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Tissue Engineering Applications in Neurology

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 Tissue Engineering
Applications in Neurology
E. L. K. Goh, H. Song, G.-Li Ming
Contents
56.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 815
56.2 Cell-Based Tissue Engineering
in CNS Repair . . . . . . . . . . . . . . . . . . . . . . 816
56.3 Applications of Tissue Engineering in
Therapeutic Approaches to Nervous System
Injury and Neurodegenerative Diseases . . . 818
56.3.1 Spinal Cord Injury . . . . . . . . . . . . . . . . . . . 818
56.3.2 Neurodegenerative Diseases . . . . . . . . . . . 819
56.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 821
References . . . . . . . . . . . . . . . . . . . . . . . . . 822
56.1
Introduction
Tissue engineering has tremendous potential to revo-
lutionize regenerative medicine. Research activities
related to tissue engineering have expanded from
the repair, replacement and regeneration of bone [1],
blood vessels [2, 3] and organs such as the kidneys
and heart [2–4] to the delicate nervous system. Initial
research efforts mainly focused on providing three-
dimensional (3D) scaffolds to facilitate the growth of
nerve cells in vitro. The ultimate goal is to generate
desirable cell types and create niches to support the
growth and regeneration of endogenous cells and/or
transplanted cells by recapitulating an in-vivo-like
environment. Advances in basic neuroscience and
56
biomaterials engineering have resulted in significant
progress in this direction over the past few years.
This chapter will discuss the potential and applica-
tions of tissue engineering approaches in regenera-
tive neurological medicine.
The nervous system comprises of two intercon-
nected but fundamentally different parts: the central
nervous system (CNS) and the peripheral nervous
system (PNS). The CNS consists of the brain and the
spinal cord, whereas the PNS consists of somatic and
autonomic nervous systems, which connect periph-
eral organs and tissues to the CNS. Neurons in the
adult PNS retain some ability to regenerate. Thus,
tissue engineering for neurological applications in
vivo has been dominated by research on develop-
ing polymeric nerve guidance conduits to facilitate
nerve regeneration and prevention of fibrous tissue
in-growth that impedes the regenerating nerve. In
contrast, adult CNS neurons are in general refractory
to regeneration upon physical or chemical damage
[5–7]. Several factors contribute to this lack of re-
generative capacity. These include reduced intrinsic
growth ability in adult neurons [8], physical barriers
formed by the scar tissue, and the presence of inhibi-
tory molecules that may exhibit further elevated ex-
pression and/or release upon injury [9]. Injuries and
degenerative neurological diseases of the CNS even-
tually lead to the loss of neurons and supporting tis-
sues. While glial cells are continuously generated in
the adult nervous system, neurons in most regions of
the brain are not. Therefore, cell replacement strat-
egies, including the transplantation of stem cells or
differentiated neural cells as well as promoting re-
generation of endogenous neurons, have been the
816
active lines of research [10–12]. The development
of a combinatory approach of biodegradable poly-
meric channels used for PNS and the incorporation
of growth factors and Schwann cells has provided
excellent foundations for advances in tissue engi-
neering for CNS regeneration [13].
56.2
Cell-Based Tissue Engineering
in CNS Repair
The main purpose of tissue engineering is to improve
or replace the biological functions of damaged or
missing tissues or organs. In order to enhance the
efficiency of tissue engineering, scaffolds with bio-
compatible and biodegradable materials are normally
used in combination with biological active molecules,
cells or tissues (Fig. 56.1).
Cell-based therapy has been attempted in inju-
ries of the CNS and in some neurological disorders
that are associated with neuronal cell death [14, 15].
