in vivo integration of poly(ε-caprolactone)/gelatin nanofibrous nerve guide seeded...

In vivo integration of poly(e-caprolactone)/gelatin nanofibrous nerveguide seeded with teeth derived stem cells for peripheral nerveregeneration

Mohammad-Hossein Beigi,1,2 Laleh Ghasemi-Mobarakeh,3 Molamma P. Prabhakaran,4

Khadijeh Karbalaie,1 Hamid Azadeh,5 Seeram Ramakrishna,4 Hossein Baharvand,6

Mohammad-Hossein Nasr-Esfahani1

1Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR,

Isfahan, Iran2Materials Engineering Department, Najafabad Branch, Islamic Azad University, Najafabad, Iran3Department of Textile Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran4Center for Nanofibers and Nanotechnology, E3-05-14, Nanoscience and Nanotechnology Initiative, Faculty of Engineering,

National University of Singapore, Singapore 1175765Department of Physiotherapy, School of Rehabilitation Science, Isfahan University of Medical Sciences, Isfahan, Iran6Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology

and Technology, ACECR, Tehran, Iran

Received 5 November 2013; revised 29 January 2014; accepted 4 February 2014

Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35119

Abstract: Artificial nanofiber nerve guides have gained huge

interest in bridging nerve gaps and associated peripheral

nerve regeneration due to its high surface area, flexibility and

porous structure. In this study, electrospun poly (e-caprolac-

tone)/gelatin (PCL/Gel) nanofibrous mats were fabricated,

rolled around a copper wire and fixed by medical grade

adhesive to obtain a tubular shaped bio-graft, to bridge 10

mm sciatic nerve gap in in vivo rat models. Stem cells from

human exfoliated deciduous tooth (SHED) were transplanted

to the site of nerve injury through the nanofibrous nerve

guides. In vivo experiments were performed in animal mod-

els after creating a sciatic nerve gap, such that the nerve gap

was grafted using (i) nanofiber nerve guide (ii) nanofiber

nerve guide seeded with SHED (iii) suturing, while an

untreated nerve gap remained as the negative control. In

vitro cell culture study was carried out for primary investiga-

tion of SHED-nanofiber interaction and its viability within the

nerve guides after 2 and 16 weeks of implantation time.

Walking track analysis, plantar test, electrophysiology and

immunohistochemistry were performed to evaluate func-

tional recovery during nerve regeneration. Vascularization

was also investigated by hematoxilin/eosine (H&E) staining.

Overall results showed that the SHED seeded on nanofibrous

nerve guide could survive and promote axonal regeneration

in rat sciatic nerves, whereby the biocompatible PCL/Gel

nerve guide with cells can support axonal regeneration and

could be a promising tissue engineered graft for peripheral

nerve regeneration. VC 2014 Wiley Periodicals, Inc. J Biomed Mater

Res Part A: 00A:000–000, 2014.

Key Words: peripheral nerve regeneration, nerve guide, electro-

spinning, stem cells

How to cite this article: Beigi M-H, Ghasemi-Mobarakeh L, Prabhakaran MP, Karbalaie K, Azadeh H, Ramakrishna S, BaharvandH, Nasr-Esfahani M-H. 2014. In vivo integration of poly(e-caprolactone)/gelatin nanofibrous nerve guide seeded with teethderived stem cells for peripheral nerve regeneration. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

The regeneration of defective or damaged peripheral nervehas been a difficult and challenging problem in reconstruc-tive surgery and is considered as a common cause of per-manent functional loss and post traumatic morbidity, mainlybecause nerve regeneration is a complex biological phenom-enon.1–3

Direct end-to-end suturing of the damaged nerve ends isa commonly utilized method for peripheral nerve regenera-tion (PNS). For PNS injuries of relatively short distances

(<5 mm), axonal regeneration occurs spontaneously. How-ever, functional recovery of nerve repair remains unsatisfac-tory especially when the nerve defect or gap is too long.4 Asa promising alternative method to suturing, implantation ofan autologous nerve graft harvested from functionally lessimportant nerves such as sural nerves, superficial cutaneousnerves or lateral and medial antebrachii cutaneous nerveshave been used traditionally to bridge peripheral nervedefects. It provides structural support for sprouting axonsand bridges the proximal and distal nerve stumps,

Correspondence to: M.-H. Nasr-Esfahani; e-mail:[email protected]

