Accelerated fracture healing in mice lacking the 5-lipoxygenase gene.
ABSTRACT Cyclooxygenase-2 (COX-2) promotes inflammation by synthesizing pro-inflammatory prostaglandins from arachidonic acid. Inflammation is an early response to bone fracture, and ablation of COX-2 activity impairs fracture healing. Arachidonic acid is also converted into leukotrienes by 5-lipoxygenase (5-LO). We hypothesized that 5-LO is a negative regulator of fracture healing and that in the absence of COX-2, excess leukotrienes synthesized by 5-LO will impair fracture healing.
Fracture healing was assessed in mice with a targeted 5-LO mutation (5-LO(KO) mice) and control mice by radiographic and histological observations, and measured by histomorphometry and torsional mechanical testing. To assess effects on arachidonic acid metabolism, prostaglandin E2, F2α, and leukotriene B4 levels were measured in the fracture calluses of control, 5-LO(KO) COX-1(KO), and COX-2(KO) mice by enzyme linked immunoassays.
Femur fractures in 5-LO(KO) mice rapidly developed a cartilaginous callus that was replaced with bone to heal fractures faster than in control mice. Femurs from 5-LO(KO) mice had substantially better mechanical properties after 1 month of healing than did control mice. Callus leukotriene levels were 4-fold higher in mice homozygous for a targeted mutation in the COX-2 gene (COX-2(KO)), which indicated that arachidonic acid was shunted into the 5-LO pathway in the absence of COX-2.
These experiments show that 5-LO negatively regulates fracture healing and that shunting of arachidonic acid into the 5-LO pathway may account, at least in part, for the impaired fracture healing response observed in COX-2(KO) mice.
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ABSTRACT: Lipid mediators regulate bone regeneration during fracture healing. Prostaglandins and leukotrienes are well-known lipid mediators that regulate inflammation and are synthesized from the Ω-6 fatty acid, arachidonic acid. Cyclooxygenase (COX-1 or COX-2) and 5-lipoxygenase (5-LO) catalyze the initial enzymatic steps in the synthesis of prostaglandins and leukotrienes, respectively. Inhibition or genetic ablation of COX-2 activity impairs fracture healing in animal models. Genetic ablation of COX-1 does not affect the fracture callus strength in mice, suggesting that COX-2 activity is primarily responsible for regulating fracture healing. Inhibition of cyclooxygenase activity with nonsteroidal anti-inflammatory drugs (NSAIDs) is performed clinically to reduce heterotopic ossification, although clinical evidence that NSAID treatment impairs fracture healing remains controversial. In contrast, inhibition or genetic ablation of 5-LO activity accelerates fracture healing in animal models. Even though prostaglandins and leukotrienes regulate inflammation, loss of COX-2 or 5-LO activity appears to primarily affect chondrogenesis during fracture healing. Prostaglandin or prostaglandin analog treatment, prostaglandin-specific synthase inhibition and prostaglandin or leukotriene receptor antagonism also affect callus chondrogenesis. Unlike the Ω-6-derived lipid mediators, lipid mediators derived from Ω-3 fatty acids, such as resolvin E1 (RvE1), have anti-inflammatory activity. In vivo, RvE1 can inhibit osteoclastogenesis and limit bone resorption. Although Ω-6 and Ω-3 lipid mediators have clear-cut effects on inflammation, the role of these lipid mediators in bone regeneration is more complex, with apparent effects on callus chondrogenesis and bone remodeling.BoneKEy reports. 04/2014; 3:517.
