Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue...

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Acta Biomaterialia 5 (2009) 2560–2569

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Aligned and random nanofibrous substrate for the in vitro cultureof Schwann cells for neural tissue engineering

Deepika Gupta a, J. Venugopal a,*, Molamma P. Prabhakaran a, V.R. Giri Dev c,Sharon Low b, Aw Tar Choon b, S. Ramakrishna a

a Nanoscience and Nanotechnology Initiative, Division of Bioengineering, National University of Singapore, Block E3, #05-12,

2 Engineering Drive 3, Singapore 117576b StemLife Sdn Bhd, 50450 Kuala Lumpur, Malaysia

c Department of Textile Technology, AC College of Technology, Anna University, Chennai, India

Received 20 August 2008; received in revised form 20 January 2009; accepted 26 January 2009Available online 5 February 2009

Abstract

The current challenge in peripheral nerve tissue engineering is to produce an implantable scaffold capable of bridging long nerve gapsthat will produce results similar to autograft without requiring the harvest of autologous donor tissue. Aligned and random polycapro-lactone/gelatin (PCL/gelatin) nanofibrous scaffolds were fabricated for the in vitro culture of Schwann cells that assist in directing thegrowth of regenerating axons in nerve tissue engineering. The average fiber diameter attained by electrospinning of polymer blend (PCL/gelatin) ranged from 232 ± 194 to 160 ± 86 nm with high porosity (90%). Blending PCL with gelatin resulted in increased hydrophilicityof nanofibrous scaffolds and yielded better mechanical properties, approaching those of PCL nanofibers. The biocompatibility of fab-ricated nanofibers was assessed for culturing and proliferation of Schwann cells by MTS assay. The results of the MTS assay and scan-ning electron microscopy confirmed that aligned and random PCL/gelatin nanofibrous scaffolds are suitable substrates for Schwann cellgrowth as compared to PCL nanofibrous scaffolds for neural tissue engineering.� 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Electrospinning; Random and aligned nanofibers; PCL; Gelatin; Schwann cells

1. Introduction

Existing tissue engineering approaches focus on evolvingan alternative route for nerve tissue regeneration usingpolymeric scaffolds and striving to find suitable biomateri-als for tissue engineering. Due to the unavailability of idealscaffold material and limited knowledge of cell and scaffoldinteraction, there have been few breakthroughs in nerve tis-sue engineering [1]. A scaffold that can mimic, and hencereplace, the structure and function of extracellular matrix(ECM), i.e., with strong regenerative and cell supportingcapacity, is central to nerve tissue engineering [2]. Nanofi-brous scaffolds have replaced conventional strategies such

1742-7061/$ - see front matter � 2009 Acta Materialia Inc. Published by Else

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* Corresponding author. Tel.: +65 6516 4272; fax: +65 6773 0339.E-mail address: [email protected] (J. Venugopal).

as autografts, allografts and xenografts [3,4] for nerve tis-sue engineering due to the limited supply of donor nervegrafts in autografts [5,6], the chances of immunorejectionin allografts and xenografts [7] and their use being limitedto bridging only small nerve gaps and defects [8,9].

Nerve tissue regeneration requires an scaffold thatshould ideally be biodegradable, biocompatible andmechanically robust, having large surface area, high poros-ity and interconnected pores [10] and also act as a bridge toregenerate growing axons across larger nerve defects. Scaf-fold is also critical for regenerating cells as it allows thecells to proliferate, migrate, produce ECM to form func-tional tissues, and provides cells with their own microenvi-ronment [11]. The high porosity of nanofibrous scaffoldsprovides more structural space for cell accommodationand enables the exchange of nutrient and metabolic wastebetween the scaffold and environment [12]. Electrospinning

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D. Gupta et al. / Acta Biomaterialia 5 (2009) 2560–2569 2561

is one of the novel techniques for achieving nanofibrousscaffolds, mimicking the structural and functional mor-phology of ECM, which includes the use of very high volt-age for the production of nanofibers [13].

