Chitosan-Coated Poly(vinyl alcohol) Nanobers For

Wound Dressings
Yun Ok Kang,1 In-Soo Yoon,2 So Young Lee,1 Dae-Duk Kim,2 Seung Jin Lee,3
Won Ho Park,4 Samuel M. Hudson5
1

Department of Nanotechnology, Chungnam National University, Daejon 305-764, South Korea

College of Pharmacy, Seoul National University, Seoul 151-742, South Korea

College of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea

Department of Advanced Organic Materials and Textile System Engineering, Chungnam National University,
Daejeon 305-764, South Korea

Department of Fiber and Polymer Science, College of Textiles, North Carolina State University, Raleigh,
North Carolina 27695-8301

Received 1 June 2009; revised 28 September 2009; accepted 1 October 2009

Published online 2 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31554

Abstract: A PVA nanofibrous matrix was prepared by electrospinning an aqueous 10 wt %

PVA solution. The mean diameter of the PVA nanofibers electrospun from the PVA aqueous
solution was 240 nm. The water resistance of the as-spun PVA nanofibrous matrix was
improved by physically crosslinking the PVA nanofibers by heat treatment at 1508C for 10
min, which were found to be the optimal heat treatment conditions determined from chemical
and morphological considerations. In addition, the heat-treated PVA (H-PVA) nanofibrous
matrix was coated with a chitosan solution to construct biomimetic nanofibrous wound
dressings. The chitosan-coated PVA (C-PVA) nanofibrous matrix showed less hydrophilic and
better tensile properties than the H-PVA nanofibrous matrix. The effect of the chitosan
coating on open wound healing in a mouse was examined. The C-PVA and H-PVA nanofibrous
matrices showed faster wound healing than the control. The histological examination and
mechanical stability revealed the C-PVA nanofibrous matrix to be more effective as a woundhealing accelerator in the early stages of wound healing than the H-PVA nanofibrous matrix.
' 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 92B: 568576, 2010

Keywords:
healing

poly(vinyl alcohol) (PVA); electrospinning; nanofiber; chitosan; coating; wound

INTRODUCTION
Chitosan is obtained by the hydrolysis of chitin, which is
the principal structural polysaccharide in arthropods (for
example, crabs and insects).1 Until now, many researchers
have examined chitosan as a promising material for biomedical applications on account of its good biocompatibility, biodegradability, cellular binding capability, antimicrobial activity and wound healing effect.2,3 On the basis
of the acceleration of wound healing and antimicrobial activity, chitosan has been applied to wound treatments in

Correspondence to: W. H. Park (e-mail: parkwh@cnu.ac.kr)

Contract grant sponsor: Korea Science and Engineering Foundation (KOSEF),
Korea government (MOST); contract grant number: 2005-00009.
' 2009 Wiley Periodicals, Inc.

568

various physical forms, such as beads, powders, gels,

sponges, tubes, lms, and bers.3
With the development of nanofabrication techniques,
chitosan has been fabricated into nanostructures, including
nanoparticles, nanotubes and nanobers to maximize its biological activity.4 In particular, a large number of studies
have been carried out on chitosan micro or nanoparticles,
most likely due to their ease of fabrication. However, there
have been few studies on the biomedical applications of a
chitosan nanobrous matrix.
Recently, electrospinning has been studied widely
because of its efciency and simplicity in fabricating of
nanobrous structures.5 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 dressing,

