OBJECTIVETo investigate the effect of pulsed electromagnetic fields (PEMs) on inducing osteoclastic like cell (OLC) formation changes and apoptosis in ovariectomized (OVX) rats bone marrow culture in vitro.

METHODSThirty healthy three-month-old female Wistar rats were either sham-operated (Sham) or ovariectomized (OVX) and randomly divided into three groups: group A (OVX + PEMs, 18 rats), group B (OVX, 6 rats) and group C (Sham, 6 rats); group A was again randomly divided into three groups: A1, A2, A3. The frequencies adopted were 1.5, 2, 75 Hz and 30 minutes for once a day. All rats were fed with normal diet for 3 months, then the bone marrow of all rats were cultured, 2 days later, group A cells (including group A1, A2, A3) were collected and exposed to different frequencies PEMs for 2 weeks (30 min/day). In order to observe the changes of osteoclasts and count their numbers, cells were taken for Wright Giemsa staining, tartrate-resistance acid phosphatase (TRAP) staining and Hoechst 33258 staining.

RESULTSTRAP staining results indicated the number of OLC in group C was the least, then was group A2, A3, A1, B. The number of OLC in group B was remarkably increased (P < 0.01; vs group C, A2). The number of OLC in group B was significantly increased (P < 0.05; vs group A1, A3). Hoechst 33258 staining results indicated the number of apoptosis of OLC in group C was more than other groups, which of group C, A2 was significantly increased (P < 0.05; vs group B).

CONCLUSIONPEMs had decreased the formation of OLC and increased the number of apoptosis of OLC in ovariectomized (OVX) rats bone marrow culture in vitro, the effects of 2 Hz was the best. PEMs would be a new way of osteoporosis therapy.

Acid Phosphatase ; metabolism ; Animals ; Apoptosis ; radiation effects ; Bone Marrow Cells ; cytology ; enzymology ; radiation effects ; Cells, Cultured ; Electromagnetic Fields ; Female ; Isoenzymes ; metabolism ; Osteoclasts ; cytology ; enzymology ; radiation effects ; Ovariectomy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Tartrate-Resistant Acid Phosphatase

This study was aimed to assess the effects of fluid shear stress on the bone resorption in rat osteoclasts. The osteoclasts were derived from the lumbar vertebrae marrow cells which were isolated from the 6-month-old female Sprague Dawley rats, cultured on the slide at 1 x 10(6) cell/ml, and induced with 1, 25 (OH)2 Dihydroxyvitamin D3. The slide containing osteoclasts was taken out on day 7 after culture and then put into the flow chamber. The loads of fluid shear stress applied to the osteoclasts were 5.97, 11.36, 16.08 and 20.54 dyne/cm2, respectively, for 30 minutes. The osteoclasts unloading fluid shear stress were used as control. The bone resorptive pits were studied by light microscopy and scanning electron microscopy. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was detected with ultraviolet spectrophotometry. The results showed that fluid shear stress can increase the activity of TRAP and significantly increase the number and area of bone resorptive pits made by osteoclasts,and the effect of fluid shear stress on the bone resorption of osteoclasts is the same as that on the activity of TRAP. The reaction of the osteoclasts to the fluid shear stress in this study also suggested that the bone resorption of osteoclasts be increased in a magnitude of fluid shear stress-dependent manner, and that the changes of TRAP activity be closely related to the changes of the number and area of resorptive pits of the osteoclasts.
Acid Phosphatase ; metabolism ; Animals ; Bone Resorption ; physiopathology ; Calcitriol ; pharmacology ; Cells, Cultured ; Female ; Isoenzymes ; metabolism ; Lumbar Vertebrae ; cytology ; Osteoclasts ; cytology ; enzymology ; physiology ; Rats ; Rats, Sprague-Dawley ; Shear Strength ; Tartrate-Resistant Acid Phosphatase

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Acid Phosphatase/metabolism ; Animals ; Blotting, Western ; Calcitriol/*pharmacology ; Calcium Channel Agonists/pharmacology ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Enzymologic/*drug effects ; Isoenzymes/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Mice ; Osteoclasts/*cytology/*enzymology ; Tetrazolium Salts ; Thiazoles

OBJECTIVETo investigate the effects of fluoride on activities of tartrate-resistant acid phosphate (TRAP) and matrix metalloproteinase-9 (MMP-9) in rat osteoclasts cultured in vitro.

