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The improvement of hemostatic and wound
healing property of chitosan by halloysite
nanotubes
Mingxian Liu,†
a
Yan Shen,†
b
Peng Ao,
a
Libing Dai,
b
Zhihe Liu
b
and Changren Zhou*
a
As tube-like natural nanomaterials, halloysite nanotubes (HNTs) have potential applications in wound healing
due to their high mechanical strength, good biocompatibility, hemostatic property, and wound healing ability.
Here, we have developed flexible 3D porous chitosan composite sponges via the addition of HNTs.
Morphological observation, mechanical property, porosity, swelling ability, and degradation behavior in
phosphate buffer of the chitosan–HNTs composite sponges were investigated by various physicochemical
methods. Compared to pure chitosan sponge, the composite sponges exhibit a similar porous morphology
with a maximum of 8.8-fold increase in compression mechanical properties. The elastic modulus,
compressive strength, and toughness of the composite sponges were simultaneously increased by HNTs.
The whole-blood clotting experiment suggests that HNTs can increase the blood clotting rates of chitosan.
The composite sponges with 67% HNTs shows an 89.0% increase in the clotting ability compared with
pure chitosan. Cytocompatibility of the composite sponges is confirmed by cell attachment and infiltration
of fibroblast and vascular endothelial cells. In vivo evaluation on full-thickness excision wounds in
experimental Sprague-Dawley rats reveal that these composite sponges enhance the wound healing
property especially at the early stage. The composite sponges show a 3.4–21-fold increase in wound
closure ratio compared to that of pure chitosan after one week. The addition of HNTs helps in faster re-
epithelialization and collagen deposition. All these data demonstrate the potential applications of the
chitosan–HNTs composite sponges for burn wounds, chronic wounds, and diabetic foot ulcers.
1. Introduction
As the largest organ of the human body, the human skin is the
rst outside barrier between thebodyandtheenvironment.
Humanskinperformsmanyfunctionssuchasprotection,
sensation, control of evaporation, storage and synthesis,
absorption, and water resistance. However, trauma or other
injuries always lead to varying degrees of damage and skin
defects. Human skin generally needs to be covered with
dressings immediately aer it is damaged. The wound healing
can proceed through regeneration and reconstruction by a
series of pathophysiological processes, which involve the
complex interactions among different types of skin cells,
cytokines and extracellular matrices. For the goal of wound
healing, many types of dressing materials such as hydrogel,
1,2
membrane,
3,4
sponge,
5
non-woven fabrics and nanobers
6,7
have been explored. Sponges are soand exible materials
with interconnected microporous structure that show many
unique characteristics such as good uid absorption capa-
bility, cell interaction and hydrophilicity. However, they have
some serious aws such as low hemostasis ability, poor
mechanical property, limited healing ability, and high fabri-
cation cost, which restrict their practical applications. Many
synthetic or natural macromolecules have been selected as
matrices for wound dressing to improve the healing process.
8
Among these, the macromolecule, chitosan, derived from
natural resources and available abundantly, is considered a
promising material for tissue regeneration.
9
The desirable
features of chitosan for use in wound healing include its
biocompatibility, biodegradability, hemostatic activity, anti-
inectional activity and property to accelerate wound heal-
ing.
4,10
Furthermore, chitosan can easily be processed into
membranes, gels, nanobers,beads,nanoparticles,scaffolds,
and sponges for wound dressing applications. Chitosan has
the functions for the acceleration of inltration of poly-
morphonuclear cells at the early stage of wound healing, fol-
lowed by the production of collagen by broblasts.
11
However,
the hemostatic performance, healing ability, and exibility of
chitosan should be further improved in order to expand its
applications as wound dressing materials.
Clays are important adjutants and supports for medical
products, since they have many physicochemical, mechanical,
a
Department of Materials Science and Engineering, Jinan University, Guangzhou
510632, China. E-mail: tcrz9@jnu.edu.cn
b
Guangzhou Institute of Traumatic Surgery, Guangzhou Red Cross Hospital Medical
College, Jinan University, Guangzhou 510220, China
†The authors contributed equally to this work.
Cite this: RSC Adv.,2014,4,23540
Received 13th March 2014
Accepted 29th April 2014
DOI: 10.1039/c4ra02189d
www.rsc.org/advances
23540 |RSC Adv.,2014,4, 23540–23553 This journal is © The Royal Society of Chemistry 2014
RSC Advances
PAPER
and biological properties such as high absorption ability,
drug-loading ability, absence of toxicity, insensitive to other
raw materials, and complex formation properties.
12,13
Among
various types of clays, one-dimensional halloysite nanotubes
(HNTs) have been used to improve the mechanical properties,
drug-loading properties, cell attachment and hemostatic
performance of polymers in recent years.
14
HNTs are natural
inorganic nanomaterials with a chemical formula of
Al
2
Si
2
O
5
(OH)
4
$nH
2
O. The length of HNTs is in the range of
0.2–1.5 mm, while the inner diameter and the outer diameter of
tubes are in the ranges of 10–40 nm and 40–70 nm, respec-
tively. The aspect ratio (L/D)ofHNTsisintherangeof10–50.
The hollow lumen microstructures and porosity of HNTs
afford them to have a high loading and absorption ability for
active compounds;
14
therefore, HNTs are usually used in drug
delivery systems and waste water treatment.
