Organogenesis

ISSN: 1547-6278 (Print) 1555-8592 (Online) Journal homepage: https://www.tandfonline.com/loi/kogg20

3D bioprinting: A new insight into the therapeutic

strategy of neural tissue regeneration

Fu-Yu Hsieh & Shan-hui Hsu

To cite this article: Fu-Yu Hsieh & Shan-hui Hsu (2015) 3D bioprinting: A new insight into
the therapeutic strategy of neural tissue regeneration, Organogenesis, 11:4, 153-158, DOI:
10.1080/15476278.2015.1123360

To link to this article: https://doi.org/10.1080/15476278.2015.1123360

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Dec 2015.
Published online: 28 Dec 2015.

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Organogenesis, 11:153–158, 2015
Ó 2015 Taylor and Francis Group, LLC
ISSN: 1547-6278 print / 1555-8592 online
DOI: 10.1080/15476278.2015.1123360

VIEWS AND COMMENTARY

3D bioprinting: A new insight into the therapeutic strategy

of neural tissue regeneration
Fu-Yu Hsieh1 and Shan-hui Hsu1,2,3,*
1
Institute of Polymer Science and Engineering; National Taiwan University;
Taipei, Taiwan
2
Research Center for Developmental Biology and Regenerative Medicine;
National Taiwan University; Taipei, Taiwan
3
Center of Tissue Engineering and 3D printing; National Taiwan University;
Taipei, Taiwan

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.

KEYWORDS. bioink, neural stem cells, neural tissue engineering, neurodegenerative diseases, 3D
bioprinting

*Correspondence to: Shan-hui Hsu; Email:shhsu@ntu.edu.tw

Received October 12, 2015; Revised November 12, 2015; Accepted November 16, 2015.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/
kogg.
153
154 F.-Y. Hsieh and S.-h. Hsu
INTRODUCTION
Acute traumatic injuries or chronic degener-
ative 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 dis-
eases are an increasingly important public
health concern. Major neurodegenerative dis-
eases with unmet medical needs include Alz-
heimer 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 treat-
ments 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 numer-
ous clinical treatments, therapeutics for fully
recovering the neural function are still in their
infancy thus leading scientists to draw inspira-
tion 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 differ-
entiate into several types of functional cells.
Based on the differentiation potential and cell
origin, stem cells used for treating neurodegen-
erative 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 gen-
erate a variety of beneficial effects such as anti-
inflammation, neural cell protection, and induc-
tion of the endogenic recovery system. How-
ever, transplantation of stem cells to the injury
sites often shows poor cell survival and engraft-
ment.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 inte-
grate biomimetic 3D scaffolds with cells for
improvement of neural tissue function. As a
critical component in successful nerve regener-
ation, 3D scaffolds provide a necessary physi-
cal support to facilitate cell function, resulting
in better host tissue engraftment and subse-
quent 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 scaf-
folds.6-8 To overcome this problem, 3D printing
technology serves as a powerful tool for build-
ing tissue and organ structures in the field of
tissue engineering. Recent advances have
enabled 3D printing of biocompatible materi-
als, cells, and supporting components into com-
plex 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 bio-
printings 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
 3D BIOPRINTING 155

for generation of several tissues for transplanta- FIGURE 1. (A) The bioprinting process for neu-
tion, including multilayered skin, bone, heart ral stem cell (NSC)-laden constructs by an
tissue, and cartilaginous structures. Relatively extrusion-based deposition system using the
few studies have focused on applying 3D bio- thermoresponsive PU as the printing ink. (B)
printing in neural tissue. A collagen hydrogel The fused deposition manufacturing (FDM)
precursor (in liquid form) as bioink was created machine.
and cell viability was remained after printing.11
In another study, an artificial neural tissue con-
taining 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 bio-
ink was a novel aqueous dispersion of PU nano-
particles, 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 con-
trollers, and 2 nozzles with heaters (Fig. 1B).
The computer was used for design of the struc-
ture, 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 ther-
moresponsive 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 mm) 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
(b-tubulin) of NSCs embedded in PU hydrogels
was stained by immunofluorescence staining.
The data are presented in Figure 2B. The
maker protein b-tubulin at 7 d was highly
expressed for NSCs embedded in 25% PU
hydrogels. These images revealed that PU
156 F.-Y. Hsieh and S.-h. Hsu
FIGURE 2. Behavior of NSCs in PU hydrogels
after being printed. (A) Images for NSCs in the
3D printed stacking fibers of PU hydrogels dur-
ing a period of 3 d. (B) The protein expression
of b-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 b-tub-
line.
hydrogels may promote the neuronal differenti-
ation of NSCs.
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 ani-
mal 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 bioprint-
ing process. Currently, the biomaterials for 3D
bioprinting are selected either base on their pre-
ferred biocompatibility or because of their
favorable extrusion and crosslinking character-
istics. 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 mecha-
nisms of the hydrogel determine its printability.
So far, only a limited range of materials can be
used, such as collagen, hyaluronic acid, algi-
nate, modified copolymers, and photocured
acrylates.
The 3D bioprinting of neural tissue regenera-
tion and the ultimate success for neural applica-
tions also largely relies on the development of
suitable bioink. Although various traditional
biomaterials have continued to be improved,
they have been limited by inadequate biomi-
metic properties that cannot satisfy the strict
requirements of 3D bioprinting for neural tissue
 3D BIOPRINTING
regrowth. This commentary reviews recent
advancements in using the materials technol-
ogy 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 regen-
eration. However, there are some limitations of
the hydrogel materials, such as lower gel modu-
lus. 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 dis-
covery, toxicity screening, and basic research.
For these applications, extensive validations
are needed to ensure that the bioprinted neural
tissues can recapitulate the key pathophysiolog-
ical features of the disease models. Although
many of the applications of 3D bioprinting are
still at the R&D stage, we expect new 3D bio-
printing 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.
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