Download Using extracellular matrix for regenerative medicine in the spinal

Biomaterials 34 (2013) 4945e4955
Contents lists available at SciVerse ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Review
Using extracellular matrix for regenerative medicine in the spinal cord
Fabio Zomer Volpato a, b,1, Tobias Führmann a,1, Claudio Migliaresi b,
Dietmar W. Hutmacher a, Paul D. Dalton a, *
a
b
Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove 4059, Australia
BIOtech Research Centre, University of Trento, 101 Via delle Regole, Trento 38123, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 January 2013
Accepted 20 March 2013
Available online 15 April 2013
Regeneration within the mammalian central nervous system (CNS) is limited, and traumatic injury often
leads to permanent functional motor and sensory loss. The lack of regeneration following spinal cord
injury (SCI) is mainly caused by the presence of glial scarring, cystic cavitation and a hostile environment
to axonal growth at the lesion site. The more prominent experimental treatment strategies focus mainly
on drug and cell therapies, however recent interest in biomaterial-based strategies are increasing in
number and breadth. Outside the spinal cord, approaches that utilize the extracellular matrix (ECM) to
promote tissue repair show tremendous potential for various application including vascular, skin, bone,
cartilage, liver, lung, heart and peripheral nerve tissue engineering (TE). Experimentally, it is unknown if
these approaches can be successfully translated to the CNS, either alone or in combination with synthetic
biomaterial scaffolds. In this review we outline the first attempts to apply the potential of ECM-based
biomaterials and combining cell-derived ECM with synthetic scaffolds.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Spinal cord injury
Tissue engineering
Extracellular matrix
Decellularization
1. Introduction
Unlike most other organs/tissues, the central nervous system
(CNS) has a limited capacity to regenerate and traumatic injuries to
the CNS are associated with a poor prognosis. Injuries to the spinal
cord lead to functional motor and sensory loss by disrupting long
distance projecting axons in white matter tracts, so far no widely
accepted treatment strategies are available [1]. The only commonly
used interventions are surgical stabilization of damaged vertebrae
and intensive rehabilitation [1]. Different experimental treatment
strategies are under investigation to promote recovery after spinal
cord injury (SCI), with cell and/or pharmaceutical delivery as the
most common approaches [2,3].
Biomedical materials are used in regenerative medicine approaches to replace or restore the anatomic structure and function
of damaged or missing tissues following any injury or disease by
combining the topographical cues of biomaterials with cells or
bioactive molecules [1]. Tissue engineered scaffolds intended for
CNS repair are often based on particular extracellular matrix (ECM)
molecules (e.g. fibrin, collagen, fibronectin [4e6]), other natural
polymers (alginate, agarose, chitosan [7e9]) or synthetic polymers
(e.g. poly(-hydroxy acids), poly(2-hydroxyethyl methacrylate),
* Corresponding author. Fax: þ61 7 3138 6030.
E-mail address: [email protected] (P.D. Dalton).
1
These authors contributed equally to this work and are first authors.
0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2013.03.057
polyethylene glycol [10e12]). While natural/synthetic polymer
blends are widely accepted as potential materials [13,14] the use of
ECM grafts is uncommon [15e19]. However decellularized tissue
grafts used for regeneration in other systems (e.g. peripheral nerves
[20,21], heart [22], and skin [23]) have shown great promise. Here
we discuss the possibility of combining orientated scaffolds of
synthetic biomaterials with cell-derived ECM to promote CNS
repair.
2. Pathophysiology of SCI
The severity of SCI depends on the level, type and intensity of
injury but includes permanent locomotor and sensory deficits, and
may lead to neuropathic pain, spasticity, urinary and respiratory
dysfunction, metabolic problems as well as psychological problems.
The primary, mechanical injury leads to damage of numerous nerve
fibre pathways in the white matter and possibly also cells in the
grey matter. The mechanical disruption of the highly organized
cytoarchitecture of the spinal cord has devastating effects on
both the primary injury zone and the injury parenchyma. The
primary injury is followed by secondary degenerative events
including apoptosis, bleeding, excitotoxicity, free-radical production, inflammation, ischemia, oedema, scarring and cystic cavitation, all of which contribute to an increase in tissue loss [24,25].
Inflammatory processes are a major contributor to secondary
degeneration, which take place over a time scale of hours, days,
weeks and even months after the injury [26]. Microglia and also
4946
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
astrocytes represent intrinsic immunocompetent cells. If the bloodspinal cord barrier is disrupted, extrinsic inflammatory cells are
recruited (neutrophils, macrophages, monocytes, lymphocytes, and
natural killer cells). These inflammatory cells express proteolytic
enzymes which degrade ECM proteins and contribute further to
bloodebrain barrier degradation and oedema. Inflammatory cells
and (to a lesser degree) astrocytes express a wide range of proinflammatory cytokines and chemokines (e.g. interleukin (IL)-a,
interferon (INF)-g, tumour necrosis factor (TNF)-a) as well as
reactive oxygen species, oxidative enzymes, and metalloproteinases, which exacerbate secondary tissue damage and the
formation of an inhibitory glial scar [27,28]. However, the immune
response may also contribute to tissue repair by clearance of
hemorrhagic and necrotic tissue, reducing the lesion size and the
release of trophic factors and cytokines which can be neuroprotective and promote axonal regeneration (e.g. interleukin (IL)10, transforming growth factor (TGF-b)) [27,29].
The final, chronic stage of SCI is characterized by Wallerian
degeneration, death of oligodendrocytes, fluid-filled cyst formation
within grey and white matter and the development of the glial scar
[3,25]. The glial scar represents a major mechanical and chemical
barrier (e.g. chondroitin sulphate proteoglycans (CSPGs)) to axonal
regeneration [30]. In lesions that spare the dura mater, the scar is
composed primarily of astrocytes, but in more severe lesions
(which open or rupture the meninges) invading connective tissue
elements (e.g. fibroblasts, endothelial cells, ependymal cells)
intermingle with the astrocytes [31,32]. Furthermore, depending on
lesion type and severity, Schwann cells migrate from the adjacent
nerve roots into the lesion side and come into close contact with the
reactive astrocytes [31,32]. In the end, this glial scar encapsulates a
cavity, which can be many times the size of the initial wound [33].
Fig. 1 shows such a fluid-filled cavity six days after a unilateral
compression injury to the rodent spinal cord. Surrounding the edge
of the cavity, an intense staining of GFAP can be seen, which
identifies the intermediate filaments within astrocytes. It is
important to note that GFAP, a common marker for astrocytes,
identifies internal filaments of this cell. The cell membrane of astrocytes, however, is markedly larger and the density of such
reactive astrocytes at the edge of the cyst is particularly intense.
Internally, such a cyst is almost exclusively filled with activated
macrophages at this time point, emphasizing the role that inflammation plays in the cyst formation. There is also recent evidence
that intermediate filaments of glial cells significantly contribute to
the local stiffness of the tissue. Kas and colleagues correlated the
GFAP intensity with the viscoelastic properties of glia-rich retinal
slices that underwent ischemia [34]. It is an important finding since
increasing the stiffness of glial scar tissue would further impede
any neurite growth into this region. Conversely, the formation of a
glial scar has a beneficial role during the acute phase (1e2 weeks)
after SCI. When reactive astrocytes were eliminated or prevented
from contributing to scar formation after CNS injury, the result was
a failure in bloodebrain barrier repair, accompanied by massive
inflammatory cell infiltration, increased loss of neurons and oligodendrocytes, and the eventual worsening of the functional
outcome [35,36].
The lack of any functionally significant axon regeneration is
mainly due to an imbalance of local axon growth-promoting and
growth-inhibitory mechanisms. Neurotrophic factors and guidance
Fig. 1. AeC: Immunohistological sections of the rodent spinal cord six days after injury showing a debris-filled cyst, surrounded by a dense glial scar, which is impermeable to
regenerating axons. B: Higher magnification of the glial scar with GFAP staining. C: Glial scar stained for CD68þ cells, which are activated macrophages/microglia. The insetin C is
luxol fast blue that stained myelin and visualizes the white and grey matter, with the red line showing the plane of sectioning for AeC. D: injection of a single astrocyte with a
florescent dye demonstrates the volume of an astrocyte. E: Glial scar at 4 weeks stained for neurocan, an ECM known for inhibiting neurite outgrowth. AeC: Unpublished Images.
D: Republished with permission of the Society for Neuroscience, from Wilhelmsson et al. [210]; permission conveyed through Copyright Clearance Center, Inc. and E: Reprinted from
Huang et al. [211] with permission from Elsevier. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
cues (e.g. from the ECM) are only poorly expressed at the lesion site,
while the presence of potent molecular (myelin inhibitors: NOGOA, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin
glycoprotein (OMgp)) and physical barriers (glial scar) prevent
axonal regeneration [31,37]. The CNS contrast with injuries of the
peripheral nervous system, which has the capacity to regenerate
significant distances; nerve gaps of approximately 4 cm in humans
can be bridged with nerve guidance channels [38]. Such a “critical
gap” equivalent for the spinal cord has been described as overcoming scar and myelin mediated inhibition[31].
3. Natural ECM of the CNS
The ECM contributes only approximately 20% to the total tissue
volume within the CNS [39]. While structural proteins such as
elastin, collagen and fibronectin are the major component of most
tissues’ ECM, the uninjured adult CNS only sparingly contains
these elements. The ECM of the spinal cord is composed mostly of
glycosaminoglycans (hyaluronic acid) and proteoglycans (agrin,
aggrecan, brevican, neurocan, phosphacan, versican) [40]. Other
components include laminin, nidogen, reelin, netrin-1, tenascin-C
and tenascin-R, Slit-1, -2, as well as attached growth factors like
fibroblast growth factor (FGF)-2 and epidermal growth factor
(EGF). A schematic drawing of the major ECM proteins of the CNS
is shown in Fig. 2. Additionally, a specialized extracellular matrix
called perineuronal nets (PNN) can be found in the maturing CNS
[41,42]. PNN are layers of condensed pericellular matrix surrounding the cell body and dendrites of many neurons. They
control and stabilize the formation of synapses and connections
[43,44]. The main components of PNN are hyaluronan (HA), link
proteins, CSPGs and tenascin-R [41,43]. It is thought, that this
structure plays an important role in CNS regeneration and its
formation is directly correlated to decrease in neuron plasticity.
