Pellicle Samples Research Articles

Impact of pH-adjusted fluoride and stannous solutions on the protective properties on the pellicle layer in vitro and in situ

This study evaluates the ideal pH for anti-erosion and anti-adherent efficacy of fluoride and stannous solutions (sodium fluoride (SF), amine fluoride (AF), sodium monofluorophosphate (SMFP), stannous fluoride (SnF2) with 500 ppm fluoride concentration each and stannous chloride (SnCl2, 1563 ppm stannous)). In vitro, solutions were tested at pH 4.5 and 5.5. The main in situ experiments were carried out at the pH of 4.5: For pellicle formation 6 volunteers wore bovine enamel slabs intraorally for 1 min, rinsed with 8 ml solution for 1 min and continued for up to 30 min/8 h. Physiological pellicle samples served as controls. After incubation in HCl (2.0, 2.3) for 2 min mineral release was determined photometrically. Bacterial counts on 8 h biofilms were determined by fluorescence microscopy (BacLight™ and DAPI with Concanavalin A). Modification of the pellicle ultrastructure was examined by TEM. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney-U tests with Bonferroni-correction (p < 0.05). SnF2 showed a significant erosion protection. AF, SnF2, and SnCl2 were most anti-adherent. SnF2 and SnCl2 caused a pronounced basal pellicle with stannous precipitates. Compared to other fluoride monosubstances, stannous ions offer greater protection against erosive acidic attacks. Stannous ions act as crucial co-factor in this process.

Effect of Homogenization Pressure on Bacterial Cellulose Membrane Characteristic Made from Pineapple Peel Waste

Many studies were conducted to maintain the environment by reducing the waste, especially pineapple peel waste. This study aims to explore the effect of various pressure of the homogenization process on bacterial cellulose membrane surface morphology and structure produced using extract of pineapple peel waste. The methods include the preparation of pellicle samples from the product of the fermentation process of Acetobacter xylinum using a medium from the extract of pineapple peel waste. Bacterial cellulose pellicles were crushed using a blender. Mashed bacterial cellulose pellicle then homogenized in High-Pressure Homogenizer with pressure variation of 0 bar, 150 bar, 300 bar, 450 bar, dan 600 bar then cast into a mold. The bacterial cellulose solutions were dried in an oven at 60°C for 8 hours. The dried bacterial cellulose membrane was analyzed using XRD for the structure and SEM analysis for the morphology. The results indicate that the crystalline properties of BCM were shifted after being treated by various pressure processing in a High-Pressure Homogenizer. It was found that the High-Pressure Homogenizer with higher pressure reduced the peak intensity, decreased crystalline index from 87% to 70%, and decreased the degree of crystalline from 88% to 77% without changing the cellulose structure. The higher pressure of the homogenization process causes the porosity of the membrane to be decreased.

The lipid composition of the in situ pellicle

ObjectiveThe present study aimed to systematically analyse the complete lipid profile of the in situ pellicle in comparison to saliva. For the first time, the modern sensitive methods GC-EI/MS and HPLC MS/MS were to be used for this purpose. DesignBovine enamel slabs were exposed to the oral cavity of 12 subjects by customized splints (3 min, 30 min or 120 min). Afterwards, the pellicle samples were obtained and further investigated in vitro. Additionally, corresponding unstimulated saliva samples were collected. GC-EI/MS was performed to qualitatively and quantitatively determine all fatty acids contained in the investigated samples. The individual lipid classes of phospholipids, triacylglycerols, glycolipids, cholesterol and cholesterol esters were analysed qualitatively by HPLC MS/MS. ResultsA characteristic fatty acid profile of the in situ pellicle was proven. Furthermore, triacylglycerols with the major fatty acids 16:0, 18:0, 18:1, 18:2, and phospholipids were detected as integral components in the pellicle. There were four groups of phospholipids: Lyso-phosphatidylcholines, phosphatidylcholines, phosphatidylethanol-amines, and phosphatidylinositols. Differences between saliva and pellicle were evident in the composition of the fatty acid- and the phospholipid profile. Glycolipids, cholesterol and cholesterol esters could neither be detected in pellicle- nor in saliva samples. ConclusionThe lipid profiles of the in situ pellicle and saliva were successfully characterised. Differences in the phospholipid and fatty acid composition between pellicle and saliva indicate a selective pellicle formation process. The results provide an important reference and core data for further investigation of the complex surface interactions in the oral cavity, especially concerning hydrophobic substances.

