Acta Biomaterialia 6 (2010) 4027–4033

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Acta Biomaterialia

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Enhancement of nerve regeneration along a chitosan nanofiber mesh tube onwhich electrically polarized b-tricalcium phosphate particles are immobilized

Wei Wang a, Soichiro Itoh b,*, Naoki Yamamoto a, Atsushi Okawa c, Akiko Nagai a, Kimihiro Yamashita a

a Department of Inorganic Materials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japanb Affiliated Facility for Clinical and Fieldwork Practices, International University of Health and Welfare, 6-1-14 Kounodai, Ichikawa-shi, Chiba 272-0827, Japanc Department of Orthopaedic and Spinal Surgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 February 2010Received in revised form 28 April 2010Accepted 30 April 2010Available online 6 May 2010

Keywords:Tissue regenerationNerve conduitChitosanb-Tricalcium phosphateElectrical polarization

1742-7061/$ - see front matter � 2010 Acta Materialdoi:10.1016/j.actbio.2010.04.027

* Corresponding author.E-mail address: itoso.gene@kaken-hp.or.jp (S. Itoh

The ability of b-tricalcium phosphate (b-TCP) particles to store electric charge was confirmed by ther-mally stimulated depolarization current measurement as well as surface potential measurement. Theefficacy of stored electrical charge on b-TCP particles in enhancing nerve regeneration was evaluated.Bridge grafting was performed into sciatic nerve defects in Wistar rats with the following tubes: chitosanmesh tubes; chitosan mesh tubes on which b-TCP particles with or without electrical polarization treat-ment had been immobilized (polarized and non-polarized tubes, respectively). As a control, isograftswere used. Both motor and sensory nerve function as well as electrophysiological recovery progressedwith time in each group. Immunofluorescence revealed rapider nerve regeneration in the polarized tubegroup compared with the non-polarized tube group. The axon density and axon area in the polarized tubegroup were significantly greater than those in the chitosan mesh tube and non-polarized group, andshowed no significant differences from the control group. These results suggest that the stored chargeon electrically polarized b-TCP particles immobilized on chitosan mesh tubes may enhance nerve regen-eration to the same extent as isografting.

� 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

The repair of peripheral nerve lesions has been attempted bybridging with a scaffold, which includes implantation and tubuli-zation techniques. At present, autogenous nerve grafting is themost successful method for nerve repair. A wide variety of bioma-terials, however, have been suggested for the production of artifi-cial devices for nerve repair, and biodegradable scaffolds may bean alternative [1]. Chitosan has a number of beneficial biologicalproperties; enhancing wound healing, suppressing bacterialgrowth and easy fabrication into various shapes, such as films,sponges and fibers. Chitosan mesh tubes with a deacetylation rate(DAc) of 93% have been developed [2]. Given the molecular charac-teristics as well as sufficient mechanical strength, these tubes maybe substitutes for autogenous nerve grafts. The functional and his-tological results, however, have revealed that nerve regenerationwas delayed in chitosan mesh tube grafts relative to autografts[2]. This result indicates that the nerve conduit must offer addi-tional advantages, such as enhanced growth of Schwann cells orthe growth cone and adsorption of bioactive molecules on the tubesurface.

ia Inc. Published by Elsevier Ltd. A

).

It is reported that negatively charged surfaces of electricallypolarized hydroxyapatite (HA) ceramics enhance bone bondingwhen transplanted into canine femora [3]. To explain this phenom-enon it was hypothesized that the polarizing charge could inducethe orientation of OH� ions in the HA structure [4,5]. Dehydroxyla-tion during the measurement of electrical conductivity in air led toan increase in conductivity due to an increase in the initial concen-tration of free protons. Proton transport polarization, however,cannot affect b-tricalcium phosphate (b-TCP), which has com-monly been used for the preparation of biodegradable compositematerials, because it does not contain protons. For this reason, ithas been believed up to now that it is impossible to electricallypolarize b-TCP ceramics. We have succeeded for the first time,however, in confirming that b-TCP can be electrically polarized.In addition to the adsorption of charge-compensatory ions to thenegatively charged surface, large positive surface charges inducedby electrical polarization may affect osteogenic cell activity, result-ing in enhanced new bone growth [6]. Recent studies indicate thatlow intensity electrical stimulation is equivalent to various growthfactors [7,8]. Moreover, it has been reported that electric and elec-tromagnetic fields enhance regeneration following nerve injury [7–10].

