Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH.

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

An in vitro study of salivary film formation at solid/liquid interfaces

The aim of this study was to use the technique of in situ null ellipsometry to study some aspects of salivary film formation at solid/liquid interfaces. Experiments were performed in a fused quartz cell, and hydrophilic plasma cleaned silica and methylated hydrophobic silica surfaces were used as substrates. Samples of unstimulated whole saliva were allowed to adsorb on the test surfaces for 30 min. After the adsorption period, rinsing was performed for 5 min. Recordings were continued for another 30 min, and then new saliva samples were added in the cell. The results showed that statistically significant higher amounts were adsorbed on the hydrophobic than on the hydrophilic surfaces. The adsorbed films on both types of tested substrates consisted of loosely associated parts which were removed after rinsing and of more tightly adsorbed fractions. A significantly larger fraction was desorbed from the films adsorbed on the hydrophobic than on the hydrophilic surfaces. When saliva was introduced again in the cell, it was shown that the amounts adsorbed attained the values obtained before the rinsings. Increase in the concentration of saliva in the cell without previous rinsing did not cause any further increase in the mass of the adsorbed film.     

Adsorption from salivary fractions at solid/liquid and air/liquid interfaces.

Ellipsometry and the drop-volume technique were used to study the interfacial behaviour of fractions obtained from unstimulated whole saliva. Fractionation was by gel filtration on a Superdex 200 Hiload column equilibrated with 10 mM potassium phosphate buffer, pH 6.8, containing 0.15 M NaCl. The fractions were reconstituted to have the same absorbance at 215 nm (estimated molecular-weight range, F1 greater than 760-460 K, F2 205-39 K, F3 14-4.5 K, F4 4.5-2.5 K, F5 1.5-0.85 K, F6 0.85 less than or equal to 0.5 K). The fractions were analysed for amino acid composition and studied by hydrophobic interaction chromatography on a Phenyl-Superose column. Fraction 3 contained the largest amounts of proline, followed by fractions 4 and 2. Fraction 3 showed the highest relative hydrophobicity. Ellipsometric measurements on negatively charged silica surfaces and methylated hydrophobic surfaces revealed that larger amounts of material adsorbed on hydrophobic than on hydrophilic surfaces. On hydrophilic surfaces the largest amounts were adsorbed from the high molecular-weight fraction 1. Fractions 4 and 6 did not give any adsorption at all on these surfaces. Fraction 3 gave the largest amounts adsorbed on the hydrophobic surfaces. Drop-volume measurements showed distinct differences in the ability of the salivary fractions to lower the surface tension. Fractions 2 and 3 showed the greatest reduction in surface tension. It was concluded that the adsorption behaviour of salivary proteins showed a wide variation among the different fractions and that it is influenced by the physicochemical characteristics of the interfaces present in the mouth.     

Quantitative and qualitative analyses of human salivary micelle-like globules.

In the present study we examined the protein proportion and amino acid profile of the salivary micelle-like globules (SMGs) of human whole saliva and parotid saliva (HWS, HPS). Saliva and SMG samples from each subject (clarified HWS and HPS from 6 subjects, and unclarified HWS from 3 subjects) were analysed for amino acids using standard acid hydrolysis procedures. HPS, clarified HWS and the respective supernatant samples (remaining after removal of the SMGs) were also measured for protein using the micro-Kjeldahl method. SMGs from clarified and unclarified HWS made up 4.7% and 19.7%, respectively, of the total salivary protein based on amino acid analyses. With the micro-Kjeldahl method SMGs from clarified HWS made up 7.3% of the total saliva protein. SMGs isolated from HPS were found in only small amounts. The amino acid profile for the SMGs was strikingly similar to that known for the 2-h pellicle, and differed significantly from HWS or HPS. The results support previous morphological studies indicating that the SMGs represent a major component of the newly formed pellicle.     

