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Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model
Bone 48 (2011) 197–201
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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model XiaoYan Tian a, Webster S.S. Jee a,⁎, Xiaodong Li b, Chris Paszty b, Hua Zhu Ke b a b
Radiobiology Division, University of Utah School of Medicine, Salt Lake City, UT, USA Metabolic Disorders, Amgen Inc., Thousand Oaks, CA, USA
a r t i c l e
i n f o
Article history: Received 11 June 2010 Revised 10 August 2010 Accepted 8 September 2010 Available online 17 September 2010 Edited by: Robert Recker Keywords: Sclerostin antibody Bone formation Resorption Bone balance Immobilization
a b s t r a c t Sclerostin monoclonal antibody (Scl-Ab) has been shown to increase bone mass and bone strength by stimulating bone formation in an ovariectomy-induced bone loss rat model. The purpose of this study was to determine the effects of Scl-Ab in a rat immobilization/disuse model in which there was both a decrease in bone formation and an increase in bone resorption. Ten-month-old female Sprague Dawley rats were divided into normal weight-bearing (normal-loaded, NL) and right hindlimb-immobilization (under-loaded, UL) groups. Both NL and UL rats were treated with vehicle or Scl-Ab at 5 or 25 mg/kg, twice per week for 4 weeks. Trabecular and cortical bone histomorphometric analyses were performed on the proximal tibial metaphysis (PTM) and tibial shaft (TS). Compared to NL controls, UL rats had reduced body and muscle weights, increased bone marrow fat cells in the PTM, increased trabecular bone resorption and periosteal mineral apposition rate (MAR) as well as decreased trabecular MAR and bone formation rate (BFR/BS). In NL bones, treatment with Scl-Ab significantly increased bone formation and decreased bone resorption, resulting in increased trabecular and cortical bone mass. In UL trabecular bone, treatment with Scl-Ab at 5 or 25 mg/kg induced significant and dose-dependent increases in trabecular bone volume and thickness, mineralized surfaces (MS/BS), MAR and BFR/BS, and a significant decrease in eroded surface (Er.S/BS) compared with UL controls. In UL cortical bone, Scl-Ab treatment induced significant increases in cortical width, periosteal and endocortical MS/BS, MAR and BFR/BS, and significant decreases in endocortical Er.S/BS compared with UL controls. Taken together, these findings suggest that antibody-mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization-induced osteopenia. © 2010 Elsevier Inc. All rights reserved.
Introduction Sclerostin deficiency in humans, together with data from sclerostin-knockout mice, suggests that sclerostin inhibition might be an attractive approach for the development of a novel bone anabolic agent [1–4]. Studies have demonstrated that inhibition of sclerostin by a sclerostin monoclonal antibody (Scl-Ab) stimulated bone formation, and increased bone mass and bone strength in the ovariectomy-induced bone loss rat [5] and gonad-intact female monkey models [6]. Dose-dependent increases in biochemical markers of bone formation and decreases in a bone resorption marker were observed following a single subcutaneous injection of Scl-Ab in healthy post-menopausal women [7]. However, it has yet to be demonstrated whether Scl-Ab can build bone in the setting of the immobilized/disuse-induced osteopenia that occurs in paraplegic conditions, space flight and prolonged bed rest. It has been reported that increases in loading of bone were associated with decreased ⁎ Corresponding author. Division of Radiobiology, University of Utah, 729 Arapeen Dr., Suite 2338, Salt Lake City, UT 84108-1218, USA. Fax: +1 801 581 7008. E-mail address: [email protected] (W.S.S. Jee). 8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2010.09.009
sclerostin expression [8] and bone loss did not occur after unloading in mice lacking sclerostin [4]. In the current study, we employed the modified Swedish adult rat right hindlimb-immobilization model [9– 12] to test the effects of Scl-Ab on the under-loaded (UL) bone. A group of normal-loaded rats were also treated with Scl-Ab as a reference for the effects of Scl-Ab on normal-loaded bone in these adult female rats. Materials and methods The Institutional Animal Care and Use Committee at the University of Utah approved all animal procedures in this study. Animals Seventy-seven Sprague Dawley intact virgin female rats (Harlan, Indianapolis, IN, USA) were purchased at 3 months of age and housed in the University of Utah, Division of Radiobiology animal facility. The animals were housed at 72 °F with a 12:12 h light/dark cycle and were allowed free access to water and a pelleted commercial natural diet (Teklad Rodent Laboratory Chow 8640, Harlan Teklad, Madison WI,
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USA) that contained 1.13% calcium, 0.94% phosphorus and 2.99 IU/g of vitamin D3. The general condition of the animals was monitored daily and body weights recorded weekly. At 5 months of age, in order to acclimate the rats to the immobilization procedure, all rats had their right hindlimb immobilized once a week for 6 weeks for a period of 24 h. They were then immobilized once every 2 weeks for 24 h for the next 8 weeks. From that time on, until the beginning of the experiment, they were no longer immobilized. Briefly, the immobilization procedure during the study consisted of the right foot and ankle being padded with cotton to prevent pressure sores and wrapped with several layers of elastic bandage around the right hind leg which has been pulled up onto the abdomen and then layers of bandage wrapped around the abdomen allowing the front legs and left hindlimb to move freely [9–12]. The immobilization procedure generated a right under-loaded (UL) hindlimb [9–12]. The rats were checked several times every day and rewrapped as needed especially if signs of swelling, movement of the leg out of position, or evidence of the wrap being chewed was present. The rats tolerated the immobilization very well, quickly returning to ambulating and feeding on 3 legs. A group of freely ambulating rats served as normal-loaded (NL) controls. At 10-months of age, 67 rats weighing an average of 270 g were randomly divided into 7 groups with 8–11 rats per group. One group of rats was sacrificed at the beginning of the experiment to serve as beginning (basal) controls. Three weight-bearing control groups and 3 right hindlimb under-loaded groups of rats were injected with xylenol orange subcutaneously at the back of the neck (90 mg/kg, Sigma Chemical Co., St. Louis, MO, USA) on Day 1. They received vehicle (0.9% saline) or a sclerostin monoclonal antibody (Scl-AbIII; Amgen, Thousand Oaks, CA, USA) at 5 mg/kg or 25 mg/kg, twice per week (Tuesday and Friday) for 4 weeks by subcutaneous injection. The Scl-AbIII antibody is a mouse antibody in which most of the mouse sequences were replaced by rat sequences in order to reduce immunogenicity. The Scl-AbIII was sent on dry ice from Amgen to the University of Utah and stored at −80 °C until thawed to make solutions to be injected. All rats (including beginning control) were injected subcutaneously with calcein (10 mg/kg; Sigma Chemical Co. St. Louis, MO, USA) on days 14, 13, and 4, 3 before sacrifice. One day after the final treatment, animals were anesthetized with Avertin (0.4 mg/kg body weight; Sigma-Aldrich Inc., St. Louis, MO, USA) and euthanized