Biomaterials 279 (2021) 121211

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Stem cell-derived neuronal relay strategies and functional electrical

stimulation for treatment of spinal cord injury
Bi-Qin Lai a, d, Xiang Zeng a, Wei-Tao Han b, Ming-Tian Che a, Ying Ding b, Ge Li a,
Yuan-Shan Zeng a, b, c, d, e, *
a
Key Laboratory for Stem Cells and Tissue Engineering (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China
b
Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China
c
Institute of Spinal Cord Injury, Sun Yat-sen University, Guangzhou, 510120, China
d
Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226001, China
e
Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan, School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China

A R T I C L E I N F O A B S T R A C T

Keywords: The inability of adult mammals to recover function lost after severe spinal cord injury (SCI) has been known for
Neuronal relay millennia and is mainly attributed to a failure of brain-derived nerve fiber regeneration across the lesion. Po­
Functional electrical stimulation tential approaches to re-establishing locomotor function rely on neuronal relays to reconnect the segregated
Stem cells
neural networks of the spinal cord. Intense research over the past 30 years has focused on endogenous and
Spinal cord injury
Neural network
exogenous neuronal relays, but progress has been slow and the results often controversial. Treatments with stem
cell-derived neuronal relays alone or together with functional electrical stimulation offer the possibility of
improved repair of neuronal networks. In this review, we focus on approaches to recovery of motor function in
paralyzed patients after severe SCI based on novel therapies such as implantation of stem cell-derived neuronal
relays and functional electrical stimulation. Recent research progress offers hope that SCI patients will one day be
able to recover motor function and sensory perception.

1. Introduction
Severe spinal cord injury (SCI), such as complete spinal cord tran­
section manifested by paraplegia or quadriplegia, causes tremendous
physical pain and mental anguish for patients while placing a heavy
financial burden on the patient’s family and society as a whole [1,2]. In
spite of hundreds of years of research into SCI repair, no breakthrough
has yet been made, and clinical demand for SCI treatment continues to
grow [3–5]. In order to improve quality of life for this population and
reduce the social burden, development of effective treatment strategies
for SCI is needed. This will require a better understanding of processes
underlying the repair of severe SCI at both the structural and functional
levels [6].
Severe SCI in adult mammals generally makes it difficult for
descending nerve fibers in the corticospinal tract (CST) to achieve long-
distance across the injured area, resulting in difficulty in restoring distal
motor function [7,8]. In the more than 30 years since the first trans­
plantation of embryonic neural tissue into an SCI site, the concept of SCI
repair using a neuronal relay has gradually been established and vali­
dated [9–12]. Several studies have proposed use of neuronal relay-based
repair strategies to provide endogenous or exogenous neuronal relays
(for example, neuronal network or spinal cord-like tissue), while
improving the microenvironment within the damaged spinal cord tissue
[8,13–15]. These strategies use novel tissue engineering technologies
involving stem cells, cytokines, and biomaterials. They are able to
receive ascending and descending neural information, and to transmit
neural information to propriospinal nerve endings at both ends of the
injured area. SCI repair using neuronal relays has transformed the
traditional idea that “ascending and descending nerve fibers in adult
mammalian spinal cord must regenerate across the SCI area with entire
complete spinal cord transection to restore spinal cord motion and
sensory function” [16–19]. In this review, we focus on recent progress in
research on SCI repair using stem cell-derived neuronal relay strategies
and functional electrical stimulation, both of which use novel interdis­
ciplinary biomedical technologies to achieve repair of motor and sen­
sory function after complete SCI.

* Corresponding author. Department of Histology and Embryology Zhongshan School of Medicine Sun Yat-sen University, 74# Zhongshan 2nd Road, Guangzhou,
510080, China.
E-mail address: zengysh@mail.sysu.edu.cn (Y.-S. Zeng).

https://doi.org/10.1016/j.biomaterials.2021.121211
Received 29 December 2020; Received in revised form 9 October 2021; Accepted 20 October 2021
Available online 22 October 2021
0142-9612/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
B.-Q. Lai et al.
2. Challenges of SCI repair
Tissue ischemia, necrosis, edema, and oxidative stress during the
acute phase of SCI can result in neuronal and glial cell necrosis and
apoptosis. Over time, the injured area continues to expand, forming a
microenvironment that is unfavorable to nerve regeneration owing to
processes such as inflammatory factor infiltration, glial scarring, fibrous
scarring, and cavity formation [20–22]. Post-SCI structural and func­
tional reconstruction is a challenge for both basic and applied research.
Problems to be urgently addressed include: (1) how to improve the
post-injury microenvironment and reconstruct the damaged neural
network after SCI [21,23], (2) how to replace dead neurons and oligo­
dendrocytes [24], and (3) how to integrate new functional cells into the
host neural network to repair the structure and restore the function of
the injured spinal cord [25]. Solving these problems depends on
in-depth knowledge of the mechanisms of SCI repair.
SCI can be subcategorized into incomplete and complete SCI. A series
of experiments have shown that early adoption of specific strategies
(such as reducing tissue edema, inhibiting inflammation, providing
neurotrophic factors, and carrying out rehabilitation therapy after
incomplete SCI) can facilitate reconnection of residual damaged neurons
via collateral sprouting of adjacent undamaged axons [21,26–29]. Use
of small molecule inhibitors to reduce the excitability of inhibitory in­
terneurons in the injured spinal cord enhances responsiveness to
descending excitatory signals and promotes functional recovery after
SCI in mice with staggered bilateral hemisections in which the lum­
barspinal cord is deprived of all direct brain-derived innervation while
retaining dormant relay circuits [30]. These findings suggest that by
establishing new synaptic connections with relay neurons, the
brain-derived descending neural information is retransmitted to target
neurons that regulate limb movement, thereby achieving functional
repair [16,31]. However, in patients with paraplegia or quadriplegia
caused by complete SCI, such reconnection via axonal collateral
sprouting is precluded; all axon bundles must traverse the lesion area.
Even using a combination of treatments, it is still difficult to regenerate
brain-derived descending nerve fibers, especially in the CST that is
responsible for governing autonomic motor function. This is mainly due
to microenvironmental factors in the area of SCI inhibiting nerve
regeneration and the inability of neuronal axons to dynamically regen­
erate [4,32]. In the past decade, researchers have made many attempts
to improve the microenvironment of the SCI site (including strength­
ening the axonal regeneration [27,33], transplanting and replacing cells
[15], and administering gene therapy [34]) and have found that mouse
CST nerve fibers can at least partially regenerate. However, in large
mammals, the truncated bundles at the CST site are unable to suffi­
ciently regenerate across the SCI area to reconnect the target neurons at
the end of the injury site [8,19].
In addition to the absence of CST regeneration, the inability to
restore motor function after SCI may also be associated with a lack of
regeneration of other descending nerve fibers that project to the spinal
cord. These descending nerve fibers include the rubrospinal tract (RST),
reticulospinal tract, 5-hydroxytryptamine (5-HT)-positive nerve fibers,
tyrosine hydroxylase (TH)-positive nerve fibers, dopaminergic nerve
fibers, and spinal cord fasciculus proprius nerve fibers [2,35,36]. Some
studies suggest that the brain-derived 5-HT- and TH-positive nerve fibers
are more likely than CST nerve fibers to regenerate into sites of SCI,
which is an important factor for improving motor function after SCI
[35–37]. Moreover, regeneration of ascending nerve fibers is essential
for restoration of locomotor coordination [38]. Therefore, it is necessary
to fully activate regeneration of brain-derived nerve fibers and sensory
nerve fibers when repairing spinal nerve circuits. Adequate consider­
ation should be given to effective integration of neuronal relay,
ascending and descending nerve fibers.
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Biomaterials 279 (2021) 121211
3. Neuronal relay function
Although brain-derived descending nerve fibers such as those in the
CST cannot easily regenerate across the SCI area to reconnect with target
neurons, it may be feasible to provide an interneuronal network con­
taining “hand-in-hand connections” for relaying neural information
through the defective area [12,16,39]. This interneuronal network could
perform an information-relaying role in the SCI area in the following
ways (Fig. 1). In one direction, it would establish synaptic connections
with nerve fibers transmitting brain-derived descending motor neuronal
signals and would deliver these signals to motor neurons in the caudal
area to the injury site of spinal cord. In the other direction, it would
establish synaptic connections with nerve fibers transmitting informa­
tion carried by sensory nerve fibers and upload this information to the
brainstem and the cerebrum. This would enable the higher central
nervous system to regulate the accuracy of limb movement according to
the afferent sensory signals it receives [35,40]. However, in order for
these neuronal relays to become functional, it is necessary for synapses
to form between the host axons extending from the rostral area and relay
neurons at the injury site (Fig. 1). To establish functionality, neuro­
transmitters must also be appropriately released from the relay neurons,
and axons must extend from them to form synapses with neurons in
areas that are caudal to the injury site [33,39,41]. Finally, axons that
traverse the lesion need to undergo remyelination [42,43]. Recent
studies have demonstrated that neuronal relays can be formed from
various cells, and can be applied to a range of SCI models with the aim of
restoring motor, sensory, and autonomic function [8,13,19]. However,
it is important that efforts to refine these techniques continue in order to
improve the specificity of neuronal connections while enhancing func­
tional outcomes and reproducibility.
An increasing number of researchers are attempting to use this relay
strategy to provide an interneuronal network at the SCI site to re-
establish neural transmission through the spinal cord (Table 1). Based
on research studies and the neuronal relay strategy most commonly used
in recent years (Fig. 2), the majority of researchers have attributed the
behavioral recovery of animals after complete SCI to synaptic connec­
tions between regenerating propriospinal nerve fibers and neurons
transplanted into the injury site [21,51,59]. These donor neurons act as
interneurons to relay neural information through the spinal cord neural
network, and are either excitatory or inhibitory [17,21,59]. Although
there are no reports to date on whether a particular type of neuron acts
as a relay interneuron, Lai et al. [35] did confirm in their previous
studies that excitatory interneurons play a major role in recovery of
motor function. The conceptual focus of this study was whether relay
neurons are able to transmit information via the host axon in a form that
is actually related to the desired motor signals. The data showed that
more than half of the donor neurons were capable of synthesizing
excitatory neurotransmitters and could integrate with host axons to
transmit descending excitatory signals from the brain. Furthermore, the
axons of donor neurons extended to the adjacent caudal segment of the
host tissue and successfully formed synaptic connections with the host
propriospinal neurons via excitatory presynaptic buttons. These mech­
anisms enabled the experimental animals to regain weight-bearing
locomotion and also to coordinate their stepping movements.
The role of inhibitory relay neurons is also not negligible, given that
canines with transplanted inhibitory relay neurons have shown signs of
coordinated motor recovery [35]. Our view is that there are changes in
synaptic plasticity as new relay neurons integrate into the propriospinal
neural network; that is, the spinal cord is able to retain functional neural
pathways in response to physical therapy or rehabilitation training
[60–63]. In the process, as the survival time of stem cell-derived donor
neurons at the injury/graft site of spinal cord increases, those neurons
and their axons that do not form functional connections are removed,
leaving only those donor relay neurons that can establish functional
connections with the host neurons. Some researchers have used a
counterevidence strategy, opting to ablate the neural network at the SCI
B.-Q. Lai et al. Biomaterials 279 (2021) 121211

