Journal of Biomaterials and Nanobiotechnology, 2011, 2, 353-360
doi:10.4236/jbnb.2011.24044 Published Online October 2011 (http://www.SciRP.org/journal/jbnb)
353
Cells Attachment Property of PVA Hydrogel
Nanofibers Incorporating Hyaluronic Acid for
Tissue Engineering
Kyu-Oh Kim1, Yaeko Akada2, Wei Kai2, Byoung-Suhk Kim2, Ick-Soo Kim2
1Department of Bioscience and Textile Technology, Shinshu University, Nagano, Japan; 2Nano Fusion Technology Research Group,
Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
Received May 20th, 2011; revised June 20th, 2011; accepted July 20th, 2011.
ABSTRACT
In this work, we report the fabrication and cell affinity studies of the poly (vinyl alcohol) (PVA)/hyaluronic acid (HA)
cross-linked nanofibers via electrospinning and post cross-linking. FT-IR and TGA analysis demonstrate that HA is not
influenced by acid environment such as HCl vapor during cross-linking, and well incorporated into PVA nanofibers.
Swelling behavior and cell adhesion of the PVA/HA hydrogel nanofibers are investigated and compared with pure PVA
hydrogel nanofibers. It is expected that the nanofibrous PVA/HA hydrogel fibers could be a promising scaffold for cell
culture and tissue engineering applications.
Keywords: Nanofiber, Electrospinning, Hydrogel, Poly(Vinyl Alcohol), Hyaluronic Acid, Cell Affinity
1. Introduction
Tissue-engineered skin has enormous potential that is
only just beginning to be realized and has delivered con-
siderable benefits to patients with chronic wounds. A
number of people need skin grafts due to dermal wounds
caused by fire, heat, electricity, chemicals, ultraviolet,
nuclear energy, or diseases. In the case of wounds that
extend entirely through the dermis, such as full-thickness
burns or deep ulcers, many skin substitutes such as xeno-
graft, allograft, isograft and autograft have been em-
ployed for wound healing. However, these approaches
have disadvantages, which are high cost, the limited
availability of skin grafts in severely burned patients, and
problems of disease transmission and immune response
[1-3]. Currently, various carrier dressing of skin repair
product have being explored to improve factors such as
ease of use, cost, transportation and variables for both the
clinician and the patients for achieving skin repair made
into bovine collagen [4], thin polymer containing acid
functional groups using the plasma polymerization [5].
As the other remedy, the nanofiber technology have great
potential of this skin dressing by advantages of the nano-
fiber such as similar morphology to a natural extracellu-
lar matrix (ECM), high surface-area, excellent air-perme-
ability and low cost. To make the electrospun hydrogel
nanofibers as a wound dressing, we used two polymers,
hyaluronic acid (HA) and poly (vinyl alcohol) (PVA).
First, HA having the unique hygroscopy could play a
bioactive role in wound healing by creating a swollen
macro- and micro-environment [6,7]. PVA was known to
get excellent biological properties, good physical pro-
perty as well as to form the hydrogel by chemical gela-
tion via γ-ray or electron-beam irradiation [8,9] using
glutaraldehyde (GA) as a cross-linker and by physical
gelation via freezing-thawing cycles [10]. However, the
fabrication of PVA cross-linked nanofibers via electro-
spinning process has a difficulty due to rapidly increase
in solution viscosity, if GA agent is used [11]. This dis-
advantage caused to be poor electrospinnability and was
also hard to control. To overcome these problems, in this
study, we use post cross-linking method of the elec-
trospun PVA/HA nanofibers using GA and HCl as the
cross-linker and catalyst, respectively, and report en-
hanced bioactivity of the resultant PVA/HA hydrogel
nanofibers, compared to pure PVA hydrogel nanofibers.
The motivation of this study is to develop and optimize
the novel PVA/HA hydrogel nanofibers. The cell culture
using MC3T3-E1 is also conducted to assess the viability
and potential application of this material as a scaffold.

