Relationships between composite roughness and Streptococcus mutans biofilm depth under shear in vitro

Streptococcus mutans is a commonly studied model cariogenic oral bacterium [1], [2], [3]. S. mutans produces interfacial biofilms anchored in the oral cavity via the production of a matrix comprising extra-cellular polymeric substances (EPS). The biofilm matrix adheres to oral cavity surfaces, such as tooth enamel, periodontal pockets, or the surface of composite, amalgam restorations, and other dental fixtures [4]. Biofilms grow as bacteria within the anchored biofilm produce additional EPS and cells. The external surface of the biofilm can be air or liquid, depending on location and dryness of the oral cavity. Biofilm attachment to the solid surface can be disrupted by brushing, shear forces, and cell death at depth within the biofilm depth, typically because of an imbalance between the diffusional flux of bacterial substrates and metabolic products [5].

Despite decades of study, scientists have yet to develop a comprehensive biological, chemical, and physical mechanistic understanding of S. mutans biofilm structure and function. Given the key role that biofilm chemodynamic behavior plays in cariogenesis, a better understanding of what factors affect S. mutans biofilm structure would be of great value.

Many studies have identified a strong link between fermentative substrate and S. mutans biofilm structure [6,7]. These findings have led to the paradigm that sucrose fermentation by S. mutans and other fermentative bacteria produces volatile fatty acid (VFAs) such as lactic and acetic acid in a deep biofilm, creating conditions that are the primary cause of dental caries [8], [9], [10]. Supporting the link between sucrose availability with enhanced cariogenicity is the finding that S. mutans biofilms are thicker and contain large concentrations of dextrans when growing on sucrose compared to biofilms when grown on other sugars such as glucose [4,[11], [12], [13], [14], [15], [16]]. Additional support for this hypothesis comes from studies of the expression of specific genes known to play a role in sugar metabolism [17,18] and how substrate concentration affects VFA production [19,20]. Together, these studies (often performed under quiescent, non-shear conditions) demonstrate a focus on the biochemical cariogenic potential of the biofilm to a greater extent than on how the combination of physical factors and chemical fluxes affect the growth of the biofilm itself. There is a comparative lack of literature providing quantitative data on microbial biokinetic rates and physical process characteristics such as hydrodynamic shear and hydraulic residence time (HRT). Given the substantial differences in S. mutans substrate utilization kinetics and yield properties when grown on sucrose compared to glucose, it is worth investigating how these biokinetic differences result in different biofilm structure in a dynamic biofilm system representative of the oral cavity.

Among physical parameters that impact biofilm formation, studies have suggested that surface roughness can influence biofilm size, more so than other factors, such as surface free energy and hydrophobicity [21], [22], [23]. Rougher surfaces facilitate the growth of thicker biofilms compared to those grown on smoother surfaces [24,25]. This is important for the field of restorative dentistry, as dental composites used to fill cavities are often polished to create a smoother surface. Many studies suggest a link between surface roughness and biofilm growth and thickness. Some studies have compared different manufactured dental composites [26] while others have polished the same dental composite to achieve different levels of surface roughness for comparison [22,27,28].

Techniques to quantitatively assess both surface roughness and biofilm thickness now exist in sufficient resolution relevant to microbial ecosystems. Roughness is commonly assessed as a scalar roughness coefficient using contact profilometry or laser interferometry. Newer techniques like confocal laser microscopy allow the quantitative assessment of biofilm growth at the microbial scale.

The goal of this study was to investigate how S. mutans biofilms are affected by physical processes relevant to oral dental systems focusing specifically on restorations. Specific studies investigate the hypothesis that biofilm structure is significantly affected by composite surface roughness and HRT while grown under hydrodynamic shear in a Centers for Disease Control (CDC) bioreactor on purportedly cariogenic and non-cariogenic substrates using profilometry and confocal microscopy. The null hypothesis is that changes in composite surface roughness, HRT, or substrate do not significantly affect biofilm thickness.

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