All procedures were performed with Hadassah Medical Center Institutional Review Board approval and in accordance with the Helsinki Declaration of ethical principles for medical research involving human subjects. Formal written informed consent was obtained from 30 donors with OA, who were undergoing total knee arthroplasty (mean age 72 years, mean body mass index (BMI) 31.5 kg/m2), prior to obtaining articular cartilage samples from their knee joints. Age-matched non-OA cartilage was obtained from NDRI, Philadelphia.
Serum and synovial fluid were obtained from non-OA donors and patients with early OA, late OA and RA, as partly described previously , following institutional approval by the local ethics committee of Justus-Liebig-University of Giessen, and obtaining written informed consent from all participants.
SF was obtained postmortem from the knee joints of 11 donors with no documented history of joint disease. The average (av) age of the non-OA donors was 22.5 years and the av BMI was 22.6. The control subjects were examined at the Institute of Forensic Medicine, Justus-Liebig-University of Giessen. Serum samples were obtained from 10 healthy donors (av age 22.5 years, BMI 22.6, 40% male).
SF (total of 32 samples) and serum (total of 20 samples) were ranked for OA severity based on the macroscopic appearance of six cartilage surfaces , namely the patella, trochlea, and medial and lateral femur and tibia, using the Outerbridge scale . Joints with an av Outerbridge score ≤2 were classified as having early OA, and joints with an av Outerbridge score of ≥2 were classified as having late OA . We collected serum from early OA (10 patients, av age 47.3 years, av BMI 27.6, 70% male), SF from early OA (17 patients, av age 44.8 years, av BMI 26.8, 75% male), serum from late OA (10 patients, av age 68.3 years, av BMI 28.2, 60% male), SF from late OA (15 patients, av age 67 years, av BMI 29.4, 69% male). SF was also collected from RA patients (10 patients, av age 64.6 years, av BMI 27.3, av serum C-reactive protein (CRP) 25.9 mg/L, 70% male) who were diagnosed by a board-certified rheumatologist, according to the American College of Rheumatology (ACR) 1987 revised criteria .
Serum samples were filtered with a 50-kDa cutoff amicon filter (Millipore, Billerica, MA, USA) and then labeled with our published cathepsin activity-based probe, GB123 (specified below) at 2 μM concentration for 3 hours. Reaction was stopped with sample buffer and 24-μg proteins were analyzed by SDS PAGE. Synovial fluids, 10 μL, were labeled with 5 μM GB123 for 3 hours, and then the reaction was stopped with sample buffer and analyzed by fluorescent SDS PAGE scanning using a Typhoon flatbed scanner (GE Healthcare: Bio-Sciences AB, Uppsala, Sweden, Excitation/Emission 630/670 nm). GB123 fluorescently labeled cathepsins B, L and S bands represent enzyme activity in proportion to band intensity, not enzyme quantity, as described previously .
Cells, tissue cultures and reagents
Freshly isolated chondrocytes were obtained from intact and degenerative regions of OA-derived cartilage, dissected and subjected to 0.25% Trypsin-EDTA solution (Beit-Haemek Kibutz, Israel) at 37°C, 60 minutes rotation, then washed with PBS twice and overnight digested with 0.02% collagenase (Worthington Biochemical, Lakewood, NJ, USA) in DMEM containing 10% FCS and 1% Penicillin-streptomycin (Biological Industries, Beit-Haemek Kibutz, Israel). On the following day the cells were filtered through 40-μm nylon mesh, washed twice with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (PBS, 1% NP-40, 0.5% Deoxycholate (DOC), 0.1% SDS). Proteins were quantified using the Bradford assay and 50 μg of proteins from each sample were mixed 1:1 with acetate buffer pH 5.5 (10 mM MgCl2, 8 mM DTT, 100 mM Acetate) and with 1 μM GB123 (final concentration) for 1 hour at 37°C for cathepsins labeling. Intact cartilage (IC) and degraded cartilage (DC) supernatants were treated with collagenase and directly labeled with 1 μM GB123 for 5 hours at 37°C. Supernatants were filtered using a 50-kDa cutoff amicon filter (Millipore, MA), proteins were precipitated with 70% acetone at −80°C. On the following day the samples were centrifuged for 15 minutes at 14,000 rpm, pellets were resuspended in 35 μl RIPA buffer, and 30 μg of proteins were separated by SDS-PAGE.
To test if cathepsin S or B were modified in the presence of collagenase digestion media, 0.5 μg recombinant human cathepsin B or S , were incubated (1h or overnight) with collagenase media at 37°C and analyzed for cathepsin activity and protein levels. GB123 potency towards human cathepsin B, K and S was determined by labeling 0.5 μM active recombinant enzyme with 1 μM GB123 for 1 hour as indicated above.
