Physiological Hypoxia as a Potent Tool for Directed Modification of the Extracellular Matrix of Multipotent...

Multipotent mesenchymal stromal cells (MSCs) are widely used in regenerative medicine. Their decellularized extracellular matrix (dECM) has emerged as a key bioactive substrate for tissue engineering. The properties of MSC-derived dECM depend on cell culture conditions. Physiological hypoxia mimics the native MSC milieu and represents a promising preconditioning strategy. This study aimed to evaluate the structural and mechanical properties of dECM derived from MSCs cultured under different oxygen levels and to assess the phenotype of MSCs recellularized on these matrices.

Human adipose-derived MSCs were permanently expanded under 20% or 5% O₂ to obtain 20-dECM and 5-dECM. Matrix architecture was visualized using scanning electron microscopy and quantitative phase contrast. Stiffness of dECM was measured by atomic force microscopy. MSCs were recellularized on dECM and cultured under 20% O₂ for 72 hours. The assessment of cell morphology, cytoskeletal organization, nuclear translocation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), integrin expression, levels of reactive oxygen species (ROS), and cell cycle analysis were assessed using confocal microscopy and flow cytometry.

In comparison with 20-dECM, 5-dECM demonstrated significant alignment of fibrillar bundles and a 1.6-fold increase in stiffness (p < 0.05). Reseeded MSCs on 5-dECM have acquired a spindle-shaped, aligned morphology, whereas cells on 20-dECM displayed a rounded morphology with multiple processes. The YAP and TAZ nuclear-to-cytoplasmic ratio was significantly lower on both dECM compared with uncoated surfaces (p < 0.0001). Total fluorescence intensity of YAP and TAZ per cell revealed divergent patterns on 5-dECM vs 20-dECM: YAP levels were increased, whereas TAZ levels were decreased. The expression of integrin α2 and α6 was higher (p < 0.01), while the expression of integrin α1 and intercellular adhesion molecule 1 (ICAM-1) was lower on both dECM vs control (p < 0.05). Intracellular ROS levels were reduced twofold on dECM (p < 0.01). The proportion of MSCs in the G2/M phase and the increase in cell number were more significant on dECM compared with uncoated plastic.

Hypoxia-derived dECM exhibits increased anisotropy and stiffness, promoting MSC alignment and cytoskeleton organization, integrin expression, altering the subcellular localization of YAP and TAZ and reducing ROS levels. Thus, it may represent a promising, effective bioactive scaffold for MSC expansion.