Key microarray findings were verified by real time PCR and additional in vitro experiments of matrix synthesis and signal transduction. We found that OP 1 BMP 7 controls numerous metabolic pathways that are not limited to its direct anabolic or anti catabolic function, but also related to cell growth, cell proliferation, differentiation, survival, example apoptosis, and death. Materials and methods Materials Dulbeccos modified Inhibitors,Modulators,Libraries Eagles medium, fetal bovine serum, gentamicin, Hams F 12, lipofectin, Opti MEM, penicillin streptomycin fungizone, 1X Platinum Quantitative PCR SuperMix UDG and Super Script III reverse transcriptase with oligo 12 18 were purchased from Invitrogen. Phos phorothioate ODN was custom synthesized by Oligos Etc. RNeasy mini kit, QIA shredder, RNase free DNase kit and QuantiTect Primer Assay were purchased Inhibitors,Modulators,Libraries from Qiagen.

Real time polymerase chain reaction pri mers were custom synthesized by Inhibitors,Modulators,Libraries Integrated DNA Technologies, Coralville, IA, USA. 10,000 X SYBR Green 1 was purchased from Cambrex, Rockland, ME, USA. Recombinant human rhOP 1 was kindly provided by Stryker Biotech. Isolation and culture of chondrocytes Full thickness articular cartilage from the talus of the talocrural joint from 12 human organ donors and from the femur of the tibiofemoral joint from two human organ donors was obtained from the Gift of Hope Organ and Tissue Donor Network with Institutional Review Board approval and appropriate consent within 24 hours of the donors death. Knee cartilage was utilized for verification Inhibitors,Modulators,Libraries of the ankle cartilage results using real time PCR.

Chondrocytes were isolated by sequential digestion with pronase for 60 minutes and collagenase P overnight. Chondrocytes were plated in high density monolayer culture and cultured for 24 hours in 50% DMEM 50% Hams F 12 supplemented with 10% FBS, 1% PSF, and gentamicin for attachment prior to treatment with either antisense or recombinant OP 1. Both treatments Inhibitors,Modulators,Libraries were administered for 48 hours in the absence of serum. Phosphorothioate ODNs Antisense ODNs were designed to be complementary to sequences in the 5 and 3 untranslated regions of the human OP 1 messenger RNA sequence as described. All verification experi ments with appropriate negative controls Treatment groups Chondrocyte cultures were divided into three experimen tal groups and treated for 48 hours as follows, 1 trans fected with OP 1 AS in the presence of 10 ug ml lipofectin, 2 treated with 100 ng ml of rhOP 1, and 3 culture control.

RNA Isolation Total cellular RNA was isolated using the RNeasy Mini Kit, following lysis of the cells with a Qia shredder and included an on column DNase digestion, according to the manufacturers instructions. All samples were stored at 80 C until analyzed. Microarray and pathway analysis Gene expression profiles download the handbook were analyzed by HG U133A gene chips from Affimetrix. At least 10 ug of RNA per experimental group was required for analysis.