Anti-adherence and bactericidal activity of sphingolipids against Streptococcus mutans.

This study evaluated the anti-biofilm activity of sphingosine, phytosphingosine (PHS), and sphinganine for: (i) anti-adherence activity on hydroxyapatite (HA) surfaces; and (ii) bactericidal activity on different Streptococcus mutans phenotypes (i.e. planktonic cells and cells from a disrupted biofilm). For this, HA discs treated with sphingolipids were incubated with S. mutans and the number of adherent cells was evaluated by both culture and confocal microscopy. Sphinganine strongly inhibited bacterial adherence by 1000-fold compared with an untreated surface. Phytosphingosine and sphingosine inhibited bacterial adherence by eight- and five-fold, respectively, compared with an untreated surface. On saliva-coated HA, sphinganine and PHS inhibited bacterial adherence by 10-fold. Bactericidal activity of sphingolipids was evaluated by culture. For biofilms, the strongest bactericidal activity was exhibited by sphingosine compared with PHS and sphinganine. At a concentration of 12.5 µg ml(-1) , PHS and sphingosine were profoundly effective against planktonic and disrupted biofilms; and sphinganine reduced the number of cells in planktonic form by 100-fold and those derived from a disrupted biofilm by 1000-fold. Atomic force microscopy studies suggested that mechanical stability does not appear to be a factor relevant for anti-fouling activity. The results suggest that sphingolipids may be used to control oral biofilms, especially those loaded with S. mutans.

Affinity states of biocides determine bioavailability and release rates in marine paints.

A challenge for the next generation marine antifouling (AF) paints is to deliver minimum amounts of biocides to the environment. The candidate AF compound medetomidine is here shown to be released at very low concentrations, ie ng ml(-1) day(-1). Moreover, the release rate of medetomidine differs substantially depending on the formulation of the paint, while inhibition of barnacle settlement is independent of release to the ambient water, ie the paint with the lowest release rate was the most effective in impeding barnacle colonisation. This highlights the critical role of chemical interactions between biocide, paint carrier and the solid/aqueous interface for release rate and AF performance. The results are discussed in the light of differential affinity states of the biocide, predicting AF activity in terms of a high surface affinity and preserved bioavailability. This may offer a general framework for the design of low-release paint systems using biocides for protection against biofouling on marine surfaces.

Sphingoid bases inhibit acid-induced demineralization of hydroxyapatite.

Calcium hydroxyapatite (HAp), the main constituent of dental enamel, is inherently susceptible to the etching and dissolving action of acids, resulting in tooth decay such as dental caries and dental erosion. Since the prevalence of erosive wear is gradually increasing, there is urgent need for agents that protect the enamel against erosive attacks. In the present study we studied in vitro the anti-erosive effects of a number of sphingolipids and sphingoid bases, which form the backbone of sphingolipids. Pretreatment of HAp discs with sphingosine, phytosphingosine (PHS), PHS phosphate and sphinganine significantly protected these against acid-induced demineralization by 80 ± 17%, 78 ± 17%, 78 ± 7% and 81 ± 8%, respectively (p < 0.001). On the other hand, sphingomyelin, acetyl PHS, octanoyl PHS and stearoyl PHS had no anti-erosive effects. Atomic force measurement revealed that HAp discs treated with PHS were almost completely and homogeneously covered by patches of PHS. This suggests that PHS and other sphingoid bases form layers on the surface of HAp, which act as diffusion barriers against H(+) ions. In principle, these anti-erosive properties make PHS and related sphingosines promising and attractive candidates as ingredients in oral care products.

Influence of substratum hydrophobicity on salivary pellicles: organization or composition?

Different physico-chemical properties (eg adsorption kinetics, thickness, viscoelasticity, and mechanical stability) of adsorbed salivary pellicles depend on different factors, including the properties (eg charge, roughness, wettability, and surface chemistry) of the substratum. Whether these differences in the physico-chemical properties are a result of differences in the composition or in the organization of the pellicles is not known. In this work, the influence of substratum wettability on the composition of the pellicle was studied. For this purpose, pellicles eluted from substrata of different but well-characterized wettabilities were examined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that substratum hydrophobicity did not have a major impact on pellicle composition. In all substrata, the major pellicle components were found to be cystatins, amylases and large glycoproteins, presumably mucins. In turn, interpretation of previously reported data based on the present results suggests that variations in substratum wettability mostly affect the organization of the pellicle components.

Efficacy and safety of an intraoral electrostimulation device for xerostomia relief: a multicenter, randomized trial.

