Modulation of proinflammatory mediators by viruses-bacteria synergism in human osteoblasts-an in vitro study.

BACKGROUND/PURPOSE: Viruses-bacteria synergistic interaction is associated with destructive periodontal diseases. However, the underlying mechanism for tissue destruction is not fully elucidated. In this study, lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) and polyinosinic-polycytidylic acid (poly I:C) were used to simulate bacteria and viruses, respectively. The possible combined effects of both molecular patterns on secretion of interleukin (IL)-6 and prostaglandin E2 (PGE2) from osteoblasts were determined. The effects of povidone-iodine (PVP-I) on the secretion of IL-6 and PGE2 were also examined. METHODS: Viability of treated osteoblastic cells (MG63) was examined by detection the mitochondrial dehydrogenase activity. Secretion of IL-6 and PGE2 was detected using the enzyme-linked immunosorbent assay (ELISA). Mitogen-activated protein kinases (MAPKs) and cyclooxygenase-2 (COX-2) were determined using the Western blotting analysis. RESULTS: Pg-LPS or poly I:C significantly enhanced the production of IL-6 and PGE2 in MG63 cells. The additive/synergistic effects of Pg-LPS/poly I:C on production of IL-6 and PGE2 were evident. The levels of phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and expression of COX-2 protein were enhanced by Pg-LPS and/or poly I:C. On the other hand, the level of phosphorylation of extracellular signal-regulated kinase (ERK) was reduced by Pg-LPS and/or poly I:C. The stimulatory secretion of PGE2 by poly I:C was significantly reduced by PVP-I. CONCLUSION: Concomitant infection of viruses and bacteria may be potentially harmful to the tooth supporting tissues by production of proinflammatory mediators. The results suggest the potential anti-inflammatory effect of PVP-I on bacterial or viral infection.

Enhancing production of herpes simplex virus type 1 in oral epithelial cells by co-infection with Aggregatibacter actinomycetemcomitans.

BACKGROUND/PURPOSE: The association between herpetic/bacterial co-infection and periodontal diseases has been reported. However, how interactions between herpesviruses and periodontal bacteria dampen periodontal inflammation is still unclear. This study determined effects of co-infection with oral bacteria, including Streptococcus sanguinis, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans, in herpes simplex virus type 1 (HSV-1)-infected oral epithelial cells. METHODS: Cell viability was determined by detection the activity of mitochondrial dehydrogenase. Viral production was measured using the plaque assay. Levels of bacterial and viral DNA were determined by real-time polymerase chain reaction. Secretion of interleukin (IL)-6 and IL-8 was measured using the enzyme-linked immunosorbent assay. RESULTS: Viability was not further reduced by bacterial co-infection in HSV-1-infected cells. Co-infection with HSV-1 and S. sanguinis or F. nucleatum reduced the viral yield whereas co-infection with HSV-1 and A. actinomycetemcomitans significantly enhanced the viral yield in oral epithelial cells. The enhancing effect of A. actinomycetemcomitans was not affected by bacterial heat-inactivation. Co-infection with HSV-1/A. actinomycetemcomitans increased intracellular levels of both viral and bacterial DNA. Secretion of IL-6 and IL-8 stimulated by A. actinomycetemcomitans infection was partly reduced by co-infection with HSV-1 in oral epithelial cells. CONCLUSION: In contrast to S. sanguinis and F. nucleatum, A. actinomycetemcomitans enhanced the yield of HSV-1. Either HSV-1 or A. actinomycetemcomitans may be benefited from co-infection, in aspects of increases in production of viral and bacterial DNA as well as reductions in cytokine secretion. These findings echoed with previous clinical studies showing co-infection of HSV and A. actinomycetemcomitans in patients with aggressive periodontitis.

The role of fibroblasts in the modulation of dental pulp inflammation.

