Modified pellicle formation and reduced in vitro bacterial adherence after surface treatment with different siloxane polymers

•

COLLOIDS SURFACES

ELS EV ] ER

Colloids and Surfaces B: Biointerfaces 5 {19951161 169

Modified pellicle formation and reduced in vitro bacterial adherence after surface treatment with different siloxane polymers Jan Olsson a . , Anette Carldn a Norman L. Burns b, Krister Holmberg b Department o[ Cariolo~y, Facuhy o[ Odontology, GSteborg University, Medicinare~atan 12. 413 90 G/it ehor~... Sweden b hlstitutefi~r Smjace Chemistry, P.O. Box 5607, S-114 86 Stockholm, Sweden Received 2 February 1995; accepted 5 April 1995

Abstract

The formation of a salivary pellicle is a prerequisite of bacterial colonization on the tooth, and the aim of this s t u d y has been to further the understanding of the role of surface properties in the formation of the salivary pellicle and subsequent adhesion of oral bacteria. Surface modification as a means of interfering with pellicle and plaque formation has been investigated. Five different silicone-containing compounds were used for the surface treatments: polydimethylsiloxane containing aminoalkyl groups {I), polydimethylsiloxane containing partially neutralized aminoalkyl groups (Ill, ethyl silicate (llt), potassium methyl siliconate (IV) and sodium silicate (V). Studies of water wetting, surface charge, oral bacterial adherence and pellicle formation were performed on glass slides and hydroxyapatite beads coated by the test compounds. No correlation was found between contact angle and surface charge, and evidently hydrophobicity, as expressed by water wetting, is not necessarily an indication of a low surface concentration of polar groups. All compounds reduced bacterial adherence after saliva contact, compound IV by around 90%. Different pauerns were seen in the adsorption of pellicle proteins on the different polysiloxanes.

Keywords: Bacterial adherence; Pellicle formation; Polysiloxanes: Surthce charge: Water welting

I. Introduction

The role of the substrate surface in bacterial adhesion has been the topic of numerous investigations during the last 10 15 years. Since the attachment of bacteria to a solid surface is virtually always preceded by the adsorption of a proteinaceous film, considerable effort has been devoted to the understanding of the driving forces for protein adsorption and the mechanism by which the bound biomolecules promote bacterial adhesion. It is by now an established fact that inert protein-repelling materials can be obtained either by surface grafting with

* ('orresponding author. 0927-7765:95/$09.50 ~ 1995 Elsevier Science B.V. All rights reserved 5'SDI 0 9 2 7 - 7 7 6 5 ( 9 5 ) 0 1 2 1 2 - 5

a non-charged, hydrophilic polymer, such as poly(ethylene glycol)(PEG) [ 1,2], or by transformation of the surface into an extremely low-energy material, e.g. a perfluorinated or a polydimethylsiloxane surface [3,4]. Both routes are being used in the development of biomedical products. Surfaces with a low tendency to adsorb proteins and bacteria are of interest for implants and catheters, in solid-phase diagnostics and in biosensor technology, just to mention a few applications [5,6]. The two types of surfaces mentioned above are clearly very different and the mechanisms by which they prevent protein adsorption are likely to be different. A densely packed and lirmly attached layer of non-charged hydrophilic polymer, such as PEG, rejects proteins due to an unfavourable