Electrospinning is a simple and efficient technique usedfor the fabrication of high-porosity nanoscale scaffolds.Such scaffolds are effective in providing a large surface areafor cell attachment, and allows cells to attain proper mor-phology [10]. The morphology, fiber diameter and porosityof electrospun nanofibrous scaffolds can be controlled byvarying parameters, such as applied electric field strength;spinneret diameter; distance between the spinneret andthe collecting substrate; temperature; feeding rate; humid-ity; air speed; and properties of the solution or melt, includ-ing the type of polymer, polymer molecular weight, surfacetension, conductivity, and viscosity, temperature, concen-tration of the polymer, etc. [14,15]. Numerous naturaland synthetic polymers have been investigated for thefabrication of nanofibrous scaffolds for nerve tissue regen-eration. Yang et al. designed nanostructured porouspoly(L-lactic acid) (PLLA) scaffolds intended for nervetissue engineering [16], while polymers such as poly(glycolicacid) (PGA) [17], poly(L-lactic acid)-co-poly(e-caprolac-tone) (PLA-CL) [18] have also been used for the regenera-tion of nerve tissue. Natural and synthetic polymers alonecannot meet all the requirements of a perfect scaffold: nat-ural polymers lose their mechanical properties very earlyduring degradation while synthetic polymers alone are lesshydrophilic, lack binding sites for cell adhesion and releaseacidic degradation products [19]. To overcome the prob-lems associated with individual polymers, hybrid materials(blends of two or more types of polymers) have beendevised by researchers that assimilate the desirable charac-teristics of component materials.

An attempt to devise a near-perfect scaffold for growingSchwann cells for nerve tissue engineering was performedin the present study by using a blend of natural and syn-thetic polymer to impart favorable characteristics to thescaffold. Gelatin, a natural polymer and a product of par-tial hydrolysis of collagen [10], was blended with polycap-rolactone (PCL), a synthetic and highly hydrophobicpolymer, to fabricate aligned and random nanofibers forculturing Schwann cells, which are the supporting cells ofperipheral nervous system (PNS) and play a key role inaxonal regeneration. Gelatin contains many glycine, pro-line and hydroxyproline residues that help in cell adhesionand differentiation but it is also a soft material and has lowtensile properties. PCL has good mechanical properties butits low hydrophilicity together with a lack of functionalgroups often results in low cell adhesion and proliferationon these scaffolds. Different types of physicochemical andpost-processing surface modification techniques have beenattempted to solve the problem of cell adhesion and prolif-eration on hydrophobic surfaces [20]. Moreover, nerveregeneration requires a scaffold with low degradation andswelling properties, in order to withstand nerve compres-sion which otherwise impedes the outgrowth of regenerat-

ing nerves. Blending PCL with gelatin has furnished abetter biocompatibility and hydrophilicity as well asmechanical properties suited for nerve tissue engineering.In an effort to closely mimic the ECM, aligned and randomnanofibers were produced as potential substrates for Schw-ann cell growth, aiming at comparing the effect of fiber ori-entation and surface morphology on proliferation ofSchwann cells. The aligned-nanofiber fabrication techniqueinvolves high rotation speed, providing thinner and closelypacked nanofibers [10,14] that help in providing guidancecues to the regenerating axons, while the random nanofi-bers acquire high porosity, an even pore size and a surfacetopography containing ridges and grooves [16]. In thisstudy the composite nanofibrous scaffold of PCL/gelatinwas fabricated and evaluated for the properties such ashydrophilicity, functional groups, tensile strength, pore sizedistribution and growth of Schwann cells for neural tissueengineering.

2. Materials and methods

2.1. Materials

PCL (Mw 80,000) and gelatin (Type A) were broughtfrom Sigma–Aldrich (St. Louis, MO, USA) and 2,2,2-tri-fluoroethanol (TFE) was obtained from Fluka (Germany).Rat Schwann cells were obtained from ATCC (USA) forcell culture studies. Dulbecco’s Modified Eagle’s Medium(DMEM) was obtained from Sigma; fetal bovine serum(FBS), antibiotics and trypsin-EDTA were purchased fromGIBCO Invitrogen (Carlsbad, CA, USA). Hexamethyldisi-lazane (HMDS) was bought from Sigma (Singapore) andused as sample drying agent for scanning electron micros-copy (SEM) studies.

2.2. Methods

2.2.1. Fabrication of nanofibrous scaffoldsSolutions of PCL and PCL/gelatin with suitable concen-

trations were prepared for electrospinning. PCL (9% w/v)solution was made by dissolving PCL in TFE. Gelatinwas dissolved first in TFE followed by the addition ofPCL pellets, to make a 10% translucent solution and sub-sequently used for electrospinning to obtain PCL/gelatinaligned and random nanofibers. For electrospinning, thepolymer solution was fed into a 3 ml standard syringeattached to a 27G blunted stainless steel needle with innerdiameter of 0.4 mm using a syringe pump (KDS 100, KDScientific, Holliston, MA) at a flow rate of 1.5 ml h�1 withan applied voltage of 17.5 kV (Gamma High VoltageResearch, USA). Random fibers were collected on a flatcollector plate wrapped with aluminum foil that was keptat a distance of 13 cm from the needle tip. Aligned nanof-ibers were formed using a rotating disk setup with the sameparameters and this rotating disk operated at a speed of4000 rpm for obtaining well-aligned nanofibers. Thesenanofibers were collected on 15 mm coverslips for cell cul-

2562 D. Gupta et al. / Acta Biomaterialia 5 (2009) 2560–2569

ture studies. The as-spun nanofibrous scaffolds were driedin vacuum at room temperature prior to their characteriza-tion and used for cell culture studies.

2.2.2. Characterization of nanofibrous scaffolds

2.2.2.1. Surface morphology of nanofibrous scaffolds. Thesurface morphology of electrospun PCL and PCL/gelatinaligned and random nanofibrous scaffolds were studiedunder field emission SEM (FEI-QUANTA 200F, TheNetherlands) at an accelerating voltage of 10 kV, aftersputter coating with gold (JEOL JFC-1200 fine coater,Japan). Diameters of the electrospun fibers were analyzedfrom the SEM images using image analysis software(Image J, National Institutes of Health, USA).

2.2.2.2. Fourier transform infrared spectroscopy (FTIR).

FTIR spectroscopic analysis of electrospun PCL andPCL/gelatin random nanofibrous scaffolds was performedon an IR-Prestige-21 (Shimadzu Europe) over a range of500–4000 cm�1 at a resolution of 4 cm�1.

2.2.2.3. Pore size and porosity. Pore size and pore size dis-tribution of the nanofibrous scaffolds were measured bycapillary flow porometer (CFP-1200-A) (Ithaca, NY,USA) by wet up/dry up method and the analysis was doneusing automated capillary flow porometer system software.The bulk density of PCL/gelatin random nanofibrousmembrane is known to be 1.2–1.3 g cm�3 [22] and that ofPCL is 1.146 g cm�3. The thicknesses of PCL and PCL/gel-atin random nanofibrous membranes were measured bymicrometer, and apparent densities and porosities were cal-culated by the following equations:

Apparent Density ðg=cm3Þ

¼ Mass of Nanofiber Membrane ðgÞMembrane thickness ðcmÞ � area ðcm2Þ ð1Þ

Porosity ð%Þ

¼ 1� Apparent Density ðg=cm3ÞBulk density of membranes ðg=cm3Þ

� �� 100%

ð2Þ

2.2.2.4. Contact angle measurement. The hydrophilic natureof the electrospun nanofibrous scaffolds was measured bysessile drop water contact angle measurement using aVCA Optima Surface Analysis system (AST products,Billerica, MA). Distilled water was used for drop forma-tion. The measured contact angle value reflected the hydro-philicity of the scaffolds.

2.2.2.5. Tensile strength measurement. Tensile properties ofnanofibrous membrane were determined at normal roomtemperature using an Instron 5845 Microtester (USA), ata cross-head speed of 5 mm min�1. Rectangular specimenswere cut from the electrospun membranes of 20–30 lmthickness and used for mechanical studies. The ends of

the rectangular specimens were mounted vertically onmechanical gripping units of the tensile tester and a loadof 10 N was applied for tensile measurements.

2.2.3. Cell culture

Schwann cell line (rat), RT4-D6P2T, was obtained fromATCC (USA) and was cultured in DMEM high glucose,supplemented with 10% FBS and 1% antibiotic and anti-mycotic solutions (Invitrogen Corp, USA.). Cells wereincubated at 37 �C in a humidified atmosphere containing5% CO2 and the culture medium was changed once in every3 days. Each of the nanofibrous scaffolds on 15 mm cover-slip was placed in 24-well plate and pressed with a stainlesssteel ring to ensure complete contact of the scaffolds withwells. The specimens were sterilized under UV light,washed thrice with phosphate buffer saline (PBS) and sub-sequently immersed in DMEM overnight before cell seed-ing. Schwann cells grown in 75 cm2 cell culture flaskswere detached by adding 1 ml of 0.25% trypsin containing0.1% EDTA. Detached cells were centrifuged, counted bytrypan blue assay using a hemocytometer and seeded onthe scaffolds at a density of 1.2 � 104 cells/well and left inincubator for facilitating cell growth. Tissue culture poly-styrene (TCP) was used as a control and PCL, PCL/gelatinaligned and random nanofibrous scaffolds were used forcomparisons. Cell proliferation was determined by MTSassay after 3, 6 and 9 days of cell culture.

2.2.4. Cell proliferation

The cell adhesion and proliferation on the scaffolds weredetermined using the colorimetric MTS assay (CellTiter96� AQueous One solution, Promega, Madison, WI,USA). The reduction of yellow tetrazolium salt in MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) to form purple forma-zan crystals by the dehydrogenase enzymes secreted bymitochondria of metabolically active cells forms the basisof this assay. The formazan dye shows absorbance at492 nm and the amount of formazan crystals formed isdirectly proportional to the number of cells. To processfor MTS assay, the samples were rinsed with PBS toremove unattached cells and incubated with 20% MTSreagent in a serum free medium for a period of 3 h at37 �C. Absorbance of the obtained dye was measured at490 nm using a spectrophotometric plate reader (FLUO-star OPTIMA, BMG lab Technologies, Germany).

2.2.5. Morphology of Schwann cells

Morphological study of the in vitro cultured Schwanncells on PCL, PCL/gelatin aligned and random nanofi-brous scaffolds were performed. After 9 and 12 days of cellproliferation, the cell-cultured scaffolds were processed forSEM studies. The scaffolds were rinsed twice with PBS andfixed in 3% glutaraldehyde for 3 h. Thereafter, the scaffoldswere rinsed in DI water and dehydrated with upgradingconcentrations of ethanol (50%, 70% 90%, 100%) twicefor 15 min each. Final washing with 100% ethanol was fol-


lowed by treating the specimens with hexamethyldisilazane(HMDS). The HMDS was air-dried by keeping the sam-ples in fume hood. Finally the scaffolds were sputter coatedwith gold and then observed under SEM.

2.2.6. Statistical analysisAll the data presented in the paper are expressed as

mean ± standard deviation (SD) and were analyzed usingStudent’s t-test for the calculation of significance level ofthe data (n = 6). Differences were considered statisticallysignificant when P 6 0.05.

3. Results

3.1. Electrospun nanofibrous scaffolds

SEM micrographs of electrospun nanofibrous scaffoldsrevealed porous, nanoscaled fibrous structures formedunder controlled parameters. Randomly oriented PCLand PCL/gelatin nanofibrous scaffolds are obtained uni-form, bead free nanofibers and interconnected pores withfiber diameter in the range of 471 ± 218 and232 ± 194 nm, respectively. Aligned PCL and PCL/gelatinnanofibers showed a consistent thickness with fiber diame-ter of 200 ± 100 and 160 ± 86 nm, respectively, compara-tively lower than the random nanofibers (Fig. 1).