Protein and mucin retention on oral mucosal surfaces in dry mouth patients

Pramanik R, Osailan SM, Challacombe SJ, Urquhart D, Proctor GB. Protein and mucin retention on oral mucosal surfaces in dry mouth patients. Eur J Oral Sci 2010; 118: 245–253. © 2010 The Authors. Journal compilation©2010 Eur J Oral Sci Oral homeostasis depends largely on proteins and mucins present in saliva that coat all oral surfaces. The present study compared the protein composition of residual fluid on mucosal surfaces in subjects with normal salivary flow with that of patients with dry mouth caused by salivary hypofunction. Samples of residual mucosal fluid were collected using paper strips and then analysed by protein electrophoresis and immunoblotting. In both patients and controls, residual fluids on mucosal surfaces (except the anterior tongue in control subjects) had higher protein concentrations than unstimulated whole‐mouth saliva. High‐molecular‐weight mucin (MUC5B) was present in greater amounts on the anterior tongue than on other surfaces in control subjects. In dry mouth patients who were unable to provide a measurable saliva sample, MUC5B was often still present on all mucosal surfaces but in reduced amounts on the anterior tongue. The membrane‐bound mucin, MUC1, was prominent on buccal and labial surfaces in patients and controls. Statherin was still present on surfaces that were dried to remove salivary fluid, suggesting that it may be adsorbed as a protein pellicle. It is concluded that oral mucosal surfaces in dry mouth patients can retain MUC5B and other salivary proteins, although the functional integrity of these proteins is uncertain.     

Interactions of chlorhexidine with salivary films adsorbed at solid/liquid and air/liquid interfaces

Chlorhexidine is a cationic compound which has been shown to bind to salivary proteins and enamel through electrostatic interactions. The aims of this study were to investigate the interaction of chlorhexidine molecules with salivary films absorbed on solid surfaces with varying physico-chemical characteristics and to investigate the effect of different concentrations of chlorhexidine on the surface tension of saliva. The interactions between 0.2% chlorhexidine digluconate with films adsorbed from whole saliva were monitored by a Rudolph Thin-film ellipsometer equipped with a He-Ne laser (632.8 nm). The films were adsorbed on hydrophilic silica surfaces which were plasma cleaned or on methylated hydrophobic surfaces. Experiments of chlorhexidine adsorption on bare surfaces were also performed. The surface tension of mixtures of whole saliva with various concentrations of (0.1%, 0.2%, 1%) chlorhexidine was monitored with a tensiometer. The results show that chlorhexidine adsorbs on both types of studied substrates. Addition of the substance followed by rinsing caused a partial desorption of the adsorbed pellicles. Furthermore, at all studied concentrations chlorhexidine reduced the interfacial tension. There are indications that the amphiphilic characteristics of the molecule play an important role in the retention of the substance in the oral cavity.     

Modulation of herpes simplex virus type 1 replication by human salivary secretions

Saliva functions to protect the oral cavity from pathogenic invasion by modulating the ability of microbes to colonize the oral surfaces or limiting their growth and/or viability. Although the role of salivary secretions in the modulation of the oral bacteria flora has received considerable attention, little is known concerning its role in viral pathogenesis. Accordingly, the purpose of this study was to assess the effect of salivary secretions on herpes simplex virus type 1 (HSV-1) replication. Initially, HSV-1 plaque and liter reduction assays were performed to determine the ability of human submandibular/sublingual (HSMSL) and parotid (HPS) salivas to inhibit the early stages of HSV-1 infection (adsorption and penetration). Our results suggested that both HSMSL and HPS possess cell-protective and virus neutralization activities, with HSMSL being more active than HPS. Additional experiments were performed to determine the effect of saliva on the yield of virus progeny. Again, HSMSL caused a greater reduction of HSV-1 replication than did HPS. A similar effect could not be obtained using vaccinia, suggesting that this inhibitory activity of human saliva is selective. Collectively, these results suggest that human salivary secretions can modulate the replication of HSV-I in vitro.     

Adsorption of human salivary mucin MG1 onto glow-discharge plasma treated acrylic resin surfaces

It has been suggested that altering the surface properties of acrylic resin material may change the nature of the adsorbed pellicle affecting denture retention and microbial adherence. This study aimed at evaluating the adsorption of salivary high molecular-weight mucins, a major component of denture pellicle, onto modified acrylic resin surfaces. (Poly) methylmethacrylate specimens were treated by glow discharge plasma technique, using hydrophilic 2-Hydroxyethylmethacrylate monomer or oxygen (O(2)) gas and hydrophobic Hexamethyldisiloxane monomer, at different discharge powers. Acrylic samples were incubated with high-molecular weight mucin, MG1 purified from saliva, the adsorbed fractions were transferred to nitrocellulose membranes by slot-blot technique, stained by periodic acid-Schiff and colour intensities were analysed by a colour densitometer. Higher amounts of mucins were adsorbed on all the surfaces modified by glow-discharge plasma treatment. Within the limitations of this study, it was concluded that glow-discharge plasma altered the surfaces of acrylic resin denture base materials and significantly increased the adsorption of high molecular-weight mucins at varying levels depending on plasma parameters.     

