Adsorption of chlorhexidine and black tea onto in vitro salivary pellicles, as studied by ellipsometry

The adsorption from 0.2% (w/w) chlorhexidine and black tea solutions onto an in vitro pellicle from whole unstimulated saliva on hydroxyapatite discs was studied by ellipsometry. It was found that chlorhexidine adsorbed to the pellicle and caused a modification of the pellicle properties, leading to a subsequent increase in adsorption of salivary and black tea components. There was a distinct order-of-addition effect, whereby chlorhexidine followed by black tea gave an overall greater adsorption of components compared with black tea followed by chlorhexidine. This increase in adsorption resulted in a concomitant increase in color or stain, as measured by a reflectance chromameter. The increase in adsorbed amounts and stain was modified, in part, by the adsorption of salivary fractions between the chlorhexidine and black tea treatments. In all cases, the chlorhexidine and black tea-modified pellicles were not readily removed by either phosphate or sodium dodecyl sulfate rinses. Thus, following exposure to chlorhexidine, the accelerated adsorption of salivary and black tea components can ultimately lead to increased staining of the pellicle.     

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.     

人唾液α-淀粉酶与红/绿茶多酚相互作用的吸附动力学研究

目的 探索茶黄素(TF)和表没食子儿茶素没食子酸酯(EGCG)与人唾液α-淀粉酶(HSA)反应所引起的口腔收敛性感觉的驱动力.方法利用表面等离子共振仪CSPR)和吸附动力学原理,测量Langmuir、Freundlich吸附等温线常数(K<,L>、K<,t>和M<,m>)和吸附反应速率及平衡常数(K<,a>、K<,d>...     

Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro

Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.     

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.     

The effect of red wine in modifying the salivary pellicle and modulating dental erosion kinetics

This study investigated the potential of red wine in modulating dental erosion kinetics in the presence or absence of salivary pellicle. Polished human enamel specimens were used in two conditions; presence or absence of acquired enamel pellicle; and subdivided according to exposure: red wine, orange juice, apple juice, or citric acid. The specimens were incubated in clarified whole human saliva (presence of acquired enamel pellicle) or in a humid chamber (absence of acquired enamel pellicle) for 2 h at 37°C, then in the test substances for 1 min, at 25°C, under shaking. This was repeated four times. Surface hardness was measured initially and after each cycle and surface reflection intensity was measured initially and after all cycles. In the presence of acquired enamel pellicle, red wine caused the least surface hardness loss, followed by orange juice, apple juice, and citric acid. Statistically significantly less surface reflection intensity loss was observed for red wine and orange juice than for apple juice and citric acid. In the absence of acquired enamel pellicle, red wine and orange juice caused less surface hardness loss than apple juice and citric acid. Orange juice showed the least surface reflection intensity loss, followed by red wine, citric acid, and apple juice. The polyphenol composition of these drinks can notably modulate the erosion kinetics.     

Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

Abrasion and stain removal by different manual toothbrushes and brush actions: studies in vitro

BACKGROUND AND AIMS: A limited amount of data using flat trim multitufted toothbrushes shows that abrasion of substrate surfaces by a standard toothpaste varies dependent on filament stiffness and configuration; soft brushes producing the most abrasion. The aims of these studies in vitro were to assess toothpaste abrasion of acrylic and stain removal by 5 proprietary medium toothbrushes with different head filament arrangements, and a prototype brush with rectangular filaments. The prototype brush had a medium texture in the long axis and soft texture at right angles to the long axis. MATERIAL AND METHOD: Optically clear acrylic was used as the substrate for abrasion by a standard toothpaste. Loss of substrate was determined by profilometry after 5000, 10000, 15000 and 20000 linear or rotary brushing actions. Stain removal was determined spectrophotometrically from optically clear acrylic specimens stained by chlorhexidine tea soaking sequences. Stained specimens were brushed with water using linear or rotary actions and measurements taken every 10 s to 60 s. RESULTS: Abrasion was progressive with increasing strokes and the pattern for each brush and brush action was to a first approximation linear. Overall, abrasion was significantly greater with linear compared to rotary action. Also overall brushes differed in the abrasion produced with both actions and particularly at greater exposure times. Within brush differences for the two motions were all significant by 20000 strokes except for the prototype brush. Stain removal was progressive over time with each brush but the pattern was non-linear. For the proprietary brushes the rotary motion removed less stain. For the prototype brush more stain was removed with the rotary action. Overall brushes differed significantly in stain removal within each motion and for each motion most differences between the proprietary and prototype brushes reached significance. CONCLUSIONS: The differences between brushes for both abrasion and stain removal must in large part relate to the filament contact area with the substrate surface. Whilst the model may not be predictive of clinical differences, it could find use to establish minimum criteria for toothbrush action.     

Adsorption of salivary and serum proteins, and bacterial adherence on titanium and zirconia ceramic surfaces

Objectives: The aim of this study was to determine the pattern of salivary and serum proteins present in pellicles formed on titanium (Ti) and zirconia ceramic (ZrO₂) surfaces, and the ability of bacterial cells to adhere to the experimental pellicles. In addition, the protein profiles and bacterial binding properties of pellicles on Ti and ZrO₂ were compared to those formed on hydroxyapatite (HA) surface.
Methods: The pellicles were formed in vitro by incubating the materials with whole saliva, serum or saliva+serum. Protein composition in each of the pellicles was investigated by SDS-PAGE and immunodetection. The adherence of radiolabeled Streptococcus mutans and Actinomyces naeslundii to uncoated surfaces and experimental pellicles was determined by means of scintillation counting. Statistical analyses were done using ANOVA and Tukey's test at significance level at P <0.05. In general, the electrophoretic analysis of the pellicles formed on HA, Ti and ZrO₂ revealed few qualitative differences of the composition of proteins of the pellicles formed on HA, Ti and ZrO₂ surfaces. Pellicle components identified included amylase, IgA, IgG, albumin, fibronectin and fibrinogen. The number of S. mutans cells adhered to uncoated Ti and ZrO₂ was significantly higher than those adhered to HA ( P <0.05). In contrast, lower number of A. naeslundii cells adhered to uncoated Ti and ZrO₂ than to HA ( P <0.05). However, the presence of saliva and saliva+serum pellicles greatly reduced the number of S. mutans cells bound to each of the surfaces. The data showed that Ti and ZrO₂ display similar pellicle protein composition and bacterial binding properties; however, significant differences were observed when both materials were compared to HA.     

Comparison of 2 chlorhexidine mouthwashes on plaque regrowth in vivo and dietary staining in vitro

Abstract Until recently, the few available chlorhexidine mouthrinse products have been 0.2% formulations. However, concentrations of 0.12% chlorhexidine appear as effective as 0.2%, if the volume of the rinse is increased to 15 ml. Since the mere incorporation of chlorhexidine in a formulation does not guarentee availability of the antiseptic, it would seem reasonable to evaluate or compare all products. This is particularly the case when other ingredients, such as fluoride are added. The 1st study compared the effect of a 0.12% chlorhexidine rinse with a 0.12% chlorhexidine/0.022% sodium fluoride rinse for effects on plaque re-growth. The study was a 7-day, blind, randomised, 2-cell cross-over design with a baseline control run in period, in which 18 subjects participated. Both chlorhexidine products significantly reduced plaque compared to control but the chlorhexidine fluoride rinse was less effective than the chlorhexidine only rinse. The 2nd study assessed the propensity of the chlorhexidine rinses to induce dietary staining in vitro. For the chlorhexidine fluoride rinse, this was less than the other 0.12% rinse and a commonly used 0.2% product. The data in vivo and in vitro suggest reduced chlorhexidine availability from the chlorhexidine fluoride product which appears to cause some loss of efficacy.     

Studies on stannous fluoride toothpaste and gel (1). Antimicrobial properties and staining potential in vitro

Abstract Stannous fluoride (SF) in a toothpaste vehicle has the potential to provide anticaries and plaque inhibitory benefits through the fluoride and antimicrobial stannous moieties respectively. Dental staining, however, can occur by precipitation of dietary chromogens onto the tooth surface by stannous ions. These studies in vitro compare the antimicrobial profile and propensity to cause tea staining of a number of stannous fluoride formulations, The formulations used were 2 SF toothpaste products (SFl. SF2). 2 experimental SF plus stannous pyrophosphate toothpastes (SFSP1. SFSP2). a SF gel (G| and a NaF toothpaste (C). Maximum inhibitory dilution values against a range of oral bacteria were determined by agar dilution. Tea staining was measured spectrophotometr-ically on saliva coated dear acrylic blocks exposed to slurries of the paste or gel. All formulations showed antimicrobial activity with the order of greatest activity downwards being C. SF2. SFl, SFSP1, SFSP2 and G.Tea staining at 10 exposures was in the following descending order of optical density SFSP1, SFSP2. G-C. SFl, SF2. water control. The antimicrobial profile of G was similar to that of SF. whereas that of the other formulations were varied but similar to a detergent profile. The difference in staining suggested considerable variation in availability of stannous ions in the formulations. However, the propensity for stannous ions to stain must be balanced against the stain removal propensity of the contained detergents in the toothpaste formulations. In conclusion, the variation in antimicrobial activity and more particularly staining activity of the formulations suggest the products will vary in activity in vivo.     

Co-adhesion and removal of adhering bacteria from salivary pellicles by three different modes of brushing

This study compares removal of pairs of co-adhering and non-co-adhering oral actinomyces and streptococci by hand, electric and sonic brushing from salivary pellicles. In addition, re-deposition of a co-adhering and non-co-adhering streptococcal strain to brushed pellicles was studied. First, actinomyces were allowed to adhere to a pellicle in a flow chamber, after which streptococci suspended in saliva were perfused through the chamber at 33 C. Pellicles with adhering bacterial pairs were brushed and the number of bacteria remaining determined. Whereas sonic brushing removed nearly all adhering bacteria, greater numbers of larger aggregates of the co-adhering pair, involving Streptococcus oralis J22, were left behind after hand and electric brushing than of the non-co-adhering pair with Streptococcus sanguis PK1889. Re-deposition of streptococci to electrically and sonically brushed pellicles, however, was two-fold higher for the co-adhering pair than for the non-co-adhering pair. This demonstrates a role for co-adhesion in de novo plaque formation. Removal by the three different modes of brushing was not affected by the presence of fluoride, indicating that fluoride is not able to disrupt calcium bridges between co-adhering pairs. In contrast, in the presence of lactose only small aggregates of co-adhering pair were left behind.     

Bacteria-binding plasma proteins in pellicles formed on hydroxyapatite in vitro and on teeth in vivo

Previous studies on dental pellicle formation and bacterial adherence have focussed on saliva and its components. The tooth surface is, however, also exposed to the plasma-derived crevicular fluid. In the present study, (i). plasma proteins in in vitro and in vivo pellicles were examined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and image analysis and (ii). the adherence of periodontopathogenic bacteria to experimental plasma and saliva pellicles was examined using radio-labelled bacteria and liquid scintillation counting. The plasma components fibrinogen, fibronectin, albumin and IgG were incorporated from plasma in the experimental pellicle and mediated the adherence of Porphyromonas gingivalis, Fusobacterium nucleatum and Actinomyces naeslundii. These proteins were also readily detected in in vivo pellicles and were found to a higher extent in pellicles formed at the gingival part of the tooth surface than at the incisal part.     

Absence of capsule reveals glycan‐mediated binding and recognition of salivary mucin MUC7 by Streptococcus pneumoniae

Salivary proteins modulate bacterial colonization in the oral cavity and interact with systemic pathogens that pass through the oropharynx. An interesting example is the opportunistic respiratory pathogen Streptococcus pneumoniae that normally resides in the nasopharynx, but belongs to the greater Mitis group of streptococci, most of which colonize the oral cavity. Streptococcus pneumoniae also expresses a serine‐rich repeat (SRR) adhesin, PsrP, which is a homologue to oral Mitis group SRR adhesins, such as Hsa of Streptococcus gordonii and SrpA of Streptococcus sanguinis. As the latter bind to salivary glycoproteins through recognition of terminal sialic acids, we wanted to determine whether S. pneumoniae also binds to salivary proteins through possibly the same mechanism. We found that only a capsule‐free mutant of S. pneumoniae TIGR4 binds to salivary proteins, most prominently to mucin MUC7, but that this binding was not mediated through PsrP or recognition of sialic acid. We also found, however, that PsrP is involved in agglutination of human red blood cells (RBCs). After removal of PsrP, an additional previously masked lectin‐like adhesin activity mediating agglutination of sialidase‐treated RBCs becomes revealed. Using a custom‐spotted glycoprotein and neoglycoprotein dot blot array, we identify candidate glycan motifs recognized by PsrP and by the putative S. pneumoniae adhesin that could perhaps be responsible for pneumococcal binding to salivary MUC7 and glycoproteins on RBCs.     

Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a non-inhibitory mode

OBJECTIVES: Our aim was to purify large quantities of human salivary cystatins S, SA and SN in order to determine whether these salivary cystatins have a stable interaction with cysteine proteases at a second binding site, other than the protease active site. This property may affect their availability to act as cysteine protease inhibitors within the oral environment. METHODS: Salivary cystatins S, SA and SN were purified from human submandibular sublingual saliva to homo- geneity by column chromatography. Formation of stable complexes between the model cysteine protease papain in the absence of reductant was assessed by SDS-PAGE and probing Western blots with antibody to human salivary cystatin SN. Proteolytic activity of the complex was determined in the gel after electrophoresis. RESULTS AND CONCLUSIONS: Only cystatin SN (14.3 kD) was found to form a stable complex with papain (22 kD) that could be separated by SDS-PAGE producing a Coomassie stained band at (37 kD). After western transfer this same band (37 kD) cross-reacted with antibody to SN. In the presence of E64, an active site inhibitor of cysteine proteases, the same complex was formed, suggesting that SN is able to bind to papain at a site other than the active site. Activity staining of the gel confirmed that this complex (-E64) retained proteolytic activity. Such complex formation between cystatin SN and cysteine proteases in a non-inhibitory mode may reduce its availability to act as an effective cysteine protease inhibitor in the oral environment.     

Probiotic bacteria affect the composition of salivary pellicle and streptococcal adhesion in vitro

Introduction:  The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria.
Methods:  Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii , were used as a model.
Results:  Probiotic bacteria that bound to saliva-coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii . Two of the proteins missing from the pellicles made of saliva-treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains.
Conclusion:  This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.     

Bacteria‐binding plasma proteins in pellicles formed on hydroxyapatite in vitro and on teeth in vivo

Previous studies on dental pellicle formation and bacterial adherence have focussed on saliva and its components. The tooth surface is, however, also exposed to the plasma‐derived crevicular fluid. In the present study, (i) plasma proteins in in vitro and in vivo pellicles were examined using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), immunoblotting and image analysis and (ii) the adherence of periodontopathogenic bacteria to experimental plasma and saliva pellicles was examined using radio‐labelled bacteria and liquid scintillation counting. The plasma components fibrinogen, fibronectin, albumin and IgG were incorporated from plasma in the experimental pellicle and mediated the adherence of Porphyromonas gingivalis, Fusobacterium nucleatum and Actinomycesnaeslundii. These proteins were also readily detected in in vivo pellicles and were found to a higher extent in pellicles formed at the gingival part of the tooth surface than at the incisal part.     

Comparison of salivary calmodulin binding proteins in Sjögren''s syndrome and healthy individuals

Background: Reduction in salivary secretion is the hallmark of Sjögren's syndrome (SS). Calmodulin (CaM) and calmodulin binding proteins (CaMBPs) play a key role in the secretory process of saliva. Recent studies have suggested that SS‐B, an autoantibody associated with SS, is a CaMBP. This finding suggests that CaMBP may contribute to the loss of saliva in SS. To better understand the role(s) of these proteins in SS, the purpose of this study was to compare salivary CaMBPs in Sjögren's patients and controls. Methods: Saliva samples were collected from 20 patients and 20 age‐, race‐, and gender‐matched controls. CaM overlay was used to identify CaMBPs in saliva of patients and controls. Results: Higher number of salivary CaMBPs was observed among patients than controls. Conclusions: The increased number of salivary CaMBPs in SS may suggest a potential role for these proteins in the pathogenesis of the disease.     

Stimulated salivary flow rate and buffer effect in schoolchildren from Greenland and Sweden: A comparative study

Objective. To compare food habits and stimulated salivary flow rate and buffer effect between schoolchildren from Greenland and Sweden and to evaluate whether the change in lifestyle concerning eating habits in Sweden during recent decades has resulted in any obvious alteration in these salivary properties. Material and methods. Fifty healthy schoolchildren from Greenland were included and compared with 50 age-matched and gender-matched Swedish children. Whole saliva stimulated by chewing was collected, and prior to sampling each participant filled in a simple questionnaire regarding their food habits. Results. Salivary flow rate and buffer effect were significantly (p<0.001) higher for the Greenlandic children. The difference in flow rate was on average 0.71 ml/min. Milk, fish/meat and fruit/vegetables were more frequently consumed by the Swedish children, while snacks, soft drinks and sweets had a higher consumption frequency on Greenland. No obvious correlation could be found between consumption frequency of the tested food products and flow rate or buffer effect of saliva. Twenty-nine Swedish children were within the age range±6 months of an age with earlier documented values of stimulated salivary flow rate. Twenty-six of these 29 Swedish children were within the 25–75 percentile range of the old values, while 22 of their Greenlandic “twins” were in or above percentile 75. Conclusions. Obvious differences in salivary flow rate and buffer effect between schoolchildren from Greenland and Sweden illustrated the importance of being cautious when exchanging reference data between different cultures/ethnic groups.     

Distribution and toxicity resulting from adenoviral vector administration to a single salivary gland in adult rats

BACKGROUND: We examined the distribution and toxicity associated with a single salivary gland administration of a recombinant adenoviral vector, AdCMVH3, encoding human histatin 3. METHODS: Adult rats received different doses of AdCMVH3 (0, 106, 3 x 107, and 109 pfu; 50 microl) via the right submandibular gland and were followed for 15 days. Food consumption, weight gain, clinical appearance, and serum chemistry were monitored, and a necropsy was performed. Vector distribution was examined by polymerase chain reaction, and selected saliva samples were tested for replication-competent adenovirus (RCA). RESULTS: All animals survived to sacrifice (days 2, 8, and 15), and appeared normal clinically. There were no differences in food consumption, weight gain, and serum chemistry. The only consistent necropsy findings were lymphoid infiltrates and necrosis in the target submandibular glands of high-dosage animals. AdCMVH3 detection was virus dose dependent, decreased with time, and at low dose preferentially observed in the targeted gland. No RCA was detected. CONCLUSIONS: Salivary gland administration of 109 pfu AdCMVH3 elicits an initial focal pathologic response and wide tissue distribution. There is no associated systemic toxicity up to 15 days, and lower doses are primarily found in glands.