Development of Electroactive and Elastic Nanofibers that contain Polyaniline and Poly(L-lactide- co...

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Development of Electroactive and ElasticNanofibers that contain Polyaniline andPoly(L-lactide-co-e-caprolactone) for theControl of Cell Adhesion

Sung In Jeong, In Dong Jun, Moon Jae Choi, Young Chang Nho,Young Moo Lee, Heungsoo Shin*

I. D. Jun, H. ShinDepartment of Bioengineering, College of Engineering, HanyangUniversity, Haengdang-dong, Seongdong-gu, Seoul 133-791,Republic of KoreaE-mail: [email protected]

In this work, electrically conductive polyaniline (PAni) doped with camphorsulfonic acid(CPSA) is blendedwith poly(L-lactide-co-e-caprolactone) (PLCL), and then electrospun to prepareuniform nanofibers. The CPSA-PAni/PLCL nanofibers show a smooth fiber structure withoutcoarse lumps or beads and consistent fiber diameters (which range from 100 to 700 nm) evenwith an increase in the amount of CPSA-PAni (from 0 to 30 wt.-%). However, the elongation atbreak decreases from 391.54� 9.20% to 207.85� 6.74% when 30% of CPSA-PAni is incorpo-rated. Analysis of the surface of the nanofibers demonstrates the presence of homogeneouslyblended CPSA-PAni. Most importantly, a four-point probe analysis reveals that electricalproperties are maintained in the nanofibers where the conductivity is significantly increasedfrom 0.0015 to 0.0138 S � cm�1 when the nanofibersare prepared with 30% CPSA-PAni. The cell adhesiontests using human dermal fibroblasts, NIH-3T3 fibro-blasts, and C2C12 myoblasts demonstrate signifi-cantly higher adhesion on the CPSA-PAni/PLCLnanofibers than pure PLCL nanofibers. In addition,the growth of NIH-3T3 fibroblasts is enhanced underthe stimulation of various direct current flows. TheCPSA-PAni/PLCL nanofibers with electrically conduc-tive properties may potentially be used as a platformsubstrate to study the effect of electrical signals oncell activities and to direct desirable cell function fortissue engineering applications.

Macromol. Biosci. 2008, 8, 627–637

� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

S. I. Jeong, M. J. Choi, Y. M. LeeSchool of Chemical Engineering, College of Engineering, HanyangUniversity, Haengdang-dong, Seongdong-gu, Seoul 133-791,Republic of KoreaY. C. NhoRadiation Application Research Division, Advanced RadiationTechnology Institute, Korea Atomic Energy Research Institute,Sinjeongdong, Jeongeup, Jeonbuk 580-195, Republic of Korea

DOI: 10.1002/mabi.200800005 627

S. I. Jeong, I. D. Jun, M. J. Choi, Y. C. Nho, Y. M. Lee, H. Shin

628

Introduction

The central strategy in tissue engineering involves a

biomaterial scaffold as a delivery carrier of cells.[1] The

ability of a scaffold to control cellular response to direct

particular repair and regeneration processes is essential for

the generation of successful tissue engineering products.

Therefore, many efforts have been made to understand

local interactions of cells with their microenvironment and

exploit these interactions to design an ideal scaffold that is

able to elicit a desirable cellular response.[2,3]

Cells in native tissue are surrounded by a complex

chemical, mechanical, and electrical microenvironment.[4–7]

While cell adhesion is mediated by integrin receptors

bound to extracellular matrix (ECM) proteins, tissue

specific mechanical stiffness and electrical signals deter-

mine cell division, development, and morphogenetic

alternation. For example, the stiffness of scaffolding

materials regulates the responses of skeletal myoblasts,

endothelial cells, and neuron cells with respect to the

formation of striated structure, induction of capillary

tubes, and neurite branch outgrowth, respectively.[8–10]

We have also reported the importance of mechanical

stimulation on the protein expression of smooth muscle

cells cultured on elastic and biodegradable poly(L-lactide-

co-e-caprolactone) (PLCL) scaffolds.[11] The influence of

electrical and electromagnetic fields on cells has also been

widely investigated, particularly on cardiac myocytes,

neuronal cells, keratinocytes, fibroblasts, and mesenchy-

mal stem cells.[12–16] Collectively, these studies have

emphasised that the mechanical and electrical properties

of scaffolds should be appropriately controlled to regen-

erate physiologically robust artificial tissue. However, the

number of studies that explore the response of cells

cultured on electroactive substrates is relatively limited

and the fabrication of electroactive synthetic scaffolds

with elastic properties is even more challenging.

