The Antimicrobial Action of Fluoride and its Role in Caries Inhibition C. VAN LOVEREN Department of Cariology & Endodontology, Academic Center for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066 EA Amsterdam, The Netherlands Despite a considerable amount of literature on the effects offluoride in dental plaque, several urgent questions remain unanswered, such as: Does the inhibiting effect offluoride on dental plaque metabolism contribute to caries prevention? Does adaptation ofplaque to fluoride affect its cariogenicity? Single applications of fluoride directly to dental plaque reduced acid production. Also, fluoride dissolving from topically treated en~m~l reduced the acid production in covering layers of oral bacteria 10 vitro. The effects of both treatments were only of short duration and may not be relevant to caries prevention i? vivo. In c~ntrast~ t!aily applications offluoride resulted in a reduction of the acidogenicity of dental plaque even 8-12 h after the treatment. Such a reduction is likely to contribute to caries prevention. But it has to be realized that when plaque reaches saturation with respect to fluoridated calcium (phosphate) precipitates, enamel becomes insoluble and any antim~ crobial effect becomes irrelevant. Still lacking are data on the antimicrobial effects offluoride regimens normally used in home care, in weekly rinsing programs in schools, or treatments applied professionally every six months. Adaptation of Streptococcus mutans to fluoride has been suggested to reduce the cariogenic potential of the cells. In vitro-induced fluoride-resistant strains were less cariogenic in rats, and the velocity of acid production in vitro was reduced at constant pH > 5.5. Despite the ability oforal bacteria to adapt to fluoride, evidence ofadaptation in dental plaque of normal subjects resulting in a reduced cariogenic potential has not yet been demonstrated.
J Dent Res 69(Spec Iss):676-681, February, 1990
Introduction. Demineralization and remineralization are the processes which determine the condition of tooth surfaces after eruption. Demineralization is caused by acidswhich are produced by bacteria in dental plaque. A microbial contributio~ to r~mi~erali~ation is not generally accepted, although remineralization will be enhanced by an elevated plaque pH resulting from t~e dewadation of nitrogenous substrates (such as urea and ammo acids) by bacterial enzymes (Kleinberg, 1967; Hayes and Hyatt, 1974; Kleinberg et al., 1977; Sissons and Cutress, 1988). Fluoride is known to inhibit the energy and biosynthetic metabolism of bacteria (Bibby and Van Kesteren, 1940; Kashket et al., 1977; Maltz and Emilson, 1982), but these antimicrobial effects of fluoride in caries prevention are often regarded as of little or no importance as compared with the direct interactions of fluoride with the hard tissue during caries lesion development and progression (Clarkson et ,al., 1988~. In fact no in vivo studies are available that quantify the antimicrobial effect in relation to the overall effect of fluoride on dental caries. In vitro studies with oral lactobacilli and streptococci have demonstrated adaptation of these organisms to fluoride resistance (Green and Dodd, 1957; Williams, 1964; Hamilton, 1969), but data on adaptation in vivo are scarce. It has been stated Presented at a Joint IADR/ORCA International Symposium on Fluorides: Mechanisms of Action and Recommendations for Use, held March 21-24, 1989, Callaway Gardens Conference Center, Pine Mountain, Georgia 676
that due to fluoride adaptation, the inhibition of acid production'is unlikely to be important as a caries-preventive action of fluoride (Van der Hoeven and Franken, 1984). On the other hand, it has been proposed that the cariogenicity of plaque adapted to fluoride will be reduced (Loesche, 1982). In this paper several topics will be reviewed and discussed concerning the effect of fluoride in dental plaque: (a) the inhibitory effect of both enamel-derived or topically applied fluoride on acid production in dental plaque; (b) the effect of plaque on the retention of fluoride in enamel; (c) the relevance of the inhibition of plaque acid production by fluoride for caries prevention; and (d) development of fluoride resistance and the consequences of that for plaque cariogenicity.
Effects of fluoride derived from enamel. In their review, Jenkins and Edgar (1977) regarded plaque as a depot of fluoride available for incorporation into the enamel, rather than enamel fluoride as a source for plaque fluoride. This conclusion was based on the work of Gren et al, (1969), who found no evidence of fluoride uptake into dental plaque from shark enamel (fluorapatite). Fluorapatite is not the only mineral which has to be considered as a source for fluoride. Application of fluoride may result in the deposition of CaFz and any other soluble or loosely incorporated fluoride in the enamel (Brudevold, 1959; Mellberg et al., 1966; Dijkman et al., 1982). Subsequent dissolution of the soluble fluoride leads to a release of fluoride into the plaque (Dijkman et al., 1983; R¢lla and 0gaard, 1986). In vitro experiments showed decreased acid production in suspensions of oral lactobacilli and ~ral streptococci in con~act with fluoridated enamel or fluorapatite (Zwemer, 1957; Briner and Francis, 1962; Luoma and Luoma, 1982; Harper and Loesche, 1986). Removing the soluble fluoride from enam~l specimens treated with a fluoride lacquer (Fluor Protector; VIvadent, Schaan, Liechtenstein) before incubation with a suspension of oral streptococci abolished almost completely the inhibitory effect of enamel fluoride on the acid production (Van Loveren et al., 1984). Luoma et al, (1984) showe? no difference in final pH in fermenting S. mutans layers either covering enamel treated with Duraphat, a NaF varnish (Woelm Pharma GmbH & Co., 3440 Eschwege, FRG), but subsequently rinsed with water for 6 h for removal of absorbed fluoride and CaFz, or covering non-fluoride-treated enamel. Interpretation of the cited in vitro experiments is hampered by the buffering effect on pH which accompanies the dissolut,ion of enamel. This buffering will support bacterial metabohsm but, because less mineral dissolves in the presence of fluoride, acid production will be reduced. This inhibitory effect was not distinguished from the action of fluoride on bacterial metabolism. Therefore, these studies were not conclusive as to whether, or to what extent, fluoride itself caused the antimetabolic effect. Inhibition of the metabolism in the cell suspensions in contact with fluoridated enamel was not permanent and disappeared rapidly (Zwemer, 1957; Van Loveren et al., 1984).
