Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Levels of Pro-Inflammatory and Bone-Resorptive Mediators in Periodontally Compromised Patients under Orthodontic Treatment Involving Intermittent Forces of Low Intensities.

During orthodontic treatment, diverse cytokines, enzymes, and osteolytic mediators produced within the teeth surrounding periodontal tissues determine the rate of alveolar bone remodeling and consequent teeth movement. In patients with teeth presenting reduced periodontal support, periodontal stability should be ensured during orthodontic treatment. Thus, therapies based on the application of low-intensity intermittent orthodontic forces are recommended. To determine if this kind of treatment is periodontally well tolerated, this study aimed to analyze the production of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), interleukin (IL)-6, IL-17A, and matrix metalloproteinase (MMP)-8 in periodontal tissues of protruded anterior teeth with reduced periodontal support and undergoing orthodontic treatment. Patients with periodontitis-associated anterior teeth migration received non-surgical periodontal therapy and a specific orthodontic treatment involving controlled low-intensity intermittent orthodontic forces. Samples were collected before periodontitis treatment, after periodontitis treatment, and at 1 week to 24 months of the orthodontic treatment. During the 2 years of orthodontic treatment, no significant differences were detected in the probing depth, clinical attachment level, supragingival bacterial plaque, and bleeding on probing. In line with this, the gingival crevicular levels of RANKL, OPG, IL-6, IL-17A, and MMP-8 did not vary between the different evaluation time-points of the orthodontic treatment. When compared with the levels detected during the periodontitis, the RANKL/OPG ratio was significantly lower at all the analyzed time-points of the orthodontic treatment. In conclusion, the patient-specific orthodontic treatment based on intermittent orthodontic forces of low intensities was well tolerated by periodontally compromised teeth with pathological migration.

Fengshi Qutong capsule ameliorates bone destruction of experimental rheumatoid arthritis by inhibiting osteoclastogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bone destruction plays a key role in damaging the joint function of rheumatoid arthritis (RA). Fengshi Qutong capsule (FSQTC) consisting of 19 traditional Chinese medicines has been used for treating RA in China for many years. Preliminary studies show that FSQTC has analgesic activity and inhibits synovial angiogenesis of collagen-induced arthritis (CIA), but its role on bone destruction of RA is still unclear. AIM OF THE STUDY: To explore the effect of FSQTC on bone destruction of RA and the possible mechanism of osteoclastogenesis in vivo and in vitro. MATERIALS AND METHODS: LC-MS system was used to detect the quality control components of FSQTC. The anti-arthritic effect of FSQTC on CIA rats was evaluated by arthritis score, arthritis incidence and histopathology evaluation of inflamed joints. The effect of treatment with FSQTC on bone destruction of joint tissues was determined with X-ray and micro-CT quantification, and on bone resorption marker CTX-I and formation marker osteocalcin in sera were detected by ELISA. Then, osteoclast differentiation and mature were evaluated by TRAP staining, actin ring immunofluorescence and bone resorption assay both in joints and RANKL-induced RAW264.7 cells. In addition, RANKL, OPG, IL-1ß and TNFα in sera were evaluated by ELISA. The molecular mechanisms of the inhibitions were elucidated by analyzing the protein and gene expression of osteoclastic markers CTSK, MMP-9 and ß3-Integrin, transcriptional factors c-Fos and NFATc1, as well as phosphorylation of ERK1/2, JNK and P38 in joints and in RANKL-induced RAW264.7 cells using western blot and/or qPCR. RESULTS: In this study, 12 major quality control components were identified. Our data showed that FSQTC significantly increased bone mineral density, volume fraction, trabecular thickness, and decreased trabecular separation of inflamed joints both at periarticular and extra-articular locations in CIA rats. FSQTC also diminished the level of CTX-I and simultaneously increased osteocalcin in sera of CIA rats. The effects were accompanied by reductions of osteoclast differentiation, bone resorption, and expression of osteoclastic markers (CTSK, MMP-9 and ß3-Integrin) in joints. Interestingly, FSQTC treatment could reduce the protein level of RANKL, increase the expression of OPG, and decrease the ratio of RANKL to OPG in inflamed joints and sera of CIA rats. In addition, FSQTC inhibited the levels of pro-inflammatory cytokines implicated in bone resorption, such as IL-1ß and TNFα in sera. When RAW264.7 cells were treated with RANKL, FSQTC inhibited the formation of TRAP + multinucleated cells, actin ring and the bone-resorbing activity in dose-dependent manners. Furthermore, FSQTC reduced the RANKL-induced expression of osteoclastic genes and proteins and transcriptional factors (c-Fos and NFATc1), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). CONCLUSION: FSQTC may inhibit bone destruction of RA by its anti-osteoclastogenic activity both in vivo and in vitro.

Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

Objective: Chronic periodontitis is a bone-destructive disease affecting periodontal support structures. Although leptin has a protective effect against periodontitis, the underlying mechanism remains unclear. Therefore, this study aimed to investigate the possible role of leptin by examining its relationship with OPG and RANKL in human gingival tissues obtained from patients with chronic periodontitis. Method: Twenty-two patients with chronic periodontitis were enrolled (10 with moderate periodontitis and 12 with severe periodontitis) in the experimental group, and 12 healthy individuals were enrolled in the control group. Gingival tissue samples were collected, and the protein levels and localization of leptin, OPG, and RANKL were studied using immunohistochemistry (IHC). The staining intensities of leptin, OPG, and RANKL were correlated with the periodontal clinical index. Moreover, real-time quantitative PCR (RT-qPCR) was used to determine OPG and RANKL mRNA levels in gingival fibroblasts stimulated with gradient concentrations of leptin protein in vitro. Result: Leptin, OPG, and RANKL were located in the cytoplasm of gingival epithelial cells and the connective tissue. Leptin was widely and significantly expressed in the control group, whereas it was lightly stained in the severe group. RANKL was lightly stained in the control group, whereas it was widely and significantly expressed in the severe group. The control and the moderate groups had similar OPG levels, which were significantly higher than that in the severe group. Leptin was positively correlated with OPG(r = 0.905, p < 0.01) and negatively correlated with RANKL (r = -0.635, p < 0.01). In vitro low concentrations of leptin led to an increased OPG/RANKL mRNA ratio, whereas the opposite effect was observed at high concentrations. Conclusion: Leptin can regulate OPG and RANKL expression in gingival fibroblasts and may thus play a role in the development of chronic periodontitis by modulating the OPG/RANKL ratio.

Uterine leiomyosarcomas with osteoclast-like giant cells associated with high expression of RUNX2 and RANKL.

Uterine leiomyosarcoma (ULMS) with osteoclast-like giant cells (OLGCs) has been reported as a rare phenomenon in ULMS, and its clinico-pathological features and tumorigenesis remain unclear. We recently reported high expression of receptor activator of nuclear factor κB ligand (RANKL) in ULMS with OLGCs. As osteoblasts produce RANKL, in this study, we analyzed the expression of Runt-related transcription factor 2 (RUNX2), a critical transcription factor for osteoblasts, and osteoclast-related proteins in three cases of ULMS with OLGCs as well as five conventional ULMSs and nine leiomyomas. Immunohistochemistry and real-time reverse transcription quantitative polymerase chain reaction analyses showed high expression of RUNX2 and RANKL in ULMS with OLGCs. In these cases, macrophages expressed receptor activator of nuclear factor κB (RANK), and OLGCs expressed osteoclast-related proteins (nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and cathepsin K). Accumulation sites of cathepsin K-positive OLGCs showed hemorrhagic appearance and degraded type IV collagen. We reviewed reported cases of ULMS with OLGCs, including ours, and found that they presented an aggressive course even at stage I. Furthermore, metastatic lesions showed similar histological features to those of OLGC association in ULMS. Here, we show that tumor cells in ULMS with OLGCs highly express RUNX2 and RANKL and that osteoclastic differentiation of macrophages occurs in the tumor tissue.

Impact of Notch signalling molecules and bone resorption regulators on clinical parameters in periodontitis.

BACKGROUND AND OBJECTIVE: Notch signalling cascade has recently been connected to alveolar bone resorption in periodontitis. Hence, the present cross-sectional study aimed to analyze the expression of Notch signalling pathway (Notch 1, Notch 2, Jagged 1, Hes 1, Hey 1) and periodontitis-related (tumor necrosis factor alpha- TNF-α, interleukin 17-IL-17, receptor activator of nuclear factor-kappa B ligand-RANKL, osteoprotegerin-OPG) molecules and correlate it with clinical parameters in aggressive (AP) and chronic (CP) periodontitis. Additionally, the aforementioned markers' expression was evaluated in periodontitis patients with different RANKL/OPG ratios. MATERIAL AND METHODS: Eighty patients were enrolled either in AP or CP group. Clinical attachment level (CAL), bleeding on probing (BOP), periodontal probing depth (PPD) and plaque index (PI) were recorded for each patient. Total RNA was extracted from gingival crevicular fluid samples. Relative gene expression of investigated markers was determined by reverse transcriptase-real-time polymerase chain reaction. RESULTS: Significantly higher values of PPD were observed in AP compared to CP (P = .010). Negative correlations between OPG and CAL, and OPG and PI, were found in AP (P = .045, P = .006, respectively), while Hey 1 and PI had a positive correlation (P = .049). In multivariate linear regression analysis, OPG and Notch 2 were predictors of CAL in AP group. TNF-α and IL-17 were higher in RANKL predominant than in OPG predominant cases (P = .007, P = .001, respectively). In RANKL predominant lesions Notch 1 and Jagged 1 were down-regulated in AP compared to CP patients (P = .010, P = .025, respectively). CONCLUSION: The present study demonstrated that changes in Notch 2 expression affected CAL in AP cases hence this molecule could be considered as a contributor to alveolar bone loss. In RANKL-activated settings, the down-regulation of Notch 1 might participate in more severe bone resorption in AP.