VC 2014 WILEY PERIODICALS, INC. 1

promoting nerve regeneration.5 Despite the well-knownbenefits of nerve autograft, limitation in tissue availabilityand shortage of graft, neuroma formation, unacceptablescarring, donor site morbidity, need for a second operation,mismatch of donor-site nerve size with the recipient site areseveral limitations associated with this method.6–8 The useof artificial nerve grafts has been explored as an alternativetechnique to autograft for repair of nerve injury.7 However,current research focus on the development of tubular nerveguides made from either degradable or non-degradable syn-thetic materials to stimulate regeneration of severed periph-eral nerve axons, assist nerve growth in the correctdirections and provide protection to the regenerating nervesby introducing both ends of injured nerve stumps into atubular nerve guide.9 Biocompatible and biodegradablepolymers have attracted more attention for fabrication ofnerve guides since nondegradable materials lead to possiblechronic foreign body reaction, secondary complaints, andimpairment of nerve function in addition to the need of asecond surgery to remove the nerve guide.9 On the otherhand, cell transplantation has been demonstrated as apromising strategy in improving nerve regeneration, whilenerve guides seeded with cells might enhance the nerveregeneration process especially because the cells can act asa source of neurotrophic factors.10–17

Tooth pulp is a major source of mesenchymal compo-nents and stem cells cultured from the dental pulp areplenty available and these cells could serve as an ideal adultcell source for autologous transplantation. Such cells canalso be made for personalized applications via cell bank-ing.18 Dental pulp stem cells (DPSCs), stem cells fromhuman exfoliated deciduous tooth (SHED), periodontal liga-ment stem cells (PDLSCs) and stem cells from apical papilla(SCAP) are different types of stem cells available in dentaltissue. Among these cells, except SHED all the other cellsare from permanent teeth. Every child loses primary teeth,which provides the gold opportunity to recover and storethis source of stem cells (SHED) to regenerate future inju-ries.19 Biological properties of these cells such as multipo-tency, high proliferation rates and accessibility make themimportant in regenerative medicine.20

Electrospinning is a simple, versatile, cost-effective andpromising method for producing nanofibers. The techniqueof electrospinning has been modified to a higher extent withthe aim of producing special structures such as tubularnanofiber mat suitable for nerve guide application. Electro-spun nanofibrous nerve guides have attracted much interestin recent days for fabrication of nerve guide as they mimicthe architecture and fibrous structure of extracellular matrixin native nerve.21 Moreover, the high surface area to volumeratio of nanofibers provides more surfaces for cell attach-ment and the highly porous structure of electrospun nano-fibers make them permeable to exchange of oxygen, entry ofnutrients and growth factors, required for cell proliferationand tissue regeneration.22–24 Meanwhile, they provide thenecessary barrier to prevent infiltration of unwanted tissuesinto the nerve guide from outside and compared to othercommon nerve guides, they do not break after implantation

due to their flexibility and are well adaptable to the livingsystem.24

Our previous study showed that electrospun poly(e-cap-rolactone) (PCL)/gelatin nanofibrous scaffolds are promisingsubstrates for nerve tissue engineering.25 Aiming to studythe potential of a nanofibrous nerve guide containing cellsfor peripheral nerve regeneration, we focused on the fabri-cation of PCL/Gel nanofibers, rolled them to obtain a nerveguide to bridge a 10-mm long nerve defect in rat sciaticnerve. SHED was transplanted to injured sciatic nervethrough nanofibrous nerve guide to evaluate their ability innerve regeneration as SHED have neural progenitor potency.

For primary investigation of cell-scaffold interaction,SHED was seeded on PCL/Gel nanofibrous scaffolds and themorphology and proliferation of SHED on electrospun nano-fibrous scaffolds were studied. Subsequently, both PCL/Gelnanofiber nerve guides with and without SHED wereapplied to bridge a 10-mm long sciatic nerve defect of rat.The functional recovery and axonal regeneration of sciaticnerve was evaluated after implantation of the nerve guidesin rat models.

EXPERIMENTAL METHODS

Fabrication and characterization of PCL/gel nanofibersElectrospun PCL/gel nanofibrous scaffolds were fabricatedby electrospinning method as described previously.25 Briefly,the polymer solution with concentration of 6 wt% was pre-pared by dissolving PCL and gelatin with a weight ratio of70:30 in hexafluoro-2-propanol (HFP) and the solution waselectrospun from a 5-mL syringe with a needle diameter of0.4 mm at a mass flow rate of 1 mL/h, and a high voltageof 12 kV was applied to the tip of the needle attached tothe syringe. Nanofibers were collected on a flat aluminumplate.

The morphology of nanofibrous scaffolds was studied byscanning electron microscopy (SEM) (JSM 5600, JEOL, Japan)at an accelerating voltage of 15 kV, after coating with goldusing a sputter coater (JEOL JFC-1200 fine coater, Japan) andthe diameter of the fibers were measured from the SEMmicrographs using image analysis software (Image J, NationalInstitutes of Health).

Electrospun nanofiber nerve guide preparationPCL/gel nanofibrous mat was cut to rectangular sheets withdimensions of 20 3 13 mm2 and rolled around a copperwire (16 guage) and fixed and secured into place with amedical grade adhesive (ethyl-2-cyanoacrylate adhesive) toform a three-dimensional tubular structure (Fig. 1). Thenanofiber nerve guides were sterilized under UV for 6 hrfor further in vivo experiments.

In vitro cell culture studyIn vitro cell culture studies were performed for primaryinvestigation on cell-scaffold interaction. For this, SHEDwere prepared by a method described earlier by Taghipouret al.18 In summary, the separated dental pulp was digestedby collagenase and the cell suspension was cultured inDMEM supplemented with 15% ES-FCS for 3 days until

2 BEIGI ET AL. PERIPHERAL NERVE REGENERATION

single colony appeared. Single colonies were separatelytreated with trypsin/EDTA and passaged further.

The nanofibrous scaffolds were exposed to UV radiationfor 2 hr, washed three times with PBS and incubated withDMEM (Gibco, 12800) supplemented with 15% ES-FCS for24 hr before cell seeding. Cells were further seeded on thenanofibrous scaffolds placed in a 24-well plate and tissueculture polystyrene (TCP) control at a density of 5 3 104

cells/well, grown in culture media and incubated at 37�C,5% CO2 incubator with 95% humidity.

The morphology of cells on PCL/gel nanofibrous scaf-folds was observed by SEM. After 7 days of cell seeding,samples were fixed with 3% glutaraldehyde for 2 hr. Speci-mens were rinsed in water and dehydrated with gradedconcentrations (50, 70, 90, 100% v/v) of ethanol. Finallythe samples were coated with gold to observe the cellmorphology.

MTS assay was used to study the cell proliferation onnanofibrous scaffolds and TCP. After 3, 5 and 7 days of cellseeding, cells were washed with PBS and incubated with20% of CellTiter 96VR AQueous One Solution reagent (MTS)reagent containing serum free medium. After 3 hr of incuba-tion at 37�C in 5% CO2, aliquots were pipetted into a 96-well plate. The absorbance of the content of each well wasmeasured at 492 nm using a spectrophotometric platereader (Fluostar Optima, BMG Lab Technologies, Germany).

In vivo implantation of nanofiber nerve guidesPCL/gel nanofibrous nerve guides were evaluated for theirefficiency to promote nerve regeneration in male Wistarrats. Rats were randomly divided into four groups to evalu-ate the nerve guide efficiency with or without cells (SHED).

The groups are: PCL/gel nanofibrous nerve guide withoutcells (group A, n5 15); PCL/gel nanofibrous nerve guidewith cells (group B, n515), end-to-end sutured sciaticnerve (group C, n5 10) and untreated defected nerve asnegative control (group D, n5 10).

For group A, nerve guides were filled with 3 mL of PBSand for group B, nerve guides were incubated in culturemedium for 1 hr and 3 mL of cell suspensions with densityof 1 3 105 cells were filled within the nerve guide.

Surgical procedureMale wistar rats (Medical experimental animal center, col-lege of Isfahan, Isfahan, Iran) with weight around 250 to300 g were used for this study. All animal care and experi-mental procedures were approved according to the Institu-tion Review Board and the Institution Ethical Committee ofRoyan Institute. Rats were anesthetized with a mixture ofketamine (90 mg/kg) and Xylazine (12.5 mg/Kg) and surgi-cal parts were shared and sterilized by povidone iodine.18

Following longitudinal incision along the posterior lateralthigh, the sciatic nerve was identified and 10 mm segmentof the nerve was removed from the center of the thigh cre-ating the dissected nerve.

Further a 13 mm nerve guide with and without SHEDwere put between the proximal and distal stump of trans-ected sciatic nerves in the rats for groups A and B. Thenerve was inserted into the conduits and 1.5 mm of thenerve end remained within the nerve guide and was fixedwith 8-0 vicryl suture. For proper fixation of the nerveguide, ethyl-2-cyanoacrylate (EPI glue, Meyer-Haake, Ger-many) was employed to both sutured ends as an adhesive.Following the implantation, the muscle incision was closed

FIGURE 1. PCL/Gel nanofiber nerve guide preparation: (a) PCL/Gel nanofiber mat is fabricated as a sheet and it can be cut to any size, (b) nanofi-

brous nerve guide rolled around a copper wire, (c) fixing of edge using medical grade adhesive, (d) nanofibrous nerve guides for implantation.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00 3

using absorbable 5–0 vicryl suture and the skin was closedwith use of a 3–0 nylon suture. For group C proximal anddistal of transected nerves were connected with 8–0 vicrylsuture and muscle incision and skin were closed in a similarway of group A and B.

Walking track analysis, plantar test, electrophysiologicalexamination and histological evaluation was carried outafter 4, 8, 12, 16 weeks of surgery to evaluate nerveregeneration.

