In vitro physicochemical characterization of five root canal sealers and their influence on an ex vivo oral multi-species biofilm community

Abstract

Aim: To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque-derived polymicrobial community.

Methodology: Dental plaque-derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio-C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy-dispersive spectroscopy. Data normality was assessed using the Shapiro-Wilk test. One-way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two-way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray-Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05).

Results: The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity.

Conclusion: Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

Keywords: antimicrobial; bacteria; biofilm; endodontic infection; physicochemical properties; root canal sealers.

FIGURE 1

Representative backscatter scanning electron micrographs showing root canal sealers' surfaces microstructure at 1000× magnification. All sealers present a regular and compact matrix embedding different‐sized particles. (a) AH Plus; (b) GuttaFlow Bioseal; (c) Endoseal MTA; (d) Bio‐C Sealer; and (e) BioRoot RCS. pH analysis revealed that calcium silicate‐based sealers induced increased alkalinization over time (f) Quantitative measurement (mean and standard deviation) of pH relative to negative control by pH analysis. Different symbols in each column indicate statistically significant differences between tested materials and negative control (*p < .05; **p < .01; ***p < .001, ****p < .0001, respectively; two‐way anova with Dunnett's multiple comparisons test)

FIGURE 2

Effects of root canal sealers on biofilm biomass and metabolic activity after 3, 6 and 18 h of incubation under 5% CO₂ at 37°C. Except for AH Plus, which induced a ~50% decrease at 18‐h post‐inoculation, most sealers did not cause impairment on biofilm biomass. Conversely, except for BioRoot RCS, all sealers decreased MTT reduction compared with the control, suggesting a negative impact in biofilm metabolic activity or viable cell numbers (a) Quantitative measurement of crystal violet staining relative to negative control as an indicator of biomass accumulation on root canal sealer specimens. (b) Quantitative measurement of plaque‐derived biofilm MTT conversion relative to negative control as an indicator of community metabolic activity. Values were significantly different compared with the control group (*p < .05; **p < .01; ***p < .001, ****p < .0001, respectively; two‐way anova and Dunnett's multiple comparisons test)

FIGURE 3

Taxonomic and principal coordinate analysis from 16S rRNA gene sequencing. The community composition of biofilms formed on sealers under 5% CO₂ and anaerobic conditions showed significant differences in alpha diversity using the Shannon index and Chao analysis of variance. (a) Relative abundances of families within biofilm communities incubated under 5% CO₂ at 37°C. (b) Relative abundances of families within biofilm communities incubated under anaerobic conditions. (c) Specimens were incubated under 5% CO₂. (d) Specimens were incubated under anaerobic conditions. Negative control (CTRL); AH Plus (AHP); GuttaFlow Bioseal (GFB); Endoseal MTA (ESM); Bio‐C Sealer (BCS); BioRoot RCS (BRR)

FIGURE 4

Biofilm morphology induced by different root canal sealers and negative control. FE‐SEM images of control biofilms (left side images; plaque‐inoculated sterile dentine discs) show several distinct morphotypes, including cocci, rods and spirals, in diverse arrangements. In the presence of the sealers (right side images; plaque‐inoculated sealer/dentine discs), the community richness decreased. Biofilms were visualized using a field‐emission gun scanning electron microscope at 5000 and 10,000× magnification