A Concise Review of Silver Diamine Fluoride on Oral Bioﬁlm

: Studies have shown that silver diamine ﬂuoride (SDF) is an effective agent to arrest and prevent dental caries due to its mineralizing and antibacterial properties. While plenty of studies have investigated the mineralizing properties, there are few papers that have examined its antibacterial effect on oral bioﬁlm. The objective of this study was to identify the effect of silver diamine ﬂuoride on oral bioﬁlm. Method: The keywords used were (silver diamine ﬂuoride OR silver diammine ﬂuoride OR SDF OR silver ﬂuoride OR AgF AND bioﬁlm OR plaque). Two reviewers screened the titles and abstracts and then retrieved the full text of the potentially eligible publications. Publications of original research investigating the effect of SDF on oral bioﬁlm were selected for this review. Results: This review included 15 laboratory studies and six clinical studies among the 540 papers identiﬁed. The laboratory studies found that SDF could prevent bacterial adhesion to the tooth surface. SDF also inhibited the growth of cariogenic bacteria, including Streptococcus mutans , Lactobacillus acidophilus , Streptococcus sobrinus , Lactobacillus rhamnosus , Actinomyces naeslundii , and Enterococcus faecalis , thus contributing to its success in caries arrest. One clinical study reported a decrease in Streptococcus mutans and Lactobacillus sp. in arrested caries after SDF treatment, and another clinical study found that SDF inhibited the growth of periodontitis microbiota, including Porphyromonas gingivalis , Tannerella forsythia , and Prevotella intermedia/nigrescens . However, three clinical studies reported no signiﬁcant change in the microbial diversity of the plaque on the tooth after SDF treatment. Moreover, one laboratory study and one clinical research study reported that SDF inhibited the growth of Candida albicans . Conclusion: Not many research studies have investigated the effects of SDF on oral bioﬁlm, although SDF has been used as a caries-arresting agent with antibacterial properties. However, a few publications have reported that SDF prevented bacterial adhesion to the teeth, inhibited the growth of cariogenic and periodontal bacteria, and possessed antifungal properties.


Introduction
The oral microbiota colonizes oral biofilm on the surface of the tooth or on the mucosa within the mouth. The accumulation of oral biofilm can lead to oral diseases such as dental caries or periodontitis [1]. Oral biofilm can lead to dental caries in two ways. First, bacteria within the oral biofilm produce acid through sugar metabolism. Second, the acid causes a subsequent decrease in the environmental pH value. Both of the above are responsible for the demineralization of the tooth surface and the formation of dental caries [2]. The amount of tooth mineral and other calcium phosphates in the plaque fluid decreases rapidly after exposure to fermentable carbohydrates. Lactic acid production and a reduction in the plaque fluid volume can result in the formation of caries [3].
Acid-base-producing bacteria, in varying numbers and proportions, are the key pathogens associated with dental caries [4]. Among the suspected acid-producing bacteria, Mutans streptococci (MS) has been confirmed, in a systematic literature review, to play a central role in the initiation of dental caries on both enamel and root surfaces [5]. This is the case for several reasons: first, MS is the most frequently isolated species from a caries 2 of 11 lesion; second, MS is highly acidogenic and aciduric [6,7]; and third, MS can produce surface antigens I/II and water-insoluble glucan, which promote bacterial adhesion to the tooth surface and to other bacteria [6]. For the purpose of halting the closed circle of the process, an intervention that can inhibit the bacteria that produce acid is needed.
Silver diamine fluoride (SDF) has been widely used to prevent and arrest caries, and "silver diamine fluoride" is the most common spelling/keyword for this compound in the dental literature. SDF was approved for use as a therapeutic agent in Japan over 50 years ago [8]. It has also been used for dental caries treatment in Australia and in some countries in Latin America such as Argentina and Brazil for many years [9]. The Food and Drug Administration approved the marketing of SDF in the United States for the treatment of human dentinal hypersensitivity in adults in 2014 [10].
SDF contains silver and fluoride, which form a complex with ammonia in a colorless alkaline solution [9]. It is not only a combination of silver salt, ammonium, and fluoride ions but also a mixed heavy-metal halide co-ordination complex. Ammonia can provide an alkaline environment, thus keeping the solution constant for a certain period of time [11]. In addition, silver compounds have been reported to have antimicrobial properties in the application of medicine and dentistry for decades [12]. Fluoride is also used in various forms to prevent and arrest caries due to its remineralization function [9]. Therefore, SDF has been hypothesized to contain the combined effects of silver and fluorides.
Fluoride can inhibit the production and metabolic activity of certain strains of bacteria within biofilm at low concentrations [13]. At high concentrations, fluoride inhibits the growth of cariogenic bacteria in dental plaque [14]. One mechanism of biofilm inhibition is to bind its ions to the bacterial cell constituents and to influence enzymes such as enolase and proton-extruding adenosine triphosphatase [15]. Another mechanism is the inhibition of the carbohydrate metabolism of acidogenic oral bacteria [16,17]. However, this may contribute to the development of bacterial tolerance and shorten the duration of the antibacterial effect [18]. Nonetheless, the dominant advantage of fluoride is that the re-mineralizing effect, particularly for the SDF solution, effectively arrests dental caries [19].
Several reviews have been conducted regarding the clinical use of SDF for preventing and arresting dental caries with significant effect [20,21]. However, the rationale behind the impact of SDF on biofilm remains unclear. The aim of this systematic review was to identify the effect of SDF on biofilm. The study question was as follows: What is the effect of SDF on biofilm?

