Long-term changes in the subgingival microbiota in patients with stage III-IV periodontitis treated by mechanical therapy and adjunctive systemic antibiotics: A secondary analysis of a randomized controlled trial.

AIM: To explore whether adjunctive antibiotics can relevantly influence long-term microbiota changes in stage III-IV periodontitis patients. MATERIALS AND METHODS: This is a secondary analysis of a randomized clinical trial on periodontal therapy with adjunctive 500 mg amoxicillin and 400 mg metronidazole or placebo thrice daily for 7 days. Subgingival plaque samples were taken before and 2, 8, 14 and 26 months after mechanical therapy. The V4-hypervariable region of the 16S rRNA gene was sequenced with Illumina MiSeq 250 base pair paired-end reads. Changes at the ribosomal sequence variant (RSV) level, diversity and subgingival-microbial dysbiosis index (SMDI) were explored with a negative binomial regression model and non-parametric tests. RESULTS: Overall, 50.2% of all raw reads summed up to 72 RSVs (3.0%) that were generated from 163 stage III-IV periodontitis patients. Of those, 16 RSVs, including Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans, changed significantly over 26 months because of adjunctive systemic antibiotics. SMDI decreased significantly more in the antibiotic group at all timepoints, whereas the 2-month differences in alpha and beta diversity between groups were not significant at 8 and 14 months, respectively. CONCLUSIONS: Mechanical periodontal therapy with adjunctive antibiotics induced a relevant and long-term sustainable change towards an oral microbiome more associated with oral health.

Effect of interleukin-17 on periodontitis development: An instrumental variable analysis.

BACKGROUND: Circulating levels of interleukin-17 (IL-17) are associated with the presence and severity of periodontitis. However, whether IL-17 is causal for disease development is unknown. We investigated the effect of genetically proxied IL-17 on periodontitis using instrumental variable analysis. METHODS: We identified 12 genetic variants from genome-wide association study (GWAS) of 7760 European descent individuals, used these variants as instrumental variables for IL-17, and linked them to a GWAS of 17,353 clinical periodontitis cases and 28,210 European controls. Generalized weighted least squares analysis accounted for linkage disequilibrium of variants. RESULTS: We found an inverse association of genetically proxied IL-17 and periodontitis (odds ratio, 0.84; 95% confidence interval: 0.75-0.94; p = 0.003), which was corroborated after sensitivity analysis for horizontal pleiotropy. CONCLUSION: The findings suggest that IL-17 protects against initial periodontitis.

Nitrate-rich diet alters the composition of the oral microbiota in periodontal recall patients.

BACKGROUND: This follow-up study evaluated microbiome changes in periodontal recall patients after consuming a nitrate-rich diet that led to a marked decrease of gingival inflammation. METHODS: Subgingival microbial samples of 37 patients suffering from gingival inflammation with reduced periodontium were taken before professional mechanical plaque removal (baseline) and subsequently after 2 weeks of regularly consuming a lettuce juice beverage (day 14) containing a daily dosage of 200 mg of nitrate (test group, n = 18) or being void of nitrate (placebo group, n = 19). Three hundred base pairs paired-end sequencing of the V3-V4 hypervariable region of the 16S rDNA was performed. RESULTS: At baseline, there were no significant differences about the bacterial diversity parameters between the groups (Mann-Whitney U test). After intervention in the test group, Rothia and Neisseria, including species reducing nitrate, increased significantly (negative binomial regression model). Alpha diversity decreased significantly from 115.69 ± 24.30 to 96.42 ± 24.82 aRSVs/sample (P = 0.04, Wilcoxon signed-rank test), accompanied by a significant change in beta diversity (P < 0.001, PERMANOVA). In the control group, however, no genus changed significantly, and alpha-, as well as beta-diversity did not change significantly. CONCLUSIONS: The decrease of gingival inflammation in periodontal recall patients induced by a nitrate-rich diet is accompanied by significant compositional changes within the subgingival microbiome.

