In Vitro morphological, optical and microbiological evaluation of nanosilver fluoride in the remineralization of deciduous teeth enamel

1 Introduction

Dental caries is one of the most prevalent and costly diseases in the world [1], and untreated dental caries in children remain a public health challenge in poor communities [2]. Control of dental caries is extremely difficult, especially because it has a multifactorial etiology. The progression or reversion of dental caries depends on the balance between demineralization and remineralization and can be visualized for clinical purposes as the caries balance. The knowledge of fluoride action on enamel demineralization and remineralization is an important advancement for the control of the disease [3]. Over the years, several fluoride preparations have been developed for topical application, with varying concentrations for dental caries control [4], [5], [6], [7], [8], [9], [10]. However, if the pathological factors, such as acid-producing bacteria, outweigh the protective factors, such as fluoride remineralization and antimicrobial use, the caries progresses [3]. Streptococcus mutans is considered the primary agent of dental caries and contributes significantly to the virulence of dental biofilms in the presence of sucrose. Silver nanoparticles may be effective for controlling S. mutans [9], [10], and the antimicrobial effect is better when the nanoparticle is only a few nanometers in diameter. Particles smaller than 10 nm are considered to have a high performance against pathogens of the dental biofilm [11].

Silver nanoparticles have shown high antibacterial properties against Gram-negative and Gram-positive bacteria [11] because of their ability to damage the cell wall, while silver nanoparticles accumulate in the bacterial membrane, which causes a significant increase in permeability, resulting in the death of the cell [11], [12], [13]. This antibacterial ability is especially important in deciduous teeth, as they have a thinner and more permeable enamel layer than permanent teeth and they are more vulnerable to the acid action of these bacteria and, therefore, more susceptible to dental caries [14].

Nanoparticles could control the formation of oral biofilm because of their biocidal and anti-adhesive microbial capabilities. As such, particles are reduced from micrometer to nanometer size, therefore altering active surface area, hardness, chemical reactivity and biological activity. For this reason, with respect to metallic nanoparticles, the biocidal effectiveness has been suggested to be due to both their size and high surface-to-volume ratio [11].

Nanosilver fluoride (NSF) and sodium fluoride (NaF) with cariostatic action were described successfully in a controlled clinical trial that showed effectiveness in paralyzing dentine caries in children from poor communities. Unlike silver diamine fluoride (SDF), the NSF did not show distinctive tissue darkening due to the oxidation of silver ions when in contact with the teeth [9]. The effectiveness of NSF in the arrestment of caries is attributed to the synergistic actions of both silver nanoparticles and fluoride. However, the mechanism of arrestment is not clear.

Therefore, the mechanism of incorporating NSF in the enamel surface needs to be better studied, as well as its action on the healthy tissues of the tooth.

To evaluate the effectiveness of dental enamel remineralization through the NaF and NSF, three different methods were used: microhardness, fluorescence spectroscopy and imaging by optical coherence tomography (OCT).

The microhardness test is an indirect test with excellent acceptance for experiments that test de-remineralization processes, as it has the ability to evaluate quantitatively the degree of mineralization through the measurement of the mineral level of the dental enamel [15], [16].

Fluorescence spectroscopy at the visible ultraviolet (UV-VIS) spectral range is a widely used tool for the evaluation of enamel demineralization due to its ability to detect minimal mineral changes with high sensitivity and specificity, providing an accurate identification of the carious lesion in the incipient stages [17], [18]. The dental enamel can fluoresce upon photon excitation, and mineralized and demineralized tissues can be differentiated. In general terms, it consists of a quantitative and non-invasive technique that captures the signal, resulting from the interaction of the tissue excited by monochromatic light, through a detector that converts the optical signal to digital signal, visualized in the spectral form, which allows differentiation between healthy and demineralized tissues when the ultraviolet excitation wavelength is used (405-nm led-induced fluorescence spectroscopy) [19], [20], [21]. This is relevant in dental practice because of the possibility of its clinical application.

