European Polymer Journal xxx (2013) xxxxxx

Contents lists available at SciVerse ScienceDirect

European Polymer Journal

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

Feature Article

Chitosan-based biomaterials for tissue engineering

Florence Croisier 1, Christine Jrme
Center for Education and Research on Macromolecules (CERM), Department of Chemistry, University of Lige, Alle de la Chimie 3, B6a, 4000 Lige, Belgium

a r t i c l e

i n f o

Article history:
Received 10 October 2012
Received in revised form 12 December 2012
Accepted 15 December 2012
Available online xxxx
Keywords:
Chitosan scaffolds
Biomaterials
Tissue engineering
Wound healing

a b s t r a c t
Derived from chitin, chitosan is a unique biopolymer that exhibits outstanding properties,
beside biocompatibility and biodegradability. Most of these peculiar properties arise from
the presence of primary amines along the chitosan backbone. As a consequence, this polysaccharide is a relevant candidate in the eld of biomaterials, especially for tissue engineering. The current article highlights the preparation and properties of innovative
chitosan-based biomaterials, with respect to their future applications. The use of chitosan
in 3D-scaffolds as gels and sponges and in 2D-scaffolds as lms and bers is discussed, with a special focus on wound healing application.
2013 Elsevier Ltd. All rights reserved.

Contents
1.
2.
3.
4.
5.

6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chitosan: structure and extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structureproperties relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Remarkable properties of chitosan as biomaterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chitosan 3D-scaffolds for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1.
Physically associated chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2.
Chitosan cross-linked by coordination complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.3.
Chemically cross-linked chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Chitosan sponges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chitosan 2D-scaffolds for wound dressing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Chitosan films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.1.
Improving properties of chitosan free-standing films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.2.
Chitosan thin films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Chitosan porous nanofiber membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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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. Jrme).

URL: http://www.cerm.ulg.ac.be (C. Jrme).
Tel.: +32 4 366 34 69; fax: +32 4 366 34 97.

0014-3057/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.eurpolymj.2012.12.009

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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 challenging part of this approach is to obtain bio-based materials
with properties equivalent to those of fully synthetic products, from a functional point of view. In this respect, chitosan is a quite unique bio-based polymer: its intrinsic
properties are so singular and valuable that chitosan possesses 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-deoxyb-D-glucan (D-glucosamine) units [2]. The structure of this
polymer is depicted in Fig. 1. As such, chitosan is not extensively present in the environment however, it can be easily 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-glucosamine to the sum of D-glucosamine and N-acetyl D-glucosamine. 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

HN

OH
HO

O
O

HO

OH

NH2

Fig. 1. Chemical structure of chitosan.

HN

OH

under severe alkaline conditions or by enzymatic hydrolysis in the presence of particular enzymes, among of chitin
deacetylase [5,6].
After cellulose, chitin is the second most abundant biopolymer [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
[79]. 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 production 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 narrower molecular mass distribution than the chitosan produced 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 preparation. Chitosan oligomers can be prepared by degradation of
chitosan using specic enzyme [15] or reagent as hydrogen
peroxide [16].
At the time of its production, it is difcult to predict
chitosan characteristics. Therefore, the current approach
consists in analyzing the resulting product composition
and properties rather than in targeting predened characteristics by controlling the process of production. The characterization of chitosan is thus quite of importance but it is
not easygoing nor straightforward. Being a natural-based
compound, chitosan may be contaminated by organic
and inorganic impurities, and presents broad polydispersity, this is why producers mainly refer to samples viscosity 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 difcult
to perform. Different methods including pH-potentiometric titration, IR-spectroscopy, 1H NMR spectroscopy, but
also UV-spectroscopy, colloidal titration and enzymatic
degradation are reported in the literature for the determination of chitosan deacetylation degree [17], while its
molecular mass is typically deduced from viscosimetry or
determined by size exclusion chromatography [2].

3. Structureproperties relationship

HO

O
O

HO
OH

HN

O
Fig. 2. Chemical structure of chitin.

