Cartilage explant culture and measurement of GAG synthesis Full-depth human articular cartilage slices were washed in serum-free DMEM-F12 medium containing antibiotics, then diced into 1–2-mm3 pieces and maintained in a 24-well culture plate (20–30 mg cartilage/well) in 900 ml of DMEM-F12 and 100 ml of PBS, containing 0.1% (w/v) BSA. After 48 h of equilibration, explants were radiolabeled with 10 mCi/ml 35S- sulfate (Perkin Elmer, Courtabœuf, France) for 6 h. Explants were then digested in papain and aliquots of 35S-labeled digest were precipitated by using cetylpyridinium chloride [18], and the sulfated GAG synthesis rate was measured by liquid scintillation counting. Protein extraction and enzymatic activity of XT-I in human cartilage In brief, cartilage explants were cryosectioned into 10-mm slices using a freezing microtome (Microm HM 440 E) at 220uC. Slices were homogenized using a glass Teflon in cold extraction buffer (500 mM NaCl, 50 mM HEPES, pH 7.2, containing proteinase inhibitor cocktail) at a ratio of 10:1 (buffer:tissue) on ice [19]. Protein extraction was carried out at 4uC overnight. The resulting mixture was clarified by centrifugation at 3,000 rpm for 5 min at 4uC. The supernatant was used for measurement of XT-I activity based on the binding of [14C]xylose to recombinant bikunin (acceptor substrate), as reported by Weilke et al [20]. GAG chain analyses by fluorophore-assisted carbohydrate electrophoresis (FACE) Disaccharide composition of chondroitin sulfate chain was examined on proteinase K-digested cartilage explants by FACE method as described previously [21]. Aliquots containing 5 mg of GAGs, as judged from uronic acid assay [22] from each cartilage sample, were digested with chondroitinase ABC and the disaccharides obtained were fluorotagged and derivatized [21]. Electrophoresis was carried out at 4uC by using miniature vertical slab gels of 29% acrylamide and 1% bisacrylamide at 5 W of constant power per gel. Fluorescent bands were immediately scanned in a UV-light box Gel Doc System (Bio-Rad, Marne la Coquette, France) and quantitated with Bio-Rad QUANTITY ONE software. CS disaccharide standards were from Dextra Laboratories (Reading, U.K.). Western blot analysis Total protein from chondrocytes transfected with pCMV-XT-I or pCMV empty vector was extracted 24 h posttransfection and quantified using Bradford method [23]. Proteins (30 mg/lane) were separated on 12% SDS-PAGE, transferred onto ImmobilonH (Millipore, Molsheim, France) membrane and then probed with anti-XT-I antibody (Sigma). Statistical analysis Since OA is a focal disease and because of the reported variations in composition and bioactivity of the cartilage depending on the site studied, the findings of 3 cartilage samples per parameter per joint per patient were averaged and considered as a representative value of that subject [24]. Differences between cartilage samples taken from the lesion, from sites adjacent to the lesion, and from sites distant from the lesion were analyzed by Student’s t-test (within individual knees) and by Wilcoxon’s matched pairs test (between groups) with values of p,0.05 were considered significant. XT-I and Proteoglycan Synthesis in Osteoarthritis Results Characterization of lesional and non-lesional areas of human OA cartilage Previous studies of human OA have compared late OA cartilage obtained from joint replacement surgeries to non-OA cartilage from trauma subjects or at autopsy. However, these samples may be collected from subjects that are not corresponding according to age, genetic background or other features, including clinical management of the disease [25]. Also, it is well known that the pattern of disease progression clinically may differ from one patient to another. An alternative method is to evaluate lesional and non-lesional cartilage from neighbouring areas of the same joint [25–27]. Three tissue subgroups were objectively segregated according to their degree of degeneration in the same joint. To assess disease severity and cartilage damage part of the cartilage biopsy was processed for histology and graded according to Mankin’s scale [15] (surface fibrillation, matrix depletion, cellularity, and the integrity of the tidemark) with a maximum score of 11. No tidemarks were present in our cartilage samples. Cartilage samples taken from degenerative lesions were designated ‘‘late disease’’ (grade 8–11). The sample taken from sites immediately adjacent to lesions was designated ‘‘early disease’’ (grade 3–7) and sample isolated from macroscopically unaffected region and remote from the lesion was termed ‘‘normal’’ (grade 1– 2). Cartilage was taken from the same topographical region of the medial condyle and a representative sample of these tissue groups is illustrated in Fig. 1. Adaptive increases in sulfated-GAG synthesis in human early OA cartilage There was a significant increase (1.5- to 3.5-fold) in the rate of sulfated-GAG synthesis in cartilage samples recovered near the site of the lesion (early disease) compared to the samples taken from Figure 1. Representative sample of femoral condyle from human OA knee and toluidine blue stained sections. (A) Femoral condyle from human OA knee joint indicating three sites biopsied for further biochemical and molecular analysis; (a) cartilage sample taken from macroscopically unaffected area and distant from the lesion was termed ‘‘normal’’ (b) cartilage sample isolated from areas immediately close to lesion was termed ‘‘Next to lesion’’; (c) cartilage specimen excised from lesion was termed ‘‘late-stage OA’’; Cartilage samples were isolated using a 6 mm-biopsy punch. (B) Representative photomicro- graphs of cartilage sections showing areas from where samples were recovered for toluidine blue staining; (a) cartilage section from a normal area showing a relatively smooth articular surface; (b) section from areas close to lesion showing dense staining for PGs in mid zone of the cartilage where chondrocyte proliferation and activation occurs; (c) cartilage section from lesions showing degraded articular surface, loss of PG staining and chondrocyte cloning (original magnification 640). doi:10.1371/journal.pone.0034020.g001 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34020