Antibacterial efficacy of a human β-defensin-3 peptide using dentin infection model and biofilm viability assay

Abstract

The aggregation of bacterial flora into sessile biofilms on root canal surfaces can be one of the causes of persistent apical periodontitis. The aim of this study was to evaluate the antibacterial efficacy of human β-defensin-3 (HBD3) peptide compared with calcium hydroxide (CH) and chlorhexidine (CHX) on Enterococcus faecalis (E. faecalis)-infected dentin blocks and multi-species biofilms. Standardized human dentin blocks were infected with E. faecalis ATCC 29212 for 3 weeks. Aqueous CH paste, a 2% CHX gel, an HBD3 peptide gel, control peptide (CP) and saline were treated in canal lumen. 12 dentin block samples were included in each group. After 1 week of medication, the dentinal samples at the depth of 200 and 400 μm were collected from medicated canal lumens. Bacterial growth was assessed by spectrophotometric analysis of optical density (OD) after 72 hours of incubation. Repeated-measures analysis of variance and Tukeys post hoc test were used for data analysis. For multispecies biofilm formation, Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and E. faecalis were cultured in a peptone-yeast-glucose broth and their culture suspensions combined in equal proportions. The mixed bacteria were inoculated on sterile cover slips placed into the wells of tissue culture plates. After incubation for three weeks, the samples were treated for 24 hours with saline, saturated CH solution, 2% CHX solution, and 50 μg/ml HBD3 solution. The percentage of dead cells was determined by viability staining and confocal laser scanning microscopy. The results were subjected to one-way analysis of variance and a post hoc test. In dentin block study, the HBD3 group was associated with significantly lower OD values (p < 0.05) than the CH or CHX groups at both depths. The CH group did not differ significantly from CP or saline group at either depth (p > 0.05). There was no significant difference (p > 0.05) in the OD values of the inner (200 μm) and outer (400 μm) dentinal samples for any group. In multispecies biofilm study, three medication groups showed a significant reduction of biovolume within the biofilms compared with the control group (p < 0.05). The HBD3-treated biofilms had a higher percentage of dead cells than the other medication groups (p < 0.05). The CH and CHX showed higher levels of bactericidal activity than saline (p < 0.05), and no significant difference between the two groups (p > 0.05). HBD3 exhibited marked antibacterial activity against E. faecalis biofilms in dentin block and multi-species biofilms, than either CH or CHX.