Multiple components contribute to ability of saliva to inhibit influenza viruses

Introduction:  Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied.
Methods:  We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro .
Results:  Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study.
Conclusions:  These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.     

Measurement of adsorption of salivary proteins onto soft denture lining materials.

STATEMENT OF PROBLEM: The adhesion of Candida albicans to soft liners is a major causative factor in denture stomatitis. It has been suggested that salivary proteins play an important role in this candidal adhesion. PURPOSE: This study measured the adsorption of salivary proteins on soft liners. MATERIAL AND METHODS: Five commercial materials and 1 experimental material were immersed in saliva, albumin solution, or milk. Proteins adsorbed on the material surfaces were measured by ATR/FT-IR spectroscopy. RESULTS: The amount of proteins adsorbed to the 6 materials varied considerably. Protein adsorption was significantly lower in the experimental fluoropolymer and polyphosphazene, and higher in acrylic resin and silicone. CONCLUSION: Different soft liners promote adsorption of varying amounts of protein.     

In vitro interactions of anionic and cationic surfactants with salivary fractions on well-defined solid surfaces.

Ellipsometry was used to study the interaction of one anionic (SDS) and one cationic (CTAB) surfactant with films adsorbed from six different salivary fractions obtained after fractionation of whole unstimulated saliva on a Superdex 200 Hiload gel filtration column. Experiments were performed on both hydrophilic silica and hydrophobic methylated silica surfaces. The results of this study indicate that the adhesive and cohesive properties of the films adsorbed from the individual fractions were strongly dependent on the surface characteristics of the substrates and that the outcome of protein/surfactant interactions was dependent on factors such as protein composition, surfactant charge, and substrate characteristics. These interactions probably involve replacement of the adsorbed proteins by surfactants or protein/surfactant complex formation. The anionic surfactant seemed to be more efficient in removing adsorbed salivary proteins than the cationic one.     

Influences of animal mucins on lysozyme activity in solution and on hydroxyapatite surfaces

The purpose of this study was to investigate the influence of animal mucins on lysozyme activity in solution and on the surface of hydroxyapatite (HA) beads. The effects of animal mucins on lysozyme activity in solution were examined by incubating porcine gastric mucin (PGM) or bovine submaxillary mucin (BSM) with hen egg-white lysozyme (HEWL) or salivary samples. HA-immobilised animal mucins or lysozyme were used to determine the influence of animal mucins on lysozyme activity on HA surfaces. Lysozyme activity was determined by turbidity measurement of a Micrococcus lysodeikticus substrate suspension. Protein concentration was determined by ninhydrin assay. PGM inhibited the activity of HEWL and salivary lysozyme in solution. The amount of inhibition was dependent on mucin concentration, incubation time and temperature, and the structural integrity of the mucin. The inhibition of salivary lysozyme activity by PGM was greater in submandibular/sublingual saliva than in parotid saliva. The inhibition of lysozyme activity by PGM was markedly dependent on pH. However, BSM did not inhibit the in-solution lysozyme activities of HEWL and clarified saliva. Both PGM and BSM bound to HA surfaces, and HA-adsorbed animal mucins increased the subsequent adsorption of lysozyme. When HA beads were exposed to a mixture of HEWL and PGM or BSM, lysozyme activity on the HA surfaces was significantly increased. The results suggest that animal mucins affect lysozyme activity, and the effects are different on HA surfaces compared with in solution. Further research is needed to determine the effect of animal mucins on lysozyme activity in vivo.     

Salivary protein adsorption and Streptococccus gordonii adhesion to dental material surfaces

Objectives

The initial adhesion of microorganisms to clinically used dental biomaterials is influenced by physico-chemical parameters like hydrophobicity and pre-adsorption of salivary proteins. Here, polymethyl methacrylate (PMMA), polyethylene (PE), polytetrafluoroethylene (PTFE), silicone (Mucopren soft), silorane-based (Filtek Silorane) and methacrylate-based (Tetric EvoCeram) dental composites, a conventional glassionomer cement as well as cobalt–chromium–molybdenum (Co28Cr6Mo) and titanium (Ti6Al4V) were tested for adsorption of salivary proteins and adhesion of Streptococcus gordonii DL1.

Methods

Wettability of material surfaces precoated with salivary proteins or left in phosphate-buffered saline was determined by the measurement of water contact angles. Amounts of adsorbed proteins were determined directly on material surfaces after biotinylation of amino groups and detection by horseradish peroxidase-conjugated avidin-D. The same technique was used to analyze for the binding of biotinylated bacteria to material surfaces.

Results

The highest amount of proteins (0.18 μg/cm²) adsorbed to hydrophobic PTFE samples, and the lowest amount (0.025 μg/cm²) was detected on silicone. The highest number of S. gordonii (3.2 × 10⁴ CFU/mm²) adhered to the hydrophilic glassionomer cement surface coated with salivary proteins, and the lowest number (4 × 10³ CFU/mm²) was found on the hydrophobic silorane-based composite. Hydrophobicity of pure material surfaces and the number of attached microorganisms were weakly negatively correlated. No such correlation between hydrophobicity and the number of bacteria was detected when surfaces were coated with salivary proteins.

Significance

Functional groups added by the adsorption of specific salivary proteins to material surfaces are more relevant for initial bacterial adhesion than hydrophobicity as a physical property.     

Adsorbed salivary acidic proline-rich proteins contribute to the adhesion of Streptococcus mutans JBP to apatitic surfaces

Experimental pellicles formed on hydroxyapatite (HA) beads from parotid or submandibular saliva promoted the adhesion of Streptococcus mutans JBP cells to a greater extent than did pellicles prepared from buffer, human plasma, or serum. The nature of the salivary components responsible was studied by the preparation of pellicles from fractions of parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Two groups of fractions promoted attachment of the organism. Components migrating in the high-molecular-weight mucin fraction were most effective, but a later-eluting fraction also possessed adhesion-promoting activity. Subfractionation of the latter material indicated that the adhesion-promoting activity was associated with the acidic proline-rich proteins (PRPs). Pellicles prepared from 10-20-micrograms/mL solutions of pure PRP-1 were effective in promoting attachment of S. mutans JBP cells. PRP-3 was less effective, while human salivary statherin, fibrinogen, fibronectin, type 1 collagen, and the amino-terminal tryptic peptide derived from PRP-1 were ineffective. The quantities of 150-residue and 106-residue PRPs and of statherin, which became incorporated into experimental pellicles prepared from saliva, were estimated with use of radiolabeled protein tracers. The data obtained suggest that these proteins compete for similar binding sites on HA, and that their ratios in saliva would therefore influence the quantity of the larger PRPs that become incorporated into the pellicle. Such competition may contribute to the variability observed in the adhesion-promoting activities of different saliva samples.     

On the rôle of human salivary micelle-like globules in bacterial agglutination

The hypothesis to be tested in this in –vitro study was that the salivary micelle like globules (SMGs) have a rôle in the agglutination of some oral bacteria. An attempt to determine the mechanisms for the interactions involved was also carried out. 4 laboratory and 4 native streptococci strains were tested. Human whole (HWS) and parotid (HPS) saliva was collected from 4 subjects, and SMGs were isolated from both salivas, and agglutination was recorded in the various bacterial suspensions over time. HPS, HWS and SMGs isolated from HPS and HWS caused typical agglutination patterns for the mutans strains. Salivary supernatants (without SMGs) caused a much delayed or no agglutination. Electron microscopy showed SMG-like structures on the surface of the agglutinated bacteria. Addition of pyrophosphate to HPS prevented agglutination, whereas guanidine HC1 prevented normal agglutination of a sanguis strain, and urea had no obvious effect. Together, these results indicate that the SMGs are important in the agglutination of streptococci, and that both calcium-dependent, electrostatic and hydrophobia interactions may be involved.     

Effect of salivary biofilm on the adherence of oral bacteria to bleached and non-bleached restorative material.

OBJECTIVE: The objective of this work was to examine the effect of in vitro salivary biofilm on the adherence of oral bacteria to bleached and non-bleached restorative material (Charisma). METHODS: Charisma samples, prepared in silicon models, were treated with either 10% carbamide peroxide (CP) or 10% hydrogen peroxide (HP). After incubation with the bleaching agent for a period of one, two or three days, the samples were coated with freshly collected human saliva. The adsorption pattern of the saliva to the restorative material was determined using gel electrophoresis coupled with computerized densitometry techniques. The amount of salivary proteins adsorbed onto the treated surfaces was measured using the Bradford method. Sucrose-dependent bacterial adhesion to the salivary-coated Charisma was tested using radio-labeled Streptococcus mutans, Streptococcus sobrinus and Actinomyces viscosus. Adhesion of each bacterium to surfaces pretreated with the bleaching agents was compared with saliva coated bleached surfaces. RESULTS: The profile of salivary proteins adsorption followed a similar pattern in Charisma samples pretreated with either CP or HP or untreated samples. However, the total amount of salivary proteins adsorbed onto the samples decreased after bleaching with CP or HP. Salivary biofilm, coating the surface of the restorative material, significantly decreased sucrose-dependent adhesion of Streptococcus sobrinus and Streptococcus mutans to the bleached and non-bleached surfaces, compared to non-coated specimens (p < 0.05). Saliva had a minor effect on adhesion of Actinomyces viscosus. SIGNIFICANCE: Our study demonstrates the importance of salivary biofilm in controlling adhesion of oral bacteria to restorative material pretreated with bleaching agents or untreated.     