Materials and Methods
Two independent reviewers (Zhang and Got) conducted a literature search using the four most commonly used databases: Medline, PubMed, Embase, and Scopus. Only papers published in English were included. The keywords used were (silver diamine fluoride OR silver diamine fluoride OR SDF OR silver fluoride OR AgF AND biofilm OR plaque). No publication date limitation was established during the search. The latest search was conducted on 30 October 2020.
The inclusion criteria were as follows: (1) papers investigating biofilm; (2) SDF solution at any concentration adopted as an intervention. The exclusion criteria were (1) studies with no information concerning biofilm and (2) reviews, case reports, and conference papers. In addition, a manual search of the reference lists of the selected papers and review articles was conducted. The two reviewers independently screened the papers and identified the relevant studies. When a disagreement occurred, another researcher (Chu) was consulted to achieve a consensus.
The results were summarized into three tables classified by the aim of the studies: Table 1 is for the studies investigating the effect of SDF on biofilm formed via microbiota, Table 2 is for the studies on the diversity and microbial change resulting from the application of SDF, and Table 3 is for the studies focused on the effect of SDF on fungus. Information regarding the following aspects of the background of the included studies was extracted and is presented in the summary tables: (1) authors and year; (2) materials studied; (3) setting of the study; (4) block (or sample) adopted in the study; (5) microbiota studied; (6) assessing method for the biofilm; (7) duration of the study; and (8) findings of the study. Abbreviations: Gp, group; CFU, colony forming unit; CLSM, confocal laser scanning microscopy (dead-to-live ratio); NaCl, sodium chloride; BL, baseline; Ca(OH) 2 , calcium hydroxide; NaOCl, sodium hypochlorite; TBC, total bacteria count; APF, acidulated phosphate fluoride; AHF, ammonium hexafluorosilicate; CPC, cetylpyridinium chloride; CHX, chlorhexidine; NaF, sodium fluoride; N/T, no treatment; NSF, nano silver fluoride; PPF, propolis fluoride; KI, potassium iodide; AgNO 3 ,silver nitrate; SP, severe periodontitis; TVC, total viable counts; PLPP, proportional levels of periodontal pathogens; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

Results
A total of 540 potentially relevant records were identified through the database search, and 194 were left after the removal of duplicates. A manual search of the references of these selected publications was conducted. One article that met the inclusion criteria was added to the list. After the titles and abstracts were screened, 133 records were excluded. The remaining 62 papers, all published in English, were retrieved for full-text reading. Among them, 20 articles did not report microbial analysis and 21 did not contain biofilm or microbiome information.
Among the 21 papers included, six were laboratory studies and 15 were clinical studies. The 21 included papers were published between 2010 and 2020. An SDF solution concentration of 38% was reported in 16 studies, followed by 3.8% SDF in three studies and 25% SDF, or 8.5 wt%, in the other studies. Figure 1 is a flowchart showing the search results.

Antibacterial Effect
A total of 15 studies reporting the antibacterial effects of SDF on bacteria are summarized in Table 1. Among them, 14 studies measured the antibacterial properties using monospecies bacteria such as Lactobacillus acidophilus, Streptococcus sobrinus, Enterococcus faecalis, and Actinomyces naeslundii. However, subgingival microbial biofilm specimens were used on 24 adults with severe periodontitis [22]. The majority of the included studies that explored bactericidal properties were operated in vitro on dentin or enamel samples, with no difference in the effect regarding the sample material. Only one research study was conducted in vivo [23]. The longest study duration was 21 days, whereas the shortest one was 14 min. They also revealed that biofilm treated with SDF had fewer bacteria compared with that treated with water or other interventions. However, one study did not detect any significant difference among the study groups [24]. Among the included studies, eight out of nine studies reported that SDF inhibited the growth of Streptococcus mutans through colony-forming unit counts. Two studies revealed that SDF also had an antibacterial function in Lactobacillus acidophilus and Actinomyces naeslundii. In addition, only one out of the two studies reported that SDF reduced the amount of Enterococcus faecalis [25] while the other reported reduced Lactobacillus rhamnosus [26]. One study reported that a 38% and a 19% SDF solution inhibited cultivable bacteria without any significant difference [27]. Only P. micra and S. constellatus belong to red-and orange-complex species recovered from SDF-treated specimens.
reading. Among them, 20 articles did not report microbial analysis and 21 did not co biofilm or microbiome information.
Among the 21 papers included, six were laboratory studies and 15 were clinical ies. The 21 included papers were published between 2010 and 2020. An SDF solution centration of 38% was reported in 16 studies, followed by 3.8% SDF in three studies 25% SDF, or 8.5 wt%, in the other studies. Figure 1 is a flowchart showing the searc sults.  Furthermore, some studies reported that the live-to-dead ratios of Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces naeslundii in biofilm were significantly lower after the application of SDF [23][24][25][26][27]. Only Al-Madi and co-workers reported that a 5.25% sodium hypochlorite solution had a significantly higher live-to-dead ratio of Enterococcus faecalis compared with the study's SDF group [27]. Only one study reported that the antibacterial effects of nano silver fluoride varnish and the potency of propolis fluoride varnish were comparable with a 38% SDF varnish. Another study compared the minimum inhibitory concentration and the minimum bactericidal concentration of propolis fluoride and nano silver fluoride for the inhibition of biofilm formation [28].

Microbiota Change in Community Diversity and/or Composition within Biofilm
A total of four studies reported on the microbiota diversity before and after SDF treatment. In terms of intervention with SDF solution, only a 38% concentration was adopted in all four studies ( Table 2). The majority of the included studies that explored bactericidal properties were operated in vivo, with one study conducted in vitro; it used extracted caries teeth in an artificial mouth model [29]. Both saliva and plaque were collected in two studies [29,30], whereas only plaque was collected in the other two studies [31,32]. Two studies were conducted on preschool children, whereas one was conducted on primary school students [29], and another was conducted on adults with a mean age of 47 years [32]. The shortest study duration was 24 h, whereas the longest was 12 weeks. Among the included studies, three out of the four used the polymerase chain reaction amplification of 16S ribosomal ribonucleic acid genes and MiSeq sequencing in diversity analyses. One study used ribosomal ribonucleic acid sequencing and quantitative polymerase chain reaction quantification [31]. The Shannon index was the most frequently used index for diversity assessment.
All four studies reported no significant change in diversity. However, one study found a significant decrease in the diversity of oral bacteria plaque within active caries, but not in arrested caries, 12 weeks after SDF treatment [30]. Furthermore, the same paper reported an alteration in the species of the bacteria both before and after the SDF treatment. Streptococcus mutans and Streptococcus sobrinus increased significantly after two and 12 weeks of the SDF treatment in the plaque within active caries. Meanwhile, Lactobacillus sp. and Rothia sp. increased significantly after two weeks. Another study reported that the carbohydrate transportation and metabolic functions in plaque were significantly reduced at 24 h and one week post-intervention [29].

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.

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.

Conclusions
The number of publications exploring the effects of SDF on oral biofilm is limited, although SDF has been used as a caries-arresting agent with antibacterial properties. The limited publications reported that SDF prevented bacterial adhesion to teeth, inhibited the growth of cariogenic and periodontal bacteria, and possessed antifungal properties.