Electron microscopic detection of salivary alpha-amylase in the pellicle formed in situ

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.     

Protective effect of the in situ formed short-term salivary pellicle

Salivary pellicle, as previously investigated, protects the enamel surface after certain processes of maturation against the influence of acidic agents. The aim of the present study was to investigate the protective effect of the short-term salivary pellicle formed in situ over periods of 3, 60 and 120 min. Six human volunteers used intraoral acrylic splints with bovine enamel samples fixed at the buccal and palatal sites of the maxillary first molars and second premolars. Enamel specimens (n = 252) with and without pellicle were immersed for 60 s in 1.0% citric acid solution under agitation. Knoop surface hardness (KHN) of uneroded polished enamel was measured as a baseline and estimated immediately after erosive treatment reflecting the microhardness loss (DeltaKHN). The amounts of calcium dissolved from the eroded enamel surface were analysed by atomic absorption spectroscopy and scored in mg/l per 10 mm2 of enamel surface area. In addition, the scanning electron microscope was used for the micromorphological examination of the erosive alterations of the enamel surface. The average microhardness loss values after erosion of the enamel samples with buccally/palatally formed pellicle layers were measured as 139.1/144.9 DeltaKHN for 3 min pellicle, 145.9/146.9 DeltaKHN for 60 min pellicle and 141.7/138.6 DeltaKHN for 120 min pellicle. Calcium release values from the specimens with buccal/palatal pellicles were amounted to 15.0/14.9, 16.5/15.9 and 15.3/17.4 mg/l per 10 mm2 for 3, 60 and 120 min-old pellicles, respectively. No significant differences were related to the pellicle formation time and intraoral site (buccal or palatal) in all tested series (ANOVA, P < 0.05). However, significant protection of the enamel surface provided by the pellicle layer was observed on all pellicle-covered surfaces if compared to the non-covered enamel samples (calcium release: 25.6 mg/l per 10 mm2; microhardness loss 187.0 DeltaKHN). These data were in accordance with the morphologic alterations caused by citric acid on the pellicle-covered and pellicle non-covered specimens. It could be concluded that salivary pellicle formed in situ within a period of 3 min offers protection of enamel against citric acid. However, pellicle does not completely inhibit the erosive action of citric acid under the conditions of the present study.     

唾液获得性膜对不同桥体材料表面自由能的影响

目的：评价唾液获得性膜对不同桥体材料表面自由能的影响。方法：采用接触角测量仪检测4种桥体材料(Co—Cr合金、Au—Pt合金、纯Ti以及Vita95瓷)表面形成唾液获得性膜前后试件的接触角并计算表面能。结果：制备唾液获得性膜后所有受测材料的表面自由能极性分量升高，自由能总量趋于一致。结论：唾液获得性膜会改变修复材料的表面属性，使不同材料表面自由能之间的差异减小。     

An in vitro model of chlorhexidine-induced tooth staining

BACKGROUND: Tooth staining is a common feature of chlorhexidine treatment for periodontal disease and there is a large variation between patients as to the degree of their tooth staining. Although the mechanism of tooth staining is uncertain, diet, smoking and oral hygiene appear probable factors. OBJECTIVES: This study investigated the role of saliva in chlorhexidine-induced tooth staining and used tea as the staining agent in an in vitro model with hydroxyapatite mimicking teeth. METHODS: Saliva has been used to create an acquired pellicle and in solution to mimic its effects in vivo. Using different combinations of tea, chlorhexidine and parotid saliva, substances binding to hydroxyapatite were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this system, tea, chlorhexidine and salivary proteins were clearly identifiable following staining by Coomassie Brilliant Blue. RESULTS: The results indicated that tea interacted with many salivary proteins, in particular proline-rich proteins and histatins. Chlorhexidine did not appear to complex with or precipitate salivary proteins nor prevent the formation of an acquired pellicle on the hydroxyapatite. In isolation, tea and chlorhexidine bound in small amounts to hydroxyapatite, but when added in combination, binding of both to hydroxyapatite was greatly increased. The acquired pellicle reduced chlorhexidine and tea binding, but conversely increased the binding of either tea or chlorhexidine alone to hydroxyapatite. CONCLUSION: In conclusion, salivary proteins play an important role in the staining process and the combination of tea and chlorhexidine appears to be a very potent staining factor.     

