Full text of DOI:10.1359/jbmr.2002.17.8.1498

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 8, 2002
©
2002 American Society for Bone and Mineral Research
Prevention of Bone Loss in Ovariectomized Rats by
Combined Treatment With Risedronate and
1␣,25-Dihydroxyvitamin D3
1
2
2
1
1
REINHOLD G. ERBEN, LIS MOSEKILDE, JESPER S. THOMSEN, KARIN WEBER, KERSTIN STAHR,
3
3
4
ALYSON LEYSHON, SUSAN Y. SMITH, and ROGER PHIPPS
ABSTRACT
Bisphosphonates inhibit bone loss through inhibition of osteoclast-mediated bone resorption. At low doses, vitamin
D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses
they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphospho-
nates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of
this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1␣,25-
dihydroxyvitamin D (calcitriol), alone and in combination, for the prevention of ovariectomy-induced bone loss in
3
rats. One hundred ten female 4-month-old Sprague–Dawley rats were used for this experiment. Ninety rats were
bilaterally ovariectomized (OVX), 10 rats were sham-operated (SHAM), and 10 rats were killed at the time of
surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of
risedronate or 0.05 ␮g or 0.1 ␮g of calcitriol/kg body weight, alone and in combination. Both compounds were
administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency–induced bone
loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a
combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and
bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol
enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive
effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedr-
onate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in
calcitriol-treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphospho-
nates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs
alone. (J Bone Miner Res 2002;17:1498–1511)
Key words: bisphosphonates, vitamin D, osteoporosis, histomorphometry, ovariectomy
(
1)
INTRODUCTION
osteoclast apoptosis. Bisphosphonates prevent estrogen
(2,3)
deficiency–induced bone loss in animals,
drugs also are very effective in the prevention and treatment
of postmenopausal osteoporosis.
and these
EWLY DEVELOPED bisphosphonates such as risedronate
and alendronate are potent inhibitors of the bone re-
sorptive capacity of mature osteoclasts and also may induce
N
(4,5)
However, although
bisphosphonates increase bone mineral density (BMD) at
several bone sites and significantly reduce spine and hip
fracture rates, they do not appear to increase cancellous
bone area (B.Ar) measured by histomorphometry in patients
Dr. Erben serves as a consultant. Dr. Phipps has financial inter-
ests in the forms of corporate appointments and stock ownership.
He also is an employee at Procter & Gamble. All other authors
have no conflict of interest.
(
6,7)
with osteoporosis.
1
Institute of Physiology, Physiological Chemistry, and Animal Nutrition, Ludwig Maximilians University, Munich, Germany.
Department of Cell Biology, Institute of Anatomy, University of Århus, Århus, Denmark.
ClinTrials BioResearch, Senneville, Quebec, Canada.
2
3
4
Procter & Gamble Pharmaceuticals, Miami Valley Labs, Cincinnati, Ohio, USA.
1
498

CALCITRIOL AND RISEDRONATE IN OVX RATS
1499
Therefore, a combination of an antiresorptive and a bone
anabolic drug may be a powerful treatment option for os-
teoporosis. However, data to support this notion are con-
flicting. A study in ewes has shown that the bisphosphonate
tiludronate blocked the bone anabolic actions of parathyroid
TABLE 1. STUDY DESIGN
Group
N
Status
Treatment
Dose
BL
1
2
3
4
5
6
7
10
Intact
BL
—
—
—
10 SHAM
Vehicle
(8)
hormone (PTH) in this animal model. Similarly, when rats
were pretreated with alendronate for 2–4 months, the bone
anabolic response to subsequent treatment with high doses
of a PTH analog was diminished in the tibia and femur
compared with animals that did not receive the bisphospho-
10
10
10
10
10
10
OVX
OVX
OVX
OVX
OVX
OVX
Vehicle
Risedronate
Risedronate
Calcitriol
Calcitriol
Risedronate
Calcitriol
Risedronate
Calcitriol
Risedronate
Calcitriol
Risedronate
Calcitriol
0.1 mg/kg per day
0.5 mg/kg per day
0.05 ␮g/kg per day
0.1 ␮g/kg per day
0.1 mg/kg per day
0.05 ␮g/kg per day
0.1 mg/kg per day
0.1 ␮g/kg per day
0.5 mg/kg per day
0.05 ␮g/kg per day
0.5 mg/kg per day
0.1 ␮g/kg per day
(9)
nate pretreatment. On the other hand, the anabolic activity
of PTH is not hampered when antiresorptive agents are
(10–13)
coadministered in rodents,
and a pilot study in a small
8
9
10
10
10
OVX
OVX
OVX
number of postmenopausal women has suggested that alen-
dronate does not block the bone anabolic response to PTH
(
14)
in humans.
1
0
The role of vitamin D metabolites in the treatment of
osteoporosis is still controversial. Some clinical studies
have suggested that therapy of patients with osteoporosis
with active vitamin D metabolites increases BMD in the
BL, baseline.
(
15–18)
spine and forearm and reduces vertebral fractures.
(
19,20)
However, other studies have provided negative results.
In addition, the use of vitamin D metabolites in patients with tum to all rats except for a period from 4 h before dosing
osteoporosis, especially at higher doses, has been hampered until 2 h after dosing with risedronate. Water was available
by the major side effects of this compound class, hypercal- ad libitum.
cemia and hypercalciuria, because of excessive stimulation
Animals were randomized into the groups (10/group)
of intestinal calcium absorption. Although the results of shown in Table 1. Animals received daily doses of both
clinical trials have been ambiguous, studies in rats have vehicles (risedronate and calcitriol, groups 1 and 2) or 0.1
shown clearly that active vitamin D metabolites can fully mg/kg or 0.5 mg/kg of risedronate and calcitriol vehicle
prevent estrogen deficiency–induced loss of cancellous and (groups 3 and 4) or 0.05 ␮g/kg or 0.1 ␮g/kg of calcitriol and
(
21–24)
cortical bone by suppression of bone resorption.
At risedronate vehicle (groups 5 and 6) or a combination of
higher doses, vitamin D metabolites are bone anabolic and risedronate and calcitriol (groups 7–10). Starting the day
increase osteoblast team performance by augmenting osteo- after surgery, both compounds and their vehicles were ad-
(23–27)
blast activity and proliferation of osteoblast precursors.
ministered orally via gavage daily for 12 weeks at a dose
The aim of this study was to compare the efficacy of volume of 10 ml/kg. Risedronate was dissolved in sterile
risedronate and of the naturally occurring vitamin D hor- saline, and calcitriol was dissolved in 0.1% Tween 20. The
mone 1␣,25-dihydroxyvitamin D (calcitriol), alone or in dosing solution for calcitriol was prepared freshly each day
3
combination, for the prevention of ovariectomy-induced from a stock solution of calcitriol dissolved in 100% etha-
bone loss in rats. The data provided by this experiment nol. To prevent interference with risedronate absorption,
indicate that combined therapy with bisphosphonates and calcitriol or calcitriol vehicle was administered in the morn-
calcitriol may offer advantages over the treatment with ing and risedronate or risedronate vehicle was administered
bisphosphonates or calcitriol alone.
in the afternoon.
Body weights were taken weekly. All animals were given
subcutaneously a single tetracycline label (25 mg/kg) on
day 1 of the study and a calcein double label (8 mg/kg) 12
days and 2 days before the rats were killed. After an
overnight period of food deprivation, all rats were killed by
MATERIALS AND METHODS
Animal procedures
All animal procedures were conducted in accordance with exsanguination from the abdominal aorta under isoflurane
the United States Department of Agriculture (USDA) Ani- anesthesia 12 weeks postovariectomy. Blood samples for
mal Welfare Regulations. One hundred ten female ϳ4- PTH measurements were obtained from the tail vein imme-
month-old Sprague–Dawley CD rats (Charles River Labo- diately before necropsy. Serum samples were stored at
ratories, Raleigh, SC, USA) weighing 250–310 g were used Ϫ80°C until assayed. Success of ovariectomy was con-
for this experiment. Ninety rats were ovariectomized (OVX) firmed by lack of ovarian tissue at necropsy and a marked
bilaterally by dorsal approach under isoflurane anesthesia, reduction in uterine weight.
1
0 rats were sham-operated (SHAM), and 10 rats were
killed at the time of surgery to serve as a baseline control.
The animals were housed individually at 22°C with a 12-h
light/12-h dark cycle and were fed a standard laboratory
Serum analysis
Serum samples were analyzed for calcium, phosphorus,
chow (PMI 5002; PMI, Montreal, Canada) containing 0.8% creatinine, urea, alkaline phosphatase, and PTH. Serum
calcium and 0.6% phosphorus. Food was available ad libi- calcium, alkaline phosphatase activity, blood urea nitrogen

1
500
ERBEN ET AL.
(BUN), creatinine, and phosphorus were analyzed on a Histology
Hitachi 717 autoanalyzer (Boehringer, Mannheim, Ger-
many). Serum PTH was measured with a rat-specific immu-
noradiometric assay reacting with both N-terminal and intact
PTH (Nichols, San Juan Capistrano, CA, USA). The intra-
and interassay variabilities of this assay were below 10%.
At necropsy, the fourth lumbar vertebrae and the proxi-
mal right tibias were defleshed and fixed in 70% ethanol at
room temperature. After fixation, the bones were embedded
undecalcified in methylmethacrylate as described previ-
(28)
ously.
Five-micrometer-thick undecalcified sections
were prepared with an HM 360 microtome (Microm, Wall-
dorf, Germany). The sections were sampled in the median
plane of the vertebrae and the midsagittal plane of the tibias
BMD measurements
BMD of the left tibias and of the L3 vertebrae was
measured by DXA using a Hologic QDR 2000 plus bone
scanner (Hologic, Inc., Waltham, MA, USA). The scans of
the tibias were analyzed for BMD of the whole tibia and of
subregions that included the proximal, central, and distal
portions of the bone. The scans of the L3 vertebra were
analyzed for BMD of the whole bone and of a central
subregion that excluded the majority of cortical bone.
(29)
and were stained with von Kossa
and toluidine blue at
(30)
acid pH.
For demonstration of cement lines the surface
(28)
staining technique described by Schenk et al.
was used.
Histomorphometry
All measurements were performed on the secondary
spongiosa of the proximal tibial metaphysis and of the
fourth lumbar vertebral body. The measurements were made
in a blind fashion and are presented as two-dimensional
Biomechanical testing
Biomechanical testing of the fifth lumbar vertebral body, histomorphometric terms. Histomorphometric parameters
the femoral neck, the femoral diaphysis, and the distal were calculated and expressed according to the recommen-
femoral metaphysis was performed in a blind fashion with a dations made by the American Society for Bone and Min-
(31)
materials testing machine (Alwetron TCT 5; Lorentzen & eral Research (ASBMR) nomenclature committee.
Wettre, Stockholm, Sweden) on bones that had been frozen Structural histomorphometric measurements were made
at Ϫ20°C in saline-soaked gauze. All specimens were kept with an automatic image analysis system (VIDAS; C. Zeiss,
in Ringer’s solution after thawing. For all tests a constant Oberkocken, Germany) connected to a Zeiss stereo micro-
deformation rate of 2 mm/minute was used. Load- scope (C. Zeiss) via a TV camera (Bosch, Stuttgart, Ger-
deformation curves were recorded and analyzed by a com- many). All measurements with the automatic image analysis
puter. Biomechanical parameters evaluated include ultimate system were performed on sections stained with von Kossa
2
load (Fmax), maximum stress (␴max ϭ Fmax/cross-sectional at a magnification of ϫ2.0. A measuring field (ϳ18 mm in
2
area), stiffness, energy absorption (Wabs), and Young’s the tibias and ϳ12 mm in the vertebrae) encompassing
modulus (maximum slope of the stress-strain curve).
most of the cancellous bone in the L4 vertebrae and the
The L5 vertebra was mounted on a wooden block, and a tibias was used. The cancellous bone within 0.5 mm from
central specimen with a height of approximately 3.9 mm the growth plates was excluded from the measurements in
was prepared using a precision parallel saw (Exakt, Norder- both the tibias and the vertebrae. All values for individual
stedt, Germany). All remaining parts of the process were animals are the mean of two measurements made on two
removed using a fine electric saw (Minimot 40 E; Proxxon, different sections that were generally ϳ100 ␮m spaced
Niersbach, Germany). The length of the specimen was from each other. The image analysis system automatically
measured with a micrometer, and the volume was deter- determined the measuring area (tissue area [T.Ar]), B.Ar,
mined by Archimedes principle with an electronic balance and bone perimeter (B.Pm). From these data, the structural
(Mettler AG245; Mettler-Toledo, N a¨ nikon-Greifensee, parameters B.Ar (B.Ar/T.Ar), B.Pm (B.Pm/T.Ar), trabecu-
Switzerland). The vertebral bodies were tested along the lar width (Tb.Wi), trabecular number (Tb.N), and trabecular
cephalocaudal axis. After biomechanical testing, the speci- separation (Tb.Sp) were calculated.
mens were ashed, and the ash content was expressed as
milligrams of ash per cubic millimeter of specimen volume histomorphometric measurements were made using a semi-
ash density). automatic system (Videoplan; C. Zeiss) and a Zeiss Axio-
The right femur was used for a three-point bending test of skop microscope with a drawing attachment. In the cancel-
Cellular, dynamic, and bone structural unit (BSU)-related
(
the diaphysis, followed by compression tests of the distal lous bone of the fourth lumbar vertebra and the proximal
2
metaphysis and the femoral neck. Biomechanical testing of tibial metaphysis ϳ30 fields (7–8 mm ) were evaluated in
the femoral neck was performed by applying vertical load each section. At least 20 mm of cancellous B.Pm was
onto the femoral head in a direction parallel to the axis of sampled in the tibia and the vertebra of each animal. The
the femoral diaphysis. A 4-mm-thick section with planopa- area within 0.5 mm from the growth plates was excluded
rallel ends consisting of metaphyseal bone was sawed from from the measurements in tibial and vertebral cancellous
the distal part of the femora just over the patellofemoral bone. Static histomorphometric parameters were measured
joint cartilage. Measurements of the section volume and in sections stained with toluidine blue, and dynamic
biomechanical testing along the proximodistal axis then fluorochrome-based parameters were measured in unstained
were performed as described for the vertebral bodies. After sections.
biomechanical testing, the distal femoral metaphyseal spec-
imens were ashed.
