Abstract

Culture of three-dimensional (3D) constructs in hypoxic conditions (1-5% O2) has been shown to increase production of extracellular matrix components in primary intervertebral disc (IVD) cells and drive chondrogenesis of human mesenchymal stem cells (hMSCs). Growing evidence suggests that two-dimensional (2D) expansion under hypoxic conditions may have an even greater influence on chondrogenesis in MSCs. This study aims to determine the effects of hypoxia during 2D expansion and subsequent 3D culture on the in vitro maturation of tissue-engineered IVDs (TE-IVDs) made with hMSCs, using a previously developed TE-IVD system. hMSCs were expanded in either hypoxic (5% O2) or normoxic (21% O2) conditions before construction of TE-IVDs. Discs were cultured in 3D in either hypoxic or normoxic conditions to create four experimental groups. Discs made from MSCs expanded in hypoxia were up to 141% stiffer than those made with normoxia-expanded MSCs. Similar patterns were seen in all mechanical properties. Increases in glycosaminoglycan content and collagen content in the nucleus pulposus (NP) were associated with 3D hypoxic culture. A boundary region between the manufactured fibrosus and NP regions developed by 2 weeks and mimicked the organization of the native disc. Hypoxic conditions in both 2D expansion and subsequent 3D culture improved the maturation of TE-IVDs made with hMSCs.