BACKGROUNDBisphosphonates (BPs) have been reported to reduce local recurrence in giant cell tumor (GCT) of bone because of their osteoclast-suppressing effect; however, the optimal mode of delivery and the dose and duration of treatment of BPs remain to be established. To address these issues, it is first necessary to clarify the manner of action of BPs on osteoclasts. We herein evaluated the osteoclast-suppressing effect of sodium ibandronate in vitro.

METHODSMouse osteoclasts (OCLs) were generated in vitro using mouse bone marrow mononuclear cells. First, various concentrations of sodium ibandronate and equal amounts of phosphate-buffered saline were added to cell culture media. The number of multinucleated cells (over three nuclei) was recorded in each group, OCL formation was compared, and the most effective concentration of sodium ibandronate was determined. Then, high concentrations of sodium ibandronate were added to the experimental cell culture media; no ibandronate was given in the control group. Comparisons were made between the two groups in terms of OCL adhesion, migration, and bone resorption.

RESULTSOCL formation was suppressed by sodium ibandronate in vitro; the most pronounced effect was observed at the concentration of 10(-5) mol/L. OCL migration and bone resorption were significantly suppressed at this concentration, though there was no effect on OCL adhesion.

CONCLUSIONSSodium ibandronate was effective in suppressing OCLs and decreasing resorption in GCT. The strong anti-OCL effectiveness at a high concentration in vitro indicates a topical mode of application.

Animals ; Bone Resorption ; Cell Movement ; drug effects ; Cells, Cultured ; Diphosphonates ; pharmacology ; Mice ; Osteoclasts ; cytology ; drug effects

Searched the articles between 2000 and 2010, found out and summarized the articles with the topic on the experiment and new techniques of traditional Chinese medicine treating to osteoporosis. The preventive and therapeutic effect to osteoporosis by traditional Chinese medicine had been developed in the past 10 years. The study on standardization of experimental drugs, and the mechanism study with modern cell culture techniques should be enhanced.
Animals ; Humans ; Medicine, Chinese Traditional ; Osteoblasts ; cytology ; drug effects ; Osteoclasts ; cytology ; drug effects ; Osteoporosis ; drug therapy ; Tissue Engineering

Through morphological observation, HE staining, TRAP staining and toluidine blue staining of bone resorption pits to identify osteoclasts which obtained by 1α, 25-(OH)2 VitD3 inducing rabbit bone marrow cells. Three indicators-TRAP staining, TRAP enzyme activity detecting and the number and area of bone resorption pits were adapted to detect the effect of Sargentodoxae caulis on the activity of osteoclasts. Culturing MC3T3-E1 Subclong 14 cells and detecting the effect of S. caulis on differentiation and proliferation of them by MTT and detecting the alkaline phosphatase in cells. The results show that all of the low, middle and high doses of water and alcohol extracts of S. caulis have significant inhibition on osteoclast differentiation and bone resorption ability in a dose-dependent manner. The low and middle doses of water and alcohol extracts of S. caulis can stimulate differentiation and proliferation of MC3T3-ElSubclone 14 cells, which indicates S. caulis can prevent osteoporosis and the function could be achieved by inhibiting osteoclast activity and promoting the proliferation and differentiation of osteoblasts.
Animals ; Bone Resorption ; drug therapy ; physiopathology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Mice ; Osteoclasts ; cytology ; drug effects ; Rabbits

This study was aimed to explore the influence of brucine on the early differentiation of osteoblasts and the metabolic pathway of osteoclast in multiple myeloma (MM) and to compare the effects of brucine and bortezomib on MM. The half inhibitory concentration (IC(50)) of brucine and bortezomib on MM cell line U266 was determined by MTT method; the mRNA levels of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and osteoprotegerin ligand (RANKL) were detected by RT-PCR after the supernatant of cultured U266 cells was added into the culture system for inducing the differentiation of osteoblast line MC3T3-E1 and culturing. The results showed that the IC(50)of bortezomib and brucine on U266 cells for 48 hours were 22.4 nmol/L and 0.16 mg/ml respectively. As compared with osteoblasts treated by supernatant of cultured MM cells alone, the mRNA levels of ALP, OC and OPG in osteoblasts treated by brucine combined with supernatant of cultured MM cells were enhanced (p < 0.05), while the RANKL mRNA level was lowered (p < 0.05), moreover the enhanced and lowered degree also was large (p < 0.05). It is concluded that the influence of brucine on metabolism of osteoblasts and osteoclasts in MM may be realized through the regulation of osteoclasts by osteoblasts. The therapeutic efficacy of brucine on MM is superior to bortezomib.
Animals ; Cell Line ; Cell Line, Tumor ; Humans ; Inhibitory Concentration 50 ; Mice ; Multiple Myeloma ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteoclasts ; cytology ; drug effects ; metabolism ; Strychnine ; analogs & derivatives ; pharmacology

OBJECTIVETo compare the effect of recombinant OPG-Fc and recombinant RANK protein on the differentiation of osteoclast precursors.

METHODSMouse osteoblasts cell lines were incubated with osteoclast precursors cell lines RAW 264.7 for 9 days with 10(-5) g/L rhRANK or rhOPG-Fc or PBS added to the coculture system. TRAP stain positive cells counting and cortical bone pit formation counting were performed in the 9th day.

RESULTSMultinuleated TRAP stain positive cells were observed in the cocluture systems after 6 days incubation,and plenty of mature osteoclasts could be observed in the 9th day. With the addition of 10(-5) g/L rhOPG-Fc or rhRANK, multinucleated giant cells and cortical bone pit formation couting decreased significantly compared with the control group, and the rhRANK group decreased more significantly than the rhOPG-Fc group.

CONCLUSIONSBoth rhOPG-Fc and rhRANK can inhabit the differentiation of osteoclast precursors and prevent them forming mature osteoclasts,moreover,the rhRANK shows the significant inhabition effect than the rhOPG-Fc.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Mice ; Osteoclasts ; cytology ; drug effects ; Osteoprotegerin ; pharmacology ; Receptor Activator of Nuclear Factor-kappa B ; pharmacology ; Recombinant Proteins ; pharmacology ; Stem Cells ; cytology ; drug effects

We hypothesized that the formation and differentialtion of osteoclasts are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow in the early stage of stroke. We randomly divided white female Sprague-Dawley (SD) rats (n = 30) into two groups, stroke (n = 15) and sham group (n = 15). On the 7th day after stroke, after cutting away the epiphyses of the femurs and tibias, diaphyseal channels were flushed using alpha-minimum essential medium (alpha-MEM) and bone marrow cells were collected. Bone marrow stem cells, which were extracted from the femur and tibia, were cultured on the 7th day after middle cerebral artery occlusion. We then estimated the ratio of non-adherent cells to total bone marrow cells that included osteoclast precursor cells. After culturing these cells separately, cells that tested positive on the tartrate resistant acid phosphatase (TRAP) were counted and bone resorption was evaluated by using the OAAS(TM) plate. In comparison to the control group, the stroke group showed a higher increase of non-adherent cells in the hemiplegic side bone marrow. In addition, after the primary culture, the stroke group showed an increased number of TRAP positive cells and a higher degree of bone resorption estimated by OAAS(TM) plate. As a result, osteoclastogenesis and osteoclast differentiation are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow and these changes are detected as early as within the first week after middle cerebral artery occlusion in SD rats.
Animals ; Bone Marrow Cells/cytology/drug effects ; Bone Resorption/*physiopathology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Female ; Femur/cytology ; Osteoclasts/cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells/cytology/metabolism ; Stroke/*metabolism/pathology ; Tartrates/pharmacology ; Tibia/cytology