Fig. 1. Schematic diagram of neuronal relay forma­

tion models for the transmission of descending and
ascending neural signals in SCI repair. A) Relay neu­
rons (green) might receive and integrate neural sig­
nals from multiple descending nerve fibers and output
these to propriospinal neurons in the injury site of the
completely transected spinal cord. B) Relay neurons
(green) may also receive and integrate neural signals
from multiple ascending nerve fibers and output these
to supraspinal neurons and propriospinal neurons.
DRG, dorsal root ganglion.

Table 1
Summary of neuronal relay strategies in animal models of complete spinal cord transection.
Strategies Target cells in the injury/ Important principles Challenges Refs
graft site

Guiding endogenous neurogenesis
In vivo direct reprogramming
Cell transplantation
Tissue-engineered neural network
tissue or spinal cord-like tissue
transplantation
Fetal spinal cord tissue transplantation
Endogenous NSCs
Cells in injured spinal cord
tissue or glial scar tissue
Stem cells or NPCs
Gene-modified cells in a
engineered tissue implant
Neural cells in a fetal spinal
cord tissue implant
Inducing neuronal differentiation of Insufficient endogenous NSCs or NSCs [12,34,44,
endogenous NSCs depleted 45]
Converting local cells directly into neurons Low reprogramming efficiency [46–48]
Inducing neuronal differentiation of Long-distance cell migration and [15,41,
exogenous stem cells or NPCs colony formation 49–52]
Integrating donor functional neurons with the Long-term survival and functional [19,25,35,
host propriospinal neural network integration with the host spinal cord 37,53–55]
tissue
Providing fetal neurons, oligodendrocytes, Sourcing and ethical limitations in [9,10,
astrocytes, NSCs, NPCs and the extracellular translational medicine 56–58]
matrix

Abbreviations: NSCs, neural stem cells; NPCs, neural precursor cells.

site and have found that motor function in the animals is again impaired
[51,64,65], which functionally confirms the existence of the neuronal
relay. Other researchers have confirmed the existence of the neuronal
relay at the structural and functional levels using trans-synaptic nerve
tracing, immunoelectron microscopy, and neurophysiological tech­
niques [35,37].
It is worth noting that, in adopting a neuronal relay strategy to repair
SCI, simple supplementation of stem cell-derived interneurons has
yielded unsatisfactory results, so the combination of multiple factors has
to be considered. Ensuring the success of the interneuronal relay
network at the SCI site requires improving the adverse microenviron­
ment of the injured area, as this microenvironment is not conducive to
the survival and integration of the network [20,66]. The latest ap­
proaches for improving the adverse conditions of the specific microen­
vironment at the site of injury include the simple addition of
neurotrophic factors, the use of specially modified materials that allow
the slow release of neurotrophic factors, or the use of drugs that inhibit
the inflammatory response or promote angiogenesis at the site of injury.
Moreover, repair attempts are made not only to support stem
cell-derived interneuron survival, but also to promote the formation of
synaptic connections between the propriospinal regenerating nerve fi­
bers and the interneurons at the SCI site, as well as to increase remye­
lination [26,43,59]. Only in this way can the newly replenished stem
cell-derived interneurons act effectively as a relay. Besides, before the
neuronal relay can be effective, the atrophy, functional silencing, and
apoptosis of the denervated spinal cord neurons or muscle has to be
prevented [30,67]. It is necessary to consider whether rehabilitation
training and physiotherapy can be used in combination with a neuronal
relay strategy to promote the formation of a greater number of correct
synaptic connections between the regenerating nerve fibers and the stem

3
B.-Q. Lai et al. Biomaterials 279 (2021) 121211

Fig. 2. Therapeutic strategies for neuronal relays. A) Transplantation of a bioactive factor-modified scaffold into the injury site in the spinal cord attracts migrating
endogenous stem cells and promotes their differentiation into relay neurons. B) Tissue-engineered neural network tissue is transplanted into the injury site in the
spinal cord, and plays a role in the neuronal relay via the relay neurons. C) Stem cells are directly injected into the injury site of spinal cord and induced to
differentiate into neurons. D) Transcription factors are directly injected into the injured spinal cord tissue and induce the reprogramming of glial cells into neurons.