METHODSOsteoclast was isolated mechanically from long bones of neonatal rats and cultured in vitro. Histochemical stain was applied to detect the effects of fluoride on activities of TRAP and in-situ hybridization was used to study the expression of MMP-9 mRNA in rat osteoclasts in vitro.

RESULTSNumber of TRAP positive cells was 154.2, 160.0, 170.6, 179.0 and 180.0 per cm(2), respectively for the rats with varied doses of fluoride, in a dose-response pattern but without statistical significance. The expression of MMP-9 mRNA increased with elevating dose of fluoride, especially in the rats with 1.00, 2.00 and 4.00 mg/L of fluoride, to 94.50, 94.64 and 104.97, respectively, significantly different from those in control group.

CONCLUSIONSFluoride can enhance the MMP-9 mRNA expression in cultured osteoclasts of rats.

Acid Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Biomarkers ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fluorides ; pharmacology ; Isoenzymes ; metabolism ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Osteoclasts ; cytology ; drug effects ; enzymology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Tartrate-Resistant Acid Phosphatase

OBJECTIVETo investigate the effect of osteoclast bone resorption supernatants on the osteogenic activity of mouse MC3T3-E1 cell line.

METHODSMouse RAW264.7 cell line was induced to osteoclast which was identified with tartrate resistant acid phosphatase (TRAP) staining and osteoclast specific gene detection. The differentiated RAW264.7 osteoclast was co-cultured with bovine milling bone specimen followed by toluidine blue staining. Then mouse MC3T3-E1 cell was cultured with supernatant from the osteoclast bone absorbent model. Methyl thiazolyl tetrazolium (MTT) method, alizarin red S staining, enzyme-linked immunosorbent assay detection of osteocalcin, and reverse transcriptase polymerase chain reaction detection were adopted to investigate the proliferation, calcification and osteogenic activity of MC3T3-E1 cells.

RESULTSTRAP staining, osteoclast specific gene detection and toluidine blue staining all indicated that RAW264.7 cell could be differentiated into functioning osteoclast. The supernatant from the osteoclast bone absorbent model could inhibit the proliferation of MC3T3-E1 cells, with the A value between 0.062 ± 0.004 and 0.405 ± 0.033 (P < 0.05). It could also increase the formation of calcification nods, promote the osteocalcin level which peaked with the tenth day's supernatant at a level of (2.965 ± 0.047) µg/L, as well as enhance the transcription of the alkaline phosphatase and Runt related transcription factor 2 gene.

CONCLUSIONSRAW264.7 osteoclast bone absorbent supernatant might influence the osteogenic activity of osteoblast-like cell by inhibiting proliferation, promoting differentiation and calcification.

Acid Phosphatase ; metabolism ; Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Bone Resorption ; Calcification, Physiologic ; Cathepsin K ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Culture Media, Conditioned ; pharmacology ; Gene Expression ; Isoenzymes ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; metabolism ; Osteoclasts ; cytology ; enzymology ; Tartrate-Resistant Acid Phosphatase ; Transcription, Genetic

The cajanine (longistylin A-2-carboxylic acid) is isolated and identified from extracts of Cajanus cajan L. (ECC) , which structure is similar to diethylstilbestrol. The regulation properties of the cajanine and other four extracts of Cajanus cajan L. (32-1, 35-1, 35-2, and 35-3) were tested in human osteoblast-like (HOS) TE85 cells and marrow-derived osteoclast-like cells. By using MTT assay to test the change of cell proliferation, 3H-proline incorporation to investigate the formation of collagen, and by measuring alkaline phosphatase (ALP) activity, bone formation in HOS TE85 cell was evaluated after pretreated for 48 hours. Bone marrow cells were cultured to examine the derivation of osteoclast cells (OLCs), which were stained with tartrate-resistant acid phosphatase (TRAP). The long term effect (pretreated for 18 days) on promoting mineralized bone-like tissue formation was tested by Alizarin red S staining in HOS TE85 cells. After the treatment with cajanine (1 x 10(-8) g x mL(-1)) for 48 hours, cell number increased significantly (57.7%). 3H-Proline incorporation also statistically increased (98.5%) in those cells. Significant change of ALP activity was also found (P < 0.01) in 35-1 and 35-3 treated cells (they were 66.2% and 82.4% in the concentration of 1 x 10(-8) g x mL(-1), respectively). The long term (18 days) effects of 32-1 and 35-3 on promoting mineralized bone-like tissue formation in HOS TE85 cell were obvious. There were much more red blots over the field of vision compared with that of control group. After the treatment of cajanine, derived-osteoclast cells appeared later and much less compared with control. The inhibition of cajanine was 22.8% while it was 37.9% in 32-1 treated cells in the dose of 1 x 10(-7) g x mL(-1). It is obvious that cajanine and ECCs promoted the osteoblast cells proliferation and mineralized bone-like tissue formation in HOS TE85 cells, while inhibited derivation of osteoclast cells. All of these suggested that cajanine has the estrogen-like action on osteoblast and osteoclast, which could be developed as anti-osteoporosis drugs.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Bone Neoplasms ; metabolism ; pathology ; Cajanus ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Diethylstilbestrol ; analogs & derivatives ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Osteoblasts ; drug effects ; Osteoclasts ; cytology ; metabolism ; Osteogenesis ; drug effects ; Osteosarcoma ; enzymology ; pathology ; Phytoestrogens ; isolation & purification ; pharmacology ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar

OBJECTIVETo develop zoledronic acid (ZA)-loaded collagen membranes, and to study its effect on osteoclast and osteoblast so as to investigate whether ZA-loaded membranes can inhibit local bone resorption and promote bone formation.

METHODSZA-loaded double-layer (Bio-Gide(®)) and single-layer (BME-10X(®)) collagen membranes were prepared and divided into eight groups according to the concentrations of ZA in the membrane, namely Group BG0, BG1, BG2, BG3 and BM0, BM1, BM2, BM3 (BG refers to Bio-Gide(®), BM refers to BME-10X(®), 0, 1, 2, 3 refer to the concentrations of ZA, 0, 1 × 10(-4), 1 × 10(-3), 1 × 10(-2) mol/L respectively). Blank control group was set without using collagen membrane. The effects of ZA-loaded membranes on osteoclast and osteoblast were assessed using in vitro cell culture models.

RESULTSIn vitro coculture of ZA-loaded membrane with osteoclast for seven days showed that the percentage of bone resorption area in BG1, BG2, BG3, BM1, BM2, BM3 were 18.80%, 14.75%, 14.28%, 20.51%, 15.77%, 15.12% respectively, which were lower than that in BG0 (31.53%) and BM0 (32.22%, P < 0.05), and the higher ZA loading was, the stronger its inhibition to osteoclast was. In vitro coculture of ZA-loaded membrane with osteoblast for four days indicated that alkaline phosphatase (ALP) activities in BG2 (154.67 U/g), BM2 (154.33 U/g), BG3 (155.33 U/g), BM3 (152.00 U/g) were higher than that in BG0 (129.33 U/g) and BM0 (127.67 U/g, P < 0.05). What's more, results from seven-day coculture showed that proliferation index in BG2 (7.00) was higher than that in BG0 (6.90).

CONCLUSIONSZA-loaded collagen membrane can not only inhibit osteoclastic bone resorption but also improve proliferation of osteoblast.

Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; pharmacology ; Bone Density Conservation Agents ; administration & dosage ; pharmacology ; Bone Resorption ; pathology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; pharmacology ; Diphosphonates ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Imidazoles ; administration & dosage ; pharmacology ; Membranes, Artificial ; Osteoblasts ; cytology ; drug effects ; enzymology ; Osteoclasts ; cytology ; drug effects ; Osteogenesis ; Rabbits

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors