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Biomaterials 33 (2012) 3860e3867

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Biomaterials

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Bridging peripheral nerve defects with a tissue engineered nerve graft composedof an in vitro cultured nerve equivalent and a silk fibroin-based scaffold

Xin Tang, Chengbin Xue, Yaxian Wang, Fei Ding, Yumin Yang**, Xiaosong Gu*

Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong JS226001, PR China

a r t i c l e i n f o

Article history:Received 16 January 2012Accepted 2 February 2012Available online 24 February 2012

Keywords:Tissue engineered nerve graftsPeripheral nerve regenerationIn vitro cultured nerve equivalentSchwann cellsDorsal root ganglia

* Corresponding author. Fax: þ86 515 85511585.** Corresponding author.

E-mail addresses: Yangym@ntu.edu.cn (Y. Yan(X. Gu).

0142-9612/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.biomaterials.2012.02.008

a b s t r a c t

Tissue engineered nerve grafts are considered as a promising alternative to autologous nerve grafts usedfor peripheral nerve repair. The differences between these two types of nerve grafts are mainly in theregenerative microenvironment established by them. To construct ideal tissue engineered nerve grafts, itis therefore required to develop a better way to introduce biochemical cues into a neural scaffold, ascompared to single or combined use of support cells and growth factors. Here, we used a co-culturesystem of dorsal root ganglia and Schwann cells to create an in vitro formed nerve equivalent, whichwas introduced into a silk fibroin-based scaffold to furnish a tissue engineered nerve graft (TENG). At4- and 12- weeks after the TENG was implanted to bridge a 10-mm-long sciatic nerve defect in rats,histological and functional assessments as well as Western blot analysis were performed to evaluate theinfluences of the TENG on peripheral nerve regeneration. We found that at an early stage of nerveregeneration, the TENG significantly accelerated axonal growth, and up-regulated expressions ofN-cadherin and PMP22. Twelve weeks after nerve grafting, the TENG produced a further improvedoutcome of nerve regeneration and functional recovery, which was more close to that of the autologousnerve graft than that of the silk fibroin-based scaffold. The introduction of an in vitro cultured nerveequivalent into a scaffold might contribute to establishing a native-like microenvironment for nerveregeneration.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Peripheral nerve defects are clinically treated with implantationof an autologous nerve graft, which by far offers the best outcomeof nerve regeneration and function restoration. This recognized“gold standard” strategy of peripheral nerve repair, however,suffers from several drawbacks, and the development of an alter-native to autologous nerve grafts motivates great endeavors ofneuroscientists and surgeons [1]. Now, tissue engineered nervegrafts provide an ideal option among various current alternatives.They are typically composed of a physical scaffold and biochemicalcues. The former is usually prepared with a wide range of bioma-terials, mainly exerting the actions of contact guidance and diffu-sion barrier for axonal regrowth. The latter is furnished by supportcells and/or growth factors, mainly aiding to create a favorablemicroenvironment for peripheral nerve regeneration [2e4].

g), neurongu@public.nt.js.cn

All rights reserved.

Schwann cells (SCs) are the main glial cells in the peripheralnervous system (PNS) and play an indispensable role in peripheralnerve regeneration. They become the first and most widely usedsupport cells for introduction to the scaffold of tissue engineerednerve grafts, thus improving nerve regeneration in experimentalstudies [5e7]. To expand the source of support cells, stem cells,especially bone marrow mesenchymal stem cells, are tried toserve as support cells within tissue engineered nerve grafts withconsiderable success in peripheral nerve repair [8e11]. On theother hand, the use of growth factors and other bioactive moleculesfor tissue engineered nerve grafts has also been demonstrated toexert positive effects on peripheral nerve regeneration [12,13].

It seems that the differences between tissue engineered nervegrafts and autologous nerve grafts are mainly in the regenerativemicroenvironment established by them. To overcome the congen-ital deficiency of tissue engineered nerve grafts and create anoptimal microenvironment for peripheral nerve regeneration, weattempted to seek a better way, as compared to single or combineduse of support cells and growth factors, to introduce biochemicalcues into a scaffold. Here we tested a hypothesis whether a co-culture system of dorsal root ganglia (DRGs) and SCs could giverise to an in vitro cultured nerve equivalent that was likely tomimic

X. Tang et al. / Biomaterials 33 (2012) 3860e3867 3861

the native nerve microenvironment, and we further integratedthese nerve-like tissues with the silk fibroin (SF)-based scaffold toengineer a nerve graft, which was implanted to bridge a 10-mmdefect in the rat sciatic nerve. The process of nerve regenerationwas monitored at an early stage of 4 weeks after grafting, and theregenerative outcomeswere evaluated at 12weeks after grafting byfunctional, morphological and histological assessments.

2. Material and methods

2.1. Construction of tissue engineered nerve grafts

2.1.1. Fabrication of SF-based scaffoldsBombyx mori SF fibers, bought from Xinyuan sericulture company (Nantong,

Jiangsu, China), were subjected to degumming treatments of boiling in Na2CO3

solution. The degummed fibers were dissolved into an aqueous solution forpreparing SF-based scaffolds as described previously [14]. In brief, a nerve guidanceconduit was fabricated from the SF aqueous solution by a method of injectionmolding, and 20 longitudinal aligned SF filaments (4 15 mm) were inserted into theconduit lumen, thus furnishing a SF-based scaffold.

2.1.2. Formation of in vitro cultured nerve equivalent inside SF-based scaffoldThe L4-6 DRGs were removed from Sprague-Dawley (SD) rat embryos between

E14.5 and E16.5, and dissociated in 0.25% Trypsin for 30min at 37 �C. The dissociatedDRGs were plated onto the SF filaments within the scaffold, which was immersed inDMEM supplemented 10% FBS (Gibco, Grand Island, NY) for 4 h. The medium wasreplaced by neurobasal mediumwith B27 supplements (Gibco), and the tissues wereincubated at 37 �C in a humidified atmosphere of 95% air and 5% CO2 for 7 days toallow the neurite extension. A fewDRG explants were inserted into either opening ofthe SF nerve guidance conduit, and each DRG explant had been pre-coated witha film (pore diameter 8 mm) that served as a barrier but permiting the neuriteoutgrowth.

The bilateral sciatic nerves of neonatal SD rats (1-2-d old) were excised outunder sterile condition. After the epineurium, connective tissue, and blood vesselswere stripped off, the nerves were sheared into small explants, which were platedonto a polylysine-coated coverslip in a 6-well tissue culture plate. DMEM supple-mented with 10% FBS was added to each well, and the explants were incubated at37 �C in a humidified atmosphere of 95% air and 5% CO2 with half of the mediumbeing replaced every other day. The dissociated SCs were maintained in DMEMsupplemented 10% FBS for 2 weeks to allow cell expansion before they were seededat a density of 2 � 105 cells/mL onto the neurites extended from DRGs within theSF-based scaffold. For cell seeding procedure, a suspension of SCs (5 � 105 cells/mL)

Fig. 1. Scanning (AeC) and transmission (D) electron micrography, obtained at 2 (A), 3 (B), anerve equivalent. Immunocytochemistry with anti-NF (green) and anti-S100 (red) antibodiDRGs (E). Scale bar, 20 (AeC and E) and 0.5 (D) mm.

was pumped in a closed bioreactor loop at the rate of 1 mL/min through the scaffoldfor 4 h and then rotated at 25 rpm for up to 3 weeks at 37 �C in a humidifiedatmosphere of 95% air and 5% CO2 [15e17]. Afterwards, both ends of the scaffoldwere sheared to remove DRG neuron bodies. In this way, a specific tissue engineerednerve graft (TENF) was generated by integrating an in vitro cultured nerve equiva-lent with a SF-based scaffold.