cell-derived interneurons [68].
3.1. Endogenous neuronal relays
As early as the 1980s, it was demonstrated that embryonic spinal
cord tissue transplanted into the SCI site can survive and promote
regeneration of brain-derived descending nerve fibers [9,10,56]. These
regenerating nerve fibers (i.e., CST nerve fibers) are capable of estab­
lishing synaptic connections with transplanted embryonic neurons,
leading to improvement of motor function. The concept of an early
neuronal relay was also introduced at that time [12,16–18,39]. Impor­
tantly, Reier et al. [9] reported their findings regarding transplantation
of fetal rat spinal cord tissue into 2–4 mm-long intraspinal cavities in
both adult and neonatal rats. The authors identified surviving donor
tissues and studied their differentiation and extent of fusion with host
spinal cords. The data showed that over 80% of the grafts survived in the
lesion sites in the spinal cords of both neonatal and adult rats for periods
of 1–16 months. Furthermore, the authors provided evidence that the
donor tissue had undergone maturation, including demonstrating
certain topographical features that are characteristic of normal spinal
cord. Many of the transplants extended over the entire length of the
lesion and were often closely apposed to the injured surfaces of the host
spinal cords without an intervening dense glial scar. In addition, the
authors identified neurons and axons within the graft using retrograde
and anterograde labeling, respectively. Taken together, these results
demonstrate a clear potential for transplanting embryonic spinal cord
tissue to replace damaged intraspinal neuronal populations in mammals.
In another study, Iwashita et al. [57] reported reconstruction of severed
spinal cords in which function was restored by replacing spinal cord
segments which then successfully connected with host spinal cord. In
this neonatal rat injury model, transplantation of embryonic spinal cord
tissue promoted robust growth and regrowth of axons across the graft,
resulting in nearly normal neural connections. Animals that had been
fitted with replacement segments were able to walk, run, and climb with
levels of hind-forelimb coordination that were almost normal. However,
given the strict ethical limitations on research in humans and the diffi­
culty of obtaining embryonic nerve tissue, this strategy cannot meet the
clinical demands of SCI treatment. Owing to the continued rapid
advancement in biomedical technology, scientists are beginning to
develop neuronal relay strategies that are more suitable for clinical
applications.
Based on the concept of creating an endogenous neuronal relay at the
SCI site (i.e., via activation and induction of neuronal differentiation
from endogenous stem cells to form a neural network), Li and colleagues
investigated a biodegradable neurotrophin-3 (NT-3)-loaded chitosan
material that could provide 14 weeks of controlled NT-3 release [44].
They found that transplantation of this material improved the micro­
environment at the SCI site, resulting in regeneration of long-distance
nerve fibers and recruitment of endogenous neural stem cells (NSCs).
These NSCs were found to proliferate and migrate into the injury/graft
site in the spinal cord, where they were able to further differentiate into
interneurons. Such stem cell-derived interneurons were able to establish
connections with propriospinal neurons and to transmit ascending and
descending neural signals to proper targets, ultimately improving motor
function in experimental animals [44,45,69,70]. Dai and colleagues
developed a functional collagen-based scaffold that could bind to

4
B.-Q. Lai et al.
different growth factors and was able to significantly promote endoge­
nous neurogenesis at the SCI site and improve motor function in rats and
canines [71–74]. In one study, the same researchers investigated the
effect of the microtubule-stabilizing agent paclitaxel (PTX) on the
endogenous differentiation of NSCs. They loaded PTX-encapsulated li­
posomes into a collagen microchannel scaffold, which released PTX in a
prolonged and sustained manner when it was implanted at the complete
transection spinal cord injury site in a rat model. This scaffold provided a
microenvironment that was suitable for neuronal differentiation of NSCs
and the extension of neuronal axons. Endogenous neurogenesis at the
site of spinal cord injury eventually led to improvements in both
motor-evoked potentials and recovery of hindlimb locomotion. In
another study, the same researchers investigated the effect of the
microtubule-stabilizing agent PTX on the endogenous differentiation of
NSCs. They loaded PTX-encapsulated liposomes into a collagen micro­
channel scaffold, which released PTX in a prolonged and sustained
manner when it was implanted at the complete transection spinal cord
injury site in a rat model. This scaffold provided a microenvironment
that was suitable for neuronal differentiation of NSCs and the extension
of neuronal axons. Endogenous neurogenesis at the site of spinal cord
injury eventually led to improvements in both motor-evoked potentials
and recovery of hindlimb locomotion. Han et al. [75] transplanted a
linear-ordered collagen scaffold bound to brain-derived neurotrophic
factor (BDNF) (using a tagged collagen-binding domain) into a canine
model of complete spinal cord transection. This linear-ordered collagen
scaffold-BDNF complex was shown to effectively promote generation,
maturation, and synaptic formation, as well as axonal remyelination, of
endogenous stem cell-derived interneurons at the SCI/graft site, thereby
restoring motor function. In addition to the studies described above,
several have also shown that manipulation of the SCI microenvironment
in order to guide endogenous neurogenesis can promote recovery of
motor function in animals with spinal cord deletions [60,76].
Collectively, these studies demonstrate that a fundamental principle
underlying formation of neuronal relays at sites of injury, along with
recovery of motor function, is initiation of endogenous neurogenesis
from NSCs. Furthermore, these studies suggest that formation of
neuronal relays, as well as recovery of function, may be more successful
if material could be implanted at the injury site that can create a pro-
regenerative microenvironment to facilitate formation of NSC progeny
and ultimately a greater number of relay neurons. These results
emphasize that the primary mechanism underlying regenerative repair
in SCI is differentiation of endogenous NSCs into interneurons that then
function as neuronal relays. Thus, when designing endogenous neuronal
relay treatments for animals with complete SCI, it is essential that
neuronal relay function be established that results in formation of
appropriate axonal reconnections with the original downstream targets
[12,44,69].
Another novel strategy for promoting the growth of endogenous in­
terneurons at the site of SCI is to reprogram glial cells (e.g., astrocytes) to
differentiate into interneurons in vivo (Fig. 2) [46–48,77]. An early
study established that the adult mammalian spinal cord lacks intrinsic
neurogenic capacity [78]. On the basis of this finding, Wang et al. [78]
attempted to generate neurons after SCI by reprogramming glial cells in
vivo. The authors showed that the p53-dependent signaling pathway
constitutes a critical checkpoint for SOX2-mediated reprogramming of
resident glial cells in the adult mouse spinal cord. They also found that
the p53 pathway promoted cell-cycle exit in SOX2-induced adult neu­
roblasts (iANBs). As such, silencing of either p53 or p21 led to a sig­
nificant increase in the overall production of iANBs. Furthermore, BDNF
and Noggin created a neurotrophic milieu that promoted maturation of
iANBs into a diverse range of neuronal cell types, although gluta­
matergic neurons predominated. The study identified critical molecular
and cellular checkpoints that can be manipulated to enhance neuro­
genesis after SCI. In another study, Zarei et al. [48] demonstrated that
ectopic expression of zinc-finger transcription factor 521 (Zfp521) re­
programs fibroblasts and astrocytes into NSCs in vitro. Additional in
5
Biomaterials 279 (2021) 121211
vivo studies showed that Zfp521 treatment also affects endogenous as­
trocytes and thus promotes further functional recovery following
experimental SCI in rats. The authors further reported that Zfp521 is
able to reprogram cultured astrocytes more efficiently than SOX2, and
that resident astrocytes within the injured spinal cord of adult rats can
be reprogrammed by Zfp521 into neurons via a neuronal progenitor
stage. Treatment with Zfp521 led to a significant enhancement in hind
limb locomotor activity, step length, toe spread, foot length, and paw
area. These reprogramming strategies have been shown to induce a high
rate of neuronal differentiation in vitro. However, due to the influence of
the complex, damaged microenvironment of the injured spinal cord,
reprogramming efficiency is lower in vivo than in vitro [79–81].
Therefore, to date, such reprogramming strategies have seldom been
used for complete SCI repair (Table 2) [47,82,83].
Analysis of papers reporting on endogenous neuronal relay strategies
published in recent years shows that the ependymal cells of the central
canal of the spinal cord have NSC-like potential in different mammals
ranging from rodents to primates [69,76,90,91]. These cells are not
active in normal spinal cord tissue, but become activated in response to
injury and migrate from the central canal into the injury site. In the
microenvironment of the injury site, these activated ependymal cells
tend to differentiate into astrocytes rather than neurons. These astro­
cytes accumulate at the edge of the lesion and form a dense glial scar,
hindering nerve fiber regeneration [32,90,92–94]. Several bioactive
factors have been shown to be capable of ameliorating this adverse
microenvironment, effectively promoting activation of ependymal cells
with NSC-like potential that have migrated into the site of spinal cord
injury (Fig. 2) [44,73,95]. Furthermore, these ependymal cells induce
the NSC-like cells to differentiate into interneurons that eventually serve
a relay role during repair of the spinal cord neuronal network [66,76].
Some studies have demonstrated that endogenous NSC-derived neurons
function as relay neurons by receiving supraspinal input and extend
their axons across the injury/graft site into the caudal spinal cord for
connectivity [28,96,97]. Even so, before using bioactive factors, at­
tempts should be made to fully consider the time course of endogenous
stem cell activation after SCI. Previous studies have revealed that
NSC-like cells in rat spinal cord reach their peak of activation on the fifth
day after injury [12,73]. Therefore, the use of bioactive factors depends
on the particular time course of endogenous stem cell activation after
SCI in different animals. In addition, the influence of the chosen carrier
and mechanism of bioactive factor release on the effectiveness of this
strategy should be considered. If bioactive factors can be loaded into a
hydrogel, they can be injected directly into the injured area of the spinal
cord [28,97,98]. Some bioactive factors can be loaded into a collagen
scaffold and slowly released during collagen degradation to exert a
long-lasting effect (Fig. 2) [66,70,86]. More importantly, in addition to
activating endogenous stem cells and allowing them to migrate into
injured spinal cord tissue to differentiate into interneurons, there is
another key issue to consider when attempting to improve motor func­
tion. This is whether bioactive factors and their carriers can effectively
support survival and functional maturation of endogenous stem
cell-derived interneurons, as well as formation of new synaptic con­
nections between these interneurons and regenerating nerve fibers. A
promising research direction is to find a combination of bioactive factors
that can inhibit inflammation while promoting nerve fiber regeneration
and differentiation of endogenous NSCs into relay interneurons. Dif­
ferentiation of endogenous NSCs into oligodendrocytes to repair
demyelinated axons also plays an important role in motor and sensory
repair. Moreover, a potentially valuable research direction with many
applications would be to develop a scaffold that loads bioactive factors
to allow for long-term stable release and provides directional guidance
for nerve fiber regeneration and remyelination (Fig. 2) [99,100]. It is
important to consider that while therapeutic strategies based on
endogenous stem cell activation may have better effects for acute SCI, in
cases of chronic SCI, aging patients with SCI, complete SCI with stem cell
depletion or insufficient mobilization, it may be necessary to adopt a
B.-Q. Lai et al. Biomaterials 279 (2021) 121211

Table 2
Summary of endogenous neuronal relay strategies and their effectiveness for complete SCI repair.
Publication Species Treatment Autonomous Electrophysi- Key outcomes in the injury/graft site of Synapselike
locomotor ological spinal cord structure in the
recovery improvement injury/graft site

2006. Ohori Rat Injection of growth factor and NR NR Replacing lost neurons and Yes
et al. [34] transcription factors oligodendrocytes
2013. Li et al. Rat Implantation of EGFR antibody- Yes NR Inducing differentiation of endogenous Yes
[71] functionalized collagen scaffolds NPCs into neurons
2014. Su et al. Mouse Injection of lentivirus NR NR Converting local astrocytes into synapse- Yes
[46] forming interneurons
2014. Guo et al. Mouse Injection of lentiviral vector Yes NR Activating and inducing endogenous NSC NR
[84] containing Sox11 differentiation into neurons, and inducing
expression of BDNF
2015. Han et al. Canine Implantation of linear-ordered Yes Yes Promoting nerve regeneration and NR
[75] collagen binding BDNF improving myelination
2015. Yang et al. Rat Implantation of NT-3-coupled Yes Yes Promoting endogenous neurogenesis Yes
[44] chitosan
2016. Li et al. Rat Implantation of collagen scaffold Yes NR Reducing cavity volume, facilitating nerve NR
[72] and administration of cAMP regeneration, and promoting neurogenesis
2017. Yang et al. Mouse Injection of substance P Yes NR Activating endogenous NSCs and enhancing NR
[85] neurogenesis
2017. Li et al. Canine Implantation of cetuximab- Yes Yes Activating endogenous neurogenesis and Yes
[73] modified collagen scaffold reducing chondroitin sulfate proteoglycan
deposition
2017. Fan et al. Rat Implantation of EGFR antibody Yes Yes Blocking myelin-associated inhibitory Yes
[86] CBD-Fab-modified collagen molecules, guiding nerve regeneration, and
scaffold enhancing neurogenesis
2016/2018. Li Canine Implantation of NT-3/fibroin- Yes Yes Promoting nerve regeneration, attenuating Yes
et al. [60,87] coated gelatin sponge scaffold inflammation, and attracting migrating
cells
2018. Rao et al. Monkey Implantation of NT-3-loaded Yes Yes Promoting nerve regeneration NR
[45] chitosan biodegradable material
2018. Zhang Rat Injection of erythropoietin Yes NR Promoting neurogenesis and NR
et al. [24] oligodendrogenesis
2018. Li et al. Rat Implantation of a collagen Yes Yes Inducing differentiation of endogenous NR
[74] microchannel scaffold carrying NSCs into neurons
paclitaxel-liposomes
2018. Zhou et al. Mouse Implantation of a conducting Yes NR Activating endogenous NSCs and eliciting NR
[88] polymer hydrogel neurogenesis
2019. Zarei- Rat Injection of zinc-finger Yes Yes Converting astrocytes into neuron-like cells NR
Kheirabadi transcription factor
et al. [48]
2019. Shang Rat Implantation of bFGF-Sodium Yes NR Reducing microglial activation, increasing NR
et al. [66] hyaluronate collagen scaffold angiogenesis, eliciting endogenous
neurogenesis, and promoting nerve
regeneration
2019. Pan et al. Rat Implantation of PLGA/GO Yes Yes Increasing neurons survival NR
[23] electrospun nanofibers
containing immobilized IGF-1
and BDNF
2019. Sperling Rat Injection of galantamine Yes NR Increasing neurogenesis and tissue cell NR
et al. [89] survival
2020. Ma et al. Mouse Injection of photo-crosslinked Yes Yes Reducing inflammation and inducing NR
[29] hydrogel and CSF1R inhibitor differentiation of endogenous NPCs into
neurons

Abbreviations: NR, not reported; EGFR, epidermal growth factor receptor; NPCs, neural precursor cells; Sox11, sex-determining region Y-box 11; NSCs, neural stem
cells; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; cAMP, cyclic adenosine monophosphate; CBD, collagen-binding domain; Fab, Fab fragment of
antibody; bFGF, basic fibroblast growth factor; GO, graphene oxide; PLGA, poly(lactic-co-glycolic acid); IGF-1, insulin-like growth factors-1; CSF1R, colony stimulating
factor-1 receptor.

therapeutic strategy of supplementing exogenous stem cells [33,50,
101].
3.2. Exogenous neuronal relays
In recent study, Li et al. [60,87] developed an NT-3/fibroin-coated
gelatin sponge scaffold with sustained NT-3 release, which demon­
strated the ability to promote nerve fiber regeneration and to activate
endogenous stem cells to differentiate into interneurons that integrate
into the propriospinal neural network; this scaffold has been used to
successfully repair SCI in rats and canines. However, for complete SCI,
the insufficient quantity of endogenous stem cells may limit the effec­
tiveness of this approach. Therefore, Abematsu et al. [51] applied
valproic acid to induce differentiation of transplanted NSCs to in­
terneurons. The transplant-derived interneurons were able to recon­
struct the damaged propriospinal neural network, to both receive and
send synaptic information from/to host neurons, and to directly
contribute to the restoration of motor function. Lu et al. [11,14,101]
adsorbed NSCs and 10 different neurotrophic factors using fibrin
matrices and co-transplanted them into the SCI area, thereby greatly
improving the microenvironment of the injury/graft site. Trans­
plantation of the NSC-derived interneurons led to remarkable axonal
growth and supported the formation of electrophysiological relays
across the injury/graft sites of the complete spinal cord transection,
resulting in functional recovery. However, Sharp et al. [52] and other
researchers have demonstrated repeatedly that the direct

6
B.-Q. Lai et al. Biomaterials 279 (2021) 121211

transplantation of human NSCs into the SCI site generates a risk of
abnormal proliferation and long-distance migration. Furthermore,
direct transplantation of NSCs might slows differentiation and matura­
tion, resulting in delayed functional recovery of the injured spinal cord
[102]. Lu et al. [15] implanted and assessed human H9 NSCs in an
immunodeficient rat model of SCI over a period of 1.5 years, with the
grafts showing evidence of continued maturation over the entire period
of assessment. Although biomarkers of neuronal maturity were first
expressed three months after grafting, the complex processes of neuro­
genesis, neuronal pruning, and neuronal enlargement continued for one
year thereafter. The authors also found that half of the axonal pro­
jections persisted for 1.5 years, and that the number of mature oligo­
dendrocyte markers did not increase until one year after grafting.
Functional recovery was first observed more than one year after the
original grafting. We analyzed a series of studies regarding the appli­
cation of exogenous neuronal relays as a repair strategy for SCI
(Table 3). Some studies have shown that, although transplanted stem
cells can survive and differentiate well into interneurons in the SCI area
and even extend their long processes into the host spinal cord, the
behavioral recovery of the animals is not noticeable [98,103]. Other
studies have indicated that the key issue in the repair of motor function
in animals may additionally relate to whether exogenous stem
cell-derived interneurons are able to produce the correct synaptic con­
nections with regenerating nerve fibers [16,31,59]. A further key point
is whether the exogenous neuronal relay can simultaneously be
replenished by a subset of oligodendrocytes to increase the remyelina­
tion of regenerating nerve fibers in the injury site [59,100,104]. In
addition, another key issue that should be considered is the abnormal
proliferation and migration of stem cells to form space-occupying lesions
after transplantation [52,105]. Successfully establishing an exogenous
neuronal relay in the SCI site in a safe and effective manner remains a
challenge.
4. Tissue-engineered neuronal relays
Research into tissue-engineered nerve regeneration and repair stra­
tegies has been rapidly developing over the past decade or so, with such
strategies considered to offer novel treatment approaches for severe
central nervous system injuries [58,110–112]. Tissue engineering is a
technique that combines cell biology and materials science to construct
biological tissues or organs in vitro or in vivo, involving a combination
of three elements: seed cells, biomaterials, and bioactive factors. The
decision of how to assemble these three elements is based on the re­
quirements for repair of the damaged tissue or organ. Repair of tissue
structure and function in a safe and effective manner is key to success­
fully applying tissue engineering techniques [4,58,113].
For complete SCI, repair strategies based on tissue engineering
should consider the massive loss of neurons and oligodendrocytes,
infiltration of inflammatory cells, and cavity and glial scar formation at
the SCI site. In such cases, the greatest advantage of the tissue engi­
neering strategy is its ability to meet the diverse needs of spinal cord
structural and functional repair by effectively combining seed cells,
biomaterials, and bioactive factors. For example, some studies have
shown that stem cells can be induced to become neurons and oligo­
dendrocytes that can then be loaded together into a functional bioma­
terial scaffold [43,100,109]. A number of studies have shown that seed
cells can be used as gene carriers to transplant a target gene of thera­
peutic interest, thus achieving the dual effects of cell replacement and
gene therapy [13,42,107,114]. Other studies have indicated that bio­
materials modified by bioactive factors can simultaneously be used for
loading cells, fighting inflammation, promoting nerve regeneration, and
increasing survival of transplanted cells in SCI repair [70,75]. Therefore,
this approach not only fills the space resulting from SCI with bio­
materials and cells but can also provide directional guidance to the
grafted neurons and regenerating nerve fibers [70,86].
Based on stem cell transplantation and tissue engineering, Zeng and
colleagues have developed a series of important components needed to
address key scientific challenges in this field, including a human NT-3
gene recombinant adenovirus [115], a three-dimensional gelatin
sponge scaffold [116], a tissue-engineered neural network [53,110],
scaffolds with chemotactic function [60,117], and decellularized nerve
scaffolds [99,100,118]. By combining these components with novel
nerve tissue engineering techniques such as use of neurotrophic factors
and their receptors, gene-modified stem cells, and biomaterials, they
constructed an exogenous stem cell-derived neural network tissue with
functional synaptic transmission [37,110]. This tissue was then trans­
planted into the gap created by complete spinal cord transection
(removal of a 2-mm segment of rat spinal cord or a 4-mm segment of
canine spinal cord). As well as improving the microenvironment at the
injury/graft site of the spinal cord, this neural network tissue was able to
promote dynamic integration and functional synergism of endogenous
stem cell-derived neurons, allowing it to function as a neuronal relay
that could repair the host propriospinal neural network (Fig. 3) [35,53,
109]. This approach avoids the uncertain differentiation of stem cells,
especially embryonic stem cells, transplanted directly into the spinal
cord, as well as reducing the risk of abnormal proliferation and
long-distance migration [52,105].
Previous studies by Lai et al. [35,54] have shown that stem
cell-derived tissue-engineered neural network transplantation is able to
act as a neuronal relay for synaptic connections between regenerating
ascending and descending neuronal nerve fibers (Fig. 3). Marchini et al.
[113] also developed an in vitro three-dimensional (3D) functional
neuronal network derived from human neural stem cells (hNSCs). They
tracked the proliferation, differentiation, and maturation of the hNSCs
into glutamatergic, cholinergic, and GABAergic neurons in
multi-functionalized hydrogels, and evaluated how these engineered
neural network tissues facilitated treatment of spinal cord injuries. They
found that transplantation of the multi-functionalized hydrogel scaffolds

Table 3
Comparison of exogenous neuronal relay strategies in different treatments of complete spinal cord transection.
Treatments Primary strategies Major advantages Major disadvantages Refs

Transplantation of NSCs or
NPCs
Transplantation of
biomaterials loaded with
NSCs or NPCs
Transplantation of engineered
neural network tissue or
spinal cord-like tissue
Transplantation of fetal spinal
cord tissue
Injecting cells into the epicenter
of the injury site
Seeding cells in functionalized
materials with drug or
neurotrophic factor
Constructing stem cell-derived
neuronal network tissue or
spinal cord-like tissue in vitro
Transplanting fetal spinal cord
tissue directly
Connectivity of donor cell-derived neuronal axons, and Abnormal proliferation, uncertain [17,20,26,
neuroprotection and immunomodulation in vivo differentiation, or long-distance 41,49,51,
migration in donor cells in vivo 52,106]
Replacing lost neurons, repairing the propriospinal Abnormal proliferation, uncertain [11,13,15,
neural network, enhancing nerve regeneration, differentiation, or long-distance 98,105,
promoting angiogenesis, and reducing cavity size in migration in donor cells in vivo 107,108]
vivo
Replacing lost neurons and oligodendrocytes, High complexity of fabrication [19,25,37,
repairing the propriospinal neural network, enhancing technique in vitro 53–55,109]
the neuronal relay effect, and promoting remyelination
in vivo
Replacing neurons and oligodendrocytes, repairing the Sourcing and ethical limitations in [9,10,
propriospinal neural network, in vivo translational medicine 56–58]

Abbreviations: NSCs, neural stem cells; NPCs, neural precursor cells.

7
B.-Q. Lai et al. Biomaterials 279 (2021) 121211

Fig. 3. Schematic diagram depicting a tissue-engineered neuronal relay. A) Fabrication process of the neural network tissue. B) Transplantation of the neural
network tissue into the injury site in completely transected spinal cord. C) Integration of the grafted neural network tissue into the host propriospinal neuronal
network, myelination of the grafted neuronal axons, and functional relay formation across the injury site in the spinal cord. SCs, Schwann cells; NSCs, neural stem
cells; 3D, three-dimensional; DRG, dorsal root ganglion; T9, 9th thoracic vertebra of the spinal cord; CST, corticospinal tract; 5-HT, 5-hydroxytryptamine; NF,
nerve fiber.

containing pre-differentiated hNSCs resulted in a greater proportion of
neuronal markers, better hNSC engraftment, and improved behavioral
recovery. Their study demonstrated that 3D cultures are better able to
mimic the conditions to which cells are normally exposed, and further
showed that neural network tissue formed by the pre-differentiated
hNSCs in vitro is able to integrate and form functional circuits in the
rat spinal cord within 6 weeks. However, this technique requires further
development as functional integration of tissue-engineered neural
network tissue into the host propriospinal neural network remains
limited, which in turn limits improvement of autonomous motor func­
tion in experimental animals. Recent literature suggests that disordered
regeneration of nerve fibers or disordered distribution of grafted neu­
rons is not conducive to repair of spinal cord structure and function
[100]. In order to improve the efficiency of functional integration be­
tween grafted and host neurons, it will be necessary to develop a new
scaffold that can guide directional axon regeneration and the distribu­
tion of transplanted cells [70,99].
Based on the hypothesis that a tissue-engineered neuronal relay can
be used to repair spinal cord defects, and given the progress made in
stem cell tissue engineering and tissue organ construction techniques,
Lai et al. [54] combined NSCs, neurotrophic factors and their receptors,
and a collagen scaffold to construct a spinal cord-like tissue (SCLT) in
vitro for the first time. This SCLT is similar to normal white matter and
gray matter of the spinal cord and has architectural, phenotypic, and
functional similarities to adult rat spinal cord. It contains the corre­
sponding major cell types (neurons, oligodendrocytes, and astrocytes),
and is composed of white matter-like and gray matter-like tissue mod­
ules. This modular construction of the SCLT offers the possibility of
structural and functional repair after complete spinal cord transection.
Transplantation of this SCLT into the gap of a complete spinal cord
transection (with a 2-mm-long defective tissue area) was able to
significantly promote regeneration of brain-derived descending nerve
fibers. NSC-derived neurons were able to establish synaptic connections
with descending nerve fibers, support transmission of brain-derived
excitatory neural information, and promote improvement of motor
function. Thus, SCLT transplantation shows promise for application to
repair of complete SCI.
It has been acknowledged that research on complete SCI repair
should not only focus on the effects of spinal nerve fiber regeneration
and the transmission of ascending and descending neural information
but also consider disuse atrophy of neurons and muscles deprived of
functional innervation [67,93,119]. The current work of Lai et al. [54]
shows that transplanted neural network tissue or SCLT can induce
brain-derived nerve fiber regeneration in the injury/graft area of a
complete spinal cord transection, achieving synaptic contacts with
transplanted stem cell-derived neurons to enable functional integration.
However, adverse effects can occur before functional integration of the
neural network tissue or the SCLT into the host spinal cord is complete.
Specifically, pathophysiological changes in the injured spinal cord can
result in functional silencing or disuse atrophy of the propriospinal
neurons at the injury/graft site, the caudal spinal cord, and the para­
lyzed limb muscles, which may limit repair of spinal cord defects [30,
40]. Therefore, rehabilitation physiotherapy (for example functional
electrical stimulation) should be used in combination with the stem
cell-derived neuronal relay strategy to restore innervation of spinal cord
motor neurons and their targeted muscle cells as early as possible [1,5,
120]. In the anterograde direction, a variety of factors (including
acetylcholine and neurotrophic factors) secreted by spinal motor neu­
rons are transported through motor nerve fibers to maintain the struc­
ture and function of target muscle cells, and to prevent muscle atrophy
[119,120]. In the retrograde direction, myogenic trophic factors
secreted by muscle cells are transported via the motor nerve fibers to the