Since the PNN are considered a specialized form of the ECM, they
will be combined and described as ECM in the context of this review. For a detailed overview on ECM and PNN see Refs. [45] and
[44], respectively.
In addition to the storage of cytokines and growth factors, the
ECM can also regulate their activity [46]. In the neural stem cell
(NSC) niche, FGF-2 binds to N-sulphated heparan sulphate proteoglycans (HSPGs) to help maintain the NSC phenotype [47].
Moreover, intracellular signalling activated by integrins can
modulate pathways activated by cytokines [48,49]. Another
mechanism to regulate cellular function by the ECM is transduction
of mechanical signals. CNS ECM molecules have also been recognized for being multivalent, which means that they can bind to
either identical or multiple other ECM ligands showing opposite
4947
effects. An example is tenascin-C that has both adhesive and antiadhesive domains, each recognized by different receptors, and
specific binding interactions may engender contrary effects (i.e.
tenascin and tenascineglycoprotein conjugates may either promote or inhibit axonal growth) [50].
It is important to appreciate that the ECM of embryonic, postnatal and adult spinal cord tissue is different. For example, PNN
appear only late in development and are related to the increase in
neural activity [44]. Furthermore, laminin (e.g. laminin 111 or
a1b1g1 e previously known as laminin-1 [51]) a widely used ECM
molecule for promoting neurite growth in vitro, is widely expressed
during early embryogenesis, but not usually in contact with neurons during adulthood [52,53]. In total, there are at least 15
different isoforms of laminin in vivo; this diversity and distribution
(both location and in time) of laminin demonstrates the complex
and specific function of this extracellular constituent.
4. ECM of the glial scar
The injury-induced glial scar is both a physical and chemical
barrier to axonal regeneration. The chemical nature of the glial scar
in particular has been the subject of considerable research, yet our
understanding is not complete [54]. The glial scar in vivo differs in
distribution and type; and its major constituents (e.g. tenascin,
HSPGs, CSPGs and keratan sulphate proteoglycans (KSPGs)) are
mainly inhibitory for neurite outgrowth [55e57]. These ECM molecules are already abundant in the normal CNS but are highly upregulated during gliosis, e.g. by astrocytes and fibroblasts [55].
Structurally, CSPG contains a single core protein and one or more
polysaccharide glycosaminoglycan (GAG) chains, which are the
main inhibitory part. Examples of CSPG implicated in hindering
regeneration after injury include aggrecan, brevican, neurocan,
neuronglial antigen 2 (NG2), phosphacan and versican [55,58].
CSPGs suppress axonal regeneration via negatively charged sulphate [59] or via binding and blocking growth-promoting effects of
some ECM molecules, such as laminin and tenascin [60,61]. Most
axonal growth inhibitors in the CNS limit axonal regeneration via
binding their interacting receptor proteins on the membrane
[62,63]. Two studies indicate that receptor protein tyrosine phosphatase s (RPTP s) acts as a receptor for CSPGs and partially mediates the ability of CSPGs to restrict regenerative neurite
outgrowth [64,65]. Most recently, the leukocyte common antigenrelated phosphatase (LAR) was recognized as a functional receptor
for CSPG axon growth inhibitors [66].
Nevertheless, axon “growth-promoting” molecules are also
expressed (e.g. by reactive astrocytes), for example laminin,
fibronectin, decorin, and poly-sialylated neural cell adhesion
Fig. 2. Schematic drawing of the major ECM proteins of the CNS.
4948
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
molecule (PSA-NCAM) have also been reported [31,67,68]. Neurotrophins (e.g. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)), embedded within the ECM, support
neuronal survival and differentiation, guide axonal growth, and
regulate synaptic plasticity in a variety of neuronal populations
in vitro and in vivo[69,70].
The effect of ECM on axonal growth is complex and understanding interactions depends on its context. In general, the growth
cone extension within the ECM environment is affected by the 1)
isoform of the ECM molecule, 2) combination of ECM molecules, 3)
age of the neuron, 4) type of the neuron and 5) recent history of the
growth cone environment.
While the ECM of the CNS can be broadly considered as
“inhibitory” or “growth promoting”, the reality is different since
ECM molecules interact with each other to control this effect. For
example, an “inhibitory” ECM molecule (such as phosphacan,
neurocan) can inhibit neurite growth, yet promote outgrowth
when combined with another ECM molecule (such as fibronectin)
in vitro. “Growth-promoting” ECM can also inhibit neurite growth
through strong adhesion, diminishing cell-membrane release and
this can be regulated during development. For example embryonic
neurons on laminin in vitro can regulate their integrin receptors
significantly, controlling the extent of adhesion, however adult
neurons are less capable of this, and growth can actually be slowed
on laminin through excessive adhesion [71,72]. Fig. 3 schematically
shows how intermediate concentrations of ECM provide the most
neurite outgrowth.
Although the expression of both growth-inhibitory and
growth-promoting ECM molecules increases after injury, overall
the environment is inhibitory to regeneration [73]. Therefore to
induce regeneration the final composition of growth-promoting
and growth-inhibitory molecules has to be balanced towards
promoting tissue regeneration. One way to achieve this could be
the transplantation of scaffolds made primarily from growthpromoting ECM.
5. Regenerative medicine strategies to promote recovery after
SCI
Since the pathophysiology of SCI is both complex and resistant
to therapeutics, many conceptually different ways to promote recovery have been attempted, including cell-based therapies, gene
therapy, drug, antibody and growth factor delivery [3,74]. Cellbased therapies include the transplantation of Schwann cells
[75,76], olfactory ensheathing cells (OECs) [77,78], astrocytes
[79,80], neural progenitor cells [81,82], mesenchymal stromal cells
[83,84], or induced pluripotent stem cells (iPS) [85,86] either alone
or in combination with a growth-promoting scaffold, into the lesion
site to promote axon regeneration or tissue sparing. Such cells are
capable to produce neurotrophic factors such as NGF, BDNF,
neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial cellderived neurotrophic factor (GDNF) and leukemia inhibitory factor
(LIF), in addition to proteins such as laminins, fibronectin, collagen
I/III and IV [87e90], all of which are able to promote recovery after
CNS injury. Schwann cells have been the most investigated in cell
transplantation studies to promote neural repair after CNS injury,
due to their crucial role in PNS repair [76,91e93]. These cell-based
strategies have been reported to promote functional improvement
in experimental models of CNS, but the mechanism(s) of action
remain poorly defined (e.g. Refs. [94,95]). Cell-based therapies are
reviewed in depth elsewhere [96,97].
Drug delivery therapies include inhibition of the inflammatory
response (e.g. inhibition of leukocyte, macrophage/monocyte, and
neutrophil infiltration [98e100], inhibition of inflammatory angiogenesis [101], administration of classical immunosuppressives
[102]), stimulation of the inflammatory response (e.g. administration
of T-cells, transplantation of stimulated homologous macrophages,
vaccination with myelin self-antigens [103e107]), suppression of
myelin-associated inhibitor molecules (e.g. NOGO-A, MAG, OMgp)
and its pathways [108e111], and CSPGs digestion (e.g. administration
of chondroitinase ABC (ChABC) or hyaluronidase) [112e114]. Reports
Fig. 3. Both ‘growth-promoting’ and ‘growth-inhibiting’ molecules may prevent migration of growth cones in regions of injury. (a) For non-neuronal cells, adhesion to the substratum increases linearly with amount of bound matrix protein, yet peak motility is only supported by a narrow range of adhesion states. Low levels of attachment/matrix inhibit
motility by reducing traction (grip) while high levels of attachment/matrix inhibit motility by reducing the ability of cells to release from the substratum. (b) In regions of CNS injury,
growth cone motility may be reduced by more than a single mechanism. Factors providing mechanical barriers to migration may physically block growth cone advance (red). Factors
that antagonize the function of cell-matrix receptors or directly induce growth cone collapse will prevent migration by reducing traction (blue). ECM molecules and receptors that
increase growth cone adhesion will prevent migration by preventing release from the substratum (orange). Finally, high levels of neurotrophic molecules (i.e. biglycan) or molecules
that recruit growth factors (in italics) may retain growth cones in regions injury due to high trophic support (yellow). Reprinted from Condic et al. [71] with permission.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
suggest that inhibition of inflammation promotes tissue sparing and
improves functional recovery in animal models of SCI [100,101,115].
Axonal growth-inhibitory molecules such as Nogo, MAG, CSPGs,
activate the Rho pathway [108e111]. Therefore inhibition of the Rho/
ROCK signalling pathway demonstrated beneficial effects through
modulation of the inflammatory response after injury [116,117].
Nevertheless, there are negative effects associated to the inhibition of
such signalling pathways, i.e. increased spinal cord tissue atrophy
[118], decreased axonal sprouting/regeneration, impaired functional
recovery [119,120], and increase astroglial activation and CSPGs
deposition [119]. The administration of ChABC has shown to effectively degrade CSPGs present in the ECM [112,113], allowing significant axon regeneration both in vitro and in vivo.
The complexity of the CNS and its response to injury dictates
that the latest advances in bioengineering and macromolecular
chemistry have to be implemented in the design of scaffolds
intended to promote CNS tissue repair. Researchers have developed
multi-therapeutic strategies, combining various elements to promote repair [76,121e123]. Biomaterials are well-positioned to
deliver such combinatorial approaches for SCI repair, matrices or
scaffolds have been used in many of the strategies outlined above.
