Carbohydrate Polymers 199 (2018) 445–460

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Chitosan based hydrogels and their applications for drug delivery in wound T
dressings: A review
⁎
Hamid Hamedi, Sara Moradi, Samuel M. Hudson, Alan E. Tonelli
Textile Engineering Chemistry and Science, Fiber & Polymer Science Program, College of Textiles, North Carolina State University, Raleigh, North Carolina 27606-8301,
United States

A R T I C LE I N FO A B S T R A C T

Keywords: Advanced development of chitosan hydrogels has led to new drug delivery systems that can release their active
Chitosan hydrogel ingredients in response to environmental stimuli. This review considers more recent investigation of chitosan
Wound dressing hydrogel preparations and the application of these preparations for drug delivery in wound dressings.
Drug delivery Applications and structural characteristics of different types of active ingredients, such as growth factors, na-
Growth factor
noparticles, nanostructures, and drug loaded chitosan hydrogels are summarized.
Nanoparticles

1. Introduction
Hydrogels are three-dimensional, cross-linked networks which can
absorb and retain significant amounts of water, without dissolving or
losing their three dimensional structures (Ahmed, 2015; Kashyap, 2005;
Y.B. et al., 2008). The gelation and biodegradation are two key factors
affecting the fate of cells (Li et al., 2012). Moreover, some hydrogels
have antibacterial and antifungal activities (Jaya kumar et al., 2011),
and these properties could be useful for wound dressings and accel-
erating the wound healing process (Ueno et al., 2001). Polymers that
form hydrogels may have hydrophilic or hydrophobic functional
groups. Hydrophilic functional groups, such as hydroxyl (−OH), amine
(NH2) and amide (−CONH−CONH2) enable the hydrogel to absorb
water leading to hydrogel expansion, which is known as swelling.
During swelling, the cross-linked structure of hydrogels prevents com-
plete destruction of the hydrogel cross-links and dissolution. Hydrogels
with hydrophobic chains such as poly lactic acid have lower water
swelling capacity than hydrophilic lattices. Hydrogels can be prepared
from synthetic or natural polymers, involving a wide range of chemical
compositions and with different mechanical, physical and chemical
properties. Natural polymers such as alginate (Balakrishnan et al.,
2005; Kamoun et al., 2015; Kim et al., 2008; Murakami et al., 2010),
chitosan (Milosavljević et al., 2010; Racine et al., 2017), hyaluronic
acid (Luo et al., 2000; Segura et al., 2005; Silva et al., 2017) cellulose
(Abd El-Mohdy, 2013; Chang et al., 2009; Maneerung et al., 2008;
Sannino et al., 2009), starch (Pal et al., 2006; Pal et al., 2006; Zhai
et al., 2002), ulvan (Alvesab et al., 2012; Morelli & Chiellini, 2010),
gelatin (Draye, Delaey, Voorde, Bulcke, Reu et al., 1998; Wang et al.,
2012), pullulan (Li et al., 2011; Wong et al., 2011) and/or synthetic
polymers like polyvinyl alcohol (Kokabi et al., 2007; Razzak, 2001;
Yang et al., 2008), polyacrylamide (Ezra et al., 2009; Risbud & Bhonde,
2000; Rosiak et al., 1983) and polyethylene glycol (Ajji et al., 2005;
Gupta et al., 2011; Lih et al., 2012) form hydrogels.
Hydrogels are classified into two categories: chemical or permanent
gels that have covalently bonded cross-linked networks (replacing hy-
drogen bonds by strong and stable covalent bonds) and physical or
reversible gels whose networks are held together by molecular en-
tanglements, and/or secondary forces including ionic, hydrogen
bonding or hydrophobic interactions (Ahmed, 2015). In physically
cross-linked gels, dissolution is prevented by physical interactions,
which exist between different polymer chains (Hennink & Nostrum,
2002). Hydrogels can be formulated in a variety of physical shapes,
including slabs, microparticles, nanoparticles, coatings, and films
(Hoare & Kohane, 2008).
The most important property of hydrogels is their biocompatibility,
which can be defined as the ability of a material to be in contact with
bodily organs with minimum damage to the surrounding tissues and
without triggering undesirable immune responses (Caló &
Khutoryanskiy, 2015). In some cases this property has been taken ad-
vantage of in wound dressings, because after healing it prevents ex-
cessive tissue granulation and scar formation (Chung et al., 1994),
though they can be hard to handle, may be difficult to load with drugs
and sterilize, and usually have low mechanical strength (Huffman,
2012). In spite of these disadvantages, hydrogels have many applica-
tions in different fields including tissue engineering, pharmaceuticals
and biomedical engineering, in the forms of wound dressings (Benamer

⁎
Corresponding author.
E-mail address: atonelli@ncsu.edu (A.E. Tonelli).