OBJECTIVE: To evaluate the efficacy and safety of an intraoral electrostimulation device, consisting of stimulating electrodes, an electronic circuit, and a power source, in treating xerostomia. The device delivers electrostimulation through the oral mucosa to the lingual nerve in order to enhance the salivary reflex. METHODS: The device was tested on a sample of patients with xerostomia due to Sjögren's syndrome and other sicca conditions in a 2-stage prospective, randomized, multicenter trial. Stage I was a double-blind, crossover stage designed to compare the effects of the electrically active device with the sham device, each used for 1 month, and stage II was a 3-month open-label stage designed to assess the long-term effects of the active device. Improvement in xerostomia severity from baseline was the primary outcome measure. RESULTS: A total of 114 patients were randomized. In stage I, the active device performed better than the sham device for patient-reported xerostomia severity (P<0.002), xerostomia frequency (P<0.05), quality of life impairment (P<0.01), and swallowing difficulty (P<0.02). At the end of stage II, statistically significant improvements were verified for patient-reported xerostomia severity (P<0.0001), xerostomia frequency (P<0.0001), oral discomfort (P<0.001), speech difficulty (P<0.02), sleeping difficulty (P<0.001), and resting salivary flow rate (P<0.01). CONCLUSION: Our findings indicate that daily use of the device alleviated oral dryness, discomfort, and some complications of xerostomia, such as speech and sleeping difficulties, and increased salivary output. The results show a cumulative positive effect of the device over the period of the study, from baseline to the end of the trial.

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics.

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.

Friction force spectroscopy as a tool to study the strength and structure of salivary films.

In this work, we employ atomic force microscopy based friction force spectroscopy to study the strength and structure of salivary films. Specifically, films formed on model hydrophobic (methylated silica) and hydrophilic (clean silica) substrata have been studied in water at pHs in the range 3.3-7. Results reveal that films formed on both types of substrata can be described in terms of two different fractions, with only one of them being able to diffuse along the underlying substrata. We also show how the protective function of the films is reduced when the pH of the surrounding medium is lowered. Specifically, lowering of pH causes desorption of some components of the films formed on hydrophobic methylated surfaces, leading to weaker layers. In contrast, at low pHs, saliva no longer forms a layer on hydrophilic silica surfaces. Instead, an inhomogeneous distribution of amorphous aggregates is observed. Our data also suggest that hydrophobic materials in the oral cavity might be more easily cleaned from adsorbed salivary films. Finally, reproducible differences are observed in results from experiments on films from different individuals, validating the technique as a tool for clinical diagnosis of the resistance to erosion of salivary films.

Adsorption of HSA, IgG and laminin-1 on model titania surfaces--effects of glow discharge treatment on competitively adsorbed film composition.

This study investigated the effect of glow discharge treatment of titania surfaces on plasma protein adsorption, by means of ellipsometry and mechanically assisted SDS elution. The adsorption and film elution of three plasma proteins, viz. human serum albumin (HSA), human immunoglobulin G (IgG) and laminin-1, as well as competitive adsorption from a mixture of the three proteins, showed that the adsorbed amount of the individual proteins after 1 h increased in the order HSA

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol.

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.

A randomized prospective clinical evaluation of two desensitizing agents on cervical dentine sensitivity. A pilot study.

The aim of this prospective clinical pilot study was to evaluate the pain alleviation effectiveness of two desensitizing agents (VivaSens and Seal&Protect) on 30 patients suffering from cervical dentine sensitivity (CDS) over a six month period. Analysis of possible differences in pain alleviation effect between the agents and over time was performed. Further,the experienced pain was registered in a questionnaire regarding to what extent the treatment improved oral health/life quality among the patients. The patients (23 female, 7 male) were randomly divided into two groups. Each group was treated with one of the two desensitizing agents. Sensitivity measurements were recorded before treatment (baseline) and after treatment at time points of one week and six months. The patients were asked to rate the sensitivity experienced in the area during air stimulation by marking on a Visual Analogue Scale (VAS). At six months, 27 patients (90%) had completed the clinical trial. The results showed that a significant reduction of CDS was achieved by using VivaSens or Seal&Protect after both one week and six months. However, there were no differences found on treatment effects between the two desensitizing agents. The results from the questionnaire showed that the patients experienced improved oral health/life quality when comparing the status before and after treatment (0.000 < or = p < or = 0.0021) and there were no statistically significant difference in treatment effects between the products. In conclusion, both desensitizing agents were effective in relieving cervical dentin hypersensitivity during the time course of the study as evaluated both by air stimulation and a questionnaire related to oral health/quality of life status.