BACKGROUND/PURPOSE: Dental pulp fibroblasts can protect dental pulp from microbial invasion. However, little is known about the interaction between pulp fibroblasts and the immune cells. In this study, the production of proinflammatory cytokines related to inflammatory cell recruitment was evaluated in tumor necrosis factor (TNF)-α-stimulated human dental pulp fibroblasts (HDPFs). The role of TNF-α-stimulated HDPFs in the cell fusion under inflammatory process was determined with the cell co-culture with peripheral blood mononuclear cells (PBMCs). METHODS: HDPFs were stimulated with various concentrations of TNF-α, and the secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 was analyzed by the enzyme-linked immunosorbent assay. The mRNA expression levels of intercellular adhesion molecule-1 (ICAM-1), macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) were determined by real-time quantitative polymerase chain reaction. TNF-α-treated HDPFs were co-cultured with PBMCs for 21 days, and characteristics of cell differentiation were assessed. RESULTS: TNF-α induced IL-6, IL-8 and MCP-1 production in HDPFs. Moreover, mRNA expression levels of ICAM-1, M-CSF and OPG were significantly increased in TNF-α-treated HDPFs. Co-culture of TNF-α-treated HDPFs and PBMCs stimulated formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and the F-actin rings were observed in these multinucleated cells. CONCLUSION: Our results indicate that under the stimulation of TNF-α, HDPFs may amplify inflammatory response by cytokines production, which in turn can modulate the differentiation of immune cells.

Effects of epigallocatechin-3-gallate and acyclovir on herpes simplex virus type 1 infection in oral epithelial cells.

BACKGROUND/PURPOSE: Herpes simplex virus type 1 (HSV-1) is the pathogenic agent of human diseases, including gingivostomatitis and herpes labialis. The anti-viral activities of the tea polyphenol, epigallocatechin-3-gallate (EGCG), have been demonstrated. This study examined the combined effects of EGCG and the antiviral drug, acyclovir (ACV), on infection of HSV-1 in oral epithelial cells. METHODS: Cell viability was examined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. Viral yields were determined using the plaque assay. Viral proteins were detected using Western blotting analysis or confocal laser scanning microscopy. Viral DNA was detected using the real-time polymerase chain reaction. RESULTS: Cytotoxic effects of HSV-1 on the viability of oral epithelial cells were evidently reduced in the presence of EGCG (25 µg/ml) or/and ACV (50 µg/ml). Viral yields were also significantly reduced by treatment of cells with EGCG or/and ACV. Expression of viral immediate early protein, infected cell protein 0 (ICP0), was greatly inhibited when cells were treated with EGCG. Combined effects of EGCG and ACV were more evident for the expression of viral thymidine kinase, ICP5 and glycoprotein D. EGCG, but not ACV, significantly reduced the levels of viral particles and viral DNA during viral entry phase. However, at 20 h post infection, the intracellular viral DNA was evidently reduced in HSV-1 infected cells treated with EGCG and ACV. Moreover, the stimulatory effects of HSV-1 on phosphorylation of c-Jun N-terminal kinase could be reduced by ACV. CONCLUSION: The results demonstrated the additive effects of EGCG and ACV on HSV-1 infection in oral epithelial cells.

Stimulatory effects of Porphyromonas gingivalis GroEL protein on interleukin-6 and interleukin-8 in human osteoblasts.

BACKGROUND/PURPOSE: Porphyromonas gingivalis is an oral pathogen associated with periodontal diseases. P. gingivalis GroEL protein is a stimulator of inflammatory cytokines in macrophages. This study inspected effects of P. gingivalis GroEL protein on production of interleukin (IL)-6 and IL-8 by human osteoblasts. METHODS: Viability of GroEL-treated osteoblasts was analyzed with 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. Secretion of IL-6 and IL-8 was analyzed using the enzyme-linked immunosorbent assay. Levels of mRNA were analyzed using the reverse transcription and real-time polymerase chain reaction. The antioxidant (curcumin), the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and the c-Jun N-terminal kinase (JNK) inhibitor (SP600125) were employed to elucidate possible signaling pathways involved. RESULTS: Treatment with GroEL did not affect morphology and viability of osteoblasts. GroEL significantly induced the secretion of IL-6 and IL-8 by osteoblasts in a concentration-dependent pattern. Moreover, the mRNA levels of IL-6 and IL-8 were stimulated by GroEL. The application of SP600125 (10 µM) significantly suppressed the induction of IL-6 and IL-8 by GroEL-treated cells. However, curcumin (20 µM) and SB203580 (20 µM) only down-regulated the stimulatory effects of GroEL on IL-6. CONCLUSION: GroEL protein stimulated the inflammatory reaction of osteoblasts, probably through the activation of p38 MAPK or JNK pathway. The findings suggest that P. gingivalis GroEL may influence the immune functions of osteoblasts and endanger the periodontal health.

Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment.

BACKGROUND/PURPOSE: Elevated monocyte chemoattractant protein-1 (MCP-1) is related to severe periodontal destruction. Furthermore, MCP-1 -2518 A/G gene polymorphism affects MCP-1 after inflammatory stimuli. This study analyzed the association between MCP-1 -2518 gene polymorphism and the outcome of nonsurgical periodontal treatment. METHODS: Forty periodontal patients were recruited and MCP-1 -2518 A/G gene polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay. The clinical periodontal parameters, including probing depth (PD), clinical attachment level (CAL), gingival index (GI), bleeding index (BI) and plaque index (PI), were recorded before and six weeks after nonsurgical periodontal therapy. Patients were divided into chronic periodontitis (CP) or aggressive periodontitis (AP). Multiple linear regression analysis was performed to investigate certain predictors of the therapy outcome. RESULTS: The frequency of MCP-1 -2518 genotype-positive (carrying allele G) was 42.5%. Poor treatment outcome in PD, GI and BI improvement could be predicted with MCP-1 -2518 A/G genotype and aggressive periodontitis status as the predictor variables. In contrast, MCP-1 -2518 A/A genotype and aggressive periodontitis status could predict better treatment response in PD and BI improvement. However, MCP-1 -2518 genotype did not affect the treatment outcome in patients with chronic periodontitis. CONCLUSION: MCP-1 -2518 A/G genotype might be useful in predicting less favorable nonsurgical treatment outcome in patients with aggressive periodontitis. However, MCP-1 -2518 gene polymorphism may not play a role in patients with chronic periodontitis. This study suggests that MCP-1 -2518 genotype may influence the outcome of nonsurgical periodontal treatment in aggressive periodontitis patients.

Levels of high-mobility group box-1 in gingival crevicular fluid in nonsmokers and smokers with chronic periodontitis.

BACKGROUND/PURPOSE: High-mobility group box-1 (HMGB1), a proinflammatory cytokine, plays a role in inflammatory disorders. Smoking is a well-established risk factor for periodontal disease. The aim of this study was to compare the levels of HMGB1 in the gingival crevicular fluid from periodontally healthy nonsmokers, chronic periodontitis nonsmokers, and chronic periodontitis smokers. Furthermore, the relationship between levels of HMGB1 and periodontal parameters was examined. METHODS: Periodontal parameters of 17 nonsmokers with chronic periodontitis, nine smokers with chronic periodontitis, and nine periodontally healthy nonsmokers were examined. Gingival crevicular fluid samples were collected, and the levels of HMGB1 were analyzed using the enzyme-linked immunosorbent assay. RESULTS: The median level of HMGB1 was statistically significantly higher in chronic periodontitis nonsmokers (37.5 ng/mL) than in chronic periodontitis smokers (9.5 ng/mL) and periodontally healthy nonsmokers (3.7 ng/mL). There was no significant difference in the levels of HMGB1 between chronic periodontitis smokers and periodontally healthy nonsmokers. Levels of HMGB1 were positively correlated with plaque index, gingival index, probing depth, and clinical attachment level of nonsmokers. However, no significant correlations were found between levels of HMGB1 and all periodontal parameters examined in chronic periodontitis smokers. CONCLUSION: Chronic periodontitis nonsmokers had elevated levels of HMGB1 in gingival crevicular fluid. Moreover, the levels of HMGB1 were correlated with severity of periodontitis. Chronic periodontitis smokers exhibited lower levels of HMGB1 than chronic periodontitis nonsmokers. Further research is needed for understanding the role of HMGB1 in smoking and pathogenesis of periodontitis.

Bacterial adhesion to antibiotic-loaded guided tissue regeneration membranes - a scanning electron microscopy study.