https://doi.org/10.1016/j.carbpol.2018.06.114
Received 19 March 2018; Received in revised form 25 June 2018; Accepted 26 June 2018
Available online 28 June 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
H. Hamedi et al.
et al., 2006; Kokabi et al., 2007; Lu et al., 2010), drug delivery vehicles
(Hoare & Kohane, 2008; Qiu & Park, 2001; Xinyin, 2003), implants
(Refojo & Leong, 1981; Stasica et al., 2000; ZZZZZ, 2018), and in-
jectable polymeric systems (J.P. & T.H., 2006; Macaya & Spector, 2001;
Shibata et al., 2010; Yu & Ding, 2008).
Chitosan is one of the most widely used materials for making hy-
drogels, and has been considered for wound dressing applications. It
has excellent biocompatibility, low toxicity and immune-stimulatory
activities (Li, Rodrigues et al., 2012; Souza et al., 2009). Due to these
properties, chitosan shows good biocompatibility and positive effects
on wound healing. It can also accelerate repair of different tissues and
facilitate contraction of wounds (Jaya kumar et al., 2011). The Cyto-
compatibility of chitosan has also been studied and shown that it does
not have acute cytotoxic effect (Gonçalves Ferreira et al., 2016). On the
other hand, as a natural based compound, chitosan may be con-
taminated by organic and inorganic impurities, and presents broad
polydispersity. Chitosan is poorly soluble, except in acidic medium,
which makes analyses difficult to perform (Croisier & Jérôme, 2013).
Chitosan also has been used in commercial wound dressings such as
ChitoSam™ (sam medical, 2018), ChitoGauze XR pro (North American
Rescue, 2018), ChitoFlex (H.M.T. Inc., 2007), and Axiostat® (AXIO,
2018) which are high-performance hemostatic dressings.
This review emphasizes on chitosan hydrogels and their applica-
tions in wound dressing and drug delivery. Initially chitosan and its
properties are described and different kinds of hydrogel preparation
methods are discussed. In the second section the utilization of chitosan
hydrogels are summarized. Finally, utilization of some drugs such as
growth factors, nano particles and chemicals in chitosan hydrogels are
described.
2. Chitosan
Due to its reactive amino groups, chitosan is the only natural ca-
tionic polymer and has many commercial applications: it accelerates
wound healing, is antimicrobial, anticoagulant, antibacterial, anti-
fungal, anti-tumor, and haemostatic [62,63]. The generic term chitosan
describes a range of presumably pure poly-(beta-1-4) N-acetyl-D-glu-
cosamine materials (shown in Fig. 1) whose properties are highly de-
pendent on its degree of deacetylation; average molecular weight,
polydispersity, and its structure.
(Zhou et al. (2008)) investigated the effect of molecular weight and
degree of deacetylation on chitosan hydrogels and concluded that these
two parameters affected the pH values, turbidity, viscosity, and ther-
mosensitive characteristics of the product hydrogels. Degree of deace-
tylation affects chitosan antimicrobial activity (Chung et al., 2004; Liu
et al., 2001); on the other hand chitosan microspheres with a high
Fig. 1. Chitosan structure.
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Carbohydrate Polymers 199 (2018) 445–460
degree of deacetylation (97.5%) lead to higher positive charge density,
which confers stronger antibacterial activity than moderate degree of
deacetylation (83.7%) against Staphylococcus aureus at pH 5.5 (Kong
et al., 2008b). It was reported that polydispersity is a useful quantity
which can clarify the degradation mechanism (Chen et al., 1997).The
decrease of polydispersity as degree of deacetylation increases could
appear as surprising since the deacetylation reaction does not degrade
chitosan chains (Schatz et al., 2003). Min et al. (Tsai et al., 2008) have
studied factors that are effective on chitosan polydispersity, such as,
solution temperature and solution concentration of chitosan. They also
explained why the polydispersities of chitosan nanoparticles obtained
by ultrasonic treatment or by mechanical shearing were similar. The
structures of chitosan hydrogels can affect their swelling behaviors,
because swelling behavior reflects the amount of network porosity and
mesh size of the network, which may also indicate its drug release
behavior (Berger et al., 2004). Porosity of chitosan membranes could be
modified by crosslinking with some materials such as glutaraldehyde
(Krajewska, 2001).
Chitosan under acidic condition also has a high capacity for en-
trapping various metal ions including (Ni2+, Zn2+, Co2+, Fe2+, Mg2+
and Cu2+) (Kurita, 1998). Rainaudo et al. (Mazeau et al., 2000) have
studied chitosan chain stiffness, and concluded that chitin and chitosan
are semi-rigid polymers characterized by a persistence length (asymp-
totic value obtained at high degree of polymerization) that depends
moderately on the degree of acetylation of the molecule.
2.1. Sources of chitosan
Chitin, poly (β-(1→4)-N-acetyl-D-glucosamine) is a natural poly-
saccharide, first identified in 1811 by French chemist Henri Braconnot
(Arias et al., 2004). Chitin forms a part of the extracellular matrix of
certain living organisms as a proteoglycan. It is the most abundant
polymer after cellulose. This biopolymer is synthesized by an enormous
number of living organisms among which are insects (cuticles) and
crustaceans (skeletons), e.g., crab, shrimp and lobster. Cephalopod by-
products are a valuable source of chitin, polyunsaturated acids, and
collagen. (Koueta, 2014). Chitin is also extracted from the exoskeleton
of cephalopod species, such as squid, cuttlefish and octopi (Arrouze
et al., 2017; Jothi & Nachiyar, 2013; Kurita et al., 1993; Shanmugam
et al., 2016). (See Fig. 1).
Chitosan, which was discovered and named in 1859 by Roget
(Patrulea et al., 2015), is obtained by partial deacetylation of chitin
under alkaline conditions and is the most important chitin derivative in
terms of applications (Rinaudo, 2006b). (See Fig. 1). The physico-
chemical characteristics of the obtained chitosan are closely related to
the taxonomy of the marine sources (Rhazi et al., 2000). Chitosan can
H. Hamedi et al.
also be produced by enzymatic rather than alkali treatment at high
temperatures (Cai et al., 2006; No & Meyers, 1995). Chitosan can be
enzymatically degraded by chitinases and chitosanases but these en-
zymes are unavailable in bulk quantities for commercial applications. It
has been reported that β-glucosidase from almond emulsin can hydro-
lyze chitin substrates owing to an existing chitinase in the enzyme
preparation (Zhang & Neau, 2011). It has been reported that chitinases
are not responsible for the in vitro degradation of chitosans in human
serum (Vårum et al., 1997). Enzymatic degradation minimizes adverse
chemical modifications of products and promotes their biological ac-
tivities. However, higher costs of hydrolytic enzymes limit the appli-
cation of enzymatic methods. To reduce this production cost, reuse of
hydrolytic enzymes may be recommended (Kim & Rajapakse, 2005).
Chitosan preparation from fungal cell walls with fermentation
technology has become an alternative economical way for the pro-
duction of this polymer. It can be found in the cell wall of certain
groups of fungi, particularly zygomycetes (Nwe et al., 2002; Tayel et al.,
2010). There are several advantages of using these fungi to produce
chitosan. The most important is that the cell wall of zygomycetous fungi
contains a large quantity of chitosan and the physicochemical proper-
ties of this chitosan can be manipulated and standardized by controlling
the parameters of fermentation. For example, different molecular
weight chitosans can be produced when these fungi are grown on at
different pHs and on media with different compositions (Tan et al.,
1996). Sufficient amounts of chitosan have been identified in Mucor
rouxii (White et al., 1979), phycomyces, and saccharomyces (Arcidiacono
& Kaplan, 1992). In another study, chitosans with high degree of dea-
cetylation (above 97%) were extracted from R.miehei and M.racemosus
(Tajdini et al., 2010).
An investigation was performed to evaluate the chitosan char-
acterization produced by several species of fungi. Comparison of fungal
chitosan with a commercial one derived from crab shells showed lower
degree of deacetylation, viscosity and molecular weight of the fungal
chitosan. Chitosan with a low molecular weight reduces the tensile
strength and elongation of the chitosan membrane, but increase its
permeability. Fungal chitosan may have potential medical and agri-
cultural applications (Pochanavanich & Suntornsuk, 2002). This is in
general agreement with the findings of Yen et al. (Yen & Mau, 2007;
Yen, Yang, and Mau, (2009)).
2.2. Solubility of chitosan
Due to the high cohesive energy of chitin, which is related to strong
intermolecular interactions from hydrogen bonds, chitin is insoluble in
many typical solvents, such as water, dilute acids and alcohols (Zargar
et al., 2015). This is a major problem that limits the development of
processing and usage of chitin.
Chitosan, the most important derivative of chitin, is soluble in dilute
aqueous acidic media at a degree of deacetylation of 50% and higher
(depending on the origin of the polymer) due to its primary amino
groups that have a pKa value of 6.3. Solubilization occurs by protona-
tion of the –NH2 group of the D-glucosamine repeating unit, whereby
the polysaccharide is converted to a polyelectrolyte in acidic media.
Solubility of chitosan is usually examined in acetic acid by dissolving it
in 1% or 0.1 M acetic acid (Rinaudo, 2006a). Rinaudo et al.. (Rinaudo,
Pavlov, and Desbrie`res, (1990)) demonstrated that the amount of acid
needed depends on the quantity of chitosan to be dissolved. The con-
centration of protons needed is at least equal to the concentration
of − NH2 units involved. As the degree of protonation is progressively
increased, so is the solubilization of chitosan. The effect of depoly-
merization on the solubility of the chitosans with different degree of
deacetylation at neutral pH-values was investigated by Vårum et al
(Varum et al., 1994). They emphasized that the solubility differences
between chitosans with different degree of deacetylation may have
profound influence on accessibility of chitosans to enzymes (many en-
zyme assays are performed at pH 6.5) and the biological effects of
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Carbohydrate Polymers 199 (2018) 445–460
chitosans (testing of chitosans in vivo and in vitro are often performed at
neutral pH-values).
Sorlier et al. (Sorlier, Viton, and Domard, (2002)) demonstrated that
when the degree of dissociation (α) in solution increases, the role of the
cationicity of the amine groups, which depends on the degree of acet-
ylation, plays a more important role on the behavior of the polymer
chains. They showed that molecular weight and degree of deacetylation
have important effect on chitosan solubility.
Solubility of chitosan will be enhanced by decreasing its molecular
weight. Molecular weight changes the content of N-acetylglucosamines
units in chitosan, which will have intramolecular as well as an inter-
molecular influences, resulting in different chitosan conformations.
However, improving solubility by controlling deacetylation comes at
the cost of low yield (Kurita et al., 1991). Sogias et al.. (Sogias, Khu-
toryanskiy, and Williams, (2010)) showed that break down of chitosan
crystallinity will expand the range of chitosan solubility. They studied
two approaches for improving chitosan solubility, physical and che-
mical methods. In their chemical approach, re-acetylation improved
chitosan solubility up to pH = 7.4. The physical approach included the
use of additives, such as urea, and guanidine hydrochloride, with the
abilities to disrupt intra- and inter macromolecular hydrogen bonding.
In the presence of 8 mol L−1 urea and 1.5 mol L−1 guanidine hydro-
chloride the solubility of chitosan shifted to pH = 8.0 and 6.5, respec-
tively.
Introducing small chemical groups to the chitosan structure, such as
alkyl (hydroxypropyl chitosan (Xie et al., 2002)) or carboxymethyl
groups can drastically increase the solubility of chitosan (Jayakumar
et al., 2010; Pillai et al., 2009). A simple and improved method of
preparing highly soluble chitosan (half N-acetylated chitosan) was de-
veloped using a series of chitosan samples of low molecular weights
(Kubota et al., 2000).
2.3. Toxicity
There are various studies on chitosan toxicity. Scientists have done
some studies on chitosan toxicity in guinea pigs (Aspden et al., 1997),
frog, human nasal palate tissue (Aspden et al., 1994; Aspden et al.,
1995), and the nasal membranes of rats (Aspden et al., 1996). In all of
the tests, toxicity was negligible. Ribeiro et al. (Ribeiro et al. (2009))
studied the in vitro cytotoxicity of chitosan hydrogels tested with dermal
fibroblasts obtained from rat skin. Their cell viability study showed that
the hydrogel and its degradation by-products are non-cytotoxic. His-
tological analysis also revealed lack of a reactive or a granulomatous
inflammatory reaction in skin lesions with chitosan hydrogels, and the
absence of pathological abnormalities in the organs supported the local
and systemic histocompatibility of this biomaterial.
in vivo chronic toxicity studies reported by Carreño-Gómez et al.
(Carreño-Gómez & Duncan, 1997) have shown that intravenous ad-
ministration of chitosan (4.5 mg/kg/day for 11 days) to rabbits did not
lead to any abnormal changes. Data on chitosan toxicity from human
studies in general are quite limited. Gades et al. (Gades & Stern, 2003)
reported that quantities exceeding 4.5 g chitosan taken daily by human
volunteers did not result in toxic effects. Even higher oral levels of up to
6.75 g were reported as safe. It has been reported that short-term
human trials of up to 12 weeks have shown no clinically significant
symptoms, including no evidence of an allergic response (Tapola et al.,
2008). Chitosan mouthwash safety was evaluated through Ames, MTT
and V79 chromosomal aberration assays. The chitosan mouthwash
possessed lower toxicity and higher antimicrobial activity than the
commercial mouthwash tested. Furthermore, while it is capable of
completely inhibiting the two selected pathogenicity markers (strep-
tococci and enterococci), contrary to the commercial mouthwash, it did
not cause major reductions in the viability of the normal oral microflora
(Costa et al., 2014).
Ravindranathan et al. (Ravindranathan, Koppolu, Smith, and
Zaharoff, (2016)) Studied purified, low endotoxin chitosan, and
H. Hamedi et al.
determined that viscosity/molecular weight and degree of deacetyla-
tion within the ranges of 20–600 CP and 80–97%, respectively, have no
impact on chitosan’s immunoreactivity. Endotoxin contamination was
expected to be a major factor influencing immunoreactivity. Their re-
sults indicated that only endotoxin content and not degree of deace-
tylation or viscosity influenced chitosan-induced immune responses.
Their data also indicated that low endotoxin chitosan (< 0.01 EU/mg)
with viscosities of 20–600 cP and 80%–97% degree of deacetylation are
essentially inert. This study highlights the need for more complete
characterization and purification of chitosan in preclinical studies be-
fore being used in clinical application.
Baldrick (Baldrick (2010)) implies that chitosan can be used as a
safe pharmaceutical excipient for non-parenteral and non-blood con-
tact. Based on the available data, safe use of chitosan as a parenteral
excipient is not clear. Its use as a medical device to control bleeding
relies on the haemostatic biological nature of the material and studies
have demonstrated local findings, such as blood coagulation, thrombus
formation and platelet adhesion/aggregation. In spite of some in vitro
studies related to cytotoxicity findings (Baldrick (2010)), there are re-
ports of non-toxic usages of chitosan in pharmaceuticals, such as to stop
bleeding (Dowling et al., 2011; Horio et al., 2010; Valentine et al.,
2010).
2.4. Biomedical properties
The degradation rate of chitosan can be controlled by changing its
degree of deacetylation and/or its molecular weight (Tomihata & Ikada,
1997). Many researches have been done to prove the degraded products
of chitosan are nontoxic, non-immunogenic, and non-carcinogenic
(MuzzareUi, 1993). Risbud et al. (Risbud & Bhonde, 2000) reported
characterization, biocompatibility, and release properties of poly-
acrylamide/chitosan hydrogels and showed that chitosan possess ex-
cellent biocompatibility and bioabsorbability. In this research, in vitro
cytotoxicity studies using a colorimetric assay for assessing cell meta-
bolic activity, membrane permeability, and lysosomal activity of cells
(MTT and NR assays) showed no toxic effects on different cells in up to
40% hydrogel extract.
The antimicrobial properties of chitosan and its derivatives have
attracted great attention from researchers (Kong et al., 2010). Anti-
microbial activity of chitosan has been demonstrated against many
bacteria, filamentous fungi, and yeasts. Chitosan has a wide spectrum of
activity and high killing rates against gram-positive and gram-negative
bacteria (Ueno et al., 1997), with low toxicity toward mammalian cells.
Antibacterial effects of chitosan are reported to be dependent on its
molecular weight (Jeon et al., 2001). To prove this idea, No et al. (No,
Park, Lee, and Meyers, (2002)) examined the antibacterial activity of
six chitosans and six chitosan oligomers with widely different molecular
weights against gram-negative and gram-positive bacteria. They
showed that chitosan markedly inhibited the growth of most bacteria
tested, although their inhibitory effects differed with molecular weight
and the particular bacterium. Chitosan generally showed stronger
bactericidal effects for gram-positive bacteria than for gram-negative
bacteria. The reason for this difference is unclear, but Y. J. Jeon et. al
(Jeon et al., 2001) showed the oligosaccharides as well as chitosan
exhibited better inhibitory effects against Gram-positive compared with
Gram-negative bacteria. Thus, it might be reasonable that chitosan
showed remarkable inhibitory effect against the growth of lactic acid
bacteria which are Gram-positive.
Sizes of chitosan particles play an important role in inflammasome
activation. The inflammasome is a cytosolic complex which is re-
sponsible for the activation of inflammatory responses, such as IL-1β.
Glycans passed through filters of defined size, showed that the smallest
size fraction (< 20 μm) induced the most cleavage of pro-IL-1β and
release of the mature cytokine. The larger-sized fractions also had some
bioactivity, which may have been due to some smaller particles that
failed to pass through the filters. Partial digestion of chitosan with
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Carbohydrate Polymers 199 (2018) 445–460
pepsin, an enzyme that breaks down proteins into smaller peptides,
enhanced the ability of the glycan to activate the inflammasome (Bueter
et al., 2011).
Biodegradability of chitosan has been studied by many researchers.
It was found that lysozyme (Onishi & Machida, 1999), proteases (Rao &
Sharma, 1997) and porcine pancreatic enzymes (Verheul et al., 2009)
are able to degrade Chitosan under specific conditions. Pectinase iso-
zyme from Aspergilus niger has also been shown to digest chitosan at
low pH providing lower Mw chitosans (Kittur et al., 2003; Kittur et al.,
2005). Connell et al. (McConnell et al., 2008) used human fecal pre-
parations and showed significant degradation of chitosan films, glu-
taraldehyde crosslinked films, and tripolyphosphate crosslinked films.
Brenner et al. (Brenner, Hostetter, and Humes, (1978)) demonstrated
that many enzymes seem to show an activity against chitosan, at least in
vitro, but derivatives could give indigestible molecules. To be clinically
viable, these compound particles should remain small enough (< 42 Å
radius for neutral molecules) to be renally excreted. The bio-distribu-
tion is controllable through the route of administration, dosage form
and chitosan characteristics, i.e., a film/dense matrix will persist at the
site of administration due to the physical characteristics. The de-
gradation of the matrix is controllable through crosslinking modifica-
tion of the amine groups. Intravenous distribution can be adjusted by
particle size (< 100 nm), and molecular weight can determine the
stability of the particle (Kean & Thanou, 2010).
2.5. pH sensitivity
The numerous amine groups on the chitosan backbone confer a pH-
dependent solubility, which has been utilized to fabricate biosensors
and drug delivery systems. Chitosan properties can be conveniently
altered by derivatization of its hydroxyl and amino groups (Zhang et al.,
2012). The amino groups of chitosan are ionized in acidic buffers,
which contribute to the electrostatic repulsion between adjacent io-
nized –NH2 groups and lead to chain expansion and eventually increase
the water absorption of the chitosan gels. The swelling degree of the
ionic pH-sensitive hydrogel is mainly determined by the degree of io-
nization (Guanghua et al., 2008). Some attempts have been made for
regulating chitosan pH-sensitivity by adding different additives or
grafting or mixing with other polymers and preparing Polyelectrolyte
complexes with anionic polymers (Krishna Rao et al., 2006; Meng et al.,
2010; Qu et al., 1999; Qu et al., 2000). The nature of the pH response of
chitosan and its derivatives has been described by several researchers.
Islam et al. (Islam & Yasin, 2012) synthesized a pH sensitive blend
based on chitosan and poly vinyl alcohol (PVA) using a nontoxic cross
linker for a drug delivery system. They concluded that the pH sensi-
tivity and biocompatibility properties of this chitosan/PVA hydrogel
made it suitable for medicinal and pharmaceutical applications. Anti-
microbial activity of chitosan is also pH dependent, it has been reported
that chitosan displayed antibacterial activity only in an acid environ-
ment (Helander et al., 2001).
3. Chitosan hydrogel preparation methods
3.1. Chemical Cross linking (permanent hydrogels)
Chemically cross-linked hydrogels are formed by covalent linking of
the chitosan macromers, where the bond formation is irreversible. The
cross-linking of natural and synthetic polymers can be achieved through
the reaction of their functional groups (such as OH, COOH, and NH2)
with cross-linkers (Liu et al., 2014; Milosavljević et al., 2010; Singh
et al., 2006; Zhang et al., 2005). There are a number of methods re-
ported in the literature to obtain chemically cross-linked permanent
hydrogels. The following section reviews the major chemical methods
used to produce chitosan hydrogels.
H. Hamedi et al.
3.1.1. Chemical cross-linkers
Cross-linkers are molecules with at least two reactive functional
groups that allow the formation of bonds between polymeric chains. To
date, the most common cross-linkers used to obtain chitosan hydrogels
are di-aldehydes, such as glutaraldehyde (Milosavljević et al., 2010;
Zhang et al., 2005), formaldehyde (Singh et al., 2006), or ethylene
glycol di-glycidyl ether (EGDE) (Liu et al., 2014), genipin (Mi et al.,
2000; Muzzarelli, 2009b) and others (Draye, Delaey, Voorde, Bulcke,
Bogdanov et al., 1998; Hennink & Nostrum, 2002; Huffman, 2012) (See
Fig. 2).
Among these mentioned cross-linkers, reaction of aldehydes with
chitosan is well-documented; the aldehyde groups form covalent imine
bonds with the amino groups of chitosan, due to the resonance estab-
lished with adjacent double ethylenic bonds (Monteiro & Airoldi,
1999). Di-aldehydes allow direct reaction in aqueous media, under mild
conditions and without the addition of auxiliary molecules, such as
reducers, which is advantageous with respect to biocompatibility.
However, the main drawback of di-aldehydes, such as glutaraldehyde,
is that they are generally considered to be toxic. It is reported that
glutaraldehyde is cytotoxic even at low doses and glutaraldehyde
polymerization may release glutaraldehyde residues during storage or
sterilization (Chang et al., 2004; MacDonald & Pepper, 1994). Glutar-
aldehyde is known to be neurotoxic, its fate in the human body is not
fully understood. Therefore, even if hydrogels are purified before ad-
ministration, the presence of free unreacted di-aldehydes in hydrogels
could not be completely excluded and may induce toxic effects. This
could impair the biocompatibility of the cross-linked products (Berger,
Reist, Mayer, Felt, Peppas et al., 2004; Chen et al., 2004).
In order to obtain non-soluble hydrogels for controlling drug release
and pharmaceutical applications, researchers have been attempting to
find a cross-linking agent with low cytotoxicity (Chen et al., 2004; Cui
et al., 2014; Delmar & Bianco-Peled, 2015). Genipin is a natural cross-
linking agent extracted from geniposide, an iridoid glucoside isolated
from the Genipa fruits (Tsai et al., 1994). Genipin has been reported to
bind with biological tissues (Sung et al., 2001) and biopolymers, such as
chitosan and gelatin (Bigi et al., 2003; Mi et al., 2005), leading to
covalent coupling. It works as an effective cross-linker for polymers
containing amino groups (mostly used for chitosan crosslinking) (Sung
et al., 1999). It has been found that the reaction rate of genipin with
amino group containing biomaterials is significantly slower than that of
glutaraldehyde (Moura et al., 2007; Sung et al., 2000). The reaction
between chitosan and genipin (See Fig. 3) is well understood for a
variety of experimental conditions and has no cytotoxicity for human
and animal cells (Muzzarelli et al., 2015). Biocompatibility,
Fig. 3. Cross linking between Genipin and Chitosan (Muzzarelli et al., 2015).
449
Carbohydrate Polymers 199 (2018) 445–460
Fig. 2. Chemical Cross Linker Structures.
biodegradability and pharmaceutical effects of the genipin-crosslinked
hydrogels were investigated, and it was concluded that genipin may be
well suited for clinical usage (Kitano, 2006; Muzzarelli, 2009a; Yaoa
et al., 2005; Zhang et al., 2006). Effects of genipin cross-linking of
chitosan hydrogels on cellular adhesion and viability were investigated.
It was found that the cross-linked hydrogels did not induce cytotoxic
effects and significantly improved the cell adhesion and viability on
hydrogel surfaces (Gao et al., 2014). Genipin has also been shown to act
significantly as an anti-inflammatory and anti-angiogenesis agent, and
protected the hippocampal neurons from the toxicity of Alzheimer’s
amyloid beta protein (Kooa, 2004).
3.1.2. Photo crosslinking
Photo-crosslinking, a type of chemical crosslinking, is performed in
the presence of ultraviolet (UV) light and a chemical photo initiator
(PI). A variety of natural and synthetic macromers have been modified
to formed hydrogels by photo activation of the coexisting photo in-
itiators, such as methacrylated or aryl azide modified macromers
(Rickett et al., 2011). The degree of crosslinking reaction depends on
UV irradiation time and increases with the increase of exposure time.
This lead to a hydrogel with higher mechanical properties and lower
swelling ratio (Ik et al., 2016). The polymerization reaction can also
controlled by adjusting the distance from the UV light source (Ahmadi,
Oveisi, Mohammadi Samani, & Amoozgar, 2015). Obara et al.. (Obara
et al. (2003)) prepared a photo cross-linkable chitosan aqueous solution
including fibroblast growth factor-2 (FGF-2) by using ultraviolet light
(UV) irradiation to result in an insoluble, flexible hydrogel. A viscous
azide-chitosan-lactose aqueous solution was converted into an insoluble
hydrogel within 10 s upon UV-irradiation at a lamp distance of 2 cm
through crosslinking of the azide and amino groups of the azide-chit-
osan-lactose molecules. The results showed that the growth factor FGF-
2 molecules remained biologically active, and were released from the
chitosan hydrogel upon in vivo biodegradation. Application of the
chitosan hydrogel significantly induced wound contraction and ac-
celerated wound closure.
Among the photo initiators, Irgacure2959 caused minimum cyto-
toxicity (cell death) over a broad range of mammalian cell types and
species (Hu & Gao, 2008). Pluronic-chitosan hydrogels used as a dres-
sing for ulcers wounds were fabricated, and the release of growth factor
was investigated. Photo-irradiation was applied to chemically cross-link
the hydrogel. Before irradiation by UV, a photo-initiator, Irgacure2959
(0.1%, w/w), was added to the hydrogel mixture to form physical hy-
drogels. Release profiles of rhEGF growth factor from hydrogel net-
works were evaluated with modification of the chitosan blend ratios
and photo-irradiation time. At the same photo-irradiation time, chit-
osan accelerated rhEGF release due to Pluronic chains (b-PPO-b-PEO-b-
PPO) inhibiting the chitosan from being tightly crosslinked because of
hydrophobic interactions. Increasing exposure time led to a tight net-
work which can control growth factor release (Choi & Yoo, 2010b).
3.1.3. Graft polymerization
Graft Polymers are segmented copolymers with a linear backbone
made of one polymer and randomly distributed branches made of an-
other polymer. Grafting of natural polymers such as chitosan containing
two types of reactive groups is of considerable interest for modification
of polymer structure. Chitosan has two types of reactive groups that can
be grafted; the free amine groups on deacetylated units and the
H. Hamedi et al.
hydroxyl groups on the C3 and C6 carbons of acetylated or deacetylated
units. Recently researchers have shown that chitosan derivatives, such
as hydroxypropyl chitosan (HPCT) and carboxymethyl chitosan sodium
(CMCTS) functionalized by grafting, improved water solubility, anti-
bacterial, and antioxidant properties (Alves & Manoa, 2008; Xie et al.,
2002; Xie et al., 2001).
3.1.3.1. Chemical grafting. Chemical grafting is initiated by generating
one or more free radicals on the chitosan chain and allowing these
radicals to react with polymerizable monomers that build the grafted
chain (Girin et al., 2012). In recent years, a number of initiators such as
Ferrous Ammonium Sulfate (FAS), Potassium PerSulfate (PPS), Ceric
Ammonium Nitrate (CAN), and Ammonium PerSulfate (APS) have been
developed to initiate grafting copolymerization.
Yazdani-Pedram et al. (Yazdani-Pedram, Retuert, and Quijada,
(2000)) modified chitosan by grafting with polyacrylic acid, a well-
known hydrogel forming polymer. The reaction was carried out in a
homogeneous aqueous phase by using PPS and FAS as the redox in-
itiators. It was observed that the efficiency of grafting depended on
monomer, initiator, and ferrous ion concentrations, as well as reaction
time and temperature. Also the level of grafting could be controlled by
varying the amount of ferrous ions used as a co-catalyst in the reaction.
Jung et al. (Jung, Chung, and Lee, (2006)) prepared a chitosan and
eugenol hydrogel to enhance and maintain antioxidant activities by
using CAN as initiator. Eugenol is generally recognized as a safe ma-
terial by the Food and Drug Administration, so it can be used in bio-
logical applications.
The graft yield increased with the concentration of CAN. Thermal
stabilities of chitosan alone and eugenol-chitosan hydrogels were
measured using TGA analysis and it was observed that the eugenol-
chitosan hydrogels have a faster thermal decomposition in comparison
with that of chitosan alone. This is likely because the introduction of
the bulky eugenol side chains inside the hydrogel decreased the crys-
talline regions of chitosan. They observed that the equilibrium–swelling
ratio of eugenol-grafted chitosan hydrogels is smaller than that of
chitosan gels and decreased with increased graft yield, because of the
hydrophobicity of the attached eugenol molecules. Because chitosan is
a cationic polymer, the swelling ratio of chitosan hydrogels decreased
with increasing pH values, but the swelling ratios of the more hydro-
phobic eugenol-grafted chitosan hydrogels were not affected by the pH
of the media, because chitosan amino groups, which were pH sensitive,
were grafted with eugenol.
The pH responsive property of chitosan hydrogels can be explained
as indicated in Fig. 4 (Zou et al., 2015). At low pH, the amide groups on
the chitosan can become protonated and the resulting electrostatic re-
pulsion between the protonated amino groups weaken the
Fig. 4. Mechanism of pH-responsive swelling behavior of chitosan (Zou et al., 2015).
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Carbohydrate Polymers 199 (2018) 445–460
intermolecular and intramolecular hydrogen bonding interaction of
chitosan molecules. Consequently, buffer solution can easily diffuse
into the network and lead to increased swelling ratios. In neutral and
basic conditions, no protonation occurs and the swelling ratio is low.
Mahdavinia et al. (Mahdavinia, Pourjavadi, Hossein zadeh, and
Zohuriaan, (2004)) introduced graft copolymerization of mixtures of
acrylic acid (AA) and acrylamide (AAm) onto chitosan by using PPS as a
free radical initiator and methylene bis-acrylamide (MBA) as a cross
linker. It was concluded that the swelling capacity of the hydrogels is
affected by the MBA cross linker concentration and monomer ratio, so
that the swelling decreases with increased MBA concentration and
AAm/AA ratio. They also concluded that this pH–sensitive super-
absorbent poly-ampholytic network may be considered as an excellent
candidate to design influential drug delivery systems.
3.1.3.2. Radiation grafting. Grafting can also be initiated by the use of
high energy radiation from gamma rays and from electron beams.
Radiation graft copolymerization is a well-established technique for
producing polymeric materials which combine chemical and physical
properties of both base polymer and grafted monomer. Grafting
copolymerization of thermal and pH-sensitive chitosan and N-
isopropyl acrylamide hydrogels were investigated by γ-radiation (Cai
et al., 2005). In this research the effects of monomer concentration and
irradiation dose on grafting percentage and efficiency were examined.
Grafting percentage and efficiency increased with increased monomer
concentration and total irradiation dose. However, γ-radiation degrades
chitosan. Sterilization of chitosan by γ-radiation leads to discoloration
and loss of amino groups and lowering of molecular weight.
Compared to other methods, radiation crosslinking doesn’t require
any additives to start the process, so the final product contains only
polymer. Moreover, ionizing radiation usually provides combination of
the synthesis and sterilization of polymeric materials in a single tech-
nological step, which leads to reduced cost and production time.
Therefore, ionizing radiation or electron beam radiation methods are an
excellent tool in the fabrication of materials for biomedical applications
(Zhao & Mitomo, 2008). A series of hydrogels were prepared from
polyvinyl alcohol (PVA) and carboxy methylated chitosan with electron
beam irradiation at room temperature. The mechanical properties and
degree of swelling improved substantially after adding chitosan into the
PVA hydrogels. These blend hydrogels, even at low concentration of
chitosan (3 wt%), exhibited satisfactory antibacterial activity against E.
coli (Zhao, 2003). In another study, N-maleoyl-chitosan was synthesized
by reaction of chitosan with maleic anhydride (MAH) in N,N-dimethyl
formamide (DMF) via electron beam irradiation. Grafting yield and
grafting efficiency increased with increasing absorbed radiation dose
and monomer amount, and then decreased. Usually, the free radicals in
H. Hamedi et al.
Fig. 5. Scheme of polyelectrolyte complexing reaction between chitosan and pectin (Archana, Dutta et al., 2013).
the reaction system increase with dose and then eventually decay. The
swelling ratio of the copolymer hydrogel was low at pH 4–5 (Fan et al.,
2009).
3.2. Physical Cross linking (reversible hydrogels)
Physically cross linked gels have attracted much interest recently
due to the absence of chemical crosslinking agents and reagents. Many
chemical crosslinking agents are toxic compounds which can be de-
tached or isolated frequently from prepared gels before application.
They can also affect the nature of the substances when entrapped (e.g.
proteins, drugs, and cells) (Kamoun et al., 2015). The main dis-
advantages of physical gels are their instability (uncontrolled dissolu-
tion may occur), low mechanical resistance, and difficult control of pore
size (Croisier & Jérôme, 2013). Also free chain ends or chain loops are
also present as transient network defects in physical gels (Huffman,
2012). Some of the most common physical cross-linking methods for
chitosan hydrogel preparation are now described.
3.2.1. Hydrophobic interaction
Polymers with hydrophobic domains can crosslink in aqueous en-
vironments via reverse thermal gelation, also known as ‘sol-gel’ chem-
istry. The hydrophobic segment is coupled to a hydrophilic polymer
segment by post-polymerization grafting or by directly synthesizing a
block copolymer to create a polymer amphiphile. These amphiphiles
are water soluble at low temperature. But by increasing the tempera-
ture, hydrophobic domains aggregate to minimize the hydrophobic
surface area contacting the bulk water, reducing the amount of struc-
tured water surrounding the hydrophobic domains, and maximizing the
solvent entropy. The temperature at which gelation occurs depends on
the concentration of the polymer, the length of the hydrophobic block,
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Carbohydrate Polymers 199 (2018) 445–460
and the chemical structure of the polymer (Hoare & Kohane, 2008).
Tien et al. (Tiena et al., 2003) studied the N-acylation of chitosan
with fatty acyl chlorides to introduce hydrophobicity for use as a matrix
for drug delivery. They concluded that hydrophobic interactions en-
hance the stability of substituted chitosan via hydrophobic self-as-
sembly. Also release of drugs depended on the acyl chain length and the
degree of acylation and could be managed by diffusion, or by swelling
followed by diffusion. An injectable in situ thermosensitive chitosan–β-
glycerophosphate (CeGP) gel formulation has been proposed for tissue
repair and drug delivery (Ruel-Gariépya et al., 2002). The sol/gel
transition can be summarized as a competition between several inter-
molecular interactions: 1) the electrostatic repulsion between positively
charged chitosan molecules, 2) a screening effect on this repulsion,
induced by the negatively charged β-glycerophosphate, and 3) attrac-
tive interactions due to hydrophobicity and hydrogen bonding between
the chitosan molecules (Supper et al., 2013). This system can release
macromolecules over a period of several hours to a few days. The re-
sults presented in this work indicated that the liposome–C–GP system
could be used for the delivery of small molecular weight hydrophilic
compounds. The release profile of the incorporated compound can be
controlled by adjusting liposome characteristics, such as size and
composition. This formulation can be a good candidate for local release
of drugs and biomedical applications which require the local and con-
trolled delivery of pharmaceuticals, such as growth factors in tissue
repair.
3.2.2. Polyelectrolyte complexation
Formation of chitosan hydrogels by polyelectrolyte complexation
(PEC) has become an alternative for cross-linking. In the last decade
there has been an increasing interest in the use of PEC gels formed by
chitosan and polyanions as carriers for drug delivery systems. PECs are
H. Hamedi et al.
generally biocompatible networks exhibiting interesting swelling
characteristics. PEC gels formed by the electrostatic attractions between
two oppositely charged polyelectrolytes in aqueous solution are known
to exhibit unique physical and chemical properties. For instance, the
electrostatic interactions within the PEC gels are considerably stronger
than most secondary binding interactions. The electrostatic attraction
between the cationic amino groups of chitosan and the anionic groups
of the other polyelectrolyte is the main interaction leading to the for-
mation of PECs (Argin-Soysal et al., 2009; Berger, Reist, Mayer, Felt,
Gurny et al., 2004). However, the main drawback of these systems is
their difficult preparation, especially in large-scale processes (Berger,
Reist, Mayer, Felt, Gurny et al., 2004; Long & Luyen, 1996).
Archana et al. (Archana, Dutta, and Dutta, (2013)) have studied a
ternary nano-dressing consisting of titanium dioxide (TiO2) nano-
particles loaded into a chitosan pectin polyelectrolyte complex that was
prepared to evaluate biocompatibility, and antimicrobial and in vivo
wound healing properties. The electrostatic attractions between the
ionized amino groups of chitosan (NH2) and the ionized carboxyl acid
groups (COO−) of pectin are the main interactions leading to the for-
mation of the pectin/chitosan PECs illustrated in Fig. 5. The tensile
strength of the blended dressing material increased with decreasing
pectin content (1:1) and incorporation of TiO2 nanoparticles. This nano-
dressing exhibited superior antimicrobial activity against five patho-
gens (E. coli, S. aereus, P. aeruginosa, B. subtilis and A. niger) and good
blood-compatible properties. The chitosan–pectin–TiO2 dressing mate-
rial could control evaporative water loss from wound beds at an optimal
level and absorb more exudates, keeping the wound beds moist without
risking dehydration or accumulation of exudates. Also the nano-TiO2
particles in the chitosan pectin matrix have been shown to decrease
cytotoxicity.
Structurally stable chitosan and γ-poly glutamic acid (γ-PGA) PEC
hydrogel formed through simple ionic complex interaction, between the
amine groups of chitosan and the carboxylic acid groups of γ-PGA, was
confirmed by FTIR spectroscopy (Tsao et al., 2010).. It was found that
the chitosan–γ-PGA PEC hydrogels show antibacterial activity and
promoted cell proliferation. Therefore, the chitosan–γ-PGA polyelec-
trolyte hydrogel appears to have potential as a new material for bio-
medical applications.
3.2.3. Freeze-Thaw processing
The mechanical properties of chitosan hydrogels are not adequate
for some biomedical applications and many researchers have tried to
improve them. In order to achieve good mechanical and biological
performance, a combination of a synthetic and a natural polymer seems
to be a good choice. These materials are known in the literature as
‘bioartificial’ polymeric materials. Among synthetic polymers, poly-
vinyl alcohol (PVA) has been widely used in biomedical and bio-
chemical applications because of its excellent chemical and physical
properties, biodegradability, easy preparation and low cost. Blends of
chitosan with PVA have been reported to have good mechanical and
chemical properties (Milosavljević et al., 2010).
Hydrogels prepared by freeze – thaw processing of poly vinyl al-
cohol (PVA) solutions have drawn extensive attention. Some ad-
vantages of physically cross-linked hydrogels obtained by freeze
thawing are the nontoxic, noncarcinogenic, and biocompatibility of the
resulting polymers, good mechanical strength and the absence of
crosslinking agents or initiators in the hydrogel synthesis (Ricciardi
et al., 2004; Smith et al., 2009; Xiaomin et al., 2008). During the
freezing step, a liquid–liquid phase separation and formation of ice
crystals in the polymer-poor phase occurs. Polymer chains in the
polymer-rich phase lead to the formation of hydrogen bonding and PVA
crystallites (See Fig. 6). In addition, the thawing procedure facilitates
the interactions and formation of crystalline regions between the re-
maining polymers, leading to formation of hydrogel networks
(Holloway et al., 2013; Zhang et al., 2013). The ice crystals act as cross-
linkers during the formation of hydrogels and leave porous structures in
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Carbohydrate Polymers 199 (2018) 445–460
the hydrogels, because of the space left from the melting ice crystals
during the thawing stages (Guan et al., 2014).
The size of ice crystals formed in the structure increases with the
number of repeated freeze/thaw cycles (Guan et al., 2014). Ricciardi
et.al (Ricciardi et al., 2004) studied the structure of physical PVA hy-
drogels prepared by freeze-thaw cycling with a systematic X-ray
powder diffraction technique, as a function of the number of cycles and
aging time. Their results showed that both the degree of crystallinity of
the gels and the size of PVA crystallites increased with increasing
number of freeze/thaw cycles as illustrated in Fig. 7. The increased
degree of crystallinity is mostly due to the increase in the crystallite
size, although formation of new crystalline aggregates could not be
excluded.
Guanghua et al. (Guanghua et al. (2008)) prepared physically cross-
linked PVA/chitosan hydrogels by the freeze/thaw technique. The
swelling results indicated that the hydrogels have good pH-sensitive
properties in acidic environments and maximum swelling rate rises
with the increase of chitosan content in the blend, but leads to more
dissolution at pH 1.0. Longer freeze/thaw cycle times result in a lower
swelling rate. They concluded that the swelling behavior of the com-
posite hydrogels could be adjusted by changing the chitosan contents
and the freezing/thawing cycle times.
PVA hydrogels including chitosan were synthesized by combined
irradiation and freeze-thaw cycling and their properties were compared
with those prepared by irradiation or freeze-thaw cycling alone. It was
found that hydrogels made by irradiation alone have large swelling
capacity and are transparent, but too weak to be used as a wound
dressing. Hydrogels made by irradiation followed by freeze-thaw have
large swelling capacity, good thermal stability, high mechanical
strength, and translucent appearance. All the hydrogels containing
chitosan showed pH sensitivity and antibacterial activity (Yang, Liu
et al., 2008).
Fig. 8 shows the effects of the hydrogel preparation method on
mechanical compression ratio (Afshari et al., 2015). The mechanical
strengths of the hydrogels prepared by the combinational method are
higher than that of the hydrogels prepared only by radiation. This is due
to the presence of physical crosslinks in addition to chemical crosslinks.
This fact suggests that in order to increase the mechanical strength of
hydrogels prepared by radiation, the freeze-thawing treatment after
irradiation is very effective [Yang et al., 2008, 2010; Boynueğri et al.,
2009].
4. Clinical studies
There are few prospective clinical trials in the area of chitosan hy-
drogels for wound repair. Some clinical studies have been reported for
using chitosan in various medical fields [Diamond et al., 2003; Méthot
et al., 2016; Mason & Clarke, 2015]. A research has been done on a
chitosan gelling fiber dressing (Kytocel®) in Staffordshire community
care to examine improved healing of patients with chronic non-healing
wounds. KytoCel® (Aspen Medical) is a highly absorbent dressing
composed of chitosan fibers which bond with wound exudate to form a
clear gel absorbing pathogens and is conformable to the wound bed.
Over a 4-week period or until complete wound healing, leaking, stri-
kethrough, pain, time, and malodour were examined. This new ad-
vanced wound dressing has the ability to gel when in contact with
wound fluid, making it less painful to remove, suitable for moderate to
high exudate, reduced bioburden, and maintain haemostasis (Mason &
Clarke, 2015). Another clinical study was conducted at three medical
centers in China. A total of 90 patients with unhealed chronic wounds
including pressure ulcers, vascular ulcers, diabetic foot ulcers, and
wounds with minor infections, or at risk of infection, were treated with
chitosan wound dressing or traditional Vaseline gauze as a control
(Foshan United Medical Technologies Ltd, Guangdong, China). After 4
weeks of treatment, the wound area reduction was significantly greater
in the test group than the control group. The average pain level in the
H. Hamedi et al.
Fig. 6. Schematic diagrams showing the mechanisms for hydrogel formation from PVA solutions by the freezing–thawing method (Afshari et al., 2015).
Fig. 7. Apparent dimensions of PVA hydrogel crystallites as a function of
freeze/thaw cycles (Ricciardi et al., 2004).
Fig. 8. Comparison of mechanical strength of hydrogels prepared by
Combinational and radiation method (Yang, Zhu et al., 2008).
test group was 1.12 ± 0.23 and 2.30 ± 0.23 in the control group. The
mean duration of the test group was 27.31 ± 5.37 days and the control
group 27.09 ± 6.44 days. Consequently, the new chitosan wound
dressing can enhance the wound healing process and reduce the pa-
tient’s pain level. Furthermore the dressing was shown to be clinically
safe and effective in the management of chronic wounds (Mo et al.,
2015).
An evaluation of healing at split skin graft donor sites, dressed half
with chitosan and half with a conventional dressing, revealed that
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Carbohydrate Polymers 199 (2018) 445–460
chitosan facilitated wound re-epithelialisation and the regeneration of
nerves within a vascular dermis. In addition, digital color separation
analysis of donor site scars demonstrated an earlier return to normal
skin color at chitosan-treated areas (Stone et al., 2000). Chitosan pre-
pared from natural biopolymer chitin and cast into membranes has
been tested as wound dressing at the skin‐graft donor site in patients.
Bactigras, a commonly used impregnated tulle gras bandage, served as
a control. The progress in wound healing was compared by clinical and
histological examination. After 10 days, the chitosan‐dressed area had
been healed more promptly as compared with the Bactigras dressed
area. Moreover, the chitosan membrane showed a positive effect on the
re‐epithelialization and the regeneration of the granular layer. The data
confirm that chitosan membrane is a potential substitute for human
wound dressings (Azad et al., 2004).
5. Chitosan application for drug delivery in wound dressings
Wound healing is a complicated process including four overlapping
phases: coagulation, inflammation, migration-proliferation (including
matrix deposition), and remodeling (Falanga, 2005; Kirsner &
Eaglstein, 1993; Velnar et al., 2009). The human body can provide all
the requirements for the healing process in normal wounds, unless there
is any kind of deficiency of skin function or massive fluid losses in large
wounds. However, providing appropriate pH, moisture, oxygen pres-
sure, and preventing microbial invasion can accelerate the wound
healing process (Field & Kerstein, 1994; Guo & DiPietro, 2010). Wound
care for non-healing wounds has recently been a growing concern of
many wound dressing applications involving various mechanisms, such
as coagulation, inflammation, angiogenesis, epithelization, contraction
and remodeling. An ideal wound dressing should protect the wound
from bacterial infection, provide a moist and healing environment, and
be biocompatible (Boateng, Matthews, Stevens, & Eccleston, 2008;
Bhuvanesh, 2010; Paul & Sharma, 2004). Because of chitosan’s pre-
viously mentioned significant characteristics, such as biocompatibility,
biodegradability, hemostatic and anti-infection activity, and the ability
to accelerate wound healing, it has attracted many researchers to be
used as a wound dressing material. Applications of chitosan in different
types of wound dressing are explained in the following sections.
5.1. Application of growth factors in chitosan-based wound dressings
Growth factors are polypeptides which control the growth, differ-
entiation, and metabolism of cells and regulate the process of tissue
repair. Despite their small amounts, they exert a powerful influence on
the process of wound repair. Use of growth factors for accelerating the
healing of wounds presents tremendous promise as a therapeutic ap-
proach in treating chronic wounds. Several peptide growth factors, such
as epidermal growth factor (EGF), platelet derived growth factor
(PDGF), fibroblast growth factor (FGF) and transforming growth factor-
beta (TGF-β), have been shown to accelerate cellular proliferation and
synthesis of the extracellular matrix (Alemdaroğlu et al., 2006; Paul &
H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Table 1 5.2. Nanoparticle and nanostructure incorporated hydrogels

The average stained cell number in Chitosan gel with and without EGF
(Yenilmez et al., 2015).
Groups The average stained cell number
3rd day 7th day 14th day
J* 1.20 ± 0.17 2.43 ± 0.61 3.10 ± 0.68
EJ* 1.63 ± 0.35 4.20 ± 0.18 4.95 ± 0.39
* Burn wounds treated by chitosan gel without (J) and with (EJ) with EGF.
Sharma, 2004). EGF has been reported to increase the rates of cellular
proliferation, growth and regeneration of tissue in numerous papers
(Choi et al., 2012; Su et al., 2014; Yenilmez et al., 2015).
Regarding EGF and chitosan properties for wound healing,
Alemdaroğlu et al. (Alemdaroğlu et al. (2006)) developed an effective
chitosan hydrogel formulation containing EGF to determine its effect on
healing of second-degree burn wounds in rats. Burns are classified ac-
cording to the depth of the injury. In superficial second-degree burns,
the epidermis and the superficial dermis are mainly affected. And these
kinds of burns are very painful. According to the in vitro release studies,
the release of EGF from the chitosan gel was found to be 97.3% after
24 h. The release kinetics from the gel formulation was found to be of
the first order, and the release rate of EGF from the gel varied with time.
The in vivo study exhibited significant increase in cell proliferation in
the EGF containing gel applied group. The average numbers of pro-
liferating cells observed on the 3rd, 7th and 14th days are presented in
Table 1. Evaluation of these immunehisto-chemically observed results
showed that the rate of healing in the EJ group was increased. Finally it
has concluded that this formulation can play an important role in burn
wound treatment.
Park et al. (Park, Clark, Lichtensteiger, Jamison, and Wagoner
Johnson, (2009)) prepared chitosan scaffolds loaded with basic fibro-
blast growth factor (bFGF) contained in gelatin micro-particles and
tested for clinical relevance in an aged mouse model. The application of
this kind of wound dressing was for accelerating the healing process of
pressure ulcers. bFGF was successfully delivered to the wound and in-
creased angiogenesis compared to chitosan alone. They showed that
levels of bFGF in wound tissue were significantly higher, indicating
successful and prolonged growth factor delivery without burst effects.
Finally they concluded that chitosan loaded with bFGF is a good can-
didate for treatment of chronic wounds and reduces the proteolytic
environment of the wound and potentiates bFGF activity, leading to
accelerated wound closure.
Table 2 Summarizes some reported chitosan hydrogels with in-
corporated growth factors for wound dressing applications.
Nanotechnology is developing as a new growing field with appli-
cations in Science and Technology for the purpose of manufacturing
new materials at the nanoscale level. Nano-particles are used not only
in fundamental chemistry and physics investigations, but also widely in
biomedical fields. Due to their good antibacterial properties, their large
surface area to volume ratios, and high microbial resistance, nanoscale
materials have emerged as novel antimicrobial agents (Sung et al.,
2010; Radhakumary et al., 2011). Different types of metal materials
have been known to be used as antibacterial and/or/ antimicrobial
agents in wound healing such as, silver, zinc oxide, titanium dioxide,
copper oxide, and graphene. However, there could be problems with
long term exposure.
Sudheesh Kumar et al. (Kumbar et al., 2003) developed a flexible
and microporous chitosan/nano zinc oxide composite hydrogel. At
neutral pH, chitosan does not show much antibacterial activity; in order
to impart that activity, it is necessary to incorporate some antibacterial
agents into it. Zinc oxide nanoparticles were used in this research due to
its antibacterial activity. in vitro cytocompatibility studies revealed that
the dressing showed enhanced cell viability and infiltration. in vivo
wound healing evaluation proved the healing ability and antibacterial
potential of the prepared hydrogel without causing toxicity to cells.
They found that these advanced dressings can be used to prevent in-
fections in burns and chronic and diabetic wounds. It is also possible to
synthesize chitosan quaternary salts that have a permanent cationic
site, at all pH.
Silver and silver ions have been known as effective antimicrobial
agents for a long time. Owing to this ability, they have been applied to a
wide range of healthcare products, such as burn dressings, scaffolds,
skin replacements and medical devices (Altiok et al., 2010).
Nano silver/gelatin/carboxymethyl (CM)-Chitosan hydrogels were
synthesized by radiation-induced reduction and crosslinking at ambient
temperature (Altiok et al., 2010). After incubation at 37 °C for 12 h,
these hydrogels clearly showed antibacterial activity against gram-ne-
gative E. coli. The hydrogel with 10 mM nano silver content inhibited
more than 99% of the E. coli. When the nano silver content decreased to
5 mM, the maximum inhibition ratio was around 50%, and when the
nano silver content was further decreased to 2 mM, the ratio decreased
to 26%. The results of this study suggest that nano silver/gelatin/CM-
Chitosan hydrogels have potential as an antibacterial and anti-in-
flammation wound dressing.
Nano-curcumin was incorporated in N,O-carboxymethyl chitosan/
oxidized alginate hydrogel to develop a novel nano-composite hydrogel
wound dressing (Gaysinsky et al., 2008). Curcumin, a natural product
of the rhizomes of Curcuma longa, has been widely used as coloring

Table 2
Applications of incorporated growth factors in chitosan hydrogels.
Growth Factors Preparation Technique Potential Application References

*
EGF Mixing chitosan gel with GF Treating second-degree burn wounds (Yenilmez et al., 2015)
EGF Semi-IPN hydrogel Wound dressing (Morones et al., 2005)
EGF-liposome – Treating second-degree burn wounds (Sudheesh Kumar et al., 2012)
EGF & VEGF* Ionotropic gelation Wound healing accelerator (Zhou et al., 2012)
rh-EGF* Photo crosslinking Diabetic ulcers wound dressing (Li, Chen et al., 2012)
FGF-1 & FGF-2* Photo crosslinking Carrier material & Wound healing accelerator (Mani et al., 2002)
FGF-2* Photo crosslinking Treating healing-impaired wounds (Obara et al., 2003)
b FGF – Treating pressure ulcers (Rai et al., 2009)
b FGF Polyelectrolyte complexes (PEC) Skin tissue regeneration (Nimal et al., 2016)
PDGF-BB* & VEGF* Visible light activated crosslinking Wound healing accelerator (Bhattarai et al., 2010)

* EGF: Epidermal growth factor, VEGF: Vascular endothelial growth factor, rh-EGF: recombinant human epidermal growth factor, FGF-1: fibroblast growth factor-
1, PDGF-BB: Platelet derived growth factor-BB.

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H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Table 3
Application of nanoparticles Chitosan hydrogels.
Drug Preparation Technique Potential Application References

Zinc oxide / Silver Genipin-crosslinking Burn dressings (Yang et al., 2017)

Silver sulfadiazine Genipin-crosslinking As a carrier dressing (Choi & Yoo, 2010a)
Silver Oxide Casting/solvent evaporation Wound dressing (Pulat et al., 2013)
Silver UV radiation Biomedical fields (Ishihara et al., 2003)
UV radiation Anti-infectious wound dressing (Ho et al., 2009)
Glutaraldehyde-crosslinking Anti-microbial wound dressing (Liu & Kim, 2012a)
Copper oxide Epichlorohydrin-crosslinking Biomedical fields (Tyliszczak et al., 2017)
Zinc oxide Casting/solvent evaporation Wound dressing (Hajji et al., 2017)
Zinc oxide Genipin-crosslinking Burn dressings (Wahid et al., 2017)
Titanium dioxide Casting/solvent evaporation Wound healing dressing (Değim et al., 2011)
Graphene oxide Schiff-base reaction* Wound dressing (Vasile et al., 2014)
Curcumin nanoformulation Casting/solvent evaporation Wound healing dressing (Archana et al., 2015)
Nano Curcumin Mixing wound dressing (Gaysinsky et al., 2008)
Tigecycline nanoparticles Mixing Infectious wound treatment (Galotto et al., 2016)

*Reaction between aldehyde and the amino group of two materials.

