OA chondrocytes were prepared from articular cartilage specimens collected from 6 end-stage OA patients (age range of 62-68) undergoing joint replacement surgery at Carolinas Medical Center with the approval of the authors’ Institutional Review Board. The need for informed consent was waived because those specimens were surgical waste, and no private patient information was collected. Articular cartilage was evaluated as described [24], and only grade 4 cartilages were used. Briefly, articular cartilage was removed from the medial tibia plateau with a surgical blade, minced into small pieces, and cultured in 60 mm plates at 37^oC in medium containing 10% serum and 0.5% antibiotic/antimycotic solution. Media was changed every 3 days. After chondrocytes reached to 70% confluence, they were passaged. Chondrocytes with 2-3 passages were used in the study.

OA chondrocytes derived from 3 OA patients were harvested from 60 mm cell culture plates, mixed, and re-plated in 60 mm plates at 90% confluence (passage 2; 9 x 10⁶ per plate). On the second day, medium containing 1% serum was added. On the third day, medium in 2 plates was replaced with medium containing 1% serum and 1 mm PC and medium in the other 2 plates were replaced with medium containing 1% serum without PC as a control. Twenty-four hours later, total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and purified using the Oligotex kit (Qiagen, Valencia, CA). The quality and integrity of these RNA samples were checked using RNA 6000 Nano Assay Reagent Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

RNA samples extracted from two independent experiments were used for microarray analyses. Briefly, double-stranded DNA was synthesized using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, San Diego, CA, USA) using 1 μg of each RNA sample. The DNA product was purified using a GeneChip sample cleanup module (Affymetrix, Santa Clara, CA, USA). CRNA was synthesized and biotin labeled using BioArray high yield RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY, USA). The cRNA product was purified using GeneChip sample cleanup module and subsequently chemically fragmented. The fragmented and biotinylated cRNA was hybridized to HG-U133_Plus_2.0 gene chip using Affymetrix Fluidics Station 400 (Affymetrix, Santa Clara, CA, USA). The fluorescent signals were quantified during two scans by Agilent Gene Array Scanner G2500A (Agilent Technologies, Palo Alto, CA, USA) and GeneChip operating Software (Affymetrix, Santa Clara, CA, USA). Genesifter (VizX Labs, Seattle, WA, USA) was used for the analysis of differential gene expression and gene ontology. The results of the two microarray experiments were similar, therefore, we focused on the results of the first microarray experiment in this study.

The RNA samples used in microarray experiments were used for real-time RT-PCR. Briefly, cDNA was synthesized using TaqMan^® Reverse Transcription Reagents (Applied Biosystems, University Park, IL) using 1 μg RNA sample. Quantification of relative transcript levels of selected genes and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed using TaqMan^® Gene Expression assay and ABI7000 Real-Time PCR system (Applied Biosystems, University Park, IL). CDNA samples were amplified with an initial Taq DNA polymerase activation step at 95^oC for 10 minutes, followed by 40 cycles of denaturation at 95^oC for 15 seconds and annealing at 60^oC for one minute. Fold change was calculated and the expression level of a specific gene was normalized to the expression level of GAPDH.

OA chondrocytes (2x10⁴) were plated in six-well cluster plates and cultured in medium containing 10% serum in the absence (triplicates) or the presence of 1 mM PC (triplets). The medium was changed every three days until the chondrocytes in the wells without PC reached to 85% confluence (it took about 14-16 days). Chondrocytes in all wells were harvested and cell numbers counted. This experiment was repeated three times using OA chondrocytes derived from 3 different OA patients. Trypan blue test was performed to examine the effect of PC on chondrocyte viability. Since foreskin fibroblasts were used in many previous studies to determine the effect of PC on crystal-induced mitogenesis and expression of MMPs [15, 25, 26], foreskin fibroblasts were also used here for comparison.

OA chondrocytes were plated in twenty-four well cluster plates at 90% confluence (7 x 10⁵ cells). On the second day, the medium was replaced with StemPro osteogenesis differentiation medium with or without 1 mm PC (triplicates). These chondrocytes were fed with StemPro osteogenesis differentiation medium with or without PC every three days. Fourteen days later, the media were removed and calcium deposition was examined using alizarin red. This experiment was repeated 6 times using OA chondrocytes derived from 6 different OA patients.

Micromasses were prepared as described previously [7, 27]. Briefly, OA chondrocytes were harvested from 60 mm culture plates and suspended in DMEM containing 10% serum. For preparing a micromass, a droplet of the cell suspension containing 2 x 10⁵ chondrocytes was placed in a well of a 24-well cluster plate. After placing all droplets, the plate was incubated for 4 hours at 37⁰C in a tissue culture incubator and each of the micromasses formed was then fed with StemPro chondrogenesis differentiation medium. Starting on the next day, these micromasses were fed with StemPro chondrogenesis differentiation medium with PC (1 mM) or without PC every three days.

Fourteen days later each well was rinsed twice with 500 μl of Hank’s balanced salt solution and two drops of eosin were added. Five minutes later, eosin was aspirated off and the micromasses were transferred to a strip of filter paper which sat on the top of an ethanol-soaked sponge within a plastic cassette. The cassettes sat in a 10% formalin solution for one hour. These micromasses then underwent routine paraffin embedding. Sections were cut at 5 μm thick and stained with picrosirius red for collagens and alcian blue for proteoglycans. This experiment was repeated 3 times using OA chondrocytes derived from 3 different patients.

Results are expressed as the mean ± standard error. The difference between the PC-treated and untreated group was analyzed using Student's t-test. It was considered significant when the p-value was < 0.01. Statistical analysis was performed using the statistical analysis tool in the Sigma Plot software, version 12 (Systat software Inc., San Jose, CA).

Of more than 50,000 transcripts, 558 transcripts displayed differential expression (more than 1.5 fold changes) in PC-treated OA chondrocytes compared to untreated OA chondrocytes. A total of 334 transcripts displayed decreased expression and 224 transcripts displayed increased expression. The genes that fell into specific biological processes implicated in OA or suspected to have a role in OA are listed in (Tables 1 and 2).

As shown in Table 1, the expression of numerous genes classified in proliferation and apoptosis was down-regulated by PC. Of the 39 differentially-expressed genes classified in cell proliferation, the expression of 30 genes, including lymphoid-specific helicase (HELLS, -2.32 fold) and interleukin 7 (IL-7, -1.82 fold), was down-regulated by PC. Of the 38 differentially-expressed genes classified in apoptosis, the expression of 30 genes, including sprouty homolog 2 (SPRY2, -1.92 fold) and baculoviral IAP repeat-containing 5 (BIRC5, -1.76 fold), was down-regulated by PC. In contrast, the expression of many genes classified in the transforming growth factor β (TGF-β) receptor signaling pathway and ossification were upregulated by PC. Of the 5 differentially-expressed genes classified in the TGF-β receptor signaling pathway, the expression of all 5 genes, including TGF-beta receptor 1 (TGFBR1, 1.74 fold), SMAD specific E3 ubiquitin protein ligase 1 (SMURF1, 1.57 fold), and SMAD family member 3 (SMAD3, 1.55 fold), was up-regulated by PC. Of the 7 differentially-expressed genes classified in ossification, the expression of 5 genes, including fibroblast growth factor receptor 2 (FGFR2, 1.99 fold), PDZ and LIM domain 7 (PDLIM7, 1.57 fold), and insulin-like growth factor binding protein 5 (IFGBP5, 1.53 fold), was up-regulated by PC. However, ectonucleotide pyrophosphatase 1 (ENPP1, -1.66 fold) and bone morphogenetic protein 2 (BMP2, -1.55 fold), two genes implicated in promoting ossification, was down-regulated by PC.

The expression of many genes classified in the inflammatory response, skeletal system development, and Wnt receptor signaling pathway was also downregulated by PC (Table 2). Of the 6 differentially-expressed genes classified in the inflammatory response, the expression of 4 genes, including fatty acid-binding protein 4 (FABP4; -2.11 fold), interleukin-1 receptor-associated kinase 2 (IRAK2, -1.71), and interleukin 1 receptor accessory protein (IL1RAP, -1.57), were down-regulated by PC of the 8 differentially-expressed genes classified in skeletal system development, the expression of 5 genes, including distal-less homeobox 2 (DLX2, -2.37 fold) and periostin (POSTN, -1.56 fold) was down-regulated by PC, of the 8 differentially-expressed genes classified in the Wnt receptor signaling pathway, the expression of 6 genes, including adenomatosis polyposis coli down-regulated 1 (APCDD1, -2.68 fold), norrie disease (NDP, -2.30 fold), and frizzled homolog 8 (FZD8, -1.58 fold), was down-regulated by PC.

Real-time RT-PCR was performed to confirm the differential expression of selected genes. As shown in Table 3, the differential expression of the genes examined was confirmed by real-time RT-PCR (P < 0.01).

The down-regulatory effect of PC on the expression of numerous genes classified in cell proliferation suggests that PC inhibits the proliferation of OA chondrocytes. Indeed, there were roughly 45% fewer OA chondrocytes in the wells containing PC compared to the wells containing no PC Fig. (1). The morphology of OA chondrocytes in the wells with and without PC was similar and trypan blue test showed little dead cells in both the untreated and PC-treated chondrocytes (not shown).

Fig. (1). Effect of PC on chondrocyte proliferation. Left bar group-numbers of foreskin fibroblasts cultured in the absence and presence of PC. No effect was noted. Right bar group - numbers of OA chondrocytes cultured in the absence and presence of PC. PC inhibited the proliferation of OA chondrocytes. * = p < 0.01 versus untreated control.

To investigate the OA chondrocyte-mediated calcification, we cultured OA chondrocytes in osteogenetic differentiation medium. As shown in Fig. (2), calcium deposits were detected in the monolayer of OA chondrocytes cultured in osteogenetic differentiation medium in the absence of PC, but not in the presence of 1 mm PC. These findings demonstrated that OA chondrocytes were capable of producing calcium crystals and that PC inhibited this calcification process.

Fig. (2). Representative images of alizarin red stained OA chondrocytes. Calcium deposits were detected in monolayer of OA chondrocytes cultured in osteogenetic differentiation medium, but not in the monolayer of OA chondrocytes cultured in osteogenetic differentiation medium containing 1 mm PC. (bar = 10 μm).

Representative images of untreated and PC-treated micromasses of OA chondrocytes are provided in Fig. (3). As shown, the intensity of picrosirius red staining in the outer layer of the PC-treated micromass of OA chondrocytes was higher than the intensity of picrosirius red staining in the outer layer of the untreated micromass, indicating that PC stimulated the production of collagens by OA chondrocytes. The intensity of alcian blue staining in the outer layer of the PC-treated micromass of OA chondrocytes was also higher than the intensity of alcian blue staining in the untreated micromass, indicating that PC also stimulated the production of proteoglycans by OA chondrocytes.

Fig. (3). Representative images of picrosirius red and alcian blue stained sections of micromasses of OA chondrocytes. Arrows – outer layers of the micromasses of OA chondrocytes. (bar = 100 μm). PC stimulated the production of collagens and proteoglycans.