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ABSTRACT: This study investigated the localization of critical enzymes involved in arachidonic acid metabolism during the initial and regenerative phases of mouse femur fracture healing. Previous studies found that loss of cyclooxygenase-2 activity impairs fracture healing while loss of 5-lipoxygenase activity accelerates healing. These diametric results show that arachidonic acid metabolism has an essential function during fracture healing. To better understand the function of arachidonic acid metabolism during fracture healing, expression of cyclooxygenase-1 (COX-1), cyclooxygenase -2 (COX-2), 5-lipoxygenase (5-LO), and leukotriene A4 hydrolase (LTA4H) was localized by immunohistochemistry in time-staged fracture callus specimens. All four enzymes were detected in leukocytes present in the bone marrow and attending inflammatory response that accompanied the fracture. In the tissues surrounding the fracture site, the proportion of leukocytes expressing COX-1, COX-2, or LTA4H decreased while those expressing 5-LO remained high at 4 and 7 days after fracture. This may indicate an inflammation resolution function for 5-LO during fracture healing. Only COX-1 was consistently detected in fracture callus osteoblasts during the later stages of healing (day 14 after fracture). In contrast, callus chondrocytes expressed all four enzymes, though 5-LO appeared to be preferentially expressed in newly differentiated chondrocytes. Most interestingly, osteoclasts consistently and strongly expressed COX-2. In addition to bone surfaces and the growth plate, COX-2 expressing osteoclasts were localized at the chondro-osseous junction of the fracture callus. These observations suggest that arachidonic acid mediated signaling from callus chondrocytes or from callus osteoclasts at the chondro-osseous junction regulate fracture healing.PLoS ONE 01/2014; 9(2):e88423. · 3.53 Impact Factor
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ABSTRACT: Lipid mediators derived from 5-lipoxygenase (5-LO) metabolism can activate both pro- and anti-inflammatory pathways, but their role in wound healing remains largely unexplored. In this study we show that 5-LO knockout (5-LO(-/-)) mice exhibited faster wound healing than wild-type (WT) animals, and exhibited upregulation of heme oxygenase-1 (HO-1). Furthermore, HO-1 inhibition in 5-LO(-/-) mice abolished the beneficial effect observed. Despite the fact that 5-LO(-/-) mice exhibited faster healing, in in vitro assays, both migration and proliferation of human dermal fibroblasts (HDF) were inhibited by the 5-LO pharmacologic inhibitor AA861. No changes were observed in expression of fibronectin, transforming growth factor (I and III), and alpha-smooth muscle actin (α-SMA). Interestingly, AA861 treatment significantly decreased ROS formation by stimulated fibroblasts. Similar to 5-LO(-/-) mice, induction of HO-1, but not superoxide dismutase-2 (SOD2), was also observed in response to 5-LO (AA861) or 5-LO activating protein (MK886) inhibitors. HO-1 induction was independent of nuclear factor (erythroid derived-2) like2 (Nrf-2), cyclooxygenase 2 (COX-2) products, or lipoxin action. Taken together, our results show that 5-LO disruption improves wound healing and alters fibroblast function by an antioxidant mechanism based on HO-1 induction. Over-expression of HO-1 in wounds may facilitate early wound resolution.Journal of Investigative Dermatology accepted article preview online, 13 November 2013. doi:10.1038/jid.2013.493.Journal of Investigative Dermatology 11/2013; · 6.19 Impact Factor
748 Acta Orthopaedica 2010; 81 (6): 748–755
Accelerated fracture healing in mice lacking the 5-lipoxygen-
Michaele B Manigrasso and J Patrick O’Connor
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School and Graduate School of Biological Sciences, Newark, NJ, USA
Submitted 10-02-22. Accepted 10-07-08
Open Access - This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use,
distribution, and reproduction in any medium, provided the source is credited.
Background and purpose Cyclooxygenase-2 (COX-2) promotes
inflammation by synthesizing pro-inflammatory prostaglandins
from arachidonic acid. Inflammation is an early response to bone
fracture, and ablation of COX-2 activity impairs fracture healing.
Arachidonic acid is also converted into leukotrienes by 5-lipoxy-
genase (5-LO). We hypothesized that 5-LO is a negative regulator
of fracture healing and that in the absence of COX-2, excess leu-
kotrienes synthesized by 5-LO will impair fracture healing.
Methods Fracture healing was assessed in mice with a tar-
geted 5-LO mutation (5-LOKO mice) and control mice by radio-
graphic and histological observations, and measured by histo-
morphometry and torsional mechanical testing. To assess effects
on arachidonic acid metabolism, prostaglandin E2, F2a, and leu-
kotriene B4 levels were measured in the fracture calluses of con-
trol, 5-LOKO COX-1KO, and COX-2KO mice by enzyme linked
Results Femur fractures in 5-LOKO mice rapidly developed a
cartilaginous callus that was replaced with bone to heal fractures
faster than in control mice. Femurs from 5-LOKO mice had sub-
stantially better mechanical properties after 1 month of healing
than did control mice. Callus leukotriene levels were 4-fold higher
in mice homozygous for a targeted mutation in the COX-2 gene
(COX-2KO), which indicated that arachidonic acid was shunted
into the 5-LO pathway in the absence of COX-2.
