Spinal Cord Explants Use Carbon Nanotube Interfaces To Enhance Neurite Outgrowth and To Fortify Synaptic Inputs

Abstract

New developments in nanotechnology are increasingly designed to modulate relevant interactions between nanomaterials and neurons, with the aim of exploiting the physical properties of synthetic materials to tune desired and specific biological processes. Carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. Recent reports show that nanotubes can sustain and promote electrical activity in networks of cultured neurons. However, such results are usually limited to carbon nanotube/neuron hybrids formed on a monolayer of dissociated brain cells. In the present work, we used organotypic spinal slices to model multilayer tissue complexity, and we interfaced such spinal segments to carbon nanotube scaffolds for weeks. By immunofluorescence, scanning and transmission electronic microscopy, and atomic force microscopy, we investigated nerve fiber growth when neuronal processes exit the spinal explant and develop in direct contact to the substrate. By single-cell electrophysiology, we investigated the synaptic activity of visually identified ventral interneurons, within the ventral area of the explant, thus synaptically connected, but located remotely, to the substrate/network interface. Here we show that spinal cord explants interfaced for weeks to purified carbon nanotube scaffolds expand more neuronal fibers, characterized by different mechanical properties and displaying higher growth cones activity. On the other hand, exploring spontaneous and evoked synaptic activity unmasks an increase in synaptic efficacy in neurons located at as far as 5 cell layers from the cell-substrate interactions.

Carbon Nanotubes: Artificial Nanomaterials to Engineer Single Neurons and Neuronal Networks

“…It is therefore suggested that CNTs affect neuronal firing activity and synaptic connectivity in the neuronal layer directly facing CNT scaffold (in agreement with previous findings on dissociated neurons 27,28,30 ), where CNTs intimately and extensively interact with membranes (Figure 4c), 10 and that this alteration is remotely translated into an amplification of synaptic signals at the level of ventral interneurons relatively far from the CNT interface.…”

“…A step beyond these considerations emerged in the same work 10 where we reported, for the first time, the impact of CNT interfaces to cell layers distant from the CNTs themselves. More explicitly, CNT scaffolds are able to modulate the functional performance of neurons spatially far from the scaffold.…”

“…We characterized the longterm impact on neuronal performance of interfacing spinal cord explants with scaffolds of purified MWCNTs. 10 In our experimental model, we cocultured spinal cord and dorsal root ganglia (DRG) slices on a film of purified, nonfunctionalized CNTs or on control glass, and we explored how explants interfaced with the CNT substrates in the long term (up to three weeks) developed; in particular, the growing ability of neurites and the functional properties of spinal neurons were assessed. In agreement with our previous reports (see above), CNTs efficiently sustained spinal neurons survival and growth in vitro.…”

Carbon nanotube interaction with extracellular matrix proteins producing scaffolds for tissue engineering

“…143 A breakthrough study from Fabbro et al developed for the first time an artificial nanomaterials-based scaffold to explore the semichronic (weeks) impact of CNT interfaces on spinal segment growth and activity. 149 They demonstrated that the long-term impact of an artificial MWNT meshwork characterized by large surface roughness and conductivity 150,151 favors neurite regrowth in spinal explants, with the appearance of increased growth-cone activity (Figure 2). 149 Ultimately, this study supports one of the emerging strategies in nanoscale engineering, which is the use of physical features alone to guide different biological responses, without the levels of sophistication required by biomolecule selective patterning.…”

“…149 They demonstrated that the long-term impact of an artificial MWNT meshwork characterized by large surface roughness and conductivity 150,151 favors neurite regrowth in spinal explants, with the appearance of increased growth-cone activity (Figure 2). 149 Ultimately, this study supports one of the emerging strategies in nanoscale engineering, which is the use of physical features alone to guide different biological responses, without the levels of sophistication required by biomolecule selective patterning. 152 CNTs have also been shown to favor the differentiation of human embryonic stem cells (hESCs) into neurons.…”