Retrospective study comparing the clinical outcomes of bar-clip and ball attachment implant-supported overdentures.

The aim of this study was to compare the clinical outcomes of implant-supported overdentures (ODs) with either bar-clip or ball attachments. The implant, prosthesis failure, and technical complications were the outcomes analyzed in this retrospective clinical study conducted in a specialty clinic. Seventy-five patients with 242 implants supported by 76 ODs (36 maxillary, 40 mandibular) were included in the study and followed up for 88.8 ± 82.9 months (mean ± standard deviation). Bar-clip and ball attachments were used in 78.9% and 21.1% of the cases, respectively. Forty-three implant failures (17.8%) in 17 prostheses (17/76; 22.4%) were observed in this study. The average period of implant failure was 43.3 ± 41.0 months, and most of them were maxillary turned implants. The bar-clip system demonstrated more complications in the attachment parts compared to the ball attachment system. Poor retention of the prosthesis was similar between the two systems. Loss of implants resulted in the failure of 10 ODs in this study. ODs opposed by natural dentition or fixed prostheses presented with more complications. The Cox proportional hazards model did not show a significant effect on prosthesis failure for any of the factors. These findings indicated that patients with ODs need constant maintenance follow-ups to address the technical complications and perform prosthodontic maintenance regardless of the attachment system used.

Validation of mechanically-assisted sodium dodecyl-sulphate elution as a technique to remove pellicle protein components from human enamel.

The salivary film, denoted the pellicle, formed on oral surfaces is of great importance for oral health and comfort. The present study describes mechanically-assisted sodium dodecyl sulphate (SDS) elution of the in vivo pellicle formed on human enamel and visualisation of the desorbed pellicle proteins using two-dimensional gel electrophoresis (2-DE). To verify this removal of the pellicle, a combined mechanical and surfactant procedure was additionally performed on an in vitro pellicle formed on human enamel, and the effectiveness was validated by mechanical removal in combination with HCl. As indicated by protein quantitation and one dimensional gel electrophoresis, rubbing with polyamide fibre pellets soaked in a 0.5% SDS solution was optimal for completely removing the adsorbed proteins from the enamel surface, and yet provided separation of the proteins by 2-DE to enable identification in future studies.

Studies on the exchange of early pellicle proteins by mucin and whole saliva.

Adsorption of small pellicle proteins statherin or proline-rich protein 1 (PRP1), respectively, and subsequent adsorption of human whole saliva (HWS) or salivary mucin MUC5B, respectively, was studied using ellipsometry and total internal reflectance fluorescence. Differences in elution (using sodium dodecyl sulphate (SDS) solutions) between mixed and single protein films were also investigated. On both hydrophilic and hydrophobized surfaces HWS and MUC5B were found to adsorb to pre-adsorbed layers of statherin and PRP1, respectively. Statherin adsorption on both substrate types showed no or minor exchange by HWS or MUC5B and no change in SDS elution between mixed and single protein films. Small amounts of PRP1 were exchanged by HWS on both surface types and the SDS elutable fractions were similar or larger for mixed films compared to single protein films. PRP1 and MUC5B in sequence showed minor exchange of PRP1 on hydrophilic surfaces, while no exchange could be established on hydrophobized substrates. SDS elutable fractions decreased for PRP1 and MUC5B mixed films compared to single protein films. In conclusion, minor amounts of statherin and PRP1 are exchanged during the time course of the experiments, which indicates that these proteins may to a large extent remain incorporated in the pellicle.

Adsorption behavior of statherin and a statherin peptide onto hydroxyapatite and silica surfaces by in situ ellipsometry.

The salivary protein statherin is known to adsorb selectively onto hydroxyapatite (HA), which constitutes the main mineral of the tooth enamel. This adsorption is believed to be crucial for its function as an inhibitor of primary (spontaneous) and secondary (crystal growth) precipitation of calcium phosphate salts present in saliva. A fragment corresponding to the first 21 N-terminus amino acids of statherin (StN21) was previously found to reduce the rate of demineralization of HA. Therefore, the interfacial properties of this peptide and statherin onto silica, hydrophobized silica and HA discs was studied by in situ ellipsometry. Their reversibility induced by dilution and elutability induced by buffer and sodium dodecyl sulfate (SDS) was also determined. The results revealed that statherin adsorbed at a greater extent onto the HA as compared to StN21, suggesting that the hydrogen bonding between the uncharged polar residues at the C-terminal region of statherin and HA contributes to its adsorption. However, on both silica surfaces the peptide adsorption appeared to proceed in a similar way. Onto the hydrophobized silica the adsorption of both peptides was suggested to occur either via multilayer formation or adsorption of aggregates from solution, while onto the hydrophilic silica adsorption of peptide aggregates from solution was the suggested mechanism. Further, both peptides were observed to be strongly adsorbed onto HA, even after SDS treatment, in comparison to the layers adsorbed onto hydrophobized silica. Both peptide layers were found to be weakly adsorbed onto the hydrophilic silica surface as they were totally removed by buffer dilution.

