Pellicle Modification Research Articles

Pellicle modification with natural bioproducts: Influence on tooth color under erosive conditions.

Salivary pellicle was modified with bioproducts and we assessed the change in tooth color and the protection of enamel to erosion. Human enamel specimens were assigned to one of three solutions: grape seed extract or black tea (bioproducts), or deionized water (negative control); after which one half the specimens underwent erosive challenges. The specimens underwent 15 cycles involving salivary pellicle formation (10min, 37°C), incubation in solution (2min, 25°C), subsequent pellicle formation (90min, 37°C). Half of the specimens was kept in a humid chamber and the other half was submitted to erosion (2min, 1% citric acid). After 15 such cycles, the pellicle was removed. Tooth color and the surface reflection intensity were assessed after every five cycles and after pellicle removal. For non-eroded specimens, the exposure to bioproducts promoted significantly greater color change than the deionized water, with increases in yellow appearance. After pellicle removal, the color was similar in all non-eroded specimens. The bioproducts increased the surface reflection intensity over cycles. For the erosion-exposed specimens, erosion itself resulted in color change. Black tea and deionized water resulted in increased yellow appearance. Exposure to the bioproducts resulted in higher relative surface reflection intensity values over time, but only grape seed extract resulted in higher relative surface reflection intensity value at the time of pellicle removal. The bioproducts caused transient staining effect, which was reduced after pellicle removal. For enamel submitted to erosion, grape seed extract resulted in less color change and better protection of enamel against erosion than black tea or water.

Salivary Pellicle Modification with Grape-seed Extract: In Vitro Study on the Effect on Bacterial Adhesion and Biofilm Formation.

Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel. The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests. GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group. AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

Salivary pellicle modification with polyphenol-rich teas and natural extracts to improve protection against dental erosion

ObjectiveTo investigate the modification of the salivary pellicle with different polyphenol-rich teas and natural extracts for the protection against dental erosion. MethodsWe performed two experiments: one with teas (Green tea, Black tea, Peppermint tea, Rosehip tea, negative control [NC]) and other with natural extracts (Grape seed, Grapefruit seed, Cranberry, Propolis, NC), where NC was deionized water. A total of 150 enamel specimens were used (n = 15/group). Both experiments followed the same design, consisting of 5 cycles of: salivary pellicle formation (30 min, 37 °C), modification with the solutions (30 min, 25 °C), further salivary pellicle formation (60 min, 37 °C) and erosive challenge (1 min, 1% citric acid, pH 3.6). Relative surface microhardness (rSMH), relative surface reflection intensity (rSRI) and amount of calcium release (CaR) were evaluated. Data were analysed with Kruskal-Wallis and Wilcoxon rank sum tests with Bonferroni correction (α = 0.05). ResultsRegarding teas, Black and Green teas showed the best protection against dental erosion, presenting higher rSMH and lower CaR than NC. Peppermint tea was not different to NC and Rosehip tea caused erosion, showing the highest CaR and greatest loss of SMH and SRI. Regarding natural extracts, Grape seed and Grapefruit seed extracts presented the best protective effect, with significantly higher rSMH and lower CaR. Cranberry caused significantly more demineralization; and Propolis did not differ from NC. ConclusionGreen tea, Black tea, Grape seed extract and Grapefruit seed extract were able to modify the salivary pellicle and improve its protective effect against enamel erosion, but Rosehip tea and Cranberry extract caused erosion. Clinical relevanceSome some bio-products, such as teas and natural extracts, improve the protective effect of the salivary pellicle against enamel erosion. More studies should be performed in order to test the viability of their use as active ingredients for oral care products.

