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Objective: The objective of this study was to determine whether differences in the abundance of mucins 5b and 7 as well as secretory IgA exist in the oral acquired pellicle between children with active caries and caries-free children., Design: Pellicle formation was performed for 10 min in-situ on ceramic slabs in the oral cavity of children (5-7 years of age) with caries (n = 15) and without signs of caries (n = 13). Furthermore, unstimulated saliva was collected. Concentrations of Muc5b, Muc7 and sIgA were measured in desorbed pellicle eluates and in saliva., Results: Significantly larger concentrations of Muc5b, Muc7 and sIgA were detected in the pellicle obtained from children with caries compared to caries-free children. However, in the salivary samples concentrations of mucins Muc5b and Muc7 as well as sIgA did not differ significantly between the two groups., Conclusions: All three pellicle components Muc5b, Muc7 as well as sIgA could be identified as potential biomarkers for early childhood caries with high sensitivity and specificity. This could contribute to a better understanding of the different caries susceptibility in children., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Extensive biofilm formation on materials used in restorative dentistry is a common reason for their failure and the development of oral diseases like peri-implantitis or secondary caries. Therefore, novel materials and strategies that result in reduced biofouling capacities are urgently sought. Previous research suggests that surface structures in the range of bacterial cell sizes seem to be a promising approach to modulate bacterial adhesion and biofilm formation. Here we investigated bioadhesion within the oral cavity on a low surface energy material (perfluorpolyether) with different texture types (line-, hole-, pillar-like), feature sizes in a range from 0.7-4.5 µm and graded distances (0.7-130.5 µm). As a model system, the materials were fixed on splints and exposed to the oral cavity. We analyzed the enzymatic activity of amylase and lysozyme, pellicle formation, and bacterial colonization after 8 h intraoral exposure. In opposite to in vitro experiments, these in situ experiments revealed no clear signs of altered bacterial surface colonization regarding structure dimensions and texture types compared to unstructured substrates or natural enamel. In part, there seemed to be a decreasing trend of adherent cells with increasing periodicities and structure sizes, but this pattern was weak and irregular. Pellicle formation took place on all substrates in an unaltered manner. However, pellicle formation was most pronounced within recessed areas thereby partially masking the three-dimensional character of the surfaces. As the natural pellicle layer is obviously the most dominant prerequisite for bacterial adhesion, colonization in the oral environment cannot be easily controlled by structural means.

Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2-10.5 nmol/cm 2 ), propionic acid (0.1-8.5 nmol/cm 2 ), glycine (0.7-3.5 nmol/cm 2 ), serine (0.08-2.3 nmol/cm 2 ), galactose (galactose + mannose; 0.035-0.078 nmol/cm 2 ), lactose (0.002-0.086 nmol/cm 2 ), glucose (0.018-0.953 nmol/cm 2 ), palmitic acid (0.26-2.03 nmol/cm 2 ), and stearic acid (0.34-1.81 nmol/cm 2 ). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.