Cells Attachment Property of PVA Hydrogel Nanofibers Incorporating Hyaluronic Acid for Tissue Engineering 354
2. Experimental Section
2.1. Materials
Poly (vinyl alcohol) (PVA) (degree of hydrolysis = 88%,
degree of polymerization (DP)  1700) was obtained
from Kuraray Co. Ltd., Japan. Glutaraldehyde (GA, 50%
in water) was purchased from Sigma-Aldrich, USA.
Hyaluronic acid (HA, SHANDONG FREDA BIOPH-
ARM Co., China) was used as received. Hydrochloric
acid (purity, 37%) and ethanol (purity, 99.5%) were pur-
chased from Wako Chem. Co., Japan. All materials were
used as-received without further purification.
2.2. Electrospinning
A high-voltage power supply (Har-100*12, Matsusada
Co., Japan), capable of generating voltages up to 100 kV,
was used as a source of electric filed. The pure PVA and
PVA/HA solutions were supplied through a 5 ml plastic
syringe attached to a capillary tip with an inner diameter
of 0.6 mm. The copper wire connected to a positive elec-
trode (anode) was inserted into each polymer solution,
and a negative electrode (cathode) was attached to a me-
tallic collector. The applied voltage was fixed at 14 kV.
The distance between the capillary tip and the collector
was fixed to be 15 cm. All solutions were electrospun
onto a rotating metallic collector at room temperature
under identical conditions.
2.3. Cross-Linking of Electrospun PVA and
PVA/HA Nanofibers
In order to achieve the cross-linking of PVA nanofibers,
GA as a cross-linker was added to both pure PVA and
PVA/HA solutions, respectively. The PVA/GA solutions
(molar ratios of GA and PVA were 3.4/1.0, 6.9/1.0 and
10.3/1.0) were dissolved in distilled water at 50˚C over-
night. On the other hand, the PVA/HA/GA solutions
(molar ratios of GA and PVA were 8.2/1.0, 16.5/1.0 and
24.8/1.0) in the presence of HA were dissolved in the
mixed water/ethanol (9/1, v/v) solvent at 50˚C overnight
to improve the solubility HA in PVA/GA solution. Here,
the concentration of PVA solution was fixed at 12 wt%.
The weight ratio of PVA and HA was 5/1. Afterwards,
the obtained electrospun nanofibers were exposed to HCl
vapor, which was produced in the desiccators at 30˚C.
The exposure time (10 s, 30 s and 60 s) of acid treatment
and molar ratio of GA and PVA were varied. After
cross-linking, the samples were found to shrink slightly
during the treatment of HCl vapor, and used carefully
after vacuum-drying at room temperature.
2.4. Characterization
To identify the cross-linking of PVA/GA and the incor-
poration of HA, FT-IR analysis was carried out using an
IRPrestige-21 (Shimadzu Co., Japan). In order to deter-
mine the amount of HA presenting in the cross-linked
PVA/HA nanofibers, thermogravimetric analysis (TGA)
(TG/DTA6200, Seiko Instruments Inc. Japan) was car-
ried out by heating from room temperature to 600˚C un-
der a continuous nitrogen purge of 20 mL/min. The mor-
phologies of electrospun PVA/HA hydrogel nanofibers
were characterized using scanning electron microscopy
(SEM, S-3000N HITACHI, Japan) on samples sputtered
with Pd-Pt. The fiber diameter and its distribution were
determined by using image J (ImageJ v1.41, Wayne
Rasband National institutes of Health, USA) software.
2.5. Swelling Behavior
After measuring the initial size and weight, dried elec-
trospun fibrous membranes were immersed into distilled
water for 5 days at room temperature to attain an equilib-
rium swelling state. Before measuring the size and
weight of the swollen samples, the excess amounts of
waters onto the surface were carefully removed by blot-
ting with filter paper. The swelling ratio was calculated
as follows [12]:

Swelling ratio
s
d
d
WW
gg W

 (1)
where Ws and Wd are the weights of swollen and dried
samples, respectively.
