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Chitosan topical gel formulation in the management of burn wounds

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DOI: 10.1016/j.ijbiomac.2009.03.010 · Source: PubMed

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 International Journal of Biological Macromolecules 45 (2009) 16–21

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Chitosan topical gel formulation in the management of burn wounds

Ibrahim A. Alsarra ∗
Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Wound healing properties of chitosan with different molecular weight and degree of deacetylation ranges
Received 25 February 2009 have been examined. The macroscopic image and histopathology were examined using chitosan, Fucidin®
Received in revised form 24 March 2009 ointment and to blank. The rate of contraction was evaluated by determination of the unclosed area as
Accepted 25 March 2009
a function of time. The treated wounds were found to contract at the highest rate with high molecu-
Available online 2 April 2009
lar weight–high degree of deacetylation chitosan-treated rats as compared to untreated, treated, and
Fucidin® ointment-treated rats. Wounds treated with high molecular weight chitosan had significantly
Keywords:
more epithelial tissue (p < 0.05) than wounds with any other treatment and the best re-epithelization
Chitosan
Wound healing
and fastest wounds closure were found with the high molecular weight chitosan treatment group. His-
Histophathological studies tological examination and collagenase activity studies revealed advanced granulation tissue formation
Fucidin® and epithelialization in wounds treated with high molecular weight chitosan (p < 0.05). High molecular
Topical applications weight with high degree of deacetylation chitosan samples therefore demonstrates potential for use as a
treatment system for dermal burns.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction biodegradability properties [3,4]. Chitosan appears to have no

Slow healing and non-healing wounds, such as ulcers, as well
as wounds caused by major or minor injuries, surgery, or burns,
represents the most widespread treatable conditions encountered
by humans and animals. Wound repair is a well highly coordinated
process that involves a series of overlapping phases: inflammation,
cell proliferation, matrix deposition and tissue remodeling. Under-
lying repair is a complex dynamic series of events including clotting,
inflammation, granulation tissue formation, epithelialization, neo-
vascularization, collagen synthesis, and wound contraction [1].
Briefly, the wound healing process consists of three major stages.
First, inflammatory cells from the surrounding tissue move towards
the lesion site. Subsequently, fibroblasts appear and begin to pro-
duce collagen connective fibers that impart tensile strength to the
regenerating tissue. Simultaneously, numerous capillaries begin to
form to supply the site with nutrients and oxygen, while the epithe-
lial cells at the edge of the wound start filing in the area under the
scab. In the third and final phase, the new epithelium forms and the
wound is considered healed [2].
Chitosan is obtained by partial deacetylation of the amines
of chitin, which yields a copolymer of N-acetyl-glucosamine and
N-glucosamine. Its use has been explored in various biomaterial
and medical applications. Chitosan has desirable qualities, such as
hemostasis, wound healing, bacteriostatic, biocompatibility, and
∗ Tel.: +966 1 4677504; fax: +966 1 4676295.
E-mail address: ialsarra@ksu.edu.sa.
adverse effects after implantation in tissues and, for this reason,
it has been used for a wide range of biomedical applications [5].
Chitosan may be used to inhibit fibroplasia in wound healing and
to promote tissue growth and differentiation in culture [6].
It is commonly accepted that the ideal wound covering should
mimic many properties of human skin. It should be adhesive, elas-
tic, durable, occlusive and impermeable to bacteria [7]. Because of
their biocompatibility, ability to absorb exudates, and film form-
ing properties, chitosan products are good candidates for burn and
wound management [8].
The main parameters influencing the characteristics of chitosan
are its molecular weight (MW) and its degree of deacetylation (DD)
[9]. Due to the diversity of the sources of chitosan, and to the fact
that it is commercially available with a wide range of DD and MW,
each of which may have an effect on chitosan properties, it is impor-
tant to take into consideration the effects of these parameters on
biomedical activity in order to optimize the desired application.
The molecular weight of chitosan is likely to be the more impor-
tant property because a minimum molecular weight is often needed
to achieve the desired property [10]. Several researchers have found
that the acceleration of wound healing by chitosan is related to its
chemical structure, whereas other reports indicated that the wound
repair effect is related to the different physical forms of the chitosan
samples used [11–13]. This discrepancy appears to result, at least in
part, from the different chemical compositions and physical forms
of the biopolymer samples investigated, making it difficult to dif-
ferentiate the relationship between chitosan structure and its effect
on the wound healing process.

