Glycolysis and Automated Plaque Regrowth Method for Evaluation of Antimicrobial Performance

Abstract

Purpose: This study explored the potential of a new in vitro method in evaluating antiplaque benefits from five sets of antimicrobial systems including cetylpyridinium chloride (CPC), stannous fluoride (SnF₂), Listerine essential oil mouthwashes (+/- alcohol), zinc chloride (ZnCl₂), and sodium fluoride. (NaF).

Methods: Gingival dental plaque was collected and propagated using sterilized tryptic soy broth and sucrose, and then allocated into separate glycolysis and regrowth recipes for antiplaque evaluations. Glycolysis measurements (in duplicate) were recorded via pH microelectrode on plaque-treatment samples thermomixed (1200 rpm, 37 °C) for 4 h. For plaque regrowth, optical densities (in duplicate) were automatically collected on plaque-treatment samples using a microplate reader (linear shaking, 37 °C) from baseline to 4 h.

Results: Calculations of percent change in pH and optical density were performed and analyzed for each set of antimicrobial treatment groups. Statistical analysis (one-way ANOVA, Student-Newman-Keuls stepwise comparison tests) revealed dose responses and significant differences (p < 0.05) among treatment groups, including between negative and clinically relevant positive controls.

Conclusions: This lab method produces results consistent with published clinical observations. This glycolysis and plaque growth method is sensitive to antimicrobial mechanisms of action, and may offer a convenient and clinically relevant screening tool in the evaluation of putative antimicrobial agents and formulations.

Keywords: CPC; antigingivitis; antimicrobial; antiplaque; chemical plaque control; essential oil; fluoride; preventive dentistry; stannous; zinc.

Figure 1

Glycolysis (via pH measurements) produced from human plaque treated with 0.001% (blue open triangles, line), 0.1% (red open circles, line), 0.03% (open black squares, line), 0.05% (closed black squares, line), 0.07% (closed red circles, line), or 0.1% (closed blue triangles, line) CPC, 50 μL sucrose, and thermomixed at 37 °C for up to four hours. The dashed line at pH 5.5 marks the dissolution of enamel.

Figure 2

Plaque regrowth (via optical density measurements) produced from human plaque treated with 0.001% (blue open triangles, line), 0.1% (red open circles, line), 0.03% (open black squares, line), 0.05% (closed black squares, line), 0.07% (closed red circles, line), or 0.1% (closed blue triangles, line) CPC at 37 °C for up to four hours.

Figure 3

Plaque regrowth response as a function of CPC weight percent, including those recommended for antiplaque/antigingivits benefits.

Figure 4

Plaque regrowth response as a function of NaF content. The range of NaF shown covers those levels in community water supplies (e.g., 0.1 ppm F) up to varnishes with 5% NaF.