**3. Results**

#### *3.1. Surface Imaging and Analysis*

To investigate the presence of toothpaste residues on enamel and RBC surfaces as well as the composition of the tested toothpastes, specimens were observed using SEM-EDS in surface-charge reduction mode without sputter-coating, using an accelerating voltage of 15 KV (Figure 2). EDS results showed that the elemental composition of both toothpastes was very similar, except that α toothpaste contained zinc, while β toothpaste contained strontium, magnesium, and fluoride (Figure 3). Enamel specimens showed a significant increase in surface zinc content after treatment with α toothpaste, and a significant increase in strontium and magnesium content after treatment with β toothpaste (Table 2). Very interestingly, EDS detected the presence of calcium and phosphate on the surfaces of RBC specimens treated with both toothpastes, as well as traces of zinc on specimens treated with α toothpaste and magnesium on specimens treated with β toothpaste (Figure 2, Table 2).

**Figure 2.** The panel, to be read horizontally, depicts the SEM backscattered electrons micrographs (500×

and 5000×, respectively) and EDS elemental maps (5000×) of the specimen surfaces. The EDS maps are additionally presented as single-channel maps to better identify the topographical presence of each element. The first two rows of the panel display the surfaces of the vacuum-dried toothpastes tested in this study: α toothpaste (containing Zn-carbonate substituted n-HAp) and β toothpaste (containing F, Mg, Sr-carbonate substituted n-HAp). Their aspect is very similar, showing silica microparticles (identified by Si signal) and clusters of n-HAp (identified by Ca and P signals). Zn and Sr signals were below the detection limits in mapping mode and were not displayed. The last two rows of the panel represent the surfaces of the tested restorative material after treatment with the toothpastes: RBC + α toothpaste and RBC + β toothpaste. The RBC composition included silica particles (identified by Si signal), and alumina and barium glass micro and nanoparticles (identified by Al and Ba signals). Ca signal was displayed as a marker of toothpaste remnants. It is noteworthy that toothpaste remnants could be associated with alumina and barium glass fillers rather than silica particles.

**Figure 3.** EDS spectra acquired from vacuum-dried tested toothpastes surfaces (α toothpaste containing Zn-carbonate substituted n-HAp, and β toothpaste containing F, Mg, Sr-carbonate substituted n-HAp). Strong Ca and P signals are identified belonging to the n-HAp, as well as the signals corresponding to the corresponding doping elements (Zn in α toothpaste and F, Mg, Sr, in β toothpaste). High counts of Si were also detected in both toothpastes, together with relatively low counts of Al and other elements. The relative amounts of n-HAp doping elements and other elements such as Al and S are below the conventionally considered detection limit of EDS (about 1 wt%). However, the presence of such elements is shown by peaks that were clearly identifiable on all acquired spectra. In this sense, the acquisition of several spectra over the surfaces of many specimens, and the use of statistical analysis on acquired data demonstrating low variability in signals among the different acquisitions (cf. Table 2) allows overcoming, to some extent, this detection limit, improving the performances of EDS analysis.



## *3.2. Microbiological Results*

The effect of the tested toothpastes on EC and BF, expressed as mean OD values ± 1 standard error, according to test setup are shown in Figures 4 and 5. ANOVA results showed an overall significant influence of the tested toothpastes on both EC and BF, with a significant interaction between the factors, toothpaste, and microbiological model (*p* = 0.017). No significant interactions were found between toothpaste and incubation time, adherence surface, or test setup, meaning that the toothpastes had the same effect on microbial viability both in EC and BF, independently from the type of bioreactor used. Most importantly, the tested toothpastes had the same effect on microbial viability, both on enamel and RBC surfaces. On the contrary, ANOVA highlighted a significant interaction (*p* < 0.0001) among adherence surface, test setup, microbiological model, and incubation time.

**Figure 4.** Biofilm formation on the surface of the tested specimens using the shaking multiwell plate test setup, according to the microbiological model (*S. mutans* monospecific biofilm or artificial oral microcosm, aerobically grown) and to the incubation time (12 h or 24 h). Sucrose-enriched sterile modified artificial saliva medium was used in all experiments. Low shear stress on specimens' surfaces was obtained by an orbital incubator, to simulate oral conditions during the night. Moreover, the closed system setup allows a progressive increase in microorganism catabolites and antimicrobial agents released from the surfaces. Results of viable biomass assay are expressed as mean OD ± SE. Different superscript letters indicate significant differences between groups (student's test, *p* < 0.05). α toothpaste contains Zn-carbonate substituted n-HAp; β toothpaste contains F, Mg, Sr-carbonate substituted n-HAp while Ctrl group was brushed with distilled water. β toothpaste significantly reduced the early colonization of the artificial oral microcosm on enamel surfaces when compared to the control.

Considering the results of the post-hoc test on the toothpaste factor, the artificial oral microcosm model showed significantly lower EC and BF on group β disks compared to the control, with group α disks showing lower EC and BF compared to control, but higher EC and BF compared to β toothpaste. Surprisingly, this pattern was found on both tested adherence surfaces, demonstrating an influence of toothpastes on EC and BF even over RBC surfaces. In the monospecific *S. mutans* model, the toothpastes did not significantly influence EC and BF.