Another method to evaluate the action of fluoride in demineralizing/remineralizing on the tooth enamel is the OCT, which allows the accurate evaluation of enamel mineral losses. It is a non-invasive, non-destructive, non-ionizing, real-time diagnostic method with high sensitivity and specificity in detecting early lesions on smooth and occlusal surfaces and follow-up of lesion progression [22]. It is a well-accepted and exploited method in dentistry [22], [23], [24], and it was chosen to analyze the effectiveness of NSF in the remineralization treatment and ratify the findings obtained by the microhardness test and fluorescence spectroscopy.

The acid action of caries pathogens is more harmful in deciduous teeth due to the histological characteristic of this enamel [14], [25]. Among these pathogens, Streptococcus mutans stands out. For this reason, bacterial control is fundamental in the process of reversion of the caries at the initial stages, and microbiological assay was performed to complement this work.

The objective of this study was to test the remineralizing action of NSF on incipient enamel caries and its antimicrobial action in relation to enamel adhesion and acid production of Streptococcus mutans.

2 Methods

This study was approved by the Federal University of Paraíba Ethics Committee (Protocol No. 0469/15 and CAAE 48033215.0.0000.5188) in accordance with the World Medical Association Declaration of Helsinki.

This study was carried out in two stages. In the first stage, microhardness test, fluorescence spectroscopy and OCT image capture were performed to evaluate the performance of NSF on the enamel remineralization process. In the second stage, microbiological tests were carried out to evaluate the action of the NSF on Streptococcus mutans.

The assay solutions were as follows: NSF [400 ppm silver nanoparticles/5000 ppm NaF] (test solution), NaF [5000 ppm] (positive control) and deionized water (negative control).

2.1 NSF synthesis and characterization

To prepare the NSF, 1.0 g of chitosan, a carrier to stabilize the nanoparticles, was dissolved in 200 ml of 2% (v/v) acetic acid solution. Then, 4.0 ml of silver nitrate solution (AgNO₃) at a concentration of 0.012 mol/l was added to 60 ml of chitosan solution and left for 30 min before adding sodium borohydride (NaBH₄). NaF was added at a concentration of 5000 ppm and stirred continuously until it was completely dissolved. Soon after the synthesis, NSF was stored in a refrigerator [26], [27]. UV-VIS spectroscopy and transmission electron microscopy (TEM) were performed for the NSF characterization.

UV-VIS analysis showed a peak at the 400-nm wavelength, confirming the presence of silver nanoparticles in the colloid, and a TEM image showed monodispersal and spherical silver particles with a diameter of 8.7±3.1 nm [28], as shown in Figure 1.

Figure 1:

NSF characterization.

2.2 Specimen preparation and experimental design

All 33 samples were randomly distributed into one of three groups (n=11) according to the treatment applied and as described in Table 1, and then submitted to a demineralization process through pH cycling. The samples in the negative control group did not receive any remineralizing solution.

Table 1:

Description of the studied groups.

Groups	Type	Remineralizing solution
G1	Positive control	NaF
G2	Experimental group	NSF
G3	Negative control	None

The samples were washed in deionized water and kept in phosphate-buffered saline (PBS) solution (8 g/l NaCl, 2 g/l KCl, 2 g/l Na₂HPO₄, 2 g/l KH₂PO₄, pH 7.0) for ionic enamel restoration until further microhardness test, fluorescence spectroscopy and OCT. Measures and images were acquired at three different times: the initial stage (T0), the demineralization stage (after the chemical caries induction of caries – T1) and the remineralization stage (post-cariogenic challenge – T2).

At T1, the application of an acid pH solution removed the minerals from the enamel to induce the formation of a subsurface lesion. To avoid the erosive process in the adamantine tissue, which is characteristic of low-pH substances, it was necessary to saturate the solution in relation to the enamel. A 0.05 m acetate buffer solution containing 1.28 mm calcium, 0.74 mm phosphate and 0.03 μg fluorine/ml solution with a pH of 5.0 was used in the specimens, which were kept individually immersed in a 2 ml/mm² volume of this solution for 16 h at 37°C [29].

