WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

OBJECTIVE: Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. METHODS: Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. RESULTS: WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. CONCLUSIONS: In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.

Human single-chain variable fragment that specifically targets arthritic cartilage.

OBJECTIVE: To demonstrate that posttranslational modification of type II collagen (CII) by reactive oxygen species (ROS), which are known to be present in inflamed arthritic joints, can give rise to epitopes specific to damaged cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA) and to establish a proof of concept that antibodies specific to ROS-modified CII can be used to target therapeutics specifically to inflamed arthritic joints. METHODS: We used a semisynthetic phage display human antibody library to raise single-chain variable fragments (scFv) specific to ROS-modified CII. The specificity of anti-ROS-modified CII scFv to damaged arthritic cartilage was assessed in vitro by immunostaining articular cartilage from RA and OA patients and from normal controls. The in vivo targeting potential was tested using mice with antigen-induced arthritis, in which localization of anti-ROS-modified CII scFv in the joints was determined. The therapeutic effect of anti-ROS-modified CII scFv fused to soluble murine tumor necrosis factor receptor II-Fc fusion protein (mTNFRII-Fc) was also investigated. RESULTS: The anti-ROS-modified CII scFv bound to damaged arthritic cartilage from patients with RA and OA but not to normal preserved cartilage. When systemically administered to arthritic mice, the anti-ROS-modified CII accumulated selectively at the inflamed joints. Importantly, when fused to mTNFRII-Fc, it significantly reduced inflammation in arthritic mice, as compared with the effects of mTNFRII-Fc alone or of mTNFRII-Fc fused to an irrelevant scFv. CONCLUSION: Our findings indicate that biologic therapeutics can be targeted specifically to arthritic joints and suggest a new approach for the development of novel treatments of arthritis.

Chondroprotection in Models of Cartilage Injury by Raising the Temperature and Osmolarity of Irrigation Solutions.

Objectives During arthroscopic or open joint surgery, articular cartilage may be subjected to mechanical insults by accident or design. These may lead to chondrocyte death, cartilage breakdown and posttraumatic osteoarthritis. We have shown that increasing osmolarity of routinely used normal saline protected chondrocytes against injuries that may occur during orthopedic surgery. Often several liters of irrigation fluid are used during an orthopedic procedure, which is usually kept at room temperature, but is sometimes chilled. Here, we compared the effect of normal and hyperosmolar saline solution at different temperatures on chondrocyte viability following cartilage injury using in vitro and in vivo models of scalpel-induced injury. Design Cartilage injury was induced in bovine osteochondral explants and the patellar groove of rats in vivo by a single pass of a scalpel blade in the presence of normal saline (300 mOsm) or hyperosmolar saline solution (600 mOsm, sucrose addition) at 4°C, 21°C, or 37°C. Chondrocytes were fluorescently labeled and visualized by confocal microscopy to assess cell death. Results Hyperosmolar saline reduced scalpel-induced chondrocyte death in both bovine and rat cartilage by ~50% at all temperatures studied (4°C, 21°C, 37°C; P < 0.05). Raising temperature of both irrigation solutions to 37°C reduced scalpel-induced cell death ( P < 0.05). Conclusions Increasing the osmolarity of normal saline and raising the temperature of the irrigation solutions to 37°C reduced chondrocyte death associated with scalpel-induced injury in both in vitro and in vivo cartilage injury models. A hyperosmolar saline irrigation solution at 37°C may protect cartilage by decreasing the risk of chondrocyte death during mechanical injury.

Identification of the molecular response of articular cartilage to injury, by microarray screening: Wnt-16 expression and signaling after injury and in osteoarthritis.

OBJECTIVE: To characterize the molecular response of adult human articular cartilage to acute mechanical injury. METHODS: An established ex vivo model was used to compare gene expression of adult human articular cartilage explants 24 hours after mechanical injury with that of uninjured controls by microarray analysis of gene expression. Confirmation for selected genes was obtained by real-time polymerase chain reaction and immunohistochemical analysis. Expression of selected genes was also investigated in preserved and osteoarthritic (OA) cartilage. RESULTS: Six hundred ninety genes were significantly regulated at least 2-fold following mechanical injury. They included genes previously reported to be differentially expressed in OA versus normal cartilage or having allelic variants genetically linked to OA. Significant functional clusters included genes associated with wound healing, developmental processes, and skeletal development. The transforming growth factor beta, fibroblast growth factor, and Wnt pathways were modulated. A systematic analysis of the Wnt signaling pathway revealed up-regulation of Wnt-16, down-regulation of FRZB, up-regulation of Wnt target genes, and nuclear localization of beta-catenin in injured cartilage. In addition, in OA, Wnt-16 and beta-catenin were barely detectable in preserved cartilage areas, but were dramatically up-regulated in areas of the same joint with moderate to severe OA damage. CONCLUSION: Our findings indicate that mechanical injury to adult human articular cartilage results in the activation of a signaling response, with reactivation of morphogenetic pathways. Therapeutic targeting of such pathways may improve current protocols of joint surface defect repair and/or prevent the evolution of such lesions into posttraumatic OA.