lable at ScienceDirect

Biomaterials 29 (2008) 4532–4539

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Electrospun poly(3-caprolactone)/gelatin nanofibrous scaffoldsfor nerve tissue engineering

Laleh Ghasemi-Mobarakeh a,b,c,d, Molamma P. Prabhakaran a, Mohammad Morshed c,Mohammad-Hossein Nasr-Esfahani d, Seeram Ramakrishna a,*

a NUS Nanoscience and Nanotechnology Initiative, National University of Singapore, 9 Engineering Drive 1, Singapore 117576b Islamic Azad University, Najafabad branch, Isfahan, Iranc Department of Textile Engineering, Isfahan University of Technology, Isfahan, Irand Department of Cell Sciences Research Center (Isfahan Campus), Royan Institute, ACECR, Tehran, Iran

a r t i c l e i n f o

Article history:Received 21 June 2008Accepted 4 August 2008Available online 30 August 2008

Keywords:Nerve tissue engineeringElectrospinningNanofiberPCLGelatin

* Corresponding author. Tel.: þ65 6516 8596; fax:E-mail address: seeram@nus.edu.sg (S. Ramakrish

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.08.007

a b s t r a c t

Nerve tissue engineering is one of the most promising methods to restore nerve systems in humanhealth care. Scaffold design has pivotal role in nerve tissue engineering. Polymer blending is one of themost effective methods for providing new, desirable biocomposites for tissue-engineering applications.Random and aligned PCL/gelatin biocomposite scaffolds were fabricated by varying the ratios of PCL andgelatin concentrations. Chemical and mechanical properties of PCL/gelatin nanofibrous scaffolds weremeasured by FTIR, porometry, contact angle and tensile measurements, while the in vitro biodegrad-ability of the different nanofibrous scaffolds were evaluated too. PCL/gelatin 70:30 nanofiber was foundto exhibit the most balanced properties to meet all the required specifications for nerve tissue and wasused for in vitro culture of nerve stem cells (C17.2 cells). MTS assay and SEM results showed that thebiocomposite of PCL/gelatin 70:30 nanofibrous scaffolds enhanced the nerve differentiation and prolif-eration compared to PCL nanofibrous scaffolds and acted as a positive cue to support neurite outgrowth.It was found that the direction of nerve cell elongation and neurite outgrowth on aligned nanofibrousscaffolds is parallel to the direction of fibers. PCL/gelatin 70:30 nanofibrous scaffolds proved to bea promising biomaterial suitable for nerve regeneration.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Recently, tissue engineering provided new medical therapy asan alternative to conventional transplantation methods usingpolymeric biomaterials with or without living precursor cells.Nerve tissue repair is a precious treatment concept in humanhealth care as it directly impacts on the quality of life [1–3]. Thefundamental approach in neural tissue engineering involves thefabrication of polymeric scaffolds with nerve cells to producea three-dimensional functional tissue suitable for implantation [4].In living systems, extracellular matrix (ECM) plays a pivotal role incontrolling cell behavior [5], while scaffold design plays a pivotalrole in tissue engineering. Nanofibrous scaffolds serve as suitableenvironment for cell attachment, and proliferation due to similarityto physical dimension of natural extracellular matrix [5–8]. Withincreasing interest in nanotechnology, development of nanofibersby using the technique of electrospinning is having a newmomentum [9]. This process involves using an electric field to

þ65 6872 0830.na).

All rights reserved.

convert polymer solution or melt into a fiber form [10–15]. Interesttowards employing electrospinning for scaffold fabrication ismainly due to the mechanical, biological and kinetic properties ofthe scaffold being easily manipulated by altering the polymercomposition and processing parameters [15]. The orientation ofnanofibers is one of the important features of a perfect tissuescaffold, because the fiber orientation greatly influences cellgrowth and related functions in cells such as nerve, smooth musclecells, etc. [5,16–20]. An established contact guidance theory illus-trates that a cell has the maximum probability of migrating inpreferred directions which are associated with chemical, structuraland/or mechanical properties of the substratum [5,21]. Therefore,aligned nanofibers could provide better contact guidance effectstowards neurite outgrowth [17,19,20].

