Meniscal explant disks were isolated from bovine menisci (from 16 to 24 month old cattle), procured from a local abattoir with authorization from the relevant meat inspectors. This study does not involve human subjects, human tissue or experimentation of animals. Up to four full thickness tissue cylinders (10 mm in diameter) per meniscus were punched perpendicular to the meniscus bottom surface. Tissue disks 1 mm in thickness were sliced including the original meniscal surface using a sterile scalpel blade, and four to five smaller explant disks (3 mm in diameter × 1 mm thick) were isolated using a biopsy punch (HEBUmedical, Tuttlingen, Germany) and cultured in DMEM (supplemented with 100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B; Sigma-Aldrich, St. Louis, MO, USA) in a 37°C, 5% CO₂ environment after measurement of wet weight. The total of up to 60 explants per animal (2 knee joints including medial/lateral menisci) were randomised among the different experimental groups matched by their anatomical location for every single experiment and cultured in the absence or presence of varying concentrations of recombinant human TNFα (R & D Systems, Minneapolis, MN, USA). In most of the experiments a concentration of 100 ng TNFα/ml was used. Three explant disks per well of a 24-well plate were cultured in 1 ml medium. After three days of culture the medium and explants were used for measurements. For inhibitory studies different tissue inhibitor of metalloproteinases (TIMPs; R & D Systems, Minneapolis, MN, USA) and the NO synthetase inhibitor L-NMMA were used. For these investigations only one meniscal explant per well was cultured for three days in 200 μl medium in 96-well plates.

The meniscal explants were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Serial sections (7 μm) were cut sagittally through the entire thickness of the explant disks, immobilised on glass slides, and deparaffinised. After incubation for 2.5 minutes in a digester at 100°C (in 0.01 M citric acid, pH 6.0), they were incubated overnight at 4°C with the primary antibody (anti-NITEGE; 1:50 dilution in 1% BSA; ABR Affinity BioReagents, Golden, CO, USA), rinsed in Tris-NaCl three times for five minutes and incubated with the secondary antibody AlexaFluor 488 goat anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA) for one hour at room temperature. After further washing, the sections were labeled for nuclear staining with bisbenzimide (Sigma, St. Louis, MO, USA), mounted with fluorescence mounting medium (Dako, Glostrup, Denmark), and visualised using the Apotome (ZEISS, Jena, Germany) fluorescence microscope.

For radiolabel incorporation the meniscal explants were placed in fresh culture medium containing 10 μCi/ml [³⁵SO₄]-sulfate (Amersham Pharmacia, GE Healthcare Europe GmbH, Munich, Germany) for six to eight hours at 37°C under free-swelling conditions right after cytokine treatment. Afterwards, the explants were washed in PBS containing 0.5 mM proline and digested overnight in 1 ml of papain solution (0.125 mg/ml (2.125 U/ml, Sigma, St. Louis, MO, USA), 0.1 M Na₂HPO₄, 0.01 M Na-EDTA, 0.01 M L-cysteine, pH 6.5) at 65°C. A 200 μl aliquot of each sample were added to 2 ml scintillation fluid (Opti Phase Hi Safe 3, Perkin Elmer, Waltham, MA, USA) and measured using a Beckmann scintillation counter (Wallac 1904. Turku, Finland). Counts were expressed in cpm/mg wet weight and normalised to the radiolabel incorporation of untreated control tissue, which was set to 100%.

For measurement of GAG release or content the media were collected after cytokine treatment or the papain-digested explants were used (see above), and GAG content was determined by DMMB dye assay photometrically at a wavelength of 520 nm (Photometer Ultraspec II, Biochrom, Cambridge, UK) using shark chondroitin-sulfate as standard. Values were presented as μg GAG per mg wet weight of the explants.

Generation of NO was determined by measuring nitrite accumulation in culture supernatants using Griess reagent (1% sulfanilamide and 0.1% N-(1-naphtyl)-ethylene diamine-dihydro-chloride in 5% H₃PO₄, Sigma-Aldrich, St. Louis, MO, USA). A 100 μl aliquot of each sample and 100 μl Griess reagent were mixed and incubated for five minutes, and the absorption was determined in an automated plate reader (SLT Reader 340 ATTC, SLT-Labinstruments, Achterwehr, Germany) at 540 nm. Sodium nitrite (NaNO₂, Merck, Darmstadt, Germany) was used to generate a standard curve for quantification.

