Journal of Biomaterials

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Electrospinning of polyvinyl alcohol/gelatin nanofiber composites and cross-linking for bone tissue
engineering application
Nguyen Thuy Ba Linh and Byong-Taek Lee
J Biomater Appl 2012 27: 255 originally published online 16 June 2011
DOI: 10.1177/0885328211401932

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Article
Journal of Biomaterials Applications
27(3) 255–266
! The Author(s) 2011
Electrospinning of polyvinyl alcohol/ Reprints and permissions:
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gelatin nanofiber composites and DOI: 10.1177/0885328211401932
jba.sagepub.com
cross-linking for bone tissue
engineering application

Nguyen Thuy Ba Linh and Byong-Taek Lee

Abstract
A three-dimensional polymer composite system consisting of polyvinyl alcohol/gelatin (PVA/GE) was fabricated via the
electrospinning method and physically cross linked by methanol treatment. The effects of cross-linking between PVA/GE
blend on physical, mechanical, and biological properties were investigated. After treating with methanol, PVA/GE mats
become dense, hard, and aggregative with increased resistance to water dissolution. Osteoblasts like MG-63 cells were
seeded on the surfaces of the cross linked PVA/GE mats and were found to attach firmly by expressing philopodial
extensions. In addition, MTT assay and Western Blot analysis confirmed that the cells readily proliferated on the cross
linked PVA/GE scaffolds. The osteoblast cell–matrix interaction demonstrated that the active biocompatibility of the
mats was facilitated by using GE and cross-linking. In conclusion, our results suggest that cross-linked PVA/GE scaffolds
hold promise for tissue engineering applications, especially in the field of artificial bone implant.

Keywords
Electrospinning, polyvinyl alcohol/gelatin, osteoblast cell, bone tissue engineering

Introduction applications, including tissue engineering of scaffolds,

Several new promising approaches in tissue engineering
have recently been developed to create biological alter-
natives for various regenerating tissues. Several require-
ments are necessary in the design of tissue engineering
scaffolds, including a high porosity, large surface area,
adequate pore size, and uniformly distributed intercon-
nected porous structures throughout the matrix.1,2
Biodegradable polymeric fiber structures can provide
a large surface area, and a relatively high porosity,
which can be optimized for specific applications. Due
to the structural similarity between electrospun poly-
meric mats and the collagen fiber arrangement in
the natural extracellular matrix component of bone,
the electrospinning process has drawn a great deal
of attention in scaffold processing for tissue
engineering.3–5 In addition, electrospinning is regularly
used to fabricate nano-fibrous structures from a
number of natural synthetic polymers, such as colla-
gen,6 chitosan,7–9 silk fibroin,10 hyaluronic acid,11
poly(L-lactide),12 and polycaprolactone,13–15 among
others. Within the last few years, this technique has
proven to be especially suitable for biomedical
wound dressings, drug delivery, and in other fields such
as medical implants.16–21
Biodegradable polymers have been proposed as a
possible alternative to other tissue engineering mate-
rials and have gained a large amount of attention.
These polymers can be easily processed into three-
dimensional porous structures with the proper degra-
dation behavior and mechanical strength.22,23 Among
the biodegradable and biocompatible polymers, polyvi-
nyl alcohol (PVA), gelatin (GE) and their blends have
extensive applications as surgical sutures, implant
materials, drug carriers, and scaffolds for tissue engi-
neering.24–29 PVA is a semi-crystalline polymer that
Department of Biomedical Engineering and Materials, College of
Medicine, Soonchunhyang University, 366-1 Ssangyong dong, Cheonan,
330-090, Korea
Corresponding author:
Byong-Taek Lee, Department of Biomedical Eng. & Mater., College of
Medicine, Soonchunhyang University, 366-1 Ssangyong dong, Cheonan,
330-090, Korea.
Email: lbt@sch.ac.kr

