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Interaction of Two Oral Streptococcal Strains with Physicochemically Characterized Fluorosilane Diffusion Gradient Surfaces Rolf Bos, Jeroen H. de Jonge, Betsy van de Belt-Gritter, Joop de Vries, and Henk J. Busscher* Department of Biomedical Engineering, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands Received July 22, 1999. In Final Form: November 5, 1999 Gradient surfaces have a defined variation in surface chemistry along their length that allow study of the influence of substratum wettability on bioadhesion phenomena along their length in relation with a controlled surface chemistry. (Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane (MCFS) diffusion gradients were made on glass and characterized by advancing and receding water contact angles and scanning X-ray photoelectron spectroscopy. Model calculations demonstrated that the hydrophobic ends of these gradients were only 50% covered by MCFS, which could be confirmed by atomic force microscopy showing hydrophobic patches. Therewith, bacteria can interact with either hydrophobic or hydrophilic patches on the hydrophobic end of a diffusion gradient, while on the hydrophilic end there is no such choice. By use of a parallel plate flow chamber, the position-bound adhesion, including initial deposition rates and numbers of adhering bacteria after 3 h, of two different oral streptococcal strains was studied along the lengths of MCFS gradients. Streptococcus oralis J22 did not show any position-bound adhesion along the length of a gradient surface. The organism also had similar adhesion behavior on homogeneous, hydrophobic FEP-Teflon as on hydrophilic glass. Streptococcus sobrinus HG1025, however, adhered 2-fold better to the hydrophobic end of a MCFS gradient than to its hydrophilic end, while also on homogeneous, hydrophobic FEP-Teflon adhesion was more extensive than on hydrophilic glass. When streptococci adhering along the length of a gradient were exposed to a passing liquid-air interface, no position-bound detachment was observed for any of the strains, but upon perfusion of the flow chamber with a detergent solution S. sobrinus HG1025 detached less from the hydrophobic end than from the hydrophilic end of the gradient. This study demonstrates, using MCFS diffusion gradients, that the sensitivity of bacterial strains to differences in substratum hydrophobicity, originating from a known chemical heterogeneity, is straindependent.
Introduction Microbial adhesion to biomaterials implants still causes severe infection problems, mostly resulting in replacement of the implant.1 To increase the biocompatibility of biomaterials, the surface physicochemistry of materials can be changed to make an implant surface more hydrophobic or hydrophilic. Despite an enormous body of literature (see ref 2 for a review, comprising 230 references), a ubiquitously accepted definition on the role of substratum hydrophobicity in microbial adhesion is absent. This is due, in part, to the difficulties involved in carrying out microbial adhesion experiments,2 the variability in cell surface properties among microbial strains,3,4 and the fact that in nearly all studies on substratum hydrophobicity and microbial adhesion, the influence of the surface chemistry responsible for a certain hydrophobicity is neglected. Surfaces may have comparable wettabilities, but their electrostatic charge, topography, and chemistry may be * To whom correspondence may be addressed. Tel: + 31 50 363 3140. Fax: + 31 50 363 3159. E-mail:
[email protected]. (1) Cristina, A. G. Biomaterial-Centered infection: microbial adhesion versus tissue integration. Science 1987, 237, 1588-1595. (2) Bos, R.; Van der Mei, H. C.; Busscher, H. J. Physico-chemistry of initial microbial adhesive interactionssits mechanisms and methods for study. FEMS Microbiol. Rev. 1999, 23, 179-229. (3) Handley, P. S. Structure, composition and functions of surface structures on oral bacteria. Biofouling 1990, 2, 239-264. (4) Pratt-Terpstra, I. H.; Weerkamp, A. H.; Busscher, H. J. On the relation between interfacial free energy-dependent and noninterfacial free energy-dependent adherence of oral streptococci to solid substrata. Curr. Microbiol. 1988, 16, 311-313.
completely different. One method to vary in a controlled way the chemical composition and accompanying changes in physical properties of a surface employs so-called chemical gradient surfaces, recently reviewed by Ruardy et al.5 Several methods have been described for the preparation of gradient surfaces.6-9 Particularly, the diffusion method is convenient because of its ease of preparation and good control of the surface chemistry and wettability steepness along its length.5,10 Protein adsorption studies on gradient surfaces11,12 demonstrated that the largest amounts of proteins adsorbed on the hydrophobic ends of gradient surfaces, probably due to a (5) Ruardy, T. G.; Schakenraad, J. M.; Van der Mei, H. C.; Busscher, H. J. Preparation and characterization of chemical gradient surfaces and their application for the study of cellular interaction phenomena. Surf. Sci. Rev. 1997, 29, 1-30. (6) Go¨lander, C. G.; Caldwell, K.; Lin, Y. S. A new technique to prepare gradient surfaces using density gradient solutions. Colloids Surf. 1989, 42, 165-172. (7) Chaudhury, M. K.; Whitesides, G. M. How to make water run uphill. Science 1992, 256, 1539-1541. (8) Lee, H. B.; Andrade, J. D. Cell adhesion on gradient surfaces. Trans. Biomater. Congr., 3rd Kyoto, Japan, 1988, 43. (9) Lee, J. H.; Kim, H. G.; Khang, G. S.; Lee, H. B.; Jhon, M. S. Characterization of wettability gradient surfaces prepared by corona discharge treatment. J. Colliod Interface Sci. 1992, 151, 563-570. (10) Elwing, H.; Askendal, A.; Lundstro¨m, I. Protein exchange interactions on solid surfaces studied with a wettability gradient method. Prog. Colloid Polym. Sci. 1987, 74, 103-107. (11) Go¨lander, C. G.; Lin, Y. S.; Hlady, V.; Andrade, J. D. Wetting and plasma-protein adsorption studies using surfaces with a hydrophobicity gradient. Colloids Surf. 1990, 49, 289-302. (12) Lin, Y. S.; Hlady, V. Human serum albumin adsorption onto octadecyldimethylsilyl-silica gradient surface. Colloids Surf., B: Biointerfaces 1994, 2, 481-491.
10.1021/la9909805 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/19/2000
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Table 1. Physicochemical Surface Properties of the Two Oral Streptococcal Strains Employed, Including Their Contact Angles by Water (w), Formamide (f), Methyleneiodide (m), and r-Bromonaphthalene (r-br), as Well as Their Zeta Potential in Adhesion Buffer and Elemental Surface Composition by XPSa
θw S. oralis J22 S. sobrinus HG1025
24 29
contact angles (deg) θf θm 31 33
θR-br
zeta potential (mV)
33 34
-9 -10
49 47
elemental surface concentration ratios O/C N/C PC 0.392 0.380
0.102 0.071
0.020 0.010
a Data taken from Bos et al.21 and Van der Mei et al. (Van der Mei, H. C.; De Soet, J. J.; De Graaff, J.; Rouxhet, P. G.; Busscher, H. J. Comparison of the physicochemical surface properties of Streptococcus rattus with those of other mutans streptococcal species. Caries Res. 1991, 25, 415-423.)
favorable interaction between hydrophobic groups on the surfaces and hydrophobic amino acids exposed at the exterior of the proteins after conformational changes .13 Desorption of fibrinogen and immunoglobulin G adsorbed to methyl diffusion gradients was effectively stimulated by nonionic Tween 20, as studied ellipsometrically, from the hydrophobic ends and the transition regions of the gradients, whereas the anionic surfactant SDS removed the proteins from all regions of the gradient.14 Cellular interactions with gradient surfaces have recently been investigated, and spreading of fibroblasts and endothelial cells was clearly minimal on the hydrophobic ends of methyl diffusion gradients.5 While several investigators have studied the interactions of proteins and cells with gradient surfaces, only one study has recently been conducted evaluating bacterial interactions with gradient surfaces.15 Treponema denticola, a spiral-shaped bacterium, adhered in four times higher numbers to the hydrophobic end of a Si-(CH3)2 diffusion gradient than to its hydrophilic end, while bacteria were oriented flat on the hydrophobic end and tip-oriented on the hydrophilic end. Since, however, gradient surfaces are based on varying the relative presence of one defined chemical group along its length, it is inevitable that a chemical heterogeneity is present along the length of the gradient too, which is eventually responsible for the position-bound wettability. Whereas the hydrophilic end may probably be considered