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ABSTRACT The aim of this study was to determine the influence of osteoprotegerin (OPG) on the differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow cells were harvested from 23-d-old Gaoyou duck embryos and cultured in the presence of different concentrations of OPG (group A: no added factors, group B: 30 ng/mL of OPG, and group C: 100 ng/mL of OPG). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, and co-staining with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were all performed to determine the number of TRAP-positive cells, bone resorption activity, and the level of apoptosis, respectively. The number of TRAP-positive cells and the net expansion of pit formations area peaked on d 7 of culture in all 3 groups. The number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly lower compared with group A (P < 0.05). At each time point, the net expansion of pit formations area correlated with the number of TRAP-positive cells. The OPG inhibited the de novo formation of filamentous (F)-actin rings and promoted the disruption of existing F-actin rings in mature osteoclasts. In addition, OPG induced apoptosis in mature osteoclasts, as demonstrated by morphological changes in the nuclei. In osteoclast precursors, OPG inhibited differentiation and downregulated the formation of F-actin rings. In mature osteoclasts, OPG suppressed activation and enhanced the development of apoptosis, observed as a decrease in the number of TRAP-positive cells, the disruption of F-actin rings and morphological changes of the nuclei.

The aim of the present study was to determine whether osteoprotegerin (OPG) influences the bone resorption activity of osteoclasts. RAW264.7 cells were induced by macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) and 0, 10, 20, 50 and 100 ng/ml OPG were added into various groups in the presence of the two cytokines. The OPG treatment was continued for 24 h. Osteoclast differentiation and activation were estimated via TRAP staining assay, TRITC-conjugated phalloidin staining, resorption activity analysis. Furthermore, the expression levels of the osteoclastic bone resorption-related genes MMP-9, cathepsin K and carbonic anhydrase II (CA II) were examined using real-time polymerase chain reaction (PCR). The data demonstrated that high concentrations of OPG could inhibit the differentiation and activation of osteoclasts. Furthermore, real-time PCR analysis illustrated that OPG decreased the expression of MMP-9 and cathepsin K in different concentrations of OPG and it decreased the expression of CA II genes at 10 and 20 ng/ml concentrations of OPG. For the time gradient study, OPG decreased the expression of MMP-9 and CA II genes but not that of the cathepsin K gene. In summary, the resorption activity of osteoclasts was suppressed by high concentrations of OPG and, at the molecular level, OPG decreased the expression of osteoclastic bone resorption-related genes.

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor κB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.

The aim of this study was to determine whether receptor activator of nuclear factor NF-kappaB ligand (RANKL), osteoprotegerin (OPG) and a calcium:phosphorus (Ca:P) ratio of 2:1 could affect survival and activation of Muscovy duck osteoclasts (OCs). Bone marrow cells were obtained from 5-day-old Muscovy ducks and cultured with (Group A) No added factors, (B) 30ng/mL soluble RANKL (sRANKL), (C) 30ng/mL sRANKL and 10ng/mL OPG, (D) 10ng/mL OPG, (E) 50ng/mL OPG, (F) 100ng/mL OPG and (G) 30ng/mL sRANKL, 6mmol/L Ca and 3mmol/L P. sRANKL promoted the survival of OCs on day 2, whereas the number of OCs decreased with addition of OPG in a dose-dependent manner. OPG and Ca:P (2:1) both inhibited OC survival induced by RANKL. RANKL stimulated bone resorption by OCs, whereas OPG, but not Ca:P (2:1), inhibited the activity of OCs induced by RANKL. RANKL promotes the survival and activation of OCs from Muscovy ducks, whereas OPG and, to a lesser extent, Ca:P (2:1) reduce the life span and inhibited the activation of OCs induced by RANKL.

The article presents a study which determines the influence of osteoprotegerin (OPG) and receptor activator NF-kB ligand (RANKL) on duck's osteoclasts formation and activation. The result showed that the area of lacunar resorption decreased with concentration and increasing of OPG-RANKL may advance osteoclasts (OC) formation and activation. On the other hand, OPG may limit bone absorbing activity through inhibiting the formation and activation of OC.