8
B.-Q. Lai et al.
spinal cord to maintain structure and function of the target motor neu­
rons, and to prevent neuronal atrophy [121].
5. Brain-spine interfaces and electrical stimulation
Based on the observation that brain-derived descending nerve fibers
such as the CST have difficulty in regenerating across a complete spinal
cord transection to reconnect with target neurons, Courtine and col­
leagues developed a brain–spine interface which could bypass the lesion
[122,123]. This strategy was able to restore control over leg muscle
activity for walking using epidural electrical stimulation (EES; Fig. 4).
Researchers interfaced motor cortex activity guiding leg movement with
EES protocols to establish a brain-spine interface that alleviated gait
deficits in a rhesus monkey with SCI. An intracortical microelectrode
array was implanted into the region of the motor cortex in the brain that
guides leg movement, and a spinal cord electrical stimulation system
composed of a pulse generator and a spatially selective epidural implant
was placed in the epidural area of the spinal cord. The design and
implementation of the control system linked online neural decoding of
flexion and extension motor states with electrical stimulation protocols,
9
Biomaterials 279 (2021) 121211
promoting weight-bearing locomotion of paralyzed legs in monkeys
[122].
The Lancet Neurology also reviewed the use of a brain–spine interface
to record and decode brain signals to restore reaching and grasping
movements via brain-controlled spinal cord stimulation [61]. These
studies revealed that repeated spinal cord electrical stimulation and
rehabilitation training could enhance autonomous movement [121,
124–126]. Harkema et al. [38] described a 23 year-old man who was
paraplegic due to a C7-T1 subluxation, with complete loss of clinically
detectable voluntary motor function with partial preservation of
sensation below the T1 cord segment. The authors placed a 16-electrode
array on the dura to allow for chronic electrical stimulation. They sub­
sequently found that the patient was able to produce standing- and
locomotor-like patterns during stimulation using standing-specific pa­
rameters and bilateral load-bearing proprioceptive input. Further
research has confirmed that EES is able to modulate the propriospinal
neural network in humans into a physiological state that enables sensory
input from standing and stepping movements to serve as a source of
neural control [22,127,128]. The above studies indicate that a brain–­
spine interface has the potential to serve as an “artificial intelligence
Fig. 4. Schematic diagram showing two approaches
to spinal cord stimulation in the brain–spine inter­
face. Intracortical microelectrode array are implanted
in the motor cortex of the cerebrum to record motor
information in the cerebral cortex; this information is
used to form the input stimulation signals to the
spinal cord, using computer-based decoding. During
this process, a pulse generator is required to generate
a connection with the input stimulation signal elec­
trodes in the spinal cord. EES: The input stimulation
signal electrodes are placed on the dural surface of
the spinal cord, in order to affect the afferent
neuronal circuits and the intrinsic interneuronal cir­
cuit in the spinal cord. ISMS: The input stimulation
signal micro-needle electrodes are accurately inserted
into the spinal cord tissue to transmit stimulation
signals to the intrinsic interneuronal circuits in the
spinal cord. EES, epidural electrical stimulation;
ISMS, intraspinal microstimulation; SCI, spinal cord
injury; DRG, dorsal root ganglion.
B.-Q. Lai et al.
relay” where brain-derived neuronal signals are first converted into an
electrical signal and then reconverted into neuronal signals in the
injured spinal cord (Fig. 4).
Another spinal cord electrical stimulation approach that is able to
restore motor function is intraspinal microstimulation (ISMS) [129,
130]. ISMS differs from EES in that it delivers the electrical signal via
microelectrodes implanted within the spinal cord [131]. To date, the use
of ISMS strategies is rare in humans, but animal experiments have
provided insights into the potential benefits of this approach [132,133].
In animal models, ISMS can elicit a wide variety of function-related
movements, such as grasping, reaching, and stepping [134–136].
When ISMS microelectrodes were implanted into the ventral spinal cord,
direct activation of ventral root nerve fibers or motor neurons was
observed, leading to single joint movement [129,137]. In contrast, when
the microelectrodes were implanted into the intermediate lamina of the
spinal cord, the electrical stimulation was most likely to subsequently
activate interneurons and nerve fibers [130,138–140]. The interneurons
in turn activated a complex propriospinal neural network, resulting in
coordinated motion. Paired with the brain–spine interface system, ISMS
may be especially useful for controlling the electrical current based on
activity-related signals from the brain that occur when an animal at­
tempts to move (Fig. 4) [134,141,142]. This kind of activity-dependent
electrical stimulation might also be used in SCI rehabilitation in the
future to strengthen cortico-spinal connections and to regulate synaptic
plasticity in the propriospinal neural network [124,143]. Many studies
suggest that EES or ISMS may be useful in directing the rehabilitation of
specific motor pathways. Research has established that the spinal cord
undergoes neural remodeling after injury, and that this process of
remodeling can lead to synaptic plasticity in neural pathways, thus
inducing beneficial motor effects [127,143]. Thus, targeted therapeutic
EES or ISMS may help to guide these remodeling mechanisms towards
the formation of functional motor pathways, particularly when the
neuromuscular system has not yet undergone atrophy following chronic
paralysis. The observed improvements in muscle mass and other phys­
iological functions support the notion that EES or ISMS can help to
counteract such deterioration [144–147]. However, there are still many
challenges, as the limitations of microelectrode implantation at this
stage mean that it is only possible to record a small amount of brain
activity and there is a risk of infection. Moreover, the microelectrode
implanted into brain or spinal cord can cause local immune responses,
induce the generation of glial scars around the electrode, and result in
the reduced efficacy of the brain–spine interface system [148]. In
addition, the implanted microelectrode also has limited longevity and
stability, with failure typically occurring approximately one year
post-implantation [149]. Although the brain–spine interface can suc­
cessfully transmit brain-derived motor nerve signals to the spinal cord,
the first generation of such artificial intelligence relay technology sys­
tems cannot return the sensory nerve signals of the paralyzed patients to
the brain [121,150]. Therefore, there is still a crucial role for biomimetic
medical techniques in repairing SCI at the structural and functional
levels.
Increasing numbers of studies have suggested that more effective SCI
repair can be achieved by a combination of multiple strategies, such as
cell and tissue transplantation, application of neurotrophic factors for
axon and myelin regeneration, delivery of neuroprotective drugs or
immunosuppressants, and regulation of neural circuit plasticity by
physical therapy and rehabilitation training [125,151]. Optimal com­
binations of the above treatment strategies will provide inspiration for
improved clinical treatments. We suggest that combinatorial treatments
using physical therapy and tissue-engineering neuronal relay could
regulate synaptic plasticity and promote effective integration of
neuronal relays and the propriospinal neural network.
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Biomaterials 279 (2021) 121211
6. Combinatorial treatments using electrical stimulation and
tissue-engineered neuronal relays
Recent studies in brain–spine interfaces and electrical stimulation
have suggested that EES or ISMS are able to stimulate functionally silent
(inactivated) descending neural pathways in injured spinal cord [129,
130,140]. Brain-spinal interfaces and electrical stimulation, by
enhancing electrical activity of cortex pyramidal neurons, can promote
regeneration of axons in the CST of a damaged spinal cord. These re­
generated CST fibers expressing protein tyrosine phosphatase-σ (PTPσ)
may specifically bind to NSC-derived neurons overexpressing NT-3 re­
ceptor tyrosine kinase C (TrkC) at the injury/graft site of a spinal cord,
enhancing the possibility of forming synaptic structures between them.
PTPσ in the presynaptic membrane binds to TrkC in the postsynaptic
membrane, thereby enhancing adhesion of the presynaptic membrane
and promoting development of excitatory synapses [152]. NT-3 can
further promote adhesion between PTPσ and TrkC [153]. It was
observed that a small number of CST fibers can regenerate into the
injury/graft site to connect with stem cell-derived neurons over­
expressing TrkC in response to an exogenous NT-3 concentration
gradient [19,25,154]. Furthermore, electrical stimulation might also
provoke relay neurons at the injury site that innervate the caudal spinal
cord to transmit their signals to target muscle cells, enabling standing
and walking movements [127,155,156].
Transcutaneous spinal cord stimulation (tcSCS) is a noninvasive
method in which electrodes are placed on the surface of the skin above
the spinal column and modulated intermediate-frequency currents
(medium frequency electrical stimulation) are used to activate dorsal
root ganglion neurons and propriospinal neurons; this method is also
used for functional repair following SCI (Fig. 5) [157–159]. Medium
frequency electrical stimulation does not generate tissue electrolysis.
Furthermore, it can overcome the electrical resistance of body tissues,
penetrate deeply into spinal tissue, and increase the excitability of
propriospinal neurons and muscles without exciting afferent nerve fibers
involved in algesthesia [160]. Emerging evidence suggests that EES and
tcSCS activate the same propriospinal neural network, and that both can
regulate the synaptic plasticity of neural networks, promote neuro­
trophic factor secretion, and improve the spinal cord microenvironment
[159,161]. Although early results from functional electrical stimulation
of the spinal cord indicate that it can play a significant role in promoting
motor recovery, tapping its full potential to elicit sustained, long-term
improvements will likely require a combination of treatments [22,162].
Rodent studies have demonstrated that functional electrical stimu­
lation may be particularly useful when combined with physical reha­
bilitation to enable activation of specific motor pathways or
strengthening of synaptic connections between neurons. Even weeks
after electrical stimulation has ceased, treated animals show lasting
motor improvements [63,120,163]. These results suggest long-term
therapeutic effects of electrical stimulation of the descending path­
ways. These functional electrical stimulation technologies might also be
combined with tissue-engineered neuronal relay strategies, stem cell
transplantation, pharmacological interventions, and training activities
[22,91,154].
The results Jin and his colleagues obtained for repair of SCI using
spinal cord electrical stimulation in combination with a tissue-
engineered neuronal relay suggest that electrical stimulation of the
tail nerve or electroacupuncture (EA) can be used to upregulate
expression of endogenous NT-3 in the spinal cord after its complete
transection [55,67]. Tail nerve electrical stimulation falls into the
category of tcSCS methods, while EA refers to the application of a pulsed
current to the needle and is based on traditional Chinese acupuncture
(acupoint treatment with a needle (Fig. 5) [164]). EA can maintain a
continuous state of stimulation in the body for long periods due to the
physiological effect of pulsed electricity, thereby providing the dual
benefits of acupoint and electrical stimulation [112,165]. The mecha­
nism of its therapeutic effect on SCI mainly involves improvement of the
B.-Q. Lai et al. Biomaterials 279 (2021) 121211

Fig. 5. Schematic diagram showing two methods of body surface electrical stimulation. EA: A pulse generator (a medical EA apparatus) generates the pulse current.
This is transmitted through the acupuncture needle at the acupoint to stimulate sensory nerve endings of the meningeal branch of the spinal nerve, affecting the
afferent neuronal circuit in the spinal cord. tcSCS: An electrode patch is placed on the surface of the skin above the spinal column, and then a pulse generator is used
to generate a modulated intermediate-frequency current to activate DRG and propriospinal neurons. EA, electroacupuncture; tcSCS, transcutaneous spinal cord
stimulation; DRG, dorsal root ganglion.