In this study the ability of b-TCP to store an electrical charge in-duced by electrical polarization was confirmed using thermally

ll rights reserved.

4028 W. Wang et al. / Acta Biomaterialia 6 (2010) 4027–4033

stimulated depolarization current (TSDC) and electrical conductionmeasurements. Furthermore, the efficacy of the stored electricalcharge on b-TCP particles in enhancing nerve regeneration wasevaluated using a nerve bridging model in rats.

2. Materials and methods

2.1. Preparation of b-TCP

b-TCP was manufactured from commercially available(NH4)2HPO4 and CaCO3 powders (Wako Pure Chemical Industries,Osaka, Japan). The initial weight ratio of (NH4)2HPO4 to CaCO3

was 1.0:1.5. After milling these powders with 70 vol.% ethanol atroom temperature for 48 h the ethanol was completely removedusing a rotary evaporator. The mixed powders were sintered at1100 �C for 24 h in air to obtain b-TCP powder, which was mixedinto a slurry with 3.0 vol.% polyvinyl alcohol (Wako Pure ChemicalIndustries) and then spray dried to spherical particles followed bysintering at 500 �C for 30 min to eliminate the polyvinyl alcoholcomponent.

2.2. Characterization of b-TCP

X-ray diffractometry (XRD) was performed on b-TCP particlesusing a diffractometer control (PW-1710, Phillips, Eindhoven,The Netherlands) fitted with a 4 kW X-ray generator, copper tar-get and graphite monochromator (each n = 3). Infrared absorptionspectra of b-TCP particles were measured by the KBr pellet meth-od using a Fourier transform infrared (FTIR) spectrophotometer(FTIR-500, JASCO, Tokyo, Japan) in the range 4000–400 cm�1

(n = 3).Electrical polarization was carried out according to a previous

study [4]. b-TCP particles were put in an alumina ring with thediameter of 2.0 cm and height of 0.5 cm, clamped between a pairof platinum electrodes and electrically polarized in a direct current(DC) field of 4 kV cm�1 at 400 �C for 1 h in air (Fig. 1). The sampleswere cooled to room temperature under electric field polarization.TSDC measurement of the electrically polarized b-TCP particleswas performed. The measurements were carried out in air fromroom temperature to >700 �C at a heating rate of 5.0 �C min�1.The stimulated current was measured with a Hewlett–Packard4140B pA meter (Palo Alto, CA) and analyzed with the appropriatecomputer software to calculate the stored electric charge. TheArrhenius equation was plotted from the graph with ln s(T) on

Fig. 1. Scheme of the method to electrically polarize b-TCP particles. The b-TCPparticles were put in an alumina ring with a diameter of 2.0 cm and height of0.5 cm, clamped between a pair of platinum (Pt) electrodes and electricallypolarized by applying a DC field of 4 kV cm�1 at 400 �C for 1 h in air. The sampleswere cooled to room temperature under the electric field.

the y-axis and T�1 (K�1) on the x-axis, where s is the orientationtime(s) and T is the absolute temperature (K). The gradient was cal-culated by the method of least squares, giving the energy of activa-tion for relaxation of the electrical polarization.

