Structure Response of Preadsorbed Saliva Pellicle to the Interaction between Dairy and Saliva Protein.

Using the surface characterization techniques of quartz crystal microbalance with dissipation, atomic force microscopy, and scanning electron microscopy, the structure of the salivary pellicle was investigated before and after it was exposed to dairy proteins, including micellar casein, skim milk, whey protein isolate (WPI), and a mixture of skim milk and WPI. We have shown that the hydration, viscoelasticity, and adsorbed proteinaceous mass of a preadsorbed salivary pellicle on a PDMS surface are greatly affected by the type of dairy protein. After interaction with whey protein, the preadsorbed saliva pellicle becomes softer. However, exposure of the saliva pellicle to micellar casein causes the pellicle to partially collapse, which results in a thinner and more rigid surface layer. This structure change correlates with the measured lubrication behavior when the saliva pellicle is exposed to dairy proteins. While previous studies suggest that whey protein is the main component in milk to interact with salivary proteins, our study indicates interactions with casein are more important. The knowledge gained here provides insights into the mechanisms by which different components of dairy foods and beverages contribute to mouthfeel and texture perception, as well as influence oral hygiene.

Hemoglobin Protects Enamel against Intrinsic Enamel Erosive Demineralization.

INTRODUCTION: This study investigated the changes in the acquired enamel pellicle (AEP) proteome when this integument is formed in vivo after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), and a statherin-derived peptide (StN15), or their combination and then exposed to an intrinsic acid challenge. The effectiveness of these treatments in preventing intrinsic erosion was also evaluated. METHODS: Ten volunteers, after prophylaxis, in 5 crossover phases, rinsed with the following solutions (10 mL, 1 min): control (deionized water-H2O) - group 1, 0.1 mg/mL CaneCPI-5 - group 2, 1.0 mg/mL HB - group 3, 1.88 × 10-5M StN15 - group 4, or a blend of these - group 5. Following this, AEP formation occurred (2 h) and an enamel biopsy (10 µL, 0.01 m HCl, pH 2.0, 10 s) was conducted on one incisor. The biopsy acid was then analyzed for calcium (Arsenazo method). The vestibular surfaces of the other teeth were treated with the same acid. Acid-resistant proteins in the residual AEP were then collected and analyzed quantitatively via proteomics. RESULTS: Compared to control, treatment with the proteins/peptide, mixed or isolated, markedly enhanced acid-resistant proteins in the AEP. Notable increases occurred in pyruvate kinase PKM (11-fold, CaneCPI-5), immunoglobulins and submaxillary gland androgen-regulated protein 3B (4-fold, StN15), Hb, and lysozyme C (2-fold, StN15). Additionally, a range of proteins not commonly identified in the AEP but known to bind calcium or other proteins were identified in groups treated with the tested proteins/peptide either in isolation or as a mixture. The mean (SD, mM) calcium concentrations released from enamel were 3.67 ± 1.48a, 3.11 ± 0.72a, 1.94 ± 0.57b, 2.37 ± 0.90a, and 2.38 ± 0.45a for groups 1-5, respectively (RM-ANOVA/Tukey, p < 0.05). CONCLUSIONS: Our findings demonstrate that all treatments, whether using a combination of proteins/peptides or in isolation, enhanced acid-resistant proteins in the AEP. However, only HB showed effectiveness in protecting against intrinsic erosive demineralization. These results pave the way for innovative preventive methods against intrinsic erosion, using "acquired pellicle engineering" techniques.

Effect of Polyphenols on the Ultrastructure of the Dentin Pellicle and Subsequent Erosion.

INTRODUCTION: Erosive tooth wear is a highly prevalent dental condition that is modified by the ever-present salivary pellicle. The aim of the present in situ study was to investigate the effect of polyphenols on the ultrastructure of the pellicle formed on dentin in situ and a subsequent erosive challenge. METHODS: The pellicle was formed on bovine dentin specimens for 3 min or 2 h in 3 subjects. After subjects rinsed with sterile water (negative control), 1% tannic acid, 1% hop extract, or tin/fluoride solution containing 800 ppm tin and 500 ppm fluoride (positive control), specimens were removed from the oral cavity. The erosive challenge was performed on half of the specimens with 1% citric acid, and all specimens were analyzed by transmission electron microscopy. Incorporation of tannic acid in the pellicle was investigated by fluorescence spectroscopy. RESULTS: Compared to the negative control, ultrastructural analyses reveal a thicker and electron-denser pellicle after application of polyphenols, in which, according to spectroscopy, tannic acid is also incorporated. Application of citric acid resulted in demineralization of dentin, but to a lesser degree when the pellicle was pretreated with a tin/fluoride solution. The pellicle was more acid-resistant than the negative control when modified with polyphenols or tin/fluoride solution. CONCLUSION: Polyphenols can have a substantial impact on the ultrastructure and acid resistance of the dentin pellicle, while the tin/fluoride solution showed explicit protection against erosive demineralization.