via exsanguinations to permit collection of blood samples by open-chest cardiac puncture. The blood was centrifuged and the sera were sent to Amgen for analysis. The soft tissue (thymus, heart, lungs, liver, spleen, kidneys, and adrenals) and muscles (soleus, gastrocnemius, and quadriceps) were weighed and collected for future analyses. Right tibiae from immobilized/under-loaded (UL) and normal-loaded (NL) rats were harvested at autopsy for histomorphometry. Bone histomorphometry The right and left proximal tibiae and tibial shafts, at the tibiofibular junction, were stained in Villanueva Osteochrome Bone Stain (Arizona Histology & Histomorphometry Center, Phoenix, AZ, USA), embedded in methyl methacrylate, and sawed and ground to 20 μm longitudinal proximal tibia sections and 30 μm tibia cross sections for analyses. Data from the left bones was only summarized. The region of interest for the proximal tibial trabecular bone was an area (2.16 mm2) one mm below the growth plate within the proximal tibial metaphysis. The region of interest for cortical bone analyses included the whole mid-tibial shaft. Static and dynamic parameters were calculated and expressed according to published methods [13–15]. Additionally, point counting with a Merz eyepiece reticule [16] was performed to measure the percent fat content of the bone marrow. The number of points superimposed over fat cells was
divided by the total number of points superimposed over bone marrow to calculate the percent marrow fat area. Statistical analysis All data were presented as group means ± standard deviation (SD). The statistical analyses were performed using the Ultimate Integrated Data Analysis and Presentation System (StatView 5.0.1, SAS Institute Inc. Cary, NC). Across group comparisons were made with a parametric analysis of variance (ANOVA) followed by a Fisher Protected Least Significant Difference (Fisher PLSD) test, and a value of p b 0.05 was considered statistically significant. Results At the end of the study, body weights in UL rats were 15% less than those of NL rats regardless of treatment. Both doses of the Scl-Ab had no effect on body weight (data not shown). Muscle weights in UL rats were significantly lower as compared with basal controls and NL controls. Specifically, the right soleus, gastrocnemius and quadriceps muscles weighed 44%, 59% and 56% less, respectively, relative to beginning controls. Treatment with 5 or 25 mg/kg Scl-Ab had no effect on muscle weight in both UL and NL rats (data not shown). The bone marrow in the proximal tibial metaphysis (PTM) of NL rats contained 10.8 ± 7.9% (Mean ± SD) fat cell area while UL rats contained 33.1 ± 12.6% fat cell area, a 218% higher fat cell content. SclAb treatment at both doses had no significant effect on fat cell content in both UL and NL bone marrow sites compared to their respective vehicle controls (data not shown). Trabecular bone in the proximal tibial metaphysis (PTM) At the end of the study there was no significant difference in all parameters listed in Table 1 in NL controls compared with basal controls (Table 1), indicating that no age-related change was observed during the 4-week experimental period for these rats. Compared with basal controls, bone formation rate (BFR/BS) was significantly lower by 23%, and eroded surface (Er.S/BS) was significantly higher by up to 44% in UL controls (Table 1), indicating that immobilization had induced lower bone formation and higher bone resorption. However, there was no significant difference in trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) or trabecular separation (Tb.Sp) between vehicle-treated UL and NL groups (Table 1), indicating immobilization for 4 weeks did not induce significant trabecular bone loss in these 10month-old female rats. Compared to their respective vehicle controls, BV/TV and Tb.Th were significantly greater in the 5 or 25 mg/kg-treated NL and UL trabecular bones and additionally, Tb.N was significantly higher and Tb.Sp was significantly lower than vehicle controls in the 25 mg/kg NL group (Table 1). Scl-Ab treatment at both doses significantly increased MS/BS, MAR, BFR/BS and significantly decreased Er.S/BS in both NL and UL trabecular bones as compared to their respective vehicle controls, indicating that Scl-Ab stimulates bone formation and decreases bone resorption in the setting of normal loading and of under-loading (Table 1 and Fig. 1). There were significant increases in Tb.Th, MS/BS, MAR and BFR/BS in UL trabecular bone treated with 25 mg/kg vs. 5 mg/kg, indicating a dose-dependent effect for these parameters. Similarly, there were significant increases in BV/TV, Tb. Th, MS/BS and BFR/BS and there was a significant decrease in Er.S/BS in NL trabecular bone treated with 25 mg/kg vs. 5 mg/kg (Table 1). Scl-Ab at 5 and 25 mg/kg were more effective in NL than UL cancellous bones. For example, at 5 mg/kg TbN, MS/BS, MAR and BFR/BS were significantly increased and Er.S/BS was decreased more in NL than UL bones. In addition, at 25 mg/kg all the selected static and
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Table 1 Selective static and dynamic cancellous bone histomorphmetric analysis of proximal tibial metaphysis (PTM). Parameters
BV/TV (%) Tb.Th (μm) Tb.N (#/mm) Tb.Sp (μm) MS/BS (%) MAR (μm/d) BFR/BS (μm3/μm2/d × 100) Er.S/BS (%)
Beginning control [basal]
Normal-loaded + Scl-Ab (mg/kg) 0
5
25
0
Under-loaded + Scl-Ab (mg/kg) 5
25
14.3 ± 4.8 45.3 ± 6.4 3.1 ± 0.7 297.8 ± 99.0 24.6 ± 7.3 0.7 ± 0.1 17.9 ± 6.1 3.2 ± 1.1
13.9 ± 2.7 44.5 ± 2.8 3.1 ± 0.6 285.2 ± 65.6 27.6 ± 4.5 0.7 ± 0.1 20.1 ± 3.1⁎ 3.4 ± 0.8⁎
24.6 ± 9.2a,b 75.5 ± 9.9a,b,d 3.2 ± 1.0 310.9 ± 271.9 55.1 ± 3.8a,b,d 1.0 ± 0.1a,b,d 55.4 ± 8.7a,b,d 1.7 ± 0.6a,b,d
34.7 ± 6.5a,b,c,e 93.6 ± 10.7a,b,c,e 3.7 ± 0.3a,b,e 180.3 ± 33.3a,b,e 69.2 ± 2.6a,b,c,e 1.1 ± 0.1a,b 75.7 ± 9.6a,b,c,e 0.8 ± 0.3a,b,c,e
13.4 ± 1.1 41.9 ± 3.3 3.2 ± 0.4 271.6 ± 36.7 25.7 ± 2.3 0.6 ± 0.1 14.1 ± 3.6⁎
19.4 ± 3.3a,b 57.9 ± 5.7a,b,d 3.4 ± 0.4 245.0 ± 42.2 44.4 ± 5.0a,b,d 0.8 ± 0.1a,b,d 37.6 ± 6.9a,b,d 3.2 ± 1.0b,d
21.2 ± 6.3a,b,e 71.3 ± 7.1a,b,c,e 2.9 ± 0.7e 300.3 ± 156.1e 56.8 ± 7.2a,b,c,e 1.0 ± 0.2a,b,c 55.7 ± 15.1a,b,c,e 2.7 ± 0.9b,e
4.7 ± 0.8a,⁎
BV/TV, trabecular bone volume / tissue volume × 100; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; MS/BS, mineralizing surface/bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate/bone surface referent; and Er.S/BS, eroded surface/bone surface. Mean ± SD. a p b 0.05 vs. beginning control. b p b 0.05 vs. respective 0 mg/kg. c p b 0.05 vs. 5 mg/kg. d p b 0.05 NL vs. UL 5 mg/kg. e p b 0.05 NL vs. UL 25 mg/kg. ⁎ p b 0.05 NL + 0 mg/kg vs. UL + 0 mg/kg.
dynamic histomorphometric parameters were significantly more effective in NL than in UL bones (Table 1). This indicated that there may be some additive effects of Scl-Ab and mechanical loading on bone. We also observed that trabecular bone of NL and UL rats treated with 5 or 25 mg/kg Scl-Ab exhibited buried xylenol orange labels given on Day 1 apposed by double calcein labels given on 14 and 15 and 24 and 25 days. This observation indicates that the Scl-Abstimulated bone formation on the trabecular bone surface may not be dependent upon activation of bone resorption (Fig. 2A). Compared with the right UL-PTM, 5 and 25 mg/kg Scl-Ab of the left PTM of the right hindlimb immobilized rats induced non-significantly increased BV/TV, Tb.Th, TB.N, MS/BS, MAR and BFR/BS and nonsignificantly decreased Tb.Sp and Er.S/BS (data not shown).