Antimicrobial peptide gene expression in medication-related osteonecrosis of the jaw.

Bisphosphonates and denosumab are commonly used antiresorptive therapies in patients with bone metastasis and osteoporosis. Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of these drugs, and infection has been recognized as a contributing factor. Current therapeutic options for MRONJ show limited effectiveness, therefore necessitating novel treatment strategies. Bisphosphonates have recently been reported to induce the expression of antimicrobial peptides (AMPs), an inherent component of the immune system. Therefore, the aim of the present study was to investigate and compare the influence of the anti-RANKL antibody denosumab and bisphosphonates on the gene expression of selected AMPs: human α-defensin-1, human α-defensin-3, human ß-defensin-1, and human ß-defensin-3. Bone specimens were collected from patients with MRONJ who had been treated with bisphosphonates (n = 6) or denosumab (n = 6), and from healthy subjects (n = 6) with no history of treatment with bone metabolism-influencing drugs. Reverse transcription-quantitative polymerase chain reaction was used to quantify the expression levels of selected AMPs. Samples from patients treated with denosumab showed significantly higher mRNA expression of human α-defensin-3 and human ß-defensin-3 than those from healthy subjects. This finding is similar to previously described upregulated expression of human defensins in patients with MRONJ after bisphosphonates treatment. This suggests that the elevated expression of defensins may be at least a part of the mechanism underlying the pathogenesis of osteonecrosis induced by antiresorptive therapies, which can serve as a new target for potential treatment of MRONJ.

Collagen Extract Derived from Yeonsan Ogye Chicken Increases Bone Microarchitecture by Suppressing the RANKL/OPG Ratio via the JNK Signaling Pathway.

Yeonsan Ogye is a traditional Korean chicken breed (Gallus domesticus, GD), with a dominant gene for fibromelanosis, showing entirely black fluffy head feathers, ear lobes, and pupils. GD collagen extract (78.6 g per 100 g total protein) was derived from the flesh of Yeonsan Ogye. The effects of GD collagen on bone mass, microarchitecture, osteogenic, osteoclastogenic differentiations, and function factor expression were investigated in ovariectomized (OVX) rats. GD collagen stimulated osteogenesis in OVX rats and increased tibial bone strength and calcium content. Micro-computed tomography analysis of tibia cross-sections revealed that GD collagen attenuated the OVX-induced changes in trabecular thickness, spacing, and number. GD collagen stimulated alkaline phosphatase activity, bone-specific matrix proteins (alkaline phosphatase (ALP), osteocalcin, collagen type I (COL-I)) and mineralization by activating bone morphogenetic protein 2 (BMP-2)/mothers against decapentaplegic homolog 5 (SMAD5)/runt-related transcription factor 2 (Runx2). GD collagen inhibited osteoclast differentiation and function gene markers (TRAP, cathepsin K) by interfering with the Wnt signaling, increasing OPG production, and reducing the expression of RANKL, TRAP, and cathepsin K. GD collagen promoted osteogenesis by activating the p38 signal pathway and prevented osteoclastogenesis by lowering the RANKL/OPG ratio and blocking the JNK signaling pathway. Dietary supplementation with GD collagen might inhibit osteoclastogenesis, stimulate osteoblastogenesis, and regulate bone metabolism.

Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats.

BACKGROUND: Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. OBJECTIVE: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano-growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. METHODOLOGY: Thirty Sprague Dawley rats were randomly divided into three groups: sham-operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2-month experiment. Enzyme-linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real-Time PCR. RESULTS: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. CONCLUSION: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.

Effects of orthodontic forces on bone turnover biomarkers in peri-miniscrew crevicular fluid: A systematic review.

OBJECTIVE: Peri-miniscrew crevicular fluid (PMCF) analysis of biomarkers representing bone formation or resorption could provide a non-invasive way to monitor bone turnover around miniscrews and the response to force loading. Our objective was to systematically investigate the relevant available evidence. MATERIALS AND METHODS: Search without restrictions in eight databases and hand searching until March 2020 took place. We searched for prospective human studies measuring the levels of markers of bone formation and resorption in PMCF under the effect of orthodontic forces. Following study retrieval and selection, relevant data was extracted and the risk of bias was assessed following the Cochrane Collaboration guidelines. RESULTS: Four studies, two randomized and two non-randomized, were finally identified, following miniscrews for a period up to 90 days. Loading of miniscrews led to a transient increase in C-telopeptide of type I collagen amounts. Temporary increases were also observed for the enzymes: alkaline phosphatase and aspartate aminotransferase. Under the effect of orthodontic loading the total amount of Receptor Activator of Nuclear Factor-κB Ligand (RANKL) in the PMCF consistently increased compared to the unloaded group, at all sampling points. These changes led to a stable decrease in the osteoprotegerin (OPG)/RANKL ratio under force application throughout the study period, as OPG in this group, together with OPG and RANKL in the unloaded group, remained mostly unchanged. No differences were detected for the total OPG quantity between the two loading groups. The levels of bone specific alkaline phosphatase and chondroitin sulfate did not change significantly during observation. All studies presented some issues of concern regarding the risk of bias. CONCLUSION: Biomarkers of bone turnover in PMCF showed variable responses following orthodontic loading. Overall, the findings were suggestive of adaptive bone alterations to physiologic force stimuli.

Impact of history of periodontitis on gene expression of bone-related factors in young patients.

Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-ß and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

The increase in bone resorption in early-stage type I diabetic mice is induced by RANKL secreted by increased bone marrow adipocytes.

Bone marrow adipose tissue (BMAT) has recently been found to induce osteoclastogenesis by secreting RANKL. Although Type 1 diabetes mellitus (T1DM) has been reported to be associated with increased BMAT and bone loss, little is known about the relationship between BMAT and osteoclasts in T1DM. We studied the role of BMAT in the alterations of osteoclast activities in early-stage T1DM, by using a streptozotocin-induced T1DM mouse model. Our results showed that osteoclast activity was enhanced in the long bones of T1DM mice, accompanied by increased protein expression of RANKL. However, RANKL mRNA levels in bone tissues of T1DM mice remained unchanged. Meanwhile, we found that BMAT was significantly increased in the long bones of T1DM mice, and both mRNA and protein levels of RANKL were elevated in the diabetic BMAT. More importantly, RANKL protein was mainly expressed on the cell membranes of the increased adipocytes, most of which were located next to the metaphyseal region. These results suggest that the enhanced bone resorption in early-stage diabetic mice is induced by RANKL derived from BMAT rather than the bone tissue itself.

Evaluation of the effect of topical and systemic ozone application in periodontitis: an experimental study in rats.

OBJECTIVE: The goal of the present study was to determine the effect of systemic and topical ozone application on alveolar bone loss (ABL) by evaluating the effect of Hypoxia-inducible factor -1 alpha (HIF-1-α) and receptor activator of NF-kB ligand (RANKL)-positive cells on histopathological and immunohistochemical changes in a rat periodontitis model. METHODOLOGY: Thirty male Wistar rats were divided into three groups: 1) Group C (control group); 2) Group SO (systemic ozone group) and 3) Group TO (topical ozone group). Experimental periodontitis was induced with a 3/0 silk suture placed at the mandibular left first molars of rats, and the suture was removed 14 days later. Ozone gas was injected intraperitoneally (0.7 mg/kg) in SO group. Topical ozone application protocol was performed using an ozone generator at 80% concentration (4th grade) 90- degree probe for the duration of 30 s. Both ozone applications were carried out for two weeks at intervals of two days. Histomorphometric and immunohistochemical analysis were performed. RESULTS: ABL was significantly lower in Group SO compared to Group C (p: 0.0052). HIF-1α- positive cells were significantly lower in Group TO than in Group C (p: 0.0043). RANKL-positive cells were significantly lower in Group SO and in Group TO compared to the control group (p: 0.0033, p: 0.0075, respectively). CONCLUSION: Both ozone applications decreased RANKL-positive cell counts, TO application decreased HIF-1-α positive cells counts, and SO application was found to be more effective in reducing ABL compared to control group.

Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats

Abstract Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. Objective: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano-growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. Methodology: Thirty Sprague Dawley rats were randomly divided into three groups: sham-operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2-month experiment. Enzyme-linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real-Time PCR. Results: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. Conclusion: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.

Impact of history of periodontitis on gene expression of bone-related factors in young patients

Abstract Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-β and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis.

