Sclerostin Antibody Reverses Bone Loss by Increasing Bone Formation and Decreasing Bone Resorption in a Rat Model of Male Osteoporosis

Sclerostin Antibody Reverses Bone Loss by Increasing Bone Formation and Decreasing Bone... Abstract Sclerostin antibody (Scl-Ab) restored bone mass and strength in the ovariectomized rat model of postmenopausal osteoporosis. Increased bone mineral density (BMD) and decreased skeletal fragility fracture risk have been reported in postmenopausal osteoporotic women receiving Scl-Ab. In males, loss of androgen leads to rapid decreases in BMD and an increased risk of fragility fractures. We hypothesized that Scl-Ab could reverse the loss of bone mass and strength caused by androgen ablation in the orchiectomized (ORX) rat model of male osteoporosis. We treated 9-month-old ORX Sprague Dawley rats (3 months after ORX) subcutaneously twice weekly with vehicle or Scl-Ab (5 or 25 mg/kg) for 6 weeks (n = 10 per group). Both doses of Scl-Ab fully reversed the BMD deficit in the lumbar spine and femur and tibia in ORX rats. Microcomputed tomography showed that the bone mass in the fifth lumbar vertebral body, femur diaphysis, and femoral neck were dose-dependently restored by Scl-Ab. The bone strength at these sites increased significantly with Scl-Ab to levels matching those of sham-operated controls and correlated positively with improvements in bone mineral content, demonstrating bone quality maintenance. Dynamic histomorphometry of the tibial diaphysis and second lumbar vertebral body demonstrated that Scl-Ab significantly increased bone formation on periosteal, endocortical, and trabecular surfaces and significantly decreased bone resorption on endocortical and trabecular surfaces. The effects of Scl-Ab on increasing bone formation and decreasing bone resorption led to restoration of bone mass and strength in androgen-deficient rats. These findings support the ongoing evaluation of Scl-Ab as a potential therapeutic agent for osteoporosis in men. Osteoporosis in men is a major public health problem, with approximately one-third of all osteoporotic fractures occurring in men (1–3). As populations continue to age and life expectancy increases, the burden of osteoporotic fractures in men is expected to greatly increase (3, 4). Aging and gonadal hormone deficiency are two important risk factors for osteoporosis in men (4, 5). The loss of androgen leads to rapid decreases in bone mineral density (BMD) and increased susceptibility to osteoporotic fractures. Aged male rats and male rats with androgen deficiency induced by orchiectomy have been used as animal models to test the efficacy of therapeutic agents aimed at preventing and treating osteoporosis in men (6–8). One important therapeutic target emerging in bone formation is sclerostin, a protein that binds to the low-density lipoprotein receptor-related protein 4/5/6 Wnt coreceptors to inhibit canonical Wnt signaling in osteoblast lineage cells (9–13). Higher bone formation and bone mass have been observed in people with a genetic disorder that results in a lifelong absence of functional sclerostin (14) and in sclerostin knockout mice (15, 16). Several monoclonal antibodies against sclerostin (Scl-Abs) have been developed as potential bone-forming agents for the treatment of conditions with a low bone mass (17). Preclinical studies have shown that Scl-Ab activates Wnt signaling in osteoblast lineage cells, thus increasing bone formation via the activation of bone-lining cells, bone matrix production by osteoblasts, and recruitment of osteoprogenitor cells (18). Furthermore, Scl-Ab induces expression changes in osteoclast mediators, thereby decreasing bone resorption (18). These effects resulted in rapid increases in trabecular and cortical bone mass and improvements in bone structure and strength in several animal models of bone loss (17, 18). In clinical trials, Scl-Abs have been shown to rapidly increase BMD and decrease the risk of osteoporotic fractures in postmenopausal women (19, 20). Although most preclinical investigations have focused on animal models relevant to postmenopausal osteoporosis, Scl-Ab has also been studied in animal models of immobilization-induced bone loss (21–25), osteogenesis imperfecta (26–28), diabetes-related bone loss (29), and radiation-induced bone loss (30). In addition, we previously showed that Scl-Ab increased bone formation, mass, and strength in an aged male rat model of osteoporosis (31). In the present study, we investigated the effects of Scl-Ab in the orchiectomized (ORX) rat, an androgen-deficient male osteoporosis model. We hypothesized that Scl-Ab treatment would reverse the loss of bone mass and strength caused by androgen ablation in the established osteopenia ORX rat model. Materials and Methods The male rats used in the present study were housed two per cage in an environmentally controlled room (temperature 20°C to 22°C, relative humidity 34% to 73%) at an Amgen animal facility (Thousand Oaks, CA). The room was kept at a 12-hour light/dark cycle and met all specifications of the Association for Assessment and Accreditation of Laboratory Animal Care International. Water and food were supplied ad libitum. The institutional animal care and use committee of Amgen Inc. (Thousand Oaks, CA) approved the protocol and procedures. Six-month-old male Sprague Dawley rats (Envigo, Indianapolis, IN) were either sham-operated (sham) or underwent orchiectomy (ORX) and were left untreated for 3 months, allowing for the development of osteopenia. The body weights were recorded, and the areal BMD was determined in vivo using dual-energy x-ray absorptiometry (DXA; Hologic QDR 4500a; Bedford, MA) before the initiation of treatment. These measurements were used to balance the groups, such that all groups had a similar mean body weight and BMD before the start of treatment. These 9-month-old ORX rats were treated with either vehicle (10 mM sodium acetate, 9% sucrose, pH 5.2, with 0.004% Tween 20) or rat sclerostin antibody (Scl-AbIII; Amgen Inc.) at 5 or 25 mg/kg, twice weekly by subcutaneous injection for 6 weeks. DXA scans were performed on isoflurane-anesthetized rats at weeks 3 and 6. One group of rats was euthanized before surgery and the sham and ORX rats were euthanized before the treatment period. Ten rats were in each subgroup. The rats were injected subcutaneously with xylenol orange (90 mg/kg) at the start of Scl-Ab treatment and with calcein (20 mg/kg) at 13 and 3 days before necropsy. The two calcein labels were used for histomorphometric analysis of cortical and trabecular bone. At necropsy, the second lumbar (L2) vertebra and the right tibia were collected from each rat for dynamic histomorphometry, and the third to fifth lumbar vertebrae and left femur were collected for microcomputed tomography (micro-CT) and strength assessment. Bone densitometry Areal BMD was determined in vivo in isoflurane-anesthetized rats using DXA, as described previously (32). The regions of interest included the lumbar 1 to 5 vertebrae and the femur–tibia (entire femur and the proximal half of the tibia). The volumetric BMD (vBMD) and microarchitecture were determined in the right femurs and fifth lumbar (L5) vertebrae using a desktop micro-CT system (GE eXplore Locus SP Specimen Scanner; GE Health Care, London, ON, Canada). Whole femurs and vertebrae were scanned, and the images were reconstructed to an isotropic voxel size of 30 and 18 μm3, respectively. For cortical diaphyseal analysis, a central region equivalent to 10% of the height of the femur (measured from the apex of the femoral head to the base of the condyles) was selected distally to the midpoint. For femoral neck analysis, the proximal femur was reoriented relative to the neck axis, and a 0.3-mm region was selected at its narrowest aspect. For distal femur analysis, a trabecular region equivalent to 10% of the femur height was selected, beginning 0.3 mm from the most proximal aspect of the growth plate. For vertebral body analysis, a central region was selected equivalent to 70% of the vertebral body height. The trabecular bone within each region was segmented from the cortex using a semiautomated contouring algorithm, and the vertebral cortex was separately examined. At each site, the volumetric bone mineral content (vBMC) and vBMD were determined without thresholding. For the microarchitectural parameters, the thresholds were applied for all samples at the L5 vertebra (480 mg/mL), distal femur (500 mg/mL), femur diaphysis (800 mg/mL), and femoral neck (500 mg/mL). At the femur diaphysis, analyses were run to generate the periosteal perimeter (Ps.Pm) and endocortical perimeter (Ec.Pm), cortical thickness (Ct.Th), cortical bone area (Ct.B.Ar), cross-sectional moment of inertia (CSMI), and tissue mineral density, as well as the distance from the centroid to the posterior edge, used in the strength calculations provided in subsequent sections. The femoral neck endpoints included the bone area, vBMC, vBMD, bone volume fraction [bone volume/total volume (BV/TV)], and Ps.Pm. The calculated trabecular parameters included the trabecular vBMD (both total and tissue), trabecular BV/TV, trabecular number (Tb.N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th), and structural model index. At the vertebra, the total, cortical, and trabecular region data are reported. Bone strength Femurs and lumbar vertebrae were harvested, wrapped in gauze, and stored in phosphate-buffered saline at −20°C. On the day of testing, the femurs were kept hydrated and removed from the saline solution only for mechanical testing. Strength testing was performed only on the groups that had completed the 6-week treatment period. The method of testing was described in our previous report (32) and is summarized in the next paragraphs. L5 vertebral bodies were tested in compression to failure using an MTS 858 Mini Bionix servohydraulic system (MTS Systems Corp., Eden Prairie, MN). Plano-parallel specimens (approximately 5 mm in height) were first prepared by removing the laminae using a diamond wire saw (model 3241; Well Diamond Wire Saws Inc., Norcross, GA), and the sample height was recorded with calipers. During compression testing at 6 mm/min, the load–displacement curve was recorded, and the extrinsic strength parameters (peak load, stiffness, and energy to peak load) and displacement to peak load were generated within Microsoft Excel (Microsoft Corp., Redmond, WA). The estimated material properties were calculated using micro-CT–derived bone area as follows: ultimate strength = peak load/bone area; modulus = stiffness/(bone area/height); toughness = energy/(bone area × height). Femur diaphyses were tested in a three-point bending configuration using the MTS servohydraulic system (MTS Systems Corp.). A preload of 5 N was applied before loading the bones to failure at a displacement rate of 3 mm/min. A load transducer of 1 kN ± 0.02 N was used to record the force measurements. The span length between supports was set to 20 mm. The load–displacement curve was recorded by the testing system, and the extrinsic strength parameters (peak load, stiffness, and energy to failure) and ultimate displacement were generated. The material properties (ultimate strength, modulus, and toughness) were calculated using the CSMI and the distance from the centroid to the posterior edge per the standard engineering approach for three-point bending tests. After diaphyseal testing, the proximal one-half of the left femur was fixed in a drill chuck for destructive shear testing of the femoral neck. The femoral head was displaced at 1 mm/s until failure occurred. The load–displacement curves were analyzed for peak load, stiffness, and energy to failure. Bone histomorphometric analysis Undecalcified parasagittal 4-µm-thick sections of the L2 vertebral bodies and 6-µm-thick transverse sections of tibia at the tibiofibular junction were prepared as described previously (32). The vertebral body sections were either stained with modified Goldner trichrome for analysis of static parameters or left unstained for collection of fluorochrome-based data. Tibial shaft sections were left unstained for collection of both fluorochrome-based and static parameter data. Histomorphometric analyses were performed using Osteomeasure bone analysis software (Osteometrics, Inc., Decatur, GA). The region of interest for the lumbar vertebral body trabecular bone included all trabecular bone within the parasagittal section. Static and dynamic parameters were calculated and expressed according to reported methods (33). Statistical analysis All results are expressed as the mean ± standard error of the mean. Longitudinal DXA data were analyzed using a two-way analysis of variance followed by the Tukey multiple comparison test. All other endpoints were subjected to one-way analysis of variance, followed by the Tukey test. These analyses were performed in GraphPad Prism software, version 5.01 (GraphPad Software Inc., San Diego, CA), with a P value of <0.05 used to identify significant pairwise group differences. Linear regression analysis was applied at each strength-tested site to correlate the peak load with the vBMC across groups. Regression coefficients (r2) are provided to demonstrate the strength of association, and positive relationships were confirmed if the slope was significantly different from zero at P < 0.05. Results Confirmation of expected organ and body weight changes after ORX ORX resulted in a significant decrease in the weight of the seminal vesicle, prostate gland, and total body weight before treatment, an effect that persisted throughout the treatment period (Table 1). Scl-Ab treatment for 6 weeks did not significantly alter any of these variables. Table 1. Effect of ORX and Scl-Ab on Seminal Vesicle, Prostate Gland, and Body Weight Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Seminal vesicle weight (g)  1.910 ± 0.133  0.232 ± 0.020a  1.748 ± 0.123  0.188 ± 0.017a  0.191 ± 0.011a  0.183 ± 0.020a  Prostate gland weight (g)  1.652 ± 0.118  0.308 ± 0.021a  1.424 ± 0.095  0.210 ± 0.016a  0.185 ± 0.015a  0.204 ± 0.016a  Terminal body weight (g)  684.0 ± 20.8  618.5 ± 15.3a  727.7 ± 26.9  628.7 ± 25.3a  601.5 ± 27.2a  630.8 ± 33.2a  Baseline body weight (g)  NA  NA  714.7 ± 26.0  654.5 ± 23.2a  631.7 ± 28.3a  656.5 ± 29.6a  Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Seminal vesicle weight (g)  1.910 ± 0.133  0.232 ± 0.020a  1.748 ± 0.123  0.188 ± 0.017a  0.191 ± 0.011a  0.183 ± 0.020a  Prostate gland weight (g)  1.652 ± 0.118  0.308 ± 0.021a  1.424 ± 0.095  0.210 ± 0.016a  0.185 ± 0.015a  0.204 ± 0.016a  Terminal body weight (g)  684.0 ± 20.8  618.5 ± 15.3a  727.7 ± 26.9  628.7 ± 25.3a  601.5 ± 27.2a  630.8 ± 33.2a  Baseline body weight (g)  NA  NA  714.7 ± 26.0  654.5 ± 23.2a  631.7 ± 28.3a  656.5 ± 29.6a  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL, baseline; NA, not applicable. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Bone densitometry Osteopenia was reflected in reduced DXA BMD at the lumbar vertebrae and femur–tibia sites in the ORX rats compared with the sham controls at 3 months after orchiectomy (12 weeks; Fig. 1). Relative to the ORX controls, Scl-Ab significant increased the BMD at the lumbar spine [Fig. 1(a)] and at the femur–tibia sites [Fig. 1(b)] after 3 and 6 weeks of treatment at 5 and 25 mg/kg (15 and 18 weeks after surgery, respectively). Scl-Ab treatment at both doses normalized the femur–tibia BMD to that of the sham control levels. Furthermore, Scl-Ab at 25 mg/kg significantly increased the lumbar BMD relative to that of the sham controls. Figure 1. View largeDownload slide Scl-Ab restored BMD at the (a) lumbar spine and (b) femur–tibia in ORX rats. In vivo DXA scans were performed before the initiation of treatment (12 weeks after surgery) and at 3 and 6 weeks after dosing (15 and 18 weeks after surgery, respectively). Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 1. View largeDownload slide Scl-Ab restored BMD at the (a) lumbar spine and (b) femur–tibia in ORX rats. In vivo DXA scans were performed before the initiation of treatment (12 weeks after surgery) and at 3 and 6 weeks after dosing (15 and 18 weeks after surgery, respectively). Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Representative micro-CT images of the L5 vertebrae for each group at the end of the treatment period are shown in Fig. 2(a). Using micro-CT, the ORX vertebrae showed a significantly lower total vBMC and total bone area compared with the sham vertebrae at 3 months after orchiectomy (Table 2). These changes stemmed from a significant decrease in the cortical region vBMC and bone area, accompanied by a nonsignificant decrease in trabecular vBMC and trabecular bone area. These effects persisted throughout the treatment period, with further reductions with ORX in the trabecular compartment. Both doses of Scl-Ab significantly increased the vBMC, vBMD, and bone area of the total region after 6 weeks of treatment (18 weeks after surgery), without affecting the vertebral body total (cross-sectional) area. In the trabecular compartment, Scl-Ab resulted in significant increases in vBMD and BV/TV relative to ORX [Table 2; Fig. 2(b)] and Tb.Th relative to both ORX and sham controls (Table 2). At both doses, Scl-Ab induced increases in the vertebral cortical region vBMC, bone area, and cortical thickness, relative to both ORX and sham controls. Figure 2. View largeDownload slide Scl-Ab reversed cortical and trabecular bone loss at the L5 vertebral body of ORX rats. (a) Representative micro-CT images of L5 vertebrae (18-µm resolution). Thicker cortex and thicker trabeculae were observed in Scl-Ab–treated ORX rats. (b) Scl-Ab (start of treatment at 12 weeks after surgery indicated by arrows) restored cortical thickness and trabecular BV/TV in ORX rats. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 2. View largeDownload slide Scl-Ab reversed cortical and trabecular bone loss at the L5 vertebral body of ORX rats. (a) Representative micro-CT images of L5 vertebrae (18-µm resolution). Thicker cortex and thicker trabeculae were observed in Scl-Ab–treated ORX rats. (b) Scl-Ab (start of treatment at 12 weeks after surgery indicated by arrows) restored cortical thickness and trabecular BV/TV in ORX rats. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Table 2. Effect of Scl-Ab on L5 Vertebral Bone Mass and Microarchitecture in ORX Rats Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Total               vBMC (mg/mm)  6.51 ± 0.32  5.75 ± 0.13a  6.69 ± 0.09  5.77 ± 0.21a  6.77 ± 0.22b  7.26 ± 0.26b   vBMD (mg/mL)  551.3 ± 16.2  516.4 ± 12.5  553.0 ± 8.8  474.6 ± 14.1a  571.8 ± 8.9b  590.6 ± 15.1b   Area (mm2)  11.79 ± 0.36  11.17 ± 0.29  12.14 ± 0.29  12.17 ± 0.27  11.82 ± 0.24  12.30 ± 0.28   Bone area (mm2)  5.67 ± 0.31  4.80 ± 0.13a  5.71 ± 0.06  4.73 ± 0.19a  5.84 ± 0.23b  6.35 ± 0.27b  Trabecular               vBMC (mg/mm)  3.10 ± 0.20  2.71 ± 0.10  3.14 ± 0.09  2.55 ± 0.12a  2.83 ± 0.15  3.09 ± 0.19   vBMD (mg/mL)  421.1 ± 18.9  382.7 ± 14.6  421.3 ± 9.7  337.3 ± 15.0a  418.2 ± 13.5b  444.3 ± 20.0b   Bone area (mm2)  7.33 ± 0.26  7.11 ± 0.22  7.47 ± 0.24  7.57 ± 0.20  6.73 ± 0.17b  6.95 ± 0.24   BV/TV (%)  32.63 ± 2.20  27.91 ± 1.53  31.91 ± 1.09  23.62 ± 1.49a  31.71 ± 1.69b  34.93 ± 2.24b   Tb.N (1/mm)  4.30 ± 0.24  3.94 ± 0.19  4.23 ± 0.13  3.44 ± 0.15a  3.51 ± 0.15a  3.48 ± 0.22a   Tb.Th (mm)  0.067 ± 0.002  0.062 ± 0.001  0.066 ± 0.001  0.060 ± 0.002  0.090 ± 0.003a,b  0.103 ± 0.004a,b   SMI  −0.05 ± 0.15  0.36 ± 0.10a  −0.04 ± 0.09  0.45 ± 0.10a  0.24 ± 0.14  0.15 ± 0.14  Cortical region               vBMC (mg/mm)  3.42 ± 0.12  3.04 ± 0.07a  3.55 ± 0.08  3.22 ± 0.09  3.95 ± 0.09a,b  4.17 ± 0.10a,b   vBMD (mg/mL)  765.3 ± 10.8  749.3 ± 9.5  762.7 ± 7.7  700.8 ± 9.9a  774.1 ± 7.1b  779.4 ± 7.9b   Area (mm2)  4.46 ± 0.12  4.06 ± 0.09a  4.67 ± 0.12  4.60 ± 0.10  5.10 ± 0.09a,b  5.35 ± 0.09a,b   Bone area (mm2)  3.27 ± 0.12  2.83 ± 0.07a  3.33 ± 0.07  2.95 ± 0.09a  3.69 ± 0.08a,b  3.92 ± 0.11a,b   Ct.Th (mm)  0.243 ± 0.007  0.219 ± 0.006a  0.248 ± 0.005  0.217 ± 0.008a  0.274 ± 0.005a,b  0.290 ± 0.008a,b  Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Total               vBMC (mg/mm)  6.51 ± 0.32  5.75 ± 0.13a  6.69 ± 0.09  5.77 ± 0.21a  6.77 ± 0.22b  7.26 ± 0.26b   vBMD (mg/mL)  551.3 ± 16.2  516.4 ± 12.5  553.0 ± 8.8  474.6 ± 14.1a  571.8 ± 8.9b  590.6 ± 15.1b   Area (mm2)  11.79 ± 0.36  11.17 ± 0.29  12.14 ± 0.29  12.17 ± 0.27  11.82 ± 0.24  12.30 ± 0.28   Bone area (mm2)  5.67 ± 0.31  4.80 ± 0.13a  5.71 ± 0.06  4.73 ± 0.19a  5.84 ± 0.23b  6.35 ± 0.27b  Trabecular               vBMC (mg/mm)  3.10 ± 0.20  2.71 ± 0.10  3.14 ± 0.09  2.55 ± 0.12a  2.83 ± 0.15  3.09 ± 0.19   vBMD (mg/mL)  421.1 ± 18.9  382.7 ± 14.6  421.3 ± 9.7  337.3 ± 15.0a  418.2 ± 13.5b  444.3 ± 20.0b   Bone area (mm2)  7.33 ± 0.26  7.11 ± 0.22  7.47 ± 0.24  7.57 ± 0.20  6.73 ± 0.17b  6.95 ± 0.24   BV/TV (%)  32.63 ± 2.20  27.91 ± 1.53  31.91 ± 1.09  23.62 ± 1.49a  31.71 ± 1.69b  34.93 ± 2.24b   Tb.N (1/mm)  4.30 ± 0.24  3.94 ± 0.19  4.23 ± 0.13  3.44 ± 0.15a  3.51 ± 0.15a  3.48 ± 0.22a   Tb.Th (mm)  0.067 ± 0.002  0.062 ± 0.001  0.066 ± 0.001  0.060 ± 0.002  0.090 ± 0.003a,b  0.103 ± 0.004a,b   SMI  −0.05 ± 0.15  0.36 ± 0.10a  −0.04 ± 0.09  0.45 ± 0.10a  0.24 ± 0.14  0.15 ± 0.14  Cortical region               vBMC (mg/mm)  3.42 ± 0.12  3.04 ± 0.07a  3.55 ± 0.08  3.22 ± 0.09  3.95 ± 0.09a,b  4.17 ± 0.10a,b   vBMD (mg/mL)  765.3 ± 10.8  749.3 ± 9.5  762.7 ± 7.7  700.8 ± 9.9a  774.1 ± 7.1b  779.4 ± 7.9b   Area (mm2)  4.46 ± 0.12  4.06 ± 0.09a  4.67 ± 0.12  4.60 ± 0.10  5.10 ± 0.09a,b  5.35 ± 0.09a,b   Bone area (mm2)  3.27 ± 0.12  2.83 ± 0.07a  3.33 ± 0.07  2.95 ± 0.09a  3.69 ± 0.08a,b  3.92 ± 0.11a,b   Ct.Th (mm)  0.243 ± 0.007  0.219 ± 0.006a  0.248 ± 0.005  0.217 ± 0.008a  0.274 ± 0.005a,b  0.290 ± 0.008a,b  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL, baseline; SMI, structural model index. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Representative micro-CT images of the femur diaphysis, femur distal metaphysis, and femoral neck for each group at the end of the treatment period are shown in Fig. 3. ORX resulted in impaired periosteal expansion, corresponding to a decrease in periosteal perimeter, bone area, and CSMI relative to sham at the femur diaphysis at 3 months after orchiectomy (Table 3). By the end of the study period, the bone area and Ct.Th were significantly lower in the ORX group compared with the sham group. Scl-Ab resulted in significant increases in cortical vBMC, Ct.B.Ar, and Ct.Th at the femur diaphysis, with no significant changes in Ec.Pm or Ps.Pm observed (Table 3). At the distal femur metaphysis, ORX induced significant loss in trabecular vBMC, trabecular region area, BV/TV, and Tb.N at 3 months after orchiectomy. Scl-Ab improved vBMD and BV/TV relative to the ORX controls and Tb.Th relative to both ORX and sham controls (Table 3). ORX did not result in significant bone loss at the femoral neck at any time point (data not shown). Significant increases in bone area, but no other parameters, were observed at this site after Scl-Ab treatment (3.69 ± 0.07 mm2 vs 4.29 ± 0.19 mm2 and 4.53 ± 0.16 mm2, ORX control vs Scl-Ab 5 and 25 mg/kg, respectively; P < 0.05). Figure 3. View largeDownload slide Representative micro-CT images of the femoral neck, diaphysis, and distal metaphysis from sham controls, ORX controls, and ORX rats treated with Scl-Ab for 6 weeks. Micro-CT was performed on right femurs (32-µm resolution) for all samples. Representative images were selected according to the group median for the bone mass parameters. A greater bone mass at all three sites was observed in the ORX rats treated with Scl-Ab at both doses. Figure 3. View largeDownload slide Representative micro-CT images of the femoral neck, diaphysis, and distal metaphysis from sham controls, ORX controls, and ORX rats treated with Scl-Ab