JADA Continuing Education
Biofilm Plaque and Hydrodynamic Effects on Mass Transfer, Fluoride Delivery and Caries
Paul Stoodley, PhD,
James Wefel, PhD,
Armin Gieseke, PhD,
Dirk deBeer, PhD and
Christiane von Ohle, DMD
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ABSTRACT
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Background. The biofilm concept of dental plaque now is widely accepted in the dental clinic, particularly with respect to its importance to oral hygiene. A number of reviews have focused on the microbial ecology of biofilm with regard to oral health; however, there has been less focus on how the interaction of biofilms and hydrodynamics with mass transfer (the movement of molecules and particulates) and physiological processes may relate to caries.
Types of Studies Reviewed. The authors reviewed reports in the microbiology and dental literature addressing microbiological, engineering and clinical aspects of biofilms with respect to mass transport and microbial physiology, with an emphasis on fluoride ions (F–).
Conclusions and Practical Implications. These data illustrate how dental plaque biofilms may affect the delivery of cariogenic agents, such as sucrose, or anticariogenic agents, such as F–, into and out of the biofilm, with subsequent consequences for the development of physio-chemical microenvironments at the tooth surface. Increasing the flow rate in an overlying fluid (such as saliva or mouthrinse) increases transport from the fluid into and through biofilms. Increasing the delivery of anticariogenic agents such as F– into the plaque biofilm, by generating strong fluid flows, may be a useful strategy for enhancing the anticaries effects of F– in areas of the mouth where complete biofilm removal is not possible with routine daily cleaning techniques.
Key Words: Biofilms; caries; fluoride; microbiology; immunologyAbbreviations: Ca2+: Calcium ion. • DO: Dissolved oxygen. • EPS: Extracellular polymeric slime. • F–: Fluoride ion. • FHA: Fluorhydroxyapatite. • MBC: Minimum bactericidal concentration. • MIC: Minimum inhibitory concentration. • NaF: Sodium fluoride. • PO43–: Phosphate ion.
Dental plaque is a dynamic community of microor-ganisms, developing continually and reshaping the microenvironment in which they live.1,2 Bacteria and other organisms in the plaque take nutrients from our saliva and the food we eat to proliferate. Immediately after tooth cleaning, bacteria left on the tooth surface and those attaching to the tooth surface from other parts of the oral cavity such as the tongue, gingivae and cheek mucosa begin to regrow. As the biofilm grows, it forms an irregular heterogeneous structure containing clusters of cells surrounded by channels through which liquid, such as saliva, can flow.3,4
Aerobic organisms on the periphery of the cell clusters remove dissolved oxygen (DO) rapidly, creating favorable microniches for pathogenic anaerobic bacteria to thrive. Thus, as the biofilm develops, it may be thought of as an ecosystem, containing many habitats and organisms. Bacteria modify the local environment through the production of acid from the fermentation of sucrose and other fermentable sugars in the diet, which then may increase demineralization of the enamel surface, leading to, or accelerating, the development of caries.5
The literature contains many excellent reviews regarding the microbial ecology and management of dental plaque biofilms.1,2,6 However, it is the goal of this review to concentrate on the effect that the interactions between biofilm and hydrodynamics have on the delivery of fluoride ion (F–) to the tooth surface, and the effect that F– might have on biofilm physiology and, consequently, the cariogenic process.
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FLUORIDE AS ANTICARIES AGENT
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To set the scene for our discussion of the interplay between F–, tooth enamel and biofilm, we first provide a brief overview of the chemistry of tooth dissolution and why F– has been such an important component in combating the caries epidemic. Wefel7 and ten Cate and Featherstone8 provided comprehensive reviews of this topic. The striking therapeutic effect of F– on dental caries was first demonstrated in Grand Rapids, Mich., in 1945 when F– was added to the drinking water.9,10 After monitoring almost 30,000 schoolchildren in various cohorts across an 11-year period after water fluoridation had begun, researchers found that the caries incidence—measured as decayed, missing or filled permanent teeth—decreased by more than 60 percent after water fluoridation.10 However, fluorosis is a potential problem when F– is added to water, particularly when the natural concentration might be high; the concentration should be optimized to maximize the anti-caries effect while minimizing the probability of fluorosis. The optimal concentration is approximately 1 milligram/liter.11
Investigators have proposed three main mechanisms to account for the observed protective effects of F– on the reduction of enamel decay.7,12 The mechanisms, in the traditionally considered order of importance, are as follows:
- – reduction in solubility of calcium hydroxyapatite;
- – balance of rates of demineralization and remineralization;
- – antimicrobial effects of F– in terms of affecting metabolism and as a killing agent.7,13
To help conceptualize the caries process, Featherstone14–16 introduced the concept of "caries balance," in which pathological factors are weighed against protective factors. Overwhelming evidence points to F– as tipping the balance with regard to the protective factors. The relative contribution of F– to the various mechanisms of protection, however, remains less clear.