Biodegradability, mechanical strength, channels/fibres, porosity,
capability of cell adhesion and electrical activity are some of the
parameters that affect the regenerative capacity of a given design
[1,124]. A number of general issues may need to be considered for
the choice of biomaterial and the design of scaffolds, including:
1. Biocompatibility, minimizing adverse tissue reactions in vivo;
2. Topographic cues for orientated tissue integration and cellular
stimulation;
3. Cytocompatibility, promoting optimal cell adhesion, migration
and axon outgrowth;
4. Physical properties (e.g. elasticity, strength, tenacity) that
approach those of the host tissue;
5. Biodegradability with the production of non-toxic by-products.
Any tissue engineered scaffold implantation would require
major surgery and might not be suitable for many types of spinal
cord injuries. Injuries where the cord is lacerated (e.g. gun or knife
injuries) are the most likely candidates for the application of such
an approach. However, there is a significant opportunity to implant
and understand fundamental tissue engineering (TE) scaffold
integration within the CNS. Therefore, the immediate need for
degradability of these scaffolds/materials remains open for discussion. While few researchers will argue for a non-degradable
clinical product, the majority of current SCI in vivo models extend
out to only 4e6 weeks. With such short time periods, rapid
degradation may induce localized responses which may the lead to
poor outcomes [125]. Since many significant challenges remain
with SCI (i.e. robust regeneration/repair, migration of fibres out of
scaffolds), degradability remains an optional requirement for
biomedical materials in the spinal cord.
One important factor when designing biomaterials is that their
mechanical properties are similar to the spinal cord. Soft, high
water content hydrogels are the more widely used materials and
significant in vivo data supports their use. As one example, the
Shoichet laboratory developed hydrogel guidance channels that
were mechanically similar to the spinal cord [11,126] to contain and
protect a particular therapy. These “guidance channels” wrap
around the proximal and distal stumps of transected spinal cord
and can be filled with a gel-like matrix or glial cells [76,92,127].
Surprisingly, these guidance channels supported significant axonal
ingrowth, which mainly occurred on the inner surface on the tubes.
However, since these channels were soft, they collapsed slightly
during implantation, and efforts were made to strengthen them.
4949
However, making the channels more resistant to collapse resulted
in syringomyelia of the spinal cord [128]. Therefore scaffolds and
biomedical materials used within the spinal cord should be soft and
compliant, with suitable mechanical properties otherwise other
complications could arise. The elastic modulus of the spinal cord
(including pia/dura) is approximately 230 kPa [11], while that of the
grey/white matter is 2e5 kPa [129].
6. ECM as scaffolds/matrices for regenerative medicine
Biomaterials science includes the implantation of artificial, 3D,
highly orientated and biodegradable scaffolds [121,130] into the
injured spinal cord. Such oriented, cell-invasive scaffolds are often
derived from natural polymers, including ECM-molecules [6,8],
synthetic polymers [11] or combinations of both [13,14]. Treatment
strategies to promote regeneration of other tissues and organs
focus also on decellularised tissues and cells intended to promote
regeneration, known as ECM-derived scaffolds (ECM-scaffolds). In
the context of this review, these scaffolds do not include scaffolds
derived from purified ECM molecules (e.g. collagen, fibrin alone).
Such ECM-scaffolds demonstrated tremendous potential in both
experimental animal studies and clinical trials in non-CNS organs
[21,131e133]. Applications of ECM-scaffolds include blood vessels
[134,135], skin [23], bone [136], cartilage [137,138], trachea [132],
lung [139,140], heart [22,141,142] and peripheral nerves [20,21].
However, only few preliminary studies tried to harvest this potential for CNS repair [17,18,143,144].
In their normal environment cells are surrounded by ECM [145],
therefore three-dimensional scaffolds derived of or functionalized
with native ECM are thought to play an important role in regenerative medicine to promote regeneration of new tissues and organs [146]. The ECM functions not only as a supporting material but
also as a regulator of cellular functions such as cell survival, proliferation, morphogenesis and differentiation [145,147]. Moreover,
the ECM can modulate signal transduction activated by various
bioactive molecules such as growth factors and cytokines [48,49].
The ECM molecules and networks change their compositions,
structures and biomechanical properties according to each specific
tissue and organ. Their compositions and properties also dynamically change and remodel during development [148]. ECM from
developing tissue is thought to have greater beneficially effects
compared to adult tissue: i.e. immature astrocytes demonstrated a
greater ability to promote neurite outgrowth and scaffold integration compared to more mature astrocytes [149,150]. Ideally, the
scaffolds and substrates used for regenerative medicine can provide
the same or similar macro- and micro-environment to cells as that
of an ECM existing in vivo and assemble the infiltrating cells and
secreted matrices into functional tissues and organs [151]. If
degradable, the scaffolds will be replaced by the cells own matrices
during regeneration.
An alternative to using the full ECM-derived matrix is the use of
purified ECM proteins (e.g. fibrin, laminin, collagen, fibronectin).
However, these approaches lack in replicating the morphological
structure of native ECM. Furthermore, usually one single protein is
applied to the scaffold production, whilst the ECM is a combination
of several proteins, growth factors and other cytokines. Various
scaffolds or hydrogels based on purified ECM proteins have been
developed [1,80,152,153] and used for treatment strategies to promote recovery of the injured spinal cord and are reviewed in more
detail elsewhere [1].
Another, even more reductive approach, is the use of short
bioactive peptide sequences, such as arginine-glycine-aspartic acid
(RGD), tyrosine-isoleucine-glycine-serine-arginine (YIGSR) and
isoleucine-lysine-valine-alanine-valine (IKVAV). Such bioactive
peptides can be used for surface modification or as part of a
4950
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
self-assembling peptide and are reviewed for neural applications
elsewhere [154]. This approach uses synthetic peptide fragments
from the active protein to specifically change cell behaviour [155e
158]. For example, neurite extension in vitro is greatest with an
optimized concentration of RGD, with low or excessive amounts
shown to reduce the extent of neurite outgrowth [159]. Peptide
combinations can induce negative, additive and synergistic effects
on neurite extension in vitro [159]. For example combinations of
RGD- and IKVAV-modified fibrin matrices reduced neurite extension compared to either peptide alone. On the other hand, other
peptide combinations can synergistically promote neurite extension e the combination of YIGSR and IKVAV promotes robust
adhesion and neurite outgrowth on fluorinated polymer surfaces
[160].
In situ gelling hydrogels have attracted significant recent interest for neural tissue engineering [161,162]. This includes selfassembling peptides, which aggregate into nanoscale structures
under certain conditions and can be combined with bioactive
peptides such as IKVAV to impart a biological effect [163e166].
Such in situ gelling materials have a high water content and have
been injected directly into [165,166] or next to the spinal cord
[162,167]. The intrathecal injection of in situ gelling hydrogels based
on hyaluronic acid/methyl cellulose has been combined with drug
delivery for enhanced release [167e170]. In situ gelling hydrogels
are reviewed extensively elsewhere [171,172].
However, tissue-ECM is derived from multiple cell types and
composed of many kinds of proteins, including growth factors and
cytokines, and has a complex structure. Although the complexity is
advantageous for promoting repair, current decellularization protocols lack the specificity to remove growth-inhibitory molecules
while retaining growth-promoting molecules. Furthermore, it
would be difficult to reconstruct a scaffold or substrate that has the
same composition as that of an in vivo ECM using purified ECM
proteins, short peptides or conventional physicochemical methods.
In addition to the difficulty of mimicking the ECM composition, the
complex microstructure of extracellular space makes accurately
reproducing this environment extremely challenging. Researchers
have therefore been focussing on natural tissues to develop novel
biomaterial scaffolds based on cell-derived ECM [151,158]. Moreover, synthetic peptides might be initially useful for the functionalization of scaffolds to promote cell adhesion however the final
product would consist of a biomaterial scaffold with cell-derived
ECM. In contrast to cell transplantation, ECM alone has the
advantage of not requiring immunosuppression treatment
[143,174].
7. Decellularized tissue intended for CNS regeneration
Treatment strategies intended to promote CNS regeneration
based on decellularized tissue use primarily peripheral nerves
[15,16,19,175], however there are examples of decellularized nonneuronal [18] tissue used in the CNS. All of these strategies are
mostly early attempts and only give a hint of the potential of ECMbased strategies. Approaches used include acellular peripheral
nerve grafts implanted after optic nerve injury [15,16,19,144] and
into the diencephalon [175] (in experimental rat and hamster
models, respectively). Interestingly, the conclusions were that the
seeding of Schwann cells into the decellularized nerves was
important for axon regeneration, with insignificant regeneration
with acellular nerves alone. Despite the lack of axonal regeneration,
acellular nerves assisted the survival of retinal ganglion cells,
possibly due to a release of neurotrophic factors [16]. However,
none of protocols to decellularize the peripheral nerves included a
step to remove myelin, which could have affected its growth promoting properties.
A recent study investigated the potential of acellular muscle,
which contains laminin, fibronectin and collagen, to promote recovery after SCI in rats [18]. The chemically extracted acellular
muscle was implanted into spinal cord after lateral hemi-section
and was able to promote neuronal survival, neovascularisation
and axonal ingrowth into the lesion site, which followed the
orientation of the ECM-scaffold. Furthermore, ED1 and glial fibrillary acidic protein (GFAP) immunoreactivity was comparable to the
injured controls, indicating that ECM-scaffolds do not exacerbate
inflammation or gliosis [18].
Finally, CNS-derived acellular scaffolds have been investigated
on their potential to promote repair. Brain, spinal cord or optic
nerve tissue was decellularized either through chemical treatments
[174] or a combination of freeze-drying and chemical treatments
[17,143]. The acellular scaffolds contained laminin, fibronectin,
myelin and growth factors (vascular endothelial growth factor
(VEGF), FGF-2) [17,174]. Decellularized CNS scaffolds promoted
angiogenesis of chick embryo chorioallantoic membranes [143],
proliferation, migration and differentiation of neuronal cells in vitro
[17,174] and elicited no immune response after subcutaneous
transplantation in vivo [174]. Similar to the acellular peripheral
nerve grafts, myelin was not completely removed, which would
hamper the benefits of CNS-derived ECM-scaffolds after transplantation into the CNS [17].
