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1.  RANKL Inhibition Through Osteoprotegerin Blocks Bone Loss in Experimental Periodontitis 
Journal of periodontology  2007;78(7):1300-1308.
Prevention of alveolar bone destruction is a clinical challenge in periodontal disease treatment. The receptor activator of nuclear factor-kappa B ligand (RANKL) inhibitor osteoprotegerin (OPG) inhibits osteoclastogenesis and suppresses bone resorption.
To study the effects of RANKL inhibition on alveolar bone loss, an experimental ligature-induced model of periodontitis was used. A total of 32 rats were administered human OPG-Fc fusion protein (10 mg/kg) or vehicle by subcutaneous delivery twice weekly for 6 weeks. Negative or positive controls received no treatment or disease through vehicle delivery, respectively. Biopsies were harvested after 3 and 6 weeks, and mandibulae were evaluated by microcomputed tomography (μCT) and histology. Serum levels of human OPG-Fc and tartrate-resistant acid phosphatase-5b (TRAP-5b) were measured throughout the study by enzyme-linked immunosorbent assay (ELISA). Statistical analyses included analysis of variance (ANOVA) and Tukey tests.
Human OPG-Fc was detected in the sera of OPG-Fc–treated animals by 3 days and throughout the study. Serum TRAP-5b was sharply decreased by OPG-Fc treatment soon after OPG-Fc delivery and remained low for the observation period. Significant preservation of alveolar bone volume was observed among OPG-Fc–treated animals compared to the controls at weeks 3 and 6 (P <0.05). Descriptive histology revealed that OPG-Fc significantly suppressed osteoclast surface area at the alveolar crest.
Systemic delivery of OPG-Fc inhibits alveolar bone resorption in experimental periodontitis, suggesting that RANKL inhibition may represent an important therapeutic strategy for the prevention of progressive alveolar bone loss.
PMCID: PMC2583091  PMID: 17608585
Bone resorption; osteoprotegerin; periodontal disease; receptor activator of nuclear factor-kappa B ligand; therapy
2.  The Cytolethal Distending Toxin Induces Receptor Activator of NF-κB Ligand Expression in Human Gingival Fibroblasts and Periodontal Ligament Cells  
Infection and Immunity  2005;73(1):342-351.
Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-κB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.
PMCID: PMC538937  PMID: 15618171
3.  Interleukin-10 Inhibits Gram-Negative-Microbe-Specific Human Receptor Activator of NF-κB Ligand-Positive CD4+-Th1-Cell- Associated Alveolar Bone Loss In Vivo  
Infection and Immunity  2006;74(8):4927-4931.
To study anti-inflammatory cytokine effects on RANKL+-T-cell-mediated osteoclastogenesis in vivo, we injected human interleukin-10 (hIL-10) into pathogen-infected HuPBL-NOD/SCID mice. The results show significantly decreased RANKL+ Th1-associated alveolar bone loss and coexpression of human gamma interferon (hIFN-γ) and human macrophage colony-stimulating factor, but not hIL-4, in RANKL+ Th cells compatible with those from successfully treated aggressive periodontitis subjects. Thus, there are critical cytokine interactions linking hIFN-γ+ Th1 cells to RANKL-RANK/OPG signaling for periodontal osteoclastogenesis in vivo.
PMCID: PMC1539589  PMID: 16861684
4.  Magnolol Ameliorates Ligature-Induced Periodontitis in Rats and Osteoclastogenesis: In Vivo and In Vitro Study 
Periodontal disease characterized by alveolar bone resorption and bacterial pathogen-evoked inflammatory response has been believed to have an important impact on human oral health. The aim of this study was to evaluate whether magnolol, a main constituent of Magnolia officinalis, could inhibit the pathological features in ligature-induced periodontitis in rats and osteoclastogenesis. The sterile, 3–0 (diameter; 0.2 mm) black braided silk thread, was placed around the cervix of the upper second molars bilaterally and knotted medially to induce periodontitis. The morphological changes around the ligated molars and alveolar bone were examined by micro-CT. The distances between the amelocemental junction and the alveolar crest of the upper second molars bilaterally were measured to evaluate the alveolar bone loss. Administration of magnolol (100 mg/kg, p.o.) significantly inhibited alveolar bone resorption, the number of osteoclasts on bony surface, and protein expression of receptor activator of nuclear factor-κB ligand (RANKL), a key mediator promoting osteoclast differentiation, in ligated rats. Moreover, the ligature-induced neutrophil infiltration, expression of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase (MMP)-1 and MMP-9, superoxide formation, and nuclear factor-κB activation in inflamed gingival tissues were all attenuated by magnolol. In the in vitro study, magnolol also inhibited the growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans that are key pathogens initiating periodontal disease. Furthermore, magnolol dose dependently reduced RANKL-induced osteoclast differentiation from RAW264.7 macrophages, tartrate-resistant acid phosphatase (TRAP) activity of differentiated cells accompanied by a significant attenuation of resorption pit area caused by osteoclasts. Collectively, we demonstrated for the first time that magnolol significantly ameliorates the alveolar bone loss in ligature-induced experimental periodontitis by suppressing periodontopathic microorganism accumulation, NF-κB-mediated inflammatory mediator synthesis, RANKL formation, and osteoclastogenesis. These activities support that magnolol is a potential agent to treat periodontal disease.