0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2009.03.010
 I.A. Alsarra / International Journal of Biological Macromolecules 45 (2009) 16–21 17

Minagawa et al. studied the effects of chitin and chitosan on gel solutions were sonicated to remove air bubbles. Each of the test
wound healing with reference to chemical properties using a lin- samples and control was filled into 4-oz tubes.
ear incisional wound model in rats. Chitin with MW of 300 kD and
chitosan with MW of 80 kD were used as polymers samples, along 2.3. Animals
with monomers and oligomers, at different concentrations to test
their effects on the acceleration of wound healing [14]. The study Healthy Wistar rats (male and female), weighing 180–220 g
revealed that, not only chitin and chitosan, but also their oligomers obtained from the Center for Laboratory Animals and Experimen-
and monomers accelerated healing. However, samples were lim- tal Surgery, College of Medicine, King Saud University were used in
ited in molecular weight and the relationship between the presence the evaluation of the wound healing properties of the tested agents.
of free amine groups and wound healing enhancement was not The animals were randomly distributed into five treatment groups
studied. (2%, w/v high, medium or low chitosan, Fucidin® ointment positive
Chitosan has been used in the forms of filament, powder, gran- control, or no treatment) and were maintained in accordance with
ule, sponge, and a composite with cotton or polyester in most the recommendations in the University Guide for the Care and Use
studies. Therefore, the effect of chitosan on the acceleration of of Laboratory Animals, approved by Animal Care and Use Commit-
wound healing could not be completely achieved, due to the rel- tee. The rats were placed in individual cages where they had free
atively low interaction between the wound site and the healing access to food and clean drinking water during the two weeks of
agent [4,15]. acclimatization and throughout the experimental period. On the
DD is a structural parameter which influences physicochemical day following the last administration, the animals were sacrificed.
properties of chitosan [16]. It also influences many biological prop-
erties, including biodegradation by lysozyme [17], wound healing
2.4. Establishment of skin burn wounds
properties [18]. In order to fully understand the interaction of MW
and DD of chitosan, it was of particular importance to study these
Procedures involving animals were conducted in accordance
effects (MW and DD) dependently. In a previous study, we have
with U.S. guidelines as found in the NIH Guide for the Care and
showed that the effects of the degree of deacetylation and molec-
Use of Laboratory Animals (NIH Publication No. 18–23, 1985). Each
ular weight are best studied using a quadratic model, an empirical
rat was anesthetized by an intramuscular injection of ketamine
mathematical model, that can accommodate linear, quadratic, and
and xylazine, at dose of 40 and 5 mg/kg body weight, respectively.
interactive predictors to better understand these effect appropri-
The surgical area was shaved with an electric razor and disinfected
ately; this practice is widely accepted in industry and becoming
using 70% ethanol. After a deep surgical plane of general anesthesia
more accepted in academia [19]. Therefore, to explain the effects
had been reached, a wound, approximately 1 cm in diameter, was
of the degree of deacetylation and molecular weight, influence of
inflicted for 15 s on the dorsal side of the rats using water-boiled,
the interaction between these characteristics must be considered
curved blade, surgical scissors. Both the epidermal and dermal lay-
[18].
ers were removed down to the panniculus carnosus muscle layer,
The present study examines the effect of chitosan, with samples
creating a full-thickness wound with minimal bleeding. The rats
covering a wide range of molecular weight and degree of deacety-
were grossly examined and photographed for measurement of
lation, on its ability to influence the wound healing process. Since
wound size reduction. The skin, including the entire wound with
chitosan is a linear polymer, this study thus also examines the effect
adjacent normal skin, was excised and histological examination was
of the chitosan polymer length on wound healing. To promote the
carried out.
interaction between the agent and the wound, chitosan samples
were applied directly on the wound site.
2.5. Treatment of wounds with chitosan gel

