Expression of Vascular Endothelial Growth Factor on Chondrocytes Increases with Osteoarthritis - An Animal Experimental Investigation

Animals, Surgery and Macroscopic Evaluation

The experiments were performed with permission of the local government (G49/2000) in accordance with National Institute of Health (NIH) guidelines as described earlier [8]. Briefly, twenty eight fully grown, female white New Zealand rabbits were used. In the experimental group (12 animals), the anterior cruciate ligament was resected to create instability of the knee. In the control group (12 animals), only a sham operation without resection of the ACL was done. Four animals of each group were killed at 3, 6, and 12 weeks postoperatively. In addition, four animals (0 weeks) did not undergo any treatment and were killed at the time of surgery. After slaying the animals, both distal femurs were prepared and immediately stored at –80°C. Macroscopic grading of arthritis was done as described previously [8].

Histological Evaluation

Sample preparation and immunostaining were performed as described earlier [8], with some modifications. Briefly, frozen tissue samples were thawed, fixed with 4% paraformaldehyde, decalcificated using buffered EDTA (20%, pH 7.4), dehydrated and embedded in paraffin. Sections (5 µm thick) were cut, mounted on poly-L-lysine coated glass slides, deparaffinized in xylene, and washed. Epitope unmasking was done using proteinase K digestion (DAKO, Germany) for 5 minutes and endogenous peroxidase activity was quenched by treating tissue sections with 3% H₂O₂ for 10 minutes, non-specific binding was blocked using 3% bovine serum albumin (BSA/TrisHCl), each followed by an excessive washing procedure. Sections thus prepared were incubated with monoclonal mouse IgG antibodies against VEGF diluted 1:50 in Tris/BSA (Calbiochem Clone JH 121) at 4°C over night. Visualization was done using EnVision+ System- HRP with AEC-chromogene (DAKO Envision Systems, Germany, K4001). As control instead of primary antibody, mouse IgG1 with irrelevant specificity (Aspergillus niger glucose oxidase, DAKO, Germany) was used. In addition, sections were incubated without primary antibody.

Image Analysis

Histological sections were photographed (CoolSnap-Pro Color A00J82025, Media Cybernetics, Silver Spring, MD, USA). The cells were counted using the Image-Pro Plus software for Windows, version 4.1 (Media Cybernetics, Silver Spring, MD, USA). The total number of chondrocytes was determined in a hematoxylin and eosin stained section and compared with the number of immunohistochemically stained cells.

Histological grade of OA was evaluated according to Mankin [9] by two independent investigators who were unaware of the source of the specimens. The sections were decalcified by using EDTA which leads to an adapted mankin score which we used in the present study.

Statistical Analysis

Statistical analysis was performed using SPSS 11.0 (SPSS GmbH, Munich, Germany). Student’s t test was used for comparison of scores, and Spearman´s coefficient was calculated for correlations. P values less then 0.05 were considered significant.

Histological Grading of OA

According to the Mankin score, the experimental group showed a highly significant increase in the grade of OA in comparison with the control group and the untreated group (Figs.3-6). In the untreated group the mean (SD) grade of OA was 1.75 (1.58). In the control group it was 3.25 (0.71) after 3 weeks, 1 (1.20) after 6 weeks, and 2.25 (1.17) after 12 weeks. In the experimental group, the scores were 7.25(3.62) after 3 weeks, 8 (2.88) after 6 weeks, and 9.83 (3.06) after 12 weeks. The experimental group had significantly higher values than the control group at every point of time (p<0.01). The average histological grade of OA increased with the number of postoperative weeks. It is remarkable that in the control group the histological grading increases within the first three weeks and drops to normal in the following time.

Immunohistochemical Staining of VEGF

Chondrocytes expressing VEGF were found in the experimental and control groups (Fig. 1). In the control group, the mean percentage (SD) of VEGF positive cells was 16.2 ± 4.7 % after 3 weeks, 11.5 ± 5.9 % after 6 weeks, and 11.6 ± 3.3 % after 12 weeks. In the experimental group, percentages were 17.7 ± 5.7 % after 3 weeks,19.4 ± 4.6 % after 6 weeks, and 21.3 ± 5.4 % after 12 weeks (Fig.1).There was no significant difference between control and experimental groups after 3 weeks (p= 0.594), but a significant difference was seen after 6 and 12 weeks (p=0.01 and p=0.05). It is remarkable that in the control group the percentage of VEGF expressing chondrocytes increases within the fist three weeks and drops to normal in the following time.

A significant correlation was found between the percentage of VEGF positive chondrocytes and the histological grade (r = 0.767, p = 0.01) as well as the macroscopic grade (r = 0.518, p = 0.02) (Fig. 2). Focusing on osteophyte formation (as part of the macroscopic grade) a positive correlation (r = 0.651; p< 0.01) was detectable. The VEGF staining occurred throughout the cartilage.