Schwann cell durotaxis can be guided by physiologically relevant stiffness gradients

Evans et al. Biomaterials Research (2018) 22:14 https://doi.org/10.1186/s40824-018-0124-z RESEARCH ARTICLE Open Access Schwann cell durotaxis can be guided by physiologically relevant stiffness gradients Elisabeth B. Evans1 , Samantha W. Brady1 , Anubhav Tripathi1,2 and Diane Hoffman-Kim1,2,3,4* Abstract Background: Successful nerve regeneration depends upon directed migration of morphologically specialized repair state Schwann cells across a nerve defect. Although several groups have studied directed migration of Schwann cells in response to chemical or topographic cues, the current understanding of how the mechanical environment influences migration remains largely understudied and incomplete. Therefore, the focus of this study was to evaluate Schwann cell migration and morphodynamics in the presence of stiffness gradients, which revealed that Schwann cells can follow extracellular gradients of increasing stiffness, in a form of directed migration termed durotaxis. Methods: Polyacrylamide substrates were fabricated to mimic the range of stiffness found in peripheral nerve tissue. We assessed Schwann cell response to substrates that were either mechanically uniform or embedded with a shallow or steep stiffness gradient, respectively corresponding to the mechanical niche present during either the fluid phase or subsequent matrix phase of the peripheral nerve regeneration process. We examined cell migration (velocity and directionality) and morphology (elongation, spread area, nuclear aspect ratio, and cell process dynamics). We also characterized the surface morphology of Schwann cells by scanning electron microscopy. Results: On laminin-coated polyacrylamide substrates embedded with either a shallow (∼0.04 kPa/mm) or steep (∼0.95 kPa/mm) stiffness gradient, Schwann cells displayed durotaxis, increasing both their speed and directionality along the gradient materials, fabricated with elastic moduli in the range found in peripheral nerve tissue. Uniquely and unlike cell behavior reported in other cell types, the durotactic response of Schwann cells was not dependent upon the slope of the gradient. When we examined whether durotaxis behavior was accompanied by a pro-regenerative Schwann cell phenotype, we observed altered cell morphology, including increases in spread area and the number, elongation, and branching of the cellular processes, on the steep but not the shallow gradient materials. This phenotype emerged within hours of the cells adhering to the materials and was sustained throughout the 24 hour duration of the experiment. Control experiments also showed that unlike most adherent cells, Schwann cells did not alter their morphology in response to uniform substrates of different stiffnesses. Conclusion: This study is notable in its report of durotaxis of cells in response to a stiffness gradient slope, which is greater than an order of magnitude less than reported elsewhere in the literature, suggesting Schwann cells are highly sensitive detectors of mechanical heterogeneity. Altogether, this work identifies durotaxis as a new migratory modality in Schwann cells, and further shows that the presence of a steep stiffness gradient can support a pro-regenerative cell morphology. Keywords: Schwann cell, Durotaxis, Peripheral nerve regeneration, Gradient, Band of Büngner, Morphodynamics *Correspondence: Department of Molecular Pharmacology, Physiology, Brown University, Providence, Rhode Island, 02912 USA 2 Center for Biomedical Engineering, Brown University, Providence, Rhode Island, 02912 USA Full list of author information is available at the end of the article 1 © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Evans et al. Biomaterials Research (2018) 22:14 Background Peripheral nerve injuries often result in significant disability and greatly reduced quality of life from persistent neuropathic pain. Further, clinical consequences of peripheral nerve injuries are long-lasting, given that the highest incidence occurs most commonly in younger healthy patients [1]. Better clinical nerve repair treatments are needed, as functional recovery in patients with current treatments is often unsatisfactory and off-site complications arise at secondary surgical sites. Physical and functional impairments following injury are the result of incomplete or aberrant nerve regeneration across the nerve defect, in which the proximal nerve end either entirely or partially fails to reconnect to its original innervation target. In contrast, successful reinnervation is dependent upon directed migration of morphologically-specialized Schwann cells across the injury site. Once Schwann cells migrate into the defect and assemble into an aligned elongated structure, severed axons can regenerate, using this transient structure for directional guidance. If Schwann cells fail to either migrate across the nerve defect or establish a direct trajectory for axonal regrowth, the process of functional nerve regeneration will not occur. Although several groups have developed biomaterials that present various guidance cues including chemotactic, haptotactic, and topographical gradients to enhance nerve repair through directed migration of Schwann cells [2], the current understanding of how the mechanical environment influences Schwann cell migration remains largely understudied and incomplete. Recent reports, including studies from our group, have begun to characterize the mechanosensitivity of Schwann cells [3–5]. Specifically, we previously observed that migratory Schwann cells variably alter their traction forces on uniform substrates tuned to different discrete stiffnesses found within the peripheral nervous system. Given that the post-injury nerve environment is mechanically dynamic and heterogeneous, in this study we cultured Schwann cells on substrates embedded with stiffness gradients to determine whether Schwann cells can display durotaxis, directed cell migration in response to a gradient of increasing stiffness [6]. The presence of mechanical gradients has previously been described in neurodevelopmental tissue niches, physiologic niches where the processes of both directional migration and morphological specialization are ubiquitous [7]. Focal adhesion kinase, which is necessary for durotaxis [8], also mediates specific physiological processes in Schwann cells, including the induction of a pro-migratory phenotype following nerve inju (...truncated)