PMCID: PMC3618931  PMID: 23573141
5.  Induction of Osteoclastogenesis and Matrix Metalloproteinase Expression by the Lipooligosaccharide of Treponema denticola  
Infection and Immunity  2003;71(1):226-233.
Alveolar bone destruction is a characteristic feature of periodontitis. Treponema denticola is known to be involved in periodontitis. To elucidate the role of T. denticola in alveolar bone destruction in periodontitis, the effects of lipooligosaccharide (LOS) from T. denticola on osteoclast formation and on expression of osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) mRNAs were examined in a coculture system by using mouse calvaria and bone marrow cells. In addition, the effect of T. denticola LOS on expression of matrix metalloproteinases (MMPs), which are involved in bone resorption, was estimated in mouse calvaria-derived osteoblastic cells. When the mouse calvaria and bone marrow cells were challenged with LOS (0.1 to 10 μg/ml) for 4 days, the number of tartrate-resistant acid phosphatase-positive multinucleated cells increased in a dose-dependent manner. The expression of ODF mRNA increased, while OPG mRNA expression decreased. Polymyxin B changed the effect of LOS (10 μg/ml) on ODF and OPG mRNA expression to the control level. LOS (10 μg/ml) stimulated prostaglandin E2 (PGE2) production in the cocultures. Adding indomethacin, an inhibitor of prostaglandin synthesis, resulted in a reduction in the number of osteoclasts induced by LOS and eliminated the effect of T. denticola LOS on ODF and OPG mRNA expression. T. denticola LOS increased the levels of mRNAs encoding MMP-3, -8, -9, -10, -13, and -14. Expression of one of these mRNAs, MMP-9 mRNA, was significantly induced by T. denticola LOS. These findings suggest that LOS from T. denticola stimulates osteoclastogenesis and MMP expression. Up-regulation of ODF and down-regulation of OPG by a PGE2-dependent mechanism were involved in the osteoclastogenesis induced by T. denticola LOS.
PMCID: PMC143274  PMID: 12496170
6.  Gamma Interferon Positively Modulates Actinobacillus actinomycetemcomitans-Specific RANKL+ CD4+ Th-Cell-Mediated Alveolar Bone Destruction In Vivo  
Infection and Immunity  2005;73(6):3453-3461.
Recent studies have shown the biological and clinical significance of signaling pathways of osteogenic cytokines RANKL-RANK/OPG in controlling osteoclastogenesis associated with bone pathologies, including rheumatoid arthritis, osteoporosis, and other osteolytic disorders. In contrast to the inhibitory effect of gamma interferon (IFN-γ) on RANKL-mediated osteoclastogenesis reported recently, alternative new evidence is demonstrated via studies of experimental periodontitis using humanized NOD/SCID and diabetic NOD mice and clinical human T-cell isolates from diseased periodontal tissues, where the presence of increasing IFN-γ is clearly associated with (i) enhanced Actinobacillus actinomycetemcomitans-specific RANKL-expressing CD4+ Th cell-mediated alveolar bone loss during the progression of periodontal disease and (ii) a concomitant and significantly increased coexpression of IFN-γ in RANKL(+) CD4+ Th cells. Therefore, there are more complex networks in regulating RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo than have been suggested to date.
PMCID: PMC1111859  PMID: 15908374
7.  Osteoprotegerin in Bone Metastases: Mathematical Solution to the Puzzle 
PLoS Computational Biology  2012;8(10):e1002703.
Bone is a common site for cancer metastasis. To create space for their growth, cancer cells stimulate bone resorbing osteoclasts. Cytokine RANKL is a key osteoclast activator, while osteoprotegerin (OPG) is a RANKL decoy receptor and an inhibitor of osteoclastogenesis. Consistently, systemic application of OPG decreases metastatic tumor burden in bone. However, OPG produced locally by cancer cells was shown to enhance osteolysis and tumor growth. We propose that OPG produced by cancer cells causes a local reduction in RANKL levels, inducing a steeper RANKL gradient away from the tumor and towards the bone tissue, resulting in faster resorption and tumor expansion. We tested this hypothesis using a mathematical model of nonlinear partial differential equations describing the spatial dynamics of OPG, RANKL, PTHrP, osteoclasts, tumor and bone mass. We demonstrate that at lower expression rates, tumor-derived OPG enhances the chemotactic RANKL gradient and osteolysis, whereas at higher expression rates OPG broadly inhibits RANKL and decreases osteolysis and tumor burden. Moreover, tumor expression of a soluble mediator inducing RANKL in the host tissue, such as PTHrP, is important for correct orientation of the RANKL gradient. A meta-analysis of OPG, RANKL and PTHrP expression in normal prostate, carcinoma and metastatic tissues demonstrated an increase in expression of OPG, but not RANKL, in metastatic prostate cancer, and positive correlation between OPG and PTHrP in metastatic prostate cancer. The proposed mechanism highlights the importance of the spatial distribution of receptors, decoys and ligands, and can be applied to other systems involving regulation of spatially anisotropic processes.
