Oxygen Consumption Rate-Defined Phases Couple Metabolism to Matrix Dynamics in Chondrocyte-Mesenchymal Stromal Cell Co-Culture

This study investigated how the ratio of human nasal chondrocytes to bone marrow-derived mesenchymal stromal cells regulates metabolism, extracellular matrix formation, and mechanical properties during engineered cartilage development. Nasal chondrocyte monocultures, MSC monocultures, and five NC:MSC co-culture ratios ranging from 3:1 to 1:3 were cultured as three-dimensional pellets for 27 days while oxygen consumption rate was measured continuously every 15 minutes. The authors identified three reproducible metabolic phases: early condensation and priming, differentiation and matrix synthesis, and late maturation and remodeling. NC-rich pellets showed earlier HIF-1α and SOX9 activity, stronger COL2A1 and ACAN induction, and greater hyaline-like cartilage matrix formation, with the 3:1 NC:MSC condition showing the strongest glycosaminoglycan synergy. In contrast, MSC-rich pellets maintained greater late-stage oxidative activity, showed increased PGC-1α, COL10A1, and MMP13 expression, and exhibited more pronounced hypertrophic and remodeling signatures. Mechanical testing revealed a different composition dependence from total GAG production: the MSC-rich 1:3 NC:MSC group achieved the highest equilibrium and peak moduli despite having lower GAG/DNA than NC-rich groups. The 1:3 condition reached an equilibrium modulus of 147.5 kPa and a peak modulus of 699.2 kPa. Overall, the study demonstrates that oxygen-consumption trajectories can serve as a non-destructive process indicator linking cellular metabolism with cartilage matrix development, mechanical maturation, and hypertrophic risk, while showing that NC:MSC ratio can be tuned to prioritize either hyaline cartilage quality or mechanical stiffness.