Introduction
Dental caries is a chronic, multifactorial, bacterial disease causing enamel demineralization and disintegration of the organic substances of the teeth (Karpiński and Szkaradkiewicz
2013). The etiology of caries includes host factors, carbohydrates intake, plaque bacteria, and time (Samaranayake
2018). A homeostasis occurs between demineralization and remineralization. However, if this balance is disturbed, demineralization overtakes remineralization leading to dental caries (García-Godoy and Hicks
2008; Salma et al.
2022; Stephan and Miller
1943; Takahashi and Nyvad
2008). Biofilm plays an essential role in the initiation and progression of dental caries (Lee et al.
2010). It metabolizes dietary carbohydrates via glycolysis to form lactic acid leading to a drop in pH levels and, consequently, causing enamel demineralization (Pandit et al.
2015).
Acidogenicity and acidurity are considered crucial factors for the survival of biofilm (Pandit et al.
2013). Another key feature is the ability of biofilm to synthesize water-insoluble glucans from glucose by the enzyme glucosyltransferases (Pandit et al.
2013).
Streptococcus mutans has both acidogenic and aciduric characteristics. Therefore, it is identified as the primary source of caries initiation (ten Cate
2006). Lactic acid is produced by
Streptococcus mutans through fermentation of dietary carbohydrates. Drop in oral pH contributes to both dominance of the
Streptococcus mutans and formation of caries. Demineralization occurs by a complex interaction between commensals, carbohydrates, and salivary components. Demineralization overtakes remineralization when the pH at the enamel surface drops below 5.5 (Loesche
1986).
Recently, early childhood caries (ECC) has become a considerable public health problem (Anil and Anand
2017; Çolak et al.
2013). Despite the decrease in dmft index in the developed countries, it is increasing in the developing world nations (Anil and Anand
2017; Folayan et al.
2020). One of the most essential factors that predispose ECC is the formation of acidogenic and aciduric biofilm of Mutans Streptococci (Carlsson
1997; Ccahuana-Vásquez and Cury
2010; Hamada et al.
1984; Hamada and Slade
1980; Seow
1998). Therefore, biofilm control is crucial for prevention of ECC. Fluoride application is one of the main strategies used to control ECC by enhancing remineralization (Cate and Featherstone
1991), preventing demineralization (Tenuta et al.
2009), and induction of anti-biofilm activities of tooth enamel (Pandit et al.
2013). Fluoride accelerates the remineralization process by adsorbing to the enamel surface and attracting phosphate and calcium ions. Additionally, fluoride substitutes the hydroxyl ions in hydroxyapatite of enamel forming fluorapatite which has greater resistance to bacterial acids (Featherstone
1999).
Many companies are globally striving to develop fluoride varnish that can adhere to the tooth surface to improve the antibacterial properties and acid resistance. Cerkamed Co., Poland, has developed Fluor defender® that comprises hydroxyethyl methacrylate which contains 0.1% fluorosilane. Fluor defender® can be used to improve remineralization, strength of enamel, and to build a protective layer on the enamel’s surface. Enamelast®, a product of Ultradent Co., USA, is a flavored, xylitol-sweetened 5% sodium fluoride in a resin carrier which produces a mechanical occlusion of the dentinal tubules in the treatment of tooth hypersensitivity. However, to the best of our knowledge, there are no reports that investigate the antibacterial activities of Fluor defender® or Enamelast® on primary teeth enamel. The aim of the current study is to evaluate the effect of Enamelast® on the formation of Streptococcus mutans biofilm, as compared to Fluor defender® on primary teeth. The null hypothesis tested in this study was that Enamelast® varnish has the same antibacterial efficacy of Fluor defender® varnish on the formation of Streptococcus mutans biofilm.
Discussion
Virulence attributes of biofilm sheathed bacteria, such as acidogenicity, acidurity, and formation of extracellular polysaccharides, induce an acidic microenvironment that causes ecological dysbiosis (Pandit et al.
2013; Philip et al.
2018). Subsequently, a shift in the homeostasis of oral bacteria in favor of cariogenic flora occurs which predisposes the teeth to dental caries (Schwendicke et al.
2016). Removal of dental plaque biofilm by mechanical cleansing is an effective means to disrupt the caries process. However, reformation of bacterial biofilm starts immediately afterward.
Enamel remineralization has been suggested as a non-invasive treatment of ECC by remineralization in the clinical management of the disease (Shen et al.
2011). This takes place when the pH rises and phosphate, calcium, and fluoride ions deposit on tooth enamel in the form of fluorapatite which is more resistant to organic acids than hydroxyapatite (Cilurzo et al.
2003). Many investigations have tested the efficacy of antimicrobials against biofilms cariogenicity (Dang et al.
2016; Kulshrestha et al.
2016; Pandit et al.
2013). Fluoride has been reported as the gold standard agent for caries control (Zero
2006). To date, no studies have focused on comparing the antibacterial efficacy of Enamelast® and Fluor defender® on biofilm formation of
Streptococcus mutans. Hence, our aim was to evaluate the effect of these fluoride varnishes on the formation of
Streptococcus mutans biofilm.
In the current study, Fluor defender® and Enamelast® were applied to enamel tooth surfaces of primary teeth. Thenceforward, the biofilm formation was detected spectrophotometrically and was observed by SEM in order to investigate the anti-biofilm activity of Fluor defender® and Enamelast®. Primary teeth specimens were used in the current study for biofilm growth. Although bovine enamel has been used in many studies (Lippert and Lynch
2014), using human enamel specimens is more clinically relevant. The growth medium used was tryptone soya broth, supplemented with sucrose to maintain the viability of
Streptococcus mutans. The same medium was documented in previous studies (Latimer et al.
