Development of New Models of Oral Mucosa to Investigate the Impact of the Structure of Transmembrane Mucin-1 on the Mucosal Pellicle Formation and Its Physicochemical Properties.

The mucosal pellicle (MP) is a biological film protecting the oral mucosa. It is composed of bounded salivary proteins and transmembrane mucin MUC1 expressed by oral epithelial cells. Previous research indicates that MUC1 expression enhances the binding of the main salivary protein forming the MP, MUC5B. This study investigated the influence of MUC1 structure on MP formation. A TR146 cell line, which does not express MUC1 natively, was stably transfected with genes coding for three MUC1 isoforms differing in the structure of the two main extracellular domains: the VNTR domain, exhibiting a variable number of tandem repeats, and the SEA domain, maintaining the two bound subunits of MUC1. Semi-quantification of MUC1 using dot blot chemiluminescence showed comparable expression levels in all transfected cell lines. Semi-quantification of MUC5B by immunostaining after incubation with saliva revealed that MUC1 expression significantly increased MUC5B adsorption. Neither the VNTR domain nor the SEA domain was influenced MUC5B anchoring, suggesting the key role of the MUC1 N-terminal domain. AFM-IR nanospectroscopy revealed discernible shifts indicative of changes in the chemical properties at the cell surface due to the expression of the MUC1 isoform. Furthermore, the observed chemical shifts suggest the involvement of hydrophobic effects in the interaction between MUC1 and salivary proteins.

Study of Serial Exposures of an Astringent Green Tea Flavonoid Extract with Oral Cell-Based Models.

It is well known that repeated exposure to phenolic compounds (PCs) raises astringency perception. However, the link between this increase and the oral cavity's interactions with salivary proteins (SPs) and other oral constituents is unknown. To delve deeper into this connection, a flavonoid-rich green tea extract was tested in a series of exposures to two oral cell-based models using a tongue cell line (HSC3) and a buccal mucosa cell line (TR146). Serial exposures show cumulative PC binding to all oral models at all concentrations of the green tea extract; however, the contribution for the first and second exposures varies. The tongue mucosal pellicle (HSC3-Mu-SP) may contribute more to first-stage astringency (retaining 0.15 ± 0.01 mg mL-1 PCs at the first exposure), whereas the buccal mucosal pellicle (TR146-Mu-SP) retained significantly less (0.08 ± 0.02 mg mL-1). Additionally, increased salivary volume (SV+), which simulates the stimulation of salivary flow brought by a food stimulus, significantly enhances PC binding, particularly for TR146 cells: TR46-Mu-SP_SV+ bound significantly higher total PC concentration (0.17 ± 0.02 mg mL-1) than the model without increased salivary volume TR146-Mu-SP_SV- (0.09 ± 0.03 mg mL-1). This could be associated with a higher contribution of these oral cells for astringency perception during repeated exposures. Furthermore, PCs adsorbed in the first exposure to cell monolayer models (+TR146 and +HSC3) change the profile of PCs bound to these models in the second exposure. Regarding the structure binding activity, PCs with a total higher number of hydroxyl groups were more bound by the models containing SP. Regarding the SP, basic proline-rich proteins (bPRPs) may be involved in the increased perception of astringency upon repeated exposures. The extent of bPRP precipitation by PCs in mucosal pellicle models for both cell lines (HSC3 and TR146) in the second exposure (76 ± 13 and 83 ± 6%, respectively) was significantly higher than in the first one (25 ± 14 and 5 ± 6%, respectively).

Molecular mechanisms of aroma persistence: From noncovalent interactions between aroma compounds and the oral mucosa to metabolization of aroma compounds by saliva and oral cells.

