ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance

Abstract

Numerous nanostructured synthetic scaffolds mimicking the architecture of the natural extracellular matrix (ECM) have been described, but the polymeric nanofibers comprising the scaffold were substantially thicker than the natural collagen nanofibers of neural ECM. Here, we report neuron growth on electrospun scaffolds of nylon-4,6 fibers with an average diameter of 60 nm, which closely matches the diameter of collagen nanofibers of neural ECM, and compare their properties with the scaffolds of thicker 300 nm nanofibers. Previously unmodified nylon was not regarded as an independent nanostructured matrix for guided growth of neural cells; however, it is particularly useful for ultrathin nanofiber production. We demonstrate that, while both types of fibers stimulate directed growth of neuronal processes, ultrathin fibers are more efficient in promoting and accelerating neurite elongation. Both types of scaffolds also improved synaptogenesis and the formation of connections between hippocampal neurons; however, the mechanisms of interaction of neurites with the scaffolds were substantially different. While ultrathin fibers formed numerous weak immature β1-integrin-positive focal contacts localized over the entire cell surface, scaffolds of submicron fibers formed β1-integrin focal adhesions only on the cell soma. This indicates that the scaffold nanotopology can influence focal adhesion assembly involving various integrin subunits. The fabricated nanostructured scaffolds demonstrated high stability and resistance to biodegradation, as well as absence of toxic compound release after 1 month of incubation with live cells in vitro. Our results demonstrate the high potential of this novel type of nanofibers for clinical application as substrates facilitating regeneration of nervous tissue.

Light-to-Heat Converting ECM-Mimetic Nanofiber Scaffolds for Neuronal Differentiation and Neurite Outgrowth Guidance

“…As we reported previously [42], nylon fibers can significantly increase the proliferation rate of neuroblastoma cells. Since even minor changes in the nanotopology of a material can cause significant changes in cell behavior [4], we tested the cytotoxicity of the nanoparticle decorated materials.…”

“…Several studies reported preparation and successful application of artificial nerve conduits based on electrospun fibrous materials in biological test systems [13,62,63]. However, relatively few works are deploying fiber nanomaterials with fiber diameters of 100 nm due to technical difficulties of manufacturing [42]. The use of composite light-converting nanomaterials demonstrates efficiency in stimulating differentiation of neural cells [59,60]; therefore, we believe that the combination of topology-mediated stimulation with thermally induced control of cellular activity is of practical interest for the creation of neurosurgical implants.…”

“…For each replicate three independent measurements were performed, in which at least 100 cells were analyzed. The length and orientation of neurites were analyzed using NeuronJ and OrientationJ plugins for ImageJ software (v. 1.5, National Institutes of Health [NIH]) as described earlier [42].…”

“…Previously, we have described neuron growth on electrospun scaffolds of nylon-4,6 aligned ultrathin (AU) fibers with an average diameter of 60 nm, which closely match the diameter of collagen nanofibers of neural ECM. This biocompatible nanomaterial has been shown to stimulate directed accelerating neurite elongation, improved synaptogenesis, and formation of connections between hippocampal neurons [42]. In this work, we present a hybrid biocompatible nanomaterial consisting of oriented polymeric nanofibers decorated with TNPs using alternative methods: internal impregnation during electrospinning process and chemical immobilization on the surface.…”

Biomaterials-based strategies for in vitro neural models

“…These anisotropic meshes provide the neural population with a path of least resistance for growth in a general direction. 187,197 This concept is useful for matrix-assisted neurite guidance which makes it possible to model nerve fibers in vitro. 198 Besides being recruited as aligned 3D scaffolds or 2D substrates, they can be used as sacrificial fibers to imprint another substrate with the anisotropic fiber pattern to guide axonal growth.…”