Cortical bone in the tibial shaft (TS) Compared with basal controls, endocortical MS/BS increased significantly in NL controls (Table 2). Periosteal MAR increased significantly in UL controls compared with basal or NL controls (Table 2). There was no significant difference in cortical bone mass (Ct.Ar), thickness (Ct.Th) and marrow areas (Ma.Ar) in NL or UL vehicle controls compared with basal controls. Scl-Ab treatment at 5 or 25 mg/kg significantly increased Ct.Ar, Ct. Th, periosteal and endocortical MS/BS, MAR and BFR/BS, and
significantly decreased Ma.Ar and endocortical Er.S/BS in both UL and NL groups compared with basal controls (Table 2). Compared with UL vehicle controls, Scl-Ab treatment at 5 or 25 mg/kg induced a non-significant increase in Ct.Ar and a nonsignificant decrease in Ma.Ar in UL cortical bone. Ct.Th increased significantly in the 25 mg/kg group and increased non-significantly in the 5 mg/kg group in UL cortical bone compared with UL vehicle controls (Table 2). Scl-Ab treatment at 5 or 25 mg/kg significantly increased periosteal and endocortical MS/BS, MAR, BFR/BS with the exception of periosteal MAR at 5 mg/kg, and significantly decreased endocortical Er.S/BS as compared with UL vehicle controls (Table 2; Fig. 3). Compared with NL vehicle controls, Scl-Ab treatment at 25 mg/kg significantly increased Ct.Ar and Ct.Th, and significantly decreased Ma. Ar, while no significant effect was found in the 5 mg/kg dose group (Table 2; Fig. 3). Similar to what was observed in UL cortical bone, Scl-Ab treatment at 5 or 25 mg/kg significantly increased periosteal and endocortical MS/BS, MAR, BFR/BS, and significantly decreased endocortical Er.S/BS in NL cortical bone as compared with NL vehicle controls (Table 2). Compared with UL treated with 5 mg/kg, there were significant increases in periosteal MS/BS, MAR, BFR/BS and endocortical MAR and BFR/BS in the UL treated with 25 mg/kg (Table 2). For NL cortical bone, Scl-Ab at 25 mg/kg induced significantly higher Ct.Th, periosteal and endocortical MS/BS, MAR, BFR/BS, and a significantly lower endocortical Er.S/BS compared with
Fig. 1. Five and 25 mg/kg Scl-Ab treatment significantly increased trabecular bone mass and width, and bone formation in both normal-loaded and under-loaded cancellous bones of the proximal tibial metaphyses. GP = growth plate. Villanueva bone stain; 10×.
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Fig. 2. Scl-Ab induced bone formation on trabecular and endocortical bone surfaces without prior bone resorption. (A) Buried xylenol orange labels (*) apposed by calcein labeling in trabecular bone. (B) Extensive buried xylenol orange labels near endocortical surface (*) followed by double calcein labeling. Xylenol orange label given on Day 1 and double calcein labels given on days 14–15 and 24–25; 100×.
the 5 mg/kg dose group (Table 2). These data demonstrated that there was a dose-dependent effect between 5 and 25 mg/kg on cortical bone of normal-loaded or under-loaded rats. Unlike in cancellous bone, there was no increase in effects of Scl-Ab and mechanical loading. Compared to right UL-tibial shaft, the 25 mg/kg Scl-Ab in left tibial shaft from the right hindlimb immobilized rats induced significantly increased periosteal MS/BS and significantly decreased endocortical MAR, BFR/BS and Er.S/BS (data not shown). Similar to what was observed for trabecular bone, we also found that the endocortical surfaces of NL and UL rats treated with 5 or 25 mg/kg Scl-Ab exhibited buried xylenol orange labels given on Day 1 apposed by double calcein labels given on 14 and 15 and 24 and 25 days. This observation indicates that the Scl-Ab stimulated bone formation on the endocortical bone surface may not be dependent upon activation of bone resorption (Fig. 2B). Discussion We studied antibody-mediated sclerostin inhibition in adult female rats with or without right hindlimb-immobilization. Scl-Ab treatment significantly induced increases in trabecular and cortical bone under both normal-loaded and under-loaded conditions. The increased bone mass with Scl-Ab treatment was due to increased bone formation and decreased bone resorption on both trabecular and endocortical bone surfaces, as well as increased bone formation on periosteal surfaces. Furthermore, histological observations indicated that Scl-Ab can increase bone formation without prior activation of bone resorption. There were several divergent findings from our previous studies [17–21] in our current use of this model. One involved the lack of inhibition of periosteal bone formation in the under-loaded (UL) tibial shaft. Instead, periosteal osteoblastic activity (mineral appositional
Fig. 3. The responses of normal-loaded and under-loaded tibial shafts to Scl-Ab. Scl-Ab at 25 mg/kg increased periosteal (P) and endocortical (E) bone formation in both normal-loaded and under-loaded tibial shafts. Villanueva bone stain; 10×.
rate) and bone formation rate were stimulated. In these older rats, apparently the immobilization by bandaging set off Frost's regional acceleratory phenomenon (RAP) [22–25]. To quote Frost, “A RAP normally accelerates all ongoing regional processes involving blood flow, temperature, cell metabolism and turnover, creating new capillaries and any ongoing modeling and remodeling and healing of hard and soft tissues.” [25]. In support, the RAP set off in the 4-point loading study by Turner et al. [26] in which the periosteum was squeezed by loading that activated periosteal woven bone formation. In addition, the RAP may have been responsible for why Scl-AbIII treatment did not induce more changes in normal-loaded than under-loaded cortical bone. The RAP response caused by under-loading by bandaging tended to increase periosteal and endocortical bone formation and decreased bone resorption in the UL-TS to the same level that occurred in the NL-TS (Table 2). Possibly, also the brief treatment period of 4 weeks to cortical bone was insufficient to overcome the functional lag in cortical bone tissue level responses. Two, the brief treatment period of 4 weeks dictated by the quantity of the available antibody and the use of older rats resulted in the lack of static bone changes in the immobilized-limb. The effects of Scl-Ab at the higher dose (25 mg/kg) on trabecular bone mass and thickness, bone formation and bone resorption, were more pronounced in normal-loaded (NL) proximal tibial metaphysis (PTM) compared with the effects noted in under-loaded (UL) PTM, indicative of an enhanced response to Scl-Ab treatment in mechanically-loaded bone. This greater anabolic response observed for normal-loaded bone vs. under-loaded bone may be due, in part, to the previously reported finding that sclerostin expression is decreased by loading and increased by unloading [4,8]. Thus in the current study, a relative excess of sclerostin in the UL bones would likely have a dampening effect on the UL Scl-Ab-mediated anabolism relative to the situation in the NL bones. In contrast to what was found for the PTM,
Table 2 Static and dynamic cortical bone histomorphometric analysis of tibial shaft. Parameters
Ct.Ar (%) Ma.Ar (%) Ct.Th (μm) Ps-MS/BS (%) Ps-MAR (μm/d) Ps-BFR/BS (μm/d × 100) Ec-MS/BS (%) Ec-MAR (μm/d) Ec-BFR/BS (μm/d × 100) Ec-Er.S/BS (%)
Beginning control [basal]
Normal-loaded + Scl-Ab (mg/kg) 0
5
25
0
5
25
80.7 ± 2.6 19.3 ± 2.6 645 ± 33 26.1 ± 7.8 0.5 ± 0.2 12.7 ± 6.5 17.3 ± 7.0 0.5 ± 0.1 9.5 ± 5.4 3.3 ± 1.0
81.7 ± 2.0 18.3 ± 2.0 658 ± 36 30.6 ± 12.6 0.5 ± 0.2⁎
83.4 ± 1.6a 16.6 ± 1.6a 677 ± 18a 46.3 ± 15.8a,b 0.7 ± 0.1a,b 34.9 ± 17.7a,b 65.1 ± 13.9a,b 1.5 ± 0.2a,b 99.5 ± 25.3a,b 0.8 ± 0.4a,b
