3D Scaffolds for tissue engineering
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In our group, we have explored the interactions of carbon nanotubes with electrically active biosystems, specifically neuronal and cardiac systems, in particular to understand how the presence of carbon nanotubes (CNTs) influences the electrical activity of neurons and cardiomyocytes, especially in terms of synaptic events and syncytia formation respectively. In our initial experiments, we found out that neonatal hippocampal neurons, laid over a film of CNTs, increases noteworthy their electrical spontaneous activity. In the following years, we were able to demonstrate that not only the number of synapses increase in the presence of CNTs, but also the integration of neuronal compartments and CNTs is very deep and generates very tight interactions of the two systems. High resolution electron microscopy investigation revealed extremely intimate contacts between regrown neuronal processes and underneath CNTs to the point that the boundary line between organic and inorganic matter is ambiguous. As a consequence, in the past decade, we have demonstrated that CNT-based substrates are indeed able to profoundly impact on neuronal physiology from the functional (electrical) point of view. We have studied CNTs as substrates for neuronal growth in the form of a mat of pure, non-functionalized multi walled carbon nanotubes (MWCNT), obtained by CNT functionalization (by 1,3-dipolar cycloaddition of azomethine ylides), deposition and thermal defunctionalization, yielding a stable and homogeneous CNT meshwork and then we have progressed with 3D substrates based on a composite made of a biocompatible polymeric matrix and carbon nanotubes. These latter materials have shown to be particularly promising both for neurons and for cardiomyocytes. We fabricated a microporous, self–standing, elastomeric scaffold whose skeleton material is made of polydimethylsiloxane (PDMS), while the micrometric cavities are generated by dissolving a sugar template embedded by PDMS (Figure 1).
Figure 1
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This polymer allows to manufacture 3D structures with a spongy appearance characterized by pores of irregular shape and dimension, interconnected by variable paths of connectivity. The maximal diameter of the pores is in the 20÷150 µm range leading to 40% PDMS sponge final porosity with a corresponding bulk density of 0.58 g/cm3, as determined by gravimetric measurements. SEM indicates that the sugar amount exceed percolation limit for such a system resulting in the generation of interconnected pores giving rise to intricate networks of channels within the PDMS scaffold (Figure 2).
Figure 2
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To exploit their effect in the three-dimensional neuronal circuits, we incorporated MWCNTs in the PDMS porous scaffold. Within the same fabrication procedure described above, we are able to generate micrometric cavities upholstered with carbon nanotubes by a sugar mold that was previously mixed to functionalized MWCNTs. This allows the formation of scaffolds with the pores layered by an irregular carbon nanotube carpet (around 100 nm thick) stably entrapped in the PDMS matrix. The porous environment is perfectly similar to the previously described PDMS scaffolds except that MWCNTs are exposed on the hole surfaces. The two scaffolds (with and without MWCNTs) did not display differences in compressibility.
We tested the biocompatibility of both 3D materials by growing hippocampal cells in vitro. In both cases, proper neuronal growth and development were documented by immunofluorescence staining and confocal microscopy (Figure 3A). Three core milestones were set up by our pioneering work: first, a three-dimensional cellular organization is per se able to induce neuronal network outputs that strongly differ from the usual 2D construct; second, we succeeded in extending from 2D to 3D the exceptionally unique capabilities of CNTs to improve and boost neuronal functionality; third, the 3D PDMS scaffold was biocompatible when implanted for 4 weeks in the rat brain (Figure 3 B and C). Together these three achievements pave the way to further develop new and innovative paradigms in the nano-neuroscience research arena.
Figure 3
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Our preliminary proof-of-concept investigations in cardiac cells grown on 2D MWCNT substrates have shown that neonatal rat ventricular miocytes (NRVMs) cultured on carbon nanotubes exhibit improved viability, proliferation, maturation, and electrical properties compared with control substrates (2D gelatin-coated tissue culture dishes). In contrast, cardiac fibroblasts showed a decrease in their proliferative capacity on these substrates, which allowed for a favorable and stable cardiomyocyte/cardiac fibroblast ratio over time. In vitro biocompatibility of 3D-PDMS and 3D-PDMS+MWCNT was demonstrated by evaluating its effect on NRVMs. As previously demonstrated on 2D scaffolds, MWCNT enriched substrates are responsible for an improvement of cardiomyocytes viability and retention and is associated with a significantly higher proliferative capacity when compared to those on control (gelatin coated dishes). Here, based on a metabolic activity assay, we found that 3D-PDMS+MWCNT scaffolds exhibit again a significant improvement in NRVMs cell retention and viability as indicated by the relative absorbance, compared to 3D-PDMS control. Another important aspect of cardiomyocyte culture on 3D scaffolds was their electrophysiological maturation. We previously found that NRVMs grown on carbon nanotube scaffolds, either 2D MWCNT substrates or 3D reverse-thermal polymers with integrated MWCNTs, had a more developed and mature electrophysiological phenotypes as shown by higher gap junction connexin 43 protein expression content, better organized in structural syncytia. Similarly, the study of intracellular communication and electrical coupling of NRVMS by immunofluorescence revealed an increase in connexin 43 expression on 3D-PDMS+MWCNTs compared to 3D-PDMS control with a more homogeneous distribution and a significant higher number of cell-junctions . Cardiomyocytes are mainly terminally differentiated cells that do not proliferate sufficiently to recover the loss of cardiomyocytes during an injury. However, it has been shown that in the early stages of neonatal life in vivo and in vitro, they maintain a low level of proliferative capacity. There is evidence from our previous investigations that carbon nanotubes interacting with NRVMs boost their proliferative capacity, as shown by the number of active cell duplications in MWCNT enriched scaffolds both in 2D and 3D environments. We also observed that carbon nanotubes inhibit cardiac fibroblast proliferation. We found that carbon nanotubes boosted the proliferation of NRVMs, which were significantly increased in 3D-PDMD +MWCNT (8%) when compared with the NRVMs cultured in the PDMS scaffolds (3.2%, p < 0.005) 72 h after seeding. Furthermore, as we previously reported for other carbon nanotube-enriched-scaffolds, no significant difference on the number of proliferating cardiac fibroblasts was observed.
The 3D-PDMS enriched by MWCNT condition exhibits mechanical and conductive properties similar to the native heart muscle and may represent an alternative to injectable polymers for distinct clinical applications, such as solid patches for congenital tissue defects or large infarcted fibrotic areas.
Highlighted contributions:
3D Carbon Nanotube-Based Composites for Cardiac Tissue Engineering.
V. Martinelli, S. Bosi, B. Peña, G. Baj, C.S. Long, O. Sbaizero, M. Giacca, M. Prato, L. Mestroni. ACS Appl. Bio Mater. 15, 1530-1537 (2018). Link
Successful Regrowth of Retinal Neurons When Cultured Interfaced to Carbon Nanotube Platforms.
G. Cellot, S. La Monica, D. Scaini, R. Rauti, S. Bosi, M. Prato, S. Gandolfi, L. Ballerini. J. Biomed. Nanotechnol., 13, 559–565 (2017). Link
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3D meshes of carbon nanotubes guide functional reconnection of segregated spinal explants.
S. Usmani, E.R. Aurand, M. Medelin, A. Fabbro, D. Scaini, J. Laishram, F.B. Rosselli, A. Ansuini, D. Zoccolan, M. Scarselli, M. De Crescenzi, S. Bosi, M. Prato, L. Ballerini. Sci. Adv. 2, e1600087 (2016). Link
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From 2D to 3D: novel nanostructured scaffolds to investigate signalling in reconstructed neuronal networks.S. Bosi, R. Rauti, J. Laishram, A. Turco, D. Lonardoni, T. Nieus, M. Prato, D. Scaini, L. Ballerini. Sci. Rep. Article n. 9562 (2015). Link
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Improving cardiac myocytes performance by carbon nanotubes platforms.
V. Martinelli, G. Cellot, A. Fabbro, S. Bosi, L. Mestroni, L. Ballerini. Front. Physiol. 4, 239 (2013). Link
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Interfacing neurons with carbon nanotubes: (re)engineering neuronal signaling.
A. Fabbro, G. Cellot, M. Prato, L. Ballerini, Prog. Brain Res. 194, 241-252 (2011). Link
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Carbon nanotubes might improve neuronal performance by favouring electrical shortcuts.
G. Cellot, E. Cilia, S. Cipollone, V. Rancic, A. Sucapane, S. Giordani, L. Gambazzi, H. Markram, M. Grandolfo, D. Scaini, F. Gelain, L. Casalis, M. Prato, M. Giugliano, L. Ballerini. Nat. Nanotechnol. 4, 126-133 (2009). Link
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Carbon Nanotube Substrates Boost Neuronal Electrical Signaling.
V. Lovat, D. Pantarotto, L. Lagostena, B. Cacciari, M. Grandolfo, M. Righi, G. Spalluto, M. Prato, L. Ballerini. Nano Lett 5, 1107-1110 (2005). Link