Chemical reagents were purchased from Sigma (Munich, Germany), unless otherwise indicated.
Samples of synovial fluid from patients with OA (n = 8) were obtained from the Department of Orthopaedic Surgery at the University Hospital Schleswig-Holstein (Kiel, Germany). Synovial fluid from healthy joints was collected from deceased donors (n = 5) at the Department of Anatomy, Christian-Albrechts-University (Kiel, Germany).
Chondrocyte monolayer culture
The human C28/I2 chondrocyte cell line was used in monolayer culture. These chondrocytes, which were immortalized using SV-40 large T-antigen, continue to express chondrocyte-specific aggrecan and collagen type II mRNA after multiple subculture [21
]. Chondrocytes were seeded (500,000 cells/25 cm2
) in DMEM supplemented with 10% foetal bovine serum, 10 mM h-(2-hydroxyethyl)-1-piperazinethansulfonacid (HEPES) buffer, 1 mM sodium pyruvate, 0.4 mM proline, 20 μg/ml ascorbic acid, 100 U/ml penicillin G, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. After reaching 80% confluence, the cells were rinsed twice with HANK's solution and placed in serum-free medium, containing 0.05% BSA, for subsequent stimulation.
Human articular cartilage explants
The tissue harvest was approved by the Ethical Commission of Christian-Albrechts-University of Kiel (Kiel, Germany). Knee cartilage was obtained from the Institute of Pathology, Christian-Albrechts-University, (Kiel, Germany) as post-mortem donor tissue. Donors who were 75 years of age or older were excluded; the average age of the donors was 54 years. Knee cartilage was scored, using a modified Collins scale [22
], for visual degeneration of grade 2 or less, otherwise the sample was rejected.
Cartilage–bone cylinders (11 mm in diameter) from the femoral condyles and femoropatellar groove were punched out perpendicular to the cartilage surface using Arthrex® (Arthrex GmbH, Karlsfeld, Germany) instruments for osteochondral transplantation (T-handle bar and punch; AR-1980D-11). The cartilage–bone samples were removed, rinsed in HANK's solution (supplemented with antibiotics; see below) and placed in a microtome holder. After creating a level surface by removing superficial tissue, the cartilage tissue was sliced at a thickness of 1 mm. Finally, up to eight explant discs (measuring 3 mm in diameter and 1 mm in thickness) were punched from each slice. In all subsequent experiments, treatment groups were location-matched by distributing the explant discs from a single slice to each of the different groups.
Cartilage explants were equilibrated for 2 days in culture medium (250 μl of high-glucose DMEM with supplements (as above) per explant in a 96-well plate) under free-swelling conditions at 37°C in a standard cell-culture environment. Then, cartilage explants were rinsed twice with HANK's solution and placed in serum-free medium, containing 0.05% BSA, for subsequent stimulation. For each group, we used eight cartilage explants in five different experiments.
PMA was used at concentrations of 5, 10 and 20 μg/ml in medium. SIN-1 concentrations were 1, 10 and 20 μM. Chondrocytes in monolayer culture were exposed to PMA or SIN-1 stimulation for 48 hours and cartilage explants were similarly stimulated for 72 hours.
Isolation of RNA and cDNA synthesis
Total RNA was extracted from immortalized chondrocytes using an RNeasy Total RNA Kit (Qiagen, Hilden, Germany). Total RNA from tissue homogenates of cartilage explants was extracted using the TriZOL Reagent (Invitrogen, Life Technologies, Karlsruhe, Germany). DNA contamination was destroyed by digestion with RNase-free DNase-I (20 minutes at 25°C; Boehringer, Mannheim, Germany), and cDNA was generated from 100 ng RNA reacting with 1 μl (20 pmol) of oligo(dT)15 primer (Amersham Biosciences, Amersham, UK) and 0.8 μl of superscript RNase H-reverse transcriptase (Gibco, Paisley, UK) in 50 μl total volume for 60 minutes at 37°C. For each sample, a control without reverse transcriptase was run in parallel to enable assessment of genomic DNA contamination.