Polymers can be used as scaffolds to promote cell adhesion,maintenance of differentiated, cell function and direct the growthof cells and help in the function of extracellular matrix. However,cell affinity towards synthetic polymers is generally poor as aconsequence of their low hydrophilicity and lack of surface cell-recognition sites [22]. Improving the hydrophilic property andincorporation of cell-recognition domains such as RGD and ECMbioactive proteins onto nanofibers are carried out to enhance

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–4539 4533

cell–scaffold interactions [23]. Polymer blending is one of the mosteffective methods for providing new, desirable biocomposite forparticular applications [24]. For example, blending synthetic andnatural polymers improves cell adhesion and the degradation rateof the blended polymeric system can be modified depending on itsapplication [15,17,22]. Poly(3-caprolactone) or PCL is a semi crys-talline linear hydrophobic polymer. Though the electrospun PCLmats mimic the identity of ECM in living tissues, its poor hydro-philicity caused a reduction in the ability of cell adhesion, migra-tion, proliferation, and differentiation [25,26]. On the other hand,gelatin is a natural biopolymer derived from collagen by controlledhydrolysis. Because of its many merits, such as its biological origin,biodegradability, biocompatibility and commercial availability atrelatively low cost, gelatin has been widely used in the pharma-ceutical and medical fields [15,23]. Therefore, gelatin can beblended with PCL to obtain a scaffold with desired cell adhesionand degradation properties. In a previous study [15], PCL/gelatinnanofibrous scaffolds were used for dermal reconstitution.However, there are no reports utilizing PCL/gelatin nanofibrousscaffolds for nerve tissue engineering.

This study is aimed at investigating the properties of PCL/gelatinnanofibrous scaffolds by FTIR, porometry, contact angle and tensilemeasurements. Differentiation and proliferation of C17.2 cells onaligned and random PCL and PCL/gelatin nanofibrous scaffoldswere also studied for understanding the effect of nanofiber align-ment and gelatin incorporation towards nerve tissue engineering.

2. Materials and methods

2.1. Materials

PCL (Mw¼ 80,000), gelatin type A from porcine skin and hexafluoro-2-propanol(HFP) were all obtained from Sigma–Aldrich (St. Louis, MO). Hexamethyldisilazane(HMDS) was purchased from Fluka. Dulbecco Modified Eagle’s Medium (DMEM),fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin–EDTA and horseserum were purchased from Gibco, Singapore. CellTiter 96� AQueous One solutionreagent (MTS), used in the cell-adhesion assay, was purchased from Promega.

2.2. Fabrication of aligned and random nanofiber scaffold

The polymer solution with concentration of 6 wt% was prepared by dissolvingPCL and gelatin with a weight ratio of 50:50 and 70:30 in HFP and stirred for 24 h atroom temperature. The solution was electrospun from a 5 ml syringe with a needlediameter of 0.4 mm and mass flow rate of 1 ml/h. A high voltage (12 kV) was appliedto the tip of the needle attached to the syringe when a fluid jet was ejected. Forcollecting aligned nanofibers, a rotating disk was used, whereas a flat aluminumplate was used for collecting random nanofibers. The linear rate of the rotating diskwas set to 1000 rpm. The resulting fibers were collected on 15-mm cover slipsplaced on respective collectors.

2.3. Characterization of scaffolds

The morphology of nanofibrous scaffolds was studied by scanning electronmicroscopy (SEM) (JSM 5600, JEOL, Japan) at an accelerating voltage of 15 kV. Beforeobservation, the scaffolds were coated with gold using a sputter coater (JEOL JFC-1200 fine coater, Japan). The diameter of the fiber was measured from the SEMmicrographs using image analysis software (Image J, National Institutes of Health,USA).

For determination of wettability (or hydrophilicity) of scaffolds, the contactangle of electrospun nonwoven materials was measured by a video contact anglesystem (VCA Optima, AST Products). The droplet size was set at 0.5 ml. Five sampleswere used for each test. The average value was reported with standard deviation(�SD).

Assessment of structural pore properties was done by Capillary Flow Porometry(1200-AEHXL capillary flow porometer, Porous Media Inc., Ithaca, NY). Capillary flowporometry provides a simple and non-destructive technique that allows rapid andaccurate measurement of pore size and distribution. In a capillary flow porometrymeasurement, a non-reacting gas flows through a dry sample and then through thesame sample after it has been wet with a liquid of known surface tension. Thechange in flow rate is measured as a function of pressure for both dry and wetprocesses. Because of the low pressure applied during the process, the porousstructure of nanofiber membranes is not distorted [27]. The pore size was calculatedby the software from Porous Media Inc (Ithaca, NY) using the equation:

D ¼ 4g cos q

Dp(1)

Where D is the pore diameter, g the surface tension of the wetting liquid, q thecontact angle of the wetting liquid, and Dp is the differential pressure.

