Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

AIM: The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. METHODOLOGY: Twenty-five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. RESULTS: Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. CONCLUSIONS: This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss.

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. The effects of lymphatic drainage dysfunction on periodontitis have not been well studied. Here we show that lymphatic vessel endothelial receptor 1 (LYVE1)+ /podoplanin (PDPN)+ lymphatic vessels (LVs) are increased in the periodontal tissues, with accumulation close to the alveolar bone surface, in two murine periodontitis models: rheumatoid arthritis (RA)-associated periodontitis and ligature-induced periodontitis. Further, PDPN+ /alpha-smooth muscle actin (αSMA)- lymphatic capillaries are increased, whereas PDPN+ /αSMA+ collecting LVs are decreased significantly in the inflamed periodontal tissues. Both mouse models of periodontitis have delayed lymph flow in periodontal tissues, increased TRAP-positive osteoclasts, and significant alveolar bone loss. Importantly, the local administration of adeno-associated virus for vascular endothelial growth factor C, the major growth factor that promotes lymphangiogenesis, increases the area and number of PDPN+ /αSMA+ collecting LVs, promotes local lymphatic drainage, and reduces alveolar bone loss in both models of periodontitis. Lastly, LYVE1+ /αSMA- lymphatic capillaries are increased, whereas LYVE1+ /αSMA+ collecting LVs are decreased significantly in gingival tissues of patients with chronic periodontitis compared with those of clinically healthy controls. Thus, our findings reveal an important role of local lymphatic drainage in periodontal inflammation-mediated alveolar bone loss. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

Objective: Chronic periodontitis is a bone-destructive disease affecting periodontal support structures. Although leptin has a protective effect against periodontitis, the underlying mechanism remains unclear. Therefore, this study aimed to investigate the possible role of leptin by examining its relationship with OPG and RANKL in human gingival tissues obtained from patients with chronic periodontitis. Method: Twenty-two patients with chronic periodontitis were enrolled (10 with moderate periodontitis and 12 with severe periodontitis) in the experimental group, and 12 healthy individuals were enrolled in the control group. Gingival tissue samples were collected, and the protein levels and localization of leptin, OPG, and RANKL were studied using immunohistochemistry (IHC). The staining intensities of leptin, OPG, and RANKL were correlated with the periodontal clinical index. Moreover, real-time quantitative PCR (RT-qPCR) was used to determine OPG and RANKL mRNA levels in gingival fibroblasts stimulated with gradient concentrations of leptin protein in vitro. Result: Leptin, OPG, and RANKL were located in the cytoplasm of gingival epithelial cells and the connective tissue. Leptin was widely and significantly expressed in the control group, whereas it was lightly stained in the severe group. RANKL was lightly stained in the control group, whereas it was widely and significantly expressed in the severe group. The control and the moderate groups had similar OPG levels, which were significantly higher than that in the severe group. Leptin was positively correlated with OPG(r = 0.905, p < 0.01) and negatively correlated with RANKL (r = -0.635, p < 0.01). In vitro low concentrations of leptin led to an increased OPG/RANKL mRNA ratio, whereas the opposite effect was observed at high concentrations. Conclusion: Leptin can regulate OPG and RANKL expression in gingival fibroblasts and may thus play a role in the development of chronic periodontitis by modulating the OPG/RANKL ratio.

Exploring the putative interactions between chronic kidney disease and chronic periodontitis.

Chronic kidney disease (CKD) and chronic periodontitis (CP) are both common diseases, which are found disproportionately comorbid with each other and have been reported to have a detrimental effect on the progression of each respective disease. They have an overlap in risk factors and both are a source of systemic inflammation along with a wide selection of immunological and non-specific effects that can affect the body over the lifespan of the conditions. Previous studies have investigated the directionality of the relationship between these two diseases; however, there is a lack of literature that has examined how these diseases may be interacting at the localized and systemic level. This review discusses how oral microorganisms have the ability to translocate and have distal effects and provides evidence for microbial involvement in a systemic disease. Furthermore, it summarizes the reported local and systemic effects of CKD and CP and discusses how the interaction of these effects may be responsible for directionality associations reported.