15,16
Recent
research has suggested that HNTs are cytocompatible and can
potentially be used as scaffold materials in tissue engi-
neering.
17,18
These unique properties of HNTs inspire us to
explore their applications in wound healing. Interestingly, in
traditional Chinese medicine, halloysite (with a Chinese
traditional medicine name “Chishizhi”)wascommonlyused
as wound dressing materials in the form of powder, which has
been conrmed by the efficacy of hemostasis and wound
healing. However, to date, there is no scienticreportonthe
wound healing applications of HNTs. In contrast, there are
numerous papers and patents on otherclaymineralsforuseas
wound dressing materials.
19–21
Chitosan porous scaffolds prepared by lyophilization or
electrospinning serve as good candidates for wound treatment
with the benet of drug/growth factors delivery.
22–25
These active
compounds can signicantly accelerate the wound healing
process; however, the preparation process of drug-loaded chi-
tosan scaffolds is complicated. Moreover, the loaded growth
factors are easily degraded by proteinases or removed by
exudate before reaching the wound bed.
22
Therefore, preparing
high healing performance chitosan dressing materials loaded
with drugs is still a challenge.
In the present work, the chitosan–HNTs composite sponges
with differentHNTsloadingswerepreparedbylyophilization
method. The inuences of HNTs on the physicochemical,
microstructure, cytocompatibility, and in vivo wound healing
abilityofchitosanspongewereinvestigated.HNTssimulta-
neously improve the mechanical properties, cell attachment,
hemostatic performance, and wound healing rate of chitosan.
The composites sponges have a maximum of 8.8-fold increase
in compression strength, an 89.0% increase in clotting ability,
anda21-foldincreaseinwoundclosureratiocomparedwith
pure chitosan sponges. Moreover, the composite sponges have
controllable porosity, swelling ratio, and degradation proper-
ties by changing the HNTs contents. In addition, the cost of
the composite sponges is much lower than that of the pure
chitosan sponges, which facilitates their commercialization.
This work opens a new area of biomedical applications of
HNTs and provides a novel routine for high performance
wound dressing materials by a simple fabrication method with
alowcost.
2. Experimental
2.1 Raw materials
Chitosan was purchased from Jinan Haidebei Marine Bioengi-
neering Co. Ltd (China). Its deacetylation and viscosity-average
molecular weight was 95% and 600 000 g mol
1
, respectively.
Raw halloysite was mined from Hunan province (China) and
puried before use. The elemental composition of puried
HNTs by X-ray uorescence (XRF) was as follows (wt%):
SiO
2
, 54.29; Al
2
O
3
, 44.51; Fe
2
O
3
, 0.63; TiO
2
, 0.006. The Bru-
nauer–Emmett–Teller (BET) surface area of the used HNTs was
50.4 m
2
g
1
. All other chemicals used in this work were of
analytical grade. Ultrapure water from Milli-Q water system was
used to prepare the aqueous solutions.
2.2 Preparation of the chitosan–HNTs composite sponge
The chitosan–HNTs composite sponges were prepared by
solution mixing and subsequently by the freeze-drying method.
The raw chitosan was rst treated with acetic acid (2%
concentration) with stirring for 10 hours. The insoluble fraction
was separated by centrifugation at 8000 rpm for 15 min, and
then the supernatant containing the chitosan was isolated. The
puried chitosan was obtained by freeze-drying. The typical
procedure to prepare the composite sponges was described
below and depicted in Fig. 1. Chitosan (2 g) was dissolved in 100
mL of 2 wt% acetic acid solution under mechanical stirring.
Then, the calculated amount of HNTs powder was added into
the chitosan solution. The mixture was continuously stirred
overnight under ambient temperature, and then treated by
ultrasonic for 30 min to obtain a good dispersion of HNTs and
interfacial adsorptions. Then, the solutions were poured into a
cylinder plastic mold. Subsequently, they were frozen into ice
at 20 C overnight in a refrigerator, and then lyophilized at
80 C using Christ freeze dryer ALPHA 1-2/LD plus. Then, the
scaffolds were immersed for 2 h in 2% NaOH to neutralize the
residual acetic acid and rinsed extensively in sterile distilled
water. Finally, the scaffolds were freeze-dried and stored for
further use. For comparison, pure chitosan sponge was also
prepared in the same way but without the addition of HNTs.
The sample codes of the composite sponge (CS2N1, CS1N1,
CS1N2, CS1N4) represented the weight ratio of chitosan (CS)
and HNTs (N). For example, in the CS1N2 sample the weight
ratio of chitosan and HNTs was 1 : 2.
2.3 Physicochemical characterization of the chitosan–HNTs
composite sponge
Scanning electron microscopy (SEM). Before SEM observa-
tion, the sponges were sectioned and sputter-coated with 10 nm
thick gold–palladium layer using a sputter coater (BALTEC SCD
005). The morphology of the sponges was observed with a Phi-
lips XL30 ESEM and Hitachi S-4800 FE-SEM (for high magni-
cation images).
Porosity. The porosity of the sponges was determined using
the reported method.
26
First, the sponges were immersed in
absolute ethanol until they were saturated. Aerwards, the
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 23540–23553 | 23541
Paper RSC Advances
sponges were weighed before and aer immersion in alcohol.
The porosity was calculated using the formula,
porosity ð%Þ¼W2W1
rV1
%
here, W
1
and W
2
are the weight of sponges before and aer
immersion in alcohol, respectively, V
1
is the volume before
immersion in alcohol and ris a constant (the density of
alcohol). All samples were triplicated in the experiment.
Swelling ratio. The equal volume sponges were immersed in
phosphate buffered saline (PBS) (pH 7.4, 37 C). The sponges
were taken out at predetermined time intervals and the water
that adhered on the surface was removed by gently blotting the
sponges with lter paper. These sponges were then immediately
weighed (W
d
), and the swelling ratio was calculated by the
following formula,
DSð%Þ¼ WwWd
Ww
100%
here, DS is the degree of swelling, and W
w
and W
d
represent the
wet and dry weight of the sponges, respectively.
In vitro biodegradation behavior. The sponge samples were
equally weighed and immersed in lysozyme (10 000 U mL
1
)
containing the medium and incubated at 37 C for 28 days. The
samples were removed aer 7, 14, 21 and 28 days from the
medium containing lysozyme and washed with deionized water
to remove ions adsorbed on surface and then freeze-dried. The
dry weight was noted as W
t
and initial weight as W
i
. The
degradation of the sponges was calculated using the formula,
degradation ð%Þ¼WiWt
Wi
%
Compression property. The compression property of pure
chitosan and the chitosan–HNTs composite sponges was
determined using Universal Testing Machine (Zwick/Roell
Z005, Germany) at 25 C according to ASTM D5024-95a. The
samples for the test were cylinder samples with a diameter of
16 mm and thickness of 14 mm. The crosshead speed was 2
mm min
1
, and up to 85% reduction in specimen height was
obtained. The stress–strain curves for every sample were
recorded automatically by the testXpert® II V2.0 soware.
Compressive modulus was calculated as the slope of the initial
linear portion of the stress–strain curves. The deformation
recovery ratio (R) was calculated by the following equation,
Rð%Þ¼hfð13Þh0
3h0
%
here, h
f
is the nal height of sample aer 30 min of the
compressive testing; h
0
is the initial height of the samples
before compressive testing; and 3is the deformation ratio when
the compression test stops (3¼85% for the present work). Five
samples were used to obtain reliable data.
2.4 Cell cultures on the chitosan–HNTs composite sponges
Fibroblasts were isolated from a human skin biopsy and used at
passages of 3–4. Endothelial cells were from a dermal micro-
vascular origin, and keratinocyte cultures were established from
human skin biopsies.
The sterile pure chitosan and chitosan–HNTs sponges were
seeded with NIH 3T3 and vascular endothelial cells in a 24-well
plate at a concentration of 1 10
5
cells per well. Aer 3 days of
incubation, the sponges were washed with PBS and xed with
2.5% glutaraldehyde for 1 h. The samples were thoroughly
washed with PBS, and then sequentially dehydrated by a series
of graded-ethanol solutions, freeze-dried, gold sputtered in
vacuum and observed by SEM.
2.5 Whole-blood clotting and platelet activation evaluation
of chitosan–HNTs composite sponges
The blood clotting study of the materials was performed
according to the literature.
24,26
Blood was drawn from human
ulnar vein using BD Discardit II sterile syringe and mixed with
anticoagulant agent acid citrate dextrose at the ratio of
85% : 15%. Triplicate samples were determined for every
sample and blood without adding materials was used as a
negative control. Blood was added to 10 mg sponges and freeze-
dried HNTs powder (from 5 wt% HNTs aqueous dispersion) that
were placed in a 6-well plate, which was followed by the addition
of 10 mL of 0.2 M CaCl
2
solutions to initiate blood clotting.
These sponges were then incubated at 37 C for 10 min. Fieen
milliliters (15 mL) of distilled water was then added dropwise
without disturbing the clot. Subsequently, 10 mL of solution
was taken from the dishes and was centrifuged at 1000 rpm for
1 min. The supernatant was collected for each sample and
maintained at 37 C for 1 hour. Two hundred microliters (200
mL) of this solution was transferred to a 96-well plate, and the
optical density was measured at 540 nm using a plate reader
(Multiskan MK3, Thermo Electron Corporation).
Fig. 1 Schematic representation of the preparation of chitosan–HNTs composite sponges.
23542 |RSC Adv.,2014,4, 23540–23553 This journal is © The Royal Society of Chemistry 2014
RSC Advances Paper
Platelet activation study was conducted as follows. Platelet-
rich plasma (PRP) was isolated from the blood by centrifugation
of blood at 2500 rpm for 5 min. One hundred microliters
(100 mL) of PRP was poured onto the sponge piece (10 mg) and
incubated at 37 C for 20 min. The sponges were then washed
three times with PBS solution and xed using 0.1% glutaral-
dehyde solution. The sponges were dried and then SEM images
were obtained.
2.6 In vivo evaluation of wound healing properties of
chitosan–HNTs composite sponges
All experimental procedures were performed according to the
Guide for the Care and Use of Laboratory Animals and were in
compliance with the guidelines specied by the Chinese Heart
Association policy on research animal use and the Public Health
Service policy on the use of laboratory animals. Sprague-Dawley
(SD) rats, weighing 200–250 g and 4–6 weeks of age, were used in
this study. The rats were divided into seven groups and each
group contains three rats (n¼3), and they were allowed to eat
normal rat food and water without restriction. On the day of
wounding, the rats were anaesthetized by intramuscular injec-
tion of 35.0 mg kg
1
ketamine and 5.0 mg kg
1
xylazine. The
dorsal area of the rats was depilated and the operative area of
skin cleaned with alcohol. Full thickness wounds (1.5 cm 1.5
cm) were prepared by excising the dorsum of the rat using
surgical scissors and forceps. The prepared wounds were then
covered with the pure chitosan sponge, chitosan–HNTs
composite sponges, commercially available adhesive wound
dressing (AWD), and oily cotton gauze (OY). Aer applying the
dressing materials, the rats were housed individually in cages at
room temperature.
The dressing materials were changed at week 1, 2, and 3.
During the changing of dressings, photographs were taken and
the wound area was measured using a soplastic sheet. The
sheet was kept on top of the wound and the area was marked
Fig. 2 SEM images of pure chitosan (a), CS2N1 (b), CS1N1 (c), CS1N2
(d), CS1N4 (e) magnified region of (c) that shows the presence of
HNTs (f).
Fig. 3 Density/porosity (a), swelling ratios in PBS at 37 C (b) and
degradation ratio (c) of pure chitosan and chitosan–HNTs composite
sponge.
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 23540–23553 | 23543
Paper RSC Advances
using a marker pen. The marked area was then transferred to
graph sheet to obtain the exact value. Aer Week 4, the wound
tissue of the rat was excised, xed with 10% formalin, and
stained with a hematoxylin–eosin (H&E) reagent for histological
observations. The amount and the type of collagen deposition
were determined by Masson and Sirius Red (SR) staining,
respectively.
3. Results and discussion
3.1 SEM observation of construction of the chitosan–HNTs
composite sponges
The morphology of the lyophilized chitosan and chitosan–HNTs
sponges was investigated by SEM (Fig. 2). All of the sponges
show honeycomb-like porous microstructures with a pore
diameter of about 200 mm and pore-wall thickness in nanome-
ters. The addition of HNTs has a slight effect on the pore
structure of the chitosan sponge even with 80 wt% HNTs
loading. Such interconnected micro-pore structures of the
sponges provide efficient channels for rapid liquid gas trans-
port, beneting their wound healing applications. In the
enlarged images of the composite sponges (Fig. 2f), it is clear
that HNTs are embedded in the chitosan matrix with indistinct
interfaces, suggesting their strong interfacial interactions
between HNTs and chitosan due to their hydrogen bonding and
electrostatic attraction.
27
It should be noted that the roughness
of the pore-wall for the sponges may be increased by the pres-
ence of the nanoparticles.
27–29
Moreover, the rough surface
benets the adhesion and growth of cells compared to smooth
surface.
29,30
This is also conrmed in this work and will be
shown in the cell experiment result below.
3.2 Physicochemical characterization of chitosan–HNTs
composite sponges
The density and porosity of the prepared sponges were deter-
mined and the results are shown in Fig. 3a. As expected, the
density of the chitosan–HNTs sponges increased linearly with
the loading of HNTs. This is attributed to the fact that the
concentration of chitosan solutions is xed with the gradual
addition of HNTs into the aqueous solution when preparing the
sponges. Therefore, in the same volume of the sponges, the
amount of materials in the composite sponges increases with
the loading of HNTs. As a result, the density of the composite
sponges increases by the addition of HNTs. The increased
density of the sponges is benecial to the improvement of
mechanical properties such as dimensional stability. A slight
decrease in porosity of the sponges is obtained with an increase
in HNTs loadings. The pure chitosan sponges have a maximum
porosity of 94.3%, while the porosity is decreased to 80.7% for
the CS1N4 sponge. The porosity of the sponges is a critical
factor in determining the gas permeability, uid absorption
capability, cell migration behavior, and mechanical perfor-
mance. The high porosity of pure chitosan sponges is helpful in
Fig. 4 Compressive stress–strain curves for chitosan–HNTs
composite sponges: (a) CS; (b) CS2N1; (c) CS1N1; (d) CS1N2; (e) CS1N4.
The inset shows the region for determining the compressive modulus
of the samples.
Table 1 Summary of the mechanical properties data (data in the parentheses indicates the standard deviations)
Sample
Elastic
modulus (kPa)
Stress at 60%
strain (kPa)
Maximum
load (N)
Deformation recovery
ratio (%)
CS 311.7 (259.0) 55.1 (5.7) 59.9 (20.9) 9.36 (6.98)
CS2N1 408.7 (239.2) 101.0 (3.1) 75.2 (9.9) 23.11 (3.72)
CS1N1 578.3 (456.1) 151.0 (15.6) 122.0 (47.5) 30.91 (4.14)
CS1N2 1428.8 (860.2) 220.0 (10.8) 260.8 (76.2) 19.33 (4.89)
CS1N4 3054.0 (1115.5) 458.0 (24.8) 870.4 (523.6) 9.85 (5.39)
Fig. 5 Images of the sponges and freeze-dried HNTs powder (a),
blood on the materials with CaCl
2
solution (b), clotted blood on the
materials after culture at 37 C for 30 min (c), and the corresponding
aqueous solution (d). From left to right and from top to bottom: CS;
CS2N1; CS1N1; CS1N2; CS1N4, HNTs powders, and blood.
23544 |RSC Adv.,2014,4, 23540–23553 This journal is © The Royal Society of Chemistry 2014
RSC Advances Paper
absorbing exudate from the wound surface and facilitating the
transfer of nutrients and medium for the cells. However, the
sponges with high porosity suffer from weak stress resistance
especially during compression. The inuence of porosity on the
mechanical performance of the sponges will be discussed in the
following section.
