Potential Roles of Specific Subclasses of Premotor Interneurons in Spinal Cord Function Recovery after Traumatic Spinal Cord Injury in Adults.

The differential expression of transcription factors during embryonic development has been selected as the main feature to define the specific subclasses of spinal interneurons. However, recent studies based on single-cell RNA sequencing and transcriptomic experiments suggest that this approach might not be appropriate in the adult spinal cord, where interneurons show overlapping expression profiles, especially in the ventral region. This constitutes a major challenge for the identification and direct targeting of specific populations that could be involved in locomotor recovery after a traumatic spinal cord injury in adults. Current experimental therapies, including electrical stimulation, training, pharmacological treatments, or cell implantation, that have resulted in improvements in locomotor behavior rely on the modulation of the activity and connectivity of interneurons located in the surroundings of the lesion core for the formation of detour circuits. However, very few publications clarify the specific identity of these cells. In this work, we review the studies where premotor interneurons were able to create new intraspinal circuits after different kinds of traumatic spinal cord injury, highlighting the difficulties encountered by researchers, to classify these populations.

Flexible metallic core-shell nanostructured electrodes for neural interfacing.

Electrodes with nanostructured surface have emerged as promising low-impedance neural interfaces that can avoid the charge-injection restrictions typically associated to microelectrodes. In this work, we propose a novel approximation, based on a two-step template assisted electrodeposition technique, to obtain flexible nanostructured electrodes coated with core-shell Ni-Au vertical nanowires. These nanowires benefit from biocompatibility of the Au shell exposed to the environment and the mechanical properties of Ni that allow for nanowires longer and more homogeneous in length than their only-Au counterparts. The nanostructured electrodes show impedance values, measured by electrochemical impedance spectroscopy (EIS), at least 9 times lower than those of flat reference electrodes. This ratio is in good accordance with the increased effective surface area determined both from SEM images and cyclic voltammetry measurements, evidencing that only Au is exposed to the medium. The observed EIS profile evolution of Ni-Au electrodes over 7 days were very close to those of Au electrodes and differently from Ni ones. Finally, the morphology, viability and neuronal differentiation of rat embryonic cortical cells cultured on Ni-Au NW electrodes were found to be similar to those on control (glass) substrates and Au NW electrodes, accompanied by a lower glial cell differentiation. This positive in-vitro neural cell behavior encourages further investigation to explore the tissue responses that the implantation of these nanostructured electrodes might elicit in healthy (damaged) neural tissues in vivo, with special emphasis on eventual tissue encapsulation.

High-Performance Implantable Sensors based on Anisotropic Magnetoresistive La0.67Sr0.33MnO3 for Biomedical Applications.

We present the design, fabrication, and characterization of an implantable neural interface based on anisotropic magnetoresistive (AMR) magnetic-field sensors that combine reduced size and high performance at body temperature. The sensors are based on La0.67Sr0.33MnO3 (LSMO) as a ferromagnetic material, whose epitaxial growth has been suitably engineered to get uniaxial anisotropy and large AMR output together with low noise even at low frequencies. The performance of LSMO sensors of different film thickness and at different temperatures close to 37 °C has to be explored to find an optimum sensitivity of ∼400%/T (with typical detectivity values of 2 nT·Hz-1/2 at a frequency of 1 Hz and 0.3 nT·Hz-1/2 at 1 kHz), fitted for the detection of low magnetic signals coming from neural activity. Biocompatibility tests of devices consisting of submillimeter-size LSMO sensors coated by a thin poly(dimethyl siloxane) polymeric layer, both in vitro and in vivo, support their high suitability as implantable detectors of low-frequency biological magnetic signals emerging from heterogeneous electrically active tissues.

Nanostructured gold electrodes promote neural maturation and network connectivity.

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 µm 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 tissues including the injured spinal cord.

Interfacing Neurons with Nanostructured Electrodes Modulates Synaptic Circuit Features.

