Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Epidermal cellular response to poly(vinyl alcohol) nanobers containing

silver nanoparticles
Ja Young Chun a,1 , Hyun Ki Kang b,1 , Lim Jeong a , Yun Ok Kang a , Ju-Eun Oh b , In-Sung Yeo c ,
Sung Youn Jung b , Won Ho Park a, , Byung-Moo Min b,
a

Department of Advance Organic Materials and Textile System Engineering, and BK21 FTIT, Chungnam National University, Daejeon 305-764, South Korea
Department of Oral Biochemistry and Program of Craniomaxillofacial Reconstruction Science, Dental Research Institute, BK21 CLS, and IBEC,
Seoul National University School of Dentistry, Seoul 110-749, South Korea
c
Department of Prosthodontics, Seoul National University School of Dentistry, Seoul 110-749, South Korea
b

a r t i c l e

i n f o

Article history:
Received 9 November 2009
Received in revised form 1 February 2010
Accepted 22 March 2010
Available online 29 March 2010
Keywords:
Poly(vinyl alcohol)
Nanobers
Silver
Nanoparticles
Ions
Epidermal cells
Cytotoxicity

a b s t r a c t
A heat-treated PVA nanobrous matrix containing silver (Ag) was prepared by electrospinning an aqueous
10 wt% PVA solution and followed by heat treatment at 150 C for 10 min. The average diameter of the asspun and heat-treated PVA nanobers was 330 nm. The heat-treated PVA nanobrous matrix containing
Ag was irradiated with UV light to transform the Ag ions in the nanobrous matrix into Ag nanoparticles.
The in vitro cytotoxicity of the Ag ions and/or nanoparticles on normal human epidermal keratinocytes
(NHEK) and broblasts (NHEF) cultures was examined. The PVA nanobrous matrix containing Ag showed
slightly higher level of attachment and spreading in the early stage culture (1 h) than the PVA nanobers
without Ag (control). However, compared with the PVA nanobers without Ag, the heat-treated and UVirradiated PVA nanobers, containing mainly Ag ions and nanoparticles, respectively, showed reduced
cell attachment and spreading. This shows that both Ag ions and Ag nanoparticles are cytotoxic to NHEK
and NHEF. There was no signicant difference in cytotoxicity to NHEK and NHEF between Ag ions and
Ag nanoparticles. NHEF appeared to be more sensitive to Ag ions or particles than NHEK. In addition, the
residual nitrate ions (NO3 ) in the PVA nanobers had an adverse effect on the culture of both cells.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Many studies have examined silver (Ag) salts or silver compounds as promising materials for wound management. With
respect to the antimicrobial activity, Ag salts or Ag compounds have
been used as wound treatments in a variety of physical forms, such
as beads, gels, lms and bers. Recently, there has been a rapid
increase in the number of commercial Ag dressings on the market,
such as silver nitrate, silver sulphadiazine, and anticoat (nanocrystalline silver), even though Ag compounds have been found to have
serious cytotoxic activity on various host cells [15]. Hidalgo et
al. [5] reported that the cell viability was reduced considerably
when cultured broblasts were exposed directly to AgNO3 in both
dose- and time-dependent manners. Practically, when antibacterial

Corresponding author. Tel.: +82 42 821 6613; fax: +82 42 823 3736.
Corresponding author at: Department of Oral Biochemistry and Program
of Craniomaxillofacial Reconstruction Science, Seoul National University School
of Dentistry, 28 Yeonkun-Dong, Chongno-Ku, Seoul 110-749, South Korea.
Tel.: +82 2 740 8661; fax: +82 2 740 8665.
E-mail addresses: parkwh@cnu.ac.kr (W.H. Park), bmmin@snu.ac.kr (B.-M. Min).
1
These authors contributed equally to this work.
0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.03.026

agents, such as Ag compounds, are applied to open or burn wounds,

it is very important to consider their potential cytotoxicity to the
target cells (broblasts, endothelial cells and keratinocytes), which
are essentially responsible for skin regeneration.
With the development of nanofabrication techniques, Ag has
been fabricated into a range of nanostructures (for example,
nanoparticle) to maximize its chemical and biological activities [6].
A large number of studies have been conducted on Ag nanoparticles,
due to its ease of formation. Ag nanoparticles are of particular interest on account of their good conductivity, chemical stability, and
catalytic and antibacterial activities [7]. In addition, Ag nanoparticles can be incorporated into polymer matrices, including polymer
nanobers. The incorporation of Ag nanoparticles into a nanobrous structure is an attractive method because Ag nanoparticles
distributed evenly in a nanobrous structure can be used in novel
specic applications in catalysis, sensors, photonics, and electronics
[812].
Electrospinning has been studied widely because of its efciency
and simplicity for the fabrication of nanobrous structures [13,14].
The nanobers exhibit outstanding characteristics, such as very
large surface area-to-volume ratio and high porosity with a very
small pore size. Therefore, electrospun nanobers are candidate
materials for many biomedical applications, such as wound dress-

J.Y. Chun et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

ing, drug delivery, and scaffolds for tissue engineering [1517]. A

combination of Ag nanoparticles and polymer nanobers can be
accomplished by electrospinning polymer solutions containing Ag
nanoparticles or Ag salts, which are then reduced into particles in
electrospun polymer nanobers.
On the other hand, poly(vinyl alcohol) (PVA) is a water-soluble,
non-toxic, biocompatible and biodegradable synthetic polymer.
Based on these features, PVA has attracted considerable in the
biomedical, plastics, and textile elds. The electrospinning of PVA
has been studied extensively, due mainly to its use of water-based
solvents [1822]. Therefore, PVA was chosen as a suitable base
polymer to construct an electrospun nanobrous structure containing Ag compounds. Although Ag nanoparticles have been studied
widely as an antiseptic agent, there are few reports on the cellular toxicity of Ag nanoparticles, particularly Ag nanoparticles
supported by a nanobrous structure.
This paper reports a PVA nanobrous matrix containing Ag
ions or nanoparticles for biomimetic wound dressings. The asspun PVA/AgNO3 nanobers containing AgNO3 were heat-treated
at 150 C for 10 min to impart water resistance. The cytotoxic effect
of the Ag ions and/or nanoparticles in the nanobrous matrix
on epidermal cells (keratinocytes and broblasts) was evaluated.
In addition, the cytotoxicity of the residual nitrate ions (NO3 )
in the nanobrous matrix was examined using a washing process.

335

2.2. Electrospinning
The electrospinning setup consisted of a hypodermic syringe
(ID = 0.495 mm), a ground electrode (d = 21.5 cm, a stainless steel
sheet on a drum with a variable rotation speed), and a high voltage supply (Chungpa EMT; CPS-40K03, Seoul, Korea). The syringe
needle was connected to the high voltage supply, which could generate positive DC voltages of up to 40 kV. For the electrospinning of
PVA bers containing Ag+ ions, PVA and AgNO3 were dissolved in
deionized water at 80 C for 2 h with constant stirring. The concentrations of PVA and AgNO3 were 10 and 1.0 wt%, respectively. The
solution was delivered by a syringe pump (Model 100, KD Scientic,
Incheon, Korea) at a ow rate of 1 mL/h. The distance between the
needle tip and ground electrode was 10 cm, and the positive voltage applied to the polymer solutions was 20 kV. All experiments
were carried out at room temperature.

2.3. Heat treatment of PVA nanobers containing Ag+ ions

For the stabilization of PVA nanobers against water, the electrospun PVA nanobrous matrix was crosslinked physically by a
thermal treatment at 150 C for 10 min.

2.4. UV irradiation of PVA nanobers containing Ag+ ions

2. Experimental
2.1. Materials
Poly(vinyl alcohol) (PVA) powder (98% hydrolyzed,
Mn =78,000 g/mol) was purchased from Polysciences Inc. (Warrington, PA, USA). Silver nitrate (AgNO3 , 99.8%) was obtained from
SigmaAldrich (Saint Louis, MO, USA) and used as received.

The photoreduction of the Ag+ ions incorporated within the PVA

nanobers was examined by electrospinning the PVA nanobers
with 1.0 wt% of AgNO3 on glass slides and irradiating them with
UV light with a maximum wave length of 254 nm using a 500 W
high-pressure Hg lamp system (StabiLight, NT-LS-HG50-SR). The
glass slide was washed with deionized water in an ultrasonicator
for 30 min and dried completely.