microenvironment of the injured area, reducing secondary injury,
reducing scar tissue formation, and promoting nerve regeneration
[166]. Several studies have reported that EA treatment can lead to
obvious improvements in hindlimb locomotor and sensory function in
SCI rats, and have concluded that EA may promote recovery of neuro­
plasticity via systematic regulation of neurotrophic factors and their
receptors [44,167,168]. Due to the action of the pulsed electric fields, a
large number of residual propriospinal neurons and ascending and
descending nerve fibers are also stimulated, overcoming inhibition due
to inactivity and helping to restore normal function. Neurons that are
partially damaged are able to recover, restoring functional connections
with the propriospinal neural network (Fig. 5) [55,154,165].
Following the transplantation of NSC-derived neural network tissues
into the gap resulting from complete spinal cord transection, Jin et al.
[55] showed that use of EA to regulate the transplanted area and its
adjacent tissue microenvironment was able to achieve the following:
promotion of cell synthesis and secretion of NT-3, activation of the
downstream TrkC/AKT/mTOR (TrkC, NT-3 receptor, AKT, AKT kinase,
mTOR, mammalian target of rapamycin) signaling pathway in target
cells, increase of survival and neuronal differentiation in transplanted
neural network tissues, maintenance of synaptic connections, and
enhanced functional integration of tissue with the host spinal neural
network. EA in Governor vessel acupoints may increases release of
calcitonin gene-related peptide (CGRP) in afferent nerve endings in the
spinal cord via stimulating the nerve endings of meningeal branch
(containing the sensory afferent fibers from the dorsal root ganglion),
acting on CGRP receptors in the spinal cord neurons. CGRP excites
L-type voltage-gated Ca2+ channels (VGCCs) on the cell membrane by
up-regulating the calcium/calmodulin-dependent protein kinase
(αCaMKII) pathway, promotes differentiation of neurons, and stimulates
secretion of NT-3 [165]. If tissue-engineered neural network tissue is
transplanted and then stimulated by EA, it is possible to promote
continuous synthesis and secretion of endogenous NT-3 in the injured
spinal cord so as to enable targeted growth of the axons of stem
cell-derived neurons, establish synaptic connections with host neurons
of the spinal cord, and improve limb motor function. In addition, EA
stimulation could increase the level of NT-3 in the rostral and caudal
areas adjacent to the injured spinal cord, and the concentration of NT-3
in both areas was higher than in the injured area itself. Therefore, gra­
dients of NT-3 concentration are formed between the rostral/caudal
areas and the injured area, which facilitates axon-targeting growth of
transplanted mesenchymal stem cell-derived neuron-like cells over­
expressing TrkC, enhance synaptic connections with host spinal neurons
secreting endogenous NT-3, and promote integration of the transplanted
stem cell-derived neural network with host neural circuits in the injured
spinal cord [55,112].
Although the results obtained from functional electrical stimulation
of the spinal cord at the locations described above for EES, ISMS, tcSCS,

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B.-Q. Lai et al. Biomaterials 279 (2021) 121211

and EA has led to substantial progress in the field (Fig. 6; Table 4), we
have limited insight into the underlying mechanisms responsible for the
functional responses of the spinal cord to electrical stimulation. Never­
theless, electrical stimulation does improve the microenvironment of the
SCI area, activates the propriospinal neuronal network, regulates syn­
aptic remodeling, and increases motor function in paralyzed patients
[67,169–171]. In future studies, continuous use of neuroregulatory
techniques in the cerebral cortex, including transcranial magnetic
stimulation (TMS), brain-spinal interface stimulation, photogenetic
stimulation, and chemogenetic stimulation may help to transform
transplanted tissue-engineered neural network tissue from a disordered
to an ordered state. Concurrent limb movement training should also help
to accelerate ordering of tissue-engineered neural network tissue
(Fig. 6), which can then better act as a neuronal relay.
7. Conclusion and future perspectives
In summary, post-SCI damage can be repaired by both endogenous
and exogenous neuronal relay strategies. Endogenous neuronal relay
strategies include: (1) compensatory neuronal relays (which take

Fig. 6. Schematic diagram showing the combined strategies using a tissue-engineered neuronal relay and electrical stimulation. Approaches are grouped by the type
of spinal cord electrical stimulation: A) EA treatment; B) tcSCS; C) EES; D) ISMS; E) Summary of the electrical stimulation pathway. tcSCS, transcutaneous spinal cord
stimulation; EA, electroacupuncture; EES, epidural electrical stimulation; ISMS, intraspinal microstimulation; DRG, dorsal root ganglion; CST, corticospinal tract; 5-
HT, 5-hydroxytryptamine; TH, tyrosine hydroxylase; NFs, nerve fibers.

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B.-Q. Lai et al. Biomaterials 279 (2021) 121211

Table 4
Treatment protocols of functional electrical stimulation strategies for spinal cord injury SCI.
Functional Treatment protocols Key outcomes Major advantages Major disadvantages Refs
electrical
stimulation

Epidural Surgical implantation of Enabling voluntary control of Modulates the propriospinal Invasive; facilitates locomotion only [122–125,
electrical microelectrode array into the walking and sustaining active neural network, activates via a narrow range of stimulation 150,156,172,
stimulation epidural surface of spinal movement silent neuronal circuits, and parameters 173]
(EES) cord promotes synaptic plasticity
Intraspinal Stimulating the propriospinal Promoting and sustaining Accurately targets specific Invasive; causes gliosis and electrode [129,130,
microstimu- locomotor neural network motor recovery, and enabling spinal locomotor circuitry damage, and eventually results in 132,133]
lation (ISMS) via an intraspinal long distance walking inefficient stimulation
microelectrode array
Transcutan-eous Placing electrodes on the skin Modulating the excitability of Enables noninvasive Targets a specific area of spinal cord [38,157,158,
spinal cord surface of the vertebral the propriospinal neural treatment, with a convenient less accurately compared with EES and 161,174]
stimulation column network and evoking rhythmic and inexpensive commercial ISMS
(tcSCS) limb movement stimulation device
Electroacu- Application of pulsed current Effectively increasing Achieves double efficacy with Targets a specific area of spinal cord [154,164,
puncture (EA) to acupuncture needles neurotrophic factor secretion, combined acupoint and less accurately compared with EES and 166,170,
inhibiting inflammation, and electrical stimulation; ISMS; mostly used in analgesia and 175]
promoting nerve regeneration minimally invasive less so for the recovery of locomotor
function

advantage of the intact propriospinal neuronal axons in the spinal cord
using the collateral sprouting method to repair the damaged neural
network), and (2) autologous stem cell-derived neuronal relays (in
which a neural network is formed by the differentiation of spinal cord
stem cells). Exogenous neuronal relays include the following: (1)
transplanted embryonic spinal cord tissue-derived neuronal relays
(which used embryonic spinal cord tissue to repair the damaged host
neural network), (2) transplanted stem cell-derived neuronal relays
(which use differentiation of transplanted stem cells in the SCI area to
form a neural network), (3) tissue-engineered neuronal relays (Fig. 3)
which involve the application of tissue-engineered neural network tis­
sues or SCLTs constructed in vitro), and (4) artificial intelligence relays
(i.e., brain–spine interfaces). Regardless of which neuronal relay strat­
egy is adopted for SCI repair, the key question is how to better achieve
functional integration with the propriospinal neural network; this re­
mains to be investigated in future studies [16,18,102,176]. In the future,
we envisage that for complete spinal cord transection or for larger spinal
cord defects, biomimetic medical techniques will be available to provide
tissue-engineered neuronal relays for the timely re-construction of
neural networks in the spinal cord. The combination of such techniques
with electrical stimulation of propriospinal neurons could enable acti­
vation of neuronal circuits that are functionally silent, owing to the loss
of brain-derived neural information (Fig. 6). Electrical stimulation
should enable regulation of synaptic plasticity in the propriospinal
neural network, and furthermore help stem cell-derived neurons from
the tissue-engineered neural network tissue or SCLT to establish more
accurate synaptic connections with the host propriospinal neurons,
achieve functional integration, and repair autonomous motor function
in paralyzed patients (Fig. 6).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by grants from the Chinese National Nat­
ural Science Foundation (81891003, 8167150879) to Y. S. Zeng; from
the National Key R&D Program of China (2017YFA0104700), the Young
Elite Scientist Sponsorship Program by CAST (YESS, 2018QNRC001),
the Fundamental Research Funds for the Central Universities
(18ykpy38), and the Chinese National Natural Science Foundation
(81971157) to B. Q. Lai; and from the Co-innovation Foundation of
Guangzhou City (201704020221), the Foundation of Guangdong Prov­
ince (2017B020210012) and the 111 Project for Academic Exchange
Program (B13037) to Y. S. Zeng and X. Zeng.
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