## 4 Discussion  
Here, we provide the first in vitro data concerning the effects of the Biorepair-PERIBIOMA toothpaste and chewing gum on the ability of oral microbiota to adhere, grow and produce biofilm onto orthodontic devices. Orthodontic therapy with fixed appliances is commonly used to treat malocclusions and teeth irregularities; in these cases, orthodontic materials such as brackets, tubes and elastics are widely employed. These materials prevent an accurate oral hygiene and promote an increase in microbial load, deposition of debris and formation of plaque. With the aim of promoting a better oral hygiene, a new toothpaste, fluorine-free, and a chewing gum, Biorepair(r) PERIBIOMATM, added with probiotics, have recently been developed; little information exists on their mechanisms of action. Among the most important components in the oral environment, saliva is an integral part of oral health; particularly, it plays a role in caries control and bacterial plaque containment [8]. Although being sterile when secreted [38], saliva immediately becomes a major vehicle of microorganisms, either resident microbiota in healthy subjects as well as pathogens in patients with oral cavity diseases. From here, our choice of using saliva from healthy donors as source of oral microbial population to be employed in an in vitro model that, by mimicking orthodontic materials contamination, would allow to evaluate the efficacy of novel tools for oral hygiene. The Biorepair(r) PERIBIOMATM gum and a traditional gum, both sugar-free, have been used to facilitate saliva collection. In line with the literature [39], we show that the microbial species, isolated by a culture-based approach from saliva A and P are mainly _S. mitis/oralis_ and _S. salivarius_, followed by _N. flava/subflava_  
Figure 3: Biofilm persistence following Tp-SUP treatment. The saliva-contaminated OE, that had been exposed to Tp-Sup or saline (at time 23 h, for 1 h) were further incubated at 37 \({}^{\circ}\)C + 5% CO\({}_{2}\) up to 48 h in fresh medium. Then, the OE were label with CFDA for 1 h to allow fluorescence staining, washed with warm saline and transferred to new wells containing fresh medium. The plate was read by the Fluoroskan microplate reader and the fluorescence signal was recorded. The depicted values represent the mean RFU/OE \(\pm\) SEM of 15 replicates obtained by 3 independent experiments. \(\mathbf{***}\)\(p<0.001\); \(\mathbf{*}\)\(p\leq 0.05\). and _S. thermophilus_. Interestingly, the number of CFU observed in saliva P is slightly higher (397 vs. 501 CFU/100 \(\mu\)L \(\times\) 10\({}^{4}\) A vs. P samples) as compared to that of saliva A; unexpectedly, the relative abundance of _S. salivarius_ appears to be enhanced in the latter group (16.88% vs. 2.99%, A vs. P samples). These results suggest that the gum P is slightly more effective in mechanically removing local microorganisms than a traditional gum. The partial fluctuation in relative abundance of some species may be due to their different susceptibly to mechanical gum-mediated detachment and/or to the different ingredients contained in each gum. Being adhesion a crucial step in microbial biofilm formation, both on oral tissues and abiotic surfaces, here we investigated the ability of microbial communities occurring in saliva A and saliva P to adhere onto OE. As indicated by the fluorescence data, adhesion occurs, irrespectively of the saliva employed; yet, the phenomenon is less pronounced when using saliva P, implying that, although slightly more numerous, this microbial population exhibits a reduced efficacy on binding to an abiotic surface, such as OE. Whether the slightly higher numbers of _S. initis/oralis_ detected in saliva P may account for the overall less efficient adhesion, remains to be established. Furthermore, we cannot exclude that, in our in vitro model, hydroxyapatite crystals and probiotics, present in both Peribioma products, remain and persist in saliva P samples and, thus, may interfere with microbial adhesion to OE.