Fig. 1. SEM images of the as-spun, heat-treated, and UV-irradiated PVA nanobers: (a) as-spun PVA only, (b) as-spun PVA with AgNO3 , (c) heat-treated PVA, and (d)
UV-irradiated PVA.

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Fig. 2. TEM images of the UV-irradiated PVA nanobers.

2.5. Measurements
The morphology of the as-spun, heat-treated PVA nanobers
containing Ag was observed by eld emission scanning electron
microscopy (FE-SEM; JSM-7000F, JEOL, Japan). The samples were
coated with platinum by ion sputtering for a few seconds to prevent surface charging during FE-SEM observations. The average
diameter and distribution were obtained by analyzing the SEM
images using a custom code image analysis program (Scope Eye
II, Masan, Korea). X-ray photoelectron spectroscopy (XPS) of the
PVA nanobers containing Ag was performed on a VG Escalab Mk2
spectrometer equipped with an unmonochromatized Mg K X-ray
source (1253.6 eV) and a hemispherical analyzer. The photoemitted core level electrons were collected at a xed takeoff angle (75
with respect to the sample surface) with electron detection at a
constant analyzer energy (CAE) mode operating at a pass energy of
20 eV. The transmission electron microscopy (TEM) images of samples deposited on carbon coated copper grids were obtained using
a Philips CM 200 transmission electron microscope. The Ag+ ions in
the PVA nanobers were extracted with water, and the Ag+ concentration was monitored using a Direct Reading Echelle inductively
coupled plasma (ICP) spectrometer (Leeman Labs Inc., DRE). Elemental analysis of the PVA nanobers after removing the NO3 ions
was carried out using an Automatic Elemental Analyzer (Thermo
Fisher Scientic, Flash EA 1112 series).
2.6. Cells and cell culture
Normal human epidermal keratinocytes (NHEK) were prepared
and maintained, as previously described [15,23]. Briey, the NHEK
were isolated from the human foreskins of patients (13 years old)
undergoing surgery. The epidermal keratinocytes were isolated
from the separated epithelial tissue by trypsinization, and the primary cultures were established in a keratinocyte growth medium
containing 0.15 mM calcium and a supplementary growth-factor
bullet kit (KGM; Clonetics, San Diego, CA, USA). Primary NHEK
(approximately 70% conuent) were plated at 1 105 cells per

Fig. 3. Expanded XPS spectra of the Ag regions of the heat-treated (a) and UVirradiated (b) PVA nanobers.

60-mm culture dish and cultured until the cells reached 70%
conuence. The second-passage keratinocytes were used in the
experiments described. Primary normal human epidermal broblasts (NHEF) were established from the explant cultures of the
foreskin connective tissue, which was excised from a patient undergoing surgery. The cells that proliferated outwardly from the
explant culture were cultured continuously in Dulbeccos modied Eagles medium (DMEM) supplemented with 10% fetal bovine
serum (FBS). The assays were performed on the cells in their fourth
passage. All procedures for sampling the human tissue specimens
were performed in accordance with the guidelines of the Institutional Review Board (IRB) on Human Subjects Research and the
Ethics Committee at Seoul National University Dental Hospital,
Seoul, Korea.
2.7. Cell attachment and spreading assays
Cell attachment was assayed using a modication of the
method reported by Mould et al. [24]. Briey, the PVA nano-

Table 1
Morphological parameters of the as-spun, heat-treated and UV-irradiated PVA nanobers containing silver.
Sample

AFDa (nm)

Total Ag content (wt%)

Oxidation state of Ag

Ag nanoparticle size (nm)

PVA only
PVA (Heat)
PVA (Heat + UV)

310 50
330 70
330 70

1.0
1.0

Ag+
Ag+ /Ag0 (40/60)

30

Average ber diameter.

