This study examined the osteogenic effect of electromagnetic fields (EMF) under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells (BMSCs) and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit (CCK-8). For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase (ALP) staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure (2 h/day) could obviously promote proliferation of BMSCs and osteoblasts, while long-term EMF (8 h/day) could promote osteogenic differentiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when cells were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differentiation of BMSCs.
Alkaline Phosphatase ; biosynthesis ; Animals ; Bone Marrow Cells ; cytology ; radiation effects ; Cell Differentiation ; genetics ; radiation effects ; Cell Proliferation ; radiation effects ; Coculture Techniques ; Electromagnetic Fields ; Mesenchymal Stromal Cells ; radiation effects ; Osteoblasts ; radiation effects ; Osteogenesis ; genetics ; radiation effects ; Rats

BACKGROUNDMechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress (FSS) is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells.

METHODSMC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm(2) using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs (miRNAs) were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase (ALP) activity assay was performed at 24th, 48th, and 72 th hours after FSS treatment, and Alizarin Red Staining was checked at day 12.

RESULTSOne hour of FSS at 12 dyn/cm(2) induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated.

CONCLUSIONThe short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be involved in FSS-induced pre-osteoblast differentiation.

Actins ; chemistry ; Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclooxygenase 2 ; genetics ; Gene Expression Profiling ; Mice ; MicroRNAs ; physiology ; Osteoblasts ; cytology ; Osteogenesis ; Stress, Mechanical ; Stress, Physiological

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
Adipogenesis/drug effects ; Alkaline Phosphatase/biosynthesis/genetics ; Antigens, Differentiation/biosynthesis/genetics ; Bone Marrow/pathology ; Cell Differentiation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/biosynthesis/genetics ; Culture Media ; Gene Expression Regulation ; Humans ; Lipoprotein Lipase/biosynthesis/genetics ; Mesenchymal Stem Cells/*drug effects/metabolism/pathology ; Osteoblasts/drug effects/*metabolism/pathology ; Osteogenesis/drug effects ; PPAR gamma/biosynthesis/genetics ; Parathyroid Hormone/*pharmacology ; Peptide Fragments/*pharmacology ; Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors

To study the role of the thyroid hormone receptor TRalphaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRalphaA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl beta-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRalphaA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRalphaA in vivo.
Alkaline Phosphatase ; genetics ; immunology ; Animals ; Antibodies ; immunology ; Escherichia coli ; genetics ; metabolism ; Flounder ; physiology ; Metamorphosis, Biological ; physiology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Thyroid Hormone Receptors alpha ; biosynthesis ; genetics ; immunology

OBJECTIVE: To investigate the effect of adiponectin on the osteoblast differentiation and its signal transduction. METHODS: Adipopnectin receptor (AdipoR) was detected by immunoblot analysis. Alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. Osteocalcin was measured by a specific radioimmunoassay kit, and the extent of mineralized matrix was determined. RNA interference was used to down-regulate the expression of AdipoR1 in human osteoblasts, and the effect of adiponectin on osteoblast differentiation was investigated. RESULTS: Only AdipoR1 protein was detected in human osteoblasts. Adiponectin could promote osteoblast differentiation, and result in a dose-dependent increase in ALP activity, osteocalcin secretion, and an increase in mineralized nodules. Suppression of AdipoR1 with siRNA could abolish the adiponectin induced ALP expression. Adiponectin could induce the activation of p38 and JNK, but not ERK1/2 in osteoblasts, and the pretreatment of osteoblasts with the p38 inhibitor (SB203580) could block the adiponectin-induced ALP activity. CONCLUSION Adiponectin can induce human osteoblast differentiation via AdipoR1/p38 pathway.
Adiponectin ; pharmacology ; Alkaline Phosphatase ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; analysis ; RNA, Small Interfering ; genetics ; Receptors, Adiponectin ; biosynthesis ; Signal Transduction

BACKGROUNDIcariine is a flavonoid isolated from a traditional Chinese medicine Epimedium pubescens and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis. But the mechanism is unclear. The aim of the study was to research the effect of Icariine on the proliferation and differentiation of human osteoblasts.

METHODSHuman osteoblasts were obtained by inducing human marrow mesenchymal stem cells (hMSCs) directionally and were cultured in the presence of various concentrations of Icariine. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of Icariine on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of calcified nodules were assayed to observe the effect on cell differentiation. The expression of bone morphogenetic protein 2 (BMP-2) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSIcariine (20 microg/ml) increased significantly the proliferation of human osteoblasts. And, Icariine (10 microg/ml and 20 microg/ml) increased the activity of ALP and the amount of calcified nodules of human osteoblasts significantly (P < 0.05). BMP-2 mRNA synthesis was elevated significantly in response to Icariine (20 microg/ml).

CONCLUSIONSIcariine has a direct stimulatory effect on the proliferation and differentiation of cultured human osteoblast cells in vitro, which may be mediated by increasing production of BMP-2 in osteoblasts.

Alkaline Phosphatase ; analysis ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Humans ; Osteoblasts ; cytology ; drug effects ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta ; biosynthesis ; genetics

OBJECTIVETo explore the effect of Herba Epimedii flavone (HEF) on the osteoblast metabolism in vitro.

METHODOsteoblast were obtained from new born rat calvaria by digestive enzymes. MTF, PNPP and RT-PCR were used to observe the proliferation, activity of ALP and mRNA expression of OPG and RANKL of cultured osteoblasts in vitro.

RESULTIt was found that HEF had the effect on stimulating cell proliferation, activity of ALP and the mRNA expression of OPG of cultured osteoblasts (P < 0.01, P < 0.05).

CONCLUSIONHEF can promote the proliferation, the differentiation and the expression of OPG mRNA of the osteoblasts cultured in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Carrier Proteins ; biosynthesis ; genetics ; Cell Proliferation ; drug effects ; Cells, Cultured ; Epimedium ; chemistry ; Flavones ; isolation & purification ; pharmacology ; Glycoproteins ; biosynthesis ; genetics ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteoprotegerin ; Plants, Medicinal ; chemistry ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics

OBJECTIVETo develop a cellular assay based on heat shock signal pathway and secreted alkaline phosphatase (SEAP) reporter gene for investigating/predicting the early toxicity of heavy metals on HeLa cells in Chinese traditional medicine (TCM).

METHODThe pHSE-SEAP plasmid was transfected into HeLa cells to build a HSE-SEAP-HeLa cell model. For validation of the model, the transfected cells were treated by either heating at 42 degrees C for 1 h or incubated with 5 mol x L(-1) CdCl2 for 4 h. Then the cells were covered in complete DMEM culture medium for 48 h and the activity of SEAP (reflecting the cellular level of heat shock protein) in cultural supernatants was measured; meanwhile, cell viability was determined by MTT assays. In addition, the cells were treated by four mercury compounds, HgCl2, merthilate sodium, HgS and cinnabar at the sub-lethal concentrations (determined by MTT assays). Then the heat shock response was detected likewise.

RESULTSignificant level of secreted alkaline phosphatase (SEAP) was found in pHSE-SEAP transfected HeLa cells treated either by heating (42 degrees C) or incubating with CdCl2. The heat shock protein was induced by CdCl2 before decrease of cell viability was observed. All four mercury compounds induced heat shock response in both time and concentration-dependant manner. However, there were big differences among the mercury compounds, suggesting potential differences for early-stage toxicity in vivo.

CONCLUSIONThe pHSE-SEAP transfected HeLa cells respond effectively to heat shock and metal stresses, and therefore provide a practical and repeatable assay for investigating/predicting the early toxicity of heavy metals and mineral-containing drugs in TCM.

Alkaline Phosphatase ; biosynthesis ; genetics ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Genes, Reporter ; HeLa Cells ; Heat-Shock Proteins ; biosynthesis ; genetics ; Humans ; Mercuric Chloride ; administration & dosage ; toxicity ; Mercury Compounds ; administration & dosage ; toxicity ; Metals, Heavy ; administration & dosage ; toxicity ; Plasmids ; Response Elements ; genetics ; Time ; Transfection

Alkaline Phosphatase ; biosynthesis ; Animals ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; pharmacology ; Cell Line ; Enzyme Activation ; Enzyme Induction ; drug effects ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Mice ; Osteoblasts ; enzymology ; Pyridines ; pharmacology ; Smad Proteins ; genetics ; Stem Cells ; enzymology ; p38 Mitogen-Activated Protein Kinases ; physiology

Bone morphogenetic protein 2(BMP-2) is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1(+) to construct hBMP-2 eukaryotic expression vector pCDNA3.1(+)-hBMP-2. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level recombinant human bone morphogenetic protein 2(rhBMP-2) was constructed by co-transfecting the expression vectors pCDNA3.1(+)-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.1 and 1 micromol/L. Western blot analyses showed a specific band of about 18 kD in reduced sample lane and a specific band of about 32 kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level (7.83 microg/24 h/10(6) cells) of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase(ALP) activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium(CM) for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.
Alkaline Phosphatase ; biosynthesis ; Animals ; Blotting, Western ; Bone Morphogenetic Protein 2 ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; CHO Cells ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Induction ; drug effects ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; Solubility