title: Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth.
abstract: The design of bioactive materials allows tailored studies probing cell-biomaterial interactions, however, relatively few studies have examined the effects of ligand density and material stiffness on neurite growth in three-dimensions. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within three-dimensional (3-D) materials. To promote neurite out-growth and better understand the effects of common biomaterial design parameters on neuronal cultures we here focused on the cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins these 3-D ELP hydrogels enabled decoupled investigations into the effects of biomechanics and biochemistry on neurite out-growth from dorsal root ganglia. Increasing the cell-adhesive RGD ligand density from 0 to 1.9×10(7)ligands μm(-3) led to a significant increase in the rate, length, and density of neurite out-growth, as quantified by a high throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite out-growth was observed in materials with the higher ligand density at all time points up to 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest out-growth, with some neurites extending over 1800μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite out-growth within defined and tunable 3-D microenvironments these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron-biomaterial interactions.
authors.affiliation: Materials Science and Engineering Department, Stanford University, Stanford, CA 94305, USA.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Lampe
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Antaris
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Heilshorn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002645:Chickens; D055119:Elastic Modulus; D004549:Elastin / Q000737:chemistry / Q000494:pharmacology*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects; D020100:Hydrogels / Q000737:chemistry / Q000494:pharmacology*; D008024:Ligands; D008969:Molecular Sequence Data; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*
chemical_list: D020100:Hydrogels; D008024:Ligands; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D004549:Elastin