as relatively homogeneous from a chemical point of view, especially the transition region, and possibly also the hydrophobic end of a gradient surface, is chemically heterogeneous with a potential impact on bacterial adhesion, since bacteria have a choice to interact with either a hydrophobic or hydrophilic region. The aim of this paper is first to characterize (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane diffusion gradients chemically by X-ray photoelectron spectroscopy (XPS) and physically by the Wilhelmy plate technique (advancing and receding contact angles) and atomic force microscopy (AFM) in order to model the heterogeneity along the length of the gradient. Second, the influence of the chemical heterogeneity along the length of the gradient surfaces on the position-bound interaction with two different oral streptococcal strains was measured in a parallel plate flow chamber.16 (13) Van Oss, C. J. Interfacial Forces in Aqueous Media; Marcel Dekker, Inc.: New York, 1994. (14) Elwing, H.; Askendal, A.; Lundstro¨m, I. Desorption of fibrinogen and globulin from solid surfaces induced by a nonionic detergent. J. Colloid Interface Sci. 1989, 128, 296-300. (15) Ellen, R. P.; Wikstro¨m, M.; Grove, D. A.; Song, M.; Elwing, H. Polar adhesion of Treponema denticola on wettability gradient surfaces. Colloids Surf., B: Biointerfaces 1998, 11, 177-186. (16) Sjollema, J.; Busscher, H. J.; Weerkamp, A. H. Real-time enumeration of adhering microorganisms in a parallel plate flow cell using automated image analysis. J. Microbiol. Methods 1989, 9, 7378.
Materials and Methods Bacterial Strains, Culture Conditions, Harvesting, and Suspending Fluids. Streptococcus oralis J22 and Streptococcus sobrinus HG1025 were cultured in Todd Hewitt Broth all at 37 °C in ambient air. For each experiment, strains were inoculated from blood agar in a batch culture. This culture was used to inoculate a second culture which was grown for 16 h prior to harvesting. The physicochemical surface properties of the strains are summarized in Table 1. Bacteria were harvested by centrifugation (5 min at 10000g), washed twice with demineralized water, and resuspended in buffer (2 mM potassium phosphate, 0.5 mM calcium chloride, and 50 mM potassium chloride, pH 6.8) for adhesion experiments. To break bacterial chains and aggregates, cells were sonicated for 30 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT). Sonication was done intermittently while the cells were cooling in an ice/water bath. These conditions were found not to cause cell lysis in any strain. Preparation of Gradient Surfaces. Wettability gradients were prepared according to the diffusion method described by Elwing et al.10 Glass slides were cleaned thoroughly by sonication in a detergent solution (2% RBS 35, Omnilabo International BV, The Netherlands), extensive rinsing in demineralized water, washing in methanol, and again rinsing with water. This procedure yielded a zero degree water contact angle. After drying in an oven at 80 °C each slide was glow discharge treated in a modified sputter coater Edwards S150B at 50 W and 10-15 mbar argon pressure for 2 min. Thereafter, the slides were immediately placed in a cuvette filled with xylene. Subsequently, a 0.05% (w/w) solution of C6F13CH2-CH2-Si-(CH3)2Cl ((tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane, MCFS) in trichloroethylene was injected under the xylene phase through a drain in the cuvette, allowing MCFS to diffuse into the xylene phase and react with the glass surface. After 1 h of diffusion at room temperature, the cuvette was drained. The slides were rinsed in subsequently trichloroethylene and methanol and stored under methanol (5 days maximum). All chemicals were purchased from Merck, Darmstadt, Germany, except for MCFS (Fluka chemie AG, Buchs, Switzerland) and were of the highest purity. Wettability Characterization and Modeling Surface Chemistry from Wettability Data. After the samples were dried, advancing and receding water contact angles were recorded along the lengths of the gradient surfaces using the Wihelmy plate method.17 Samples were connected to a microbalance (Lauda tensiometer, Ko¨nigshofen, Germany), and the resulting force was continu(17) Ruardy, T. G.; Schakenraad, J. M.; Van der Mei, H. C.; Busscher, H. J. Adhesion and spreading of human skin fibroblasts on physicochemically characterized gradient surfaces. J. Biomed. Mater. Res. 1995, 29, 1415-1423.
