*Article* **Substituted Nano-Hydroxyapatite Toothpastes Reduce Biofilm Formation on Enamel and Resin-Based Composite Surfaces**

#### **Andrei C. Ionescu 1,\*, Gloria Cazzaniga 1, Marco Ottobelli 1, Franklin Garcia-Godoy 2 and Eugenio Brambilla 1**

1 Oral Microbiology and Biomaterials Laboratory, Department of Biomedical, Surgical and Dental Sciences, University of Milan, via Pascal 36, 20133 Milan, Italy; gloria.cazzaniga@yahoo.it (G.C.); marco.ottobelli@unimi.it(M.O.);eugenio.brambilla@unimi.it(E.B.)


Received: 3 April 2020; Accepted: 21 May 2020; Published: 1 June 2020

**Abstract:** *Background*: Toothpastes containing nano-hydroxyapatite (n-HAp) substituted with metal ions provide calcium and phosphate ions to dental hard tissues, reducing demineralization, and promoting remineralization. Few data are available about the effect of these bioactive compounds on oral microbiota. *Methods*: This in vitro study evaluated the influence of two commercially-available substituted n-HAp-based toothpastes ( α: Zn-carbonate substituted n-HAp; β: F, Mg, Sr-carbonate substituted n-HAp) on early colonization (EC, 12 h) and biofilm formation (BF, 24 h) by oral microbiota. Controls were brushed with distilled water. Artificial oral microcosm and *Streptococcus mutans* biofilms were developed using human enamel and a resin-based composite (RBC) as adherence surfaces. Two test setups, a shaking multiwell plate and a modified drip-flow reactor (MDFR), were used to simulate clinical conditions during the night (low salivary flow and clearance) and daytime, respectively. Energy-dispersive X-ray spectrometry (EDS) was used to evaluate specimens' surfaces after toothpaste treatment. Fluoride release from β toothpaste was evaluated. Viable adherent biomass was quantified by MTT assay, and biofilms' morphology was highlighted using confocal microscopy. *Results*: EDS showed the presence of remnants from the tested toothpastes on both adherence surfaces. β toothpaste showed significantly lower EC and BF compared to control using the artificial oral microcosm model, while α toothpaste showed lower EC and BF compared to control, but higher EC and BF compared to β toothpaste. The effect shown by β toothpaste was, to a minimal extent, due to fluoride release. Interestingly, this result was seen on both adherence surfaces, meaning that the tested toothpastes significantly influenced EC and BF even on RBC surfaces. Furthermore, the effect of toothpaste treatments was higher after 12 h than 24 h, suggesting that toothbrushing twice a day is more effective than brushing once. *Conclusions*: The efficacy of these treatments in reducing microbial colonization of RBC surfaces may represent a promising possibility in the prevention of secondary caries.

**Keywords:** fluoride(s); biofilm(s); *Streptococcus mutans*; bioreactor(s); enamel; composite materials; biomaterials; hydroxyapatite; nanostructured materials; dental
