Effect of xylitol on salivary β‐glucosidase in humans

Dental biofilm – in which a diverse set of microorganisms are embedded in a complex polysaccharide matrix that adheres to oral components – is one of the most complex microbial communities in the human body. As biofilm formation is related to oral infections, such as caries and periodontal diseases, strategies for biofilm control are crucial for maintaining oral health. Xylitol, a synthetic sugar used as a sucrose substitute, has been shown to reduce biofilm formation. However, its precise mechanism of action on biofilm reduction has so far not been elucidated. Previous studies demonstrate that bacterial β‐glucosidase action is crucial for biofilm formation. Here, we investigated the correlation between salivary β‐glucosidase activity and dental plaque occurrence. We found a positive correlation between enzymatic activity and the presence of dental biofilm. We observed that xylitol inhibits β‐glucosidase in human saliva. Kinetic studies also confirmed that xylitol acts as a mixed type inhibitor of salivary β‐glucosidase. Based on our data, we suggest that xylitol impairs oral biofilm formation by the inhibition of bacterial β‐glucosidase, which is essential for biofilm formation in the oral cavity.     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

Effects of d‐valine on periodontal or peri‐implant pathogens: Porphyromonas gingivalis biofilm

1 Background

When presented with a surface or an interface, bacteria often grow as biofilms in which cells are held together by an extracellular matrix. Biofilm formation on implants is an initiating factor for their failure. Porphyromonas gingivalis is the primary etiologic bacteria of initiation and progression of periodontal disease. This microorganism is also the risk factor of many systemic diseases, such as cardiovascular disease, diabetes, and pulmonary infection. To date, no medication that can remove such biofilm has been accepted for clinical use. D ‐valine (D‐val) can reportedly inhibit the formation of biofilm and/or trigger the scattering of mature biofilm. Accordingly, this study investigated the effects of d ‐val on single‐species P. gingivalis biofilms in vitro.

2 Methods

P. gingivalis grown in brain heart infusion culture with or without d ‐val was inoculated in 24‐ or 96‐well plates. After incubation for 72 hours, biomass via crystal violet staining, extracellular polysaccharide production by biofilms, and scanning electron microscopy (SEM) were used to determine the d ‐val concentration that can effectively prevent P. gingivalis biofilm formation.

3 Results

Experimental results showed that d ‐val effectively inhibited biofilm formation at concentrations ≥50 mM (mMol/L), and that d ‐val inhibition increased with increased concentration. Moreover, at high concentrations, the bacterial form changed from the normal baseball form into a rodlike shape. d ‐val also notably affected extracellular polysaccharide production by P. gingivalis.

4 Conclusions

d ‐val can inhibit P. gingivalis biofilm formation, and high concentrations can affect bacterial morphology.     

Influence of Streptococcus mutans on Enterococcus faecalis Biofilm Formation

IntroductionAn important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of E. faecalis in the presence of other microorganisms.MethodsEight clinical strains of E. faecalis were tested for biofilm formation on hydroxyapatite disks in the presence and absence of a Streptococcus mutans biofilm.ResultsSignificantly more E. faecalis viable cells were found in biofilms in the presence of S. mutans. This phenomenon was, however, strain-dependent. Of the 8 strains tested, biofilm formation of strains AA-OR34, ER5/1, and V583 was not influenced by S. mutans biofilms.ConclusionsThe results from this study, especially the strain difference, underline the importance of studying biofilm formation in a more realistic multispecies setting.     

Characterisation of the efficacy of endodontic medications using a three‐dimensional fluorescent tooth model: An ex vivo study

The purpose of this study was to establish a three‐dimensional fluorescent tooth model to investigate bacterial viability against intra‐canal medicaments across the thickness and surface of root dentine. Dental microbial biofilms (Enterococcus faecalis and Streptococcus mutans) were established on the external root surface and bacterial kill was monitored over time against intra‐canal medicament (Ca(OH)₂) using fluorescent microscopy in conjunction with BacLight SYTO9 and propidium iodide stains. An Olympus digital camera fitted to SZX16 fluorescent microscope captured images of bacterial cells in biofilms on the external root surface. Viability of biofilm was measured by calculating the total pixel area of green (viable bacteria) and red (non‐viable bacteria) for each image using ImageJ® software. All data generated were assessed for normality and then analysed using a Mann–Whitney t‐test. The viability of S. mutans biofilm following Ca(OH)₂ treatment showed a significant decline compared with the untreated group (P = 0.0418). No significant difference was seen for E. faecalis biofilm between the Ca(OH)₂ and untreated groups indicating Ca(OH)₂ medicament is ineffective against E. faecalis biofilm. This novel three‐dimensional fluorescent biofilm model provides a new clinically relevant tool for testing of medicaments against dental biofilms.     

Phospholipase C‐γ1 expression correlated with cancer progression of potentially malignant oral lesions

Background: Phospholipase C‐γ1 (PLCγ1) is required for cellular migration during tumor progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to determine immunoexpression pattern of PLCγ1 in oral potentially malignant lesions (OPLs) and evaluate PLCγ1 usefulness as a biomarker for predicting clinical behavior in the carcinogenesis of OPL. Methods: In a retrospective follow‐up study, the expression pattern of PLCγ1 protein was determined using immunohistochemistry in samples from 68 patients, including untransformed cases (n = 38) and malignant‐transformed cases (n = 30). The corresponding post‐malignant lesions (OSCCs) were also performed. Results: We observed that elevated expression of PLCγ1 in 40 of 68 (59%) general OPLs and 23 of 30 (77%) OSCCs compared with that in normal oral mucosa. Kaplan–Meier analysis revealed that patients with PLCγ1 positivity had a significantly higher incidence of OSCC than those with PLCγ1 negativity. Cox regression analysis revealed that PLCγ1 expression patterns were significantly associated with increased risk of malignant progression. In addition, the correlation between PLCγ1 expression in pre‐malignant OPL and that in post‐malignant OSCC was significant (P = 0.004). Conclusion: These data indicate that PLCγ1 expression in OPL correlated with oral cancer progression, and PLCγ1 may serve as a useful marker for the identification of high‐risk OPL into OSCC.     

The effect of sodium hypochlorite on Enterococcus faecalis when grown on dentine as a single‐ and multi‐species biofilm

Enterococcus faecalis is often involved in the aetiology of apical periodontitis after endodontic treatment. This project aimed to establish, on dentine in vitro, a multi‐species biofilm containing E. faecalis, and to determine if the organism had an increased resistance to sodium hypochlorite compared with an axenic biofilm. Biofilms were established on dentine discs in flow cells with either E. faecalis alone (axenic) or together with Fusobacterium nucleatum and Streptococcus sanguinis. Following treatment with either 0.9% sodium hypochlorite or saline, the viability of E. faecalis was determined by serial plating and qualitative analysis was performed by scanning electron microscopy and confocal laser scanning microscopy. Viable counts indicated that 0.9% NaOCl is highly effective against E. faecalis grown alone and as part of a multi‐species biofilm (P = 0.0005 and P = 0.001, respectively). No significant difference in its survival in the two biofilm types was found (P = 0.8276).     

Antibacterial efficacy of casein-derived peptides against Enterococcus faecalis

Background : The aim of this study was to test a casein peptide in its glycosylated form (kappa‐casein glycopeptide, KCGP) and its non‐glycosylated form (kappa‐casein peptide, KCP) for antibacterial efficacy against Enterococcus faecalis in planktonic and biofilm cultures. Methods : E. faecalis strain JKD 15036 was exposed to different concentrations of KCGP and KCP in a 96‐well culture plate. The effect of the peptides on the growth of E. faecalis in planktonic culture was monitored by measuring optical density over 7 hours. Biofilm formation was measured after 24 hours using a crystal violet assay. All experiments were performed in triplicate. Results : KCGP and KCP inhibited growth of E. faecalis in planktonic culture with no significant difference in activity between the peptides. KCGP at 0.16% w/v was significantly better at inhibiting E. faecalis biofilm formation than KCP at the same concentration and significantly better than NaOCl at 1.0% w/v. Conclusions : KCGP effectively inhibited E. faecalis biofilm formation and may have potential to augment the efficacy of traditional antiseptic agents.     

Steroid 5α‐reductase activity of Treponema denticola

Introduction: Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5α‐reductase (5α‐R) and 3β‐ and 17β‐hydroxysteroid dehydrogenase activity. A gene matching the 3‐oxo‐5α‐steroid 4‐dehydrogenase family protein (gene ID: 2739284; locus tag: TDE2697) has been identified in T. denticola ATCC 35405. The aim of the work presented here was to optimize assay conditions and determine steroid substrate specificities for the 5α‐R activity of T. denticola ATCC 33520. Methods: 5α‐R activity of cell‐free preparations was assayed with radioactive steroid substrates. 5α‐R‐reduced products were identified using thin‐layer chromatography and a radioisotope scanner. Assay conditions were optimized for co‐factor, buffer and pH requirements. Apparent substrate specificities were determined for progesterone, 4‐androstenedione, testosterone and corticosterone. The time–course for metabolism of radiolabelled progesterone and cholesterol substrates was investigated with anaerobic cultures. Results: The optimum pH for 5α‐R was 5.5 and the preferred co‐factor was NADPH. The order of the steroids with respect to their 5α‐R substrate specificities was (in descending order): progesterone, 4‐androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5α‐dihydrocholesterol from cholesterol. Conclusion: These results suggest that the 3‐oxo‐5α‐steroid 4‐dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5α‐R isoenzyme 2 with regard to co‐factor requirement and pH optimum.     

Antibacterial Effect of Synthetic Peptide LyeTxI and LyeTxI/β‐Cyclodextrin Association Compound Against Planktonic and Multispecies Biofilms of Periodontal Pathogens

Background: Antimicrobial peptides (AMPs) have shown rapid and potent effect against planktonic bacteria. However, control of periodontopathic biofilms is a challenge even for AMPs. Thus, the present study evaluates in vitro antimicrobial activity of synthetic peptide LyeTxI and association compound LyeTxI/β‐cyclodextrin (βCD) against multispecies biofilms. Methods: Sensibility to LyeTxI and LyeTxI/βCD was determined for planktonic Gram‐negative periodontopathogens. Time‐kill kinetic assay was performed at minimum inhibitory concentrations (MICs) in all planktonic strains. Multispecies biofilms were grown on pegs using a biofilm device and studied by scanning electron microscopy at 2, 5, and 10 days. Minimal biofilm eradication concentration (MBEC) was determined for 2‐ and 4‐day multispecies biofilms. Metabolic activity of biofilms was determined by fluorometry study. Results: Biofilms showed reproducible cell density on pegs of the biofilm device. LyeTxI and LyeTxI/βCD were active against all strains tested at concentrations ≤62.5 μg/mL. Kinetic assays showed rapid bactericidal effect of LyeTxI against all periodontopathogens. MBECs of LyeTxI and LyeTxI/βCD against multispecies 2‐day biofilms were two‐fold higher than MICs of cells shed from biofilms. LyeTxI was able to reduce multispecies 2‐day metabolic activity by 90%. Multispecies 4‐day biofilms were tolerant to all agents tested. Conclusions: LyeTxI and LyeTxI/βCD are active against periodontopathic bacteria, showing rapid bactericidal effect and may be used to prevent biofilm development. In the future, AMPs could be therapeutic tools for treatment of periodontitis.     

Psychological stress has no association with salivary levels of β‐defensin 2 and β‐defensin 3

J Oral Pathol Med (2010) 39 : 765–769 Background: Recent studies suggest that stress can predispose an individual to the development of periodontal disease, but the exact biological mechanism is unknown. Considering that psychological stress can down‐regulate the production of β‐defensins (antimicrobial peptides produced in the oral cavity), the aim of the present study was to evaluate the association between stress and salivary levels of β‐defensin 2 (HBD‐2) and β‐defensin 3 (HBD‐3). Methods: For this purpose, seventy five volunteers, classified as periodontally healthy, were submitted to a psychological evaluation using a validated questionnaire (Questionnaire of Lipp‐ISS). Following analysis of the questionnaires, the subjects were divided in two groups (Group A: Absence of stress and Group B: Presence of stress). Unstimulated saliva samples were collected and the concentration of total protein was determined using the BCA method, and the concentrations of HBD‐2 and HBD‐3 were determined by ELISA. Results: The levels of total protein did not show a statistically significant difference between the groups. Analyses of HBD‐2 and HBD‐3 concentrations indicate that the stress condition was not associated with the levels of either peptide in saliva (P = 0.3664 for HBD‐2 and P = 0.3608 for HBD‐3). Conclusion: In periodontally healthy subjects, HBD‐2 and HBD‐3 levels are not influenced by stress.     

Assessment of antimicrobial susceptibility of Enterococcus faecalis isolated from chronic periodontitis in biofilm versus planktonic phase

Background: Enterococci are often associated with chronic and recurrent infectious diseases because of their antimicrobial resistance. The aim of this study is to assess antimicrobial susceptibility of Enterococcus faecalis in chronic periodontitis. Methods: Antimicrobial susceptibility was determined on 23 E. faecalis strains isolated from patients with chronic periodontitis. Ampicillin, erythromycin, gentamicin, tetracycline, triclosan, and vancomycin were prepared in two‐fold serial dilution up to 8,192 μg/mL. Enterococcal biofilm was established by a biofilm device and observed by confocal laser microscopy and scanning electron microscopy. The minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration were determined by spectrophotometer at optical density₆₅₀. Results: A few patches of monolayer early biofilm were observed on the surfaces of biofilm device pegs. The colony‐forming units of biofilm per peg were 1.2 × 10³ to 1.7 × 10⁴ and 0 to 20 post‐triclosan treatment. The MIC₅₀ was higher than the MIC epidemiologic cut‐off for tetracycline and the MIC₉₀ was higher than the cut‐off for erythromycin and tetracycline, respectively. In biofilm, minimum biofilm eradication concentrations were extremely high for all of the drugs except triclosan. Conclusions: The E. faecalis strains of chronic periodontitis exhibited weak biofilm formation ability at the early stage. Over 50% of the strains were resistant to tetracycline, and a few strains were highly resistant to erythromycin or gentamicin. E. faecalis cells in biofilm were hardly eradicated by most of the agents, even in high concentrations. Triclosan was effective in inhibiting E. faecalis growth in both biofilm and planktonic phase.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Lactobacillus plantarum Lipoteichoic Acid Inhibits Oral Multispecies Biofilm

Introduction

Apical periodontitis is an inflammatory disease in the periradicular region of teeth that results from infection by multispecies bacterial biofilm residing in the root canal system. In this study, we investigated whether Lactobacillus plantarum lipoteichoic acid (Lp.LTA) could inhibit multispecies oral pathogenic bacterial biofilm formation.

Inhibiting effects of Streptococcus salivarius on competence‐stimulating peptide‐dependent biofilm formation by Streptococcus mutans

Introduction: The effects of Streptococcus salivarius on the competence‐stimulating peptide (CSP)‐dependent biofilm formation by Streptococcus mutans were investigated. Methods: Biofilms were grown on 96‐well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. Results: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC‐3 and FSC‐3ΔglrA in separate dual‐species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP‐binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene‐dependent quorum sensing systems. Conclusion: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell‐to‐cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.     

Inhibitory effects of tea catechin epigallocatechin-3-gallate against biofilms formed from Streptococcus mutans and a probiotic lactobacillus strain

ObjectiveEffects of tea catechin epigallocatechin-3-gallate (EGCG) against biofilm formation by Streptococcus mutans and probiotic Lactobacillus casei in Yakult^® (LcY) were examined.DesignBiofilms were formed by S. mutans alone (Sm) and co-culture of S. mutans and LcY (Sm + LcY) in the absence or presence of EGCG. The biomass of biofilms, which were sonicated or not, was measured by the crystal violet assay. Biofilm morphology was observed by scanning electron microscopy. Bacterial viability and extracellular polysaccharides were determined by SYTO9/propidium iodide and dextran-conjugated fluorescein staining, respectively, and confocal microscopy. Gene expression of glucosyltransferase was determined by quantitative polymerase chain reaction.ResultsWhile 250 μg/ml EGCG significantly decreased the biomass and acid production of Sm biofilms, 500 μg/ml EGCG was required to inhibit Sm + LcY biofilm formation and acid production. EGCG decreased the amount of live bacteria present in both Sm and Sm + LcY biofilms. The level of dead bacteria in Sm + LcY biofilms was higher than in Sm biofilms when formed in the presence of 250 μg/ml EGCG. EGCG decreased levels of extracellular polysaccharides in Sm and Sm + LcY biofilms. The extent of biofilm removal by sonication was not different between Sm and Sm+LcY biofilms formed in the absence or presence of 62.5 or 125 μg/ml EGCG. The level of Sm gtfB and gtfD expression in Sm + LcY biofilms was higher than those in the Sm biofilms when formed in the presence of EGCG at 250 μg/ml.ConclusionThe results indicated that LcY might interfere the inhibitory effects of EGCG against biofilm formation by S. mutans.     

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca2+‐promoted biofilms

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca²⁺ utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca²⁺, the additive effects of Ca²⁺‐promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca²⁺‐promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl₂ exhibited enhanced cell aggregation and increased levels of cell‐associated Ca²⁺. Biofilm‐derived cells grown in the presence of Ca²⁺ exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca²⁺‐promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca²⁺‐promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.     

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

Chotjumlong P, Khongkhunthian S, Ongchai S, Reutrakul V, Krisanaprakornkit S. Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E ₂ synthesis in human gingival fibroblasts. J Periodont Res 2010; 45: 464–470. © 2010 John Wiley & Sons A/S Background and Objective: Oral epithelial cells express three antimicrobial peptide human β‐defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) synthesis in non‐immune cells, such as human gingival fibroblasts. Material and Methods: Cultured fibroblasts were treated with different concentrations of hBD‐1, ‐2, ‐3 or interleukin‐1β, as a positive control, for various times, in the presence or absence of NS‐398, a specific COX‐2 inhibitor. The levels of COX‐1 and COX‐2 mRNA expression were analyzed using RT‐PCR and real‐time PCR. Whole cell lysates were analyzed for COX‐1 and COX‐2 protein expression by western blotting. Cell‐free culture supernatants were assayed for PGE₂ levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. Results: Ten and 40 μg/mL of hBD‐3 up‐regulated COX‐2 mRNA and protein expression, consistent with COX‐2 up‐regulation by interleukin‐1β, whereas hBD‐1 and hBD‐2 did not. However, COX‐1 mRNA and protein were constitutively expressed. The time‐course study revealed that hBD‐3 up‐regulated COX‐2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX‐2 up‐regulation, 10 and 40 μg/mL of hBD‐3 significantly increased PGE₂ levels in cell‐free culture supernatants (p < 0.05), and this was inhibited by NS‐398 in a dose‐dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. Conclusion: These findings indicate that epithelial human β‐defensin‐3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.     

Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites

Objectives

The aim of this study was to evaluate the acid production, acid tolerance and composition of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites.

Methods

S. mutans biofilms were formed on saliva-coated discs prepared from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing materials (Filtek Z350, GRADIA DIRECT and hydroxyapatite). To assess the level of acid production and acid tolerance, glycolytic pH drop and proton permeability assays were performed using 94 h old S. mutans biofilms. To evaluate the biofilm composition, the biomass (total dry-weight), colony forming unit (CFU), water-insoluble extracellular polysaccharides (EPS), water-soluble EPS and intracellular iodophilic polysaccharides (IPS) of 94 h old S. mutans biofilms were analysed. The amount of fluoride of old culture medium released from the materials during the experimental period was also determined. Each assay was performed in duplicate in at least four different experiments (n = 8).

Results

All biofilms showed similar initial rates of acid production (0.083–0.089 pH drop/min) and proton permeability (0.025–0.036 pH increase/min), irrespective of fluoride release from the materials. On the other hand, the amount of biomass, water-insoluble EPS and IPS of the biofilms on Unifil Flow, which releases a larger amount of fluoride in the early stages of biofilm formation, were significantly lower than those on the other materials (up to 27%, 38% and 36% reduction in biomass, water-insoluble and IPS, respectively).

Conclusions

Our finding suggests that fluoride releasing resin composites might contribute to the decrease in cariogenic composition of S. mutans biofilms if an appropriate amount of fluoride is released in the early stages of biofilm formation.