BACKGROUND/PURPOSE: Bacterial contamination of sites undergoing guided tissue regeneration (GTR) therapy may reduce the efficiency of periodontal regeneration. This study compared bacterial adhesion onto various GTR membranes incorporated with antibiotics. METHODS: Three barrier membranes, including expanded polytetrafluoroethylene (ePTFE) membrane, collagen membrane, and glycolide fiber membrane, were loaded with tetracycline or amoxicillin. The adhesion of Streptococcus mutans and Aggregatibacter actinomycetemcomitans onto the GTR membranes with or without antibiotics was analyzed using the scanning electron microscopy (SEM) analysis. RESULTS: The SEM analysis showed no apparent alteration in the physical structure of the membranes loaded with antibiotics. Both S. mutans and A. actinomycetemcomitans attached best on the collagen membranes, followed by the ePTFE membranes, and then the glycolide fiber membranes without antibiotics. Moreover, higher numbers of bacteria were observed on the fibril areas than on the laminar areas of the ePTFE membranes. The amounts of attached bacteria on the GTR membranes increased after longer incubation. Incorporation of tetracycline or amoxicillin greatly reduced the adhesion of S. mutans and A. actinomycetemcomitans onto all of the GTR membranes examined. CONCLUSION: Incorporation of tetracycline or amoxicillin greatly reduced adhesion of S. mutans or A. actinomycetemcomitans on the ePTFE, glycolide fiber, or collagen membranes. This finding indicates that it is valuable and effective to use the antibiotic-loaded GTR membranes for periodontal regeneration therapy.

Antiherpetic potential of 6-bromoindirubin-3'-acetoxime (BIO-acetoxime) in human oral epithelial cells.

Glycogen synthase kinase 3 (GSK-3) functions in the regulation of glycogen metabolism, in the cell cycle, and in immune responses and is targeted by some viruses to favor the viral life cycle. Inhibition of GSK-3 by 6-bromoindirubin-3'-acetoxime (BIO-acetoxime), a synthetic derivative of a compound from the Mediterranean mollusk Hexaplex trunculus, protects cells from varicella infection. In this study, we examined the effects of BIO-acetoxime against herpes simplex virus-1 (HSV-1) infection in human oral epithelial cells, which represent a natural target cell type. The results revealed that BIO-acetoxime relieves HSV-1-induced cytopathic effects and apoptosis. We also found that BIO-acetoxime reduced viral yields and the expression of different classes of viral proteins. Furthermore, addition of BIO-acetoxime before, simultaneously with or after HSV-1 infection significantly reduced viral yields. Collectively, BIO-acetoxime may suppress viral gene expression and protect oral epithelial cells from HSV-1 infection. These results suggest the possible involvement of GSK-3 in HSV-1 infection.

Comparison of clinical outcomes following guided tissue regeneration treatment with a polylactic acid barrier or a collagen membrane.

This prospective, randomized, controlled clinical trial compared the clinical outcomes for a polylactic acid barrier versus a collagen membrane in conjunction with guided tissue regeneration (GTR). Thirty patients with chronic periodontitis and at least one intrabony defect of a minimum 4 mm were enrolled. Following full-mouth scaling, GTR using a collagen membrane or a polylactic acid barrier was performed at one site in each patient. At 1 week before and 6 months after surgery, probing pocket depth (PPD), clinical attachment level (CAL), marginal tissue recession, and bone fill were assessed. A significant reduction in PPD (2.50 +/- 1.35 mm for the polylactic acid barrier and 2.60 +/- 1.08 mm for the collagen membrane) was obtained, in addition to gains in CAL (2.40 +/- 1.17 mm for the polylactic acid barrier and 2.60 +/- 1.26 mm for the collagen membrane) and bone fill (0.33 +/- 1.89 mm for polylactic acid barrier and 2.57 +/- 1.64 mm for collagen membrane), for each group compared to baseline. Significantly, the results from 6 months after surgery showed that there was greater bone fill when the collagen membrane was used compared to the polylactic acid barrier.

Areca nut extract-treated gingival fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2.

BACKGROUND: Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases. This study investigated the potential effects of areca nut extract (ANE) on human gingival fibroblasts and the consequential impacts on inflammatory pathogenesis. METHODS: Analyses of senescence marker, cell viability, changes of the cell cycle, and cell granularity in gingival fibroblasts together with an assessment of the invasiveness of polymorphonuclear (PMN) leukocytes after treatment with the supernatant of ANE-treated gingival fibroblasts were performed to characterize the phenotypic impacts. Western blotting and gelatin zymography were used to assay the expression and activity of MMP-2. RESULTS: Chronic subtoxic (<10 microg/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated beta-galactosidase activity and various other senescence-associated phenotypes in gingival fibroblasts. Gingival fibroblasts established from older individuals had a higher propensity to become ANE-induced senescent gingival fibroblasts. An activation of MMP-2 was identified in senescent cells. PMN leukocytes treated with the supernatant of ANE-induced senescent cells exhibited a significant increase in invasiveness, which was abrogated by both a MMP-2 blocker and a MMP-2 nullifying antibody. CONCLUSIONS: This study provides evidence whereby MMP-2 secreted from ANE-induced senescent gingival fibroblasts would facilitate the invasiveness of PMN leukocytes, which could be associated with the oral inflammatory process in areca chewers.

Bacterial penetration through antibiotic-loaded guided tissue regeneration membranes.

BACKGROUND: This study compared bacterial penetration through guided tissue regeneration (GTR) membranes impregnated with antibiotics. METHODS: Three barrier membranes, expanded polytetrafluoroethylene (ePTFE) membrane, collagen membrane, and glycolide fiber composite membrane, were loaded with amoxicillin or tetracycline. The penetration of Streptococcus mutans and Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) through the GTR membranes was achieved using a device consisting of an inner tube and an outer bottle filled with culture media. RESULTS: The penetration of S. mutans or A. actinomycetemcomitans into the inner tubes significantly decreased with all of the antibiotic-loaded membranes compared to membranes without antibiotics. However, differences were found in the behavior of the three membranes. The antibiotic-loaded ePTFE membranes showed the best barrier effect. Moreover, the inhibitory effect of tetracycline on S. mutans was greater than that of amoxicillin for all GTR membranes. Furthermore, the inhibitory effect of tetracycline on A. actinomycetemcomitans was lower than that of amoxicillin with the glycolide fiber membrane. CONCLUSIONS: The results showed that penetration of S. mutans and A. actinomycetemcomitans through amoxicillin- or tetracycline-loaded ePTFE membrane, glycolide fiber membrane, and collagen membrane was delayed and/or reduced. Thus, incorporation of an antibiotic into the membrane may be of value when controlling membrane-associated infection during GTR therapy.

Inhibitory effects of safrole on phagocytosis, intracellular reactive oxygen species, and the activity of myeloperoxidase released by human polymorphonuclear leukocytes.

BACKGROUND: Safrole, a component of Piper betle inflorescence, inhibits bactericidal activity and the release of superoxide anion (O(2)(-)) by polymorphonuclear leukocytes (PMNs). This in vitro study further investigated the effects of safrole on phagocytic activity, the intracellular production of reactive oxygen species (ROS), and the activity of the lysosomal enzyme myeloperoxidase (MPO), which is released by human PMNs. METHODS: The possible effects of safrole on the phagocytic activity of PMNs against Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) were determined using flow cytometry. PMNs were treated with various concentrations of safrole, which was followed by treatment with Hanks balanced salt solution with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). Intracellular ROS was determined using 2',7'-dichlorofluorescein diacetate and a fluorometer, whereas MPO activity was determined using a substrate assay. RESULTS: Safrole significantly inhibited the phagocytic activity of PMNs in a dose-dependent manner. Approximately 50% of the phagocytic activity of PMNs was affected when 10 mM safrole was used. Exposure of the PMNs to safrole (up to 5 mM) did not directly affect the intracellular levels of ROS and the extracellular activity of MPO. However, the ability of CB/fMLP to trigger production of intracellular ROS and the activity of MPO released by human PMNs was significantly suppressed by safrole. CONCLUSIONS: Safrole reduced the uptake of A. actinomycetemcomitans by human PMNs. Safrole also impaired the normal activation activity of PMNs. Alterations in the defensive properties of PMNs by safrole might promote bacterial colonization, and this could result in periodontal infection.

Inhibition of Streptococcus mutans by a commercial yogurt drink.

BACKGROUND/PURPOSE: Studies have been focused on using probiotics to prevent caries. The lactobacillus probiotic bacteria in Yakult® (LcY) has been shown to inhibit the growth or biofilm formation of Streptococcus mutans. However, sucrose in Yakult® raised concerns. The purpose of this study was to determine effects of Yakult® on the growth and adhesion of S. mutans. MATERIALS AND METHODS: S. mutans was grown in serial diluted Yakult®, filtered Yakult® or 20% heated Yakult®. S. mutans was co-cultured with LcY in media with or without diluted filtered Yakult®, or in LcY grown in media with or without sugars. Colony forming units and pH values of bacterial cultures were determined. SYTO 9-stained adhered bacteria were observed. RESULTS: Yakult® inhibited the growth of S. mutans. Filtering or heating Yakult® reduced its inhibitory ability against S. mutans. The inhibitory effect of LcY against S. mutans was enhanced when cultured in the presence of 20% filtered Yakult®. LcY cultured in sucrose media for 24 h inhibited the growth of S. mutans, but this effect was less evident when LcY was grown for 48 h. LcY grown in glucose or lactose media similarly reduced S. mutans growth. Culturing S. mutans with LcY grown in sucrose or glucose media reduced bacterial adhesion. However, co-culturing S. mutans with LcY grown in the lactose media did not decrease bacterial adhesion. CONCLUSION: Yakult® and its probiotic content may inhibit S. mutans growth and the effect may be moderated by the type of sugar added for LcY cultivation.

Eugenol inhibited the antimicrobial functions of neutrophils.

Eugenol-containing restorative materials are commonly used for vital pulp therapy. A well-regulated host defense response is pivotal for the success of vital pulp therapy. The present study was to assess the effects of eugenol on the antimicrobial functions of polymorphonuclear leukocytes (neutrophils). Treatment with eugenol (< or = 1.25 mmol/L) for 30 minutes did not significantly affect the viability of neutrophils. However, preincubation of neutrophils with eugenol (1.25 mmol/L and 2.5 mmol/L) abolished their bactericidal activity against oral pathogens Streptococcus mutans and Actinobacillus actinomycetemcomitans. In addition, through the suppression of the extracellular release of myeloperoxidase and the intracellular production of reactive oxygen species, eugenol at sufficient concentrations impaired the activation of neutrophils by cytochalasin B and fMet-Leu-Phe (CB/fMLP). These results suggested that the antimicrobial functions of neutrophils were interfered by eugenol, and the inhibitory effects of eugenol (< or = 1.25 mmol/L) were not due to direct killing of neutrophils.

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

OBJECTIVE: Effects of tea catechin epigallocatechin-3-gallate (EGCG) against biofilm formation by Streptococcus mutans and probiotic Lactobacillus casei in Yakult® (LcY) were examined. DESIGN: Biofilms 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. RESULTS: While 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. CONCLUSION: The results indicated that LcY might interfere the inhibitory effects of EGCG against biofilm formation by S. mutans.

Osteoblastic response to collagen scaffolds varied in freezing temperature and glutaraldehyde crosslinking.

Collagen sponges are widely used scaffolds in bone engineering. To form bone, the osteoblastic cells undergo proliferation, differentiation, and mineralization stages in the scaffold. Crosslinking and freezing temperature are two important variables in fabricating collagen sponges. The purpose of this study was to examine the osteoblastic responses to collagen sponges prepared with or without glutaraldehyde crosslinking at different freezing temperatures (-20 degrees C or -80 degrees C). MC3T3-E1 osteoblastic cells were cultured in differently prepared sponges. Osteoblastic responses examined included cell numbers, osteocalcin expression, and calcium deposition. Cell numbers were measured by DNA content. Osteocalcin expression was determined by RT-PCR and real-time RT-PCR. Calcium deposition was assayed by ortho-cresophthalein complexone method and von Kossa stain. The osteoblastic cells grown in all collagen sponges did not show apparent signs of cytotoxicity. Collagen sponges differed in freezing temperatures resulted in similar osteoblastic responses. Glutaraldehyde-crosslinked sponges demonstrated less cell-mediated contraction and more cell numbers at day 7 (p < 0.005). However, they showed lower osteocalcin expression at day 7 (p < 0.05) and less calcium deposition at day 21 (p < 0.001). In summary, different freezing temperatures played a minor role in osteoblastic responses. Glutaraldehyde crosslinking process, though improved the dimensional stability of collagen sponges, might compromise the osteoblastic differentiation and mineralization.

Eugenol suppressed the expression of lipopolysaccharide-induced proinflammatory mediators in human macrophages.

Eugenol is commonly used as an analgesic agent during acute pulpitis and is a major component of root canal sealers. Despite the frequent applications of eugenol in the practice of dentistry, little is known about the role of eugenol under the status of inflammation. This study was aimed to investigate the influence of eugenol on human macrophages (U937) under the stimulation of lipopolysaccharide (LPS). Eugenol was shown to block the release of the bone resorbing mediators, including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E2 from LPS-stimulated macrophages. In contrast, eugenol alone did not alter the expression levels of these proinflammatory mediators in macrophages. Consistent with downregulation of bone-resorbing mediators, eugenol suppressed the messenger RNA expression of LPS-induced IL-1beta, TNF-alpha, and cyclooxygenase-2 in macrophages. The results suggest a potential anti-inflammatory effect of eugenol in the acute inflamed pulps and apical periodontitis.

Eugenol modulates cyclooxygenase-2 expression through the activation of nuclear factor kappa B in human osteoblasts.

Because eugenol is a major component of root canal sealers and retrograde filling materials, its effects on periapical bone healing are therefore of concern. In this study, the effects of eugenol on the activation of nuclear factor kappa B (NF-kappaB) and the expression of cyclooxygenase-2 (COX-2) in human osteoblasts were investigated. The results showed that eugenol activated the nuclear translocation of NF-kappaB. In addition, COX-2 protein expression in osteoblasts was induced by eugenol in a dose-dependent manner. Furthermore, the eugenol-modulated COX-2 expression was inhibited by an NF-kappaB inhibitor, N-acetylcysteine. Taken together, eugenol might induce COX-2 expression through the activation of NF-kappaB in human osteoblasts. These results suggest that eugenol might be involved in periapical healing by impairing the functions of osteoblasts.

Enhancing effects of areca nut extracts on the production of interleukin-6 and interleukin-8 by peripheral blood mononuclear cells.

BACKGROUND: The habit of chewing areca quid (AQ) has been implicated in oral pathogenesis, including periodontal disease. Little is understood about the roles of AQ in the cytokine secretion by immune cells. The study examined the effects of areca nut, the major ingredient of AQ, on the production of interleukin (IL)-6 and IL-8 by peripheral blood mononuclear cells (PBMC), the immunocompetent cells. The possible role of oxidative stress of areca nut was also examined. METHODS: Extracts of ripe areca nut (rANE) and tender areca nut (tANE) were examined for their cytotoxic effects on human PBMC using the trypan blue exclusion test. The production of IL-6 and IL-8 by ANE-treated PBMC was analyzed using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. Effects of an antioxidant, pyrrolidine dithiocarbamate (PDTC), on ANE-induced cytokine secretion were also studied. RESULTS: At the experimental conditions, 20 micro g/ml rANE decreased cell viability significantly, whereas no significant effect of tANE (< or =80 micro g/ml) was observed. Both rANE (< or =20 micro g/ml) and tANE (< or =160 micro g/ml) significantly increased the secretion of IL-6 and IL-8 by PBMC in a dose- and time-dependent manner. The altered mRNA expression of IL-6 by rANE and tANE was also observed. Moreover, the stimulating effects of rANE on cytokine expression in PBMC could be attenuated by PDTC, suggesting that the oxidative stress of rANE may play a role. CONCLUSIONS: Markedly enhancing effects of ANE on PBMC-released inflammatory cytokines might cause a sustained cytokine-rich inflammatory milieu in oral cavity of AQ chewers. These excessive cytokines from ANE-treated immune cells may impair periodontal health.