The overall goal of our study is to synthesize a nano-

structured three-dimensional scaffold for regeneration of

tissue whose function can be significantly controlled by

mechanical and electrical stimulation, such as muscle,

neuron, and skin. The nanofibrous scaffold, which struc-

turally mimics the ECM, is generated by an electrospinning

method. This method has been widely utilized to fabricate

nanometer-scaled and three-dimensional interconnected

networks of natural and synthetic polymers.[17,18] The

electroactive and elastic properties of the nanofibers were

achieved by blending the conducting polymer, polyaniline

(PAni), with an elastic PLCL matrix using 1,1,1,3,3,3-

hexafluoropropan-2-ol (HFP) as a solvent. PAni has been

previously explored in several biomedical applications

including biosensors and scaffolds in tissue engineering,

and demonstrates biocompatibility in both in vitro and

in vivo analysis.[17,19,20]



Here, our primary objectives were to investigate cell

response to nanofibrous substrates prepared from the

mixture of PLCL and PAni using camphorsulfonic acid

(CPSA) as the dopant. First, the effect of incorporated PAni

on the morphology, mechanical strength, surface char-

acteristics, and conductivity of the fabricated nanofibers

was examined. The cellular response to the nanofibers was

then studied by investigation of the effect of PAni on the

adhesion and viability of three model cell types: primary

fibroblasts, NIH-3T3 fibroblasts, and C2C12 myoblasts.

Finally, we developed a house-made bioreactor to apply

electrical stimuli, and analyzed the metabolic activity of

the cells cultured on CPSA-PAni/PLCL scaffolds under

electrical stimulation.

Experimental Part

Materials

Polyaniline (PAni), camphorsulfonic acid (CPSA), 1,1,1,3,3,3-hexa-

fluoropropan-2-ol (HFP), tris-Base, triton X-100, toluene, and

e-caprolactone were purchased from Sigma Chemical Co. (St.

Louis, MO, USA). Reagents for the synthesis of PLCL were dried or

purified as previously described.[11,21] Fetal bovine serum (FBS),

Dulbecco’s modified eagle’s medium (DMEM), Dulbeccos phos-

phate buffered saline (PBS), Trypsin-EDTA, and penicillin-

streptomycin were purchased from Gibco BRL (Rockville, MD,

USA). Sucrose, L-ascorbic acid, aprotinin, leupeptin, and phenyl

methyl sulfonyl fluoride (PMSF) were purchased from Amresco Co.

(Solon, OH, USA). NaCl and MgCl2 were purchased from Junsei

chemical Co. (Tokyo, Japan). Water was distilled and deionized

(DDW) using the Milli-Q System (Millipore, MA, USA). Microscope

glass cover-slips (diameter: 12 mm) were purchased from

Marienfeld Co. (GmbH, Germany).

Synthesis and Preparation of Electrospun CPSA-PAni/

PLCL Nanofibers

The synthesis of PLCL was as described previously.[11,21] Briefly,

L-lactide (36 g) and e-caprolactone (28.5 g) were polymerized at

150 8C for 24 h in a 50 mL glass ampoule that contained 0.028 M

stannous octoate solution (2.381 g). The molar ratio of L-lactide,

e-caprolactone, and stannous octoate was 1:1:0.01. After poly-

merization, the PLCL was dissolved in chloroform and filtered

using a 0.45 mm pore membrane filter. The polymer was

precipitated into an excess amount of methanol, filtered off,

and dried under vacuum.

CPSA-PAni was prepared by mixing both 30 mg of PAni and

CPSA in 10 mL of HFP. PLCL (1 g) was also dissolved in 10 mL of HFP

by stirring for 4 h at room temperature (RT). The CPSA-PAni/PLCL

blends were prepared by mixing each solution with two different

ratios (PAni/PLCL: 15/85 and 30/70 v/v). The molecular structure

of PAni and PLCL is shown in Figure 1. For electrospinning, the

resulting solutions (10 mL) were loaded into a 20 mL glass syringe

(Hamilton, NV, USA) equipped with a blunt 23 gauge needle. The

glass syringe was then placed in a syringe pump (KD Scientific

DOI: 10.1002/mabi.200800005

Elong

Development of Electroactive and Elastic Nanofibers that contain Polyaniline and . . .