After three days of incubation, quantitative real-time RT-PCR was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene to determine gene expression levels. Meniscal explants (approximately 100 mg from each group) were frozen immediately in liquid nitrogen. Total RNA was extracted after pulverisation of the tissue using the TRIZOL reagent (1 ml/100 mg wet weight tissue; Invitrogen, Carlsbad, CA, USA) followed by extraction with chloroform and isopropanol precipitation. The concentration of extracted RNA was quantified spectro-photometrically at OD₂₆₀/OD₂₈₀ nm. Before real-time RT-PCR was performed using the Qiagen QuantiTect SYBR^® Green RT-PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions the extracted RNA was digested with DNase (65°C for 10 minutes; Promega, Madison, WI, USA) to remove any traces of DNA. Bovine primers were designed using Primer3 Software 25 and used at a concentration of 0.5 μM (Table 1). Conditions for real-time RT-PCR were as specified by manufacturer's description: reverse transcription 30 minutes at 50°C; PCR initial activation step 15 minutes at 95°C; denaturation 15 seconds at 94°C; annealing 30 seconds at 60°C; extension 30 seconds at 72°C; optional: data acquisition 30 seconds at melting temperature 70 to 78°C. Differences of mRNA levels between control and stimulated samples were calculated using the ΔΔC_T-method. ΔC_T represents the difference between the C_T (cycle of threshold) of a target gene and the reference gene (GAPDH). The ΔΔC_T value is calculated as the difference between ΔC_T from the stimulated samples and the control.

Protein levels of MMPs were assayed in conditioned media by gelatin and casein zymography. Equal volumes of medium samples and loading buffer (2 mM EDTA, 2% (w/v) SDS, 0.02% (w/v) bromophenol blue, 20 mM Tris-HCl, pH 8.0) were mixed, subjected to electrophoresis using 0.1% (w/v) gelatin and 0.2% (w/v) casein as substrate in 4.5 to 15% gradient SDS-PAGE, washed in 2.5% (v/v) Triton X-100, rinsed in distilled water and incubated for 16 hours at 37°C in 50 mM Tris-HCL (pH 8.5) containing 5 mM CaCl₂. Gels were stained with 0.1% (w/v) Coomassie brilliant blue R250 (Serva, Heidelberg, Germany) and destained with 10% (v/v) acetic/50% (v/v) methanol and with 10% (v/v) acetic acid/10% (v/v) methanol. MMPs were identified by molecular weight and substrate specificity as clear bands against a blue background of undigested substrate. Additionally, samples were incubated with 1 mM 4-aminophenylmercuric acetate (APMA; Sigma-Aldrich, St. Louis, MO, USA) for three hours at 37°C to activate MMP-pro-forms prior to loading.

Quantitative data are presented as mean ± standard error of the mean, n represents the number of independent experiments. Statistical analysis of data was made using a one-way analysis of variance (ANOVA) indicating significant differences, and comparisons among the various experimental groups were made using the two-tailed Student's t-test. Differences were considered significant if P ≤ 0.05.

We have established an in vitro model for the investigation of bovine meniscal tissue destruction where tissue explant disks (3 mm in diameter and 1 mm thick) were isolated from the meniscal bottom surface (facing the tibial articular cartilage). Mean GAG content of freshly isolated explants was 14.2 ± 0.8 μg/mg wet weight (n = 8). After three days of culture, 4.8 ± 0.3 μg/mg of GAG was released into the media in control explants (normalised to the mean GAG content of fresh explants about one-third of explant GAG is being released during culture). Stimulation with TNFα induced a dose-dependent increase in GAG release: using a concentration of 1 ng/ml caused an additional but non-significant increase in GAG release of approximately 8.8 ± 3.7% compared with control release. With 10 ng TNFα/ml, GAG release increased significantly by 30 ± 12% (n = 11), and 100 ng TNF α/ml (chosen for all subsequent experiments; Figure 1a) increased GAG release significantly by 24 ± 10% (n = 11). In order to distinguish between the release of existing GAG or newly synthesised GAG, radiolabeled sulfate was incorporated after cytokine treatment. TNFα induced a significant reduction in sulfate uptake (controls: 100 ± 12% vs TNFα: 55 ± 11%; n = 4), suggesting that the TNFα-dependent increase in media GAG content must be predominantly the result of an increased matrix degradation, rather than an increased biosynthetic activity.

