Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1–34) on trabecular and cortical bone in mice

Bone 43 (2008) 238–248

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Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1–34) on trabecular and cortical bone in mice Toshihiro Sugiyama ⁎, Leanne K. Saxon, Gul Zaman, Alaa Moustafa, Andrew Sunters, Joanna S. Price, Lance E. Lanyon Department of Veterinary Basic Sciences, The Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, UK

a r t i c l e

i n f o

Article history: Received 18 December 2007 Revised 11 March 2008 Accepted 13 April 2008 Available online 1 May 2008 Edited by: S. Ralston Keywords: Mechanical loading Adaptation Parathyroid hormone Mouse Micro-computed tomography

a b s t r a c t The separate and combined effects of intermittent parathyroid hormone (iPTH) (1–34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1–34) at low (20 μg/kg/day), medium (40 μg/kg/day) or high (80 μg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 μm. Treatment with iPTH (1–34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1–34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1–34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1–34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1–34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1–34) or either level of loading alone, whereas the combination of iPTH (1–34) and the “sufficient” level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1–34) in trabecular and cortical bone. © 2008 Elsevier Inc. All rights reserved.

Introduction Fragility fracture associated with osteoporosis is a major health problem especially for women [1–3]. Although anti-resorptive agents such as bisphosphonates have played a central role in the management of osteoporosis, anabolic therapies have the potential to strengthen the fragile skeleton by enhancing bone formation [4]. At present, intermittent parathyroid hormone (iPTH) [5–15] is the only treatment licensed for this purpose. Patients who fail anti-resorptive therapy and are at high risk of fracture, would be candidates for such therapy. However, one possible concern [16–18] is that the effects of iPTH might be attenuated under conditions of low physical activity, which is itself a risk factor for fracture of the hip in postmenopausal women [19].

⁎ Corresponding author. E-mail address: [email protected] (T. Sugiyama). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.04.012

Mechanical loading is the major natural anabolic factor that influences bone structure and strength [20–22]. However, prescribing osteogenic levels of load-bearing exercise for osteoporotic patients already at high risk of fracture is problematic. There would therefore be benefit if iPTH could reduce the loading level necessary to stimulate a loading-related anabolic effect [23]. Clinical evidence shows that treatment with iPTH increases cortical width at some skeletal sites [24,25]. This site specificity has been attributed to differences in the bones' local mechanical environment [25]. Since iPTH increases remodelling turnover within the bone, it is associated with a reduction in overall bone mineralization [25,26]. This decrease could contribute to the stimulus for cortical expansion by a compensatory effect through increased mechanical strain from loading [27,28]. If this were so, it would suggest that in the human skeleton iPTH is stimulating an osteogenic response, at least in part, as a secondary effect. No such secondary effect is possible in vitro, where PTH promotes the response of bone-forming osteoblastic cells to mechanical

T. Sugiyama et al. / Bone 43 (2008) 238–248

stimulation [29,30]. Consistent with this in vitro observation, several experimental in vivo studies using rats have shown that iPTH and mechanical loading act synergistically in both trabecular and cortical bone [31–36]. These experiments include the effects of loading on the vertebra of the tail [32,36]; mediolateral bending of the tibia [34,35]; and axial compression of the ulna [35]. Although, to our knowledge, there is no direct evidence of such synergistic actions in mice, iPTH treatment has been reported to increase periosteal bone formation and cortical thickness in the tibia but not in the vertebral body in this species. The authors ascribed this regional difference to the different mechanical environment at the two sites [37]. Since, in rodents such as rat and mouse, intracortical bone remodelling only occurs at very low levels, any potential effects of iPTH on mineralization due to increased remodelling can probably be discounted in these animals. This means that any possible secondary effect on bone's response to loading such as that mentioned above can be excluded. In this situation, the responses of the skeleton of wildtype and genetically modified mice to iPTH and mechanical loading should provide significant mechanistic insights into their primary and combined effects. C57BL/6 mice have been extensively used as the background of genetically modified animals in the field of bone research [38] and also show a good response to mechanical loading [39]. The present study was therefore designed to assess the effects of mechanical loading on trabecular and cortical sites of long bones in female C57BL/6 mice treated with low, medium or high dose of iPTH (1–34). These mice were pre-treated with iPTH (1–34) before loading in order to approximate to the most likely clinical situation where iPTH would have been prescribed and exercise then introduced to provide an additional osteogenic stimulus. It would be unlikely for both exercise and iPTH therapy to be introduced together, or exercise before iPTH therapy. The tibia and ulna on one side were subjected to noninvasive, dynamic axial loading using methods previously reported

