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Progress in the clinical application of recording and stimulation devices for neural diseases is still limited, mainly because of suboptimal material engineering and unfavorable interactions with biological entities. Nanotechnology is providing upgraded designs of materials to better mimic the native extracellular environment and attain more intimate contacts with individual neurons, besides allowing for the miniaturization of the electrodes. However, little progress has been done to date on the understanding of the biological impact that such neural interfaces have on neural network maturation and functionality. In this work, we elucidate the effect of a gold (Au) highly ordered nanostructure on the morphological and functional interactions with neural cells and tissues. Alumina-templated Au nanostructured electrodes composed of parallel nanowires of 160 nm in diameter and 1.2 mu m in length (Au-NWs), with 320 nm of pitch, are designed and characterized. Equivalent non-structured Au electrodes (Au-Flat) are used for comparison. By using diverse techniques in in vitro cell cultures including live calcium imaging, we found that Au-NWs interfaced with primary neural cortical cells for up to 14 days allow neural networks growth and increase spontaneous activity and ability of neuronal synchronization, thus indicating that nanostructured features favor neuronal network. The enhancement in the number of glial cells found is hypothesized to be behind these beneficial functional effects. The in vivo effect of the implantation of these nanostructured electrodes and its potential relevance for future clinical applicability has been explored in an experimental model of rat spinal cord injury. Subacute responses to implanted Au-NWs show no overt reactive or toxic biological reactions besides those triggered by the injury itself. These results highlight the translational potential of Au-NWs electrodes for in vivo applications as neural interfaces in contact with central nervous, Union Europea (Programa FP 7), Marie Sklodowska-Curie grant, Ministerio de Ciencia e Innovación (España), Project BiSURE, 'Severo Ochoa’ Programme for Centres of Excellence, Comunidad de Madrid, Depto. de Física de Materiales, Fac. de Ciencias Físicas, TRUE, pub

Graphene oxide (GO) assists a diverse set of promising routes to build bioactive neural microenvironments by easily interacting with other biomaterials to enhance their bulk features or, alternatively, self-assembling toward the construction of biocompatible systems with specific three-dimensional (3D) geometries. Herein, we first modulate both size and available oxygen groups in GO nanosheets to adjust the physicochemical and biological properties of polycaprolactone–gelatin electrospun nanofibrous systems. The results show that the incorporation of customized GO nanosheets modulates the properties of the nanofibers and, subsequently, markedly influences the viability of neural progenitor cell cultures. Interestingly, the partially reduced GO (rGO) nanosheets with larger dimensions trigger the best cell response, while the rGO nanosheets with smaller size provoke an accentuated decrease in the cytocompatibility of the resulting electrospun meshes. Then, the most auspicious nanofibers are synergistically accommodated onto the surface of 3D-rGO heterogeneous porous networks, giving rise to fibrous-porous combinatorial architectures suitable for enhancing adhesion and differentiation of neural cells. By varying the chemical composition of the nanofibers, it is possible to adapt their performance as physical crosslinkers for the rGO sheets, leading to the modulation of both pore size and structural/mechanical integrity of the scaffold. Importantly, the biocompatibility of the resultant fibrous-porous systems is not compromised after 14 days of cell culture, including standard differentiation patterns of neural progenitor cells. Overall, in light of these in vitro results, the reported scaffolding approach presents not only an indisputable capacity to support highly viable and interconnected neural circuits but also the potential to unlock novel strategies for neural tissue engineering applications.

Neural diseases at the central nervous system including spinal cord injury (SCI) remain therapeutic challenges. Graphene materials are being delineated as alternative tools for neural repair. Herein, the regenerative ability of reduced graphene oxide (rGO) scaffolds to support pivotal features of neural repair at 4 months after SCI is assessed by an interdisciplinary approach. 3D randomly porous foams have been prepared in mechanical compliance with neural cells and tissues (Young's modulus of 1.3 ± 1.0 kPa) as demonstrated by atomic force microscopy techniques applied ex vivo. After implantation, the significant increase in Young's modulus caused by massive cell/protein infiltration does not alter the mechanical performance of the contralateral spinal cord but provides mechanical stability to the lesion. These aerogels appear fully vascularized and populated with neurites, some of them being myelinated excitatory axons. Clinically-inspired magnetic resonance imaging studies demonstrate that the scaffolds significantly reduce perilesional damage with respect to rats without implants and cause no compressive damage in the contralateral hemicord and rostral/caudal regions. The rGO implants do not either alter the rat spontaneous behaviour or induce toxicity in major organs. Finally, preliminary data suggest hints of rGO sheets dissociation and eventual degradation at the injured spinal cord for the first time. In summary, these 3D porous rGO scaffolds are able to induce, without any further biological functionalization, a compilation of positive effects that have been rarely described before, if ever, for any other material implanted in the injured spinal cord to date.

Spinal cord injury (SCI) remain a therapeutic challenge. In this work, we have explored the regenerative ability of reduced graphene oxide (rGO) scaffolds to support pivotal features of neural repair at 4 months after SCI. 3D randomly porous foams have been prepared in mechanical compliance with neural cells and tissues. After 4 months, these scaffolds are fully vascularized and populated with neurites, some of them identified as myelinated excitatory axons. Magnetic resonance imaging studies demonstrate reduce perilesional damage in the presence of the scaffolds. In summary, these 3D porous rGO scaffolds are able to induce a compilation of positive effects that have been rarely described before, if ever, for any other material implanted in the injured spinal cord. The techniques used in this study include: immunofluorescence, scanning electron microscopy, atomic force microscopy, transmission electron microscopy, confocal laser scanning microscopy, histology, behavioral tests, magnetic resonance imaging, and X-ray photoelectron spectroscopy.

The attractiveness of graphene-derived materials (GDMs) for neural applications has fueled their exploration as components of biomaterial interfaces contacting the brain and the spinal cord. In the last years, an increasing body of work has been published on the ability of these materials to create biocompatible and biofunctional substrates able to promote the growth and activity of neural cells in vitro and positively interact with neural tissues when implanted in vivo. Encouraging results in the central nervous tissue might impulse the study of GDMs towards preclinical arena. In this mini-review article, we revise the most relevant literature on the interaction of GDMs with the spinal cord. Studies involving the implantation of these materials in vivo in the injured spinal cord are first discussed, followed by models with spinal cord slides ex vivo and a final description of selected results with neural cells in vitro. A closing debate of the major conclusions of these results is presented to boost the investigation of GDMs in the field.

Neural tissue engineering approaches show increasing promise for the treatment of neural diseases including spinal cord injury, for which an efficient therapy is still missing. Encouraged by both positive findings on the interaction of carbon nanomaterials such as graphene with neural components and the necessity of more efficient guidance structures for neural repair, we herein study the potential of reduced graphene oxide (rGO) microfibers as substrates for neural growth in the injured central neural tissue. Compact, bendable, and conductive fibers are obtained. When coated with neural adhesive molecules (poly-L-lysine and N-cadherin), these microfibers behave as supportive substrates of highly interconnected cultures composed of neurons and glial cells for up to 21 days. Synaptic contacts close to rGO are identified. Interestingly, the colonization by meningeal fibroblasts is dramatically hindered by N-cadherin coating. Finally, in vivo studies reveal the feasible implantation of these rGO microfibers as a guidance platform in the injured rat spinal cord, without evident signs of subacute local toxicity. These positive findings boost further investigation at longer implantation times to prove the utility of these substrates as components of advanced therapies for enhancing repair in the damaged central neural tissue including the injured spinal cord.

Attractive physic-chemical features of graphene oxide (GO) and promising results in vitro with neural cells encourage its exploration for biomedical applications including neural regeneration. Fueled by previous findings at the subacute state, we herein investigate for the first time chronic tissue responses (at 30 days) to 3D scaffolds composed of partially reduced GO (rGO) when implanted in the injured rat spinal cord. These studies aim to define fibrotic, inflammatory and angiogenic changes at the lesion site induced by the chronic implantation of these porous structures. Injured animals receiving no scaffolds show badly structured lesion zones and more cavities than those carrying rGO materials, thus pointing out a significant role of the scaffolds in injury stabilization and sealing. Notably, GFAP cells and pro-regenerative macrophages are evident at their interface. Moreover, rGO scaffolds support angiogenesis around and, more importantly, inside their structure, with abundant and functional new blood vessels in whose proximities inside the scaffolds some regenerated neuronal axons are found. On the contrary, lesion areas without rGO scaffolds show a diminished quantity of blood vessels and no axons at all. These findings provide a foundation for the usefulness of graphene-based materials in the design of novel biomaterials for spinal cord repair and encourage further investigation for the understanding of neural tissue responses to this kind of materials in vivo.

The increasing prevalence and high sanitary costs of lesions affecting the central nervous system (CNS) at the spinal cord are encouraging experts in different fields to explore new avenues for neural repair. In this context, graphene and its derivatives are attracting significant attention, although their toxicity and performance in the CNS in vivo remains unclear. Here, the subacute tissue response to 3D flexible and porous scaffolds composed of partially reduced graphene oxide is investigated when implanted in the injured rat spinal cord. The interest of these structures as potentially useful platforms for CNS regeneration mainly relies on their mechanical compliance with neural tissues, adequate biocompatibility with neural cells in vitro and versatility to carry topographical and biological guidance cues. Early tissue responses are thoroughly investigated locally (spinal cord at C6 level) and in the major organs (i.e., kidney, liver, lung, and spleen). The absence of local and systemic toxic responses, along with the positive signs found at the lesion site (e.g., filler effect, soft interface for no additional scaring, preservation of cell populations at the perilesional area, presence of M2 macrophages), encourages further investigation of these materials as promising components of more efficient material-based platforms for CNS repair., Ministerio de Economía y Competitividad (MINECO), Instituto de Salud Carlos III (ISCIII) / FEDER, ISCIII-MINECO (España), Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Fac. de Ciencias Biológicas, TRUE, pub

The increasing prevalence and high sanitary costs of lesions affecting the central nervous system (CNS) at the spinal cord are encouraging experts in different fields to explore new avenues for neural repair. In this context, graphene and its derivatives are attracting significant attention, although their toxicity and performance in the CNS in vivo remains unclear. Here, the subacute tissue response to 3D flexible and porous scaffolds composed of partially reduced graphene oxide is investigated when implanted in the injured rat spinal cord. The interest of these structures as potentially useful platforms for CNS regeneration mainly relies on their mechanical compliance with neural tissues, adequate biocompatibility with neural cells in vitro and versatility to carry topographical and biological guidance cues. Early tissue responses are thoroughly investigated locally (spinal cord at C6 level) and in the major organs (i.e., kidney, liver, lung, and spleen). The absence of local and systemic toxic responses, along with the positive signs found at the lesion site (e.g., filler effect, soft interface for no additional scaring, preservation of cell populations at the perilesional area, presence of M2 macrophages), encourages further investigation of these materials as promising components of more efficient material-based platforms for CNS repair.