2. Experimental
Treatment, which started shortly after wound was produced,
consisted of applying each of the tested samples on the wound.
2.1. Materials
Group I animals were treated with 2% (w/v) low molecular weight
chitosan (CH-L); Group II with 2% (w/v) medium molecular weight
High molecular weight chitosan (CH-H) with a 2,000,000 MW
chitosan (CH-M); Group III with 2% (w/v) high molecular weight
and 92% DD, medium molecular weight chitosan (CH-M) with
chitosan (CH-H) and Group IV animals (positive controls) were
a 750,000 MW and 75% DD, and low molecular weight chitosan
treated by applying 2% Fucidin® ointment (FU); while Group V
with a 70,000 MW and 63% DD were obtained from Fluka Chemie
(untreated group) received no treatment.
AG (Buchs, Switzerland). The degree of deacetylation was mea-
Once daily for 12 days, the test samples were applied topically
sured using a circular dichroism method [20]. Curity® non-adhering
after cleaning the wound with a dilute solution of Dettol® . The
dressing and Curity® Sheer Bandages were purchased from Kendall
wounds were then covered with a Curity® non-adhering dress-
Company (Mansfield, MA, USA), and Autoclips® were from Bec-
ing, bandage with a Curiy® Sheer Bandage, secured with 9-mm
ton Dickinson (Sparks, MA, USA). Type I collagenase measurement
Autoclips® and allowed to heal [20,21]. The bandages were replaced
kit (YU-16001) purchased from Yagai Company Limited (Yama-
every other day. Similar procedures were also applied to the control
gata, Japan), while fusidic acid (Fucidin® ) was obtained form local
group animals.
market. All other chemicals used were of analytical grade and the
highest quality available and were considered pure for these stud-
ies. 2.6. Wound size reduction

The wound area of each animal was measured on days 4, 8, and

2.2. Preparation of chitosan gels 12 post-surgery. The wound size measurements taken at the time of
surgery and at the time of biopsy were used to calculate the percent
Gels were prepared by dissolving 2% (w/v) of high, medium wound contraction, using Eq. (1):
or low molecular weight chitosan in 1% (v/v) aqueous acetic acid.
Methyl paraben sodium salt (0.1%, w/w) as a preservative was added A0 − At
%wound contraction = × 100 (1)
to the tested samples. The samples were stirred and the resulting At
18 I.A. Alsarra / International Journal of Biological Macromolecules 45 (2009) 16–21

Table 1
The compositions of various topical formulations.

Formula code Description Composition (w/w)

Blank Untreated group –

CH-H High molecular weight chitosan 2%
CH-M Medium molecular weight chitosan 2%
CH-L Low molecular weight chitosan 2%
Positive control Fucidin® ointment 2%

where A0 is the original wound area, and At is the area of wound at

the time of biopsy.

2.7. Histological examination

Excised wound sites were formalin fixed, and then processed

and embedded in paraffin. Thick sections (3–5 ␮m) were stained
with haematoxylin and eosin stain and photographed under 40×
magnification. Histological examination included a quantitative
measurement of the percent of re-epithelialization, a quantitative
measurement of the inflammatory response employing the follow-
ing Eq. (2):

D0 − DB
%re-epithelialiazation = × 100 (2)
DB

where D0 is the original wound diameter, DB is the length of un-

epithelialized tissues at the time of biopsy.
Fig. 2. Photographs of macroscopic appearances of wound excised from rats that
were untreated (control), treated with high molecular weight chitosan (CH-H), or
2.8. Collagenase activity treated with Fucidin® ointment (FU).

