Menisci can be classified into two regions with respect to blood supply and healing potential: (1) the peripheral third, with good vascularity and good healing potential (the vascular region) and (2) the central two-thirds, without vascularity and healing capacity (the avascular region)1. The lack of sufficient vascularity in the avascular meniscal region is assumed to be the key factor in the absence of healing in this region2.
To induce vascularity and thus healing in the avascular meniscal region, several techniques have been performed, including flipping synovial flaps in the lesion3, meniscal trepanation4, and even adding fibrin clots5.
In recent years, growth factors have become a promising tool for inducing angiogenesis and healing, e.g., to improve the osseous ingrowth of anterior cruciate ligament grafts6 or patellar tendon repair7. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenetic growth factors. Applying high concentrations of VEGF165 to avascular tissue like cornea has consistently induced angiogenesis8. In a previous study involving a rabbit model, we observed that an artificially created meniscal lesion in the avascular region caused a significant increase in VEGF during the first two weeks following the injury9; however, this amount of VEGF did not induce sufficient angiogenesis for meniscal healing. Previously, we reported increased Factor VIII staining after locally applied VEGF in sheep menisci without distinguishing between the avascular and vascular zones10. Thus, the purpose of the current study was to evaluate the effect of locally applied VEGF165 in both the vascular and avascular meniscal regions with respect to vascularization, the amount of VEGF mRNA, and VEGF expression. Additionally, we analyzed the release kinetics of the VEGF165-coated sutures.
Toward this end, three hypotheses were developed: (1) the local application of VEGF165 improves healing of lesions in the avascular region of menisci, (2) the local application of VEGF165 induces angiogenesis in both the vascular and avascular regions of the menisci, and (3) the local application of VEGF165 increases the amount of VEGF mRNA and VEGF in menisci.
Materials and Methods
The VEGF family includes VEGF-A through VEGF-E and placenta growth factor (PLGF). VEGF-A has several isoforms such as VEGF121, VEGF165, VEGF189, and VEGF206, with VEGF165 being one of the most potent angiogenetic isoforms11.
VEGF165 Release Kinetics from VEGF/PDLLA (Poly[D,L–Lactide])-Coated Sutures
To evaluate the VEGF165 release pattern from the coated sutures, one VEGF/PDLLA (Poly[D,L-Lactide])-coated suture was inserted in 5 mL of phosphate-buffered saline solution at 37°C at 100% humidity. On Days 3, 9, 12, 15, 18, and 21, the phosphate-buffered saline solution was completely replaced. The VEGF165 concentration in the phosphate-buffered saline solution and on the suture material (on Day 21) was analyzed with use of a Luminex assay (Luminex, Austin, Texas) according to standard protocols using LiquiChip200 (Qiagen, Valencia, California). For the detection of VEGF165, the Milliplex human cytokine immunoassay kit (MPXHCYTO-60K-01; Millipore, Billerica, Massachusetts) was used.
The local government’s animal rights protection authorities approved this study in accordance with the National Institutes of Health guidelines for the use of laboratory animals. Eighteen adult female Merino sheep were used as the use of sheep is an established large-animal model for meniscal healing12,13. The animals were on average three years of age and had a mean weight (and standard deviation) of 60 ± 4 kg.
Preoperatively and through the second postoperative day, antibiotic prophylaxis, analgesia, and thrombosis prophylaxis were given. After sedation (ketamine; Serumwerke, Bernburg, Germany), anesthesia was initiated with intravenous injections of 8 mL of 1% propofol (Leiras Oy, Turku, Finland) and 10 mL of 0.5-mg fentanyl (Janssen-Cilag, Neuss, Germany). Anesthesia was continued with endotracheal administration of isoflurane (CuraMED, Karlsruhe, Germany). Open surgery was performed under sterile conditions. The medial meniscus was cut longitudinally in the avascular region for a length of about 15 mm from the anterior horn to the middle portion with use of a number-15 scalpel blade. The meniscal lesions were repaired with two sutures with use of a modified outside-in technique (Fig. 1).