**Figure 5.** Biofilm formation on the surface of the tested specimens using the MDFR bioreactor test setup, according to the microbiological model (*S. mutans* monospecific biofilm or artificial oral microcosm, aerobically grown) and to the incubation time (12 h or 24 h). Sucrose-enriched sterile modified artificial saliva medium was used in all experiments, being pumped through the flow-cells of the bioreactor. High hydrodynamic stress conditions that occur during the daytime can thus be simulated. An elution of microorganism catabolites and antimicrobial agents released from the surfaces can also occur. Results of viable biomass assay are expressed as mean OD ± SE. Different superscript letters indicate significant differences between groups (student's test, *p* < 0.05). α toothpaste contains Zn-carbonate substituted n-HAp; β toothpaste contains F, Mg, Sr-carbonate substituted n-HAp while Ctrl group was brushed with distilled water. β toothpaste significantly reduced early colonization (enamel and RBC) and biofilm formation (RBC) of the artificial oral microcosm when compared to the control. Interestingly, the adherence surface showed a more considerable influence than toothpaste treatment on early colonization and biofilm formation, independent of the microbiological model or test setup applied. The effect of the toothpaste on RBC surfaces was not expected and opens the possibility to control microbial colonization on RBCs, and, ultimately, secondary caries prevention, by such treatments.

Considering the role of the adherence surface, the oral microcosm model showed significantly higher EC and BF on enamel than on the tested RBC in both test setups (shaking multiwell plate and MDFR). *S. mutans* showed significantly higher EC and BF on the tested RBC than on enamel in the shaking multiwell plate test setup and significantly higher BF on the tested RBC than on enamel in the MDFR.

Regarding incubation time, as expected, there was an overall significant increase in biomass over time. The artificial oral microcosm grown in the MDFR on enamel surfaces showed no significant difference in viable adherent biomass between 12 and 24 h incubation (*p* = 0.0711). Likewise, *S. mutans* grown in the MDFR on both adherence surfaces showed no significant difference in viable adherent biomass between 12 and 24 h incubation (*p* = 0.5665 and *p* = 0.7691 for enamel and RBC, respectively).

From the test setup point of view, MDFR showed an overall significantly higher EC and BF than the shaking multiwell plate, except for BF of artificial oral microcosm on enamel surfaces.

Considering the microbiological model, the artificial oral microcosm showed higher EC and BF than *S. mutans* on enamel surfaces in both test setups, whereas *S. mutans* showed higher EC and BF than the artificial oral microcosm in the shaking multiwell plate on RBC surfaces.

A set of additional enamel and RBC specimens were made and treated with the toothpastes or with the control, as described in the Methods section, to provide a morphological view of the microbial colonization. The most biologically meaningful result was found to be the one obtained using the MDFR test setup and the artificial oral microcosm model for 12 h (EC, Figure 5). Therefore, this particular situation was furtherly investigated using confocal laser-scanning microscopy and live/dead staining. The results are illustrated in Figure 6. Enamel specimens showed a uniform microbial layer colonizing all surfaces, with multilayered structures starting to develop. The aspect is in keeping with the viable biomass results, showing early colonization of β toothpaste < α toothpaste = control. On enamel specimens treated with β toothpaste, a slightly higher amount of dead cells and microcolonies can be seen. RBC specimens showed much lower colonization than enamel, showing early colonization of β toothpaste < α toothpaste < control. The surfaces of the specimens provided an intense red fluorescence that was not wholly subtracted by parallel scanning in the near-UV 405 nm channel. This artifact was not due to dead cells, but rather to autofluorescence coming from the RBC, and also to an a ffinity of the propidium iodide dye with RBC surfaces, as previously shown [22].

**Figure 6.** CLSM results of the adherence surfaces treated with the control and the tested toothpastes using the MDFR test setup and the artificial oral microcosm model for 12 h. Scans were analyzed 3D reconstructions obtained using Drishti software. Enamel surfaces provided a much higher early colonization than RBC surfaces. In the background of the enamel control specimen, a central microcolony shows a long tail detached from the surface and oriented horizontally downstream (to the right). This feature is typical of biofilms that develop on surfaces in the presence of relatively high shear stress and is also a means to colonize downstream surfaces rapidly. This feature demonstrates the good morphological resemblance of bioreactor-grown biofilms with in vivo ones. Enamel specimens treated with β toothpaste showed a higher amount of dead cells compared to the other groups. RBC specimens treated with β toothpaste showed the lowest early colonization overall, consistently with viable biomass results.

#### *3.3. Fluoride Release Results*

The fluoride release from the disks treated with the fluoride-hydroxyapatite containing toothpaste, and the fluoride content of the biofilms after 12 and 24 h are displayed in Figure 7. A significant albeit shallow release of fluoride was seen from enamel specimens treated with β toothpaste. Contrary to RBC specimens, enamel specimens showed biofilms containing significant amounts of fluoride at both incubation times. After 12 h, treatment with β toothpaste significantly decreased fluoride presence in the overlying biofilm compared to the control (*p* < 0.001).

**Figure 7.** Immediate fluoride release (ppb/mm<sup>2</sup> ± SE) after disks' brushing and fluoride presence (ppm ± SE) in the biofilms grown over the disks' surfaces after 12 and 24 h. Different superscript letters indicate significant differences between groups (Student's test, *p* < 0.05). β toothpaste contains F, Mg, Sr-carbonate substituted n-HAp while Ctrl group was brushed with distilled water. β toothpaste did not increase the baseline fluoride presence in biofilms grown over enamel surfaces despite showing a low but significant immediate fluoride release. The reduction in fluoride presence seen in enamel specimens after 12 h of incubation when compared to the control may be due to an uptake of fluoride by the tested toothpaste.