Acquired enamel pellicle and biofilm engineering with a combination of acid-resistant proteins (CaneCPI-5, StN15, and Hemoglobin) for enhanced protection against dental caries - in vivo and in vitro investigations.

OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.

Proteomic profile of the acquired enamel pellicle of children with early childhood caries and caries-free children.

Acquired enamel pellicle plays an important role in the pathogenesis of early childhood caries (ECC), working as a protective interface between the tooth and the oral cavity. The aim of this cross-sectional in vivo proteomic study was to compare the acquired enamel pellicle protein profile of 3-5-year-old children with ECC (n = 10) and caries-free children (n = 10). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). In total, 241 proteins were identified. Basic salivary proline-rich protein 1 and 2, Cystatin-B, and SA were found only in the caries free group. When comparing caries free and ECC groups, lower protein levels were found in the caries free group for hemoglobin subunit beta, delta, epsilon, gamma-2, globin domain-containing protein and gamma-1, neutrophil defensin 3, serum albumin, protein S100-A8, and S100-A9. The proteins histatin-1, statherin, salivary acidic proline-rich phosphoprotein ½, proline-rich protein 4, submaxillary gland androgen-regulated protein 3B, alpha-amylase 1 and 2B were found at higher levels in the caries free group. The exclusive and the proteins found at higher levels in the caries free group might have protective functions that play a role in the prevention of caries, besides providing important insights to be evaluated in future studies for the possible development of new therapeutic strategies for ECC.

Salivary pellicle modulates biofilm formation on titanium surfaces.

OBJECTIVES: The present study aimed to evaluate the potential of the salivary pellicle (SP) formed on titanium (Ti) surfaces to modulate the formation of a biofilm composed of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. MATERIALS AND METHODS: Ti substrates were incubated for 2 h with a pool of saliva samples obtained from 10 systemically and periodontally healthy subjects. Enamel substrates were included as a biological reference. Scanning electron microscopy (SEM) and Raman spectroscopy analysis were used to analyze the formation of the salivary pellicle. After the SP formation, the surfaces were incubated for 12 h with a mix of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. The number of bacterial cells attached to each surface was determined by the XTT assay while bacterial viability was analyzed by fluorescence microscopy using the LIVE/DEAD® BacLightTM kit. RESULTS: The SEM and Raman spectroscopy analysis confirmed the presence of a salivary pellicle formed on the tested surfaces. Regarding the biofilm formation, the presence of the SP decreases the number of the bacterial cells detected in the test surfaces, compared with the uncover substrates. Even more, the SP-covered substrates showed similar bacterial counts in both Ti and enamel surfaces, meaning that the physicochemical differences of the substrates were less determinant than the presence of the SP. While on the SP-uncover substrates, differences in the bacterial adhesion patterns were directly related to the physicochemical nature of the substrates. CONCLUSIONS: The salivary pellicle was the main modulator in the development of the biofilm consisting of representative oral bacteria on the Ti substrates. CLINICAL RELEVANCE: The results of this study provide valuable information on the modulatory effect of the salivary pellicle on biofilm formation; such information allows us to understand better the events involved in the formation of oral biofilms on Ti dental implants.

Salivary Pellicle Formed on Dental Composites Evaluated by Mass Spectrometry-An In Situ Study.

