www.afm-journal.de FULL PAPER © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1 www.MaterialsViews.com wileyonlinelibrary.com Amy M. Hopkins, Laura De Laporte, Federico Tortelli, Elise Spedden, Cristian Staii, Timothy J. Atherton, Jeffrey A. Hubbell, and David L. Kaplan* 1. Introduction Spinal cord and traumatic brain injuries (TBI) are sustained by millions of people each year, with almost half a million emer- gency department visits for TBI made annually by children. [1] Research efforts toward neural tissue engi- neering are in the early stages of discovery, with investigations of regeneration of cen- tral nervous system (CNS) tissues, [2,3] soft biomaterials to decrease inflammation from implantable devices, [4–7] and non- invasive drug delivery to the CNS. [6,8–10] Previous efforts to develop biomaterials for neural tissue repair have focused on anti-inflammatory coatings for implant- able devices [10–13] or construction of nerve guide conduits for peripheral and central nerve repair. [14–22] With increased focus on advanced, three-dimensional biomaterial systems, progress is being made toward the engineering of soft neural tissues ex vivo for modeling and implantation. It is evident that substrate stiffness sig- nificantly affects cell attachment, survival, and growth and therefore should be taken into consideration when engineering scaf- folds for neural cell culture. Traditionally, neuronal cells cultured in vitro are plated on laminin (LN)-, fibronectin (FN)-, poly- lysine-coated glass or tissue culture plastic (TCP) providing integrin binding sites for cell attachment and neurite extension. [23–26] Neural tissues are inherently soft, with stiffness values generally less than 100 kPa (ranging from <1 kPa for brain slices [27] and single brain cells [28] to ≈90–230 kPa for spinal cord, [29,30] as compared to ≈70 GPa for glass). This has prompted studies of neural cell growth and function on substrates of varying stiffness. For example, Koch et al. found that peripheral nerve outgrowth is more sensitive to changes in substrate stiffness as compared to CNS hippocampal neural extension. [31] This is perhaps due to the increased diversity in mechanical and biochemical environment of peripheral nervous system extracellular matrices (ECMs) versus those of the CNS, where connective tissue consists mostly of glial cells. [31] There is also evidence that effects of substrate stiffness on neuron extension and differentiation vary with the presence of particular ECM molecules and enzyme expression. [32] In addition, biochemical cues are integral to reconstruction of neural tissue in vitro, specifically for nerve growth guidance toward the target effector without collateral sprouting. Many groups have reported on the soluble and contact-mediated ECM factors responsible for axonal guidance including both hapto- tactic cues (i.e. collagen, LN, FN, and cell adhesion molecules) and chemotactic cues (i.e. neurotrophic factors). [33–37] However, Silk Hydrogels as Soft Substrates for Neural Tissue Engineering There is great need for soft biomaterials that match the stiffness of human tissues for tissue engineering and regeneration. Hydrogels are frequently employed for extracellular matrix functionalization and to provide appropriate mechanical cues. It is challenging, however, to achieve structural integrity and retain bioactive molecules in hydrogels for complex tissue formation that may take months to develop. This work aims to investigate mechanical and biochemical characteristics of silk hydrogels for soft tissue engineering, spe- cifically for the nervous system. The stiffness of 1 to 8% silk hydrogels, meas- ured by atomic force microscopy, is 4 to 33 kPa. The structural integrity of silk gels is maintained throughout embryonic chick dorsal root ganglion (cDRG) explant culture over 4 days whereas fibrin and collagen gels decrease in mass over time. Neurite extension of cDRGs cultured on 2 and 4% silk hydrogels exhibit greater growth than softer or stiffer gels. Silk hydrogels release <5% of neurotrophin-3 (NT-3) over 2 weeks and 11-day old gels show maintenance of growth factor bioactivity. Finally, fibronectin- and NT-3-functionalized silk gels elicit increased axonal bundling suggesting their use in bridging nerve injuries. These results support silk hydrogels as soft and sustainable biomate- rials for neural tissue engineering. DOI: 10.1002/adfm.201300435 A. M. Hopkins, Prof. D. L. Kaplan Department of Biomedical Engineering Tufts University Medford, MA 02155, USA E-mail: david.kaplan@tufts.edu A. M. Hopkins, Dr. L. De Laporte, Dr. F. Tortelli Prof. J. A. Hubbell Institute of Bioengineering School of Life Sciences and Institute for Chemical Sciences and Engineering, School of Basic Sciences École Polytechnique Fédérale de Lausanne (EPFL) 1015 Lausanne, Switzerland E. Spedden, Prof. C. Staii, Prof. T. J. Atherton Department of Physics and Astronomy and Center for Nanoscopic Physics Tufts University Medford, MA 02155, USA Adv. Funct. Mater. 2013, DOI: 10.1002/adfm.201300435