Validation of mechanically-assisted sodium dodecyl-sulphate elution as a technique to remove pellicle protein components from human enamel

The salivary film, denoted the pellicle, formed on oral surfaces is of great importance for oral health and comfort. The present study describes mechanically-assisted sodium dodecyl sulphate (SDS) elution of the in vivo pellicle formed on human enamel and visualisation of the desorbed pellicle proteins using two-dimensional gel electrophoresis (2-DE). To verify this removal of the pellicle, a combined mechanical and surfactant procedure was additionally performed on an in vitro pellicle formed on human enamel, and the effectiveness was validated by mechanical removal in combination with HCl. As indicated by protein quantitation and one dimensional gel electrophoresis, rubbing with polyamide fibre pellets soaked in a 0.5% SDS solution was optimal for completely removing the adsorbed proteins from the enamel surface, and yet provided separation of the proteins by 2-DE to enable identification in future studies.     

Validation of mechanically-assisted sodium dodecyl-sulphate elution as a technique to remove pellicle protein components from human enamel

The salivary film, denoted the pellicle, formed on oral surfaces is of great importance for oral health and comfort. The present study describes mechanically-assisted sodium dodecyl sulphate (SDS) elution of the in vivo pellicle formed on human enamel and visualisation of the desorbed pellicle proteins using two-dimensional gel electrophoresis (2-DE). To verify this removal of the pellicle, a combined mechanical and surfactant procedure was additionally performed on an in vitro pellicle formed on human enamel, and the effectiveness was validated by mechanical removal in combination with HCl. As indicated by protein quantitation and one dimensional gel electrophoresis, rubbing with polyamide fibre pellets soaked in a 0.5% SDS solution was optimal for completely removing the adsorbed proteins from the enamel surface, and yet provided separation of the proteins by 2-DE to enable identification in future studies.     

SIgA Binding to Mucosal Surfaces Is Mediated by Mucin-Mucin Interactions

The oral mucosal pellicle is a layer of absorbed salivary proteins, including secretory IgA (SIgA), bound onto the surface of oral epithelial cells and is a useful model for all mucosal surfaces. The mechanism by which SIgA concentrates on mucosal surfaces is examined here using a tissue culture model with real saliva. Salivary mucins may initiate the formation of the mucosal pellicle through interactions with membrane-bound mucins on cells. Further protein interactions with mucins may then trigger binding of other pellicle proteins. HT29 colon cell lines, which when treated with methotrexate (HT29-MTX) produce a gel-forming mucin, were used to determine the importance of these mucin-mucin interactions. Binding of SIgA to cells was then compared using whole mouth saliva, parotid (mucin-free) saliva and a source of purified SIgA. Greatest SIgA binding occurred when WMS was incubated with HT29-MTX expressing mucus. Since salivary MUC5B was only able to bind to cells which produced mucus and purified SIgA showed little binding to the same cells we conclude that most SIgA binding to mucosal cells occurs because SIgA forms complexes with salivary mucins which then bind to cells expressing membrane-bound mucins. This work highlights the importance of mucin interactions in the development of the mucosal pellicle.     

Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches

Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.     

The composition of enamel salivary films is different from the ones formed on dental materials

This study utilized two-dimensional gel electrophoresis (2DE) to illustrate the compositional differences between in vitro salivary conditioning films (denoted pellicles) formed on human enamel as well as on the dental materials titanium and poly(methyl methacrylate). The salivary pellicles were formed by immersing each surface in individual tubes containing small volumes of freshly collected whole saliva. Saliva remaining in the tubes after the pellicle formation for 2 h was visualized by means of 2DE and silver staining. The results showed that the protein patterns in 2DE of the liquid phase of saliva left after the exposure to the respective surfaces, regarding proteins <100 kDa in size, were different depending on the surface used. Several protein groups and/or individual proteins were shown to be distinct for each surface used.     

Human parotid and submandibular glands express and secrete surfactant proteins A,B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.     

Quantitative proteomic analysis of the effect of fluoride on the acquired enamel pellicle

The acquired enamel pellicle (AEP) is a thin film formed by the selective adsorption of salivary proteins onto the enamel surface of teeth. The AEP forms a critical interface between the mineral phase of teeth (hydroxyapatite) and the oral microbial biofilm. This biofilm is the key feature responsible for the development of dental caries. Fluoride on enamel surface is well known to reduce caries by reducing the solubility of enamel to acid. Information on the effects of fluoride on AEP formation is limited. This study aimed to investigate the effects of fluoride treatment on hydroxyapatite on the subsequent formation of AEP. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition of AEP proteins. Hydroxyapatite discs were randomly divided in 4 groups (n = 10 per group). Each disc was exposed to distilled water (control) or sodium fluoride solution (1, 2 or 5%) for 2 hours. Discs were then washed and immersed in human saliva for an additional 2 hours. AEP from each disc was collected and subjected to liquid chromatography electrospray ionization mass spectrometry for protein identification, characterization and quantification. A total of 45 proteins were present in all four groups, 12 proteins were exclusively present in the control group and another 19 proteins were only present in the discs treated with 5% sodium fluoride. Relative proteomic quantification was carried out for the 45 proteins observed in all four groups. Notably, the concentration of important salivary proteins, such as statherin and histatin 1, decrease with increasing levels of fluoride. It suggests that these proteins are repulsed when hydroxyapatite surface is coated with fluoride. Our data demonstrated that treatment of hydroxyapatite with fluoride (at high concentration) qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms.     

Proteomic Evaluation of Acquired Enamel Pellicle during In Vivo Formation

Acquired enamel pellicle (AEP) is a protein film that forms on the enamel surface of teeth by selective adsorption of proteins and peptides present in the mouth. This protein film forms the interface between enamel and the damage oral biofilm, which modulates the attachment of bacteria found in oral biofilm. The overall goal of this study was to gain insight into the biological formation of the human in vivo AEP. This study hypothesized that AEP is created by the formation of successive protein layers, which consist of initial binding to enamel and subsequent protein-protein interactions. This hypothesis was examined by observing quantitative and qualitative changes in pellicle composition during the first two hours of AEP formation in the oral cavity. Quantitative mass spectrometry approaches were used to generate an AEP protein profile for each time-point studied. Relative proteomic quantification was carried out for the 50 proteins observed in all four time-points. Notably, the abundance of important salivary proteins, such as histatin 1, decrease with increasing of the AEP formation, while other essential proteins such as statherin showed constant relative abundance in all time-points. In summary, this is the first study that investigates the dynamic process to the AEP formation by using proteomic approaches. Our data demonstrated that there are significant qualitative and quantitative proteome changes during the AEP formation, which in turn will likely impact the development of oral biofilms.     

Identification of protein components in in vivo human acquired enamel pellicle using LC-ESI-MS/MS

The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC-ESI-MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed.     

Competitive Adsorption of Lecithin and Saliva at the O/W Interface in Relation to the Oral Processing of Lipid Continuous Foods

This research is relevant to oral processing of lipid continuous foods. During this first step of food digestion, lipid continuous foods such as chocolate or margarine phase invert into oil-in-water emulsions stimulated through the mechanical action of tongue and teeth in combination with the change in temperature and the high surface activity of salivary proteins. These are hypothesised to stabilise the newly formed interface in competition with surfactants or surface active molecules released from the food if present. Here competitive adsorption between mechanically stimulated human whole saliva (HWS) and lecithin dissolved in sunflower oil freed of interfacially active contaminants was investigated in-vitro using a pendant drop tensiometer for dynamic interfacial tension and interfacial rheological measurements. Initially, it was validated that the interfacial properties of HWS samples remained unaffected by frozen storage at ?80 °C during 6 weeks. Protein concentration affected the absolute values of interfacial tension and in particular the dilatational elastic modulus. Competitive adsorption studies revealed a mixed interface and it follows that emulsion stabilisation during oral processing involves both salivary proteins and lecithin present in the oil phase.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.     

The Impact of Stannous,Fluoride Ions and Its Combination on Enamel Pellicle Proteome and Dental Erosion Prevention

Objectives

To compare the effects of stannous (Sn) and fluoride (F) ions and their combination on acquired enamel pellicle (AEP) protein composition (proteome experiment), and protection against dental erosion (functional experiment).

Methods

In the proteome experiment, bovine enamel specimens were incubated in whole saliva supernatant for 24h for AEP formation. They were randomly assigned to 4 groups (n=10), according to the rinse treatment: Sn (800ppm/6.7mM, SnCl₂), F (225ppm/13mM, NaF), Sn and F combination (Sn+F) and deionized water (DIW, negative control). The specimens were immersed 3× in the test rinses for 2min, 2h apart. Pellicles were collected, digested, and analyzed for protein content using liquid chromatography electrospray ionization tandem mass spectrometry. In the functional experiment, bovine enamel specimens (n=10) were similarly treated for pellicle formation. Then, they were subjected to a five-day erosion cycling model, consisting of 5min erosive challenges (15.6 mM citric acid, pH 2.6, 6×/d) and 2min treatment with the rinses containing Sn, F or Sn+F (3×/d). Between the treatments, all specimens were incubated in whole saliva supernatant. Surface loss was determined by profilometry.

Results

Our proteome approach on bovine enamel identified 72 proteins that were common to all groups. AEP of enamel treated with Sn+F demonstrated higher abundance for most of the identified proteins than the other groups. The functional experiment showed reduction of enamel surface loss for Sn+F (89%), Sn (67%) and F (42%) compared to DIW (all significantly different, p<0.05).

Conclusion

This study highlighted that anti-erosion rinses (e.g. Sn+F) can modify quantitatively and qualitatively the AEP formed on bovine enamel. Moreover, our study demonstrated a combinatory effect that amplified the anti-erosive protection on tooth surface.     