CHITOSAN-COATED POLY(VINYL ALCOHOL) NANOFIBERS FOR WOUND DRESSINGS

drug delivery and scaffolds for tissue engineering.68 Chitosan is an attractive material for electrospinning. However,
chitosan is difcult to electrospin into a nanobrous structure because it has a polycationic character in acidic aqueous solutions due to the many amino groups in its
backbone. The polycationic nature of chitosan increases the
surface tension of the solution considerable. A strong electrical force is needed to produce electrospun chitosan nanobers, and particles are often formed during the
electrospinning process, which is likely due to the repulsive
forces between the ionic groups in the chitosan backbone
in an acidic solution.9 In a few studies, brous structures
were formed successfully by electrospinning chitosan solutions using environmentally harmful and toxic solvents,
such as triuoroacetic acid (TFA) or TFA/dichloromethane
(DCM).911 Unfortunately, the electrospinning conditions
are relatively limited in terms of concentration, molecular
weight, and degree of deacetylation.12 In addition, the
resulting chitosan bers need to be cross-linked in order to
maintain their structural integrity because they can readily
swell in aqueous solutions.13 Since the electrospinning of
chitosan itself is difcult, chitosan has been mixed with
other synthetic or natural polymers.1418
Considering this aspect, a surface coating by chitosan
can be an alternative approach to applying chitosan to
nanobers because its benecial functions occurs mainly
on the surface of the nanobers. Chitosan-coated nanobrous structures have potential for a variety of biomedical
applications because of their chemical similarity to the glycosaminoglycans in the extracellular matrix (ECM) and
their morphological proximity to brous collagen structures
in the ECM on the nanometer scale.
On the other hand, poly(vinyl alcohol) (PVA) is a
water-soluble polymer as well as a nontoxic, biocompatible, and biodegradable synthetic polymer. Therefore, PVA
has been studied widely in biomedical, plastics, and textile
elds. The electrospinning of PVA has been studied extensively due to use of water-based solvents.1923 Therefore,
PVA was chosen as a suitable base polymer to construct an
electrospun nanobrous structure.
This article reports a chitosan-coated PVA nanobrous
matrix for biomimetic wound dressings. First, the as-spun
PVA nanobers were heat-treated at different temperatures
to provide water resistance and optimize the heat treatment
condition. Second, the heat-treated PVA nanobrous matrix
was coated with chitosan to improve the wound healing
properties. The wound healing behavior on open wound
healing in mice was also investigated to determine the
effect of the chitosan coating.

MATERIALS AND METHODS

Materials

Poly(vinyl alcohol) (PVA) powder (98% hydrolyzed, Mn 5

78,000 g/mol) was obtained from Polysciences Inc. (WarJournal of Biomedical Materials Research Part B: Applied Biomaterials

569

rington, PA). Chitosan (80% deacetylated, Mv 5 220,000)

was purchased from Kumho Chemical Co. (Seoul, Korea).
Glacial acetic acid (Duksan Pure Chemical Co., Korea) and
1N sodium hydroxide (Dc Chemical Co., Korea) were supplied by Sigma-Aldrich (Saint Louis, MO).
Electrospinning

The electrospinning setup used in this study consisted of a

hypodermic syringe (ID 5 0.495 mm), a ground electrode
(d 5 21.5 cm, a stainless steel sheet on a drum whose rotation speed could be varied), 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 up to 40 kV. For the
electrospinning of PVA bers, a PVA solution (10 wt %)
was prepared by dissolving PVA powder in deionized
water at 808C with constant stirring for 2 h. The solution
was then delivered through a syringe pump (Model 100,
KD Scientic, Incheon, Korea) at a ow rate of 1 mL/h.
The distance between the needle tip and the ground electrode was 10 cm, and the positive voltage applied to the
polymer solutions was 22 kV. All experiments were carried
out at room temperature.
Heat Treatment of the Electrospun PVA Nanobers

For the stabilization of PVA against water, the electrospun

PVA nanobrous matrix was physically crosslinked with a
thermal treatment at various temperatures from 1108C to
1908C for 10 min.
Preparation of PVA Nanobrous Matrix Coated
With Chitosan

The chitosan solution (1.0 wt %) for coating the PVA

nanobrous matrix was prepared by dissolving chitosan
powder in an aqueous 1% acetic acid solution at room temperature. The heat-treated PVA nanobrous matrix was
coated by immersion in a chitosan solution at 308C for 1 h.
For xation, the chitosan-coated PVA matrix was treated
with a 1N sodium hydroxide solution after the chitosan
coating, and then washed with deionized water until pH
was neutral. The chitosan-coated PVA matrix was dried in
a vacuum oven at room temperature for 24 h.
Water Uptake Capacity

The water uptake capacity of the heat-treated and chitosancoated PVA nanobers (200 mg) was determined by
immersing the bers in distilled water for 1 h at room temperature.24 The hydrated samples were then taken and immediately weighed after removing the surface water with
lter paper. The water content (WC, %) was calculated as
follows: WC (%) 5 (W 2 W0)/W0 3 100, where W0 and
W denote the weight of the sample before and after immersion in water for 1 h, respectively.