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FLUORIDE AND REDUCED ENAMEL SOLUBILITY UNDER ACIDIC CONDITIONS
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Tooth enamel is composed largely of calcium hydroxyapatite [Ca10(PO4)6(OH)2]. However, it is referred to often as calcium-deficient carbonate-containing apatite to reflect impurities from the time of formation. Apatites in general are good ion exchangers, and topically supplied F– enters the enamel lattice, replacing hydroxyl groups to produce fluorhydroxyapatite (FHA) and fluorapatite (which is 100 percent replacement of hydroxyl ions with fluoride ions), which have a lower solubility at a low pH than does calcium hydroxyapatite. For example, Tenuta and colleagues17 calculated that fluorapatite would not dissolve until the pH dropped below approximately 4.4. However, researchers now believe that it is not the reduction of solubility of calcium hyroxyapatite by F– alone that is the main mechanism attributed to caries reduction, but rather the effect of F– on the balance between the rates of demineralization (dissolution of enamel) and remineralization (deposition of enamel) that is most important.7,13
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FLUORIDE AND DEMINERALIZATION/ REMINERALIZATION RATES UNDER ACIDIC CONDITIONS
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In terms of illustrating the role of acid in caries formation, it is a useful simplification to use the concept of a so-called "critical pH," below which demineralization dominates, resulting in a net dissolution of tooth enamel.18 The "critical pH" depends on the calcium ion (Ca2+) and phosphate ion (PO43–) concentration in the fluid overlying the tooth surface, which varies between individuals and over time as a result of saliva chemistry, diet and eating habits, but generally it is considered to be approximately 5.5 for most people.19 The tooth surface always is in contact with fluid (such as saliva, biofilm plaque, food stuffs) and there is a constant exchange of Ca2+, PO43– and other ions between the tooth enamel and the fluid.20,21 Fluoride ions in oral fluids overlying an enamel surface (that is, saliva, mouthrinse or plaque) increase the rate of remineralization of calcium hydroxyapatite and FHA and reduce the rate of demineralization, as shown for calcium phosphate and enamel22 and, more recently, in artificial sub-surface carious lesions in human enamel.23 The extent of the "fluoride effect" was related directly to the concentration of F–, as well as to the concentration of Ca2+ and PO43–
Bacteria in the biofilm can produce lactic acid through the fermentation of dietary sugars such as sucrose and carbohydrates. In addition, oral streptococci can metabolize sucrose to produce insoluble glucans that promote the formation of the biofilm extracellular polymeric slime (EPS) matrix and, consequently, more voluminous biofilms.24,25 Thus, sucrose has a negative synergy with respect to caries in that it promotes both biofilm formation and acid production by cariogenic bacteria such as Streptococcus mutans. If the exposure to acid is short, the saliva will raise the pH naturally so that the enamel loss can be repaired through remineralization. However, if exposure to acid is prolonged (for example, by sipping sugar-containing soft drinks or sucking on sugar-containing candies), the remineralization rate may not repair the loss from demineralization, thus increasing the probability of caries developing.26
An important factor in caries formation and control is the cycling time between exposures to low pH. The cycling time, therefore, is related directly to intake of sucrose and other fermentable carbohydrates, which cause acid generation.27 The first evidence of this emerged from the Vipeholm studies in the 1950s.28 More recently, Marshall and colleagues29 reported a positive correlation between the incidence of caries and the "frequency and length of eating events" of sugar-containing foods.