The salivary mucin MUC5B and lactoperoxidase can be used for layer-by-layer film formation.

In situ ellipsometry was used to study layer-by-layer film formation on hydrophilic and hydrophobized silica surfaces by alternating sequential adsorption of human mucin MUC5B and cationic proteins lysozyme, lactoferrin, lactoperoxidase or histatin 5, respectively. The stability of the multilayers was investigated by addition of sodium dodecyl sulfate solution (SDS). Atomic force microscopy was employed to investigate morphological structures on the surfaces during the layer-by-layer film build-up. It was clearly shown that, on both hydrophilic and hydrophobized silica, only MUC5B and lactoperoxidase showed the ability for multilayer formation, resulting in an approximately linear increase in adsorbed amount and film thickness with each deposition cycle. The net increase in amounts per cycle was larger on the hydrophilic silica. Further, MUC5B needs to be adsorbed first on the hydrophilic substrates to obtain this fast build-up behavior. Generally, addition of SDS solution showed that a large fraction of the adsorbed film could be desorbed. However, films on the hydrophobized silica were more resistant to surfactant elution. In conclusion, MUC5B-cationic protein multilayers can be formed on hydrophilic and hydrophobized silica, depending on the choice of the cationic protein as well as in which order the build-up is started on hydrophilic silica. Additionally, SDS disrupts the layer-by-layer film formed by MUC5B and lactoperoxidase.

Adsorption behaviour and surfactant elution of cationic salivary proteins at solid/liquid interfaces, studied by in situ ellipsometry.

Adsorption of the cationic salivary proteins lactoferrin, lactoperoxidase, lysozyme and histatin 5 to pure (hydrophilic) and methylated (hydrophobized) silica surfaces was investigated by in situ ellipsometry. Effects of concentration (/=lactoperoxidase>lysozyme>/=histatin 5. On hydrophilic silica, the adsorption was most likely driven by electrostatic interactions, which resulted in adsorbed amounts of lactoferrin that indicated the formation of a monolayer with both side-on and end-on adsorbed molecules. For lactoperoxidase the adsorbed amounts were somewhat higher than an end-on monolayer, lysozyme adsorption showed amounts corresponding to a side-on monolayer, and histatin 5 displayed adsorbed amounts in the range of a side-on monolayer. On hydrophobized substrata, the adsorption was also mediated by hydrophobic interactions, which resulted in lower adsorbed amounts of lactoferrin and lactoperoxidase; closer to side-on monolayer coverage. For both lysozyme and histatin 5 the adsorbed amounts were the same as on the hydrophilic silica. The investigated proteins exhibited fast adsorption kinetics, and the initial kinetics indicated mass transport controlled behaviour at low concentrations on both types of substrates. Buffer rinsing and SDS elution indicated that the proteins in general were more tightly bound to the hydrophobized surface compared to hydrophilic silica. Overall, the surface activity of the investigated proteins implicates their importance in the salivary film formation.

Layer-by-layer assembly of mucin and chitosan--Influence of surface properties, concentration and type of mucin.

Bovine submaxillary mucin (BSM) and chitosan were used to build layer-by-layer structures on solid substrates. The build-up was monitored using in situ ellipsometry to obtain time resolved values of the thickness and adsorbed amount. Additionally surface morphology during build-up was studied by atomic force microscopy (AFM). It was found that the adsorbed amount of the film increases approximately linearly with each deposition cycle on hydrophobized silica whereas construction on silica was found not to be possible at the experimental conditions used. We conclude that sufficient amount of the first mucin layer is crucial for the subsequent multilayer formation. The complex build-up kinetics on hydrophobized silica is characterized by adsorption and redissolution processes and the overall growth is the sum of both processes. AFM imaging on hydrophobized silica also confirmed the presence of redissolution processes and chitosan addition led to a reduction both in the number of surface aggregates and in the roughness of the surface. The present work also shows that by adjusting the relative concentrations of the polyelectrolytes it is possible to change the growth rate considerably. The final structures after deposition of 8 bilayers were found to have a high content of water and film stability test revealed that a substantial amount dissolves when increasing electrolyte concentration or pH of the ambient solution. Human mucin from saliva (MUC5B) was also used to create multilayers with chitosan on hydrophobized silica and it was revealed that no redissolution appears to be present in this system.

