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.

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.     

Iron staining of the acquired enamel pellicle after exposure to tannic acid or chlorhexidine: preliminary report

Abstract – Extrinsic discoloration of teeth following a large consumption of tannin-containing beverages or a prolonged use of chlorhexidine mouthrinses is a well known observation. Tannins as well as chlorhexidine are denaturing agents. Based on preliminary studies revealing the presence of iron in chlorhexidine discolored pellicle material, the ability of iron to stain the integument after pretreatmentwith the two denaturants was studied in a human model. The denaturing effect of an acidic environment was also included. Enamel slabs fixedto acrylie appliances were carried in the oral cavity and alternately exposed to the test solutions in different sequences in vitro. Pretreatment with chlorhexidine or tannic acid led to marked discolorations upon iron application during 5-d tests, whereas the compounds individually had no such effect. A large content of the metal was found in the stained material. Stannous fluoride appeared to reduce the formation of the pigments, and strong oxidation completely bleached the established color. Possible mechanisms underlying the phenomena observed are discussed.     

Further studies on the composition of the acquired enamel pellicle

Abstract – In the present study pellicle material was collected from human teeth 2, 4 and 6 hr after cleaning. The material obtained was examined by gel filtration, ion exchange chromatography on CM-Sephadex and DEAE-Sephadex with subsequent amino acid analyses of the major anionic component. No major changes were observed to occur either in the overall composition or in the main anionic component of the pellicle during the first 6 hr.     

Desorption of acquired enamel pellicle in vivo by pyrophosphate

Abstract – Soluble pyrophosphate (PP) has been introduced in dentifrices to inhibit the formation of dental calculus. The mechanism of inhibition is probably an adsorption of the pyrophosphate ions to the Ca-sites on the enamel surfaces and a blocking of the active sites for crystal growth. It has been shown in a recently published study that PP reduced the protein adsorption to hydroxyapatite (HA) in vitro and also inhibited the pellicle formation in vivo. The aim of the present study was to examine the desorption potential of pyrophosphate on the acquired enamel pellicle in vivo. Enamel fragments were carried in the mouth to collect pellicle material and some of the enamel surfaces were then treated with PP. Pellicle formation was examined by SEM of the enamel surfaces. The results showed that pyrophosphate desorbed the acquired enamel pellicle effectively. The clinical consequences of this effect is unknown, but it could possibly explain some aspects of hypersensitivity of teeth observed in some individuals using dentifrices containing PP.     

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 plasma on composition of human enamel and cementum pellicle

Abstract – Slabs of human enamel and cementum were incubated with plasma alone or with various mixtures of plasma and saliva. Proteins and glycoproteins that adsorbed to the surface of the slabs in 0 to 60 min were labeled by lactoperoxidase-catalyzed ¹²⁵I-iodination and by mild periodate oxidation followed by NaB³H₄ reduction. The labeled components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography or fluorography. From plasma alone, a 58 and a 66 kDa protein (probably albumin) were adsorbed to the enamel surface in relatively equal amounts, but no ¹²⁵I-labeled components were detected on the cementum surface in the absence of saliva. Adding 10% saliva to the incubation mixture promoted the adsorption of the 58 and 66 kDa components to cementum. In addition, another set of proteins, including components of 44, 47, 29, and 25 kDa, was adsorbed to both cementum and enamel in the presence of saliva. These six proteins were the major ¹²⁵I-labeled species in all of the pellicles formed from mixtures of plasma and saliva. The electrophoretic mobility of the major 120 and 140 kDa ³H-labeled sialoglycoproteins adsorbed to both cementum and enamel was similar to that of the low-molecular-weight mucin of submandibular/sublingual saliva.     

Gel filtration, ion exchange chromatography and chemical analysis of macromolecules present in acquired anamel pellicle (2-hour-pellicle)

Abstract – Proteins obtained from the surface of human teeth in vivo were solubilized in EDTA and subjected to gel permeation, ionic exchange chromatography and amino acid analysis. It was found that the main component was anionic and was eluted between albumin and iysozyme on Sepharose. It contained abundant amounts of serine, glycine and glutamic acid. A salivary phosphoprotein of similar amino acid composition has previously been purified.     

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.     

Enzymes in the acquired enamel pellicle

The acquired pellicle is a biofilm, free of bacteria, covering oral hard and soft tissues. It is composed of mucins, glycoproteins and proteins, among which are several enzymes. This review summarizes the present state of research on enzymes and their functions in the dental pellicle. Theoretically, all enzymes present in the oral cavity could be incorporated into the pellicle, but apparently enzymes are adsorbed selectively onto dental surfaces. There is clear evidence that enzymes are structural elements of the pellicle. Thereby they exhibit antibacterial properties but also facilitate bacterial colonization of dental hard tissues. Moreover, the immobilized enzymes are involved in modification and in homeostasis of the salivary pellicle. It has been demonstrated that amylase, lysozyme, carbonic anhydrases, glucosyltransferases and fructosyltransferase are immobilized in an active conformation in the pellicle layer formed in vivo. Other enzymes, such as peroxidase or transglutaminase, have been investigated in experimental pellicles. Despite the depicted impact of enzymes on the formation and function of pellicle, broader knowledge on their properties in the in vivo-formed pellicle is required. This might be beneficial in the development of new preventive and diagnostic strategies.     

