Melatonin Receptor Agonists as the "Perioceutics" Agents for Periodontal Disease through Modulation of Porphyromonas gingivalis Virulence and Inflammatory Response.

AIM: "Perioceutics" including antimicrobial therapy and host modulatory therapy has emerged as a vital adjunctive treatment of periodontal disease. Melatonin level was significantly reduced in patients with periodontal diseases suggesting melatonin could be applied as a potential "perioceutics" treatment of periodontal diseases. This study aims to investigate the effects of melatonin receptor agonists (melatonin and ramelteon) on Porphyromonas gingivalis virulence and Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation. METHODS: Effects of melatonin receptor agonists on Porphyromonas gingivalis planktonic cultures were determined by microplate dilution assays. Formation, reduction, and viability of Porphyromonas gingivalis biofilms were detected by crystal violet staining and MTT assays, respectively. Meanwhile, biofilms formation was also observed by confocal laser scanning microscopy (CLSM). The effects on gingipains and hemolytic activities of Porphyromonas gingivalis were evaluated using chromogenic peptides and sheep erythrocytes. The mRNA expression of virulence and iron/heme utilization was assessed using RT-PCR. In addition, cell viability of melatonin receptor agonists on human gingival fibroblasts (HGFs) was evaluated by MTT assays. After pretreatment of melatonin receptor agonists, HGFs were stimulated with Pg-LPS and then release of cytokines (IL-6 and lL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Melatonin and ramelteon did exhibit antimicrobial effects against planktonic culture. Importantly, they inhibited biofilm formation, reduced the established biofilms, and decreased biofilm viability of Porphyromonas gingivalis. Furthermore, they at sub-minimum inhibitory concentration (sub-MIC) concentrations markedly inhibited the proteinase activities of gingipains and hemolysis in a dose-dependent manner. They at sub-MIC concentrations significantly inhibited the mRNA expression of virulence factors (kgp, rgpA, rgpB, hagA, and ragA), while increasing the mRNA expression of ferritin (ftn) or hemolysin (hem). They did not show obvious cytotoxicity toward HGFs. They inhibited Pg-LPS-induced IL-6 and IL-8 secretion, which was reversed by luzindole, the melatonin receptor antagonist. CONCLUSION: Melatonin receptor agonists can inhibit planktonic and biofilm growth of Porphyromonas gingivalis by affecting the virulent properties, as well as Pg-LPS-induced inflammatory response. Our study provides new evidence that melatonin receptor agonists might be useful as novel "perioceutics" agents to prevent and treat Porphyromonas gingivalis-associated periodontal diseases.

[Exploring the stem cell surface markers expressed in human dental pulp stem cells].

PURPOSE: To explore the stem cell surface markers expressed in human dental pulp stem cells which were selected and isolated by magnetic beads. METHODS: Human dental pulp cells (hDPCs) were separated and cultured from dental pulp of healthy third molars for orthodontic purpose. HDPSCs were isolated from cultured hDPCs by magnetic-activated cell sorting's (MACS) indirect magnetic cell labeling and positive selection strategy with antibody STRO-1 in the 2nd generation. Then the stem cell surface markers (CD73, CD90, CD105, CD166 and STRO-1) were respectively detected in 3, 4, 5, 6, 7 and 8 generation of dental pulp stem cells. HDPSCs were induced to differentiation by adipogenic medium and osteogenic medium in the 3rd generation. Adipogenic differentiation was assessed by oil red O staining in day 21, and osteogenic differentiation was assessed by alizarin red staining in day 21. RESULTS: HDPSCs could differentiate into adipocyte and osteoblasts. Oil red O staining and alizarin red staining were positively expressed after induction of HDPSCs. STRO-1's expression was decreased with the increase of generation. The expressions of CD73, CD90, CD105 and CD166 were relatively stable. CONCLUSIONS: The expression of STRO-1 is declined with the increase of generation, and the expressions of CD73, CD90, CD105 and CD166 are relatively stable with the changes of generation. Supported by National Natural Science Foundation of China (81070826/81371143) and Shanghai Rising-Star Program (12QH1401400).

[The efficacy of cold-light tooth bleaching on the formation of bacteria biofilm on the enamel surfaces].