85.3 ± 2.2a,b 14.7 ± 2.2a,b 723 ± 43a,b,c 85.9 ± 11.0a,b,c 1.1 ± 0.2a,b,c,e 95.9 ± 27.9a,b,c 84.9 ± 12.5a,b,c 1.7 ± 0.1a,b,c 148.9 ± 27.0a,b,c 0.3 ± 0.2a,b,c
81.6 ± 3.2 18.4 ± 3.2 651 ± 52 24.0 ± 8.8 0.9 ± 0.3a,⁎ 22.0 ± 12.7 19.3 ± 2.3 0.5 ± 0.2 10.2 ± 2.7 4.4 ± 2.5
84.1 ± 1.5a 15.9 ± 1.5a 686 ± 37a 48.2 ± 13.7a,b 1.0 ± 0.3a 48.6 ± 18.4a,b 51.0 ± 10.0a,b 1.3 ± 0.3a,b 67.8 ± 23.2a,b 1.2 ± 1.2a,b
84.7 ± 2.6a 15.3 ± 2.6a 723 ± 44a,b 72.7 ± 10.1a,b,c 1.7 ± 0.2a,b,c,e 120.0 ± 26.1a,b,c 84.5 ± 9.7a,b 1.7 ± 0.2a,b,c 141 ± 26a,b,c 0.5 ± 0.2a,b
17.1 ± 11.2 25.1 ± 6.9a 0.6 ± 0.2 16.7 ± 8.7 3.6 ± 1.2
Under-loaded + Scl-Ab (mg/kg)
Ps — Perisoteal; Ec — Endocortical; Ct.Ar, cortical bone area / total tissue area × 100; Ma.Ar, marrow cavity area / total tissue area × 100; Ct.Th, cortical thickness; MS/BS, mineralizing surface/bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate/bone surface referent; and Er.S/BS, eroded surface/bone surface. ap b 0.05 vs. beginning control, b p b 0.05 vs. respective 0 mg/kg, cp b 0.05 vs. 5 mg/kg, dp b 0.05 NL vs. UL 5 mg/kg, e p b 0.05 NL vs. UL 25 mg/kg, ⁎p b 0.05 NL + 0 mg vs. UL + 0 mg.
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for cortical bone at the mid-tibial shaft there was no difference regardless of mechanical loading in response to Scl-Ab at both the lower and the higher doses. The difference between trabecular and cortical bone in response to the interaction of mechanical loading and sclerostin inhibition in this short-term (4 weeks) study is currently not well understood. One possible explanation is that immobilization for 4 weeks did not induce significant changes in cortical bone surface with the exception of an increase in periosteal MAR, while it induced significant decreases in bone formation and increases in bone resorption in trabecular bone. It has been well established that a longer duration was required to induce cortical bone changes in this rat immobilization model [11]. Thus, a longer-term study would be required to test whether there are differences in the cortical bone response to sclerostin antibody treatment under different mechanical loading conditions (i.e. NL vs. UL). With Scl-Ab administration it was not surprising to find the dramatic dual effect of increased cancellous and cortical bone formation, and decreased bone resorption resulting in increased trabecular bone mass and thickness in normal- and under-loaded bone sites because earlier studies reported similar dramatic responses in aged OVX rats, cynomolgus monkeys and humans [5–7]. In our current study we found that the 25 mg/kg dose of Scl-Ab increased bone formation surfaces more than that observed for the 5 mg/kg dose. The histological findings of xylenol orange labeling given on Day 1 separated from double calcein labels given on days 14–15 and days 24– 25 indicate that the bone formation induced by Scl-Ab treatment can take place on bone surfaces without prior activation of bone resorption. These sites can be interpreted as bone formation occurring at ongoing bone formation surfaces, occurring from the overfilling of bone remodeling sites or onto adjacent quiescent surfaces or from bone lining cells initiating minimodeling, observed previously in PTH stimulated bone formation via modeling and bone remodeling [27,28]. In conclusion, short-term administration of 5 and 25 mg/kg sclerostin neutralizing antibody in an adult female rat model of right hindlimb-immobilization and normally loaded ambulated rats resulted in a dramatic increase in bone formation and a decrease in bone resorption that led to increased trabecular and cortical bone mass in normal- and under-loaded cancellous and cortical bone sites. The rapid increase in bone mass was dominated by robust bone formation on trabecular, periosteal and endocortical bone surfaces with greater trabecular bone gain in normal-loaded than underloaded bones. Finally, the bone building effects obtained in this immobilized-limb rat study suggests that antibody-mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization-induced osteopenia. Conflict of interest Tian and Jee have no conflict of interest. Li, Paszty and Ke have corporate appointments with Amgen, Inc. Acknowledgments The authors acknowledge the support of Rebecca B. Setterberg and Min Chen for their excellent technical assistance. The funding for this study was supported by Amgen Inc. and UCB. The authors thank all members of the Amgen and UCB (Slough, United Kingdom) sclerostin team for their support of this study.
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References [1] Beighton P. Sclerosteosis. J Med Genet 1988;25:200–3. [2] Hamersma H, Gardner J, Beighton P. The natural history of sclerosteosis. Clin Genet 2003;63:192–7. [3] Li X, Ominsky MS, Niu QT, Sun N, Daugherty B, D'Agostin D, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008;23:860–9. [4] Lin C, Jiang X, Dai Z, Guo X, Weng J, Wang J, et al. Sclerostin mediates bone response to mechanical loading through anatogonizing Wnt/B-catenin signaling. J Bone Miner Res 2009;24:1651–61. [5] Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 2009;24:578–88. [6] Ominsky MS, Vlasseros F, Jolette J, Smith SY, Stouch B, Doeligast G, et al. Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength. J Bone Miner Res 2010;25:948–59. [7] Padhi D, Stouch B, Jang G, Fang L, Darling M, Glise H, et al. Anti-sclerostin antibody increases markers of bone formation in healthy postmenopausal women. J Bone Miner Res 2007;22:S37 suppl. [8] Robling AG, Niziolek PJ, Baldridge LA, Condon KW, Allen MR, Alam I, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/ sclerostin. J Biol Chem 2008;283:5866–75. [9] Lindgren U. The effect of thyroparathyroidectomy: development of disuse osteoporosis in adult rats. Clin Orthop Relat Res 1976;118:251–6. [10] Li XJ, Jee WSS, Chow SY, Woodbury DM. Adaptation of cancellous bone to aging and immobilization in the rat. A single photon absorptiometry and histomorphometry study. Anat Rec 1990;227:12–24. [11] Li XJ, Jee WSS. Adaptation of diaphyseal structure to aging and decreased mechanical loading in adult rats: a densitometric and histomorphometry study. Anat Rec 1991;229:291–7. [12] Jee WSS, Li XJ. Adaptation of cancellous bone to overloading in the adult rat: a single photon absorptiometry study. Anat Rec 1990;227:418–26. [13] Parfitt AM, Matthews CH, Villanueva AR, Kleerekoper M, Frame B, Rao DS. Relationship between surface, area and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the micronatomic and cellular mechanisms of bone loss. J Clin Invest 1983;72:1396–409. [14] Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols and units. Report to the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595–610. [15] Li XJ, Jee WSS, Ke HZ, Mori S, Akamine T. Age-related changes of cancellous and cortical bone histomorphometry in female Sprague-Dawley rats. Cells Mater 1992 (Suppl 1):25–35. [16] Merz WA, Shenk RK. Quantitative structural analysis of human cancellous bone. Acta Anat (Basel) 1970;75:54–66. [17] Li XJ, Jee WSS, Chow SY, Woodbury DM. Adaptation of cancellous bone to aging and immobilization in the rat. A single photon absorptiometry and histomorphometry study. Anat Rec 1990;227:12–24. [18] Li XJ, Jee WSS. Adaptation of diaphyseal structure to aging and decreased mechanical loading in adult rats: a densitometric and histomorphometry study. Anat Rec 1991;229:291–7. [19] Jee WSS, Inoue J, Jee KW, Haba T. Histomorphometric assay of the growing long bone. In: Takahashi H, editor. Handbook of Bone Morphology. Niigata City: Nishimura Co., Ltd.; 1983. p. 101–22. [20] Jee WSS, Ke HZ, Li XJ. Long-term anabolic effects of prostaglandin-E2 on tibial diaphyseal bone in male rats. Bone Miner 1991;15:33–55. [21] Jee WSS, Mori S, Li XJ, Chan S. Prostaglandin E2 enhances cortical bone mass and activates intracortical bone remodeling in intact and ovariectomized female rats. Bone 1991;11:253–66. [22] Frost HM. The Utah paradigm of skeletal physiology. Bone and bones (and associated problems)Athens: ISMNI; 2002. p. 140. [23] Frost HM. Bone dynamics in osteoporosis and osteomalacia. Springfield: Charles C. Thomas; 1966. [24] Frost HM. The regional acceleratory phenomenon. A review. Henry Ford Hosp Med J 1983;31:3–9. [25] Frost HM. Intermediary organization of the skeleton. Vols. I & II. Boca Raton: CRC Press; 1986. [26] Turner CH, Forwood MR, Pho JY, Yoshikawa T. Mechanical loading thresholds for lamellar and woven bone formation. J Bone Miner Res 1994;9:87–96. [27] Ma YL, Zeng Q, Donley DW, Ste-Marie LG, Gallagher JC, Dalsky GP, et al. Teriparatide increases bone formation in modeling and remodeling osteons and enhances IGF-II immunoreactivity in postmenopausal women with osteoporosis. J Bone Miner Res 2006;21:855–64. [28] Lindsay R, Cosman F, Zhou H, Bostrom MP, Shen VW, Cruz JD, et al. A novel tetracycline labeling scheduling for longitudinal evaluation of the short-term effects of anabolic therapy with a single iliac crest bone biopsy: early actions of teriparatide. J Bone Miner Res 2006;21:366–73.
Contents lists available at ScienceDirect
Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model XiaoYan Tian a, Webster S.S. Jee a,⁎, Xiaodong Li b, Chris Paszty b, Hua Zhu Ke b a b
Radiobiology Division, University of Utah School of Medicine, Salt Lake City, UT, USA Metabolic Disorders, Amgen Inc., Thousand Oaks, CA, USA
a r t i c l e
i n f o
Article history: Received 11 June 2010 Revised 10 August 2010 Accepted 8 September 2010 Available online 17 September 2010 Edited by: Robert Recker Keywords: Sclerostin antibody Bone formation Resorption Bone balance Immobilization
a b s t r a c t Sclerostin monoclonal antibody (Scl-Ab) has been shown to increase bone mass and bone strength by stimulating bone formation in an ovariectomy-induced bone loss rat model. The purpose of this study was to determine the effects of Scl-Ab in a rat immobilization/disuse model in which there was both a decrease in bone formation and an increase in bone resorption. Ten-month-old female Sprague Dawley rats were divided into normal weight-bearing (normal-loaded, NL) and right hindlimb-immobilization (under-loaded, UL) groups. Both NL and UL rats were treated with vehicle or Scl-Ab at 5 or 25 mg/kg, twice per week for 4 weeks. Trabecular and cortical bone histomorphometric analyses were performed on the proximal tibial metaphysis (PTM) and tibial shaft (TS). Compared to NL controls, UL rats had reduced body and muscle weights, increased bone marrow fat cells in the PTM, increased trabecular bone resorption and periosteal mineral apposition rate (MAR) as well as decreased trabecular MAR and bone formation rate (BFR/BS). In NL bones, treatment with Scl-Ab significantly increased bone formation and decreased bone resorption, resulting in increased trabecular and cortical bone mass. In UL trabecular bone, treatment with Scl-Ab at 5 or 25 mg/kg induced significant and dose-dependent increases in trabecular bone volume and thickness, mineralized surfaces (MS/BS), MAR and BFR/BS, and a significant decrease in eroded surface (Er.S/BS) compared with UL controls. In UL cortical bone, Scl-Ab treatment induced significant increases in cortical width, periosteal and endocortical MS/BS, MAR and BFR/BS, and significant decreases in endocortical Er.S/BS compared with UL controls. Taken together, these findings suggest that antibody-mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization-induced osteopenia. © 2010 Elsevier Inc. All rights reserved.
Introduction Sclerostin deficiency in humans, together with data from sclerostin-knockout mice, suggests that sclerostin inhibition might be an attractive approach for the development of a novel bone anabolic agent [1–4]. Studies have demonstrated that inhibition of sclerostin by a sclerostin monoclonal antibody (Scl-Ab) stimulated bone formation, and increased bone mass and bone strength in the ovariectomy-induced bone loss rat [5] and gonad-intact female monkey models [6]. Dose-dependent increases in biochemical markers of bone formation and decreases in a bone resorption marker were observed following a single subcutaneous injection of Scl-Ab in healthy post-menopausal women [7]. However, it has yet to be demonstrated whether Scl-Ab can build bone in the setting of the immobilized/disuse-induced osteopenia that occurs in paraplegic conditions, space flight and prolonged bed rest. It has been reported that increases in loading of bone were associated with decreased ⁎ Corresponding author. Division of Radiobiology, University of Utah, 729 Arapeen Dr., Suite 2338, Salt Lake City, UT 84108-1218, USA. Fax: +1 801 581 7008. E-mail address: [email protected] (W.S.S. Jee). 8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2010.09.009
sclerostin expression [8] and bone loss did not occur after unloading in mice lacking sclerostin [4]. In the current study, we employed the modified Swedish adult rat right hindlimb-immobilization model [9– 12] to test the effects of Scl-Ab on the under-loaded (UL) bone. A group of normal-loaded rats were also treated with Scl-Ab as a reference for the effects of Scl-Ab on normal-loaded bone in these adult female rats. Materials and methods The Institutional Animal Care and Use Committee at the University of Utah approved all animal procedures in this study. Animals Seventy-seven Sprague Dawley intact virgin female rats (Harlan, Indianapolis, IN, USA) were purchased at 3 months of age and housed in the University of Utah, Division of Radiobiology animal facility. The animals were housed at 72 °F with a 12:12 h light/dark cycle and were allowed free access to water and a pelleted commercial natural diet (Teklad Rodent Laboratory Chow 8640, Harlan Teklad, Madison WI,
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USA) that contained 1.13% calcium, 0.94% phosphorus and 2.99 IU/g of vitamin D3. The general condition of the animals was monitored daily and body weights recorded weekly. At 5 months of age, in order to acclimate the rats to the immobilization procedure, all rats had their right hindlimb immobilized once a week for 6 weeks for a period of 24 h. They were then immobilized once every 2 weeks for 24 h for the next 8 weeks. From that time on, until the beginning of the experiment, they were no longer immobilized. Briefly, the immobilization procedure during the study consisted of the right foot and ankle being padded with cotton to prevent pressure sores and wrapped with several layers of elastic bandage around the right hind leg which has been pulled up onto the abdomen and then layers of bandage wrapped around the abdomen allowing the front legs and left hindlimb to move freely [9–12]. The immobilization procedure generated a right under-loaded (UL) hindlimb [9–12]. The rats were checked several times every day and rewrapped as needed especially if signs of swelling, movement of the leg out of position, or evidence of the wrap being chewed was present. The rats tolerated the immobilization very well, quickly returning to ambulating and feeding on 3 legs. A group of freely ambulating rats served as normal-loaded (NL) controls. At 10-months of age, 67 rats weighing an average of 270 g were randomly divided into 7 groups with 8–11 rats per group. One group of rats was sacrificed at the beginning of the experiment to serve as beginning (basal) controls. Three weight-bearing control groups and 3 right hindlimb under-loaded groups of rats were injected with xylenol orange subcutaneously at the back of the neck (90 mg/kg, Sigma Chemical Co., St. Louis, MO, USA) on Day 1. They received vehicle (0.9% saline) or a sclerostin monoclonal antibody (Scl-AbIII; Amgen, Thousand Oaks, CA, USA) at 5 mg/kg or 25 mg/kg, twice per week (Tuesday and Friday) for 4 weeks by subcutaneous injection. The Scl-AbIII antibody is a mouse antibody in which most of the mouse sequences were replaced by rat sequences in order to reduce immunogenicity. The Scl-AbIII was sent on dry ice from Amgen to the University of Utah and stored at −80 °C until thawed to make solutions to be injected. All rats (including beginning control) were injected subcutaneously with calcein (10 mg/kg; Sigma Chemical Co. St. Louis, MO, USA) on days 14, 13, and 4, 3 before sacrifice. One day after the final treatment, animals were anesthetized with Avertin (0.4 mg/kg body weight; Sigma-Aldrich Inc., St. Louis, MO, USA) and euthanized via exsanguinations to permit collection of blood samples by open-chest cardiac puncture. The blood was centrifuged and the sera were sent to Amgen for analysis. The soft tissue (thymus, heart, lungs, liver, spleen, kidneys, and adrenals) and muscles (soleus, gastrocnemius, and quadriceps) were weighed and collected for future analyses. Right tibiae from immobilized/under-loaded (UL) and normal-loaded (NL) rats were harvested at autopsy for histomorphometry. Bone histomorphometry The right and left proximal tibiae and tibial shafts, at the tibiofibular junction, were stained in Villanueva Osteochrome Bone Stain (Arizona Histology & Histomorphometry Center, Phoenix, AZ, USA), embedded in methyl methacrylate, and sawed and ground to 20 μm longitudinal proximal tibia sections and 30 μm tibia cross sections for analyses. Data from the left bones was only summarized. The region of interest for the proximal tibial trabecular bone was an area (2.16 mm2) one mm below the growth plate within the proximal tibial metaphysis. The region of interest for cortical bone analyses included the whole mid-tibial shaft. Static and dynamic parameters were calculated and expressed according to published methods [13–15]. Additionally, point counting with a Merz eyepiece reticule [16] was performed to measure the percent fat content of the bone marrow. The number of points superimposed over fat cells was
divided by the total number of points superimposed over bone marrow to calculate the percent marrow fat area. Statistical analysis All data were presented as group means ± standard deviation (SD). The statistical analyses were performed using the Ultimate Integrated Data Analysis and Presentation System (StatView 5.0.1, SAS Institute Inc. Cary, NC). Across group comparisons were made with a parametric analysis of variance (ANOVA) followed by a Fisher Protected Least Significant Difference (Fisher PLSD) test, and a value of p b 0.05 was considered statistically significant. Results At the end of the study, body weights in UL rats were 15% less than those of NL rats regardless of treatment. Both doses of the Scl-Ab had no effect on body weight (data not shown). Muscle weights in UL rats were significantly lower as compared with basal controls and NL controls. Specifically, the right soleus, gastrocnemius and quadriceps muscles weighed 44%, 59% and 56% less, respectively, relative to beginning controls. Treatment with 5 or 25 mg/kg Scl-Ab had no effect on muscle weight in both UL and NL rats (data not shown). The bone marrow in the proximal tibial metaphysis (PTM) of NL rats contained 10.8 ± 7.9% (Mean ± SD) fat cell area while UL rats contained 33.1 ± 12.6% fat cell area, a 218% higher fat cell content. SclAb treatment at both doses had no significant effect on fat cell content in both UL and NL bone marrow sites compared to their respective vehicle controls (data not shown). Trabecular bone in the proximal tibial metaphysis (PTM) At the end of the study there was no significant difference in all parameters listed in Table 1 in NL controls compared with basal controls (Table 1), indicating that no age-related change was observed during the 4-week experimental period for these rats. Compared with basal controls, bone formation rate (BFR/BS) was significantly lower by 23%, and eroded surface (Er.S/BS) was significantly higher by up to 44% in UL controls (Table 1), indicating that immobilization had induced lower bone formation and higher bone resorption. However, there was no significant difference in trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) or trabecular separation (Tb.Sp) between vehicle-treated UL and NL groups (Table 1), indicating immobilization for 4 weeks did not induce significant trabecular bone loss in these 10month-old female rats. Compared to their respective vehicle controls, BV/TV and Tb.Th were significantly greater in the 5 or 25 mg/kg-treated NL and UL trabecular bones and additionally, Tb.N was significantly higher and Tb.Sp was significantly lower than vehicle controls in the 25 mg/kg NL group (Table 1). Scl-Ab treatment at both doses significantly increased MS/BS, MAR, BFR/BS and significantly decreased Er.S/BS in both NL and UL trabecular bones as compared to their respective vehicle controls, indicating that Scl-Ab stimulates bone formation and decreases bone resorption in the setting of normal loading and of under-loading (Table 1 and Fig. 1). There were significant increases in Tb.Th, MS/BS, MAR and BFR/BS in UL trabecular bone treated with 25 mg/kg vs. 5 mg/kg, indicating a dose-dependent effect for these parameters. Similarly, there were significant increases in BV/TV, Tb. Th, MS/BS and BFR/BS and there was a significant decrease in Er.S/BS in NL trabecular bone treated with 25 mg/kg vs. 5 mg/kg (Table 1). Scl-Ab at 5 and 25 mg/kg were more effective in NL than UL cancellous bones. For example, at 5 mg/kg TbN, MS/BS, MAR and BFR/BS were significantly increased and Er.S/BS was decreased more in NL than UL bones. In addition, at 25 mg/kg all the selected static and
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Table 1 Selective static and dynamic cancellous bone histomorphmetric analysis of proximal tibial metaphysis (PTM). Parameters
BV/TV (%) Tb.Th (μm) Tb.N (#/mm) Tb.Sp (μm) MS/BS (%) MAR (μm/d) BFR/BS (μm3/μm2/d × 100) Er.S/BS (%)
Beginning control [basal]
Normal-loaded + Scl-Ab (mg/kg) 0
5
25
0
Under-loaded + Scl-Ab (mg/kg) 5
25
14.3 ± 4.8 45.3 ± 6.4 3.1 ± 0.7 297.8 ± 99.0 24.6 ± 7.3 0.7 ± 0.1 17.9 ± 6.1 3.2 ± 1.1
13.9 ± 2.7 44.5 ± 2.8 3.1 ± 0.6 285.2 ± 65.6 27.6 ± 4.5 0.7 ± 0.1 20.1 ± 3.1⁎ 3.4 ± 0.8⁎
24.6 ± 9.2a,b 75.5 ± 9.9a,b,d 3.2 ± 1.0 310.9 ± 271.9 55.1 ± 3.8a,b,d 1.0 ± 0.1a,b,d 55.4 ± 8.7a,b,d 1.7 ± 0.6a,b,d
34.7 ± 6.5a,b,c,e 93.6 ± 10.7a,b,c,e 3.7 ± 0.3a,b,e 180.3 ± 33.3a,b,e 69.2 ± 2.6a,b,c,e 1.1 ± 0.1a,b 75.7 ± 9.6a,b,c,e 0.8 ± 0.3a,b,c,e
13.4 ± 1.1 41.9 ± 3.3 3.2 ± 0.4 271.6 ± 36.7 25.7 ± 2.3 0.6 ± 0.1 14.1 ± 3.6⁎
19.4 ± 3.3a,b 57.9 ± 5.7a,b,d 3.4 ± 0.4 245.0 ± 42.2 44.4 ± 5.0a,b,d 0.8 ± 0.1a,b,d 37.6 ± 6.9a,b,d 3.2 ± 1.0b,d
21.2 ± 6.3a,b,e 71.3 ± 7.1a,b,c,e 2.9 ± 0.7e 300.3 ± 156.1e 56.8 ± 7.2a,b,c,e 1.0 ± 0.2a,b,c 55.7 ± 15.1a,b,c,e 2.7 ± 0.9b,e
4.7 ± 0.8a,⁎
BV/TV, trabecular bone volume / tissue volume × 100; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; MS/BS, mineralizing surface/bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate/bone surface referent; and Er.S/BS, eroded surface/bone surface. Mean ± SD. a p b 0.05 vs. beginning control. b p b 0.05 vs. respective 0 mg/kg. c p b 0.05 vs. 5 mg/kg. d p b 0.05 NL vs. UL 5 mg/kg. e p b 0.05 NL vs. UL 25 mg/kg. ⁎ p b 0.05 NL + 0 mg/kg vs. UL + 0 mg/kg.
dynamic histomorphometric parameters were significantly more effective in NL than in UL bones (Table 1). This indicated that there may be some additive effects of Scl-Ab and mechanical loading on bone. We also observed that trabecular bone of NL and UL rats treated with 5 or 25 mg/kg Scl-Ab exhibited buried xylenol orange labels given on Day 1 apposed by double calcein labels given on 14 and 15 and 24 and 25 days. This observation indicates that the Scl-Abstimulated bone formation on the trabecular bone surface may not be dependent upon activation of bone resorption (Fig. 2A). Compared with the right UL-PTM, 5 and 25 mg/kg Scl-Ab of the left PTM of the right hindlimb immobilized rats induced non-significantly increased BV/TV, Tb.Th, TB.N, MS/BS, MAR and BFR/BS and nonsignificantly decreased Tb.Sp and Er.S/BS (data not shown).
Cortical bone in the tibial shaft (TS) Compared with basal controls, endocortical MS/BS increased significantly in NL controls (Table 2). Periosteal MAR increased significantly in UL controls compared with basal or NL controls (Table 2). There was no significant difference in cortical bone mass (Ct.Ar), thickness (Ct.Th) and marrow areas (Ma.Ar) in NL or UL vehicle controls compared with basal controls. Scl-Ab treatment at 5 or 25 mg/kg significantly increased Ct.Ar, Ct. Th, periosteal and endocortical MS/BS, MAR and BFR/BS, and
significantly decreased Ma.Ar and endocortical Er.S/BS in both UL and NL groups compared with basal controls (Table 2). Compared with UL vehicle controls, Scl-Ab treatment at 5 or 25 mg/kg induced a non-significant increase in Ct.Ar and a nonsignificant decrease in Ma.Ar in UL cortical bone. Ct.Th increased significantly in the 25 mg/kg group and increased non-significantly in the 5 mg/kg group in UL cortical bone compared with UL vehicle controls (Table 2). Scl-Ab treatment at 5 or 25 mg/kg significantly increased periosteal and endocortical MS/BS, MAR, BFR/BS with the exception of periosteal MAR at 5 mg/kg, and significantly decreased endocortical Er.S/BS as compared with UL vehicle controls (Table 2; Fig. 3). Compared with NL vehicle controls, Scl-Ab treatment at 25 mg/kg significantly increased Ct.Ar and Ct.Th, and significantly decreased Ma. Ar, while no significant effect was found in the 5 mg/kg dose group (Table 2; Fig. 3). Similar to what was observed in UL cortical bone, Scl-Ab treatment at 5 or 25 mg/kg significantly increased periosteal and endocortical MS/BS, MAR, BFR/BS, and significantly decreased endocortical Er.S/BS in NL cortical bone as compared with NL vehicle controls (Table 2). Compared with UL treated with 5 mg/kg, there were significant increases in periosteal MS/BS, MAR, BFR/BS and endocortical MAR and BFR/BS in the UL treated with 25 mg/kg (Table 2). For NL cortical bone, Scl-Ab at 25 mg/kg induced significantly higher Ct.Th, periosteal and endocortical MS/BS, MAR, BFR/BS, and a significantly lower endocortical Er.S/BS compared with
Fig. 1. Five and 25 mg/kg Scl-Ab treatment significantly increased trabecular bone mass and width, and bone formation in both normal-loaded and under-loaded cancellous bones of the proximal tibial metaphyses. GP = growth plate. Villanueva bone stain; 10×.
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Fig. 2. Scl-Ab induced bone formation on trabecular and endocortical bone surfaces without prior bone resorption. (A) Buried xylenol orange labels (*) apposed by calcein labeling in trabecular bone. (B) Extensive buried xylenol orange labels near endocortical surface (*) followed by double calcein labeling. Xylenol orange label given on Day 1 and double calcein labels given on days 14–15 and 24–25; 100×.
the 5 mg/kg dose group (Table 2). These data demonstrated that there was a dose-dependent effect between 5 and 25 mg/kg on cortical bone of normal-loaded or under-loaded rats. Unlike in cancellous bone, there was no increase in effects of Scl-Ab and mechanical loading. Compared to right UL-tibial shaft, the 25 mg/kg Scl-Ab in left tibial shaft from the right hindlimb immobilized rats induced significantly increased periosteal MS/BS and significantly decreased endocortical MAR, BFR/BS and Er.S/BS (data not shown). Similar to what was observed for trabecular bone, we also found that the endocortical surfaces of NL and UL rats treated with 5 or 25 mg/kg Scl-Ab exhibited buried xylenol orange labels given on Day 1 apposed by double calcein labels given on 14 and 15 and 24 and 25 days. This observation indicates that the Scl-Ab stimulated bone formation on the endocortical bone surface may not be dependent upon activation of bone resorption (Fig. 2B). Discussion We studied antibody-mediated sclerostin inhibition in adult female rats with or without right hindlimb-immobilization. Scl-Ab treatment significantly induced increases in trabecular and cortical bone under both normal-loaded and under-loaded conditions. The increased bone mass with Scl-Ab treatment was due to increased bone formation and decreased bone resorption on both trabecular and endocortical bone surfaces, as well as increased bone formation on periosteal surfaces. Furthermore, histological observations indicated that Scl-Ab can increase bone formation without prior activation of bone resorption. There were several divergent findings from our previous studies [17–21] in our current use of this model. One involved the lack of inhibition of periosteal bone formation in the under-loaded (UL) tibial shaft. Instead, periosteal osteoblastic activity (mineral appositional
Fig. 3. The responses of normal-loaded and under-loaded tibial shafts to Scl-Ab. Scl-Ab at 25 mg/kg increased periosteal (P) and endocortical (E) bone formation in both normal-loaded and under-loaded tibial shafts. Villanueva bone stain; 10×.
