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.     

On the adsorption behaviour of saliva and purified salivary proteins at solid/liquid interfaces

Salivary proteinaceous substances are known to play important roles in the formation of the salivary pellicle. The aim of this study was to investigate some aspects of the interfacial behaviour of selected purified salivary proteins, as well as human saliva secretions, using time-resolved in situ ellipsometry. Hydrophobic methylated silica and hydrophilic pure silica were used as test substrates. Experiments were performed in vitro, preferentially in the low concentration range, with samples of fresh human whole resting saliva, parotid resting saliva and submandibular/sublingual resting saliva. The protein fractions investigated were human MUC5B, PRP-1, PRP-3 and statherin, as well as bovine submaxillary mucin (BSM). The results indicate that the amount of material adsorbed was strongly related to the protein concentration in the range investigated for both pure proteins and secretions. Generally, a larger amount of material was adsorbed onto hydrophobic surfaces than onto hydrophilic ones. However, pure PRP-1 adsorbed in similar amounts to both hydrophilic and hydrophobic surfaces in the concentration range investigated and BSM adsorbed in larger amounts at high concentrations on hydrophilic surfaces. Comparison of the observed adsorption rates for salivary secretions and calculated diffusion rates for individual proteins suggested initial adsorption of low-molecular-weight proteins/peptides. On hydrophilic surfaces the data indicate adsorption of proteins with diffusion rates corresponding to those of statherin, PRP-3 and PRP-1. MUC5B adsorbs at a later stage from both HWS and the individual secretions, which can be explained by a "Vroman effect"-like phenomenon. On hydrophobic surfaces, adsorption rates were found to be faster than those calculated for any of the proteins, and thus smaller proteins/peptides appear to be involved. The similar adsorption behaviour of PRP-1 and parotid saliva (HPS) on hydrophilic surfaces may suggest that long aPRPs account for a substantial portion of the film-forming capacity of HPS. Effects of added electrolyte could be explained by general screening effects and specific Ca2+ binding to serine phosphates in aqueous solutions, but were complex in phosphate buffer. Inter-individual differences in amounts adsorbed from HWS, HPS and HSMSLS, respectively, were not found to be statistically significant.     

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.     

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.     

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.     

Reflectometry: a new method for quantitative determinations of intraoral film formation

A simple optical method - reflectometry - is described to determine the thickness and mass of salivary films formed on solid surfaces at oral exposure. Reflectometry is based on the fact that p-polarized light is reflected with minimum intensity at an interface when the angle of incidence equals the so-called pseudo-Brewster angle. Hydrophilic and hydrophobized silica surfaces were used as substrates and the reflectometer was calibrated against a null ellipsometer. A linear relation was found between the square of the ellipsometrically measured thickness of silicon oxides on silicon substrates and the reflectometrically registered intensity of the reflected light at these surfaces. Thirty volunteers participated in the study of the thickness of films formed on the test substrates at oral exposure. The test silica surfaces were positioned in the vestibular sulcus of each test subject for periods of 1, 5, 15, 30, 60, and 120 min. The results show that films formed on hydrophobic surfaces leveled-off to a thickness value of 140 Å after 60 min. Films formed on hydrophilic surfaces, however, reached a plateau value of approximately 100 Å after only 300 min. Reflectometry seems to be an accurate and valid, yet inexpensive and quick method for quantitative investigations and thickness measurements of early salivary pellicles in large subject groups.     

The effect of ionic surfactants on salivary proteins adsorbed on silica surfaces.

The adsorption onto silicon oxide surfaces from water and 0.1 M acetate buffer containing 10% parotid saliva at 25 degrees C and 35 degrees C and at pH 6 was monitored in situ using ellipsometry. The silicon oxide surface was used as a model for dental enamel. The adsorption kinetics and the reversibility on rinsing were determined, and the desorbable fraction was found not to change after either 30 or 120 min of adsorption. Addition of sodium dodecyl sulfate after 30 or 120 min of saliva adsorption caused strong desorption. Rinsing 30 min after surfactant addition caused some redeposition if saliva was present, whereas continued desorption occurred in the absence of saliva. Cetyltrimethylammonium bromide caused either an increase or a slight decrease in the amount adsorbed when added after 30 min and 120 min, respectively. For both times, rinsing caused desorption, left the same amount adsorbed, and was not affected by the presence or absence of saliva in solution. No major effect from temperature and ionic strength was found.     

Film-forming properties and viscosities of saliva substitutes and human whole saliva

Hypo-salivation, related to medical remedies, is an increasing clinical problem. Studies report a weak correlation between subjective mouth dryness and objective sialometry. This indicates that both quantity and quality of saliva are important for the surface-associated functions of saliva, such as lubrication and hydration, to be expressed. Film-forming properties and viscosities of three saliva substitutes were compared to human saliva. Adsorption to surfaces was measured by ellipsometry, infrared spectroscopy and drop-volume technique. Viscosity measurements were carried out using an oscillating rheometer. Saliva, with the lowest viscosity value and the highest protein content, presented superior film retention on both hydrophilic and hydrophobic surfaces. The carboxymethylcellulose-based MAS 84 showed intermediate values of viscosity, poorest ability to reduce surface tension, and negligible film-forming capacity. The porcine mucin-based Saliva Orthana showed about twice the viscosity of saliva and film-forming capability on preferably hydrophobic substrates. Salinum, a linseed extract, possessed the highest viscosity value and an initial surface tension close to that of saliva. The film retention on hydrophilic surfaces was not as effective as for saliva. The results indicate that the film-forming capacity of saliva substitutes is a property also to be considered in the exploration of clinically effective artificial salivas.     

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.     

Influence of liquid films on mercury vapor loss from dental amalgam.

OBJECTIVES: The aim of this study was to determine the effect of aqueous liquid films on Hg emission from of dental amalgam. METHODS: Amalgam specimens (4 mm dia. x 4 mm long) made from ten alloys were uniformly abraded on wet ASTM 600 grit SiC paper, quickly dried and covered by liquid films of a thickness that approximated the thickness of saliva films on tooth surfaces in vivo. Distilled water and two formulations of artificial saliva were tested. After abrasion and film placement, the specimens were immediately inserted in a plastic tube which was then closed. After 30 min, the Hg vapor that had collected in the tube was measured using a Jerome 411Hg analyzer. Hg emission from abraded surfaces exposed to air was also measured and used for comparative purposes. The test results were compared using ANOVA and Tukey's contrast test (p=0.05). RESULTS: Hg emission from abraded surfaces under liquid films was one to two orders of magnitude less than Hg emission from abraded surfaces exposed to air. SIGNIFICANCE: In vitro measurement of Hg vapor emission from abraded surfaces exposed to air should not be used to estimate directly the Hg vapor release from dental amalgam restorations in vivo.     

Gas-liquid chromatographic analysis of amino acids in pellicle formed on tooth surface and plastic film in vivo

The amino acid composition of 2-h-old pellicle formed in vivo on tooth enamel and on plastic film (polyvinylchloride/polyvinylacetate) applied on tooth surfaces was studied. For comparison, saliva samples were analysed. On 5 consecutive mornings, the plastic film was applied on the buccal surfaces of 12 teeth in each of 4 subjects. After 2 h, the films were removed and material from the buccal surfaces of the 12 contralateral teeth was scraped off with a curette and pooled with material collected in the same way in the afternoon. A sample of whole unstimulated saliva was collected in the afternoon. After acid hydrolysis, N-trifluoro-acetyl-n-butyl derivatives of the amino acids were analysed by gas-liquid chromatography. The amount of protein in the pellicle samples (20–300 μg in the hydrolysates) allowed analysis to be performed well above the detection limit of the method. Thirteen amino acids were separated; the predominant ones in the film samples were in order: glutamic acid, glycine, aspartic acid, leucine and serine, and in the tooth samples: glutamic acid, glycine, serine, aspartic acid, leucine. Between film and tooth samples, statistically significant differences were observed in the amounts of glycine, isoleucine, serine and lysine. In the saliva samples, the predominant amino acids were in order: glutamic acid, glycine, proline, aspartic acid and leucine. Tyrosine and phenylalanine were in small amounts in all samples.     

Measurements of the wettability of protein-covered hydroxyapatite surfaces.

We developed a new method (dropping time method, DTM) to investigate the wettability of a surface of a protein layer adsorbed on glass plates in aqueous solution. However, the previous setup of DTM can only be utilized for optically transparent materials. In this study, we have extended the method to optically nontransparent materials such as hydroxyapatite plates. DTM is based on measuring the dropping time of a liquid film along a protein-covered surface when this surface is instantaneously vertically removed from the protein solution. The intensity of the reflected light beam depends on the presence of a liquid film on the surface. This allows to estimate the movement of the liquid film along the sorbent surface. Thus, the extended DTM can be used for determining the wettability of optically nontransparent solid plates. The adsorption behavior of four proteins (albumin, lysozyme, beta-lactoglobulin, ovalbumin) on a hydrophobic hydroxyapatite plate in water was studied by this method. When adsorbed from a protein solution of high concentration, the surfaces of adsorbed proteins, except ovalbumin, were fairly hydrophilic; this hydrophilicity was already attained at the initial stage of the adsorption process. The surface of ovalbumin on hydroxyapatite was more hydrophobic than those of the other proteins, and the hydrophilicity increased with the protein adsorption process. At low protein concentration, the hydrophilicity increased in the course of the adsorption process. The change in hydrophilicity with time depends on the kind of protein. Hen's egg lysozyme is more hydrophilic and the time to reach saturation is shorter than for the other proteins. The processes of increasing hydrophilicity of the surface of human serum albumin, beta-lactoglobulin and ovalbumin are similar. However, for beta-lactoglobulin hydrophobicity at adsorption saturation is stronger than for human serum albumin and ovalbumin. Thus, using DTM it is shown that the hydrophilicity of the surface of adsorbed protein on hydroxyapatite depends strongly on the kind of protein.     

