RESOURCES

Abstract
Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications.

Abstract
Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of −80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.

Abstract
The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.

Abstract
Development of a biomimetic tubular scaffold capable of recreating developmental neurogenesis using pluripotent stem cells offers a novel strategy for the repair of spinal cord tissues. Recent advances in 3D printing technology have facilitated biofabrication of complex biomimetic environments by precisely controlling the 3D arrangement of various acellular and cellular components (biomaterials, cells and growth factors). Here, we present a 3D printing method to fabricate a complex, patterned and embryoid body (EB)-laden tubular scaffold composed of polycaprolactone (PCL) and hydrogel (alginate or gelatine methacrylate (GelMA)). Our results revealed 3D printing of a strong, macro-porous PCL/hydrogel tubular scaffold with a high capacity to control the porosity of the PCL scaffold, wherein the maximum porosity in the PCL wall was 15%. The method was equally employed to create spatiotemporal protein concentration within the scaffold, demonstrating its ability to generate linear and opposite gradients of model molecules (fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) and rhodamine). 3D bioprinting of EBs-laden GelMA was introduced as a novel 3D printing strategy to incorporate EBs in a hydrogel matrix. Cell viability and proliferation were measured post-printing. Following the bioprinting of EBs-laden 5% GelMA hydrogel, neural differentiation of EBs was induced using 1 μM retinoic acid (RA). The differentiated EBs contained βIII-tubulin positive neurons displaying axonal extensions and cells migration. Finally, 3D bioprinting of EBs-laden PCL/GelMA tubular scaffold successfully supported EBs neural differentiation and patterning in response to co-printing with 1 μM RA. 3D printing of a complex heterogeneous tubular scaffold that can encapsulate EBs, spatially controlled protein concentration and promote neuronal patterning will help in developing more biomimetic scaffolds capable of replicating the neural patterning which occurs during neural tube development.

Abstract
Abstract Electrically conductive materials that mimic physical and biological properties of tissues are urgently required for seamless brain–machine interfaces. Here, a multinetwork hydrogel combining electrical conductivity of 26 S m−1, stretchability of 800%, and tissue-like elastic modulus of 15 kPa with mimicry of the extracellular matrix is reported. Engineering this unique set of properties is enabled by a novel in-scaffold polymerization approach. Colloidal hydrogels of the nanoclay Laponite are employed as supports for the assembly of secondary polymer networks. Laponite dramatically increases the conductivity of in-scaffold polymerized poly(ethylene-3,4-diethoxy thiophene) in the absence of other dopants, while preserving excellent stretchability. The scaffold is coated with a layer containing adhesive peptide and polysaccharide dextran sulfate supporting the attachment, proliferation, and neuronal differentiation of human induced pluripotent stem cells directly on the surface of conductive hydrogels. Due to its compatibility with simple extrusion printing, this material promises to enable tissue-mimetic neurostimulating electrodes.

Abstract
Abstract Surface topography has a profound effect on the development of the nervous system, such as neuronal differentiation and morphogenesis. While the interaction of neurons and the surface topography of their local environment is well characterized, the neuron–topography interaction during the regeneration process remains largely unknown. To address this question, an anisotropic surface topography resembling linear grooves made from poly(ethylene-vinyl acetate) (EVA), a soft and biocompatible polymer, using nanoimprinting, is established. It is found that neurons from both the central and peripheral nervous system can survive and grow on this grooved surface. Additionally, it is observed that axons but not dendrites specifically align with these grooves. Furthermore, it is demonstrated that neurons on the grooved surface are capable of regeneration after an on-site injury. More importantly, these injured neurons have an accelerated and enhanced regeneration. Together, the data demonstrate that this anisotropic topography guides axon growth and improves axon regeneration. This opens up the possibility to study the effect of surface topography on regenerating axons and has the potential to be developed into a medical device for treating peripheral nerve injuries.

Abstract
Neural tissue engineering (TE), an innovative biomedical method of brain study, is very dependent on scaffolds that support cell development into a functional tissue. Recently, 3D patterned scaffolds for neural TE have shown significant positive effects on cells by a more realistic mimicking of actual neural tissue. In this work, we present a conductive nanocellulose-based ink for 3D printing of neural TE scaffolds. It is demonstrated that by using cellulose nanofibrils and carbon nanotubes as ink constituents, it is possible to print guidelines with a diameter below 1 mm and electrical conductivity of 3.8 × 10−1 S cm−1. The cell culture studies reveal that neural cells prefer to attach, proliferate, and differentiate on the 3D printed conductive guidelines. To our knowledge, this is the first research effort devoted to using cost-effective cellulosic 3D printed structures in neural TE, and we suppose that much more will arise in the near future.