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256
possesses good chemical and thermal stability.30 PVA is
a nonhazardous material, has no negative effect on ani-
mals, and does not cause any injuries to the skin upon
contact. Due to their excellent biocompatibility and
biodegradability, GE fibers are widely used in a variety
of biomedical applications,31 including wound or burn
dressings, surgical treatments, and tissue engineering of
bone, skin, and cartilage.24,27,32 Furthermore, GE fibers
can be easily extended into three-dimensional structures
of woven, knitted, and nonwoven structures. This is
mainly derived from the fact that GE is a natural poly-
mer that has well-known wound healing abilities and a
biological aptitude for stimulating cell proliferation.
PVA/GE nano-fibrous membranes with different com-
positions could have different clinical applications,
especially the controlled release of drugs and bone
tissue engineering.25
The most common strategy employed to engineer
bone is to use a scaffold combined with osteoblast
cells, or cells that can mature/differentiate into osteo-
blasts and regulatory factors that promote cell attach-
ment, differentiation, and mineralized bone
formation.23,33 However, the requirements for design-
ing and production of an ideal scaffold for bone regen-
eration are very complex and not yet fully understood.
It is generally agreed that the scaffold must be a bio-
compatible, porous (more than 90% and pore sizes
between 100 and 350 mm), interconnected, and perme-
able structure in order to permit the ingress of cells and
nutrients.14,33 Clearly, structures designed with biode-
gradable fibers can meet all these criteria and may serve
as a scaffold for the engineering of bone. Many differ-
ent fiber-based polymeric matrices have been tested
with different cell types to create a bone
construct.14,24,33,34
The fabrication of a composite material from PVA
and GE nano-fibers for using in tissue engineering or in
guided tissue regeneration, particularly for bone regen-
eration, has already been reported earlier.35 In that
study, nano-structured scaffolds of PVA/GE blends
were fabricated via electrospinning and analyzed
in vitro. The hydrophilic of this biocomposite resulted
in the ability to support osteoblast cell MG-63 attach-
ment and proliferation.36 However, due to their good
solubility at room or body temperature, hydrophilic
scaffolds made from PVA/GE blends were easily dis-
solved in water or cell culture mediums. Therefore, a
cross-linking step was needed prior to in vitro analysis
to increase the resistance of the scaffold to dissolution
in water. The purpose of this study was to better under-
stand the effect of cross-linking the PVA/GE blends.
These effects were assessed using a number of tech-
niques, including SEM, DSC, tensile strength, and con-
tact angle. Moreover in order to understand the initial
behavior of osteoblast cells, cellular adhesion,
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Journal of Biomaterials Applications 27(3)
proliferation, and toxicity were assessed in vitro using
osteoblast like MG-63 cell line.
Materials and methods
Materials
Poly(vinyl alcohol) (PVA, 99þ% hydrolyzed and
number average Mw of 115,000 g/mol) was obtained
from Aldrich Chemical Co (USA). Polymers of GE
Type A (Approx. 300 Bloom, Sigma, St. Louis, MO)
from porcine skin were obtained in powder form.
Acetic acid (CH3COOH, glacial, 99.0%) and methanol
were purchased from Duksan Pure Chemical Co.,
Korea.
The human osteoblast-like cells, MG-63 cell line,
derived from human osteosarcoma, were obtained
from Korean Cell Bank (KCB). Dulbecco’s Modified
Eagle’s Medium (DMEM, HyClone, Logan, UT), fetal
bovine serum (FBS, Grand Island, NY), penicillin/
streptomycin (antibiotics), and trypsin-EDTA were
purchased from GIBCO (Carlsbad, CA).
Glutaraldehyde was obtained from DeaJung Co.,
Korea. Hexamethyldisilazane (HDMS, Sigma, USA),
Dimethylsulfoxide (DMSO 99.0%, Samchun Pure
Chemical Co., LTD, Korea), ethanol (Merck,
Germany) were used as received. For Western Blot
Analysis, antibodies (anti-PCNA and anti-beta actin)
were purchased from AnaSpec, Inc.
Electrospinning setting
Aqueous PVA solutions (12 w/v %) were dissolved in
deionized water at 80 C with constant stirring for at
least 3 h and electrospun to obtain the PVA mat.
12 w/v % GE solutions in a solvent consisting of deio-
nized water: acetic acid ¼ 1:9 were prepared at room
temperature under gentle stirring for 30 min37 and sub-
jected to electrospinning. To obtain the PVA/GE blend,
similar concentrations were used for both PVA and GE
solutions. Then, the GE solution was added to the PVA
solution at specific volumes to obtain a PVA/GE
weight ratio of 2/8. Owing to the excellent biocompat-
ibility of GE, the PVA/GE ¼ 2/8 biocomposite was
chosen for further investigation. The mixture was
then stirred for an additional 15 min before
electrospinning.
For electrospinning (eS-robot, Electrospinning/
Spray system), the solutions were placed into a 10 mL
syringe fitted to a needle with a tip (diameter of
25 gages, inner diameter 0.25 mm), a syringe pump
(lure-lock type, Korea) was set to a constant feeding
rate of 0.5–1.0 mL/h, and a grounded cylindrical stain-
less steel mandrel was used to collect the mat. The elec-
trospinning voltage was directly supplied by a high DC
Linh and Lee
voltage power supply (NNC–30 kV–2 mA portable
type, Korea). A piece of aluminum foil, which was
used as the collector, was located 10 cm from the cap-
illary tip. The electrostatic field was controlled at 22 kV
and electrospinning was performed at room
temperature.
Electrospun PVA mats have been stabilized against
disintegration in water by treatment with methanol.38
In this study, Electrospun PVA/GE mats were stabi-
lized against disintegration in water through treatment
with methanol for several h (8-24 h; 24 h is typical) by
immersing the mats in methanol. Then, the methanol-
treated PVA/GE mat was dried in a ventilated hood at
room temperature overnight.
Characterization
Morphology analysis. The morphology of the electro-
spun fibers and fiber diameters were examined by scan-
