## 8 Discussion
[20] reported that the effect of fluoride activity on bacterial cells is dependent not only on the amount of ions but also on the pH of the material at the time of bonding, which might explain the differences in findings obtained with the materials tested. [PERSON] et al. [37] compared the antibacterial potential of Fuji II LC and a giomer and reported that the antibacterial effect of the giomer against _S. Mutans_ was superior to Fuji II LC. Conversely, [PERSON] [15] reported no antibacterial activity of Fuji IX against the tested bacteria. The anticariogenic effect of a fluoride-releasing material depends on the sustainability of fluoride release and the level of reduction in fluoride release over time [20]. Previous studies emphasized that GICs are capable of recharge when treated with fluoride-containing mouth rinses or dentifrices [38,39]. To the extent of the authors' knowledge, the effect of recharging the fluoride-releasing restorative materials with fluoride on their antibacterial activities has not yet been investigated. In the present study, as none of the tested fluoride-releasing materials showed antibacterial activity, all specimens were treated with a fluoride (900 ppm)-containing topical agent to provide additional fluoride supply or to recharge. However, for all materials, again, no inhibition zones were observed after topical fluoride application. Biofilm on the surface of restorative material leads to the development of secondary caries that will endanger the long-term survival of the restoration. Therefore, a restorative material having a low sensitivity to bacterial adhesion is always preferred [40]. Biofilm formation can be measured quantitatively and qualitatively with different methods such as CFU counting, SEM, and CLSM after staining of the bacteria [41,42, 26]. Nevertheless, all these techniques have some limitations and disadvantages. In the present study, crystal violet assay was used to evaluate biofilm formation. In vivo and in vitro studies conducted on bacterial adhesion to dental restoratives have also shown some differences [42,43]. These differences were reported to be related to the chemical structure and surface properties of the restorative materials [40]. In the present study, the fluoride-releasing materials were also tested in terms of 24 h biofilm formation. The same amount of inoculum and equal quantities were valid for all the specimens, but significant differences were detected in the biofilm formation of the materials. Equia Forte HT Fil either with or without coating showed the lowest biofilm formation. It was thought that, apart from fluoride, factors related to the materials have been involved in _S. mutans_ biofilm formation on the tested restorative materials. Therefore, the second null hypothesis was rejected. Although it has been well proven that the interaction of surface properties of a restorative material and biofilm formation can be influenced by the exploratory conditions of an in vitro study, it might also be assumed that the surface roughness has a significant effect on initial microbial adhesion [44,45]. Generally, rough surfaces are very attractive for bacteria with adhesion ability, as the contact area between the bacterial cell and the material is increased, as well as contribution to micromechanical retention [46]. A roughness (Ra) of 0.2 \(\upmu\)m was reported to be the threshold for maximum reduction in bacterial adhesion on the surface of restorations [45]. However, bacterial adhesion or biofilm formation dependson not only the surface roughness of the material, but also on the shape and size of the bacterial cells and other environmental variables [45]. In the present study, the surface roughness values of all fluoride-releasing materials were higher than the 0.2 \(\upmu\)m threshold with significant differences, leading to rejection of third null hypothesis. Therefore, it was not possible to establish a correlation between biofilm formation and surface roughness.