Osteoporos Int (2007) 18:743–750 DOI 10.1007/s00198-006-0299-3
ORIGINAL ARTICLE
Differences of bone mass and bone structure in osteopenic rat models caused by spinal cord injury and ovariectomy S.-D. Jiang & C. Shen & L.-S. Jiang & L.-Y. Dai
Received: 23 July 2006 / Accepted: 22 November 2006 / Published online: 11 January 2007 # International Osteoporosis Foundation and National Osteoporosis Foundation 2007
Abstract Summary Both spinal cord injury and ovariectomy can result in ostepenia in rats. SCI induces more deterioration of cortical geometric structure and trabecular microstructure in the proximal tibial metaphysis than OVX. The proximal tibial metaphysis microstructure significantly correlates with its biomechanical properties. Introduction The purpose of the present study was to compare the effects of spinal cord injury (SCI) and ovariectomy (OVX) on bone gain in young female rats. Methods Thirty young female Sprague-Dawley rats were randomized into three groups: age-matched intact control (CON), OVX and SCI. The tibiae were assessed for DXA and micro-CT analysis, biomechanical testing, the upper tibial epiphyseal plate height, and blood samples for biochemical analysis. Results SCI rats showed lower aBMD in the proximal tibiae as compared with OVX rats. Cortical geometric structural parameters of the tibial midshaft in SCI rats were significantly lower than OVX rats. SCI or OVX induced significant changes in all trabecular microstructural parameters in the proximal tibial metaphysis. The trabecular separation (Tb.Sp) and structure mode index (SMI) in SCI rats were significantly higher than in OVX rats. BV/TV explained 84% of the variation of ultimate load of the proximal tibial metaphysis. There was no difference of the upper tibial epiphyseal plate height between SCI and OVX rats. Serum NTX level in SCI rats was significantly higher than in OVX rats. S.-D. Jiang : C. Shen : L.-S. Jiang : L.-Y. Dai (*) Department of Orthopaedic Surgery, Xinhua Hospital, 1665 Kongjiang Road, Shanghai 200092, People’s Republic of China e-mail:
[email protected]
Conclusions SCI induces more deterioration of cortical bone geometric structure and trabecular microstructure in the proximal tibial metaphysis than OVX. Keywords Osteoporosis . Ovariectomy . Spinal cord injury
Introduction Both gonadal hormones and innervation play an important role in the maintenance of bone mass. Ovariectomy (OVX) has long been associated with bone loss in rats [1–3]. In young rats OVX leads to increased bone formation and resorption [4, 5]. The importance of estrogen in the pathogenesis of this bone loss has been clearly demonstrated, since osteoporosis may be prevented by estrogen replacement therapy [6, 7]. The existence of a bone remodeling regulatory arm with neuronal characteristics was first suggested from clinical observations reporting patients with head trauma and post-injury increase in osteogenic activity [8]. Recent immunocytochemistry studies have shown a dense innervation of bone, and numerous in vitro and in vivo studies have shown roles for neuromediators in bone cell functions [9–13]. Spinal cord injury (SCI) leads to the denervation of bones and results in great bone loss in rats [14, 15]. The pathogenesis of osteopenia may differ between SCI and OVX, so the different prophylaxis regime may be applied in osteoporosis after SCI. However, treatment interventions of osteoporosis after SCI are most based on the regime applied to postmenopausal women. Thus, there is a need to directly compare the effects of denervation caused by SCI and estrogen deficiency caused by OVX on cortical bone and trabecular bone. Our purpose was to examine the differences in patterns of deterioration with regard to cortical geometry
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and trabecular microstructure as a result of denervation and hormonal alteration.
Osteoporos Int (2007) 18:743–750
inter- and intra-analyzer variations for regional BMDs were less than 2% in our laboratory. Micro-CT analysis
Methods Animal protocols The protocols for animal experimentation described in this article were approved previously by the Animal Research Committee of the University School of Medicine. All subsequent animal experiments adhered to the “Guidelines for Animal Experimentation” of the University. Thirty female Sprague-Dawley rats, at 6 weeks of age, were randomly divided into three groups of ten rats each, treated as follows: age-matched intact control (CON), OVX and SCI. Rats in the SCI and OVX group were anaesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). The lower thoracic cord of rats in the SCI group was exposed by a laminectomy over T10-12 segments, and then the cord was transected. The urinary bladder of SCI rats was emptied manually at least four times a day for the first 1–2 weeks until the bladder emptied automatically and then they were monitored at least twice a day. Rats in the OVX group were bilaterally ovariectomized. The success of the ovariectomy was confirmed by inspecting marked atrophy of the uterus. All the rats were maintained on commercial rat chow available ad libitum with 0.95% calcium and 0.67% phosphate. Rats were housed in a controlled environment at 22°C with a 12-hour light/dark cycle. No rats died during the observation period, and all animals were anesthetized with ketamine/xylazine anesthesia and killed by exsanguination from the abdominal aorta after three weeks of feeding. Blood samples were obtained in the morning for biochemical analysis (osteocalcin and NTX) according to the protocol previously described [15]. The left femora were collected for the measurement of length, and the left tibiae were obtained for the measurement of BMD by dual-energy X-ray absorptiometry (DXA) and structure analysis by micro-CT. The right tibiae were fixed in 10% phosphatebuffered formalin, and demineralized in 10% nitric acid. The proximal half of the tibia was sliced mid-sagittally, dehydrated, embedded in paraffin, and sectioned at 5 μm. All sections were stained with hematoxylin/eosin, and the height of the upper tibial epiphyseal plate was recorded. Absorptiometry BMD of the proximal tibiae was determined using a Hologic QDR Discovery A machine equipped with the software for high-resolution analysis of small-animal images. A region of interest was drawn, and BMD of this region was computed. The percent coefficients (%CVs) of
For a detailed qualitative and quantitative 2-D and 3-D evaluation, whole tibial bones were examined by a desktop micro-CT system. The different scans of each sample were reoriented in three dimensions to match the scans, using image registration techniques [16]. Since the growth plate of the rats in our study did not close, the epiphysis and metaphysis were registered separately to avoid mismatches due to growth by apposition at the growth plate. This method resulted in accurate matches of the consecutive scans [16]. The registered data sets were segmented into binary data sets using a specially developed algorithm based on local thresholds [17]. Each scan yielded an image data set of 20 1024×1024 2-D axial slices through the midshaft region of the tibia. These 2-D images had an element size of 12.5 μm in all three spatial dimensions, and were segmented into bone and marrow regions by applying a visually chosen, fixed threshold for all samples, after smoothing the image with a 3-D gaussian low-pass filter. The outer contour of the bone was found automatically with the built-in Scanco iterative contouring tool. Total area (TA) was calculated by counting all voxels within the contour, cortical bone area (Ct.Ar) by counting all voxels that were segmented as cortical bone, and marrow area (MA) was calculated as TA-Ct.Ar. This calculation was performed on all 20 slices, using the average for the final calculation. The outer and inner perimeter of the cortical midshaft was determined by a 3-D triangulation of the bone surface (BS) of the 20 slices, and cortical thickness was calculated by the formula Ct.Th = 1/2 * BS/BV. The above formula can be explained by: area of a ring=thickness of ring * length of middle line=thickness * (outer circumference+inner circumference)/2. In addition, the average areal moment of inertia (MOI) was calculated in x and y direction as well as the polar moment of inertia (J). An eigen-value analysis of the MOI tensor gave the principal directions and minimal and maximal I values (Imin and Imax). Maximal radial extents in the directions perpendicular to Imax and Imin were calculated (Cmin and Cmax, short for “Cmin for Imax” and “Cmax for Imax”). Section modul, Imax/Cmax and Imin/Cmin, were determined and they were used as determinants of bending strength. The polar moment of inertia (J; mm4) was calculated as Imax + Imin. The moment of inertia calculations were performed in 2-D mode; thus, it was assumed that the bones were aligned with the axis of the scanner. Cortical geometric structure and trabecular microstructure of the proximal tibial metaphysis were also analyzed using micro-CT. The spatial resolution for specimen scanning was also set to 12.5 μm. Trabecular and cortical bone were
Osteoporos Int (2007) 18:743–750
separated by drawing regions of interests, using software provided with the scanner. Three-dimensional trabecular microstructural analysis was performed on the cancellous bone area 1 to 5 mm distal to the growth plate. Bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were calculated as measures of trabecular bone mass and its distribution [18], while trabecular architecture was quantified by calculating structure model index (SMI) and connectivity of the trabecular network (Conn.D) [19, 20]. SMI indicates whether the trabeculae are more rod-like (SMI = 3) or more plate-like (SMI = 0). Conn.D was obtained by calculating the connectivity of the trabecular network and normalized by dividing the connectivity by bone volume (BV/TV). Cortical area and thickness were calculated according to the aforementioned formula. This calculation was performed on all slices, using the average for the final calculation. These measurements were used to confirm the effects of SCI and OVX on cortical bone and cancellous bone. Biomechanical testing Mechanical strength of the proximal tibial metaphysis was evaluated by a compression test using a material testing machine (MZ-500D, Maluto, Tokyo, Japan). The tibiae were cut using an electric saw at a site 10 mm distal to the proximal end, and then it was placed on the test apparatus with the anterior side up [21]. A compression force was applied to the proximal tibial metaphysis at a speed of 1 mm/min until a fracture occurred. Ultimate load was determined from the load-displacement curve by a connected computer. Statistical analysis Comparisons among groups were performed using the ANOVA test. The values of P
