A Biomaterials Approach to Peripheral Nerve Regeneration

7/28/2019 A Biomaterials Approach to Peripheral Nerve Regeneration

1/21

published online 16 November 2011J. R. Soc. Interface

W. Daly, L. Yao, D. Zeugolis, A. Windebank and A. Pandit

recoverybridging the peripheral nerve gap and enhancing functionalA biomaterials approach to peripheral nerve regeneration:

References

ref-list-1

http://rsif.royalsocietypublishing.org/content/early/2011/11/16/rsif.2011.0438.full.html# This article cites 121 articles, 8 of which can be accessed free

P


2/21

REVIEW

A biomaterials approach to peripheralnerve regeneration: bridging theperipheral nerve gap and enhancing

functional recoveryW. Daly 1, L. Yao 1, D. Zeugolis 1, A. Windebank 2 and A. Pandit 1, *

1 Network of Excellence for Functional Biomaterials (NFB ), National University of Ireland,Newcastle Road, Dangan, Galway, Republic of Ireland

2 Mayo Clinic, Rochester, MA, USA

Microsurgical techniques for the treatment of large peripheral nerve injuries (such as the goldstandard autograft) and its main clinically approved alternativehollow nerve guidance con-duits (NGCs)have a number of limitations that need to be addressed. NGCs, in particular,are limited to treating a relatively short nerve gap (4 cm in length) and are often associatedwith poor functional recovery. Recent advances in biomaterials and tissue engineeringapproaches are seeking to overcome the limitations associated with these treatment methods.This review critically discusses the advances in biomaterial-based NGCs, their limitationsand where future improvements may be required. Recent developments include the incorpor-ation of topographical guidance features and / or intraluminal structures, which attempt toguide Schwann cell (SC) migration and axonal regrowth towards their distal targets. The useof such strategies requires consideration of the size and distribution of these topographicalfeatures, as well as a suitable surface for cellmaterial interactions. Likewise, cellular andmolecular-based therapies are being considered for the creation of a more conductive nervemicroenvironment. For example, hurdles associated with the short half-lives and low stabilityof molecular therapies are being surmounted through the use of controlled delivery systems.Similarly, cells (SCs, stem cells and genetically modied cells) are being delivered with bioma-terial matrices in attempts to control their dispersion and to facilitate their incorporation withinthe host regeneration process. Despite recent advances in peripheral nerve repair, there are anumber of key factors that need to be considered in order for these new technologies to reachthe clinic.

Keywords: peripheral nerve conduit; topographical guidance; molecular therapy;Schwann cells; stem cells; neurotrophic factors

1. INTRODUCTION: PERIPHERAL NERVEINJURY AND REPAIR

Peripheral nerve injury is a large-scale problemannually affecting more than one million people world-wide. These injuries often result in painful neuropathiesowing to reduction in motor function and sensory per-ception. Peripheral nerve injuries are common in bothcivil and military environments and are primarily theresult of transection injuries or burns, but may alsoarise from degenerative conditions [ 1,2]. Over relativelyshort nerve gaps, spontaneous natural regeneration mayoccur. However, over larger gaps, microsurgical repair isessential for nerve repair [ 35].

Currently, there are a variety of microsurgical repairmethods available, including direct repair, autograft /allograft transplantation and the use of hollow nerve gui-dance conduit (NGC) repair [ 35]. Direct nerve repair(also known as end-to-end suturing, endend repair,end-to-end neurorrhaphy or end-to-end coaptation) is thepreferred method of treatment for peripheral nerve repair[6]. This method of treatment, however, is limited to thetreatment of short nerve defects requiring tension-freesuturing of the injury site [ 6]. For optimal regeneration,the nerve stumps must be correctly aligned and repairedwith minimal tissue damage, using the minimal number

of sutures. This repair method is limited to nerve gapsshorter than 5 mm [ 7]. Beyond this relatively short gap,injuries areprecluded from primary repair, and alternativetissue engineering strategies are the main option.*Author for correspondence ( [email protected] ).

J. R. Soc. Interface doi:10.1098/ rsif.2011.0438

Published online

Received 5 July 2011Accepted 28 October 2011 1 This journal is q 2011 The Royal Society

on March 11, 2013rsif.royalsocietypublishing.orgDownloaded from
mailto:[email protected]://rsif.royalsocietypublishing.org/http://rsif.royalsocietypublishing.org/mailto:[email protected]


3/21

1.1. Autograft: the limited gold standard

For patients precluded from direct repair, autograft isthe current gold standard and has remained so for thelast 50 years [79]. These grafts being taken primarilyfrom the sural nerve of the treated patient and havedemonstrated a success rate of only 50 per cent onpatients treated [ 6,10]. These grafts are primarily sen-sory, owing to the unavailability of motor nerves,limiting their potential to repair pure motor nerve def-

icits (tibial) and mixed nerve injuries (sciatic) and maybe one of the primary reasons for the poor functionalrecovery rates associated with autografts [ 11,12].The use of sensory nerves for the treatment of motornerve decits causes morphometric mismatches inthe native environments, mismatch in axonal size,distribution and alignment [ 12,13]. Motor neurons areprimarily in the range of 3 20 mm, whereas sensory neur-ons range from 0.2 to 15 mm [14]. If sensory nerve graftsare therefore used to treat a pure motor nerve injury,there is a great potential for size mismatch, potentiallylimiting regeneration. Secondary to this limitation, theuseof autograft hasa numberof disadvantages, including

donor site morbidity, the requirement for a second surgi-cal site, a very limited supply, donor site mismatch andthe possibility of painful neuroma formation and scarring[15]. The use of autograft also requires secondary removalof degenerated axons and myelin by the host from thegraft itself, increasing the healing time [ 16]. Similarly inrecent studies, it has been shown that sensory andmotor neurons have different Schwann cell (SC) modal-ities and if placed in the incorrect microenvironment,may limit their regenerative ability [ 17].

Autograft use is currently limited to a critical nervegap of approximately 5 cm in length and beyondthis distance requires the use of allograft [ 2]. Allograft

however requires the use of extensive immune sup-pression up to 18 months post implantation, andpatients become susceptible to opportunistic infections,occasionally resulting in tumour formation [ 18]. The

combinatorial effects of these limitations may be theprimary cause for the limited recovery associated withautograft and allograft treatment. In efforts to addressthe limitations of these nerve grafting techniques, theprimary alternative is the use of hollow NGCs.

1.2. The development of nerve guidance conduits

The use of hollow NGCs was originally proposed for use

for nerve repair as early as 1881 with the rst successfulapplication occurring in 1882, where a hollow bone tubewas used to bridge a 30 mm nerve gap in a dog [ 19].Today, the use of hollow NGCs is the clinicallyapproved alternative to autograft repair. These con-duits have a number of advantages for nerve repair,including limited myobroblast inltration, reducedneuroma and scar formation, reduction in collateralsprouting and no associated donor site morbidity, andfacilitates the accumulation of a high concentration of neurotrophic factors; ultimately guiding regeneratingnerves to their distal targets [ 20]. However, the use of hollow NGCs is currently limited to a critical nerve

gap of approximately 4 cm [ 21]. These NGCs allowthe creation of a controlled microenvironment for theregeneration of nerve bres and have shown some clini-cal success [22,23]. Current clinically translated NGCSare primarily made from synthetic materials such aspoly-glycolic acid (PGA), polylactide-caprolactone(PLCL), various combinations of the PGA or PLCL,or from animal extracted collagen ( table 1 ) [6,22,23].

Despite some success in nerve repair, these hollowNGCs fail to match the regenerative levels of autograftand show poor functional recovery [ 24]. Early attemptsof improvements for NGCs involved variations inmaterial design and fullling a number of criteria for

the ideal hollow conduit. These criteria included:(i) limiting scar inltration, while allowing diffusion of nutrients into the conduit and wastes to exit theconduit; (ii) providing sufcient mechanical properties

Table 1. Current clinically approved and upcoming nerve guidance conduits.

product company composition degradation time max length

Neurogen Integra Neurosciences,Plainsboru, NJ, USA

collagen type I 4 years 3 cm

NeuraWrap Integra Neurosciences,Plainsboru, NJ, USA

collagen type I 4 years 4 cm

Neuromend Collagen Matrix, Inc.,Franklin Lakes, NJ, USA collagen type I 48 months 2.5 cm

Neuromatrix / Neuroex Collagen Matrix, Inc.,Franklin Lakes, NJ, USA

collagen type I 48 months 2.5 cm

Neurotube Synovis Micro CompaniesAlliance, Birmingham, AL,USA

woven polyglycolic acid(PGA)

6 12 months 3 cm

Neurolac Polyganics Inc., TheNetherlands

poly(DL-lactic-co-1-caprolactone) (PLCL)

23 years 3 cm

Salubridge / Hydrosheathor Salutunnel

Salumedica LLC, Atlanta,GA, USA

Salubriapolyvinyl alcohol(PVA) hydrogel

non-biodegradable 6.35 cm

Surgisis Nerve Cuff /Axoguard

Cook Biotech Products, WestLafayette, IN, USA

porcine small intestinalsubmucosa (SIS) matrix

not reported 4 cm

AxonScaff / Cellscaff /

StemScaff (ling for CEand FDA approval)

Axongen, Umea, Sweden polyhydroxybuturate

(PHB)

not reported not reported

2 Review. Peripheral nerve regeneration W. Daly et al.

J. R. Soc. Interface

http://rsif.royalsocietypublishing.org/http://rsif.royalsocietypublishing.org/


4/21

for structural support; (iii) exhibiting a low immuneresponse; and (iv) biodegradability, to remove the needfor secondary surgery and to prevent chronic inam-mation and pain caused by nerve compression due tothe eventual collapse of implanted NGCs [ 25]. For therst criterion, adequate nutrient exchange and wasteremoval in an NGC can be achieved, if the material is

permeable with a molecular weight limit of approxi-mately 50 kDa [2628]. For the remaining criteria, anumber of different materials both biological (e.g. col-lagen, small intestinal submucosa) and synthetic (e.g.polyhydroxybuturate, polyvinyl alcohol, PGA) havebeen considered throughout the years [ 3,5,29,30].These past studies have shown some improvements innerve regeneration and functional recovery; however,certain key elements are missing with the use of hollowNGCs alone.

1.3. Regeneration within a hollow nerve guidance conduit

Understanding the natural regenerative process occurr-ing within hollow NGCs is a prerequisite for improvednerve regeneration. Briey this regenerative process canbe divided into ve main phases: (i) the uid phase;(ii) the matrix phase; (iii) the cellular migration phase;(iv) the axonal phase; and (v) the myelination phase(gure 1) [7]. In the initial uid phase, there is aninux of plasma exudate from both the proximal anddistal nerve stumps, which is lled with neurotrophic fac-tors and extracellular matrix (ECM) precursor molecules(e.g. brinogen and factor XIII), which peak in concen-tration after 36 h; the time-course mentioned refers to

nerve conduit repair occurring within a rat modelacross a non-critical 10 mm gap [ 1,7,31]. This initialuid phase is followed by the formation of an acellularbrin cable between the proximal and distal stumps,formed from the former inuxed ECM precursor mol-ecules [7,31,32]. This brin cable usually forms withinone week of NGC repair and forms an ECM bridge forthe next stage of regeneration. During the second weekof repair, SCs from the proximal and distal nervestumps, as well as some endothelial cells and broblasts,migrate along this brin cable [ 1,7]. These SCs sub-sequently proliferate and align, forming an aligned SCcable, i.e. the glial bands of Bu ngner. This biological

tissue cable provides a trophic and topographical tissuecable for the axonal phase of repair. During this axonalphase of repair, new regenerative axonal sprouts,guided by their individual growth cones, use this biologi-cal cable tissue as a guidance mechanism to ultimatelyreach their distal target.

These regenerating axons reach the aforementionedtargets after approximately 2 4 weeks [ 1,7,31]. It isworth pointing out that during the weeks of the cellularand axonal phase, the brin cable, which has a degra-dation time of approximately two weeks, has most likelydegraded, having fullled its role for cellular migration[33,34]. Following the axonal phase, SCs switch from

the more proliferative regenerative phenotype to a pre-sumably more mature myelinating phenotype [ 29].These mature SCs subsequently wrap around the largerregenerated axons to form the myelin sheath (a mature

myelinated axon), resulting in some functional repairof nerve bres; this usually occurs 6 16 weeks afterrepair and longer in some larger animal models [ 35,36].This regenerative sequence takes place within hollowNGCs up to a critical nerve gap of approximately 4 cmin humans and approximately 1.5 cm in a rat sciaticnerve model, after which regeneration is limited or

absent [3,29,37,38]. Functional recovery however remainspoor across all nerve gaps [38,39].

2. GUIDED NERVE REGENERATION: THEUSE OF STRUCTURAL GUIDANCE CUES

Insufcient levels of regeneration in a hollow NGC,especially across critical nerve gaps, may be attributedto the inadequate formation of ECM componentsduring the initial stagesof regeneration, i.e. the formationof the brin cable [13,40]. Without the formation of thisaligned ECM bridge, there is a limitedmigration of nativeSCs into the site of the lesion, from both proximal and

distal nerve stumps and consequently a reduction in theformation of glial bands of Bu ngner, the essential trophicand topographical guidance structure for regeneratingaxons [1,7,13,32]. In attempts to replace the supportand guidance provided by this ECM tissue cable, anumber of strategies for nerve repair have focused onthe addition or manipulation of structure in NGCs(gure 2).

2.1. Intraluminal guidance structures: replacing or supporting the brin cable

One current strategy for nerve repair is the addition of

structural intraluminal guidance cues, which may act asa replacement for the unformed or incomplete brincable, or act as an additional anchor for its formation[38,41]. These intraluminal guidance channels act asa platform for SC migration and proliferation andsimultaneously can provide additional topographicalguidance cues to regenerating axons. Ultimately, theaddition of intraluminal channels aim to recapitulatethe hierarchical organization and biological functionof the native ECM [ 13].

In early studies by Matsumoto et al . [36], theaddition of laminin-coated collagen bres to a PGANGC was shown to bridge a gap of 8 cm within a

canine peroneal nerve model far exceeding that of acritical nerve gap. However, functional recovery wasnot characterized. This concept was further exploredby Yoshii and co-workers [ 42,43], using bundles of col-lagen bres alone without the use of an externalconduit structure. Gaps of 20 and 30 mm were consecu-tively bridged in successive studies; however, functionalrecovery remained poor. Despite this, the addition of intraluminal llers clearly showed the ability toextend the regeneration limits of hollow NGCs [ 42,43].Over the years, a number of variations of these intra-luminal guidance structures have been used withinhollow NGCs in attempts to bridge a critical nerve

gap or to enhance functional recovery ( table 2 ). Similarstudies by Ngo et al . highlighted the importance of packing density (or void fraction) [ 36], as well asthe distribution of intraluminal structures, as essential

Review. Peripheral nerve regeneration W. Daly et al. 3




5/21

considerations for their incorporation within hollowNGC [39,41,52]. Another study observed that high den-sities (approx. 1530% of the cross-sectional area) of poly( L-lactide) (PLLA) microlaments inhibited nerveregeneration [ 47]. At lower densities (approx. 3.757.5% of the cross-sectional area), regeneration was

increased and the ability to bridge a critical nervegap in a rat sciatic nerve model was demonstrated.These were taken as the optimal packing densities forthe introduction of intraluminal structures, as lower

densities resulted in the bres settling to the bottomof the conduit, while higher densities resulted in theinhibition of regenerating nerves [ 47]. This inhibitionwas similarly seen by Stang et al . [52], where theaddition of a dense collagen sponge within a hollowNGC was shown to inhibit regeneration entirely.

Ngo et al . [47] demonstrated that axonal regener-ation was further reduced when intraluminal breswere juxtaposed. One instance showed that bres clus-tered in the centre of the conduit resulted in complete

(1) fluid phase

(2) matrix phase

(3) cellular phase

(4) axonal phase

(5) myelination phase

regeneration within a tube

accumulation of neurotrophic factors and ECMmolecules

fibrin cable formation

Schwann cell migration, proliferation andalignment and tissue cable formation

growth of daughter axons (proximal todistal) across de novo tissue cable

myelination of regenerated immature axonsforming mature axonal fibres

Figure 1. Regenerative sequence occurring within a hollow NGC. Figure adapted from Belkas et al . [7]. This regenerative process

occurs in ve main phases: (1) the uid phase: plasma exudate lls the conduit resulting in accumulation of neurotrophic factorsand ECM molecules; (2) the matrix phase: an acellular brin cable forms between the proximal and distal nerve stumps; (3) thecellular phase: Schwann cells, endothelial cells and broblasts migrate (from the proximal and distal nerve stumps), align andproliferate along the brin cable forming a biological tissue cable; (4) axonal phase: re-growing axons use this biological tissuecable to reach their distal targets; (5) myelination phase: Schwann cells switch to a myelinating phenotype and associatedwith regenerated axons forming mature myelinated axons.





6/21

regeneration failure. This result highlights the necessityfor the correct positioning of intraluminal llers withina hollow NGC and when used with the appropriatematerial combinations, as seen in a later study wherePLLA intraluminal laments were incorporated intoa permeable poly(lactic acid) (PLA) NGC. A furtherincrease in their regenerative potential was seen in vivo in contrast to that of the original impermeablesilicone NGC [53].

A number of similar studies were carried out usingdifferent combinations of intraluminal guidance struc-tures and outer conduit materials and are summarizedin table 2 . These intraluminal guidance structures

include gels, sponges, lms, laments and bres, whichhave been used alone, or in combination with a numberof supportive factors. One approach taken is the addi-tion of nano-scale guidance cues to micrometre-scaleintraluminal guidance structures.

These nano-scale features were successfully incorpor-ated into both lm [ 39,54] and lament guidancestructures [ 13]. The use of aligned polymeric brouslms serves as one interesting alternative to the use of intraluminal bres / laments. A critical nerve gap of approximately 17 mm was bridged using aligned electro-spun thin lms of poly(acrylonitrile-co-methylacrylate;PAN-MA) bres [ 54]. These aligned sub-micrometre-

scale bres (400600 nm in diameter) showed a signi-cant increase in nerve regeneration in contrast to thatof control unaligned lms and in later studies showedthe ability to be arranged into a variety of congurations.

These electrospun lms have the advantages of ahigh surface area-to-volume ratio, a compact alignedtopography, controlled packing congurations and alow packing density (approx. 0.6% of the NGC cross-sectional area) [ 39]. These intraluminal lms allowcontrolled positioning of guidance structures, eliminat-ing the problem of bre overlap associated with theuse of intraluminal bres / laments. Despite the advan-tages of such a concept, a single lm placed along themidline of the conduit showed the most promisingresults. The addition of further lms (in various con-gurations) limited regeneration-creating areas devoidof axonal growth. The author noted the disadvantage

of creating zones within the conduit itself. Thesezones allowed symmetrical mismatches of migration of supportive cells from the proximal and distal nervestumps. SCs could be seen migrating in the upperzone proximally, while distilling migrating in a lowerzone of the conguration. This misalignment resultedin the incorrect formation of an aligned tissue cable[39]. The use of such a system therefore requires carefulpositioning of each lm within the conduit to create acontrolled environment for repair.

The use of nano-scale topographies was similarlyachieved by Koh et al . [13] through the use of micro-metre-scale intraluminal laments that were composed

of aligned electrospun nanobrous yarns. These intra-luminal laments consisted of poly(lactic-co-glycolicacid; PLGA) nanobres (between 200 and 600 nm indiameter) and have a diameter of approximately

intraluminalguidance

micro-groovedluminal design

electrospun fibrous outerconduit

combinatorialapproaches

surfacefunctionalization

variations inconduit design

Figure 2. Summarized schematic of the structural repair strategies used for improving existing hollow nerve guidance conduits.Repair strategies include the use of intraluminal guidance structures and micro-grooved luminal designs to provide additionalstructure support and topographical guidance to regenerating axons and migrating Schwann cells. A similar strategy involvesusing electrospun brous conduits with the advantages of high exibility and porosity, a high surface area-volume and bresthat can be aligned for guided Schwann cell migration and proliferation and axonal growth. Variations in conduit design includethe use of multi-channel conduits for control of axonal dispersion, as well as designs which optimize nutrient exchange or intro-duce external stimuli. These designs may be used alone or in combination, but also require further surface functionalization.These surface modications can increase cell adhesion, migration, alignment and proliferation.





7/21

25 mm (approx. 10% of the NGC cross-sectional area).These laments, combined with surface functionaliza-tion and growth factor delivery, successfully bridged anerve gap of approximately 15 mm after a period of 12 weeks. Further such intraluminal structures com-bined with a bi-layered outer conduit were shown toachieve similar levels of regeneration and functional

recovery to that of autograft across a large criticalnerve gap [13].

2.2. Luminal wall guidance features: enhancing porosity and increasing guided cell migration

A number of physical alterations to the luminal wallhave also been considered to introduce physical gui-dance signals within a hollow NGC. These physicalguidance features range from micrometre-scale featu-res to the more biomimetic nano-scale topographies(table 3 ) [4] and primarily involve either the incorpor-

ation of longitudinal micro-channels on the innerlumen of an NGC or luminal walls composed of orien-tated and non-orientated electrospun micrometre-scaleto nano-scale bres. It has been shown that the use of micrometre-scale features induces a guidance effect onneurites of re-growing neurons. These neurites showincreasing alignment as features approach the size of regenerating axons, which are of approximately thesame width as glial bands of Bu ngner or smaller [8].Depending on nerve type and anatomical location,these axons may have a diameter of 25 mm (Ad) or1520 mm (Aa ) [8,14]. Overall, neurites show increas-ing alignment as features decrease in width from 500

to 5 mm [4,8,32,59,60]. To take advantage of the gui-dance effect of the aforementioned topographicalfeatures micro and nano-scale structures are currentlybeing incorporated into a number of NGCs designswith the luminal walls displaying longitudinally orderedguidance structures to regenerating axons and similarlyto that of migrating and proliferating SCs ( table 3 ).One such example was shown by Rutkowski et al . [55]across a nerve gap of approximately 10 mm in a rat scia-tic nerve model using a micro-patterned laminin-coated(poly (D,L-lactic acid)) PDLLA conduit. This studyhighlighted that over a non-critical nerve gap, theinclusion of micro-channels alone had no signicant

effect on the level of nerve regeneration, as against con-trol hollow non-micro-grooved conduits. However, theaddition of micro-channels, when assessed over a criti-cal 1.5 cm nerve gap, exhibited a signicant increasein nerve regeneration and functional recovery versuscontrol NGCs [56]. Similarly, Hu et al ., using a uni-directional freezing method, followed by freeze drying,produced a collagenchitosan conduit with longitudinalorientated micro-channels, in the range of 25 55 mm,within the luminal wall. This produced a hollow NGCwith topographical guidance features, while maintainingstructural integrity and a high degree of porosity, andwas successfully used to bridge a 15 mm critical nerve

gap. This longitudinal micro-channelled conduit showeda similar level of regeneration and functional recovery tothat of autografts at 12 weeks post implantation. It alsoshowed the ability of an NGC with micro-scale

T a b l e 2 . E x a m p l e s o f i n t r a l u m i n a l g u i d a n c e s t r u c t u r e s .

s t r u c t u r e

m o d e l

g a p

t i m e

s i g n i c a n t o u t c o m e

r e f e r e n c e s

l a m i n i n / Y I G S R c o l l a g e n b r e s ( u

, 1 0 0

1 5 0

m m )

r a t s c i a t i c

1 5 m m

e i g h t w e e k s

l a m i n i n / Y I G S R c o a t e d b r e g r o u p s s i g n i c a n t l y i n c r e a s e d a x o n a l d e n s i t y

v e r s u s u n c o a t e d b r e s .

I t o h e t a l . [

4 4 ]

2 0 0 0 c o l l a g e n l a m e n t s ( u

, 2 0

m m )

r a t s c i a t i c

2 0 m m

f o u r , e

i g h t

w e e k s

c r i t i c a l g a p b r i d g e d . N o s i g n i c a n t d i f f e r e n c e v e r s u s a u t o g r a f t a t e i g h t

w e e k s

.

Y o s h i i & O k a

[ 4 5 ]

8 0

l a m i n i n - c o a t e d c o l l a g e n b r e s

( u , 5

0 m m ) / s p o n g e

c a n i n e

p e r o n e a l

8 0 m m

1 2 m o n t h s

n o s i g n i c a n t d i f f e r e n c e i n n e r v e r e g e n e r a t i o n o r f u n c t i o n a l r e c o v e r y s e e n

b e t w e e n g r o u p s .

T o b a e t a l . [

4 6 ]

v a r i o u s d e n s i t i e s o f P L L A ( u

, 4 0 1 0 0

m m )

m i c r o l a m e n t s

r a t s c i a t i c

1 0 , 1

4 , 1 8 m m

1 0 w e e k s

h i g h l a m e n t d e n s i t i e s i n h i b i t e d n e r v e r e g e n e r a t i o n . L o w l a m e n t d e n s i t i e s

i n c r e a s e d n e r v e r e g e n e r a t i o n .

N g o e t a l . [

4 7 ]

2 0 0 0

P G A ( u

, 1 4

m m ) l a m e n t s

d o g s c i a t i c

3 0 m m

s i x m o n t h s

c r i t i c a l g a p b r i d g e d w i t h s i m i l a r f u n c t i o n a l r e c o v e r y t o a u t o g r a f t .

W a n g e t a l . [

4 8 ]

c o l l a g e n g e l

r a t p e r o n e a l

1 5 m m

1 2 w e e k s

a c r i t i c a l n e r v e g a p b r i d g e d w i t h o u t t h e a d d i t i o n o f n e u r o t r o p h i c f a c t o r s .

L e e e t a l . [

4 9 ]

1 / 3 P A N - M A b r o u s l m c o n g u r a t i o n s

( u , 4

0 0 6 0 0 n m ) .

r a t t i b i a l

1 4 m m

s i x , 1

3 w e e k s

f u n c t i o n a l n e r v e r e g e n e r a t i o n w a s s i g n i c a n t l y g r e a t e r i n t h e 1 l m c o n d u i t

v e r s u s t h a t o f t h e 3 l m c o n d u i t .

C l e m e n t s e t a l .

[ 3 9 ]

b r o u s ( u , 2 2 0

m m ) k e r a t i n h y d r o g e l

m o u s e t i b i a l 4 m m

s i x w e e k s

k e r a t i n g r o u p s h o w e d s i g n i c a n t l y g r e a t e r c o n d u c t i o n d e l a y t h a n a u t o g r a f t

g r o u p .

S i e r p i n s k i e t a l .

[ 5 0 ]

1 0 0 0

P L G A b r e s ( u

, 1 4

m m ) M S C s

d o g s c i a t i c

5 0 m m

s i x m o n t h s

c r i t i c a l g a p b r i d g e d . F u n c t i o n a l r e c o v e r y s i g n i c a n t l y g r e a t e r t h a n a h o l l o w

c o n d u i t a n d l e s s t h a n a u t o g r a f t .

D i n g e t a l . [

5 1 ]





8/21

topographical features to successfully bridge a criticalnerve gap without the addition of neurotrophic factors,cells or similar molecular therapies [ 56].

Another similar luminal wall guidance strategyinvolves the use of electrospun brous conduit ( table 3 )[25,27,40,5558,61]. The use of these aligned electrospuntubes has a number of advantages over continuous tube

strategies: (i) the materials are highly exible andporous, and these are well adapted for use within biologi-cal systems; (ii) nano- and micro-scale bres have a highsurface area-to-volume ratio increasing the area availablefor protein absorption, SC migration and regeneration of axons; (iii) bres that can be preferentially aligned result-ing in increased SC alignment, proliferation and growth,and the promotion of guided axonal growth [ 13,40,62].The use of wall guidance avoids the problem of unevenbre distribution, seen in the use of low-density intra-luminal guidance structures [ 40,47]. This eliminates theproblem of potential growth inhibition from overlappingbres or compartmentalization, which have adverse

effects on nerve regeneration [ 39,47,52].In an interesting study by Chew et al . [40], the use of micro-scale electrospun copolymer of caprolactone andethyl ethylene phosphate (PCLEEP) bres successfullybridged a 15 mm nerve gap. The aligned electrospunbres showed an increase in functional recovery versuscontrol non-brous PCLEEP conduits showing anincreasing trend in nerve regeneration with the sub-sequent addition of exogenous growth factors [ 40].Interestingly, in this study, regeneration occurred atboth the periphery and at the centre of NGC lumen[40]. This was reported to be due to the slippage of PCLEEP bres from the wall into the centre of the

lumen and possibly highlights the need for additionalintraluminal guidance structures [ 40]. In a recent in vitro study by Madduri et al . [4], the effects of topogra-phical guidance of electrospun bres is elegantly shown,through the use of silk broin nanobres. These electro-spun bres, in the range of 400500 nm, successfullyencapsulated neurotrophic factors (glial-derived neuro-trophic factor; GDNF and nerve growth factor; NGF)to provide synergistic topographical and trophicsupport to re-growing axons [ 4]. The silk broin mem-branes were subsequently assessed with chick dorsalroot ganglion cells (primarily sensory neurons andSCs) and chicken embryonic spinal cord explants (pri-

marily motor neurons and SCs) [ 4]. Interestingly, itwas shown that there was a signicant increase in neur-ite length and alignment, and promotion of glial cellmigration and alignment, in the case of aligned elec-trospun nano-scale bres [ 4]. This combination of topography and trophic support shows potential forthe treatment of critical nerve gaps and increasing func-tional recovery. It also highlights the different modalityof SCs and axons that need to be targeted for mixednerve repair.

However, it seems that luminal wall guidance alonedoes not exhibit similar levels of axonal guidance as dointraluminal llers when bridging a critical nerve gap

[40]. To complement these luminal wall guidance featuresand increase regeneration across a critical nerve gap, anumber of approaches need to be considered. A studyby Koh et al . [13] combined a number of strategies

T a b l e 3 . L u m i n a l g u i d a n c e f e a t u r e s a n d v a r i a t i o n s i n m a t e r i a l d e s i g n .

f e a t u r e

m o d e l

g a p

t i m e

s i g n i c a n t r e s u l t s

r e f e r e n c e s

m i c r o - g r o o v e d

/ m i c r o - c

h a n n e l

l e d l u m i n a l

f e a t u r e s

m i c r o - c

h a n n e l l e d P D L L A N G C S C s ( g r o o v e w i d t h

1 0 m m , d e p t h 4 . 3

m m )

r a t s c i a t i c

1 0 m m

e i g h t w e e k s

m i c r o - c

h a n n e l s h a d n o s i g n i c a n t e f f e c t o n r e g e n e r a t i o n ;

a d d i t i o n o f S C s i n c r e a s e d f u n c t i o n a l r e c o v e r y

R u t k o w s k i e t a l . [

5 5 ]

m i c r o - c

h a n n e l l e d c o l l a g e n c h i t o s a n c o n d u i t ( g r o o v e

w i d t h 2 5 5 5

m m )

r a t s c i a t i c

1 5 m m

f o u r , 1 2 w e e k s

s i m i l a r l e v e l o f r e g e n e r a t i o n a n d f u n c t i o n a l r e c o v e r y t o

a u t o g r a f t a t 1 2 w e e k s .

H u e t a l . [

5 6 ]

e l e c t r o s p u n n a n o - a n

d m i c r o -

b r o u s c o n d u i t s

e l e c t r o s p u n P C L E E P b r o u s c o n d u i t w i t h G D N F ( u

( 3 . 9

6 +

0 . 1 4 ) m m )

r a t s c i a t i c

1 5 m m

1 2 w e e k s

s i g n i c a n t i n c r e a s e i n f u n c t i o n a l r e c o v e r y c o u l d b e

s e e n v e r s u s c o n t r o l c o n d u i t s

C h e w e t a l . [

4 0 ]

s i l k b r o i n

p o l y ( L - l a c t i c a c i d - c o - 1 - c a p r o l a c t o n e ) ( P ( L L A -

C L ) ) b r o u s N G C

r a t s c i a t i c

1 0 m m

f o u r , e i g h t w e e k s s i g n i c a n t i n c r e a s e i n f u n c t i o n a l n e r v e r e g e n e r a t i o n

v e r s u s P ( L L A - C

L ) N G C s .

W a n g e t a l . [

5 7 ]

v a r i a t i o n s i n c o n d u i t d e s i g n :

m u l t i - c h a n n e l l e d c o l l a g e n c o n d u i t ( 1 - ,

2 - , 4 - ,

7 - c h a n n e l

c o n d u i t s )

r a t s c i a t i c

1 0 m m

1 6 w e e k s

4 - c h a n n e l c o n d u i t s s i g n i c a n t l y d e c r e a s e d a x o n a l

d i s p e r s i o n v e r s u s c o n t r o l s i n g l e c h a n n e l c o n d u i t s

.

Y a o e t a l . [

5 8 ]

e n h a n c i n g n u t r i e n t e x c h a n g e a n

d t h e i n t r o d u c t i o n o f e x t e r n a l s t i m u l i

P L G A c o n d u i t w i t h a s y m m e t r i c

/ s y m m e t r i c p o r e s

r a t s c i a t i c

1 0 m m

f o u r , e i g h t w e e k s P L G A c o n d u i t s w i t h a s y m m e t r i c p o r e s s h o w e d h i g h e r n e r v e

r e g e n e r a t i o n t h a n P L G A c o n d u i t s w i t h s y m m e t r i c p o r e s .

C h a n g & H s u [ 2 7 ]

b i - l a y e r e d m i c r o a n d n a n o p o r o u s P L G A / p l u r o n i c F 1 2 7

N G C

U S

r a t s c i a t i c

1 0 m m

o n e , e

i g h t w e e k s

a b i - l a y e r e d N G C U S i n c r e a s e d n e r v e r e g e n e r a t i o n r a t e s

v e r s u s n o U S g r o u p s ( 0

. 7 2 m m d 1

V 0 . 4 8 m m d 1 )

P a r k e t a l . [

2 5 ]





9/21

within their conduit design. A bi-layered laminin-coatedPLLA conduit, used in combination with intra-luminal PLGA bres, was demonstrated to enhance themodied outer NGC. This bi-layered conduit consistedof an outer layer of randomly aligned electrospun nano-bres and an inner layer of longitudinally alignednanobres that were in the range 2501000 nm in diam-

eter. It was proposed that the longitudinally alignedinner layer provided topographical cues for regeneratingaxons and migrating SCs, while the outer layer provi-ded structural support to the conduit structure whilemaintaining the porosity of the tube. This conduitwas successfully used to bridge a critical nerve gap of 15 mm and exhibited functional recovery comparable toautografts [ 13].

2.3. Optimizing conduit design and the introduction of external stimuli

Alternative strategies for enhancing nerve repair involve

reconsidering the overall conduit design ( table 3 ).These approaches have been used to limit axonal dis-persion [58], optimize nutrient exchange [ 25,63] and tomore closely resemble the micro-architecture of the per-ipheral nerve environment [ 64]. Some of these designshave been successfully combined with non-invasive clini-cal approaches (i.e. ultrasound) and have shown thepotential to enhance peripheral nerve repair [ 25,61].

The use of a multi-channel conduit is one promisingalternative for peripheral nerve repair [ 21,58,64,65]. Amulti-channel PLGA was originally investigated as analternative to conventional NGC, which was closelyimitating native nerves architecture [ 64]. Using a

foam-processing technique, conduits with multiplemicro-channels were manufactured. The primary pre-mise for this design was the controlled introduction of allogenic SCs by increasing the overall surface area forSC adherence and distribution. From this basis, a vechannel conduit was then successfully used to bridge ashort 7 mm rat sciatic nerve gap. This design howeverhad a very low cross-section available for nerve regener-ation, making comparison with the control autograftgroup difcult [ 64]. It was later put forward by deRuiter et al . [66] and by Yao et al . [58] that thismulti-channel design could be used to limit axonal dis-persion within NGC. It was later put forward by de

Ruiter et al . that a single and seven channel PLGANGCs were used to bridge a 10 mm nerve gap in a ratsciatic nerve model. At 12 weeks, there was no signi-cant difference between single and multi-channelconduits with regard to nerve regeneration. However,using a simultaneous retrograde tracing technique,there was a signicant decrease in axonal dispersionversus control single channel conduits. The use of thisconduit however showed that these results in only50 per cent of the groups assessed, primarily due toswelling of the PLGA tube, resulting in occlusion of anumber of the channels and the consequences of theseresults were not denitive [ 66]. In order to improve

this design, Yao et al . [58] showed that the use of multi-channelled collagen nerve conduits could simi-larly be used to bridge a 10 mm rat sciatic nerve gap,without the structural instability seen in previous

studies. This studyshowed similarresults fornerve regen-eration to previous work; however there was a signicantdecrease in overall axonal dispersion / misdirection usingthis multi-channel design. Using this multi-channeldesign in combination with additional factors, such asguidance structures or molecular / cell-based therapies,could be an interesting approach for future nerve

repair, and potentially could reduce signicantly the mis-direction of re-growing axons.Another approach is the use of a bi-layered PLGA /

pluronic F127 asymmetrically porous conduit thathas been shown to have a number of features [ 25,63]to increase regeneration within a hollow nerve conduit.This conduit contains two distinct layers: an innersurface with nano-pores of 50 nm in diameter asym-metrically aligned, which allows the diffusion of nutrients and neurotrophic factors but reduces scarinltration; and an outer surface consisting of micro-pores approximately 50 mm in diameter, which permitsvascular ingrowth into the conduit [ 25,63]. The use of

asymmetric pores over non-asymmetric pores had pre-viously been shown to increase early stage nerveregeneration [ 27,63]. This, in combination with thepluronic F127 coating, increases the hydrophilicity of the conduit, resulting in an increase in the regenerationrate of regenerating axons versus that of control con-duits [63]. In later studies, these bi-layered coatedconduits were combined with external ultrasoundstimulation (US), a novel non-invasive approach. Theuse of low-intensity US indicated a signicant increasein nerve regeneration rates (0.72 mm d 1 in the US-treated group versus 0.48 mm d 1 in the non-treatedgroup) [25]. Likewise, US resulted in increased myelina-

tion, axon diameter and thicker regenerative nervecable [25]. The effects of US stimulation have exhibitedcomparable results in a number of studies and may holdpotential to improve current clinical nerve therapiesespecially when used in combination with additionalregenerative factors, i.e. neurotrophic factors, growthfactors or cell-based therapies.

2.4. Surface modications and peptide mimetics

The addition of topographical guidance cues and struc-tural features to a conduit may require additionalsurface modications of the biomaterial surface,

depending on the base material. Numerous forms of surface modications have been used with bothsynthetic biodegradable materials (polycaprolactone(PCL), PLA and PLLA) and numerous naturalmaterials (collagen, chitosan and brin) [ 36,46,6771]. These materials while they exhibit the requiredstructural cues for guided cell growth, SC adhesion andmigration, their surface characteristics may not be suchas to induce the required effects; these materials tend tobe hydrophilic or hydrophobic reducing their applica-bility for nerve repair [ 72]. Consequently, a number of surface modication techniques have been employedto increase cell adhesion, proliferation and migration.

These modications may take the form of full proteincoatings, chemical and physical treatments, or theaddition of protein mimetics onto the surface of thematerial [ 72].





10/21

Numerous ECM proteins have been considered as can-didates for surface functionalization including collagen,bronectin and laminin [ 13,73,74]. Laminin, a complextrimeric glycoprotein, is a major component of the basallaminaof SCsandhas positiveeffects on neurite extensionand SC adhesion, proliferation and migration, and overallhave exhibited the ability to improve nerve regeneration

[13,46,70]. This trimeric glycoprotein has been demon-strated to interact with SC integrins, which may resultin activation of myelination needed for successfulgrowth and repair [ 13]. Numerous studies have shownits ability to enhance Schwann proliferation andmigration, as well as itsdirect effects on neurite outgrowth[13,36,70]. Laminin has been used most frequently forsurface modication for NGC and for their respectivestructural components [ 13,36,46,55,68,70,74,75]. OtherECM molecules, such as collagen and bronectin, havethe ability to signicantly increase SC adhesion as wellas proliferation, and enhance neurite outgrowth however,although results have been shown to be signicantly lower

than that of laminin [ 13,73,74]. A number of studies haveconjugated laminin to their respective material or usedthem to enhance the aforementioned intraluminal l-lers [36,46,68,70,76]. Each respective study notablyshowed a signicant increase in nerve regeneration com-pared with that of uncoated bres [ 36,46,68,70]. Yu &Bellamkonda [ 74] presented a combination of lamininand slow-releasing NGF from an agarose hydrogel. Thecombined effect of these two factors yielded nerveregeneration and functional recovery similar to that of autograft [ 74]. Similarly, in a recent study by Koh et al .[13], the incorporation of a laminin coating, combinedwith PLGA intraluminal guidance structures, success-

fully bridged a critical nerve gap of 15 mm, and showedsuperior functional recovery to that of autograft. Thesesame ECM molecules can similarly be used as a luminalwall coating, increasing cell adhesion and proliferationas well as increasing guided axonal outgrowth and mayserve to enhance some of the luminal wall guidancefeatures mentioned previously [ 55,67,77].

Large ECM molecules, such as laminin, have a largemolecular weight (about 900 kDa), making them quitedifcult to synthesize [ 67,70]. One alternative to the useof these largeglycoproteinsis the useof shortchainproteinpeptide mimetics ( table4 ). These peptides have a numberof advantages over large proteins, including (i) high stab-

ility; (ii) low immune response; (iii) high surface densityand orientation for ligandreceptor interaction and celladhesion; (iv) a relatively low molecular weight; and (v)the ability to be used in high concentrations [ 70,72]. Anumber of these peptides have been used in the contextof peripheral nerve repair, including RGD (ArgGlyAsp), a peptide found in bronectin, laminin and otherECM molecules; IKVAV (IleLysValAlaVal) andYIGSR (TyrIle Gly Ser Arg) of the laminin bchain, RNIAEIIKDI (ArgAsnIleAlaGluIleIleLysAspIle) peptides of the laminin g chain and theprimary cell binding domains of laminin; as well as similarpeptide sequences such as HAV (HisAlaVal), a

mimetic of the N-cadherin regulatory protein that ispresent on both neurons and glial cells [ 67,7072,77].A range of these peptides was successfully assessed, in

vitro and in vivo , by Schense et al . [71] within a brin

matrix.Thesevarious peptide sequencesexhibited a signi-cant increase in regenerationcompared with that of controluncoated brin matrix in vitro . Noteworthy was the syner-gistic effect of multiple peptides on neurite outgrowth,where the combined effect of four individual laminin pep-tides was greater than the sum of neurite extension forindividual peptide alone. In an in vivo study, using a

4 mm dorsal root model, an NGC lled with a peptide-loadedbrinmatrix was successfully implanted. The incor-poration of individual peptides within a brin matrixshowed no signicant difference versus brin alone; how-ever the synergistic effects of the four laminin peptidesshowed a signicant increase in nerve regenerationversus control brin matrices [ 71]. Similarly, in a work car-ried out by Yao et al . [60], a human laminin ve peptide(PPFLMLLKGSTR (Pro Pro Phe LeuMet LeuLeuLys GlySer ThrArg)) coating was shown toexhibit similar levels of neurite outgrowth to that of a col-lagen-coated substrate in vitro and successfully used incombination with micro-structured templates, enhancing

neurite growth and alignment.These peptides have been used in a number of similarstudies and results have shown levels of regenerationequivalent to whole proteins [ 44,67,86]. Itoh etal . [44] suc-cessfully coated collagen intraluminal llers with eitherlaminin or the YIGSR peptide, and compared regener-ation with uncoated collagen bres. It was shown thatboth the laminin-coated and peptide-coated bres indi-cated a signicant increase in nerve regeneration versusuncoated collagen bres. In particular, there was no sig-nicant difference in nerve regeneration between thewhole glycoprotein and the peptide mimetic [ 44]. Inmore recent studies, Santiago et al . [67] modied the

inner surface of a PCL scaffold with a peptide sequence(RGD) as a means to enhance axonal interactions aswell asSC adhesion,and to increase adhesionof implantedadipose-derived stem cells (ASCs). Wang & Huang [ 86]incorporated a CYIGSR (Cys TyrIle GlySerArg)peptide (YIGSR peptide with a glycine spacer) with abi-layered micro / nanobrous conduit, resulting inincreased nerve regeneration. These surface modications,including large glycoproteins or their peptide memetics,canbe seen as key factors for enhancing structural featuresof current NGCs.

3. MOLECULAR DELIVERY THERAPIES:THE CREATION OF A CONDUCTIVEMICROENVIRONMENT

The addition of structural features to hollow NGCs isone approach to improve nerve regenerationin par-ticular, across critical nerve gap [ 36,43,45,49,51]. Theaddition of these features alone is insufcient toincrease functional recovery. In efforts to improve func-tional regeneration in both critical and non-criticalgaps, the creation of a more conductive microenviron-ment is of high importance. The reduction infunctional nerve regeneration over these challengingnerve gaps can be attributed to a variety of factors.

These include inadequate ECM formation (mentionedearlier), insufcient neurotrophic support, inadequateSchwann numbers, reduction in SC migration and pro-liferation, and possible reduction in the neurotrophic





11/21

effects of the distal nerve stump [ 1,7,31]. In efforts toenhance functional nerve regeneration, advances havebeen made to create a more conductive environmentfor repair. Strategies include the use of exogenousgrowth factors (e.g. vascular endothelial growth factor(VEGF), broblast growth factor (bFGF)), neuro-trophic factors (e.g. neurotrophin-3 (NT3), NGF) or

cell-based therapies (e.g. SCs, stem cells; gure 3)[9,7885,8791].Neurotrophic factors enhance functional regener-

ation, by supporting axonal growth, SC migration andproliferation, and increasing neuroprotection throughreceptor-mediated activation of specic intrinsic sig-nalling pathways [ 92]. These neurotrophic factorsprimarily belong to three distinct families: (i) the neuro-trophins; (ii) the glial-cell-line-derived neurotrophicfactor family ligands (GFLs); and (iii) the neuropoeiticcytokines [93]. Each family has distinct functionalcharacteristics with some overlapping cellular respon-ses [93]. Neurotrophins include NGF, brain-derived

neurotrophic factor (BDNF), neurotrophin-3 (NT3)and neurotrophin-4 (NT4) [ 94]; GFLs include GDNF,and neuropoeitic cytokines include cilary neurotro-phic factor (CNTF) [ 93]. These neurotrophic factors havebeen used alone or in combination so as to harness themost effective response for nerve regeneration ( table 4 ).

An ethylene vinyl acetate (EVA) conduit used forthe release of either NGF or GDNF bridged a 15 mmnerve gap through the addition of these respective neu-rotrophic factors alone [ 87]. The GDNF conduit groupexhibited four times the number of myelinated axonsthan that of the NGF group, showing its potential forperipheral nerve repair [ 87]. Neurotrophic factors deliv-

ered alone however have shown limited functionalrecovery, and efforts have been made recently for thesynergistic delivery of these neurotrophic factors [ 9].One such study, carried out by Madduri et al . [9],involved co-delivery of both NGF and GDNF, using aluminal diffusion-based delivery system. This studyargued that co-delivery was essential for increased func-tional regeneration, as peripheral nerve containeddifferent neuronal and glial subpopulations (both motorand sensory) [4,9]. NGF, which acts through the high-afnity TrkA receptor, is primarily found on sensoryneurons, shown to promote axon regeneration andre-innervate sympathetic axons following nerve injury [ 9].

The failure of single growth factor delivery may also beattributed to poor release kinetics, with some delivery sys-tems exhibiting a high initial burst release [ 88]. In effortsto improve release, delivery systems that alter theserelease kinetics are being considered. One such strategyinvolves the use of physical cross-linking methods usedin combination with a polymer coating [ 9]. This combi-nation was shown to limit the initial burst release of growth factors, indicating a signicant increase in nerveregeneration versus a PLGA polymer coating alone. Theeffects of this system on late stage functional recoveryremain to be seen, but early results seem promising.

One alternative, for controlled delivery of neuro-

trophic factors, is the use of an afnity-based deliverysystem [83,89]. This system encloses growth factorswithin a brin-based matrix for intraluminal deliveryof growth factors. This avoids the initial burst release

T a b l e 4 . M o l e c u l a r t h e r a p i e s f o r t h e c r e a t i o n o f a c o n d u c t i v e m i c r o e n v i r o n m e n t .

N G C

d e l i v e r y m e t h o d

m o d e l

g a p

t i m e


r e f e r e n c e s

G D N F / N T - 3

E V A N G C

l u m i n a l r e l e a s e

r a t f a c i a l

5 m m

s i x w e e k s

N G C G

D N F s h o w e d h i g h e s t n e r v e r e g e n e r a t i o n

l e v e l .

B a r r a s e t a l . [

7 8 ]

V E G F

s i l i c o n e N G C

s u s p e n s i o n ( m a t r i g e l )

r a t s c i a t i c

1 0 m m

o n e , t w o , f o u r a n d

2 6 w e e k s

V E G F i n c r e a s e d a n g i o g e n e s i s , S C m i g r a t i o n a n d

n e r v e r e g e n e r a t i o n .

H o b s o n [ 7 9 ]

N G F

p o l y ( p h e n y l e n e

e t h y n y l e n e ) ( P P E )

P P E

m i c r o s p h e r e

r e l e a s e

r a t s c i a t i c

1 0 m m

1 2 w e e k s

N G F i n c r e a s e d n e r v e r e g e n e r a t i o n v e r s u s c o n t r o l

c o n d u i t s

.

X u e t a l . [

8 0 ]

G D N F

c o l l a g e n P L G A N G C


( v a r i o u s r a t e s )

r a t p e r o n e a l 3 m m

1 2 w e e k s

n u m b e r o f m y e l i n a t e d b r e s t r i p l e d f o r a l l r a t e s

v e r s u s n o G D N F g r o u p .

P i q u i l l o u d e t a l .

[ 8 1 ]

N G F

p o r o u s P C L c o n d u i t

s u s p e n s i o n / l u m i n a l

r e l e a s e

r a t s c i a t i c

1 2 m m

f o u r , e

i g h t w e e k s

N G F l u m i n a l r e l e a s e s h o w e d s u p e r i o r r e g e n e r a t i o n

t o s u s p e n s i o n .

C h a n g [ 8 2 ]

G D N F N G F c o l l a g e n P L G A N G C


r a t s c i a t i c

1 0 m m

t w o w e e k s

s i g n i c a n t i n c r e a s e i n e a r l y p e r i p h e r a l n e r v e

r e g e n e r a t i o n .

M a d d u r i e t a l .

[ 9 ]

G D N F / N G F

s i l i c o n e N G C

a f n i t y - b a s e d b r i n

m a t r i x

r a t s c i a t i c

1 3 m m

f o u r , e

i g h t

, 1 2 w e e k s 1 2 w e e k s m o t o r r e c o v e r y o f G D N F g r e a t e r t h a n

i s o g r a f t .

W o o d e t a l . [

8 3 ]

C N T F

c h i t o s a n / P L G A N G C


d o g t i b i a l

2 5 m m

1 2 w e e k s

f u n c t i o n a l r e c o v e r y c o m p a r a b l e t o t h a t o f

a u t o g r a f t .

S h e n e t a l . [

8 4 ]

N G F

P L L A - C

L b r o u s N G C

c o r e s h e l l n a n o b r o u s

r e l e a s e

r a t s c i a t i c

1 0 m m

1 2 w e e k s

N G C s h o w e d s i m i l a r f u n c t i o n a l r e c o v e r y

r e g e n e r a t i o n t o a u t o g r a f t .

L i u e t a l . [

8 5 ]





12/21

seen in some diffusion-based systems and allows thecontrolled release of growth factors by cell-based degra-dation of the delivery system and the surrounding brinmatrix. Using this system, GDNF or NGF were succes-sfully delivered within the lumen of a silicone conduit,increasing the early stage regenerative response andsuccessfully bridging a 13 mm gap in a rat sciaticnerve model [83]. For functional recovery, GDNF, incombination with this diffusion-based delivery system,exhibited a higher level functional recovery than thatof the control allograft groups. This may be partiallyattributable to an increase in the number of large mye-

linated axons, increased early stage regeneration andthe ability of the brin matrix to act as an intraluminalguidance structure for early stage cell migration. Thisstudy successfully incorporates a brin-based intra-luminal guidance structure for enhanced contactguidance while synergistically creating a more conductivemicroenvironment for functional nerve regeneration [ 83].GDNF, in particular, contributes to this signicantincrease in functional recovery, owing to its ability toact on both motor and sensory neurons [ 90]. This samecombinatorial effect was seen in the use of nanobrousconstructs that were successfully combined with GDNFdelivery, resulting in a similar increase in functional

recovery [40]. However, the nerve regeneration responseas a whole is stimulated by a number of factorsthat act synergistically to improve nerve repair. If thedevelopment of the nervous system is considered in its

entirety, there is a dened synergy between angiogenesisand neurogenesis [ 91]. The addition of VEGF indicated asignicant increase in angiogenesis and also exhibited asimilar increase in overall nerve regeneration [ 79].Although the addition of neurotrophic factors hasshown advantages for nerve repair, their use has somelimitations, including unintentional activation of multiple signalling pathways resulting in undesired bio-logical effects, e.g. aberrant sprouting associated withthe use of NGF, and short half-lives and poor stab-ilitylasting literally minutes upon release in serumconditions [9496]. These limitations may be overcome

through increasing our knowledge of neurotrophicfactor and growth factor delivery reducing unintentionaleffects. Similarly, by optimizing the release kinetics of neurotrophic factors delivery (using new biomaterialtechnologies) the disadvantage of their limited half-lives may be overcome [9496]. New emerging deliveryapproaches being developed in our own laboratoryinclude the use of biological collagen/ brin microspheres,microbres and hydrogels, as well as synthetic polymericcarriers for the creation of a sustained system for viablegrowth factor delivery [ 9799].

4. SCHWANN CELLS: THE GOLDSTANDARD FOR CELL-BASED REPAIR

During nerve regeneration, SC migration and prolifer-ation can be seen as prerequisites for successful nerve

neurotrophic factors/

growth factors

Schwann cells stem cells

-differentiated-undifferentiated

genetically

modified cells

means of delivery

diffusion-basedrelease

suspension affinity-baseddelivery

microsphereencapsulation

Figure 3. Schematic of cellular and molecular-based therapies used for the creation of a more conductive nerve microenvironment.Examples of molecular therapies include growth factors (VEGF and bFGF) and neurotrophic factors (NT3 and NGF). Likewisecell therapies involve the use of SCs, stem cells (ASCs and MSCs) and genetically modied cells (SCs overexpressing GDNF).These can be delivered by a number of means including: (i) suspension within solution or a biomaterial matrix (hydrogel,sponge), (ii) released via a diffusion-based systems (controlled released via cross-linking, slow degrading polymer coatings etc.from luminal wall), (iii) the use of afnity-based delivery systems (factors conjugated to a brin matrix), and (iv) microsphere(e.g. collagen, brin) encapsulation which can either be suspended within the lumen or released from the luminal wall.





13/21

repair, seen in the cellular phase of successful nerverepair [1,7,28,31]. SCs that remain after Walleriandegeneration migrate and proliferate to form alignedglial bands of Bu ngner, during the cellular phase of NGC repair [ 7,28,29,31]. At this stage, SCs haveswitched to a regenerative phenotype, are activelysecreting neurotrophic factors, and laying down basal

lamina, and importantly their numbers have increasedto 4 17 times the original number seen in normalnerve (approx. 2010 6 cells ml

2 1 ) [28,37]. However, asgap length increases, SC migration, proliferation andalignment decrease and SC numbers may be deemedinsufcient for the creation of a conductive nerveenvironment [ 1]. In attempts to aid the regenerative cel-lular response to injury, cellular-based therapies arebeing considered as an alternate means for repair(table 5 ) [33,51,61,67,90,100102].

One suggested approach to improve functional recov-ery and nerve regeneration, and as an alternative toneurotrophic factor delivery, is the use of autologous

or allogenic SCs [37,90,101]. If autograft is taken asthe current gold standard for peripheral nerve repair,similarly the addition of autologous SCs to NGCs canbe taken as the current gold standard of cellular-basedtherapies. The use of SCs have the advantage of produ-cing a number of neurotrophic factors, building theirown basal lamina, expressing cell adhesion moleculesand at a later stage are actively involved in the re-myelination of regenerating nerve bres [ 90,101]. Theintroduction of additional SCs would therefore assistin the creation of a conductive nerve microenvironment,especially across a critical nerve gap [ 50,90].

SCs may be introduced into the conduit via a number

of methods. These include injection, suspension withinan intraluminal hydrogel, distributed along intraluminalguidance structures or released from the luminal wall[33,61,67,101]. The implanted SCs can be successfullyincorporated into the regenerative process and is nicelyshown through the use of retrovirally labelled allogenicSCs (harvested from neonatal rats) [ 37]. At the optimumconcentration (80 106 cells ml

2 1 ), these labelled cellswere shown to be successfully incorporated into thehost regenerative process, and furthermore doubled therate of regeneration versus that of control hollow siliconeNGCs [37]. On the basis of these studies, SCs have beensuccessfully implanted in a number of studies with vary-

ing effects on nerve regeneration and functional recovery.In a study by Di Summa et al . [101], SCs were seededwithin a hollow brin conduit and implanted in a10 mm rat sciatic nerve model. These brin-SC conduitsshowed a signicant increase in nerve regeneration,versus control hollow conduits, conduits seeded with dif-ferentiated bone marrow-derived mesenchymal stem cells(dBMSCs), or conduits seeded with differentiatedadipose-derived mesenchymal stems cells (dADSCs)[101]. Recently, this same system, was used to bridge a10 mm rat sciatic nerve gap over a period of 16 weeks[103]. This later study highlighted the benets of alter-nate cell therapies, showing functional recovery levels

comparable to that of autograft (discussed later). A simi-lar study using a polyhydroxybuturate (PHB) conduitlled with a brin matrix and seeded with SCs ordBMSCS was shown to increase early nerve regeneration

T a b l e 5 . E x a m p l e s o f c e l l - b a s e d a p p r o a c h e s f o r p e r i p h e r a l n e r v e r e p a i r .

c e l l t y p e

N G C

m e t h o d o f d e l i v e r y

m o d e l

g a p


r e f e r e n c e s

S C s U S

h o l l o w P L G A /

s i l i c o n e

N G C

w a l l r e l e a s e ( 3

1 0 5

c e l l s m l 2 1 )

r a t s c i a t i c

1 0 m m

i n P L G A N G C s S C S U S n e r v e r e g e n e r a t i o n s i g n i c a n t l y i n c r e a s e d .

C h a n g e t a l . [

6 1 ]

S C s d B M S C s

P H B c o n d u i t b r i n

m a t r i x

b r i n m a t r i x ( 8

1 0 7

c e l l s m l 2 1 )

r a t s c i a t i c

1 0 m m

i n c r e a s e d n e r v e r e g e n e r a t i o n d i s t a n c e v e r s u s a h o l l o w P H B a n d b r i n

a l o n e .

K a l b e r m a t t e n e t a l .

[ 3 3 ]

h u m a n u A S C s

P C L c o n d u i t R G D

p e p t i d e

s u s p e n s i o n ( 2

1 0 8

c e l l s m l 2 1 )

r a t s c i a t i c

6 m m

i n c r e a s e d f u n c t i o n a l r e c o v e r y a n d

r e g e n e r a t i o n v e r s u s c o n t r o l g r o u p s .

S a n t i a g o e t a l . [

6 7 ]

S C s

c e l l u l o s e c o n d u i t B D

h y d r o g e l

s u s p e n s i o n ( 8

1 0 7

c e l l s m l 2 1 )

r a t s c i a t i c

1 0 m m

S C s h y d r o g e l i n c r e a s e d r e g e n e r a t i o n d i s t a n c e . E

f f e c t s l o s t a t 1 6

w e e k s

.

M c G r a t h e t a l . [

9 0 ]

u B M S C s

P C L c o n d u i t

i n j e c t i o n i n t o l u m e n

( 5

1 0 8

c e l l s m l 2 1 )

m o u s e

m e d i a n

3 m m

i n c r e a s e d n u m b e r o f m y e l i n a t e d b r e s a n d a n g i o g e n e s i s v e r s u s c o n t r o l

g r o u p

O l i v e i r a e t a l . [

1 0 0 ]

u B M S C s

c h i t o s a n

c o n d u i t P L G A b r e s


( 8

1 0 7 c e l l s m l 2 1 )

d o g s c i a t i c 5 0 m m

i n c r e a s e d r e g e n e r a t i o n a n d f u n c t i o n a l r e c o v e r y v e r s u s n o n B M S C

g r o u p .

D i n g e t a l . [

5 1 ]

S C s , d A S C

s

d B M S C s

b r i n g l u e c o n d u i t


( 4

1 0 7

c e l l s m l 2 1 )

r a t s c i a t i c

1 0 m m

d B M S C s / d A S C s i n c r e a s e r e g e n e r a t i o n d i s t a n c e a n d S C m i g r a t i o n

v e r s u s h N G C

. S C r e m a i n e d s u p e r i o r t o a l l g r o u p s .

D i S u m m a e t a l .

[ 1 0 1 ]

d B M S C s S C s

c o l l a g e n c o n d u i t

( N e u r a g e n )

w a l l r e l e a s e ( 8

1 0 5

c e l l s m l 2 1 )

r a t s c i a t i c

1 2 m m

i n c r e a s e i n n e r v e r e g e n e r a t i o n i n S C d B M S C N G C s v e r s u s h o l l o w

g r o u p .

L a d a k e t a l . [

1 0 2 ]





14/21

(two weeks), unlike that of control hollow PHB conduitsand conduits lled with matrix alone [ 33]. Howevereffects on late stage functional recovery remain to be seen.

The use of autologous SCs has a number of disadvan-tages associated with their use: culture times are longand difcult; the extraction of SCs from the host isoften painful and requires sacrice of host nerve tissue

[101]. The sacrice of this tissue has the same disadvan-tages as those of autograft, i.e. donor site morbidity,and the need for a secondary surgical site. Similarlyfor the use of allogenic SCs, an extensive immuneresponse, requiring further immune suppression, similarto that associated with the use of allografting, is exhib-ited [104]. Alternative extraction methods and cellulartherapies are now being considered, including stemcells and the use of gene therapy approaches.

4.1. Stem cells: a possible alternative to autologous Schwann cells

One cellular-based alternative is the use of stem cells toenhance the host regenerative response. In a numberof studies, these cell types have been considered toenhance nerve regeneration ( table 5 ). These stem cellscome from numerous sources but many studies are con-centrating on the use of either BMSCs or ASCs[51,67,101]. Autologous BMSCs can be easily derivedby aspirating from the bone marrow of patients [ 51].Likewise, ASCs can be easily extracted using con-ventional liposuction techniques [ 101]. These cellsconform to the criteria for ideal transplantable cells:are easily extracted, proliferate rapidly in culture,have a relatively low cost, raise no ethical issues associ-

ated with their use and have the ability to differentiatealong multiple cells lines, in particular neural andassociated glial cell lineages [51,100,101,105]. BothASCs and BMSCs have the advantage of exhibitingthe ability to secrete multiple neurotrophic factors,including GDNF, NGF, NT-3 and BDNF [ 106108].These cells have been used in a number of studies inboth the differentiated and undifferentiated states inorder to investigate their effect on peripheral nerveregeneration ( table 5 ).

The advantage of using mesenchymal stem cells(MSCs) in their undifferentiated state in vivo allowsthese multi-potent cells to be stimulated by advancing

axons and native SCs, differentiating the MSCs alongmultiple pathways. This can aid in the creation of a con-ductive environment for nerve regeneration [ 100,101].This differentiation in vivo may be caused by thefusion of implanted MSCs with host cells, rather thanby directly differentiating into known cell types [ 51].These MSCs have the capacity to differentiate direc-tly or indirectly into glial-like cells, possibly secretinga variety of neurotrophic factors. Alternatively, theimplanted MSCs have showed the capacity to differen-tiate into other supportive cells, such as endothelial-likecells, smooth muscle cells or pericytes [100]. Theseendothelial-like cells can produce a variety of growth

factors, such as VEGF, which has been shown to havea simultaneous effect on angiogenesis, neuritogenesisand neuroregeneration, which translates to positiveeffects on nerve regeneration in vivo [79,91,100].

In a very interesting study by Oliveira et al . [100], theaddition of undifferentiated BMSCs were shown to sig-nicantly increase functional recovery in a mousemedian nerve model. Using the earlier mentionedmodel, a PCL NGC with suspended undifferentiatedBMSCs was successfully implanted and regenerationwas evaluated up to 12 weeks post implantation. At

the dened endpoint, there was a signicant increasein the number of myelinated bres and angiogenesis,versus control conduits [ 100]. The authors hypothesizedthat this could be attributed to the multi-potent natureof the BMSCs and their known ability to secretemultiple neurotrophic factors. Similarly, in a studycarried out by Ding et al. [51] a combination of intra-luminal llers and undifferentiated BMSCs showedthat a signicant increase in functional recovery acrossa critical nerve gap of approximately 50 mm in acanine nerve model versus that of the control group.This functional recovery approached that of autografts.

However, the mechanisms of the enhanced regenera-

tive response are largely unknown, with very fewBMSCs seen differentiating along an SC-like lineage[100]. This variability was highlighted in an early studyby Santiago et al ., where the use of undifferentiatedASCs showed no trans -differentiation to an SC-like phe-notype. A signicant increase in nerve thickness was seenin the cell-based group versus that of hollow NGCs [ 67].The variability of using these undifferentiated MSCsmay limit their future clinical applications, and furthercharacterization is needed to assess their suitability forperipheral nerve repair.

Owing to these possible limitations of undifferen-tiated MSCs, one interesting alternative is the use of

their differentiated counterparts. BMSCs and ASCscan be differentiated in vitro through combinations of various neurotrophic and growth factors, into a moreglial or neural cell lineage [102,104106,108]. Thesedifferentiated MSCs (dMSCs) have the advantage of being differentiated in a controlled manner, withboth bone marrow and adipose-derived cells showingthe ability to differentiate into SC-like cells[101,102,104,106]. These SC-like cells have a positiveeffect on neurite outgrowth on sensory dorsal rootganglion neurons in vitro [104,106] and, in recentstudies, have been shown to have benecial effects in vivo [102]. In the case of dA

A Biomaterials Approach to Peripheral Nerve Regeneration

Documents

Transcript of A Biomaterials Approach to Peripheral Nerve Regeneration