2015; Lotfy et al.
2018; Zhang et al.
2015).
Compared with the negative control group at 48 and 72 h, Enamelast® and Fluor defender®-treated group showed significantly (
p < 0.001) slight adhered bacterial cells as revealed by the absorbance and SEM as well. This emphasizes the antimicrobial effect of both types as attributed to fluoride content which interfered with bacterial metabolism and inhibited bacterial growth (Bradshaw et al.
2002). However, no significant difference was observed in the bacterial adherence in any of three groups up to 24 h following experimental treatment. In this context, we emphasize that tooth-brushing behavior should not be carried out at least 24 h following the application of fluoride varnish to avoid reducing the amount of attached varnish on teeth surfaces.
The fluoride varnishes, Fluor defender®, and Enamelast® were able to protect the under-treatment area against biofilm formation by
Streptococcus mutans. Although Enamelast® has an enhanced retention on the tooth surface allowing higher fluoride uptake (Godoi et al.
2019). Nevertheless, the absorbance from the Enamelast®-treated group, respectively, showed 7- and 16.5-fold increase up to 48 and 72 h after exposure when compared to the Fluor defender®-treated group (
p < 0.001). Moreover, in the SEM images, there were visibly fewer cells of
Streptococcus mutans attached to the enamel surfaces from 48 to 72 h after exposure to Fluor defender® than Enamelast®. The number of bacterial cells adhered to enamel surfaces in the Fluor defender®-treated group was significantly (
p < 0.001) fewer than the Enamelast®-treated group by approximately 36.55% and 20.62% up to 48 and 72 h after exposure, respectively.
The noticeable low antimicrobial performance of Enamelast® could be attributed to its hydrophobic resinous content, which causes weak release of fluoride (Fernández et al.
2014). This assumption is consistent with Al Dehailan et al. who related the difference in composition of varnishes to their mechanism of action of releasing and deposition of fluoride on the outer layers of enamel lesions (Al Dehailan et al.
2016). Enamelast® contains higher concentration of fluoride, 22,600 ppm while Fluor defender® contains 1600 ppm fluoride. In this regard, no relation was detected between the fluoride concentration in the varnish and the fluoride release or its antimicrobial effect. This is consistent with the results obtained by Bolis et al. who compared different brands of varnish and found that Duraphat® varnish released the lowest and MI varnish™ the highest amount of fluoride while enamel fluoride uptake by both materials was not statistically different (Bolis et al.
2015). Additionally, lower viscosity of Fluor defender® than that of Enamelast® may have promoted greater release of fluoride with its antimicrobial effect. According to Carvalho et al., the lower viscosity of certain varnishes may boost stronger retention on enamel, provide greater contact, and allow greater release of fluoride (Carvalho et al.
2015).
Fluor defender® contains 0.1% fluorosilane in its formulation (a polyurethane-based compound) that may act by inhibiting the adhesion of
Streptococcus mutans cells to the enamel surface and promoting fluoride release which inhibits demineralization (Baygin et al.
2014; Byeon et al.
2016; Punathil et al.
2018) and promotes remineralization (Yadav et al.
2019). Moreover, the protective quality of Fluor defender® is also attributed to the mechanical barrier provided by preventing direct contact of acids on the surface. The anti-streptococcal biofilm activity of Fluor defender® is basically linked to the fluoride incorporation into the crystalline lattice of enamel and formation of calcium fluoride after 24 h (Harding et al.
1994; Seppä
2004). A previous study by Erdem et al. reported that Fluor Protector® showed a better antibacterial effect when compared to Bifluoride 12 varnish. Although Bifluoride 12 had higher content of fluoride, they attributed the results to the Fluor Protector® silane content (Erdem et al.
2012). The latter has similar polyurethane-based compound; difluorosilane and a similar low fluoride concentration to Fluor defender®. The implication of this study supports the view that the higher antibacterial activity of Fluor defender® is attributed to its formulation. Additionally, Bezerra et al. (
2022) studied the anti-cariogenic effect of Fluor Protector®, hybrid coatings, and a combination of stannous chloride and sodium fluoride using confocal microscopy. They reported that Fluor Protector® showed greater protection against
Streptococcus mutans UA159 on bovine enamel (Bezerra et al.
2022). On the other hand, the fluoride content in Enamelast® may have hindered the effect of xylitol and consequently reduced the defensive effects of the varnish as compared to Fluor defender® (Cardoso et al.
2014; Mohd Said et al.
2017).
Some mandatory limitations were encountered in the current study. Since the caries process has a multifactorial nature, we could not cover all its aspects in our study.
Streptococcus mutans was chosen in our model as it represents the primary source of caries initiation. We believe that a significant antimicrobial effect against
Streptococcus mutans was obtained by Fluor defender®. However, a cariogenic challenge is recommended using other types of cariogenic flora such as lactobacilli. The lack of acquired salivary pellicle formation is another limitation of the study which would have influenced the interaction between fluoride and minerals on enamel surface (Souza et al.
2010). Moreover, polishing the specimens might have affected the varnish’s surface retention compared with clinical conditions (Rios et al.
2006) though it was imperative for standardization of specimens. Autoclaving of disk models was another limitation of the current study, but likewise it was a mandatory procedure in the methodology.
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