Synthesis and characterization of an experimental primer containing chitosan nanoparticles - Effect on the inactivation of metalloproteinases, antimicrobial activity and adhesive strength.

OBJECTIVE: The aim of this study was to synthesize and characterize an experimental primer containing cationic lipid nanoparticles (NPL-chitosan) and to evaluate its properties. DESIGN: The NPL-chitosan were synthesized by emulsion and sonication method. The experimental primers were applied in dentin surface of fifty human molars. The experimental groups were: 1) application of commercial primer; 2) Primer containing 2% of Chlorhexidine (CHX) 3); Primer with 2% NPL-chitosan 4); Primer with 0.6 % of NPL-chitosan 5); Primer with 0.4 % of NPL-chitosan. A composite resin plateau was used for the analysis, where sections were made for making the dentin beams. The effect of experimental primer with cationic nanoparticles in the inhibition of matrix metalloproteinase (MMP) activity was carrying out by in situ zymography. For the Resin-Dentin Adhesive Strength and in situ Zymography analysis, was used the One-way analysis of variance (ANOVA) with significance level of 95 %. RESULTS: Spherical NPL-chitosan presented size below 220 nm, polydispersity index of 0.179 and zeta potential positive and was stable over 75 days. These nanoparticles showed antibacterial activity agsainst S. mutans with MIC of the 0.4 % and MBC of 0.67 %. In the Microtensile Strength, no statistical difference was observed between the experimental groups (p = 0.9054). The in situ zymography assay showed that the group with 2% of NPL-chitosan presented higher inactivation activity of MMPs compared to the other groups (p < 0.05). CONCLUSION: The experimental primer containing NPL-chitosan has antimicrobial activity, does not alter the adhesive resistance and inactivates MMPs present in dentin.

Antimicrobial effect of a local release system containing metronidazole against a Porphyromonas gingivalis biofilm.

The aim of this study was to evaluate a semi-solid system containing metronidazole (MDZ) in presence of challenging conditions for drug release, as well its antimicrobial effect against Porphyromonas gingivalis biofilm. Biofilms grown in culture medium were exposed to a formulation containing MDZ or its vehicle. After 24, 48, and 72 h, biofilm viability were analyzed while MDZ was quantified in culture medium and buffer solution (control). MDZ formulation reduced bacterial viability when compared to control groups. The vehicle formulation also affected bacterial viability in relation to control at all periods. Culture medium impaired MDZ release compared to buffer solution at 24 h. The semi-solid system reported herein is able to release MDZ and maintain its levels at concentrations that control viability of P. gingivalis in 1- to 3-day-old biofilms.

Disruption of Staphylococcus aureus biofilms using rhamnolipid biosurfactants.

Staphylococcus aureus is an important pathogen that has shown ability to establish biofilm communities that can represent a source of contamination and resistance in food processing. Rhamnolipids (RL) have attracted attention as candidates to replace synthetic surfactants, exhibiting high surface activity combined with its microbial origin, biodegradability, and low toxicity. In this work, an RL biosurfactant was evaluated regarding its ability to disrupt or remove S. aureus biofilms established on polystyrene plates using nutrient broth and skim milk as the growth media. Rhamnolipid treatment was performed at different surfactant concentrations and temperatures. Rhamnolipid removes up to 88.9% of milk-based biofilms, whereas for nutrient medium 35% removal was attained. The RL concentration affects the disruption of nutrient medium-based biofilms. High carbohydrate content of milk-based biofilms favors disruption by RL and the organization of RL molecules in solution showed a predominance of aggregates from 1 to 10 and 100 to 1,000 nm in all conditions studied. Biofilm disruption activity of RL is nutrient-specific and dependent on biofilm matrix composition. Staphylococcus aureus biofilms established in milk were significantly reduced using RL at low concentrations and temperatures. These findings suggest potential application of RL in milk (dairy) processing industries where low temperatures are applied.

Local antibiotic delivery in periodontitis: drug release and its effect on supragingival biofilms.

The effect of a drug-delivery system containing antibacterial metronidazole (MDZ) prescribed for periodontitis on supragingival biofilm was evaluated, and possible interference by this biofilm in the drug release profile was investigated. Streptococcus mutans biofilms were grown and exposed to a controlled-release formulation of MDZ or the same formulation without MDZ (vehicle control). Untreated biofilms were used as a negative control (NC). Biofilms and culture medium (containing detached cells) were collected 24, 48, 72, and 96 h after first exposure to treatments. The biomass of the MDZ group was lower than that of the NC group at all times. Although MDZ yielded low drug-release rates in the presence of the biofilm, it was sufficient for reducing viability for 24 h and affecting bacterial metabolism for 48 h. These results suggest that MDZ appears to destabilize supragingival biofilm. This biofilm may interfere with MDZ release from the formulation.

Effects of low-level laser therapy combined with toluidine blue on polysaccharides and biofilm of Streptococcus mutans.

The aim of this study was to evaluate the effect of a low-level laser therapy in combination with toluidine blue on polysaccharides and biofilm of Streptococcus mutans. S. mutans biofilms were formed on acrylic resin blocks. These biofilms were exposed eight times/day to 10 % sucrose, and two times/day, they were subjected to one of the following treatments: G1, 0.9 % NaCl as a negative control; G2, 0.12 % chlorhexidine digluconate (CHX) as a positive antibacterial control; and G3 and G4 antimicrobial photodynamic therapy (aPDT) combined with toluidine blue using dosages of 320 and 640 J/cm(2), respectively. The experiment was performed in triplicate. The biofilm formed on each block was collected for determination of the viable bacteria and concentration of insoluble extracellular polysaccharides (IEPS) and intracellular polysaccharides (IPS). CHX and aPDT treatments were able to inhibit bacterial growth in comparison with negative control (p < 0.05). The aPDT treatment reduced the number of viable bacteria formed in the S. mutans biofilm, in a dose-dependent manner (p < 0.05). The concentration of IEPS and IPS in the biofilms formed in presence of aPDT did not differ each other or in comparison to CHX (p > 0.05). The results suggest that low-level laser therapy presents effects on biofilm bacteria viability and in polysaccharides concentration.

Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans.

The aim of this study was to examine the effects of Dorstenia asaroides extracts on cariogenic properties of the most cariogenic bacteria, Streptococcus mutans. Hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr) extracts obtained from D. asaroides rhizomes were submitted to chemical analyses, Minimal Inhibitory Concentrations (MIC), glycolysis assay and S. mutans 12-h-old initial biofilms. Chemical characterization showed that all the extracts present furanocoumarins. The MIC values were 80 (HFr and CFr) and 50 µg/mL (EFr). Acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group in order to 20, 30 and 25% respectively (p < 0.01). The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans.

A procedure for characterizing glucans synthesized by purified enzymes of cariogenic Streptococcus mutans.

Extracellular polysaccharide is a virulence factor of the most cariogenic bacteria, Streptococcus mutans. We describe a procedure for chemical analysis of this polysaccharide of S. mutans, using a well-known glucan synthesized by purified enzymes of cariogenic streptococci. Soluble and insoluble glucan from glucosyltransferase D and B respectively, were prepared and analyzed by nuclear magnetic resonance spectroscopy and methylation. Soluble alpha-glucan contained 60% of (1-->6)-linkages and 17% of branching while insoluble alpha-glucan was composed of 57% of (1-->3)- and 28% of (1-->6)-linkages with 8% branching. The described procedure could be important focusing future studies on in vivo biofilm.

Effect of starch and sucrose on dental biofilm formation and on root dentine demineralization.

The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch --> sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtfB and gtfC mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.

Influences of starch and sucrose on Streptococcus mutans biofilms.

INTRODUCTION: The combination of starch and sucrose has been shown to be potentially more cariogenic than either alone. The aim of this study was to examine the influence of starch and sucrose, alone or in combinations, on formation, polysaccharide composition, gene expression, and acidogenicity of Streptococcus mutans biofilms. METHODS: S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs in batch culture for 5 days in the presence of 1% (weight/volume) starch, 1% sucrose, 1% starch plus 1% sucrose, 1% starch plus 0.5% fructose plus 0.5% glucose, or 1% sucrose plus 1% glucose. RESULTS: Amylase activity from sHA disks was detected up to 48 h, thereby increasing the availability of reducing sugars and acidogenicity in the early stages of biofilm development. S. mutans grown in the presence of sucrose alone or in combinations formed well-defined and tightly adherent biofilms comprised of mostly water-insoluble polysaccharides (INS); in contrast, the presence of starch or starch + glucose + fructose resulted in little biofilm formation with minimal amounts of INS. However, the combination of starch + sucrose produced biofilms with more biomass and acidogenicity, and a higher content of INS than those grown in sucrose or sucrose + glucose (P < 0.05). The INS extracted from biofilms formed in the presence of starch + sucrose displayed a higher percentage of 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) compared to those from biofilms grown in sucrose or sucrose + glucose. Furthermore, biofilms grown in starch + sucrose expressed significantly higher levels of gtfB messenger RNA than sucrose-grown or sucrose + glucose-grown biofilms (P < 0.05). CONCLUSION: The combination of starch and sucrose has profound effects not only on the composition and structure of the polysaccharide matrix but also on gene expression of S. mutans within biofilms, which may enhance the cariogenic potential of dental biofilms.

Effect of sucrose concentration on dental biofilm formed in situ and on enamel demineralization.

The relationship between sucrose concentration and cariogenic potential was studied in situ. Adult volunteers wore intraoral palatal appliances containing human dental enamel blocks, which were extraorally submitted 8 times a day for 14 days, to the treatments: deionized distilled water and sucrose solutions from 1 to 40%. The biofilm formed was analyzed with respect to acidogenicity and biochemical composition; enamel demineralization was evaluated by microhardness. The results showed that 1% sucrose is less cariogenic than 5% or higher concentrations, although sucrose solution at 40% was still able to increase the concentration of insoluble polysaccharide in the biofilm formed. The findings suggest that the threshold of sucrose solution concentration for the formation of a cariogenic biofilm is 5%, which provided the same cariogenic potential as that observed for 10 and 20% sucrose solution.

Effect of a lactose-containing sweetener on root dentine demineralization in situ.

Since lactose has been used as an excipient in powdered sweeteners, we evaluated its effect on dentine demineralization, in an in situ crossover study. Twelve adult volunteers wore palatal appliances containing 4 blocks of bovine root dentine, which were submitted 4 times a day to: distilled water (negative control), aqueous solution of powdered sweetener (Zero Cal); experimental group), 1.5% lactose solution (active group), 1.5% sucrose solution (positive control). After 15 days, microhardness was determined on dentine surface (SMH) and on the sectioned blocks (CSMH). All groups presented statistical differences in SMH before and after the treatments; water increased, while sweetener, lactose and sucrose decreased SMH. By CSMH, the only significant difference was observed after sucrose treatment. The data suggest that sweeteners containing lactose as a bulking agent may be cariogenic for root dentine.