Mass Transport of Macromolecules within an In Vitro Model of Supragingival Plaque

The aim of this study was to examine the diffusion of macromoleculesthrough an in vitro biofilm model of supragingival plaque. Polyspeciesbiofilms containing Actinomyces naeslundii, Fusobacterium nucleatum,Streptococcus oralis, Streptococcus sobrinus, Veillonella dispar,and Candida albicans were formed on sintered hydroxyapatitedisks and then incubated at room temperature for defined periodswith fluorescent markers with molecular weights ranging from3,000 to 900,000. Subsequent examination by confocal laser scanningmicroscopy revealed that the mean square penetration depthsfor all tested macromolecules except immunoglobulin M increasedlinearly with time, diffusion coefficients being linearly proportionalto the cube roots of the molecular weights of the probes (range,10,000 to 240,000). Compared to diffusion in bulk water, diffusionin the biofilms was markedly slower. The rate of diffusion foreach probe appeared to be constant and not a function of biofilmdepth. Analysis of diffusion phenomena through the biofilmssuggested tortuosity as the most probable explanation for retardeddiffusion. Selective binding of probes to receptors presentin the biofilms could not explain the observed extent of retardationof diffusion. These results are relevant to oral health, asselective attenuated diffusion of fermentable carbohydratesand acids produced within dental plaque is thought to be essentialfor the development of carious lesions.

INTRODUCTION

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Introduction

The structure of microbial biofilms, consisting of single cells,cell aggregates, and microcolonies embedded in an exocellularpolymeric hydrogel, has been the subject of intense experimentaland theoretical scrutiny over the past decade (10, 20, 38, 46).Biofilm anatomy and the physiological status of the cells containedwithin biofilms have profound consequences for clinical, industrial,and environmental microbiology. The exocellular space constitutesa primitive microcirculatory system that enables two-way transportbetween biofilm constituents and their surroundings and thatfacilitates intrabiofilm communication.

Knowledge of the kinetics of mass transport within biofilmsis essential for understanding how they achieve their characteristicarchitectures and for optimizing strategies to control or eradicatebiofilms. Transport phenomena in biofilms have been studiedby using microelectrodes (3, 41, 42) fiberoptic microsensors(2, 43), nuclear magnetic resonance spectroscopy (1, 52), infraredspectroscopy combined with Raman microscopy (47), fluorescencerecovery after photobleaching (4, 5), fluorescence correlationspectroscopy (FCS) (18), and confocal laser scanning microscopy(CLSM) (11, 25, 26). Indirect methods predicated upon nuclearmagnetic resonance spectroscopy, infrared spectroscopy-Ramanmicroscopy, fluorescence recovery after photobleaching, FCS,or CLSM are preferred for measuring mass transport in biofilms,since invasive procedures (e.g., those involving microelectrodesor microsensors) are likely to compromise structural integrityand therefore perturb the delicate systems that investigatorsseek to monitor.

Dental plaques are naturally occurring biofilms involved inthe development of caries and periodontal diseases. The diffusionof carbohydrates into supragingival plaque and of metaboliteswithin biofilms into the exterior milieu is crucial for cariouslesion formation (8, 12, 30, 32, 48). In this study, we soughtto quantify mass transport within a recently described polyspeciesbiofilm model of supragingival plaque (16).

MATERIALS AND METHODS

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Materials and Methods

Preparation of biofilms.
Actinomyces naeslundii OMZ 745, Veillonella dispar ATCC 17748^T(OMZ 493), Fusobacterium nucleatum KP-F2 (OMZ 596), Streptococcussobrinus OMZ 176, Streptococcus oralis SK248 (OMZ 607), andCandida albicans OMZ 110 were used as inocula for biofilm formation(16, 45). Biofilms were grown on sintered hydroxyapatite (HA)disks (diameter, 10.6 mm) in 24-well polystyrene cell cultureplates as described previously (16), except that the ratio ofsaliva to modified fluid universal medium (mFUM) was 70:30 (vol/vol)instead of 50:50 (vol/vol) (45); mFUM is fluid universal medium(15) modified by the addition of 67 mmol of Sørensenbuffer/liter [final pH, 7.2]). Pellicle-coated disks (16, 45)were covered with 1.6 ml of 70% saliva-30% mFUM. The carbohydrateconcentration in mFUM was 0.3% (wt/vol), and the carbohydratewas either glucose (biofilm cultivation from 0 to 16.5 h) ora 1:1 (wt/wt) mixture of glucose and sucrose (biofilm cultivationfrom 16.5 h to 64.5 h). Wells were inoculated with mixed cellsuspensions (200 µl) prepared from equal volumes of thevarious species (optical density at 550 nm of each species,1.0 ± 0.05 [range of acceptible values]) and incubatedanaerobically at 37°C. The medium was replenished afterdipping (see below) at 16.5 and 40.5 h by aspirating spent mediumand adding fresh medium (1.6 ml of 70:30 [vol/vol] saliva-mFUM).At 16.5, 20.5, 24.5, 40.5, 44.5, 48.5, and 64.5 h, biofilmswere washed by three consecutive dips in 2 ml of sterile physiologicalsaline (1 min per dip, room temperature [20 to 23°C]). Afterthe 64.5-h dip, biofilms were stored in physiological salineuntil further use.

Staining of bacteria and EPS in biofilms.
Cells in biofilms were stained by incubation at room temperaturefor 30 min with a 0.3% solution of Syto13 (Molecular ProbesB. V., Leiden, The Netherlands) in physiological saline. Exocellularpolymeric substances (EPS) were stained by incubating biofilmsfor 3 h with Calcofluor (Sigma Chemical Co., Buchs, Switzerland;10 µg ml^-1 in 10 mM sodium phosphate [pH 7.5]).

Incubation of biofilms with fluorescent mass transport probes.
Rinsed HA disks covered with 64.5-h biofilms were immersed insolutions of the following fluorescent probes at room temperature:neutral Texas red (TR)-conjugated dextrans (TR-dextrans; MolecularProbes; 0.2 mg/ml) with mean molecular weights of 3,000 (3K-Dex;product no. D-3329), 10,000 (10K-Dex; product no. D-1828), 40,000(40K-Dex; product no. D-1829), and 70,000 (70K-Dex; productno. D-1819); TR-conjugated whole immunoglobulin G (IgG) witha molecular weight of $~$ 150,000 (150K-Ig; Milan Analytica S.A.,La Roche, Switzerland; product no. 115-075-003; 0.03 mg/ml);TR-conjugated F(ab')₂ fragments of IgG with a molecular weightof $~$ 100,000 (100K-Ig; Milan Analytica; product no. 115-076-003;0.03 mg/ml); and R-phycoerythrin with a molecular weight $~$ 240,000(240K-PE; Molecular Probes; product no. P-801; 4 mg/ml). Immediatelyafter incubation with the fluorescent probes for 2, 60, 120,300, or 600 s, disks were washed by two 60-s dips in physiologicalsaline at room temperature. All experiments were repeated threetimes with biofilms produced on separate occasions.

Additional experiments were performed with IgM monoclonal antibody396AN1 (molecular weight, $~$ 900,000; 900K-Ig) (50) conjugatedto Alexa Fluor594 (Molecular Probes; product no. A20185), Nilered-coated polystyrene microspheres (diameter, 0.02 µm;Molecular Probes; product no. F-8784; 0.2 mg/ml), and anionicfluorescein-conjugated dextrans with molecular weights of 4,000and 70,000 (Fluka Chemie AG, Buchs, Switzerland; product no.46944 and 46945, respectively; 0.2 mg/ml). 900K-Ig and Nilered-coated microspheres were incubated with biofilms for 90min.

To test for the binding of dextrans to biofilms, biofilms werepreincubated with unlabeled dextran (molecular weight, 1,000;Fluka Chemie; product no. 31416) for 10 min and washed by two60-s dips in physiological saline. Biofilms then were reincubatedwith TR-dextrans and processed as described above.

After dip washing, excess saline was aspirated gently from thedisks without touching the biofilms, and the disks were embeddedupside down in 20 µl of Mowiol (17). Embedded disks werestored at room temperature in the dark for at least 6 h priorto microscopic examination. Dip washing failed to displace dyesthat had already penetrated the biofilm, and embedding in Mowiolblocked further diffusion of probes through the biofilm.

Microscopy and image analysis.
Stained biofilms were examined using a DM IRB E inverted microscope(Leica Mikroskopie GmbH, Wetzlar, Germany) fitted with a UVlaser (Coherent Inc., Santa Clara, Calif.), an He-Ne laser (UniphaseVertriebs GmbH, Eching/Munich, Germany), an Ar laser (CoherentInc.), and a TCS SP2 computer-operated confocal laser scanningsystem (Leica Lasertechnik GmbH, Heidelberg, Germany). Filterswere set to 400 to 490 nm for detection of Calcofluor, 520 to540 nm for fluorescein and Syto13, and 595 to 660 nm for TR,Alexa Fluor594, Nile red, and R-phycoerythrin. Confocal imageswere obtained by using x40 (numeric aperture, 1.25; zoom factor,2.5) and x100 (numeric aperture, 1.4) oil immersion objectives.Each biofilm was scanned at five randomly selected positionsnot close to the disk edge. z series were generated by verticaloptical sectioning at each of these positions; the thicknessof each section was 1.018 µm. Image acquisition was donein the x6 line average mode, and the data were processed ona Silicon Graphics (Mountain View, Calif.) 320 visual workstationfitted with Windows NT version 4.0. Scans were recombined byusing Imaris 3.1 software (Bitplane AG, Zürich, Switzerland).The depths of probe penetration into biofilms were calculatedby multiplying the number of stained z sections by 1.018 µm.

Viscometry.
The viscosity of the 70:30 saliva-mFUM biofilm cultivation mediumwas measured at room temperature by using an Ubbelohde capillaryviscometer (Schott capillary no. 0a; Digitana AG, Horgen, Switzerland)according to the instructions of the manufacturer.

Diffusion coefficients.
The calculation of diffusion coefficients is based on the followingtheoretical background. Diffusion results from the stochasticmovement of molecules due to thermal vibrations. The mean squaredisplacement (²) for rigid spheres over abrief time interval ( ${Delta}$ t) due to Brownian motion was shown independentlyby Einstein (14) and von Smoluchowski (51) to be

(1)
where k is Boltzmann's constant, r is the hydrodynamicradius of the molecule, T is the absolute temperature, and ${eta}$ is the shear viscosity of the medium.

According to Fick's first law of diffusion, the flux (J) ofmolecules passing through a defined surface during a given timeis proportional to the concentration gradient ( ${partial}$ C/ ${partial}$ z) normal tothat surface, as follows:

(2)
whereD is the diffusion coefficient for the molecule of interest,C is the concentration of the diffusing substance, and z isthe distance from the diffusion surface. Einstein (14) showedthat ² is related to D by the equation

(3)

Combining equations 1 and 3 yields the Stokes-Einstein equation

(4)

It is assumed, as a first approximation, that the biofilm coveringa disk is cylindrical. The surface available for entry of thedye consists of the vertical border and the top of the cylinder.Since HA disks have a diameter of 10.6 mm and 64.5-h biofilmshave a maximum height of 50 µm, it can be calculated that>98% of the dye must enter the biofilm through the top surface.Therefore, net mass transport of dye into the biofilm can beregarded as a unidimensional phenomenon parallel to the surfaceof the biofilm. Experimental biofilm diffusion coefficients(D_bf) for each probe were determined by using weighted least-squaresregression (49) of ² versus ${Delta}$ t.

RESULTS

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Results

Figure 1 provides an impression of the composition of the artificialsupragingival plaque biofilm at 64.5 h and the distributionsof cells, EPS, and interstitial voids. The biofilm thicknessat 64.5 h was 30 ± 10 µm (mean and standard deviation).

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FIG. 1. CLSM images showing perpendicular xy, xz, and yz planes through a randomly selected point of a 64.5-h biofilm. The biofilm was stained with Syto13 for microorganisms (green), with Calcofluor for EPS (blue), and with 10K-Dex for interstitial spaces (red). Images were obtained by using a x40 oil immersion objective.

Images of the penetration of 10K-Dex into artificial supragingivalplaque as a function of time are shown in Fig. 2. After 2 s,the probe was confined largely to the superficial layer of thebiofilm, with progressive migration of the probe to greaterdepths with longer incubation periods. After 120 s, the probeappeared to have extended approximately half way through thebiofilm, and by 600 s, 10K-Dex was distributed throughout muchof the volume of the biofilm in an anisotropic manner consistentwith the structural heterogeneity revealed in Fig. 1. Probepenetration into substratal regions occurred to a lesser extentthan did that into superficial regions, and throughout the biofilm,"lakes" and "holes" corresponding to areas of high and low levelsof probe penetration, respectively, were readily apparent.

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FIG. 2. Images of xz sections of 64.5-h biofilms showing the diffusion of 10K-Dex as a function of time (up to 600 s). Images were obtained by using a x100 oil immersion objective.

The diffusion into biofilms of probes ranging in molecular weightfrom 3,000 (3K-Dex) to 240,000 (240K-PE) is depicted in Fig.3 (incubation time for 3K-Dex, 120 s; incubation time for theother probes, 600 s). Whereas 3K-Dex was distributed throughoutthe whole biofilm by 120 s and 10K-Dex was so distributed by600 s, both 40K-Dex and 70K-Dex were largely confined to theupper half of the biofilm by 600 s. Only slight penetrationwas observed for the 100K-Ig, 150K-Ig, and 240K-PE probes by600 s, although small lakes of 240K-PE were seen by 600 s insubstratal regions. However, even after 90 min, neither AlexaFluor594-conjugated 900K-Ig nor Nile red-coated microspheres(0.02 µm) succeeded in penetrating beyond the upper surfaceof the biofilm (data not shown). To evaluate electrostatic effectson diffusion, biofilms were exposed to fluorescein-conjugateddextrans bearing a net negative charge. The diffusion coefficients(see below) of these anionic probes with molecular weights of4,000 and 70,000 were 4.97 and 0.36 µm² s^-1, respectively,and were nearly identical to those seen for their neutral TR-dextrancounterparts (see Table 2).

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FIG. 3. xz sections of 64.5-h biofilms showing the diffusion of macromolecules with different molecular weights after 120 s (3K-Dex) or 600 s (probes with molecular weights of 10,000 to 240,000 [10K to 240K]).

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TABLE 2. Diffusion coefficients at 25°C of fluorescent probes in water and biofilms and calculated tortuosity indices

The mean depths of penetration (²) of TR-dextrans,100K-Ig, 150K-Ig, and 240K-PE into biofilms during the timeintervals examined are presented in Table 1. Fifteen independentmeasurements of ² for each incubation period( ${Delta}$ t) for each fluorescent probe were plotted according to equation3 (data not shown). These scattergrams yielded indices of determination(r²) for ² versus ${Delta}$ t of 0.90, 0.84, 0.90,0.93, 0.88, 0.68, and 0.83 for the 3K-Dex, 10K-Dex, 40K-Dex,70K-Dex, 100K-Ig, 150K-Ig, and 240K-PE probes, respectively,indicating that diffusion rates for each probe were more orless constant and did not vary substantially with biofilm depth.The data in Table 1 were used to calculate D_bf values (Table2). On theoretical grounds (19), it may be shown that diffusivityis linearly proportional to the cube root of the molecular weight.Therefore, when D_bf was plotted against the cube root of themolecular weight, a linear relationship (r² = 0.94) was obtainedover the molecular weight range of 10,000 through 240,000, buta dramatic increase in diffusivity occurred as the probe molecularweight decreased from 10,000 to 3,000 (Fig. 4).

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TABLE 1. Mean distances of penetration of fluorescent probes into artificial supragingival plaque as a function of time

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FIG. 4. Plot of D_bf versus the cube root of the molecular weight of fluorescent probes over the molecular weight range of 3,000 to 240,000. A linear relationship (r² = 0.94) between D_bf and the cube root of the molecular weight was seen for the molecular weight range of 10,000 to 240,000.

In additional control experiments, biofilms preincubated withunlabeled dextran (molecular weight, 1,000) and then reincubatedwith TR-dextrans showed diffusion coefficients for the fluorescentprobes nearly identical to those obtained for biofilms not treatedwith unlabeled dextran. Differences in measured diffusion coefficientsfor 3K-Dex, 10K-Dex, 40K-Dex, and 70K-Dex with and without unlabeleddextran pretreatment were -0.42, -0.01, -0.02, and -0.04 µm²s^-1, respectively. Furthermore, extensive washing (60 min) ofbiofilms following incubation with TR-dextrans resulted in nearlycomplete removal of the dye.

The D_bf values were strikingly lower than the diffusion coefficientsseen in bulk water or agar gels (D_w) (Table 2). Such retardeddiffusion could have been due to several factors (discussedbelow). To assess the impact of the convoluted paths traversedby the macromolecules during biofilm penetration, tortuosityindices were calculated. The tortuosity index, defined as thesquare root of D_w/D_bf, is a measure of probe percolation throughinterstitial space (36). Plotting tortuosity indices for allprobes (Table 2) versus probe molecular weights yielded a linefor which r² was 0.76 (Fig. 5).

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FIG. 5. Plot of tortuosity index [ ${lambda}$ _Mr (D)] versus probe molecular weight (3,000 to 240,000). The approximately linear correspondence between the tortuosity index and molecular weight is evidence for the role of tortuosity in the retardation of probe diffusion through biofilms.

DISCUSSION

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Discussion

In the present study, we sought to quantify diffusion into andwithin a polyspecies biofilm model of supragingival plaque byinvestigating penetration of fluorescent probes under stagnantconditions, where convective effects on intrabiofilm transportare negligible (21). Although composed of only six microorganisms,the model was characterized by considerable morphological complexity,illustrated through the use of dyes to highlight microorganisms(Syto13), EPS (Calcofluor), and interstitial spaces (10K-Dex)(Fig. 1). Structural heterogeneity within the biofilms was expectedto result in regional differences in macromolecule diffusion;therefore, diffusion measurements were always performed by usingtriplicate biofilms and examining five randomly selected spotsper biofilm (for a total of 15 independent analyses per probeper time point). Our measurements showed that for molecularweights of up to 240,000, probe penetration depths increasedlinearly (or nearly so) with time (Table 1), enabling us tocalculate D_bf values for each of the probes. The 240K-PE probepenetrated poorly into biofilms, whereas 900K-Ig collected onthe surface and hardly penetrated into biofilms at all. Fromreported hydrodynamic radii for R-phycoerythrin (5.5 nm) (29)and IgM (14.0 nm) (40), the limiting width of these biofilmlumens can be estimated to be slightly greater than 11 nm. Thefinding that IgM was unable to penetrate biofilms contrastswith previously reported data (17). However, in this earlierstudy, a growth medium with a 50:50 (vol/vol) ratio of salivato mFUM was used, whereas in the present study, the ratio wasshifted to 70:30 (vol/vol). This change in the saliva/mFUM ratioled to a more rigid biofilm with apparently different diffusionproperties. Thus, altered growth conditions can profoundly affectbiofilm diffusivity.

The linear relationships seen in plots of ²versus ${Delta}$ t attest to the high measurement quality of each dataset and indicate that there was no change in the resistanceto diffusion to the extent that the probes migrated throughthe biofilms. Curiously, a difference of 1 order of magnitudewas observed between 3K-Dex and 10K-Dex for D_bf (Fig. 4), whereason theoretical grounds, a D_bf value of only about 1.7 was expected(Table 2). This discrepancy suggested a possible sieving effectin the biofilm brought about by a preponderance of EPS "pore"diameters wider than 2.6 nm (estimated diameter of 3K-Dex) (37)but narrower than 4.6 nm (estimated diameter of 10K-Dex) (37).

In the absence of mitigating circumstances, the diffusion coefficientof molecules in a bulk solution should decrease with the cuberoot of the molecular weight (19). Except for 3K-Dex, this relationshipwas observed for our biofilms, although the D_bf values indicatedthat diffusion rates in biofilms were much lower than thosein bulk water; this attenuation effect was more pronounced athigher probe molecular weights (Table 2). Retarded diffusionhas been reported for a variety of biological systems (Table3) as well as for artificial test systems, such as hydroxypropylcellulose(7) or gel matrices consisting of cross-linked polymers (28).Lawrence et al. (26), assessing dextran diffusion in mixed-culturesoil biofilms, reported D_bf values for 40K-Dex and 70K-Dex probescomparable to those obtained in our model of supragingival plaque.Likewise, Guiot and coworkers (18), using FCS under two-photonexcitation, reported that apparent diffusion coefficients formacromolecules in monospecies biofilms were up to 50 times smallerthan those in bulk water.

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TABLE 3. Diffusion coefficients determined with various biological systems

To what extent discrepancies between D_bf values determined fordifferent biofilm models (Table 3) can be explained by methodologicalor analytical differences remains to be explored. Retarded diffusioncould be related to increased viscosity within the biofilm mass,to selective binding of probe molecules to bacteria and/or theEPS matrix, to a highly tortuous biofilm microstructure, toentropic trapping (28), or to any combination of these factors.The viscosity of the biofilm growth medium at 25°C was determinedto be 1.062 ± 0.004 (mean and standard deviation) cP,similar to that of water at the same temperature (0.890 cP).The physicochemical properties of the interstitial fluid inour biofilm model are not known but likely closely resemblethose of the medium in which the biofilm is grown (18). Thus,viscosity, which appears in the denominator of the Stokes-Einsteinequation (equation 4), can hardly account for the observed retardationof diffusion.

Selective binding of dextran (glucans) to biofilms containingoral streptococci, in particular, S. sobrinus, is well known(33), and such binding conceivably could lead to an apparentretardation of the diffusion of dextrans. Interestingly, Birminghamet al. (4), who used fluorescence photobleaching recovery methods(Table 3) to measure diffusion, detected an apparent increasein the rate of diffusion of dextrans in S. sobrinus-containingbiofilms grown in the presence of sucrose. They explained theobserved increase in the diffusion rate in terms of selectivetemporary binding of dextrans to enzymatic and/or nonenzymaticglucan-binding proteins, leading to significant fluorescencequenching and an apparent but physically nonexistent increasein the diffusion rate. Preincubation with unlabeled dextranfailed to impede the transit of TR-dextrans through our biofilms,and the fact that the dextran, immunoglobulin, and phycoerythrinprobes used all yielded reasonably linear relationships betweenD_bf and probe molecular weight indicated that selective bindingof probes to EPS or microorganisms is not a significant factorin the retardation of diffusion. This conclusion is confirmedby the fact that extensive washing of the stained biofilms resultedin nearly complete removal of the dye. In addition, the twostreptococcal species present in our biofilm model produce largequantities of ${alpha}$ (1-6)- and ${alpha}$ (1-3)-linked glucans from sucrose (23).It is very likely that these glucans continuously bind to andsaturate whatever streptococcal glucan-binding sites may existin the biofilm.

Tortuosity seems the most likely explanation for the observeddiscrepancy between the diffusion of probes in biofilms andthat in bulk water: a molecule traversing a highly convolutedthree-dimensional route laid out by interstitial voids in amatrix will be delayed in comparison to a molecule showing freediffusion in bulk water. The extracellular space of the biofilmcomprises an EPS-rich hydrogel of variable porosity; the largerthe probe, the more often it will encounter a pore too narrowto allow it to pass. In such cases, diffusion into the biofilm(along the z axis) can proceed only if the probe backtracksuntil it encounters a pore sufficiently wide to permit penetrationdeeper into the biofilm.

The relationship between the incidence of dental caries andthe properties of supragingival plaque is complex. The formationof gelatinous biofilms on teeth is a necessary condition forthe formation of carious lesions in vivo. The same is true forlesions formed in vitro with exogenous acids (24). Such observationsled to the empirical conclusion that selective retardation ofdiffusion is a prerequisite for carious demineralization ofenamel (13). The diffusion properties of dental plaque are governedmainly by two interactive factors: microbial composition anddiet (composition and frequency of consumption). It has beenpostulated that the nature and amounts of extracellular glucansproduced by oral streptococci from sucrose in dental plaqueare major pathogenic determinants retarding acid diffusion (22).Subjects intolerant of high fructose levels cover their carbohydrateintake through starch and avoid sweet-tasting mono- and disaccharides;compared to subjects without this intolerance, their cariesincidence is low (31). Epidemiological findings point in thesame direction. Before the massive increase in sucrose consumptionthat started in Europe in about 1850, starch was the main dietarycarbohydrate and the incidence of dental caries was modest (34).The low cariogenic potential of starch was explained by itsslow diffusion into plaque, after which the starch polymersmust be cleaved by salivary amylase to maltotriose, maltose,or glucose to become accessible to fermenting bacteria and tomanifest their full acidogenic potential (27, 44). Questionsrelated to the effect of diffusion in dental plaque may nowbe addressed experimentally with biofilm models or models ofdental plaque formed in situ. If measurements of enamel demineralizationare used as additional experimental parameters, further relevantinsights into the pathogenic mechanisms of dental caries canbe expected.

ACKNOWLEDGMENTS

We thank T. Bächi and M. Höchli at the Central ElectronMicroscopy Laboratory of the University of Zürich for useof the confocal laser scanning microscope, G. Stark of the Universityof Konstanz (Konstanz, Germany) for useful discussions on diffusionand critical review of the manuscript, and A. Meier and V. Osterwalderfor excellent technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Oral Microbiology and General Immunology, University of Zürich, Plattenstr. 11, CH-8028 Zürich, Switzerland. Phone: 41 1 634 3256. Fax: 41 1 634 4310. E-mail: thurnher{at}zzmk.unizh.ch.

Present address: Basilea Pharmaceutica AG, CH-4002 Basel, Switzerland.

REFERENCES

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