AIMTo study the effects of fluoride (F-) on ionized calcium ([Ca2+]i) and calcium channel in osteoblast-like cell (OBLs).

METHODSInvestigating [Ca2+]i was by fluorospectrophotometry and recording the calcium currents of OBLs was using whole patch-clamp technique.

RESULTSThe addition of fluoride to the medium made a rapid and significant increase in free [Ca2+]i, especially in 100 ng F- /ml group. There were significances between control group and 50, 100 ng F- /ml groups. And 25 ng/ml F- could stimulate OBLs calcium channel open. Compared with control group, fluoride could significantly increase the amplitudes of calcium currents (P < 0.01), furthermore the activation was in dose-dependent manner.

CONCLUSIONFluoride can make calcium channel open and then increase the concentrations of [Ca2+]i.

Calcium ; physiology ; Calcium Channels ; physiology ; Cell Line ; Fluorides ; toxicity ; Humans ; Osteoblasts ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques

OBJECTIVETo study influence of serum containing Liuwei Dihuang decoction (see text) on proliferation and differentiation of osteoblast form neonatal SD rats cultured in vitro at different times and different stretch stress.

METHODSAfter osteoblast cultured for 24 hours in the serum containing Liuwei Dihuang decoction (see text) and serum in control group, the 0.5 Hz frequency, 6% and 12% stretch-stress were added. The MTT1 and the activity of ALP were measured at the 12th and 24th hours, and the data were analyzed.

RESULTS1. In the environment of stretch stress to the frequency of 0.5 Hz, and stretched for 24 hours, the osteoblast was stimulated under elongation rate of 6% and 12%; the proliferation and differentiation of osteoblast was more active under elongation rate of 12% than that of 6%. 2. There were no stimulating effects on osteoblast proliferation and differentiation of serum containing Liuwei Dihuiang decoction (see text) acted on osteoblast cells of SD rats cultured in vitro for a shot time.

CONCLUSIONStretch stress environment can enhance osteoblast proliferation and differentiation cultured in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Male ; Osteoblasts ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Serum ; Stress, Mechanical

OBJECTIVETo investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process.

METHODSMC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h).

RESULTSThe level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control.

CONCLUSIONSPe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Animals ; Lipopolysaccharides ; pharmacology ; Macrophage Colony-Stimulating Factor ; drug effects ; physiology ; Mice ; NF-kappa B ; metabolism ; Nitriles ; Osteoblasts ; drug effects ; metabolism ; Porphyromonas endodontalis ; RNA, Messenger ; Signal Transduction ; Sulfones

OBJECTIVETo study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro.

METHODSType I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28.

RESULTSThe signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the alkaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblasts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity.

CONCLUSIONNative bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Matrix ; metabolism ; Bone Morphogenetic Proteins ; pharmacology ; Cattle ; Cell Line ; DNA ; metabolism ; Gene Expression Regulation ; physiology ; Mice ; Osteoblasts ; drug effects ; metabolism

Na+ -Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+](ER)) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+](i)). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+](i). In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.
Alkaline Phosphatase/metabolism ; Animals ; Calcium/*metabolism ; Cell Membrane/metabolism ; Cell Survival ; Cells, Cultured ; Cytoplasm/metabolism ; Endoplasmic Reticulum/metabolism ; Intracellular Space/metabolism ; Oligodeoxyribonucleotides, Antisense/pharmacology ; Osteoblasts/drug effects/*physiology ; Rats ; Signal Transduction ; Sodium/physiology ; Sodium-Calcium Exchanger/*physiology

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein (ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1 (DDK-1, 100 μg/L) was added in ox-LDL 72-h group. The expression of a-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of a-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups (P<0.05 for all); β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group (P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion (P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group (P<0.05). The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.
Actins ; metabolism ; Animals ; Aortic Valve ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Myofibroblasts ; drug effects ; Osteoblasts ; physiology ; Phenotype ; Swine ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo investigate the molecular mechanism of TFE (total flavone of epimedium) in the treatment of osteoporosis, and then provide experimental evidence for modernization and further development of TFE as an traditional Chinese medicine.