Objective: This study investigated, for the first time, pellicle enzymes with respect to their activity, distribution and fluorescence pattern in children with different caries experience., Design: In-situ pellicles were collected from 41 children (aged 4-6 years) with different caries status; 17 of them were caries-free (dmf = 0), 12 had dental restorations but no current caries (dmf ≥ 2) and 12 had at least two carious lesions (dmf ≥ 2). Bovine enamel samples were fixed on individual upper jaw braces for pellicle formation. After 30 min of intraoral exposure, the pellicle and saliva samples were analysed for the activities of amylase, lysozyme, peroxidase and glucosyltransferase (GTF). The distribution of these enzymes, including GTF-isoforms B, C and D, and the pellicle ultrastructure were examined by gold-immunolabelling and transmission electron microscopy (TEM). Furthermore, interactions between pellicle enzymes and adherent bacteria were visualised using combined fluorescence and immunofluorescence labelling., Results: There were no significant differences in the pellicle enzyme activities between the study groups. TEM analysis revealed the absence of GTF C and D in the pellicle of caries-active children. Amylase, peroxidase and GTF-isoforms showed a random distribution within the pellicle layer; lysozyme was found in the form of clusters. A similar ultrastructural pattern was observed for all subjects. Fluorescence labelling technique enabled visualisation of all enzymes, except for GTF B., Conclusion: Pellicle enzyme activities and ultrastructure are not associated with children's caries status. Further investigation is needed to assess the influence of individual GTF-isoforms on caries susceptibility in children., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The proteinaceous pellicle layer, which develops upon contact with saliva on the surface of teeth, is important for the formation of oral biofilms and for the protection of teeth from abrasion and chemically induced erosion. Astringent food ingredients comprising polyphenols, cationic macromolecules, and multivalent metal salts are known to interact with the pellicle. However, astringent-induced changes in the physicochemical properties of the tooth-saliva interphase are not yet completely understood. Here we provide comprehensive insights into interfacial charging, ultrastructure, thickness, and surface roughness of the pellicles formed on the model substrates silicon oxide (SiO 2 ), Teflon ® AF, and hydroxyapatite, as well as on bovine enamel before and after incubation with the astringents epigallocatechin gallate, tannic acid, iron(III) salt, lysozyme, and chitosan. Quartz crystal microbalance with dissipation monitoring demonstrated viscous behavior of untreated pellicles formed in vitro on the different materials. Electrokinetic (streaming current) measurements revealed that cationic astringents reverse the charge of native pellicles, whereas polyphenols did not change the charge under physiological pH condition. In addition, transmission electron microscopy and atomic force microscopy showed a concentration-dependent increase in average film thickness and pellicle surface roughness as induced by astringents. These multifaceted alterations of the salivary pellicle may come along with an increase in roughness perceived on the teeth, which is part of the complex sensations of oral astringency., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bioadhesion and bio-adsorption of proteins, glycoproteins and other biomolecules are ubiquitous phenomena in the oral cavity. While the protective role of the adsorbed salivary biomolecules on teeth (the acquired enamel pellicle) is well established, it has yet to be defined whether comparable processes occur on the desquamating oral soft tissues. The general term for these layers is pellicle, but due to the different characteristics of the coated surfaces the enamel pellicle and mucosal pellicle are their own entities. There is considerable information on the enamel pellicle, whereas only limited data are available on the mucosal pellicle. This can be attributed to the difficult standardized preparation of this biological structure. Based on the present knowledge the abundant and characteristic components of the mucosal pellicle include secreted soluble mucins (MUC5B, MUC7), membrane-associated epithelial mucins (MUC1), and to a lesser degree CA VI, sIgA, and cystatin. However, it seems to be of completely different ultrastructure as compared with the enamel pellicle. Since it is comprised of larger glycoproteins retaining water, it might be considered as a hydrogel, and it appears to have a lower tenacity than the enamel pellicle. Maturation and turnover are influenced by the delivery of salivary proteins, by the flow of saliva and the underlying desquamating oral epithelium. Its probable functions include lubrication and moisture retention. In general, the mucosal pellicle can be regarded as an underestimated key player in oral physiology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated., Methods: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4',6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle's ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM)., Results: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense., Conclusion: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion., (© 2016 S. Karger AG, Basel.)

Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle., Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4',6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples., Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern., Conclusion: The enzyme activities as well as the pellicle's ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals., (© 2017 S. Karger AG, Basel.)

Objectives: The present in situ study investigated the effect of Inula viscosa tea on the pellicle's acid protective properties and on initial oral biofilm formation., Design: Biofilm formation was performed on bovine enamel slabs on individual maxillary splints. Following 1min of pellicle formation, eight subjects rinsed for 10min with Inula viscosa tea and the splints remained for 8h intraorally. Samples carried after 1-min rinsing with CHX (0.2%) or without rinse served as controls. BacLight™ staining, 4',6-diamidino-2-phenylindole (DAPI)-staining and fluorescence in situ hybridization (FISH) were used for fluorescence microscopic detection of adherent bacteria. For investigation of acid protective properties, three subjects rinsed for 10min with Inula viscosa tea after 1min pellicle formation and kept the splints intraorally for further 19min. Physiological 30-min pellicles and native enamel samples served as controls. After HCl incubation of the samples ex-vivo over 120s (pH 2.0, 2.3, 3.0) calcium- and phosphate release were quantified photometrically. Potential influences on the pellicle's ultrastructure by Inula viscosa tea were evaluated by transmission electron microscopy (TEM)., Results: Application of Inula viscosa tea yielded a significant reduction of adherent bacteria on all enamel samples as detected by fluorescence microscopy. For calcium- and phosphate release no significant effect was recorded. TEM investigation indicated a modification of the pellicle's ultrastructure, but no enhanced protection against erosive noxae., Conclusion: Rinsing with Inula viscosa tea influences the bacterial colonization on enamel in situ over 8h but has no impact on acid protective properties of the pellicle., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: Antiadherent and antibacterial effects of certain plant extracts have been proven to be beneficial in preventive dentistry. In the present in situ/in vitro crossover study, the impact of plant extracts rich in polyphenols on the erosion-protective properties of the in situ pellicle was evaluated., Methods: Individual splints were prepared for 12 subjects for intraoral exposure of bovine enamel specimens. Following formation of a 1-min pellicle, watery plant extracts (leaves of the wild form of Ribes nigrum, the wild form of Origanum as well as a combination of both) were administered for 10 min in situ. Alternatively, a mouth rinse with fluorides (Elmex Kariesschutz) was performed for 1 min. After further oral exposure for 19/28 min, respectively, slabs were removed and incubated with HCl in vitro over 120 s (pH 2, 2.3, 3). The resulting calcium and phosphate release was quantified photometrically. Slabs with and without a 30-min in situ pellicle served as controls. The modification of pellicle ultrastructure was evaluated by transmission electron microscopy (TEM)., Results: Plant extracts modulated the erosion-protective properties of the native in situ pellicle in all test groups in a pH-dependent manner. The combination of R. nigrum leaves and Origanum enhanced the protective properties of the pellicle at all pH values; the administration of this preparation was comparable, yet superior, to the effect of the fluoridated mouth rinse. TEM images indicated that rinsing with R. nigrum leaves/Origanum yielded a distinctly thicker and more electron-dense pellicle., Conclusion: The combination of certain plant extracts offers a novel approach to the complementary prevention of dental erosion., (© 2015 S. Karger AG, Basel.)

All tooth surfaces exposed to the oral environment are naturally coated by the acquired salivary pellicle. The pellicle is composed of adsorbed macromolecular components from saliva, gingival crevicular fluid, blood, bacteria, mucosa and diet. The pellicle (formed in situ/in vivo) functions as a semipermeable network of adsorbed salivary macromolecules and provides partial protection against acidic challenges; however, it cannot completely prevent demineralization of the tooth surface. The physiological pellicle reduces calcium and phosphate release from the enamel, and much less from the dentinal surface. With high probability, calcium- and phosphate-binding peptides and proteins adsorbed in the basal pellicle layer are of main relevance for the erosion-reducing effects of the natural salivary pellicle. Improvement of the pellicle's protective properties by dietary components (e.g. polyphenolic agents) might be a promising erosion-preventive approach that, however, needs validation by in situ experiments., (© 2014 S. Karger AG, Basel.)

The first step of bioadhesion on dental surfaces is the formation of the acquired pellicle. This mainly acellular layer is formed instantaneously on all solid surfaces exposed to oral fluids. It is composed of proteins, glycoproteins and lipids. However, information on the lipid composition is sparse. The aim of the present study was to evaluate the fatty acid (FA) profile of the in-situ pellicle for the first time. Furthermore, the impact of rinses with safflower oil on the pellicle's FA composition was investigated. Pellicles were formed in situ on bovine enamel slabs mounted on individual upper jaw splints. The splints were carried by ten subjects over durations of 3-240 min. After comprehensive sample preparation, gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS) was used in order to characterize qualitatively and quantitatively a wide range of FA (C12-C24). The relative FA profiles of the pellicle samples gained from different subjects were remarkably similar, whereas the amount of FA showed significant interindividual variability. An increase in FA in the pellicle was observed over time. The application of rinses with safflower oil resulted in an accumulation of its specific FA in the pellicle. Pellicle formation is a highly selective process that does not correlate directly with salivary composition, as shown for FA.

The acquired pellicle is a tenacious organic layer covering the surface of teeth, protecting the underlying dental hard tissues. Lipids account for about one quarter of the pellicle's dry weight and are assumed to be of considerable importance for their protective properties. Nevertheless, only preliminary information is available about the nature of lipids in the pellicle. Gas chromatography coupled with electron impact ionization mass spectrometry was used to establish a convenient analytical protocol in order to obtain a qualitative and quantitative characterization of a wide range of FAs (C(12)-C(22)). In situ biofilm formation was performed on bovine enamel slabs mounted on individual splints carried by 10 subjects. A modified Folch extraction procedure was adopted to extract the lipids from the detached pellicle, followed by transesterification to fatty acid methyl esters using methanol and concentrated hydrochloric acid. Tridecanoic and nonadecanoic acid were used as internal standards suitable and reliable for robust, precise and accurate measurements. The present study demonstrates, for the first time, a procedure based on a combination of innovative specimen generation and convenient sample preparation with sensitive GC-MS analysis for the determination of the fatty acid profile of the initial oral biofilm.

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.

One of the greatest challenges in life sciences and biomaterials research is adhesion of biomolecules and bacteria to solid surfaces in aqueous solutions. An example concerning everybody is biofilm formation in the oral cavity on dental materials and dental hard substances, respectively. The main characteristics typical for any bioadhesion can be observed excellently in the oral cavity. Initially, a proteinaceous layer termed pellicle is formed. It mediates the interactions between solid substrata, oral fluids and microorganisms. Numerous different materials with differing physico-chemical properties and possible impact on the acquired pellicle are present in the oral cavity such as enamel, dentine, restorative materials or dental implants. Despite the fact that in vitro studies demonstrate considerable differences of experimental pellicles formed on these materials, the in situ pellicles seem to be relatively similar and level off the different properties of the underlying substrates. However, the bacterial colonisation of pellicle-coated surfaces under in vivo conditions differs considerably. Long-range forces and detachment of biofilm layers may account for this phenomenon despite the masking effect of the pellicle. Accordingly, low-energy surfaces are desirable for restorative materials exposed to the oral cavity to minimise bacterial adhesion. The oral cavity is an easy accessible in vivo model for understanding bioadhesion and for investigation of protein-surface interactions noninvasively. For evaluation of biofilm formation on dental materials, in situ or in vivo studies are preferable.

Lysozyme is among the most protective enzymes in the pellicle layer. The aim of the present study was to establish a precise fluorimetric assay for determination and characterisation of lysozyme activity immobilised in the initial in situ formed pellicle. For in situ pellicle formation, bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 min) on buccal and palatal sites. The enzymatic assay was based on hydrolysis of cell walls from Micrococcus lysodeicticus linked to a fluorogenic substance. When the substrate is hydrolysed, a fluorescing product is released. Furthermore, the effects of chlorhexidine and black tea on lysozyme in the in situ pellicle were investigated. The fluorimetric method allowed direct determination of the enzyme activity with the slab inside the well of a microtiter plate. The mean immobilised activity over all samples amounted to 68.67 +/- 27.35 U/cm(2) (desorbed activity = 46.76 +/- 21.18 U/cm(2)). The enzyme activity exposed at the pellicles' surfaces increased in a time-dependant manner and showed a Michaelis-Menten kinetic. Chlorhexidine and black tea reduced lysozyme activity of the in situ pellicle significantly. After rinsing with tea or chlorhexidine, V(max) was reduced, whereas K(m) remained unaffected indicating a negative allosteric effect of the V type. The fluorimetric method is appropriate for determination of pellicle lysozyme activity. The influence of effectors on immobilised lysozyme activity can be monitored.

