Transplantation of Nanostructured
Composite Scaffolds Results in the
Regeneration of Chronically Injured
Spinal Cords
Fabrizio Gelain,†,‡,* Silvia Panseri,† Stefania Antonini,†,‡ Carla Cunha,†,‡ Matteo Donega,†,‡ Joseph Lowery,§
Francesca Taraballi,†,‡ Gabriella Cerri,
Marcella Montagna,
Fausto Baldissera,
and Angelo Vescovi‡,§,*
†Biotechnology and Biosciences Department, University of Milan-Bicocca, Milan 20126, Italy, ‡Center for Nanomedicine and Tissue Engineering - A.O. Niguarda Ca’
Granda, Milan 20162, Italy, §Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States,
Department of
Human Physiology, University of Milan, Milan 20133, Italy, and §IRCCS Casa Sollievo della Sofferenza, Opera di San Pio da Pietrelcina, San Giovanni Rotondo 71013, Italy
A complex cascade of harmful eventsunderlies the establishment ofchronic spinal cord injury (SCI). The
complexity of this cascade has been de-
scribed in some detail, from the events fol-
lowing the initial contusion1 to those estab-
lishing chronic injuries.2 When developing
treatments for SCI, the complexity of the
task at hand varies with the stage of the le-
sion, peaking in chronic injuries. Key patho-
physiological factors, such as lack of neu-
rotrophic stimulation or permissive
substrates,3 inhibitory environment,4 sec-
ondary inflammatory damage,5 and intrin-
sic growth deficiency in adult neurons,6
have been targeted with a good degree of
success in acute and subacute SCI.3,7 Unfor-
tunately, despite significant progress,
chronic injuries still present us with a com-
plex scenario. Over time, damaged axons
undergo atrophy,8 retrograde degeneration
and retraction,9 while genes required for re-
generation are down regulated.10 This is
compounded by the eventual establish-
ment of a refractory barrier of extracellular
matrix and glial scarring at the injury site.7
Complementary approaches, including cell
therapy,11,12 digestion of glial scarring,7,11
neurotrophic factor delivery,13 and electri-
cal stimulation of the tissue surrounding the
injury site14 as well as clinical rehabilita-
tion,15 are being developed to accomplish
nerve fiber regeneration and functional res-
toration in SCI. Intriguingly, Schwann cell
transplantation can produce partial recov-
ery in subacute contusive lesions,16 and ef-
fective bridging and functional improve-
ments have recently been accomplished in
chronic SCI by combinatorial approaches,
modifying both neuronal-intrinsic and
-extrinsic growth mechanisms17 and ex-
ploiting neural progenitors transplanta-
tion.18
While the scenario is now turning on
the brighter side, the breadth of destruc-
tion of the nervous tissue observed in
chronic SCI in humansOwith entire sec-
tions of the spinal cord being replaced by
fluid-filled cystsOremains a critical concern.
Within these regions, the mechanical sub-
strates that provide physical support for ax-
onal regeneration and three-dimensional
positional information as well as the cytoar-
chitectural organization required for effec-
tive nerve regrowth have gone perma-
nently lost. Hence, a most pressing issue in
chronic SCI is to warrant a suitable level of
*Address correspondence to
fabrizio.gelain@unimib.it,
angelo.vescovi@unimib.it.
Received for review September 20,
2010 and accepted December 13, 2010.
Published online December 28, 2010.
10.1021/nn102461w
© 2011 American Chemical Society
ABSTRACT The destruction and hollowing of entire tissue segments represent an insurmountable barrier to
axonal regeneration and therapeutics in chronic spinal cord injury. To circumvent this problem, we engineered
neural prosthetics, by assembling electrospun nanofibers and self-assembling peptides into composite guidance
channels and transplanted them into the cysts of a postcontusive, chronic spinal cord injury rat model, also
providing delivery of proregenerative cytokines. Six months later conspicuous cord reconstruction was observed.
The cyst was replaced by newly formed tissue comprising neural and stromal cells. Nerve fibers were interspersed
between and inside the guidance channels, spanning the lesion, amidst a well-developed vascular network,
basal lamina, and myelin. This was accompanied by a significant improvement in the activity of ascending and
descending motor pathways and the global locomotion score. Thus by engineering nanostructured matrices into
neuroprosthetics, it is possible to recreate an anatomical, structural, and histological framework, which leads to
the replacement of large, hollow tissue gaps in the chronically injured spinal cord, fostering axonal regeneration
and neurological recovery.
KEYWORDS: self-assembling peptide · spinal cord injury · tissue
engineering · electrospinning · evoked potentials
ARTICLE
www.acsnano.org VOL. 5 ▪ NO. 1 ▪ 227–236 ▪ 2011 227

anatomical, histological, and cellular reconstruction at
the lesion site. Thus, the scar tissue and hollow cysts
should be replaced with new neural tissue, permissive
for both axonal regrowth and lesion bridging. Pioneer-
ing studies on acute injuries show the usefulness of
various artificial or biological materials in acute and sub-
acute SCI.19,20 In particular Teng and colleagues ob-
tained significant functional recovery in poly(lactic-co-
glycolic acid) (PLGA) scaffolds seeded with neural stem
cells.21 In animal models of complete transection,
Bunge’s group obtained significant axonal regenera-
tion and myelination within implanted resorbable poly-
meric scaffolds seeded with Schwann cells.22 Unfortu-
nately, similar studies on chronicized contusions in the
spinal cord, the most frequent situation in clinics, are
few. Biomatrix bridges, mixed with cells of olfactory ep-
ithelial origin, were explored but failed to sustain
chronically injured cortico-spinal axons.23 Better results
were obtained through the use of longitudinal channels
within poly(
-caprolactone) (PCL) foam implants, that
supported axonal regrowth, albeit for a limited time.24
Self-assembling peptides (SAPs) and electrospun
constructs, both characterized by their nanoscale archi-
tecture, are now being used in regenerative
medicine,25,26 including neuroregeneration27,28 and
slow delivery of cytokines.29 Here we tackled the issue
of anatomical and functional reconstruction in chronic
SCI by using SAPs in a combinatorial approach, in which
they were assembled into electrospun microchannel
guidance constructs made of PLGA and PCL blended fi-
bers. The aim was to establish a “neuro-prosthetic”
which would improve directional axonal regeneration
while supplying critical mechanical compliance, bio-
compatibility and degradation, and high porosity and
functionalization with biologically active motifs.30 These
microconduits, alone or imbued with a mix of regenera-
tive cytokines,31,32 were transplanted into the large
cysts that formed in the postcontusive spinal cord one
month after injury. Six months later, we found that,
while cysts persisted in control and sham-operated ani-
mals, they had been replaced by neo-formed tissue in
animals transplanted with microconduits. The latter
contained and was intermingled with bundles of regen-
erating axons and myelin, deposition of extracellular
matrix (ECM) and stromal cells, and well developed neo-
vascular structures, in the absence of considerable in-
flammatory response. This was matched by significant
improvement in motor function on the Basso, Beattie,
and Bresnahan (BBB) scale and in amplitudes and laten-
cies of evoked responses in ascending tracts. Thus,
transplantation of combined electrospun fibers/SAP
scaffolds neuro-prosthetics supports broad anatomical
and histological reconstruction and significant func-
tional recovery in the chronically injured, hollowed spi-
nal cord.
RESULTS
Surgery and Scaffold Implantation. We investigated a
postcontusion, chronic SCI rat model characterized by
centrally located cavities (cysts)33 and surrounded by
gliotic scar tissue, four weeks following a standardized,
weight-drop trauma34 (Figure 1A).
Various biological or synthetic materials have been
proposed in order to develop therapies for the injured
spinal cord,19,20 although most attempts have focused
on acute transection lesions rather than the postcontu-
sion damage most frequent in humans.35 These com-
prise the use of a combination of synthetic guidance
channels, hydrogel fillers, growth factors, and fetal,
schwann, or bone marrow cells in acute SC transection
studies.20,22,36,37
Here, we attempted to reinstate an appropriate
structural and environmental regenerative milieu us-
ing SAP-assembled tridimensional nanostructures (Fig-
ure 1B).
Using electrospinning, we generated hollow chan-
nels of nanofibers, composed of blended PCL and PLGA
at a weight ratio of 5.5/4. The PLGA provides short-
term strength, and the PCL provides long-term stabil-
ity.38 By this, we achieved a long-lasting reabsorption
Figure 1. Injury model and scaffold preparation. (A) hematoxylin-
eosin of a one-month old spinal cord lesion in longitudinal section;
arrows point at the cyst margins containing tissue debris (magnifi-
cation 5X). SEM images of self-assembled RADA16-I-BMHP1 (B)
and of an electropsun, PCL/PLGA microguidance channel (C); a
high-power magnification is shown in the inset. (D) Schematic rep-
resentation of scaffold implantation. Conduits, presoaked in PBS
and filled with RADA16-I-BMHP1 self-assembling peptide via
microsyringe injection (inset), were placed singularly within the
cavity following scar ablation. An electrospun lamina was sutured
and glued to the meninges.
AR
TI
CL
E
VOL. 5 ▪ NO. 1 ▪ GELAIN ET AL. www.acsnano.org228