METHODSSixty healthy female SD rats with aged 4 months were randomly divided into three groups (including control group in which rats received sham surgery, OVX group in which ovariectomized rats didn't give any medicine after the removal of ovaries and TFE group in which ovariectomized rats administrated TFE), 20 rats in each group. Compared bone mineral density (BMD) between before operation and at 4th week after operation in order to verify the establishment of osteoporotic model (criteria: BMD decreased more than 20% at 4th week after operation). The rats in TEF group were administrated total flavone of epimedium(concentration 30 mg/ml, 10 ml/kg, qd) orally for 4 weeks. After this, killed rats to harvest the lower part of the femur and detected BMD again. Applying the reverse transcriptase-polymerase chain reaction technique (RT-PCR) to detect expression of OPG, OPGL mRNA in bone tissue.

RESULTS(1) At 4th week after ovariectomy, the mean BMD of lumbar vertebra in TFE group fell to (0.084 +/- 0.020) g/cm2. Administrated with TFE for 4 weeks,the BMD increased to (0.112 +/- 0.009) g/cm2. There was significant improvement compare with the OVX group (P < 0.05). (2) Compared between OVX group and TFE group, The OPG mRNA expression of TFE group obviously enhanced. There was significant difference in statistics (P < 0.05). However,the promotion for OPGL mRNA expression were detected between OVX group and TFE group,there was no significant difference in statistics (P > 0.05).

CONCLUSIONThis study showed that TFE could inhibit differentiation and maturation of osteoclast through enhancing OPG mRNA expression, accordingly,to treat osteoporosis.

Animals ; Bone Density ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Female ; Flavones ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; Osteoblasts ; drug effects ; metabolism ; physiology ; Osteoprotegerin ; genetics ; Ovariectomy ; RANK Ligand ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats

Extracellular matrix (ECM) keeps cell's shape, protects and nourishes cells; it plays a great role in cell proliferation and differentiation. Therefore, ECM is very important in cell and tissue engineering. In this study, after primary mouse osteoblasts and fibroblasts maintained at confluence in vitro were removed, their ECM coated on cell culture plate was prepared, and bone morphogenetic proteins 2 (BMP-2) was detected in the osteoblasts ECM. MC3T3-E1 preosteoblasts cells were seeded on cell culture plates covered with fibroblasts ECM and osteoblasts ECM respectively. The proliferative activity of the cells cultured on fibroblasts ECM was higher than that on osteoblasts ECM and the control group. The alkaline phosphatase activity, relative protein levels of BMP-2 and osteopontin, secreted calcium of the cells cultured on osteoblasts ECM were all the highest. The results indicate that the two different ECMs produced in vitro had different bioactivities, the fibroblasts ECM coated on cell culture plates could accelerate MC3T3-E1 cells proliferation, and the osteoblasts ECM could promote cells osteogenic differentiation.
3T3 Cells ; Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Bone Morphogenetic Protein 2 ; metabolism ; Calcium ; metabolism ; Cell Differentiation ; physiology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Matrix ; metabolism ; physiology ; Mice ; Osteoblasts ; cytology ; Osteopontin ; metabolism

Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
Animals ; Cell Differentiation ; physiology ; Core Binding Factor Alpha 1 Subunit ; Homeodomain Proteins ; metabolism ; Mesenchymal Stem Cells ; metabolism ; Mice ; Mice, Nude ; Osteoblasts ; metabolism ; Osteocalcin ; drug effects ; Osteogenesis ; physiology ; Transcription Factors ; metabolism ; Up-Regulation

OBJECTIVETo establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field.

METHODSThree 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining.

RESULTSCompared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01).

CONCLUSION4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Chloral Hydrate ; pharmacology ; Cilia ; drug effects ; enzymology ; physiology ; Male ; Osteoblasts ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley