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Title: Neuron networking with nano bridges via the synthesis and integration of funcionalized carbon nanotubes
Authors: Villari, Ambra
Supervisore/Tutore: Ballerini, Laura
Issue Date: 22-Mar-2013
Publisher: Università degli studi di Trieste
Regenerative medicine is a broad interdisciplinary field, tremendously grown in the last decades, which encompasses several different research areas, such as biomaterial sciences and tissue engineering, whose unifying concept holds an enormous therapeutic potential, being that of restoring impaired organs or tissue functions, resulting from congenital defects, trauma or disease (Greenwood et al.; 2006; Mason and Dunnill, 2008).
The main challenge faced by tissue engineering is the need to have a renewable source of cells and biomaterials possessing the right mechanical, chemical and biological features, to create constructs resembling native tissues. The design of scaffolds able to support and promote tissue regeneration and/or functional restore, in particular, is a critical step for the success of an implant, as it should recapitulate the complex architecture of the physiological microenvironment, e.g. the appropriate extracellular matrix, which has been shown to actively direct the behaviour of cells, through both chemical and physical cues (Daley et al., 2008; Place et al., 2009; Rozario and DeSimone, 2010). In this context, nanotechnology tools may greatly enhance the success of tissue engineering strategies, by providing the chance of producing surfaces and materials with topographical features that mimic the natural ones, in addition to the possibility to functionalize nanomaterials with bioactive molecules (Gelain et al., 2008; Zhang and Webster , 2009; Dvir et al., 2011; Koh et al., 2008).
Among nanomaterials, carbon nanotubes (CNTs) stood out, since their discovery, for their outstanding mechanical, electrical and thermal properties, like their extraordinary strength coupled with remarkable flexibility, or their high electrical conductivity, which make them a well-suited platform technology for biomedical applications. Recently, several works have been published, which support the use of CNT-based scaffolds to promote neuronal attachment, differentiation and growth (Mattson et al., 2000; Hu et al., 2004; Hu et al., 2005; Galvan-Garcia et al., 2007). Moreover, in the last decade, our group showed that CNT/neuronal hybrid networks show a boost in synaptic transmission (Lovat et al., 2005; Mazzatenta et al., 2007) and that the direct contact established between single CNT and neuronal membranes affect single neuron integrative abilities (Cellot et al., 2009), besides promoting network connectivity and synaptic plasticity phenomena in cortical cultured circuits (Cellot et al., 2011).
Here, to extend our knowledge about interactions between CNT and neurons, we long-term cultured organotypic spinal explants, possessing a complex multilayered cytoarchitecture, with highly purified MWCNT scaffolds and then investigated, via a multidisciplinary approach, their growth and synaptic activity. Our aim was to verify whether and how a CNT-induced effect on neuronal performance could be transferred to network locations, which are far from the neuronal/MWCNT layer of interaction, but sinaptically communicating with it.
We documented, via TEM investigations, the presence of tight connections established between the neuronal membranes of neurons belonging to the bottom layer of the spinal tissue and the CNT meshwork underlying it. By means of confocal microscopy, SEM and AFM techniques, we showed, for the first time, that the long-term interfacing of spinal cord explants to CNTs induced an increase in the number and length of peripheral neuronal fibres outgrowing the spinal tissue, associated to changes in growth cone activity and in fibre elastomechanical features.
We also demonstrated, via patch-clamp recordings performed from interneurons in the ventral (premotor) area of the explants, that both spontaneous and evoked synaptic currents displayed a potentiation in the presence of the CNT scaffold, detected as an increase in current amplitude in neurons which were as far as 5 cell layers from the tissue/substrate site of interaction.
We speculate that these two effects (the increased fibres growth and the boosting in synaptic activity) rely upon two different mechanisms, a direct and a remote one, by which CNTs affect the spinal tissue development. Indeed, the first exerted on fibres directly adhering to the CNT substrate, while the second is likely to be mediated by alterations occurring at the tissue layer integrated with CNTs, which are transmitted, through a remote effect, to distant network locations, synaptically communicating with such a layer.
These results support the hypothesis that CNTs may be employed to boost spinal neurite re-growth and functional spinal performance, in the perspective of re-establishing the physical and functional communication between disconnected spinal segments, We therefore decided to implement a model in which two organotypic spinal explants grow together on the same support, as a useful model for neuronal reconnection investigations and to test the possibility that a CNT-based scaffold, interposed between the two explants, may act as a bridge to promote the physical and electrical communication between the two spinal segments.
By means of immunostaining experiments and confocal microscopy we reported the presence of a huge amount of fibres, projecting from the two spinal slices and integrating in a complex network, especially localized in the DRG regions, while very few fibres seemed to directly connect the two explanted tissues at the level of the explants cores. When, via voltage-clamp pair recordings, we looked for the presence of an electrical reconnection between explants, we found a small percentage of co-cultured explants displaying a complex coupled behaviour, detected as a strongly correlated bursting activity.
These preliminary data seem to confirm the goodness of such an in vitro model to investigate the intrinsic reparative potential of spinal cord tissue and to improve such ability via nanotechnological tools.
Ciclo di dottorato: XXV Ciclo
Keywords: Nerve tissue engineering
Carbon nanotubes
Organotypic cultures
Spinal cord
Language: en
Type: Doctoral Thesis
Settore scientifico-disciplinare: BIO/09 FISIOLOGIA
NBN: urn:nbn:it:units-9953
Appears in Collections:Scienze biologiche

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