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.     

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.     

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.     

Chemical analysis and scanning electron microscopy of acquired pellicle formed in vivo on stannous fluoride treated enamel.

Stannous fluoride (SnF2) has been shown to be an effective caries preventive agent. After topical treatment of enamel surfaces, two reaction products have been demonstrated to precipitate on the surfaces, a larger type of globules, probably a calcium fluoride like product, and a smaller type of globules, probably a tin phosphate. The aim of the present study was to examine the amino acid composition and the formation of the acquired pellicle on SnF2-treated enamel in vivo. The chemical composition was examined by amino acid analysis of pellicle material collected in vivo from SnF2-treated enamel surfaces. Pellicle formation was examined by scanning electron microscopy on SnF2-treated enamel fragments carried in the mouth for 2 h. The results showed that pellicle material was formed in abundant amounts and covered the globular surfaces following the SnF2 treatment. The chemical analyses showed amino acid profiles with high content of acidic and neutral amino acids. The profiles were different from known amino acid profiles obtained from analyses of pellicle material collected from untreated enamel surfaces.     

Chemical analysis and scanning electron microscopy of acquired pellicle formed in vivo on stannous fluoride treated enamel

Abstract – Stannous fluoride (SnF₂) has been shown to be an effective caries preventive agent. After topical treatment of enamel surfaces, two reaction products have been demonstrated to precipitate on the surfaces, a larger type of globules, probably a calcium fluoride like product, and a smaller type of globules, probably a tin phosphate. The aim of the present study was to examine the amino acid composition and the formation of the acquired pellicle on SnF₂-treated enamel in vivo. The chemical composition was examined by amino acid analysis of pellicle material collected in vivo from SnF₂-treated enamel surfaces. Pellicle formation was examined by scanning electron microscopy on SnF₂-treated enamel fragments carried in the mouth for 2 h. The results showed that pellicle material was formed in abundant amounts and covered the globular surfaces following the SnF₂ treatment. The chemical analyses showed amino acid profiles with high content of acidic and neutral amino acids. The profiles were different from known amino acid profiles obtained from analyses of pellicle material collected from untreated enamel surfaces.     

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.     

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.     

Protein adsorption to hydroxyapatite and to calcium fluoride in vitro and amino acid analyses of pellicle formed on normal enamel and on calcium-fluoride-covered enamel in vivo

Fluoride treatment of enamel has been reported to result in the formation of a layer of a CaF2-like material on the enamel surface. Protein adsorption to enamel is a specific process dependent on the nature of the surface, and little is known about protein adsorption to CaF2. Albumin and lysozyme were adsorbed to hydroxyapatite (HA) and CaF2 powder in vitro, and protein adsorption patterns constructed. In vivo pellicle was collected from three volunteers from fluoride-treated enamel and from normal enamel, and the amino acid compositions analyzed separately. The results showed that CaF2 took up small amounts of proteins as compared with HA. When the CaF2 was pretreated with a phosphate buffer, pH 6.8, the protein adsorption increased markedly. The amino acid analyses showed no major differences in the amino acid compositions between pellicle collected from CaF2-covered enamel and pellicle collected from normal enamel. This lack of difference is presumably due to the adsorption of phosphate ions to the CaF2 crystals and hence changed surface properties.     

Characteristics of an in vitro dental pellicle.

Procedures developed for in vitro pellicle formation in intact enamel proved useful for relating qualitiative characteristics of dental pellicle to a number of factors. Coronal surfaces of extracted human molars from experimental and control groups were pumiced, sterilized, and incubated for two hours at 37 C in parotid saliva and distilled water, respectively. Pellicle proteins were desorbed sequentially with water and 0.2 M sodium phosphate, with a pH of 7.0. Polyacrylamide disc electrophoresis of the desorbates yielded distinct patterns, indicating selective adsorption of proteins from saliva, varying affinity to enamel, and the presence of proteins not acquired in vitro from saliva. Certain pellicle components, including amylase and IgA, showed a relatively weak affinity for enamel and were eluted in part by water; other proteins were desorbed only by phosphate buffer. Anionic electropherograms of the phosphate desorbates showed an increase in the two most anodic proteins relative to corresponding salivary bands. An intense anodic protein and two minor bands were eluted by water or buffer from the surface of control as well as experimental teeth but not from teeth coated with sealants. Serum albumin and alkaline phosphatase were identified as components of the extra-salivary material. Further investigation of the sources and functions of the constituents of the protein layer generally considered as "acquired" dental pellicle appears warranted.     

External radiolabelling of components of pellicle on human enamel and cementum

Enamel and cementum pellicles form by different adsorption of salivary and serum components to the tooth surface. The authors compared the constituents of surface pellicle formed on human enamel and cementum under three conditions: (1) natural pellicle, present on extracted teeth, which was formed by prolonged exposure to human salivary and serum components in vivo; (2) short-term in-vivo pellicle, formed by exposing enamel and cementum slabs to the oral environment for 0-60 min; (3) in-vivo pellicle, formed by incubating enamel and cementum slabs in a 1:1 mixture of parotid and submandibular/sublingual saliva for 0-60 min. Pellicle composition was characterized by external radiolabelling techniques specific for exposed carbohydrate (sialic acid and galactose) and amino-acid (tyrosine) residues. There were differences between cementum and enamel in the electrophoretic profiles of natural-pellicle components; notably, a major 180 kda 3H-labelled sialoglycoprotein, unique to the cementum pellicle, had the same electrophoretic mobility as the low-molecular-weight mucin from human submandibular/sublingual saliva. After alkaline-borohydride treatment, 3H-labelled natural-pellicle oligosaccharides chromatographed in the di- to tetrasaccharide region of a Bio-Gel P-2 column. The most prominently labelled components of short-term enamel and cementum pellicles in vivo and in vitro had the same electrophoretic mobility as the low-molecular-weight salivary mucin. The pellicle components formed in vitro, unlike those formed for the same period of time in vivo, were rapidly desorbed from the cementum, but not from the enamel surface. We conclude that: (1) external labelling techniques are useful for obtaining a profile of pellicle components; (2) submandibular/salivary mucins are major constituents of salivary pellicles on tooth surfaces; (3) glycoproteins that carry low-molecular-weight, sialic-acid-containing saccharides are important determinants of pellicle surface properties [corrected].     

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.     

Chemical composition of salivary integuments formed in vivo on solids with some established surface characteristics.

The amino acid composition of salivary integuments formed on three artificial surfaces in vivo was investigated. The test solids Mylar^® and Melinex^® had the same overall chemical composition but differed in their critical surface tensions. The critical surface tension of the polymers coincided approximately with that of human enamel. The third solid, glass, had a critical surface tension and a chemical composition different from both the other solids and from human enamel. The results indicated that the chemical properties of the surfaces of Mylar and Melinex were different, presumably owing to different processes of manufacturing. The results of the amino acid analyses showed that the chemical compositions of the adsorbed integuments were different and also that they differed from the previously reported amino acid composition of the pellicle formed in 2 hr on human enamel in vivo. It is concluded that the chemical composition of the surface of solids has an important influence on the type of proteins which they adsorb.     

The effect of red wine in modifying the salivary pellicle and modulating dental erosion kinetics

This study investigated the potential of red wine in modulating dental erosion kinetics in the presence or absence of salivary pellicle. Polished human enamel specimens were used in two conditions; presence or absence of acquired enamel pellicle; and subdivided according to exposure: red wine, orange juice, apple juice, or citric acid. The specimens were incubated in clarified whole human saliva (presence of acquired enamel pellicle) or in a humid chamber (absence of acquired enamel pellicle) for 2 h at 37°C, then in the test substances for 1 min, at 25°C, under shaking. This was repeated four times. Surface hardness was measured initially and after each cycle and surface reflection intensity was measured initially and after all cycles. In the presence of acquired enamel pellicle, red wine caused the least surface hardness loss, followed by orange juice, apple juice, and citric acid. Statistically significantly less surface reflection intensity loss was observed for red wine and orange juice than for apple juice and citric acid. In the absence of acquired enamel pellicle, red wine and orange juice caused less surface hardness loss than apple juice and citric acid. Orange juice showed the least surface reflection intensity loss, followed by red wine, citric acid, and apple juice. The polyphenol composition of these drinks can notably modulate the erosion kinetics.     

Hydroxyapatite-reactive salivary protein revealed by iso-electrofocusing electrophoresis.

Salivary protein involvement in the formation of acquired enamel pellicle, so far, has been discussed in terms of hydroxyapatite (HA)-reactive salivary proteins only from the parotid gland. This study was undertaken to seek this type of protein in the human whole (mixed) saliva and to investigate its normal and pathological variations. Several kinds of hydroxyapatite, either biogenous or synthesized by solid phase reaction, were used as a powder (250 mesh). HA was incubated with concentrated whole saliva at 25 degrees for 30 min. After centrifugation and filtration salivary proteins were analysed on a Multiphor isoelectrofocusing gel electrophoresis. The control salivary proteins were separated into three major groups; acidic (A1-A8), neutral neutral (N1-N4), and basic (B1-B3) isoelectric point (pI). In the HA incubated sample, one of the major neutral bands (NI) preferentially disappeared at about pI 7.5. This NI band was missing or scarce in the parotid saliva and had an amino acid composition rich in glycine, lysine, serine, glutamic acid, aspartic acid, and histidine. This protein was considered to be one of the major HA-reactive proteins in human whole saliva.     

Characterization of in vivo salivary-derived enamel pellicle

Salivary proteins and glycoproteins that participate in the formation of 2-h in vivo enamel pellicle were determined utilizing polyacrylamide gel electrophoresis [sodium dodecyl sulphate (SDS)-PAGE and anionic PAGE]/Western transfer analyses, and specific radiolabelling/SDS-PAGE fluorography. The sensitivity of these methods permitted the identification of individual members of different salivary protein families. The major components of this pellicle were salivary alpha-amylase, cysteine-containing phosphoprotein (CCP or cystatins), salivary mucin and sIgA. Glycosylated amylase was present in larger quantity than the non-glycosylated species. Only CCP1 (cystatin SA-I) of the cysteine-containing phosphoprotein family was identified. The higher molecular-weight salivary mucin (MG1), but not the lower molecular-weight species (MG2), was detected. These results extend earlier observations regarding the selective nature of salivary protein adsorption to enamel surface by demonstrating that only specific members of salivary protein families are involved in 2-h in vivo enamel pellicle formation. The findings also suggest that individual family members may have different functions in the mouth.     

New in vitro model for the acquired enamel pellicle: pellicles formed from whole saliva show inter-subject consistency in protein composition and proteolytic fragmentation patterns

The present investigation was undertaken to investigate the variability of proteins in whole saliva which adsorb to hydroxyapatite and are thus likely to be precursors of the acquired enamel pellicle. Whole-saliva proteins from 18 subjects were absorbed to hydroxyapatite, and the gel filtration patterns of released proteins revealed four major peaks and three minor peaks eluting between the major peaks. Amino acid analysis indicated that minor peaks contained fragments of proteins in major peaks, and this was confirmed by sequence analysis. Major peaks comprised 95% and minor peaks comprised 5% of protein absorbed to hydroxyapatite, suggesting a limited proteolytic capacity of whole saliva. HPLC elution patterns of components in minor peaks suggested that proteolysis is not totally random but is an orderly and consistent process. These studies suggest that whole saliva may be suitable as a model system for the investigation of post-secretory modifications of salivary proteins important for the formation of the acquired enamel pellicle.     