2.2. Immunohistochemistry

The sections from DRG-SC co-culture inside a SF-based scaffold were allowed toincubate with mouse anti-neurofilament (NF) 200 (1:500 dilution, Sigma, St. Loius,MO) and polyclonal antibody rabbit anti-S100 (1:500 dilution, Sigma) at 4 �C for24 h. Then the sections were incubated with the FITC labeled secondary antibodygoat anti-mouse IgG (1:200 dilution, Santa Cruz, Santa Cruz, CA) or TRITC labeledsecondary antibody goat anti-rabbit IgG (1:200 dilution, Santa Cruz) at 4 �C over-night. After labeled with Hochest 33342 (5 mg/ml), the sections were mounted influorescent mounting medium and observed under a confocal laser scanningmicroscope (Leica, Heidelberg, Germany).

Four or twelve weeks post-implantation, tissues at the implantation place wereharvested, post-fixed and sectioned. The procured sections were subjected toimmunohistochemistry with anti-NF200 and anti-S100 using the procedures similarto the above mentioned.

2.3. Electron microscopy

After 2-, 3- or 4-week in vitro culture, a nerve equivalent was fixed in 4%glutaraldehyde, post-fixed with 1% OsO4, and dehydrated stepwise in increasingconcentrations of ethanol. A portion of samples were dried in a critical point drier(Hitachi, Tokyo, Japan) for coating with gold, followed by observation undera scanning electron microscope (JEM-T300, JEOL, Japan). And other samples wereembedded in Epon 812 epoxy resin to make ultrathin sections, which were stainedwith lead citrate and uranyl acetate, followed by observation under a transmissionelectron microscope (JEM-1230, JEOL, Japan).

Four or twelve weeks post-implantation, tissues in the implantation place wereharvested, post-fixed and sectioned. The procured tissue sections were subjected totransmission electron microscopy using the procedures similar to the abovementioned.

2.4. Animals and surgical procedures

All experimental procedures involving animals were performed as per theinstitutional animal care guidelines and approved ethically by the AdministrationCommittee of Experimental Animals, Jiangsu Province, China.

nd 4 (C and D) weeks after co-culture of SCs and DRGs to generate an in vitro culturedes depicting that nerve-like structure occurred at 3 weeks after co-culture of SCs and

X. Tang et al. / Biomaterials 33 (2012) 3860e38673862

Adult SD rats, weighing 200-220 g, were randomized into 3 grafted groups anda non-grafted group (n ¼ 11 each). Animals were anesthetized by intraperitonealinjection of 3% sodium pentobarbital (30 mg/kg body weight) before the sciaticnervewas exposed bymaking a skin incision and splitting the underlying muscles inthe left lateral thigh. A segment of sciatic nerve was excited and removed, leavinga 10-mm-long defect following retraction of the nerve ends. In scaffold and TENFgroups, the sciatic nerve defect was bridged by a SF-based scaffold and a TENFconsisting of a nerve equivalent inside a SF-based scaffold, respectively; in autograftgroup, the cut nerve segment was re-implanted into the sciatic nerve defect (posi-tive control); in non-grafted group, the sciatic nerve defect was left un-bridged(negative control). After surgery, animals in all groups were housed and fedroutinely, and monitored for changes in their general conditions and locomotionactivities.

Walking track analysis was performed to all animals at 4, 6, 8, 10, 12 weeks asdescribed previously [18], and the sciatic function index (SFI) value was calculatedby the formula proposed by Bain et al. [19].

2.5. Electrophysiological assessment

Twelve weeks after nerve grafting, the sciatic nerve at the injured side was re-exposed under anesthesia. Electrical stimuli were applied to the nerve trunk at itsdistal or proximal portion, and compound muscle action potentials (CMAPs) wererecorded on the gastrocnemius belly at the ipsilateral side. The normal CMAP wasmeasured at the contralateral, uninjured side. The motor nerve conduction velocity(MCV) was calculated from the CMAP amplitude and the distance between the distaland proximal stimulated sites.

Fig. 2. Immunohistochemistry (AeC) with anti-NF (green) and anti-S100 (red) antibodieautograft (A, D), TENG (B, E), and scaffold (C, F) groups at 4 weeks after nerve grafting, respesheath thickness (G) and number of myelin lamellae (H) among regenerating nerves taken fgroup.

2.6. Masson trichrome staining

Twelve weeks after nerve grafting, the gastrocnemius muscle was harvestedfrom the mid-belly of the injured and contralateral uninjured limbs in rats. Themuscle samples were post-fixed and cut on a cryostat into sections, whichwere thensubjected to Masson trichrome staining.

2.7. Western blot analysis

Four weeks after nerve grafting, tissues in the implantation place were har-vested and analyzed for protein expression levels of N-cadherin and peripheralmyelin protein-22 (PMP22) by Western blot analysis as described previously [20].The primary antibodies were polyclonal antibody rabbit anti-N-cadherin (1:1000dilution, Abcam) and polyclonal antibody rabbit anti-PMP22 (1:1000 dilution,Abcam). The secondary antibody was HRP-conjugated goat anti-rabbit IgG (1:2000dilution) and HRP-conjugated goat anti-mouse IgG (1:2000 dilution). And the HRPactivity was detected using an ECL kit (Pierce, Rockford, IL). The image was scannedwith GS800 Densitometer Scanner (Bio-Rad, Hercules, CA), and the optical densitywas analyzed using PDQuest 7.2.0 software (Bio-Rad). Beta-actin (1:4000) served asan internal control.

2.8. Statistics

The data were expressed as means � SEM. Statistical analysis was performedwith one-way ANOVA and Scheffe’s post hoc test. Values of p< 0.05were consideredstatistically significant.

s and transmission electron micrography (DeF) for regenerating nerves taken fromctively. Scale bar, 20 (AeC) and 2 (DeF) mm. Histrograms comparing the average myelinrom different groups at 4 weeks after nerve grafting, respectively. *p < 0.05 vs. scaffold

X. Tang et al. / Biomaterials 33 (2012) 3860e3867 3863

3. Results

3.1. Characterization of an in vitro cultured nerve equivalent

Electron microscopy and immunohistochemistry were used tocharacterize the nerve equivalent generated by co-culture of DRGand SCs. Scanning electron microscopy showed the extracellularmatrix (ECM)-like tissues appeared along longitudinal aligned SFfilaments, and SCs with a spindle or spherical shape encircled theneurites of DRGs (Fig. 1AeC). Transmission electron microscopyindicated the establishment of myelin sheaths following SCswrapping DRG axons, and clearly demonstrated the lamella struc-ture (Fig. 1D). Immunocytochemistry further displayed that thereexisted S-100 positive and NF200 positive cells, identified as SCsand DRG neurites, which attached to and encircled SF-based scaf-fold in terms of their relatively strong red and green fluorescenceemission (Fig. 1E). The features of TENFs, developed in this study,manifested themselves in the inclusion of the above characterizednerve equivalent into the SF-based scaffold.

3.2. Nerve regeneration at 4 weeks after nerve grafting

Four weeks after nerve grafting, immunohistochemistry withanti-NF200 and anti-S100 demonstrated that more number of

Fig. 3. The protein levels of N-cadherin and PMP22, detected by Western blot analysis, in renerve grafting, respectively. *p < 0.05 and **p < 0.01 vs. scaffold group. Also shown is the

myelinated nerve fibers appeared in TENF group than in scaffoldgroup (Fig. 2AeC); transmission electron micrography comparedthe different characteristics of myelin sheaths formed in 3 graftedgroups, mainly the differences in the thickness of myelin sheathsand number of myelin lamellae (Fig. 2DeH).