162

J. Olsson et al./Colloids Sur[hces B." BiointetJhces5 (1995) 161 169

entropy change upon compression of the conformationally random, heavily hydrated polymer chains. in addition, the polymer chains as such, being strongly hydrophilic and free of charges, will give a minimum driving force for adsorption since both hydrophobic interactions and double-layer attractive forces are absent [7]. The protein-repelling character of the low-energy surface, on the other hand, may be seen as a wetting effect: spreading on very low-energy surfaces is energetically unfavourable for all molecules, including proteins. In a series of papers from our group, the "PEG route" to prevent salivary proteins and oral bacteria binding to hydroxyapatite and natural tooth surfaces has been explored [8 11]. Excellent in vitro results have been obtained by this approach. The clinical experiments, however, have not been convincing. The lack of proper in vivo effect is most likely due to poor durability of the thin layer of grafted polymer chains. In a recent study we compared salivary protein adsorption and oral bacterial adhesion to hydrophilic and hydrophobic surfaces, both in vitro and in vivo [ 12]. The hydrophilic surface was a silicone wafer modified by plasma polymerization of allyl alcohol-acrylic acid followed by adsorption of a PEG grafted copolymer. The hydrophobic surface was the same silicone wafer modified by plasma polymerization of hexamethyldisiloxane. Plasma polymerization of the latter species is known to give highly branched Si O Si polymers [13]. It was found that the hydrophobization procedure gave more adsorption of model proteins but less in vitro adherence of oral bacteria and much less plaque accumulation than the hydrophilization. These results were intriguing, and prompted this study in which bacterial adhesion of surfaces treated by different siloxane polymers is investigated. The aim of the study is to further the understanding of the role of surface properties in the formation of the salivary pellicle and subsequent adhesion of oral bacteria. 2. Material and methods 2.1. Compounds used for surface treatment

Five different polysiloxanes were used. Compound I, an aqueous solvent-free emulsion of

polydimethylsiloxane containing aminoalkyl substituents, was kindly supplied by Wacker-Chemie, Munich, Germany (Wacker silicone BS 1306). Compound I was diluted 10 times with water prior to use. Compound II, a microemulsion of polydimethylsiloxane containing partially neutralized aminoalkyl groups, was obtained from Wacker-Chemie (Wacker silicone 1311). Compound II was diluted 10 times with water before use. Compound IIl, a low molecular weight ethyl silicate, was dissolved in ethanol in the proportions ethyl silicate/ethanol 75:25 by weight. Compound IlI, which contains di-n-butyltindilaurate as curing catalyst, was used as received from Wacker-Chemie (Wacker Stone Strengthener OH). Compound IV, an aqueous low molecular weight potassium methyl siliconate of 42% dry content, was obtained from Wacker-Chemie (Wacker silicone BS 15). Compound IV was diluted 10 times with water prior to use. Compound V, sodium silicate (water glass) with a SiO2:Na20 ratio of 3.3, was obtained from Eka Nobel, Surte, Sweden. Compound V was diluted with water 10 times before use. 2.2. Test sulT['aces

Glass slides, approximately 20mm x 10ram, were cut out of microscope slides (76 mm x 26 ram, Kebo Lab, Stockholm, Sweden). Prior to use, the slides were cleaned in concentrated potassium dichromate/sulfuric acid and thoroughly rinsed in distilled water. Spheroidal hydroxyapatite beads (HA), which have previously been described [8], were also used. 2.3. Surface modification and characterization

The surface treatments were performed by incubation in solutions of compounds I-V for 60 min followed by drying overnight at room temperature. The advancing contact angle was recorded for distilled water on the surface-modified glass slides. Droplets of size 10/~1 were used and the contact angle was determined by viewing through a gonioscope. The values given are the mean of three recordings.

J. Olsson et aL/('olloids Surflwes"B." BiointetJDces5 (1995) 161 16~)

Charge vs. pH profiles for the surface-modified glass slides were determined by measuring the pH dependence of electroosmosis at macroscopic surfaces. For the measurement of electroosmosis, sample plates (10 mm × 20 mm) are clamped onto either side of a hollow spacer forming the upper and lower walls of a rectangular chamber. The spacer, made of optical quality poly(methyl methacrylate}, forms a chamber of dimension 2 × 5 × 15 mm 3. Blank platinum electrode wires mounted inside the spacer provide for induced electroosmolic fluid at the sample plates and consequent hydrodynamic fluid flow in the chamber. Polystyrene latex particles of zero electrophoretic mobility fire observed through the side of the spacer using a standard Rank apparatus (Rank Bros.. Cambridge, UK). Electroosmotic fluid flow induced at the sample plates is determined from the latex particle velocity measured in a region where sidewall effects are negligible. A more detailed discussion of the method is given elsewhere [ 14]. Measurements were performed in 1 mM NaC1 fit constant ionic strength, adjusting the pH with l m M HCI or l mM NaOH. Values of electrokinetic potential (~') and effective surface charge density t aol (Table 2) were calculated from electroosmosis values using the Smoluchowski equation U~o