At T2, the pH cycling was carried out over 14 days as shown in Figure 2. The daily procedure included immersing the samples in demineralizing solution for 6 h, followed by 18 h of immersion in remineralizing solution. The demineralizing solution was composed of 2.0 mmol/l Ca, 2.0 mmol/l P and 75 mmol/l acetate buffer and had a pH of 4.4, while the remineralizing solution was composed of 1.5 mmol/l Ca, 0.9 mmol/l P, 130 mmol/l KCl and 20 mmol/l sodium cacodylate buffer and had a pH of 7.0. All samples were individually washed with deionized water for 1 min prior to the application of the tested substance (positive control, experimental solution or negative control). All samples were washed again with deionized water before being immersed in the solution for the new cycle. The solutions were maintained at a temperature of 37°C in a biological stove [30]. All the solutions of this study were produced by the department of chemical engineering in the Federal University of Pernambuco (Brazil).

Figure 2:

Routine for 24-h pH cycling stages, repeated for 14 days.

3 Results

Table 3 describes the results of the microhardness tests and fluorescence spectroscopy for the NSF, NaF and negative control groups at T0, T1 and T2. It was observed that there was a statistically significant difference for all variables in relation to the negative control group.

Figure 3 shows the performance of all the groups at T0, T1 and T2 through the fluorescence (intensity peak and total intensity) and microhardness tests: all the enamel samples were demineralized at T1 and only the NaF and NSF groups were remineralized at T2.

Figure 3:

Fluorescence spectroscopy in (A) intensity peak, (B) total intensity (in arbitrary units) and (C) microhardness measurements (in Vickers hardness) obtained from dental enamel at T0 (before pH cycling), T1 (post-caries induction) and T2 (post-enamel treatment with NaF, NSF and deionized water).

The principle of using a laser beam for diagnosis relies on the fact that an altered mineralized surface irradiated by a longitudinal light wave emits fluorescent radiation. If the emission of light occurs uniformly on the surface of the sound enamel, it is not possible to observe spots that denounce the presence of caries. In the microhardness test, a diamond pyramidal indentation for Vickers hardness measurement was observed on the enamel surface.

Figure 4 shows a representative image of a sound tooth sample before submitting it to pH cycling, during the fluorescence and microhardness tests.

Figure 4:

Presentation of the fluorescence in sound enamel deciduous tooth (A) and diamond pyramid indentation for Vickers hardness measurement (B).

Figure 5 shows a representative image of a sound tooth sample before submitting it to pH cycling. Note the enamel layer over the dentin and the dark line between them, which represents the dentin-enamel junction (DEJ). An A-scan (yellow curve) is shown, in which the tissue interfaces are evidenced by the peaks in the graph.

Figure 5:

OCT image showing the enamel and dentin layers and also the DEJ. These anatomical structures are confirmed by the A-scan of a selected point, presented in the form of yellow lines, showing the peaks of various tissue layers due to the differences in their refractive indexes.

The OCT analysis was also performed at T0, T1 and T2. As already described in the literature [22], [24], differences can be observed in the teeth submitted to a cariogenic challenge, as shown in Figure 6. For instance, in the T1 images (Figures 6B, E), it is not possible to visualize the DEJ: this is probably due to the higher backscattering of the demineralized enamel, which does not allow light to reach the dentin.

Figure 6:

OCT images acquired from representative samples of the NSF group (A, B and C) and negative control (D, E and F) at T0, T1 and T2 phases.

4 Discussion

The mechanism of action of fluoride ions in the remineralization process occurs when there are interactions capable of decreasing the solubility of dental enamel [5], [34], [35]. Fluorine accelerates remineralization by adsorbing itself to the crystal surface attracting calcium and phosphate ions, resulting in a new mineral with a composition between hydroxyapatite and fluorapatite [36]. Fluorine acts as a catalyst by increasing the amount of hydroxyapatite and forming the fluorinated hydroxyapatite when the surface is exposed for at least 1 min to fluorine [34], [36]. To evaluate the mineral alterations, quantitative tests were performed in this study, which allowed the analysis of the deremineralization process in the enamel of the deciduous teeth tested.

NSF was tested as a dental enamel remineralizing agent. It was compared with NaF and a negative control through the microhardness test, fluorescence spectroscopy and OCT imaging.

