LAUSR

Monolayer culture is integral to many cell‐based cartilage repair strategies, but chondrocytes lose regenerative potential with increasing duration in vitro. This coincides with elevated reactive oxygen species (ROS) levels and a bioenergetic transformation characterized by increasing mitochondrial function. This study investigates ROS as stimuli for bioenergetic reprogramming and the effect of antioxidants on the propensity of chondrocytes to regenerate a cartilaginous matrix. Articular chondrocytes were cultured in monolayer under a 2% O2 atmosphere. Oxidative stress was increased using 50 μm H2O2 or a 20% O2 culture atmosphere, or decreased using the antioxidant N‐acetyl‐cysteine (NAC). Mitochondrial function was characterized using 200 nm Mitotracker green and an oxygen biosensor. After two population doublings ± NAC, chondrocytes were encapsulated in alginate beads (1 × 107 cells/ml) for an additional 10 days before DMB assay of glycosaminoglycan content. The beads were cultured under both 20% O2 and the more physiological 5% O2 condition. Chondrocytes expanded in 20% O2 exhibited elevated mitochondrial mass and functional capacity, which was partially mimicked by the exogenous ROS, H2O2. Oligomycin treatment revealed that the increased oxygen consumption was coupled to oxidative phosphorylation. NAC limited these markers of bioenergetic reprogramming during culture‐expansion with no significant effect on subsequent GAG production under 20% O2. However, NAC treatment in monolayer abolished the hypoxic induction of GAG in alginate beads. This supports the hypothesis of a causal relationship between exposure to ROS and acquired mitochondrial function in chondrocytes. Additionally, mitochondrial function may be required for the hypoxic induction of GAG synthesis by chondrocytes. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.