Calcified Tissue International

, Volume 85, Issue 2, pp 146–157 | Cite as

Does Exercise Modify the Effects of Zoledronic Acid on Bone Mass, Microarchitecture, Biomechanics, and Turnover in Ovariectomized Rats?

  • E. Lespessailles
  • C. Jaffré
  • H. Beaupied
  • P. Nanyan
  • E. Dolléans
  • C. L. Benhamou
  • D. Courteix
Article

Abstract

Regular activity has effects on bone size, shape, and density, resulting in an increase in mechanical strength. The mechanism of action that underlies this improvement in bone strength is mainly linked to an increase in bone formation. Zoledronic acid (Z), in contrast, may prevent bone strength changes in ovariectomized (OVX) rodents by its potent antiresorptive effects. Based on these assumptions we hypothesized that combined effects of exercise (E) and Z may produce higher benefits on bone changes resulting from estrogen deficiency than either intervention alone. At 6 months of age, 60 female Wistar rats were OVX or sham operated (SH) and divided into five groups: SH, OVX, OVX-E, OVX-Z, and OVX-ZE. OVX rats were treated with a single IV injection of Z (20 μg/kg) or vehicle and submitted or not to treadmill exercise (15 m/min, 60 min/day, 5 days/week) for 12 weeks. Whole-body BMD and bone turnover markers were analyzed longitudinally. At sacrifice, femurs were removed. BMD by DXA, three-point bending test, and μCT were performed to study biomechanical and trabecular structure parameters, respectively. After 12 weeks, bone volume fraction decreased in OVX rats, whereas bone turnover rate, trabecular spacing, and structure model index increased compared with those in the SH group (P < 0.05). Zoledronic acid prevented the ovariectomy-induced trabecular bone loss and its subsequent trabecular microarchitectural deterioration. Treadmill exercise running was shown to preserve the bone strength and to induce bone turnover changes in favor of bone formation. However, the combined effects of zoledronic acid and running exercise applied simultaneously did not produce any synergetic or additive effects.

Keywords

Zoledronic acid Treadmill exercise Ovariectomized rats Trabecular microarchitecture Bone strength 

With the increasing life expectancy of the population, osteoporosis and osteoporotic fractures have been more and more diagnosed worldwide [1]. Current strategies for the prevention and treatment of osteoporosis have benefited from the development of new bisphosphonates [2]. Recently, it has been shown that zoledronic acid infusions given at a 1-year interval for 3 years had a proven efficacy against fractures in postmenopausal women [3]. Bisphosphonates are potent inhibitors of bone resorption. They act mainly on osteoclasts by inhibiting the bone resorptive capacity of mature osteoclasts. They also may induce osteoclast apoptosis [4]. In addition to the accelerated rate of bone loss as a result of menopause, it has been reported that the estrogen deficiency may also be involved in the decrease of lean mass, leading to sarcopenia [5]. As regular physical activity may prevent the decline in bone and muscle losses associated with menopause [6, 7], many public health officials encourage or recommend that individuals must engage in daily exercise [8, 9]. The specific physiological mechanisms that underlie the effect of physical activity on bone are not completely understood. However, the triad of osteoblast, osteocyte, and bone lining cells may have a key role in the mechanisms implicated in the response to osteogenic exercise stimuli [10]. Therefore, a combination of antiresorptive therapy like zoledronic acid (whose target cell is the osteoclast) and a bone anabolic intervention induced by mechanical loading (whose target cells are resident osteocytes, osteoblasts, and bone lining cells) may be a powerful treatment option for osteoporosis. This strategy of combining the osteogenic effects of exercise and the antiresorptive activity of bisphosphonates or estrogen has already been suggested [11]. In animal studies, data to support a beneficial effect of the combination between antiresorptive drugs and anabolic therapy are conflicting [12, 13, 14, 15]. It has been found in ewes that the bone anabolic action of parathyroid hormone was blocked by tiludronate [12]. In rats there was also an interaction between alendronate and PTH analogue, as the anabolic response of the latter was different in animals pretreated or not by alendronate [13]. However, the two studies to our knowledge aiming to combine the potential osteogenic effect of exercise with the antiresorptive effect of bisphosphonates have found that this combination may offer advantages over treatment with either intervention alone [14, 15].

Based on these assumptions, the aim of this study was to assess the efficacy of a single injection of zoledronic acid and of treadmill running exercise, alone or in combination, for the prevention of ovariectomy-induced bone loss in rats.

Materials and Methods

Rat Groups and Treatment

Sixty 6-month-old Wistar female rats (Animal Production Center, Olivet, France) were maintained and acclimatized for 2 weeks on a 12-h light/12-h dark cycle at 22 ± 2°C during the experiment. Rats were housed in standard cages (two animals by cage) with ad libitum access to food (a commercial standard diet [A04, SAFE, France], phosphorus = 5.9 mg/g, calcium = 8.3 mg/g, and vitamin D3 = 1 IU/g) and water. Rats were randomized to either sham-operated (sham; n = 12) or bilateral OVX (n = 48). We did not ration OVX rats in order to mimic real life conditions in postmenopausal women, knowing that this is usually recommended, as obesity induced by OVX may protect against osteopenia [16]. Among the rats randomly selected for the OVX group, a second randomization procedure was done, leading to four groups of 12 animals each in addition to the sham-operated group (SH): OVX sedentary controls (OVX), OVX-exercise (OVX-E), OVX-zoledronic acid (OVX-Z), and OVX- zoledronic acid exercise (OVX-ZE). Based on a previous dose-ranging study [17], we selected a single i.v. dose of 20 μg/kg zoledronic acid, as it has been demonstrated to be the minimum dose required to provide long-term bone protection against the effects of OVX [17]. We injected the drug or a physiological saline solution 2 days before the OVX surgery. Sham operations were performed by exteriorizing the ovaries. Bilateral ovariectomies were performed under ketamine-xylasine anesthesia (80 + 10 mg/kg). This experiment was conducted in conformity with the Public Health Service Policy on Human Care and Use of Laboratory Animals. The procedure for the care and killing of animals was in accordance with the European Community Standards on the care and use of laboratory animals (Ministère de l’Agriculture, France; Authorization Inserm 45–001).

Exercise Regimen

The exercise regimen consisted in running in a custom-made, motor-driven treadmill, which allowed eight rats to exercise together. The running program was initiated gradually. The exercise regimen consisted in daily training. During the first 4 weeks, the speed of the treadmill and the duration of each running episode were gradually increased from 8 m/min for 3–30 min to 12 m/min for the first 2 weeks. In the following 2 weeks, the speed and duration of the running session were gradually increased to 15 m/min for 60 min. This speed and duration were maintained for 5 days per week for the rest of the experiment (8 more weeks).

Bone Turnover Biomarkers

At baseline and at the end of the study blood was obtained by veinous puncture at the tail. Blood samples were allowed to clot at 4°C for 20 min before centrifugation at 3,000 rpm for 10 min. Sera were stored at −20°C during 24 h and then archived at −80°C until analysis.

Osteocalcin (OC) and C-terminal collagen cross-links (CTX), which are, respectively, biomarkers of bone formation and resorption, were assayed in duplicate by ELISA (Nordic Bioscience Diagnostics, Herlev Hoved-Gade, Denmark). The within-assay and between-assay CVs were <10% in our laboratory. An uncoupling index (UI) was calculated to assess the relative balance of the formation and resorption processes of bone remodeling, as previously described [18]. First, the mean ± SE of the baseline CTX and OC values were determined in each rat. Using the values of the SHAM group, the OC and CTX Z-score [(rat value—mean baseline)/SD baseline] were calculated by subtracting the Z-score of the resorption marker from the Z-score of the formation marker. A positive UI indicates bone remodeling unbalanced in favor of bone formation, whereas a negative UI reflects an imbalance favoring bone resorption.

Body Weight, Fat Mass, Lean Mass, and Bone Mineral Content (BMC) Measurements

All analyses were conducted using the Discovery A densitometer (Hologic, Inc., Bedford, MA) calibrated daily in accordance with the manufacturer’s recommendations. The rat whole body (WB) module was used to provide WB fat mass and WB lean mass. To perform in vivo DXA analysis of rat femurs, the anesthetized rat was placed on a thin plastic sheet where the outline of a typical rat had been drawn to ensure that adequate positioning precision was maintained. Rats were placed in the supine position and appendages were positioned flat to the table using adhesive plaster.

The regional high-resolution mode of the small animal scan protocol of the Discovery A densitometer was used to assess bone density measurement of the excised rat femurs. As a thin plastic sheet was placed beneath the rat for an exam, this was also placed beneath the small animal step phantom during the quality control procedure. In addition, as we studied small animals (small regions of interest), we performed and compared step phantom analyses between the rat group measurements in order to detect any potential drift of the instrumentation. Thereafter, the phantom was analyzed daily before animal testing for quality control purposes. For scanning, the tail of the rat was curled alongside the animal in order to image it entirely. The default scan width for a rat WB study is 17.8 cm. For in vivo and ex vivo analysis of the rat femur, a regional high-resolution study was used. The excised femur was placed in the center of a thin-walled plastic container with a smooth uniform bottom. The container, as recommended by the manufacturer, was sufficiently wide (8.3 cm) to allow 2.5 cm of water on both sides of the excised femurs. The container was filled with 3 cm of water. For all measurements, a constant depth of the water in the container was controlled carefully using a water fill line on the container.

At baseline and at 12 weeks (4 weeks of gradually increased exercise and 8 weeks of full regimen, i.e., see the exercise regimen described above), lean and fat masses were measured by DXA. The root-mean square CV of in vivo WBBMC, WBBMD, and WB fat mass were 1.2%, 0.87%, and 3.8%, respectively. These CVs were determined from two repeated measures with repositioning in 30 animals.

Excised rat femurs were measured at the end of the study. The left femur was bathed in saline solution during DXA measurement. In addition to the total femur measurement, two subregions were determined. The first subregion (R1) corresponded to the femoral distal metaphysis and excluded the growth plate area. The second subregion was located more distally and corresponded to a diaphyseal zone (R2) (Fig. 1) [19]. The R1 region is considered to contain more trabecular bone than the R2 one, which is richest in cortical bone [19]. The root-mean square CVs in vitro of BMC and BMD of the total excised femur and of the two generated subregions were 1.15% and 0.39% for the total excised femur, 5.73% and 2.64% for the R1 subregion, and 4.09% and 1.55% for the R2 subregion. These CVs were determined from four repeated measures on nine animals.
Fig. 1

The two subregions in the femur. The first subregion (R1) corresponds to the femoral distal metaphysis and excludes the growth plate area. The second subregion is located more distally and corresponds to a diaphyseal zone (R2)

Micro-CT (μCT) of Trabecular Bone Microarchitecture

The microarchitecture of the distal femur was studied using μCT (Skyscan 1072; Skyscan, Aartselaar, Belgium). The X-ray source was set at 75 kV and 100 μA, with a pixel size of 11 μm. Four hundred projections were acquired over an angular range of 180° (angular step of 0.45°). Image slices were reconstructed using the cone-beam reconstruction software version 2.6 based on the Feldkamp algorithm. The registered data sets were segmented into binary images. Because of the low noise and relatively good resolution of the data sets, simple global thresholding methods were used [20]. On the femur, 250 slices were selected from the distal growth plate to the shaft proximally. The trabecular bone was extracted by drawing ellipsoid contours with CT analyzer software (Skyscan). The following parameters were measured for each sample in the experimental groups.
  • The bone volume/tissue volume, BV/TV (%), corresponding to the bone proportion

  • The ratio of bone surface to bone volume, BS/BV (mm2/mm3), measured based on a faceted surface of the marching cubes volume model [21]

  • The trabecular thickness, Tb·Th (μm), and the trabecular spacing, Tb·Sp (μm), measured from three-dimensional (3D) μCT images by the sphere method developed by Hildebrand and Ruegsegger [22]

In addition to the morphological parameters, the following parameters were calculated for each sample.
  • The trabecular number (Tb·N; mm−1) was calculated from the following equation considering the parallel plate model but using a direct 3D measurement of thickness [23]:

    $$ {\text{Tb}}.{\text{N}} = {\frac{\left( {{\text{BV}}/{\text{TV}}} \right)}{{\text{Tb}}.{\text{Th}}}} $$
  • The degree of anisotropy (DA; no units) was calculated using the mean intercept length (MIL) and eigen vector analysis. An ellipsoid was fitted to the 3D distribution of MILs measured from 128 random 3D directions of a spherical volume of interest. Values of DA vary from 1 (fully isotropic) to infinity (fully anisotropic).

  • The structure model index (SMI; no units) indicated the relative proportion of rods and plates in the sample. SMI was calculated by a differential analysis of the triangulated surface of trabecular bone [24].

The trabecular bone pattern factor (TBPf) was calculated. This parameter is used to indicate the connectivity of trabecular bone [25]. The TBPf is an indicator of the concavoconvex structure of the surface of the trabecular bone. A higher TBPf value implies a poorly connected state of trabeculae.

Biomechanical Testing

Mechanical properties of the left femur diaphysis were assessed by three-point bending tests. Four hours before mechanical testing, the bones were thawed at room temperature. Each bone was secured on the two lower supports on the anvil of a Universal Testing Machine (Instron 4501; Instron, Canton, MA). The diameter of these supports was 4 mm and the distance between the two supports was 20 mm.The cross-head speed for all tests was 1 mm/min. Load-displacement curves were recorded using specialized software (Instron 4501). Biomechanical properties were calculated from these curves: ultimate force (the maximum force supported by the bone before fracture, Fult [N]); energy to ultimate force (energy required to fracture the bone, U [N m]); and stiffness (extrinsic rigidity of the femur, S [N/m]). Because of the irregular shape of the femoral diaphysis, the femoral diameter used in the calculation was the mean of the mediolateral and the anteroposterior femoral middiaphysis diameters. Ultimate stress (σu [MPa]) and Young’s modulus (E [MPa]; modulus of elasticity) were determined by the equations previously described by Turner and Burr [26]. To ensure good reproducibility between measurements, the femur was always mounted so that the cross-head could be applied just in the middle of the bone [26].

Statistical Analysis

Data are presented as mean ± standard deviation (SD) for body weight and body mass as mean ± standard error of the mean (SE) for serum chemistry. Normal distributions (Gaussian) of the data were assessed by Kolmogorov Smirnov test. The effect of ovariectomy was determined by comparing the sham-OVX and OVX groups using Student’s t-test. To compare the main and combined effects of exercise and zoledronate between the OVX groups for longitudinal data, a two-way ANOVA with repeated measures was performed. In the event of a significant interaction, exercise and zoledronate effects were considered synergistic. A one-way ANOVA with post hoc, pairwise comparisons using Fisher’s protected least significant difference [27] was used in case of significant interactions. In the event of a nonsignificant interaction, the main effect for each intervention (zoledronate, exercise) was explored by Newman–Keuls test and intervention effects were considered additive. The level of significance was set at P ≤ 0.05. All statistical analyses were performed with PCSM software (OPTIMA-Deltasoft, France).