The following primary parameters were determined at
magnification ϫ200: B.Pm, osteoid perimeter, osteoblast

CALCITRIOL AND RISEDRONATE IN OVX RATS
1501
perimeter, eroded perimeter, osteoclast perimeter, number
of osteoclasts, calcein double-labeled perimeter, and
tetracycline-labeled perimeter. Osteoid seams with a width
Ͻ2 ␮m were excluded from the measurements. Osteoblasts
were defined as mononuclear, basophilic cells in contact
with osteoid. Osteoclasts were defined as large, irregularly
shaped cells with a foamy, slightly metachromatic cyto-
RESULTS
Unless otherwise specified, effects of ovariectomy are
from comparisons of OVX controls versus SHAM controls.
Effects of risedronate or calcitriol are from comparisons of
OVX treatment groups versus OVX controls.
plasm containing one or more nuclei and located within Body weight and serum biochemistry
Howship’s lacunae. Osteoid width was determined directly
OVX rats showed increased body weight by the end of
at magnification ϫ400, sampling each osteoid seam every
0 ␮m. Osteoid seams with a width Ͻ2 ␮m were not
the experiment (Table 2). Treatment with risedronate or
calcitriol had no influence on the ovariectomy-induced in-
crease in body weight. Calcitriol given alone produced a
decrease in BUN levels, a dose-dependent increase in serum
calcium, and a suppression of serum PTH concentrations.
Risedronate, especially at the high dose of 0.5 mg/kg,
tended to suppress the increase in serum calcium and the
subsequent inhibition of PTH secretion induced by calcit-
riol.
5
included. For the measurement of eroded perimeter, only
erosions with a depth of at least 3 ␮m were included into the
measurements.
The interlabel width between calcein double labels was
measured at magnification ϫ400, sampling each double
label every 50 ␮m. The mineral apposition rate (MAR)
was given by the arithmetic mean of the width measure-
ments, divided by the interlabel period (10 days). Bone
formation rate (BFR/B.Pm) was calculated by multiply-
ing mineralizing perimeter (double-labeled B.Pm) by the
MAR. The wall width of completed cancellous bone
structural units was measured at magnification ϫ200.
The distance between the reversal line and the bone
surface was measured at four equidistant points in at least
BMD measurements
Ovariectomy reduced BMD in the whole tibia, proximal
tibia, whole L3 vertebra, and L3 vertebra core (data only
partially shown; Fig. 1). The central tibia and the distal tibia
did not show significant loss of BMD in response to ovari-
ectomy in this study (data not shown). Changes in BMD
were very similar in the whole tibia compared with the
proximal tibia as well as in the whole L3 vertebra compared
with the L3 vertebral core. Therefore, effects on BMD in
proximal tibia and L3 vertebral core only are presented.
Both risedronate and calcitriol given alone provided dose-
dependent protection against ovariectomy-induced loss of
BMD at all sites. Generally, combined treatment with
risedronate and calcitriol resulted in higher BMD values at
all sites when compared with the individual treatment com-
ponents. In the animals treated with low-dose risedronate
1
5 remodeling sites in each animal. Values for MAR,
osteoid width, and wall width were not corrected for
obliquity of the plane of sectioning.
Statistical analysis
The biochemical and BMD data for the baseline control
and the vehicle-treated SHAM (group 1) and OVX (group
2
) groups were analyzed using one-way ANOVA. When the
ANOVA performed over all groups indicated a significant
p Ͻ 0.05) difference among the groups, statistical differ-
(
ences between individual groups were subsequently evalu-
ated using Tukey’s multiple comparison test as a post hoc
test. Because histomorphometry was not performed on the
baseline group, statistical comparisons between the SHAM
ϩ vehicle group and the OVX ϩ vehicle group were made
using a two-sided t-test for unpaired samples.
The biochemical, histomorphometric, and BMD data for
the OVX groups (groups 2–10) were analyzed by ANOVA
followed by Tukey’s multiple comparison test. Results for
Tukey’s test are reported for the comparison between indi-
vidual treatment groups and the OVX vehicle control, as
well as for the comparison between combination treatments
and the respective individual treatment components, for
(
␮
0.1 mg/kg) in conjunction with high-dose calcitriol (0.1
g/kg), BMD at the whole tibia, proximal tibia, whole L3
vertebra, and L3 vertebral core was significantly greater
than that of the group receiving low-dose risedronate alone.
On the other hand, the increase in BMD with calcitriol was
not significantly augmented by low-dose risedronate at any
site. High-dose risedronate (0.5 mg/kg) in combination with
low- (0.05 ␮g/kg) or high-dose calcitriol resulted in a sig-
nificant increase in BMD in whole and proximal tibia as
well as in L3 vertebra and L3 vertebral core compared with
the group treated with high-dose risedronate alone. In con-
trast with the results obtained for combined treatment with
low-dose risedronate, combination of high-dose risedronate
example, 0.1 mg/kg of risedronate plus 0.05 ␮g/kg of cal- with low- or high-dose calcitriol significantly enhanced the
citriol (group 7) versus 0.1 mg/kg of risedronate (group 3) positive effects of calcitriol on BMD at the whole and
and versus 0.05 ␮g/kg of calcitriol (group 5).
proximal tibia but not in the vertebra.
Because most of the biomechanical data were not distrib-
High-dose risedronate and high-dose calcitriol as well as
uted normally, these data were analyzed by the nonpara- all combination treatments (with the exception of low-dose
metric Kruskal-Wallis test, including a multiple comparison risedronate plus high-dose calcitriol) also increased BMD in
procedure.
distal tibia relative the OVX controls (data not shown). We
The values of p Ͻ 0.05 were considered significant for all did not observe any significant treatment effects in the
statistical analyses. The data are presented as means Ϯ SD. central tibia (data not shown).

1
502
ERBEN ET AL.
TABLE 2. BODY WEIGHT AND SERUM BIOCHEMISTRY IN OVX RATS TREATED WITH RISEDRONATE AND CALCITRIOL 12
WEEKS POSTOVARIECTOMY
OVX
Risedronate (mg/kg per day)
0
.1
0.5
0
0
0
0.1
0.1
0.5
0.5
0.1
Calcitriol (␮g/kg per day)
Variable
Body weight
BL
SHAM
Vehicle
0
0.05
0.1
0.05
0.1
0.05
275
(11)
55.3
(19.3)
16.4
(2.5)
0.58
(0.04)
10.4
(0.4)
8.5
324*
(20)
387
(22)
50.7
(16.3)
18.1
(3.1)
0.64
(0.05)
9.6
(0.2)
7.4
(0.8)
175
386
(28)
45.3
(9.2)
15.7
(2.3)
0.65
(0.05)
9.4
(0.2)
6.6
(0.9)
167
417
(34)
48.6
(12.3)
15.0
(2.2)
0.67
(0.05)
9.7
(0.3)
5.8*
(0.9)
146
405
(33)
388
(31)
390
(33)
388
(34)
38.3
(6.7)
16.0
(2.8)
0.68
(0.06)
10.0
(0.2)
7.2
373
(25)
391
(22)
51.7
(12.9)
17.1
(2.1)
0.67
(
g)
Serum ALP
U/l)
BUN
28.2*
(6.9)
18.3
(4.1)
0.74*
(0.05)
9.7
(0.4)
5.3*
(1.7)
228
42.7
(10.0)
14.0*
(1.7)
0.66
(0.07)
10.4*
(0.4)
8.0
45.1
(9.7)
14.1*
(2.0)
0.65
(0.05)
10.7*
(0.2)
7.8
42.7
(7.0)
16.2
(2.8)
0.66
(0.07)
10.3*
(0.5)
7.2
40.0
(9.5)
16.8
(3.3)
0.70
(0.08)
(
(
mg/dl)
Serum Crea
(
mmol/l)
Serum Ca
mg/dl)
Serum P
mg/dl)
Serum PTH
pg/ml)
(0.07)
#
Þ
¶
9.7
9.8
(
(0.4)
6.6
(1.0)
85
(0.4)
¥
7.4
(
(0.8)
—
(—)
(1.2)
79*
(69)
(0.8)
29*
(24)
(1.7)
(1.0)
(1.1)
56
(63)
#
#
54*
38*
(
(180)
(102)
(81)
(63)
(43)
(25)
(43)
All values are means Ϯ SD (in parentheses).
BL, baseline; Crea, creatinine; Ca, calcium; P, phosphorus.
#
¥
*
p Ͻ 0.05 versus OVX ϩ vehicle; p Ͻ 0.05 versus OVX ϩ 0.1 mg/kg of risedronate; p Ͻ 0.05 versus OVX ϩ 0.5 mg/kg
Þ
¶
of risedronate; p Ͻ 0.05 versus OVX ϩ 0.05 ␮g/kg of calcitriol; p Ͻ 0.05 versus OVX ϩ 0.1 ␮g/kg of calcitriol by Tukey’s test.
Biomechanics
but Tb.Wi was unchanged. The ovariectomy-induced ver-
tebral and tibial osteopenia was associated with elevated
static and dynamic indices of bone formation. Osteoclast
numbers were higher in OVX animals. However, this effect
reached statistical significance only in the tibia. Wall width
of completed remodeling units was higher in tibial and
vertebral cancellous bone of OVX rats relative to SHAM
controls (Fig. 4). Furthermore, ovariectomy induced a three-
to fourfold increase in activation frequency in tibial and
vertebral cancellous bone.
Effects of risedronate treatment: Risedronate signifi-
cantly increased tibial cancellous B.Ar and prevented dete-
rioration of trabecular bone architecture induced by ovari-
ectomy (Fig. 2). Relative to OVX vehicle controls,
however, risedronate reduced Tb.Wi in the tibia. Although
risedronate had dose-dependent positive effects on vertebral
cancellous bone architecture (Tb.N and Tb.Sp), it did not
result in a statistically significant increase in vertebral can-
cellous B.Ar (Fig. 2). Trabecular thinning due to risedronate
treatment was not observed in vertebral bone (Fig. 3).
Risedronate dose-dependently decreased activation fre-
quency as well as static and dynamic histomorphometric
Ovariectomy did not induce a significant reduction in
biomechanical properties of the fifth lumbar vertebral body
(Fig. 1), or any other site tested (Table 3). Generally,
treatment with calcitriol alone or in combination with
risedronate resulted in significantly higher values for max-
imum load compared with OVX controls in the L5 vertebra
(Fig. 1), the femoral diaphysis, and the femoral neck. How-
ever, low-dose risedronate tended to diminish the increase
in Fmax in the L5 vertebra seen with calcitriol treatment.
Risedronate alone had significant effects on load values
only at the femoral diaphysis. Animals receiving high-dose
calcitriol together with high-dose risedronate had signifi-
cantly higher maximum load values compared with individ-
ual risedronate treatment in the L5 vertebra, the femoral
diaphysis, and the femoral neck. None of the treatment
regimens showed a significant effect at the distal femoral
metaphysis.
Histomorphometry
Effects of ovariectomy: In comparison with SHAM con- indices of vertebral and tibial bone formation (Fig. 4).
trols, ovariectomy resulted in a 61% reduction in tibial Moreover, risedronate treatment dose-dependently reduced
cancellous B.Ar and a 22% reduction in vertebral cancellous wall width (Fig. 5) and osteoclast numbers in tibial and
B.Ar (Figs. 2 and 3; Tables 4 and 5). The tibial and vertebral vertebral cancellous bone (Fig. 4). High-dose risedronate,
cancellous bone osteopenia in OVX rats was accompanied alone or in combination with calcitriol, produced changes in
by increased discontinuity of the trabecular bone structure, osteoclast morphology typical of those seen with bisphos-

CALCITRIOL AND RISEDRONATE IN OVX RATS
1503
(
Figs. 2 and 3). The effects of calcitriol on B.Ar were more
pronounced in the vertebrae, where high-dose calcitriol
increased cancellous B.Ar 21% above the level observed in
SHAM controls. The increase in vertebral cancellous B.Ar
in calcitriol-treated OVX animals was accompanied by a
dose-dependent increase in Tb.Wi and wall width (Figs. 4
and 5). A similar but nonsignificant trend for increased
Tb.Wi and wall width was observed in the tibia (Table 4).
At the higher dose, calcitriol significantly reduced oste-
oclast number in tibial cancellous bone. A similar but non-
significant trend was observed in the vertebra (Fig. 4). The
effects of calcitriol on bone formation and activation fre-
quency were different in the tibia and vertebra. In the
vertebra, calcitriol had no significant effect on MAR, min-
eralizing perimeter, BFR, or activation frequency (Fig. 4).
On the other hand, in the tibia, calcitriol treatment induced
dose-dependent decreases in MAR, mineralizing perimeter,
BFR, and activation frequency. High-dose calcitriol in-
creased osteoid width but had no effect on osteoid matura-
tion time.
Effects of combination treatment: The combination of
risedronate and calcitriol therapy had additive effects on
cancellous B.Ar in the tibia. With the exception of animals
treated with the two low doses (0.1 mg/kg of risedronate and
0
.05 ␮g/kg of calcitriol), combined administration of risedr-
onate and calcitriol to OVX animals resulted in a signifi-
cantly higher cancellous B.Ar compared with the groups
receiving the respective individual risedronate or calcitriol
doses alone (Fig. 2). Compared with SHAM controls, tibial
cancellous B.Ar was increased by 28% with low-dose cal-
citriol and high-dose risedronate and by 46% with high-dose
calcitriol and high-dose risedronate. Calcitriol coadminis-
tration counteracted the decrease in Tb.Wi and also, par-
tially, the decrease in wall width observed with risedronate
alone. Furthermore, the combination of calcitriol and risedr-
onate tended to suppress osteoclast numbers more than each
of the drugs administered alone. The prevailing influence on
histomorphometric parameters of bone formation and on
activation frequency in animals cotreated with risedronate
and calcitriol was the dose of risedronate administered (Fig.
FIG. 1. BMD measured by DXA (A) in the proximal tibia and (B)
in the third lumbar vertebral core, and (C) maximum load (Fmax) values
of the fifth lumbar vertebral body measured by a compression test in
SHAM and OVX rats orally treated with different doses of risedronate
4
). There were no additive effects of the two drugs on these
parameters in tibial bone.
(
Ris) or calcitriol (VitD), alone or in combination, 12 weeks post-OVX.
