CALCIUM-REGULATING
HORMONES
Effect of Bisphosphonates on the Rapidly Growing
Male Murine Skeleton
Eric D. Zhu, Leeann Louis, Daniel J. Brooks, Mary L. Bouxsein,
and Marie B. Demay
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
02114
Bisphosphonates are effective for preventing and treating skeletal disorders associated with hyperresorption. Their safety and efficacy has been studied in adults where the growth plate is fused
and there is no longitudinal bone growth and little appositional growth. Although bisphosphonate use in the pediatric population was pioneered for compassionate use in the treatment of
osteogenesis imperfecta, they are being increasingly used for the treatment and prevention of
bone loss in children at risk of hyperresorptive bone loss. However, the effect of these agents on
the growing skeleton in disorders other than osteogenesis imperfecta has not been systematically
compared. Studies were, therefore, undertaken to examine the consequences of bisphosphonate
administration on the growth plate and skeletal microarchitecture during a period of rapid
growth. C57Bl6/J male mice were treated from 18 to 38 days of age with vehicle, alendronate,
pamidronate, zoledronate, or clodronate at doses selected to replicate those used in humans.
Treatment with alendronate, pamidronate, and zoledronate, but not clodronate, led to a decrease
in the number of chondrocytes per column in the hypertrophic chondrocyte layer. This was not
associated with altered hypertrophic chondrocyte apoptosis or vascular invasion at the growth
plate. The effects of pamidronate on trabecular microarchitecture were less beneficial than those
of alendronate and zoledronate. Pamidronate did not increase cortical thickness or cortical area/
total area relative to control mice. These studies suggest that bisphosphonate administration does
not adversely affect skeletal growth. Long-term investigations are required to determine whether
the differences observed among the agents examined impact biomechanical integrity of the growing skeleton. (Endocrinology 155: 1188 –1196, 2014)
isphosphonates have been shown to be safe and effective for the treatment of disorders associated with increased bone resorption (1). However, the large randomized controlled studies that establish the safety and
efficacy of these agents have focused on adults in whom
the growth plate is fused. Thus, their actions have been
investigated in a setting where there is no longitudinal
bone growth and little to no appositional bone growth
(2– 4). Although bisphosphonate administration in the pediatric population was initially pioneered for compassionate use in children with severe osteogenesis imperfecta
(OI) (5), these medications are being increasingly used for
B
other disorders (6), ranging in severity from spontaneous
disuse fractures in patients with cerebral palsy (7) to the
prevention of steroid-induced osteoporosis in ambulatory
children (8) and prevention of bone loss in children with
hypercalciuria (9). A review of several case reports and
clinical trials suggested that further evaluation of bisphosphonates in children with secondary osteoporosis is
needed (6) and that available data do not support recommendation of bisphosphonates as standard therapy in
children with secondary osteoporosis (10).
Technicium, 99mTc-labeled 1-hydroxy-methyledene
bisphosphonate, a compound closely related to clodro-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received October 30, 2013. Accepted January 7, 2014.
First Published Online January 14, 2014
Abbreviations: BMD, bone mineral density; CD31, cluster differentiation 31; ColX, type X
collagen; ␮CT, microcomputed tomography; Ct.Ar, cortical bone area; Ct.Po, cortical
porosity; Ct.Th, cortical thickness; Imax, maximum moment of inertia; Imin, minimum moment of inertia; MMP-9, matrix metalloproteinase-9; OI, osteogenesis imperfecta; pErk1/2,
phospho-Erk1/2; pMOI, polar moment of inertia; SMI, structural model index; Tb.N, trabecular number; Tb.Sp, trabecular separation.
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Endocrinology, April 2014, 155(4):1188 –1196
doi: 10.1210/en.2013-1993
doi: 10.1210/en.2013-1993
nate, is used for bone scans. In children and growing animals, this agent is avidly taken up in the region of the
growth plate where cartilage is being replaced by bone
(11). Injection of 14C zoledronate into normal rats also
demonstrates localization to this region of the growth
plate in addition to bone (12). These observations raise the
question as to whether these agents can interfere with normal growth plate maturation at the chondro-osseous junction. The consequences of bisphosphonate treatment on
growth plate histology of long bones are unknown. However, studies in pamidronate-treated children with OI
demonstrate the presence of radiodense bands parallel to
the growth plate (5) correlating temporally with pamidronate infusions. Iliac crest biopsies demonstrate that in the
band closest to the apophyseal growth plate of the iliac
crest, approximately one quarter of the mineralized tissue
is calcified cartilage (13). This suggests that pamidronate
may alter hypertrophic chondrocyte apoptosis in vivo, because these bands of retained mineralized cartilage contain
cells that have not undergone the normal progression of
chondrocyte maturation that culminates in programmed
cell death.
