In situ elasticity modulation with dynamic substrates to direct cell phenotype

Abstract

Microenvironment elasticity influences critical cell functions such as differentiation, cytoskeletal organization, and process extension. Unfortunately, few materials allow elasticity modulation in real time to probe its direct effect on these dynamic cellular processes. Here, a new approach is presented for the photochemical modulation of elasticity within the cell's microenvironment at any point in time. A photodegradable hydrogel was irradiated and degraded under cytocompatible conditions to generate a wide range of elastic moduli similar to soft tissues and characterized using rheometry and atomic force microscopy (AFM). The effect of the elastic modulus on valvular interstitial cell (VIC) activation into myofibroblasts was explored. In these studies, gradient samples were used to identify moduli that either promote or suppress VIC myofibroblastic activation. With this knowledge, VICs were cultured on a high modulus, activating hydrogel substrate, and uniquely, results show that decreasing the substrate modulus with irradiation reverses this activation, demonstrating that myofibroblasts can be de-activated solely by changing the modulus of the underlying substrate. This finding is important for the rational design of biomaterials for tissue regeneration and offers insight into fibrotic disease progression. These photodegradable hydrogels demonstrate the capability to both probe and direct cell function through dynamic changes in substrate elasticity.

Introduction

Fabrication of cell culture substrates with different elastic moduli has been explored for the past decade, as researchers identified that cell contractile forces and related cell functions, such as motility [1], cytoskeletal organization [2], and differentiation [3], [4], are influenced by the elasticity of the underlying substrate [5], [6]. For example, in response to an injury, fibroblasts are known to differentiate into myofibroblasts, a wound healing phenotype responsible for repairing and replacing damaged extracellular matrix (ECM) in injured tissues and organs [7]. As ECM is secreted, the elastic modulus of the cell microenvironment increases. Once the desired matrix modulus is achieved, the myofibroblasts deactivate and undergo a number of cellular processes, including senescence [8] and apoptosis [7], the exact pathways of which are not well understood. If this de-activation is misregulated and the myofibroblast phenotype persists, ECM secretion continues, increasing the matrix modulus and effecting fibrosis [9]. Understanding the role of the matrix modulus in dynamic cellular signaling processes such as these is important for treatment of fibrotic diseases, as well as in the design of tissue regeneration strategies [3]. Many researchers have thus sought to explore the influence of substrate elasticity on cell function by the development of materials with highly regulated properties.

Researchers have used discrete poly(acrylamide)-, poly(ethylene glycol) (PEG)-, or poly(dimethyl siloxane)-based gels with different moduli to explore cellular processes such as myotube differentiation [10], embryonic cardiomyocyte beating [11], and neuronal cell process extension [12]; smooth muscle cell (SMC) proliferation and focal adhesion formation [13]; and myofibroblastic activation [14], respectively. These materials are useful but require the preparation of a unique formulation for each gel (e.g., different crosslinker concentration) to vary the modulus, and more importantly, the material properties are fixed upon formation. To address some of these limitations, advanced processing techniques have been developed to create modulus gradients within hydrogels [15], [16]. For example, gradient hydrogels formed from mixtures of acrylamide and bis-acrylamide have been used to study fibroblast migration [17]. Improved gradient fabrication techniques, such as using microfluidics with poly(acrylamide)-hydrogels [18] and advanced patterning with poly(dimethylsiloxane) (PDMS) [19] gels, have been used to examine vascular SMC spreading and cytoskeletal organization [18] and fibroblast and endothelial cell migration [19], respectively. While these approaches have been proven versatile, the material properties are static. Recently, Frey and Wang developed a polyacrylamide-based photodegradable hydrogel whose modulus can be decreased 20–30% of its initial value with irradiation in the presence of NIH 3T3 fibroblasts [20]. This dynamic modulation of substrate rigidity was used to study the influence of modulus on 3T3 cell morphology and migration. In parallel to this work, we were interested in probing the influence of dynamic changes in substrate modulus on cell function (e.g., differentiation), which often requires a large variation in modulus. Specifically, we have developed a photodegradable monomer for synthesizing PEG-based hydrogels that degrade in response to light, and the chemistry is compatible with cell encapsulation and 3D cell culture [21]. Here, we exploit this chemistry to create a 2D cell culture platform whose elasticity can be tuned over a wide range of moduli and subsequently varied in real time by exposure to light. Experiments were designed to answer how in situ changes in substrate modulus might influence the fibroblast–myofibroblast differentiation process, especially how myofibroblast de-differentiation might be directed by a step change in the elasticity of its microenvironment. In general, a cell culture substrate that allows on-demand creation of materials with variable elasticity, either in a discrete or gradient fashion, would allow the design of unique experiments to further explore the influence of elasticity on cellular functions. Moreover, such materials would improve the understanding of how cells respond to dynamic microenvironmental changes (e.g., the role of matrix elasticity in promoting or suppressing fibrosis).