The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1alpha,25(OH)2D3-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1alpha,25(OH)2D3-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1alpha,25(OH)2D3-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor kappaB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1alpha,25(OH)2D3. Knockdown of Runx2 inhibited 1alpha,25(OH)2D3-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1alpha,25(OH)2D3-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.
Bone Remodeling ; Coculture Techniques ; Down-Regulation ; Osteoblasts ; RANK Ligand ; Sucrose

OBJECTIVETo explore certain principle of how osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) take part in the periodontal tissues remodeling under the combined influence of inflammation and orthodontic force.

METHODSThe positive signals of OPG and OPGL mRNA were measured with in situ hybridzation after orthodontic tooth movement in the experimental periodontitis groups and control ones.

RESULTSThe OPG and OPGL mRNA expression intensity in the experimental group showed difference from control. All their optical density index reached a peak in day 2, respectively.

CONCLUSIONOPG and OPGL play important roles in the periodontal reconstruction induced by inflammation irritation and orthodontic force, and complex interaction could exist between the two factors.

Humans ; Osteoprotegerin ; Periodontitis ; RANK Ligand ; RNA, Messenger ; Tooth Movement Techniques

Osteocytes may function as mechanotransducers by regulating local osteoclastogenesis. Reduced availability of oxygen, i.e. hypoxia, could occur during disuse, bone development, and fracture. Receptor activator of nuclear factor-kappaB ligand (RANKL) is an osteoblast/stromal cell derived essential factor for osteoclastogenesis. The hypoxia induced osteoclastogenesis via increased RANKL expression in osteoblasts was demonstrated. Hypoxic regulation of gene expression generally involves activation of the hypoxia-inducible factor (HIF) transcription pathway. In the present study, we investigated whether hypoxia regulates RANKL expression in murine osteocytes and HIF-1alpha mediates hypoxia-induced RANKL expression by transactivating RANKL promoter, to elucidate the role of osteocyte in osteoclastogenesis in the context of hypoxic condition. The expression levels of RANKL mRNA and protein, as well as hypoxia inducible factor-1alpha (HIF-1alpha) protein, were significantly increased in hypoxic condition in MLO-Y4s. Constitutively active HIF-1alpha alone significantly increased the levels of RANKL expression in MLO-Y4s under normoxic conditions, whereas dominant negative HIF-1alpha blocked hypoxia-induced RANKL expression. To further explore to find if HIF-1alpha directly regulates RANKL transcription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity, whereas mutations of putative HIF-1alpha binding elements in RANKL promoter prevented this hypoxia-induced RANKL promoter activity in MLO-Y4s. These results suggest that HIF-1alpha mediates hypoxia-induced up-regulation of RANKL expression, and that in osteocytes of mechanically unloaded bone, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in osteocytes.
Anoxia* ; Bone Development ; Gene Expression Regulation ; Luciferases ; Osteoblasts ; Osteocytes* ; Oxygen ; RANK Ligand* ; RNA, Messenger ; Up-Regulation

Receptor activator of nuclear factor-κB ligand (RANKL) is an osteoblast/stromal cell-derived essential factor for osteoclastogenesis. During endochondral bone formation, hypertrophic chondrocytes calcify cartilage matrix that is subsequently resorbed by osteoclasts in order to be replaced by new bone. Hypoxia-induced upregulation of RANKL expression has been previously demonstrated in an in vitro system using osteoblasts; however, the involved mechanism remains unclear in chondrocytes. In the present study, we investigated whether hypoxia regulates RANKL expression in ATDC5 cells, a murine chondrogenic cell line, and hypoxia-inducible factor-1α (HIF-1α) mediates hypoxia-induced RANKL expression by transactivating the RANKL promoter. The expression levels of RANKL mRNA and protein, as well as HIF-1α protein, were significantly increased in ATDC5 cells under hypoxic condition. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression under normoxic conditions, whereas dominant negative HIF-1α reduced hypoxia-induced RANKL expression. HIF-1α increased RANKL promoter reporter activity in a HIF-1α binding element-dependent manner in ATDC5 cells. Hypoxia-induced RANKL levels were much higher in differentiated ATDC5 cells, as compared to proliferating ATDC5 cells. These results suggested that under hypoxic conditions, HIF-1α mediates induction of RANKL expression in chondrocytes; in addition, hypoxia plays a role in osteoclastogenesis during endochondral bone formation, at least in part, through the induction of RANKL expression in hypertrophic chondrocytes.
Anoxia* ; Cartilage ; Cell Line ; Chondrocytes* ; Osteoblasts ; Osteoclasts ; Osteogenesis ; RANK Ligand ; RNA, Messenger ; Up-Regulation

