Materials Science & Engineering C 110 (2020) 110741

Contents lists available at ScienceDirect

Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Review

3D bioprinting applications in neural tissue engineering for spinal cord T

injury repair
⁎
Tuba Bedira,b, Songul Ulaga,c, Cem Bulent Ustundagb, Oguzhan Gunduza,c,
a
Center for Nanotechnology & Biomaterials Application and Research (NBUAM), Marmara University, Turkey
b
Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University, Turkey
c
Department of Metallurgical and Materials Engineering, Faculty of Technology, Marmara University, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: Spinal cord injury (SCI) is a disease of the central nervous system (CNS) that has not yet been treated suc-
Neural tissue engineering cessfully. In the United States, almost 450,000 people suffer from SCI. Despite the development of many clinical
3D bioprinting treatments, therapeutics are still at an early stage for a successful bridging of damaged nerve spaces and com-
Scaffolds plete recovery of nerve functions. Biomimetic 3D scaffolds have been an effective option in repairing the da-
Stem cells
maged nervous system. 3D scaffolds allow improved host tissue engraftment and new tissue development by
Spinal cord injury
supplying physical support to ease cell function. Recently, 3D bioprinting techniques that may easily regulate the
dimension and shape of the 3D tissue scaffold and are capable of producing scaffolds with cells have attracted
attention. Production of biologically more complex microstructures can be achieved by using 3D bioprinting
technology. Particularly in vitro modeling of CNS tissues for in vivo transplantation is critical in the treatment of
SCI. Considering the potential impact of 3D bioprinting technology on neural studies, this review focus on 3D
bioprinting methods, bio-inks, and cells widely used in neural tissue engineering and the latest technological
applications of bioprinting of nerve tissues for the repair of SCI are discussed.

1. Introduction surgical operation for stabilizing, decompressing the spinal cord and
multisystem medical administration, hypothermia, and rehabilitative
Vital and healthy functions of the body are controlled by a quite medical care [5,6]. Despite the development of many clinical therapies,
complicated system called the Central Nervous System (CNS) [1,2]. therapeutics are still in the early stages for the successful bridging of
Degenerations in the CNS structure caused by physical damages or damaged nerve gaps and complete recovery of nerve functions [11,12].
neurological diseases often cause loss of neuronal cell bodies, axons and Recent advances in nanotechnology and tissue engineering offer
glia support [3]. The CNS has a low capacity to replace neurons lost effective strategies to repair CNS diseases. Neural tissue engineering is
during damage or diseases [4]. One of the CNS diseases, spinal cord focused on the improvement of an appropriate environment that con-
injury (SCI), breaks nerve contacts among the brain and other parts of nects the biomimetic scaffold with cells to repair and restore neural
the body. It causes sensory loss and paralysis under the level of damage. tissue function. Physical support for successful nerve regeneration is
11,000 new incidences emerge every year only in the US, with ap- provided by three-dimensional (3D) scaffold and scaffold-like materials
proximately 450,000 patients living with SCI [5,6]. Traffic accidents, [13,14]. This results in better host tissue engraftation, followed by new
acts of violence, falls, and sports injuries constitute most of the injuries tissue development to facilitate cell function [13,15]. An ideal scaffold
[7]. SCI affects patients' quality of life, forces the families, and causes for neural tissue engineering should meet the following criteria: (i)
socio-economic impacts on healthcare systems worldwide [8]. Cur- biocompatibility is an important feature for scaffold to provide cell
rently, one of the treatments for SCI is the application of methyl- adhesion, proliferation and differentiation in the lack of immune and
prednisolone in overdoses to reduce secondary damage processes [5,9]. cytotoxic reactions; (ii) biodegradability is essential for degradation of
However, this therapy is disputable as it causes many critical side ef- the scaffold at a close-matched rate of new tissue formation and finally
fects, containing sepsis, wound infection, gastric bleeding and pneu- scaffold removed from the system; (iii) neural transmission is based on
monia. Furthermore, it has just minor progress in neurological im- the potential of action generated in the synapse. An electrically con-
provement [6,10]. Other therapy options applied for SCI contain the ductive scaffold can support neurite growth and neural regeneration;

⁎
Corresesponding author at: Center for Nanotechnology & Biomaterials Application and Research (NBUAM), Marmara University, Turkey.
E-mail address: [email protected] (O. Gunduz).

https://doi.org/10.1016/j.msec.2020.110741
Received 7 November 2019; Received in revised form 5 February 2020; Accepted 10 February 2020
Available online 19 February 2020
0928-4931/ © 2020 Elsevier B.V. All rights reserved.
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

Fig. 1. Schematic illustration of design criteria for neural scaffold.

(iv) the scaffold must have appropriate mechanical properties so that it products [25]. Laser-assisted bioprinting can create high-resolution
does not increase stress in the lesion region or collapse throughout structures using lasers as energy [26]. However, there are considerable
regular motion; and (v) the scaffold with porous interconnection imi- advantages and limitations of these technologies in the production of
tates the extracellular matrix (ECM) of natural tissue, vascularizations, tissue structures such as the neural scaffold mentioned above.
allowing the cells to disperse well and exchange of waste and nutrients. In this review article, various 3D bioprinting techniques such as
(Fig. 1) [14,15]. Various methods such as gas foaming, melt moulding, extrusion, inkjet, laser, plotting used in neural tissue engineering will
electrospinning and phase separation have been used in the production be discussed in detail. Furthermore, bio-inks and cells available for
of 3D scaffolds made of synthetic and natural polymers [16,17]. bioprinting will be described. Finally, bioprinting applications of neural
However, the scaffold shape or inner channel configuration and pore tissues for the repair of SCI will be reported.
size in the scaffold cannot be exactly adjusted by these scaffold man-
ufacturing methods. Besides, these techniques cannot allow fabrication 2. 3D bioprinting methods for neural tissue applications
of the scaffolds with cells due to harsh processing conditions.
Recently, 3D bioprinting methods have been noted, which can ea- Among the 3D bioprinting methods inkjet, microextrusion, bio-
sily arrange the dimension and shape of 3D scaffold and fabricate plotting, stereolithography, and fused deposition modeling have at-
scaffolds with cells [18]. 3D printing was described first by Charles W. tracted more attention in the neural tissue engineering studies.
Hull in 1986 [19]. 3D bioprinting also called additive manufacturing
(AM) is the use of rapid prototyping (RP) systems for the printing of
2.1. Inkjet bioprinting
cells, growth factors, and different biomaterials in layers form. With
this technology, biological structures that highly mimic natural tissue/
Inkjet bioprinting is a cost-effective technique. Bio-inks can be dis-
organ properties can be fabricated [20]. 3D bioprinting utilizes 3D
persed well by using inkjet bioprinting which has controllable manner.
modeling program (e.g. computer-aided design (CAD) or computer to-
It permits simultaneous and contactless deposition of cells in certain
mography (CT) scan images) to make 3D solid or gelation objects [21].
directions in micrometer resolution [27]. Since bio-ink does not have
Generally, 3D bioprinting system comprises an X-, Y-, Z-axis drive de-
high viscosity in this technique, generally results in structures with low
vice, 3D modeling program, computers, and bioprinting ingredients/
mechanical properties. Besides, the small nozzle dimension and flow
inks. Firstly, a model is formed by utilization CAD or CT images. Then a
rate limit the volume accumulated per drop (< 10 pL). This means that
3D scaffold structure is generated by bioprinting equipment attached to
high cell concentrations (> 5 million cells/ mL) must be seeded to
a computer [22]. In 3D bioprinting, bio-inks are an essential compo-
maximize the probability that each bio-ink drop will contain a cell [28].
nent. Bio-inks consist of biomaterials that can be used cell encapsula-
There are two types of inkjet printers: thermal and piezoelectric. In
tion and biomolecule incorporation. Before printing, the basic features
thermal inkjet printers, the temperature provides to heat the printer
of a bio-ink such as cross-linking, viscosity and gelation must be re-
head and this forms air pressure pulses. These pulses enable droplets to
garded. These features have an effect on printing quality, morphology,
push from the nozzle [27]. This method has high printing speed and a
cell viability and proliferation [23]. According to the working princi-
resolution between 20 and 100 μm. It has the potential to print droplets
ples, three basic 3D bioprinting technologies are defined: extrusion,
in picoliter volume for obtaining better resolution [29]. In piezoelectric
inkjet and laser-assisted (Fig. 2) [24]. Within these technologies, ex-
inkjet printing, droplets are formed by applying a piezoelectric ac-
trusion bioprinting can generate 3D structures in large scale-up by
tuator. Applied voltage excites the piezoelectric crystal, which is lo-
utilization hydrogels with cell-laden. Inkjet bioprinting generally
cated in the printer head. Deformation occurs both rapid and reversely
adapted from the commercial two dimensional (2D) printing system
due to this voltage. The speed and dimension of the droplets ejected can
and prints liquid droplets with cell-laden for rapid and minor-scale
be controlled by changing factors such as time, pulse and amplitude

2
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

Fig. 2. Schematic illustration of three significant 3D bioprinting technologies according to working principles. (A) Micro-extrusion bioprinting (pneumatic, piston,
screw); (B) inkjet bioprinting (thermal, piezoelectric); (C) laser-assisted bioprinting.