2.3. Preparation of the chitosan nanofiber mesh tube

The chitosan mesh tube (inner diameter 1.2 mm, thickness –0.3–0.5 mm) was prepared by electrospinning [2]. Polarized ornon-polarized b-TCP particles were immobilized on the tube(15 mg cm�2) by spreading the ethanol suspension. The surfacepotentials of chitosan nanofiber mesh tubes and those with polar-ized or non-polarized b-TCP particles were measured using an ori-ginal Kelvin probe apparatus. Measurements were performed atthree evenly separated points along the axis of each tube (n = 3for each tube).

The prepared chitosan nanofiber mesh tubes with polarized ornon-polarized b-TCP particles were fixed in 2.5 vol.% glutaralde-hyde in 0.1 M phosphate-buffered saline (PBS), post-fixed with1% OsO4 buffered with 0.1 M PBS and dehydrated through a gradedethanol series. Specimens were dried in a critical point dryingapparatus (hcp-2, Hitachi, Tokyo, Japan) with liquid CO2, ion sput-ter coated with platinum and examined by scanning electronmicroscopy (SEM) (Model S-4500, Hitachi). The size and densityof the b-TCP particles immobilized on the chitosan mesh tubeswere measured on the SEM images of the composite tubes usingImage Pro Plus 6.0 software (Media Cybernetics, Carlsbad, CA) forWindows (n = 3 for each group).

2.4. Implantation of the chitosan nanofiber mesh tubes

Male Wistar rats weighing 180–200 g were anesthetized withan intraperitoneal injection of sodium pentobarbital (50 mg kg�1

body weight). The right sciatic nerve was exposed and a 10 mmlong section was excised at the center of the thigh. A graft of15 mm in length was performed by end to end suturing with 8–0monofilament nylon to connect nerve ends with the followingchitosan tubes (n = 8 in each group): chitosan mesh tubes with aDAc of 93%; chitosan mesh tubes on which b-TCP particles withor without electrical polarization treatment were immobilized(polarized tube and non-polarized tube, respectively). As a control,portions of sciatic nerve 15 mm in length were harvested fromother Wistar rats and grafted into the gap resulting from the resec-tion of a 10 mm segment of the right sciatic nerve in eight rats(isografts).

2.5. Assessment of function recovery

To assess the recovery of motor and sensory function associatedwith the sciatic nerve, the static toe spread factor (STSF) and vonFrey hair test, respectively, were evaluated every 4 weeks until12 weeks post-implantation.

The test animals were placed on a transparent plastic plate un-der which a digital camera (Exilim-Z55, Casio, Tokyo, Japan) wasset at a distance of 10 cm beneath the surface. After an intervalto permit the animal to adapt to the new environment, threeframes of both hind feet were taken for each rat. The images werethen transferred to a personal computer and the distance betweenthe spread first and fifth toes was determined. Measurements weretaken from three consecutive images and averaged for each side.The results were assessed using a simplified formula from the sta-tic sciatic index (SSI: scale 1–3):

STSF ¼ ðlength between the 1st and 5th toes on the normal side

� length between the 1st and 5th toes on the experimental side� length between the 1st and 5th toes on the normal side:

W. Wang et al. / Acta Biomaterialia 6 (2010) 4027–4033 4029

The test rats were placed on a wire mesh plate and permitted toadapt to the new environment. Nylon monofilaments (Touch-TestSensory Evaluator, North Coast Medical, CA) were then used tostimulate the sole of the hind feet from below. The size of themonofilament was recorded when a paw withdrawal or lick reac-tion was repeatedly observed. Both sides were tested and the re-sults were calculated using the formula:

ðsize on the experimental side� size on the normal sideÞ� size on the normal side:

2.6. Electrophysiological evaluation

Electrophysiological evaluations were carried out at 12 weekspost-implantation (n = 5 for each group) under anesthesia achievedby intraperitoneal injection of ketamine hydrochloride (40–50 mg kg�1 body weight). A bipolar stimulating electrode wasplaced at the sciatic notch, proximal to the anastomosed sitearound the exposed sciatic nerve, and compound muscle actionpotentials (CMAPs) evoked in response to 1 Hz square wave pulses(superthreshold intensity, 0.2 ms duration, provided by a Neuro-pack 8, Nihon Kohden Co., Tokyo, Japan) were recorded from thetriceps surae muscle. To evaluate motor nerve recovery the ampli-tudes of the CMAPs were measured peak to peak. The results wereassessed using the formulae:

amplitude ¼ ðamplitude on the normal side

� amplitude on the experimental side� amplitude on the normal side;

latency ¼ ðlatency on the experimental side

� latency on the normal side� latency on the normal side:

