IOP PUBLISHING JOURNAL OF NEURAL ENGINEERING J. Neural Eng. 4 (2007) 26–34 doi:10.1088/1741-2560/4/2/003 Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity Jennie B Leach 1,3 , Xin Q Brown 1 , Jeffrey G Jacot 1,4 , Paul A DiMilla 1,2 and Joyce Y Wong 1 1 Department of Biomedical Engineering, Boston University, 44 Cummington St., Boston, MA 02215, USA 2 Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave, Boston, MA 02115, USA E-mail: jleach@umbc.edu and jywong@bu.edu Received 24 July 2006 Accepted for publication 15 November 2006 Published 24 January 2007 Online at stacks.iop.org/JNE/4/26 Abstract Rationally designed matrices for nerve tissue engineering and encapsulated cell therapies critically rely on a comprehensive understanding of neural response to biochemical as well as biophysical cues. Whereas biochemical cues are established mediators of neuronal behavior (e.g., outgrowth), physical cues such as substrate stiffness have only recently been recognized to influence cell behavior. In this work, we examine the response of PC12 neurites to substrate stiffness. We quantified and controlled fibronectin density on the substrates and measured multiple neurite behaviors (e.g., growth, branching, neurites per cell, per cent cells expressing neurites) in a large sample population. We found that PC12 neurons display a threshold response to substrate stiffness. On the softest substrates tested (shear modulus ∼10 Pa), neurites were relatively few, short in length and unbranched. On stiffer substrates (shear modulus ∼10 2 –10 4 Pa), neurites were longer and more branched and a greater percentage of cells expressed neurites; significant differences in these measures were not found on substrates with a shear modulus >10 2 Pa. Based on these data and comparisons with published neurobiology and neuroengineering reports of neurite mechanotransduction, we hypothesize that results from studies of neuronal response to compliant substrates are cell-type dependent and sensitive to ligand density, sample size and the range of stiffness investigated. (Some figures in this article are in colour only in the electronic version) Introduction The stiffness of a cell’s environment impacts cell adhesion, proliferation, migration, differentiation and phenotype [1–11]. 3 Current Affiliation: Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA. 4 Current Affiliation: Department of Bioengineering, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA. Seminal work in 1997 by Wang et al [3] first demonstrated that fibroblast spreading and migration are greater on stiffer compared to more compliant polyacrylamide hydrogels. In subsequent years, the spread area and motility of myoblasts, osteoblasts, vascular smooth muscle cells and endothelial cells have also been demonstrated to correlate with substrate stiffness [2, 5, 12–14]. As a consequence many researchers have proposed a correlation between matrix stiffness and cellular processes in injury [15], disease [16] and cancer [17]. 1741-2560/07/020026+09$30.00 © 2007 IOP Publishing Ltd Printed in the UK 26