Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells—including fibroblasts, myocytes, neurons, and other cell types—sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.

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We develop a model for gels and entangled solutions of semiflexible biopolymers such as F-actin. Such networks play a crucial structural role in the cytoskeleton of cells. We show that the rheologic properties of these networks can result from nonclassical rubber elasticity. This model can explain a number of elastic properties of such networks {\em in vitro}, including the concentration dependence of the storage modulus and yield strain.Comment: Uses RevTeX, full postscript with figures available at http://www.umich.edu/~fcm/preprints/agel/agel.htm

Many cell types alter their morphology and gene expression profile when grown on chemically equivalent surfaces with different rigidities. One expectation of this change in morphology and composition is that the cell's internal stiffness, governed by cytoskeletal assembly and production of internal stresses, will change as a function of substrate stiffness. Atomic force microscopy was used to measure the stiffness of fibroblasts grown on fibronectin-coated polyacrylamide gels of shear moduli varying between 500 and 40,000 Pa. Indentation measurements show that the cells' elastic moduli were equal to, or slightly lower than, those of their substrates for a range of soft gels and reached a saturating value at a substrate rigidity of 20 kPa. The amount of cross-linked F-actin sedimenting at low centrifugal force also increased with substrate stiffness. Together with enhanced actin polymerization and cross-linking, active contraction of the cytoskeleton can also modulate stiffness by exploiting the nonlinear elasticity of semiflexible biopolymer networks. These results suggest that within a range of stiffness spanning that of soft tissues, fibroblasts tune their internal stiffness to match that of their substrate, and modulation of cellular stiffness by the rigidity of the environment may be a mechanism used to direct cell migration and wound repair.

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Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.