Fig. 2. Representative TSDC spectrum of the polarized b-TCP particles. The TSDCcurve increased at 450 �C, reaching a maximum at 650 �C. The stored charge in b-TCP was 13.0 lC cm�2.

2.7. Histomorphological evaluation

For each experimental group three and five rats, respectively,were killed 4 and 12 weeks post-implantation by intraperitonealinjection of a high dose of sodium pentobarbital. Samples were ta-ken from the middle third of the grafted tube for each group andsectioned transversely.

Samples harvested 4 weeks post-implantation were fixed in 4%paraformaldehyde in PBS, embedded in paraffin and sliced trans-versely to 4 lm thick sections for immunofluorescence staining.The sections were incubated overnight at 4 �C with a mixture ofrabbit anti-S100 protein antibody (1:100, Sigma, St. Louis, MO)and mouse anti-nuerofilament 160 k monoclonal antibody(1:100, clone NN18, SigmaA) for primary incubation and for 1 hat room temperature with a mixture of goat anti-mouse IgG FITCfor NF160 and goat anti-rabbit IgG TRITC for S-100 (Sigma) at finaldilutions of 1:300 for secondary incubation. After washing, cover-slips were mounted on glass slides with 20 vol.% glycerol/10 vol.%polyvinyl alcohol in 0.1 M Tris–HCl buffer, pH 8.0. The tissue spec-imens were then examined under a microscope equipped withultraviolet, fluorescein and rhodamine optics (AX80TR, Olympus,Tokyo, Japan).

The samples harvested 12 weeks post-implantation were sec-tioned transversely and embedded in Epon 812 resin after fixationwith 2.5 vol.% glutaraldehyde in 0.1 M PBS, followed by post-fixa-tion in 1% OsO4 in 0.1 M PBS. Thin transverse sections (1 lm thick)were stained with toluidine blue for light microscopy, causing themyelin of the myelinated axons to be stained blue (BH-2, Olym-pus). Images of whole sections of the implanted tubes and isografts

were captured to measure the average axon diameter, density andarea excluding myelin. The Image Pro Plus 6.0 software (MediaCybernetics) was employed for image analysis.

These experiments were carefully conducted by veterinarians inaccordance with the Guidelines for Animal Experimentation (To-kyo Medical and Dental University) as well as the National Insti-tutes of Health (NIH) Guidelines of the USA regarding the careand use of animals in experimental procedures (NIH PublicationsNo. 80-23, 1996 revision).

2.8. Statistical analysis

The variance among the experimental and control groups wasdetermined and evaluated for statistical significance using theBartlett test. Differences were assessed using one-way analysis ofvariance (ANOVA). Thereafter, statistical significance was evalu-ated using the Bonferroni/Dunn’s multiple comparison test.

3. Result

3.1. Characterization of b-TCP

The XRD patterns of the synthesized b-TCP particles showed therepresentative peaks of b-TCP; the lattice parameters a-axis = 10.429 Å and c-axis = 37.38 Å matched those of b-TCP. TheFTIR patterns of the synthesized particles also showed the repre-sentative peaks of b-TCP; the peaks attributable to v1PO4, v3PO4

and v4PO4 matched well with those of b-TCP.

3.2. Electrical polarization of b-TCP

A representative TSDC spectrum of the polarized b-TCP powderis shown in Fig. 2. The slope of the TSDC curve increased at 450 �C,reaching a maximum at 650 �C. The stored charge on b-TCP was13.0 lC cm�2.

