To investigate whether the effect of an sinusoidal electromagnetic field on the differentiation and maturation of osteoblasts is mediated by the NO-cGMP-protein kinase G signal pathway.
Extremely low frequency electromagnetic fields are used as a method of treatment for some orthopedic diseases, including osteoporosis. However, the exact role of the field in the management of osteoporosis is still unclear. For this reason, eight groups of cell cultures were examined: 1.) control group, 2.) exposure group, 3.) treatment with L-NAME (nitric oxide synthase inhibitor), 4.) exposure + L-NAME, 5.) treatment with ODQ (inhibitor of soluble guanylyl cyclase, blocks the increase in cGMP levels), 6.) exposure + ODQ, 7.) treatment with PDE5 (phosphodiesterase type 5, abolishes cGMP) and 8.) exposure + PDE5. There were five samples in each group and all experiments were conducted twice.
| Exposure | Parameters |
|---|---|
|
Exposure 1:
50 Hz
Exposure duration:
continuous for 30 min per day for 1, 9, 14, or 21 days; or for 12 h (see add. information)
|
|
cells were treated in the following eight groups: i) control ii) exposure to EMF iii) 100 µM L-NAME added iv) 100 µM L-NAME added 6 h before EMF treatment + exposure to EMF v) 20 µM ODQ added vi) 20 µM ODQ added 4 h before EMF treatment + exposure to EMF vii) 0.02 U PDE5 added viii) 0.02 U PDE5 added 0.1 h before EMF treatment + EMF exposure
| Frequency | 50 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | continuous for 30 min per day for 1, 9, 14, or 21 days; or for 12 h (see add. information) |
| Exposure source | |
|---|---|
| Setup | solenoids consisting of 50 cm long hollow cylinders with 20 cm diameter, placed in an incubator with 5% CO2 and 37 °C; culture dishes positioned in the center of the solenoid; magnetic field parallel to surface of the plates |
| Additional info | exposure durations are not clearly documented for the different endpoints investigated (remark EMF-Portal), but it seems that the following durations were studied: osterix gene expression following 12 h of exposure ALP activity following (up to?) 9 days of exposure mineralization following up to 14 or 21 days of exposure (30 min/day) all other parameters seemed to be investigated following a single exposure of 30 min. |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| magnetic flux density | 1.8 mT | - | - | - | - |
30 minutes after exposure, the enzyme activity of the NOS and the level of NO were significantly higher than those in the control group. After these 30 minutes, the nitric oxide activity decreased in the exposure group, but remained higher than in the control group during the observed 4 hours.
In the exposed group, the protein expression of iNOS, eNOS and sGC were significantly up-regulated compared to the control group. However, an addition of L-NAME to the exposed cell cultures decreased the expression of iNOS, eNOS and sGC in comparison to the exposed group without L-NAME. If ODQ was given to the exposed cell cultures a decreased expression of sGC and a decreased amount of cGMP compared to the exposed cell cultures without ODQ was found. An addition of PDE5 to the exposed cell cultures led to a decreased expression level of PKG and a decreased amount of cGMP compared to the exposed cell cultures without PDE5.
The gene expression level of the transcription factor Sp7 was significantly increased in the exposed samples in comparison to the control cells. An addition of L-NAME, ODQ or PDE5 blocked this exposure effect.
The activity of ALP was increased in the exposed group in comparison to the control. An addition of L-NAME, ODQ and PDE5 to the exposure cell cultures led to a lower ALP activity in comparison to the exposed samples without the inhibitors.
Regarding the mineralization, the exposed cell cultures showed significantly more calcified areas than the control cultures. However, an addition of L-NAME, ODQ or PDE5 decreased the level of mineralization in the exposed samples compared to the exposed ones without inhibitors.
The authors conclude that the NO-cGMP-PKG pathway is activated by a sinusoidal electromagnetic field exposure, because the stimulatory effect of this kind of exposure on the differentiation and mineralization of osteoblasts was attenuated when the pathway was blocked by the added substances.
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