Schwann cell durotaxis can be guided by physiologically relevant stiffness gradients

Abstract

BackgroundSuccessful 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.MethodsPolyacrylamide 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.ResultsOn 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (10.1186/s40824-018-0124-z) contains supplementary material, which is availab...

Mechanical Response of Neural Cells to Physiologically Relevant Stiffness Gradients

“…We fabricated collagen substrates with stiffnesses of physiological relevance to the PNS by varying the collagen concentration using bespoke 3D‐printed moulds and the RAFT‐stabilization process. Two different collagen gradients (Lower and Higher) were created and compared to control plain RAFT‐stabilized gels with no gradient (Control).…”

“…Our primary goal was to design a flat substrate with a characterized rigidity gradient to study cell responses to mechanical cues. We created physiologically relevant stiffness gradients within the range of modulus values reported previously for PNS tissue using tissue engineering techniques to control collagen matrix density . Collagen gels were generated and characterized, exhibiting a flat surface topography and continuous linear gradients of defined density, which correlated to the stiffness .…”

“…By contrast, the approach used here does not affect the microstructure of the collagen fibrils and the banding pattern is conserved after gel stabilization, offering a way to make gradients purely of collagen, in a simple, controlled, and reproducible way. Previous studies have reported the effects of similar substrate stiffness profiles for other types of cells (including human adipose‐derived stem cells, human mesenchymal stem cells, vascular smooth muscle cells, fibroblasts, and Schwann cells). Focusing on the improvement of nerve repair solutions, Evans et al, have shown that a 0.95 kPa mm −1 gradient induces Schwann cell durotaxis.…”

“…Cells exert forces on, and sense the stiffness of the surrounding extracellular matrix (ECM) . The mechanical properties of the microenvironment have important implications in cell differentiation, proliferation, and migration . For nervous system cells, neurite outgrowth and branching patterns have been shown to depend on the substrate stiffness .…”

“…Koser et al have highlighted cellular durotactic responses to a stiffness gradient, by showing that axon bundles were orientated toward the softer side of their growth substrate. Therefore, biomimetic mechanical gradients have the potential to be used in the construction of nerve repair materials, to improve axon pathfinding and accelerate nerve repair by controlling the orientation of neurite regeneration …”