Advances in 3D tissue models for neural engineering: self-assembled versus engineered tissue models.

Neural tissue engineering has emerged as a promising field that aims to create functional neural tissue for therapeutic applications, drug screening, and disease modelling. It is becoming evident in the literature that this goal requires development of three-dimensional (3D) constructs that can mimic the complex microenvironment of native neural tissue, including its biochemical, mechanical, physical, and electrical properties. These 3D models can be broadly classified as self-assembled models, which include spheroids, organoids, and assembloids, and engineered models, such as those based on decellularized or polymeric scaffolds. Self-assembled models offer advantages such as the ability to recapitulate neural development and disease processes in vitro, and the capacity to study the behaviour and interactions of different cell types in a more realistic environment. However, self-assembled constructs have limitations such as lack of standardised protocols, inability to control the cellular microenvironment, difficulty in controlling structural characteristics, reproducibility, scalability, and lengthy developmental timeframes. Integrating biomimetic materials and advanced manufacturing approaches to present cells with relevant biochemical, mechanical, physical, and electrical cues in a controlled tissue architecture requires alternate engineering approaches. Engineered scaffolds, and specifically 3D hydrogel-based constructs, have desirable properties, lower cost, higher reproducibility, long-term stability, and they can be rapidly tailored to mimic the native microenvironment and structure. This review explores 3D models in neural tissue engineering, with a particular focus on analysing the benefits and limitations of self-assembled organoids compared with hydrogel-based engineered 3D models. Moreover, this paper will focus on hydrogel based engineered models and probe their biomaterial components, tuneable properties, and fabrication techniques that allow them to mimic native neural tissue structures and environment. Finally, the current challenges and future research prospects of 3D neural models for both self-assembled and engineered models in neural tissue engineering will be discussed.

Connectomic reconstruction of a female Drosophila ventral nerve cord.

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.

Validation of a Cleanroom Compliant Sonication-Based Decellularization Technique: A New Concept in Nerve Allograft Production.

Defects of the peripheral nervous system are extremely frequent in trauma and surgeries and have high socioeconomic costs. If the direct suture of a lesion is not possible, i.e., nerve gap > 2 cm, it is necessary to use grafts. While the gold standard is the autograft, it has disadvantages related to its harvesting, with an inevitable functional deficit and further morbidity. An alternative to autografting is represented by the acellular nerve allograft (ANA), which avoids disadvantages of autograft harvesting and fresh allograft rejection. In this research, the authors intend to transfer to human nerves a novel technique, previously implemented in animal models, to decellularize nerves. The new method is based on soaking the nerve tissues in decellularizing solutions while associating ultrasounds and freeze-thaw cycles. It is performed without interrupting the sterility chain, so that the new graft may not require post-production γ-ray irradiation, which is suspected to affect the structural and functional quality of tissues. The new method is rapid, safe, and inexpensive if compared with available commercial ANAs. Histology and immunohistochemistry have been adopted to evaluate the new decellularized nerves. The study shows that the new method can be applied to human nerve samples, obtaining similar, and, sometimes better, results compared with the chosen control method, the Hudson technique.

3D bioprinted silk fibroin hydrogels for tissue engineering.

The development of biocompatible and precisely printable bioink addresses the growing demand for three-dimensional (3D) bioprinting applications in the field of tissue engineering. We developed a methacrylated photocurable silk fibroin (SF) bioink for digital light processing 3D bioprinting to generate structures with high mechanical stability and biocompatibility for tissue engineering applications. Procedure 1 describes the synthesis of photocurable methacrylated SF bioink, which takes 2 weeks to complete. Digital light processing is used to fabricate 3D hydrogels using the bioink (1.5 h), which are characterized in terms of methacrylation, printability, mechanical and rheological properties, and biocompatibility. The physicochemical properties of the bioink can be modulated by varying photopolymerization conditions such as the degree of methacrylation, light intensity, and concentration of the photoinitiator and bioink. The versatile bioink can be used broadly in a range of applications, including nerve tissue engineering through co-polymerization of the bioink with graphene oxide, and for wound healing as a sealant. Procedure 2 outlines how to apply 3D-printed SF hydrogels embedded with chondrocytes and turbinate-derived mesenchymal stem cells in one specific in vivo application, trachea tissue engineering, which takes 2-9 weeks.