A systematic review on bacterial community changes after periodontal therapy with and without systemic antibiotics: An analysis with a wider lens.

BACKGROUND: This systematic review aimed to provide a comprehensive view on microbial community shifts after periodontal therapy with and without systemic antibiotics, conducted in randomized controlled trials (RCTs). METHODS: Search functions in PubMed, Scopus, the Web of Knowledge, and the Cochrane Oral Health Library databases were used to locate studies published up to December 2018 that reported at least two bacteria before and after periodontal therapy. Gray literature and manual searching were done. Information about reported bacteria in those studies were extracted, and a descriptive microbial community analysis was conducted to observe trends and influencing factors on microbial dynamics. Methodological aspects were examined, including the bacterial detection method, heterogeneity of procedures, and risk of bias (RoB) of the studies. RESULTS: The 30 included studies reported 130 different bacterial genera. Four different detection methods were reported: cultivation, polymerase chain reaction, DNA-DNA-checkerboard hybridization, and 16S rDNA amplicon sequencing. No general compositional change between the antibiotic and placebo groups could be found after therapy on the community level. Fifty-five bacteria were reported in two or more studies. Of those, 24 genera decreased and 13 increased more frequently after antibiotic use. Great heterogeneity between procedures and variability in RoB were found among the studies. CONCLUSIONS: Microbial shifts occurred regardless of the use of antibiotics. Antibiotic therapy seems to induce more changes in single bacteria. The heterogeneity in methods and reporting of the included studies preclude clinical recommendations on the use or not of adjunctive antibiotics. The present results may guide further research on the topic.

Significant Short-Term Shifts in the Microbiomes of Smokers With Periodontitis After Periodontal Therapy With Amoxicillin & Metronidazole as Revealed by 16S rDNA Amplicon Next Generation Sequencing.

The aim of this follow-up study was, to compare the effects of mechanical periodontal therapy with or without adjunctive amoxicillin and metronidazole on the subgingival microbiome of smokers with periodontitis using 16S rDNA amplicon next generation sequencing. Fifty-four periodontitis patients that smoke received either non-surgical periodontal therapy with adjunctive amoxicillin and metronidazole (n = 27) or with placebos (n = 27). Subgingival plaque samples were taken before and two months after therapy. Bacterial genomic DNA was isolated and the V4 hypervariable region of the bacterial 16S rRNA genes was amplified. Up to 96 libraries were normalized and pooled for Illumina MiSeq paired-end sequencing with almost fully overlapping 250 base pairs reads. Exact ribosomal sequence variants (RSVs) were inferred with DADA2. Microbial diversity and changes on the genus and RSV level were analyzed with non-parametric tests and a negative binomial regression model, respectively. Before therapy, the demographic, clinical, and microbial parameters were not significantly different between the placebo and antibiotic groups. Two months after the therapy, clinical parameters improved and there was a significantly increased dissimilarity of microbiomes between the two groups. In the antibiotic group, there was a significant reduction of genera classified as Porphyromonas, Tannerella, and Treponema, and 22 other genera also decreased significantly, while Selenomonas, Capnocytophaga, Actinomycetes, and five other genera significantly increased. In the placebo group, however, there was not a significant decrease in periodontal pathogens after therapy and only five other genera decreased, while Veillonella and nine other genera increased. We conclude that in periodontitis patients who smoke, microbial shifts occurred two months after periodontal therapy with either antibiotics or placebo, but genera including periodontal pathogens decreased significantly only with adjunctive antibiotics.

[Design and quality control of the oral health status examination in the German National Cohort (GNC)]. / Design und Qualitätskontrolle der zahnmedizinischen Untersuchung in der NAKO Gesundheitsstudie.

