AIMTo investigate the effects of genistein on bone mineralization in ovariectomized rats.

METHODSForty-seven Wistar rats were randomly allocated into six groups: sham-operated (sham), ovariectomized (ovx), ovariectomized supplied with diethyl stilbestrol (E, 20 microg x kg bw(-1) x d(-1)) or genistein (25, 50, 100 mg x kg bw(-1) x d(-1)). After the rats had been fed for three months, analysis of the bone mineral density, parameters related to mineralization, bone content of Ca, P, Mg, Mn and Zn and serum concentration of parathyroid calcitonin and estrogen was performed.

RESULTSBone mineral density, bone Ca, P, Zn and Mg content and serum estrogen concentration in ovariectomized rats were significantly decreased, but mean osteoid width increased, mineralization lag time and osteoid maturation period prolonged compared with sham animals. After three months supplementation to ovariectomized rats, bone Ca, P and Mg content increased, mean osteoid width decreased, mineralization lag time and osteoid maturation period shortened compared with ovariectomized animals.

CONCLUSIONGenistein promotes bone mineralization by increasing bone Ca, P, Mg and adjusting serum calcitonin to prevent osteoporosis.

Animals ; Bone Density ; drug effects ; Bone Resorption ; Calcification, Physiologic ; drug effects ; Female ; Genistein ; pharmacology ; Ovariectomy ; Rats ; Rats, Wistar

Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P <0.01 or P <0.05); while the mRNA expressions of Collagen-1 and BMP-2 in PEMF group were significantly higher than those in SEMF group. After 6 days treatment, the activity of ALP in PEMF group was significantly higher than that in blank control group (P<0.05), while such difference was not observed in SEMF group (P0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all P<0.05), but the difference between PEMF and SEMF groups was not significant (P0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all P<0.01), and the stained area was bigger in PEMF group than that in SEMF group (P<0.05). After 12 days treatment, calcium nodules were increased in PEMF and SEMF groups compared with that in blank control group (all P<0.01), and more calcium nodules were observed in PEMF group than SEMF group (P<0.05). Conclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.
Animals ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; Cells, Cultured ; Electromagnetic Fields ; Gene Expression Regulation, Developmental ; radiation effects ; Magnetic Fields ; Osteoblasts ; cytology ; radiation effects ; Rats ; Skull ; drug effects

OBJECTIVETo assess the effect of puerarin, a natural flavonoid found in Chinese Pueraria Lobata (Wild.) Ohwi, on promotion of new bone formation.

METHODSOsteoblasts isolated from calvarial of newborn rats were cultured in vitro in the presence of puerarin at various concentrations. The viability of osteoblasts and alkaline phosphotase activity and mineral node formation were determined. In addition, osteoblasts seeded in the β-tricaclium phosphate scalfolds as bone substitute were implanted in rat dorsal muscles. Half -of the recipient rats received intramuscular injection of puerarin at 10 mg/(kg·d) for 7 days. Osteogenesis was analyzed by examining the histology after 4 weeks of implantation.

RESULTSThe viability of osteoblasts treated with puerarin at either 40 or 80 μmol/L was significantly higher than that of the control (P<0.05 and P<0.01, respectively). Alkaline phosphatase and mineral modules were significantly increased in osteoblasts cultured with puerarin at 40 or 80 mol/L when compared with that of the untreated cells. The puerarin-treated rats had a higher rate of bone formation in the osteoblast implants than the control rats (6.35% vs. 1.32%, respectively, P<0.05).

CONCLUSIONPuerarin was able to affect osteoblast proliferation and differentiation, and promote the new bone formation in osteoblast implants.

Alkaline Phosphatase ; metabolism ; Animals ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Implants, Experimental ; Isoflavones ; pharmacology ; Male ; Microscopy, Electron, Scanning ; Osteoblasts ; cytology ; drug effects ; enzymology ; Osteogenesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Tissue Scaffolds

The RGD-containing peptide was used to modify the surface of porous PLGA-[ASP-PEG], and was incubated in the modified simulated body fluid (mSBF) for two weeks. The mineralization of PLGA-[ASP-PEG] was explored. The active peptide was used to modify PLGA-[ASP-PEG] through cross-linker (Sulfo-LC-SPDP), characterized by X-ray photoelectron spectroscopy (XPS) the peptide-modified PLGA-[ASP-PEG] (Experiment group, EG) and PLGA-[ASP-PEG] without modification (Control group, CG) were all incubated in mSBF for two weeks, confirmed by observation of Scanning electron microscope(SEM) and measurements of Energy dispersive analysis system of X-ray (EDS) and X-ray diffractometry (XRD). XPS indicated that the binding energy of sulphur in EG was 164eV, and the ratio of carbon to sulphur in EG was 99.746 : 0.1014, however, sulphur was not detected in CG; SEM analysis demonstrated that the mineralization layers were more consecutive and compact in EG than in CG. The results of EDS and XRD indicated that the main component of mineral was hydroxyapatite, and the ratio of Ca/P was 1.60 in EG, and 1.52 in CG. RGD-containing peptide provided enough functional groups for mineralization; the mineralized peptide- modified PLGA-[ASP-PEG] possessed the bonelike microstructure.
Biocompatible Materials ; chemistry ; Bone Substitutes ; Bone and Bones ; metabolism ; Calcification, Physiologic ; Lactic Acid ; chemistry ; Oligopeptides ; chemistry ; Osteogenesis ; drug effects ; Peptides ; chemical synthesis ; pharmacology ; Polyglycolic Acid ; chemistry ; Surface Properties

It is well known that estrogen can inhibit bone absorption, decrease bone turnover and preserve bone mass. Some studies indicated that the effect of estrogen on calcium and bone is relative to vitamin D system, while others also reported that this effect of estrogen is independent of vitamin D. The genomic effect of 1alpha, 25(OH)(2)D(3)is mediated by the nuclear vitamin D receptor (VDR) in a ligand-dependent manner. Hypocalcemia, hyperparathyroidism and osteomalacia are developed in VDR gene knockout mice. To determine whether the effect of estrogen on calcium and bone is dependent on VDR, this study examined the effect of exogenous estrogen on calcium and bone homeostasis in VDR gene knockout mice. Male and female wild type (WT) and VDR gene knockout heterozygous mice were mated each other and the genotyping of their offsprings were determined by PCR. At age of 21-day, WT and knockout mice were weaned and treated by one of three different regimens: (1) WT-vehicle group: the WT mice were injected with normal saline; (2) VDR KO-vehicle group: the VDR gene knockout mice were injected with normal saline; (3) VDR KO-E group: the VDR gene knockout mice were subcutaneously injected with estradiol, 0.2 mug per mouse, once daily for 1 month. The bone mineral density (BMD) of mice was measured using dual-energy X-ray absorptiometry. All mice were sacrificed at age of 50-day. Blood was taken by heart puncture under anesthesia and serum calcium was measured by autoanalyser.Tibiae were removed, fixed and embedded with the methylmethacrylate (MMA), and undecalcified sections were cut. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue. Static histomorphometric analyses were performed on those stained sections. The results showed that the serum calcium level was (2.10+/-0.37) mmol/L in the VDR KO-vehicle mice and rose to (2.80+/-0.41) mmol/L in the VDR KO-E mice although it was still lower than WT-vehicle mice [(3.10+/-0.48) mmol/L]. BMD and mineralized trabeculer volume were increased significantly in VDR KO-E group compared with that in VDR KO-vehicle group. These results suggest that exogenous estrogen can improve calcium absorption and skeletal mineralization in a VDR-independent manner.
Animals ; Bone Density ; Calcification, Physiologic ; drug effects ; Calcium ; metabolism ; Estrogens ; pharmacology ; Female ; Gene Knockout Techniques ; Homeostasis ; Mice ; Mice, Knockout ; Receptors, Calcitriol ; genetics

OBJECTIVETo investigate the effect of naringin on the proliferation, differentiation and matrix mineralization of MC3T3-E1 cells in vitro.

METHODMC3T3-E1 cell lines were taken in vitro model. CCk-8 method was used to observe the proliferation of MC3T3 cells. Lactic acid dehydrogenase cytotoxicity (LDH) test was used to observe the cell toxicity. Bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP) and osteocalcin (OC) were used to observe the cell differentiation. Von kossa calcification staining method was used to observe the cell calcification.

RESULTSThe high dosages of the naringin could promote the proliferation of MC3T3-E1 cells at both 12 h and 24 h. While, low dosages did not show the same capability. LDH test showed that the cytotoxicity percentages in all six naringin treated groups were quite low. BMP-2 cytoimmunochemistry test showed that the three naringin treated group (10, 1, 0.1 micromol x L(-1)) showed higher brown coloration in cytoplasm than the control group at both 24 h and 48 h. 1, 0.1 micromol x L(-1) naringin raised ALP activity of MC3T3-E1 cells at 48 h (P < 0.05). Meanwhile, 0.1 micromol x L(-1) naringin increased the ALP activity at 72 h (P < 0.05). 10 and 1 micromol x L(-1) naringin increased the capability of MC3T3-E1 cell to synthesize osteocalcin during 8th - 12th dsince adding the medicine (P < 0.05). Naringin did not show the positive effects on cell calcification.

CONCLUSIONSNaringin could promote proliferation and differentiation of MC3T3-E1 cells.

Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Flavanones ; pharmacology ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; metabolism

OBJECTIVETo explore the effects of Drynaria fortunei J. Smith (DFS) on promoting the proliferation, differentiation and calcification of cells.

METHODTwo DFS preparations were extracted with distilled water (DFS aqueous-extract) and 95% ethanol (DFS ethanol-extract), respectively. A mouse osteoblast cell line MC3T3-E1 was used as a cell model for screening potency of DFS. MTT and Flow cytometry were applied to determine proliferation of the cell promoted by DFS aqueous-and ethanol-extracts at different dosages. Differentiating effects of the two extracts with different concentrations in the cell were evaluated through the examinations of alkali phosphate (ALP) activities and osteocalcin levels. Von kossa staining method was used to understand the effects of the two extracts promoting calcification of the cell.