First insights into chlorhexidine retention in the oral cavity after application of different regimens

ObjectivesThis in situ study aimed to determine and compare the chlorhexidine (CHX) retention in the oral cavity after the application of different CHX pharmaceutical regimens.MethodsFive volunteers used different CHX treatment regimens including mouth rinses, dental spray and toothpaste gel. After the application of the different CHX regimens, 2-μl samples were taken from saliva and buccal mucosa pellicle as well as the dental pellicle samples formed on standardized enamel surfaces. Sample collection was conducted at six time points within 12 h. Retention of CHX was measured using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.ResultsCHX retention values in the oral mucosa pellicle were significantly higher than those in saliva. CHX remained in the mucosal pellicle at microgrammes per millilitre levels for 12 h after mouth rinsing, 10 h after spray application and 2 h after using the toothpaste. CHX was detected in the dental pellicle for at least 12 h after application of mouth rinsing and spray. Retention of CHX after mouth rinsing or spray application was significantly higher than the retention after using toothpaste.ConclusionsOral mucosa was the favourable site for CHX retention. Higher mouth rinse concentration and longer rinsing time produced a slight increase in CHX retention. CHX spray provided considerable retention values, whereas toothpaste gel delivered the lowest retention after application. MALDI-TOF was a sensitive method with excellent limits of quantification for CHX detection.

Proteomic profile of the acquired enamel pellicle of professional wine tasters with erosive tooth wear.

The aim of this study was to compare the acquired enamel pellicle protein profile of professional wine tasters with mild and moderate erosive tooth wear. Twelve professional wine tasters participated (3 from a low tooth wear group; 9 from a high tooth wear group). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). The acquired enamel pellicle proteomic profile was different between the groups. The proteins found exclusively in the low tooth wear group were histatins 1 and 3 and mucins 7 and 21. When comparing the wear groups, proteins with higher levels in the low tooth wear group included neutrophil defensins (1 and 3), lysozyme C, lysozyme, myeloperoxidase, and squalene monooxygenase. In conclusion, the findings indicate that the proteins found at higher levels in the low tooth wear group and proteins exclusively found in the low tooth wear group might be protective and, therefore, could be good candidates for further studies regarding their potential to be added to dental products to protect professional wine tasters from extrinsic erosive tooth wear.

Modification of the Lipid Profile of the Initial Oral Biofilm In Situ Using Linseed Oil as Mouthwash.

Lipids are of interest for the targeted modification of oral bioadhesion processes. Therefore, the sustainable effects of linseed oil on the composition and ultrastructure of the in situ pellicle were investigated. Unlike saliva, linseed oil contains linolenic acid (18:3), which served as a marker for lipid accumulation. Individual splints with bovine enamel slabs were worn by five subjects. After 1 min of pellicle formation, rinses were performed with linseed oil for 10 min, and the slabs’ oral exposure was continued for up to 2 or 8 h. Gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS) was used to characterize the fatty acid composition of the pellicle samples. Transmission electron microscopy was performed to analyze the ultrastructure. Extensive accumulation of linolenic acid was recorded in the samples of all subjects 2 h after the rinse and considerable amounts persisted after 8 h. The ultrastructure of the 2 h pellicle was less electron-dense and contained lipid vesicles when compared with controls. After 8 h, no apparent ultrastructural effects were visible. Linolenic acid is an excellent marker for the investigation of fatty acid accumulation in the pellicle. New preventive strategies could benefit from the accumulation of lipid components in the pellicle.

Efficacy of mouthrinses with bovine milk and milk protein isolates to accumulate casein in the in situ pellicle.