Fig. 1. SEM micrographs of nanofibers: (a) aligned PCL nanofibers; (b) aligngelatin nanofibers. Arrows represents direction of alignment in aligned nanofi

3.2. Fourier transform infrared spectroscopy

FTIR analysis of PCL/gelatin random nanofibrous scaf-folds illustrated the characteristic peaks of gelatin (Fig. 2).Typical bands of N–H stretch for amide A and C–H stretchfor amide B were obtained at 3194 and 3032 cm�1, respec-tively. The appearance of such characteristic peaks sug-gested the presence of amine groups on the surface ofPCL/gelatin nanofibers. Amide I band of gelatin forC@O stretch, which is the measure of secondary structureof proteins, was obtained at 1650 cm�1 in PCL/gelatinand was very prominent. Amide II peak for N–H bend cou-pled with C–N stretch was obtained at 1527 cm�1 andamide III region for N–H bend which is associated with tri-ple helix structure of gelatin was obtained between 1200and 1250 cm�1.

3.3. Porosity and pore size

Structural pore properties assessed by capillary flowporometer for PCL and PCL/gelatin random nanofibrousscaffolds showed the largest and smallest pore diameterobtained was 2.95 and 1.05 lm, respectively. The plots ofpore size distribution vs. average diameter of pore shownin Fig. 3 indicate maximum number of pores with an aver-age diameter in the range of 1.1–1.25 lm for PCL/gelatinand 1.5 lm for PCL scaffolds per unit area. The porosities

ed PCL/gelatin nanofibers; (c) random PCL nanofibers; (d) random PCL/bers.

Fig. 2. FTIR spectroscopy of PCL and PCL/gelatin random nanofibers.

Fig. 3. Pore size distribution curve of PCL and PCL/gelatin random nanofibers.

Table 1Comparison of nanofiber properties of PCL and PCL/gelatin randomnanofibers.

Properties PCL PCL/gelatin

Fiber diameter 471 ± 218 nm 232 ± 194 nmPorosity 86 ± 5% 90 ± 5%Contact angle 112� 0�Wetability Highly hydrophobic Highly hydrophilic


of PCL and PCL/gelatin random nanofibrous scaffoldswere calculated and it was as high as 86% and 90%, respec-tively, which is an indicative of the fact that the nanofi-brous scaffolds were highly porous structure.

3.4. Hydrophilic characteristics

Contact angle studies of PCL and PCL/gelatin nanofi-brous scaffolds revealed the surface hydrophilic nature.The contact angle obtained for PCL scaffolds was about112� which implies that these scaffolds were highly hydro-phobic and nonadsorbent to water. The PCL/gelatin ran-dom nanofibrous scaffolds were extremely hydrophilic withtheir water contact angle being zero showing 100% wettabil-ity by the water droplet signifying the presence of hydrophilicgroups on the surface of the scaffold. A comparison betweenthe fiber properties of PCL and PCL/gelatin random nanofi-brous scaffolds has been illustrated in Table 1.

3.5. Mechanical properties of electrospun nanofibers

PCL and PCL/gelatin random nanofibers showed a typi-cal non-linear stress–strain curve as illustrated in Fig. 4. The

tensile properties of PCL/gelatin (1:1 wt.%) blended nanofi-bers were very near to PCL nanofibers. PCL/gelatin randomnanofibrous scaffold could bear a strain of 71.25% which wasclose to the strain endured by PCL (72.5%). Elastic modulus,which is a measure of resistance to deformation, was high forPCL/gelatin random nanofibers as compared to PCL. Table2 documents the mechanical properties of PCL and PCL/gel-atin random nanofibrous scaffolds with respect to tensilestress, strain and elastic modulus.