ning electron microscopy (SEM, JSM-7401 F). The
diameter of at least 10 different positions on the fiber
mat was measured. A small section of the fiber mat
was placed on the SEM sample holder and sputter
coated with platinum (Cressington 108 Auto). An
accelerating voltage of 15 kV was used to acquire the
SEM images.
Differential scanning calorimetry. DSC has been
widely used to examine the miscibility of polymer
blends and cross-linkable polymers by measuring ther-
mal properties.39 The crystal melting temperature (Tm)
of the membranes was measured using DSC
(METTLER TOLEDO KOREA – DSC822e) with a
sample weight of 3–5 mg under a nitrogen atmosphere.
The samples were heated from 0 to 250 C at a scanning
speed of 10 C/min.
Contact angles measurement. In order to track
changes in water resistant of cross-linked PVA/GE
mat, water contact angles of uncross-linked and cross-
linked PVA/GE nanofibers mat were measured using a
EasyDrop Contact Angle Measuring System (DSA1,
KRUSS GmbH) apparatus. A droplet of water was
placed on the uncross-linked and cross-linked PVA/
GE nanofibers mat located on a sample table. For con-
tact angle measurement, the thin needle for sessile
drops was used. The drop is usually produced at the
tip of the needle and then picked up by turning the
sample table upwards and back downwards again.
The drop is illuminated from one side and a camera
at the opposite side records an image of the drop.
The drop image was transferred to a computer and
shown on the monitor. The DSA software contains
time-prove tools for analyzing the drop image with
whose help it is possible to calculate the contact angle.
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257
Tensile strength. The mechanical properties of the
scaffolds were assessed using tensile tests of thin mem-
branes. In order to use the as-fabricated fibers in the
tensile tests, a method to handle and align the ultrafine
fibers is required. The frame was cut into rectangular
pieces along the vertical lines. The gage length (20 mm)
of the electrospun fibers was determined by the gap
between the parallel strips of the frame. The card-
board partitions were cut along the discontinu-
ous lines before the fiber was stretched. The tensile
strength of the electrospun fibrous membranes was
determined using a universal testing machine
(R&B UNITECH – T, Korea). All samples prepared
from the electrospun fibrous membranes were in the
form of a rectangular shape with dimensions of
width  length ¼ 4 mm  20 mm.35 The thicknesses of
the samples were measured using a digital micrometer
that had a precision of 1 mm. At least five samples were
stretched to failure for each type of electrospun fibrous
membrane. The measurements were taken with a
500 g.f load cell. Load–deformation data were recorded
at a deforming speed of 0.5 mm/s, and the stress–strain
curve of the nano-fibrous structure was constructed
from the load–deformation curve.
Cells and cell culture
MG-63 cells were maintained and suspended in a
humidified incubator at 37 C and a 5% CO2 atmo-
sphere (incubator, ASTEC, Japan) in DMEM supple-
mented with 10% FBS, 2 g/mL of glutamine (Sigma),
100 units of penicillin–streptomycin as an antibiotics,
100 mg/mL streptomycin (Sigma) and 0.25 mg/mL of
fungizone (Bio-Whittaker).
Cell viability assay
Cellular survivability or toxicological evaluation
against cross linked PVA, GE, and PVA/GE blend
has been evaluated by using MTT (3-[4, 5-dimethylthia-
zol-2-yl]-2, 5-diphenyltetrazolium bromide) assay.40
This standard test method is based on exposing the
cells to the fluid extract of the test materials and control
materials. The extract solution of the PVA, GE, or
PVA/GE blend was prepared according to the protocol
as outlined in ISO 10993.41 In brief, to determine the
cytotoxicity of osteoblast-like MG-63 cells on the elec-
trospun samples, samples were incubated with DMEM
media at 37 C for 72 h in a shaking incubator at
100 RPM (Rotation per Minute). The extract solutions
were diluted serially with media (0%, 12.5%, 25%,
50%, and 100%). For the control, 100% media was
used to compare among the dilution, and then 200 mL
of extracted and diluted solution was added in a 96-well
plate. The 96-well plate was previously (24 h before
258
adding the extract solutions) incubated and coated with
osteoblast like MG-63 cells (1  104 cells/well). The
plate was then incubated in a CO2 incubator at 37 C
for 1 day, 3 days, and 5 days. In MTT, assay cell sur-
vivability is measured through the production of purple
colors by the reaction of dehydrogensae enzymes of
living cells and MTT. Metabolically active and viable
cells produce mitochondrial dehydrogenase enzymes
during incubation in media which can be read out
through the change of color intensity by a spectrophot-
ometer. The optical density (OD) corresponds to the
viable cell numbers. Therefore, cell survival and prolif-
eration at the different dilutions and time points were
quantified by adding 100 mL of the MTT solution
(20 mg/100 mL) to each of the wells. After 4 h of incu-
bation, the OD values of the solution were measured
using an ELISA reader (EL, 312, Biokinetics reader,
Bio-Tek instruments) at a wavelength of 595 nm.
Optical microscope study
In order to observe directly whether viable cell numbers
increase after treating the osteoblast like MG-63 cells
with samples extract at day 1, 3, and 5, an inverted light
microscope (Olympus, 1 X 71) attached with LCD
monitor (Samsung) was used. After 70–80% confluent
growth in the subculture flask, cells were trypsinized
(0.25% Trypsin-EDTA), detached, and counted.
Approximately 1  103 cells/mL media was pipetted
into the wells of a microtiter plate containing 24
wells. The plate was then incubated in a CO2 incubator
(5% CO2, 37 C) for 24 h. After seeding cells into the
wells of microtiter plates, media was removed carefully
and replaced with the extract solution of the samples
and the microtiter plate was kept back into the CO2
incubator either for 1 day, 3 days, or 5 days. After
finishing each of the incubation periods, cell growth
in the microtiter plate was observed using an inverted
