All tissue samples were graded according to a modified Mankin scale 25, with a score of less than 3 points being normal and a score of greater than 5 representing OA 26. Normal articular cartilage was harvested from femoral condyles and tibial plateaus of human tissue donors under approval of the Scripps Human Subjects Committee. Osteoarthritic cartilage was obtained from patients undergoing knee replacement surgery. The thickness of these cartilages ranged from 1.5 to 2.8 mm. Once cartilage surfaces were rinsed with saline, scalpels were used to cut parallel sections 5 mm apart, vertically from the cartilage surface onto the subchondral bone. These cartilage strips were then resected from the bone. Human chondrocytes were isolated and cultured as described previously 27. The cartilage tissue was incubated with trypsin at 37°C for 10 minutes. After the trypsin solution was removed, the tissue slices were treated for 12 to 16 hours with type IV clostridial collagenase in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal calf serum. After initial isolation, the cells were kept in high-density cultures in DMEM (high glucose) supplemented with 10% CS, L-glutamine, and antibiotics and allowed to attach to the surface of the culture flasks. After the cells had grown to confluence, they were split once (passage 1) and grown to confluence again for use in the experiments.

RNA was isolated from fresh frozen cartilage by homogenizing the tissue in a freezer mill (Spex CertiPrep, Inc., Metuchen, NJ, USA) and extracting the homogenate in Trizol (Life Technologies, Inc., now part of Invitrogen Corporation, Carlsbad, CA, USA). The samples were extracted with chloroform and centrifuged at 15,000 g for 20 minutes, and the aqueous phase was collected. An equal volume of 70% ethanol was added, mixed, and applied to RNeasy columns (Qiagen Inc., Valencia, CA, USA). RNA concentrations were determined using RiboGreen reagent (Molecular Probes Inc., now part of Invitrogen Corporation). Total RNA was isolated from chondrocyte cultures plated at confluence at 3 × 10⁶ cells per 100-mm plate using the RNeasy kit (Qiagen Inc.) with on-column DNA digestion. Complementary DNA was produced using the SuperScript III First-Strand kit (Invitrogen Corporation) with random hexamers.

Sulf-1 and Sulf-2 primers and conditions for reverse transcription-polymerase chain reaction (RT-PCR) were based on the protocol of Morimoto-Tomita and colleagues 17. Real-time RT-PCR with SYBR green detection was performed using an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as follows: 2 minutes at 50°C and then 10 minutes at 95°C for initial denaturation, followed by 40 cycles at 95°C (15 seconds), 60°C (1 minute), followed by the measurement of fluorescence at the end of each cycle. Each run included a melting curve to determine the correct response of the primers 28. The following primers were used: Sulf-1: forward 5'-AGACCTAAGAAT CTTGATGTTGGAA-3', reverse 5'-CCATCCCATAACTGTCCTCTG-3'(74 base pairs [bp], NM15170), Sulf-2: forward 5'-TGAGGGAAGTCCGAGGTCAC-3', reverse 5'-CTTGCGGAGTTTCTTCTTGC-3' (194 bp, NM018837, NM198596), glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward 5'-ACCCACTCCTCCACCTTTGA-3', reverse 5'-ATGAGGTCCACCACCCTGTT-3'.

Primers were selected in consideration of the low homology between the sequences of Sulf-1 and Sulf-2. Furthermore, human Sulf-2 primers were designed to detect both Sulf-2 splice variants, NM 018837 and NM 198596. The specificity of detection of Sulf-1 and Sulf-2 was confirmed by sequencing the PCR products after isolation with the QIAquick gel extraction kit (Qiagen Inc.). Changes in Sulf gene expression were calculated relative to GAPDH.

Cartilage tissues were fixed with 4% paraformaldehyde and stained with safranin O. Sulf antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Paraffin-fixed samples were first deparaffinized in xylene substitute Pro-Par Clearant (Anatech Ltd., Battle Creek, MI, USA), ethanol then water for rehydration. After washing with PBS, sections were blocked with 0.1% Tween20 with 3% normal goat serum for 30 minutes at room temperature. Sulf-1 and Sulf-2 antibodies (2 μg/mL) and normal mouse IgG (1 μg/mL) as negative control were applied and incubated overnight at 4°C. After washing with PBS, sections were incubated with biotinylated goat anti-mouse secondary antibody for 30 minutes (1:200; Vector Laboratories Inc., Burlingame, CA, USA) and then incubated with Vectastain ABC-AP kit (AK-5000; Vector Laboratories Inc.) for 30 minutes at room temperature. Finally, sections were stained with an alkaline phosphatase substrate kit (Vector Laboratories Inc.).

Sulf-1 and Sulf-2 localization throughout each cartilage zone was assessed systematically by counting positive and negative cells in a 50 × 50 μm grid (using a ×40 field objective) starting from the cartilage surface to the deep zone. This was repeated a minimum of five times for each section. The identification of each zone was based on previously reported characteristics that comprise cell shape, morphology, orientation, and pericellular matrix (PM) deposition 29. Thus, superficial zone (SZ) cells were characterized by their elongated shape, their parallel orientation relative to the surface, and lack of extensive PM. These cells predominate within the first 50 μm. The middle zone (MZ) was distinguishable by rounded cells that did not exhibit an organized orientation relative to the surface, that have ECM rich in proteoglycans, and that show the presence of PM. Conversely, deep zone (DZ) cells were recognized with an extensive PM deposition and organized in columns of chondron groups of three or more cells. The depth of each zone was recorded for each section for comparative analysis on the frequency of positive signal in each zone. The frequency of positive cells was expressed as a percentage relative to the total number of cells counted in each zone.