PURPOSE: To evaluate the efficacy of Beyond cold-light tooth bleaching on the formation of main cariogenic bacteria biofilm on enamel surfaces. METHODS: Twenty enamel discs with the size of 4 mm×4 mm×1 mm in size, were made. The enamel discs were divided into 4 groups randomly: cold-light bleaching group, bleaching gel group, cold-light group and control group. Five discs were in each group. Cold-light bleaching group was whitened 3 times with bleaching gel and cold-light, and 12 min per session. Bleaching gel was smeared on the surface of enamel in bleaching gel group for 3 times and 12 min per session. Enamel discs of cold-light group were treated with cold-light for 12 min and 3 sessions. Control group was treated without any processing. The 4 groups were incubated in mixed bacteria liquid, including Streptococcus mutans(SM), Actinomyces viscosus (Av) and Fusobacterium nucleatum (Fn), within the artificial oral cavity model. After 36 h, the samples were observed under confocal laser scanning microscopy(CLSM). The data was analyzed with SAS8.2 software package. RESULTS: The biofilms in 3 experimental groups were sparser than the control group under CLSM, and the thickness significantly decreased after treatment (P<0.05), while no significant difference was found among 3 experimental groups (P>0.05).Compared with the control group, the percentage of vital bacteria in biofilm of the experimental groups decreased significantly after treatment (P<0.001). CONCLUSIONS: Cold-light tooth bleaching can inhibit the formation of mixed bacteria biofilm, damage the structure of biofilm and reduce the number of vital bacteria. Supported by Research Fund of Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine (2013-06).

[Immortalization of human dental pulp stem cells caused by transferring hTERT gene].

PURPOSE: To establish an immortalized human dental pulp stem cell line used for basic and clinical research of oral science. METHODS: Human telomerase reverse transcriptase (hTERT) cDNA was transferred into human dental pulp stem cells (hDPSCs) by lentivirus. The resultant stable clones reproduced successively and the expression of hTERT was identified. RESULTS: The hTERT gene was transferred into human dental pulp stem cells successfully. The transformed cells expressed telomerase activity and divided vigorously. p35 had been obtained so far. CONCLUSIONS: The hDPSCs can be immortalized by transferring exogenous hTERT gene to constitute telomerase activity.

The impact of various time intervals on the supragingival plaque dynamic core microbiome.

OBJECTIVE: The aim of this study was to examine the influence of various time intervals on the composition of the supragingival plaque microbiome, especially the dynamic core microbiome, and to find a suitable observation interval for further studies on oral microbiota. METHODS AND MATERIALS: Eight qualified volunteers whose respective age ranges from 25 to 28 years participated in the present study. The supragingival plaque was collected from the buccogingival surface of the maxillary first molar at eight time slots with different intervals (day 0, 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, and 3 months). Bioinformatic analyses was performed based on 16S rDNA pyrosequencing (454 sequencing platform) targeting at the hypervariable V4-V5 region, in order to assess the diversity and variation of the supragingival plaque microbiome. RESULTS: A total of 359,565 qualified reads for 64 samples were generated for subsequent analyses, which represents 8,452 operational taxonomic units identified at 3% dissimilarity. The dynamic core microbiome detected in the current study included five phyla, 12 genera and 13 species. At the genus level, the relative abundance of bacterial communities under the "1 day," "1 month," and "3 months" intervals was clustered into sub-category. At the species level, the number of overlapping species remained stable between the "1 month" and "3 months" intervals, whereas the number of dynamic core species became stable within only 1 week. CONCLUSIONS: This study emphasized the impact of different time intervals (days, weeks and months) on the composition, commonality and diversity of the supragingival microbiome. The analyses found that for various types of studies, the time interval of a month is more suitable for observing the general composition of the supragingival microbiome, and that a week is better for observing the dynamic core microbiome.

[Complementation and function of methyl-metabolic pathway in Streptococcus mutans luxS null strain].

PURPOSE: To complement the activated methyl cycle (AMC) pathway at an AI-2 defect background in Streptococcus mutans (S. mutans) luxS null strain. METHODS: A sahH gene was amplified from Pseudomonas aeruginosa and introduced into the S. mutans luxS null strain to complement the methyl-metabolic disruption at an AI-2 defect background. Western blot, reverse-transcription PCR and AI-2 bioassay were performed to confirm the heterogenous expression of SahH in S. mutans luxS null strain. The data was statistically analyzed by SAS8.0 software package. RESULTS: LuxS and SahH were detected to express in Escherichia coli BL21 as well as their mRNA were confirmed to be successfully transcribed in S. mutans luxS null strain. AI-2 production was found in wide type S. mutans and its luxS-introduced luxS null strain but not found in the luxS null strain and its sahH and empty plasmid-introduced strains. CONCLUSIONS: A new S. mutans derivative with the AMC pathway complements while the AI-2 defect is constructed.

[Effect of different stress conditions on growth and biofilm formation capability of Enterococcus faecalis].