Biodegradable and biocompatible graphene-based scaffolds for functional neural tissue engineering: A strategy approach using dental pulp stem cells and biomaterials.

Neural tissue engineering aims to restore the function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of three-dimensional (3D) graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that the addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p < 0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of the scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly- l-lysine was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favorable effects on the biological responses of DPSCs.

Histological, Biomechanical, and Biological Properties of Genipin-Crosslinked Decellularized Peripheral Nerves.

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Fabrication of 3D Scaffolds Displaying Biochemical Gradients along Longitudinally Oriented Microchannels for Neural Tissue Engineering.

Biochemical and physical guidance cues are both pivotal for axonal guidance and nerve regeneration. However, fabrication of a platform that can integrate biochemical gradients and topographical guidance cues remains challenging, especially in a three-dimensional (3D) scaffold that closely mimics in vivo axonal outgrowth conditions and ready to be used for in vivo nerve repair. In this study, a new method was introduced to construct 3D scaffolds displaying continuous biochemical gradients along longitudinally oriented microchannels by combining the modified 3D printing and directional freezing techniques. Fluorescence analysis and ELISA results demonstrated that a continuous biochemical gradient was formed, and scanning electron microscopy revealed a longitudinally oriented microstructure. Dorsal root ganglia explants seeded on the longitudinal sections of the newly developed scaffold (scaffold with nerve growth factor gradient along oriented microstructure, G-NGF + OS) showed that 81.3 ± 4.5% of neurites oriented within ±10°, 0.3 ± 0.1 of guidance ratio, and 1.5-fold of the average length of neurites on the high-nerve growth factor (NGF) concentration side compared to that on the low-NGF concentration side, which were significantly higher than those in the other groups. In addition, the G-NGF + OS scaffold was used to repair a 15 mm sciatic nerve defect in rats. Immunofluorescence staining, Fluoro-Gold retrograde tracing, and transmission electron microscopy results confirmed that the G-NGF + OS scaffold enhanced nerve regeneration to the distal target and promoted myelination of regenerated axons. More importantly, the sciatic functional index and the von Frey test demonstrated that the G-NGF + OS scaffold accelerated sensory and motor functional recovery. These results provide new insights into the importance of combining topographical guidance cues with bioactive molecule gradient cues for neural tissue engineering. The 3D scaffold displaying biochemical gradients along longitudinally oriented microchannels represents a promising platform for the development of advanced devices for severe nervous system injuries.

Generation of Vascularized Neural Organoids by Co-culturing with Mesodermal Progenitor Cells.

Organoids are three-dimensional (3D) constructs generated in stem cell cultures and are thought to mimic tissue and organ development in situ. However, until recently, they often exclusively recapitulated the development of the organ`s parenchyma without the major components of the organ stroma. Here, we describe a protocol to incorporate stromal components, first of all blood vessels, by co-culturing with induced pluripotent stem cell-derived mesodermal progenitor cells. For complete details on the use and execution of this protocol, please refer to Wörsdörfer et al. (2019).

Fibroblasts Colonizing Nerve Conduits Express High Levels of Soluble Neuregulin1, a Factor Promoting Schwann Cell Dedifferentiation.

Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both express NRG1ß. These data suggest that sNRG1 might be mainly expressed by fibroblasts colonizing nerve conduit before Schwann cells. Immunohistochemistry analysis confirmed NRG1 and fibroblast marker co-localization. These results suggest that fibroblasts, releasing sNRG1, might promote Schwann cell dedifferentiation to a "repair" phenotype, contributing to peripheral nerve regeneration.

Mechanical properties of the sciatic nerve following combined transplantation of analytically extracted acellular allogeneic nerve and adipose-derived mesenchymal stem cells.

PURPOSE: To investigate the effects of Chemically Extracted Acellular Nerves (CEANs) when combined with Adipose-Derived mesenchymal Stem Cell (ADSC) transplantation on the repair of sciatic nerve defects in rabbits. METHODS: A total of 71 six-month-old Japanese rabbit were used in this study. Twenty rabbits served as sciatic nerve donors, while the other 51 rabbits were randomly divided into Autologous Nerve Transplantation Group (ANT, n=17), CEAN group (n=17) and CEAN-ADSCs group (n=17). In all these groups, the rabbit's left sciatic nerves were injured before the experiment, and the uninjured sciatic nerves on their right side were used as the control (CON). Electrophysiological tests were carried out and sciatic nerves were prepared for histomorphology and stretch testing at 24 weeks post-transplant. RESULTS: There were significant differences between ANT and Con groups in amplitude (AMP): P=0.031; motor nerve conduction velocity (MNCV): P=0.029; Maximum stress: P=0.029; and Maximum strain P=0.027. There were also differences between the CEAN and CEAN+ADSCs groups in AMP: P=0.026, MNCV: P=0.024; Maximum stress: P=0.025 and Maximum strain: P=0.030. No significant differences in these parameters were observed when comparing the ANT and CEAN+SACN groups (MNCV: P=0.071) or the CEAN and ANT groups (Maximum stress: P=0.069; Maximum strain P=0.077). CONCLUSION: Addition of ADSCs has a significant impact on the recovery of nerve function, morphology, and tensile mechanical properties following sciatic nerve injury.

Effect of the Cell Alive System on nerve tissue cryopreservation.

Effective cellular cryopreservation while maintaining high cell viability is achieved by preventing intracellular and extracellular ice crystal formation using the Cells Alive System (CAS), a programmed freezer that applies a magnetic field. Here, the optimal temperature settings of the CAS were determined using rat sciatic nerves as a model tissue. Firstly, it was found that Schwann cell survival was increased by pre-cooling the samples in the ice crystal formation zone, increasing the freeze-thaw speed, and freezing-thawing in a magnetic field. Secondly, the setting (intensity and frequency) of the magnetic field at freezing-thawing was changed, and the optimum magnetic field strength was determined by evaluating cell viability. At the set temperature excluding previous studies, the minimum temperature was set to - 50 °C and kept frozen for 15 min, and then thawed immediately. The highest cell viability (27%) was achieved at 0.67 mT (intensity 3 [29.6 V] and frequency setting 10 [60 Hz]). The effects of the freeze-thaw program were assessed using transplanted sciatic nerve tissues removed after 2, 4, and 8 weeks. Anterior tibial muscle wet weight increased at 8 weeks in the control (without freezing) and after freezing-thawing in a magnetic field, compared to that without a magnetic field. Fluorescence staining of the sciatic nerve with anti-S100 antibodies revealed that Schwann cell counts increased at the transplanted site (at 8 weeks) of nerves that were freeze-thawed in a magnetic field. Overall, the CAS prevented ice crystal formation in rat sciatic nerves and could be used to maintain cell viability during cryopreservation.

Integrating Organs-on-Chips: Multiplexing, Scaling, Vascularization, and Innervation.

Organs-on-chips (OoCs) have attracted significant attention because they can be designed to mimic in vivo environments. Beyond constructing a single OoC, recent efforts have tried to integrate multiple OoCs to broaden potential applications such as disease modeling and drug discoveries. However, various challenges remain for integrating OoCs towards in vivo-like operation, such as incorporating various connections for integrating multiple OoCs. We review multiplexed OoCs and challenges they face: scaling, vascularization, and innervation. In our opinion, future OoCs will be constructed to have increased predictive power for in vivo phenomena and will ultimately become a mainstream tool for high quality biomedical and pharmaceutical research.

Biodegradable cell-laden starch foams for the rapid fabrication of 3D tissue constructs and the application in neural tissue engineering.

