in: the sciatic nerve: blocks, injuries and regeneration ... · in: the sciatic nerve: blocks,...

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withdrawn from a healthy sensory nerve during an extra surgical incision that commonly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


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


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


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


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


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


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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