*3.2. Application of Silver Diamine Fluoride on Fungus*

Two studies explored the anti-candidal effect of SDF [16,33] (Table 3). One study of *Candida albicans* on a dentin block taken from human teeth reported that the anti-candidal effect was a dose-related property. No significant differences were found between 3.8% and 38% SDF solutions for the colony-forming unit counts, the colorimetric quantification of *Candida*, the real-time polymerase chain reaction-based quantification, and the scanning electron microscopy. The 38% SDF caused severe damage to candida cell walls, whereas the 3.8% SDF caused only partial damage. In another study, *Candida albicans*, *Candida glabrata*, *Candida parapsilosis*, *Candida tropicalis*, *Candida krusei*, and *Candida dubliniensis* were isolated from the carious lesions of preschool children. Zones of growth inhibition, a multiplex polymerase chain reaction, and minimal inhibitory concentrations (MIC50 and MIC90) were adopted for assessing the anti-candidal effect of SDF and the controls. *C. tropicalis* was more resistant to SDF based on the diameter of the inhibitory zone of the culture growth. Meanwhile, *C. krusei* and *C. glabrata* were more sensitive to SDF.

#### **4. Discussion**

This review is the first review concerning the effect of SDF applied directly to biofilm. It is based on the count of bacteria and fungi as well as the diversity within the biofilm. A large number of reviews have reported the effectiveness of SDF for preventing and arresting caries in clinical studies, and its effect was reported as extraordinary, mostly due to its antibacterial function and remineralization property [20].

Although no limitation was set regarding the publication year during the search, the earliest study found was published in 2010. This shows a new trend where research is being turned into laboratory work exploring the reason for SDF's effectiveness in preventing and arresting caries. The majority (15/21, 71%) of the included studies were published between 2016 and 2020. SDF solutions featuring different concentrations [22,34] or combined with different materials [17,35,36] were adopted to investigate their potential use for antibacterial purposes.

SDF has an antibacterial function because both the silver ions and the fluoride contained in SDF appear to have the ability to inhibit the formation of cariogenic biofilm [37]. A 38% SDF solution contains approximately 253,870 ppm silver and 44,800 ppm fluoride ions [38]. The microorganisms can be killed and silver ions can interfere with metabolic processes [12]. A three-pronged approach exists for silver ions to kill the microbiota: damage the cell wall structure of bacteria, influence metabolic processes and inhibit enzyme activities, and, finally, inhibit the replication of bacterial deoxyribonucleic acid [12]. In addition, it has been suggested that silver ions at a concentration of 20 ppm can inhibit the growth of *Streptococcus mutans* [39]. It has also been reported that the antimicrobial effect stems from the silver ions, especially at low concentrations [40]. Furthermore, the antimicrobial function of a high concentration of fluoride cannot be ignored [41]. The reason for this is that a high concentration of fluoride can influence enzymes' carbohydrate metabolism and sugar uptake and bind the bacterial cellular components, resulting in

the inhibition of biofilm formation [37]. However, only one study reported no significant difference in the count of *Lactobacillus rhamnosus* among the 38% SDF group and the other groups (35% chlorhexidine varnish, 5% sodium fluoride varnish, 500 ppm sodium fluoride solution + 0.1% chlorhexidine solution, and the blank control) [26]. The possible reason for this may be that the formation of the SDF adopted in the study was a varnish rather than a solution, which indicates that the reduction in bioactive silver ions was not the same as that in the 38% SDF solution.

According to this review, even though SDF inhibits some oral microbiota species, the diversity of the biofilm does not seem to change before and after SDF application. One possible reason for this is that SDF only reduces the number of carious species, for example, *Streptococcus mutans* and *Lactobacillus*, rather than inhibiting all of the microbiota species. In the caries process, *Streptococci*, *Lactobacilli*, and *Actinomycetes* are treated as the initial bacterial invasion species. Among them, *Streptococcus mutans* is one of the most important pathogens associated with the initiation and progression of caries [42]. *Lactobacillus acidophilus* and *Lactobacillus rhamnosus* are routinely found in deep and superficial carious lesions, which indicates that they are the most abundant bacteria species [43,44]. *Actinomyces naeslundii* is related to root caries, which have the potential to invade dentinal tubules [15]. After the results of the studies were analyzed, it was found that the possible mode of action of SDF can be related to its antibacterial properties on cariogenic bacteria rather than a change in the diversity of the biofilm. The microbiota appeared to reach a new balance, but with fewer carious species [45].

According to this review, SDF also has antifungal properties. As discussed above, the bioactive form of silver in SDF in its ionized form is "Ag+". It has been reported that silver particles can inhibit the growth of *C. albicans* under high concentrations [46]. One of the possible reasons for this is that silver ions can also suppress extracellular phospholipase production, which plays a crucial role in the pathogenicity of *C. albicans* [47]. Phospholipase is associated with the development of hyphae, which plays an essential role in the biofilm's adherence and formation [48]. The blocking transformation from a yeast form to a hyphal form could stop colonization and initiation or pathogenesis. However, variations in inhibition still exist among different species in the yeast. Another possible explanation for this could be the avidity of the biological ligands of the yeast to SDF [49].

Based on the review, the most frequently used approach for assessing biofilm is still the colony-forming unit count and the live-to-dead ratio in the traditional culture method. New molecular biological technology is being adopted into assessments, usually combined with traditional ones. The majority of the studies included in this review were conducted in vitro. As the duration of the selected laboratory studies was relatively short, the long-term caries-arresting effect and the periodicity of the SDF application could not be evaluated. It was not the objective of this review to judge the quality of the studies or to discuss the limitations of each study. This should be taken into consideration when interpreting the results as well as the conclusions of this review.

This concise review was intended to provide the best evidence regarding SDF's antimicrobial effect, as presented in 21 selected papers, to obtain a definitive answer to a research question involving antibacterial function. It provides an overview and updated information about the antimicrobial effect of SDF on oral biofilm, as well as its known role in the inhibition of cariogenic bacteria. The major lines of investigation in this review involved 15 laboratory studies and six clinical studies. Some data are quite limited due to the lack of studies on certain topics such as the antibacterial effect of SDF on periodontal pathogens. As a whole, this field shows significant gaps, with only one study so far providing limited information [22]. In addition, only two published papers have shown promising results for SDF's antifungal effect against candida [16,33]. However, overall, this review provides strong evidence that SDF can, indeed, inhibit the growth of cariogenic bacteria and prevent bacterial adhesion to the tooth surface. This review—the first concise review of the effect of SDF applied directly to biofilm—can be used to guide future research in this area.

Even though SDF has been used for decades, its properties remain under-investigated. Thus far, most laboratory studies have focused on its remineralization properties rather than its antimicrobial functions within biofilm. It has been suggested that the concentrations of antibacterial agents required to inhibit biofilm are more than 100 times higher than those needed to inhibit planktonic bacteria, as biofilm is more resistant to antimicrobial agents than planktonic bacteria are [12]. More studies exploring the rationale behind these materials are still needed.
