表面活性剂与实验性获得性膜相互作用的体外研究

为了探索表面活性剂影响细菌对牙面粘附的机理，本文比较了５种表面活性剂与实验性获得性膜的相互作用，结果表明，实验用５种表面活性剂中，阴离子表面活性剂ＺｏｎｙｌＦＳＡ使吸附的唾液蛋白量明显增加，而其余４种阳离子表面活性剂能明显地移去吸附的唾液蛋白，本研究结果提示，表面活性剂与实验性获得性膜的相互作用与其所带的电荷有关，表面活性剂对获得性膜的影响是其菌斑的可能机理之一。     

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.     

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.     

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 of human salivary proteins to hydroxyapatite: a comparison between whole saliva and glandular salivary secretions.

The protein compositions of in vitro pellicles formed from whole saliva and parotid and submandibular secretions were determined by use of synthetic hydroxyapatite as a model for dental enamel. The adsorbed and unadsorbed protein fractions were analyzed by amino acid analysis and both anionic and cationic discontinuous polyacrylamide gel electrophoresis. For further characterization of the in vitro pellicle, the adsorbed fractions were subjected to gel filtration on Sephadex G-100 and reversed-phase chromatography on C18 columns. Amylase, acidic and glycosylated proline-rich proteins, statherins, and histatins were identified in the parotid-derived pellicle. Detailed analysis of the statherin-containing fractions resulted in the observation of several statherin-like proteins. The use of cationic gel electrophoresis allowed for the identification of histatin 3 and histatin 5, which have not been previously detected in pellicle formed in vitro. The protein composition of submandibular-derived pellicle was similar to that of parotid-derived pellicle except for the presence of cystatins and the absence of glycosylated proline-rich proteins. In contrast, in vitro pellicle derived from whole saliva exhibited a vastly different composition, consisting primarily of amylase, acidic proline-rich proteins, cystatins, and proteolytically-derived peptides. The results indicate that acidic phosphoproteins as well as neutral and basic histatins from pure secretions selectively adsorb to hydroxyapatite, whereas in whole saliva some of these proteins are proteolytically degraded, dramatically changing its adsorption pattern.     

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.     

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.     

Fractionation of salivary micelle-like structures by gel chromatography

Globular structures have been demonstrated in human parotid saliva by transmission electron microscopy and photon correlation spectroscopy. The aim of this study was to fractionate these salivary globular structures for analytical and preparative purposes using a gel-filtration material capable of separating spherical particles up to 300-400 nm in diameter. Freshly obtained parotid saliva was applied to a Sephacryl S-1000 column. Peak fractions were collected and prepared for transmission electron microscopy (TEM) or for amino acid analysis. Bovine milk was included as the casein micelles by TEM appear to be similar to the salivary aggregates and their elution profiles are known. The salivary globular structures were eluted in one major peak. TEM of negatively stained samples from the peak fractions demonstrated globular protein aggregates consistent with the salivary structures in parotid saliva. Amino acid analysis showed characteristic amino acid profiles with unusual high levels of proline, 40-45%. The casein micelles were eluted in one major peak and separated from the whey proteins. This study indicates that the salivary globular structures can be isolated by gel chromatography. The amino acid analysis indicates that proline-rich proteins may be an important fraction of the salivary globular structures.     

Relationship of the basic glycoprotein to the basic proline-rich proteins in human parotid saliva

The relationship was investigated by chemically deglycosylating the glycoprotein and comparing the protein moiety so obtained with non-glycosylated proline-rich proteins. Deglycosylation of the purified salivary basic proline-rich glycoprotein resulted in three protein moieties. After purification, these fractions were compared biochemically to the non-glycosylated basic proline-rich proteins by amino acid composition, cationic and sodium dodecyl sulphate gel electrophoreses, end groups and peptide mapping. The findings indicate that none of the basic proline-rich proteins are precursor proteins to the basic glycoprotein.     

Adsorption of salivary proteins and serum proteins to titanium.

Titanium (Ti) is very effective as a dental implant material owing to its low toxicity and high biocompatibility. I carried out in vitro experiments on the adsorption of salivary proteins and serum proteins to Ti to better understand how this material reacts in the oral cavity. I found that when Ti that had adsorbed serum proteins was exposed to salivary proteins, there was an exchange of the proteins on the Ti surface. The same thing happened when Ti that had adsorbed salivary proteins was exposed to serum proteins. These results suggest that both human salivary proteins and serum proteins adsorb to Ti with different affinity, and that protein already adsorbed to Ti might mask binding sites for other proteins.     

Investigation of initial pellicle formation on modified titanium dioxide (TiO2) surfaces by reflectometric interference spectroscopy (RIfS) in a model system.

OBJECTIVES: Plaque accumulation, leading to inflammatory processes and bone loss, is one of the main reasons for failure of dental implants. Pellicle formation plays a key role in bacterial adhesion and plaque accumulation. The influence of experimental bioactive implant surface coatings on the initial process of pellicle formation was investigated in a model system. METHODS: TiO2-films were modified by covalent binding of laminin and human epidermal growth factor (EGF) to promote adhesion of epithelial cells. Adsorption and dissociation behavior of bovine serum albumin (BSA) and salivary proteins on these surfaces were monitored by time-resolved reflectometric interference spectroscopy (RIfS). RESULTS: The thickness of the irreversibly adsorbed salivary protein layer was reduced from 2.78 +/- 0.71 nm on unmodified TiO2 to 0.78 +/- 0.22 nm on laminin-coated surfaces and to 1.18 +/- 0.29 nm on EGF-coated surfaces. The percentage of initially adsorbed proteins remaining irreversibly bound was reduced from 51 +/- 8% on titanium to 23 +/- 5% by laminin coating and to 44 +/- 11% on EGF-coated surfaces. The highest reduction of protein adsorption (layer thickness lower than 0.05 nm) was achieved on DC-PEG-layers used as spacer for protein coupling. SIGNIFICANCE: Laminin and EGF were shown to be promising candidates for use as biological coatings on the transmucosal part of titanium dental implants where the objective is to enhance epithelial adhesion and inhibit adsorption of salivary proteins and bacteria.     

Does irradiation affect the protein composition of saliva?

The purpose of this study was to compare the relative amount of low molecular weight salivary proteins in patients with head and neck tumours treated with radiotherapy and healthy subjects. Reverse-phase high-pressure liquid chromatography was used for protein separation. Nine protein fractions (including acidic and basic proline-rich proteins (PRPs), cystatins, histatins and statherin) were identified in saliva from irradiated patients as well as healthy subjects. However, compared with non-irradiated healthy subjects, the fraction of acidic PRPs was significantly reduced in irradiated patients. These data indicate an alteration of the relative amount of low molecular weight salivary proteins in irradiated patients besides the reduction of salivary flow.     

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.     

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.     

Effects of acidic phosphate fluoride on the protein compositions of acquired pellicle in vitro

The human enamel powders treated with acidic phosphate fluoride (APF) were used to react with human mixed saliva and purified proteins. Before and after treated with APF, the changes of fluoride content in the enamel and the salivary proteins adsorbed on the enamel surfaces were noticed. The results are as follows: 1. The post-treated enamel fluoride is increased, and the production is fluorapatite besides a little CaF2. 2. No effect of APF on the kind of salivary proteins adsorbed on the enamel is found. 3. The quantity of salivary proteins adsorbed on the enamel is increased in different percentage and the proportion of the proteins on the enamel surface may be changed. Therefore, we believe that these changes must be one of the APF's mechanisms on caries prevention.     

Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite

The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.     

Bioadhesion--a phenomenon with multiple dimensions

This paper reviews some relevant citations regarding the non-specific forces that must be considered in oral bioadhesive events. These range from forces related to restorative dentistry to those related to prevention and molecular biology. Types of interactions discussed are: 1. Van der Waal's forces; 2. electrostatic double-layer forces; 3. solvent-dependent interactions; 4. hydrogen bonding; 5. hydrophobic interactions; 6. hydration forces; 7. steric forces; and 8. covalent bonds. Examples are given of the various types of interaction that occur at different surface separation (< or =400 A) between adsorbed films of a pure salivary protein fraction (PRP1).     

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.     

A further study of the factors enhancing glycolysis in human saliva

The glycolysis-enhancing factors of high molecular weight in saliva were fractionated by gel-filtration chromatography. Some correlation was found between the enhancing activity and the protein content of the fractions obtained. It appears that many of the proteins in saliva enhance the glycolysis of the salivary sediment. The enhancement per unit of protein was of the same order for most of the protein fractions. However, one fraction containing peptides or small proteins effected twice as much enhancement per unit of protein as did the other protein-containing fractions.