Results

Anthropometric and Densitometry Parameters

Body Weight

The data presented in Table 1 show that the initial body weights in all groups were not statistically different. However, body weight in the OVX group was significantly higher than in sham-operated rats 12 weeks postsurgery: 386 ± 33 vs. 333 ± 22 g (P = 0.002). There was a significant difference in body weight between the OVX-Z group and the two exercised groups (Table 1).
Table 1

Changes in whole-body anthropometric and bone DXA parameters in sham-operated (SH) or ovariectomized (OVX) mature rats treated or not treated with zoledronic acid (Z; a single injection, 20 μg/kg) and practicing or not practicing treadmill running exercise (E) for 12 weeks: mean ± SD

Parameter

Group

SH

OVX

OVX-Z

OVX-E

OVX-ZE

Pre

Post

Pre

Post

Pre

Post

Pre

Post

Pre

Post

BW (g)

300.3 ± 27.3

333.1 ± 21.7

310.5 ± 20.7

386.4 ± 32.6a

320.8 ± 23.9

416.5 ± 40.7

319.9 ± 25.7

370.1 ± 35.5b

327.9 ± 30.2

370.8 ± 34.4b

LBM (g)

242.0 ± 13.5

241.1 ± 13.7

245.0 ± 16.6

259.2 ± 10.7a

255.1 ± 16.9

260.7 ± 17.2

245.8 ± 16.9

260.6 ± 21.4

248.7 ± 21.1

270.7 ± 25.0

% FM

17.8 ± 3.5

19.0 ± 4.1

17.3 ± 3.6

27.8 ± 7.3a,b

16.6 ± 3.5

28.9 ± 3.8

19.5 ± 4.4

24.0 ± 7.4b

20.3 ± 5.2

23.0 ± 6.0b

WBBMC (g)

11.4 ± 0.7

12.4 ± 0.8

11.7 ± 0.8

12.9 ± 0.7b

12.1 ± 0.5

13.9 ± 0.9

11.8 ± 0.8

12.2 ± 1.0b

11.9 ± 0.7

13.0 ± 0.8b

WBBMD (g/cm²)

0.700 ± 0.007

0.172 ± 0.008

0.171 ± 0.007

0.172 ± 0.003

0.172 ± 0.004

0.180 ± 0.007

0.173 ± 0.006

0.171 ± 0.005

0.171 ± 0.007

0.176 ± 0.008

BW body weight, LBM lean body mass, FM fat mass, WBBMC whole-body bone mineral content, WBBMC whole-body bone mineral density

aOVX differences versus SH

bDifferences versus OVX-Z

Lean and Fat Mass

Percentage fat mass was significantly higher in the OVX-Z group compared to the other OVX groups (Table 1). There was a higher percentage fat mass in the OVX group (27.7%) compared to the SH group (19%; P < 0.005). There were no significant differences observed between the groups for WB lean mass.

Whole-Body and Excised Femur BMC

We found a significant difference in WB BMC between the OVX-Z group and the OVX, OVX-E, and OVX-ZE groups (Table 1). Whereas we did not find any significant differences in BMD or BMC for total excised femurs, we observed statistical differences at the two subregions as indicated in Table 2. There was no significant effect of exercise but there was one of zoledronic acid, with a higher BMD in the R1 region in the OVX-ZE and OVX-Z groups than in the OVX-E and OVX groups. In the R2 subregion there was also a significant effect of zoledronic acid on BMC, with higher results in the groups treated with zoledronic acid than in the OVX or OVX-E groups.
Table 2

Changes in bone densitometric parameters at the femur and in subregions R1 and R2 in sham-operated (SH) or ovariectomized (OVX) mature rats treated or not treated with zoledronic acid (Z; a single injection, 20 μg/kg) and practicing or not practicing treadmill running exercise (E) for 12 weeks: mean ± SD

Parameter

Group

SH

OVX

OVX-Z

OVX-E

OVX-ZE

Femur

    BMC (g)

0.499 ± 0.046

0.469 ± 0.029

0.514 ± 0.055

0.455 ± 0.037

0.521 ± 0.036

    BMD (g/cm²)

0.258 ± 0.12

0.242 ± 0.011

0.269 ± 0.016

0.240 ± 0.011

0.267 ± 0.014

R1

    BMC (g)

0.061 ± 0.006

0.059 ± 0.010

0.066 ± 0.011

0.055 ± 0.007c

0.068 ± 0.006b,d

    BMD (g/cm²)

0.226 ± 0.013

0.203 ± 0.014a

0.236 ± 0.026b

0.198 ± 0.018a,c

0.234 ± 0.016b,c,d

R2

    BMC (g)

0.85 ± 0.005

0.079 ± 0.010

0.091 ± 0.007b

0.083 ± 0.007a,c

0.093 ± 0.005b,d

    BMD (g/cm²)

0.207 ± 0.014

0.199 ± 0.008

0.220 ± 0.008a,b

0.204 ± 0.010c

0.219 ± 0.013a,b,d

BMC bone mineral content, BMD bone mineral density, R1 submetaphyseal area (richest in trabecular bone), R2 diaphyseal area (richest in cortical bone)

aOVX differences versus SH

bDifferences versus OVX

cDifferences versus OVX-Z

dDifferences versus OVX-E

Micro-CT Trabecular Bone Microarchitecture

Effects of Ovariectomy

At the end of the study (12 weeks after ovarian removal), we observed significant differences in bone volume fraction (−37.50%), trabecular spacing (+31.4%), and structure model index (+51.6%) in the OVX group compared to the SH group. The femoral cancellous bone osteopenia induced by the ovariectomy was also characterized by changes in connectivity indexes, with significantly higher TBPf and BS/TV values in the OVX group than in the SH group as reported in Table 3.
Table 3

Changes in microarchitecture parameters in sham-operated (SH) or ovariectomized (OVX) mature rats treated or not treated with zoledronic acid (Z; a single injection, 20 μg/kg) and practicing or not practicing treadmill running exercise (E) for 12 weeks: mean ± SD

Parameter

Group

SH

OVX

OVX-Z

OVX-E

OVX-ZE

BV/TV (%)

26.05 ± 5.64

16.31 ± 6.76a

24.94 ± 8.12b

14.4 ± 5.68c

25.47 ± 6.76b,d

BS/BV (mm2/mm3)

0.031 ± 0.002

0.037 ± 0.005a

0.032 ± 0.003

0.039 ± 0.004c

0.034 ± 0.004b,d

Tb·Th (μm)

110.74 ± 5.37

97.38 ± 9.83a

107.30 ± 8.33b

96.04 ± 6.20c

104.69 ± 8.23b,d

Tb·N (1/mm)

0.002 ± 0.0004

0.002 ± 0.001a

0.002 ± 0.001b

0.001 ± 0.001c

0.002 ± 0.001b,d

Tb·Sp (μm)

519.83 ± 224.78

683.56 ± 288.32

539.76 ± 215.30b

704.37 ± 237.77c

502.96 ± 219.06b,d

TBPf

0.001 ± 0.002

0.006 ± 0.004a

0.002 ± 0.003b

0.008 ± 0.003c

0.002 ± 0.003b,d

SMI

0.841 ± 0.340

1.275 ± 0.420a

0.974 ± 0.368b

1.610 ± 0.313c

1.033 ± 0.321b,d

DA

1.920 ± 0.200

2.290 ± 0.300

2.214 ± 0.258

1.970 ± 0.293

1.911 ± 0.184b,c,d

BV/TV bone volume/tissue volume, BS/BV ratio of bone surface to bone volume, Tb·Th trabecular thickness, Tb·Sp trabecular spacing, TBPf trabecular bone pattern factor

aOVX differences versus SH

bDifferences versus OVX

cDifferences versus OVX-Z

dDifferences versus OVX-E

In this study we did not find any additive interaction between zoledronic acid treatment and treadmill running exercise on trabecular bone microarchitecture parameters assessed by μCT.

Effects of Zoledronic Acid

Both zoledronic acid and zoledronic acid plus exercise were fully protective against estrogen-dependent bone loss. Indeed, OVX animals showed a −37.5% overall reduction in trabecular bone volume fraction versus SH animals, whereas the BV/TV was not significantly different in the OVX-Z and OVX-ZE groups versus the SH group, at 24.9%, 25.5%, and 26%, respectively. Trabecular spacing values were significantly higher in the OVX and OVX-E groups than in the OVX-Z and OVX-ZE groups. There were significant differences concerning Tb·Th, BS/BV, Tb·N, TBPf, DA, and SMI between the OVX-Z and the OVX-ZE groups, on one hand, and between the OVX and the OVX-E groups, on the other hand, as indicated in Table 3.

Effects of Exercise

Exercise alone did not prevent the loss of cancellous bone induced by ovariectomy in this study, as BV/TV, BS/BV, Tb·Sp, Tb·N, Tb·Th, and TBPf, values were not statistically different between the OVX and the OVX-E groups. Only for DA were significant differences between sedentary rat groups and exercise rat groups observed (Table 2). However, we did not find any significant inhibition or enhancement of the effect of zoledronic acid, by exercise, on trabecular bone parameters, and only for DA did we observe a positive effect with the combination of exercise and zoledronic acid, with a lower DA in the OVX-ZE group compared to the OVX-E, OVX-Z, and OVX groups (Table 3).

Biomechanics

In this study, ovariectomy did not induce a significant decrease in biomechanical properties as shown by the bending test at the femur. In contrast, we observed an effect of exercise, with a significantly higher ultimate force in the OVX-E and OVX-ZE groups than in the sedentary rat groups (OVX and OVX-Z). There was an additive effect of zoledronic acid and exercise, with a significantly higher ultimate force in the OVX-ZE group than in the OVX and OVX-Z groups: 143 versus 120.4 N (P < 0.05) and 143 versus 124.4 N (P < 0.05), respectively. We found a statistically significant effect of zoledronic acid on the energy to failure (Table 4). A higher energy to failure was observed in the OVX-Z group compared to the OVX and OVX-E groups (P < 0.05), and a higher energy to failure in the OVX-ZE group compared to the OVX-E and OVX groups (P < 0.05). There was no difference between OVX-Z and OVX-ZE.
Table 4

Changes in biomechanical properties in sham-operated (SH) or ovariectomized (OVX) mature rats treated or not treated with zoledronic acid (Z; a single injection, 20 μg/kg) and practicing or not practicing treadmill running exercise (E) for 12 weeks: mean ± SD

Parameter

Group

SH

OVX

OVX-Z

OVX-E

OVX-ZE

Ultimate force (N)

133.06 ± 20.82

120.39 ± 13.98

124.44 ± 16.95

138.25 ± 16.96b,c

143.06 ± 15.94b,c

Cross-sectional area (mm²)

6.69 ± 0.64

6.35 ± 0.54a

6.63 ± 0.61

7.04 ± 0.55b,c

7.10 ± 0.63b,c,d

Moment of inertia (mm4)

5.64 ± 0.94

5.55 ± 0.80

5.49 ± 0.89

6.06 ± 1.14

6.15 ± 1.43

Stress (N/mm²)

185.83 ± 19.21

172.72 ± 26.72

177.87 ± 22.90

184.75 ± 23.05

189.48 ± 18.61

Energy max (N-m)

68.66 ± 12.37

55.06 ± 15.57a

64.48 ± 12.22b

56.76 ± 16.16c

67.66 ± 8.56b,d

Stiffness (N/m)

238.76 ± 46.86

257.37 ± 24.78

227.43 ± 35.06

245.68 ± 35.27

262.35 ± 33.43*

Young modulus (MPa)

7176.48

7910.05

7071.23

6925.87

7388.97

* Interaction between treatment and exercise

aOVX differences versus SH

bDifferences versus OVX

cDifferences versus OVX-Z

dDifferences versus OVX-E

Bone Turnover

The CTX level was 27.7% higher in the OVX group (23.8 ng/ml) than in the SH group (18.6 ng/ml; P < 0.0005). CTX levels were significantly lower in the OVX-Z and OVX-E groups compared to the OVX group (P < 0.05). However, CTX levels in the OVX-ZE group were not statistically different from those in the OVX group but were significantly higher than in the OVX-Z and OVX-E groups (Fig. 2). In contrast, at the end of the study, serum osteocalcin levels were not significantly different between the SH group and the OVX group. The OVX-ZE group displayed a higher osteocalcin level than did the OVX, OVX-Z, and OVX-E groups. The osteocalcin level was significantly higher in the OVX-E group (178.27 ng/ml) versus the OVX and OVX-Z groups (117.83 and 139.32 ng/ml, respectively) (Fig. 2). As expected the UI decreased from −0.26 at baseline to −4.43 at the end of the study in the OVX group. In the OVX-E group the UI increased from −0.22 at baseline to +3.42 at the end of the study. The UI increased from −0.31 to +0.45 in the OVX-Z group.
Fig. 2

Changes in bone turnover parameters and uncoupling index in sham-operated (SH) or ovariectomized (OVX) mature rats treated or not treated with zoledronic acid (Z; a single injection, 20 μg/kg) and practicing or not practicing treadmill running exercise (E) for 12 weeks. a OVX differences versus SH, b differences versus OVX, c differences versus OVX-Z, d differences versus OVX-E and e differences versus OVX-ZE. *Interactions between treatment and exercise

Discussion

The salient findings of the present study are as follows.
  • First, we found that a single i.v. injection of zoledronic acid, 20 μg/kg, prior to OVX protected ovariectomized mature rats against estrogen-dependent trabecular bone loss and microarchitectural deterioration at 12 weeks.

  • Second, in the present study, the running exercise, which was characterized by 60 min of daily exercise on a treadmill, at a speed of 15 m/min for 12 weeks, did not prevent the trabecular bone loss induced by ovariectomy but had a significant effect on the ultimate force and on bone remodeling.

  • Third, although higher values for ultimate force and energy to failure were found in rats treated with zoledronic acid and undergoing the running exercise, we did not observe any synergistic interaction between zoledronic acid and exercise.