Human saliva forms a complex film structure on alumina surfaces

Films formed from saliva on surfaces are important for the maintenance of oral health and integrity by protection against chemical and/or biological agents. The aim of the present study was to investigate adsorbed amounts, thickness, and structure of films formed from human whole saliva on alumina surfaces by means of in situ ellipsometry, neutron reflectivity, and atomic force microscopy. Alumina (Al2O3, synthetic sapphire) is a relevant and interesting substrate for saliva adsorption studies as it has an isoelectric point close to that of tooth enamel. The results showed that saliva adsorbs rapidly on alumina. The film could be modeled in two layers: an inner and dense thin region that forms a uniform layer and an outer, more diffuse and thicker region that protrudes toward the bulk of the solution. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates.     

In vitro hydroxyapatite adsorbed salivary proteins

In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified.     

Adherence, accumulation, and cell division of a natural adherent bacterial population.

Developing dental bacterial plaques formed in vivo on enamel surfaces were examined in specimens from 18 adult volunteers during the first day of plaque formation. An intraoral model placing enamel pieces onto teeth was used to study bacterial plaque populations developing naturally to various cell densities per square millimeter of surface area of the enamel (W. F. Liljemark, C. G. Bloomquist, C. L. Bandt, B. L. Philstrom, J. E. Hinrichs, and L. F. Wolff, Oral Microbiol. Immunol. 8:5-15, 1993). Radiolabeled nucleoside incorporation was used to measure DNA synthesis concurrent with the taking of standard viable cell counts of the plaque samples. Results showed that in vivo plaque formation began with the rapid adherence of bacteria until ca. 12 to 32% of the enamel's salivary pellicle was saturated (ca. 2.5 x 10(5) to 6.3 x 10(5) cells per mm2). The pioneer adherent species were predominantly those of the "sanguis streptococci." At the above-noted density, the bacteria present on the salivary pellicle incorporated low levels of radiolabeled nucleoside per viable cell. As bacterial numbers reached densities between 8.0 x 10(5) and 2.0 x 10(6) cells per mm2, there was a small increase in the incorporation of radiolabeled nucleosides per cell. At 2.5 x 10(6) to 4.0 x 10(6) cells per mm2 of enamel surface, there was a marked increase in the incorporation of radiolabeled nucleosides per cell which appeared to be cell-density dependent. The predominant species group in developing dental plaque films during density-dependent growth was the sanguis streptococci; however, most other species present showed similar patterns of increased DNA synthesis as the density noted above approached 2.5 x 10(6) to 4.0 x 10(6) cells per mm2.     

The fine structure of the gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida)

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200-300+ nm long, 75-100 nm wide, and have a height of approximately 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 microm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.     

Lubricating Properties of the Initial Salivary Pellicle — an AFM Study

The role of saliva in the oral cavity is manifold; an important function is to serve as lubricant between hard (enamel) and soft (mucosal) tissues. Intraoral lubrication is of crucial importance in order to maintain functions such as deglutition, mastication and the faculty of speech. A large number of people suffer from impaired salivary functions, displaying symptoms such as ‘dry mouth’. This results in a need for methods to assess the lubricating properties of both native saliva and potential artificial saliva formulations. Here, normal as well as lateral forces, acting between adsorbed salivary films, have been measured for the first time by means of colloidal probe atomic force microscopy (AFM). It was found that the presence of salivary pellicles between hard surfaces reduces the friction coefficient by a factor of 20. This reduction of friction is consistent with the long-range purely repulsive nature of the normal forces acting between the salivary films. The lubricating mechanism is presumably based on a full separation of the sliding surfaces by the salivary films. The friction between salivary films has been investigated at normal loads that cover the clinical jaw closing forces, and it can be concluded that the lubricating properties are maintained within this load interval. The present study indicates the usefulness of colloidal probe AFM, which offers a direct and quantitative measure of lubrication at a molecular level, in the study of biotribological phenomena. In particular, the results obtained here may have implications for the development of saliva substitutes.     

A comprehensive method for determination of fatty acids in the initial oral biofilm (pellicle)

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.     

Lubricating properties of the initial salivary pellicle--an AFM study

The role of saliva in the oral cavity is manifold; an important function is to serve as lubricant between hard (enamel) and soft (mucosal) tissues. Intraoral lubrication is of crucial importance in order to maintain functions such as deglutition, mastication and the faculty of speech. A large number of people suffer from impaired salivary functions, displaying symptoms such as 'dry mouth'. This results in a need for methods to assess the lubricating properties of both native saliva and potential artificial saliva formulations. Here, normal as well as lateral forces, acting between adsorbed salivary films, have been measured for the first time by means of colloidal probe atomic force microscopy (AFM). It was found that the presence of salivary pellicles between hard surfaces reduces the friction coefficient by a factor of 20. This reduction of friction is consistent with the long-range purely repulsive nature of the normal forces acting between the salivary films. The lubricating mechanism is presumably based on a full separation of the sliding surfaces by the salivary films. The friction between salivary films has been investigated at normal loads that cover the clinical jaw closing forces, and it can be concluded that the lubricating properties are maintained within this load interval. The present study indicates the usefulness of colloidal probe AFM, which offers a direct and quantitative measure of lubrication at a molecular level, in the study of biotribological phenomena. In particular, the results obtained here may have implications for the development of saliva substitutes.