Background/Purpose

Osteoimmunology is a field of emerging interest in which bone formation and resorption are understood in the context of the immune system. In rheumatoid arthritis (RA) uncontrolled joint inflammation alters the balance between osteoclasts and osteoblasts resulting in bone resorption. The inflamed joint in RA contains multiple factors contributing to the activation of osteoclasts, among these RANKL, M-CSF, TNFα and IL-17 secreted from activated fibroblast-like cells and T-cells. In this study, we aimed to investigate the potential of synovial fluid mononuclear cells (SFMCs) to develop into functional osteoclasts in vitro. Methods

Synovial fluid was collected from inflamed joints of chronic RA patients at the out patient clinic at Aarhus University Hospital. SFMCs were isolated using ficoll paque and cultured in DMEM (+10% FCS + 2% penicillin/streptomycin + 1% glutamin) at a concentration of 0.5 x 10⁶ cells/cm² (n=5). Following 21 days in culture, cells were TRAP stained and examined in light microscopy or lysed for qPCR for the common osteoclast genes calcitoninR, cathepsinK and beta3 integrin. To investigate functionality, SFMCs were also cultured on dentin plates for 21 days. To potentially increase the osteoclast differentiation in the SFMC cultures, culture medium was supplemented with RANKL (50ng/ml) and M-CSF (25ng/ml). As a control, conventional osteoclasts where cultured from healthy control monocytes with RANKL (50ng/ml) and M-CSF (25ng/ml).

Results

SFMCs cultured for 21 days differentiate into both multinucleated TRAP positive osteoclasts (6.7%, SD 4.8%) and mononuclear TRAP positive pre-osteoclasts (43.2%, SD 0.45%) (Fig 1). These cells expressed the common osteoclast genes calcitonin receptor, cathepsin K and beta3 integrin. Both the percentage of TRAP positive multinucleated cells (8.3%) and gene expression were comparable with conventional osteoclasts. Adding RANKL and M-CSF to SFMC cultures increased the percentage of multinucleated TRAP positive cells to 15.3% (SD 1.5%). SFMCs cultured on dentin plates finally confirmed the osteoclast phenotype of these cells, as the dentin was digested in the same manner as observed with conventional osteoclasts cultured on dentin plates.

Conclusion

Here we provide a new and simple method for generating functional osteoclasts from RA SFMCs. This spontaneous differentiation of osteoclasts from cells of the arthritic joint provides a new understanding of the inflamed joint and could explain the increased bone resorption observed in RA. Because osteoclasts are one of the ultimate effector cells in RA, this method could be a new tool to evaluate the effect of novel signaling molecules or the effectiveness of new drugs.

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Fig1: TRAP+ multinucleated cells differentiated from RA SFMCs.