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.     

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.     

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.     

Effect of silicone oil on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Abstract – Silicone oil has been introduced in a dentifrice for smokers because of its effect as a polishing agent. Silicone oils are hydrophobic in character and have low surface tensions and good wetting properties. Due to the low surface tension, silicone oils may spread readily on solid surfaces and cover them with a thin, water-repellant film. Introduced via dentifrices silicone oil may thus well be able to adsorb to enamel surfaces and to interfere with surface characteristics such as protein adsorption. The aim of the present study was to examine the effect of silicone oil on protein adsorption to hydroxyapatite (HA) in vitro and on pellicle formation in vivo. The effect on protein adsorption to HA in vitro was studied by adsorption of albumin to either untreated or silicone oil treated HA powders. Ion exchange chromatography was also used with either untreated or silicone oil treated HA as bed materials. The effect on pellicle formation in vivo was studied using enamel fragments carried in the mouth to acquire pellicle material. The chemical composition of the acquired pellicle was studied by collection and chemical analysis of pellicle material formed on enamel surfaces in vivo. The study showed that silicone oil treated HA took up less protein and that the adsorbed protein was bound to hydroxyapatite by a different mechanism as compared to untreated controls. The results indicated that hydrophobic interactions could be involved in binding of proteins to silicone oil treated hydroxyapatite. Silicone oil treated enamal fragments carried in the mouth showed a slower rate of pellicle formation as compared to untreated fragments. The amino acid composition of the acquired pellicle collected in vivo from silicone oil treated enamel surfaces was also different from pellicle material collected from untreated enamel.     

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.     

Effect of silicone oil on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Silicone oil has been introduced in a dentifrice for smokers because of its effect as a polishing agent. Silicone oils are hydrophobic in character and have low surface tensions and good wetting properties. Due to the low surface tension, silicone oils may spread readily on solid surfaces and cover them with a thin, water-repellent film. Introduced via dentifrices silicone oil may thus well be able to adsorb to enamel surfaces and to interfere with surface characteristics such as protein adsorption. The aim of the present study was to examine the effect of silicone oil on protein adsorption to hydroxyapatite (HA) in vitro and on pellicle formation in vivo. The effect on protein adsorption to HA in vitro was studied by adsorption of albumin to either untreated or silicone oil treated HA powders. Ion exchange chromatography was also used with either untreated or silicone oil treated HA as bed materials. The effect on pellicle formation in vivo was studied using enamel fragments carried in the mouth to acquire pellicle material. The chemical composition of the acquired pellicle was studied by collection and chemical analysis of pellicle material formed on enamel surfaces in vivo. The study showed that silicone oil treated HA took up less protein and that the adsorbed protein was bound to hydroxyapatite by a different mechanism as compared to untreated controls. The results indicated that hydrophobic interactions could be involved in binding of proteins to silicone oil treated hydroxyapatite. Silicone oil treated enamel fragments carried in the mouth showed a slower rate of pellicle formation as compared to untreated fragments. The amino acid composition of the acquired pellicle collected in vivo from silicone oil treated enamel surfaces was also different from pellicle material collected from untreated enamel.     

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.     

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.     

Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period

The purpose of the present in vivo study was to examine salivary pellicle formation on enamel surfaces at two different intraoral sites for periods of 1 min up to 24 h by means of transmission electron microscopy. Bovine enamel specimens were attached to the buccal and lingual surfaces of the upper first molars in three subjects using removable intraoral splints. Specimens were carried over periods of 1, 10, 30 and 60 min, 2, 6 and 24 h and were processed for transmission electron microscopy. After 1 min, an electron dense pellicle layer, 10–20 nm thick, was observed on the enamel surfaces. The subsequent adsorption of salivary biopolymers was governed by local influences of the oral cavity. Specimens located on the lingual aspect were covered within 2 h by a 20- to 80-nm-thick, homogeneous, predominantly granular-structured pellicle. The thickness of the surface coatings that were adsorbed on lingually carried specimens increased to 100–200 nm after 24 h. In contrast, on the buccally mounted specimen surfaces, a variably structured pellicle with granular and globular components could be detected after intraoral exposure for 2 h. The thickness of the 2-h buccal pellicles ranged between 200 and 700 nm. After 24 h, the buccally positioned specimens were covered by a dense globular pellicle layer varying in thickness from 1000 to 1300 nm. It is suggested that in vivo pellicle formation is initiated by adsorption of an electron-dense layer of salivary proteins. Further adsorption of salivary biopolymers leads to the formation of an outer loosely arranged pellicle layer. Under oral conditions, the locally available salivary biopolymers and the influence of locally effective shearing forces are of significance for the ultrastructural pattern and extent of pellicle formation. Received: 8 March 1999 / Accepted: 19 April 1999     

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.     

Adsorption of human salivary proteins on synthetic hydroxyapatite

Protein adsorption of mixed saliva obtained after Millipore filtration on synthetic hydroxyapatite was studied as a function of variable concentrations of proteins for a constant weight of hydroxyapatite and for a constant protein concentration as a function of variable quantities of hydroxyapatite. Similar experiments were conducted on calcium-precipitable salivary glycoprotein. The salivary proteins and the calcium-precipitable salivary glycoprotein showed respectively an adsorption of 25 and 100 per cent. Selective adsorption of the salivary proteins remaining in the supernatant after hydroxyapatite adsorption was studied with acrylamide gel electrophoresis. The quantitative study of the gels by densitometry showed extinction only of the band corresponding to the calcium-precipitable salivary glycoprotein, indicating that this glycoprotein is selectively adsorbed. It is suggested that this salivary glycoprotein is concerned in the exogenous pellicle formation.     

Adsorption of chlorhexidine and black tea onto in vitro salivary pellicles, as studied by ellipsometry

The adsorption from 0.2% (w/w) chlorhexidine and black tea solutions onto an in vitro pellicle from whole unstimulated saliva on hydroxyapatite discs was studied by ellipsometry. It was found that chlorhexidine adsorbed to the pellicle and caused a modification of the pellicle properties, leading to a subsequent increase in adsorption of salivary and black tea components. There was a distinct order-of-addition effect, whereby chlorhexidine followed by black tea gave an overall greater adsorption of components compared with black tea followed by chlorhexidine. This increase in adsorption resulted in a concomitant increase in color or stain, as measured by a reflectance chromameter. The increase in adsorbed amounts and stain was modified, in part, by the adsorption of salivary fractions between the chlorhexidine and black tea treatments. In all cases, the chlorhexidine and black tea-modified pellicles were not readily removed by either phosphate or sodium dodecyl sulfate rinses. Thus, following exposure to chlorhexidine, the accelerated adsorption of salivary and black tea components can ultimately lead to increased staining of the pellicle.     

Interindividual and longitudinal studies of amino acid composition of pellicle collected in vivo

Abstract – The formation of the acquired enamel pellicle is due to the adsorption of salivary proteins to the enamel surface. This adsorption is assumed to be specific and is dependent on the chemical characteristics of the surface. The aim of the present study was to investigate the consistency of the chemical composition of the acquired pellicle collected in vivo. Inter- and intraindividual differences in the chemical composition of pellicle material were examined during 2 yr in three different individuals. The amino acid profiles obtained from pellicle analyses were compared to hydrolyzed whole saliva collected at the same time as the pellicle material. The results showed that the amino acid composition of pellicle was consistent both between and within the individuals. The amino acid profiles obtained from the analyses of the saliva samples were different from the pellicle profiles, illustrating the selective nature of pellicle formation. This supports the contention that the adsorption of salivary proteins to dental enamel is a very specific process.     

Interindividual and longitudinal studies of amino acid composition of pellicle collected in vivo

The formation of the acquired enamel pellicle is due to the adsorption of salivary proteins to the enamel surface. This adsorption is assumed to be specific and is dependent on the chemical characteristics of the surface. The aim of the present study was to investigate the consistency of the chemical composition of the acquired pellicle collected in vivo. Inter- and intraindividual differences in the chemical composition of pellicle material were examined during 2 yr in three different individuals. The amino acid profiles obtained from pellicle analyses were compared to hydrolyzed whole saliva collected at the same time as the pellicle material. The results showed that the amino acid composition of pellicle was consistent both between and within the individuals. The amino acid profiles obtained from the analyses of the saliva samples were different from the pellicle profiles, illustrating the selective nature of pellicle formation. This supports the contention that the adsorption of salivary proteins to dental enamel is a very specific process.     

