Tyrosine-protein phosphatase non-receptor type 2 inhibits alveolar bone resorption in diabetic periodontitis via dephosphorylating CSF1 receptor.

Tyrosine-protein phosphatase non-receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25-Hydroxyvitamin D3 (25(OH)2D3 ) on diabetic periodontitis. 25(OH)2D3 photo-affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D3 , which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony-stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss-related diseases.

Gingival Tissue Inflammation Promotes Increased Matrix Metalloproteinase-12 Production by CD200Rlow Monocyte-Derived Cells in Periodontitis.

Irreversible tissue recession in chronic inflammatory diseases is associated with dysregulated immune activation and production of tissue degradative enzymes. In this study, we identified elevated levels of matrix metalloproteinase (MMP)-12 in gingival tissue of patients with the chronic inflammatory disease periodontitis (PD). The source of MMP12 was cells of monocyte origin as determined by the expression of CD14, CD68, and CD64. These MMP12-producing cells showed reduced surface levels of the coinhibitory molecule CD200R. Similarly, establishing a multicellular three-dimensional model of human oral mucosa with induced inflammation promoted MMP12 production and reduced CD200R surface expression by monocyte-derived cells. MMP12 production by monocyte-derived cells was induced by CSF2 rather than the cyclooxygenase-2 pathway, and treatment of monocyte-derived cells with a CD200R ligand reduced CSF2-induced MMP12 production. Further, MMP12-mediated degradation of the extracellular matrix proteins tropoelastin and fibronectin in the tissue model coincided with a loss of Ki-67, a protein strictly associated with cell proliferation. Reduced amounts of tropoelastin were confirmed in gingival tissue from PD patients. Thus, this novel association of the CD200/CD200R pathway with MMP12 production by monocyte-derived cells may play a key role in PD progression and will be important to take into consideration in the development of future strategies to diagnose, treat, and prevent PD.

Increased citrullination and expression of peptidylarginine deiminases independently of P. gingivalis and A. actinomycetemcomitans in gingival tissue of patients with periodontitis.

BACKGROUND: A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. METHODS: Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. RESULTS: Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. CONCLUSION: Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.

Bismuth subsalicylate nanoparticles with anaerobic antibacterial activity for dental applications.

In recent years, nanomaterials have been used in the medical-dental field as new alternative antimicrobial agents. Bismuth subsalicylate (BSS) has been used as an antimicrobial agent, but the effect of BSS in the form of nanoparticles (BSS-nano) as a potential antimicrobial agent has not been tested, in specific against bacteria responsible for periodontal disease. The aim of this study was to evaluate the antibacterial effect of BSS-nano against oral anaerobic bacteria and to assess the safety of BSS-nano by evaluating their cytotoxicity in human gingival fibroblast (HGF-1) cells. BSS-nano were synthesized by laser ablation and were previously physico-chemically characterized using in vitro assays. The antibacterial activity was measured using the tetrazolium-based XTT assay, and cytotoxicity was determined using lactate dehydrogenase (LDH) and MTS assays in HGF-1 cells. Transmission electron microscopy of HGF-1 exposed to BSS-nano was also performed. BSS-nano was shown to have a primary size of 4-22 nm and a polygonal shape. Among the tested bacterial strains, those with a greater sensitivity to BSS-nano (highest concentration of 21.7 µg ml-1) were A. actinomycetemcomitans, C. gingivalis, and P. gingivalis. BSS-nano at a concentration of 60 µg ml-1 showed low cytotoxicity (6%) in HFG-1 cells and was mainly localized intracellularly in acidic vesicles. Our results indicate that the concentration of BSS-nano used as an effective antibacterial agent does not induce cytotoxicity in mammalian cells; thus, BSS-nano can be applied as an antibacterial agent in dental materials or antiseptic solutions.

Diabetes may affect the expression of matrix metalloproteinases and their inhibitors more than smoking in chronic periodontitis.

BACKGROUND AND OBJECTIVE: No previous study has directly compared the levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) between smokers and individuals with diabetes mellitus (DM) with periodontitis. Therefore, the aim of this study was to evaluate the gene expression of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 in tissues with chronic periodontitis (ChP) of smokers and individuals with type 2 DM. MATERIAL AND METHODS: Gingival biopsies were harvested from: non-smokers and non-diabetic individuals with ChP (n = 18) (ChP group); non-diabetic smokers (≥ 10 cigarettes per day for at least the past 5 years) with ChP (n = 18) (SChP group); non-smoking individuals with type 2 diabetes (glycated hemoglobin levels ≥ 7.5%) and ChP (n = 18) (DMChP group). The tissue levels of mRNA of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 were evaluated by quantitative real-time polymerase chain reaction. RESULTS: The MMP-8 expression was the lowest in the ChP group (p < 0.05). The DMChP group presented increased mRNA levels of MMP-2 and MMP-9, when compared to the SChP group (p < 0.05). MMP-1 expression and the MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-8/TIMP-1, MMP-9/TIMP-1, MMP-1/TIMP-2 and MMP-2/TIMP-2 ratios were higher in the DMChP group than in the ChP and SChP groups (p < 0.05). The DMChP group presented lower mRNA levels of TIMP-1 than the ChP group (p < 0.05). The MMP-8/TIMP-2 ratio was the highest in the SChP group (p < 0.05). CONCLUSION: Uncontrolled type 2 DM upregulates the ratio of MMP/TIMPs in sites with ChP more than smoking, which may contribute to a greater extracellular matrix degradation and periodontal breakdown in DM-related periodontitis.

Gene-expression analysis of matrix metalloproteinases 1 and 2 and their tissue inhibitors in chronic periapical inflammatory lesions.

BACKGROUND: Periapical inflammatory lesions have been investigated previously, but understanding of pathogenesis of these lesions (granulomas and radicular cysts) at the molecular level is still questionable. Matrix metalloproteinases (MMPs) are enzymes involved in the development of periapical pathology, specifically inflammation and tissue destruction. To elucidate pathogenesis of periapical granulomas and radicular cysts, we undertook a detailed analysis of gene expression of MMP-1, MMP-2 and their tissue inhibitors, TIMP-1 and TIMP-2. METHODS: A total of 149 samples were analyzed using real-time PCR (59 radicular cysts, 50 periapical granulomas and 40 healthy gingiva samples as controls) for expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 genes. The determination of best reference gene for expression analysis of periapical lesions was done using a panel of 12 genes. RESULTS: We have shown that ß-actin and GAPDH are not the most stable reference controls for gene expression analysis of inflammatory periapical tissues and healthy gingiva. The most suitable reference gene was determined to be SDHA (a succinate dehydrogenase complex, subunit A, flavoprotein [Fp]). We found that granulomas (n = 50) and radicular cysts (n = 59) exhibited significantly higher expression of all four examined genes, MMP-1, MMP-2, TIMP-1, and TIMP-2, when compared to healthy gingiva (n = 40; P < 0.05). CONCLUSION: This study has confirmed that the expression of MMP-1, MMP-2, TIMP-1, and TIMP-2 genes is important for the pathogenesis of periapical inflammatory lesions. Since the abovementioned markers were not differentially expressed in periapical granulomas and radicular cysts, the challenge of finding the genetic differences between the two lesions still remains.

Noncanonical activation of ß-catenin by Porphyromonas gingivalis.

Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on ß-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of ß-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for ß-catenin processing. The ß-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3ß were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that ß-catenin fragments were translocated to the nucleus. The accumulation of ß-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the ß-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis-induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of ß-catenin and disassociation of the ß-catenin destruction complex by gingipain-dependent proteolytic processing. ß-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype.

Expression of cathepsin K in periodontitis and in gingival fibroblasts.

OBJECTIVE: To study non-osteoclastic sources of cathepsin K in periodontitis. MATERIALS AND METHODS: Tissue samples were obtained from 10 otherwise healthy periodontitis pati-ents during routine periodontal flap operations and 10 systemically and periodontally healthy individuals who underwent extraction operations for retained third molars. Methods used were immunohistochemistry, image analysis, immunofluorescence double-staining, gingival fibroblast culture, tumour necrosis factor-α (TNF-α) stimulation and Western blotting. RESULTS: Macrophage-like cells, fibroblast-like cells, vascular endothelial cells and gingival epithelial cells were more intensively stained for cathepsin K and also more frequent in periodontitis than in controls (665 ± 104 vs 258 ± 40 cells mm(-2) , P < 0.01). Some cathepsin K(+) cells in periodontal tissues were CD68(+) , but some were CD68(-) and probably fibroblasts. Indeed, in gingival fibroblast culture, resting fibroblasts released cathepsin K, more 43 kD procathepsin K than 29 kD active cathepsin K. TNF-α increased the release of the activated cathepsin K 4- to 5-fold. CONCLUSIONS: Results suggest that GCF-cathepsin K is not only osteoclast-derived, but in periodontitis, also other cells contribute to it. GCF-cathepsin K, perhaps together with intracellular, lysosomal collagenolytically active cathepsin K in fibroblasts, macrophages and gingival epithelial cells, can contribute to the loss of attachment and destruction of the periodontal ligament.

A dual role for ß1 integrin in an in vitro Streptococcus mitis/human gingival fibroblasts co-culture model in response to TEGDMA.

AIM: To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY: ß1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase ß (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS: When HGFs are co-cultured with S. mitis, ß1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS: ß1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.

Low-dose combination of alendronate and atorvastatin reduces ligature-induced alveolar bone loss in rats.

BACKGROUND AND OBJECTIVE: Atorvastatin (ATV) has bone anabolic properties, and alendronate (ALD) is an important antiresorptive drug. This study aimed to evaluate the effects of the combination of ALD and ATV on ligature-induced alveolar bone loss in rats. MATERIAL AND METHODS: Periodontitis was induced by ligature in 78 Wistar rats. Groups of six rats prophylactically received 0.9% saline (SAL), ALD (0.01 or 0.25 mg/kg subcutaneously) or ATV (0.3 or 27 mg/kg by gavage). Then, groups of six rats received the combination of ALD+ATV (0.25 mg/kg + 27 mg/kg, 0.01 mg/kg + 0.3 mg/kg, 0.25 mg/kg + 0.3 mg/kg or 0.01 mg/kg + 27 mg/kg) prophylactically. An extra group of six rats received therapeutic SAL or a lower-dose combination of ALD+ATV (0.01 mg/kg + 0.3 mg/kg, respectively) therapeutically. Three extra groups of six rats each received SAL or a lower-dose combination of ALD+ATV (0.01 mg/kg + 0.3 mg/kg, respectively) prophylactically or therapeutically for histometric and immunohistochemical analyses. The rats were killed on day 11 after ligature placement, and the maxillae were removed and processed for macroscopic, histomorphometric and TRAP immunohistochemical analyses. Gingival samples were collected to evaluate myeloperoxidase (MPO) activity. Blood samples were collected to measure serum bone-specific alkaline phosphatase (BALP) and transaminase levels and for hematological studies. Rats were weighed daily. RESULTS: All combined therapies prevented alveolar bone loss when compared with SAL or low doses of monotherapy (ALD or ATV) (p < 0.05). The lower-dose combination of ALD+ATV (0.01 mg/kg + 0.3 mg/kg, respectively), administered either prophylactically (39.0%) or therapeutically (53.5%), prevented alveolar bone loss. Decreases in bone and cementum resorption, in leukocyte infiltration and in immunostaining for TRAP and MPO activity corroborated the morphometric findings. The lower-dose combination of ALD+ATV (0.01 mg/kg + 0.3 mg/kg, respectively) prevented BALP reduction (p < 0.05) and did not alter the level of serum transaminases. Moreover, the lower-dose combination of ALD+ATV (0.01 mg/kg + 0.3 mg/kg, respectively) also reduced neutrophilia and lymphomonocytosis and did not cause weight loss when compared with administration of SAL. CONCLUSION: The lower-dose combination of ALD+ATV (0.01 mg/kg + 0.3 mg/kg, respectively) demonstrated a protective effect on alveolar bone loss.

Regulation of NAMPT in human gingival fibroblasts and biopsies.

Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1ß and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.

Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

OBJECTIVES: The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). MATERIALS AND METHODS: HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. RESULTS: At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each < 0.001). Scratch assay demonstrated an increased migration ability for HGF on e.max CAD HT, Empress CAD and Mark II (p each < 0.001), whereas HOK showed a significantly decreased migration ability on all tested materials at all points of measurement (between -36 % and -71 %; p each < 0.001). At ToxiLight assay, only small cytotoxic effects of the all-ceramic materials could be investigated. CONCLUSIONS: This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CLINICAL RELEVANCE: CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

The effects of cytotoxicity and genotoxicity induced by 2,2-bis[4-(acryloxypropoxy)phenyl]propane via caspases in human gingival fibroblasts.

The toxicity of dental materials has raised public concern over the past years. One of the most commonly used methacrylic monomers for building the three-dimensional structure of the dental resin composites is 2,2-bis[4-(acryloxypropoxy)phenyl]propane (BAPP). The purpose of this study is to evaluate the potential toxicological implication of BAPP on human gingival fibroblasts (HGFs). Flow cytometric, fluorometric, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assays were used to detect the mode of cell death, caspase activities, and cell viability, respectively. In addition, alkaline single-cell gel electrophoresis (COMET) and cytokinesis block micronucleus (MN) assays were applied to evaluate the genotoxicity. According to the results BAPP demonstrated a cytotoxic effect on HGFs in a dose- and time-dependent manner. With increasing concentrations of BAPP, the mode of cell death shifted from apoptosis to necrosis, and the activities of caspases 3, 8, and 9 were also significantly induced. Moreover, a dose-related increase in the number of micronucleus and DNA strand breaks hinted at the expression of genotoxicity by BAPP. In conclusion, the results gathered from this study had demonstrated that BAPP-induced cytotoxicity and genotoxicity on HGFs were mediated by DNA damage and the activation of caspases 3, 8, and 9.

Requirement for active glycogen synthase kinase-3ß in TGF-ß1 upregulation of connective tissue growth factor (CCN2/CTGF) levels in human gingival fibroblasts.

Connective tissue growth factor (CCN2/CTGF) mediates transforming growth factor-ß (TGF-ß)-induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3-kinase (PI3K) pathway in mediating TGF-ß1-stimulated CCN2/CTGF expression in primary human adult gingival fibroblasts and human adult lung fibroblasts was compared. Data indicate that PI3K inhibitors attenuate upregulation of TGF-ß1-induced CCN2/CTGF expression in human gingival fibroblasts independent of reducing JNK MAP kinase activation. Pharmacologic inhibitors and small interfering (si)RNA-mediated knockdown studies indicate that calcium-dependent isoforms and an atypical isoform of protein kinase C (PKC-δ) do not mediate TGF-ß1-stimulated CCN2/CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3ß (GSK-3ß) can undergo phosphorylation by the PI3K/pathway, the effects of GSK-3ß inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGF-ß1-mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK-3ß, similarly inhibits TGF-ß1-stimulated CCN2/CTGF expression. In contrast, inhibition of GSK-3ß by Wnt3a does not inhibit, but modestly stimulates, CCN2/CTGF levels in primary human adult lung fibroblasts and is ß-catenin dependent, consistent with previous studies performed in other cell models. These data identify a novel pathway in gingival fibroblasts in which inhibition of GSK-3ß attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK-3ß modestly stimulates TGF-ß1-regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue specificity of drug-induced gingival overgrowth.

Temporal expression of metalloproteinase-8 and -13 and their relationships with extracellular matrix metalloproteinase inducer in the development of ligature-induced periodontitis in rats.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinases (MMPs) play important roles in extracellular matrix degradation and may be regulated by extracellular matrix metalloproteinase inducer (EMMPRIN). The aim of this study was to investigate the temporal expression and localization of MMP-8 and MMP-13 during the development of ligature-induced periodontitis in rats, and to analyze the correlations of EMMPRIN with MMP-8 and MMP-13 in periodontitis. MATERIAL AND METHODS: Periodontitis was simulated in rats by ligaturing the cervix of the lower first molars, as described in our previous method. The rats were killed 0, 3, 5, 7, 11, 15 and 21 d after ligation. Micro-computed tomography examinations were performed to detect alveolar bone loss. Semiquantitative western blotting was used to assess the temporal changes in the levels of MMP-8, MMP-13 and EMMPRIN proteins in gingival tissue. Immunohistochemistry was applied to detect the expression and locations of MMP-8 and MMP-13 in gingival tissue and alveolar bone. RESULTS: Alveolar bone loss showed an exponential increase from days 3 to 11, followed by a slower rate of loss at subsequent study time points. MMP-8 showed a rapid increase of expression from baseline to a peak on day 3, a gradual decrease from days 5 to 7 and then stabilized thereafter. MMP-8 was predominantly located in neutrophil-like cells. Statistically, the expression of MMP-8 was not correlated with the expression of EMMPRIN. The expression of MMP-13 and of EMMRPIN increased from days 3 to 7, and showed a moderate decrease thereafter. The immunoreactivity of MMP-13 was mainly detected in monocytes/macrophages, on the alveolar bone surface, in osteoclasts and in gingival epithelial cells. Statistically, MMP-13 had a strong, positive correlation with EMMPRIN (r = 0.855, p < 0.01). CONCLUSION: The levels of expression of MMP-8 and MMP-13 are temporally varied at different periods during the development of experimental periodontitis. The level of expression of EMMPRIN is closely associated with the expression of MMP-13, but not with the expression of MMP-8. In addition, MMP-13 might be involved in alveolar bone destruction, as well as in physiological bone remodeling.

Over-expression and potential role of cyclophilin A in human periodontitis.

BACKGROUND AND OBJECTIVE: We previously demostrated that EMMPRIN participates in the periodontitis and its interaction with Cyclophilin A possibly exists in animal periodontitis models. This study is aimed to address the expression and potential role of cyclophilin A (CypA) in human periodontitis. MATERIAL AND METHODS: Gingival tissues and peripheral blood were collected from patients with moderate to severe periodontitis or from healthy donors. Western blotting and immunohistochemistry were performed to detect the expression and distribution of CypA in the gingival tissues. Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated from the peripheral blood by Ficoll-Paque density-gradient centrifugation. Chemotaxis assays were applied to evaluate the effects of different concentrations of CypA (100, 300 and 500 ng/mL) on the migration of PBMCs and neutrophils. Supernatants of human THP-1 cells were collected after treatment with 200 ng/mL of CypA for different periods of time (1, 3, 6, 12 and 24 h) to detect the levels of interleukin (IL)-1ß, IL-8 and tumor necrosis factor alpha (TNF-α) by ELISA. RESULTS: Western blot analyses revealed an increase of CypA expression in inflamed gingival tissues compared with healthy tissues. Immunohistochemistry identified that the over-expressed CypA was localized in the infiltrating cells and/or in the extracellular matrix in the inflamed gingival connective tissues. The positive infiltrating cells contained mononuclear cells and lobulated-nuclei neutrophils. Chemotactic assays showed that 300 ng/mL of CypA apparently facilitated the chemotaxis of PBMCs/neutrophils from healthy donors, compared with the no-treatment control (p < 0.01 for PBMCs, p < 0.05 for neutrophils), whereas 100 and 500 ng/mL of CypA only weakly enhanced the chemotaxis of PBMCs/neutrophils (p > 0.05 for PBMCs/neutrophils, not significant). The PBMCs/neutrophils from patients with periodontitis exhibited a stronger ability to migrate when stimulated with 300 ng/mL of CypA than did PBMCs/neutrophils from healthy donors (p < 0.05 for PBMCs, p < 0.01 for neutrophils). ELISA revealed that the level of TNF-α secreted by THP-1 cells was elevated after treatment with 200 ng/mL of CypA for 12 h compared with the no-treatment 0-h control (p < 0.05). The IL-8 level was sharply raised after 3 h of stimulation with 200 ng/mL of CypA (p < 0.01 compared with 0 h), but no significant change was observed at the other time points (p > 0.05). There was no statistical difference at any of the treatment time points for the secretion of IL-1ß (p > 0.05 for 1, 3, 6, 12 and 24 h compared with 0 h). CONCLUSIONS: CypA participates in the pathogenesis of human periodontitis. It may be involved in the inflammatory response of periodontal tissues through inducing the chemotaxis of PBMCs/neutrophils and the secretion of TNF-α/IL-8.

Cytokine and matrix metalloproteinase expression in fibroblasts from peri-implantitis lesions in response to viable Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. METHODS: Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases (MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. RESULTS: Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin (IL)-1ß, IL-8, and monocyte chemotactic protein (MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1ß, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1ß, MCP-1, and MMP-1 compared to periodontitis fibroblasts. CONCLUSIONS: Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP-1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis.

Lipopolysaccharide-induced indoleamine 2,3-dioxygenase expression in the periodontal ligament.

BACKGROUND AND OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-oxidizing enzyme with immune-inhibitory effects. The aim of this study was to investigate the expression of IDO by lipopolysaccharide (LPS), a component of gram-negative bacteria, in human periodontal ligament (PDL) cells. MATERIAL AND METHODS: Human PDL cells and gingival fibroblasts (GFs) were prepared from explants of human PDLs and from gingival tissues of clinically healthy donors, respectively. Real-time RT-PCR, western blotting and the IDO enzyme assay were performed to determine the expression of IDO following LPS treatment of cells. LPS was injected into mice tail veins to evaluate the effects of LPS in vivo in the maxillary first molar. Immunofluorescence staining and histological analysis were followed to localize IDO in mouse PDL. RESULTS: The level of expression of IDO mRNA in primary human PDL cells after LPS treatment was increased in a dose-dependent manner, reaching a peak 8 h after LPS treatment. The expression and activities of IDO protein were significantly increased in comparison with those of the control. In addition, the increased production of kynurenine in culture medium was observed 72 h after LPS treatment. In the immunofluorescence findings, stronger immunoreactivities were shown in PDL than in gingival tissues in the maxillae. In accordance with the immunofluorescence findings, LPS treatment induced a strong up-regulation of IDO mRNA in human PDL cells, whereas human GFs showed only a weak response to LPS. CONCLUSION: These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.

Inhibitory effect of Zingiber cassumunar extracts on lipopolysaccharide-induced cyclooxygenase-2 and matrix metalloproteinase expression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Lipopolysaccharides (LPS) induce the production of proinflammatory mediators such as prostaglandins and matrix metalloproteinases (MMPs) in human gingival fibroblasts (HGFs). Zingiber cassumunar is a medicinal plant that possesses anti-inflammatory properties. The aim of this study was to determine the effects of the Z. cassumunar extract on the expression of cyclooxygenase (COX)-1, COX-2 and MMP-2 in HGFs challenged with LPS. MATERIAL AND METHODS: HGFs were treated with LPS in the presence or absence of Z. cassumunar extracts. The levels of expression of COX-1, COX-2 and MMP-2 mRNAs and of COX-1, COX-2 and MMP-2 proteins were detected by reverse transcription-polymerase chain reaction and western blotting, respectively. MMP-2 activities in cell-culture supernatants were determined using gelatin zymography. MAPK activation was evaluated by western blotting. RESULTS: LPS treatment of HGFs resulted in the activation of ERK1/2, p38 and JNK. Z. cassumunar extracts significantly inhibited the phosphorylation of ERK1/2 and JNK in HGFs stimulated with LPS. A lesser inhibitory effect was observed for the phosphorylation of p38. RT-PCR and western blot analyses showed that Z. cassumunar extracts inhibited the LPS-induced expression of COX-2 mRNA and COX-2 protein, respectively, but not of COX-1 mRNA or COX-1 protein. Pretreatment of HGFs with Z. cassumunar also attenuated the induction of MMP-2 with LPS. CONCLUSION: Our results indicate that Z. cassumunar extracts inhibit COX-2 and MMP-2 production by LPS-activated human gingival fibroblasts through blocking the proinflammatory signaling pathway involving ERK1/2, JNK and p38.

pPKC α regulates through integrin ß 1 human gingival fibroblasts/Streptococcus mitis adhesion in response to HEMA.

AIM: To investigate in coculture of human gingival fibroblasts (HGFs) and Streptococcus mitis, the molecular mechanisms driving the response to 2-hydroxyethyl methacrylate (HEMA) in terms of eukaryotic/prokaryotic cell adhesion, signal transduction and apoptosis. METHODOLOGY: The clinical strain S. mitis DS12, cultured in Trypticase soy broth was added to HGFs, obtained from fragments of healthy marginal gingival tissue and cultured in DMEM, treated with 3 mmol L(-1) 2-hydroxyethyl methacrylate (HEMA) for 48 h and processed for microscopic, western blotting and flow cytometric analyses. RESULTS: 2-hydroxyethyl methacrylate (HEMA) treatment increased the adhesion between S. mitis and HGFs, which seemed to be mediated by the PKC α/integrin ß 1 signalling system, improved by the presence of saliva. It also reduced the viability and the adhesion of HGFs to polypropylene substrate in terms of procollagen I and MMP3 expression. The presence of saliva and S. mitis reduced the number of necrotic HGFs and upregulated the expression of both procollagen I and MMP3. CONCLUSIONS: These results shed more light on the biological and molecular events occurring in vitro in a coculture model that mimics the environment of the oral cavity with HEMA treatment. The key role played by oral bacteria and saliva in preventing inflammatory and toxic processes that occur in vivo in human gingival fibroblasts upon the release of dental material monomers is confirmed.