=

-

e~,"q

163

is actually a measure of diffuse double layer charge adjacent to the plane of shear, i.e. the equations consider no separate treatment for a compact double layer region. 2.4. Adherence experiments

With minor modifications [8], the adherence experiments were performed as described by Clark et al. [16] using spheroidal hydroxyapatite (HA) beads and a -~sS-labelled clinical isolate of Streptococcus sanguis [17]. Apatitic pellicles (SHA) were formed by incubation of 40 mg of HA beads in tubes with 1.0ml of saliva diluted 1:2 with adhesion buffer (1.0 mM potassiumphosphate, 5 0 m M KCI, 1.0 mM CaCI2, and 0.1 mM MgCI2, pH 6.5). The tubes were rotated end-over-end for 60 min and, after washing, the beads were incubated with 1.0 ml of bacteria suspended ira buffer with 0.5% albumin (Sigma Chemical Corp., St Louis, MO). The number of bound bacteria was determined by scintillation counting (1215 Rackbeta, Wallac, Sweden) of the beads after thorough washing. The total activity added in the assay was determined and the adherence was expressed as the percentage of bound cells of the total number of cells added. Bacterial binding experiments were performed on surfacemodified HA and on modified HA after treatment with saliwt.

and the Poisson Boltzmann equation for a 1:1 electrolyte

2.5. Characterization ol'pelliclejbrmation hv sodium dodecyl sulphate-polyacrylamide .¢el electrophoresis ( SDS-PA GE )

ao = (8cd,'Tt 1'2 sinh \2~]Tj

Proteins incorporated in the experimental pellicles on hydroxyapatite (SHA) were desorbed by heating 40 mg of SHA in 50 t-tl of sample buffer (50raM Tris HAc (pH7.5k 2.5% SDS, 5% 2-mercaptoethanol, 0.01% Bromophenol Bluel. The samples, together with high and low molecular mass protein standards (Pharmacia). were run on precast 4 15% gradient gels. The proteins were blotted onto parallel nitrocellulose membranes, one of which was silverstained according to Kovarik et al. [ 18]. The others were analyzed for bacteria-binding [ 19-21 ] high molecular weight {HMWI agglutinins and proline-rich proteins

for electroosmotic fluid mobility at the electrokinetic plane of shear (U~o), bulk electrolyte concentration (c), permittivity of the medium (e), temperature IT), the Boltzmann constant (k), coloumbic charge (e}, and viscosity of the medium 01) [15]. It should be kept in mind that derivations of the above equations treat ions in solution as point charges in a continuous dielectric medium and assume an equilibrium Boltzmann distribution of counterions outside a well-defined electrokinetic plane of shear'. As such, this effective surface charge

164

J. Olsson et al.,,'Colloid9 Sur/'aces B: Biointerfiwes 5 (1995) 161 169 Table 1 Advancing contact angle of water on untreated glass and on glass slide surfaces treated with compounds 1 V

(PRP) by immunoblotting with anti-agglutinin and anti-PRP antibodies (kindly supplied by Dr. Malamud and Dr. Bennick, respectively). SDS-PAGE and blotting were performed on a Pharmacia automated PhastSystem TM (Pharmacia Biotechnology, Uppsala, Sweden). Antigen reactions were detected using anti-mouse (agglutinin) and anti-goat-Ig (PRP) biotin conjugate ( 1:20 000) followed by streptavidin-conjugated phosphatase (1:30000) (TAGO Inc., Burlingame, U.S.A.). Staining was performed with 5-bromo-4-chloro-3indolylphosphate (BCIP) and 4-nitro blue tetrazoliumchloride (NBT), as recommended by the manufacturer (Boehringer Mannheim Biochemica, Germany).

