Matrix stiffness changes affect astrocyte phenotype in an in vitro injury model

Abstract Injury to the central nervous system (CNS) usually leads to the activation of astrocytes, followed by glial scar formation. The formation of glial scars from active astrocytes in vivo has been found to be dependent on the cell microenvironment. However, how astrocytes respond to different microenvironmental cues during scar formation, such as changes in matrix stiffness, remains elusive. In this work, we established an in vitro model to assess the responses of astrocytes to matrix stiffness changes that may be related to pathophysiology. The investigated hydrogel backbones are composed of collagen type I and alginate. The stiffness of these hybrid hydrogels can be dynamically changed by association or dissociation of alginate chains through adding crosslinkers of calcium chloride or a decrosslinker of sodium citrate, respectively. We found that astrocytes obtain different phenotypes when cultured in hydrogels of different stiffnesses. The obtained phenotypes can be switched in situ when changing matrix stiffness in the presence of cells. Specifically, matrix stiffening reverts astrogliosis, whereas matrix softening initiates astrocytic activation in 3D. Moreover, the effect of matrix stiffness on astrocytic activation is mediated by Yes-associated protein (YAP), where YAP inhibition enhances the upregulation of GFAP and contributes to astrogliosis. To investigate the underlying mechanism of matrix stiffness-dependent GFAP expression, we also developed a mathematical model to describe the time-dependent dynamics of biomolecules involved in the matrix stiffness mechanotransduction process of astrocytes. The modeling results further indicate that the effect of matrix stiffness on cell fate and behavior may be related to changes in the cytoskeleton and subsequent activity of YAP. The results from this study will guide researchers to re-examine the role of matrix stiffness in reactive astrogliosis in vivo and inspire the development of a novel therapeutic approach for controlling glial scar formation following injury, enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.