The present study aims to reveal the molecular mechanisms underlying aroma persistence, as it plays a major role in food appreciation and quality. A multidisciplinary approach including ex vivo experiments using a novel model of oral mucosa and saliva as well as in vivo dynamic instrumental and sensory experiments was applied. Ex vivo results showed a reduction in aroma release between 7 and 86% in the presence of the thin layer of salivary proteins covering the oral mucosa (mucosal pellicle). This reduction was explained by hydrophobic interactions involving the mucosal pellicle and by the ability of oral cells and saliva to metabolize specific aroma compounds. The in vivo evaluation of exhaled air and perception confirmed the ex vivo findings. In conclusion, this work reveals the need to consider physiological reactions occurring during food oral processing to better understand aroma persistence and open new avenues of research.

Oral mucosal pellicle as an immune protection against micro-organisms in patients with recurrent aphthous stomatitis: A hypothesis.

Recurrent Aphthous Stomatitis (RAS) is the most common ulcerative diseases of oral mucosa affecting an estimate of 20% of the world's population. Majority of the people affected by RAS are under 30 years of age. RAS is located on the lining (non-keratinized) oral mucosa, i.e. buccal mucosa, lateral side of the tongue, soft palate, lip mucosa, or the floor of mouth. An aphthous ulcer develops when lymphocytic cells infiltrate into the epithelium and cause an edema due to transient inflammatory stimuli. Bacteria, viruses and fungi have been suggested to cause aphthous lesions, but findings regarding oral pathogens are conflicting. Prior consensus has been that RAS is a multifactorial condition, with microbes, allergies, nutritional deficiencies, genetic factors, certain illnesses, immunodeficiency, hormonal changes, trauma and stress among others, contributing to the condition. In spite of many suggestions and investigations, the etiology and pathophysiology of RAS remains uncertain. Our hypothesis focuses on mucin proteins that have been shown to play a role in the formation of protective mucosal pellicle, which serves as the first line of defense between oral epithelium and pathogens within the oral cavity. Mucins, including transmembrane mucin 1 (MUC1), and salivary mucins MUC5B and MUC7 form a protein network that is strongly retained to oral epithelium. The role of the mucosal pellicle in pathophysiology of RAS is unknown. Structural variations have been found in the salivary MUC7 terminal end oligosaccharides in RAS patients, rendering the protein unable to agglutinate pathogens. Furthermore, low levels of MUC1 fail to provide a scaffold for assembly of salivary mucins. We introduce a new hypothesis, the alterations in the structure of these glycoproteins could have a profound impact on the oral mucosal barrier function. On the other hand, micro-organisms secreting their mucolytic enzymes destroy the mucosal pellicle causing oral ulcers.

Salivary Factors that Maintain the Normal Oral Commensal Microflora.

The oral microbiome is one of the most stable ecosystems in the body and yet the reasons for this are still unclear. As well as being stable, it is also highly diverse which can be ascribed to the variety of niches available in the mouth. Previous studies have focused on the microflora in disease-either caries or periodontitis-and only recently have they considered factors that maintain the normal microflora. This has led to the perception that the microflora proliferate in nutrient-rich periods during oral processing of foods and drinks and starves in between times. In this review, evidence is presented which shows that the normal flora are maintained on a diet of salivary factors including urea, lactate, and salivary protein degradation. These factors are actively secreted by salivary glands which suggests these factors are important in maintaining normal commensals in the mouth. In addition, the immobilization of SIgA in the mucosal pellicle indicates a mechanism to retain certain bacteria that does not rely on the bacterial-centric mechanisms such as adhesins. By examining the salivary metabolome, it is clear that protein degradation is a key nutrient and the availability of free amino acids increases resistance to environmental stresses.

Proteomic characterization of the mucosal pellicle formed in vitro on a cellular model of oral epithelium.