The effects of combination therapy in the vertebra dif-
fered from those in the tibia. In contrast to the tibia, admin-
istration of risedronate together with calcitriol did not aug-
Each data point represents the mean Ϯ SD of 10 animals. *p Ͻ 0.05
#
versus OVX ϩ vehicle; p Ͻ 0.05 versus OVX ϩ 0.1 of mg/kg
¥
þ
risedronate; p Ͻ 0.05 versus OVX ϩ 0.5 mg/kg of risedronate; p Ͻ
0
0
¶
.05 versus OVX ϩ 0.05 ␮g/kg of calcitriol; p Ͻ 0.05 versus OVX ϩ ment further cancellous B.Ar in comparison to treatment
.1 ␮g/kg of calcitriol by Tukey’s test (BMD data) or Kruskal-Wallis with calcitriol alone (Fig. 2). Nevertheless, lumbar vertebral
test (biomechanical data).
cancellous B.Ar was significantly higher in OVX rats re-
ceiving low- or high-dose risedronate in combination with
high-dose calcitriol relative to treatment with risedronate
phonates (increased cell size, increased number of nuclei, alone. Moreover, risedronate did not impair the positive
and altered morphology of nuclei). effects of calcitriol on Tb.Wi and on wall width of com-
Osteoid maturation time, that is, the mean time interval pleted remodeling units in the vertebra. Similar to tibial
between the onset of matrix deposition and the beginning of bone, coadministration of risedronate and calcitriol signifi-
mineralization at individual bone-forming sites, showed a cantly enhanced the suppressive effect on osteoclast number
slight, dose-dependent increase in both tibial and vertebral and osteoclast perimeter induced by each compound, espe-
bone of risedronate-treated rats. However, risedronate had cially at the highest doses. This finding of an augmented
no effect on osteoid width.
antiresorptive effect in OVX animals cotreated with risedr-
Effects of calcitriol treatment: Calcitriol dose- onate and calcitriol was corroborated by increased preser-
dependently increased tibial and vertebral cancellous B.Ar vation of the tetracycline label administered on day 1 of the

1
504
ERBEN ET AL.
TABLE 3. MAXIMUM LOAD VALUES (FMAX) FOR DIFFERENT SKELETAL SITES IN OVX RATS TREATED WITH RISEDRONATE
AND CALCITRIOL 12 WEEKS POSTOVARIECTOMY
OVX
Risedronate (mg/kg per day)
0
.1
0.5
0
0
0
0.1
0.1
0.5
0.5
Calcitriol (␮g/kg per day)
Site
L5 vertebra
SHAM
Vehicle
0
0.05
0.1
0.05
0.1
0.05
0.1
Þ
¶
¥
270
(55)
151
(14)
524
(115)
80
283
(65)
160
(11)
546
(129)
73
320
(61)
175*
(18)
443
(109)
75
(11)
297
(70)
154
(14)
466
(138)
76
353*
(58)
171
(16)
544
(158)
83*
(15)
385*
(37)
175*
(18)
607
(159)
87*
(12)
290
338*
374*
(103)
164
(25)
591
400*
(
N)
Fem diaphysis
N)
Dist fem metaphysis
N)
Fem neck
N)
(51)
167
(10)
502
(87)
(78)¶
(85)
¥
160
179*
(17)
(
(15)
557
(163)
85*
536
(
(217)
(184)
#
¥
¥
90*
92*
91*
(
(11)
(8)
(15)
(15)
(10)
(9)
(13)
All values are means Ϯ SD (in parentheses).
Fem, femoral; dist, distal.
#
¥
*
p Ͻ 0.05 versus OVX ϩ vehicle; p Ͻ 0.05 versus OVX ϩ 0.1 mg/kg of risedronate; p Ͻ 0.05 versus OVX ϩ 0.5 mg/kg
Þ
¶
of risedronate; p Ͻ 0.05 versus OVX ϩ 0.05 ␮g/kg of calcitriol; p Ͻ 0.05 versus OVX ϩ 0.1 ␮g/kg of calcitriol by Dunn’s test.
study in vertebral cancellous bone. Furthermore, when
risedronate was given together with calcitriol, vertebral
BFR (Fig. 4), mineralizing perimeter, and osteoblast perim-
eter were significantly lower relative to treatment with cal-
citriol alone. However, in comparison with risedronate
given alone, high-dose calcitriol partially antagonized the
suppressive effect of risedronate on MAR, mineralizing
perimeter, BFR, and activation frequency (Fig. 4).
Combined therapy with risedronate and calcitriol had
different effects on bone mineralization in the vertebra
compared with the tibia. In the vertebra, high-dose calcitriol
reduced the elevated osteoid maturation time in animals
receiving high-dose risedronate to approximately OVX ve-
hicle control levels. However, in the tibia high-dose risedr-
onate together with low- or high-dose calcitriol increased
osteoid maturation time to a significantly higher level com-
pared with the groups receiving each of the drugs alone.
DISCUSSION
(2)
In agreement with a previous long-term study, this
experiment has shown that prophylactic administration of
risedronate to OVX rats over a 12-week period can prevent
estrogen depletion–induced bone loss in the tibia and in the
lumbar vertebra by depressing the elevated bone turnover.
In both tibial and vertebral bone, risedronate treatment
dose-dependently suppressed osteoblast perimeter, mineral-
izing perimeter, MAR, and wall width, clearly indicating
that risedronate decreased osteoblast recruitment and also
FIG. 2. Cancellous B.Ar in the (A) proximal tibial metaphysis and
the (B) fourth lumbar vertebral body in SHAM and OVX rats orally
treated with different doses of risedronate (Ris) or calcitriol (VitD),
alone or in combination, 12 weeks post-OVX. Each data point repre-
sents the mean Ϯ SD of 10 animals. *p Ͻ 0.05 versus OVX ϩ vehicle;
(32)
osteoblast team performance in OVX rats.
Recently,
it has been suggested that bisphosphonates inhibit
glucocorticoid-induced osteoblast and osteocyte apoptosis
#
¥
p Ͻ 0.05 versus OVX ϩ 0.1 mg/kg of risedronate; p Ͻ 0.05 versus
þ
OVX ϩ 0.5 mg/kg of risedronate; p Ͻ 0.05 versus OVX ϩ 0.05 ␮g/kg
(33)
¶
in vivo in mice.
Increased cell survival under the influ- of calcitriol; p Ͻ 0.05 versus OVX ϩ 0.1 ␮g/kg of calcitriol by
ence of bisphosphonates may enhance the performance of Tukey’s test.

CALCITRIOL AND RISEDRONATE IN OVX RATS
1505
FIG. 3. Fourth lumbar vertebral bodies of (A) a SHAM rat, (B) a vehicle-treated OVX rat, and (C) OVX rats treated with high-dose (0.5 mg/kg
per day) risedronate, (D) high-dose (0.1 ␮g/kg per day) calcitriol, or (E) combined treatment with the high doses of both drugs, 12 weeks
post-OVX. (C) Risedronate alone largely protected against estrogen deficiency–induced bone loss and deterioration of cancellous bone structure.
(
D) High-dose calcitriol had pronounced bone anabolic effects and increased cancellous B.Ar in OVX rats to levels beyond that observed in
SHAM animals. (E) Coadministration of risedronate did not impair the bone anabolic effect of calcitriol. Note the increase in Tb.Wi in the OVX
animals receiving high-dose calcitriol alone or in combination with risedronate (5-␮m-thick undecalcified sections; Von Kossa stain; original
magnification ϫ15).
individual osteoblast teams, resulting in increased wall
The daily dose of 0.05 ␮g/kg of calcitriol provided com-
thickness and a positive remodeling balance. In line with plete protection against postovariectomy vertebral cancel-
this idea, it has been reported that wall thickness and re- lous bone loss in this study, and 0.1 ␮g/kg of calcitriol had
modeling balance may increase under treatment with risedr- bone anabolic effects on vertebral bone in OVX animals,
(
34)
onate in dogs.