Mechanical properties of different scaffolds were determined using a tabletopuniaxial testing machine (INSTRON 3345) using a 10-N load cell under a cross-headspeed of 10 mm/min at ambient conditions. All samples were prepared in the formof rectangular shape with dimensions of 50�10 mm2 from the electrospun fibrousmembranes. At least five samples were tested for each type of electrospun fibrousmembrane.

Chemical analysis of PCL and PCL/gelatin nanofibrous scaffolds were performedby ATR-FTIR spectroscopy over a range of 4000–400 cm�1. ATR-FTIR spectra of PCLand PCL/gelatin nanofibrous scaffolds were obtained on a Nicolet spectrometersystem with an Avatar Omni Sampler accessory.

Electrospun nanofibrous scaffolds were placed in 24-well plate containing 1 mlof a phosphate buffer solution (PBS; pH 7.4) in each well and were incubated in vitroat 37 �C for different periods of time (3, 7, 10, 14 days). After each degradation period,the samples were washed and subsequently dried in a vacuum oven at roomtemperature for 24 h. SEM of scaffolds were performed to understand the change innanofibers morphology during this period.

2.4. Cell culture study

The nanofibrous scaffolds were exposed to UV radiation for 2 h, washed 3 timeswith PBS for 20 min each and incubated with DMEM/F12 1:1 mixture containing N2supplement for 24 h before cell seeding. Neonatal mouse cerebellum C17.2 stemcells were cultured in DMEM supplemented with 10% FBS, 5% horse serum and 1%penicillin/streptomycin. After reaching 70% confluency, the cells were detached bytrypsin–EDTA and viable cells were counted by trypan blue assay. Cells were furtherseeded onto nanofibrous scaffolds, placed in a 24-well plate and tissue culturepolystyrene (TCP as control) at a density of 10�103 cells/well and cultured withDMEM/F12 1:1 mixture containing N2 supplement at 37 �C, 5% CO2 and 95%humidity.

The morphology of C17.2 cells on PCL and PCL/gelatin nanofibrous scaffolds wasobserved by SEM. After 6 days of cell seeding, samples were fixed with 3% glutar-aldehyde for 2 h. Specimens were rinsed in water and dehydrated with gradedconcentrations (50, 70, 90, 100% v/v) of ethanol. Subsequently the samples weretreated with HMDS and kept in a fume hood for air drying. Finally the samples werecoated with gold for the observation of cell morphology.

To study the cell proliferation on different substrates, viable cells were deter-mined by using the colorimetric MTS assay. After 2, 4 and 6 days of cell seeding in24-well plate, cells were washed with PBS and incubated with 20% of MTS reagentcontaining serum free medium. After 3 h of incubation at 37 �C in 5% CO2, aliquotswere pipetted into a 96-well plate. The absorbance of the content of each well wasmeasured at 492 nm using a spectrophotometric plate reader (Fluostar Optima,BMG Lab Technologies, Germany).

2.5. Statistical analysis

All data presented are expressed as mean� standard deviation (SD). Statisticalanalysis was carried out using single-factor analysis of variance (ANOVA). A value ofp� 0.05 was considered statistically significant.

3. Results

3.1. Morphology and properties of PCL and PCL/gelatin electrospunnanofibrous scaffolds

Fig. 1 shows the SEM micrographs of random and alignedelectrospun PCL and PCL/gelatin 50:50 and 70:30, along with theirdiameter distribution. As can be seen from Fig. 1, by decreasingthe gelatin content, a greater degree of alignment can be obtained.In the production of aligned nanofibers using electrospinningmethod, the polymer solution is continuously extruded from thetip of the needle while drawing the polymer solution towardsthe collector continues under the influence of the electric field. Ifthe electrospinning jet is not interrupted, a long continuous fiberstrand is produced as the solvent evaporates and higher alignmentcan be obtained [28]. A longer continuous fiber strand can beelectrospun as the gelatin content is decreased. With increasinggelatin content, the probability of fiber breakage increases dueto the decrease in solution viscosity (data not shown) and,therefore, the fiber alignment also decreased. Moreover, gelatinconcentrations of more than 30% in the blend were found to be

Fig. 1. SEM micrographs of (A) random and aligned PCL/gelatin 50:50, (B) random and aligned PCL/gelatin 70:30 (C), and random and aligned PCL nanofibers.