The comparison of the swelling ratio of pure chitosan and
chitosan–HNTs sponges in PBS solutions is given in Fig. 3b.
On day 1, the sponges have the swelling ratios in the range of
8.8–16.4. With the extension of immersing time, all of the
samples exhibit an increasing trend in the swelling ratio. For
example, the swelling ratio of CS1N1 sponges increases from
14.7 on day 1 to 17.2 on day 7. With respect to the differences in
swelling ratios among the samples, apart from those of pure
chitosan, the swelling ratios of the composite sponges decrease
with the loading of HNTs. The hydrophilicity of the materials
and the pore structure of the sponges affect their swelling
ratios. The decreasing degree of swelling is attributed to the low
porosity, as shown in Fig. 3a, and the relatively low water
absorption ability of HNTs (3–5.3%)
31
compared with the same
quality of chitosan (48%)
32
in the composite sponges. The
pure chitosan sponge has a moderate swelling ratio among the
samples, indicating that the water retention of the sponges can
be adjusted by the addition of HNTs. The hydrophilicity of the
prepared sponges is expected to accelerate the blood coagula-
tion process and enhance cell attachment and proliferation
during the tissue regeneration process.
The structural integrity of the sponges under biologically
relevant pH and ion concentrations is vital to ensure sufficient
maintenance of mechanical strength and porosity for cell
interactions. The weight losses of the pure chitosan and chito-
san–HNTs composite sponges were monitored as a measure of
degradation in biological buffer (PBS) over 28 days (Fig. 3c). It
can be seen that with the increase in the contact time, all of the
samples decrease in weight in PBS. With incorporation of the
HNTs, chitosan sponges show a decreased weight loss ratio.
This is attributed to the primary degradation of the sample
related to the chitosan chain breakage while HNTs almost do
not degrade in PBS solutions. With the increase in the loading
of HNTs, the relative contents of the chitosan in the samples
decrease. As a result, the weight loss ratio of the composites is
lower than that of pure chitosan. Furthermore, the interfacial
interactions between HNTs and chitosan can constrain the
molecular mobility of chitosan. Note that HNTs play the role of
protecting chitosan against the attack by the medium. However,
when the loading of HNTs is increased to 80 wt% (CS1N4), the
weakened interfacial interactions may increase the chance of
exposure of the chitosan chain in the medium. As a result, the
CS1N4 shows a slight increase in the weight loss ratio compared
with that of CS1N2. The decreased degradation rate of chitosan
by the nanollers has also been reported.
26,33,34
Fig. 6 Whole-blood clotting evaluation of the pure chitosan, HNTs
and the chitosan–HNTs composite sponges.
Fig. 7 SEM images of platelet activation on pure chitosan (a), CS1N1 (b), and CS1N4 (c) sponges.
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 23540–23553 | 23545
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3.3 Mechanical property of chitosan–HNTs composite
sponges
The inuences of HNTs on the mechanical properties of chitosan
sponges were investigated via the compression test. Fig. 4 shows
typical compressive stress–strain curves for pure chitosan and
chitosan–HNTs composite sponges. Table 1 summarizes the data
on the mechanical properties of the samples. HNTs can effec-
tively increase the compressive modulus and strength of chito-
san, and the increasing trend is proportional to the HNTs
loadings. For example, the elastic modulus of CS1N4 is 3054 kPa,
which is an 8.8-fold increase relative to that of pure chitosan. The
reinforcing ability of HNTs for chitosan is attributed to both the
high strength of the tubes and the good interfacial interactions in
the composite systems as illustrated before.
17,27
Moreover, the
decrease in porosity by the incorporation of HNTs also helps in
improving the compression properties. On the other hand, ex-
ibleness iscritical for the practical application of wound dressing
materials as well as their compression strength.
35
Amaterialwith
proper exibleness is benecial for close contact with the
wounded surface. Due to the difficulty in direct determination of
impact toughness of the sponges, we employed the deformation
recovery ratio to compare their exibleness. In Table 1, all the
composite sponges exhibit higher deformation recovery ratio
compared with pure chitosan. The maximum deformation
recovery ratio of the composite sponges (CS1N1) is three-fold
compared to pure chitosan, suggesting the good elasticity of the
samples. The lowered deformation recovery ratio of the
composite sponges at relatively high HNTs loading (CS1N2 and
CS1N4) is attributed to both the decrease of the chitosan content
and the weakened interfacial interactions. All these results
demonstrate that the chitosan–HNTs composite sponges can
fulll the essential requirements for dressing materials to be
used on wound healing under high stresses and can provide
mechanical support for the protection of wound surfaces and
facilitation of cells attachment.
3.4 Evaluation of whole-blood clotting and platelet
activation
In order to evaluate the inuence of HNTs on the blood clotting
behavior of chitosan sponges, whole blood was kept in contact
Fig. 8 SEM images of fibroblast (NIH3T3) cultured on CS–HNTs nanocomposite scaffolds after 3 days: (a) CS; (b) CS2N1; (c) CS1N1; (d) CS1N2; (e)
CS1N4; (f) enlarged image of CS1N2 sample with artificial staining showing the cells.
