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In: The Sciatic Nerve: Blocks, Injuries and Regeneration ISBN: 978-1-61122-916-5
Editors: David J. Fonseca and Joanne L. Martins ©2011 Nova Science Publishers, Inc.
Chapter 10
Experimental Approaches on Tissue Engineering Repair of Peripheral
Nerve Gaps
Kirsten Haastert-Talini*
Hannover Medical School, Institute of Neuroanatomy,
Hannover, Germany
Abstract
Peripheral nerve research is highly dynamic and a current major focus is the
development of cell based supportive therapies as well as bioengineered nerve conduits to
overcome the challenges in reconstructing long peripheral nerve defects. Whenever
primary nerve repair cannot be performed without regeneration impairing tension at the
suture sites, autologous nerve grafting or nerve tubulization are standard surgical repair
techniques. However, functional recovery after severe nerve lesions is generally partial
and unsatisfactory. Furthermore, nerve grafting is accompanied with several
disadvantages. Autologous nerve material is only of limited availability as it has to be
withdrawn from a healthy sensory nerve during an extra surgical incision that commonly
results in sensory residual deficits. Reconstruction of long nerve defects remains a
challenge since the gold standard of autologous nerve grafting as well as transplantation
of clinically approved artificial resorbable nerve conduits result in a success rate of
approximately 69% for nerve defects not exceeding a length of 3 cm only. This chapter
reviews basic science studies from several laboratories evaluating tissue engineering
nerve repair strategies especially with regard to the cellular and molecular composition of
artificial nerve grafts. The possibility to attach Schwann cells or Schwann cell-like cells
to biodegradable nerve guides for cellular support of the regeneration processes or for ex
vivo gene therapy is of similar interest as the opportunity to functionalize the biomaterial
with bioactive molecules through physicochemical modification or to allow long term
release of regeneration promoting molecules during biodegradation. Peripheral nerve
* Corresponding author: Kirsten Haastert-Talini: Hannover Medical School, Institute of Neuroanatomy, D-30623
Hannover, Germany, Email: [email protected], Tel: 0049-511-532-2891; Fax: 0049-511-532-
2880
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Kirsten Haastert-Talini 210
tissue engineering holds promise to provide future support and orchestration of
regeneration processes across long nerve gaps to a similar or even higher extend an
autologous nerve graft does today.
Introduction
Peripheral nerves that have been transected are able to regenerate across distances;
provided the gap is short and tension-free coaptation of proximal and distal nerve stumps by
end-to-end sutures can be achieved. However, these ideal conditions for peripheral nerve
regeneration are usually not present when lesions follow trauma or extended surgical
resection and are thus accompanied by massive loss of nerve tissue. In Europe approximately
300 000 cases of peripheral nerve injury occur per year [1]. In the United States more than
50 000 surgical peripheral nerve repair procedures are performed each year and the number of
injuries that deserve treatment has been estimated to be much higher [2]. Severe sciatic nerve
lesions with poor long term recovery could result from hip surgery [3].
Therapeutic repair strategies depend on the length of the gap between the transected
nerve stumps [4]. The clinical gold standard to overcome larger nerve gaps (20 mm or longer
in humans) is the transplantation of sensory nerve autografts, mainly harvested from sural and
medial and lateral antebrachial cutaneous nerves [5-7]. Nerve autografting is, however,
accompanied by side effects like functional loss of the donor nerve, size and quality (sensory
versus motor nerves) mismatch with the host nerve, and putative formation of painful
neuroma at the donor site [6, 8, 9]. Examples for long nerve gaps also include brachial plexus
lesions, where reinnervation of the hand muscles will take 800 days and more under optimal
conditions [10]. An autologous nerve graft provides an immunogenically inert nerve bridge
which contains viable Schwann cells and appropriate neurotrophic support for axonal
regeneration. However, even given the best achievable outcome, functional regeneration of
peripheral nerves after nerve autotransplantation is often disappointing.
Even before nerve autotransplantation became the gold standard, the use of tubular
implants to bridge the gap between transected peripheral nerve stumps (nerve tubulization)
was thought to support regeneration. First reports on this technique using hollow cylinders of
bone in animal models can be traced back to the end of the 19th century [11]. Due to increased
possibilities on the material sector, research through the last decades concentrated on the
development of alternative three-dimensional scaffold materials that should guide and
promote the regeneration process [12]. The rat sciatic nerve is a commonly used model for
investigation of peripheral nerve regeneration and therefore, this chapter was included to this
book. The tissue engineering of peripheral nerves combines the following strategies: the
investigation of biomaterials, the transplantation of regeneration promoting cells or tissue, as
well as ex vivo and in vivo gene therapy approaches [13]. Furthermore transplantation of
innovative tissue engineered constructs should be done using highly developed microsurgical
techniques and should eventually be combined with accompanying physical strategies to
enhance the regeneration outcome [13]. An ideal biomimetic nerve conduit would shield the
regenerating nerve against the environment and support generation of a permissive
environment, e.g., via transplanted Schwann cells within tubular implants, and in the ultimate
case it would degrade with ongoing regeneration processes [12].
Experimental Approaches on Tissue Engineering Repair … 211
Figure 1 summarizes the concept of tissue engineering of peripheral nerves and the
different aspects which will be addressed in this chapter.
Figure 1: Experimental strategies for the development of innovative peripheral nerve reconstruction
approaches. Regeneration processes in axotomized neurons have to be triggered and coordinated for
effective regeneration across long nerve gaps. This is supposed to be achievable (1) by physical therapy
e.g., electrical or phototherapeutical stimulation of the proximal nerve stump together with the
reconstructive surgery. Instead of autotransplants, (2) biodegradable scaffolds could bridge the defects.
These scaffolds should provide (3) guidances cues for regenerating fibers such as electrospun
nanofibers and a container for (4) seeding of therapeutic cells, e.g., Schwann cells. Regeneration
promoting substances/factors should either be delivered from the biomaterial or be presented at its inner
surface or be secreted by transplanted genetically modified Schwann cells (ex vivo gene therapy).
Current research aims in the development of mainly tubular or multichannel biomimetic
materials, which will allow seeding of therapeutic cells as well as a timely coordinated
delivery of regeneration promoting substances. Furthermore physical therapies, such as
electrical or phototherapeutic stimulation of the proximal nerve stump at the time of
reconstructive surgery have to be tested in combination with the biomimetic grafts to achieve
a synchronized regeneration activity resulting in improved functional recovery.
Physical Therapy Approaches for the Combination with Nerve Reconstructive Surgery
Severely damaged or transected peripheral nerves can be reconstructed by microsurgical
techniques [4, 14]. The repair techniques can roughly be defined as direct nerve repair for
sharp cuts, which allow approximation of the stumps with minimal tension, or nerve grafting
whenever nerve defects > 3 cm occur [5]. Peripheral nerve reconstruction generally aims at
restoration of function as promptly and completely as possible, while minimizing donor site
and systemic morbidity. In cases where a tension-free primary end-to-end neurorrhaphy is not
Kirsten Haastert-Talini 212
possible, several alternatives exist which have been comprehensively addressed in three
recent reviews [5, 9, 15]. Nerve autotransplantation still has the best outcome regarding nerve
tissue regeneration and achievable functional recovery [8]. However, full functional
restoration especially with regard to motor recovery is rarely seen because regeneration of
axons to the appropriate targets remains a challenge with inappropriate reinnervation being an
impediment to full recovery [12, 16]. Insufficient functional recovery is presumably related to
at first variable time points after nerve transection at which axonal sprouts begin to elongate
(―staggered outgrowth‖) [17] and at second to reinnervation of inappropriate pathways [18].
Therefore, supplementary therapeutic strategies are needed which result in an expedition of
regenerating axons across surgical repair sites and in an increased regeneration accuracy [15].
Two strategies have shown high potential during the last decades not only in
experimental but also first clinical studies. Low-power laser irradiation (phototherapy)
applied transcutaneously on consecutive 14 to 21 days after surgery resulted in significantly
faster functional sensory as well as motor recovery in animal models as well as few clinical
studies [19-21]. The mechanism by which phototherapy accelerates the regeneration process
is poorly understood [15]. But it has been demonstrated that phototherapy alters the
regeneration activity of the neurons which correspond to the injury in a way that the neuronal
production of neurotrophic factors is increased to support neurite outgrowth [20]. A similar
mechanism is discussed for the regeneration promoting effect of 1 hour of low-frequency (20
Hz) electrical stimulation of the proximal nerve stump (ESTIM) at the time of nerve
reconstruction. Direct ESTIM of the proximal nerve stump accelerates axonal outgrowth
across the lesion site and expedites functional motor recovery in animal models of peripheral
nerve lesion and repair as well as human patients, which underwent carpal tunnel release
surgery [22-26]. Most investigations were done in animal models using end-to-end nerve
suture or small gap repair, two studies, however, reported an acceleration of axonal and
functional recovery by applying ESTIM together with reconstruction of 10 mm rat femoral
nerve gaps with autotransplantation [27] or 15 mm rat sciatic nerve gaps with longitudinally
oriented microchannels [28]. Furthermore our own studies in the rat sciatic nerve model
clearly demonstrated that ESTIM successfully promotes functional sensory and motor
recovery after 13 mm sciatic nerve autotransplantation [29]. Evidences exist that ESTIM does
not account for better overall and long-term outcome of motor function but that it accelerates
short term recovery [22]. Faster recovery in turn reduces the time of impairment and increases
the life quality. Furthermore it provides a promising platform for investigations of other
supplementary approaches. Although the mechanisms by which the described physical
therapy approaches trigger regeneration processes are not fully understood, first clinical
studies with positive outcome have already been performed [20, 26] and furthered the
suggestion that phototherapy and electrical stimulation should find their clinical position as
innovative therapeutic approaches to support functional outcome.
Seeing the improvement in regeneration outcome when commonly used nerve
reconstructive surgery is combined with supplementary physical therapy, one should keep the
later in mind also for a combination with tissue engineered biomimetic nerve grafts (see
Fig.1) which are the main focus of this chapter. Although autologous nerve grafts are the gold
standard for nerve gap repair, the use of tissue engineered nerve grafts offers several
advantages in comparison to autotransplants. Beside the avoidance of donor site morbidity,
the repair technique is supposed to be relatively easier plus the inner diameter of the tubular
implants can be tailor-made to fit the cut nerve stumps. Nonetheless artificial conduits
Experimental Approaches on Tissue Engineering Repair … 213
provide a vehicle or container for administering trophic factors, extracellular matrix proteins,
guidance cues such as cell adhesion molecules and supporting cells which will further support
the regeneration process [30, 31].
Biomaterials for Tissue Engineered Nerve Grafts
As already mentioned above, autologous nerve tissue still provides the best properties for
bridging nerve defects and for best achievable functional restoration. Therefore, the first
choice to replace autologous nerve grafts avoiding donor nerve morbidity and immune-
incompatibility would be other autologous biological tissues [15]. Nerve conduits that have
been engineered by enriching autologous vein segments with fresh autologous skeletal muscle
fibers showed an almost similar performance as autologous nerve grafts with regard to axonal
and functional regeneration over 10 mm nerve gaps in rat [13, 32]. Furthermore, also rather
promising clinical outcome has been reported from the autologous muscle-in-vein approach
[13].
Despite investigations on biological nerve conduits, much interest has also been given to
the development of biodegradable nerve conduits which could be used for tubulization of
transected nerves without the need of tissue transplantation. Today a considerable amount of
viable synthetic or biologic nerve conduits is approved for clinical use and commercially
available [7, 9, 15, 33]. The advantages of these products are their unlimited supply which
avoids donor site morbidity. On the other hand, their use shows variable outcome and results
comparable or superior to nerve autotransplantation are restricted to the reconstruction of
short-gap, small diameter nerve injuries [7, 9]. The current state-of-the-art for production of
bioartificial nerve grafts as well as the perspectives for their clinical use have been recently
addressed in four comprehensive reviews [7, 9, 15, 33]. Both synthetic and biological
materials could be used for the construction of bioartificial nerve conduits [7, 33]. Pure
biological materials such as muscle-in-vein grafts [32] have already been mentioned above. In
the following two further aspects will be elucidated which have been outlined in the concept
of tissue engineering shown in Fig. 1. Collagen is one example of a natural polymer which is
present in the extracellular matrix of many tissues including the peripheral nerve [8, 15, 33,
34]. Earlier studies led to the development of single channel collagen conduits [35, 36] which
are, among other products, commercially available for short gap repair of small diameter
nerves. Resembling natural endothelial tubes in nerve conduits is supposed to reduce
dispersion of axonal sprouts and inappropriate target reinnervation, which should in the future
also allow long nerve gap repair. Different techniques for manufacturing of collagen-based
multichannel conduits have been recently described [37-39]. Collagen-based nerve conduits
are further suitable for biofunctional modification either by seeding of Schwann cells [40] or
incorporation or surface-linkage of various regeneration promoting neurotrophic factors [15,
34, 41-43]. Among collagen and classical neurotrophic factors also other extracellular matrix
proteins and growth factors are discussed as potential additives for tissue engineered nerve
grafts [15, 34]. Synthetic polymers are as well available for biofunctionalization and do
further allow tailor-made biodegradation [7, 33]. Aliphatic polyesters and co-polyesters have
frequently been evaluated as possible building blocks for nerve conduits [15].
Poly-caprolactone is one of the biocompatible and biodegradable candidate synthetic
Kirsten Haastert-Talini 214
polymers for which promising in vivo data have been reported [15, 44, 45]. The
electrospinning technique enables fabrication of nanofibers from poly-caprolactone
biofunctionalized with regeneration promoting factors [44, 46-48]. As outlined in Fig. 1,
nanofibers are supposed to direct axonal elongation across long nerve gaps. Using the
nanotechnique of electrospinning, 2D- and 3D-matrixes or scaffolds can be designed as
axonal guidance cues by either subdividing the lumen of a nerve conduit [48] or by providing
a microstructured nerve regeneration scaffold [46, 47], respectively. Structuring the lumen of
hollow nerve conduits was shown to increase the regeneration outcome in comparison to
single channel grafts. But the ultimate goal of creating biocompatible devices that incorporate
multiple cues and closely mimic an autograft remains to be achieved [49]. Incorporation of
different regeneration promoting cues could also be obtained by filling the nerve conduit
lumen with biomimetic 3D-hydrogels [50]. Porosity, degradation time and ingredients of
hydrogels can be chemically modified. This enables the incorporation of molecules which are
known to be important guidance cues during developmental fiber tract formation, as e.g.,
polysialic acid [51, 52]. Exogenous polysialic acid [53] or polysialic acid glycomimetics [54]
are interesting candidates with regard to support of appropriate target reinnervation, however,
more research is needed to demonstrate relevance of their presence in tissue engineered nerve
grafts.
Several additives to the lumen of biomimetic nerve conduits have been positively
evaluated to be beneficial for the regeneration outcome [12, 34, 50]. As depicted in Figure 1,
beside extracellular matrix proteins, regeneration promoting neurotrophic factors and axonal
guidance cues such as cell adhesion molecules also the transplantation of supporting cells is
an important issue [30, 31].
Transplantation of Therapeutic Cells
In response to peripheral nerve injury, Schwann cells switch their function from
myelination of electrically active axons to growth support for regenerating axons. They
dedifferentiate, proliferate, and line up to form bands of Büngner, which guide the
regenerating axons in a proximodistal direction to the denervated targets [55]. Furthermore,
they produce and accumulate growth factors, which provide a regeneration-promoting
environment [56-58], and are thus necessary for peripheral nerve regeneration. The
regeneration process is completed by the remyelination of newly formed axons [55]. Adding
Schwann cells to artificial tubular nerve grafts enables administration of a combination of
regeneration supporting factors including several neurotrophic factors, cell adhesion
molecules and basement membrane components [30]. Microstructuring luminal nerve
conduits as described above is suggested not only to provide guidance cues for regenerating
axons by itself but also to provide valid substrates for seeding of transplanted cells. Such a
cell transplantation approach would in turn mimic the presence of Schwann cell tubes (bands
of Büngner) for axon guidance [59-61].
Because the success of nerve autotransplantation is mainly attributed to the presence of
Schwann cells as well [2, 8, 49], those are the most obvious cellular tools for transplantation
within tissue engineered nerve conduits. Adult Schwann cells from different species like
rodents, dog, primates and man have been employed for transplantation purposes and various
Experimental Approaches on Tissue Engineering Repair … 215
protocols exist for their preparation for experimental transplantation purposes [62-71]. The
transplantation of Schwann cells within tissue engineered nerve grafts evidently leads to
better axonal regeneration. Although several protocols have been developed to make adult
human Schwann cells available for their clinical use [62, 64, 66, 72], it still remains an open
question whether autologous Schwann cells will be widely used for tissue engineering of
nerve conduits.
Therefore studies have been initiated to elucidate the potential of alternative, maybe more
easy to obtain, cell sources. Olfactory ensheathing cells, glial cells form the olfactory bulb,
have almost similar in vitro properties as Schwann cells as shown with cells from canine
origin [73]. Further more olfactory ensheathing cells showed regeneration promoting
properties in animal models of peripheral nerve repair which have been recently reviewed
[74].
Beside the described glia cells, stem cells have gained more and more attention as
cellular components in tissue engineered nerve grafts. Mesenchymal stem cells derived from
bone-marrow, skin or adipose tissue can be transdifferentiated in vitro into
Schwann cell-like cells [75-83]. These cells are more easily available from autologous
sources as primary autologous Schwann cells and increasing evidence demonstrates their
safety and efficiency to promote peripheral nerve regeneration [77, 79-81].
Not only naïve Schwann cells or Schwann cell-like cells are interesting cellular tools for
peripheral nerve tissue engineering purposes. These cells could be also subjected to genetic
modification to increase their regeneration promoting potential.
Gene Therapy
Several neurotrophic factors and cell adhesion molecules have been well characterized to
promote peripheral nerve regeneration when present at the lesion site or within the nerve graft
[31, 57, 58, 84-86]. Administration of recombinant proteins is generally impaired by short
serum half-life; therefore, gene therapy as an alternative tool could lead to continuous
endogenous expression of the respective gene products. Basically, gene transfer can be
subdivided into ex vivo and in vivo approaches [87-89]. Ex vivo gene transfer includes cell
harvest, genetic modification and re-transplantation of cells expressing the target
gene product. For in vivo gene transfer genetic material is directly delivered to
peripheral nerve cells or muscle cells aiming in survival promoting activities of the delivered
genes on the regenerating neurons as well as in growth promoting activities of the proteins on
axonal regeneration. Non-viral and viral methods are used to insert genetic material into
living cells. Non-virally the plasmid DNA is inserted by transfection which allows DNA
uptake through transient pores in the cell membrane. Viral vectors, modified viruses without
the ability to replicate but able to insert foreign genetic material into target cells, are used for
transduction of living cells [88, 89].
Our own group established protocols and performed basic in vivo experiments to enable
transplantation of in vitro genetically modified adult autologous Schwann cells from several
species (rat, dog and man) [67, 90-93]. In a rat model of peripheral nerve regeneration
(15 mm gap, adult sciatic nerve) we demonstrated that transplantation of genetically modified
Schwann cells over-expressing fibroblast growth factor-2 (FGF-2) within silicone tubes
Kirsten Haastert-Talini 216
results in a high rate of tissue regeneration and in long distance myelination of regenerated
axons. This was especially seen after transplantation of Schwann cells, which over-expressed
high molecular weight FGF-2, FGF-221/23kD
. However, recovery of motor function [90, 91] as
well as recovery after facial nerve repair [94] was less successful than expected.
Over-expression of glia derived neurotrophic factor (GDNF) [89, 95-98] is another example
for neurotrophic factor delivery by genetically modified transplanted Schwann cells, which
demonstrated increased functional and morphological regeneration [89, 98]. Regeneration
across peripheral nerve gaps has also been found to be increased by transduction of Schwann
cells to express sialyl-transferase-X (STX), an enzyme which is known to transform the
neural cell adhesion molecule N-CAM into its polysialylated form. Upon polysialylation of
NCAM a wide variety of contact-dependent cell interactions is down-regulated. After
establishing stable contacts nerves lose expression of polysialic acid and the molecule
reappears after nerve injury accounting for selective reinnervation of motor targets [99].
Transplanting genetically modified STX-expressing Schwann cells in an animal model for
peripheral nerve repair did increase axonal and functional regeneration [100].
In vivo gene therapy approaches enable local elevation of gene products such as again
neurotrophic factors. Direct injection of viral-vectors encoding for neurotrophic factors into
the injured peripheral nerve resulted in the transduction of local Schwann cells and fibroblasts
which delivered the induced regeneration promoting proteins to the axons and neurons [88,
98]. Very recently it has been shown that nerve growth factor (NGF) gene therapy of the
transected and resutured rat femoral nerve increase appropriate reinnervation of sensory
targets when applied at the deviation point between the sensory and the muscle branch of the
nerve [101].
Gene therapy therefore enables injury specific support of neuronal survival, axonal
regeneration and appropriate re-establishment of functional circuits. Due to this fact, not only
the introduction of genes directly into the neuronal or glial target cells with the aim to support
their survival and functionality but also the transplantation of genetically modified supportive
cells have reached the level of clinical research [98, 102].
Conclusion
Several attempts resulting in improvement of the regeneration outcome after peripheral
nerve reconstruction have been described in this chapter. However, all recent results
demonstrate that only a combination of different promising approaches will lead to nerve
grafts that are as qualified as nerve autotransplants or even more qualified to provide optimal
conditions for structural and functional peripheral nerve regeneration [34]. Tissue engineering
of peripheral nerve grafts has to combine innovations from the field of material science,
delivery of regeneration promoting substances from the materials and optimized cellular
composition. Also supplementary physical therapy like phototherapy or electrical stimulation
has to be considered.
Future research will hopefully result in the development of tubular transplants which
allow regenerating nerve fibers an appropriate reconnection to their distal targets.
Furthermore, the biodegradable biomaterial used should be functionalized with regeneration
promoting substances or should deliver those during the degradation process. Additional
Experimental Approaches on Tissue Engineering Repair … 217
seeding of genetically modified cells into the nerve grafts or direct gene transfer will enable
gene therapeutic support of peripheral nerve regeneration. Keeping eye on the ultimate goal
of replacing nerve autografts, innovative nerve guidance channels will not only have to be
guiding and expediting regrowing nerve fibers but also enhancing their accuracy to
reinnervate appropriate targets and synchronizing the regeneration processes by a
temporospatially organized delivery of regeneration promoting substances.
Acknowledgment
I wish to thank all my co-workers from different institutions as well as all colleagues,
doctoral students and technicians from the Institute of Neuroanatomy and its director, Prof.
Dr. Claudia Grothe, for an inspiring working atmosphere. Our own work was financially
supported by the German Research Foundation (DFG), the International Foundation
Neurobionic, the Kogge-Foundation for Veterinary Science, the Foundation for Neurosurgical
Research (German Society of Neurosurgery, DGNC) and the Hannover Medical School
(HilF).
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