J.Y. Chun et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

brous matrix containing Ag was cut out with a punch (14-mm

in diameter) and placed onto 24-well culture plates (Nunc, Denmark). The 24-well culture plates containing the PVA nanobrous
matrix with Ag+ were soaked in serum-free medium for 1 h at
room temperature, followed by rinsing with phosphate-buffered
saline (PBS). The cells were detached by a treatment with 0.05%
trypsin and 0.53 mM ethylenediaminetetraacetic acid (EDTA) in
PBS, resuspended in the culture medium (1 105 cells/500 l), and
incubated for 1 h at 37 C. The unattached cells were removed
by rinsing twice with PBS. The attached cells were xed with
10% formalin in PBS for 15 min and rinsed twice with PBS.
The cells attached to the PVA matrix were stained with hematoxylin and eosin, and the wells were rinsed gently three times
with double-distilled water. The PVA matrix containing Ag+ was
mounted, and the cells that attached to the matrix were photographed.
The extent of cell spreading was analyzed using the photographs
of the cell attachment assay. In order to ensure a representative
count, each nanobrous sample was divided into quarters, and
two elds per quarter were photographed using an Olympus BX51
microscope at 150. The cells that adopted a attened, polygonal shape with lopodia- and lamellipodia-like extensions were
considered to be spreading cells. In contrast, the cells that resisted
washing and remained tethered to the plate surface were classied
as non-spreading cells. For this, each spreading cell was determined
using a computer equipped with CellProler cell image analysis
software (Broad Institute, Cambridge, MA). A minimum of 100 cells
were counted on each occasion. The mean percentage and standard
deviation were calculated from four independent experiments. The
percentage of cells showing a spreading morphology were quantied by dividing the number of spreading cells by the total number
of bound cells.

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2.8. Scanning electron microscopy

SEM was used to examine the morphological characteristics of
the cells cultured on the PVA nanobrous matrix containing Ag. The
matrix was cut out with a punch (14-mm in diameter) and placed
onto 24-well culture plates (Nunc, Denmark). The 24-well culture
plates containing the PVA nanobrous matrix with Ag were soaked
in serum-free medium for 1 h at room temperature, followed by
rinsing with PBS. NHEK and NHEF were detached by a treatment
with 0.05% trypsin and 0.53 mM EDTA in PBS and resuspended in
the culture medium (1 105 cells/500 l). The cells were plated at
5 104 cells onto each matrix and cultured for 1, 3, or 7 days at 37 C
in 5% CO2 . The medium was changed every 2 days during the culture
period. The remaining bound cells were xed in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.4) for 10 min. The xative
was then aspirated. After washing in the buffer, the PVA matrix
was dehydrated in a graded series of ethanol. After critical point
drying (Polaron model 5400; Bio-Rad, Hercules, CA), the sample
was sputtered with gold using a SEM coating system (Bio-Rad), and
the probes were examined by SEM (JEOL, JSM 840A, Japan). Each
matrix sample was divided into quarters, and four elds per quarter were photographed by SEM at 1000. The cells that adopted
a attened, polygonal shape with lopodia- and lamellipodia-like
extensions were regarded as spreading cells. In contrast, the cells
that resisted washing and remained tethered to the matrix surface
were regarded as non-spreading cells.
2.9. Statistics and data analysis
Cell adhesion onto the PVA matrix containing Ag was compared
by an analysis of the variance (ANOVA), using the STATISTICA 6.0
software package. When signicant differences were found, pair-

Fig. 4. (a) Photographs and numbers of NHEK and NHEF adhering to the heat-treated and UV-irradiated PVA nanobers. (b) Images and percentage of cell spreading of the
NHEK and NHEF plated onto the heat-treated and UV-irradiated PVA nanobers. PVA (Heat): heat-treated PVA nanober; PVA (Heat + UV): UV-irradiated PVA nanober. The
data is expressed as the mean SD (n = 4). ANOVA: P < 0.05. Pairwise comparisons: *P < 0.05.

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J.Y. Chun et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

wise comparisons were performed using a Scheffes adjustment or

Students t-test. P values <0.05 were considered signicant.
3. Results and discussion
3.1. Characterization of PVA nanobrous matrices containing Ag
The PVA nanobrous matrix containing Ag+ ions was prepared
by electrospinning an aqueous 10 wt% PVA solution. In order to
improve the water resistance of the as-spun PVA nanobrous
matrix, the PVA nanobers were crosslinked physically by heat
treatment at 150 C for 10 min [2527]. Fig. 1 shows SEM images
of the as-spun, heat-treated and UV-treated PVA nanobers containing Ag (1.0 wt%), along with images of the pure PVA nanobers
without Ag. The as-spun pure PVA and PVA/AgNO3 nanobers
were quite uniform with an average diameter of 310 50 nm and
330 70 nm, respectively. In addition, the morphology and ber
diameter of the PVA nanobers containing Ag+ ions remained
relatively unchanged after heat treatment or UV irradiation. Interestingly, the addition of AgNO3 did not affect the average diameter
of PVA nanobers signicantly due to the inherent polarity of the
PVA solution.

