Dental caries is a common and costly infectious disease that depends on biofilms in the oral cavity. It is mainly caused by the interaction of specific bacteria with diet constituents within dental biofilms. Microbial adhesion to solid surfaces is a crucial step in biofilm formation, including on tooth surfaces, where it serves as a base for subsequent colonization (Kolenbrander et al., 2010). The complex physical and chemical interactions between oral bacteria and tooth surfaces during biofilm formation can significantly impact the composition and function of dental biofilms, ultimately affecting oral health (Bernabe et al., 2017). Therefore, understanding the interactions that influence dental biofilm formation is essential for developing strategies to control dental caries by modulating biofilms.

Various environmental factors, such as surface characteristics, salivary proteins, and oral conditions, can influence the interactions between tooth surfaces and microbial cells (Han et al., 2016). Among these factors, substratum-related factors such as surface charge, surface hydrophobicity, and surface roughness have been extensively studied for their influence on microbial adhesion during the initial step of dental biofilm formation (Zheng et al., 2021). A recent study has also revealed that carious lesions are increasingly localized to specific tooth sites (Carvalho, 2014). In children and adolescents, occlusal surfaces are more vulnerable to dental caries compared to proximal surfaces, suggesting that the direction of tooth surfaces (substratum) can be an important factor affecting the interaction between microbial cells and tooth surfaces. Our previous study also reported that the direction of tooth surfaces significantly impacts the formation and pathogenicity of Streptococcus mutans biofilms (Dang et al., 2018). Specifically, the upward direction of substratum surfaces (occlusal surfaces of mandibular teeth) showed the highest colony-forming unit (CFU) counts, extracellular polysaccharide (EPS) amounts, biofilm density, and acid production of the biofilms. However, this study only examined biofilms composed of a single species, S. mutans, and did not simulate the complex microbial community found in dental biofilms.

The types and properties of bacteria also influence the interactions between tooth surfaces and microbial adhesion. Numerous studies have shown that bacteria adhere to surfaces based on their specific characteristics, such as hydrogen-bonding capability and ability to form ligand-receptor bonds (Gibbons et al., 1985, Nobbs et al., 2011). Moreover, a previous study demonstrated that initial colonizers of dental hard surfaces exhibited stronger adhesion forces to saliva-coated enamel than cariogenic strains (Mei et al., 2009), which may further impact the formation and microbial composition of dental biofilms. However, few studies have investigated the impact of substratum surface direction on the adhesion ability of different types of oral bacteria and their biofilm formation.

In this study, we investigated the differences in microbial adhesion and biofilm formation in dental biofilms according to the direction of tooth surfaces using a multi-species biofilm model. The multi-species biofilm model was composed of S. mutans, Streptococcus oralis, and Actinomyces naeslundii on three different saliva-coated hydroxyapatite (sHA) disc direction groups: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in the opposite direction of gravity) to mimic tooth surfaces in the mouth.