2.6. Cell Culture
The pure PVA and PVA/HA hydrogel nanofibers (col-
lected on round cover glass slips of 15mm in diameter)
were immersed in 80% ethanol for 2 hrs for sterilization
purposes. The hydrogel nanofibers were washed with
phosphate buffered saline (PBS) thrice followed by cul-
ture medium thrice to eliminate any residual ethanol.
MC3T3-E1 osteoblast-like cells, which were obtained
from the RIKEN Cell Bank (Tsukuba, Japan), were cul-
tured until passage 7 and seeded on pure PVA and PVA/
HA hydrogel nanofibers at a cell concentration of 3 × 104
cells/well. Cells were incubated at 37˚C, in a 5% CO2
atmosphere incubator, using α-modified minimal essen-
tial medium (α-MEM; GIBCO). The medium comprised
of 10% heat-inactivated fetal bovine serum (FBS), 100
U/ml penicillin, 100 U/ml streptomycin and 0.1% β-gly-
cerophosphate was used to induce osteoblastic differen-
tiation. For all cell investigations, cells cultured on TCDs
(tissue culture dishes, high-grade polystyrene Nunc™
Dishes, Thermo Fisher Scientific, Denmark) were evalu-
ated as controls. The medium was changed every two
days to ensure that there was an adequate supply of nu-
trients present in the culture plate. The hydrogel nanofi-
brous scaffolds, which were inoculated with MC3T3-E1
(cell culture for 1, 6 and 24 hrs), were fixed in 10% for-
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Cells Attachment Property of PVA Hydrogel Nanofibers Incorporating Hyaluronic Acid for Tissue Engineering 355
maldehyde solution overnight at 4˚C. Next, the scaffolds
were dehydrated in a series of 5 cycles (1 cycle: 5 min)
with increasing concentrations of ethanol: 50%, 60%,
70%, 80%, 90% and 100% (twice) respectively. Finally,
the samples were freeze-dried overnight and scanning
electron microscopy (SEM, S-3000N HITACHI, Japan)
analysis was done to view the morphology of the at-
tached cells on the nanofiber scaffolds.
reduced, compared with pure PVA nanofibers, indicating
the successful cross-linking reaction between –OH group
in PVA and –CHO in GA. Furthermore, assuming that
the C=O contents, whose intensity do not change from
the region between 1788 - 1639 cm−1 during cross-linking
reaction, are set to constant intensity [13], it was esti-
mated that loss contents of hydroxyl group in the PVA
cross-linked nanofibers (molar ratio of GA and PVA ~
10.3:1.0) was about 5.84%, which was ascribed to a pos-
sible formation of acetal bridges from C-H stretching
related to aldehyde group [14]. In addition, in case of the
PVA/HA cross-linked nanofibers, N-H bending was
clearly appeared at near 1635 cm−1, suggesting that HA
was not influenced by acid environment such as HCl
vapor during cross-linking.
2.7. Cell Adhesive
Cells were seeded and cultured as under the same con-
ditions in “Cytotoxicity assay and live/dead cell staining”
section. Cells in culture medium were counted (Nm) af-
ter 1, 3, 6, and 24 hrs of incubation. The cell adhesion
ratio for each condition was calculated using the follow-
ing equation: 3.2. TGA Study

4
Adhesionratio (%)13.010100Nm (2) Figure 2 shows TGA curves of pure PVA nanofibers,
pure PVA cross-linked nanofibers (molar ratio of GA
and PVA ~ 10.3:1.0), and PVA/HA cross-linked nanofi-
bers (molar ratio of GA and PVA ~ 24.8:1.0). Firstly,
pure PVA showed three-step degradation behaviors. That
is, the first degradation step below 150˚C was aware of
the removal of moisture, physisorbed, and chemisorbed
water molecules. The second degradation step was asso-
ciated with the degradation of PVA backbond. The third
degradation step around 600˚C was depicted to the deg-
radation of vinyl acetate group of PVA. Majority weight
loss of pure PVA nanofibers was occurred around 300˚C,
while pure PVA cross-linked nanofibers showed higher
All data reported were the mean of three examinations.
