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This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8: 5 µg/ml, Gal-3: 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-κB activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression.

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Purpose: N-Glycan profiling provides an indicator of the cellular potential for functional pairing with tissue lectins. Following the discovery of galectin expression by chondrocytes as a factor in osteoarthritis pathobiology, mapping of N-glycans upon their phenotypic dedifferentiation in culture and in fibroblast-like synoviocytes is a step to better understand glycobiological contributions to disease progression. Experimental design: The profiles of cellular N-glycans of human osteoarthritic chondrocytes and fibroblast-like synoviocytes were characterized by mass spectrometry. RT-qPCR experiments determined mRNA levels of 16 glycosyltransferases. Responsiveness of cells to galectins was quantified by measuring the mRNA level for interleukin-1β. Results: The shift of chondrocytes to a fibroblastic phenotype (dedifferentiation) is associated with changes in N-glycosylation. The N-glycan profile of chondrocytes at passage 4 reflects characteristics of synoviocytes. Galectins-1 and -3 enhance expression of interleukin-1β mRNA in both cell types, most pronounced in primary culture. Presence of interleukin-1β leads to changes in sialylation in synoviocytes that favor galectin binding. Conclusions and clinical relevance: N-Glycosylation reflects phenotypic changes of osteoarthritic cells in vitro. Like chondrocytes, fibroblast-like synoviocytes express Nglycans that are suited to bind galectins, and these proteins serve as inducers of proinflammatory markers in these cells. Synoviocytes can thus contribute to disease progression in osteoarthritis in situ.

This study demonstrates that the combination of high- and low-frequency supraglottic jet ventilation via a jet ventilation laryngoscope provides a better pulmonary gas exchange and allows more accurate airway pressure monitoring during microlaryngeal surgery than subglottic monofrequent jet ventilation via an endotracheal catheter.