Modulation of bacterial binding to salivary pellicle by treatment with hydrophilizing compounds

Archs oral Bid. Vol. 35, Suppl., pp. 137%14OS, 1990 Printed in Great Britain. All rights reserved

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MODULATION OF BACTERIAL BINDING TO SALIVARY PELLICLE BY TREATMENT WITH HYDROPHILIZING COMPOUNDS J. OLSSON,‘*A. CARLBNIand K. HOLMBERG~ ’ Department of Cariology, Faculty of Odontology, Giiteborg and *Berol Nobel AB, Stenungsund, Sweden Summary-Two hydrophilizing agents, a branched polyethylene glycol derivative and a non-ionic cellulose ether (EHEC) bind to buffer-treated hydroxyapatite and prevent attachment of Streptococcus mufans. EHEC gives a more efficient surface modification, presumably due to a more complete surface coverage. Neither of the 2 hydrophilizing agents were effective on hydroxyapatite which had been pre-treated with saliva. In a small clinical trial, EHEC was found to be moderately effective in preventing plaque formation. Key words: salivary pellicle, bacterial adhesion, hydrophilizing agent, Streptococcus

INTRODUCTION Chemical modification of the tooth surface may cause a release of receptor molecules from the pellicle or prevent them from binding. The receptor function may also be disturbed by chemical treatment such that the bacteria-binding properties are destroyed. Indeed chemical modification of the tooth surface offers an attractive, non-bactericidal basis for preventing dental plaque formation. Non-charged, hydrophilic surfaces typically display low bioadhesiveness and both proteins and cells are effectively rejected (Golander et al., 1986; Humphries et al., 1987). Polyethylene glycol derivatives and polysaccharides are examples of agents forming highly hydrophilic surfaces. Our working hypothesis has been that a densely packed hydrophilic layer of this type on the tooth surface would impart bacteria-rejecting properties, provided a virtually permanent surface modification could be achieved. We have now investigated two polymeric compounds for hydrophilizing the tooth surface. A branched polyethylene glycol derivative with phosphate end-groups and a non-ionic cellulose ether (EHEC) were studied for adsorption to hydroxyapatite and their effect on the adherence of Streptococcus mutans to hydroyapatite. The effect of EHEC on plaque formation in vivo was also studied. MATERIALSANDMETHODS Microorganisms

A fresh isolate of a hydrophobic Strep. mutans [strain LK, serotype c (Westergren and Olsson, 1983)], later confirmed as serotype e (8 Hamada, personal communication) was used. Tritium-labelled bacteria were obtained by spreading a loopful of *Address correspondence to: Dr Jan Olsson, Department of Cariology, Faculty of Odontology, Box 33070, 400 33 Goteborg, Sweden. Abbreviufion : EHEC, ethyl hydroxyethyl cellulose.

mutans.

bacteria in 100~1 of a [methyl-3H]-thymidine solution, 0.5 mCi/ml (Amersham, Poole, England) on a blood agar plate. The cells were harvested after 16 h of anaerobic growth at 37”C, washed twice in 1.0 mmol/l potassium phosphate buffer with 50 mmol/l KCl, 1.0 mmol/l CaCl, and 0.1 mmol/l MgCl,, pH 6.5 and adjusted to an optical density of 0.35 (A,,,nm), which corresponds to 5 x 10’ cells per ml. Saliva

Parotid saliva, lightly stimulated with citric acid, was collected via Lashley cups into an ice-chilled tube, early in the morning, immediately before use, and diluted 1: 1 with potassium phosphate buffer in the assay. Saliva from two donors was used, one of which supported binding of Strep. mutans (donor No. 1) and one which did not (donor No. 2). Hydrophilizing agents

The polyethylene glycol derivative was synthesized by propoxylation (130 mol of propylene oxide) and ethoxylation (25 mol of ethylene oxide) of glycerol using KOH as initiator, followed by introduction of phosphate groups with polyphosphorous acid. “Clabelled material was made by using radioactive ethylene oxide. The compound was applied as a 2.5% solution of a mixture of isopropanol (15 parts) and water (85 parts). The cellulose derivative, EHEC, had a cloud point of 37°C in distilled water and 35°C in saliva in 1% solutions. EHEC was labelled by methylation with [ i4C]-methyl chloride. The EHEC derivative was applied as 0.5% and 0.1% solutions in water. Bacterial adherence to hydroxyapatite beads

The effect of the test substance was investigated by measuring the binding of [ 3HI-bacteria to hydroxyapatite beads (BDH Chemicals Ltd, Poole, England) first treated with buffer or saliva, followed by treatment with the test substance. The effect of treating with the test substance before saliva was also investigated. Numbers of unbound bacteria and bacteria

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binding to the tube walls were determined by scintillation counting. A detailed description of the experimental procedure has been given by Olsson, Carlen and Holmberg (1990).

Donor No 1

x

Pilot clinical trial

The efficacy of EHEC in preventing plaque formation was compared with that of a placebo solution. The trial had a double-blind cross-over design and 7 dental students (4 males and 3 females) were randomly assigned to treatment with either test or placebo. One millilitre of paraffin-stimulated whole saliva was collected for microbiological analysis. After disclosing with erythrosine (Diaplac Rondell’, Astra Sweden), the teeth were cleaned carefully with pumice and dental floss. The subjects were instructed to avoid all oral hygiene for the next four days and asked to rinse their mouths for 2 min 3 times daily with the contents of a coded bottle. The bottles contained either a 0.2% solution of EHEC or 0.2% Tween 20 in water. Tween 20 was chosen as placebo because it had foaming characteristics similar to EHEC. At the end of the rinsing period 1 mg of plaque was collected from the buccal surface of the maxillary molars and premolars and transferred to RTF transport fluid (Syed and Loesche, 1972) for microbiological examination. After disclosing with erythrosine, intraoral colour photographs were obtained from the buccal surfaces of the maxillary canines, lateral and central incisors. Evaluation of the effect on plaque formation was made by a panel of 9 observers who independently compared pairs of photographs without knowing the sequence of treatment. The examiner had to decide whether the teeth in the first series had more (scores + 2 and + 1) or less (scores- 1 and -2) plaque than the teeth in the second series.

RESULTS

Polyethylene glycol derivative

Figure 1 shows the effect of the derivative on Strep. to buffer- and saliva-treated hydroxyapatite. Two different salivas were used, one promoting bacterial binding (No. 1) and one nonpromoting (No. 2). The effect of hydrophilization was considerable in the buffer system and largely retained also after post-addition of saliva. However, when saliva was added before the polyethylene glycol derivative, inhibition of adherence did not occur on the hydroxyapatite surface. Adhesion of Strep. mutans on the tube surface was effectively prevented by the derivative regardless of the order of addition. mutans adherence

‘;’ .i? 2

100 80

x g

60

2g

40

8 &

20

m

a 0

E3u

S

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A

B

C

S

-

A

B

C

Donor No 2

3100

2

g

80

z z$ m

60

;

40

8 b a

20 0

Bu

Fig. 1. Adherence of Strep. mutans to hydroxyapatite treated with buffer (Bu), saliva (S), polyethylene glycol (PEG) derivative (A), PEG/saliva (B) and saliva/PEG (C).

The amount of polyethylene glycol derivative adsorbed on the hydtoxyapatite and on the tube surface was determined using “C-labelled compound. The result is shown in Table 1. When the hydrophilizing agent was added before saliva approximately 20% (equivalent to 5 mg) of the amount added in the assay bound to 40 mg of hydtoxyapatite. This value is only slightly lower than the control, i.e. hydroxyapatite without post-treatment with saliva. However, 10 times less polyethylene glycol derivative bound when the hydroxyapatite had been pre-treated with saliva. Only small amounts of the derivative bound to the tube walls. Pre-treatment with saliva enhanced binding to the tube. Cellulose derivative

The effect of EHEC on Strep. mutans adherence is shown in Fig. 2. Only the adherence-promoting saliva (No. 1) was used in these experiments. As can be seen,

Table I. Binding of “C-labelled polyethyleneglycol (PEG) derivative to hydroxyapatite and tube walls in buffer or treated with PEG followed by parotid saliva (PEG-PS) or in the reverse order (PS-PEG) at room temperature Saliva No. 1 Hydroxyapatite Buffer PEG-PS PS-PEG

6 k 0.2* 5 f 0.2 0.5 f 0.2

Wall 0.5 + 0.01 0.5 * 0.1 1 L-0.03

Saliva No. 2 -~ Wall Hydroxyapatite 6 + 0.2 6 + 0.2 0.3 + 0.01

0.5 + 0.08 0.5 &-0.03 1 + 0.3

*Values are mg PEG derivative (mean k SEM) bound to 40 mg hydroxyapatite or tube wall.

Modulation of bacterial binding to teeth

100

m

Wall

m

Unbound

0

Hydroxyapatlte

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Table 3. Effect on plaque formation of rinsing four days with 0.2% EHEC (test) or 0.2% Tween 20 (placebo)

-bound

-bound

Subjects

Less plaque after:

1 2 3 4

Test Test Test Test

5 6 7

Test Placebo Placebo

18°C t

Plaque score (mean difference) 0.1 0.8 1.1 0.7 0.8 1.1 0.7

much adsorption as the 0.1% solution. Rinsing with EHEC gave a reduction in plaque score in 5 out of the 7 test subjects (Table 3). There were no marked differences between test and placebo in the numbers of bacteria that could be cultured from plaque and saliva samples obtained at the end of the mouthrinsing periods.

DISCUSSION

-

l3u

s

-

A

B

C

-

Eu

S

Fig. 2. Adherence of Strep. mutans to hydroxyapatite treated with buffer (Bu), saliva (S). EHEC 0.5% (A), EHEC/saliva (B) and saliva/EHEC (C) at 18°C and 37°C respectively.

EHEC at 0.5% concentration was very effective in preventing binding of bacteria, both to hydroxyapatite and to the tube surface, provided that saliva was absent during the hydrophilization. A good effect was also obtained when EHEC was applied as a 0.1% solution (data not shown). Like the polyethylene glycol derivative EHEC was not effective when hydroxyapatite was treated with saliva before application of EHEC. Bacterial adherence to the tube walls was almost negligible under conditions tested. Table 2 shows the results from studies of EHEC adsorption using “C-labelled material. Approximately the same amount of EHEC adsorbed to the surfaces regardless of whether saliva was absent, post- or pre-added. The temperature did not significantly affect the amount adsorbed. The higher concentration of EHEC gave a higher loading of material on the surface (data shown only for 0.25% EHEC). This was particularly the case for the hydroxyapatite surface, the 0.5% solution giving about 5 times as

Ethyl hydroxyethyl cellulose (EHEC) is a non-ionic cellulose ether that consists of a cellulose backbone substituted with ethyl groups and short oligoethylene glycol chains. Like many other polymers containing oxyethylene chains, EHEC shows a reversed temperature-dependent phase behaviour. Hence, at low temperatures aqueous EHEC solutions are clear, isotropic l-phase systems, whereas at higher temperatures the polymer becomes water insoluble and separation into 2 liquid phases occur. As the 2-phase system scatters light, the temperature at which phase separation occurs is easily detectable and is commonly referred to as the cloud point. The cloud point of EHEC is governed by the molecular weight and by the degree of substitution of the two substituents. It has previously been found that on hydrophobic surfaces adsorption of EHEC is at maximum at around the cloud point. Furthermore, EHEC when adsorbed on surfaces retains its hydrophilic and repellent properties at temperatures up to at least 5°C above the cloud point (Malmsten et al., 1990). We have used EHEC with a cloud point in saliva of 35’C. Such a polymer will be completely water soluble at ambient temperatures, i.e. at the temperature of application, for instance as a mouth rinse. In the mouth the cloud point of the polymer is approached and adsorption on the tooth surface will take place. When adsorbed, the EHEC molecule is likely to orientate its hydrophilic oligo-ethylene glycol chains towards the water phase and provide protein-rejecting properties to the tooth surface. Thus, we had

