Three-dimensional carbon nanotube-polymer composite promotes functional reconnection of organotypic spinal slices

The development of novel microporous materials, able to support neuronal cells growth and development in the three-dimensions, is triggered by the increasing necessity to make available to the medical community new tools for neuro-regenerative and neuro-prosthetic applications. A three-dimensional structure could provide cells with the physical and mechanical cues and framework to build tissue-like formations and more natural cell-to-cell interactions. Anyhow, to be truly effective and not limit just in providing the appropriate geometry, these materials need to be further modified to provide either the (micro)morphological or the interactive chemical and signaling environment necessary for complex functional tissue formation.
In this context carbon nanotubes (CNT) have repeatedly been shown to provide an electrically favorable environment to increase/improve neuronal signaling improving the probability of a neuron to fire an action potential. Here we developed and characterized a composite 3D elastomeric material incorporated with CNT and tested as neuronal reconnecting promotor using organotypic spinal slices. 
A micro-porous, free-standing, PDMS scaffold, composed by PDMS surface functionalized by means of CNT, was developed following a sugar mold dissolution procedure. Scanning electron microscopy analysis of the composite scaffold revealed a “sponge-like” morphology (Figure 1a), and the presence of a carpet of CNT lining inner facets of the pores (see insets). Three-dimensional X-ray microtomography (µCT) was performed at the SYRMEP beamline of Elettra to point out the inner pore and channel organization of the material (Figure 1b). Pores and throats were analyzed following a skeleton analysis and using the concept of maximal inscribed sphere. The scaffold is characterized by pores of irregular shape and dimension having a mean fill-up sphere diameter of 18±11 µm, interconnected by interconnection throats 9±6 µm in size. To compare effects of material 3D morphology on the spread of spinal neurons from organotypic spinal cord slices, we pointed out neuronal fibers by means of anti-β-Tubulin III immunolabeling and confocal microscopy to visualize the spatial distribution of neuronal cells and processes (Figure 2a). Image stacks demonstrate that neurites were distributed in complex network formations across the surface of the substrates, and to varying depths into the substrates (red filaments). The primary interest of this study was to investigate the in vitroeffects of the PDMS+CNT porous composite on the electrophysiological properties of spinal slices and possible (re)formation of functional physiological connections between slice pairs. Slices were cultured side-by-side in pairs at distances known to prevent effective functional reconnection in basal conditions.
 

Figure 1.  Morphological analysis of the PDMS+CNT composite scaffolds designed for culturing organotypic spinal slices.In (a) SEM micrograph of the material pointing out its “sponge-like” aspect, consequence of sugar grains dissolution. A high-resolution image of a pore surface portion (inset), reveals carbon-nanotubes decorating pore’s walls and resulting in a rough surface. Low magnification SEM images scale bar: 100 µm. Inset scale bars: 1 µm.  In (b) a µCT volumetric reconstructions of scaffolds’ pores and channelization in a ROI of 5123 voxels of the total volume. In blue and green the maximal filling sphere for pore and throat diameters determination, respectively, and in red the interconnection path skeleton. Samples show that all empty spaces inside the material are interconnected, giving rise to a single, branched out volume.

Spinal slice functional activity was assessed by performing extracellular dorsal stimulations and recordings of local field potential from ventral regions of each slice (Figure 2b, above). Interestingly, the frequency of electrical bursts from the two slices, was significantly higher in spinal slices cultured on the PDMS+CNT composite compared with controls suggesting material ability to improve the strength of neuronal signaling and synapses. 
To determine if there was cross communication between the two separated explants we evaluated if any synchrony in the activity between the spinal slice pairs exists. We stimulated at a defined frequency one of the two slices, inducing a rhythmic response in it, while the activity of the second slice was simultaneously recorded. The two trains of electrical activity were subsequently analyzed in terms of cross-correlation (CCF) to evaluate the synchronicity degree: higher CCF perhaps indicates presence of functional contacts between the two slices. The percentage of correlated spinal slice pairs was significantly higher on PDMS+CNT when compared with controls (81%, n=6, compared with 37.8%, n=8, respectively; p<0.05, Figure 2b, below), suggesting that this composite material have the ability to increase functional (re)connections between the spinal slices interfaced with it.

Figure 2.  In (a) the three-dimensional distribution of β-Tubulin III immunolabeled neuronal processes (in red) form vast networks when cultured on our PDMS+CNT porous composite matrix. Scale bars: 50 µm. In (b) a schematic drawing depicting the experimental setup for the characterization of the electrophysiological activity of spinal slices when plated on PDMS+CNT scaffolds (above). Below some representative traces depict the bursting activity of the slices: frequency of events is significantly higher in the presence of CNTs on PDMS surfaces, compared with spinal slices cultured on controls (PDMS without CNTs). Moreover, a higher synchronous activity between the two slices is present, suggesting a functional reconnection.

Our data suggest that the incorporation of multi-walled carbon nanotubes into a three-dimensional polymer substrate confers increased activity and synchronization, suggesting increased connectivity, of two organotypic spinal slices integrated with the scaffold. Here we demonstrate that the benefit is not simply due to the three-dimensionality, but that carbon nanotubes can contribute an added benefit to improve material functionality.

This research was conducted by the following research team:

Emily R. Aurand¹, Sadaf Usmani¹, Federica B. Rosselli¹, Jummi Laishram¹, Laura Ballerini¹,Manuela Medelin², Denis Scaini², Susanna Bosi², Maurizio Prato², Sandro Donato³ and Giuliana Tromba³

¹ Scuola Internazionale Superiore di Studi Avanzati, Via Bonomea, 265 - 34136 Trieste, ITALY
² University of Trieste, Via Giorgieri, 1 – 34127 Trieste ITALY
³ Elettra-Sincrotrone Trieste S.C.p.A., AREA Science Park, 34149 Basovizza, Trieste ITALY

Contact persons:

Denis Scaini, email: