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Deposition of Oral Bacteria and Polystyrene Particles to Quartz and Dental Enamel in a Parallel Plate and Stagnation Point Flow Chamber
Journal of Colloid and Interface Science 220, 410 – 418 (1999) Article ID jcis.1999.6539, available online at http://www.idealibrary.com on
Deposition of Oral Bacteria and Polystyrene Particles to Quartz and Dental Enamel in a Parallel Plate and Stagnation Point Flow Chamber Junlin Yang,* Rolf Bos,* Gerald F. Belder,† Jan Engel,‡ and Henk J. Busscher* ,1 *Department of Biomedical Engineering, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands; †Philips Research Laboratories, Professor Holstlaan 4, 5656 AA Eindhoven, The Netherlands; and ‡Centre for Quantitative Methods (CQM), Building HCZ-3, P.O. Box 414, 5600 AK Eindhoven, The Netherlands Received June 4, 1999; accepted September 10, 1999
mous and their cell surfaces are both structurally and chemically very heterogeneous, with a likely impact on adhesion. Dental enamel surfaces resemble hydroxylapatite, as far as its mineral component is concerned (8), but in dental enamel hydroxylapatite crystallites are embedded in an organic matrix (9), with an impact on the adhesive properties of its surface and causing a typical surface heterogeneity uncommon to nonbiological model surfaces employed, like germanium prisms, glass, quartz, or even hydroxylapatite (10). The adhesion of colloidal particles as well as of microorganisms to solid substrata is of great practical importance both in industrial and in biomedical applications (11–14). As a result, different flow chamber devices have been developed to study particle deposition and adhesion under controlled mass transport, such as the parallel plate (PP) flow chamber (15, 16) and the stagnation point (SP) flow chamber (1, 17). Mass transport in a parallel plate flow chamber is slow and the convective-diffusion equation for the parallel plate flow chamber is difficult to solve as compared to, e.g., the stagnation point flow chamber. On the other hand, the parallel plate flow chamber is conceptually simpler than the stagnation point flow chamber. In the PP flow chamber, mass transport is through convection parallel to the substratum surfaces, usually constituting the top and/or bottom plates of the chamber, while the actual transport toward the substratum, needed for adhesion to occur, is through diffusion, which makes the PP flow chamber a kinetically slow one. Alternatively, in the SP flow chamber, convective mass transport is directed toward the substratum surface bending hyperbolically in a parallel direction close to the surface and creating a stagnation point on the substratum in the middle of the flow. Recently, we compared deposition of polystyrene particles to quartz in a PP and SP flow chamber and concluded that SP experiments were more reproducible than PP experiments, due to a stronger influence of interaction forces in the SP flow chamber as compared to a stronger influence of chance processes, like collisions between flowing and adhering particles and diffusion in the PP flow chamber. The aim of this paper is to compare the deposition of three initially colonizing bacterial strains of tooth surfaces in vivo
The aim of this paper is to determine to what extent (i) deposition of oral bacteria and polystyrene particles, (ii) onto quartz and dental enamel with and without a salivary conditioning film, (iii) in a parallel plate (PP) and stagnation point (SP) flow chamber and at common Peclet numbers are comparable. All three bacterial strains showed different adhesion behaviors, and even Streptococcus mitis BMS, possessing a similar cell surface hydrophobicity as polystyrene particles, did not mimic polystyrene particles in its adhesion behavior, possibly as a result of the more negative z potentials of the polystyrene particles. The stationary endpoint adhesion of all strains, including polystyrene particles, was lower in the presence of a salivary conditioning film, while also desorption probabilities under flow were higher in the presence of a conditioning film than in its absence. Deposition onto quartz and enamel surfaces was different, but without a consistent trend valid for all strains and polystyrene particles. It is concluded that differences in experimental results exist, and the process of bacterial deposition to enamel surfaces cannot be modeled by using polystyrene particles and quartz collector surfaces. © 1999 Academic Press
Key Words: deposition; parallel plate flow chamber; stagnation point flow chamber; bacteria; polystyrene particles; convectivediffusion; pair distribution function.
INTRODUCTION
Physico– chemical approaches have been used by several groups in an attempt to model biological adhesion processes. For instance, it is often assumed that inert polystyrene particles mimic bacteria in their adhesion to surfaces, that glass (1, 2), germanium (3, 4), or quartz (3) surfaces behave in adhesion studies, similarly to dental enamel, and that bathing of collector surfaces in biological fluids (5), like saliva, urine, or seawater, has little influence. Although mechanistically, the same fundamental forces, like Lifshitz–Van der Waals, electrostatic, and acid– base forces, govern adhesion of polystyrene particles and of bacteria (6, 7), the diversity in the bacterial world is enor1
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with the deposition of polystyrene particles in a PP and SP flow chamber, both to enamel and quartz collector surfaces and with and without a so-called salivary conditioning film. MATERIALS AND METHODS
Bacterial Strains, Polystyrene Particles, and Cell Surface Characterization For each adhesion experiment, Streptococcus oralis J22 was precultured from blood agar plates in a batch culture of Todd Hewitt broth for 24 h at 37°C in ambient air. Streptococcus mitis BMS was precultured from a blood agar plate for 24 h at 37°C in Todd Hewitt broth with 0.5% (w/v) glucose added, also in ambient air. Actinomyces naeslundii T14V was precultured from blood agar for 24 h in Schaedler’s broth supplemented with 0.01 g L 21 of hemin at 37°C in atmosphere air of 10% H 2, 85% N 2, and 5% CO 2. These cultures were used to inoculate second cultures which were allowed to grow for 16 h. Bacterial cells were harvested by centrifugation (5 min at 6500g) and washed twice with adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride, and 1 mM calcium chloride at pH 6.8). To break up bacterial aggregates, bacteria were sonicated for 30 –50 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT). Sonication was carried out intermittently while cooling in an ice/water bath. These procedures were found not to cause cell lysis in any strain. Subsequently, the cells were counted in a Bu¨rker-Turk counting chamber. Thereafter, the cells were suspended in an adhesion buffer to a density of 1.4 3 10 8 cells ml 21. Polystyrene particles (Vs2A, AKZO Research, Arnhem, The Netherlands) with a diameter of 783 nm were prepared as described by Brouwer and Zsom (18). The particles were washed twice by centrifugation in demineralized water and suspended in the adhesion buffer to the same density as the bacterial strains. Bacterial cell surfaces were characterized by their z potentials, as measured in an adhesion buffer employing particulate microelectrophoresis (19). Also, z potentials of the polystyrene particles were measured for comparison. Water contact angles of sessile droplets were measured on bacterial lawns, deposited on cellulose acetate membrane filters (pore diameter, 0.45 mm), and dried in ambient air to a so-called “plateau state” (20). The plateau state of drying the lawns was determined in an earlier experiment after the onset of drying, as the time span during which a constant value for the water contact angle is maintained. To determine the elemental surface composition of the bacteria, X-ray photoelectron spectroscopy (XPS) was performed on freeze-dried bacterial pellets (21). Collector Materials and Surface Characterization Quartz collector surfaces were cleaned thoroughly by sonication for 2 min in a 2% solution of surfactant RBS 35 (Fluka Chemie AG, Buchs, Switzerland), followed by extensively
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rinsing with methanol and demineralized water. The labial surfaces of bovine dental incisors were ground under running tap water with abrasive paper (800 and 1200 grit) and polished with a slurry of Al 2O 3 powder (particle diameter, 0.05 mm) in distilled water. Afterward, the polished surface was cleaned with demineralized water under sonication two times for 20 s. Subsequently, the polished labial surface was cut into a regular shape and polished to a thickness of approximately 800 mm and glued to a glass plate using a drop of nail polish. Thereafter, the exposed side of the enamel slice was ground successively with 400-, 800-, and 1200-grit abrasive paper until a thickness of 25–35 mm was obtained, polished with a slurry of Al 2O 3 powder, and cleaned ultrasonically again. Finally, the enamel pieces were removed from the glass and stored at a temperature of 8°C for further use in a moist environment. For the experiments in the parallel flow chamber, an enamel piece was glued to an already cleaned and dried quartz plate with a small drop of nail polish. z potentials of the quartz and enamel surfaces with and without an adsorbed salivary conditioning film were derived from streaming potential measurements in an adhesion buffer, as described by Van Wagenen and Andrade (22). Water contact angles on both collector materials were measured by the sessile drop technique as employed for contact angle measurements on bacterial lawns, while again collector materials with an adsorbed salivary conditioning film were dried in ambient air to a so-called plateau state prior to contact angle measurements (20). The elemental surface concentrations of the surfaces were obtained by XPS (23). Saliva Collection and Preparation Human whole saliva from 10 healthy volunteers of both sexes was collected into ice-cooled beakers after stimulation by chewing Parafilm. After the saliva was pooled and centrifuged at 1200g for 20 min at 4°C, 0.2 M phenylmethylsulfonylfluoride (PMSF) as a protease inhibitor was added to a final concentration of 1 mM to reduce protein breakdown. Afterward, the solution was centrifuged again at 10,000g for 20 min at 4°C, dialyzed overnight at 4°C against distilled water, and freeze-dried for storage. For coating the collector surfaces, the freeze-dried saliva was dissolved at a concentration of 1.5 mg ml 21 in adhesion buffer. This solution was centrifuged at 10,000g for 5 min and the supernatant was used to flow in the system under consideration, i.e., the PP or SP flow chamber. Flow Chamber and Image Analysis The SP and PP flow chambers used are schematically presented in Fig. 1, together with their essential dimensions. Briefly, for the parallel plate flow chamber, deposition was observed on the center of the bottom plate with a CCD-MXR camera (High Technology, Eindhoven, The Netherlands) mounted on a phase contrast microscope (Olympus BH-2), whereas with the stagnation flow chamber deposition was
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FIG. 1. Schematic presentations of the PP and SP flow chambers used in this study. In the SP flow chamber, the stagnation point is exactly opposite of the fluid inlet channel.