Real-time interaction of oral streptococci with human salivary components

Oral streptococci are present in large numbers in dental plaque and several types interact with the enamel salivary pellicle to form a biofilm on tooth surfaces. The respective affinity of individual streptococci for salivary components has an influence on the etiologic properties of oral biofilm in the development of dental caries. We studied real-time biospecific interactions between oral streptococci and salivary components utilizing biosensor technology to analyze surface plasmon resonance. Streptococcus sanguis and Streptococcus mutans showed significant responses for binding to salivary components, in comparison with other bacteria. Further, the association rates (4.1 x 10-11/bacterium) and dissociation rate (5.7 +/- 0.9 x 10-3 Second(s)-1) were higher for S. sanguis than for S. mutans (2.4 x 10-11 and 2.9 +/- 0.8 x 10-3) and Streptococcus mitis (1.3 x 10-11 and 3.5 +/- 1.3 x 10-3). However, the association equilibrium constants (8.2 S/bacterium) for S. mutans was 2 times higher in than S. mitis (3.8) and slightly higher than S. sanguis (7.2). These findings may provide useful information regarding the mechanism of early biofilm formation by streptococci on the tooth surface.     

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.     

The effects of pellicle formation on streptococcal adhesion to human enamel and artificial substrata with various surface free-energies

The influence of a pellicle on streptococcal adhesion was studied. A "ripened" two-hour salivary pellicle and an "early" five-minute salivary pellicle were formed on human enamel and artificial solid substrata with varying surface free-energies. Three strains of oral streptococci, also with widely different surface free-energies, were used for adhesion studies. Pellicle formation and streptococcal adhesion took place at a constant shear rate of 21 s-1. Adhesion of S. mitis BMS to bare and pellicle-covered enamel was low and not significantly affected by the presence of a pellicle (0.7 x 10(6) and 0.6 x 10(6) cells.cm-2, resp.), whereas the numbers of S. sanguis 12 and S. mutans NS adhering to bare enamel (4.2 x 10(6) cells.cm-2 and 13.8 x 10(6) cells, cm-2, resp) were significantly reduced by the presence of a pellicle. This reduction was almost complete after only five minutes of salivary protein adsorption (1.9 x 10(-6) and 1.1 x 10(6) cells.cm-2 for S. sanguis and S. mutans, resp.) but further reduced for S. sanguis adhering to a ripened pellicle (0.7 x 10(6) cells.cm-2). The numbers of streptococci adhering at equilibrium to bare enamel could be fitted to a thermodynamically based model, which was previously described for bacterial adhesion to homogenous artificial substrata. Streptococcal adhesion to artificial substrata exposed to saliva was low, and the differences among uncoated materials were markedly reduced even after only five minutes' exposure to saliva.     

External radiolabelling of components of pellicle on human enamel and cementum

Enamel and cementum pellicles form by different adsorption of salivary and serum components to the tooth surface. The authors compared the constituents of surface pellicle formed on human enamel and cementum under three conditions: (1) natural pellicle, present on extracted teeth, which was formed by prolonged exposure to human salivary and serum components in vivo; (2) short-term in-vivo pellicle, formed by exposing enamel and cementum slabs to the oral environment for 0-60 min; (3) in-vivo pellicle, formed by incubating enamel and cementum slabs in a 1:1 mixture of parotid and submandibular/sublingual saliva for 0-60 min. Pellicle composition was characterized by external radiolabelling techniques specific for exposed carbohydrate (sialic acid and galactose) and amino-acid (tyrosine) residues. There were differences between cementum and enamel in the electrophoretic profiles of natural-pellicle components; notably, a major 180 kda 3H-labelled sialoglycoprotein, unique to the cementum pellicle, had the same electrophoretic mobility as the low-molecular-weight mucin from human submandibular/sublingual saliva. After alkaline-borohydride treatment, 3H-labelled natural-pellicle oligosaccharides chromatographed in the di- to tetrasaccharide region of a Bio-Gel P-2 column. The most prominently labelled components of short-term enamel and cementum pellicles in vivo and in vitro had the same electrophoretic mobility as the low-molecular-weight salivary mucin. The pellicle components formed in vitro, unlike those formed for the same period of time in vivo, were rapidly desorbed from the cementum, but not from the enamel surface. We conclude that: (1) external labelling techniques are useful for obtaining a profile of pellicle components; (2) submandibular/salivary mucins are major constituents of salivary pellicles on tooth surfaces; (3) glycoproteins that carry low-molecular-weight, sialic-acid-containing saccharides are important determinants of pellicle surface properties [corrected].     

Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period

The purpose of the present in vivo study was to examine salivary pellicle formation on enamel surfaces at two different intraoral sites for periods of 1 min up to 24 h by means of transmission electron microscopy. Bovine enamel specimens were attached to the buccal and lingual surfaces of the upper first molars in three subjects using removable intraoral splints. Specimens were carried over periods of 1, 10, 30 and 60 min, 2, 6 and 24 h and were processed for transmission electron microscopy. After 1 min, an electron dense pellicle layer, 10–20 nm thick, was observed on the enamel surfaces. The subsequent adsorption of salivary biopolymers was governed by local influences of the oral cavity. Specimens located on the lingual aspect were covered within 2 h by a 20- to 80-nm-thick, homogeneous, predominantly granular-structured pellicle. The thickness of the surface coatings that were adsorbed on lingually carried specimens increased to 100–200 nm after 24 h. In contrast, on the buccally mounted specimen surfaces, a variably structured pellicle with granular and globular components could be detected after intraoral exposure for 2 h. The thickness of the 2-h buccal pellicles ranged between 200 and 700 nm. After 24 h, the buccally positioned specimens were covered by a dense globular pellicle layer varying in thickness from 1000 to 1300 nm. It is suggested that in vivo pellicle formation is initiated by adsorption of an electron-dense layer of salivary proteins. Further adsorption of salivary biopolymers leads to the formation of an outer loosely arranged pellicle layer. Under oral conditions, the locally available salivary biopolymers and the influence of locally effective shearing forces are of significance for the ultrastructural pattern and extent of pellicle formation. Received: 8 March 1999 / Accepted: 19 April 1999     

Ellipsometry analysis of the in vitro adsorption of tea polyphenols onto salivary pellicles

The adsorption of components from black tea and of purified tea polyphenols onto a whole unstimulated salivary pellicle-like protein layer, formed in vitro on hydroxyapatite discs, was studied by in situ ellipsometry. It was found that components from black tea and the purified polyphenols epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and theaflavin readily adsorbed onto the pellicle. Further investigations showed that under the experimental conditions of this study, no black tea- or purified polyphenol-modified pellicles were eluted by either phosphate buffer or sodium dodecyl sulphate rinses. Therefore, black tea and its polyphenol components are indicated to have a profound effect on in vitro pellicle modification. Similar effects were observed for tannic acid.     

Influence of salivary pellicle formation time on enamel demineralization – an in situ pilot study

This study assessed the protective potential of salivary pellicles formed in situ over periods ranging from 2 to 24 h. Pellicles were produced on enamel slabs mounted on the palatal aspect of removable acrylic splints and exposed to the oral environment in three subjects for 2, 6, 12 and 24 h. Enamel specimens with and without pellicles were immersed in citric acid (1%) for 60 s, and the amount of dissolved calcium was measured by atomic absorption spectroscopy. In addition, specimens were processed for transmission electron microscopy (TEM). Mean values (standard deviations) for calcium release (mg/l related to the specimen's surface area of 5×5 mm²) were: 2-h pellicle 6.94 (1.55); 6-h pellicle 6.69 (2.05); 12-h pellicle 6.57 (2.31); 24-h pellicle 5.71 (2.46); enamel without pellicle 8.95 (1.66). There were no significant differences in calcium release that were dependent on pellicle formation time, but in comparison to enamel specimens without pellicle, significantly less (p <0.05) demineralization of the enamel was observed in pellicle-covered specimens. TEM showed that the pellicle was partly, but not completely dissolved following acid exposure. It is concluded that even a 2-h in-situ-formed pellicle layer protects the enamel surface to a certain extent against demineralization.     

Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle

Salivary carbonic anhydrase (CA VI) appears to protect teeth from caries via mechanisms other than direct regulation of salivary pH and buffering capacity. To elucidate whether CA VI acts in the local microenvironment of the tooth surface, we studied the location and activity of the enzyme in the human enamel pellicle. The study was performed using a specific rabbit antiserum to human CA VI in conjunction with immunostaining and immunoblot techniques. CA activity was demonstrated using a histochemical staining method. CA VI immunostaining of extracted teeth having in vivo formed pellicle showed that the enzyme is present in the enamel pellicle. Immunostaining for salivary alpha-amylase, which is known to be present in the pellicle, showed a similar staining pattern. The presence of CA VI in the enamel pellicle was confirmed by immunoblotting of in vivo formed pellicle proteins. In vitro studies showed that CA VI binds to polished enamel surfaces from both saliva and solutions of purified enzyme. The intensity of the CA VI immunostaining on the enamel surface was dependent on the concentration of the applied enzyme. The histochemical staining of in vitro formed enamel pellicle confirmed that the bound enzyme retains its enzymatic activity. The presence of active CA VI in the human enamel pellicle suggests that it may accelerate the removal of acid by functioning locally in the pellicle layer on dental surfaces.     