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ously recorded while the plate was immersed into water at a constant speed of 27 mm/min. The contact angle θ was calculated as a function of the position along the length of the gradient from
F ) pγ cos θ + (m - V ∆F)g
(1)
where F is the force on the slide, p the perimeter, γ liquid surface tension, m the mass of the slide, ∆F the liquidvapor density difference, g the acceleration of gravity, and V the volume displaced. The surface chemistry along the length of the gradient was subsequently modeled using the Cassie equation, which relates equilibrium contact angles on solid surfaces with the fractional coverage of the surface by microscopic domains with known contact angles18
cos θe ) f1 cos θ1 + f2 cos θ2
(2a)
where θe is the equilibrium contact angle on a heterogeneous surface and f1 and f2 are the fractions of the surface with a equilibrium contact angle of θ1 and θ2, respectively. Equilibrium contact angles were calculated from the average between the advancing and receding angles.19 Since FEP-Teflon is the most fluoridated material available, θ1 was assumed to equal to 107° (average between advancing and receding contact angles on FEPTeflon) for a completely fluorinated surface. Furthermore, it was assumed that the equilibrium water contact angle on clean glass can be calculated from the average between advancing and receding water contact angles on the hydrophilic ends of the gradients. On the basis of these assumptions, the fractional surface coverage with silane groups along the length of the gradients can be calculated from eq 2a employing
cos θe ) fθ cos 107° + (1 - fθ) cos θglass
(2b)
in which fθ is the position-bound fluorosilane fractional surface coverage and θe the position-bound equilibrium water contact angle along the gradient. Scanning X-ray Photoelectron Spectroscopy (XPS) and Modeling Surface Chemistry from XPS Data. Scanning XPS was carried out using an S-probe apparatus (Surface Science Instruments, Mountain View, CA). The residual pressure in the spectrophotometer was approximately 10-7 Pa. An aluminum anode was used for the X-ray production (10 kV, 22mA) at a spot size of 250 × 1000 µm2. The distance between consecutive spot areas was 1 mm. Relative atomic percentages of silicon, oxygen, fluorine, and carbon were calculated from peak areas of Si2p, O1s, F1s, and C1s (each peak 1 min accumulation in the unscanned mode) using the instrumental sensitivity factors as supplied by the manufacturer. Spectra were taken at a low resolution of 150 eV and a photoemission angle of 35°. A patchy overlayer model20 was used to calculate the fractional surface coverage of MCFS along the lengths of the gradient surfaces in which maximal surface coverage by the fluorosilane was assumed to correspond with an F1s electron count as observed for FEP-Teflon. (18) Johnson, R. E.; Dettre, R. H. Wettability and contact angles. In Surface and Colloid Science; Matijevic, E., Ed.; Wiley-Interscience: New York, 1969; Vol. 2, p 85. (19) Cassie, A. B. D. Contact angles. Discuss Faraday Soc. 1948, 3, 11-15. (20) Chatelier R. C.; Xie, X.; Gengenbach, T. R.; Griesser, H. J. Quantitative analysis of polymer surface restructuring. Langmuir 1995, 11, 2576-2584.
Atomic Force Microscopy. Topographic images of homogeneously MCFS coated (0.05% (w/w), with a reaction time of 1 h) and bare glass surfaces (results not shown) were produced with an atomic force microscope (Park Scientific Instruments) operated in the contact mode and noncontact mode. Images were obtained in air with standard triangular silicon nitride cantilevers (spring constant 0.06 nm-1). At least three separately prepared samples were analyzed at different scan sizes, directions, and speeds. Parallel Plate Flow Chamber and Image Analysis. The parallel plate flow chamber (length × width × height: 7.6 × 3.8 × 0.06 cm) and image analysis system have been described in detail.16,21 Gradients and FEP-Teflon surfaces (5.5 × 3.8 cm) were used as bottom plates of the flow chamber, while unmodified glass slides were used as top plates. FEP-Teflon surfaces were prepared by affixing thin FEP-Teflon sheets (0.2 mm) with double sided sticky tape to a thicker (1.8 mm) Perspex plate. Deposition on the bottom plate of the parallel plate flow chamber was observed with a CCD-MXR camera (High Technology, Eindhoven, The Netherlands) mounted on a phase contrast microscope (Olympus BH-2) equipped with a 40× ultralong working distance objective (Olympus ULWD-CD Plan 40 PI). The camera was coupled to an image analyzer (TEA, Difa, Breda, The Netherlands). Image acquisition and processing were done as previously described.16 Briefly, live images were Laplace filtered after subtraction of an out-of-focus image. Thereafter, adhering bacteria were discriminated from the background by single gray value thresholding. This yields binary black and white images which were subsequently stored on disk. In this setup, one image covers a surface area of 0.016 mm2. Deposition Protocol and Data Analysis. Before each deposition experiment, buffer was circulated through the system by hydrostatic pressure for at least 30 min at a wall shear rate of 10 s-1. Subsequently, the microbial suspension (3 × 108 cells mL-1) was circulated through the system at a wall shear rate of 10 s-1 for 3 h, and images were grabbed at three locations at regular time intervals on the hydrophobic and hydrophilic ends of a gradient as well as on the transition region. In addition, bacterial deposition experiments were carried out on hydrophobic FEP-Teflon and on hydrophilic glass, as homogeneous surfaces. After 3 h, the experiment was continued in two different ways to see whether detachment could be induced by a chemical or physical stimulus. The chemical stimulus was provoked by flushing the chamber with demineralized water and subsequently a 1% (w/w) sodium dodecyl sulfate (SDS) solution at a shear rate of 10 s-1. A physical stimulus, i.e., passage of a liquid-air interface, was created by emptying the flow chamber by hydrostatic pressure. The effects of these stimuli were evaluated by comparing images taken at the specific locations prior to and after a particular stimulus. Results Wettability Characterization. Figure 1 presents the advancing and receding water contact angles along the length of a gradient surface together with the positionbound fractional surface coverage by fluorosilane groups, as calculated from eq 2b. At the hydrophobic end, surface coverage is maximal but far from complete (approximately (21) Bos, R.; Van der Mei, H. C.; De Vries, J.; Busscher, H. J. The role of physicochemical and structural surface properties in co-adhesion of microbial pairs in a parallel-plate flow chamber. Colloids Surf., B: Biointerfaces 1996, 7, 101-112.
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Figure 1. Advancing (ΘA) and receding (ΘR) water contact angles as a function of the distance from the hydrophobic end of a MCFS gradient surface and the position-bound fractional surface coverage (2) by fluorosilane groups calculated from the wettability data with the aid of eq 2b.
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Figure 3. Position-bound fractional surface coverage by fluorosilane groups calculated from the XPS data relative to the one of FEP-Teflon as a function of the distance from the hydrophobic end.
Figure 2. Elemental surface compositions in terms carbon (O), oxygen (1), fluoride (3), and silicon (b) over the length of the MCFS gradient as a function of the distance from the hydrophobic end.
0.5) and decreases sharply over the length of the gradients to virtually zero at the hydrophilic end. The water contact angle hysteresis varied from 45° on the hydrophobic end up to 80° in the transition zone and reduced to 30° on the hydrophilic end. Scanning XPS. Figure 2 presents the variation of the elemental surface concentration of carbon, oxygen, fluorine, and silicon over the length of the gradient. The fluorine surface concentration decreased sharply over the length of gradient from 13% on the hydrophobic end to background levels on the hydrophilic end. Simultaneously, the oxygen surface concentration increased from approximately 49% on the hydrophobic end to 56% on the hydrophilic end. Like for carbon, the silicon surface concentration was nearly constant over the length of the gradient. Note that the decrease in the fluorine surface concentration occurs approximately 30 mm from the hydrophobic end, as does the decrease in water contact angles. Figure 3 shows the position-bound fractional surface coverage calculated from the XPS data over the length of the gradient. The transition zone from XPS modeling is located at the same distance from the hydrophobic end as that calculated from contact angles (compare Figure 2), but the fractional surface coverage calculated for the hydrophobic end (0.20) is lower than that calculated from contact angles (compare Figure 1). Atomic Force Microscopy. Figure 4 presents a topographic image of a homogeneously MCFS-coated glass
Figure 4. Topographic AFM image (noncontact mode) of a homogeneous MCFS-coated glass surface.