The collagenase activity in the tissue was assayed with type I col-
lagenase measurement kit (YU-16001, Yagai Co., Yamagata, Japan).
Each sample was applied topically at wounding [22,23]. The colla- 3. Results
genase activity was expressed in units per microgram of protein in
the tissue (mean ± s.d.; n = 3). The compositions of different tested topical formulations are
listed in Table 1, including chitosan of different molecular weights
and fucidin as a commercial wound healing product.
2.9. Analysis of data

All data are presented as the mean ± the standard deviation
(s.d.). Data were analyzed by one-way analysis of variance (ANOVA)
using the SPSS® statistical package (version 10, 1999, SPSS Inc.,
Chicago, IL). Statistical differences yielding P ≤ 0.05 were consid-
ered significant. Tukey’s multiple-comparison post hoc test was
applied when necessary.
Fig. 1. Percent of wound contraction for in vivo wound healing experiments
(mean ± s.d., n = 6).
3.1. In vivo wound healing experiments
In our wound healing model, a single full thickness skin wound
(about 1 cm) was made on the back of rats. The rate of contraction
of control and treatment wounds has been evaluated by determi-
nation of the unclosed area as a function of time (Figs. 1 and 2).
The treated wounds were found to contract at the highest rate
with CH-H-treated rats as compared to untreated, treated, and
FU-treated rats. By day 4, wounds treated with CH-H had sig-
nificantly more epithelial tissue (p < 0.05) than wounds with any
other treatment. At days 8 and 12, a notable difference occurred
because the % wound contraction of CH-H (Fig. 1) was signif-
icantly higher than any of the treated groups (p < 0.05). Eighty
percent wound closure could be achieved within 8 days for those
groups treated with CH-H. In contrast, control wounds with no
treatment healed more slowly and about 40% wound closure was
achieved only after 12 days (Fig. 2). There was no significant
difference between CH-M and FU treated groups (70% wound clo-
sure by day 12) at any time point suggesting the wound healing
property of CH-M is comparable to that of FU. The findings indi-
cate the CH-H had the superiority over other tested groups and
wound healing in both CH-H and FU treated groups occurred
faster than the control wound. There was a significant reduction
in wound size from day 4 onwards in rats treated with CH-H
and FU (compared to CH-M and CH-L) and FU and also, on later
days, the closure rate is much faster when compared to con-
trols.
 I.A. Alsarra / International Journal of Biological Macromolecules 45 (2009) 16–21
Table 2
Rate of wound re-epithelialization at 4, 8 and 12 days post wounding (n = 6).
Days of observation Wound re-epithelialization ± s.d. (%)
Untreated FU
4 27 ± 6 42 ± 3
8 56 ± 7 73 ± 5
12 72 ± 6 84 ± 6
3.2. Wound re-epithelialization
The percentage of re-epithelialization with the CH-H-treated
rats was compared to that of rats treated with low and medium
chitosan, or FU, or that received no treatment (Table 2). Superfi-
cially, control wounds showed a slight reduction in defect area at
day 4 (Fig. 1). On measuring the wound epithelium, it was found
that wounds had started healing. However, due to scab forma-
tion, the rate of epithelialization was significantly lower (Table 2)
in control wounds than wounds treated with either CH-H or FU
(p < 0.05). At 8 days, the rate of re-epithelialization increased to
85 ± 4% and 73 ± 5% for test wounds (CH-H and FU, respectively). By
days 12 post-surgery, CH-H treated groups had almost completely
re-epithelialized (97 ± 3%) as compared to the groups treated with
FU (84 ± 6% re-epithelialization) (p < 0.05).
3.3. Histological observations
The healing pattern of the wounds was studied by examining
the histology of the treatment and control samples at 4, 8 and 12
day post wounding (Fig. 3). The inflammatory phase is a normal and
necessary prerequisite to healing. This can be initiated by numerous
causes, one of which is injury [24]. Therefore, during the early stage
of wound healing, it is difficult to assess whether the inflammatory
response is part of normal healing process or due to the effect of
the test material [25].
On day 4, for the untreated group, the epidermis is com-
pletely destroyed and interrupted and the wound gap is filled by
necrotic material and acute inflammatory exudates. The underly-
ing subcutaneous tissue shows sever inflammatory filtrate with mid
proliferation of blood vessels forming granulation tissue (Fig. 3).
CH-L-treated rats exhibited a large wound gap filled with necrotic
Fig. 3. Histopathological photographs of the burn epithelial tissues stained with hematoxylin and eosin. Bars: 100 ␮m. The first column corresponds to tissues from untreated
group; the second to those treated with low molecular weight chitosan (CH-L); the third to those treated with medium molecular weight chitosan (CH-M); the fourth to
those treated with high molecular weight chitosan (CH-H); the fifth to those treated with Fucidin® ointment (FU) (×40).
19
debris and fibrin with underlying acute inflammatory exudates. No
significant granulation tissue is identified. There was no histologi-
cal difference between the groups treated with FU and CH-L (Fig. 3).
CH-H The photographs for CH-M reveal that there is necrotic debris with
extensive acute inflammatory exudates and fibrin, with underly-
52 ± 5
85 ± 4
ing early formation of neovasculariazation surrounded by fibrinous
97 ± 3 material. As for CH-H groups, the wound gap is filled by necrotic
debris and acute inflammatory exudates. The base of the wound
gap shows early formation of granulation tissue. The skin of the
wound edge contracted markedly.
At 8 days, in untreated wounds, the necrotic material and acute
inflammatory exudate is much less. The underlying area now shows
fibrosis with proliferation of fibroblasts that are replacing the gran-
ulation tissue and starting to fill up the gap (Fig. 3). CH-L treated
wounds showed residual acute inflammatory exudate. Fibrinous
materials are still seen filling the gap, surrounded by granulation
tissue. However, necrotic debris and fibrin were still seen superfi-
cially with underlying granulation tissue surrounded by fibrosis and
early scar formation with CH-M-treated wounds. The defect area in
CH-H wounds improved dramatically and become small (Fig. 3). The
epidermal re-growth is now complete with intact epidermis and
underlying scar tissue. Only minimal granulation is seen at subep-
ithelial level (Fig. 3). Acute inflammatory infiltrate is still evident
with FU-treated wounds (as compared to CH-H). The gap between
edges of the epidermis is smaller and the wound is filled by mature
granulation tissue.
3.4. Collagenase activity
Collagenase activities in each of the treatment samples were
increased significantly compared to that of the untreated sam-
ples. Among the chitosan-treated wounds, the activity was the
highest in CH-H-treated wounds (Fig. 4) suggesting a relationship
between the molecular weight and the collagenase activity; the
higher molecular weight chitosan was the most effective at stimu-
lating macrophage activation. Suzuki et al. indicated that a chitosan
sample with a higher DD induced a strong complementary activity
compared to a sample with a lower DD [26]. Fig. 4 also shows that
the granulation was more accelerated by CH-H on the 8th and 12th
day, and the amount of collagen was larger than that of the control
(Fig. 4). From these results, it was evident that CH-H also promotes
the granulation phase of healing.
20 I.A. Alsarra / International Journal of Biological Macromolecules 45 (2009) 16–21
Fig. 4. Effect of chitosan molecular weight on collagenase activity. On day 8 after
wounding, each sample was assayed for collagenase measurement. Results are
expressed as mean ± s.d. (n = 3). The first bar corresponds to the untreated group; the
second to the low molecular weight chitosan group; the third to the medium molec-
ular weight chitosan group; and the fourth to the high molecular weight chitosan
group.
4. Discussion
Burns remain a public health issue, especially in terms of
morbidity and long-term disability, throughout the world, but par-
ticularly in developing countries [27]. Burn wounds have a complex
healing process, cause severe discomfort, and are prone to infection
and other complications.
The study of the physicochemical interactions of chitosan with
the most important components of living matter appears to be
an essential point for the interpretation of biological responses
induced by the presence of chitosan in living media. Although
several studies have investigated the accelerating effects of open
wound healing by chitin and chitosan in multiple animal models,
most of the results were considered inconclusive. The resultant
wound healing effects were, in many cases, attributed to the
synergistic effect of the numerous wound dressing materials:
antimicrobial agents [28,29], alginate [30,31], gelatin [24,32] or
heparin [15,33,34].
The scope of this study, however, was to determine the effect of
chitosan samples with different molecular weight on the extent of
wound healing acceleration. To ascertain chitosan’s wound heal-
ing acceleration superiority, CH-H with high DD was compared
to a commercially available Fucidin® ointment that has an anti-
bacterial agent and is used to treat a number of bacterial skin
infections, skin irritation, and burning. It acts in the bacterial cell