Might smoking assuage the pro-inflammatory effect of diabetes in periodontal sites?

OBJECTIVES: This study evaluated the effects of type 2 diabetes mellitus (DM), smoking, and these two factors combined on gingival crevicular fluid levels and ratios of pro-/anti-inflammatory cytokines. Associations between cytokines with each other and with key periodontal pathogens in periodontal sites under the challenge of one or both of these risk factors were also assessed. METHODS: A total of 102 subjects with periodontitis were included in this cross-sectional study and assigned to one of the following groups: non-diabetic non-smokers (control group, n = 25), non-smokers with DM (DM group, n = 30), non-diabetic smokers (S group, n = 26), and smokers with DM (S + DM group, n = 21). The levels of 13 pro-inflammatory (IFN-γ, TNF-α, MIP-1α, GM-CSF, IL-1ß, IL-2, IL-6, IL-7, IL-8, IL-12, IL-17, IL-21, and IL-23) and 5 anti-inflammatory (IL-4, IL-5, IL-10, IL-13, and TGF-ß) cytokines were assessed in healthy and diseased sites, using multiplex immunoassay. Ratios of pro-/anti-inflammatory cytokines were obtained in all possible permutations. The levels of 7 key periodontal pathogens were evaluated by qPCR. RESULTS: Overall, the ratios of pro-/anti-inflammatory cytokines were higher in healthy and diseased sites of the DM group and in healthy sites of the S + DM group, and lower in diseased sites of the S group, compared with the control (p < .05). The proportion of the pro-inflammatory cytokines in relation to the 18 cytokines studied was higher in the DM group and lower in the S group, whereas the proportion of the anti-inflammatory cytokines was lower in both diabetic groups and higher in the S group, compared to the control (p < .05). A cluster of six common cytokines (IL-4, IL-5, IL-12, IL-13, IL-21, and IL-23) was observed in the diseased sites of all groups studied. Eight common cytokines (IL-4, IL-5, IL-12, IL-13, IL-17, IL-21, IL-23, and IFN-γ) grouped closely in the healthy sites of both diabetic groups. Significant associations between pathogens and cytokines occurred mainly in the diseased sites of the S + DM group (p < .05). CONCLUSION: Diabetes mellitus induced an overall pro-inflammatory state, while smoking mainly stimulated immunosuppression in periodontal sites. When the two risk factors overlapped, smoking seemed to partially assuage the hyperinflammatory effect of DM.

Macrophage polarization in human gingival tissue in response to periodontal disease.

OBJECTIVE: Although accumulating evidence indicates that macrophages are central players in the destructive and reparative phases of periodontal disease, their polarization states at different stages of periodontal inflammation remain unclear. METHODS: We collected gingival biopsies from patients with chronic periodontitis (P group), gingivitis (G group), or periodontally healthy individuals (H group). Polarized macrophages were identified through immunofluorescence. M1- and M2-related cytokines were detected by immunohistochemistry. RESULTS: Compared with the H group, the P group had more M1 cells (higher M1/M2 ratio) and significantly higher TNF-α, IFN-γ, IL-6, and IL-12 levels. Although the G group also exhibited higher TNF-α and IL-12 levels than the H group, they had similar M1/M2 ratios. The M1/M2 ratio and IFN-γ and IL-6 levels were significantly higher in the P than the G group. Among M2-related cytokines, IL-4 levels were significantly higher in the G than the H group. The M1/M2 ratio was positively correlated with clinical probing depth (PD), and both were positively correlated with IFN-γ and IL-6. PD was negatively correlated with IL-4. CONCLUSION: Macrophage polarization in gingival tissue may be responsible for the development and progression of inflammation-induced tissue destruction, and modulating macrophage function may be a potential strategy for periodontal disease management.

Upregulation of proteins of the NLRP3 inflammasome in patients with periodontitis and uncontrolled type 2 diabetes.

OBJECTIVES: To evaluate the expression of proteins related to activation of the NLRP3 inflammasome in patients with chronic periodontitis (CP) and type 2 diabetes mellitus (T2D), and to determine whether the exacerbated periodontal pathological process observed in diabetic patients is related to its upregulation. MATERIALS AND METHODS: We performed an observational, analytical, cross-sectional study in three study groups: individuals systemically and orally healthy, and patients with CP with and without T2D. Gingival biopsies were taken from the three study groups. The expression of mRNAs for CASP1, NLRP3 and ASC was detected using real-time PCR, and the expression of NLRP3 and ASC proteins was determined by immunohistochemistry. The quantification of IL-18 and IL-1ß was determined in the gingival crevicular fluid using ELISA. The results were analysed by ANOVA followed by Tukey's test to compare differences between individual groups. RESULTS: Patients with CP and uncontrolled T2D presented severe periodontal disease and inflammation (PPD, p = 0.0072; CAL, p = 0.0480; bone loss, p = 0.0088), higher levels of CASP1 mRNA expression (p = 0.0026), a stronger pattern of staining for NLRP3 and ASC proteins in the epithelium and connective tissues, and significantly higher production of IL-18 (p = 0.0063) and IL-1ß (p = 0.0018) in comparison with healthy or CP subjects. CONCLUSION: The upregulation of genes and proteins involved in the activation of the NLRP3 inflammasome components in patients with periodontitis and uncontrolled T2D suggests a possible role in the more severe pathological processes leading to destruction of periodontal tissues observed in these patients.

Association of initial mucogingival status with clinical outcome of non-surgical periodontal therapy: A retrospective analysis of 204 patients.

BACKGROUND/PURPOSE: This study was conducted to evaluate the influence of mucogingival parameters, including keratinized mucosa (KM) and attached gingiva (AG), on the outcome of non-surgical periodontal therapy (NSPT). METHODS: A total of 204 non-smoking patients with generalized chronic periodontitis who received NSPT between 2012 and 2014 were included. The Mantel-Haenszel chi-square test was used to assess the associations between initial mucogingival parameters and initial clinical parameters on the buccal aspect, and the associations between initial mucogingival parameters and outcome clinical parameters on the buccal aspect of the sites with severe periodontal destruction. The generalized liner model was used to evaluate the contribution of initial clinical parameters to the outcome of NSPT. RESULTS: KM ≥ 3 mm was associated with greater probing pocket depth (PD), less gingival recession (REC), and less clinical attachment level (CAL), and AG < 1 mm was associated with greater PD, REC, and CAL before NSPT. At the sites with severe periodontal destruction, KM ≥ 3 mm was associated with greater PD reduction (0.25 ± 0.08 mm) and CAL gain (0.25 ± 0.09 mm), and AG < 1 mm was associated with greater CAL gain (0.15 ± 0.08 mm) after NSPT. Initial PD ≥ 7 mm and non-molar teeth showed greater contribution to the outcome of NSPT. CONCLUSION: Less AG (<1 mm) was associated with greater periodontal destruction at baseline. At the sites with severe periodontal destruction, greater KM (≥3 mm) and less AG (<1 mm) resulted in better outcomes of NSPT.

Disease severity, debridement approach and timing of drug modify outcomes of adjunctive azithromycin in non-surgical management of chronic periodontitis: a multivariate meta-analysis.

BACKGROUND: Past meta-analyses have shown adjunctive systemic Azithromycin (AZI) to provide minor clinical benefits in scaling and root surface debridement (S/RSD). However, these have not considered the covariance of key outcome parameters; probing pocket depth (PPD) and Clinical Attachment Level (CAL) or systematically examined some potential sources of heterogeneity. AIM: To jointly synthesize 6-month outcomes of systemic AZI as adjunctive to S/RSD in chronic periodontitis and investigate 3 potential sources of heterogeneity. METHODS: Four databases were searched for suitable randomized controlled clinical trials (RCTs). Standardized mean differences (SMD) in PPD and CAL between AZI + S/RSD and S/RSD alone, at 6-month follow-up were computed. Within-study covariances of PPD and CAL were derived from reported multiple time-point data. A multivariate meta-analysis with random effects jointly modelled PPD and CAL, factoring in their covariance. This model included 3 moderators with interaction effects; timing of AZI initiation (pre-therapy/post-therapy), type of S/RSD [full-mouth debridement (FMD)/partial-mouth debridement (PMD)], and baseline study-level mean values of PPD/CAL. RESULTS: Among 276 abstracts, 11 observations from 9 RCTs qualified for meta-analysis. Within-study correlation-coefficients of PPD with CAL significantly increased with increasing study-level baseline mean values (Spearman's r = 0.79, p < 0.01). The full multivariate meta-analysis model showed significant effects for the 3 moderators (Q statistic = 150.03, p < 0.01), retained significant residual heterogeneity (Q statistic = 88.50, p < 0.01) but outperformed (Likelihood- ratio statistic = 102.95, p < 0.01,) a null-model with no moderators (Q statistic = 201.5, p < 0.01). A significant effect was seen only on the SMD for PPD (estimate = 1.16 mm, 95% CI: 0.27 mm-2.07 mm mm, p = 0.01) but not CAL (estimate = 0.17 mm, 95% CI: -0.92 mm-1.26 mm, p = 0.76). SMD in PPD positively interacted with study baseline value (estimate = 0.11, 95% CI: 0.08-0.15, p < 0.01). Significant negative interactions of SMD in PPD with PMD (estimate = - 1.25 mm, 95% CI: -1.73 mm- -0.78 mm, p < 0.01) and pre-therapy drug initiation (estimate = - 1.18 mm, 95% CI: -1.48 mm--0.87 mm, p < 0.01) were evident. CONCLUSION: Joint synthesis of PPD and CAL showed, at 6-months, AZI + S/RSD provided a benefit over S/RSD alone for PPD alone when correlation with CAL was accounted for. Deeper study-level baseline PPD, FMD type of S/RSD, and post-therapy drug initiation associated with greater PPD reduction.

Immunohistochemical and Morphological Study of Periodontal Tissues in Predicting the Results of Dental Implantation in Patients with Chronic Periodontitis.

Morphological and immunohistochemical examination was made on 24 gum biopsies obtained from 35- to 60-year-old patients with diagnosis of partial secondary adentia, chronic generalized moderate to severe periodontitis (19 patients), as well as on the biopsy samples from five patients without pathological periodontal changes who underwent dental implantation. Serial paraffin sections were treated with antibodies against Ki-67, VEGF, and SMA. In patients with severe chronic periodontitis, a high proliferative activity of epithelium indicative of hyperplastic changes was observed, as well as a reduced number of the SMA-positive cells and actual absence of the SMA-positive cell couplings associated with the "growth zones" in tissues, which testifies indirectly to a lower tissue regenerative capacity. Hence, before dental implantation, additional anti-inflammatory and pro-regenerative treatment is required.

Genome-wide association study of biologically informed periodontal complex traits offers novel insights into the genetic basis of periodontal disease.

Genome-wide association studies (GWAS) of chronic periodontitis (CP) defined by clinical criteria alone have had modest success to-date. Here, we refine the CP phenotype by supplementing clinical data with biological intermediates of microbial burden (levels of eight periodontal pathogens) and local inflammatory response (gingival crevicular fluid IL-1ß) and derive periodontal complex traits (PCTs) via principal component analysis. PCTs were carried forward to GWAS (∼2.5 million markers) to identify PCT-associated loci among 975 European American adult participants of the Dental ARIC study. We sought to validate these findings for CP in the larger ARIC cohort (n = 821 participants with severe CP, 2031-moderate CP, 1914-healthy/mild disease) and an independent German sample including 717 aggressive periodontitis cases and 4210 controls. We identified six PCTs with distinct microbial community/IL-1ß structures, although with overlapping clinical presentations. PCT1 was characterized by a uniformly high pathogen load, whereas PCT3 and PCT5 were dominated by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. We detected genome-wide significant signals for PCT1 (CLEC19A, TRA, GGTA2P, TM9SF2, IFI16, RBMS3), PCT4 (HPVC1) and PCT5 (SLC15A4, PKP2, SNRPN). Overall, the highlighted loci included genes associated with immune response and epithelial barrier function. With the exception of associations of BEGAIN with severe and UBE3D with moderate CP, no other loci were associated with CP in ARIC or aggressive periodontitis in the German sample. Although not associated with current clinically determined periodontal disease taxonomies, upon replication and mechanistic validation these candidate loci may highlight dysbiotic microbial community structures and altered inflammatory/immune responses underlying biological sub-types of CP.

Comparative analysis of immune cell subsets in peripheral blood from patients with periodontal disease and healthy controls.

Periodontitis is a chronic inflammatory disease caused by the colonization of teeth by the bacterial plaque biofilm and the resultant host immune responses in adjacent periodontal tissues. Disease severity can vary dramatically between patients with periodontitis, with some subjects displaying inflammation without bony destruction (gingivitis), while others experience chronic progressive or rapidly aggressive gingival connective tissue damage and bone loss. To determine whether peripheral immune dysregulation is associated with periodontitis, we performed extensive analysis of immune cell subsets in peripheral blood from patients with chronic or aggressive periodontitis versus periodontally healthy control subjects. Peripheral blood mononuclear cells (PBMC) from patients with chronic periodontitis or aggressive periodontitis and from periodontally healthy controls were analysed by 8-10-colour flow cytometry for the frequencies of various lymphocyte subsets, including interleukin (IL)-17-, interferon (IFN)-γ-, tumour necrosis factor (TNF)-α- and IL-10-producing cells, and the frequencies and phenotype of monocytes. Cytokine levels in serum from the different groups were determined by Luminex assay. We found no significant differences in the frequencies of major immune cell populations [CD4+ T cells, CD8+ T cells, γδ T cells, CD4+ CD45RO+ CD25+ CD127low regulatory T cells (Tregs ), CD19+ B cells, CD14+ monocytes] or of cytokine-producing T cells, or in the phenotype of CD14+ monocytes in peripheral blood from these patient cohorts. Additionally, no significant differences were observed in serum levels of prototypical inflammatory cytokines. These results suggest that the local gingival inflammatory response is not reflected by obvious changes in major blood immune cell subset frequencies.

Detection of antibodies against Aggregatibacter actinomycetemcomitans in serum and saliva through ELISA in periodontally healthy individuals and individuals with chronic periodontitis.

BACKGROUND: Periodontitis is a persistent polymicrobial infection, which leads to chronic inflammation in the tooth supporting tissues. Aggregatibacter actinomycetemcomitans are normal commensals of oral cavity but are low in number in periodontally healthy subjects. They are one of the major pathogens aetiologically linked to periodontal disease. Plasma and salivary antibody measurement may be useful to support diagnosis, disease activity, classification and prognosis of periodontitis. The aim of the present study was to investigate the association between the serum and salivary antibody levels to A. actinomycetemcomitans and therefore, to find whether this association was varying in different grades of periodontitis. METHOD: Total of 50 periodontally healthy and 50 chronic periodontitis subjects (35-65 years) of both sexes were included for the study. 2 ml of un-stimulated saliva and 5 ml of venous blood was collected under sterile conditions. The detection of antibodies against A. actinomycetemcomitans in periodontally healthy individuals and individuals with chronic periodontitis was performed using indirect ELISA. RESULTS: Results showed serum IgG, IgA mean levels against A. actinomycetemcomitans were higher in chronic periodontitis subjects compared to mean levels in periodontally healthy subjects. Similarly, salivary IgG, IgA levels were also raised in chronic periodontitis patients as compared in healthy subjects. Also the mean levels of serum IgG and salivary IgA were increased as the severity of disease increased. CONCLUSION: Antibody titer using saliva and serum could be useful tool for screening of patients with chronic periodontitis. Further, monitoring the various phases of treatment outcome using saliva could be a useful, non-invasive, prognostic indicator.

Morphological characterization of para- and proinflammatory neutrophil phenotypes using transmission electron microscopy.

BACKGROUND AND OBJECTIVE: Bacterial challenge is constant in the oral cavity. To contain the commensal biofilm, partly activated neutrophils are continuously recruited as part of a normal physiologic process, without exposing the host to the harmful effect of a fully active neutrophil response. This intermediate immune state has been termed para-inflammation, as opposed to the fully activated proinflammatory state in oral disease. Directly visualizing these cells and their components via transmission electron microscopy (TEM) enhances our understanding of neutrophil activation state differences in oral health and disease, as obtained from molecular studies. The aim of this study was to describe the morphology of the para-inflammatory phenotype displayed by oral neutrophils in health, and compare it to the morphology of the naïve blood neutrophil, and the proinflammatory oral neutrophils in chronic periodontitis. This morphology was characterized by differences in granule content, phagosome content and cytoplasm and nuclear changes. We also examined the morphological changes induced in naïve neutrophils, which were stimulated in vitro by bacteria, and in oral neutrophils in full tissue samples in vivo. MATERIAL AND METHODS: Neutrophils were isolated from blood and saliva samples of patients with chronic periodontitis and healthy individuals. The cells were viewed under TEM and analyzed in imaging software examining granularity, cytoplasm density, euchromatin amount in the nucleus and phagosome content. A separate cohort of blood neutrophils was incubated with Streptococcus oralis and analyzed under TEM in the same manner. Gingival tissue samples were obtained from patients with chronic periodontitis and viewed under TEM, with the neutrophils present analyzed in the same manner. RESULTS: The proinflammatory cells showed less granulation, lighter cytoplasm and higher amount of nuclear euchromatin. These changes were accentuated in the proinflammatory oral chronic periodontitis neutrophils compared to the para-inflammatory oral health neutrophils. The oral chronic periodontitis neutrophils also contained more phagosomes and had more phagosomes containing undigested bacteria. These changes were partially reproduced in the naïve blood cells after exposing them to S. oralis. The neutrophils in the gingival tissues displayed naïve morphology when viewed in the blood vessels and gradually showed proinflammatory morphological changes as they traveled through the connective tissue into the epithelium. CONCLUSION: Oral neutrophils display morphological changes consistent with partial or full activation, corresponding to their para- or proinflammatory states. These changes can also be induced in naïve cells by incubating them with commensal bacteria. Neutrophils change their morphology towards an activated state as they travel through the gingival tissue.

The role of extracellular matrix metalloproteinase inducer glycosylation in regulating matrix metalloproteinases in periodontitis.

BACKGROUND AND OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that may induce activation of matrix metalloproteinases (MMPs) and lead to the destruction of periodontal tissue. The level of EMMPRIN glycosylation might be involved in this process. This study aims to investigate the role of EMMPRIN glycosylation in regulating MMP-2 and MMP-9 during the progression of periodontitis. MATERIAL AND METHODS: Gingival tissues were collected from patients with chronic periodontitis and from patients undergoing crown-lengthening procedures (healthy gingival tissue). Tissues were used for immunohistochemistry and double immunofluorescence. A human immortalized oral epithelial cell (HIOEC) line was stably transfected by an N-acetylglucosaminyltransferase-V (GnT-V) RNA interference (RNAi) lentivirus to suppress EMMPRIN glycosylation. Gene silence efficiency was detected by western blot, quantitative real-time PCR and immunofluorescence (IF) staining. An HIOEC/human gingival fibroblast (HGF) co-culture model and an individual culture model were used in this study. After exposure of cells to Porphyromonas gingivalis lipopolysaccharide (Pg. LPS), the expression of EMMPRIN, MMP-2 and MMP-9 were assessed by western blot, quantitative real-time PCR and IF, and the secretion of MMP-2 and MMP-9 were detected by gelatin-degradation assays. RESULTS: Compared with the periodontally healthy group, patients with periodontitis showed increased expression of EMMPRIN on the gingival epithelial cell membrane. GnT-V, a key regulator of EMMPRIN glycosylation, was co-expressed with EMMPRIN in gingival epithelial cells in patients with periodontitis. Knockdown of GnT-V reduced the level of EMMPRIN glycosylation in HIOECs. Furthermore, in the HIOEC/HGF co-culture model, stimulation with Pg. LPS (10 µg/mL, 4 hours) promoted EMMPRIN glycosylation and increased the activities of MMP-2 and MMP-9, while suppression of EMMPRIN glycosylation by GnT-V knockdown reduced the synthesis and activities of MMP-2 and MMP-9 under Pg. LPS stimulation. Moreover, the gelatin-degradation assay showed that inhibition of EMMPRIN glycosylation suppressed the Pg. LPS-induced degradation of gelatin in the co-culture model. CONCLUSION: We conclude that EMMPRIN glycosylation participates in the regulation of MMP-2 and MMP-9 production through mediating the interaction of HIOECs and HGFs. Inhibiting EMMPRIN glycosylation can reduce the activation of MMP-2 and MMP-9 and suppress the degradation of extracellular matrix (ECM) in the HIOEC/HGF co-culture model. Therefore, this study suggests that EMMPRIN glycosylation may affect the host immune-inflammatory response by regulating MMPs in periodontitis.

Strontium ion attenuates lipopolysaccharide-stimulated proinflammatory cytokine expression and lipopolysaccharide-inhibited early osteogenic differentiation of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease characterized by the destruction of the periodontium. The strontium ion (Sr2+ ) can prevent the bone loss associated with periodontitis and promote the regeneration of the bone. The mechanisms by which the Sr2+ works remain poorly understood. We aim to investigate the effects of the Sr2+ ion on cell proliferation, inflammatory regulation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) in pathological conditions. MATERIAL AND METHODS: hPDLCs were obtained from premolars that came from the orthodontic extraction. The hPDLCs were treated with Sr2+ and/or lipopolysaccharide (LPS), which was applied as the pathological condition of periodontitis. The effect of the dose of Sr2+ on cell proliferation was analyzed using a Cell Counting Kit-8 assay. The gene and protein expression of proinflammatory cytokines were detected by the real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The osteogenic differentiation and mineralization were assessed by the real-time polymerase chain reaction, alkaline phosphatase activity assay and alizarin red staining. RESULTS: Results demonstrated that Sr2+ in a range of concentrations from 0.02 to 2.5 mmol/L significantly improved the proliferation of hPDLCs. Sr2+ reversed LPS-stimulated proinflammatory cytokine expressions such as tumor necrosis factor alpha, interleukin (IL)-1ß, IL-6 and IL-8. Moreover, Sr2+ rescued the LPS-inhibited gene expression of osteogenic differentiation. Although it appeared to suppress the late mineralization, Sr2+ can reverse the LPS-inhibited early osteogenic differentiation of hPDLCs. CONCLUSION: These results indicated that Sr2+ could attenuate the LPS-stimulated proinflammatory molecule expression and inhibit early osteogenic differentiation of hPDLCs.

The effect of horizontal and vertical furcation involvement on molar survival: A retrospective study.

BACKGROUND: Periodontal involvement of the furcation area (furcation involvement, FI) has been reported to confer molars a higher risk of tooth loss. AIMS: The aim of this retrospective analysis was to assess the effect of FI on disease progression and tooth loss in molars of patients with chronic periodontitis undergoing supportive periodontal therapy (SPT) in a UK private practice setting. MATERIALS AND METHODS: Six-hundred and thirty-three molars were analysed in 100 chronic periodontitis patients treated with active periodontal therapy (APT) and followed up in SPT for at least 5 years. Molars were treated with a combination of resective, regenerative or conservative approaches, according to the different clinical needs. RESULTS: Twenty-three molars were extracted during APT and a further 23 were lost during SPT. Multivariable analysis showed that both horizontal FI and vertical furcation component were associated with increased risk of tooth loss during SPT (OR 5.26, 95% CI: 1.46-19.03, p = .012 and OR 9.83, 95% CI: 1.83-50.11, p = .006, respectively). CONCLUSION: Attention should be placed on both horizontal and vertical FI in molars, owing to their association with tooth loss during SPT.

A retrospective study on the crestal bone loss associated with different implant surfaces in chronic periodontitis patients under maintenance.

OBJECTIVE: To analyze retrospectively interproximal crestal bone loss (CBL) on external-hex "non-identical" (NI) dental implants with different surface topography, when placed in the same intra-oral location in patients with a history of chronic periodontitis following maintenance care. MATERIAL AND METHODS: The patient population consisted of 206 consecutive patients with a history of chronic periodontitis who underwent implant surgery between 2007 and 2010; 755 NI implants with different implant surfaces were placed at posterior mandibular sites: 72 machined, 145 acid-etched/machined (hybrid), and 538 anodized. Crestal bone loss measurements were carried out analyzing the calibrated digital X-rays taken at 1-year intervals as part of the maintenance program, being the time of this retrospective examination from 1 to 3 years. RESULTS: At 3 years (51 patients), the mean CBL was 1.36/1.35 mm at implant/patient level (range: 0-6 mm). A higher CBL was observed at anodized surface implants, when compared to machined and hybrid implants, being the mean CBL 1.48, 0.96, and 0.77 mm, respectively (p = .005). CBL between 2 and 3.9 mm was found in 10.6% of the implants at 3 years (95% CI: 6%-15%), ≥4 mm in 5% (95% CI: 2%-8%). Anodized surface implants had CBL >2 mm at 2 years of 9% (95% CI: 6%-12%) and at 3 years of 18% (95% CI: 12%-24%), which were 2.5 times higher than hybrid implants with 3.6% (95% CI: 0.8%-4%) and 7.4% (95% CI: 0%-17%), respectively. CONCLUSIONS: A higher CBL was observed in the anodized surface implants group, when compared to the hybrid implants group in patients with a history of chronic periodontitis followed during 1-3 years.

[Association between chronic periodontitis and carotid intima-media thickness of middle-aged and older adults in Shijingshan community of Beijing].

OBJECTIVE: To investigate the relationship between periodontal disease and subclinical atherosclerosis in middle-aged and older adults in Shijingshan community of Beijing. METHODS: In 2005-2010, a total of 830 middle-aged and older adults were recruited from Shijingshan community of Beijing, who were divided into two groups by severity of periodontitis. A questionnaire, periodontal examination, blood biochemical examination, carotid intima-media thickness (CIMT), including common carotid artery (CCA-IMT), internal carotid artery (ICA-IMT) and carotid bifurcation (CB-IMT), were measured of each subject. The associations of periodontitis with CIMT was evaluated by multivariable Logistic regression analysis and analysis of covariance, adjusted for age, gender, education level, hypertension, hyperlipidemia, obesity, smoking, drinking, and diabetes. And then anther definition of periodontitis (mild periodontitis: percentage of AL≥3 mm <10%; moderate periodontitis: percentage of AL≥3 mm 10%-30%; severe periodontitis: percentage of AL≥3 mm ≥30%) was used to investigate the hypotheses that different classification of periodontitis would affect results. RESULTS: The subjects with moderate-severe periodontitis were characterized by significantly higher levels of CCA-IMT, ICA-IMT, CB-IMT and mean CIMT than the mild group. In the univariate analysis, moderate-severe periodontitis was associated with an increased risk of ICA-IMT>0.9 mm (adjusted OR=1.230, 95% CI: 1.058-1.452, P=0.031). Furthermore, moderate periodontitis was associated with an increased risk of CB-IMT>0.9 mm (adjusted OR: 1.297, 95%CI: 1.117-1.753, P=0.011) and severe periodontitis was associated with an increased risk of CB-IMT>0.9 mm (adjusted OR=1.771, 95%CI: 1.176-3.503, P=0.042) according to another classification of periodontitis. CONCLUSION: Our data provided evidence that periodontitis was associated with CIMT among middle-aged and older adults in Shijingshan community of Beijing and different periodontitis classification would affect the results.

[Cytological characteristic of the buccal cells in chronic generalized periodontite.]

The research is devoted to the study of the occurrence of abnomal cells in buccal epithelium in patients with chronic generalize periodontitis. Buccal cytograms of 102 patients were examined. It was established that cells with karyopycnosis, karyorexis and karyolysis are a sensitive index of the initial stage (mild severity) in periodontitis. The number of buccal cells with these features was increased in 2.1; 3.0; 13.4 times for chronic generalized periodontitis of mild, moderate and severe degree, respectively. The obtained data shown the accumulation of cytological anomalies (micronucleus, protrusion of the nucleus and other) in patients with chronic generalized periodontitis.