The adsorption of bovine milk caseins on the tooth surface might have a positive impact on the prevention of dental diseases. Therefore, the present study aimed to investigate the efficacy of mouthrinses with different types of bovine milk and milk protein isolates to accumulate caseins in the pellicle. An indirect enzyme-linked immunosorbent assay (ELISA) was established to quantify the amount of caseins adsorbed into the in situ pellicle. In situ pellicle samples were collected from 2 volunteers on ceramic specimens (A = 8cm2). After 10min of pellicle formation, different types of bovine milk, 3% micellar casein in synthetic milk ultrafiltrate (SMUF) or 3% non-micellar caseinate in SMUF, were used as mouthrinses for 10min. The pellicle material was harvested after 30min in situ and examined for caseins by the indirect ELISA. Selected pellicle samples were subjected to TEM analysis. All mouthrinses accumulated caseins in the in situ pellicle (2.0 ± 0.7-20 ± 1.7μg/ml) that, under native conditions, expressed no casein signal. Micellar protein association increased the adsorption of casein into the pellicle. Milk homogenization also had an influence on the casein accumulation in the pellicle. TEM analysis confirmed the integration of micellar casein into the pellicle. The mouthrinses altered the protein composition and the ultrastructure of the in situ pellicle to a different extent: bovine milk with 3.8% fat content and 3% micellar casein in SMUF being particularly effective. The study provides interesting perspectives for innovative prevention strategies in dentistry.

Targeted metabolomics of pellicle and saliva in children with different caries activity

Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2–10.5 nmol/cm2), propionic acid (0.1–8.5 nmol/cm2), glycine (0.7–3.5 nmol/cm2), serine (0.08–2.3 nmol/cm2), galactose (galactose + mannose; 0.035–0.078 nmol/cm2), lactose (0.002–0.086 nmol/cm2), glucose (0.018–0.953 nmol/cm2), palmitic acid (0.26–2.03 nmol/cm2), and stearic acid (0.34–1.81 nmol/cm2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.

Characterization of the in situ pellicle ultrastructure formed under the influence of bovine milk and milk protein isolates

ObjectivesThe present study aimed to investigate if bovine milk or milk protein isolates, respectively, alter the ultrastructure of thein situ pellicle and might therefore have an influence on oral health. MethodsIn situ pellicle samples were formed on bovine enamel slabs exposed in the oral cavity of three subjects for 6, 30, 60 or 120 min. After 3 min of pellicle formation, mouthrinses were performed for 3 min with (non-)homogenized UHT- or fresh milk (0.3% or 3.8% fat), 30% UHT-treated cream or different types of casein- or milk protein isolates containing preparations. The specimens were removed after the exposure times and transmission electron microscopy (TEM) was performed. Native pellicle samples served as controls. ResultsTopical ultrastructural pellicle modifications were detected after mouthrinses with all types of homogenized UHT- or fresh milk and after the application of a 3% native casein micelles containing experimental solution. Atypical globular protein structures, identified as casein micelles, were temporarily adsorbed onto the pellicle. They were closely associated with lipid droplets. Furthermore, the mouthrinses occasionally affected the morphology of salivary bacteria. However, no notable ultrastructural alterations remained after 120 min of pellicle formation. ConclusionFor the first time, bovine milk- and micellar casein-induced pellicle modifications were revealed by TEM. The adsorption of micellar casein is possibly due to its molecular interactions. Clinical significanceBovine milk or micellar caseins provide some potential for the development of preventive strategies against bacterial biofilm formation or erosive processes at the tooth surface.

Impact of customary fluoride rinsing solutions on the pellicle\u2019s protective properties and bioadhesion in situ

This study investigated the impact of customary fluoride based mouthrinses on the ultrastructure and the functional properties of the in situ pellicle, considering the prevention of erosion (8 volunteers) and initial biofilm formation (12 volunteers). Bovine enamel slabs were carried intraorally. After 1 min of pellicle formation, the subjects rinsed with elmex Kariesschutz (A), Dontodent Med Care (B), meridol (C) or elmex Zahnschmelzschutz Professional (D) for 1 min. In situ pellicle formation was continued up to 30 min/8 h before processing the slabs in vitro. Erosion was simulated by incubating the specimens in HCl (pH 3.0, 2.3, 2.0) for 120 s, measuring the kinetics of calcium/phosphate release photometrically; representative samples were evaluated by TEM and EDX. Bacterial adhesion was visualized fluorescence microscopically (DAPI/BacLight). Native enamel slabs or physiological pellicle samples served as controls. All investigated mouthrinses enhanced the erosion preventive pellicle effect in dependence of the pH-value. A significant decrease of Ca/P release at all pH values was achieved after rinsing with D; TEM/EDX confirmed ultrastructural pellicle modifications. All mouthrinses tendentially reduced bacterial adherence, however not significantly. The mouthrinse containing NaF/AmF/SnCl2 (D) offers an effective oral hygiene supplement to prevent caries and erosion.