Vol. 69 Special Issue
677
ANTIMICROBIAL ACTION OF FLUORIDE
Therefore, it is not likely that an antimetabolic effect is relevant for caries prevention in regimens where fluoride is topically applied only at six-month intervals.
Influence of plaque on fluoride uptake by enamel. Non-fermenting Streptococcus mutans plaque did not reduce the fluoride uptake by underlying enamel after application of an aqueous fluoride solution in vitro (Joyston-Bechal et al., 1976). Metabolically active plaque enhanced fluoride uptake by enamel in vivo after a single application with a NaF solution (Bruun and Stoltze, 1976), or after NaF or MFP rinses (Hellwig et al., 1987). These results have not been reproduced after a single application of a MFP solution (0gaard et al., 1985), Duraphat (Seppa, 1983), an APF gel (Tinanoff et al., 1974), or when an amine fluoride solution was applied (Bruun and Stoltze, 1976; Von Klimek et at., 1982). In these experiments, a limited plaque thickness or a long application time may have guaranteed an almost complete equilibrium between the concentration of fluoride in the plaque fluid at the plaque-enamel interface and the application solution. In a 100-J-Lm layer of plaque, a 90% equilibrium would be completed after 20 s, but in a 500-J-Lm layer a 90% equilibrium would be reached only after 500 s, assuming no chemical or bacterial interactions (McNee et al., 1980). Under these conditions, equilibrium ~ould be app:oxim~te~y 64% complete after an application time of four rmn. This Illustrates the effect of plaque thickness on the availability of fluoride at the plaque-enamel interface ~nd indicates that thick layers of plaque, as possibly present in fissures and interproximal spaces, may interfere with the uptake of fluoride by enamel. Plaque has been shown to interfere with the mechanism of fluoride uptake by enamel. After application, MFP was re~ov~re~ from clean enamel, but not from plaque-coveredenamel, indicating that MFP was rapidly hydrolyzed by dental plaque (0~aard et al., 1985? H~llwig et al., 19~7). After the application of Duraphat zn vitro, the formation of alkali-soluble fluoride precipitates (CaF2) was inhibited by the presence of pl~que, .although the total amount of fluoride taken up by demineralized enamel was not reduced (Hellwig et al., 1985). Fluoride uptake after an application with an APF solution is supposed to be affected similarly (Joyston-Bechal et al., 1976).
differences. In the experiments of Shields and Miihlemann (1975), they reduced plaque thickness by cleaning the entrance of the interproximal space. . A significant inhibitory effect on the Stephan curves (see FIg. 1) was observed when 48 mmol/L NaF (912 ppm fluoride) was added to sucrose rinses (Geddes and McNee, 1982). Lactate production was inhibited by approximately 50% immediately after a 1% NaF gel (238 mmol/L fluoride) application, but one hour after the application the observed inhibition lacked significance (Brown et al., 1981). In conclusion, a single application of fluoride reduces the acid production in dental plaque. A fluoride concentration in the plaque as low as 2.5 ppm (0.13 mmollL) appeared to be effective. To have an effect in interproximal plaque, concentrations as high as 250 ppm (132 rnrnol/L) in the application fluid were needed. However, a long-term residual effect is not to be expected after one single application.
Effects of continuous or repeated applications of fluoride. After the introduction of fluoridation of the drinking water, plaque pH drops produced in vitro from sucrose were shown to be reduced (Edgar et at., 1970). The mean final pH increased.by 0.13 units to pH 5.07. The increase appeared to be proportional to the DMFT of the individuals. The mean ionic fluoride concentration in the plaque increased to 0.64 ppm (34
pH
7
Effect of a single application of fluoride. By exposing collected plaque to a fluoride and sucrose challenge, Bibby and Fu (1986) showed that 10 ppm (0.53 mmoll L) fluoride applied either as NaF or MFP restricted the pHminimum to 4.51 and 4.74, respectively, whereas in the control experiments the pH dropped to 4.31 and 4.39. Using a comparable method, Eisenberg et at. (1985) found that 5 ppm (0:26 :n:nol/L) fluoride}n a glucose solution decreased plaque aciduricity so that the final pH was 4.77, an increase of about 1 pH unit compared with the control experiments. Jenkins and Edgar (1969) showed 2.5 ppm (0.13 mmollL) of fluoride to have a significant effect on the acid production by plaque in vitro, increasing the final pH by 0.06 pH unit to 5.02. The effect of low concentrations in situ was studied by Neff (1967), who found an inhibitory effect of 5 ppm (0.26 mmol/ L) fluoride in a sucrose solution on the pH drop in buccal plaque. In order for an inhibitory effect to be obtained in interproximal plaque, fluoride concentrations of 250 ppm (13.2 mmol/L; Shields and Miihlemann, 1975) or 1000 ppm (52.6 mmol/L; Jensen, 1985) were required. A variation of plaque thickness in these experiments may possibly account for these
5
o
4
8
12
16
20
time, min. Fig. 1-Mean values of plaque pH from measurements of Stephan curves induced by a sucrose rinse (adapted from Geddes and McNee, 1982, by permission of Archives of Oral Biology). --e= control experiments, - -0- - = during a period of daily mouth-rinsing with 48 mmol/L NaF; the measurements were performed 8-12 h after the last fluoride rinse (estimated values), • = 48 mrnol/L NaF incorporated in the sucrose rinse; the measurements were performed at least two months after the fluoride-rinse period.
