicle), A (AH Plus), B (BC Sealer), C (Calcium Hydroxide), Control (untreated). (B) Principal coordinate analysis of Bray–Curtis dissimilarity matrices. Differences between untreated controls and AH Plus (ANOSIM R = 0.674, p < 0.001, Bonferroni‐corrected ɑ = 0.003). Vehicle (PG), control (untreated) (C). Distribution of abundant genera among groups. Genera with a mean abundance < 1.5% among all groups were consolidated. Vehicle = propylene glycol.