Mimicking the hypoxic environment (1%~7% O
) of native cartilage results in improved secretion of extracellular matrix (ECM) by chondrocytes in monolayer culture. However, investigations of the role of hypoxia in long-term three-dimensional (3D) development of biomaterial-based cartilage constructs are limited. The major challenge to overcome is the insufficient oxygen delivery to the inner core of cell-scaffold constructs, which may result in anoxic conditions (<<1%) that are linked to nitric oxide (NO)-induced damage to ECM and is associated with diseases such as osteoarthritis. To evaluate the interaction between hypoxia and hydrodynamic stimuli, chondrocyte-seeded PGA scaffolds were cultured in a petri dish (static) or a wavy-walled bioreactor (dynamic) under ambient (21%) or hypoxic (5%) conditions for 28 days, followed by biochemical and immunohistochemical evaluation. We found that sulfated glycosaminoglycan (GAG) production increased under 5% O
in the presence of hydrodynamic forces, whereas total collagen content was down-regulated by hypoxia in both static and dynamic groups. Greater amounts of soluble GAG were released into media under hypoxia and under mixing. In addition, expression of inducible nitric oxide synthase (iNOS) increased under static and under hypoxic conditions, revealing that NO production was elevated. Significantly decreased collagen content of Hypoxic-Static constructs may indicate that suboptimal oxygen and NO levels might be reached. These findings suggest that hydrodynamic forces are required for 3D regeneration of articular cartilage under hypoxia, but further study is required to enhance retention of soluble GAG within the construct and to minimize NO damage to collagen fibrils.