BACKGROUND: Caries and periodontitis are highly prevalent worldwide. Because detailed data on these oral diseases were collected within the framework of the German National Cohort (GNC), associations between oral and systemic diseases and conditions can be investigated. OBJECTIVES: The study protocol for the oral examination was designed to ensure a comprehensive collection of dental findings by trained non-dental staff within a limited examination time. At the mid-term of the GNC baseline examination, a first quality evaluation was performed to check the plausibility of results and to propose measures to improve the data quality. MATERIALS AND METHODS: A dental interview, saliva sampling and oral diagnostics were conducted. As part of the level­1 examination, the number of teeth and prostheses were recorded. As part of the level­2 examination, detailed periodontal, cariological and functional aspects were examined. All examinations were conducted by trained non-dental personnel. Parameters were checked for plausibility and variable distributions were descriptively analysed. RESULTS: Analyses included data of 57,967 interview participants, 56,913 level­1 participants and 6295 level­2 participants. Percentages of missing values for individual clinical parameters assessed in level 1 and level 2 ranged between 0.02 and 3.9%. Results showed a plausible distribution of the data; rarely, implausible values were observed, e.g. for measurements of horizontal and vertical overbite (overjet and overbite). Intra-class correlation coefficients indicated differences in individual parameters between regional clusters, study centres and across different examiners. CONCLUSIONS: The results confirm the feasibility of the study protocol by non-dental personnel and its successful integration into the GNC's overall assessment program. However, rigorous dental support of the study centres is required for quality management.

Periodontal Health and Use of Oral Health Services: A Comparison of Germans and Two Migrant Groups.

A cross-sectional study was performed with 251 individuals, consisting of 127 Germans, 68 migrants from Turkey, and 56 resettlers (migrants from the former Soviet Union with German ancestors) to compare periodontal health status, with a special focus on associations with lifestyle and anthropometric factors, and use of dental health services. Maximal pocket depth was used as a clinical surrogate marker for periodontitis. Other variables were obtained by questionnaires administered by a Turkish or Russian interpreter. The age- and sex-adjusted prevalence of periodontitis was significantly higher in Turks (odds ratio (OR) 2.84, 95% CI = 1.53-5.26) and slightly higher in resettlers (OR = 1.33, 95% CI = 0.71-2.49). These differences are partly explained by a differential distribution of known risk factors for periodontitis. A full model showed a higher prevalence of maximal pocket depth above 5 mm in Turks (OR = 1.97, 95% CI = 0.99-3.92). Use of oral health services was significantly lower in the two migrant groups. Individuals who reported regular visits to a dentist had significantly less periodontitis, independent of migrant status. A reasonable conclusion is that, since oral health causes major chronic diseases and has a major effect on total health system expenditures, public health efforts both generally and specifically focused on migrant groups are warranted.

Methodological evaluation of reviews that support recommendations from three consensus workshops in periodontology.

OBJECTIVE: This study aimed to evaluate comprehensiveness and reproducibility of reviews that support consensus guidelines in periodontology. METHODS: We included the reviews that support consensus guidelines from three workshops in periodontology, which were overseen by likely the two most important organisations in the field: the European Federation of Periodontology and the American Academy of Periodontology. We independently evaluated the comprehensiveness of literature searches by determining whether authors had searched reference lists, journals, registries and grey literature and whether the searches were limited to only one or a few languages. We evaluated whether review authors reported the eligibility criteria, the search strategies, and the list of included/excluded articles. We tested whether the search and selection of articles in one major database was reproducible. RESULTS: Twenty-nine reviews were evaluated. Two (7%) reviews reported grey literature searches, and more than two-thirds of the reviews did not report hand-searching. Almost half of the reviews did not report whether there was language restriction for the literature searches. Two-thirds of the reviews reported the use of keywords only (without Boolean operators). One-fourth of the reviews reported the presence of a list of excluded articles after the full-text assessment. None of the reviews reported a detailed list of excluded articles after screening of titles/abstracts. None of the reviews reported enough information to allow reproduction of the findings of the PubMed search. CONCLUSIONS: There is room to improve the reporting of the methodologies that are used in reviews that support periodontology consensus guidelines, although heterogeneity in reporting was found across all the reviews.

