SUPPLEMENT ARTICLE

Advances in the Establishment of Dened Mouse Models

for the Study of Fracture Healing and Bone Regeneration
J. H. Holstein, MD,* P. Garcia, MD,* T. Histing, MD,* A. Kristen, MD,* C. Scheuer, PhD,
M. D. Menger, MD, and T. Pohlemann, MD*
Summary: The availability of a broad spectrum of antibodies and
gene-targeted animals caused an increasing interest in mouse models
for the study of molecular mechanisms of fracture healing and bone
regeneration. In most murine fracture models, the tibia or the femur is
fractured using a 3-point bending device (closed models) or is
osteotomized using an open surgical approach (open models). For
fracture studies in mice, the tibia has to be considered less appropriate
compared with the femur because the stabilization of the fracture is
more difcult due to its triangular, distally declining caliber and its
bowed longitudinal axis. Biomechanical factors critically inuence
the bone healing process. Thus, the use of stable osteosynthesis
techniques is also of interest in murine fracture models. To achieve
stable xation, several biomechanically standardized implants have
recently been introduced, including a locking nail and an intra-
medullary compression screw. Other implants, such as a pin-clip, an
external xator, and a locking plate, additionally allow the
stabilization of fractures with distinct gap sizes. This enables the
study of healing of critical size defects and nonunions. The use of
these implants further allows a rigid xation of fractures in bridle
bones, which is essential for fracture studies in animals suffering from
metabolic bone diseases like osteoporosis. In general, the analysis of
bone healing in these models includes different imaging techniques
and histologic, immunohistochemical, biomechanical, and molecular
methods. To evaluate the impact of different osteosynthesis
techniques on physical activity and rehabilitation, gait analysis
may additionally be performed. By this, the gait of the animals can be
visualized and quantitatively analyzed using modied running wheels
and dynamic high-resolution radiography systems. Taken together,
a variety of different murine femur fracture models have become
available, providing dened biomechanical conditions for fracture
research. The use of these mouse models may now allow studying the
inuence of fracture stabilization techniques on molecular mecha-
nisms of bone healing.
Key Words: animal model, bone repair, mice, fracture stabilization
(J Orthop Trauma 2009;23:S31S38)
THE VALUE OF MURINE FRACTURE MODELS
Although in vitro studies and clinical trials provide
adequate techniques and instruments to improve our knowl-
edge on bone repair, animal studies still represent an essential
tool to analyze the biology of fracture healing.
1
Accordingly,
numerous mammalian species, ranging from the mouse to
the horse, have been introduced as models to study fracture
healing.
28
Nonetheless, differences in the anatomy and
metabolism of animals compared with humans must be con-
sidered in the experimental setup and in the interpretation of
experimental results. In addition, the transfer of the experi-
mental data to the clinical situation must be interpreted in light
of these limitations.
Because of a more primitive bone structure without
a haversian system, small rodents, like mice and rats, are
thought to be less appropriate for bone healing studies
compared with larger animals. In contrast to larger rodents and
other mammalians, small rodents use resorption cavities for
bone remodeling during fracture healing, and this process of
remodeling has been shown similar to the haversian
remodeling in larger animals.
1
There are several reasons why small animal models and
in particular mouse models have become of increasing interest
for fracture healing studies. The expenses for purchase,
breeding, and holding of mice are relatively low. Compared
with larger animals, the space necessary for holding and the
time necessary for bone healing are reduced. Thus, the
implementation of larger groups in the experimental study
design is more feasible. Finally and of utmost importance,
a broad spectrum of antibodies and gene-targeted animals is
available for mice, allowing mechanistic studies on molecular
mechanisms of bone healing and regeneration.
911
Thus, the
murine model is considered as the ideal species for bone
healing and regeneration studies.
The establishment of standardized fracture models in
mice requires sophisticated surgical skills and ne technical
developments. For biomechanical testing, highly sensitive
testing devices are necessary to detect minor differences in the
biomechanical properties of the fragile mouse bones. In
addition, the accurate positioning and xation of the small
mouse bones in the testing machine is demanding and
represents a prerequisite because minor deviations in the
testing setup may signicantly affect the test results.
Previous studies have shown that the age of the animals
has a critical impact on fracture healing.
12
Therefore, animals
with an age of completed bone growth should be used to
mimic fracture healing in adults. In humans, the ratio of age at
Accepted for publication January 27, 2009.
From the *Department of Trauma, Hand and Reconstructive Surgery;
Institute for Clinical & Experimental Surgery; and Collaborative
Research Center AO Foundation, University of Saarland, Homburg/Saar,
Germany.
Disclosure: The authors report no conicts of interest.
Reprints: J. H. Holstein, MD, Department of Trauma, Hand and
Reconstructive Surgery, University of Saarland, Homburg/Saar D-66421,
Germany (e-mail: joerg.holstein@uks.eu).
Copyright 2009 by Lippincott Williams & Wilkins
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S31
growth plate closure and life expectancy is about 20%. This
ratio is quite comparable to that of mice, whereas it markedly
differs from that of other species of established models such as
rats (30%), rabbits, dogs, and sheep (5%10%).
13
ANATOMIC AND SURGICAL CONSIDERATIONS
IN MURINE FRACTURE MODELS
The small size of mice is challenging for the de-
velopment of a fracture model. Therefore, particularly large
long bones, such as the femur and the tibia, are thought to be
most appropriate for studies on fracture healing.
6,14
Nonethe-
less, previous studies have also used the rib, the radius, the
ulna, the mandible, and the calvaria to investigate bone
repair.
1520
However, these bones are unfavorable for bio-
mechanical testing due to their small size and their anatomic
conguration.
The tibia fracture model, which was rst described by
Hiltunen et al
6
in 1993, is supposed to be the most established
murine fracture model. However, due to the distally declining
caliber of the tibia, slightly different fracture sites in closed
models may result in markedly different callus sizes, which
limits both standardization and comparability. The triangular
conguration and the bowed longitudinal axis further affords
a more sophisticated design for implants, guaranteeing stable
xation. In addition, the biomechanical test accuracy is limited
due to the irregular shape of bone (Fig. 1). Although the tibia is
relatively easy to fracture because of its thin soft tissue cover,
this anatomic condition is disadvantageous when analyzing the
role of soft tissue in bone repair. The last issue that has to be
considered when using tibia fracture models is the inuence
of the bula. In closed tibial midshaft fractures, the bula
may also break, which results in either 2 different calluses or
1 combined callus (Fig. 2). To avoid a fracture of the bula,
the tibia has to be broken very distally and closely to the
metaphysis, which, however, is not feasible in a standardized
fashion in closed models.
FIGURE 1. The mouse tibia with bula (A) and the mouse
femur (B). Note the triangular, distally declining caliber and the
bended longitudinal axis of the tibia. In contrast to the tibia,
the mouse femur is a tubular bone with a relatively consistent
inner and outer diameter.
FIGURE 2. X-ray of a mouse tibia at day 28 after closed fracture
and stabilization with an intramedullary pin (Hiltunen et al
6
).
Note the combined callus of the tibia and the distal bula. A
more proximal fracture of the tibia may create even 2 calluses,
one of the tibia and another of the bula. To avoid a fracture of
the bula, the tibia has to be broken very distally and closely to
the metaphysis. Because an exact positioning of the fracture
is not possible in closed tibia fracture models, the callus
formation is highly variable and not predictable.
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In contrast to the tibia, the mouse femur is a tubular bone
(length about 15 mm; Fig. 1) with a relatively consistent inner
and outer diameter (outer diameter about 1.5 mm). Accord-
ingly, different fracture sites within the diaphyseal part of the
bone show comparable callus responses. Because of the
straight longitudinal axis, standardized fracture stabilization is
easier in the femur than in the tibia. Unlike the tibia, the femur
is capable of rotation testing, and also 3- and 4-point bending
tests provide more consistent results in the femur than in the
tibia. As mentioned above, the relatively bulky muscle cover of
the femur is challenging when creating a closed fracture. But
on the other hand, the muscle cover is an important element to
investigate the role of soft tissue during bone repair. Thus, in
our opinion, the femur has to be considered more appropriate
for fracture studies compared with the tibia.
To analyze bone repair, both closed and open fracture
models have been used. Closed femur fracture models in mice
are based on the model described by Manigrasso and
OConnor,
14
which is a modication of the rat femur fracture
model reported by Bonnarens and Einhorn.
3
A standardized
transverse fracture pattern associated with minor soft tissue
trauma is accomplished in these models by a 3-point bending
device. Using a parapatellar approach, the patella is dislocated
to insert the intramedullary implant retrogradely at the
intercondylar notch. The advantage of closed models is that
the parapatellar approach and the closed fracture production
cause only a minor soft tissue trauma. However, the
intramedullary implant might affect the endosteum and the
bone marrow. In open models, the femur is fractured or
osteotomized visibly using a lateral approach.
21,22
Thereby, the
entire lateral muscle layer has to be split to expose the full
length of the femur from the greater trochanter to the lateral
femoral condyle. In fact, the open surgery procedure produces
a major soft tissue trauma, which may have a critical impact on
the vascularization of the femur. In contrast to closed models,
which use intramedullary implants, open models use extra-
medullary implants, which preserve the endosteum and the
bone marrow. However, care has to be taken during the
surgical procedure to avoid damage to the periosteum.
THE RELEVANCE OF BIOMECHANICALLY
STANDARDIZED OSTEOSYNTHESIS IN MURINE
FEMUR FRACTURE MODELS
Because of the lack of stable xation techniques, most of
the former femur fracture studies in mice were conducted with
an unstable intramedullary pin xation
14,23,24
or even without
fracture stabilization.
2527
However, the course of fracture
healing has been demonstrated to depend on the stability of the
osteosynthesis technique.
2831
In large animal models, it has
been shown that interfragmentary movements lead to an
increased formation of brocartilage, which is associated with
a signicantly decreased bone formation.
30,32
Thereby, bone
mineral density and bending rigidity are strongly reduced in
fractures that experience shear compared with those that are
exposed to axial compression.
28
It has been further demon-
strated that interfragmentary motion disturbs angiogenesis.
30,33
In fact, the expression of angiogenic and osteogenic cytokines
is affected by the biomechanical environment in the fracture
gap.
33,34
Murine studies have illustrated that mechanical fac-
tors during fracture healing alter the chronology of chondro-
genic and osteogenic cytokine induction, the differentiation of
mesenchymal cells, and the size and tissue composition of the
callus.
22,26
Standardized osteosynthesis techniques that guarantee
dened biomechanical and biologic conditions for fracture
research are a matter of course in large animal models.
35
In
contrast, it is commonly argued that murine fracture models do
not require biomechanical standardization because the pre-
dominance of research using mouse models has focused on the
molecular aspects of fracture healing. However, as outlined
above, the cellular and molecular mechanisms of fracture
healing are critically affected by the mechanical environment
in the fracture gap.
EXPERIMENTAL FIXATION TECHNIQUES TO
STABILIZE FEMUR FRACTURES IN MICE
Several femoral fracture models have been developed in
mice.
14,21,22,3638
Additional models have been introduced for
the analysis of bone repair in segmental bone defects.
39,40
Due
to the recent advances in osteosynthesis in mice, standardized
stabilization may also become feasible in osteoporotic bones.
41
Intramedullary Pin
The intramedullary pin provides a simple but unstable
closed fracture stabilization (Fig. 3A).
14
Using a medial para-
patellar incision, the patella is dislocated to ream the intra-
medullary canal at the intercondylar notch. A stainless steel
wire (diameter 0.25 mm) is applied for intramedullary fracture
stabilization. Migration of the pin is prevented by an additional
wedge (length 2 mm and diameter 0.3 mm) at the distal end of
the intramedullary canal. The diaphyseal fracture is produced
by a 3-point bending device after the insertion of the implant.
Intramedullary Locking Nail
To overcome the lack of rotational stability observed
with pin stabilization, a locking nail has been developed
(Fig. 3B).
36
The nail system consists of a modied injection
needle (diameter 0.55 mm) and a tungsten guide wire
(diameter 0.1 mm). Rotation stability is accomplished by
attening the proximal and distal ends of the needle. Using the
same surgical approach as described for the pin stabilization,
the guide wire is inserted into the intramedullary canal. After
the production of a closed diaphyseal fracture, the locking nail
is pushed over the guide wire. The guide wire is then removed.