Table 2. Binding of “‘C-1abelled EHEC (0.5%) to hydroxyapatite and tube walls at 18’C and 37°C in buffer or treated with EHEC followed by_ parotid saliva (EHEC-PS) or in the reverse order (PS-EHEC) _ Hydroxyapatite Buffer EHEC-PS PS-EHEC

0.2 * o.o+ 1 + 0.003 0.3 * 0.01

18°C

Hydroxyapatite

37°C

0.2 f 0.0 0.05 * 0.003 0.1 + 0.008

*Values are mg EHEC (Mean ri: SEM) bound to 40 mg hydroxyapatite

Wall 18°C

Wall 37°C

0.2 f 0.0 0.2 * 0.009 0.2 + 0.002

0.3 + 0.0 0.2 + 0.003 0.3 & 0.02

or tube wall.

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expected the layer of EHEC to be more densely packed when adsorbed at 37°C which is slightly above the cloud point, than when adsorbed at 18°C. The adsorption at 37°C would then lead to the best results in terms of prevention of bacterial adhesion. However, neither the measurements of EHEC adsorption nor the determinations of Strep. mutans adherence showed indications of a temperature dependence. The results with both the polyethylene glycol derivative and EHEC show that non-charged hydrophilic agents can prevent bacterial attachment to buffer-treated hydroxyapatite. EHEC was particularly effective in interfering with bacterial binding to hydroxyapatite post-treated with saliva. When hydroxyapatite was first treated with saliva neither of the two hydrophilizing compounds gave any effect. There are two possibilities to account for the reduced effect of the polyethylene glycol derivative when saliva is post-added. The binding of the hydrophilizing agent to the hydroxyapatite surface may be of such low affinity that significant amounts are replaced by salivary components, which can bind to apatite with high affinity (Ericson, 1968). Alternatively, salivary components bind directly to the hydrophilized surface, presumably to patches not covered by the hydrophilizing agent. The results from the studies with labelled material indicate that the polyethylene glycol derivative is not replaced by salivary components. This indicates that adsorption of that derivative to hydroxyapatite does not give a complete surface coverage. EHEC seems to give a more efficient surface modification because adherence to the EHEC-treated hydroxyapatite surface was not affected by post-addition of saliva. Neither of the 2 hydrophilizing agents seemed capable of replacing salivary components already attached to the apatite surface. It is noticeable that

EHEC evidently binds onto the layer of salivary constituents, unfortunately without preventing bacterial adhesion. The inability of EHEC to function on hydroxyapatite previously treated with saliva is reflected in the relatively poor result obtained with this compound in the pilot clinical study. investigation was supported partly by the Swedish Medical Research Council (project Acknowledgements-This

No. 8697) and the Swedish National Board for Technical Development (project No. 89-01945P). REFERENCES Ericson T. (1968) Salivary glycoproteins, composition and adsorption to hydroxylapatite in relation to the formation of dental pellicles and calculus. Acta odont. stand. 26, 3-21.

Giilander C.-G., Jiinsson S., Vladkova T., Stenius P. and Eriksson J. C. (1986) Preparation and protein adsorption properties of photopolymerized hydrophilic films containing N-vinylpyrrolidone (NVP), acrylic acid (AA) or ethyleneoxide (EO) units as studied by ESCA. Colloids Surfaces 21, 149-165. Humphries M., Jaworzyn J., Cantwell J. and Eakin A. (1987) The use of nonionic ethoxylated and propoxylated surfactants to prevent the adhesion of bacteria to solid surfaces. FEMS Microbial. L..etts 42, 91-101. Malmsten M., Claesson P., Pezron E. and Pezron I. (1990) Temperature dependent forces between hydrophobic surfaces coated with ethyl(hydroxyethyl)llulose. (To be published.) Olsson J., CarlCn A. and Holmberg K. (1990) Inhibition of Streptococcus mutans adherence by means of surface hydrophilization. J. dent. Res. (In press). Syed S. A. and Loesche W. J. (1972) Survival of human dental plaque flora in various transport media. Appl. Microbial.

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Westergren G. and Olsson J. (1983) Hydrophobicity and adherence of oral streptococci after repeated subculture in vitro. Infect. Immun. 40, 432-435.