Effects of local administration of vascular endothelial growth factor on properties of the in situ frozen-thawed anterior cruciate ligament in rabbits.

Abstract

In the autogenous tendon for anterior cruciate ligament reconstruction, intrinsic fibroblasts are necrotized immediately after surgery, and repopulation and revascularization occur. Vascular endothelial growth factor is considered to be a potent mediator of angiogenesis.
An application of vascular endothelial growth factor significantly enhances angiogenesis in the in situ frozen anterior cruciate ligament, and the application significantly affects mechanical properties of the in situ frozen anterior cruciate ligament.
Controlled laboratory study.
Right anterior cruciate ligaments from 66 rabbits underwent the freeze-thaw treatment, and animals were then divided into 3 groups. Group I served as a freeze-thaw but otherwise untreated control. In group II, 0.2 mL phosphate-buffered saline alone was applied. In group III, 30 mug vascular endothelial growth factor was applied. The groups were compared on the basis of histologic revascularization examinations using the Chalkley score, an indicator of the microvessel density, and mechanical evaluations, which included the anterior-posterior translation of the tibia relative to the femur during +/- 10 N of anterior-posterior load and the mechanical properties of the anteromedial bundle of the anterior cruciate ligament.
Group III's Chalkley score was significantly greater than that of groups I and II. The tensile strength and the tangent modulus of anterior cruciate ligaments in groups I, II, and III were significantly lower than those of a normal anterior cruciate ligament, although there were no significant differences among groups I, II, and III.
Vascular endothelial growth factor, as administered in this study, significantly promoted angiogenesis in the devitalized anterior cruciate ligament with in situ freeze-thaw treatment, but it did not affect the mechanical properties of the in situ frozen-thawed anterior cruciate ligament in the rabbit model.
An application of the recombinant anterior cruciate ligament is a potential future strategy to enhance revascularization of the autograft in anterior cruciate ligament reconstruction.

Full text

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0BEffects of Local Administration of Vascular Endothelial
Growth Factor on Properties of the in Situ Frozen-
Thawed Anterior Cruciate Ligament in Rabbits


Young-Jin Ju, MD*,, Harukazu Tohyama, MD, PhD*, Eiji Kondo, MD, PhD*, Toshikazu
Yoshikawa, MD, PhD*, Takeshi Muneta, MD, PhD , Kenichi Shinomiya, MD, PhD and
Kazunori Yasuda, MD, PhD*
From the * Department of Sports Medicine and Joint Reconstruction Surgery, Hokkaido University School of Medicine, Sapporo, Japan, the
Section of Orthopedic Surgery, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan, and the Department of
Orthopaedic Surgery, Tokyo Medical and Dental University School of Medicine, Tokyo, Japan. Address correspondence to Harukazu
Tohyama, MD, PhD, Department of Sports Medicine and Joint Reconstruction Surgery, Hokkaido University School of Medicine, Kita-15,
Nishi-7, Kita-ku, Sapporo 060-8638, Japan (e-mail: HUtohyama@med.hokudai.ac.jpUH).




ABSTRACT


Background: In the autogenous tendon for anterior cruciate ligament reconstruction,
intrinsic fibroblasts are necrotized immediately after surgery, and repopulation and
revascularization occur. Vascular endothelial growth factor is considered to be a potent
mediator of angiogenesis.

Hypothesis: An application of vascular endothelial growth factor significantly enhances
angiogenesis in the in situ frozen anterior cruciate ligament, and the application significantly
affects mechanical properties of the in situ frozen anterior cruciate ligament.

Study Design: Controlled laboratory study.

Methods: Right anterior cruciate ligaments from 66 rabbits underwent the freeze-thaw
treatment, and animals were then divided into 3 groups. Group I served as a freeze-thaw but
otherwise untreated control. In group II, 0.2 mL phosphate-buffered saline alone was applied.
In group III, 30 µg vascular endothelial growth factor was applied. The groups were
compared on the basis of histologic revascularization examinations using the Chalkley score,
an indicator of the microvessel density, and mechanical evaluations, which included the
anterior-posterior translation of the tibia relative to the femur during ± 10 N of anterior-
posterior load and the mechanical properties of the anteromedial bundle of the anterior
cruciate ligament.

Results: Group III’s Chalkley score was significantly greater than that of groups I and II. The
tensile strength and the tangent modulus of anterior cruciate ligaments in groups I, II, and III
were significantly lower than those of a normal anterior cruciate ligament, although there
were no significant differences among groups I, II, and III.

Conclusion: Vascular endothelial growth factor, as administered in this study, significantly
promoted angiogenesis in the devitalized anterior cruciate ligament with in situ freeze-thaw
treatment, but it did not affect the mechanical properties of the in situ frozen-thawed anterior
cruciate ligament in the rabbit model.

Clinical Relevance: An application of the recombinant anterior cruciate ligament is a
potential future strategy to enhance revascularization of the autograft in anterior cruciate
ligament reconstruction.

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Key Words: angiogenesis • anterior cruciate ligament reconstruction • biomechanical
properties • vascular endothelial growth factor (VEGF)




INTRODUCTION


The vascularization of the grafted tendon for ACL reconstruction has been examined.H2
Initially, nutrition of the graft is thought to occur through diffusion of nutrients from the
adjacent synovial tissue and synovial fluid.H1
the midsubstance of the grafts while vascular invasions are started in the periphery of the
graft after ACL reconstruction.H2
reconstruction case in which the core portion of the patellar tendon graft remained necrotic at
18 months after surgery. With damage to any tissue, cell infiltration from the blood system is
thought to be required for tissue healing.H7
graft may induce degeneration or microruptures of the grafted tendon during the
postoperative period. On the other hand, there is also a possible risk that the
neovascularization may subsequently increase the mechanical deterioration of the graft
matrix because newly formed vessels in the graft may cause soft
23
e graft. th

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis that involves
activation, migration, and proliferation of endothelial cells in various pathologic conditions.H8
Petersen et alHU20
after ACL reconstruction in sheep. Our recent experimental study demonstrated that extrinsic
cells newly proliferated in the necrotized tendon graft express VEGF at 2 weeks after surgery
when revascularization has not yet occurred.HU29
mediates angiogenesis in the intra-articular tendon graft for the ACL reconstruction. Corral et
alH5
also wound healing in rabbit skin. Therefore, there is a high possibility that an application of
VEGF to the necrotized tendon graft will enhance angiogenesis in the graft. No studies,
however, have been conducted to clarify the effect of an application of VEGF to the
necrotized tendon graft after ACL reconstruction, and it is still unknown how stimulating
ngiogenesis affects the mechanical properties of the ACL graft. a

To investigate these issues, we have conducted a biomechanical study using the in situ
frozen-thawed ACL in the rabbit model. The in situ frozen-thawed ACL, which is anatomical
but acellular, has been established as an ideal ACL graft model.HU13
that an application of VEGF may significantly enhance angiogenesis in the in situ frozen ACL
and that the application may significantly affect mechanical properties of the in situ frozen
CL. The purpose of this study was to test these hypotheses.
A

H,
H3
H
H Thus regions of ischemic necrosis are seen in
H,
H3
H Recently, Delay et alH6
H reported a human ACL
H Therefore, the lack of vascularity within the ACL
tissues "flaws" that weaken
HU UH

H
UH reported that VEGF is expressed in the autologous tendon graft at 6 months
UH These findings have suggested that VEGF
H reported that an application of VEGF significantly enhances not only angiogenesis but
UH,
HU14
UH,
HU17
UH,
HU22
UH We hypothesized



MATERIALS AND METHODS


Experimental Design
A total of 66 mature female Japanese White rabbits weighing 3.4 ± 0.2 kg (mean and
standard deviation) were used in the present study. After the freeze-thaw treatment was
applied in the right ACL to kill intrinsic fibroblasts, all animals were randomly divided into 3
groups. In group I (n = 22), no additional treatments were applied. In group II (n = 22), 0.2
mL of phosphate-buffered saline (PBS) was injected into the right knee joint. In group III (n =

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22), 30 µg VEGF mixed with 0.2 mL PBS was injected into the right knee joint. In each
group, we sacrificed 5 rabbits by a lethal dose of pentobarbital injection to evaluate the effect
on angiogenesis at 3, 6, and 12 weeks, respectively, after surgery. For biomechanical
examinations, 7 rabbits were sacrificed at 12 weeks in each group. To obtain normal control
data, 12 left knees harvested from group I underwent the same evaluations as performed in
e treated right knees. th

