Ji-Eun Jung

Differential effect of oxidative stress on the apoptosis of early and late passage human diploid fibroblasts: implication of heat shock protein 60

Since an attenuated response to stress is a characteristic of senescence, a cellular senescence model was used to examine the mechanism of resistance against oxidative stress using human diploid fibroblasts (HDF). With increasing passage, the HDF showed increased production of reactive oxygen species (ROS). Late passage HDF were resistant to the lethal effects of oxidative stress, showing less cleavage of pro‐caspase‐3 and PARP than those of early ones. Since heat shock proteins (Hsps) are not only cytoprotective but also interfere with the apoptotic cascade, the expression patterns of Hsps during cellular senescence were next examined. Oxidative stress induced a decrease in the mitochondrial Hsp60 levels with a concomitant increase in the cytosolic Hsp60 levels in the early passage HDF, but not in late ones. To show that the resistance to oxidative stress is a specific effect of Hsp60, the levels of Hsp60 were knocked down by siRNA. As expected the Hsp60 knock‐down cells were more resistant to oxidative stress. These findings show that Hsp60 is a key player in the resistance mechanism against oxidative stress and aging. Copyright

Evaluation of Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity

OBJECTIVES The aim of this study was to evaluate Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity. METHODS After producing fluoride varnish-coated hydroxyapatite discs using Fluor Protector (FP), Bifluorid 12 (BIF), Cavity Shield (CASH), or Flor-Opal Varnish White (FO), S. mutans biofilms were formed on the discs. To assess S. mutans adhesion to the discs, 4-h-old biofilms were analysed. To investigate the change in biofilm accumulation during subsequent biofilm formation, the biomass, colony forming units (CFU), and water-insoluble extracellular polysaccharides (EP) of 46-, 70-, and 94-h-old biofilms were analysed. To investigate the change in acidogenicity, pH values of the culture medium were determined during the experimental period. The amount of fluoride in the culture medium was also determined during the experimental period. RESULTS BIF, CASH, and FO affected S. mutans adhesion (67-98% reduction) and subsequent biofilm accumulation in 46-, 70-, and 94-h-old biofilms. However, the reducing effect of the fluoride varnishes on the biomass, CFU count, water-insoluble EP amount, and acid production rate of the biofilms decreased as the biofilm age increased. These results may be related to the fluoride-release pattern of the fluoride varnishes. Of the fluoride varnishes tested, FO showed the highest reducing effect against the bacterial adhesion and subsequent biofilm accumulation. CONCLUSIONS Our findings suggest that if the results of these experiments are extrapolable to the in vivo situation, then reduced clinical benefit of using fluoride varnishes may occur with time. CLINICAL SIGNIFICANCE Fluoride varnish application can affect cariogenic biofilm formation but the anti-biofilm activity may be reduced with time.

Effect of 1-Minute Fluoride Treatment on Potential Virulence and Viability of a Cariogenic Biofilm.

Fluoride is a well-studied and widely used agent for the prevention of dental caries. Although dental caries is strongly related to cariogenic biofilms, the effect of brief fluoride treatment on the virulence properties of biofilms has not been well studied. This study evaluated the effect of a 1-min fluoride treatment on the virulence properties and viability of cariogenic biofilms, using a Streptococcus mutans biofilm model. For this study, 46-hour-old S. mutans biofilms were formed on saliva-coated hydroxyapatite discs and were treated with fluoride (0, 30, 300, 1,000, and 2,000 ppm F-) for 1 min. Viability and changes in acidogenicity, aciduricity and extracellular polysaccharide (EPS) formation of the biofilms were analyzed using biochemical and microbiological methods (pH drop, H+ permeability, acid killing, and bacterial colony-forming unit assays). Laser scanning confocal fluorescence microscopy study was also performed. After the 1-min fluoride treatment, acidogenicity, aciduricity, and EPS formation of 46-hour-old S. mutans biofilms were significantly reduced when treated with concentrations ≥300 ppm F-. The antivirulence activities of the 1-min fluoride treatment increased in a concentration-dependent pattern. However, the 1-min fluoride treatments did not affect viability, biovolume, and microcolony appearance of biofilm bacteria, even at high concentrations. These results suggest that the brief treatment with fluoride at concentrations ≥300 ppm F- is an effective measure for controlling cariogenic biofilms.

Functional Relationship between Sucrose and a Cariogenic Biofilm Formation.

Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Abstract Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.

Effects of combined oleic acid and fluoride at sub-MIC levels on EPS formation and viability of Streptococcus mutans UA159 biofilms.

Despite the widespread use of fluoride, dental caries, a biofilm-related disease, remains an important health problem. This study investigated whether oleic acid, a monounsaturated fatty acid, can enhance the effect of fluoride on extracellular polysaccharide (EPS) formation by Streptococcus mutans UA159 biofilms at sub-minimum inhibitory concentration levels, via microbiological and biochemical methods, confocal fluorescence microscopy, and real-time PCR. The combination of oleic acid with fluoride inhibited EPS formation more strongly than did fluoride or oleic acid alone. The superior inhibition of EPS formation was due to the combination of the inhibitory effects of oleic acid and fluoride against glucosyltransferases (GTFs) and GTF-related gene (gtfB, gtfC, and gtfD) expression, respectively. In addition, the combination of oleic acid with fluoride altered the bacterial biovolume of the biofilms without bactericidal activity. These results suggest that oleic acid may be useful for enhancing fluoride inhibition of EPS formation by S. mutans biofilms, without killing the bacterium.