Salivary non-immunoglobulin agglutinin inhibits human leukocyte elastase digestion of acidic proline-rich salivary proteins

Saliva contains acidic proline-rich salivary proteins that are involved in the formation of the salivary pellicle coating supragingival tooth surfaces. However, human leukocyte elastase, arriving in gingival exudates from inflamed periodontal tissues, degrades the acidic proline-rich salivary proteins, preventing binding to hydroxylapatite surfaces. Here it is reported that high-molecular-weight non-immunoglobulin salivary agglutinin inhibited the proteolytic action of human leukocyte elastase on purified acidic proline-rich salivary proteins. Inhibition was eliminated with monoclonal antibody to a protein determinant on the salivary agglutinin. The addition of antibody against salivary agglutinin blocked the inhibitory effect of parotid saliva on exogenously applied human leukocyte elastase, allowing for the elastase-mediated digestion of the salivary acidic proline-rich salivary proteins. Salivary agglutinin, therefore, is a physiologically important inhibitor of human leukocyte elastase and is able to inhibit elastase-mediated digestion of salivary acidic proline-rich proteins.     

Salivary secretion in health and disease

Saliva is a complex fluid produced by 3 pairs of major salivary glands and by hundreds of minor salivary glands. It comprises a large variety of constituents and physicochemical properties, which are important for the maintenance of oral health. Saliva not only protects the teeth and the oropharyngeal mucosa, it also facilitates articulation of speech, and is imperative for mastication and swallowing. Furthermore, saliva plays an important role in maintaining a balanced microbiota. Thus, the multiple functions provided by saliva are essential for proper protection and functioning of the body as a whole and for the general health. A large number of diseases and medications can affect salivary secretion through different mechanisms, leading to salivary gland dysfunction and associated oral problems, including xerostomia, dental caries and fungal infections. The first part of this review article provides an updated insight into our understanding of salivary gland structure, the neural regulation of salivary gland secretion, the mechanisms underlying the formation of saliva, the various functions of saliva and factors that influence salivary secretion under normal physiological conditions. The second part focuses on how various diseases and medical treatment including commonly prescribed medications and cancer therapies can affect salivary gland structure and function. We also provide a brief insight into how to diagnose salivary gland dysfunction.     

Protective properties of salivary pellicles from two different intraoral sites on enamel erosion

The purpose of this study was to investigate the protective effect and ultrastructure of salivary pellicles formed in vivo near the orifices of the ducts of parotid and submandibular/sublingual salivary glands. Pellicles were formed by exposing bovine enamel slabs to the oral environment at the buccal aspect of the upper first molars and at the lingual aspect of the lower incisors in 3 subjects over periods of 24 h. Enamel specimens with and without 24-hour pellicles were immersed in citric acid (0.1 and 1%) for periods ranging from 30 s to 5 min, and processed for measurement of surface microhardness (SMH) and transmission electron microscopy (TEM). In comparison to uncovered enamel specimen significantly less decrease in SMH due to acid exposure was observed in pellicle-coated enamel specimens. Pellicles formed at the buccal aspect of the upper molars were less effective in protecting the enamel against acid-induced softening as compared to pellicles formed at the lingual aspect of the lower incisors only after 5 min exposure in 1% citric acid. TEM analysis showed that pellicle layers were dissolved continuously due to acid exposure. However, even after 5 min exposure to 1% citric acid, a residual pellicle layer could be detected on the enamel surface. In conclusion, site-dependent differences of buccally and lingually in vivo formed 24-hour pellicles have minor importance concerning the pellicle-induced protection of the enamel surface against erosive changes.     

Non-destructive visualisation of protective proteins in the in situ pellicle

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.     

Adsorption from black tea and red wine onto in vitro salivary pellicles studied by ellipsometry

The adsorption of black tea and red wine components onto a pellicle-like protein layer formed in vitro by adsorption from whole unstimulated saliva on hydroxyapatite discs were studied by in situ ellipsometry. It was found that components from black tea readily adsorbed to the pellicle. Subsequent exposure to saliva led to further adsorption of salivary components to give an overall increase in the amounts adsorbed. The amounts adsorbed increased still further following a third tea and saliva exposure. Components of red wine gave significantly greater amounts of adsorption to the pellicle than black tea. The adsorption of components of black tea gave a concomitant increase in colour or stain as measured by a reflectance chromameter. In all cases, the black tea- and red wine-modified pellicles were not eluted by either phosphate buffer or sodium dodecyl sulphate (SDS) rinses. Thus, black tea and red wine components have been shown to have a profound effect on in vitro pellicle maturation, causing thickened layers of stained material to build up, which are not readily removed.     

Transmission electron microscopy comparison of methods for collecting in situ formed enamel pellicle

The in vivo formed salivary pellicle is composed of an outer globular and a densely structured basal layer. This study developed a method for selective recovering of these pellicle layers from the enamel surface. Two-hour in situ pellicles were formed by intraoral exposure of enamel specimens in two adults. Pellicle-covered enamel specimens were treated either mechanically (scraping with scaler, curette or razor blade, or rubbing with a sponge) or chemically (phosphate buffer, NaCl, NaOCl, CaCl₂, NaSCN, urea, tetrahydrofurane, guanidine, SDS, HCl, or EDTA with or without additional ultrasonication). Specimens were processed for transmission electron microscopic analysis to detect pellicle residues remaining on the enamel surface after the different treatments. Most of the chemical treatments caused partial, incomplete removal of the globular layer. Complete removal of the globular layer without disruption of the basal layer was obtained by sponge rubbing or by CaCl₂ combined with ultrasonication, whereas scraping caused partial disruption of the basal layer. Removal of the basal layer was observed after treatment with HCl, EDTA, or NaOCl combined with ultrasonication. Electrophoretical analysis of recovered pellicle fractions indicate that combination of sponge-rubbing followed by EDTA treatment can be recommended for stepwise removal of the globular and basal pellicle layers.     

Salivary proteins: protective and diagnostic value in cariology?

Saliva is essential for a lifelong conservation of the dentition. Various functions of saliva are implicated in the maintenance of oral health and the protection of our teeth: (i) The tooth surface is continuously protected against wear by a film of salivary mucins and proline-rich glycoprotein. (ii) The early pellicle proteins, proline-rich proteins and statherin, promote remineralization of the enamel by attracting calcium ions. (iii) Demineralization is retarded by the pellicle proteins, in concert with calcium and phosphate ions in saliva and in the plaque fluid. (iv) Several salivary (glyco)proteins prevent the adherence of oral microorganisms to the enamel pellicle and inhibit their growth. (v) The salivary bicarbonate/carbonate buffer system is responsible for rapid neutralization of acids. An overview is presented on the major antimicrobial systems in human saliva. Not only the well-known major salivary glycoproteins, including mucins, proline-rich glycoprotein and immunoglobulins, but also a number of minor salivary (glyco)proteins, including agglutinin, lactoferrin, cystatins and lysozyme, are involved in the first line of defense in the oral cavity. Besides, small cationic antimicrobial peptides, e.g. defensins, cathelicidin and the histatins, have come into focus. These are potentially suited as templates for the design of a new generation of antibiotics, since they kill a broad spectrum of microorganisms, while hardly evoking resistance, in contrast to the classical antibiotics.     

Quantitative determination of salivary components in the pellicle on PMMA denture base material

The formation of a salivary pellicle is a protective mechanism of the body for all surfaces in the oral cavity. The nature of the substrate may influence the composition of the pellicle. The aim of this study was to investigate the quantitative composition and individual variation of the salivary pellicle formed on denture base material (PMMA). Cylindrical specimens of PMMA were carried in the mouth and then desorbed with a 0.5-M sodium chloride solution. The solution was analysed for total protein, alpha-amylase, total proteases, protease inhibitors, secretory immunoglobulin A, immunoglobulin G, peroxidases, thiocyanate, lysozyme, and calcium content. All investigated salivary components could be found unequivocally in the desorption solution, indicating that a salivary pellicle had formed on the surface of the PMMA. Large coefficients of variation indicate large individual variations in the adsorbed amounts. The data also point to large intraindividual variations for the bound salivary components. Only the protease inhibitors revealed a strong positive correlation of the bound activity to the salivary activity. It is hypothesised that differences in the bound amounts of antimicrobial components might influence the microbial colonisation of denture bases and that protease inhibitors could be meaningful for the spread of the yeast Candida albicans from denture base material to the oral mucosa and thus might be an explanation for different susceptibility to denture base stomatitis.