At an early stage after nerve grafting, 3 grafted groups weresubjected toWestern blot analysis to examine the protein level of 2nerve regeneration-related proteins in the tissues at the graft place.We found that theN-cadherin expressionwas significantly higher inTENF group than in scaffold group starting from 4weeks after nervegrafting, while the PMP22 expression was significantly higher inTENF group than in scaffold group at 2, 3, and 4 weeks after nervegrafting (Fig. 3). On the other hand, no significant difference in theexpression of either proteinwas observed between TENF group andautograft group at all designated time points.

3.3. Regenerative outcomes at 12 weeks after nerve grafting

3.3.1. Functional recovery evaluationWalking track analysis was performed to assess the recovery of

locomotor function in rats. The SFI value varies from 0 to 100, with0 corresponding to normal function and 100 to complete dysfunc-tion. Immediately after nerve injury, the SFI value for all animalsdecreased dramatically to the lowest level. Afterwards, the animals

generating nerves taken from scaffold, TENG and autograft groups at 2, 3, 4 weeks afterrepresentative Western blot image.

Fig. 4. Curve graph showing sciatic function index (SFI) values of rats in 3 graftedgroups and a non-grafted group at 4, 6, 8, 10, 12 weeks after nerve grafting. *P < 0.05vs. scaffold group at the same time point, and #P < 0.05 vs. other 3 groups at the sametime point.

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in 3 grafted groups demonstrated a time-dependent increase in theSFI value due to partial recovery of locomotor function, while non-grafted group showed no obvious change in the SFI level withina 12-week period after nerve injury. The scaffold group displayed anelevation in the SFI value from 8 weeks after grafting, followedby gradual increase of SFI value until 12 weeks after grafting. Incontrast, autograft andTENF groups keptelevation in the SFI value ata steadily rapid rate during 6e12weeks after nerve graftingwithoutsignificant difference between two groups. Interestingly, the SFIvalue was significantly higher in TENF group than in scaffold groupwith significant difference (Fig. 4).

Fig. 5. Representative CMAP recordings, obtained 12 weeks after nerve grafting, at the inuninjured side (D). Histograms showing CMAP recovery ratio for 3 grafted groups (E) and mothe contralateral uninjured side (F) 12 weeks after nerve grafting. *P < 0.05 vs. scaffold gr

Twelve weeks after nerve grafting, the CMAP amplitude valuemeasured at the injured side in each grafted groups was signifi-cantly less than that measured at the contralateral uninjured side,and no CMAP was recorded at the injured side in non-graftedgroup. The ratio of CMAP amplitude between the injured sideand the contralateral uninjured side was calculated for differentgroups. The CMAP amplitude ratio showed no significant differ-ence between TENF and autograft groups, but CAMP amplituderatio was significantly larger in either TENF or autograft groupthan in scaffold group. The MCV value exhibited no significantdifference between 3 grafted groups, although, as expected, theMCV value detected at the injured side in each grafted group wassignificantly less than that detected at the contralateral uninjuredside (Fig. 5)

3.3.2. Histological investigation and morphomethric analysisIn TENF group, immunochemistry with anti-NF and anti-S100

antibodies confirmed the regeneration of myelinated nerve fiberswhich occurred as massive bundles at the TENF place (Fig. 6AeD).In scaffold group, a less number of regenerated myelinated nervefibers were noticeable only at the scaffold place. In autograft group,most of nerve fibers were regenerated along the autograft with aneven distribution.

Transmission electron microscopy indicated that in 3 graftedgroups, besides numerous unmyelinated fibers, the regeneratedmyelinated fibers were dispersed in clusters, and the myelinatedaxon was surrounded by a clear, thick, and electron-dense myelinsheath, despite the thinner myelin sheaths as compared to themyelinated axon existing in contralateral, uninjured sciatic nerve.The myelin lamellar structures provided further evidence forthis observation. By comparing the morphometric data regardingmyelinated nerve fibers, as obtained from transmission electronmicroscopy,wenoted that either sheath thicknessormyelin lamellae

jured side in autograft (A), TENG (B) and scaffold (C) groups and at the contralateraltor nerve conduction velocity detected at the injured side in for 3 grafted groups and atoup, and #P < 0.05 vs. other 3 groups.

