Augmentation of integrin-mediated mechanotransduction by hyaluronic acid.

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Substrate stiffness regulates solubility of cellular vimentin.

The intermediate filament protein vimentin is involved in the regulation of cell behavior, morphology, and mechanical properties. Previous studies using cells cultured on glass or plastic substrates showed that vimentin is largely insoluble. Although substrate stiffness was shown to alter many aspects of cell behavior, changes in vimentin organization were not reported. Our results show for the first time that mesenchymal stem cells (hMSCs), endothelial cells, and fibroblasts cultured on different-stiffness substrates exhibit biphasic changes in vimentin detergent solubility, which increases from nearly 0 to 67% in hMSCs coincident with increases in cell spreading and membrane ruffling. When imaged, the detergent-soluble vimentin appears to consist of small fragments the length of one or several unit-length filaments. Vimentin detergent solubility decreases when these cells are subjected to serum starvation, allowed to form cell-cell contacts, after microtubule disruption, or inhibition of Rac1, Rho-activated kinase, or p21-activated kinase. Inhibiting myosin or actin assembly increases vimentin solubility on rigid substrates. These data suggest that in the mechanical environment in vivo, vimentin is more dynamic than previously reported and its assembly state is sensitive to stimuli that alter cellular tension and morphology.

Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors.

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.

The hard life of soft cells.

Cells are mechanical as well as chemical machines, and much of the energy they consume is used to apply forces to each other and to the extracellular matrix around them. The cytoskeleton, the cell membrane, and the macromolecules composing the extracellular matrix form networks that in concert with the forces generated by the cell create dynamic materials with viscoelastic properties unique to each tissue. Numerous recent studies suggest that the forces that cells create and are subjected to, as well as the mechanical properties of the materials to which they adhere, can have large effects on cell structure and function that can act in concert with or override signals from soluble stimuli. This brief review summarizes recent studies of the effects of substrate mechanics on cell motility, differentiation, and proliferation, and discusses possible mechanisms by which a cell can probe the stiffness of its surroundings. Cell Motil. Cytoskeleton, 2009. (c) 2009 Wiley-Liss, Inc.

Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering.

Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(l,d-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3 +/- 58.0 microm) rather than smaller (11.15 +/- 11.08 microm) microspheres, generated pores on the order of 200 microm as compared to 20 microm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.