The Effect of Bovine Trypsin on the Adhesion and pH of Dental Plaque Biofilms: An In Vitro Study

Abstract

Objective: The aim of this study was to investigate the effect of bovine trypsin on the adhesion and pH of dental plaque biofilms.

Methods: A multispecies dental plaque biofilm model and a single-species dental plaque biofilm model were established in vitro. Three groups were tested: (1) blank control group (aseptic ultrapure water); (2) negative control group (1M Tris-HCl buffer, pH = 7.4); and (3) experimental group (bovine trypsin). Adhesion ability was measured using an automatic microplate reader and visualised by confocal laser scanning microscopy (CLSM). The pH was measured using a pH meter. The expression of gtfB, gtfC, and gtfD was analysed using quantitative real-time polymerase chain reaction.

Results: Adhesion ability in the experimental group was significantly lower than that in the blank group and the negative control group (P < .05); readhesion ability in the experimental group was inhibited for a certain period of time (24-hour multispecies biofilms were inhibited from 4 to 8 hours, and the 48- and 72-hour multispecies biofilms were inhibited from 2 to 6 hours; P < .05). The decrease in pH was inhibited for a certain period of time (24-hour multispecies biofilms were inhibited from 2 to 8 hours, and the 48- and 72-hour multispecies biofilms were inhibited from 1 to 8 hours; P < .05). Expression levels of gtfB, gtfC, gtfD, and ldh in the experimental group were significantly lower than those in the blank group (P < .05).

Conclusions: Bacterial adhesion, and readhesion, decreasd pH, and expression of adhesion- and acid-related genes by Streptococcus mutans in biofilms could be reduced by bovine trypsin for a certain period of time.

Keywords: Adhesion; Bovine trypsin; Dental caries; Dental plaque biofilms; pH.

Fig. 1

Flowchart of the adhesion experiment.

Fig. 2

Confocal laser scanning microscopy 3D images of the 24-hour, 48-hour, and 72-hour multispecies biofilms (A) and 24-hour single-species biofilms (B).

Fig. 3

Confocal laser scanning microscopy 3D images of the adhesion ability of multispecies biofilms treated with bovine trypsin at 24 hours (A), 48 hours (B), and 72 hours (C).

Fig. 4

Confocal laser scanning microscopy 3D images of the readhesion ability of multispecies dental plaque biofilms treated with bovine trypsin at 24 hours (A), 48 hours (B), and 72 hours (C).

Fig. 5

The quantitative analysis of the effect of bovine trypsin on the readhesion of multispecies dental plaque biofilms at 24 hours (A), 48 hours (B), and 72 hours (C). * Indicates a statistically significant difference compared with the blank control group (P < .05), and ^# indicates a statistically significant difference compared with the negative control group (P < .05).

Fig. 6

Quantitative analysis of the effect of bovine trypsin on multispecies biofilms on pH at 24 hours (A), 48 hours (B), and 72 hours (C). * Indicates a statistically significant difference compared with the blank control group (P < .05).

Fig. 7

Quantitative analysis of the effect of bovine trypsin on the expression levels of gtfB, gtfC, gtfD, and ldh in 24-hour Streptococcus mutans biofilms. A, Quantitative analysis of the expression levels of gtfB, gtfC, gtfD, and ldh in 24-hour single-species Streptococcus mutans biofilms immediately after treatment with bovine trypsin. B, Quantitative analysis of the expression levels of gtfB, gtfC, gtfD, and ldh in 24-hour single-species Streptococcus mutans biofilms cultured in brain–heart infusion for 6 hours. C, Quantitative analysis of the expression levels of gtfB, gtfC, gtfD, and ldh in 24-hour single-species Streptococcus mutans biofilms added to fresh bacterial liquid for 6 hours. * Indicates a statistically significant difference compared with the blank control group (P < .05).