Michele E. Barbour

Interaction of Candida albicans Cell Wall Als3 Protein with Streptococcus gordonii SspB Adhesin Promotes Development of Mixed-Species Communities

ABSTRACT Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3− mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.

Inhibition of hydroxyapatite dissolution by whole casein: the effects of pH, protein concentration, calcium, and ionic strength

Formulating drinks with reduced erosive potential is one approach for reducing dental erosion. In this study, whole casein was added to citric acid solutions representative of soft drinks, and the hydroxyapatite dissolution rate was assessed. Adding 0.02% (w/v) casein to acid solutions significantly reduced the hydroxyapatite dissolution rate by 51 +/- 4% at pH values of 2.80, 3.00, 3.20, 3.40, and 3.60, although the baseline dissolution rates of course varied as a function of pH. The protein concentration [0.002, 0.02, and 0.2% (w/v) casein] had no significant effect on dissolution inhibition. Adding both casein and calcium to citric acid resulted in a further reduction in the dissolution rate at low and intermediate calcium concentrations (5 and 10 mM) but not at higher calcium concentrations (20 and 50 mM). Ionic strength had no significant impact on the efficacy of casein. Casein also significantly reduced the hydroxyapatite dissolution rate when the hydroxyapatite was coated with a salivary pellicle. The reduction in dissolution rate is ascribed to firmly adsorbed casein on the hydroxyapatite surface, which stabilizes the crystal surface and inhibits ion detachment.

Enamel dissolution as a function of solution degree of saturation with respect to hydroxyapatite: a nanoindentation study

The objective of this study was to investigate human enamel dissolution as a function of degree of saturation (DS) of the surrounding solution with respect to hydroxyapatite. Nanoindentation was used to compare changes in enamel nanomechanical properties due to dissolution by two solutions. Citric acid solution (DS=0.000, pH 3.30) and citric acid solution containing calcium (299 mg/l) and phosphate (54.0 mg/l) (DS=0.032, pH 3.30) were compared with a control mineral water (DS=0.673, pH 7.48). Exposure times were 0, 120, 300, 600, 900, and 1200 s. Compared to untreated enamel, there was a statistically significant change in enamel hardness after 120 s exposure to both citric acid solutions, and in elastic modulus after 300 s exposure. The rate of change of both variables decreased with exposure time. This suggests that dissolution rate is diffusion-limited under these conditions, in agreement with previous studies. There was no statistically significant difference between the hardness or elastic modulus of enamel exposed to the two citric acid solutions at any time. This may be due to a change in solution composition during contact with the enamel.

Screening and prediction of erosive potential

The literature on the erosive potential of drinks and other products is summarised, and aspects of the conduct of screening tests as well as possible correlations of the erosive potential with various solution parameters are discussed. The solution parameters that have been suggested as important include pH, acid concentration (with respect to buffer capacity and concentration of undissociated acid), degree of saturation, calcium and phosphate concentrations, and inhibitors of erosion. Based on the available data, it is concluded that the dominant factor in erosion is pH. The effect of buffer capacity seems to be pH dependent. The degree of saturation probably has a non-linear relationship with erosion. While calcium at elevated concentrations is known to reduce erosion effectively, it is not known whether it is important at naturally occurring concentrations. Fluoride at naturally occurring concentrations is inversely correlated with erosive potential, but phosphate is probably not. Natural plant gums, notably pectin, do not inhibit erosion, so they are unlikely to interfere with the prediction of erosive potential. The non-linearity of some solution factors and interactions with pH need to be taken into account when developing multivariate models for predicting the erosive potential of different solutions. Finally, the erosive potential of solutions towards enamel and dentine might differ.

