following degradation by intraarticular injection of papa¨ın in rat knee, PG synthesis was increased concomitantly to up-regulation of gene expression of the xylosyltransfersase I (XT-I) responsible for the initiation of biosynthesis of the chondroitin sulfate and heparan sulfate GAG chains of PGs. shRNA inhibition of XT-I expression during recovery phases prevent cartilage repair indicating that XT-I play a central role in cartilage repair process [13]. Human studies relative to this issue, however, are lacking at present, and such information is essential in furthering our understanding of cartilage degeneration as well as to stimulate cartilage repair in OA patients. In this study, we examined the role of XT-I in OA disease. We found that XT-I regulates GAG synthesis in human cartilage during early onset and late stage of OA disease and that expression of XT-I gene is regulated by the proinflammatory cytokine IL-1b and by the cartilage anabolic factor TGF-b1. Interestingly, forced expression of this enzyme by gene transfer in late stage OA cartilage enhanced GAG synthesis and stimulate cartilage repair. Collectively, our results provide important insights into regulatory factors driving the changes in GAG synthesis that occur with the onset and progression of cartilage degeneration. Materials and Methods Human osteoarthritis cartilage samples, histology and scoring disease severity Articular cartilage samples (femoral condyles and tibial plateau) were obtained from eight patients with OA (mean age 6569 yr) undergoing total knee joint replacement. All OA patients were examined by a certified rheumatologist and were diagnosed as having OA, using the criteria of the American College of Rheumatology for OA [14]. This study was approved by our local Research Institution review board (Comission de la Recherche Clinique; registration number UF 9757 – CPRC 2004 – Cellules souches et chondroge´ne`se). The protocol conforms to the ethical guidelines of the Declaration of Helsinki and written informed consent has been obtained from each patient. Cartilage specimens (within 1 h of operation) were dissected using sterile techniques, and full-thickness cartilage was removed from all of the femoral condyle using a 6 mm biopsy punch. Extreme care was taken to avoid subchondral bone, osteophytic cartilage and fibrocartilage. Cartilage samples were fixed for 24 h in 4% paraformaldehyde at 4uC, rinsed in running tap water for 1 minute, decalcified with RDO (Apex Engineering Products Corporation), in a volume of decalcifier equal to at least 10-times the tissue volume for 2 h. Samples were checked every 30 min and decalcified tissue was rinsed in running tap water for 5 min and further fixed in 4% paraformaldehyde. Samples were embedded in paraffin, and tissue sections (5 mm thick) were cut. Histological sections were rehydrated in a graded series of ethanol and stained with toluidine blue. Cartilage sections were graded histologically in line with Mankin’s histological grading system for OA cartilages [15] to assess disease severity and cartilage damage based on surface fibrillation, matrix depletion and cellularity, and the integrity of the tidemark. No tidemarks were present in our cartilage samples. Construction of XT-I expression vector, transfection of chondrocyte and GAG synthesis A human XT-I cDNA has been cloned and ligated into the unique HindIII-NotI sites of mammalian expression vector pCMV (Clontech, Mountain View, CA, USA) to generate pCMV-XT-I. Primary chondrocytes were isolated by sequential enzymatic digestion with pronase and collagenase of human femoral condyle XT-I and Proteoglycan Synthesis in Osteoarthritis cartilage [16] and maintained in DMEM-F12 mix medium in 6- well plates at 37uC in a humidified atmosphere supplemented with 5% CO2. Cells were then transfected with 1 mg of pCMV-XT-I expressing vector or pCMV empty vector at 80% confluence using Exgen 500 (Euromedex, Souffelweyersheim, France). At 48 h posttransfection, cells were labeled with 10 mCi/ml 35S-sulfate for the last 6 h of experimental period and PGs were extracted from cell layer and culture medium [17] with 4 M guanidine hydrochloride, precipitated using cetylpyridinium chloride, and sulfated GAG synthesis rate was measured by liquid scintillation counting [18]. Real-time Quantitative PCR All reagents and instruments used for RNA extraction were RNase-free. Total RNA from human cartilage samples was extracted by homogenization in Trizol solution and purified based on the manufacturer’s instruction for the RNAeasy Kit (Qiagen, Cologne, Germany). Briefly, sterile scalpel was used for slicing of cartilage samples on ice to very small pieces (1 mm pieces) before homogenization process. Homogenization was performed in a 1.5 ml microcentrifuge tube on ice using a power homogenizer (Yellowline, DI25) with homogenization time of 10 s intervals for up to 1 min. The ratio of cartilage to Trizol used was 2% (w/v). In the case of cultured chondrocytes total RNA was prepared using RNAeasy kit. cDNA was synthesized from 0.2–1 mg of total RNA using oligodT primer and Power Script Reverse Transcriptase (Clontech). Quantitative PCR was performed using LightCycler system (Roche, Mannheim, Germany) and SYBR green detector (QIAGEN GmbH, Hilden, Germany). Transcripts were normal- ized to ribosomal protein S29. RT2 PCR primer sets for human XT-I, Aggrecan, IL-1R1, TGF-b1 and ribosomal protein S29 were from SuperArray (Frederick, MD, USA). PCR cycling parameters were 15 min at 95uC; 40 cycles of 10 s at 95uC, 35 s at 65uC, and 15 s at 72uC. Expression levels of target genes were normalized to ribosomal protein S29 RNA level. XT-I shRNA Transfection Four shRNA vectors for XT-I and control were obtained from (SuperArray). The shRNA vectors were generated by ligating the hairpin oligonucleotides of the target sequences A: CCGGAGGTTTGTAGAATATGT, B: CGGGACAATG- CAAGGTTCATT, C: GAGAGGCTATTCCGCAACTTT and D: TACCTTCTTTGCCCGCAAGTT, into SureSilencingTM shRNA plasmid. Each vector contains the shRNA under the control of the U1 promoter. The two vectors expressing shRNA of the sequences B and D were selected for their ability to significantly inhibit XT-I gene expression in primary chondro- cytes. Full thickness cartilage (6 mm-biopsy punch) cultured in DMEM-F12 medium in 6-well plates at 37uC were transfected with 5 mg of shRNA-XT-I using the transfection reagent Exgen 500. Total RNA were extracted 72 h after transfection and expression level of XT-I gene was analyzed by qPCR, as described below. In parallel, 72 h after shRNA-XT-I transfection, cartilage explants were pulsed with 35S-sulfate (5 mCi/ml) for the last 6 h prior to GAG analysis [18]. Cytokine Treatments Cartilage explants were cultured in six-well plates in DMEM- F12 medium at 37uC in a humidified atmosphere supplemented with 5% CO2. After 48 h, explants were serum-starved for 24 h and treated or not (control) with 10 ng/ml of IL-1b or TGF-b1 (Sigma-Aldrich, Saint Quentin, France) for 24 h. At 6 h before the end of the treatment, explants were pulsed with 35S-sulfate (10 mCi/ml) prior to GAG analysis [18]. PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34020