OBJECTIVE: To study the changes of growth and biofilm formation capability of Enterococcus faecalis (Ef) in different stress conditions. METHODS: The changes of growth of Ef in stress conditions were observed by measuring the A600 value with ultraviolet spectrophotometer. Ef was incubated on glass slide in stress conditions, biofilm formation capability of cells was investigated by colony-forming unit (CFU) counting of the culturable bacteria and fluorescence confocal laser scanning microscopy. RESULTS: Ef couldn't growth under the conditions of 2%, 5%NaClO, pH = 11 and 12, the A600 value was unchanged in 96 hours. But the growth curve changed at different levels in other stress conditions: under 1%NaClO, the A600 value peaked at 1.461 at 16 hour (the peaked level was 1.238 at 6 hours in control group) ; under 0,0.05%,0.15% glucose, it peaked at 0.645,0.890, 1.173, respectively, at 6 hour (it was maximized to 1.195 at 6 hours in control group); the A600 value peaked at 1.704 at 6 hours at pH = 9 and 1.225 at 10 hours at pH = 10 (the peak level was 1.732 at 6 hours at pH = 7) . Biofilm assay showed that Ef were able to form biofilm in these stress conditions except 5%NaClO and pH = 12. CONCLUSIONS: Ef could growth and form biofilms in energy starvation, low concentrations of sodium hypochlorite and weak alkaline stress.

[Effect of compound Chinese traditional medicine on periapical periodontitis in dogs and cytotoxic assay].

PURPOSE: To assess the efficacy of compound Chinese traditional medicine(CTM), which is composed of gallic acid, magnolol and polysaccharide of Bletilla, against apical periodontitis in dogs and cytotoxic assay. METHODS: A animal model of apical periodontitis was built, CTM was then used to disinfect the root canal. The effect of the restoration of periapical bone in dogs was investigated after regular root canal filling. SAS6.12 software package was used for statistical analysis, and MTT was used to test cell toxicity of CTM. RESULTS: CTM can cure inflammation effectively, and CTM had no cytotoxic effect on periodontal ligament cells at 5-week. CONCLUSIONS: The compound Chinese traditional medicine may be an effective disinfecting drug for root canal disinfection.

[Observation of genetic diversity in dental plaque of elder people with root caries].

PURPOSE: Bacterial community in dental plaque of elder people was analyzed to learn about the microhabitat composition and diversity. METHODS: Dental plaque samples were collected from 25 elders. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to evaluate the microbial diversity by displaying PCR-generated 16SrDNA fragments that migrate at different distances, reflecting the different sequence of fragment. SPSS12.0 software was used to analyze the variance of genotypes between different groups of bacteria. RESULTS: Genotypes of bacteria in dental plaques in the root caries group was significantly more than the other two groups. Crown caries group and caries-free group had no significant difference. CONCLUSIONS: The genetic diversity of the dental plaque microflora in the root caries group is significantly higher than coronal caries group and caries-free group.

[Influence of Streptococcus mutans luxS gene on polysaccharide matrix metabolism].

PURPOSE: To investigate the effect of Streptococcus mutans luxS gene on polysaccharide matrix metabolism. METHODS: Based on the immobilization of magnetic beads by adherent cells,an assay of biofilm quantitative analysis was developed for the kinetic quantification of biofilm formation. S.mutans luxS gene mutant strain and wild-type strain were compared for their ability of utilizing exogenous carbohydrate to form extracellular polysaccharide matrix. SPSS 10.0 software package was used for statistical analysis. Dunnet t two-side test of one factor analysis of variance was performed. RESULTS: Both luxS mutant strain and wild-type strain could use exogenous carbohydrate to form polysaccharide matrix.With 1% sucrose added ,both strains completed their biofilm formation within one hour.When adding 1% glucose,these strains also accelerated the formation of biofilm,and this was more significant in the mutant strain. CONCLUSIONS: The luxS gene of S. mutans can regulate its extracellular polysaccharide matrix metabolism. Moreover, the regulation of this gene on biofilm formation is more probably via polysaccharide matrix pathway. Supported by National Natural Science Foundation of China(30872886), Research Fund of Science and Technology Commission of Shanghai Municipality(08DZ2271100),Shanghai Leading Academic Discipline Project(S30206) and Youth Phosphor Program of Science and Technology Commission of Shanghai Municipality (09QA1403700).

[Analysis of community composition in dental plaque of elder people with root caries].

OBJECTIVE: To analyze the community in dental plaque of elder people with root caries. METHODS: Total DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification. RESULTS: The dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114). CONCLUSIONS: There were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

[Effect of compound Chinese traditional medicine on infected root canal bacteria biofilm].

PURPOSE: To assess the efficacy of compound Chinese traditional medicine(CTM), which composed of gallic acid, magnolol and polysaccharide of Blettila striata, against the infected root canal bacterial biofilm. METHODS: Actinomyces viscosus (Av), Enterococcus faecalis (Ef), Fusobacterium nucleatum (Fn) were composed to form biofilm, then confocal laser scan microscope (CLSM) was used to observe and study the bacterial activity. SAS6.12 software package was used for statistical analysis. RESULTS: The biofilm thickness reduced after treatment by both CTM and ZnO (P>0.05),while there was a significant decrease of the percentage of vital bacterias after treatment by CTM (P<0.01). CONCLUSIONS: The compound Chinese traditional medicine is effective on biofilm control, so that it would be an effective disinfecting drug for root canal sealers. Supported by Research Fund of Bureau of Traditional Chinese Medicine of Shanghai Municipality (Grant No.2008L008A).

[The antibacterial activity of plasma sprayed silver-containing hydroxyapatite coatings].

PURPOSE: To study the antibacterial activity of plasma sprayed silver-containing hydroxyapative(HA) coatings. METHODS: Silver-containing HA coatings were prepared on titanium substrated by vacuum plasma spraying (VPS). The samples were divided into 4 groups according to weight percent of the antimicrobial: group HA0 (0%),group HA1 (1%),group HA3 (3%) and group HA5 (5%). The antimicrobial properties against Streptococcus mutans were assayed with the pellicle-sticking method. RESULTS: When the weight percent of the silver was >3%, the silver-containing HA coatings exhibited significant anti-bacterial effects against Streptococcus mutans. CONCLUSION: The silver-containing HA coating has good inhibitory effect on Streptococcus mutans.

[Study on the biofilm early formation of Streptococcus mutans luxS gene mutation].

OBJECTIVE: To investigate the effect of Streptococcus mutans luxS mutarotation on the early biofilm formation. METHODS: Based on the immobilization of magnetic beads by adherent cells, an assay of biofilm quantitative analysis was developed for the kinetic quantification of biofilm formation in this study. Streptococcus mutans luxS mutant strain was constructed and subject to this biofilm luxS mutant strain were compared. RESULTS: The delta luxS mutant started to form a biofilm from the 6th hour (delta BFI = 2.015), and the delta BFI of luxS mutant increased more quickly than that of the wild type strain, until reaching a complete immobilization of the beads after 10 hours (delta BFI = 7.025). The wild-type strain start to form a biofilm from the 10 th hour (delta BFI = 1.875) and the beads were completely immobilized between 12 and 14 hours. CONCLUSIONS: The luxS mutation can accelerate biofilm on a polystyrene surface during the mid-exponential growth phase. And a luxS-dependent signal may play an important role in the early biofilm formation of Streptococcus mutans.

[The disinfecting effect of ozone on four kinds of bacteria].

PURPOSE: To evaluate the disinfecting effect of ozone on 4 kinds of bacteria: Staphylococcus aureus, Streptococcus mutans, Staphylococcus epidermidis and Streptococcus pneumoniae. METHODS: The concentration of ozone that was transmitted by ozone generator at different time was determined by using iodine titrimetric method. According to the bactericidal assay of quantitative vehicle, the bacteria on the ozonized vehicles and unozonized vehicles was washed, 50 microl eluant was seeded on the TSA plates. The TSA plates were put into the anaerobiotic incubator (90% N(2),10% CO(2),37 degrees centigrade). After 24 to 48 hours, the CFU on the plate was counted.The data was analyzed by one-way ANOVA with SAS 6.12 software package. RESULTS: The sterilization effect depended on the ozone concentration and the treatment time. When the 4 kinds of bacteria were treated with 2.73 mg/L ozone for 45 minutes, there was no bacteria alive. CONCLUSIONS: Ozone has obvious disinfecting effect on the 4 kinds of bacteria and the effect is correlated with the concentration of ozone.

[Effects of garlic on oral bacteriostasis and root detoxification].

PURPOSE: To evaluate the pharmacologic effect of garlic on oral bacteriostasis and root detoxification. METHODS: Eight representative oral anaerobes (58 strains) including Streptococcus (9), Peptococcus (4), Lactobacillus (6), Actinomyces (3), Veillonella (10), Porphyromonas (12), Prevatella (3), Fusobacterium (11) to garlic juice were systematically studied by classical tube susceptibility test. The periodontally involved roots (12) were selected and the slices (6 mm x 3 mm x 1 mm) were prepared on the exposed root surface. Six slices were randomly treated by garlic juice for 5 minutes; the other six slices were treated by normal saline with same method as control. The attachment of cultured L-fibroblasts to the treated root surface was observed under phase contrast microscope and electron scanning microscope. The attachment cells were statistically analyzed using SPSS10.0 software package for Student's t test. RESULTS: The minimal inhibitory concentration of garlic juice on most oral anaerobes (87.93%) was 1:64-1:512. The attachment L-fibroblasts to the garlic treated group increased significantly vs. the control group (P<0.05). CONCLUSION: The study shows that garlic juice could effectively inhibit oral anaerobes and reestablish the root biological adaptation.

[Inhibition of extracts from 17 Chinese herbs on periodontal pathogenic microbes].

PURPOSE: To evaluate the efficiency of 17 Chinese herbs on periodontal pathogenic microbes. METHODS: 17 efficient substances from Chinese herbs were purchased from Chinese Drug Identification Bureau, including magnesium lithospermate B, magnolol, tetramethyl pyrazine, matrine, dycyrrhizin, gentiopicrin, aloperin, baicalin, oleanolic acid, ginkgo seed, total glucosides of paeony capsules, anisldehyde, archin, cablin patchouli, hydrochloric acid Berberine, forsythin, and kakonein. Antimicrobial sensitivity tests of broth microdilution methods on 96-microwell plate were carried out for identification of the antimicrobial activity of extracts against six species of microorganisms: Actinobacillus actinomycete mitans(Aa) Y4, Actinomycetes viscosus(Av) 19246, Porphyromonas gingivalis(Pg) 33277, Fusobacterium necrophorum(Fn) 25286, Actinomyces naeslundii(An) wvl 45 and Prevotella nigrescens(Pn). RESULTS: It was found that magnesium lithospermate B and magnolol showed the most efficient inhibition on microorganism of Pn and Fn, with the MIC being 0.053 and 0.313 mg/ml for Pn and Fn, respectively. Tetramethyl pyrazine, matrine, dycyrrhizin, gentiopicrin, aloperin, baicalin, and oleanolic acid had better inhibition than total glucosides of paeony capsules, anisldehyde, archin, cablin patchouli, hydrochloric acid berberine, forsythin, and kakonein. CONCLUSION: The Chinese herbs, magnesium lithospermate B and magnolol are efficient agents for inhibition against periodontal pathogenic microbes.

[A comparative study of root smear layer removal using different pH EDTA salts].

OBJECTIVE: To evaluate the effect of different pH EDTA salts on removing root canal smear layers. METHODS: Sixty human teeth with single root were instrumented using step-back technique, then were irrigated with several irrigating solutions including A: 0.9% saline; B: 5.25% NaOCl +3% H2O2; C: 15% EDTA (pH = 6.5); D: 15% EDTA (pH = 13); E: 15% EDTA (pH = 6.5) 25% NaOCl +3% H2O2; F: 15% EDTA (pH = 13) 25% NaOCl +3% H2O2. After the teeth were split, the root canal walls were examined with scanning electron microscopy at the coronal, middle and apical thirds for smear layer removal. RESULTS: Except A and B group, all the groups were effective on removing smear layer at the coronal, middle thirds of the root canal, group C had a stronger effect to remove smear layer than group D (P < 0.05), group E was the most effective among these groups. However, these groups were all ineffective on removing smear layer at the apical thirds of root canal. CONCLUSIONS: 15% EDTA (pH = 6.5) 25% NaOCl +3% H2O2 was the most effective irrigation on removing smear layer.

[Comparison of effect on S.mutans producing acid ability between organic weak acid and fluoride].

OBJECTIVE: To figure out the effects of organic weak acids, benzoate, citric acid and malic acid on the main cariogenic bacteria S.mutans Ingbritt. To evaluate the possibility of above weak acids as anti-caries reagents. METHODS: S.mutans Ingbritt was inoculated into tryptic soy broth containing different concentrations of fluoride, benzoate, citric acid and malic acid, grew in anaerobic conditions at 37 degrees centigrade for 48h. The final pH values of suspension were measured. RESULTS: Fluoride had best inhibitory effect on S.mutans Ingbritt in low concentrations, whereas other 3 weak acids, benzoate, citric acid and malic acid could inhibit S.mutans Ingbritt in a higher concentrations. The order of effectiveness was fluoride>malic acid>benzoate>citric acid. CONCLUSION: Fluoride has the best inhibitory effect on S.mutans producing acid ability. Though weak acids benzoate, malic acid and citric acid in high concentration had different inhibitory effects on S.mutans Ingbritt, however high concentration of organic acids can make enamel demineralization.