3. Results and Discussion
3.1. FT-IR Study
Figure 1 shows FT-IR spectra of pure PVA nanofibers,
pure PVA cross-linked nanofibers (molar ratio of GA
and PVA ~ 10.3:1.0), and PVA/HA cross-linked nanofi-
bers (molar ratio of GA and PVA ~ 24.8:1.0). All spectra
show characteristic PVA bands at hydroxyl group (3300
cm-1), alkyl group (2906 - 2908 cm-1) and acetyl group
(1730 cm-1), respectively. After cross-linking, the inten-
sity of hydroxyl group at 3685-3000 cm-1 was relatively
Figure 1. FT-IR spectra of pure PVA nanofibers, pure PVA cross-linked nanofibers (molar ratio of GA and PVA ~ 10.3:1.0),
and PVA/HA cross-linked nanofibers (molar ratio of GA and PVA ~ 24.8:1.0). The concentration of PVA solution is 12 wt%.

Cells Attachment Property of PVA Hydrogel Nanofibers Incorporating Hyaluronic Acid for Tissue Engineering 356
Figure 2. TGA curves of pure PVA nanofibers, pure PVA
cross-linked nanofibers (molar ratio of GA and PVA ~
10.3:1.0), and PVA/HA cross-linked nanofiber s (molar ratio
of GA and PVA ~ 24.8:1.0). The concentration of PVA solu-
tion is 12 wt%.
thermal stability than pure PVA due to cross-linking [15].
Moreover, the PVA/HA cross-linked nanofibers exhi-
bited weight loss of 4% at about 215˚C by the decompo-
sition of hyaluronic acid, suggesting that HA was well
incorporated into the PVA cross-linked nanofibers. The
weight residue of pure PVA nanofibers around 600˚C
was ca. 5.1% due to the various carbonaceous matters,
whereas both pure PVA and PVA/HA cross-linked nano-
fibers showed much higher char values of 12.2% and
17.0% than that of pure PVA, respectively, due to the
alkyl group of GA.
3.3. Morphologies
Electrospinnability was strongly influenced by solvents
and solution viscosity during electrospinning process
[16-21]. Here, mixed solvent (water/ethanol = 9/1, v/v)
was used in order to improve the solubility and the cor-
responding electrospinning of the PVA/HA solution.
Figure 3 shows SEM images of pure PVA (a), PVA/GA
(b), PVA/HA (c), and PVA/HA/GA (d) electrospun na-
nofibers before cross-linking. As evidenced by SEM ana-
lysis, the fiber diameters were regular with an average
diameter of approximately 430 ± 50 nm, 400 ± 60 nm,
200 ± 50 nm, 180 ± 50 nm, and its distribution was nar-
rower. It can be seen that GA did not influence the di-
ameters of the resulting nanofibers, whereas the incor-
poration of HA (PVA/HA and PVA/HA/GA) resulted in
smaller diameters than that of pure PVA nanofiber due to
an enhanced conductivity by hyaluronic acid. Further-
more, the investigation of the fiber morphologies and the
pores in aqueous media are important for applications as
wound dressings.
After cross-linking, we have also studied the changes
in the morphologies and swelling behavior of both pure
PVA and PVA/HA cross-linked nanofibers after im-
mersing in distilled water for 72 hrs. As seen in Figures
4 and 5, it was observed that the fiber morphologies of
Figure 3. SEM images of pure PVA (a), PVA/GA (b), molar
ratio of GA and PVA ~ 10.3:1.0), PVA/HA (c), and PVA/
HA/GA (d, molar ratio of GA and PVA ~ 24.8:1.0) nanofi-
bers before cross-linking. The concentration of PVA solu-
tion is 12 wt%.