Understanding neural physiopathology requires advances in nanotechnology-based interfaces, engineered to monitor the functional state of mammalian nervous cells. Such interfaces typically contain nanometer-size features for stimulation and recording as in cell-non-invasive extracellular microelectrode arrays. In such devices, it turns crucial to understand specific interactions of neural cells with physicochemical features of electrodes, which could be designed to optimize performance. Herein, versatile flexible nanostructured electrodes covered by arrays of metallic nanowires are fabricated and used to investigate the role of chemical composition and nanotopography on rat brain cells in vitro. By using Au and Ni as exemplary materials, nanostructure and chemical composition are demonstrated to play major roles in the interaction of neural cells with electrodes. Nanostructured devices are interfaced to rat embryonic cortical cells and postnatal hippocampal neurons forming synaptic circuits. It is shown that Au-based electrodes behave similarly to controls. Contrarily, Ni-based nanostructured electrodes increase cell survival, boost neuronal differentiation, and reduce glial cells with respect to flat counterparts. Nonetheless, Au-based electrodes perform superiorly compared to Ni-based ones. Under electrical stimulation, Au-based nanostructured substrates evoke intracellular calcium dynamics compatible with neural networks activation. These studies highlight the opportunity for these electrodes to excite a silent neural network by direct neuronal membranes depolarization.

3D Reduced Graphene Oxide Scaffolds with a Combinatorial Fibrous-Porous Architecture for Neural Tissue Engineering.

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.

Combined Magnetoliposome Formation and Drug Loading in One Step for Efficient Alternating Current-Magnetic Field Remote-Controlled Drug Release.

We have developed a reproducible and facile one step strategy for the synthesis of doxorubicin loaded magnetoliposomes by using a thin-layer evaporation method. Liposomes of around 200 nm were made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and iron oxide nanoparticles (NPs) with negative, positive, and hydrophobic surfaces that were incorporated outside, inside, or between the lipid bilayers, respectively. To characterize how NPs are incorporated in liposomes, advanced cryoTEM and atomic force microscope (AFM) techniques have been used. It was observed that only when the NPs are attached outside the liposomes, the membrane integrity is preserved (lipid melt transition shifts to 38.7 °C with high enthalpy 34.8 J/g) avoiding the leakage of the encapsulated drug while having good colloidal properties and the best heating efficiency under an alternating magnetic field (AMF). These magnetoliposomes were tested with two cancer cell lines, MDA-MB-231 and HeLa cells. First, 100% of cellular uptake was achieved with a high cell survival (above 80%), which is preserved (83%) for doxorubicin-loaded magnetoliposomes. Then, we demonstrate that doxorubicin release can be triggered by remote control, using a noninvasive external AMF for 1 h, leading to a cell survival reduction of 20%. Magnetic field conditions of 202 kHz and 30 mT seem to be enough to produce an effective heating to avoid drug degradation. In conclusion, these drug-loaded magnetoliposomes prepared in one step could be used for drug release on demand at a specific time and place, efficiently using an external AMF to reduce or even eliminate side effects.

Graphene Oxide Microfibers Promote Regenerative Responses after Chronic Implantation in the Cervical Injured Spinal Cord.

Spinal cord injury (SCI) is characterized by the disruption of neuronal axons and the creation of an inhibitory environment for spinal tissue regeneration. For decades, researchers and clinicians have been devoting a great effort to develop novel therapeutic approaches which include the fabrication of biocompatible implants that could guide neural tissue repair in the lesion site in an attempt to recover the functionality of the nervous tissue. In this context, although fiberlike structures have been hypothesized to serve as a topographical guidance for axonal regrowth, work on the exploration of this type of materials is still limited for SCI. Aiming to develop such guidance platforms, we recently designed and explored in vitro reduced graphene oxide materials in the shape of microfibers (rGO-MFs). After preliminary studies to assess the feasibility of their implantation at the injured spinal cord in vivo, no evident signs of subacute local toxicity were noticed (10 days of implantation). In this work, we specifically examine for the first time the regenerative potential of these scaffolds, slightly modified in their fabrication for improved reproducibility, when chronically interfaced with a cervical spinal cord injury. After extensive characterization of their physicochemical properties and in vitro experiments with neural progenitor cells, their neural regenerative capacity in vivo is investigated in a rat experimental model of SCI after 4 months of implantation (chronic state). Behavioral tests involving the use of forelimbs are performed. Immunofluorescence studies evidence that rGO-MFs scaffolds foster the presence of neuronal structures along with blood vessels both within the epicenter and in the surroundings of the lesion area. Moreover, the inflammatory response does not worsen by the presence of this material. These findings outline the potential of rGO-MF-based scaffolds to promote regenerative features at the injured spinal cord such as axonal and vascular growth. Further studies including biological functionalization might improve their therapeutic potential by a synergistic effect of topographical and chemical cues, thus boosting neural repair after SCI.

Myelinated axons and functional blood vessels populate mechanically compliant rGO foams in chronic cervical hemisected rats.

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 ±â€¯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.

Graphene-Derived Materials Interfacing the Spinal Cord: Outstanding in Vitro and in Vivo Findings.

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.