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

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

To investigate the effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor (MC3T3-E1) cells.miR-705 mimics, inhibitors and negative control were transfected into MC3T3-E1 cells. Alkaline phosphates (ALP) staining were performed and quantified after 7 days of osteogenic medium induction. The mRNA and protein expression levels of runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by real-time RT-PCR and Western blot after 14 days of osteogenic induction. Alizarin red staining was performed and quantified in MC3T3-E1 cells after 21 days of osteogenic induction.After 7 days of osteogenic induction, ALP staining showed that overexpression of miR-705 significantly reduced ALP activity, whereas knockdown of miR-705 increased ALP activity (all<0.05). Consistently, after 14 days of osteogenic induction, mRNA and protein expressions of Runx2 and OCN were suppressed by overexpression of miR-705, whereas they were promoted by knockdown of miR-705 (all<0.05). After 21 days of osteogenic induction, alizarin red staining showed that overexpression of miR-705 significantly reduced the formation of mineralized node, the opposite results were found in miR-705 knockdown group (all<0.05).miR-705 can inhibit the osteogenic differentiation of MC3T3-E1 cells.
Alkaline Phosphatase ; drug effects ; genetics ; Animals ; Calcification, Physiologic ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Core Binding Factor Alpha 1 Subunit ; drug effects ; genetics ; Down-Regulation ; drug effects ; genetics ; Fetal Stem Cells ; Mice ; MicroRNAs ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; drug effects ; genetics ; Osteogenesis ; drug effects ; genetics

OBJECTIVETo evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to explore the possible application on the alveolar bone regeneration.

METHODSTo determine the optimum concentration, the effects of ginsenoside Rg-1 ranging from 10 to 100 μmol/L were evaluated by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2(I) chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction.

RESULTSCompared with the control group, a certain concentration (10 μmol/L) of the Rg-1 solution significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs (P<0.05). However, concentrations that exceeds 100 μmol/L led to cytotoxicity whereas concentrations below 10 nmol/L showed no significant effect as compared with the control.

CONCLUSIONGinsenoside Rg-1 can enhance the proliferation and osteogenic differentiation of hPDLSCs at an optimal concentration of 10 μmol/L.

Adolescent ; Alkaline Phosphatase ; metabolism ; Biomarkers ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Separation ; Cell Shape ; drug effects ; Cells, Cultured ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects ; genetics ; Periodontal Ligament ; cytology ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; drug effects ; enzymology ; Time Factors ; Young Adult

AIM: Osteoblasts are key functional cells in the process of bone metabolic balance. Phytoestrogens have an important influence on the proliferation and differentiation of osteoblasts. Puerarin, a plant estrogen, has a wide range concentration in vitro on the function of osteoblasts. The current study investigates the effect of the phytoestrogen puerarin on the proliferation, differentiation, and mineralization of osteoblasts in vitro. METHODS: The calvaria bone of eight-ten Wistar rats which were born within 24 h were obtained in aseptic condition. After enzyme digestion, isolation, purified osteoblasts of rats were cultured for further study. The cells of the first to third generation were divided into a control group and a puerarin-treated group with 10(-3)-10(-10) mol·L(-1) puerarin. The cells were exposed to the medium containing a low level of carbohydrates, 10% (V/V) FBS for 24 h. After 1 to 4 days of culture, the OD values on the proliferation of osteoblasts in each group were determined by microplate reader. The cells were cultured in the medium containing 50 μg·mL(-1) vitamin C, 10(-2) mol·L(-1) sodium glycerophosphate, 10% FBS and the medium was changed every 3 to 4 days. After 2 to 8 days of culture, expression of alkaline phosphatase were tested and compared by microplate reader. The mineral nodes of osteoblasts were dyed using alizarin red or improved Von Kossa way after four weeks. RESULTS: Compared with those in the 10(-5)-10(-9) mol·L(-1) puerarin, the proliferation of osteoblasts, the expression of alkaline phosphatase, and the number of mineral nodes of osteoblasts were significantly decreased in the control group. The increase was the fastest in the third day, while on the fourth day it was decreased, and arrived at statistical significance compared with the alkaline phosphatase activities and control group. The 10(-6) mol·L(-1) group was the most distinct, and formed the most mineralized nodule. Compared with the 10(-3) mol·L(-1) puerarin group, those changes were markedly increased in the control group. CONCLUSIONS Puerarin has proliferation, differentiation, and mineralization effects on osteoblasts in a dose-dependent manner, and has a double-way effect on the osteoblasts in vitro. A low-dose showed positive effects on the development of osteoblasts, and high-dose puerarin could inhibit the formation of bone.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Density ; Bone and Bones ; cytology ; drug effects ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Female ; In Vitro Techniques ; Isoflavones ; pharmacology ; Osteoblasts ; drug effects ; Phytoestrogens ; pharmacology ; Rats, Wistar

OBJECTIVETo evaluate the roles or effects of oviductus ranae (OR) or oviductus ranae eggs (ORE) in preventing and treating postmenopausal osteoporosis.

METHODSIn vivo experiment: Sixty female adult Wistar rats were randomly divided into 5 groups of 12. To provide an osteoporosis model 4 groups of rats were ovariectomized (OVX), with the 5th being sham operated. Medication commenced 7 days after the operation and lasted continuously for 12 weeks. Sham operated and OVX groups were given equivalent volumes of 5% Tween-80. The other three groups intragastrically received conjugated estrogens (CE), OR or ORE of the corresponding doses. At the 12th week, serum estrogen, bone gla protein (BGP), serum calcium, phosphorus, and alkaline phosphatase (ALP) were assayed; bone mineral densities (BMD) were measured and bone scanning was conducted; uteri were weighed, and weight, volume and length of the femoral bones were determined; and cortical thickness of femoral heads and area of bone trabecula were measured by image analyzer. In vitro experiment: Eighty 10-month old SD rats, with equal numbers of males and females, were randomly divided into 8 groups. Osteoblasts were isolated from neonatal rat calvariae, and the cells were exposed to various concentrations of serum from OR and ORE groups to study the impact of these sera on osteoblastic proliferation, ALP activity and mineralization. Osteoclastic numbers were determined using tartrate resistant acid phosphatase (TRAP).

RESULTSIn vivo experiment: The body weight of the four OVX groups increased significantly (P<0.01). Uterine weight of the CE group was the highest (P<0.01); Compared with the model group, estrogen level, BMD, bone scanning/bone imaging index weight of the femoral bones, cortical thickness of femoral heads in the OR and ORE groups increased significantly (P<0.05, P<0.01); femoral volume in the ORE group increased significantly (P<0.05); and the content of osteocalcin, phosphorus, and ALP in serum decreased significantly (P<0.05, P<0.01). In vitro experiment: Sera from OR and ORE groups had notable effects on the proliferation of osteoblasts (P<0.05 and P<0.01, repsectively) and stimulated the formation of calcium nodes (P<0.05, P<0.01), while the enhancement of ALP activity in osteoblasts was significant (P<0.05, P<0.01). The number of TRAP-positive cells was significantly reduced as well (P<0.01).

CONCLUSIONSOR and its eggs could effectively suppress OVX-induced osteoporosis in rats, and increase bone turnover possibly by both an increase in osteoblastic activity and a decrease in osteoclastic activity. The present study provides evidence that OR and its eggs could be considered a complementary and alternative medicine for the treatment of postmenopausal osteoporosis.

Acid Phosphatase ; metabolism ; Alkaline Phosphatase ; metabolism ; Animals ; Biomarkers ; blood ; Body Weight ; drug effects ; Bone Density ; drug effects ; Bone and Bones ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Count ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Female ; Femur ; drug effects ; metabolism ; pathology ; Isoenzymes ; metabolism ; Male ; Materia Medica ; pharmacology ; therapeutic use ; Organ Size ; drug effects ; Osteoblasts ; drug effects ; enzymology ; pathology ; Osteoclasts ; drug effects ; enzymology ; pathology ; Osteoporosis ; blood ; drug therapy ; metabolism ; physiopathology ; Ovariectomy ; Ovum ; metabolism ; Rats ; Rats, Wistar ; Tartrate-Resistant Acid Phosphatase ; Uterus ; drug effects ; pathology

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

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

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 aim of this study is to investigate a new method for preparing a biomimetic bone material-surface modified sintered bovine cancellous bone, and to improve its bioactivity as a tissue engineering bone. The prepared sintered bovine cancellous bones with the same size were randomly divided into two groups, immersing in 1 and 1. 5 times simulated body fluid (SBF), respectively. The three time periods of soak time were 7, 14, and 21 days. After sintered bone was dried, the surface morphology of sintered bone and surface mineralization composition were observed under scanning electron microscopy (SEM). By comparing the effect of surface modification of sintered bone materials, we chose the most ideal material and studied its pore size, the rate of the porosity, the compress and bend intensity. And then the material and the sintered bone material without surface modification were compared. The study indicated that sintered bone material immersed in SBF (1.5 times) for 14 days showed the best effect of surface modification, retaining the original physico-chemical properties of sintered bone.
Animals ; Biocompatible Materials ; chemical synthesis ; Biomimetic Materials ; chemical synthesis ; Bone Substitutes ; Bone and Bones ; chemistry ; drug effects ; Calcification, Physiologic ; physiology ; Cattle ; Chemical Phenomena ; Hydroxyapatites ; chemistry ; Porosity ; Surface Properties ; Tissue Engineering ; methods