Biomedical engineering approaches were initially
applied in vitro, largely to optimize cell culture pro-
cesses in order to obtain the desired cell types suit-
Fig. 56.1 Nerve conduits for regeneration of the PNS and CNS
E. L. K. Goh, H. Song, G.-Li Ming
able for cell therapy. One particularly promising area
involves the use of stem cells. Different types of
scaffolds that can influence the survival, prolifera-
tion, differentiation, and fate determination of stem
cells for various applications have been engineered
[16–18]. Different types of stem cells, including em-
bryonic stem cells, neural stem cells, hematopoietic
stem cells and mesenchymal stem cells, have all been
considered as transplantable cell sources for cell re-
placement therapy owing to their capability to dif-
ferentiate into different cell types [10, 19–23]. The
differentiation and regenerative potential of these
stem cells have been extensively reviewed elsewhere
[22, 23] and will not be elaborated upon here. Em-
bryonic stem cells have been considered to be an
ideal cell type for regeneration therapy due to their
pluripotency, i.e., the potential to differentiate into
almost all cell types found in adult tissues [24–30].
However, widespread clinical use of embryonic stem
cells requires overcoming various practical hurdles
and scientific and ethical issues associated with the
acquisition of these cells. Therefore, efforts have also
focused on finding sources of expandable and trans-
plantable adult stem cells. Recent studies have indeed
shown the existence of stem cells in many parts of
the body, such as the brain, blood, skin, adipose tis-
sue, bone, testis and amniotic fluid [10]. Other than
Chapter 56 Tissue Engineering Applications in Neurology
these stem cell sources, the induced pluripotent stem
cells (iPSC) derived from reprogramming of somatic
cells has tremendous clinical potential for cell trans-
plantations [31]. The potentially unlimited supply
of somatic cells and also the potential of autologous
transplantations are the two main advantages among
many. However, issues such low reprogramming ef-
ficiency and the concern of genetic alterations will
need to be solved before iPSC can be used for regen-
erative medicine.
Studies over the past decade have firmly estab-
lished that new neurons are continuously generated
in restricted regions of the adult mammalian brain,
including the subventricular zone of the lateral ven-
tricles and dentate gyrus of the hippocampus [32–
37]. Through a process termed adult neurogenesis,
new neurons from adult neural stem cells go through
a sequential process of neuronal development and
eventually become incorporated into the existing
neuronal circuitry [37] and are believed to contribute
to specific brain functions [38]. Other than these two
regions, the generation of new neurons in the adult
CNS appears to be very limited or non-existing [11,
39, 40]. On the other hand, a variety of adult neural
stem cells have now been identified and isolated not
only from regions with active neurogenesis but also
from other brain regions, including the cortex and
spinal cord [41–53]. With the novel tools and strate-
gies developed, we are now able to manipulate adult
neural stem cells from experimental animals and
from humans in vitro [10, 37, 50, 54], and to study
how these cells develop in vivo in animal transplan-
tation models [55]. While there is a potential of us-
ing adult neurogenesis for regenerative applications,
how these endogenous progenitor or stem cells could
be mobilized or utilized in clinical settings remains
to be explored.
One major challenge in developing stem cell-
based transplantation therapy relates to the number
and types of transplantable cells that can be realisti-
cally derived from adult stem cells expanded in cul-
ture [56, 57]. Tissue availability, suitable (preferably
autologous) stem cell types and their expandability
in culture are major issues that are currently being
explored. There are also many obstacles associated
with stem cell transplantation. Immune acceptance
of allogenic grafts, effective means of transplanta-
tion, and the survival, differentiation and integration
of transplanted cells, are all critical issues that need
to be addressed. Advances in bioengineering could
817
greatly facilitate efforts in developing stem cell-
based therapy by providing permissive graft environ-
ments [17]. For example, neural stem cells supported
by suitably engineered scaffolds were able to interact
with host tissue when implanted into the infarct cavi-
ties of mouse brains from induced hypoxia-ischemia
[58] and promote functional recovery after traumatic
spinal cord injury [59].
In another aspect of cell-based therapy, the effec-
tive delivery of biological factors is also a main focus.
Many biological factors, including neurotransmitters
and neurotrophic factors, are known to influence the
survival, neurite outgrowth, guidance and cellular
function of implanted cells or endogenous cells in
the nervous system. During development, axons and
dendrites can be guided to their targets by a wide
array of guidance cues, possibly present in the de-
veloping CNS as concentration gradients [60–62].
Multiple membrane-associated and soluble factors
work in synergy to guide axons to their precise
target. This guidance environment that exists dur-
ing development is, however, lost in the adult CNS.
Neurotrophic factors have been applied in animal
models of CNS injury or degenerative neurological
diseases to promote cell survival, axon regeneration
and guidance [63–65]. In addition, micro- and nano-
patterning have been explored in the bioengineering
of scaffolds for neurite guidance in vitro (Fig. 56.1).
For example, axons of peripheral neurons could be
guided by nanopatterns on polymethylmethacrylate
(PMMA) or protein-micropatterned surfaces in
vitro [66]. While some positive effects on neuronal
survival and neurite outgrowth have been observed
upon delivery of these factors, either via osmotic
pump or through implantation of silicon reservoirs,
there are several caveats. These include potential
failure of devices, infections, inflammations and
poor in-vivo stability of the protein factors. In most
cases, these factors can only be effectively delivered
through transplanted cells engineered to release
them in order to circumvent the blood-brain barrier
(BBB) [67].
The immune response of the host is also a major
issue in implantation/transplantation therapy. Cell or
protein encapsulation can potentially overcome this
problem [68]. The membrane of the capsules provides
a barrier to prevent the immune system from neutral-
izing the proteins or killing the implanted cells. In ad-
dition, this approach also allows a slow and sustained
release of molecules from the capsules [69].
818
In summary, cell-based engineering approaches
serve to provide physical support, suitable microen-
vironment for survival, proliferation and appropriate
differentiation as well as efficient cell and growth
factor delivery in vivo. At the same time, these
approaches also work on minimizing problems as-
sociated with the invasiveness of transplantation, in-
cluding the host immune response.
56.3
Applications of Tissue Engineering
in Therapeutic Approaches
to Nervous System Injury
and Neurodegenerative Diseases
The development of suitable biomaterials is expected
to play an important role in the feasibility of thera-
peutic success, and is therefore a main engineering
activity that would aid regenerative medicine. Some
of these biomaterials developed for various appli-
cations in the nervous system are briefly discussed
below.
Nerve guidance channels (NGCs) have been
shown to facilitate axonal regeneration after transec-
tion injury to the peripheral nerve [13]. The guidance
channels, as the name implies, serve as a physical
guide for nerve growth and also confine the migra-
tion of cells within the channels. NGCs with growth
factors incorporated onto the walls or within the ma-
trix of their inner lumen were engineered to enhance
regeneration [70, 71]. Neurotrophic factors (such as
nerve growth factor) that promote neuronal survival
and axonal outgrowth have been shown to improve
regeneration in the PNS when used in combination
with the channels [72]. Although NGCs have been
shown to be able to repair short gaps, axonal regen-
eration over longer distances remains incomplete [4,
73]. One of the few reports showing significant re-
generation over the distance of 30 mm in the PNS
was published in 2000 [73]. In that study, 80-mm
conduits consisting of a polyglycolic acid (PGA) col-
lagen tube filled with laminin coated collagen fibers
were implanted in the peroneal nerve in canine sub-
jects. Numerous myelinated nerves were observed
across the long gap and significantly enhanced func-
tional recovery was recorded over controls.
