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Targeting osteoclasts with zoledronic acid prevents bone destruction in collagen-induced arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 7, July 2004, pp 2338–2346
DOI 10.1002/art.20382
© 2004, American College of Rheumatology
Targeting Osteoclasts With Zoledronic Acid Prevents
Bone Destruction in Collagen-Induced Arthritis
Natalie A. Sims,1 Jonathan R. Green,2 Markus Glatt,2 Stephen Schlict,3 T. John Martin,4
Matthew T. Gillespie,5 and Evan Romas1
Objective. To study the effect of zoledronic acid
(ZA) on synovial inflammation, structural joint damage,
and bone metabolism in rats during the effector phase
of collagen-induced arthritis (CIA).
Methods. CIA was induced in female dark agouti
rats. At the clinical onset of CIA, rats were assigned to
treatment with vehicle or single subcutaneous doses of
ZA (1.0, 10, 50, or 100 ␮g/kg). Clinical signs in all 4
paws were scored on a daily basis. After 2 weeks, the
joints in the hind paws were assessed using plain
radiographs, microfocal computed tomography (microCT), histologic scoring, and histomorphometry, and the
serum levels of type I collagen crosslinks were measured
by enzyme-linked immunosorbent assay.
Results. Although ZA mildly exacerbated synovitis, it effectively suppressed structural joint damage. At
doses of >10 ␮g/kg, ZA significantly reduced radiographic bone erosions, Larsen scores, and juxtaarticular trabecular bone loss as quantified by micro-CT. ZA
prevented increased type I collagen (bone) breakdown
in CIA and diminished histologic scores of focal bone
erosion by up to 80%. Increases in the percentage of
eroded surface, osteoclast surface, and osteoclast num-
bers associated with CIA were prevented by ZA, even
though synovitis scores were unchanged.
Conclusion. Single doses (>10 ␮g/kg) of ZA strikingly reduced focal bone erosions and juxtaarticular
trabecular bone loss, although synovitis was mildly
exacerbated. Targeting osteoclasts with ZA may therefore be an effective strategy for preventing structural
joint damage in rheumatoid arthritis.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive destruction
of synovial joints. Osteoclasts, the cells responsible for
bone degradation, play a pivotal role in the joint destruction seen in RA (1–3) as well as animal models of RA
(4–7).
Interactions between activated T cells and macrophages drive the process of bone destruction that occurs
in RA by releasing proinflammatory cytokines, especially tumor necrosis factor ␣ (TNF␣), interleukin-1
(IL-1), and IL-17, which transform myeloid precursor
cells and synovial fibroblasts into tissue-destructive effector cells (8,9). TNF␣ from activated macrophages, in
the presence of permissive levels of RANKL (which is
produced by osteoblasts, synovial fibroblasts, and activated T cells), stimulates production of osteoclasts (10).
The importance of osteoclasts in arthritic bone
destruction has been verified by administration of osteoprotegerin (OPG; a decoy receptor for RANKL) in
spontaneously arthritic TNF␣-transgenic mice and models such as collagen-induced arthritis (CIA) and
adjuvant-induced arthritis, as well as a serum transfer
model of arthritis using RANKL-deficient mice, all of
which demonstrated reduced bone damage and dissociated synovial inflammation from bone destruction
(4,6,11,12). Osteoclasts were also directly implicated in
TNF␣-mediated bone loss, because crossing spontaneously arthritic human TNF␣-transgenic mice with osteopetrotic c-fos–deficient mice lacking osteoclasts resulted
Supported by the University of Melbourne Early Career
Research Grant Scheme, the National Health and Medical Research
Council (NHMRC; project grant 247909), and the Arthritis Foundation of Australia. Dr. Sims’ work was supported by an NHMRC Career
Development Award.