Interaction of Polyelectrolytes with Salivary Pellicles on Hydroxyapatite Surfaces under Erosive Acidic Conditions.

The modification of acidic beverage formulations with food-approved, nonhazardous substances with antierosive properties has been identified as a key strategy for counteracting the prevalence of dental erosion, i.e., the acid-induced dissolution of hydroxyapatite (HA, the main mineral component of tooth surfaces). While many of such substances have been reported, very little is known on how they interact with teeth and inhibit their acid-induced dissolution. With the aim of filling this gap in knowledge, we have studied under acidic conditions the interaction between two polyelectrolytes of differing ionic character, carboxymethyl cellulose (CMC) and chitosan, and saliva-coated hydroxyapatite, i.e., a model for the outer surface of teeth. These studies were performed by means of ellipsometry, quartz crystal microbalance with dissipation monitoring, and atomic force microscopy. We also studied, by means of pH variations, how dissolution of saliva-coated HA is affected by including these polyelectrolytes in the erosive solutions. Our results confirm that salivary films protect HA from acid-induced dissolution, but only for a limited time. If the acid is modified with CMC, this polyelectrolyte incorporates into the salivary films prolonging in time their protective function. Eventually, the CMC-modified salivary films are removed from the HA surfaces. From this moment, HA is continuously coated with CMC, but this offers only a weak protection against erosion. When the acid is modified with the cationic chitosan, the polyelectrolyte adsorbs on top of the salivary films. Chitosan-modified salivary films are also eventually replaced by bare chitosan films. In this case both coatings offer a similar protection against HA dissolution, which is nevertheless notably higher than that offered by CMC.

On the adsorption behaviour of saliva and purified salivary proteins at solid/liquid interfaces.

Salivary proteinaceous substances are known to play important roles in the formation of the salivary pellicle. The aim of this study was to investigate some aspects of the interfacial behaviour of selected purified salivary proteins, as well as human saliva secretions, using time-resolved in situ ellipsometry. Hydrophobic methylated silica and hydrophilic pure silica were used as test substrates. Experiments were performed in vitro, preferentially in the low concentration range, with samples of fresh human whole resting saliva, parotid resting saliva and submandibular/sublingual resting saliva. The protein fractions investigated were human MUC5B, PRP-1, PRP-3 and statherin, as well as bovine submaxillary mucin (BSM). The results indicate that the amount of material adsorbed was strongly related to the protein concentration in the range investigated for both pure proteins and secretions. Generally, a larger amount of material was adsorbed onto hydrophobic surfaces than onto hydrophilic ones. However, pure PRP-1 adsorbed in similar amounts to both hydrophilic and hydrophobic surfaces in the concentration range investigated and BSM adsorbed in larger amounts at high concentrations on hydrophilic surfaces. Comparison of the observed adsorption rates for salivary secretions and calculated diffusion rates for individual proteins suggested initial adsorption of low-molecular-weight proteins/peptides. On hydrophilic surfaces the data indicate adsorption of proteins with diffusion rates corresponding to those of statherin, PRP-3 and PRP-1. MUC5B adsorbs at a later stage from both HWS and the individual secretions, which can be explained by a "Vroman effect"-like phenomenon. On hydrophobic surfaces, adsorption rates were found to be faster than those calculated for any of the proteins, and thus smaller proteins/peptides appear to be involved. The similar adsorption behaviour of PRP-1 and parotid saliva (HPS) on hydrophilic surfaces may suggest that long aPRPs account for a substantial portion of the film-forming capacity of HPS. Effects of added electrolyte could be explained by general screening effects and specific Ca2+ binding to serine phosphates in aqueous solutions, but were complex in phosphate buffer. Inter-individual differences in amounts adsorbed from HWS, HPS and HSMSLS, respectively, were not found to be statistically significant.

Salivary pellicles.

The salivary pellicle is a thin acellular organic film that forms on any type of surface upon exposure to saliva. The role of the pellicle is manifold, and it plays an important role in the maintenance of oral health. Its functions include not only substratum protection and lubrication, but also remineralization and hydration. It also functions as a diffusion barrier and possesses buffering ability. Not only the function, but also the formation, composition and stability of the pellicle are known to be highly influenced by the physicochemical properties of both substrata and ambient media. In this chapter, we discuss these aspects of salivary pellicles, an area where research has boomed in the past years partly because of the application of experimental techniques often reserved for more traditional surface science studies.