Oral astringent stimuli alter the enamel pellicle’s ultrastructure as revealed by electron microscopy

ObjectivesThis electron microscopic study aimed at investigating effects of oral astringent stimuli on the enamel pellicle’s morphology. MethodsPellicles were formed in situ within 30min on bovine enamel slabs, fixed to individuals’ upper jaw splints. The pellicle-coated specimens were immersed in vitro in seven diverse astringent solutions and subsequently analyzed by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, as well as transmission electron microscopy (TEM). Four biocompatible astringents, namely the polyphenol epigallocatechin gallate, the metal salt iron(III) sulfate, the basic protein lysozyme, and the aminopolysaccharide chitosan, were additionally applied in situ. After rinsing the oral cavity with these compounds, the pellicle’s ultrastructure was imaged by SEM and TEM, respectively. Untreated pellicle samples served as controls. ResultsExposure to polyphenols and lysozyme induced particularly thicker and electron-denser pellicles in comparison to the control pellicle with similar characteristics in vitro and in situ. In contrast, acidic chitosan and metal salt solutions, respectively, revealed minor pellicle alterations. The incorporation of Fe and Al into the pellicles treated with the corresponding inorganic salts was verified by EDX analysis. ConclusionsAstringent-induced pellicle modifications were for the first time visualized by TEM. The ultrastructural alterations of the dental pellicle may partly explain the tooth-roughening effect caused by oral astringent stimuli. Clinical significanceAstringents might modify the pellicle’s protective properties against dental erosion, attrition, as well as bacterial adhesion, and by this means may influence tooth health. The findings may thus be particularly relevant for preventive dentistry.

Fatty Acid Profile of the Initial Oral Biofilm (Pellicle): an In‐Situ Study

The first step of bioadhesion on dental surfaces is the formation of the acquired pellicle. This mainly acellular layer is formed instantaneously on all solid surfaces exposed to oral fluids. It is composed of proteins, glycoproteins and lipids. However, information on the lipid composition is sparse. The aim of the present study was to evaluate the fatty acid (FA) profile of the in-situ pellicle for the first time. Furthermore, the impact of rinses with safflower oil on the pellicle's FA composition was investigated. Pellicles were formed in situ on bovine enamel slabs mounted on individual upper jaw splints. The splints were carried by ten subjects over durations of 3-240min. After comprehensive sample preparation, gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS) was used in order to characterize qualitatively and quantitatively a wide range of FA (C12-C24). The relative FA profiles of the pellicle samples gained from different subjects were remarkably similar, whereas the amount of FA showed significant interindividual variability. An increase in FA in the pellicle was observed over time. The application of rinses with safflower oil resulted in an accumulation of its specific FA in the pellicle. Pellicle formation is a highly selective process that does not correlate directly with salivary composition, as shown for FA.

Targeted immobilisation of lysozyme in the enamel pellicle from different solutions

Mouthwashes containing protective enzymes are required especially for patients suffering from xerostomia. The present study aimed to investigate the possibilities of modulating the immobilisation of lysozyme in the in situ pellicle layer. In situ formed pellicles were incubated in vitro for 10min with various enzymatic buffer solutions containing lysozyme and additive enzymes such as transglutaminase or trypsin as well as polyphenolic compounds (cistus tea). After the rinses, the pellicle samples were incubated in collected whole saliva or in desorption solutions for 0, 20 and 40min and the enzyme activities were measured. Furthermore, accumulation of lysozyme in the pellicle was visualised in ultrathin sections of the pellicle using the gold immunolabelling technique and transmission electron microscopy. Hen egg white lysozyme was accumulated in the in situ pellicle tenaciously. Up to 2.8-fold higher activities than in controls were observed. The addition of transglutaminase did not enhance the immobilisation of lysozyme activity, whereas the polyphenolic compound had no adverse effect. Accumulation of lysozyme in the acquired pellicle was confirmed by gold immunolabelling. Targeted and tenacious immobilisation of lysozyme in the acquired pellicle is possible. Poylphenolic compounds might be a relevant additive for mouthwashes containing lysozyme.

