ARTICLE IN PRESS

ORBABLE GLASS FIBRES FACILITATE PER

BIORES IPHERALNERVE REGENERATION

S. BUNTING, L. DI SILVIO, S. DEB and S. HALL

From the Wolfson Centre for Age-Related Diseases, King’s College London, UK, and Guy’s Tower, London Bridge, London UK

This is a proof of principle report showing that fibres of Bioglasss 45S5 can form a biocompatible

scaffold to guide regrowing peripheral axons in vivo. We demonstrate that cultured rat Schwanncells and fibroblasts grow on Bioglasss fibres in vitro using SEM and immunohistochemistry, andprovide qualitative and quantitative evidence of axonal regeneration through a Silastic conduitfilled with Bioglasss fibres in vivo (across a 0.5 cm interstump gap in the sciatic nerves of adultrats). Axonal regrowth at 4 weeks is indistinguishable from that which occurs across an autograft.Bioglasss fibres are not only biocompatible and bioresorbable, which are absolute requirements ofsuccessful devices, but are also amenable to bioengineering, and therefore have the potential for usein the most challenging clinical cases, where there are long inter-stump gaps to be bridged.Journal of Hand Surgery (British and European Volume, 2005) 30B: 3: 242–247

Keywords: bioresorbable glass fibres, peripheral nerve regeneration

INTRODUCTION

Repairing a traumatically injured peripheral nerve canbe a protracted and frequently incomplete process; theclinical outcome is often unsatisfactory and there israrely a complete return of function (Birch et al., 1998;Lundborg, 2000). When severed nerve ends cannot bereapposed by tension-free sutures, these injuries areconventionally managed using an interfascicular orgroup fascicular autograft. The latter contain acutelydenervated, ‘‘axon-responsive’’ Schwann cells, lyingwithin a scaffold of longitudinally aligned basal laminae,and provide a microenvironment that is known tofacilitate axonal regrowth (Hall, in press). However,although they remain the gold standard, autografts offera less than ideal solution to the problem of bridging aninter-stump gap, particularly when the tissue defect isextensive (Shirley et al., 1996). Complications associatedwith their use include limited tissue volume andfascicular and modality mismatches between host anddonor nerves, secondary donor site morbidity, neuromaformation and numbness within the distribution of thedonor nerve.

The continuing search for an alternative to a nerveautograft has taken two main routes. In broad terms,either the nerve stumps are enclosed (entubulated)within some type of non-neural tube, fashioned fromnatural or synthetic material (McDonald and Zochodne,2003) or a suitably prepared peripheral nerve allograft,used in combination with low-grade immunosuppres-sion, is implanted into the inter-stump gap (Grand et al.,2002; Udina et al., 2004). Thus far, none of these optionshas outperformed an autograft when used in a shortgap, and none has proved effective in bridging longgaps.

An extensive literature search suggests that the designspecifications for an alternative to a nerve graft (whetherautograft or allograft) must include the provision of a

242

biocompatible and bioresorbable scaffold that supportsoutgrowing axons and their associated cells and withinwhich the microenvironment of a peripheral nerve fibrecan be replicated. Very similar generic requirementshave driven the search for alternatives to autografts inreplacing bone defects: research in this field has movedwith speed in the last decade and has benefited greatlyfrom the development and manipulation of bioactiveglasses and ceramics. Bioglasss is a widely used melt-derived bioactive glass (Hench, 1998; Hench et al., 2004;Kaufmann et al., 2000; Loty et al., 2001; Maquet et al.,2004; Verrier et al., 2004; Xynos et al., 2000, 2001) andits surface is chemically active. Biomaterials have beendeveloped that take advantage of the ability to tailor itssurface to specific applications (Lu et al., 2003).

We report here, in a proof of principle study, thatfibres of Bioglasss entubulated within silastic conduitsprovide a biocompatible and bioresorbable scaffold thatsupports axonal regeneration across 0.5 cm gaps in thesciatic nerves of adult rats: the axonal regrowth isqualitatively and quantitatively indistinguishable fromthat seen using an autograft.

MATERIALS AND METHODS

Bioglass 45S5 fibre preparation

Bioglass 45S5 (45% SiO2, 24% Na2O, 24.5% CaO and6% P2O5 in weight percent) was prepared by melting ina BLF-1700 high-temperature furnace at 1250 and1350 1C for 45 minutes each. The platinum cruciblewas removed from the furnace, and placed on a thermo-resistant asbestos plate. A Bioglass 45S5 monolith rodof 10 mm diameter was immersed in the liquid bioglassand removed at a rate of approximately 1 metre/second.The attached viscous bioglass 45S5 formed a continuous

ARTICLE IN PRESS

BIORESORBABLE GLASS FOR NERVE REGENERATION 243

fibre, whose diameter could be altered by varying theremoval speed. The resulting solid fibres were cut into10 cm-long sections, visually inspected at � 20 magni-fication for imperfections such as inclusions and stresscracks and then sorted into diameter sizes by micro-scopical sorting on a micrometer grid. Fibres werewashed twice using 70% methanol and allowed to airdry under a stream of sterile air before use. Asepticprocedures were used during subsequent handling of thefibres to ensure that they remained sterile.

Interactions of Bioglasss with cells in vitro

Preparation of Schwann cellsMale Wistar rats (225 g) were killed by CO2 asphyxia-tion. Their sciatic nerves were exposed in mid-thighthrough a muscle-splitting incision, and all majorbranches of the common peroneal and tibial nerveswere dissected out and washed twice in Hank’s BalancedSalt Solution (GIBCO) supplemented with gentamycin(50 mg/ml; Sigma Chemical Co., St. Louis, MO). Theepineurium and perineurium were stripped off with fineforceps under a dissecting microscope. Desheathednerves were blotted with sterile filter paper, choppedinto 1mm segments using a McIlwain tissue chopperand placed in 35 mm dishes containing Dulbecco’sModified Eagle’s Medium (DMEM) (GIBCO, GrandIsland, NY), supplemented with 10% fetal calf serum(FCS), (DMEM/FCS), and 50 mg/ml gentamycin. Ex-plants were left floating in this medium for 4 days at37 1C. Collagenase/dispase (0.1%, Boehringer Man-nheim Biochemicals, Indianapolis, IN) was added tothe medium 18 hours before dissociation, and explantswere dissociated on day 4 by gentle trituration through aflame-narrowed pipette (0.5–1 mm bore). Cells werestained with trypan blue (to differentiate them frommyelin debris) and the number of viable cells wascounted using a haemocytometer. To determine theproportions of the constituent cell types, aliquotscontaining 104 cells were plated onto poly-l-lyseinecoated 13 mm diameter coverslips and covered with 1 mlDMEM/FCS. After 12 hours in culture the coverslipswere removed, fixed for 10 minutes using 4% parafor-madehyde (PFA) (Sigma), and immunostained asdescribed below.

Determination of glass fibre biocompatibilityFive fibres (�10 mm in length; �150–200 mm diameter)were placed into each well of a 24-well cell culture plate.One millilitre of DMEM/FCS containing 2� 104 cellswas added to each well. After plating for 24 hours, theculture medium was removed from each well and thecells fixed with 250 ml of either 4% PFA for 1 hour.Several glass fibres fixed with PFA were stained withtoluidine blue, mounted in aqueous mounting medium(Serotec) and inspected. The remainder were immunos-tained with anti-S100 (DAKO; 1:100; 1 hour at RT)

which was visualized with anti-rabbit FITC (Sigma;1:100; 1 hour at RT). Fibres were mounted in Citifluorusing a CoverWell (Grace Biolabs) and viewed on aProvis AX70 microscope (Olympus).

Scanning electron microscopySamples were fixed in culture dishes with 4% gluter-aldehyde (TAAB) for 3 hours, washed 3� 5minutes in0.1 M sodium cacodylate buffer pH7.2, followed by90 minutes 1% osmium tetroxide in water, 2� 10 min-utes water washes, then dehydrated in an ascendingseries of ethanols, followed by 100% ethanol overnight.They were next transferred to 100% acetone in criticalpoint drying holders and critical point dried usingcarbon dioxide in a Polaron E3000 critical point dryer,mounted on stubs with rapid araldite and sputter coatedwith gold in a Polaron E5100 sputter coater. Specimenswere examined and images recorded using a PhilipsSEM501B scanning electron microscope fitted with aDeben Pixie 3000 digital scan generator and imagingsystem and operated at 15 kV.

Interactions of Bioglasss with regrowing axons in vivo

Surgery and subsequent histologyAll animals were handled ethically in accordance withthe guidelines of King’s College Health Guide for theCare and Use of Laboratory Animals. The Home Officefor Animal Care Committee approved the experimentalprocedures. Twenty four male Wistar rats were rando-mized into 4 groups (A–D) and deeply anaesthetizedusing Isoflourane by administered inhalation. Under adissecting microscope, the left sciatic nerve was trans-ected in mid-thigh and a 0.5 cm gap was created betweenthe proximal and distal nerve stumps. In group A, asilastic conduit (S.F. Medical, Hudson, MA) (internaldiameter 1.98 mm, external diameter 3.17 mm, length8mm) containing an average of 10 Bioglasss fibres(0.5 cm long; �25 mm diameter) was sutured between thestumps using 10/0 sutures (Ethicon). Groups B–D werecontrol groups. In group B, an empty silastic conduitwas sutured between the nerve stumps, in group C a0.5 cm section of the sciatic nerve was removed, reversedand sutured in place (i.e., an autograft), and in group Da piece of nerve was excised to leave a 0.5 cm inter-stump gap which was not treated further. In all cases,wounds were closed and animals were allowed torecover for 2 or 4 weeks. Animals were sacrificed atthese times by CO2 asphyxiation ðn ¼ 3Þ; and 3 cmlengths of nerve containing either the constructs (A, B),the autograft (C), or the tissue bridge that crossed theinitially empty inter-stump gap (D), were harvestedunder a dissecting microscope and immersion-fixedovernight in 4% PFA. After fixation, and under adissecting microscope, each silastic conduit in group Awas gently separated from its contents. Most of the glassfibres were removed from the tissue thus exposed using

ARTICLE IN PRESS

THE JOURNAL OF HAND SURGERY VOL. 30B No. 3 JUNE 2005244

fine forceps and the minimum of force: where resistancewas encountered they were left in situ. Extracted glassfibres were stained with toluidine blue to assess thenature of any adherent material.

Tissue processing and immunohistochemistryProcessed tissue was embedded in polyester wax (Steed-man, 1957). Each nerve was blocked out in a proximo-distal sequence in order to sample the proximal anddistal stumps and their associated outgrowth zones, andthe mid inter-stump gap. Sections cut 1 cm distal to theproximal end of the distal stump were used forquantitative analysis of the extent of axonal regenera-tion. Transverse sections if 10 mm were immunostainedwith the following antibodies: anti-EHS laminin (1:200,Sigma), anti-S-100 (1:200, DAKO), anti-200kD neuro-filament (1:400, Serotec), and ED1 (1:200, Serotec) toreveal basal laminae, Schwann cells, axons and recruitedmacrophages, respectively. Primary antibodies werevisualized using anti-mouse FITC or anti-rabbit TRITC(both Sigma 1:100). All immunostained samples weremounted in Citifluor and viewed on a Provis AX70microscope (Olympus). Images were captured using anAxiocam H100 and Axiovision software (Carl Zeiss).

QuantificationS-100 positive Schwann cell tubes and neurofilamentpositive axons were counted in double immunolabelledtransverse sections of each distal stump 1 cm distal to itsproximal end, 4 weeks after surgery. The number ofreinnervated Schwann cell tubes was expressed as apercentage of the total number of Schwann cell tubes ineach section. Regeneration in group A was comparedstatistically to that seen in groups B–D using one-wayANOVA and Bonferroni’s multiple comparison post-tests (Prism 3.0, Graph Pad software).

RESULTS

Interactions of Bioglasss with Schwann cells in vitro

Primary cell cultures obtained from digested rat sciaticnerves contained �50% Thy-1 immunoreactive fibro-blasts and �50% S-100 immunoreactive Schwann cells(data not shown). Phase microscopy and SEM revealedthat bipolar spindle-shaped cells with a typical Schwanncell morphology and large fibroblast-like flattened cellshad attached to both the fibres and the coverslips24 hours after plating cells onto Bioglasss fibres orpoly-l-lysine coated coverslips. (Fig 1a). The bipolarcells were S-100 positive (Fig 1b). Cell attached to allcalibres of glass fibre: Schwann cells were sometimesaligned in longitudinal chains on the smallest calibrefibres. The culture medium contained debris from thenerve digestion, but visual inspection revealed noobvious difference in the number of dead cells betweenthe Bioglasss and control groups.

Interactions of Bioglasss with regrowing axons in vivo

In groups A, B and D, a tissue bridge extendedbetween the proximal and distal stumps: the bridgewas markedly thicker at 4 weeks than at 2 weeks in allanimals, and was narrowest at both time points ingroup D. Regenerating axons grew within pre-existingSchwann cell tubes in the autografted nerve (group C),and in minifasicles in groups A, B and D. (A minifascicletypically contains a small group of o20 myelinated andunmyelinated regrowing axons and their associatedSchwann cells, the whole surrounded by one or twolayers of perineurial cells: such structures mayconveniently be identified by immunostaining thebasal laminae delineating the Schwann cell tubesand the perineurial cells.) In group A, the minifasciclesgrew in well-vascularized connective tissue that was fullof ‘‘holes’’ of varying sizes (Figs 1c and d). Presumablythe holes had housed the Bioglasss fibres prior totheir extraction at tissue harvesting. Although mostholes appeared empty, some contained what appeared tobe flakes of autofluorescent material, which weassume represented an early stage in the dissolution ofthe Bioglasss, and which may possibly correlate withthe tesselated appearance of the fibres seen in SEM(Fig 1a). It was notable that this material did notcause sectioning artefacts, suggesting that it wassofter than the preimplantation glass fibres, becausethis could not be sectioned satisfactorily. We suggestthat these persisting fibres were the ones that hadproved resistant to manual extraction. The connectivetissue surrounding the holes contained occasionalisolated, randomly oriented, S-110 positive Schwanncells: these cells were not usually associated with axons.Extraction of the glass fibres at harvesting appearedto cause minimal disruption to the surrounding tissueas assessed on tissue sections (Fig 1d): only smallproteinaceous fragments were observed on thesurface of extracted rods that had been stained withtoluidine blue.

ED1 positive macrophages were present in theepineurium (groups A–D), the interstump tissue bridges(groups A, B, D), and the endoneurium of the autografts(group C).

Quantification of axonal regrowth through constructs

Axonal outgrowth from the proximal nerve stumps andthe degree of penetration of the distal nerve stumps byregenerating axons was significantly more robust at both2 and 4 weeks in groups A and C than in groups B andD (Fig 2). Statistical analysis revealed there was nosignificant difference in re-innervation between groupsA and C ðP40:05Þ; but that re-innervation wassignificantly greater in group A than in either of groupsB or D (0:054P40:005 in both cases).

ARTICLE IN PRESS

Fig 1 (a and b) Representative fields from the in vitro assessment of the interaction of non-neuronal cells and Bioglasss fibres, after 24 hours in

culture. (a) SEM of a bipolar cell with the morphological characteristics of a Schwann cell adhering to Bioglasss fibre; (b) S-100

immunopositive Schwann cells adhering to Bioglasss fibre, note Hoescht stained nuclei of (predominantly) fibroblasts also adhering to the

fibre. Figs 1(c) and (d) are representative fields of immunostained transverse sections of tissue extracted from the lumen of a conduit in

experimental group A, 4 weeks after implantation of conduits. Sections were double imunostained with anti-laminin 1 antibody (visualized

with FITC) and anti-200kD neurofilament antibody (visualized with TRITC). In (c) autofluorescent material remains within the spaces

formerly occupied by the Bioglasss fibres, BG,� 225. The field in (d) shows the vascular connective connective tissue surrounding a ‘‘hole’’,

BG; v ¼ blood vessel; mf ¼ minifascicle: � 450.

BIORESORBABLE GLASS FOR NERVE REGENERATION 245

DISCUSSION

We have demonstrated in this proof of principle studythat fibres of Bioglasss ensheathed within a silastic tubecan support axonal regrowth across a 0.5 cm gap in anadult rat sciatic nerve. We used a well-established testsystem because we wished to examine the behaviour ofthe Bioglasss fibers and not the conduit. Quantitatively,the axonal regrowth that occurred in 4 weeks wasstatistically indistinguishable from that seen using anautograft and was at least ten times greater than theregrowth achieved in this time across either an emptysilastic tube or an unrepaired gap.

Our results are consistent with previous findings thatlongitudinally aligned substrates facilitate axonal regen-eration, whether natural, e.g. tendon (Brandt et al., 1999)

or acellular sarcolemmal tubes (Enver and Hall, 1994;Hall, 1997), or artificially constructed, e.g. magneticallyaligned type I collagen gel (Ceballos et al., 1999), orbundles of type I collagen filaments (Yoshii and Oka,2001). On the basis of extrapolations from in vitro results(Dubey et al., 1999), the underlying mechanism isbelieved to be contact guidance of outgrowing axonsand/or their associated non-neuronal cells.

Bioglasss has been shown to support the growth anddifferentiation of osteoblasts and osteoblast-like cells invitro (Kaufmann et al., 2000; Loty et al., 2001; Lu et al.,2003). We have shown here for the first time thatBioglasss fibres support the attachment and spreadingof Schwann cells in vitro: we believe that these fibres alsoprovided a scaffold that was invaded by cells growingout from proximal and distal nerve stumps in vivo (Hall,

ARTICLE IN PRESS

Sila

stic

+F

ibre

s (A

)

Sila

stic

(B

)

Gra

ft (C

)

Gap

(D

)0

25

50

75

Control or conduit type used

% S

100

imm

un

ore

acti

vetu

bes

co

nta

inin

g N

Fim

mu

no

reac

tive

axo

ns

Fig 2 The percentage of S-100 positive Schwann cell tubes containing

neurofilament positive axons 1 cm distal to the proximal end of

the distal stump 4 weeks after implantation. There was no

significant difference in re-innervation between groups A and C

ðP40:05Þ: Re-innervation was significantly greater in group A

than in either of groups B or D (0:054P40:005 in both cases).

THE JOURNAL OF HAND SURGERY VOL. 30B No. 3 JUNE 2005246

2004; Weis et al., 1994; Whitworth et al., 1995; Williams,1987), and that these cells subsequently secreted anextracellular matrix that facilitated axonal regrowth.

That rodent peripheral axons can cross an interstumpgap of �0.5 cm is well documented: the phenomenonhas been reported in a variety of experimental modelssince the introduction of entubulation as a research toolover 20 years ago. However, we believe that our resultsare noteworthy for several reasons. Axonal regrowthwas comparable with that seen using an autograft,suggesting that the lag time before axons and Schwanncells started to grow out from the proximal stumps wasminimal and reinforcing the biocompatibility of theBioglasss fibres. We noted that some of the Bioglasss

fibres were already beginning to break down, which is animportant consideration given that biocompatibility andbioresorbability are absolute requirements of successfultissue-engineered devices. We tested the fibres within anon-resorbable tube, the properties of which are wellestablished. An optimized device would use a tube thatelicited minimal tissue response in the wound bed andwas bioresorbable, such as a biodegradable controlledrelease inorganic polymer glass (Gilchrist et al., 1998;Lenihan et al., 1998).

Recent evidence suggests that Bioglasss compositeswith a biodegradable polymeric phase may be useful asscaffolds in bone and lung tissue engineering (Verrier etal., 2004). In the context of nerve repair, bioengineeredBioglasss fibres could be used to deliver growth factorsand/or adherent cells within the lumen of a nerveconduit. From a clinical perspective, the fibres can bemany centimetres long, packed into conduits of varying

internal diameters, and their manufacture is indepen-dent of the time of use: Bioglasss fibres therefore havethe potential to be used in the most challenging cases,where inter-stump gaps are longer and nerves are widerthan those used in this study.

Acknowledgements

We are grateful to Dr Tony Brain, Centre for Ultrastructural Imaging,King’s College London, for expert technical assistance with SEM andto Garrit Koller, GKT Dental Institute, for preparation of theBioglasss fibres.

References

Birch R, Bonney G, Wynn Parry CB. Surgical disorders of theperipheral nerves. Churchill Livingstone, Edinburgh, 1998.

Brandt J, Dahlin LB, Kanje M, Lundborg G (1999). Spatiotemporalprogress of nerve regeneration in a tendon autograft used forbridging a peripheral nerve defect. Experimental Neurology, 160:386–393.

Ceballos D, Navarro X, Dubey N, Wendelschafer-Crabb G, KennedyWR, Tranquillo RT (1999). Magnetically aligned collagen gelfilling a collagen nerve guide improves peripheral nerve regenera-tion. Experimental Neurology, 158: 290–300.

Dubey N, Letourneau PC, Tranquillo RT (1999). Guided neuriteelongation and schwann cell invasion into magnetically alignedcollagen in simulated peripheral nerve regeneration. ExperimentalNeurology, 158: 338–350.

Enver MK, Hall SM (1994). Are Schwann cells essential for axonalregeneration into muscle autografts?. Neuropathology and AppliedNeurobiology, 20: 587–598.

Gilchrist T, Glasby MA, Healy DM, Kelly G, Lenihan DV, McDowallKL, Miller IA, Myles LM (1998). In vitro nerve repair – in vivo.The reconstruction of peripheral nerves by entubulation withbiodegradeable glass tubes – a preliminary report. British Journalof Plastic Surgery, 51: 231–237.

Grand AG, Myckatyn TM, Mackinnon SE, Hunter DA (2002).Axonal regeneration after cold preservation of nerve allografts andimmunosuppression with tacrolimus in mice. Journal of Neuro-surgery, 96: 924–932.

Hall S (1997). Axonal regeneration through acellular muscle grafts.Journal of Anatomy, 190: 57–71.

Hall S. Mechanisms of repair after traumatic injury. In: Dyck P,Thomas PK (Eds). Peripheral neuropathy. Amsterdam, Elsevier,2005, Vol 4, Ch 58 (in press).

Hench LL (1998). Bioceramics. Journal of the American CeramicSociety, 81: 1705–1728.

Hench LL, Xynos ID, Polak JM (2004). Bioactive glasses for in situtissue regeneration. Journal of Biomaterial Science PolymerEdition, 15: 543–562.

Kaufmann EABE, Ducheyne P, Shapiro IM (2000). Evaluation ofosteoblast response to porous bioactive glass (45S5) substrates byRT-PCR analysis. Tissue Engineering, 6: 19–28.

Lenihan DV, Carter AJ, Gilchrist T, Healy DM, Miller IA, Myles LM,Glasby MA (1998). Biodegradable controlled release glass in therepair of peripheral nerve injuries. Journal of Hand Surgery, 23B:588–593.

Loty C, Sautier JM, Tan MT, Oboeuf M, Jallot E, Boulekbache H,Greenspan D, Forest N (2001). Bioactive glass stimulates in vitroosteoblast differentiation and creates a favorable template for bonetissue formation. Journal of Bone and Mineral Research, 16: 231–239.

Lu HH, El-Amin SF, Scott KD, Laurencin CT (2003). Three-dimensional, bioactive, biodegradable, polymer-bioactive glasscomposite scaffolds with improved mechanical properties supportcollagen synthesis and mineralization of human osteoblast-like cellsin vitro. Journal of Biomedical Materials Research, 64A: 465–474.

ARTICLE IN PRESS

BIORESORBABLE GLASS FOR NERVE REGENERATION 247

Lundborg G (2000). A 25-year perspective of peripheral nerve surgery:evolving neuroscientific concepts and clinical significance. Journalof Hand Surgery, 25A: 391–414.

Maquet V, Boccaccini AR, Pravata L, Notingher I, Jerome R (2004).Porous poly(alpha-hydroxyacid)/Bioglass composite scaffolds forbone tissue engineering. I: preparation and in vitro characterisa-tion. Biomaterials, 25: 4185–4194.

McDonald DS, Zochodne DW (2003). An injectable nerve regenera-tion chamber for studies of unstable soluble growth factors.Journal of Neuroscience Methods, 122: 171–178.

Shirley DM, Williams SL, Covey JF, Santos PM (1996). A functionalmodel of nerve repair. Archives of Otolaryngology and Head andNeck Surgery, 122: 785–788.

Steedman H (1957). Polyester wax. A new ribboning embeddingmedium for histology. Nature, 179 (4574): 1345–1346.

Udina E, Gold BG, Navarro X (2004). Comparison of continuous anddiscontinuous FK506 administration on autograft or allograftrepair of sciatic nerve resection. Muscle & Nerve, 29: 812–822.

Verrier S, Blaker JJ, Maquet V, Hench LL, Boccaccini AR (2004).PDLLA/bioglass composites for soft-tissue and hard-tissue en-gineering: an in vitro cell biology assessment. Biomaterials, 25:3013–3021.

Weis J, May R, Schroder JM (1994). Fine structural and immunohis-tochemical identification of perineurial cells connecting proximaland distal stumps of transected peripheral nerves at early stages ofregeneration in silicone tubes. Acta Neuropathologica, 88:159–165.

Whitworth IH, Dore C, Hall S, Green CJ, Terenghi G (1995).Different muscle graft denaturing methods and their use for nerverepair. British Journal of Plastic Surgery, 48: 492–499.

Williams LR (1987). Exogenous fibrin matrix precursors stimulate thetemporal progress of nerve regeneration within a silicone chamber.Neurochemical Research, 12: 851–860.

Yoshii S, Oka M (2001). Peripheral nerve regeneration along collagenfilaments. Brain Research, 888: 158–162.

Xynos ID, Edgar AJ, Buttery LDK, Hench LL, Polak JM (2001).Gene-expression profiling of human osteoblasts following treat-ment with the ionic products of Bioglass 45S5 dissolution. Journalof Biomedical and Material Research, 55: 151–157.

Xynos ID, Hukkanen MVJ, Batten JJ, Buttery LD, Hench LL, PolakJM (2000). Bioglass 45S5 stimulates osteoblast turnover andenhances bone formation in vitro: implication and applicationsfor bone tissue engineering. Calcified Tissue International, 67:321–329.

Received: 2 September 2004Accepted after revision: 5 November 2004Professor Susan Standring, Wolfson Centre for Age-Related Diseases, King’s College London,Hodgkin Building, Guy’s campus. London SE1 1UL, UK. Tel.: +20 7848 6083.E-mail: susan.standring@kcl.ac.uk

r 2005 The British Society for Surgery of the Hand. Published by Elsevier Ltd. All rightsreserved.doi:10.1016/j.jhsb.2004.11.003 available online at http://www.sciencedirect.com