Figure 4. SEM images of PVA hydrogel nanofibers with
different cross-linking time (a, at constant molar ratios of
GA and PVA of 10.3:1.0) and various molar ratios of GA
and PVA (b, at constant soaking time of 30 s) before and
after soaking in distilled water for 3 days.
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Cells Attachment Property of PVA Hydrogel Nanofibers Incorporating Hyaluronic Acid for Tissue Engineering357
Figure 5. SEM images of PVA/HA hydrogel nanofibers with
different cross-linking time (a, at constant molar ratios of
GA and PVA of 24.8:1.0) and various molar ratios of GA
and PVA (b, at constant soaking time of 30 s) before and
after soaking in distilled water for 3 days.
pure PVA and PVA/HA hydrogel nanofibers soaked in
distilled water was strikingly different depending on the
cross-linking time and the GA concentration (that is, the
cross-linking density). The optimum cross-linking time
and GA concentration on both pure PVA and PVA/HA
hydrogel nanofibers were 30 sec and 30 m (corre-
sponding to molar ratio of GA and PVA ~ 16.5:1.0), re-
spectively, showing clear fibrous morphologies. Obvi-
ously, shorter cross-linking time and lesser GA concen-
tration did not maintain fiber morphologies after soaking
in distilled water and then resulted in fused film-like
morphologies, due to an incomplete cross-linking.
3.4. Swelling Behavior
Hydrogels are soft and wet and look like a solid material,
but are capable of undergoing large deformation [22,23],
and thus are often used as swelling controlled-release
devices, for which absorption of water leads to polymer
expansion, which directly affects the diffusivity of drugs.
The degree of swelling is related to the ability of solute
molecules to move through the gels networks and the
diffusion of fluid in the polymer as well as its macromo-
lecular relaxation [24]. Figure 6 shows swelling ratio of
pure PVA and PVA/HA hydrogel nanofibers as a func-
tion of soaking time at room temperature. In principle,
the swelling extent of hydrogels is determined by a ba-
Figure 6. Swelling ratio of pure PVA hydrogel (molar ratio
of GA and PVA ~ 10.3:1.0) and PVA/HA hydrogel (molar
ratio of GA and PVA ~ 24.8:1.0) nanofibers as a function of
soaking time at room temperature. The concentration of
PVA solution is 12 wt%.
lance between the driving force of an exothermic mixing
enthalpy (ΔHswell < 0), resulting from favorable interact
tions between hydrophilic groups and water molecules,
and a mediating loss of polymer chain conformation en-
tropy (ΔSswell < 0) during the same swelling. This beha-
vior is well described by Flory-Rehner theory [25]. As
seen in Figure 6, the pure PVA and the PVA/HA hy-
drogel nanofibers reached swelling ratios of ca. 6.2 and
ca. 7.2 at the soaking time of 1 hr, indicating a fast
swelling via a capillary force due to the nanoporous
structures, and then gradually increased as increasing
soaking time. Moreover, the swelling ratio of the PVA/
HA hydrogel nanofibers showed always higher values
than those of pure PVA hydrogel nanofibers because of a
strong hydrophilic HA. We could also see that the hy-
drogel nanofibers became transparent after swelling (see
inset in Figure 6), indicating the water molecules dif-
fusing into the sample to hydrate the polar/hydrophilic
groups, leading to volumetric expansion that comes to an
equilibrium swelling ratio. Evidently, the size of swollen
sample also became larger, compared to the dried sam-
ple.