(1) Background: In the oral environment, sound enamel and dental restorative materials are immediately covered by a pellicle layer, which enables bacteria to attach. For the development of new materials with repellent surface functions, information on the formation and maturation of salivary pellicles is crucial. Therefore, the present in situ study aimed to investigate the proteomic profile of salivary pellicles formed on different dental composites. (2) Methods: Light-cured composite and bovine enamel samples (controls) were exposed to the oral cavity for 30, 90, and 120 min. All samples were subjected to optical and mechanical profilometry, as well as SEM surface evaluation. Acquired pellicles and unstimulated whole saliva samples were analyzed by SELDI-TOF-MS. The significance was determined by the generalized estimation equation and the post-hoc bonferroni adjustment. (3) Results: SEM revealed the formation of homogeneous pellicles on all test and control surfaces. Profilometry showed that composite surfaces tend to be of higher roughness compared to enamel. SELDI-TOF-MS detected up to 102 different proteins in the saliva samples and up to 46 proteins in the pellicle. Significant differences among 14 pellicle proteins were found between the composite materials and the controls. (4) Conclusions: Pellicle formation was material- and time-dependent. Proteins differed among the composites and to the control.

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 (10 min, 37°C), incubation in solution (2 min, 25°C), subsequent pellicle formation (90 min, 37°C). Half of the specimens was kept in a humid chamber and the other half was submitted to erosion (2 min, 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.

Acquired Pellicle Engineering Using a Combination of Organic (Sugarcane Cystatin) and Inorganic (Sodium Fluoride) Components against Dental Erosion.

This study evaluated the combination of a sugarcane cystatin (CaneCPI-5) and sodium fluoride (NaF) in acquired pellicle engineering for the prevention of dental erosion in vitro. Seventy-five human enamel specimens were prepared and divided into 5 treatment groups (n = 15/group): Deionized water (Control); Elmex™ (SnCl2/NaF/AmF); 0.1 mg/mL CaneCPI-5; 500 ppm NaF; and CaneCPI-5+NaF (Combination). The specimens were individually treated (200 µL; 2 min; 37°C), then incubated in human saliva (200 µL; 1 h, at 37°C) for acquired pellicle formation. Afterward, the specimens were submitted to an erosive challenge (1% citric acid [CR], pH 3.6, 10 mL, 2 min, 25 °C). This sequence was conducted 5 times. Percentage of surface microhardness change (%SMC), relative surface reflection intensity (rSRI), and calcium released to the CR were measured and analyzed by one-way ANOVA followed by Tukey's test (p < 0.05). In general, all the treatments (SnCl2/NaF/AmF, CaneCPI-5, NaF, and Combination) significantly protected the enamel when compared the control group. Regarding %SMC and rSRI, the Combination was the most effective treatment, reducing the %SMC significantly (p < 0.01) when compared to all the other treatments, although this difference was not significant in the CR analysis. All treatments demonstrated a protective effect on enamel against dental erosion; however, the combination of CaneCPI-5 with NaF showed a greater protection.

Ultrastructure of the Dentin Pellicle and the Impact of Erosion.

While the ultrastructure of the enamel pellicle and its erosion protective properties are well studied, the dentin pellicle is still neglected in dental research. Therefore, the ultrastructure and erosion protective properties of a pellicle formed on bovine dentin specimens were investigated in the present study. The dentin pellicle was formed in situ for 3, 30, 120, and 360 min at buccal or palatal oral sites of 3 subjects and analyzed by transmission electron microscopy. In order to clarify the impact of an erosive challenge to the ultrastructure of the pellicle and the underlying dentin, specimens were exposed to the oral cavity and eroded in vivo with 0.1% or 1% citric acid either immediately or after 30 min of pellicle formation. Specimens that were eroded without exposure to the oral cavity served as control. In another trial, specimens with a 30-min pellicle were exposed to the oral cavity for a further 60 min after the erosive challenge to investigate the effect of saliva on the impaired pellicle and dentin. Transmission electron micrographs reveal a globular and granular structured pellicle layer, which was thicker when the pellicle was formed buccally or with longer formation times. Erosion with citric acid reduced the thickness of the pellicle and interrupted its continuity. The dentin was also affected by erosion, which was represented by a lower electron density and formation of demineralized lacunae. These were infiltrated by a granular structured material when specimens were exposed to the oral cavity. After further intraoral exposure, the infiltration was more pronounced, indicating a significant impact of saliva on the demineralized dentin. A reformation of the dentin pellicle on the other hand did not occur. In conclusion, the dentin pellicle is neither acid-resistant nor able to effectively protect dentin from erosion.

