Effect of HIF-1α inhibitor LW6 on chondrogenic properties of mesenchymal stromal cells and chondrocytes in cell sheets under physioxia

Hypoxia-inducible factor 1 (HIF-1) plays a central role in cartilage homeostasis, and its modulation may influence the performance of cell-based therapies. LW6, an inhibitor of HIF-1α and a potential anti-cancer therapeutic, has not been previously evaluated in cartilage regeneration-related studies and chondrogenic cell sheet systems. The aim of this study was to investigate how LW6 affects the intracellular functions and chondrogenic potential of human bone marrow–derived mesenchymal stromal cells (BMMSCs) and chondrocytes cultured in 2D, and as scaffold-free cell sheets under physioxic (5% O₂) or normoxic (21% O₂) conditions. Physioxia enhanced proliferation, suppressed mitochondrial and malate dehydrogenase 2 activity, reduced glycolysis, lowered intracellular calcium but stabilized HIF-1α and increased the expression of chondrogenic genes (SOX9, COL2A1, ACAN) in both cell types. LW6 further reduced metabolic activity and calcium levels, degraded HIF-1α and downregulated chondrogenic gene expression, particularly in chondrocytes. Physioxia also improved cell sheet integration with human cartilage explants and ECM deposition. Physioxic conditions support cartilage-forming potential and improve biointegration of BMMSC and chondrocyte cell sheets with human cartilage explants, while HIF-1α modulator LW6 negatively affects chondrogenic and metabolic pathways. These findings provide new insights into how HIF-1-targeting compounds may influence cell–based cartilage engineering and highlight the need for careful consideration of HIF-1 modulation in regenerative applications.