The central nervous system (CNS) has a limited capacity to grow new cells to repair the loss or damage of neurons and axonal pathways that accompany conditions such as traumatic brain injury (TBI), stroke, spinal cord injury, and neurodegenerative diseases 1–4. Neurological disorders (such as stroke, dementia, meningitis, etc.) are among the leading causes of disability (~ 276 million) and deaths (~ 9 million in 2016) which continue to increase each year 5. Neurogenesis is a multi-step process of neural stem cell differentiation in the CNS; however, it is restricted to a limited number of areas in the brain (e.g. hippocampus dentate gyrus, etc.), hampering the restoration of the lost neurons 6,7. Additionally, regeneration of lost axonal pathways in the CNS is insufficient due to the lack of directed guidance as well as inhibitors like glial scar or myelin 8,9. Conventional regenerative strategies to treat neurological diseases, including gene therapy, stem cell transplantation/engraftment or growth factor delivery, have shown limited success in creating functional tissues due to the low cell engraftment and lack of growth factors localization 10,11. Animal model, as a traditional alternative, is extensively employed for the regenerative medicine experiments and CNS disorder therapies 12,13. However, the differences in animal models compared to human body, in addition to the high costs and related ethical issues, have posed serious concerns on the applicability of animal models 14. Regarding these critical issues, numerous measures have been implemented to develop reliable in vitro disease models 15–17.
Different in vitro techniques such as soft lithography 18, laser texturing 19, electrospinning 20, and electrospraying 21, have been employed to generate 2D cell-laden constructs. However, these techniques fail to accurately mimic the mechanical properties, architecture and physiology of brain-specific structure and function 22. In fact, in 2D culture methods, the functionality of neural cells (including morphology, proliferation, and differentiation) is either limited or completely lost 23,24. In contrast to the 2D methods, three-dimensional (3D) bioprinted structures have demonstrated new prospective in mimicking the functional native-like tissue microenvironment 25,26. In 3D bioprinting, cells, biomaterial, and bioactive moieties can be precisely positioned to mimic in vivo conditions 27,28. This holds a great promise for future advancements toward having a cost-effective, fast and anatomically accurate representation to address the current issues of CNS disorder therapies 29–31.
Generally, 3D bioprinting techniques can be divided into optical-based and nozzle-based methods 32. In optical-based methods, the major step is the scaffold photopolymerization where a high energy radiation is exposed to the cell-containing precursors 33. This can significantly reduce the cell viability rate, as compared to nozzle-based methods 34. Although the optical-based methods have higher printing resolution, the simplicity and the low cost of nozzle-based methods have made the them more prevalent. Amongst the nozzle-based bioprinting approaches, extrusion bioprinters have the advantage of generating low shear stress on living cells, making them more cytocompatible 34. Another issue in mimicking neural tissues is the complex microenvironment where neurons, glial cells, blood vessels, pericytes, and extracellular matrix are elaborately bundled 35. This requires manipulation of different cell types and printing materials simultaneously 36. For that reason, coaxial extrusion methods can crosslink different biocompatible hydrogels (e.g. core-shell) at the same time through printing a fiber-like construct 37,38. In fact, there are two major superiorities of coaxial extrusion printing over other methods to be used for neural tissue disease models: (i) it provides guidelines for a controlled cell proliferation and axon outgrowth (fiber structure); and (ii) it can delicately position more than one cell type in a 3D tubular structure (core-shell structure).
Regarding the cell proliferation guidelines, a common approach is to use aligned polymer nanofibers to fabricate a 3D scaffold as a culture microenvironment 39,40. The electrospun nanofibers can provide a controlled neurite outgrowth and cell alignment replicating the native neuroanatomical architecture 41. As an example, a 3D scaffold based on a bacterial nanocellulose (BNC) can prove a biocompatible environment for neural cells (neuroblastoma, SH-SY5Y) 42. The nanofibers can also mix with carbon nanotubes to create a conductive 3D composite scaffold in which an exceptional cell (SH-SY5Y) attachment and proliferation along the guidelines can occur 43. In addition to nanofibers, other strategies such as chemical surface treatment, mechanical stimulation, and surface patterning can be implemented for a directional cell spreading and growth 44. Although these methods can provide a relatively acceptable cell guiding platform, the complexity engaged in simultaneously manipulating of different cell types as well as the higher fabrication costs are still their ongoing challenges.
The fiber constructs when fabricated in a core-shell form provides even a broader course of in vitro applications. The structure of coaxial constructs can robustly recapitulate the luminal/tubular configuration of cell bundles of neurovascular/neuromuscular tissues in a 3D environment, rather than a planner organ-on-chip 35,45. In core-shell constructs, cells located in the core/shell are protected by the outer hydrogel shell that provides both stability and the possibility of cell-cell interactions within the environment 46. A longitudinal fiber-based scaffold fabricated to investigate the drug resistance in Glioblastoma, with human glioma stem cell (GSC23) in the shell and glioma cell line (U118) in the core, showed the superiority of core-shell constructs to mimic the native microenvironment of the neural tissues 47. A recent study with neural stem cells (core) and Schwann cells (shell) showed an enhancement in cell proliferation and differentiation that can be implemented for spinal cord injury research 48. In neurodegenerative disease models, however, longitudinal scaffolds do not necessarily provide a native-like microenvironment where neural cells are highly paralleled with the aligned Schwann cells 44,49. In other words, growing highly aligned neural cells even in the core-shell fibers is still a challenge. Therefore, a neural tissue model should be able to align cells in an parallel orientation where the synaptic electrophysiology, plasticity, and neural vesicle trafficking can occur 44.
Specifically under the topic of scaffold activated directional neural growth, only a few studies are conducted. As an instant, rat cortical cells and mouse neural stem cell were encapsulated in an ECM (extracellular matrix) protein hydrogel using the coaxial printing method. Despite the extensive investigation on different cell types in this study, the neural outgrowth and directional neural outgrowth is not quantitively measured 50. Glia cells together with primary cortical neurons are also printed using a coaxial printing strategy that utilizes Gellan gum-RGD as the bioink 37. Rather than the directed neurite outgrowth of the cells, networking of these types of cells was investigated in that study. Human derived cortical neurons and glial cells (derived from Human-Induced Pluripotent Stem Cells) were used in another bioprinting study 51. Likewise, the cellular alignment was not the focus in that study.
In addition to CNS related diseases, peripheral nerve injuries and regeneration of neural fibers are other important applications of coaxial bioprinting 52. In fact, the importance of growing neural cells along the desired direction can be realized in the regeneration of the peripheral nervous system 53. In most of the studies on peripheral neural on-chip models, fabrication of a guiding conduit for directed axonal growth is of interest.
As mentioned above, in vitro cell alignment in neural tissue engineering plays a critical role to faithfully model the neurodegenerative diseases. Herein, we fabricated a core-shell hydrogel scaffold with a superior neural cell alignment within the construct. The hybrid scaffold microenvironment is comprised of gelatin methacryloyl (GelMA)/gelatin and hydrogel as the core, and an alginate as the shell. Human neuroblastoma cells (SH-SY5Y) are cultured in the fiber core with a high cell proliferation and more than 85% cell viability. Cell alignment is achieved with a standard deviation of less than 6 in cellular orientation. The microcylindrical 3D scaffold structure is shown to recapitulate the structure of the native extracellular matrix (ECM), as the neural growth is highly aligned with the fiber guidelines.