678
VAN LOVEREN
urnol/L) fluoride (Edgar et al., 1970). An average increase of 0.11 to 0.20 pH unit in the pH minimum has been found in dental plaque collected from individuals who rinsed daily with a 48 mmol/L NaF solution for a period of 1-2 months (see Fig. 1). The fluoride rinses were more effective in the individuals exhibiting the largest pH drops at the control measurements. The results did not depend on the fluoridation of the drinking water in the residential areas of the subjects. The mean total fluoride concentration in the plaque was found to be 55 ppm (2.9 mmol/L; Geddes and McNee, 1982), of which < 5% is ionized (Tatevossian, 1989). An approximate 60% decrease in sucrose-induced lactate production in plaque has been demonstrated after one to three weeks of daily five-minute 1% NaF gel (238 mmol/L fluoride) applications. After the plaque was suspended in TISAB (Total Ionic Strength Activity Buffer, Orion Electrode 96-09 and Ionanalyzer Manual IM96, Orion Research, Inc., Cambridge, MA), the fluoride concentration in plaque was found to be 125 ppm (6.6 mrnol/L; Brown et al., 1983). Daily application of a 2% NaF solution (476 mmol/L fluoride) reduced the pH drop in plaque by 0.5 pH unit (Woolley and Rickles, 1971). The inhibitory effect of fluoride disappeared within 4-10 days after termination of the fluoride administrations (Woolley and Rickles, 1971; Geddes and McNee, 1982). Several explanations have been proposed for the mechanism by which continuous exposure to fluoride causes a reduced acid production in dental plaque. The bacterial metabolism is inhibited by fluoride taken up by the bacteria (Edgar et al., 1981). Furthermore, Edgar et al, (1981) have speculated that the relation between fluoride and pH depends on an effect of plaque thickness. Plaque fluoride is inversely related to the amount or thickness of plaque (Hardwick, 1970; Agus et al., 1976), which in turn is related to its metabolic activity (Charlton et al., 1974). Rugg-Gunn et al, (1981) showed that a positive relation between plaque fluoride and final plaque pH after sugar incubation in children using fluoridated school milk lost significance when the data were standardized on weight. It has also been suggested that oral streptococci which have adapted to fluoride are less cariogenic than the parent cells (Loesche, 1982). In the abovementioned studies (Woolley and Rickles, 1971; Geddes and McNee, 1982; Brown et al., 1983), the plaque acidogenicity was assessed between 8 and 12 h after the last fluoride treatment. Assuming a direct inhibiting effect of the fluoride ion, greater effects on plaque acidogenicity would have been detected at a shorter time interval after the fluoride applications (see Fig. 1). Therefore, daily fluoride treatments should be performed shortly before the cariogenic challenges in order for an optimal caries-preventive effect to be obtained.
Relevance to caries prevention. All experiments described have shown an inhibitory effect of fluoride on pH fall or acid production in plaque after a sucrose or glucose challenge, but none of them showed the relevance of this effect for caries inhibition. In our laboratory, we tried to separate the antimicrobial effect from the effect of fluoride on enamel solubility in an in vitro study. In a continuous demineralization model consisting of a S. mutans suspension covering an enamel specimen, the effect of fluoride was compared with the effect of nigericin, an ionophore which inhibits acid production but does not affect enamel solubility. Under our experimental conditions, inhibition of the metabolism by 0.5 mmol/L fluoride accounted for 75% of the reduction in enamel demineralization (Van Loveren et al., 1987). The effects of pH and fluoride during demineralization were
J Dent Res February 1990
studied extensively in non-bacteriological models (Ten Cate and Duijsters, 1983; Arends et al., 1983; Margolis et al., 1986). These studies have indicated that the concentration of the fluoride ion in solution required to inhibit demineralization is determined by the solubility products of fluoridated calcium (phosphate) precipitates. The minimum inhibitory concentration in a solution containing Ca and P at concentrations as present in dental plaque (Moreno and Margolis, 1988) can be estimated to fall in the range of 1 to 2 ppm (50 to 100 umol/ L). In vivo, higher concentrations of the fluoride ion may be necessary, because of remineralization inhibitory factors in dental plaque. If the fluoride ion concentration in plaque rises above this minimum inhibitory concentration, the underlying enamel becomes insoluble, and therefore the antimicrobial effect of the fluoride will be irrelevant. Lower concentrations will also contribute to caries prevention, because of their inhibiting effect on plaque metabolism.