Due to the application of the guide wire, it is possible to
produce the fracture without prenailing the bone. This avoids
alterations to the implant and also more closely mimics the
clinical setting.
Intramedullary Compression Screw
Recently, an intramedullary compression screw (length
18 mm and diameter 0.5 mm) has been engineered (Fig. 3C).
37
It provides not only rotational but also axial stability after
osteosynthesis of closed femoral fractures in mice. The
technique uses also a guide wire to allow the production of the
fracture before insertion of the implant. Rotational and axial
stability after fracture xation is achieved by means of
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Volume 23, Number 5 Supplement, May/June 2009 Murine Fracture Models
interfragmentary compression through the screw, which
consists of a proximal cone-shaped head and a distal thread.
According to a pilot study in senescence accelerated mice,
strain P6 (SAMP 6), osteoporotic bones can be stabilized by
the use of the intramedullary compression screw (Holstein JH,
Histing T, Garcia P et al, unpublished data).
External Fixator
The application of an external xator also allows stable
fracture xation (Fig. 3D).
21
In addition, segmental bone
defects might be stabilized by an external xator. As an
external stabilization device, the xator preserves the
endosteum and the bone marrow. However, a traumatizing
surgery with a signicant injury of the soft tissue is necessary
to insert the xator pins (diameter 0.3 mm) and to fracture the
bone. Furthermore, the bulky design of the external xator
might restrict the physiologic activity and gait of the animals.
Pin-clip Device
The pin-clip device represents an open model using an
intramedullary pin in combination with an extramedullary clip
for fracture xation (Fig. 3E).
22,39
Of interest, the pin-clip
technique permits also the stabilization of segmental bone
defects. In accordance, it is possible to create different gap
sizes and thereby to affect the healing process resulting in
critical size defects or even nonunions. Compared with the
bulky external xator, this internal stabilization device causes
no obvious alteration of the animals gait. In contrast to the
external xator, however, the intramedullary pin may affect
the endosteal bone repair. Also, the insertion of the pin-clip
device requires a traumatizing surgical approach.
Locking Plate
The locking plate is a third implant that enables a stable
fracture xation in the mouse femur but does require open
surgery (Fig. 3F).
38,40
The plate is xed to the bone by
4 interlocking screws. The plate is designed to minimize
the implant to bone contact area, avoiding major damage to the
periosteum. Nonetheless, the plate xation may affect the
external callus formation at the implant site. Instead of a rigid
plate, a exible plate can be used in which the medial part is
replaced by 2 elastic splinting wires. These 2 plate models
allow either stable fracture xation or standardized exible
stabilization of the mouse femur. Comparable to the pin-clip
device, the plate also permits the stabilization of different gap
sizes. As a common drawback of open fracture models, the
insertion of the locking plate is also associated with
a traumatizing surgical approach, which affects the soft tissue
cover of the bone. As the intramedullary compression screw,
the locking plate can also be used in osteoporotic bones
(Histing T, Holstein JH, Garcia P et al, unpublished data).
BIOMECHANICAL EX VIVO TESTING OF
OSTEOSYNTHESIS DEVICES
To evaluate the biomechanical properties of different
osteosynthesis devices, biomechanical ex vivo testings have
been performed. For these analyses, murine cadaver femora
are osteotomized and stabilized by the implant of interest.
Using highly sensitive testing devices, the rotational or axial
stiffness of the osteosynthesis devices can be analyzed. Of
interest, these analyses revealed highly signicant differences
in rotation stiffness of the different devices. The locking plate
showed a rotational stiffness, which was comparable to that of
FIGURE 3. X-rays of fractured mouse
femurs after stabilization with (A) an
intramedullary pin (Manigrasso and
OConnor
14
), (B) an intramedullary
locking nail (Holstein et al
36
), (C) an
intramedullary compression screw
(Holstein et al
37
), (D) an external
xator (Cheung et al
21
), (E) a pin-clip
(Garcia et al
22
; Garcia et al
39
), and
(F) a locking plate (Grongroft et al
40
;
Matthys and Perren
38
). X-rays in
gures B, C, and F were taken at
the day of fracture and those in
gure A, D, and E were taken at day
7, day 21, or 35 after fracture.