Walking track analysisWalking track analysis was performed on a monthly basison all rats after nerve guide implantation for a period of 16weeks. Rats were put in a 100 3 20 cm2 walking track witha piece of white paper at the bottom of the track. The hindfeet of rats were put in ink in which the damaged paw (leftpaw) and undamaged paw (right paw) were dipped ingreen and purple inks respectively. From footprints pat-terns, distance from the first to the fifth toe, the toe spread(TS), the distance from the heel to the third toe, the printlength (PL) and distance from the second to the fourth toe,the intermediary toe spread (ITS) were all measured andSFI was calculated as below:10,17

SFI5 ð238:3 3 ððEPL2NPL Þ=NPL ÞÞ1 ð109:53 ððETS2NTS Þ=NTS ÞÞ1 13:3 3 EIT – NITð Þ=NITð Þð Þ – 8:8

where, the prefix E is used for defected paw and N for thenormal nondefected paw. In each walking track three foot-prints were analyzed by a single examiner and the averageof the measurements was reported as SFI.

Plantar testPlantar test was used for evaluation of heat hypersensitivityand sensory functional recovery of rats. The animals weretested at week 4, 8, 12, and 16. In brief, each animal wasplaced in a clear Plexiglas box and radiant heat was applied

to both right and left hind paw. Time, in seconds, from ini-tial heat source activation until paw withdrawal to avoidheat pain was recorded. The test was repeated in 10 mininterval and cut-off time was 33.1 s.

Electrophysiological examinationAfter 4, 8, 12, and 16 weeks postoperation, the rats wereanesthetized under room temperature (37�C) and an inci-sion was performed by surgical procedures. Sciatic nervewas stimulated by monopolar needle electrode on the nerveas cathode electrode, and cup electrode (anode) on theshaved skin was placed 2 cm away from the cathode.Recording active and reference cup electrodes were placedin the mid of gastrocinemius and its tendon respectively,while compound muscle action potentials (CMAPs) of gas-trocinemius muscle of each rat was recorded. The latency(LAT) was measured in millisecond (ms) from stimulationsite to onset of the response. The amplitude (AMP) ofCMAPs was also measured in millivolt (mV).

Cell attachment and viability on implanted nerve guideHematoxilin/eosine (H&E) and immunohistochemicalanalysis. The sciatic nerve was dissected from surroundingtissues and excised, including several millimeters proximaland distal to the implanted device after 2 and 16 weeks ofnerve guide implantation. The specimen was fixed by immer-sion in 4% paraformaldehyde for 24 hr at 4�C and then cryo-preserved in 30% sucrose solution for 96 hr at 4�C and wassectioned (8 mm cross direction) and placed on tissue adher-ing slides. These slides were stored at 280�C and mountedon cover slips coated with polylysine for analysis.

The specimens were stained by H&E and observedunder light microscope equipped with a camera (OlympusBX5, Japan) for evaluation of viability of transplanted SHEDin nanofiber nerve guides with cells after 2 and 16 weeks.Vascularization was also investigated around the regener-ated sciatic nerve grafted with nanofiber nerve guidesimplanted with or without cells.

FIGURE 2. Morphology of PCL/Gel nanofiber mat.

FIGURE 3. Morphology of SHED on TCP after 4 days of cell seeding.

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


Immunofluorescent labeling for HNu, OCT4, b-Tubulinand P75 respective to human cells, human stem cells,human neuronal phenotype and rat neuronal phenotypewere performed during this study.

For immunohistochemistry, after permeabilization ofthe cytoplasmic proteins with Triton X100, the nonspe-cific sites were blocked by using 10% goat serum and 1mg/m: bovine serum albumin followed by incubation withHNu, P75, OCT4, b-Tubulin antibodies overnight at 4�C.Subsequently, sections were incubated with FITC andTRITC conjugated secondary antibodies for 1 hr at 37�C.This incubation was followed by 3 min incubation in DAPI(1:100) solution. Finally, the sections were washed, dried,and mounted on cover-slips for evaluation. The sectionswere observed by fluorescence microscope (OlympusBX51, Japan) equipped with Olympus DP70 camera.

Statistical analysisAll data presented are expressed as mean6 standard devia-tion (SD). Statistical analysis was carried out using single-factor analysis of variance (ANOVA). A value of p�0.05 wasconsidered statistically significant.

RESULTS

Nanofiber nerve guidesFigure 2 shows the results of SEM evaluation, whereby themorphology of PCL/gel nanofibrous mat were investigatedand the fiber diameter was obtained as 189656 nm.

In vitro cell culture studyFigure 3 shows the spindle-shape morphology of SHEDseeded on TCP which was similar to the morphology ofSHED reported by other researchers.20

MTS assay was carried out to evaluate the cell prolifera-tion of SHED on PCL/gel nanofibrous scaffolds. As shown inFigure 4, the proliferation of cells on PCL/gel nanofibrousscaffolds was higher than that on TCP, indicating that thePCL/gel nanofibrous scaffolds serve as a suitable substratefor cell proliferation. Although cell proliferation was foundhigher on the nanofibrous scaffolds compared to TCP, thisdifference was only significant after 7 days of cell seeding(p�0.05).