Development of fluoride resistance. Development of fluoride resistance by oral lactobacilli (Green and Dodd, 1957) and streptococci (Williams, 1964; Hamilton, 1969) in vitro has been demonstrated to occur after serial passage of cells through increasingly higher levels of fluoride. Continuous culture experiments showed that oral streptococci (Hamilton and Bowden, 1982) and lactobacilli (Hamilton et al., 1985) grown at low pH with or without fluoride developed resistance. The resistance of the oral streptococci was characterized by the ability of the cells to grow on solid media with an increased fluoride content (growth adaptation) and by decreased sensitivity of glycolysis to the inhibitor (metabolic adaptation). The adaptation was accompanied by a reduced acidogenicity at constant pH 7 in a fluoride-free environment. The resistance was lost when the inducing factors were removed from the culture media. Data on growth adaptation to fluoride by oral micro-organisms in vivo are scarce. Hamilton and Bowden (1988) were not able to show differences in inherent fluoride resistance of strains of S. mutans from plaque from fluoridated and nonfluoridated areas after one subculture on a fluoride-free medium at pH 6.5. The fluoride resistance of growth of S. mutans in rat dental plaque was not increased by exposure to 60-ppm fluoride in the diet (3.16 mmol/kg) and in drinking water (3.16 mrnol/L), or by daily brushing with 10,000 ppm fluoride (526 mrnol/L; Van Loveren and Ten Cate, 1988). In contrast, Brown et al. (1983) demonstrated phenotypic and genotypic growth adaptation to fluoride by S. mutans in xerostomia patients who had applied a 1% NaF gel (238 rnrnol/ L fluoride) daily for one to three years. Eisenberg and Spitz (1988) also isolated fluoride-resistant mutans streptococci from xerostomia patients being treated with a fluoride gel. It is questionable whether such an adaptation would occur in normal subjects. The oral conditions in xerostomia patients may promote adaptation to fluoride resistance. Higher and prolonged retention of topically applied fluoride (Billings et al., 1988) will increase the pressure on the bacteria. Also, continuous non-cariogenic conditions may be selective for less acidogenic strains, which are expected to be more fluoride-resistant. There are no reports which demonstrate metabolic adaptation to fluoride by oral micro-organisms in human dental plaque, but it has been suggested that the acidogenicity of cells is reduced due to adaptation to fluoride (Loesche, 1982; Hamilton and Bowden, 1988). This suggestion was based on the apparent paradox that caries progression in xerostomia patients was inhibited by fluoride treatment, even though high proportions of S. mutans and Lactobacillus persisted in the dental
Vol. 69 Special Issue
679
ANTIMICROBIAL ACTION OF FLUORiDE
plaque (Brown et al., 1983). Reduced acidogenicity can be explained as a mechanism by which cells are protected against fluoride. The sensitivity to fluoride under constant pH (7.2) conditions was shown to be inversely related to the rate of glycolysis (Hamilton, 1977). Furthermore, the fluoride sensitivity of cells is potentiated by a low pH environment (Kashket et al., 1977). If cells are not able to lower the extracellular pH, higher fluoride concentrations are needed for inhibition.
Possible role of adaptation in caries prevention. In vitro-induced fluoride-resistant mutant cells of oral streptococci have been shown to be less cariogenic in rats when compared with parent cells (Rosen et al., 1978; Van Loveren et al., 1989). Reduced cariogenicity may be caused by a slower glycolytic rate in the rat dental plaque. A reduced velocity of acid production caused by mutation to fluoride resistance has been demonstrated in vitro (Eisenberg and Spitz, 1987; Van Loveren, unpublished data). At constant pH 7.0, fluoride-resistant mutants of oral mutans streptococci produced acid less rapidly than their parent cells. When the environmental pH was lowered, the differences in the velocity of acid production between the fluoride-resistant mutants and the parent cells decreased or even reversed (Fig. 2). This complicates the idea that adaptation to fluoride in vivo would compromise plaque cariogenicity. In the study by Brown et al, (1983), only 25% of the isolated mutans streptococci showed increased fluoride resistance after five years of daily fluoride gel application. Therefore, it is conceivable that, when adaptation occurs in vivo, only a small proportion of the mutans population develops resistance. The resistant cells will be in the plaque community next to the parent cells. In such a situation, the parent cells may still cause the pH drop, whereas the resistant cells may be capable of continuing acid production at low pH. This may even result in a more cariogenic plaque. The mechanisms of adaptation to fluoride and of fluoride resistance are still unknown. Especially when the in vitro mutation is induced by a mutagen or UV light (Rosen et al., 1978; Eisenberg and Spitz, 1987), the mutations may be different from adaptations that may occur in vivo. For this reason, care should be taken when the results of the in vitro experiments are extrapolated to the in vivo situation.
(Geddes and McNee, 1982). Both studies indicated that human plaque exposed to one of the two most commonly used modes of fluoride administration remains sensitive to additional fluoride treatments.
Conclusions. It has been shown clearly that the acid production in dental plaque is inhibited by fluoride. However, the clinical importance of this inhibition can be questioned and may depend on the kind of application. In dental plaque grown over enamel treated with fluoride, acid production will be inhibited. Since this inhibition is only observed for a short period of time, it will not play an important role in caries reduction by topical fluorides professionally applied at six-month intervals. For the same reason, the clinical importance of the inhibition of plaque metabolism after a single application of fluoride to dental plaque is negligible. Daily or more frequent exposure of plaque to fluoride will result in a reduced acidogenicity for at least 8 to 12 h without necessarily increasing plaque fluoride to a level at which the enamel solubility will be strongly decreased. To serve an optimal antimicrobial effect of fluoride, daily fluoride treatments should be performed shortly before the cariogenic challenges. It has been suggested that an adaptation to fluoride may reduce the cariogenic potential of dental plaque, but such an adaptation has never been demonstrated to occur in vivo. Ex-
200
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.... .... 150
....