Cartilage was cut into 1-mm-thin slices, and 200 to 1,000 mg of frozen cartilage was pulverized in a liquid nitrogen-cooled freezer mill for two cycles of 1.5 minutes at the rate of maximum impact frequency. Dry weight of normal and OA cartilage was measured and the same amount of protein was resuspended in SDS gel loading buffer (50 mM Tris pH 6.8, 10% glycerol, 4% sodium dodecyl sulfate, 10% 2-mercaptoethanol, and 0.001% bromophenol blue) and mixed for 2 hours at room temperature. Centrifugation at 14,000 rpm was performed for 30 minutes and then supernatants were harvested and heated at 80°C for 10 minutes. The concentrated samples were then adjusted for equal volumes before resolution on 12% Tris-Glycine gels (Invitrogen Corporation). Protein was transferred to nitrocellulose membranes (Invitrogen Corporation), blocked with 5% dry milk in Tris-buffered saline–Tween (TBST), and blotted with mouse polyclonal antibody specific for Sulf-1 or Sulf-2 (Abcam Inc.) for 1 hour. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 hour. Afterwards, the membranes were washed three times with TBST and developed using the enhanced chemiluminescent substrate from Pierce (Rockford, IL, USA).

All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at The Scripps Research Institute (La Jolla, CA, USA). Sulf-1 and Sulf-2 expression was analyzed by immunohistochemistry in temporomandibular (TM) joints and knee joints of 1-, 6-, 9-, and 12-month-old C57BL/6J mice. Each mouse joint was cut in half along the mid-sagittal plane and fixed in 10% zinc-buffered formalin (Z-Fix; Anatech Ltd.) for 2 to 3 days and then decalcified in Shandon TBD-2 decalcifier (Fisher Scientific Pittsburgh, PA, USA) for 2 to 3 weeks. Three-millimeter serial sections (from posterior to anterior) were cut and immunostained for Sulf-1 and Sulf-2 as described above.

Statistically significant differences between two groups were determined with t tests. The results are reported as mean ± standard deviation. P values of less than 0.05 were considered significant.

Sulf-1 and Sulf-2 mRNA expression in eight OA donors (49 to 68 years old; Mankin score: 7 to 10 points) was significantly higher than in eight young donors (19 to 37 years old; Mankin score: 0 to 2 points) as determined by real-time PCR (Figure 1).

Young and old normal samples as seen on safranin O staining (Figure 2a, d, g) had only a few Sulf-positive cells in the superficial zone (Figure 2b, c, e, f, h, i) and no positive cells in the middle and deep zones. In general, the expression of Sulf-2 appeared more intense than Sulf-1 in normal cartilage. In OA cartilage, many positive cells were detected, especially in chondrocyte clusters (Figure 3g, h, k, l). The representative example of 65-year-old cartilage had both normal areas (Mankin score: 2) (Figure 3a, b) and OA areas with fibrillations and cluster formation (Mankin score: 8) (Figure 3c, d). The normal appearing areas from OA joints had 18.5% Sulf-1-positive and 31.9% Sulf-2-positive cells in the superficial zone (Figure 3e, f, i, j), which was greater than in normal cartilage (Figure 2). On the other hand, OA areas had 75.3% Sulf-1-positive and 73.2% Sulf-2-positive cells (Figure 3g, h, k, l).

Figure 4 shows quantitative analysis of the zonal distribution of Sulf-1- and Sulf-2-expressing cells in eight normal (17 to 37 years old) and eight OA (43 to 82 years old) donors. In OA, the superficial zone was already eroded. The middle zone in OA cartilage had significantly more positive cells than normal (*P < 0.01). Moreover, the number of Sulf-2-positive cells in the superficial and middle zones was greater than Sulf-1-expressing cells (P = 0.02).

Western blotting was performed to visualize Sulf proteins and determine differences in the expression between normal and OA. In total protein extracts from normal cartilage, Sulf-1 and Sulf-2 were not detectable. In contrast, high levels of Sulf-1 and Sulf-2 protein were detected in OA cartilage (Figure 5). The major Sulf-1 and Sulf-2 protein bands migrated at approximately 72 kDa, which is the molecular mass of the secreted proteins 173031.

TM joints from normal C57BL/6J mice (n = 6) were analyzed with safranin O staining (Figure 6a–c) and immunohistochemistry for Sulf-1 and Sulf-2 (Figure 6d–i). Histology showed thinning and reduced cell density in articular cartilage with increasing age (Figure 6c). In 6-month-old mice, only a few cells were positive for Sulf-1 but Sulf-2-positive cells were present throughout the cartilage. There was a marked increase in Sulf-2 expression at 9 months and in Sulf-1 expression at 12 months. In murine knee joints (n = 8), there was high Sulf expression at 1 month of age, followed by a decrease with joint maturation. Increased expression of Sulf-1 and Sulf-2 was seen in the articular cartilage of murine knee joints by 12 months of age, when early OA-like changes become apparent (Figure 7).