Figure 5 illustrates the morphology of cells on the scaf-folds revealing cell attachment, and spreading on the nanofi-brous scaffolds which is consistent with the results of ourcell proliferation test.

In vivo studyAforementioned results showed the in vitro integration ofSHED with scaffolds, suggesting the suitability of these scaf-folds for in vivo applications, which was evaluated here fornerve tissue regeneration. After converting nanofibrous matto nerve guide tubes, they were implanted in sciatic nerveand in vivo studies were performed.

Functional recovery assessment. Walking track analysiscalled the “sciatic functional index” (SFI) is a frequentlyused method to investigate peripheral nerve regeneration,where it provides information about nerve motor/ sensoryfunction and muscle force.26,27 SFI is a simple, repeatableand useful tool for evaluating the functional condition of sci-atic nerve and varies from 0 to 2100, where 0 and 2100are corresponding to normal function and complete dys-function, respectively.10,27 Figure 6 indicates the SFI resultsobtained from all animal groups. During this study, the SFI

FIGURE 4. MTS results of SHED on PCL/gel nanofibrous scaffolds after 3, 5 and 7 days of cell seeding. *Difference significant at p� 0.05, ** dif-

ference not significant (p> 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


value increased over time for all the study groups. No signifi-cant difference was observed for SFI value up to 8 weeks(p>0.05) for all groups of this study. After 8 weeks of sur-gery, the SFI value for sutured sciatic nerve group anduntreated group was found significantly lower compared tothe nanofiber nerve guide groups with and without cells,indicating the positive effect of nanofiber nerve guidestowards nerve regeneration and functional recovery. The SFIvalue was found higher for nanofiber nerve guide with SHEDcompared to that for nanofiber nerve guide group devoid ofcells, but the difference was not significant (p> 0.05).

Sensory functional recovery assessment. Figure 7 showsthe latency of response to hot stimulus for different groupsas well as the control group (uninjured rat). Similar to theresults of SFI analysis, no significant differences were

observed in sensory functional recovery of all groups after8 weeks of surgery (p> 0.05). Although latency of responseto the hot stimulus did not reach to normal value after 16weeks, the nanofiber nerve guide groups with and withoutcells showed a progressive decrease in latency of responseto the hot stimulus revealing the improvement of sensoryfunctional recovery. Latency of response to the hot stimulusfor nanofiber nerve guide was found significantly lower fornanofiber nerve guide with cells compared to that withoutSHED (p� 0.05). No improvement was however observedfrom the sensory functional recovery analysis for suturedand untreated groups after 16 weeks time period.

Electrophysiology of regenerated nerve. Table I shows theamplitude of compound muscle action potentials (CMAPs)and latency for nanofiber nerve guide groups with and

FIGURE 5. Morphology of SHED on PCL/gel nanofibrous scaffolds after 7 days of cell seeding (magnification: 125 (a) and 1000 (b)).

FIGURE 6. SFI results of different animal groups (mean 6 SD, n 5 3). [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


without cells after 4, 8, 12, and 16 weeks of implantationand Table II compares the AMP and LAT of different groupsafter 16 weeks. It is also known that the higher the ampli-tude, the better is the axonal regeneration of the targetedmuscle, namely the garstrocinemius. No CMAPs value wasrecorded for the untreated group after 16 weeks surgeryand low AMP was obtained for sutured group after 16weeks (0.5 mV). As it is noticeable from Table I, no signifi-cant differences in AMP values between the nanofiber nerveguide groups with or without cells were observed after 12weeks of implantation. AMP value was found significantlyhigher for nanofiber nerve guide group with cells comparedto the nanofiber nerve guide group without cells after 16weeks of implantation (p� 0.05). However it was lower forboth nanofiber nerve guide groups with and without cells,in comparison to the normal (uninjured) rat. Latent periodwas found to decrease over time for both nanofiber nerveguides with and without cells. Shorter LAT was observedfor nanofiber nerve guides with cells compared to that with-out cells. However, the difference was not significant(p> 0.05). As can be seen in Table II, AMP and LAT did notreach to normal value and the group implanted with nano-

fiber nerve guides with cells showed the closest values tonormal one.

Figure 8 illustrates the electrophysiology wave of differentanimal groups in comparison to the wave of a normal rat after16 weeks. Higher amplitude indicates more axonal regenera-tion of sciatic nerve and as observed from Figure 8, the ampli-tude of the wave was very short for sutured group revealingno significant improvement while the waves from the nano-fiber nerve guide with and without cells had more amplitude.

Histological assessment of transplanted cells throughnanofiber nerve guide. In order to evaluate the viability ofSHED seeded inside the nerve guides namely for group A andgroup B, transverse sections of nerve guides were obtainedafter 2 weeks of in vivo implantation and sections were alsostained for H&E evaluation. Figure 9 shows the results of theviability of cells inside the nerve guides. Moreover, Figure 9indicates the penetration of SHED cells into the graft core.

