Pellicle-coated Tooth Surfaces Research Articles

Design and Characterization of a Novel p1025 Peptide-Loaded Liquid Crystalline System for the Treatment of Dental Caries.

Dental caries, mainly caused by the adhesion of Streptococcus mutans to pellicle-coated tooth surfaces, is an important public health problem worldwide. A synthetic peptide (p1025) corresponding to residues 1025–1044 of the adhesin can inhibit this binding. Peptides are particularly susceptible to the biological environment; therefore, a p1025 peptide-loaded liquid crystalline system (LCS) consisting of tea tree oil as the oil phase, polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol as the surfactant, and water or 0.5% polycarbophil polymer dispersions as the aqueous phase was employed as a drug delivery platform. This system exhibited anticaries and bioadhesive properties and provided a protective environment to p1025 at the site of action, thereby modulating its action, prolonging its contact with the teeth, and decreasing the frequency of administration. LCSs were characterized by polarized light microscopy (PLM), small-angle X-ray scattering (SAXS), and rheological, texture, and bioadhesive tests. PLM and SAXS revealed the presence of hexagonal liquid crystalline phases and microemulsions. Rheological analyses demonstrated that the addition of polymer dispersions favored characteristics such as shear thinning and thixotropy, hence improving buccal application. Bioadhesion tests showed that polymer dispersions contributed to the adhesion onto the teeth. Taken together, LCS could provide a novel pharmaceutical nanotechnology platform for dental caries treatment.

Dynamic Changes in the Initial Colonization of Actinomyces naeslundii and Streptococcus gordonii Using a New Animal Model

Actinomyces naeslundii and Streptococcus gordonii are the predominant bacteria and initial colonizers of oral microflora. The binding of A. naeslundii and S. gordonii and the interaction between them on the salivary pellicle-coated tooth surface play an important role in the biofilm development. Recently, we reported that NOD/SCID.e2f1(-) mice are a useful model for studying oral biofilm formation by Streptococcus mutans on the tooth surface. In this study, we aimed to determine whether NOD/SCID.e2f1(-) mice can be used for studying oral colonization of A. naeslundii and S. gordonii. Colonization of A. naeslundii in mice fed with 1% sucrose water for 24 h before inoculation was higher than that among mice fed with sucrose water for 1 h. A. naeslundii colonization using mixed species-inoculation was lower than that using single-species inoculation 30-90 min after inoculation; however, the colonization was higher 120-180 min after inoculation. The mixed inoculation induced better colonization of S. gordonii than single-species inoculation 60-180 min after inoculation. Polyclonal and fluorescein isothiocyanate-labeled antibody stained bacteria showed better colonization of S. gordonii when a mixed culture is used in vivo. NOD/SCID.e2f1(-) mice were useful for studying the initial colonization of A. naeslundii and S. gordonii. Long-term supply of sucrose water creates a favorable environment for the initial colonization of A. naeslundii that, in turn, supports the colonization of S. gordonii.

The oral disposition of zinc following the use of an anticalculus toothpaste containing 0.5% zinc citrate.

Zinc is retained in the mouth after use of a toothpaste containing 0.5% zinc citrate. More than one third of the dose was found to be retained after normal brushing. Elevated zinc levels were also found in plaque. Saliva zinc levels were significantly above background for at least 2 h after brushing. In-vitro experiments demonstrated that zinc can bind to the pellicle-coated tooth surface and can subsequently adsorb into saliva. Plaque can calcify to form calculus containing appreciable levels of hydroxyapatite. Zinc adsorbs to hydroxyapatite inhibiting crystal growth. Levels of zinc in plaque were found to be considerably higher than those taken up by hydroxyapatite in an in-vitro test of crystal growth inhibition indicating the potential of zinc to inhibit calculus formation.

In vitro anti‐biofilm activity of macelignan isolated from Myristica fragrans Houtt. against oral primary colonizer bacteria

In early dental plaque formation, oral primary colonizers such as Streptococcus mutans, Streptococcus sanguis and Actinomyces viscosus are initially attached to the pellicle-coated tooth surface to form a biofilm. The study aimed to determine the efficacy of macelignan, isolated from nutmeg (Myristica fragrans Houtt.), in removing each single oral primary biofilm in vitro on a polystyrene 96-well microtiter plate. Four biofilm growth phases (4, 12, 20 and 24 h) were evaluated in this study after treatment with macelignan at various concentrations (0.2, 2 and 10 microg/mL) and exposure times (5, 10 and 30 min). Anti-biofilm activity of macelignan was measured as the percentage of the remaining biofilm absorbance after macelignan treatment in comparison with the untreated control. At 24 h of biofilm growth, S. mutans, A. viscosus and S. sanguis biofilms were reduced by up to 30%, 30% and 38%, respectively, after treatment with 10 microg/mL macelignan for 5 min. Increasing the treatment time to 30 min resulted in a reduction of more than 50% of each of the single primary biofilms. The results indicate that macelignan is a potent natural anti-biofilm agent against oral primary colonizers.