Figure 1. Chemical structure of a) PAni and CPSA and b) PLCL.

Single-Syringe Infusion Pump, Fisher, MA, USA) and the needle

was connected to the positive output of a high voltage power

supply (NanoNC, Seoul, Korea). The collector was wrapped with

aluminum foil and located at a fixed distance of 20 cm from the

needle. The flow rate of the solution, applied voltage, and spinning

time were fixed at 20 mL �min�1, 18–20 kV, and 24 h at RT,

respectively. A PLCL only nanofiber was prepared as a negative

control. The prepared nanofibers on the foil were dried for

overnight at RT.

Characterization of the Nanofibers

The morphology of the nanofibrous CPSA-PAni/PLCL scaffolds was

examined using scanning electron microscopy (SEM, JEOL

JSM-6300, Japan). To measure the mean diameter of each scaffold,

the images were captured and imported to Photoshop (Photoshop

CS, Adobe), and the diameters of fibers from 30 randomly selected

fields of each SEM image were manually measured.

The tensile properties of rectangular samples (10�30 mm2,

n¼5) were measured using Instron (Model 5567, Canton, MA,

USA) as previously described.[22] The thickness of the samples is

given in Table 1. A 10-Newton-maximum load cell was used at a

cross-head speed of 1 mm �min�1. Calculations were performed

based on stress–strain curves of each sample; the tensile strength,

elongation, and Young’s modulus (E) were calculated in the

following manner:

Tab

Sam

PAC

PAC

PAC

a)CPSA

Macrom

� 2008

Tensile strength ðMPaÞ ¼ F=A (1)

le 1. Composition, thickness, contact angle, and electrical conductivity of CPSA-PAni/PL

ple code CPSA-PAnia):PLCLb) Thickness Contact

%, v/v mm deg

L-1 0:100 0.33� 0.48 86.10�L-2 15:85 0.26� 0.51 86.88�L-3 30:70 0.21� 0.42 87.42�

-doped PAni was prepared by mixing both 30 mg of PAni and CPSA in 10 mL of HF

ol. Biosci. 2008, 8, 627–637

WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

where F is the maximum load of force and A

is the cross-sectional area of the nanofiber

specimen.

CL nan

angle

.

0.66

1.31

1.24

P; b)C

ationð%Þ ¼ ½ðL0 � LÞ=L0� � 100 (2)

where L0 refers to the initial length of the

sample and L is the elongated length at the

break age of the sample. The Young’s

modulus (E) was calculated from the slope

of the straight line of the stress–strain curve

in the elastic region.

For the characterization of surface prop-

erties, an attenuated total reflectance Fourier-

transform infrared (ATR FT-IR) spectro-

scopic investigation was carried out using

a Bruker TENSOR 37 (Bruker, MA, USA)

spectrophotometer with a scanning range between 2 400 and

800 cm�1. Raman spectra were recorded with a Jasco NRS-3100

spectrometer (Jasco, Tokyo, Japan) at wavelengths ranging from

800 to 2 000 cm�1. A holographic notch filter was used to filter out

the Raleigh radiation. The Raman radiation was dispersed using

an 1 800 groove per mm grating and detected by a politer cooled

charge coupled device array. X-Ray photoelectron spectroscopy

(XPS) investigations were carried out with an ESCA LAB 220I

(Thermo VG Scientific, MA, USA) spectrometer with a magnesium

anode source that produced Mg Ka (1 253.6 eV photons) radiation

with a pass energy of 20 eV for high-resolution narrow scans.

Thermal gravimetric analysis (SDT2960, TA Instrument, CA, USA)

was carried out to confirm the presence of CPSA-PAni within the

PLCL matrix. Ten milligrams of each sample, including pure PAni

and CPSA-PAni (negative control), was placed in the measurement

chamber and the scanning range was from RT to 900 8C with an

ascending rate of 5 8C �min�1.

The conductivity (s, S � cm�1) of the electrospun CPSA-PAni/

PLCL scaffolds (10�40 mm2, n¼5) was measured using a four

point probe analysis and calculated based on the equation: s¼ l/

(RsS) (where l is the distance between reference electrodes, Rs is the

bulk resistance of the membrane sample derived from an

impedance analyzer, and S is the cross-sectional area of the

sample) as previously reported.[23] The thermo- and hydro-

controlled chamber was used to avoid the influence of electro-

magnetic noise. Ohmic resistance (R) was measured by four-point

probe alternating current (ac) impedance spectroscopy using an

electrode system connected with an impedance/gain-phase

ofiber scaffolds.