RESULT0.01 mg x L(-1) and 1 mg x L(-1) of DFS aqueous-extracts showed the proliferative promotion for MC3T3-E1 cell (P < 0.05). Both the 0.01 mg x L(-1) and 1 mg x L(-1) of the two DFS extracts increased the percentages of S-phase cells whereas the percentages of G1-phase cells were decreased. 1 and 100 mg x L(-1) of the ethanol-extract remarkably increased the synthesis and secretion of osteocalcin of the cell (P < 0.001). 1 mg x L(-1) of the aqueous-extract and 0.01 mg x L(-1) of the ethanol-extract were able to promote the cell calcification (P < 0.05).

CONCLUSIONIn the DFS aqueous-and ethanol-extracts, there are some of the components promoting the proliferation, differentiation and calcification of osteoblast.

Animals ; Calcification, Physiologic ; drug effects ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Mice ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; biosynthesis ; Plants, Medicinal ; chemistry ; Polypodiaceae ; chemistry

OBJECTIVETo investigate the effects of total flavonoid extract of Epimedium sagittatum (TFE) on the proliferation and differentiation of newborn rat calvarial osteoblasts (ROB).

METHODTFE was supplemented into the culture medium of ROB at 0. 1, 1, 10 and 100 microg x mL(-1) respectively. The serum of rats administered TFES (SRAT) was also added into the medium in a parallel treatment at 2.5%, 5% and 10% respectively. Their effects on cell proliferation and differentiation was studied by MTT and the analysis of osteogenic differentiation marks.

RESULTTFE had no appreciable and on cell proliferation and differentiation at any concentration. However, 2.5% and 5% SRAT stimulated cell proliferation strongly and, 5% SRAT significantly promoted the maturation and function of osteoblast by improving the alkaline phosphatase activity, osteocalcin secretion, calcium deposition and the number of mineralized nodular structures.

CONCLUSIONThe metabolites of TFE should be the anti-osteoporosis constitutes of Epimedium sagittatum.

Animals ; Calcification, Physiologic ; drug effects ; Calcium ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Flavonoids ; blood ; pharmacology ; Osteoblasts ; cytology ; drug effects ; metabolism ; Rats ; Rats, Wistar

Objective: To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. Methods: The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. Results: Real time RT-PCR showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related genes in icaritin group were significantly increased (P<0.05 or P<0.01); After AMD3100 treatment, the relative expression of CXCR4 gene was decreased (P<0.05). Western blot showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related proteins in the icaritin group were significantly increased (all P<0.01), but were decreased after AMD3100 was added (all P<0.01). The ALP activity of icaritin group was significantly higher than that of blank control group (all P<0.01) on d3 and d6 after drug treatment, while the activity of ALP was significantly decreased after AMD3100 treatment (all P<0.01). At d14 after drug treatment, compared with the blank control group, the area of alizarin red staining was increased in the icaritin group, while it was significantly reduced after the addition of AMD3100. Conclusion: Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.
3T3 Cells ; Animals ; Calcification, Physiologic ; drug effects ; Chemokine CXCL12 ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Osteoblasts ; cytology ; drug effects ; Receptors, CXCR4 ; metabolism ; Signal Transduction ; drug effects

Zebrafish was selected as model animal, and glucocorticoid dexamethasone was used as a model compound to establish a rapid and high efficient osteopenia model. Zebrafish larvae at 4 days post fertilization (dpf) were exposed to a serial concentrations of dexamethasone solutions, and 0.5% DMSO was selected as the vehicle control group. All groups were incubated in 24-well plates (28.5 degrees C) until 9 dpf. In addition, effects of 10 micromol x L(-1) dexamethasone on preventing against osteopenia induced by etidronate disodium were also investigated. Zebrafish bones at 9 dpf were stained with alizarin red. Quantitative analysis of the stained area was performed by microscopic inspection and digital imaging methods to reflect the amount of bone mineralization. Results showed that dexamethasone group at 2.5, 10 and 25 micromol x L(-1) can decrease the staining area and the staining optical density values of zebrafish head bones when compared with the vehicle control group (0.5% DMSO), which suggested that dexamethasone can significantly reduce the zebrafish mineralized bone and the bone mineral density. Results also showed that 15 and 30 microg x mL(-1) etidronate disodium can increase the mineralized matrix of zebrafish head bone and prevent against osteopenia induced by dexamethasone. In conclusion, the study indicated that zebrafish can be an idea osteopenia model induced by dexamethasone.
Animals ; Bone Density ; drug effects ; Bone Density Conservation Agents ; pharmacology ; therapeutic use ; Bone Diseases, Metabolic ; chemically induced ; prevention & control ; Calcification, Physiologic ; drug effects ; Dexamethasone ; toxicity ; Disease Models, Animal ; Etidronic Acid ; pharmacology ; therapeutic use ; Larva ; drug effects ; growth & development ; Zebrafish