Identification of linoleic acid, a main component of the n-hexane fraction from Dryopteris crassirhizoma, as an anti-Streptococcus mutans biofilm agent

Dryopteris crassirhizoma is a semi-evergreen plant. Previous studies have shown the potential of this plant as an agent for the control of cariogenic biofilms. In this study, the main antibacterial components of the plant were identified by correlating gas chromatography–mass spectrometry data with the antibacterial activity of chloroform and n-hexane fractions and then evaluating the activity of the most potent antibacterial component against Streptococcus mutans UA159 biofilms. The most potent antibacterial component was linoleic acid, a main component of the n-hexane fraction. Linoleic acid reduced viability in a dose dependent manner and reduced biofilm accumulation during initial and mature biofilm formation. Furthermore, when the biofilms were briefly treated with linoleic acid (10 min/treatment, a total of six times), the dry weight of the biofilms was significantly diminished. In addition, the anti-biofilm activity of the n-hexane fraction was similar to that of linoleic acid. These results suggest that the n-hexane fraction of D. crassirhizoma and linoleic acid may be useful for controlling cariogenic biofilms.

Terrein reduces age-related inflammation induced by oxidative stress through Nrf2/ERK1/2/HO-1 signalling in aged HDF cells.

This study investigated whether multiple bioactivity of terrein such as anti‐inflammatory and anti‐oxidant inhibits age‐related inflammation by promoting an antioxidant response in aged human diploid fibroblast (HDF) cells. HDF cells were cultured serially for in vitro replicative senescence. To create the ageing cell phenotype, intermediate stage (PD31) HDF cells were brought to stress‐induced premature senescence (SIPS) using hydrogen peroxide (H2O2). Terrein increased cell viability even with H2O2 stress and reduced inflammatory molecules such as intracellular adhesion molecule‐1 (ICAM‐1), cyclooxygenase‐2 (COX‐2), interleukin‐1beta (IL‐1β) and tumour necrosis factor‐alpha (TNF‐α). Terrein reduced also phospho‐extracellular kinase receptor1/2 (p‐EKR1/2) signalling in aged HDF cells. SIPS cells were attenuated for age‐related biological markers including reactive oxygen species (ROS), senescence associated beta‐galactosidase (SA β‐gal.) and the aforementioned inflammatory molecules. Terrein induced the induction of anti‐oxidant molecules, copper/zinc‐superoxide defence (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and heme oxygenase‐1 (HO‐1) in SIPS cells. Terrein also alleviated reactive oxygen species formation through the Nrf2/HO‐1/p‐ERK1/2 pathway in aged cells. The results indicate that terrein has an alleviative function of age‐related inflammation characterized as an anti‐oxidant. Terrein might be a useful nutraceutical compound for anti‐ageing. Copyright

Recovery of Acid Production in Streptococcus mutans Biofilms after Short-Term Fluoride Treatment

Fluoride is commonly used as an ingredient of topical oral hygiene measures. Despite the anti-acidogenic activities of fluoride against cariogenic biofilms, the recovery of the biofilms from fluoride damage is unclear. Herein, we investigated the recovery of acid production in Streptococcus mutans biofilms after short-term or during periodic 1-min fluoride treatments. For this study, 46-hour-old S. mutans biofilms were treated with fluoride (0-2,000 ppm F-) for 1-8 min and then incubated in saliva for 0-100 min. The 74-hour-old biofilms were also periodically treated with the fluoride concentration during biofilm formation (1 min/treatment). Changes in acidogenicity and viability were determined via pH drop and colony-forming unit assays, respectively. In this study, acid production after a 1-min fluoride treatment was recovered as saliva incubation time increased, which followed a linear pattern of concentration dependence (R = 0.99, R2 = 0.98). The recovery pattern was in a biphasic pattern, with an initial rapid rate followed by a second slow recovery. Furthermore, recovery from fluoride damage was retarded in a concentration-dependent manner as treatment time increased. In periodic 1-min fluoride treatments, acid production in the biofilms was not diminished during the non-fluoride treatment period; however, it was reduced in a concentration-dependent manner during the fluoride treatment period. The viability of the biofilm cells did not change, even at high fluoride concentrations. Collectively, our results suggest that brief fluoride treatment does not sustain anti-acidogenic activity against S. mutans in biofilms since the damage is recoverable with time.

Long-term anti-cariogenic biofilm activity of glass ionomers related to fluoride release

OBJECTIVES The aim of this study was to evaluate the difference between anti-cariogenic biofilm activities of glass ionomers (G-Is) during the initial and second fluoride release phases and to define relationships between the anti-biofilm activities and fluoride release. METHODS Fluoride release of three commercially available G-Is in a buffer was evaluated for 770 h, and then 70-h-old Streptococcus mutans UA159 biofilms were formed on the G-Is that had been immersed in the buffer for 0, 100, 200, or 700 h. The dry weight, bacterial cell number, water-insoluble extracellular polysaccharides (EPSs), and accumulated fluoride concentration of the 70-h-old biofilms and fluoride release and acid production rates during biofilm formation were determined. Relationships between the experimental variables and fluoride release rate were also evaluated using linear regression analysis. RESULTS In this study, fluoride release of the tested G-Is did not exhibit a biphasic pattern during biofilm formation. The release was sustained or did not rapidly decrease even over long immersion periods and was strongly correlated with an increase in accumulated fluoride concentration of the biofilms (R=0.99, R(2)=0.98) and reductions in dry weight, water-insoluble EPSs, and acid production rate of the biofilms (R=-0.99 to -0.96, R(2)=0.92-0.98). CONCLUSIONS This study suggests that G-Is can effectively affect acid production, EPS formation, and accumulation of cariogenic biofilms even during the second fluoride release phase, and that the anti-cariogenic biofilm activity is strongly correlated with fluoride release, which may be enhanced by acid production of cariogenic biofilms. CLINICAL SIGNIFICANCE G-Is can affect cariogenic biofilm formation even during the second fluoride release phase.