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FLUORIDE AS MODULATOR OF BACTERIAL ACID PRODUCTION
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In addition to the physicochemical interactions between the chemistry of the overlying fluid (Ca2+, PO43– F–, pH) and the mineralogy of the solid-phase calcium hydroxyapatite in the enamel, F– may tip the caries balance by reducing acid production by the acidogenic (acid-producing) bacteria: lactobacilli and mutans streptococci. The efficacy of F– from a microbiological context is explained by two main mechanisms: the killing of biofilm plaque bacteria and the inhibition of metabolic pathways, including the fermentation pathway, that lead to lactic acid production.30
S. mutans is a cariogenic, aerotolerant anaerobic bacterium that is used commonly as a model for studying acid production and the subsequent events in the initial stages of caries formation. Interestingly, S. mutans can consume DO, apparently not for energy,5,31 but rather as a defense from competitive streptococci that are thought to use DO as a substrate for hydrogen peroxide production, presumably to attack other bacteria.32 Oxygen also has been shown to hinder the early (48 hours) development of biofilm formation by S. mutans,33 and it is possible that DO consumption by S. mutans on the outside of a biofilm may create more favorable conditions for growth of those bacteria deeper in the biofilm, as hypothesized by Thomas and Pera.34 In preliminary studies in our laboratory (unpublished data, July 2007) with DO microelectrodes (Figure 1A
35), we found that an S. mutans biofilm grown for three days on calcium hydroxyapatite–coated coupons in a 5 percent carbon dioxide environment, as described by Adams and colleagues36 and Heersink and colleagues,37 can cause an anaerobic region (defined as < 0.5 percent of DO saturation38) to develop at a depth of approximately 65 micrometers within the biofilm when stimulated with 10 percent sucrose (Figure 1B) (A.G., D.de B., C.von O., P.S., unpublished data, 2007).
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Figure 1. Development of anaerobic zones in dental biofilms. A. Stereomicroscopic image of a microelectrode (black pointer) positioned within a Streptococcus mutans biofilm (open arrow) grown on a hydroxyapatite-coated slide. Measurements were made by placing the biofilms in a flow cell with aerated recirculating 10 percent weakly buffered saliva and 10 percent sucrose35 (as described in von Ohle and colleagues35). B. Dissolved oxygen profile for an S. mutans biofilm. C. Dissolved oxygen profile for a natural human ex vivo dental plaque biofilm.35 mm: Millimeters.
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In four-day-old ex vivo biofilms grown on human enamel, von Ohle and colleagues35 reported that anaerobic regions developed at a depth of approximately 150 µm in 300-µm biofilms after they added 10 percent sucrose (Figure 1C
). Deng and colleagues39 used micro-electrodes to measure the kinetics of acid production after sucrose addition and found that the pH dropped from 7 to below 5 in approximately 10 minutes in an in vitro biofilm plaque model. The development of anaerobic and acidic regions within only a few tens to hundreds of micrometers from the top of the biofilm (defined as the surface farthest away from the enamel) creates a favorable environment for anaerobic acidophiles, thereby accelerating the process of cariogenic biofilm development. F– may attenuate this effect.
F– is highly electronegative and can bind to positive charges in a number of enzymes, rendering them inactive.30 Of specific relevance with regard to an immediate influence on caries is a study conducted by Bradshaw and colleagues40; they found that 10 mg/L sodium fluoride (NaF) decreased acidification (as measured in the overlying biofilm plaque fluid) so that when glucose was added, the pH went down from 6.8 to 5.5, rather than to 4.5 without NaF. Pratten and colleagues41 described a similar effect for biofilms grown on milk with F– (5 mg/L) or without F–, but only after 168 hours of growth. The longer period for the drop in pH to be manifested may be related to the lower concentration of F– and the high buffering capacity of milk. Another possibility is that F– binds with Ca2+ to form calcium. fluoride, which has very low solubility. In a combined in vitro biofilm study and an in vivo clinical study, a 1.5-minute mouthrinse with 58 mil-limolar (2,436 mg/L) NaF had no effect on the viability of plaque bacteria or lactic acid production,42 but NaF at 2 mM (84 mg/L) did reduce lactic acid formation in planktonic and biofilm S. mutans cultures by approximately 25 and 13 percent, respectively.
Similarly, Gerardu and colleagues43 did not observe any reduction in either plaque formation or lactic acid formation as a function of long-term use of an amine fluoride mouthrinse two days after cessation of the rinsing regimen. However, Damen and colleagues44 reported a reduction in acidification for up to eight hours after subjects used a mouthrinse containing amine fluoride and stannous fluoride. Some of these conflicting data have led researchers to question whether the effect of F– on microbial physiology is of clinical relevance.21,45 However, like the effect of acidification on caries, it might be the cycling frequency, duration of F– exposure and F– concentration that are more important.
Indeed, in preliminary microelectrode studies, we showed that 1,000 mg/L NaF can significantly reduce DO consumption (Figure 2A) and acidification (Figure 2B) by an S. mutans biofilm with 10 percent sucrose added (A.G., D.de B., C.von O., P.S., unpublished data, 2007). The F– effect was transient and rapid so that the acidification was suppressed within 1.5 minutes of the NaF addition, and the pH resumed to a steady-state value five minutes after we replaced the medium with NaF-free 10 percent sucrose medium (data not shown). The minimum inhibitory concentration (MIC) of NaF on various strains of streptococci ranges from 128 mg/L to 512 mg/L.46 Maltz and Emilson47 reported a minimum bactericidal concentration (MBC) of greater than 800 mg/L NaF as needed to kill all of the 20 strains of various streptococci and lactobacilli tested.
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Figure 2. Influence of sucrose and fluoride on a Streptococcus mutans biofilm grown in vitro on a hydroxyapatite-coated microscope slide, measured via dissolved oxygen (DO) consumption (A) and acid production (B). Oxygen and pH were measured with microelectrodes in a flow cell with recirculating 10 percent saliva in a weakly buffered phosphate solution (as described by von Ohle and colleagues35). The depiction and position of the biofilm structure were based on confocal images. With 10 percent saliva only (white circles), the base of the biofilm was 50 percent DO saturated. A small pH decline within the biofilm indicated that some acid fermentation occurred. When 2 percent sucrose (black triangles) was added, the DO consumption rate increased, causing the biofilm to become anaerobic at a depth of 200 micrometers (see Figure 1B). The sucrose also caused the pH to drop rapidly (within 10 minutes) from 7.4 to 5.8 at the hydroxyapatite surface, consistent with the expected production of lactic acid via fermentation. When 1,000 parts per million sodium fluoride was added with 2 percent sucrose (white triangles), the DO consumption rate decreased, so the biofilm again was fully penetrated by DO. The pH increased to 6.8, suggesting that acid fermentation was inhibited.
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The bactericidal effect of NaF is highly dependent on pH, as shown by a strain of S. mutans, which had an MBC of approximately 1,000 mg/L at pH 7 but only 30 mg/L at pH 5.48 Because Fraises pH, there might be an antagonistic influence between modulating physiology and killing in the presence of fermentable sugars (that is, as the F– causes the pH to rise, the bacteria become less susceptible to F–). However, these data were for exposure times of 24 hours; for exposure times of five minutes, these authors48 reported no bactericidal activity of F– against S. mutans aggregates, even at the saturated concentration of 34,000 mg/L at pH 3.5.49 Taken together, our data and those of others suggest that the influence of NaF on microbiology probably is not due directly to killing during the short exposure times at MIC and MBC F– concentrations to dentifrices or mouthrinses, but probably is due more to the effects on physiology, which, in turn, may influence the microenvironment of the dental plaque. This results in a more aerated, less acidic environment, at least periodically.
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DELIVERY OF FLUORIDE TO PLAQUE BIOFILM AND ENAMEL SURFACE
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Regardless of the mode of action of F–, it is becoming generally accepted that topical application rather than systemic delivery is most effective in reducing caries.50,51 Therefore, it is important to consider how F– and other anticaries agents are delivered to the plaque biofilm and tooth surface. The plaque biofilm acts as a reservoir for F–, Ca2+, PO43– and other ions,23 possibly allowing for a greater period of exchange between these ions and the tooth enamel, thus enhancing the length of the remineralization period. The effect of the retention time of F– in plaque on demineralization/remineralization has been estimated to be less than one hour (on the basis of modeled and measured literature values of release kinetics of F– [that is, the time-dependent manner in which F– is released] from biofilm plaque and saliva).52
However, Duckworth and colleagues53 reported a significant increase in measured plaque F– from approximately 1 nanogram of F–/mg wet weight in subjects using a mouthrinse containing 0 parts per million NaF to 3.5 ngF–/mg wet weight in subjects using a mouthrinse containing 1,000 ppm NaF after at least 18 hours had elapsed since they used the mouthrinse. The authors collected these data after 10 days of daily mouthrinsing, when monitoring of salivary F–levels showed that steady-state F– concentrations had been achieved. Thus, plaque that is not removed readily during brushing actually may offset some of the negative effects with regard to caries, because F– may remain in the plaque.
Bacterial cell walls and EPS constituents usually are negatively charged owing to the presence of teichoic acids (gram-positive) and lipopolysac-charides (gram-negative) in the cell walls and acidic sugars and extracellular DNA in the biofilm matrix54; therefore, they would be expected to repel F– ions. However, owing to this electronegativity, biofilms tend to bind multivalent cations such as calcium through cross-linking, thus increasing cohesiveness55,56; in addition, the possibility exists of an electrostatic interaction between bound calcium in the EPS and the highly electronegative F–.