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KANG ET AL.

Wound Healing Test

An in vivo wound healing test using hairless mice (n 5 3)

was conducted to evaluate the effect of the chitosan-coated
PVA nanobrous matrix in the regeneration of damaged
skin. The skin from the back of four-week-aged hairless
mice was removed in a round-shape using a sterile dermal
biopsy punch (6 mm). Consequently, one hairless mouse
had three wound sites on its back. For comparison, two of
these wounds were covered with the PVA nanobrous matrix (1 cm 3 1 cm) and chitosan-coated PVA nanobrous
matrix (1 cm 3 1 cm), and the other wound was untreated
as a control. A piece of TegadermTM (3M, USA) was
applied to the top of the wound dressing. Healing wounds
were observed on the 1st, 3rd, 5th, and 7th days. The
degree of wound healing was expressed as the wound contraction ratio (WCR) as follows: WCR (%) 5 (A0 2 At)/A0
3 100, where A0 and At indicate the initial wound area and
the wound area at time t, respectively. The wound area was
measured using a slide caliper. The wound tissues were dissected, xed with 10% phosphate-buffered formalin, and
stained with Massons and hematoxylin & eosin (HE)
reagents for the histological observations of epithelialization and granulation.25
Measurements

The morphology of the as-spun, heat-treated and chitosancoated PVA nanobers was observed by eld emission
scanning electron microscopy (FE-SEM; JSM-7000F,
JEOL, Japan). Prior to the observations, the samples were
coated with platinum by ion sputtering for a few seconds.
The average diameter and diameter distribution were determined by analyzing the SEM images using a custom code
image analysis program (Scope Eye II, Masan, Korea). The
differential scanning calorimetry (DSC) measurements were
carried out using a Perkin Elmer DSC7 instrument in a
nitrogen atmosphere. A 10-mg sample was sealed in an
aluminum pan for the measurements, and heated from 308C
to 2508C at a rate of 108C/min. The crystalline structure of
the samples was analyzed using a wide-angle X-ray diffractometer (model D/max-IIB, Rigaku International Corp., Japan). The water contact angle (WCA) was measured using
a DSA 100 drop shape analysis system (Kruss Gmbh, Germany). The volume of the water droplet for each measurement was kept at 3 3 1029 m3. The XPS spectra of the
PVA nanobrous matrices were recorded on a VG Escalab
Mk2 spectrometer equipped with an unmonochromatized
Mg Ka X-ray source (1253.6 eV) and hemispherical analyzer. The photoemitted core level electrons were collected
at a xed takeoff angle (758 with respect to the sample surface) with electron detection in constant analyzer energy
(CAE) mode operating at a pass energy of 20 eV.
The tensile properties of PVA nanobrous matrices were
measured by Instron1 8511 (Instron, Canton, MA). The
PVA nanobrous matrices (0.2 mm thick) were cut into a
50 mm 3 10 mm dumbbell-shape according to ASTM D

Figure 1. (a) SEM image of the as-spun PVA nanobers, (b) photograph, and (c) SEM image of the as-spun PVA nanobers after
immersion in water for 5 min.

882-97. A load cell of 500 N was hammered vertically

onto the specimen at a speed of 1 mm/min. The samples
were tested at 258C and 50% humidity (n 5 10).
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CHITOSAN-COATED POLY(VINYL ALCOHOL) NANOFIBERS FOR WOUND DRESSINGS

571

Figure 2. DSC thermograms (a) and XRD patterns (b) of PVA nanobers with heat treatment temperature.

RESULTS
Morphology of As-Spun PVA Nanobers

Figure 1(a) shows a SEM image of the as-spun (i.e.,

untreated) PVA nanobers under a magnication of
80003. The as-spun PVA nanobers consisted of a very
uniform matrix of bers with a smooth surface. Image
analysis showed that the bers had a mean diameter of
240 6 40 nm. However, when the as-spun PVA nanobers
were immersed in water for only 5 min, they swelled easily
or partially dissolved [Figure 1(b)], and dissolved completely after a further few minutes. The PVA nanobrous
structure was changed immediately into a dense membrane
structure in an aqueous system, even though an alcoholwater mixed solution with a small amount of water was
used [Figure 1(c)]. Therefore, it was essential to stabilize
the as-spun PVA bers using an appropriate method.

increasing heat treatment temperature, indicating that heat

treatment induced the crystallization of PVA samples.
The solubility of the heat-treated PVA nanobrous matrix was determined by weighing the dried sample before
and after immersion in boiling water for 2 h. Figure 3
shows the change in the soluble fraction (%) of the PVA
sample as a function of the heat treatment temperature. The
PVA samples heat-treated to 1508C were almost soluble
(soluble fraction, 100%), indicating that physical crosslinking was introduced by heat treatment without chemical
changes. The soluble fraction decreased abruptly, as the
heat treatment temperature was increased further. For
example, the PVA samples heat-treated at 1708C and
1908C showed soluble fractions of 76% and 14%,
respectively.

Stabilization of the PVA Nanobers

Figure 2 shows DSC thermograms and XRD patterns of the

heat-treated PVA nanobrous matrices. In the DSC thermograms [Figure 2(a)], the Tm of the as-spun PVA nanobers
appeared at 2138C. The Tm of the heat-treated PVA samples shifted slightly toward a higher temperature at a heat
treatment temperature of 1708C. In contrast, the abrupt
decrease in Tm and peak broadening were observed in the
PVA sample heat-treated at 1908C for 10 min.
In the XRD patterns [Figure 2(b)], the as-spun PVA
nanobers showed a broad peak at approximately 20.48,
that is, low crystallinity. After heat treatment, there were
three typical peaks at 2h 5 10.78, 20.48, and 23.08 corresponding to the (100), (101), and (200) planes, respectively.
The intensity of these peaks increased gradually with
Journal of Biomedical Materials Research Part B: Applied Biomaterials

Figure 3. Change in the soluble fraction (%) of PVA nanobers with

heat treatment temperature.

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KANG ET AL.

a SEM image of the PVA nanobrous matrix after heat

treatment at 1508C for 10 min. After heat treatment, there
was no signicant change in ber morphology and ber diameter. In addition, the heat-treated PVA nanobrous matrix were not nearly swelled or dissolved after immersion
in distilled water [Figure 4(b)] for 48 h, and the SEM
image of PVA nanobers dried after the immersion showed
similar ber morphology [Figure 4(c)].

Chitosan Coating of PVA Nanobrous Matrix

Figure 5 shows the ber morphology of C-PVA. As shown

in Figure 5, the ber morphology changed into slightly collapsed ber morphology during the chitosan coating. This
might be due to the H-PVA sample being swelled somewhat in the acidic aqueous solution during coating.
The surface and compositional analyses were carried out
to conrm the chitosan coating on the H-PVA nanobrous
matrix. Figure 6 shows the XPS spectra of the C-PVA samples produced with different chitosan solution concentration. The intensity of the peak at 395.0 eV corresponding
to the N1s binding energy increased gradually with increasing concentration of the chitosan solution from 0.1 to 1.0
wt %. Table I lists the elemental analysis results of the HPVA and C-PVA nanobrous matrices. From elemental
analysis, the nitrogen content (%) of the C-PVA sample
was 0.68%, while the H-PVA sample did not contain any
nitrogen. The degree of coating calculated using this value
was 10 wt%. In addition, the change in hydrophilicity of
the H-PVA sample after coating was measured using two
parameters: the water content (WC, %) and water contact
angle (WCA, 8). The C-PVA sample showed a lower water
content (350%) and higher contact angle (658) than the
H-PVA sample, as shown in Table I. This suggests that
the H-PVA nanobrous matrix became less hydrophilic
after the chitosan coating because PVA has higher hydrophilicity.
Table II summarizes the tensile properties of the H-PVA
and C-PVA nanobrous matrices. The chitosan coating
improved the tensile stability of the H-PVA matrix. Both
the tensile strength and modulus of the C-PVA matrix