[30]. Cell viability is higher since bio-ink is not exposed to heat and been many studies by using the FDM technique for both musculoske-
pressure in this method [31]. Inkjet printing has high potential in tissue letal (cells seeded on the structures) and neural tissue applications
engineering applications and regenerative medicine [28]. (encapsulated cells with structure) [28].

2.2. Fused deposition modeling (FDM) 2.3. Stereolithography (SLA)

The FDM technique utilizes filament, which is made up from ther- The stereolithography method consists of an ultraviolet (UV) laser
moplastic polymer to construct a 3D structure. With the temperature and has the functionality to focus the laser on a hydrogel or resin that
application, the filament is heated in the nozzle to get the printable has been photo-sensitized by the addition of a photoinitiator [37]. UV
form (semi-liquid state) and extruded onto the platform [32]. This laser provides to solidify the liquid, especially, covalent bonds between
method is not appropriate for cell printing directly since high tem- neighboring polymer chains are created by the energy supplied by the
perature is used. Therefore, the cells are cultured onto the construct laser [38]. The layers in the 3D structure are obtained by immersing the
after the printing process [33]. The use of a thermoplastic polymer fi- stage in the tank of liquid and changing its movement from up to down
lament is an easy way to fuse the filaments during printing and after the to equal the distance to the height of the layer [38,39]. SLA bioprinters
printing process, it provides to solidify at room temperature. Layer offer a very high print resolution, considering factors such as laser
thickness, width values, and direction of the filaments in addition to the power, time of exposure, size of the laser spot, light wavelength value
air gap within the identical layer or between layers affect the me- [39,40]. However, the SLA method is somewhat slow, costly and has
chanical behaviour of the printed structures [32]. Inter-layer defects are restricted types of materials for 3D printing applications. Furthermore,
the main cause of mechanical weakness [34]. Since any solvents are not the kinetics of the reaction and the curing treatment are complex. The
necessary for the FDM method, it provides to the convenience in the thickness of each layer depends on the light source energy and time of
way of material fabricating and handling. This method allows con- exposure [41]. Desired mechanical performance can be obtained by
tinuous production without having to change the feedstock [35]. The applying post-treatment processes such as heating or photo-curing to
FDM is a simple process and it has low cost and high-speed properties as fabricated parts [42]. The SLA technique can be applied effectively to
the main advantage. However, it has disadvantages such as poor me- produce the additional complex nanocomposites [43]. It can print
chanical features, layer-by-layer appearance, poor surface property photosensitive polymeric material polymerizing to soft substrate ma-
[36] and the limited amount of thermoplastic polymer [32]. There have terials, which generally have mechanical markings similar to neural

3
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

tissue types, thereby assisting the differentiation of seeded cells into to regain its functionality [63]. This scaffold and cell combination re-
neuronal subtypes [44]. generate growth potential by developing scaffold integration [64]. A
hydrogel in the form of the biomaterials is water-swollen crosslinked
2.4. Micro-extrusion bioprinting polymer networks that can mimic mechanical and to some extent, the
architecture of soft spinal cord tissue [65]. Injectable hydrogels are that
Micro-extrusion-based bioprinting comprises of extruding a con- has an important role in delivering cells and growth factors into an
tinuous bio-ink stream [45]. The continuous accumulation of bio-ink injury with less invasive operative interferences by constructing a 3D
provides excellent structural integrity in this technique [46]. In micro- matrix imitating native extracellular matrix to adjust cell fate [63].
extrusion printing, the cells are mixed into the bio-ink, and then the Some features of the hydrogels that maybe include: porosity, degrada-
biomaterials are dispensed through nozzles or needles [45]. The micro- tion, functionality, biocompatibility, in situ gelation and elastic modulus
extrusion head accumulates the material into the substrate as beads to optimize their applications in spinal cord repair [66–68]. Hydrogels
using instructions come from CAD-CAM program. First, the beads are are under consideration as a bio-ink due to their ability to form 3D
put in the x-y direction, and then the extrusion head is moved along the hydrophilic polymer networks [69]. Therefore, bioprinting methods
z-axis to fabricate complex 3D constructs [47]. In this method, since mainly utilize the hydrogels to print in a bio-ink form [70]. Hydrogels
high viscosity bio-ink can be fabricated with micro nozzle sizes, there is used as bio-ink can be formed with synthetic and natural polymers.
a broad range of bio-ink usage [48]. The piston, screw or pneumatic Natural polymers such as collagen, hyaluronic acid, chitosan, gelatin,
pressures are the parts and driven forces of the extrusion bioprinting agarose, alginate, fibrin, and synthetic polymers such as Poly D, L-lactic
[49]. The major advantage of this process is that the cell density with acid (PLA), Poly-ϵ-caprolactone (PCL), Poly-lactic-co-glycolic acid
high values can be fabricated with a rapid production rate [50]. (PLGA), Poly-glycolic acid (PGA), Polyethylene glycol (PEG) are widely
However, there is a problem which is the shear stress occurs in the cells. used in neural tissue engineering for the repair of SCI. These bioma-
The applied pressure can affect the viability in a pressure-based system, terials are given in Table 1 in detail [71–98].
and nozzle diameter is another parameter that affects the viability [51].
On the other hand, optimization parameters such as the concentration 3.1. Cell types
of the components, pressure and diameter of the nozzle are the critical
values to solve these problems. The micro-extrusion technique has been Cell selection is an important factor in tissue and organ printing for
successfully applied to develop constructs for tissue engineering [52]. the proper function of the produced structure. Cells selected for printing
For example, aortic valves [53], tumor models [54] and vascular tissues must imitate the physiological situation of the cells in vivo also sustain
[55] were printed with this technique. Also, cell viability in biological their functions under optimized conditions [99]. Different viable cell
structures developed by micro-extrusion is reported to be higher than types have been used to promote neural regeneration. These can con-
90% [56]. tain Schwann cells, neural stem cells (NSCs), olfactory ensheathing cells
(OECs) and mesenchymal stem cells (MSCs). Viable cells may be em-
2.5. 3D bioplotting bedded into the scaffold or placed in the printing medium during the
fabrication operation of the scaffold [62].
In the 3D bioplotting technique, dropwise extrusion of a viscous Activated Schwann cells have a crucial role in directing axon
liquid medium into a gel-like material using a pressure syringe [57]. elongation by forming new myelin after peripheral nerve injury. In this
The loaded material is extruded from the micro-sized needle and dis- context, it may be a useful approach to assemble Schwann cells on
tributed as micro-strand [58]. Using a crosslinking agent or UV radia- tissue engineering scaffolds. Gu et al. [100] developed scaffolds mod-
tion, the hydrogel material is solidified over time. The key point of this ified with Schwann cell-derived ECM to connect a 10 mm sciatic nerve
technique is the 3D dispersion of a viscous printing material into a li- space. In this study, they observed that the number and size of re-
quid medium having a similar density. 3D bioplotting can use a broad generative neural fibers were higher in the Schwann cell-derived ECM
range of different materials such as polymer melts, thermoset resins, than in the cell-free scaffold [100]. NSCs are capable of producing
polymer solutions, high fillers, cement and bioactive proteins [57]. neurons and glia cells. Also, engrafted NSCs may differentiate into as-
However, these materials can be too stiff or have low elasticity in- trocytes that facilitate glial scar formation at the injury site [101]. It
appropriate for neural tissue engineering. 3D bioplotter can print co- was supported by some works that MSCs increased differentiation of
cultured scaffolds and tissue structures [28]. Scaffolds produced by this NSCs to oligodendroglia [102]. When transplanted into the injured
technique generally demonstrate a smooth surface due to the extrusion spinal cord, OECs can penetrate astrocytic scars and a result ease axonal
and hardening procedure. Studies have demonstrated that scaffold growth along a wound barrier [103,104]. Furthermore, OECs may
surface topology greatly affects cell adhesion and proliferation [59]. myelinate growing axons, thus improve axonal transmission [105].
Accordingly, it is necessary to create surface roughness by scaffold Latest works have investigated the effect of embryonic stem cells (ESCs)
surface modification to increase cell adhesion on bioplotted structures. in the therapy of SCI in animal models. Keirstead et al. [106] showed
that ESCs have differentiation potential into mature oligodendrocytes
3. Biomaterials and hydrogels for neural tissue engineering and human ESCs can support healing following SCI in rats. Baharvand
et al. [107] observed an enhancement in the locomotor function of rats
SCI contusions result in an irregularly shaped cavity surrounded by after transplantation of collagen scaffolds containing human ESCs in
preserved white matter. The ideal way of inserting a biomaterial for SCI rats. Another type of cell used in neural tissue applications is
repair is to place it into the cavity, which would reduce extra damage to pluripotent stem cells [62]. These versatile cells have large differ-
the nerve tissue. Thus, a large number of research groups have high- entiation potential and their viability has been examined in different
lighted injectable, in situ gelling substrates for spinal cord repair clinical studies [62,108]. Since undifferentiated pluripotent stem cells
[60,61]. 3D bioprinting is a novel process to construct complex struc- can cause the formation of teratoma, stringent differentiation proce-
tures on the biomimetic scale [62]. The scaffold material has a sig- dures have to be performed to generate certain cellular phenotypes
nificant effect on printability and cell growth. In addition to tradi- before in vivo studies [62].
tionally used biomaterials, 3D bioprinting technology has shown
advances to the development of the ideal printable biomaterials which 4. 3D bioprinting applications of neural tissue
provide to mimic the natural structure of the ECM. Biomaterial-based
scaffolds enable microenvironment for cells. Also, these materials pro- Several studies have been tried with nerve scaffolds for CNS re-
vide to axonal regrowth in lesion sites and reinstate the neural circuitry generation. Table 2 summarizes 3D bioprinting applications for neural

