Keratin Nanofibers as a Biomaterial - ipcbee

2010 International Conference on Nanotechnology and Biosensors
IPCBEE vol.2 (2011) © (2011) IACSIT Press, Singapore
Keratin Nanofibers as a Biomaterial
Zhi-Cai Xing, Jiang Yuan, Won-Pyo Chae, and
Inn-Kyu Kang *
Department of Polymer Science and Engineering,
Kyungpook National University
Daegu 702-701, South Korea
e-mail: ikkang@knu.ac.kr
Suk-Young Kim
School of Materials Science and Engineering,
Yeungnam University
Gyongbuk 712-749, South Korea
Abstract—Keratin is one of the most abundant proteins which
can be used in a variety of biomedical applications due to its
biocompatibility and biodegradability. However, keratin is
insoluble in solvents and soluble in few solvent systems, which
lead to a limitation in its processability for further utilization.
In this study, keratin was extracted and chemically modified
by reacting sulfide side group with iodoacetic acid to enhance
its solubility in organic solvent. The modified keratin (mkeratin)
was then blended with poly (ethylene oxide) (PEO) in
different proportions, dissolved in 2,2,2,-trifluoroethanol
(TFE), and electrospun to produce nanofibrous mats. The m-
keratin nanofibers were obtained by crosslinking the m-
keratin/PEO nanofibrous mats with glutaraldehyde vapor
(25%) and washed with distilled water for 3 times to remove
PEO. The morphology, diameter distribution, biodegradation,
and interaction of NIH 3T3 cells with nanofibrous mats were
investigated. The results showed that the crosslinked m-keratin
nanofibrous mats have a potential to be used in tissue
engineering and wound dressing.
Keywords- biodegradability, NIH3T3 cells, keratin, nanofiber,
poly(ethylene oxide)
I. INTRODUCTION
In recent years, much attention has been focused on the
electrospinning of biopolymers such as silk fibroin [1-2],
collagen [3], fibrinogen [4], gelatin [5] and elastin [6].
However, keratin has received poor attention even though it
is one of the most abundant proteins, being the major
component of hair, feathers, wool, nails and horns of
mammals, reptiles and birds. Keratin is well known as
biocompatible and biodegradable proteins [7], which can
accelerate the growth of fibroblast [8]. Thus, keratin is
expected to be applicable for biomedical use in a similar
manner to collagen and fibroin. Unfortunately, the poor
mechanical properties of regenerated keratin hinder its
processability and restrict its practical applications to
blending with appropriate polymers having better structural
properties. In the previous work, Lee at al [9] blended keratin
and fibroin and found that the films composed of silk fibroin
and S-carboxymethyl kerateine showed lower blood
coagulation compared to silk fibroin or keratin alone [10].
Yuan et al. [11] fabricated the poly(hydroxybutylate-cohydroxyvalerate)
(PHBV)/keratin composite nanofibrous
mats and concluded the resulted keratin nanofibers contained
many beads due to the broad molecular weight distribution
and low dissolvability of keratin. Aluigi et al [12] fabricated
composite nanofibers consisted of keratin and poly (ethylene
oxide) (PEO) using water as a solvent. As a result, regularly
shaped nanofibers could be obtained at the ratio of 50/50 and
polymer concentration of 7-10%. They only extracted the
keratin from wool and studied the chemical, physical and
rheological characteristics of the electrospun PEO/keratin
mats [13].
PEO is an amphiphilic, water-soluble, and nondegradable
polymer with good biocompatibility [14] and low
toxicity [15]. To produce keratin nanofibers, PEO has been
added to keratin solution with different ratio to improve the
processability of the keratin itself because it can be
electrospun without defects from solutions [12, 13, 16].
In this study, keratin was chemically modified with
iodoacetic acid for enhancing its solubility in organic solvent.
The modified keratins (m-keratin) were mixed with PEO at
different ratio, dissolved in 2,2,2,-trifluoroethanol (TFE) and
electrospun to produce m-kerain/PEO composite nanofiber
mats. The m-keratin nanofibers were obtained by
crosslinking the m-keratin//PEO nanofiber mats with
glutaraldehyde vapor followed by removal of PEO by
washing with water. The morphology of the nanofibrous
mats were studied with field-emission scanning electron
microscope (FE-SEM). Biological performances of the
nanofibrous mats including biodegradation and cell-scaffold
interaction were also studied.
II.