Intrinsic enzymatic crosslinking and maturation of the in situ pellicle

Aim The acquired enamel pellicle is a proteinaceous layer formed on all solid substrata exposed to the oral cavity. It has been supposed that the pellicle undergoes maturation after protein adsorption. The aim of the present study was to investigate enzyme activities with an impact on intrinsic maturation processes in in situ formed pellicles. Methods Bovine enamel specimens were exposed to the oral cavity in six subjects to allow in situ pellicle formation over 3, 30 and 120 min. The slabs were fixed on the buccal and palatal surfaces of individual splints fixed with silicone impression material. After rinsing with deionised water, the pellicle samples were tested fluorimetrically for transglutaminase, protease and elastase activity. Phosphatase activities were tested photometrically. Separate samples were used for each of the enzymes tested. Results Transglutaminase was detected in in situ pellicle (16.7 ± 21.2 mU/cm 2) as was alkaline phosphatase activity (0.87 ± 0.99 mU/cm 2). For both enzymes, there was no correlation of enzyme activities with time or localisation of pellicle formation. Acidic phosphatase- and protease-activities were not detectable. Only traces of elastase activity were found in 57% of the samples. Conclusion Transglutaminase and phosphatase activity are detectable within in situ pellicle. Enzymatic crosslinking and dephosphorylation appear more important for intrinsic maturation of the acquired enamel pellicle than proteolysis.

Gas-liquid chromatographic analysis of amino acids in pellicle formed on tooth surface and plastic film in vivo

The amino acid composition of 2-h-old pellicle formed in vivo on tooth enamel and on plastic film (polyvinylchloride/polyvinylacetate) applied on tooth surfaces was studied. For comparison, saliva samples were analysed. On 5 consecutive mornings, the plastic film was applied on the buccal surfaces of 12 teeth in each of 4 subjects. After 2 h, the films were removed and material from the buccal surfaces of the 12 contralateral teeth was scraped off with a curette and pooled with material collected in the same way in the afternoon. A sample of whole unstimulated saliva was collected in the afternoon. After acid hydrolysis, N-trifluoro-acetyl- n-butyl derivatives of the amino acids were analysed by gas-liquid chromatography. The amount of protein in the pellicle samples (20–300 μg in the hydrolysates) allowed analysis to be performed well above the detection limit of the method. Thirteen amino acids were separated; the predominant ones in the film samples were in order: glutamic acid, glycine, aspartic acid, leucine and serine, and in the tooth samples: glutamic acid, glycine, serine, aspartic acid, leucine. Between film and tooth samples, statistically significant differences were observed in the amounts of glycine, isoleucine, serine and lysine. In the saliva samples, the predominant amino acids were in order: glutamic acid, glycine, proline, aspartic acid and leucine. Tyrosine and phenylalanine were in small amounts in all samples.

Amino Acid Composition of Experimental Salivary Pellicles

The amino acid compositions of pellicles formed in vitro from submandibular, parotid, and mixed saliva were determined and compared. Samples of the total pellicles as well as the relatively acid-soluble supernates and acid-insoluble sediments were collected and analyzed. In each instance striking differences in composition between the pellicle and the saliva from which it was formed indicate that pellicle formation is a highly selective process. The similarity in composition of the total and fractionated submandibular-parotid pellicle samples indicated a representative dissolution of the total mixed pellicle in the collection acid, rather than a preferential dissolution of part of the total pellicle. In the case of the pellicles formed from the isolated submandibular and parotid salivas, the supernate and the total pellicle were quite similar in composition, but differed somewhat from the sediment. Those results indicate that most of the salivary pellicle is soluble in the dilute acid used to collect the pellicle, and that a portion of the total submandibular or parotid salivary pellicle dissolves preferentially. The compositions of the total parotid pellicle and the parotid supernate and sediment were remarkable similar to the counterparts formed from submandibular saliva alone, suggesting similar origins and mechanisms of formation.