In vivo studies in genetic and diet-induced rachitic
mouse models demonstrate that hypophosphatemia impairs hypertrophic chondrocyte apoptosis, leading to the
development of rickets (14). The nitrogen-containing
(amino-) bisphopshonate, alendronate has been shown to
inhibit phosphate uptake by avian chondrocytes as well as
phosphate-mediated cell death in this cell culture model
(15). Studies with first generation bisphosphonates (ethane-1-hydroxy-1,1-diphosphonate and dichloromethylene diphsophonate), performed in growing rats more than
4 decades ago, demonstrated expansion of the growth
plate and persistence of columns of calcified cartilage (16,
17), suggesting impairment of programmed cell death.
More recently, alendronate treatment of a mouse model of
OI resulted in increased length of the growth plate due to
expansion of the hypertrophic cartilage layer (18). Although apoptosis was not examined in these investigations, the authors speculate that expansion of the hypertrophic chondrocyte layer was due to impairment of
apoptosis and vascular invasion. These findings suggest
that administration of bisphosphonates to individuals
whose growth plate has not fused may alter growth plate
maturation and, thus, have implications for the use of
bisphosphonates in the treatment of skeletal disorders in
children. Although bisphosphonates improve growth and
decrease morbidity in children with OI (5, 9, 19 –24), their
impact on growth, when used to treat or prevent accelerated bone loss from other causes in children, has not been
systematically examined. Due to the invasiveness and potential growth disruption that may result from biopsy of
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long bone growth plates, the effect of different bisphosphonates on long bone growth plate histology and maturation has not been methodically analyzed. Furthermore,
studies of the bone consequences have been limited to examining cortical bone from iliac crest biopsies and vertebral bone density (13, 25). Studies of long bones are limited to traditional radiography. Thus, the consequences of
treatment with these agents on the microarchitectural
properties of bones that are growing appositionally and
longitudinally has not been systematically examined.
Therefore, we investigated the acute effects of selected
bisphosphonates used in clinical practice, including the
nonamino-bisphosphonate, clodronate (26), and those of
the amino-bisphosphonates, alendronate, pamidronate
(20), and zoledronate (27), on growth plate maturation,
bone density, and microarchitecture in growing male
mice.
Materials and Methods
Cell culture
Primary chondrocytes were isolated from ventral rib cages of
newborn mice by sequential collagenase II digestions and plated
onto gelatin-coated plates at a density of 3 ⫻ 105/cm2 as previously described (14, 28). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 25-mg/mL ascorbic acid at 37°C and 5% CO2. To
evaluate Erk1/2 phosphorylation, bisphosphonates were added
to the culture 1 hour before the addition of 7mM sodium phosphate or sodium sulfate, in DMEM with 0.5% fetal bovine serum. Erk1/2 phosphorylation was evaluated 30 minutes later.
Western blot analyses
Chondrocytes were lysed in Tris-buffered saline containing
2% SDS, 2% Triton X-100, 1mM EDTA, 1mM sodium fluoride,
20mM ␤-glycerol phosphate, 2mM sodium orthovanadate, and
protease inhibitor mixture (Roche Applied Science). Lysates
were subjected to 2 freeze/thaw cycles. Protein concentration
was calculated using the bicinchoninic acid protein assay, after
which 10 ␮g of protein were subjected to SDS-PAGE. Erk1/2
phosphorylation was evaluated using primary polyclonal antibodies against Erk1/2 and phospho-Erk1/2 (Cell Signaling), a
horseradish peroxidase-conjugated secondary antibody, and visualized using ECL Plus (Amersham Biosciences) according to
the manufacturer’s instructions.
Animal studies
Animals were maintained in a virus- and parasite-free barrier
facility and exposed to a 12-hour light, 12-hour dark cycle. Animal procedures were approved by the institutional animal care
and use committee at the Massachusetts General Hospital.
C57BL/6J male mice were weaned onto a diet containing 1.0%
calcium and 0.6% phosphate at 18 days of age. Bisphosphonate
doses and administration schedules were chosen to parallel those
used to treat OI or osteoporosis in humans. Mice were treated sc
from 18 to 38 days with vehicle, clodronate, alendronate,
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Bisphosphonate Effects During Growth
pamidronate, or zoledronate. Specifically, clodronate (Sigma)
was administered continuously by Alzet minipump at a dose of
0.5 mg/kg䡠d to approximate the 1600-mg/d human dose (⬃23
mg/kg䡠d) and the documented 2.1% absorption of this drug (29).
Alendronate (Sigma) was administered sc twice weekly based on
the rapid growth of mice compared with humans, who receive
weekly treatment. The dose of alendronate selected (100 mcg/kg
twice weekly) was the same as the dose administered parenterally
for studies in rapidly growing oim/oim mice (200 mcg/kg䡠wk), a
model of OI (30). Pamidronate (3 mg/kg; Sigma and American
Reagent, Inc) and zoledronate (0.07 mg/kg; Novartis) were injected at day 18, because they are administered to humans quarterly and annually, respectively. The pamidronate dose of 3
mg/kg is based on the use of 3 mg/kg for the treatment of children
with OI (25). The zoledronate dose of 0.07 mg/kg reflects the
human dose of 5 mg (average weight 70 kg) and approaches that
used for children with secondary osteoporosis (0.1 mg/kg䡠y) (31).
To permit evaluation of the acute effects of these agents on the
growth plate, all amino-bisphosphonates were administered to
mice 24 hours before killing (d 38).
Histology
Immunohistochemical detection of phospho-Erk1/2 was performed as previously described (32). Apoptosis was evaluated
using the terminal deoxynucleotidyl transferase 2⬘-deoxyuridine, 5⬘-triphosphate nick end labeling (TUNEL)-based in situ
cell death detection kit (Roche Diagnostics) and an antibody to
cleaved caspase 3 (Cell Signaling) (14). The presence of endothelial cells was evaluated using an anti-cluster differentiation 31
(CD31) antibody (BD Biosciences) (33). In situ hybridization
was performed on fixed frozen or paraffin sections using 35Suracil tri-phosphate-labeled antisense RNA probes as previously
described (34).
Microcomputed tomography (␮CT)
␮CT imaging was performed on the distal metaphysis and
middiaphysis of the femur using a high-resolution desktop imaging system (␮CT40; Scanco Medical AG). Scans were acquired
using a 6-␮m3 isotropic voxel size, 70-kVp peak X-ray tube
potential, 200-ms integration time, and were subjected to Gaussian filtration. Trabecular bone microarchitecture was evaluated
in the distal metaphysis in a region that began 0.54 mm proximal
to the growth plate and extended proximally 2.1 mm. Cortical
bone morphology was evaluated in the middiaphysis in a region
that started 55% of the bone length below the femoral head and
extended 0.60 mm distally. Thresholds of 316 and 733 mg HA/
cm3 were used to segment trabecular and cortical bone, respectively, from surrounding soft tissue based on adaptive-iterative
thresholding performed on the control group. Trabecular bone
outcomes included trabecular bone volume fraction (%), trabecular thickness (mm), trabecular number (Tb.N, mm⫺1), trabecular separation (Tb.Sp, mm), connectivity density (mm⫺3), and
structural model index (SMI). Cortical bone outcomes included
cortical thickness (Ct.Th, mm), cortical bone area (Ct.Ar, mm2),
Ct.Ar fraction (%), cortical porosity (Ct.Po, %), and polar moment of inertia (pMOI, mm4), maximum moment of inertia
(Imax, mm4), and minimum moment of inertia (Imin, mm4). Scan
acquisition and analyses were conducted in accordance with
guidelines for use of ␮CT in rodents (35).
Endocrinology, April 2014, 155(4):1188 –1196
Results
Pamidronate decreases basal ERK1/2
phosphorylation in hypertrophic chondrocytes
Previous investigations have demonstrated that extracellular phosphate leads to hypertrophic chondrocyte apoptosis by activating the mitochondrial apoptotic pathway (14). Treatment of hypertrophic chondrocytes with
phosphate leads to rapid phosphorylation of Erk1/2,
which is required for phosphate-mediated hypertrophic
chondrocyte apoptosis (33). Based on studies demonstrating that alendronate impairs phosphate-induced cell death
of cultured avian chondrocytes (36), investigations were
undertaken to determine whether bisphosphonates alter
basal or phosphate-induced Erk1/2 phosphorylation in
hypertrophic chondrocytes. Primary murine costal chondrocytes were cultured under differentiating conditions
for 10 –14 days. Clodronate and the amino-bisphosphonates, alendronate, pamidronate, and zoledronate, were
added to the media 1 hour before the addition of phosphate. At the concentrations examined (10⫺6M, 10⫺7M,
and 10⫺8M), none of the bisphosphonates altered Erk1/2
phosphorylation in response to 3mM, 5mM, or 7mM
phosphate (data not shown). However, pamidronate, at
concentrations of 10⫺6M to 10⫺8 M, decreased basal
Erk1/2 phosphorylation (Figure 1).
Bisphosphonates do not impair hypertrophic
chondrocyte apoptosis in vivo
To evaluate whether bisphosphonate treatment alters
Erk1/2 phosphorylation in hypertrophic chondrocytes in
vivo, the effect of these treatment regimens on phosphoErk1/2 (pErk1/2) immunoreactivity was examined in the
growth plate of treated and control mice (Figure 2A). Immunohistochemical analyses for pErk1/2 demonstrated
homogeneous staining of the hypertrophic chondrocyte
layer in control mice as well as in clodronate-treated mice.
However, the domain of pErk1/2 immunoreactive cells
was attenuated in the clodronate-treated animals. A
smaller region of nonuniform pErk1/2 immunoreactivity
was observed in mice treated with alendronate, pamidro-
Figure 1. Effect of bisphosphonates on basal Erk1/2 phosphorylation.
Primary murine hypertrophic chondrocytes were treated with
bisphosphonates at the dose indicated (⫺, none; or 10⫺6M, 10⫺7M, or
10⫺8M), before the addition of 7mM sodium sulfate. Cell lysates were
prepared 30 minutes later to evaluate total (lower panel) Erk1/2 and
pErk1/2 (upper panel). Doses of bisphosphonates are indicated: ⫺,
none; ⫺6, 10⫺6; ⫺7, 10⫺7; ⫺8, 10⫺8. Data are representative of those
obtained from 3 independent primary chondrocyte preparations.
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Figure 2. Effect of bisphosphonates on pErk1/2, cleaved caspase 3
immunoreactivity, and hypertrophic chondrocyte apoptosis in vivo. The
growth plates of mice treated or not with bisphosphonates, from day
18 to 38, were subjected to immunohistochemical analyses for pErk1/2
(A) and cleaved caspase 3 (B). TUNEL was performed to identify
apoptotic hypertrophic chondrocytes along a 1600 ␮m width of the
late hypertrophic chondrocyte layer (C). The number of TUNEL labeled
hypertrophic chondrocytes per section was counted on at least 6 tibial
sections from 3 mice for each treatment condition (D). Data are
expressed as average ⫾ SEM. C, control; Alen, alendronate; Clo,
clodronate; Pam, pamidronate; ZA, zoledronate. Immunohistochemical
and TUNEL images are representative of data obtained from 2 sections
from each of 3 mice for each treatment condition.
nate, and zoledronate. Cleavage of caspase 3 is the final
step in programmed cell death. Immunohistochemical
analyses for cleaved caspase 3 demonstrated uniform
staining in both control and clodronate-treated mice.
Analogous to pErk1/2 immunoreactivity, nonhomogenous cleaved caspase 3 immunoreactivity was observed
in the growth plates of mice treated with alendronate,
pamidronate, and zoledronate (Figure 2B). To determine
whether the abnormalities in the growth plates of the bisphosphonate-treated mice were associated with alterations in hypertrophic chondrocyte apoptosis, TUNEL was
performed. These studies demonstrated a slight but nonsignificant increase in apoptotic hypertrophic chondrocytes in the pamidronate-treated mice (Figure 2, C and D).
Amino-bisphosphonates decrease hypertrophic
chondrocyte column length
To further characterize the effects of bisphosphonates
on the maturing growth plate, growth plate morphology
was evaluated by hematoxylin and eosin staining (Figure
3A). The number of chondrocytes per column in the hypertrophic chondrocyte layer was reduced in mice treated
with the nitrogen-containing bisphosphonates alendronate (3.0 ⫾ 0.4, P ⫽ .012 vs control), pamidronate (3.6 ⫾
0.2, P ⫽ .002 vs control), and zoledronate (3.3 ⫾ 0.2, P ⫽
.002 vs control) compared with controls (5.3 ⫾ 0.3).