Aim: Peroxidase is the main salivary antioxidant. The aim of the present study was to detect and to characterise peroxidase in the in situ enamel pellicle., Methods: Bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 and 120 min) on buccal and palatal sites. Pellicle bound peroxidase activity was determined fluorimetrically using 2',7'-dichlorofluorescin as a substrate. The peroxidase molecules present in the pellicle were visualised with the gold-immunolabelling technique and evaluated by TEM. Furthermore, effects of polyphenols and hydrogen peroxide on peroxidase and its enzymatic activity were examined., Results: All pellicles which were tested revealed peroxidase activity and labelled peroxidase molecules were detected in all samples. The numbers of gold-labelled peroxidase molecules detectable in cross-sections of the pellicles were correlated significantly with the pellicle formation time. After 3 min, 0.50+/-1.01 labelled molecules were detected (30 min: 1.42+/-1.98; 120 min: 4.15+/-4.13, ANOVA, p<0.001). The mean immobilised peroxidase activity exposed at the surface amounted to 24.4+/-27.7 mU/cm2; no continuous increase of activity with formation time was found. Hydrogen peroxide and polyphenolic beverages inactivated peroxidase activity of the pellicle. Despite these inhibiting effects, considerable amounts of peroxidase molecules were still detectable by gold-immunolabelling. After contact with the inhibiting agents in situ, peroxidase activity of the pellicle reconstituted slowly., Conclusion: Peroxidase activity is present in the pellicle already after 3 min of formation time, but is inhibited by the substrate and polyphenolic beverages.

Objectives: Polyphenols are expected to have antibacterial properties. Cistus is a tea rich in polyphenols. The aim of the present in situ study was to investigate the effect of Cistus-tea on the pellicle and on the initial oral biofilm., Methods: For in situ pellicle formation and initial biofilm formation, bovine enamel slabs were fixed on maxillary splints and carried by four subjects at buccal sites for up to 2 h. Bacteria present in 120-min pellicles were determined with DAPI-staining and fluorescence in situ hybridization with and without a 10 min rinse with Cistus-tea performed 1 min after incorporation of the slabs. In addition, amylase, lysozyme, glucosyltransferase and peroxidase activities immobilised in the pellicle layer were measured before and after rinsing for 10 min with Cistus-tea., Results: The amount of bacteria detected in the 120-min biofilm was reduced significantly, if a 10 min rinse with Cistus-tea was performed one min after insertion of the enamel slabs. DAPI-staining yielded 13.2+/-3.5 for controls and 6.5+/-1.1 x 10(4) bacteria/cm(2), if a rinse with Cistus-tea was applied. Lysozyme, amylase and glucosyltransferase activities immobilised in the pellicle were not affected following a rinse with Cistus-tea. However, peroxidase activity was reduced significantly., Conclusions: Cistus-tea may be used to reduce the initial bacterial adhesion in the oral cavity.

Amylase and lysozyme are components of the salivary pellicle, exposing considerable enzymatic activity in the immobilized state. The purpose of the present study was to elucidate the influence of different solid substrata on the amount and distribution of amylase and lysozyme exposed on the surface of the salivary pellicle formed in situ. Slabs of titanium, feldspar ceramic, and bovine enamel were fixed on the buccal sites of individual splints worn by three subjects for 3 or 30 min, respectively, to allow pellicle formation. Subsequently, slabs were removed from the splints and rinsed with running water. Detection of amylase and lysozyme was performed by FEI-SEM after gold-immunolabeling of the enzymes. Both enzymes were found to be distributed randomly at the pellicle surface. Irrespective of formation time and substratum, significantly more labeled lysozyme molecules (5.23 +/- 4.5 microm(-2)) were detected compared with amylase (3.4 +/- 2.9 microm(-2)). Neither the substratum nor the pellicle formation time had significant impact on the amount of the respective enzyme that could be detected. This study for the first time provides evidence, that amylase and lysozyme are exposed at the surface of the salivary pellicle formed in situ on titanium and ceramics. Both enzymes are distributed randomly on the surface of the pellicle, irrespective of the underlying substratum.

Several salivary anti-microbial and buffering components are part of the acquired in vivo pellicle. The purpose of the present in situ study was to visualise these proteins within the in situ formed pellicle and to investigate their distribution with respect to pellicle formation time and intra-oral localisation. Bovine enamel slabs were fixed on individual splints. They were carried by 6 subjects buccally and palatally in the region of the upper first molar teeth over 30 and 120 min, respectively, for in situ pellicle formation. After intra-oral exposure, enamel specimens were processed for transmission electron microscopy. Secretory immunoglobulin A (sIgA), lactoferrin, lysozyme, carbonic anhydrase (CA) I and II were visualised successfully in the in situ pellicle layer by gold immuno-labelling. All components were found to be distributed randomly within all layers of the pellicle. Significantly higher amounts of the proteins were detected after 120 min of formation time. Furthermore, significantly more labelled lactoferrin and lysozyme were found on buccal surfaces compared with palatal sites. For CA I, CA II and sIgA, no significant influence of the localisation was detected. All investigated anti-bacterial and buffering proteins are distributed randomly in the in situ formed pellicle layer and thus could contribute to its protective properties as an early defence barrier.

