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Subgingival microbiome dysbiosis promotes the development of periodontitis, an irreversible chronic inflammatory disease associated with metabolic diseases. However, studies regarding the effects of a hyperglycemic microenvironment on host–microbiome interactions and host inflammatory response during periodontitis are still scarce. Here, we investigated the impacts of a hyperglycemic microenvironment on the inflammatory response and transcriptome of a gingival coculture model stimulated with dysbiotic subgingival microbiomes. HGF-1 cells overlaid with U937 macrophage-like cells were stimulated with subgingival microbiomes collected from four healthy donors and four patients with periodontitis. Pro-inflammatory cytokines and matrix metalloproteinases were measured while the coculture RNA was submitted to a microarray analysis. Subgingival microbiomes were submitted to 16s rRNA gene sequencing. Data were analyzed using an advanced multi-omics bioinformatic data integration model. Our results show that the genes krt76, krt27, pnma5, mansc4, rab41, thoc6, tm6sf2, and znf506 as well as the pro-inflammatory cytokines IL-1β, GM-CSF, FGF2, IL-10, the metalloproteinases MMP3 and MMP8, and bacteria from the ASV 105, ASV 211, ASV 299, Prevotella, Campylobacter and Fretibacterium genera are key intercorrelated variables contributing to periodontitis-induced inflammatory response in a hyperglycemic microenvironment. In conclusion, our multi-omics integration analysis unveiled the complex interrelationships involved in the regulation of periodontal inflammation in response to a hyperglycemic microenvironment.

BACKGROUND There is a growing literature on the potential of digital technologies for improving access, continuity, quality of healthcare and to strengthen health systems. Some authors reported the cost-effectiveness of teledentistry, its reliability for remote dental screening, diagnosis, consultation, and treatment planning. Despite these benefits, the current evidence suggests that teledentistry implementation faces many challenges and is not yet adopted by dental health care providers (DHCPs). Developing strategies aiming to improve teledentistry adoption require an understanding of the factors, which promote or hinder its successful implementation. Therefore, we will perform a systematic review on the barriers and enablers to implementing teledentistry. OBJECTIVE The review purpose aims to synthesize evidence on DHCPs’ perceptions on barriers and enablers to use teledentistry using the Theoretical Domain Framework. METHODS This protocol has been written according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P). A search strategy will be performed through the following databases: PubMed, Cochrane Library, Web of Science, CINAHL, Embase and PsycINFO. We will perform additional searches in Google, Google Scholar, and in Dissertations & Theses Global (ProQuest). We will check references of included studies to capture additional relevant studies. Moreover, we will contact authors of studies if we need more details. We will consider studies using qualitative, quantitative, and mixed methods. There will be no restrictions on the publication date and dental setting. We will include studies published in French and English. Two independent reviewers will carry out the study selection, data extraction, and the assessment of the methodological quality using the checklist of mixed methods appraisal tool (MMAT). We will conduct a descriptive and thematic content analysis. We will synthesize and categorize the barriers and enablers using the theoretical domains framework (TDF). We will present a narrative synthesis of results using tables, figures, and quotes. RESULTS By October 2022, the literature search has retrieved 7355 publications. We will identify the range of barriers and enablers to implementing teledentistry through DHCPs’ perspectives. Considering the critical need for theory-based implementation interventions to improve the use of evidence-informed practices, we will synthesize the factors influencing adoption of teledentistry according to the domains of the Theoretical Domains Framework (TDF) and the three essential conditions predicting the behavior change according to Behavior change wheel (BCW) model. We will identify behaviour change techniques (BCTs) related to these determinants. As needed, we will add additional determinants if not included in the TDF. We expect to complete the review by July 2023. CONCLUSIONS Teledentistry is slow to be adopted by DHCPs. This review will be of interest to patients, families, DHCPs, researchers, academic and professional decision-makers and policy makers. The results of the systematic review could be used to improve the teledentistry implementation in dental settings. CLINICALTRIAL PROSPERO CRD42021293376; https://www.crd.york.ac.uk/prospero/#searchadvanced

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species, increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

Oral cancer is one of the major public health problems. The aim of this study was to evaluate the effects of anethole, 1-methoxy-4-[(E)-1-propenyl]-benzene, on growth and apoptosis of oral tumor cells, and to identify the signaling pathways involved in its interaction with these cancer cells. Cancer gingival cells (Ca9-22) were treated with different concentrations of anethole. Cell proliferation and cytotoxic effects were measured by MTT and LDH assays. Cell death, autophagy and oxidative stress markers were assessed by flow cytometry while cell migration was determined by a healing capacity assay. The effect of anethole on apoptotic and pro-carcinogenic signaling pathways proteins was assessed by immunoblotting. Our results showed that anethole selectively and in a dose-dependent manner decreases the cell proliferation rate, and conversely induces toxicity and apoptosis in oral cancer cells. This killing effect was mediated mainly through NF-κB, MAPKinases, Wnt, caspase 3, 9 and PARP1 pathways. Anethole showed an ability to induce autophagy, decrease reactive oxygen species (ROS) production and increased intracellular glutathione (GSH) activity. Finally, anethole treatment inhibits the expression of oncogenes (cyclin D1) and up-regulated cyclin-dependent kinase inhibitor (p21WAF1), increases the expression of p53 gene, but inhibits the epithelial-mesenchymal transition markers. These results indicate that anethole could be a potential molecule for the therapy of oral cancer.

The aim of this work was to compare the duration of treatment between orthodontic treatment combined with piezocorticision (OT-PC) and conventional orthodontic treatment (COT), as well as to evaluate the safety, inflammatory process, periodontal health, and soft tissue healing in the OT-PC group.Twelve patients were included in each group, and their treatment times were compared for preliminary bracket alignment (PBA) and for overall treatment. In the OT-PC group, the inflammatory process was evaluated by quantifying cytokines in the gingival crevicular fluid. A calibrated examiner measured the probing depth (PD), the distance between the gingival margin and the cementoenamel junction (GM-CEJ), and the clinical attachment level (CAL), before and after OT-PC. The presence of gingival scars was evaluated. Bone and root injuries were assessed with the use of cone-beam computed tomography.The treatment time was significantly reduced in the OT-PC group for PBA in both maxilla (45%; P = 0.001) and mandible (31%; P = 0.023), as well as for overall treatment (52%; P 0.0001). Although not statistically significant, several inflammatory mediators demonstrated peaks at 3-5 and 16 weeks. There were not significant changes in PD and GM-CEJ after OT-PC treatment, unlike CAL (0.09 ± 0.12 mm; P = 0.024); 47.5% of piezocorticisions caused gingival scars. Only one patient showed no scars. Four cortical bones did not heal completely, and 2 roots had piezoelectric injuries.OT-PC was effective at reducing the orthodontic treatment time.

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma (OC-SCC) were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species (ROS), increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

Enamel matrix derivatives (EMDs) have been used clinically for more than a decade for the regeneration of periodontal tissues. The aim of the present study is to analyze the effect on cell growth of EMDs in a gel carrier in comparison to EMDs in a liquid carrier. EMDs in a liquid carrier have been shown to adsorb better to bone graft materials.Primary human osteoblasts and periodontal ligament (PDL) cells were exposed to EMDs in both gel and liquid carriers and compared for their ability to induce cell proliferation and differentiation. Alizarin red staining and real-time polymerase chain reaction for expression of genes encoding collagen 1, osteocalcin, and runt-related transcription factor 2, as well as bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-β1, and interleukin (IL)-1β, were assessed.EMDs in both carriers significantly increased cell proliferation of both osteoblasts and PDL cells in a similar manner. Both formulations also significantly upregulated the expression of genes encoding BMP2 and TGF-β1 as well as decreased the expression of IL-1β. EMDs in the liquid carrier further retained similar differentiation potential of both osteoblasts and PDL cells by demonstrating increased collagen and osteocalcin gene expression and significantly higher alizarin red staining.The results from the present study indicate that the new formulation of EMDs in a liquid carrier is equally as potent as EMDs in a gel carrier in inducing osteoblast and PDL activity. Future study combining EMDs in a liquid carrier with bone grafting materials is required to further evaluate its potential for combination therapies.

To examine associations between periodontal disease severity and clinical and microbiological measures of caries in adults. A cross-sectional study of 94 healthy adults ((mean ± SD) 55.4 ± 13.0 years) was conducted. Data were collected by means of questionnaire and a clinical examination that included the Decayed, Missing, Filled teeth Surfaces (DMFS) index, probing depth (PD), clinical attachment level (CAL), and gingival bleeding and plaque scores. Supra- and subgingival plaque samples were collected to assess the presence of Streptococcus mutans and six periodontal pathogens. Participants were subsequently categorized using Center for Disease Control and Prevention/American Academy of Periodontology (CDC-AAP) definitions and tertiles of percentage of sites with CAL ≥ 3mm. Significant positive associations were found between the periodontal disease severity (CDC-AAP) and the DMFS (aOR = 1.03; 95% CI 1.01–1.05) and DS indices (aOR = 1.18; 95% CI 1.05–1.32) as well as between the tertiles of percentage of sites with CAL ≥ 3 mm and DMFS (aOR = 1.03; 95% CI 1.00–1.05) and DS indices (aOR = 1.12; 95% CI 1.00–1.25). A significant positive association was also found between oral levels of F. nucleatum and S. mutans (aOR = 6.03; 95% CI 1.55–23.45). A small but positive association was found between clinical measures of caries and periodontal disease severity. Further research is warranted to examine the association between these two common oral diseases. Periodontal diseases and caries are the two most common oral diseases. There was a positive association between clinical and microbiological markers of both diseases. Therefore, strategies in oral health education should involve both caries and periodontitis prevention.

OBJECTIVES Since the original description of osteoinduction in the early 20th century, the study and development of innovative biomaterials has emerged. Recently, novel synthetic bone grafts have been reported with potential to form ectopic bone in vivo. However, their full characterization in comparison with other leading bone grafts has not been investigated. The aim of this study was to determine the osteoinductive potential of bone grafts by comparing autogenous bone grafts, demineralized freeze-dried bone allografts (DFDBA), a commonly utilized natural bone mineral (NBM) from bovine origin (Bio-Oss), and a newly developed biphasic calcium phosphate (BCP). MATERIALS AND METHODS Grafts were compared in vitro for their ability to stimulate bone marrow stromal cell (BMSC) migration, proliferation, and differentiation as assessed by quantitative real-time PCR for genes coding for bone markers including Runx2, collagen I, and osteocalcin. Furthermore, bone grafts were implanted in the calf muscle of 12 beagle dogs to determine their potential to form ectopic bone in vivo. RESULTS The in vitro results demonstrate that both autografts and DFDBA show potential for cell recruitment, whereas only autografts and BCP demonstrated the ability to differentiate BMSCs toward the osteoblast lineage. The in vivo ectopic bone model demonstrated that while NBM particles were not osteoinductive and autogenous bone grafts were resorbed quickly in vivo, ectopic bone formation was reported in DFDBA and in synthetic BCP grafts. CONCLUSION The modifications in nanotopography and chemical composition of the newly developed BCP bone grafts significantly promoted ectopic bone formation confirming their osteoinductive potential. In conclusion, the results from this study provide evidence that synthetic bone grafts not only serve as a three-dimensional scaffold but are also able to promote osteoinduction.

OBJECTIVES Research across many fields of medicine now points towards the clinical advantages of combining regenerative procedures with platelet-rich fibrin (PRF). This systematic review aimed to gather the extensive number of articles published to date on PRF in the dental field to better understand the clinical procedures where PRF may be utilized to enhance tissue/bone formation. MATERIALS AND METHODS Manuscripts were searched systematically until May 2016 and separated into the following categories: intrabony and furcation defect regeneration, extraction socket management, sinus lifting procedures, gingival recession treatment, and guided bone regeneration (GBR) including horizontal/vertical bone augmentation procedures. Only human randomized clinical trials were included for assessment. RESULTS In total, 35 articles were selected and divided accordingly (kappa = 0.94). Overall, the use of PRF has been most investigated in periodontology for the treatment of periodontal intrabony defects and gingival recessions where the majority of studies have demonstrated favorable results in soft tissue management and repair. Little to no randomized clinical trials were found for extraction socket management although PRF has been shown to significantly decrease by tenfold dry sockets of third molars. Very little to no data was available directly investigating the effects of PRF on new bone formation in GBR, horizontal/vertical bone augmentation procedures, treatment of peri-implantitis, and sinus lifting procedures. CONCLUSIONS Much investigation now supports the use of PRF for periodontal and soft tissue repair. Despite this, there remains a lack of well-conducted studies demonstrating convincingly the role of PRF during hard tissue bone regeneration. Future human randomized clinical studies evaluating the use of PRF on bone formation thus remain necessary. CLINICAL RELEVANCE PRF was shown to improve soft tissue generation and limit dimensional changes post-extraction, with little available data to date supporting its use in GBR.

Periodontitis (PD) has emerged as a risk factor in a number of health conditions including rheumatoid arthritis (RA) (1). Sharing both morphologic and histopathologic similarities with RA (2), PD is an inflammatory disease initiated by bacterial infection resulting in soft and hard tissue destruction and ultimately leading to tooth loss. In addition to shared inflammatory pathways, PD and RA share risk factors for susceptibility and progression, most notably cigarette smoking and, possibly, shared epitope-containing HLA-DRB1 alleles, the latter associated with localized aggressive periodontitis (3–10). Although a causal link between these conditions has not been established, several reports have demonstrated an increased PD prevalence in RA patients compared to controls (11–18). Growing evidence suggests that pathogens associated with PD could play a role in RA propagation. Chief among the organisms of interest is Porphyromonas gingivalis (P. gingivalis) (19). P. gingivalis is the only known pathogen expressing peptidylarginine deiminase (PPAD). Similar to its human counterpart, P. gingivalis-expressed PAD catalyzes the citrullination of arginine-containing peptides. This is noteworthy because citrullinated antigens are thought to drive adaptive immune responses that are nearly exclusive to RA. The potential role of P. gingivalis in RA pathogenesis has been borne out in epidemiologic investigations. Concentrations of circulating antibody to P. gingivalis have been demonstrated to be associated with the expression of anti-citrullinated peptide antibody (ACPA) (20–22). More recently, our group has shown that antibody to P. gingivalis is associated with the presence of RA-related autoantibody (a combination of rheumatoid factor [RF] and/or ACPA) among individuals at increased risk for disease but who have not yet developed RA symptoms (23), underscoring the potential role of this pathogen in RA development. As part of the present study, we conducted a large case-control investigation to examine the relationship of PD with established RA. We sought to examine the degree to which this relationship is impacted by shared genetic and/or environmental factors. We also sought to elucidate the degree to which the relationship of PD with RA may be related to infection and/or colonization with P. gingivalis. By using a rigorously selected control population, we attempted to mitigate issues of bias or unmeasured confounding that may have impacted other efforts often using healthy volunteers as comparators (16–18). Finally, using a multiplex approach, we examined the associations of PD and P. gingivalis with autoreactivity to several citrullinated autoantigens that have been implicated in RA disease pathogenesis.

OBJECTIVES The aim of the present study was to investigate the effects of Osteogain, a new formulation of enamel matrix derivative (EMD) in combination with a grafting material on a wide variety of genes for cytokines, transcription factors and extracellular matrix proteins involved in osteoblast differentiation. MATERIALS AND METHODS Primary human periodontal ligament (PDL) cells were seeded on natural bone mineral (NBM) particles coated with Osteogain for 24 h and analyzed for regulated gene expression using a human osteogenesis gene super-array kit. Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation as well as gene products representing extracellular matrix molecules, transcription factors and cell adhesion molecules. RESULTS Osteogain significantly upregulated the expression of over 20 of the 100 genes examined including bone morphogenetic protein 2 (BMP2), TGFβ1, fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) as well as some of their associated receptors. Osteogain also promoted gene expression of a number of osteoblast differentiation markers including collagen1α2 and alkaline phosphatase as well as cell adhesion molecules including fibronectin and a variety of integrin binding proteins. Interestingly, Osteogain promoted calcitonin receptor 55-fold and also promoted annexin A5 gene expression over 12-fold. CONCLUSION The present study demonstrates that Osteogain is capable of either upregulating or downregulating the expression of a wide variety of genes including those for growth factors and cytokines when combined with a bone grafting material. CLINICAL RELEVANCE The results from the present study demonstrate the large and potent effect of addition of Osteogain in combination to a bone grafting material over a wide variety of genes supporting osteogenesis.

OBJECTIVES Recently, novel biphasic calcium phosphate (BCP) scaffolds have emerged as a new class of bone grafts with osteoinductive potential demonstrating the ability to form ectopic bone in extra-skeletal sites. The aim of the present study was to perform an osteogenic gene array to target possible genes responsible for eliciting the changes in cell expression responsible for inducing osteoblast differentiation. MATERIALS AND METHODS Human MG63 osteoblast-like cells were seeded for 24 h on tissue culture plastic or osteoinductive BCP particles and analyzed for upregulated genes using an osteogenesis super-array. Osteoblast-related genes including those transcribed during bone mineralization, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules were investigated. RESULTS An upregulation of genes transcribing biglycan (1.7-fold), bone morphogenetic proteins 1, 2, 4, 6, and 7 (1.5-2.1-fold), various collagen isoforms including 1a1, 1a2, 2a1, and 5a1 (1.73-2.72-fold), colony stimulating factor 2 (2.59-fold), fibroblast growth factor receptor 2 (1.79-fold), fibronectin (2.56-fold), integrin alpha 1, 2, and 3 (1.82-2.24-fold), SOX9 (2.75-fold), transforming growth factor beta receptor 2 (1.72-fold), vitamin D (1.89-fold), and vascular endothelial growth factor A and B (2.00, 1.75-fold) were all significantly (p

OBJECTIVES Guided bone regeneration (GBR) aims to predictably restore missing bone that has been lost due to trauma, periodontal disease or a variety of systemic conditions. Critical to this procedure is the ability of a bone grafting material to predictably serve as a 3-dimensional scaffold capable of inducing cell and bone tissue in-growth at the material surface. Although all bone grafts are osteoconductive to bone-forming osteoblasts, only a small number of commercially available bone grafts with FDA approval are osteoinductive including demineralized freeze-dried bone allographs (DFDBA) and scaffolds containing bone morphogenetic proteins (BMPs). Recently, a class of synthetic bone grafts fabricated from biphasic calcium phosphate (BCP) sintered at a low temperature have been shown to form ectopic bone formation in non-skeletal sites without the use of growth factors. Therefore, the present study aimed to compare the osteoinductive potential of this group of synthetic BCP alloplasts with autografts, allografts and xenografts. MATERIALS AND METHODS In the present study, 4 types of bone grafting materials including autogenous bone harvested with a bone mill, DFDBA (LifeNet, USA), a xenograft derived from bovine bone mineral (NBM, BioOss, Geistlich, Switzerland) and a novel synthetic biphasic calcium phosphate (BCP, Straumman, Switzerland) were implanted into intramuscular pouches of 24 rats and analysed histologically for their ability to form ectopic bone formation around grafting particles. A semi-quantitative osteoinductive score was used to quantify the osteoinductive ability of each bone graft. RESULTS The results from the present study reveal that (1) autogenous bone resorbed rapidly in vivo, (2) the xenograft showed no potential to form ectopic bone formation and (3) both DFDBA and BCP were able to stimulate ectopic bone formation. CONCLUSION These studies demonstrate that these newly developed synthetic bone grafts have potential for inducing ectopic bone formation similar to DFDBA. Future clinical testing is necessary to reveal their bone-inducing properties in clinical scenarios including GBR procedures and in combination with implant dentistry. CLINICAL RELEVANCE Novel BCP scaffolds are able to induce ectopic bone formation without the use of osteoinductive growth factors such as BMP2 and thus demonstrate a large clinical possibility to further enhance bone formation for a variety of clinical procedures.

Background: The findings from the studies on the relationship between periodontal disease and preeclampsia are inconsistent. The objective of this study is to examine the relationship between periodontal disease and preeclampsia.Methods: A multicenter case-control study was conducted in Quebec, Canada. Preeclampsia was defined as blood pressure ≥140/90 mm Hg and ≥1+ proteinuria after 20 weeks of gestation. Periodontitis was defined as the presence of ≥4 sites with a probing depth ≥5 mm and a clinical attachment loss ≥3 mm at the same sites.Results: A total of 92 preeclamptic women and 245 controls were analyzed. The percentage of periodontal disease was 18.5% in preeclamptic women and 19.2% in normotensive women (crude odds ratio [OR] = 0.96, 95% confidence interval [CI] = 0.52 to 1.77). After adjusting for confounding variables, periodontitis remained not associated with preeclampsia (adjusted OR = 1.13, 95% CI = 0.59 to 2.17).Conclusion: This study does not support the hypothesis of an association betwe...

Membrane-anchored complement regulatory proteins (CRPs), including CD46, CD55, and CD59, protect host cells from complement attack. In the present study, we investigated whether periodontopathogen lipopolysaccharide and proinflammatory cytokines modulate CRP gene/protein expression in human oral epithelial cells. The lipopolysaccharide of Treponema denticola and Tannerella forsythia were the most potent for increasing the gene expression of CD55 and CD59, and to a lesser extent CD46, after a 48-h stimulation. An lipopolysaccharide-induced upregulation of epithelial cell-surface CRP was also demonstrated. The stimulation of epithelial cells with lipopolysaccharide was associated with interleukin-6 (IL-6) and IL-8 secretion. Although these two cytokines had no effect on CD46 and CD55 gene expression in epithelial cells, IL-1β and tumor necrosis factor-α induced a significant upregulation. The cell-surface expression of CRP was also increased by the stimulation of epithelial cells with cytokines. The CD46, CD55, and CD59 gene/protein expression was upregulated by periodontopathogen lipopolysaccharide and proinflammatory cytokines. It can be hypothesized that, when faced with bacterial challenges and inflammatory conditions associated with active periodontal sites, oral epithelial cells may respond by increasing CRP gene/protein expression to avoid cell lysis by the complement system, which is activated during periodontitis.

Background: Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Methods: Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Results: Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum–bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. Conclusions: The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction

In the past decade, cranberry extracts have been attracting ever-growing attention by dental researchers. The potential benefits of cranberry components in reducing oral diseases, including dental caries and periodontitis, are discussed in this review. A non-dialysable cranberry fraction enriched in high molecular weight polyphenols has very promising properties with respect to cariogenic and periodontopathogenic bacteria, as well as to the host inflammatory response and enzymes that degrade the extracellular matrix. Cranberry components are potential anti-caries agents since they inhibit acid production, attachment, and biofilm formation by Streptococcus mutans. Glucan-binding proteins, extracellular enzymes, carbohydrate production, and bacterial hydrophobicity, are all affected by cranberry components. Regarding periodontal diseases, the same cranberry fraction inhibits host inflammatory responses, production, and activity of enzymes that cause the destruction of the extracellular matrix, biofilm formation, and adherence of Porphyromonas gingivalis, and proteolytic activities and coaggregation of periodontopathogens. The above-listed effects suggest that cranberry components, especially those with high molecular weight, could serve as bioactive molecules for the prevention and/or treatment of oral diseases.

Vascular disruption and bleeding during periodontitis likely increase the levels of hemoglobin in gingival crevicular fluid. The aim of this study was to investigate the effect of hemoglobin on the inflammatory responses of human macrophages stimulated with lipopolysaccharides (LPS) isolated from periodontopathogens. The production of interleukin-1 beta (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) by macrophages following challenges with Porphyromonas gingivalis and Fusobacterium nucleatum LPS in the presence or absence of human hemoglobin was analyzed by ELISA. The effect of hemoglobin on LPS-binding to macrophages was evaluated with 3H-LPS. Hemoglobin and LPS from periodontopathogens acted in synergy to stimulate the production of high levels of IL-1β, IL-6, IL-8, and TNF-α by macrophages. Hemoglobin also enhanced LPS-binding to macrophages. This study suggests that hemoglobin contributes to increases in the levels of pro-inflammatory mediators in periodontal sites by acting in synergy with LPS from periodontopathogens, thus favoring the progression of periodontitis.

Resume Les parodontites sont des infections mixtes qui sont a l'origine de la destruction des tissus de soutien de la dent, incluant le ligament parodontal et l'os alveolaire. Parmi plus de 500 especes bacteriennes presentes dans la cavite buccale, un complexe bacterien nomme « complexe rouge » et compose de Porphyromonas gingivalis , Treponema denticola et Tannerella forsythia a ete fortement associe aux lesions parodontales avancees. Bien que les bacteries parodontopathogenes soient le facteur etiologique primaire des parodontites, la destruction des tissus mous et durs resulte essentiellement de la reponse immunitaire de l'hote face a l'agression bacterienne. Les especes du complexe rouge sont des bacteries anaerobies a Gram negatif exprimant de nombreux facteurs de virulence qui leur permettent de coloniser l'espace sous-gingival, de perturber le systeme de defense de l'hote, d'envahir et de detruire les tissus parodontaux ou encore de promouvoir la reponse immunodestructrice de l'hote. Cet article passe en revue les connaissances actuelles des mecanismes pathogeniques des bacteries du complexe rouge contribuant aux destructions tissulaires et osseuses observees au cours des parodontites.

Periodontitis is a chronic inflammatory disease that affects the tooth supporting tissues. Gingival fibroblasts are the most abundant cells in periodontal tissues and participate actively in the host inflammatory response to periodontopathogens, which is known to mediate local tissue destruction in periodontitis. The aim of this study was to investigate the effect of a proanthocyanidin-enriched cranberry fraction, prepared from cranberry juice concentrate, on inflammatory mediator production by gingival fibroblasts stimulated by the lipopolysaccharide (LPS) of Aggregatibacter actinomycetemcomitans. Interleukin (IL)-6, IL-8, and prostaglandin E(2) (PGE(2)) production by fibroblasts treated with the cranberry fraction and stimulated by A. actinomycetemcomitans LPS was evaluated by enzyme-linked immunosorbent assay. Changes induced by A. actinomycetemcomitans LPS and the cranberry fraction in the expression and phosphorylation state of fibroblast intracellular signaling proteins were characterized by antibody microarrays. The LPS-induced IL-6, IL-8, and PGE(2) responses of gingival fibroblasts were inhibited by treatment with the cranberry fraction. This fraction was found to inhibit fibroblast intracellular signaling proteins, a phenomenon that may lead to a down-regulation of activating protein-1 activity. Cranberry components also reduced cyclooxygenase 2 expression. This study suggests that cranberry juice contains molecules with interesting properties for the development of new host-modulating therapeutic strategies in the adjunctive treatment of periodontitis.

Background Periodontal disease has been associated with systemic inflammation and adverse pregnancy outcomes, including preeclampsia and preterm birth. Objective To examine the relationship between periodontal disease in early pregnancy and the risk of amniotic inflammation, preterm birth, and preeclampsia. Methods We performed a prospective cohort study of women undergoing amniocentesis for fetal karyotype between 15 and 24 weeks' gestation. Participants underwent periodontal examination by a certified dentist, and a sample of amniotic fluid was collected. Periodontal disease was defined as the presence of one or more sites with probing depths ≥ 4 mm and ≥ 10% bleeding on probing. Matrix metalloproteinase-8 and interleukin-6 concentrations in the amniotic fluid were measured. Medical charts were reviewed for perinatal outcomes. Univariate and multivariate logistic regression analyses were used to assess the association between periodontal disease and adverse pregnancy outcomes. Results We recruited 273 women at a median gestational age of 16 weeks (range 15 to 24), and 258 (95%) agreed to undergo periodontal examination. Periodontal disease was observed in 117 of the participants (45%). We observed no significant association between periodontal disease and preterm birth (relative risk [RR] 2.27; 95% CI 0.74 to 6.96) or spontaneous preterm birth (RR 0.90; 95% CI 0.20 to 4.11). However, women with periodontal disease were more likely to develop preeclampsia, and this association remained significant after adjustment for potential confounders (adjusted RR 5.89; 95% CI 1.24 to 28.05). Periodontal disease was not associated with significant differences in the intra-amniotic concentration of matrix metalloproteinase-8 (13.0 ± 46.6 vs 5.7 ± 10.4 ng/mL, P = 0.098) or interleukin-6 (3.3 ± 20.3 vs 1.0 ± 1.6 ng/mL, P = 0.23), although a non-significant trend was observed. Conclusion Periodontal disease is associated with preeclampsia but not with spontaneous preterm birth. The current study cannot exclude an association between periodontal disease and intra-amniotic inflammation.

OBJECTIVES Bone replacement grafting materials play an important role in regenerative dentistry. Despite a large array of tested bone-grafting materials, little information is available comparing the effects of bone graft density on in vitro cell behavior. Therefore, the aim of the present study is to compare the effects of cells seeded on bone grafts at low and high density in vitro for osteoblast adhesion, proliferation, and differentiation. MATERIALS AND METHODS The response of osteoblasts to the presence of a growth factor (enamel matrix derivative, (EMD)) in combination with low (8 mg per well) or high (100 mg per well) bone grafts (BG; natural bone mineral, Bio-Oss®) density, was studied and compared for osteoblast cell adhesion, proliferation, and differentiation as assessed by real-time PCR. Standard tissue culture plastic was used as a control with and without EMD. RESULTS The present study demonstrates that in vitro testing of bone-grafting materials is largely influenced by bone graft seeding density. Osteoblast adhesion was up to 50 % lower when cells were seeded on high-density BG when compared to low-density BG and control tissue culture plastic. Furthermore, proliferation was affected in a similar manner whereby cell proliferation on high-density BG (100 mg/well) was significantly increased when compared to that on low-density BG (8 mg/well). In contrast, cell differentiation was significantly increased on high-density BG as assessed by real-time PCR for markers collagen 1 (Col 1), alkaline phosphatase (ALP), and osteocalcin (OC) as well as alizarin red staining. The effects of EMD on osteoblast adhesion, proliferation, and differentiation further demonstrated that the bone graft seeding density largely controls in vitro results. EMD significantly increased cell attachment only on high-density BG, whereas EMD was able to further stimulate cell proliferation and differentiation of osteoblasts on control culture plastic and low-density BG when compared to high-density BG. CONCLUSION The results from the present study demonstrate that the in vitro conditions largely influence cell behavior of osteoblasts seeded on bone grafts and in vitro testing. CLINICAL RELEVANCE These results also illustrate the necessity for careful selection of bone graft seeding density to optimize in vitro testing and provide the clinician with a more accurate description of the osteopromotive potential of bone grafts.

OBJECTIVES Enamel matrix derivative (EMD) has been successfully used for the regeneration of periodontal tissues including new cementum, periodontal ligament, and alveolar bone. Combination of EMD with bone grafting materials has however generated variable clinical results. Recently, we have demonstrated that a new formulation of EMD in a liquid carrier system (Osteogain®) has improved physicochemical properties for the adsorption of EMD to a bone grafting material. The aim of the present study was to investigate the regenerative potential of Osteogain®, in combination with a bone graft, on new bone formation in a rat femur defect model. MATERIALS AND METHODS Fifty-four critically sized femur defects (3 mm in diameter) were created bilaterally in 27 rats and treated following the group allocation: (1) drilled unfilled control, (2) a natural bone mineral (NBM), and (3) NBM + Osteogain®. All defects were histologically analyzed at 2, 4, and 8 weeks after surgical intervention. Micro-CT analysis, hematoxylin and eosin (H&E) staining, and Safranin O staining were performed to quantify new bone formation. RESULTS Significantly more new bone formation was observed in defects treated with NBM + Osteogain® at both 4 and 8 weeks when compared to NBM alone and the control unfilled defects (P

BACKGROUND The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

Periodontitis is a chronic inflammatory disease affecting oral tissues. The continuous, high production of cytokines by host cells triggered by periodontopathogens is thought to be responsible for the destruction of tooth-supporting tissues. Macrophages play a critical role in this host inflammatory response to periodontopathogens. The aim of this study was to investigate the effect of non-dialyzable material prepared from cranberry juice concentrate on the pro-inflammatory cytokine response of macrophages induced by lipopolysaccharides (LPS) from Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum subsp. nucleatum, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli. Interleukin-1 beta (IL-1β), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), and Regulated on Activation Normal T-cell Expressed and Secreted (RANTES) production by macrophages treated with the cranberry fraction prior to stimulation by LPS was evaluated by ELISA. Our results clearly indicate that the cranberry fraction was a potent inhibitor of the pro-inflammatory cytokine and chemokine responses induced by LPS. This suggests that cranberry constituents may offer perspectives for the development of a new therapeutic approach to the prevention and treatment of periodontitis.

Received 15 December 2005; returned 8 January 2006; revised 20 January 2006; accepted 21 January 2006Background: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are three majoraetiologicalagentsofchronicperiodontitis.Thestrongproteolyticactivitiesofthesebacteriaarecriticaltotheir survival since their energy source is obtained from peptides and amino acids derived from proteins.In addition, proteases are important factors contributing to periodontal tissue destruction by a variety ofmechanisms, including direct tissue degradation and modulation of host inflammatory responses.Objectives: The aim of this study was to investigate the effect of non-dialysable material (NDM) preparedfromcranberryjuiceconcentrateontheproteolyticactivitiesofP.gingivalis,T.forsythiaandT.denticola.Methods: The effect of NDM on gingipain and dipeptidyl peptidase IV (DPP IV) activities of P. gingivalis,trypsin-like activity of T. forsythia and chymotrypsin-like activity of T. denticola was evaluated usingsynthetic chromogenic peptides. In addition, the capacity of P. gingivalis to degrade fluorescein-labelledtype I collagen and fluorescein-labelled transferrin in the presence of NDM was evaluated by fluorometry.Results:NDM dose-dependently inhibited the proteinases of P. gingivalis,T. forsythia and T.denticola aswell as type I collagen and transferrin degradation by P. gingivalis.Conclusions: These results suggest that NDM has the potential to reduce either the proliferation ofP.gingivalis,T.forsythia andT.denticola inperiodontalpockets or their proteinase-mediateddestructiveprocess occurring in periodontitis.Keywords:Porphyromonasgingivalis,Tannerellaforsythia,Treponemadenticola,periodontitis,proteaseinhibitors

Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.

SummaryPeriodontitis is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. Leukocytes play a major role in the host response to Porphyromonas gingivalis, a major aetiological agent of chronic periodontitis. Secretion of high levels of inflammatory mediators, including cytokines and prostaglandins, by leucocytes is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of an ex vivo whole blood model to P. gingivalis stimulation. The production of interleukin-1 beta (IL-1β), IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IFN-γ-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), Regulated on Activation Normal T cell Expressed and Secreted (RANTES) and prostaglandin E2 (PGE2) were quantified by enzyme-linked immunosorbent assays. P. gingivalis induced the secretion of the pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IFN-γ, the chemokines IL-8, RANTES and MCP-1 and the inflammatory mediator PGE2 in an ex vivo human whole blood model. The secretion levels were dependent on the strain and the infectious dose used. While the mediator profiles were comparable between six healthy subjects, a high interindividual variability in the levels of secreted mediators was observed. This study supports the view that P. gingivalis, by inducing high levels of inflammatory mediators from a mixed leucocyte population, can contribute to the progression of periodontitis.

Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis . Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis , inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis , including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.

Over 15 years have passed since an enamel matrix derivative (EMD) was introduced as a biologic agent capable of periodontal regeneration. Histologic and controlled clinical studies have provided evidence for periodontal regeneration and substantial clinical improvements following its use. The purpose of this review article was to perform a systematic review comparing the eff ect of EMD when used alone or in combination with various types of bone grafting material.A literature search was conducted on several medical databases including Medline, EMBASE, LILACS, and CENTRAL. For study inclusion, all studies that used EMD in combination with a bone graft were included. In the initial search, a total of 820 articles were found, 71 of which were selected for this review article. Studies were divided into in vitro, in vivo, and clinical studies. The clinical studies were subdivided into four subgroups to determine the eff ect of EMD in combination with autogenous bone, allografts, xenografts, and alloplasts.The analysis from the present study demonstrates that while EMD in combination with certain bone grafts is able to improve the regeneration of periodontal intrabony and furcation defects, direct evidence supporting the combination approach is still missing.Further controlled clinical trials are required to explain the large variability that exists amongst the conducted studies.

Streptococcus salivarius , a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 10 6 to 40 × 10 6 Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus .

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.

The aim of this study was to combine immunomagnetic capture and a polymerase chain reaction (PCR) followed by hybridization with a DNA probe for the detection of Bacteroides forsythus. Magnetic beads were coated with the immunoglobulin G fraction of an antiserum specific for B. forsynthus. Aliquots were incubated with various concentrations of a suspension of B. forsythus or with a suspension containing 16 bacterial species, at a concentration of 10(10) cells/ml, spiked with dilutions of B. forsythus. Beads with bound bacteria were boiled, and the target DNA in the supernatant was amplified to generate a 392-bp PCR fragment specific for B. forsythus. The amplified product was detected by dot-blot hybridization with a digoxigenin-labeled 392-bp probe. The detection limit was determined to be 10 cells/ml using immunocapture on a suspension of B. forsythus and 100 on spiked bacterial suspensions. Subgingival plaque samples were obtained from 39 Bolivian individuals with poor oral hygiene. Each sample was analyzed by the above procedure and by immunofluorescence. The overall prevalence of individuals harboring B. forsythus was 62% by immunofluorescence and 82% by PCR-DNA probe assay. The immunocapture, PCR. DNA-probe procedure should be useful for the detection of B. forsythus, particularly in false-negative samples obtained by less sensitive techniques.

The mechanisms by which Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is pathogenic for the periodontium remain largely hypothetical. Invasion of host tissues by P. gingivalis is believed to require adhesion of the bacterium to host cells. The aim of this study was to use monoclonal antibodies (MAbs) to characterize the bacterial cell surface component(s) acting as a ligand binding to a receptor on epithelial cells. Surface antigens of P. gingivalis ATCC 33277 were obtained as a glass bead-EDTA extract (GBE), and antiserum against the GBE was produced in rabbits. Epithelial cell membrane proteins (ECMP) were prepared from a homogenate of the SK-MES-1 cell line with Triton X-100. The antigen/ligand profile of GBE was resolved by crossed immunoaffinity electrophoresis by using ECMP in the first-dimension gel. The migration of one immunoprecipitate (IP) was retarded, indicating a ligand-receptor interaction between a surface antigen of P. gingivalis and a complementary binding site on the epithelial cell membrane. The corresponding IP in the GBE/anti-GBE immunoelectrophoresis profile was excised from replicate gels to immunize mice for production of MAbs specific for the bacterial ligand. Five MAbs were obtained and tested for reactivity with GBE in immunoblots and for inhibition of the interaction between GBE and ECMP. Immunoblots revealed polypeptides at 28, 42, 43, and 49 kDa. Inhibition tests were positive for all five MAbs. These results are conclusive evidence that the MAbs recognize functional epitopes involved in the adherence of P. gingivalis to epithelial cells and that the adhesins are likely associated with fimbriae and the hemagglutinating adhesin HA-Ag2.

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2-5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.

While the adhesive properties of Porphyromonas gingivalis are known to allow colonization of the subgingival tissues, the roles of fimbriae and adhesin molecules in hemagglutination remain unclear. The purpose of this study was to analyze the antigenic, structural, and functional relationships of these two components. Five populations of monoclonal antibodies were produced against (i) the hemagglutinating adhesin HA-Ag2 resolved by crossed immunoelectrophoresis (CIE), (ii) native fimbriae, and (iii) each of the three immunoprecipitates, Ag8a, Ag8b, and Ag8c, that define fimbriae by CIE. The tests used for characterization of the monoclonal antibodies included immunoblot reactivity, inhibition of hemagglutination, capacity to dissociate immunoprecipitates by CIE, localization of recognized epitopes by immunoelectron microscopy, and epitope mapping by competition enzyme-linked immunosorbent assay. The results from the different immunochemical tests clearly showed a close antigenic relationship between fimbriae and the hemagglutinating adhesin HA-Ag2. We were able to establish that the epitopic domain H1 of HA-Ag2 is hemagglutinin specific and that domain F2 is fimbria specific. Our data indicate that the polymeric structural unit of fimbriae must be complexed to HA-Ag2, the adhesin, to confer hemagglutination activity to the bacterial cells.

To evaluate the potential oral origin of Fusobacterium nucleatum found in amniotic fluid of women at high risk of preterm birth.A transversal study nested into a cohort study of women with preterm labor and/or preterm premature rupture of membranes was undergone. Women with the presence of F. nucleatum in the amniotic fluid and their respective partners were invited to be examined for their periodontal health after delivery, and samples of saliva and subgingival plaque were collected. For each couple, specific PCR detection of Fusobacterium species was performed on each oral sample, and the DNA sequences were compared with the one obtained from amniotic fluid.Three women, all in preterm labor with intact membranes, were included. Intra-amniotic sludge was observed in all of them. A strain of F. nucleatum with 100% sequence identity with the strain detected in the amniotic fluid was found in the oral samples of one of them and of two partners.This study suggests that intra-amniotic F. nucleatum could originate from the patient's or the partner's oral microflora.

Objective.Anticitrullinated protein antibodies (ACPA) are relatively specific for rheumatoid arthritis (RA), and predate disease. The oral pathogenPorphyromonas gingivalismay play a role in breaking immune tolerance to citrullinated antigens. We studied a cohort of patients with RA and their relatives looking for associations between anti-P. gingivalisantibodies and ACPA.Methods.Patients with RA (n = 82) and their relatives (n = 205) from a North American Native (NAN) population were studied, along with 47 NAN and 60 non-NAN controls. IgM and IgA rheumatoid factor (RF) were tested by nephelometry and ELISA. Second-generation anticyclic citrullinated peptide (anti-CCP2) isotypes and IgG anti-P. gingivalislipopolysaccharides were tested by ELISA. HLA-DRB1 typing was performed by sequencing. Oral hygiene and smoking habits were assessed by questionnaires.Results.Autoantibody frequency in patients with RA and relatives: ACPA 91% vs 19%, respectively; IgM RF 82% vs 17%; IgA RF 48% vs 22%. Anti-P. gingivalislevels were higher in patients with RA compared to relatives and controls (p = 0.005) and higher in ACPA-positive patients with RA than in ACPA-negative patients with RA (p = 0.04) and relatives (p < 0.001), but comparable in RF-positive and RF-negative patients and relatives. Poor oral hygiene and smoking were prevalent, but with no clear association with autoantibodies. Relatives with 2 shared-epitope alleles were more likely to be ACPA-positive (OR 2.5, p = 0.02).Conclusion.In a genetically predisposed population of NAN patients with RA and their relatives, anti-P. gingivalisantibodies were associated with ACPA. These findings suggest that immune responses toP. gingivalismay be involved in breaking immune tolerance to citrullinated antigens.

International audience; The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

International audience; Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

For some decades now, bacteria known as probiotics have been added to various foods because of their beneficial effects for human health. The mechanism of action of probiotics is related to their ability to compete with pathogenic microorganisms for adhesion sites, to antagonize these pathogens or to modulate the host"s immune response. The potential application of probiotics for oral health has recently attracted the attention of several teams of researchers. Although only a few clinical studies have been conducted so far, the results to date suggest that probiotics could be useful in preventing and treating oral infections, including dental caries, periodontal disease and halitosis. This article summarizes the currently available data on the potential benefits of probiotics for oral health.