23546 |RSC Adv.,2014,4, 23540–23553 This journal is © The Royal Society of Chemistry 2014
RSC Advances Paper
with the prepared sponges. For comparison, the freeze-dried
HNTs powder was selected as control. The appearance of the
blood clot caused by the sponges and HNTs powder is shown in
Fig. 5. It can be seen that the chitosan–HNTs composite
sponges have much higher blood clotting ability compared with
pure chitosan or neat HNTs. When the blood is dripped onto
the composite sponges, the blood can be rapidly absorbed in
the porous composite sponges. However, for the pure chitosan
sponges and HNTs powder, the blood seems to have hardly
inltrated the material. To quantify the clotting ability of the
samples, red blood cells (RBCs) that were not trapped in the
sponges and the HNTs were hemolyzed with water, and the
absorbance of the resulting hemoglobin solution was measured
(Fig. 6). A higher absorbance value of the hemoglobin solution
thus indicates a slower clotting performance. All of the
composite sponges exhibit lowered absorbance than that of the
pure chitosan sponge. For example, the CS2N1 and CS1N2
sponges show an 82.2% and 89.0% decrease in the absorbance
value, respectively, compared with pure chitosan, suggesting
their high clotting ability. However, in a similar research,
adding nano-sized ZnO has nearly no effect on the clotting
properties of chitosan and b-chitin.
26,36
Incorporation of nano
chondroitin sulfate into chitosan–hyaluronan blend can lead to
50% decrease in the absorbance value of the hemoglobin
solution.
37
Therefore, HNTs are superior to other nanoparticles
in view of clotting ability.
Generally, the clotting behavior of wound dressing is related
to the chemical composition, morphological features, and 3D
microstructure of the materials. Chitosan is a hemostat, which
can help in natural blood clotting and blocks nerve endings and
hence reduces pain. Chitosan's hemostasis ability can be
attributed to the attraction of negatively charged residues on
red blood cell membranes by protonated amine groups and the
adsorption of brinogen and plasma proteins. On the other
hand, HNTs are porous inorganic nanomaterials with hollow
lumen structure, resulting in a high absorption ability for many
types of active compounds. From the clotting experiment
results, we can speculate that HNTs can shorten both the time
lag for initial thrombin generation as well as the time to peak
thrombin generation. Therefore, HNTs can accelerate the
Fig. 9 SEM images of vascular endothelial cell cultured on CS–HNTs nanocomposite scaffolds after 3 days: (a) CS; (b) CS2N1; (c) CS1N1; (d)
CS1N2; (e) CS1N4; (f) enlarged image of CS1N2 sample.
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 23540–23553 | 23547
Paper RSC Advances
production of sufficient amounts of thrombin to support earlier
brin generation. Due to the interactions between HNTs and
chitosan and the 3D pore structures of the sponges, the
composite sponges could trap more RBCs to enlarge and
solidify the growing thrombus, leading to more rapid and stable
clotting compared with pure chitosan. With respect to the HNTs
powder, the low clotting ability can be attributed to the highly
aggregated state of the tubes. The clotting ability of the
sponges was further conrmed by the platelet adhesion exper-
iment via the SEM observation (Fig. 7). It can be seen that the
platelets exhibit spread morphology on the sponge surfaces,
suggesting that the materials can activate platelets during
wound healing.
Overall, the results of the hemostatic assays show that the
CS1N2 sponges is the best among the samples for enhancing
hemostasis, since it leads to the fastest blood clotting
and platelet adhesion. Since the swelling ratio in PBS solu-
tions by CS1N2 is comparable to that of pure chitosan,
the enhanced blood absorption can be attributed to
specic attractions of blood proteins and other blood
components with HNTs. The ability of the composite sponges
to absorb more blood should assist in stopping high ow
hemorrhage and removing excess exudates at the wound
interface.
3.5 Cell attachment and spread on the chitosan–HNTs
composite sponges
The inuence of HNTs on the cytocompatibility of chitosan
sponges was assessed using the broblasts and endothelial
cells. The morphology, adhesion, and spreading of the cells on
pure chitosan and chitosan–HNTs composite sponges were
observed by SEM as a result of the opacity of the composite lms
(Fig. 8 and 9). The two types of cells can spread on all of the
samples aer culturing for 3 days. SEM examination at
higher magnication of the cell morphology (Fig. 8f) shows
cellular extensions interacting closely with tubular HNTs in
the composite sponges even when HNTs loading is as high as
80 wt%, indicating good cytocompatibility of the inorganic
nanotubes. It can also be seen that the cells on the pure chi-
tosan fail to spread completely. This is not a result from the
possible toxicity of chitosan, but can be attributed to the
smooth pore wall structures. From the morphology results
above, the pore-walls of the composites are rougher than that of
chitosan, therefore leading to the better spreading of the cells
on them. The cell experiment results demonstrate a promotion
effect of HNTs for the cell attachment and growth due to their
high surface roughness of the composites and biocompatibility
of HNTs. In our previous study, the cytocompatibility of HNTs
was conrmed using the osteoblasts and broblasts in polyvinyl
Fig. 10 Appearance of wounds treated with oily cotton gauze (OY), adhesive wound dressing (AWD), pure chitosan sponge, and chitosan–HNTs
composite sponge.
23548 |RSC Adv.,2014,4, 23540–23553 This journal is © The Royal Society of Chemistry 2014
RSC Advances Paper
alcohol (PVA)–HNTs nanocomposites.
38
The morphological,
physicochemical, blood-clotting properties and in vitro cell
attachment and spread results of the chitosan–HNTs composite
sponges indicate the possibility of them being used as wound
dressing materials. These results stimulated us to evaluate their
in vivo biological properties using full-thickness skin wound in
an animal model.
3.6 In vivo wound healing evaluation of the chitosan–HNTs
composite sponges
In vivo study conducted in SD rats demonstrates the enhanced
wound healing ability of chitosan sponges by HNTs. Fig. 10
shows the images of the wound healing process aer treatment
with different materials. On the day of surgery, no visible
difference in wound appearance is found. Obviously, for all the
groups the wound shows granulation tissue formation with the
extension of time. Except for the wounds treated by AWD, the
wounds in the rats are nearly completely closed aer a 4 week
treatment. The regenerated skin is smooth and similar to
normal skin without scar formation aer 4 weeks, indicating
the good healing ability for the skin tissue by the materials
used. With respect to the differences among the groups, the
unhealed area of the AWD group is much larger than that of
other groups. Moreover, the composite sponges have much
higher wound healing rate and contraction ability than those of
pure chitosan sponges. The extent of wound closure was
quantied at different time points and the results are shown in
Fig. 11. Aer one week, the composite sponges show a 3.4–21-
fold increase in closure ratio compared with the pure chitosan.
Especially, the CS1N4 sponges show the highest closure ratio of
22%. Aer two weeks, apart from the AWD and OY groups, the
wounds treated with the chitosan–HNTs composite sponges
exhibit a linear increase in closure ratio with the loading of
Fig. 11 Evaluation of the wound area closure treated by different
dressing materials.
Fig. 12 Photomicrographs of hematoxylin and eosin (H&E)-stained normal skin (a), OY treated wounds (b), AWD treated wound (c), pure chi-
tosan sponge treated wound (d), and chitosan–HNTs composite sponge treated wound ((e–h), (e) CS2N1; (f) CS1N1; (g) CS1N2; (h) CS1N4). The
GT represents the granulation tissue and NE represents the neoepidermis.
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 23540–23553 | 23549
Paper RSC Advances
HNTs. For example, the wound closure of CS1N4 sponges is
85%, which is 32% higher than that of the pure chitosan
sponges. For all groups, the data of closure aer three and four
weeks are slightly different, suggesting that wound healing is
achieved aer 3 weeks. The maximum closure at 28 day is
98.0%, which corresponds to the CS1N1 group. The data is
substantially higher than that of pure chitosan groups, which is
only 87%. It should also be noted that although OY can effec-
tively repair the wound, the possibility of secondary damage
upon removal limits their applications in skin regeneration.
From the in vivo healing experiment results, we can conclude
that the incorporation of HNTs into chitosan can accelerate
wound healing especially at the initial stage. This is attributed
to the synergistic effect of chitosan and the HNTs. There are
numerous reports on wound dressing materials based on chi-
tosan.
25
The promotion of wound healing and scar prevention is
realized by the stimulation of the inammatory cell aggregation
and promotion of the cell migration into the wound area by the
porous scaffolds.
39
The improvement of wound healing ability
of chitosan sponges by HNTs may be attributed to their
intrinsic, high hemostasis properties and promotion of cell
migration into the wound areas. In fact, the raw HNTs were
commonly used as wound treatment materials in ancient
China, although the mechanism for wound healing was not
clear. The relevant mechanism will be discussed in the
following section. Above all, the prepared wound dressing
composite sponges have advantages such as rapid hemostasis,
accelerated tissue regeneration, promoted cell attachment, and
low cost. Furthermore, HNTs in the composite sponges can be
used as a vehicle for biopharmaceuticals, antimicrobials,
growth factors, and functional gene to wounds owing to their
perfect lumen structures. From a practical point of view, the
composite sponges are not dissolved or adhered to during the
application period to the wound and are easy to remove without
ripping the skin. Therefore, the chitosan–HNTs sponges have
promising applications as wound dressing materials in skin or
other organ regeneration.
3.7 Histological observation in the wound area
The nal goal for the wound dressing of the skin is to restore the
structural and functional properties to the levels of normal
tissue, involving the re-epithelialization and orchestrated
regeneration of all the skin appendages.
40
Fig. 12 shows the
histological observations for the growth status and structure of
epithelial tissue in each group at week 4 aer the operation. It
can be seen that for all the groups the wounds are closed
without signicant difference in the surrounding normal skin
tissue. The areas of epithelialization and granulation tissues,
including dense broblast deposition in the composite sponges
groups, are found to be larger than those in AWD and OY
groups. Re-epithelialization on the granulation tissue in open
wound can form a barrier between the wound and the envi-
ronment, which is very important for wound healing. The
Fig. 13 Photomicrographs of Masson-stained normal skin (a), OY treated wounds (b), AWD treated wound (c), pure chitosan sponge treated
wound (d), and chitosan–HNTs composite sponge treated wound ((e–h), (e) CS2N1; (f) CS1N1; (g) CS1N2; (h) CS1N4).
23550 |RSC Adv.,2014,4, 23540–23553 This journal is © The Royal Society of Chemistry 2014
RSC Advances Paper
epithelialization rate and the deposition of collagen in the
dermis are increased by HNTs. The wound treated by the CS1N2
sponge shows the minimum wound area among the various
groups. Fully differentiated epidermic cells, closely arranged
basal cells, and the horny layer and large amounts of hair and
sebum are observed in the wound treated by the composite
sponges, suggesting a similar epithelial structure to the normal
skin. These results suggest HNTs can accelerate the prolifera-
tion of neonatal granulation tissue and offer optimum condi-
tions for epithelial cell migration during wound healing, as well
as the production of collagen by broblasts.
As shown in the HE staining results above, collagen depo-
sition is observed in all the groups. Moreover, the amount and
the type of collagen bers are important for the keloids and
hypertrophic scar formation. In the present study, Masson and
SR staining were used to analyse the collagen deposition and
remolding in the regenerated tissues. Fig. 13 shows the Masson
staining images of the wound for different groups, in which red
denotes keratin and muscle bers, blue or green denotes
collagen and bone, light red or pink denote cytoplasm, and dark
brown or black denotes cell nuclei. The collagen bers are ne
and matured in the wound treated by the composite sponges,
and their arrangement is similar to that of native skins. Fig. 14
shows the polarizing microscope images of collagen bers by SR
staining, in which the yellow/red color denotes collagen type I,
red/white denotes collagen II, green denotes collagen type III
and light yellow denotes collagen type IV. It can be seen that the
type I collagen is the main component for the native skin. At
week 4 aer operation, the regenerated tissue in all the groups
consists chiey of collagen type I with a small amount of other
types of collagen interspersed in the tissue, which is close to the
prole of native skin. This also suggests the good healing ability
of the composite sponges as well as the chitosan sponges.
Wound healing is a complex process, which consists of a
series of coordinated overlapping biological events, involving
acute and chronic inammations, cell division, and extracel-
lular matrix (ECM) synthesis. From the histological studies by
the different staining, expect the AWD, all dressing materials
exhibit good healing ability for the wound. Especially, the chi-
tosan–HNTs composite sponges show improved wound healing
ability. The mechanisms involved in benecial healing activity
should be attributed to the unique characteristics of HNTs.
HNTs in the composite markedly increase the nano-roughness
of the pore-wall for the sponges, which could (i) favor the
trapping of factors detrimental to the repair process when
present in excess (such as proteases, or reactive oxygen species),
(ii) stimulate the progressive release of active fragments, which
are reported to recruit and activate leukocytes and mesen-
chymal cells, and (iii) increase the surface available for protein
coating and cell adhesion. Moreover, the enhancement in the
mechanical property of the chitosan sponges by HNTs strongly
regulates the phenotype and the differentiation process of the
cells. In short, the highly porous structure and good mechanical
properties of the chitosan–HNTs composite sponges allow
Fig. 14 Polarimicroscope images of SR-stained normal skin (a), OY treated wounds (b), AWD treated wound (c), pure chitosan sponge treated
wound (d), and chitosan–HNTs composite sponge treated wound ((e–h), (e) CS2N1; (f) CS1N1; (g) CS1N2; (h) CS1N4).
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 23540–23553 | 23551
Paper RSC Advances
gaseous and uid exchanges, stop bleeding and absorb excess
exudates, facilitate the attachment and spread of the cells, and
promote the healing of the wounds, indicating that they are
one of the most suitable wound dressing materials. Among
the samples, the CS1N2 sponges with 33 wt% chitosan and
66 wt% HNTs exhibit the best overall performance. They show
an 89.0% increase in clotting ability and 12%, 29.3%, 7.1% and
3.1% increase in the wound closure ratio aer 1, 2, 3, and 4
weeks, respectively, compared with those of the pure chitosan
sponges.
4. Conclusions
The chitosan–HNTs composite sponges with different HNTs
loadings are prepared by lyophilization. The addition of HNTs
slightly affects the pore structure of the chitosan sponges even
with 80 wt%. A slight decreasing trend in porosity and weight
loss in PBS of the composite sponges is obtained with the
incorporation of the HNTs. The swelling ratios of the pure
chitosan and chitosan–HNTs sponges in PBS solutions are
comparable. HNTs can simultaneously enhance the elastic
modulus, compressive strength, and toughness of the chitosan
sponges. The broblasts and endothelial cells can spread well
in the composite sponges, indicating their good cytocompati-
bility. The composite sponges show enhanced blood clotting
and platelet activation ability. The composite sponges with 67%
HNTs show an 89.0% increase in clotting ability compared with
that of pure chitosan. In vivo wound healing evaluation
conrms the enhanced healing ability of the chitosan sponges
by HNTs. The composite sponges show a 3.4–21-fold increase in
wound closure ratio compared with that of pure chitosan aer
one week. The addition of HNTs helps in faster re-epithelial-
ization and collagen deposition. All these are attributed to the
unique characteristics of HNTs and the synergistic effect of
chitosan and the HNTs. Overall results demonstrate that these
advanced chitosan–HNTs composite sponges have many
potential applications for burn, chronic, and diabetic wound
infections.
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (81272222), the Guangdong
natural science funds for distinguished young scholar
(S2013050014606), the foundation for the author of Guangdong
excellent doctoral dissertation (sybzzxm201220), Guangdong
science and technology project (2011B031300026), Guangzhou
science and technology project (2013J4100099), Guangzhou
applied basic research project (2013J4100100), the project of
regional demonstration for Guangdong ocean economic inno-
vative development (GD2012-B03-009), the research fund for
the doctoral program of higher education of China
(20114401120003), and the key project of department of
education of Guangdong province (cxzd1108). The authors also
thank Dr Hau-To Wong for reading and revising of the
manuscript.
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