~ \ \
Q
o
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+-
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The effects of fluoride in adapted plaque. Van der Hoeven and Franken (1984) have concluded that S. mutans cells colonizing the mouths of gnotobiotic rats receiving fluoride-supplemented diet (0.48 mmol/kg) and drinking water (0.48 mmollL) develop metabolic resistance to 20 mmol/ L fluoride rinses when compared with those colonizing the mouths of control animals, The adapted plaque produced 20 nmol lactic acid/ug DNA 15 min after a sucrose rinse containing 20 mmollL fluoride. The non-adapted plaque produced 16.6 nmol lactic acid/ug DNA. These results do not necessarily indicate an adaptation to fluoride. Because of a lower initial acid concentration and slower glycolytic rate in the adapted plaque, more acid may have been formed before plaque pH dropped to the level at which the plaque became sensitive to the fluoride. The conclusion of Van der Hoeven and Franken (1984) has not been confirmed in vivo. As little as 0.26 mmol/ L fluoride decreased the acidogenicity of pooled plaque samples from persons using fluoridated toothpastes containing at least 1000 ppm (52.6 mmollL) fluoride (Eisenberg et ai., 1985). Also, the sucrose-mediated pH minimum of plaque of persons living in an area with fluoridated drinking water increased during a period of daily rinsing with 48 rnrnol/L NaF solution
....J
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7.0
6.5
6.0
5.5
5.0
4.5
• 4.0
constant pH Fig. 2-The effect of in vitro mutation to fluoride resistance on the velocity of acid production at various constant pH-conditions.- o-" - . - = Streptococcus sobrinus 6715 and its fluoride-resistant mutant LSF-2 (Eisenberg and Spitz, 1987~, ......... = S. mutans C180·2 and its fluoride-resistant mutant C180-2 FR. The values were significantly different at each pH~5 (Student'S t test: p
J Dent Res 69(Spec Iss):676-681, February, 1990
Introduction. Demineralization and remineralization are the processes which determine the condition of tooth surfaces after eruption. Demineralization is caused by acidswhich are produced by bacteria in dental plaque. A microbial contributio~ to r~mi~erali~ation is not generally accepted, although remineralization will be enhanced by an elevated plaque pH resulting from t~e dewadation of nitrogenous substrates (such as urea and ammo acids) by bacterial enzymes (Kleinberg, 1967; Hayes and Hyatt, 1974; Kleinberg et al., 1977; Sissons and Cutress, 1988). Fluoride is known to inhibit the energy and biosynthetic metabolism of bacteria (Bibby and Van Kesteren, 1940; Kashket et al., 1977; Maltz and Emilson, 1982), but these antimicrobial effects of fluoride in caries prevention are often regarded as of little or no importance as compared with the direct interactions of fluoride with the hard tissue during caries lesion development and progression (Clarkson et ,al., 1988~. In fact no in vivo studies are available that quantify the antimicrobial effect in relation to the overall effect of fluoride on dental caries. In vitro studies with oral lactobacilli and streptococci have demonstrated adaptation of these organisms to fluoride resistance (Green and Dodd, 1957; Williams, 1964; Hamilton, 1969), but data on adaptation in vivo are scarce. It has been stated Presented at a Joint IADR/ORCA International Symposium on Fluorides: Mechanisms of Action and Recommendations for Use, held March 21-24, 1989, Callaway Gardens Conference Center, Pine Mountain, Georgia 676
that due to fluoride adaptation, the inhibition of acid production'is unlikely to be important as a caries-preventive action of fluoride (Van der Hoeven and Franken, 1984). On the other hand, it has been proposed that the cariogenicity of plaque adapted to fluoride will be reduced (Loesche, 1982). In this paper several topics will be reviewed and discussed concerning the effect of fluoride in dental plaque: (a) the inhibitory effect of both enamel-derived or topically applied fluoride on acid production in dental plaque; (b) the effect of plaque on the retention of fluoride in enamel; (c) the relevance of the inhibition of plaque acid production by fluoride for caries prevention; and (d) development of fluoride resistance and the consequences of that for plaque cariogenicity.
Effects of fluoride derived from enamel. In their review, Jenkins and Edgar (1977) regarded plaque as a depot of fluoride available for incorporation into the enamel, rather than enamel fluoride as a source for plaque fluoride. This conclusion was based on the work of Gren et al, (1969), who found no evidence of fluoride uptake into dental plaque from shark enamel (fluorapatite). Fluorapatite is not the only mineral which has to be considered as a source for fluoride. Application of fluoride may result in the deposition of CaFz and any other soluble or loosely incorporated fluoride in the enamel (Brudevold, 1959; Mellberg et al., 1966; Dijkman et al., 1982). Subsequent dissolution of the soluble fluoride leads to a release of fluoride into the plaque (Dijkman et al., 1983; R¢lla and 0gaard, 1986). In vitro experiments showed decreased acid production in suspensions of oral lactobacilli and ~ral streptococci in con~act with fluoridated enamel or fluorapatite (Zwemer, 1957; Briner and Francis, 1962; Luoma and Luoma, 1982; Harper and Loesche, 1986). Removing the soluble fluoride from enam~l specimens treated with a fluoride lacquer (Fluor Protector; VIvadent, Schaan, Liechtenstein) before incubation with a suspension of oral streptococci abolished almost completely the inhibitory effect of enamel fluoride on the acid production (Van Loveren et al., 1984). Luoma et al, (1984) showe? no difference in final pH in fermenting S. mutans layers either covering enamel treated with Duraphat, a NaF varnish (Woelm Pharma GmbH & Co., 3440 Eschwege, FRG), but subsequently rinsed with water for 6 h for removal of absorbed fluoride and CaFz, or covering non-fluoride-treated enamel. Interpretation of the cited in vitro experiments is hampered by the buffering effect on pH which accompanies the dissolut,ion of enamel. This buffering will support bacterial metabohsm but, because less mineral dissolves in the presence of fluoride, acid production will be reduced. This inhibitory effect was not distinguished from the action of fluoride on bacterial metabolism. Therefore, these studies were not conclusive as to whether, or to what extent, fluoride itself caused the antimetabolic effect. Inhibition of the metabolism in the cell suspensions in contact with fluoridated enamel was not permanent and disappeared rapidly (Zwemer, 1957; Van Loveren et al., 1984).
Vol. 69 Special Issue
677
ANTIMICROBIAL ACTION OF FLUORIDE
Therefore, it is not likely that an antimetabolic effect is relevant for caries prevention in regimens where fluoride is topically applied only at six-month intervals.