Electrical conductivity

S � cmS1

0.0015� 0.0001

0.0077� 0.0001

0.0138� 0.0002

oncentration of PLCL: 10% (w/v).

www.mbs-journal.de 629


630

analyzer (Solatron 1260) and an electrochemical interface

(Solatron 1287, Farnborough Hampshire, ONR, UK).[24]

in vitro Cell Culture Study

The human fibroblasts, NIH-3T3 (CRL-1658, American Type

Culture Collection (ATCC), Rockville, MD, USA), and C2C12

(CRL-1772, ATCC) were used to study the biocompatibility of

the scaffolds. The human fibroblasts were isolated from foreskin

biopsies donated by the Dermatology Department (Dongguk

University Hospital in Goyang, Korea). The human fibroblasts,

NIH-3T3, and C2C12 cells were cultured in DMEM supplemented

with 10% FBS and 1% penicillin-streptomycin under standard

culture conditions (37 8C and 5% CO2). When the cells were

cultured with 70–80% confluency, they were detached from the

culture flasks using a 0.05% Trypsin-EDTA solution. Following

three washes, the cells were re-suspended at 106 cells per mL and

were seeded onto the surface of the CPSA-PAni/PLCL nanofiber

scaffolds at a density of 2� 104 cells per cm2.

For the cell culture, circular samples were prepared by

punching out the electrospun nanofibers (diameter: 10 mm),

and all samples were sterilized by UV irradiation for 5 min,

immersed in 70% ethanol, washed three times with PBS, and then

placed in 24-well tissue culture plates (Corning, NY, USA) (under

the sterilization conditions, we did not observe shrinkage or other

deformation of the scaffolds). On day one after seeding, samples

were rinsed with PBS, and 70 mL of 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (12�10�3 m) was added to each sample. After 4 h of incubation at

37 8C, the MTT solution was removed, and any insoluble formazan

crystals formed were dissolved with 100 mL of dimethyl sulfoxide.

The absorbance of the formazan product was measured at 540 nm

using a 96-well plate spectrophotometer (spectraMax M2e,

Molecular Devices, Ontario, Canada). Results were expressed as

a percentage of the absorbance of the sample to the negative

control.

To investigate the morphological changes of the cultured cells

on the scaffolds, samples were fixed in 3.7% formaldehyde in PBS

for 10 min. Following a wash in PBS, the samples were incubated

for 60 min at 37 8C with rhodamine-phalloidin (Invitrogen, NY,

USA), a specific dye for F-actin, and Hoechst 33258 (Invitrogen, NY,

USA) for a nuclear stain and then mounted on glass slides. The

samples were then visualized on a Nikon (TE2000-E, Nikon, Tokyo,

Japan) inverted microscope equipped with the appropriate

fluorescence filters. Digital images were acquired using a Nikon

camera (Digital sight DS-SWc, Nikon, Tokyo, Japan).

For the electrical stimulation experiment, the NIH-3T3 cells

were seeded on nanofibers (diameter: 18 mm, 1.5� 104 cells per

sample, n¼3). The cell-seeded scaffolds were placed between the

stainless steel electrodes connected to a direct current (DC) power

supply within the electrical stimulation chamber (diameter:

130 mm) filled with 100 mL of DMEM, and were cultured at 5% CO2

and 37 8C for 2 d under electrical stimulation. Various electrical

currents were applied through the voltage DC power supply (ED

Laboratory, ED-330, GyeongGiDo, Korea) ranging from 0 to 200

mA. The fresh culture media was changed each day. After culture,

viable cells were measured using an MTT assay and their

morphology was examined by F-actin staining.



Statistical Analysis

All the quantitative results were obtained from triplicate samples.

Data were expressed as a mean� standard deviation (SD).

Statistical analysis was carried out using paired Student’s t-test,

and a value of p< 0.05 was considered to be statistically

significant.

Results and Discussion

Characterization of Electrospun CPSA-PAni/PLCLNanofibers

We prepared the composite nanofiber scaffolds by

electrospinning physical blends of PLCL and CPSA-PAni

with two different volume ratios of PLCL to PAni, i.e., 85:15

and 70:30 (PACL-2 and PACL-3), respectively. As a control

group, pure PLCL was also electrospun at a concentration of

10% in HFP (PACL-1). Figure 2 shows SEM images, and the

distribution and mean value fiber diameters of the

prepared nanofibers. Since the color of the CPSA-PAni

solution was inherently dark blue, the color of the

corresponding CPSA-PAni/PLCL nanofibers became homo-

geneously darker as shown in Figure 2a–c as the amount of

PAni in the blend increased. Both CPSA-PAni and PLCL were

favorably dissolved in HFP and electrospun to form

smooth nanofibers. There was no indication of lumps or

coarse roughening on the surface of nanofibers by the

incorporation of PAni, as confirmed by SEM images. The

non-woven fibers were randomly distributed throughout

the scaffolds and formed interconnected pores. Intercon-

nected pores and a large pore volume of nanofiber

scaffolds are the most important factors that determine

cell adhesion and proliferation.[25] Image analysis of

approximately 50 fibers from three samples shows that

the diameters of the electrospun nanofibers range from

100 to 700 nm. Although increasing the volume ratio of

PAni to PLCL decreases the total concentration of PLCL in

the mixture, the mean fiber diameters of each sample were

consistent (430� 116, 423� 100, and 382� 102 nm for

PACL-1, PACL-2, and PACL-3, respectively). However, with

the incorporation of an increased amount of CPSA-PAni

solution, i.e., greater than 30%, we observed a decrease in

the nanofiber size with bead formation instead of the

generation of uniform nanofibers. Previous reports

demonstrated that the fiber diameter distribution of

electrospun nanofibers was affected by solvent concentra-

tions and viscosities of polymers.[17,26] Therefore, it is

important to optimise these parameters to obtain uniform

sized nanofibers. Our results suggest that bead-free

nanofibers can be reproducibly generated within a blend

with a concentration of PAni of less than 30%.

The control of fiber size is critical in our experiment to

investigate the effect of the presence of electroconductive

DOI: 10.1002/mabi.200800005


Figure 2. Morphologies and fiber diameters of CPSA-PAni/PLCL nanofiber scaffolds. a–c) Macroscopics, d–f) SEM micrographs, g) averagefiber diameters, and h) distribution of fiber diameters of CPSA-PAni/PLCL nanofiber scaffolds.

functional groups on cell adhesion and morphology. Since

the fiber diameters of the CPSA-PAni/PLCL nanofibers were

within a similar range, we could exclude the possibility

that cellular responses were influenced by the size of the

fibers. Besides chemical properties, it has been established

that the size of nanofibers regulates adhesion, prolifera-

tion, and differentiation of NIH-3T3 fibroblasts, chondro-

cytes, and osteoblasts.[27–30]

The representative mechanical parameters of CPSA-

PAni/PLCL nanofibers calculated from stress–strain curves

are presented in Figure 3. Overall, PACL-1 shows the

highest tensile strength, Young’s modulus, and elongation

at break. For example, the tensile strength of PACL-1 was

1.04� 0.04 MPa, which decreased to 0.82� 0.04 and

0.69� 0.03 MPa for PACL-2 and PACL-3, respectively. In

particular, incorporation of PAni into PLCL significantly



reduces the elasticity of the nanofiber as shown in

Figure 3c. The elongation at break for pure PLCL was

391.54� 9.20%, which decreased to 309.41� 7.22% and

207.85� 6.74% when the volume ratio of PAni to PLCL was

increased to 15 and 30%, respectively. The elastic property

of the CPSA-PAni/PLCL scaffolds decreased with increasing

CPSA-PAni content. These characteristics may be a result of

the decrease in the concentration of total PLCL polymer

solution with the increased amount of CPSA-PAni in the

blend. Despite the reduced mechanical properties, elastic

properties of CPSA-PAni/PLCL nanofibers are much higher

than those of electroactive electrospun scaffolds from

other synthetic or natural polymers (PLGA and gelatin

exhibit a strain of 96% and 12.5%, and even native skin

extends between 35% and 115%).[25,31] Our results suggest

that the elastic properties of electroactive nanofibers can



Figure 3.Mechanical properties of CPSA-PAni/PLCL nanofiber scaffolds. a) Tensile strength, b) Young’s modulus, c) elongation at break, andd) stress–strain curve of CPSA-PAni/PLCL nanofiber scaffolds.

632

be enhanced by electrospinning with elastic polymers,

thereby allowing for more realistic mimicry of the

mechanical and electrical characteristics of the natural

ECM of soft tissue such as skin, skeletal muscle, and blood

vessels.

The conductivity of electrospun CPSA-PAni/PLCL nano-

fibers was measured at various ratios of CPSA-PAni and

PLCL in blend. As show in Table 1, PACL-1 without

incorporation of PAni shows the minimal conductivity

(0.0015 S � cm�1). Incorporation of PAni in the nanofiber

significantly increases the conductivity as a function of

increased amount of PAni where the conductivity of

PACL-2 and PACL-3 is 0.00765 and 0.0138 S � cm�1,

respectively. The main factor to determine the conductiv-

ity of PAni (more specifically emeraldine base) is known to

be the oxidation status originated by the interactions

between the organic acid and PAni. Furthermore, the

expanded structure of the coil-like PAni chains affects the

conductivity, which is influenced by the interactions of

the dopant (usually an organic acid) with the solvent.[17]

Previous reports have demonstrated that the conductivity

of PAni tremendously differs by selection of appropriate

solvents; the CPSA-PAni had a conductivity of 0.1 S � cm�1



in chloroform, which became four orders of magnitude

greater in m-cresol.[32,33] Considering the increased con-

ductivity of the PAni/PLCL composite nanofibers, we

assumed that CPSA, dissolved in HFP, protonated the

emeraldine base of PAni, thereby forming the polyelec-

trolyte salt within the organic matrix of PLCL. Our results

indicate that HFP was effective for the formation of a

CPSA-PAni salt within the PLCL matrix, which makes the

chain conformation of PAni conductive.[17] The result is

consistent with a previous study that used HFP for the

fabrication of nanofibers from a mixture of gelatin and

PAni. More importantly, the increased conductivity of the

CPSA-PAni/PLCL nanofibers with a higher amount of PAni

indicates that the expanded chains of CPSA-PAni are

homogeneously distributed in the PLCL.

We then examined the presentation of surface func-

tional groups of the nanofibers using ATR FT-IR and Raman

spectroscopy, and XPS measurements. As shown in

Figure 4, all the characterization methods support the

presence of CPSA-PAni on the surface of the composite

nanofibers. The ATR FT-IR spectra present the major

characteristic peaks of CPSA-PAni at 1 652, 1 543, 1 280, and

1 192 cm�1. All nanofibers show the characteristic ester

DOI: 10.1002/mabi.200800005


Figure 4. Surface properties of CPSA-PAni/PLCL nanofiber scaffolds. a) ATR FT-IR, b) Raman, and c) XPS spectra of the samples.

stretching of PLCL at 1 751cm�1.[34] The characteristic

stretching peaks of CPSA-PAni are observed at 1 652cm�1

(C––O) and 1 543 cm�1 (N–H) in the PACL-2 and PACL-3

nanofiber samples, while the same peak disappears for the

PACL-1 nanofiber.[35] The results strongly suggested that

CPSA-PAni is present at the surface of the nanofibers. In

addition, the intensity of the corresponding peaks

appeared to increase as the amount of PAni in the

nanofibers increased. The ATR FT-IR spectra are similar to

those of PAni-blended electrically conductive materials

that include PAni-chitosan and PAni-polyurethane sub-

strates.[36,37]

The Raman spectra strengthened our early conclusion of

the presence of PAni on the surface of the nanofiber

composites. The characteristic two peaks for CPSA-PAni are

observed at 1 330 cm�1 (C–N) and 1 601 cm�1 (phenyl C–C),

which are present in spectra for PACL-2 and PACL-3



nanofibers, whereas both peaks disappear from the spectra

of the PACL-1 nanofibers. The presence of these character-

istic peaks is in a good agreement with previous

reports.[38,39] Notably, the intensity of these two bands in

PACL-3 is greater than that in PACL-2, which indicates that

the amount of CPSA-PAni may be correspondingly greater

at the nanofibers surface. The presence of nitrogen at the

surface of the composite nanofibers was doubly confirmed

by XPS, where the N 1s peak is observed at 399 eV for

PACL-2 and PACL-3 nanofibers only, while no nitrogen

peak is found in PACL-1.[35] PLCL shows only C 1s and O 1s

peaks present at binding energies of 291 and 530 eV,

respectively.[40] Collectively, these results suggest that the

electrospinning process of CPSA-PAni/PLCL blends is

successful for the presentation of electrically conducting

domains at the surface, which may be effective to transfer

electrical signals.