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[40,41], and two levels of peak load were used; one sufficient to engender an osteogenic response and the other insufficient to do so. This is the first study, of which we are aware, concerning the separate and combined effects of iPTH and in vivo mechanical loading on the mouse skeleton. Materials and methods Experimental design Virgin, female C57BL/6 mice at 7 weeks of age were purchased from Charles River Laboratories, Inc. (Margate, UK) and group-housed (n = 3–5) in sterilized polypropylene cages with free access to water and a maintenance diet containing 0.73% calcium, 0.52% phosphorus, and 3.5 IU/g vitamin D (RM1; Special Diet Services Ltd., Witham, UK) in a 12-h light/dark cycle, with room temperature at 21 ± 2 °C. Body weight was measured once a week. In an initial “Introductory iPTH” experiment, the mice were randomised into four groups at 13 weeks of age and received subcutaneously 4-weeks of intermittent treatment with vehicle (99.7% saline, 0.2% bovine serum albumin [Sigma Chemical Co., St. Louis, Missouri, USA], and 0.1% hydrochloric acid) or human PTH (1–34) (Bachem Biosciences, Inc., King of Prussia, Pennsylvania, USA) at low (20 μg/kg/day), medium (40 μg/kg/day) or high (80 μg/kg/day) dose (7 days/week). At 17 weeks of age (day 29), the mice were euthanized and both tibiae and ulnae collected as described below. In a second “iPTH/loading” experiment, the mice were randomised into four groups at 13 weeks of age and subcutaneously received 6-weeks of intermittent treatment with vehicle or human PTH (1–34) at low (20 μg/kg/day), medium (40 μg/kg/day) or high (80 μg/kg/day) dose (7 days/week). During the last 2 weeks, i.e., from 17 to 19 weeks of age, the right tibiae and ulnae of these mice were subjected to external mechanical loading under isoflurane-induced anesthesia (approximately 7 min/day, 3 alternate days/week; tibia: Mon, Wed and Fri, ulna: Tue, Thu and Sat). Loading was carried out between 30 and 40 min after the vehicle or PTH (1–34) injection. Normal cage activity was allowed and the left tibiae and ulnae were used as non-loaded controls. Double calcein (30 mg/kg body weight; Sigma Chemical Co.) and single alizarin (30 mg/kg body weight; Sigma Chemical Co.) labels were injected intraperitoneally on the first days of iPTH (1–34) treatment (day 1) and loading (day 29), and the last day of loading (day 41), respectively. At 19 weeks of age (day 43), the mice were euthanized and bilateral tibiae and ulnae were collected as described below.

Fig. 1. Direction of mechanical loading in the right tibia and ulna and their representative transverse μCT images at the analyzed sites in a 17 week old female C57BL/6 mouse. Levels of peak load: sufficient to engender an osteogenic response in the tibia and insufficient to do so in the ulna.

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All procedures complied with the UK Animals (Scientific Procedures) Act 1986 and were reviewed and passed by the ethics committee of The Royal Veterinary College (London, UK). In vivo mechanical loading The apparatus and protocol for dynamically loading the mouse tibia and ulna have been reported previously [40,41]. Dynamic cyclical loads are applied between the flexed knee and ankle or the flexed elbow and wrist. The flexed joints are positioned in concave cups; the upper cup, into which the knee or wrist is positioned, is attached to the actuator arm of a servo-hydraulic loading machine (Model HC10; Zwick Testing Machines Ltd., Leominster, UK) and the lower cup to a dynamic load cell. The servo-hydraulic mechanism of the loading machine operates to apply controlled dynamic compressive loads axially to the tibia/fibula or ulna/ radius (Fig. 1). In the “Introductory iPTH” experiment, the right fore and hind limbs including all tissues were stored at −20 °C in sealed, air-tight plastic bags immediately after sacrifice. When ready for measurement, these limbs were put in saline solution at room temperature for at least 3 h. Soft tissues were dissected to expose the medial surface of the proximal tibia (37% along the shaft) and the lateral surface of the ulna mid-shaft, and trimmed single element strain gauges (EA-06-015DJ-120; Vishay Measurements Group, Basingstoke, UK) were bonded to these surfaces in longitudinal alignment. The gauges were connected to a strain conditioner/amplifier system and mechanical loadinduced strains were measured as previously described [40,41]. In the “iPTH/loading” experiment, the right tibia and ulna of each mouse was held in the upper and lower cups of the loading apparatus and dynamic axial loads (trapezoidal-shaped pulse period = 0.1 s [loading 0.025 s, hold 0.05 s and unloading 0.025 s]; rest time between pulses = 10 s; cycles/day = 40) were applied between the knee and ankle or the elbow and wrist. In the mice treated with vehicle, peak loads of 12.0 N (engendering approximately 1200 µε at the medial surface of the tibiae 37% along the bone shafts from their proximal end) and 2.5 N (engendering approximately 1350 µε at the lateral surface of the ulna midshafts) were applied to the tibia and ulna, respectively. These peak loads were sufficient and insufficient, respectively, to stimulate an osteogenic response in the present loading regimen [40,41]. The peak loads applied to the iPTH (1–34)-treated bones were adjusted to induce similar peak levels of bone strain by using data of the bone length and

“average” polar mean moment of inertia at proximal (25%), middle (50%) and distal (75%) sites in the “Introductory iPTH” experiment. As a result, in mice treated with iPTH (1–34) at low, medium or high dose, peak loads for the tibiae sufficient to engender osteogenesis, and for the ulnae insufficient to do so were 13.7, 14.7 and 15.8 N, and 2.8, 3.0 and 3.3 N, respectively. High-resolution micro-computed tomography (μCT) analysis The left tibiae and ulnae in the “Introductory iPTH” experiment and the bilateral tibiae and ulnae in the “iPTH/loading” experiment were stored in 70% ethanol. These tibiae and ulnae were scanned at high-resolution μCT (SkyScan 1172; SkyScan, Kontich, Belgium) with a pixel size of 5 μm. The images of the whole bones were reconstructed by the SkyScan software (version 1.4.4) and their lengths were measured. The analyzed regions were 1) in trabecular bone of the proximal tibiae, between 0.01 and 0.25 mm (containing primary spongiosa) and 0.25 and 1.25 mm (secondary spongiosa) distal to growth plate, 2) in cortical bone of the tibiae, 0.5 mm long centered across the proximal (25% and 37%), middle (50%) and distal (75%) sites, and 3) in cortical bone of the ulnae, 0.5 mm long sections across the proximal (25%), middle (50%) and distal (75%) sites (Fig. 1). In the tibiae, the 37% proximal cortical bone site was selected because this was where the strain gauges were positioned. Three-dimensional structural analysis for these trabecular and cortical bone sites were performed by the SkyScan software (version 1.6.1.1). Percent bone volume, trabecular number and trabecular thickness in the trabecular bone, and periosteally-enclosed volume, cortical bone volume, medullary volume and cross-sectional polar mean moment of inertia, a parameter of structural bone strength, in the cortical bones were evaluated. Calcein and alizarin labels imaging by confocal microscopy In the “iPTH/loading” experiment, both left non-loaded and right loaded tibiae and ulnae were maintained in 70% ethanol after scanning by μCT. These bones were then dehydrated, cleared and embedded in methyl methacrylate as previously described [42], and 500 μm-thick serial transverse segments of the whole tibiae and ulnae were obtained by cutting with an annular diamond saw. Images of calcein and alizarin labelled transverse bone sections were visualized using argon 488 nm laser and HeNe 543 nm laser, respectively, of a confocal laser scanning microscope (LSM

Table 1 Structural parameters in the tibia and ulna of 17 week old female C57BL/6 mice treated with 4-weeks of intermittent parathyroid hormone (iPTH) (1–34) Dose of PTH (1–34)

0 μg/kg/day

20 μg/kg/day

40 μg/kg/day

80 μg/kg/day

Trabecular bone of proximal tibia Secondary spongiosa Percent bone volume (%) Trabecular number (mm− 1) Trabecular thickness (mm)

10.9 ± 0.4 2.09 ± 0.08 0.0523 ± 0.0012

16.0 ± 0.4a 3.24 ± 0.05a 0.0494 ± 0.0007

17.8 ± 0.3a,b 3.81 ± 0.09a,b 0.0468 ± 0.0005a

29.2 ± 1.3a,b,c 6.68 ± 0.34a,b,c 0.0438 ± 0.0008a,b

2.88 ± 0.04 1.55 ± 0.04 1.32 ± 0.02

3.10 ± 0.04a 1.81 ± 0.02a 1.30 ± 0.03

3.22 ± 0.08a 1.84 ± 0.05a 1.39 ± 0.04

3.34 ± 0.06a,b 1.92 ± 0.05a 1.43 ± 0.03b

2.04 ± 0.02 1.20 ± 0.02 0.832 ± 0.022

2.12 ± 0.04 1.27 ± 0.02 0.856 ± 0.024

2.18 ± 0.04a 1.31 ± 0.02a 0.872 ± 0.018

2.24 ± 0.04a 1.38 ± 0.03a,b 0.868 ± 0.018

1.53 ± 0.02 1.08 ± 0.02 0.450 ± 0.018

1.58 ± 0.02 1.16 ± 0.02a 0.424 ± 0.016

1.63 ± 0.03 1.22 ± 0.04a 0.402 ± 0.018

1.69 ± 0.03a,b 1.30 ± 0.04a,b 0.388 ± 0.022

0.886 ± 0.020 0.814 ± 0.016 0.0726 ± 0.0052

0.916 ± 0.012 0.846 ± 0.012 0.0708 ± 0.0052

0.962 ± 0.016a 0.886 ± 0.016a 0.0754 ± 0.0056

0.984 ± 0.010a,b 0.904 ± 0.010a,b 0.0788 ± 0.0076

0.584 ± 0.010 0.484 ± 0.008 0.101 ± 0.007

0.616 ± 0.010 0.528 ± 0.006a 0.088 ± 0.005

0.648 ± 0.012a 0.546 ± 0.010a 0.101 ± 0.004

0.672 ± 0.012a,b 0.568 ± 0.010a,b 0.104 ± 0.007

0.408 ± 0.006 0.360 ± 0.008 0.0486 ± 0.0024

0.460 ± 0.010a 0.406 ± 0.006a 0.0520 ± 0.0030

0.456 ± 0.006a 0.412 ± 0.004a 0.0450 ± 0.0038

Cortical bone of tibia Proximal (25%) Periosteally-enclosed volume (mm3) Cortical bone volume (mm3) Medullary volume (mm3) Middle (50%) Periosteally-enclosed volume (mm3) Cortical bone volume (mm3) Medullary volume (mm3) Distal (25%) Periosteally-enclosed volume (mm3) Cortical bone volume (mm3) Medullary volume (mm3) Cortical bone of ulna Proximal (25%) Periosteally-enclosed volume (mm3) Cortical bone volume (mm3) Medullary volume (mm3) Middle (50%) Periosteally-enclosed volume (mm3) Cortical bone volume (mm3) Medullary volume (mm3) Distal (25%) Periosteally-enclosed volume (mm3) Cortical bone volume (mm3) Medullary volume (mm3)

Mean ± S.E. (n = 8–10). a p b 0.05 vs 0 μg/kg/day (one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test). b p b 0.05 vs 20 μg/kg/day (one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test). c p b 0.05 vs 40 μg/kg/day (one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test).