Surgical Procedure
Animal experimentation was conducted under the Rules and Regulations of the Animal Care
and Use Committee, Hokkaido University School of Medicine. Surgery was performed under
anesthesia (intravenous injection of pentobarbital, 25 mg/kg). In the right knee, the ACL was
exposed through a medial parapatellar approach. First, we froze the ACL using a previously
developed cryoprobe.HU22
joint cavity. This freeze-thaw procedure was repeated 3 times for each ACL. Our previous
study revealed that 95% to 100% of the cells in the ACL are destroyed by this procedure.HU14
The incised joint capsule was then tightly closed with 3-0 nylon sutures so that liquid did not
escape from the knee joint. In group I, no additional treatment was applied to the joint. In
group II, 0.2 mL of PBS was injected into the right knee joint. In group III, 30 µg VEGF mixed
with 0.2 mL PBS was injected into the right knee joint. The dose of VEGF was chosen
according to the in vivo study, which showed that 30 µg exogenous VEGF165 improved
wound healing produced by ischemia in rabbit skin.H5
closed with 3-0 nylon sutures, and an antiseptic spray dressing was applied. No
immobilization was applied after surgery, and the rabbits were allowed unrestricted daily
ctivities in their cages (52 cm in width, 35 cm in height, and 33 cm in depth). a

Evaluation of Angiogenesis
For histologic observation, the femur-ACL-tibia complex was resected and fixed in a buffered
10% formalin solution, decalcified, and cast in paraffin blocks. From each ACL, 3 paraffin
sections aligned to the ligament axis were made and immunostained with a monoclonal
antibody against CD31 (DAKO Co, Carpinteria, Calif), which is a marker for vascular
endothelial cells. Two independent observers evaluated angiogenesis with the Chalkley
scoring method, which was established for the evaluation of tumor angiogenesis.HU10
Chalkley score is known as a rapid method of quantifying tumor angiogenesis and is based
on the microvessel density in the cancer.HU10
group and the postoperative period of the specimens. Briefly, the 3 most vascular areas (hot
spots) with the highest number of microvessel profiles were chosen by scanning each ACL
section at low power (x50). The Chalkley eyepiece graticule (Leitz Orthoplan; Leica
Microsystems AG, Wetzlar, Germany)H4
hot spot and oriented so that the maximum number of points at x200 magnification was on or
within the areas of highlighted microvessel profiles. This number of points on or within the
areas of microvessels among 25 points in the eyepiece graticule was defined as the
Chalkley score of each graticule. Therefore, the greatest number of points for the Chalkley
score was 25. The Chalkley score for an individual specimen was taken as the mean value
of 18 graticule counts (the 2 independent observers made counts at 3 hot spots at 3 ACL
ections). s

Biomechanical Evaluation
Both hind limbs were resected immediately after the animals were sacrificed. Each hind limb
was stored at –32°C until testing.HU25
overnight at 4°C. Anterior-posterior translation testing of the knee was performed as follows:
The femur and the tibia were separately cast in cylindrical aluminum tubes using
polymethylmethacrylate resin. The knee joint was mounted onto a specially designed testing
device with 3 degrees of freedom (anterior-posterior, medial-lateral, and proximal-distal
translations), which was then attached to a tensile tester (RTC-1210, Orientec, Oakabe,
UH The ACL was then thawed by 25°C saline solution poured into the
UH
H In each group, the skin wound was
UH The
UH The observers were blinded to the treatment
H has 25 random points and was employed over each
UH Before mechanical testing, each knee was thawed

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Japan). This testing device allowed unrestricted motion of the tibia relative to the femur in the
proximal-distal direction using a frictionless X-Y table. Therefore, no compressive load was
generated during the anterior-posterior displacement measurements. Four cycles of anterior-
posterior loads of 10 N were applied to the knee specimen at 30°, 60°, and 90° of knee
flexion. Load-displacement curves were drawn with an X-Y recorder (Model 3023,
Yokogawa, Tokyo, Japan). The displacement of the femur relative to the tibia with the 10 N
anterior-posterior load was defined as the anterior-posterior translation of the knee at each
angle of knee flexion. We performed the reproducibility analysis of the anterior-posterior
displacement measurement of 5 normal rabbit knees. In this analysis, we measured the
anterior-posterior displacement in a sequential order, at 30°, 60°, 90°, and 30° (as a
repeated measure), respectively. The difference in the anterior-posterior displacement
etween the first 30° and the repeated 30° measurements was 0.1 ± 0.3 mm. b

Next, the joint capsule and all ligaments except for the ACL were carefully dissected in each
specimen. The cross-sectional area of each ACL specimen was measured by an optical
noncontact method using a video dimension analyzer (HTV-C1170; Hamamatsu Photonics,
Tokyo, Japan).HU28
femoral condyle anterior to the ACL insertion were resected for visualization with the video
dimension analyzer. The femur was attached to the stepping motor, and a constant tensile
load of 0.5 N was applied to the ACL by suspending a weight from the tibia. The femur was
rotated with the stepping motor at 5° angular increments through 180°, and the
corresponding profile width of the ACL was recorded with the video dimension analyzer. The
ross-sectional shape of the ACL was reconstructed using a computer algorithm.28
c

After measurement of the cross-sectional area of the whole ACL, the posterolateral bundle of
the ACL was resected using a stainless-steel razor blade. The resection of the posterolateral
bundle was required to determine the material properties of the ACL because intrasubstance
failure rarely occurs when the femur–intact ACL–tibia complex undergoes tensile testing.HU26
We measured the cross-sectional area of the anteromedial (AM) bundle after the removal of
the posterolateral bundle from the femur-ACL-tibia complex, applying the same method as
the measurement of the cross-sectional area of the whole ACL. Then, the prepared femur–
AM bundle–tibia complex specimen was mounted onto a conventional tensile tester (PTM-
250W; Orientec, Tokyo, Japan). The tibia was flexed 45° against the femur. The knee was
rotated approximately 90° medially to remove the normal distortion of the AM bundle so that
all portions of the bundle were uniformly loaded during tensile testing.HU14
lines were drawn transversely on the surface using nigrosine stains as gauge-length markers
for strain measurement. The distance between the 2 lines was approximately 5 mm. Before
the tensile test, the specimen was preconditioned with a static preload of 0.5 N for 5 minutes,
followed by 10 cycles of loading and unloading (3% strain) with a crosshead speed of 5
mm/min. Then, each specimen was stretched to failure at a crosshead speed of 20 mm/min.
Elongation in the ligament substance was determined with a video dimension analyzer using
the gauge-length markers. We determined the tensile strength and the modulus of the ACL
AM bundle based on the data of cross-sectional area of the AM bundle, a load cell, and a
ideo dimension analyzer. v

Statistical Analysis
One-way analysis of variance (ANOVA) and post hoc tests with the Fisher protected least
significant difference test were performed to compare anterior-posterior translation at each
knee flexion angle and the mechanical properties of the AM band–ACL complexes among
the experimental and the control specimens. For the angiogenesis evaluation, 2-way ANOVA
was used to detect the effects of the treatment over time on the Chalkley score. When a
significant effect was obtained, Fisher protected least significant diffe
c

UH Briefly, a medial femoral condyle and an anterior portion of the lateral
HUUH

UH
UH,
HU22
UH,
HU27
UH Two parallel
rence tests were
onducted for multiple comparisons. Significance level was set at P = .05.

Page 5

RESULTS


At the time of sacrifice, we found no signs of infection, such as purulent effusion, rubefaction,
or local heat, in the knee joint. There were no arthritic changes in the articular cartilage. In
experimental knees of group III, massive vessel-abundant tissues were formed arou
t find such vessel-abundant tissues in group I or group II. fa pad, whereas we did not

Angiogenesis Evaluation
Generally, highlighted vascular endothelial cells were observed predominantly at the
superficial area in the experimental ACLs in all groups, whereas we did not find obvious
differences in the distribution of highlighted vascular endothelial cells among proximal,
middle, and distal portions (Figures 1 , 2 , and 3 ). In groups I and II, few CD31 positive
vascular endothelial cells were found in the ACL at 3 weeks after the in situ freeze-thaw
treatment (Figure 1 ); vascular formation with CD31 positive cells was observed at the
superficial portion of the ACL at 6 and 12 weeks (Figures 2 and 3 ). In group III, vascular
formation with CD31 positive cells was observed at the superficial portion of the AC
xperimental periods after the in situ freeze-thaw treatment (Figures 1e


nd the
L at all
, 2 , and 3 ).


Figure 1.
vascular endothelial cells in the ACL 3 weeks after the in situ
freeze-thaw treatment. A, group I x100; B, group I x400; C,
group II x100; D, group II x400; E, group III x100; F, group III
x400.
Immunohistochemistry for CD31 to identify





Figure 2.
vascular endothelial cells in the ACL 6 weeks after the in situ
freeze-thaw treatment. A, group I x100; B, group I x400; C,
group II x100; D, group II x400; E, group III x100; F, group III
x400.
Immunohistochemistry for CD31 to identify