No differences in microbiome changes between anti-adhesive and antibacterial ingredients in toothpastes during periodontal therapy.

AIM: This subgroup analysis of a 12-week randomized, double-blind, and two-center trial aimed to evaluate whether two different toothpaste formulations can differentially modulate the dental microbiome. MATERIAL AND METHODS: Forty one mild to moderate periodontitis patients used as an adjunct to periodontal treatment either a toothpaste with anti-adhesive zinc-substituted carbonated hydroxyapatite (HA) or with antimicrobial and anti-adhesive amine fluoride/stannous fluoride (AmF/SnF2 ) during a 12-week period. Plaque samples from buccal/lingual, interproximal, and subgingival sites were taken at baseline, 4 weeks after oral hygiene phase, and 8 weeks after periodontal therapy. Samples were analyzed with paired-end Illumina Miseq 16S rDNA sequencing. The differences and changes on community level (alpha and beta diversity) and on the level of single agglomerated ribosomal sequence variants (aRSV) were calculated with analysis of covariance (ANCOVA) and likelihood ratio test (LRT). RESULTS: Interproximal and subgingival sites harbored predominately Fusobacterium and Prevotella species associated with periodontitis, whereas buccal/lingual sites harbored mainly Streptococcus and Veillonella species associated with periodontal health. Alpha and beta diversity did not change noticeably differently between both toothpaste groups (P > 0.05, ANCOVA). Furthermore, none of the aRSVs showed a noticeably different change between the tested toothpastes during periodontal therapy (Padj . > 0.05, LRT). CONCLUSION: The use of a toothpaste containing anti-adhesive HA did not induce statistically noticeably different changes on microbial composition compared to an antimicrobial and anti-adhesive AmF/SnF2 formulation.

Do we treat our patients or rather periodontal microbes with adjunctive antibiotics in periodontal therapy? A 16S rDNA microbial community analysis.

Empiric antibiotics are often used in combination with mechanical debridement to treat patients suffering from periodontitis and to eliminate disease-associated pathogens. Until now, only a few next generation sequencing 16S rDNA amplicon based publications with rather small sample sizes studied the effect of those interventions on the subgingival microbiome. Therefore, we studied subgingival samples of 89 patients with chronic periodontitis (solely non-smokers) before and two months after therapy. Forty-seven patients received mechanical periodontal therapy only, whereas 42 patients additionally received oral administered amoxicillin plus metronidazole (500 and 400 mg, respectively; 3x/day for 7 days). Samples were sequenced with Illumina MiSeq 300 base pairs paired end technology (V3 and V4 hypervariable regions of the 16S rDNA). Inter-group differences before and after therapy of clinical variables (percentage of sites with pocket depth ≥ 5mm, percentage of sites with bleeding on probing) and microbiome variables (diversity, richness, evenness, and dissimilarity) were calculated, a principal coordinate analysis (PCoA) was conducted, and differential abundance of agglomerated ribosomal sequence variants (aRSVs) classified on genus level was calculated using a negative binomial regression model. We found statistically noticeable decreased richness, and increased dissimilarity in the antibiotic, but not in the placebo group after therapy. The PCoA revealed a clear compositional separation of microbiomes after therapy in the antibiotic group, which could not be seen in the group receiving mechanical therapy only. This difference was even more pronounced on aRSV level. Here, adjunctive antibiotics were able to induce a microbiome shift by statistically noticeably reducing aRSVs belonging to genera containing disease-associated species, e.g., Porphyromonas, Tannerella, Treponema, and Aggregatibacter, and by noticeably increasing genera containing health-associated species. Mechanical therapy alone did not statistically noticeably affect any disease-associated taxa. Despite the difference in microbiome modulation both therapies improved the tested clinical parameters after two months. These results cast doubt on the relevance of the elimination and/or reduction of disease-associated taxa as a main goal of periodontal therapy.