However, the number of hypertrophic chondrocytes per
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Figure 3. Effect of bisphosphonates on molecular markers of
hypertrophic chondrocytes. A, Hematoxylin and eosin staining of the
growth plate of control and treated mice. In situ hybridization for ColX
(B), osteopontin (OP) (C), and MMP-9 (D). E, CD31
immunohistochemistry for endothelial cells. C, control; Alen,
alendronate; Clo, clodronate; Pam, pamidronate; ZA, zoledronate.
Images are representative of data obtained from 2 sections from each
of 3 mice for each treatment condition.
column in the growth plates of mice treated with the nonnitrogen-containing bisphosphonate, clodronate (5.0 ⫾
0.3, P ⫽ .25 vs control) was not significantly different
from that observed in control mice. In situ hybridization
analyses were performed to evaluate the effects of these
treatments on molecular markers of hypertrophic chondrocytes. The domain of type X collagen (ColX)-expressing hypertrophic chondrocytes was reduced in the mice
treated with nitrogen-containing bisphosphonates. The
uniform columnar pattern of ColX-expressing cells observed in the control mice was severely disrupted in growth
plates of the pamidronate-treated mice and to a lesser extent in those of mice treated with clodronate, alendronate,
and zoledronate (Figure 3B). To determine whether this
reflected an increase in terminal differentiation of hypertrophic chondrocytes, in situ hybridization was performed
for osteopontin, which is expressed in terminally differentiated hypertrophic chondrocytes and osteoblasts.
These studies demonstrated that the homogenous region
of osteopontin-expressing, terminally differentiated hypertrophic chondrocytes was disrupted in the pamidronate- and zoledronate-treated animals (Figure 3C).
Bisphosphonates have been shown to alter matrix metalloproteinase-9 (MMP-9) levels. In particular, zoledronate
suppresses MMP-9 expression (37), whereas pamidronate
increases MMP-9 mRNA levels in cells of the monocyte/
macrophage lineage, and clodronate has no effect (38). Based
on these data, in situ hybridization analyses for MMP-9 were
performed to evaluate the presence of osteoclasts/chondroclasts at the chondro-osseous junction. These studies demonstrated a decrease and nonuniformity in the domain of
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Endocrinology, April 2014, 155(4):1188 –1196
MMP-9-expressing cells in the pamidronate-treated mice
compared with control mice and mice treated with the other
3 agents (Figure 3D).
Terminally differentiated hypertrophic chondrocytes
signal vascular invasion, and the resultant blood vessels
bring in osteoclasts and osteoblasts, which form the primary spongiosa. Amino-bisphosphonates have been
shown to exhibit antiangiogenic properties (18, 37, 39).
However, in the current study, vascular invasion, assessed
by CD31 immunoreactivity for endothelial cells, was not
altered by bisphosphonate treatment (Figure 3E).
Alendronate and zoledronate decrease
metaphyseal osteoclast number
Tartrate resistant acid phosphatase staining was performed to evaluate the effect of bisphosphonate treatment
regimens on metaphyseal cortical bone osteoclasts (Figure
4). The number of osteoclasts in this region (350 ␮m) was
significantly decreased in the mice treated with alendronate and zoledronate relative to control mice (2.3 ⫾ 0.6
and 3.3 ⫾ 0.5, respectively, vs 7.5 ⫾ 1.5 in control mice;
P ⬍ .002 for both). Pamidronate and clodronate treatment
resulted in a small and insignificant decrease in cortical
osteoclasts in this region relative to control animals (6.0 ⫾
1.6 and 7.0 ⫾ 1.2, respectively).
Bisphosphonates effects on bone
Based on the more marked histological abnormalities observed in the growth plate of the amino-bisphosphonatetreated mice, investigations were undertaken to determine
the effectiveness of the 4 bisphosphonate treatment regimens
at increasing bone mass and microarchitecture. Femoral
length did not differ between control and treated mice, nor
among treatment groups (data not shown). A two-dimensional sagittal reconstruction of representative femurs from
treated mice is shown in Figure 5A. Arrows point to regions
of incompletely remodeled trabecular bone, corresponding
to the day-18 injections of pamidronate and zoledronate.