for 6 weeks. Micro-CT was performed on right femurs (32-µm resolution) for all samples. Representative images were selected according to the group median for the bone mass parameters. A greater bone mass at all three sites was observed in the ORX rats treated with Scl-Ab at both doses. Table 3. Effect of Scl-Ab on Femur Bone Mass and Microarchitecture in ORX Rats Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Femur diaphysis               Ct. vBMC (mg/mm)  9.11 ± 0.17  8.62 ± 0.19  9.61 ± 0.14  8.49 ± 0.20  9.00 ± 0.21b  9.38 ± 0.28b   Ct. TMD (mg/mL)  1093.0 ± 3.4  1122.1 ± 4.0  1104.4 ± 5.7  1098.5 ± 2.1  1096.0 ± 7.0  1095.4 ± 9.4   Ct. bone area (mm2)  8.43 ± 0.15  7.77 ± 0.19a  8.81 ± 0.12  7.82 ± 0.17a  8.31 ± 0.20b  8.66 ± 0.24b   Ct.Th (mm)  0.80 ± 0.01  0.78 ± 0.01  0.83 ± 0.02  0.76 ± 0.01a  0.82 ± 0.01  0.84 ± 0.02b   CSMI (mm4)  10.70 ± 0.55  8.60 ± 0.66a  11.18 ± 0.46  9.29 ± 0.59  9.52 ± 0.61  10.66 ± 0.59   Ec.Pm (mm)  9.11 ± 0.19  8.37 ± 0.18a  8.93 ± 0.23  8.84 ± 0.27  8.54 ± 0.21  8.51 ± 0.17   Ps.Pm (mm)  14.00 ± 0.17  13.25 ± 0.19a  14.06 ± 0.16  13.59 ± 0.23  13.61 ± 0.22  13.78 ± 0.17  Distal femur metaphysis               Tb.vBMC (mg/mm)  3.04 ± 0.29  2.10 ± 0.11a  3.17 ± 0.13  2.08 ± 0.11a  2.45 ± 0.17a  2.58 ± 0.15a   Tb.vBMD (mg/mL)  230.20 ± 18.01  190.54 ± 9.69  238.59 ± 9.01  180.11 ± 9.08a  211.44 ± 10.57  232.34 ± 13.92b   Tb. region area (mm2)  13.08 ± 0.48  11.07 ± 0.42a  13.34 ± 0.49  11.55 ± 0.34a  11.52 ± 0.50b  11.17 ± 0.41b   BV/TV (%)  9.71 ± 1.92  5.21 ± 0.71a  10.04 ± 0.86  4.77 ± 0.47a  8.27 ± 0.86b  10.19 ± 1.08b   Tb.N. (1/mm)  1.56 ± 0.27  0.84 ± 0.13a  1.55 ± 0.13  0.72 ± 0.11a  1.06 ± 0.13a  1.17 ± 0.14   Tb.Th. (mm)  0.048 ± 0.002  0.048 ± 0.002  0.050 ± 0.002  0.048 ± 0.001  0.068 ± 0.003a,b  0.080 ± 0.003a,b   SMI  2.36 ± 0.12  2.81 ± 0.06a  2.28 ± 0.10  2.62 ± 0.09  2.38 ± 0.08  2.34 ± 0.14  Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Femur diaphysis               Ct. vBMC (mg/mm)  9.11 ± 0.17  8.62 ± 0.19  9.61 ± 0.14  8.49 ± 0.20  9.00 ± 0.21b  9.38 ± 0.28b   Ct. TMD (mg/mL)  1093.0 ± 3.4  1122.1 ± 4.0  1104.4 ± 5.7  1098.5 ± 2.1  1096.0 ± 7.0  1095.4 ± 9.4   Ct. bone area (mm2)  8.43 ± 0.15  7.77 ± 0.19a  8.81 ± 0.12  7.82 ± 0.17a  8.31 ± 0.20b  8.66 ± 0.24b   Ct.Th (mm)  0.80 ± 0.01  0.78 ± 0.01  0.83 ± 0.02  0.76 ± 0.01a  0.82 ± 0.01  0.84 ± 0.02b   CSMI (mm4)  10.70 ± 0.55  8.60 ± 0.66a  11.18 ± 0.46  9.29 ± 0.59  9.52 ± 0.61  10.66 ± 0.59   Ec.Pm (mm)  9.11 ± 0.19  8.37 ± 0.18a  8.93 ± 0.23  8.84 ± 0.27  8.54 ± 0.21  8.51 ± 0.17   Ps.Pm (mm)  14.00 ± 0.17  13.25 ± 0.19a  14.06 ± 0.16  13.59 ± 0.23  13.61 ± 0.22  13.78 ± 0.17  Distal femur metaphysis               Tb.vBMC (mg/mm)  3.04 ± 0.29  2.10 ± 0.11a  3.17 ± 0.13  2.08 ± 0.11a  2.45 ± 0.17a  2.58 ± 0.15a   Tb.vBMD (mg/mL)  230.20 ± 18.01  190.54 ± 9.69  238.59 ± 9.01  180.11 ± 9.08a  211.44 ± 10.57  232.34 ± 13.92b   Tb. region area (mm2)  13.08 ± 0.48  11.07 ± 0.42a  13.34 ± 0.49  11.55 ± 0.34a  11.52 ± 0.50b  11.17 ± 0.41b   BV/TV (%)  9.71 ± 1.92  5.21 ± 0.71a  10.04 ± 0.86  4.77 ± 0.47a  8.27 ± 0.86b  10.19 ± 1.08b   Tb.N. (1/mm)  1.56 ± 0.27  0.84 ± 0.13a  1.55 ± 0.13  0.72 ± 0.11a  1.06 ± 0.13a  1.17 ± 0.14   Tb.Th. (mm)  0.048 ± 0.002  0.048 ± 0.002  0.050 ± 0.002  0.048 ± 0.001  0.068 ± 0.003a,b  0.080 ± 0.003a,b   SMI  2.36 ± 0.12  2.81 ± 0.06a  2.28 ± 0.10  2.62 ± 0.09  2.38 ± 0.08  2.34 ± 0.14  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL, baseline; SMI, structural model index; Tb, trabecular; TMD, tissue mineral density. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Bone strength The observed decreases in bone mass after ORX corresponded to losses in whole bone strength, most notably in the L5 vertebrae (Table 4). Across the L5 vertebra, femur diaphysis, and femoral neck, the strength parameters were lower in the ORX rats than in the sham controls (Table 4). Scl-Ab significantly increased all the L5 vertebra whole bone strength parameters relative to the ORX controls at both doses (Table 4), and the peak load and stiffness were also increased at the femur diaphysis at the 25-mg/kg dose. At the femoral neck, the peak load was significantly increased for both Scl-Ab doses, with no differences in the other whole bone strength parameters (Table 4). Table 4. Effect of Scl-Ab on L5 Vertebra Bone Strength in ORX Rats Site  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  L5 vertebra           Specimen height  3.71 ± 0.01  3.74 ± 0.02  3.74 ± 0.01  3.71 ± 0.01   Peak load (N)  459.8 ± 22.2  317.4 ± 23.5a  520.9 ± 30.9b  565.0 ± 36.9b   Stiffness (N/mm)  3739 ± 141  2820 ± 247  4514 ± 358b  4667 ± 287b   Energy at peak load (N*mm)  41.2 ± 3.6  27.5 ± 2.5a  46.5 ± 2.8b  50.5 ± 3.5b   Displacement at peak load (mm)  0.20 ± 0.01  0.19 ± 0.01  0.18 ± 0.01  0.19 ± 0.01   Ultimate strength (MPa)  80.7 ± 4.1  66.4 ± 2.8a  89.0 ± 3.0b  88.7 ± 4.0b   Modulus (MPa)  2432 ± 83  2208 ± 159  2888 ± 179b  2741 ± 152   Toughness (MPa)  1.95 ± 0.17  1.53 ± 0.08  2.12 ± 0.07b  2.13 ± 0.09b  Femur diaphysis           Peak load (N)  241.4 ± 8.7  204.9 ± 5.2a  229.7 ± 6.4  254.2 ± 13.6b   Stiffness (N/mm)  713.6 ± 32.5  624.7 ± 23.3  678.9 ± 22.2  743.7 ± 34.9b   Energy (N*mm)  118.1 ± 12.0  83.2 ± 7.2  98.6 ± 10.2  116.1 ± 11.1   Ultimate displacement (mm)  0.72 ± 0.06  0.61 ± 0.03  0.65 ± 0.05  0.67 ± 0.03   Ultimate strength (MPa)  199.8 ± 8.0  204.3 ± 11.3  221.4 ± 8.0  218.2 ± 8.1   Elastic modulus (MPa)  10708 ± 466  11542 ± 703  12128 ± 459  11755 ± 476   Toughness (MPa)  5.38 ± 0.56  4.45 ± 0.40  5.01 ± 0.38  5.40 ± 0.45  Femoral neck           Peak load (N)  190.3 ± 11.8  162.8 ± 5.5  205.6 ± 15.0b  228.6 ± 7.5a,b   Stiffness (N/mm)  757.8 ± 46.2  718.2 ± 52.6  710.6 ± 38.4  725.3 ± 15.1   Energy (N*mm)  49.1 ± 6.2  42.9 ± 3.4  70.6 ± 13.3  77.0 ± 8.8  Site  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  L5 vertebra           Specimen height  3.71 ± 0.01  3.74 ± 0.02  3.74 ± 0.01  3.71 ± 0.01   Peak load (N)  459.8 ± 22.2  317.4 ± 23.5a  520.9 ± 30.9b  565.0 ± 36.9b   Stiffness (N/mm)  3739 ± 141  2820 ± 247  4514 ± 358b  4667 ± 287b   Energy at peak load (N*mm)  41.2 ± 3.6  27.5 ± 2.5a  46.5 ± 2.8b  50.5 ± 3.5b   Displacement at peak load (mm)  0.20 ± 0.01  0.19 ± 0.01  0.18 ± 0.01  0.19 ± 0.01   Ultimate strength (MPa)  80.7 ± 4.1  66.4 ± 2.8a  89.0 ± 3.0b  88.7 ± 4.0b   Modulus (MPa)  2432 ± 83  2208 ± 159  2888 ± 179b  2741 ± 152   Toughness (MPa)  1.95 ± 0.17  1.53 ± 0.08  2.12 ± 0.07b  2.13 ± 0.09b  Femur diaphysis           Peak load (N)  241.4 ± 8.7  204.9 ± 5.2a  229.7 ± 6.4  254.2 ± 13.6b   Stiffness (N/mm)  713.6 ± 32.5  624.7 ± 23.3  678.9 ± 22.2  743.7 ± 34.9b   Energy (N*mm)  118.1 ± 12.0  83.2 ± 7.2  98.6 ± 10.2  116.1 ± 11.1   Ultimate displacement (mm)  0.72 ± 0.06  0.61 ± 0.03  0.65 ± 0.05  0.67 ± 0.03   Ultimate strength (MPa)  199.8 ± 8.0  204.3 ± 11.3  221.4 ± 8.0  218.2 ± 8.1   Elastic modulus (MPa)  10708 ± 466  11542 ± 703  12128 ± 459  11755 ± 476   Toughness (MPa)  5.38 ± 0.56  4.45 ± 0.40  5.01 ± 0.38  5.40 ± 0.45  Femoral neck           Peak load (N)  190.3 ± 11.8  162.8 ± 5.5  205.6 ± 15.0b  228.6 ± 7.5a,b   Stiffness (N/mm)  757.8 ± 46.2  718.2 ± 52.6  710.6 ± 38.4  725.3 ± 15.1   Energy (N*mm)  49.1 ± 6.2  42.9 ± 3.4  70.6 ± 13.3  77.0 ± 8.8  Data presented as mean ± standard error of the mean (n = 10 per group). a P < 0.05 vs time-matched sham control by one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Correlations between the bone mass (vBMC) and bone strength were examined to determine the change in bone material properties. Across the L5 vertebra, femur diaphysis, and femoral neck, Scl-Ab treatment maintained the significant and positive correlations between the vBMC and peak load observed in controls (Fig. 4), indicating that Scl-Ab treatment maintained bone quality in this ORX rat model. Figure 4. View largeDownload slide Linear regressions of vBMC to peak load at (a) L5 vertebra, (b) femur diaphysis, and (c) femoral neck. The peak load generated from each test was correlated with the vBMC across groups, demonstrating a consistent positive relationship. All correlations were significantly different from zero (P < 0.0001). Figure 4. View largeDownload slide Linear regressions of vBMC to peak load at (a) L5 vertebra, (b) femur diaphysis, and (c) femoral neck. The peak load generated from each test was correlated with the vBMC across groups, demonstrating a consistent positive relationship. All correlations were significantly different from zero (P < 0.0001). Histomorphometric analysis Consistent with the micro-CT results, ORX resulted in significantly lower BV/TV (−26%) and Tb.N (−18%) and higher Tb.Sp (+37%) compared with sham controls at L2 at the end of the study period (Table 5). These changes corresponded to a significant increase in the ratio of eroded surface to bone surface (ES/BS), a bone resorption index, in the ORX controls compared with the sham controls. Both doses of Scl-Ab restored BV/TV to the sham control levels owing to robust increases in Tb.Th. In the Scl-Ab–treated ORX rats, these increases were associated with greater bone formation [i.e., mineralizing surface/BS, mineral apposition rate (MAR), and bone formation rate (BFR)/BS, and with lower bone resorption (ES/BS)] [Fig. 5(a) and 5(b)]. Table 5. Histomorphometric Analysis of Trabecular Bone at L2 Vertebral Body Parameter  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  BV/TV (%)  33.1 ± 0.8  24.4 ± 1.2a  34.5 ± 2.2b  33.2 ± 1.7b  Tb.Th (µm)  92.9 ± 2.6  82.0 ± 2.0  121.4 ± 5.9b  129.5 ± 8.5b  Tb.Sp (µm)  188.8 ± 7.3  258.9 ± 12.6a  235.1 ± 15.6  260.4 ± 12.7  Tb.N (1/mm)  3.58 ± 0.12  2.97 ± 0.11a  2.85 ± 0.12a  2.59 ± 0.09a  MS/BS (%)  26.8 ± 1.6  27.6 ± 2.1  44.3 ± 2.0a,b  38.0 ± 3.2a,b  MAR (µm/d)  1.02 ± 0.04  1.13 ± 0.08  1.39 ± 0.07a,b  1.25 ± 0.08  BFR/BS (µm3/µm2/d)  0.274 ± 0.020  0.320 ± 0.036  0.619 ± 0.043a,b  0.482 ± 0.054a,b  ES/BS (%)  4.04 ± 0.29  7.04 ± 0.33a  2.78 ± 0.38a,b  2.30 ± 0.31a,b  Parameter  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  BV/TV (%)  33.1 ± 0.8  24.4 ± 1.2a  34.5 ± 2.2b  33.2 ± 1.7b  Tb.Th (µm)  92.9 ± 2.6  82.0 ± 2.0  121.4 ± 5.9b  129.5 ± 8.5b  Tb.Sp (µm)  188.8 ± 7.3  258.9 ± 12.6a  235.1 ± 15.6  260.4 ± 12.7  Tb.N (1/mm)  3.58 ± 0.12  2.97 ± 0.11a  2.85 ± 0.12a  2.59 ± 0.09a  MS/BS (%)  26.8 ± 1.6  27.6 ± 2.1  44.3 ± 2.0a,b  38.0 ± 3.2a,b  MAR (µm/d)  1.02 ± 0.04  1.13 ± 0.08  1.39 ± 0.07a,b  1.25 ± 0.08  BFR/BS (µm3/µm2/d)  0.274 ± 0.020  0.320 ± 0.036  0.619 ± 0.043a,b  0.482 ± 0.054a,b  ES/BS (%)  4.04 ± 0.29  7.04 ± 0.33a  2.78 ± 0.38a,b  2.30 ± 0.31a,b  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: MS/BS, mineralizing surface/bone surface. a P < 0.05 vs time-matched sham control by one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Figure 5. View largeDownload slide Effect of Scl-Ab on trabecular bone histomorphometry at the L2 vertebral body. (a) ORX rats treated with either dose of Scl-Ab showed more active mineralization surface (arrows) than did ORX controls. (b) Mineralizing surface/bone surface (MS/BS) and BFR/BS were significantly greater for both doses compared with those in the sham and ORX controls. MAR was significantly greater in the 5-mg/kg group compared with that in the sham and ORX controls. In addition, ES/BS, an index of bone resorption, was significantly decreased for both doses compared with that in the sham and ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group) *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 5. View largeDownload slide Effect of Scl-Ab on trabecular bone histomorphometry at the L2 vertebral body. (a) ORX rats treated with either dose of Scl-Ab showed more active mineralization surface (arrows) than did ORX controls. (b) Mineralizing surface/bone surface (MS/BS) and BFR/BS were significantly greater for both doses compared with those in the sham and ORX controls. MAR was significantly greater in the 5-mg/kg group compared with that in the sham and ORX controls. In addition, ES/BS, an index of bone resorption, was significantly decreased for both doses compared with that in the sham and ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group) *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. At the tibia diaphysis, ORX resulted in significantly lower periosteal bone formation (Table 6). At the endocortical surface, ORX increased the bone formation parameters before treatment and increased ES/BS at the end of treatment. Both doses of Scl-Ab resulted in significant increases in bone formation on both periosteal and endocortical surfaces (Fig. 6), with reduced endocortical ES/BS relative to that of the ORX controls. Table 6. Histomorphometric Analysis of Cortical Bone at Tibial Shaft Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Ct.B.Ar (mm2)  5.82 ± 0.20  5.42 ± 0.18  6.03 ± 0.17  5.48 ± 0.15  5.52 ± 0.12  5.80 ± 0.14  Ct.Th (µm)  1457 ± 32  1374 ± 28  1463 ± 22  1376 ± 24  1388 ± 17  1447 ± 27  Ps.Pm (mm)  10.15 ± 0.16  10.02 ± 0.15  10.48 ± 0.15  10.12 ± 0.13  10.13 ± 0.12  10.21 ± 0.11  Ec.Pm (mm)  3.71 ± 0.16  3.86 ± 0.10  3.92 ± 0.12  4.26 ± 0.13  3.77 ± 0.09b  3.66 ± 0.07b  Ps.MS/BS (%)  13.5 ± 4.0  1.1 ± 0.2a  41.0 ± 8.0  6.1 ± 2.1a  51.7 ± 7.8b  54.7 ± 4.0b  Ps.MAR (µm/day)  0.41 ± 0.13  0.08 ± 0.06a  0.76 ± 0.14  0.20 ± 0.12a  0.88 ± 0.15b  1.27 ± 0.16b  Ps.BFR/BS (µm3/µm2/d)  0.093 ± 0.040  0.002 ± 0.002a  0.389 ± 0.093  0.020 ± 0.012a  0.527 ± 0.125b  0.725 ± 0.092b  Ec.MS/BS (%)  17.4 ± 1.9  28.8 ± 3.6a  28.4 ± 3.5  28.4 ± 2.3  83.7 ± 6.1a,b  87.1 ± 3.3a,b  Ec.MAR (µm/d)  0.08 ± 0.06  0.27 ± 0.07  0.50 ± 0.06  0.43 ± 0.09  1.10 ± 0.04a,b  1.10 ± 0.05a,b  Ec.BFR/BS (µm3/µm2/d)  0.013 ± 0.010  0.089 ± 0.027a  0.146 ± 0.024  0.131 ± 0.029  0.932 ± 0.086a,b  0.965 ± 0.065a,b  Ec.ES/BS (%)  10.47 ± 1.86  14.18 ± 2.41  8.21 ± 1.71  13.67 ± 2.02a  0.50 ± 0.34a,b  0.87 ± 0.64a,b  Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Ct.B.Ar (mm2)  5.82 ± 0.20  5.42 ± 0.18  6.03 ± 0.17  5.48 ± 0.15  5.52 ± 0.12  5.80 ± 0.14  Ct.Th (µm)  1457 ± 32  1374 ± 28  1463 ± 22  1376 ± 24  1388 ± 17  1447 ± 27  Ps.Pm (mm)  10.15 ± 0.16  10.02 ± 0.15  10.48 ± 0.15  10.12 ± 0.13  10.13 ± 0.12  10.21 ± 0.11  Ec.Pm (mm)  3.71 ± 0.16  3.86 ± 0.10  3.92 ± 0.12  4.26 ± 0.13  3.77 ± 0.09b  3.66 ± 0.07b  Ps.MS/BS (%)  13.5 ± 4.0  1.1 ± 0.2a  41.0 ± 8.0  6.1 ± 2.1a  51.7 ± 7.8b  54.7 ± 4.0b  Ps.MAR (µm/day)  0.41 ± 0.13  0.08 ± 0.06a  0.76 ± 0.14  0.20 ± 0.12a  0.88 ± 0.15b  1.27 ± 0.16b  Ps.BFR/BS (µm3/µm2/d)  0.093 ± 0.040  0.002 ± 0.002a  0.389 ± 0.093  0.020 ± 0.012a  0.527 ± 0.125b  0.725 ± 0.092b  Ec.MS/BS (%)  17.4 ± 1.9  28.8 ± 3.6a  28.4 ± 3.5  28.4 ± 2.3  83.7 ± 6.1a,b  87.1 ± 3.3a,b  Ec.MAR (µm/d)  0.08 ± 0.06  0.27 ± 0.07  0.50 ± 0.06  0.43 ± 0.09  1.10 ± 0.04a,b  1.10 ± 0.05a,b  Ec.BFR/BS (µm3/µm2/d)  0.013 ± 0.010  0.089 ± 0.027a  0.146 ± 0.024  0.131 ± 0.029  0.932 ± 0.086a,b  0.965 ± 0.065a,b  Ec.ES/BS (%)  10.47 ± 1.86  14.18 ± 2.41  8.21 ± 1.71  13.67 ± 2.02a  0.50 ± 0.34a,b  0.87 ± 0.64a,b  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL = baseline; Ec., endocortical; MS/BS, mineralizing surface/bone surface; Ps., periosteal. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Figure 6. View largeDownload slide Effects of Scl-Ab on cortical bone histomorphometry at the tibial shaft. (a) More calcein-labeled surface was observed on periosteal (arrowheads) and endocortical (arrows) surfaces in Scl-Ab–treated ORX rats. Bone formation on (b) periosteal and endocortical surfaces was greater for both Scl-Ab doses compared with that in ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 6. View largeDownload slide Effects of Scl-Ab on cortical bone histomorphometry at the tibial shaft. (a) More calcein-labeled surface was observed on periosteal (arrowheads) and endocortical (arrows) surfaces in Scl-Ab–treated ORX rats. Bone formation on (b) periosteal and endocortical surfaces was greater for both Scl-Ab doses compared with that in ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Discussion This experiment demonstrates the efficacy of Scl-Ab in restoring bone mass and strength in an androgen-deficient, orchiectomized rat model with established osteopenia. Scl-Ab increased bone formation on periosteal, endocortical, and trabecular surfaces and decreased bone resorption on the endocortical and trabecular surfaces. These effects led to a rapid restoration of BMD by only 3 weeks of treatment in the lumbar spine and by 6 weeks of treatment in the long bones (tibia and femur). By the end of the study, the decreased bone strength (peak load) in the ORX rats had been completely restored to that of the sham control level in the L5 vertebra and the femur diaphysis and to greater than the sham control level in the femoral neck. The consistent and positive correlations between bone mass and bone strength indicated the maintenance of bone quality with Scl-Ab treatment. In the present study, adult male rats at 6 months of age were subjected to ORX to induce androgen deficiency. Osteopenia was established in both the lumbar spine and the long bones (tibia and femur) at 3 months after ORX, as demonstrated by a significant decrease in DXA BMD at these sites. Micro-CT analysis of L5 confirmed the decreases in bone mass indexes at 3 and 4.5 months after ORX, an effect reflected in the deterioration of both trabecular and cortical microarchitecture. Histomorphometric analysis of cortical bone in the tibial shaft showed a 98% reduction in periosteal BFR in ORX baseline controls compared with the sham baseline controls, supporting the effect of androgen deficiency in reducing periosteal bone formation. A significant increase in eroded surface, a bone resorption index, on the trabecular surfaces of L2 and endocortical surfaces of the tibial shafts at the end of the study in the ORX controls indicated the effects of androgen deficiency in increasing bone resorption. The increase in bone resorption and decrease in bone formation in these ORX rats are in agreement with observations in hypogonadal men, as reviewed by Khosla et al. (4). The combined effects of ORX on bone formation and bone resorption led to significantly lower bone mass and strength in these rats. Therefore, the model used in the present study mimics the conditions observed in male osteoporotic patients. The effects of Scl-Ab on trabecular bone mass and bone formation that we observed are similar to those reported in intact aged male rats (31). In both male osteopenic models, Scl-Ab administration increased the trabecular mineralizing surface, MAR, and BFR. These effects led to increases in the trabecular bone volume and Tb.Th. Also, the effect of Scl-Ab in increasing trabecular bone formation has been consistently observed in various animal models of osteopenia (23, 24, 31, 32). In our ORX rat study, and consistent with the ovariectomized (OVX) rat and the normal-loaded and underloaded aged female rat models, Scl-Ab induced significant decreases in the bone resorption parameters (24, 32). The reported absence of such an effect in aged male rats (31) is likely attributable to low bone turnover indexes in this aging model. The mechanism of inhibition of bone resorption by Scl-Ab is an area of continued investigation. The expression of osteoclast mediators might be altered by treatment with Scl-Ab, as reviewed by Ominsky et al. (18). The inhibition of bone resorption by Scl-Ab has also been shown in human clinical trials, in which Scl-Ab significantly decreased serum markers of bone resorption in both postmenopausal women with osteoporosis and healthy men (19, 20, 34). In the present study, treatment with Scl-Ab significantly increased the periosteal BFR more than 26- and 36-fold in the 5- and 25-mg/kg groups, respectively, compared with vehicle treatment in the ORX rats. This resulted from significant increases in both the periosteal mineralizing surface and the MAR. On the endocortical surface, the increased bone formation and decreased bone resorption induced by Scl-Ab led to a significant decrease in Ec.Pm. These effects contributed to the significant increases in cortical vBMD, Ct.B.Ar, and Ct.Th, as determined by micro-CT of the femur diaphysis. In general, the effects of Scl-Ab on the periosteal and endocortical surfaces of cortical bone were similar to those reported for aged OVX rats, aged male rats, and normal-loaded and underloaded female rats (24, 31, 32). Scl-Ab did not induce a significant increase in Ps.Pm, as BFR was dramatically increased on this surface in all these short-term rodent studies. In a longer-term, 12-month study in OVX rats, Scl-Ab significantly increased Ps.Pm (18), indicating that longer-term treatment might be required to observe this effect. In conclusion, in a rat model of male osteoporosis, we found that Scl-Ab treatment increased bone formation and decreased bone resorption, leading to restoration of bone mass and strength to levels similar to those observed in the sham controls. Increased bone mass and improved bone architecture at multiple skeletal sites after Scl-Ab treatment led to increased trabecular and cortical bone strength, as demonstrated by the significant dose-dependent increases in peak load in the lumbar vertebral body, femur diaphysis, and femoral neck. More importantly, increases in bone mass and bone strength remained well correlated across all groups, demonstrating that Scl-Ab significantly increased bone mass and maintained bone quality in this androgen-deficient, established osteopenia animal model. These results support the ongoing evaluation of sclerostin antibodies as therapeutic agents to restore bone mass and strength and thereby potentially reduce skeletal fragility fractures in hypogonadal and aged men with osteoporosis. Abbreviations: BFR bone formation rate BMD bone mineral density BS bone surface BV/TV bone volume/total volume CSMI cross-sectional moment of inertia Ct.B.Ar cortical bone area Ct.Th cortical thickness DXA dual-energy x-ray absorptiometry Ec.Pm endocortical perimeter ES/BS eroded surface/bone surface L5 fifth lumbar MAR mineral apposition rate micro-CT microcomputed tomography ORX orchiectomized OVX ovariectomized Ps.Pm periosteal perimeter Scl-Ab sclerostin antibody sham sham-operated Tb.N trabecular number Tb.Sp trabecular spacing Tb.Th trabecular thickness vBMC volumetric bone mineral content vBMD volumetric bone mineral density. Acknowledgments The authors thank the members of the sclerostin antibody project team at Amgen and UCB Pharma for their support and helpful discussions. The authors acknowledge Louise Profit, Gardiner-Caldwell Communications, Macclesfield, UK, for editorial assistance, which was funded by UCB Pharma. FinancialSupport: This study was supported by Amgen and UCB Pharma. Current Affiliation: M.S. Ominsky’s current affiliation is Radius Health, Inc., Waltham, Massachusetts 02451. K.S. Villasenor’s current affiliation is BRB Pet Products, Murrieta, California 92562. Q.-T. Niu’s current affiliation is Comparative Biology and Safety Science, Amgen Inc., Thousand Oaks, California 91320. X. Xia’s current affiliation is Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200030, China. Disclosure Summary: X.L. is an employee of Amgen and owns Amgen stocks/stock options; M.S.O. is a stockholder and former employee of Amgen, and a current employee and stockholder at Radius Health, Inc.; M.G. is a former employee of Amgen. K.S.V., Q.-T.N., F.J.A., and W.S.S. are former employees of Amgen and own Amgen stocks/stock options. H.Z.K. is an employee of UCB Pharma and owns Amgen and UCB stocks/stock options. T.J.W. owns Amgen stock. The remaining author has nothing to disclose. References 1. 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Sclerostin Antibody Reverses Bone Loss by Increasing Bone Formation and Decreasing Bone Resorption in a Rat Model of Male Osteoporosis

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00794
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Abstract