The PVA nanobers containing Ag nanoparticles were prepared

by reducing Ag+ ions by UV irradiation. When the heat-treated PVA
nanobers with AgNO3 were irradiated with UV light at 254 nm for
4 h, the color of the as-spun PVA nanobers was changed gradually to brown with increasing irradiation time, indicating that
the Ag+ ions in the PVA nanobers had rapidly photoreduced and
aggregated into Ag nanoparticles. Ag nanoparticles were generated preferentially on the surface of the PVA nanobers and had
an almost spherical shape, as shown in Fig. 2. The size distribution
of the Ag nanoparticles was quite narrow with an average diameter of 30 nm. It was reported that some Ag nanoparticles were
generated during the heat treatment of PVA nanobers containing
Ag+ ions [27]. However, no Ag nanoparticles were observed in the
heat-treated PVA sample, possibly because the heat treatment time
was only 10 min (data not shown).
The oxidation state of Ag ions and nanoparticles in the PVA
nanobers was examined by XPS. Fig. 3 shows the expanded XPS
spectra of the Ag regions for PVA nanobers with Ag ions or
nanoparticles. The heat-treated PVA nanobers with mainly Ag+
ions showed doublet peaks at 367.6 and 373.6 eV, corresponding to
binding energies of Ag 3d5/2 and Ag 3d3/2 , respectively. On the other
hand, the UV-irradiated PVA nanobers with mainly Ag nanoparti-

Fig. 5. Cell attachment and spreading of NHEK and NHEF plated onto the heat-treated and UV-irradiated PVA nanobers. (a) SEM images showing the interaction between
NHEK and PVA nanobers. (b) SEM images showing the interaction between NHEF and PVA nanobers.

J.Y. Chun et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

339

cles showed doublet peaks at 368.0 and 374.0 eV. The Ag 3d5/2 and
Ag 3d3/2 binding energies at 367.6 and 373.6 eV, corresponding to
Ag+ ions, were shifted slightly to 368.0 and 374.0 eV corresponding to metallic Ag0 [28]. This shift indicates that the Ag+ ions had
transformed to Ag nanoparticles.
The relative contents (%) of Ag ions and nanoparticles were
obtained by curve tting, as listed in Table 1. The peak deconvolution revealed the presence of a second component displaced
0.4 eV towards lower binding energy, which can be easily identied
as Ag+ ions. The UV-irradiated PVA nanobers contained 60% Ag
nanoparticles and 40% Ag+ ions. The in vitro cytotoxicity of three
kinds of PVA nanobers (PVA only, PVA (Heat), PVA (Heat + UV))
against epidermal cells (NHEK and NHEF) was examined.
3.2. The cytotoxic effect of silver nanoparticles in the PVA
nanobrous matrix on cell adhesion and spreading of NHEK and
NHEF
Ag nanoparticles are being used increasingly in wound dressings, catheters, and range of household products due to their
antimicrobial activity. Although silver compounds have been
exploited for their medicinal properties for centuries, the use of
Ag as an antimicrobial agent has been limited its toxicity to normal
cells. Therefore, a study on the cytotoxicity of a PVA nanobrous
matrix containing Ag ions/nanoparticles would be particularly
interesting in terms of its possible use as a wound dressing material. The response of NHEK and NHEF forming these tissues was
analyzed in vitro because the epidermis and connective tissue play
important roles in reconstructing skin defects. Firstly, the initial
cell attachment and spreading of PVA nanobers containing Ag
nanoparticles were examined because initial cell attachment and
spreading may be important factors in the early stages of the wound
healing process. To assess cell attachment, the heat-treated and
UV-irradiated PVA matrix containing Ag ions/nanoparticles were
seeded with NHEK and NHEF. To observe and compare the cytotoxicity of the PVA matrices themselves, the cells were seeded onto
PVA nanobrous matrices that had just been soaked in serum-free
medium for 1 h.
Fig. 4(a) shows representative images of the NHEK and NHEF
attached to the PVA matrices. A relatively large number of the cells
tested adhered to the heat-treated PVA matrix containing mainly
Ag+ ions, compared to the PVA only (no Ag) and UV-treated PVA
matrices. One possible explanation is that the heat-treated PVA
matrix contains a higher concentration of Ag+ ions than the UVtreated PVA matrix. A higher number of NHEK and NHEF might
be attached to the heat-treated PVA matrix through electrostatic
interactions between anionic cell surfaces and cationic Ag+ ions
on the matrix. Ag+ ions are highly reactive, and bind readily to
the negatively charged cell wall [29]. This study further examined whether the adhered cells were only tethered to the substrate
(non-spreading) or had spread over the substrate (spreading). Photographs of the NHEK and NHEF adhering to the PVA nanobrous
matrices were taken and used for the spreading assay. Interestingly,
cell spreading showed a somewhat different pattern (Fig. 4(b)). The
initial spreading of the NHEK and NHEF was similar but slightly
higher in the UV-treated PVA matrix. This suggests that the cytotoxicity of Ag+ ions for both NHEK and NHEF is not serious in the
early stages of cell culture.
The cell morphology and interaction between the cells and PVA
matrices were examined in vitro over a 7 day period. Fig. 5 shows
representative images of the cells attached to the PVA nanobrous
matrices. In the case of the heat- and UV-treated PVA matrices containing Ag, NHEK and NHEF attached to the matrices did not spread
for 7 days. On the other hand, the pure PVA matrix promoted the
growth of NHEK and NHEF. Fig. 6 shows the comparative data for
the attachment and spreading of NHEK and NHEF for 7 days. The