Interpretation These experiments show that 5-LO negatively
regulates fracture healing and that shunting of arachidonic acid
into the 5-LO pathway may account, at least in part, for the
impaired fracture healing response observed in COX-2KO mice.
The molecular events that initiate and maintain bone fracture
healing are poorly understood. Inflammation is an early physi-
ological response to fracture and it has been postulated that
signaling molecules produced during inflammation initiate the
tissue regeneration pathway (Mountziaris and Mikos 2008,
Cottrell et al. 2009).
Cyclooxygenase-2 (COX-2) is a critical, enzymatic regula-
tor of inflammation (Simmons et al. 2004). Loss of COX-2
function dramatically impairs fracture healing in mice and
rats (Simon et al. 2002). Fracture healing in COX-2 knock-out
mice is characterized by formation of a small cartilaginous
fracture callus with delayed, reduced, or no endochondral
bone formation (Zhang et al. 2002). Pharmacological inhibi-
tion of COX-2 also severely impairs fracture healing (Simon
and O’Connor 2007). Human studies examining the effects of
non-steroidal anti-inflammatory drugs on fracture healing sup-
port the conclusions from animal studies (Burd et al. 2003).
Inflammatory stimuli activate phospholipase A2 to release
arachidonic acid from membrane stores (Capper and Marshall
2001). In turn, arachidonic acid is converted into prostaglan-
dins by COX-2 or into leukotrienes by 5-lipoxygenase (5-LO)
(Murphy and Gijon 2007). By inhibiting COX-2, arachidonic
acid can be shunted into the 5-LO pathway to produce abnor-
mally high levels of leukotrienes (Hudson et al. 1993, Mar-
tel-Pelletier et al. 2004, Marcouiller et al. 2005). Conversely,
ablation of 5-LO function can lead to abnormally high levels
of prostaglandins (Byrum et al. 1997). Since COX-2 activity
is critical for successful fracture healing, loss of COX-2 activ-
ity may alter fracture callus prostaglandin levels and lead to
excess leukotriene synthesis. In turn, these excess amounts of
leukotrienes could contribute to the impaired healing observed
in mice lacking COX-2 or in animals treated with COX-2
inhibitors by altering the inflammatory response.
To test this hypothesis, we measured fracture healing in
mice homozygous for a targeted mutation in the 5-LO gene
(5-LOKO mice) as loss of 5-LO activity would prevent leu-
kotriene synthesis and potentially lead to accelerated healing.
Fracture healing was assessed by radiography, histomorphom-
etry, and torsional mechanical testing. Levels of fracture callus
prostaglandin (PG) E2, PGF2a and leukotriene (LT) B4 were
measured to determine how loss of COX-1, COX-2, or 5-LO
activity alters the synthesis of these lipid signaling molecules
during fracture healing.
Acta Orthopaedica 2010; 81 (6): 748–755 749
Materials and methods
All animal experiments were approved by the Institutional
Animal Care and Use Committee of New Jersey Medi-
cal School. Experimental mice homozygous for inactivat-
ing mutations in the 5-lipoxygenase, cyclooxygenase-1, or
cyclooxygenase-2 gene (5-LOKO, COX-1KO, and COX-2KO,
respectively) were obtained from breeding colonies. Founder
mice were obtained from Jackson Laboratories (Alox5tm1Fun
(5-LOKO); Bar Harbor, ME) or from Taconic (Ptgs1tm1Unc
and Ptgs2tm1Unc (COX-1KO and COX-2KO); Germantown,
NY) (Chen et al. 1994, Langenbach et al. 1995, Morham et
al. 1995). The 5-LOKO mice had been backcrossed into the
C57BL/6 background and so C57BL/6 mice purchased from
Jackson Laboratories were used as the control for the 5-LOKO
mice. The COX-1KO and COX-2KO mice have a B6;129P2
mixed genetic background and so wild-type (B6;129P2) and
COX-2 heterozygous (COX-2Het) mice obtained from the
COX-2KO breeding colony were used as controls for the COX-
1KO and COX-2KO strains. Mouse genotypes were confirmed
by PCR analysis of tail biopsy DNA samples using recom-
binant Pwo polymerase (Dabrowski and Kur 1998). Experi-
ments were performed on female mice only. Mice were 10–12
weeks old at the time of fracture.
