**4. Discussion**

The adhesion of streptococci to solid surfaces has been described as a two-step process, where the first step is influenced predominantly by macroscopic substratum properties such as surface roughness and surface free energy [33]. High substratum surface roughness has been shown to induce increased plaque formation in vivo [34,35], as the initial adhesion of microorganisms preferably starts at locations that provide shelter against shear forces. The surface roughness of di fferently prepared lithia disilicate ceramic restorations (pressed (Press), milled (CAD), fluorapatite layered (ZirPress/Ceram), and glazed (Ceram Glaze)) is closely related to the adherence of *S. mutans* [27]. The application of a SiC coating on the surfaces of ceramic significantly minimized biofilm adhesion on the surface. A possible explanation is that SiC altered the surface roughness of the ceramic. SEM 3D analysis of ceramic surfaces before and after coating with SiC (Figure 2A–F) revealed a distinct di fference in surface roughness between the two surfaces. The SiC coating achieved planarization of the ceramic surface, essentially minimizing surface roughness that bacteria can adhere to. Even when compared to a glazed ceramic surface, the SiC coating produced a smoother surface (Figure 2B,C). This change in surface roughness was further confirmed by a di fference in surface topography of coated and non-coated surfaces, as observed by AFM (Figure 1).

Numerous studies have also demonstrated that surfaces with high surface free energy foster microbial adherence in vitro and in vivo [35–38]. In addition, bacteria with high cell surface free energy appear to adhere preferentially to surfaces with high surface free energy.

Bacterial adhesion to a substrate and the initial biofilm composition is also related to surface hydrophobicity, and communication between existing microorganisms [28,37,38]. Although the literature shows that bacteria adhere better to hydrophobic surfaces, biofilm formation depends not only on the surface topography but also on the bacteria species involved. Evidence suggests that the presence of polysaccharides on the cell surface of Gram-positive bacteria, such as *S. mutans* and *S. sanguinis*, tends to make the bacterial cell more hydrophilic [35,38]. Additionally, the cell shape and size, including surface characteristics such as extracellular substances such as flagella and fimbriae, as well as the production of proteins, are believed to have a significant influence on the process of bacterial adhesion [33,34].

On the basis of contact angle measurements conducted in this study, we found that coated SiC surfaces were more hydrophobic than non-coated surfaces; however, we found lower colonized bacteria. This might sugges<sup>t</sup> that SiC-coated samples can cause the detachment of bacteria from the biofilm after 24 h.

In vitro studies have been performed on bacterial adhesion to various ceramic materials [28,39–43]. In the present study, we verified the adhesion of streptococcal species and the accumulation of complex biofilms, which are of critical importance to predict the e ffect of biofilms on the surface ceramics. *Streptococcus mutans* and *Streptococcus sanguinis* were used for this study because these bacteria are predominant members in dental plaque, which is often described as a dual-species biofilm model [36,44]. The association of these microorganisms can interfere in the development of each other and promote di fferent results between polymicrobial and monomicrobial biofilms.

Lower adhesion of polymicrobial biofilm of *S. mutans* and *S. sanguinis* on the surface of fluorapatite glass-ceramic coated with SiC was demonstrated. Additionally, we found a significant reduction in their monomicrobial biofilm. Two studies found significant di fferences in adhesion of di fferent streptococcal species among di fferent ceramic materials. These di fferences were observed in the bacterial surface coverage, as well as in the thickness of the biofilm between the tested ceramic materials. The lowest surface coverage (19.0%) and biofilm thickness (1.9 mm) were determined on the HIP Y-TZP ceramic; the highest mean values were identified with the lithium disilicate glass-ceramic (46.8%, 12.6 mm) [41,42]. On the other hand, Meier et al. [40] did not observe any significance. Our study showed that the percentage of polymicrobial biofilm coverage was five times less for the ceramic coated with SiC (16.9%) compared with the uncoated group (91.8%). Ceramic coating also presented significant reductions of CFU/mL in monomicrobial biofilms of *S. mutans* and *S. sanguinis*. This may be related to the alteration of the surface energy and slightly smoother surface of coated SiC surfaces, which can decrease the bacteria adhesion.

Because significant differences in bacteria adhesion were identified among the groups, the hypothesis that SiC coatings on ceramic will decrease monomicrobial and polymicrobial biofilm adhesion on the surface of the ceramic has to be accepted.

Furthermore, a study by Auschill et al. [45] found that biofilms form a very thin layer on ceramic, but demonstrated the highest viability compared with biofilms on amalgam, gold, and composite resin. Dal Piva et al. [28] found a lower value of bacterial adhesion for zirconia reinforced lithium silicate (ZLS) ceramic compared to zirconia partially stabilized by yttrium (YZHT) ceramic. Lithium disilicate glass-ceramics (LDS) have lower *S. mutans* adhesion than fully stabilized zirconia (FSZ) and partially stabilized zirconia (PSZ), but the difference was not reflected in early biofilm formation [46]. Polished feldspathic ceramics showed less *Candida albicans* adherence, whereas the adherence of streptococci was greater than *C. albicans* in all conditions [39].

Ceramic materials may exhibit positive or negative results in terms of cell viability, according to the ceramic composition [37,38]. Studies have demonstrated a significant decrease in cell viability for computer-assisted design (CAD) block feldspathic ceramics [47]. Feldspathic ceramic glass and glass-ceramics based on the 3CaO·P2O5-SiO2-MgO system were considered not cytotoxic by MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [39] and neutral red analysis [48], respectively. Additionally, lithia–silica glass-ceramic was biocompatible, promoting cell adhesion and proliferation after 21 days [38]. However, zirconia-reinforced lithium silicate (ZLS) ceramic showed severe initial cytotoxicity when in contact with fibroblasts for 24 h [28]. This study demonstrated that fluorapatite glass-ceramic with or without SiC coating presented no cytotoxicity on human periodontal ligament cells by CellTiter-Blue. SiC-coated surfaces seeded with HPdLF cells may foster cell survival similar to surfaces that were not coated with SiC. Additionally, cells appeared elongated and intimately attached to both ceramic surfaces by SEM images. Some cell-to-cell interaction on the surface of ceramics was observed (Figure 8). Because the biocompatibility of the materials can be evaluated by the direct interaction between the ceramic surface and the cells, we can affirm that SiC coating is biocompatible.

This study confirmed that SiC coating can decrease bacterial adhesion on the surface of ceramic veneers while maintaining biocompatibility with other cells. SiC recently demonstrated optimized color properties and corrosion resistance [21,22]. This coating can be applied to dental materials to improve their clinical performance.