RT-PCR for VEGF splice variants
For PCR, 4 μl of cDNA was incubated with 30.5 μl water, 4 μl 25 mM MgCl2, 1 μl deoxynucleoside-triphosphate, 5 μl 10 × PCR buffer, 0.5 μl (2.5 U) Platinum Taq DNA polymerase (Gibco) and 2.5 μl (10 pmol) of each primer pair. The following primers and conditions were applied: VEGF splice variants, 5'-CCA-TGA-ACT-TTC-TGC-TGT-CTT-3' (sense) and 5'-TCG-ATC-GTT-CTG-TAT-CAG-TCT-3' (antisense), with 40 cycles performed at a 55°C annealing temperature. A glyceraldehyde-3-phosphate-dehydrogenase (G3PDH)-specific primer pair (5'-ATC-AAG-AAG-GTG-GTG-AAG-CAGG-3' (sense) and 5'-TGA-GTG-TCG-CTG-TTG-AAG-TCG-3' (antisense), with 40 cycles at 58°C) served as the internal control (983 bp).
Quantitative real-time RT-PCR for VEGF, VEGFR-1 and VEGFR-2
Real-time RT-PCR was carried out using a one-step system, according to the manufacturer's instructions (QuantiTect SYBR Green RT-PCR; Qiagen), with 100 ng of total RNA in an i-Cycler (Biorad, Munich, Germany). The temperature profile included an initial denaturation for 15 minutes at 95°C, followed by 37 cycles of denaturation at 95°C for 15 seconds, annealing at a temperature of 60°C for 30 seconds, elongation at 72°C (the elongation time depended on the size of the fragment, that is the number of bp divided by 25 yielded the time in seconds) and fluorescence monitoring at 72°C. Each cDNA sample was analysed for expression of the gene of interest, in addition to G3PDH, with the fluorescent TaqMan 5'-nuclease assay, using 2 × TaqMan Master Mix (Applied Biosystems, Foster City, CA, USA) and 20 × assay-on-demand TaqMan primers and probes in a total volume of 20 μl. Each plate included no-template controls (NTCs). TaqMan human primers and probes had the following identification numbers: VEGF, Hs00173626_m1; VEGFR-1, Hs00176573_m1; VEGFR-2, Hs00176676_m1; and G3PDH, Hs99999905_m1. The cycle of threshold (CT) for each sample was averaged and normalized to G3PDH. The results were then analysed by comparative ΔΔCT method (2(-ΔΔCT)) for relative quantification of gene expression:
ΔΔCT = ΔCT (sample) - ΔCT (control)
ΔCT (sample) = CT (sample; target) - CT (sample; G3PDH)
ΔCT (control) = CT (control; target) - CT (control; G3PDH)
After stimulation with PMA or SIN-1, the conditioned medium supernatant of each chondrocyte monolayer or cartilage explant culture was collected. Aliquots were analysed using a sandwich ELISA (R&D Systems, Minneapolis, MN, USA) to detect VEGF, and signals were identified by a chemoluminescence reaction (ECL-Plus; Amersham-Pharmacia, Uppsala, Sweden). Human recombinant VEGF165 (Repro Tech, Rocky Hill, NJ, USA) served as an internal standard. Aliquots of synovial fluid samples from OA patients were analysed by an identical procedure. VEGF concentrations were normalized using Bradford reagent (Roti-Quant; Roth, Karlsruhe, Germany).
Concentrations of nitrite, the stable end product of NO, were analysed in the culture medium using Griess reagent, according to the protocol described by Ailland and coworkers [23
]. Results were corrected for the nitrite content of pure medium with or without (blanks) the PMA or SIN-1 stimulants. Data were calculated according to the amount of medium and normalized to the number of cells or cartilage wet weight (monolayer or tissue explants, respectively) and control group, which was set at 100%.
All data are shown as mean ± standard error of the mean (SEM), unless indicated otherwise. Differences between analysed data were tested using the Student t test. Significance was set to p value of < 0.05.