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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 − 5 M 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 I n vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H 2 O-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). I n 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.

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.

BackgroundAll soft and solid surface structures in the oral cavity are covered by the acquired pellicle followed by bacterial colonization. This applies for natural structures as well as for restorative or prosthetic materials; the adherent bacterial biofilm is associated among others with the development of caries, periodontal diseases, peri-implantitis, or denture-associated stomatitis. Accordingly, there is a considerable demand for novel materials and coatings that limit and modulate bacterial attachment and/or propagation of microorganisms.Objectives and findingsThe present paper depicts the current knowledge on the impact of different physicochemical surface characteristics on bioadsorption in the oral cavity. Furthermore, it was carved out which strategies were developed in dental research and general surface science to inhibit bacterial colonization and to delay biofilm formation by low-fouling or “easy-to-clean” surfaces. These include the modulation of physicochemical properties such as periodic topographies, roughness, surface free energy, or hardness. In recent years, a large emphasis was laid on micro- and nanostructured surfaces and on liquid repellent superhydrophic as well as superhydrophilic interfaces. Materials incorporating mobile or bound nanoparticles promoting bacteriostatic or bacteriotoxic properties were also used. Recently, chemically textured interfaces gained increasing interest and could represent promising solutions for innovative antibioadhesion interfaces. Due to the unique conditions in the oral cavity, mainly in vivo or in situ studies were considered in the review.ConclusionDespite many promising approaches for modulation of biofilm formation in the oral cavity, the ubiquitous phenomenon of bioadsorption and adhesion pellicle formation in the challenging oral milieu masks surface properties and therewith hampers low-fouling strategies.Clinical relevanceImproved dental materials and surface coatings with easy-to-clean properties have the potential to improve oral health, but extensive and systematic research is required in this field to develop biocompatible and effective substances.

(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.

Objective This study aimed to examine and compare the efficacy of mouthwashes containing different proteins and peptide on the prevention of enamel erosion in vivo, as well as to evaluate the participants’ satisfaction with the formulations. Methods Twelve participants were selected and underwent five cross-over mouthwash phases: Water (control); 0.1 mg/mL CaneCPI-5; 0.5 mg/mL MaquiCPI-3; 0.1 mg/mL CsinCPI-2; and 0.037 mg/mL Stn15pSpS. After prophylaxis, the participants rinsed (1 min), followed by the acquired enamel pellicle (AEP) formation (2 h). An erosive challenge was made (biopsy, citric acid 1%, 15s) on the buccal surface of the central maxillary incisors. The Relative Surface Reflection Intensity (%SRI) was assessed and analyzed by ANOVA/Tukey’s tests. The calcium release in acid was measured by the Arsenazo method and verified by Kruskal-Wallis/Dunn’s tests. The Spearman’s correlation was used between analyses. A questionnaire evaluated the satisfaction of participants. Results For both analyses, the results showed that mouthwashes containing the proteins or peptide were significantly more effective in preventing enamel erosion compared to deionized water, with no significant differences among the active ingredients ( p  < 0.05). Also, there was a significant negative correlation between %SRI and calcium released ( r =-0.5754). The questionnaire revealed that the volunteers were satisfied with the taste of the products. In addition, the experimental procedures were well tolerable, and no side effects were reported. Conclusion All mouthwashes containing proteins or peptide were acceptable and effective in protecting enamel against initial dental erosion in vivo. Clinical significance: This study highlights the potential of these pioneer organic components for the development of mouthwashes designed for people with risk of erosive tooth wear.

To compare the effects of stannous (Sn) and fluoride (F) ions and their combination on acquired enamel pellicle (AEP) protein composition (proteome experiment), and protection against dental erosion (functional experiment). In the proteome experiment, bovine enamel specimens were incubated in whole saliva supernatant for 24h for AEP formation. They were randomly assigned to 4 groups (n=10), according to the rinse treatment: Sn (800ppm/6.7mM, SnCl2), F (225ppm/13mM, NaF), Sn and F combination (Sn+F) and deionized water (DIW, negative control). The specimens were immersed 3× in the test rinses for 2min, 2h apart. Pellicles were collected, digested, and analyzed for protein content using liquid chromatography electrospray ionization tandem mass spectrometry. In the functional experiment, bovine enamel specimens (n=10) were similarly treated for pellicle formation. Then, they were subjected to a five-day erosion cycling model, consisting of 5min erosive challenges (15.6 mM citric acid, pH 2.6, 6×/d) and 2min treatment with the rinses containing Sn, F or Sn+F (3×/d). Between the treatments, all specimens were incubated in whole saliva supernatant. Surface loss was determined by profilometry. Our proteome approach on bovine enamel identified 72 proteins that were common to all groups. AEP of enamel treated with Sn+F demonstrated higher abundance for most of the identified proteins than the other groups. The functional experiment showed reduction of enamel surface loss for Sn+F (89%), Sn (67%) and F (42%) compared to DIW (all significantly different, p<0.05). This study highlighted that anti-erosion rinses (e.g. Sn+F) can modify quantitatively and qualitatively the AEP formed on bovine enamel. Moreover, our study demonstrated a combinatory effect that amplified the anti-erosive protection on tooth surface.

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

We aimed to comprehensively compare two compartmented oral proteomes, the salivary and the dental pellicle proteome. Systematic review and datamining was used to obtain the physico-chemical, structural, functional and interactional properties of 1,515 salivary and 60 identified pellicle proteins. Salivary and pellicle proteins did not differ significantly in their aliphatic index, hydrophaty, instability index, or isoelectric point. Pellicle proteins were significantly more charged at low and high pH and were significantly smaller (10–20 kDa) than salivary proteins. Protein structure and solvent accessible molecular surface did not differ significantly. Proteins of the pellicle were more phosphorylated and glycosylated than salivary proteins. Ion binding and enzymatic activities also differed significantly. Protein-protein-ligand interaction networks relied on few key proteins. The identified differences between salivary and pellicle proteins could guide proteome compartmentalization and result in specialized functionality. Key proteins could be potential targets for diagnostic or therapeutic application.

ObjectiveThis study evaluated the protective capacity of a sugarcane-derived cystatin (CaneCPI-5) in different vehicles (1-solution and 2-chitosan gel) against erosive dentin wear in situ.MethodsIn part-1, 15 volunteers participated in a crossover protocol (solutions): Water; Elmex™ and CaneCPI-5. The volunteers wore an appliance with 4 dentin samples for 5 days. These samples were treated with a drop of the solutions for 1 min (4X/d), then the acquired pellicle (AP) was formed and the samples were subjected to erosive challenges (EROSION: citric acid, for 90 s, 4X/day). 2X/day, half of the samples were also abraded for 15 s (ABRASION). In part-2, 16 volunteers participated in a crossover protocol (gel): No gel, Chitosan gel, Chitosan gel + NaF and Chitosan gel + CaneCPI-5. The volunteers also wore an appliance. The samples were treated once/day with the gel or not for 4 min, then the AP was formed and the samples were subjected to erosive and abrasive challenges, as reported in part-1. Dentin wear was measured by profilometry. Data were analyzed by two-way RM-ANOVA and Sidak’s tests (p < 0.05).ResultsPart-1: Elmex™ and CaneCPI-5 significantly reduced dentin loss in comparison with Water for the EROSION/ABRASION conditions (p < 0.05). Part-2, all the treated groups significantly reduced the dentin loss in comparison to the No gel. The greatest reduction was found for the gel + CaneCPI-5 group for the EROSION/ABRASION (p < 0.05).ConclusionThe solution and chitosan gel containing CaneCPI-5 protected against erosive dentin wear in situ.Clinical relevanceThese different vehicles are probably sufficient for protecting people with high risk of developing erosive dentin wear.

This study investigates the differences in the lithographic impact of particles on the pellicle surface depending on the type of extreme ultraviolet (EUV) mask pattern. Using an EUV ptychography microscope, we analyzed how mask imaging performance is affected by locally obstructed mask diffraction caused by a 10 μm × 10 μm patterned tin particle intentionally fabricated on the pellicle surface. The resulting critical dimension variations were found to be approximately three times greater in line-and-space patterns than in contact hole patterns. Based on these findings, we recommend defining the critical size of particles according to the mask pattern type to optimize lithographic quality.