Rose and Turner57 showed that F– doubled the Ca2+ binding capacity and increased the diffusion of calcium through an S. mutans biofilm. These experiments were performed at a pH of 5 and 7. The pKa (pKa = – log10 [Ka], where Ka = the dissociation constant) of carboxyl, which is an important component of various EPS molecules such as proteins and uronic acids, has been estimated to be 4.8 in an environmental biofilm.58 At the lower pH caused by acid fermentation, which may develop under sucrose metabolism, we would expect acidic components in the biofilm (such as teichoic acids in the cell wall of gram-positive bacteria and acidic sugars in the EPS) to be protonated, thus neutralizing the negative charges of the acid group that occur when protons are dissociated. In a similar study, Whitford and colleagues59 found a relationship between plaque Ca2+ and retained F–, and they concluded that Ca2+ facilitated the retention. Clearly, the chemistry between biofilm components and dissolved ions such as Ca2+ and F– is not trivial, and more studies are required to elucidate these interactions over a full range of physiological conditions.
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HYDRODYNAMICS AND FLUORIDE DELIVERY
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The more effective delivery of anticaries agents such as F– to biofilm plaque may be particularly important for plaque growing in inaccessible places such as fissures and interproximal spaces where toothbrush bristles and dental floss may not make contact. Although powered brushing can project fluid and entrained bubbles in a turbulent jet beyond the bristles to remove biofilm plaque not in direct contact with bristles, the efficacy of the removal diminishes with distance.35,36 However, the fluid motion caused by powered brushing might increase the delivery of anticaries agents into and through the plaque biofilm to the tooth surface. Biofilms often are heterogeneous and consist of cell clusters made up of bacteria embedded in an EPS matrix, with channels running through the biofilm (Figure 3).3,4 Several authors35,60,61 have reported the presence of similar structures with fluid-filled voids and channels in dental plaque biofilms grown in vivo.
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Figure 3. In vitro Streptococcus mutans biofilm grown on a hydroxyapatite-coated slide in a drip flow reactor (as described by Heersink and colleagues37). The bacterial cells were stained with the nucleic acid stain Syto 9 (Invitrogen, Carlsbad, Calif.) and individual cocci appear green. The enveloping extracellular polymeric slime (EPS) matrix was stained with calcofluor white (0.05 percent, Difco, Becton Dickinson, Franklin Lakes, N.J.) and imaged with fluorescent and reflected laser light (blue). The EPS in the foreground was made transparent by using software (Imaris 3D rendering software, Bitplane, St. Paul, Minn.), allowing the bacteria inside to be visible. In some places, channels (white arrow) protruded all the way through the biofilm to the hydroxyapatite underneath.
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Investigators have used particle tracers in laboratory studies to demonstrate that water can flow through the biofilm channels, and microelectrodes showed that this fluid could transport nutrients such as DO.3,62 The velocity of the water flow through the channels depended on the velocity of the water flow above the biofilm.62 In quiescent conditions, little or no flow occurred in the channels, and the channels did not increase nutrient delivery into the biofilm. Although these studies3,62 measured DO, the same principles apply to F–.
Salivary flow rate varies according to location in the mouth and between individuals, and it has biological rhythms. Dawes and colleagues63 measured unstimulated salivary flow velocities, which ranged from 0.8 millimeter/minute to 7.6 mm/ minute, at various lingual and buccal sites. The authors estimated a salivary film thickness of approximately 0.1 mm. The corresponding Reynolds numbers will be less than 0.01, which predicts laminar creeping flow.
Results of laboratory studies showed that the biofilm channels only increased the transport of DO to the biofilm when the Reynolds number was greater than 74.64 Therefore, most likely the principal mechanism of transport through the dental plaque biofilm is via diffusion. Because the diffusion coefficient of F– in biofilm clusters is approximately 43 percent of the diffusion coefficient in water,65,66 the biofilm does not represent a significant impediment to diffusion alone, but it does represent an obstacle to transport by increasing the diffusion distance. Across small distances, the transport of molecules by diffusion is relatively fast, but the time to reach a certain concentration at the base of the biofilm (or any stagnant fluid with no convective fluid flow) increases with the square of the thickness of the stagnant layer.67 Because biofilm cell clusters do not allow fluid flow through the clusters themselves, they essentially create a stagnant layer through which molecules such as F– have to diffuse to get into, and through, the biofilm to reach the tooth surface.68
Watson and colleagues66 reported that a 30- or 120-second exposure to 1,000 mg/L NaF (under stagnant conditions) resulted in only limited delivery of the NaF in an ex vivo plaque (measured as F–) of approximately 60 and 80 mg/L, respectively. They also found that there was no significant loss of F–from the biofilm after a 30-second rinse, suggesting that the F–was bound in the biofilm. However, these experiments were performed under quiescent conditions, designed to mimic diffusion into stagnant sites inaccessible to brushing; rinsing experiments appeared to increase F– transport into the biofilm (C. Robinson, FMedSci, written communication, Nov. 24, 2007).
In vitro studies have shown that powered brushing with both sonic and rotary action significantly increased the transport of F– through S. mutans biofilm–colonized membranes compared with diffusion alone, illustrating the potential importance of convective transport for the delivery of F–.61 This effect may explain the results of a clinical study by Sjögren and colleagues,69 who found that sonic brushing for four days increased the retained F– concentration in the plaque by more than 40 percent compared with the results of other treatments (that is, manual brushing, manual brushing and flossing, and powered rotary brushing), suggesting that sonic brushing increased the delivery of F– into the interproximal plaque.
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CONCLUSIONS
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Although at first glance the physicochemistry and biology of the local environment at the tooth surface appears straightforward, the interactions are extremely complex. There are essentially three compartments to consider: saliva and overlying oral fluids, the plaque biofilm and the tooth enamel (Figure 4). There is an exchange (and reaction) of Ca2+, PO43– and F– between (and within) each of these compartments, and we must consider them across a wide range of transient physiological conditions, ranging from pH 4 to 7 and fully aerobic to fully anoxic over small timescales (seconds to minutes) and distances (micrometers to millimeters).
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Figure 4. Influence of fluid flow on exchange of ions (fluoride [F–] and calcium [Ca2+]) between the three relevant compartments: oral fluid and possible dentifrice (light blue), plaque biofilm (green) and tooth surface (white). Convective flow resulting from powered brushing in the overlying fluid and biofilm channels is indicated by blue curved arrows. Diffusion through the biofilm cell clusters and into and out of the tooth enamel is indicated by the black arrows.
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Microelectrodes are one of a number of promising tools that allow the real-time interrogation of these interactions, at least in in vitro models and in ex vivo specimens. Although the hydrodynamics will not change the thermodynamics of the biological and chemical reactions, the hydrodynamics may be used to influence the rates of these reactions by increasing delivery of anticaries agents such as F–. Thus, fluid flow caused by powered brushing (or other means) may be optimized for two effects. First, the main goal is the removal of biofilm plaque in locations outside of bristle contact. Second, for those remote areas in which plaque cannot be removed completely, increased delivery of anticaries agents should be such that they can be delivered on a timescale that is relevant to routine self-administered oral hygiene regimens, with clinically proven benefits.
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FOOTNOTES
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Dr. Stoodley is an associate professor, Immunology and Microbiology, Drexel University College of Medicine, Pittsburgh Campus, and Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, 320 E. North Ave., Pittsburgh, Pa. 15212, e-mail "pstoodley{at}gmail.com". Address reprint requests to Dr. Stoodley.
Dr. Wefel is the director, Dows Institute for Dental Research, and a professor, Department of Pediatric Dentistry, The University of Iowa College of Dentistry, Iowa City.
Dr. Gieseke is a research scientist, Max Planck Institute for Marine Microbiology, Bremen, Germany.
Dr. deBeer is a group leader, Microsensor Group, Max Planck Institute for Marine Microbiology, Bremen, Germany.
Dr. von Ohle is a chief senior clinician, Dental Clinic, Department of Conservative Dentistry, University of Tübingen, Germany.
Disclosure. Dr. Stoodley has been a consultant for Philips Oral Healthcare, Snoqualmie, Wash., and served on its scientific advisory board. Dr. Wefel has served as a consultant for Philips Oral Healthcare. The other authors did not report any disclosures.
Funding for the study described in this article was provided by Philips Oral Healthcare.
The authors thank Nebojsa Milanovich, Marcelo Aspiras and Marko deJager, Philips Oral Healthcare, for their technical input. They also thank Gaby Eickert, Ines Schroeder and Karin Hohmann from the Max Planck Institute for Marine Microbiology, Bremen, Germany, for microelectrode fabrication.
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ABSTRACT
FLUORIDE AS ANTICARIES AGENT