0.478 ± 0.006a 0.428 ± 0.006a 0.0496 ± 0.0030

T. Sugiyama et al. / Bone 43 (2008) 238–248 Table 2 Relative “average” polar mean moment of inertia at proximal (25%), middle (50%) and distal (25%) sites of the tibia and ulna in female C57BL/6 mice treated with 4-weeks of intermittent parathyroid hormone (iPTH) (1–34), or 6-weeks of iPTH (1–34) and 2weeks of mechanical loading Dose of PTH (1–34)

0 μg/kg/day

20 μg/kg/day

17 week old after 4-weeks of iPTH (1–34) Tibia (%) 100 ± 2 114 ± 3a Ulna (%) 100 ± 3 113 ± 3a 19 week old after 6-weeks of iPTH (1–34) and 2-weeks of Tibia (%) Left control 100 ± 3 124 ± 5a Right loaded 100 ± 2 120 ± 3a Ulna (%) Left control 100 ± 3 122 ± 5a Right loaded 100 ± 3 118 ± 6a

40 μg/kg/day

80 μg/kg/day

122 ± 4a 120 ± 3a

131 ± 4a,b 131 ± 3a,b

loading 142 ± 5a 142 ± 4a,b

145 ± 5a 142 ± 4a,b

139 ± 5a 135 ± 4a,b

137 ± 5a 142 ± 4a,b

Mean ± S.E. (n = 5–10). a p b 0.05 vs 0 μg/kg/day (one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test). b p b 0.05 vs 20 μg/kg/day (one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test). 510; Carl Zeiss MicroImaging GmbH, Jena, Germany) at the similar regions as the μCT analysis. Statistical analysis All data are shown as mean ± S.E. Statistical analysis was performed by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test, two-way ANOVA or paired t-test using SPSS 15.0 software (SPSS Inc., Chicago, Illinois, USA). p b 0.05 was considered as statistically significant.

Results Effects of iPTH (1–34) alone The effects of 4-weeks of pre-treatment with iPTH (1–34) in the “Introductory iPTH” experiment are shown in Tables 1 and 2. In trabecular bone of the proximal tibiae, 4-weeks of iPTH (1–34) resulted in a marked, dose-dependent increase in percent bone volume in the secondary spongiosa (Table 1). This was achieved primarily through an increase in trabecular number which transformed the whole appearance of the trabecular bone structure (Fig. 2). In cortical bones of the tibiae and ulnae, 4-weeks of iPTH (1–34) also

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induced significant increases in periosteally-enclosed volume and cortical bone volume at proximal, middle and distal sites (Table 1). The “average” polar mean moment of inertia at these sites was significantly increased in a dose-related manner (Table 2). In the “iPTH/loading” experiment, 6-weeks of treatment with iPTH (1–34) modified trabecular and cortical bone structure in a timedependent manner, compared with the 4-weeks of iPTH (1–34) treatment in the “Introductory iPTH” experiment. In trabecular bone of the proximal tibiae, the additional 2-weeks of iPTH (1–34) resulted in further dose-dependent increases in percent bone volume and trabecular number in the secondary spongiosa, although these effects in the primary spongiosa were similar at all doses of iPTH (1–34) (Table 3, Fig. 3). In cortical bone of the tibiae and ulnae, the additional 2-weeks of iPTH (1–34) induced further increases in periosteally-enclosed volume and cortical bone volume at proximal, middle and distal sites (Tables 4 and 5), and the “average” polar mean moment of inertia at these sites was markedly increased in a dose-related manner (Table 2). Effects of mechanical loading alone In trabecular bone of the proximal tibiae, 2-weeks of mechanical loading sufficient by itself to stimulate an osteogenic response, was associated with a 18.6% increase in percent bone volume in the primary spongiosa (from 21.0% to 24.9%; Table 3, Figs. 3 and 4). In the secondary spongiosa, this level of loading resulted in a 31.9% increase in percent bone volume (from 11.3% to 14.9%), a 13.1% increase in trabecular number (from 2.22 to 2.51 mm− 1) and a 15.8% increase in trabecular thickness (from 0.0513 to 0.0594 mm) (Table 3, Figs. 3 and 4). In cortical bone of the tibiae, the “sufficient” level of loading also produced increases in periosteally-enclosed volume, cortical bone volume and polar mean moment of inertia at the proximal and middle, but not the distal, sites (Table 4, Fig. 5). In cortical bone of the ulnae, as expected, 2-weeks of mechanical loading insufficient by itself to engender an osteogenic response, had no discernible effect on any measure of bone structure (Table 5, Fig. 5). Combined effects of iPTH (1–34) and mechanical loading In the primary spongiosa of the proximal tibiae, mechanical loading sufficient by itself to stimulate an osteogenic response resulted in an increase in trabecular thickness at low and high doses of iPTH (1–34)

Fig. 2. Representative transverse μCT images of the trabecular bone in 17 week old female C57BL/6 mice treated with 4-weeks of intermittent parathyroid hormone (1–34).

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Table 3 Trabecular structural parameters in the proximal tibia of 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (iPTH) (1–34) and 2-weeks of mechanical loading Dose of PTH (1–34) Primary spongiosa Percent bone volume (%) Left control Right loaded Trabecular number (mm− 1) Left control Right loaded Trabecular thickness (mm) Left control Right loaded Secondary spongiosa Percent bone volume (%) Left control Right loaded Trabecular number (mm− 1) Left control Right loaded Trabecular thickness (mm) Left control Right loaded

0 μg/kg/day

20 μg/kg/day

40 μg/kg/day

80 μg/kg/day

iPTH (1–34)

p valuea Loading

Interaction

21.0 ± 1.5 24.9 ± 1.5

43.3 ± 0.8 44.4 ± 0.8

47.5 ± 1.1 46.9 ± 1.5

46.9 ± 0.7 48.8 ± 1.4

b 0.001

0.088

0.347

4.6 ± 0.4 4.9 ± 0.3

10.3 ± 0.2 9.9 ± 0.2

11.5 ± 0.2 10.9 ± 0.2

11.7 ± 0.1 11.5 ± 0.2

b 0.001

0.158

0.221

0.0457 ± 0.0017 0.0507 ± 0.0008

0.0423 ± 0.0010 0.0447 ± 0.0011

0.0414 ± 0.0007 0.0432 ± 0.0012

0.0400 ± 0.0006 0.0424 ± 0.0008

b 0.001

b 0.001

0.426

11.3 ± 0.7 14.9 ± 1.1

18.0 ± 1.0 22.4 ± 1.3

30.0 ± 1.3 31.2 ± 1.3

39.0 ± 0.8 41.8 ± 1.8

b 0.001

0.002

0.539

2.22 ± 0.16 2.51 ± 0.20

3.71 ± 0.24 3.97 ± 0.27

6.18 ± 0.27 5.81 ± 0.32

8.69 ± 0.28 8.50 ± 0.49

b 0.001

0.999

0.567

0.0513 ± 0.0018 0.0594 ± 0.0009

0.0486 ± 0.0009 0.0567 ± 0.0015

0.0486 ± 0.0008 0.0539 ± 0.0008

0.0450 ± 0.0010 0.0494 ± 0.0012

b 0.001

b 0.001

0.281

Mean ± S.E. (n = 5–8). a Two-way ANOVA.

(Table 3, Figs. 3 and 4). This level of loading had no anabolic effects on percent bone volume or trabecular number at any dose of iPTH (1–34). At low dose of iPTH (1–34), it induced increases in percent bone volume, trabecular number and trabecular thickness in the secondary spongiosa; however, except for trabecular thickness, there were no significant effects at medium or high dose of iPTH (1–34) (Table 3, Figs. 3 and 4). At the proximal and middle cortex of the tibiae, the “sufficient” level of loading resulted in increases in periosteally-enclosed volume and cortical bone volume at all doses of iPTH (1–34) (Table 4, Fig. 5). At high dose of iPTH (1–34), this level of loading also induced a

significant decrease in medullary volume at the 37% proximal and middle sites of the tibiae (Table 4, Fig. 5). Polar mean moment of inertia at proximal and middle sites of the tibiae was markedly increased (Table 4, Fig. 5), and the relative “average” polar mean moment of inertia was similar between the left non-loaded and right loaded tibiae at each dose of iPTH (1–34) (Table 2). At high dose of iPTH (1–34), there was a quantitative synergistic response with the “sufficient” level of loading in cortical bone volume at the 37% proximal site of the tibiae (p b 0.01 [interaction by two-way ANOVA]; Fig. 6). Woven bone was observed at both endosteal and periosteal surfaces of the proximal tibial cortices that underwent this level of

Fig. 3. Representative transverse μCT images of the trabecular and cortical bone in 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (1– 34) and 2-weeks of mechanical loading. Level of peak load: sufficient to engender an osteogenic response.

T. Sugiyama et al. / Bone 43 (2008) 238–248

loading combined with all doses of iPTH (1–34), whereas either alone did not induce woven bone formation (Fig. 3). In contrast, the distal cortical bone of the tibiae showed no response to loading at the peak loads used at any dose of iPTH (1–34) (Table 4). In the distal cortical region of the ulnae, when combined with high, but not low or medium, dose of iPTH (1–34), mechanical loading insufficient by itself to engender an osteogenic response produced significant increases in periosteally-enclosed volume and cortical bone volume, a decrease in medullary volume, and an increase in polar mean moment of inertia (Table 5, Fig. 5). In addition, there was a quantitative synergistic relationship in increased cortical bone volume

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between the “insufficient” level of loading and high-dose iPTH (1–34) (p = 0.04 [interaction by two-way ANOVA]; Fig. 6). The relative “average” polar mean moment of inertia was similar between the left non-loaded and right loaded ulnae at each dose of iPTH (1–34) (Table 2). In contrast to the tibiae, no woven bone was observed at the ulnae, and the proximal and middle regions of the ulnae showed no response to loading at the peak loads used at any dose of iPTH (1–34) (Table 5). Confocal microscope images of fluorescently labelled bones are shown in Fig. 7. In trabecular bone of the proximal tibiae, the “sufficient” level of mechanical loading produced an increase in bone

Table 4 Cortical structural parameters in the tibia of 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (iPTH) (1–34) and 2-weeks of mechanical loading Dose of PTH (1–34) Proximal (25%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded Proximal (37%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded Middle (50%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded Distal (25%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded Mean ± S.E. (n = 5–8). a Two-way ANOVA.

0 μg/kg/day

20 μg/kg/day

40 μg/kg/day

80 μg/kg/day

iPTH (1–34)

p valuea Loading

Interaction

2.82 ± 0.04 3.16 ± 0.06

3.26 ± 0.08 3.56 ± 0.04

3.46 ± 0.08 3.86 ± 0.08

3.48 ± 0.12 3.88 ± 0.06

b 0.001

b0.001

0.889

1.56 ± 0.02 1.82 ± 0.02

1.82 ± 0.04 2.26 ± 0.04

1.98 ± 0.04 2.44 ± 0.04

2.02 ± 0.08 2.48 ± 0.06

b 0.001

b0.001

0.145

1.28 ± 0.03 1.33 ± 0.04

1.43 ± 0.05 1.31 ± 0.04

1.48 ± 0.04 1.41 ± 0.05

1.45 ± 0.06 1.40 ± 0.04

0.013

0.151

0.291

0.358 ± 0.011 0.442 ± 0.013

0.465 ± 0.018 0.571 ± 0.016

0.542 ± 0.021 0.670 ± 0.021

0.559 ± 0.026 0.677 ± 0.020

b 0.001

b0.001

0.661

2.44 ± 0.04 2.78 ± 0.06

2.80 ± 0.06 3.12 ± 0.04

2.96 ± 0.04 3.38 ± 0.06

2.96 ± 0.06 3.44 ± 0.06

b 0.001

b0.001

0.376

1.44 ± 0.04 1.72 ± 0.04

1.64 ± 0.04 2.02 ± 0.02

1.78 ± 0.04 2.20 ± 0.04

1.74 ± 0.06 2.32 ± 0.06

b 0.001

b0.001

0.018

1.02 ± 0.02 1.06 ± 0.03

1.17 ± 0.03 1.10 ± 0.04

1.18 ± 0.04 1.20 ± 0.04

1.22 ± 0.03 1.13 ± 0.03

b 0.001

0.300

0.142

0.295 ± 0.012 0.378 ± 0.011

0.379 ± 0.018 0.463 ± 0.012

0.420 ± 0.015 0.539 ± 0.017

0.413 ± 0.018 0.551 ± 0.018

b 0.001

b0.001

0.262

2.00 ± 0.02 2.18 ± 0.02

2.20 ± 0.06 2.32 ± 0.04

2.34 ± 0.04 2.52 ± 0.04

2.34 ± 0.04 2.50 ± 0.04

b 0.001

b0.001

0.844

1.20 ± 0.02 1.39 ± 0.01

1.32 ± 0.03 1.54 ± 0.02

1.40 ± 0.02 1.65 ± 0.02

1.50 ± 0.07 1.75 ± 0.03

b 0.001

b0.001

0.721

0.796 ± 0.016 0.792 ± 0.018

0.872 ± 0.034 0.790 ± 0.020

0.934 ± 0.020 0.858 ± 0.020

0.844 ± 0.034 0.750 ± 0.034

b 0.001

0.001

0.270

0.140 ± 0.004 0.174 ± 0.003

0.170 ± 0.008 0.200 ± 0.006

0.191 ± 0.006 0.234 ± 0.006

0.199 ± 0.009 0.238 ± 0.008

b 0.001

b0.001

0.759

1.49 ± 0.04 1.51 ± 0.03

1.64 ± 0.04 1.61 ± 0.03

1.73 ± 0.03 1.75 ± 0.03

1.70 ± 0.02 1.71 ± 0.02

b 0.001

0.889

0.863

1.07 ± 0.03 1.09 ± 0.02

1.22 ± 0.04 1.23 ± 0.03

1.40 ± 0.05 1.36 ± 0.03

1.51 ± 0.04 1.54 ± 0.04

b 0.001

0.947

0.775

0.420 ± 0.020 0.422 ± 0.020

0.414 ± 0.022 0.380 ± 0.022

0.334 ± 0.034 0.394 ± 0.030

0.198 ± 0.038 0.174 ± 0.028

b 0.001

0.946

0.309

0.083 ± 0.004 0.084 ± 0.004

0.101 ± 0.004 0.098 ± 0.004

0.116 ± 0.004 0.117 ± 0.004

0.114 ± 0.003 0.115 ± 0.003

b 0.001

0.898

0.925

244

T. Sugiyama et al. / Bone 43 (2008) 238–248

Table 5 Cortical structural parameters in the ulna of 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (iPTH) (1–34) and 2-weeks of mechanical loading Dose of PTH (1–34) Proximal (25%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded Middle (50%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded Distal (25%) Periosteally-enclosed volume (mm3) Left control Right loaded Cortical bone volume (mm3) Left control Right loaded Medullary volume (mm3) Left control Right loaded Polar mean moment of inertia (mm4) Left control Right loaded

0 μg/kg/day

20 μg/kg/day

40 μg/kg/day

80 μg/kg/day

iPTH (1–34)

p valuea Loading

Interaction

0.88 ± 0.01 0.86 ± 0.02

0.95 ± 0.02 0.93 ± 0.02

1.02 ± 0.01 0.98 ± 0.02

1.00 ± 0.02 0.98 ± 0.02

b 0.001

0.083

0.917

0.804 ± 0.018 0.794 ± 0.020

0.884 ± 0.020 0.864 ± 0.024

0.944 ± 0.020 0.918 ± 0.018

0.928 ± 0.018 0.920 ± 0.016

b 0.001

0.244

0.971

0.0764 ± 0.0060 0.0670 ± 0.0046

0.0674 ± 0.0066 0.0712 ± 0.0016

0.0722 ± 0.0104 0.0592 ± 0.0050

0.0710 ± 0.0060 0.0630 ± 0.0050

0.803

0.155

0.614

0.0481 ± 0.0013 0.0464 ± 0.0020

0.0529 ± 0.0018 0.0508 ± 0.0023

0.0613 ± 0.0017 0.0576 ± 0.0019

0.0590 ± 0.0022 0.0578 ± 0.0018

b 0.001

0.122

0.905

0.578 ± 0.008 0.608 ± 0.022

0.676 ± 0.020 0.656 ± 0.022

0.716 ± 0.016 0.706 ± 0.016

0.716 ± 0.016 0.716 ± 0.016

b 0.001

0.974

0.534

0.492 ± 0.008 0.492 ± 0.004

0.560 ± 0.010 0.552 ± 0.010

0.598 ± 0.012 0.584 ± 0.010

0.600 ± 0.014 0.610 ± 0.010

b 0.001

0.691

0.696

0.085 ± 0.009 0.115 ± 0.019

0.115 ± 0.013 0.103 ± 0.012

0.118 ± 0.009 0.122 ± 0.008

0.115 ± 0.008 0.106 ± 0.012

0.387

0.680

0.276

0.0174 ± 0.0002 0.0175 ± 0.0007

0.0221 ± 0.0010 0.0205 ± 0.0012

0.0252 ± 0.0010 0.0232 ± 0.0008

0.0254 ± 0.0010 0.0247 ± 0.0010

b 0.001

0.125

0.673

0.412 ± 0.012 0.414 ± 0.008

0.470 ± 0.012 0.464 ± 0.012

0.496 ± 0.012 0.502 ± 0.008

0.492 ± 0.012 0.524 ± 0.006

b 0.001

0.238

0.266

0.370 ± 0.010 0.374 ± 0.006

0.422 ± 0.010 0.432 ± 0.010

0.446 ± 0.008 0.464 ± 0.006

0.450 ± 0.010 0.490 ± 0.006

b 0.001

0.004

0.133

0.0412 ± 0.0030 0.0404 ± 0.0036

0.0472 ± 0.0024 0.0320 ± 0.0034

0.0504 ± 0.0040 0.0384 ± 0.0032

0.0428 ± 0.0032 0.0340 ± 0.0018

0.243

b 0.001

0.178

0.0068 ± 0.0004 0.0068 ± 0.0002

0.0088 ± 0.0004 0.0086 ± 0.0004

0.0098 ± 0.0004 0.0101 ± 0.0003

0.0097 ± 0.0004 0.0110 ± 0.0002

b 0.001

0.197

0.217

Mean ± S.E. (n = 6–8). a Two-way ANOVA.

formation in the vehicle-treated mice; in contrast, where high-dose iPTH (1–34) was associated with markedly increased bone formation, this level of loading did not result in a further apparent increase in bone formation. In cortical bone of the 37% proximal tibiae, the “sufficient” level of loading induced increases in both endosteal and periosteal bone formation in the vehicle-treated mice, and these effects were markedly promoted in the high-dose iPTH (1–34)-treated mice. In cortical bone of the distal ulnae, confocal microscopic images of fluorescently labelled bones confirmed that the synergism between high-dose iPTH (1–34) and the “insufficient” level of mechanical loading involved both endosteal and periosteal bone formation (Fig. 7). Discussion In the present study, vehicle or PTH (1–34) at low (20 μg/kg/day), medium (40 μg/kg/day) or high (80 μg/kg/day) dose was intermittently administered to female C57BL/6 mice, and the tibiae and ulnae were subjected to levels of dynamic axial loading “sufficient” and “insufficient” to by themselves engender an osteogenic response. In agreement with previous in vivo studies using rats [34,35], not only “sufficient” but also “insufficient” levels of loading reacted synergistically with high-dose iPTH (1–34) to stimulate osteogenesis in cortical regions of the tibiae and ulnae. In contrast to previous reports

in rats [32,36], however, we found no such synergism of response in trabecular bone. This may be because any synergistic effects of loading were obscured by the dramatic dose-related effects that iPTH (1–34) alone had on trabecular bone. These effects of iPTH (1–34) could not be separated from those of growth, although at 13 weeks of age the growth had passed its peak [43,44]. The “Introductory iPTH” experiment was designed to establish the minimum-effective dose of iPTH (1–34) and the dose-related effects of 4-weeks of pre-treatment with iPTH (1–34) on the trabecular and cortical bone structure. The former is important in order to relate the results in animals to the clinical situation in humans. The latter was necessary to correct the magnitudes of applied mechanical load for the “iPTH/loading” experiment. As expected, 4-weeks of iPTH (1–34) resulted in a dose-dependent increase in percent bone volume in trabecular bone of the proximal tibiae. This regimen also induced an increase in cortical bone volume at the proximal, middle and distal sites in both the tibiae and ulnae. Since low-dose iPTH (1–34) failed to produce a significant increase in cortical bone volume at some sites analyzed, a dose of 20 μg/kg/day would be considered as the minimum-effective osteogenic dose for both the trabecular and cortical bones in the present protocol. In both the tibiae and ulnae, 4-weeks of pre-treatment with iPTH (1–34) resulted in a dose-related increase in the “average” polar

T. Sugiyama et al. / Bone 43 (2008) 238–248

Fig. 4. Two-week mechanical load-induced changes ([right loaded − left control] / left control) of the trabecular structural parameters in 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (iPTH) (1–34). Level of peak load: sufficient to engender an osteogenic response. Mean ± S.E. (n = 5–8). ⁎p b 0.05 by paired t-test between left control and right loaded. #p b 0.05 by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test among four doses of PTH (1–34).

mean moment of inertia, a parameter of resistance to bending, at the proximal, middle and distal sites. These data as well as those for bone length were used to establish the necessary magnitudes of mechanical load to produce the required bone strains. Since the additional 2-weeks of treatment with iPTH (1–34) stimulated a further increase in the “average” polar mean moment of inertia in a dose-related manner, the corrected levels of mechanical load would result in relatively lower peak strain in the bones of the iPTH (1–34)treated mice than in those treated with vehicle during the latter duration of loading. Thus, the effects of mechanical loading are likely to be under rather than overestimated. Nevertheless, the similar changes in the relative “average” polar mean moment of inertia between the left non-loaded and right loaded tibiae and ulnae at each dose of iPTH (1–34), as shown in Table 2, provides a validation of the corrected levels of applied mechanical load in the present study. In the “iPTH/loading” experiment, iPTH (1–34) and mechanical loading at a level sufficient by itself to stimulate an osteogenic response were both associated with an increase in trabecular percent bone volume in the proximal tibia. However, these effects

245

were produced in a different way; iPTH (1–34) increased trabecular number but had no anabolic effect on trabecular thickness, whereas the “sufficient” level of loading increased both trabecular number and thickness. This level of loading resulted in an additive increase in percent bone volume of the secondary spongiosa in mice also treated with low-dose iPTH (1–34), but we did not detect the synergistic effects between iPTH (1–34) and loading reported in previous in vivo studies in rats using axial compression of the tail vertebrae [32,36]. However, since these rats were not treated with iPTH before loading [32,36], the anabolic actions of mechanical loading are likely to be restricted owing to the marked modification of trabecular structure induced by the 4-weeks of pre-treatment with iPTH (1–34). In cortical regions of the tibiae, iPTH (1–34) and the “sufficient” level of mechanical loading resulted in additive or synergistic increases in periosteally-enclosed volume and cortical bone volume at the proximal and middle sites; iPTH (1–34) induced an osteogenic response at all sites, whereas this level of loading had no effect at the distal site. At the 37% proximal site of the tibiae where there was a synergistic effect in cortical bone volume between high-dose iPTH (1– 34) and the “sufficient” level of loading, woven bone was produced at both endosteal and periosteal surfaces in response to this level of loading when combined with iPTH (1–34) at all doses but in no situation in response to either stimulus alone. Since there is a strain threshold above which woven bone formation occurs [45], these results suggest that iPTH (1–34) sensitizes bone cells to mechanical stimulation and thus lowers the bone-modelling threshold of mechanical strain. This concept is consistent with the confocal microscope images and previous findings in rats [34,35], both of which showed that iPTH (1–34) and mechanical loading synergistically increased endosteal and periosteal bone formation. As expected, in cortical regions of the ulna, mechanical loading at a level insufficient by itself to stimulate an osteogenic response did not change cortical bone structure in the vehicle-treated mice. In contrast, in mice treated with high, but not low or medium, dose of iPTH (1–34), this level of loading induced increases in the periosteally-enclosed volume and cortical bone volume and a decrease in the medullary volume at the distal site. Together, these changes resulted in an increase in the polar mean moment of inertia. Consistent with the data from the proximal tibia, the synergistic relationship between high-dose iPTH (1–34) and the “insufficient” level of loading for cortical bone volume in the distal ulna also supports the concept that iPTH (1–34) lowers the bone-modelling threshold of mechanical strain. In the present study, the synergistic effects of iPTH (1–34) and different levels of mechanical loading were observed at the 37% proximal tibia and distal ulna in mice treated with iPTH (1–34) at a dose of 80 μg/kg/day, but not 20 or 40 μg/kg/day, suggesting that iPTH (1–34) sensitizes bone cells to mechanical stimulation only at higher dose. This possibility is supported by a previous report in rats that treatment with iPTH (1–38) at a dose of 80 μg/kg/day, but not 30 μg/ kg/day, induced a higher periosteal bone formation rate, which resulted in larger cross-sectional total and bone areas, in remobilized limbs than in immobilized limbs [33]. On the other hand, the distal tibiae as well as the proximal and middle ulnae showed no response to mechanical loading at levels applied in the present study. The shape of the skeleton is controlled by its mechanical environment, and the differences among bone sites in response to any one loading configuration are likely to be determined by how different this loading environment is from that to which the site in question is habituated. In conclusion, the present study in female C57BL/6 mice shows that iPTH (1–34) and mechanical loading independently stimulate osteogenesis in both trabecular and cortical regions of long bones. The dose-dependent effects of iPTH (1–34) on trabecular bone are particularly marked, and the pre-treatment with iPTH (1–34) at

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T. Sugiyama et al. / Bone 43 (2008) 238–248

Fig. 5. Two-week mechanical load-induced changes ([right loaded − left control] / left control) of the cortical structural parameters in 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (iPTH) (1–34). Levels of peak load: sufficient to engender an osteogenic response in the tibia and insufficient to do so in the ulna. Mean ± S.E. (n = 5–8). ⁎p b 0.05 by paired t-test between left control and right loaded. #p b 0.05 by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test among four doses of PTH (1–34).

Fig. 6. Relative effect of 6-weeks of high-dose intermittent parathyroid hormone (iPTH) (1–34) and 2-weeks of mechanical loading alone or in combination on cortical bone volume at the proximal (37%) tibia and distal ulna in 19 week old female C57BL/6 mice. Levels of peak load: sufficient to engender an osteogenic response in the tibia and insufficient to do so in the ulna. Mean ± S.E. (n = 5–8). Interaction between high-dose iPTH (1–34) and mechanical loading by two-way ANOVA.

T. Sugiyama et al. / Bone 43 (2008) 238–248

247

Fig. 7. Representative transverse confocal microscope images of the trabecular and cortical bone in 19 week old female C57BL/6 mice treated with 6-weeks of intermittent parathyroid hormone (iPTH) (1–34) and 2-weeks of mechanical loading. Levels of peak load: sufficient to engender an osteogenic response in the tibia and insufficient to do so in the ulna. Green: double calcein labels injected on the first days of iPTH (1–34) treatment (day 1) and mechanical loading (day 29). Red: single alizarin label injected on the last day of mechanical loading (day 41).

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