Pellicle Modification with Casein and Mucin Does Not Affect Surface Loss from Erosion and Abrasion

Aim: A combination of the proteins casein and mucin is known to modify the salivary pellicle and improve its protection of the underlying enamel from erosion. It is so far not known if this protection is confined solely to erosion, or if it also extends to abrasion, and this in vitro study aimed at investigating this question. Methods: A total of 72 human enamel specimens were prepared and randomly assigned to four groups: pellicle (P), casein/mucin (CM), pellicle + casein/mucin (PCM), and control (Ctrl). Each specimen underwent five cycles, each cycle consisting of a pellicle/treatment part, an erosion part (3 min in 1% citric acid, pH 3.6, 25°C, 70 rpm), and an abrasion part (50 toothbrush strokes within 25 s in toothpaste slurry with a 200-g load). The pellicle/treatment part consisted of 2 h of incubation in whole human saliva for group P, 2 h of incubation (25°C, 70 rpm) in a protein mixture of 1% casein and 0.27% mucin for group CM, and 2 h of incubation in saliva followed by 2 h of incubation in the protein mixture for group PCM. The fourth group (Ctrl) served as the control and was kept in a humid chamber without saliva or protein treatment. The enamel surfaces were scanned with an optical profilometer initially and after the final cycle, and surface loss was analyzed. Furthermore, the surface microhardness (SMH) was measured initially, after each pellicle/treatment part and each erosion cycle, and after the final abrasion cycle. The results were analyzed with Kruskal-Wallis and Wilcoxon tests with Bonferroni corrections. Results: The different treatments did not show differences in surface loss and therefore did not protect enamel from surface loss by abrasion. Nonetheless, we observed differences in the SMH values, namely the Ctrl group being significantly softer than the experimental groups. Conclusion: The observed differences in SMH suggest that a different abrasion protocol could lead to differences in surface loss, and further investigation of whether and under which conditions pellicle modification leads to increased resistance to abrasion remains worthwhile.

Pellicle Modification with Casein and Mucin Does Not Promote In Vitro Bacterial Biofilm Formation.

During biofilm formation, bacterial species do not attach directly onto the enamel surface, but rather onto the salivary pellicle. Salivary pellicle modification with casein and mucin can hinder erosive demineralisation of the enamel, but it should also not promote bacterial adhesion. The aim of our study was to assess whether salivary pellicle modification with casein, or mucin, or a mixture of both proteins (casein and mucin) influence bacterial adhesion, biofilm diversity, metabolism and composition, or enamel demineralisation, after incubation in: (a) a single bacterial model; (b) a five-species biofilm model; or (c) biofilm reformation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Enamel specimens were prepared from human molars. Whole-mouth stimulated human saliva was used for pellicle formation. Four pellicle modification groups were established: control (non-modified pellicle); casein - modified with 0.5% casein; mucin - modified with 0.5% mucin; casein and mucin - modified with 0.5% casein and 0.5% mucin. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 6 h or 24 h. After 24 h incubation in the five-species biofilm model, the mucin group presented significantly lower biofilm mass than the control (p = 0.028) and the casein and mucin (p = 0.030) groups. No other differences between the groups were observed in any of the other experimental procedures. Pellicle modification with casein and mucin does not promote in vitro bacterial biofilm formation.

Casein phosphopeptide combined with fluoride enhances the inhibitory effect on initial adhesion of Streptococcus mutans to the saliva-coated hydroxyapatite disc

BackgroundRecent preventive strategies for dental caries focus on targeting the mechanisms underlying biofilm formation, including the inhibition of bacterial adhesion. A promising approach to prevent bacterial adhesion is to modify the composition of acquired salivary pellicle. This in vitro study investigated the effect and possible underlying mechanism of pellicle modification by casein phosphopeptide (CPP) on Streptococcus mutans (S. mutans) initial adhesion, and the impact of fluoride on the efficacy of CPP.MethodsThe salivary pellicle-coated hydroxyapatite (s-HA) discs were treated with phosphate buffered saline (negative control), heat-inactivated 2.5% CPP (heat-inactivated CPP), 2.5% CPP (CPP) or 2.5% CPP supplemented with 900 ppm fluoride (CPP + F). After cultivation of S. mutans for 30 min and 2 h, the adherent bacteria were visualized by scanning electron microscopy (SEM) and quantitatively evaluated using the plate count method. Confocal laser scanning microscopy (CLSM) was used to evaluate the proportions of total and dead S. mutans. The concentrations of total, free, and bound calcium and fluoride in the CPP and fluoride-doped CPP solutions were determined. The water contact angle and zeta potential of s-HA with and without modification were measured. The data were statistically analyzed using one-way ANOVA followed by a Turkey post hoc multiple comparison test.ResultsCompared to the negative control group, the amount of adherent S. mutans significantly reduced in the CPP and CPP + F groups, and was lowest in the CPP + F group. CLSM analysis showed that there was no statistically significant difference in the proportion of dead S. mutans between the four groups. Water contact angle and zeta potential of s-HA surface significantly decreased in the CPP and CPP + F groups as compared to the negative control group, and both were lowest in the CPP + F group.ConclusionsPellicle modification by CPP inhibited S. mutans initial adhesion to s-HA, possibly by reducing hydrophobicity and negative charge of the s-HA surface, and incorporating fluoride into CPP further enhanced the anti-adhesion effect.