Abstract Sclerostin antibody (Scl-Ab) restored bone mass and strength in the ovariectomized rat model of postmenopausal osteoporosis. Increased bone mineral density (BMD) and decreased skeletal fragility fracture risk have been reported in postmenopausal osteoporotic women receiving Scl-Ab. In males, loss of androgen leads to rapid decreases in BMD and an increased risk of fragility fractures. We hypothesized that Scl-Ab could reverse the loss of bone mass and strength caused by androgen ablation in the orchiectomized (ORX) rat model of male osteoporosis. We treated 9-month-old ORX Sprague Dawley rats (3 months after ORX) subcutaneously twice weekly with vehicle or Scl-Ab (5 or 25 mg/kg) for 6 weeks (n = 10 per group). Both doses of Scl-Ab fully reversed the BMD deficit in the lumbar spine and femur and tibia in ORX rats. Microcomputed tomography showed that the bone mass in the fifth lumbar vertebral body, femur diaphysis, and femoral neck were dose-dependently restored by Scl-Ab. The bone strength at these sites increased significantly with Scl-Ab to levels matching those of sham-operated controls and correlated positively with improvements in bone mineral content, demonstrating bone quality maintenance. Dynamic histomorphometry of the tibial diaphysis and second lumbar vertebral body demonstrated that Scl-Ab significantly increased bone formation on periosteal, endocortical, and trabecular surfaces and significantly decreased bone resorption on endocortical and trabecular surfaces. The effects of Scl-Ab on increasing bone formation and decreasing bone resorption led to restoration of bone mass and strength in androgen-deficient rats. These findings support the ongoing evaluation of Scl-Ab as a potential therapeutic agent for osteoporosis in men. Osteoporosis in men is a major public health problem, with approximately one-third of all osteoporotic fractures occurring in men (1–3). As populations continue to age and life expectancy increases, the burden of osteoporotic fractures in men is expected to greatly increase (3, 4). Aging and gonadal hormone deficiency are two important risk factors for osteoporosis in men (4, 5). The loss of androgen leads to rapid decreases in bone mineral density (BMD) and increased susceptibility to osteoporotic fractures. Aged male rats and male rats with androgen deficiency induced by orchiectomy have been used as animal models to test the efficacy of therapeutic agents aimed at preventing and treating osteoporosis in men (6–8). One important therapeutic target emerging in bone formation is sclerostin, a protein that binds to the low-density lipoprotein receptor-related protein 4/5/6 Wnt coreceptors to inhibit canonical Wnt signaling in osteoblast lineage cells (9–13). Higher bone formation and bone mass have been observed in people with a genetic disorder that results in a lifelong absence of functional sclerostin (14) and in sclerostin knockout mice (15, 16). Several monoclonal antibodies against sclerostin (Scl-Abs) have been developed as potential bone-forming agents for the treatment of conditions with a low bone mass (17). Preclinical studies have shown that Scl-Ab activates Wnt signaling in osteoblast lineage cells, thus increasing bone formation via the activation of bone-lining cells, bone matrix production by osteoblasts, and recruitment of osteoprogenitor cells (18). Furthermore, Scl-Ab induces expression changes in osteoclast mediators, thereby decreasing bone resorption (18). These effects resulted in rapid increases in trabecular and cortical bone mass and improvements in bone structure and strength in several animal models of bone loss (17, 18). In clinical trials, Scl-Abs have been shown to rapidly increase BMD and decrease the risk of osteoporotic fractures in postmenopausal women (19, 20). Although most preclinical investigations have focused on animal models relevant to postmenopausal osteoporosis, Scl-Ab has also been studied in animal models of immobilization-induced bone loss (21–25), osteogenesis imperfecta (26–28), diabetes-related bone loss (29), and radiation-induced bone loss (30). In addition, we previously showed that Scl-Ab increased bone formation, mass, and strength in an aged male rat model of osteoporosis (31). In the present study, we investigated the effects of Scl-Ab in the orchiectomized (ORX) rat, an androgen-deficient male osteoporosis model. We hypothesized that Scl-Ab treatment would reverse the loss of bone mass and strength caused by androgen ablation in the established osteopenia ORX rat model. Materials and Methods The male rats used in the present study were housed two per cage in an environmentally controlled room (temperature 20°C to 22°C, relative humidity 34% to 73%) at an Amgen animal facility (Thousand Oaks, CA). The room was kept at a 12-hour light/dark cycle and met all specifications of the Association for Assessment and Accreditation of Laboratory Animal Care International. Water and food were supplied ad libitum. The institutional animal care and use committee of Amgen Inc. (Thousand Oaks, CA) approved the protocol and procedures. Six-month-old male Sprague Dawley rats (Envigo, Indianapolis, IN) were either sham-operated (sham) or underwent orchiectomy (ORX) and were left untreated for 3 months, allowing for the development of osteopenia. The body weights were recorded, and the areal BMD was determined in vivo using dual-energy x-ray absorptiometry (DXA; Hologic QDR 4500a; Bedford, MA) before the initiation of treatment. These measurements were used to balance the groups, such that all groups had a similar mean body weight and BMD before the start of treatment. These 9-month-old ORX rats were treated with either vehicle (10 mM sodium acetate, 9% sucrose, pH 5.2, with 0.004% Tween 20) or rat sclerostin antibody (Scl-AbIII; Amgen Inc.) at 5 or 25 mg/kg, twice weekly by subcutaneous injection for 6 weeks. DXA scans were performed on isoflurane-anesthetized rats at weeks 3 and 6. One group of rats was euthanized before surgery and the sham and ORX rats were euthanized before the treatment period. Ten rats were in each subgroup. The rats were injected subcutaneously with xylenol orange (90 mg/kg) at the start of Scl-Ab treatment and with calcein (20 mg/kg) at 13 and 3 days before necropsy. The two calcein labels were used for histomorphometric analysis of cortical and trabecular bone. At necropsy, the second lumbar (L2) vertebra and the right tibia were collected from each rat for dynamic histomorphometry, and the third to fifth lumbar vertebrae and left femur were collected for microcomputed tomography (micro-CT) and strength assessment. Bone densitometry Areal BMD was determined in vivo in isoflurane-anesthetized rats using DXA, as described previously (32). The regions of interest included the lumbar 1 to 5 vertebrae and the femur–tibia (entire femur and the proximal half of the tibia). The volumetric BMD (vBMD) and microarchitecture were determined in the right femurs and fifth lumbar (L5) vertebrae using a desktop micro-CT system (GE eXplore Locus SP Specimen Scanner; GE Health Care, London, ON, Canada). Whole femurs and vertebrae were scanned, and the images were reconstructed to an isotropic voxel size of 30 and 18 μm3, respectively. For cortical diaphyseal analysis, a central region equivalent to 10% of the height of the femur (measured from the apex of the femoral head to the base of the condyles) was selected distally to the midpoint. For femoral neck analysis, the proximal femur was reoriented relative to the neck axis, and a 0.3-mm region was selected at its narrowest aspect. For distal femur analysis, a trabecular region equivalent to 10% of the femur height was selected, beginning 0.3 mm from the most proximal aspect of the growth plate. For vertebral body analysis, a central region was selected equivalent to 70% of the vertebral body height. The trabecular bone within each region was segmented from the cortex using a semiautomated contouring algorithm, and the vertebral cortex was separately examined. At each site, the volumetric bone mineral content (vBMC) and vBMD were determined without thresholding. For the microarchitectural parameters, the thresholds were applied for all samples at the L5 vertebra (480 mg/mL), distal femur (500 mg/mL), femur diaphysis (800 mg/mL), and femoral neck (500 mg/mL). At the femur diaphysis, analyses were run to generate the periosteal perimeter (Ps.Pm) and endocortical perimeter (Ec.Pm), cortical thickness (Ct.Th), cortical bone area (Ct.B.Ar), cross-sectional moment of inertia (CSMI), and tissue mineral density, as well as the distance from the centroid to the posterior edge, used in the strength calculations provided in subsequent sections. The femoral neck endpoints included the bone area, vBMC, vBMD, bone volume fraction [bone volume/total volume (BV/TV)], and Ps.Pm. The calculated trabecular parameters included the trabecular vBMD (both total and tissue), trabecular BV/TV, trabecular number (Tb.N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th), and structural model index. At the vertebra, the total, cortical, and trabecular region data are reported. Bone strength Femurs and lumbar vertebrae were harvested, wrapped in gauze, and stored in phosphate-buffered saline at −20°C. On the day of testing, the femurs were kept hydrated and removed from the saline solution only for mechanical testing. Strength testing was performed only on the groups that had completed the 6-week treatment period. The method of testing was described in our previous report (32) and is summarized in the next paragraphs. L5 vertebral bodies were tested in compression to failure using an MTS 858 Mini Bionix servohydraulic system (MTS Systems Corp., Eden Prairie, MN). Plano-parallel specimens (approximately 5 mm in height) were first prepared by removing the laminae using a diamond wire saw (model 3241; Well Diamond Wire Saws Inc., Norcross, GA), and the sample height was recorded with calipers. During compression testing at 6 mm/min, the load–displacement curve was recorded, and the extrinsic strength parameters (peak load, stiffness, and energy to peak load) and displacement to peak load were generated within Microsoft Excel (Microsoft Corp., Redmond, WA). The estimated material properties were calculated using micro-CT–derived bone area as follows: ultimate strength = peak load/bone area; modulus = stiffness/(bone area/height); toughness = energy/(bone area × height). Femur diaphyses were tested in a three-point bending configuration using the MTS servohydraulic system (MTS Systems Corp.). A preload of 5 N was applied before loading the bones to failure at a displacement rate of 3 mm/min. A load transducer of 1 kN ± 0.02 N was used to record the force measurements. The span length between supports was set to 20 mm. The load–displacement curve was recorded by the testing system, and the extrinsic strength parameters (peak load, stiffness, and energy to failure) and ultimate displacement were generated. The material properties (ultimate strength, modulus, and toughness) were calculated using the CSMI and the distance from the centroid to the posterior edge per the standard engineering approach for three-point bending tests. After diaphyseal testing, the proximal one-half of the left femur was fixed in a drill chuck for destructive shear testing of the femoral neck. The femoral head was displaced at 1 mm/s until failure occurred. The load–displacement curves were analyzed for peak load, stiffness, and energy to failure. Bone histomorphometric analysis Undecalcified parasagittal 4-µm-thick sections of the L2 vertebral bodies and 6-µm-thick transverse sections of tibia at the tibiofibular junction were prepared as described previously (32). The vertebral body sections were either stained with modified Goldner trichrome for analysis of static parameters or left unstained for collection of fluorochrome-based data. Tibial shaft sections were left unstained for collection of both