Human minor and major gland saliva proteins and ability to mediate Actinomyces naeslundii adherence

Bacteria-binding components and the ability to mediate bacterial adhesion to the tooth surface have been thoroughly studied in major salivary gland secretions. Our knowledge on the bacteria binding activity in minor gland saliva is, however, limited. In this study, proteins were examined in parallel in minor (palatal, buccal and labial) and major (parotid and submandibular/sublingual) salivary gland secretions in one subject using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The adherence of early colonizing Actinomyces naeslundii to pellicles formed from the secretions on hydroxyapatite beads was also examined. Amylase, IgA, proline-rich proteins and the high-molecular-weight glycoproteins, agglutinins, were detected in all saliva tested. Carbohydrate-reactive antibodies recognized the low-molecular weight mucin, MUC 7 in submandibular/sublingual saliva only. A. naeslundii strain 12104 adhered to all pellicles and especially to the buccal gland saliva pellicles. Strain LY7 adhered in highest numbers to the submandibular/sublingual saliva pellicles. It also bound in considerable numbers to parotid and palatal saliva pellicles but not to the ones formed from buccal and labial gland saliva. Our findings indicate that several bacteria-binding components are secreted in both minor and major gland saliva. The adherence-promoting ability of the various gland secretions differs, however.     

Analysis of residual saliva and minor salivary gland secretions in patients with dry mouth

The importance of oral mucosal wetness in the condition of dry mouth and the role of salivary proteins in proper oral function are acknowledged. A negative correlation between mucosal wetness and the protein concentration of residual saliva has been reported in normosalivators. Here, to examine the suggestion that a reduction in residual salivary volume leads to a concomitant elevation of its protein concentration, the amount of residual saliva and minor salivary gland secretions, and their protein concentrations, were measured in hyposalivators and normosalivator controls. A Periotron 8000 micro-moisture meter was used to measure the thickness of the mucosal film at six selected mucosal surfaces and the minor salivary gland secretion rate at two mucosal surfaces. The unstimulated whole salivary flow rate was measured by the spitting method. The total protein concentration of all salivary samples was measured by bicinchoninic acid assay. The hyposalivators had significantly lower amounts of residual saliva and minor salivary gland secretions than the normosalivators at all selected mucosal sites except the soft palate. In both groups, the site with the thinnest coat of residual saliva was the anterior hard palate and the wettest site was the anterior dorsal mucosa of the tongue. The protein concentration of residual saliva was significantly higher in hyposalivators than normosalivators. In the minor salivary gland secretions there was no significant difference in protein concentration between the normo- and hyposalivators. When the hyposalivators were divided into two subgroups according to their severity of dryness, the reduction of residual salivary volume and the elevation of protein concentration were more apparent in the group with the more severe dry mouth. Collectively, these results indicate that oral mucosal wetness is associated with the flow rate of unstimulated whole saliva. The function of the minor salivary glands was less affected and relatively well preserved in patients with dry mouth. The increased protein concentration of residual saliva in the hyposalivators appeared to be the result of decreased salivary volume.     

Adsorption from black tea and red wine onto in vitro salivary pellicles studied by ellipsometry

The adsorption of black tea and red wine components onto a pellicle-like protein layer formed in vitro by adsorption from whole unstimulated saliva on hydroxyapatite discs were studied by in situ ellipsometry. It was found that components from black tea readily adsorbed to the pellicle. Subsequent exposure to saliva led to further adsorption of salivary components to give an overall increase in the amounts adsorbed. The amounts adsorbed increased still further following a third tea and saliva exposure. Components of red wine gave significantly greater amounts of adsorption to the pellicle than black tea. The adsorption of components of black tea gave a concomitant increase in colour or stain as measured by a reflectance chromameter. In all cases, the black tea- and red wine-modified pellicles were not eluted by either phosphate buffer or sodium dodecyl sulphate (SDS) rinses. Thus, black tea and red wine components have been shown to have a profound effect on in vitro pellicle maturation, causing thickened layers of stained material to build up, which are not readily removed.