surface. On the homogeneously MCFS-coated glass, which we assume to similar to the hydrophobic end of a gradient, a regular pattern of patches is visible. Although it was not attempted to quantitatively derive a surface coverage from the AFM image, it can be estimated that the surface coverage by the hydrophobic patches on the homogeneously MCFS-coated, hydrophobic surface is approximately 0.5. Bacterial Interaction. Table 2 summarizes the adhesion of the streptococci to FEP-Teflon and glass, including the intial deposition rate, the number of bacteria adhering after 3 h, and the detachment induced by the passage of a liquid-air interface or 1% SDS. Adhesion of S. oralis J22 to hydrophilic glass and hydrophobic FEP-Teflon surfaces is surprisingly similar, but for S. sobrinus HG1025, however, initial deposition rates and numbers of adhering bacteria after 3 h are approximately 2-fold lower on glass than on FEP-Teflon. For both strains, detachment stimulated by the passage of a liquid-air interface is comparable with the detachment achieved by perfusion of the flow chamber with an SDS detergent solution. However, S. sobrinus HG1025 was more easily stimulated to detach from hydrophilic glass than was S. oralis J22. In general, the interaction of S. sobrinus HG1025 appeared more sensitive to differences in substratum hydrophobicity than the one of S. oralis J22.
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Table 2. Deposition and Detachment of S. Oralis J22 and S. Sobrinus HG1025 for Glass and FEP-Teflon Expressed as an Initial Deposition Rate (j0), Number of Bacteria Adhering after 3 h Deposition (n3h), and the Percentage of Bacteria Detached from the Surface by the Passage of a Liquid-Air Interface or Perfusion of the Flow Chamber with a 1% SDS Detergent Solutiona j0
strains S. oralis J22 S. sobrinus HG1025 S. oralis J22 S. sobrinus HG1025 S. oralis J22
(cm-2 s-1)
n3h (106 cm-2)
Glass 848 650
% induced detachment liquid-air SDS
9.3 5.9
35 70
44 100
FEP-Teflon 991 9.0 1146 12.0 848 9.3
18 b 35
9 5 44
a All data are results from duplicate experiments with separately cultured bacteria and coincided within, on average, 20%, except for the liquid-air induced detachment data which represents single run data. b Microorganisms could not be enumerated after passage of an air bubble through the flow chamber, due to clumping of laterally displaced bacteria.
Figure 6. Percentage of (9) S. sobrinus HG1025 and (0) S. oralis J22 detached by the passage of a liquid-air interface or perfusion of the flow chamber with a 1% SDS solution from the hydrophobic end, transition region (middle), and the hydrophilic end of a MCFS gradient.
preference for FEP-Teflon. The initial deposition rate of S. sobrinus HG1025 decreases gradually from the hydrophobic to the hydrophilic end of the gradient. Similar remarks can be made with regard to the number of bacteria adhering after 3 h. Figure 6 summarizes the streptococcal detachment from gradient surfaces induced by a liquid-air interface (top) or a 1% SDS solution (bottom). No position-bound detachment of any of the two strains by the passage of a liquidair interface was observed, but detachment by the SDS detergent solution of S. sobrinus HG1025 was less on the hydrophobic end than on the hydrophilic end. Discussion Figure 5. Initial deposition rates (top) and numbers of adhering (bottom) (9) S. sobrinus HG1025 and (0) S. oralis J22 after 3 h on the hydrophobic end, the transition region (middle), and the hydrophilic end of a MCFS gradient. All results are averages of duplicate runs, with separately cultured bacteria, and coincided within 20%.
