PlaqueGuard: Investigating the Effects of Polyphenols on Dental Plaque Formation

Dental caries are a widespread health issue, affecting nearly 3.7 billion people worldwide, leading to pain, infection, and eventual tooth loss1,2,3. Many popular mouthwashes address this using alcohol to nonselectively kill bacteria, including nonpathogens, disrupting the natural balance of the oral microbiome and leading to side effects like irritation, staining, and potentially increased cancer risks4,5,6,7. These limitations display the need for more targeted strategies that preserve a stable oral environment.

As I explored this further, I learned that dental caries are caused by plaque, a biofilm that forms on the salivary pellicle, containing binding sites for bacterial colonisation8,9,10. Particularly, the bacteria Streptococcus mutans produces glucosyltransferase, which catalyses the conversion of adhered sugars into sticky glucans, facilitating further adhesion of sugars and bacteria through a positive feedback loop1,2,3,9.

Motivated by my interest in how biochemical interactions and molecular structures influence biological systems, I was led to research a class of natural compounds called polyphenols, found in substances like green tea and cranberries, known for influencing microbial interactions and enzyme activity11,12,13,14.

In particular, catechins and proanthocyanidins may interfere with adherence mechanisms in plaque formation. Their abundance of hydroxyl groups allows hydrogen bonding with sugars and salivary pellicle proteins, potentially disrupting sugar aggregation and surface interactions15,16,17. In addition, studies suggest these polyphenols may inhibit glucosyltransferase18.

Based on these properties, I developed my research question, "To what extent can polyphenols disrupt sugar adherence mechanisms, reducing early plaque formation?”

This study focuses on isolating the initial adhesion phase of sugars in plaque formation, leading to reduced localized substrate availability for glucan synthesis, diverging from popular nonselective bactericidal methods.

Peer reviewed literature was first covered to understand plaque structure and formation, as well as unique properties of polyphenols which could affect the biochemical interactions leading to the formation of this biofilm.

A sucrose solution was prepared by dissolving table sugar in distilled water. Treatment groups were then

prepared, including extracts of green tea_(containing catechin polyphenols), cranberry_(containing proanthocyanidin polyphenols), as well as a combined solution of both.

The primary experiment used microscope slides to simulate the tooth surface. Slides were coated with egg white as a proxy for the salivary pellicle, and then exposed to the sucrose solution. Initial weight measurements were recorded, followed by application of the treatment being tested(green tea, cranberry, combined, and control). Final weight measurements were taken, and the difference was calculated to find the reduction of adhered sugar. Each treatment was tested 3 times to improve reliability and generalization of the results.

A second experiment was conducted using a dental model to support image analysis for validation of the original results. This model was similarly coated with egg white as a salivary pellicle proxy, and applied with a dyed sucrose solution so areas with adhered sugar would have clear visibility. After drying, each corresponding treatment was applied.

Images were taken before and after each treatment application. To analyze the adhered sugar, image analysis was performed through a custom python program. The tooth area was isolated, the coloured sugar layer was identified through a binary function, and percentage reduction in coverage was calculated by comparing the before and after images.

Variables were kept consistent, such as drying time, solution volume, and application method to ensure experimental reliability.

The results of this project showed a clear and consistent trend of each treatment on sucrose adhesion across both experiments. The control (distilled water) and Listerine showed roughly similar residual sucrose mass remaining, with Listerine being approximately 7.76% more effective on average. In contrast, the natural extract treatments with polyphenols displayed strong effectiveness with significantly reduced adhesion. In the weight based experiment, green tea reduced sugar adhesion by approximately 31.5% relative to control, cranberry about 41.3%, and the combined treatment showing the greatest reduction at about 53.9%.

This trend was independently validated in experiment 2 using image based analysis of a dental model. Listerine had a moderate removal efficiency of about 23.9% relative to the control. All of the polyphenols again demonstrated strong effectiveness, with green tea at 41.1%, cranberry at 50.4%, and the combined treatment reaching the highest removal efficiency at about 62.3% compared to the control. The consistency of the results between the weight based measurements and image analysis strengthens the reliability of these findings, as two independent analysis methods produced similar trends and consistent outcomes.

