gms | German Medical Science

Objectives: During osteoarthritis (OA) pathogenesis, articular chondrocyte phenotype becomes unstable and cells dedifferentiate. However, the exact mechanisms inducing dedifferentiation are still not known. Recently, increased levels of norepinephrine (NE) were detected in the synovial fluid of knee trauma and OA patients. It is also known that OA articular cartilage expresses adrenergic receptors and NE can modulate IL-1ß-induced changes in chondrocyte function. Under normoxia, IL-1ß promotes chondrocyte dedifferentiation but these effects under physiological hypoxic conditions are not yet investigated. Therefore, the aim of this study was to analyse the response of human articular OA chondrocytes to NE in the absence or presence of IL-1ß under hypoxia.

Methods: Articular cartilage samples were obtained from OA patients undergoing total knee replacement surgery. After isolation, cells were seeded in a density of 20.000 cells/cm² and cultured in a humidified atmosphere at 37°C and in 2% O₂ or 20% O₂, respectively. Cells were treated with different concentrations of NE (10^-8 M, 10^-6 M) and/or IL1-ß (low dose: 0.5 ng/ml). Cell viability was determined in supernatants using an LDH assay. Adrenergic receptor (AR) profile of freshly isolated chondrocytes was analysed by PCR. At day 7, cell morphology was documented by phase contrast microscopy and sulphated proteoglycans (sGAG) in the extracellular matrix were visualized via DMMB staining. Quantitative PCR was preformed to analyse Col1A1, Col2A1, COMP, Sox-9 expression.

Results and conclusion: Chondrocytes express α2A-, α2C- and ß2-AR. At day 7, untreated chondrocytes showed the characteristic shift towards a fibroblast-like shape under normoxia and hypoxia. NE alone did not affect morphology, but in combination with IL-1ß markedly accelerated this shift. We observed moderate DMMB staining in untreated and NE-treated cells, while IL-1ß alone and in combination with NE strongly decreased sGAGs.

Under normoxia, untreated cells expressed slightly increased Col1A1, decreased Sox-9, unchanged Col2A1, and slightly decreased COMP levels. Hypoxia decelerated dedifferentiation with stable Sox-9 and COMP and markedly increased Col2A1, only Col1A1 expression increased significantly. IL-1ß alone did not induce further changes in Col1A1 expression, but caused a slight Sox-9 and a highly significant Col2A1 and COMP decrease under normoxia and hypoxia. NE alone did not influence gene expression and did not modulate IL-1ß-mediated effects.

This study shows that articular chondrocyte dedifferentiation is decelerated under hypoxia and that IL-1ß completely reversed this effect. NE was not able to exhibit any modulating effect. Thus, low-grade inflammation exerts a dominant effect during OA pathogenesis and should be targeted early in OA therapy. Analysing the major sources of IL-1ß in the joint and further molecular effects of mild inflammation during OA pathogenesis will be the focus of our future work.