3D bioprinting: A new insight into the therapeutic strategy of neural tissue regeneration

ABSTRACT

Acute traumatic injuries and chronic degenerative diseases represent the world’s largest unmet medical need. There are over 50 million people worldwide suffering from neurodegenerative diseases. However, there are only a few treatment options available for acute traumatic injuries and neurodegenerative diseases. Recently, 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. In this commentary, the newly developed 3D bioprinting technique involving neural stem cells (NSCs) embedded in the thermoresponsive biodegradable polyurethane (PU) bioink is reviewed. The thermoresponsive and biodegradable PU dispersion can form gel near 37°C without any crosslinker. NSCs embedded within the water-based PU hydrogel with appropriate stiffness showed comparable viability and differentiation after printing. Moreover, in the zebrafish embryo neural deficit model, injection of the NSC-laden PU hydrogels promoted the repair of damaged CNS. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden constructs. Therefore, the newly developed 3D bioprinting technique may offer new possibilities for future therapeutic strategy of neural tissue regeneration.

Introduction

Acute traumatic injuries or chronic degenerative diseases of the nervous system typically lead to the loss of central nervous system (CNS) function. Traumatic injury of the CNS may include traumatic brain injuries (TBI) and spinal cord injuries (SCI),¹ which have had a significant impact on the national healthcare system and the overall quality of life of patients around the world. On the other hand, because of the aging society, neurodegenerative diseases are an increasingly important public health concern. Major neurodegenerative diseases with unmet medical needs include Alzheimer disease, Parkinson disease, multiple sclerosis, and Huntington's disease. There are only a few treatment options available for some of these diseases, and several of these treatments provide only symptomatic relief.² The large economic costs of CNS disorders include not only the cost of treatment, but also the lost productivity of patients and their caregivers, for whom looking after chronically disabled family members can represent an enormous source of emotional, practical, and financial burden. In spite of the development of numerous clinical treatments, therapeutics for fully recovering the neural function are still in their infancy thus leading scientists to draw inspiration from neural tissue engineering strategies in the repairing the CNS disorders.^3,4

Stem cells in treating CNS degenerative diseases

For the past a few decades, the stem cell replacement has attracted much attention as a promising therapeutic option for the treatment of various neurodegenerative diseases.² Stem cells have the capacity to self-renew and differentiate into several types of functional cells. Based on the differentiation potential and cell origin, stem cells used for treating neurodegenerative diseases include the embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adipose-derived adult stem cells (ADASs), mesenchymal stem cells (MSCs), and neural stem cells (NSCs). In addition to the direct cell replacement, stem cells can secrete various cytokines and growth factors that generate a variety of beneficial effects such as anti-inflammation, neural cell protection, and induction of the endogenic recovery system. However, transplantation of stem cells to the injury sites often shows poor cell survival and engraftment.⁵ To support the physiological function of stem cells in the implanted site of tissue, the use of 3 dimensional (3D) scaffolds that mimic the biologically functional and organizational complexity of the tissue is an important approach.

3D bioprinting of neural tissue

The purpose of neural tissue engineering is to develop of biological substitutes that integrate biomimetic 3D scaffolds with cells for improvement of neural tissue function. As a critical component in successful nerve regeneration, 3D scaffolds provide a necessary physical support to facilitate cell function, resulting in better host tissue engraftment and subsequent new tissue development.

The conventional 3D scaffold fabrication has yielded favorable effects in repairing nerve injuries. Intrinsic limitations, however, exist with regards to adequate control of the external shape and internal architecture of the scaffolds.^6-8 To overcome this problem, 3D printing technology serves as a powerful tool for building tissue and organ structures in the field of tissue engineering. Recent advances have enabled 3D printing of biocompatible materials, cells, and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation.^9,10 3D bioprintings can produce complex tissue scaffolds directly with precise spatial distribution and biomimetic architecture. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. So far, 3D bioprinting has been used for generation of several tissues for transplantation, including multilayered skin, bone, heart tissue, and cartilaginous structures. Relatively few studies have focused on applying 3D bioprinting in neural tissue. A collagen hydrogel precursor (in liquid form) as bioink was created and cell viability was remained after printing.¹¹ In another study, an artificial neural tissue containing vascular endothelial growth factor (VEGF), murine NSCs, collagen, and fibrin gel was printed,¹² where NSCs showed viability after printing. In a most recent work, retinal glia cells were printed with cell culture media.¹³ In all the above investigations, the bioprinted neural constructs are still relatively thin layers of neural cells or NSCs within liquid hydrogel polymer or precursor. Choices of "ink "for printing the neural cells/tissues are very limited.