Analytical and ultrastructural studies of pellicle on primary teeth

The pellicle on permanent enamel has been thoroughly studied. The aims of this study were to compare the chemical composition, rate of formation, and ultrastructural appearance of pellicle formed on deciduous enamel in children with those on permanent teeth. This was done by amion acid analyses, Auger analyses, and transmisiion electron microscopy. The amino acid composition of 2-h pellicle on deciduous and permanent enamel had an overall similar pattern, but the contents of serine, glycine, and tyrosine were statistically significantly different. An initially slower pellicle formation and a thinner 2-h pelliced without a globular structured second layer was observed on deciduous enamel. The results indicated therefore distinct differences in chemical composition, rate of formation, and ultrastructural appearance between pellicle on primary teeth and that on permanent teeth.     

Amino acid composition of experimental salivary pellicles.

The amino acid compositions of pellicles formed in vitro from submandibular, parotid, and mixed saliva were determined and compared. Samples of the total pellicles as well as the relatively acid-soluble supernates and acid-insoluble sediments were collected and analyzed. In each instance striking differences in composition between the pellicle and the saliva from which it was formed indicate that pellicle formation is a highly selective process. The similarity in composition of the total and fractionated submandibular-parotid pellicle samples indicated a representative dissolution of the total mixed pellicle in the collection acid, rather than a preferential dissolution of part of the total pellicle. In the case of the pellicles formed from the isolated submandibular and parotid salivas, the supernate and the total pellicle were quite similar in composition, but differed somewhat from the sediment. Those results indicate that most of the salivary pellicle is soluble in the dilute acid used to collect the pellicle, and that a portion of the total submandibular or parotid salivary pellicle dissolves preferentially. The compositions of the total parotid pellicle and the parotid supernate and sediment were remarkable similar to the counterparts formed from submandibular saliva alone, suggesting similar origins and mechanisms of formation.     

The interaction of human parotid salivary proteins with hydroxyapatite

The interactions responsible for the formation of the acquired integuments of the human dental enamel surface take place in a complex and poorly, understood milieu. One interaction of particular interest is the selective adsorption of certain salivary proteins on to the hydroxyapatite surface. The purpose of this study was to examine this process in isolation from other events occurring in vivo on the tooth surface and in the saliva. To this end the interaction between various hydroxyapatites and parotid saliva was examined.

Hydroxyapatite and dental enamel powders were found to selectively adsorb seven parotid salivary components from the stimulated and the unstimulated secretion. The two components showing the greatest affinity for these mineral surfaces have been shown, in parallel studies, to be acidic peptides or small proteins of unusual composition. A further four of these components were a group of previously described proline rich proteins, these being characterized by high levels of dicarboxylic amino acids, proline and glycine. The seventh component showed a marked resemblance to the latter materials and appeared to be a sub-group of related proteins. Possible mechanisms underlying the observed interactions are discussed.     

Compositional analysis of human acquired enamel pellicle by mass spectrometry

Relatively little is known about the formation of the acquired enamel pellicle other than that it involves the selective adsorption of specific proteins from oral fluids. Previous studies on the identification of pellicle components have relied largely on immunological or enzymatic detection and have been hampered by the fact that only minute quantities of pellicle can be removed from tooth surfaces. The present work describes an improved method of harvesting pellicle that combines mechanical and chemical removal; this approach was used to investigate systematically the desorption of in vitro pellicle components with different solutions. Eleven major in vitro pellicle proteins were identified by using a combination of electrophoretic separation and matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. A similar analysis of in vivo-formed pellicle revealed the presence of intact statherin, lysozyme, albumin and amylase. Further analysis of in vivo pellicle by liquid chromatography-electrospray ionization mass spectrometry suggested the presence of numerous low molecular-weight fragments of precursor proteins. The protein composition of in vitro whole-salivary pellicle adsorbed to hydroxyapatite and that of in vivo enamel pellicle differed for proline, the result of a reduction in the content of acidic proline-rich proteins in the in vivo samples. Unique features of the oral environment such as enzymatic activities or mineral surface properties may account for these differences between in vivo and in vitro pellicle formation.