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Objective To explore the effects of electromagnetic fields (EMFs) on osteogenesis during co-culture of bone mesenchymal stem cells (BMSCs) with osteoblasts in rats.Methods BMSCs and osteoblasts were isolated from Sprague-Dawley rats and cultured.Sub-cultured osteoblasts and BMSCs were seeded in transwell cell-culture-chamber polyester inserts to establish the co-culture system.The co-cultures were then randomly divided into a normal co-culture group and a group exposed to an EMF.Single-cultured BMSCs and osteoblasts were set as a single culture group.The EMF group was exposed to an EMF for 4 hours per day.On the 14th day,cell culture plates or inserts were randomly selected for total RNA extraction and measurement of the mRNA expression levels of Runt-related transcription factor 2,transcription factor 7,alkaline phosphatase,collagen type Ⅰ,bone morphogenetic protein 2 (BMP-2) and bone gamma-carboxyglutamate protein (Osteocalcin gene,OC gene) using real-time PCR assays.Cell culture dishes or inserts were also randomly chosen for Alizarin red staining to detect mineralized nodules.Results The level of osteogenic gene expression in single-cultured BMSCs and osteoblasts was low,while it was much higher in the co-culture group.The level of gene expression in the EMF-exposed and co-cultured group was even higher.Alizarin red staining also showed that calcium mineralized modules had increased in the stimulated,co-cultured system compared with the unstimulated,co-cultured cells.Conclusion EMF exposure can promote osteogenic differentiation of BMSCs and osteoblasts when they are co-cultured.BMP2-mediated cellular interaction might play an important role in osteogenic differentiation induced by EMF exposure.
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Objective To explore in vitro the best time window for using sinusoidal electromagnetic fields to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were isolated and cultured from 4-week-old Sprague-Dawley rats (male and female,80-120g).The BMSCs (from passage 3) were exposed 0,1,4 or 8h/d for 7d,14,or 28d,respectively,to 15Hz sinusoidal electromagnetic fields with a maximum amplitude of lmT.Those exposed 0h/d served as the control.The relative expressions of runt related gene-2 (RUNX2),bone sialoprotein (BSP) and osteopontin (OPN) were determined using real-time,quantitative reverse transcription-polymerase chain reactions (RT-PCRs).The level of RUNX2 protein was determined by Western blotting after 14d.Alizarin red staining was used to compare calcium distribution in each group.Results Obvious promotion of differentiation to osteoblasts was observed after 7 days of exposure to the15 Hz sinusoidal electromagnetic fields,most obviously manifested by an outstanding increase of the early osteogenic index RUNX2 in those exposed 4h/d.After 14 days of intervention,the 1h/d exposure showed to be most effective,especially in inducing the changes of the late osteogenic index OPN.The trends of changes in RUNX2 protein were similar in all groups.After stimulating 1h/d for 14 and 28days,calcium deposition increased to the greatest extent.Conclusions Exposure to sinusoidal electromagnetic fields induces osteogenic differentiation to osteoblasts in rat BMSCs in vitro.There is an apparent window effect.The best results are observed with more days of exposure and shorter exposure time (1h) every day.
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Objective To study the impacts of dynamic compressive stress on the mRNA expression of osteopontin ( OPN ),runt related gene 2 ( Runx2 ),osteocalcin ( OC ),osterix,alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP-2) in the osteoblasts of Sprague-Dawley (SD) rats. Methods Osteoblasts extracted from skull periosteum tissue of neonatal SD rats were digested using trypsin and collagenase （Ⅰ）,then were subcultured and amplified in vitro.ALP staining and alizarin red staining were performed to identify the purified cells.The cells were treated with compressive stress at 20,50 or 100 mmHg for 24 h.The expression levels of OPN,Runx-2,OC,osterix,ALP and BMP-2 were measured and quantitatively analysed using a real-time quantitative polymerase chain reaction. Results Under 20 mmHg of dynamic compressive stress the expression levels of OPN,Runx2,OC,osterix,ALP and BMP-2 all were elevated compared with the control group.The peak expression oecured under 50 mmHg pressure. The expression levels did not change significantly compared with the control group under 100 mmHg pressure. Conclusions Moderate dynamic compressive stress can promote the expression of OPN,Runx-2,OC,osterix,ALP and BMP-2 mRNA in osteoblasts,which might be an important mechanism for promoting the union of fractures.
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Objective To investigate the biological effect of 50 Hz sinusoidal electromagnetic fields at 1 mT on the proliferation of the human osteosarcoma cell line MG-63.Methods Osteosarcrma MG-63 cells were divided into control and experimental groups.The control group was incubated without an electromagnetic field; the experimental group was incubated in a 50 Hz,1.0 mT sinusoidal electromagnetic field.On the 2nd,4th and 6th day,their proliferation was determined using a cell counting kit-8(CCK-8)assay.Variations in the cell cycle were detected with flow cytometry(FCM).Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to measure cyclin B1 and cyclin D1 mRNA.Results Compared with the control group,proliferation of the experimental group cells was reduced significantly.The percentage of cells at G0-G1 phase increased,and the mRNA expression of cyclin B1 and cyclin D1 was significantly reduced.Conclusions A 50 Hz sinusoidal electromagnetic field at 1.0 mT can inhibit the proliferation of osteosarcoma cell line MG-63 significantly.
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Objective To investigate the effects of 620nm noncoherent red light emitted from light-emitting diode (LED) on proliferation and osteogenic differentiation of rat osteoblasts.Methods Osteoblasts were isolated from SD rat and cultured in vitro.The LED was placed at 2cm above the cell layer.Cells were divided into four groups and each group was irradiated for 0s, 150s, 300s and 600s at energy dose of 0, 1, 2, and 4J/cm2, respectively.Cells were irradiated once every day.Cellular proliferation activities were evaluated by using cell counting kit-8 ( CCK-8 ) at 2nd and 4th d.The alkaline phosphatase (ALP) ratio activity was evaluated by alkaline phosphatase activity kit at the 9th d, and expressions of osteoblast master genes ( Collα1, Bglap and Runx2) were monitored as indicators of osteoblast differentiation by reverse transcription-polymerase chain reaction (RT-PCR) technique.Results In the group irradiated with 1 and 2 J/cm2 proliferation activities of osteoblasts enhanced significantly compared with the control group at the 4th d ( P < 0.05 ).In the group irradiated with 4 J/cm2 ALP ratio activity elevated significantly compared with the control group at the 9th d ( P <0.05 ).In the group of red light irradiation expressions of Bglap and Runx2 enhanced significantly.Conclusion The 620nm nonconherent red light emitted from LED can promote proliferation of osteoblasts and enhance osteogenic differentiation significantly.
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Objective To study the effects of an electromagnetic field (EMF) on the expression of fibro-blast growth factor (FGF-2) and it' s receptor (FGFR-2) mRNA in rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Rat BMSCs were isolated and cultured in vitro. The subcultured cells were divided into different groups to be EMF stimulated at 1.0 mT. The expression of FGF-2 and FGFR-2 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results Different frequencies and durations of 1.0 mT EMF exposure induced FGF-2 and FGFR-2 mRNA expression in comparison to blank controls. The expression of FGF-2 mRNA reached a peak after stimulation at 15 Hz for 10 min, 50 Hz for 60 min and 75 Hz for 30 min. And the expression of FGFR-2 mRNA reached a peak after 30 minutes at all frequencies. At 1.0 mT with 30 min exposure, the expression of FGF-2 mRNA peaked after 50 Hz stimulation, and the expression of FGFR-2 mRNA peaked after stimulation at 75 Hz. Conclusions Moderate EMF stimulation can significantly increase the expression of FGF-2 and FGFR-2 mRNA in rat BMSCs in vitro.
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Objective To study the effects of electromagnetic field (EMF) exposure on osteoporosis in ovariectomized mice. Methods Sixty 8-week-old female Kunming mice were divided into four groups at random: a sham operation group (group A), an ovariectomized group (group B), an EMF and ovariectomized group (group C) and a nilestriol and ovariectomized group (group D). Bilateral ovariectomies were performed on all mice except those in group A. The mice of group C were exposured to a 15 Hz, 1.0 mT electromagnetic field. The mice of group D were given at nilestriol 1.5 mg/kg/week. The bone mineral density (BMD) of the lumbar vertebrae was measured before the mice were sacrificed at the 12th week. Blood specimens were collected every two weeks to measure the ac-tivity of alkaline phosphatase (ALP) and the concentration of bone gamma-carboxyglutamic-acid-containing proteins (BGP), calcium and estradiol in the serum. Histological sections were taken to examine and analyze the changes in bone trabeculae in the lumbar vertebrae after 6 and 12 weeks. Results EMF at 15 Hz and 1.0 mT intensity signifi-cantly increased the activity of ALP and the concentrations of BGP and calcium in the serum. In addition, the absorp-tion of bone trabeculae in the lumbar vertebrae was significantly restrained. Conclusions EMF at 15 Hz and 1.0 mT can restrain the development of osteoporosis in ovariectomized mice.