A novel bioink for 3D bioprinting for neural tissue repair

Recently, the printable synthetic water-based polyurethane dispersion (PU) was reported for the first time to heal CNS disorders.¹⁶ The bioink was a novel aqueous dispersion of PU nanoparticles, which may form hydrogel upon heating without any crosslinker.¹⁸ The modulus of the hydorgel could be easily adjusted by the solid content of the dispersion to mimic the stiffness of neural tissue. The NSC-embedded PU hydrogel constructs were printed by a self-developed fused deposition manufacturing (FDM) equipment.¹⁹ The self-developed FDM equipment integrates a personal computer, an x–y–z motion platform with temperature controllers, and 2 nozzles with heaters (Fig. 1B). The computer was used for design of the structure, planning of manufacturing paths, and motion control of the platform. The computer automatically sends commands to the platform and the pressure and temperature control units so that the nozzle can inject biocompatible materials to form the designed shape and inner structure of the scaffolds. The 3D bioprinting process of NSC-laden constructs using the thermoresponsive PU ink is illustrated in Figure 1A. NSCs in culture medium were mixed with the PU nanoparticle dispersion so the final mixture contained 4×10 ⁶ cells/ml and 25% solid content of PU nanoparticles in the culture medium. The cell-containing “ink” was filled in a barrel and printed through the nozzle (250 μm) of the 3D printer as stacking fibers into a petri dish placed on the platform in 37°C. The fibers can be stacked up for 8 layers (~1.5 mm thickness) without severe collapse. The dimension of the 3D-printed NSC-laden constructs construct is 1.5 cm×1.5 cm×1.5 mm (W×D×H). Figure 2A displays that NSCs may be printed with these hydrogels and show comparable viability. The mature neuron maker (β-tubulin) of NSCs embedded in PU hydrogels was stained by immunofluorescence staining. The data are presented in Figure 2B. The maker protein β-tubulin at 7 d was highly expressed for NSCs embedded in 25% PU hydrogels. These images revealed that PU hydrogels may promote the neuronal differentiation of NSCs.

Figure 1.

(A) The bioprinting process for neural stem cell (NSC)-laden constructs by an extrusion-based deposition system using the thermoresponsive PU as the printing ink. (B) The fused deposition manufacturing (FDM) machine.

Figure 2.

Behavior of NSCs in PU hydrogels after being printed. (A) Images for NSCs in the 3D printed stacking fibers of PU hydrogels during a period of 3 d. (B) The protein expression of β-tubline analyzed by immunofluorescence staining for NSCs embedded in PU hydrogels for 0 d (d) and 7 d after being printed. The red fluorescence indicates the presence of β-tubline.

In addition to cellular-level investigations, the study has investigated the capacity of bioprinted grafts in recovering the motor function. Zebrafish (Danio rerio) neural injury models were employed to evaluate the potential of the NSC-laden biodegradable PU constructs in CNS repair.¹⁶ The zebrafish (Danio rerio) is an important vertebrate animal model in neuroscience, which is also a suitable preclinical model for the damaged CNS.¹⁷ In the first zebrafish model, the developmental zebrafish embryos were exposed to ethanol to induce CNS deficits. Rescue of the neural deficits by various treatments was evaluated such as in-chorion coiling contraction (an index of motor function (Hz)) and hatching rate (an index for CNS function (%)) of the embryos. Results showed the recovery of in-chorion coiling contraction (wild type: ~0.075 Hz; NSC-laden PU: ~0.065 Hz) and hatching rate (wild type: ~95%; NSC-laden PU: 60%) after receiving NSC-laden PU gel, suggesting that the NSC-laden PU gel may rescue the function of impaired nervous system in the embryonic period. A second model, adult zebrafish with traumatic brain injury, was employed to evaluate the potential of the NSC-laden PU constructs in CNS repair. The adult zebrafish with traumatic brain injury and without treatment were either immobile or imbalance in swimming, but those implanted with the 3D-printed NSC-laden PU constructs had the locomotor function recovery and a low mortality rate. These observations supported the potential of 3D-printed NSC-laden constructs in repairing the CNS diseases.

Conclusion and future challenges

Many challenges in the 3D bioprinting field are related to the materials used in the bioprinting process. Currently, the biomaterials for 3D bioprinting are selected either base on their preferred biocompatibility or because of their favorable extrusion and crosslinking characteristics. An ideal material for bioprinting should be biocompatible, and at the same time can be easily manipulated into complex 3D structures, and maintain cell survival and function. The rheoloical properties and crosslinking mechanisms of the hydrogel determine its printability. So far, only a limited range of materials can be used, such as collagen, hyaluronic acid, alginate, modified copolymers, and photocured acrylates.

The 3D bioprinting of neural tissue regeneration and the ultimate success for neural applications also largely relies on the development of suitable bioink. Although various traditional biomaterials have continued to be improved, they have been limited by inadequate biomimetic properties that cannot satisfy the strict requirements of 3D bioprinting for neural tissue regrowth. This commentary reviews recent advancements in using the materials technology and 3D bioprinting platform as novel tools to fabricate bioactive constructs for improving neural regeneration. The thermoresponsive water-based biodegradable PU hydrogel not only provides a suitable environment for neural cell growth, but also geometrically directs CNS repair and regeneration. The 3D-printed NSC-laden PU constructs may be a potential strategy to fabricate patient-specific complex neural grafts where NSCs can be readily incorporated and synergistically employed for neural regeneration. However, there are some limitations of the hydrogel materials, such as lower gel modulus. This low modulus may lead to gel collapse if more layers have to be deposited during 3D printing. 3D-bioprinted neural tissue constructs are being developed not only for transplantion and regeneration but also for use in drug discovery, toxicity screening, and basic research. For these applications, extensive validations are needed to ensure that the bioprinted neural tissues can recapitulate the key pathophysiological features of the disease models. Although many of the applications of 3D bioprinting are still at the R&D stage, we expect new 3D bioprinting techniques and bioink formulae that combine to mimic native cells and their ECM may foster for a next generation of neural tissue repair and regeneration.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.