Closed femur fractures were produced in the right hind
limb of mice using an established procedure (Manigrasso and
O’Connor 2004). Mice were anesthetized with a mixture of
ketamine and xylazine. A 0.254-mm diameter stainless steel
pin was inserted retrograde into the femoral canal and wedged
in place with the tip of a 0.255-mm diameter needle. The
fracture was produced using a custom-made 3-point bending
device. The mice were allowed to ambulate freely following
fracture and were provided food and water ad libitum. They
were killed with an inhaled overdose of halothane anesthesia.
Torsional mechanical testing
The mechanical strength of the mouse femurs was determined
by torsional mechanical testing to failure as described previ-
ously (Manigrasso and O’Connor 2004). Testing was accom-
plished using a servohydraulic testing machine (MTS Test
Star, Eden Prairie, MN), a 20-Nm reaction torque cell (Inter-
face, Scottsdale, AZ), at an actuator head displacement rate
of 1 degree per second. Peak torque, rigidity, maximum shear
stress, and shear modulus were calculated from the torque-
displacement curves and callus dimensions as previously
described (Manigrasso and O’Connor 2004). Femur cortical
bone thickness was measured from cross sections of intact
femurs from C57BL/6 (0.166 (0.004) mm, n = 9) and 5-LOKO
(0.174 (0.003), n = 9) that were approximately 14 weeks old.
The fractured and contralateral femurs from each mouse were
tested at 4 and 12 weeks after fracture. Values for each frac-
tured femur were normalized to the corresponding contralat-
eral femur as a percentage. The normalized values for each
parameter were compared between the 5-LOKO and C57BL/6
mice using Student’s t-tests at each time point.
Radiography, histology, and histomorphometry
All animals were examined by radiography immediately after
fracture to ensure fracture quality, and after killing to assess
healing and any morbid conditions. Three 5-LOKO and three
C57BL/6 mice were examined by radiography periodically
throughout healing. Radiographs were taken using a Model
804 Packard Faxitron (Field Emission Corp., McMinnville,
OR) and Kodak MINR2000 mammography film (Eastman
Kodak, Rochester, NY).
For histology and histomorphometry, fractured femurs
were embedded in polymethylmethacrylate using standard
procedures (Baron et al. 1983). Specimens were sectioned
longitudinally using a diamond wafering saw and polished
with alumina grit prior to staining with van Gieson’s picro-
fuchsin (where mineralized tissue stains red) and Stevenel’s
blue (where proteoglycan stains deep blue) (Maniatopoulos
et al. 1986). Digital images of each specimen were captured
and analyzed using Image Pro software version 5 (Media
Cybernetics, Bethesda, MD). Callus, cartilage, and mineral-
ized tissue area were measured. The cartilage and mineralized
tissue area were normalized to callus area as a percentage for
each specimen. Data for callus area, per cent cartilage, and
per cent mineralized tissue were initially analyzed by 2-way
ANOVA using genotype and time after fracture as the inde-
pendent variables. The analysis found differences for callus
area (p = 0.02), per cent cartilage (p < 0.001), and per cent
mineralized tissue (p = 0.04). Fisher’s least significant differ-
ence tests were used for post-hoc comparisons between geno-
types and time points for callus area, per cent cartilage, and
per cent mineralized tissue.
Fracture callus eicosanoid levels
To determine how loss of COX-1, COX-2, and 5-LO activ-
ity affects eicosanoid synthesis during fracture healing, callus
eicosanoid levels were measured 4 days after fracture during
the inflammatory phase of healing. To inhibit all cyclooxygen-
ase activity, some COX-1KO and COX-2KO mice were treated,
respectively, with the COX-2 selective inhibitor rofecoxib (30
mg/kg VIOXX; Merck, West Point, PA), or the COX-1 selec-
tive inhibitor SC-560 (30 mg/kg; Cayman Chemicals, Ann
Arbor, MI) by oral gavage using 1% methylcellulose as carrier
2 h before killing on day 4 after fracture (Smith et al. 1998,
Chan et al. 1999). Each fracture callus was rapidly resected
and flash frozen in liquid nitrogen. Eicosanoids were isolated
by extracting the pulverized sample into M-PER buffer (Pierce
Biotechnology, Inc., Rockford, IL) and then purifying the eico-
sanoid fraction by sequential methanol and C18 solid phase
extractions as described previously (Simon and O’Connor
2007). PGE2, PGF2a, and LTB4 levels were measured using
enzyme-linked immunoassays (Cayman Chemicals) and nor-
malized to protein levels. Extracted protein concentration was
750 Acta Orthopaedica 2010; 81 (6): 748–755
determined using the bicinchoninic acid assay method and an
aliquot of the M-PER extract (Smith et al. 1985). Values for
the C57BL/6 and 5-LOKO samples were compared using Stu-
dent’s t-tests because of genetic background differences with
the cyclooxygenase-deficient strains, which includes loss of
secreted phospholipase A2 activity in C57BL/6 mice (Kennedy
et al. 1995). Values for the other strains and the rofecoxib and
SC-560 treatment groups were tested for significance using
ANOVA (p < 0.001) before using Fisher’s least significant dif-
ference tests to identify differences between strains and the
rofecoxib and SC-560 treatment groups.
Accelerated and enhanced fracture healing in
Fracture healing was initially assessed in the 5-LOKO mice by
radiography (Figure 1). We observed a mineralized callus at
the periphery of the fracture site by 7 days after fracture in the
5-LOKO mice as compared to 10 days in the C57BL/6 mice.
After 10 days of healing, a large mineralized callus was appar-
ent in the 5-LOKO mice, which was similar in appearance to
the 14-day post-fracture callus of the C57BL/6 mice. Fracture
bridging was apparent by 14 days after fracture in the 5-LOKO
mice but not until 21 days after fracture in the C57BL/6
mice. Fracture callus size appeared reduced after 21 days of
healing in the 5-LOKO mice as compared to 28 days in the
C57BL/6 mice. While the temporal pattern of fracture heal-
ing in the C57BL/6 mice was similar to what was previously
observed in outbred (ICR) and inbred (C57BL/6, DBA/2, and
C3H) strains of mice, healing appeared to be accelerated in the
5-LOKO mice (Manigrasso and O’Connor 2004, Manigrasso
and O’Connor 2008).
Fractured femurs from C57BL/6 and 5-LOKO mice were
harvested at 7, 10, 14, and 21 days after fracture for histo-
morphometric analysis (n = 5 for each genotype at each time
point). Callus morphology appeared relatively normal in the
5-LOKO mice, with cartilage juxtaposed between the frac-
ture site and newly formed bone at the periphery of the callus
(Figure 2). These observations indicate that fractures heal via
endochondral ossification in 5-LOKO mice. For both mouse
strains, callus area peaked at 14 days after fracture (p < 0.005
vs. 21 days) (Figure 3A). The proportion of callus cartilage in
the 5-LOKO mice was almost 4 times greater than that from
the C57BL/6 mice at 7 days after fracture (Figure 3B). By 10
days after fracture, the proportion of callus cartilage declined
in the 5-LOKO mice, but peaked in the C57BL/6 mice. Further
reductions in callus per cent cartilage were noted at 14 and
21 days after fracture for both mouse strains. The proportion
of callus mineralized tissue was almost 2-fold higher in the
5-LOKO mice at 7 days after fracture and remained higher at
10 days after fracture as compared to the C57BL/6 samples
(Figure 3C). By 14 and 21 days after fracture, no statistically
significant difference in the percentage of callus mineralized
tissue was found between the mouse strains. The data indicate
that bone regeneration occurs through an accelerated endo-
chondral ossification process in the 5-LOKO mice.
Fracture callus mechanical properties were measured in
the 5-LOKO and C57BL/6 mice after 4 and 12 weeks of heal-
ing (Figure 4). Contralateral femurs from each mouse were
also mechanically tested to failure in torsion (Table). Despite
having the same genetic background, femur morphology was
different between the C57BL/6 and 5-LOKO strains (Table).
The better structural properties of the unfractured, contralat-
eral C57BL/6 femurs probably relates to the larger diameter of
these bones as compared to their 5-LOKO counterparts. Because
of the baseline differences in femoral mechanical properties,
Figure 1. Radiographic examination of fracture healing in 5-LOKO mice
and control C57BL/6 mice. Shown are serial dorsal-ventral radio-
graphs made from a C57BL/6 mouse (column A) and a 5-LOKO mouse
(column B) of identical age and genetic background at 7, 10, 14, 21,
and 28 days after fracture, as indicated.
Figure 2. Fractures heal by endochondral ossification in 5-LOKO mice.
Shown are fracture callus specimens from control C57BL/6 mice (A),
and 5-LOKO mice (B) 7 days after fracture. Specimens were embed-
ded in polymethylmethacrylate, sectioned, and stained with Stevenel’s
blue and van Gieson’s picrofuchsin to identify mineralized tissue (Bn;
red) and proteoglycan-rich cartilage (Ca; deep blue). The fracture site
(Fx) is in the lower, left quadrant of each image. Digital images were
collected at the same magnification and the white bar in panel A cor-
responds to 500 μm. In the 5-LOKO specimen, a portion of the femoral
cortical bone was lost during preparation (M).
Acta Orthopaedica 2010; 81 (6): 748–755 751
fracture callus mechanical properties were normalized to the
contralateral femur values as a percentage. Normalized peak
torque and maximum rigidity of the 5-LOKO fracture calluses
were approximately 20% and 40% higher than the C57BL/6
values after 4 weeks of healing. Similarly, normalized maxi-
mum shear stress and shear modulus were approximately 40%
and 70% higher in the 5-LOKO mice after 4 weeks of healing.
By 12 weeks, normalized peak torque and maximum shear
stress remained higher in the 5-LOKO mouse fractures than in
the C57BL/6 mice. The mechanical testing data demonstrate
that the 5-LOKO fracture callus regains structural and material
properties sooner than the fracture callus in control C57BL/6
Fracture callus eicosanoid levels in COX-1KO, COX-
2KO, and 5-LOKO mice
The effects of lost COX-1, COX-2, and 5-LO activity on
PGE2, PGF2a, and LTB4 synthesis was measured at day 4
after fracture (Figure 5). Surprisingly, callus PGE2 levels were
more than 3-fold higher in the COX-1KO and COX-2KO callus
samples than in the COX-2HET samples (Figure 5A). Treat-
ment of the COX-1KO mice with rofecoxib to inhibit COX-2
dramatically reduced callus PGE2 levels and, similarly, treat-
ment of the COX-2KO mice with SC-560 to inhibit COX-1
dramatically reduced callus PGE2 levels. No statistically sig-
nificant effect on callus PGE2 levels was seen in the 5-LOKO
mice (p = 0.1 vs. C57BL/6).
Figure 3. Endochondral ossification is accelerated in 5-LOKO mice.
Callus area (A), percent cartilage (B), and percent mineralized tissue
(C) were measured for femoral fracture specimens at 7, 10, 14, and
21 days after fracture. Group size was 5 at each time point for the
C57BL/6 strain (blue) and 5-LOKO strain (green). Each rectangle repre-
sents the 25th and 75th percentiles and the median value is indicated
with a line. Differences between the C57BL/6 and 5-LOKO samples are
indicated with the corresponding p-values above the x-axis.
Contralateral mechanical values for unfractured femurs
A B C
F G H I J
B Age (weeks)
C Sample size
D Length (mm)
E Max. diameter (mm)
F Min. diameter (mm)
G Peak torque (Nmm)
H Max. rigidity (Nmm2/rad)
I Max. shear stress (MPa)
J Shear modulus (Ga)
752 Acta Orthopaedica 2010; 81 (6): 748–755
In contrast to PGE2, callus PGF2a levels were significantly
lower in the COX-2KO mice as compared to the other mouse
strains, and treatment with SC-560 to inhibit COX-1 caused
no further reduction in PGF2a levels (p = 0.9). Callus PGF2a
levels were also reduced in the COX-1KO mice, but they were
also higher than in the COX-2KO mice (p = 0.04). Treatment
of COX-1KO mice with rofecoxib to inhibit COX-2 further
reduced callus PGF2a levels (p = 0.04) to values similar to
those found in the COX-2KO mice (p = 0.7). No significant
effect on callus PGF2a level was detected in the 5-LOKO mice
(p = 0.4 vs. C57BL/6).
As expected, callus LTB4 levels were low in the 5-LOKO
mice. Conversely, they were almost 2-fold higher in the COX-
1KO callus samples (p = 0.02) than in the B6;129P2 samples,
while COX-2KO callus LTB4 levels were over 4-fold higher
than in the B6;129P2 samples (p < 0.001) and over 2-fold
higher than in the COX-1KO samples (p < 0.001). Treatment
of COX-1KO mice with rofecoxib had no significant effect on
callus LTB4 levels, but treatment of the COX-2KO mice with
SC-560 caused a 3.5-fold decrease in callus LTB4 levels.
The normal functions of 5-LO in bone metabolism are not well
described. Genetic loss of 5-LO activity alters bone morphol-
ogy (Table) and increases cortical bone thickness (Bonewald
et al. 1997). Leukotrienes produced via 5-LO can stimulate
osteoclast activity, suggesting that the increased cortical thick-
ness found in 5-LOKO mice may be associated with reduced
osteoclast activity (Gallwitz et al. 1993, Garcia et al. 1996,
Flynn et al. 1999).
Our findings indicate that endochondral ossification during
fracture healing is accelerated in 5-LOKO mice, leading to
reduced bridging time and enhanced biomechanical proper-
ties of the callus (Figures 1-4). The fracture calluses of the
5-LOKO mice obtained greater maximum shear stress and
shear modulus sooner than the C57BL/6 controls. Whether
the enhanced material properties are a direct consequence of
lost 5-LO activity or an indirect consequence of earlier bridg-
ing remains to be determined. These experimental results sup-
port the hypothesis that 5-LO metabolites such as LTB4 can
Figure 4. Fracture calluses from 5-LOKO mice have enhanced mechanical properties. Fractured and contralateral
femurs after 28 days (blue) and 84 days (green) of healing from control C57BL/6 mice (n = 11 at 28 and 84 days)
and 5-LOKO mice (n = 11 and 8 at 28 and 84 days, respectively) were mechanically tested to failure in torsion. Peak
torque (panel A), maximum rigidity (panel B), maximum shear stress (panel C), and shear modulus (panel D) were
calculated from the torque to angular displacement curves and callus dimensions. Fractured femur values were nor-
malized to values from the contralateral femur of that mouse as a percentage. The limits of each rectangle represent
the 25th and 75th percentile of the normalized values. The median value is indicated in each rectangle. The 5th and
95th percentile values are shown below and above each rectangle for those with group sizes of 11. Normalized values
were compared between genotypes at each time point and p-values are indicated in each graph above the x-axis.
Acta Orthopaedica 2010; 81 (6): 748–755 753
impede fracture healing. Indeed, in vitro studies have shown
that LTB4 treatment can impede osteoblast activity (Ren and
Dziak 1991, Traianedes et al. 1998).
Measurement of eicosanoid levels in fracture callus sup-
ports the initial hypothesis that loss of COX-2 activity can
lead to shunting of arachidonic acid into the 5-LO pathway,
since 4-fold more LTB4 was found in the COX-2KO fracture
callus (67 pg/mg of protein) than in the B6;129P2 controls
(15 pg/mg of protein) (Figure 5). While the 2-fold increase
in COX-1KO callus LTB4 (29 pg/mg of protein) also supports
an arachidonic acid shunting mechanism, the magnitude of
callus LTB4 levels in the COX-1KO mice may not be sufficient
to impair fracture healing. Unexpectedly, callus PGE2 levels
were dramatically higher in the COX-1KO callus (3-fold) and
COX-2KO callus (4-fold) than in the B6;129P2 controls. Thus,
impaired fracture healing in COX-2KO mice does not appear
to be caused by lack of PGE2. This suggests that therapies
designed to activate the EP2 or EP4 receptors for PGE2 may
be enhancing healing by a non-specific mechanism (Li et al.
2003, 2005, Paralkar et al. 2003, Tanaka et al. 2004).
Perhaps more intriguing was the observation that PGF2a syn-
thesis appears to be dependent upon COX-2 activity. This sug-
gests that COX-2-dependent PGF2a synthesis may be critical
for endochondral ossification during fracture healing. Indeed,
glycosaminoglycan synthesis and the expression of type II col-
lagen and aggrecan were stimulated in human articular chon-
drocyte pellet cultures treated with PGF2a, to a lesser extent
by PGD2, but not by PGE2 (Jakob et al. 2004). PGF2a treat-
ment also stimulated replication and sulfate incorporation by
rat RCJ3.1C5.18 chondrocytes at levels 10–100 fold lower
than PGE2 (Lowe et al. 1996). Analysis of tissue prostaglandin
levels during demineralized bone matrix-induced heterotopic
ossification in rats showed that chondrocyte differentiation
and progression into hypertrophy correlated with peak PGF2a
levels (Wientroub et al. 1983). Thus, lack of PGF2a correlates
well with impaired fracture healing in COX-2KO mice. Unlike
COX-2KO mice, PGF2a levels in COX-1KO mice appear to be
sufficient since fracture healing proceeds normally in COX-
1KO mice (Simon et al. 2002). Why PGF2a levels were lower
in the COX-1KO callus as compared to the B6;129P2 controls
but higher than in COX-2KO callus is not known. The differ-
ences could relate to preferential coupling of PGF2a synthase
to heterodimers of COX-1 and COX-2 as compared to COX-2
homodimers (which would be the only available form of cyclo-
oxygenase in COX-1KO mice), and failure of PGF2a synthase
to couple with COX-1 homodimers (which would be the only
available cyclooxygenase form in COX-2KO mice) (Yu et al.
2006, Yuan et al. 2006). Genetic ablation of COX-1 or COX-2
may also alter the temporal pattern of prostaglandin synthesis.
We only measured prostaglandin levels at a single time point
after fracture; additional experiments are needed to extend
these results and confirm when and to what level the synthesis
of each prostaglandin peaks during fracture healing. It is likely
that the bioactive lipids synthesized via COX-2 and 5-LO have
a multiplicity of effects during tissue regeneration or repair.
The cell types that synthesize and respond to prostaglandins
or leukotrienes during bone healing are not known. Prosta-
glandins can affect osteoblasts, chondrocytes, and osteoclasts
(Raisz 1999, Zhang et al. 2004, Clark et al. 2005). It has also
been suggested that prostaglandins can affect fracture callus
Figure 5. Loss of COX-1, COX-2, or 5-LO function alters callus eico-
sanoid levels. Rectangles represent the 25th and 75th percentiles
with median values shown for fracture callus PGE2 levels (A), PGF2a
levels (B), and LTB4 levels (C) measured 4 days after fracture in control
B6;129P2 mice (n = 6), COX-2HET mice (n = 6), COX-1KO mice (n = 4),
COX-1KO mice treated with 30 mg/kg of rofecoxib (n = 3), COX-2KO
mice (n = 6), COX-2KO mice treated with 30 mg/kg of SC-560 (n = 5),
control C57BL/6 mice (n = 6), and 5-LOKO mice (n = 6). Significant
differences are indicated as a, p < 0.001 vs. COX-2HET; b, p < 0.001
vs. COX-1KO; c, p < 0.001 vs. COX-2KO; d, p = 0.04 vs. COX-1KO; e, p
= 0.02 vs COX-2HET; and f, p = 0.002 vs. C57BL/6. Red bars indicate
values from mice with the mixed B6;129P2 background and yellow
bars indicate values from mice with the C57BL/6 background.
754 Acta Orthopaedica 2010; 81 (6): 748–755
mesenchymal cells (Zhang et al. 2002). Much less is known
about effects of leukotrienes on these cell types (Ren and
Dziak 1991, Traianedes et al. 1998). Prostaglandins also have
proangiogenic effects; thus, reduced angiogenesis may under-
lie—at least in part—the impaired fracture healing response
caused by loss of COX-2 function (Form and Auerbach 1983,
Seno et al. 2002). The expression of 5-LO and synthesis of
leukotrienes occurs primarily in cells of the myeloid lineage
(Steinhilber 1994). However, 5-LO expression in primary
cultures of human osteoblasts has been detected (Maxis et
al. 2006). It is likely that the bioactive lipids synthesized via
COX-2 and 5-LO have multiple effects during tissue regenera-
tion or repair.
Our study shows that 5-LO negatively regulates fracture
healing. In contrast, previous studies have shown that COX-2
is a positive regulator of fracture healing (Simon et al. 2002,
Simon and O’Connor 2007). Thus, two enzymes that use the
same lipid substrate to produce different bioactive lipids have
diametric effects on bone regeneration. Consistent with these
results, inhibition of 5-LO with an orally delivered drug has
been shown to accelerate and enhance fracture healing in
another animal model (Cottrell and O’Connor 2009). Phar-
macological or genetic manipulation of the arachidonic acid
metabolic and signaling pathways to alter COX-2 and 5-LO
activity may be a means to accelerate and enhance tissue
repair or regeneration.
MBM performed the experiments and also data analysis. JPOC developed the
hypothesis and experimental design, performed data analysis, and wrote the
Research concerning the COX-1KO and COX-2KO mice was funded in part by
a grant to JPOC from the Arthritis Foundation. MBM has no competing inter-
ests. JPOC has filed patents to protect intellectual property disclosed in this
manuscript, founded Accelalox Inc. to commercialize this technology, is an
owner of Accelalox Inc., and serves as Chief Scientific Officer at Accelalox.
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