3.6. Cell proliferation

The proliferation of Schwann cells on PCL/gelatinnanofibrous scaffolds was significantly (P 6 0.05) higher

Fig. 4. Stress–strain curve of PCL and PCL/gelatin random nanofibers.

Table 2Tensile properties of electrospun PCL/gelatin random nanofibers.

Nanofibrousscaffolds

Elastic modulus(MPa)

Tensile stress(MPa)

Tensile strain(%)

PCL 2.5 1.42 72. 5PCL/gelatin 6.0 1.26 71.25


than the cell proliferation on PCL nanofibrous scaffolds(Fig. 5). On day 9, PCL/gelatin random nanofibers showeda cell proliferation of 43% higher than PCL random nanof-ibers while PCL/gelatin aligned nanofibers showed anincrease of 28% compared to PCL aligned nanofibers.The cell proliferations on both aligned and random PCL/gelatin nanofibers were found 80% higher compared toPCL nanofibers on day 9. The results obtained fromMTS assay indicated that PCL/gelatin nanofibrous scaf-folds are suitable substrates than PCL nanofibers, in termsof cell attachment and proliferation. However, the prolifer-ation of cells was significantly (P 6 0.05) increased in ran-dom nanofibers than aligned nanofibrous scaffold.

Fig. 5. MTS assay for Schwann cell proliferation on PCL and PCL/gelatin aligned and random nanofibrous scaffolds and TCP. Bar representsmean ± standard deviation. Asterisks indicate significance level of prolif-eration as compared to PCL scaffolds obtained by t-test: **P 6 0.05;*P 6 0.001. A, aligned nanofibers; R, random nanofibers.

3.7. Morphological studies of Schwann cells

SEM images observed for day 9 (Fig. 6) and 12 (Fig. 7)of cell culture showed normal morphology of Schwanncells on PCL/gelatin aligned and random nanofibrous scaf-folds. Aligned nanofibers of both PCL and PCL/gelatin(Figs. 6a,b and 7a,b) showed cells oriented along the direc-tion of the fibers and clustered around the aligned fibers ina longitudinal fashion while the random fibers showed cellsoriented different directions of the nanofibers. Cells onPCL/gelatin random nanofibrous scaffolds (Figs. 6d and7d) showed normal bipolar and tripolar extensions andspindle-shaped morphology similar to the cell morphologyon tissue culture polystyrene (Fig. 6e). PCL random nano-fibrous scaffolds displayed cells with bipolar processes buttheir morphology was rounded with flattened cells and vis-ibly less proliferation (Figs. 6c and 7c).

4. Discussion

4.1. Electrospun nanofibers

Electrospinning is being successfully used for generat-ing fibers to mimic ECM-like properties such as nanoscaledimension, high porosity, interconnected pores and largesurface area for tissue engineering. Its flexibility in con-trolling fiber size and fiber orientation makes it superiorto other methods of scaffold formation such as self-assem-bly, phase separation and solvent casting [14]. Also large-scale production of three-dimensional aligned and randomnanofibers could not be created by any other technique[21]. A variety of natural and synthetic polymers havebeen investigated for the growth of Schwann cells fornerve tissue regeneration. Synthetic polymers such asPLLA scaffolds fabricated by Yang et al. for nerve stemcell culture [16], PLGA scaffolds fabricated by Li et al.for Schwann cells and neural stem cells [22], and naturalpolymers such as collagen [23], alginates [24], chitosan[25], and silk fibroin [26] scaffolds for nerve repair, havebeen investigated but most of the natural or syntheticpolymer alone proves to be appropriate for nerve tissueengineering, thus leading researchers to explore possibili-ties of blending natural and synthetic polymers that caneliminate the shortcomings of individual polymers. Sch-nell et al. devised collagen/PCL nanofibers as guidancestructures for axonal regeneration [27]. Similarly, Pra-bhakaran et al. fabricated chitosan/PCL biocompositenanofibers for neural cell culture, where chitosan/PCLcontained suitable properties that supported cell growth[28]. In this study PCL/gelatin (10%) was prepared inTFE solvent and electrospun for producing beadless andrelatively thin fibers of 200–400 nm. Similar results wereobtained by Zhang et al. by electrospinning 10% solutionof PCL/gelatin mixed in a ratio of 50:50 in TFE for bonemarrow stromal cell culture [29]. PCL/gelatin scaffoldshave also been used in wound healing and dermal recon-stitution by Chong et al. [30] and culturing endothelial

Fig. 6. SEM micrographs of Schwann cells on nanofibrous scaffolds obtained after day 9 of cell culture: (a) aligned PCL nanofibers; (b) aligned PCL/gelatin nanofibers; (c) random PCL nanofibers; (d) random PCL/gelatin; (e) TCP.

Fig. 7. SEM micrographs of Schwann cells on nanofibrous scaffolds obtained after day 12 of cell culture: (a) aligned PCL nanofibers; (b) aligned PCL/gelatin nanofibers; (c) random PCL; (d) random PCL/gelatin; (e) TCP.


cells for blood vessel tissue engineering by Ma et al. [31].However, the use of PCL/gelatin nanofibrous scaffolds aspotential substrates for nerve tissue engineering and com-

parison of PCL/gelatin aligned and random nanofibersfor Schwann cell growth, proliferation and alignment isreported in the present study.


4.2. Surface properties and characterization of nanofibrous

scaffolds

PCL has been extensively used for tissue engineeringdue to its high tensile strength, non-toxic nature and bio-degradability [19,32] but its hydrophobic character isundesirable for in vitro cell culture. Blending of syntheticpolymer with biopolymers is one of the methods to intro-duce functional groups such as amine, hydroxyl and car-boxyl groups to provide scaffolds with more hydrophiliccharacter, and previous studies have shown that scaffoldswith hydrophilic surfaces show better cell adhesion andgrowth [33]. SEM micrographs of random and alignednanofibrous scaffolds (Fig. 1) showed that the fiber sizewas polydispersed in both the types of nanofibers. Useof high speed rotating disk setup for the production ofaligned nanofibers imparted a smooth surface morphol-ogy with highly aligned and fine fibers. Random fibersobtained on the static collector plate showed a surfacemorphology with grooves, ridges and pores impartingrough surface topography that makes better scaffolds forcell attachment and proliferation [16]. The FTIR analysisof PCL/gelatin random fibers showed peaks for N–H andC–H stretch at 3194 and 3032 cm�1, respectively, whichconfirmed the presence of amino groups on the surfaceof the hybrid material. Presence of amide III bandshowed that the gelatin has a triple helix structure similarto that of collagen, which is an integral part of ECM,hence gelatin greatly favors cell adhesion and prolifera-tion [34]. The results of contact angle measurement pro-vided evidence for increased hydrophilicity of PCL/gelatin scaffolds and the presence of functional groupsto form hydrogen bonds with water molecules thatincreased wettability of the nanofibers. Porosity of 86%and 90% for PCL and PCL/gelatin random nanofibers,respectively, showed that the scaffolds were highly porousdue to the formation of very fine fibers imparting largesurface area and more room for cell adhesion and migra-tion. Pore size in the range of 1–2 lm (Fig. 3) obtainedfor these scaffolds was desirable since large pore size helpsin cell migration and nutrient flow thus enhancing theircell supporting capacity [35]. The direction of neurite out-growth and cellular elongation are parallel to the direc-tion of aligned nanofibrous scaffolds, and thisphenomenon of ‘‘contact guidance” is considered impor-tant for peripheral nerve regeneration. Our SEM resultsalso showed Schwann cell growth in an oriented fashionalong the alignment of nanofibers. The growth of Schw-ann cells on aligned nanofibers provides better guidancecues for cell growth, axonal extension and thereforeresults in better nerve repair [36].

4.3. Characterization of mechanical properties

Tensile stress obtained for random nanofibers of PCL/gelatin was 1.26 MPa and for PCL was 1.42 MPa, andtensile strain for PCL/gelatin was 71.25% and for PCL

72.5% (Table 2). The mechanical properties of PCL/gel-atin nanofibers were almost nearing to PCL and impliedthat blending PCL with gelatin provided a scaffold withsuitable biological properties and mechanical strength.Mechanical property of gelatin alone is very weak andblending with PCL produced a nanofibrous scaffold withintermediary mechanical strength better for nerve regen-eration. Similar effect was also observed by Lee et al.with the addition of 10–39 wt.% of gelatin, resulting inenhanced tensile properties and elongation at break ofPLCL/gelatin scaffolds [37].

4.4. Cell proliferation and cell-scaffold interaction

PCL/gelatin nanofibrous scaffolds were suitable forSchwann cell growth compared to PCL nanofibrousscaffolds observing a significant level (P 6 0.05) ofincrease in cell proliferation after day 6 of culture anda percentage increase upto 80% after day 9 of culture(Fig. 5). The proliferation of Schwann cells wasincreased on random nanofibers than on aligned fibers,a possible reason for this being that random fibers con-tain lots of interconnected pores, rough surfaces thatassist in adhesion and proliferation of more number ofcells [16]. Hydrophilic nature of PCL/gelatin scaffoldscould be another reason for the Schwann cells showingbetter cell adhesion and proliferation. Suwantong et al.proved Schwann cells attached more on poly (3-hydroxybutyrate) films are hydrophilic than on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film havinghydrophobic surfaces [38].

SEM micrographs of Schwann cells obtained on day 9and 12 of culture on nanofibrous scaffolds showed nor-mal cell morphology in PCL/gelatin nanofibers (Figs. 6and 7). Schwann cells possess bi-polar and tri-polarextensions with spindle-shaped morphology [39]. Kimet al. studied Schwann cell migration and orientationon uniaxially aligned poly(acrylonitrile-co-methylacrylate)(PAN-MA) fiber films for the peripheral nerve repair[40]. Normal bi-polar extension was found on TCP anda similar morphology was observed with bi and tri-polarcells in PCL/gelatin nanofibers. Schwann cells showednormal morphology on PCL/gelatin nanofibrous scaf-folds while on PCL scaffolds the morphology wasrounded and flat; it could be inferred that gelatin favoredthe growth and normal morphology of Schwann cells. Asimilar result was obtained by Pego et al.: trimethylenecarbonate (co) polymer (TMC) and gelatin scaffoldsseeded with Schwann cells showed typical bipolar mor-phology on gelatin scaffolds while rounded morphologyon TMC copolymer scaffolds [41]. This observation couldbe attributed to the fact that the gelatin contains manyintegrin binding sites similar to that found on collagen,facilitating cell adhesion and differentiation [37]. Thusblending gelatin with PCL provides the nanofibrous scaf-folds with a novel biocompatibility, bioactivity and cellaffinity for neural tissue engineering.


5. Conclusions

Natural and synthetic polymer mix is a simple and effec-tive approach towards fabrication of novel compositematerials with tuned properties suitable for nerve tissueengineering. PCL/gelatin nanofibrous scaffolds showedsome prospects as they possess desirable properties suchas increased hydrophilicity, porosity, tensile strength, andbiocompatibility. The enhanced affinity of Schwann cellsfor PCL/gelatin aligned and random nanofibrous scaffoldsconfirmed from cell adhesion and SEM studies suggestedthat surface hydrophilicity and functionalization of thefibers play a fundamental role in cell adhesion, prolifera-tion and healthy cell morphology. Although the prolifera-tion of Schwann cells was lower on aligned nanofibersthan on random nanofibers of PCL/gelatin, it was notice-ably more than PCL nanofibrous scaffolds, thus confirmingthat PCL/gelatin nanofibrous scaffolds are potential sub-strates for nerve tissue engineering.

Acknowledgments

This study was supported by the Office of Life Sciencesin the National University of Singapore and StemLife SdnBhd, 50450 Kuala Lumpur, Malaysia.

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Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue...

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