Figures AC represent closed femur
fracture models and gures DF
show open models.
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unfractured control femora. In contrast, the conventional pin
failed to provide any rotationally stable fracture xation.
22,37
METHODS FOR THE ANALYSIS OF BONE
REPAIR IN MURINE FEMUR FRACTURE
Imaging Techniques
To study femur and tibia fracture healing in mice, the
animals have in most cases to be killed at different time points
after fracture. This has to be done to resect and prepare the
bones for further radiologic, histologic, biomechanical, cyto-
logic, and molecular analyses.
14,42
However, beside these post-
mortem analyses, noninvasive imaging techniques have been
introduced during the last years to evaluate bone repair,
including micro-positron emission tomography and micro-
magnetic resonance imaging.
43,44
In addition, a variety of
molecular imaging techniques, such as bioluminescence, near-
infrared uorescence, and nuclear and magnetic resonance
imaging, have been applied for noninvasive real-time studies
on gene expression, protein degradation, cell migration, and
cell death in living animals.
45
Nevertheless, the most established imaging techniques
to study murine fracture healing are high-resolution radiog-
raphy and 2-dimensional and 3-dimensional microcomputer
tomography (CT). Using conventional x-ray techniques, the
size and radiologic density of the fracture callus can be
analyzed in living animals and in resected bones.
22,39,46
Although micro-CT scans can be applied also in vivo, ex
vivo micro-CT scans reveal a signicantly higher resolution
than in vivo scans.
47,48
Common parameters that are evaluated
by micro-CT are tissue mineral density, total callus volume,
and bone volume fraction of the callus.
24,49
By the postmortem
injection of a chromate-based contrast agent, the vasculature of
the callus can also be visualized and quantitatively assessed by
3-dimensional micro-CT techniques.
50
Histologic Analysis
The quantitative histologic analysis of the fracture callus
(histomorphometry) has been proven to be an important
parameter to assess bone repair.
51
Thereby, the American
Society of Bone and Mineral Research has provided recom-
mendations regarding a standardized nomenclature, appro-
priate indices for assessment, and methodologic approaches
for histomorphometric analyses.
52
In addition to histomor-
phometric studies, immunohistochemical analyses allow the in
situ detection of different proteins like cytokines and cell
markers within the fracture callus.
46,53
Because the callus is
a heterogeneous, 3-dimensional structure comprising several
different types of tissue, it is a great challenge to accurately
analyze the different tissue areas using 2-dimensional histo-
logic sections. Therefore, it is crucial to dene representative,
standardized longitudinal or transverse bone sections that
allow a reproducible calculation of the size and tissue com-
position of the callus.
51
From the technical aspect, decalcied
and undecalcied bone sections are used for a variety of
different staining methods.
51
In general, the implant has to be
removed; however, when applying the sawing and grinding
technique, the implant can also be left in place.
54
Biomechanical Analysis
Three-point and 4-point bending tests and torsion tests
have proven to be applicable for biomechanical studies on
bone repair in murine femur fracture.
40,46,55
In addition, axial
testing, which has been reported for the ex vivo analysis of
osteosynthesis devices, might be additionally appropriate for
the evaluation of bone repair.
37
As mentioned above, the small
size of the murine femur is a great challenge for biomechanical
tests because an inaccurate, nonstandardized xation of the
bones in the testing machine critically affects the testing
results. As a matter of course, highly sensitive testing devices
are required for biomechanical tests in mouse bones. Using
nondestructive 3-point and 4-point testing devices, usually the
bending stiffness of the healed bone is assessed from load
displacement curves. When applying destructive devices, the
ultimate load at failure of the bone is determined.
55
In
accordance, torsion stiffness and ultimate torque and angle
at failure can be quantitatively analyzed in torsion testing
procedures.
46
In general, the results of the biomechanical
analysis of the healing bone are expressed as percentage of that
of the contralateral intact bone to account for individual
differences of the animals.
46,55
Cytologic and Molecular Analysis
Beside the new exciting eld of molecular imaging,
conventional techniques are available to investigate cellular
and molecular aspects of bone repair. Numerous proteins like
cytokines, cell metabolism markers, and cell surface proteins
are detectable in situ by immunohistochemical methods.
46,53
Results of immunohistochemical assessments can be sup-
ported by semiquantitative protein analyses using biochemical
methods such as Western blotting and enzyme-linked immu-
nosorbent assay techniques.
46,53
In situ hybridization studies
provide further information on the corresponding messenger
RNA expression in the different cells.
56,57
Also, the assessment
of the in situ messenger RNA expression can be additionally
supported by semiquantitative techniques such as Northern blot
analysis and reverse transcriptionpolymerase chain reaction
(RT-PCR).
50,57
Furthermore, cell counting methods like FACS
analysis have been reported to be effective to evaluate the effect
of different bone marrow cells on bone repair.
58
Cell apoptosis
can be assessed by in situ labeling of nuclear DNA fragments
using the TdT-mediated duTP-biotin nick end labeling
(TUNEL) technique.
59
Finally, cells of the fracture callus can
be harvested for further cell culture studies.
60
METHODS TO EVALUATE THE IMPACT OF
OSTEOSYNTHESIS ON
ANIMAL REHABILITATION
As indicated above, bone repair is dependent on the local
mechanical environment within the fracture gap.
61
Although
mechanical loading during the rst days after fracture (stage of
inammation) seems to disturb bone healing, controlled loading
stimulates endochondral ossication and bone remodeling
during the later stages of healing.
62,63
Of interest, in 1977,
Sarmiento et al
64
demonstrated that weight bearing is capable
of accelerating bone repair in rats. The animals activity and
capability of weight bearing, which might be affected by the
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osteosynthesis technique used, has therefore always to be
addressed when analyzing bone repair. In large animal models,
gait analysis has proven to be a powerful tool for monitoring
the course of fracture healing. Thereby, a signicant corre-
lation between the stiffness of the osteosynthesis technique,
the mineralization of the callus, and the amount of weight
bearing of the animals could be demonstrated.
65
First gait analyses in mice were reported in 1974.
66
Using simultaneous video and reaction force analyses, Clarke
and Still provided a great review of the murine gait.
42,67
The
gait of mice has been further analyzed using treadmills and
high-speed video cameras combined with cineuoroscopic
equipment.
68
In addition, treadmills were used for the
evaluation of the animals running speed.
69
To assess
locomotor alterations in arthritic mice, paw print pattern
analysis was included in the gait analysis.
70
Recently, we
developed a novel approach for gait analysis during murine
fracture healing (Fig. 4). Using modied radiolucent running
wheels, we were able to visualize the articulating bones of
mice by dynamic high-resolution radiography. Thereby, we
analyzed characteristic gait parameters such as the tibiofe-
moral angle of the fractured limb. Of interest, we observed
signicant alterations of the mouse gait when using different
osteosynthesis devices for fracture stabilization (Histing et al.,
unpublished data). In fact, we are convinced that this analysis
method represents an additional powerful tool to evaluate
healing and rehabilitation of murine femur fractures.
SUMMARY AND RECOMMENDATIONS
Taken together, a variety of different murine femur
fracture models have recently been introduced. These models
allow distinct evaluation of the molecular aspects of bone
repair. Due to its anatomy, the mouse femur is more
appropriate for fracture studies compared with the mouse
tibia. Depending on the study, open or closed fracture models
with external or internal osteosynthesis devices can be used.
The standardization of the experimental setup is a precondition
for every animal study and should also include the fracture
stabilization. Therefore, biomechanical parameters of fracture
stabilization have to be assessed not only in large animals but
also in mouse models. A large number of analytical methods,
ranging from conventional histology and biomechanics to
novel molecular imaging techniques, can be used to evaluate
bone repair in mice. In addition, new methods for gait analyses
are applicable, which can be either used as a further approach
to indirectly assess the fracture healing process or to evaluate
the impact of different osteosynthesis devices on physical
activity and rehabilitation of the animals.
In conclusion, current murine femur fracture models
provide standardized conditions with regard to the fracture
creation, the character of soft tissue trauma, and the
osteosynthesis technique for fracture xation. So far, the
mouse has been of interest particularly to analyze molecular
and genetic aspects of bone repair. Due to the great progress in
the standardization of murine fracture, however, mouse models
have become also a powerful tool for research on general
aspects of bone repair.
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