Zoledronic acid is a heterocyclic bisphosphonate which has been found in in vitro assays to be the most potent antiresorptive agent by inhibition of osteoclast-mediated bone resorption. It has been shown that a single dose of zoledronic acid, 100 μg/kg i.v., was able to maintain for 180 days the compressive bone strength at the vertebrae and the degree of bone mineralization in old (18 months) Fisher 344 ovariectomized rats [28]. In a model of acute stroke in 12-week-old female Sprague-Dawley rats, a 30 μg/kg i.v. injection of zoledronic acid preserved the bone mass and biomechanical bone strength at 3 weeks after stroke, whereas a 6 μg/kg i.v. injection did not [29]. In vivo, μCT examination has been used to assess the efficacy of zoledronic acid by a single 20 μg/kg s.c. injection either as a preventive (early) or as a recovering treatment of OVX-induced bone loss [30]. In the latter experiment, 30-week-old retired breeding Wistar rats were ovariectomized and studied at 16 weeks. Although early treatment of OVX rats led to preservation of structural parameters, no significant differences between groups emerged in the three-point bending test [30]. An improvement in biomechanical parameters was found in the bending strength of the femoral shaft in the study by Gasser et al. [31]. A 20 μg/kg dose was shown to improve the ultimate strength, but not the toughness, in their OVX rats and they concluded that only a 100 μg/kg dose of zoledronic acid fully prevented the loss of bone strength induced by OVX [31]. In our study we found a significant effect of zoledronic acid on the energy to failure compared to the OVX and OVX-E groups at the femur, whereas it was assessed at the tibia in the study by Browers et al. [30]. Our study is also in agreement with the latter experiment; indeed, we did not found any significant differences between the SH group and the zoledronic acid-treated groups for any 3D structural parameters. In their experiment Lee et al. [29] did not find any statistical significant differences in osteocalcin levels among their groups of rats 3 weeks after induction of stroke (two groups were treated with zoledronic acid). In contrast, zoledronic acid (30 μg/k) inhibited the increase in CTX value observed in their stroke vehicle group.

We cannot strictly compare the latter results to our study, as the rats involved in these studies were twofold younger, of different species, and, above all, not ovariectomized. However, we also found an inhibition by zoledronic acid on the elevation in CTX levels observed in the OVX group, with no statistical difference between the OVX-Z and the SH groups. Gasser et al. [31], in their long-term assessment of zoledronic acid effects on ovariectomized rats, found an increase in plasma osteocalcin levels after OVX. In our study this was not the case, but we also did not find a suppression of bone formation activity, as the plasma osteocalcin levels were not significantly different between SH-operated rats and OVX animals treated with zoledronic acid. In contrast, we confirmed the antiresorptive effects of zoledronic acid [29, 31], as the elevated osteoclast activity reflected by the rise in CTX levels in OVX rats was prevented.

In the present study, serum osteocalcin levels did not rise after OVX; in contrast, running exercise significantly increased serum osteocalcin. In addition, the uncoupling index (UI) increased from baseline to the end of the study, confirming that treadmill running exercise stimulated bone formation. These results are in accordance with previous studies where the exercise regimen included treadmill running [32] or jumping training [33]. We found a significant main effect for exercise on femoral ultimate force and cross-sectional area as assessed by the three-point bending test of the femur. This is in line with the data of Fuchs et al. [14], who found a beneficial effect of treadmill running on ultimate force, modulus, and stiffness in 7-month-old virgin female Fisher 344 ovariectomized rats. The exercise protocol regimen applied to our rats did not result in preservation of BMC after OVX during the 12-week study period. Despite the lack of change in BMC and any significant improvement or preservation in trabecular bone parameters assessed by 3D μCT produced by our running exercise experiment, exercise training had a positive effect on cross-sectional area at the femur. This effect on geometry might explain in part the higher ultimate force observed in exercised rats compared to sedentary ones.

Moreover, it has been reported that exercise may have a greater impact on architectural and geometric properties of bone rather than on BMD [34, 35].

In most published reports, treadmill running exercise was shown to be beneficial to the rat skeleton [36]. However, bone adaptation to treadmill exercise might vary according to several factors: age, gender, animal species, gonadal status (ovariectomized or not), other models of osteopenia, experimental period, frequency, duration, and intensity of exercise. In our study we chose the exercise regimen according to previous studies indicating that a 15 m/min running speed, 60 min/day, 5 days/week, for 12 weeks, was osteogenic in ovariectomized mature rats [36, 37]. The running speed and the corresponding VO2 max (maximum oxygen consumption) [38] or maximal aerobic speed [39] are critical to obtain a beneficial effect on the skeleton. It has been demonstrated that overly vigorous exercise (VO2 max > 80%) is less effective than more moderate exercise (50–70% VO2 max) [38]. The number of days per week on which the rats undergo running exercise is another variable [40]. A moderate running load at a frequency of 4 or 5 days per week has been shown to be beneficial to BMD in 8-week-old female Wistar rats [40].

The bone site at which the experiment is assessed may also produce different results. In our study, we did not observe an effect of exercise on BMC at the femur. Numerous exercise studies produced more significant effects at specific bone sites, at the tibia more than at the femur or vertebrae [15, 39, 40, 41, 42]. For instance, gain in BMC was shown to be higher at the distal tibia versus the distal femur in rats taking voluntary exercise [43]. The femur is likely to receive less impact (mechanical loading) than the tibia, as it is located more distally from the ground impact [44, 45]. Regional differences in the skeletal response to the exercise protocol and treatment may be observed [46].