4
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

Table 1
Biomaterials for treatment of injured spinal cord.
Materials Appropriate properties of the materials for the nervous Ref.

system

Collagen -Low immune response after transplantation. [71–74]

-Enables binding sites to reinforce cell adhesion, migration, proliferation, and differentiation.
-Shows enhanced astrocyte migration, oriented axonal growth and decreased scar invasion in the injury site, in a
transection SCI model in rats.
Hyaluronic acid -Highly compatible and supportive material to design scaffolds for SCI. [75–77]
-Exhibits alleviated inflammatory cell infiltration and decreased glial scar formation in vivo studies.
Chitosan -Supports the re-fabrication of spinal tissue, and modifies the inflammatory response in SCI. [75,78,79]
-Promotes axonal growth and cell adhesion.
-Increases differentiation of neural stem cell (NSC) in vivo.
Gelatin and gelatin-based biomaterials -Have the ability for filling the cavity, inhibiting inflammation, and diminishing necrosis and apoptosis for SCI repair. [75]
Agarose -Uses to regulate the cell adsorption and neurite outgrowth. [75,80–82]
-Promotes differentiation of neural progenitor cell (NPC) to oligodendrocytes.
Alginate -Can fill the injured spinal cord. [75,83,84]
-Promotes axonal outgrowth.
Fibrin -Has no toxic effects or contrary reactions to the recipient. [75,85]
-Conducts reconstructed blood vessels and axonal regeneration in the SCI model in rats.
Self-assembling peptides -Good biocompatibility. [75]
-Promote NSC growth and migration.
Acellular scaffolds -Act as a skeleton structure for cell attachment, proliferation, migration, and differentiation. [75,86]
-Can provide functional recovery through the facilitation of axonal regeneration.
Poly D, L-lactic acid (PLA) -Provides the direction to improve axonal expansion in vitro. [87–89]
-Decreases cavity volume in SCI models.
-Can be used as a drug carrier.
Poly-ϵ-caprolactone (PCL) -High mechanical property. [75,90]
-Slow degradation rate.
-Induces cell differentiation and neuronal extension.
Poly-lactic-co-glycolic acid (PLGA) -Enhances the therapeutic profits in animal models by binding the cells. [75,91,92]
-Encourages axonal outgrowth and NCS viability more than PCL and PLA.
Poly-glycolic acid (PGA) -Has a nerve-like structure with 3D scaffolds. [93–95]
-Mostly limited to bind a short nerve cavity because of unstable structure.
Polyethylene glycol (PEG) -Can repair the injured neuronal membrane. [75,96–98]
-Diminishes the mitochondrial-derived oxidative stress to save the damaged spinal cord.
-Supports NPC differentiation, adhesion and proliferation.
-Demonstrates good therapeutic impact in rat SCI models.

tissue engineering. manufacturing (FDM) technique. As a result of the study, improved cell
Wong et al. [109] developed scaffolds in different architectures proliferation and differentiation were observed. Scaffold showed great
using a 3D printer and investigated their effects on spinal cord re- success in the neural injury of zebrafish. Cell-loaded PU has the po-
generation. Porous PCL scaffolds were fabricated in macro-designs of tential to repair the function of damaged CNS. Tse et al. [113] printed
the cylinder, hollow tube, five-channel, open-path with core, and open- neuronal NG 108–15 cells and Schwann cells by utilization a piezo-
path with no core. These scaffolds implanted to transection rat SCI electric model inkjet printer and produced neural tissue. After printing,
model for 1 and 3 months. In vivo studies have shown that the open- 86% and 90% neuronal and glial cell viability was observed. Also,
path architectures resulted in less scar and provided elongation of nerve printed cells showed similar proliferation rates than unprinted cells.
fibers across the length of the defect in the scaffold. On the other hand, This study demonstrated that inkjet bioprinting supplies perfect cell
cylinder, tube and channel designs had a double defect length and have viability and has the potential to form a neural connection for printed
not provided a favorable environment for spinal cord regeneration. Lee structures. Furthermore, Gu et al. [114] performed micro-extrusion
et al. [110] produced collagen hydrogel scaffold with murine neural bioprinting of frontal cortical NSCs supplemented with alginate, car-
stem cells (NSCs, C17.2) and vascular endothelial growth factor boxymethyl chitosan and agarose hydrogel to obtain neural mini-tissue
(VEGF)-containing fibrin gel using inkjet bioprinting technique. As a structure. With this study, it was observed that the cells could survive in
result of printing, C17.2 cells demonstrated > 92% cell viability. When the structure and become functional neurons in situ. These differ-
C17.2 cells were printed in VEGF-fibrin gel, they demonstrated sig- entiated neurons may create synaptic connections and show an in-
nificant morphological alterations, multiplication and migration com- creased calcium response due to bicuculline. Wei et al. [115] used
pared to cells in the control group. This technique may be efficient to stereolithography bioprinting to promote nerve regeneration. In the
evaluate cellular behaviour and nerve tissue regeneration applications. study, a new nano-bioink consisting of gelatin methacrylamide, bioac-
In another work, Owens et al. [111] produced cellular nerve graft by 3D tive graphene nanoplatelets and NSCs was developed, and its applic-
bioprinting. Firstly, mouse bone marrow stem cells (BMSCs) and ability was evaluated. The results showed that the cells were distributed
Schwann cells were formed as cylindrical units as bio-ink components. homogeneously throughout all scaffolds and neurites spread from soma
Then, they were printed in layers to create a nerve graft. As a result of after 14 days of culture. Huang et al. [116] produced a biodegradable
the work, in vivo tests have shown that cellular nerve graft restores both polyurethane hydrogel containing graphene or graphene oxide. NSC
motor and sensory functions to certain levels. This work approved that loaded hydrogel was printed using a 3D bioprinter. Cell viability, dif-
the bioprinted graft was superior to an autologous graft for neural da- ferentiation, gene expression and oxygen metabolism were observed in
mages. Hsieh et al. [112] developed thermo-responsive polyurethane the scaffold with a low rate of graphene. This study supports the use of
(PU) hydrogel scaffold for traumatic brain injury in zebrafish. Firstly, 3D tissue structures containing cells in neural tissue engineering ap-
they produced PU hydrogel and then embedded NSCs into the hydrogel. plications. In another study, Zhu et al. [117] produced GelMA and
Then, they printed the PU hydrogel scaffold by fused deposition PEGDA scaffold via stereolithography-based bioprinting. Next, NSCs

5
 T. Bedir, et al.

Table 2
3D bioprinting applications of neural tissue by different printing methods and various cell types and bio-inks.
Bio-ink Cell type Printing method In vitro/in vivo Outcome Ref.