MATERIALS AND METHODS
A. Preparation of m-keratin/PEO blend solutions
Keratin was extracted and chemically modified according
to the method previously reported [11]. In brief, raw keratin
(MP Biomedical Company, Germany) was first mixed with
urea, sodium dodecyl sulfate (SDS), 2-mercaptoethanol and
water. The mixture was stirred for 12 h at 60ºC and then
filtered. Subsequently, the filtrate was dialysed against
deionized water to afford a colorless solution. The dialysate
(unmodified keratin solution) was allowed to react with
iodoacetic acid for modification. Finally, S-(carboxymethyl)
keratin was lyophilized to obtain modified keratin (mkeratin).
PEO powder with a viscosity-average molecular weight
of ca. 9×10 5 g/mol (Sigma–Aldrich, St. Louis, MO) was
dissolved in 2,2,2,-trifluoroethanol (TFE) at the room
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temperature for about 12 h. The concentration was adjusted
at 2 wt %.
The m-keratin/PEO blend solutions were prepared by
adding m-kerain to the PEO solution and stirring for 12 h at
room temperature. The blend solutions of the m-keratin/PEO
were adjusted at the concentration of 2 wt% and the ratio of
m-keratin and PEO was changed from 50:50 to 90:10.
B. Preparation of m-keratin/PEO blend nanofibers
The blend solution was delivered to a metal needle
connected to a high-voltage power supply. Upon applying a
high voltage, a fluid jet was ejected from the needle. As the
jet accelerated towards a grounded collector, the solvent
evaporated and a charged polymer fiber was deposited on the
collector in the form of a nanofibrous mat. The typical
parameters for electrospinning were as follows: 9 kV
(voltage), 12 cm (distance between tip and receptor),
1.0mLh −1 (feed rate), 60% (humidity) and 25ºC
(temperature). For analysis of the morphology of the
electrospun fibers, the samples were sputter-coated with gold,
and examined using FE-SEM (Hitachi S-4300, Japan). The
diameters of the electrospun nanofibres were measured at
100 different points from SEM pictures for each sample
produced.
C. Preparation of m-keratin nanofibers
The m-keratin/PEO nanofiber mats need to be
crosslinked to reduce their solubility in water. The
electrospun m-keratin/PEO nanofibrous mats were
crosslinked by treating them with glutaraldehyde vapor and
saturated with a 25% glutaraldehyde aqueous solution at
room temperature for 4 h. This was followed by treatment
with 0.1 M glycine aqueous solution to block unreacted
aldehyde groups. The crosslinked m-keratin/PEO
nanofibrous mat was then washed with distilled water for
three times (10 minutes each) to produce m-keratin nanofiber
mats. To examine the presence of PEO in the crosslinked m-
keratin/PEO mat after removal by water, fluorescein-tagged
PEO (F-PEO) was synthesized by reacting hydroxyl end
group of PEO with group of fluorescein isothiocyanate
(FITC) [20].
D. In vitro biodegradation
The m-keratin/PEO and m-keratin nanofiber mats were
cut into rectangles (20 × 20 × 0.05 mm) for in vitro
degradation testing. Each specimen was placed in a test tube
containing 10 ml of phosphate-buffered saline (PBS, pH 7.0,
Gibco) and incubated for 12 h at 37ºC. After incubation, the
samples were washed and lyophilized for 24 h. In order to
measure the enzymatic degradation of nanofibrous mats, the
samples were incubated in a PBS containing trypsin (10
mg/ml) at 37ºC. After incubation for a requisite time (2 h or
12 h), the samples were washed with distilled water and then
lyophilized for 24 h. Morphological changes were observed
with a FE-SEM.
E. Cell adhesion
In order to examine the interaction of nanofiber mats
with cells (NIH 3T3), the circular nanofibrous mats were
fitted in a 24-well culture plate and subsequently immersed
in a DMEM medium containing 10% fetal bovine serum
(FBS, Gibco) and 1% penicillin G-streptomycin. To seed the
cells, 1 ml of NIH 3T3 cell solution (3×10 4 cells) was added
and incubated in a humidified atmosphere of 5% CO 2 at 37ºC.
After incubation for a 4 h, the medium solution was removed.
These samples were washed twice with the PBS, and fixed
by 2.5% glutaraldehyde aqueous solution for 20 min. The
sample mats were then dehydrated in a graded concentration
of ethanol (25, 50, 75, 90, and 100) for 10 min each. Finally,
the sample mats were air dried in a fume hood overnight.
Dry cellular structures were sputter-coated with gold and
observed with a FE-SEM.
