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First published on October 6, 2005, doi:10.1177/0363546505278700
This version was published on January 1, 2006
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The American Journal of Sports Medicine 34:84-91 (2006)
© 2006 American Orthopaedic Society for Sports Medicine

Effects 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*,{dagger}, Harukazu Tohyama, MD, PhD*,{ddagger}, Eiji Kondo, MD, PhD*, Toshikazu Yoshikawa, MD, PhD*, Takeshi Muneta, MD, PhD{dagger}, 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 {dagger} 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

{ddagger} 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: tohyama{at}med.hokudai.ac.jp).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The vascularization of the grafted tendon for ACL reconstruction has been examined.2,3 Initially, nutrition of the graft is thought to occur through diffusion of nutrients from the adjacent synovial tissue and synovial fluid.1 Thus regions of ischemic necrosis are seen in the midsubstance of the grafts while vascular invasions are started in the periphery of the graft after ACL reconstruction.2,3 Recently, Delay et al6 reported a human ACL 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.7 Therefore, the lack of vascularity within the ACL 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 tissues "flaws" that weaken the graft.23

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis that involves activation, migration, and proliferation of endothelial cells in various pathologic conditions.8 Petersen et al20 reported that VEGF is expressed in the autologous tendon graft at 6 months 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.29 These findings have suggested that VEGF mediates angiogenesis in the intra-articular tendon graft for the ACL reconstruction. Corral et al5 reported that an application of VEGF significantly enhances not only angiogenesis but 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 angiogenesis affects the mechanical properties of the ACL graft.

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.13,14,17,22 We hypothesized 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 ACL. The purpose of this study was to test these hypotheses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 = 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 the treated right knees.

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.22 The ACL was then thawed by 25°C saline solution poured into the 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.14 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.5 In each group, the skin wound was 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 activities in their cages (52 cm in width, 35 cm in height, and 33 cm in depth).

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.10 The Chalkley score is known as a rapid method of quantifying tumor angiogenesis and is based on the microvessel density in the cancer.10 The observers were blinded to the treatment 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)4 has 25 random points and was employed over each 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 sections).

Biomechanical Evaluation
Both hind limbs were resected immediately after the animals were sacrificed. Each hind limb was stored at –32°C until testing.25 Before mechanical testing, each knee was thawed 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, 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 between the first 30° and the repeated 30° measurements was 0.1 ± 0.3 mm.

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).28 Briefly, a medial femoral condyle and an anterior portion of the lateral 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 cross-sectional shape of the ACL was reconstructed using a computer algorithm.28

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.26 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.14,22,27 Two parallel 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 video dimension analyzer.

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 difference tests were conducted for multiple comparisons. Significance level was set at P = .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 around the fat pad, whereas we did not find such vessel-abundant tissues in group I or group II.

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 1Go, 2Go, and 3Go). 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 1Go); vascular formation with CD31 positive cells was observed at the superficial portion of the ACL at 6 and 12 weeks (Figures 2Go and 3Go). In group III, vascular formation with CD31 positive cells was observed at the superficial portion of the ACL at all experimental periods after the in situ freeze-thaw treatment (Figures 1Go, 2Go, and 3Go).



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  		Figure 1. Immunohistochemistry for CD31 to identify 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.

 


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  		Figure 2. Immunohistochemistry for CD31 to identify 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.

 


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  		Figure 3. Immunohistochemistry for CD31 to identify vascular endothelial cells in the ACL 12 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.

 
The Chalkley score in the normal ACL was 0.27 ± 0.28. Two-way ANOVA demonstrated significant effects of the treatment and time on the Chalkley score (treatment, P = .001; time, P = .001; interaction effect, P = .890). The Chalkley score of group III (3.40 ± 0.96, 5.07 ± 1.04, and 2.60 ± 0.83 at 3, 6, and 12 weeks, respectively) was significantly higher than that of group I (1.87 ± 0.80, 3.53 ± 1.07, and 1.40 ± 0.55 at 3, 6, and 12 weeks, respectively) and group II (2.13 ± 0.77, 3.13 ± 1.39, and 1.20 ± 0.38 at 3, 6, and 12 weeks, respectively) (vs group I, P = .001; vs group II, P = .001), although there was no significant difference between groups I and II (P = .740) (Figure 4Go). These findings suggest that the microvessel density was higher in group III than in groups I and II. The Chalkley score determined at 6 weeks was significantly higher than those at 3 weeks and 12 weeks (vs 3 weeks, P = .001; vs 12 weeks, P = .030). The Chalkley score at 12 weeks was significantly lower than that at 3 weeks (P = .030).



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  		Figure 4. Quantification of angiogenesis using the Chalkley scoring method. The Chalkley score of group III was significantly higher than that of group I and group II, whereas there were no significant differences between groups I and II. The Chalkley score at 6 weeks was significantly higher than those at 3 weeks and 12 weeks. The count at 12 weeks was significantly lower than that at 3 weeks.

 
Biomechanical Evaluation
Concerning the anterior-posterior translation, mean values in group I were 2.5 mm at 30°, 2.1 mm at 60°, and 1.1 mm at 90°. Mean anterior-posterior translation values in group II were 2.6 mm at 30°, 2.2 mm at 60°, and 1.0 mm at 90°. The mean values of group III were 2.8 mm at 30°, 2.2 mm at 60°, and 1.0 mm at 90°. These mean values were slightly more than the normal control values of 1.9 mm at 30°, 1.6 mm at 60°, and 0.8 mm at 90°, although we did not find any significant differences (30°: vs group I, P = .191; vs group II, P = .171; vs group III, P = .066) (60°: vs group I, P = .264; vs group II, P = .238; vs group III, P = .225) (90°: vs group I, P = .251; vs group II, P = .374; vs group III, P = .310) One-way ANOVA did not show any significant differences among the groups, including the control group (30°: P = .195, 1 – ß = 0.378; 60°: P = .548, 1 – ß = 0.177; 90°: P = .643, 1 – ß = 0.147) (Figure 5Go).



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  		Figure 5. Anterior-posterior translation of the tibia relative to the tibia between ± 10 anterior-posterior load at 30°, 60°, and 90° of knee flexion. Two-way analysis of variance failed to demonstrate a significant effect of the treatment or the knee flexion angle.

 
In groups I, II, and III, the cross-sectional areas of the ACLs were significantly greater than that of the normal ACL (Table 1Go); there were no significant differences among the 3 experimental groups (Figure 6AGo). During tensile failure tests, all specimens failed at the midsubstance of the AM band–ACL without avulsion failures. The tensile strength and the tangent modulus of groups I, II, and III were significantly lower than those of the normal ACL; there were no significant differences among the 3 experimental groups (Table 1Go) (Figures 6 B and CGo). Concerning the strain at failure, there were no significant differences among the 4 groups, including the control group (Figure 6DGo).


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  		TABLE 1 Statistical Comparisons of Biomechanical Parametersa
 


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  		Figure 6. Mechanical evaluation. A, the cross-sectional area of the whole ACL; B, the tensile strength of the antero-medial bundle of the ACL; C, the tangent modulus of the anteromedial bundle of the ACL; D, the strain at failure of the anteromedial bundle of the ACL. There were no significant differences among the 3 experimental groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent evidence suggests that recombinant VEGF therapy may provide added stimulus to the healing of certain types of injuries, such as chronic wounds. For example, Corral et al5 reported that an application of 30 µ g VEGF165 as well as VEGF121 improved granulation tissue formation in both normal and ischemic dermal ulcers in a standardized model with a rabbit ear. Using a rat model, Zhang et al32 showed that tensile strength was statistically higher in the incisional wound group and in the ischemic flap wound group with 10 µ g VEGF165 treatment compared with the ischemic flaps with no treatment at 1 week. They also reported that ischemic wound repair with VEGF treatment had a significantly higher level of microvessel density than did normal incisional wounds and ischemic wounds without treatment. These studies have suggested that the administration of VEGF can accelerate the remodeling process in devitalized soft tissues. The present study has added important information that intra-articular administration of 30 µg VEGF165 significantly enhanced the Chalkley score, indicating the microvessel density, in the ACL devitalized in situ by the freeze-thaw treatment.

In vitro VEGF stimulates endothelial cells to migrate, proliferate, and form tubes.8 In vivo VEGF also functions as an endogenous stimulator of both angiogenesis and increased vascular permeability.11 For example, VEGF is expressed in developing blood vessels, and its receptors are found exclusively on endothelial cells.18 Nissen et al19 also reported that VEGF mediates angiogenic activity during the proliferative phase of wound healing. Corral et al5 found that induction of VEGF messenger RNA was increased 6-fold by wounding in ischemic conditions in contrast to basic fibroblastic growth factor, and concluded that VEGF seems to be more important than basic fibroblastic growth factor during ischemic wound healing. Ishii et al12 reported that oxygen injection leads to down-regulation of VEGF messenger RNA expression in the ligament after the surgical laceration. Therefore, the expression of VEGF is considered to be potentiated in response to ischemia.31 In addition, a recent study demonstrated that mechanical tensile stress promotes VEGF expression in ligament fibroblasts in vitro.30 However, the role of VEGF in healing of the ACL graft has been unclear. We previously investigated the VEGF expression and angiogenesis in the graft using the rabbit ACL reconstruction model with patellar tendon autograft,29 which was a different model from that in the present study. In this previous study, we observed the VEGF expression in the graft 2 weeks after the ACL reconstruction, whereas no VEGF expression existed at 1 week. Petersen et al20 reported that VEGF expression is found in the free tendon graft at 6 weeks after surgery and diminishes 52 weeks after ACL reconstruction in the sheep. The current study showed that an exogenous application of VEGF stimulates revascularization in the ACL after necrosis. Although infiltrative cells into the ACL graft secrete endogenous VEGF, this endogenous VEGF may not be enough for enhancement of early revascularization in the ACL graft.

With damage to tendon tissues, cell infiltration from the blood system is thought to be required to provide the necessary reparation for tissue healing.7 However, degeneration and subsequent rupture of tendons have been associated with hypovascularity of specific regions within certain tendons.9,21 The lack of vascularity within the ACL graft may result in microdamage of the graft during the postoperative period. Therefore, there was the possibility that stimulating angiogenesis in response to exogenous VEGF might prevent the mechanical deterioration of the ACL after necrosis. On the other hand, Shrive et al23 found that the amount of tissue flaws (blood vessels, fat cells, hypercellular areas, loose matrix, disorganized matrix, or a combination of these) in scar tissue is inversely correlated with the mechanical properties of the scar tissue in the rabbit medial collateral ligament injury model. Concerning the ACL graft, previous studies have demonstrated negative correlation between vascularity of the ACL graft and mechanical strength of the graft after ACL reconstruction in animal experiments.16,24 Therefore, there was the possibility that the stimulation of angiogenesis by VEGF application deteriorates the mechanical properties of the frozen-thawed ACL. The present study showed that, interestingly, 30 µ g VEGF165 application did not affect the mechanical properties of the in situ frozen-thawed ACL, although 30 µ g VEGF165 treatment significantly promoted angiogenesis in the ACL after the in situ freeze-thaw treatment.

We do not know exactly why our VEGF application did not affect the mechanical properties of the in situ frozen-thawed ACL. The first possibility was that our VEGF application might have had too little effect to change the ACL after necrosis because of the dose, the molecular format of VEGF, the delivery method, or the animal model. However, our vascularity analysis showed that our VEGF application significantly increased blood vessel invasion in the ACL after the necrosis.

The second possibility was that the time of mechanical evaluation might have been too early or too late to detect the effect of VEGF application. However, we observed a significant effect of VEGF on neovascularization in the necrotized ACL at 3 weeks after VEGF application. Therefore, the mechanical evaluation was thought to be not too early. Our previous study, using the same model, showed that the in situ freeze-thaw procedure without growth factor application does not affect the mechanical properties of the ACL until 6 weeks after the procedure and then the mechanical properties deteriorate at 12 weeks. Therefore, it was unlikely that mechanical evaluation at 12 weeks was too late.

The third possibility was that the resolution of our method for mechanical evaluation was too low to detect the effect of the VEGF application on mechanical properties of the ACL. However, we previously examined the effect of transforming growth factor (TGF)/endothelial growth factor (EGF) application on the rabbit in situ frozen-thawed ACL,22 which was the same model as the present study, using the same evaluation methods as the present study. We found that local administration of 4 ng TGF-ß1/100 ng EGF significantly improved mechanical properties of the AM bundle 12 weeks after the in situ freeze-thaw treatment. Concerning mechanical properties of the ACL after the in situ freeze-thaw treatment, the effect of the intra-articular injection of 30 µg VEGF was considered to be less than that of the local administration of 4 ng TGF-ß1/100 ng EGF.

The fourth possibility was that additional effects of VEGF, other than stimulation of angiogenesis, might have compensated for mechanical deterioration of the ACL in response to the abundant tissue flaws that an exogenous application of VEGF induces. Zhang et al33 reported that an exogenous application of VEGF significantly upregulates TGF-ß expression in the injured tendon. The authors previously found that a TGF-ß application significantly inhibits the mechanical deterioration of the ACL that underwent the in situ freeze-thaw treatment.17

The fifth possibility was that vascular conditions of the ACL graft might not have significantly reflected mechanical properties of the ACL graft, if the anatomical orientation and the physiologic tension of the ACL graft were preserved. Jackson et al13 showed that the in situ freeze-thaw treatment, which was a similar procedure to that in our present study, did not significantly affect mechanical strength of the goat ACL, although they observed a marked synovial vascular reaction surrounding the ACL.

There are some limitations in the present study. The first limitation is that the in situ frozen-thawed ACL is not a true model of ACL reconstruction by use of a free tendon graft. Biologic differences must exist between the frozen-thawed ACL and the intra-articular grafted tendon after ACL reconstruction because bone marrow–derived cells contribute to a graft that is placed in a bone tunnel. A further study using a large animal model should be conducted before its clinical application to clarify if recombinant VEGF application deteriorates the mechanical properties of the grafted tendon after ACL reconstruction. Second, we applied the single dose, 30 µg, of VEGF in the present study, and we showed that this dose of VEGF significantly stimulated the angiogenesis of the ACL after the necrosis. We should examine the dose-dependent effects on the ACL after the necrosis. The third limitation is that we did not examine the material properties of the posterolateral bundle of the ACL. Therefore, this study cannot refer to the effect of VEGF application on the posterolateral bundle of the ACL. The fourth limitation is that we did not attempt to evaluate the effect of VEGF on the structural properties of the intact ACL because previous studies have shown that intrasubstance failure rarely occurs in the rabbit model when the femur–intact ACL–tibia complex undergoes tensile testing.26 The fifth limitation is that we did not measure changes in the VEGF concentration in the joint cavity after its administration. The sixth limitation is that we have no information to assess whether the stimulating effect of VEGF application on angiogenesis in the ACL after necrosis might or might not be clinically significant. In spite of these limitations, however, we believe that the present study has provided useful information concerning the basic science of ACL reconstruction.

As to clinical relevance, VEGF is widely used for patients with extensive tissue ischemia in whom primary vascular reconstruction procedures are not feasible or had previously failed in clinical trials.15 Early clinical data provide evidence that VEGF application can achieve beneficial angiogenesis, with minimal side effects. The present study implies that an application of recombinant VEGF therapy can enhance revascularization of the graft after ACL reconstruction without significant adverse effects on mechanical properties of the graft. Many investigators have shown that the vascularity within the ACL graft is not sufficient at the early period after ACL reconstruction.1,2,3,16 Poor vascularity within the graft may result in microdamage of the graft during the postoperative period because cellular infiltration from the blood system is theoretically necessary for tissue healing after the damage of the graft.7 The recombinant VEGF therapy may be beneficial to prevent microdamage of the graft during the early rehabilitation period after ACL reconstruction. However, it still remains unknown how the application of VEGF affects the remodeling of the ACL graft or the clinical outcome after ACL reconstruction. Therefore, a great deal more research should be conducted before clinical application of VEGF to ACL reconstruction.


   FOOTNOTES
 
No potential conflict of interest declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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