PCL – 3D printing In vivo (transection rat SCI The open-path scaffold designs preserved their length of defect up to 3 months and [109]
model) increased successfully spinal cord regeneration by comparison to the cylinder, tube
and channel designs.
Collagen and fibrin, fibrin loaded with NSCs Inkjet bioprinting In vitro 92% high cell viability observed after printing. [110]
VEGF Cells demonstrated morphological changes, migration and proliferation in VEGF-
fibrin gel.
Collagen BMSCs and Schwann cells 3D bioprinting In vivo Cellular nerve graft repaired both motor and sensory function. [111]
Polyurethane NSCs Fused deposition manufacturing In vitro Great cell growth and differentiation were observed on the scaffold. [112]
In vivo (zebrafish TBI PU showed improved motor function and survival rate in adult zebrafish with
model) induced TBI.
Cell suspension Schwann cells and neuronal NG Piezoelectric inkjet bioprinting In vitro 86% and 90% neuronal and glial cell viability was observed. [113]
108-15 cells
Alginate, carboxymethyl chitosan, Cortical NSCs Micro-extrusion bioprinting In vitro Cell viability and differentiation were observed. [114]

6
and agarose Differentiated neurons showed an increased calcium response due to bicuculline.
GelMa, graphene nanoplatelets NSCs Stereolithography In vitro Cells distribution throughout all scaffolds homogeneously was observed and neurites [115]
spread from soma after 14 days of culture.
Polyurethane and graphene or NSCs 3D bioprinting In vitro Cell viability, differentiation, the expression of gene and oxygen metabolism were [116]
graphene oxide observed in the scaffold with a low rate of graphene.
GelMa and PEGDA NSCs Stereolithography In vitro Light stimulation increased NSC neuronal differentiation and inhibited the [117]
generation of glial cells.
Carbon nanotube nanocellulose Neuroblasto ma cells 3D bioprinting In vitro The nanosized scaffold had good electrical conductivity. [118]
Cell growth and adhesion were observed.
PEDOT:PSS Dorsal root ganglia (DRG) cells Stereolithography In vitro Conductive hydrogel improved regulation and stimulation of cell behaviour. [119]
Alginate, HA and fibrin Schwann cells 3D bioplotting In vitro High viability of SCs (> 90%) was observed. [120]
SPCL and gellan gum Oligodendrocyte-like cells 3D bioplotting In vivo (hemisection rat Scaffolds were well fused within injury and did not trigger chronic inflammatory [121]
SCI model) processes.
Collagen–heparin sulfate NSCs 3D bioprinting In vivo (rat SCI model) Improved locomotor function was observed. [122]
Gelatin/fibrin and GelMa NPCs OPCs and Micro-extrusion bioprinting In vitro Bioprinted NPCs differentiated and extended axons throughout microscale scaffold [123]
channels.
PEGDA-GelMa NPCs Micro-scale continuous projection In vivo (rat SCI model) Injured host axons were regenerated in the scaffolds and formed synapse onto NPCs [124]
printing (μCPP) implanted into the scaffold.
Materials Science & Engineering C 110 (2020) 110741
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

a b

c d

Fig. 3. Production of BFO doped PLA scaffold by the modified 3D printer (a), BFO doped PLA scaffold (infill 70%) (b), SEM images of BFO doped PLA scaffold (infill
70%) (c,d).

were transplanted onto the scaffold. The study was performed using low this study have demonstrated the applicability of bioprinted scaffolds
rate light treatment to the recovery of injured nerve tissues. Thus, the that has low-viscosity bio-inks and proper for cell migration in the
cells were further stimulated and achieved a greater curative effect. scaffolds to repair neural damage. Similarly, Silva et al. [121] devel-
This study demonstrated that bioprinted neural scaffold with low rate oped a new biphasic tubular scaffold via 3D bioplotting method to
light treatment enhances cell growth and expression of the neural support regeneration in SCI sites. This scaffold consists of a mixture of
marker. Besides, neural stem cells can be further differentiated into starch = poly-ε-caprolactone (SPCL) and gellan gum. As a result of the
neural cells by using the laser. The use of this technique in neural re- in vitro study, it was observed that combined scaffolds could promote
generation can be effective. Kuzmenko et al. [118] developed a carbon oligodendrocyte-like cell culture. Furthermore, in vivo studies con-
nanotube nanocellulose scaffold using the 3D printing method. Then, ducted in a hemisection rat SCI model demonstrated that the scaffolds
neuroblastoma cells were embedded on the printed scaffold. This are well fused within the wound and did not induce chronic in-
scaffold that nano-sized demonstrated high electrical conductivity. flammatory processes. This study demonstrated that these 3D structures
Also, increased cell growth and binding were observed in cells cultured could be used in future SCI regeneration approaches. Chen et al. [122]
on the scaffold. The results showed that composite and electrically performed crosslinking of collagen using a natural polysaccharide he-
active scaffolds have the potential for neural tissue regeneration. Zhang parin sulfate. NSCs seeded on the 3D bioprinted collagen–heparin sul-
et al. [119] fabricated a conductive hydrogel for the treatment of fate scaffolds. This study improved a strategy to promote axonal re-
neurological diseases using stereolithography bioprinting for biological generation and functional recovery for a spinal cord injury. Research on
signal recording and inducement of living tissues. The electrical con- Sprague-Dawley rats reported that improvements in locomotor function
ductivity to the scaffold was provided by a poly(3,4-ethylene diox- according to electrophysiological examinations. Joung et al. [123] de-
ythiophene) PEDOT: polystyrene sulfonate (PSS) solution during veloped a scaffold for SCI using an extrusion-based bioprinter. Then,
printing. In this study, a 3D hydrogel scaffold that conductive has been induced pluripotent stem cells (iPSC)-derived neural progenitor cells
developed for regulation and stimulation of cell behaviour. This study (NPCs) and oligodendrocyte progenitor cells (OPCs) were implanted
supports applications for regeneration of neural tissue. Ning et al. [120] into the printed scaffolds. Cell position was checked up via a point-
produced hydrogels, containing alginate, HA and fibrin by the sub- dispensing printing method. The results showed that NPCs differentiate
merged cross-linking method. Then, they printed the hydrogel scaffold and elongate axons along the micro-scaffold channels. Properly bio-
including Schwann cells using 3D bioplotting method. The results of printing of OPCs with NPCs supports multicellular neural tissue