F. Determination of cell viability
A standard Live/Dead assay was used to image cell
survival, adhesion, and spatial organization. After 6 days
incubation, cells were collected by centrifugation and
incubated in calcein-AM (1 mM in PBS) and ethidium
homodimer-1 (2.5 mg/ml PBS) solution for 15 min. Cells
with compromised membranes exhibit red-fluorescence from
the live-cell impermanent nucleic acid stained with ethidium
homodimer-1. Cells with intact membranes are able to use
nonspecific cytosolic esterases to convert nonfluorescent
calcein-AM into bright green-fluorescent calcein. Cells were
observed under a fluorescence microscope using a band-pass
filter (Nikon Eclipse E600-POL, Japan).
Cell viability was measured after 2, 4 and 6 days of
culture using a commercially available MTT assay kit
(Sigma). After incubation of certain time, the medium was
replaced with a (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide (MTT) solution and incubated for
further 4h. Mitochondrial dehydrogenases of viable cells
cleave the tetrazolium ring, yielding purple formazan crystals.
Formazan crystals were then dissolved in PBS solution. The
optical density (OD) of the solvent is proportional to the
mitochondrial activity of the cells on the surface. OD was
measured at 570 nm using a kinetic microplate reader (EL 9
800, Bio, Tek, Instruments, Inc, Highland Park, USA).
Background absorbance at 690 nm was subtracted from the
measured absorbance.
G. Statistical analysis
Results are displayed as mean ± standard deviation.
Statistical differences were determined by a student’s twotailed
t-test. Scheffe’s method was used for multiple
comparison tests at a level of 95%.
III.
RESULTS AND DISCUSSION
A. Nanofiber morphology
Figure 1 shows the SEM images of nanofibers obtained
from electrospinning of the m-keratin/PEO blend solutions.
The electrospinning of a 2 wt% PEO pure solution produced
nanofibers without defects at the condition of a flow rate of
1ml/h and a voltage of 9 kV. However the diameter
distribution of PEO nanofibers was so broad, ranging from
200 nm to 2000 nm. The diameter distribution of PEO
became narrow with an increase of m-keratin content (Figure
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1b,c,d). Therefore, it could be say that the blend composition
plays an important role in determining the diameter
distribution of the nanofibers. In addition, the average
diameter of the blend nanofibers gradually decreased from
950±50 to 400±30 nm as the m-keratin content increases
(Figure 1b and Figure 1d). The viscosity of m-keratin/PEO
(50/50) blend solution (224 cP) was significantly decreased
down to 34 cP with the increase of m-keratin (90/10). The
conductivity of m-keratin/PEO blend solution also increased
with an increase of the m-keratin data not shown. In fact, it is
known that lower viscosity promotes the formation of finer
nanofibers [17] and higher charge density carried by jet
forms smoother and finer nanofibers because the stronger
whipping instability of the jet enhances the filament
stretching [18,19].
B. Crosslinking of m-keratin/PEO nanofibers
The m-keratin/PEO blend nanofibers can be easily
dissolved in water. Therefore, the nanofibers need to be
crosslinked to reduce their solubility. The most popular
crosslinking reagent used in proteins is glutaraldehyde vapor.
Glycine solution was used to block residual aldehyde group
after treatment of m-keratin/PEO nanofibers with
glutaraldehyde vapor. Figure 2 showed the morphology of
m-keratin/PEO (90/10) nanofibrous mats before and after the
crosslinking with glutaraldehyde vapor for 4h. Obviously,
the m-keratin/PEO nanofiber mat lost its fibrous morphology
slightly after the crosslinking. Figure 3 showed the
fluorescence images of the m-keratin/F-PEO(90/10) mat
(Figure 3a) and the m-keratin nanofibrous mat obtained by
removal of F-PEO by water. As a result, the m-keratin/F-
PEO showed the image of green color due to the presence of
F-PEO. However, the green color was almost disappeared
after washing the crosslinked m-keratin/F-PEO nanofibrous
mats with water (Figure 3b). It is revealed, from the data of
fluorescence image, that m-keratin nanofiber mat could be
obtained by crosslinking the m-keratin/PEO mat with
glutaraldehyde vapor followed by washing with water.
Figure 2. SEM images of the m-keratin/PEO (90/10) nanofibrous mats
before (a) and after (b) crosslinking with glutaraldehyde vapor for 4h.
C. In vitro biodegradation
The image of the mat biodegraded was examined with
FE-SEM. Figure 4 illustrates the morphological changes of
the electrospun mats during in vitro degradation. The
crosslinked m-keratin nanofibrous mats lost their nanofibrous
form after 2h degradation in trypsin aqueous solution, and
the mat seriously degraded and remained as debris after 12h
incubation. Yuan et al [11] reported on biodegradation of m-
keratin/PHBV nanofibrous mats by trypsin solution. As a
result, fibrous morphology almost not changed when the m-
keratin/PHBV mat was incubated in trypsin agueous solution
for 24 h. It is concluded that m-keratin/PEO nanofibrous
mats underwent a higher biological degradation than the
keratin/PHBV nanofibrous mats. This result suggests that the
m-keratin mats studied in this study are suitable for further
biomedical and biotechnological applications [21].
Figure 3. Fluorescence images of the crosslinked m-keratin/F-PEO mat
before (a) and after (b) removal of F-PEO by water nanofibours mats.
Figure 4. SEM images of the crosslinked m-keratin nanofibrous mats
incubated in trypsin solution for 2h (a) and 12h (b).
Figure 1. SEM images of electrospun nanofibers with different ratio of m-
keratin and PEO (a) pure PEO, (b) 50/50, (c) 70/30 and (d) 90/10.
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D. Cell-scaffold interaction.
To evaluate cellular behavior on electrospun fibers,
fibroblasts were seeded and cultivated on the crosslinked m-
keratin nanofibers and the tissue culture polystyrene
(control). As shown in Figure 5, cells were more adhered to
the surface of the crosslinked m-keratin nanofiber mat, and
showed much more spread morphology than the tissue
culture polystyrene. The viability of NIH 3T3 cells on the
surface of crosslinked m-keratin nanofibrous mat and the
tissue culture polystyrene were also investigated. NIH 3T3
survival was assessed through live/dead fluorescence
staining. Images taken on a fluorescence microscope indicate
that the surface of crosslinked m-keratin nanofiber mat was a
favorable template for cell adhesion. NIH 3T3 cells seeded
on crosslinked m-keratin nanofibers displayed a high level of
viability as assessed using a standard MTT assay (Figure 7).
The viability of cells on the surface of crosslinked m-keratin
nanofiber is significantly higher than that on the tissue
culture polystyrene after incubation of 6 days, indicating that
the NIH 3T3 cells seeded on the crosslinked m-keratin
nanofiber surface are healthy and there are no cytotoxic
effects (Figure 6). Further, fluorescence images of live cells
seeded on surface of crosslinked m-keratin nanofiber showed
higher degree of spreading compared to that on the tissue
culture polystyrene. These results suggest that the
crosslinked m-keratin nanofiber mat is a good scaffold for
the adhesion and spread of NIH3T3 cells compared to tissue
culture polystyrene.
Figure 5. SEM images of NIH 3T3 cells cultured for 4 h on the tissue
culture polystyrene (a) and the crosslinked m-keratin nanofibrous mats (b).
Figure 7. MTT assay, Formozan absorbance expressed as a measure of
cell viability from the NIH 3T3 cells cultured on the tissue culture
polystyrene and the crosslinked m-keratin/PEO nanofibrous mats (Data are
expressed as means ± SD (n=6) for the specific absorbance, * p < 0.05,
values are significantly different from those of the previous group).
IV. CONCLUSIONS
The m-keratin/PEO blend solutions prepared in 2,2,2,-
trifluoroethanol (TFE) were electrospun to produce
nanofibers. All blend solutions were electrospun successfully.
Morphological investigation showed that as the m-keratin
amount in the blend solution increased, the nanofibers
became thinner and more homogeneous. In addition to being
biocompatible and biodegradable, crosslinked m-keratin
nanofibers induced an enhanced NIH 3T3 cells response.
The results demonstrated that the crosslinked m-keratin
nanofibers enhanced NIH 3T3 cells adhesion and
proliferation as compared to the tissue culture polystyrene.
The performance of the crosslinked m-keratin nanofiber
warrants future work aimed at in vivo characterization and
fabrication of 3-D mats.
ACKNOWLEDGMENTS
This research was supported by the research grants of the
Biotechnology development project (2009-0090907) and by
the grant of 2010-0011125 from Ministry of Education,
Science and Technology of Korea.
Figure 6. Fluorescence images of NIH 3T3 cells cultured for 6days on the
tissue culture polystyrene (a) and the crosslinked m-keratin nanofiber mat
(b).
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