1
Natalie A. Sims, PhD, Evan Romas, MBBS, PhD: St. Vincent’s Hospital, University of Melbourne, Melbourne, Victoria, Australia; 2Jonathan R. Green, PhD, Markus Glatt, PhD: Novartis
Pharma, Basel, Switzerland; 3Stephen Schlict, MBBS: St. Vincent’s
Hospital, Melbourne, Victoria, Australia; 4T. John Martin, MD, DSc:
University of Melbourne, Melbourne, Victoria, Australia; 5Matthew T.
Gillespie, PhD: St. Vincent’s Institute, Melbourne, Victoria, Australia.
Address correspondence and reprint requests to Evan Romas,
MBBS, PhD, Department of Medicine, University of Melbourne, St.
Vincent’s Hospital, 41 Victoria Parade Fitzroy 3065, Melbourne,
Victoria, Australia. E-mail: romase@svhm.org.au.
Submitted for publication October 7, 2003; accepted in revised form April 13, 2004.
2338
ZOLEDRONATE PREVENTION OF BONE EROSIONS IN CIA
in inflammatory arthritis without bone destruction (7).
Together, these lines of evidence indicate that suppressing osteoclast numbers or function can be a strategy for
preserving skeletal integrity in inflammatory arthritis.
The bisphosphonates inhibit osteoclast formation, function, and survival at least in part by inhibiting
the mevalonate pathway enzyme, farnesyl diphosphate
synthase (13–15). Zoledronic acid (ZA; Zoledronate) is
a third-generation bisphosphonate (16,17) that is reported to have the greatest suppressive effect on bone
resorption. Although it was shown to be beneficial in
clinical trials of osteoporosis (18), Paget’s disease of
bone (19), renal transplantation–related bone loss (20),
and metastatic and osteolytic bone disease (21,22), ZA
has not been rigorously studied for its potential to
prevent bone destruction in models of RA.
The present study was designed to investigate the
effects of ZA on synovial inflammation, joint structure, and
bone resorption in rats during the effector phase of CIA.
The results show that single doses of ZA (ⱖ10 ␮g/kg)
administered at the onset of arthritis are sufficient to
prevent focal and juxtaarticular bone loss caused by arthritis, in the absence of therapeutic effects on synovitis.
MATERIALS AND METHODS
Reagents. Lyophilized native bovine type II collagen
(Sigma, St. Louis, MO) was dissolved at a concentration of 2
mg/ml in 0.01 moles/liter acetic acid. ZA was supplied by
Novartis Pharma AG (Basel, Switzerland).
Experimental animals. Female 9-week-old dark agouti
rats (n ⫽ 46) were used as the model for arthritis. Six rats
served as nonarthritic controls, and CIA was induced in the
remaining 40 (8 rats per treatment group). All animals were
fed standard rodent food and given water and 0.9% NaCl for
drinking ad libitum. The animals were weighed and monitored
daily for signs of arthritis. All experimental procedures conformed to the National Health and Medical Research Council
guidelines and were approved by an institutional animal ethics
committee. The rats were maintained at 22°C in an animal
room that was illuminated for 12 hours daily. After a 1-week
acclimatization period, the animals were used for experiments.
Induction of CIA. CIA was induced as previously
described (5,6). CIA developed in all of the immunized rats
after a mean (⫾SEM) interval of 19.5 ⫾ 0.5 days; the day of
arthritis onset was similar in all treatment groups.
Clinical assessment of CIA. Arthritis was monitored
daily by a macroscopic scoring system (range 0–4 for each
paw), as previously described (5,6). The scoring system is as
follows: 0 ⫽ no arthritis, 1 ⫽ swelling and/or redness of 1–2
interphalangeal (IP) joints, 2 ⫽ involvement of 3–4 IP joints or
1 larger joint, 3 ⫽ more than 4 joints red/swollen, and 4 ⫽
severe arthritis of an entire paw. Use of this system yielded a
score between 0 and 16 (maximum possible score) per animal.
In addition, the width of each paw was measured, using digital
calipers, at baseline and 3 times weekly beginning at the onset
2339
of arthritis. The percentage of change in paw thickness for each
paw was calculated from baseline measurements and was
expressed as an average for each rat, for statistical analysis.