Aim: The acquired pellicle is well known as an anti-erosive proteinaceous layer on enamel, but its protective properties on dentin have not been investigated in detail until now. The aim of the present ex vivo study was to evaluate the erosive effects on pellicle coated dentin., Methods: Bovine dentin slabs were exposed to the oral cavity of one subject for 120 min for in situ pellicle formation. Subsequently, the slabs were incubated with HCl (pH 2.3) in vitro for 5 min and erosive calcium-release was measured photometrically. In addition, the acid treated specimens were evaluated by transmission electron microscopy (TEM). Pellicle free samples served as controls., Results: Calcium erosion from the pellicle coated dentin slabs amounted to 23.5+/-2.9 microg Ca/min (pellicle free samples: 32.2+/-4.2 microg Ca/min). The difference was statistically significant (p < or = 0.05). In pellicle coated as well as in uncoated dentin samples, TEM-evaluation showed a demineralised dentinal surface layer which thickness ranged between 3 and 6 microm. The pellicle itself was partially dissolved but not removed by hydrochloric acid treatment., Conclusion: The protective properties of the acquired pellicle against an erosive challenge of the dentinal surface are limited. The dentinal pellicle functions like an ion permeable network rather than a barrier.

Unlabelled: Lysozyme is one of the most abundant enzymatic components in the salivary pellicle. The purpose of the present in situ study was to determine if and to which extent lysozyme immobilised in pellicles exposes enzymatic activity. Influence of different oral sites and pellicle formation time on enzyme activity was also evaluated. Bovine enamel slabs (5mm diameter) were fixed on buccal and oral sites of individual trays worn by six subjects for 3 and 30 min on different days. After pellicle formation, slabs were removed from the trays and rinsed with running water. Afterwards, pellicle-bound lysozyme activity was determined via lysis of Micrococcus lysodeicticus photometrically in two steps. In a first step, lysozyme was desorbed in phosphate buffer and dissolved activity was measured. In a second step, slabs were incubated in phosphate buffer with the substrate and remaining immobilised activity was determined. All investigated pellicles exhibited lysozyme activity. Great intra- and inter-individual differences were observed. Mean desorbed activity of 3 min-pellicles amounted to 26.06+/-17.81 U/cm(2) (30 min; 26.79+/-17.48). The remaining immobilised activity was 13.54+/-11.42 for 3 min-pellicles and 16.08+/-12.81 for 30 min-pellicles. Pellicle derived lysozyme showed a Michaelis type kinetic., Conclusion: In situ pellicle exposes lysozyme activity even after a 3 min formation period. Exposed enzyme activity is neither influenced by pellicle formation time nor by the site of pellicle formation. It shows great inter- and intra-individual differences.

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.

Immunological and biochemical analyses have shown that alpha-amylase is an essential component of the acquired pellicle. After adsorption, this enzyme might act as a receptor for bacterial adherence. However, data indicating that amylase is bound to the pellicle surface in vivo and thus available for adhering bacteria are rare. Therefore, the present study focused on alpha-amylase within the pellicle formed in situ, using gold-immunolabeling electron microscopic techniques. Pellicles were formed by intra-oral exposure of enamel specimens for 30 and 120 min in six subjects. The results obtained by transmission electron microscopy indicate that amylase was randomly distributed in the pellicle layer without any preferential localization within the pellicle. Thus, salivary alpha-amylase might be considered as an important structural component that is even involved in the early stages of pellicle formation. The findings of field emission in-lens scanning electron microscopy provided evidence that the enzyme is located on the pellicle surface. It could be concluded that alpha-amylase might act as a receptor for bacterial adherence to the pellicle in vivo., (Copyright Eur J Oral Sci, 2004.)

Unlabelled: 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.