Neurite outgrowth and β1-integrin signaling pathways in 3D systems

Andreia Ribeiro¹, Carla Guarraia¹, Erin L. Voss¹, Elizabeth M. Powell^2,3 and Jennie B. Leach¹

¹Department of Chemical & Biochemical Engineering, University of Maryland, Baltimore County, ²Department of Anatomy & Neurobiology, University of Maryland, Baltimore, ³Program in Neuroscience, University of Maryland, Baltimore

Translating information from two-dimensional (2D) culture into three-dimensional (3D) systems has been a major hurdle in the use of biopolymers for tissue engineering, including neuronal repair, applications. Environmental cues are critical for cellular maturation and function and in vivo, extracellular matrix (ECM) molecules, growth factors and adhesion molecules are presented in a 3D environment.  While 2D substrates have been incredibly valuable in revealing intricacies of cell biology, current studies demonstrate that non-neuronal cells dramatically alter signaling pathways when placed in a 3D environment.  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. Preliminary results show that 3D culture demonstrates more diffuse integrin mediated signaling as neurons are embedded in Type I collagen, a major component of the ECM and ligand for β1-integrin.  We test our model using dorsal root ganglion (DRG) neurons isolated from E13.5 mouse embryos embedded in Type I collagen gels.  We further test our model using a synthetic polyethylene-glycol (PEG) crosslinked hydrogel.  PEG represents a pioneering biomaterial with highly tunable properties including the ability to modulate both the mechanical properties of the material, including stiffness, and introduction of specific ligands.  We examine the expression patterns and phosphorylation of downstream molecules including Rho GTPases and focal adhesion kinase (FAK).  Culturing neurons in 3D, 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.

Keywords: tissue engineering, neuronal repair, cell response, neurite outgrowth, β1-integrin signaling, 3D culture, DRG neurons, collagen gels, PEG hydrogels