Whereas it was an attractive hypothesis that a concurrent combination of exercise and zoledronic acid would be superior to either intervention alone, we found that this intervention did not produce any significant synergistic interaction. However, the highest values of BV/TV, energy to failure, ultimate force, and cross-sectional area, although nonsignificant statistically, were found in the OVX-ZE group.

Direct comparison between rat experimental protocols where bisphosphonates and exercise have been combined [14, 15] may be hazardous because the exercise regimen, experimental animals, age at ovariectomy, and bisphosphonates tested were different across studies. Tamaki et al. [14] assessed the effect of combining treadmill running exercise and etidronate in ovariectomized rats. In that study, rats were split into five groups as in our study. Using histomorphometry, the results showed an interaction between etidronate treatment and subsequent physical exercise, as evidenced by the increased trabecular bone area of the proximal tibia. A significant interaction was also observed for BMD at the proximal femur of rats, but there was no interaction dependence at the mid and distal regions either in the femur or in the measurement of osteoclast and osteoblast numbers. Alendronate and treadmill running exercise have been combined to examine their effects on bone mass and strength [15] in ovariectomized rats. In that experiment, using a 2 × 2 factorial design, the authors found the combined intervention of alendronate and treadmill running exercise to be superior in preserving the WB and proximal femur BMC and maintaining bone strength at the hip than either intervention alone. In our study we failed to demonstrate a synergistic interaction or significant additive effect when combining zoledronic acid and treadmill exercise running. This lack of additive effect of the combination of exercise and zoledronic acid on BMC, trabecular bone microarchitecture, and bone strength might be explained by the potency of the antiresorptive effect of zoledronic acid. The response to the osteogenic effects of exercise might have been blunted by the powerful effect of zoledronic acid. This hypothesis has been mentioned to explain the lack of efficacy of combining parathyroid hormone with alendronate, the capacity of the antiresorptive drug being superior to the capacity of formation associated with PTH treatment [47]. In their experimental protocol, Tamaki et al. [14] administered etidronate before the exercise training of the rat. We propose that, to permit an anabolic window due to the osteogenic effects of exercise, the bisphosphonate could be given a second time, to obtain an in-sequence treatment rather than a simultaneous one. This is in agreement with the study by Rhee et al. [48], who found, in an experiment studying the lose, restore, and maintain concept, that sequential therapy with zoledronic acid after rhPTH(1–84) was consistently effective in treating ovariectomy-induced bone loss.

Any conclusions with regard to human use should be drawn with caution, as running exercise in rats and humans may result in different loading patterns and responsiveness. Besides, in humans, both studies which have tested this hypothesis to obtain additive effects by combining bisphosphonate and exercise training failed to demonstrate a superior action of the combined interventions compared to either separate treatment [49, 50].

Chilibeck et al. set up a randomized trial to study the combined effects of etidronate (ETI) and exercise (E) in postmenopausal women [49]. Forty-eight women, with a mean age of 57 years, were divided into four groups: ETI + E, ETI only, E only, and placebo without E. Etidronate was taken over 12 months at a dose of 400 mg/day over 14 days, every 90 days. Resistance training was carried out three times a week, for 45 min. When the data for all ETI groups were pooled, WB and lumbar spine bone density gains were significantly greater than those of the placebo group. However, no interaction or additive effect between ETI and exercise emerged. Uusi Rasi et al. [50] studied the association of physical exercise with a daily intake of 5 mg alendronate in 164 postmenopausal women (age: 54 ± 2 years). The daily exercise protocol comprised 20 min of jumping activities and 15 min of stretching and other nonimpact activities. It appeared that alendronate treatment increased the BMD at the lumbar spine and femoral neck but that exercise alone had no effect. Besides, no interaction or additive effect between alendronate and exercise could be shown.

Overall, the findings of the present study suggest that zoledronic acid prevented the ovariectomy-induced trabecular bone loss and its subsequent trabecular microarchitectural deterioration. Treadmill exercise running was shown to preserve bone strength and to induce bone turnover changes in favor of bone formation. However, the combined effects of zoledronic acid and running exercise applied simultaneously did not produce any synergistic or additive effects.

Notes

Acknowledgment

This work was supported by a grant from Novartis France.

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • E. Lespessailles
    • 1
  • C. Jaffré
    • 1
    • 2
  • H. Beaupied
    • 1
  • P. Nanyan
    • 1
  • E. Dolléans
    • 1
  • C. L. Benhamou
    • 1
  • D. Courteix
    • 3
  1. 1.INSERM U658Laboratoire de Caractérisation du Tissu Osseux par Imagerie: Techniques et ApplicationsOrleansFrance
  2. 2.UFR STAPSUniversité d’OrléansOrleans Cedex 2France
  3. 3.Laboratoire de Biologie des APS, UFR STAPSUniversité Blaise PascalAubière CedexFrance

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