observed in the area very close to the stagnation point with a dark-field microscope (Leitz) equipped with A CCD-LDH camera (Philips, Eindhoven, The Netherlands). Live images were grabbed with a PC-Vision 1 frame grabber and Gauss and Sharp filtered after subtraction of an out-of-focus image. Thereafter, deposited particles were discriminated from the background by single gray value thresholding. This yielded binary black and white images which were subsequently stored on a disk for later analysis. Experiments were carried out three times at a flow rate of 0.05 cm 3 s 21 for the PP and 0.00883 cm 3 s 21 for the SP flow chamber, corresponding with a wall shear rate of 22.5 and 17.3 s 21, respectively, and a common Peclet number of 3.95 3 10 23. Deposition Protocol and Data Analysis Before each experiment, all tubes and the flow chamber were filled with buffer, while air bubbles were carefully removed from the system. Flasks containing bacterial suspensions, buffer, and human whole saliva were all connected to the flow chamber. First, flow was switched to saliva when appropriate, for about 100 min to create a salivary conditioning film. Thereafter, flow was switched to buffer for 10 min for removal of all remnants of saliva from the system and finally switched to the bacterial suspension. The number of bacteria or particles adhering to the substratum was determined in time. All experiments were done in triplicate with separate bacterial cultures at room temperature. The total number of adhering bacteria or particles per unit area, n(t), were recorded as a function of time by image sequence analysis (24). The initial deposition rate, j 0 , of the depositing bacteria or particles was determined from the initial increase of n(t) with time, while the later stages of the deposition process were assumed to be described by n~t! 5 j 0 t ~1 2 e 2t/ t !,
[1]
1/ t 5 j 0 A l 1 b ,
[2]
where A l is the area blocked by an adhering bacterium and particle, as can be calculated from the radial pair distribution
function g(r) and b is the desorption probability, assumed to be independent of the residence time in the present study. The expression 1/t can be calculated directly from the measured initial deposition rates and the number of bacteria or particles adhering in a stationary endpoint, while subsequently combination with the initial deposition rate and blocked area yields the desorption probability b. Blocked areas A l are assumed to be similar to the depletion zones with area A g(r) that can be derived from the radial pair distribution function g(r) which describes the relative density of adhering particles around a given particle as a function of interparticle distance,
g~r! 5
r ~r, dr! , r0
[3]
where r is the radius of a shell, r (r, dr) is the density of adhering particles or bacteria in a shell with thickness dr, and r 0 is the average density of the total field of view. By determination of the radius, g(r) , 1 was taken. Statistical Analyses Univariate ANOVA was used to test the significance of the differences observed between the main factors studied (i.e., quartz or enamel collector surfaces, the absence or presence of a salivary conditioning film, polystyrene particle, or bacterial involvement) on the deposition parameters j 0 , n ` , t , A l, and b. Separate analyses were made for the PP and SP cells. Multivariate ANOVA was carried out to determine the significance of the differences observed between the main factors on the deposition process as a whole. To this end, the deposition parameters were grouped together as a vector and the analysis was conducted with the vector rather as with the separate parameters. Finally, a comparison was made between the two systems involved in this study, i.e., the real system, consisting of enamel and bacteria and the model system, consisting of the quartz collector surface in combination with polystyrene particles. Statistical significance was accepted at P , 0.05. RESULTS
Bacterial Cell and Collector Surface Characterization Table 1 lists the z potentials of the bacteria and polystyrene particles, together with their water contact angles and elemental surface concentration ratios. The bacterial strains are all less negatively charged than the polystyrene particles, while the intrinsic cell surface hydrophobicity of the organisms varies from hydrophilic (S. oralis J22) to extremely hydrophobic and, in fact, S. mitis BMS is equally hydrophobic as polystyrene. XPS analysis demonstrates that bacterial cell surface hydrophobicity is caused by nitrogen-rich groups, most notably
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TABLE 1 z Potentials of Polystyrene Particles Vs2A, Bacteria (S. oralis J22, S. mitis BMS, and A. naeslundii T14V), of Powdered Dental Enamel by Particulate Microelectrophoresis, and of Quartz Surfaces with (1) or without (2) a Salivary Conditioning Film (SCF) by Streaming Potential Measurements in Adhesion Buffer (pH 6.8), Together with Water Contact Angles and Elemental Surface Concentration Ratios by XPS Elemental surface concentration ratio Particle type
z potential (mV)
PS S. oralis J22 S. mitis BMS A. naeslundii T14V
250 6 4 22 6 3 210 6 7 23 6 4
Quartz Enamel
2 1 2 1
SCF SCF SCF SCF
225 6 1 210 6 5 218 6 2 217 6 2
Water contact angle (°)
O/C
N/C
P/C
S/C
2 4 4 4
0.042 0.392 0.312 0.313
0 0.102 0.124 0.090
0 0.020 0.009 0.005
26 6 2 18 6 2 45 6 10 29 6 6
5.357 0.350 1.738 0.358
0 0.172 0.052 0.205
0 0.012 0.369 0.029
90 6 24 6 100 6 64 6
proteins, while cell surface hydrophilicity is due to oxygenand phosphorus-rich groups. The collector surfaces are also negatively charged (see also Table 1) and the z potential of quartz is more negative (225 mV) than that of enamel (218 mV) in the absence of a salivary conditioning film. Salivary protein adsorption yields less negative z potentials. Quartz surfaces are more hydrophilic than enamel surfaces, while salivary protein adsorption converges their water contact angles to values characteristic for hydrophilic surfaces. The quartz surface had a minor carbon contamination as detected by XPS (11.2%), with 60.0% oxygen and 28.9% silicon measured. Note that also enamel without a salivary conditioning film shows a small amount of nitrogen, resulting from the surface exposure of organic matrix proteins. Adsorption of salivary components on both types of collector surfaces is accompanied by an increase in the amount of surface nitrogen and a reduction in the amount of collector surface elements exposed, i.e., Si or Ca for quartz and enamel, respectively. Deposition Experiments Figure 2 shows the deposition kinetics for S. oralis J22 to quartz and enamel surfaces with and without a salivary conditioning film in a PP flow chamber and to quartz surfaces in a SP flow chamber. The differences between the deposition kinetics with and without a salivary conditioning film are considerably smaller in the PP flow chamber than in the SP flow chamber. Note that in the SP flow chamber the use of enamel appeared impossible due to the turbidity of the enamel samples, yielding image problems. Quantitative features of the deposition kinetics, including initial deposition rates j 0 , numbers adhering in a stationary endpoint n ` , blocked area A g(r) , and desorption probabilities b of the bacteria and polystyrene particles are summarized in Tables 2 and 3 for the PP and SP flow chamber, respectively.