TNFα induced a dose-dependent (not shown) and significantly increased production of NO in meniscal explants which increased about four-fold in comparison to the un-stimulated control (Figure 1b). The NO-synthetase inhibitor L-NMMA reduced the basal NO production of the tissue significantly and prevented the TNFα-mediated increase in NO completely.

It has been described that proteoglycan degradation in cartilage tissues can be mediated by both the production of NO and the involvement of matrix-degrading enzymes. We therefore studied the influence of the NO-synthetase inhibitor L-NMMA on meniscal tissue. L-NMMA had no significant influence on the basal GAG release and did not reduce the TNFα-induced effect (Figure 1a). There was a slight, but not significant, increase of GAG release instead. In order to support the hypothesis that aggrecanases are involved in TNFα-dependent GAG release, we studied the influence of TIMP-1, -2 and -3. TIMPs are known as specific inhibitors of MMPs, but it has been reported that TIMP-3 has the additional ability to inhibit the aggrecanases ADAMTS-4 and -5 2627. TIMPs did not affect the GAG release in control cultures (not shown). However, the TNFα-induced GAG release was significantly reduced by TIMP-3 by approximately 52% (Figure 1c), whereas TIMP-1 and TIMP-2 showed a trend to increase the TNFα-induced GAG release, although this effect was not significant.

To further determine the mechanisms of TNFα-dependent GAG release, the mRNA of meniscal explants was analyzed after a three-day incubation by quantitative RT-PCR. GAPDH had been used as a reference gene, and it had been tested that there is no significant alteration in the C_T values of GAPDH expression under the influence of TNFα (control: 27.1 ± 1.7 versus TNFα: 27.3 ± 0.9; n = 4 independent experiments). Additionally, GAPDH expression had been tested in relation to another housekeeping gene, 18sRNA: the ratio of GAPDH expression remained unaffected under the influence of TNFα (1.03).

The mRNA levels of most of the genes tested were quite variable under the influence of TNFα except for the matrix-degrading enzymes MMP-3 and ADAMTS-4 (see below). Collagen type I mRNA was decreased in all cases (0.75 ± 0.15), while aggrecan and collagen type II as well as MMP-1 and MMP-13 showed both increases and decreases depending on the experiment. ADAMTS-5 was not detectable in some cases or not increased by TNFα. MMP-3 and ADAMTS-4 showed a mean TNFα-dependent 6.9 ± 2.1 and 3.7 ± 0.8-fold increase of mRNA expression (Figure 1d). Comparing delta-C_T-values (C_TGAPDH - C_{Tgene of interest}) of controls and TNFα-stimulated meniscal explants allows a statistical analysis and showed a significant mean change of about 2.5 ± 0.58 for MMP-3 and 1.86 ± 0.16 for ADAMTS-4, indicating a clear up-regulation of these enzymes in all four independent experiments. The TNFα-dependent MMP-3 expression was also detectable in the supernatants of the cultures by casein zymography (Figure 2). There was only one band detectable in the gels, which was missing or expressed at lower levels in controls, but strong in TNFα-stimulated cultures. This band was not visible in gelatin zymograms (not shown), and had a molecular size of about 57 kDa (typical size for MMP-3, 28). TIMP-3 as well as L-NMMA had no influence on the band intensity. However, the enzyme activator substance APMA altered the size of the band, indicating that most of the enzyme was expressed as a pro-form 28.

Immunostaining of the aggrecanase activity-specific aggrecan neoepitope NITEGE showed very low signals in control tissue with a clear TNFα-dependent increase in staining in all meniscal tissue areas that could be characterised as fibrous cartilage (Figures 3a and 3d). Co-incubation with the NO-synthetase inhibitor L-NMMA failed to influence the TNFα-dependent NITEGE formation (Figures 3c and 3f), whereas TIMP-3 clearly inhibited this effect (Figures 3b and 3e).