Cell-matrix tension contributes to hypoxia in astrocyte-seeded viscoelastic hydrogels composed of collagen and hyaluronan

Astrocyte activation is crucial for wound contraction and glial scar formation following central nervous system injury, but the mechanism by which activation leads to astrocyte contractility and matrix reorganization in the central nervous system (CNS) is unknown. Current means to measure cell traction forces within three-dimensional scaffolds are limited to analyzing individual or small groups of cells, within extracellular matrices, whereas gap junctions and other cell-cell adhesions connect astrocytes to form a functional syncytium within the glial scar. Here, we measure the viscoelastic properties of cell-seeded hydrogels to yield insight into the collective contractility of astrocytes as they exert tension on the surrounding matrix and change its bulk mechanical properties. Our results indicate that incorporation of the CNS matrix component hyaluronan into a collagen hydrogel increases expression of the intermediate filament protein GFAP and results in a higher shear storage modulus of the cell/matrix composite, establishing the correlation between astrocyte activation and increased cell contractility. The effects of thrombin and blebbistatin, known mediators of actomyosin-mediated contraction, verify that cell-matrix tension dictates the hydrogel mechanical properties. Viability assays indicate that increased cell traction exacerbates cell death at the center of the scaffold, and message level analysis reveals that cells in the hyaluronan-containing matrix have a ~ 3-fold increase in HIF-1α gene expression. Overall, these findings suggest that astrocyte activation not only increases cell traction, but may also contribute to hypoxia near sites of central nervous system injury.