Ca/C
Si/C
0.006 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0.528 0.033
2.580 0.045 0 0
Both in the PP as well as in the SP flow chamber, initial deposition rates j 0 of bacteria and polystyrene particles are slightly higher in the presence of a salivary conditioning film as compared to those in its absence. Initial deposition rates are on average two-fold higher in the SP flow chamber than in the PP flow chamber, but differences among strains, including the polystyrene particles, are minor. Furthermore, Table 2 shows that initial deposition rates on enamel surfaces are generally different from those on the quartz collector surfaces. The number of bacteria and particles adhering in a stationary endpoint to a salivary conditioning film are smaller than those adhering to the collector surfaces without saliva, while here it appears that bacteria generally adhere in significantly higher numbers than the polystyrene particles. Most notably, the blocked areas for A. naeslundii T14V are much larger than those for the other bacterial strains and polystyrene particles. Due to the turbidity of the enamel samples, no blocked areas could be derived from the spatial arrangement of the bacteria and particles adhering on enamel. The desorption probabilities in the presence of a salivary conditioning film are larger than those measured in the absence of a salivary conditioning film. Statistical Analyses The results of the statistical analyses are summarized in Tables 4 – 6. For the PP flow chamber, A l and b were omitted from the analysis, since no values were available for enamel. For the SP flow chamber, all five parameters were included, but the collector material was omitted as a factor, since data for enamel were not available. Table 4 gives an overview of the effects of the main factors (collector material, salivary conditioning film, and particle type) on the different deposition parameters, both for the PP and the SP flow chamber. All factors appear to have a significant influence on the deposition parameters, except collector material on stationary endpoint numbers (PP flow chamber),
FIG. 2. Examples of the deposition kinetics for S. oralis J22 on quartz (a) and enamel (b) in a parallel plate flow chamber and on a quartz collector surface near the stagnation point in a stagnation point flow chamber with (c) (1 SCF) or without (2 SCF) a salivary conditioning film in potassium phosphate adhesion buffer (pH 6.8). 414
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TABLE 2 Initial Deposition Rates j 0, a Number of Bacteria and Polystyrene Particles Adhering in a Stationary Endpoint n `, Blocked Areas A g(r) Calculated from the Radial Pair Distribution Functions and Desorption Probabilities b on Quartz and Enamel Collector Surfaces with (1) and without (2) a Salivary Conditioning Film (SCF) in Adhesion Buffer (pH 5 6.8) Obtained in a Parallel Flow Chamber (6 Denotes Standard Deviation over Triplicate Experiments) Particle type
n (10 6 cm 22)
A g(r) (mm 2)
b (10 25 s 21)
57 44 85 91
4.4 6 1.0 1.7 6 0.7 4.1 6 0.6 1.9 6 0.5
3.0 6 0.5 2.5 6 1.0 nd a nd
7.7 6 2.9 23.2 6 12.0 nd nd
2 1 2 1
490 6 85 605 6 94 657 6 93 687 6 128
6.0 6 0.9 3.3 6 0.5 5.5 6 1.0 3.8 6 0.5
1.4 6 0.4 1.0 6 0.5 nd nd
6.7 6 2.2 17.7 6 4.2 nd nd
2 1 2 1
525 6 673 6 582 6 656 6
45 50 66 80
5.4 6 0.5 3.0 6 0.4 5.0 6 0.7 3.1 6 0.4
2.6 6 0.6 1.7 6 0.5 nd nd
8.3 6 1.3 21.9 6 4.2 nd nd
2 1 2 1
563 6 128 610 6 35 496 6 97 531 6 91
5.0 6 0.9 4.5 6 1.2 3.9 6 1.3 3.8 6 1.0
4.3 6 0.8 4.6 6 2.6 nd nd
9.3 6 4.1 11.4 6 3.2 nd nd
SCF
Quartz
2 1 2 1
394 6 411 6 549 6 606 6
PS
Enamel S. mitis BMS
Quartz Enamel
S. oralis J22
Quartz Enamel
A. naeslundii T14V
Quart Enamel
a
j 0 (cm 22 s 21)
Substratum
nd is “not determined” due to poor image quality.
particle type on 1/t (PP flow chamber), and the presence of a salivary conditioning film on blocked areas (SP flow chamber). Table 4 also indicates factor interactions: the influence that a change in one factor setting may have on the effect of another factor. Most interactions are significant, especially in the SP flow chamber. Moreover, there is a clear interaction between the presence of a salivary conditioning film and particle type, indicating that the effect of the presence of a salivary conditioning film on the deposition depends on the particle type. In Table 5, each deposition parameter is ordered according to an increasing mean value per particle type. In the PP flow cham-
ber, polystyrene particles invariably show the lowest values for all deposition parameters. The deposition parameters for the bacteria show no significant differences between themselves. In the SP flow chamber, the results are somewhat different and break up more with respect to particle type. Multivariate analyses were performed on the complete set of deposition parameters and account for the overall deposition behavior and correlations between parameters. Multivariate ANOVA was carried out on the vector ( j 0 , n ` , b ) for the PP flow chamber, and on the vectors ( j 0 , n ` , t ) and ( j 0 , n ` , t , A l, b) for the SP flow chamber. All factors and their interactions
TABLE 3 Initial Deposition Rates j 0, a Number of Bacteria and Polystyrene Particles Adhering in a Stationary Endpoint n `, Blocked Areas A g(r) Calculated from the Radial Pair Distribution Functions, and Desorption Probabilities b on a Collector Surface with (1) or without (2) a Salivary Conditioning Film (SCF) in an Adhesion Buffer (pH 6.8) near the Stagnation Point in a Stagnation Point Flow Chamber (6 Denotes Standard Deviation over Triplicate Experiments) SCF
j 0 (cm 22 s 21)
n ` (10 6 cm 22)
A g(r) (mm 2)
b (10 25 s 21)
PS
2 1
840 6 41 1100 6 150
8.7 6 0.8 2.6 6 0.2
1.9 6 0.2 2.5 6 0.4
6.8 6 0.9 29.2 6 5.2
S. mitis BMS
2 1
1002 6 189 1210 6 130
10.8 6 0.9 7.7 6 0.4
1.7 6 0.3 1.2 6 0.2
6.7 6 1.6 10.8 6 2.1
S. oralis J22
2 1
935 6 67 1486 6 71
10.5 6 1.0 8.2 6 1.1
1.7 6 0.1 1.5 6 0.3
4.1 6 0.6 10.6 6 1.7
A. naeslundii T14V
2 1
1105 6 30 1162 6 33
7.4 6 1.0 6.2 6 0.3
4.8 6 0.7 4.9 6 0.9
8.5 6 2.3 11.7 6 0.1
Particle type
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TABLE 4 Statistically Significant Effects (P < 0.05) of the Main Factors (Collector Material, COL; Salivary Conditioning Film, SCF, and Particle Type, Part) and Their Interactions on the Deposition Parameters (x Indicates Significant and 0 Not Significant; na Is Not Available) Factors
TABLE 6 Pairwise Comparison According to Bonferroni (25) (The Lines Indicate Groups in Which Particles Do Not Differ Significantly)
Factor interactions
PP chamber
Col
SCF
Part
Col/SCF
Col/Part
SCF/Part
j0 n` t SP chamber j0 n` t Al b
x 0 x Col na na na na na
x x x SCF x x x 0 x
x x 0 Part x x x x x
0 0 x Col/SCF na na na na na
x 0 0 Col/Part na na na na na
0 x x SCF/Part x x x 0 x
univariate ANOVA. The initial deposition rates j 0 of the two systems differ significantly, irrespective of the presence or absence of a salivary conditioning film. If the latter is present, there is also a significant difference between the numbers of particles in a stationary endpoint. DISCUSSION
turn out to have a significant effect, both for the PP and the SP flow chamber. In Table 6 the particles are grouped together based on their vectors. The order in which the particles are shown is arbitrary in this case, unlike in Table 5, since there is no mean value for ordering the particles. Based on the vector ( j 0 , n ` , t ), the polystyrene particles are clearly distinct from the bacteria. Using the full vector ( j 0 , n ` , t , A l, b ) for the SP flow chamber data, A. naeslundii T14V is distinct from the other bacterial strains and the polystyrene particles. Finally, an overall comparison was made between the real (bacteria and enamel collector surfaces) and the model system (polystyrene particles and a quartz collector surface), using TABLE 5 Pairwise Comparison of the Deposition Results According to Tukey (25) (For Each Parameter, the Particles Are Ordered According to Increasing Mean Value; the Lines Indicate Groups in Which Particles Do Not Differ Significantly)