3.5. Cell Adhesion and Its Morphology
HA can directly communicate with proteins and cells
present in tissues if HA is contained onto the surface of
matrix well [26-29]. The adhesion ratio of MC3T3-E1
cells [30] on the pure PVA and PVA/HA hydrogel nano-
fibers was shown in Figure 7. Comparison of initial ad-
hesion ratio (at 0 hr) for the PVA hydrogel nanofibers in
the presence and absence of HA has probed that HA
containing PVA hydrogel nanofibers showed relatively
higher adhesion ratio, indicating that the PVA/HA hy-

Cells Attachment Property of PVA Hydrogel Nanofibers Incorporating Hyaluronic Acid for Tissue Engineering 358
drogel nanofibers could acquire excellent interfacial
biocompatibility. Moreover, they quickly reached as high
as about 100% after 6 hrs’ cultivation without difference.
For cell spreading study, Figure 8 shows SEM images of
MC3T3-E1 grown onto TCD, pure PVA and PVA/HA
hydrogel nanofibers with various cell-culture times. It
clearly appeared that MC3T3-E1 cells were well adhered
to the PVA/HA hydrogel nanofibers unlike pure PVA
hydrogel nanofibers, which is also well coincided with
the results of adhesion ratio, probably due to the remark-
able lower toxicity of HA. Also, it could be obviously
seen that vigorous cell migration occurred on PVA/HA
hydrogel nanofibers and a number of microvilli on the
surfaces of nanofiber webs can be observed due to pro-
bably specific interactions between HA and cells. After
Figure 7. Adhesion ratio of MC3T3-E1 cells cultured on
TCD, pure PVA hydrogel (molar ratio of GA and PVA ~
10.3:1.0), and PVA/HA hydrogel (molar ratio of GA and
PVA ~ 24.8:1.0) nanofibers. The concentration of PVA so-
lution is 12 wt%.
Figure 8. SEM images of MC3T3-E1 cells cultured on TCD,
pure PVA hydrogel (molar ratio of GA and PVA ~ 10.3:1.0),
and PVA/HA hydrogel (molar ratio of GA and PVA ~
24.8:1.0) nanofibers as a function of cell-culture time. The
concentration of PVA solution is 12 wt%. The scale bar
indicates 100 nm.
the cell culture for 24 hr, in particular, MC3T3-E1 cells
grown onto PVA/HA hydrogel nanofibers became huge
cells via cell-cell interactions, suggesting an enhanced
cell migration for cell organization.
4. Conclusions
We have successfully prepared the pure PVA and PVA/
HA cross-linked nanofibers via electrospinning and post
cross-linking. FT-IR and TGA analysis demonstrated
that HA was not influenced by acid environment such as
HCl vapor during cross-linking, and also well incorpo-
rated into PVA nanofibers. As evidenced by SEM analy-
sis, the fiber diameters of the pure PVA, PVA/GA, PVA/
HA, PVA/HA/HA electrospun nanofibers before cross-
linking were regular with an average diameter of appro-
ximately 430 ± 50 nm, 400 ± 60 nm, 200 ± 50 nm, 180 ±
50 nm, respectively. It was found that the fiber mor-
phologies of pure PVA and PVA/HA hydrogel nanofi-
bers soaked in distilled water was strikingly different
depending on the cross-linking time and the GA concen-
tration (that is, the cross-linking density). The pure PVA
and the PVA/HA hydrogel nanofibers reached swelling
ratios of ca. 6.2 and ca. 7.2 at the soaking time of 1 hr,
indicating a fast swelling via a capillary force. The swell-
ing ratio of the PVA/HA hydrogel nanofibers showed
always higher values than those of pure PVA hydrogel
nanofibers because of a strong hydrophilic HA. Com-
parison of initial adhesion ratio (at 0 hr) for the PVA
hydrogel nanofibers in the presence and absence of HA
has probed that HA containing PVA hydrogel nanofibers
showed relatively higher adhesion ratio, indicating that
the PVA/HA hydrogel nanofibers have an excellent in-
terfacial biocompatibility.
5. Acknowledgements
The authors acknowledge the support of Shinshu Univer-
sity Global COE Program “International Center of Ex-
cellence on Fiber Engineering”.
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