Desorption of acquired enamel pellicle in vivo by pyrophosphate

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.     

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.     

Influence of in vivo formed salivary pellicle on enamel erosion.

This study assessed the protective effect of the salivary pellicle formed in vivo during 24 h or 7 days against demineralization of bovine enamel caused by citric acid. In addition, the influence of acid treatment on the behavior of the pellicle was investigated. Enamel specimens with and without in vivo pellicles were immersed in citric acid (0.1, 1.0%) over 30, 60, and 300 s, and processed for scanning (SEM) and transmission electron microscopy (TEM), as well as for measurement of surface microhardness (SMH). Specimens coated with the in vivo formed pellicles revealed less extensive erosive demineralization of the enamel surface compared to uncovered enamel specimens. SEM analysis and SMH results did not indicate distinct differences between erosive surface alterations on enamel slabs covered with 24-hour pellicles and on those covered with 7-day pellicles. TEM analysis showed that the pellicle layer was dissolved in part from the enamel surface due to acid exposure. However, pellicle residues could be detected by TEM in all specimens, even after 5-min exposure to 1.0% citric acid. It is concluded that the in vivo salivary pellicle can resist the acidic action to some extent and provides protection to the underlying enamel surface against erosive destruction caused by short-term action of citric acid.     

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

Two hour in vivo formed enamel pellicle samples and paraffin wax-stimulated saliva samples were collected from 10 volunteers for analyses of glycosyltransferase 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.     

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.     

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.     

Variations in the initial formation of pellicle and plaque on enamel in vivo.

Four subjects wore acrylic baseplates with small pieces of enamel from unerupted teeth for periods of 24 and 48 hours. The rate of colonization of the pellicle with bacteria varied among the subjects in 2 of whom bacteria were not seen on the surface at 24 hours. Etching the enamel did not enhance the rate of colonization. Chlorhexidine treatment did not prevent bacterial adherence after 2 days, but etched enamel retained the antibacterial effect of chlorhexidine longer than normal enamel.     

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.     

Proteomic profiles of the acquired enamel pellicle formed in vitro,in situ,or in vivo

This study compared the protein profile of the acquired enamel pellicle (AEP) formed under three conditions: in vitro, in situ, and in vivo. Nine volunteers participated in all procedures. In the in vitro condition, the volunteers donated saliva, in which specimens were incubated to form the AEP. In the in situ condition, the volunteers used an oral device containing specimens where the AEP was formed. In the in vivo condition, the AEP was collected from the volunteers own teeth. All AEPs were formed for 120 min, collected and processed by mass spectrometry. Overall, a total of 321 proteins were identified, among which 37 proteins are commonly considered typical in the AEP. For each of the in vitro, in situ, and in vivo conditions, respectively, 66, 174, and 170 proteins were identified. For the in vitro condition, 17 pellicle-typical proteins were not identified. Furthermore, several proteins with important functions within the AEP presented differences in expression in the three conditions. The qualitative profile of the proteins, especially the typical ones, is different in the in vitro condition. In addition, there are important quantitative differences that may interfere when attempting to extrapolate in vitro results to an in situ and in vivo condition.     

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.     

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.     

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.     

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.     

Targeted immobilisation of lysozyme in the enamel pellicle from different solutions

Mouthwashes containing protective enzymes are required especially for patients suffering from xerostomia. The present study aimed to investigate the possibilities of modulating the immobilisation of lysozyme in the in situ pellicle layer. In situ formed pellicles were incubated in vitro for 10 min with various enzymatic buffer solutions containing lysozyme and additive enzymes such as transglutaminase or trypsin as well as polyphenolic compounds (cistus tea). After the rinses, the pellicle samples were incubated in collected whole saliva or in desorption solutions for 0, 20 and 40 min and the enzyme activities were measured. Furthermore, accumulation of lysozyme in the pellicle was visualised in ultrathin sections of the pellicle using the gold immunolabelling technique and transmission electron microscopy. Hen egg white lysozyme was accumulated in the in situ pellicle tenaciously. Up to 2.8-fold higher activities than in controls were observed. The addition of transglutaminase did not enhance the immobilisation of lysozyme activity, whereas the polyphenolic compound had no adverse effect. Accumulation of lysozyme in the acquired pellicle was confirmed by gold immunolabelling. Targeted and tenacious immobilisation of lysozyme in the acquired pellicle is possible. Poylphenolic compounds might be a relevant additive for mouthwashes containing lysozyme.     

Impact of acquired enamel pellicle modification on initial dental erosion

The acquired enamel pellicle that forms on the tooth surface serves as a natural protective barrier against dental erosion. Numerous proteins composing the pellicle serve different functions within this thin layer. Our study examined the effect of incorporated mucin and casein on the erosion-inhibiting potential of the acquired enamel pellicle. Cyclic acidic conditions were applied to mimic the erosive environment present at the human enamel interface during the consumption of soft drinks. One hundred enamel specimens were prepared for microhardness tests and distributed randomly into 5 groups (n = 20) that received the following treatment: deionized water, humidity chamber, mucin, casein, or a combination of mucin and casein. Each group was exposed to 3 cycles of a 2-hour incubation in human saliva, followed by a 2-hour treatment in the testing solution and a 1-min exposure to citric acid. The microhardness analysis demonstrated that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. The addition of individual proteins did not statistically impact the function of the pellicle. These data suggest that protein-protein interactions may play an important role in the effectiveness of the pellicle to prevent erosion.     

Effect of salivary pellicle on enamel subsurface demineralization in vitro.

Diffusion fluxes of two anionic species through hydroxyapatite membranes were found to be reduced up to 50% by the presence of adsorbed salivary pellicles developed on the membrane surfaces. By contrast, water fluxes were only marginally affected, indicating that salivary pellicles display ionic permselectivity. This property is used to explain a remarkable protection of the enamel observed when salivary pellicles were developed on extracted teeth before exposure to acid lactate buffers.     

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.     

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.