H&E staining was also performed for transverse sectionsof nerve guides with and without cells for comparison ofvascularization in these groups and the results showedmore vascularization in the vicinity of sciatic nerve guide

FIGURE 7. Latency of response to the hot stimulus for different groups and control (normal rat) (mean 6 SD, n 5 3). [Color figure can be viewed

in the online issue, which is available at wileyonlinelibrary.com.]

TABLE I. Electrophysiology Results, Amplitude (AMP), and Latency (LAT) of Nanofiber Nerve Guide Groups with and Without

Cells After 4, 8, 12, and 16 Weeks Nerve Guide Implantation

GROUPS AMP (mV) Statistical Results LAT (ms) Statistical Results

Nanofiber nerve guides(4 weeks) 0.5 6 0.1 p>0.05 18.1 6 2.3 p>0.05Nanofiber nerve guide with cells(4 weeks) 0.5 6 0.1 17.4 6 2.9Nanofiber nerve guides(8 weeks) 1.6 6 0.3 p>0.05 4.8 6 1.2 p>0.05Nanofiber nerve guide with cells(8 weeks) 1.5 6 0.4 3.9 6 1.5Nanofiber nerve guides(12 weeks) 2.9 6 0.8 p>0.05 4.1 6 0.9 p>0.05Nanofiber nerve guide with cells(12 weeks) 3.1 6 1 3.1 6 0.6Nanofiber nerve guides(16 weeks) 4.7 6 1.9 p�0.05 2.9 6 0.5 p>0.05Nanofiber nerve guide with cells(16 weeks) 8.1 6 1.6 2.2 6 0.4

ORIGINAL ARTICLE


for nanofiber nerve guides with cells (group B) compared tothat for nanofiber nerve guide without cells (group A) after16 weeks (Fig. 10).

Histological assessment. Transversely cut sections were alsostained with a specific marker for human cells (HNu), a markerof human neural cells (b-Tubulin) and human stem cell-specific

FIGURE 8. Electrophysiology results, waves recorded for different animal groups after 16 weeks of nerve guide implantation. (a) sutured group,

(b) nanofibrous nerve guide without cells, (c) nanofibrous nerve guides with SHED, (d) normal rat. Mild increasing in amplitude in nerve guides with

cells compare to that for nanofiber nerve guide alone could be due to neural differentiation of cells and this is inferred by present of higher ampli-

tude of CMAPs in this group which is nearer to normal. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

TABLE II. Electrophysiology Results, Amplitude (AMP), and Latency (LAT) of Nanofiber Nerve Guide Groups with and Without

Cells and Sutured Groups in Comparison with Normal Rat After 16 Weeks Nerve Guide Implantation(n 5 9)

Normal (N)

NanofiberNerve Gide

with Cells (B)

NanofiberNerve

Guides (C) Sutured (D) Statistical Results

AMP (mV) 13.8 6 2.8 8.1 6 1.6 4.7 6 1.9 0.5 6 0.1 NBa, NCa, NDa, BCa, BDa, CDa

LAT (ms) 1.6 6 0.1 2.2 6 0.4 2.9 6 0.5 7.3 6 2.1 NB, NCa, NDa, BC, BDa, CDa

aShows that difference is significant (p� 0.05) between different groups labeled as A, B, C, and D.


marker (OCT-4) after 2 weeks of nerve guide implantation to fol-low the seeded cells inside the nerve guide. Figure 11 shows thepositive expression of b-Tubulin, OCT-4 and HNu by cells withinthe nanofiber nerve guides seeded with SHED revealing survivalof the transplanted cells after 2 weeks of implantation.

The histological analysis after 16 weeks showed P75positive cells in both nanofiber nerve guides with and with-out cells indicating the integration of rat’s own nerve cells,

and regeneration of the nerve defect through the nerveguides (Figs. 12 and 13). The positive staining patterns ofHNu, b-tubulin, and OCT-4 were also observed for nerveguides group with cells. Higher expression of b-tubulin andlower expression of OCT-4 were observed on 16 week anal-ysis, compared to that after 2 weeks which could be attrib-uted to the differentiation of SHED to NSCs after 16 weeksof transplantation period.

FIGURE 9. H&E staining of SHED seeded in nanofibrous nerve guide after 2 weeks of implantation (Scale bars 5 1000 lm (a) and 500 lm (b)).