The relationship of log s to 1000/T obtained from TSDC mea-surement of the b-TCP particles is shown in Fig. 3. The approximateformula of this curve was y = 19.1 � �14.4. The energy of activa-tion for relaxation of the electrical polarization was 1.65 eV.

Fig. 3. The relationship of log s and 1000/T obtained from the TSDC measurementsof the b-TCP particles. The approximate formula of this curve is y = 19.1 � –14.4with ln s(T) on the y-axis and T�1 (K�1) on the x-axis. The energy of activation forrelaxation of the electrical polarization was 1.65 eV.

Table 1The static toe spread factor STSF (means ± SD).

Tube Non-polarized tube Polarized tube Isograft

4w 0.68 ± 0.04 0.63 ± 0.07 0.65 ± 0.08 0.63 ± 0.058w 0.63 ± 0.04 0.58 ± 0.04 0.59 ± 0.38 0.58 ± 0.0512w 0.58 ± 0.04 0.54 ± 0.08 0.53 ± 0.01 0.51 ± 0.03

Table 2von Frey hair test (means ± SD).

Tube Non-polarized tube Polarized tube Isograft

4w 0.30 ± 0.06 0.29 ± 0.04 0.22 ± 0.04 0.22 ± 0.038w 0.19 ± 0.02 0.20 ± 0.03 0.17 ± 0.04 0.18 ± 0.0312w 0.14 ± 0.06 0.17 ± 0.02 0.13 ± 0.02 0.11 ± 0.02

Table 3Electrophysiological evaluation using the amplitudes and latencies of CMAPs(means ± SD).

Tube Non-polarized tube Polarized tube Isograft

Amplitude 0.40 ± 0.05 0.40 ± 0.03 0.35 ± 0.05 0.33 ± 0.12Latency 0.61 ± 0.19 0.50 ± 0.20 0.44 ± 0.24 0.40 ± 0.22

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3.3. Properties of the chitosan nanofiber mesh tubes

The surface potential of polarized b-TCP particles immobilizedon the chitosan mesh tubes varied along the axis in the range�200 to +200 mV, while the non-polarized and non-powdergroups presented in the range �15 to + 15 mV.

The SEM images of the chitosan mesh tubes revealed that b-TCPparticles were dispersed evenly on the mesh of the tube outer sur-face, forming spherical clusters of b-TCP particles (Fig. 4). The meansize and density of b-TCP particles immobilized on the chitosanmesh tubes were 36 ± 7.4 lm and 391 ± 83 mm�2, respectively.

3.4. Recovery of sensory and motor function after sciatic nerve injury

The von Frey hair test and STSF values indicated that both themotor and sensory nerve function had recovered with time in each

Fig. 4. SEM images of the chitosan mesh tube immobilized with polarized b-TCP particle360 lm. (B) Enlargement of the chitosan mesh tube wall shown in (A). Spherical b-TCP150 lm. (C) Enlargement of the spherical b-TCP particles shown in (B). The polarized b-

group after implantation (Tables 1 and 2). There were no signifi-cant differences in function recovery among these groups.

3.5. Electrophysiological evaluation

The CMAPs were recorded in five rats in each group. There wereno significant differences in amplitude and latency among thegroups (Table 3).

3.6. Histology of the regenerated nerve in the implanted tube

The inner space of the chitosan nanofiber mesh tube was pre-served and, while b-TCP particles attached to the outer surface ofthe tube until 12 weeks after implantation. A mild inflammatoryresponse occurred around the tube wall. Regenerating axons under

s. (A) The chitosan mesh tube on which b-TCP particles were immobilized. Scale barparticles were dispersed evenly on the mesh of the tube outer surface. Scale bar

TCP particles formed spherical clusters. Scale bar 25 lm.

Fig. 5. Histological views of b-TCP particles immobilized on chitosan mesh tubes with and without electrical polarization 4 weeks after implantation. Toluidine blue stain. (A)Polarized tube. Scale bar 500 lm. (B) Enlargement of the frame shown in (A) Regenerating axons under myelinization (arrow) elongated along the inner wall of the chitosanmesh and were accompanied by abundant newly formed blood vessels (V). Scale bar 50 lm. (C) Non-polarized tube. Scale bar 500 lm. (D) Enlargement of the frame shown in(C). Non-myelinated axons and immature myelinated axons were predominantly found. Scale bar 50 lm.

W. Wang et al. / Acta Biomaterialia 6 (2010) 4027–4033 4031

myelinization elongated along the inner wall of the chitosan meshand were accompanied by abundant newly formed blood vessels asearly as 4 weeks after implantation in the polarized tubes (Fig. 5Aand B). Non-myelinated axons and immature myelinated axonswere predominantly found in the non-polarized tubes (Fig. 5Cand D). Immunofluorescence revealed many myelinated axonsaccompanied by Schwann cells in the polarized tubes (Fig. 6Aand C). In the non-polarized tubes, however, the numbers of mye-

Fig. 6. Immunofluorescence staining of the implanted b-TCP tubes shown in Fig. 5. (A) Powere predominantly found in the polarized tube, compared with the non-polarized tubewere accompanied by Schwann cells stained red with anti-s100. Scale bar 200 lm.

linated axons were few. Many myelinated axons had matured tomass on a large monofascicle in each group 12 weeks after implan-tation (Fig. 7A–D).

At 12 weeks after implantation there was no significant differ-ence in average axon diameter of myelinated axons among thegroups (Table 4). The axon density and area in the polarized groupwere significantly larger, however, than those in the tube (P < 0.05)and non-polarized group (P < 0.01). The axon density and area in

larized tube. (B) Non-polarized tube. Myelinated axons stained green with NF 160 k. Scale bar 100 lm. (C) Enlargement of the frame shown in (A). Regenerating axons

Fig. 7. Histological views at 12 weeks after implantation. Toluidine blue stain. (A) Polarized tube. (B) Non-polarized tube. (C) Chitosan mesh tube. (D) Isograft. Many large andsmall myelinated axons had matured to mass on a large monofascicle in each group. Scale bar 50 lm.

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the isograft group were significantly larger than those in the tubeand non-polarized groups (each P < 0.01).

4. Discussion

The ability of b-TCP to store an electric charge was confirmed bythe TSDC measurements, as well as the surface potential measure-ments. Although the mechanism remains to be ascertained, electri-cal polarization of the ceramics was stable and ongoing [5]. Ratherthan proton transport polarization by inducing orientation of OH�

ions as in the HA structure, a charge carrier such as Ca2+ or O2� isassumed for b-TCP. Oxide ion conduction has already been provenwith an oxygen concentration cell in ceramic oxyhydroxyapatite(Ca10 � xYx(PO4)6(OH)2 � xOx), where Ca2+ is partially substitutedby Y3+, giving vacancies at OH� lattice sites for charge compensa-tion [11], and carbonate apatite [12]. As the lattice structure of b-TCP contains some Ca2+ vacancies, the conduction of Ca2+ ions ispossible.

The inner space of the chitosan nanofiber mesh tube was pre-served without a prominent inflammatory response throughoutthe observation period. Functional recovery of both motor and sen-

Table 4Histological analysis of the grafted tubes and isograft harvested at 12 weeks afterimplantation (means ± SD).

Tube Non-polarizedtube

Polarizedtube

Isograft

Axon diameter (lm) 1.72 ± 0.09 1.65 ± 0.13 1.72 ± 0.13 1.63 ± 0.14Axon density

(� 102 lm�2)3.21 ± 0.42 2.77 ± 0.79 4.71 ± 0.44a,b 5.31 ± 0.73b,b

Axon area (%) 14.0 ± 1.8 13.4 ± 1.8 21.8 ± 2.8a,b 25.5 ± 3.2b,b

The axon density and axon area in the polarized tube group were significantlygreater than those in the tube (P < 0.05) and non-polarized group (P < 0.01). Theaxon density and axon area in the isograft group were significantly greater thanthose in the tube and non-polarized group (each P < 0.01).

a P < 0.05.b P < 0.01.

sory nerves occurred with time after implantation without signifi-cant differences among the groups. Electrophysiological recoveryalso progressed in each group. These results suggest that chitosannanofiber mesh tubes are suitable conduits for the sprouting andelongation of myelinated axons accompanied by Schwann cellmigration, followed by axonal maturation. Immunofluorescencerevealed more rapid nerve regeneration in the polarized tubegroup compared with the non-polarized tube group. The axon den-sity and area in the polarized tube group were significantly largerthan in the chitosan mesh tube and non-polarized groups, andthere were no significant differences from the control group. Theseresults suggest that the stored charge in the electrically polarizedb-TCP particles enhanced nerve regeneration comparably with iso-grafting. Dramatic up-regulation of mRNA encoding brain-derivedneurotrophic factor (BDNF) and TrkB in response to electrical stim-ulation has been reported [13]. This observation is consistent withevidence implicating BDNF in the stimulation of both central ner-vous system [14–16] and peripheral nervous system [17–19]regeneration. It was also reported that transient post-transectionloss of target-derived nerve growth factor (NGF) can affect growthfactor activity and levels, and that pulsed electromagnetic fieldsmight promote nerve regeneration by amplifying an early post-in-jury decline in NGF activity [20]. Furthermore, it was found that in-creased adsorption of the cell adhesion molecule fibronectin withelectrical stimulation enhanced neurite out-growth on polypyrrole[9]. These reports suggest the possibility of enhancing nerve regen-eration by electromagnetic stimulation without the addition ofeither neurotrophic factors or cell adhesion molecules.

5. Conclusion

It is suggested that chitosan nanofiber mesh tubes are suitablescaffolds for nerve conduits and that the stored charge in electri-cally polarized b-TCP particles may enhance nerve regenerationthat matches isografting. Thus a novel localized electrical stimula-tion method combining artificial biomaterials and induction byelectrically polarized b-TCP particles to promote nerve regenera-

W. Wang et al. / Acta Biomaterialia 6 (2010) 4027–4033 4033

tion has emerged. The intensity of the electrical field could be con-trolled by modifying the conditions of polarization, the particlesize and the method of immobilization of the b-TCP particles.The biomaterial has a limited lifespan depending on the bioabsorb-ability of b-TCP and, therefore, any concerns about possible side-ef-fects are minimized. The b-TCP particle size selected was largeenough to prevent phagacytosis by macrophages and the contentof b-TCP particles fixed on the chitosan mesh tube was, as far asis possible, set in this study. These two parameters should be opti-mized, however, for nerve regeneration in further studies.

Acknowledgements

This study was supported in part by a Grant-in-Aid for ScientificResearch. The chitosan nanofiber mesh tubes were kindly providedby Hokkaido soda Co. Ltd., Hokkaido, Japan.

Appendix. Figures with essential colour discrimination

Certain figures in this article, particularly Figures 5, 6 and 7, aredifficult to interpret in black and white. The full colour images canbe found in the on-line version, at doi:10.1016/j.actbio.2010.04.027.

References

[1] Itoh S, Yamaguchi I, Matsuda A, Kobayashi H, Taguchi T, Shinomiya K, et al.Development of the biomaterials for nerve scaffold and immobilization oflaminin peptides to enhance nerve regeneration. In: Tanaka J, Itoh S, Chen G,editors. Surface design and modification of biomaterials for clinicalapplication. Trivandrum: Transworld Research Network; 2008. p. 205–25.

[2] Wang W, Itoh S, Matsuda A, Ichinose S, Shinomiya K, Hata Y, et al. Influences ofmechanical properties and permeability on chitosan nano/microfiber meshtubes as a scaffold for nerve regeneration. J Biomed Mater Res A 2008;84:557–66.

[3] Kobayashi T, Nakamura S, Yamashita K. Enhanced osteobonding by negativesurface charges of electrically polarized hydroxyapatite. J Biomed Mater Res2001;57:477–84.

[4] Nakamura S, Takeda H, Yamashita K. Proton transport polarization anddepolarization of hydroxyapatite ceramics. J Appl Phys 2001;89:5386–92.

[5] Ueshima M, Nakamura S, Yamashita K. Huge millicoulomb charge storage inceramic hydroxyapatite by bimodal electric polarization. Adv Mater2002;14:591–5.

[6] Itoh S, Nakamura S, Kobayashi T, Shinomiya K, Yamashita K. Effect of electricalpolarization of hydroxyapatite ceramics on new bone formation. Calcif TissueInt 2006;78:133–42.

[7] Sisken BF, Walker J, Orgel M. Prospects on clinical applications of electricalstimulation for nerve regeneration. J Cell Biochem 1993;51:404–9.

[8] Gordon T, Brushart TM, Chan KM. Augmenting nerve regeneration withelectrical stimulation. Neurol Res 2008;30:1012–22.

[9] Kotwal A, Schmidt CE. Electrical stimulation alters protein adsorption andnerve cell interactions with electrically conducting biomaterials. Biomaterials2001;22:1055–64.

[10] Lagoa N, Ceballosa D, Rodrigueza FJ, Stieglitzb T, Navarro X. Long termassessment of axonal regeneration through polyimide regenerative electrodesto interface the peripheral nerve. Biomaterials 2005;26:2021–31.

[11] Tanaka Y, Iwasaki T, Nakamura M, Nagai A, Katayama K, Yamashita K.Polarization and microstructural effects of ceramic hydroxyapatite electrects. JAppl Phys 2010;107:014107.

[12] Yamashita K. Effects of electrical polarization on B-type carbonatedhydroxyapatite. Phos Res Bull 2004;17:126–9.

[13] Al-Majed AA, Brushart TM, Gordon T. Electrical stimulation accelerates andincreases expression of BDNF and trkB rnRNA in regenerating rat femoralmotoneurons. Eur J Neurosci 2000;12:4381–90.

[14] Kobayashi NR, Fan DP, Giehl KM, Bedard AM, Wiegand SJ, Tetzlaff W. BDNF andNT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy,stimulate GAP-43 and T 1-tubulin mRNA expression, and promote axonalregeneration. J Neurosci 1997;17:9583–95.

[15] Menei P, Montero-Menei C, Whittemore SR, Bunge RP, Bunge MB. Schwanncells genetically modified to secrete human BDNF promote enhanced axonalregrowth across transected adult rat spinal cord. Eur J Neurosci1998;10:607–21.

[16] Kohmura E et al. BDNF atelocollagen mini-pellet accelerates facial nerveregeneration. Brain Res 1999;849:235–8.

[17] Utley DS, Lewin SL, Cheng ET, Verity AN, Sierra D, Terris DJ. Brain-derivedneurotrophic factor and collagen tubulization enhance functional recoveryafter peripheral nerve transection and repair. Arch Otolaryngol Head NeckSurg 1996;122:407–13.

[18] Lewin SL, Utley DS, Cheng ET, Verity AN, Terris DJ. Simultaneous treatmentwith BDNF and CNTF after peripheral nerve transaction and repair enhancesrate of functional recovery compared with BDNF treatment alone.Laryngoscope 1997;107:992–9.

[19] Zhang JY, Luo XG, Xian CJ, Liu ZH, Zhou XF. Endogenous BDNF is required formyelination and regeneration of injured sciatic nerve in rodents. Eur JNeurosci 2000;12:4171–80.

[20] Longo FM et al. Electromagnetic fields influence NGF activity and levelsfollowing sciatic nerve transaction. J Neurosci Res 1999;55:230–7.