Influence of plaque on fluoride uptake by enamel. Non-fermenting Streptococcus mutans plaque did not reduce the fluoride uptake by underlying enamel after application of an aqueous fluoride solution in vitro (Joyston-Bechal et al., 1976). Metabolically active plaque enhanced fluoride uptake by enamel in vivo after a single application with a NaF solution (Bruun and Stoltze, 1976), or after NaF or MFP rinses (Hellwig et al., 1987). These results have not been reproduced after a single application of a MFP solution (0gaard et al., 1985), Duraphat (Seppa, 1983), an APF gel (Tinanoff et al., 1974), or when an amine fluoride solution was applied (Bruun and Stoltze, 1976; Von Klimek et at., 1982). In these experiments, a limited plaque thickness or a long application time may have guaranteed an almost complete equilibrium between the concentration of fluoride in the plaque fluid at the plaque-enamel interface and the application solution. In a 100-J-Lm layer of plaque, a 90% equilibrium would be completed after 20 s, but in a 500-J-Lm layer a 90% equilibrium would be reached only after 500 s, assuming no chemical or bacterial interactions (McNee et al., 1980). Under these conditions, equilibrium ~ould be app:oxim~te~y 64% complete after an application time of four rmn. This Illustrates the effect of plaque thickness on the availability of fluoride at the plaque-enamel interface ~nd indicates that thick layers of plaque, as possibly present in fissures and interproximal spaces, may interfere with the uptake of fluoride by enamel. Plaque has been shown to interfere with the mechanism of fluoride uptake by enamel. After application, MFP was re~ov~re~ from clean enamel, but not from plaque-coveredenamel, indicating that MFP was rapidly hydrolyzed by dental plaque (0~aard et al., 1985? H~llwig et al., 19~7). After the application of Duraphat zn vitro, the formation of alkali-soluble fluoride precipitates (CaF2) was inhibited by the presence of pl~que, .although the total amount of fluoride taken up by demineralized enamel was not reduced (Hellwig et al., 1985). Fluoride uptake after an application with an APF solution is supposed to be affected similarly (Joyston-Bechal et al., 1976).
differences. In the experiments of Shields and Miihlemann (1975), they reduced plaque thickness by cleaning the entrance of the interproximal space. . A significant inhibitory effect on the Stephan curves (see FIg. 1) was observed when 48 mmol/L NaF (912 ppm fluoride) was added to sucrose rinses (Geddes and McNee, 1982). Lactate production was inhibited by approximately 50% immediately after a 1% NaF gel (238 mmol/L fluoride) application, but one hour after the application the observed inhibition lacked significance (Brown et al., 1981). In conclusion, a single application of fluoride reduces the acid production in dental plaque. A fluoride concentration in the plaque as low as 2.5 ppm (0.13 mmollL) appeared to be effective. To have an effect in interproximal plaque, concentrations as high as 250 ppm (132 rnrnol/L) in the application fluid were needed. However, a long-term residual effect is not to be expected after one single application.
Effects of continuous or repeated applications of fluoride. After the introduction of fluoridation of the drinking water, plaque pH drops produced in vitro from sucrose were shown to be reduced (Edgar et at., 1970). The mean final pH increased.by 0.13 units to pH 5.07. The increase appeared to be proportional to the DMFT of the individuals. The mean ionic fluoride concentration in the plaque increased to 0.64 ppm (34
pH
7
Effect of a single application of fluoride. By exposing collected plaque to a fluoride and sucrose challenge, Bibby and Fu (1986) showed that 10 ppm (0.53 mmoll L) fluoride applied either as NaF or MFP restricted the pHminimum to 4.51 and 4.74, respectively, whereas in the control experiments the pH dropped to 4.31 and 4.39. Using a comparable method, Eisenberg et at. (1985) found that 5 ppm (0:26 :n:nol/L) fluoride}n a glucose solution decreased plaque aciduricity so that the final pH was 4.77, an increase of about 1 pH unit compared with the control experiments. Jenkins and Edgar (1969) showed 2.5 ppm (0.13 mmollL) of fluoride to have a significant effect on the acid production by plaque in vitro, increasing the final pH by 0.06 pH unit to 5.02. The effect of low concentrations in situ was studied by Neff (1967), who found an inhibitory effect of 5 ppm (0.26 mmol/ L) fluoride in a sucrose solution on the pH drop in buccal plaque. In order for an inhibitory effect to be obtained in interproximal plaque, fluoride concentrations of 250 ppm (13.2 mmol/L; Shields and Miihlemann, 1975) or 1000 ppm (52.6 mmol/L; Jensen, 1985) were required. A variation of plaque thickness in these experiments may possibly account for these
5
o
4
8
12
16
20
time, min. Fig. 1-Mean values of plaque pH from measurements of Stephan curves induced by a sucrose rinse (adapted from Geddes and McNee, 1982, by permission of Archives of Oral Biology). --e= control experiments, - -0- - = during a period of daily mouth-rinsing with 48 mmol/L NaF; the measurements were performed 8-12 h after the last fluoride rinse (estimated values), • = 48 mrnol/L NaF incorporated in the sucrose rinse; the measurements were performed at least two months after the fluoride-rinse period.