The binding of delmopinol and chlorhexidine to Streptococcus mutans and Actinobacillus actinomycetemcomitans strains with varying degrees of surface hydrophobicity

This study evaluated the binding of chlorhexidine and the new surface-active anti-plaque agent delmopinol hydrochloride to Streptococcus mutans and Actinobacillus actinomycetemcomitans cells with various cell surface hydrophobicities. The influence of saliva concentration on the binding of these compounds was also investigated. The radiolabeled compounds were incubated with bacteria and the cells were recovered using a centrifugal filtering technique. Delmopinol had higher binding to the hydrophilic variant strains than to the hydrophobic parent strains; chlorhexidine had higher binding to hydrophobic than to hydrophilic A. actinomycetemcomitans strains and higher binding to hydrophilic than to hydrophobic S. mutans strains. The presence of salivary films decreased the binding of both compounds. Both delmopinol and chlorhexidine had stronger affinity to A. actinomycetemcomitans cells than to S. mutans cells. At equimolar concentrations, delmopinol had a lower binding to all strains tested than chlorhexidine. The high reversibility of the delmopinol binding might be related to a higher diffusion rate and solubility compared with that of chlorhexidine. The amphiphilicity of both molecules is an important feature in their retention to S. mutans and A. actinomycetemcomitans strains of varying hydrophobicities and could play an important role in the substantivity of delmopinol or chlorhexidine in the oral cavity.     

The stability of nonporous and macroporous titania thin films in aqueous electrolyte solutions

A simple electrochemical approach was used to evaluate the stability and porosity of titania and silica thin films spin coated on electrode surfaces. This approach involved monitoring the magnitude of the Faradaic current of diffusing redox probes at the modified electrode surfaces over the course of a week to 4.5 months. Relatively nonporous films were examined as well as films templated with polystyrene latex spheres. The results show that templated titania films were significantly more porous compared to non-templated films. After the defect sites in the templated films were blocked, their long-term stability in aqueous electrolyte was evaluated. For titania, blocking was done by spin coating a dilute titania sol on the top of the film whereas for silica, the film was soaked in octyltrimethoxysilane. Both types of titania films (templated and non-templated) were found to be significantly more stable than the corresponding silica films, showing no signs of deterioration in simple electrolyte solutions during the entire evaluation period. In contrast, silica films showed significant deterioration in as little as 3 days. The enhanced stability of the titania films relative to silica films in near neutral electrolyte solutions was attributed to the differences in the point of zero charge of the oxide films.     

Effect of amine/sodium fluoride rinsing on toothbrush abrasion of softened enamel in situ

The aim of this study was to test the effect of fluoride rinsing on the prevention of toothbrush abrasion of softened enamel in situ. For softening, the samples were immersed in 0.1 M citric acid (pH 3.5) for 3 min. Eight test subjects had to make 5 runs in which 4 slabs per run were attached to intraoral appliances. They were as follows: (1) no softening, no fluoride rinsing (control 1); (2) softening, no fluoride rinsing (control 2); (3) softening, rinsing in situ with a sodium/amine fluoride rinsing solution (250 ppm F) for 30 s; (4) rinsing in situ with the sodium/amine fluoride rinsing solution (250 ppm F) for 30 s, softening; (5) softening, rinsing in situ with an experimental amine fluoride-containing rinsing solution (250 ppm F) for 30 s. After exposure for 60 min to the oral milieu, the volunteers brushed the samples for 30 s with toothpaste and the loss of tooth substance was determined. For each test person, the secretion rate of resting and paraffin-stimulated saliva, buffering capacity and pH were measured. Toothbrush abrasion in situ was not significantly lower using the fluoride rinsing solutions before or after softening the enamel compared to no rinsing (p > 0.05). Multiple linear regression analyses revealed that 57% of the variation in toothbrush abrasion could be attributed to the severity of softening (p < 0.001) and the pH of stimulated saliva (p < 0.001). It was concluded that a single rinse for 30 s had no statistically significant effect on the prevention of toothbrush abrasion of softened enamel.     