fluorochrome-based and static parameter data. Histomorphometric analyses were performed using Osteomeasure bone analysis software (Osteometrics, Inc., Decatur, GA). The region of interest for the lumbar vertebral body trabecular bone included all trabecular bone within the parasagittal section. Static and dynamic parameters were calculated and expressed according to reported methods (33). Statistical analysis All results are expressed as the mean ± standard error of the mean. Longitudinal DXA data were analyzed using a two-way analysis of variance followed by the Tukey multiple comparison test. All other endpoints were subjected to one-way analysis of variance, followed by the Tukey test. These analyses were performed in GraphPad Prism software, version 5.01 (GraphPad Software Inc., San Diego, CA), with a P value of <0.05 used to identify significant pairwise group differences. Linear regression analysis was applied at each strength-tested site to correlate the peak load with the vBMC across groups. Regression coefficients (r2) are provided to demonstrate the strength of association, and positive relationships were confirmed if the slope was significantly different from zero at P < 0.05. Results Confirmation of expected organ and body weight changes after ORX ORX resulted in a significant decrease in the weight of the seminal vesicle, prostate gland, and total body weight before treatment, an effect that persisted throughout the treatment period (Table 1). Scl-Ab treatment for 6 weeks did not significantly alter any of these variables. Table 1. Effect of ORX and Scl-Ab on Seminal Vesicle, Prostate Gland, and Body Weight Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Seminal vesicle weight (g)  1.910 ± 0.133  0.232 ± 0.020a  1.748 ± 0.123  0.188 ± 0.017a  0.191 ± 0.011a  0.183 ± 0.020a  Prostate gland weight (g)  1.652 ± 0.118  0.308 ± 0.021a  1.424 ± 0.095  0.210 ± 0.016a  0.185 ± 0.015a  0.204 ± 0.016a  Terminal body weight (g)  684.0 ± 20.8  618.5 ± 15.3a  727.7 ± 26.9  628.7 ± 25.3a  601.5 ± 27.2a  630.8 ± 33.2a  Baseline body weight (g)  NA  NA  714.7 ± 26.0  654.5 ± 23.2a  631.7 ± 28.3a  656.5 ± 29.6a  Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Seminal vesicle weight (g)  1.910 ± 0.133  0.232 ± 0.020a  1.748 ± 0.123  0.188 ± 0.017a  0.191 ± 0.011a  0.183 ± 0.020a  Prostate gland weight (g)  1.652 ± 0.118  0.308 ± 0.021a  1.424 ± 0.095  0.210 ± 0.016a  0.185 ± 0.015a  0.204 ± 0.016a  Terminal body weight (g)  684.0 ± 20.8  618.5 ± 15.3a  727.7 ± 26.9  628.7 ± 25.3a  601.5 ± 27.2a  630.8 ± 33.2a  Baseline body weight (g)  NA  NA  714.7 ± 26.0  654.5 ± 23.2a  631.7 ± 28.3a  656.5 ± 29.6a  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL, baseline; NA, not applicable. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Bone densitometry Osteopenia was reflected in reduced DXA BMD at the lumbar vertebrae and femur–tibia sites in the ORX rats compared with the sham controls at 3 months after orchiectomy (12 weeks; Fig. 1). Relative to the ORX controls, Scl-Ab significant increased the BMD at the lumbar spine [Fig. 1(a)] and at the femur–tibia sites [Fig. 1(b)] after 3 and 6 weeks of treatment at 5 and 25 mg/kg (15 and 18 weeks after surgery, respectively). Scl-Ab treatment at both doses normalized the femur–tibia BMD to that of the sham control levels. Furthermore, Scl-Ab at 25 mg/kg significantly increased the lumbar BMD relative to that of the sham controls. Figure 1. View largeDownload slide Scl-Ab restored BMD at the (a) lumbar spine and (b) femur–tibia in ORX rats. In vivo DXA scans were performed before the initiation of treatment (12 weeks after surgery) and at 3 and 6 weeks after dosing (15 and 18 weeks after surgery, respectively). Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 1. View largeDownload slide Scl-Ab restored BMD at the (a) lumbar spine and (b) femur–tibia in ORX rats. In vivo DXA scans were performed before the initiation of treatment (12 weeks after surgery) and at 3 and 6 weeks after dosing (15 and 18 weeks after surgery, respectively). Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Representative micro-CT images of the L5 vertebrae for each group at the end of the treatment period are shown in Fig. 2(a). Using micro-CT, the ORX vertebrae showed a significantly lower total vBMC and total bone area compared with the sham vertebrae at 3 months after orchiectomy (Table 2). These changes stemmed from a significant decrease in the cortical region vBMC and bone area, accompanied by a nonsignificant decrease in trabecular vBMC and trabecular bone area. These effects persisted throughout the treatment period, with further reductions with ORX in the trabecular compartment. Both doses of Scl-Ab significantly increased the vBMC, vBMD, and bone area of the total region after 6 weeks of treatment (18 weeks after surgery), without affecting the vertebral body total (cross-sectional) area. In the trabecular compartment, Scl-Ab resulted in significant increases in vBMD and BV/TV relative to ORX [Table 2; Fig. 2(b)] and Tb.Th relative to both ORX and sham controls (Table 2). At both doses, Scl-Ab induced increases in the vertebral cortical region vBMC, bone area, and cortical thickness, relative to both ORX and sham controls. Figure 2. View largeDownload slide Scl-Ab reversed cortical and trabecular bone loss at the L5 vertebral body of ORX rats. (a) Representative micro-CT images of L5 vertebrae (18-µm resolution). Thicker cortex and thicker trabeculae were observed in Scl-Ab–treated ORX rats. (b) Scl-Ab (start of treatment at 12 weeks after surgery indicated by arrows) restored cortical thickness and trabecular BV/TV in ORX rats. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 2. View largeDownload slide Scl-Ab reversed cortical and trabecular bone loss at the L5 vertebral body of ORX rats. (a) Representative micro-CT images of L5 vertebrae (18-µm resolution). Thicker cortex and thicker trabeculae were observed in Scl-Ab–treated ORX rats. (b) Scl-Ab (start of treatment at 12 weeks after surgery indicated by arrows) restored cortical thickness and trabecular BV/TV in ORX rats. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Table 2. Effect of Scl-Ab on L5 Vertebral Bone Mass and Microarchitecture in ORX Rats Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Total               vBMC (mg/mm)  6.51 ± 0.32  5.75 ± 0.13a  6.69 ± 0.09  5.77 ± 0.21a  6.77 ± 0.22b  7.26 ± 0.26b   vBMD (mg/mL)  551.3 ± 16.2  516.4 ± 12.5  553.0 ± 8.8  474.6 ± 14.1a  571.8 ± 8.9b  590.6 ± 15.1b   Area (mm2)  11.79 ± 0.36  11.17 ± 0.29  12.14 ± 0.29  12.17 ± 0.27  11.82 ± 0.24  12.30 ± 0.28   Bone area (mm2)  5.67 ± 0.31  4.80 ± 0.13a  5.71 ± 0.06  4.73 ± 0.19a  5.84 ± 0.23b  6.35 ± 0.27b  Trabecular               vBMC (mg/mm)  3.10 ± 0.20  2.71 ± 0.10  3.14 ± 0.09  2.55 ± 0.12a  2.83 ± 0.15  3.09 ± 0.19   vBMD (mg/mL)  421.1 ± 18.9  382.7 ± 14.6  421.3 ± 9.7  337.3 ± 15.0a  418.2 ± 13.5b  444.3 ± 20.0b   Bone area (mm2)  7.33 ± 0.26  7.11 ± 0.22  7.47 ± 0.24  7.57 ± 0.20  6.73 ± 0.17b  6.95 ± 0.24   BV/TV (%)  32.63 ± 2.20  27.91 ± 1.53  31.91 ± 1.09  23.62 ± 1.49a  31.71 ± 1.69b  34.93 ± 2.24b   Tb.N (1/mm)  4.30 ± 0.24  3.94 ± 0.19  4.23 ± 0.13  3.44 ± 0.15a  3.51 ± 0.15a  3.48 ± 0.22a   Tb.Th (mm)  0.067 ± 0.002  0.062 ± 0.001  0.066 ± 0.001  0.060 ± 0.002  0.090 ± 0.003a,b  0.103 ± 0.004a,b   SMI  −0.05 ± 0.15  0.36 ± 0.10a  −0.04 ± 0.09  0.45 ± 0.10a  0.24 ± 0.14  0.15 ± 0.14  Cortical region               vBMC (mg/mm)  3.42 ± 0.12  3.04 ± 0.07a  3.55 ± 0.08  3.22 ± 0.09  3.95 ± 0.09a,b  4.17 ± 0.10a,b   vBMD (mg/mL)  765.3 ± 10.8  749.3 ± 9.5  762.7 ± 7.7  700.8 ± 9.9a  774.1 ± 7.1b  779.4 ± 7.9b   Area (mm2)  4.46 ± 0.12  4.06 ± 0.09a  4.67 ± 0.12  4.60 ± 0.10  5.10 ± 0.09a,b  5.35 ± 0.09a,b   Bone area (mm2)  3.27 ± 0.12  2.83 ± 0.07a  3.33 ± 0.07  2.95 ± 0.09a  3.69 ± 0.08a,b  3.92 ± 0.11a,b   Ct.Th (mm)  0.243 ± 0.007  0.219 ± 0.006a  0.248 ± 0.005  0.217 ± 0.008a  0.274 ± 0.005a,b  0.290 ± 0.008a,b  Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Total               vBMC (mg/mm)  6.51 ± 0.32  5.75 ± 0.13a  6.69 ± 0.09  5.77 ± 0.21a  6.77 ± 0.22b  7.26 ± 0.26b   vBMD (mg/mL)  551.3 ± 16.2  516.4 ± 12.5  553.0 ± 8.8  474.6 ± 14.1a  571.8 ± 8.9b  590.6 ± 15.1b   Area (mm2)  11.79 ± 0.36  11.17 ± 0.29  12.14 ± 0.29  12.17 ± 0.27  11.82 ± 0.24  12.30 ± 0.28   Bone area (mm2)  5.67 ± 0.31  4.80 ± 0.13a  5.71 ± 0.06  4.73 ± 0.19a  5.84 ± 0.23b  6.35 ± 0.27b  Trabecular               vBMC (mg/mm)  3.10 ± 0.20  2.71 ± 0.10  3.14 ± 0.09  2.55 ± 0.12a  2.83 ± 0.15  3.09 ± 0.19   vBMD (mg/mL)  421.1 ± 18.9  382.7 ± 14.6  421.3 ± 9.7  337.3 ± 15.0a  418.2 ± 13.5b  444.3 ± 20.0b   Bone area (mm2)  7.33 ± 0.26  7.11 ± 0.22  7.47 ± 0.24  7.57 ± 0.20  6.73 ± 0.17b  6.95 ± 0.24   BV/TV (%)  32.63 ± 2.20  27.91 ± 1.53  31.91 ± 1.09  23.62 ± 1.49a  31.71 ± 1.69b  34.93 ± 2.24b   Tb.N (1/mm)  4.30 ± 0.24  3.94 ± 0.19  4.23 ± 0.13  3.44 ± 0.15a  3.51 ± 0.15a  3.48 ± 0.22a   Tb.Th (mm)  0.067 ± 0.002  0.062 ± 0.001  0.066 ± 0.001  0.060 ± 0.002  0.090 ± 0.003a,b  0.103 ± 0.004a,b   SMI  −0.05 ± 0.15  0.36 ± 0.10a  −0.04 ± 0.09  0.45 ± 0.10a  0.24 ± 0.14  0.15 ± 0.14  Cortical region               vBMC (mg/mm)  3.42 ± 0.12  3.04 ± 0.07a  3.55 ± 0.08  3.22 ± 0.09  3.95 ± 0.09a,b  4.17 ± 0.10a,b   vBMD (mg/mL)  765.3 ± 10.8  749.3 ± 9.5  762.7 ± 7.7  700.8 ± 9.9a  774.1 ± 7.1b  779.4 ± 7.9b   Area (mm2)  4.46 ± 0.12  4.06 ± 0.09a  4.67 ± 0.12  4.60 ± 0.10  5.10 ± 0.09a,b  5.35 ± 0.09a,b   Bone area (mm2)  3.27 ± 0.12  2.83 ± 0.07a  3.33 ± 0.07  2.95 ± 0.09a  3.69 ± 0.08a,b  3.92 ± 0.11a,b   Ct.Th (mm)  0.243 ± 0.007  0.219 ± 0.006a  0.248 ± 0.005  0.217 ± 0.008a  0.274 ± 0.005a,b  0.290 ± 0.008a,b  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL, baseline; SMI, structural model index. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Representative micro-CT images of the femur diaphysis, femur distal metaphysis, and femoral neck for each group at the end of the treatment period are shown in Fig. 3. ORX resulted in impaired periosteal expansion, corresponding to a decrease in periosteal perimeter, bone area, and CSMI relative to sham at the femur diaphysis at 3 months after orchiectomy (Table 3). By the end of the study period, the bone area and Ct.Th were significantly lower in the ORX group compared with the sham group. Scl-Ab resulted in significant increases in cortical vBMC, Ct.B.Ar, and Ct.Th at the femur diaphysis, with no significant changes in Ec.Pm or Ps.Pm observed (Table 3). At the distal femur metaphysis, ORX induced significant loss in trabecular vBMC, trabecular region area, BV/TV, and Tb.N at 3 months after orchiectomy. Scl-Ab improved vBMD and BV/TV relative to the ORX controls and Tb.Th relative to both ORX and sham controls (Table 3). ORX did not result in significant bone loss at the femoral neck at any time point (data not shown). Significant increases in bone area, but no other parameters, were observed at this site after Scl-Ab treatment (3.69 ± 0.07 mm2 vs 4.29 ± 0.19 mm2 and 4.53 ± 0.16 mm2, ORX control vs Scl-Ab 5 and 25 mg/kg, respectively; P < 0.05). Figure 3. View largeDownload slide Representative micro-CT images of the femoral neck, diaphysis, and distal metaphysis from sham controls, ORX controls, and ORX rats treated with Scl-Ab for 6 weeks. Micro-CT was performed on right femurs (32-µm resolution) for all samples. Representative images were selected according to the group median for the bone mass parameters. A greater bone mass at all three sites was observed in the ORX rats treated with Scl-Ab at both doses. Figure 3. View largeDownload slide Representative micro-CT images of the femoral neck, diaphysis, and distal metaphysis from sham controls, ORX controls, and ORX rats treated with Scl-Ab for 6 weeks. Micro-CT was performed on right femurs (32-µm resolution) for all samples. Representative images were selected according to the group median for the bone mass parameters. A greater bone mass at all three sites was observed in the ORX rats treated with Scl-Ab at both doses. Table 3. Effect of Scl-Ab on Femur Bone Mass and Microarchitecture in ORX Rats Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Femur diaphysis               Ct. vBMC (mg/mm)  9.11 ± 0.17  8.62 ± 0.19  9.61 ± 0.14  8.49 ± 0.20  9.00 ± 0.21b  9.38 ± 0.28b   Ct. TMD (mg/mL)  1093.0 ± 3.4  1122.1 ± 4.0  1104.4 ± 5.7  1098.5 ± 2.1  1096.0 ± 7.0  1095.4 ± 9.4   Ct. bone area (mm2)  8.43 ± 0.15  7.77 ± 0.19a  8.81 ± 0.12  7.82 ± 0.17a  8.31 ± 0.20b  8.66 ± 0.24b   Ct.Th (mm)  0.80 ± 0.01  0.78 ± 0.01  0.83 ± 0.02  0.76 ± 0.01a  0.82 ± 0.01  0.84 ± 0.02b   CSMI (mm4)  10.70 ± 0.55  8.60 ± 0.66a  11.18 ± 0.46  9.29 ± 0.59  9.52 ± 0.61  10.66 ± 0.59   Ec.Pm (mm)  9.11 ± 0.19  8.37 ± 0.18a  8.93 ± 0.23  8.84 ± 0.27  8.54 ± 0.21  8.51 ± 0.17   Ps.Pm (mm)  14.00 ± 0.17  13.25 ± 0.19a  14.06 ± 0.16  13.59 ± 0.23  13.61 ± 0.22  13.78 ± 0.17  Distal femur metaphysis               Tb.vBMC (mg/mm)  3.04 ± 0.29  2.10 ± 0.11a  3.17 ± 0.13  2.08 ± 0.11a  2.45 ± 0.17a  2.58 ± 0.15a   Tb.vBMD (mg/mL)  230.20 ± 18.01  190.54 ± 9.69  238.59 ± 9.01  180.11 ± 9.08a  211.44 ± 10.57  232.34 ± 13.92b   Tb. region area (mm2)  13.08 ± 0.48  11.07 ± 0.42a  13.34 ± 0.49  11.55 ± 0.34a  11.52 ± 0.50b  11.17 ± 0.41b   BV/TV (%)  9.71 ± 1.92  5.21 ± 0.71a  10.04 ± 0.86  4.77 ± 0.47a  8.27 ± 0.86b  10.19 ± 1.08b   Tb.N. (1/mm)  1.56 ± 0.27  0.84 ± 0.13a  1.55 ± 0.13  0.72 ± 0.11a  1.06 ± 0.13a  1.17 ± 0.14   Tb.Th. (mm)  0.048 ± 0.002  0.048 ± 0.002  0.050 ± 0.002  0.048 ± 0.001  0.068 ± 0.003a,b  0.080 ± 0.003a,b   SMI  2.36 ± 0.12  2.81 ± 0.06a  2.28 ± 0.10  2.62 ± 0.09  2.38 ± 0.08  2.34 ± 0.14  Site  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Femur diaphysis               Ct. vBMC (mg/mm)  9.11 ± 0.17  8.62 ± 0.19  9.61 ± 0.14  8.49 ± 0.20  9.00 ± 0.21b  9.38 ± 0.28b   Ct. TMD (mg/mL)  1093.0 ± 3.4  1122.1 ± 4.0  1104.4 ± 5.7  1098.5 ± 2.1  1096.0 ± 7.0  1095.4 ± 9.4   Ct. bone area (mm2)  8.43 ± 0.15  7.77 ± 0.19a  8.81 ± 0.12  7.82 ± 0.17a  8.31 ± 0.20b  8.66 ± 0.24b   Ct.Th (mm)  0.80 ± 0.01  0.78 ± 0.01  0.83 ± 0.02  0.76 ± 0.01a  0.82 ± 0.01  0.84 ± 0.02b   CSMI (mm4)  10.70 ± 0.55  8.60 ± 0.66a  11.18 ± 0.46  9.29 ± 0.59  9.52 ± 0.61  10.66 ± 0.59   Ec.Pm (mm)  9.11 ± 0.19  8.37 ± 0.18a  8.93 ± 0.23  8.84 ± 0.27  8.54 ± 0.21  8.51 ± 0.17   Ps.Pm (mm)  14.00 ± 0.17  13.25 ± 0.19a  14.06 ± 0.16  13.59 ± 0.23  13.61 ± 0.22  13.78 ± 0.17  Distal femur metaphysis               Tb.vBMC (mg/mm)  3.04 ± 0.29  2.10 ± 0.11a  3.17 ± 0.13  2.08 ± 0.11a  2.45 ± 0.17a  2.58 ± 0.15a   Tb.vBMD (mg/mL)  230.20 ± 18.01  190.54 ± 9.69  238.59 ± 9.01  180.11 ± 9.08a  211.44 ± 10.57  232.34 ± 13.92b   Tb. region area (mm2)  13.08 ± 0.48  11.07 ± 0.42a  13.34 ± 0.49  11.55 ± 0.34a  11.52 ± 0.50b  11.17 ± 0.41b   BV/TV (%)  9.71 ± 1.92  5.21 ± 0.71a  10.04 ± 0.86  4.77 ± 0.47a  8.27 ± 0.86b  10.19 ± 1.08b   Tb.N. (1/mm)  1.56 ± 0.27  0.84 ± 0.13a  1.55 ± 0.13  0.72 ± 0.11a  1.06 ± 0.13a  1.17 ± 0.14   Tb.Th. (mm)  0.048 ± 0.002  0.048 ± 0.002  0.050 ± 0.002  0.048 ± 0.001  0.068 ± 0.003a,b  0.080 ± 0.003a,b   SMI  2.36 ± 0.12  2.81 ± 0.06a  2.28 ± 0.10  2.62 ± 0.09  2.38 ± 0.08  2.34 ± 0.14  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL, baseline; SMI, structural model index; Tb, trabecular; TMD, tissue mineral density. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Bone strength The observed decreases in bone mass after ORX corresponded to losses in whole bone strength, most notably in the L5 vertebrae (Table 4). Across the L5 vertebra, femur diaphysis, and femoral neck, the strength parameters were lower in the ORX rats than in the sham controls (Table 4). Scl-Ab significantly increased all the L5 vertebra whole bone strength parameters relative to the ORX controls at both doses (Table 4), and the peak load and stiffness were also increased at the femur diaphysis at the 25-mg/kg dose. At the femoral neck, the peak load was significantly increased for both Scl-Ab doses, with no differences in the other whole bone strength parameters (Table 4). Table 4. Effect of Scl-Ab on L5 Vertebra Bone Strength in ORX Rats Site  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  L5 vertebra           Specimen height  3.71 ± 0.01  3.74 ± 0.02  3.74 ± 0.01  3.71 ± 0.01   Peak load (N)  459.8 ± 22.2  317.4 ± 23.5a  520.9 ± 30.9b  565.0 ± 36.9b   Stiffness (N/mm)  3739 ± 141  2820 ± 247  4514 ± 358b  4667 ± 287b   Energy at peak load (N*mm)  41.2 ± 3.6  27.5 ± 2.5a  46.5 ± 2.8b  50.5 ± 3.5b   Displacement at peak load (mm)  0.20 ± 0.01  0.19 ± 0.01  0.18 ± 0.01  0.19 ± 0.01   Ultimate strength (MPa)  80.7 ± 4.1  66.4 ± 2.8a  89.0 ± 3.0b  88.7 ± 4.0b   Modulus (MPa)  2432 ± 83  2208 ± 159  2888 ± 179b  2741 ± 152   Toughness (MPa)  1.95 ± 0.17  1.53 ± 0.08  2.12 ± 0.07b  2.13 ± 0.09b  Femur diaphysis           Peak load (N)  241.4 ± 8.7  204.9 ± 5.2a  229.7 ± 6.4  254.2 ± 13.6b   Stiffness (N/mm)  713.6 ± 32.5  624.7 ± 23.3  678.9 ± 22.2  743.7 ± 34.9b   Energy (N*mm)  118.1 ± 12.0  83.2 ± 7.2  98.6 ± 10.2  116.1 ± 11.1   Ultimate displacement (mm)  0.72 ± 0.06  0.61 ± 0.03  0.65 ± 0.05  0.67 ± 0.03   Ultimate strength (MPa)  199.8 ± 8.0  204.3 ± 11.3  221.4 ± 8.0  218.2 ± 8.1   Elastic modulus (MPa)  10708 ± 466  11542 ± 703  12128 ± 459  11755 ± 476   Toughness (MPa)  5.38 ± 0.56  4.45 ± 0.40  5.01 ± 0.38  5.40 ± 0.45  Femoral neck           Peak load (N)  190.3 ± 11.8  162.8 ± 5.5  205.6 ± 15.0b  228.6 ± 7.5a,b   Stiffness (N/mm)  757.8 ± 46.2  718.2 ± 52.6  710.6 ± 38.4  725.3 ± 15.1   Energy (N*mm)  49.1 ± 6.2  42.9 ± 3.4  70.6 ± 13.3  77.0 ± 8.8  Site  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  L5 vertebra           Specimen height  3.71 ± 0.01  3.74 ± 0.02  3.74 ± 0.01  3.71 ± 0.01   Peak load (N)  459.8 ± 22.2  317.4 ± 23.5a  520.9 ± 30.9b  565.0 ± 36.9b   Stiffness (N/mm)  3739 ± 141  2820 ± 247  4514 ± 358b  4667 ± 287b   Energy at peak load (N*mm)  41.2 ± 3.6  27.5 ± 2.5a  46.5 ± 2.8b  50.5 ± 3.5b   Displacement at peak load (mm)  0.20 ± 0.01  0.19 ± 0.01  0.18 ± 0.01  0.19 ± 0.01   Ultimate strength (MPa)  80.7 ± 4.1  66.4 ± 2.8a  89.0 ± 3.0b  88.7 ± 4.0b   Modulus (MPa)  2432 ± 83  2208 ± 159  2888 ± 179b  2741 ± 152   Toughness (MPa)  1.95 ± 0.17  1.53 ± 0.08  2.12 ± 0.07b  2.13 ± 0.09b  Femur diaphysis           Peak load (N)  241.4 ± 8.7  204.9 ± 5.2a  229.7 ± 6.4  254.2 ± 13.6b   Stiffness (N/mm)  713.6 ± 32.5  624.7 ± 23.3  678.9 ± 22.2  743.7 ± 34.9b   Energy (N*mm)  118.1 ± 12.0  83.2 ± 7.2  98.6 ± 10.2  116.1 ± 11.1   Ultimate displacement (mm)  0.72 ± 0.06  0.61 ± 0.03  0.65 ± 0.05  0.67 ± 0.03   Ultimate strength (MPa)  199.8 ± 8.0  204.3 ± 11.3  221.4 ± 8.0  218.2 ± 8.1   Elastic modulus (MPa)  10708 ± 466  11542 ± 703  12128 ± 459  11755 ± 476   Toughness (MPa)  5.38 ± 0.56  4.45 ± 0.40  5.01 ± 0.38  5.40 ± 0.45  Femoral neck           Peak load (N)  190.3 ± 11.8  162.8 ± 5.5  205.6 ± 15.0b  228.6 ± 7.5a,b   Stiffness (N/mm)  757.8 ± 46.2  718.2 ± 52.6  710.6 ± 38.4  725.3 ± 15.1   Energy (N*mm)  49.1 ± 6.2  42.9 ± 3.4  70.6 ± 13.3  77.0 ± 8.8  Data presented as mean ± standard error of the mean (n = 10 per group). a P < 0.05 vs time-matched sham control by one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Correlations between the bone mass (vBMC) and bone strength were examined to determine the change in bone material properties. Across the L5 vertebra, femur diaphysis, and femoral neck, Scl-Ab treatment maintained the significant and positive correlations between the vBMC and peak load observed in controls (Fig. 4), indicating that Scl-Ab treatment maintained bone quality in this ORX rat model. Figure 4. View largeDownload slide Linear regressions of vBMC to peak load at (a) L5 vertebra, (b) femur diaphysis, and (c) femoral neck. The peak load generated from each test was correlated with the vBMC across groups, demonstrating a consistent positive relationship. All correlations were significantly different from zero (P < 0.0001). Figure 4. View largeDownload slide Linear regressions of vBMC to peak load at (a) L5 vertebra, (b) femur diaphysis, and (c) femoral neck. The peak load generated from each test was correlated with the vBMC across groups, demonstrating a consistent positive relationship. All correlations were significantly different from zero (P < 0.0001). Histomorphometric analysis Consistent with the micro-CT results, ORX resulted in significantly lower BV/TV (−26%) and Tb.N (−18%) and higher Tb.Sp (+37%) compared with sham controls at L2 at the end of the study period (Table 5). These changes corresponded to a significant increase in the ratio of eroded surface to bone surface (ES/BS), a bone resorption index, in the ORX controls compared with the sham controls. Both doses of Scl-Ab restored BV/TV to the sham control levels owing to robust increases in Tb.Th. In the Scl-Ab–treated ORX rats, these increases were associated with greater bone formation [i.e., mineralizing surface/BS, mineral apposition rate (MAR), and bone formation rate (BFR)/BS, and with lower bone resorption (ES/BS)] [Fig. 5(a) and 5(b)]. Table 5. Histomorphometric Analysis of Trabecular Bone at L2 Vertebral Body Parameter  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  BV/TV (%)  33.1 ± 0.8  24.4 ± 1.2a  34.5 ± 2.2b  33.2 ± 1.7b  Tb.Th (µm)  92.9 ± 2.6  82.0 ± 2.0  121.4 ± 5.9b  129.5 ± 8.5b  Tb.Sp (µm)  188.8 ± 7.3  258.9 ± 12.6a  235.1 ± 15.6  260.4 ± 12.7  Tb.N (1/mm)  3.58 ± 0.12  2.97 ± 0.11a  2.85 ± 0.12a  2.59 ± 0.09a  MS/BS (%)  26.8 ± 1.6  27.6 ± 2.1  44.3 ± 2.0a,b  38.0 ± 3.2a,b  MAR (µm/d)  1.02 ± 0.04  1.13 ± 0.08  1.39 ± 0.07a,b  1.25 ± 0.08  BFR/BS (µm3/µm2/d)  0.274 ± 0.020  0.320 ± 0.036  0.619 ± 0.043a,b  0.482 ± 0.054a,b  ES/BS (%)  4.04 ± 0.29  7.04 ± 0.33a  2.78 ± 0.38a,b  2.30 ± 0.31a,b  Parameter  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  BV/TV (%)  33.1 ± 0.8  24.4 ± 1.2a  34.5 ± 2.2b  33.2 ± 1.7b  Tb.Th (µm)  92.9 ± 2.6  82.0 ± 2.0  121.4 ± 5.9b  129.5 ± 8.5b  Tb.Sp (µm)  188.8 ± 7.3  258.9 ± 12.6a  235.1 ± 15.6  260.4 ± 12.7  Tb.N (1/mm)  3.58 ± 0.12  2.97 ± 0.11a  2.85 ± 0.12a  2.59 ± 0.09a  MS/BS (%)  26.8 ± 1.6  27.6 ± 2.1  44.3 ± 2.0a,b  38.0 ± 3.2a,b  MAR (µm/d)  1.02 ± 0.04  1.13 ± 0.08  1.39 ± 0.07a,b  1.25 ± 0.08  BFR/BS (µm3/µm2/d)  0.274 ± 0.020  0.320 ± 0.036  0.619 ± 0.043a,b  0.482 ± 0.054a,b  ES/BS (%)  4.04 ± 0.29  7.04 ± 0.33a  2.78 ± 0.38a,b  2.30 ± 0.31a,b  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: MS/BS, mineralizing surface/bone surface. a P < 0.05 vs time-matched sham control by one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Figure 5. View largeDownload slide Effect of Scl-Ab on trabecular bone histomorphometry at the L2 vertebral body. (a) ORX rats treated with either dose of Scl-Ab showed more active mineralization surface (arrows) than did ORX controls. (b) Mineralizing surface/bone surface (MS/BS) and BFR/BS were significantly greater for both doses compared with those in the sham and ORX controls. MAR was significantly greater in the 5-mg/kg group compared with that in the sham and ORX controls. In addition, ES/BS, an index of bone resorption, was significantly decreased for both doses compared with that in the sham and ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group) *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 5. View largeDownload slide Effect of Scl-Ab on trabecular bone histomorphometry at the L2 vertebral body. (a) ORX rats treated with either dose of Scl-Ab showed more active mineralization surface (arrows) than did ORX controls. (b) Mineralizing surface/bone surface (MS/BS) and BFR/BS were significantly greater for both doses compared with those in the sham and ORX controls. MAR was significantly greater in the 5-mg/kg group compared with that in the sham and ORX controls. In addition, ES/BS, an index of bone resorption, was significantly decreased for both doses compared with that in the sham and ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group) *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. At the tibia