nerve repait tissue engineering

7/30/2019 Nerve Repait Tissue Engineering

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BIOMEDICAL ENGINEERING-

APPLICATIONS, BASIS & COMMUNICATIONS

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1. INTRODUCTION

Five per cent of all open wounds in theextremities, due to i.e. sports and road accidents arecomplicated by peripheral nerve trauma. Also duringcomplicated births, peripheral nerves may be disruptedby traction [1], the mean incidence of these lesions

being 0.12% of all births [2]. Each year approximately10,000 Americans sustain spinal cord injuries (SCI).Functional deficits following SCI result from damageto or severance of axons, loss of neurons and glia, anddemyelination. SCI pathology is determined not only

ABSTRACT

Nerve regeneration is a complex biological phenomenon. Once the nervous system is impaired,its recovery is difficult and malfunctions in other parts of the body may occur because matureneurons don't undergo cell division. To increase the prospects of axonal regeneration and functionalrecovery, researches have focused on designing "nerve guidance channels" or "nerve conduits". Fordeveloping tissue engineered nerve conduits, four components come to mind, including a scaffold foraxonal proliferation, supporting cells such as Schwann cells, growth factors, and extracelluar matrix.This article reviews the nervous system physiology, the factors that are critical for nerve repair, andthe advanced technologies that are explored to fabricate nerve conduits. Furthermore, we alsointroduce a new method we developed to create longitudinally oriented channels withinbiodegradable polymers, Chitosan and PLGA, using a combined lyophilizing and wire-heatingprocess. This innovative method using Ni-Cr wires as mandrels to create nerve guidance channels.The process is easy, straightforward, highly reproducible, and could easily be tailored to otherpolymer and solvent systems. These scaffolds could be useful for guided regeneration aftertransection injury in either the peripheral nerve or spinal cord.

Biomed Eng Appl Basis Comm, 2006(June); 18: 100-110.Keywords: nerve regeneration; nerve guidance channels; nerve conduits; lyophilizing; wire-heatingprocess

Received: May 3, 2005; Accepted: April 3, 2006Correspondence: Yi-You Huang, ProfessorInstitute of Biomedical Engineering, National TaiwanUniversity, No. 1, Section 1, Jen-Ai Road, Taipei 100,TaiwanE-mail: [email protected]

TISSUE ENGINEERING FOR NERVE REPAIR

YI-CHENG HUANG, YI-YOU HUANG

Institute of Biomedical Engineering, College of Medicine and Engineering,

National Taiwan University, Taipei, Taiwan

by the initial mechanical insult, but also by secondaryprocesses including ischemia, anoxia, free-radicalformation, and excitotoxicity that occur over hours anddays following injury [3]. Over the recent years,knowledge of the factors influencing a nerve

reconstruction has increased, and also new surgicaltechniques and equipment have been developed. Still,functional outcome of peripheral nerve trauma andspinal cord injuries are often disappointing and thisurges to optimise therapeutical intervention. Fortargeting, the unique challenges to bioengineeringresearch addressing nerve injuries are the physiologyof the nervous system.


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2. PHYSIOLOGY OF THE NERVOUSSYSTEM

The nervous system consists of two parts,peripheral nervous system (PNS) and central nervoussystem (CNS) that differ in their physiology andfunction. The PNS consists of sensory neurons runningfrom stimulus receptors that inform the CNS of thestimuli and motor neurons running from the CNS tothe muscles and glands, called effectors that takeaction. (Fig. 1) The CNS is made up of spinal cord andbrain. The CNS is surrounded by bone-skull andvertebrate. Fluid and tissue also insulate the brain andspinal cord.

Nervous tissue is composed of two main cel ltypes: neurons and glial cells. Neurons transmit nervemessages. Gial are in direct contact with neurons andoften surround them. Neurons are the basic structuraland functional elements of the nervous system andconsist of a cell body (soma) and its extensions (axons

and dendrites). Glial cells, or neuroglia, are supportcells that aid the function of neurons and includeSchwann cells in the PNS and astrocytes andoligodendrocytes in the CNS. Glial cells are moreabundant than neurons, and unlike neurons whichcannot undergo mitosis, glial cells have some capacityfor cell division. Although neurons cannot divide bymitosis, they can regenerate a severed portion or sproutnew processes under certain conditions.

A peripheral nerve consists of motor and sensoryaxons bundled together by support tissue into ananatomically defined trunk. A peripheral neuronconsists of a cell body and a long process, or axon,which may reach one meter in length. (Fig.2) Short

segments of the axon are wrapped with an insulatingmyelin sheath formed by Schwann cells, which alsoserve several important roles in the axon-regenerationprocess. Axons are grouped together into fascicles,several of which are enclosed in the epineurium toform a peripheral nerve.

For CNS, both the spinal cord and the brainconsist of white matter and gray matter. White matteris bundles of axons, each coated with a sheath ofmyelin. Gray matter is masses of the cell bodies anddendrites, each covered with synapses. In the spinalcord, the white matter is at the surface, the gray matter

Sensory neurons Sensory neurons

Internal Central External

Environment nervous Environment

System

Motor neurons Motor neurons

Fig. 1. The nervous system.

Fig. 2. Peripheral motor neuron (a) the cell body (b)

a cross section of the neuron. Figure adapted from

reference 108.

Fig. 3. (a) Diagrammatic transverse section of

spinal cord with ventral and dorsal roots, showing

the attachment zones (AZ) of their rootlets, together

with the extents of both dorsal and ventral rootlet

transitional zones (TZ). The former TZ contains a

central tissue projection (CTP). (b, c) Enlargement

of areas outlined in (a) showing the thickened glia

limitans (arrows) at the TZ. (d) Schematic diagram

of ventral rootlet TZ showing an individual

myelinated fibre (arrowhead) and a bundle of

nonmyelinated axons (asterisk) traversing the

thickened glia limitans (arrow) in individual

tunnels. Figure adapted from reference 107.

inside. In the brain of mammals, this pattern isreversed. Axons project from the white matter inbundles, exiting the encasing bone of the spinal cord,travel through the PNS-CNS transition zone, and enterthe PNS (Fig. 3). The transition zone is clearly definedregion where the glial cells in the CNS are separated

from those in the PNS.


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highly differing success rates [11-12]. Many differentmethods [13-14], including thermal techniques [15],radiations [16-17], and chemical processes, have beenused to remove or destruct the immunogenic cells andpreserve the ECM components that are essentiallyconserved between species.

Some natural-based materials have alsoinvestigated for nerve repair, including ECMmolecules (i.e. laminin, collage, and fibronectin) [18-19], hyaluronic [20], fibrinogen [21], fibrin gels [22],self-assembling peptide scaffolds [23], alginate [24],agarose [25-26], and chitosan [27]. Current studies areto further modify these materials to enhance neuriteextension. [28-31]

4.2. Artificial Nerve Grafts

Avoiding the problems of availability and immunerejection, a promising alternative for extending thelength over which nerve can successfully regenerate isthe artificial nerve graft. The materials used must bemodified to be more cell friendly . Variations inthe chemical and physical properties of artificialmaterial are attractive, allowing alterations in thegeometric configuration, biocompatibility, porosity,degradation, electrical conductivity, and mechanicalstrength. The variation in the above parameters can

3. CHALLENGES FOR NERVEREPAIR

The key difference between PNS and CNS afterinjuries is shown in Fig. 4. In PNS, the proximalsegment may be able to regenerate and reestablishnerve function. Proliferating Schwann cells,macrophages, and monocytes work together to removemyelin debris, release neruotrophins, and lead axonstoward their synaptic targets, resulting in restoredneuronal function. To improve recovery, severednerves can be surgically sutured end-to-end over smallgaps. However, end-to-end suturing is only effective ifthe nerve ends directly adjacent and can be connectedwithout causing tension. Special attentions should begiven to the alignment of the fascicles.[4] If, however,

the gap between the nerve stumps is too large, a deviceis needed to bridge the gap in order to guide theoutgrowing nerve fibers and to prevent the formationof neuroma. Thus, bioengineering strategies has focuson the developing alternative treatments to the nervegraft, especially for larger defects, and improvingrecovery rates and functional outcome.

In CNS, an impermeable glial scar composed ofmyelin, cellular debris, astrocytes, oligodendrocytes,and microglia formed. Regeneration neurons areblocked fro m rea ch ing thei r syna ptic target .Researches have shown that both embryonic spinalcord grafts and peripheral nerve tissue grafts can

support regeneration fibers in the CNS, but growingback across the PNS-CNS transition zone is still adifficult. [5-6] In addition, some other factors i.e.Nerve growth factor(NGF), Laminin, Schwann cell andso on have been focused to improve nerve recovery.

In brief, the challenge for nerve repair is designingnerve guides or nerve guidance channels to

direct axons sprouting from the proximal nerve end,provide a conduit for the diffusion of growth factorssecreted by the injured nerve ends, and reduce theinfiltration of scar tissue.

4. NERVE GRAFTS

4.1. Nature Nerve Grafts

To increase the prospects of axonal regenerationand functional recovery, numerous strategies have beenused. These have included implantation of autografts,allografts, and xenografts [7-8]. The best choice is theautograft, a segment of nerves removed from anotherbody. This has inevitable disadvantages, limited supplyof available nerve graft material and permanent loss ofthe donor nerve function [9-10]. Allografts andxenografts have also been used, but are accompaniedby the need for immunosuppressive therapy and have

Fig. 4. Nerve regeneration process after injuries. (a)

in peripheral nervous system (b) in central nervous

system. Figure adapted from reference 109.


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A variety of resorbable artificial nerve grafts havebeen used for aiding nerve regeneration. (Table )Polyesters, such as PGA, PLA, PLGA, and PHB were

some of the first synthetic polymers studied because oftheir availability, ease of processing, biodegradationcharacters and approval by the FDA. Moreover, betterresults have been obtained by using these materials.Other materials in table have also shown thecapacities for guiding regeneration.

4.3. Advanced Fabrication Techniques

In order to accurately mimic natural repair in thebody, various fabrication techniques have been used tocreating three-dimensional channels and unique poresor fiber structures. The guidance channels arefabricated in a number of ways, including magneticpolymer fiber alignment [56-57], injection molding[62-63], phase separation [64], solid free-formfabrication [65-66], ink-jet liquid polymer printing[67], micropatterning [68-71], lyophilizing and wire-heating process [72].

Ner ve gui da nce chann el s hav e bee npredominantly fabricated as hollow tubes or as porousfoam rods because of the ease in manufacturing thesedevices. According to all of the research, incorporationof an oriented intraluminal framework into the channellumen, which qualitatively models biologicalautogeneous materials, has been a significant

dramatically alter the ability of axons to proliferate.The properties of the ideal nerve guidance channelhave shown in Fig. 5, including a biodegradable and

porous channel wall, the ability to deliver bioactivefactors, the incorporation of support cells, an internaloriented matrix to support cell migration, intraluminalchannels to mimic the structure of nerve fascicles, andelectrical activities.

Fig. 5. Properties of the ideal nerve conduits. Figure

adapted from reference 32.

Material Animal Gap length (mm) Refs

Polyglycolic acid (PGA) Monkey 1-1.5 33

Monkey 30 34

Rat 5 35, 36

Human 5-30 37

Rabbit 30 38

Polylactic acid (PLA) Mouse 4-5 39

polyglactin Rabbit 7-9, 10 40, 41

Poly(organo)phosphazine Rat 10 42

polyorthoester Cat 15 43

Glycolide trimethylene carbonate Rat 5 44Polyurethane Rat 8 45

Methacrylate-based hydergels Rat 10 46

Poly-3-hydroxybutyrate (PHB) Rabbit 2-4 cm 47

Poly(lactic-co-glycolic acid) (PLGA) Rat 10, 12 48, 49

Poly(l-lactide-co-caprolactone) Rat 10 50-52

Polyglycolic-acid mesh coated with collagen Cat 25 53

Poly(glycosaminoglycan-co-collagen) Rat 10,15 54

Poly(l-lactide-co- -caprolactone)(inner) Rat 7 55

a mixture of polyurethane and

polylactic acid (outer)

Table . Bioresorbable materials used in peripheral-nerve grafts


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advancement. These channels have demonstratedsuperior regeneration because the intraluminal matrixsupports cell attachment/migration and guides

regenerating axons. A variety of matrix strategies havebeen invest igated , such as fi lament s of : al ignedcollagen [56-58], carbon [59], poly (l-lactide) [60] orpolyamide [61]. However, the arrangement of thefilaments within the guidance channels is irregular anddifficult to reproduce. Magnetic fields have been usedto orient polymers for matrix alignment and alsoimprove upon processing conditions [56-57]. In adifferent approach, a novel foam-processing technique,utilizing low-pressure injection molding, createdhighly porous conduits from poly (lactic-co-glycolicacid) with continuous longitudinal channels [62]. 1, 5,16, 45 or more longitudinally aligned channels have

constructed within the lumen of channels. In similarstudies, poly (lactic-co-glycolic acid) was subjected toinjection molding followed by a thermally inducedphase transition process to produce conduits withlongitudinally aligned internal channels [63]. As partof a separate study from this same laboratory, a two-phase conduit was fabricated for spinal cord repair[64]. We have also devised a new approach: thelyophilizing and wire-heating process [72], to form ascaffold with microtubular architecture to provide alarger surface area for extending axons in order toimprove and enhance axonal elongation. This strategypromises to be more effective and reproducible.

Solid freeform fabrication and ink-jet liquid

polymer printing have even more opportunities for thedesign of intricate devices for peripheral nerve andspinal cord repair [65-67]. Recently, a novel fabricatedtechnique, micropatterning, is used to guide nerveregeneration. Micropatterning is a common techniqueused to pat te rn g lass sur faces with ink of alkylsilanes [68-71] as well as peptides [73-75] andpr ot eins [76- 79 ] to cont ro l ce llu lar at tachmen t.Micropatterned polymer substrates on which lamininadhered have used to control alignment of Schwanncells to direct neuronal regeneration in those researches[75, 80-81]. It means that not only physical guidance:micropatterned substrates, but also chemical cues:laminin and supporting cells: schwann cells areimportant factors for nerve repair.

5. LYOPHILIZING AND WIRE-HEATING PROCESS

We have developed a method to createlongitudinally oriented channels within biodegradablepolymers using a combined lyophilizing and wire-heating process for nerve tissue regeneration. Theprimary goal of this investigation was to design andcharacterize a scaffold with oriented, longitudinal

channels that would provide increased surface areawithin a tubular structure for use in neural tissueengineering applications. Such a scaffold could support

and guide extending axons subsequent to nerve injuryin a cell-adhesive scaffold. To develop a safe, effectiveand reproducible method for scaffold formation, weused Ni-Cr wires as mandrels to create longitudinaloriented channels in scaffold. The scaffolds were madeof the biodegradable polymers, Chitosan and Poly (d,l-lactide-co-glycolide) (PLGA), because of theiravailability, ease of process, proved by FDA, and lowinflammatory response. In addition to biodegradabilityand biocompatibility issues, the degree of permeabilityof the nerve guide may also influence nerveregeneration. The size of the tube wall porous and itsstability over time seem to be important factors to

determine the flow of different constituents that maypromote or inhibit regenerat ion. According to ourexperiments, the high permeability and porousstructure of Chitosan make it a better material of nerveguide than PLGA.

Experimental results showed that the scaffolds ofporous po lymer nerve condui ts manufactured bylyophilizing and wire-heating process werehomogeneous and well-defined. The scanning electronmicrographs (Fig.6) revealed that the scaffolds wereconsistent along the longitudinal axis, with thechannels distributed throughout the scaffolds. Therewas no evidence to suggest merging or splitting ofindividual channels. The diameter of the channel is

about 100um, almost the same as the Ni-Cr wire(115um).

Regulating the size and quantity of the Ni-Crwires allow us to control the number and diameter ofthe channels. Furthermore, the pattern of the scaffolddepends on the arrangement of the wires. Comparedwith previous studies, the fabrication process isstraightforward and the resultant scaffolds are highlyreproducible.

The neutralizing processes have significantlyinfluenced the porous structure of biopolymers. The

Fig. 6. Scaffold with longitudinal oriented channels.


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porous structure is more uniform by using weak acid(NaHCO3 0.1M). (Fig. 7) Using strong acid (NaOH0.1M) as neutralizing agent makes the pores variable,

so it 's difficult to control the pore size. Thereproducibility of the scaffold isn't as well.

6. OTHER COMPONENTS FORNERVE REGENERATION

6.1. Support Cells

Although the exact underlying mechanismsregulating dynamic axon/schwann cell apposition areunknown, experiments support the concept thatSchwann cells offer a highly preferred substrate foraxon migration and release bioactive factors thatfurther enhance nerve migration [82-84]. Schwanncells produce structural and adhesive extracellular

matrix (ECM) molecules such as laminin and collagen,and express many cell adhesion molecules andreceptors, including L1, N-cadherin, gamma1integrins, and the neural cell adhesion molecule (N-CAM). Moreover, Schwann cells synthesize andsecrete a cocktail of neurotrophic molecules such asnerve growth factor (NGF), brain derived neurotrophicfactor (BDNF), and ciliary neurotrophic factor(CNTF). The substances Schwann cells secret enhancenerve regeneration; they form an endoneurial sheath,

which serves as a guide for axonal growth; they aid inclearing debris and create a suitable environment fornerve growth; they myelinate axons [85]. Experiments

have shown that Schwann cells stimulate axons toelongate faster and over long distance than is possibleon acellular matrices [86]. Schwann cells have playeda critical role in leading peripheral axons to the distalnerve stump and in synapse formation [87]. The abilityof Schwann cells to promote nerve regeneration hasbee n a re sea rch ar ea of inten se fo cu s [5 7, 88].However, there are still several challenges facingSchwann cell therapies for spinal cord repair [89-91].

Recently, a new cell type known as stem cell ornerve progenitor cell has been isolated from variousregions of the adult or embryonic CNS of mice, rat andhuman for nerve regeneration application [92-93].

These cells are unique in that they are multipotent, i.e.,they can give rise to the three cell lineages of the CNS:neurons, astrocytes and oligodendrocytes. The inherentplasticity of neuron stem cells has generated interest inwhether there cells can be used to replace cells in themammalian CNS. Indeed, some studies have alsofound that stem cells implanted into injured spinal corddifferentiate into neuroms and glial cells [94-95].

6.2. Growth Factors

As described in the earlier section, neurotrophicfactors are important in neural generation. Theinfluence of these factors in neural development,

survival, outgrowth, and branching has been exploredin various levels, from molecular interactions tomacroscopic tissue responses [88]. No matter wherethe injuries happened, PNS or CNS, neurotrophicfactors can promote neural response. There are variouscompounds of neurotrophic factors, and one of them isneurotrophins, including nerve growth factor (NGF),bra in- de rive d ne uro tro phi c fa ctor (B DN F) ,neurotrophin-3 (NT-3), and neutotrophin-4/5 (N/T-4/5).Moreover, cytokines and insulin derivatives, ciliary

Fig. 7. Chitosan scaffold were neutralized by (a,

b)NaHCO3 0.1M (c) NaOH 0.1M.

a b c

Neural response promoted Neurotrophic factors

Motor neuron survival BDNF, NT-3, NT-4/5, CNTF, GDNF

Motor neuron outgrowth BDNF, NT-3, NT-4/5, CNTF, GDNF

Sensory neuron survival NGF, NT-4/5, GDNF

Sensory neuron outgrowth NGF, BDNF, NT-3

Spinal cord regeneration NGF, NT-3, CNTF, FGFs

Peripheral nerve regeneration NGF, NT-3, NT-4/5, CNTF, GDNF, FGFs

Sensory nerve growth across the NGF, NT-3, GDNF, FGFs

PNS-CNS transition zone

Table Neural responses to neurotrophic factors


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neurotrophic factor (CNTF), glial cell line-derivedgrowth factor (GDNF), and acidic and basic fibroblastgrowth factor (aFGF, bFGF) are also neurotrophic

factors of importance [96-99] (Table ). However, invivo response can vary due to the method of deliveringthe growth factors. A new search field has focused onthe delivery method [100].

Controlled release is one means of supplyingfactors to enhance nerve generation. Bioresorbablegrafts can be made to release growth or trophic factorstrapped in or adsorbed to the polymer as they degrade[116]. With controlled release, compounds with shortin vivo half-lives can be supplied slowly to theregeneration axons over the life of the graft. There areseveral delivery devices use in neural application,including polymer matrices [101], microspheres [102-

103], viral vectors [104-105] and liposome [106].Nevertheless, providing the necessary quantities andtypes of neurotrophic compounds at the rates mostconducive to regeneration is still surely a challenge.

6.3. Extracellular Matrix

Insoluble extracellular matrix molecules such aslaminin, fibronectin, and some forms of collagen,promote axonal extension and, therefore, are excellentcandidates for incorporation into the lumen ofguidance channels. Placing these molecules into thelumen of the nerve grafts by physical coating, plasmatreating, chemical bonding through adhesion mediators

[110-112], and some other similar processes is aresearch area of intense focus. As described earlier, its an important chemical cues deeply affecting theprocess of neural regeneration. Variable results havebeen demons trated by the incorporat ion of thesemolecules within channels, however, it appears that byreducing the concentration of protein gel or shortingthe oligopeptide sequence, axonal proliferation can bepromoted. [113-115]

7. CONCLUSION

In order to successful regeneration of nerve tissue,nerve conduit was introduced to guide axonaloutgrowth between damaged nerve ends. However,employment of simple physical conduits is notsufficient to reconnect damaged tissue across a criticalsize defect. Successful nerve regeneration requirestissue-engineered scaffolds that provide not onlymechanical support for growing neurites andprevention from ingrowth of fibrous scar tissue, butalso biological signals to direct the axonal growth coneto the distal stump. Various fabrication techniques havebeen used to develop tissue engineered nerve conduits.

We herein manufacture porous polymer nerveconduits by lyophilizing and wire-heating process.Using Ni-Cr wires as mandrels to create longitudinal

oriented channels is an innovative method to fabricatenerve guides. The process is easy, straightforward andthe resultant scaffold is highly reproducible.Furthermore, this technique is versatile and can easilybe tailored to other polymer and solvent systems tomanufacture nerve conduits or other microporoustubular system.

However, the functional recovery of nerveregeneration is still complex, and we are still hinderedby a lack of knowledge of the underlying mechanismfor axonal proliferation. The combined efforts ofscientists and engineers from a variety of disciplines,experimental work in this field has made great

progress. The understanding of the related areas inbiomaterials, drug delivery, and cell culture is on theincrease. It is able to apply the therapies more efficient.Regardless of the challenges happened, the repair ofperipheral and central nerves will continue to improvebecause of the contributions of many workers in thisfield.

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