Cells encapsulation by biomaterials has been widely studied as a strategy of building tissue construct in tissue engineering. Conventional encapsulation of cells using hydrogels often needs the polymerization process or relatively complex molding process. In this study, we developed a facile strategy for the in situ fabrication of biodegradable cell-laden starch foams. By utilizing the unique gelatinization property of starch, cell-laden starch foams with tunable architecture were rapidly prepared in a green and biological-friendly process. The bubble size and stiffness of starch foams could be tuned by controlling the content of premixed starch in the cell culture medium. Cells were encapsulated in situ during the foaming process, and the resultant starch foams could be used as building blocks to fabricate three-dimensional tissue construct. The potential application of the cell-laden starch foams in neural tissue engineering was also validated. RSC96 Schwann cells were encapsulated in the starch foams and revealed good viability. Due to the serum-induced degradation of the starch, RSC96 Schwann cells could be released from the starch foams in a controlled manner while remaining high viability. Dorsal root ganglion (DRG) neurons co-cultured with the cell-laden starch foams extended significantly longer neurites compared with neurons cultured in minimum Eagle's medium (664.88 ± 190.39 µm vs. 311.19 ± 105.25 µm). DRG neurons retained high viability even after encapsulation in the starch foams for 3 days. This facile strategy of rapidly fabricating cell-laden starch foams can be further extended to construct centimeter-scale micro-tissue for tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:104-116, 2020.

Electrospun natural polymer and its composite nanofibrous scaffolds for nerve tissue engineering.

Attributed to the excellent biocompatibility and desirable mechanical properties to natural tissue, natural polymer-based electrospun nanofibers have drawn extensive research interests in tissue engineering. Electrospun nanofibers have been explored as scaffolds in tissue engineering to modulate cellular behavior. Also, electrospun nanofiber matrices have morphological similarities to the natural extra-cellular matrix (ECM). Natural polymer and its composite nanofiber mats are the promising candidates in governing nerve cells growth and nerve regeneration due to their unique characteristics such as high permeability, stability, porosity, suitable mechanical performance and excellent biocompatibility. In this review, the progress in electrospun natural polymers and its composite nanofibers scaffold for neural tissue engineering are presented. The influences of fiber orientation and electrical stimulation on the nerve cell behavior and neurite growth are systematically summarized. Furthermore, the current application of natural polymer composite scaffold as in vivo implantable device for nerve regeneration is also discussed (see Figure 1).

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach.

Bees are important pollinators that help to maintain the biodiversity of wild and cultivated plants. However, the increased and inappropriate use of agrochemicals has caused an imbalance in the populations of these insects visiting flowers for pollen and nectar collection. Therefore, new research methods for understanding the mechanisms of action of pesticides and their impacts on the brains of bees, such as neurotoxicity and cellular changes, in response to different active characteristics and dosages of insecticides are necessary. Thus, with the aim of developing tests with greater specificity at the level of cells or tissues, this study sought to standardize a method for the in vitro culture of the nervous tissue of Apis mellifera. For this purpose, the brains of six foragers bees were transferred to three different insect cell culture media and it supplementation with 10% foetal bovine serum (FBS): Grace, Schneider, Leibovitz, Grace + FBS, Schneider + FBS and Leibovitz + FBS media for each collection time. Nervous tissue was collected after 1, 6, 12 and 24 h of incubation in a humidified CO2 incubator at 32 °C, and histological sections of the organs were analysed. The results showed that Leibovitz medium and Leibovitz medium + serum are potential culture media for the cultivation of nervous tissue, since they resulted in less tissue spacing and tissue disarrangement. Therefore, additional supplements are necessary to obtain an ideal medium for the cultivation of A.mellifera nervous tissue.

Engineering geometrical 3-dimensional untethered in vitro neural tissue mimic.

Formation of tissue models in 3 dimensions is more effective in recapitulating structure and function compared to their 2-dimensional (2D) counterparts. Formation of 3D engineered tissue to control shape and size can have important implications in biomedical research and in engineering applications such as biological soft robotics. While neural spheroids routinely are created during differentiation processes, further geometric control of in vitro neural models has not been demonstrated. Here, we present an approach to form functional in vitro neural tissue mimic (NTM) of different shapes using stem cells, a fibrin matrix, and 3D printed molds. We used murine-derived embryonic stem cells for optimizing cell-seeding protocols, characterization of the resulting internal structure of the construct, and remodeling of the extracellular matrix, as well as validation of electrophysiological activity. Then, we used these findings to biofabricate these constructs using neurons derived from human embryonic stem cells. This method can provide a large degree of design flexibility for development of in vitro functional neural tissue models of varying forms for therapeutic biomedical research, drug discovery, and disease modeling, and engineering applications.