Administration of ZA. At the clinical onset of arthritis
(the first day on which the clinical score was ⱖ2), rats were
randomly assigned to receive a single subcutaneous injection of
phosphate buffered saline (PBS) (vehicle) or ZA (1.0, 10, 50,
or 100 ␮g/kg).
Tissue collection and specimen preparation. Two
weeks after the onset of arthritis, blood was collected by
cardiac puncture exsanguination. The hind paws of each rat
were dissected and fixed in 4% paraformaldehyde/PBS overnight at 4°C, then stored in 70% ethanol for radiographic and
microfocal computed tomographic (micro-CT) analyses. Following these procedures, individual toes of the hind paws were
dissected and decalcified with 15% EDTA in 0.5%
paraformaldehyde/PBS at 4°C for 1 week before standard
processing for paraffin wax embedding. Serial 5-␮m sagittal
sections were obtained through the digits and stained with
hematoxylin and eosin (H&E) for general histologic evaluation
or toluidine blue for the specific assessment of cartilage. To
identify osteoclasts, sections were stained for tartrate-resistant
acid phosphatase (TRAP) using an acid phosphatase kit
(Sigma).
Radiographic examination. Contact radiographs of
both hind feet were obtained using a Faxitron x-ray cabinet
(Faxitron, Wheeling, IL) and high-resolution mammography
film (Eastman Kodak, Rochester, NY). Destruction of bone
and cartilage was classified and scored according to the Larsen
method (23), without knowledge of the assigned treatment
groups.
Micro-CT. Left hind paws were analyzed with a ␮CT
20 scanner (Scanco Medical, Bassersdorf, Switzerland). The
paws were scanned perpendicular to their longitudinal axis by
a fixed x-ray fan beam (10 ␮m spot-size tube, 0.1 mA, 50 kVp)
while the holder rotated along its axis. For 3-dimensional (3-D)
data accumulation, the sample was moved incrementally along
its axis after each turn. A total of 300 slices (1,024 ⫻
1,024–pixel matrix per slice) of 13 ␮m thickness were determined, yielding a voxel size of 13 ⫻ 13 ⫻ 13 ␮m3. The resulting
scan length of 3.9 mm was sufficiently long to cover the distal
metatarsal joint area of the third metatarsal bone. The distal
growth plate (“visible” as empty clefts) was used to position a
core volume of interest for 3-D reconstruction. A cancellous
core of 100 slices of 13 ␮m thickness (31.3-mm length) was
finally evaluated with MicroCT software (Scanco Medical) for
bone morphometry. The primary spongiosa was excluded from
measurement. The spatial resolution with the given settings of
the instrument was 24 ␮m; the precision of repeated measurements was ⬍1%. Threshold settings were optimized with
histomorphometric methods. Total volume, bone volume,
bone surface, trabecular number, trabecular separation, and
trabecular thickness were measured in the volume of interest
after removal of cortical bone using a semiautomatic morphing
procedure. Trabecular number, trabecular separation, and
trabecular thickness denote model-independent 3-D calculations (24).
Enzyme-linked immunosorbent assay (ELISA) for
type I collagen crosslinks. Serum was isolated by centrifugation and stored at ⫺80°C before analysis. The serum concentration of type I collagen crosslinks, a measure of bone
2340
resorption breakdown products, was measured by ELISA
(RatLaps ELISA; Nordic Bioscience, Herlev, Denmark).
Histopathologic assessment. The histopathologic assessment focused on the hind paw digits that show the most
severe effects of joint destruction. Because the distal IP (DIP)
joints were the first to become inflamed, followed by the
proximal IP (PIP) and metatarsophalangeal (MTP) joints,
each joint was scored separately to allow analysis of the effects
of ZA at different stages of inflammation and arthritic destruction. For standardization, sections bisecting the digits were
used to ensure that the proximal, middle, and distal phalanges
with their opposing cortices and cartilage ends were present.
At least 2 digits were randomly selected from each rat for
histologic assessment, without knowledge of specific interventions. H&E-stained sections were scored by 2 independent
observers, at low power for inflammation and pannus, and at
low (⫻10) and high (⫻100 or ⫻200) power for bone erosion,
with consultation to TRAP-stained sections when required.
Sequential toluidine blue–stained sections were then
scored for cartilage damage and proteoglycan loss. Inflammation was scored 0–4 according to the following criteria: 0 ⫽
normal, 1 ⫽ minimal inflammatory infiltration, 2 ⫽ mild
infiltration, 3 ⫽ moderate infiltration with lymphoid aggregates, 4 ⫽ marked infiltration with lymphoid aggregates and
edema. Pannus formation was defined as synovial proliferation
adjacent to cartilage and filling the joint space and was scored
0–3 as follows: 0 ⫽ none, 1 ⫽ minimal, 2 ⫽ moderate (invasion
of ⬍50% of the cartilage surface), 3 ⫽ severe (invasion of
ⱖ50% of the cartilage surface). Articular cartilage damage was
scored 0–4 according to the following criteria: 0 ⫽ normal, 1 ⫽
minimal (loss of toluidine blue staining only, indicating proteoglycan loss but no tissue damage), 2 ⫽ mild (loss of
toluidine blue staining, and cartilage thinning of the superficial
zone only), 3 ⫽ moderate (loss of cartilage to the second
articular cartilage zone or beyond), 4 ⫽ marked (regions of
exposed bone below the articular cartilage). Bone resorption
was scored 0–4 according to the following criteria: 0 ⫽ none,
1 ⫽ minimal (1–2 sites of resorption, visible only at high
magnification), 2 ⫽ mild (at least 3 sites of resorption, visible
only at high magnification), 3 ⫽ moderate (obvious foci of
resorption, visible at low power), 4 ⫽ marked (large erosions
extending through to the marrow space).
To enumerate osteoclasts, histomorphometric analysis
was performed on masked H&E-stained sections by adapting
standard procedures using the OsteoMeasure analysis system
(OsteoMetrics, Decatur, GA). Osteoclasts were defined using
the standard criterion of multinucleated cells residing in
resorptive lacunae on the bone surface. Both IP and MTP
joints were included in the analysis, which was carried out on
the periosteal surface (the cortical bone surface facing the
external environment) over a region 300 ␮m from the chondroosseus junction toward the midshaft. At each joint, the eroded
surface (ES), osteoclast surface (OcS), osteoclast number
(NOc), and osteoclast area were expressed as a ratio in relation
to the bone surface (BS), bone perimeter (BPm), or ES.
Statistical analysis. Differences between groups were
analyzed by one- or two-way analysis of variance followed by
Fisher’s post hoc test. P values less than 0.05 were considered
significant.
SIMS ET AL
Figure 1. A, Inflammation scores in rats treated with or without
zoledronic acid (ZA). Arthritis was evaluated daily by a clinical score
(maximum score per animal 16; see Materials and Methods). B, Effect
of ZA on paw swelling. For each hind paw, the footpad thickness was
measured and expressed as a percentage of baseline thickness; an
average value was determined for each rat. Values are the mean ⫾
SEM. ⴙ ⫽ first day at which the P value was ⬍0.05 versus nonarthritic
rats; ⴱ ⫽ P ⬍ 0.05 versus collagen-induced arthritis (CIA)–untreated
rats at the same time point. Z ⫽ zoledronic acid.
RESULTS
Changes in clinical markers of incidence and
severity of arthritis. The rate of progression of CIA after
onset was not significantly altered by ZA treatment
(Figure 1A). In rats treated with 100 ␮g/kg of ZA, the
arthritis score was higher on days 10–13 compared with
untreated rats with CIA (Figure 1A). Paw thickness
peaked 1 week after the onset of CIA. All doses of ZA
enhanced paw swelling (Figure 1B); the most severe
exacerbation was detected in rats treated with 100 ␮g/kg
of ZA 4–7 days after disease onset. These differences in
paw thickness between groups were no longer present at
2 weeks.