Effects of pH and acid concentration on erosive dissolution of enamel, dentine, and compressed hydroxyapatite

The aims of this study were to determine the effects of pH and acid concentration on the dissolution of enamel, dentine, and compressed hydroxyapatite (HA) in citric acid solutions (15.6 and 52.1 mmol l(-1) ; pH 2.45, 3.2, and 3.9), using a pH-stat system. After an initial adjustment period, the dissolution rates of enamel and HA were constant, while that of dentine decreased with time. The dissolution rate increased as the pH decreased, and this was most marked for enamel. To compare substrates, the rate of mineral dissolution was normalized to the area occupied by mineral at the specimen surface. For a given acid concentration, the normalized dissolution rate of HA was always less than that for either dentine or enamel. The dissolution rate for dentine mineral was similar to that for enamel at pH 2.45 and greater at pH 3.2 and pH 3.9. The concentration of acid significantly affected the enamel dissolution rate at pH 2.45 and pH 3.2, but not at pH 3.9, and did not significantly affect the dissolution rates of dentine or HA at any pH. The variation in response of the dissolution rate to acid concentration/buffer capacity with respect to pH and tissue type might complicate attempts to predict erosive potential from solution composition.

Inhibition of hydroxyapatite dissolution by ovalbumin as a function of pH, calcium concentration, protein concentration and acid type.

Hydroxyapatite is the main constituent of the dental hard tissues, and in vivo its dissolution in acids leads to the pathological condition of dental erosion. Food proteins which inhibit hydroxyapatite dissolution may find application as erosion-reducing agents in food and drink products. The aim of this study was to investigate the egg protein ovalbumin as a potential inhibitor of hydroxyapatite dissolution in acidic solutions, with conditions representative of dental erosion. The dissolution rate of hydroxyapatite discs was measured in an acidic solution as a function of pH, calcium concentration, ovalbumin concentration and acid type. All experiments were performed in triplicate. 0.2% w/v ovalbumin significantly reduced the dissolution rate in citric acid by 50–75% over the pH range 2.80–4.00, and by 45–60% in solutions with calcium concentrations of up to 20 mM (p < 0.05). The effect was persistent for several rinses after the initial exposure to the protein. 0.02% w/v ovalbumin significantly reduced the dissolution of hydroxyapatite in citric acid by 30–55%. Ovalbumin did not, however, statistically significantly reduce the hydroxyapatite dissolution rate in malic or lactic acids. The effect is ascribed to adsorption and partial, reversible denaturation of ovalbumin on the hydroxyapatite surface. There may be some interaction between ovalbumin and the citrate ion which promotes the adsorption of protein in the presence of citric acid. Ovalbumin shows promise as a potential erosion-reducing additive to citrus-based drinks.

Enamel erosion in dietary acids: inhibition by food proteins in vitro.

The aim of this study was to investigate the effects of two common food proteins on human enamel erosion in vitro. Erosion was measured by non-contact profilometry in citric, malic and lactic acids at pH 2.8, 3.2 and 3.8 and five commercially available soft drinks, in the presence of a salivary pellicle. Whole milk casein or hen egg ovalbumin was added to the acid solutions and drinks at 0.2% w/v, and the effect on erosion was determined by comparison with the corresponding solution without protein. Casein significantly reduced erosion in all but two solutions. The effects of the individual subfractions of casein in citric acid at pH 3.2 were similar to that of whole casein. Ovalbumin reduced erosion in some solutions, but the magnitude of the reduction was less than that with casein. A greater proportional reduction in erosion was seen in citric acid than in malic or lactic acids. We postulate that the mechanism involves adsorption of proteins to the pellicle or the enamel surface, forming a protein film with enhanced erosion-inhibiting properties. The citrate ion may play an active stabilising role, since erosion reduction was less in the other acids. In conclusion, casein and, to a lesser extent, ovalbumin show promise as potential anti-erosive additives to drinks.

Inhibition of dental erosion by casein and casein-derived proteins.

The application of milk-derived proteins such as casein as anti-erosion agents in oral healthcare products is of current interest. The aim of this study was to investigate the potential of 3 commercially available, milk-derived proteins as agents to inhibit enamel erosion. Aqueous solutions of 0.5% w/v casein, casein phosphopeptide (CPP) or glycomacropeptide (GMP) with and without 300 ppm fluoride (F, as NaF) were investigated with regard to enamel softening and tissue loss, in comparison with a deionised water (DIW) negative control and 300 ppm F positive control. Casein and F reduced enamel surface softening compared to DIW, but CPP and GMP did not (DIW: 58.2% reduction in hardness; F: 13.3%; casein: 21.8%; CPP: 50.8%; GMP: 62.4%). Similar results were obtained with solutions containing protein and F, and the effects were statistically indistinguishable from protein alone (casein + F: 19.1%; CPP + F: 48.2%; GMP + F: 66.1%). By contrast, all protein solutions and F significantly reduced tissue loss (p < 0.050; DIW: 25.8 µm tissue loss; F: 21.6 µm; casein: 20.3 µm; CPP: 20.5 µm; GMP: 20.0 µm). Solutions containing protein and F reduced erosion more than protein alone, but this difference was only significant from protein alone for casein (casein + F: 12.2 µm; CPP + F: 17.3 µm; GMP + F: 18.2 µm). Casein and casein-derived proteins may therefore have the potential to act as agents to reduce or prevent enamel erosion. Furthermore, the erosion-reducing efficacy is not reduced by F, and is in some cases enhanced.

Effects of buffering properties and undissociated acid concentration on dissolution of dental enamel, in relation to pH and acid type

To quantify the relationships between buffering properties and acid erosion and hence improve models of erosive potential of acidic drinks, a pH-stat was used to measure the rate of enamel dissolution in solutions of citric, malic and lactic acids, with pH 2.4-3.6 and with acid concentrations adjusted to give buffer capacities (β) of 2-40 (mmol·l-1)·pH-1 for each pH. The corresponding undissociated acid concentrations, [HA], and titratable acidity to pH 5.5 (TA5.5) were calculated. In relation to β, the dissolution rate and the strength of response to β varied with acid type (lactic > malic ≥ citric) and decreased as pH increased. The patterns of variation of the dissolution rate with TA5.5 were qualitatively similar to those for β, except that increasing pH above 2.8 had less effect on dissolution in citric and malic acids and none on dissolution in lactic acid. Variations of the dissolution rate with [HA] showed no systematic dependence on acid type but some dependence on pH. The results suggest that [HA], rather than buffering per se, is a major rate-controlling factor, probably owing to the importance of undissociated acid as a readily diffusible source of H+ ions in maintaining near-surface dissolution within the softened layer of enamel. TA5.5 was more closely correlated with [HA] than was β, and seems to be the preferred practical measure of buffering. The relationship between [HA] and TA5.5 differs between mono- and polybasic acids, so a separate analysis of products according to predominant acid type could improve multivariate models of erosive potential.

Development of a novel antimicrobial-releasing glass ionomer cement functionalized with chlorhexidine hexametaphosphate nanoparticles.

BackgroundGlass ionomer cements (GICs) are a class of dental biomaterials. They have a wide range of uses including permanent restorations (fillings), cavity linings, fissure sealants and adhesives. One of the most common reasons for replacing a dental restoration is recurrent bacterial tooth decay around the margins of the biomaterial. Therefore, a dental biomaterial which creates a sustained antimicrobial environment around the restoration would be of considerable clinical benefit. In this manuscript, the formulation of a GIC containing novel antimicrobial nanoparticles composed of chlorhexidine hexametaphosphate at 1, 2, 5, 10 and 20% powder substitution by mass is reported. The aim is to create GICs which contain chlorhexidine-hexametaphosphate nanoparticles and characterize the nanoparticle size, morphology and charge and the release of chlorhexidine and fluoride, tensile strength and morphology of the GICs.ResultsThe GICs released chlorhexidine, which is a broad spectrum antimicrobial agent effective against a wide range of oral bacteria, over the duration of the experiment in a dose-dependent manner. This was not at the expense of other properties; fluoride release was not significantly affected by the substitution of antimicrobial nanoparticles in most formulations and internal structure appeared unaffected up to and including 10% substitution. Diametral tensile strength decreased numerically with substitutions of 10 and 20% nanoparticles but this difference was not statistically significant.ConclusionA series of GICs functionalized with chlorhexidine-hexametaphosphate nanoparticles were created for the first time. These released chlorhexidine in a dose-dependent manner. These materials may find application in the development of a new generation of antimicrobial dental nanomaterials.