Animal Procedures and Disposition
In the present study, female Sprague-Dawley rats received celecoxib once a
day by means of oral gavage. Celecoxib has a plasma elimination half-life of
fourteen hours in female rats, compared with 3.73 hours in male
rats41. In humans,
the elimination half-life of celecoxib is approximately eleven
hours42. Thus,
celecoxib was chosen as the experimental COX-2-selective nonsteroidal
anti-inflammatory drug and female rats were chosen as the animal model because
the pharmacokinetics of celecoxib in female rats closely mimics those in
humans.
Three hundred and seventy-seven female Sprague-Dawley rats weighing an
average (and standard deviation) of 272 ± 7 g were used in the study.
Ninety-eight of these rats were excluded from the study because of fracture
destabilization, anesthesia-related death, poor fracture quality, infection,
or experimental error (Tables I
and II). Fracture
destabilization was considered to have occurred if the intramedullary rod that
was used to stabilize the fracture (described below) had dislodged from the
proximal part of the femur and moved distally such that adequate fracture
reduction was not assured. A number of rats were used only for radiographic
analysis because the fracture was too proximal or distal to the midpoint of
the femur and thus was not acceptable for mechanical testing but was adequate
for radiographic scoring or because an error that occurred during the
mechanical testing precluded use of the mechanical data but not the
radiographic data.
The rats were divided randomly into three study groups: a dose-dependent
study group, a time-dependent study group, and a time-delay study group. The
animals in the three study groups were treated with celecoxib or vehicle (1%
methylcellulose) by means of oral gavage once a day. In addition, eighteen
animals were randomly selected for the determination of the effect of
nonsteroidal anti-inflammatory drug treatment on fracture callus prostaglandin
levels. Three treatment groups comprising six animals each received either
vehicle (control), celecoxib (4 mg/kg), or diclofenac (5 mg/kg) and were
analyzed at four days after fracture. One rat that was treated with diclofenac
died prior to the end point. The overall success rate for rats reaching their
experimental end point and successful data acquisition was 74%. The New Jersey
Medical School Institutional Animal Use and Care Committee approved all animal
procedures. Diclofenac was purchased from Cayman Chemical (Ann Arbor,
Michigan), celecoxib was obtained in the form of Celebrex capsules (Pfizer,
New York, NY), and methylcellulose was purchased from Sigma (St. Louis,
Missouri).
The dose-dependent study involved 154 female rats. These animals were
randomly selected into four treatment groups that received (1) vehicle only
(the control group) (forty-eight rats), (2) celecoxib at a dose of 2 mg/kg
(thirty-two rats), (3) celecoxib at a dose of 4 mg/kg (thirty-eight rats), and
(4) celecoxib at a dose of 8 mg/kg (thirty-six rats). The animals were treated
once daily beginning four hours after the fracture and continuing for fifteen
days. The time-dependent study involved 139 rats. These animals were selected
randomly into four groups that were treated with 4 mg/kg of celecoxib once a
day for (1) five days (thirty-seven rats), (2) ten days (forty-four rats), (3)
twenty-one days (twenty rats), or (4) twenty-eight days (thirty-eight rats)
beginning four hours after the fracture. The time-delay study involved
sixty-six rats. These animals were selected randomly into three groups that
received (1) a five-day pretreatment (twenty-three rats), (2) a seven to
twenty-eight-day treatment (twenty rats), or (3) a fourteen to
twenty-eight-day treatment (twenty-three rats). The drug treatment was started
either five days before the fracture or on the seventh or fourteenth day after
the fracture, depending on the experimental group. In the fourteen to
twenty-eight-day treatment group and the seven to twenty-eight-day treatment
group, treatment was continued until the twenty-eighth day after the fracture.
In the pretreatment group, a femoral fracture was induced in each rat four
hours after the fifth and final celecoxib dose.