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–45394534

less suitable for the production of oriented nanofibers. Our resultsare consistent with those reported by Schnell et al., where theyfound that concentrations more than 25% of collagen in the blendof PCL/collagen is less suitable for the production of orientedfibers [17].

Fiber diameter was estimated to be 113� 33, 189� 56, 431�118for PCL/gelatin 50:50, 70:30 and PCL, respectively. Fiber diameterwas found to decrease with increasing the concentration of gelatinin PCL/gelatin blend polymeric system. No significant differences(p� 0.05) in the diameter were observed for random compared to

aligned nanofibers for the respective PCL and PCL/gelatinnanofibers.

Porosity is an important parameter when selecting the scaffoldfor cell culture experiments. The results of pore size measurementsshown in Table 1 indicate that with increasing gelatin content, thepore diameter decreased. This can be attributed to the reduction infiber diameter by incorporation of gelatin into the electrospinningsolution. More layers of fibers might overlap with each other,especially when the fiber diameter is smaller, resulting in smallerpore diameter. However, the pore size of PCL/gelatin 50:50

Table 1Contact angle and pore size measurement of PCL and PCL/gelatin 50:50 and 70:30nanofibers

Substrate Contact angle (�) Pore size (mm)

PCL 118� 6 1.70� 0.39PCL/gelatin 70:30 32� 8 1.00� 0.35PCL/gelatin 50:50 0 0.80� 0.20

Fig. 3. Typical stress–strain curves of PCL, PCL/gelatin 50:50 and PCL/gelatin 70:30nanofibers.

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–4539 4535

nanofibers was not significantly different (p� 0.05) compared toPCL/gelatin 70:30 nanofibers.

Table 1 shows the results of contact angle measurement for PCLand PCL/gelatin nanofibrous scaffolds. The hydrophilicity of PCL/gelatin 50:50 nanofibers was higher than PCL/gelatin 70:30,proving that the hydrophilicity of scaffolds increased withincreasing gelatin concentration.

ATR-FTIR analysis was carried out for surface characterization ofPCL and PCL/gelatin nanofibers. Fig. 2 shows the FTIR spectra of PCLand PCL/gelatin 70:30 nanofibrous scaffolds. FTIR spectra of PCL/gelatin 50:50 nanofibers were nearly same as that of PCL/gelatin70:30. Infrared spectra for PCL-related stretching modes wereobserved for PCL and PCL/gelatin scaffolds. These include2949 cm�1 (asymmetric CH2 stretching), 2865 cm�1 (symmetricCH2 stretching), 1727 cm�1 (carbonyl stretching), 1293 cm�1 (C–Oand C–C stretching) and 1240 cm�1 (asymmetric COC stretching)[29]. Common bands of protein appeared at approximately1650 cm�1 (amide I) and 1540 cm�1 (amide II), corresponding tothe stretching vibrations of C]O bond, and coupling of bending ofN–H bond and stretching of C–N bonds, respectively. The amide Iband at 1650 cm�1 was attributable to both a random coil anda-helix conformation of gelatin [30].

Fig. 3 shows the stress–strain behavior of electrospun PCL, PCL/gelatin 50:50 and PCL/gelatin 70:30 nanofibers. As shown in thisfigure, blending of PCL with gelatin caused a reduction inmechanical strength. It is suggested that this phenomenon is likelydue to the weak physical properties of gelatin. The % elongation ofPCL/gelatin 70:30 nanofibers were higher compared to PCL indi-cating a good deformability and flexibility. However, the % elon-gation of PCL/gelatin 50:50 decreased significantly due to highergelatin concentration in this blended polymeric system.

3.2. In vitro degradation of gelatin-containing nanofibrousscaffolds

Fig. 4 illustrates the morphological changes in electrospun PCL/gelatin 70:30 and 50:50 nanofibers during in vitro degradation after2-week period. No significant morphological changes in PCL wereobserved during 2-week study. As shown in Fig. 4B significant

Fig. 2. FTIR spectra of PCL and PCL/gelatin nanofibers.

morphological changes were observed for electrospun PCL/gelatin50:50 but not for electrospun PCL/gelatin 70:30. PCL/gelatin 70:30nanofibers was found swelling, though significant morphologicalchanges were not observed for PCL/gelatin 70:30 (Fig. 4D). Gelatincan be easily dissolved in water at a temperature of 40 �C [31] andhence by increasing gelatin content the biodegradability of PCL/gelatin nanofibrous scaffolds increased in PBS over 2-week period.

3.3. Cell–scaffold interaction

Aforementioned results revealed that the electrospun PCL/gelatin nanofibers with ratio of 50:50 had fastest degradation rateand weakest mechanical properties. The use of PCL/gelatin withratio of 70:30 was found to exhibit the most balanced properties tomeet all the required specifications for tissue engineering andespecially for nerve tissue engineering. Therefore, electrospun PCL/gelatin 70:30 nanofibers were selected and chosen for our cellculture experiments hereafter.

In vitro studies were carried out using C17.2 nerve stem cells.These cells can be used as neuron precursors since they areinvolved in the normal development of cerebellum, embryonicneocortex and other structures upon implantation into mousegerminal zones [16]. Figs. 5 and 6 show the SEM micrographs of cellinteraction with random and aligned PCL and PCL/gelatin electro-spun nanofibrous scaffolds after 6 days of cell culture. As shown inFig. 5, the blending of PCL with gelatin was found to promoteneurite extension more than PCL nanofibers, and neurites on PCLwere shorter than on gelatin-containing nanofibers after the sameperiod of incubation. These observations indicate a better integra-tion of cells with PCL/gelatin nanofibrous scaffolds. Cultures ofneurons on aligned nanofibers have shown that nerve cells candirect neurite outgrowth along the direction of fiber orientation(Fig. 6). However, the fiber alignment of PCL/gelatin nanofibrousscaffolds is less than PCL nanofiber scaffolds, but it can be seen thatthe cells elongated parallely along the fiber orientation.

3.4. Viability of nerve cells

MTS assay was carried out to evaluate the cell proliferation ofC17.2 cells on PCL and PCL/gelatin nanofibrous scaffolds. As shownin Fig. 7, the proliferation of cells on gelatin-containing scaffolds ishigher than PCL scaffolds, indicating that the gelatin-containing

Fig. 4. Morphology of (A) PCL/gelatin 50:50 before biodegradability test, (B) PCL/gelatin 50:50 after 2-week biodegradability test, (C) PCL/gelatin 70:30 before biodegradability test,and (D) PCL/gelatin 70:30 nanofibers after 2-week biodegradability test.

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–45394536

scaffolds might have accelerated the proliferation and differentia-tion of C17.2 cells. In comparison to random nanofibers, cellproliferation is significantly higher on aligned nanofibrous scaffolds(p� 0.05).

4. Discussion

Among the numerous attempts to integrate tissue-engineeringconcepts into strategies to repair nearly all parts of the body,neuronal repair stands out. This is partially due to the complexity ofthe nervous system anatomy, functioning and the inefficiency ofconventional repair approaches, which were based upon singlecomponents of either biomaterials, or cells alone [32]. Recentlyelectrospun nanofibrous scaffolds have been used for nerve tissueengineering [1,4,5,16,17,19,20].

Natural and synthetic materials can be used for fabrication ofnanofibrous scaffolds for nerve regeneration [33]. One limitation ofelectrospun scaffolds fabricated with synthetic polymers such asPCL is low cell affinity towards them due to low hydrophilicity andlack of surface cell-recognition sites. A major drawback of naturalpolymers such as gelatin is their weak mechanical property.Production of composite scaffolds containing heterogeneous fibersof natural and synthetic polymers provided a scaffold with desir-able properties. Using such biocomposites, the synthetic polymericmaterials can function as a fibrous backbone providing mechanicalproperties while natural polymer component can act as promotersof cellular attachment and growth. Reports by a few researchersdemonstrated that gelatin enhances the attachment and prolifer-ation of cells [15,34–39]. However, there are no reports utilizingPCL/gelatin nanofibrous scaffolds for nerve tissue engineering. Inthis study, electrospinning of a blended solution of PCL and gelatin

was found to be an efficient technique to modify PCL nanofibrousscaffolds for nerve regeneration. The incorporation of gelatinimproves the hydrophilicity of PCL/gelatin nanofibrous scaffolds.This might be attributed to the amine and carboxylic functionalgroups in gelatin structure whereas such functional groups are notpresent in PCL structure. The appearance of amide group in the FTIRspectra of PCL/gelatin nanofibrous scaffolds indicates that the PCLchains were chemically bonded to gelatin sidewalls and it leads tothe introduction of functional groups such as NH2 and COOH on thesurface of PCL/gelatin scaffolds. Although the scaffolds with higherconcentration (50%) of gelatin were found to be better substrates toprovide enhanced hydrophilicity, this result did not correlate to thebetter strength of nanofibrous scaffolds. Moreover, the combinationof PCL and gelatin with ratio of 50:50 gave rise to loose and weakstructure and fastened the degradation rate compared to pure PCLand PCL/gelatin 70:30 nanofibers. Materials for fabrication of nerveguides are required to be slowly degrading with low degree ofswelling, as well as suitable mechanical properties for the bearingof stresses during the surgical procedure (handling and suturing)and implantation time (movements of the patient) [33]. Therefore,PCL/gelatin 50:50 nanofibers are not favorable for nerve tissueengineering due to their high degradation rate and weakmechanical properties. Therefore, we selected PCL/gelatin 70:30 forour in vitro studies due to its slow degradation rate and bettermechanical properties compared to PCL/gelatin 50:50. Both theSEM and MTS assay showed that the nerve cells can attach andgrow on PCL/gelatin and PCL nanofibrous scaffolds, but blending ofPCL/gelatin improves cell proliferation compared with the poor cellgrowth on unmodified PCL nanofibrous scaffolds. The neuriteextension of nerve cells was more pronounced on PCL/gelatinscaffolds than PCL scaffolds. Similar results were also reported by

Fig. 5. Morphology of C17.2 cells on PCL/gelatin (magnification: 3000 (A) and 6000 (B)) and PCL (magnification: 3000 (C) and 6000 (D)) after 6 days of cell culture.

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–4539 4537

Schnell et al. [17]. They successfully produced nanofibers of PCL andPCL/collagen blends by electrospinning, where they founda biochemical interaction between cells and collagen exposed onthe surface of the nanofibers. Such interaction is known to bemediated by the ECM glycoprotein fibronectin, which binds tocollagen I and to integrin receptors on cell membranes [17]. Gelatinis a natural biopolymer derived from collagen by controlledhydrolysis and has structural similarity to collagen. Therefore, it canbe concluded that the inclusion of gelatin into PCL enhances theinteraction of the cells with the scaffolds by engaging cell-adhesionmolecules which then permit the cells to exert/generate higherinteraction forces as required for cell motility.

Fig. 6. Morphology of C17.2 cells on aligned (A) PCL and (B) P

The results of contact angle measurement of PCL nanofibrousscaffolds showed the contact angle of 118� indicating that PCLnanofibrous scaffolds are hydrophobic. Generally, the hydrophilic/hydrophobic characteristic of scaffold is important in tissue cultureand can influence the initial cell adhesion and cell migration toa higher extent [23–26]. According to previous literatures, hydro-phobic surfaces lead to lower cell adhesion in the initial step of cellculture. TCP is normally treated by oxygenated gas plasma to createa more hydrophilic oxidized polymer surface and provides a surfacechemistry that absorbs sufficient amounts of trace ECM proteinsfrom serum-supplemented media to promote cell attachment [4].Therefore, it is not surprising that a greater percentage of cells

CL/gelatin (70:30) nanofibers after 6 days of cell culture.

Fig. 7. MTS results of C17.2 on PCL and PCL/gelatin nanofibers after 2, 4 and 6 days ofcell seeding.

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–45394538

adhered to TCP (control) as compared to electrospun PCL nano-fibrous scaffolds.

Aligned PCL and PCL/gelatin nanofibers fabricated by electro-spinning process provided a controlled orientation of nerve cellsproving that the aligned electrospun nanofibers are capable ofcontrolling the orientation of neurons by a mechanism calledcontact guidance [5,16–20]. Results of our study also showed thatthe direction of nerve cell elongation and neurite outgrowth isparallel to the direction of fiber alignment. The direction of neuriteelongation was largely orientated in parallel to the fiber alignmentand such contact guidance might enhance the nerve regenerationprocess. The proliferation of C17.2 was higher on aligned nano-fibrous scaffolds in comparison to random nanofibrous scaffolds forboth PCL and PCL/gelatin. It can be concluded that the arrangementof cells in controlled architecture has beneficial effects on cellproliferation, especially for nerve regeneration.