Effects of Human Milk on Adhesion of Streptococcus mutans to Saliva-Coated Hydroxyapatite in vitro

Adhesion of bacteria to pellicle-coated tooth surfaces is the first step in biofilm formation on teeth. The aim of this study was to explore whether human milk prevents or promotes adhesion of cariogenic Streptococcus mutans to saliva-coated hydroxyapatite (HA) using an in vitromodel system. S. mutans binding to HA coated with human parotid saliva (s-HA) or human milk was studied, in addition to binding inhibition to s-HA by human milk. S. mutans did not bind to HA coated with milk. S. mutans binding to s-HA was inhibited by milk from 15 (71 %) of 21 women, whereas milk from the remaining 6 mothers enhanced binding of S. mutans to s-HA. Inhibition of S. mutans binding correlated with bacterial binding to s-HA (r = 0.76). Human milk does not mediate adhesion of S. mutans to HA in vitro, but affects adhesion in an individually varying fashion. Phenotypic variations in milk and saliva glycosylation may explain the inhibitory capacity and possibly affect susceptibility to colonization by S. mutans in childhood.

Supragingival calculus: formation and control.

Dental calculus is composed of inorganic components and organic matrix. Brushite, dicalcium phosphate dihydrate, octacalcium phosphate, hydroxyapatite, and whitlockite form the mineral part of dental calculus. Salivary proteins selectively adsorb on the tooth surface to form an acquired pellicle. It is followed by the adherence of various oral micro-organisms. Fimbriae, flagella, and some other surface proteins are essential for microbial adherence. Microbial co-aggregation and co-adhesion enable some micro-organisms, which are incapable of adhering, to adhere to the pellicle-coated tooth surface. Once organisms attach to the tooth surface, new genes could be expressed so that mature dental plaque can form and biofilm bacteria assume increased resistance to antimicrobial agents. Supersaturation of saliva and plaque fluid with respect to calcium phosphates is the driving force for plaque mineralization. Both salivary flow rate and plaque pH appear to influence the saturation degree of calcium phosphates. Acidic phospholipids and specific proteolipids present in cell membranes play a key role in microbial mineralization. The roles of crystal growth inhibitors, promoters, and organic acids in calculus formation are discussed. Application of biofilm culture systems in plaque mineralization is concisely reviewed. Anti-calculus agents used--centering on triclosan plus polyvinyl methyl ether/maleic acid copolymer, pyrophosphate plus polyvinyl methyl ether/maleic acid copolymer, and zinc ion-in commercial dentifrices are also discussed in this paper.

Affinity and specificity of the interactions between Streptococcus mutans antigen I/II and salivary components.

Adherence to salivary pellicle-coated tooth surfaces and aggregation by salivary components of Streptococcus mutans involves a major cell surface protein termed antigen (Ag) I/II. The objectives of this study were to evaluate the affinity and specificity of the interactions between AgI/II and human saliva in assays of 125I-AgI/II binding to saliva-coated hydroxyapatite (SHA) and of S. mutans aggregation by salivary agglutinin (SAG), monitored turbidimetrically. 125I-AgI/II binding to SHA followed saturation kinetics, and Scatchard plot analysis indicated two binding sites with dissociation constants of the order of 10(-10) mol/L and 10(-9) mol/L. The binding to SHA of the C-terminal one-third of AgI/II which corresponds to AgII was less than one-fifth that of the whole molecule and did not show evidence of saturation. The binding of 125I-AgI/II was inhibited by native or recombinant fragments that mapped in the N-terminal part of the molecule and that contained the alanine-rich repeat region, whereas fragments mapping at the central or C-terminal one-third had no effect. As with binding to SHA, the regions of AgI/II which inhibited aggregation mapped at the N-terminal part of the molecule, but, in addition, a recombinant segment mapping at the central part and containing the proline-rich repeat region was also inhibitory. The S. mutans-aggregating activity of SAG or whole saliva was inhibited by amino compounds, and most strongly by L-lysine and analogues possessing omega-primary amine groups. These data support the role of AgI/II as an adhesin with high-affinity binding for SHA receptors, mediated by the N-terminal part of the molecule. This region is also involved in SAG-induced S. mutans aggregation, which is sensitive to amino compounds.