Denosumab, a specific inhibitor of RANK ligand, is a novel therapy for postmenopausal osteoporosis and related disorders. An extensive clinical development program has evaluated the clinical efficacy and safety of denosumab with several thousand patients being followed for up to 10 years. Combined with more than six years of postmarketing experience, these studies provide substantial confidence that denosumab is a convenient and appropriate treatment for patients, including Asians, at high risk for fracture. This review will summarize the clinical development of denosumab and lessons learned since its approval for clinical use in 2010.
Asian Continental Ancestry Group ; Denosumab* ; Female ; Humans ; Osteoporosis* ; Osteoporosis, Postmenopausal ; RANK Ligand ; Treatment Outcome

OBJECTIVETo investigate the effect of gradually induced disordered occlusion (GIDO) on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in mandibular condylar cartilage of rats.

METHODSTotally 48 rats, aged 8 weeks were included, and were divided into experimental and control groups randomly at 4 time points, with same gender distribution (n=3). By inserting elastic rubber band the right side mandibular first molar and the left side maxillary first molar were moved mesially. Four weeks later, the right side mandibular third molar and the left side maxillary third molar were moved distally with same method. In this way, the GIDO was established in rats. The rats were sacrificed at the end of 2th, 4th, 6th and 8th week respectively after the application of the GIDO. The expression of OPG and RANKL in condylar cartilage was examined with immunohistochemical method and calculated by the area of positive cell percentage.

RESULTSOPG and RANKL expressed predominantly in condylar cartilage hypertrophic layer. The rats in experimental group expressed a higher OPG level in all of the 4 time points than their age-matched controls (P<0.05), while RANKL were higher in 2, 6, 8 weeks subgroups (P<0.05), but not in 4 weeks subgroup. No differences were found between male and female subgroups.

CONCLUSIONThe present results suggest that both OPG and RANKL take part in the condylar cartilage remodeling procedure in the present rat model.

Animals ; Cartilage ; Female ; Humans ; Male ; Mandibular Condyle ; Molar ; Osteoprotegerin ; RANK Ligand ; Rats

OBJECTIVETo investigate the exerted force in different phases of the female menstrual cycle, as well as the changes in estrogen (E2), osteocalcin (OCN), receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) levels in the gingival crevicular fluid (GCF) during orthodontic tooth movement, to provide a theoretical basis for the selection of the best opportunity for efficient tooth movement.

METHODSTwelve women (aged 18 years to 28 years) with extracted first premolars had been selected. Six women in the group were randomly selected as the menstrual period group, whereas the remaining six were assigned to the ovulation period group. Right canines were retracted with 1.5 N NiTi close coil spring. GCF samples were collected prior to the force exertion experiments at 0 (T0), 15 (T1), 30 (T2), and 45 d (T3). The levels of E2, OCN, OPG and RANKL in GCF were measured by chemiluminescence and enzymelinked immunosorbent assay.

RESULTSThe E2 and OCN levels were significantly higher in the ovulation period group than in the menstrual period group (P < 0.05). No significant differences were found in RANKL and OPG levels between the two groups (P > 0.05). Finally, no significant difference was found in RANKL/OPG ratio between the two groups (P > 0.05).

CONCLUSIONExerted force on teeth during the menstrual period may promote rapid tooth movement.

Female ; Gingival Crevicular Fluid ; Humans ; Menstrual Cycle ; Osteoprotegerin ; RANK Ligand ; Tooth Movement Techniques

OBJECTIVEThis study aims to explore the effect of Notch signaling depression on the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells.

METHODSMice RAW264.7 cells were cultured and differentiated into osteoclasts with the induction of RANKL. The expressions of Notch1, Notch2, Deltal, Jagged1, Hes1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K genes during osteoclastogenesis were analyzed using real-time polymerase chain reaction. Osteoclast formation was analyzed using TRAP assay with suppression of Notch receptors by a selective γ-secretase inhibitor (GSI).