5. Conclusion

In this study, we produced a biocomposite PCL/gelatin nano-fibrous scaffolds with weight ratios of 50:50 and 70:30. The resultsreported in this study demonstrated that the properties of nano-fibrous scaffolds were strongly influenced by the concentration ofgelatin in the biocomposite. PCL/gelatin 70:30 nanofibers wereselected for cell culture study as it provided better mechanical andbiodegradation properties than PCL/gelatin 50:50. It was found thatPCL/gelatin 70:30 enhanced the nerve differentiation and prolif-eration compared to PCL nanofibrous scaffolds. Although randomlyoriented nanofibrous scaffolds are useful in tissue engineering, theresults showed that aligned nanofibers highly supported the nervecells and improved the neurite outgrowth and cell differentiationprocess.

Acknowledgements

We would like to express our thanks to National MedicalResearch Council (grant number: NMRC/1015/2005, R397000625213),Singapore for their financial support.

References

[1] Yang F, Murugan R, Ramakrishna S, Wang X, Mac YX, Wang S. Fabrication ofnano-structured porous PLLAscaffold intended for nerve tissue engineering.Biomaterials 2004;25:1891–900.

[2] Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair andregeneration. Annual Review of Biomedical Engineering 2003;5:293–347.

[3] Bellamkonda R, Aebischer P. Review: tissue engineering in the nervoussystem. Biotechnology and Bioengineering 1994;43:543–54.

[4] Yang F, Xu CY, Kotaki M, Wang S, Ramakrishna S. Characterization of neuralstem cells on electrospun poly(L-lactic acid) nanofibrous scaffold. Journal ofBiomaterials Science, Polymer Edition 2004;15:1483–97.

[5] Xu CY, Inai R, Kotaki M, Ramakrishna S. Aligned biodegradable nanofibrousstructure: a potential scaffold for blood vessel engineering. Biomaterials2004;25:877–86.

[6] Ma PX. Scaffolds for tissue fabrication. Materials Today 2004;7:30–40.[7] Smith LA, Ma PX. Nano-fibrous scaffolds for tissue engineering. Colloids and

Surfaces B: Biointerfaces 2004;39:125–31.[8] Ma Z, Kotaki M, Inai R, Ramakrishna S. Potential of nanofiber matrix as tissue-

engineering scaffolds. Tissue Engineering 2005;11:101–9.[9] Ashammakhi N, Ndreu A, Yang Y, Ylikauppila H, Nikkola L. Nanofiber-based

scaffolds for tissue engineering. European Journal of Plastic Surgery 2008[available online].

[10] Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang ZM. Electrospinningof gelatin fibers and gelatin/PCL composite fibrous scaffolds. Journal ofBiomedical Materials Research, Part B: Applied Biomaterials 2005;72B:156–65.

[11] Pham Q, Sharma U, Mikos A. Electrospinning of polymeric nanofibers for tissueengineering applications: a review. Tissue Engineering 2006;12:1197–211.

[12] Zong X, Kim K, Fang D, Ran S, Hsiao B, Chu B. Structure and process rela-tionship of electrospun bioabsorbable nanofiber membranes. Polymer2002;43:4403–12.

[13] Zeng J, Chen X, Xu X, Liang Q, Bian X, Yang L, et al. Ultrafine fibers electrospunfrom biodegradable polymers. Journal of Applied Polymer Science2003;89:1085–92.

[14] Yarin AL, Koombhongse S, Renrker DH. Bending instability in electrospinningof nanofibers. Journal of Applied Physics 2001;90:4836–46.

[15] Chong EJ, Phan TT, Lim IJ, Zhang YZ, Bay BH, Ramakrishna S, et al. Evaluation ofelectrospun PCL/gelatin nanofibrous scaffold for wound healing and layereddermal reconstitution. Acta Biomaterialia 2007;3:321–30.

[16] Bini TB, Gao S, Wang S, Ramakrishna S. Poly(L-lactide-co-glycolide) biode-gradable microfibers and electrospun nanofibers for nerve tissue engineering:an in vitro study. Journal of Materials Science 2006;41:6453–9.

[17] Schnell E, Klinkhammer K, Balzer S, Brook G, Kleeb D, Dalton P, et al. Guidanceof glial cell migration and axonal growth on electrospun nanofibers of poly-3-caprolactone and a collagen/poly-3-caprolactone blend. Biomaterials 2007;28:3012–25.

[18] Lee CH, Shin HJ, Cho IH, Kang YM, Kim IA, Park KD, et al. Nanofiber alignmentand direction of mechanical strain affect the ECM production of human ACLfibroblast. Biomaterials 2005;26:1261–70.

[19] Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/microscale poly(L-lactic acid) aligned fibers and their potential in neural tissueengineering. Biomaterials 2005;26:2603–10.

[20] Murugan R, Ramakrishna S. Design strategies of tissue engineering scaffoldswith controlled fiber orientation. Tissue Engineering 2007;13:1845–66.

[21] Lackie JM. The dictionary of cell and molecular biology. 4th ed. San Diego:Academic Press; 2007.

[22] Ciardelli G, Chiono V, Vozzi G, Pracella M, Ahluwalia A, Barbani N, et al. Blendsof poly(3-caprolactone) and polysaccharides in tissue engineering applica-tions. Biomacromolecules 2005;6:1961–76.

[23] Koh HS, Yong T, Chan CK, Ramakrishna S. Enhancement of neurite outgrowthusing nano-structured scaffolds coupled with laminin. Biomaterials 2008;29:3574–82.

[24] Cheng M, Deng J, Yang F, Gong Y, Zhao N, Zhang X. Study on physical prop-erties and nerve cell affinity of composite films from chitosan and gelatinsolutions. Biomaterials 2003;24:2871–80.

[25] Kim CH, Khil MS, Kim HY, Lee HU, Jahng KY. An improved hydrophilicity viaelectrospinning for enhanced cell attachment and proliferation. Journal ofBiomedical Materials Research Part B: Applied Biomaterials 2006;78B:283–90.

[26] Li WJ, Cooper Jr , Mauck RL, Tuan RS. Fabrication and characterization of sixelectrospun poly(a-hydroxyester)-based nanofibrous scaffolds for tissueengineering applications. Acta Biomaterialia 2006;2:377–85.

[27] Li D, Frey MW, Joo YL. Characterization of nanofibrous membranes withcapillary flow porometry. Journal of Membrane Science 2006;286:104–14.

[28] Teo WE, Kotaki M, Mo XM, Ramakrishna S. Porous tubular structures withcontrolled fibre orientation using a modified electrospinning method. Nano-technology 2005;16:918–24.

[29] Catledge SA, Clem WC, Shrikishen N, Chowdhury S, Stanishevsky AV,Koopman M, et al. An electrospun triphasic nanofibrous scaffold for bonetissue engineering. Biomedical Materials 2007;2:142–50.

[30] Ki CS, Baek DH, Gang KD, Lee KH, Um IC, Park YH. Characterization of gelatinnanofiber prepared from gelatin–formic acid solution. Polymer 2005;46:5094–102.

[31] Huang ZM, Zhang YZ, Ramakrishna S, Lim CT. Electrospinning and mechanicalcharacterization of gelatin nanofibers. Polymer 2004;45:5361–8.

[32] Zhang N, Yan H, Wen X. Tissue-engineering approaches for axonal guidance.Brain Research Reviews 2005;49:48–64.

L. Ghasemi-Mobarakeh et al. / Biomaterials 29 (2008) 4532–4539 4539

[33] Ciardelli G, Chiono V. Materials for peripheral nerve regeneration. Macro-molecular Bioscience 2006;6:13–26.

[34] Bordenave L, Caix J, Basse-Cathalinat B, Baquey C, Midy D, Baste JC, et al.Experimental evaluation of a gelatin coated polyester graft used as an arterialsubstitute. Biomaterials 1989;10:235–42.

[35] Warocquier-Clerout R, Lee YS, Penhoat J, Sigot-Luizard MF. Comparativebehaviour of L-929 fibroblastic and human endothelial cells onto crosslinkedprotein substrates. Cytotechnology 1990;3:259–69.

[36] Ma Z, Gao C, Gong Y, Ji J, Shen J. Immobilization of natural macromolecules onpoly-L-lactic acid membrane surface in order to improve its cytocompatibility.Journal of Biomedical Materials Research 2002;63:838–47.

[37] Takahashi Y, Yamamoto M, Tabata Y. Osteogenic differentiation of mesen-chymal stem cells in biodegradable sponges composed of gelatin and b-tri-calcium phosphate. Biomaterials 2005;26:3587–96.

[38] Ma Z, He W, Yong T, Ramakrishna S. Grafting of gelatin on electrospunpoly(caprolactone) nanofibers to improve endothelial cell spreading andproliferation and to control cell orientation. Tissue Engineering 2005;11:1149–58.

[39] Li M, Mondrinos MJ, Chen X, Gandhi MR, Ko FK, Lelkes PI. Co-electrospunpoly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineeringscaffolds. Journal of Biomedical Materials Research Part A 2006;79A:963–73.