Fig. 6. Cell attachment and spreading of NHEK and NHEF plated onto the heattreated and UV-irradiated PVA nanobers. (a) Level of cell attachment of the NHEK
and NHEF plated onto heat-treated and UV-irradiated PVA nanobers. (b) Amount
of cell spreading of the NHEK and NHEF plated onto heat-treated and UV-irradiated
PVA nanobers. The data is expressed as the mean SE (n = 4). ANOVA: P < 0.05.
Pairwise comparisons: *P < 0.05.

number of attached NHEK and NHEF of the heat- and UV-treated

PVA matrices containing Ag was considerably lower than that of
the pure PVA matrix, and decreased gradually with increasing culture time. In addition, the spreading area of the NHEK and NHEF
of the heat- and UV-treated PVA matrices containing Ag was only
approximately 30% compared to the pure PVA matrix, in which the
spreading area of NHEF increased signicantly with culture time.
Overall, these results show that a PVA matrix containing Ag is cytotoxic to both NHEK and NHEF cells.
This conclusion is supported partly by a previous study [4],
which reported that Ag is highly toxic to both keratinocytes and
broblasts in a monolayer culture and that the broblasts appear to
be more sensitive to Ag than keratinocytes. Nevertheless, it is possible that differences in the growth media used for NHEK and NHEF
cultures could cause different responses of the cells to the toxic
effects of the Ag. In addition, it was reported that Ag nanoparticles
reduce the ATP content of a cell causing damage to the mitochondria and increasing the production of reactive oxygen species in a
dose-dependent manner. This causes cell cycle arrest in the G2 /M
phase possibly due to the repair of damaged DNA [30]. Therefore,
they suggest a possible mechanism of toxicity, which involves disrupting the mitochondrial respiratory chain by Ag nanoparticles

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J.Y. Chun et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

leading to production of reactive oxygen species and an interruption of ATP synthesis, which in turn causes DNA damage.
3.3. The cytotoxic effect of nitrate ions in PVA nanobrous matrix
on cell attachment and spreading of NHEK and NHEF
Silver nitrate (AgNO3 , 0.5%) is the standard and most popular Ag
salt solution used for topical burn wound therapy. Silver nitrate has
a high Ag concentration but no residual activity necessitating very
frequent applications, up to 12 times per day. However, the effect
of nitrate anions (NO3 ) dissociated from AgNO3 on the epidermal
cells and wound healing needs to be claried.
In order to determine the effect of residual nitrate ions in the
PVA matrix, the UV-irradiated PVA matrix containing mainly Ag
nanoparticles was washed with deionized water for 1 h (Washing). The heat-treated PVA nanobers with mainly Ag+ ions were
excluded because Ag+ ions could be also removed from the PVA
matrix during washing. The initial cell attachment and spreading
of the PVA nanobers with or without nitrate ions (Washing or
No washing) were examined. From elemental analysis, the nitrogen contents of the un-washed and washed PVA nanobers were
0.127% and 0.016%, respectively. This suggests that approximately
90% of nitrate ions are removed by washing.
Similarly, the attachment of cultured NHEK and NHEF was evaluated using a cell adhesion assay in serum-free medium. Fig. 7
shows representative images of NHEK and NHEF attached to the
PVA matrices in the early stage of culture. In the case of NHEK, the

