*2.2. Toothpastes*

Two toothpastes containing n-HAp were tested (Table 1). The toothpastes were inserted into test tubes coded by the Greek letter corresponding to the first two groups (<sup>α</sup>, β). In this way, the experimenters performing microbiological procedures were blinded regarding their composition. Each toothpaste was mixed to a slurry using one part of toothpaste and two parts of distilled water. Then, enamel and RBC disks of groups α and β were manually brushed by a single operator with the corresponding toothpaste for 2 min and rinsed with distilled water for 1 min. Disks belonging to the control group were brushed with distilled water for 2 min, then rinsed for 1 min with distilled water. All disks were then sterilized using a chemical peroxide-ion plasma sterilizer (STERRAD, ASP, Irvine, CA, USA).


**Table 1.** Label, manufacturer and composition of the tested substituted n-Hap-containing toothpastes.

#### *2.3. Surface Imaging and Analysis*

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis were performed (n = <sup>4</sup>/group, Figure 1) using a TM4000Plus Tabletop scanning electron microscope (Hitachi, Schaumburg, IL, USA) equipped with an EDS probe (Q75, Bruker, Berlin, Germany) to investigate the presence of toothpaste residues on the specimen surface. Dry specimens were observed in surface-charge reduction mode without sputter-coating, using an accelerating voltage of 15 KV. Three randomly selected fields were acquired for each specimen at 500× magnification and were analyzed using the EDS probe in full-frame mode using an acquisition time of 150 s. SEM micrographs (backscattered electrons mode) and EDS elemental maps of the surfaces at 5000× magnification were also obtained. The acquired data represent the elemental composition of the ≈1 μm superficial layer.

#### *2.4. Saliva Collection*

Whole saliva was collected from five healthy volunteers, according to a previously described protocol [18]. They refrained from oral hygiene for 24 h, did not have any active dental disease, and did not have antibiotic therapy for at least three months prior to the experiment. Chilled test tubes were used for saliva collection. Saliva was then pooled, heated to 60 ◦C for 30 min and centrifuged (27,000× *g*, 4 ◦C, 30 min). The sterile supernatant was collected into sterile tubes and stored at −20 ◦C. Saliva was thawed at 37 ◦C for 1 h before use. The Institutional Review Board of the University of Milan approved the use of saliva (codename: SALTiBO-2017), and written, informed consent was obtained from all volunteers.

#### *2.5. Microbiological Procedures*

Two microbiological models were used in this study: a monospecific *S. mutans* culture and an artificial oral microcosm based on mixed oral flora (Figure 1).

A pure suspension of *S. mutans* strain ATCC 35668 was obtained as described elsewhere [19]. Briefly, Mitis Salivarius Bacitracin agar (MSB agar) plates were inoculated with the *S. mutans* strain and incubated aerobically in a 5% CO2-supplemented atmosphere at 37 ◦C for 48 h. After transferring an inoculum in Brain Heart Infusion (BHI) broth and further incubating in a 5% CO2-supplemented atmosphere at 37 ◦C for 12 h, a pure culture of the microorganism was obtained. Cells were harvested by centrifugation (2200× *g*, 19 ◦C, 5 min), washed twice with sterile phosphate-buffered saline (PBS), and resuspended in the same buffer., The suspension was sonicated using low-energy output (7W for 30 s, B-150 Sonifier, Branson, Danbury, CT, USA) to disperse bacterial chains. Finally, the suspension was adjusted to an optical density of 1.0 on the McFarland scale, corresponding to a concentration of approximately 6.0 × 10<sup>8</sup> cells/mL.

A mixed oral flora inoculum was obtained from fresh, pooled saliva expectorated from the same five donors previously described, filtered through sterile glass wool, vigorously stirred for 2 min, and immediately used. Two test setups were used in this study: a shaking multiwell plate and a modified drip-flow reactor (MDFR).

#### 2.5.1. Shaking Multiwell Plate

Disks were placed in 48-well sterile plates and incubated at 37 ◦C for 24 h in sterile saliva; then, the saliva was removed by gentle aspiration. Each well was inoculated with 100 μl of either *S. mutans* suspension or mixed oral flora, and 900 μl of sterile modified artificial saliva medium. The medium composition was the following: 10.0 g/<sup>L</sup> sucrose, 2.5 g/<sup>L</sup> mucin (type II, porcine gastric), 2.0 g/<sup>L</sup> bacteriological peptone, 2.0 g/<sup>L</sup> tryptone, 1.0 g/<sup>L</sup> yeas<sup>t</sup> extract, 0.35 g/<sup>L</sup> NaCl, 0.2 g/<sup>L</sup> KCl, 0.2 g/<sup>L</sup> CaCl2, 0.1 g/<sup>L</sup> cysteine hydrochloride, 0.001 g/<sup>L</sup> hemin, and 0.0002 g/<sup>L</sup> vitamin K1 [20]. Plates were then incubated aerobically in an orbital shaker at 37 ◦C and 100 rpm. After either 12 h (colonization) or 24 h (biofilm formation), adherent viable biomass assessment was performed.