Initial deposition rates (j0) and number of microorganisms adhering after 3 h of flowing (n3h) at the hydrophilic, transition, and hydrophobic region of the MCFS gradients are presented in Figure 5. No position-bound adhesion patterns are observed for S. oralis J22, but for S. sobrinus HG1025 the initial deposition rate on the hydrophobic end of gradient is approximately 2-fold higher than on the hydrophilic end, in analogy with the bacterium’s
Diffusion gradient surfaces are frequently employed to study the influence of one particular substratum surface property upon bioadhesion phenomena. For diffusion gradients, the variation in wettability along the length of a gradient is concurrent with a change in one well-defined chemical group, which implies that the physical surface properties must vary as a result of an incomplete and varying surface composition along the length of a gradient. The modeling of this surface chemical heterogeneity of MCFS gradients on glass obtained from contact angles suggests slightly higher surface coverages of MCFS on the hydrophobic ends of the gradients than those obtained from scanning XPS. This is probably due to the assumption used in the XPS model that adsorbed silane molecules adapt a perpendicular configuration with respect to surface. However, in the high vacuum environment of the
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XPS the silane molecules will probably adapt a more flattened configuration. Consequently, the thickness of the silane patches is overestimated, which results in an underestimation of the fractional surface coverage. Moreover, the AFM experiments clearly demonstrate the existence of hydrophobic patches of approximately 0.1 µm in size with similar spacing between patches with an estimated fractional surface coverage of 0.5. Surface chemical heterogeneity along the length of gradient surfaces poses a complication in the interpretation of bioadhesion phenomena on diffusion gradients. Proteins are able to adsorb either to the hydrophobic patches or to the hydrophilic surface on which the gradients are made, as their dimensions are within the range of the size of the hydrophobic patches and their spacing. Yet, despite having hydrophobic and hydrophilic substratum choices available, proteins interact differently along the length of a diffusion gradient.11,12 Fibroblasts and endothelial cells are orders of magnitude larger than the chemical heterogeneities along a diffusion gradient, and still their spreading varies along its length.9,17 Bacteria interacting with a gradient likely also have a choice to interact with either hydrophobic or hydrophilic substratum regions on a gradient, as their dimensions are in the micrometer size while interacting parts of the cell surface may well be in the sub-micrometer size. S. oralis J22 was relatively indifferent in its choice for hydrophobic or hydrophilic substrata (see Table 2), and its adhesion was similar along the length of a gradient surface. S. sobrinus HG1025 on the other hand, despite having nearly identical physicochemical cell surface properties as S. oralis J22 (see Table 1), preferentially adhered to hydrophobic substrata. Pratt-Terpstra et al.4 suggested in this respect to distinguish between surface free energy sensitive and surface free energy insensitive strains in microbial adhesion, the degree of surface free energy sensitivity being dependent on the absence or presence of structural cell surface appendages3 or the presence of hydrophobic moieties on the cell surface that
Bos et al.
could remove interfacial water.22 As obvious from this study, S. sobrinus HG1025 clearly represents a surface free energy sensitive strain in its adhesion to surfaces. Since Treponema denticola also demonstrated a different behavior along the length of a gradient surface, this strain too must be classified as a surface free energy sensitive strain .15 The surface free energy sensitive S. sobrinus HG1025 clearly has a choice to adhere on the hydrophobic patches or on uncoated, hydrophilic glass, as abundantly present also on the hydrophobic end of a gradient. If the initial deposition of S. sobrinus HG1025 would be random over the hydrophobic and hydrophilic regions on a gradient, it can be calculated that for the hydrophobic end of the gradient (surface coverage by hydrophobic regions equal to 0.5), the initial deposition rate should be the weighted average of the deposition rates observed on FEP-Teflon and glass, i.e., 900 cm-2 s-1. Since the initial deposition rate on the hydrophobic end of a MCFS gradient is considerably higher, it can be concluded that when the bacterium is given a choice it adheres preferentially on hydrophobic patches. The implications of this may be far reaching, because all real life surfaces have some degree of surface chemical heterogeneity. Recently, it has been demonstrated that polystyrene particles adhere preferentially on certain spots on a glass substratum and that, moreover, after removal by a liquid-air interface passage, the same spots are again preferentially occupied.23 In conclusion, S. oralis J22 is insensitive to the chemical heterogeneities along the length of a MCFS diffusion gradient, opposite to S. sobrinus HG1025, adhering preferentially to hydrophobic regions. LA9909805 (22) Busscher, H. J.; Van der Mei, H. C. Physicochemical interactions in initial microbial adhesion and relevance for biofilm formation. Adv. Dent. Res. 1997, 11, 24-32. (23) Wit, P. J.; Busscher, H. J. Site selectivity in the deposition and redeposition of polystyrene particles to glass. J. Colloid Interface Sci. 1989, 208, 351-352.