678
VAN LOVEREN
urnol/L) fluoride (Edgar et al., 1970). An average increase of 0.11 to 0.20 pH unit in the pH minimum has been found in dental plaque collected from individuals who rinsed daily with a 48 mmol/L NaF solution for a period of 1-2 months (see Fig. 1). The fluoride rinses were more effective in the individuals exhibiting the largest pH drops at the control measurements. The results did not depend on the fluoridation of the drinking water in the residential areas of the subjects. The mean total fluoride concentration in the plaque was found to be 55 ppm (2.9 mmol/L; Geddes and McNee, 1982), of which < 5% is ionized (Tatevossian, 1989). An approximate 60% decrease in sucrose-induced lactate production in plaque has been demonstrated after one to three weeks of daily five-minute 1% NaF gel (238 mmol/L fluoride) applications. After the plaque was suspended in TISAB (Total Ionic Strength Activity Buffer, Orion Electrode 96-09 and Ionanalyzer Manual IM96, Orion Research, Inc., Cambridge, MA), the fluoride concentration in plaque was found to be 125 ppm (6.6 mrnol/L; Brown et al., 1983). Daily application of a 2% NaF solution (476 mmol/L fluoride) reduced the pH drop in plaque by 0.5 pH unit (Woolley and Rickles, 1971). The inhibitory effect of fluoride disappeared within 4-10 days after termination of the fluoride administrations (Woolley and Rickles, 1971; Geddes and McNee, 1982). Several explanations have been proposed for the mechanism by which continuous exposure to fluoride causes a reduced acid production in dental plaque. The bacterial metabolism is inhibited by fluoride taken up by the bacteria (Edgar et al., 1981). Furthermore, Edgar et al, (1981) have speculated that the relation between fluoride and pH depends on an effect of plaque thickness. Plaque fluoride is inversely related to the amount or thickness of plaque (Hardwick, 1970; Agus et al., 1976), which in turn is related to its metabolic activity (Charlton et al., 1974). Rugg-Gunn et al, (1981) showed that a positive relation between plaque fluoride and final plaque pH after sugar incubation in children using fluoridated school milk lost significance when the data were standardized on weight. It has also been suggested that oral streptococci which have adapted to fluoride are less cariogenic than the parent cells (Loesche, 1982). In the abovementioned studies (Woolley and Rickles, 1971; Geddes and McNee, 1982; Brown et al., 1983), the plaque acidogenicity was assessed between 8 and 12 h after the last fluoride treatment. Assuming a direct inhibiting effect of the fluoride ion, greater effects on plaque acidogenicity would have been detected at a shorter time interval after the fluoride applications (see Fig. 1). Therefore, daily fluoride treatments should be performed shortly before the cariogenic challenges in order for an optimal caries-preventive effect to be obtained.
Relevance to caries prevention. All experiments described have shown an inhibitory effect of fluoride on pH fall or acid production in plaque after a sucrose or glucose challenge, but none of them showed the relevance of this effect for caries inhibition. In our laboratory, we tried to separate the antimicrobial effect from the effect of fluoride on enamel solubility in an in vitro study. In a continuous demineralization model consisting of a S. mutans suspension covering an enamel specimen, the effect of fluoride was compared with the effect of nigericin, an ionophore which inhibits acid production but does not affect enamel solubility. Under our experimental conditions, inhibition of the metabolism by 0.5 mmol/L fluoride accounted for 75% of the reduction in enamel demineralization (Van Loveren et al., 1987). The effects of pH and fluoride during demineralization were
J Dent Res February 1990
studied extensively in non-bacteriological models (Ten Cate and Duijsters, 1983; Arends et al., 1983; Margolis et al., 1986). These studies have indicated that the concentration of the fluoride ion in solution required to inhibit demineralization is determined by the solubility products of fluoridated calcium (phosphate) precipitates. The minimum inhibitory concentration in a solution containing Ca and P at concentrations as present in dental plaque (Moreno and Margolis, 1988) can be estimated to fall in the range of 1 to 2 ppm (50 to 100 umol/ L). In vivo, higher concentrations of the fluoride ion may be necessary, because of remineralization inhibitory factors in dental plaque. If the fluoride ion concentration in plaque rises above this minimum inhibitory concentration, the underlying enamel becomes insoluble, and therefore the antimicrobial effect of the fluoride will be irrelevant. Lower concentrations will also contribute to caries prevention, because of their inhibiting effect on plaque metabolism.
Development of fluoride resistance. Development of fluoride resistance by oral lactobacilli (Green and Dodd, 1957) and streptococci (Williams, 1964; Hamilton, 1969) in vitro has been demonstrated to occur after serial passage of cells through increasingly higher levels of fluoride. Continuous culture experiments showed that oral streptococci (Hamilton and Bowden, 1982) and lactobacilli (Hamilton et al., 1985) grown at low pH with or without fluoride developed resistance. The resistance of the oral streptococci was characterized by the ability of the cells to grow on solid media with an increased fluoride content (growth adaptation) and by decreased sensitivity of glycolysis to the inhibitor (metabolic adaptation). The adaptation was accompanied by a reduced acidogenicity at constant pH 7 in a fluoride-free environment. The resistance was lost when the inducing factors were removed from the culture media. Data on growth adaptation to fluoride by oral micro-organisms in vivo are scarce. Hamilton and Bowden (1988) were not able to show differences in inherent fluoride resistance of strains of S. mutans from plaque from fluoridated and nonfluoridated areas after one subculture on a fluoride-free medium at pH 6.5. The fluoride resistance of growth of S. mutans in rat dental plaque was not increased by exposure to 60-ppm fluoride in the diet (3.16 mmol/kg) and in drinking water (3.16 mrnol/L), or by daily brushing with 10,000 ppm fluoride (526 mrnol/L; Van Loveren and Ten Cate, 1988). In contrast, Brown et al. (1983) demonstrated phenotypic and genotypic growth adaptation to fluoride by S. mutans in xerostomia patients who had applied a 1% NaF gel (238 rnrnol/ L fluoride) daily for one to three years. Eisenberg and Spitz (1988) also isolated fluoride-resistant mutans streptococci from xerostomia patients being treated with a fluoride gel. It is questionable whether such an adaptation would occur in normal subjects. The oral conditions in xerostomia patients may promote adaptation to fluoride resistance. Higher and prolonged retention of topically applied fluoride (Billings et al., 1988) will increase the pressure on the bacteria. Also, continuous non-cariogenic conditions may be selective for less acidogenic strains, which are expected to be more fluoride-resistant. There are no reports which demonstrate metabolic adaptation to fluoride by oral micro-organisms in human dental plaque, but it has been suggested that the acidogenicity of cells is reduced due to adaptation to fluoride (Loesche, 1982; Hamilton and Bowden, 1988). This suggestion was based on the apparent paradox that caries progression in xerostomia patients was inhibited by fluoride treatment, even though high proportions of S. mutans and Lactobacillus persisted in the dental
Vol. 69 Special Issue
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ANTIMICROBIAL ACTION OF FLUORiDE
plaque (Brown et al., 1983). Reduced acidogenicity can be explained as a mechanism by which cells are protected against fluoride. The sensitivity to fluoride under constant pH (7.2) conditions was shown to be inversely related to the rate of glycolysis (Hamilton, 1977). Furthermore, the fluoride sensitivity of cells is potentiated by a low pH environment (Kashket et al., 1977). If cells are not able to lower the extracellular pH, higher fluoride concentrations are needed for inhibition.