The oral mucosal pellicle is a thin lubricating layer generated by the binding of saliva proteins on epithelial oral cells. The protein composition of this biological structure has been to date studied by targeted analyses of specific salivary proteins. In order to perform a more exhaustive proteome characterization of pellicles, we used TR146 cells expressing or not the transmembrane mucin MUC1 and generated pellicles by incubation with human saliva and washing to remove unbound proteins. A suitable method was established for the in vitro isolation of the mucosal pellicle by "shaving" it from the cells using trypsin. The extracts, the washing solutions and the saliva used to constitute the pellicles were analyzed by LC MS/MS (data are available via ProteomeXchange with identifier PXD017268). Comparison of pellicle and saliva compositions evidenced the adsorption of proteins not previously reported as pellicle constituents such as proteins of the PLUNC family. Pellicles formed on TR146 and TR146/MUC1 were also analyzed and compared by protein label-free quantification. The two types of samples appeared as distinct clusters in multivariate analyses, but the discriminant proteins (Welch test p < .05, FDR < 0.1) were cellular rather than salivary proteins. SIGNIFICANCE: The oral mucosal pellicle is made of salivary proteins tightly bound to oral epithelial cells. It is essential to oral health, with biological functions depending largely on its protein constituents. Characterizing its proteome is difficult due to the intimate association of this protein layer to cell membranes. In this work, we report a trypsin "shaving" protocol which enabled to sample the pellicle formed on an in vitro cellular model of oral epithelium. Analyzing such samples by high-resolution mass spectrometry provided novel information on the mucosal pellicle composition. This work is therefore a good starting point for further characterization of this biological structure.

Understanding retention and metabolization of aroma compounds using an in vitro model of oral mucosa.

The mechanism leading to aroma persistence during eating is not fully described. This study aims at better understanding the role of the oral mucosa in this phenomenon. Release of 14 volatile compounds from different chemical classes was studied after exposure to in vitro models of oral mucosa, at equilibrium by Gas-Chromatography-Flame Ionization Detection (GC-FID) and in dynamic conditions by Proton Transfer Reaction- Mass Spectrometry (PTR-MS). Measurements at equilibrium showed that mucosal hydration reduced the release of only two compounds, pentan-2-one and linalool (p < 0.05), and suggested that cells could metabolize aroma compounds from different chemical families (penta-2,3-dione, trans-2-hexen-1-al, ethyl hexanoate, nonan- and decan-2-one). Dynamic analyses for pentan-2-one and octan-2-one evidenced that the constituents of the mucosal pellicle influenced release kinetics differently depending on molecule hydrophobicity. This work suggests that mucosal cells can metabolize aroma compounds and that non-covalent interactions occur between aroma compounds and oral mucosa depending on aroma chemical structure.

Biological films adhering to the oral soft tissues: Structure, composition, and potential impact on taste perception.

The role of free-flowing saliva in taste perception is increasingly recognized, but saliva is also present in the mouth as films intimately associated to soft or hard tissues. On mucosal surfaces, particularly on the tongue, the structure and composition of such films (including its microbial constitutive part) may play a particular role in the sense of taste due to their proximity with the taste anatomical structures. This review compiles the current knowledge on the structure of biological films adhering to oral mucosae and on their biochemical and microbiological composition, before presenting possible implications for taste perception. PRACTICAL APPLICATIONS: The understanding of the role of oral biological films on taste perception may provide new avenues of research and development for the industry or academia interested broadly in chemosensation.

The mucosal pellicle - An underestimated factor in oral physiology.

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.

The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium.

OBJECTIVES: The mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium. DESIGN: MUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells. RESULTS: The cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivary mucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p<0.05) or by immunocytochemistry (+44%, p<0.001). CONCLUSION: The membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivary MUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.

Histatin 5 inhibits adhesion of C. albicans to Reconstructed Human Oral Epithelium.

Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. The formation of a saliva-derived protein pellicle on the mucosa surface can provide protection against C. albicans on oral epithelial cells, and narrow information is available on the mucosal pellicle composition. Histatins are one of the most abundant salivary proteins and presents antifungal and antibacterial activities against many species of the oral microbiota, however, its presence has never been studied in oral mucosa pellicle. The objective of this study was to evaluate the potential of histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37°C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37°C at 5% CO2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. HOET pre-incubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization on epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis.