Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.

Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.

Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm²*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.

The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.