7
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

engineering applications. The capability to direct the modeling and lessons from preclinical studies, J. Tissue Eng. Regen. Med. 11 (2017) 321–333,
combination of transplanted cells may be useful in reconstructing ax- https://doi.org/10.1002/term.2052.
[9] V. Cheung, R. Hoshide, V. Bansal, E. Kasper, C.C. Chen, Methylprednisolone in the
onal links along with the CNS injury sites. This work may be used to management of spinal cord injuries: lessons from randomized, controlled trials,
create new biomimetic scaffolds that model CNS structure in vitro and to Surg. Neurol. Int. 6 (2015) 142, https://doi.org/10.4103/2152-7806.163452.
develop novel clinical approaches to cure neurological defects, con- [10] Z. Liu, Y. Yang, L. He, M. Pang, C. Luo, B. Liu, L. Rong, High-dose methyl-
prednisolone for acute traumatic spinal cord injury, Neurology 93 (2019) 1–10,
taining SCI. Koffler et al. [124] fabricated 3D biomimetic PEGDA- https://doi.org/10.1212/WNL.0000000000007998.
GelMa scaffolds using a micro-scale continuous projection printing [11] M. Schmidt, H.C. Neural tissue engineering, Biomaterials 22 (2001) 1015–1193.
method (μCPP) to form a complicated CNS structure for regenerative [12] A. Constans, Neural tissue engineering, Scientist. 18 (2004) 40–42.
[13] W. Zhu, N.J. Castro, L.G. Zhang, Nanotechnology and 3D bioprinting for neural
medicine applications in the spinal cord. μCPP can print the hydrogel tissue regeneration, 3D Bioprinting and Nanotechnology in Tissue Engineering and
scaffolds adapted to the size of the rodent spinal cord in 1.6 s. Also, it Regenerative Medicine, Elsevier, London, 2015, pp. 307–331.
can be scaled to human spinal cord dimensions and lesion geometries. [14] C. Cunha, S. Panseri, S. Antonini, Emerging nanotechnology approaches in tissue
engineering for peripheral nerve regeneration, Nanomedicine 7 (2011) 50–59,
After that, they loaded NPCs onto the 3D-printed scaffolds to support
https://doi.org/10.1016/j.nano.2010.07.004.
axon regeneration and form new ‘neural relays’ across sites of complete [15] A. Subramanian, U.M. Krishnan, S. Sethuraman, Development of biomaterial
spinal cord injury in vivo in rodents [125,126]. As a result of the study, scaffold for nerve tissue engineering: biomaterial mediated neural regeneration, J.
they found that the damaged host axons were regenerated into the Biomed. Sci. 16 (2009) 108–1108, https://doi.org/10.1186/1423-0127-16-108.
[16] T. Cheng, H. Qu, G. Zhang, X. Zhang, Osteogenic and antibacterial properties of
scaffolds. Also, these axons synapse onto NPCs placed into the device vancomycin-laden mesoporous bioglass/PLGA composite scaffolds for bone re-
and that implanted NPCs, in turn, elongate axons out of the scaffold and generation in infected bone defects, Artif Cells Nanomed Biotechnol 46 (2018)
into the host spinal cord below the damage to repair the synaptic 1935–1947, https://doi.org/10.1080/21691401.2017.1396997.
[17] S.S. Liao, F.Z. Cui, W. Zhang, Q.L. Feng, Hierarchically biomimetic bone scaffold
conduction and develop functional results. This study demonstrates that materials: nano-HA/collagen/PLA composite, J Biomed Mater Res B Appl
3D biomimetic scaffolds are an effective option to increase CNS re- Biomater 69 (2004) 158–165, https://doi.org/10.1002/jbm.b.20035.
generation [124]. [18] B.K. Gu, D.J. Choi, S.J. Park, M.S. Kim, C.M. Kang, C.H. Kim, 3-dimensional bio-
printing for tissue engineering applications, Biomaterials Research 20 (2016),
Our laboratory has carried out studies using 3D printing technology https://doi.org/10.1186/s40824-016-0058-2.
for applications of tissue engineering. In a recent study, we successfully [19] J.P. Kruth, M.C. Leu, T. Nakagawa, Progress in additive manufacturing and rapid
produced PLA scaffold with bismuth ferrite (BFO) nanoparticles on a prototyping, ClRP Annals Manufacturing Technology 47 (1998) 525–540, https://
doi.org/10.1016/S0007-8506(07)63240-5.
modified 3D printer. This produced biocompatible and electrically ac- [20] A.P. Haring, H. Sontheimer, B.N. Johnson, Microphysiological human brain and
tive scaffold has excellent potential for use in applications of neural neural systems-on-a-chip: potential alternatives to small animal models and
tissue engineering (Fig. 3). emerging platforms for drug discovery and personalized medicine, Stem Cell Rev.
Rep. 13 (2017) 381–406, https://doi.org/10.1007/s12015-017-9738-0.
[21] H. Bikas, P. Stavropoulos, G. Chryssolouris, Additive manufacturing methods and
5. Conclusion modeling approaches: a critical review, Int. J. Adv. Manuf. Technol. 83 (2016)
389–405, https://doi.org/10.1007/s00170-015-7576-2.
SCI is a CNS disease that does not have an adequate treatment yet [22] K.V. Wong, A. Hernandez, A review of additive manufacturing, Inter Scholar
Resear Net 2012 (2012) 10, https://doi.org/10.5402/2012/208760.
and affects the individual's life negatively. Disruptions in the CNS [23] J. Malda, J. Visser, F.P. Melchels, T. Jüngst, W.E. Hennink, W.J.A. Dhert, J. Groll,
structure result in loss of neuronal cell bodies, axons, and associated D.W. Hutmacher, 25th anniversary article: engineering hydrogels for biofabrica-
glia support. Neural tissue engineering is focused on the improvement tion, Adv. Mater. 25 (2013) 5011–5028, https://doi.org/10.1002/adma.
201302042.
of an appropriate environment that connects the biomimetic scaffold [24] X. Wang, Y. Yan, R. Zhang, Rapid prototyping as tool for manufacturing bioarti-
with cells to repair and restore neural tissue function. 3D bioprinting is ficial livers, Trends Biotechnol. 25 (2007) 505–513, https://doi.org/10.1016/j.
an attractive technology for the production of complicated neural grafts tibtech.2007.08.010.
[25] J. Hendriks, C.W. Visser, S. Henke, J. Leijten, D.B. Saris, C. Sun, D. Lohse,
where different bioactive factors and cells can be easily combined and M. Karperien, Optimizing cell viability in droplet-based cell deposition, Sci. Rep. 5
used synergistically for developed neural regeneration. On the other (2015) 11304, https://doi.org/10.1038/srep11304.
hand, the production of complicated constructions with multiple cell [26] I.T. Ozbolat, M. Hospodiuk, Current advances and future perspectives in extrusion-
based bioprinting, Biomaterials 76 (2016) 321–343, https://doi.org/10.1016/j.
types still a challenge for this technology. In this review article, various
biomaterials.2015.10.076.
3D bioprinting methods such as extrusion, inkjet, laser, plotting utilized [27] Q. Zheng, J. Lu, H. Chen, L. Huang, J. Cai, Z. Xu, Application of inkjet printing
in applications of neural tissue have been discussed in detail. technique for biological material delivery and antimicrobial assays, Anal.
Biochem. 410 (2011) 171–176, https://doi.org/10.1016/j.ab.2010.10.024.
Furthermore, bio-inks and cells available for bioprinting have been
[28] C. O’Brien, B. Holmes, S. Faucett, L. Zhang, Three-dimensional printing of nano-
explained. Finally, applications for models of 3D neural tissue designed material scaffolds for complex tissue regeneration, Tissue Eng. Part B. 21 (2015)
by bioprinting methods, especially for the repair of spinal cord injury 103–114, https://doi.org/10.1089/ten.teb.2014.0168.
has been described. [29] C.C. Chang, E.D. Boland, S.K. Williams, Direct-write bioprinting three-dimensional
biohybrid systems for future regenerative therapies, J. Biomed. Mater. Res. B Appl.
Biomater. 98 (2011) 160–170, https://doi.org/10.1002/jbm.b.31831.
References [30] S.V. Murphy, A. Atala, 3D bioprinting of tissues and organs, Nat. Biotechnol. 32
(2014) 773–785, https://doi.org/10.1038/nbt.2958.
[31] X. Cui, D. Dean, Z.M. Ruggeri, Cell damage evaluation of thermal inkjet printed
[1] D. Purves, G.J. Augustine, D. Fitzpatrick, W.C. Hall, A.S. Lamantia, Chinese hamster ovary cells, Biotechnol. Bioeng. 106 (2010) 963–969, https://doi.
J.O. Mcnamara, S.M. Williams, Neuroscience Third Edition, Sinauer Associates org/10.1002/bit.22762.
Inc, USA, 2004. [32] O.A. Mohamed, S.H. Masood, J.L. Bhowmik, Optimization of fused deposition
[2] L.R. Squire, F.E. Bloom, N. Spitzer, S. Lac, A. Ghosh, D. Berg, Fundamental modeling process parameters: a review of current research and future prospects,
Neuroscience, Elsevier Inc, USA, 2008, p. 1127. Adv. Manuf. 3 (2015) 42–53, https://doi.org/10.1007/s40436-014-0097-7.
[3] O. Lindvall, Z. Kokaia, Stem cells for the treatment of neurological disorders, [33] H. Cui, M. Nowicki, J.P. Fisher, L.G. Zhang, 3D bioprinting for organ regeneration,
Nature 441 (2006) 1094–1096, https://doi.org/10.1038/nature04960. Adv. Healthcare Mater. 6 (2017) 1601118, , https://doi.org/10.1002/adhm.
[4] Y. Zhong, R.V. Bellamkonda, Biomaterials for the central nervous system, J. R. 201601118.
Soc. Interface 5 (2008) 957–975, https://doi.org/10.1098/rsif.2008.0071. [34] A.K. Sood, R.K. Ohdar, S.S. Mahapatra, Parametric appraisal of mechanical
[5] T. Qian, X. Guo, A.D. Levi, S. Vanni, R.T. Shebert, M.L. Sipski, High-dose me- property of fused deposition modelling processed parts, Mater. Des. 31 (2010)
thylprednisolone may cause myopathy in acute spinal cord injury patients, Spinal 287–295, https://doi.org/10.1243/09544054JEM1565.
Cord 43 (2005) 199–203, https://doi.org/10.1038/sj.sc.3101681. [35] K.F. Leong, C.M. Cheah, C.K. Chua, Solid freeform fabrication of three-dimen-
[6] D.C. Baptiste, M.G. Fehlings, Pharmacological approaches to repair the injured sional scaffolds for engineering replacement tissues and organs, Biomaterials 24
spinal cord, J. Neurotrauma 23 (2006) 318–334, https://doi.org/10.1089/neu. (2003) 2363–2378, https://doi.org/10.1016/S0142-9612(03)00030-9.
2006.23.318. [36] J.S. Chohan, R. Singh, K.S. Boparai, R. Penna, F. Fraternali, Dimensional accuracy
[7] L.H. Sekhon, M.G. Fehlings, Epidemiology, demographics, and pathophysiology of analysis of coupled fused deposition modeling and vapour smoothing operations
acute spinal cord injury, Spine 26 (2001) 2–12, https://doi.org/10.1097/ for biomedical applications, Compos B Eng 117 (2017) 138–149, https://doi.org/
00007632-200112151-00002. 10.1016/j.compositesb.2017.02.045.
[8] C. Venugopal, S. Chandanala, H.C. Prasad, D. Nayeem, R.R. Bhonde, [37] S. Knowlton, B. Yenilmez, S. Anand, S. Tasoglu, Photocrosslinking-based bio-
A. Dhanushkodi, Regenerative therapy for hippocampal degenerative diseases: printing: examining crosslinking schemes, Bioprinting 5 (2017) 10–18, https://

8
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

doi.org/10.1016/j.bprint.2017.03.001. [64] S. Liu, T. Schackel, N. Weidner, R. Puttagunta, Biomaterial-supported cell trans-

[38] T. Billiet, M. Vandenhaute, J. Schelfhout, S. Van Vlierberghe, P. Dubruel, A review plantation treatments for spinal cord injury: challenges and perspectives, Front.
of trends and limitations in hydrogel-rapid prototyping for tissue engineering, Cell. Neurosci. 11 (2017), https://doi.org/10.3389/fncel.2017.00430.
Biomaterials 33 (2012) 6020–6041, https://doi.org/10.1016/j.biomaterials.2012. [65] J. Zhu, R.E. Marchant, Design properties of hydrogel tissue-engineering scaffolds,
04.050. Expert Rev Med Devices 8 (2011) 607–626, https://doi.org/10.1586/erd.11.27.
[39] P.W.F. Melchels, J. Feijen, D.W. Grijpma, A review on stereolithography and its [66] D. Macaya, M. Spector, Injectable hydrogel materials for spinal cord regeneration:
applications in biomedical engineering, Biomaterials 31 (2010) 6121–6130, a review, Biomed. Mater. 7 (2012) 22, https://doi.org/10.1088/1748-6041/7/1/
https://doi.org/10.1016/j.biomaterials.2010.04.050. 012001.
[40] J.S. Choi, H.W. Kang, I.H. Lee, T.J. Ko, D.W. Cho, Development of micro- [67] A.E. Haggerty, M. Oudega, Biomaterials for spinal cord repair, Neurosci. Bull. 29
stereolithography technology using a UV lamp and optical fiber, Int. J. Adv. (2013) 445–459, https://doi.org/10.1007/s12264-013-1362-7.
Manuf. Technol. 41 (2009) 281–286, https://doi.org/10.1007/s12541-015- [68] M. Spector, T.C. Lim, Injectable biomaterials: a perspective on the next wave of
0179-x. injectable therapeutics, Biomed. Mater. 11 (2016) 014110, , https://doi.org/10.
[41] S.A. Skoog, P.L. Goering, R.J. Narayan, Stereolithography in tissue engineering, J. 1088/1748-6041/11/1/014110.
Mater. Sci. Mater. Med. 25 (2014) 845–856, https://doi.org/10.1007/s10856- [69] S. Knowlton, S. Anand, T. Shah, S. Tasoglu, Bioprinting for neural tissue en-
013-5107-y. gineering, Trends Neurosci. 41 (2018) 31–46, https://doi.org/10.1016/j.tins.
[42] Z.C. Eckel, C. Zhou, J.H. Martin, A.J. Jacobsen, W.B. Carter, T.A. Schaedler, 2017.11.001.
Additive manufacturing of polymer-derived ceramics, Science 351 (2016) 58–62, [70] P. Rider, Ž. Kačarević, S. Alkildani, S. Retnasingh, M. Barbeck, Bioprinting of
https://doi.org/10.1126/science.aad2688. tissue engineering scaffolds, Journal of Tissue Engineering 9 (2018) 1–16, https://
[43] J.Z. Manapat, Q. Chen, P. Ye, R.C. Advincula, 3D printing of polymer nano- doi.org/10.1177/2041731418802090.
composites via stereolithography, Macromol. Mater. Eng. 302 (2017), https://doi. [71] S. Guan, X.L. Zhang, X.M. Lin, T.Q. Liu, X.H. Ma, Z.F. Cui, Chitosan/gelatin porous
org/10.1002/mame.201600553. scaffolds containing hyaluronic acid and heparan sulfate for neural tissue en-
[44] J. Edgar, M. Robinson, S. Willerth, Fibrin hydrogels induce mixed dorsal/ventral gineering, J Biomater Sci Polym Ed 24 (2013) 999–1014, https://doi.org/10.
spinal neuron identities during differentiation of human induced pluripotent stem 1080/09205063.2012.731374.
cells, Acta Biomater. 51 (2017) 237–245, https://doi.org/10.1016/j.actbio.2017. [72] A.R. Murphy, A. Laslett, C.M. O’Brien, N.R. Cameron, Scaffolds for 3D in vitro
01.040. culture of neural lineage cells, Acta Biomater. 54 (2017) 1–20, https://doi.org/10.
[45] A. Panwar, L.P. Tan, Current status of bioinks for micro-extrusion-based 3D bio- 1016/j.actbio.2017.02.046.
printing, Molecules 21 (2016) 685, https://doi.org/10.3390/molecules21060685. [73] M.H. Spilker, I.V. Yannas, S.K. Kostyk, T.V. Norregaard, H.P. Hsu, M. Spector, The
[46] F.P.W. Melchels, M.A.N. Domingos, T.J. Klein, Additive manufacturing of tissues effects of tubulation on healing and scar formation after transection of the adult
and organs, Prog. Polym. Sci. 37 (2012) 1079–1104, https://doi.org/10.1016/j. rat spinal cord, Restor. Neurol. Neurosci. 18 (2001) 23–38.
progpolymsci.2011.11.007. [74] S. Liu, P. Peulve, O. Jin, N. Boisset, J. Tiollier, G. Said, M. Tadie, Axonal regrowth
[47] S.M. Peltola, F.P. Melchels, D.W. Grijpma, A review of rapid prototyping techni- through collagen tubes bridging the spinal cord to nerve roots, J. Neurosci. Res. 49
ques for tissue engineering purposes, Ann. Med. 40 (2008) 268–280, https://doi. (1997) 425–432, https://doi.org/10.1002/(SICI)1097-4547(19970815)
org/10.1080/07853890701881788. 49:4<425::AID-JNR4>3.0.CO;2-A.
[48] C.M. Smith, A.L. Stone, R.L. Parkhill, R.L. Stewart, M.W. Simpkins, A.M. Kachurin, [75] S. Liu, Y.Y. Xie, B. Wang, Role and prospects of regenerative biomaterials in the
Three-dimensional bioassembly tool for generating viable tissue engineered con- repair of spinal cord injury, Neural Regen. Res. 14 (2019) 1352–1363, https://doi.
structs, Tissue Eng. 10 (2004) 1566–1576, https://doi.org/10.1089/ten.2004.10. org/10.4103/1673-5374.253512.
1566. [76] Z.K. Zin, D.M. Brian, E.V. Jennifer, K.S. Stephanie, J.G. Raymond, E.S. Christine,
[49] F. Pati, J. Jang, J.W. Lee, Extrusion bioprinting, Essentials of 3D Biofabrication High molecular weight hyaluronic acid limits astrocyte activation and scar for-
and Translation, 2015, pp. 123–152, , https://doi.org/10.1016/B978-0-12- mation after spinal cord injury, J. Neural Eng. 8 (2011) 046033, , https://doi.org/
800972-7.00007-4. 10.1088/1741-2560/8/4/046033.
[50] E.S. Bishop, S. Mostafa, M. Pakvasa, 3-D bioprinting technologies in tissue en- [77] C.M. Lin, J.W. Lin, Y.C. Chen, H.H. Shen, L. Wei, Y.S. Yeh, Y.H. Chiang, R. Shih,
gineering and regenerative medicine: current and future trends, Genes Dis 4 P.L. Chiu, K.S. Hung, L.Y. Yang, W.T. Chiu, Hyaluronic acid inhibits the glial scar
(2017) 185–195, https://doi.org/10.1016/j.gendis.2017.10.002. formation after brain damage with tissue loss in rats, Surg. Neurol. 72 (2009)
[51] K. Nair, M. Gandhi, S. Khalil, K.C. Yan, M. Marcolongo, K. Barbee, 50–54, https://doi.org/10.1016/j.wneu.2009.09.004.