Inhibition of Streptococcus mutans adherence to saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II: reversal by IgA1 protease cleavage.

The effect of human secretory immunoglobulin A (S-IgA) and serum antibodies to surface protein antigen (Ag) I/II on the adherence of Ag I/II-bearing Streptococcus mutans and of free Ag I/II to saliva-coated hydroxyapatite (SHA) was investigated. The inhibition by S-IgA of binding of both S. mutans and free Ag I/II to SHA was dependent on antibody to Ag I/II. Essentially no difference was found between S-IgA1 and S-IgA2 with respect to antibody-dependent inhibition of Ag I/II binding to SHA, but S-IgA1 inhibited S. mutans adherence more effectively than did either serum immunoglobulin A1 (IgA1) or IgG antibodies. The antiadherence effect of S-IgA was abrogated after cleavage by IgA1 protease. Purified Fab alpha fragments containing Ag I/II-binding activity enhanced the binding of free Ag I/II to SHA and showed greater binding to SHA than did intact S-IgA1. Despite its relative inability to interact with precoated SHA, S-IgA1 containing antibody to Ag I/II was readily incorporated into the salivary pellicle during coating, but this did not promote Ag I/II binding. These data suggest that S-IgA antibodies can inhibit the initial adherence of S. mutans to salivary pellicle-coated tooth surfaces in an adhesin-specific fashion, but the presence in the oral cavity of bacterial IgA1 proteases would potentially interfere with this antiadherence mechanism.

Delivery of Antiplaque Agents from Dentifrices, Gels, and Mouthwashes

Antiplaque agents delivered from toothpastes, gels, or mouthrinses can augment mechanical oral hygiene procedures to control the formation of supragingival plaque and the development of early periodontal disease. Clinically effective antiplaque agents are characterized by a combination of intrinsic antibacterial activity and good oral retention properties. The overall oral retention of an antiplaque agent is determined by the strength and rate of association of the agent with its receptor sites and the accessibility of these sites. The substantivity of an antiplaque agent and its clearance from the oral cavity are determined by the rate of dissociation of the agent from the receptor sites and the salivary composition and flow rate. Positively charged and non-charged organic molecules, metal ions, enzymes, and surface-active agents have all been considered as antiplaque agents. To exert clinical antiplaque activity, an antimicrobial agent must be formulated in a chemically compatible delivery vehicle to give optimal release and uptake to the sites of action in a biologically active form during its time of application. In principle, antiplaque activity may be enhanced by combining antimicrobial agents with broadly similar, but complementary, modes of action. Alternatively, the activity of a single agent may be increased by use of a retention aid to enhance oral substantivity. Substantial evidence exists to demonstrate the validity of the first approach. However, there are few data, as yet, to support the effectiveness of the second. The oral mucosa is the bulk retention site for all clinically proven antiplaque agents. Plaque, the pellicle-coated tooth surface, and saliva are probably all sites of biological action. A detailed understanding of the interactions between agents and the various receptor sites, and of the importance of these receptor sites to biological activity, is generally lacking.

Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis.

