Variations in rigidity and ligand density influence neuronal response in methylcellulose-laminin hydrogels

Cells are continuously sensing their physical and chemical environment, generating dynamic interactions with the surrounding microenvironment and neighboring cells. Specific to neurons, neurite outgrowth is influenced by many factors, including the mechanical properties and adhesive signals of the growth substrata. In designing biomaterials for neural regeneration, it is important to understand the influence of substrate material, rigidity and bioadhesion on neurite outgrowth. To this end, we developed and characterized a tunable 3-D methylcellulose (MC) hydrogel polymeric system tethered to laminin-1 (MC-x-LN) across a range of substrate rigidities (G* range = 50-565 Pa) and laminin densities. Viability and neurite outgrowth of primary cortical neurons plated within 3-D MC hydrogels were used as cell outcome measures. After 4 days in culture, neuronal viability was significantly augmented with increasing rigidity for MC-x-LN as compared to control non-bioactive MC; however, neurite outgrowth was only observed in MC hydrogels with complex moduli of 565 Pa. Varying LN while maintaining a constant MC formulation (G* = 565 Pa) revealed a threshold response for neuronal viability, whereas a direct dose-dependent response to LN density was observed for neurite outgrowth. Collectively, these data demonstrate the synergistic play between material compliance and bioactive ligand concentrations within MC hydrogels. Such results can be used to better understand the adhesive and mechanical factors that mediate neuronal response to MC-based, tissue-engineered materials.