| Background and objective With the increasing of the number of aged people and the use of mechanized transportation, disuse osteoporosis (DOP) is becoming a significant problem in clinical medicine. It is reported that the bone mass density (BMD) of astronauts during 6-month exposure to microgravity decreases up to 25%. The fracture risk of DOP patient induced by spinal cord injury, hemiplegia, and so on, increases 7-fold with 1 year of hospitalization. DOP causes more and more economic burden for families and the society. Many treatments, such as a regimen of calcium, vitamin D, and hormones supplements, are effective in treating primary osteoporosis, but it is still not clear about their effects on disuse osteoporosis. At the same time, these treatments have some adverse effects, such as hypercalcemia and breast cancer, which vary in severity with different individuals. For this reason, alternative regimens like pulsed electromagnetic fields (PEMF) are proposed.PEMF have been used to deal with different kinds of osteoporosis over the past decades, as a biological and physical method. In 1957, it was reported an electrical effect in bones similar to the classic piezoelectricity, which was found in certain crystals. When a pressure is exerted to the bone surface, it can generate electrical signal. As a result, interactions of electricity with bone biological processes becomes the subject of increasing investigational activities. In 1980, American FDA approved the first PEMF device for the very narrow application of healing fractures that had not healed for at least four months. And in 1989, Bassett predicted bravely that PEMF would play an important role in the therapy of osteoporosis. Indeed, the effects of PEMF on primary osteoporosis have been demonstrated by many researchers, but relatively little is known about its effects on disuse osteoporosis and the mechanisms involved.BMD measurements have provided the basis for making the diagnosis of osteoporosis, predicting the risk of osteoporotic fracture and evaluating the therapeutic effects of drugs. BMD is highly correlated with bone strength and it is generally assumed that changes in BMD affect bone strength. It is well known that various local factors influence bone remodeling, including bone resorption induced by osteoclast and bone formation induced by osteoblast. Meanwhile, local factors themselves can reflect the level of bone remoding. Specifically, interleukin-6 (IL-6) enhances bone resorption through stimulating the development and activities of osteoclasts. The first action of IL-6 is to stimulate osteoblastic production of downstream effectors that activate osteoclasts, among which (receptor activator of NF-kappa B ligand) RANKL is concerned. On the other hand, transforming growth factor-beta 1 (TGF-β1) is also a potent stimulator of bone formation. TGF-β1 can promote the cell proliferation and differentiation of osteoblasts.In the present study, we decided to observe the preventive effects of PEMF on disuse osteoporosis assayed with cancellous BMD, and investigate the underlying mechanism through the observation of serum IL-6 and TGF-β1 concentrations quantified with enzyme linked immunosorbent assay (ELISA), so as to provide theoretical basis for the clinical application of PEMF.Methods1. Eighty 4-month old female Sprague Dawley (SD) rats were divided into four groups by randomized block design according weight: intact (INT) group, disuse osteo- porosis (DOP) group, calcitonin-treated (CT) group, and pulsed electromagnetic fields (PEMF) stimulation group. The right hindlimbs of all the rats were immobilized by tibia-tail fixation except those of the rats in INT group. Rats in CT group were injected with calcitonin (2 IU/kg, ip, once a day) and rats in the PEMF group were subjected to PEMF exposure for 2 hours after the surgery immediately. Five rats of each group were killed and the experimental samples were collected 1, 2, 4, and 8 weeks after treatment.2. The proximal ends of the right tibiae were stained with hematoxylin and eosin (HE). Histological observation was performed under an Olympus microscope.3. The BMD of the proximal end of the femur was measured with dual-energy X-ray absorptiometry (small animal software).4. Rat blood was collected by cardiac puncture. The serum IL-6 and TGF-β1 concentrations were quantified with ELISA.Results1. All experimental rats survived well, without occurrence of accidental death or infection. The body weight of the INT group increased more than that of the immobilized rats, but there was no significant difference (P >0.05).2. At the first week, the bone trabecula of the proximal half of right tibiae in all groups was all arranged densely. Two weeks after surgical procedure, the trabecula was rare and broke off, even vanished at some regions in the DOP group. However, it was more tight and well-arranged in the CT group. From 4 weeks after surgical procedure, the trabecula in the PEMF rats became compact compared with that of the DOP rats. Eight weeks after surgical procedure, the trabecula in DOP group rats became much rare, and disappeared in large range. Meanwhile, there was a significant increase in the density of trabecula of PEMF and CT groups compared to DOP group, and the trabecula of PEMF group is more dense than CT group.3. There was no significant difference of BMD values of the proximal end of femur between groups one week after surgical procedure (P >0.05). The BMD of the rats in the DOP group was reduced obviously (T=-2.54, P <0.01) compared with the INT and CT groups 2 weeks after surgical procedure. The BMDs in the CT and PEMF groups were considerably higher (P < 0.05) than that of the DOP group from 4 weeks after treatment. After 8 weeks of respective intervention, the BMD of DOP group rats significantly decreased constrast to INT group(T=-2.73, P <0.01), and the BMD in the PEMF group became higher significantly (P <0.05) than that of the CT group.4. Immobilization of right hindlimbs could significantly promote IL-6 production throughout the experimental period after surgery, and the IL-6 concentration maintained at a high level from 2 weeks. One week after surgery, the IL-6 concentration in the CT group was obviously lower than that of the DOP group and PEMF group (P <0.05). From 2 weeks after surgery, PEMF stimulation and calcitonin treatment all significantly decreased (P <0.05) IL-6 concentration constrast to DOP group. After 8 weeks, IL-6 in the CT group was lower than that of PEMF group (P <0.05) and INT group. However, there was no remarkable difference between the CT group and INT group (P >0.05).5. A biphasic change of TGF-β1 after surgical procedure was observed. It raised significantly (P <0.05) than normal level at the first week, then reduced gradually, and obviously lower from 4 weeks after surgical procedure (P <0.05). There was no significant difference of TGF-β1 concentration between DOP, CT and PEMF groups at 1, 2, 4 weeks. The TGF-β1 concentration in the PEMF group was much higher than those of the CT and DOP groups 8 weeks after surgical procedure (P <0.01).Conclusion1. Modified right hindlimb immobilization is a successful and convenient new way to make animal model of disuse osteoporosis in rats.2. The expression of IL-6 shows a continuous high level after surgery procedure, and the serum concentration of the TGF-β1 in DOP rats experiences biphasic changes. Bone loss due to disuse is primarily result from elevated bone resorption, the bone formation level gradually decreased after increasing in initial stage of immobilization.3. The present study demonstrated that both PEMF and calcitonin may be useful in the preventive therapy of disuse osteoporosis. Calcitonin takes effect quickly, but the effects of PEMF are more obvious at later stage.4. PEMF could affect many bone cells, besides inhibiting osteoclasts to suppress bone resorption through decreasing IL-6 expression, it also stimulates osteoblastic progenitor cell to secrete TGF-β1 and enhances the bone formation induced by osteoblasts. |
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