Bioadhesion on Textured Interfaces in the Human Oral Cavity-An In Situ Study.

Extensive biofilm formation on materials used in restorative dentistry is a common reason for their failure and the development of oral diseases like peri-implantitis or secondary caries. Therefore, novel materials and strategies that result in reduced biofouling capacities are urgently sought. Previous research suggests that surface structures in the range of bacterial cell sizes seem to be a promising approach to modulate bacterial adhesion and biofilm formation. Here we investigated bioadhesion within the oral cavity on a low surface energy material (perfluorpolyether) with different texture types (line-, hole-, pillar-like), feature sizes in a range from 0.7-4.5 µm and graded distances (0.7-130.5 µm). As a model system, the materials were fixed on splints and exposed to the oral cavity. We analyzed the enzymatic activity of amylase and lysozyme, pellicle formation, and bacterial colonization after 8 h intraoral exposure. In opposite to in vitro experiments, these in situ experiments revealed no clear signs of altered bacterial surface colonization regarding structure dimensions and texture types compared to unstructured substrates or natural enamel. In part, there seemed to be a decreasing trend of adherent cells with increasing periodicities and structure sizes, but this pattern was weak and irregular. Pellicle formation took place on all substrates in an unaltered manner. However, pellicle formation was most pronounced within recessed areas thereby partially masking the three-dimensional character of the surfaces. As the natural pellicle layer is obviously the most dominant prerequisite for bacterial adhesion, colonization in the oral environment cannot be easily controlled by structural means.

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.

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.

Rinsing with Statherin-Derived Peptide Alters the Proteome of the Acquired Enamel Pellicle.

Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10-5 M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS.

Preventive Applications of Polyphenols in Dentistry-A Review.

Polyphenols are natural substances that have been shown to provide various health benefits. Antioxidant, anti-inflammatory, and anti-carcinogenic effects have been described. At the same time, they inhibit the actions of bacteria, viruses, and fungi. Thus, studies have also examined their effects within the oral cavity. This review provides an overview on the different polyphenols, and their structure and interactions with the tooth surface and the pellicle. In particular, the effects of various tea polyphenols on bioadhesion and erosion have been reviewed. The current research confirms that polyphenols can reduce the growth of cariogenic bacteria. Furthermore, they can decrease the adherence of bacteria to the tooth surface and improve the erosion-protective properties of the acquired enamel pellicle. Tea polyphenols, especially, have the potential to contribute to an oral health-related diet. However, in vitro studies have mainly been conducted. In situ studies and clinical studies need to be extended and supplemented in order to significantly contribute to additive prevention measures in caries prophylaxis.

Proteomic profiles of the acquired enamel pellicle formed in vitro, in situ, or in vivo.

This study compared the protein profile of the acquired enamel pellicle (AEP) formed under three conditions: in vitro, in situ, and in vivo. Nine volunteers participated in all procedures. In the in vitro condition, the volunteers donated saliva, in which specimens were incubated to form the AEP. In the in situ condition, the volunteers used an oral device containing specimens where the AEP was formed. In the in vivo condition, the AEP was collected from the volunteers own teeth. All AEPs were formed for 120 min, collected and processed by mass spectrometry. Overall, a total of 321 proteins were identified, among which 37 proteins are commonly considered typical in the AEP. For each of the in vitro, in situ, and in vivo conditions, respectively, 66, 174, and 170 proteins were identified. For the in vitro condition, 17 pellicle-typical proteins were not identified. Furthermore, several proteins with important functions within the AEP presented differences in expression in the three conditions. The qualitative profile of the proteins, especially the typical ones, is different in the in vitro condition. In addition, there are important quantitative differences that may interfere when attempting to extrapolate in vitro results to an in situ and in vivo condition.

Comparison of protein profiles of the pellicle, gingival crevicular fluid, and saliva: possible origin of pellicle proteins.