In this study, we compared the deposition of three initially colonizing strains of dental enamel surfaces and polystyrene particles both on quartz and enamel surfaces with or without a salivary conditioning film in a parallel plate and in a stagnation point flow chamber at a common Peclet number. Measurements in the SP flow chamber were done slightly next to the stagnation point because otherwise adhering bacteria could not be visualized. The effects of a salivary conditioning film are more pronounced in the SP flow chamber than in the PP chamber, which confirms our previous suggestion that deposition in the SP flow chamber is more strongly controlled by interaction forces than that in the PP chamber (26). The effect of the salivary conditioning film depends on the type of particle, which is to be expected given the widely varying surface properties summarized in Table 1. The reproducibility in the SP chamber seems superior to the one that could be observed in the PP chamber, as also discussed in our previous work (26). With a comparison of Tables 2 and 3, it can be seen that the initial deposition rates as well as the numbers of bacteria and particles adhering in a stationary endpoint in the SP flow chamber are higher than those in the PP flow chamber within the timescale of the experiments, presumably because convective mass transport is directed toward the collector surface in the SP flow chamber. A comparison of theoretical initial deposition rates on the basis of the Smoluchowski–Levich approach (27) indicates an average factor of 4.5 between initial deposition rates in both flow chamber devices, which is much higher than what can be obtained from the experimental results, yielding a ratio of 2.1. This is probably due to the fact that the theoretical SP deposition rate was calculated for the stagnation point, while experiments were done slightly next to the stagnation point. With regard to the use of quartz or glass as a model for dental enamel surfaces, it can be concluded, from the present
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results, that in the absence of a salivary conditioning film especially the initial deposition rates differ between both materials. The higher initial deposition rates on enamel (see Table 2) might be due to the typical surface heterogeneity, causing the exposure of proteins from the interprismatic space, holding together the hydroxylapatite crystallites at the surface. Proteincovered collector surfaces, quartz and enamel, appear to have higher initial deposition rates than bare surfaces, which could be due to a train and loop conformation of adsorbed salivary proteins, in which particles can become more easily trapped. Interestingly, differences in initial deposition rates persist between both materials, also when a salivary conditioning film is adsorbed to these collector materials, indicating that selective protein adsorption and conformational changes of adsorbed proteins may be governed by differences in surface properties between quartz and enamel. Protein adsorption and their conformational state have been shown before to depend on the hydrophobicity of the surfaces (28). From a mechanistic point of view, it is interesting that the polystyrene particles have lower initial deposition rates and stationary endpoint adhesion numbers than S. mitis BMS, despite their being similarly hydrophobic. This may be partly attributed to the higher negative surface charge of the polystyrene particles, yielding stronger electrostatic repulsion, while also the presence of a variety of surface appendages on the streptococcal cell surface assists adhesion (29, 30). For S. mitis BMS, protein-rich fibrils with lengths up to 1 mm have been observed electron microscopically (31). The presence of structural and chemical surface heterogeneities on bacterial cell surfaces is the major obstacle for a straightforward physico– chemical interpretation of bacterial adhesion data. Nevertheless, on ideally smooth and chemically homogeneous substrata, as constituted by a hexadecane–water interface, DLVO analyses of adhesion data have been fairly successful (32). The application of a physiological, relatively high ionic strength and a complex buffer system for suspension of the bacteria decreases differences between the bacterial z potentials as found in low ionic strength solutions (33) and z potentials of the different bacterial strains are likely too small to become reflected in the initial deposition rates. Also, in a stationary endpoint, a number of adhering bacteria are by no means related to their intrinsic cell surface hydrophobicity. Stationary endpoint adhesion, both of bacteria as well as of polystyrene particles in the presence of a salivary conditioning film, is reduced compared with adhesion to bare collector surfaces, in agreement with previous findings for streptococcal adhesion to enamel and artificial substrata (34), indicating a protective role of salivary conditioning films in the oral cavity, as also described for salivary conditioning films on tumor epithelial cells and Candida albicans adhesion (35). Desorption probabilities of polystyrene particles and bacteria from quartz collector surfaces with a salivary conditioning film are higher than those from bare quartz surfaces, despite the fact that it was anticipated for the bacteria that binding to a
salivary conditioning film would be less reversible due to irreversible specific interactions, which are absent on bare collector surfaces. This possibly indicates detachment of bacteria adhering to salivary conditioning films through cohesive failure in the conditioning films or interfacial failure of adsorbed proteins at the collector surface. Similar suggestions were made before by Busscher et al. when adhering bacteria were exposed to excessively high external shear forces (36) that exceed the shear forces in the present paper by several orders of magnitude. Summarizing, this study for the first time compares bacterial deposition in a PP and SP flow chamber, demonstrating that the SP flow chamber shows larger influences of the properties of the interacting surfaces than those found in the PP flow chamber also when bacterial cell surfaces are involved, while furthermore SP experimental data on bacterial adhesion are more reproducible than PP data. However, the use of a natural collector surface, i.e., dental enamel, appeared impossible in the SP flow chamber as imaging was hampered too strongly by the turbidity of thin enamel slabs, and enamel as a collector material could only be used in the PP flow chamber. Although the use of polystyrene particles as a model of oral bacteria, and the use of quartz as a model of dental enamel, allows one to observe some features of bacterial deposition to enamel, major differences in experimental results became obvious, especially where the initial deposition rate is concerned, warranting the conclusion that the process of bacterial deposition to enamel surfaces cannot be modeled by using polystyrene particles and quartz collector surfaces. REFERENCES 1. Xia, Z., Woo, L., and Van de Ven, T. G. M., Biorheology 23, 359 (1989). 2. Pratt-Terpstra, I. H., Weerkamp, A. H., and Busscher, H. J., J. Colloid Interface Sci. 129, 568 (1989). 3. Christersson, C. E., Glantz, P. O., and Baier, R. E., J. Dent. Res. 96, 91 (1988). 4. Morisaki, H., J. Gen. Microbiol. 137, 2649 (1991). 5. Schneider, R. P., and Marshall K. C., Colloid Surf., B 2, 387 (1994). 6. Rutter, P. R., and Vincent, B., in “The Adhesion of Microorganisms to Surfaces: Physico–Chemical Aspects” (R. C. W. Berkeley, J. W. Lynch, J. Melling, P. R. Rutter, and B. Vincent, Eds.), p. 79. Chichester: Ellis Horwood, 1980. 7. Mozes, N., Marchal, F., Hermesse, H. P., Van Haecht, J. L., Reuliaux, L., Le´onard, A. J., and Rouxhet, P. G., Biotechnol. Bioeng. 30, 433 (1987). 8. Weatherell, J., and Robinson, C., in “The Inorganic Composition of Teeth” (I. Zipkin, Ed.), p. 43. Wiley, New York, 1973. 9. Jongebloed, W. L., Molenaar, I., and Arends, J., Calcif. Tissue Res. 19, 109 (1975). 10. Cowan, M. M., Taylor, K. G., and Doyle, R. J., J. Bacteriol. 169, 2995 (1987). 11. Lalande, M., Tissier, J. P., and Corrieu, G., J. Dairy Res. 51, 557 (1984). 12. Dankert, J., Hogt, A. M., and Feijen, J., Rev. Biocompat. 2, 219 (1986). 13. Chang, H. H., Chang, C. P., Chang, J. C., Dung, S. Z., and Lo, S. J., J. Biomed. Sci. 4, 235 (1997). 14. Chen. C. C., and Hang, S. H., Am. Ind. Hyg. Assoc. J. 59, 227 (1998). 15. Adamczyk, Z., and Van de Ven, T. G. M., J. Colloid Interface Sci. 80, 340 (1981).
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16. Sjollema, J., and Busscher, H. J., Colloid Surf. 47, 382 (1989). 17. Adamczyk, Z., Zembala, M., Siwek, B., and Czarnecki, J., J. Colloid Interface Sci. 10, 188 (1986). 18. Brouwer, W. M., and Zsom, R. L., J. Colloid Surf. 24, 195 (1987). 19. James, A. M., “Charge Properties of Microbial Cell Surfaces in Microbial Cell Surface Analysis-Structure and Physicochemical Methods” (N. Mozes, P. S. Handley, H. J. Busscher, and P. G. Rouxhet, Eds.), p. 221. VCH Publishers, Inc., New York, 1991. 20. Van Oss, C. J., and Gillman, C. F., J. Reticul. Soc. 12, 283 (1972). 21. Amory, D. E., Genet, M. J., and Rouxhet, P. G., Surf. Interface Anal. 11, 478 (1988). 22. Van Wagenen, R. A., and Andrade, J. D., J. Colloid Interface Sci. 76, 305 (1980). 23. Landa, A. S., Van der Mei, H. C., and Busscher, H. J., Biofouling 9, 327 (1996). 24. Wit, P. J., and Busscher, H. J., Colloid Surf, A 125, 85 (1995). 25. Miller, R. G., “Simultaneous Statistical Inference.” Springer Verlag, New York, 1981. 26. Yang, J., Bos, R., Poortinga, A., Wit, P. J., Belder, G. F., and Busscher, H. J., Langmuir 15, 4671 (1999). 27. Elimelech, M., Gregory, J., Jia, X., and William, R., “Particle Deposition
28. 29. 30.