Findings on plain radiographic examination. Although ZA mildly exacerbated arthritis, the treated rats
exhibited clear protection against structural joint damage. In fact, doses of ⱖ10 ␮g/kg were sufficient to block
the significant increase in Larsen scores observed in
ZOLEDRONATE PREVENTION OF BONE EROSIONS IN CIA
2341
Figure 2. Zoledronic acid (ZA; Zol) prevents structural damage in collageninduced arthritis (CIA) model. A, Plain radiographs of the hind paw obtained 2
weeks after arthritis onset in a normal rat (left), a rat with CIA that did not receive
ZA (middle), and a rat with CIA that received 100 ␮g/kg of ZA (right). Note the
damage highlighted in the boxed area (middle) and prevention of damage by ZA
treatment (right). B, Scoring of the tarsometatarsal and metatarsophalangeal joints
using the Larsen method showed prevention of arthritic joint destruction by ZA
doses of ⱖ10 ␮g/kg. max ⫽ maximum. C, Microfocal computed tomographic images
showing dorsal surface reconstruction of the hind paws from a normal rat (left), an
untreated arthritic rat (middle), and an arthritic rat treated with 100 ␮g/kg of ZA
(right). Note the joint destruction at the distal third metatarsal joint (open arrow)
and periosteal new bone formation due to periostitis at the tarsus and proximal
metatarsal joints. D, Percentage of trabecular bone volume (Vol) ascertained at the
distal third metatarsal bone, as described in Materials and Methods. Values are the
mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus no CIA; ⴱⴱ ⫽ P ⬍ 0.01 versus no CIA; ⴙⴙⴙ
⫽ P ⬍ 0.001 versus untreated CIA.
untreated CIA rats at the hind paw, because this parameter of disease severity was reduced by ⬃50% compared with untreated CIA rats (Figures 2A and B).
Findings on micro-CT analysis. To further elucidate the effects of ZA on periarticular bone integrity,
micro-CT analysis was performed on arthritic rats treated
with or without ZA. Micro-CT imaging demonstrated that
ZA prevented focal bone erosions at the tarsal, MTP, and
IP joints (Figure 2C). The new periosteal bone formation
observed in the tarsus has been described previously in
CIA (25,26), and, to avoid confounding effects, the tarsal
region was not included in the micro-CT analysis.
To reveal the effect of ZA on juxtaarticular bone,
the trabecular bone volume at the distal end of the
middle metatarsal bone was evaluated by quantitative
imaging. Trabecular bone volume at this site was reduced by ⬎60% in the untreated CIA group; ZA not
only abrogated this loss but led to trabecular bone
2342
SIMS ET AL
Table 1. ZA-induced increase in serum type I collagen crosslinks
(RatLaps) associated with CIA*
Treatment group
RatLaps, ng/ml
No CIA
Untreated CIA
CIA ⫹ 1 ␮g/kg ZA
CIA ⫹ 10 ␮g/kg ZA
CIA ⫹ 50 ␮g/kg ZA
CIA ⫹ 100 ␮g/kg ZA
19.1 ⫾ 2.8
31.2 ⫾ 4.0†
16.9 ⫾ 5.2
18.9 ⫾ 4.3
22.1 ⫾ 3.6
20.5 ⫾ 5.2
* Values are the mean ⫾ SEM for 6–8 rats per group. Serum samples
were collected 2 weeks after the onset of clinical arthritis symptoms.
ZA ⫽ zoledronic acid; CIA ⫽ collagen-induced arthritis.
† P ⬍ 0.05 by one-way analysis of variance followed by Fisher’s post
hoc test, versus controls (no CIA).
accrual despite continued inflammation (Figure 2D).
ZA maintained trabecular bone volume by preventing
CIA-induced reductions in both trabecular number and
thickness (data not shown).
Changes in systemic bone resorption. To ascertain the degree of bone resorption occurring in the
whole animal, the levels of circulating type I collagen
crosslinks were quantitated by ELISA. At 2 weeks, the
mean level of type I collagen crosslinks was almost
2-fold higher in the arthritic rats compared with the
nonarthritic rats (Table 1). ZA abrogated the increased
concentration of type I collagen crosslinks in CIA, at all
doses tested.
Histopathology of arthritis. To elucidate the
effects of ZA treatment on joint structure at the histologic level, inflamed joints were scored with semiquantitative grading scales. In the toes, inflammatory synovitis was most consistent and severe in the IP and MTP
joints, which therefore were assessed in detail.
Bone erosions.The most striking effects of ZA were
reflected in the bone erosion scores. Arthritic rats treated
with ZA had significantly lower bone damage scores compared with vehicle-treated rats (Figure 3B). At a dose of
100 ␮g/kg, ZA reduced bone erosion scores by at least 80%
compared with vehicle. A dose-dependent relationship was
Figure 3. A, Joint regions used for histologic evaluation of B, bone erosion, C, cartilage
damage, D, pannus formation, and E, synovial inflammation in rats with collageninduced arthritis (CIA). The distal interphalangeal (DIP), proximal IP (PIP), and
metatarsophalangeal (MTP) joints from normal (nonarthritic) rats and arthritic rats
treated with or without zoledronic acid (ZA; Z) were scored using a semiquantitative
system (described in Materials and Methods). Results are the mean and SEM score.
max ⫽ maximum; ⴙ ⫽ P ⬍ 0.05 versus control (nonarthritic) rats; ⴙⴙ ⫽ P ⬍ 0.01 versus
control (nonarthritic) rats; ⴙⴙⴙ ⫽ P ⬍ 0.001 versus control (nonarthritic) rats; ⴱ ⫽ P
⬍ 0.05 versus untreated rats; ⴱⴱ ⫽ P ⬍ 0.01 versus untreated rats; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus
untreated rats.
ZOLEDRONATE PREVENTION OF BONE EROSIONS IN CIA
2343
Figure 4. Effects of zoledronic acid (ZA) on osteoclasts and bone erosion sites in
collagen-induced arthritis (CIA). Low-power images of an untreated joint (A) and a joint
treated with 10 ␮g/kg of ZA (B) clearly show sites of reduced erosion in ZA-treated
joints (white arrows). Area boxed in black (A) indicates region of new periosteal bone
formation. Higher-power images (C and D) of regions boxed in green in A and B show
that although numerous osteoclasts (black arrows) can be seen in erosion sites in
untreated CIA (C), these were rarely detected in joints from rats treated with 10 ␮g of
ZA (D). Histomorphometric analysis (E) (see Materials and Methods) demonstrated
significantly reduced erosion sites (eroded surface/bone surface [ES/BS]), osteoclast
surface (OcS/BS), and osteoclast number (NOc/bone perimeter [BPm]) per unit of joint
surface in CIA rats treated with 10 ␮g, 50 ␮g, and 100 ␮g/kg of ZA. In contrast, the
number of osteoclasts per unit of eroded surface (NOc/eroded perimeter [EPm]) was
significantly increased in rats with CIA that were treated with 50 ␮g and 100 ␮g of ZA,
indicating that an increased number of osteoclasts is required for the same unit of eroded
surface (reduced osteoclast activity). Values are the mean and SEM. ⴙⴙⴙ ⫽ P ⬍ 0.001
versus control (nonarthritic) rats; ⴱ ⫽ P ⬍ 0.05 versus untreated rats with CIA; ⴱⴱⴱ ⫽
P ⬍ 0.001 versus untreated rats with CIA. Bar ⫽ 500 ␮m in A; 100 ␮m in C.
present at the DIP joints, with 100 ␮g/kg of ZA providing
the greatest bone protection, and a dose of 1 ␮g/kg
providing no significant protection.
Cartilage damage. Arthritic rats exhibited similar
degrees of cartilage damage regardless of the assigned
treatment group (Figure 3C).
2344
SIMS ET AL
Synovial inflammation and pannus. Consistent
with the lack of clinical antiinflammatory effect, ZA in
doses ranging from 1–100 ␮g/kg did not influence histologic scores for synovial inflammation or pannus invasion at either the IP or MTP joints (Figures 3D and E).
Bone histomorphometry. To further quantify the
effects of ZA, histomorphometric analysis of the bone
surface at the joint was performed (Figure 4). This
analysis showed that CIA was associated with increased
eroded surface (ES/BS), osteoclast surface (OcS/BS),
and osteoclast numbers (NOc/BPm) in the vicinity of
inflamed joints, and all of these parameters were inhibited by ZA at doses of ⱖ10 ␮g/kg (Figure 4). Furthermore, in arthritic rats treated with ⱖ50 ␮g/kg of ZA, the
number of osteoclasts per unit of eroded surface (NOc/
ES) was significantly increased (Figure 4). This increase
in the NOc/ES quotient suggests reduced osteoclast
activity in ZA-treated rats, because a greater number of
osteoclasts are required to produce the same unit of
eroded surface. Finally, there was no significant change
in osteoclast size or the number of nuclei per osteoclast
with either CIA or with ZA treatment at any dose (data
not shown).
DISCUSSION
The ability of bisphosphonates to regulate bone
turnover by suppressing osteoclast activity, together with
their selective localization to bone, have inspired their
widespread use for osteoporosis, Paget’s disease of bone,
humoral hypercalcemia of malignancy, and metastatic
cancer in bone (18–22). RA is characterized by multifaceted bone pathology, specifically generalized and
juxtaarticular osteoporosis, as well as focal bone erosions at the joint (2,9,27). Experimental models of RA
provide compelling verification that these focal bone
erosions are dependent on osteoclastic activity that in
turn is regulated by proinflammatory cytokines such as
IL-1, TNF␣, and IL-17, as well as RANKL (4,6,7,11,13).
We therefore hypothesized that ZA, as an osteoclast
inhibitor, might be effective in preventing structural
damage in arthritis.
ZA is a third-generation bisphosphonate with
inhibitory effects on bone resorptive activity that are
100-fold to 10,000-fold stronger than those of the
second- and first-generation bisphosphonates pamidronate and etidronate, respectively (16,17). In the
ovariectomized rat, ZA administered once weekly at a
dose of 0.3 ␮g/kg slowed this bone loss, and at a dose of
1.5 ␮g/kg, bone loss was completely prevented (16).
Similarly, 4 mg of ZA administered intravenously once
per year to postmenopausal women with osteoporosis
reduced biochemical markers of bone metabolism and
increased bone mineral density (18).
In the present study, ZA dose-dependently suppressed bone erosions in CIA even though inflammation
was not reduced. In fact, at early time points, arthritis
scores and paw swelling were exacerbated, but this did
not result in worsening of the joint damage. This dissociation of synovial inflammation from bone erosion was
also shown with the osteoclastogenesis inhibitor, OPG
(4,6,12). Both the histopathologic and radiographic analyses revealed a significant bone protective effect of ZA
in CIA. This was particularly clear in the histomorphometric and micro-CT analyses. This protection was less
obvious in the Larsen damage scores, probably because
of the lower resolution of plain radiographs, the later
onset of inflammation in the ankle region, and the
confounding factor of new periosteal bone formation
that exists in this model (25,26). The effectiveness of ZA
in suppressing bone erosions was comparable with the
effects of OPG injections, highlighting the preeminence
of osteoclastic activity in mediating these bone lesions
(4,6,9,12). The effects of ZA in CIA are consistent with
the inhibition of osteoclastic bone resorption by this
agent in other models of clinical bone loss, such as
osteoporosis in the ovariectomized rat and rhesus monkey (16).