8. 3D hybrid-scaffolds: combining synthetic biomaterials
with natural ECM
Outside to the CNS, several studies demonstrated the capacity of
scaffolds with in vitro deposited ECM to promote tissue repair (e.g.
in cartilage, skin and bone [177e180]). This type of hybrid scaffold,
schematically shown in Fig. 4, is initially produced though conventional or additive manufacturing principles then temporarily
seeded with cells to produce ECM that partially remains after
decellularization. Following a similar logic, the same concept could
be applied to the CNS. The shape and morphology of the 3D scaffold
could then be controlled to provide mechanical stability and a
guidance structure for the seeded cells, as orientation guides axonal
growth [121,130,154]. Studies that focus on both synthetic and
natural polymers for SCI regeneration, such as polycaprolactone
[13], poly(lactic-co-glycolic acid) [83,181], fibrin [152,182,183],
collagen [13,184,185] and hydrogels [186,187] could be used as
underlying substrates for natural ECM deposition. A schematic for
the combinations of such hybrid-scaffolds is shown in Fig. 4.
The potential source of ECM proteins and growth factors for CNS
applications is diverse, with many cells secreting ECM proteins and
molecules that are able to enhance axonal regeneration and neuroprotection [31,68,154,173]. Since the ECM-derived from cells
might contain inhibitory elements it is important to chose a cell
type and developmental stage where the ECM is overall more
growth promoting than inhibitory. For example, only mature oligodendrocytes produce myelin, together with its inhibitory factors
[188], while oligodendrocyte progenitor cells produce neurotrophic
factors like BDNF [189]. Other cell types, which have overall
growth-promoting properties, include immature astrocytes,
Schwann cells, stem cells and olfactory ensheathing cells (OECs).
ECM produced by such cells contains neurotrophic factors such as
NGF, BDNF, neurotrophin-3 (NT-3), ciliary neurotrophic factor
(CNTF), glial cell-derived neurotrophic factor (GDNF) and leukemia
inhibitory factor (LIF), in addition to proteins such as laminins,
fibronectin, collagen I/III and IV [87e90].
Schwann cells have been investigated in cell transplantation
studies to promote neural repair after CNS injury, due to their crucial
role in PNS repair [76,91e93]. Schwann cells have been shown to
express the surface adhesion molecules N-CAM, Ng-CAM/L1 and J1
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
4951
Fig. 4. Schematic diagram of a hybrid-scaffold containing synthetic fibers deposited by additive manufacture technologies and cell-derived ECM. The hybrid-scaffold is decellularized to minimize immunological response, whilst maintaining the growth-promoting molecules (e.g. laminin, collagen IV, NT-3, BDNF).
[190,191]; neurotrophic factors such as NGF, BDNF, GDNF and
insulin-like growth factor (IGF) [192,193]; and the ECM molecules
laminin and fibronectin [154,194], all of which are able to promote
recovery after CNS injury. Furthermore certain cytokines (e.g.
interleukin (IL)-1 alpha, beta, IL-6, TNF-alpha), which can be preserved during decellularisation [195] are known to influence the
inflammatory response and are able to modulate the secondary
injury [196]. Therefore Schwann cells would make a good starting
point for future investigations.
One current challenge with transplantation of live Schwann
cells is that they form boundaries with astrocytes in vitro and
in vivo, so the bridges will not integrate with the spinal cord
[176,197]. These sharp boundaries that form between Schwann
cells and host tissue are a barrier to regenerating axons, particularly
axons attempting to leave the growth-promoting Schwann cell
environment and reenter the host spinal cord [176]. One possibility
is that Schwann cell-derived ECM is highly growthpromoting,
preventing axons to reenter the host spinal cord, especially if the
matrix is slowly degraded.
Another challenge of cell transplantation into chronic lesions is
poor cell survival [208]. Since scaffolds or matrices do not depend
on cell survival, this is an alternative approach to promote repair in
a chronic lesion [209]. Therefore, it is envisioned that ECM-scaffolds
will be beneficial for acute, sub-acute and chronic lesions. Different
time points of implantation could be fine-tuned to target different
aspects of the pathophysiology of spinal cord injury, e.g. while
neuroprotection is a good target for scaffolds used in the acute
phase, chronic lesions may benefit more from factors promoting
axonal regeneration.
The efficiency of such hybrid-scaffolds depends largely on the
availability of growth-promoting ECM molecules. Several groups
have reported that ECM-scaffolds rapidly degrade after implantation by enzymatic (collagenase and matrix metalloproteinases
(MMP’s)) and cellular mechanisms [198e201]. Leukocytes, such as
neutrophils, macrophages, monocytes, lymphocytes and microglia,
as well as astrocytes all express such proteolytic enzymes. In tendon
replacement, approximately 60% of the ECM-scaffold is degraded
within 4 weeks, and completely in 3 months [200]. The mechanical
properties of the matrix rapidly decreased due to the nature of its
components. However, the kinetics behind ECM degradation are
still largely unknown, and further studies of the inflammatory
response to transplanted ECM and how it will affect its composition
4952
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
and bio-availability are necessary. Decellularization and sterilization protocols can also affect the biomechanical [202e204] and
biological (e.g. strength and functionality of growth factors)
[205,206] properties of the scaffolds. However, different crosslinking methods are able to reduce the degradation kinetics and
prolong scaffold stability [198,207]. Furthermore, after implantation, the scaffold is in a dynamic environment which is not only
characterized by the degradation of the ECM-scaffold but also by
the migration of host cells onto the scaffold, which can deposit new
ECM [199,200]. Further complementary therapies might be combined with the hybrid-scaffold, i.e. to reduce the effect of the inflammatory response and associated ECM degradation.
9. Conclusions
The lack of functionally significant axon regeneration after SCI is
mainly due to an imbalance of local axon growth-promoting and
growth-inhibitory mechanisms. Over the past decade, biomaterials
and regenerative medicine research has diversified and developed
rapidly. While the use of decellularized tissue has been widely
embraced in non-CNS tissues, its efficacy in the spinal cord is still
not fully understood and has been partially restricted by the
inhibitory ECM molecules that remain after processing. The native
ECM is a complex combination of proteins and polysaccharides that
play an important role in cellular behaviour such as attachment,
differentiation, and proliferation. Therefore a recent approach
where cells are temporarily grown on synthetic scaffolds to deposit
growth-promoting ECM could assist in controlling the regeneration
and integration of tissue engineering scaffolds. Synthetic scaffolds
with naturally deposited ECM are an emerging area of research and
could assist in tissues, like the CNS, where an inhibitory environment exists.
Acknowledgements
We appreciate the financial support from the European Union,
Seventh Framework Programme [FP7/2007-2013] under grant
agreement [Marie Curie FP7 e PCOFUND-GA-2008-226070, acronym
“progetto Trentino”] and the US Army Grant MS090180P1.
References
[1] Fuehrmann T, Gerardo-Nava J, Brook GA. Central nervous system. Tissue
engineering: from lab to clinic. Berlin: SpringereVerlag Berlin; 2011. p.221e
44.
[2] Madigan NN, McMahon S, O’Brien T, Yaszemski MJ, Windebank AJ. Current
tissue engineering and novel therapeutic approaches to axonal regeneration
following spinal cord injury using polymer scaffolds. Resp Physiol Neurobi
2009;169:183e99.
[3] Bauchet L, Lonjon N, Perrin FE, Gilbert C, Privat A, Fattal C. Strategies for
spinal cord repair after injury: a review of the literature and information.
Ann Phys Rehabil Med 2009;52:330e51.
[4] Liu S, Said G, Tadie M. Regrowth of the rostral spinal axons into the caudal
ventral roots through a collagen tube implanted into hemisected adult rat
spinal cord. Neurosurgery 2001;49:143e50 [discussion 50e1].
[5] King VR, Henseler M, Brown RA, Priestley JV. Mats made from fibronectin
support oriented growth of axons in the damaged spinal cord of the adult rat.
Exp Neurol 2003;182:383e98.
[6] Tsai EC, Dalton PD, Shoichet MS, Tator CH. Matrix inclusion within synthetic
hydrogel guidance channels improves specific supraspinal and local axonal
regeneration after complete spinal cord transection. Biomaterials 2006;27:
519e33.
[7] Talac R, Friedman JA, Moore MJ, Lu L, Jabbari E, Windebank AJ, et al. Animal
models of spinal cord injury for evaluation of tissue engineering treatment
strategies. Biomaterials 2004;25:1505e10.
[8] Li X, Yang Z, Zhang A, Wang T, Chen W. Repair of thoracic spinal cord injury
by chitosan tube implantation in adult rats. Biomaterials 2009;30:1121e32.
[9] Luo Y, Shoichet MS. A photolabile hydrogel for guided three-dimensional cell
growth and migration. Nat Mater 2004;3:249e53.
[10] Shi R, Borgens RB, Blight AR. Functional reconnection of severed mammalian
spinal cord axons with polyethylene glycol. J Neurotraum 1999;16:727e38.
[11] Dalton PD, Flynn L, Shoichet MS. Manufacture of poly(2-hydroxyethyl
methacrylate-co-methyl methacrylate) hydrogel tubes for use as nerve
guidance channels. Biomaterials 2002;23:3843e51.
[12] Giannetti S, Lauretti L, Fernandez E, Salvinelli F, Tamburrini G, Pallini R.
Acrylic hydrogel implants after spinal cord lesion in the adult rat. Neurol Res
2001;23:405e9.
[13] Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P, et al. Guidance
of glial cell migration and axonal growth on electrospun nanofibers of polyepsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend. Biomaterials 2007;28:3012e25.
[14] Gerardo-Nava J, Fuhrmann T, Klinkhammer K, Seiler N, Mey J, Klee D, et al.
Human neural cell interactions with orientated electrospun nanofibers
in vitro. Nanomedicine 2009;4:11e30.
[15] Berry M, Hall S, Follows R, Rees L, Gregson N, Sievers J. Response of axons
and glia at the site of anastomosis between the optic nerve and cellular or
acellular sciatic nerve grafts. J Neurocytol 1988;17:727e44.
[16] Berry M, Rees L, Hall S, Yiu P, Sievers J. Optic axons regenerate into sciaticnerve isografts only in the presence of Schwann-cells. Brain Res Bull
1988;20:223e31.
[17] Crapo PM, Medberry CJ, Reing JE, Tottey S, van der Merwe Y, Jones KE, et al.
