**1. Introduction**

Biofilms colonizing dental surfaces represent the habitat of a wide range of microorganisms. In healthy conditions, this microbial community provides fundamental benefits to host surfaces, including homeostasis of critical environmental parameters such as pH, as well as protection against

> 163

colonization by exogenous species. However, loss of this equilibrium can result in dysbiosis, leading to the onset of oral diseases, including dental caries [1,2].

Mechanical biofilm control can be beneficially associated with the use of active compounds to reduce biofilm formation, positively a ffecting the biological equilibrium of the ecosystem [3]. The search for new bioactive compounds to prevent dental caries development and progression has led researchers to focus their attention on the use of nanotechnologies, especially hydroxyapatite (HAp), metals, and metal oxide nanoparticles [4–6]. Nanotechnology applications o ffer the opportunity to modulate the formation of dental biofilms using nanoparticles with bioactive e ffects. Indeed, their possible use in biofilm control is related to some peculiar characteristics that materials acquire when nano-sized: shape, surface-to-volume ratio, chemical reactivity, and ability to interact with the bacterial cell wall directly [6–8]. Considerable e fforts have been made to clarify the transfer mechanisms of such nanoparticles through biofilms, indicating that nanoparticles can di ffuse through water channels inside biofilms, interacting both with the microbial cells and with the hard tissue surface.

Toothpastes containing nano-hydroxyapatite (n-HAp) and its precursors can provide calcium and phosphate ions to dental hard tissues, reducing demineralization, and promoting remineralization. N-HAp may also form a protective layer on dental hard tissues with chemical characteristics very close to natural tissues, showing biomimetic properties [9,10]. Biomimetic n-HAp or other calcium phosphate-based nanosystems have been shown to actively shift the ionic balance toward remineralization [11,12]. Recently, n-HAp-based toothpastes containing metal ions such as Zn, F, Mg, and Sr have been introduced to improve the remineralization processes. The small quantity of metal ions with which nHAp is doped modulates the properties of the compound already from its deposition, influencing crystal growth and length, and influencing its solubility and strength. They are firmly embedded inside the lattice and are not supposed to be released. Most studies, therefore, provide data about the remineralization potential of these compounds on surface lesions, with few reporting their influence on early microbial colonization (EC) and biofilm formation (BF) [13–16].

Resin-based composites (RBCs) provide an example of artificial surfaces intensely colonized by cariogenic biofilms due to their lack of bu ffering e ffect [17]. This characteristic has been put into relation with the high occurrence of secondary caries in the sound dental tissues surrounding an RBC restoration [17]. The possibility of influencing microbial colonization of RBCs, therefore, can be seen as a means to prevent the occurrence of such lesions. For this reason, RBCs are a potential target of preventive measures exploiting the potential of nanotechnologies in order to reduce secondary caries occurrence.

The aim of this study was to evaluate the in vitro effect of two n-Hap-based toothpastes on early colonization (EC) (12 h) and biofilm formation (BF) (24 h) of human enamel and RBC surfaces. The study was performed by aerobically culturing the monospecies *Streptococcus mutans* and an artificial oral microcosm using both a static and a continuous flow setup. The aim of the two models was to assess the e ffect of the tested toothpastes under environments simulating clinical conditions during the night (low salivary flow and clearance) and day (high salivary flow and clearance). The null hypothesis was that toothpaste treatment would not influence EC and BF on sound enamel surfaces in the tested models. An additional null hypothesis was that toothpaste treatment would not influence EC and BF on RBC surfaces in the tested models.

#### **2. Materials and Methods**

#### *2.1. Preparation of Specimens*

Reagents, culture media, and disposables used in this study were obtained from E. Merck AG (Darmstadt, Germany). A total of 110 human teeth extracted for clinical reasons were obtained and stored at −20 ◦C until use. The Institutional Review Board of the University of Milan approved the protocol (codename: SALTiBO-2017). Enamel disks 6.0 mm in diameter and 1.5 mm thick were cut from the teeth surfaces by means of custom-made water-cooled diamond trephine burs (Indiam, Carrara, Italy). A total of 213 enamel disks were prepared for the study, using this procedure to obtain more than one disk from each tooth.

A total of 213 disks (6.0 mm diameter and 1.5 mm thickness) of a nanohybrid resin-based composite (RBC, Clearfil Majesty ES-2, shade A2, Kuraray Europe GmbH, Hattersheim am Main, Germany) were obtained by packing an excess of uncured material into a custom-made PTFE mold. The top and bottom surfaces of the RBC were covered with a cellulose acetate strip (Mylar) and condensed against a glass plate by applying a load of 1 kg for 20 s. The specimens were then irradiated for 40 s by placing the tip of a light-curing unit (MiniLED, Satelec, Acteon Group, Merignac, France, 420–480 nm emission, 1250 mW/cm<sup>2</sup> irradiance) into direct contact with the acetate strip. To remove most of leachates, RBC specimens were then stored under light-proof conditions in phosphate-buffered saline (PBS) for 6 days at 37 ◦C, with the buffer replaced twice a day.

Both enamel and RBC disks were polished using grinding paper (1000, 2000, and 4000 grit) to obtain a surface roughness with Ra < 0.2 μm. They were then randomly divided into three groups (<sup>α</sup>, β, and control, n = <sup>71</sup>/group, Figure 1).

**Figure 1.** Diagram of specimens processing for the present study. After brushing with the tested toothpastes or with distilled water, enamel and RBC specimens were rinsed, sterilized, then subjected to microbiological analysis and surface imaging (SEM, CLSM) and analysis (EDS). The microbiological analysis included two test setups, two microbiological models, and two incubation times.
