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.

这项工作旨在研究 OA 软骨细胞颗粒中半乳糖凝集素的失调网络，并评估它们最近发现的作为功能性生物标志物的分子开关的活性是否会导致体外细胞外基质的降解。建立了人类 OA 软骨细胞的无支架 3D 颗粒培养物。相对于二维培养物或临床组织切片，通过 RT-qPCR、免疫组织化学和 ELISA 监测半乳糖凝集素 (Gal)-1、-3 和 -8 的表达和分泌。将 2D 和 3D 培养物暴露于类似体内的半乳糖凝集素混合物（Gal-1 和 Gal-8：5 µg/ml，Gal-3：1 µg/ml），然后评估颗粒大小、免疫组织化学基质染色、和/或 MMP-1、-3 和 -13 的定量。NF-κB 活化抑制剂的应用探讨了干预半乳糖凝集素诱导的基质降解的潜力。半乳糖凝集素分析显示颗粒培养物中 Gal-1、-3 和 -8 的持续失调，类似于原位 OA 情况。半乳糖凝集素混合物的存在促进了颗粒大小的显着减小和富含 II 型胶原的细胞外基质的损失，同时伴随着 MMP-1、-3 和 -13 的上调。咖啡酸苯乙酯对 p65 磷酸化的抑制有效地减轻了半乳糖凝集素的不利影响，导致 MMP 分泌下调、基质分解减少和颗粒大小增加。这项研究表明 OA 软骨中失调的半乳糖凝集素网络导致细胞外基质分解，并提供了令人鼓舞的证据，证明可以抑制半乳糖凝集素引发的活动。OA 软骨细胞颗粒有可能作为体外疾病模型，用于进一步研究 OA 发病和进展中的半乳糖凝集素。