Fimbriae are important in the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas (Bacteroides) gingivalis fimbriae appear to mediate adherence to oral epithelial cells and the pellicle-coated tooth surface. The role and contribution of fimbriae in the binding of P. gingivalis to hydroxyapatite (HAP) coated with saliva as a model for the pellicle-coated tooth surface were investigated. 3H-labeled P. gingivalis or the radioiodinated purified fimbriae were incubated with 2 mg of HAP beads coated with whole human saliva (sHAP) and layered on 100% Percoll to separate unbound from sHAP-bound components. The radioactivity of the washed beads was a measure of the bound components. The binding of P. gingivalis 2561 (381) cells and that of purified fimbriae were concentration dependent and saturable at approximately 10(8) cells and 40 micrograms of fimbriae added, respectively. The addition of fimbriae inhibited binding of P. gingivalis to sHAP beads by 65%, while the 75-kDa protein, which is another major surface component of P. gingivalis 2561, did not show significant inhibition, suggesting that the fimbriae are important in adherence. Encapsulated and sparsely fimbriated P. gingivalis W50 did not bind to sHAP beads. On the basis of the predicted sequence of the fimbrillin, a structural subunit of fimbriae, a series of peptides were synthesized and used to localize the active fimbrillin domains involved in P. gingivalis adherence to sHAP beads. Peptides from the carboxyl-terminal one-third of the fimbrillin strongly inhibited P. gingivalis binding to sHAP beads. Active residues within the sequence of inhibitory peptide 226-245 (peptide containing residues 226 to 245) and peptide 293-306 were identified by using smaller fragments prepared either by trypsin cleavage of the peptide 226-245 or by synthesis of smaller segments of peptide 293-306. Hemagglutinin activity, lectinlike binding, and ionic interaction did not seem to be involved in this binding since lysine, arginine, carbohydrates, and calcium ions failed to affect the binding of P. gingivalis. The observation that poly-L-lysine, bovine serum albumin, and defatted bovine serum albumin, even at high concentrations, only partially blocked the binding of P. gingivalis indicates that hydrophobic interactions are not the major forces involved in P. gingivalis binding to sHAP beads. Protease inhibitors such as EDTA, leupeptin, pepstatin, 1,10-phenanthroline, and phenylmethylsulfonyl fluoride did not interfere with the binding of P. gingivalis. However, the binding of P. gingivalis to trypsin- or chymotrypsin-pretreated sHAP beads was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic control of serum antibody responses of inbred mice to type 1 and type 2 fimbriae from Actinomyces viscosus T14V

Antibodies reactive with type 1 and type 2 fimbriae from Actinomyces viscosus T14V specifically inhibit the adherence of A. viscosus T14V to salivary pellicle-coated tooth surfaces and other bacteria, and these antibodies are thought to modulate colonization by this microorganism. These studies were done to determine whether previously noted differences in the antibody responses of inbred mice to type 1 and type 2 fimbriae might be under genetic control. The serum immunoglobulin G (IgG) and IgM antibody responses of inbred, F1 hybrid, and H-2 congenic mice, immunized with A. viscosus T14V cells, were analyzed by enzyme-linked immunosorbent assays for antibodies reactive with A. viscosus T14V whole-cell type 1 and type 2 fimbriae. The results confirmed earlier findings and indicated striking variations in the amounts of IgG anti-type 1 (23-fold) and anti-type 2 (48-fold) fimbria antibodies elicited. The responses of the 17 inbred strains tested showed a relatively continuous distribution from high to low, as well as marked differences in the responses of H-2 and Igh-C identical strain pairs. An analysis of the responses of F1 hybrid and H-2 congenic mice indicated dominance of the low-responder gene(s) and control by H-2-linked genes. Antisera from two high-responder strains inhibited in vitro bacterial adherence to a much greater degree than antisera from a low-responding strain. These data suggest polygenic control of the magnitude of the IgG anti-type 1 and anti-type 2 fimbria antibody responses by H-2-linked genes as well as background genes not associated with H-2 or Igh-C loci.

Interaction between surface protein antigens of Streptococcus mutans and human salivary components.

The potential involvement of surface antigens (Ags) I/II and III of Streptococcus mutans in its adherence to salivary pellicle-coated tooth surfaces was investigated. The binding of radiolabelled Ag I/II to hydroxyapatite was increased by pretreating the mineral with human parotid saliva, and binding was maintained in the continuous presence of saliva. Binding of Ag III to hydroxyapatite was inhibited by pretreatment with, or in the presence of, saliva. Various aminohexoses, and also tris, inhibited the binding of Ag I/II. When Ags I/II and III were tested for their ability to bind to salivary components separated by SDS gel electrophoresis, several proteins capable of binding Ag I/II were identified, notably 2 proteins of apparent relative molecular mass 28,000 and 38,000. Analysis of these proteins, isolated by micro-preparative electrophoresis, indicated high proportions of proline, glycine, and glutamic acid, and overall compositions similar to basic proline-rich salivary proteins.

Virulence factors of Streptococcus mutans and dental caries prevention.

Streptococcus mutans possesses the abilities to adhere to pellicle-coated tooth surfaces and to form acids - two characteristics associated with the cariogenicity of this micro-organism. De novo synthesis of insoluble glucan by S. mutans glucosyltransferase from sucrose is essential in the adherence process. Therefore, agents which interfere with the adherence ability of S. mutans would be useful for controlling dental caries. In the present report, we have summarized our recent findings concerning virulence factors of S. mutans and means for prevention of S. mutans-induced dental caries.