This study was directed to the effects of macrophage-colony stimulating factors (M-CSF) concentration, recerptor activator of nuclear factor kappaB ligand (RANKL) concentration and M-CSF preinduction on osteoclastogenesis and the related resorption function. Bone marrow mononuclear cells were isolated and were divided into 4 groups. Group A underwent osteoclastogenic induction with the use of 30 ng/ml M-CSF and 50 ng/ml RANKL, while Group B received 50 ng/ml M-CSF and 100 ng/ml RANKL treatment. Both C and D Group underwent preinduction with the use of 30 ng/ml M-CSF for 3days, and then they were treated with 30 ng/ml M-CSF and 50 ng/ml RANKL, 50 ng/ml M-CSF and 100 ng/ml RANKL, respectively. Osteoclastogenesis was examined by TRAP staining 6 days after induction, and dentin resorption lacunae were detected by Scanning Electron Microscope 9 days after induction. TRAP positive multinuclear cells were observed in all groups of cells, and resorption lacunae were formed in all of them. However, more TRAP positive multinuclear cells were observed and more large resorption lacunae were detected in groups B and D than in groups A and C, respectively. The number of TRAP positive cells, number of resorption lacunae and lacuna areas in groups C and D were also greater than those in groups A and B, respectively. Higher concentration of M-CSF and RANKL and preinduction with M-CSF may benefit osteoclastogenesis and increase resorption function of osteoclast.
Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; pharmacology ; Leukocytes, Mononuclear ; cytology ; Macrophage Colony-Stimulating Factor ; pharmacology ; Mice ; Osteoclasts ; cytology ; RANK Ligand ; pharmacology

The purpose of this study was to determine whether the Ca2+ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappaB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca2+ concentration [Ca2+]i and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca2+]i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca2+ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca2+ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.
Animals ; Calcium/*metabolism ; *Calcium Signaling ; *Cell Differentiation/drug effects ; Cell Line ; Cell Survival/drug effects ; Gene Expression Regulation/drug effects ; Macrophage Colony-Stimulating Factor/metabolism ; Mice ; Osteoclasts/*cytology/*drug effects/*metabolism ; Osteoprotegerin/*pharmacology ; RANK Ligand/metabolism

BACKGROUNDThe cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.

METHODSRAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.

RESULTSAM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.

CONCLUSIONAM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Animals ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Indoles ; pharmacology ; Mice ; Osteoclasts ; cytology ; drug effects ; metabolism ; RANK Ligand ; pharmacology ; Receptor, Cannabinoid, CB2 ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

OBJECTIVETo study the effect of combination rhOPG-Fc and alendronate on mature osteoclasts.

METHODSRecombinant human osteoprotegerin secretory expression in P. pastoris was performed. Osteoblasts were got from new born mouse skeletal bone and proved by ALP staining and incubated together with osteoclasts precursor cell line Raw 264.7 in 96 well plate. After 9 d, 10 micromol/L ALN, 10(-5) g/L rhOPG-Fc, 10 micromol/L ALN + 10(-5) g/L rhOPG-Fc, 5 micromol/L ALN + 5 x 10(-6) g/L rhOPG-Fc were added to these coculture systems. Osteoblasts cultured without the drugs mentioned above served as controls. TRAP stain positive cells counting and cortical bone pit formation counting were preformed in the following the 3rd and 7th d.

RESULTSSDS-PAGE and Western blot showed that molecular weight of the expressed protein was about 55 KD, and it could reach specifically with anti-IgG antibody. Many multi-nuclear TRAP stain positive cells were found in the coculture control group after 9 d incubation, and proved to be mature osteoclasts by TRAP stain. In the 3rd and 7th d after the addition of rhOPG-Fc, ALN or both, TRAP stain positive cells counting and cortical bone pit formation counting decreased significantly in the rhOPG-Fc, ALN or both groups than in the control group, and the combine group (10(-5) g/L rhOPG-Fc + 10 micromol/L ALN) decreased most significantly when compared with rhopG-FC or ALN single.

CONCLUSIONSrhOPG-Fc can decrease the number of osteoclasts and inhibit their function. The combination of both rhOPG-Fc and ALN shows the significant inhibition effect on mature osteoclasts.

Alendronate ; pharmacology ; Animals ; Drug Synergism ; Humans ; In Vitro Techniques ; Mice ; Mice, Inbred Strains ; Osteoclasts ; cytology ; drug effects ; Osteoprotegerin ; biosynthesis ; pharmacology ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; pharmacology