Study On The Effects And Mechanism Of Sinusoidal Electromagnetic Fields On The Differentiation And Maturation Of Osteoblast

Osteoporosis is one of the most common chronic age-related diseases in both the Western and Asian populations, which affects one in two postmenopausal women and one out of eight men in their lifetime. Having osteoporosis puts people at higher risk of fractures that are painful, and can reduce their ability to lead active lives. If diagnosed early, the fractures associated with the disease can often be prevented. Unfortunately, osteoporosis frequently remains undiagnosed until a fracture occurs. Twenty percent of seniors who break a hip will die within one year. Many of those who survive will need long-term nursing home care. Osteoporotic fractures cause greater economic burden for families and society. Clinically, many treatments, such as a regimen of calcium, vitamin D, hormone replacement therapy, are effective in treating primary osteoporosis, but these drugs mainly alleviate symptoms, expensive and with many adverse effects. Recently, sinusoidal electromagnetic fields (SEMFs) have been taken as a potential candidate for the treatment of osteoporosis. However, different parameters of EMFs have been used in study by different authors. Moreover, the detailed mechanism of EMFs for the management of osteoporosis is still unclear.To investigate the effects and detailed mechanism of SEMFs on the differentiation and maturation in rat osteoblast and find out the intensity with the best therapeutic efficacy, a series intensity of SEMFs with constant frequency of 50Hz is used in this study.Rat bone marrow mesenchymal stem cells (rBMMSCs) were obtained from Wistar rats and screened by the adhesive method. They were exposed to sinusoidal electromagnetic fields with 50Hz frequency and 0.0mT, 1.4mT, 1.6mT, 1.8mT, 2.0mT, 2.2mT intensity respectively, 30min per day. The osteogenic differentiation markers including ALP activity, calcium deposition, mineralized bone modulus and collagenâ… expression were compared among the exposed groups and the control. The total cellular RNA was extracted after 6, 12, 24 and 48 hours .The gene expression of Osterix and IGF-1 was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The absorbance of exposed groups was suppressed significantly in comparison with control group. 1.8mT intensity strongly enhances the osteogenic differentiation of rMSCs, indicated by significantly improved ALP activity, calcium deposition, collagenâ… expression and the number of mineralized bone nodules compared to the control and other groups. Osterix and IGF-1 were also significantly improved (P<0.05). 50Hz and 1.4~2.2mT sinusoidal electromagnetic fields enhance the osteogenic differentiation of rMSCs but inhibite their proliferation in the presence of 0.1% serum culture. Among them, 1.8mT has the strongest activity, indicating that it may be the optimal intensity for the clinical application.Nitric oxide (NO) is an important intracellular and intercellular messenger which has important effects on bone metabolism. The second purpose of this research was to investigate whether the effects of sinusoidal electromagnetic field (SEMF) stimulation on the differentiation and maturation of osteoblast are mediated by the NO-cGMP-PKG signal pathway. In the experiment, we first investigated whether SEMF had an effect on nitric oxide synthase (NOS) activity after SEMF treatment. Secondly, L-NAME, the inhibitor which blocks nitric oxide synthase, prevented the SEMF-stimulated increase in NOS activity and NO content. An inhibitor of soluble guanylyl cyclase (ODQ) blocked the SEMF-stimulated increase in cGMP content. The inhibitor PDE5, which hydrolyzes 3',5'-cyclic-GMP to 5'-GMP, prevented the SEMF-stimulated increase in PKG. Thirdly, the NO-cGMP-PKG signal pathway was blocked to observe whether the maturation and mineralization of osteoblast were stimulated by SEMF were inhibited, which evaluated by measuring alkaline phosphatase (ALP)activity, Osterix gene expression and mineralized bone modulus. After treatment with SEMF, the NOS activity in SEMF group increased in comparison with the normal control group (P<0.01), reaching the highest level after 0.5 h. The gene expression of Osterix, ALP activity and mineralized bone modulus in the SEMF group were also increased significantly. However, these effects were partially blocked in the L-NAME group. Surprisingly, all the osteogenic markers in the SEMF plus L-NAME group were slightly higher than those of the control group, but lower than those of the SEMF group. In conclusion, the NO-cGMP-PKG signal pathway was activated by SEMF treatment. The stimulatory effects of SEMF on the differentiation and mineralization of osteoblast were attenuated when NO-cGMP-PKG signal pathway was blocked.