Characterization of cell viability during bioprinting processes, Biotechnol. J. 4 [78] T. Freier, R. Montenegro, H. Shan Koh, M.S. Shoichet, Chitin-based tubes for tissue
(2009) 1168–1177, https://doi.org/10.1002/biot.200900004. engineering in the nervous system, Biomaterials 26 (2005) 4624–4632, https://
[52] X. Zhang, Y. Zhang, Tissue engineering applications of three-dimensional bio- doi.org/10.1016/j.biomaterials.2004.11.040.
printing, Cell Biochem. Biophys. 72 (2015) 777–782, https://doi.org/10.1007/ [79] T. Zahir, H. Nomura, X.D. Guo, H. Kim, C. Tator, C. Morshead, M. Shoichet,
s12013-015-0531-x. Bioengineering neural stem/progenitor cell-coated tubes for spinal cord injury
[53] S. Hsu, W.C. Chang, C.T. Yen, Novel flexible nerve conduits made of water-based repair, Cell Transplant. 17 (2008) 245–254, https://doi.org/10.3727/
biodegradable polyurethane for peripheral nerve regeneration, J. Biomed. Mater. 096368908784153887.
Res. A 105 (2017) 1383–1392, https://doi.org/10.1002/jbm.a.36022. [80] Y. Luo, M.S. Shoichet, A photolabile hydrogel for guided three-dimensional cell
[54] B. Dhariwala, E. Hunt, T. Boland, rapid prototyping of tissue-engineering con- growth and migration, Nat. Mater. 3 (2004) 249–253, https://doi.org/10.1038/
structs, using photopolymerizable hydrogels and stereolithography, Tissue Eng. 10 nmat1092.
(2004) 1316–1322, https://doi.org/10.1089/ten.2004.10.1316. [81] A. Jain, Y.T. Kim, R.J. McKeon, R.V. Bellamkonda, In situ gelling hydrogels for
[55] A. Skardal, J. Zhang, G.D. Prestwich, Bioprinting vessel-like constructs using conformal repair of spinal cord defects, and local delivery of BDNF after spinal
hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetra- cord injury, Biomaterials 27 (2006) 497–504, https://doi.org/10.1016/j.
crylates, Biomaterials 31 (2010) 6173–6181, https://doi.org/10.1016/j. biomaterials.2005.07.008.
biomaterials.2010.04.045. [82] Y. Aizawa, N. Leipzig, T. Zahir, M.S. Shoichet, The effect of immobilized platelet
[56] Y.J. Seol, H.W. Kang, S.J. Lee, Bioprinting technology and its applications, Eur. J. derived growth factor AA on neural stem/progenitor cell differentiation on cell-
Cardiothorac. Surg. 46 (2014) 342–348, https://doi.org/10.1093/ejcts/ezu148. adhesive hydrogels, Biomaterials 29 (2008) 4676–4683, https://doi.org/10.1016/
[57] A. Pfister, R. Landers, A. Laib, U. Hübner, R. Schmelzeisen, R. Mülhaupt, j.biomaterials.2008.08.018.
Biofunctional rapid prototyping for tissue-engineering applications: 3D bioplotting [83] Y. Suzuki, M. Kitaura, S. Wu, K. Kataoka, K. Suzuki, K. Endo, Y. Nishimura, C. Ide,
versus 3D printing, J. Polym. Sci. A Polym. Chem. 42 (2004) 624–638, https://doi. Electrophysiological and horseradish peroxidase-tracing studies of nerve re-
org/10.1002/pola.10807. generation through alginate-filled gap in adult rat spinal cord, Neurosci. Lett. 318
[58] R. Landers, R. Mülhaupt, Desktop manufacturing of complex objects, prototypes (2002) 121–124, https://doi.org/10.1016/S0304-3940(01)02359-X.
and biomedical scaffolds by means of computer-assisted design combined with [84] K. Kataoka, Y. Suzuki, M. Kitada, K. Ohnishi, K. Suzuki, M. Tanihara, C. Ide,
computer-guided 3D plotting of polymers and reactive oligomers, Macromol. K. Endo, Y. Nishimura, Alginate, a bioresorbable material derived from brown
Mater. Eng. 282 (2000) 17–21, https://doi.org/10.1002/1439-2054(20001001) seaweed, enhances elongation of amputated axons of spinal cord in infant rats, J.
282:1<17::AID-MAME17>3.0.CO;2-8. Biomed. Mater. Res. 54 (2001) 373–384, https://doi.org/10.1002/1097-
[59] C.Y. Huang, K.H. Hu, Z.H. Wei, Comparison of cell behavior on pva/pva-gelatin 4636(20010305)54:3<373::aid-jbm90>3.0 (co;2-q).
electrospun nanofibers with random and aligned configuration, Sci. Rep. 6 (2016), [85] Z.A. Yao, F.J. Chen, H.L. Cui, T. Lin, N. Guo, H.G. Wu, Efficacy of chitosan and
https://doi.org/10.1038/srep37960. sodium alginate scaffolds for repair of spinal cord injury in rats, Neural Regen.
[60] K. Suzuki, Y. Suzuki, K. Ohnishi, K. Endo, M. Tanihara, Y. Nishimura, Res. 13 (2018) 502–509, https://doi.org/10.4103/1673-5374.228756.
Regeneration of transected spinal cord in young adult rats using freeze-dried al- [86] Y. Hong, T. Jiang, X. Deng, M. Yu, H. Xing, X. Ren, A cellular spinal cord scaffold
ginate gel, Neuroreport 10 (1999) 2891–2894, https://doi.org/10.1097/ seeded with rat adipose-derived stem cells facilitates functional recovery via en-
00001756-199909290-00003. hancing axon regeneration in spinal cord injured rats, Mol. Med. Rep. 17 (2018)
[61] H. Nomura, C.H. Tator, M.S. Shoichet, Bioengineered strategies for spinal cord 2998–3004, https://doi.org/10.3892/mmr.2017.8238.
repair, J. Neurotrauma 23 (2006) 496–507, https://doi.org/10.1089/neu.2006. [87] J.M. Corey, D.Y. Lin, K.B. Mycek, Q. Chen, S. Samuel, E.L. Feldman, D.C. Martin,
23.496. Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite
[62] S.J. Lee, T. Esworthy, S. Stake, S. Miao, Y.Y. Zuo, B.T. Harris, L.G. Zhang, growth, J Biomed Mater Res Part A 83 (2007) 636–645, https://doi.org/10.1002/
Advances in 3D bioprinting for neural tissue engineering, Adv. Biosys. 2 (2018) jbm.a.31285.
1700213, , https://doi.org/10.1002/adbi.201700213. [88] B.W. Han, M.E. Mullins, J.M. Cregg, A. Hurtado, M. Oudega, M.T. Trombley,
[63] R.C. Assuncao-Silva, E.D. Gomes, N.A. Silva, A.J. Salgado, Nanoengineered bio- R.J. Gilbert, Creation of highly aligned electrospun poly-L-lactic acid fibers for
materials for spinal cord regeneration, Micro and Nano Technologies (2019) nerve regeneration applications, J. Neural Eng. 6 (2008), https://doi.org/10.
167–185, https://doi.org/10.1016/B978-0-12-813355-2.00008-9. 1088/1741-2560/6/1/016001.