rate) and bone formation rate were stimulated. In these older rats, apparently the immobilization by bandaging set off Frost's regional acceleratory phenomenon (RAP) [22–25]. To quote Frost, “A RAP normally accelerates all ongoing regional processes involving blood flow, temperature, cell metabolism and turnover, creating new capillaries and any ongoing modeling and remodeling and healing of hard and soft tissues.” [25]. In support, the RAP set off in the 4-point loading study by Turner et al. [26] in which the periosteum was squeezed by loading that activated periosteal woven bone formation. In addition, the RAP may have been responsible for why Scl-AbIII treatment did not induce more changes in normal-loaded than under-loaded cortical bone. The RAP response caused by under-loading by bandaging tended to increase periosteal and endocortical bone formation and decreased bone resorption in the UL-TS to the same level that occurred in the NL-TS (Table 2). Possibly, also the brief treatment period of 4 weeks to cortical bone was insufficient to overcome the functional lag in cortical bone tissue level responses. Two, the brief treatment period of 4 weeks dictated by the quantity of the available antibody and the use of older rats resulted in the lack of static bone changes in the immobilized-limb. The effects of Scl-Ab at the higher dose (25 mg/kg) on trabecular bone mass and thickness, bone formation and bone resorption, were more pronounced in normal-loaded (NL) proximal tibial metaphysis (PTM) compared with the effects noted in under-loaded (UL) PTM, indicative of an enhanced response to Scl-Ab treatment in mechanically-loaded bone. This greater anabolic response observed for normal-loaded bone vs. under-loaded bone may be due, in part, to the previously reported finding that sclerostin expression is decreased by loading and increased by unloading [4,8]. Thus in the current study, a relative excess of sclerostin in the UL bones would likely have a dampening effect on the UL Scl-Ab-mediated anabolism relative to the situation in the NL bones. In contrast to what was found for the PTM,
Table 2 Static and dynamic cortical bone histomorphometric analysis of tibial shaft. Parameters
Ct.Ar (%) Ma.Ar (%) Ct.Th (μm) Ps-MS/BS (%) Ps-MAR (μm/d) Ps-BFR/BS (μm/d × 100) Ec-MS/BS (%) Ec-MAR (μm/d) Ec-BFR/BS (μm/d × 100) Ec-Er.S/BS (%)
Beginning control [basal]
Normal-loaded + Scl-Ab (mg/kg) 0
5
25
0
5
25
80.7 ± 2.6 19.3 ± 2.6 645 ± 33 26.1 ± 7.8 0.5 ± 0.2 12.7 ± 6.5 17.3 ± 7.0 0.5 ± 0.1 9.5 ± 5.4 3.3 ± 1.0
81.7 ± 2.0 18.3 ± 2.0 658 ± 36 30.6 ± 12.6 0.5 ± 0.2⁎
83.4 ± 1.6a 16.6 ± 1.6a 677 ± 18a 46.3 ± 15.8a,b 0.7 ± 0.1a,b 34.9 ± 17.7a,b 65.1 ± 13.9a,b 1.5 ± 0.2a,b 99.5 ± 25.3a,b 0.8 ± 0.4a,b
85.3 ± 2.2a,b 14.7 ± 2.2a,b 723 ± 43a,b,c 85.9 ± 11.0a,b,c 1.1 ± 0.2a,b,c,e 95.9 ± 27.9a,b,c 84.9 ± 12.5a,b,c 1.7 ± 0.1a,b,c 148.9 ± 27.0a,b,c 0.3 ± 0.2a,b,c
81.6 ± 3.2 18.4 ± 3.2 651 ± 52 24.0 ± 8.8 0.9 ± 0.3a,⁎ 22.0 ± 12.7 19.3 ± 2.3 0.5 ± 0.2 10.2 ± 2.7 4.4 ± 2.5
84.1 ± 1.5a 15.9 ± 1.5a 686 ± 37a 48.2 ± 13.7a,b 1.0 ± 0.3a 48.6 ± 18.4a,b 51.0 ± 10.0a,b 1.3 ± 0.3a,b 67.8 ± 23.2a,b 1.2 ± 1.2a,b
84.7 ± 2.6a 15.3 ± 2.6a 723 ± 44a,b 72.7 ± 10.1a,b,c 1.7 ± 0.2a,b,c,e 120.0 ± 26.1a,b,c 84.5 ± 9.7a,b 1.7 ± 0.2a,b,c 141 ± 26a,b,c 0.5 ± 0.2a,b
17.1 ± 11.2 25.1 ± 6.9a 0.6 ± 0.2 16.7 ± 8.7 3.6 ± 1.2
Under-loaded + Scl-Ab (mg/kg)
Ps — Perisoteal; Ec — Endocortical; Ct.Ar, cortical bone area / total tissue area × 100; Ma.Ar, marrow cavity area / total tissue area × 100; Ct.Th, cortical thickness; MS/BS, mineralizing surface/bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate/bone surface referent; and Er.S/BS, eroded surface/bone surface. ap b 0.05 vs. beginning control, b p b 0.05 vs. respective 0 mg/kg, cp b 0.05 vs. 5 mg/kg, dp b 0.05 NL vs. UL 5 mg/kg, e p b 0.05 NL vs. UL 25 mg/kg, ⁎p b 0.05 NL + 0 mg vs. UL + 0 mg.
X. Tian et al. / Bone 48 (2011) 197–201
for cortical bone at the mid-tibial shaft there was no difference regardless of mechanical loading in response to Scl-Ab at both the lower and the higher doses. The difference between trabecular and cortical bone in response to the interaction of mechanical loading and sclerostin inhibition in this short-term (4 weeks) study is currently not well understood. One possible explanation is that immobilization for 4 weeks did not induce significant changes in cortical bone surface with the exception of an increase in periosteal MAR, while it induced significant decreases in bone formation and increases in bone resorption in trabecular bone. It has been well established that a longer duration was required to induce cortical bone changes in this rat immobilization model [11]. Thus, a longer-term study would be required to test whether there are differences in the cortical bone response to sclerostin antibody treatment under different mechanical loading conditions (i.e. NL vs. UL). With Scl-Ab administration it was not surprising to find the dramatic dual effect of increased cancellous and cortical bone formation, and decreased bone resorption resulting in increased trabecular bone mass and thickness in normal- and under-loaded bone sites because earlier studies reported similar dramatic responses in aged OVX rats, cynomolgus monkeys and humans [5–7]. In our current study we found that the 25 mg/kg dose of Scl-Ab increased bone formation surfaces more than that observed for the 5 mg/kg dose. The histological findings of xylenol orange labeling given on Day 1 separated from double calcein labels given on days 14–15 and days 24– 25 indicate that the bone formation induced by Scl-Ab treatment can take place on bone surfaces without prior activation of bone resorption. These sites can be interpreted as bone formation occurring at ongoing bone formation surfaces, occurring from the overfilling of bone remodeling sites or onto adjacent quiescent surfaces or from bone lining cells initiating minimodeling, observed previously in PTH stimulated bone formation via modeling and bone remodeling [27,28]. In conclusion, short-term administration of 5 and 25 mg/kg sclerostin neutralizing antibody in an adult female rat model of right hindlimb-immobilization and normally loaded ambulated rats resulted in a dramatic increase in bone formation and a decrease in bone resorption that led to increased trabecular and cortical bone mass in normal- and under-loaded cancellous and cortical bone sites. The rapid increase in bone mass was dominated by robust bone formation on trabecular, periosteal and endocortical bone surfaces with greater trabecular bone gain in normal-loaded than underloaded bones. Finally, the bone building effects obtained in this immobilized-limb rat study suggests that antibody-mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization-induced osteopenia. Conflict of interest Tian and Jee have no conflict of interest. Li, Paszty and Ke have corporate appointments with Amgen, Inc. Acknowledgments The authors acknowledge the support of Rebecca B. Setterberg and Min Chen for their excellent technical assistance. The funding for this study was supported by Amgen Inc. and UCB. The authors thank all members of the Amgen and UCB (Slough, United Kingdom) sclerostin team for their support of this study.
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