Author Summary
Breast and prostate cancers commonly metastasize to bone. To create more space for their expansion, metastatic tumors activate osteoclasts, the only cells capable of bone destruction. The main osteoclast stimulator is the cytokine RANKL, while osteoprotegerin (OPG) acts as a RANKL inhibitor. Systemic application of OPG leads to a decrease in tumor-associated bone destruction, but surprisingly, OPG produced locally by metastasizing cancer cells can enhance bone destruction and tumor growth. Here, we provide a novel explanation for these apparently contradictory experimental results: the osteolysis-promoting effect of OPG is due to a local reduction in RANKL levels, resulting in a spatial RANKL gradient oriented from tumor towards bone tissue. At low rates of OPG expression by cancer cells, such gradients result in the correct orientation of osteoclast movement and intensified bone resorption. We positively test our hypothesis by means of a partial differential equations model, and further substantiate our results with a meta-analysis of gene expression. Even though developed for the specific problem of bone metastases, our model naturally applies to other systems operating within a geometrically anisotropic environment.
PMCID: PMC3475686  PMID: 23093918
8.  Platelet-Activating Factor Receptor Blockade Ameliorates Aggregatibacter actinomycetemcomitans-Induced Periodontal Disease in Mice 
Infection and Immunity  2013;81(11):4244-4251.
Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by oral biofilm-producing microorganisms, such as Aggregatibacter actinomycetemcomitans. The levels of the phospholipid platelet-activating factor (PAF) in the saliva, gingival crevicular fluid, and periodontal tissues are significantly increased during inflammatory conditions, such as PD, but the exact mechanism that links PAF to alveolar bone resorption is not well understood. In the current study, alveolar bone resorption was induced by experimental PD through the oral inoculation of A. actinomycetemcomitans in wild-type (WT) and PAF receptor knockout (Pafr−/−) mice. In vitro experiments using A. actinomycetemcomitans lipopolysaccharide (LPS)-stimulated RAW 264.7 cells treated with a PAF receptor antagonist (UK74505) were also performed. The expression of lyso-PAF acetyltransferase in periodontal tissues was significantly increased 3 h after A. actinomycetemcomitans LPS injection in mice. WT and Pafr−/− mice that were subjected to oral inoculation of A. actinomycetemcomitans presented neutrophil accumulation and increased levels of CXCL-1 and tumor necrosis factor alpha (TNF-α) in periodontal tissues. However, Pafr−/− mice presented less alveolar bone loss than WT mice. The in vitro blockade of the PAF receptor impaired the resorptive activity of A. actinomycetemcomitans LPS-activated osteoclasts. In conclusion, this study shows for the first time that the blockade of PAF receptor may contribute to the progression of PD triggered by A. actinomycetemcomitans by directly affecting the differentiation and activity of osteoclasts.
PMCID: PMC3811845  PMID: 24002061
9.  Dysregulated B Cell Expression of RANKL and OPG Correlates with Loss of Bone Mineral Density in HIV Infection 
PLoS Pathogens  2014;10(11):e1004497.
HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction.
Author Summary
HIV infection causes significant bone loss and skeletal deterioration, leading to fractures that are often devastating and incur significant financial burden on patients and their families. HIV-infected individuals have up to a five-fold higher risk of bone fractures, and the increasing average age of people living with HIV/AIDS has triggered fears of an impending epidemic of bone fractures in this population. Antiretroviral therapy, used to manage HIV infection, fails to prevent, but rather paradoxically accelerates skeletal decline. The underlying mechanisms of HIV-induced bone loss are poorly understood. The aim of this study was to clarify the mechanisms of bone loss in HIV-infected patients, in an effort to better understand how bone loss and fractures occur, and consequently how it can be prevented in this population. The cytokine RANKL (Receptor Activator of Nuclear Factor kappa-B Ligand) helps induce bone loss. We show that RANKL expression was increased in immune cells in HIV-infected individuals. Another cytokine, osteoprotegerin (OPG), counteracts the activity of RANKL, and therefor helps prevent bone loss. OPG expression by the same immune cells was decreased in HIV-infected individuals. We conclude that disrupted immune cell expression of RANKL and OPG in HIV-infected patients contributes to bone loss.
PMCID: PMC4231117  PMID: 25393853
10.  A dominant function of p38 Mitogen Activated Protein Kinase signaling in Receptor Activator of Nuclear Factor-κB Ligand expression and osteoclastogenesis induction by A. actinomycetemcomitans and E. coli LPS 
Journal of periodontal research  2008;43(2):201-211.
Evaluate the role of p38 p38 mitogen activated protein kinase (MAPK) signaling pathway in lipopolysaccharide (LPS)-induced receptor activator of nuclear factor-κB ligand (RANKL) expression by murine periodontal ligament (PDL) cells.
LPS from Gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response leading to tissue destruction observed in periodontitis.
Expression of RANKL and osteoprotegerin (OPG) mRNA was studied by reverse transcription polymerase chain reaction (RT-PCR) upon stimulation with LPS from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of p38 MAPK signaling pathway to LPS-induced RANKL and OPG expression. Stable cell lines expressing dominant negative forms of MAKPK kinase (MKK)-3 and MKK6 were generated to confirm the role of p38 MAPK pathway. An osteoclastogenesis assay using a co-culture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by LPS-stimulated PDL was correlated with RANKL expression.
Inhibiting p38 MAPK previous to LPS stimulation resulted in a significant decrease of RANKL mRNA expression. OPG mRNA expression was not affected by LPS or p38 MAPK. LPS-stimulated PDL cells increased osteoclast differentiation, an effect that was completely blocked by OPG and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from mPDL cultures did not increase osteoclast differentiation, indicating that PDL cells produced membrane-bound RANKL.
LPS resulted in a significant increase on RANKL in PDL cells. p38 MAPK pathway is required for LPS-induced membrane-bound RANKL expression in these cells.
PMCID: PMC3086662  PMID: 18302623
osteoclastogenesis; RANKL; PDL cells; p38 MAPK
11.  Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease 
PLoS Pathogens  2012;8(3):e1002601.
Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the host's immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis.
Author Summary
Periodontal disease is a common bacterial-induced inflammatory process in which F. nucleatum and P. gingivalis infections lead to the destruction of the teeth supporting attachment apparatus. Previous reports demonstrated that immune cells aggravate the severity of the disease. However, whether NK cells in general and NKp46 (a major killer receptor expressed by NK cells) in particular, play a protective or destructive role in this disease is unknown. Using mice deficient in NCR1 (the mouse orthlogue of NKp46), we demonstrate that oral infection of mice with F. nucleatum, but not with P. gingivalis results in an NCR1-dependent alveolar bone loss. In addition, we show that F. nucleatum is recognized by NCR1 and NKp46 directly and that this recognition leads to the secretion of TNF-α, a central cytokine critically involved in the pathogenesis of periodontal destruction. Collectively, we show that NCR1 and NKp46 play a critical role in the pathogenesis of F. nucleatum-mediated periodontitis.
PMCID: PMC3310798  PMID: 22457623
12.  Bis-Enoxacin Blocks Rat Alveolar Bone Resorption from Experimental Periodontitis 
PLoS ONE  2014;9(3):e92119.
Periodontal diseases are multifactorial, caused by polymicrobial subgingival pathogens, including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. Chronic periodontal infection results in inflammation, destruction of connective tissues, periodontal ligament, and alveolar bone resorption, and ultimately tooth loss. Enoxacin and a bisphosphonate derivative of enoxacin (bis-enoxacin) inhibit osteoclast formation and bone resorption and also contain antibiotic properties. Our study proposes that enoxacin and/or bis-enoxacin may be useful in reducing alveolar bone resorption and possibly bacterial colonization. Rats were infected with 109 cells of polymicrobial inoculum consisting of P. gingivalis, T. denticola, and T. forsythia, as an oral lavage every other week for twelve weeks. Daily subcutaneous injections of enoxacin (5 mg/kg/day), bis-enoxacin (5, 25 mg/kg/day), alendronate (1, 10 mg/kg/day), or doxycycline (5 mg/day) were administered after 6 weeks of polymicrobial infection. Periodontal disease parameters, including bacterial colonization/infection, immune response, inflammation, alveolar bone resorption, and systemic spread, were assessed post-euthanasia. All three periodontal pathogens colonized the rat oral cavity during polymicrobial infection. Polymicrobial infection induced an increase in total alveolar bone resorption, intrabony defects, and gingival inflammation. Treatment with bis-enoxacin significantly decreased alveolar bone resorption more effectively than either alendronate or doxycycline. Histologic examination revealed that treatment with bis-enoxacin and enoxacin reduced gingival inflammation and decreased apical migration of junctional epithelium. These data support the hypothesis that bis-enoxacin and enoxacin may be useful for the treatment of periodontal disease.
PMCID: PMC3956892  PMID: 24638087
13.  Antibody to Receptor Activator of NF-κB Ligand Ameliorates T Cell-Mediated Periodontal Bone Resorption ▿  
Infection and Immunity  2010;79(2):911-917.
Activated T and B lymphocytes in periodontal disease lesions express receptor activator of NF-κB ligand (RANKL), which induces osteoclastic bone resorption. The objective of this study was to evaluate the effects of anti-RANKL antibody on periodontal bone resorption in vitro and in vivo. Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) and A. actinomycetemcomitans lipopolysaccharide (LPS) were injected into 3 palatal gingival sites, and Omp29-specific T clone cells were transferred into the tail veins of rats. Rabbit anti-RANKL IgG antibody or F(ab′)2 antibody fragments thereof were injected into the palatal sites in each rat (days −1, 1, and 3). Anti-RANKL IgG antibody significantly inhibited soluble RANKL (sRANKL)-induced osteoclastogenesis in vitro, in a dose-dependent manner, but also gave rise to a rat antibody response to rabbit IgG in vivo, with no significant inhibition of periodontal bone resorption detected. Lower doses (1.5 and 0.15 μg/3 sites) of F(ab′)2 antibody were not immunogenic in the context of the experimental model. Periodontal bone resorption was inhibited significantly by injection of the anti-RANKL F(ab′)2 antibody into gingivae. The sRANKL concentrations for the antibody-treated groups were decreased significantly compared to those for the untreated group. Osteoclasts on the alveolar bone surface were also diminished significantly after antibody injection. Gingival sRANKL concentration and bone loss showed a significant correlation with one another in animals receiving anti-RANKL F(ab′)2 antibody. These results suggest that antibody to RANKL can inhibit A. actinomycetemcomitans-specific T cell-induced periodontal bone resorption by blockade and reduction of tissue sRANKL, providing an immunological approach to ameliorate immune cell-mediated periodontal bone resorption.
PMCID: PMC3028846  PMID: 21078845
14.  Salivary soluble receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio in periodontal disease and health 
The receptor activator of nuclear factor kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system plays a significant role in osteoclastogenesis, activation of osteoclasts, and regulation of bone resorption. This study aimed to evaluate the use of the salivary soluble RANKL (sRANKL)/OPG ratio as a diagnostic marker for periodontitis in nonsmokers.
Twenty-five patients with chronic periodontitis and 25 individuals with a healthy periodontium were enrolled in this study. Samples containing 5 mL of unstimulated saliva were obtained from each subject. Salivary sRANKL and OPG concentrations were determined using a standard enzyme-linked immunosorbent assay. Statistical analysis was performed using SPSS ver. 18.0.
The levels of sRANKL and OPG were detectable in all of the samples. Positive relationships were found between the plaque index and clinical attachment level and both the salivary concentration of sRANKL and the salivary sRANKL/OPG ratio (P<0.05). The salivary concentration of sRANKL and the sRANKL/OPG ratio were significantly higher in the periodontitis group than in the healthy group (P=0.004 and P=0.001, respectively). In contrast, the OPG concentration showed no significant differences between the groups (P=0.455).
These findings suggest that the salivary sRANKL/OPG ratio may be helpful in the screening and diagnosis of periodontitis. However, longitudinal studies with larger populations are needed to confirm these results.
PMCID: PMC3825990  PMID: 24236245
Biological markers; Osteoprotegerin; Periodontitis; RANK ligand; Saliva
15.  TLR2 signaling and Th2 responses drive Tannerella forsythia-induced periodontal bone loss1 
Periodontal disease (PD) is a chronic inflammation of the tooth supporting soft tissue and alveolar bone due to infection by a select group of gram negative microbes, and leads to tooth loss if untreated. Since mice deficient in CD4+ cells are resistant to infection-induced alveolar bone loss, Th cells have been implicated in bone destructive processes during PD. However, the extent to which different Th-cell subtypes play roles in pathogenesis or host protection remains to be defined, and is likely to vary depending on the dominant microorganism involved. By far the best studied periodontal microbe in PD is Porphyromonas gingivalis. Even though the gram negative anaerobe Tannerella forsythia is also a vital contributor to periodontal bone loss, almost nothing is known about immune responses to this organism. Previous studies from our laboratory have revealed that T. forsythia induces periodontal bone loss in mice, and that this bone loss depends on the bacterially-expressed BspA protein. In this study, we show that T. forsythia activates murine APCs primarily through TLR2-dependent signaling via BspA. Furthermore, T. forsythia infection causes a pronounced Th2 bias, evidenced by T cell expression of IL-5 but not IFN-γ or IL-17 in draining LN. Consistently, deficiencies in TLR2 or STAT6 result in resistance to T. forsythia-induced alveolar bone loss. Thus, TLR2 signaling and Th2 cells play pathogenic roles in T. forsythia-induced alveolar bone destruction.
PMCID: PMC3119786  PMID: 21632710
16.  Periodontal inflammation and bone loss in aged mice 
Journal of periodontal research  2010;45(4):574-578.
Background and objective
Young mice do not develop measurable periodontal bone loss, unless heavily infected with human periodontal pathogens. However, mice with genetically altered immune system are unable to control their own oral flora and develop periodontitis early in life. Based on the potential of the indigenous oral microbiota to cause periodontitis, we hypothesized that normal mice may ultimately develop inflammatory periodontal bone loss, i.e., as a function of age. If confirmed, this could serve as an aging model of chronic periodontitis.
Materials and methods
Periodontal bone levels were measured as the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC), in young (8-10 weeks of age), old (≥ 18 months of age), and mice of intermediate ages. Differential expression of inflammatory mediators in the gingivae of young and old mice was determined by quantitative real-time PCR.
In comparison to young mice, old mice displayed significantly (p < 0.05) increased periodontal bone loss, accompanied by elevated expression of proinflammatory cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-17A) and innate immune receptors involved in the induction or amplification of inflammation (Toll-like receptor 2, CD14, CD11b, CD18, complement C5a receptor, and triggering receptor expressed on myeloid cells-3).
Mice develop naturally-induced periodontal bone loss as a function of age. This aging model of periodontitis represents a genuinely chronic model to study mechanisms of periodontal tissue destruction.
PMCID: PMC2894296  PMID: 20337897
Animal model; Alveolar bone; Chronic periodontitis; Inflammation; Innate immunology
17.  The role of RANK ligand/osteoprotegerin in rheumatoid arthritis 
In the complex system of bone remodeling, the receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) pathway is the coupling factor between bone formation and bone resorption. RANKL binds to the RANK receptor of pre-osteoclasts and mature osteoclasts and stimulates their activation and differentiation. The production of RANKL/OPG by osteoblasts is influenced by hormones, growth factors and cytokines, which each have a different effect on the production of RANKL and OPG. Ultimately, the balance between RANKL and OPG determines the degree of proliferation and activity of the osteoclasts. In rheumatoid arthritis (RA), bone erosions are the result of osteoclastic bone resorption at the sites of synovitis, where RANKL expression is also found. Furthermore, magnetic resonance imaging (MRI) bone edema in RA indicates the presence of active inflammation within bone and the presence of osteitis, which is also associated with the expression of RANKL. Bone loss has been documented in the cortical and trabecular bone in the joints of the hand of RA patients. Both synovitis and periarticular bone involvement (osteitis and bone loss) are essential components of RA: they occur early in the disease and both are predictive for the occurrence and progression of bone damage. RANKL knockout mice and mice treated with OPG did not develop focal bone loss, in spite of persistent joint inflammation. Inhibition of osteoclasts by denosumab, a humanized antibody that selectively binds RANKL, has revealed in patients with RA that the occurrence of erosions and periarticular bone loss can be halted, however without affecting synovial inflammation. This disconnect between inflammation and bone destruction opens new ways to separately focus treatment on inflammation and osteoclastogenesis for preventing and/or minimizing the connection between joints and subchondral bone and bone marrow.
PMCID: PMC3403250  PMID: 22859921
RANK ligand; osteoprotegerin; rheumatoid arthritis; bone erosions; osteitis; bone loss
18.  Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid 
Dental Research Journal  2012;9(6):752-757.
Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls.
Materials and Methods:
In this analytical study, GCF was obtained from healthy (n = 10), mild chronic periodontitis (n = 18), moderate chronic periodontitis (n = 18), severe chronic periodontitis (n = 20), and generalized aggressive periodontitis (n = 20) subjects. RANKL and OPG concentrations were measured by enzyme-linked immunosorbent assay. Statistical tests used were Kruskal–Wallis test, Mann–Whitney U rank sum test, and Spearman's rank correlation analysis. The level of statistical significance was set at P < 0.05.
Mean RANKL concentration showed no statistically significant differences between groups (P = 0.58). There were also no significant differences between mean OPG concentration in the five groups (P = 0.0.56). Moreover, relative RANKL/OPG ratio did not reveal a significant difference between the three study group subjects: healthy, chronic periodontitis (mild, moderate, severe), and aggressive periodontitis (P = 0.41). There was statistically significant correlation between the concentration of sRANKL and Clinical Attachment Level (CAL) in moderate chronic periodontitis patients (R = 0.48, P = 0.04). There was also negative correlation between OPG concentration and CAL in moderate chronic periodontitis patients, although not significant (R = −0.13).
RANKL was prominent in periodontitis sites, especially in moderate periodontitis patients, whereas OPG was not detectable in some diseased sites with bleeding on probing, supporting the role of these two molecules in the bone loss developed in this disease.
PMCID: PMC3612226  PMID: 23559954
Enzyme-linked immunosorbent assay; gingival crevicular fluid; Osteoprotegerin; RANKL; Receptor activator of nuclear factor kappa B ligand
19.  Induction of retinol-binding protein 4 and placenta-specific 8 expression in human prostate cancer cells remaining in bone following osteolytic tumor growth inhibition by osteoprotegerin 
International Journal of Oncology  2013;43(2):365-374.
New drugs that inhibit the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL)/RANK pathway have demonstrated efficacy for the treatment of bone metastasis. Toxicities induced by these drugs, however, including osteonecrosis of the jaw and hypocalcemia, may adversely affect therapy. The aim of this study was to identify additional therapeutic targets that can be combined with OPG/RANKL/RANK pathway inhibition in the treatment of prostate cancer bone metastasis. We established a stable transfectant that produces high levels of OPG mRNA and protein from PC-3 human prostate cancer cells (PC3-OPG). The culture medium of PC3-OPG cells significantly inhibited the differentiation of mouse monocytes into mature osteoclasts. Furthermore, when PC3-OPG cells were injected into the bones of nude mice, bone destruction and tumor-induced osteoclast formation were reduced. Injection into bone of the mixtures containing equal amounts of green fluorescent protein (GFP)-expressing PC-3 cells (PC3-GFP) and PC3-OPG cells also reduced bone destruction, compared to the control mixture. PC3-GFP cells were subsequently isolated from bone tumors and used for microarray analysis to assess changes in gene expression following osteolytic tumor growth inhibition by OPG. We selected the top 10 upregulated genes based on results from microarrays and confirmed mRNA expression of each gene by RT-PCR. The expression patterns of retinol-binding protein 4 (RBP4) and placenta-specific 8 (PLAC8) were consistent with microarray results. Expression of these genes was also increased in the bone tumors of PC3-GFP/PC3-OPG-injected mice. Knockdown of both RBP4 and PLAC8 by siRNA inhibited the growth of PC-3 cells in vitro. Thus, RBP4 and PLAC8 may become new therapeutic targets for prostate cancer bone metastasis, in combination with OPG/RANKL/RANK pathway inhibition.
PMCID: PMC3775580  PMID: 23708710
osteoprotegerin; retinol binding protein 4; placenta-specific 8; bone metastasis; prostate cancer
20.  Gingival crevicularfluid osteoprotegerin levels in Indian population 
Dental Research Journal  2012;9(6):774-782.
Initial research indicated that higher concentration of osteoprotegerin (OPG) is associated with healthy periodontium (protective) and its concentration decreases as the periodontal disease progresses. However, till date, there are no studies to investigate the levels of OPG in gingival crevicular fluid (GCF) after the treatment of periodontitis. Hence, the present study was carried out to assess its concentration in GCF to find out their association if any, and to explore its possible use as a ‘novel bone marker’ of the host modulation of periodontal disease.
Materials and Methods:
Sixty-four subjects were divided into 4 groups (16 each), based on clinical attachment loss (CAL) and radiological parameters (bone loss); healthy (group I), gingivitis (group II), slight periodontitis (group III), and moderate-to-severe periodontitis (group IV). Moderate-to-severe periodontitis subjects, after nonsurgical periodontal treatment, (SRP) constituted group V. GCF samples were collected to estimate the levels of OPG using enzyme-linked immunosorbent assay (ELISA). The Kruskal-Wallis, Man-Whitney U test, and Wilcoxon signed-rank tests were carried out to compare OPG levels among groups. The Spearman rank correlation test was used to correlate OPG levels between the study groups and the clinical parameters; P < 0.05 was considered significant.
The highest mean OPG concentration in GCF was obtained for group I (162.47 ± 51.171 pg/ μL) and the least for group IV (10.92 ± 1.913 pg/μL), suggesting a negative correlation between OPG concentration and CAL. OPG concentrations in GCF after the treatment of group IV increased from 10.92 ± 1.913 pg/μL to 15.63 ± 4.679 pg/μL.
OPG concentration in GCF was inversely proportional to CAL and not an active progression factor for periodontal disease. Further, after the treatment of moderate-to-severe periodontitis subjects (group IV), OPG concentrations increased. Hence, it can be concluded that OPG could be considered as a ‘novel bone marker’ the host modulation of periodontal disease.
PMCID: PMC3612230  PMID: 23559958
Gingival crevicular fluid; Osteoprotegerin; periodontal disease
21.  Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK 
Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa β ligand /receptor activator of NF-kappa β (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK.
In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics.
In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients ≥ 70 years (Fishers exact p < 0.01).
Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was assayed in this study, but transmembrane RANKL may also be important in this system. Macrophages subjected to pressure (both alone and in co-culture) stained more strongly for RANK on immunohistochemstry than non-pressurized controls and 1,25-dihydroxyvitamin D3 (1,25 D3) further increased this. Immunocytochemical staining also demonstrated that more cells in pressurized co-cultures exhibited osteoclast markers (tartrate-resistant acid phosphatase, vitronectin receptor and multinuclearity) than did unpressurized controls.