The rats were anesthetized with an intraperitoneal injection of ketamine
(40 mg/kg) and xylazine (5 mg/kg). The right hindlimb was shaved and was
washed with a povidoneiodine solution. Under aseptic conditions, a medial
parapatellar incision (0.4 to 0.5 cm) was created. The patella was dislocated
laterally and the medullary canal was entered through the intercondylar notch
and was reamed with a 21-gauge needle. A 0.7-mm stainless steel pin (Small
Parts, Miami Lakes, Florida) was then inserted through the medullary canal
into the proximal part of the greater trochanter and was tapped into place.
The distal portion of the pin was cut flush with the femoral condyles so as
not to interfere with knee function. The patellar dislocation was reduced, and
the soft tissue and skin were closed with 4-0 Vicryl resorbable sutures in two
layers. After the incision was closed, the middle of the diaphysis of the
pinned femur was fractured with use of a custom-made three-point bending
device (BBC Specialty Automotive Center, Linden, New Jersey) as described
previously28,43.
The animals were caged in pairs and were allowed to walk freely after
surgery.
Radiographic Analysis
All animals were examined with mediolateral radiographs immediately after
the fracture and at the time of death. Radiographs were made with use of a
Packard Faxitron (model 804; Field Emission, McMinnville, Oregon) and Kodak
Min-R 2000 film (Eastman Kodak, Rochester, New York). In addition, radiographs
were made of femora that were designated for mechanical testing after
resection. Blinded samples of the eight-week post-resection radiographs were
independently examined by three observers (including one of the authors
[J.P.O'C.]) and were graded by assigning a score ranging from 0 to 4 as
described
previously44. The
grading scheme was based on the bridging of the fracture by callus and the
cortical bone at the fracture site. One point was assigned to each aspect of
the fracture (that is, the right callus and cortex and the left callus and
cortex) that appeared bridged. A score of 0 represented the absence of
radiographic bridging among all four aspects of the fracture, indicating a
fracture nonunion. A score of 4 represented a fully bridged fracture.
Mechanical Testing
Animals within each treatment group were killed at eight weeks after the
fracture by means of CO2 asphyxiation. Animals with an oblique
fracture, a comminuted fracture, or an infection involving the femur were
excluded from mechanical testing. Femora were resected and cleaned of all soft
tissue, with the fracture callus being left intact. Femoral length, maximum
and minimum fracture callus diameters, and maximum and minimum mid-diaphyseal
diameters of the intact (contralateral) femur were measured with use of
digital calipers. The intramedullary rod was left in place because, as a
result of the smaller size of the animals, removal of the rod would damage the
condyles and also the integrity of the fracture callus. It was postulated that
the intramedullary rod would not interfere with the mechanical testing because
it lay along the neutral axis of the torsional test. The femoral ends were
potted in 1-in (2.54-cm) hexagonal nuts with use of a low-melt-temperature
metal (Wood's metal; Alfa Aesar, Ward Hill, Massachusetts). The samples were
wrapped in saline solution-soaked gauze to prevent dehydration between
steps.
Once the femoral ends had been potted, the gauge length of the sample was
measured and torsional testing was conducted with use of a servohydraulic
testing machine (MTS, Eden Prairie, Minnesota) with a 20-Nm reaction torque
cell (Interface, Scottsdale, Arizona). The testing was carried out to failure
at an actuator head displacement rate of 2° per second and a data
recording rate of 20 Hz. The fractured and intact femora were tested in
internal rotation and proper anatomic orientation. The failure mode of each
femur during the mechanical testing procedure was determined by means of
visual inspection and was designated as union (indicating that the femur had
failed spirally), incomplete union (indicating that the femur had some osseous
bridging but had failed principally along the original fracture line), or
nonunion (indicating that the femur had failed along the original fracture
line and had no evidence of osseous bridging). The results were recorded with
an Olympus C-3040 Zoom digital camera (Olympus Imaging America, Center Valley,
Pennsylvania).
The peak torque and the angle at the time of failure were obtained from the
load-deformation curves. Internal callus dimensions were measured after
torsional testing. The femoral dimensions were used to calculate shear stress,
shear modulus, and torsional
rigidity45,46.
The femora were modeled as hollow ellipses, and the polar moment of inertia
was
calculated45,47.
Fracture Callus Prostaglandin Level
Animals within each treatment group were killed one hour after the final
drug dose was administered on the fourth day after the fracture. The femora
were resected and the fracture callus was isolated and flash frozen with use
of liquid nitrogen. The samples were then weighed and pulverized with a mortar
and pestle. The pulverized callus was extracted with five volumes of M-PER
reagent (Pierce, Rockford, Illinois) that was supplemented with protease
inhibitors (Sigma Aldrich, St. Louis, Missouri). The samples were placed on a
mixer at 4°C for thirty minutes. Insoluble material was removed from the
extract by means of centrifugation (10,000 RPM for ten minutes). The
supernatant (clarified extract) was collected and stored at —80°C.
Two milliliters of ethanol was added to 0.5 mL of clarified extract, and the
precipitated proteins were removed by centrifugation at 3000 times gravity for
ten minutes. The supernatant was dried in a vacuum and then was dissolved in
1-M citrate buffer (pH 4). The samples then were applied to 500-mg C18 columns
(Waters Sep-Pak; Waters, Milford, Massachusetts) that had been preactivated by
means of methanol and water washes. The columns were washed with water and
then hexane. The eicosanoids were eluted with 5 mL of ethyl acetate containing
1% methanol. The eluted eicosanoids were dried in a vacuum and were
resuspended in EIA buffer (0.1-M sodium phosphate, pH 7.4; 0.4-M sodium
chloride; 0.1% bovine serum albumin; 1-mM ethylenediaminetetraacetic acid; and
0.01% sodium azide). Prostaglandin E2 (PGE2) and
prostaglandin F2a (PGF2a) concentrations
were determined by means of enzyme-linked immunoassay (Cayman Chemical). The
assays and analyses were performed according to the manufacturer's
instructions. The PGE2 and PGF2a concentrations
were normalized to total protein concentration measured with use of
bicinchoninic acid (BCA Protein Assay;
Pierce)48.
Statistical Methods
SigmaStat software (version 3.0; SPSS, Chicago, Illinois) was used for all
statistical analyses. The radiographic analysis, mechanical testing data, and
prostaglandin levels were evaluated for significant difference with use of a
one-way analysis of variance and post hoc Holm-Sidak tests. The failure mode
was compared between groups with use of Fisher exact tests by comparing the
proportion of nonunions in a treatment group with that in the control group.
Data from specific treatment groups, such as the control group, are
repetitively shown in the figures and tables for clarity. However, each
outcome parameter (radiographic scores, peak torque, rigidity, maximum shear
stress, and shear modulus) was compared between all of the treatment groups as
a single statistical analysis because only one control group was used for
these experiments. Differences between treatment groups were considered to be
significant when the p value was =0.05. Analysis of variance indicated that
statistical differences existed between the tested outcome parameters with p
values of <0.001 for all tested outcomes and with statistical power of
>0.9 (range, 0.94 to 0.999) for each test at an alpha value of 0.05.
Effects of Celecoxib Dose on Fracture-Healing
Inhibition of fracture-healing was detected in association with all doses
of celecoxib used (Fig. 1; see
Appendix). Rats were treated with celecoxib once per day, for fifteen days,
beginning four hours after the fracture. The celecoxib doses were 2, 4, and 8
mg/kg. Healing was measured at eight weeks after the fracture by means of
radiography and mechanical testing. Radiographic scoring showed that whereas
the 2-mg/kg celecoxib dose had no significant effect on healing, the 4 and
8-mg/kg doses significantly impaired healing
(Fig. 1, panel A). Torsional
mechanical testing of the healing femora also demonstrated that celecoxib
treatment impaired healing (Fig.
1, panels B and C). While a dose-dependent decrease in peak torque
was observed, the differences between the control and celecoxib-treated
samples were significantly different only in the 4 and 8-mg/kg celecoxib
treatment groups (see Appendix). In contrast, the rigidity
(Fig. 1, panel B), shear stress
(Fig. 1, panel C), and shear
modulus (see Appendix) of the healing femora from the celecoxib-treated rats
were all significantly reduced relative to the healing femora from control
rats. Finally, all femora were examined after torsional mechanical testing to
determine if the femur failed through the callus as a spiral fracture
(indicating union), principally along the original fracture with some
bone-bridging evident (indicating incomplete union), or along the original
fracture with no bone-bridging apparent (indicating nonunion). All
thirty-seven femora from the control rats failed as unions (twenty-nine
femora) or incomplete unions (eight femora). Celecoxib treatment, however,
significantly increased the number of nonunions in the 2, 4 and 8-mg/kg
celecoxib treatment groups (with nonunion occurring in five of fourteen,
thirteen of twenty-three, and four of thirteen femora, respectively)
(Fig. 1, panel D).
These data show that, in rats, celecoxib treatment within the therapeutic
range used by humans can significantly impair fracture-healing. On the basis
of these data, subsequent experiments employed the 4-mg/kg celecoxib dose to
determine the effects of treatment regime on fracture-healing.
Effects of Celecoxib Treatment Duration on Fracture-Healing
Continuous pharmacological inhibition of COX-2 or ablation of the COX-2
gene dramatically impairs
fracture-healing28.
However, this is unlike the common clinical scenario when patients use
analgesics for the first one or two weeks following a fracture. Thus, it is
possible that inhibition of COX-2 activity during the early phases of
fracture-healing may have little or no deleterious effects on ultimate healing
outcomes and that it is only sustained inhibition of COX-2 or inhibition of
COX-2 during a critical later phase of healing that impairs
fracture-healing.
To address this question, rats were treated with a human therapeutic dose
of celecoxib (4 mg/kg) for five, ten, fifteen, twenty-one, or twenty-eight
days, beginning four hours after the fracture.
The treatment periods spanned phases of fracture-healing from the early
inflammatory phase to normal fracture-bridging (approximately twenty-eight
days). Fracture-healing was assessed at eight weeks by means of radiography
and mechanical testing (Fig. 2;
see Appendix). Radiographic scoring at eight weeks after the fracture showed
significant impairment of fracture-healing when celecoxib treatment had lasted
fifteen days or longer (Fig. 2,
panel A). In contrast, torsional mechanical testing demonstrated that
celecoxib treatment significantly reduced peak torque (see Appendix), rigidity
(Fig. 2, panel B), maximum
shear stress (Fig. 2, panel C),
and shear modulus at all treatment periods (see Appendix). Post-mechanical
testing assessment of the failure mode showed that five days of celecoxib
treatment significantly increased the prevalence of nonunion after eight weeks
of healing (with nonunion occurring in nine of twenty-five femora)
(Fig. 2, panel D). The
proportion of nonunions after ten days of celecoxib treatment was lower (with
nonunion occurring in four of twenty-six femora), but it increased to >50%
after fifteen, twenty-one, or twenty-eight days of celecoxib treatment (with
nonunion occurring in thirteen of twenty-three, eight of twelve, and twelve of
twenty femora, respectively; Fig.
2, panel D). These data suggest that the duration of COX-2
inhibition therapy correlates with the inhibition of fracture-healing.
Interestingly, after ten days of celecoxib treatment, peak torque (see
Appendix) and maximum shear stress (Fig.
2, panel C) were not significantly different from control values
but rigidity (Fig. 2, panel B)
and shear modulus (see Appendix) were significantly less.
Effects of Delayed or Previous Celecoxib Treatment on
Fracture-Healing
Additional clinical scenarios of nonsteroidal anti-inflammatory drug
therapy were tested for potential negative effects on fracture-healing.
Specifically, we sought to determine whether nonsteroidal anti-inflammatory
drug use prior to fracture affects healing as might occur in a chronic
nonsteroidal anti-inflammatory drug user, such as a patient with arthritis. We
also sought to determine when nonsteroidal anti-inflammatory drug use can be
resumed following a fracture. To mimic these clinical scenarios, rats were
treated with celecoxib (4 mg/kg/day) for five days, receiving the final
celecoxib dose four hours prior to the femoral fracture (pre-5 days). In the
other groups, celecoxib treatment (4 mg/kg/day) was initiated seven or
fourteen days after the fracture and was continued to the twenty-eighth day
after the fracture, when fracture bridging normally occurs in the rat.
Prior celecoxib treatment had little negative effect on fracture-healing.
Radiographic scoring, peak torque, maximum shear stress, shear modulus, and
the prevalence of nonunion (zero of fourteen) were similar to control values
(Fig. 3; see Appendix). One
exception was that femoral torsional rigidity was significantly lower after
eight weeks of healing in the pretreated rats
(Fig. 3, panel B).
Delayed celecoxib treatment was associated with improvement in many healing
outcomes as compared with celecoxib treatment that began immediately after the
fracture, although most values were still less than the control values.
Continuous celecoxib treatment for twenty-eight days after the fracture was
associated with the lowest radiographic and mechanical scores
(Fig. 3; see Appendix).
Delaying celecoxib treatment until seven days after the fracture was
associated with a higher radiographic score, although it was still
significantly less than the control value
(Fig. 3, panel A). Rats that
were treated with celecoxib from the seventh through the twenty-eight day also
had reduced rigidity (Fig. 3,
panel B) and shear modulus (see Appendix), but the values for peak torque (see
Appendix) and maximum shear stress (Fig.
3, panel C) were similar to control values. The proportion of
nonunions in the seven to twenty-eight-day celecoxib treatment group (four of
fifteen) was reduced compared with that in the group that received continuous
celecoxib treatment (Fig. 3,
panel D). Delaying celecoxib treatment until fourteen days after the fracture
was associated with normalized radiographic scores
(Fig. 3, panel A), peak torque
(see Appendix), and maximum shear stress
(Fig. 3, panel C), with a lower
proportion of nonunions (two of twelve) compared with the group that received
continuous celecoxib treatment (Fig.
3, panel D), although the values for torsional rigidity
(Fig. 3, panel B) and shear
modulus (see Appendix) remained significantly lower than control values.
Effects of Nonsteroidal Anti-Inflammatory Drug Therapy on Fracture
Callus Prostaglandin Levels
The straightforward hypothesis indicated by these experimental observations
is that celecoxib treatment reduces COX-2 activity and prostaglandin levels
that are essential for normal fracture-healing to proceed. To test this
hypothesis, rats were treated with carrier (1% methylcellulose), celecoxib (4
mg/kg), or diclofenac (5 mg/kg) once a day for four days after the fracture.
One hour after drug dosing on the fourth day, the rats were killed,
eicosanoids were extracted from the callus, and PGE2 and
PGF2a were quantified. Celecoxib or diclofenac treatment
reduced callus PGE2 levels by >60%
(Fig. 4, panel A). Similarly,
PGF2a levels were reduced significantly (by >75%) in the
fracture calluses of the rats that had been treated with nonsteroidal
anti-inflammatory drug therapy (Fig.
4, panel B). These data indicated that nonsteroidal
anti-inflammatory drug therapy reduced prostaglandin levels at the fracture
site.
With use of a rodent model, the effects of celecoxib (a COX-2-selective
nonsteroidal anti-inflammatory drug) on fracture-healing were investigated in
detail. The data suggest that higher doses
(Fig. 1) and longer periods of
celecoxib treatment (Fig. 2)
are more detrimental to fracture-healing than lower doses and shorter periods
of celecoxib treatment are. However, rats that were treated with a modest dose
of celecoxib (4 mg/kg/day) for five days following a fracture had
significantly worse outcomes after eight weeks of healing than control rats
did. These experimental observations suggest that nonsteroidal
anti-inflammatory drug therapy following a fracture may adversely affect
healing in humans. We are unaware of any prospective human studies that
support this contention. However, recent human retrospective studies have
supported these experimental
findings49,50.
In the present study, 43% (sixty-eight) of 160 rats that were treated with
celecoxib after the fracture appeared to have union after eight weeks of
healing, 19% (thirty-one) showed some evidence of new bone bridging the
fracture site, and 38% (sixty-one) appeared to have a nonunion. In contrast,
82% (forty-two) of fifty-one control rats or rats that had been treated with
celecoxib prior to fracture had union, 18% (nine) showed some evidence of new
bone bridging the fracture site, and 0% were observed to have a nonunion. This
dichotomy in the healing pattern of the celecoxib-treated rats creates
considerable variation within the experimental results and could lead to
discrepancies in how results were interpreted in studies that have used fewer
animals or employed a limited number of outcome
measures28,51,52.
The large number of animals used in the present study shows that
COX-2-selective nonsteroidal anti-inflammatory drug therapy impairs
fracture-healing.
Five days of treatment with celecoxib at a dose of 4 mg/kg/day
significantly impaired fracture-healing, with 36% (nine) of twenty-five rats
having nonunion after eight weeks. In female Sprague-Dawley rats, bridging of
a fracture site is normally observed by four weeks after a fracture. Since
celecoxib is nominally inhibiting COX-2
(Fig. 4), these observations
indicate that inhibiting the early inflammation phase of fracture-healing can
ultimately impair fracture-healing at later times.
In the group in which celecoxib treatment was delayed for two weeks after
the fracture, certain mechanical testing values were less than those in
control animals (Fig. 3) and
nonunion still developed in two (17%) of twelve rats. These findings suggest
that COX-2 may have an additional function during fracture-healing beyond
those associated with inducing or enhancing inflammation.
A curious observation was that five or fifteen days of celecoxib therapy
was more deleterious than ten days of treatment after the fracture
(Fig. 2). The rigidity of the
femora of the rats that had been treated with celecoxib for ten days was
significantly less than that in the control group, and the proporation of
nonunions was signficantly higher than that in the control group. However,
other outcome parameters were similar to control values. One potential
explanation for this observation may be that a bounce-back effect occurs when
COX-2 expression is still high and that discontinuing celecoxib treatment
leads to a burst of prostaglandin synthesis that rescues healing. In support
of this explanation, it was previously shown that COX-2 expression peaks on
the third day after a fracture, declines by the fifth day, peaks again on the
tenth day, and declines to background levels by the twenty-first day during
fracture-healing in
rats51. When
celecoxib therapy is stopped on the fifth day, the fifteenth day, or later,
the level of COX-2 expression may not be sufficient to provide a bounce-back
effect of sufficient magnitude to rescue healing as may occur when celecoxib
therapy is stopped on the tenth day.
The mechanistic functions of COX-2 during fracture-healing are not known;
thus, the mechanism by which celecoxib inhibits fracture-healing is not
understood. There are a number of possible mechanisms, and it is likely that
multiple cellular and molecular mechanisms involved in fracture-healing are
impaired by COX-2 inhibition. For instance, angiogenesis is essential for
successful fracture-healing and endochondral
ossification6-9.
Prostaglandins are known to promote
angiogenesis53,54.
Furthermore, inhibition of COX-2 has been shown to inhibit angiogenesis in
animal tumor
models55-57.
Thus, one likely mechanism to account for impaired fracture-healing in
celecoxib-treated rats is that celecoxib treatment reduces angiogenesis at the
fracture site. It is conceivable that because of a reduction in prostaglandin
levels at the fracture site, the initial inflammatory phase is blunted and the
local production and release of cytokines and growth factors that recruit
mesenchymal cells to the fracture site are reduced. In due course, this leads
to failed healing in some animals. An additional possibility is that
inhibition of COX-2 alters the normal temporal pattern of cellular and
molecular events, leading to an uncoordinated healing response. Ultimately,
inhibition of cyclooxygenase activity that reduces PGE2 levels by
>60% and PGF2a levels by >75% is sufficient to impair
fracture-healing in a large proportion of animals.
Prostaglandins have direct effects on chondrocytes and osteoblasts. In
vitro, PGF2a increases chondrocyte proliferation and matrix
synthesis58,59.
PGE2 does not enhance chondrocyte matrix synthesis but inhibits the
expression of genes associated with the terminally differentiated phenotype of
chondrocytes (type-X collagen, vascular endothelial growth factor [VEGF], and
alkaline phosphatase), and only enhances chondrocyte proliferation at very
high
concentrations58-60.
Osteoblasts respond to prostaglandins by increasing cell division, elaborating
more osteoid, and enhancing osteoclast
activity61-68.
Thus, one could expect that chondrocyte as well as osteoblast metabolism would
be affected by a decrease in fracture callus prostaglandin levels.
Other experimental evidence also indicates that COX-2 is necessary for
fracture repair. For example, the treatment of fractures with PGE2
can enhance fracture-healing, but the PGE2 must be administered
locally and continuously to be
effective69.
Similarly, compounds that stimulate the EP2 or EP4 PGE2 receptors
also have been shown to stimulate osteogenesis and fracture
repair70-72.
There have been few human studies concerning the function of COX-2 or its
metabolites in fracture-healing. Recent retrospective studies have correlated
nonsteroidal anti-inflammatory drug use with impaired
fracture-healing49,50.
In particular, Burd and colleagues performed a retrospective study of the
effects of indomethacin therapy on the prevalence of nonunions of long-bone
fractures among patients who had also sustained an acetabular
fracture50. Data
for the analysis by Burd and colleagues came from a prospective study
comparing the efficacy of localized radiation therapy with that of
indomethacin therapy for the prevention of heterotopic ossification following
an acetabular fracture. They found that patients who had received localized
radiation therapy had a 7% rate of nonunion (five nonunions among 118
fractures in seventy-four patients), whereas those who had received
indomethacin therapy had a 29% rate of nonunion (eleven nonunions among
seventy-two fractures in thirty-eight patients). Several studies have
indicated that nonsteroidal anti-inflammatory drug use reduces the prevalence
and severity of heterotopic ossification in humans, again indicating a role
for COX-2 in human
osteogenesis73-78.
The present study demonstrates that even short-term treatment with a COX-2
inhibitor can impair ultimate fracture-healing outcomes. In our previous
study, we found that long-term administration of celecoxib at a dose of 4
mg/kg/day showed impaired fracture-healing in male rats on the basis of
radiographs and histological findings but that no difference was noted between
the mechanical properties of the healing femora from the control and
celecoxib-treated
rats28. We suggest
that the difference between the previous experiments and the present study is
the difference in pharmacokinetics of celecoxib between male and female rats.
In male rats, the half-life is approximately four hours whereas in female rats
it is approximately fourteen hours, which is similar to the half-life of
celecoxib in
humans41,42.
Therefore, a 4 mg/kg/day dose of celecoxib in female rats should provide for
substantially longer COX-2 inhibition over a twenty-four-hour cycle than the
same dose in male rats. We theorize that this accounts for the difference in
results between this study, our previous study, and another study that
examined the effects of celecoxib on fracture-healing in male rats treated
with a 3 mg/kg/day dose of
celecoxib52.
The conclusions of the present study also differ from those reported by
Gerstenfeld et
al.51. In their
study, Gerstenfeld et al. found that parecoxib treatment (at a dose of 0.3 or
1.5 mg/kg/day) ultimately did not impair fracture-healing in male rats.
Unfortunately, the pharmacokinetics of parecoxib are not well described in the
literature. However, in other studies, parecoxib has been used at doses of 6.4
and 10 mg/kg/day to assess the effects on tendon-healing and soft-tissue
injury in
rats79,80.
Additional experiments examining the efficacy of valdecoxib, the active form
of parecoxib, on models of pain and inflammation in rats have shown effective
doses (ED50) of 5.9 mg/kg for edema and 14 mg/kg for
hyperalgesia81.
Thus, it is likely that equivalence in fracture-healing between control and
parecoxib-treated rats as described was due to insufficient inhibition of
COX-2 by the parecoxib dose used.
The data from the present study indicate that nonsteroidal
anti-inflammatory drug use probably should be avoided by patients during
fracture-healing, especially during the immediate and early inflammatory
phases of fracture-healing. Conversely, nonsteroidal anti-inflammatory drug
use prior to a fracture does not appear to have a negative effect on
healing.
Additional research is needed to confirm these animal findings in humans
and to define the mechanistic role of COX-2 in bone regeneration.