The microhardness test and the fluorescence spectroscopy were used to evaluate dental enamel demineralization due to their high sensibility and specificity to the detection of minimal mineral changes [15]. Table 3 and Figure 3 show the performance of each group at T0, T1 and T2. It was observed that at phase T2, the NaF and NSF groups presented similar behavior with no significant difference between the remineralization pattern through fluorescence spectroscopy (total intensity) and microhardness test (p>0.005). This can be explained by the ionic stability of the silver nanoparticles, which do not react when inserted in the various biomaterials [11] with no interference on the fluoride ability to reverse the de-remineralization process observed in the NSF group. Otherwise, the negative control group was not able to promote remineralization in the enamel and presented inferior performance compared to the NSF and NaF groups in both results of the presented tests (fluorescence spectroscopy p<0.001 and microhardness p=0.001), with the result expected due to the absence of remineralizing agent.

The OCT presents limitations for study of great depths, for example, in dentin, because a greater scattering can further reduce image quality [23], [37]. However, in this study, the thickness of the enamel was about 1–2 mm, which is quite appropriate for the analysis. Thus, parameters such as central wavelength, bandwidth and optical instrumentation must be adjusted so that the advantages of this system overcome the disadvantages [23].

The OCT images presented in Figure 6 show the light propagation from the enamel surface to the dentin at the phases for the NSF group and the negative control group. All the enamel layer, the enamel/dentin junction and the dentin, in phases T0 (Figure 6A, D) and T2 (Figure 6C), can be observed. In the Figure 6B, E and F, this distinction between layers can no longer be seen. This behavior can be explained by enamel demineralization. In this situation, there is an intense increase in backscattering in this region, so the light does not reach the deeper layers, where the dentine is found. This is justified by the induction of caries in the enamel at phase T1 for all the three groups. This fact corroborates the findings in the literature suggesting that in the presence of caries, there is a greater scattering due to the modification of the scattering properties in carious enamel, which can further reduce image quality at greater depths [23].

Whereas in the NSF and NaF groups, there was a reversal in that backscattering at T2 (Figure 6C), in the negative control group, a lesser reduction of backscattering (Figure 6F) was observed, precisely because this group was not able to remineralize the induced caries lesions.

The findings from the OCT images corroborate the data found from microhardness testing and fluorescence spectroscopy regarding the ability of NSF to remineralize primary tooth enamel. This result becomes especially important in the enamel surface of the deciduous tooth because it is more porous and permeable than the enamel surface of the permanent tooth, which implies a greater susceptibility to demineralization due to the acid action of caries pathogens [14]. However, this increased permeability of the deciduous tooth enamel favors the action of fluorides, which diffuse about 150 times more than in the permanent tooth [25].

In addition to the remineralization capacity, NSF is an antimicrobial agent. In this study, the antimicrobial action of the silver nanoparticle colloid on Streptococcus mutans in relation to its enamel adhesion and its acid production was tested. Studies have reported the antimicrobial action of silver nanoparticles on Gram-positive and Gram-negative bacteria, showing bactericidal effects on microorganisms, such as Escherichia coli [33], [38], [39], Staphylococcus aureus [38], Enterococcus faecalis [39], and S. mutans [38], [40]. Furthermore, silver nanoparticles–fluoride colloids are potential options for the control of multidrug-resistant microorganisms and do not represent substantial risks to human health [40], [41]. S. mutans is considered the principal microorganism associated with dental caries, and for that reason, it was chosen in this assay. Spherical silver nanoparticles have bactericidal action on S. mutans, and that action depends on the particle size: the smaller the diameter, the lower the minimum inhibitory concentration for the microorganism [42]. Concentrations of 4.86±2.71 μg/ml of silver nanoparticles, with an average size of 25 nm, are sufficient to inhibit the growth of S. mutans [43]. The action of NSF on the formation of biofilms containing S. mutans was assessed in vitro, and it was observed that, compared to SDF, NSF prevented biofilm formation on tooth enamel, confirming its antimicrobial potential even in lower concentrations than the SDF [9], [38].

The results corroborated those findings. The concentration of NSF was 400 ppm with a particle size of 8.7±3.1 nm, which was capable of showing antimicrobial action. It is suggested that the inhibitory power of the silver ions is due to their interaction with thiol groups of the protein and phospholipid portion of bacterial membrane [44], since silver ions have a large chemical affinity for compounds containing nitrogen, sulfur and phosphorus. When formed into nanoparticles, silver ions interact more strongly with other organic and inorganic molecules due to their larger surface area [45]. Thus, silver nanoparticles also act on bacterial membranes by altering their permeability and causing rupture, and, inside the cell, interact with nucleic acids, which likely prevents the replication process [41], [42], [43]. Therefore, these mechanisms of action may interfere with bacterial metabolism, including adhesion and acidogenicity.

The NSF prevented bacterial adhesion to the sound enamel, and its effectiveness was statically significantly higher than NaF. The percentage of adsorption inhibition of NSF adhesion was more than double that presented by NaF, indicating its greater power in preventing bacterial adhesion. This fact may be related to the ability of silver ions to present bacterial biofilm formation [39] and because the synthesis of glucans by the bacteria is responsible for their adhesion to the enamel surface [10], perhaps that synthesis could be impaired by NSF. Furthermore, chitosan used as a stabilizing agent of the colloid [28], [46] also has antimicrobial properties [28], [47], [48], including inhibition of Streptococcus mutans adhesion to the enamel [49], [50], which may confer a synergistic effect.

Fluoride causes inhibition of bacterial activity [51] and acid production of Streptococcus mutans [40]. In this study, acid production of S. mutans AU159 was inhibited by NSF, but NaF did not show efficacy by inhibiting bacterial acid production. Samples treated with NaF showed a negative percentage of pH variation, meaning that all the samples showed a drop in the pH of the medium because the NaF was not able to inhibit bacterial acid production. The samples treated with NSF showed a positive percentage of pH variation, increasing on average the pH of the medium from 6.6 to 6.8, indicating that the colloid could prevent acid production by S. mutans.

Clinical development of dental caries involves the simultaneous demineralization and remineralization, and the effect of fluorides in those processes is well known. Zhi et al. [51] reported that silver ions (non-nanoparticles) may increase mineral density during the remineralization of enamel caries. However, silver ions have little effect on the prevention of enamel demineralization, unlike fluorides, which are deposited on the enamel surface as CaF₂, which is inclined for remineralization [41]. This study focused on investigating silver nanoparticles and the effect of fluoride-containing colloids on preventing the formation of enamel demineralization damage, which also differs from the studies mentioned above because the enamel blocks were submitted to a pH cycling challenge. Hence, the results showed a similar effect (non-statistically significant difference) of both NSF and NaF on enamel demineralization even though the NaF presented a lower percentage of microhardness variation. All enamel blocks were submitted to a pH challenge for 14 days, and all the samples had some demineralization, but NSF and NaF avoided higher demineralization, such as the negative control-treated samples. Furthermore, NSF-treated samples did not show black stains like SDF does [9]. Since silver particles were reduced to nanometer size, their chemical reactivity was altered and the colloid was not able to stain the teeth black. Chitosan may have acted as an adjuvant in preventing enamel mineral loss during pH cycling because it can positively affect the in vitro enamel demineralization, preventing the loss of ions, especially P, from hydroxyapatite [50]. These results are a satisfactory outcome because NaF is considered the reference standard for the prevention of enamel demineralization. NSF is a potential agent to be used in public health programs because it has great advantages over SDF and better action than NaF on the virulence factors of Streptococcus mutans as analyzed in this study.

5 Conclusions

Microhardness, fluorescence spectroscopy and OCT tests indicated that NSF is as efficient as NaF for decidous enamel remineralization. Besides that, NSF presented higher interference on Streptococcus mutans AU159 adhesion to the enamel surface and higher inhibition on S. mutans AU159 acidogenicity than NaF. Therefore, NSF may be more effective than conventional fluorides for incipient caries lesion treatment due to its remineralization and antibacterial actions.