RESULTSNotch1, Notch2, Delta1, Jagged1, and Hes1 expressions in RAW264.7 cells were upregulated following 50 ng · mL-RANKL stimulation for 3 d, concomitant with the expression of the osteoclast differentiation markers TRAP and Cathepsin K. Notch2 and Jagged1 had the most remarkable increase in the Notch family members. GSI inhibited RANKL-induced osteoclastogenesis of RAW264.7 cells and Hes1 expression dose-dependently.

CONCLUSIONNotch signaling activation may promote RANKL-induced osteoclastogenesis of RAW264.7 cells.

Animals ; Cathepsin K ; Cell Differentiation ; In Vitro Techniques ; Mice ; Osteoclasts ; Osteogenesis ; RANK Ligand

INTRODUCTION: Diabetes mellitus, as a major health problem for the elderly has been shown to alter the properties of the bone and impair bone healing around a titanium implant in both humans and animals. The aim of this study was to examine the effect of adipose-derived stem cells on the healing process around a titanium implant in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Thirteen rats were divided into two groups: adipose-derived stem cells injected group and a control group. A titanium screw implant (diameter: 2.0 mm, length: 3.5 mm) was placed into both tibia of 13 rats: 13 right tibia as the control group and 13 left tibia as the experimental group. The rats were sacrificed at different intervals (1, 2, and 4 weeks) after implantation for histopathology observations and immunohistochemistric analysis. RESULTS: The histopathological findings revealed earlier new formed bone in the experimental group than the control group. In particular, at 1 week after implantation, the experimental group showed more newly formed bone and collagen around the implant than the control group. In immunohistochemistric analysis, osteoprotegerin (OPG) expression in the experimental group increased early compared to that of the control group until 2 weeks after implantation. However, after 2 weeks, OPG expression in the experimental group was similar to OPG expression in the control group. The receptor activator of nuclear factor kappaB ligand (RANKL) expression in the experimental group increased early compared to that of the control group, and then decreased at 2 weeks. After 2 weeks, the level of RANKL expression was similar in both groups. CONCLUSION: These results suggest that adipose-derived stem cells in implantation can promote bone healing around titanium, particularly in diabetes mellitus induced animals.
Aged ; Animals ; Collagen ; Dental Implants ; Diabetes Mellitus ; Humans ; Osteoprotegerin ; RANK Ligand ; Rats ; Stem Cells ; Tibia ; Titanium

PURPOSE: To investigate the pathogenesis of articular prosthesis osteolysis, and to clarify the role of OPG, RANKL and RANK on osteolysis in aseptic loosening of hip prostheses. MATERIALS AND METHODS: We examined the mRNAs of OPG, RANKL and RANK from cultured peripheral mononuclear cells and the tissue surrounding failed hip prostheses by RT-PCR and gel electrophoresis and performed immunohistochemistry for RANKL and RANK in the periprosthetic tissue of revised hip replacement therapy. RESULTS: RANKL was detected in 32% and RANK was detected in 20% of the periprosthetic tissues of failed hip prostheses. Proliferative responses of cultured PBMCs occurred in cells with the titanium, cobalt and LPS, and highest response was observed in cells with cobalt particles. The mRNAs of RANKL and OPG were expressed in the periprosthetic tissues of loosened hip prostheses, but RANK mRNA was not detected. OPG mRNA was not detected in cultured PBMCs with any particles, RANKL mRNA was detected in cultured PBMCs with titanium and cobalt particles by RT-PCR, and RANK mRNA was detected in PBMCs with cobalt particles, but not with titanium. CONCLUSION: Osteolysis around the failed hip prosthesis may be related to activation of the OPG-ANKLRANK system. It is suggested that OPG, RANKL and RANK are major mediators of osteolysis in failed hip prosthesis.
Cobalt ; Electrophoresis ; Hip Prosthesis* ; Hip* ; Immunohistochemistry ; Osteolysis ; Osteoprotegerin* ; Prostheses and Implants ; RANK Ligand* ; RNA, Messenger ; Titanium