European Polymer Journal 49 (2013) 780–792

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European Polymer Journal

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

Feature Article

Chitosan-based biomaterials for tissue engineering

Florence Croisier 1, Christine Jérôme ⇑
Center for Education and Research on Macromolecules (CERM), Department of Chemistry, University of Liège, Allée de la Chimie 3, B6a, 4000 Liège, Belgium

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

Article history: Derived from chitin, chitosan is a unique biopolymer that exhibits outstanding properties,
Received 10 October 2012 beside biocompatibility and biodegradability. Most of these peculiar properties arise from
Received in revised form 12 December 2012 the presence of primary amines along the chitosan backbone. As a consequence, this poly-
Accepted 15 December 2012
saccharide is a relevant candidate in the field of biomaterials, especially for tissue engi-
Available online 23 January 2013
neering. The current article highlights the preparation and properties of innovative
chitosan-based biomaterials, with respect to their future applications. The use of chitosan
Keywords:
in 3D-scaffolds – as gels and sponges – and in 2D-scaffolds – as films and fibers – is dis-
Chitosan scaffolds
Biomaterials
cussed, with a special focus on wound healing application.
Tissue engineering Ó 2013 Elsevier Ltd. Open access under CC BY-NC-ND license.
Wound healing

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
2. Chitosan: structure and extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
3. Structure–properties relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
4. Remarkable properties of chitosan as biomaterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
5. Chitosan 3D-scaffolds for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
5.1. Chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
5.1.1. Physically associated chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
5.1.2. Chitosan cross-linked by coordination complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
5.1.3. Chemically cross-linked chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
5.2. Chitosan sponges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
6. Chitosan 2D-scaffolds for wound dressing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
6.1. Chitosan films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
6.1.1. Improving properties of chitosan free-standing films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
6.1.2. Chitosan thin films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
6.2. Chitosan porous nanofiber membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

⇑ Corresponding author. Tel.: +32 4 366 34 91; fax: +32 4 366 34 97.
E-mail addresses: fcroisier@ulg.ac.be (F. Croisier), C.Jerome@ulg.ac.be (C. Jérôme).
URL: http://www.cerm.ulg.ac.be (C. Jérôme).
1
Tel.: +32 4 366 34 69; fax: +32 4 366 34 97.

0014-3057 Ó 2013 Elsevier Ltd. Open access under CC BY-NC-ND license.

http://dx.doi.org/10.1016/j.eurpolymj.2012.12.009
 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792
1. Introduction
Over the last years, many attempts have been made to
replace petrochemical products by renewable, biosourced
components. Abundant naturally occurring polymers – as
starch, collagen, gelatin, alginate, cellulose and chitin [1]
– represent attractive candidates as they could reduce
the actual dependence on fossil fuels, and consequently
have a positive environmental impact. The most challeng-
ing part of this approach is to obtain bio-based materials
with properties equivalent to those of fully synthetic prod-
ucts, from a functional point of view. In this respect, chito-
san is a quite unique bio-based polymer: its intrinsic
properties are so singular and valuable that chitosan pos-
sesses no actual petrochemical equivalent. Consequently,
the inherent characteristics of chitosan make it exploitable
directly for itself.
2. Chitosan: structure and extraction
Chitosan is a linear, semi-crystalline polysaccharide
composed of (1 ? 4)-2-acetamido-2-deoxy-b-D-glucan
(N-acetyl D-glucosamine) and (1 ? 4)-2-amino-2-deoxy-
b-D-glucan (D-glucosamine) units [2]. The structure of this
polymer is depicted in Fig. 1. As such, chitosan is not exten-
sively present in the environment – however, it can be eas-
ily derived from the partial deacetylation of a natural
polymer: the chitin (Fig. 2). The deacetylation degree
(DD) of chitosan, giving indication of the number of amino
groups along the chains, is calculated as the ratio of D-glu-
cosamine to the sum of D-glucosamine and N-acetyl D-glu-
cosamine. To be named ‘‘chitosan’’, the deacetylated chitin
should contain at least 60% of D-glucosamine residues [3,4]
(which corresponds to a deacetylation degree of 60). The
deacetylation of chitin is conducted by chemical hydrolysis
O compound, chitosan may be contaminated by organic
OH HN
O HO
O O
HO O to perform. Different methods including pH-potentiomet-
NH2 OH
Fig. 1. Chemical structure of chitosan.
O determined by size exclusion chromatography [2].
OH HN
O HO
O O
HO O
HN OH
O
Fig. 2. Chemical structure of chitin.
781
under severe alkaline conditions or by enzymatic hydroly-
sis in the presence of particular enzymes, among of chitin
deacetylase [5,6].
After cellulose, chitin is the second most abundant bio-
polymer [2] and is commonly found in invertebrates – as
crustacean shells or insect cuticles – but also in some
mushrooms envelopes, green algae cell walls, and yeasts
[7–9]. At industrial scale, the two main sources of chitosan
are crustaceans and fungal mycelia; the animal source
shows however some drawbacks as seasonal, of limited
supplies and with product variability which can lead to
inconsistent physicochemical characteristics [10]. The
mushroom source offers the advantage of a controlled pro-
duction environment all year round that insures a better
reproducibility of the resulting chitosan [11], the physical
properties of extracted chitosan being notably related to
the growth substrate composition. Moreover, the vegetable
source is generally preferred to the crustacean one from an
allergenic point of view – the produced chitosan being
safer for biomedical and healthcare applications [12]. The
mushroom-extracted chitosan typically presents a nar-
rower molecular mass distribution than the chitosan pro-
duced from seafood [12], and may also differ in terms of
molecular mass, DD and distribution of deacetylated
groups [11,13]. Chitosan DD greatly varies between 60
and 100% while its molecular weight typically ranges from
300 to 1000 kDA [1], depending on the source and prepara-
tion. Chitosan oligomers can be prepared by degradation of
chitosan using specific enzyme [15] or reagent as hydrogen
peroxide [16].
At the time of its production, it is difficult to predict
chitosan characteristics. Therefore, the current approach
consists in analyzing the resulting product composition
and properties rather than in targeting predefined charac-
teristics by controlling the process of production. The char-
acterization of chitosan is thus quite of importance but it is
not easygoing nor straightforward. Being a natural-based
and inorganic impurities, and presents broad polydisper-
sity, this is why producers mainly refer to samples viscos-
ity rather than molecular mass. Chitosan is also poorly
soluble, except in acidic medium (as it will be discussed
in the following paragraph) which makes analyzes difficult
ric titration, IR-spectroscopy, 1H NMR spectroscopy, but
also UV-spectroscopy, colloidal titration and enzymatic
degradation are reported in the literature for the determi-
nation of chitosan deacetylation degree [17], while its
molecular mass is typically deduced from viscosimetry or
3. Structure–properties relationship
The presence of amino groups in the chitosan structure
(Fig. 1) differentiates chitosan from chitin, and gives to this
polymer many peculiar properties. Indeed, the amino
groups of the D-glucosamine residues might be protonated
providing solubility in diluted acidic aqueous solutions
(pH < 6) [14]. In contrast, practical applications of chitin
are extremely limited due to its poor solubility, if any
782 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792
[18]. Interestingly, the aqueous solubility of chitosan is pH-
dependent allowing processability under mild conditions
[4], which opens prospects to a wide range of applications,
particularly in the field of cosmetics [2].
Due to these amino groups, chitosan also efficiently
complexes various species, such as metal ions, and there-
fore is often used for the treatment of waste waters, puri-
fying them by recovering heavy metals [2]. The complexing
ability of chitosan is further exploited for beverages (wine,
juices. . .) clarification [19].
Chitosan with protonated amino groups becomes a
polycation that can subsequently form ionic complexes
with a wide variety of natural or synthetic anionic
species [4], such as lipids, proteins, DNA and some nega-
tively charged synthetic polymers as poly(acrylic acid)
[4,18,20,21]. As a matter of fact, chitosan is the only posi-
tively charged, naturally occurring polysaccharide [18].
As a polyelectrolyte, chitosan can notably be employed
for the preparation of multilayered films, using layer-by-
layer deposition technique [22]. This particular case will
be detailed later on.
The amino and alcohol functions along chitosan chains
enable this polysaccharide to form stable covalent bon-
dings with other species. Non-specific reactions can be
performed on the alcohol groups, as etherification and
esterification reactions [2] but remarkably, the amino
group of D-glucosamine residues may be specifically quat-
ernized or reacted with aldehyde functions under mild
conditions through reductive amination [2]. Various func-
tionalizations can be introduced along chitosan backbone
by this technique to further extend chitosan field of
applications.
Besides, chitosan exhibits other remarkable intrinsic
properties: this polysaccharide exhibits antibacterial activ-
ity [23,24], along with antifungal [9], mucoadhesive [25],
analgesic [9] and haemostatic properties [26]. It can be bio-
degraded into non-toxic residues [27,28] – the rate of its
degradation being highly related to the molecular mass
of the polymer and its deacetylation degree – and has
proved to some extent biocompatibility with physiological
medium [29,30]. All these singular features make chitosan
an outstanding candidate for biomedical applications.
4. Remarkable properties of chitosan as biomaterial
As already mentioned, several remarkable properties of
chitosan offered unique opportunities to the development
of biomedical applications. The elucidation of their mecha-
nism will lead to a better understanding of chitosan med-
ical and pharmaceutical interest.
The presence of the protonable amino group along
D-glucosamine residues allows to elucidate most of chito-
san properties. Indeed, the mucoadhesion of chitosan for
example, can be explained by the presence of negatively
charged residues (sialic acid) in the mucin – the glycopro-
tein that composes the mucus. In acidic medium, chitosan
amino groups are positively charged and can thus interact
with the mucin. This mucoadhesion is directly related to
the DD of chitosan: actually, if chitosan DD increases, the
number of positive charges also increases, which leads to
improved mucoadhesive properties [31].
The haemostatic activity of chitosan can also be related
to the presence of positive charges on chitosan backbone.
Indeed, red blood cells membranes are negatively charged,
and can thus interact with the positively charged chitosan.
Besides, chitin shows less effective haemostatic activity
than chitosan, which tends to confirm this explanation
[32–34].
Due to its positive charges, chitosan can also interact
with the negative part of cells membrane, which can lead
to reorganization and an opening of the tight junction pro-
teins, explaining the permeation enhancing property of
this polysaccharide. As for mucoadhesion, if chitosan DD
increases, the permeation ability also increases [35].
The case of chitosan antimicrobial activity is slightly
more complex; two main mechanisms have been reported
in the literature to explain chitosan antibacterial and
antifungal activities. In the first proposed mechanism,
positively charged chitosan can interact with negatively
charged groups at the surface of cells, and as a conse-
quence, alter its permeability. This would prevent essential
materials to enter the cells or/and lead to the leaking of
fundamental solutes out of the cell. The second mechanism
involves the binding of chitosan with the cell DNA (still via
protonated amino groups), which would lead to the inhibi-
tion of the microbial RNA synthesis. Chitosan antimicrobial
property might in fact result from a combination of both
mechanisms [23,36].
The polycationic nature of chitosan also allows explain-
ing chitosan analgesic effects. Indeed, the amino groups of
the D-glucosamine residues can protonate in the presence
of proton ions that are released in the inflammatory area,
resulting in an analgesic effect [37].
Now, to explain chitosan biodegradability, it is impor-
tant to remember that chitosan is not only a polymer bear-
ing amino groups, but also a polysaccharide, which
consequently contains breakable glycosidic bonds. Chito-
san is actually degraded in vivo by several proteases, and
mainly lysozyme [9,38,39]. Till now, eight human chitinas-
es have been identified, three of them possessing enzy-
matic activity on chitosan [40]. The biodegradation of
chitosan leads to the formation of non-toxic oligosaccha-
rides of variable length. These oligosaccharides can be
incorporated in metabolic pathways or be further excreted
[41]. The degradation rate of chitosan is mainly related to
its degree of deacetylation, but also to the distribution of
N-acetyl D-glucosamine residues and the molecular mass
of chitosan [42–44].
To explain the relationship between chitosan biodegra-
dation and DD, it is important to remember that chitosan is
a semi-crystalline polymer; crystallinity is indeed maximum
for a DD equal to 0 or 100% (chitin or fully deacetylated
chitosan, respectively), and decreases for intermediate
DD. Yet, as polymer crystallinity is inversely related to
the biodegradation kinetic, when chitosan DD decreases
(close to 60%), its crystallinity also decreases, which results
in an increase of the biodegradation rate. Besides, the dis-
tribution of acetyl residues along chitosan will also affect
its crystallinity, and consequently the biodegradation rate.
Finally, it can be reasonably assumed that smaller chitosan
chains will be more rapidly degraded into oligosaccharides
than chitosan with higher molecular mass.
 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792
Considering all the aforementioned properties, it is not
surprising that chitosan was, is and will be tested in many
biomedical and pharmaceutical applications, notably for
sutures, dental and bone implants and as artificial skin
[2]. Within the framework of these tests, chitosan has
shown biocompatibility [9,45] and was notably approved
by the Food and Drug Administration (FDA) for use in
wound dressings [46].
However, the compatibility of chitosan with physiolog-
ical medium depends on the preparation method (residual
proteins could indeed cause allergic reactions) and on the
DD – biocompatibility increases with DD increase. Chito-
san actually proved to be more cytocompatible in vitro
than chitin. Indeed, while the number of positive charges
increases, the interaction between cells and chitosan in-
creases as well, which tends to improve biocompatibility
[47].
Besides, some chemical modifications of chitosan struc-
ture could induce toxicity [38].
After describing chitosan main properties in the previ-
ous paragraphs, the following will focus on the potential
applications of chitosan as a biomaterial, and more pre-
cisely on its processing methods for uses as biomedical
3D-scaffolds for tissue engineering, and 2D-scaffolds espe-
cially for wound healing purposes.
5. Chitosan 3D-scaffolds for tissue engineering
During the fabrication of implantable scaffolds particu-
lar attention should be paid to body compatibility,
mechanical properties, scaffold morphology and porosity,
as well as healing and tissue replacement capacity [48].
According to literature, the main requirements for the
elaboration of tissue engineering scaffolds also include that
the scaffold should not induce acute or chronic response,
should be biodegradable so that the cured tissue will be
able to replace the biomaterial, possess surface properties
that will promote cell attachment, differentiation and pro-
liferation, have suitable mechanical properties for handling
and to mimic the damaged tissue, and finally, be manufac-
tured into variety of shapes [49,50].
Due to its aforementioned remarkable properties, chito-
san appears thus as a relevant candidate for the prepara-
tion of such biomaterials, which could substitute for
missing or damaged tissue and organ [48], and allow cell
attachment and proliferation [51] provided that 3D-scaf-
folds might be produced. Such development relies thus
on robust processing methods for chitosan making able
to adjust the scaffold properties at best to the diseased tis-
sue requirements. Therefore, methodologies have been
developed to finely shape chitosan hydrogels and foams
(or sponges) as pertinent 3D-scaffolds applicable for tissue
engineering. These will be reviewed in the following
sections.
5.1. Chitosan hydrogels
Gels are composed of a solid phase, typically represent-
ing less than 10% of the total volume of the gel, and a liquid
phase. In hydrogels, the liquid phase is water (and some-
times adjuvants). The solid phase ensures the consistency
783
of the gel, making it able to soak up/absorb large quantities
of water while remaining insoluble in the liquid phase [12].
Hydrogels are interesting biomaterials as their high water
content makes them compatible with a majority of living
tissues. Moreover, they are soft and bendable, which min-
imizes the damage to the surrounding tissue during and
after implantation in the patient [38]. The mechanical
properties of hydrogels tend to mimic those of the soft
body-tissues, which allows the gels to insure both func-
tional and morphological characteristics of the tissue to
be repaired [38]. This is why hydrogels are often used as
biomedical scaffolds for tissue replacements, but also for
other biomedical applications such as drug and growth fac-
tor delivery [52–54].
Three main types of chitosan hydrogels have been
developed, presenting either reversible or irreversible gela-
tion. Chitosan can indeed be either physically associated,
coordinated with metal ions or irreversibly/chemically
cross-linked into hydrogels [38].
5.1.1. Physically associated chitosan hydrogels
The formation of ‘‘physical’’ hydrogels is based on the
reversible interactions that can occur between polymer
chains. Those interactions have a non-covalent nature,
such as electrostatic interactions, hydrophobic interactions
or hydrogen bondings [55,56].
Those interactions can be dependent of various param-
eters as pH, concentration, temperature. . ., which make
them not very stable, exhibiting reversible gelation. The
swelling of those hydrogels can be tuned by adjusting the
nature and the quantities of each component, in order to
increase or decrease the number of interactions. As a rule,
fewer interactions will lead to a softer gel while a higher
number of interactions will give a tighter and stiffer gel.
It is remarkable to observe that chitosan is able to form
a gel, all by itself, without the need of any additive. The
process is based on the neutralization of chitosan amino
groups and thus the inhibition of the repulsion between
chitosan chains. The formation of the hydrogel then occurs
via hydrogen bonds, hydrophobic interactions and chito-
san crystallites [38,70], as illustrated in Fig. 3.
Further on, chitosan hydrogels can be formed by blend-
ing chitosan with other water-soluble non-ionic polymers,
as poly(vinyl alcohol) (PVA) [55,71]. Thermo-sensitive
chitosan hydrogels can be obtained by blending chitosan
with polyol salts, as glycerol phosphate disodium salt
[72]. Chitosan structure can also be modified to form phys-
ical hydrogels: chitosan-g-poly(ethylene glycol) (PEG)
graft copolymer is able of self-organization depending on
the temperature to form stable hydrogels [73].
Thanks to the polycationic nature of chitosan in acidic
conditions, formation of chitosan hydrogels through elec-
trostatic interactions involving small-size polyanions but
also polyelectrolytes appears straightforward (Fig. 4a).
Positively charged chitosan will interact with nega-
tively charged molecules, such as phosphates, sulfates
and citrates ions [57,58], to form hydrogels. Varying con-
centration and size of the anionic species as well as the
numbers of D-glucosamine units vs. N-acetyl-D-glucosa-
mine units allows fine tuning of the swelling of the ob-
tained hydrogel.
784 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792

H bonds
Hydrophobic interactions
Crystallites

Fig. 3. Schematic representation of physical chitosan hydrogel, without any additives.

Non-covalent cross-linking (a) Coordination complex cross-linking (b) Covalent cross-linking (c)

Via electrostatic interactions with anions

(as phosphates, sulfates and citrates) or
polyanions (as proteins (gelatin and Via amide or ester bonds formation
Via coordination bonds with metal ions
collagen…) and anionic polysaccharides (with glyoxal, glutaraldehyde and
(as Pd(II), Pt (II) and Mo(VI)).
(hyaluronic acid…)). Genipin…).
Also via H bonds, hydrophobic
interactions and crystallization

Fig. 4. Schematic representation of three types of chitosan hydrogels, prepared in presence of additives. Gels can be formed through non-covalent cross-
linking (a), coordination complex cross-linking (b) and covalent cross-linking (c).

Larger negatively charged molecules, natural and syn-
thetic polyanions, can also give gels with chitosan. In the
natural polyanion category, proteins (as gelatin, collagen,
keratin, albumin and fibroin) [59–61], anionic polysaccha-
rides (as hyaluronic acid, alginate, pectin, heparin, xanthan,
dextran sulfate, chodroitin sulfate, fucoidan) [62–65],
carboxymethyl cellulose and glycosaminoglycans [62,66]
have been reported for such purpose. Synthetic polyanions
– as poly(acrylic acid) (PAA) – were also used to form
hydrogels [67]. The charge density of chitosan and of the
polyanion will play a major role in the swelling and stabil-
ity of the formed hydrogels. Proper adjustment of the pH of
the medium is thus important.
It should be noted that electrostatic interactions can oc-
cur with other secondary interactions, as for example
hydrogen bonding [68,69]. However, the interactions be-
tween charged chitosan and anions or polyanions are
stronger than these secondary bonding. The preparation
of ionic chitosan hydrogels avoids the use of catalyst or
toxic reacting agent, which is a prerequisite for biomedical
applications.
The easiness of gelation without the need of toxic addi-
tives, in conditions compatible with the human body led to
the development of injectable solutions that exhibit a
sol/gel transition upon injection in the body, and offer
the unique opportunity to shape the gel within the diseased
 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792
tissue used as mold or template, perfectly adjusting the
scaffold to the defect. However, the mechanical resis-
tance/strength of the aforementioned hydrogels is quite
limited and uncontrolled dissolution of the gels can occur
[74]. Besides, it is difficult to accurately control the size
of the hydrogel pores. Therefore, irreversible chemical
cross-linking of the hydrogels have been investigated.
5.1.2. Chitosan cross-linked by coordination complex
Coordinate-covalent bonds can also occur with chitosan
via metal ions as Pt (II), Pd (II), and Mo (VI) [68,69] leading
to the formation of another type of hydrogels, but less sui-
ted for biomedical use. This gelation is depicted in Fig. 4b.
5.1.3. Chemically cross-linked chitosan hydrogels
The formation of those hydrogels occurs via covalent
bonding between polymer chains (Fig. 4c). The obtained
hydrogels are much more stable than the previous physi-
cally associated hydrogels since the gelation is irreversible.
However, this approach requires the chemical modification
of the primary structure of chitosan, which could alter its
initial properties, particularly if amino groups are involved
in the reaction. In addition, the involved reaction might be
a source of contamination by toxic residual reactants or
catalyst traces.
Both the amino and the hydroxyl groups of chitosan can
form a variety of linkages, as amide and ester bonding, but
also Schiff base formation [55,75], which can lead to chito-
san hydrogels formation. Small multifunctional molecules
or polymeric cross-linker can both be used to react with
chitosan and induce cross-linking. Chitosan can also be
firstly modified by activated functional groups before
inducing cross-linking by photo-induced reaction or enzy-
matic catalyzed reactions [38].
A straightforward way to obtain irreversible chitosan
hydrogels is the use of dialdehyde cross-linkers, such as
glyoxal and glutaraldehyde, which fastly react with the
amino groups of the chitosan D-glucosamine units and, to
a lesser extent, with the hydroxyl groups of chitosan
[62]. Tripolyphosphate, ethylene glycol, diglycidyl ether
and diisocyanate can also play the role of cross-linker to
obtain chitosan hydrogels. However, all the aforemen-
tioned cross-linkers may impart toxicity to the formed
hydrogels, which is a major concern given the future appli-
cation of the hydrogels as biomaterials [76].
Lately, a natural derived cross-linker, the genipin
(C11H14O5, isolated from Genipa Americana in 1960 and
then from Gardenia jasminoides Ellis) has been used as
an alternative cross-linking agent [62]. This cross-linker
degrades more slowly than other cross-linkers (such as
glutaraldehyde) and its toxicity has not yet been estab-
lished [62,77]. Genipin can react with primary amine and
thus cross-link polymers containing such amino groups
[77,78], like proteins and chitosan.
The properties of the resulting hydrogels will rely on
the cross-linking density and the ratio of cross-linker mol-
ecules to the moles of polymer repeating units [79].
It is important to note that chemical cross-linking of
chitosan that involves chemical modifications of chitosan
primary amine might induce modifications of chitosan
properties. However, these groups being more reactive
785
100 µm
Fig. 5. SEM image of a chitosan sponge prepared by freeze-drying.
than the hydroxyl groups, they are mostly used for this
purpose since the cross-linking degree might consume
only few of the amino groups initially present on chitosan.
Chitosan with high DD is then of interest as starting
material.
5.2. Chitosan sponges
Sponges are nothing else than foams with an open
porosity. These solid structures are able to absorb high
amount of fluids (more than 20 times their dry weight),
due to their micro-porosity. They typically offer good cell
interaction, still being soft and flexible [80].
Chitosan sponges are mainly used as wound healing
materials, as they can soak up the wound exudates, while
helping the tissue regeneration. Chitosan sponges also find
application in bone tissue engineering, as a filling material
[81].
These sponges are mainly obtained by freeze drying
(lyophilization), a simple efficient process consisting in
freezing a solution of chitosan followed by sublimation of
the solvent under reduced pressure (a SEM image of a
chitosan sponge prepared by this technique is presented
in Fig. 5). For example, chitosan [54], chitosan/tricalcium
phosphate (TCP) [81,82], and chitosan/collagen sponges
[83] were prepared by this technique and used as scaffolds
for bone regeneration. Chitosan–ZnO composite sponges
also prepared by freeze-drying [80] showed good swelling,
antibacterial and haemostatic activities, confirming their
potential healing in wound dressing application.
Cross-linked chitosan sponges loaded with a model of
