Selective axonal growth of embryonic hippocampal neurons according to topographic features of various sizes and shapes

Abstract

Purpose Understanding how surface features influence the establishment and outgrowth of the axon of developing neurons at the single cell level may aid in designing implantable scaffolds for the regeneration of damaged nerves. Past studies have shown that micropatterned ridge-groove structures not only instigate axon polarization, alignment, and extension, but are also preferred over smooth surfaces and even neurotrophic ligands. Methods Here, we performed axonal-outgrowth competition assays using a proprietary four-quadrant topography grid to determine the capacity of various micropatterned topographies to act as stimuli sequestering axon extension. Each topography in the grid consisted of an array of microscale (approximately 2 μm) or submicroscale (approximately 300 nm) holes or lines with variable dimensions. Individual rat embryonic hippocampal cells were positioned either between two juxtaposing topographies or at the borders of individual topographies juxtaposing unpatterned smooth surface, cultured for 24 hours, and analyzed with respect to axonal selection using conventional imaging techniques. Results Topography was found to influence axon formation and extension relative to smooth surface, and the distance of neurons relative to topography was found to impact whether the topography could serve as an effective cue. Neurons were also found to prefer submicroscale over microscale features and holes over lines for a given feature size. Conclusion The results suggest that implementing physical cues of various shapes and sizes on nerve guidance conduits and other advanced biomaterial scaffolds could help stimulate axon regeneration.

Electroconductive polymeric nanowire templates facilitates in vitro C17.2 neural stem cell line adhesion, proliferation and differentiation

“…As such, there has been great interest in the field of tissue engineering for solutions aimed at such disorders. Neural cells on scaffolds with unique nanotopography have shown the most promising results for tissue engineering approaches to cure spinal cord diseases and injuries[9, 17, 33-37]. Thus, in this work, we have developed polymeric scaffolds with nanowire surfaces that were bio-functionalized with an electro-conductive polymer capable of providing physiological levels of electrical stimulation to NSCs.…”

Production of Highly Aligned Collagen Scaffolds by Freeze-drying of Self-assembled, Fibrillar Collagen Gels

“…Several studies have investigated the influence of feature size on cell alignment. 44–49 Many groups have identified nanoscale features as a critical determinant for guiding cell alignment, which led us to believe that the randomly oriented nanotopography would direct cell alignment in our scaffolds instead of the 10–100 µM microfeatures. 45–47 We found that both chick DRG explants and rat dermal fibroblasts could sense and respond to the microscale aligned topographical cues on the scaffold surface.…”

Substrate Topography Determines Neuronal Polarization and Growth In Vitro

“…Examples of in vivo guiding topography are the already formed glial processes and pre-existing axons, along which new axons migrate to establish connectivity [12]–[15]. Many studies have sought to explain how cellular and, more specifically, neuronal morphology are determined by topography [10], [16]–[19]. An important observation was that neurons sense isotropic micro-fabricated pillar surfaces, since they align to the pillar geometry.…”

“…With the use of interrupted microstructures (holes/pillars) this type of contact guidance is more readily realized [10], [11]. In a recent publication Fozdar et al [16] compared different shapes (lines and holes of (i) 300 nm and (i) 2 µm) for their ability to attract axons. The results show that for both dimension sizes, holes –or in general, interrupted features- are a more potent topographic cue to attract axonal specification, with over 70% of neurons extending to the holes vs grooves.…”