The oral cavity is a complex ecosystem in which a large number of microorganisms coexist and interact. In addition to bacteria, this microbial community includes viruses, bacteriophages and fungi (1) forming biofilms that adhere to the tooth surface (2). However, before a biofilm can attach and grow, a pellicle (3) forms on the tooth surface by binding proteins and other molecules from saliva. Early colonizers such as streptococci and actinomyces species adhere to the pellicle forming an initial dental biofilm (4, 5). Even before the term was coined (6), scanning electron microscopy of what we now call “biofilm” revealed bacteria embedded in a polysaccharide-like matrix (7). Extracellular polysaccharides produced by oral bacteria such as S. mutans make up an important part of biofilm in the form of dental plaque (4). Depending on their composition, biofilms can shift toward an acid-producing and acid-tolerant environment in which acidophilic pathogenic microorganisms become dominant (2). Fermentation of dietary carbohydrates by these acidophilic species leads to an increase in acidic metabolites resulting in a drop in biofilm pH. This acidic pH causes demineralization of dental hard tissue at the tooth surface and the development of caries (8). Over time, therapeutic approaches to caries have evolved from preventive to operative therapy (9). Fluoride is the most widely used and well-studied caries-preventive agent (10, 11). Despite the caries decline since the introduction of fluoride, caries remains the most prevalent condition globally, particularly in disadvantaged groups (12, 13). As fluorides in systemic and local application forms are widespread and only a significant minority actively avoids fluoride use (14, 15), there is a need to develop and investigate alternative caries preventive strategies. While some bactericidal agents such as chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) are highly effective against oral bacteria, there are concerns about their repeated use due to potential development of resistance and shifts in oral microbiota towards caries or periodontitis (16, 17).

Cerium is a lanthanide, a rare earth element (18). As a trivalent ion, the atomic radius of cerium III is 1.01 Å and that of divalent calcium is 1.00 Å, which means that Ce III may be capable of substituting for Ca²⁺ (18). EDX analyses showed that application of CeCl₃ resulted in the incorporation of cerium into bovine enamel (19). The replacement of Ca²⁺ by cerium in hydroxyapatite is thought to create a more stable crystal, resulting in greater acid resistance (20). Human enamel treated with CeCl₃ has been shown to have increased resistance to demineralization (21, 22). Bacteriostatic effects on planktonic bacteria have also been demonstrated for CeCl₃ when added directly to the culture medium (22, 23). Cerium compounds are not currently used clinically for caries or periodontitis treatment, and available safety data are limited to animal laboratory studies (24). A recent study found no difference in cytotoxicity for cerium salts including CeCl₃ and ionic fluorides (NaF, NH₄F) (25). To our knowledge, the effects of cerium chloride pretreatment of hydroxyapatite surfaces, rinsed off prior to biofilm formation, have not yet been tested.

The primary aim of this study was to evaluate the effect of cerium III chloride (50%CeCl₃) pretreatment of hydroxyapatite discs on the subsequent growth of an initial 3-species caries biofilm in vitro. A secondary aim was to determine whether cerium remains incorporated on the hydroxyapatite surface following a brief treatment and rinse protocol.

In the present study, pretreatment of HA-Discs with 50% CeCl₃ reduced initial biofilm formation compared to 0.02% CHX, or ultrapure water (control) treated samples. The 3 species caries model used here contains the cariogenic species S. odontolytica and S. mutans in addition to A. naeslundii, which functions as a bridging organism, facilitating co-adhesion and structural maturation in early biofilms (29, 30). This three-species model has been used previously to investigate multispecies biofilm formation (29, 31), although in this case we specifically investigated 4 h old initial bacterial colonization (32). The CHX treated and untreated controls show formation of microcolonies characteristic of initial biofilm formation.

SEM was used for qualitative assessment of surface morphology, not to generate quantitative data. As is standard in SEM-based surface analyses, reproducibility was assessed by examining multiple independent imaging fields across several HA discs per condition. Because SEM sample preparation is destructive, identical technical replicates of the same specimen are not feasible. SEM micrographs revealed a marked reduction in bacterial adhesion on the CeCl₃ treated discs and characteristic precipitates on all samples in the CeCl₃ group. These spherical precipitates have previously been described in cerium doped hydroxyapatite and on cerium treated human and bovine enamel and dentin surfaces (19, 23, 33, 34).

CHX was included as an antibacterial control for immediate surface effects. Even after rinsing, CHX exhibits significant substantivity on oral surfaces making it suitable for comparison as an antibacterial in initial-attachment assays (35, 36). Fluoride on the other hand, was not included as a control in this study because its primary mechanisms of action are enamel remineralisation, fluorapatite formation and reduction of enamel solubility, which were not tested here.

EDX analyses confirmed the presence of cerium in all CeCl₃ treated samples both in the precipitates and in areas free of precipitates, consistent with previous studies showing similar precipitations after cerium-salt application on enamel with and without pellicle or dentin (19, 23, 34). A significant negative correlation of cerium to calcium was found indicating a true lattice substitution rather than just surface adsorption.

