Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) are major contributors to the composition and function of synovial fluid (SF). In disease, changes to important SF molecules such as hyaluronic acid (HA), lubricin, and numerous inflammatory markers contribute to a loss of SF functional properties. Previous studies characterized the ability of FLS to produce SF molecules in short-term cultures using continuous cytokine supplementation. This study assessed the HA, lubricin, and matrix metalloproteinase-2 (MMP-2) secretion profile of FLS over 12 days of culture. FLS were subjected to continuous, intermittent, and sequential cytokine treatments of interleukin-1 beta (IL-1ß), tumour necrosis factor-alpha (TNF-α), and transforming growth factor-beta 1 (TGF-ß1). HA was assessed by an enzyme-linked immunosorbent assay (ELISA) for content and agarose gel electrophoresis for molecular weight distribution. Relative lubricin content was determined by western blot. Pro MMP-2 and active MMP-2 were quantified by gelatin zymography. All intermittent and sequential treatments significantly increased secretion of high-molecular-weight (>3 MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL-1ß and TNF-α for 2 days followed by TGF-ß1 for 1 day reduced active MMP-2 to unstimulated control levels. These data provide important information on the long-term functional potential of cytokine-stimulated FLS and suggest that temporal regulation of cytokine exposure can be a powerful tool to guide healthy synovial secretions.

Cartilage Metabolism is Modulated by Synovial Fluid Through Metalloproteinase Activity.

Synovial fluid (SF) contains various cytokines that regulate chondrocyte metabolism and is dynamically associated with joint disease. The objective of this study was to investigate the effects of diluted normal SF on catabolic metabolism of articular cartilage under inflammatory conditions. For this purpose, SF was isolated from healthy bovine joints, diluted, and added to cartilage explant cultures stimulated with interleukin-1 (IL-1) for 12 days. The kinetic release of sulfated glycosaminoglycan (sGAG) and collagen, as well as nitric oxide and gelatinase matrix metalloproteinases were analyzed in the supernatant. Chondrocyte survival and matrix integrity in the explants were evaluated with Live/Dead and histological staining. Diluted synovial fluid treatment suppressed sGAG and collagen release, downregulated the production of nitric oxide and matrix metalloproteinases, reduced IL-1-induced chondrocyte death, and rescued matrix depletion. Our results demonstrate that normal SF can counteract inflammation-driven cartilage catabolism. This study reports on the protective function of healthy SF and the therapeutic potential of recapitulation of SF for cartilage repair.

High-throughput cell aggregate culture for stem cell chondrogenesis.

Cell aggregate culture is a widely used, reliable system for promoting chondrogenic differentiation of stem cells. A high-throughput cell pellet culture enables screening of various soluble factors for their effects on stem cell function and chondrogenesis. In this protocol, we report a platform that allows the formation of stem cell aggregates in a 96-well plate format. Specifically, stem cells are centrifuged to form high-density pellets, mimicking mesenchymal condensation. The cell aggregates can be differentiated into chondrocytes when cultured in chondrogenic medium for 4 weeks. Such a technique is compatible for high-throughput screening and can be very useful for optimizing conditions for cartilage tissue engineering.