Biologic scaffolds composed of central nervous system extracellular matrix.
Biomaterials 2012;33:3539e47.
[18] Zhang X-Y, Xue H, Liu J-M, Chen D. Chemically extracted acellular muscle: a
new potential scaffold for spinal cord injury repair. J Biomed Mater Res A
2012;100:578e87.
[19] Dezawa M, Nagano T. Contacts between regenerating axons and the
Schwann cells of sciatic nerve segments grafted to the optic nerve of adult
rats. J Neurocytol 1993;22:1103e12.
[20] Kim BS, Yoo JJ, Atala A. Peripheral nerve regeneration using acellular nerve
grafts. J Biomed Mater Res A 2004;68A:201e9.
[21] Karabekmez FE, Duymaz A, Moran SL. Early clinical outcomes with the use of
decellularized nerve allograft for repair of sensory defects within the hand.
Hand 2009;4:245e9.
[22] Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusiondecellularized matrix: using nature’s platform to engineer a bioartificial
heart. Nat Med 2008;14:213e21.
[23] Chen RN, Ho HO, Tsai YT, Sheu MT. Process development of an acellular
dermal matrix (ADM) for biomedical applications. Biomaterials 2004;25:
2679e86.
[24] Bunge RP, Puckett WR, Becerra JL, Marcillo A, Quencer RM. Observations on
the pathology of human spinal cord injury. A review and classification of 22
new cases with details from a case of chronic cord compression with
extensive focal demyelination. Adv Neurol 1993;59:75e89.
[25] Schwab ME, Bartholdi D. Degeneration and regeneration of axons in the
lesioned spinal cord. Physiol Rev 1996;76:319e70.
[26] Schwab JM, Brechtel K, Mueller C-A, Failli V, Kaps H-P, Tuli SK, et al.
Experimental strategies to promote spinal cord regenerationean integrative
perspective. Prog Neurobiol 2006;78:91e116.
[27] Donnelly DJ, Popovich PG. Inflammation and its role in neuroprotection,
axonal regeneration and functional recovery after spinal cord injury. Exp
Neurol 2008;209:378e88.
[28] Fitch MT, Doller C, Combs CK, Landreth GE, Silver J. Cellular and molecular
mechanisms of glial scarring and progressive cavitation: in vivo and in vitro
analysis of inflammation-induced secondary injury after CNS trauma.
J Neurosci 1999;19:8182e98.
[29] Chan CCM. Inflammation: beneficial or detrimental after spinal cord injury?
Recent Pat CNS Drug Discov 2008;3:189e99.
[30] Yiu G, He Z. Glial inhibition of CNS axon regeneration. Nat Rev Neurosci
2006;7:617e27.
[31] Silver J, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci
2004;5:146e56.
[32] Beattie MS, Bresnahan JC, Komon J, Tovar CA, Van Meter M, Anderson DK,
et al. Endogenous repair after spinal cord contusion injuries in the rat. Exp
Neurol 1997;148:453e63.
[33] Balentine JD. Pathology of experimental spinal cord trauma. I. The necrotic
lesion as a function of vascular injury. Lab Invest 1978;39:236e53.
[34] Lu YB, Iandiev I, Hollborn M, Korber N, Ulbricht E, Hirrlinger PG, et al.
Reactive glial cells: increased stiffness correlates with increased intermediate filament expression. Faseb J 2011;25:624e31.
[35] Faulkner JR, Herrmann JE, Woo MJ, Tansey KE, Doan NB, Sofroniew MV.
Reactive astrocytes protect tissue and preserve function after spinal cord
injury. J Neurosci 2004;24:2143e55.
[36] Okada S, Nakamura M, Katoh H, Miyao T, Shimazaki T, Ishii K, et al. Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes
after spinal cord injury. Nat Med 2006;12:829e34.
[37] Horner PJ, Gage FH. Regenerating the damaged central nervous system.
Nature 2000;407:963e70.
[38] Daly W, Yao L, Zeugolis D, Windebank A, Pandit A. A biomaterials approach
to peripheral nerve regeneration: bridging the peripheral nerve gap and
enhancing functional recovery. J R Soc Interface 2012;9:202e21.
[39] Sobel RA. The extracellular matrix in multiple sclerosis lesions. J Neuropath
Exp Neur 1998;57:205e17.
[40] DeQuach JA, Yuan SH, Goldstein LSB, Christman KL. Decellularized porcine
brain matrix for cell culture and tissue engineering scaffolds. Tissue Eng Part
A 2011;17:2583e92.
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
[41] Kwok JCF, Carulli D, Fawcett JW. In vitro modeling of perineuronal nets:
hyaluronan synthase and link protein are necessary for their formation and
integrity. J Neurochem 2010;114:1447e59.
[42] Carulli D, Rhodes KE, Brown DJ, Bonnert TP, Pollack SJ, Oliver K, et al.
Composition of perineuronal nets in the adult rat cerebellum and the cellular
origin of their components. J Comp Neurol 2006;494:559e77.
[43] Koppe G, Bruckner G, Hartig W, Delpech B, Bigl V. Characterization of
proteoglycan-containing perineuronal nets by enzymatic treatments of rat
brain sections. Histochem J 1997;29:11e20.
[44] Kwok JCF, Dick G, Wang DF, Fawcett JW. Extracellular matrix and perineuronal nets in CNS repair. Dev Neurobiol 2011;71:1073e89.
[45] Dityatev A, Seidenbecher CI, Schachner M. Compartmentalization from the
outside: the extracellular matrix and functional microdomains in the brain.
Trends Neurosci 2010;33:503e12.
[46] Moreno M, Munoz R, Aroca F, Labarca M, Brandan E, Larrain J. Biglycan is a
new extracellular component of the Chordin-BMP4 signaling pathway. Embo
J 2005;24:1397e405.
[47] Kerever A, Schnack J, Vellinga D, Ichikawa N, Moon C, Arikawa-Hirasawa E,
et al. Novel extracellular matrix structures in the neural stem cell niche
capture the neurogenic factor fibroblast growth factor 2 from the extracellular milieu. Stem Cells 2007;25:2146e57.
[48] Rahman S, Patel Y, Murray J, Patel KV, Sumathipala R, Sobel M, et al. Novel
hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling
pathway in endothelial cells. Bmc Cell Biol 2005;6.
[49] Comoglio PM, Boccaccio C, Trusolino L. Interactions between growth factor
receptors and adhesion molecules: breaking the rules. Curr Opin Cell Biol
2003;15:565e71.
[50] Gotz B, Scholze A, Clement A, Joester A, Schutte K, Wigger F, et al. Tenascin-C
contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting
sites for neurons. J Cell Biol 1996;132:681e99.
[51] Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D,
Ekblom P, et al. A simplified laminin nomenclature. Matrix Biol 2005;24:
326e32.
[52] Colognato H, Yurchenco PD. Form and function: the laminin family of heterotrimers. Dev Dynam 2000;218:213e34.
[53] Sosale A, Robson JA, Stelzner DJ. Laminin distribution during corticospinal tract development and after spinal cord injury. Exp Neurol
1988;102:14e22.
[54] Fitch MT, Silver J. CNS injury, glial scars, and inflammation: Inhibitory
extracellular matrices and regeneration failure. Exp Neurol 2008;209:
294e301.
[55] Grimpe B, Silver J. The extracellular matrix in axon regeneration. Prog Brain
Res 2002;137:333e49.
[56] Jones LL, Sajed D, Tuszynski MH. Axonal regeneration through regions of
chondroitin sulfate proteoglycan deposition after spinal cord injury: a
balance of permissiveness and inhibition. J Neurosci 2003;23:9276e88.
[57] McKeon RJ, Jurynec MJ, Buck CR. The chondroitin sulfate proteoglycans
neurocan and phosphacan are expressed by reactive astrocytes in the
chronic CNS glial scar. J Neurosci 1999;19:10778e88.
[58] Morgenstern DA, Asher RA, Fawcett JW. Chondroitin sulphate proteoglycans
in the CNS injury response. Prog Brain Res 2002;137:313e32.
[59] Gilbert RJ, McKeon RJ, Darr A, Calabro A, Hascall VC, Bellamkonda RV. CS-4,6
is differentially upregulated in glial scar and is a potent inhibitor of neurite
extension. Mol Cell Neurosci 2005;29:545e58.
[60] Sherman LS, Back SA. A ‘GAG’ reflex prevents repair of the damaged CNS.
Trends Neurosci 2008;31:44e52.
[61] Burg MA, Tillet E, Timpl R, Stallcup WB. Binding of the NG2 proteoglycan to
type VI collagen and other extracellular matrix molecules. J Biol Chem
1996;271:26110e6.
[62] Klein R. Eph/ephrin signaling in morphogenesis, neural development and
plasticity. Curr Opin Cell Biol 2004;16:580e9.
[63] Liu BP, Cafferty WBJ, Budel SO, Strittmatter SM. Extracellular regulators of
axonal growth in the adult central nervous system. Philos Trans R Soc Lond B
Biol Sci 2006;361:1593e610.
[64] Shen Y, Tenney AP, Busch SA, Horn KP, Cuascut FX, Liu K, et al. PTPsigma is a
receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration. Science 2009;326:592e6.
[65] Fry EJ, Chagnon MJ, Lopez-Vales R, Tremblay ML, David S. Corticospinal tract
regeneration after spinal cord injury in receptor protein tyrosine phosphatase sigma deficient mice. Glia 2010;58:423e33.
[66] Fisher D, Xing B, Dill J, Li H, Hoang HH, Zhao Z, et al. Leukocyte common
antigen-related phosphatase is a functional receptor for chondroitin sulfate
proteoglycan axon growth inhibitors. J Neurosci 2011;31:14051e66.
[67] Minor KH, Bournat JC, Toscano N, Giger RJ, Davies SJ. Decorin, erythroblastic
leukaemia viral oncogene homologue B4 and signal transducer and activator
of transcription 3 regulation of semaphorin 3A in central nervous system
scar tissue. Brain 2011;134:1140e55.
[68] Ridet JL, Malhotra SK, Privat A, Gage FH. Reactive astrocytes: cellular and
molecular cues to biological function. Trends Neurosci 1997;20:570e7.
[69] McAllister AK, Katz LC, Lo DC. Neurotrophins and synaptic plasticity. Annu
Rev Neurosci 1999;22:295e318.
[70] Thoenen H. Neurotrophins and neuronal plasticity. Science 1995;270:593e8.
[71] Condic ML, Lemons ML. Extracellular matrix in spinal cord regeneration:
getting beyond attraction and inhibition. Neuroreport 2002;13:A37e48.
4953
[72] Condic ML, Letourneau PC. Ligand-induced changes in integrin expression
regulate neuronal adhesion and neurite outgrowth. Nature 1997;389:
852e6.
[73] McKeon RJ, Schreiber RC, Rudge JS, Silver J. Reduction of neurite outgrowth
in a model of glial scarring following CNS injury is correlated with the
expression of inhibitory molecules on reactive astrocytes. J Neurosci
1991;11:3398e411.
[74] Nomura H, Tator CH, Shoichet MS. Bioengineered strategies for spinal cord
repair. J Neurotraum 2006;23:496e507.
[75] Kohama I, Lankford KL, Preiningerova J, White FA, Vollmer TL, Kocsis JD.
Transplantation of cryopreserved adult human Schwann cells enhances
axonal conduction in demyelinated spinal cord. J Neurosci 2001;21:944e50.
[76] Xu XM, Zhang SX, Li H, Aebischer P, Bunge MB. Regrowth of axons into the
distal spinal cord through a Schwann-cell-seeded mini-channel implanted
into hemisected adult rat spinal cord. Eur J Neurosci 1999;11:1723e40.
[77] Ramon-Cueto A, Nieto-Sampedro M. Regeneration into the spinal cord of
transected dorsal root axons is promoted by ensheathing glia transplants.
Exp Neurol 1994;127:232e44.
[78] Ramon-Cueto A, Plant GW, Avila J, Bunge MB. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory
ensheathing glia transplants. J Neurosci 1998;18:3803e15.
[79] Davies JE, Proschel C, Zhang N, Noble M, Mayer-Proschel M, Davies SJA.
Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted
precursors have opposite effects on recovery and allodynia after spinal cord
injury. J Biol 2008;7:24.
[80] Joosten EAJ, Veldhuis WB, Hamers FPT. Collagen containing neonatal astrocytes stimulates regrowth of injured fibers and promotes modest locomotor
recovery after spinal cord injury. J Neurosci Res 2004;77:127e42.
[81] Olson HE, Rooney GE, Gross L, Nesbitt JJ, Galvin KE, Knight A, et al. Neural
stem cell- and Schwann cell-loaded biodegradable polymer scaffolds support
axonal regeneration in the transected spinal cord. Tissue Eng Part A 2009;15:
1797e805.
[82] Parr AM, Kulbatski I, Zahir T, Wang X, Yue C, Keating A, et al. Transplanted
adult spinal cord-derived neural stem/progenitor cells promote early functional recovery after rat spinal cord injury. Neuroscience 2008;155:760e70.
[83] Kang KN, Lee JY, Kim DY, Lee BN, Ahn HH, Lee B, et al. Regeneration of
completely transected spinal cord using scaffold of poly(D, L-lactide-co-glycolide)/small intestinal submucosa seeded with rat bone marrow stem cells.
Tissue Eng Part A 2011;17:2143e52.
[84] Neuhuber B, Timothy Himes B, Shumsky JS, Gallo G, Fischer I. Axon growth
and recovery of function supported by human bone marrow stromal cells in
the injured spinal cord exhibit donor variations. Brain Res 2005;1035:73e85.
[85] Tsuji O, Miura K, Okada Y, Fujiyoshi K, Mukaino M, Nagoshi N, et al. Therapeutic potential of appropriately evaluated safe-induced pluripotent stem
cells for spinal cord injury. P Natl Acad Sci U S A 2010;107:12704e9.
[86] Nori S, Okada Y, Yasuda A, Tsuji O, Takahashi Y, Kobayashi Y, et al. Grafted
human-induced pluripotent stem-cell-derived neurospheres promote motor
functional recovery after spinal cord injury in mice. P Natl Acad Sci U S A
2011;108:16825e30.
[87] White RE, Jakeman LB. Don’t fence me in: harnessing the beneficial roles of
astrocytes for spinal cord repair. Restor Neurol Neurosci 2008;26:197e214.
[88] Ramon-Cueto A, Avila J. Olfactory ensheathing glia: properties and function.
Brain Res Bull 1998;46:175e87.
[89] Fortun J, Hill CE, Bunge MB. Combinatorial strategies with Schwann cell
transplantation to improve repair of the injured spinal cord. Neurosci Lett
2009;456:124e32.
[90] Wright LS, Li J, Caldwell MA, Wallace K, Johnson JA, Svendsen CN. Gene
expression in human neural stem cells: effects of leukemia inhibitory factor.
J Neurochem 2003;86:179e95.
[91] Bunge MB, Pearse DD. Transplantation strategies to promote repair of the
injured spinal cord. J Rehabil Res Dev 2003;40:55e62.
[92] Xu XM, Chen A, Guenard V, Kleitman N, Bunge MB. Bridging Schwann cell
transplants promote axonal regeneration from both the rostral and caudal
stumps of transected adult rat spinal cord. J Neurocytol 1997;26:1e16.
[93] Raisman G. Use of Schwann cells to induce repair of adult CNS tracts. Rev
Neurol 1997;153:521e5.
[94] Eftekharpour E, Karimi-Abdolrezaee S, Fehlings MG. Current status of
experimental cell replacement approaches to spinal cord injury. Neurosurg
Focus 2008;24:E19.
[95] Noble M, Davies JE, Mayer-Proschel M, Proschel C, Davies SJA. Precursor cell
biology and the development of astrocyte transplantation therapies: lessons
from spinal cord injury. Neurotherapeutics 2011;8:677e93.
[96] Reier PJ. Cellular transplantation strategies for spinal cord injury and
translational neurobiology. NeuroRx 2004;1:424e51.
[97] Ruff CA, Wilcox JT, Fehlings MG. Cell-based transplantation strategies to
promote plasticity following spinal cord injury. Exp Neurol 2012;235:78e90.
[98] Ghirnikar RS, Lee YL, Eng LF. Chemokine antagonist infusion attenuates
cellular infiltration following spinal cord contusion injury in rat. J Neurosci
Res 2000;59:63e73.
[99] Taoka Y, Okajima K. Role of leukocytes in spinal cord injury in rats.
J Neurotraum 2000;17:219e29.
[100] Taoka Y, Okajima K, Uchiba M, Murakami K, Harada N, Johno M, et al.
Activated protein C reduces the severity of compression-induced spinal cord
injury in rats by inhibiting activation of leukocytes. J Neurosci 1998;18:
1393e8.
4954
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
[101] Wamil AW, Wamil BD, Hellerqvist CG. CM101-mediated recovery of walking
ability in adult mice paralyzed by spinal cord injury. P Natl Acad Sci U S A
1998;95:13188e93.
[102] Bavetta S, Hamlyn PJ, Burnstock G, Lieberman AR, Anderson PN. The effects
of FK506 on dorsal column axons following spinal cord injury in adult rats:
neuroprotection and local regeneration. Exp Neurol 1999;158:382e93.
[103] Yoles E, Hauben E, Palgi O, Agranov E, Gothilf A, Cohen A, et al. Protective
autoimmunity is a physiological response to CNS trauma. J Neurosci
2001;21:3740e8.
[104] Rapalino O, Lazarov-Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M,
et al. Implantation of stimulated homologous macrophages results in partial
recovery of paraplegic rats. Nat Med 1998;4:814e21.
[105] Schwartz M. Protective autoimmunity as a T-cell response to central nervous
system trauma: prospects for therapeutic vaccines. Prog Neurobiol 2001;65:
489e96.
[106] Hauben E, Butovsky O, Nevo U, Yoles E, Moalem G, Agranov E, et al. Passive
or active immunization with myelin basic protein promotes recovery from
spinal cord contusion. J Neurosci 2000;20:6421e30.
[107] Hauben E, Ibarra A, Mizrahi T, Barouch R, Agranov E, Schwartz M. Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury
promotes recovery via a T-cell-mediated neuroprotective response: comparison with other myelin antigens. P Natl Acad Sci U S A 2001;98:15173e8.
[108] Niederost B, Oertle T, Fritsche J, McKinney RA, Bandtlow CE. Nogo-A and
myelin-associated glycoprotein mediate neurite growth inhibition by
antagonistic regulation of RhoA and Rac1. J Neurosci 2002;22:10368e76.
[109] Yamashita T, Higuchi H, Tohyama M. The p75 receptor transduces the signal
from myelin-associated glycoprotein to Rho. J Cell Biol 2002;157:565e70.
[110] Borisoff JF, Chan CCM, Hiebert GW, Oschipok L, Robertson GS, Zamboni R,
et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003;22:405e16.
[111] Monnier PP, Sierra A, Schwab JM, Henke-Fahle S, Mueller BK. The Rho/ROCK
pathway mediates neurite growth-inhibitory activity associated with the
chondroitin sulfate proteoglycans of the CNS glial scar. Mol Cell Neurosci
2003;22:319e30.
[112] Yick LW, Cheung PT, So KF, Wu W. Axonal regeneration of Clarke’s neurons
beyond the spinal cord injury scar after treatment with chondroitinase ABC.
Exp Neurol 2003;182:160e8.
[113] Zuo J, Neubauer D, Dyess K, Ferguson TA, Muir D. Degradation of chondroitin
sulfate proteoglycan enhances the neurite-promoting potential of spinal
cord tissue. Exp Neurol 1998;154:654e62.
[114] Frischknecht R, Heine M, Perrais D, Seidenbecher CI, Choquet D,
Gundelfinger ED. Brain extracellular matrix affects AMPA receptor lateral
mobility and short-term synaptic plasticity. Nat Neurosci 2009;12:897e904.
[115] Anderson AJ. Mechanisms and pathways of inflammatory responses in CNS
trauma: spinal cord injury. J Spinal Cord Med 2002;25:70e9.
[116] Nakayama M, Amano M, Katsumi A, Kaneko T, Kawabata S, Takefuji M, et al.
Rho-kinase and myosin II activities are required for cell type and environment specific migration. Genes Cells 2005;10:107e17.
[117] Hara M, Takayasu M, Watanabe K, Noda A, Takagi T, Suzuki Y, et al. Protein
kinase inhibition by fasudil hydrochloride promotes neurological recovery
after spinal cord injury in rats. J Neurosurg 2000;93:94e101.
[118] Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances
axonal regeneration in the injured CNS. J Neurosci 2003;23:1416e23.
[119] Chan CC, Khodarahmi K, Liu J, Sutherland D, Oschipok LW, Steeves JD, et al.
Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632
on axonal sprouting and functional recovery after rat spinal cord injury. Exp
Neurol 2005;196:352e64.
[120] Sung JK, Miao L, Calvert JW, Huang L, Louis Harkey H, Zhang JH. A possible
role of RhoA/Rho-kinase in experimental spinal cord injury in rat. Brain Res
2003;959:29e38.
[121] Patist CM, Mulder MB, Gautier SE, Maquet V, Jerome R, Oudega M. Freezedried poly(D, L-lactic acid) macroporous guidance scaffolds impregnated with
brain-derived neurotrophic factor in the transected adult rat thoracic spinal
cord. Biomaterials 2004;25:1569e82.
[122] Fouad K, Schnell L, Bunge MB, Schwab ME, Liebscher T, Pearse DD.
Combining Schwann cell bridges and olfactory-ensheathing glia grafts with
chondroitinase promotes locomotor recovery after complete transection of
the spinal cord. J Neurosci 2005;25:1169e78.
[123] Vavrek R, Pearse DD, Fouad K. Neuronal populations capable of regeneration
following a combined treatment in rats with spinal cord transection.
J Neurotraum 2007;24:1667e73.
[124] Orive G, Anitua E, Pedraz JL, Emerich DF. Biomaterials for promoting brain
protection, repair and regeneration. Nat Rev Neurosci 2009;10:682e92.
[125] Ziats NP, Miller KM, Anderson JM. In vitro and in vivo interactions of cells
with biomaterials. Biomaterials 1988;9:5e13.
[126] Tsai EC, Dalton PD, Shoichet MS, Tator CH. Synthetic hydrogel guidance
channels facilitate regeneration of adult rat brainstem motor axons after
complete spinal cord transection. J Neurotraum 2004;21:789e804.
[127] Montgomery CT, Tenaglia EA, Robson JA. Axonal growth into tubes
implanted within lesions in the spinal cords of adult rats. Exp Neurol
1996;137:277e90.
[128] Nomura H, Katayama Y, Shoichet MS, Tator CH. Complete spinal cord transection treated by implantation of a reinforced synthetic hydrogel channel
results in syringomyelia and caudal migration of the rostral stump. Neurosurgery 2006;59:183e92.
[129] Ozawa H, Matsumoto T, Ohashi T, Sato M, Kokubun S. Comparison of spinal
cord gray matter and white matter softness: measurement by pipette aspiration method. J Neurosurg 2001;95:221e4.
[130] Huang YC, Huang YY, Huang CC, Liu HC. Manufacture of porous polymer
nerve conduits through a lyophilizing and wire-heating process. J Biomed
Mater Res B 2005;74B:659e64.
[131] Costantino PD, Wolpoe ME, Govindaraj S, Chaplin JM, Sen C, Cohen M, et al.
Human dural replacement with acellular dermis: clinical results and a review of the literature. Head Neck 2000;22:765e71.
[132] Baiguera S, Jungebluth P, Burns A, Mavilia C, Haag J, De Coppi P, et al. Tissue
engineered human tracheas for in vivo implantation. Biomaterials 2010;31:
8931e8.
[133] Kehoe S, Zhang XF, Boyd D. FDA approved guidance conduits and wraps for
peripheral nerve injury: a review of materials and efficacy. Injury 2011;43:
553e72.
[134] Conklin BS, Richter ER, Kreutziger KL, Zhong DS, Chen C. Development and
evaluation of a novel decellularized vascular xenograft. Med Eng Phys
2002;24:173e83.
[135] Schmidt CE, Baier JM. Acellular vascular tissues: natural biomaterials for
tissue repair and tissue engineering. Biomaterials 2000;21:2215e31.
[136] Woods T, Gratzer PF. Effectiveness of three extraction techniques in the
development of a decellularized bone-anterior cruciate ligament-bone graft.
Biomaterials 2005;26:7339e49.
[137] Cheng NC, Estes BT, Awad HA, Guilak F. Chondrogenic differentiation of
adipose-derived adult stem cells by a porous scaffold derived from
native articular cartilage extracellular matrix. Tissue Eng Part A 2009;15:
231e41.
[138] Yang Q, Peng J, Guo QY, Huang JX, Zhang L, Yao J, et al. A cartilage ECMderived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials 2008;29:2378e87.
[139] Cortiella J, Niles J, Cantu A, Brettler A, Pham A, Vargas G, et al. Influence of
acellular natural lung matrix on murine embryonic stem cell differentiation
and tissue formation. Tissue Eng Part A 2010;16:2565e80.
[140] Price AP, England KA, Matson AM, Blazar BR, Panoskaltsis-Mortari A.
Development of a decellularized lung bioreactor system for bioengineering
the lung: the matrix reloaded. Tissue Eng Part A 2010;16:2581e91.
[141] Korossis SA, Booth C, Wilcox HE, Watterson KG, Kearney JN, Fisher J, et al.
Tissue engineering of cardiac valve prostheses II: biomechanical characterization of decellularized porcine aortic heart valves. J Heart Valve Dis
2002;11:463e71.
[142] Rieder E, Kasimir MT, Silberhumer G, Seebacher G, Wolner E, Simon P,
et al. Decellularization protocols of porcine heart valves differ importantly
in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. J Thorac Cardiovasc Surg 2004;127:
399e405.
[143] Ribatti D, Conconi MT, Nico B, Baiguera S, Corsi P, Parnigotto PP, et al.
Angiogenic response induced by acellular brain scaffolds grafted onto the
chick embryo chorioallantoic membrane. Brain Res 2003;989:9e15.
[144] Hall S, Berry M. Electron microscopic study of the interaction of axons and
glia at the site of anastomosis between the optic nerve and cellular or
acellular sciatic nerve grafts. J Neurocytol 1989;18:171e84.
[145] Adachi E, Hopkinson I, Hayashi T. Basement-membrane stromal relationships: interactions between collagen fibrils and the lamina densa. Int Rev
Cytol 1997;173:73e156.
[146] Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological
scaffold material: structure and function. Acta Biomater 2009;5:1e13.
[147] Giancotti FG, Ruoslahti E. Transduction e integrin signaling. Science
1999;285:1028e32.
[148] Rauch U. Brain matrix: structure, turnover and necessity. Biochem Soc T
2007;35:656e60.
[149] Smith GM, Miller RH, Silver J. Changing-role of forebrain astrocytes during
development, regenerative failure, and induced regeneration upon transplantation. J Comp Neurol 1986;251:23e43.
[150] Smith GM, Rutishauser U, Silver J, Miller RH. Maturation of astrocytes in vitro
alters the extent and molecular basis of neurite outgrowth. Dev Biol
1990;138:377e90.
[151] Hoshiba T, Lu HX, Kawazoe N, Chen GP. Decellularized matrices for tissue
engineering. Expert Opin Biol Th 2010;10:1717e28.
[152] Taylor SJ, Sakiyama-Elbert SE. Effect of controlled delivery of neurotrophin-3
from fibrin on spinal cord injury in a long term model. J Control Release
2006;116:204e10.
[153] Mothe AJ, Tam RY, Zahir T, Tator CH, Shoichet MS. Repair of the injured
spinal cord by transplantation of neural stem cells in a hyaluronan-based
hydrogel. Biomaterials 2013/03/08 ed2013.
[154] Dalton PD, Mey J. Neural interactions with materials. Front Biosci 2009;14:
769e95.
[155] Barker TH. The role of ECM proteins and protein fragments in guiding cell
behavior in regenerative medicine. Biomaterials 2011;32:4211e4.
[156] Bellis SL. Advantages of RGD peptides for directing cell association with
biomaterials. Biomaterials 2011;32:4205e10.
[157] Collier JH, Segura T. Evolving the use of peptides as components of biomaterials. Biomaterials 2011;32:4198e204.
[158] Ratcliffe A. Difficulties in the translation of functionalized biomaterials into
regenerative medicine clinical products. Biomaterials 2011;32:4215e7.
F.Z. Volpato et al. / Biomaterials 34 (2013) 4945e4955
[159] Schense JC, Bloch J, Aebischer P, Hubbell JA. Enzymatic incorporation of
bioactive peptides into fibrin matrices enhances neurite extension. Nat
Biotechnol 2000;18:415e9.
[160] Tong YW, Shoichet MS. Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers. Biomaterials
2001;22:1029e34.
[161] Zhang SG. Emerging biological materials through molecular self-assembly.
Biotechnol Adv 2002;20:321e39.
[162] Gupta D, Tator CH, Shoichet MS. Fast-gelling injectable blend of hyaluronan
and methylcellulose for intrathecal, localized delivery to the injured spinal
cord. Biomaterials 2006;27:2370e9.
[163] Cui HG, Webber MJ, Stupp SI. Self-assembly of peptide amphiphiles: from
molecules to nanostructures to biomaterials. Biopolymers 2010;94:1e18.
[164] Silva GA, Czeisler C, Niece KL, Beniash E, Harrington DA, Kessler JA, et al.
Selective differentiation of neural progenitor cells by high-epitope density
nanofibers. Science 2004;303:1352e5.
[165] Gelain F, Cigognini D, Caprini A, Silva D, Colleoni B, Donega M, et al. New
bioactive motifs and their use in functionalized self-assembling peptides
for NSC differentiation and neural tissue engineering. Nanoscale 2012;4:
2946e57.
[166] Tysseling-Mattiace VM, Sahni V, Niece KL, Birch D, Czeisler C, Fehlings MG,
et al. Self-assembling nanofibers inhibit glial scar formation and promote
axon elongation after spinal cord injury. J Neurosci 2008;28:3814e23.
[167] Shoichet MS, Tator CH, Poon P, Kang C, Baumann MD. Intrathecal drug delivery strategy is safe and efficacious for localized delivery to the spinal cord.
Prog Brain Res 2007;161:385e92.
[168] Kang CE, Tator CH, Shoichet MS. Poly(ethylene glycol) modification enhances
penetration of fibroblast growth factor 2 to injured spinal cord tissue from
an intrathecal delivery system. J Control Release 2010;144:25e31.
[169] Wang YF, Lapitsky Y, Kang CE, Shoichet MS. Accelerated release of a sparingly soluble drug from an injectable hyaluronan-methylcellulose hydrogel.
J Control Release 2009;140:218e23.
[170] Kang CE, Poon PC, Tator CH, Shoichet MS. A new paradigm for local and
sustained release of therapeutic molecules to the injured spinal cord for
neuroprotection and tissue repair. Tissue Eng Part A 2009;15:595e604.
[171] Nisbet DR, Williams RJ. Self-assembled peptides: characterisation and in vivo
response. Biointerphases 2012;7:1e4.
[172] Macaya D, Spector M. Injectable hydrogel materials for spinal cord regeneration: a review. Biomed Mater 2012;7:012001.
[173] Watabe K, Fukuda T, Tanaka J, Honda H, Toyohara K, Sakai O. Spontaneously
immortalized adult mouse Schwann cells secrete autocrine and paracrine
growth-promoting activities. J Neurosci Res 1995;41:279e90.
[174] Guo SZ, Ren XJ, Wu B, Jiang T. Preparation of the acellular scaffold of the
spinal cord and the study of biocompatibility. Spinal Cord 2010;48:576e81.
[175] Smith GV, Stevenson JA. Peripheral-nerve grafts lacking viable Schwanncells fail to support central nervous-system axonal regeneration. Exp Brain
Res 1988;69:299e306.
[176] Afshari FT, Kwok JC, White L, Fawcett JW. Schwann cell migration is integrindependent and inhibited by astrocyte-produced aggrecan. Glia 2010;58:
857e69.
[177] Lee SJ, Lee IW, Lee YM, Lee HB, Khang G. Macroporous biodegradable natural/synthetic hybrid scaffolds as small intestine submucosa impregnated
poly(D, L-lactide-co-glycolide) for tissue-engineered bone. J Biomat Sci Polym
E 2004;15:1003e17.
[178] Munirah S, Kim SH, Ruszymah BHI, Khang G. The use of fibrin and
poly(lactic-co-glycolic acid) hybrid scaffold for articular cartilage tissue engineering: an in vivo analysis. Eur Cells Mater 2008;15:41e51.
[179] Lu H, Hoshiba T, Kawazoe N, Koda I, Song M, Chen G. Cultured cell-derived
extracellular matrix scaffolds for tissue engineering. Biomaterials 2011;32:
9658e66.
[180] Liao J, Guo X, Grande-Allen KJ, Kasper FK, Mikos AG. Bioactive polymer/
extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for
cartilage tissue engineering. Biomaterials 2010;31:8911e20.
[181] Kang KN, Kim DY, Yoon SM, Lee JY, Lee BN, Kwon JS, et al. Tissue engineered
regeneration of completely transected spinal cord using human mesenchymal stem cells. Biomaterials 2012;33:4828e35.
[182] Johnson PJ, Tatara A, McCreedy DA, Shiu A, Sakiyama-Elbert SE. Tissueengineered fibrin scaffolds containing neural progenitors enhance functional
recovery in a subacute model of SCI. Soft Matter 2010;6:5127e37.
[183] Johnson PJ, Parker SR, Sakiyama-Elbert SE. Fibrin-based tissue engineering
scaffolds enhance neural fiber sprouting and delay the accumulation of
reactive astrocytes at the lesion in a subacute model of spinal cord injury.
J Biomed Mater Res A 2010;92:152e63.
[184] Liu T, Houle JD, Xu JY, Chan BP, Chew SY. Nanofibrous collagen nerve conduits for spinal cord repair. Tissue Eng Part A 2012;18:1057e66.
[185] Kassar-Duchossoy L, Duchossoy Y, Rhrich-Haddout F, Horvat JC. Reinnervation of a denervated skeletal muscle by spinal axons regenerating through a
collagen channel directly implanted into the rat spinal cord. Brain Res
2001;908:25e34.
4955
[186] Zuidema JM, Pap MM, Jaroch DB, Morrison FA, Gilbert RJ. Fabrication and
characterization of tunable polysaccharide hydrogel blends for neural repair.
Acta Biomater 2011;7:1634e43.
[187] Nisbet DR, Crompton KE, Horne MK, Finkelstein DI, Forsythe JS. Neural tissue
engineering of the CNS using hydrogels. J Biomed Mater Res B 2008;87B:
251e63.
[188] Baumann N, Pham-Dinh D. Biology of oligodendrocyte and myelin in the
mammalian central nervous system. Physiol Rev 2001;81:871e927.
[189] Zhang YW, Denham J, Thies RS. Oligodendrocyte progenitor cells derived
from human embryonic stem cells express neurotrophic factors. Stem Cells
Dev 2006;15:943e52.
[190] Noble M, Albrechtsen M, Moller C, Lyles J, Bock E, Goridis C, et al. Glial
cells express N-CAM/D2-CAM-like polypeptides in vitro. Nature 1985;316:
725e8.
[191] Seilheimer B, Schachner M. Studies of adhesion molecules mediating interactions between cells of peripheral nervous-system indicate a major role
for L1 in mediating sensory neuron growth on Schwann-cells in culture.
J Cell Biol 1988;107:341e51.
[192] Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve
regeneration. Mol Neurobiol 1997;14:67e116.
[193] Ferguson TA, Son YJ. Extrinsic and intrinsic determinants of nerve regeneration. J Tissue Eng 2011;2:1e12.
[194] Frostick SP, Yin Q, Kemp GJ. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery 1998;18:397e405.
[195] Hoganson DM, Owens GE, O’Doherty EM, Bowley CM, Goldman SM,
Harilal DO, et al. Preserved extracellular matrix components and retained
biological activity in decellularized porcine mesothelium. Biomaterials
2010;31:6934e40.
[196] Gaudet AD, Popovich PG, Ramer MS. Wallerian degeneration: gaining
perspective on inflammatory events after peripheral nerve injury.
J Neuroinflamm 2011;8.
[197] Golding JP, Bird C, McMahon S, Cohen J. Behaviour of DRG sensory neurites at
the intact and injured adult rat dorsal root entry zone: postnatal neurites
become paralysed, whilst injury improves the growth of embryonic neurites.
Glia 1999;26:309e23.
[198] Gratzer PF, Lee JM. Control of pH alters the type of cross-linking produced by
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) treatment of acellular matrix vascular grafts. J Biomed Mater Res A 2001;58:172e9.
[199] Badylak SF, Gilbert TW. Immune response to biologic scaffold materials.
Semin Immunol 2008;20:109e16.
[200] Gilbert TW, Stewart-Akers AM, Simmons-Byrd A, Badylak SF. Degradation
and remodeling of small intestinal submucosa in canine Achilles tendon
repair. J Bone Jt Surg Am 2007;89A:621e30.
[201] Record RD, Hillegonds D, Simmons C, Tullius R, Rickey FA, Elmore D, et al.
In vivo degradation of C-14-labeled small intestinal submucosa (SIS) when
used for urinary bladder repair. Biomaterials 2001;22:2653e9.
[202] Freytes DO, Badylak SF, Webster TJ, Geddes LA, Rundell AE. Biaxial strength
of multilaminated extracellular matrix scaffolds. Biomaterials 2004;25:
2353e61.
[203] Freytes DO, Stoner RM, Badylak SF. Uniaxial and biaxial properties of
terminally sterilized porcine urinary bladder matrix scaffolds. J Biomed
Mater Res B 2008;84B:408e14.
[204] Gouk SS, Lim TM, Teoh SH, Sun WQ. Alterations of human acellular tissue
matrix by gamma irradiation: histology, biomechanical property, stability,
in vitro cell repopulation, and remodeling. J Biomed Mater Res B 2008;84B:
205e17.
[205] Hoganson DM, O’Doherty EM, Owens GE, Harilal DO, Goldman SM,
Bowley CM, et al. The retention of extracellular matrix proteins and angiogenic and mitogenic cytokines in a decellularized porcine dermis. Biomaterials 2010;31:6730e7.
[206] McDevitt CA, Wildey GM, Cutrone RM. Transforming growth factor-beta1 in
a sterilized tissue derived from the pig small intestine submucosa. J Biomed
Mater Res A 2003;67:637e40.
[207] Powell HM, Boyce ST. EDC cross-linking improves skin substitute strength
and stability. Biomaterials 2006;27:5821e7.
[208] Nishimura S, Yasuda A, Iwai H, Takano M, Kobayashi Y, Nori S, et al. Timedependent changes in the microenvironment of injured spinal cord affects
the therapeutic potential of neural stem cell transplantation for spinal cord
injury. Mol Brain 2013;6:3.
[209] Hejcl A, Sedy J, Kapcalova M, Toro DA, Amemori T, Lesny P, et al. HPMA-RGD
hydrogels seeded with mesenchymal stem cells improve functional outcome
in chronic spinal cord injury. Stem Cells Dev 2010;19:1535e46.
[210] Wilhelmsson U, Li LZ, Pekna M, Berthold CH, Blom S, Eliasson C, et al.
Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy
of astrocytic processes and improves post-traumatic regeneration. J Neurosci
2004;24:5016e21.
[211] Huang X, Kim JM, Kong TH, Park SR, Ha Y, Kim MH, et al. GM-CSF inhibits
glial scar formation and shows long-term protective effect after spinal cord
injury. J Neurol Sci 2009;277:87e97.