Additive effects of combined treatment with etidronate and alfacalcidol on bone mass and mechanical properties in ovariectomized rats

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Additive Effects of Combined Treatment With Etidronate and Alfacalcidol on Bone Mass and Mechanical Properties in Ovariectomized Rats T. NISHIKAWA,1 S. OGAWA,2 K. KOGITA,2 N. MANABE,1 T. KATSUMATA,2 K. NAKAMURA,1 and H. KAWAGUCHI1 1

Department of Orthopaedic Surgery, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Sumitomo Pharmaceuticals Research Center, Osaka, Japan

2

Introduction The present study was undertaken to evaluate the effects of combined treatment with intermittent cyclical etidronate and daily alfacalcidol on the mass and the mechanical properties of bone in ovariectomized rats, and to compare the effects with those of single treatments. Seventy 14-week-old female rats underwent ovariectomy (ovx) or sham operation, and were assigned to seven groups (n ⴝ 10 each): sham-operated; ovx; ovx treated with etidronate; ovx treated with 0.1 ␮g/kg alfacalcidol; ovx treated with 0.2 ␮g/kg alfacalcidol; ovx treated with etidronate and 0.1 ␮g/kg alfacalcidol; and ovx treated with etidronate and 0.2 ␮g/kg alfacalcidol. One week after the operation, etidronate (4 mg/kg per day) was intermittently injected into rats for 2 weeks followed by a 10 week period of no treatment, and alfacalcidol was administered orally every day. After 24 weeks of treatment, all single and combined treatments increased the bone mineral densities (BMDs) of the proximal tibiae, midfemurs, and the fourth and fifth vertebral bodies, which had been decreased by ovx. Combined treatment groups showed higher BMDs than single treatment groups, and the effects were almost equal to the addition of those of respective single treatment groups. The combined treatment also showed additive effects on the mechanical properties of both midfemurs and L4 vertebral bodies. The increases in mechanical properties were proportional to those in BMDs. Analyses of microcomputed tomography images and histology confirmed the strong effects of combined treatments on both trabecular and cortical bone mass without impairment of mineralization or connectivity. We conclude that the combined treatment with etidronate and alfacalcidol additively increases the mass of bone with normal quality, resulting in bone strengthening in ovx rats. (Bone 27:647– 654; 2000) © 2000 by Elsevier Science Inc. All rights reserved.

Among individuals who may develop osteoporosis, women are considered to be at highest risk due to estrogen deficiency that occurs abruptly at menopause. At present, postmenopausal osteoporosis is treated with several drugs with bone-sparing effects, and combinations of two different drugs are sometimes used clinically. However, the effects of combined treatments have been rarely investigated, although accumulated evidence has revealed the effects of a single treatment with these drugs on postmenopausal bone loss. Bisphosphonates, potent antiresorptive agents, are known to be among the most reliable and prevalent drugs in osteoporosis treatment. Studies have suggested that intermittent cyclical administration of the first generation of bisphosphonates, etidronate (disodium ethane-1-hydroxy-1,1-bisphosphonate), can significantly improve bone mass in postmenopausal women7,15,26,27,41,44,46 and in corticosteroid-induced osteoporosis patients.1 Long-term treatment with intermittent cyclical etidronate also preserves trabecular structure without negatively affecting bone mineralization.43 Intermittent cyclical etidronate has also been reported to increase bone mass47 and mechanical properties21 in ovariectomized (ovx) rats. The modes of action of etidronate include the inhibition of osteoclastic bone resorption3,4 and suppression of the differentiation of osteoclast precursors,16 resulting in a decrease in bone turnover. Recent studies have shown that bisphosphonates that closely resemble pyrophosphate (such as etidronate and clodronate) can be metabolically incorporated into nonhydrolyzable analogs of adenosine triphosphate (ATP) that may inhibit ATP-dependent intracellular enzymes, whereas the more potent nitrogen-containing bisphosphonates can inhibit enzymes of the mevalonate pathway.39 There is also increasing evidence for the role of vitamin D and vitamin D analogs in treating osteoporosis,32 and the activated form of vitamin D [calcitriol, 1,25(OH)2D3] has been shown to significantly improve bone mass and reduce vertebral fracture rates in postmenopausal women.5,11,45 Alfacalcidol [1␣(OH)D3], a prodrug of calcitriol, is metabolized into 1,25(OH)2D3 not only in the liver but also in osteoblastic cells locally.17,34 This agent exhibits a bone-sparing effect similar to that of calcitriol, and is prevalently used for the treatment of postmenopausal osteoporosis, especially in Japan and Europe.8,10,25,37,38 Alfacalcidol has been reported to prevent bone loss and increase mechanical properties in ovariectomized rats, although its mechanism is still controversial.2,18,23,36,40 Alfacal-

Key Words: Combined treatment; Etidronate; Alfacalcidol; Ovariectomy (ovx); Osteoporosis; Bone.

Address for correspondence and reprints: Hiroshi Kawaguchi, M.D., Department of Orthopaedic Surgery, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.

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8756-3282/00/$20.00 PII S8756-3282(00)00386-0

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cidol as well as calcitriol is known to increase bone mass, possibly by modulating bone turnover or increasing the intestinal absorption of calcium.12 These two agents, etidronate and alfacalcidol, which seem to show different actions in preventing bone loss, are major drugs used in the clinical treatment of postmenopausal osteoporosis. In this study, we investigate the effects of combined treatment with etidronate and alfacalcidol on the mass and mechanical properties of bone in ovx rats, and compare these effects with those of single treatment with each drug. Materials and Methods Experimental Design All animal experiments were performed according to the guidelines of the International Association for the Study of Pain.48 In addition, the experimental work was reviewed by the committees of Tokyo University and Sumitomo Pharmaceuticals charged with confirming ethics. Seventy female Wistar rats, 12 weeks of age, were purchased (Charles River Japan, Tokyo) and acclimated to conditions for 2 weeks before the experiment. The rats underwent ovariectomy (n ⫽ 60) or sham operation (n ⫽ 10) at the age of 14 weeks, and were assigned to one of seven groups: sham-operated (sham, n ⫽ 10); ovariectomized (ovx, n ⫽ 10); ovx treated with etidronate (E, n ⫽ 10); ovx with 0.1 ␮g/kg alfacalcidol (LD, n ⫽ 10); ovx with 0.2 ␮g/kg alfacalcidol (HD, n ⫽ 10); ovx with etidronate and 0.1 ␮g/kg alfacalcidol (E ⫹ LD, n ⫽ 10); or ovx with etidronate and 0.2 ␮g/kg alfacalcidol (E ⫹ HD, n ⫽ 10). Treatments began 1 week after operation. The duration of each cycle of the therapeutic regimen for etidronate was 12 weeks. Four milligrams per kilogram body weight (b.w.) of etidronate (E, E ⫹ LD, and E ⫹ HD) or distilled water vehicle (sham, ovx, LD, and HD) was subcutaneously injected 5 days each week for 2 weeks, followed by a drug-free period of 10 weeks. The total duration of the experiment, which consisted of two cycles of the regimen, was 24 weeks. Alfacalcidol (Wako Pure Chemical, Tokyo) or medium-chain triglyceride vehicle alone was administered orally at the respective doses of 0.1 or 0.2 ␮g/kg (b.w.) 5 days each week for 24 weeks. Standard rat chow containing 1.25% calcium, 1.06% phosphorus, and 200 IU/100 g of vitamin D3 (CE-2; Japan Clea Corp., Tokyo) was fed to all animals. The rats were housed in plastic cages at a room temperature of 23 ⫾ 2°C and humidity of 55 ⫾ 10%, and were allowed free access to the diet and water during the experimental period. At the end of this period, the animals were anesthetized with ether, killed by exsanguination, and samples of blood were taken. The femur, tibia, and fourth and fifth lumbar (L4 and L5) vertebral bodies were removed.

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dual-energy X-ray absorptiometry (DEXA; DCS-600, Aloka, Tokyo). For tibial and femoral specimens, BMDs of the proximal region (7 mm in length from the proximal end) and the middiaphyseal region (13 mm in length spanning the midportion) were measured, respectively. The L4 and L5 vertebral bodies were isolated by removing posterior elements. BMD of the L5 vertebral body was measured with anteroposterior application of the radiation beams to the specimen. For normalization of the mechanical properties by BMD, the BMD of the L4 vertebral body was measured in a different manner. The L4 vertebral body specimen was fixed with a clump at the bases of the transverse processes in the holder of a diamond band saw (Exakt, Norderstedt, Germany). By removing the cranial and caudal ends of the specimens, a central cylinder with planoparallel ends and a height of approximately 5 mm was obtained.29 Radiographs were taken of the specimens to confirm the removal of the cartilaginous growth plates and the primary spongiosa. After trimming, the BMD of the L4 vertebral body was measured by craniocaudal application of radiation beams to the specimen. Mechanical Properties After measuring the BMD, a three-point bending test was performed on the midfemur as previously described28 using a load torsion tester (Tensilon RTA-500, Orientic, Tokyo). The femur was placed on a special holding device with supports located 13 mm apart, with the lesser trochanter proximal to, and in contact with, the proximal transverse bar. The bending force was applied by the crosshead at a speed of 2 mm/min until fracture occurred. The L4 vertebral body specimen was trimmed and its BMD was measured as described earlier. The vertebral body samples were placed in the center of the smooth surface of a steel disk (10 cm in diameter) attached to the load torsion tester, and a craniocaudal compression force was applied to the specimen by a steel disk (2 cm in diameter) at a nominal deformation rate of 2 mm/ min.30,31 For both bones, the breaking strength (newtons) and structural stiffness (newtons per millimeter) were determined as previously reported from the load-deformation curve, and continually recorded in the computerized monitor linked to the tester.33 Relationship Between BMD and Mechanical Properties

Blood samples were obtained at the end of the treatment period. The serum calcium level was measured by the orthocresolphthalein complexon method and serum phosphorus by the method of Fiske–Subbarow. The serum osteocalcin level was measured by radioimmunoassay using goat anti-rat osteocalcin antibody (Biomedical Technologies, Inc., Stoughton, MA).

For the normalization of bone strength by bone density, the breaking strength values of the midfemur and L4 vertebral body were divided by respective BMD values for each rat, and mean values of the groups were compared. Power-law regression analyses of the relationships of the breaking strength to BMD values of the L4 vertebral body were also made. The relationship between the values of the breaking strength and BMD was analyzed by the equations: S ⫽ C ⫻ Da, where S is the breaking strength, D is the BMD, and a is the experimentally derived value for the power relationship, and C is the experimentally derived constant value. Because this equation can also be written as logS ⫽ a ⫻ logD ⫹ logC, the regression analyses were performed between logS and logD. The values for regression slope (a), correlation coefficient (r), and correlation significance (p) were obtained.

Bone Mineral Density

Microcomputed Tomography Analyses

After removing the adhering soft tissues, the lengths of the tibia and femur and the heights of the L4 and L5 vertebral bodies were measured with a micrometer. Bone mineral densities (BMDs; milligrams per square centimeter) of the proximal tibia, the midfemur, and L4 and L5 vertebral bodies were measured by

A microcomputed tomography (micro-CT) scan of the femur and L4 vertebral body specimens of each group was taken using a composite X-ray analysis system (NS-Elex, Tokyo). Femur specimens were evaluated at the midportion and 10 mm from the distal end by cross-sectional analysis, and L4 vertebral body

Blood Chemistry

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Table 1. Body weight and blood chemistry data (mean ⫾ SEM, n ⫽ 10/group) Body weight Pre (g)

Post (g)

Serum calcium (mg/100 mL)

259.4 ⫾ 4.6 270.7 ⫾ 4.0 269.0 ⫾ 2.9 272.2 ⫾ 4.0 267.9 ⫾ 4.0 270.0 ⫾ 4.4 268.2 ⫾ 3.8

335.9 ⫾ 7.9b 464.0 ⫾ 13.6 459.0 ⫾ 9.3 453.4 ⫾ 14.5 409.9 ⫾ 8.5d 414.1 ⫾ 12.9c 415.7 ⫾ 16.6

11.0 ⫾ 0.19 10.6 ⫾ 0.18 10.6 ⫾ 0.12 12.1 ⫾ 0.48d 12.9 ⫾ 0.36d 11.9 ⫾ 0.26d,e 11.9 ⫾ 0.14d,e,f

Group Sham Ovx E LD HD E ⫹ LD E ⫹ HD

Serum inorganic phosphorus (mg/100 mL)

Serum osteocalcin (ng/mL)

4.62 ⫾ 0.15 4.32 ⫾ 0.15 4.29 ⫾ 0.26 6.81 ⫾ 0.40d 7.40 ⫾ 0.36d 6.34 ⫾ 0.25d,e 6.65 ⫾ 0.29d,e

21.0 ⫾ 1.26a 25.0 ⫾ 1.31 21.3 ⫾ 0.73c 31.3 ⫾ 2.33 32.6 ⫾ 1.71c 31.1 ⫾ 2.12e 39.6 ⫾ 3.90d,e

KEY: E, etidronate; HD, high-dose alfacalcidol; LD, low-dose alfacalcidol; ovx, ovariectomy. Significantly different vs. ovx: ap ⬍ 0.05, bp ⬍ 0.01 (Student’s t-test); Significantly different vs. ovx: cp ⬍ 0.05, dp ⬍ 0.01 (analysis of variance [ANOVA] Tukey–Kramer). Analysis of combined treatment groups vs. respective single treatment groups: significantly different vs. E: ep ⬍ 0.01 (ANOVA, Tukey–Kramer); significantly different vs. HD: fp ⬍ 0.05 (ANOVA, Tukey–Kramer).

specimens were measured at the center by sagittal section. For the quantitative analysis of trabecular bone, the trabecular bone area (BV/TV; percent) and trabecular thickness (Tb.Th; microns) were measured on CT images of distal femurs. For structural analyses of the trabecular bone, the numbers of nodes (N.Nd/TV) and termini (N.Tm/TV) of the bone were counted on the images, and specimens were analyzed in terms of the connectivity of trabecular bones represented by the ratio of nodes to termini (N.Nd/N.Tm) according to the method of Garrahan et al.13 The cortical bone was analyzed by measuring the cross-sectional total area and bone area on CT images of the midfemurs. Histology Tibiae of ovx, E, E ⫹ LD, and E ⫹ HD rats were fixed with 100% ethanol for 7 days and immersed in Villanueva Goldner stain for 14 days. They were then embedded in methylmethacrylate, sectioned in 10 ␮m slices, and observed by light microscopy (⫻20). Statistical Analysis Data were expressed as the means ⫾ standard error of the mean (SEM). The differences between the values in sham and ovx groups were compared by Student’s t-test. The intergroup differences among ovx-treated groups were evaluated by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test. The relationship between the strength and each apparent

density value of the vertebral body was assessed using regression analysis. p ⬍ 0.05 was considered significant. Results Body Weight and Blood Chemistry There was no difference in the body weights of rats among any of the groups at the beginning of the experiment (Table 1). At the end of the experiment the mean body weight in the ovx group was significantly greater than that in the sham group, and none of the single or combined treatments fully restored the balance although treatments with HD and E ⫹ LD partially decreased it. Ovx did not affect the serum calcium and phosphorus levels significantly. Alfacalcidol treatment (LD, HD, E ⫹ LD, and E ⫹ HD) increased both serum levels, whereas etidronate did not affect them. The serum osteocalcin level was increased by ovx and reversed by etidronate to the level of sham; however, it was further increased by a single treatment with a high dose of alfacalcidol (HD) and by the combined treatments (E ⫹ LD and E ⫹ HD). Interestingly, etidronate did not decrease the serum osteocalcin level stimulated by alfacalcidol in the combined treatment groups. Bone Mineral Density There were no significant differences in the lengths of tibia and femur or in the heights of L-4 and L-5 vertebral bodies among

Table 2. Bone mineral densities of proximal tibia, midfemur, and vertebral body (mean ⫾ SEM, n ⫽ 10/group) Lumbar vertebral body Group

Proximal tibia

Midfemur

L-4

Sham Ovx E LD HD E ⫹ LD E ⫹ HD

171.3 ⫾ 2.0 152.7 ⫾ 3.0 177.4 ⫾ 3.8d 175.4 ⫾ 3.6d 186.7 ⫾ 5.5d 199.5 ⫾ 5.1d,e,f 227.3 ⫾ 6.7d,e,g

141.8 ⫾ 1.1 136.3 ⫾ 2.4 146.7 ⫾ 2.1c 149.8 ⫾ 1.7d 156.6 ⫾ 3.6d 157.3 ⫾ 4.3d 171.0 ⫾ 3.1d,e,g

76.9 ⫾ 1.8 64.4 ⫾ 1.7 83.0 ⫾ 1.1d 77.0 ⫾ 2.3d 83.5 ⫾ 2.8d 90.9 ⫾ 3.8d,f 95.3 ⫾ 2.6d,e,g

b

a

L-5 b

79.3 ⫾ 0.9b 68.1 ⫾ 2.2 83.2 ⫾ 1.5d 76.3 ⫾ 1.0c 87.6 ⫾ 4.2d 92.7 ⫾ 3.5d,f 96.2 ⫾ 4.1d,e

See Table 1 for abbreviations. Significantly different vs. ovx: ap ⬍ 0.05, bp ⬍ 0.01 (Student’s t-test); Significantly different vs. ovx: cp ⬍ 0.05, d p ⬍ 0.01 (ANOVA, Tukey–Kramer). Analysis of combined treatment groups vs. respective single treatment groups: Significantly different vs. E: ep ⬍ 0.01 (ANOVA, Tukey–Kramer); significantly different vs. LD: fp ⬍ 0.01 (ANOVA, Tukey–Kramer); significantly different vs. HD: gp ⬍ 0.01 (ANOVA, Tukey–Kramer).

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Table 3. Mechanical parameters of midfemur and L4 vertebral body (mean ⫾ SEM, n ⫽ 10/group) Midfemur Group Sham Ovx E LD HD E ⫹ LD E ⫹ HD

L4 vertebral body

Breaking strength (N)

Structural stiffness (N/mm)

Breaking strength (N)

Structural stiffness (N/mm)

131.4 ⫾ 2.7a 118.5 ⫾ 3.8 138.2 ⫾ 3.6d 134.8 ⫾ 2.4c 152.0 ⫾ 4.9d 147.6 ⫾ 4.2d 168.5 ⫾ 3.7d,f,g

436.1 ⫾ 13.9a 398.4 ⫾ 11.1 438.4 ⫾ 11.2c 431.7 ⫾ 17.3 482.7 ⫾ 14.3d 475.5 ⫾ 21.4c 509.5 ⫾ 21.9d,e

163.7 ⫾ 12.3 161.6 ⫾ 11.2 184.7 ⫾ 17.4 191.3 ⫾ 11.8 265.5 ⫾ 24.5d 243.0 ⫾ 17.0d 301.2 ⫾ 31.1d,f

1239 ⫾ 111 1233 ⫾ 98 1249 ⫾ 161 1196 ⫾ 109 1871 ⫾ 218c 1573 ⫾ 192 1888 ⫾ 232c

See Table 1 for abbreviations. Significantly different vs. ovx: ap ⬍ 0.05, bp ⬍ 0.01 (Student’s t-test); Significantly different vs. ovx: cp ⬍ 0.05, dp ⬍ 0.01 (ANOVA, Tukey–Kramer). Analysis of combined treatment groups vs. respective single treatment groups: Significantly different vs. E: ep ⬍ 0.05, fp ⬍ 0.01 (ANOVA, Tukey–Kramer); Significantly different vs. HD: gp ⬍ 0.05 (ANOVA, Tukey–Kramer).

the groups (data not shown). To examine the effects of single and combined treatments on bone mass, the BMDs of the proximal tibia, the midfemur, and L4 and L5 vertebral bodies were measured (Table 2). BMDs of these bones in the ovx group were significantly lower than those in the sham group, suggesting that estrogen deficiency for 24 weeks affected both trabecular and cortical bones. BMDs of all single treatment groups (E, LD, and HD) were significantly higher than those of the ovx group, and were similar to or greater than those of the sham group. No significant differences were observed among these three single treatment groups. Combined treatment groups (E ⫹ LD and E ⫹ HD) showed higher BMDs than single treatment groups and the sham group, and the effects were almost equal to the addition of those of respective single treatment groups. The additive effects of combined treatments as well as the effects of single treatments were seen not only in proximal tibiae and vertebral bodies but also in midfemurs, indicating that these treatments are beneficial for both trabecular and cortical bones. Similar results were obtained for BMDs of L4 and L-5 vertebral bodies whose sample preparation and direction of radiation beam were different. Mechanical Properties Effects of these treatments on bone strength of cortical and trabecular bones were examined by measuring the mechanical properties of the midfemur as determined by the three-point bending test and those of the L4 vertebral body as determined by the compression test (Table 3). The breaking strength and structural stiffness of the midfemur in the ovx group were significantly decreased compared with those in the sham group. Single treatments with etidronate or a high dose of alfacalcidol (E and HD) and combined treatments (E ⫹ LD and E ⫹ HD) significantly increased both parameters of the midfemur. Combined treatment groups showed higher mechanical properties than single treatment groups and the sham group, and the effects were almost equal to or slightly less than the addition of those of the respective single treatment groups. On the other hand, neither the breaking strength nor the structural stiffness of the L4 vertebral body was affected by ovx. A high dose of alfacalcidol and combined treatments, however, significantly increased the breaking strength in ovx rats. The effects of combined treatments on breaking strength were more than additive of those by respective single treatments. No treatment except that with a high dose of alfacalcidol (HD and E ⫹ HD) showed significant stimulation on the structural stiffness of the L4 vertebral body, although

combined treatments seemed to exhibit stronger effects than other treatments. Relationship Between BMD and Mechanical Properties To study the contribution of the effects of these treatments on BMD to those on mechanical properties in ovx rats, we compared the breaking strength values normalized by BMD values among the groups both in the midfemur and in the L4 vertebral body (Table 4). No significant difference in these normalized values was seen, suggesting a good correlation of increase in bone strength with that of bone density in both single and combined treatment groups. To investigate further the relationship between BMD and mechanical properties, power-law regression analyses of the relationship between breaking strength and BMD of the L4 vertebral body were carried out. In addition to the analysis of all groups, separate analyses were performed on the nontreated group (sham and ovx), etidronate-treated groups (E, E ⫹ LD, and E ⫹ HD), and alfacalcidol-treated groups (LD, HD, E ⫹ LD, and E ⫹ HD). In all groups, the etidronate-treated groups and alfacalcidol-treated groups, there were significant correlations between the values of BMD and the breaking strength of the L4 vertebral body (Figure 1 and Table 5). These results strongly suggest that the increase in bone strength is mainly due to the increase in bone mass by single and combined treatments with etidronate and alfacalcidol.

Table 4. Normalized breaking strength with bone mineral density in midfemur and L4 vertebral body Breaking strength/bone mineral density Group Sham Ovx E LD HD E ⫹ LD E ⫹ HD

Midfemur

L4 vertebral body

0.93 ⫾ 0.02 0.87 ⫾ 0.02 0.94 ⫾ 0.03 0.90 ⫾ 0.02 0.97 ⫾ 0.03 0.94 ⫾ 0.02 0.98 ⫾ 0.04

2.13 ⫾ 0.15 2.51 ⫾ 0.16 2.22 ⫾ 0.29 2.44 ⫾ 0.10 3.13 ⫾ 0.20 2.66 ⫾ 0.14 3.14 ⫾ 0.27

Data are expressed as mean ⫾ SEM (n ⫽ 10/group). No significant difference was seen among groups.

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Figure 1. Relationship between bone strength and BMD of the L4 vertebral body in all experimental groups, including the etidronate-treated groups (E, E ⫹ LD, and E ⫹ HD) and alfacalcidol-treated groups (LD, HD, E ⫹ LD, and E ⫹ HD). The L4 vertebral body specimen was trimmed to remove the cranial and caudal ends of the specimens, and its BMD was measured as described in Materials and Methods. A craniocaudal compression force was applied to the specimen by a steel disk, and the breaking strength was obtained from the monitored load-deformation curve. The correlation is expressed as the equation on each graph. The values of the regression slope (a), correlation coefficient (r), and correlation significance (p) are described in Table 5.

Micro-CT Analysis Representative pictures of micro-CT images of the distal metaphysis and the midportion of the femur, and those of the L4 vertebral body are shown in Figure 2. The decreases in trabecular bones were seen in both the distal femur (Figure 2A) and the vertebral body (Figure 2C) of ovx rats as compared with those of sham rats. All single and combined treatments increased trabecular bones in the distal femur and the vertebral body to levels similar to or greater than those in sham rats. In the combined treatment groups, the density of the trabecular bones was greater than in the other groups and parts of the bone marrow space of the distal femur appeared to be filled with compact bone. Morphometric analysis of CT images of the distal femur confirmed these findings (Table 6). Ovx decreased the bone volume (BV/ TV), and all single and combined treatments reversed it to levels similar to or greater than that of sham rats. The effects of combined treatments were significantly stronger than those of single treatments and were almost additive of those of single treatments with the respective agents. This result shows good Table 5. Regression analysis of strength of the L4 vertebral body as a function of bone mineral density

Group All groups Sham-ovx groups Etidronate-treated groups (E, E ⫹ LD, and E ⫹ HD) Alfacalcidol-treated groups (LD, HD, E ⫹ LD, and E ⫹ HD)

Exponent Correlation power coefficient Significance (a) (r) (p) 1.64 0.48 2.23

0.672 0.217 0.622

⬍0.0001 n.s. ⬍0.0001

1.84

0.765

⬍0.0001

KEY: n.s., not significant; ovx, ovariectomy.

correspondence to the results of BMD of trabecular bones in the proximal tibia and vertebral body (Table 2). No significant difference was seen in trabecular thickness except that HD and E ⫹ HD treatment increased it. The structural analysis determined by the ratio of the number of nodes to termini (N.Nd/ N.Tm) revealed that there was no significant difference in connectivity of trabecular bones among the groups. The total cross-sectional area of the midfemur did not differ among the groups, whereas the bone area and cortical thickness were decreased by ovx and reversed by all treatments (Figure 2B and Table 6). However, no significant difference was observed between single and combined treatments. Histology We examined whether or not the single and combined treatments with intermittent cyclical etidronate (E, E ⫹ LD, and E ⫹ HD) would impair normal mineralization (Figure 3). Histological analysis of the proximal tibiae by Villanueva Goldner staining revealed that these treatments did not impair the mineralization determined by both trabecular bones stained green, and growth plate thickness.