by preventing the production of essential proteins necessary for
the growth and survival of the bacteria. This medicine is available
in several preparations, as a cream, gel, ointment and impregnated
dressing.
The primary factor in the acceleration of the wound healing pro-
cess can be attributed to the presence of N-acetyl-d-glucosamine
[29]. The wound healing depends not only on the quantitative pres-
ence of N-acetyl-d-glucosamine, but even more on the molecular
structure of which it is a component. The solubility of N-acetyl-d-
glucosamine and its rapid absorption by the tissues when it was
applied to topically to the wounds allowed far too short a residence
time in the wounds to enable it to exert a major effect on the healing
process [14].
Catalysis of chitosan degradation is known to be caused by
lysozyme, an enzyme transported to the wound sites by the
inflammatory cells (polymorphonuclear leucocytes). The abundant
lysozyme at the wound site would break chitosan to the active N-
acetyl-d-glucosamine dimer and provide for its sustained release.
Because lysozyme acts slowly on chitosan, chitosan becomes a con-
tinuous source of N-acetyl-d-glucosamine dimers as long as the
wound contains inflammatory cells releasing lysozyme. The inflam-
matory reactions and therefore the release of lysozyme, continues
until the wound is finally healed. Therefore, the more N-acetylated
monomer presents, the greater the expected healing acceleration,
as seen with rats treated with CH-H.
It is apparent that the effect of treatment of the burns with high
molecular weight (high DD) chitosan is statistically different from
either CH-L or CH-M. It is clear that the size of chitosan molecule
plays some role, with the high molecular weight samples being the
most effective. It was also found that CH-H with high DD sample
is statistically superior to that obtained with Fucidin® ointment.
Therefore it is evident that d-glucosamine has a major effect on
the rate of healing of surgical burns. It is also clear that treatment
with CH-H enabled the burned skin to more successfully resist the
adverse effects of the trauma and infection.
Chitosan was found to promote the migration of the inflamma-
tory cells which are capable of the production and secretion of a
large repertoire of pro-inflammatory products and growth factors
at a very early phase of healing [30]. FU-treated rats revealed a
site that was not completely healed but more improved and with
a smaller lesion than that of untreated groups. In comparison with
CH-H-treated wounds (after 12 days), the wound site was so per-
fectly healed that it was difficult to distinguish it from normal skin
(Fig. 2).
The enhanced collagenase activity is related to the remodeling
in the wound healing process. This enzyme is produced mainly by
fibroblasts and inflammatory cells. In the histology findings, more
activated fibroblasts and less inflammatory cells were observed at
the wound site, which suggests that mainly fibroblasts provide col-
lagenase.
The present study provided evidence that degree of deacetyla-
tion and molecular weight influenced wound healing acceleration.
As a result, it can be presumed that the amino residue is strongly
related to wound healing acceleration [14]. N-acetylglucosamine
may serve as a substratum for reinforcement of wounded tissues
without excessive inflammatory reaction. Chitosan induced the for-
mation of granulation tissue or re-epithelialization in the early
stages of wound healing.
5. Conclusion
High molecular weight with high degree of deacetylation chi-
tosan was found to be superior to medium or low chitosan
in wound healing. Histological findings indicate that chitosan
accelerates the reformation of connective tissues. This study has
directed high molecular weight chitosan to be forefront, offer-
ing truly valuable material in the field of wound healing and
certainly opening new avenues for future research and develop-
ment.
Acknowledgments
The author would like to thank Dr. Abdulmalik Al-Sheikh, Chair-
man, Department of Pathology, King Khalid University Hospital,
King Saud University for his kind help in the histological study and
the valuable scientific discussion. Gratitude is also extended to Dr.
Bander Al-Mahameed and Dr. Saleh Al-Harby, Center for Labora-
tory Animals and Experimental Surgery, College of Medicine, for
their help with the experimental animals. The author is grateful
to the Research Center, College of Pharmacy, King Saud Univer-
sity, for the financial support and the facilities to carry out this
study.
 I.A. Alsarra / International Journal of Biological Macromolecules 45 (2009) 16–21
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