Characterization of the immunologic responses to human in vivo acquired enamel pellicle as a novel means to investigate its composition

Human acquired enamel pellicle is formed by molecules selectively adsorbed onto tooth surfaces. The present work describes the use of monoclonal antibody (mAb) technology as a novel approach to identify micro amounts of components present in pellicle. MAbs were obtained with reactivities against statherin, histatin, mucous glycoprotein 1(MGI), albumin, amylase and human immunoglobulins (Igs), indicating that these are pellicle components, which was further confirmed by immunoblotting. No mAbs against proline-rich proteins (PRPs), lysozyme, mucous glycoprotein 2 (MG2), carbonic anhydrase, lactoferrin or peroxidase were obtained, suggesting that these components are absent, present in low amounts, or exhibit low antigenicity. Further characterization of the binding epitopes of some of th e obtained anti-MGO, anti-statherin and anti-histatin mAbs were carried out and the biological relevance is discussed. The results open up the possibility that immunization with human pellicle and mAbs production can be employed to identify hitherto unknown constituents of pellicle.     

The acquired pellicle: enzyme and antibody activities

abstract — Assays for enzyme and antibody activities have been applied to evaluate functional properties of proteins in 2-h, experimental acquired pellicles. Enzyme activity was demonstrated for human salivary lysozyme and amylase in pellicles grown in vivo as well as in vitro , and antibody activity was demonstrated for rabbit immunoglobulins incorporated in pellicles grown in vitro . Desorption of pellicles prior to functional tests variously affected the activity of the proteins under study, thereby implying differences in the tenacity of protein binding to the pellicles.     

Immobilisation and activity of human alpha-amylase in the acquired enamel pellicle

Amylase is an important salivary component and structural element of the acquired enamel pellicle. Aim of the study was to establish a method for precise and direct determination of pellicle bound amylase activity in order to analyse kinetics and activity of the immobilised enzyme. Six bovine enamel slabs (5mm diameter) were fixed on individual maxillary trays and worn by five subjects for different times (3, 30 and 120 min) on buccal and palatal sites on different days. Slabs were removed from the trays and rinsed with aqua dest. Afterwards, pellicle bound amylase activity was determined directly with a photometric method using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltotriosid (GalG2CNP) as substrate yielding the coloured product chloronitrophenolate (CNP). All investigated pellicles exhibited immobilised amylase activity. Mean activity was 1.39 +/- 187 mU/cm(2) (n=87, range 0.14-11.5 mU/cm(2)). Product formation of CNP by immobilised amylase was linear over time. Pellicle bound amylase showed a Michaelis type kinetic (Km = 3.3 x 10(-3) M). Immobilised activity on buccal surfaces ranged between 0.25 and 11.1 mU/cm(2) (palatal slabs: 0.14-3.06 mU/cm(2)). Thirty minutes pellicles formed on buccal sites exhibited significantly higher immobilised amylase activity (2.85 +/- 3.65 mU/cm(2)) than palatal ones (0.63 +/- 0.32 mU/cm(2)). Amylase activity showed great intraindividual variability when comparing same positions on different days. CONCLUSION: Pellicle bound amylase activity can be determined directly with GalG2CNP and shows a Michaelis Menten kinetic. Enzyme activity of the amylase immobilised in the in situ pellicle reveals great intra- and interindividual differences.     

Small molecular weight proteins/peptides present in the in vivo formed human acquired enamel pellicle

Objective

The aim of this study was to investigate the type and the nature of peptides present in the in vivo formed human acquired enamel pellicle.

Design

Pellicle material was collected from 10 volunteers and subjected to sample preparations consisting of centrifugal filtration using a 10 kDa molecular weight cut-off membrane and high-resolution gel filtration chromatography. The fractions containing peptides <10 kDa obtained by both methods were analyzed by LC-ESI-MS/MS.

Results

78 natural pellicle peptides with molecular weights ranging from 766.9 Da to 3981.4 Da were identified originating from 29 different proteins.

Conclusions

The number of peptides present in acquired enamel pellicle appears to be large and this is likely to enhance the functional spectrum of this protein film. The presence of small peptides in pellicle may be functionally important since structure/function studies of many salivary proteins have shown that specific domains within these native proteins retain or even exhibit enhanced biological activities. The data present the basis for determining the precise function of these pellicle peptides and for gaining insights into the role pellicle plays in the oral cavity.     

Sulfated glycoproteins in the acquired pellicle and in plaque from Macaca fascicularis demonstrated with labeled sulfate

abstract – A sulfated glycoprotein was demonstrated in 2-h pellicle material collected from monkeys. Furthermore, a sulfated macromolecule present in plaque extracts was identified as a sulfate glycoprotein by electrophoresis on cellulose acetate strips. The present data indicate that acidic proteins are selectively adsorbed to tooth surfaces in. vivo .     

Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva

Abstract – Two hour in vivo formed enamel pellicle samples and paraffin wax-stimulated saliva samples were collected from 10 volunteers for analyses of glucosyltransferase activity (GTF). GTF activity was recorded by monitoring incorporation of radioactivity from ¹⁴C-glucose labeled sucrose into glucan. Pellicle and saliva samples from all 10 subjects demonstrated GTF activity. The GTF activity in the pellicle samples was highest in subjects, with high GTF activity-producing adhesive glucan in saliva.     

Impact of the in situ formed salivary pellicle on enamel and dentine erosion induced by different acids

Objective. To investigate and compare the protective impact of the in situ formed salivary pellicle on enamel and dentine erosion caused by different acids at pH 2.6. Methods. Bovine enamel and dentine samples were exposed for 120 min in the oral cavity of 10 healthy volunteers. Subsequently, enamel and dentine pellicle-covered specimens were extraorally immersed in 1 ml hydrochloric, citric or phosphoric acid (pH 2.6, 60 s, each acid n=30 samples). Pellicle-free samples (each acid n=10) served as controls. Calcium release into the acid was determined by atomic absorption spectroscopy. The data were analysed by two-way ANOVA and Tukey's test (α=0.05). Results. Pellicle-covered samples showed significantly less calcium loss compared to pellicle-free samples in all acid groups. The mean (SD) pellicle protection (% reduction of calcium loss) was significantly better for enamel samples [60.9 (5.3)] than for dentine samples [30.5 (5.0)], but revealed no differences among the acids. Conclusion. The efficacy of the in situ pellicle in reducing erosion was 2-fold better for enamel than for dentine. Protection of the pellicle was not influenced by the kind of acid when enamel and dentine erosion was performed at pH 2.6.     

Enamel roughness and depth profile after phosphoric acid etching of healthy and fluorotic enamel

Background: Dental fluorosis requires aesthetic treatment to improve appearance and etching of enamel surfaces with phosphoric acid is a key step for adhesive restorations. The aim of this study was to evaluate surface roughness and a depth profile in healthy and fluorotic enamel before and after phosphoric acid etching at 15, 30 and 60 seconds. Methods: One hundred and sixty enamel samples from third molars with no fluorosis to severe fluorosis were evaluated by atomic force microscopy. Results: Healthy enamel showed a statistically significant difference (p < 0.05) between mean surface roughness at 15 seconds (180.3 nm), 30 seconds (260.9 nm) and 60 seconds (346.5 nm); depth profiles revealed a significant difference for the 60 second treatment (4240.2 nm). For mild fluorosis, there was a statistically significant difference (p < 0.05) between mean surface roughness for 30 second (307.8 nm) and 60 second (346.6 nm) treatments; differences in depth profiles were statistically significant at 15 seconds (2546.7 nm), 30 seconds (3884.2 nm) and 60 seconds (3612.1 nm). For moderate fluorosis, a statistically significant difference (p < 0.05) was observed for surface roughness for 30 second (324.5 nm) and 60 second (396.6 nm) treatments. Conclusions: Surface roughness and depth profile analyses revealed that the best etching results were obtained at 15 seconds for the no fluorosis and mild fluorosis groups, and at 30 seconds for the moderate fluorosis group. Increasing the etching time for severe fluorosis decreased surface roughness and the depth profile, which suggests less micromechanical enamel retention for adhesive bonding applications.     

An in vivo model for the identification of serum proteins in the acquired subgingival pellicle

The present study describes an in vivo model for the collection of the subgingival pellicle adsorbed to tooth surface, and the identification of some serum proteins within this layer. Clean dentin slabs were prepared from freshly extracted teeth, and then placed subgingivally for 2 h. The dentin slabs with their adsorbed pellicle layer were processed for transmission electron microscopy. Thin sections were made from the specimens, and treated with antisera to human immunoglobulins and albumin. The reactions were visualized by means of protein A-gold complex, which allowed semiquantification of the serum proteins. The indicator proteins were all identified within the pellicle material, but their amounts and distribution varied. Albumin demonstrated higher amounts in the pellicle layer than other proteins, followed by IgA, IgG, and IgM in descending order. The model described seems useful for studying the acquired subgingival pellicle under varying degrees of disease and health.     

Deposition of alkali-soluble fluoride on enamel surface with or without pellicle

Abstract – Deposition of alkali-soluble fluoride on enamel in the presence and absence of an acquired pellicle was studied in vitro during a single topical application of a 2% sodium fluoride solution at neutral pH. Five experimental groups of repeated measures design were established. The amount of alkali-soluble fluoride formed on enamel (i.e., calcium fluoride) was measured by chemical assessments and by SEM. The results showed that there were no differences among the groups demonstrating the incapacity of pellicle to interfere with deposition of alkali-soluble fluoride on enamel. The fluoride ions are thus able to reach the enamel in spite of the presence of the pellicle.