## 4 Discussion
In line with these data, we show that, after 24 h incubation, the Saliva P/saline group had 1 log lower microbial CFU than the counterpart samples (Saliva A/saline); once again, we may hypothesize that the hydroxyapatite, contained in gum P and likely persisting in Saliva P samples, may have limited microbial growth. To a similar extent, the Saliva P significantly reduces biofilm formation onto the OE (time 24 h). This phenomenon is also evident at a later time, indicating that saliva collected after chewing the gum P consistently affects biofilm persistence more than saliva A (17.42 vs. 33.84 RFU, respectively; at time 48 h). Here, we have used the Tp-SUP as a simplified and easy-to-use tool, for assessing toothpaste effects against oral microorganisms _in vitro_. In particular, we show that the Tp-SUP exerts relevant antimicrobial activity, regardless of the saliva used to contaminate the orthodontic devices; specifically, both parameters, namely microbial growth and biofilm formation, are significantly impaired. The Tp-SUP-mediated microbial abatement ranges between 1 and 2 logs, which is already evident at time 24 h, when using the CFU assay. In contrast, at later times (48 h), microbial re-growth occurs comparably in all the groups, irrespective of the gum initially used for saliva collection and independently upon the in vitro Tp-SUP treatment. Furthermore, it should be noted that, at that time, the microbial composition greatly varies upon Tp-SUP exposure; in particular, the streptococci mostly detected in saliva A and saliva P/saline groups (>90%) are drastically displaced by Lactobacillus in saliva A and saliva P/Tp-SUP groups (>90%). This finding may be explained considering that the Biorepair(r) PERIBIOMA\({}^{\text{TM}}\) products also contain probiotics, such as _L. reuteri_, _L. salivarius_, _L. plantarum_ and _Bifidobacterium_. The reason for adding probiotics to such oral care products is that such microbial species may adhere to dental tissues and become part of the oral plaque; in turn, they may compete with cariogenic and periodontal pathogens for nutrients and space, thus preventing caries formation and gingivitis [40; 41]. In our hands, the late detection of Lactobacillus is in line with previous results (data not shown) showing that, indeed, Tp-SUP culture allowed growth of colonies subsequently identified as Lactobacillus. The efficacy of Tp-SUP is further emphasized by the fact that also biofilm persistence is significantly affected after such treatment: the extent of the inhibition is similar both in saliva A and saliva P contaminated OE. Overall, by an in vitro model, we provide the first evidence on the efficacy of the Biorepair(r) PERIBIOMA\({}^{\text{TM}}\) toothpaste and gum in impairing adhesion, growth and biofilm formation/persistence by oral microorganisms onto orthodontic devices. We favor the idea that, by different pathways, the hydroxyapatite crystals and the probiotics, abundantly present in such oral care products, may act in concert controlling local microbial communities. Moreover, based on the present in vitro data,we may envisage that the combined use of toothpaste and gum can ameliorate oral daily care, thus clinically reducing the risk of developing oral diseases, especially those related to microbial agents.