Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biomedical Engineering

Supervisor

Siqueira, Walter L.

2nd Supervisor

Cury, Jaime A.

Affiliation

University of Campinas

Joint Supervisor

Abstract

Dental biofilm is formed onto dental surfaces covered by a layer of specific salivary proteins and peptides named acquired enamel pellicle (AEP). It was previously demonstrated that the statherin- and histatin-derived engineered salivary peptides DR9-DR9 and DR9-RR14 were able to reduce enamel demineralization and displayed antimicrobial activity against Streptococcus mutans, respectively. However, these studies were carried under experimental conditions that did not mimic caries development induced by biofilm and sucrose exposure, the most cariogenic dietary sugar. In this thesis we assessed the effect of the engineered salivary peptides on 1) the adherence of S. mutans to hydroxyapatite (HAp), and 2) the reduction of enamel demineralization, using a validated S. mutans cariogenic biofilm model. We hypothesized that DR9-DR9 and DR9-RR14 would reduce S. mutans adherence and the cariogenicity of the biofilms, protecting enamel against demineralization. To test this hypothesis, we conducted two in vitro studies. In the first study, hydroxyapatite discs were treated with the peptides to induce AEP formation and then inoculated with S. mutans to quantify adhered bacteria, and to evaluate the total bacterial proteomes after adhesion periods of 2 h, 4 h, and 8 h. In the second study, a validated in vitro cariogenic S. mutans biofilm model was used to test the effect of DR9-DR9 and DR9-RR14 on enamel demineralization under two conditions 1) adsorbed onto the enamel surface forming the AEP, and 2) used to treat the biofilms 2x/day. Biofilms were grown for 144 h and the effect of the peptides on enamel demineralization, biomass, and total and extracellular matrix bacterial proteomes were evaluated. Results showed that DR9-DR9 reduced S. mutans adherence to HAp, but DR9-DR9 and DR9-RR14 did not have a significant effect on the development of the cariogenic biofilms formed. The engineered peptides did not modulate the bacterial proteome, neither when adhered to AEP nor when used to treat the biofilms during their formation. However, DR9-DR9 and DR9-RR14 significantly reduced enamel demineralization, suggesting that they control caries development by a physicochemical mechanism. The combinatory effect of reducing bacteria adhesion and limiting the mineral loss from the dental structure under high cariogenic challenge, suggests that the engineered peptides DR9-DR9 and DR9-RR14 represent a new translational approach for the prevention/treatment of dental caries.

Summary for Lay Audience

Tooth decay continues to affect more than 2.5 billion people worldwide, calling to search for new therapeutic strategies to control this disease. Saliva has properties to modulate dental decay and we have recently found that small fragments (peptides) originated from the salivary proteins statherin and histatin are present on tooth surfaces where the oral bacteria stick before promoting tooth decay under dietary sugar consumption. Based on the differential biological properties that statherin and histatin display, we designed and engineered two peptides expecting to have a better effect on tooth decay prevention than their precursor salivary proteins. In the present study, we extended the promising results previously found because we showed that our engineered peptides were able to reduce the bacterial attachment and the dissolution of tooth enamel (tooth decay) provoked by biofilms formed from Streptococcus mutans, the most cariogenic oral bacteria, under sucrose exposure, the most cariogenic dietary sugar. The engineered peptides worked on enamel mineral balance without killing S. mutans bacteria, in agreement with the latest knowledge of the role of the oral microflora in health and disease. Our results may contribute to the creation of a novel and contemporary biotechnological strategy for the prevention/treatment of tooth decay.

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