Comparison of periodontitis-associated oral biofilm formation under dynamic and static conditions

Abstract

Purpose: The purpose of this study was to compare the characteristics of single- and dual-species in vitro oral biofilms made by static and dynamic methods.

Methods: Hydroxyapatite (HA) disks, 12.7 mm in diameter and 3 mm thick, were coated with processed saliva for 4 hours. The disks were divided into a static method group and a dynamic method group. The disks treated with a static method were cultured in 12-well plates, and the disks in the dynamic method group were cultured in a Center for Disease Control and Prevention (CDC) biofilm reactor for 72 hours. In the single- and dual-species biofilms, Fusobacterium nucleatum and Porphyromonas gingivalis were used, and the amount of adhering bacteria, proportions of species, and bacterial reduction of chlorhexidine were examined. Bacterial adhesion was examined with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).

Results: Compared with the biofilms made using the static method, the biofilms made using the dynamic method had significantly lower amounts of adhering and looser bacterial accumulation in SEM and CLSM images. The proportion of P. gingivalis was higher in the dynamic method group than in the static method group; however, the difference was not statistically significant. Furthermore, the biofilm thickness and bacterial reduction by chlorhexidine showed no significant differences between the 2 methods.

Conclusions: When used to reproduce periodontal biofilms composed of F. nucleatum and P. gingivalis, the dynamic method (CDC biofilm reactor) formed looser biofilms containing fewer bacteria than the well plate. However, this difference did not influence the thickness of the biofilms or the activity of chlorhexidine. Therefore, both methods are useful for mimicking periodontitis-associated oral biofilms.

Keywords: Bacterial adhesion; Biofilms; Chlorhexidine; Electron microscope tomography; Periodontitis.

Figure 1

Bacterial composition of the dual-species biofilms. P. gingivalis comprised a larger proportion than F. nucleatum, and this difference was more pronounced in the dynamic method group than in the static method group. No statistically significant differences were observed in the composition of each species within the 2 groups of biofilms.

Figure 2

Scanning electron microscopic images of biofilm formation on HA disks (original magnification: ×10,000). In the single-species groups, disks from both the static group and the dynamic group were covered by F. nucleatum, instead of P. gingivalis (A-D). However, in the dual-species groups, the biofilms from the static method group showed a denser accumulation of F. nucleatum than those from the dynamic method group (E, F). HA: hydroxyapatite, CDC: Center for Disease Control and Prevention.

Figure 3

Dual-species biofilms with higher magnification (original magnification: ×30,000). (A) Biofilm in well plate (static method). (B) Biofilm in CDC biofilm reactor (dynamic method). The arrow indicate F. nucleatum and the arrowhead indicate P. gingivalis. CDC: Center for Disease Control and Prevention.

Figure 4

Confocal laser scanning microscope images of biofilm formation on HA disks (Scale bar=50 µm). The values in each image represent the distance from the surface of the HA disks. (A) F. nucleatum biofilm in a well plate. (B) F. nucleatum biofilm in the CDC biofilm reactor. (C) P. gingivalis biofilm in a well plate. (D) P. gingivalis biofilm in the CDC biofilm reactor. (E) Dual-species biofilm in a well plate. (F) Dual-species biofilm in the CDC biofilm reactor. HA: hydroxyapatite, CDC: Center for Disease Control and Prevention.