Fig. 6. Immunohistochemistry (AeD) with anti-NF (green) and anti-S100 (red) antibodies and transmission electron micrography (EeH) for regenerated nerves taken fromautograft (A, E), TENG (B, F), scaffold (C, G), and non-grafted (D, H) groups 12 weeks after nerve grafting, respectively. Scale bar, 20 (AeD) and 2 (EeH) mm. Histograms showing theaverage myelin sheath thickness (I) and number of myelin lamellae (J) for regenerated nerves taken from different groups at 12 weeks after nerve grafting, respectively. *p < 0.05 vs.scaffold group.

X. Tang et al. / Biomaterials 33 (2012) 3860e3867 3865

number in autograft or the TENF groupwas significantly greater thanthat in scaffold group, respectively (Fig. 6EeJ).

Motor nerve injury leads to denervation of target muscles andfinal muscle atrophy, which can be attenuated upon re-innervation,accompanied by gradual functional recovery. Masson trichromestaining indicated that the gastrocnemius muscle atrophy was lessserious in 3 grafted groups than in non-grafted group, although thealleviation in muscle atrophy was the greatest in autograft group(Fig. 7).

4. Discussion

In this study, TENFs that consisted of an in vitro cultured nerveequivalent residing in a SF-based scaffold were used to bridgea 10-mm sciatic nerve defect in rats. At 12 weeks after nervegrafting, a series of measurements were performed to evaluate theregenerative capacity of these TENFs. The recovery in the motorfunction of the injured hindlimb in TENF group, as indexed by theSFI value, was close to that in autograft group without significantdifference between each other, and prevailed over that in scaffoldgroup. The restoration of electrophysiological properties for 3grafted groups was reflected in detectable CAMP data, which rep-resented an important measure for the conduction function ofperipheral nerves. The comparison in the CMAP amplitudebetween 3 grafted groups provided further evidence that functional

recovery in TENF group was more close to that in autograft groupthan that in scaffold group. Histological analysis showed that eitherthe regenerated nerve or target gastronomius muscle achieved thesimilar reconstruction, both in qualitative and quantitative aspects,between TENF and autograft groups, and these similar results weresignificantly better than those in scaffold group. Our findings sug-gested that more axons might successfully grow through ourdeveloped TENF to reach the distal stumps for re-innervation oftarget muscle. In other words, the incorporation of a nerve equiv-alent into SF-based scaffold led to an enhanced repair capacity forperipheral nerve injuries.

As is known, themicroenvironment surrounding an injury site inPNS is oftenmore permissive to axonal regeneration as compared tothat in the central nervous system [1]. Although peripheral nerveregeneration is ultimately determined by quality and speed ofaxonal outgrowth [21], SCs also plays a critical role in the estab-lishment of the regenerative microenvironment. It is the specialimportance of SCs in PNS that is responsible for the effectiveness ofusing SCs as support cells for the generation of TENGs [6]. Myeli-nation of axons seems to be the most basic function of SCs becausemyelin sheath, as a unique component of the nervous system, canincrease axonal conduction, especially saltatory conduction, thusallowing fast and efficient saltatory propagation of action potentialsalong the nerve [22e24]. On the other hand, the development ofmyelinated nerve fibers in PNS depends on complex interactions

Fig. 7. Light micrographs of the transverse-sectioned gastrocnemius muscle following Masson trichrome staining (AeD) and cholinesterase staining of the motor endplate (EeH)obtained at the contralateral, uninjured side (a) and the injured side in autograft (A, E), TENG, (B, F), scaffold (C, G) groups and non-grafted group (D, H), respectively. Scale bar,50 mm (AeD), 20 mm (EeH). Histograms showing the cross-sectional area of muscle fibers (I) and average percentage of collagen fiber area for gastrocnemius muscle samples in 4different groups, respectively. *P < 0.05 vs. scaffold group, and #P < 0.05 vs. other 3 groups.