Acquired Enamel Pellicle Modification with Casein and Mucin in Different Concentrations and its Impact on Initial Dental Erosion

Casein and mucin have been shown to improve the erosion-protective properties of the pellicle when applied in combination. The aim of this in vitro study was to optimize the concentrations of these 2 proteins to achieve a maximum protective effect. For the 2 parts of this study, we prepared a total of 195 human enamel specimens and randomly assigned them to 13 groups, corresponding to 11 different casein-mucin concentration-combinations tested and 2 negative control groups (humid chamber). They underwent 5 cycles, consisting of pellicle formation from human whole saliva (2 h, 30°C), modification of the pellicle with casein and mucin in different concentrations (immersion in protein solutions for 2 h, 30°C), and erosion for 1 min in citric acid (0.65%, pH 3.5, 30°C). Surface microhardness (SMH), surface reflection intensity (SRI), and in the first part also calcium release were monitored during the cycling process, and analyzed with Kruskal-Wallis and post hoc Dunn’s tests. The results suggest that the best concentrations to achieve the highest erosion-protective effect are 3.0% casein and 0.81% mucin, which lead to a significant protection as measured by SMH as well as SRI compared to the unmodified pellicle. For the calcium release, no significant differences were found. This concentration combination corresponds to a general raise of the protein concentrations and a change in the molar ratio of the proteins as compared to earlier studies. Casein and mucin could now be incorporated at the determined concentration as natural ingredients in oral care products designed to protect against erosion.

Oil droplet formation on pellicle covered tooth surfaces studied with environmental scanning electron microscopy.

Lipophilic components are known to modulate the process of bioadhesion on the tooth surface. However, the presence of lipid droplets at the acquired pellicle under oral conditions has not been demonstrated, yet. The purpose of the present study was to establish a method for direct visualisation of lipids on the surface of hydrated, pellicle covered tooth samples by environmental scanning electron microscopy (ESEM), and to use this technique for studying the effects of rinsing with edible oils on the acquired pellicle under in vivo conditions. In situ pellicle formation was performed by 3 min exposure of enamel and dentin specimens in the oral cavity of volunteers. Subsequently, the volunteers rinsed in vivo with safflower oil or linseed oil for 30 s, and the specimens were further carried intraorally for periods from 0 min up to several hours. After intraoral exposure the specimens were treated by osmium tetroxide vapour, and were subsequently analysed by ESEM. This technique was capable to directly visualise the presence of lipid droplets at the pellicle's surface under hydrated conditions. ESEM analyses revealed that surface bound nano- and micro-sized lipid droplets were present at the acquired pellicle's surface even several hours after rinsing with edible oils indicating that these droplets had tightly adhered to the pellicle surface. Pellicle modification by edible oil rinsing as demonstrated in the present study might have the potential to be beneficial as an adjunct in dental prophylaxis.

Impact of honey on dental erosion and adhesion of early bacterial colonizers

The aim was to investigate if honey causes erosion and if salivary pellicle modified with honey, or its components, or the by-product propolis has a protective effect against dental erosion and adhesion of early bacterial colonizers. The tested substances were: 3 types of honey, methylglyoxal (MGO), hydrogen peroxide, propolis. First in the erosion experiment, 120 human enamel specimens were covered with salivary pellicle and modified with the substances. Then they were eroded with 1% citric acid, pH 3.6 for 2 min, before surface hardness was measured. In the microbiological assay, the enamel specimens (n = 126) covered with modified salivary pellicle were contaminated with bacterial suspensions. The antimicrobial activity of each substance and their effect on early bacterial colonizer adhesion and biofilm formation were determined. Despite a low pH, honey did not cause erosion. On the other hand, pellicle modification with the tested solutions did not protect the enamel from erosion. Microbiologically, the 3 honeys inhibited species-specific growth of oral bacteria. Propolis decreased initial attachment of Streptococcus gordonii, while one honey inhibited demineralization of enamel by biofilm. In conclusion, pellicle modification with honey, or its components, or propolis did neither protect against erosion nor promote it. Propolis presented some bacterial adhesion inhibition.

Impact of Acquired Pellicle Modification on Adhesion of Early Colonizers

New preventive approaches against dental erosion caused by acidic drinks and beverages include fortification of beverages with natural polymers. We have shown that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. This study aimed to investigate the effect of pellicle modification by casein, mucin and a casein-mucin mixture on the adhesion of early bacterial colonizers. Test specimens of human tooth enamel were prepared, covered with saliva and coated with 0.5% aqueous (aq.) casein, 0.27% aq. mucin or with 0.5% aq. casein-0.27% aq. mucin, after which the adhesion of Streptococcus gordonii, Streptococcus oralis, and Actinomyces odontolyticus was measured after incubation for 30 min and 2 h. log₁₀ colony-forming units were compared by nonparametric tests. All three bacterial strains adhered in higher number to pellicle-coated enamel than to native enamel. The protein modifications of pellicle all decreased the counts of adhering bacteria up to 0.34 log₁₀/mm², the most efficient being the casein-mucin mixture. In addition to the recently shown erosion-reducing effect by casein-mucin, modification of the pellicle may inhibit bacterial adherence compared to untreated human pellicle.

Effect of Safflower Oil on the Protective Properties of the in situ Formed Salivary Pellicle

Aim: The prevalence of dental erosion is still increasing. A possible preventive approach might be rinsing with edible oils to improve the protective properties of the pellicle layer. This was tested in the present in situ study using safflower oil. Methods: Pellicle formation was carried out in situ on bovine enamel slabs fixed buccally to individual upper jaw splints (6 subjects). After 1 min of pellicle formation subjects rinsed with safflower oil for 10 min, subsequently the samples were exposed in the oral cavity for another 19 min. Enamel slabs without oral exposure and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After pellicle formation in situ, slabs were incubated in HCl (pH 2; 2.3; 3) for 120 s, and kinetics of calcium and phosphate release were measured photometrically (arsenazo III, malachite green). Furthermore, the ultrastructure of the pellicles was evaluated by transmission electron microscopy (TEM). Results: Pellicle alone reduced erosive calcium and phosphate release significantly at all pH values. Pellicle modification by safflower oil resulted in an enhanced calcium loss at all pH values and caused an enhanced phosphate loss at pH 2.3. TEM indicated scattered accumulation of lipid micelles and irregular vesicle-like structures attached to the oil-treated pellicle layer. Acid etching affected the ultrastructure of the pellicle irrespective of oil rinsing. Conclusion: The protective properties of the pellicle layer against extensive erosive attacks are limited and mainly determined by pH. The protective effects are modified and reduced by rinses with safflower oil.

Impact of Acquired Enamel Pellicle Modification on Initial Dental Erosion

The acquired enamel pellicle that forms on the tooth surface serves as a natural protective barrier against dental erosion. Numerous proteins composing the pellicle serve different functions within this thin layer. Our study examined the effect of incorporated mucin and casein on the erosion-inhibiting potential of the acquired enamel pellicle. Cyclic acidic conditions were applied to mimic the erosive environment present at the human enamel interface during the consumption of soft drinks. One hundred enamel specimens were prepared for microhardness tests and distributed randomly into 5 groups (n = 20) that received the following treatment: deionized water, humidity chamber, mucin, casein, or a combination of mucin and casein. Each group was exposed to 3 cycles of a 2-hour incubation in human saliva, followed by a 2-hour treatment in the testing solution and a 1-min exposure to citric acid. The microhardness analysis demonstrated that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. The addition of individual proteins did not statistically impact the function of the pellicle. These data suggest that protein-protein interactions may play an important role in the effectiveness of the pellicle to prevent erosion.

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.