BACKGROUND: The pellicle, the acellular organic material deposited on the surface of tooth enamel, has been thought to be derived from saliva. In this study, protein compositions of the pellicle, gingival crevicular fluid, and saliva collected from healthy adults were compared to elucidate the origin of pellicle proteins. RESULTS: The pellicle, gingival crevicular fluid, and saliva from the parotid gland or mixed gland were collected; subsequently, protein expression in samples from the respective individual was compared by SDS-PAGE and mass spectrometry. Following SDS-PAGE, proteins in the major bands were identified by mass spectrometry. The band pattern of pellicle proteins appeared different from those of gingival crevicular fluid, or saliva samples. Using mass spectrometry, 13 proteins in these samples were identified. The relative abundance of the proteins was quantitatively analyzed using mass spectrometry coupled with stable isotope labeling and by western blot. Cystatin S and α-amylase detected in pellicle were enriched in saliva samples, but not in gingival crevicular fluid, by western blot, and their abundance ratios were high in saliva and low in gingival crevicular fluid when analyzed by stable isotope labeling. Serotransferrin, however, was found only in the pellicle and gingival crevicular fluid by western blot and its abundance ratio was low in saliva. CONCLUSIONS: Our study revealed that the gingival crevicular fluid appears to contribute to pellicle formation in addition to saliva.

In vitro adherence of Candida albicans to zirconia surfaces.

OBJECTIVES: This study aimed to characterize surface properties such as roughness (Ra) and surface-free energy (SFE) of glazed and polished yttria-stabilized zirconia and to evaluate in vitro adherence of fungus Candida albicans and salivary bacteria, Staphylococcus epidermidis, mixed with C. albicans to these substrata. Additionally, the influence of salivary proteins (albumin, mucin and α-amylase) on yeast adhesion was studied. MATERIAL AND METHODS: Ra and SFE of glazed and polished zirconia discs were measured. Specimens were wetted with saliva and salivary proteins prior to incubation with C. albicans and mixed suspension of C. albicans and S. epidermidis for 24 hr, respectively. Microbial adhesion was quantified by counting colony-forming units (CFU). Differences in physicochemical properties were proved by t test. "Linear mixed model" with the factors "type of surface" and "wetting media" was applied to analyse the effects on fungal adhesion (p < .05). RESULTS: SFE and Ra of glazed specimens were significantly higher than corresponding values of polished ones. The wetting media significantly changed the fungal binding (p = .0016). Significantly higher quantities of adhering fungi were found after mucin incubation compared to saliva (p = .004). For the factor "surface" as well as the interaction between "surface" and "wetting media," no statistically significant differences have been found. In mixed suspension, the growth of Candida was completely prevented. CONCLUSIONS: Glazed and polished zirconia differs in terms of physicochemical surface properties. These differences appear to be modulated by pellicle coating affecting the biomass of adhered Candida. Mucin seems to be good binding sites for adhesion of C. albicans.

Differences in the Natural Enamel Surface and Acquired Enamel Pellicle following Exposure to Citric or Hydrochloric Acid.

OBJECTIVES: The aim of this study was to investigate variations in the interaction between enamel, that is, the acquired enamel pellicle (AEP) and citric or hydrochloric acid. MATERIALS AND METHODS: A 24-h AEP was formed on natural enamel specimens (n = 40) from pooled whole mouth human saliva. Samples were randomly allocated to citric (0.3%, pH 3.2) or hydrochloric (HCl) acid (0.01 M, pH 2.38) exposure for 30 or 300 s. The total protein concentration (TPC), and phosphorous and calcium concentrations of the pellicle were determined before and after acid exposure, and again after re-immersion in saliva. Surface roughness and tandem scanning confocal microscopy imaging were used to assess enamel changes. RESULTS: After 300 s of citric acid exposure, the mean ± SD TPC reduced from 5.1 ± 1.1 to 3.5 ± 1.1 mg/mL (p < 0.05). In contrast, after 300 s of HCl exposure, the mean TPC did not reduce significantly from baseline (6.6 ± 1.1 to 5.7 ± 0.7 mg/mL) but was significantly reduced in the reformed pellicle to 4.9 ± 1.2 mg/mL (p < 0.001). This reduction occurred after significant release of calcium and phosphorous from the enamel surface (p < 0.001). Thirty seconds of exposure to either acid had no obvious effect on the AEP. The surface roughness of the enamel decreased after acid exposure but no differences between groups was observed. CONCLUSIONS: These findings indicate that citric acid interacted with proteins in the AEP upon contact, offering enamel protection. In contrast, HCl appeared to bypass the pellicle, and reduced protein was observed only after changes in the enamel chemical composition.

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.