OBJECTIVE: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis. METHODOLOGY: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed. RESULTS: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups. CONCLUSION: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

Tenofovir Has Minimal Effect on Biomarkers of Bone Health in Youth with HIV Receiving Initial Antiretroviral Therapy.

Both HIV infection and tenofovir disoproxil fumarate (TDF) treatment adversely impact bone metabolism and may lead to osteopenia, which has critical implications for youth with HIV (YWH). This study evaluates changes in the biomarkers of bone metabolism and inflammation among YWH receiving initial treatment with TDF- and non-TDF-containing antiretroviral therapies (ARTs). YWH [n = 23, median age 21 years (range 18-24), 87% male, 61% African American] were assessed for inflammatory and bone metabolism biomarkers at enrollment, after 48 weeks of TDF-containing ART, and 96 weeks of ART without TDF with continued viral suppression. Spearman's rank correlation evaluated biomarker associations. Bone alkaline phosphatase, parathyroid hormone, and osteopontin increased after TDF treatment. All fell after TDF was discontinued. Levels of RANKL and osteoprotegerin did not change throughout the study. There was little correlation between biomarkers of bone metabolism and either macrophage or lymphocyte activation at any time point. Our results establish baseline associations between bone metabolism and immune biomarkers for this population, and find that before CD4 T cell decline chronic inflammation does not perturb biomarkers of bone metabolism among YWH. The adverse effects of TDF on bone health may be marginal for YWH at the early stages of disease.

RANK-RANKL Signaling in Cancer of the Uterine Cervix: A Review.

RANK ligand (RANKL) is a member of the tumor necrosis factor alpha superfamily of cytokines. It is the only known ligand binding to a membrane receptor named receptor activator of nuclear factor-kappa B (RANK), thereby triggering recruitment of tumor necrosis factor (TNF) receptor associated factor (TRAF) adaptor proteins and activation of downstream pathways. RANK/RANKL signaling is controlled by a decoy receptor called osteoprotegerin (OPG), but also has additional more complex levels of regulation. The existing literature on RANK/RANKL signaling in cervical cancer was reviewed, particularly focusing on the effects on the microenvironment. RANKL and RANK are frequently co-expressed in cervical cancer cells lines and in carcinoma of the uterine cervix. RANKL and OPG expression strongly increases during cervical cancer progression. RANKL is directly secreted by cervical cancer cells, which may be a mechanism they use to create an immune suppressive environment. RANKL induces expression of multiple activating cytokines by dendritic cells. High RANK mRNA levels and high immunohistochemical OPG expression are significantly correlated with high clinical stage, tumor grade, presence of lymph node metastases, and poor overall survival. Inhibition of RANKL signaling has a direct effect on tumor cell proliferation and behavior, but also alters the microenvironment. Abundant circumstantial evidence suggests that RANKL inhibition may (partially) reverse an immunosuppressive status. The use of denosumab, a monoclonal antibody directed to RANKL, as an immunomodulatory strategy is an attractive concept which should be further explored in combination with immune therapy in patients with cervical cancer.

Beneficial effects of (R)-ketamine, but not its metabolite (2R,6R)-hydroxynorketamine, in the depression-like phenotype, inflammatory bone markers, and bone mineral density in a chronic social defeat stress model.

Inflammatory bone markers may play a role in the antidepressant actions of (R)-ketamine in susceptible mice after chronic social defeat stress (CSDS). In this study, we compared the effects of (R)-ketamine and its final metabolite (2R,6R)-hydroxynorketamine (HNK) in depression-like phenotypes, inflammatory bone markers and bone mineral density (BMD) in CSDS susceptible mice. We measured plasma levels of inflammatory bone markers, which included osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL), and osteopontin after behavioral tests. (R)-ketamine, but not (2R,6R)-HNK, elicited rapid and sustained antidepressant effects in CSDS susceptible mice. Furthermore, (R)-ketamine, but not (2R,6R)-HNK, significantly improved the increased plasma levels of RANKL and decreased OPG/RANKL ratio in CSDS susceptible mice. Moreover, (R)-ketamine, but not (2R,6R)-HNK, significantly attenuated the decreased BMD in CSDS susceptible mice. These findings demonstrate that (R)-ketamine may have beneficial effects in depression-like phenotype and abnormalities in bone functions of CSDS susceptible mice. It is, therefore, likely that (R)-ketamine would be a potential therapeutic drug for abnormalities in bone metabolism in depressed patients.

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model.

OBJECTIVE: The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. MATERIAL AND METHODS: Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. RESULTS: Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1ß, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. CONCLUSIONS: This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.