31. 32. 33. 34. 35. 36.
& Aggregation. Measurement, Modelling and Simulation,” p. 98. Butterworth-Heinemann Ltd., Woburn, MA, 1995. Doyle, R. J., Nesbitt, W. E., and Taylor, K. G., FEMS Microbiol. Lett. 15, 1 (1982). Handley, P. S., Biofouling 2, 239 (1990). Handley, P. S., in “Microbial Cell Surface Analysis-Structural and Physicochemical Methods” (N. Mozes, P. S. Handley, H. J. Busscher, and P. G. Rouxhet, Eds.), p. 63. VCH Publishers Inc., New York, 1991. Uyen, H. M., Van der Mei, H. C., Weerkamp, A. H., and Busscher, H. J., Appl. Environ. Microbiol. 54, 837 (1988). Busscher, H. J., Van de Belt-Gritter, B., and Van der Mei, H. C., Colloid Surf., B 5, 111 (1995). Satou, J., Fukunaga, A., Satou, N., Sintani, H., and Okuda, K., J. Dent. Res. 67, 558 (1988). Pratt-Terpstra, I. H., Weerkamp, A. H., and Busscher, H. J., J. Dent. Res. 68, 463 (1989). Umazume, M., Ueta, E., and Osaki, T., J. Clin. Microbiol. 33, 432 (1995). Busscher, H. J., Bos, R., and Van der Mei, H. C., FEMS Microbiol. Lett. 128, 229 (1995).
Deposition of Oral Bacteria and Polystyrene Particles to Quartz and Dental Enamel in a Parallel Plate and Stagnation Point Flow Chamber Junlin Yang,* Rolf Bos,* Gerald F. Belder,† Jan Engel,‡ and Henk J. Busscher* ,1 *Department of Biomedical Engineering, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands; †Philips Research Laboratories, Professor Holstlaan 4, 5656 AA Eindhoven, The Netherlands; and ‡Centre for Quantitative Methods (CQM), Building HCZ-3, P.O. Box 414, 5600 AK Eindhoven, The Netherlands Received June 4, 1999; accepted September 10, 1999
mous and their cell surfaces are both structurally and chemically very heterogeneous, with a likely impact on adhesion. Dental enamel surfaces resemble hydroxylapatite, as far as its mineral component is concerned (8), but in dental enamel hydroxylapatite crystallites are embedded in an organic matrix (9), with an impact on the adhesive properties of its surface and causing a typical surface heterogeneity uncommon to nonbiological model surfaces employed, like germanium prisms, glass, quartz, or even hydroxylapatite (10). The adhesion of colloidal particles as well as of microorganisms to solid substrata is of great practical importance both in industrial and in biomedical applications (11–14). As a result, different flow chamber devices have been developed to study particle deposition and adhesion under controlled mass transport, such as the parallel plate (PP) flow chamber (15, 16) and the stagnation point (SP) flow chamber (1, 17). Mass transport in a parallel plate flow chamber is slow and the convective-diffusion equation for the parallel plate flow chamber is difficult to solve as compared to, e.g., the stagnation point flow chamber. On the other hand, the parallel plate flow chamber is conceptually simpler than the stagnation point flow chamber. In the PP flow chamber, mass transport is through convection parallel to the substratum surfaces, usually constituting the top and/or bottom plates of the chamber, while the actual transport toward the substratum, needed for adhesion to occur, is through diffusion, which makes the PP flow chamber a kinetically slow one. Alternatively, in the SP flow chamber, convective mass transport is directed toward the substratum surface bending hyperbolically in a parallel direction close to the surface and creating a stagnation point on the substratum in the middle of the flow. Recently, we compared deposition of polystyrene particles to quartz in a PP and SP flow chamber and concluded that SP experiments were more reproducible than PP experiments, due to a stronger influence of interaction forces in the SP flow chamber as compared to a stronger influence of chance processes, like collisions between flowing and adhering particles and diffusion in the PP flow chamber. The aim of this paper is to compare the deposition of three initially colonizing bacterial strains of tooth surfaces in vivo
The aim of this paper is to determine to what extent (i) deposition of oral bacteria and polystyrene particles, (ii) onto quartz and dental enamel with and without a salivary conditioning film, (iii) in a parallel plate (PP) and stagnation point (SP) flow chamber and at common Peclet numbers are comparable. All three bacterial strains showed different adhesion behaviors, and even Streptococcus mitis BMS, possessing a similar cell surface hydrophobicity as polystyrene particles, did not mimic polystyrene particles in its adhesion behavior, possibly as a result of the more negative z potentials of the polystyrene particles. The stationary endpoint adhesion of all strains, including polystyrene particles, was lower in the presence of a salivary conditioning film, while also desorption probabilities under flow were higher in the presence of a conditioning film than in its absence. Deposition onto quartz and enamel surfaces was different, but without a consistent trend valid for all strains and polystyrene particles. It is concluded that differences in experimental results exist, and the process of bacterial deposition to enamel surfaces cannot be modeled by using polystyrene particles and quartz collector surfaces. © 1999 Academic Press
Key Words: deposition; parallel plate flow chamber; stagnation point flow chamber; bacteria; polystyrene particles; convectivediffusion; pair distribution function.
INTRODUCTION
Physico– chemical approaches have been used by several groups in an attempt to model biological adhesion processes. For instance, it is often assumed that inert polystyrene particles mimic bacteria in their adhesion to surfaces, that glass (1, 2), germanium (3, 4), or quartz (3) surfaces behave in adhesion studies, similarly to dental enamel, and that bathing of collector surfaces in biological fluids (5), like saliva, urine, or seawater, has little influence. Although mechanistically, the same fundamental forces, like Lifshitz–Van der Waals, electrostatic, and acid– base forces, govern adhesion of polystyrene particles and of bacteria (6, 7), the diversity in the bacterial world is enor1
To whom correspondence should be addressed. Fax: (131)-50-3633159. E-mail: [email protected]. 0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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with the deposition of polystyrene particles in a PP and SP flow chamber, both to enamel and quartz collector surfaces and with and without a so-called salivary conditioning film. MATERIALS AND METHODS
Bacterial Strains, Polystyrene Particles, and Cell Surface Characterization For each adhesion experiment, Streptococcus oralis J22 was precultured from blood agar plates in a batch culture of Todd Hewitt broth for 24 h at 37°C in ambient air. Streptococcus mitis BMS was precultured from a blood agar plate for 24 h at 37°C in Todd Hewitt broth with 0.5% (w/v) glucose added, also in ambient air. Actinomyces naeslundii T14V was precultured from blood agar for 24 h in Schaedler’s broth supplemented with 0.01 g L 21 of hemin at 37°C in atmosphere air of 10% H 2, 85% N 2, and 5% CO 2. These cultures were used to inoculate second cultures which were allowed to grow for 16 h. Bacterial cells were harvested by centrifugation (5 min at 6500g) and washed twice with adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride, and 1 mM calcium chloride at pH 6.8). To break up bacterial aggregates, bacteria were sonicated for 30 –50 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT). Sonication was carried out intermittently while cooling in an ice/water bath. These procedures were found not to cause cell lysis in any strain. Subsequently, the cells were counted in a Bu¨rker-Turk counting chamber. Thereafter, the cells were suspended in an adhesion buffer to a density of 1.4 3 10 8 cells ml 21. Polystyrene particles (Vs2A, AKZO Research, Arnhem, The Netherlands) with a diameter of 783 nm were prepared as described by Brouwer and Zsom (18). The particles were washed twice by centrifugation in demineralized water and suspended in the adhesion buffer to the same density as the bacterial strains. Bacterial cell surfaces were characterized by their z potentials, as measured in an adhesion buffer employing particulate microelectrophoresis (19). Also, z potentials of the polystyrene particles were measured for comparison. Water contact angles of sessile droplets were measured on bacterial lawns, deposited on cellulose acetate membrane filters (pore diameter, 0.45 mm), and dried in ambient air to a so-called “plateau state” (20). The plateau state of drying the lawns was determined in an earlier experiment after the onset of drying, as the time span during which a constant value for the water contact angle is maintained. To determine the elemental surface composition of the bacteria, X-ray photoelectron spectroscopy (XPS) was performed on freeze-dried bacterial pellets (21). Collector Materials and Surface Characterization Quartz collector surfaces were cleaned thoroughly by sonication for 2 min in a 2% solution of surfactant RBS 35 (Fluka Chemie AG, Buchs, Switzerland), followed by extensively
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rinsing with methanol and demineralized water. The labial surfaces of bovine dental incisors were ground under running tap water with abrasive paper (800 and 1200 grit) and polished with a slurry of Al 2O 3 powder (particle diameter, 0.05 mm) in distilled water. Afterward, the polished surface was cleaned with demineralized water under sonication two times for 20 s. Subsequently, the polished labial surface was cut into a regular shape and polished to a thickness of approximately 800 mm and glued to a glass plate using a drop of nail polish. Thereafter, the exposed side of the enamel slice was ground successively with 400-, 800-, and 1200-grit abrasive paper until a thickness of 25–35 mm was obtained, polished with a slurry of Al 2O 3 powder, and cleaned ultrasonically again. Finally, the enamel pieces were removed from the glass and stored at a temperature of 8°C for further use in a moist environment. For the experiments in the parallel flow chamber, an enamel piece was glued to an already cleaned and dried quartz plate with a small drop of nail polish. z potentials of the quartz and enamel surfaces with and without an adsorbed salivary conditioning film were derived from streaming potential measurements in an adhesion buffer, as described by Van Wagenen and Andrade (22). Water contact angles on both collector materials were measured by the sessile drop technique as employed for contact angle measurements on bacterial lawns, while again collector materials with an adsorbed salivary conditioning film were dried in ambient air to a so-called plateau state prior to contact angle measurements (20). The elemental surface concentrations of the surfaces were obtained by XPS (23). Saliva Collection and Preparation Human whole saliva from 10 healthy volunteers of both sexes was collected into ice-cooled beakers after stimulation by chewing Parafilm. After the saliva was pooled and centrifuged at 1200g for 20 min at 4°C, 0.2 M phenylmethylsulfonylfluoride (PMSF) as a protease inhibitor was added to a final concentration of 1 mM to reduce protein breakdown. Afterward, the solution was centrifuged again at 10,000g for 20 min at 4°C, dialyzed overnight at 4°C against distilled water, and freeze-dried for storage. For coating the collector surfaces, the freeze-dried saliva was dissolved at a concentration of 1.5 mg ml 21 in adhesion buffer. This solution was centrifuged at 10,000g for 5 min and the supernatant was used to flow in the system under consideration, i.e., the PP or SP flow chamber. Flow Chamber and Image Analysis The SP and PP flow chambers used are schematically presented in Fig. 1, together with their essential dimensions. Briefly, for the parallel plate flow chamber, deposition was observed on the center of the bottom plate with a CCD-MXR camera (High Technology, Eindhoven, The Netherlands) mounted on a phase contrast microscope (Olympus BH-2), whereas with the stagnation flow chamber deposition was
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FIG. 1. Schematic presentations of the PP and SP flow chambers used in this study. In the SP flow chamber, the stagnation point is exactly opposite of the fluid inlet channel.
observed in the area very close to the stagnation point with a dark-field microscope (Leitz) equipped with A CCD-LDH camera (Philips, Eindhoven, The Netherlands). Live images were grabbed with a PC-Vision 1 frame grabber and Gauss and Sharp filtered after subtraction of an out-of-focus image. Thereafter, deposited particles were discriminated from the background by single gray value thresholding. This yielded binary black and white images which were subsequently stored on a disk for later analysis. Experiments were carried out three times at a flow rate of 0.05 cm 3 s 21 for the PP and 0.00883 cm 3 s 21 for the SP flow chamber, corresponding with a wall shear rate of 22.5 and 17.3 s 21, respectively, and a common Peclet number of 3.95 3 10 23. Deposition Protocol and Data Analysis Before each experiment, all tubes and the flow chamber were filled with buffer, while air bubbles were carefully removed from the system. Flasks containing bacterial suspensions, buffer, and human whole saliva were all connected to the flow chamber. First, flow was switched to saliva when appropriate, for about 100 min to create a salivary conditioning film. Thereafter, flow was switched to buffer for 10 min for removal of all remnants of saliva from the system and finally switched to the bacterial suspension. The number of bacteria or particles adhering to the substratum was determined in time. All experiments were done in triplicate with separate bacterial cultures at room temperature. The total number of adhering bacteria or particles per unit area, n(t), were recorded as a function of time by image sequence analysis (24). The initial deposition rate, j 0 , of the depositing bacteria or particles was determined from the initial increase of n(t) with time, while the later stages of the deposition process were assumed to be described by n~t! 5 j 0 t ~1 2 e 2t/ t !,
[1]
1/ t 5 j 0 A l 1 b ,
[2]
where A l is the area blocked by an adhering bacterium and particle, as can be calculated from the radial pair distribution
function g(r) and b is the desorption probability, assumed to be independent of the residence time in the present study. The expression 1/t can be calculated directly from the measured initial deposition rates and the number of bacteria or particles adhering in a stationary endpoint, while subsequently combination with the initial deposition rate and blocked area yields the desorption probability b. Blocked areas A l are assumed to be similar to the depletion zones with area A g(r) that can be derived from the radial pair distribution function g(r) which describes the relative density of adhering particles around a given particle as a function of interparticle distance,
g~r! 5
r ~r, dr! , r0
[3]
where r is the radius of a shell, r (r, dr) is the density of adhering particles or bacteria in a shell with thickness dr, and r 0 is the average density of the total field of view. By determination of the radius, g(r) , 1 was taken. Statistical Analyses Univariate ANOVA was used to test the significance of the differences observed between the main factors studied (i.e., quartz or enamel collector surfaces, the absence or presence of a salivary conditioning film, polystyrene particle, or bacterial involvement) on the deposition parameters j 0 , n ` , t , A l, and b. Separate analyses were made for the PP and SP cells. Multivariate ANOVA was carried out to determine the significance of the differences observed between the main factors on the deposition process as a whole. To this end, the deposition parameters were grouped together as a vector and the analysis was conducted with the vector rather as with the separate parameters. Finally, a comparison was made between the two systems involved in this study, i.e., the real system, consisting of enamel and bacteria and the model system, consisting of the quartz collector surface in combination with polystyrene particles. Statistical significance was accepted at P , 0.05. RESULTS
Bacterial Cell and Collector Surface Characterization Table 1 lists the z potentials of the bacteria and polystyrene particles, together with their water contact angles and elemental surface concentration ratios. The bacterial strains are all less negatively charged than the polystyrene particles, while the intrinsic cell surface hydrophobicity of the organisms varies from hydrophilic (S. oralis J22) to extremely hydrophobic and, in fact, S. mitis BMS is equally hydrophobic as polystyrene. XPS analysis demonstrates that bacterial cell surface hydrophobicity is caused by nitrogen-rich groups, most notably
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TABLE 1 z Potentials of Polystyrene Particles Vs2A, Bacteria (S. oralis J22, S. mitis BMS, and A. naeslundii T14V), of Powdered Dental Enamel by Particulate Microelectrophoresis, and of Quartz Surfaces with (1) or without (2) a Salivary Conditioning Film (SCF) by Streaming Potential Measurements in Adhesion Buffer (pH 6.8), Together with Water Contact Angles and Elemental Surface Concentration Ratios by XPS Elemental surface concentration ratio Particle type
z potential (mV)
PS S. oralis J22 S. mitis BMS A. naeslundii T14V
250 6 4 22 6 3 210 6 7 23 6 4
Quartz Enamel
2 1 2 1
SCF SCF SCF SCF
225 6 1 210 6 5 218 6 2 217 6 2
Water contact angle (°)
O/C
N/C
P/C
S/C
2 4 4 4
0.042 0.392 0.312 0.313
0 0.102 0.124 0.090
0 0.020 0.009 0.005
26 6 2 18 6 2 45 6 10 29 6 6
5.357 0.350 1.738 0.358
0 0.172 0.052 0.205
0 0.012 0.369 0.029
90 6 24 6 100 6 64 6
proteins, while cell surface hydrophilicity is due to oxygenand phosphorus-rich groups. The collector surfaces are also negatively charged (see also Table 1) and the z potential of quartz is more negative (225 mV) than that of enamel (218 mV) in the absence of a salivary conditioning film. Salivary protein adsorption yields less negative z potentials. Quartz surfaces are more hydrophilic than enamel surfaces, while salivary protein adsorption converges their water contact angles to values characteristic for hydrophilic surfaces. The quartz surface had a minor carbon contamination as detected by XPS (11.2%), with 60.0% oxygen and 28.9% silicon measured. Note that also enamel without a salivary conditioning film shows a small amount of nitrogen, resulting from the surface exposure of organic matrix proteins. Adsorption of salivary components on both types of collector surfaces is accompanied by an increase in the amount of surface nitrogen and a reduction in the amount of collector surface elements exposed, i.e., Si or Ca for quartz and enamel, respectively. Deposition Experiments Figure 2 shows the deposition kinetics for S. oralis J22 to quartz and enamel surfaces with and without a salivary conditioning film in a PP flow chamber and to quartz surfaces in a SP flow chamber. The differences between the deposition kinetics with and without a salivary conditioning film are considerably smaller in the PP flow chamber than in the SP flow chamber. Note that in the SP flow chamber the use of enamel appeared impossible due to the turbidity of the enamel samples, yielding image problems. Quantitative features of the deposition kinetics, including initial deposition rates j 0 , numbers adhering in a stationary endpoint n ` , blocked area A g(r) , and desorption probabilities b of the bacteria and polystyrene particles are summarized in Tables 2 and 3 for the PP and SP flow chamber, respectively.