Oral electrochemical action after soft drink rinsing and consumption of sweets

Abstract – The aim of this study was to investigate the influence of pH changes in dental plaque and saliva on the magnitude of the galvanic current created when amalgam restorations make contact in the oral cavity. Ten persons with 46 contacts between amalgam fillings in all participated in the experiments. Potential, polarization, and pH measurements were performed before and after Coca-Cola and orange juice rinsing and intake of sweets, which were used as test products. Distilled water was used as a control. The measurements were performed 2, 5, 10, 20, and 30 min after the rinsing or intake. There was no statistically significant difference in the current magnitude after any test product or between the time intervals after the different products. The results indicated that changes of the plaque and saliva pH for a short time after food and soft drink intake do not influence the magnitude of the galvanic current flowing between amalgam restorations in contact.     

Oral electrochemical action after soft drink rinsing and consumption of sweets

The aim of this study was to investigate the influence of pH changes in dental plaque and saliva on the magnitude of the galvanic current created when amalgam restorations make contact in the oral cavity. Ten persons with 46 contacts between amalgam fillings in all participated in the experiments. Potential, polarization, and pH measurements were performed before and after Coca-Cola and orange juice rinsing and intake of sweets, which were used as test products. Distilled water was used as a control. The measurements were performed 2, 5, 10, 20, and 30 min after the rinsing or intake. There was no statistically significant difference in the current magnitude after any test product or between the time intervals after the different products. The results indicated that changes of the plaque and saliva pH for a short time after food and soft drink intake do not influence the magnitude of the galvanic current flowing between amalgam restorations in contact.     

Effect of rinsing with ethanol-containing mouthrinses on the production of salivary acetaldehyde

It has been suggested that the use of alcohol-containing mouthrinses could lead to the presence of acetaldehyde in saliva. In this cross-over study, salivary acetaldehyde levels and microbial profiles were determined before and after rinsing with ethanol-containing mouthrinses with essential oils (EO) and cetyl pyridinium chloride (CPC) as the active ingredients, and with 21.6% ethanol and water controls. After rinsing with all ethanol-containing rinses, acetaldehyde was detected in saliva after 30 s but declined to low levels after 5 min. The highest peak levels were seen with the ethanol control (median = 82.9 μM at 2 min) and were significantly higher than those seen at the same time after rinsing with the EO rinse (43.1 μM). There was no correlation between microbial counts or plaque scores and acetaldehyde levels, although dividing the subjects on the basis of a peak acetaldehyde salivary concentration of > 90.8 μM after the ethanol rinse revealed that the high responders were highly significantly more likely to harbour salivary yeasts than were the low responders. Rinsing with ethanol-containing mouthrinses causes a rapid, but transient, increase in salivary acetaldehyde levels.     

Effect of critical surface tension on retention of oral microorganisms

Abstract – The effect of critical surface tension on the initial retention of microorganisms from unstimulated human saliva was tested in a flow cell system. Prior to each experiment the total numbers and the morphotypes of microorganisms present in saliva were recorded. The test surfaces were prepared to display known increasing critical surface tensions, as verified and standardized by contact angle measurements. Surfaces of initially low (20–22 mN/m), medium (35–38 mN/m) and high (>50 mN/m) critical surface tension were exposed to saliva at a flow rate of 1 ml/min. Microbiota and biofilm material associated with the test surface after 15 min of salivary exposure, were then subjected to standard detachment forces, by introducing a cell-free rinsing fluid at two different shear rates. Both the attachment and the detachment phases were executed at room temperature or 37°C. The retained population was counted in three different zones of the test surfaces with a light microscope and statistically tested for correlation to the main variables (critical surface tension, flow rate and temperature) and interactions. Retention success was significantly dependent on the initial critical surface tension and the flow rate. Surfaces of medium critical surface tension, representative of human tooth surfaces and most restorative dental materials, retained the highest numbers of microorganisms in comparison with the other surfaces tested, with no statistically verified selectivity in proportions of retained coccoid and rodshaped microorganisms for any surface. A 30-fold increase of the flow rate resulted in a 70–80% reduction of the retention success, with a higher relative number of cocci present on all the test surfaces. These results demonstrate that initial retention of microorganisms to surfaces is non-specific with regard to morphotypes, but is strongly related both to the mechanical removal forces and the surface energetic state of the solid surface exposed. Retention of microbial populations at interfaces might, advance selection of the critical surface tensions and predicted if shear forces at given sites are known.