However, a 3-year study in dogs treated increasing cancellous B.Ar and bone strength beyond the
with high doses of alendronate did not reveal any changes in level observed in SHAM controls. Thus, at the dosages used
(35)
cancellous bone volume or architecture. In addition, can- in this experiment, calcitriol had more pronounced positive
cellous bone volume and wall thickness were unchanged effects on vertebral cancellous bone in OVX rats compared
after 1 year of alendronate treatment in patients with with risedronate. However, both drugs had comparable
(7)
glucocorticoid-induced osteoporosis. In postmenopausal bone-protective activity in the tibia. Previous experi-
(22,25,36)
women with osteoporosis, an increase in wall thickness was ments
have suggested also that the skeletal effects of
observed after 2 years but not after 3 years of alendronate vitamin D metabolites are more pronounced in the axial
(
6)
treatment. Cancellous bone volume remained unchanged skeleton compared with the appendicular skeleton. The rea-
in the latter study. Even in the presence of a down-regulated son for this discrepancy is not known but may be related to
activation frequency, one would expect a rise in Tb.Wi and a higher extent of remodeling activity in the axial skeleton
(37)
cancellous bone volume in long-term studies in the case that in the rat.
bisphosphonates increase remodeling balance. Therefore, The major mechanism of the bone-sparing effect of cal-
neither this study in rats nor the available human data citriol in tibial cancellous bone of OVX rats in this study
provide evidence to support the hypothesis that bisphospho- appeared to be a reduction in bone turnover and activation
nates have a positive effect on wall thickness and osteoblast frequency. However, in vertebral cancellous bone, calcitriol
team performance.
did not significantly suppress histomorphometric parame-

1
506
ERBEN ET AL.
TABLE 4. HISTOMORPHOMETRIC DATA IN THE CANCELLOUS BONE OF THE PROXIMAL TIBIAL METAPHYSIS IN SHAM AND
OVX RATS TREATED WITH RISEDRONATE AND CALCITRIOL 12 WEEKS POSTOVARIECTOMY
OVX
Risedronate (mg/kg per day)
0
.1
0
0.5
0
0
0
0.1
0.1
0.5
0.5
0.1
Calcitriol (␮g/kg per day)
Variable
T.Ar
SHAM
Vehicle
0.05
0.1
0.05
0.1
0.05
17.8
(1.2)
70
18.6
(1.5)
67
19.1
(1.1)
57
18.3
(0.9)
54*
18.2
(1.9)
72
19.2
(1.2)
74
19.9
(1.1)
58
18.6
(1.4)
68
19.5
(0.83)
65
18.8
(1.5)
69
2
(
mm )
Þ
¥
Tb.Wi
(
␮m)
(6)
(6)
1.77
(0.61)
567
(190)
3.14
(5)
(6)
(8)
2.34
(0.74)
401*
(117)
3.02
(9)
(6)
4.35*
(0.86Þ)
195*
(61)
(11)
(8)
(10)
#
Þ
#¶
¥Þ
¥¶
Tb.N
(
Tb.Sp
(
4.38*
(0.51)
176*
(31)
3.09*
(0.65)
294*
(81)
4.68*
(1.01)
182*
(60)
3.42*
(1.03)
264*
(118)
3.26
4.72*
(0.65)
163*
(36)
6.05*
(0.70Þ)
117*
(24)
6.51*
(0.80)
no./mm)
¶
102*
␮m)
(21)
¥
O.Wi
3.23
3.27
2.90
2.82
3.33
2.84
3.64
(
␮m)
(0.34)
5.24*
(2.05)
3.41*
(2.25)
(0.31)
3.60
(1.32)
12.79
(7.21)
(0.45)
5.87
(1.59)
4.35*
(3.55)
(0.30)
7.86*
(2.82)
1.42*
(0.98)
(0.24)
4.09
(0.99)
7.87*
(3.32)
(0.33)
5.10
(1.17)
6.93*
(2.02)
(0.44)
6.52
(1.97)Þ
1.82*
(1.48)
(0.65)
6.91
(2.03)
2.77*
(1.31)
(0.38)
(0.86)
¥
Þ
¥¶
Omt
(
Ob.Pm/
B.Pm
13.24*
(4.66)Þ
0.34*
12.90*
(4.98)
days)
¶
1.09*
(1.53)
(0.32)
(
%)
E.Pm/
B.Pm
%)
N.Oc/
Md.Pm
no./mm)
21.0
(5.1)
21.2
(5.2)
17.7
(2.7)
20.8
(3.7)
22.1
(4.1)
19.9
(5.4)
20.3
(3.1)
17.0
(3.3)
20.4
(4.2)
20.0
(2.8)
(
Þ
Þ
¶
1.84*
(0.44)
2.61
(0.69)
1.99
(0.47)
1.67*
(0.31)
2.71
(0.76)
1.81
(0.73)
1.63*
1.32*
(0.50)
1.35*
0.97*
(0.40)
(0.41)
(0.42)
(
Þ
Þ
¶
M.Pm/
B.Pm
5.94*
(4.25)
21.00
(11.04)
4.91*
(3.04)
1.51*
(0.72)
13.75*
(5.93)
8.71*
(3.98)
2.61*
3.23*
(1.53)
0.63*
1.52*
(2.19)
(1.78)
(0.40)
(
%)
Þ
Þ
¶
MAR
(
BFR/B.Pm
(
0.68*
(0.21)
0.047*
(0.040)
0.96
(0.33)
0.231
0.58*
(0.13)
0.032*
(0.030)
0.40*
(0.11)
0.006*
(0.005)
0.77
0.66*
(0.13)
0.058*
(0.030)
0.45*
0.51*
(0.16)
0.016*
(0.007)
0.23*
0.33*
(0.16)
0.008*
(0.014)
␮m/day)
(0.14)
0.109*
(0.061)
(0.09)Þ
0.013*
(0.010)
(0.07)Þ
0.002*
(0.002)
2
␮m /␮m
(0.169)
per day)
W.Wi
␮m)
Þ
¶
Þ
¥¶
21.1*
(1.5)
35
(18)
830
(1767)
0.51*
(0.40)
25.1
(2.6)
28
(8)
105
(91)
2.16
(1.65)
22.1
(1.6)
39
(7)
326
(332)
0.54*
(0.60)
18.9*
(1.2)
51
25.6
(2.6)
35
(8)
141
(52)
0.93*
(0.46)
27.5
(2.0)
43
(10)
162
(74)
0.70*
(0.22)
20.4*
23.6
21.7*
24.0
(
(2.2)
46
(2.0)
49
(13)
(2.1)
(2.2)
¥
Þ
¥
¥¶
FP
100*
(31)
86*
(
days)
(15)
(9)
695
(35)
4364
(5963)
0.10*
(0.17)
Þ
Rm.P
1141
(1055)
0.13*
(0.10)
422
14950*
(
days)
(501)
0.17*
(0.13)
(310)
0.30*
(0.21)
(14710)
Þ
Ac.F
0.02*
(
no./year)
(0.02)
All values are means Ϯ SD (in parentheses).
O.Wi, osteoid width; Omt, osteoid maturation time; E.Pm, eroded perimeter; N.Oc, no. of osteoclasts; Md.Pm, mineralized perimeter;
M.Pm, mineralizing perimeter; W.Wi, wall width; FP, formation period; Rm.P, remodeling period; Ac.F, activation frequency.
#
¥
*
p Ͻ 0.05 versus OVX ϩ vehicle; p Ͻ 0.05 versus OVX ϩ 0.1 mg/kg of risedronate; p Ͻ 0.05 versus OVX ϩ 0.5 mg/kg
Þ
¶
of risedronate; p Ͻ 0.05 versus OVX ϩ 0.05 ␮g/kg of calcitriol; p Ͻ 0.05 versus OVX ϩ 0.1 ␮g/kg of calcitriol by Tukey’s test.
(26)
ters of bone formation and bone resorption. Earlier studies the performance of individual osteoblast teams.
There-
have suggested that the bone-preserving activity of low- fore, the anabolic effect of calcitriol in vertebral bone of
dose calcitriol in OVX rats is caused by primarily an anti- OVX rats may be based on a positive remodeling balance
(21)
resorptive effect and a reduction in bone turnover,
because of augmented osteoblast recruitment and osteoblast
whereas calcitriol at higher doses can increase the recruit- team performance. This hypothesis is supported by the
ment of osteoblast precursor cells from bone marrow and increase in wall width of completed remodeling units and

CALCITRIOL AND RISEDRONATE IN OVX RATS
1507
TABLE 5. HISTOMORPHOMETRIC DATA IN THE CANCELLOUS BONE OF THE FOURTH LUMBAR VERTEBRA IN SHAM AND OVX
RATS TREATED WITH RISEDRONATE AND CALCITRIOL 12 WEEKS POSTOVARIECTOMY
OVX
Risedronate (mg/kg per day)
0
.1
0.5
0
0
0
0.1
0.1
0.5
0.5
0.1
Calcitriol (␮g/kg per day)
Variable
T.Ar
SHAM
Vehicle
0
0.05
0.1
0.05
0.1
0.05
12.0
(1.2)
87
12.6
(1.4)
87
(11)
3.91
(0.47)
191
(32)
3.43
(0.23)
3.99
(0.60)
7.94
(3.11)
19.8
(3.2)
12.63
(5.04)
0.88
(0.15)
3.14
12.0
(0.9)
86
11.8
(1.3)
80*
12.0
(1.6)
95
12.2
(1.6)
108*
(9)
4.89*
(1.03)
120*
(14)
4.07*
(0.61)
4.45
(0.87)
8.90
(3.20)
17.6
(6.1)
14.10
(3.79)
0.93
(0.12)
6.35
(2.16)
30.0
12.9
(1.4)
90
11.9
(1.3)
11.4
(1.12)
91
11.2
(0.5)
101*
(12)
2
(
mm )
#
¥
Tb.Wi
103*
(12)
5.11*
(␮m)
(6)
(6)
(7)
(9)
(7)
(5)
#
Þ
Tb.N
4.99*
(0.41)
133*
(19)
3.44
(0.29)
4.09
(0.56)
2.45*
(0.77)
22.2
(4.9)
4.73*
(2.32)
0.85
(0.12)
3.57
(1.69)
26.0
4.50*
(0.44)
157*
(23)
3.48
(0.17)
5.02
(0.88)
3.62*
(1.19)
21.0
(3.0)
4.52*
(1.62)
0.71
(0.11)
4.01
(3.37)
32.6
4.90*
(0.39)
142*
(20)
4.64*
(0.70)
145*
(39)
3.35
(0.27)
4.02
(0.79)
4.93
(1.68)
22.4
(4.7)
11.78
(3.69)
0.85
(0.14)
5.64
5.04*
(0.32)
128*
(15)
3.54
(0.18)
5.16
(0.72)
2.77*
(1.41)
18.8
(4.0)Þ
4.07*
(1.27)
0.70
(0.09)
6.20
(1.72)
34.3
5.26*
(0.34)
119*
5.28*
(0.32)
111*
(19)
(
no./mm)
(0.30)
#
Tb.Sp
116*
(
␮m)
(17)
3.69
(0.71)
4.67
(13)
3.55
¥
O.Wi
3.16
4.12
(␮m)
(0.30)
5.56*
(0.94)
1.69*
(0.74)
19.5
(3.8)
2.04*
(1.11)
0.58*
(0.09)
3.60
(1.92)
37.3*
(6.0)
449*
(207)
(0.17)Þ
6.31*
(1.08)Þ
1.18*
(0.64)
17.1
(4.9)Þ
2.15*
(0.89)Þ
0.58*
(0.09)
7.64*
(4.49)Þ
41.3*
(5.15Þ)
675*
(0.59)
4.78
Omt
(
days)
(0.69)¶
4.02*
(2.89)
(1.62)
¶
Ob.Pm/B.Pm
4.51*
(3.52)
(
%)
E.Pm/B.Pm
%)
M.Pm/B.Pm
%)
MAR
#
¥¶
12.7*
9.5*
(
(5.1)¶
5.90*
(2.68)
0.81
(0.23)
7.71*
(2.45)
33.7
(4.3)
5.42*
¶
(
(3.22)
¥
0.90
(␮m/day)
(0.16)
¥
Tc.Lb/B.Pm
9.35*
(
%)
(1.44)
28.0
(6.2)
135
(1.76)
29.3
(6.7)
158
(4.72)
29.8
FP
(
days)
(5.2)
270
(126)
(5.7)
217
(72)
(4.0)
100
(55)
(4.7)
306
(177)
(7.2)
268
(326)
(7.3)
¥
Rm.P
days)
120
(
(163)
(49)
(307)
(115)
All values are means Ϯ SD (in parentheses).
O.Wi, osteoid width; Omt, osteoid maturation time; E.Pm, eroded perimeter; M.Pm, mineralizing perimeter; Md.Pm, mineralized
perimeter; Tc.Lb, tetracycline-labeled perimeter; FP, formation period; Rm.P, remodeling period.
#
¥
*
p Ͻ 0.05 versus OVX ϩ vehicle; p Ͻ 0.05 versus OVX ϩ 0.1 mg/kg of risedronate; p Ͻ 0.05 versus OVX ϩ 0.5 mg/kg
Þ
¶
of risedronate; p Ͻ 0.05 versus OVX ϩ 0.05 ␮g/kg of calcitriol; p Ͻ 0.05 versus OVX ϩ 0.1 ␮g/kg of calcitriol by Tukey’s test.
the concomitant increase in Tb.Wi observed in calcitriol- which rats were treated with daily injections of high-dose
treated OVX rats. However, it is important to note that the calcitriol, osteoclast numbers were found to be increased in
bone anabolic potential of vitamin D analogs is seen only at tibial cancellous bone on day 1 but were diminished by day
(41)
high doses. Because of the calcemic side effects, such a 6 of the study.
Under chronic treatment with active
treatment probably is not feasible in humans with vitamin D vitamin D analogs, the initial rise in bone resorption is
compounds of equally calcemic potential as calcitriol.
In vitro, calcitriol is a potent stimulator of bone resorp- tion through increased intestinal calcium absorption.
thought to be overridden by the suppression of PTH secre-
(21,27)
tion, increasing the proliferation, differentiation, and fusion PTH is the most important hormonal regulator of bone
of preosteoclasts and augmenting the activity of mature turnover under physiological circumstances. However, cal-
(
38,39)
osteoclasts.
Similarly, acute administration of pharma- citriol also may have direct antiresorptive effects indepen-
cologic amounts of calcitriol in vivo results in a transient dent of PTH suppression. Experiments using parathyroid-
(40,41)
increase in osteoclast activity and recruitment.
This ectomized rats infused with human PTH-related peptide
acute calcitriol-induced stimulation of bone resorption can 1-34 as a model of hypercalcemia of malignancy have
(42)
be blocked by bisphosphonates.
However, chronic cal- shown that calcitriol lowers hypercalcemia and bone resorp-
citriol administration inhibits bone resorption as measured tion in a dose-dependent manner in this model, strongly
by osteoclast numbers and/or biochemical markers in implying an additional direct antiresorptive action of calcit-
(
21,22,27,43)
(18,44,45)
(46)
rats
and humans.
In an experiment in riol on bone cells.

1
508
ERBEN ET AL.
Therefore, the available data suggest that the hypercalce-
mia and hypercalciuria under chronic treatment with vita-
min D analogs is caused mainly by augmented intestinal
calcium absorption and that dietary calcium is a major
modulator of their toxicity. In agreement with this idea,
hypercalcemia in a patient with ovarian carcinoma and
abnormally high blood levels of calcitriol could not be
(47)
controlled by treatment with different bisphosphonates.
Interestingly, however, high-dose risedronate normalized
hypercalcemia and decreased suppression of PTH serum
levels in calcitriol-treated OVX rats in this study, suggesting
that calcium mobilization from the skeleton may have ac-
counted partially for the hypercalcemia induced by calcit-
riol. On the other hand, cancellous and cortical bone mass
and strength in the axial and appendicular skeleton in-
creased in OVX rats receiving calcitriol alone or in combi-
nation with risedronate, indicating that the bone balance is
positive under calcitriol treatment. Similar to our results,
combined therapy with calcitriol and estrogen corrected the
rise in serum ionized calcium seen under treatment with
calcitriol alone in a recent 3-year trial in postmenopausal
(18)
women.
Thus, although calcitriol acts as a mild antire-
sorptive drug, calcium release via osteoclastic bone resorp-
tion appears to contribute to the development of hypercal-
cemia with this compound. In light of the importance of
hypercalcemia for the clinical monitoring of patients with
osteoporosis treated with vitamin D analogs, it clearly war-
rants further study whether this side effect could be con-
trolled partially by coadministration of antiresorptive agents
such as bisphosphonates or estrogen.
Previous investigations have shown that chronic admin-
istration of calcitriol to rats at dosages higher than ϳ0.1–0.2
(43,48)
␮
g/kg can result in impaired bone mineralization.
However, at the dosages used in this study, calcitriol did not
adversely affect bone mineralization. Risedronate produced
a slight increase in osteoid maturation time, but this was
caused by a decrease in MAR and not by an increase in
osteoid width. In the absence of an accumulation of osteoid
and an increase in osteoid width, these findings are not
indicative of defective mineralization. In rats receiving
combination therapy, high-dose calcitriol antagonized the
increase in osteoid maturation time seen in rats treated with
high-dose risedronate in vertebral bone. However, in the
tibia calcitriol at both dosages used resulted in a significant
increase in osteoid maturation time in animals receiving
high-dose risedronate. The reason for the discrepancy be-
tween vertebral and tibial cancellous bone is not known.
An interesting finding in this study was that combined
treatment of OVX rats with risedronate and calcitriol re-
sulted in a significant enhancement of the suppressive ef-
fects on histomorphometric parameters of bone resorption
compared with administration of each of the compounds
alone. The antiresorptive effects of both drugs are mediated
through different pathways. Bisphosphonates inhibit the
FIG. 4. (A) BFR, (B) osteoclast number, (C) wall width, and (D)
activation frequency in vertebral cancellous bone in SHAM and OVX
rats orally treated with different doses of risedronate (Ris) or calcitriol
(
VitD), alone or in combination, 12 weeks post-OVX. Each data point
represents the mean Ϯ SD of 10 animals. *p Ͻ 0.05 versus OVX ϩ
(1)
#
¥
bone resorptive capacity of mature osteoclasts, and chron-
ically administered calcitriol inhibits osteoclast recruitment
vehicle; p Ͻ 0.05 versus OVX ϩ 0.1 mg/kg of risedronate; p Ͻ 0.05
þ
versus OVX ϩ 0.5 mg/kg of risedronate; p Ͻ 0.05 versus OVX ϩ
(27)
¶
0
.05 ␮g/kg of calcitriol; p Ͻ 0.05 versus OVX ϩ 0.1 ␮g/kg of mainly through suppression of PTH secretion. Therefore,
calcitriol by Tukey’s test.
this investigation suggests that combined treatment with
risedronate and calcitriol may result in additive or even
overadditive suppressive effects on bone resorption.

CALCITRIOL AND RISEDRONATE IN OVX RATS
1509
FIG. 5. Representative completed remodeling units in vertebral cancellous bone of (A) a vehicle-treated OVX rat and (B) OVX rats that have
been treated for 12 weeks with high-dose (0.5 mg/kg per day) risedronate or (C) with high-dose (0.1 ␮g/kg per day) calcitriol. The reversal lines
are marked with arrows. It is evident that risedronate decreases the wall width of completed remodeling units compared with the OVX vehicle
control. In contrast, administration of calcitriol results in a pronounced increase in wall width beyond the level seen in vehicle-treated OVX rats.
The convex surface of the remodeling unit shown in panel C is suggestive of a positive remodeling balance caused by overfilling of the resorption
cavity. Toluidine blue surface stain for demonstration of cement lines viewed under polarized light (original magnification ϫ400).
Combined administration of risedronate and calcitriol performance. Risedronate did not reduce the anabolic effect
caused additive effects on BMD and cancellous B.Ar in the of calcitriol, and at the high dose it normalized hypercalce-
tibia. However, in the vertebra combination therapy offered mia in calcitriol-treated OVX rats. Therefore, this study in
no advantage in terms of cancellous B.Ar, BMD, or bone OVX rats suggests that combined therapy with risedronate
strength compared with calcitriol administration alone. The and calcitriol may offer advantages over the treatment with
different effects of combination therapy in the axial and bisphosphonates or calcitriol alone.
appendicular skeleton may be related to the higher rates of
longitudinal bone growth in the proximal tibia as compared
(37)
with the vertebra.
It is well known that antiresorptive
ACKNOWLEDGMENTS
drugs increase cancellous bone mass in growing bone
(
49)
sites.
Therefore, enhanced suppression of osteoclastic
The authors thank Siglinde Lutz, Claudia Bergow, Karin
Begsteiger, and Birthe Gylling-Jørgensen for excellent tech-
nical assistance.
bone resorption by coadministration of risedronate and cal-
citriol is likely to increase cancellous bone mass in an
additive fashion in a fast-growing bone site such as the
proximal tibia. In a slowly growing bone site such as the
fourth lumbar vertebra, an increase in cancellous B.Ar in
OVX rats beyond the level observed in SHAM controls
cannot solely be explained by an antiresorptive effect in this
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Reinhold G. Erben, M.D., D.V.M.
Institute of Animal Physiology
University of Munich
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Veterinaerstrasse 13
D-80539 Munich, Germany
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1