Ca/C
Si/C
0.006 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0.528 0.033
2.580 0.045 0 0
Both in the PP as well as in the SP flow chamber, initial deposition rates j 0 of bacteria and polystyrene particles are slightly higher in the presence of a salivary conditioning film as compared to those in its absence. Initial deposition rates are on average two-fold higher in the SP flow chamber than in the PP flow chamber, but differences among strains, including the polystyrene particles, are minor. Furthermore, Table 2 shows that initial deposition rates on enamel surfaces are generally different from those on the quartz collector surfaces. The number of bacteria and particles adhering in a stationary endpoint to a salivary conditioning film are smaller than those adhering to the collector surfaces without saliva, while here it appears that bacteria generally adhere in significantly higher numbers than the polystyrene particles. Most notably, the blocked areas for A. naeslundii T14V are much larger than those for the other bacterial strains and polystyrene particles. Due to the turbidity of the enamel samples, no blocked areas could be derived from the spatial arrangement of the bacteria and particles adhering on enamel. The desorption probabilities in the presence of a salivary conditioning film are larger than those measured in the absence of a salivary conditioning film. Statistical Analyses The results of the statistical analyses are summarized in Tables 4 – 6. For the PP flow chamber, A l and b were omitted from the analysis, since no values were available for enamel. For the SP flow chamber, all five parameters were included, but the collector material was omitted as a factor, since data for enamel were not available. Table 4 gives an overview of the effects of the main factors (collector material, salivary conditioning film, and particle type) on the different deposition parameters, both for the PP and the SP flow chamber. All factors appear to have a significant influence on the deposition parameters, except collector material on stationary endpoint numbers (PP flow chamber),
FIG. 2. Examples of the deposition kinetics for S. oralis J22 on quartz (a) and enamel (b) in a parallel plate flow chamber and on a quartz collector surface near the stagnation point in a stagnation point flow chamber with (c) (1 SCF) or without (2 SCF) a salivary conditioning film in potassium phosphate adhesion buffer (pH 6.8). 414
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TABLE 2 Initial Deposition Rates j 0, a Number of Bacteria and Polystyrene Particles Adhering in a Stationary Endpoint n `, Blocked Areas A g(r) Calculated from the Radial Pair Distribution Functions and Desorption Probabilities b on Quartz and Enamel Collector Surfaces with (1) and without (2) a Salivary Conditioning Film (SCF) in Adhesion Buffer (pH 5 6.8) Obtained in a Parallel Flow Chamber (6 Denotes Standard Deviation over Triplicate Experiments) Particle type
n (10 6 cm 22)
A g(r) (mm 2)
b (10 25 s 21)
57 44 85 91
4.4 6 1.0 1.7 6 0.7 4.1 6 0.6 1.9 6 0.5
3.0 6 0.5 2.5 6 1.0 nd a nd
7.7 6 2.9 23.2 6 12.0 nd nd
2 1 2 1
490 6 85 605 6 94 657 6 93 687 6 128
6.0 6 0.9 3.3 6 0.5 5.5 6 1.0 3.8 6 0.5
1.4 6 0.4 1.0 6 0.5 nd nd
6.7 6 2.2 17.7 6 4.2 nd nd
2 1 2 1
525 6 673 6 582 6 656 6
45 50 66 80
5.4 6 0.5 3.0 6 0.4 5.0 6 0.7 3.1 6 0.4
2.6 6 0.6 1.7 6 0.5 nd nd
8.3 6 1.3 21.9 6 4.2 nd nd
2 1 2 1
563 6 128 610 6 35 496 6 97 531 6 91
5.0 6 0.9 4.5 6 1.2 3.9 6 1.3 3.8 6 1.0
4.3 6 0.8 4.6 6 2.6 nd nd
9.3 6 4.1 11.4 6 3.2 nd nd
SCF
Quartz
2 1 2 1
394 6 411 6 549 6 606 6
PS
Enamel S. mitis BMS
Quartz Enamel
S. oralis J22
Quartz Enamel
A. naeslundii T14V
Quart Enamel
a
j 0 (cm 22 s 21)
Substratum
nd is “not determined” due to poor image quality.
particle type on 1/t (PP flow chamber), and the presence of a salivary conditioning film on blocked areas (SP flow chamber). Table 4 also indicates factor interactions: the influence that a change in one factor setting may have on the effect of another factor. Most interactions are significant, especially in the SP flow chamber. Moreover, there is a clear interaction between the presence of a salivary conditioning film and particle type, indicating that the effect of the presence of a salivary conditioning film on the deposition depends on the particle type. In Table 5, each deposition parameter is ordered according to an increasing mean value per particle type. In the PP flow cham-
ber, polystyrene particles invariably show the lowest values for all deposition parameters. The deposition parameters for the bacteria show no significant differences between themselves. In the SP flow chamber, the results are somewhat different and break up more with respect to particle type. Multivariate analyses were performed on the complete set of deposition parameters and account for the overall deposition behavior and correlations between parameters. Multivariate ANOVA was carried out on the vector ( j 0 , n ` , b ) for the PP flow chamber, and on the vectors ( j 0 , n ` , t ) and ( j 0 , n ` , t , A l, b) for the SP flow chamber. All factors and their interactions
TABLE 3 Initial Deposition Rates j 0, a Number of Bacteria and Polystyrene Particles Adhering in a Stationary Endpoint n `, Blocked Areas A g(r) Calculated from the Radial Pair Distribution Functions, and Desorption Probabilities b on a Collector Surface with (1) or without (2) a Salivary Conditioning Film (SCF) in an Adhesion Buffer (pH 6.8) near the Stagnation Point in a Stagnation Point Flow Chamber (6 Denotes Standard Deviation over Triplicate Experiments) SCF
j 0 (cm 22 s 21)
n ` (10 6 cm 22)
A g(r) (mm 2)
b (10 25 s 21)
PS
2 1
840 6 41 1100 6 150
8.7 6 0.8 2.6 6 0.2
1.9 6 0.2 2.5 6 0.4
6.8 6 0.9 29.2 6 5.2
S. mitis BMS
2 1
1002 6 189 1210 6 130
10.8 6 0.9 7.7 6 0.4
1.7 6 0.3 1.2 6 0.2
6.7 6 1.6 10.8 6 2.1
S. oralis J22
2 1
935 6 67 1486 6 71
10.5 6 1.0 8.2 6 1.1
1.7 6 0.1 1.5 6 0.3
4.1 6 0.6 10.6 6 1.7
A. naeslundii T14V
2 1
1105 6 30 1162 6 33
7.4 6 1.0 6.2 6 0.3
4.8 6 0.7 4.9 6 0.9
8.5 6 2.3 11.7 6 0.1
Particle type
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YANG ET AL.