Compound

Contact angle (deg)

Untreated I II III IV V

40 80 83 24 90 33

lI1 and V, wettability is improved compared to untreated (cleaned) glass. From the values of surface charge given in Table 2, compounds II and IV stand out from the rest in that compound II produced a surface of significant positive charge at low pH and IV a surface of very low charge at all pH values. The positive charges of compound I I can be attributed to amino groups present as substituents on the polysiloxane backbone. The differences among compounds 1 V in terms of surface charge are also illustrated graphically in Fig. 1. An interesting observation that can be made from the tables of contact angle and surface charge

3. Results and discussion

3.1. Wettability and smface charge Tables 1 and 2 show values of the contact angle and surface charge for untreated glass and for the glass slides surface modified by compounds l-V. As can be seen, compounds I, II and IV give a surface of poor wettability while with compounds

0.5

0

0,5

-1

•

Untreated

0

Compound I

•

Compound II Compound III

1.5

-2-

[]

Compound IV

•

Compound V

-2.5 -

-3 0

!

2

I

4

I

I

6

8

l

I0

112

14

pH Fig. 1. The pH dependence of surface charge for glass surfaces treated with compounds I-V. Cleaned but untreated glass was used as a control. The values are taken from Table 2.

J. OL~son et aL,,ColloMs Sut~htces B." Bioinler/itce.v 5 i 1995,, 161 169

165

Table 2 ('ontact angles and electrokinetic measurement results for the pH dependence of eleclroosmosis in l m M glass surfaces lreatmcnl

pH

Electroosmosis fi_tmcmV l s ~1

Zeta potential

NaC1 with treated

Surface charge t~tC'cm :1

ImV)

t !ntrcaled

3.00 3.74 4.58 5.8(I 7.40 9.17 10.11

2.61 3.42 3.95 4.94 7.48 7.98 8.42

- 33.6 44.(t 501,I 63.5 9~.2 103 108

0.260 0.379 0.429 0.584 I. I 8 1.35 1,49

('cunpou nd 1

3.00 3.71 4.55 5.80 7.98 8.89 10.10

- 1.14 0.12 2.32 2.85 5.41 6.52 7.37

14.7 1.5 29.8 36.7 -69.6 83.8 94.8

0.108 0.011 0.0227 0288 0.671 0.911 1.14

Compound 11

3.(10 3.81 4.79 5.80 7.59 9.10 1(I.06

-- 4.28 - 4.25 -2.41 1.01 0.76 3.69 4.07

3.00 3.77 4.74 5.8(I 7.37 9.12 10.11

1.31 0.55 3.06 4.26 5.03 8.76 10.42

I ().X 7.1 3').4 54.S -- 64.7 111 134

O. 123 0.051 i).313 0.475 fl.~0 I

Compound IV

3.(/0 3,77 4,56 5,8(I 7,42 8.84 10.22

- 1.2(I -0.92 --0.30 0.65 0.28 1.36 1.66

15.4 11 .N 3.9 S.4 3.6 - 17.5 21.3

ft. I 13 0.08(~ 0.028 0.061 0.(126 0.129 (). 158

Coin pound V

3.00 3.86 4.73 5.80 7.62 9.10 10.29

1.07 2.79 2.94 3.44 4.31 5.43 5.94

13.8 - 35.9 37.8 44.2 55.4 69.8 - 76.4

I). 10 I fl.281 !).298 I).360 0.4~2 O.674 fl.778

( ' o m p o u n d 111

(Tables 1 and 2, see also Fig. 11 is that there is little correlation between the two. A m o n g the surface treatments that give poor wettability and

