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

OBJECTIVES: 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. MATERIALS/METHODS: 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 = 8 cm2). After 10 min 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 10 min. The pellicle material was harvested after 30 min in situ and examined for caseins by the indirect ELISA. Selected pellicle samples were subjected to TEM analysis. RESULTS: All mouthrinses accumulated caseins in the in situ pellicle (2.0 ± 0.7-20 ± 1.7 µg/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. CONCLUSION: 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. CLINICAL RELEVANCE: The study provides interesting perspectives for innovative prevention strategies in dentistry.

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.

Influence of Calcium Phosphate and Apatite Containing Products on Enamel Erosion.

For the purpose of erosion prevention the present study aimed to compare the efficacy of two biomimetic products and a fluoride solution to optimize the protective properties of the pellicle. After 1 min of in situ pellicle formation on bovine enamel slabs, 8 subjects adopted CPP-ACP (GC Tooth Mousse), a mouthwash with hydroxyapatite microclusters (Biorepair), or a fluoride based mouthwash (elmex Kariesschutz) for 1 min each. Afterwards, samples were exposed in the oral cavity for 28 min. Native enamel slabs and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After oral exposure, slabs were incubated in HCl (pH values 2, 2.3, and 3) for 120 s and kinetics of calcium and phosphate release were measured photometrically; representative samples were evaluated by SEM and TEM. The physiological pellicle reduced demineralization at all pH values; the protective effect was enhanced by fluoride. The biomimetic materials also reduced ion release but their effect was less pronounced. SEM indicated no layer formation after use of the different products. However, TEM confirmed the potential accumulation of mineral components at the pellicle surface. The tested products improve the protective properties of the in situ pellicle but not as effectively as fluorides.

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 conventional mouthrinses on initial bioadhesion to enamel and dentin in situ.

AIM: The study aimed to investigate the effect of a customary fluoride solution, containing sodium fluoride and amine fluoride, on initial biofilm formation on enamel and dentin in situ compared directly to chlorhexidine. METHODS: Bovine enamel and dentin specimens were mounted on maxillary splints carried by 9 subjects. After 1 min of pellicle formation, rinses with tap water (control), chlorhexidine (meridol med CHX 0.2%, GABA) and a fluoride mouthrinse (elmex, GABA) were performed for 1 min. Subsequently, the slabs were carried for another 8 h. The adherent bacteria were determined by DAPI staining, live-dead staining and determination of colony-forming units after desorption; glucan formation was visualized with concanavalin A. Additionally, energy-dispersive X-ray spectroscopy (EDX) analysis of the in situ biofilm layers was conducted, and contact angle measurements were performed. Statistical evaluation was performed by means of the Kruskal-Wallis test followed by the Mann-Whitney U test (p < 0.05). RESULTS: In the control group, significantly higher amounts of adherent bacteria were detected on dentin (4.8 × 10(6) ± 5.4 × 10(6) bacteria/cm(2)) than on enamel (1.2 × 10(6) ± 1.5 × 10(6) bacteria/cm(2), DAPI). Chlorhexidine significantly reduced the amount of adherent bacteria (dentin: 2.8 × 10(5) ± 3.4 × 10(5) bacteria/cm(2); enamel: 4.2 × 10(5) ± 8.7 × 10(5) bacteria/cm(2)). Rinses with the fluoride solution also significantly reduced bacterial adherence to dentin (8.1 × 10(5) ± 1.5 × 10(6) bacteria/cm(2)). Fluoride could not be detected by EDX analysis of the biofilms. Fluoride mouthrinsing did not influence the wettability of the pellicle-covered enamel surface. CONCLUSION: In addition to the reduction of demineralization and antibacterial effects, fluorides inhibit initial biofilm formation on dental hard tissues considerably, especially on dentin.

Lipids in preventive dentistry.

OBJECTIVES: There is still a great demand for the improvement of oral prophylaxis methods. One repeatedly described approach is rinsing with edible oils. The aim of the present review paper was to analyze the role of lipids in bioadhesion and preventive dentistry. MATERIALS AND METHODS: Despite limited sound scientific data, extensive literature search was performed to illustrate possible effects of lipids in the oral cavity. RESULTS: It is to be assumed that lipophilic components modulate the process of bioadhesion to the oral hard tissues as well as the composition and ultrastructure of the initial oral biofilm or the pellicle, respectively. Thereby, lipids could add hydrophobic characteristics to the tooth surface hampering bacterial colonization and eventually decreasing caries susceptibility. Also, a lipid-enriched pellicle might be more resistant in case of acid exposure and could therefore reduce the erosive mineral loss. Furthermore, anti-inflammatory effects on the oral soft tissues were described. However, there is only limited evidence for these beneficial impacts. Neither the lipid composition of saliva and pellicle nor the interactions of lipids with the initial oral biofilm and the pellicle layer have been investigated adequately until now. CONCLUSION: Edible oils might qualify as mild supplements to conventional strategies for the prevention of caries, erosion, and periodontal diseases but further research is necessary. CLINICAL RELEVANCE: Against the background of current scientific and empirical knowledge, edible oils might be used as oral hygiene supplements but a decisive benefit for the oral health status is questionable.

Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ.

OBJECTIVE: The aim of the present study was to investigate the efficacy of a new preparation in dental prophylaxis containing zinc-carbonate hydroxyapatite microclusters (Biorepair) for oral biofilm management. METHODS AND MATERIALS: Initial biofilm formation was carried out in situ with bovine enamel slabs fixed to individual upper jaw splints worn by six subjects. Rinses with the customary preparation as well as with subfractions (hydroxyapatite microclusters in saline solution; liquid phase without particles) were adopted for 1 min in situ after 1 min of pellicle formation, and the bacterial colonization was recorded after 6 h and 12 h, respectively. Rinses with chlorhexidine served as a reference. The adherent microorganisms were quantified and visualized using DAPI staining and live-dead staining (BacLight). Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight). RESULTS: Application of the customary preparation and of the separate components distinctly reduced the initial bacterial colonization of the enamel surface in situ as visualized and quantified with all techniques. After 12 h, 1.3 × 10(7) ± 2.0 × 10(7) bacteria/cm² were detected on unrinsed control samples with DAPI staining; 2.4 × 10(6) ± 3.3 × 10(6) after application of Biorepair (12 h after CHX-rinse; 1.3 × 10(5) ± 9.2 × 10(4)). Also, pure hydroxyapatite microclusters in saline solution (2.1 × 10(6) ± 3.0 × 10(6)) as well as the liquid phase without particles (5.1 × 10(5) ± 3.3 × 10(5)) reduced the amount of adherent bacteria. Furthermore, antimicrobial effects on S. mutans were observed in vitro. CONCLUSION: The preparation is an effective compound for biofilm management in the oral cavity due to antiadherent and antibacterial effects. CLINICAL RELEVANCE: The tested mouthrinse seems to be a reasonable amendment for dental prophylaxis.

Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm.

OBJECTIVES: The aim of the present study was to investigate different fluorescence-based, two-color viability assays for visualization and quantification of initial bacterial adherence and to establish reliable alternatives to the ethidium bromide staining procedure. MATERIALS AND METHODS: Bacterial colonization was attained in situ on bovine enamel slabs (n = 6 subjects). Five different live/dead assays were investigated (fluorescein diacetate (FDA)/propidium iodide (PI), Syto 9/PI (BacLight®), FDA/Sytox red, Calcein acetoxymethyl (AM)/Sytox red, and carboxyfluorescein diacetate (CFDA)/Sytox red). After 120 min of oral exposure, analysis was performed with an epifluorescence microscope. Validation was carried out, using the colony-forming units for quantification and the transmission electron microscopy for visualization after staining. RESULTS: The average number of bacteria amounted to 2.9 ± 0.8 × 10(4) cm(-2). Quantification with Syto 9/PI and Calcein AM/Sytox red yielded an almost equal distribution of cells (Syto 9/PI 45% viable, 55% avital; Calcein AM/Sytox red 52% viable, 48% avital). The live/dead ratio of CFDA/Sytox red and FDA/Sytox red was 3:2. An aberrant dispersal was recorded with FDA/PI (viable 34%, avital 66%). The TEM analysis indicated that all staining procedures affect the structural integrity of the bacterial cells considerably. CONCLUSION: The following live/dead assays are reliable techniques for differentiation of viable and avital adherent bacteria: BacLight, FDA/Sytox red, Calcein AM/Sytox red, and CFDA/Sytox red. These fluorescence-based techniques are applicable alternatives to toxic and instable conventional assays, such as the staining procedure based on ethidium bromide. CLINICAL RELEVANCE: Differentiation of viable and avital adherent bacteria offers the possibility for reliable evaluation of different mouth rinses, oral medication, and disinfections.

Erosion behaviour of human, bovine and equine dental hard tissues.

Dental hard tissues from different species are used in dental research, but little is known about their comparability. The aim of this study was to compare the erosive behaviour of dental hard tissues (enamel, dentin) obtained from human, bovine and equine teeth. In addition, the protective effect of the pellicle on each hard tissue under erosive conditions was determined. In situ pellicle formation was performed for 30 min on enamel and dentin samples from all species in four subjects. Calcium and phosphate release was assessed during 120 s of HCl incubation on both native and pellicle-covered enamel and dentin samples. SEM and TEM were used to examine surface changes in native enamel and dentin samples after acid incubation and the ultrastructure of the pellicle before and after erosive exposure. In general, bovine enamel and dentin showed the highest degree of erosion after acid exposure compared to human and equine samples. Erosion of human primary enamel tended to be higher than that of permanent teeth, whereas dentin showed the opposite behaviour. SEM showed that eroded equine dentin appeared more irregular than human or bovine dentin. TEM studies showed that primary enamel appeared to be most susceptible to erosion.

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.

Nanotechnology and its role in caries therapy.

The purpose of this review is to highlight recent nanotechnological developments for remineralization of incipient caries lesions as well as biomimetic strategies for enamel synthesis based on the application of nanotechnology. Analysis of in vitro data indicates that apatite nanoparticles might be effective in reversing lesion progression in the outer but not in the deeper part of early caries lesions. To control caries-induced demineralization, investigators have developed calcium and phosphate or fluoride ion-releasing nanofillers, enabling resin composites to release ions, if the pH decreases under in vitro conditions. Extensive in vitro investigations of apatite crystallization have been performed to mimic the hierarchical topology of natural enamel. Strategies for formation of highly organized biomineralized structures include oriented aggregation of nanocrystallites or the assembly of apatite nanoparticles mediated by organic scaffolds. Despite all these promising in vitro experiments, the effectiveness of such strategies for the control of demineralization processes as well as for caries therapy still needs validation by clinical studies.

The lipid composition of the in situ pellicle.

OBJECTIVE: The 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. DESIGN: Bovine 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. RESULTS: A 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. CONCLUSION: The 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.

Failure analysis and survival rate of post and core restorations under cyclic loading.

AIM: To investigate in a laboratory setting the influence of (i) post material (ii) preparation design and (iii) luting agent on the survival probability of root filled teeth, restored with all-ceramic restorations. METHODOLOGY: The crowns of 80 extracted single-rooted human teeth were removed, and root canal treatment was performed including canal filling with Gutta-percha without sealer (crown-down-pressureless technique). The root fillings were removed and the root canal enlarged with a reamer up to size 110. Prefabricated zirconia (CeraPost) or glass-fibre-reinforced posts (DentinPost) were luted using either Ketac Cem or Panavia F 2.0. A core build-up was applied (Clearfil Photocore), and the teeth were prepared with or without a 2-mm ferrule design (n=10 per experimental group). The prepared teeth were scanned (Cerec 3D) and crowns fabricated. After luting of the crowns (Ketac Cem), teeth were subjected to thermocycling (×4000, 5-55 °C) and cyclic loading (1.5 million cycles, 90 N). After load cycling, the teeth were immersed in methyleneblue solution for 24 h and subsequently sectioned in three segments for a dye penetration test. Kaplan-Meyer analysis was performed to assess the survival probability followed by a Cox regression analysis (α=5%). RESULTS: Teeth prepared using the ferrule design as well as the teeth with DentinPosts exhibited a significantly higher survival probability (P<0.05). The luting agent was of minor importance (P>0.05). Most common failure was debonding of posts (CeraPost) and post fracture (DentinPost). The majority of the teeth showed dye penetration after cyclic loading. CONCLUSIONS: Post material and ferrule design were of paramount importance regarding the survival probability of the post and core restorations using pre-fabricated posts. DentinPosts showed superior results versus CeraPosts.

Effects of Octenidine on the Formation and Disruption of Dental Biofilms: An Exploratory In Situ Study in Healthy Subjects.

Dental biofilms are highly structured, complex multispecies communities that, if left untreated, lead to severe oral complications such as caries and periodontal diseases. Therefore, antibiofilm agents are often recommended for both preventive and therapeutic measures. However, biofilm management can be challenging due to the low sensitivity of biofilms to antimicrobial treatments. Octenidine dihydrochloride (OCT) is a highly effective antibacterial agent. Because the OCT antibiofilm efficacy has not been studied in situ, this exploratory crossover study aimed to evaluate the effects of OCT mouth rinsing on biofilm formation and on the disruption of mature biofilms. Moreover, a comparison to the gold-standard chlorhexidine (CHX) was conducted. The biofilms were formed intraorally by 5 healthy volunteers on enamel specimens fixed to acrylic splints. For biofilm formation analysis, OCT, CHX, or water rinses were applied for 30 s every 12 h. The samples evaluation took place at 24-and 48-h time points. For biofilm disruption analysis, sample assessment was performed before and directly after the first OCT or CHX rinse on 48-h mature biofilms. A second rinse was carried out 12 h later. The last assessment was applied to 72-h mature biofilms. The biofilms were analyzed by fluorescence microscopy and transmission electron microscopy. The results showed OCT significantly reducing biofilm formation and bacterial vitality in situ. Simultaneously, the biofilm thickness was strongly decreased. Moreover, a single application of OCT to a 48-h mature biofilm induced substantial biofilm disruption. In addition, the efficacy of OCT compared favorably to CHX. These findings show that OCT rinses prevent biofilm formation and disrupt preexisting mature biofilms formed by healthy subjects. This work suggests that OCT might be used for dental biofilm management as a part of the medical treatment of oral diseases. Future studies with a larger subject heterogeneity and number are needed to confirm the observed OCT effects.

Long-term Clinical Performance of Composite Resin or Ceramic Inlays, Onlays, and Overlays: A Systematic Review and Meta-analysis.

CLINICAL RELEVANCE: Composite resin or ceramic inlays, onlays, and overlays can achieve high long-term survival and success rates.

Extracellular neutrophil traps in periodontitis.

BACKGROUND AND OBJECTIVE: Chronic periodontitis, the chronic inflammatory disease of the periodontium, is caused by bacteria and is characterized by an influx of neutrophils into the gingival crevice. Recently, a 'new' extracellular neutrophil defense mechanism - neutrophil extracellular traps - has been described. However, their role in periodontitis has not yet been investigated. MATERIAL AND METHODS: Clinical examinations, transmission and scanning electron microscopy, as well as cytology and confocal laser-scanning microscopy, were employed to analyze gingiva biopsies and crevicular exudate from patients with chronic periodontitis. RESULTS: An abundance of neutrophil extracellular traps and some phagocytic neutrophils was found on the gingival pocket surface and in the purulent crevicular exudate. Finding neutrophil extracellular traps in the spontaneously effused purulent crevicular exudate clearly indicated that they are flushed from the pocket by the crevicular exudate. In cases of dispersal of subgingival plaque bacteria, their trapping by neutrophil extracellular traps in purulent crevicular exudate and on the gingival surface was demonstrated. CONCLUSION: Trapping the crevicular bacteria prevents their adhesion to and invasion of the gingiva. The combination of neutrophil extracellular traps and crevicular exudate outflow appears to be a 'novel' defense mechanism for the clearance of crevicular bacteria in chronic periodontitis.

Effect of fluoride mouthrinses and stannous ions on the erosion protective properties of the in situ pellicle.

The particular feature of this study is the investigation of effects of pure fluoride- or stannous ions based mouthrinses on the erosion protective properties and the ultrastructure of the in situ pellicle (12 volunteers). Experimental solutions were prepared either from 500 ppm NaF, SMFP, AmF or SnF2 or 1563 ppm SnCl2, respectively. After 1 min of in situ pellicle formation on bovine enamel slabs, rinses with one of the preparations were performed for 1 min and intraoral specimens' exposure was continued for 28 min. Native enamel slabs and rinses with bidestilled water served as controls. After oral exposure, slabs were incubated in HCl (pH 2; 2.3; 3) for 120 s and kinetics of calcium- and phosphate release were measured photometrically; representative samples were analysed by TEM and EDX. All mouthrinses reduced mineral loss compared to the native 30-min pellicle. The effect was pH-dependent and significant at all pH values only for the tin-containing mouthrinses. No significant differences were observed between the SnF2- and the SnCl2-containing solutions. TEM/EDX confirmed ultrastructural pellicle modifications. SnF2 appears to be the most effective type of fluoride to prevent erosive enamel demineralisation. The observed effects primarily have to be attributed to the stannous ions' content.

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

OBJECTIVES: The 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. METHODS: In 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. RESULTS: Topical 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. CONCLUSION: For 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 SIGNIFICANCE: Bovine milk or micellar caseins provide some potential for the development of preventive strategies against bacterial biofilm formation or erosive processes at the tooth surface.

Whitening Efficacy of Chewing Gum Containing Sodium Metaphosphate on Coffee Stain: Placebo-controlled, Double-blind In Situ Examination.

This study aimed to evaluate the ability of chewing gum containing sodium metaphosphate (SMP) to remove coffee stains from enamel in situ. This was a double-blind (subjects, evaluators), parallel-group, crossover, randomized clinical trial with 30 healthy adult volunteers. Each participant held an appliance with a hydroxyapatite (HA) pellet on the lower lingual side of his or her mouth for two hours to allow pellicle formation. The appliances were subsequently immersed in coffee solution at 37°C for 48 hours. The color of the HA pellet before and after coffee immersion was measured using a spectrophotometer. The participant set the appliance and chewed two pieces of test gum, which contained 7.5 mg of SMP per piece, or control gum without SMP. Each cycle included five minutes of exposure to chewing gum, after which the appliances were placed in 100% relative humidity at room temperature for a 30-minute incubation. This cycle was repeated five times for each gum type. The color of the HA pellet was measured after each chewing cycle using the spectrophotometer. In addition, ΔE* values, which indicate the change in pellet color after each chewing cycle compared with after coffee immersion, were calculated. Data were analyzed using the paired t-test with Bonferroni adjustment to compare ΔE* values of control and test gum after each chewing cycle. The ΔE* values of test gum were significantly higher than those of control gum after all chewing cycles, excluding the first cycle (p<0.05). This finding indicates that test gum containing SMP was more effective at removing coffee stains from the HA pellet than control gum. We conclude that chewing gum containing SMP can effectively remove coffee stains from HA pellets. Thus, SMP is a promising agent to be further explored in tooth-cleaning studies.

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.