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Effect of clodronate treatment on established bone loss in ovariectomized rats
Bone Vol. 23, No. 4 October 1998:333–342
Effect of Clodronate Treatment on Established Bone Loss in Ovariectomized Rats K. KIPPO,1 R. HANNUNIEMI,1 L. LAURE´N,1 Z. PENG,2 P. KUURTAMO,1 T. VIRTAMO,1 P. ISAKSSON,1 ¨ STERMAN,1 H. K. VA ¨A ¨ NA ¨ NEN,3 and R. SELLMAN1 T. O 1
Leiras Oy, Biomedical Research Center, Turku, Finland Department of Anatomy and Biocenter, University of Oulu, Oulu, Finland 3 Department of Anatomy, University of Turku, Turku, Finland 2
Key Words: Clodronate; Osteopenia; Histomorphometry; Biomechanics; Adult rat.
The ability of clodronate to prevent bone loss and weakening of bone strength was studied in adult rats with established osteopenia. Six-month-old female Sprague Dawley rats were randomized into 13 groups. One group was killed at the start of the study, nine groups were ovariectomized (ovx), and three groups sham-operated (sham). After 4 months, the ovx rats were given either clodronate or vehicle subcutaneously (s.c.), once a week for 3 or 6 months, the cumulative doses of both dosing regimens being 36, 84, and 300 mg/kg. Clodronate reduced the increase in bone turnover as evidenced by serum osteocalcin and urinary deoxypyridinoline. Cancellous bone loss was more severe in distal femur than in lumbar vertebral body already at 4 months after ovx. Cortical osteopenia of femoral middiaphysis was significant at 7 and 10 months after operation and was in accordance with the impaired bending strength of the femoral shaft. In the tibia, the bending strength was, by contrast, increased at each timepoint after ovx. In distal femur, higher values of cancellous bone volume (BV/TV) were found after 6 months of clodronate treatment than in ovx/vehicle-treated rats. In lumbar vertebrae, only the lowest dose of clodronate slightly counteracted the ovx-induced further decrease in BV/TV, but reduced, at all dosages, the impairment of lumbar vertebral compression strength. The maximum load of femoral neck did not differ between vehicle-treated ovx and sham groups after clodronate treatment, but clodronate reduced the weakening of femoral shaft. A further increase in the bending strength of the tibia was found after clodronate treatment. There was a positive correlation between bending strength and ash weight in both the tibia and the femur. Histomorphometry further showed that long-term use of clodronate does not impair bone mineralization or affect modelingdependent bone formation. In conclusion, clodronate treatment clearly slows down the progress of bone loss and prevents further weakening of bone strength in femoral shaft and vertebrae, even though it cannot completely reverse the effects of ovariectomy-induced changes in established osteopenia. (Bone 23:333–342; 1998) © 1998 by Elsevier Science Inc. All rights reserved.
Introduction In animals and humans, loss of ovarian function causes dramatic changes in bone mass, due to an imbalance between the amount of resorbed bone and that formed at each remodeling site. Internal microarchitecture and strength are also impaired, leading to increased bone fragility, especially in metaphyseal regions.23 Osteoclastic perforation and removal of trabecular plates have been shown to be the primary mechanisms of postmenopausal bone loss in early phases of estrogen deficiency in humans28 and in a rat model of postmenopausal osteoporosis.9 Inhibition of osteoclastic bone resorption forms the basis of the effectiveness of antiresorptive compounds, such as bisphosphonates, in preventing the rapid phase of postmenopausal bone loss. In the treatment of established disease, antiresorptive therapy should slow down further bone loss and stabilize the bone mass.7,14 The treatment of established osteopenia with bisphosphonates, alendronate, risedronate, tiludronate, and pamidronate has been studied widely in ovx rats1,2,18,26,30,36,37 with treatment starting 4 weeks to 12 months after ovariectomy. Alendronate showed a preserving effect on cancellous bone volume and the strength of the lumbar spine and femoral diaphysis, but not on that of the femoral neck.18,37 Pamidronate induced, in ovx rats, an increase in areal bone mineral density of the femoral neck, which was not associated with changes in ultimate strength.2 Furthermore, bone mineral density was increased in ovx rats following tiludronate treatment at the lumbar spine and proximal tibia.1 Risedronate was capable of maintaining vertebral bone mass and bone biomechanical competence at control levels in sexually mature, slightly osteopenic ovx rats.26 However, no differences in femoral neck maximum load were found after 5 or 15 weeks of risedronate therapy, compared with sham-operated or ovx vehicle-treated rats.36 On the other hand, treatment of aged ovx rats with risedronate alone failed to restore lost cancellous bone in the proximal tibia or the first lumbar vertebra.30 Clodronate and other bisphosphonates have been shown to inhibit spontaneous bone resorption in young growing rats and thus dose-dependently increase metaphyseal mineralized tissue mass of long bones.10,24,25,33,35 The efficacy of clodronate in preventing estrogen deficiency-induced osteopenia in rats has been shown previously in growing and aged ovx animals.16,17,19 The present study was carried out to study the effect of clodronate on established osteopenia in adult rats. Histomorphomet-
Address for correspondence and reprints: Katriina Kippo, Leiras Oy, Biomedical Research Center, P.O. Box 415, 20101 Turku, Finland. © 1998 by Elsevier Science Inc. All rights reserved.
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Figure 1. Experimental procedure.
ric studies of the distal femur and lumbar vertebral body, and cross sections of tibial and femoral diaphysis and femoral midneck, as well as bone strength measurement, were carried out. In addition, changes in the biochemical markers of bone turnover were followed. Materials and Methods A total of 308 female 6-month-old Sprague Dawley rats (mean 6 SEM: 284 6 1 g) were used in this study. Eight rats served as the baseline group, and the remaining animals were divided into 12 groups (n 5 25 animals/group). Nine groups were bilaterally ovariectomized (ovx) according to Waynforth,40 and three were sham operated (sham). Before surgery, the rats were subcutaneously (s.c.) given atropine (Atropin, Orion, Finland) in a dose of 0.05 mg/kg. Then they were anesthetized with an s.c. injection of Hypnorm (Janssen Pharmaceuticals, Oxford, UK) with a dose of 1.5 mL/kg. After 15 min, an intraperitoneal (i.p.) injection of pentobarbital (Mebunat, Orion, Finland) was given to every animal in a dose of 15 mg/kg. The rats were fed standard small-animal laboratory food (R/M1, SDS, Witham, UK) containing 0.71% calcium, 0.50% phosphorus, and 0.60 IU/g vitamin D3, and they had free access to tap water. The rats were housed in individual cages at constant temperature (21 6 1°C) and humidity (45%–55%), using a 12 h light/dark cycle. Their body weights were measured at the time of operation and weekly thereafter. The success of ovariectomy was confirmed by serum estradiol assay and macroscopic evaluation of the uterus at autopsy. The study protocol was approved by the local ethics committee. All rats were left untreated for the first 4 months after surgery to allow establishment of cancellous osteopenia. Sham and ovx animals were killed at the age of 10 months to establish basal histomorphometric and bone strength values before initiation of
treatment. The remaining ten groups received once per week s.c. treatment with either disodium clodronate (C) (Bonefos, Leiras Oy, Finland) at doses of 3, 7, or 25 mg/kg or vehicle (Veh) (0.9% NaCl) for 3 months, or at doses of 1.5, 3.5, or 12.5 mg/kg for 6 months. Clodronate dosage was adjusted to body weight changes once per week, the cumulative doses of both regimens being 36, 84, and 300 mg/kg (Figure 1). For dynamic histomorphometry, eight animals from each group were double labeled with fluorochromes. Intraperitoneal injections of oxytetracycline (Terramycin/LA, Pfizer, Sandwich, UK) and calcein (Sigma, Deisenhof, Germany) were given at doses of 25 mg/kg at 21 and 7 days before killing in the baseline group (interlabel time [ILT] 14 days), and at 35 and 14 days before killing in all other groups (ILT 21 days). This regimen resulted in deposition of a double-fluorochrome label at bone surfaces that were undergoing active mineralization throughout the labeling period. In addition, the same animals in all other groups, except the baseline control group, were labeled i.p. with calcein (25 mg/kg) also on the day after operation, the ILT between the first and second calcein injections being 100, 187, and 267 days for the follow-up times of 4, 7, and 10 months, respectively. At the end of the study, 24 h urine was collected in metabolic cages from all animals fasting during urine collection for the determination of deoxypyridinoline (DPD) (Metra Biosystems) and creatinine (Merckotest, Germany). Blood samples were collected from all animals under slight anesthesia with CO2. Serum osteocalcin (OC) (Biomedical Technologies) and estradiol (Diagnostic Products) were determined by radioimmunoassay. After dissection, both femurs and the right tibia and lumbar vertebrae were cleaned from soft tissue. The bones intended for biomechanical tests were wrapped in saline-saturated gauze and placed in 220°C until analysis. Bone histomorphometry included the baseline group and bone samples
Bone Vol. 23, No. 4 October 1998:333–342
from all the groups were put into 4°C ethanol (40% v/v). Distal and proximal ends of the femur and fourth lumbar vertebral specimens were then processed undecalcified as described elsewhere.16,19 Longitudinal 5-mm-thick sections of the distal femoral metaphysis were cut with a Polycut S heavy-duty microtome (Leica Instruments, Germany) at the midsagittal level and at a level spaced 600 mm in the lateral direction, and stained using the von Kossa method with hematoxylin/McNeal tetrachrome counterstain.34 From the L-4 vertebral body, two 20-mm-thick longitudinal sections were microground at levels spaced 500 mm apart. A 5-mm-high diaphyseal cylinder of the distal tibiofibular junction was fresh stained by Villanueva osteochrome stain (Polysciences, Warrington, PA) and a 5-mm-high femoral cylinder was cut at the midshaft. Then 20-mm-thick cross sections of both long bone cylinders and the femoral midneck were processed as described before.19 After dynamic histomorphometry, cross sections of the long bones as well as L-4 sections were surface stained with toluidine blue and with Masson–Goldner trichrome,34 respectively. Bone Strength Measurement and Histomorphometry Measurement of femoral neck cantilever bending, three-point bending of tibial and femoral shafts, and compression of the fifth lumbar vertebra (L-5) have been described elsewhere.19,29 Ash weight of the femur and tibia was determined after the femur and tibia had been burned at 600°C for 24 h. A digitizing image-analysis system (MCID, Imaging Research, St. Catherines, ON, Canada) was used for cortical and cancellous bone histomorphometry. Total mineralized cancellous bone volume (BV) and surface (BS) were measured at distances .1.0 mm from the epiphyseal growth plate of the distal femur and from the caudal growth plate of the L-4 vertebra. A mean total tissue volume (TV) of 11.0 mm2 was analyzed per distal femoral metaphysis and a volume of 5.85 mm2 per L-4 vertebra. Cancellous bone volume (BV/TV) and trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) were calculated.28 In a cross section of the femoral midneck, the percent total bone area (%TB.Ar), including regions of cortical and cancellous bone, and the cancellous bone volume were calculated as percentages of the total cross-sectional tissue area and of the entire area within the endocortical envelope, respectively, as described previously.41 Variables measured to determine structural geometric properties of tibial and femoral cortical bone included total crosssectional tissue area (T.Ar), cortical bone area (Ct.Ar), marrow area (Ma.Ar), cortical thickness (Ct.Th), and periosteal and endocortical diameters of the femoral cross section in the vertical and horizontal directions.19 The measured variables were used to calculate the percentages of cortical bone area (%Ct.Ar), marrow area (%Ma.Ar), and cross-sectional second moment of inertia (Ix, femoral diaphysis and midneck) as described elsewhere.8,12,27 To study kinetic variables, the following were measured: single- and double-layered surfaces and interlabel thickness at the secondary spongiosa of the L-4 vertebra and along periosteal surfaces of long bone cross sections, and periosteal osteoid seam thickness (O.Th, tibial diaphysis). The percent mineralizing surface (MS/BS), mineralization lag time (Mlt, tibial diaphysis), mineral apposition rate (MAR), and surface-referent bone formation rate (BFR/BS) were then calculated.12,27 Furthermore, in tibial and femoral diaphyses, the interlabel thickness between the first and second calcein labels was measured for the entire experimental period at each follow-up (4, 7, and 10 months after operation), and MAR2 was calculated for corresponding interlabel periods. Dynamic histomorphometry of the L-4 vertebral
K. Kippo et al. Effects of clodronate on established osteopenia
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body was carried out in vehicle-treated sham and ovx groups only at 10 months after ovx, and in ovx rats treated subcutaneously by clodronate 1.5 mg/kg once per week for 6 months (cumulative dose 36 mg/kg). Statistical Analyses Body weight data were analyzed separately for groups killed at various timepoints with one-way analysis of variance (ANOVA). If ANOVA revealed a significant difference between groups, analysis was continued by performing appropriate pairwise comparisons. All other variables were analyzed in three phases to study the effects of aging, operation, and clodronate treatment. The effect of aging from 6 (baseline group) to 10 months (sham at 4 months after operation) before the onset of treatment was analyzed with the two-sample t-test. The effects of ovariectomy and aging were studied by analyzing basal (at 4 months after operation) and vehicle-treated sham and ovx groups by two-way ANOVA. In the case of significant operation 3 time interaction, the effect of ovariectomy was studied by comparing sham and ovx groups separately at each timepoint. Correspondingly, the effects of clodronate and time (aging, duration of treatment) were studied by analyzing the sham/vehicle, ovx/vehicle, and ovx/ clodronate groups at 7 and 10 months after operation, with two-way ANOVA. If ANOVA revealed a significant group effect without significant group 3 time interaction, appropriate pairwise comparisons were performed using combined means of timepoints and, in the case of interaction, separately for each timepoint. All comparisons were carried out with linear contrasts generated by the ANOVA model. If the distribution was not normal, either transformation or the Kruskal–Wallis test was used. Simple correlation analysis was used to describe the relationship between maximum load in three-point bending and ash weight of the tibia and femur. p , 0.05 was considered significant. Statistical analyses were carried using BMDP statistical software.5 Results Ovariectomy increased the change in body weight in rats from age 6 –10 months before the onset of the treatment. Body weight was significantly higher in all ovx groups at 4 and 7 months after operation and in ovx clodronate groups also at 10 months after operation as compared with sham groups (data not shown). Age-Related Changes in Bone Before Treatment In tibial diaphysis, decreases in the periosteal MAR1 and BFR/ BS, as well as increases in the mineralization lag time (Mlt) and %Ct.Ar were found during 4 months of follow-up from 6 (baseline group) to 10 months of age (sham group) (Table 1, data only partially shown). In the femoral middiaphysis, periosteal MAR1 decreased with age, but otherwise there were no agerelated changes in structural geometrical properties of femoral cross sections (Table 2). In the distal femoral metaphysis, significant age-related decreases in BV/TV, Tb.Th, and Tb.N, as well as an increase in Tb.Sp, were seen. The respective mean (SEM) values were 35.4 (1.5%) for BV/TV, 72 mm (3 mm) for Tb.Th, 4.9/mm (0.1/mm) for Tb.N, and 132 mm (5 mm) for Tb.Sp in the baseline group and 24.5% (1.7%), 60 mm (5 mm), 4.1/mm (0.2/mm), and 186 mm (9 mm) for the sham group at 4 months after operation. In the L-4 vertebra, BV/TV and Tb.N were found to decrease and Tb.Sp to increase with age before the onset of treatment (Figure 2A–D). In a cross section of the femoral midneck, there were no age-related changes in bone morphometry from 6 to 10 months of age (Table 3) or in the
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Bone Vol. 23, No. 4 October 1998:333–342
Table 1. Histomorphometry of tibial diaphyseal cross section
Group
Time after operation (months)
Baseline
0
Periosteal surface MAR1 (mm/d)
Static morphometry
MAR2 (mm/d)
1.07 (0.12)
—
aa
Ct.Th (mm)
%Ct.Ar (%)
0.66 (0.01)
82.5 (0.6) a
%Ma.Ar (%) 17.3 (0.6)
Sham Ovx
4 4
0.54 (0.09) 0.47 (0.04)
0.46 (0.03) 0.71 (0.05)bbb
0.67 (0.01) 0.68 (0.01)
84.3 (0.5) 82.4 (0.4)bbb
15.7 (0.5) 17.5 (0.4)bbb
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
7 7 7 7 7
0.60 (0.10) 0.46 (0.07) 0.38 (0.07) 0.52 (0.04) 0.59 (0.02)
0.34 (0.03) 0.44 (0.04)bbb 0.46 (0.04)bbb 0.50 (0.04)bbb 0.52 (0.06)bbb
0.67 (0.02) 0.65 (0.02) 0.65 (0.02)c 0.73 (0.02)bb,ccc 0.74 (0.01)bbb,ccc
83.7 (0.6) 79.2 (1.0)bbb 79.4 (0.9)bbb 82.2 (0.6)c 82.5 (0.9)cc
16.1 (0.6) 20.7 (1.0)bbb 20.4 (0.9)bbb 17.8 (0.6)c 17.4 (0.9)cc
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
10 10 10 10 10
0.52 (0.07) 0.42 (0.07) 0.47 (0.05) 0.45 (0.05) 0.47 (0.08)
0.23 (0.03) 0.39 (0.03)bbb 0.36 (0.04)bbb 0.38 (0.02)bbb 0.40 (0.05)bbb
0.64 (0.02) 0.62 (0.01) 0.69 (0.01)c 0.68 (0.01)bb,ccc 0.71 (0.01)bbb,ccc
80.6 (1.0) 78.4 (0.7)bbb 81.5 (0.7)cc 81.6 (0.9)cc 83.3 (0.7)b,ccc
19.1 (1.0) 21.3 (0.7)bbb 18.0 (0.8)cc 18.4 (0.9)c 16.5 (0.7)b,ccc
KEY: MAR, mineral apposition rate; Ct.Th, cortical thickness; %Ct.Ar, percent cortical bone area; %Ma.Ar, percent marrow area; MAR1, interlabel time during the 21 days between oxytetracycline and the second calcein injections, in the baseline group 14 days; MAR2, timeframe of the entire experimental period between the first and second calcein labels. Data expressed as mean (SEM), n 5 5– 8 per group. Sham (at 4 months after operation) vs. baseline: ap , 0.05; aap , 0.01. Ovx or ovx/C vs. sham: bp , 0.05; bbp , 0.01; bbbp , 0.001. Ovx/C vs. ovx: cp , 0.05; ccp , 0.01; cccp , 0.001.
cancellous bone variables BV/TV, Tb.Th, Tb.N, or Tb.Sp (data not shown). Ovx-Related Changes in Bone Ovariectomy decreased (p , 0.001) the ash weight of the whole femur at each follow-up timepoint after operation, the respective mean (SEM) values being 433 mg (8 mg) in the
sham group and 409 mg (5 mg) in the ovx group at 4 months after ovx. The maximum loads of both femoral neck and L-5 vertebra also decreased due to estrogen deficiency at each follow-up timepoint after operation, but the maximum load in the three-point bending of the femoral shaft decreased only at 7 and 10 months after operation. In the tibia, the bending strength was, by contrast, increased at each timepoint after ovx (Figure 3A–D).
Table 2. Histomorphometry of femoral middiaphyseal cross section
Group Baseline
Periosteal surface
Time after operation (months)
MAR1 (mm/d)
0
0.94 (0.09)
MAR2 (mm/d)
a
Static morphometry Ct.Th (mm)
%Ct.Ar (%)
%Ma.Ar (%)
Ix (mm4)
—
0.69 (0.01)
66.5 (1.1)
33.4 (1.0)
3.91 (0.31)
0.59 (0.04) 0.83 (0.05)bbb
0.70 (0.02) 0.73 (0.01)
66.3 (1.3) 68.3 (0.6)
33.5 (1.3) 31.6 (0.6)
4.28 (0.30) 4.16 (0.13)
Sham Ovx
4 4
0.62 (0.04) 0.59 (0.03)
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
7 7 7 7 7
0.53 (0.05) 0.57 (0.12) 0.47 (0.02) 0.50 (0.02) 0.54 (0.04)
0.50 (0.03) 0.55 (0.04) 0.52 (0.04) 0.55 (0.04) 0.55 (0.04)
0.74 (0.01) 0.68 (0.01)b 0.70 (0.01)bbb,c 0.72 (0.01)b,ccc 0.73 (0.01)ccc
68.0 (1.2) 60.6 (1.5)bbb 62.7 (1.1)bbb 64.5 (0.9)bb,cc 64.9 (0.5)bb,cc
31.7 (1.2) 39.1 (1.4)bbb 37.0 (1.1)bbb 35.1 (1.0)bb,cc 34.9 (0.5)bb,cc
4.57 (0.30) 5.66 (0.38) 5.83 (0.24)bbb 5.52 (0.19)bb 5.81 (0.18)bb
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
10 10 10 10 10
0.51 (0.07) 0.39 (0.04) 0.42 (0.04) 0.38 (0.03) 0.43 (0.02)
0.53 (0.03) 0.52 (0.03) 0.50 (0.04) 0.55 (0.03) 0.51 (0.02)
0.77 (0.02) 0.66 (0.02)bbb 0.70 (0.01)bbb,c 0.73 (0.01)b,ccc 0.73 (0.02)ccc
70.6 (0.8) 63.2 (1.3)bbb 63.5 (1.6)bbb 66.6 (1.0)bb,cc 66.1 (1.1)bb,cc
29.3 (0.8) 36.6 (1.3)bbb 36.0 (1.6)bbb 33.1 (1.0)bb,cc 33.7 (1.0)bb,cc
4.61 (0.33) 4.70 (0.30) 5.34 (0.29)bbb 5.10 (0.25)bb 5.05 (0.20)bb
KEY: MAR, mineral apposition rate; Ct.Th, cortical thickness; %Ct.Ar, percent cortical bone area; %Ma.Ar, percent marrow area; Ix, cross-sectional moment of inertia; MAR1, interlabel time during the 21 days between oxytetracycline and the second calcein injections, in the baseline group 14 days; MAR2, timeframe of the entire experimental period between the first and second calcein labels. Data expressed as mean (SEM), n 5 6 – 8 per group. Sham (at 4 months after operation) vs. baseline: ap , 0.01. Ovx or ovx/C vs. sham: bp , 0.05; bbp , 0.01; bbbp , 0.001. Ovx/C vs. ovx: cp , 0.05; ccp , 0.01; cccp , 0.001.
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Figure 2. Cancellous bone variables of the L-4 vertebral body. (A) Bone volume (BV/TV), (B) trabecular number (Tb.N), (C) trabecular thickness (Tb.Th), and (D) trabecular separation (Tb.Sp). Sham (at 4 months after operation) vs. baseline: ap , 0.05; aap , 0.01. Ovx or ovx/C vs. sham: *p , 0.05; **p , 0.01; ***p , 0.001; ovx/C vs. ovx: #p , 0.05; ##p , 0.01; ###p , 0.001. The values are expressed as mean 6 SEM, n 5 7 or 8/group. The mean of baseline values at the age of 6 months is marked by a dotted line in each figure.
There were no significant ovariectomy-induced effects on periosteal O.Th, MS/BS, Mlt, MAR1, or BFR/BS of the tibial diaphysis (Table 1, data only partially shown; Figure 4) or on MAR1 in femoral diaphysis at any follow-up timepoint (Table 2). In contrast, Ps.MAR2, analyzed for the entire experimental period, was higher at each follow-up timepoint in the tibial diaphysis, but only at 4 months after ovx in the femoral diaphysis (Tables 1 and 2). The cross-sectional tissue and marrow area of the tibia and femur were greater in ovx than in sham rats (data not shown). Tibial %Ct.Ar decreased and %Ma.Ar increased as a result of estrogen deficiency (Table 1). Ovariectomy was also found to increase the femoral cross-sectional %Ma.Ar and to decrease Ct.Th and %Ct.Ar, resulting in cortical osteopenia at 7 and 10 months after operation. However, the cross-sectional second moment of inertia in the femoral middiaphysis did not change due to estrogen deficiency (Table 2). Ovariectomy decreased the BV/TV of the distal femoral metaphysis by 48% already at 4 months after operation, which
was manifested by reduced Tb.Th and Tb.N, and increased Tb.Sp. The respective mean (SEM) values were 24.5% (1.7%) for BV/TV, 60 mm (5 mm) for Tb.Th, 4.1/mm (0.2/mm) for Tb.N, and 186 mm (9 mm) for Tb.Sp in the sham group and 12.8% (0.5%), 51 mm (2 mm), 2.5/mm (0.1/mm), and 365 mm (15 mm) in the ovx group. In the L-4 vertebra, ovariectomy decreased BV/TV and Tb.Th, and increased Tb.Sp, but the change in Tb.N was nonsignificant (Figure 2). Cancellous osteopenia proceeded in ovx rats at 7 and 10 months after operation. Estrogen deficiency of 10 months decreased %TB.Ar, %Ct.Ar, and Ct.Th, and increased %Ma.Ar in the femoral midneck (Table 3). Within the endocortical envelope of femoral midneck, there was a significant decrease in Tb.N and an increase in Tb.Sp at each follow-up timepoint after ovariectomy, although the decrease in BV/TV was not significant. The respective mean (SEM) values were 38.9% (2.1%) for BV/TV, 4.1/mm (0.1/mm) for Tb.N, and 148 mm (7 mm) for Tb.Sp in the sham
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Table 3. Bone morphometry of femoral midneck cross section
Group
Time after operation (months)
%TB.Ar (%)
%Ma.Ar (%)
%Ct.Ar (%)
Ct.Th (mm)
Ix (mm4)
Ps.MS/BS (%)
Baseline
0
88.0 (1.5)
10.5 (1.5)
79.2 (2.7)
0.59 (0.02)
0.85 (0.06)
28.0 (5.4)
Sham Ovx
4 4
89.5 (0.8) 90.8 (1.3)
9.0 (0.7) 8.2 (1.2)
83.3 (1.0) 86.1 (2.1)
0.61 (0.02) 0.65 (0.04)
0.71 (0.06) 0.86 (0.07)
30.0 (6.6) 29.9 (6.7)
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
7 7 7 7 7
87.3 (2.0) 86.4 (0.6) 87.7 (1.2) 86.2 (0.8) 84.0 (0.6)
11.4 (1.7) 12.8 (0.7) 11.2 (1.2) 12.9 (0.7) 15.3 (0.6)a
79.7 (3.0) 78.8 (0.4) 77.8 (2.8) 79.9 (1.8) 75.0 (1.4)
0.60 (0.04) 0.60 (0.01) 0.56 (0.02) 0.61 (0.02) 0.55 (0.01)
0.93 (0.10) 1.12 (0.11) 1.02 (0.15) 1.05 (0.07) 1.15 (0.07)
17.6 (3.7) 19.9 (4.8) 10.4 (3.1)a,b 7.8 (3.0)aa,bb 1.0 (0.7)aaa,bbb
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
10 10 10 10 10
90.4 (1.6) 84.9 (0.4)aa 86.3 (1.2)a 88.1 (1.2)b 88.9 (2.1)b
8.8 (1.6) 14.1 (0.7)aa 12.3 (1.2)a 11.3 (1.3) 10.2 (1.7)b
86.3 (1.6) 78.8 (1.3)aa 81.0 (1.3) 83.4 (1.6) 83.8 (4.0)
0.70 (0.02) 0.57 (0.01)aa 0.60 (0.02)aa 0.62 (0.02)a 0.64 (0.05)
1.07 (0.13) 0.92 (0.08) 0.94 (0.09) 0.91 (0.10) 0.95 (0.06)
17.2 (6.0) 14.4 (4.2) 5.5 (2.3)a,b 5.5 (2.0)aa,bb 3.5 (2.2)aaa,bbb
KEY: %TB.Ar, percent total bone area (includes cortical and cancellous bone); %Ma.Ar, percent marrow area; %Ct.Ar, percent cortical bone area; Ct.Th, cortical thickness; Ix, cross-sectional moment of inertia; Ps.MS/BS, periosteal mineralizing surface. %TB.Ar, %Ct.Ar, and %Ma.Ar are calculated as percentages of total cross-sectional tissue area. Sham (at 4 months after operation) vs. baseline not significant. Ovx or ovx/C vs. sham: ap , 0.05; aap , 0.05; aaap , 0.001. Ovx/C vs. ovx: bp , 0.05; bbp , 0.01; bbbp , 0.001. Data expressed as mean (SEM), n 5 4 – 8 per group.
group and 33.0% (4.5%), 3.3/mm (0.2/mm), and 210 mm (24 mm) in the ovx group at 4 months after operation. At 10 months after ovariectomy, double labeling of oxytetracycline and calcein could be found at cancellous bone surfaces of the L-4 vertebra in five of eight sham rats and in all ovx rats (n 5 8), resulting, however, in nonsignificant change in MS/BS (the mean [SEM] values were 6.4% [1.0%] and 6.9% [0.9%] for vehicle-treated sham and ovx groups, respectively). The endosteal MAR of the L-4 was significantly lower in ovx rats (0.39 mm/d [0.05 mm/d]) than in sham rats (0.60 mm/d [0.09 mm/d]). However, no significant differences occurred in BFR/BS between sham and ovx rats at 10 months after operation (data not shown). Effect of Clodronate on Established Osteopenia Clodronate increased the total ash weight of the femur and tibia (data not shown). In the femoral neck, the maximum load after clodronate treatment did not differ between vehicle-treated agematched ovx and sham groups, but in the L-5 vertebra all doses of clodronate improved the ovariectomy-induced impairment of compressive strength (Figure 3A,B). Clodronate also clearly prevented the ovx-induced decrease in the bending strength of the femoral shaft at all doses, the values being on a level with sham rats, and in the tibia clodronate further increased the ovx-induced increase in tibial bending strength (Figure 3C,D). There was a positive linear correlation between maximum load in three-point bending and ash weight in both the tibia (Figure 5) and the femur (n 5 196, r 5 0.84, p , 0.001). Clodronate did not induce any significant changes in terminal periosteal O.Th, MS/BS, Mlt, MAR1, or BFR/BS in tibial diaphysis (Table 1 data only partially shown; Figure 4) or in terminal MAR1 or MAR2 in femoral diaphysis (Table 2). Tibial periosteal MAR2 values were on a level with vehicle-treated ovx groups after each clodronate dose after 3 and 6 months of treatment (Table 1). Clodronate had no significant effect on the ovx-induced increase in tibial or femoral cross-sectional tissue
area (data not shown). However, clodronate did increase tibial and femoral %Ct.Ar in comparison to vehicle-treated ovx rats and, after 6 months of treatment, the highest cumulative dose (300 mg/kg) had even increased the tibial %Ct.Ar to above the level of sham controls (Tables 1 and 2). Furthermore, clodronate inhibited the ovx-induced enlargement of %Ma.Ar in tibial and femoral diaphysis (Tables 1 and 2). After the cumulative doses of clodronate (84 and 300 mg/kg), tibial Ct.Th was higher than in vehicle-treated sham or ovx groups, and the decrease in Ct.Th of femoral diaphysis was inhibited compared with the vehicletreated ovx rats (Tables 1 and 2). After 6 months of clodronate treatment, significantly higher BV/TV and Tb.N, and lower Tb.Sp, were seen in the distal femur at each dose level when compared with vehicletreated ovx rats. The mean (SEM) values of BV/TV were 7.8% (0.7%) for the vehicle-treated ovx group and 12.1% (1.0%), 11.9% (0.8%), or 11.9% (0.8%) for the clodronatetreated groups (cumulative doses 36, 84, or 300 mg/kg), respectively. After 3 months of treatment, this effect was not yet seen (data not shown). In the L-4 vertebra, clodronate (cumulative dose 36 mg/kg) slightly counteracted the ovariectomy-induced decrease in the BV/TV as a result of increased Tb.N and decreased Tb.Sp (Figure 2A–D). In femoral midneck, after 6 months of clodronate treatment, %TB.Ar was significantly higher (cumulative doses 84 and 300 mg/kg) and %Ma.Ar lower (cumulative dose 300 mg/kg) than in vehicle-treated ovx rats (Table 3). After 3 or 6 months of clodronate treatment, periosteal MS/BS was significantly lower than in age-matched vehicle-treated ovx or sham rats, due to reduced double labeling of fluorochromes (Table 3). In cancellous bone of the femoral midneck, Tb.N and Tb.Sp, after clodronate treatment for 3 or 6 months, did not significantly differ from the vehicle-treated ovx and sham groups (data not shown). Percent mineralizing surface (n 5 8) was lower (mean [SEM] 2.2% [0.3%]) in the secondary spongiosa of the L-4 vertebra after 6 months of clodronate treatment (cumulative dose 36
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Figure 3. Maximum loads of femoral neck cantilever bending (A), L-5 vertebral compression (B), and three-point bending of femoral (C) and tibial shafts (D). Ovx or ovx/C vs. sham: *p , 0.05; **p , 0.01; ***p , 0.001; ovx/C vs. ovx: ##p , 0.01; ###p , 0.001. The values are expressed as mean 6 SEM, n 5 14 –25/group.
mg/kg, which was the only dose level tested) than in the agematched vehicle-treated ovx or sham groups. The decreased Es.MS/BS was due to reduced double labeling of fluorochromes along cancellous bone surfaces after clodronate treatment, and interlabel thickness could be measured and MAR calculated in only two of eight rats. As a result, BFR/BS values of cancellous bone were also lower after clodronate treatment compared with vehicle groups (data not shown). Oxytetracycline and calcein occurred only as single labels on cancellous bone surfaces of the L-4 vertebra after higher doses of clodronate. Biochemical Variables Ovariectomy caused increases in serum osteocalcin and urinary excretion of deoxypyridinoline, which were prevented by all doses of clodronate (Figure 6A,B). After clodronate administration at cumulative doses of 84 and 300 mg/kg, the values of both variables were lower than in sham rats. Ovariectomy decreased serum estradiol at each follow-up timepoint after operation (data not shown).
Discussion Our results from the present study show that clodronate suppressed, at all dosage levels, the ovariectomy-induced increase in bone turnover in adult rats. Urinary excretion rates of deoxypyridinoline and serum osteocalcin decreased to normal after the lowest dosage regimen of clodronate, and below the level of sham rats after higher doses of clodronate. Clodronate did not induce any significant change in the indices of bone formation or mineralization at the periosteal surfaces of long bones, but depressed cancellous bone formation of L-4 vertebra, suggesting that the effect of clodronate on bone formation is exerted only through remodeling. This is in accordance with earlier observations with pamidronate, risedronate, and alendronate.4,6,32 The biomechanical behavior of a bone reflects its material and geometric properties. Both the cross-sectional area and the moment of inertia, which expresses the distribution of material around a given axis, are important for the mechanical behavior of long bones.11,38 Our study showed that bone geometry was altered after ovx as a result of increased periosteal apposition and
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Figure 4. Fluorescent micrographs of periosteal surface (Ps) of tibial diaphysis at 10 months after operation in ovx rats treated subcutaneously once per week for 6 months with vehicle (A) or clodronate at a dose level of 1.5 mg/kg (B). First and second calcein labels (filled arrows), one tetracycline label (open arrow), and osteoid seam (O) can be seen. Villanueva osteochrome bone stain. Bar 5 50 mm.
endocortical resorption in long bones. Cortical bone loss and weakening of the femoral shaft reached significant levels only at 7 and 10 months after ovariectomy, which is in accordance with a previous study by Toolan et al.37 In the tibia, the bending strength was, by contrast, increased at each timepoint (4, 7, and 10 months) after ovx. An obvious reason for this finding is the ovx induced periosteal bone growth in tibial diaphysis, as judged by the higher mineral apposition rate determined for the entire experimental period in ovx rats than in the age-matched sham rats. This resulted in continued expansion of the tibial cross-
Figure 5. Relationship between maximum load in three-point bending and ash weight of the tibia (n 5 290).
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Figure 6. Serum osteocalcin (A) and urinary excretion of deoxypyridinoline per creatinine (B). Ovx or ovx/C vs. sham: *p , 0.05; ***p , 0.001; ovx/C vs. ovx: ###p , 0.001. Values are expressed as mean 6 SEM, n 5 22–25/group.
sectional area, which is an important factor in determining the bending strength of the tibial shaft. In the femoral diaphyseal cross section, clodronate treatment increased the moment of inertia at 7 and 10 months after operation compared with age-matched sham controls, indicating that the increased diameter of the bone distributed the material further from the neutral axis and improved the resistance of the bone to bending loads. Furthermore, increases in tibial cortical bone area and thickness as effects of clodronate treatment produced the greatest mechanical advantage to the maximum load of the tibial shaft. Clodronate treatment increased the ash weight of long bones, suggesting that the treatment affects cortical as well as cancellous bone. Furthermore, it should be noted that a significant correlation between the ash weight and maximum load in three-point bending was found in both the femur and the tibia. This suggests that the increase in bone mineral content is reflected in mechanical strength. Changes in the maximum load at the femoral neck revealed a deteriorating effect of ovariectomy already at 4 months after operation, as described previously,29,36 but changes did not differ significantly between clodronate-treated ovx rats when compared with vehicle-treated ovx and sham groups. Similarly, in earlier studies with other bisphosphonates (alendronate, pamidronate, and risedronate), no beneficial effect on femoral neck strength was found.2,18,36,37 The reason for this unexpected finding is unknown. Although both decreased bone mass and impaired trabecular architecture would produce a structurally weaker femoral neck, the exact contributions of cortical and cancellous bone
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on mechanical strength in the rat femoral neck are not yet completely understood. The irreversible changes that were found in the femoral midneck cancellous bone had already taken place before the onset of the clodronate treatment. This was evidenced by decreased trabecular number and increased separation in ovx rats, in agreement with previous studies.3,15,20,21,41 After clodronate treatment, the values of trabecular number and separation did not significantly differ from the vehicle-treated ovx and sham groups. Cortical bone and inner trabeculae are connected in the femoral neck, as shown in the present study and by others,3,13,20,21,41 and the reduced endocortical-trabecular connectivity will probably also contribute to the deterioration of the mechanical properties of the femoral neck.3 Cortical osteopenia including reduced cortical thickness, and enlargement of marrow area, developed at later stages of estrogen deficiency. The effect was significant only at 10 months after ovx, as also shown by others.13,20,21 Similarly, the relative area of total bone (trabecular bone and cortical shell) significantly decreased at 10 months after ovx, and clodronate treatment (84 or 300 mg/kg) prevented this bone loss. However, the cross-sectional moment of inertia in the femoral midneck did not differ between groups in the present study, showing equal distribution of effective bone mass in rat femoral neck among the groups. Furthermore, the fact that clodronate does not induce any change in femoral neck width of ovx rats15 also partially explains why it could not improve the resistance of the femoral neck to bending and compressive loads in cantilever bend testing in the osteopenia model presented. Because of the high surface:volume ratio of cancellous bone, disorders of bone remodeling, such as osteoporosis, commonly affect trabecular sites earlier and more profoundly than cortical sites.14 This was also shown here, as evidenced by cancellous bone loss of 48% in the distal femur at 4 months after operation, the loss being much faster than in the proximal femur. This may be due to variations in mechanical loading and longitudinal bone growth rate between distal and proximal sites of the femur. With respect to severe osteopenia, 3 months of clodronate treatment could not prevent cancellous bone loss in the distal femur at any dose. However, 6 months treatment was efficient in this respect at each dose level. The trabecular number was higher and trabecular separation lower than in vehicle-treated ovx rats, indicating less damage to trabecular microarchitecture after longterm clodronate treatment. In the lumbar vertebral body, clodronate treatment for 3 or 6 months could not restore lost cancellous bone. Only the lowest clodronate dose slightly counteracted the ovariectomy-induced further decrease in cancellous bone volume, which was due to a higher trabecular number and lower trabecular separation in clodronate-treated ovx rats than in the corresponding ovx vehicle group. It is of interest, however, that the ovariectomy-induced impairment of lumbar vertebral compression strength was significantly improved at all clodronate dosages after 3 and 6 months of treatment. Compression testing of the vertebral body is presently used, although the exact contribution of cortical shell and cancellous bone to the strength of rat vertebral body is uncertain.37,38 The thickness of the vertebral cortex has been shown to be an important determinant of compressive strength of the whole vertebral body31,39 and, in patients with osteoporosis, vertebral cortex assumes more significance in bearing loads.22 The ability of clodronate to improve the impaired vertebral strength in established osteopenia can thus be due mostly to the beneficial effect of clodronate on cortical bone. In summary, the present study has shown that clodronate treatment starting 4 months after ovariectomy and continued for 3 to 6 months inhibited bone resorption as assessed by urinary deoxypyridinoline:creatinine ratio. It also inhibited bone turn-
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over in regions of remodeling as assessed by endosteal bone formation rate in the lumbar vertebra and by serum osteocalcin levels. However, there was no evidence of direct inhibition of modeling-dependent bone formation or impairment of mineralization as a result of clodronate treatment. This suggests that long-term clodronate treatment can be used as a safe and effective method for inhibiting bone resorption and suppressing increased bone turnover due to estrogen deficiency. The results further suggest that clodronate, like other bisphosphonates, is more effective in the prevention of experimental osteopenia than in the treatment of established disease, the latter requiring restoration of skeletal mass and bone microarchitecture.
Acknowledgments: The authors gratefully acknowledge the expert technical assistance of the staff of Preclinical Research at Leiras Oy and Allan Haimakainen. Also, we are grateful to Juhani Tuominen, Ph.Lic., for consulting in statistics.
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Bone Vol. 23, No. 4 October 1998:333–342 30. Qi, H., Li, M., and Wronski, T. J. A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats. J Bone Miner Res 10:948 –955; 1995. 31. Rockoff, S. D., Sweet, E., and Bleustein, J. The relative contribution of trabecular and cortical bone to the strength of human lumbar vertebrae. Calcif Tissue Res 3:163–175; 1969. 32. Sato, M., Bryant, H. U., Iversen, P., Helterbrand, J., Smietana, F., Bemis, K., Higgs, R., Turner, C. H., Owan, I., Takano, Y., and Burr, D. B. Advantages of raloxifene over alendronate or estrogen on nonreproductive and reproductive tissues in the long-term dosing of ovariectomized rats. J Pharmacol Exp Ther 279:298 –305; 1996. 33. Schenk, R., Merz, W. A., Mu¨hlbauer, R., 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. 34. Schenk, R. K., Olah, A. J., and Herrmann, W. Preparation of calcified tissues for light microscopy. In: Dickson, G. R., Ed. Methods of Calcified Tissue Preparation. Amsterdam: Elsevier; 1984; 1–56. 35. Shinoda, H., Adamek, G., Felix, R., Fleisch, H., Schenk, R., and Hagan, P. Structure-activity relationships of various bisphosphonates. Calcif Tissue Int 34:87–99; 1983. 36. Søgaard, C. H., Wronski, T. J., McOsker, J. E., and Mosekilde, L. The positive effect of parathyroid hormone on femoral neck bone strength in ovariectomized rats is more pronounced than that of estrogen or bisphosphonates. Endocrinology 134:650 – 657; 1994. 37. Toolan, B. C., Shea, M., Myers, E. R., Borchers, R. E., Seedor, J. G., Quartuccio, H., Rodan, G., and Hayes, W. C. Effects of 4-amino-1-hydroxybutylidene bisphosphonate on bone biomechanics in rats. J Bone Miner Res 7:1399 –1406; 1992. 38. Turner, C. H. and Burr, D. B. Basic biomechanical measurements of bone: A tutorial. Bone 14:595– 608; 1993. 39. Vesterby, A., Mosekilde, Li., Gundersen, H. J. G., Melsen, F., Mosekilde, Le., Holme, K., and Sørensen, S. Biologically meaningful determinants of the in vitro strength of lumbar vertebrae. Bone 12:219 –224; 1991. 40. Waynforth, H. B. Experimental and Surgical Technique in the Rat. San Diego, CA: Academic; 1980. 41. Yamamoto, N., Jee, W. S. S., and Ma, Y. F. Bone histomorphometric changes in the femoral neck of aging and ovariectomized rats. Anat Rec 243:175–185; 1995.
Date Received: November 13, 1997 Date Revised: June 1, 1998 Date Accepted: June 1, 1998
Effect of Clodronate Treatment on Established Bone Loss in Ovariectomized Rats K. KIPPO,1 R. HANNUNIEMI,1 L. LAURE´N,1 Z. PENG,2 P. KUURTAMO,1 T. VIRTAMO,1 P. ISAKSSON,1 ¨ STERMAN,1 H. K. VA ¨A ¨ NA ¨ NEN,3 and R. SELLMAN1 T. O 1
Leiras Oy, Biomedical Research Center, Turku, Finland Department of Anatomy and Biocenter, University of Oulu, Oulu, Finland 3 Department of Anatomy, University of Turku, Turku, Finland 2
Key Words: Clodronate; Osteopenia; Histomorphometry; Biomechanics; Adult rat.
The ability of clodronate to prevent bone loss and weakening of bone strength was studied in adult rats with established osteopenia. Six-month-old female Sprague Dawley rats were randomized into 13 groups. One group was killed at the start of the study, nine groups were ovariectomized (ovx), and three groups sham-operated (sham). After 4 months, the ovx rats were given either clodronate or vehicle subcutaneously (s.c.), once a week for 3 or 6 months, the cumulative doses of both dosing regimens being 36, 84, and 300 mg/kg. Clodronate reduced the increase in bone turnover as evidenced by serum osteocalcin and urinary deoxypyridinoline. Cancellous bone loss was more severe in distal femur than in lumbar vertebral body already at 4 months after ovx. Cortical osteopenia of femoral middiaphysis was significant at 7 and 10 months after operation and was in accordance with the impaired bending strength of the femoral shaft. In the tibia, the bending strength was, by contrast, increased at each timepoint after ovx. In distal femur, higher values of cancellous bone volume (BV/TV) were found after 6 months of clodronate treatment than in ovx/vehicle-treated rats. In lumbar vertebrae, only the lowest dose of clodronate slightly counteracted the ovx-induced further decrease in BV/TV, but reduced, at all dosages, the impairment of lumbar vertebral compression strength. The maximum load of femoral neck did not differ between vehicle-treated ovx and sham groups after clodronate treatment, but clodronate reduced the weakening of femoral shaft. A further increase in the bending strength of the tibia was found after clodronate treatment. There was a positive correlation between bending strength and ash weight in both the tibia and the femur. Histomorphometry further showed that long-term use of clodronate does not impair bone mineralization or affect modelingdependent bone formation. In conclusion, clodronate treatment clearly slows down the progress of bone loss and prevents further weakening of bone strength in femoral shaft and vertebrae, even though it cannot completely reverse the effects of ovariectomy-induced changes in established osteopenia. (Bone 23:333–342; 1998) © 1998 by Elsevier Science Inc. All rights reserved.
Introduction In animals and humans, loss of ovarian function causes dramatic changes in bone mass, due to an imbalance between the amount of resorbed bone and that formed at each remodeling site. Internal microarchitecture and strength are also impaired, leading to increased bone fragility, especially in metaphyseal regions.23 Osteoclastic perforation and removal of trabecular plates have been shown to be the primary mechanisms of postmenopausal bone loss in early phases of estrogen deficiency in humans28 and in a rat model of postmenopausal osteoporosis.9 Inhibition of osteoclastic bone resorption forms the basis of the effectiveness of antiresorptive compounds, such as bisphosphonates, in preventing the rapid phase of postmenopausal bone loss. In the treatment of established disease, antiresorptive therapy should slow down further bone loss and stabilize the bone mass.7,14 The treatment of established osteopenia with bisphosphonates, alendronate, risedronate, tiludronate, and pamidronate has been studied widely in ovx rats1,2,18,26,30,36,37 with treatment starting 4 weeks to 12 months after ovariectomy. Alendronate showed a preserving effect on cancellous bone volume and the strength of the lumbar spine and femoral diaphysis, but not on that of the femoral neck.18,37 Pamidronate induced, in ovx rats, an increase in areal bone mineral density of the femoral neck, which was not associated with changes in ultimate strength.2 Furthermore, bone mineral density was increased in ovx rats following tiludronate treatment at the lumbar spine and proximal tibia.1 Risedronate was capable of maintaining vertebral bone mass and bone biomechanical competence at control levels in sexually mature, slightly osteopenic ovx rats.26 However, no differences in femoral neck maximum load were found after 5 or 15 weeks of risedronate therapy, compared with sham-operated or ovx vehicle-treated rats.36 On the other hand, treatment of aged ovx rats with risedronate alone failed to restore lost cancellous bone in the proximal tibia or the first lumbar vertebra.30 Clodronate and other bisphosphonates have been shown to inhibit spontaneous bone resorption in young growing rats and thus dose-dependently increase metaphyseal mineralized tissue mass of long bones.10,24,25,33,35 The efficacy of clodronate in preventing estrogen deficiency-induced osteopenia in rats has been shown previously in growing and aged ovx animals.16,17,19 The present study was carried out to study the effect of clodronate on established osteopenia in adult rats. Histomorphomet-
Address for correspondence and reprints: Katriina Kippo, Leiras Oy, Biomedical Research Center, P.O. Box 415, 20101 Turku, Finland. © 1998 by Elsevier Science Inc. All rights reserved.
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Figure 1. Experimental procedure.
ric studies of the distal femur and lumbar vertebral body, and cross sections of tibial and femoral diaphysis and femoral midneck, as well as bone strength measurement, were carried out. In addition, changes in the biochemical markers of bone turnover were followed. Materials and Methods A total of 308 female 6-month-old Sprague Dawley rats (mean 6 SEM: 284 6 1 g) were used in this study. Eight rats served as the baseline group, and the remaining animals were divided into 12 groups (n 5 25 animals/group). Nine groups were bilaterally ovariectomized (ovx) according to Waynforth,40 and three were sham operated (sham). Before surgery, the rats were subcutaneously (s.c.) given atropine (Atropin, Orion, Finland) in a dose of 0.05 mg/kg. Then they were anesthetized with an s.c. injection of Hypnorm (Janssen Pharmaceuticals, Oxford, UK) with a dose of 1.5 mL/kg. After 15 min, an intraperitoneal (i.p.) injection of pentobarbital (Mebunat, Orion, Finland) was given to every animal in a dose of 15 mg/kg. The rats were fed standard small-animal laboratory food (R/M1, SDS, Witham, UK) containing 0.71% calcium, 0.50% phosphorus, and 0.60 IU/g vitamin D3, and they had free access to tap water. The rats were housed in individual cages at constant temperature (21 6 1°C) and humidity (45%–55%), using a 12 h light/dark cycle. Their body weights were measured at the time of operation and weekly thereafter. The success of ovariectomy was confirmed by serum estradiol assay and macroscopic evaluation of the uterus at autopsy. The study protocol was approved by the local ethics committee. All rats were left untreated for the first 4 months after surgery to allow establishment of cancellous osteopenia. Sham and ovx animals were killed at the age of 10 months to establish basal histomorphometric and bone strength values before initiation of
treatment. The remaining ten groups received once per week s.c. treatment with either disodium clodronate (C) (Bonefos, Leiras Oy, Finland) at doses of 3, 7, or 25 mg/kg or vehicle (Veh) (0.9% NaCl) for 3 months, or at doses of 1.5, 3.5, or 12.5 mg/kg for 6 months. Clodronate dosage was adjusted to body weight changes once per week, the cumulative doses of both regimens being 36, 84, and 300 mg/kg (Figure 1). For dynamic histomorphometry, eight animals from each group were double labeled with fluorochromes. Intraperitoneal injections of oxytetracycline (Terramycin/LA, Pfizer, Sandwich, UK) and calcein (Sigma, Deisenhof, Germany) were given at doses of 25 mg/kg at 21 and 7 days before killing in the baseline group (interlabel time [ILT] 14 days), and at 35 and 14 days before killing in all other groups (ILT 21 days). This regimen resulted in deposition of a double-fluorochrome label at bone surfaces that were undergoing active mineralization throughout the labeling period. In addition, the same animals in all other groups, except the baseline control group, were labeled i.p. with calcein (25 mg/kg) also on the day after operation, the ILT between the first and second calcein injections being 100, 187, and 267 days for the follow-up times of 4, 7, and 10 months, respectively. At the end of the study, 24 h urine was collected in metabolic cages from all animals fasting during urine collection for the determination of deoxypyridinoline (DPD) (Metra Biosystems) and creatinine (Merckotest, Germany). Blood samples were collected from all animals under slight anesthesia with CO2. Serum osteocalcin (OC) (Biomedical Technologies) and estradiol (Diagnostic Products) were determined by radioimmunoassay. After dissection, both femurs and the right tibia and lumbar vertebrae were cleaned from soft tissue. The bones intended for biomechanical tests were wrapped in saline-saturated gauze and placed in 220°C until analysis. Bone histomorphometry included the baseline group and bone samples
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from all the groups were put into 4°C ethanol (40% v/v). Distal and proximal ends of the femur and fourth lumbar vertebral specimens were then processed undecalcified as described elsewhere.16,19 Longitudinal 5-mm-thick sections of the distal femoral metaphysis were cut with a Polycut S heavy-duty microtome (Leica Instruments, Germany) at the midsagittal level and at a level spaced 600 mm in the lateral direction, and stained using the von Kossa method with hematoxylin/McNeal tetrachrome counterstain.34 From the L-4 vertebral body, two 20-mm-thick longitudinal sections were microground at levels spaced 500 mm apart. A 5-mm-high diaphyseal cylinder of the distal tibiofibular junction was fresh stained by Villanueva osteochrome stain (Polysciences, Warrington, PA) and a 5-mm-high femoral cylinder was cut at the midshaft. Then 20-mm-thick cross sections of both long bone cylinders and the femoral midneck were processed as described before.19 After dynamic histomorphometry, cross sections of the long bones as well as L-4 sections were surface stained with toluidine blue and with Masson–Goldner trichrome,34 respectively. Bone Strength Measurement and Histomorphometry Measurement of femoral neck cantilever bending, three-point bending of tibial and femoral shafts, and compression of the fifth lumbar vertebra (L-5) have been described elsewhere.19,29 Ash weight of the femur and tibia was determined after the femur and tibia had been burned at 600°C for 24 h. A digitizing image-analysis system (MCID, Imaging Research, St. Catherines, ON, Canada) was used for cortical and cancellous bone histomorphometry. Total mineralized cancellous bone volume (BV) and surface (BS) were measured at distances .1.0 mm from the epiphyseal growth plate of the distal femur and from the caudal growth plate of the L-4 vertebra. A mean total tissue volume (TV) of 11.0 mm2 was analyzed per distal femoral metaphysis and a volume of 5.85 mm2 per L-4 vertebra. Cancellous bone volume (BV/TV) and trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) were calculated.28 In a cross section of the femoral midneck, the percent total bone area (%TB.Ar), including regions of cortical and cancellous bone, and the cancellous bone volume were calculated as percentages of the total cross-sectional tissue area and of the entire area within the endocortical envelope, respectively, as described previously.41 Variables measured to determine structural geometric properties of tibial and femoral cortical bone included total crosssectional tissue area (T.Ar), cortical bone area (Ct.Ar), marrow area (Ma.Ar), cortical thickness (Ct.Th), and periosteal and endocortical diameters of the femoral cross section in the vertical and horizontal directions.19 The measured variables were used to calculate the percentages of cortical bone area (%Ct.Ar), marrow area (%Ma.Ar), and cross-sectional second moment of inertia (Ix, femoral diaphysis and midneck) as described elsewhere.8,12,27 To study kinetic variables, the following were measured: single- and double-layered surfaces and interlabel thickness at the secondary spongiosa of the L-4 vertebra and along periosteal surfaces of long bone cross sections, and periosteal osteoid seam thickness (O.Th, tibial diaphysis). The percent mineralizing surface (MS/BS), mineralization lag time (Mlt, tibial diaphysis), mineral apposition rate (MAR), and surface-referent bone formation rate (BFR/BS) were then calculated.12,27 Furthermore, in tibial and femoral diaphyses, the interlabel thickness between the first and second calcein labels was measured for the entire experimental period at each follow-up (4, 7, and 10 months after operation), and MAR2 was calculated for corresponding interlabel periods. Dynamic histomorphometry of the L-4 vertebral
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body was carried out in vehicle-treated sham and ovx groups only at 10 months after ovx, and in ovx rats treated subcutaneously by clodronate 1.5 mg/kg once per week for 6 months (cumulative dose 36 mg/kg). Statistical Analyses Body weight data were analyzed separately for groups killed at various timepoints with one-way analysis of variance (ANOVA). If ANOVA revealed a significant difference between groups, analysis was continued by performing appropriate pairwise comparisons. All other variables were analyzed in three phases to study the effects of aging, operation, and clodronate treatment. The effect of aging from 6 (baseline group) to 10 months (sham at 4 months after operation) before the onset of treatment was analyzed with the two-sample t-test. The effects of ovariectomy and aging were studied by analyzing basal (at 4 months after operation) and vehicle-treated sham and ovx groups by two-way ANOVA. In the case of significant operation 3 time interaction, the effect of ovariectomy was studied by comparing sham and ovx groups separately at each timepoint. Correspondingly, the effects of clodronate and time (aging, duration of treatment) were studied by analyzing the sham/vehicle, ovx/vehicle, and ovx/ clodronate groups at 7 and 10 months after operation, with two-way ANOVA. If ANOVA revealed a significant group effect without significant group 3 time interaction, appropriate pairwise comparisons were performed using combined means of timepoints and, in the case of interaction, separately for each timepoint. All comparisons were carried out with linear contrasts generated by the ANOVA model. If the distribution was not normal, either transformation or the Kruskal–Wallis test was used. Simple correlation analysis was used to describe the relationship between maximum load in three-point bending and ash weight of the tibia and femur. p , 0.05 was considered significant. Statistical analyses were carried using BMDP statistical software.5 Results Ovariectomy increased the change in body weight in rats from age 6 –10 months before the onset of the treatment. Body weight was significantly higher in all ovx groups at 4 and 7 months after operation and in ovx clodronate groups also at 10 months after operation as compared with sham groups (data not shown). Age-Related Changes in Bone Before Treatment In tibial diaphysis, decreases in the periosteal MAR1 and BFR/ BS, as well as increases in the mineralization lag time (Mlt) and %Ct.Ar were found during 4 months of follow-up from 6 (baseline group) to 10 months of age (sham group) (Table 1, data only partially shown). In the femoral middiaphysis, periosteal MAR1 decreased with age, but otherwise there were no agerelated changes in structural geometrical properties of femoral cross sections (Table 2). In the distal femoral metaphysis, significant age-related decreases in BV/TV, Tb.Th, and Tb.N, as well as an increase in Tb.Sp, were seen. The respective mean (SEM) values were 35.4 (1.5%) for BV/TV, 72 mm (3 mm) for Tb.Th, 4.9/mm (0.1/mm) for Tb.N, and 132 mm (5 mm) for Tb.Sp in the baseline group and 24.5% (1.7%), 60 mm (5 mm), 4.1/mm (0.2/mm), and 186 mm (9 mm) for the sham group at 4 months after operation. In the L-4 vertebra, BV/TV and Tb.N were found to decrease and Tb.Sp to increase with age before the onset of treatment (Figure 2A–D). In a cross section of the femoral midneck, there were no age-related changes in bone morphometry from 6 to 10 months of age (Table 3) or in the
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Table 1. Histomorphometry of tibial diaphyseal cross section
Group
Time after operation (months)
Baseline
0
Periosteal surface MAR1 (mm/d)
Static morphometry
MAR2 (mm/d)
1.07 (0.12)
—
aa
Ct.Th (mm)
%Ct.Ar (%)
0.66 (0.01)
82.5 (0.6) a
%Ma.Ar (%) 17.3 (0.6)
Sham Ovx
4 4
0.54 (0.09) 0.47 (0.04)
0.46 (0.03) 0.71 (0.05)bbb
0.67 (0.01) 0.68 (0.01)
84.3 (0.5) 82.4 (0.4)bbb
15.7 (0.5) 17.5 (0.4)bbb
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
7 7 7 7 7
0.60 (0.10) 0.46 (0.07) 0.38 (0.07) 0.52 (0.04) 0.59 (0.02)
0.34 (0.03) 0.44 (0.04)bbb 0.46 (0.04)bbb 0.50 (0.04)bbb 0.52 (0.06)bbb
0.67 (0.02) 0.65 (0.02) 0.65 (0.02)c 0.73 (0.02)bb,ccc 0.74 (0.01)bbb,ccc
83.7 (0.6) 79.2 (1.0)bbb 79.4 (0.9)bbb 82.2 (0.6)c 82.5 (0.9)cc
16.1 (0.6) 20.7 (1.0)bbb 20.4 (0.9)bbb 17.8 (0.6)c 17.4 (0.9)cc
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
10 10 10 10 10
0.52 (0.07) 0.42 (0.07) 0.47 (0.05) 0.45 (0.05) 0.47 (0.08)
0.23 (0.03) 0.39 (0.03)bbb 0.36 (0.04)bbb 0.38 (0.02)bbb 0.40 (0.05)bbb
0.64 (0.02) 0.62 (0.01) 0.69 (0.01)c 0.68 (0.01)bb,ccc 0.71 (0.01)bbb,ccc
80.6 (1.0) 78.4 (0.7)bbb 81.5 (0.7)cc 81.6 (0.9)cc 83.3 (0.7)b,ccc
19.1 (1.0) 21.3 (0.7)bbb 18.0 (0.8)cc 18.4 (0.9)c 16.5 (0.7)b,ccc
KEY: MAR, mineral apposition rate; Ct.Th, cortical thickness; %Ct.Ar, percent cortical bone area; %Ma.Ar, percent marrow area; MAR1, interlabel time during the 21 days between oxytetracycline and the second calcein injections, in the baseline group 14 days; MAR2, timeframe of the entire experimental period between the first and second calcein labels. Data expressed as mean (SEM), n 5 5– 8 per group. Sham (at 4 months after operation) vs. baseline: ap , 0.05; aap , 0.01. Ovx or ovx/C vs. sham: bp , 0.05; bbp , 0.01; bbbp , 0.001. Ovx/C vs. ovx: cp , 0.05; ccp , 0.01; cccp , 0.001.
cancellous bone variables BV/TV, Tb.Th, Tb.N, or Tb.Sp (data not shown). Ovx-Related Changes in Bone Ovariectomy decreased (p , 0.001) the ash weight of the whole femur at each follow-up timepoint after operation, the respective mean (SEM) values being 433 mg (8 mg) in the
sham group and 409 mg (5 mg) in the ovx group at 4 months after ovx. The maximum loads of both femoral neck and L-5 vertebra also decreased due to estrogen deficiency at each follow-up timepoint after operation, but the maximum load in the three-point bending of the femoral shaft decreased only at 7 and 10 months after operation. In the tibia, the bending strength was, by contrast, increased at each timepoint after ovx (Figure 3A–D).
Table 2. Histomorphometry of femoral middiaphyseal cross section
Group Baseline
Periosteal surface
Time after operation (months)
MAR1 (mm/d)
0
0.94 (0.09)
MAR2 (mm/d)
a
Static morphometry Ct.Th (mm)
%Ct.Ar (%)
%Ma.Ar (%)
Ix (mm4)
—
0.69 (0.01)
66.5 (1.1)
33.4 (1.0)
3.91 (0.31)
0.59 (0.04) 0.83 (0.05)bbb
0.70 (0.02) 0.73 (0.01)
66.3 (1.3) 68.3 (0.6)
33.5 (1.3) 31.6 (0.6)
4.28 (0.30) 4.16 (0.13)
Sham Ovx
4 4
0.62 (0.04) 0.59 (0.03)
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
7 7 7 7 7
0.53 (0.05) 0.57 (0.12) 0.47 (0.02) 0.50 (0.02) 0.54 (0.04)
0.50 (0.03) 0.55 (0.04) 0.52 (0.04) 0.55 (0.04) 0.55 (0.04)
0.74 (0.01) 0.68 (0.01)b 0.70 (0.01)bbb,c 0.72 (0.01)b,ccc 0.73 (0.01)ccc
68.0 (1.2) 60.6 (1.5)bbb 62.7 (1.1)bbb 64.5 (0.9)bb,cc 64.9 (0.5)bb,cc
31.7 (1.2) 39.1 (1.4)bbb 37.0 (1.1)bbb 35.1 (1.0)bb,cc 34.9 (0.5)bb,cc
4.57 (0.30) 5.66 (0.38) 5.83 (0.24)bbb 5.52 (0.19)bb 5.81 (0.18)bb
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
10 10 10 10 10
0.51 (0.07) 0.39 (0.04) 0.42 (0.04) 0.38 (0.03) 0.43 (0.02)
0.53 (0.03) 0.52 (0.03) 0.50 (0.04) 0.55 (0.03) 0.51 (0.02)
0.77 (0.02) 0.66 (0.02)bbb 0.70 (0.01)bbb,c 0.73 (0.01)b,ccc 0.73 (0.02)ccc
70.6 (0.8) 63.2 (1.3)bbb 63.5 (1.6)bbb 66.6 (1.0)bb,cc 66.1 (1.1)bb,cc
29.3 (0.8) 36.6 (1.3)bbb 36.0 (1.6)bbb 33.1 (1.0)bb,cc 33.7 (1.0)bb,cc
4.61 (0.33) 4.70 (0.30) 5.34 (0.29)bbb 5.10 (0.25)bb 5.05 (0.20)bb
KEY: MAR, mineral apposition rate; Ct.Th, cortical thickness; %Ct.Ar, percent cortical bone area; %Ma.Ar, percent marrow area; Ix, cross-sectional moment of inertia; MAR1, interlabel time during the 21 days between oxytetracycline and the second calcein injections, in the baseline group 14 days; MAR2, timeframe of the entire experimental period between the first and second calcein labels. Data expressed as mean (SEM), n 5 6 – 8 per group. Sham (at 4 months after operation) vs. baseline: ap , 0.01. Ovx or ovx/C vs. sham: bp , 0.05; bbp , 0.01; bbbp , 0.001. Ovx/C vs. ovx: cp , 0.05; ccp , 0.01; cccp , 0.001.
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Figure 2. Cancellous bone variables of the L-4 vertebral body. (A) Bone volume (BV/TV), (B) trabecular number (Tb.N), (C) trabecular thickness (Tb.Th), and (D) trabecular separation (Tb.Sp). Sham (at 4 months after operation) vs. baseline: ap , 0.05; aap , 0.01. Ovx or ovx/C vs. sham: *p , 0.05; **p , 0.01; ***p , 0.001; ovx/C vs. ovx: #p , 0.05; ##p , 0.01; ###p , 0.001. The values are expressed as mean 6 SEM, n 5 7 or 8/group. The mean of baseline values at the age of 6 months is marked by a dotted line in each figure.
There were no significant ovariectomy-induced effects on periosteal O.Th, MS/BS, Mlt, MAR1, or BFR/BS of the tibial diaphysis (Table 1, data only partially shown; Figure 4) or on MAR1 in femoral diaphysis at any follow-up timepoint (Table 2). In contrast, Ps.MAR2, analyzed for the entire experimental period, was higher at each follow-up timepoint in the tibial diaphysis, but only at 4 months after ovx in the femoral diaphysis (Tables 1 and 2). The cross-sectional tissue and marrow area of the tibia and femur were greater in ovx than in sham rats (data not shown). Tibial %Ct.Ar decreased and %Ma.Ar increased as a result of estrogen deficiency (Table 1). Ovariectomy was also found to increase the femoral cross-sectional %Ma.Ar and to decrease Ct.Th and %Ct.Ar, resulting in cortical osteopenia at 7 and 10 months after operation. However, the cross-sectional second moment of inertia in the femoral middiaphysis did not change due to estrogen deficiency (Table 2). Ovariectomy decreased the BV/TV of the distal femoral metaphysis by 48% already at 4 months after operation, which
was manifested by reduced Tb.Th and Tb.N, and increased Tb.Sp. The respective mean (SEM) values were 24.5% (1.7%) for BV/TV, 60 mm (5 mm) for Tb.Th, 4.1/mm (0.2/mm) for Tb.N, and 186 mm (9 mm) for Tb.Sp in the sham group and 12.8% (0.5%), 51 mm (2 mm), 2.5/mm (0.1/mm), and 365 mm (15 mm) in the ovx group. In the L-4 vertebra, ovariectomy decreased BV/TV and Tb.Th, and increased Tb.Sp, but the change in Tb.N was nonsignificant (Figure 2). Cancellous osteopenia proceeded in ovx rats at 7 and 10 months after operation. Estrogen deficiency of 10 months decreased %TB.Ar, %Ct.Ar, and Ct.Th, and increased %Ma.Ar in the femoral midneck (Table 3). Within the endocortical envelope of femoral midneck, there was a significant decrease in Tb.N and an increase in Tb.Sp at each follow-up timepoint after ovariectomy, although the decrease in BV/TV was not significant. The respective mean (SEM) values were 38.9% (2.1%) for BV/TV, 4.1/mm (0.1/mm) for Tb.N, and 148 mm (7 mm) for Tb.Sp in the sham
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Table 3. Bone morphometry of femoral midneck cross section
Group
Time after operation (months)
%TB.Ar (%)
%Ma.Ar (%)
%Ct.Ar (%)
Ct.Th (mm)
Ix (mm4)
Ps.MS/BS (%)
Baseline
0
88.0 (1.5)
10.5 (1.5)
79.2 (2.7)
0.59 (0.02)
0.85 (0.06)
28.0 (5.4)
Sham Ovx
4 4
89.5 (0.8) 90.8 (1.3)
9.0 (0.7) 8.2 (1.2)
83.3 (1.0) 86.1 (2.1)
0.61 (0.02) 0.65 (0.04)
0.71 (0.06) 0.86 (0.07)
30.0 (6.6) 29.9 (6.7)
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
7 7 7 7 7
87.3 (2.0) 86.4 (0.6) 87.7 (1.2) 86.2 (0.8) 84.0 (0.6)
11.4 (1.7) 12.8 (0.7) 11.2 (1.2) 12.9 (0.7) 15.3 (0.6)a
79.7 (3.0) 78.8 (0.4) 77.8 (2.8) 79.9 (1.8) 75.0 (1.4)
0.60 (0.04) 0.60 (0.01) 0.56 (0.02) 0.61 (0.02) 0.55 (0.01)
0.93 (0.10) 1.12 (0.11) 1.02 (0.15) 1.05 (0.07) 1.15 (0.07)
17.6 (3.7) 19.9 (4.8) 10.4 (3.1)a,b 7.8 (3.0)aa,bb 1.0 (0.7)aaa,bbb
Sham Ovx Ovx/C36 Ovx/C84 Ovx/C300
10 10 10 10 10
90.4 (1.6) 84.9 (0.4)aa 86.3 (1.2)a 88.1 (1.2)b 88.9 (2.1)b
8.8 (1.6) 14.1 (0.7)aa 12.3 (1.2)a 11.3 (1.3) 10.2 (1.7)b
86.3 (1.6) 78.8 (1.3)aa 81.0 (1.3) 83.4 (1.6) 83.8 (4.0)
0.70 (0.02) 0.57 (0.01)aa 0.60 (0.02)aa 0.62 (0.02)a 0.64 (0.05)
1.07 (0.13) 0.92 (0.08) 0.94 (0.09) 0.91 (0.10) 0.95 (0.06)
17.2 (6.0) 14.4 (4.2) 5.5 (2.3)a,b 5.5 (2.0)aa,bb 3.5 (2.2)aaa,bbb
KEY: %TB.Ar, percent total bone area (includes cortical and cancellous bone); %Ma.Ar, percent marrow area; %Ct.Ar, percent cortical bone area; Ct.Th, cortical thickness; Ix, cross-sectional moment of inertia; Ps.MS/BS, periosteal mineralizing surface. %TB.Ar, %Ct.Ar, and %Ma.Ar are calculated as percentages of total cross-sectional tissue area. Sham (at 4 months after operation) vs. baseline not significant. Ovx or ovx/C vs. sham: ap , 0.05; aap , 0.05; aaap , 0.001. Ovx/C vs. ovx: bp , 0.05; bbp , 0.01; bbbp , 0.001. Data expressed as mean (SEM), n 5 4 – 8 per group.
group and 33.0% (4.5%), 3.3/mm (0.2/mm), and 210 mm (24 mm) in the ovx group at 4 months after operation. At 10 months after ovariectomy, double labeling of oxytetracycline and calcein could be found at cancellous bone surfaces of the L-4 vertebra in five of eight sham rats and in all ovx rats (n 5 8), resulting, however, in nonsignificant change in MS/BS (the mean [SEM] values were 6.4% [1.0%] and 6.9% [0.9%] for vehicle-treated sham and ovx groups, respectively). The endosteal MAR of the L-4 was significantly lower in ovx rats (0.39 mm/d [0.05 mm/d]) than in sham rats (0.60 mm/d [0.09 mm/d]). However, no significant differences occurred in BFR/BS between sham and ovx rats at 10 months after operation (data not shown). Effect of Clodronate on Established Osteopenia Clodronate increased the total ash weight of the femur and tibia (data not shown). In the femoral neck, the maximum load after clodronate treatment did not differ between vehicle-treated agematched ovx and sham groups, but in the L-5 vertebra all doses of clodronate improved the ovariectomy-induced impairment of compressive strength (Figure 3A,B). Clodronate also clearly prevented the ovx-induced decrease in the bending strength of the femoral shaft at all doses, the values being on a level with sham rats, and in the tibia clodronate further increased the ovx-induced increase in tibial bending strength (Figure 3C,D). There was a positive linear correlation between maximum load in three-point bending and ash weight in both the tibia (Figure 5) and the femur (n 5 196, r 5 0.84, p , 0.001). Clodronate did not induce any significant changes in terminal periosteal O.Th, MS/BS, Mlt, MAR1, or BFR/BS in tibial diaphysis (Table 1 data only partially shown; Figure 4) or in terminal MAR1 or MAR2 in femoral diaphysis (Table 2). Tibial periosteal MAR2 values were on a level with vehicle-treated ovx groups after each clodronate dose after 3 and 6 months of treatment (Table 1). Clodronate had no significant effect on the ovx-induced increase in tibial or femoral cross-sectional tissue
area (data not shown). However, clodronate did increase tibial and femoral %Ct.Ar in comparison to vehicle-treated ovx rats and, after 6 months of treatment, the highest cumulative dose (300 mg/kg) had even increased the tibial %Ct.Ar to above the level of sham controls (Tables 1 and 2). Furthermore, clodronate inhibited the ovx-induced enlargement of %Ma.Ar in tibial and femoral diaphysis (Tables 1 and 2). After the cumulative doses of clodronate (84 and 300 mg/kg), tibial Ct.Th was higher than in vehicle-treated sham or ovx groups, and the decrease in Ct.Th of femoral diaphysis was inhibited compared with the vehicletreated ovx rats (Tables 1 and 2). After 6 months of clodronate treatment, significantly higher BV/TV and Tb.N, and lower Tb.Sp, were seen in the distal femur at each dose level when compared with vehicletreated ovx rats. The mean (SEM) values of BV/TV were 7.8% (0.7%) for the vehicle-treated ovx group and 12.1% (1.0%), 11.9% (0.8%), or 11.9% (0.8%) for the clodronatetreated groups (cumulative doses 36, 84, or 300 mg/kg), respectively. After 3 months of treatment, this effect was not yet seen (data not shown). In the L-4 vertebra, clodronate (cumulative dose 36 mg/kg) slightly counteracted the ovariectomy-induced decrease in the BV/TV as a result of increased Tb.N and decreased Tb.Sp (Figure 2A–D). In femoral midneck, after 6 months of clodronate treatment, %TB.Ar was significantly higher (cumulative doses 84 and 300 mg/kg) and %Ma.Ar lower (cumulative dose 300 mg/kg) than in vehicle-treated ovx rats (Table 3). After 3 or 6 months of clodronate treatment, periosteal MS/BS was significantly lower than in age-matched vehicle-treated ovx or sham rats, due to reduced double labeling of fluorochromes (Table 3). In cancellous bone of the femoral midneck, Tb.N and Tb.Sp, after clodronate treatment for 3 or 6 months, did not significantly differ from the vehicle-treated ovx and sham groups (data not shown). Percent mineralizing surface (n 5 8) was lower (mean [SEM] 2.2% [0.3%]) in the secondary spongiosa of the L-4 vertebra after 6 months of clodronate treatment (cumulative dose 36
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Figure 3. Maximum loads of femoral neck cantilever bending (A), L-5 vertebral compression (B), and three-point bending of femoral (C) and tibial shafts (D). Ovx or ovx/C vs. sham: *p , 0.05; **p , 0.01; ***p , 0.001; ovx/C vs. ovx: ##p , 0.01; ###p , 0.001. The values are expressed as mean 6 SEM, n 5 14 –25/group.
mg/kg, which was the only dose level tested) than in the agematched vehicle-treated ovx or sham groups. The decreased Es.MS/BS was due to reduced double labeling of fluorochromes along cancellous bone surfaces after clodronate treatment, and interlabel thickness could be measured and MAR calculated in only two of eight rats. As a result, BFR/BS values of cancellous bone were also lower after clodronate treatment compared with vehicle groups (data not shown). Oxytetracycline and calcein occurred only as single labels on cancellous bone surfaces of the L-4 vertebra after higher doses of clodronate. Biochemical Variables Ovariectomy caused increases in serum osteocalcin and urinary excretion of deoxypyridinoline, which were prevented by all doses of clodronate (Figure 6A,B). After clodronate administration at cumulative doses of 84 and 300 mg/kg, the values of both variables were lower than in sham rats. Ovariectomy decreased serum estradiol at each follow-up timepoint after operation (data not shown).
Discussion Our results from the present study show that clodronate suppressed, at all dosage levels, the ovariectomy-induced increase in bone turnover in adult rats. Urinary excretion rates of deoxypyridinoline and serum osteocalcin decreased to normal after the lowest dosage regimen of clodronate, and below the level of sham rats after higher doses of clodronate. Clodronate did not induce any significant change in the indices of bone formation or mineralization at the periosteal surfaces of long bones, but depressed cancellous bone formation of L-4 vertebra, suggesting that the effect of clodronate on bone formation is exerted only through remodeling. This is in accordance with earlier observations with pamidronate, risedronate, and alendronate.4,6,32 The biomechanical behavior of a bone reflects its material and geometric properties. Both the cross-sectional area and the moment of inertia, which expresses the distribution of material around a given axis, are important for the mechanical behavior of long bones.11,38 Our study showed that bone geometry was altered after ovx as a result of increased periosteal apposition and
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Figure 4. Fluorescent micrographs of periosteal surface (Ps) of tibial diaphysis at 10 months after operation in ovx rats treated subcutaneously once per week for 6 months with vehicle (A) or clodronate at a dose level of 1.5 mg/kg (B). First and second calcein labels (filled arrows), one tetracycline label (open arrow), and osteoid seam (O) can be seen. Villanueva osteochrome bone stain. Bar 5 50 mm.
endocortical resorption in long bones. Cortical bone loss and weakening of the femoral shaft reached significant levels only at 7 and 10 months after ovariectomy, which is in accordance with a previous study by Toolan et al.37 In the tibia, the bending strength was, by contrast, increased at each timepoint (4, 7, and 10 months) after ovx. An obvious reason for this finding is the ovx induced periosteal bone growth in tibial diaphysis, as judged by the higher mineral apposition rate determined for the entire experimental period in ovx rats than in the age-matched sham rats. This resulted in continued expansion of the tibial cross-
Figure 5. Relationship between maximum load in three-point bending and ash weight of the tibia (n 5 290).
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Figure 6. Serum osteocalcin (A) and urinary excretion of deoxypyridinoline per creatinine (B). Ovx or ovx/C vs. sham: *p , 0.05; ***p , 0.001; ovx/C vs. ovx: ###p , 0.001. Values are expressed as mean 6 SEM, n 5 22–25/group.
sectional area, which is an important factor in determining the bending strength of the tibial shaft. In the femoral diaphyseal cross section, clodronate treatment increased the moment of inertia at 7 and 10 months after operation compared with age-matched sham controls, indicating that the increased diameter of the bone distributed the material further from the neutral axis and improved the resistance of the bone to bending loads. Furthermore, increases in tibial cortical bone area and thickness as effects of clodronate treatment produced the greatest mechanical advantage to the maximum load of the tibial shaft. Clodronate treatment increased the ash weight of long bones, suggesting that the treatment affects cortical as well as cancellous bone. Furthermore, it should be noted that a significant correlation between the ash weight and maximum load in three-point bending was found in both the femur and the tibia. This suggests that the increase in bone mineral content is reflected in mechanical strength. Changes in the maximum load at the femoral neck revealed a deteriorating effect of ovariectomy already at 4 months after operation, as described previously,29,36 but changes did not differ significantly between clodronate-treated ovx rats when compared with vehicle-treated ovx and sham groups. Similarly, in earlier studies with other bisphosphonates (alendronate, pamidronate, and risedronate), no beneficial effect on femoral neck strength was found.2,18,36,37 The reason for this unexpected finding is unknown. Although both decreased bone mass and impaired trabecular architecture would produce a structurally weaker femoral neck, the exact contributions of cortical and cancellous bone
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on mechanical strength in the rat femoral neck are not yet completely understood. The irreversible changes that were found in the femoral midneck cancellous bone had already taken place before the onset of the clodronate treatment. This was evidenced by decreased trabecular number and increased separation in ovx rats, in agreement with previous studies.3,15,20,21,41 After clodronate treatment, the values of trabecular number and separation did not significantly differ from the vehicle-treated ovx and sham groups. Cortical bone and inner trabeculae are connected in the femoral neck, as shown in the present study and by others,3,13,20,21,41 and the reduced endocortical-trabecular connectivity will probably also contribute to the deterioration of the mechanical properties of the femoral neck.3 Cortical osteopenia including reduced cortical thickness, and enlargement of marrow area, developed at later stages of estrogen deficiency. The effect was significant only at 10 months after ovx, as also shown by others.13,20,21 Similarly, the relative area of total bone (trabecular bone and cortical shell) significantly decreased at 10 months after ovx, and clodronate treatment (84 or 300 mg/kg) prevented this bone loss. However, the cross-sectional moment of inertia in the femoral midneck did not differ between groups in the present study, showing equal distribution of effective bone mass in rat femoral neck among the groups. Furthermore, the fact that clodronate does not induce any change in femoral neck width of ovx rats15 also partially explains why it could not improve the resistance of the femoral neck to bending and compressive loads in cantilever bend testing in the osteopenia model presented. Because of the high surface:volume ratio of cancellous bone, disorders of bone remodeling, such as osteoporosis, commonly affect trabecular sites earlier and more profoundly than cortical sites.14 This was also shown here, as evidenced by cancellous bone loss of 48% in the distal femur at 4 months after operation, the loss being much faster than in the proximal femur. This may be due to variations in mechanical loading and longitudinal bone growth rate between distal and proximal sites of the femur. With respect to severe osteopenia, 3 months of clodronate treatment could not prevent cancellous bone loss in the distal femur at any dose. However, 6 months treatment was efficient in this respect at each dose level. The trabecular number was higher and trabecular separation lower than in vehicle-treated ovx rats, indicating less damage to trabecular microarchitecture after longterm clodronate treatment. In the lumbar vertebral body, clodronate treatment for 3 or 6 months could not restore lost cancellous bone. Only the lowest clodronate dose slightly counteracted the ovariectomy-induced further decrease in cancellous bone volume, which was due to a higher trabecular number and lower trabecular separation in clodronate-treated ovx rats than in the corresponding ovx vehicle group. It is of interest, however, that the ovariectomy-induced impairment of lumbar vertebral compression strength was significantly improved at all clodronate dosages after 3 and 6 months of treatment. Compression testing of the vertebral body is presently used, although the exact contribution of cortical shell and cancellous bone to the strength of rat vertebral body is uncertain.37,38 The thickness of the vertebral cortex has been shown to be an important determinant of compressive strength of the whole vertebral body31,39 and, in patients with osteoporosis, vertebral cortex assumes more significance in bearing loads.22 The ability of clodronate to improve the impaired vertebral strength in established osteopenia can thus be due mostly to the beneficial effect of clodronate on cortical bone. In summary, the present study has shown that clodronate treatment starting 4 months after ovariectomy and continued for 3 to 6 months inhibited bone resorption as assessed by urinary deoxypyridinoline:creatinine ratio. It also inhibited bone turn-
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over in regions of remodeling as assessed by endosteal bone formation rate in the lumbar vertebra and by serum osteocalcin levels. However, there was no evidence of direct inhibition of modeling-dependent bone formation or impairment of mineralization as a result of clodronate treatment. This suggests that long-term clodronate treatment can be used as a safe and effective method for inhibiting bone resorption and suppressing increased bone turnover due to estrogen deficiency. The results further suggest that clodronate, like other bisphosphonates, is more effective in the prevention of experimental osteopenia than in the treatment of established disease, the latter requiring restoration of skeletal mass and bone microarchitecture.
Acknowledgments: The authors gratefully acknowledge the expert technical assistance of the staff of Preclinical Research at Leiras Oy and Allan Haimakainen. Also, we are grateful to Juhani Tuominen, Ph.Lic., for consulting in statistics.
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Date Received: November 13, 1997 Date Revised: June 1, 1998 Date Accepted: June 1, 1998