Tumor Necrosis Factor‐α and Porphyromonas gingivalis Lipopolysaccharides Decrease Periostin in Human Periodontal Ligament Fibroblasts

Background: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease‐associated pathogen‐related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. Methods: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor‐α [TNF‐α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF‐β inducible gene clone H3 (βIGH3) mRNA expression from cell lysates were analyzed. Periostin and βIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). Results: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non‐loaded controls (higher levels of periostin in the supernatant in the non‐loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on βIGH3 levels, and no particular pattern was clearly evident. Conclusions: Inflammatory mediators (TNF‐α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.     

Stimulation of epithelial cell matrix metalloproteinase (MMP‐2, ‐9, ‐13) and interleukin‐8 secretion by fusobacteria

Background/aims: Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin‐8 (IL‐8) secretion upon exposure to fusobacteria. Methods: Eight different oral and non‐oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP‐13), gelatinase A (MMP‐2), gelatinase B (MMP‐9), and IL‐8 secretion by epithelial cells. Results: All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP‐9 and MMP‐13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP‐2 secretion. F. nucleatum and F. necrophorum also increased IL‐8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL‐8 production, suggesting that their expression is differently regulated. Conclusion: We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP‐9, MMP‐13, and IL‐8 from epithelial cells.     

Focal adhesion kinase activation is required for TNF‐α‐induced production of matrix metalloproteinase‐2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF‐α) and its effects on interleukin (IL)‐6 and IL‐8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP‐2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real‐time PCR. Tumor necrosis factor alpha dose‐dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF‐α‐induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL‐6, IL‐8, and MMP‐2 in a dose‐dependent manner. Knockdown of FAK significantly suppressed TNF‐α‐induced expression of IL6 and IL8 mRNA and release of IL‐6 and IL‐8 protein in HPDLFs. Similarly, MMP‐2 down‐regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF‐α‐induced IL‐6, IL‐8, and MMP‐2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.     

Modulatory effect of curcumin analogs on the activation of metalloproteinases in human periodontal stem cells

Periodontitis progresses due to increased levels of active metalloproteinases (MMPs) and the imbalance between MMPs and their tissue inhibitors (TIMPs). Natural curcumin limits the lytic activity of MMPs but has low cellular uptake. Use of synthetic curcumin analogs could be a means of overcoming this limitation of treatment efficiency. Human periodontal stem cells were isolated from gingival tissue, gingival ligament fibers, periodontal ligament, and alveolar bone. The effect of five synthetic curcumin analogs was compared with that of natural curcumin by assessing cytotoxicity [by 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay], the cellular uptake (by fluorometry), the proteolytic activities of MMP‐2 and ‐9 (by zymography), and the levels of TIMP‐1 (by ELISA). Our results indicated increased cytotoxicity of synthetic curcumins for doses between 100 and 250 μM. At a concentration of 10 μM, cellular uptake of synthetic curcumins varied depending on their chemical structure. The curcumin compounds modulated pro‐MMP‐2 levels and increased TIMP‐1 production. There was no detectable synthesis of pro‐MMP‐9 and no activation of MMPs 2 and 9. Gingival tissue and gingival ligament fiber stem cells were most responsive to treatment, showing inverse correlations between pro‐MMP‐2 and TIMP‐1 levels. In conclusion, synthetic curcumins influenced the balance between pro‐MMP‐2 and TIMP‐1 in human periodontal stem cells in vitro, and this could open perspectives for their application as adjuvants in periodontal therapy.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Down‐regulation of interleukin‐1α‐induced matrix metalloproteinase‐13 expression via EP1 receptors by prostaglandin E2 in human periodontal ligament cells

In the present study, we investigated the effect of prostaglandin (PG) E₂ on matrix metalloproteinase (MMP)‐13 production in human periodontal ligament cells stimulated with interleukin (IL)‐1α. IL‐1α enhanced both MMP‐13 and PGE₂ production. Indomethacin, a nonselective cyclooxygenase inhibitor, and NS‐398, a specific cyclooxygenase‐2 (COX‐2) inhibitor, significantly enhanced IL‐1α‐induced MMP‐13 production in periodontal ligament cells, although both the agents completely inhibited IL‐1α‐induced PGE₂ production. Exogenous PGE₂ reduced IL‐1α‐induced MMP‐13 mRNA and protein production in a dose‐dependent manner. 17‐phenyl‐ω‐trinor PGE₂, a selective EP₁ receptor agonist, mimicked the inhibitory effect of PGE₂ on IL‐1α‐induced MMP‐13 mRNA and protein production. On the basis of these data, we suggest that COX‐2‐dependent PGE₂ down‐regulates IL‐1α‐elicited MMP‐13 production via EP₁ receptors in human periodontal ligament cells. PGE₂ may be involved in the regulation of destruction of extracellular matrix components in periodontal lesions.     

Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain‐induced proliferation of periodontal ligament fibroblasts

Laaksonen M, Salo T, Vardar‐Sengul S, Atilla G, Han Saygan B, Simmer JP, Baylas H, Sorsa T. Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain‐induced proliferation of periodontal ligament fibroblasts. J Periodont Res 2010; 45: 353–360. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Emdogain^® (EMD), consisting mostly of amelogenin, is used in periodontal therapy to regenerate lost connective tissue. Emdogain is applied onto periodontally affected root surfaces, where it becomes exposed to proteolytic enzymes. In this study, we aimed to find out whether gingival crevicular fluid or matrix metalloproteinases (MMPs) could degrade EMD, and whether this degradation has consequences for in vitro cell proliferation. Material and Methods: We studied the effects of 156 gingival crevicular fluid samples collected from subjects with different stages of periodontal disease and from healthy control subjects and the effects of MMP‐1, ‐2, ‐8, ‐9, ‐13 and ‐14 on the degradation of EMD using EMD‐embedded zymography. The effects of gingival crevicular fluid with or without EMD and the effects of amelogenin on the proliferation of cultured periodontal ligament fibroblasts were studied by cell proliferation enzyme‐linked immunosorbent assay kit. Results: Degradation of Emdogain induced by gingival crevicular fluid was greater in samples from all stages of periodontal diseases compared with healthy control samples. Of the MMPs studied, only MMP‐2 and MMP‐8 showed limited EMD‐degrading activities. One hundred micrograms per millilitre of EMD increased proliferation of periodontal ligament fibroblasts on average by 24% (confidence interval 0.60–0.64) and at 200 μg/mL by 30% (confidence interval 0.62–0.68) compared with control fibroblasts (confidence interval 0.48–0.52). However, gingival crevicular fluid (10 μg/mL) together with 100 μg/mL EMD induced the proliferation only by 6% (confidence interval 0.51–0.55) and with 200 μg/mL EMD by 12% (confidence interval 0.54–0.58). Amelogenin at 200 μg/mL decreased the proliferation of periodontal ligament fibroblasts by 54% (confidence interval 0.22–0.25). Conclusion: We suggest that diseased gingival crevicular fluid containing various proteases leads to degradation of EMD and decreased proliferation of periodontal ligament fibroblasts.     

Orthodontic Force Increases Interleukin‐1β and Tumor Necrosis Factor‐α Expression and Alveolar Bone Loss in Periodontitis

Background: The present study aims to evaluate the effects of orthodontic movement (OM) on the periodontal tissues of rats with ligature‐induced periodontal disease. Methods: Eighty‐eight rats were divided into four groups: 1) negative control (sham operated); 2) periodontal disease; 3) OM; and 4) periodontal disease followed by OM (OMP). Rats were sacrificed 3 hours or 1, 3, or 7 days after OM commencement. Bone volume fraction (BVF) and bone mineral density (BMD) were assessed in hemimaxillae by microcomputed tomography analysis. Expression of the proinflammatory cytokines interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α were evaluated in gingival samples by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, and in the furcation region by immunohistochemistry analysis (IHC). Results: The OMP group had lower BVF and BMD levels compared to the other groups at day 7 (P <0.05). Maximum messenger ribonucleic acid expression of both cytokines was observed in the OMP group at day 1 (P <0.05). In the same period, all proteins were expressed in high levels for all test groups compared to the control group. The number of cells positive for IL‐1β and TNF‐α by IHC was highest in the OMP group at day 1, with progressive reduction thereafter. Conclusion: The results suggest that OM acts synergistically with periodontal disease in periodontal breakdown through upregulation of proinflammatory cytokines.     

Proteolytic roles of matrix metalloproteinase (MMP)‐13 during progression of chronic periodontitis: initial evidence for MMP‐13/MMP‐9 activation cascade

Aim: Matrix metalloproteinases (MMP)‐13 can initiate bone resorption and activate proMMP‐9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP‐13 activity and TIMP‐1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy‐terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP‐13 could potentiate gelatinase activation in diseased gingival tissue. Materials and Methods: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP‐13 activity and TIMP‐1. Diseased gingival explants were cultured, treated or not with MMP‐13 with or without adding CL‐82198, a synthetic MMP‐13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. Results: Active sites demonstrated increased ICTP levels and MMP‐13 activity (p<0.05) in progression subjects. The MMP‐9 activation rate was elevated in MMP‐13‐treated explants (p<0.05) and MMP‐13 inhibitor prevented MMP‐9 activation. Conclusions: MMP‐13 could be implicated in the degradation of soft and hard supporting tissues and proMMP‐9 activation during progression of chronic periodontitis. MMP‐13 and ‐9 can potentially form an activation cascade overcoming the protective TIMP‐1 shield, which may become useful for diagnostic aims and a target for drug development.     

Saliva and Serum Levels of B‐Cell Activating Factors and Tumor Necrosis Factor‐α in Patients With Periodontitis

Background: B‐lymphocytes play a central and critical role in the adaptive immune response against invading pathogens. This study evaluates saliva and serum levels of APRIL (a proliferation‐inducing ligand), B‐cell activating factor (BAFF), tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐6, and IL‐10 in patients with chronic periodontitis (CP) or aggressive periodontitis (AgP) and periodontally healthy individuals. Methods: Twenty‐five patients with AgP, 20 patients with CP, and 20 periodontally healthy individuals were included. Smoking status was recorded, and all individuals were divided into non‐smokers and smokers. Saliva and serum samples were collected before clinical periodontal measurements. APRIL, BAFF, TNF‐α, IL‐6, and IL‐10 levels in serum and saliva samples were determined by enzyme‐linked immunosorbent assay. Statistical analysis was performed using multivariate analysis of variance and bivariate correlation. Results: Serum and saliva levels of TNF‐α, APRIL, BAFF, IL‐6, and IL‐10 were similar in CP and AgP groups. Serum levels of TNF‐α, APRIL, and BAFF and saliva levels of BAFF were significantly higher in periodontitis groups than healthy controls (P <0.05). Non‐smokers with CP or AgP had lower levels of saliva TNF‐α and APRIL and serum APRIL and IL‐6 than smokers with CP or AgP (P <0.05). Saliva APRIL and serum TNF‐α and IL‐6 levels were significantly higher in healthy smokers than healthy non‐smokers (P <0.05). Clinical periodontal parameters correlated positively with TNF‐family cytokines and negatively with IL‐10 (P <0.05). Conclusions: Within the limits of this study, it may be suggested that elevated salivary and serum TNF‐α, APRIL, and BAFF in patients with periodontitis may contribute to the dominance of B cells in periodontitis lesions. Moreover, higher levels in healthy smokers than non‐smoking counterparts may play a role in detrimental effects of smoking on periodontal tissues.     

A trend of increase in periodontal interleukin-6 expression across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases

Background and Objective: Epidemiological studies have established that patients with diabetes have increased prevalence and severity of periodontal disease. However, the periodontal expression of inflammatory cytokines and matrix metalloproteinases (MMPs) in diabetic patients has not been well characterized. The objective of this study was to determine the difference in the periodontal expression of MMP‐1, MMP‐8, interleukin‐6, tumor necrosis factor‐α and interleukin‐1β between diabetic and nondiabetic patients. Material and Methods: Periodontal tissue specimens were collected from nine nondiabetic patients without periodontal disease (group 1), from 11 nondiabetic patients with periodontal disease (group 2) and from seven diabetic patients with periodontal disease (group 3). The expression of MMP‐1, MMP‐8, interleukin‐6, tumor necrosis factor‐α and interleukin‐1β was quantified using real‐time polymerase chain reaction. Results: The nonparametric Kruskal–Wallis test showed that the difference in interleukin‐6 expression among the groups was statistically significant (p = 0.04). Furthermore, the generalized Kruskal–Wallis nonparametric linear‐by‐linear association test showed a statistically significant trend of increase in the expression of interleukin‐6 from group 1 to group 2 to group 3 (p = 0.02) and a suggestion of such a trend for MMP‐1 (p = 0.05). No increase in MMP‐8 expression was observed in patients in group 3 compared to patients in groups 1 and 2. Although the average expression levels of MMP‐1, interleukin‐1β and tumor necrosis factor‐α were increased from group 1 to group 3, the differences were not statistically significant. Conclusion: A trend of increased interleukin‐6 expression in periodontal tissues was observed across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.     

Effects of loss of occlusal contact on the expression of matrix metalloproteinase‐2, membrane type 1‐MMP,tissue inhibitor of the MMP‐2, eruption rate,organization and resistance of collagen fibers of the rat incisor periodontal ligament

Background and Objective

This study investigated the effect of occlusal contact loss (induced by tooth shortening), on matrix metalloproteinase (MMP)‐2, membrane type 1‐MMP (MT1‐MMP) and tissue inhibitor of the MMP‐2 (TIMP‐2) expressions in the periodontal ligament of the rat incisor, as well as eruption rate, resistance and collagen organization.

Material and Methods

Male Wistar rats were distributed into a control group, denominated normofunctional group, whose lower teeth underwent a normal eruption process; and a hypofunctional group, whose lower left incisor teeth were shortened every 2 days during 14 days. Parameters were evaluated on the first, seventh and 14th days and the eruption rate was determined according to the size of the incisor during the eruption process. MMP‐2 activity was determined by zymography and the expressions of the MT1‐MMP and TIMP‐2 proteins were quantitated by western blot. Collagen protein organization, as indicated by the birefringence of the periodontal ligament, was analyzed under polarized light and the periodontal ligament's resistance was determined from the load necessary to inject the incisor into its alveolar space, before extraction.

Results

Loss of occlusal contact, in rats submitted to hypofunctional eruption, increased MMP‐2 activity and eruption rate, but decreased MT1‐MMP and TIMP‐2 expression and disrupted collagen organization in the periodontal ligament, consequently reducing periodontal ligament resistance.

Conclusion

We conclude that, after incisor eruption, occlusal contact may be an important factor for regulating the remodeling and the physiological resistance of the periodontal ligament against the continuous eruption process observed in rat incisors.     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament

Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309–316. © 2009 John Wiley & Sons A/S Background and Objective: MMP‐2 can degrade type IV collagen and MMP‐14 can activate pro MMP‐2. The present study was undertaken to examine the expression of MMP‐2 and MMP‐14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum‐free medium. The distribution and expression of MMP‐2 and MMP‐14 were analysed using immunohistochemistry, in situ hybridization and RT‐PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP‐2. Results: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP‐2 and MMP‐14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP‐2 mRNA while putative epithelial rests of Malassez cells expressed MMP‐14 mRNA at the interface. The RT‐PCR analysis showed that the expression of MMP‐2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP‐2 was detected at higher levels in the conditioned medium of the co‐cultured cells. Conclusion: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP‐2 in human periodontal ligament fibroblasts. Up‐regulated proMMP‐2 bound by MMP‐14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.