Figure 4. (a) SEM image of the heat-treated PVA nanobers, (b)

photograph, and (c) SEM image of the heat-treated PVA nanobers
after immersion in water for 48 h.

Heat treatment at 1508C for 10 min was chosen as the

optimized condition considering the crystallinity and solubility of the heat-treated PVA matrices. Figure 4(a) shows

Figure 5. SEM image of the chitosan-coated PVA nanobers. The

arrows indicate welding at the cross-points and neighboring-points.
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CHITOSAN-COATED POLY(VINYL ALCOHOL) NANOFIBERS FOR WOUND DRESSINGS

TABLE II. Tensile Properties
Nanobrous Matrices

Sample
H-PVA
C-PVA

Figure 6. Expanded XPS spectra in the region of N1s with the concentration of chitosan coating solution: (a) none, (b) 0.1 wt %, (c)
0.3 wt %, (d) 0.7 wt %, and (e) 1.0 wt %.

increased signicantly, whereas the breaking elongation

decreased from 80% to 47%.
Wound Healing and Histological Examination of
Chitosan-Coated PVA Nanobrous Matrix

In the open wound healing test, three circular wound sites

were made on the back of each hairless mouse. Figure 7
shows the wound contraction ratios (WCR, %) of the
H-PVA and C-PVA nanobrous matrices with time in the
early healing stage. The WCR of the H-PVA and C-PVA
nanobrous matrices increased gradually in a similar manner and reached 89.0 6 5.6% and 90.7 6 4.3%, respectively, after seven days, whereas the control wound reached
only 75.2 6 3.5%.
Figure 8 shows the healing patterns of the control
(untreated), H-PVA and C-PVA-treated wounds after ve
and seven days of treatment. In the control group after ve

Tensile Strength
(gf/mm2)
6.8 6 0.4
8.3 6 0.6

of

the

H-PVA

Elongation
at Break (%)
80.5 6 4.7
47.2 6 5.7

and

C-PVA

Modulus
(gf/mm2)
222.5 6 8.3
315.3 6 21.1

days, the wound surface was covered with brinous debris,

and a dense inltration of polymorphonuclear leukocytes
and proliferation of broblasts were observed below the
surface layer. However, the surface tissue debris disappeared from the wound surface in the H-PVA and C-PVA
groups after ve days, and there was prominent proliferation of young capillaries and broblasts. In the H-PVA and
C-PVA groups, epithelialization of the wound was complete after seven days. However, H&E staining did not
show a denite difference in the wounds.
With a view to observing the collagenous components,
the control (untreated), H-PVA and C-PVA-treated wounds
were stained using a Massons trichrome staining method.
Figure 9 shows representative photomicrographs of wound
healing. The collagenous components stained a blue color
and the cytoplasm appeared as varying shades of red. The
density of collagen in the H-PVA and C-PVA groups was
higher than control group at seven days because H-PVA
and C-PVA groups can provide both mechanical strength
and a three-dimensional structure for cell attachment,
growth, and migration.
DISCUSSION
In this study, continuous PVA nanobers were obtained at
a concentration of 10% by weight, which is a concentration
that appears to correspond to the onset of signicant chain

TABLE I. Characterization Results of the H-PVA and C-PVA

Nanobrous Matrices

Elemental Analysis
Sample

WC (%)a

WCA (8)b

H-PVA
C-PVA

0.68

51.70
50.50

40.34
40.29

477.5 6 3.0
349.3 6 12.1

0
65.2 6 4.4

a
b

WC, water content.

WCA, water contact angle.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

Figure 7. Wound contraction ratios (%) of H-PVA and C-PVA nanoborus matrices as a function of time.

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KANG ET AL.

Figure 8. Representative micrographs of wound healing of mouse skin (H&E staining).

entanglement. The solution concentration of 10% by weight

was chosen to fabricate a nanobrous matrix of randomly
arranged PVA bers with nanometer scale diameters. The
stabilization of PVA bers against water can be accomplished using various physical and chemical crosslinking
methods. The chemical crosslinking of PVA usually
involves a change in chemical structure, particularly
hydroxyl groups, which affect its benecial properties, such
as non-toxicity, biocompatibility, and biodegradability.
Therefore, physical crosslinking by heat treatment was chosen to avoid the chemical change in the PVA nanobrous
matrix by a cross-linking reaction. The PVA nanobous
matrix was heat treated for 10 min at various temperatures
ranging from 1108C to 1908C, which are located between
glass transition temperature (Tg) and crystalline melting
temperature (Tm). However, the PVA sample heat-treated at
1908C showed a light brown color. This might be due to
the thermal decomposition of PVA by excessive heat treatment.26 Overall, the heat treatment at the appropriate temperature (1508C) can provide the water resistance of the
PVA nanobers without any thermal decomposition. The
decrease in the soluble fractions of the PVA samples
treated at higher temperature (1708C or higher) might be

due to the change in chemical structure of PVA by the excessive heat treatment. It is unclear if the insoluble fraction
originated from the crosslinking and/or dehydration of the
PVA chains during the thermal decomposition process. The
PVA sample undergoes thermal decomposition and crystallization under a certain range of heating conditions [refer
to Figures 2(b) and Figure 3].
To improve the wound healing properties, the heattreated PVA nanobrous matrix (H-PVA) was coated with
a chitosan solution to prepare the chitosan-coated PVA
nanobers matrix (C-PVA). After coating, the color of HPVA changed from white to light yellow, possibly due to
the inherent color of chitosan. Interestingly, new joint
welding of the bers at their neighboring points and at their
cross-points was observed in the C-PVA sample (Figure 5).
This welding played an important role in improving the
mechanical stability of C-PVA. As described previously,
this increase in mechanical stability of the C-PVA nanobrous matrix might be due to the new interber bonds
(joint weldings) provided by the chitosan coating.
The nanobrous matrices (C-PVA and H-PVA) accelerated wound healing compared to the control (Figure 7).
This accelerated healing may be due to the high surface
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CHITOSAN-COATED POLY(VINYL ALCOHOL) NANOFIBERS FOR WOUND DRESSINGS

575

Figure 9. Representative micrographs of wound healing of mouse skin (Massons staining).

area and microporous structure of the nanobrous matrices,

which are advantageous in promoting cell attachment and
proliferation in wound healing. In addition, the collagen
regenerated in the C-PVA-covered wound was denser than
the H-PVA-covered wound, and the epidermis of the
C-PVA group was thicker than the H-PVA group, indicating that chitosan accelerated wound healing (Figure 9).
This study was intended to generate proof of concept pilot
or feasibility data and therefore not powered with sufcient
animals for in vivo evaluation.
CONCLUSIONS
PVA nanobers were electrospun and then stabilized by
heat treatment. The heat-treated PVA (H-PVA) nanobrous
matrix was stable under aqueous conditions, which is likely
due to physical crosslinking. The H-PVA nanobrous matrix was coated with chitosan to improve the wound healing
effect and mechanical stability. The open wound healing
test and histological observations conrmed that the CPVA matrix effectively regenerated the damaged skin,
although the difference to the H-PVA matrix was not signicant. In addition, the tensile properties of C-PVA matrix
Journal of Biomedical Materials Research Part B: Applied Biomaterials

were higher than those of H-PVA matrix, likely due to new

interber bonds by chitosan coating. These results suggest
that C-PVA nanobers may be a good candidate for biomedical applications, such as wound dressings for skin
regeneration and scaffolds for tissue engineering.

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