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Effects of exercise training and etidronate treatment on bone mineral density and trabecular bone in ovariectomized rats
Bone Vol. 23, No. 2 August 1998:147–153
Effects of Exercise Training and Etidronate Treatment on Bone Mineral Density and Trabecular Bone in Ovariectomized Rats H. TAMAKI,1,2 T. AKAMINE,1,3 N. GOSHI,2 H. KURATA,2 and T. SAKOU1 1 2
Department of Orthopaedic Surgery, Faculty of Medicine, Kagoshima University, Kagoshima, Japan Department of Physiological Sciences and 3Department of Health Education, National Institute of Fitness and Sports, Kagoshima, Japan
stimulation of bone formation should be employed at least to prevent the bone loss resulting from estrogen deficiency. Bisphosphonates have a high binding affinity for hydroxyapatite, and inhibit osteoclastic bone resorption. They also interfere with the differentiation, fusion, and migration of osteoclast precursors to the bone surface.3,13,19 It is, in fact, reported that the bisphosphonates increase bone mineral density (BMD) in all skeletal sites and reduce the incidence of bone fracture in estrogendeficient women.23 Furthermore, it has been reported that longterm treatment inhibits the mineralization of the bone, and several studies have demonstrated effective protocols of the administration of bisphosphonates on the restoration and maintenance of bone mass. For example, intermittent cyclic administration of bisphosphonates has been shown to preserve the mass, architecture, and mechanical properties of ovariectomized rats.21 Jee et al.20 employed the lose, restore, and maintain (LRM) concept that uses ovariectomy to induce bone loss, an anabolic agent to stimulate bone formation, and then switches to an antiresorptive agent to inhibit bone resorption. Moreover, it has been reported that exercise training enhances bone formation in rats and humans.2,6,12 Exercise therapy that loads the mechanical stress to the bone is also effective in maintaining bone mineral density in early postmenopausal women.11 It has been suggested that run training increases the BMD by improving mechanical stress on the bone and inducing hypertrophy of the cortical bone in experimental animals.26,28 Therefore, it is conceivable that the therapy that switches the treatment from etidronate to exercise training might be effective in preventing bone loss caused by an estrogen deficiency in ovariectomized rats. The purpose of this study was to assess the effects of exercise training following an etidronate treatment on BMD of the femur and the trabecular bone of the tibia in ovariectomized rats.
This study was designed to assess the effects of exercise training (Tr) following an etidronate treatment (E) on bone mineral density (BMD) of the femur and trabecular bone of the tibia in ovariectomized (ovx) rats. Female Wistar rats were ovariectomized (ovx) or sham-operated (sham) at 15 weeks of age and divided into five experimental groups: sham; ovx; ovx 1 E; ovx 1 Tr; ovx 1 E 1 Tr. Etidronate treatment of 5 mg/kg, 5 days/week was administered for 2 weeks and exercised on a treadmill for 30 m/min, 60 min/day, 5 days/week for 10 weeks. BMD of the femur and the trabecular bone area of the proximal tibia were significantly (p < 0.05) higher in E and/or Tr compared to ovx groups. However, the cortical region was not affected significantly by ovariectomy. The area partially filled with the trabecular bone at the constant width was observed only in the E rats. The number of osteoclasts in E group was significantly lower (p < 0.05) than in the ovx and ovx 1 Tr groups. The ovx 1 Tr rats had a higher number of osteoblasts (p < 0.05) than the ovx and ovx 1 E groups. There was a significant interaction between ovx 1 Tr and ovx 1 E on BMD in the proximal region of the femur (p < 0.05) and trabecular bone area of the tibia (p < 0.001). These results suggest that the etidronate treatment for 2 weeks beforehand influenced the effects of subsequent exercise training on maintaining the BMD in the proximal femur and the trabecular bone area of the tibia. (Bone 23:147–153; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: Etidronate; Exercise; Ovariectomy; Bone mineral density; Osteoclast; Osteoblast. Introduction Postmenopausal osteoporosis is a serious problem in elderly women and is characterized by a decrease in bone mass, leading to fractures and imbalanced turnover of the bone.24 Ovariectomized (ovx) rats exhibit a decrease in mineral density, volume, and strength of bone and an increase in bone turnover rate seen in women suffering from osteoporosis.21 Therapy that involves either inhibition of bone resorption or
Materials and Methods Experimental Animals Thirty-five female Wistar rats were maintained under constant temperature (25 6 2°C) and humidity (55% 6 5%), and under 12 h/12 h light– dark cycles. The rats were housed individually in standard cages and provided with a commercial standard diet (Kyudo, Japan) containing 1.2% calcium and 0.8% phosphorus. The food consumption of the rats was controlled to minimize the difference among groups in body weight associated with ovariectomy and exercise training. The food consumption of the rats
Address for correspondence and reprints: Hiroyuki Tamaki, National Institute of Fitness and Sports, Department of Physiological Sciences, 1 Shiromizu, Kanoya, Kagoshima 891-23, Japan. © 1998 by Elsevier Science Inc. All rights reserved.
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Table 1. The body weight and uterine weight in each group Group Body weight (g)
15w 28w
Uterine weight (mg)
Sham
Ovx
Ovx 1 Tr
Ovx 1 E
Ovx 1 E 1 Tr
263.3 6 4.7 324.3 6 5.7 601.5 6 81.5
271.7 6 6.5 338.3 6 8.8 206.6 6 33.9*
271.3 6 8.7 333.7 6 12.1 220.6 6 38.2*
266.0 6 4.8 343.3 6 6.4 191.8 6 48.4*
277.7 6 6.8 339.0 6 8.8 203.2 6 70.5*
*p , 0.0001, significantly different from the sham group (ANOVA). Sham: sham-operation; OVX, ovariectomy; E: etidronate treatment; Tr: exercise training.
that showed a tendency for higher increases in body weight after ovariectomy was restricted.30,31 At 15 weeks of age, animals were either ovx or sham-operated (sham). Bilateral ovariectomies were performed under anesthesia with sodium nembutal administered intraperitoneally. At necropsy, the uterus of each rat was removed and weighed. No detection of ovarian tissue and atrophy of the uterine horns were observed in the ovariectomized rats.31 (Table 1) Sham operations were performed by exteriorizing the ovaries. Ovx rats were divided into four groups based on treatment with either etidronate (E) or physiological saline and exercise training (Tr) as follows: physiological saline-treated group (ovx); etidronate-treated group (ovx 1 E); physiological saline-treated and exercise-trained group (ovx 1 Tr); and ovariectomized plus etidronate-treated and exercise-trained group (ovx 1 E 1 Tr). All procedures using animals were carried out in accordance with the guidelines presented in the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, published by the Physiological Society of Japan, and were approved by the National Institute of Fitness and Sports Animal Care Committee. Etidronate and Saline Treatment Etidronate disodium was dissolved in a saline vehicle. Etidronate and saline treatment were initiated 1 week after surgery. The rats in the ovx 1 E and ovx 1 E 1 Tr groups were treated 5 days/week for 2 weeks with subcutaneous injections of etidronate (Sumitomo Pharmaceuticals, Japan) at a dose of 5 mg/kg body weight.7,21,31 The rats in the ovx, ovx 1 Tr, and sham-operated groups were injected subcutaneously 5 days/week for 2 weeks with the saline vehicle used to dissolve the etidronate (Figure 1). Training Program The rats in the training group were given a program of running exercise on a motor-driven treadmill (Model SN-460, Shinano,
Japan) for a total of 10 weeks (18 –28 weeks of age). The time of this daily exercise and the running speed were increased gradually during the first 2 weeks of training. Following this adaptation period, the training was carried out under constant conditions (speed: 30 m/min; time: 60 min/day; frequency: 5 days/ week) until the end of the training period (Figure 1). Bone Mineral Density Measurements The bone mineral density of the femurs was measured using dual energy X-ray absorptiometry (Norland XR-26, Ft. Atkinson, WI). Each femur was placed on an acrylic board (25 mm in thickness). Scans were performed at random between groups at a scan speed of 10 mm/s and repeated three times to assess the reproducibility of the measurement system. The BMD of the femurs was measured and three regions of the femurs (proximal third, middle third, and distal third) were assigned for analysis to assess any regional differences in bone characteristics. Each scan was analyzed by using small animal software (Norland). Measurements using a lumbar spine anthropomorphic photon were performed to check the quality of the device. The coefficient of variation for this measurement technique in our laboratory was 0.9%. These measured values of bone mineral density were obtained and analyzed by the same examiner and on the same day. Tissue Preparation and Bone Histology The rats were anesthetized with sodium nembutal and immediately perfusion-fixed through the abdominalis aortae with a mixture of 1% glutaraldehyde, 1% formaldehyde, and 0.05% CaCl2 in 0.1 mol/L sodium cacodylate buffer (pH. 7.3) for 30 min at room temperature. After fixation, the femurs were removed and preserved in physiological saline until the determination of bone mineral density. The tibias were demineralized in 0.1 mol/L disodium ethylenediaminetetraacetic acid (pH 7.3) for 6 weeks at 4°C, dehydrated through a graded ethanol series, and
Figure 1. Experimental protocol. Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
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then embedded in paraffin. Longitudinal sections of 5 mm thickness were cut using a microtome. Specimens were stained with the media of azocalmin–anilinblue (Azan), tartrate resistant acid phosphatase (TRAP), or methylgreen-pyronine for counting the volume of bone, the number of osteoclasts, or the number of osteoblasts at the secondary spongiosa. The trabecular bone area was determined manually using a grid-point counting technique, and was expressed as a percentage (points on bone / points on tissue inside the measurement area).14 The micrographs used for the measurement of trabecular bone area were taken at a magnification of 3400.30 Trabecular bone measurements were performed at a 6-mm2 area, 1 mm distal from the growth plate– cancellous bone junction of the proximal tibia to exclude the primary spongiosa.30 The numbers of osteoclasts, identified on the bone surface by TRAP activity, were counted per square millimeter of bone area. The number of osteoblasts, characterized by a centrally located negative Golgi image within the basophylic cytoplasm and eccentrically located nucleus, was counted within a 6-mm2 area under the microscope at a magnification of 340. Statistics Results are expressed as the mean 6 standard deviation (SD). The significance of differences between groups was calculated by an analysis of variance (ANOVA), followed by Fisher’s PLSD test. A two-factor ANOVA was used to examine the individual effect and interaction between the exercise and etidronate treatment. The level of significance was set at p , 0.05. Results Bone Mineral Density The influences of the ovariectomy, etidronate treatment, and/or exercise training on BMD in the total and regional femur are shown in Figure 2. In the proximal and distal region, the BMD was significantly lower (p , 0.05) in the ovx than in the sham group. BMD levels were significantly higher (p , 0.05) in the ovx 1 E 1 Tr, ovx 1 E, and ovx 1 Tr groups compared with the ovx group. A two-factor ANOVA showed that the effects of exercise and etidronate treatment on BMD in the proximal femur were significant (p , 0.01), and the interaction between exercise and etidronate factors was significant (p , 0.05). In the middle region, BMD levels in the ovx 1 E 1 Tr, ovx 1 E, and ovx 1 Tr groups were significantly higher (p , 0.05) than that in the ovx group. The effects of exercise and etidronate treatment on BMD in the middle femur were also significant (p , 0.05), but there was no interaction dependence. In the distal region, ovx 1 E 1 Tr, ovx 1 E, and ovx 1 Tr groups exhibited significantly higher levels (p , 0.05) of BMD compared with the ovx group. Exercise affected the BMD in the distal femur (p , 0.05), but no interaction was observed between the exercise and etidronate treatment factors. Trabecular Bone Area The different histological areas of the proximal tibia sections from sham, ovx, ovx 1 E, ovx 1 Tr, and ovx 1 E 1 Tr rats are depicted in Figure 3. As expected, the loss of trabecular bone was observed in ovariectomized rats, and was most obvious in the central metaphysial region. The area partially filled with trabecular bone (the thick region) was observed only in the etidronate-treated rats, which exhibited bone of a constant width. In the etidronate-treated rats, the trabecular bone in the area between the growth plate–metaphysial junction and the thick
Figure 2. Effects of etidronate treatment and exercise training on bone mineral density in the proximal (A), middle (B), and distal (C) femur of ovariectomized rats. The values expressed as the mean 6SD. *p , 0.05, significantly different from the ovx group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
region of the exercise-trained rats was maintained compared with that of the nontrained rats. Conversely, the loss of trabecular bone in the area between the growth plate–metaphysial junction and the thick region was remarkable in the nontrained rats. The trabecular bone area (%) was measured and compared among the five experimental groups (Figure 4). As expected, the trabecular bone area was significantly (p , 0.05) lower in the ovx than in the sham group. However, the trabecular bone area in the etidronate-treated and/or exercise-trained rats was maintained at the level observed in the sham-operated rats. The combination of both the etidronate treatment and exercise training resulted in the greatest level of trabecular bone area of all of the groups. The trabecular bone area in the ovx 1 E 1 Tr group was significantly (p , 0.05) greater than in the ovx 1 Tr group. The effects of exercise and etidronate treatment on the trabecular bone area in the tibia were significant (p , 0.0001), as was the interaction between these factors (p , 0.001). The osteoclasts located on trabecular bone surfaces and the mean number of osteoclasts per unit area (mm2) of trabecular bone in the five experimental groups are shown in Figures 5 and 6. The number of osteoclasts per bone area was affected by ovariectomy or by etidronate treatment and exercise training of
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Figure 3. Photographs of proximal tibia mataphysis from the sham (A), ovx (B), ovx 1 exercise training (C), ovx 1 etidronate treatment (D), and ovx 1 exercise training 1 etidronate treatment (E) groups. A dramatic bone loss was observed in the ovx (B) compared with the sham (A) group. Azan stain, original magnification 320. Bar 5 1 mm.
the ovx rats. There was a significant difference (p , 0.05) between the ovx group and the sham group. The decreases in osteoclast numbers were caused by treatment with etidronate and exercise training in the ovx rats. The number of osteoclasts in the ovx 1 E and the ovx 1 E 1 Tr groups was significantly fewer (p , 0.05) than in the ovx group. However, exercise training did not affect the number of osteoclasts dramatically. The ovx 1 Tr group exhibited a significantly greater number of osteoclasts (p , 0.05) compared with both the ovx 1 E and the ovx 1 E 1 Tr groups, but did not significantly differ from the sham group. The number of osteoblasts was significantly higher (p , 0.05) in the ovx 1 Tr and the ovx 1 E 1 Tr groups than in the ovx and ovx 1 E groups (Figure 7). The effects both of etidronate treatment on osteoclast number (p , 0.001) and exercise training on osteoblast number were significant (p , 0.01), but there was no interaction between the exercise and etidronate treatment factors. Discussion Regional differences in the response to ovx were observed. The proximal region of the femur showed a lower BMD in ovx rats, but a nonsignificant influence in the middle region of the femur.
Figure 4. Effects of etidronate treatment and exercise training on trabecular bone area in the proximal tibia of ovariectomized rats. The values expressed as the mean 6SD. ap , 0.05, significantly different from the ovx group; bp , 0.05, significantly different from ovx 1 Tr group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
Similar results have been reported in adult ovx rats.15 Long bones, such as the femur, have a greater proportion of trabecular bone in their proximal and distal regions and a greater proportion of cortical bone in their middle region. Ovx may have more of an effect on the trabecular bone region than on the cortical bone region on BMD in the initial period following ovariectomy. In aged rats, however, a clear reduction in the cortical bone regions has been observed following ovx.8 It is conceivable that there is a time lag for the response to ovariectomy in the different regions of the long bone. Many drugs that prevent such ovx-induced bone loss have been investigated. Bisphosphonates are known to strongly inhibit bone resorption. In postmenopausal women, bisphosphonate treatment has been shown to increase BMD in all skeletal sites, and to decrease the incidence of vertebral fractures.23 In ovariectomized rats, treatment with bisphosphonate etidronate preserves trabecular bone volume and BMD in the vertebrae, femur, and tibia.21 In the study reported here, ovariectomized rats treated with etidronate maintained BMD in the femur and trabecular bone in the tibia to levels observed in sham-operated rats. Pharmacologically, the bisphosphonates possess a high binding affinity for skeletal calcium and prevent bone crystal dissolution.13 In addition to their indirect effects on osteoclasts, these drugs impair the resorptive function of osteoclasts. It has been suggested that bisphosphonates interfere with the differentiation, migration, and fusion of immature osteoclasts.3 These reports support our results that the number of osteoclasts were increased after ovariectomy, but that etidronate treatment caused a reduction in their number below the level observed in sham-operated rats. Bisphosphonates may act to prevent the trabecular bone loss and reductions in BMD through inhibition of osteoclast-mediated resorption and the prevention of bone crystal dissolution. On the other hand, an adverse effect of the etidronate treatment has been reported: long-term doses inhibit calcium phosphate crystallization and result in an inhibition of mineralization of the bone and the development of fractures.27 In the present study, etidronate treatment influenced the BMD of the femur, the trabecular bone area of the tibia, and the number of osteoclasts. It is possible that the etidronate treatment in this study maintained the BMD of the femur and trabecular bone of the tibia by preventing bone resorption. The level of physical activity is one of the important factors
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Figure 5. Light micrographs showing proximal metaphysis of tibias in the sham (A), ovx (B), ovx 1 Tr (C), ovx 1 E (D), and ovx 1 E 1 Tr (E) rats. The osteoclast (arrows) number appears to be greater in ovx rats compared with that in the sham. TRAP stain, original magnification 3200. Bar 5 50 mm.
that determines bone mass. For example, weightlessness resulting from spaceflight also decreases bone mineral density and bone mass and remarkably inhibits bone formation in rats.29 Conversely, it has been reported that the degree of bone mineral content is extremely high and is closely correlated to the amount of exercise performed by athletes.17 In postmenopausal women, those who are physically active also have a higher bone mineral content than those who are inactive.11 These reports suggest that mechanical stress to the bone caused by weight loading and muscle contractions strongly affects bone formation and resorption. Mechanical stress to the bone can generate strain-related potentials that affect bone formation.9 Rubin and Lanyon26 have reported that the application of consecutive external loads to a functionally isolated long bone in vivo prevents disuse atrophy of the cortical bone and increases bone mineral content, and that further increases in bone formation did not occur no matter how much mechanical stress was imposed. On the other hand, exercise-induced endocrine changes also may have an effect on bone metabolism. Physical activity such as running or weight lifting stimulates the secretion of sex hormones, growth hormones, and catecholamines,18 and increases the bone formation resulting from an increase in osteoblastic recruitment and in the activity of
Figure 6. Effects of etidronate treatment and exercise training on the number of osteoclasts per unit area in the proximal tibia. The values expressed as the mean 6SD. ap , 0.05, significantly different from the ovx group; bp , 0.05, significantly different from ovx 1 Tr group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
individual osteoblasts in rats and humans.2,6,12 It can be speculated that the increase in mechanical stress and/or secretion of hormones stimulating bone formation caused by running exercise might prevent ovariectomy-induced trabecular bone loss observed in the present study. However, it is not clear whether the exercise training effect was due to an increase in bone formation resulting from the higher activity of individual osteoblasts. Partial effects of exercise on preventing the loss of trabecular bone and calcium content have been reported.25,32 The estimated exercise intensity based on treadmill speed and grade correspond ˙ O2max (maximum oxygen consumption) to approximately 50% V in these study.6,22 Some studies report the relationship between exercise intensity and bone mass. It is reported that high-intensity ˙ O2max) reduces longitudinal bone growth and exercise (80% V increases bone loss in rats.5 The rats trained at low intensity ˙ O2max) showed a decreased trabecular bone volume of (40% V the femur.4 Thus, Bourrin et al.5,6 suggested that exercise inten˙ O2max and above 80% V ˙ O2max do not sities below 40% V improve bone loss in rats. Exercise intensity during our experi˙ O2max, according to the measurement would be about 60% V
Figure 7. Effects of etidronate treatment and exercise training on the number of osteoblasts per unit area in the proximal tibia. The values expressed as the mean 6SD. ap , 0.05, significantly different from Sham group; bp , 0.05, significantly different from the ovx group; cp , 0.05, significantly different from ovx 1 E group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
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ment of Bourrin et al.6 Barengolts et al.1 reported that biomechanical and morphometric properties of the femur and bone mineral of the tibia and femur in ovx and exercised (approxi˙ O2max) rats maintained the level of sham-operated mately 60% V rats. The setting of the exercise intensity might be an important factor to obtain an optimal effect of exercise training even within the effective range of exercise intensity. In addition to the individual efficacies of the therapy of the etidronate or the exercise alone, it is important to understand the interaction of the two. Jee et al.20 employed the LRM concept, which utilizes the anabolic agent to restore bone mass and then switches to the antiresorptive agent to maintain bone mass in ovx rats through the lose phase, for 5 months. This therapy was successful in maintaining trabecular bone mass and structure in the proximal tibia. It is reported that the combined exercise and bisphosphonate treatment reduces the calcium loss and prevents some of the changes in mineral metabolism in humans.16 The present study employed therapy that used exercise training subsequent to etidronate treatment in ovx rats. Etidronate treatment for 2 weeks preserved trabecular bone, as shown in the bone area filled with trabecular bone (the thick region) of the proximal tibia, and resulted in maintaining the trabecular bone structure. Ovx rats that were exercise trained for 10 weeks after etidronate treatment maintained their trabecular bone in the area between the growth plate–metaphysial junction and the thick region; those rats that were not trained did not. The ovariectomized rats that were treated with both etidronate and exercise training (ovx 1 E 1 Tr) exhibited the highest BMD in the proximal and middle regions of the femur and trabecular bone area of the tibia of all of the experimental groups in the present study. The effect of subsequent exercise training on maintaining the trabecular bone of the tibia was influenced by etidronate treatment beforehand in ovx rats. Taking into account these results and those of the report showing a close correlation between BMD and mechanical strength,10 it would be conceivable that a therapy consisting of exercise training following etidronate treatment would maintain bone mechanical strength and lead to a decrease in the incidence of bone fractures in postmenopausal women. In conclusion, our results indicate that the individual treatment of etidronate or exercise training prevented the loss of trabecular bone of the tibia and BMD in some regions of the femur induced by ovx. Exercise training influenced the number of osteoblasts, but not the number of osteoclasts. Furthermore, there were significant interactions between exercise and etidronate treatment on BMD in the proximal region of the femur and trabecular bone area of the tibia. This indicates that, in ovx rats, the effect of subsequent exercise training on maintaining BMD and trabecular bone in some regions is influenced by administering etidronate treatment for 2 weeks prior to exercise training.
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Acknowledgments: The authors sincerely appreciate the invaluable comments and encouragement from Dr. V. Reggie Edgerton of University of California, Los Angeles. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.
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bisphosphonates on osteoclastic resorption of mineralized matrix. Bone Miner 1:27–39; 1986. Bourrin, S., Ghaemmaghami, F., Vico, L., Chappard, D., Gharib, C. and Alexandre, C. Effect of five-week swimming program on rat bone: A histomorphometric study. Calcif Tissue Int 51:137–142; 1992. Bourrin, S., Genty, C., Palle, S., Gharib, C. and Alexandre, C. Adverse effects of strenuous exercise: A densitometric and histomorphometric study in the rat. J Appl Physiol 76:1999 –2005; 1994. Bourrin, S., Palle, S., Pupier, R., Vico, L., and Alexandre, C. Effects of physical training on bone adaptation in three zones of the rat tibia. J Bone Miner Res 10:1745–1752; 1995. Boyce, R. W., Wronski, T. J., Ebert, D. C., Stevens, M. L., Paddock, C. L., Youngs, T. A., and Gundersen, H. J. B. Direct stereological estimation of three-dimensional connectivity in rat vertebrae: Effect of estrogen, etidronate, and risedronate following ovariectomy. Bone 16:209 –213; 1995. Chen, H. K., Ke, H. Z., Jee, W. S. S., Ma, Y. F., Pirie, C. M., Simmons, H. A., and Thompson, D. D. Droloxifene prevents ovariectomy-induced bone loss in tibiae and femora of aged female rats: A dual-energy X-ray absorptiometric and histomorphometric study. J Bone Miner Res 10:1256 –1262; 1995. Cochran, G. V. B., Pawluk, R. J., and Bassett, C. A. L. Stress generated electric potentials in the mandible and teeth. Arch Oral Biol 12:917–920; 1967. Dalen, N., Hellstrom, L. G., and Jacobson, B. Bone mineral content and mechanical strength of the femoral neck. Arch Orthop Scand 47:503–508; 1976. Dalsky, G. P., Stocke, K. S., Ehsani, A. A., Slatopolsky, E., Lee, W. C., and Birge, S. J., Jr. Weight bearing training and lumbar bone mineral content in postmenopausal women. Ann Intern Med 108:824 – 828; 1988. Eliakim, A., Raisz, L. G., Brasel, J. A., and Cooper, D. M. Evidence for increased bone formation following a brief endurance-type training intervention in adolescent males. J Bone Miner Res 12:1708 –1713; 1997. Fleisch, J., Russell, R. G. G., and Francis, M. D. Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science 165:1262–1264; 1969. Frost, H. M. Bone histomorphometry: Analysis of trabecular bone dynamics. In: Recker R. R., ed. Bone Histmorphometry, Techniques and Interpretation. Boca Raton, FL: CRC; 1983; 109 –131. Frost, H. M. and Jee, W. S. S. On the rat model of human osteopenias. Bone 18:227–236; 1992. Grigoriev, A. I., Morukov, B. V., Oganov, V. S., Rakhmanov, A. S., and Buravkova, L. B. Effect of exercise and bisphosphonate on mineral balance and bone density during 360 day antiorthostatic hypokinesia. J Bone Miner Res 7:S449 –S455; 1992. Granhed, H., Jonson, R., and Hansson, T. The loads on the lumbar spine during extreme weight lifting. Spine 12:146 –149; 1986. Ha¨kkinen, K. and Pakarinen, A. Acute hormonal responses to two different fatiguing heavy-resistance protocols in male athletes. J Appl Physiol 72:882– 887; 1993. Hurghes, D. E., MacDonald, B. R., Russell, R. G. G., and Gowen, M. Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. J Clin Invest 83:1930 –1935; 1989. Jee, W. S. S., Tang, L., Ke, H. Z., Setterberg, R. B., and Kimmel D. B. Maintaining restored bone with bisphosphonate in the ovariectomized rat skeleton: Dynamic histomorphometry of changes in bone mass. Bone 14:493– 498; 1993. Katsumata, T., Nakamura, T., Ohnishi, H., and Sakurama, T. Intermittent cyclical etidronate treatment maintains the mass, structure, and the mechanical property of bone in ovariectomized rats. J Bone Miner Res 10:921–931; 1995. Lawler, J. M., Powers, S. K., Hammeren, J., and Martin, D. Oxygen cost of treadmill running in 24-month-old Fischer-344 rats. Med Sci Sports Exerc 25:1259 –1264; 1993. Liberman, U. A., Weiss, S. R., Broll, J., Minne, H. W., Quan, H., Bell, N. H., Rodriguez-Portales, J., Downs, Jr, R. W., Dequeker, J., Favus, M., Seeman, E., Recker, R. R., Capizzi, T., Santora II, A. C., Lomberdi, A., Shah, R. V., Hirsch, L. J., and Karpf, D. B. Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. N Engl J Med 333:1437–1443; 1995. Lindsay, R., Hart, D. M., Aitken, J. M., MacDonald, E. B., Anderson, J. B., and Clarke, A. C. Long-term prevention of postmenopausal osteoporosis by estrogen. Lancet 1:1038 –1041; 1976. Pohlman, R. L., Darby, L. A., and Lechner, A. J. Morphometry and calcium contents in appendicular and axial bones of exercised ovariectomized rats. Am J Physiol 248:R12–R17; 1985.
Bone Vol. 23, No. 2 August 1998:147–153 26. Rubin, C. T. and Lanyon, L. Regulation of bone formation by applied dynamic loads. J Bone Joint Surg 66A:397– 403; 1984. 27. Schenk, R., Merz, W. A., Mu¨hlbauer, R. C., Russell, R. G. G., and Fleisch H. Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res 11:196 – 214; 1973. 28. Woo, S. L. Y., Kuel, S. C., Amiel, D. Gomez, M. A., Hayes, W., White, F. C., and Akeson, W. H. The effect of prolonged physical training on the properties of long bone: A study of Wolff’s law. Bone Joint Surg 63A:780 –787; 1981. 29. Wronski, T. J. and Morey, E. R. Effect of spaceflight on periosteal bone formation in rats. Am J Physiol 244:R305–R309; 1983. 30. Wronski, T. J., Schenck, P. A., Cintro´n, M., and Walsh, C. C. Effect of body weight on osteopenia in ovariectomized rats. Calcif Tiss Int 40:155–159; 1987.
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31. Wronski, T. J., Yen, C. F., and Scott, K. S. Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats. J Bone Miner Res 6:387–394; 1991. 32. Yeh, K. J., Liu, C. C., and Aloia, J. F. Additive effect of treadmill exercise and 17 beta-estradiol replacement on prevention of tibial bone loss in adult ovariectomized rat. J Bone Miner Res 8:677– 683; 1993.
Date Received: October 9, 1997 Date Revised: April 2, 1998 Date Accepted: April 7, 1998
Effects of Exercise Training and Etidronate Treatment on Bone Mineral Density and Trabecular Bone in Ovariectomized Rats H. TAMAKI,1,2 T. AKAMINE,1,3 N. GOSHI,2 H. KURATA,2 and T. SAKOU1 1 2
Department of Orthopaedic Surgery, Faculty of Medicine, Kagoshima University, Kagoshima, Japan Department of Physiological Sciences and 3Department of Health Education, National Institute of Fitness and Sports, Kagoshima, Japan
stimulation of bone formation should be employed at least to prevent the bone loss resulting from estrogen deficiency. Bisphosphonates have a high binding affinity for hydroxyapatite, and inhibit osteoclastic bone resorption. They also interfere with the differentiation, fusion, and migration of osteoclast precursors to the bone surface.3,13,19 It is, in fact, reported that the bisphosphonates increase bone mineral density (BMD) in all skeletal sites and reduce the incidence of bone fracture in estrogendeficient women.23 Furthermore, it has been reported that longterm treatment inhibits the mineralization of the bone, and several studies have demonstrated effective protocols of the administration of bisphosphonates on the restoration and maintenance of bone mass. For example, intermittent cyclic administration of bisphosphonates has been shown to preserve the mass, architecture, and mechanical properties of ovariectomized rats.21 Jee et al.20 employed the lose, restore, and maintain (LRM) concept that uses ovariectomy to induce bone loss, an anabolic agent to stimulate bone formation, and then switches to an antiresorptive agent to inhibit bone resorption. Moreover, it has been reported that exercise training enhances bone formation in rats and humans.2,6,12 Exercise therapy that loads the mechanical stress to the bone is also effective in maintaining bone mineral density in early postmenopausal women.11 It has been suggested that run training increases the BMD by improving mechanical stress on the bone and inducing hypertrophy of the cortical bone in experimental animals.26,28 Therefore, it is conceivable that the therapy that switches the treatment from etidronate to exercise training might be effective in preventing bone loss caused by an estrogen deficiency in ovariectomized rats. The purpose of this study was to assess the effects of exercise training following an etidronate treatment on BMD of the femur and the trabecular bone of the tibia in ovariectomized rats.
This study was designed to assess the effects of exercise training (Tr) following an etidronate treatment (E) on bone mineral density (BMD) of the femur and trabecular bone of the tibia in ovariectomized (ovx) rats. Female Wistar rats were ovariectomized (ovx) or sham-operated (sham) at 15 weeks of age and divided into five experimental groups: sham; ovx; ovx 1 E; ovx 1 Tr; ovx 1 E 1 Tr. Etidronate treatment of 5 mg/kg, 5 days/week was administered for 2 weeks and exercised on a treadmill for 30 m/min, 60 min/day, 5 days/week for 10 weeks. BMD of the femur and the trabecular bone area of the proximal tibia were significantly (p < 0.05) higher in E and/or Tr compared to ovx groups. However, the cortical region was not affected significantly by ovariectomy. The area partially filled with the trabecular bone at the constant width was observed only in the E rats. The number of osteoclasts in E group was significantly lower (p < 0.05) than in the ovx and ovx 1 Tr groups. The ovx 1 Tr rats had a higher number of osteoblasts (p < 0.05) than the ovx and ovx 1 E groups. There was a significant interaction between ovx 1 Tr and ovx 1 E on BMD in the proximal region of the femur (p < 0.05) and trabecular bone area of the tibia (p < 0.001). These results suggest that the etidronate treatment for 2 weeks beforehand influenced the effects of subsequent exercise training on maintaining the BMD in the proximal femur and the trabecular bone area of the tibia. (Bone 23:147–153; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: Etidronate; Exercise; Ovariectomy; Bone mineral density; Osteoclast; Osteoblast. Introduction Postmenopausal osteoporosis is a serious problem in elderly women and is characterized by a decrease in bone mass, leading to fractures and imbalanced turnover of the bone.24 Ovariectomized (ovx) rats exhibit a decrease in mineral density, volume, and strength of bone and an increase in bone turnover rate seen in women suffering from osteoporosis.21 Therapy that involves either inhibition of bone resorption or
Materials and Methods Experimental Animals Thirty-five female Wistar rats were maintained under constant temperature (25 6 2°C) and humidity (55% 6 5%), and under 12 h/12 h light– dark cycles. The rats were housed individually in standard cages and provided with a commercial standard diet (Kyudo, Japan) containing 1.2% calcium and 0.8% phosphorus. The food consumption of the rats was controlled to minimize the difference among groups in body weight associated with ovariectomy and exercise training. The food consumption of the rats
Address for correspondence and reprints: Hiroyuki Tamaki, National Institute of Fitness and Sports, Department of Physiological Sciences, 1 Shiromizu, Kanoya, Kagoshima 891-23, Japan. © 1998 by Elsevier Science Inc. All rights reserved.
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Table 1. The body weight and uterine weight in each group Group Body weight (g)
15w 28w
Uterine weight (mg)
Sham
Ovx
Ovx 1 Tr
Ovx 1 E
Ovx 1 E 1 Tr
263.3 6 4.7 324.3 6 5.7 601.5 6 81.5
271.7 6 6.5 338.3 6 8.8 206.6 6 33.9*
271.3 6 8.7 333.7 6 12.1 220.6 6 38.2*
266.0 6 4.8 343.3 6 6.4 191.8 6 48.4*
277.7 6 6.8 339.0 6 8.8 203.2 6 70.5*
*p , 0.0001, significantly different from the sham group (ANOVA). Sham: sham-operation; OVX, ovariectomy; E: etidronate treatment; Tr: exercise training.
that showed a tendency for higher increases in body weight after ovariectomy was restricted.30,31 At 15 weeks of age, animals were either ovx or sham-operated (sham). Bilateral ovariectomies were performed under anesthesia with sodium nembutal administered intraperitoneally. At necropsy, the uterus of each rat was removed and weighed. No detection of ovarian tissue and atrophy of the uterine horns were observed in the ovariectomized rats.31 (Table 1) Sham operations were performed by exteriorizing the ovaries. Ovx rats were divided into four groups based on treatment with either etidronate (E) or physiological saline and exercise training (Tr) as follows: physiological saline-treated group (ovx); etidronate-treated group (ovx 1 E); physiological saline-treated and exercise-trained group (ovx 1 Tr); and ovariectomized plus etidronate-treated and exercise-trained group (ovx 1 E 1 Tr). All procedures using animals were carried out in accordance with the guidelines presented in the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, published by the Physiological Society of Japan, and were approved by the National Institute of Fitness and Sports Animal Care Committee. Etidronate and Saline Treatment Etidronate disodium was dissolved in a saline vehicle. Etidronate and saline treatment were initiated 1 week after surgery. The rats in the ovx 1 E and ovx 1 E 1 Tr groups were treated 5 days/week for 2 weeks with subcutaneous injections of etidronate (Sumitomo Pharmaceuticals, Japan) at a dose of 5 mg/kg body weight.7,21,31 The rats in the ovx, ovx 1 Tr, and sham-operated groups were injected subcutaneously 5 days/week for 2 weeks with the saline vehicle used to dissolve the etidronate (Figure 1). Training Program The rats in the training group were given a program of running exercise on a motor-driven treadmill (Model SN-460, Shinano,
Japan) for a total of 10 weeks (18 –28 weeks of age). The time of this daily exercise and the running speed were increased gradually during the first 2 weeks of training. Following this adaptation period, the training was carried out under constant conditions (speed: 30 m/min; time: 60 min/day; frequency: 5 days/ week) until the end of the training period (Figure 1). Bone Mineral Density Measurements The bone mineral density of the femurs was measured using dual energy X-ray absorptiometry (Norland XR-26, Ft. Atkinson, WI). Each femur was placed on an acrylic board (25 mm in thickness). Scans were performed at random between groups at a scan speed of 10 mm/s and repeated three times to assess the reproducibility of the measurement system. The BMD of the femurs was measured and three regions of the femurs (proximal third, middle third, and distal third) were assigned for analysis to assess any regional differences in bone characteristics. Each scan was analyzed by using small animal software (Norland). Measurements using a lumbar spine anthropomorphic photon were performed to check the quality of the device. The coefficient of variation for this measurement technique in our laboratory was 0.9%. These measured values of bone mineral density were obtained and analyzed by the same examiner and on the same day. Tissue Preparation and Bone Histology The rats were anesthetized with sodium nembutal and immediately perfusion-fixed through the abdominalis aortae with a mixture of 1% glutaraldehyde, 1% formaldehyde, and 0.05% CaCl2 in 0.1 mol/L sodium cacodylate buffer (pH. 7.3) for 30 min at room temperature. After fixation, the femurs were removed and preserved in physiological saline until the determination of bone mineral density. The tibias were demineralized in 0.1 mol/L disodium ethylenediaminetetraacetic acid (pH 7.3) for 6 weeks at 4°C, dehydrated through a graded ethanol series, and
Figure 1. Experimental protocol. Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
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then embedded in paraffin. Longitudinal sections of 5 mm thickness were cut using a microtome. Specimens were stained with the media of azocalmin–anilinblue (Azan), tartrate resistant acid phosphatase (TRAP), or methylgreen-pyronine for counting the volume of bone, the number of osteoclasts, or the number of osteoblasts at the secondary spongiosa. The trabecular bone area was determined manually using a grid-point counting technique, and was expressed as a percentage (points on bone / points on tissue inside the measurement area).14 The micrographs used for the measurement of trabecular bone area were taken at a magnification of 3400.30 Trabecular bone measurements were performed at a 6-mm2 area, 1 mm distal from the growth plate– cancellous bone junction of the proximal tibia to exclude the primary spongiosa.30 The numbers of osteoclasts, identified on the bone surface by TRAP activity, were counted per square millimeter of bone area. The number of osteoblasts, characterized by a centrally located negative Golgi image within the basophylic cytoplasm and eccentrically located nucleus, was counted within a 6-mm2 area under the microscope at a magnification of 340. Statistics Results are expressed as the mean 6 standard deviation (SD). The significance of differences between groups was calculated by an analysis of variance (ANOVA), followed by Fisher’s PLSD test. A two-factor ANOVA was used to examine the individual effect and interaction between the exercise and etidronate treatment. The level of significance was set at p , 0.05. Results Bone Mineral Density The influences of the ovariectomy, etidronate treatment, and/or exercise training on BMD in the total and regional femur are shown in Figure 2. In the proximal and distal region, the BMD was significantly lower (p , 0.05) in the ovx than in the sham group. BMD levels were significantly higher (p , 0.05) in the ovx 1 E 1 Tr, ovx 1 E, and ovx 1 Tr groups compared with the ovx group. A two-factor ANOVA showed that the effects of exercise and etidronate treatment on BMD in the proximal femur were significant (p , 0.01), and the interaction between exercise and etidronate factors was significant (p , 0.05). In the middle region, BMD levels in the ovx 1 E 1 Tr, ovx 1 E, and ovx 1 Tr groups were significantly higher (p , 0.05) than that in the ovx group. The effects of exercise and etidronate treatment on BMD in the middle femur were also significant (p , 0.05), but there was no interaction dependence. In the distal region, ovx 1 E 1 Tr, ovx 1 E, and ovx 1 Tr groups exhibited significantly higher levels (p , 0.05) of BMD compared with the ovx group. Exercise affected the BMD in the distal femur (p , 0.05), but no interaction was observed between the exercise and etidronate treatment factors. Trabecular Bone Area The different histological areas of the proximal tibia sections from sham, ovx, ovx 1 E, ovx 1 Tr, and ovx 1 E 1 Tr rats are depicted in Figure 3. As expected, the loss of trabecular bone was observed in ovariectomized rats, and was most obvious in the central metaphysial region. The area partially filled with trabecular bone (the thick region) was observed only in the etidronate-treated rats, which exhibited bone of a constant width. In the etidronate-treated rats, the trabecular bone in the area between the growth plate–metaphysial junction and the thick
Figure 2. Effects of etidronate treatment and exercise training on bone mineral density in the proximal (A), middle (B), and distal (C) femur of ovariectomized rats. The values expressed as the mean 6SD. *p , 0.05, significantly different from the ovx group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
region of the exercise-trained rats was maintained compared with that of the nontrained rats. Conversely, the loss of trabecular bone in the area between the growth plate–metaphysial junction and the thick region was remarkable in the nontrained rats. The trabecular bone area (%) was measured and compared among the five experimental groups (Figure 4). As expected, the trabecular bone area was significantly (p , 0.05) lower in the ovx than in the sham group. However, the trabecular bone area in the etidronate-treated and/or exercise-trained rats was maintained at the level observed in the sham-operated rats. The combination of both the etidronate treatment and exercise training resulted in the greatest level of trabecular bone area of all of the groups. The trabecular bone area in the ovx 1 E 1 Tr group was significantly (p , 0.05) greater than in the ovx 1 Tr group. The effects of exercise and etidronate treatment on the trabecular bone area in the tibia were significant (p , 0.0001), as was the interaction between these factors (p , 0.001). The osteoclasts located on trabecular bone surfaces and the mean number of osteoclasts per unit area (mm2) of trabecular bone in the five experimental groups are shown in Figures 5 and 6. The number of osteoclasts per bone area was affected by ovariectomy or by etidronate treatment and exercise training of
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Figure 3. Photographs of proximal tibia mataphysis from the sham (A), ovx (B), ovx 1 exercise training (C), ovx 1 etidronate treatment (D), and ovx 1 exercise training 1 etidronate treatment (E) groups. A dramatic bone loss was observed in the ovx (B) compared with the sham (A) group. Azan stain, original magnification 320. Bar 5 1 mm.
the ovx rats. There was a significant difference (p , 0.05) between the ovx group and the sham group. The decreases in osteoclast numbers were caused by treatment with etidronate and exercise training in the ovx rats. The number of osteoclasts in the ovx 1 E and the ovx 1 E 1 Tr groups was significantly fewer (p , 0.05) than in the ovx group. However, exercise training did not affect the number of osteoclasts dramatically. The ovx 1 Tr group exhibited a significantly greater number of osteoclasts (p , 0.05) compared with both the ovx 1 E and the ovx 1 E 1 Tr groups, but did not significantly differ from the sham group. The number of osteoblasts was significantly higher (p , 0.05) in the ovx 1 Tr and the ovx 1 E 1 Tr groups than in the ovx and ovx 1 E groups (Figure 7). The effects both of etidronate treatment on osteoclast number (p , 0.001) and exercise training on osteoblast number were significant (p , 0.01), but there was no interaction between the exercise and etidronate treatment factors. Discussion Regional differences in the response to ovx were observed. The proximal region of the femur showed a lower BMD in ovx rats, but a nonsignificant influence in the middle region of the femur.
Figure 4. Effects of etidronate treatment and exercise training on trabecular bone area in the proximal tibia of ovariectomized rats. The values expressed as the mean 6SD. ap , 0.05, significantly different from the ovx group; bp , 0.05, significantly different from ovx 1 Tr group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
Similar results have been reported in adult ovx rats.15 Long bones, such as the femur, have a greater proportion of trabecular bone in their proximal and distal regions and a greater proportion of cortical bone in their middle region. Ovx may have more of an effect on the trabecular bone region than on the cortical bone region on BMD in the initial period following ovariectomy. In aged rats, however, a clear reduction in the cortical bone regions has been observed following ovx.8 It is conceivable that there is a time lag for the response to ovariectomy in the different regions of the long bone. Many drugs that prevent such ovx-induced bone loss have been investigated. Bisphosphonates are known to strongly inhibit bone resorption. In postmenopausal women, bisphosphonate treatment has been shown to increase BMD in all skeletal sites, and to decrease the incidence of vertebral fractures.23 In ovariectomized rats, treatment with bisphosphonate etidronate preserves trabecular bone volume and BMD in the vertebrae, femur, and tibia.21 In the study reported here, ovariectomized rats treated with etidronate maintained BMD in the femur and trabecular bone in the tibia to levels observed in sham-operated rats. Pharmacologically, the bisphosphonates possess a high binding affinity for skeletal calcium and prevent bone crystal dissolution.13 In addition to their indirect effects on osteoclasts, these drugs impair the resorptive function of osteoclasts. It has been suggested that bisphosphonates interfere with the differentiation, migration, and fusion of immature osteoclasts.3 These reports support our results that the number of osteoclasts were increased after ovariectomy, but that etidronate treatment caused a reduction in their number below the level observed in sham-operated rats. Bisphosphonates may act to prevent the trabecular bone loss and reductions in BMD through inhibition of osteoclast-mediated resorption and the prevention of bone crystal dissolution. On the other hand, an adverse effect of the etidronate treatment has been reported: long-term doses inhibit calcium phosphate crystallization and result in an inhibition of mineralization of the bone and the development of fractures.27 In the present study, etidronate treatment influenced the BMD of the femur, the trabecular bone area of the tibia, and the number of osteoclasts. It is possible that the etidronate treatment in this study maintained the BMD of the femur and trabecular bone of the tibia by preventing bone resorption. The level of physical activity is one of the important factors
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Figure 5. Light micrographs showing proximal metaphysis of tibias in the sham (A), ovx (B), ovx 1 Tr (C), ovx 1 E (D), and ovx 1 E 1 Tr (E) rats. The osteoclast (arrows) number appears to be greater in ovx rats compared with that in the sham. TRAP stain, original magnification 3200. Bar 5 50 mm.