9
T. Bedir, et al. Materials Science & Engineering C 110 (2020) 110741

[89] J.A. Roman, I. Reucroft, R.A. Martin, A. Hurtado, H.Q. Mao, Local release of pa- stem cells, Stem Cell Res Ther 2 (2011) 17, https://doi.org/10.1186/scrt58.
clitaxel from aligned, electrospun microfibers promotes axonal extension, Adv [109] D.Y. Wong, J.C. Leveque, H. Brumblay, P.H. Krebsbach, S.J. Hollister, F. LaMarca,
Healthc Mater 5 (2016) 2628–2635, https://doi.org/10.1002/adhm.201600415. Macro-architectures in spinal cord scaffold implants influence regeneration, J.
[90] J. Xie, S.M. Willerth, X. Li, M.R. Macewan, A. Rader, S.E. Sakiyama-Elbert, Y. Xia, Neurotrauma 25 (2008) 1027–1037, https://doi.org/10.1089/neu.2007.0473.
The differentiation of embryonic stem cells seeded on electrospun nanofibers into [110] Y.B. Lee, S. Polio, W. Lee, G. Dai, L. Menon, R.S. Carroll, S.S. Yoo, Bio-printing of
neural lineages, Biomaterials 30 (2009) 354–362, https://doi.org/10.1016/j. collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture, Exp.
biomaterials.2008.09.046. Neurol. 223 (2010) 645–652, https://doi.org/10.1016/j.expneurol.2010.02.014.
[91] X. Yi, J.X. Zhu, Z.Y. Fang, C.G. Zeng, C. Zhang, G.L. Qi, M.H. Li, W. Zhang, [111] C.M. Owens, F. Marga, G. Forgacs, C.M. Heesch, Biofabrication and testing of a
D.P. Quan, J. Wan, Coseeded Schwann cells myelinate neurites from differentiated fully cellular nerve graft, Biofabrication 5 (2013), https://doi.org/10.1088/1758-
neural stem cells in neurotrophin-3-loaded PLGA carriers, Int. J. Nanomedicine 7 5082/5/4/045007.
(2012) 1977–1989, https://doi.org/10.2147/IJN.S30706. [112] F.Y. Hsieh, H.H. Lin, S. Hsu, 3D bioprinting of neural stem cell-laden
[92] S.H. Bhang, J.S. Lim, C.Y. Choi, Y.K. Kwon, B.S. Kim, The behavior of neural stem Thermoresponsive biodegradable polyurethane hydrogel and potential in central
cells on biodegradable synthetic polymers, J. Biomater. Sci. Polym. Ed. 18 (2007) nervous system repair, Biomaterials 71 (2015) 48–57, https://doi.org/10.1016/j.
223–239, https://doi.org/10.1163/156856207779116711. biomaterials.2015.08.028.
[93] R. Boni, A. Ali, A. Shavandi, A.N. Clarkson, Current and novel polymeric bioma- [113] C. Tse, R. Whiteley, T. Yu, J. Stringer, S. MacNeil, J.W. Haycock, P.J. Smith, Inkjet
terials for neural tissue engineering, J. Biomed. Sci. 25 (2018), https://doi.org/10. printing Schwann cells and neuronal analogue NG108-15 cells, Biofabrication 8
1186/s12929-018-0491-8. (2016) 015017, , https://doi.org/10.1088/1758-5090/8/1/015017.
[94] T. Komiyama, Y. Nakao, Y. Toyama, C.A. Vacanti, M.P. Vacanti, R.A. Ignotz, Novel [114] Q. Gu, E. Tomaskovic-Crook, R. Lozano, Y. Chen, R.M. Kapsa, Q. Zhou,
technique for peripheral nerve reconstruction in the absence of an artificial con- G.G. Wallace, J.M. Crook, Functional 3D neural mini-tissues from printed gel-
duit, J. Neurosci. Methods 134 (2004) 133–140, https://doi.org/10.1016/j. based bioink and human neural stem cells, Adv. Healthc. Mater. 5 (2016)
jneumeth.2003.11.020. 1429–1438, https://doi.org/10.1002/adhm.201600095.
[95] A.M. Moore, R. Kasukurthi, C.K. Magill, H.F. Farhadi, G.H. Borschel, [115] W. Zhu, B.T. Herris, L.G. Zhang, Gelatin methacrylamide hydrogel with graphene
S.E. Mackinnon, Limitations of conduits in peripheral nerve repairs, Hand 4 nanoplatelets for neural cell-laden 3D bioprinting, Conf. Proc. IEEE Eng. Med.
(2009) 180–186, https://doi.org/10.1007/s11552-008-9158-3. Biol. Soc. (2016) 4185–4188, https://doi.org/10.1109/EMBC.2016.7591649.
[96] A. Nehrt, K. Hamann, H. Ouyang, R. Shi, Polyethylene glycol enhances axolemmal [116] C.T. Huang, L.K. Shrestha, K. Ariga, S.H. Hsu, A graphene–polyurethane composite
resealing following transection in cultured cells and in ex vivo spinal cord, J. hydrogel as a potential bioink for 3D bioprinting and differentiation of neural stem
Neurotrauma 27 (2010) 151–161, https://doi.org/10.1089/neu.2009.0993. cells, J. Mater. Chem. B 5 (2017) 8854–8864, https://doi.org/10.1039/
[97] S. Kouhzaei, I. Rad, S. Mousavidoust, H. Mobasheri, Protective effect of low mo- C7TB01594A.
lecular weight polyethylene glycol on the repair of experimentally damaged [117] W. Zhu, J.K. George, V.J. Sorger, L.G. Zhang, 3D printing scaffold coupled with
neural membranes in rat’s spinal cord, Neurol. Res. 35 (2013) 415–423, https:// low level light therapy for neural tissue regeneration, Biofabrication 9 (2017),
doi.org/10.1179/1743132812Y.0000000133. https://doi.org/10.1088/1758-5090/aa6999.
[98] M. Pang, T. Shu, R.Q. Chen, C. Liu, L. He, Y. Yang, A.S.A. Bardeesi, C.K. Lin, [118] V. Kuzmenko, E. Karabulut, E. Pernevik, P. Enoksson, P. Gatenholm, Tailor-made
L.M. Zhang, X. Wang, Neural precursor cells generated from induced pluripotent conductive inks from cellulose nanofibrils for 3D printing of neural guidelines,
stem cells with gelatin sponge-electrospun PLGA/PEG nanofibers for spinal cord Carbohydr. Polym. 189 (2018) 22–30, https://doi.org/10.1016/j.carbpol.2018.
injury repair, Int. J. Clin. Exp. Med. 9 (2016) 17985–17994. 01.097.
[99] F.S. Ambesi-Impiombato, L.A. Parks, H.G. Coon, Culture of hormone-dependent [119] D.N. Heo, S.J. Lee, R. Timsina, X. Qiu, N.J. Castro, L.G. Zhang, Development of 3D
functional epithelial cells from rat thyroids, Proc. Natl. Acad. Sci. U. S. A. 77 printable conductive hydrogel with crystallized PEDOT: PSS for neural tissue en-
(1980) 3455–3459, https://doi.org/10.1073/pnas.77.6.3455. gineering, Mater. Sci. Eng. C. 99 (2019) 582–590, https://doi.org/10.1016/j.
[100] Y. Gu, J. Zhu, C. Xue, Z. Li, F. Ding, Y. Yang, X. Gu, Chitosan/silk fibroin-based, msec.2019.02.008.
Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat [120] L. Ning, N. Zhu, F. Mohabatpour, M.D. Sarker, D.J. Schreyer, X. Chen, Bioprinting
sciatic nerve gaps, Biomaterials 35 (2014) 2253–2263, https://doi.org/10.1016/j. schwann cell-laden scaffolds from low-viscosity hydrogel compositions, J. Mater.
biomaterials.2013.11.087. Chem. B 7 (2019) 4538–4551, https://doi.org/10.1039/c9tb00669a.
[101] L.N. Novikova, L.N. Novikov, J.O. Kellerth, Differential effects of neurotrophins on [121] N.A. Silva, A.J. Salgado, R.A. Sousa, J.T. Oliveira, A.J. Pedro, H.L. Almeida,
neuronal survival and axonal regeneration after spinal cord injury in adult rats, J. R. Cerqueira, A. Almeida, F. Mastronardi, J.F. Mano, N.M. Neves, N. Sousa,
Comp. Neurol. 452 (2002) 255–263, https://doi.org/10.3727/ R.L. Reis, Development and characterization of a novel hybrid tissue en-
000000006783464381. gineering–based scaffold for spinal cord injury repair, Tissue Eng. A 16 (2010)
[102] C.T. van Velthoven, A. Kavelaars, F. van Bel, C.J. Heijnen, Mesenchymal stem cell 45–54, https://doi.org/10.1089/ten.TEA.2008.0559.
treatment after neonatal hypoxic-ischemic brain injury improves behavioral out- [122] C. Chen, M. Zhao, R. Zhang, G. Lu, C. Zhao, F. Fu, H. Sun, S. Zhang, Y. Tu, X. Li,
come and induces neuronal and oligodendrocyte regeneration, Brain Behav. Collagen/heparin sulfate scaffolds fabricated by a 3D bioprinter improved me-
Immun. 24 (2010) 387–393, https://doi.org/10.1016/j.bbi.2009.10.017. chanical properties and neurological function after spinal cord injury in rats, J.
[103] S.C. Barnett, J.S. Riddell, Olfactory ensheathing cells (OECs) and the treatment of Biomed. Mater. Res. A 105 (2017) 1324–1332, https://doi.org/10.1002/jbm.a.
CNS injury: advantages and possible caveats, J. Anat. 204 (2004) 57–67, https:// 36011.
doi.org/10.1111/j.1469-7580.2004.00257.x. [123] D. Joung, V. Truong, C.C. Neitzke, S.Z. Guo, P.J. Walsh, J.R. Monat, F. Meng,
[104] M.A. Dombrowski, M. Sasaki, K.L. Lankford, J.D. Kocsis, C. Radtke, Myelination S.H. Park, J.R. Dutton, A.M. Parr, M.C. McAlpine, 3D printed stem-cell derived
and nodal formation of regenerated peripheral nerve fibers following transplan- neural progenitors generate spinal cord scaffolds, Adv. Funct. Mater. 28 (2018),
tation of acutely prepared olfactory ensheathing cells, Brain Res. 1125 (2006) 1–8, https://doi.org/10.1002/adfm.201801850.
https://doi.org/10.1016/j.brainres.2006.09.089. [124] J. Koffler, W. Zhu, X. Qu, O. Platoshyn, J.N. Dulin, J. Brock, L. Graham, P. Lu,
[105] Q. Ao, A.J. Wang, G.Q. Chen, S.J. Wang, H.C. Zuo, X.F. Zhang, Combined trans- J. Sakamoto, M. Marsala, S. Chen, M.H. Tuszynski, Biomimetic 3D-printed scaf-
plantation of neural stem cells and olfactory ensheathing cells for the repair of folds for spinal cord injury repair, Nat. Med. 25 (2019) 263–269, https://doi.org/
spinal cord injuries, Med. Hypotheses 69 (2007) 1234–1237, https://doi.org/10. 10.1038/s41591-018-0296-z.
1016/j.mehy.2007.04.011. [125] K. Kadoya, P. Lu, K. Nguyen, C. Lee-Kubli, H. Kumamaru, L. Yao, J. Knackert,
[106] H.S. Keirstead, G. Nistor, G. Bernal, M. Totoiu, F. Cloutier, K. Sharp, O. Steward, G. Poplawski, J. Dublin, H. Strobl, Y. Takashima, J. Biane, J. Conner, S.C. Zhang,
Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants M. Tuszynski, Spinal cord reconstitution with homologous neural grafts enables
remyelinate and restore locomotion after spinal cord injury, J. Neurosci. 25 (2005) robust corticospinal regeneration, Nat. Med. 22 (2016) 479–487, https://doi.org/
4694–4705, https://doi.org/10.1523/JNEUROSCI.0311-05.2005. 10.1038/nm.4066.
[107] M. Hatami, N.Z. Mehrjardi, S. Kiani, K. Hemmesi, H. Azizi, A. Shahverdi, [126] P. Lu, Y. Wang, L. Graham, K. McHale, M. Gao, D. Wu, J. Brock, A. Blesch,
H. Baharvand, Human embryonic stem cell-derived neural precursor transplants in S. Ephron, A.H. Leif, B. Zheng, J.M. Conner, M. Marsala, M.H. Tuszynski, Long-
collagen scaffolds promote recovery in injured rat spinal cord, Cytotherapy 11 distance growth and connectivity of neural stem cells after severe spinal cord
(2009) 618–630, https://doi.org/10.1080/14653240903005802. injury, Cell 150 (2012) 1264–1273, https://doi.org/10.1038/nm.4502.
[108] S.M. Willerth, Neural tissue engineering using embryonic and induced pluripotent

10