Background and Objective
Antimicrobial agents provide valuable adjunctive therapy for prevention and control of oral diseases. Limitations in their prolonged use have stimulated the search for new natural occurring agents with more specific activity and fewer adverse effects. Here we sought to determine the anti-bacterial properties of blackberry extract (BBE) in vitro against oral bacterial commensals and periodontopathogens.
Material and Methods
Effects of whole and fractionated BBE on the metabolism of 10 different oral bacteria were evaluated by colorimetric water-soluble tetrazolium-1 (WST-1) assay. Bactericidal effects of whole BBE against F. nucleatum were determined by quantitating colony forming units (CFUs). Cytotoxicity was determined in oral epithelial (OKF6) cells.
BBE at 350-1,400 μg/mL reduced the metabolic activity of P. gingivalis, F. nucleatum and S. mutans. The reduced metabolic activity observed for F. nucleatum corresponded to a reduction in CFUs following exposure to BBE for as little as 1 hour, indicative of its bactericidal properties. An anthocyanin-enriched fraction of BBE reduced the metabolic activity of F. nucleatum but not P. gingivalis or S. mutans, suggesting the contribution of species specific agents in the whole BBE. Oral epithelial cell viability was not reduced following ≤ 6 h exposures to whole BBE (2.24-1400 μg/mL).
BBE alters the metabolic activity of oral periodontopathogens while demonstrating minimal effect on commensals. The specific antibacterial properties of BBE shown in this study along with its anti-inflammatory and antiviral properties previously demonstrated make this natural extract a promising target as an adjunct for prevention and/or complementary therapy of periodontal infections.
Blackberry; oral bacteria; antibacterial effect; periodontitis; periodontopathogens; Fusobacterium nucleatum; topical antimicrobial
Background and Objectives
Bmp2-induced osteogenic differentiation has been shown to occur through the canonical Wnt/β-catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibited cell differentiation and promoted cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with Bmp2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway.
Materials and Methods
SVF4 cells were stimulated with Bmp2, and the following assays were carried out: 1) Wnt/β-catenin pathway activation assessed by western blot, β-catenin/TCF reporter assay, and gene expression of lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2, and 2) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp) by qPCR after Wnt3a treatment and knockdown of β-catenin.
Wnt3a induced β-catenin nuclear translocation and upregulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with Wnt3a suppressed Bmp2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that Bmp2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. Wnt3a down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared to untreated cells. In contrast, Bmp2 induction of Bsp transcripts occurred independent of Wnt/β-catenin signaling.
These data suggest that stabilization of β-catenin by Wnt-3a treatment inhibits Bmp2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although Bmp2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.
dental follicle cells; Wnt; cementoblast; maturation; BMP
To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from generalized aggressive periodontitis (GAgP) and periodontaly healthy (PH) subjects.
Material and Methods
GAgP (n=15) and PH (n=15) subjects were recruited and their clinical periodontal parameters were evaluated. Subgingival plaque samples were collected (9 samples/subject) and analyzed for the levels of 10 bacterial taxa, including cultivated and uncultivated/unrecognized microorganisms using the RNA-oligonucleotide quantification technique (ROQT). Differences in the levels of the test taxa between groups were sought using the Mann-Whitney test.
GAgP subjects showed significantly higher mean counts of Porphyromonas gingivalis, Selenomonas sputigena and Selenomonas oral clone CS002 (Human Oral Microbial Database (HOMD) Oral Taxon 131), while Actinomyces gerencseriae and Streptococcus sanguinis were found in higher mean counts in PH subjects (p<0.01). Selenomonas EW084 (HOMD OT 146) was only detected in the GAgP group. In the GAgP group, levels of P. gingivalis and S. sputigena were higher in sites with probing depth (PD) ≥5mm than in shallow sites (PD ≤3mm) (p<0.01). Furthermore, sites with PD≤3mm in GAgP subjects harbored higher levels of these two species than sites in PH subjects. There were positive correlations between PD and levels of P. gingivalis (r=0.77; p<0.01), S. sputigena (r=0.60; p<0.01) and Selenomonas sp. EW076 (OT 139) (r=042, p<0.05).
S. sputigena, Selenomonas sp. oral CS002 (OT 131) and Selenomonas sp. oral clone EW084 (OT 146) may be associated with the pathogenesis of GAgP, and their role in the onset and progression of this infection should be further investigated.
Selenomonas sputigena; molecular biology; 16S rRNA; generalized aggressive periodontitis; not-yet-cultivated species
The subgingival microbiota in Down syndrome and non-Down syndrome (non-DS) adults receiving periodic dental care was examined for 40 bacterial species with checkerboard DNA-DNA hybridization, and related to clinical periodontal attachment loss (AL).
A total of 44 Down syndrome, 66 non-DS mentally retarded, and 83 mentally normal adults were clinically evaluated, with subgingival specimens removed and pooled per subject from three interproximal sites on different teeth, and assessed for the presence and levels of 40 bacterial species using species-specific whole genomic DNA probes and checkerboard DNA-DNA hybridization. Significant group differences in species proportions averaged across subjects were evaluated using the Kruskal-Wallis test, and associations between subgingival species and mean subject AL within Down syndrome and non-DS subject groups were quantified with Pearson correlation and multiple linear regression analysis.
Down syndrome subjects exhibited greater AL than non-DS subjects (P = 0.05). Down syndrome subjects yielded most microbial species at levels similar to non-DS subjects, except for higher proportions of Selenomonas noxia, Propionibacterium acnes, Streptococcus gordonii, Streptococcus mitis, and Streptococcus oralis as compared to non-DS study subjects, higher Treponema socranskii than non-DS mentally retarded subjects, and higher Streptococcus constellatus relative to mentally normal subjects. Down syndrome adults classified with periodontitis revealed higher subgingival levels of T. socranskii than non-periodontitis Down syndrome subjects (P = 0.02). Higher subgingival proportions of S. constellatus, Fusobacterium nucleatum subsp. nucleatum, S. noxia and Prevotella nigrescens showed significant positive correlations (r = 0.35 to 0.42), and Actinomyces naeslundii II and Actinomyces odontolyticus negative correlations (r = −0.36 to −0.40), with increasing mean subject AL in Down syndrome adults.
Individuals with DS show higher levels of some subgingival bacterial species and specific associations between certain subgingival bacterial species and loss of periodontal attachment. These findings are consistent with the notion that certain subgingival bacteria may contribute to the increased level of periodontal disease seen in DS individuals, and raise the question as to the reason for increased colonization in DS.
subgingival microbiota; Down syndrome; periodontitis; periodontal
Background and objective
The field of salivary diagnostics lacks an accepted and validated biomarker of alveolar bone remodeling. To address this we examined levels of salivary biomolecules specifically associated with biological aspects of bone remodeling in subjects with chronic periodontitis in a case-control study.
Levels of macrophage inflammatory protein (MIP)-1α, osteoprotegerin (OPG), C-telopeptide pyridinoline cross-links of type I collagen (ICTP), and β-C-terminal type I collagen telopeptide (β-CTX) in unstimulated whole saliva of 80 subjects (40 subjects with moderate to severe chronic periodontitis and 40 gender- and age-matched healthy control subjects) were measured using enzyme immunosorbent assays. Saliva was collected before clinical examination that included probing depth (PD), clinical attachment loss (CAL), and bleeding on probing (BOP).
The mean level of MIP-1α in periodontitis subjects was 18-fold higher than in healthy subjects (p < 0.0001). Clinical periodontal indices significantly correlated with MIP-1α levels (p < 0.0001). MIP-1α, of the biomolecules examined, demonstrated the highest ability to discriminate between periodontal disease and health as determined by area under the curve (AUC = 0.94) and classification and regression tree analysis (sensitivity 94%, specificity 92.7%). OPG levels were elevated 1.6-fold (P = 0.055), whereas ICTP and β-CTX levels were below the level of detection in the majority of subjects.
These findings suggest that the chemokine MIP-1α may aid in identifying periodontitis. Future longitudinal studies are warranted to determine whether this biomarker can help to ascertain progression of bone loss in subjects with periodontal disease.
periodontal health, periodontal disease; saliva; biomarkers; bone remodeling; macrophage inflammatory protein (MIP)-1α; osteoprotegerin (OPG); C-telopeptide pyridinoline cross-links of type I collagen (ICTP), β-C-terminal type I collagen telopeptide (β-CTX)
Background and Objectives
Our previous study showed protease inhibitors were attenuated by periopathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize fewer protease inhibitors would be present in more advanced periodontal sites where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor (SLPI, ELAFIN, SCCA) levels in gingival crevicular fluid (GCF) and the number of P. gingivalis in subgingival plaque.
Materials and Methods
Plaque samples from subjects without (n=18) and with moderate to advanced periodontitis (n=41) were used to quantify P. gingivalis using real-time PCR. Protease inhibitor levels in the GCF of all the subjects were determined by ELISA.
P. gingivalis was detected in 68.3% of subjects with periodontitis, while 16.7% of patients without periodontitis had detectable level of P. gingivalis. Subjects with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p<0.001) compare to control subjects without periodontitis. SLPI was also reduced (p<0.05) in GCF of periodontal patients without detectable level of P. gingivalis. Periodontal patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations.
The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may further help in finding pharmacological targets directed against periodontal inflammation.
protease inhibitors; periodontitis; Porphyromonas gingivalis; gingival crevicular fluid
Background and Objective
Gingival crevicular fluid (GCF) has been of major interest for many decades as valuable body fluid that may serve as a source of biomarkers for both periodontal and systemic diseases. Because of its very small sample size, sub-μl level, identification of its protein composition by classical biochemical methods has been limited. The advent of highly sensitive mass spectrometric technology has permitted large-scale identification of protein components of many biological samples. This technology has been employed to identify protein composition of GCF from inflamed and periodontal sites. In this report we present a proteome dataset of GCF from healthy periodontium sites.
A combination of periopaper collection method with application of multidimensional protein separation and mass spectrometric (MS) technology led to a large-scale documentation of the proteome of GCF from healthy periodontium sites.
The approaches utilized have culminated in identification of 199 proteins in GCF of periodontally healthy sites. The current GCF proteome from healthy sites was compared and contrasted with those proteomes of GCF from inflamed and periodontal sites as well as serum. The cross-correlation of the GCF and plasma proteomes permitted dissociation of the 199 identified GCF proteins into, 105 proteins (57%) that can be identified in plasma and 94 proteins (43%) which are distinct and unique to GCF microenvironment. Such analysis also revealed distinctions in protein functional categories between serum proteins and those specific to GCF microenvironment.
Firstly, the data presented herein provide the proteome of GCF from periodontally healthy sites through establishment of innovative analytical approaches for effective analysis of GCF from periopapers both at the level of complete elusion and removal of abundant albumin which restricts identification of low abundant proteins. Secondly, it adds significantly to the knowledge of GCF composition and highlights new groups of proteins specific to GCF microenvironment.
Gingival crevicular fluid; mass spectrometry; proteomics; saliva; oral; biomarkers; diagnostics
To determine the order of bacterial species succession in re-developing supra and subgingival biofilms.
Supra and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from 7 teeth in randomly selected quadrants after 1, 2, 4 and 7 days of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. % DNA probe counts were averaged within subjects at each time point. Ecological succession was determined using a modified moving window analysis.
Succession in supragingival biofilms from periodontitis and health was similar. At 1 day, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1–4 days. At 4–7 days, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 days by Capnocytophaga sputigena and P. nigrescens. No significant increase in proportions of periodontal pathogens was observed in any of the clinical groups or locations.
There is a defined order in bacterial species succession in early supra and subgingival biofilm re-development after professional cleaning.
ecology; succession; oral bacteria; periodontal; periodontitis; biofilms; supragingival; subgingival
Background and Objective
Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT.
Material and Methods
Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed.
Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits.
Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.
Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; enzyme-linked immunosorbent assay (ELISA); immunoglobulin G; serotype
Whole genomic and randomly-cloned DNA probes for two fastidious periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus were labeled with digoxigenin and detected by a colorimetric method. The specificity and sensitivity of the whole genomic and cloned probes were compared. The cloned probes were highly specific compared to the whole genomic probes. A significant degree of cross-reactivity with Bacteroides species. Capnocytophaga sp. and Prevotella sp. was observed with the whole genomic probes. The cloned probes were less sensitive than the whole genomic probes and required at least 106 target cells or a minimum of 10 ng of target DNA to be detected during hybridization. Although a ten-fold increase in sensitivity was obtained with the whole genomic probes, cross-hybridization to closely related species limits their reliability in identifying target bacteria in subgingival plaque samples.
Porphyromonas gingivalis; Bacteroides forsythus; DNA probes; non-isotopic labeling
Adaptive properties of the bone-PDL-tooth complex have been identified by changing the magnitude of functional loads using small-scale animal models such as rodents. Reported adaptive responses as a result of lower loads due to softer diet include decreased muscle development, change in structure-function relationship of the cranium, narrowed PDL-space, changes in mineral level of the cortical bone and alveolar jaw bone, and glycosaminoglycans of the alveolar bone. However, the adaptive role of the dynamic bone-PDL-cementum complex due to prolonged reduced loads has not been fully explained to date, especially with regards to concurrent adaptations of bone, PDL and cementum. Hence, the temporal effect of reduced functional loads on physical characteristics such as morphology and mechanical properties, and mineral profiles of the bone-periodontal ligament (PDL)-cementum complex using a rat model was investigated.
Materials and Methods
Two groups of six-week-old male Sprague-Dawley rats were fed nutritionally identical food with a stiffness range of 127–158N/mm for hard pellet or 0.32–0.47N/mm for soft powder forms. Spatio-temporal adaptation of the bone-PDL-cementum complex was identified by mapping changes in: 1) PDL-collagen orientation and birefringence using polarized light microscopy, bone and cementum adaptation using histochemistry, and bone and cementum morphology using micro X-ray computed tomography, 2) mineral profiles of the PDL-cementum and PDL-bone interfaces by X-ray attenuation, and 3) microhardness of bone and cementum by microindentation of specimens at ages six, eight, twelve, and fifteen weeks.
Reduced functional loads over prolonged time resulted in 1) altered PDL orientation and decreased PDL collagen birefringence indicating decreased PDL turnover rate and decreased apical cementum resorption; 2) a gradual increase in X-ray attenuation, owing to mineral differences, at the PDL-bone and PDL-cementum interfaces without significant differences in the gradients for either group; 3) significantly (p<0.05) lower microhardness of alveolar bone (0.93±0.16 GPa) and secondary cementum (0.803±0.13 GPa) compared to the higher load group (1.10±0.17 GPa and 0.940±0.15 GPa respectively) at fifteen weeks indicating a temporal effect of loads on local mineralization of bone and cementum.
Based on the results from this study, the effect of reduced functional loads for a prolonged time could differentially affect morphology and mechanical properties, and mineral variations and of the local load-bearing sites in a bone-PDL-cementum complex. These observed local changes in turn could help explain the overall biomechanical function and adaptations of the tooth-bone joint. From a clinical translation perspective, our study provides an insight into modulation of load on the complex for improved tooth function during periodontal disease, and/or orthodontic and prosthodontic treatments.
functional loads; tissue interfaces; cementum; bone-tooth biomechanics; alveolar bone; periodontal ligament
Background and Objective
Diabetes predisposes to periodontal disease. However, the cellular and molecular mechanisms linking the two conditions are not clear. The impact of chronic hyperglycemia on leukocyte margination and macromolecule extravasation was determined in gingival vessels in vivo.
Materials and Methods
Gingival intravital microscopy was employed to measure extravasation of fluorescein isothiocyanate (FITC)–dextran in diabetic Akita and healthy wild-type (WT) mice. Rhodamine 6G and FITC–LY6G were injected for nonspecific and polymorphonuclear-specific leukocyte labeling, respectively. Surface expression of leukocyte adhesion molecules was determined with flow cytometry and western blotting.
Vascular permeability was significantly increased in Akita gingival vessels compared with WT [permeability index (PI): WT, 0.75 ± 0.05; Akita, 1.1 ± 0.03: p < 0.05). Wild-type gingival vessels reached comparable permeability 2 h after intragingival injection of tumor necrosis factor α (TNFα), used here as positive control (PI, 1.17 ± 0.16). The number of rolling leukocytes was significantly elevated in diabetic gingiva (WT, 25 ± 3.7 cells/min; Akita, 42 ± 8.5 cells/min; p < 0.03). Similar rolling cell counts were obtained in WT after intragingival injection of TNFα (10 ng TNFα, 47 ± 1.3 cells/min; 100 ng TNFα, 57.5 ± 5.85 cells/min). The number of leukocytes firmly attached to the endothelium was similar in WT and Akita mice. Leukocyte cell-surface expression of P-selectin glycoprotein ligand-1 and CD11a was increased in Akita mice, while L-selectin remained unchanged when compared with WT. Moreover, P-selectin expression in Akita gingival tissues was elevated compared with that of WT.
Chronic hyperglycemia induces a proinflammatory state in the gingival microcirculation characterized by increased vascular permeability, and leukocyte and endothelial cell activation. Leukocyte-induced microvascular damage, in turn, may contribute to periodontal tissue damage in diabetes.
diabetes; leukocyte; intravital microscopy; selectin; gingiva; microcirculation
Curcumin is a plant-derived dietary spice with various biological activities, including anti-tumoral and anti-inflammatory. Its therapeutic applications have been studied in a variety of conditions, including rheumatoid arthritis, colon cancer and depression; but no studies evaluated the effects of curcumin on periodontal disease in vivo.
Experimental periodontal disease was induced in rats by placing cotton ligatures around both lower first molars. Curcumin was given to the rats intragastrically daily in two doses (30 and 100 mg/Kg) during 15 days. Control animals received ligatures but only the corn oil vehicle by gavage and no treatment negative control animals were included. Bone resorption was assessed by microcomputer tomography and the inflammatory status was evaluated by stereometric analysis. RT-qPCR and ELISA were used to determine the expression of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and prostaglandin E2 (PGE2) synthase on the gingival tissues. Modulation of p38 mitogen-activated protein kinase (MAPK) and NK-kB activation was assessed by western blot.
Bone resorption was effectively induced in the experimental period, but it was not affected by either dose of curcumin. Curcumin effectively inhibited cytokine gene expression at mRNA and protein levels and dose-dependently inhibited activation of NF-kB in the gingival tissues. p38 MAPK activation was not inhibited by curcumin. Curcumin-treated animals also presented a marked reduction on the inflammatory cell infiltrate and increased collagen content and fibroblastic cell numbers.
Curcumin did not prevent alveolar bone resorption, but its potent anti-inflammatory effect suggests it may have a therapeutic potential in periodontal diseases.
Curcumin; inflammation; periodontal disease; NF-kB; p38 MAPK
Background and objective
The mechanism by which periodontal pathogens dominate at disease sites is not yet understood. One possibility is that these late colonizers antagonize quorum-sensing systems of early colonizers and render those early colonizers less resistant to environmental factors. In this study, we utilized Streptococcus mutans, a well-documented oral Streptococcus with many quorum-sensing dependent properties, as an example of targeted earlier colonizers that are antagonized by periodontal pathogens.
Material and Methods
S. mutans NG8, LT11, and BM71 were used in this study for assessment of transformation and bacteriocin production, respectively. The effects of Porphyromonas gingivalis and Treponema denticola on these competence-stimulating peptide (CSP)-dependent properties were evaluated in mixed broth assays.
Both P. gingivalis (either live bacteria or membrane vesicles) and T. denticola antagonized transformation in S. mutans NG8 and LT11. S. mutans BM71 bacteriocin production was also inhibited by P. gingivalis and T. denticola. Boiling of these late colonizers before mixing the broth cultures abolished their ability to inhibit S. mutans transformation and bacteriocin production. P. gingivalis and T. denticola inactivated S. mutans exogenous CSP, whereas the boiled bacteria did not.
This study demonstrated that periodontal pathogens antagonized S. mutans quorum-sensing properties. This may render S. mutans less virulent and less resistant to environmental antibacterial factors.
periodontal pathogen; Porphyromonas gingivalis; Treponema denticola; Streptococcus mutans; quorum sensing
Background and objectives
Epidemiological studies have established that patients with diabetes have an increased prevalence and severity of periodontal disease. Interleukin (IL)-6, a multifunctional cytokine, plays a role in the tissue inflammation that characterizes periodontal disease. Our recent study has shown a trend of increase in periodontal IL-6 expression at the mRNA level across patients with neither periodontal disease nor diabetes, patients with periodontal disease alone and patients with both diseases. However, the periodontal IL-6 expression at the protein level in these patients has not been investigated.
Material and Methods
Periodontal tissue specimens were collected from eight patients without periodontal disease and diabetes (group 1), from 17 patients with periodontal disease alone (group 2) and from 10 patients with both periodontal disease and diabetes (group 3). The frozen sections were prepared from these tissue specimens and IL-6 protein expression was detected and quantified.
The nonparametric Kruskal-Wallis test showed that differences in IL-6 protein levels among the three groups were statistically significant (p = 0.035). Nonparametric analysis using Jonckheere-Terpstra test showed a tendency of increase in periodontal IL-6 protein levels across group 1 to group 2 to group 3 (p = 0.006). Parametric analysis of variance (ANOVA) on IL-6 protein levels showed that neither age nor gender significantly affected the difference of IL-6 levels among the groups.
Periodontal IL-6 expression at the protein level is increased across patients with neither periodontal disease nor diabetes, patients with periodontal disease alone, and patients with both diseases.
Periodontal diseases; Diabetes mellitus; IL-6; Gene expression
Background and Objective
The extracellular matrix (ECM) plays a key role in signaling necessary for tissue remodeling and cell survival. However, signals from disease-altered ECMs, as that present in inflammatory diseases like periodontitis and arthritis, may lead to apoptosis or programmed cell death of resident cells. Previously, we found that a disease-associated fibronectin fragment triggers apoptosis of primary human periodontal ligament (PDL) cells via a novel apoptotic pathway in which the tumor suppressor, p53, is transcriptionally downregulated.
Materials and Methods
We used immunofluorescence, transfection assays, western blotting and ELISAs to show that p53 is degraded by a proteasomal pathway in response to a proapoptotic disease-associated fibronectin fragment.
We now show that under these same apoptotic conditions p53 is further downregulated by post-translational ubiquitination and subsequent targeting to the proteasome for degradation. Pretreatment of cells with the proteasomal inhibitors MG132 and lactacystin rescued the cells from apoptosis. p53 levels in cells transfected with ubiquitin siRNA were resistant to degradation induced by the proapoptotic fibronectin fragment, showing that ubiquitination is important for the proapoptotic fibronectin fragment-induced degradation of p53.
These data show that a proapoptotic fibronectin matrix induces ubiquitination and degradation of p53 in the proteasome as part of a novel mechanism of apoptosis associated with inflammatory diseases.
Extracellular Matrix; Fibronectin; Ubiquitin; p53; Proteasome; Periodontitis
Background and objective
Young mice do not develop measurable periodontal bone loss, unless heavily infected with human periodontal pathogens. However, mice with genetically altered immune system are unable to control their own oral flora and develop periodontitis early in life. Based on the potential of the indigenous oral microbiota to cause periodontitis, we hypothesized that normal mice may ultimately develop inflammatory periodontal bone loss, i.e., as a function of age. If confirmed, this could serve as an aging model of chronic periodontitis.
Materials and methods
Periodontal bone levels were measured as the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC), in young (8-10 weeks of age), old (≥ 18 months of age), and mice of intermediate ages. Differential expression of inflammatory mediators in the gingivae of young and old mice was determined by quantitative real-time PCR.
In comparison to young mice, old mice displayed significantly (p < 0.05) increased periodontal bone loss, accompanied by elevated expression of proinflammatory cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-17A) and innate immune receptors involved in the induction or amplification of inflammation (Toll-like receptor 2, CD14, CD11b, CD18, complement C5a receptor, and triggering receptor expressed on myeloid cells-3).
Mice develop naturally-induced periodontal bone loss as a function of age. This aging model of periodontitis represents a genuinely chronic model to study mechanisms of periodontal tissue destruction.
Animal model; Alveolar bone; Chronic periodontitis; Inflammation; Innate immunology
Evaluate the role of p38 p38 mitogen activated protein kinase (MAPK) signaling pathway in lipopolysaccharide (LPS)-induced receptor activator of nuclear factor-κB ligand (RANKL) expression by murine periodontal ligament (PDL) cells.
LPS from Gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response leading to tissue destruction observed in periodontitis.
Expression of RANKL and osteoprotegerin (OPG) mRNA was studied by reverse transcription polymerase chain reaction (RT-PCR) upon stimulation with LPS from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of p38 MAPK signaling pathway to LPS-induced RANKL and OPG expression. Stable cell lines expressing dominant negative forms of MAKPK kinase (MKK)-3 and MKK6 were generated to confirm the role of p38 MAPK pathway. An osteoclastogenesis assay using a co-culture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by LPS-stimulated PDL was correlated with RANKL expression.
Inhibiting p38 MAPK previous to LPS stimulation resulted in a significant decrease of RANKL mRNA expression. OPG mRNA expression was not affected by LPS or p38 MAPK. LPS-stimulated PDL cells increased osteoclast differentiation, an effect that was completely blocked by OPG and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from mPDL cultures did not increase osteoclast differentiation, indicating that PDL cells produced membrane-bound RANKL.
LPS resulted in a significant increase on RANKL in PDL cells. p38 MAPK pathway is required for LPS-induced membrane-bound RANKL expression in these cells.
osteoclastogenesis; RANKL; PDL cells; p38 MAPK
Background and objective
18beta-glycyrrhetinic acid (GA) is a natural anti-inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study.
Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in IL-10 deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on LPS-stimulated macrophages, T cell proliferation, and osteoclastogenesis was also examined in vitro.
GA administered either prophylactically or therapeutically dramatically reduced infection-induced bone loss in IL-10 deficient mice, which are highly disease-susceptible. Although GA has been reported to exert its anti-inflammatory activity via down-regulation of 11-beta hydroxysteroid dehydrogenase-2 (HSD2), which converts active glucocorticoids (GC) to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, under GC-free conditions, GA potently inhibited LPS-stimulated proinflammatory cytokine production and RANKL-stimulated osteoclastogenesis, both of which are NF–κB-dependent. GA furthermore suppressed LPS- and RANKL-stimulated phosphorylation of NF–κB p105 in vitro.
These findings indicate that GA inhibits periodontitis by inactivation of NF–κB in an IL-10 and GC-independent fashion.
18beta-glycyrrhetinic acid; periodontal disease; NF–κB; IL-10 deficient mouse
Background and objective
Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically contain also gelatin-binding gelatinases that may cleave FN, reduce its stability, and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN.
Materials and Methods
Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 M urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration.
Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose whereas subsequent elution with 4 M urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN which was more stable than FN eluted by urea only. FN degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly.
The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared to intact FN.
Fibronectin purification; gelatinase; human gingival fibroblast; dimethyl sulfoxide; cell attachment; cell migration
Chronic inflammatory bowel disease (IBD) demonstrates some similarities of dysregulated chronic immunoinflammatory lesion of periodontitis. Trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulphate (DSS) administered to rodents have been shown to elicit inflammatory responses that undermine the integrity of the gut epithelium similar to IBD in humans. The objective of this study was to evaluate the ability of these chemicals to elicit periodontal inflammation as a novel model for alveolar bone loss.
Mice were treated by oral application of TNBS 2 times/week, or with DSS in the diet over a period of 18 weeks. Alveolar bone loss was assessed on defleshed skull using morphometric measures for area of bone resorption.
TNBS-treated animals tolerated oral administration with no clinical symptoms and gained weight similar to normal controls. In contrast, DSS exerted a systemic response including shortening of colonic tissue and liver enzyme changes. Both TNBS and DSS caused a localized action on periodontal tissues with alveolar bone loss observed in both maxilla and mandibles with progression in a time dependent manner. Bone loss was detected as early as week 7, with more severe periodontitis increasing over the 18 weeks (p<0.001). Young (7 month) and old (12 month) SCID mice were treated with TNBS for a period of 7 weeks and did not develop significant bone loss.
These data show that oral administration of TNBS and DSS provoke alveolar bone loss in concert with the autochthonous oral microbiota.
To test the hypothesis that tooth location affected the microbial composition of supragingival plaque beyond the effect due to plaque mass as reflected by total DNA probe count.
Supragingival plaque samples were taken from the mesiobuccal aspect of each tooth in 187 subjects at baseline (N samples = 4,745). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Significance of differences in mean species counts and proportions were determined among tooth surfaces and 6 tooth type categories: molars, bicuspids, incisors/canines in the mandible and maxilla separately using the Kruskal Wallis test. Stepwise multiple linear regression was employed to examine the relationship between species proportions and total DNA probe count, tooth location, periodontal and smoking status, age and gender.
All species differed significantly among tooth types and among the 6 tooth categories. Higher plaque levels were seen at molars and lower incisors. Some differences observed between tooth types could be partially explained by the level of plaque. Teeth with high plaque mass exhibited high levels of Capnocytophaga gingivalis, Actinomyces naeslundii genospecies 2, Campylobacter rectus and Campylobacter showae. However, certain species such as Veillonella parvula and Streptococcus sanguinis differed significantly at different tooth locations despite similarities in plaque mass. Twenty of the test species exhibited a significant association with tooth location after adjusting for total DNA probe count and subject level factors.
While plaque mass was associated with differences in proportions of many species in supragingival biofilms, tooth location also was strongly associated with species proportions in both univariate and multivariate analyses.
Bacteria; supragingival plaque; tooth type; plaque mass
To examine the relationship between total DNA probe counts of supragingival biofilm samples, clinical parameters and supragingival biofilm composition.
Supragingival plaque samples were taken from 187 systemically healthy adult subjects at baseline (N samples = 4,745). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. The relationship between total DNA probe counts and microbial composition was examined by sub-setting the data into 10 groups based on 10 percentile increments of the total DNA probe counts. Differences among groups in terms of species counts and proportions were sought as well as relationships of total plaque DNA probe count and clinical parameters.
There was a wide distribution in mean total DNA probe counts among the 187 subjects. With increasing total plaque levels there was a change in the proportions of individual species and microbial complexes. “Small plaques” were characterized by high proportions of species in the yellow, orange, purple and “other” complexes; plaques of moderate mass were characterized by high proportions of Actinomyces and purple complex species, while “large plaques” exhibited increased proportions of green and orange complex species. Measures of gingival inflammation, pocket depth and recession were significantly positively associated with total DNA probe counts. Increased plaque numbers were related to increased pocket depth irrespective of presence or absence of gingival inflammation.
The proportions of individual species and microbial complexes in supragingival biofilms are influenced by the total numbers of organisms in the biofilm.
Microbiology; bacteria; supragingival plaque; ecology; plaque mass
Mucosal inflammatory responses are orchestrated largely by proinflammatory chemokines. We aimed to evaluate the expression of the chemokine GCP-2 in clinically healthy vs. diseased gingival tissues and to explore possible correlations with clinical and microbiological markers of periodontitis.
The chemokine granulocyte chemotactic protein 2 (GCP-2/CXCL6) is involved in neutrophil recruitment and migration. Previous studies showed that GCP-2 is upregulated in mucosal inflammation, e.g., in inflammatory bowel disease, similarly to the functionally and structurally related chemokine interleukin 8 (IL-8). Nevertheless, unlike IL-8, a role of GCP-2 in gingival inflammation has not yet been demonstrated.
Gene expression in 184 ‘diseased’ and 63 ‘healthy’ gingival tissue specimens from 90 periodontitis patients was analyzed using Affymetrix U133Plus2.0 arrays. GCP-2 expression was further confirmed by real time RT-PCR, western blotting and ELISA while the localization of GCP-2 protein in the gingival tissues was analyzed by immunohistochemistry. Plaque samples from the adjacent periodontal pockets were collected and evaluated for 11 periodontal species using checkerboard DNA-DNA hybridizations.
Among all known chemokines, GCP-2 mRNA was the most expressed (3.8 fold, p<1.1×10−16) in ‘diseased’ vs. ‘healthy’ tissue, as compared to a 2.6 fold increased expression of IL-8 mRNA (p<1.2×10−15). Increased expression of GCP-2 correlated with higher levels of ‘red’ and ‘orange’ complex pathogens and with increased probing depth, but not with attachment loss. Immuno-histochemistry showed that GCP-2 was expressed in gingival vascular endothelium.
GCP-2 correlates with the severity of periodontitis and appears to act as a hitherto unrecognized functional adjunct to IL-8 in diseased gingival tissues.
Periodontal disease; inflammatory mediator; Chemokines; Chemotaxis; Matrix metalloproteinase; Microarray
Background and Objective
Saliva has been proposed as a non-invasive diagnostic fluid that could be used in the diagnosis of oral and systemic diseases. The levels of salivary biomarkers such as cytokines could potentially be used as a surrogate to distinguish periodontally healthy from periodontitis subjects. Therefore, the goal of the present investigation was to determine if the levels of 10 cytokines in saliva would differ between a group of periodontally healthy and periodontitis subjects. Correlations between the concentration of these 10 cytokines and clinical parameters of periodontal disease were also examined.
Material and Methods
In this cross-sectional study, 74 chronic periodontitis and 44 periodontally healthy individuals were periodontally examined and had the levels of GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-γ and TNF-α measured in whole saliva using a multiplexed bead immunoassay (Luminex). Significance of statistical differences in the levels of salivary cytokines between groups was determined using non-parametric ANCOVA adjusting for age and smoking status. The Spearman rank correlation coefficient was used to explore associations between mean levels of salivary cytokines and mean clinical parameters.
There were no statistically significant differences between groups for any of the cytokines. There were weak statistically significant positive associations between salivary IL-8 and PD (rs=0.2, p<0.05) and BOP (rs=0.2, p<0.05) and weak negative correlations between salivary IL-10 and AL (rs=−0.2, p<0.05) and BOP (rs=−0.3, p<0.001).
Mean salivary levels of GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-γ and TNF-α could not discriminate between periodontal health and disease.
saliva; cytokines; diagnosis; chronic periodontitis; periodontal health