Hypoxic chondrogenic differentiation of human embryonic stem cells enhances cartilage protein synthesis and biomechanical functionality

SummaryBackgroundEngineering musculoskeletal cartilages with stem cells remains a challenge because researchers must control many factors, including differentiation and cartilage matrix synthesis, particularly collagen II production. Hypoxia has effects on many cellular processes, though few investigations with hypoxia provide quantitative functional data on engineered cartilage.ObjectiveThis study investigated the effects of hypoxia on chondrogenesis with human embryonic stem cells (hESCs).MethodsThe experiment comprised two phases, embryoid body (EB) differentiation for 3 wks followed by a scaffold-less tissue engineering strategy called self-assembly for 4 wks. During each phase, hypoxic conditions (2% O2) or normoxic conditions (20% O2) were applied, and engineered constructs were analyzed for cellular, morphological, biochemical, and biomechanical properties.ResultsHypoxic conditions significantly altered the chondrogenic differentiation process, whereby cells cultured in these conditions had an enhanced ability to produce collagen II (up to 3.4-times), collagen I (up to 2.9-times), and glycosaminoglycans (GAGs) (up to 1.9-times), resulting in better biomechanical functionality (up to three times in tensile modulus and up to four times in compressive properties). Hypoxic cells had a different expression profile than normoxic cells for cluster of differentiation (CD)44, CD105, and platelet derived growth factor receptor (PDGFR)α, further emphasizing that hypoxia altered hESC differentiation and suggesting that these markers may be used to predict an hESC-derived cell population's chondrogenic potential. Also, normoxic self-assembly outperformed hypoxic self-assembly in tensile and compressive biomechanical characteristics.ConclusionsThese results show that oxygen availability has dramatic effects on the differentiation and synthetic potentials of hESCs and may have important implications for the development of strategies to engineer cartilage.