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between SCs and axons. Axons can in turnpromote the deposition ofthe basal lamina by SCs, which is required for the ensheathment ofaxons and the subsequent differentiation of SCs, and thematurationof fully myelinating SCs depends on contact and signaling fromaxons [25,26].

Based on the insights into the interactions between SCs andaxons, in this study, a unique scheme for incorporating biochemicalcues into neural scaffold was adopted to establish an optimalregenerative microenvironment. This scheme was different fromthe previously frequently used way in which most TENGs wereconstructed by means of support cells and/or growth factors. Ourdeveloped TENFs were featured by the replacement of commonbiochemical cues with a nerve equivalent that was furnished byin vitro co-culture of DRGs and SCs.

To characterize the in vitro cultured nerve equivalent, electronmicroscopy and immunohistochemistry indicated that this nerveequivalent possessed the characteristics of nerve-like tissues, andencompassed appropriate guiding structures andmolecular factors,thus providing an adequate microenvironment for axon-SC inter-actions and nerve-like tissue formation.

We further investigated the changes of TENFs at 4 weeks afterthey were implantated to bridge a 10-mm sciatic defect in rats.Histological investigation confirmed that TENFs were impactingnerve regeneration inawaysimilar to thatof autografts.Westernblotanalysis compared the expressions of 2 nerve regeneration-relatedproteins among autograft, TENF and scaffold groups, indicating that

N-cadherin and PMP22 expressions were higher in autograft andTENF groups than in scaffold group.

N-cadherin is a member of the cadherin gene family encodeproteins that mediate Ca2þ-dependent adhesion. Together withb-catenin and aN-catenin, N-cadherin forms the cadherin/catenincomplex, a main complex linking the extracellular environmentto the actin cytoskeleton [27]. The cadherin/catenin complex ispresent at high levels in axons, forming the adherens junctionsin epithelial cells and synaptic junctions in neurons. N-cadherinpromotes cell migration during tissue morphogenesis and axonguidance [28,29]. PMP22 is a key component of the myelin sheathof peripheral nerves [30]. Successful myelination of PNS dependson induction of major protein components of myelin includingPMP22, and myelin stability is also sensitive to PMP22 levels [31].The high expressions of N-cadherin and PMP22 meant that in ourdeveloped nerve equivalent, as in the autologous nerve, dynamicinteractions between axons and SCs contributed to the establish-ment of an ideal microenvironment for nerve regeneration via theincreased expression of several bioactive molecules.

5. Conclusions

Wedeveloped a TENF consisting of a nerve equivalent, formed byco-culture of DRGs and SCs, residing in a SF-based scaffold. The TENFwas implanted to bridge a 10-mm-long sciatic nerve defect inrats. At an early stage after nerve grafting, the TENF significantly

X. Tang et al. / Biomaterials 33 (2012) 3860e3867 3867

accelerated axonal growth, and up-regulated N-cadherin andPMP22 expressions. Twelve weeks after nerve grafting, TENFs yiel-ded an improved outcome of nerve regeneration and functionalrecovery, whichwasmore close to that of nerve autografts than thatof SF-scaffolds. The introduction of an in vitro cultured nerveequivalent into a scaffold might contribute to establishing a native-like microenvironment for nerve regeneration.

Acknowledgements

This study was supported by Hi-Tech Research and Develop-ment Program of China (863 Program, Grant No. 2012AA020502),National Natural Science Foundation of China (Grant Nos.81130080, 81000678, and 81171457), and the Priority AcademicProgram Development of Jiangsu Higher Education Institutions(PAPD). We thank Professor Jie Liu for assistance in manuscriptpreparation.

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