number of cells attached to the washed PVA matrix with a very low
nitrate ion concentration was approximately ve times higher than
that of the PVA matrix (No washing). On the other hand, washing
did not appear to have an effect on the attachment and spreading
of NHEF. This suggests that the cytotoxicity of nitrate ions to NHEK
was serious in the early stages of the NHEK culture.
The cell morphology and interactions between the cells and PVA
matrices with or without nitrate ions were examined in vitro over
a 7 day period. Fig. 8 shows representative images of the cells
attached to the PVA nanobrous matrices after 1, 3 and 7 days.
Both NHEK and NHEF cells did not spread for 7 day culture. Fig. 9
shows comparative data for the attachment and spreading of NHEK
and NHEF for 7 days. The number of attached cells on the matrices
decreased abruptly after 1 day of culture. In addition, NHEK were
more sensitive to nitrate ions in terms of cell attachment. A comparison of the two PVA samples (Washing and No washing) showed
that the nitrate ions adversely affected the attachment of cells in
the early stages of culture. This indicates that the PVA matrix containing nitrate ions is also cytotoxic to both NHEK and NHEF. It was
reported that a silver nitrate solution at concentrations >5 103 %
was a lethal to human keratinocyte cultures after as little as 3 h
incubation, but that concentrations <3.7 104 % has no effect on
broblast culture in both the optimal broblast culture medium
and keratinocyte culture medium [30]. Therefore, the use of a PVA
matrix containing Ag nanoparticles as potential tissue engineering
scaffolds should be approached with caution, even if the surface is
modied chemically with a biocompatible material [31].

Fig. 7. (a) Photographs and numbers of NHEK and NHEF adhering to the UV-irradiated PVA nanobers with (No washing) or without (Washing) the nitrate ions. (b) Images
and percentage of cell spreading for the NHEK and NHEF plated onto the UV-irradiated PVA nanobers with or without nitrate ions. The data is expressed as the mean SD
(n = 4). *P < 0.05.

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341

Fig. 8. Cell attachment and spreading of NHEK and NHEF plated onto the UV-irradiated PVA nanobers with (No washing) or without (Washing) the nitrate ions. (a) SEM
images showing the interactions between the NHEK and UV-irradiated PVA nanobers. (b) SEM images showing the interactions between the NHEF and UV-irradiated PVA
nanobers.

Fig. 9. Cell attachment and spreading of NHEK and NHEF plated onto the UV-irradiated PVA nanobers with (No washing) or without (Washing) nitrate ions. (a) Level of cell
attachment of NHEK and NHEF plated onto UV-irradiated PVA nanobers. (b) Amount of cell spreading of NHEK and NHEF plated onto UV-irradiated PVA nanobers. The
data is expressed as the mean SE (n = 4). ANOVA: P < 0.05. Pairwise comparisons: *P < 0.05.

4. Conclusions
PVA nanobers containing 1 wt% AgNO3 were electrospun and
stabilized by heat treatment. The heat-treated PVA nanobrous
matrix containing Ag+ ions was reduced using UV light to generate Ag nanoparticles. The in vitro cytotoxicity of the Ag ions
and/or nanoparticles on normal human keratinocytes and broblasts was examined. Both Ag ions and Ag nanoparticles showed
similar cytotoxicity to the NHEK and NHEF. The NHEF appeared
to be more sensitive to Ag ions or particles than NHEK. In addi-

tion, NO3 adversely affected the attachment of cells in the early

stages of culture. The NHEK cells were more sensitive to the
nitrate ions than NHEF. Therefore, an antimicrobial Ag-containing
matrix should be designed to minimize the damage to epidermal
cells.
Acknowledgements
This study was supported nancially by the Biotechnology
Development Program (grant number 850-20080090) funded by

342

J.Y. Chun et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 334342

the Ministry of Education, Science, and Technology (MEST) of

Korea.
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