Effect of extremely low frequency magnetic field in prevention of spinal cord injury-induced osteoporosis.
Spinal cord injury (SCI)-induced osteoporosis is reported as the most rapid and severe form of osteoporosis . Post-SCI, bone loss has been detected only in the distal metaphysis of the femur on day 10, while bone loss has been detected at 3 wk in the metaphysis, epiphysis, and diaphysis of both the femur and tibia of rats [2-3]. A decrease in dry and ash weight are reported as early as 3 wk, with wet weight decreased at 6 wk [3-4]. Deterioration in bone mass and microarchitecture have been detected at 3 wk , and increase in water content has been detected at 6 mo . A recent report attributes bone mass deterioration to dysfunction of osteoclast in addition to suppression of osteoblast activity in the development of post-SCI osteoporosis  as reflected by their respective markers. The bone formation markers in the serum include osteocalcin (OC), alkaline phosphatase (ALP), and procollagen, while bone resorption markers include carboxyterminal telopeptide of type I collagen and in urine as hydroxyproline, collagen type I telopeptide, etc. ALP is produced by osteoblasts together with the collagenous bone matrix and extruded into the extracellular matrix, where the enzyme is present prior to mineralization. Ambiguity regarding ALP's activity remains in patients with SCI [5-6], while in rats no change in ALP activity is reported after 8 to 12 d of stromal cell culture from post-SCI (3 wk) tissue  and in serum at >6 mo . Serum OC, a small hydroxyapatite-binding protein, increases in urine and serum 3 mo post-SCI in patients [7-8] and 3 wk in rats [3,9], thereby indicating a predominance of osteoblast activity. Serum concentration of carboxy-terminal propeptide of type I collagen and amino-terminal propeptides of type I procollagen is an objective measure of newly formed type I collagen. Substantial increases in the serum type I collagen C-telopeptide at 3 mo and urinary hydroxyproline/creatinine ratios at 1 mo are reported in patients with SCI [8,10], while serum N-terminal telopeptide of type I collagen are increased at 3 wk to 6 mo in rats with SCI [4,11-12]. Intensive exercise or standing regimens may partially prevent bone loss in the femoral shaft but not at the proximal hip, while functional electrical stimulation [7,13-15] and pharmacological agents (etidronate, alendronate) are reported to be ineffective in patients with SCI [16-17], probably because of an exorbitant sublesional bone turnover and osteoclastic activity at all the resorption pits .
Pulsed electromagnetic field (PEMF) therapy is reported to promote peripheral nerve regeneration, osteogenesis, and probably recovery from SCI because of increases in nicotinamide adenine dinucleotide (NAD)-specific isocitrate dehydrogenase activity, acetyl cholinesterase at the motor plate, sparing of white matter, number of motor neurons reestablishing connections , maturation of bone trabecula, bone volume, bone formation, and decrease in lesion volume [19-21]. PEMF induces the differentiation of cartilage cells and enhances ALP activity in rat osteoblasts . The beneficial effect of PEMF on other models of osteoporosis, namely disuse osteoporosis (15 Hz, 8 ms for 2h/d x 8 wk)  and ovariectomy-induced osteoporosis (1-2 mV/cm, 0.3 ms, 7.5 Hz for 8h/d x 30 wk)  has been reported in avians and rats, respectively. A recent report suggests intensive electrical stimulation for 1 h/d for 6 wk can reduce femoral bone mineral density (BMD) loss induced by acute SCI  and benefits of chronic extremely low frequency magnetic field (ELF-MF; 50 Hz, 1.5 [micro]T, 4 h/d x 6 mo) in ovariectomy-induced osteoporosis in rats .
However, to the best of our knowledge, there is no report regarding ELF-MF in SCI-induced osteoporosis. Therefore, we report the efficacy of chronic ELF-MF (50 Hz, 17.96 [micro]T, 2 h/d x 8 wk) on bone mineral content (BMC; total calcium [Ca], phosphorus [P], and carbon [C]), BMD, and biochemical status (OC, collagen I, ALP) of SCI-induced osteoporosis in adult male rats.
Adult male Wistar rats (body weight 230-250 g) were individually housed in a room at 24 [+ or -] 2[degrees]C on a 14:10 h light:dark cycle and were provided with standard laboratory food pellets and fresh tap water ad libitum daily. Rats (n = 24) were equally divided into sham group, SCI group, and magnetic field (MF)-exposed SCI rats (SCI+MF group).
Extremely Low Frequency Magnetic Field Exposure Chamber
The MF stimulator used has electromagnetic coils mounted on a stand, a movable platform for the rat cage, and a current regulator that maintains constant current through the coils . The coils (2 outer coils, each with 18 turns; 2 inner coils, each with 8 turns) are connected in series and provide a uniform ELF-MF (17.96 [micro]T, 50 Hz) of the modified Helmholtz coil in the central area where the eight rats were kept separately in a specially designed polypropylene cage (Figure 1).
Spinal Cord Injury Method
Under deep anesthesia (ketamine + xylazine; 60 +10 mg/kg body weight, intramuscular), the dorsum of the animals were shaved and a longitudinal midline incision was made through the skin. A selective laminectomy (thoracic [T]10-T12 vertebrae) was done to only expose the spinal cord (sham group). After the laminectomy, complete spinal cord transection (T11 vertebra corresponding to T13 spinal cord) was done with microscissors (SCI group) under magnifying lenses. Both stumps of the spinal cord were gently lifted away to create a 1 to 2 mm gap, which was filled with sponge gel. The muscle fascia and skin were sutured, and the rats were returned to their home cages. After surgery, rats received a bolus of Lactate Ringers solution (5 mL, intraperitoneal) to compensate for blood loss, and antibiotic cover (systemic, gentamycin 50 mg/kg body weight, intramuscular; local neosporin ointment) was provided. Manual evacuation of the bladder was regularly done 3 times/day until reflex micturition was restored.
Assessment of Locomotor Functions
The quality of locomotion was assessed by Basso, Beattie, and Bresnahan (BBB) locomotor rating score .
Determination of Volumetric Bone Mineral Content and Bone Mineral Density
The volume of fresh bones was determined by submersion method. After the bone was freeze-dried, dry weight was determined and percent of water was calculated. The bone was ground and kept (50 mg) in muffle furnace (700[degrees]C) for 8 h to obtain bone ash, which was weigthed (BMC/50 mg), while BMD (g/[cm.sup.3]) was calculated from the ratio BMC/bone volume .
Determination of Element Content (Ca, P, C)
Total Ca content was determined by atomic absorption method as described elsewhere . Briefly, the lyophilized bone powder (50 mg) was digested in aqua regia, diluted with distilled water, and combined with lanthanum chloride (0.5%). The concentration of Ca was calculated from the standard curve of Ca.
P content was determined using the Vanado-Molybdo-phosphoric acid colorimetric method. Lyophilized bone powder (50 mg) was digested in a mixture of [H.sub.2]S[O.sub.4] and HN[O.sub.3], heated to remove HN[O.sub.3], neutralized with double-distilled water, and combined with a Vanadate-molybdate reagent.
Total C content in lyophilized bone sample (50 mg) kept in a sterile ceramic boat was determined by a C analyzer (CS 500, ELTRA; Haan, Germany) at 1,200[degrees]C.
A bone sample (50 mg) was digested with acetic acid (2 mL, 0.5 M, pH 2.0) for 48 h . Soluble collagen was separated by centrifugation; supernatant was transferred to a microcentrifuge tube, and the residue was digested with pepsin (1 mg/10 mg bone powder) in acetic acid for 48 h. The pepsin-soluble collagen was separated by centrifugation, whereas the pepsin-insoluble bone matrix was extracted by guanidine hydrochloride (4.0 M) in Tris-HCl (0.05 M, pH 7.5) for 48 h and separated by centrifugation. The supernatants were pooled for salt precipitation (NaCl, 2.6 M, pH 7.4) and dissolved in acetic acid containing sirius red (1%), and stained collagen was separated by centrifugation (15,000 g, 45 min). The residue was dissolved in acetic acid and the collagen I concentrations were measured in a microplate reader using collagen type I standard (C3867, Sigma-Aldrich; St. Louis, Missouri).
Bone powder was decalcified in ethylenediamine tetra-acetic acid, (10% weight per volume, pH 7.2) and extracted by stirring at 4[degrees]C for 48 h. OC in the supernatant was measured by using a rat sandwich enzyme-linked immunosorbent assay kit (BPB Biomedical; Stroughton, Massachusetts).
Alkaline Phosphatase Activity
The ALP activity in lyophilized bone powder was determined with the plasma ALP kit. Briefly, the bone powder was sonicated in MgCl2, 10 mM and Triton X-100, 0.1 percent. ALP activity was determined colorimetrically using p-nitrophenylphosphate substrate. The total protein content was measured using a protein assay kit (Bio-Rad Laboratories; Hercules, California) utilizing bovine serum albumin as the protein standard.
Scanning Electron Microscopy
The proximal diaphysis sections from the femur and tibia and the acetabulum of the femur were cut for scanning electron microscope (SEM) study in every rat. The bone samples were dried and mounted on circular disc stubs with adhesive. Gold coatings were applied at a thickness of about 20 nm with the help of a sputter coater. SEM images were obtained on low vacuum SEM Leo 435 VP (Cambridge, England) at the National Facilities of Electron Microcopy, All India Institute of Medical Sciences, New Delhi. The cortical area, interpolated polygon area, and perimeter of porosity was obtained utilizing Olympus imaging software (Center Valley, Pennsylvania.
The rats of the ELF-MF group received MF exposure consecutively for 2 h/d x 8 wk (10:00 am-12:00 pm) from post-SCI day 1, while those of sham and SCI groups were similarly treated but without MF exposure. BBB score was noted weekly pre- and post-SCI for 8 wk. Rats were sacrificed by decapitation under deep ether anesthesia. Tibia and femur bones were freed from soft tissues on both the sides and stored at -20[degrees]C until analysis.
Data are presented as mean [+ or -] standard deviation and compared between groups using one-way analysis of variance followed by post hoc analysis by Bonferroni test. P-values less than 0.05 were accepted as significant.
Histology confirmed complete transection of the spinal cord in the SCI group and partial restoration in the SCI+MF group at 8 wk post-SCI (Figure 2).
Basso, Beattie, and Bresnahan Score
BBB score decreased (p < 0.001) in the SCI versus sham groups at all time-points, which was restored to the baseline (p < 0.001) post-SCI week 2 onwards in the SCI+MF group. However, the BBB score was significantly lower in the SCI+MF group versus sham group after SCI and before MF exposure (Figure 3).
Bone Water Content, Bone Mineral Content, and Bone Mineral Density
Water content of both bones was higher (p < 0.001), while BMC and BMD were lower post-SCI versus sham group. As compared with SCI group, the SCI+MF group had less water content and higher BMC and BMD, excluding BMC in the femur. There was no statistically significant difference in the bones of the SCI+MF group versus sham group (Table 1 ).
Total Calcium, Phosphorus, and Carbon Content
In both the tibia and femur bones, Ca, P, and C contents significantly decreased post-SCI versus sham group, all of which recovered after MF except for P and C in the femur and tibia, respectively. There was no statistically significant difference in the SCI+MF group versus sham group except for C content in both the bones (Table 2).
Biochemical Status (Collagen I, Osteocalcin, and Alkaline Phosphatase)
Post-SCI, the levels of collagen I, OC, and ALP decreased (p < 0.001) in both the bones versus sham group, all of which were recovered by MF, except collagen I in the femur. In the SCI group versus the SCI+MF group, the biochemical levels were higher in both the bones, except collagen I in tibia (Table 3).
Scanning Electron Microscope of Bones
SEM images in the transverse section of bone samples show the microstructural changes in bones (Figures 4-6). The femoral neck part and acetabular head showed more compactness and less porosity in SCI+MF versus SCI rat groups (Figure 4). The trabecular bone, the frets (intertrabeculae) of proximal epiphysis in SCI rat, were absorbed and reformed after SCI+MF; however, connectivity was less than control (Figure 5 and Table 4). Cortical thickness was significantly more (Table 5) and bone marrow was attached to the cortex in the SCI+MF versus SCI rat groups (Figure 6). The SEM study indicates lesser mineral deposition and greater porosity in SCI bones, while mineral deposition was recovered in the SCI+MF group.
The present study was proposed to investigate the efficacy of ELF-MF in an SCI model of osteoporosis. SCI produced a significant decrease in BBB score, BMC, BMD, Ca, P, C, and biochemical parameters (collagen I, OC, and ALP) of weight-bearing bones (femur, tibia) as compared with a sham group of rats. Exposure (2 h/d x 8wk) to ELF-MF (17.96 [micro]T, 50 Hz) restored these parameters, except C content in both the bones and partial recovery in BBB score. Therefore, our results indicate that exposure to ELF-MF has a therapeutic potential in the management of SCI-induced osteoporosis.
Complete SCI at the lower thoracic cord of rats is widely used to study pathophysiology of SCI-induced osteoporosis [3-4,6-7]. It produces the most rapid and severe forms of osteoporosis, resistant even to exercise. Several factors, including unloading, neural lesion, and endocrine disturbances, contribute toward osteoporosis, although the precise mechanism is not known . Mechanical stimulation--which is sensed by mechanosensory cells (osteocytes), transmitted via intracellular and extracellular signals to induce bone formation by osteoblasts--during weight bearing is crucial for bone remodeling. SCI contributes toward osteoporosis directly by denervation of bone or indirectly by disrupting vasoregulation. Hormones also participate in post-SCI osteoporosis, possibly via negative Ca2+ balance caused by increased excretion.
Nonetheless, it is critical to precisely assess and monitor the progression of osteoporosis to limit the incidence of fracture. BMD and BMC are currently the gold standard for objective assessment of osteoporosis and are the best surrogate of fracture risk in vivo of 50 to 80 percent variance in bone strength . The methods used for in situ clinical assessment of osteoporosis are micro-computed tomography and dual-energy X-ray absorptiometry (DXA), while for in vitro analysis only ash weight is used. DXA-derived BMC values are on average higher than the ash weight and may lead to a dangerous underestimation of fracture risk . Lochmuller et al. have suggested that the ash weight, Ca, and P content provide a better estimate of the femur fracture risk than in situ DXA-derived BMC . Since the aim of this study was to explore the efficacy of ELF-MF in the prevention of SCI-induced osteoporosis, we chose to determine BMC and BMD from the bone ash, which has the advantage of providing direct sensitive evidence of osteoporosis.
However, osteoporosis is established in patients with SCI on the basis of the biochemical markers reflecting either an increase in osteoclastic or a decrease in osteoblastic activity. Formation, maturation, and destruction products of bone collagen I in serum and urine indicate either osteoblastic or osteoclastic activity. Collagen constitutes about 90 percent of the structural protein of the bone matrix and is secreted by osteoblasts, which are stiffened by integration of the mineral phase . In our study, the water content in the femur and tibia were increased after SCI, which indicates demineralization of these bones. We further support the loss of bone by their microarchitecture.
Post-SCI 3 wk to 6 mo, the serum level of bone collagen I telopeptide was reported to be higher, indicating predominant osteoclastic activity [3-4]. In consonance with our objective and the limited value of direct translation of circulating/urinary markers into the magnitude of bone gain and resorption, it was pertinent to determine the status of bone activity in the bone itself. In our rats, the concentration of collagen I was significantly reduced in both the sublesional bones, thereby supporting enhanced osteoclastic activity.
A higher serum OC concentration in patients with SCI (immediately, 3-6 mo) and rats (3-4 wk) has been reported [3,7-8,10]. OC serum concentration correlates with static and dynamic parameters of bone formation, but any correlation with matrix synthesis or mineralization is not evidenced. Therefore, we estimated OC concentration directly in the bones to support the possible efficacy of ELF-MF in the management of SCI-induced osteoporosis. Our study showed that SCI caused a significant decrease in OC concentration in the bones, thereby signifying a probable decrease in bone matrix synthesis in our rats.
Osteoblasts express robust ALP activity, which is anchored to the external surface of the plasma membrane and contributes to bone mineralization . ALP is considered to be an early differentiation marker of osteoblast maturation, while collagen and OC formation represent the end of differentiation. Therefore, ALP in matrix and osteoblast is a good indicator of bone formation and matrix mineralization . On the contrary, no effect on serum bone ALP post-SCI and total ALP activity in culture exist in the literature [6,10]. This discrepancy is explainable since the serum pool consists of several dimeric isoforms, which originate from various tissues besides bones . Our study provides evidence of variation in ALP activity of the bone per se, wherein SCI led to a significant decrease in ALP level in weight-bearing bones, reflecting a low osteoblastic activity.
It is clear from our study that ELF-MF exposure favored osteogenesis, although the specific mechanism is not precisely understood. Nonetheless, the literature suggests a positive effect of PEMF on bone formation in delayed-union bone fractures, failed joint fusions, congenital pseudoarthroses, neurectomy, and ovariectomy-induced osteoporosis [20,37-40]. These processes also lead to an alteration in the expression of genes leading to osteoblast proliferation. Osteoblasts proliferate and differentiate by modification of ion channel activity and enhance the synthesis of matrix formation by PEMF in other models of osteoporosis [41-42]. ALP expression is considered a good early indicator of osteoblast phenotype. In our SCI+MF rats, local concentration of ALP in bone was higher, indicating predominance of osteoblastic activity. ELF-MF has been suggested to promote the formation of matrix protein, namely collagen, OC, and ALP in bone cell cultures [43-45]. In our model of SCI-induced osteoporosis, ELF-MF also increased the concentration of collagen I, OC, and ALP, indicating the formation of matrix protein in the bones, wherein collagen I specifically provides a site for mineral precipitation and OC and ALP for its mineralization . An increased mineralization in sublesional bones in our ELF-MF exposed rats with SCI is further supported by their increase in BMC and BMD, with a concomitant decrease in water content and microarchitecture. Nonetheless, for a greater magnitude of effect, exposure sessions may be further increased.
Besides promoting in bone formation, MF stimulation possibly improves bone status by restricting the resorption process. Cytokines required for osteoclast formation--namely, receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor--bind to the transmembrane receptor of osteoclast to promote its differentiation and induce osteoclast proliferation, respectively. RANKL also binds to osteoprotegerin (OPG), a decoy receptor, which reduces resorption. PEMF is reported to increase OPG concentration, decrease RANKL concentration, and increase osteoclast apoptosis [47-48], and therefore it is probable that ELF-MF not only promotes bone formation but also limits bone resorption.
ELF-MF exposure in our rats could have possibly supported recovery by resumption of weight bearing as revealed by BBB score. Unloading is one of the important causes in the genesis of osteoporosis induced by SCI [18-19,49-52].
The present study suggests that SCI-induced osteoporosis in our rats could be limited by chronic (2 h/d x 8 wk) exposure to ELF-MF (17.96 [micro]T, 50 Hz) as revealed by BBB score, BMC, BMD, mineral element contents, and biochemical parameters pertaining to the sublesional bones.
JRRD at a Glance
Spinal cord injury (SCI)-induced osteoporosis is reported as one of the most rapid and severe forms of osteoporosis. Electromagnetic stimulation has shown to be effective in aiding bone healing in a variety of orthopedic conditions and provides a base of benefits for osteoporosis therapy. We report the efficacy of extremely low frequency magnetic field (ELF-MF) on SCI-induced bone loss. Our results suggest that chronic (2 h/d x 8 wk) exposure to ELF-MF is effective in attenuating SCI-induced osteoporosis.
Abbreviations: ALP = alkaline phosphatase; BBB = Basso, Beattie, and Bresnahan; BMC = bone mineral content; BMD = bone mineral density; C = carbon; Ca = calcium; DXA = dual-energy X-ray absorptiometry; ELF-MF = extremely low frequency magnetic field; MF = magnetic field; OC = osteocalcin; OPG = osteroprotegerin; P = phosphorus; PEMF = pulsed electromagnetic field; RANKL = receptor activator of nuclear factor kappa-B ligand; SCI = spinal cord injury; SEM = scanning electron microscope; T = thoracic.
Study concept and design: J. Manjhi, R. Mathur, J. Behari.
Acquisition of data: J. Manjhi, S. Kumar.
Analysis and interpretation of data: J. Manjhi, S. Kumar.
Drafting of manuscript: J. Manjhi, S. Kumar.
Critical revision of manuscript for important intellectual content: R. Mathur.
Obtained funding: R. Mathur, J. Behari.
Study supervision: R. Mathur, J. Behari.
Financial Disclosures: The authors have declared that no competing interests exist.
Funding/Support: This material is based on work supported by the Department of Science and Technology, New Delhi, India.
Institutional Review: All protocols for animal procedure were reviewed and approved by the Institutional Animal Ethics Committee of All India Institute of Medical Sciences, New Delhi, India, and followed by the Committee for the Purpose of Control and Supervision on Experiments on Animals, India.
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Submitted for publication January 2, 2012. Accepted in revised form May 17, 2012.
This article and any supplementary material should be cited as follows:
Manjhi J, Kumar S, Behari J, Mathur R. Effect of extremely low frequency magnetic field in prevention of spinal cord injury-induced osteoporosis. J Rehabil Res Dev. 2013;50(1):17-30. http://dx.doi.org/10.1682/JRRD.2011.12.0248
Jayanand Manjhi, PhD; (1) Suneel Kumar, MSc; (1) Jitendra Behari, PhD; (2) Rashmi Mathur, PhD (1) *
(1) Department of Physiology, All India Institute of Medical Sciences, New Delhi, India; (2) School of Environmental Sciences, Jawaharlal Nehru University (JNU), New Delhi, India
* Address all correspondence to Rashmi Mathur, PhD; Professor, Department of Physiology, All India Institute of Medical Sciences, New Delhi-110029, India; +91-11-26594243; fax: +91-11-26588641, 26588663.
Table 1. Effect of spinal cord injury (SCI) and magnetic field (MF) exposure on water content, bone mineral content (BMC), and bone mineral density (BMD) in femur and tibia. Parameter Bone Group Sham Water Content Femur 21.90 [+ or -] 5.72 (% of wet weight) Tibia 16.42 [+ or -] 3.31 BMC (mg) Femur 407.83 [+ or -] 16.4 Tibia 276.97 [+ or -] 16.9 BMD (mg/mL) Femur 1,019.99 [+ or -] 112.2 Tibia 780.12 [+ or -] 56.85 Parameter Bone Group SCI Water Content Femur 55.64 [+ or -] 4.6 (% of wet weight) Tibia 34.94 [+ or -] 8.8 BMC (mg) Femur 328.51 [+ or -] 51.45 Tibia 211.60 [+ or -] 30.67 BMD (mg/mL) Femur 707.11 [+ or -] 102.8 Tibia 552.38 [+ or -] 80.95 Parameter Bone Group SCI+MF Water Content Femur 32.55 [+ or -] 5.92 (% of wet weight) Tibia 23.14 [+ or -] 10.96 BMC (mg) Femur 366.38 [+ or -] 60.34 Tibia 268.59 [+ or -] 14.24 BMD (mg/mL) Femur 971.47 [+ or -] 308.9 Tibia 763.06 [+ or -] 65.57 Parameter Bone p-Value Sham Sham vs SCI vs vs SCI SCI+MF SCI+MF Water Content Femur <0.001 0.75 0.03 (% of wet weight) Tibia <0.001 0.2 0.001 BMC (mg) Femur 0.01 0.27 0.36 Tibia <0.001 >0.99 <0.001 BMD (mg/mL) Femur 0.02 >0.99 0.04 Tibia <0.001 >0.99 <0.001 Note: p-value of difference between groups of animals (sham vs SCI, sham vs SCI+MF, SCI vs SCI+MF) was calculated using one-way analysis of variance. Table 2. Effect of spinal cord injury (SCI) and magnetic field (MF) exposure on total calcium (Ca), phosphorus (P), and carbon (C) contents of femur and tibia. Parameter Bone Group Sham SCI Ca (mg) Femur 254.65 [+ or -] 14.64 211.42 [+ or -] 7.56 Tibia 231.58 [+ or -] 7.60 193.06 [+ or -] 8.20 P (mg) Femur 133.25 [+ or -] 12.98 117.40 [+ or -] 7.48 Tibia 115.96 [+ or -] 8.84 101.59 [+ or -] 8.23 C (% of Femur 183.91 [+ or -] 6.44 149.65 [+ or -] 1.17 dry Tibia 178.99 [+ or -] 2.98 147.74 [+ or -] 8.69 weight) Parameter Bone Group p-Value SCI+MF Sham Sham vs SCI vs vs SCI SCI+MF SCI+MF Ca (mg) Femur 244.16 [+ or -] 6.78 <0.001 0.16 <0.001 Tibia 219.84 [+ or -] 15.25 <0.001 0.13 <0.001 P (mg) Femur 130.67 [+ or -] 10.75 0.02 >0.99 0.06 Tibia 113.29 [+ or -] 6.13 0.004 >0.99 0.02 C (% of Femur 174.37 [+ or -] 5.34 <0.001 0.02 <0.001 dry Tibia 154.81 [+ or -] 4.66 <0.001 <0.001 0.08 weight) Note: p-value of difference between groups of animals (sham vs SCI, sham vs SCI+MF, SCI vs SCI+MF) was calculated using one-way analysis of variance. Table 3. Effect of spinal cord injury (SCI) and magnetic field (MF) exposure on total collagen, osteocalcin, and alkaline phosphatase (ALP) activity of femur and tibia. Parameter Bone Group Sham SCI Collagen I Femur 296.59 [+ or -] 34.80 213.33 [+ or -] 22.73 (mg/g) Tibia 274.83 [+ or -] 28.62 221.07 [+ or -] 36.99 Osteocalcin Femur 790.14 [+ or -] 76.71 459.74 [+ or -] 52.99 ([micro]/g) Tibia 850.37 [+ or -] 54.49 530.85 [+ or -] 51.26 ALP (IU/g) Femur 134.68 [+ or -] 8.84 93.10 [+ or -] 11.57 Tibia 124.53 [+ or -] 7.32 106.17 [+ or -] 3.80 Parameter Bone Group SCI+MF Collagen I Femur 255.17 [+ or -] 33.83 (mg/g) Tibia 254.31 [+ or -] 34.51 Osteocalcin Femur 705.06 [+ or -] 84.21 ([micro]/g) Tibia 785.28 [+ or -] 73.35 ALP (IU/g) Femur 121.28 [+ or -] 11.50 Tibia 115.67 [+ or -] 9.13 Parameter Bone p-Value Sham Sham vs SCI vs vs SCI SCI+MF SCI+MF Collagen I Femur <0.001 0.04 0.04 (mg/g) Tibia 0.01 0.7 0.18 Osteocalcin Femur <0.001 0.08 <0.001 ([micro]/g) Tibia <0.001 0.13 <0.001 ALP (IU/g) Femur <0.001 0.06 <0.001 Tibia <0.001 0.06 0.04 Note: p-value of difference between groups of animals (sham vs SCI, sham vs SCI+MF, SCI vs SCI+MF) was calculated using one-way analysis of variance. Table 4. Porosity quantification in different parts of proximal femur and tibia bones of scanning electron microscope micrograph. Sham Group Bone Area Interpolated Interpolated Polygon Area Polygon Perimeter ([mm.sup.2]) (mm) Trabecular Part of 155.59 [+ or -] 14.54 7.09 [+ or -] 0.35 Proximal Femur Acetabullar Head 90.58 [+ or -] 5.75 5.66 [+ or -] 0.19 of Femur Trabecular Part of 118.03 [+ or -] 6.15 7.03 [+ or -] 0.15 Proximal Tibia Proximal Part of 6,176.30 [+ or -] 23.22 9.47 [+ or -] 0.25 Tibia SCI Group Bone Area Interpolated Interpolated Polygon Area Polygon Perimeter ([mm.sup.2]) (mm) Trabecular Part of 530.47 [+ or -] 32.30 * 16.41 [+ or -] 0.99 * Proximal Femur ([dagger]) ([dagger]) Acetabullar Head 163.43 [+ or -] 4.14 * 10.44 [+ or -] 0.41 * of Femur ([section]) ([paragraph]) Trabecular Part of 210.24 [+ or -] 10.86 * 8.25 [+ or -] 0.16 * Proximal Tibia ([paragraph]) ([paragraph]) Proximal Part of 1,064.79 [+ or -] 147.17 * 11.96 [+ or -] 0.12 * Tibia ([dagger]) ([paragraph]) SCI+MF Group Bone Area Interpolated Interpolated Polygon Area Polygon Perimeter ([mm.sup.2]) (mm) Trabecular Part of 306.91 [+ or -] 16.51 12.03 [+ or -] 0.48 * Proximal Femur ([dagger])([section]) ([dagger]), ([double dagger]) ([paragraph]) Acetabullar Head 152.12 [+ or -] 4.36 7.83 [+ or -] 0.21 of Femur ([double dagger]) ([double dagger]) ([section]) ([paragraph]) Trabecular Part of 19.81 [+ or -] 1.47 8.71 [+ or -] 0.04 Proximal Tibia ([dagger]) ([dagger]) ([double dagger]) ([double dagger]) Proximal Part of 476.92 [+ or -] 101.91 * 9.80 [+ or -] 1.60 * Tibia ([dagger]) ([dagger]) * Comparison with sham group. ([dagger]) p < 0.05. ([double dagger]) Comparison with SCI group. ([section]) p < 0.001. ([paragraph]) < 0.01. MF = magnetic field, SCI = spinal cord injury. Table 5. Cortical area quantification ([mm.sup.2]) in femur and tibia cortex scanning electron microscope micrograph in different animal groups. Bone Sham Group SCI Group Cortical Bone 4,746.25 [+ or -] 829.45 972.65 [+ or -] 53.99 * in Femur ([dagger]) Cortical Bone 6,597.36 [+ or -] 403.25 3,760.44 [+ or -] 297.38 * in Tibia ([section]) Bone SCI+MF Group Cortical Bone 3,819.10 [+ or -] 172.38 ([double dagger])([section]) in Femur Cortical Bone 4,858.77 [+ or -] 438.55 ([double dagger])([section]) in Tibia * Comparison with sham group. ([dagger]) p < 0.001. ([double dagger]) Comparison with SCI group. ([section]) p < 0.01. MF = magnetic field, SCI = spinal cord injury.
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|Author:||Manjhi, Jayanand; Kumar, Suneel; Behari, Jitendra; Mathur, Rashmi|
|Publication:||Journal of Rehabilitation Research & Development|
|Date:||Jan 1, 2013|
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