Effect of smoking on concentrations of receptor activator of nuclear factor κ B ligand and osteoprotegerin in human gingival crevicular fluid

Aim: To compare the levels of the soluble receptor activator of nuclear factor κ B ligand (sRANKL), osteoprotegerin (OPG) and their relative ratio in gingival crevicular fluid (GCF) among periodontitis patients with varying smoking histories.
Material and Methods: GCF samples were collected from 149 periodontitis patients who were never smokers ( n =58), former smokers ( n =39) and current smokers ( n =52). sRANKL and OPG concentrations in GCF were measured by enzyme-linked immunosorbent assays.
Results: sRANKL, OPG and their relative ratio were not statistically significant among the never smokers, former smokers and current smokers. However, OPG was significantly reduced and subsequently the sRANKL:OPG ratio was significantly increased in the high pack-years group as compared with never smokers. The positive correlation between pack-years and the sRANKL:OPG ratio remained statistically significant after adjusting for age and current smoking status.
Conclusion: Increased lifetime exposure to cigarette smoking above a minimum threshold suppresses OPG production and leads to increased sRANKL:OPG. This may partially explain increased bone loss in smoking-related periodontitis.     

Differential expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin mRNA in periodontal diseases

BACKGROUND AND OBJECTIVE: Receptor activator of nuclear factor-kappaB ligand (RANKL) is responsible for the induction of osteoclastogenesis and bone resorption, whereas its decoy receptor, osteoprotegerin, can directly block this action. Because this dyad of cytokines is crucial for regulating the bone remodelling process, imbalances in their expression may cause a switch from the physiological state to enhanced bone resorption or formation. This study investigated the mRNA expression of RANKL and osteoprotegerin, as well as their relative ratio, in the gingival tissues of patients with various forms of periodontal diseases. MATERIAL AND METHODS: Gingival tissue was obtained from nine healthy subjects and 41 patients, who had gingivitis, chronic periodontitis, generalized aggressive periodontitis, and chronic periodontitis and were receiving immunosuppressant therapy. Quantitative real-time polymerase chain reaction was employed to evaluate the mRNA expression of RANKL and osteoprotegerin in these tissues. RESULTS: Compared with healthy individuals, patients in all periodontitis groups, but not those with gingivitis, exhibited stronger RANKL expression and a higher relative RANKL/osteoprotegerin ratio. In addition, osteoprotegerin expression was weaker in patients with chronic periodontitis. When patients with generalized aggressive periodontitis and chronic periodontitis were compared, the former exhibited stronger RANKL expression, whereas the latter exhibited weaker osteoprotegerin expression, and there was no difference in their relative ratio. When chronic periodontitis patients were compared with chronic periodontitis patients receiving immunosuppressant therapy, osteoprotegerin, but not RANKL, expression was stronger in the latter. CONCLUSION: This study demonstrates that RANKL and osteoprotegerin expression are differentially regulated in various forms of periodontitis, and the relative RANKL/osteoprotegerin ratio appears to be indicative of disease occurrence. This information may confer diagnostic and therapeutic value in periodontitis.     

Expression of receptor activator of NF-kappa B ligand and osteoprotegerin in culture of human periodontal ligament cells

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins implicated in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament (PDL) cells and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated remarkably by application of 10-8 m one-alpha, 25-dihydroxyvitamin D3[1,25-(OH)2D3] and 10-7 m dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated decrease of OPG by the application of 1,25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase-positive multinuclear cells were markedly induced when the PDL cells were cocultured with mouse bone marrow cells in the presence of an anti-OPG antibody together with 1,25-(OH)2D3 and Dex. These results indicate that PDL cells synthesize both RANKL and OPG and that inactivation of OPG may play a key role in the differentiation of osteoclasts.     

Expression of receptor activator of nuclear factor κB ligand in ligature-induced periodontitis in osteoporotic and non-osteoporotic rats

Allam E, Draz A, Hassan A, Neamat A, Galal M, Windsor LJ. Expression of receptor activator of nuclear factor κB ligand in ligature‐induced periodontitis in osteoporotic and non‐osteoporotic rats. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01210.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: This study investigated the expression of a key mediator that regulates differentiation of osteoclasts, receptor activator of nuclear factor κB ligand (RANKL), in rats with or without osteoporosis and periodontitis, to provide a better understanding of the association between these two diseases. Material and Methods: Forty adult Albino rats were divided into four groups: (1) control group; (2) experimentally induced periodontitis group; (3) experimentally induced osteoporosis group; and (4) experimentally induced osteoporosis and periodontitis group. At the end of the experimental period, blood samples were obtained and animals were sacrificed. Serum alkaline phosphatase (ALP) activity levels were measured. Histological evaluation and immunohistochemical detection of RANKL in the periodontal ligament and bone tissues were performed. Results: There were significantly higher ALP levels in all of the experimental groups than in the control group. The pathology observed in the histological sections from group 4 was more severe than in either group 2 or group 3. The percentage of RANKL‐immunoreactive cells in both the periodontal ligament and bone tissues in group 4 (16.8 ± 5.1 and 11.2 ± 5.2%, respectively) was significantly higher (p < 0.001) than in the other groups. In the periodontal ligament, the percentage of RANKL‐immunoreactive cells in group 2 (10.1 ± 1.9%) was significantly higher (p < 0.001) than in group 3 (5.3 ± 2.7%) and the control group (4.12 ± 1.5%). Conclusion: The increased bone loss observed in group 4 compared with either group 2 or group 3 supports the existence of an additive pathological effect of the two disease conditions. This is consistent with the increased RANKL expression observed in group 4.     

Receptor activator NF kappaB ligand (RANKL) and osteoprotegerin (OPG) protein expression in periodontitis

OBJECTIVES AND BACKGROUND: This study investigated the expression of key mediators that regulate differentiation of osteoclasts, receptor activator of nuclear factor kappaB ligand (RANKL), and its natural inhibitor, osteoprotegerin (OPG), in periodontitis. We aimed to compare the levels of the RANKL and OPG in the granulomatous tissue adjacent to areas of alveolar bone loss from patients with periodontitis to that present in tissue from patients without periodontitis. In addition, we aimed to determine the types of cells expressing these factors in these tissues and to demonstrate the expression of the osteoclastic markers, RANK and tartrate-resistant acid phosphatase (TRAP), in periodontitis. MATERIALS AND METHODS: Frozen biopsy specimens were analysed using specific monoclonal antibodies and were evaluated by semiquantitative analysis and digital image analysis to compare levels of RANKL and OPG protein expression. Double labelling of frozen sections with antibodies to different cell lineage specific markers was used to determine the types of cells expressing these proteins. In situ hybridization was used to detect cells expressing RANK mRNA. RESULTS: Semiquantitative image analysis demonstrated that significantly higher levels of RANKL protein (P < 0.05) were expressed in the periodontitis tissue. Conversely, OPG protein was significantly lower (P < 0.05) in the periodontitis tissues. RANKL protein was associated with lymphocytes and macrophages. OPG protein was associated with endothelial cells in both tissues. Many leukocytes expressing RANK mRNA and TRAP were observed in periodontitis tissues. CONCLUSION: The change in the levels of these key regulators of osteoclast differentiation may play a major role in the bone loss seen in periodontitis.     

破骨细胞核因子κB受体活化因子配基和骨保护因子在种植体周围软组织及骨组织的表达

目的研究破骨细胞核因子κB受体活化因子配基（RANKL）及其伪受体骨保护因子（OPG）在非负荷期种植体周围软组织和骨组织中的表达变化及时间分布特点。方法选用6只1～2岁龄的雄性Beagle犬建立种植义齿动物模型,分别观测种植体植入后3、7、15、30、60、90 d种植体周围的骨改建情况。取种植体周围软组织进行实时荧光定量聚合酶链反应（PCR）;取犬下颌骨进行大体标本观察拍摄X线片;取种植体周围骨组织进行免疫组化染色,检测RANKL和OPG随时间的表达变化及分布特点。结果骨改建的最活跃期为种植体植入后的第7天,种植体周围软组织中的RANKL和OPG mRNA及骨组织中的RANKL和OPG均随种植体植入时间的增加而增加,第7天达高峰,而后均逐渐降低。RANKL和OPG在种植体周围软组织及骨组织中的表达变化规律一致。结论OPG和RANKL能在种植体周围软组织中表达,且变化规律与种植体周围骨组织改建过程一致。种植体周围组织可以通过OPG/RANKL系统参与破骨细胞的形成,调节骨质吸收,影响骨组织代谢微环境。     

骨保护蛋白-核因子-κB受体活化因子配体-核因子-κB受体活化因子系统及其在颞下颌关节中的作用

骨保护蛋白(OPG)是骨代谢中重要的调节因子,已成为近年来的研究热点。它与核因子-κB受体活化因子配体(RANKL)和核因子-κB受体活化因子(RANK)构成一个关联系统,即OPG-RANKL-RANK系统,对于骨、软骨的形成、发育以及发病等起着很重要的调节作用。颞下颌关节对于维持口腔颌面部的正常形态和功能至关重要,而骨、软骨结构又是颞颌关节的重要组成成分,因此,OPG-RANKL-RANK系统对于颞颌关节的影响也不容忽视。     

Cross-reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent periodontal bone resorption in a mouse model

INTRODUCTION: The present study examined whether induction of an adaptive immune response to orally colonizing non-pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. METHODS: BALB/c mice harboring P. pneumotropica (P. pneumotropica(+) mice) in the oral cavity or control P. pneumotropica-free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T-cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor-kappaB ligand (RANKL) -positive T cells in gingival tissue. RESULTS: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29-kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross-reactivity with P. pneumotropica OmpA compared to results in the control non-immunized mice. The A. actinomycetemcomitans-immunized P. pneumotropica(+) mice developed remarkable periodontal bone loss in a RANKL-dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin-Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double-color confocal microscopy demonstrated that the frequency of RANKL(+) T cells in the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice was remarkably elevated compared to control mice. CONCLUSION: The induction of an adaptive immune response to orally colonizing non-pathogenic P. pneumotropica results in RANKL-dependent periodontal bone loss in mice.     

The effect of estrogen deficiency on receptor activator of nuclear factor kappa B ligand and osteoprotegerin synthesis in periapical lesions induced in rats

The aim of this research was to study the effects of estrogen deficiency on the receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) synthesis in the periapical lesions that were induced in ovariectomized (OVX) and sham-ovariectomized (Sham) rats. After mandibles were collected, the enzyme histochemical test for tartrate-resistant acid phosphatase and immunohistochemical examination for RANKL and OPG were performed. On days 0, 7, and 28, the numbers of osteoclasts in the periapical areas of the OVX rats were significantly more than those of the Sham group. The data had statistical differences on days 7, 21, and 28 for RANKL-positive cells between the 2 groups. The number of OPG-positive cells of the OVX rats was more than that of the Sham group at the early stage of estrogen deficiency (day 7). Estrogen deficiency-induced severe periapical bone resorption might be mediated by overexpression of RANKL. At the early stage of estrogen deficiency, the osteoblastogenesis was also accelerated, which was shown as a kind of reactive protective response.     

抑制核因子-κB受体活化因子及其配体通路治疗牙周炎的研究进展

核因子-κB受体活化因子（RANK）及其配体（RANKL）在牙周炎患者的牙槽骨吸收中具有重要的作用,抑制RANKL／RANK通路,可有效抑制破骨细胞的分化和激活,从而抑制牙槽骨的吸收。骨保护蛋白（OPG）和RANKL结合,干扰RANKL和RANK的结合,从而防止骨组织的过度破坏。本文就RANKL／RANK／OPG轴、RANKL／RANK／OPG与牙周炎、抑制RANKL／RANK通路治疗牙周炎的可行性等研究进展作一综述。     

Cross‐reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor‐κB ligand (RANKL)‐dependent periodontal bone resorption in a mouse model

Introduction: The present study examined whether induction of an adaptive immune response to orally colonizing non‐pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. Methods: BALB/c mice harboring P. pneumotropica (P. pneumotropica⁺ mice) in the oral cavity or control P. pneumotropica‐free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T‐cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor‐κB ligand (RANKL) ‐positive T cells in gingival tissue. Results: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29‐kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross‐reactivity with P. pneumotropica OmpA compared to results in the control non‐immunized mice. The A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice developed remarkable periodontal bone loss in a RANKL‐dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin‐Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double‐color confocal microscopy demonstrated that the frequency of RANKL⁺ T cells in the gingival tissue of A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice was remarkably elevated compared to control mice. Conclusion: The induction of an adaptive immune response to orally colonizing non‐pathogenic P. pneumotropica results in RANKL‐dependent periodontal bone loss in mice.     

Transient receptor potential vanilloid‐1 regulates osteoprotegerin/RANKL homeostasis in human periodontal ligament cells

Background and Objective: Increasing evidence has shown the presence of transient receptor potential vanilloid‐1 (TRPV1) in a variety of nonneuronal tissues; however, the function of TRPV1 in these cells is not well understood. In this study, we aimed to investigate the expression and function of TRPV1 in human periodontal ligament (HPDL) cells. As HPDL cells are known to play an important role in the bone‐remodeling process, we hypothesized that TRPV1 might be implicated in the regulation of osteoprotegerin (OPG) and RANKL expression. Material and Methods: TRPV1 expression was examined by western blot analysis. The function of TRPV1 was studied using capsaicin, a well‐known TRPV1 agonist. RT‐PCR was performed to study the expression of OPG and RANKL mRNAs. The expression of OPG and RANKL proteins was analyzed by ELISA and western blotting, respectively. The mechanisms of capsaicin‐induced OPG expression in HPDL cells were studied using inhibitors. Results: In this study we found that TRPV1 was present in HPDL cells. Treatment with capsaicin induced OPG expression in a dose‐dependent manner but did not affect the expression of RANKL. The increase of the OPG/RANKL ratio was also found in human osteoblasts, but not in MC3T3‐E1 cells, a mouse osteoblastic cell line, suggesting species specificity. Capsazepine, the competitive TRPV1 antagonist, significantly abolished the effect of capsaicin on OPG expression in HPDL cells. In addition, studies investigating the effects of a calcium chelator and a phospholipase C inhibitor indicated that calcium ions and phospholipase C were required for the induction. Interestingly, capsaicin was able to increase the OPG/RANKL ratio, even in the presence of prostaglandin E2, a potent inducer of RANKL. Conclusion: Our study demonstrates that activation of TRPV1 leads to an increase of the OPG/RANKL ratio in HPDL cells. These findings suggest the novel function of TRPV1 in periodontal tissues, at least, as the regulator of the OPG/RANKL axis.     

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.