Three subject groups were established according to the suture material used for repair: (1) six menisci with uncoated sutures (Ethibond 2-0; Ethicon, Norderstedt, Germany), (2) six menisci with sutures coated with human VEGF165 (tebu-bio, Offenbach, Germany) and 30-kDa Poly-(D,L-Lactide) (PDLLA; Boehringer, Ingelheim am Rhein, Germany), and (3) six menisci with PDLLA-coated sutures to evaluate any possible effect of the carrier material for VEGF165. A fourth group, which included the eighteen healthy medial menisci of the contralateral knee, served as the control group. The sheep were randomly assigned to one of the suture material groups. The sutures were tied, and the wounds were closed in routine fashion with use of nonabsorbable sutures to avoid immune reactions. Full weight-bearing was permitted without restriction of range of motion.
The mean concentration of VEGF165 was about 104 ng/100 mm per suture. Approximately 20 mm of coated suture material was incorporated into the meniscus, with an average surface area of about 20 mm2, thereby placing an intrameniscal amount of about 20.8 ng of VEGF165. The sutures were coated according to the method described by Schmidmaier et al.14.
After 59 ± 1.4 days, the sheep were killed and meniscal samples were collected. This time period was chosen because the postoperative rehabilitation phase after meniscal repair in humans commonly ends after six to eight weeks. Samples were collected under sterile conditions. The medial meniscus, cartilage, and synovial membrane from both knees of each sheep were assessed macroscopically. The operatively-treated menisci were grossly examined, and the grade of healing was classified as complete healing (>90% of vertical area healed), partial healing (50% to 90% healed), or no healing (<50% healed)4. Meniscal samples harvested for real-time reverse transcription-polymerase chain reaction (RT-PCR) were immediately put in RNAlater (Qiagen) and were frozen at –70°C. Furthermore, meniscal and lung samples were harvested for immunohistochemical staining in 4% paraformaldehyde. The lung samples were used as negative and positive controls for VEGF staining. Conventional light microscopy was performed with use of standard hematoxylin and eosin as well as toluidine blue.
Immunohistochemical Staining of VEGF and Factor VIII
To evaluate angiogenesis, Factor VIII was immunohistochemically stained to visualize endothelial cells15. Four regions of the operatively treated menisci were semiquantitatively investigated: (1) tissue immediately surrounding the suture in the avascular meniscal region, (2) tissue immediately surrounding the suture in the vascular region, (3) tissue approximately 10 mm from the suture in the vascular region, and (4) tissue approximately 10 mm from the suture in the avascular region (Fig. 2, a). From the contralateral meniscus, one transmural sample was harvested from a meniscal portion similar to the operatively treated meniscus for analysis (Fig. 2, b). To evaluate the VEGF expression, a transmural segment of meniscal tissue immediately surrounding the suture was harvested, was stained immunohistochemically to visualize VEGF, and was investigated semiquantitatively.
Tissue samples were processed in a conventional manner (slice thickness, 7 μm). Specimens were blocked with serum from the host species of the secondary antibodies (X0902 [rabbit serum] and X0901[pork serum]; Dako, Glostrup, Denmark). Afterward, specimens were incubated with biotinylated primary antibodies against VEGF-A (sc-7269 [mouse monoclonal IgG2a]; Santa Cruz Biotechnology, Santa Cruz, California) and Factor VIII (A 0082 [rabbit polyclonal antibody]; Dako). Secondary biotinylated antibodies were added against VEGF primary antibody (Z0259 [rabbit anti-mouse antibody]; Dako) and against Factor VIII primary antibody (E0353 [swine anti-rabbit antibody]; Dako). Specimens were incubated in streptavidin-biotinylated-peroxidase-complex (K0377; Dako). AEC chromogen (3-amino-9-ethylcarbazole; Dako) was added. Nuclei were counterstained with Mayer hemalum. Digital images were made at tenfold magnification. The specimens were analyzed blindly with use of ImagePro Plus 5.0 (MediaCybernetics, Bethesda, Maryland). The field of interest was 500 × 500 μm. The red intensity range was set at 155 to 255. The area of this color was measured in the field of interest. Five different areas of each sample slice were analyzed and averaged.
To verify our VEGF immunostaining technique, we used lung tissue. The positive control was immunostained as described above, and the negative control was stained without using the first antibody.
Real-Time RT-PCR of All VEGF Splice Variants
The influence of locally-applied VEGF on intrameniscal autogenic synthesis of VEGF was evaluated by means of quantitative measurement of VEGF mRNA with use of one-step real-time RT-PCR. Because of the relatively large amount of tissue necessary for this method, menisci were not separated into the subregions described above. Instead, complete tissue sections of the operatively-treated and contralateral menisci, including the vascular and avascular regions, were analyzed (Fig. 2, c and d). The frozen samples were chopped in an agate stone mortar with use of liquid nitrogen (–196°C). DNA was destroyed with use of RNase-free DNase (Qiagen, Hilden, Germany). For mRNA isolation, 1 mL of Trizol (Invitrogen, Carlsbad, California) was added. Then the sample was further chopped with an Ultra Turrax device (IKA, Staufen, Germany). Sense VEGF primer was 5′–ATG-GCA-GAA-GGA-GGG-CAG-CAT–3′–TTG-GTG-AGG-TTT-GAT-CCG-CAT-CAT–3′. Spectrophotometry was performed. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used for normalization.
Assuming a nonparametric distribution, the Kruskal-Wallis test was used for independent, numerical variables, and a chi-square test was used for independent, binary variables. The Wilcoxon matched-pairs test was used for paired samples. The alpha level for significance was set at p < 0.05. Data are presented as the mean and the standard deviation, and, where appropriate, the median and the range are reported. To minimize individual variations, the results of the operatively-treated menisci regarding Factor VIII and VEGF mRNA were subtracted from the results of the contralateral menisci as an intra-individual control. These data were used for plotting and for statistical analysis.
Source of Funding
No payments or benefits were received from a commercial entity related to this work. The funding from AGA, DFG, and Karl Storz was used for materials.
None of the animals showed any complication after surgery. There was primary wound-healing of the skin, and no signs of prolonged disabilities were observed. The initial postoperative limping resolved within the first two weeks.
The local application of VEGF165 did not improve the healing rate in the avascular meniscal region. There was no significant difference in healing between the three suture material groups (p = 0.393) (Table I). None of the VEGF-treated menisci healed completely. Interestingly, gelatinous tissue was found around the VEGF-coated sutures (Fig. 3, c). These findings were not observed around the uncoated or PDLLA-coated sutures (Fig. 3, a and b). No cartilage lesions were identified. Synovitis was observed in two sheep with VEGF-coated sutures.
VEGF165 Release Kinetics from VEGF/PDLLA-Coated Suture
The released VEGF165 concentration peaked on Day 3 (17.6 ng/mL), decreased on Day 6 to 1.5 ng/mL, and was nearly zero on Day 9 (0.02 ng/mL). From Day 9 to Day 21, the concentration did not change notably (Fig. 4). There was also almost no VEGF left on the suture on Day 21 (<0.001 ng/mL). In total, 104.2 ng of VEGF165 were coated on a VEGF/PDLLA suture.
Immunohistochemical Staining of Factor VIII
Factor VIII, as a marker of endothelial cells, did not show any significant difference between the suture materials in the four meniscal regions, as calculated with the corrected values (the value for the operatively treated meniscus minus the value for the contralateral meniscus) (Fig. 5).
Furthermore, the VEGF application did not influence the amount of Factor VIII in the contralateral meniscus. In the contralateral menisci, no significant difference was found between the three suture types with regard to the amount of Factor VIII (p = 0.13).
The positive control showed positive VEGF staining in type-II pneumocytes around alveoli (red), and the negative control did not show any positive staining for VEGF.
Real-Time RT-PCR of VEGF mRNA
Local VEGF treatment did not influence the amount of meniscal VEGF mRNA. There was no difference between the material groups with regard to the amount of VEGF mRNA (p = 0.94). Instead, surgery by itself had a significant influence on the amount of VEGF mRNA after eight weeks. The operatively treated menisci had significantly more VEGF mRNA in comparison with the contralateral, healthy menisci, regardless of the sutures used (p = 0.007) (Fig. 6).
Furthermore, to evaluate for a VEGF effect on the native contralateral menisci, the VEGF mRNA concentration was measured. There was no difference in VEGF mRNA concentration in the contralateral menisci between the three suture types (p = 0.56). VEGF application did not influence the amount of VEGF mRNA in the contralateral meniscus.
Immunohistochemical Staining of VEGF
VEGF application did not influence the meniscal VEGF expression at the time of death. No difference was observed with respect to the suture materials (p = 0.59). Interestingly, VEGF was still detectable immunohistochemically on the VEGF/PDLLA-coated sutures at the time of death (Fig. 7).
In the current study, the rate of healing of menisci with a lesion in the avascular region was not improved by treatment with the VEGF165-coated sutures. The artificially elevated VEGF concentration was intended to induce angiogenesis and thus healing, given the fact that naturally elevated concentrations of VEGF do not induce angiogenesis and healing in menisci9. However, VEGF165 did not increase angiogenesis in the avascular or the vascular region in this model, in the tissue immediately surrounding the VEGF-coated sutures, or in the tissue approximately 10 mm away. Previously published data have shown an increased amount of Factor VIII in VEGF-treated menisci. However, in the aforementioned study10, the vascular and avascular regions were not distinguished and the contralateral meniscus was not used as a control to normalize the Factor VIII results.
Angiogenesis is a highly complex biologic process with precise coordination. It starts with vasodilation and increased permeability. The basal membrane and the extracellular matrix are degraded gradually by chymase, heparinase, cathepsin, and matrix metalloproteinases (MMP). One of the most important metalloproteinases is MMP-2. VEGF increases the amount of MMP-216, which could be an explanation for the macroscopic degradation around the VEGF-coated sutures. It is unclear why locally applied VEGF did not increase the amount of endothelial cells, despite the proangiogenetic role of MMP-2. Interestingly, MMP-2 has an anti-angiogenetic effect at high concentrations17, which could be induced by increased VEGF concentrations that we saw during the first three days of our in vitro test.
Usually, high MMP concentrations are downregulated by tissue inhibitors of metalloproteinases (TIMPs). Interestingly, VEGF downregulates TIMP-1 and TIMP-2, which would additionally increase the amount of MMP-2 with the above-mentioned consequences18,19. Furthermore, MMP-2 has the ability to generate angiostatin from free circulating plasminogen20. Angiostatin is an important anti-angiogenetic cytokine that inhibits the proliferation, migration, differentiation, and tube-formation of endothelial cells21. We did not evaluate the influence of VEGF on the amount and the timing of these factors, but there was no angiogenesis in the avascular meniscal region. Instead, the result was a degraded area around the sutures of the VEGF-treated menisci.
This degraded area also could be related to an increased immune response, which is linked to VEGF22. Thus, the artificially increased VEGF165 concentration during the first days may be overly stimulating an immune response around the VEGF-coated sutures by promoting the chemotactic response and attracting monocytes, macrophages, and T-lymphocytes23,24. While these cells are known to play an important role in angiogenesis, they might have a detrimental effect on tissue when they are overstimulated. In our histological examinations, immune cells were found only sporadically, but we harvested our specimens fifty-nine days after VEGF application. It is unclear whether immune cells were present before this time.
Recently, a factor responsible for the avascularity of the cornea was discovered: soluble VEGF-R1 (sVEGF-R1). This soluble receptor binds VEGF but inhibits its effect25 and is upregulated by VEGF26. As far as we know, sVEGF-R1 has not yet been evaluated in menisci. In the current study, increased concentrations of VEGF were administered and may be partially responsible for the lack of vascularity in the avascular regions afterward. This overbalance of anti-angiogenetic factors in the meniscus, such as sVEGF-R1, MMP-2, and angiostatin, might be responsible for the lack of angiogenesis and healing.
Angiogenesis is also a precisely-timed process. The above-mentioned processes might have started after the application of VEGF, but the duration of the VEGF effect seems to be limited. Our in vitro testing showed supraphysiologic concentrations of VEGF165 released from the coated suture through Day 6, which should have caused an overbalance of proangiogenetic factors. The highest concentration was found on Day 3 (17.6 ng/mL). On Day 9, the concentration declined below the physiologic concentration (0.02 ng/mL). Of note, the physiologic VEGF concentration in serum is about 0.5 ng/mL27. If the in vivo VEGF release kinetics are similar to the in vitro kinetics, which has already been shown for insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1) coated on metallic implants14, the supraphysiologic VEGF concentration applied in the present study should induce angiogenesis. However, no angiogenesis was found in the VEGF165-treated sheep, in either the vascular or the avascular region. One of the reasons might be that the total amount of VEGF used in the current study was still too low (approximately 20.8 ng incorporated in the meniscus). In angiogenesis studies of the cornea, increased amounts of VEGF were successfully used for the induction of angiogenesis. The cornea is an established model in angiogenesis research for two reasons: (1) relatively uncomplicated access to corneal tissue, and (2) avascularity of the corneal tissue in almost all animals, except for Trichechus manatus latirostris28 (more commonly known as the Florida manatee).
In a rabbit study, angiogenesis of the cornea was induced with at least 200 ng of VEGF released from a slow-release polymer8. Induced angiogenesis was found after two and seven days. In a chicken model, 500 ng of VEGF were necessary to induce angiogenesis in a chorioallantoic chicken membrane29. However, in the current study, VEGF was still detectable on the VEGF-coated sutures at time of death (after fifty-nine days).
The lack of healing in the current study contrasts with the results of another study in sheep, in which synovial flaps were sutured to meniscal lesions12. Meniscus healing was found in 83% of the meniscal lesions after three months. On the basis of that study, a flipped synovial flap with a maintained vascularity seems to be superior in comparison with a single application of one growth factor. According to these results, one may presume that other factors such as IGF and TGF might be crucial to meniscal healing as well30. Furthermore, there is a controversy with regard to whether the knee should be immobilized after meniscal repair. A canine study involving synovial flaps or removal of a cylindrical core from the meniscus to improve meniscal healing demonstrated superior results in knees that were immobilized with an external fixator for eight weeks as compared with knees that were not immobilized31. In contrast, a more recent rabbit study demonstrated increased meniscal vascularity and healing in non-immobilized knees after four weeks32. In the current study, the operatively treated legs were not immobilized because it was our intention to mimic standard human treatment.
The local application of VEGF165 also did not significantly influence the amount of VEGF mRNA eight weeks after application. Interestingly, the operatively treated menisci showed a significantly higher amount of VEGF mRNA in comparison with the non-operatively-treated, contralateral menisci. Tissue damage caused by the cut and the cannula punctures necessary for meniscal suturing during surgery may have upregulated VEGF mRNA expression, similar to findings reported during wound-healing under ischemic conditions in rabbits9.
Although the desired effect of increased VEGF concentrations is to induce angiogenesis, there are several undesired effects, which must be taken into account. For instance, a prolonged elevated VEGF concentration and increased vascularization may impair the biomechanical characteristics of the meniscus. This effect was shown in a sheep study evaluating anterior cruciate ligament grafts, in which locally-applied VEGF enhanced angiogenesis over twelve weeks but decreased the stiffness of the graft33.
A limitation of the present study was the artificial creation of the meniscal lesions with use of a scalpel blade. However, this model mimics the traumatic lesion as closely as possible and guarantees a similar lesion in each subject. Another limitation was the use of human VEGF165 instead of sheep VEGF165, which is still unavailable and could lead to a different outcome. However, sheep also have VEGF16534, and human VEGF165 has been used in another sheep study, where it successfully increased angiogenesis35. Furthermore, in the current study, we included six animals in each group. This number was based on several studies that have used a group size of six sheep35-37 as well as for logistical reasons. However, we did not perform a previous power calculation.
Future studies using sheep VEGF165 in sufficiently high doses and for longer durations may produce different results.
NOTE: Financial support of the AGA (Deutschsprachige Arbeitsgemeinschaft für Arthroskopie) is gratefully acknowledged. Part of this work was funded by the Deutsche Forschungsgemeinschaft (DFG PU214/3-2; 4-2; 5-2). Part of this work was funded by Karl Storz. The authors certify that they did not receive payments or benefits from a commercial entity related to this work. Additionally, they wish to thank S. Echterhagen, B. Facompre, F. Lichte, C. Jaeschke, M. Nicolau, M. Pradella, A. Rüben, L. Shen, and R. Worm for their excellent technical assistance.
Investigation performed at the Department of Orthopaedic Surgery, Otto-von-Guericke-University, Magdeburg; the Department of Anatomy, Christian-Albrechts-University, Kiel; the Department of Trauma, Hand and Reconstructive Surgery, Westfälische Wilhelms University, Münster; and the Department of Anatomy and Cell Biology, RWTH Aachen University, Aachen, Germany
Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants in excess of $10,000 from AGA (Deutschsprachige Arbeitsgemeinschaft für Arthroskopie) and DFG (Deutsche Forschungsgemeinschaft; PU2143/3-2; 4-2; 5-2). In addition, one or more of the authors or a member of his or her immediate family received, in any one year, payments or other benefits of less than $10,000 or a commitment or agreement to provide such benefits from a commercial entity (Karl Storz).
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