light microscope.
Immunoblotting
Western blot analysis is one of the most commonly
used methods to evaluate cell proliferation. The cells
were rinsed and harvested using a lysis buffer (Tris
50 mM, pH 7.4, NaCl 40 mM, EDTA 1 mM, Triton
X-100 0.5%, Na3VO4 1.5 mM, NaF 50 mM, sodium
pyrophosphate 10 mM, glycerolphosphate 10 mM,
PMSF 1 mM, Protease inhibitor cocktail 10 mM), vor-
texed, and centrifuged for 10 min at 13,000 rpm at 4 C.
Aliquots (30 mg) of the proteins were analyzed via
Western blotting using a 1:1000 dilution of monoclonal
anti-PCNA. An anti-b actin antibody was used as the
loading control. The immunocomplexes were visualized
using the enhanced chemiluminescence reagent
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Journal of Biomaterials Applications 27(3)
(Amersham) in accordance with the manufacturer’s
instructions.
Cell seeding on the PVA/GE scaffolds
Electrospun nano-fibrous PVA/GE were initially steril-
ized under UV light for 6 h per side, sterilized further
with 70% ethanol (30 min), washed with phosphate-
buffered saline (PBS) and finally soaked in DMEM
for 1 h. MG-63 cells were seeded on 1 mm of the
PVA/GE scaffold (20 mm in diameter), and tissue cul-
ture plate (TCP-control) at a density of 104 cells/cm2 in
24-well plate culture medium with 10% FBS and 1%
PS. The growth behaviors of MG-63 were observed on
1, 3, and 5 days by SEM after seeding on the PVA/GE
scaffolds. For SEM (JSM-5410LV JEOL) observation,
the cells were fixed in 2% glutaraldehyde (v/v) and
dehydrated in ethanol solutions (50%, 70%, 90%,
and 100%). The dehydrated cells were dried in
Hexamethyldisilazane (HMDS, 99%, Sigma) and
coated by gold sputtering.
Statistical analysis
All statistical analyses were performed using SPSS
(Statistical Package for the Social Sciences version 16,
SPSS Inc., USA). Results are expressed as
means  standard error (SE). Student’s t-test was used
to compare among different treatment groups with sig-
nificance assigned at p < 0.05.
Results and discussion
Characterization of composite nanofibers
Surface morphology examination. Thin and regular
PVA/GE fibers were obtained using previously pub-
lished PVA and GE concentrations and electrospinning
parameters.35,37 Figure 1 shows SEM images of the
PVA, GE, and PVA/GE blend prepared by electrospin-
ning and the electrospun PVA/GE cross-linked blend
by methanol. As shown in Figure 1, nano-scale PVA,
GE, and PVA/GE blends were produced with diame-
ters ranging from 50  10 to 250  20 nm. PVA and GE
are water-soluble biopolymers, and they should be
cross-linked to be water-resistant before use as biomed-
ical materials. As shown in Figure 1(d), the nano-fiber
structure of the PVA/GE blend was not altered by
cross-linking and dense fibers were obtained.
Treatment of the PVA/GE fiber mats with methanol
for 24 h stabilized the fibers against disintegration
when in contact with water. This may have occurred
due to the removal of residual water within the fibers by
the alcohol, allowing PVA–water hydrogen bonding
and GE–water amine bonding to be replaced by
Linh and Lee
Figure 1. Appearance of electrospun PVA (a), GE (b), uncross-linked PVA/GE blend (c), and cross-linked PVA/GE blend (d) in SEM
imaging.
intermolecular polymer hydrogen bonding resulting in
additional crystallization. When an electrospun PVA/
GE mat (which is white because of light scattering from
the fibrous structure) was immersed in water, the mat
instantaneously shrunk and beame a clear, gelatinous
material. Thus, the unique nanofibrous structure of the
electrospun material was lost in an aqueous environ-
ment. However, we have found an apparently new
and simple method to physically cross-link electrospun
materials using lower alcohols such as methanol.
Soaking the electrospun PVA/GE mat in methanol
for 24 h preserved the integrity of the mat when it
was immersed in water. Methanol resulted in an effec-
tive way to fabricate PVA/GE water-resistant mem-
brane and make it possible to be used for tissue
engineering scaffolds.
The amount of GE added to the PVA solution was
up to 80%. Acetic acid, which was used as the solvent
for GE, has been shown to reduce the surface tension of
a solution.37 Therefore, the surface tension of the PVA/
GE blend might be decreased with the addition of GE
solution in this study. Because of the effect of surface
tension, the diameter of the PVA/GE nano-fibers was
smaller than the PVA nano-fibers and larger than GE
nano-fibers (as observed by the SEM). The average
diameters of the PVA/GE blends before and after
cross-linking by methanol were 100  10 and
150  10 nm, respectively. Moreover, the nano-fibers
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259
had a fine morphology without the presence of beads.
Because of the nano-size of the PVA/GE scaffolds and
their overall porous structure, the electrospun fibers
had a high, specific surface area, which is beneficial
for tissue engineering applications.
Differential scanning calorimetry. Figure 2 shows the
DSC thermograms of the PVA, GE, and PVA/GE
blend and cross-linked PVA/GE blend prepared by
electrospinning. Two peaks were observed in the DSC
scans of the blend membranes, one corresponds to PVA
(221 C) and the other due to GE (88 C). These results
clearly showed that the blend fibers of PVA and GE
were immiscible in the as-spun state due to the limited
solubility of one in the other. As demonstrated in these
experiments, it is difficult to form miscible polymer
blends using a 99þ% hydrolyzed PVA because of the
high surface tension and high viscosity.42,43 As shown
in Figure 2, the two peaks of the cross-linked PVA/GE
blend increased up to 227 C and 95 C compared with
the uncross-linked PVA/GE blend.
These results clearly indicate that the blend fibers of
PVA and GE were immiscible in the as-spun state due
to the limited solubility of one in the other. At a com-
position of 20% PVA and 80% GE, the methanol treat-
ment promoted the formation of fiber aggregates on the
PVA/GE scaffold. Methanol treatment not only pre-
served the fibrous structure of the PVA/GE mat but
260 Journal of Biomaterials Applications 27(3)