Notably, all of the bisphosphonate treatments influenced
trabecular bone volume and altered bone microarchitecture
(Table 1 and Figure 5B). Compared with controls, trabecular
bone volume was 3-fold higher in alendronate-treated mice,
2-fold higher in zoledronate-treated mice, and 1.3- to 1.6fold higher in clodronate- and pamidronate-treated mice.
The higher trabecular bone volume was due to increased
Tb.N and decreased Tb.Sp, with no effects on trabecular
thickness, except for an increase in the alendronate-treated
group. At the middiaphysis, bone size, as assessed by femoral
cross-sectional area, was increased in alendronate and zoledronate-treated mice compared with controls. Ct.Th and
Ct.Ar fraction were increased by clodronate, zoledronate,
and alendronate treatment. Remarkably, pamidronate was
Figure 4. Effect of bisphosphonates on cortical osteoclasts. TRAP
staining was performed to identify osteoclasts in the cortical bone of
the metaphyses. Magnified representations of the cortical bone are
shown on the right. C, control; Alen, alendronate; Clo, clodronate;
Pam, pamidronate; ZA, zoledronate. Images are representative of
those obtained from 3 mice for each treatment condition.
the only agent that failed to increase Ct.Th and Ct.Ar when
compared with untreated mice. Neither pamidronate nor
clodronate treatment led to improvement of inferred biomechanical parameters (pMOI, Imax, and Imin).
Discussion
These investigations were undertaken to determine the
effects of short-term bisphosphonate administration on
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1193
Figure 5. Effect of bisphosphonates on bone microarchitecture. A, Images of the distal femur of control and bisphosphonate-treated mice.
Arrows point to regions of retained bone associated with injection of zoledronate and pamidronate at day 18. B, BMD, Tb.N, Tb.Sp, Ct.Th, Ct.Ar/
tissue area (Ct.BA/TA), and Ct.Po values obtained from control and bisphosphonate-treated mice. Data represent the average ⫾ SEM of values
obtained from 3 femurs in each group. Significant differences are indicated in Table 1. C, control; Alen, alendronate; Clo, clodronate; Pam,
pamidronate; zol, zoledronate.
the growing murine skeleton. They were designed to address whether bisphosphonates alter growth plate maturation and longitudinal and appositional growth. They
also examined whether the skeletal phenotype of mice
treated with the nitrogen-containing bisphosphonates,
alendronate, pamidronate, and zoledronate, would differ
from that of mice treated with the nonnitrogen-containing
bisphosphonate, clodronate. The question of whether intermittent high-dose (zoledronate and pamidronate) amino-bisphosphonate administration would lead to a different phenotype, relative to that seen in mice undergoing
chronic low-dose treatment (alendronate and clodronate),
was also of interest.
Studies of the growth plate revealed only modest
changes that did not translate into altered long bone
growth. Interestingly, the nonnitrogen-containing bisphosphonate, clodronate, did not alter the number of hypertrophic chondrocytes per column and led to fewer growth
plate changes than the nitrogen-containing bisphosphonates. Based on our investigations in primary chondrocyte
cultures, which demonstrated no change in phosphatemediated Erk1/2 phosphorylation in response to acute bisphosphonate treatment, and a decrease in basal pErk1/2
in pamidronate-treated cells, it is unlikely that the modest
changes observed in vivo in the growth plate are a direct
effect of these agents on the apoptotic pathway. Histological analyses of diaphyseal radiodense band corresponding to the day-18 infusion of pamidronate and zoledronate
(arrows in Figure 5) did not reveal retained chondrocytes
but rather trabecular bone (data not shown), confirming
that these agents, as administered, do not prevent hypertrophic chondrocyte apoptosis in vivo. Furthermore, bisphosphonate treatment did not significantly increase the
number of TUNEL positive hypertrophic chondrocytes in
the maturing growth plate. In contrast to our findings,
administration of high-dose alendronate (1 mg/kg every
other day) to growing rat pups led to a marked expansion
of the hypertrophic chondrocyte layer, suggesting that
even at doses 17.5 times that used in our studies, alendronate treatment does not lead to hypertrophic chondrocyte
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Table 1.
Bisphosphonate Effects During Growth
␮CT Analyses of Femurs of Bisphosphonate-Treated and Control Mice
C
BMD (mg
HA/cm3)
BV/TV (%)
Tb.N (per
mm)
Tb.Sp (mm)
Conn.D
(mm⫺3)
Ct.Th (mm)
Ct.BA/TA
SMI
Ct.Po (%)
pMOI (mm4)
Imax (mm4)
Imin (mm4)
Endocrinology, April 2014, 155(4):1188 –1196
Clo
C
Zol
C Clo Alendronate
C
Clo Zol Pamidronate
113.83 ⫾ 4.40 149.87 ⫾ 5.91
a
204.27 ⫾ 11.94
a
a
313.97 ⫾ 17.84
a
a
a
174.21 ⫾ 6.84
a
12.76 ⫾ 0.35
4.51 ⫾ 0.04
a
23.01 ⫾ 1.4
8.58 ⫾ 0.27
a
a
a
a
a
a
a
20.28 ⫾ 0.70
6.71 ⫾ 0.26
a
a
37.99 ⫾ 2.50
11.63 ⫾ 0.63
a
a
0.109 ⫾ 0.004
628.63 ⫾ 26.67
a
a
a
a
a
a
a
0.147 ⫾ 0.006
503.17 ⫾ 50.37
a
a
0.083 ⫾ 0.004
1097.72 ⫾ 85.83
a
a
0.112 ⫾ 0.003
30.57 ⫾ 0.45
1.558 ⫾ 0.069
2.48 ⫾ 0.39
0.328 ⫾ 0.028
0.198 ⫾ 0.016
0.129 ⫾ 0.013
a
NS
NS
NS
NS
0.117 ⫾ 0.001
32.12 ⫾ 0.73
0.524 ⫾ 0.173
3.80 ⫾ 0.58
0.291 ⫾ 0.013
0.176 ⫾ 0.008
0.115 ⫾ 0.004
a
NS
a
a
NS
NS
NS
NS
a
a
a
0.092 ⫾ 0.003
25.715 ⫾ 0.85
1.603 ⫾ 0.650
2.36 ⫾ 0.16
0.203 ⫾ 0.019
0.130 ⫾ 0.013
0.074 ⫾ 0.006
17.15 ⫾ 0.67
6.47 ⫾ 0.22
a
0.224 ⫾ 0.003 0.150 ⫾ 0.006
303.06 ⫾ 6.01 472.17 ⫾ 21.79
a
0.095 ⫾ 0.004
26.77 ⫾ 0.45
1.875 ⫾ 0.038
4.87 ⫾ 0.78
0.202 ⫾ 0.004
0.132 ⫾ 0.003
0.070 ⫾ 0.002
a
0.109 ⫾ 0.004
28.93 ⫾ 0.67
1.649 ⫾ 0.047
1.55 ⫾ 0.50
0.239 ⫾ 0.024
0.152 ⫾ 0.016
0.087 ⫾ 0.008
a
a
NS
a
NS
NS
NS
a
a
a
a
a
a
a
a
a
NS NS
a
NS
a
NS
a
a
NS
a
NS
NS
C
a
a
a
a
NS
NS
NS
BV/TV, bone volume/total volume; Conn.D, connectivity density; Ct.BA/TA, Ct.Ar/total area; C, control; Clo, clodronate; Zol, zoledronate.
Pamidronate treatment was inferior to Zol and alendronate for all parameters listed with the exception of Ct.Po. The SMI and BMD of femurs from
pamidronate-treated mice were superior to those from Clo-treated mice. However, the Ct.Th of the Clo-treated femurs was significantly greater
than that of the pamidronate-treated bones. Other parameters were not significantly different between Clo and pamidronate treatment groups.
a
Significant differences when compared with C mice and those treated with Clo or Zol.
death (40) and may, in fact, impair this process. Other
studies (18) have also demonstrated expansion of the hypertrophic chondrocyte layer when alendronate is administered to growing animals. However, the doses used in
these investigations were significantly higher than those
used in our studies, which were selected to approximate
those used in clinical practice.
In rapidly growing mice, all the bisphosphonate regimens administered improved trabecular bone microarchitecture at the distal femoral metaphysis relative to controls, evidenced by greater trabecular bone volume and
Tb.N, as well as reduced Tb.Sp. Cross-sectional area and
Ct.Th at the midfemoral diaphysis were also improved by
zoledronate and alendronate treatment. Although
pamidronate did increase Tb.N and decrease Tb.Sp, it was
clearly not as effective as the other amino-bisphosphonates. The Ct.Ar moments of inertia, indirect indices of
bone strength, of the pamidronate-treated mice did not
differ from those of the control or clodronate-treated
mice. However, zoledronate and alendronate significantly
improved these parameters. Data from the pamidronatetreated mice were replicated using preparations obtained
from 2 independent manufacturers to insure that these
findings did not reflect manufacturing problems.
The results of these studies demonstrate mild histological abnormalities in the growth plate of bisphosphonatetreated mice. They do not, however, demonstrate a
marked abnormality in growth plate maturation, nor a
defect in vascular invasion. However, evaluation of the
bones of bisphosphonate-treated mice revealed significant
differences among the agents examined. Although the
doses and treatment regimens chosen for our investigations cannot precisely reproduce those used in humans,
those of pamidronate and zoledronate administered are
identical to those recommended for use in humans. Due to
the short duration of these studies, the dosing interval of
these agents was not adjusted.
The finding that the microarchitectural parameters of
the femurs of pamidronate-treated mice were not as favorable as those of the alendronate- and zoledronatetreated mice, and that the Ct.Th and Ct.Ar/total area were
not significantly different than those of control mice, was
not anticipated. Pamidronate has been widely reported to
improve bone density and decrease morbidity in children
with OI. In these studies, pamidronate increased spine
bone density in affected patients (5, 20, 21) and has been
reported to improve spine and distal femoral bone density
in children with quadriplegic cerebral palsy (7) and osteoporosis (41). However, these investigations did not examine the effects of pamidronate on the cortex of long bones.
In patients with OI treated with pamidronate, an increase
in cortical bone width was observed on iliac crest biopsy
(25), suggesting that it has beneficial effects on cortical
bone in this population. However, cortical bone mineral
density (BMD) was not examined. Based on the decrease
in fragility fractures in pamidronate-treated children with
symptomatic osteoporosis (42) and severe OI (5),
pamidronate does have beneficial effects in these populations. However, the results of our investigations suggest
that, for the treatment and prevention of secondary osteoporosis in children, alternative bisphosphonates may
improve outcomes. Although alendronate appeared to be
the superior agent in our studies, the dose administered
was chosen to reflect that administered to rapidly growing
oim/oim mice (30) and was 40% of that used in subsequent investigations in oim/oim and wild-type mice (43).
doi: 10.1210/en.2013-1993
Considering that the absorption of alendronate is approximately 0.6%, the dose administered in our studies is approximately double the dose used to treat children with OI
(1 mg/kg䡠d) (19) and exceeds that used for postmenopausal osteoporosis (70 mg/wk). However, the dose of
pamidronate administered in our studies (3 mg/kg) is that
used to treat children with OI (25) and is higher than that
used to treat symptomatic osteoporosis in children (1
mg/kg every 4 mo) (42). Thus, its modest effects, relative
to those of zoledronate and clodronate, was unanticipated. It is possible that, in our studies, more frequent
dosing of pamidronate would have improved its effects on
skeletal microarchitecture. However, higher cumulative
doses might be expected to exhibit more detrimental effects on the growth plate, notably, further reduction in
osteopontin-expressing cells and perhaps a significant increase in hypertrophic chondrocyte apoptosis.
Although longer-term studies are required to determine
whether there is a progressive growth plate phenotype
observed with chronic bisphosphonate treatment, to evaluate the consequences of the different bisphosphonate
treatments on bone length, and to assess the effects of the
different agents on biomechanical integrity of the skeleton, these short-term analyses suggest that administration
of bisphosphonates does not adversely effect the growth
plate or skeletal growth. The conclusions of the current
investigations are limited by the short duration of therapy,
the imprecise conversion of dose equivalents of oral to
parenteral medications, and potentially by different pharmacodynamics of these agents in mice vs humans. However, much preclinical data are obtained in rodent models,
and the observations that the effects of pamidronate on
bone microarchitecture are not as beneficial as those observed with other agents examined should prompt more
detailed analyses of the effects of amino-bisphosphonates
on cortical bone and growth in the pediatric population.
Acknowledgments
Address all correspondence and requests for reprints to: Marie
B. Demay, Endocrine Unit, Thier 11, Massachusetts General
Hospital, 50 Blossom Street, Boston, MA 02114. E-mail:
[email protected].
This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR061376 (to
M.B.D.).
Disclosure Summary: The authors have nothing to disclose.
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