Abstract
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 H2 O2 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, H2 O2 . 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.