Hyaluronan-mediated mononuclear leukocyte binding to gingival fibroblasts.

OBJECTIVES: Binding of mononuclear leukocytes to hyaluronan cable structures is a well-known pathomechanism in several chronic inflammatory diseases, but has not yet described for chronic oral inflammations. The aim of this study was to evaluate if and how binding of mononuclear leukocytes to pathologic hyaluronan cable structures can be induced in human gingival fibroblasts. MATERIAL AND METHODS: Experiments were performed with human gingival fibroblasts and peripheral blood mononuclear cells (PBMCs) from three healthy blood donors. Gingival fibroblasts were stimulated with (1) tunicamycin, (2) polyinosinic/polycytidylic acid (Poly:IC), and (3) lipopolysaccharides (LPS) to simulate (1) ER stress and (2) viral and (3) bacterial infections, respectively. Fibroblasts were then co-incubated with PBMCs, and the number of bound and fluorescently labeled PBMCs was assessed using a fluorescence reader and microscopy. For data analysis, a linear mixed model was used. RESULTS: Hyaluronan-mediated binding of PBMCs to gingival fibroblasts was increased by tunicamycin and Poly(I:C) but not by LPS. Hyaluronidase treatment and co-incubation with hyaluronan transport inhibitors reduced this binding. CONCLUSIONS: Results suggest that hyaluronan-mediated binding of blood cells might play a role in oral inflammations. A potential superior role of viruses needs to be confirmed in further clinical studies. CLINICAL RELEVANCE: The linkage between pathological hyaluronan matrices and oral infections opens up potential applications of hyaluronan transport inhibitors in the treatment of chronic oral inflammations.

Clustering of Subgingival Microbiota Reveals Microbial Disease Ecotypes Associated with Clinical Stages of Periodontitis in a Cross-Sectional Study.

Periodontitis is characterized by chronic inflammation associated with alteration of the oral microbiota. In contrast to previous microbiome studies focusing a priori on comparison between extreme phenotypes, our study analyzed a random sample of 85 people. The aim of this study was to link microbial differences to disease's prevalence and severity. Using next generation sequencing of 16S rRNA amplicons and cluster analysis, we observed that the population can be divided into two major ecotypes: One mainly contained periodontal healthy/mild periodontitis individuals whereas the second ecotype showed a heterogeneous microbial distribution and clustered into three distinct sub-ecotypes. Those sub-ecotypes differed with respect to the frequency of diseased patients and displayed a gradual change in distinct subgingival microbiota that goes along with clinical disease symptoms. In ecotype 2, the subgroup with no clinical signs of disease was linked to an increase of F. nucleatum vincentii but also several other species, while only in "end-stage" dysbiosis classical red complex bacteria gained overweight. Therefore, the microbial disease ecotypes observed in our population can lead to an establishment of an early microbial risk profile for clinically healthy patients.

Prevalence of TMD symptoms in Turkish migrants and re-settlers from the former Soviet Union in comparison to a German group.

OBJECTIVES: The objective of this study was to investigate the prevalence of temporomandibular disorder (TMD) symptoms among Turks and re-settlers with German origin from Russia and to compare those findings with a German group from the same area. METHOD: Sixty-nine Turkish migrants, 50 re-settlers, and 96 Germans were clinically examined according to a short version of the Research Diagnostic Criteria (RDC/TMD) protocol. The subjects participated in a feasibility study of the German National Cohort and were recruited from the study center Heidelberg/Mannheim of the cluster Baden-Württemberg/Saarland. RESULTS: Significant differences emerged between the three ethnic groups for unassisted opening without pain, maximum unassisted opening, and overbite, with highest values for the German group. No significant differences were found for muscle pain on palpation or muscle and joint pain during opening. DISCUSSION: As the authors identified significant differences between the different ethnic groups for metric measurements, it might be beneficial to include questions concerning the ethnicity to the German version of the RDC/TMD for further research, to make the results more comparable.

Pocket depth and bleeding on probing and their associations with dental, lifestyle, socioeconomic and blood variables: a cross-sectional, multicenter feasibility study of the German National Cohort.

BACKGROUND: To investigate the periodontal disease status in a multi-center cross-sectional study in Germany. Associations of dental, socio-economic, blood and biomedical variables with periodontal outcome parameters were evaluated. METHODS: From 4 different centers N = 311 persons were included, drawn randomly from the registration offices. Maximal pocket depth (PD) was used as primary indicator for periodontitis. It was classified as: no/mild ≤3 mm, moderate 4-5 mm, severe ≥6 mm. Associations between socioeconomic (household income, education), lifestyle, and biomedical factors and PD or bleeding on probing (BOP) per site ("Yes"/"No") was analyzed with logistic regression analysis. RESULTS: Mean age of subjects was 46.4 (range 20-77) years. A significantly higher risk of deeper pockets for smokers (OR = 2.4, current vs. never smoker) or persons with higher BMI (OR = 1.6, BMI increase by 5) was found. Severity of periodontitis was significantly associated with caries lesions (p = 0.01), bridges (p < .0001), crowns (p < .0001), leukocytes (p = 0.04), HbA1c (p < .0001) and MCV (p = 0.04). PD was positively correlated with BOP. No significant associations with BOP were found in regression analysis. CONCLUSIONS: Earlier findings for BMI and smoking with severity of PD were confirmed. Dental variables might be influenced by potential confounding factors e.g. dental hygiene. For blood parameters interactions with unknown systemic diseases may exist.

Is frequency of tooth brushing a risk factor for periodontitis? A systematic review and meta-analysis.

OBJECTIVES: The epidemiology of periodontitis regarding oral-hygiene practices particularly the frequency of tooth brushing has been the subject of relatively few dedicated studies. This paper provides a systematic review of available relevant epidemiological studies and a meta-analysis of the effect of tooth brushing frequency on periodontitis. To review and to quantify the risk for periodontitis associated with frequency of tooth brushing. METHODS: Systematic literature search was conducted in nine online resources (PUBMED, ISI and 7 additional databases). Related and cross-referencing publications were reviewed. Papers published until end of March 2013 reporting associations between tooth brushing frequency and periodontitis were considered. A meta-analysis was performed to quantify this association. RESULTS: Fourteen studies were identified. The test of heterogeneity for cross-sectional studies was not significant (P = 0.31). A fixed-effects model yielded a significant overall odds ratio estimate of 1.41 (95%CI: 1.25-1.58, P < 0.0001) for infrequent compared to frequent tooth brushing. For all fourteen studies, there was a slight indication for heterogeneity (I² = 48%, P = 0.02) and the corresponding result with a random-effects model was 1.44 (95%CI: 1.21-1.71, P < 0.0001). CONCLUSIONS: There are relatively few studies evaluating the association between tooth brushing frequency and periodontitis. A clear effect was observed, indicating that infrequent tooth brushing was associated with severe forms of periodontal disease. Further epidemiological studies are needed to precisely estimate the effect of key risk factors for periodontitis and their interaction effects.

Compliance with infection control practices in an university hospital dental clinic.

