Lateral boundary mechanosensing by adherent cells in a collagen gel system Hamid Mohammadi a, * , Paul A. Janmey b , Christopher A. McCulloch a a Matrix Dynamics Group, University of Toronto, Toronto, ON, Canada b Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA article info Article history: Received 30 July 2013 Accepted 19 October 2013 Available online 8 November 2013 Keywords: Cell adhesion Strain stiffening Cell extensions b1 Integrin abstract Cell adhesion responses to in-depth physical properties such as substrate roughness and topography are well described but little is known about the influence of lateral physical cues such as tissue boundaries on the function of adherent cells. Accordingly, we developed a model system to examine remote cell sensing of lateral boundaries. The model employs floating thin collagen gels supported by rigid grids of varying widths. The dynamics, lengths, and numbers of cell extensions were regulated by grid opening size, which in turn determined the distance of cells from rigid physical boundaries. In smaller grids (200 mm and 500 mm wide), cell-induced deformation fields extended to, and were resisted by, the grid boundaries. However, in larger grids (1700 mm wide), the deformation field did not extend to the grid boundaries, which strongly affected the mean length and number of cell extensions (w60% reduction). The generation of cell extensions in collagen gels required expression of the b1 integrin, focal adhesion kinase and actomyosin activity. We conclude that the presence of physical boundaries interrupts the process of cell-mediated collagen compaction and fiber alignment in the collagen matrix and enhances the formation of cell extensions. This new cell culture platform provides a geometry that more closely approximates the native basement membrane and will help to elucidate the roles of cell extensions and lateral mechanosensing on extracellular matrix remodeling by invasion and degradation. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The composition and structure of extracellular matrices vary widely and depend on tissue type and the state of health or disease of the tissue [1,2]. In health and disease, the extracellular matrix in which cells reside provides microenvironments with diverse me- chanical properties [3,4] that vary on a length scale of microns to mms. Biophysical approaches for modulating the mechanical properties of the matrix have shown that substrate elasticity, topography and roughness influence cellular processes such as spreading, migration, phagocytosis and differentiation [5e8]. The ability of cells to sense and respond to the mechanical properties of the extracellular matrix is dependent in part on application of actomyosin-dependent contractile forces [9]. The responses of cells to the mechanical properties of the matrix and force-induced matrix deformation can be measured by characterizing the defor- mation fields created in the extracellular matrix [10]. Unlike syn- thetic hydrogels (e.g. polyacrylamide gels) that exhibit nearly ideal linear elastic deformation, naturally-occurring matrix biopolymers demonstrate complex mechanical behavior after application of forces. For example, collagen gels display non-linear viscoelastic behavior when subjected to cell-generated forces and may undergo strain-stiffening [11,12], fiber alignment [13], or irreversible network compaction [14] depending on the magnitude and dura- tion of deformation. Elastic or inelastic matrix deformations enable adherent cells to detect inhomogeneous physical properties of the matrix, such as the presence of a rigid foundation [15,16] subjacent to the matrix or of adjacent cells that are located several hundred microns distant [17,18]. Notably, adherent cells can mechanosense relatively farther on fibrillar matrices than on elastic hydrogels [19]. This marked difference may be explained by the non-linear strain-stiffening behavior of fibrillar matrices [18] and/or by fiber alignment in collagen gel networks [20]. Notably, stiff fibrillar matrices such as cross-linked collagen exhibit lower rates of contraction and defor- mation than soft matrices [21]. * Corresponding author. Room 243, Fitzgerald Building, University of Toronto, 150 College Street, Toronto, ON M5S 3E2, Canada. Tel.: þ1 416 978 1258; fax: þ1 416 978 5956. E-mail address: hamid.mohammadi@utoronto.ca (H. Mohammadi). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.059 Biomaterials 35 (2014) 1138e1149