that determines bone mass. For example, weightlessness resulting from spaceflight also decreases bone mineral density and bone mass and remarkably inhibits bone formation in rats.29 Conversely, it has been reported that the degree of bone mineral content is extremely high and is closely correlated to the amount of exercise performed by athletes.17 In postmenopausal women, those who are physically active also have a higher bone mineral content than those who are inactive.11 These reports suggest that mechanical stress to the bone caused by weight loading and muscle contractions strongly affects bone formation and resorption. Mechanical stress to the bone can generate strain-related potentials that affect bone formation.9 Rubin and Lanyon26 have reported that the application of consecutive external loads to a functionally isolated long bone in vivo prevents disuse atrophy of the cortical bone and increases bone mineral content, and that further increases in bone formation did not occur no matter how much mechanical stress was imposed. On the other hand, exercise-induced endocrine changes also may have an effect on bone metabolism. Physical activity such as running or weight lifting stimulates the secretion of sex hormones, growth hormones, and catecholamines,18 and increases the bone formation resulting from an increase in osteoblastic recruitment and in the activity of
Figure 6. Effects of etidronate treatment and exercise training on the number of osteoclasts per unit area in the proximal tibia. The values expressed as the mean 6SD. ap , 0.05, significantly different from the ovx group; bp , 0.05, significantly different from ovx 1 Tr group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
individual osteoblasts in rats and humans.2,6,12 It can be speculated that the increase in mechanical stress and/or secretion of hormones stimulating bone formation caused by running exercise might prevent ovariectomy-induced trabecular bone loss observed in the present study. However, it is not clear whether the exercise training effect was due to an increase in bone formation resulting from the higher activity of individual osteoblasts. Partial effects of exercise on preventing the loss of trabecular bone and calcium content have been reported.25,32 The estimated exercise intensity based on treadmill speed and grade correspond ˙ O2max (maximum oxygen consumption) to approximately 50% V in these study.6,22 Some studies report the relationship between exercise intensity and bone mass. It is reported that high-intensity ˙ O2max) reduces longitudinal bone growth and exercise (80% V increases bone loss in rats.5 The rats trained at low intensity ˙ O2max) showed a decreased trabecular bone volume of (40% V the femur.4 Thus, Bourrin et al.5,6 suggested that exercise inten˙ O2max and above 80% V ˙ O2max do not sities below 40% V improve bone loss in rats. Exercise intensity during our experi˙ O2max, according to the measurement would be about 60% V
Figure 7. Effects of etidronate treatment and exercise training on the number of osteoblasts per unit area in the proximal tibia. The values expressed as the mean 6SD. ap , 0.05, significantly different from Sham group; bp , 0.05, significantly different from the ovx group; cp , 0.05, significantly different from ovx 1 E group (ANOVA). Sham: sham operation; OVX: ovariectomy; E: etidronate treatment; Tr: exercise training.
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ment of Bourrin et al.6 Barengolts et al.1 reported that biomechanical and morphometric properties of the femur and bone mineral of the tibia and femur in ovx and exercised (approxi˙ O2max) rats maintained the level of sham-operated mately 60% V rats. The setting of the exercise intensity might be an important factor to obtain an optimal effect of exercise training even within the effective range of exercise intensity. In addition to the individual efficacies of the therapy of the etidronate or the exercise alone, it is important to understand the interaction of the two. Jee et al.20 employed the LRM concept, which utilizes the anabolic agent to restore bone mass and then switches to the antiresorptive agent to maintain bone mass in ovx rats through the lose phase, for 5 months. This therapy was successful in maintaining trabecular bone mass and structure in the proximal tibia. It is reported that the combined exercise and bisphosphonate treatment reduces the calcium loss and prevents some of the changes in mineral metabolism in humans.16 The present study employed therapy that used exercise training subsequent to etidronate treatment in ovx rats. Etidronate treatment for 2 weeks preserved trabecular bone, as shown in the bone area filled with trabecular bone (the thick region) of the proximal tibia, and resulted in maintaining the trabecular bone structure. Ovx rats that were exercise trained for 10 weeks after etidronate treatment maintained their trabecular bone in the area between the growth plate–metaphysial junction and the thick region; those rats that were not trained did not. The ovariectomized rats that were treated with both etidronate and exercise training (ovx 1 E 1 Tr) exhibited the highest BMD in the proximal and middle regions of the femur and trabecular bone area of the tibia of all of the experimental groups in the present study. The effect of subsequent exercise training on maintaining the trabecular bone of the tibia was influenced by etidronate treatment beforehand in ovx rats. Taking into account these results and those of the report showing a close correlation between BMD and mechanical strength,10 it would be conceivable that a therapy consisting of exercise training following etidronate treatment would maintain bone mechanical strength and lead to a decrease in the incidence of bone fractures in postmenopausal women. In conclusion, our results indicate that the individual treatment of etidronate or exercise training prevented the loss of trabecular bone of the tibia and BMD in some regions of the femur induced by ovx. Exercise training influenced the number of osteoblasts, but not the number of osteoclasts. Furthermore, there were significant interactions between exercise and etidronate treatment on BMD in the proximal region of the femur and trabecular bone area of the tibia. This indicates that, in ovx rats, the effect of subsequent exercise training on maintaining BMD and trabecular bone in some regions is influenced by administering etidronate treatment for 2 weeks prior to exercise training.
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Acknowledgments: The authors sincerely appreciate the invaluable comments and encouragement from Dr. V. Reggie Edgerton of University of California, Los Angeles. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.
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Date Received: October 9, 1997 Date Revised: April 2, 1998 Date Accepted: April 7, 1998