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© 2008 SAGE Publications Strategies to Enhance the Biological Effects of Fluoride on Dental BiofilmsPresented at a symposium entitled "Fluoride and Caries Decline", sponsored by the IADR Cariology Research, Behavioral, Epidemiologic & Health Services Research, and Pharmacology/Therapeutics/Toxicology Groups, presented during the 35th Annual Meeting of the American Association for Dental Research and the 83rd Annual Session of the American Dental Education Association, March 9, 2006, Orlando, Florida, USA, and supported by the Colgate-Palmolive Co.
Eastman Department of Dentistry and Center for Oral Biology, University of Rochester Medical Center, 625 Elmwood Ave., Box 683, Rochester, NY 14620, USA; Hyun_Koo{at}urmc.rochester.edu
Key Words: Dental caries • Streptococcus mutans • glucans • fluoride • biofilm • acid production • glucosyltransferases Fluoride, in a variety of modalities, is the most effective anti-caries agent known. Nevertheless, dental caries remains one of the most prevalent and costly oral infectious diseases worldwide. Dental caries results from the interaction of cariogenic bacteria with constituents of the diet within a biofilm on teeth known as dental plaque. Fluoride exerts its major effect by reducing demineralization and enhancing remineralization of early carious lesions; it also affects the biological activities of cariogenic streptococci such as Streptococcus mutans. The effectiveness of fluoride could be enhanced by additional substances which affect the virulence of cariogenic bacteria and/or enhance the antibacterial effects of fluoride. This paper provides an overview of the influence of fluoride on microbial physiology, particularly on S. mutans, and strategies to enhance fluoride’s biological effects.
Oral diseases related to dental biofilms continue to affect the majority of the world’s population. Among them, dental caries is the most prevalent and costly oral infectious disease (NIH, 2001; Marsh, 2003). Dental caries results from the interaction of specific bacteria with constituents of the diet within a biofilm formed on teeth and known as dental plaque (Bowen, 2002). Sucrose is considered to be the "arch criminal" from the dietary aspect, because it is fermentable, and also serves as a substrate for the synthesis of extracellular (EPS) and intracellular (IPS) polysaccharides in dental biofilm (Bowen, 2002); however, it is important to emphasize that other sugars and starch can also contribute to the pathogenesis of dental caries (Bowen et al., 1980; Firestone et al., 1982; Ribeiro et al., 2005). Streptococcus mutans is generally regarded as the primary microbial culprit, although additional acidogenic micro-organisms may be involved (Hamada and Slade, 1980; Loesche, 1986; Beighton, 2005). This bacterium is acidogenic and acid-tolerant; thus, it survives and carries out glycolysis at low pH values existing within the matrix of the biofilms, which causes demineralization of the adjacent dental enamel (Belli and Marquis, 1991; Bowen, 2002). Furthermore, S. mutans also produces EPS through glucosyltransferases (GTFs) and fructosyltransferase (FTF). EPS, especially glucans, are of central importance for adherence of S. mutans to the tooth surface, and contribute to the formation and structural integrity of the matrix of dental biofilms (Yamashita et al., 1993). This paper focuses on the potential ability of fluoride to affect the biological activities of S. mutans, and strategies to enhance fluoride-related effects on this cariogenic organism.
The first clinical evidence of the interaction between bacteria and diet is the appearance of dental plaque. Dental plaque is a biofilm composed of a diverse community of bacteria and salivary constituents embedded in a polysaccharide matrix and tightly adherent to the tooth surface (as reviewed in Marsh, 2003). If dental biofilm is allowed to remain on tooth surfaces with a frequent consumption of carbohydrates (especially sucrose), S. mutans and other acidogenic bacteria, as members of the microbial community, will metabolize the sugars to organic acids and synthesize polysaccharides. The resulting low pH environment favors the growth of cariogenic aciduric streptococci (S. mutans, for example), and the elevated amounts of EPS promote biochemical and structural changes in the matrix of the biofilm. The persistence of this acidic condition triggers a shift in the biofilm community toward the dominance of acidogenic and aciduric bacteria, such as S. mutans, because of their ability to survive at low pH values (Quivey et al., 2000; Marsh, 2003); the low pH microenvironment in the biofilm’s matrix results in dissolution of enamel. The EPS provide bulk and structural integrity, and affect the porosity and inorganic composition of the biofilm’s matrix (Colman et al., 1977; Zero et al., 1986; Dibdin and Shellis, 1988; Cury et al., 2000; Thurnheer et al., 2003). These polysaccharides also protect the bacteria from harmful influences of antimicrobials and other environmental assaults (Lewis, 2001). In addition, the ability of S. mutans to utilize some exopolysaccharides and IPS (a glycogen-like storage polymer) as sources of carbohydrates offers an additional ecological benefit (Hamilton, 1976). These polysaccharides can be metabolized when exogenous fermentable substrate has been depleted in the oral cavity, which increases the amount of acid production and the extent of acidification, thus contributing to the pathogenesis of dental caries (Loesche and Henry, 1967; Tanzer et al., 1976; Spatafora et al., 1995). These observations show clearly that EPS (e.g., glucans) and the ability of S. mutans to produce and tolerate acids could be primary targets for chemotherapeutic intervention to prevent the development of cariogenic biofilms.
Fluoride, in various modalities, is the most effective known anti-caries agent (Clarkson, 2000; NIH, 2001). Nevertheless, dental caries remains a public health problem in many countries, including the United States, and continues at a high level in susceptible subpopulations, especially among economically underprivileged children (NIH, 2001). Fluoride exerts its major effect by reducing demineralization and enhancing remineralization of early caries lesions (Dawes and ten Cate, 1990). However, there is a plethora of evidence which shows that fluoride, even at low concentrations, can affect the physiology of microbial cells, including cariogenic streptococci (as reviewed by Hamilton, 1990; van Loveren, 2001; Marquis et al., 2003). Fluoride is substantive in plaque and persists long after initial exposure (Duckworth et al., 1987, 1989; Creeth et al., 1993). Normal concentrations of fluoride in dental biofilm (plaque) can reach millimolar range (0.1–0.5 mM), and its level in biofilm matrix becomes elevated after the use of fluoride-containing products (Duckworth et al., 1987; Vogel et al., 2000). The levels of fluoride in plaque are sufficient to have significant effects on bacterial metabolism (Marquis et al., 2003). Some of the biological effects are shown in Table 1  .
In general, there are two major pathways by which fluoride affects microbial cells: (1) direct inhibition of a variety of enzymes in intact cells, either directly or in the form of metal complexes at sub-millimolar levels (Marquis et al., 2003); and (2) enhancing proton permeability of cell membranes by acting in the form of HF as a transmembrane proton carrier, which discharges  pH across the cell membrane. It appears that the latter is the major action leading to inhibition of acid production by intact bacterial cells at low pH values attained within biofilms (Marquis et al., 2003). Fluoride acts to inhibit proton extrusion by F-ATPases by bringing excreted proton back into the cell through movements of HF, to which the cell is some 107 times more permeable than to F– (Marquis et al., 2003). Once HF is in the relatively alkaline cytoplasm, it dissociates to yield F– and H+, which acts to acidify the cytoplasm and inhibit glycolytic enzymes. Lowering of pH by fluoride compromises the energetic status of the cell, because by increasing re-entry of protons across the cell membrane, it augments the demand on ATP for acid-base regulation. The net result is similar to acidification or starvation stresses on the cell (Svensäter et al., 2000). In addition, fluoride in combination with aluminum inhibits the activity of proton-translocating F-ATPase (Sturr and Marquis, 1990), which contributes to diminishing pH across the cell membrane. Cytoplasmic acidification caused by fluoride would disrupt the glycolytic acid production, and the formation and catabolism of intracellular iodophilic polysaccharides (IPS) (Hamilton, 1990). By disrupting the intracellular pH, fluoride affects both the acid production and acid-tolerance of S. mutans. Bradshaw et al.(2002) showed that fluoride at concentrations as low as 0.53 mM can exert subtle antimicrobial effects in microbial communities within biofilms by reducing the extent and rate of acid production (in terms of [H+]), thereby reducing the key driving force for the selection of cariogenic and acid-tolerant species, such as S. mutans (Bradshaw et al., 1990, 2002).
Furthermore, fluoride may have an additional mechanism to disrupt biofilm formation. It has been demonstrated that mutans streptococci growing in the presence of fluoride (at 3.7 mM) resulted in changes in the composition of the EPS produced (Bowen and Hewitt, 1974). A recent study showed that fluoride at low concentrations (up to 0.2 mM) partially inhibited the production and secretion of GTF by Streptococcus mutans growing in suspension cultures (Koo et al., 2006b); the inhibitory activity did not involve growth inhibition or starvation (Table 2
Clearly, fluoride, at levels found in plaque, displays biological activity on critical virulence factors of S. mutans in vitro, such as acid production and glucan synthesis. The question remains as to whether the subtle biological effects of fluoride alone are sufficient to contribute to its anti-caries properties in vivo, and whether additives can augment the efficacy of fluoride against cariogenic bacteria (van Loveren, 2001).