Possible role of adaptation in caries prevention. In vitro-induced fluoride-resistant mutant cells of oral streptococci have been shown to be less cariogenic in rats when compared with parent cells (Rosen et al., 1978; Van Loveren et al., 1989). Reduced cariogenicity may be caused by a slower glycolytic rate in the rat dental plaque. A reduced velocity of acid production caused by mutation to fluoride resistance has been demonstrated in vitro (Eisenberg and Spitz, 1987; Van Loveren, unpublished data). At constant pH 7.0, fluoride-resistant mutants of oral mutans streptococci produced acid less rapidly than their parent cells. When the environmental pH was lowered, the differences in the velocity of acid production between the fluoride-resistant mutants and the parent cells decreased or even reversed (Fig. 2). This complicates the idea that adaptation to fluoride in vivo would compromise plaque cariogenicity. In the study by Brown et al, (1983), only 25% of the isolated mutans streptococci showed increased fluoride resistance after five years of daily fluoride gel application. Therefore, it is conceivable that, when adaptation occurs in vivo, only a small proportion of the mutans population develops resistance. The resistant cells will be in the plaque community next to the parent cells. In such a situation, the parent cells may still cause the pH drop, whereas the resistant cells may be capable of continuing acid production at low pH. This may even result in a more cariogenic plaque. The mechanisms of adaptation to fluoride and of fluoride resistance are still unknown. Especially when the in vitro mutation is induced by a mutagen or UV light (Rosen et al., 1978; Eisenberg and Spitz, 1987), the mutations may be different from adaptations that may occur in vivo. For this reason, care should be taken when the results of the in vitro experiments are extrapolated to the in vivo situation.
(Geddes and McNee, 1982). Both studies indicated that human plaque exposed to one of the two most commonly used modes of fluoride administration remains sensitive to additional fluoride treatments.
Conclusions. It has been shown clearly that the acid production in dental plaque is inhibited by fluoride. However, the clinical importance of this inhibition can be questioned and may depend on the kind of application. In dental plaque grown over enamel treated with fluoride, acid production will be inhibited. Since this inhibition is only observed for a short period of time, it will not play an important role in caries reduction by topical fluorides professionally applied at six-month intervals. For the same reason, the clinical importance of the inhibition of plaque metabolism after a single application of fluoride to dental plaque is negligible. Daily or more frequent exposure of plaque to fluoride will result in a reduced acidogenicity for at least 8 to 12 h without necessarily increasing plaque fluoride to a level at which the enamel solubility will be strongly decreased. To serve an optimal antimicrobial effect of fluoride, daily fluoride treatments should be performed shortly before the cariogenic challenges. It has been suggested that an adaptation to fluoride may reduce the cariogenic potential of dental plaque, but such an adaptation has never been demonstrated to occur in vivo. Ex-
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The effects of fluoride in adapted plaque. Van der Hoeven and Franken (1984) have concluded that S. mutans cells colonizing the mouths of gnotobiotic rats receiving fluoride-supplemented diet (0.48 mmol/kg) and drinking water (0.48 mmollL) develop metabolic resistance to 20 mmol/ L fluoride rinses when compared with those colonizing the mouths of control animals, The adapted plaque produced 20 nmol lactic acid/ug DNA 15 min after a sucrose rinse containing 20 mmollL fluoride. The non-adapted plaque produced 16.6 nmol lactic acid/ug DNA. These results do not necessarily indicate an adaptation to fluoride. Because of a lower initial acid concentration and slower glycolytic rate in the adapted plaque, more acid may have been formed before plaque pH dropped to the level at which the plaque became sensitive to the fluoride. The conclusion of Van der Hoeven and Franken (1984) has not been confirmed in vivo. As little as 0.26 mmol/ L fluoride decreased the acidogenicity of pooled plaque samples from persons using fluoridated toothpastes containing at least 1000 ppm (52.6 mmollL) fluoride (Eisenberg et ai., 1985). Also, the sucrose-mediated pH minimum of plaque of persons living in an area with fluoridated drinking water increased during a period of daily rinsing with 48 rnrnol/L NaF solution
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constant pH Fig. 2-The effect of in vitro mutation to fluoride resistance on the velocity of acid production at various constant pH-conditions.- o-" - . - = Streptococcus sobrinus 6715 and its fluoride-resistant mutant LSF-2 (Eisenberg and Spitz, 1987~, ......... = S. mutans C180·2 and its fluoride-resistant mutant C180-2 FR. The values were significantly different at each pH~5 (Student'S t test: p
