Human arthritic cartilage and e perimental osteoarthritis Human OA cartilage was sourced from individuals under going arthroplasty. selleckchem FTY720 Human cartilage was kindly pro vided by Dr Churl Hong Chun of Wonkwang University. The Institutional Review Board of the Wonkwang University Hospital approved the use of these materials, and all individuals provided written informed consent to be donors before undergoing surgery. Spontaneous OA in STR ort mice was e amined at 28 weeks of age, with CBA CaCrl mice used as controls. Aging studies were performed in 12 month old mice, and e perimental OA was induced in mice by destabilization of the medial meniscus surgery or by intra articular injection of collagenase in 8 week old male mice and in in Lrp5 mice and their wild type lit termates.

Sham operated and phosphate buffered saline injected mice were used as controls for the DMM and collagenase injected models, respectively. Mice were ana lyzed at 8 weeks after DMM surgery or 4 weeks after col lagenase injection. Micromass culture and primary culture of articular chondrocytes Mesenchymal cells were derived from the limb buds of ICR mouse embryos 11. 5 days postcoitus and main tained as micromass cultures for induction of chondro genesis as described previously. Mouse articular chondrocytes were isolated from knee cartilage obtained from postnatal day 5 mice. The articular cartilage was preincubated for 2 hours at 37 C with 0. 2% trypsin and 0. 2% type II collagenase and further digested with 0. 2% type II collagenase for 90 minutes. On culture day 3, the cells were treated with recombinant interleukin 1B, Wnt3a or Wnt7a for 24 hours.

Apoptosis was induced by treatment with an anti Fas antibody. Briefly, chondrocytes from articular cartilage of WT or Lrp5 mice were incubated in the presence or absence of IL 1B for 24 hours, then e posed to the anti Fas antibody and recombinant protein G for an additional 6 hours. Hamster immunoglobulin G2 was used as a control. The cells were stained with fluorescein isothiocyanate conjugated anne in V, and apoptotic chondrocytes were quantified by fluo rescence activated cell sorting analysis. Immunofluorescence microscopy and immunohistochemistry Chondrocytes were cultured on glass coverslips, fi ed with 3. 5% paraformaldehyde and permeabilized with 0. 1% Triton 100.

The cells were incubated for 1 hour with an antibody against type II collagen followed by incubation for 1 hour with an Ale a 488 conjugated secondary anti body. Ectopic e pression of LRP5 was determined Dacomitinib by labeling with an anti LRP5 antibody and an Ale a 555 conjugated secondary anti body. Apoptosis of chondrocytes in cartilage tissue was determined by terminal deo ynucleotidyl transferase deo yuridine triphosphate nick end labeling staining using a kit purchased from Roche Diagnostics. Specimens were visualized under an I 81 inverted fluorescence micro scope driven by MetaMorph imaging software.