Human chondrocytes were isolated from intact cartilage zones as indicated above and cultured to reach 90% confluence (passage 0 or 1) in DMEM media containing 10% FCS, 1% Penicillin-streptomycin at 37°C, and 5% CO2. Medium was then replaced with BIO-MPM (Biological Industries), a defined and enriched growth medium containing Penicillin-streptomycin with or without 50 ng/mL TNFα and/or 5 ng/mL IL1β (PeproTech, Rockey Hill, NJ, USA) for 24 hours. Cells were pretreated with a specific cathepsin inhibitor GB111-NH2  or vehicle for one hour prior to direct labeling with 5 μM GB123. Then the cells were lysed in RIPA buffer and medium proteins were precipitated with 70% acetone as indicated above. Samples were analyzed by SDS PAGE or immunoprecipitated (IP) with cathepsin S antibody (R&D Systems, Minneapolis, MN, USA, cat#AF1183). To examine cathepsin B labeling (that is, 32 kDa), cells were lysed in RIPA and an equal amount of proteins were pre-incubated for 1 hour with 5 μM CA-074 (Sigma Aldrich, St Louis, MO, USA), a specific cathepsin B inhibitor, or DMSO vehicle at 37°C. Then 1 μM GB123 was added for an additional hour at 37°C. The reaction was stopped by adding sample buffer, followed by SDS PAGE. All samples were separated on a 12.5% SDS PAGE gel and scanned for fluorescence using a Typhoon flatbed scanner.
Three-dimensional alginate cultures were obtained with passage-1 human chondrocytes, treated with cytokines and processed for mRNA isolation, as previously described . Explants cultures were obtained from intact or degenerated regions of cartilage derived from OA patients and dissected perpendicular to subchondral bone into round 4-mm-diameter cartilage tissue samples (weight 6 to 9 mg each) and analyzed for GB123 fluorescence.
Cell pellets were suspended with RIPA lysis buffer with complete inhibitor cocktail (Roche, Basel, Switzerland), quantified with Bradford reagent (Sigma-Aldrich, St Louis, MO, USA), run on standard 10% SDS PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes for immunoblot (IB). The following antibodies were used for IB: β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA; cat#47778), cathepsin S (Abcam, Cambridge, UK cat#134157), cathepsin B (Abcam cat# ab58802); Secondary antibodies: Anti-Mouse-alkaline-phosphatase (AP)-conjugated (Sigma-Aldrich, cat#, A3562), Anti-rabbit-AP conjugated (Sigma-Aldrich, cat#, A3687). All IBs were scanned at high resolution and band intensity was determined using Image J software (NIH, USA). After subtracting the background, each band was normalized to the corresponding housekeeping protein (that is, β-actin) appearing on the blot. Band intensity was presented in arbitrary units (A.U.) adjacent to the representative IB.
Fluorescent labeling of explant sections
Explants were embedded in optical cutting temperature (OCT) and snap-frozen in liquid nitrogen prior to cryosection; and 10-μm sections were obtained and embedded on glass slides, fixed with 4% paraformaldehyde (diluted with PBS) for 30 minutes at 4°C, followed by washing three times with PBS for 5 minutes. Sections were labeled with GB123 (1 μΜ, 37°C, 30 minutes) with or without pre-incubation with GB111-NH2 inhibitor (1 μΜ, 37°C, 30 minutes). Slides were washed twice with PBS and mounted with 4′: 6-diamidino-2-phenylindole (DAPI)-fluoromount-G (SouthernBiotech, Birmingham, AL, USA). The stained sections were visualized under an inverted fluorescent microscope with DAPI and Cy5 lasers and filters (Olympus FV10i inverted microscope, Tokyo, Japan).
IC/DC tissues were fixed in 4% formalin and decalcified for 3 days in 4% formic acid/4% HCl and embedded in paraffin: 5-μm sections were digested with 1 mg/mL Hyaluronidase (Sigma Aldrich, St Louis, MO, USA) in PBS (pH = 6) for 1 hour at 37°C, and stained with anti-cathepsin S antibody and DAB substrate kit (Zytomed systems, Berlin, Germany).
Quantitative polymerase chain reaction (qPCR) analysis
mRNA was isolated using RNeasy mRNA purification columns (Qiagen, Hilden, Germany). cDNA was then prepared using the OneStep RT-PCR kit according to the manufacturers guidelines (Invitrogen Carlsbad, CA, USA). Real-time PCR reactions were performed using an ABI qPCR model 7300 or 7900 (Applied Biosystems, Foster City, CA, USA) with purified samples containing a Syber Green mix (Applied Biosystems) in accordance to the manufacturers’ guidelines. Primers for quantitative PCR were prepared for the following human genes: Cathepsin S(CAT), forward: 5′-GACTGGAGAGAGAAAGGGTGTGTT-3′, reverse: 5′-CAGCTTTCCTGTTTTCAGCTTCA-3′, hβ2MG-F: ACCCCCACTGAAAAAGATGAG; reverse: ATCTTCAAACCTCCATGATGC; GAPDH-F: TACTAGCGGTTTTACGGGCG; R: TCGAACAGGAGCAGAGAGCGA.
All experiments were performed on multiple donor samples as indicated in each figure legend. Data points represent average and standard deviation, unless otherwise indicated. Statistical analysis was obtained using non-parametric Mann-Whitney analysis (two-tailed). P-values for plotted data ≤0.05 were considered statistically significant. Correlation between serum and SF cathepsin bands (imageJ, A.U.) were drawn for age per clinical group (that is, healthy/non-OA, early OA, late OA and RA) and amongst all groups, based on the Pearson coefficient (1 ≥ p ≥ −1), considering P ≤0.05 to be statistically significant. The values were also assessed by linear regression (R