New bioresorbable wraps based on oxidized polyvinyl alcohol and leukocyte-fibrin-platelet membrane to support peripheral nerve neurorrhaphy: preclinical comparison versus NeuraWrap.

Nerve wrapping improves neurorrhaphy outcomes in case of peripheral nerve injuries (PNIs). The aim of this preclinical study was to assess the efficacy of two novel biodegradable wraps made of a synthetic 1% oxidized polyvinyl alcohol (OxPVA) and a natural leukocyte-fibrin-platelet membrane (LFPm) versus the commercial product NeuraWrap. After rats sciatic nerve transection and neurorrhaphy, the wraps were implanted and compared for functional outcome, by sciatic function index assessment; structural characteristics, by histological/immunohistochemical analysis; ultrastructural features, by transmission electron microscopy. Moreover, a morphometric study was also performed and collagen distribution was observed by Second Harmonic Generation microscopy. After 12 weeks from implantation, all wraps assured nerve function recovery; no scar tissue/neuromas were visible at dissection. LFPm wraps were completely resorbed, while residues of OxPVA and NeuraWrap were observed. In all groups, biocompatibility was confirmed by the absence of significant inflammatory infiltrate. According to histological/immunohistochemical analysis and morphometric findings, OxPVA and LFPm wraps were both effective in preserving nerve integrity. These results assess that bioengineered OxPVA and LFPm wraps successfully guarantee favorable lesion recovery after PNI/neurorrhaphy and, in future, may be considered an interesting alternative to the commercial NeuraWrap.

Muscle cells, nerves, fibroblasts and vessels in the detrusor of the rat urinary bladder.

All the cells of rat detrusor muscle fall into one of five ultrastructural types: muscle cells, fibroblasts, axons and glia, and vascular cells (endothelial cells and pericytes). The tissue is ~79% cellular and 21% non-cellular. Muscle cells occupy 72%, nerves ~4% (1/3 axons, 2/3 glia), and fibroblast >3% of space. Muscle cells (up to 6 µm across and ~600 µm long, packed to almost 100,000 per mm2) have surface-to-volume ratio of 2.4 µm2/µm3 ~93% of cell volume is contractile apparatus, 3.1% mitochondria and 2.5% nucleus. Cell profiles are irregular but sectional area decreases regularly towards either end of the cell. Muscle cells are gathered into bundles (the mechanical units of detrusor), variable in length and size, but of constant width. The musculature is highly compact (without fascia or capsule) with smooth outer surfaces and extensive association and adhesion between its cells. Among many types of intercellular contact and junction, digitations are very common, each muscle cell issuing minute finger-like processes that abut on adjacent cells. Sealed apposition are wide areas of specialized contact, possibly forming a chamber between two muscle cells, distinct from the extracellular space at large (stromal space). The innervation is very dense, virtually all intramuscular axons being varicose (including afferent ones). There are identifiable neuro-muscular junctions on each muscle cell, often several junctions on a single cell. There are also unattached terminals. Fibroblasts (involved in the production of collagen), ~1% of the total number of cells, do not make specialized contacts.

An electroactive alginate hydrogel nanocomposite reinforced by functionalized graphite nanofilaments for neural tissue engineering.

To address the need to biodegradable, electroactive conduits accelerating nerve regeneration, here we develop a nanocomposite hydrogel made of alginate reinforced by citric acid functionalized graphite nanofilaments. The green, simple functionalization enhances the nanofillers distribution and their biocompatibility, as verified using mesenchymal stem cells in vitro. The uniformly distributed nanofilaments raise mechanical stability of the nanocomposite hydrogel versus the neat one up to three times. Also, the nanofilaments enable electrical contact and intercellular signaling thereby stimulating their biological activity. In vitro studies proved the biocompatibility of the nanocomposite hydrogel whereon PC12 cells proliferate and spread evidently. In vivo tests also supported applicability of the nanocomposite hydrogel for implantation within body, and the samples showed no adverse reaction and no inflammatory responses after 14 days. Conclusively, the results certify that the developed electroactive nanocomposite hydrogel is able to stimulate nerve generation and could be confidently used as a nerve conduit material.