E. L. K. Goh, H. Song, G.-Li Ming
56.3.1
Spinal Cord Injury
Despite the tremendous development and suc-
cess of NGC in promoting the regeneration of the
PNS, functional regeneration of the CNS remains
very limited due to its physically and molecularly
unfavorable environment [5–7]. The application of
blockers of growth-inhibitor signaling, antibodies to
inhibitory molecules, digestive enzymes of the glial
scar or anti-inflammatory agents that prevent the
formation of glial scar have shown some promises
in improving the regeneration of CNS neurons [9,
74]. Tissue engineering using a variety of polymers
has been evaluated for their biocompatibility as
potential implants and cell carriers for the repair
of spinal cord injury (SCI). These include natural
polymers (such as alginate hydrogel and collagen),
synthetic biodegradable materials [such as matrigel,
fibronectin, fibrin glue, polyethylene glycol and poly
(α-hydroxy acids)], synthetic non-biodegradable
polymers [such as NeuroGel and poly 2-hydroxyethyl
methacrylate (PHEMA) and poly 2-hydroxyethyl
methacrylate-co-methyl methacrylate (PHEMM)]
[75, 76]. Encouraging results have been observed
from procedures implanting composite biosynthetic
conduits combining the more rigid scaffolds with
hydrogel or extracellular matrix molecules [77]. The
rigid scaffolds mainly serve as a physical support,
while the hydrogel can be impregnated with growth
and guidance factors and also serve as extracellular
matrix for implanted cells. For example, PHEMA
soaked with brain-derived neurotrophic factor
(BDNF) [78] or N-(2-hydroxypropyl) methacryl-
amide (HPMA) incorporated with the cell-adhesive
region of fibronectin Arg-Gly-Asp (RGD) tripep-
tide sequence [77, 78] and implanted into lesion
optic tract or cerebral cortex [78] of the adult rats
promoted angiogenesis and axonal penetration into
the gel and within the microstructure of the tissue
network. Similar observations were made with poly-
[N-(2-hydroxypropyl)-methacrylamide] (PHPMA)-
RGD [79] implanted into injured spinal cord [79]. In
addition, the hydrogel reduced nerve tissue necrosis
and cavitation in the adjacent white and gray matter
[79]. RGD coupled to HPMA hydrogels also encour-
aged growth of glial tissue when implanted into rat
brains [80].
Chapter 56 Tissue Engineering Applications in Neurology
Fig. 56.2a–c The various systems for the regeneration of the
CNS. a Growth factors or proteins of interest incorporated in
biomaterials made up the scaffold that support growth and
survival of the cells. b Encapsulation of cells genetically engi-
neered to release growth factors or proteins of interest can also
It is now widely accepted that a combinatory ap-
proach is necessary to facilitate functional recovery
from SCI. This means a combination of scaffold ar-
chitecture, transplanted cells and locally delivered
molecular agents (Fig. 56.2). Different cell types
have been used as cell replacements, to release
growth-promoting factor and to facilitate myelina-
tion. A multi-channel, biodegradable scaffold [poly-
lactic-co-glycolic acid (PLGA) with copolymers]
containing Schwann cells implanted after SCI was
shown to contain regenerating axons 1 month post-
operation [59]. In a sophisticated study, neural stem
cells were seeded into a multicomponent polymer
scaffold modeled after the intact spinal cord. Implan-
tation of this scaffold-neural stem cells unit into an
adult rat hemisection model of SCI promoted long-
term functional improvement (persistent for 1 year
in some animals) [81]. The regrowth of axons within
injured CNS is also promoted by implanted hydrogel
matrices containing BDNF and ciliary neurotrophic
819
avoid an immune response in vivo. c The combination of the
biomaterial scaffold and cell encapsulation approach allows
more sophisticated manipulation of microenvironment that in-
creases the efficiency of implantation
factor (CNTF) producing fibroblasts [82]. As men-
tioned earlier, encapsulation has been suggested to
alleviate the host immune response. Indeed, a study
using alginate encapsulated BDNF-producing fibro-
blast grafts showed it to permit recovery of function
without immune suppression [83, 84].
56.3.2
Neurodegenerative Diseases
56.3.2.1
Parkinson’s Disease (PD)
PD is generally characterized by the loss of dop-
aminergic neurons at the substantia nigra pars com-
pacta (SN), ultimately resulting in non-motor and
820
motor-related symptoms such as tremor, rigidity and
bradykinesia [85]. The current treatments for PD
include conventional drug replacement therapy, sur-
gical treatment, and cell transplantation [85]. The use
of cell transplantation in patients with PD has proven
to be the most successful treatment cases of all neu-
rodegenerative diseases to date. The loss of dop-
aminergic neurons in PD patients can be overcome
by cell replacement therapy using cells engineered to
release dopamine. The major drawbacks associated
with this treatment are poor survival of these trans-
planted cells and the lack of effective control in the
amount of dopamine released by these cells [85].
Tissue engineering approaches have been used
to improve the means of drug and cell delivery to
the CNS. Cultured astrocytes grown on polyethylene
terephthalate (PET) matrices have been evaluated as
a therapeutic treatment for PD because PET supports
high-density growth of astrocytes with stable glial cell
line-derived neurotrophic factor (GDNF) production
over a period of 18 days in vitro [86]. GDNF has been
shown to promote survival and fiber outgrowth of the
degenerating dopaminergic neurons [87]. Intrastriatal
injections of GDNF in the striatum of PD-rats after
transplantation of fetal dopaminergic cells results in
significant behavioral improvement [88–90]. In fact,
GDNF infused unilaterally into the putamen for 6–12
months has also been shown to significantly improve
motor function and quality of life measures in pa-
tients with PD in a phase-I trial [91]. GDNF-loaded
PLGA microspheres, when implanted in the brains
of PD rats, were well tolerated and induced sprout-
ing of the preserved dopaminergic fibers with synap-
togenesis, accompanied by functional improvement
[92]. GNDF-loaded pharmacologically active micro-
carriers (PAM) carrying fetal ventral mesencephalic
cells also improve the survival of grafted cells and
fiber outgrowth [93]. Another study demonstrated
that intraventricular chronic infusion of low-dose
GDNF from encapsulated genetically engineered
mouse myoblast cells (C2C12) demonstrated effica-
cious benefits on the “Parkinsonian” baboons [93].
These baboons showed transient recovery of motor
deficits (hypokinesia), significant protection of in-
trinsic striatal dopaminergic function in the immedi-
ate vicinity of the site of implantation of the capsule
in the caudate nucleus, and significant long-lasting
neurotrophic properties at the nigral level with an in-
E. L. K. Goh, H. Song, G.-Li Ming
creased volume of the cell bodies. All these studies
involve the transplantation of cells (mostly fetal SN
cells) into the stratium where dopamine is released
and not into the SN where dopaminergic neurons
normally reside. Therefore, the next desirable ad-
vance will be to reconstruct a functional dopaminer-
gic nigrostriatal pathway using a PD animal model,
and in the future, in human patients with PD.
56.3.2.2
Alzheimer’s Disease (AD)
AD is characterized by dementia with progressive
cognitive deterioration mainly due to neuronal loss
or atrophy, principally involving cells in the hip-
pocampus and temporoparietal cortex, as well as
the frontal cortex in advance stages. This atrophy is
often accompanied by inflammatory response with
the deposition of amyloid plaques and neurofibril-
lary tangles [94]. Earlier strategies for treatment of
AD include delivery of drugs, neuroprotective and
anti-amyloidogenic agents [94]. Many of the existing
FDA-approved drugs for the treatment of AD have a
short half-life and adverse side effects at higher doses
[95–100]. In order to prolong the half-life and also
to enable sustained release of drugs, encapsulation
of drugs by biodegradable polymers was desirable.
The pharmacokinetics of several commonly used
drugs encapsulated in microparticles had been evalu-
ated both in vitro and in vivo. For example, Tacrine
encapsulated by poly-(D,L-lactide-co-glycolide)
(PLG), huperzine A by PLG or PLGA and denepezil
by PLGA were tested in animal models of AD and
showed sustained release over a few weeks [95–100].
The tissue engineering pioneered by developments in
PD treatment discussed above may help to develop
therapies for other neurodegenerative diseases,
including AD. While preliminary and animal-based
results are encouraging, the ultimate goal for the
treatment of AD is the replacement of lost neurons
and the restoration of cognitive function. Cell therapy
involving the use of neural stem cells can potentially
replace these lost cells in regions of the brain [12,
40]. One can also attempt to modulate neurogenesis
in AD patients to replace the lost neurons. Modulat-
Chapter 56 Tissue Engineering Applications in Neurology
ing neurogenesis, in combination with strategies
targeting the underlying causes of AD, including the
formation and accumulation of amyloid plaques, may
be most effective in the treatment of AD.
56.3.2.3
Amytrophic Lateral Sclerosis (ALS)
ALS results from a gradual loss of motor neurons
throughout the brain and spinal cord, resulting in mus-
cle weakness, atrophy and eventual death. Currently,
there are limited choices for treating ALS patients.
While studies have suggested that CNTF and GDNF
can promote motor neuron survival [101–103], the
short half-life and lack of effective ways for their de-
livery into the CNS, had limited the clinical use of
these factors. The Aebischer laboratory has reported
the use of polymer-encapsulated cells engineered to
produce CNTF or GDNF constitutively [104]. The
authors demonstrated that GDNF significantly re-
duced the loss of facial motoneurons [105]. In addi-
tion, using models of progressive motoneuronopathy
(pmn) mice displaying motoneuron degeneration, it
was shown that CNTF but not GDNF increases the
life span of these animals [106].
Treatment with neurotrophic factors, while able to
delay the progression of the disease, is not useful for
late-stage ALS. Recently, motor neurons were suc-
cessfully derived from embryonic stem cells [107],
and paralyzed adult rats transplanted with these stem
cell-derived motor neurons showed partial recovery
from paralysis [108]. This restoration of function
was greatly enhanced in the presence of various fac-
tors, such as phosphodiesterase type 4 inhibitor and
dibutyryl cyclic adenosine monophosphate (cAMP),
which could overcome myelin-mediated repulsion.
In addition, GDNF has been shown to attract trans-
planted embryonic stem cell-derived axons toward
skeletal muscle targets [109].
The main challenges in stem cell-based treatment
of ALS patients are the availability of the cells, the
ability to maintain cell survival and recognizing the
appropriate factors that encourage nerve growth
and guidance at the strategic location of transplant.
Improvement of tissue engineering approaches will
821
hopefully be able to refine and bring current research
and treatment for ALS to the next level.
56.3.2.4
Huntington’s Disease (HD)
HD is an autosomal dominant inherited neurological
disorder [110]. The genetic mutation affects neurons
in the basal ganglia and results in devastating clinical
effects on cognitive, psychological, and motor func-
tions. These clinical symptoms primarily relate to the
progressive loss of medium-spiny GABAergic neu-
rons of the striatum. There is currently no treatment
available, but likely therapeutic candidates include
the use of several neurotrophic factors that are able
to prolong the survival of striatal neurons. The use
of CNTF as gene therapy for HD has been examined
in different animal models, such as monkeys [111,
112] and mice [64]. The encouraging results in ani-
mal models have led to a phase-I clinical study. Baby
hamster kidney cells (BHK) cells engineered to syn-
thesize human CNTF were encapsulated by a semi-
permeable membrane and implanted into the lateral
ventricle. Improvements in electrophysiological re-
sults were observed in subjects with higher amounts
of CNTF detected, and with no obvious toxicity over
a period of 2 years, indicating the safety and feasibil-
ity of the approach. However, the heterogeneity in
terms of cell survival and levels of CNTF secreted
remains to be refined.
56.4
Conclusion
The human nervous system is exceedingly complex,
and is therefore perhaps the most difficult to repair
after injury and degeneration. Damage to the nervous
system by injury or diseases is highly resistant to
treatments and there is a lack of effective therapies.
Advances in tissue engineering and basic neurosci-
ence have brought new hopes and strategies for repair
and regeneration. The field of CNS regeneration has
822
still a long way to go compared with the regeneration
of other tissues or organs such as the liver. An ideal
situation for the regeneration and treatment of inju-
ries of the nervous system or neurological disorders
is to activate the endogenous regenerative capability
and allow self-regulated regrowth and repair. How-
ever, we do not yet have a sufficient understanding
of adult neurogenesis, and future studies focusing
on uncovering the mechanisms involved in the ac-
tivation of endogenous repair are highly desired. In
addition, one can create artificial microniches where
transplanted cells can grow, develop and differentiate
in vivo. Bioengineering approaches play an important
part in both the design and the construction of such
“pseudo in-vivo” systems, which may serve a variety
of different purposes. One would hope to eventually
build a scaffold that could provide physical support
for different types of neural cells to grow on, and
which would encapsulate proteins factors that would
ensure the cells survive, form connections and inte-
grate into the host tissue.
Acknowledgments
The research in the authors’ laboratory was supported
by grants from the Culpeper Scholarship in Medical
Science, March of the Dimes, Klingenstein Fellow-
ship Award in the Neuroscience, Adelson Medical
Research Foundation and National Institute of Health
and Maryland Stem Cell Research Fund.
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