AIM: Compliance with infection control practices is the key to quality care and excellence in dentistry. Infection control remains one of the most cost-beneficial interventions available. However, implementing control procedures requires full compliance of the whole dental team. The aim of our study was to measure the compliance in daily clinical practice. METHODS: The compliance with infection control practices in dentistry by dental health care personnel (DHCP) in a German university dental clinic was observed during clinical work. In addition, a survey was conducted to assess the individual knowledge about infection control procedures. Contamination of the workplace during invasive dental procedures was tested, as well. RESULTS: A total of 58 invasive dental treatments implying close contacts between HCWs and patients were scrutinized. All HCWs (100%) wore gloves during dental work, but in some cases (female dentists: 14.3%; dental assistants: 28.6%) gloves were neither changed nor hands were disinfected between different activities or patient contacts (female dentists: 68.6%; male dentists: 60.9%; dental assistants: 93%). Only 31.4% of female and 39.1% of male dentists carried out adequate hygienic hand disinfection after removing gloves. Male dentists wore significantly more often (100%) protective eyewear compared to 77.1% of female dentists (p<0.05). In addition, most of female dentists (62.9%) and dental assistants (80.7%) wore jewelry during dental procedures. CONCLUSION: Despite the knowledge of distinct hygiene procedures only a small percentage of dental staff performs hygiene practices according to recommended guidelines. Strict audit is clearly needed in the dental setting to ensure compliance with infection control guidelines to prevent transmission of pathogens. Our results provide insights for the development of a targeted education and training strategy to enhance compliance of dental staff especially of dental assistants with infection control procedures.

Periodontal pathogens and associated factors in aggressive periodontitis: results 5-17 years after active periodontal therapy.

OBJECTIVES: To assess the association between presence of periodontal pathogens and recurrence of disease in patients with aggressive periodontitis (AgP) after active periodontal therapy (APT) and further influencing factors. MATERIAL & METHODS: Microbiological samples were taken from 73 patients with AgP 5-17 years after APT at 292 sites (deepest site per quadrant). Real-time polymerase chain reactions were used to detect the periodontal pathogens Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. Uni- and multivariate analyses evaluated the associations between pathogens and recurrence of disease, smoking and adjunctive antibiotic therapy. RESULTS: At re-examination A. actinomycetemcomitans could be detected in six patients (8.2%), P. gingivalis in 24 (32.9%), T. forsythia in 31 (42.5%) and T. denticola in 35 (48.0%). Increased levels of T. forsythia and T. denticola at re-examination were significantly associated with recurrence of disease in multivariate analyses (OR: 12.72, p < 0.001; OR 5.55, p = 0.002 respectively). Furthermore, high counts of T. denticola were found in patients with increased percentage of sites with clinical attachment levels (CAL) ≥ 6 mm compared to those with low counts (13.8% versus 3.2%, p = 0.005). CONCLUSION: In patients with recurrence of disease T. forsythia and T. denticola were detected more frequently and in higher counts. Furthermore, T. denticola was found more frequently in patients with increased CAL.

Adsorption of glycosaminoglycans to the cell surface is responsible for cellular donnan effects.

In previous publications, we showed that extracellular glycosaminoglycans reduced the membrane potential, caused cell blebbing and swelling and decreased the intracellular pH independently of cell surface receptors. These phenomena were explained by Donnan effects. The effects were so large that they could not be attributed to glycosaminoglycans in solution. Therefore, we tested the hypothesis that glycosaminoglycans were concentrated on the cell membrane and analysed the mechanism of adsorption by fluorescent hyaluronan, chondroitin sulphate and heparin. The influence of the CD44 receptor was evaluated by comparing CD44 expressing human fibroblasts with CD44 deficient HEK cells. Higher amounts of glycosaminoglycans adsorbed to fibroblasts than to HEK cells. When the membrane potential was annihilated by substituting NaCl by KCl in the medium, adsorption was reduced and intracellular pH decrease was abolished. To eliminate other cellular interfering factors, potential-dependent adsorption was demonstrated for hyaluronan which adsorbed to inert gold foils in physiological salt concentrations at pH 7.2 and surface potentials up to 120 mV. From these results, we conclude that large cellular Donnan effects of glycosaminoglycans results from receptor mediated, hydrophobic and ionic adsorption to cell surfaces.

Hyaluronan export through plasma membranes depends on concurrent K+ efflux by K(ir) channels.

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K(+) channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K(+) channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.

Depolarization of the membrane potential by hyaluronan.

The membrane potential is mainly maintained by the K(+) concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high-molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration-dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high-molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high-molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high-molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism.