Sequential process in brain‐derived neurotrophic factor‐induced functional periodontal tissue regeneration

We recently demonstrated that brain‐derived neurotrophic factor (BDNF) promotes periodontal tissue regeneration. The purpose of this study was to establish an essential component of a rational approach for the clinical application of BDNF in periodontal regenerative therapy. Here, we assessed the sequence of early events in BDNF‐induced periodontal tissue regeneration, especially from the aspect of cementum regeneration. Brain‐derived neurotrophic factor was applied into experimental periodontal defects in Beagle dogs. The localization of cells positive for neurotrophic tyrosine kinase, receptor, type 2, proliferating cell nuclear antigen, osteopontin, integrin αVβ3, and integrin α2β1 was evaluated by immunohistochemistry. The effects of BDNF on adhesion of cultured human periodontal ligament cells was examined by an in vitro study. The results suggest that BDNF could induce rapid cementum regeneration by stimulating adhesion, proliferation, and differentiation of periodontal ligament cells in the early regenerative phase, resulting in enhancement of periodontal tissue regeneration.     

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.     

interleukin-1β and tumor necrosis factor-α stimulate synergistically the expression of monocyte chemoattractant protein-1 in fibroblastic cells derived from human periodontal ligament

The present study demonstrates that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induce and synergistically stimulate monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts from human periodontal ligament. IL-1β and TNF-α induced in a dose-dependent manner the expression of the MCP-1 gene in the fibroblasts from the human periodontal ligament. However, such an inducing effect was not observed with IL-6 and interferon-γ. The peak expression of the MCP-1 gene by IL-1β or TNF-α was observed at 3 h after initiation of their treatment. Furthermore, IL-1β in combination with TNF-α synergistically stimulated the MCP-1 gene expression in the cells. We also observed significant chemotactic activity for human monocytes in conditioned medium of fibroblasts from the human periodontal ligament treated with both cytokines. The stimulated chemotactic activity induced by these cytokines depended on both dose and treatment time. The chemotactic activity in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was neutralized by antiserum specific for MCP-1 protein. The MCP-1 gene product in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was shown to have a molecular mass of 11,000 Da by immunoprecipitation with the specific antiserum, and IL-1β also stimulated synergistically MCP-1 protein expression in combination with TNF-α. These results suggest that IL-1β and TNF-α may contribute to the infiltration of monocytes into inflammatory sites of periodontal ligament tissues via the MCP-1 gene product produced by fibroblasts from the human periodontal ligament.     

Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease

Da? A, F?rat ET, Kadiro?lu AK, Kale E, Y?lmaz ME. Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease. J Periodont Res 2010; 45: 445–450. © 2010 John Wiley & Sons A/S Background and Objective: The prevalence of chronic renal disease in industrialized countries is increasing, and chronic renal disease and periodontitis can have significant, reciprocal effects. The aim of this study was to evaluate the associations between specific clinical parameters and the levels of tumor necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) in the gingival crevicular fluid of hemodialysis (HD) patients with periodontal disease. Material and Methods: Forty‐three HD patients and 43 systemically healthy subjects were enrolled in this study. Plaque index (PI), gingival index (GI) and pocket depth were used to determine periodontal status. Venous blood samples were obtained from each patient in the morning before the dialysis session and analyzed to determine the levels of inflammatory, biochemical and hematological parameters. Gingival crevicular fluid was collected from all subjects, and the levels of TNF‐α and IL‐8 were determined in the gingival crevicular fluid samples. Results: The following results were obtained from HD patients and controls: TNF‐α (pg/mL), 31.40 ± 1.46 and 3.06 ± 0.15 (p < 0.001); IL‐8 (pg/mL), 90.98 ± 94.03 and 35.05 ± 16.86 (p < 0.001); PI, 1.69 ± 1.02 and 0.04 ± 0.02 (p < 0.001); GI, 0.82 ± 0.06 and 0.04 ± 0.02 (p < 0.001); and pocket depth, 2.23 ± 0.63 and 1.51 ± 0.05 (p < 0.001), respectively. In addition, there were positive correlations between TNF‐α and PI (r = 0.642, p < 0.001), between TNF‐α and GI (r = 0.565, p < 0.001), between TNF‐α and pocket depth (r = 0.522, p < 0.001), between IL‐8 and PI (r = 0.402, p = 0.002), between IL‐8 and GI (r = 0.396, p = 0.002), and between IL‐8 and pocket depth (r = 0.326, p = 0.012). There were negative correlations between albumin and PI (r = ?0.491, p < 0.001), albumin and GI (r = ?0.406, p < 0.001), albumin and pocket depth (r = ?0.464, p < 0.001) and albumin and CRP (r = ?0.467, p = 0.002) and between the gingival crevicular fluid levels of TNF‐α and IL‐8, TNF‐α and hemoglobin (r = ?0.745, p < 0.001; r = ?0.285, p < 0.05) (respectively). Conclusion: The levels of TNF‐α and IL‐8 in gingival crevicular fluid were significantly higher in HD patients than in controls. There were strong, positive correlations between clinical periodontal parameters and the levels of inflammatory cytokines in gingival crevicular fluid from the HD patients.     

Major surface protein complex of Treponema denticola induces the production of tumor necrosis factor α, interleukin‐1β, interleukin‐6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes

Gaibani P, Caroli F, Nucci C, Sambri V. Major surface protein complex of Treponema denticola induces the production of tumor necrosis factor α, interleukin‐1β, interleukin‐6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes. J Periodont Res 2010; 45: 361–366. © 2010 John Wiley & Sons A/S Background and Objective: Treponema denticola is a micro‐organism that is involved in the pathogenesis of periodontitis. Major surface protein complex (MSPc), which is expressed on the envelope of this treponeme, plays a key role in the interaction between T. denticola and gingival cells. The peptidoglycan extracted from T. denticola induces the production of a large variety of inflammatory mediators by macrophage‐like cells, suggesting that individual components of T. denticola cells induce the inflammatory response during periodontal disease. This study was designed to demonstrate that MSPc of T. denticola stimulates release of proinflammatory mediators in primary human monocytes. Material and Methods: Primary human monocytes were separated from the blood of healthy donors and incubated for up to 24 h with varying concentrations of MSPc. The production of tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and matrix metalloproteinase 9 (MMP‐9) was measured at different time points with commercially available enzyme‐linked immunosorbent assays. Results: T. denticola MSPc induced the synthesis of TNF‐α, IL‐1β, IL‐6 and MMP‐9 in a dose‐ and time‐dependent manner. Similar patterns of TNF‐α, IL‐1β and IL‐6 release were observed when cells were stimulated with 100 and 1000 ng/mL of MSPc. The production of MMP‐9 was significant only when cells were treated with 1000 ng/mL of MSPc. Conclusion: These results indicate that T. denticola MSPc, at concentrations ranging from 100 ng/mL to 1.0 μg/mL, activates a proinflammatory response in primary human monocytes.     

Effects of tobacco smoke on the secretion of interleukin‐1β, tumor necrosis factor‐α, and transforming growth factor‐β from peripheral blood mononuclear cells

Alterations of the host response caused by short‐term exposure to high levels of smoke during the act of smoking (acute smoke exposure) as well as long‐term exposure to lower levels of tobacco substances in the bloodstream of smokers (chronic smoke exposure) may play a role in the pathogenesis of periodontal diseases in smokers. In this study, we examined the secretion of three cytokines [interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, and transforming growth factor (TGF)‐β] from mononuclear blood cells from current smokers and non‐smokers exposed to in vitro tobacco smoke (which may be comparable to in vivo acute smoke exposure) and mononuclear blood cells from current smokers not exposed to further in vitro smoke (which may be comparable to chronic smoke exposure). Peripheral blood mononuclear cells were isolated from eight healthy current smokers and eight healthy non‐smokers, plated in culture wells, exposed in vitro for 1–5 min to cigarette smoke in a smoke box system or not exposed (baseline controls), and then incubated without further smoke exposure for another 24 h. Supernatants from each well were then collected and assayed for the concentrations of the three cytokines by enzyme‐linked immunosorbent assay (ELISA). At baseline, mean IL‐1β levels were higher in smokers than in non‐smokers (mean: 10.6 vs. 5.9 pg/ml, anova : P < 0.05). In both smokers and non‐smokers, secreted levels of IL‐1β increased from 0 to 5 min of in vitro smoke exposure (mean: 5.9–9.9 pg/ml, t‐test: P < 0.05 for non‐smokers only) with levels in smokers higher than in non‐smokers (P > 0.05). Mean TNF‐α levels increased from 0 to 2 min of smoke exposure and decreased from 2 to 5 min in smokers and non‐smokers, with higher levels in non‐smokers than smokers at all time‐points (P > 0.05). Mean TGF‐β levels were higher in smokers than in non‐smokers at all time‐points (mean: 180.5 vs. 132.0 pg/ml, P < 0.05 at 5 min only) with no significant alteration of the pattern of secretion with cigarette smoke exposure. These observed alterations in the secretion of cytokines from mononuclear blood cells in smokers, relative to non‐smokers, and with in vitro smoke exposure may play a role in the pathogenesis of periodontal diseases in smokers.     

Redefining the induction of periodontal tissue regeneration in primates by the osteogenic proteins of the transforming growth factor‐β supergene family

The molecular bases of periodontal tissue induction and regeneration are the osteogenic proteins of the transforming growth factor‐β (TGF‐β) supergene family. These morphogens act as soluble mediators for the induction of tissues morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented ligament fibers into newly formed cementum. Human TGF‐β₃ (hTGF‐β₃) in growth factor‐reduced Matrigel^® matrix induces cementogenesis when implanted in class II mandibular furcation defects surgically prepared in the non‐human primate Chacma baboon, Papio ursinus. The newly formed periodontal ligament space is characterized by running fibers tightly attached to the cementoid surface penetrating as mineralized constructs within the newly formed cementum assembling and initiating within the mineralized dentine. Angiogenesis heralds the newly formed periodontal ligament space, and newly sprouting capillaries are lined by cellular elements with condensed chromatin interpreted as angioblasts responsible for the rapid and sustained induction of angiogenesis. The inductive activity of hTGF‐β₃ in Matrigel^® matrix is enhanced by the addition of autogenous morcellated fragments of the rectus abdominis muscle potentially providing myoblastic, pericytic/perivascular stem cells for continuous tissue induction and morphogenesis. The striated rectus abdominis muscle is endowed with stem cell niches in para/perivascular location, which can be dominant, thus imposing stem cell features or stemness to the surrounding cells. This capacity to impose stemness is morphologically shown by greater alveolar bone induction and cementogenesis when hTGF‐β₃ in Matrigel^® matrix is combined with morcellated fragments of autogenous rectus abdominis muscle. The induction of periodontal tissue morphogenesis develops as a mosaic structure in which the osteogenic proteins of the TGF‐β supergene family singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. In primates, the presence of several homologous yet molecularly different isoforms with osteogenic activity highlights the biological significance of this apparent redundancy and indicates multiple interactions during embryonic development and bone regeneration in postnatal life. Molecular redundancy with associated different biological functionalities in primate tissues may simply represent the fine‐tuning of speciation‐related molecular evolution in anthropoid apes at the early Pliocene boundary, which resulted in finer tuning of the bone induction cascade.     

18β‐Glycyrrhetinic acid inhibits periodontitis via glucocorticoid‐independent nuclear factor‐κB inactivation in interleukin‐10‐deficient mice

Sasaki H, Suzuki N, AlShwaimi E, Xu Y, Battaglino R, Morse L, Stashenko P. 18β‐Glycyrrhetinic acid inhibits periodontitis via glucocorticoid‐independent nuclear factor‐κB inactivation in interleukin‐10‐deficient mice. J Periodont Res 2010; 45: 757–763. © 2010 John Wiley & Sons A/S Background and Objective: 18β‐Glycyrrhetinic acid (GA) is a natural anti‐inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study. Material and Methods: Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in interleukin‐10‐deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on lipopolysaccharide (LPS)‐stimulated macrophages, T cell proliferation and osteoclastogenesis was also examined in vitro. Results: 18β‐Glycyrrhetinic acid administered either prophylactically or therapeutically resulted in a dramatic reduction of infection‐induced bone loss in interleukin‐10‐deficient mice, which are highly disease susceptible. Although GA has been reported to exert its anti‐inflammatory activity via downregulation of 11β‐hydroxysteroid dehydrogenase‐2 (HSD2), which converts active glucocorticoids to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, in glucocorticoid‐free conditions, GA potently inhibited LPS‐stimulated proinflammatory cytokine production and RANKL‐stimulated osteoclastogenesis, both of which are dependent on nuclear factor‐κB. Furthermore, GA suppressed LPS‐ and RANKL‐stimulated phosphorylation of nuclear factor‐κB p105 in vitro. Conclusion: These findings indicate that GA inhibits periodontitis by inactivation of nuclear factor‐κB in an interleukin‐10‐ and glucocorticoid‐independent fashion.