RESUMEN
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.
Asunto(s)
Cartílago Articular/lesiones , Cartílago Articular/metabolismo , Osteoartritis/metabolismo , Transducción de Señal , Proteínas Wnt/biosíntesis , Proteínas Wnt/fisiología , Humanos , Análisis por Micromatrices , Técnicas de Cultivo de TejidosRESUMEN
The present study demonstrates that the best way to enhance DNA microarray assays, both in terms of analysis speed and in final spot intensity, is to dissolve the available molar amount of sample in the smallest possible buffer volume and to subsequently convect this solution continuously across the surface of the array. The presently proposed shear-driven flow system is pre-eminently suited for this task, as it allows to induce strongly enhanced lateral transport rates, independently of the degree of miniaturization of the hybridization chamber. This transport enhancement method, however, only increases the hybridization rate and not the final spot intensity, as neither can any of the other transport enhancement methods already proposed in literature. A series of experiments with synthetic single-stranded (ssDNA) samples and an accompanying mass balance analysis are presented to demonstrate these points.