55.(/ 54.7 31.0 17.0 9.77 47.5 52.3

which

would

consequently

0.477 0.474

().23,~ 0.11949 0.(1710 o.394 0.446

I.¢~5 2.5

be regarded

phobic, there are large variations Compound

I gives a pronounced

as hydro-

in s u r f a c e c h a r g e . negative

charge

166

J. Olsson eta/,/Colloids" SmJaces B." Biointer[aces5 (1995) 161 169

over almost the entire pH range studied, compound II has considerable negative and positive charges above and below pH 7, respectively, and compound IV has very low charge density over the whole pH range. Hydrophobicity, as expressed by water wetting, may not necessarily be an indication of a low surface concentration of polar groups. The same phenomenon has recently been observed in the spreading of a sizing agent ( A K D wax) on paper. By electron spectroscopy for chemical analysis (ESCA) it could be demonstrated that a surface coverage of the sizing agent as low as 15% was enough to make the paper totally hydrophobic [22]. One should keep in mind, though, that the charge densities at the surfaces in this work are low. For surfaces of higher charge density there may be a reasonable correlation between hydrophobicity and charge.

Non-treated hydroxyapatite and hydroxyapatite treated only with saliva are used as references. As can be seen from Fig. 2, all surface treatments lead to a considerable reduction in saliva-mediated S. sanguis adherence. The most efficient treatment, by compound IV, gave a reduction in bacterial adherence after saliva contact by around 90%. It is interesting to note that the control value for "buffer only", i.e. untreated HA without saliva contact, is very low and that the values for surfacemodified HA are also relatively low, except for compound II-treated HA. There are very little differences between compounds I and IV, which are hydrophobic, and compounds III and V, which are hydrophilic (as is HA itself). Evidently, surface hydrophilicity, as manifested by contact angle measurements, is not decisive of S. sanguis adherence in the absence of saliva. As revealed by the electroosmosis experiments, compound II is unique among the substances tested in that it gives a significant positive charge below pH 7. It is then reasonable to assume that attraction between the negatively charged bacterial surface [23] and amino groups at the modified solid surface is responsible for the high adherence values obtained

3.2. Bacterial adherence

Fig. 2 shows adherence of the oral bacterium S. sanguis on hydroxyapatite beads after surface modification with compounds I - V and after surface modification followed by treatment with saliva. Percentof addedactivity

Saliva only

8

7 6

5 4

CompoundI-V followedby saliva

3 2

CompoundI-V only

1

0 I

II

III

lV

V

I

II

III

IV

V

Ctrl Ctrl

Fig. 2. Effect on S. sanguis adherence of treating HA with compounds 1 V and saliwl. The bars "Compounds 1 V followed by saliva""should be compared with the bar for "'Salivaonly".

,I. Olsson et al./Colloids Surfaces B." Biointetj'aces 5 (1995) 161 169

with compound I1. The high value for "saliva only" treatment is a good illustration of how saliva mediates bacterial binding. This effect has been documented before [24,25].

pounds III and V had an abundance of agglutinin, were poor in PRP and showed a reduced bacterial binding. The amount of bacteria-binding salivary components present in pellicles on modified surfaces and the way such components are incorporated and presented at the saliva pellicle interface may determine the binding properties. Ellipsometry measurements have convincingly demonstrated that a relatively thick (around 5 mg m -2) biofilm is rapidly formed on very hydrophobic surfaces after contact with saliva [25,26]. Therefore, it seems unlikely that the reduction in bacterial binding is due to poor adsorption of saliva constituents at surface-treated HA. It is more likely that the decrease in S. sanguis adhesion is related to an unnatural salivary pellicle being formed on the surface-modified hydroxyapatite particles. It is postulated that the effect of saliva constituents on the binding of oral bacteria to the tooth surface is partly of general nature, e.g. by imparting charges or hydrophobicity to the surface [27]. It has been demonstrated by Christersson et al. that the level of adsorption of oral bacteria in the presence of saliva depends on the critical surface tension of the substrate [28,29]. Surfaces of medium critical surface tension (30 38 mN m -1) retained higher numbers of microorganisms than both higher- and lower-energy surfaces. Bacterial adherence and colonization of the tooth surface seem also to be dependent on more specific binding mechanisms. Bacteria attach by means of special surface appendages, so-called adhesins, to salivary

3.3. Pellicle composition Both the amounts and the composition of the pellicles varied considerably between'the different surfaces (Fig. 31. The most hydrophilic, strongly negatively charged surface (compound V) seemed to attract the highest amounts of protein (Fig. 3A). Lower amounts were recovered from the most hydrophobic surface (compound IV), which also had the lowest surface charge. A potential experimental error is that the recovery process may not remove all adsorbed protein from the hydroxyapatite surface. Ill future work we plan to monitor the efficiency of the desorption process by ESCA analysis. Strongly stained immunoblots indicated high amounts of certain bacteria-binding components (PRP, approximately 30 kDa, agglutinin, approximately 300 kDa) in pellicles from the untreated HA surfaces, which mediated the most pronounced binding of S. sanguis (Figs. 3B, 3C and 2). These components were weakly stained in the low binding pellicles recovered from HA modified with compound IV (Figs. 2, and 3B, 3C). Pellicles at compound 1- and compound II-modified surfaces were characterized by relatively high amounts of PRP and agglutinin and low S. sanguis binding potential. Pellicles from surfaces modified by cornA

167

B

C 212~.

76 •

53 ~-

i I

11

III

IV

V

I

H

III

IV

V

I

11

III

IV

V

Fig. 3. Salivary components in pellictes formed on untreated HA {column - ) and HA modified by compounds I V after SDS-PAGE and blotting onto nitrocellulose. (A) Silver-stained proteins; (B) proteins stained in immunoblots with anti-PRP, (Ci proteins stained in immunoblots with anti-agglutinin antibodies. Molecular mass proteins indicated {kDal are myosin (212), transferrin 176 I, glutamic dehydrogenase i 5 3 / a n d carbonic anhydrase (30).

168

J. Olsson et al./Colloids Surjitces B: Biointerfaces 5 (1995) 161 169

components carrying specific receptors. The proline-rich proteins (PRPs) and H M W agglutinins are examples of salivary components which promote bacterial adherence specifically [ 19,21,30]. The substance that gives the most pronounced reduction of S. sanguis adherence, compound IV, is the one that gives the most uncharged surface over the pH range studied (Fig. 1). An uncharged, hydrophobic surface is likely to attract proteins mainly by hydrophobic interactions which, in turn, means that the adsorbed layer on such a surface could be very different with regard to both composition and structure from that on the highly charged hydroxyapatite surface. The fact that S. sanguis binding was strongly reduced on SHA modified by compound IV implies that components with receptor activity for S. sanguis were not included in the pellicle. The finding that both the H M W agglutinins and the PRPs were present in low concentrations in the pellicle of compound IV lends support to this hypothesis (Figs. 3B and 3C). The effect on pellicle formation by silicone modification of hydroxypatite has been studied by Rykke and R611a [311. Hydroxyapatite powder treated with polydimethylsiloxane was exposed to albumin solutions. Considerably less protein adsorbed to treated HA than to untreated HA. The same tendency of reduced protein adsorption was also observed by scanning electron microscope examination of enamel fragments carried in the mouth to acquire pellicles. The silicone-oil-treated enamel exhibited a slower rate of pellicle formation than untreated enamel. Amino acid analysis showed that the chemical composition of the pellicle material collected in vivo from the treated enamel was different from that collected from untreated enamel. This indicates that different salivary proteins bind to the silicone layer than to the untreated surface. A similar finding was revealed in the present series of experiments (Fig. 3).

4. Conclusions The technique used in this work to measure electrically induced fluid flow at macroscopic surfaces is new, allowing for the characterization of a wide variety of surfaces in terms of surface charge.

We have in previous work studied the surface charge of modified HA particles and the effect on S. mutans adherence, and have found that low net charge resulted in a very low degree of bacterial binding [10]. Here, we see the same effect on the macroscopic surfaces. Hydrophobicity, as expressed by water wetting, appears not to be indicative of low numbers of polar groups in a surface, since the contact angle and surface charge are not found to correlate. Compound IV, which gave the most pronounced reduction of S. sanguis adherence (less than 90%), also gave the most uncharged surface. Effects on bacterial adherence as a result of hydrophobation of the surface seems to be due to changes in the composition or structure of the salivary pellicle rather than inhibition of its formation.

Acknowledgements This work was supported by Swedish Medical Research Council Project #5607 and Patentmedelsfonden f6r odontologisk profylaxforskning.

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J. Olsson et aL/Colloids Surfaces B: Biointerjaces 5 (1995) 161 169 [9] J. Olsson, A. Carlan and K. Holmberg, Arch. Oral Biol., 35 11990) 137. [10] J. Olsson, A. CatiOn and K. Holmberg, Caries Res., 25 (1991) 51. [ 111 J. Olsson, M. Hellsten and K. Holmberg, Colloid Polym. Sci., 269 ( 1991 ) 1295. [12] B. Lassen, K. Hohnberg, C. Brink, A. Carl6n and J. Olsson. Colloid Polym. Sci., 272 (1994) 1143. [13] B. Lassen, C.-G. G61ander. A. Johansson and H. Elwing, clm. Mater., I 1 (1992) 99. [14] N.L. Burns, J. Colloid Interface Sci., submitted for publication. [15] R.J. Hunter, Zeta Potential in Colloid Science, Academic Press, London, 198l. [16] W.B. Clark, L i . Bammann and R.J. Gibbons, Infect. Immun., 19 (1978) 846. [ 17] A. Leonhardt, J. Olsson and G. Dahl6n, J. Dent. Res., 74 (1995) in press. [18] A. Kovarik, K. Hlubinova, A. Vrebenska and J. Prachar, Folia. Biol. (Prague), 33 (1987) 253. [1911 R.J. Gibbons and D.I. Hay, in L. Switalski, M. H66k and E. Beachey (Eds.), Molecular Mechanisms of Bacterial Adhesion, Springer-Verlag, New York, 1989, p. 143.

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[20] J. Rundegren, Infect. lmmun., 53 11986i 173. [21] A. Carldn and J. Olsson, J. Dent. Res., 74 ( 19951 1040. [221 G. Str/Sm, G. Carlsson and M. Kiaer, Wochenblatt for Papierfabrikation, 120 (1992) 606. [23] J. Olsson, P.-O. Glantz, and B. Krasse, Arch. Oral Biol., 21 119761 605. [24] H.J. Busscher, M.M. Cowan and tI.C. van der Mei, FEMS Microbiol., 88 (1992) 199. [251 N. Vassilakos, T. Arnebram and P.-O. Glantz, Scand. J. Dent. Res., 101 (1993) 133. [261 N. Vassilakos. P.-O. Glantz and T. Arnebrant, Scand. J. Dent. Res.. 101 (1993i 339. [27] l.H. Pratt-Terpstra, A.H. Weerkamp and H.J. Busscher, J. Dent. Res., 68 119891 463. [28] C.E. Christersson, R.G, Dunford, P.-O. Glantz and R.E. Baier, Scand. J. Dent. Res., 97 11989i 247. [29] C.E. Christersson and P.-O. Glantz, Stand. J. Dent. Res.. 100 (1992) 98. [301 R.J. Gibbons and D.I. Hay, Infect. Immun., 5611988)439. [31] M. Rykke and G. R611a, Stand. J. Dent. Res., 98 (1990) 40 I.