diaphysis, ORX resulted in significantly lower periosteal bone formation (Table 6). At the endocortical surface, ORX increased the bone formation parameters before treatment and increased ES/BS at the end of treatment. Both doses of Scl-Ab resulted in significant increases in bone formation on both periosteal and endocortical surfaces (Fig. 6), with reduced endocortical ES/BS relative to that of the ORX controls. Table 6. Histomorphometric Analysis of Cortical Bone at Tibial Shaft Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Ct.B.Ar (mm2)  5.82 ± 0.20  5.42 ± 0.18  6.03 ± 0.17  5.48 ± 0.15  5.52 ± 0.12  5.80 ± 0.14  Ct.Th (µm)  1457 ± 32  1374 ± 28  1463 ± 22  1376 ± 24  1388 ± 17  1447 ± 27  Ps.Pm (mm)  10.15 ± 0.16  10.02 ± 0.15  10.48 ± 0.15  10.12 ± 0.13  10.13 ± 0.12  10.21 ± 0.11  Ec.Pm (mm)  3.71 ± 0.16  3.86 ± 0.10  3.92 ± 0.12  4.26 ± 0.13  3.77 ± 0.09b  3.66 ± 0.07b  Ps.MS/BS (%)  13.5 ± 4.0  1.1 ± 0.2a  41.0 ± 8.0  6.1 ± 2.1a  51.7 ± 7.8b  54.7 ± 4.0b  Ps.MAR (µm/day)  0.41 ± 0.13  0.08 ± 0.06a  0.76 ± 0.14  0.20 ± 0.12a  0.88 ± 0.15b  1.27 ± 0.16b  Ps.BFR/BS (µm3/µm2/d)  0.093 ± 0.040  0.002 ± 0.002a  0.389 ± 0.093  0.020 ± 0.012a  0.527 ± 0.125b  0.725 ± 0.092b  Ec.MS/BS (%)  17.4 ± 1.9  28.8 ± 3.6a  28.4 ± 3.5  28.4 ± 2.3  83.7 ± 6.1a,b  87.1 ± 3.3a,b  Ec.MAR (µm/d)  0.08 ± 0.06  0.27 ± 0.07  0.50 ± 0.06  0.43 ± 0.09  1.10 ± 0.04a,b  1.10 ± 0.05a,b  Ec.BFR/BS (µm3/µm2/d)  0.013 ± 0.010  0.089 ± 0.027a  0.146 ± 0.024  0.131 ± 0.029  0.932 ± 0.086a,b  0.965 ± 0.065a,b  Ec.ES/BS (%)  10.47 ± 1.86  14.18 ± 2.41  8.21 ± 1.71  13.67 ± 2.02a  0.50 ± 0.34a,b  0.87 ± 0.64a,b  Parameter  BL-Sham  BL-ORX  Sham + Vehicle  ORX + Vehicle  ORX + Scl-Ab 5 mg/kg  ORX + Scl-Ab 25 mg/kg  Ct.B.Ar (mm2)  5.82 ± 0.20  5.42 ± 0.18  6.03 ± 0.17  5.48 ± 0.15  5.52 ± 0.12  5.80 ± 0.14  Ct.Th (µm)  1457 ± 32  1374 ± 28  1463 ± 22  1376 ± 24  1388 ± 17  1447 ± 27  Ps.Pm (mm)  10.15 ± 0.16  10.02 ± 0.15  10.48 ± 0.15  10.12 ± 0.13  10.13 ± 0.12  10.21 ± 0.11  Ec.Pm (mm)  3.71 ± 0.16  3.86 ± 0.10  3.92 ± 0.12  4.26 ± 0.13  3.77 ± 0.09b  3.66 ± 0.07b  Ps.MS/BS (%)  13.5 ± 4.0  1.1 ± 0.2a  41.0 ± 8.0  6.1 ± 2.1a  51.7 ± 7.8b  54.7 ± 4.0b  Ps.MAR (µm/day)  0.41 ± 0.13  0.08 ± 0.06a  0.76 ± 0.14  0.20 ± 0.12a  0.88 ± 0.15b  1.27 ± 0.16b  Ps.BFR/BS (µm3/µm2/d)  0.093 ± 0.040  0.002 ± 0.002a  0.389 ± 0.093  0.020 ± 0.012a  0.527 ± 0.125b  0.725 ± 0.092b  Ec.MS/BS (%)  17.4 ± 1.9  28.8 ± 3.6a  28.4 ± 3.5  28.4 ± 2.3  83.7 ± 6.1a,b  87.1 ± 3.3a,b  Ec.MAR (µm/d)  0.08 ± 0.06  0.27 ± 0.07  0.50 ± 0.06  0.43 ± 0.09  1.10 ± 0.04a,b  1.10 ± 0.05a,b  Ec.BFR/BS (µm3/µm2/d)  0.013 ± 0.010  0.089 ± 0.027a  0.146 ± 0.024  0.131 ± 0.029  0.932 ± 0.086a,b  0.965 ± 0.065a,b  Ec.ES/BS (%)  10.47 ± 1.86  14.18 ± 2.41  8.21 ± 1.71  13.67 ± 2.02a  0.50 ± 0.34a,b  0.87 ± 0.64a,b  Data presented as mean ± standard error of the mean (n = 10 per group). Abbreviations: BL = baseline; Ec., endocortical; MS/BS, mineralizing surface/bone surface; Ps., periosteal. a P < 0.05 vs time-matched sham control by t test (BL groups) or one-way analysis of variance plus Tukey test (week 6 treatment groups). b P < 0.05 vs time-matched ORX control by one-way analysis of variance plus Tukey test (week 6 treatment groups). View Large Figure 6. View largeDownload slide Effects of Scl-Ab on cortical bone histomorphometry at the tibial shaft. (a) More calcein-labeled surface was observed on periosteal (arrowheads) and endocortical (arrows) surfaces in Scl-Ab–treated ORX rats. Bone formation on (b) periosteal and endocortical surfaces was greater for both Scl-Ab doses compared with that in ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Figure 6. View largeDownload slide Effects of Scl-Ab on cortical bone histomorphometry at the tibial shaft. (a) More calcein-labeled surface was observed on periosteal (arrowheads) and endocortical (arrows) surfaces in Scl-Ab–treated ORX rats. Bone formation on (b) periosteal and endocortical surfaces was greater for both Scl-Ab doses compared with that in ORX controls. Data are expressed as the mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs sham controls; †P < 0.05 vs ORX controls. Discussion This experiment demonstrates the efficacy of Scl-Ab in restoring bone mass and strength in an androgen-deficient, orchiectomized rat model with established osteopenia. Scl-Ab increased bone formation on periosteal, endocortical, and trabecular surfaces and decreased bone resorption on the endocortical and trabecular surfaces. These effects led to a rapid restoration of BMD by only 3 weeks of treatment in the lumbar spine and by 6 weeks of treatment in the long bones (tibia and femur). By the end of the study, the decreased bone strength (peak load) in the ORX rats had been completely restored to that of the sham control level in the L5 vertebra and the femur diaphysis and to greater than the sham control level in the femoral neck. The consistent and positive correlations between bone mass and bone strength indicated the maintenance of bone quality with Scl-Ab treatment. In the present study, adult male rats at 6 months of age were subjected to ORX to induce androgen deficiency. Osteopenia was established in both the lumbar spine and the long bones (tibia and femur) at 3 months after ORX, as demonstrated by a significant decrease in DXA BMD at these sites. Micro-CT analysis of L5 confirmed the decreases in bone mass indexes at 3 and 4.5 months after ORX, an effect reflected in the deterioration of both trabecular and cortical microarchitecture. Histomorphometric analysis of cortical bone in the tibial shaft showed a 98% reduction in periosteal BFR in ORX baseline controls compared with the sham baseline controls, supporting the effect of androgen deficiency in reducing periosteal bone formation. A significant increase in eroded surface, a bone resorption index, on the trabecular surfaces of L2 and endocortical surfaces of the tibial shafts at the end of the study in the ORX controls indicated the effects of androgen deficiency in increasing bone resorption. The increase in bone resorption and decrease in bone formation in these ORX rats are in agreement with observations in hypogonadal men, as reviewed by Khosla et al. (4). The combined effects of ORX on bone formation and bone resorption led to significantly lower bone mass and strength in these rats. Therefore, the model used in the present study mimics the conditions observed in male osteoporotic patients. The effects of Scl-Ab on trabecular bone mass and bone formation that we observed are similar to those reported in intact aged male rats (31). In both male osteopenic models, Scl-Ab administration increased the trabecular mineralizing surface, MAR, and BFR. These effects led to increases in the trabecular bone volume and Tb.Th. Also, the effect of Scl-Ab in increasing trabecular bone formation has been consistently observed in various animal models of osteopenia (23, 24, 31, 32). In our ORX rat study, and consistent with the ovariectomized (OVX) rat and the normal-loaded and underloaded aged female rat models, Scl-Ab induced significant decreases in the bone resorption parameters (24, 32). The reported absence of such an effect in aged male rats (31) is likely attributable to low bone turnover indexes in this aging model. The mechanism of inhibition of bone resorption by Scl-Ab is an area of continued investigation. The expression of osteoclast mediators might be altered by treatment with Scl-Ab, as reviewed by Ominsky et al. (18). The inhibition of bone resorption by Scl-Ab has also been shown in human clinical trials, in which Scl-Ab significantly decreased serum markers of bone resorption in both postmenopausal women with osteoporosis and healthy men (19, 20, 34). In the present study, treatment with Scl-Ab significantly increased the periosteal BFR more than 26- and 36-fold in the 5- and 25-mg/kg groups, respectively, compared with vehicle treatment in the ORX rats. This resulted from significant increases in both the periosteal mineralizing surface and the MAR. On the endocortical surface, the increased bone formation and decreased bone resorption induced by Scl-Ab led to a significant decrease in Ec.Pm. These effects contributed to the significant increases in cortical vBMD, Ct.B.Ar, and Ct.Th, as determined by micro-CT of the femur diaphysis. In general, the effects of Scl-Ab on the periosteal and endocortical surfaces of cortical bone were similar to those reported for aged OVX rats, aged male rats, and normal-loaded and underloaded female rats (24, 31, 32). Scl-Ab did not induce a significant increase in Ps.Pm, as BFR was dramatically increased on this surface in all these short-term rodent studies. In a longer-term, 12-month study in OVX rats, Scl-Ab significantly increased Ps.Pm (18), indicating that longer-term treatment might be required to observe this effect. In conclusion, in a rat model of male osteoporosis, we found that Scl-Ab treatment increased bone formation and decreased bone resorption, leading to restoration of bone mass and strength to levels similar to those observed in the sham controls. Increased bone mass and improved bone architecture at multiple skeletal sites after Scl-Ab treatment led to increased trabecular and cortical bone strength, as demonstrated by the significant dose-dependent increases in peak load in the lumbar vertebral body, femur diaphysis, and femoral neck. More importantly, increases in bone mass and bone strength remained well correlated across all groups, demonstrating that Scl-Ab significantly increased bone mass and maintained bone quality in this androgen-deficient, established osteopenia animal model. These results support the ongoing evaluation of sclerostin antibodies as therapeutic agents to restore bone mass and strength and thereby potentially reduce skeletal fragility fractures in hypogonadal and aged men with osteoporosis. Abbreviations: BFR bone formation rate BMD bone mineral density BS bone surface BV/TV bone volume/total volume CSMI cross-sectional moment of inertia Ct.B.Ar cortical bone area Ct.Th cortical thickness DXA dual-energy x-ray absorptiometry Ec.Pm endocortical perimeter ES/BS eroded surface/bone surface L5 fifth lumbar MAR mineral apposition rate micro-CT microcomputed tomography ORX orchiectomized OVX ovariectomized Ps.Pm periosteal perimeter Scl-Ab sclerostin antibody sham sham-operated Tb.N trabecular number Tb.Sp trabecular spacing Tb.Th trabecular thickness vBMC volumetric bone mineral content vBMD volumetric bone mineral density. Acknowledgments The authors thank the members of the sclerostin antibody project team at Amgen and UCB Pharma for their support and helpful discussions. The authors acknowledge Louise Profit, Gardiner-Caldwell Communications, Macclesfield, UK, for editorial assistance, which was funded by UCB Pharma. FinancialSupport: This study was supported by Amgen and UCB Pharma. Current Affiliation: M.S. Ominsky’s current affiliation is Radius Health, Inc., Waltham, Massachusetts 02451. K.S. Villasenor’s current affiliation is BRB Pet Products, Murrieta, California 92562. Q.-T. Niu’s current affiliation is Comparative Biology and Safety Science, Amgen Inc., Thousand Oaks, California 91320. X. Xia’s current affiliation is Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200030, China. Disclosure Summary: X.L. is an employee of Amgen and owns Amgen stocks/stock options; M.S.O. is a stockholder and former employee of Amgen, and a current employee and stockholder at Radius Health, Inc.; M.G. is a former employee of Amgen. K.S.V., Q.-T.N., F.J.A., and W.S.S. are former employees of Amgen and own Amgen stocks/stock options. H.Z.K. is an employee of UCB Pharma and owns Amgen and UCB stocks/stock options. T.J.W. owns Amgen stock. The remaining author has nothing to disclose. References 1. 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EndocrinologyOxford University Press

Published: Jan 1, 2018

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