agent and spice in food. But studies have shown that curcumin could
significantly accelerate the healing of wounds and enhance wound re-
pair in diabetic impaired healing (Gaysinsky, 2007). Nano-curcumin
could be released slowly from hydrogel to stimulate fibroblast pro-
liferation, capillary formation and collagen production which sig-
nificantly has effect on the healing phases. So, the nano-curcumin/
chitosan/alginate hydrogel might have potential application in wound
healing.
Tigecycline nanoparticles were loaded into chitosan–platelet-rich
plasma hydrogel for wound infection treatment. The tigecycline nano-
particle incorporated chitosan hydrogel showed a significant anti-
bacterial activity against S. aureus. This system can be an effective
medium for antibiotic delivery and applied on the infection sites to
effectively prevent various skin infections caused by S. aureus (Galotto
et al., 2016).
Table 3 Summarizes some reported Chitosan hydrogels containing
nanoparticles used for wound dressing applications.
5.3. Drug incorporated hydrogels
Controlled delivery systems provide an alternative approach to
regulate bioavailability of therapeutic agents. In controlled drug de-
livery systems, an active therapeutic is incorporated into a polymeric
network structure, and the drug is released from the structure in a
predefined manner. Depending on the drug delivery formulation and
application, the drug release time may be from a few hours to several
months (Vimala et al., 2011). Different approaches have been devel-
oped for drug incorporation. The method of drugs loading directly
impacts the availability of the drugs during release.
According to the application of wound dressings and the types of
wounds, various kinds of drugs can be loaded in wound dressings. Sung
et al. (Zhang et al., 2015) developed a minocycline-loaded wound
dressing made with a PVA and Chitosan blended hydrogel made using
the freeze-thaw method. Minocycline was used in this study because it
has been used in the treatment of superficial infections of the skin. The
minocycline-loaded wound dressing composed of 5% PVA, 0.75%
chitosan and 0.25% drug was more swellable, flexible and elastic than
the PVA gel alone, because of the relatively weak cross-linking
interaction of chitosan with PVA. According to their histologicaly ex-
amined data presented in Table 4, hydrogels prepared with drug re-
sulted in less inflammatory cells, more numerous collagen prolifera-
tions, and microvessels in rats than the conventional product or the
control (sterile gauze). The healing effect of minocycline-loaded hy-
drogel was great, indicating the potential healing effect of minocycline.
Finally they concluded that the aforementioned minocycline-loaded
wound dressing is a potential wound dressing with excellent forming
and enhanced wound healing characteristics.
A thermo responsive and cytocompatible chitosan based hydrogel
consisting of thiolated chitosan with poly (N-isopropyl acrylamide)
loaded with ciprofloxacin was introduced by Radhakumary et al
(Mishra et al., 2017). They reported that polymers with thiol groups
provide enhanced adhesive properties in comparison with many mu-
coadhesive polymers, and the strong cohesive properties of thiolated
chitosan make it highly suitable for controlled drug release. Cipro-
floxacin is a quinolone antibiotic drug recognized for treatment of skin
and skin structure infections. Their prepared film exhibited appropriate
mechanical properties for a wound/burn dressing and was found to be
cytocompatible and antibacterial as they released the entrapped anti-
biotic in a controlled manner for a period of more than 48 h.
Hydrogel microspheres of polyacrylamide-grafted‐chitosan crosslinked
with glutaraldehyde were prepared to encapsulate indomethacin, a non-
steroidal anti‐inflammatory drug. The microspheres were produced by the
water/oil emulsion technique and encapsulation of indomethacin was car-
ried out before crosslinking of the matrix. The initial release of in-
domethacin is due to the polymer chain relaxation process, but at longer
times, the release occurs from the fully swollen polymer and is controlled
mainly by the molecular diffusion phenomenon. This study suggests that
the hydrogel microspheres prepared from chitosan‐based polymers can be
useful in the delivery of indomethacin‐like drugs (Zhang et al., 2018).
Additional drug-loaded chitosan hydrogels reported for wound
dressing applications are presented in Table 5.
Essential oils and herbal extracts are natural complex mixtures of
volatile, lipophilic substances obtained from different parts of plants by
steam distillation and solvent extraction. In recent decades, interest in
essential oils for use in pharmaceutical and biomedical field has risen.
Essential oils have been shown to possess antibacterial, antifungal,

Table 4
Histomorphometrical Values for a Minocycline-loaded wound dressing made with a PVA and Chitosan blended hydrogel (Zhang et al., 2015).
Groups histomorphometry Gauze (control) Conventional product Hydrogel without drug Hydrogel with drug

Numbers of microvessels (vessels/1 mm2 of field) 4.40 ± 1.67 20.80 ± 6.72a 3.20 ± 2.28 23.40 ± 8.99
Numbers of inflammatory cells (cells/1 mm2 of field) 2106.00 ± 934.45 250.00 ± 217.17a 2447.00 ± 527.81 179.00 ± 227.74
Percentages of collagen tissue occupied regions (%/1 mm2 of field) 36.73 ± 8.96 66.21 ± 10.07a 33.41 ± 4.50 75.81 ± 9.44

455
H. Hamedi et al. Carbohydrate Polymers 199 (2018) 445–460

Table 5
Application of drug-loaded Chitosan hydrogels.
Drug Preparation Technique Potential Application References

*
Gentamicin sulfate EDC/NHS -crosslinking Anti-bactericidal wound dressing (Chen et al., 2017)
Gentamicin sulfate EDC/NHS*-crosslinking Anti-bactericidal wound dressing (Patel et al., 2017)
Tetracycline hydrochloride Emulsion-crosslinking Anti-bacterial wound dressing (Yang et al., 2018)
Tetracycline hydrochloride Mixing Scar preventive wound healing (Fan et al., 2016)
Tetracycline hydrochloride / silver sulfadiazine Casting/solvent evaporation Anti-infection wound dressing (Gao et al., 2016)
Naproxen Casting/solvent evaporation Anti-infection wound dressing (Ma et al., 2017)
Superoxide dismutase Polyelectrolyte complexes Antioxidant wound dressing (Rocasalbas et al., 2013)
Lignin Freeze-thaw Wound dressings (Morgado et al., 2017)
Amoxicillin Freeze-thaw Antibiotic delivery (Akıncıbay et al., 2007)
Ibuprofen Not mentioned Wound dressings (Perchyonok et al., 2014)
Metronidazole benzoate Mixing Treatment of chronic periodontitis (Ribeiro et al., 2013)
Clarithromycin, Tramadol and Heparin Genipin-crosslinking Pharmaceutical applications (Archana et al., 2013)
Nystatin Mixing Wound healing application (Liu & Kim, 2012b)
Acidic oxyphenbutazone & glafenine Coacervation techniques Anti-inflammatory drugs (Harris et al., 2008)
Essential oils or herbal extracts
Nerolidol – Wound healing dressing (Gonçalves Ferreira et al., 2016)
Thymol NVP-crosslinking* Wound dressing (Qin et al., 2006)
Apigenin PEG-crosslinking* Diabetic wound dressing (Kim et al., 2015)
Lupeol Glutaraldehyde-crosslinking Wound dressing (Risbud et al., 2000)
Polyphenolic Laccase- crosslinking Chronic wound dressing (Li et al., 2012)

* EDC: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiiminde, NHS: N-hydroxysuccinimide, PEG: Poly ethylene glycol.

NVP: 1-vinyl-2-pyrrolidone.

antiviral, insecticidal and antioxidant properties due to their biologi-
cally active compounds (See Table 5. for their applications in wound
dressings). Chitosan films loaded with thyme oil were prepared by a
solvent casting method (Jiang et al., 2016). Thyme oil has been shown
to have inhibitory activities against various bacteria and yeasts (Shukla
et al., 2016). Thymol, the major component of thyme oil (Anjum et al.,
2016), has also been shown to exhibit antimicrobial activity against
several bacteria and fungi (Koosehgol et al., 2017). The prepared films
showed both antibacterial and antioxidant activities. Results of this
research revealed that the thyme oil has a good potential to be in-
corporated into chitosan to make antibacterial and permeable films for
wound healing applications.
6. Conclusions
Wound healing is a complex and dynamic process of replacing de-
vitalized and missing cellular structures and tissue layers. Many efforts
have been focused on wound care with an emphasis on new therapeutic
approaches and development of technologies for wound management.
In recent decades, smart wound dressings with accelerated wound
healing properties have attracted much interest.
This review summarized the potential applications of chitosan hy-
drogels for pharmaceutical and biomedical uses, particularly with re-
gard to drug delivery in wound dressings. Chitosan is an excellent
candidate due to its non-toxicity, stability, biodegradability, and bio-
compatibility. Also chitosan hydrogel structures may be loaded with
various types of drugs in different physical forms, such as nanoparticles,
essential oils, and solubilized drugs. These properties have made chit-
osan a very versatile material with extensive applications in controlled
release formulations for treatment of acute and chronic wounds like
burns and pressure and diabetic ulcers. Therefore it can be concluded
that chitosan-based hydrogels are expected to become a promising
matrix for use in regenerative medicine, drug delivery, and wound
dressing applications.
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