The trivalent cerium ion and the divalent calcium ion have nearly identical ionic radii which facilitates substitution into the hydroxyapatite lattice (18). However, substituting a divalent ion with a trivalent ion leads to a positive charge imbalance, which must be compensated within the lattice, typically by permitting calcium vacancies, incorporating anions or lattice distortion (37, 38). Kaur et al. found that replacing Ca²⁺ with the lanthanide Ce³⁺ results in the replacement of OH⁻ with O²⁻, which stabilizes the lattice by decreasing the distance between ions and lowering the crystal energy (39). Predoi et al. found trivalent cerium in cerium doped hydroxyapatite next to tetravalent cerium. Surface property changes such as shifts in zeta potential or conductivity may explain reduced initial biofilm attachment to Ce doped hydroxyapatite (33, 37). Physical surface properties play a central role in initial biofilm formation by influencing protein adsorption, bacterial attachment, and binding of extracellular polymeric substances (EPS) (40). We exposed samples to CeCl₃ for just 1 min and then rinsed twice using ultrapure water to prevent any direct bactericidal effects from residual cerium remaining in the biofilm media. In contrast, CHX treatment of samples, which were also rinsed, did not completely prevent bacterial adhesion despite its known high substantivity and bactericidal properties (17, 41).

Another antimicrobial assay showed that cerium doped hydroxyapatite inhibited the growth of colony forming units for all tested strains (33). Also, a suspension of cerium and hydroxyapatite showed a biocidal effect against E. coli and C. albicans after 72 h incubation (33). S. mutans is a key acidogenic and aciduric species in cariogenic biofilms. The SEM observations in our study indicate that initial attachment is reduced on CeCl₃ treated surfaces compared to CHX treated or untreated controls. This aligns with earlier work showing that initial attachment is essential in S. mutans colonization and subsequent EPS dependent biofilm maturation (42, 43). Our findings suggest that CeCl₃ treatment may disrupt S. mutans as an early colonizer in a multispecies biofilm. Hydrolyzed Ce(IV) salts have been shown to reduce adhesion of S. mutans biofilm formation by about 40% but have very little effect on planktonic growth, indicating that cerium has a specific effect on initial adhesion rather than a direct bactericidal effect on S. mutans (44). Similarly, catheter surfaces with a slight positive charge have been shown to inhibit biofilm formation over 60- and 120-min growth periods for different microorganisms, compared to uncharged samples (45). However, Terada et al. showed that an 8-h-old E. coli biofilm had 23 times higher cell density on a positively charged diethylaminoethyl methacrylate surface than on a negatively charged sodium styrene sulfonate surface (46). An in vitro study could show that the cell walls of both Gram-positive and Gram-negative bacteria have been shown to have a negative charge (47). In another study, Gottenbos et al. demonstrated that positively charged surfaces can inhibit the growth of bacteria, particularly Gram-negative bacteria. Negatively charged surfaces resulted in slower initial adhesion of bacteria, though they did not affect the growth of Gram-negative or Gram-positive bacteria (48). Therefore, the interaction between bacteria and the surface of cerium-enriched HA could be due to the negatively charged bacterial wall and the positively charged surface of the HA. This could explain why the initial biofilm attachment is lower in cerium-enriched HA than in HA treated with chlorhexidine (CHX) or water. Kurniawan et al. demonstrated that biofilm formations show both negative and positive parts (49). A recent study found cerium oxide nanoparticles enhanced the antibacterial activity of chlorhexidine (50). Synergy depends strongly on the specific cerium compound and formulation, these findings cannot be assumed for the soluble CeCl₃ used in the present study. It is important to examine CeCl₃ separately to clarify its own surface-related mechanisms during early biofilm formation. While testing of interactions with other antibacterials and cariostatics was beyond the scope of the present study, it is a topic that could be explored in future studies.

In the present study we examined a three-species caries biofilm. To our knowledge, the effect of CeCl₃ pretreatment of HA discs has never been tested using multispecies biofilms. Although CeCl₃ application has been shown to result in increased surface roughness (51, 52) due to the acidic pH and subsequently more surface artifacts, less bacterial attachment was visible in the present study. Future studies should investigate whether the inhibitory effects we found also extend to more complex microcosm biofilms and in adapted lanthanide-salt containing formulations with reduced concentrations or adapted pH-values. We chose a 4-h incubation time in order to examine early biofilm attachment, but longer culture times are necessary to determine effects on mature biofilms. Combining CeCl₃ pretreatment with fluoride exposure in future studies would also more closely mimic clinical situations. Our study provides qualitative scanning electron microscopy (SEM) data. Further studies should investigate the quantitative effects on biofilm growth.

Within the limitations of the study, it can be concluded that CeCl₃ treatment before pellicle formation results in the development of precipitates on the surface of HA and appears to have potential to inhibit initial biofilm growth compared to CHX-treated or untreated controls. Further investigations are needed to identify the mechanisms involved and to test these effects under conditions closer to the clinical situation.