Introduction

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Osteoporosis is a metabolic bone disease in which there is both decrease in the skeletal mass and disturbance in bone microarchitecture so that the risk of fractures is increased. Osteopenia induced by OVX in rats has been widely used as a model of postmenopausal osteoporosis in humans (Kalu et al., 1989; Wronski et al., 1989). Our previous studies of s.c. administration of clodronate to mature growing OVX rats indicated an inhibition of osteopenia, measured histomorphometrically, and an inhibition of the weakening of bone strength due to estrogen deficiency in both appendicular and axial skeleton (Kippo et al., 1995; Lepola et al., 1996).

pQCT has recently been used to measure changes in BMD in rats (Turner et al., 1994; Rosen et al., 1995; Sato et al., 1995, 1996; Breen et al., 1996). This system allows a three-dimensional analysis of BMD and can differentiate between trabecular and cortical bone. pQCT also offers a way to estimate bone mechanical properties noninvasively (Ferretti, 1995; Ferretti et al., 1995a, 1996; Gasser, 1995).

This study was carried out to measure over time in vivo, as well as at the end of the study ex vivo, the preventive effects of clodronate administered p.o. on changes in BMD and bone geometry in OVX adult rats using a densitometric technique. In addition, bending strength of tibial shaft and ash weight of femur and tibia were determined after 12 weeks of treatment.

	Materials and Methods

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Animals

Sixty female Sprague-Dawley rats, with a mean weight of 270 g (S.E. 2), were used. At the start of the study, the animals were 6 months old. They were fed a commercial small-animal food (SDS R/M1, Witham, UK), and they had free access to tap water during the study. Rats were housed in individual cages at constant temperature (21 ± 1°C) and humidity (45-55%), using a 12-h light and darkness cycle. The animals were randomized into groups so that animals undergoing operation on the same day were divided into various test groups. Bilateral ovariectomies were carried out according to Waynforth (1988) in 45 rats, and the remaining animals (n = 15) were subjected to sham operation. Success of ovariectomy was confirmed by serum estradiol measurement and macroscopic evaluation of the uterus at autopsy. Body weight and food consumption were measured once a week. The study protocol was approved by the local ethical committee.

Administration of the Test Compound

Three OVX groups (n = 15/ group) were administered p.o. either clodronate (100 mg/kg/d or 500 mg/kg/d) or vehicle. The SHAM group (n = 15) received the vehicle. Clodronate and vehicle were administered five times a week from Monday to Friday. Treatments started on the day of OVX and continued for 3 months. Solutions of the test substance and vehicle were administered by gavage (5 ml/kg).

Sample Collecting

At the time of sacrifice, the bones (left femur and left tibia) were removed, cleaned, wrapped in gauze saturated with physiological saline and stored at 20°C. After measurement of bone mineral density, the tibias were tested for biomechanical strength, and the ash weight of tibia and femur was then determined.

Bone Densitometry with pQCT

BMD and bone geometry were measured in vivo and ex vivo by pQCT (XCT-960A, Stratec, Pforzheim, Germany). pQCT also offers a way to calculate, from cross-sectional images, the stability of bone against bending or torsion (SSI or density-weighted polar moment of resistance). SSI takes into account both cross-sectional moment of inertia and cortical BMD. To control the stability of measurement, the instrument was calibrated every day against a phantom of known mineral content. The coefficient of variation was previously determined in our laboratory, and it was 1% for total BMD in proximal tibial metaphyses in vivo and 2% and 1.5% for trabecular and cortical BMD, respectively.

In vivo measurement. The BMD of proximal tibia (2 mm below the growth plate) was measured in vivo 1 day before OVX and 6 and 12 weeks after OVX. For measurement, the rats were anesthetized with a s.c. injection of 15 mg/1.0 ml/kg Hypnorm (Janssen Pharmaceutical Ltd, Oxford, UK), and they were lying on their backs. Six 1-mm-thick sections of the proximal tibia were scanned at 0.5-mm intervals using a voxel size of 0.197 × 0.197 × 1.0 mm. From week 6 onward, a voxel size of 0.295 × 0.295 × 1.0 mm was used because of the larger size of the leg. The total BMD and trabecular BMD (mg/cm³) were measured using a threshold of 0.700 cm¹.

Ex vivo measurement. Before scanning, the length of the excised femur or tibia was measured with a micrometer, and the bone was placed anterior side up in the measuring tube containing water. We measured the BMD of various sites, including proximal tibia (2 mm below the growth plate), distal femur (2 mm above the growth plate), proximal femur (intertrochanter region containing the base of the femoral neck and the base of the greater trochanter) and midshaft of tibia and femur. Six 1-mm-thick sections of the distal femur and proximal tibia and three sections of the proximal femur and midshaft of tibia and femur were scanned at 0.8-mm intervals using a voxel size of 0.148 × 0.148 × 1.0 mm and a threshold of 0.700 cm¹. We chose a threshold of 0.930 cm¹ for cortical bone in proximal tibial metaphysis and in tibial and femoral midshaft. BMD (mg/cm³) and BMC were measured, and geometric parameters (cross-sectional area, cortical thickness, periosteal and endocortical perimeters) with calculated SSI (density-weighted polar moment of resistance, mm³) were defined.

Biomechanical Testing and Ash Weight

Bone strength measurement was carried out from left tibiae. The strength of the tibial diaphysis was tested by the three-point bending method as described earlier in detail by Peng et al. (1994). Thereafter, we determined the ash weight of the tibia and the femur after ashing them at 600°C for 24 h.

Statistical Analyses

Statistical analyses were carried out with the SAS system. A P value lower than .05 was considered statistically significant. The data were analyzed with one-way analysis of variance. If ANOVA revealed a significant difference between groups, the pairwise comparisons between groups were performed with linear contrasts of means. Total and cortical BMD values were chosen as primary variables, and the 90% confidence intervals for the ratios of means between each OVX group and SHAM group were calculated for these variables. Simple correlation analysis was used to describe the relationship between polar moment of resistance and bending strength of tibial shaft.

	Results

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No differences were found between groups in initial body weight. The final body weight and body weight gain were higher in the OVX vehicle group and in both clodronate groups than in the SHAM group (data not shown).

BMD and geometry. OVX induced a marked decrease in total and trabecular BMD at the proximal tibial metaphysis over a 3-month period. This decrease was prevented by clodronate treatment (fig. 1). Furthermore, pQCT images of proximal tibial metaphysis revealed a decrease in the cortical BMD and a thinning in the cortical bone thickness after OVX, changes that were most evident in the anterior cortex opposite to the fibula. After clodronate treatment, pQCT images showed cortical BMD and thickness similar to those in SHAM rats (fig. 2).

View larger version (130K): [in this window] [in a new window]   Fig. 1.   pQCT images of proximal tibia in vivo (week 12). The top row shows the tibial metaphysis from a SHAM rat (left) and an OVX rat (right). The bottom row shows equivalent regions from OVX rats treated with clodronate at doses of 100 (left) and 500 mg/kg/d (right).

View larger version (111K): [in this window] [in a new window]   Fig. 2.   pQCT images of proximal tibia in vivo (week 12). The top row shows the cortical bone (threshold 0.930 cm1) in tibial metaphysis from a SHAM rat (left) and an OVX rat (right). The bottom row shows equivalent regions from OVX rats treated with clodronate at doses of 100 (left) and 500 mg/kg/d (right). The image of the OVX rat shows that in the anterior cortex opposite to the fibula, cortical BMD was lower than the threshold used.

Ash weight of femur and tibia. OVX induced a decrease in the total ash weight of femur and tibia, and this effect was prevented by clodronate at both used dosages. The total ash weight of femur in the SHAM group was 465 (S.E. 10) mg, in the OVX vehicle group was 432 (7) mg and in the clodronate groups was 457 (6) mg (100 mg/kg/d) and 462 (6) mg (500 mg/kg/d). The respective values in total ash weight of tibia were 320 (6), 301 (5), 317 (4) and 325 (5) mg.

Biomechanical bone properties. There were no significant differences between the groups in the bending strength, deformation, rigidity, or energy absorption area of the tibia (data not shown). However, a positive correlation (n = 58, r = 0.51, P < .001) was found between the measured maximum load in the three-point bending test of tibial shaft and the density-weighted polar moment of resistance (SSI) calculated by pQCT in tibial shaft (fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Correlation between the polar moment of resistance calculated by pQCT and the results of three-point bending test of tibial shaft.

	Discussion

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Measurement of BMD in vivo at the proximal tibia using pQCT showed that after OVX, a marked decrease occurred in total and trabecular BMD in proximal tibial metaphysis. Ex vivo bone densitometry demonstrated that OVX caused a decrease in total BMD that was, on average, from 21 to 28% at proximal tibial metaphysis. In distal femoral metaphysis and in the intertrochanteric region of the proximal femur, the decreases in total BMD in the OVX vehicle group were from 21 to 31% and from 7 to 12%, respectively. This bone loss was inhibited by clodronate treatment and was completely prevented by the higher dose. The results of the present study showed that clodronate was effective in preserving BMD as well as bone geometry. These findings are in good agreement with previous histomorphometric studies on secondary spongiosa of proximal tibial and distal femoral metaphysis after the s.c. administration of clodronate (Kippo et al., 1995; Lepola et al., 1996). In an effect consistent with the densitometric results, the decreases in the total ash weight of tibia and femur after OVX were also prevented by clodronate.

At tibial and femoral diaphysis, OVX did not induce a decrease in cortical BMD or any change in bone geometry or SSI. Neither were any changes found in bending strength of the tibial shaft. The lack of any osteopenic response in cortical bone is probably due to the relatively short follow-up time after OVX. The age of the animals at the time of OVX may also play a critical role. Chen et al. (1995), who used 19-month-old rats in their study, reported decreased BMD 8 weeks after OVX in distal femoral metaphysis and proximal femur as well as in femoral diaphysis assessed by dual-energy x-ray absorptiometry (DXA). On the other hand, we recently found that OVX carried out in 14-month-old rats had not induced histomorphometrically measured cortical osteopenia or mechanical weakening of femoral shaft at 3 months after OVX (Kippo et al., 1997).

It has been suggested previously that BMC and geometry account for most of the variance in bone strength (Ferretti et al., 1993; Turner and Burr, 1993). Recently, Ferretti et al. (1996) have shown a linear correlation between the product of cross-sectional moment of inertia and volumetric cortical BMD (assessed by pQCT) and the actual breaking force of the rat femoral shaft. According to their study, either the cross-sectional moment of inertia or the cortical BMD alone was less predictive of breaking force. In the present study, although we found no difference between the groups in calculated density-weighted polar moment of resistance at tibial mid-diaphysis, there was a significant positive correlation between this and the actual bending strength of the tibial shaft. This supports the assumption that density-weighted polar moment of resistance can be used to estimate the mechanical strength of diaphyseal bone. These results further suggest that clodronate treatment causes no deleterious changes in the material properties of cortical bone.

The polar moment of resistance has earlier been determined in the cortical metaphyseal region of proximal tibia in rat (Gasser, 1995), and the SSI has been determined in distal radius of human (Ferretti et al., 1995b). The metaphyseal region is easily accessible for measurement by pQCT and is of particular interest because the majority of fractures in humans occur in this region. In agreement with the study by Gasser (1995), the polar moment of resistance in the present study was decreased in proximal tibial and distal femoral metaphysis in OVX rats. This deterioration in bone geometry was prevented by clodronate. However, because no appropriate biomechanical tests exist for this specific region of bone in small animals, it is not possible to validate the significance of the observed polar moment of resistance in this location by comparing it directly with the mechanical strength. In agreement with the study by Sato et al., (1995), the pQCT images of proximal tibial metaphysis in OVX animals revealed a decrease in cortical BMD and a thinning in cortical bone thickness, effects that were most evident in the anterior cortex opposite to the fibula. Moreover, the results of the present study showed no difference in periosteal perimeter between groups, but the endocortical perimeter was significantly higher in OVX than in other groups, which indicates endocortical resorption. All these changes were prevented by clodronate treatment.

In conclusion, our results indicate that pQCT measures changes in rat bone with high precision and that clodronate administered p.o. in adult rats prevents changes due to estrogen deficiency in BMD and bone geometry at proximal tibia and at distal and proximal femur.