The main perturbation of bone metabolism in
RA is increased osteoclastic bone resorption (28), which
is correlated to high inflammatory disease activity (2).
Focal bone destruction is strongly associated with generalized osteoporosis, indicating a common mechanism
between these types of bone loss (27). A high bone
turnover rate and decreased bone mineral density were
also demonstrated in the periarticular bone of rats with
experimental arthritis (4,28–32). In our study, micro-CT
analysis clearly demonstrated that ZA prevented juxtaarticular trabecular bone loss.
At doses of ⬎1.0 ␮g/kg, ZA inhibited the increase in eroded surface (ES/BS), osteoclast surface
(OcS/BS), and osteoclast number (NOc/BPm) that is
associated with CIA. Furthermore, in arthritic rats
treated with 50 ␮g/kg or 100 ␮g/kg of ZA, the number of
osteoclasts per millimeter of eroded surface (NOc/ES)
was significantly elevated, indicating a feedback mechanism responding to reduced osteoclast activity. Thus,
ZA may exert its beneficial effect by down-regulating
both osteoclast numbers and osteoclast activity. Like
other bisphosphonates, ZA may induce osteoclast apoptosis (13–15) and suppress osteoclast function via accessory cells, such as osteoblasts (33). Aminobisphos-
ZOLEDRONATE PREVENTION OF BONE EROSIONS IN CIA
phonates also interact with the ␥/␦ T cell receptor, and
their effects on osteoclastic resorption may be secondary
to action on T cells (34), which are critical for osteoclastogenesis in RA (4,9). ZA also stimulates the release of
OPG by osteoblasts (35), and OPG is expressed by
synoviocytes in CIA and RA (6), suggesting that several
distinct mechanisms of osteoclast inhibition might contribute to the actions of ZA in arthritis.
It is important to note that in our study, ZA at
doses as low as 1.0 ␮g/kg suppressed increased levels of
bone type I collagen breakdown products, but higher
doses (ⱖ10 ␮g/kg) were necessary for protection against
focal bone erosions. Other third-generation bisphosphonates, such as minodronic acid, have also been reported
to suppress bone turnover markers and bone mineral
density loss caused by CIA (28).
The basis for the mild exacerbation of CIA
observed with high-dose ZA may be related to proinflammatory cytokine release, as described previously for
both ZA and ibandronate (36). In the context of
bisphosphonate studies in experimental arthritis, the
suppressive effect of single doses of ZA on focal bone
destruction is noteworthy. The earlier-generation
bisphosphonates usually reduced markers of increased
bone resorption and physiologic bone resorption at the
growth plate, although evidence of inhibition of focal
bone erosions was not compelling (37–42). A notable
exception was pamidronate, which reduced the size of
radiographic bone erosions in spontaneously arthritic
TNF␣-transgenic mice to an extent similar to that
observed with the TNF inhibitor infliximab (12).
Clinical trials of bisphosphonates in RA generally
demonstrated suppressive effects on generalized bone
loss but not on focal bone erosions (43–46). In fact,
protection against structural joint damage was reported
only with high-dose pamidronate (44). Factors such as
the absence of an extensive bone surface at the joint,
lack of drug potency, or underdosing may explain why
clinical trials of bisphosphonates have thus far failed to
demonstrate modification of structural joint damage
(47). The most potent bisphosphonates (e.g., ZA), which
are associated with the most profound reductions in
bone turnover markers, have not yet been examined in
sufficiently powered controlled clinical trials.
In conclusion, single doses of ZA effectively
suppressed focal bone erosions and juxtaarticular bone
loss in CIA, despite continuing synovitis. Targeting
osteoclasts with ZA may be an effective adjunctive
strategy for preventing structural joint damage and
generalized osteoporosis in RA.
2345
ACKNOWLEDGMENTS
We sincerely thank Ingrid Kriechbaum, Jeanette Dickens, and William Paspaliaris for expert technical assistance.
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