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. 2020 Jun 1;11(2):36.
doi: 10.3390/jfb11020036.

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 , Eugenio Brambilla  1
Affiliations

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

Andrei C Ionescu et al. J Funct Biomater. .

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

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

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.
Figure 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
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.
Figure 4
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.
Figure 5
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.
Figure 6
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.
Figure 7
Figure 7
Immediate fluoride release (ppb/mm2 ± 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.
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