OA synovial fluid induces proliferation and dedifferentiation of non-OA human articular chondrocytes

Abstract

Purpose: A major hallmark of osteoarthritis (OA) is cartilage degeneration. Synovial fluid is in direct contact with the articular cartilage and other joint tissues, functioning as a source of nutrients and morphogens. Synovial fluid plays a crucial role in regulating cartilage homeostasis and repair, and alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Chondrocytes residing in cartilage from OA patients are described to adopt hypertrophic and fibrochondrocytic phenotypes. Whether OA synovial fluid has a role in directing chondrocytes into such phenotypic changes has been poorly studied. Therefore, we aimed to investigate the response of non-OA chondrocytes when cultured in vitro for an extended period of time with synovial fluid derived from end-stage OA patients. Methods: A pool of Human knee Articular Chondrocytes (HACs) from four non-OA donors was cultured in 10% (v/v) FCS, 10% (v/v) knee OA synovial fluid (OA-SF) (pool of 24 donors, mean age = 60 ±13 years old) or 10% (v/v) synovial fluid (pool of 8 donors, mean age = 57±10 years old) derived postmortem from macroscopically unaffected knees (non-OA). Cells were formalin-fixated and fluorescently stained with DAPI/Hoechst33342 for DNA quantification. In parallel, cells were harvested for RNA isolation. Expression of chondrocyte marker genes was determined by RT-qPCR and normalized to PPIA. Full ethics for use of HACs and SF are in place. Results: Cell proliferation was significantly increased by OA-SF compared to non-OA synovial fluid after two days stimulation (Figure 1A). In addition, chondrocyte proliferation was monitored for thirteen days and was strongly induced by OA-SF when compared to FCS (Figure 1B). In the first days both FCS and OA-SF induced proliferation, followed by a proliferation halt in the presence of FCS, while OA-SF supported a continued induction of chondrocyte proliferation up until day 13. Expression of genes associated with cartilage extracellular matrix, like COL2A1 (Figure 1C) and COMP, were specifically downregulated by OA-SF in a time-dependent manner. PRG4 expression was gradually decreased for both OA-SF as well as FCS. ACAN expression was maintained in both conditions. Similar to COL2A1 and COMP expression dynamics, SOX9 was strongly downregulated by OA-SF. Expression of dedifferentiation-associated genes COL1A1 (Figure 1D) and COL3A1 remained stable over time. Moreover, SERPINF1, a gene associated with chondrocyte proliferation and a fibrochondrocytic phenotype was steadily upregulated over time. Expression of genes related to chondrocyte hypertrophy and inflammation demonstrated variable responses over time after OA-SF exposure. Conclusions: Our data demonstrate that synovial fluid from end-stage knee OA patients induces a profound change in chondrocyte proliferation and their phenotype that resembles dedifferentiation. This was supported by decreased expression of cartilage-specific ECM genes, maintenance of dedifferentiation markers COL1A1 and COL3A1, induced expression of fibrochondrocyte marker SERPINF1, and sustained proliferative capacity. Notably, the effect of OA-SF on proliferation was greater than non-OA synovial fluid. Interestingly, ACAN expression was not affected by OA-SF treatment. This suggests that the expression of this particular gene is differently regulated compared to other major cartilage ECM-encoding genes. Further research should be undertaken to explore what OA-SF-induced signaling pathways are responsible for the phenotypic shift towards dedifferentiation.