Figure 5. Thermal gravimetric properties of CPSA-PAni/PLCL nano-fiber scaffolds. When the temperature exceeded 800 8C, approxi-mately 12 and 21% relative to the original weight remained forPACL-2 and PACL-3, respectively.

634

The TGA profiles of CPSA-PAni/PLCL in Figure 5 show the

dependence of thermal stabilities of the nanofibers on

CPSA-PAni concentration. The thermal decomposition of

PAni followed two step processes, where the initial broad

decrease in the sample weight was observed from

approximately 150 to 400 8C and the second stage occurred

from approximately 550 to 750 8C. On the other hand,

PACL-1 was degraded before the maximum tested

temperature (900 8C) with trace amounts of unknown

impurities. When the volume ratio of PAni to PLCL

increased from 15 to 30%, the corresponding weight loss

at 900 8C was approximately 90 and 80%, respectively. The

remaining weight was attributed to the presence of PAni,

which decomposed by 20 over 900 8C. These TGA profiles

are similar to those of conductive and non-conductive

composite materials, which include polyaniline-

polyurethane and polyaniline-chitosan substrates.[36,37]

Figure 6. in vitro cellular response to CPSA-PAni/PLCL nanofiberscaffolds. Mitochondria metabolic activity of a) human dermalfibroblasts, b) NIH-3T3 fibroblasts, and c) mouse skeletal musclecells (C2C12) on CPSA-PAni /PLCL nanofibers prepared by theelectrospinning process, respectively.

in vitro Cell Response

To investigate the effect of PAni on cell adhesion, we

seeded three different types of cells on the CPSA-PAni/PLCL

nanofibers and measured mitochondria metabolic activity

using MTT assay. On day one after seeding, each cell type

showed different levels of enzymatic activity. The general

trend was observed that the relative amount of living cells

cultured on the nanofibers significantly increased in a PAni

concentration-dependent manner (Figure 6). For example,

the relative activity of human primary fibroblasts cultured

on PACL-3 was 186.54� 3.79, which was almost three

times greater that that on PACL-1 (63.55� 2.22%). Mouse

skeletal muscle cells (C2C12) showed the same behavior

where the relative activity increased from 35.95� 4.41% to



100.82� 11.82 when the volume ratio of PAni to PLCL

increased from 0 to 30%. The morphology and cytoskeletal

structure of adherent cells on the CPSA-PAni/PLCL

nanofibers were also assessed by staining F-actin follow-

ing the fixation of cells 1 day after seeding (Figure 7), which

DOI: 10.1002/mabi.200800005


Figure 7. Representative SEM images (a–c) and fluorescence micrographs (d–f) demonstrating the formation of F-actin filaments in thehuman fibroblasts, NIH-3T3 fibroblast, and C2C12 cells cultured on CPSA-PAni/PLCL (PACL-3) nanofiber scaffolds for 1 d (scale bar¼ 100 mm).

indicates that the cells cultured on nanofibers maintain

normal morphologies and spread.

The biocompatibility of PAni has been reported from

other studies. PAni films and composites of PAni with

natural polymers allowed favorable attachment of H9c2

cardiac myoblasts, which remained viable and prolifer-

ated.[17,41] In our study, we re-confirmed the biocompat-

ibility of PAni-containing nanofibers using several cell

lines, and demonstrated that the incorporation of PAni

improved the metabolic activity of all cell types treated in

a concentration-dependent manner. Since PLCL has been

reported to allow limited cell adhesion, the composite

scaffold may be beneficial for enhanced cell compatibility

of PLCL. However, it is not clear whether the enhanced cell

adhesion is a result of either electroconductive properties

of the scaffolds or modified surface chemistry by

incorporated PAni, or both. Since the contact angle

measurement showed that all types of nanofiber scaffold

had similar values as shown in Table 1, the hydrophobi-

city/hydrophilicity of the surface may not be the primary

contributor for the observed phenomena. This would be

the subject of further investigations.

Finally, we used nanofibers as a model system to carry

out a preliminary study to investigate the combined

effects of electrical stimulus and an electrically conducting

substrate on the mitochondria metabolic activity of

NIH-3T3 cells. Cells were attached to PACL-1 and PACL-3

nanofibers. Subsequently, various electrical currents

were continuously applied to a cell/nanofiber scaffold



constructs for 2 days and their metabolic activities were

measured by MTT assay. As shown in Figure 8, the

mitochondria metabolic activity of NIH-3T3 cells

responded to the electrical stimulus. It was evident that

application of 200 mA was detrimental to the cells, which

showed minimal metabolic activity. At 0 and 2 mA, the

mitochondria metabolic activity of the NIH-3T3 cells was

similar to all the tested groups. However, 20 mA of

electrical current significantly increased the mitochondria

metabolic activity of NIH-3T3 cells cultured on PACL-3 as

compared to that on PACL-1.

The effect of electrical stimulation on cellular activities

has been reported using different cell types. The most

widely utilized conductive polymer is polypyrrole and its

derivatives. Schmidt and colleagues reported that one of

the murine neuron precursor cells, PC-12 cells, demon-

strated an increase in neurite outgrowth on polypyrrole

substrates when stimulated at 100 mA for 2 h.[13,42] On a

similar substrate, keratinocyte viability was increased by

20% upon stimulation at 100 mA as compared to a

non-stimulated control.[14] Recently, an electrically active

composite scaffold comprised of poly(e-caprolactone) and

PAni has been fabricated, which showed enhanced

calcification of osteoblasts under electrical stimulation.[43]

Although direct comparison between previous and current

studies may not be appropriate because our nanofibrous

scaffolds possess different chemical and morphological

properties, it is clear that PAni/PLCL composite scaffolds

may have transferred additive signals to adherent cells by



Figure 8. in vitro cellular response to CPSA-PAni/PLCL nanofiber scaffolds under electrical stimulation. Fluorescence micrographs demon-strating the formation of F-actin filaments of the NIH-3T3 fibroblast cultured on CPSA-PAni/PLCL (PACL-3) nanofiber scaffolds for 2 d underan electrical current of a) 20 and b) 200 mA, and c) mitochondria metabolic activity of NIH-3T3 fibroblasts (scale bar¼ 100 mm).

636

their unique electroconductive nature and thereby modu-

lated proliferation of the fibroblasts.

The PAni/PLCL scaffolds possess advantageous features

over previously fabricated electrically conductive polymer

composite systems: 1) the PLCL domain provides mecha-

nical stability, in particular the elastic properties of the

scaffolds, 2) polymer components can be easily dissolved

with HFP and then electrospun to form uniform fibers with

diverse diameter ranges, and 3) incorporated PAni can

enhance the biocompatibility of the composite scaffolds.

Our future studies will involve the use of PAni/PLCL

composite scaffolds to investigate the effects of electrical

stimulation and electroconductive properties of substrate

materials on the functional response of neural precursor

cells or muscle cells, which transfer most of their signals by

electrochemical processes.



Conclusion

In this study, we prepared electroactive CPSA-PAni/PLCL

nanofiber scaffolds using the electrospinning technique.

SEM analysis of the scaffolds showed that a nanoscaled

structure was successfully formed with a high pore

volume, interconnective pores, and a uniform mean fiber

diameter irrespective of PAni incorporation. Moreover, the

electrical conductivity of the CPSA-PAni/PLCL scaffolds

increased with increasing CPSA-PAni content. Analysis

with ATR FT-IR, Raman, and XPS spectra demonstrated the

presence of CPSA-PAni on the surface of the nanofiber

scaffolds. The adhesion of human dermal fibroblasts,

NIH-3T3 fibroblasts, and C2C12 cells on to the scaffolds

was dependent on the CPSA-PAni content. The growth of

NIH-3T3 cultured on the CPSA-PAni/PLCL scaffolds was

DOI: 10.1002/mabi.200800005


controlled by the application of a medium range of

electrical stimulation. The CPSA-PAni/PLCL nanofiber

scaffolds combine the advantages of mechanical strength,

electrical conductivity, and the nanometer-scale features

of a native ECM, and may hold a great promise as an

electrically conductive scaffold for many tissue engineer-

ing applications under a mechano-electrically stimulated

microenvironment.

Acknowledgements: This work was supported by the Seoul R &BD Program funded by Seoul Development Institute (to Y.M. Leeand H. Shin). S.I.J. is very grateful to the BK21 Project for afellowship.

Received: January 8, 2008; Revised: February 21, 2008; Accepted:February 21, 2008; DOI: 10.1002/mabi.200800005

Keywords: conducting polymers; electrospinning; nanofibers;polyaniline; poly(L-lactide-co-e-caprolactone)

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