These data show that in co-cultures of osteoblasts and macrophages the ratio of OPG : sRANKL was decreased by pressure in younger patients but increased in older patients. As falls in this ratio promote bone resorption, this finding may be important in explaining the relatively high incidence of osteolysis around orthopaedic implants in young patients. The finding that secretion of OPG and sRANKL by osteoblasts in monoculture was sensitive to hydrostatic pressure, and that hydrostatic pressure stimulated the differentiation of macrophages into cells exhibiting osteoclast markers indicates that both osteoblasts and preosteoclasts are sensitive to cyclic pressure. However, the effects of pressure on cocultures were not simply additive and coculture appears useful to examine the interaction of these cell types.
These findings have implications for future therapies for aseptic loosening and for the development of tests to predict the development of this condition.
PMCID: PMC1475864  PMID: 16519799
22.  Porphyromonas gingivalis Infection-Associated Periodontal Bone Resorption Is Dependent on Receptor Activator of NF-κB Ligand 
Infection and Immunity  2013;81(5):1502-1509.
Porphyromonas gingivalis is one of the oral microorganisms associated with human chronic periodontitis. The purpose of this study is to determine the role of the receptor activator of nuclear factor-κB ligand (RANKL) in P. gingivalis infection-associated periodontal bone resorption. Inbred female Rowett rats were infected orally on four consecutive days (days 0 to 3) with 1 × 109 P. gingivalis bacteria (strain ATCC 33277). Separate groups of rats also received an injection of anti-RANKL antibody, osteoprotegerin fusion protein (OPG-Fc), or a control fusion protein (L6-Fc) into gingival papillae in addition to P. gingivalis infection. Robust serum IgG and salivary IgA antibody (P < 0.01) and T cell proliferation (P < 0.05) responses to P. gingivalis were detected at day 7 and peaked at day 28 in P. gingivalis-infected rats. Both the concentration of soluble RANKL (sRANKL) in rat gingival tissues (P < 0.01) and periodontal bone resorption (P < 0.05) were significantly elevated at day 28 in the P. gingivalis-infected group compared to levels in the uninfected group. Correspondingly, RANKL-expressing T and B cells in rat gingival tissues were significantly increased at day 28 in the P. gingivalis-infected group compared to the levels in the uninfected group (P < 0.01). Injection of anti-RANKL antibody (P < 0.05) or OPG-Fc (P < 0.01), but not L6-Fc, into rat gingival papillae after P. gingivalis infection resulted in significantly reduced periodontal bone resorption. This study suggests that P. gingivalis infection-associated periodontal bone resorption is RANKL dependent and is accompanied by increased local infiltration of RANKL-expressing T and B cells.
PMCID: PMC3648000  PMID: 23439308
23.  Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease 
Journal of Oral Microbiology  2010;2:10.3402/jom.v2i0.5532.
Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider (1) the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and (2) the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria.
PMCID: PMC3084575  PMID: 21523224
periodontal pathogenic bacteria; RANKL; bone resorption; osteoimmunology
24.  Induction of Bone Loss by Pathobiont-Mediated Nod1 Signaling in the Oral Cavity 
Cell host & microbe  2013;13(5):595-601.
Periodontitis is a common disease that is characterized by resorption of the alveolar bone and mediated by commensal bacteria that trigger host immune responses and bone destruction through unidentified mechanisms. We report that Nod1, an innate intracellular host receptor for bacterial peptidoglycan-related molecules, is critical for commensal-induced periodontitis in a mouse model. Mice lacking Nod1 exhibit reduced bone resorption as well as impaired recruitment of neutrophils to gingival tissues and osteoclasts to the alveolar bone, which mediate tissue and bone destruction. Further analysis showed that accumulation of a Nod1-stimulating commensal bacterium, NI1060, at gingival sites was sufficient to induce neutrophil recruitment and bone resorption. Genomic sequencing revealed that NI1060 is a mouse-specific bacterium that is related to bacteria associated with the development of aggressive periodontitis in humans. These findings provide insight into commensal-host interactions contributing to periodontitis and identify a potential target for preventing this common oral disease.
PMCID: PMC3721316  PMID: 23684310
25.  Regeneration of bone and periodontal ligament induced by Recombinant amelogenin after periodontitis 
Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth-supporting tissues, alveolar bone, periodontal-ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial enamel matrix proteins are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant amelogenin protein (rHAM+), induced in-vivo regeneration of all tooth-supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first two weeks after application, rHAM+ induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio-temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on recombinant amelogenin proteins.
PMCID: PMC2889159  PMID: 19228267
Amelogenin; periodontal disease; bone regeneration; cementum regeneration; PDL regeneration; CD105; STRO-1

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