Discussion This study demonstrates that single and combined treatments with etidronate and alfacalcidol increase both bone density and mechanical properties of trabecular and cortical bones in ovx rats. The effects of combined treatments were greater than those of single treatments, and were approximately additive of those of respective single treatments. Because the increase in the mechanical properties was proportional to that in BMD, and the connectivity of trabecular bones was identical in all groups, the stimu-

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Figure 2. Representative features of micro-CT images of cross sections of distal metaphysis (A) and midshaft of femur (B) and sagittal section of the L4 vertebral body (C). Femur specimens were evaluated at the level 10 mm from the distal end and at the midportion by cross-sectional analysis, and L4 vertebral body specimens were measured at the center by sagittal section. Morphometric parameters of the distal metaphysis and the midportion of femurs are described in Table 6.

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latory effects of these treatments on bone strength are probably ascribable primarily to those on bone mass. Because this study was not designed to elucidate the mechanism of action of the combined treatment, the precise cellular mechanism of its action remains unclear. However, analysis of blood chemistry revealed that alfacalcidol significantly increased the serum levels of calcium, phosphorus, and osteocalcin, whereas etidronate slightly decreased only the serum osteocalcin level. These findings may imply the difference of functions of these two agents in preventing bone loss in ovx rats; that is, alfacalcidol might increase intestinal calcium and phosphorus absorption and bone formation/bone turnover, whereas etidronate might decrease bone turnover. 1,25(OH)2D3 is known to stimulate bone formation directly and bone resorption indirectly by acting on osteoblastic cells.5,42 It may be that etidronate blocks the catabolic action, but not the anabolic action, of alfacalcidol in combined treatment. In fact, a previous study has shown that combined alendronate and prostaglandin estradiol (PGE2) treatment prevents the resorption induced by PGE2, but not the stimulation of bone formation on endocortical and periosteal surfaces, resulting in a significant increase in cortical thickness in ovx rats.22 However, combined treatments with bisphosphonates and other anabolic agents do not always lead to beneficial effects on bone. Combined treatment with tiludronate and parathyroid hormone (PTH) has been reported to reduce bone mass in elderly female sheep, although its mechanism is unclear.6 The possible mechanism of action of each agent should at least be taken into consideration when combined therapy is applied for clinical use. The doses of alfacalcidol used in this study (0.1 and 0.2 ␮g/kg per day for 24 weeks) were much higher than those used clinically, whereas the dose of etidronate (4 mg/kg per day) used was close to clinical doses. In fact, the alfacalcidol-treated groups all showed hypercalcemia. These are, however, common doses that have been used in previous studies showing the bone anabolic effect of alfacalcidol on ovx rats with normal calcium diet.18,23,36,40 Shiraishi et al. reported the anabolic effect of alfacalcidol at the same doses with a significant increase in the serum calcium level, although their treatment period was shorter than ours (12 weeks).40 Izawa et al. also reported hypercalcemia with 0.2 ␮g/kg per day of alfacalcidol treatment for 6 months.18 On the other hand, Lindgren et al.23 and Okumura et al.36 reported no significant elevation of serum calcium level by alfacalcidol treatments (0.25 ␮g/kg per day for 6 months and 0.1 ␮g/kg per day for 12 week, respectively). These discrepancies in serum calcium level might be due to differences in the method of statistical analysis. In the latter two reports, in which hypercalcemia was not seen, the Scheffe test was performed for the

Table 6. Morphometric analyses on cross-sectional microcomputed tomography images of distal metaphysis and midportion of femurs (mean ⫾ SEM, n ⫽ 10/group) Distal metaphysis Group Sham Ovx E LD HD E ⫹ LD E ⫹ HD

Midportion 2

BV/TV (%)

Tb.Th (␮m)

N.Nd/N.Tm

Total area (mm )

Bone area (mm2)

Cortical thickness (mm)

38.4 ⫾ 2.1a 19.4 ⫾ 1.7 44.9 ⫾ 1.4c 37.3 ⫾ 2.2c 41.3 ⫾ 3.0c 65.8 ⫾ 4.2c,d,e 66.2 ⫾ 5.8c,d,f

79.2 ⫾ 5.6 92.9 ⫾ 7.0 99.3 ⫾ 4.2 97.4 ⫾ 6.7 124.1 ⫾ 10.3b 108.3 ⫾ 5.8 126.7 ⫾ 9.5c

0.397 ⫾ 0.037 0.440 ⫾ 0.043 0.419 ⫾ 0.044 0.499 ⫾ 0.080 0.495 ⫾ 0.066 0.472 ⫾ 0.051 0.502 ⫾ 0.077

7.32 ⫾ 0.24 7.40 ⫾ 0.17 6.98 ⫾ 0.14 7.42 ⫾ 0.29 7.59 ⫾ 0.31 7.05 ⫾ 0.19 7.53 ⫾ 0.21

4.51 ⫾ 0.13a 3.43 ⫾ 0.09 4.33 ⫾ 0.10c 4.60 ⫾ 0.18c 4.47 ⫾ 0.20c 4.42 ⫾ 0.08c 4.66 ⫾ 0.14c

1.81 ⫾ 0.31a 1.53 ⫾ 0.10 1.75 ⫾ 0.22c 1.85 ⫾ 0.13c 1.80 ⫾ 0.10c 1.78 ⫾ 0.25c 1.87 ⫾ 0.14c

KEY: BV/TV, bone volume/total volume; Tb.Th, trabecular thickness; N.Nd/N.Tm, number of nodes/number of termini. See Table 1 for remaining abbreviations. Significantly different vs. ovx: ap ⬍ 0.01 (Student’s t-test); significantly different vs. ovx: bp ⬍ 0.05, cp ⬍ 0.01 (ANOVA, Tukey–Kramer). Analysis of combined treatment groups vs. respective single treatment groups: significantly different vs. E: dp ⬍ 0.01 (ANOVA, Tukey–Kramer); significantly different vs. LD: ep ⬍ 0.01 (ANOVA, Tukey–Kramer); significantly different vs. HD: fp ⬍ 0.01 (ANOVA, Tukey–Kramer).

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Figure 3. Histological features of the proximal tibiae of ovx, E, E ⫹ LD, and E ⫹ HD rats. Tibiae of ovx, E, E ⫹ LD, and E ⫹ HD rats were fixed with 100% ethanol and stained with Villanueva Goldner. Original magnification ⫻20.

statistical analysis. This method is known to be less sensitive in detecting a statistical difference as compared with the Tukey– Kramer test and Dunnett test used in other reports including this one. When the serum calcium level was analyzed by the Scheffe test in this study, a significant increase vs. the ovx group was detected only in the HD group (p ⬍ 0.05), but not in the LD, E ⫹ LD, or E ⫹ HD groups. In addition, because the E ⫹ HD group showed a significantly lower calcium level than the HD group (p ⬍ 0.05), the combination with etidronate may possibly decrease the serum calcium level that was increased by alfacalcidol alone. It is true, however, that further studies using lower doses of alfacalcidol that do not cause hypercalcemia will be optimal from the clinical therapeutic standpoint. BMDs of proximal tibiae and lumbar vertebral bodies (Table 2) showed good correspondence with those of bone volume on micro-CT of distal femurs (Table 6), which confirms the stronger effects of combined vs. single treatment on trabecular bones. However, in the analyses of midfemurs, combined treatments exhibited higher BMDs (Table 2), but almost identical bone areas on micro-CT (Table 6) when compared with single treatment groups. This might be because a small change of cortical thickness can significantly influence bone density. Otherwise, one may speculate that the quality or density of cortical bone may be regulated by these factors. Another issue is that the effects of single and combined treatments on mechanical properties appeared to be less in the L4 vertebral body than in the midfemur. This may be due to the insufficient decrease in mechanical properties in the L4 vertebral body of ovx rats as compared with sham rats at 24 weeks after the surgery. The observation period of this study was from 15 to 39 weeks of age when bone growth and maturation have almost been completed in rats according to previous studies by Kalu et al.19,20 Other reports, however, have shown that neither ovx nor estrogen replacement had a significant effect on the mechanical properties of trabecular bones earlier than 9 months after the surgery.14,35 Further studies over longer periods might reveal stronger effects of these treatments on trabecular bone strength. Taken together, the results of the present study demonstrate that the combination of intermittent cyclic etidronate and daily alfacalcidol may be promising in the treatment or prevention of

postmenopausal osteoporosis. In fact, one clinical study revealed that a combination of cyclical etidronate and calcitriol was more effective than cyclical etidronate alone in terms of changes in BMD at the spine and femoral neck of postmenopausal women.24 Combined treatment with calcitriol and another bisphosphonate, alendronate, has also been reported to be more beneficial for bone mass than treatment with calcitriol or alendronate alone in postmenopausal women.9 We thus propose that a combination of two conventional drugs showing different functions in preventing bone loss could achieve a stronger effect than novel bonesparing drugs. Acknowledgments: The authors thank the hard tissue research team at Kureha Chemical Co., Ltd., for technical assistance. This study was funded by a Bristol-Myers Squibb/Zimmer unrestricted research grant and the Uehara Memorial Foundation.

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Date Received: March 24, 2000 Date Revised: July 3, 2000 Date Accepted: July 11, 2000