Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces.

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

Influence of the stiffness of three-dimensional alginate/collagen-I interpenetrating networks on fibroblast biology.

Wound dressing biomaterials are increasingly being designed to incorporate bioactive molecules to promote healing, but the impact of matrix mechanical properties on the biology of resident cells orchestrating skin repair and regeneration remains to be fully understood. This study investigated whether tuning the stiffness of a model wound dressing biomaterial could control the behavior of dermal fibroblasts. Fully interpenetrating networks (IPNs) of collagen-I and alginate were fabricated to enable gel stiffness to be tuned independently of gel architecture, polymer concentration or adhesion ligand density. Three-dimensional cultures of dermal fibroblasts encapsulated within matrices of different stiffness were shown to promote dramatically different cell morphologies, and enhanced stiffness resulted in upregulation of key-mediators of inflammation such as IL-10 and COX-2. These findings suggest that simply modulating the matrix mechanical properties of a given wound dressing biomaterial deposited at the wound site could regulate the progression of wound healing.