TABLE 4 Statistically Significant Effects (P < 0.05) of the Main Factors (Collector Material, COL; Salivary Conditioning Film, SCF, and Particle Type, Part) and Their Interactions on the Deposition Parameters (x Indicates Significant and 0 Not Significant; na Is Not Available) Factors
TABLE 6 Pairwise Comparison According to Bonferroni (25) (The Lines Indicate Groups in Which Particles Do Not Differ Significantly)
Factor interactions
PP chamber
Col
SCF
Part
Col/SCF
Col/Part
SCF/Part
j0 n` t SP chamber j0 n` t Al b
x 0 x Col na na na na na
x x x SCF x x x 0 x
x x 0 Part x x x x x
0 0 x Col/SCF na na na na na
x 0 0 Col/Part na na na na na
0 x x SCF/Part x x x 0 x
univariate ANOVA. The initial deposition rates j 0 of the two systems differ significantly, irrespective of the presence or absence of a salivary conditioning film. If the latter is present, there is also a significant difference between the numbers of particles in a stationary endpoint. DISCUSSION
turn out to have a significant effect, both for the PP and the SP flow chamber. In Table 6 the particles are grouped together based on their vectors. The order in which the particles are shown is arbitrary in this case, unlike in Table 5, since there is no mean value for ordering the particles. Based on the vector ( j 0 , n ` , t ), the polystyrene particles are clearly distinct from the bacteria. Using the full vector ( j 0 , n ` , t , A l, b ) for the SP flow chamber data, A. naeslundii T14V is distinct from the other bacterial strains and the polystyrene particles. Finally, an overall comparison was made between the real (bacteria and enamel collector surfaces) and the model system (polystyrene particles and a quartz collector surface), using TABLE 5 Pairwise Comparison of the Deposition Results According to Tukey (25) (For Each Parameter, the Particles Are Ordered According to Increasing Mean Value; the Lines Indicate Groups in Which Particles Do Not Differ Significantly)
In this study, we compared the deposition of three initially colonizing strains of dental enamel surfaces and polystyrene particles both on quartz and enamel surfaces with or without a salivary conditioning film in a parallel plate and in a stagnation point flow chamber at a common Peclet number. Measurements in the SP flow chamber were done slightly next to the stagnation point because otherwise adhering bacteria could not be visualized. The effects of a salivary conditioning film are more pronounced in the SP flow chamber than in the PP chamber, which confirms our previous suggestion that deposition in the SP flow chamber is more strongly controlled by interaction forces than that in the PP chamber (26). The effect of the salivary conditioning film depends on the type of particle, which is to be expected given the widely varying surface properties summarized in Table 1. The reproducibility in the SP chamber seems superior to the one that could be observed in the PP chamber, as also discussed in our previous work (26). With a comparison of Tables 2 and 3, it can be seen that the initial deposition rates as well as the numbers of bacteria and particles adhering in a stationary endpoint in the SP flow chamber are higher than those in the PP flow chamber within the timescale of the experiments, presumably because convective mass transport is directed toward the collector surface in the SP flow chamber. A comparison of theoretical initial deposition rates on the basis of the Smoluchowski–Levich approach (27) indicates an average factor of 4.5 between initial deposition rates in both flow chamber devices, which is much higher than what can be obtained from the experimental results, yielding a ratio of 2.1. This is probably due to the fact that the theoretical SP deposition rate was calculated for the stagnation point, while experiments were done slightly next to the stagnation point. With regard to the use of quartz or glass as a model for dental enamel surfaces, it can be concluded, from the present
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results, that in the absence of a salivary conditioning film especially the initial deposition rates differ between both materials. The higher initial deposition rates on enamel (see Table 2) might be due to the typical surface heterogeneity, causing the exposure of proteins from the interprismatic space, holding together the hydroxylapatite crystallites at the surface. Proteincovered collector surfaces, quartz and enamel, appear to have higher initial deposition rates than bare surfaces, which could be due to a train and loop conformation of adsorbed salivary proteins, in which particles can become more easily trapped. Interestingly, differences in initial deposition rates persist between both materials, also when a salivary conditioning film is adsorbed to these collector materials, indicating that selective protein adsorption and conformational changes of adsorbed proteins may be governed by differences in surface properties between quartz and enamel. Protein adsorption and their conformational state have been shown before to depend on the hydrophobicity of the surfaces (28). From a mechanistic point of view, it is interesting that the polystyrene particles have lower initial deposition rates and stationary endpoint adhesion numbers than S. mitis BMS, despite their being similarly hydrophobic. This may be partly attributed to the higher negative surface charge of the polystyrene particles, yielding stronger electrostatic repulsion, while also the presence of a variety of surface appendages on the streptococcal cell surface assists adhesion (29, 30). For S. mitis BMS, protein-rich fibrils with lengths up to 1 mm have been observed electron microscopically (31). The presence of structural and chemical surface heterogeneities on bacterial cell surfaces is the major obstacle for a straightforward physico– chemical interpretation of bacterial adhesion data. Nevertheless, on ideally smooth and chemically homogeneous substrata, as constituted by a hexadecane–water interface, DLVO analyses of adhesion data have been fairly successful (32). The application of a physiological, relatively high ionic strength and a complex buffer system for suspension of the bacteria decreases differences between the bacterial z potentials as found in low ionic strength solutions (33) and z potentials of the different bacterial strains are likely too small to become reflected in the initial deposition rates. Also, in a stationary endpoint, a number of adhering bacteria are by no means related to their intrinsic cell surface hydrophobicity. Stationary endpoint adhesion, both of bacteria as well as of polystyrene particles in the presence of a salivary conditioning film, is reduced compared with adhesion to bare collector surfaces, in agreement with previous findings for streptococcal adhesion to enamel and artificial substrata (34), indicating a protective role of salivary conditioning films in the oral cavity, as also described for salivary conditioning films on tumor epithelial cells and Candida albicans adhesion (35). Desorption probabilities of polystyrene particles and bacteria from quartz collector surfaces with a salivary conditioning film are higher than those from bare quartz surfaces, despite the fact that it was anticipated for the bacteria that binding to a
salivary conditioning film would be less reversible due to irreversible specific interactions, which are absent on bare collector surfaces. This possibly indicates detachment of bacteria adhering to salivary conditioning films through cohesive failure in the conditioning films or interfacial failure of adsorbed proteins at the collector surface. Similar suggestions were made before by Busscher et al. when adhering bacteria were exposed to excessively high external shear forces (36) that exceed the shear forces in the present paper by several orders of magnitude. Summarizing, this study for the first time compares bacterial deposition in a PP and SP flow chamber, demonstrating that the SP flow chamber shows larger influences of the properties of the interacting surfaces than those found in the PP flow chamber also when bacterial cell surfaces are involved, while furthermore SP experimental data on bacterial adhesion are more reproducible than PP data. However, the use of a natural collector surface, i.e., dental enamel, appeared impossible in the SP flow chamber as imaging was hampered too strongly by the turbidity of thin enamel slabs, and enamel as a collector material could only be used in the PP flow chamber. Although the use of polystyrene particles as a model of oral bacteria, and the use of quartz as a model of dental enamel, allows one to observe some features of bacterial deposition to enamel, major differences in experimental results became obvious, especially where the initial deposition rate is concerned, warranting the conclusion that the process of bacterial deposition to enamel surfaces cannot be modeled by using polystyrene particles and quartz collector surfaces. REFERENCES 1. Xia, Z., Woo, L., and Van de Ven, T. G. M., Biorheology 23, 359 (1989). 2. Pratt-Terpstra, I. H., Weerkamp, A. H., and Busscher, H. J., J. Colloid Interface Sci. 129, 568 (1989). 3. Christersson, C. E., Glantz, P. O., and Baier, R. E., J. Dent. Res. 96, 91 (1988). 4. Morisaki, H., J. Gen. Microbiol. 137, 2649 (1991). 5. Schneider, R. P., and Marshall K. C., Colloid Surf., B 2, 387 (1994). 6. Rutter, P. R., and Vincent, B., in “The Adhesion of Microorganisms to Surfaces: Physico–Chemical Aspects” (R. C. W. Berkeley, J. W. Lynch, J. Melling, P. R. Rutter, and B. Vincent, Eds.), p. 79. Chichester: Ellis Horwood, 1980. 7. Mozes, N., Marchal, F., Hermesse, H. P., Van Haecht, J. L., Reuliaux, L., Le´onard, A. J., and Rouxhet, P. G., Biotechnol. Bioeng. 30, 433 (1987). 8. Weatherell, J., and Robinson, C., in “The Inorganic Composition of Teeth” (I. Zipkin, Ed.), p. 43. Wiley, New York, 1973. 9. Jongebloed, W. L., Molenaar, I., and Arends, J., Calcif. Tissue Res. 19, 109 (1975). 10. Cowan, M. M., Taylor, K. G., and Doyle, R. J., J. Bacteriol. 169, 2995 (1987). 11. Lalande, M., Tissier, J. P., and Corrieu, G., J. Dairy Res. 51, 557 (1984). 12. Dankert, J., Hogt, A. M., and Feijen, J., Rev. Biocompat. 2, 219 (1986). 13. Chang, H. H., Chang, C. P., Chang, J. C., Dung, S. Z., and Lo, S. J., J. Biomed. Sci. 4, 235 (1997). 14. Chen. C. C., and Hang, S. H., Am. Ind. Hyg. Assoc. J. 59, 227 (1998). 15. Adamczyk, Z., and Van de Ven, T. G. M., J. Colloid Interface Sci. 80, 340 (1981).
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