also significantly increased the formation of fiber aggre-
gates on the PVA/GE scaffold. When immersed in
water, methanol treated PVA/GE mats swelled signifi-
cantly, thus softening the material while still maintain-
ing the characteristics of a mechanically stable
hydrogel. Importantly, the electrospun PVA/GE fibers
can be stabilized against disintegration in water by
simply soaking in methanol. It was concluded that
methanol treatment increased the degree of crystallinity
of the PVA/GE fibers, thereby increasing the number of
physical crosslinks responsible for fiber stabilization in
water.
The methanol treated mat appeared to retain a sig-
nificant degree of its fibrous character. Without meth-
anol treatment, even though the PVA/GE electrospun
1 PVA/GE blend was measured to be 2.2  0.3 . Both
PVA
GE
PVA/GE blend
0 Crosslinked PVA/GE blend
–1
–2
Endo heat flow
mat does not dissolve in water, it completely lost its
mechanical integrity, forming a soft, gelatinous mass.
In contrast, the water-swollen, methanol-treated PVA/
GE mat remained. These observations can be verified
by examining individual fibers using a light microscope.
Thereby, we confirmed that methanol can be used as
a physical cross-linking agent for PVA/GE nano-fibers.
In addition, the GE component of the PVA/GE scaf-
fold was gradually dissolved during cell culture, which
created more space for cell migration because of the
immiscibility of PVA and GE.
Contact angle measurement. The specific contact
angle for both uncross-linked and cross-linked PVA/
GE is shown in Figure 3 to observe and predict how
water dissolute on the surfaces before and after cross
linking of the mat. The contact angle of uncross-linked
PVA and GE are very soluble to water and their phys-
ical blending by electrospinning did not alter the solu-
bility in water so much. This might be because of the
poor aggregation of the fibers and high hydrophilicity
of the mat. However, after cross-linking with methanol
treatment, the solubility of the mat in water decreased
and the contact angle increased dramatically. The con-

tact angle of the cross-linked PVA/GE was found to be

–3 52.5  0.5 . From scanning electron images, it was
found that after cross-linking, fibers become more
dense and aggregative (Figure 1). Also the surface
–4
area of the mat increased after cross linking.
Therefore more air might be trapped in the cross
–5 linked fiber mat than the uncross-linked fiber which
at the same time contributes to decrease the actual con-
–6 tact area of a water droplet on a cross-linked mat. The
–50 0 50 100 150 200 250 300 high contact angle of the cross-linked PVA/GE blend
Temperature (°C) made the fiber mat less hydrophilic than that of the
uncross-linked PVA/GE.
Figure 2. Thermal behaviors of electrospun PVA, GE, uncross-
linked PVA/GE blend, and cross-linked PVA/GE blend in DSC Tensile strength. A representative stress–strain curve
measurements. was constructed from the load–deformation curve and

Figure 3. Water contact angle images of uncross-linked PVA/GE (a) and cross-linked PVA/GE (b).

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Linh and Lee 261

is illustrated in Figure 4. In these experiments, the
blending of PVA and GE was shown to result in an
increasing stress compared with PVA alone and
increasing strain compared with GE alone. On the
other hand, the toughness of PVA may have compen-
sated for the brittle characteristics of GE. The tensile
modulus of the uncross-linked and cross-linked PVA/
GE nano-fibers were around 3.6 and 4.2 MPa, respec-
tively, and the tensile strains were 19% and 31%,
respectively. Clearly, using methanol for cross-linking,
improved the mechanical properties of the PVA/GE
blends. The data in Figure 4 indicates that the tensile
strain of the cross-linked PVA/GE nanofibers was suf-
ficient to support proliferation and mineralization of
osteoblast cells.
PVA
GE
6 PVA/GE before crosslinking
PVA/GE after crosslinking
4 Cell viability on nano-fibers. The MTT assay, which is
Stress (MPa)
Yao et al reported that the mechanical strength of
the PVA mat was increased due to increased crystallin-
ity following post-spinning treatment with methanol.
By removing residual water within the fibers by the
alcohol treatment, the degree of crytallinity was
increased, which in turn increased the physical cross-
links and mechanical properties, making the fiber mat
stable from disintegration in water.38 As the cross-link-
ing by methanol in PVA/GE blend was found to pro-
duce a stable scaffold system in water, there is the
possibility that high crystallinity might result in high
mechanical properties. Yang et al. reported that the
tensile strength of 100% PVA and 10% PVA was
11.6 MPa and 2.4 MPa, respectively.25 However, in
this study, a simple cross-linking step resulted in a ten-
sile strength of 4.20  0.40 MPa (Table 1). Moreover,
for bone applications, multiple mat layers should be
hot pressed together, which is assumed to further
increase the mechanical properties of the scaffold
system.
Biocompatibility and bioactivity of MG-63 on
PVA/GE scaffold
a rapid, standardized, sensitive, and inexpensive

method to determine cellular viability and proliferation

or whether a material contains significant quantities of
2
biologically harmful extracts, is the first step used to
screen the biocompatibility of a biomaterial. The
effect of PVA, GE, and cross-linked PVA/GE nano-
0
fibers on cell viability (cytotoxicity) and proliferation
of MG-63 were examined using the MTT assay,
which is a colorimetric assay that measures the meta-
bolic activity of viable cells. The cytotoxictities of
–2 MG-63 on the electrospun PVA, GE, and cross-
0 20 40 60 80 100 120 linked PVA/GE are shown in Figure 5. Based on the
Strain (%) cytotoxicity results, a fairly acceptable viability of the
cells was observed on GE (>90% at 100% extract solu-
Figure 4. Mechanical properties of electrospun PVA, GE, tion) and PVA/GE (>85% at 100% extract solution)
uncross-linked PVA/GE blend, and cross-linked PVA/GE blend
while PVA membrane showed a slightly lower viability
in tensile strength tests.
(70% at 100% extract solution). Although the

Table 1. Properties of electrospun PVA, GE, uncross-linked and

cross-linked PVA/GE fibrous membranes.

Average diameter Ultimate Straina,* Water contact

of fibers* (nm) strength* (MPa) (%) angle (degree)*

PVA 250  20 0.85  0.60 98.0  5.0 –

GE 50  10 3.70  0.50 15.0  3.0 –
PVA/GE 100  10 3.55  0.50 19.0  3.0 2.2  0.3
Cross-linked PVA/GE 150  10 4.20  0.40 31.0  5.0 52.5  0.5
a
At ultimate strength.
*All data are expressed as mean  standard deviation.

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262
120
PVA
100
80
Cell viability (%)
60
40
20
0
0 12.5
Figure 5. Cell viability of electrospun PVA, GE, and cross-linked PVA/GE blend. The MTT assay was used to measure the viability
of MG-63 cells at various dilute extract solution concentrations. Media at 100% was used as a CONTROL. Standard errors were
expressed as bar diagram.
biocompatibility of PVA was low, the general survival
rate of the cells for PVA/GE scaffold was satisfactory;
therefore, the PVA/GE scaffolds were not toxic to the
MG-63 cells and could be used for tissue engineering
scaffolds.
Proliferation of osteoblast cells MG-63 on PVA/GE
blend nano-fibers. The proliferation of osteoblasts
on PVA, GE, and cross-linked PVA/GE blend was
evaluated using MTT assay after 1, 3, and 5 day(s) of
incubation as shown in Figure 6. A significantly higher
cell proliferation (day 5) in GE and PVA/GE blend
compared to PVA was exhibited in Figure 6. After
1 day, the number of cells had exceeded the number
of cells initially seeded (104 cells/well). As clearly
shown in Figure 6, the number of osteoblasts that
grew was greater than the number of cells initially
seeded even after only 3 days in culture. From 1 day
to 5 days in culture, the MG-63 cells continually pro-
liferated on including the cross-linked PVA/GE.
Cellular viability assay by MTT was used to find out
the percentage of viable cell numbers per extract dilu-
tions after certain periods. However, to observe the
increased cell numbers and their real time morpholo-
gies, light microscopic studies were conducted. Figure 7
shows the light microscopic images of cells treated with
extract solutions for day 1, 3, and 5. At day 1, only a
little amount of cells were observed. But the amount of
cell number was found increasing gradually at day 3
and day 5 for all samples. Especially, at day 5, cellular
growth was almost confluent. However, in case of
cross-linked PVA/GE, cellular growth pattern was
better than that of the uncross-linked PVA/GE. In
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Journal of Biomaterials Applications 27(3)
GE Uncross PVA/GE Crosslinked PVA/GE
25 50 100
Dilute extract solution (%)
fact, this result corresponds with the cell viability and
proliferation results very well. Cell morphology was
also found very well with typical spindle shape of oste-
oblasts with an average diameter of 10–20 mm.
In order to determine on the molecular scale if the
MG-63 cells were proliferating on the scaffolds, the
proliferating cell nuclear antigen (PCNA), which is a
marker for the S phase (synthesis phase) of the cell
cycle,44 was used as a marker for cell proliferation in
this study (Figure 8). Because the synthesis phase in the
cell cycle is related to cell proliferation, an increase in
the level of protein expression can be used as a measure
of the status of DNA synthesis in the S phase of cell
cycle.44 The expression of PCNA was evaluated using
western blot method after collecting total proteins from
lysed cells at day 1, 3, and 5. The PCNA protein bands
were found to significantly decrease in case of the PVA
alone (Figure 8); PVA, Lane 0–5). The expression of
this protein in GE was similar throughout 1, 3, and 5
days of cell culture. However, for the PVA/GE cross-
linked scaffold, the intensity of the band slightly
decreased at day 5, suggesting that cross-linked scaffold
promoted cell proliferation by reducing the level of tox-
icity of PVA alone or in other words, the addition of
GE was beneficial to the scaffold system containing
PVA. This result is in agreement with the MTT assay
where it was shown that cells on the PVA/GE cross-
linked scaffold proliferated better than that on the PVA
(Figure 6).
Osteoblast seeding on PVA/GE scaffolds. Various
pore diameters were formed within the nano-fiber struc-
tures and the interconnected porous structure provided
Linh and Lee 263