antibiotic drug – the norfloxacin – were prepared by sol-
vent evaporation technique [84]. Fibrillar structure was
obtained and those sponges showed promising use for
wound dressing application.
Recently, efforts were dedicated to the use of supercrit-
ical carbon dioxide (scCO2) as a green medium to induce
porosity to chitosan scaffolds [85].
The scCO2 method allows the preparation of porous
chitosan scaffolds, suitable for cell cultures directly upon
depressurization.
6. Chitosan 2D-scaffolds for wound dressing
In the particular case of wound dressing, specific
requirements have to be met to encounter efficiency. The
786 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792
wound dressing should be non-toxic and non-allergenic,
allow gas exchanges, keep moist environment, protect
the wound against microbial organisms and absorb wound
exudates [80]. As mentioned above, chitosan sponges have
been found to fit quite well these requirements. Neverthe-
less, as far as skin tissues are concerned, more 2D-shaped
scaffolds, such as films and porous membranes, are supple-
mentary candidates, spreading the panel of structures
available to tackle the challenges addressed in skin repair.
6.1. Chitosan films
Chitosan films can be easily prepared by wet casting
from chitosan salt solutions, followed by drying (typically
using oven or infrared (IR) drying [86]). For example, Hem-
ConÒ bandage is an engineered chitosan acetate prepara-
tion designed as a haemostatic dressing [80].
6.1.1. Improving properties of chitosan free-standing films
To improve the properties of those films, some physico-
chemical processes have been tested. For examples, (i)
nitrogen or argon plasma [87] treatment of chitosan mem-
branes increases the film surface roughness and improves
the cell adhesion and proliferation (especially for films
treated with nitrogen plasma at 20 W during 20 min), (ii)
ozone or UV irradiation promotes surface modification of
chitosan films leading to depolymerization of chitosan
[88], (iii) by introducing silica particles or poly(ethylene
glycol) [89] into the chitosan films, their porosity can be
artificially modified with macro and micro-pores (pores
size ranging 0.5–100 lm).
As far as mechanical properties are concerned, blending
and chemical modifications of chitosan have proven effi-
ciency. To improve the films ductility, chitosan can be
blended (or copolymerized) with poly(ethylene glycol)
(PEG), which results in a decrease of the modulus with
an increase of the strain at break (for 50/50 chitosan/PEO
blended film, the decrease of the modulus was 56%, while
the increase of the strain at break was 125%, in comparison
to pure chitosan film) [90]. Amidation of the chitosan films
(up to 50%) can occur with thermal treatment; it leads to
significant strengthening of the films and to a decrease of
the film solubility in aqueous media [91]. Chitosan films
prepared in presence of dialdehyde starch, that plays the
role of cross-linking agent, show improved mechanical
properties and better water-swelling (best values were ob-
tained with a dialdehyde starch content of 5%, with a ten-
sile strength of 113.1 MPa and an elongation at break of
27%) [92]. via Michael addition reaction, chitosan/polyeth-
ylene glycol diacrylate (PEGDA) blended films were devel-
oped [93], showing enhanced swelling (the maximum
swelling was observed at a ratio of CS/PEGDA equal to
40/60) and good mechanical properties (the maximum val-
ues of tensile strength and modulus were obtained at a ra-
tio of CS/PEGDA equal to 70/30).
Other noteworthy developments are also the prepara-
tion of: (i) phosphorylated chitosan films (prepared from
the reaction of orthophosphoric acid and urea in DMF),
which present higher ionic conductivity than normal chito-
san films (conductivity of pure chitosan film being equal to
8.3  105 S cm1, and reaching 1.2  103 S cm1 for a
chitosan film containing 87.31 mg/m2 of phosphorus –
NB: conductivity measurements were realized after
hydratation of the films) [94], (ii) chitosan blends with
minocycline hydrochloride, to prepare films that accelerate
the healing of burns (reduction of the wound size) [95], (iii)
chitosan/poly(vinyl alcohol)/alginate films by casting/
solvent evaporation method, in order to create a moist
environment within the framework of wound healing
[96], and (iv) non-adherent film of b-glucan and chitosan
[97,98].
Heterogeneous chemical modification of the chitosan
film can tune the surface properties of the film; for in-
stance, when a stearoyl group was attached to the chitosan
films, they became more hydrophobic (contact angle of
pure chitosan film being 89 ± 6°, while 101 ± 4° was found
for N-stearoylchitosan film) and promoted proteins
adsorption. When chitosan films were reacted with succi-
nic anhydride or phthalic anhydride, it resulted in more
hydrophilic films (contact angles of 56 ± 2° and 51 ± 7°
were found for N-succinylchitosan and N-phthalylchitosan
films, respectively), which promoted lysozyme adsorption
[99]. Interestingly, a thermo-sensitive film was prepared
by combining thiolated chitosan, poly(N-isopropyl acryl-
amide) and ciprofloxacin, so that by decreasing the tem-
perature, the film can easily be removed from wound
[100].
Finally, incorporation of inorganic particles in these
films allows synergistic effects of both materials leading
to high performance healing. Ag nanoparticles possess
identified antimicrobial activity, notably used in silver-
based wound dressings [101]. Those Ag particles can be
added to chitosan film, to further avoid microorganism
proliferation [102–105]. Polyphosphate procoagulant can
also be incorporated to the chitosan/Ag films [24]. Beside
Ag, zinc oxide (ZnO) nanoparticles are non-toxic and pres-
ent antibacterial and good photocatalytic activities [106].
Like the Ag nanoparticles, they can be incorporated in
chitosan films. Complex films containing chitosan, Ag
nanoparticles and ZnO particles were prepared [107]. They
possess pronounced antibacterial activity (especially the
film containing 0.1 wt.% Ag and 10 wt.% ZnO), thus promis-
ing application in wound dressing field.
6.1.2. Chitosan thin films
The properties of the wound dressing surface in contact
to the diseased body are particularly of importance to pro-
vide efficient healing. Multilayer coatings and nanostruc-
tured thin films have thus also been investigated in order
to tune the surface properties of various medical devices.
Two methods to design chitosan thin films in order to
modify the surface of a performed substrate will be shortly
described: Langmuir–Blodgett (LB), and the layer-by-layer
(LBL) deposition techniques. These two processes are of
importance since both of them allow to control parameters
such as film thickness, composition, morphology, and even
roughness [18].
To form a Langmuir [108] monolayer at the air–water
interface, a solution in an organic volatile solvent of an
amphiphilic water-insoluble (macro)molecule is spread
on the surface of water. When the organic solvent is evap-
orated, the hydrophilic part of the amphiphile is anchored
 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792 787

Fig. 6. Schematic representation of Langmuir film (a) (left) and Langmuir–Blodgett process (b) (right).

Fig. 7. Schematic representation of LBL process.

to the aqueous subphase, while the hydrophobic part is di-
rected toward air [18] (as depicted in Fig. 6a). The Lang-
muir–Blodgett (LB) process [109] consists in transferring
this Langmuir monolayer onto a solid support. This can
be done by immersing (or emerging) a solid support in
(or from) the aqueous subphase to recover the monolayer
(Fig. 6b). By repeating the process, multilayered films can
be obtained.
Chitosan being poorly soluble in organic solvents,
chemical modification is required for this process to be
performed. Chitosan modified with long alkyl chains at-
tached to the primary hydroxyl and amino groups, was
the first example of LB chitosan films formation [18,110],
this derivatization making the resulting chitosan soluble
in chloroform. Derivatives of chitosan pentamers were
used to produce LB films [111,112], while other amphi-
philic chitosans, namely, N,N-dialkyl-chitosans, were also
reported [113]. All these films find application mainly in
drug release and drug delivery systems, since the amphi-
philic films are able to entrap various drugs with efficacy.
To facilitate the optical characterization of the LB films,
amphiphilic chitosans bearing an alkyl group combined
with a cinnamoyl chromophore were synthesized [114].
Mixed films of cholesterol and chloroform-soluble O,O-di-
palmitoyl chitosan are an example of two-component LB
films [115].
On the other hand, the layer-by-layer (LBL) deposition
technique is based on the alternated adsorption of materi-
als bearing complementary charged or functional groups
[22,112], in aqueous medium. A schematic representation
of LBL process is presented in Fig. 7. A large number of mol-
ecules can be used, including synthetic and natural poly-
electrolytes [111], nanotubes [116] and biomolecules
[117]. Such polyelectrolyte multilayered films find many
applications in the field of sensors [118], filters, but also
as biomaterial for tissue engineering [119].
The polycationic nature of chitosan makes this polymer
well-suited for LBL processes. As a consequence, LBL chito-
san-based films are involved in many applications as sen-
sors, drug delivery systems, haemostatic devices, bone
implants and wound dressings [18].
Chitosan can be LBL co-deposited with several species
on metallic devices [18]. For sensing applications, en-
zymes, antigens, nucleic acids, carbon nanotubes, phthalo-
788 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792
cyanines, and porphyrins can be trapped by this process in
a chitosan matrix that helps maintaining the biomolecule
activity. For coronary stents, chitosan/heparin LBL films
are particularly interesting for their anticoagulation prop-
erties [18].
To improve mechanical properties of chitosan LBL films,
which might be especially important when deposited in
soft substrates, chitosan can be combined in the film with
clays such as Na-montmorillonite [120], with carbon nano-
tubes [121], or with cellulose nanowhiskers [122].
LBL films entirely made of polysaccharides, such as
chitosan and hyaluronic acid [123], and of chitosan and
alginate [119] offer great prospect for wound dressing pur-
pose, as they have shown abilities to accelerate the healing.
6.2. Chitosan porous nanofiber membranes
There are several ways to produce chitosan fibers; the
first example was reported as early as 1926 [124,125].
Fibers were notably produced using dry [126] and wet
spinning [127] from acetic acid solution. Lithium chloride/
N,N-dimethylacetamide was also used as a solvent [128].
In order to decrease production cost and to improve fiber
properties, blends of chitosan with other polymers were
also considered: sodium alginate [129], tropocollagen
[130], cellulose, sodium hyaluronate, sodium heparin,
sodium chondroitin sulfate [124], poly(acrylic acid) [131]
were thereby employed.
In recent years, electrospinning (ESP) [132] has distin-
guished as a versatile technique for the preparation of
polymer fibers from a few nanometers to microns in diam-
eter, depending on the processing conditions. ESP uses a
high voltage to create an electrically charged jet of polymer
solution or melt which can lead to fibers formation. Fig. 8
shows a schematic representation of this technique. The
mats of fibers produced by this method possess high
porosity, high specific surface area, and are able to mimic
the natural extracellular matrix, which makes these mats
great candidates for biomaterial applications. Chitosan
(nano)fibers find many applications for the preparation of
wound dressing and for tissue engineering [133].
However, as chitosan is a polyelectrolyte in acidic
medium, its electrospinning turns out to be tricky. Indeed,
Fig. 8. Schematic representation of the electrospinning set-up.
during ESP, repulsive forces between charged species
within polymer backbone arise as a consequence of the
application of an important electric field. This phenomenon
restricts the formation of continuous fibers and often leads
to the creation of particles [133,134].
A few examples of pure electrospun chitosan fibers are
reported; chitosan (nano)fibers were successfully pro-
duced from trifluoroacetic acid (TFA) and dichloromethane
(DCM) mixtures [135,136]. It was suggested [135] that the
amino groups of the chitosan form salts with TFA [137],
which destroy the interactions between chitosan mole-
cules and facilitate the ESP process. In addition, the use
of DCM allows improving the homogeneity of the produced
fibers. Another group used TFA in conjugation with glutar-
aldehyde, to produce cross-linked chitosan fibers [138]. Fi-
nally, the use of concentrated aqueous acetic acid was
considered [139,140].
To facilitate the electrospinning of chitosan, some
authors report on the use of modified chitosan such as
hexanoyl chitosan [141] and PEGylated chitosan [142].
Nevertheless, the easiest way to produce chitosan (nano)-
fibers is the electrospinning of chitosan as a blend with an-
other polymer. Many examples of such chitosan blend
fibers produced by ESP are reported in the literature.
The electrospinning of chitosan in presence of poly(eth-
ylene oxide) (PEO) [143–147] and ultrahigh-molecular-
weight poly(ethylene oxide) (UHMWPEO) [148] are
commonly reported. A SEM image of chitosan/PEO nanofibers
is presented in Fig. 9. Poly(vinyl alcohol) (PVA) [149–151]
can also be used to form chitosan (nano)fibers. The uses
of poly(ethylene terephtalate (PET) [152], poly(vinyl
pyrrolidone) (PVP) [153] and poly(lactic acid) (PLA) [154]
were also reported.
Chitosan/PEO nanofibers prepared by ESP exhibit cellu-
lar biocompatibility [133] [147]. The structure of the fiber
mats was found to promote the attachment of human cells,
while preserving their morphology and viability [147]. The
addition of UHMWPEO to chitosan ESP solution allows
forming fibers ranging from less than an hundred nanome-
ters to a few tens micrometers [148], while adding PVP al-
lows decreasing the diameter of chitosan-based fibers
[153]. These blends thus offer great prospects for designing
tissue engineering scaffolds [133]. The joint use of PVA and
chitosan allows the preparation of fibers without beaded
10 µm
Fig. 9. SEM image of electrospun chitosan/PEO blended fibers.
 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792 789

Table 1
Main advantages, disadvantages and applications of the chitosan biomaterials presented.

Biomaterial type Specifications Main advantage(s) Main disadvantage(s) Main biomedical application(s)
Hydrogels (3D)  Physically associ- Soft flexible non-toxic Not stable (uncontrolled Tissue replacements/engineering
ated (reversible) dissolution may occur) drug/growth factor delivery
Low mechanical resistance
Pore size difficult to control
 Chemically cross- Soft flexible stable May be toxic
linked controlled pore size
(irreversible)
Crosslinking may affect
chitosan intrinsic properties
Sponges (3D) High porosity May shrivel Tissue engineering (filling material)
 Free-standing Soft Low porosity Wound dressings skin substitutes
Films (2D)  Thin (LB) Material coating Laborious for the Coatings for a variety of scaffolds
construction of multilayers wound dressings skin substitutes
 Thin (LBL) Material coating Many steps
Multilayer
construction
Porous Membranes (2D) Nanofibers High porosity Coatings for a variety of scaffolds
Mimic skin ESP of pure chitosan difficult wound dressings skin substitutes
extracellular matrix

defects; chitosan/PVA nanofibers are notably used for a Acknowledgements

variety of biomedical applications, as they also proved bio-
compatible [149–151]. Chitosan/PET nanofibers present,
besides biocompatibility, enhanced antibacterial proper-
ties, in comparison to pure PET fibers or chitin/PET fibers
[148].
The addition of another polymer to facilitate the ESP
process is actually a way to improve the properties of the
resulting fibers such as the mechanical properties, the bio-
compatibility, and the antibacterial behavior [133]. Elec-
trospun chitosan-based nanofiber mats appear thus today
as an emerging biomaterial in wound dressing applications
sustained by the apparition of products such as ChitoflexÒ
on the market.
7. Conclusions
Chitosan is a quite unique biosourced polymer charac-
terized by primary amines along the backbone. Such struc-
ture imparts to this polysaccharide not only highly
valuable physico-chemical properties but also particular
interactions with proteins, cells and living organisms. In
addition, this polycation (upon protonation in acidic condi-
tions) shows improved solubility in aqueous media which
allows the processing of this material in very mild condi-
tions in a wide variety of shapes. Soft gels and thin films,
so as stronger sponges and nanofiber mats are thus easily
made available with good control on their composition,
function and morphology. The main advantages and disad-
vantages of the chitosan biomaterials presented in this
article are pointed out in Table 1, with respect to their bio-
medical applications. Therefore, this polymer is very
attractive for biomedical uses, notably in tissue engineer-
ing. The wide panel of chitosan properties and processed
materials gives to this biosourced polymer a quite promis-
ing future as biomaterial as demonstrated by emerging
products on the market notably in the wound dressing
field.
F.C. is grateful to the Belgian ‘‘Fonds pour la Formation à
la Recherche dans l’Industrie et dans l’Agriculture’’ (FRIA)
for financial support. The present work has been supported
by the Science Policy Office of the Belgian Federal Govern-
ment (IAP 7/05). CERM thanks the Walloon Region for sup-
porting researches on chitosan in the frame of CHITOPOL,
TARGETUM and GOCELL projects.
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Florence Croisier received her master’s degree
in chemical sciences from the University of
Liège (ULg, Belgium) in 2007. She is currently
finishing her Ph.D. under the supervision of
Professor Christine Jérôme in the Center for
Education and Research on Macromolecules
(ULg, Belgium). Her research focuses on the
preparation of chitosan-based nanofibers
with multilayered structure, using a combi-
nation of electrospinning and layer-by-layer
deposition techniques.
Christine Jérôme completed her PhD on con-
jugated polymers for aqueous waste treat-
ments in 1998 at the University of Liege,
Belgium and then worked as a post-dosctoral
researcher on developing the electrografting
process of acrylates. In 2000, she joined the
University of Ulm in Germany as a recipient of
the Humboldt scholarship and studied the
synthesis of functional magnetic nanohybrids.
She returned to the University of Liege in
2001 as Research Associate of the National
Foundation of the Scientific Research in the
group of Professor R. Jérôme, where she became Professor in 2006 and
director of the Center for Education and Research on Macromolecules in
2007. Today full Professor, her research interests include biosourced
polymers, functional macromolecular systems and advanced biomateri-
als.