Chitosan adsorption to salivary pellicles

The salivary pellicle is a negatively charged protein film, to which oral bacteria readily adhere. Chitosans are cationic biomolecules with known antimicrobial properties that can be modified in different ways to enhance its antimicrobial activity. Here, we determined the changes in surface chemical composition using X-ray photoelectron spectroscopy (XPS), in hydrophobicity by analyzing water contact angles, in charge through measuring streaming potentials, and evaluated morphology using atomic force microscopy (AFM), of salivary pellicles upon adsorption of different chitosans. The adsorption of chitosans to pellicles was chemically evident from altered carbon functionalities and the presence of an N(1s) peak at 401.1 eV as a result of protonated amines in XPS. Chitosan adsorption made the pellicle (zeta potential of untreated pellicles 29 mV) positively charged and more hydrophobic. A chemically modified chitosan (CL) and an unmodified chitosan (UC) caused aggregation of adsorbed salivary proteins, and AFM revealed clumps of protein after treatment with these chitosans, yielding an increase in pellicle surface roughness from 5.1 nm to between 16.3 and 35.6 nm for CL and UC, respectively. In summary, chitosans have a clear tendency to adsorb to salivary pellicles with a profound effect on the surface properties of the pellicle. Therefore, chitosans may provide anchoring molecules to affix antimicrobials to pellicle surfaces.     

Amino acid composition of acquired enamel pellicle collected in vivo after 2 hours and after 24 hours

Abstract— The adsorption of salivary proteins to dental enamel during pellicle formation has been shown to be a specific process and dependent on the chemical composition of the surfaces. Most studies on the amino acid composition of the acquired enamel pellicle have, however, been performed on the "2-h-pellicle"under controlled experimental conditions. This may have eliminated some natural factors involved in pellicle formation. The aim of the present study was to investigate the effect of extended time of formation and diet on the pellicle formation. Pellicle material was collected from the same subject after 2 and after 24 h when food and beverages were avoided, and after 24 h with the intake of a normal diet. The collected pellicle materials were hydrolyzed and amino acid analyzed. The results showed that pellicle material collected after 24 h and fasting had a chemical composition similar to the "2-h-pellicle", whereas pellicle material collected after 24 h and a normal diet was different, indicating a dietary contribution to pellicle formation or a bacterial degradation of the pellicle.     

Pellicle formation on hydroxyapatite splints attached to the human dentition: Morphologic confirmation of the concept of adsorption

The formation of pellicle was studied using epoxy resin splints with hydroxyapatite crystals at the surface. The splints were attached to the buccal surfaces of molar and premolar teeth of human test subjects and removed after 2 hr. Specimens were processed for transmission and scanning electron microscopy. An incomplete pellicle of uneven thickness characterized by small projections of organic material partly covered the hydroxyapatite areas of the splint surface. Only traces of such pellicle occurred on the resin surfaces. The findings support the concept that the first stage in the formation of an acquired pellicle occurs as an adsorption of salivary components to hydroxyapatite crystals on the tooth surface.     

Amino acid composition of acquired enamel pellicle collected in vivo after 2 hours and after 24 hours.

The adsorption of salivary proteins to dental enamel during pellicle formation has been shown to be a specific process and dependent on the chemical composition of the surfaces. Most studies on the amino acid composition of the acquired enamel pellicle have, however, been performed on the "2-h-pellicle" under controlled experimental conditions. This may have eliminated some natural factors involved in pellicle formation. The aim of the present study was to investigate the effect of extended time of formation and diet on the pellicle formation. Pellicle material was collected from the same subject after 2 and after 24 h when food and beverages were avoided, and after 24 h with the intake of a normal diet. The collected pellicle materials were hydrolyzed and amino acid analyzed. The results showed that pellicle material collected after 24 h and fasting had a chemical composition similar to the "2-h-pellicle", whereas pellicle material collected after 24 h and a normal diet was different, indicating a dietary contribution to pellicle formation or a bacterial degradation of the pellicle.     

Quantitative determination of salivary components in the pellicle on PMMA denture base material

The formation of a salivary pellicle is a protective mechanism of the body for all surfaces in the oral cavity. The nature of the substrate may influence the composition of the pellicle. The aim of this study was to investigate the quantitative composition and individual variation of the salivary pellicle formed on denture base material (PMMA). Cylindrical specimens of PMMA were carried in the mouth and then desorbed with a 0.5-M sodium chloride solution. The solution was analysed for total protein, alpha-amylase, total proteases, protease inhibitors, secretory immunoglobulin A, immunoglobulin G, peroxidases, thiocyanate, lysozyme, and calcium content. All investigated salivary components could be found unequivocally in the desorption solution, indicating that a salivary pellicle had formed on the surface of the PMMA. Large coefficients of variation indicate large individual variations in the adsorbed amounts. The data also point to large intraindividual variations for the bound salivary components. Only the protease inhibitors revealed a strong positive correlation of the bound activity to the salivary activity. It is hypothesised that differences in the bound amounts of antimicrobial components might influence the microbial colonisation of denture bases and that protease inhibitors could be meaningful for the spread of the yeast Candida albicans from denture base material to the oral mucosa and thus might be an explanation for different susceptibility to denture base stomatitis.