3.5
Control
3 PVA
GE
2.5

OD absorbance
Uncross PVA/GE
2 Cross linked PVA/GE

1.5

0.5

0
0 1 3 5

Figure 6. Osteoblast cellular proliferation of electrospun PVA, GE, and cross-linked PVA/GE blend after 1, 3, and 5 days using the
MTT assay. Standard errors were expressed in the bar diagram. Media at 100% was used as a CONTROL and Optical Density (OD)
corresponded to the cell survivability after 1, 3, and 5 days.

Figure 7. Osteoblast cellular proliferation of electrospun PVA/GE, and cross-linked PVA/GE blend after 1 (a, d), 3 (b, e), and 5 (c, f)
days using the optical microscope observation.

scaffolds. Both PVA and GE are hydrophilic polymers,

GE PVA/GE PVA
0 1 3 5 0 1 3 5 0 1 3 5
Figure 8. Identification of PCNA (Proliferating Cell Nuclear
Antigen) at 1, 3, and 5 days by Western Blot Analysis.
a large surface area for cell residence and proliferation,
and effectively simplified the exchange of nutrients
between the scaffold environments. Osteoblasts cul-
tured on composite nano-fibrous scaffolds showed
higher proliferation rates and formed a multilayer of
cells on the surface of the composite nano-fibrous
which is a beneficial attribute for cell attachment. The
days surface topography of nano-fibers plays an important
role in cell behavior, including cell adhesion.45 It is
IB: PCNA known that human cells can attach and organize them-
selves well around fibers with diameters smaller than
IB: b actin
those of the cells.46 This might explain the behavior
of the MG-cells on the combined scaffolds, which had
a nano-scale structure.
Figure 9 shows the confluent growth of the MG-63
osteoblast-like cell line on the PVA/GE scaffolds and
controlled substrate 1, 3, and 5 days after seeding.
These scaffolds provided a surface for cell residence,
interaction, and growth. After 1 day of culture, the
MG-63 cells tended to adopt a normal phenotypic
shape and adhered according to the nano-fiber orienta-
tion. Cell shape was observed to be round to oval with

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264
Figure 9. Morphology of MG-63 cells on the tissue control plate and on cross-linked PVA/GE membrane after 1 (a,d), 3 (b,e), and
5 (c,f) days seeding in SEM imaging.
philopotial extension. After 3 days of culture, the cells
exhibited a flat morphology over the membrane and
integrated with the surrounding fibers. After 5 days of
culture, the cell number reached a plateau, possibly
because the cells may have occupied all of the available
spaces on the prepared scaffold. The number of cells
seemed to grow slowly after 5 days of seeding due to
the degradation of the hydrophilic PVA/GE. As PVA/
GE degraded the fiber structure inside and on the sur-
face of the scaffold dissolved, retarding cell growth.
Overall, this result indicates that nano-fibrous PVA/
GE positively promoted cell–matrix and cell–cell
interactions.
Conclusion
PVA/GE fibers were produced using the electrospin-
ning technique. A cross-linking treatment with metha-
nol was used to decrease the dissolution of the fibers
in water. The mechanical tests showed that the fibers
had a high enough tensile strength for using as scaffolds
in tissue engineering applications. The cytotoxicity
assays confirmed that the composite materials were
nontoxic. Fibers could also be formed into three-
dimensional structures, which have the potential
for use as tissue engineering scaffolds. We also found
that the mesh structures of the scaffold were suitable for
cell growth. This was confirmed by the results of the
in vitro cell culture studies. In addition, osteoblasts
that grew over the scaffold surfaces exhibited the
appropriate morphology and displayed good
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Journal of Biomaterials Applications 27(3)
proliferation, suggesting that the developed scaf-
folds might be used for bone tissue engineering
applications.
Acknowledgement
We are grateful for the support by the National Research
Foundation of Korea (NRF) grant funded by the ea govern-
ment (MEST) (No. 2009 - 0092808).
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