A one way ANOVA test was performed to determine whether there were statistically significant differences between the treatment groups. Following the ANOVA result, Tukey HSD post hoc analysis with Bonferroni correction was applied to identify which specific group comparisons drove that significance.

The ANOVA showed significant differences of treatment on sucrose adhesion(F(4,10) = 166.25, p < 0.01), indicating that at least one group mean differed significantly from the others. While low mass values can increase relative impact of measurement resolution, the consistently small standard deviations and error bars indicate that experimental variability stayed low relative to the magnitude of treatment effects. The treatments had direct, consistent effects and the differences between groups were large relative to internal variability.

A Tukey HSD post hoc analysis was then conducted across all 10 pairwise comparisons among the 5 groups. Bonferroni correction was subsequently applied to adjust the p values for multiple comparisons and determine the level of significance for each result. The combined treatment was shown to have statistically significant reduction compared to the control (p = 0.001), Listerine (p = 0.002). Individually, green tea and cranberry also showed statistically significant reductions compared to the control (p = 0.005 and p = 0.002 respectively), and cranberry additionally showed a significant difference compared to Listerine (p = 0.008).

Both experiments showed the same pattern, polyphenol based treatments reduced sucrose adhesion significantly more effectively than the control and Listerine, with the combined treatment producing the largest observed reduction.

The results of this experiment suggest that polyphenols could have a significant effect on interference of sucrose adhesion to teethlike surfaces, targeting disruption of early stages in plaque formation. All treatment groups containing natural extracts showed measurable reductions relative to the control, with the combined extract reducing the most adhered sugar.

In contrast, Listerine had relatively less of an effect in this experiment, having significantly lower performance than the natural extracts. This is likely because it is designed to be antimicrobial, while this project currently solely targets isolation of the sugar adhesion phase4,5.

Speculatively, differences between individual polyphenol treatments may be related to structural variations. The stronger reduction effect of proanthocyanidins may be associated with a larger, polymeric structure, possibly allowing for extensive surface protein interactions, and having a higher amount of hydroxyl groups allows for more potential hydrogen bonds17,18. In contrast, smaller catechins may interact differently with the sugars and proteins, potentially influencing adhesion through faster or alternative binding behaviour19.

The enhanced effect of the combined treatment suggests possible additive or synergistic effects between the interactions of the two polyphenol compounds, supporting the idea of variations due to structure.

This research demonstrates strong effectiveness of polyphenols on the adhesion phases in plaque formation. With further investigation, this alternate approach could be a cost effective, and accessible solution involving the targeted prevention of plaque growth. Diverging from popular nonselective methods, it provides a way to avoid the risk of harming beneficial microorganisms, and maintain a balanced oral environment.

I am currently working to improve this project by incorporating bacterial cultures such as Streptococcus mutans to better model real plaque formation. Also, an in vitro test could be conducted, testing different polyphenols as an allosteric inhibitor to glucosyltransferase, which is the enzyme responsible for catalyzing the reaction between sugar to extracellular glucans. This would display the effectiveness of polyphenols to reduce further bacterial adhesion from residual sugar. Coupled with this, extraction of the pure natural compound could be done. Increasing the amount of replications for the experiment(n > 5-10) would increase robustness of statistical analysis.

I would like to thank my family, teachers, and friends for their support and encouragement throughout the journey for this project. Their motivation has played an important role in helping me stay focused and committed.

I am incredibly grateful to the regional fair coordinators, Ms. Susan Lindsay, and the delegates, for organizing the regional fair and supporting the trip to the CWSF. Their hard work is what made these opportunities possible.

In addition, I would like to thank the past finalist Isabella Bu, for her advice regarding lab opportunities, which has helped me explore research environments to further develop my project.

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