Matrix Dimensionality Alters Integrin Signaling and Neurite Outgrowth
Andreia S. Ribeiro1, Erin Voss1, Elizabeth M. Powell2, and Jennie B. Leach1. (1) Chemical and Biochemical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, (2) Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201
Translating information from two-dimensional (2D) culture into three-dimensional (3D) systems has been a major hurdle in the use of tissue engineered biopolymers for neuronal repair applications. The ultimate goal of peripheral nerve tissue engineering is to provide rationally designed alternatives to grafted tissue through a deeper understanding of the dynamic 3D interactions between the cellular and extracellular environment. Neurons sense ligands in the surrounding extracellular matrix (ECM) and respond through integrin signaling pathways which influence cell fate and process outgrowth. Neural signaling response controls neurite outgrowth and is thought to be different in 2D and 3D systems, with in vitro 3D environments being a better representation of in vivo systems. While 2D substrates have been incredibly valuable in revealing intricacies of cell biology, previous studies with non-neuronal cells demonstrate that signaling pathways are dramatically altered when cells are placed in a 3D matrix. For example, integrin expression in migrating cells in 3D substrates more closely resembles the in vivo pattern. We hypothesize that 3D culture imposes changes in matrix ligand organization and alters neuronal behavior by modulating β1-integrin cytoskeletal signaling. We test our model using dorsal root ganglion (DRG) neurons isolated from E13.5 mouse embryos cultured in Type I collagen, a major component of the ECM and ligand for β1-integrin. We examine the expression patterns and phosphorylation of downstream molecules including Rho GTPases and focal adhesion kinase (FAK). Preliminary results show that 3D culture demonstrates more diffuse integrin mediated signaling as neurons are embedded in collagen gels when compared to the punctate signaling complexes formed in 2D culture in the location of point contacts. Tissue engineering principles are also being implemented to develop a new in vitro model system based on a synthetic polyethylene-glycol (PEG) crosslinked hydrogel that allows for independent tuning of the mechanical properties of the material (e.g. stiffness) and introduction of specific biological ligands. Culturing neurons in 3D in tunable materials offers vast potential for deciphering both the morphogenic and molecular response to the in vivo ECM. These results provide a foundation to design optimal biomaterials for controlled nerve growth which is critical for the development of therapeutics for nerve repair.