FIGURE 10. H&E staining of cross-sectioned nerve guide after 16 weeks of implantation, (a) nanofiber nerve guide without cells, (b) nanofiber

nerve guides with SHED, (c) boxed area in section b is shown at a higher magnification (arrows in section b and c shows vascularization). [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


DISCUSSION

Peripheral nerve injury is a common and serious troublecaused by accidents or injury, and reconstruction and func-tional recovery of injured nerve remains a huge challengefor clinicians and researchers.11,28

Recently artificial nerve guides has been considered as apromising technique to guide axonal re-growth and facilitatenerve regeneration after peripheral nerve injury.12,23 Duringthis study, electrospun nanofibrous mat was fabricated androlled to a tube by rotating around a copper wire and theedges were fixed using medical grade adhesive (Fig. 1). Themethod which we applied in this study is a convenient pro-cedure to fabricate nerve guides without limitation in choos-ing the desired tube diameter and dimension which isusually encountered during the fabrication of nerve guidetubes by other methods. In addition, no special and addi-tional accessories are required to the electrospinning set-up

for fabrication of such nanofiber tubes. Previous studies byPanseri et al. used electrospun poly(L-lactide-co-glycolide)(PLGA)/PCL nerve guides to regenerate a 10-mm nerve gapin a rat sciatic nerve and their results showed that thesetubes are promising tools for functional nervous regenera-tion.24 PLGA nanofiber nerve guides were also used by Biniet al. for peripheral nerve regeneration. After implantationof PLGA nanofiber nerve guide to the injured sciatic nerveof rats, no inflammatory response was observed uponimplantation and their findings showed successful nerveregeneration approximately 1 month after implantation.28

Electrospun PCL/collagen nerve guides has been shown asa useful and promising alternative for autologous nervegrafts for peripheral nerve regeneration.29 Li et al. success-fully connected the proximal and the distal stumps of sev-ered sciatic nerve of rats using PLGA/silk fibroin nanofibernerve guide recently.30 In yet another study, a fully

FIGURE 11. Immunohistochemical results for SHED cells seeded on nanofibrous nerve guide after 2 weeks of implantation. b-Tub, OCT-4 and

HNu positive cells are representative of SHED grafted cells. Scale bars 5 200 mm. [Color figure can be viewed in the online issue, which is avail-

able at wileyonlinelibrary.com.]


integrated bi-layered nerve guide was developed includinglongitudinally aligned nanofibers along the inner side andrandomly oriented nanofibers over the outer side and thisengineered nanostructure demonstrated a significantimpact towards neural tissue regeneration.31 Moreover, theefficacy of nerve guides can be improved by incorporationof growth factors such as nerve growth factor, cells orother biomolecules into nerve guide.24 Koh et al. incorpo-rated laminin and nerve growth factor into nanofibrousnerve guide to evaluate their efficacy in in vivo nerveregeneration and their findings showed that laminin andnerve growth factor could improve the bridging of periph-eral nerve gaps.32

Cell transplantation has attracted more interest for treat-ment of nerve injuries and it has been found to significantlyimprove the motor and sensory functional recovery of sciaticnerve.33,34 Several cell types such as bone marrow mesenchy-mal stem cells (BMMSCs), mesenchymal stem cells (MSCs),Schwann cells, co-culture of dorsal root ganglia and Schwanncells and neural stem cells (NSCs) have been used for theimprovement of peripheral nerve regeneration through theirseeding in conventional nerve guides and the results showedfaster improvement of injured nerve and positive impact ofthese cells on nerve regeneration.11–17,35–38 However, the useof NSCs from other tissues is considered invasive, and theclinical use of BMMSCs is also limited due to its painful

FIGURE 12. Immunohistochemical results of SHED cells seeded on nanofibrous nerve guide after 16 weeks of implantation. b-Tub, OCT-4, and

HNu positive cells are representative of SHED grafted cells. P75 positive cells indicate the presence of rat neural cells. Scale bars 5 200 mm.


ORIGINAL ARTICLE


recovery as well as the availability of limited number ofharvested cells from BMMSCs.20,35

Previous studies have shown that SHED are a source ofmultipotent stem cells with ability to differentiate intodifferent cell types including odontoblasts, chondrocytes,endothelocytes, adipocytes, smooth muscle cells, andosteoblasts.18,20

Easy isolation with minimal invasiveness compared toother sources of MSCs, high proliferation rate and multipo-tency of these cells provide a unique and available popula-tion of stem cells from an unexpected tissue resource.39,40

Every kid loses primary teeth which provide great opportu-nity to restore this great source of stem cells for treatmentof future injuries or diseases.40

Very few reports are available on cell transplantationthrough nanofiber nerve guides for peripheral nerve regen-eration. Unlike previous researches who used nanofibrousnerve guides without cell seeding, the present workemployed a biodegradable PCL/gel nanofiber nerve guideseeded with SHED to assess the efficacy of nerve guide sup-ported with cells towards peripheral nerve regeneration. Tothe best of our knowledge there is no study on the trans-plantation of SHED through nanofiber or other types ofnerve guides for treatment of nerve injury.

In vitro cell culture study showed good interaction ofSHED with PCL/gel nanofibers and their proliferation onnanofibrous substrate was found higher than that on TCP.Our previous study showed that a hydrophilic surface withthe presence of amine and carboxylic functional groups onthe surfaces of PCL/gel nanofibers, provide an environmentthat supports cell adhesion and proliferation.25 The higherproliferation of SHED on the nanofibrous scaffolds com-pared to TCP is not surprising and it confirms the feasibilityof further in vivo application of these nanofibers as nerveguide to bridge a 10 mm sciatic nerve gap in rats.

An SFI value of 85 was observed during this study forall groups after 1 week surgery, and this was similar to thereports by Zheng and Cui where they transplanted BMSCsthrough chitosan nerve guide for bridging an 8-mm longneural gap.10 SFI value did not increase notably for suturedand untreated group at 16 weeks after the implantationwhile it increased in both nanofiber nerve guide with andwithout SHED to 220 and 225 (Fig. 6) revealing the effi-

ciency of nerve guide either with or without SHED for nerveregeneration.

No significant electrophysiological improvement wasnoticed up to 12 weeks (0.2 mV AMP and 23 ms LAT) forthe sutured group, and no significant recovery was observedeven after 16 weeks (0.5 mV AMP and 7.3 ms LAT) timeperiod. Electrophysiology results demonstrated that theamplitudes of CMAPs were greater and latency period wasshorter for nanofiber nerve guides with SHED than thenanofiber nerve guides alone, which yet again support theresults of the SFI analysis.

H&E staining showed the viability of seeded SHED insidethe nerve guide after 2 and 16 weeks of implantation whichsuggests that the nanofiber nerve guides could hold the trans-planted cells during the early stages of nerve regeneration.Presence of blood vessels in the vicinity of the nerve guidewas observed for both nerve guide group with or withoutcells. However, more vascularization was observed for nano-fiber nerve guide groups with cells compared to that withoutcells revealing that the blood vessel regeneration was pro-moted by SHED. Expression of P75 marker by immunohisto-chemistry results showed the axonal regeneration pertinencefor both nanofiber nerve guide group with or without cells.Our overall results showed the significant effect of SHED inaccelerating the peripheral nerve regeneration. Sasaki et alalso fabricated silicone and poly-D,L-lactide-co-glycolide(PLGA) tubes filled with DPSCs and their results showed thatDPSCs enhanced facial nerve regeneration in rats.35,41

Previous studies showed that the dental pulp cells pro-duce neurotrophic factors such as the nerve growth factor,brain-derived neurotrophic factor and glial cell line-derivedneurotrophic factor, that enable peripheral nerve regenera-tion and protect from facial motor neuron death.39,41

Regarding the ability of SHED to produce neurotrophic fac-tors, these cells can be beneficial for the treatment of neuro-degenerative diseases and can be as a good cell source fortransplantation in injured nerve.19

It can be concluded that PCL/gel nanofiber nerve guideseeded with SHED offer a more mimicking micro and macroenvironment for peripheral nerve regeneration, by providingan excellent physical substrate to guide naturally regenerat-ing axons to the distal section while being supported by theneurotrophic factors released from the SHED.

FIGURE 13. Immunohistochemical results of the cross-sectioned nerve guide without cells after 16 weeks of implantation. P75-positive cells show

the presence of rat neural cells. Scale bars 5 200 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


More b-tubulin expression compared to OCT-4 after 16weeks for nanofiber nerve guide with SHED (Fig. 12) couldbe due to in vivo differentiation of these cells to neural likecells. A possible explanation can be that the dental pulp isderived from neural crest tissue, giving a predisposition ofin vivo differentiation towards neural lineages.42 Nour-bakhsh et al. evaluated the in vitro differentiation of SHEDto nerve cells and their results provided evidence thatSHED can differentiate into neural cells by expression of acomprehensive set of genes and proteins related to neural-like cells.40 They also showed that SHED inherently expressneural markers as they are derived from cranial neural crestcells. In yet another study SHED was able to express pro-teins on their cell surfaces that allowed them to differenti-ate into neural cells.19 However, further studies are neededto ensure whether SHED can be differentiated to neural likecells after transplantation to injured sciatic nerve.

CONCLUSION

In this study, we successfully fabricated tubular nerveguides from electrospun PCL/gel nanofibers and the effectof the nerve guides with and without SHED on sciatic nerveregeneration was evaluated. For comparison, nerve regener-ation in sutured sciatic nerve and untreated injured sciaticnerve were also examined. While both nanofiber nerveguides with or without cells supported axonal regenerationacross the nerve gap, regeneration through nanofiber nerveguides with SHED was found superior in terms of nerve re-growth, functional and sensory recovery and histologicalassessment. Higher nerve regeneration in nerve guide groupseeded with SHED could be attributed to the expression ofneurotrophic factors which is related to neural-crest originof these cells. In conclusion, the tabulation of injured sciaticnerve using nanofiber nerve guide seeded with SHED enablebridging of longer nerve gaps, a promising solution in thefield of neuroscience.

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