According to an NIH Consensus Statement on Diagnosis and Management of Dental Caries Throughout Life, the effectiveness of fluoride could be enhanced when combined with additional cariostatic agents (NIH, 2001). In addition, inclusion of agents that enhance the effectiveness may possibly result in preparations with lower concentrations of fluoride, thus reducing the risk of fluorosis. Chemical agents that have biological activities against biofilms would be potential candidates to be used in combination with fluoride; such compound(s) should have one or more of the following effects: (1) inhibition of bacterial adherence and colonization; (2) inhibition of EPS (e.g., glucans) synthesis; (3) inhibition of sugar metabolism (e.g., glycolysis); (4) inhibition of expression of virulence genes; (5) disruption of established/mature biofilms; (6) modification of biochemical composition of biofilms; and (7) selective suppression of oral pathogens. Thus, enhancement of the protective effects of fluoride by including substances in preparations, which modulate the cariogenicity of S. mutans and its ability to form biofilms, offers an attractive route to reduce the prevalence of dental caries. However, most of the chemotherapeutic strategies to enhance the biological activity of fluoride are based on the use of broad-spectrum antimicrobials, such as chlorhexidine, triclosan, and metal ions/cations, which could suppress the resident flora.
Our laboratory has been focusing on therapeutic approaches using agents with specific activities against S. mutans virulence in which fluoride has effects (e.g., glucan synthesis and acid production). Recently, apigenin and tt-farnesol, two naturally occurring compounds, have shown biological activities against cariogenic properties of S. mutans in vitro and in vivo (Koo et al., 2002, 2003a,b, 2005). Apigenin (4',5, 7-trihydroxyflavone) is a bioflavonoid found in fruits and vegetables, and tt-farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) is a sesquiterpene alcohol found in essential oils of citrus fruits (Koo et al., 2002). Apigenin is a unique compound that affects both the activity and expression of GTFs (Koo et al., 2002, 2006a). tt-Farnesol disrupts the proton-permeability of the S. mutans membrane, and inhibits acid production and glucan synthesis by S. mutans within biofilms (Koo et al., 2003b, 2005). However, these compounds are not lethal to S. mutans biofilms at the highest concentration we tested (5 mM or 0.1%, w/v) (Koo et al., 2003a, 2005). Topical application of apigenin and tt-farnesol reduced the incidence of dental caries, with minimal effects on the viability of oral flora populations in rats (Koo et al., 2003a). Thus, apigenin and tt-farnesol could enhance the biological effects of fluoride against S. mutans by simultaneously modulating specific virulence attributes associated with EPS matrix formation and acid production, thereby reducing the development and virulence of cariogenic biofilms. Initially, we have examined the effectiveness of various concentrations and combinations of the agents on S. mutans biofilms in vitro. The combination of 1 mM apigenin and 5 mM tt-farnesol with 250-ppm fluoride was the most effective in reducing the biomass, polysaccharide content, and acidogenicity of S. mutans biofilms (Koo et al., 2005). The reduction in the amounts of insoluble glucans and IPS of the S. mutans biofilm matrix without displaying bactericidal effects was one of the most relevant biological effects of our chemotherapeutic approach (Table 3
The reduction of IPS content in the biofilms was likely due to cytoplasmic acidification caused by the agents. Fluoride and tt-farnesol increase the proton-permeability of the S. mutans membrane, disrupting the pH across the cell membrane; apigenin may also contribute to this effect by inhibiting the activity of F-ATPase, on which fluoride also has some inhibitory effects. It is noteworthy that the biofilms with the least amount of IPS had the highest pH values in the biofilm matrix, especially those treated with the combination of agents (Table 3 ).
Furthermore, the effectiveness of the combination of the agents has been evaluated in vivo in a rat model of dental caries (Koo et al., 2005). The agents were applied topically twice daily and their effects compared with positive (0.12% chlorhexidine + 250 ppm fluoride, w/v) and negative (25% ethanol containing 1.25% DMSO) controls. The combination Apigenin+Farnesol+Fluoride displayed the maximum therapeutic effect in vivo without significantly affecting the viability of the oral flora populations in rats, and its potency was comparable with that of positive control (Table 4
The addition of agents that enhance and/or complement the biological effects of fluoride is a potentially useful alternative approach to the current chemotherapeutic strategies based on the use of broad-spectrum antimicrobials, which require elevated concentrations of the agents due to reduced susceptibility of micro-organisms in biofilms (Lewis, 2001). Results from our studies support this concept (Koo et al., 2003a,b, 2005). Future studies are necessary to examine the effectiveness of this approach at the clinical level, and to investigate whether other bioactive compounds can also enhance the physiologic actions of fluoride on S. mutans and other oral pathogens.
The authors are grateful to Dr. William Bowen for critical reading of the manuscript prior to submission. This research was supported by USPHS Research Grant 1R03 DE015441-01 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
Advances in Dental Research, Vol. 20, No. 1,
17-21 (2008)
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Introduction
The Role of S....