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

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[18]. Interestingly, the aqueous solubility of chitosan is pHdependent allowing processability under mild conditions
[4], which opens prospects to a wide range of applications,
particularly in the eld of cosmetics [2].
Due to these amino groups, chitosan also efciently
complexes various species, such as metal ions, and therefore is often used for the treatment of waste waters, purifying them by recovering heavy metals [2]. The complexing
ability of chitosan is further exploited for beverages (wine,
juices. . .) clarication [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 negatively charged synthetic polymers as poly(acrylic acid)
[4,18,20,21]. As a matter of fact, chitosan is the only positively charged, naturally occurring polysaccharide [18].
As a polyelectrolyte, chitosan can notably be employed
for the preparation of multilayered lms, using layer-bylayer 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 bondings with other species. Non-specic reactions can be
performed on the alcohol groups, as etherication and
esterication reactions [2] but remarkably, the amino
group of D-glucosamine residues may be specically quaternized or reacted with aldehyde functions under mild
conditions through reductive amination [2]. Various functionalizations can be introduced along chitosan backbone
by this technique to further extend chitosan eld of
applications.
Besides, chitosan exhibits other remarkable intrinsic
properties: this polysaccharide exhibits antibacterial activity [23,24], along with antifungal [9], mucoadhesive [25],
analgesic [9] and haemostatic properties [26]. It can be biodegraded 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 mechanism will lead to a better understanding of chitosan medical and pharmaceutical interest.
The presence of the protonable amino group along
D-glucosamine residues allows to elucidate most of chitosan properties. Indeed, the mucoadhesion of chitosan for
example, can be explained by the presence of negatively
charged residues (sialic acid) in the mucin the glycoprotein 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 conrm this explanation
[3234].
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 proteins, 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 rst proposed mechanism,
positively charged chitosan can interact with negatively
charged groups at the surface of cells, and as a consequence, 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 inhibition 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 explaining 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 inammatory area,
resulting in an analgesic effect [37].
Now, to explain chitosan biodegradability, it is important to remember that chitosan is not only a polymer bearing amino groups, but also a polysaccharide, which
consequently contains breakable glycosidic bonds. Chitosan is actually degraded in vivo by several proteases, and
mainly lysozyme [9,38,39]. Till now, eight human chitinases have been identied, three of them possessing enzymatic activity on chitosan [40]. The biodegradation of
chitosan leads to the formation of non-toxic oligosaccharides 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 [4244].
To explain the relationship between chitosan biodegradation 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 distribution 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.

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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 articial 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 physiological medium depends on the preparation method (residual
proteins could indeed cause allergic reactions) and on the
DD biocompatibility increases with DD increase. Chitosan actually proved to be more cytocompatible in vitro
than chitin. Indeed, while the number of positive charges
increases, the interaction between cells and chitosan increases as well, which tends to improve biocompatibility
[47].
Besides, some chemical modications of chitosan structure could induce toxicity [38].
After describing chitosan main properties in the previous paragraphs, the following will focus on the potential
applications of chitosan as a biomaterial, and more precisely on its processing methods for uses as biomedical
3D-scaffolds for tissue engineering, and 2D-scaffolds especially for wound healing purposes.
5. Chitosan 3D-scaffolds for tissue engineering
During the fabrication of implantable scaffolds particular 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 proliferation, have suitable mechanical properties for handling
and to mimic the damaged tissue, and nally, be manufactured into variety of shapes [49,50].
Due to its aforementioned remarkable properties, chitosan appears thus as a relevant candidate for the preparation of such biomaterials, which could substitute for
missing or damaged tissue and organ [48], and allow cell
attachment and proliferation [51] provided that 3D-scaffolds 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 tissue requirements. Therefore, methodologies have been
developed to nely 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 representing less than 10% of the total volume of the gel, and a liquid
phase. In hydrogels, the liquid phase is water (and sometimes adjuvants). The solid phase ensures the consistency

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 minimizes 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 functional 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 factor delivery [5254].
Three main types of chitosan hydrogels have been
developed, presenting either reversible or irreversible gelation. 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 parameters 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 chitosan crystallites [38,70], as illustrated in Fig. 3.
Further on, chitosan hydrogels can be formed by blending 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 modied to form physical 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 electrostatic interactions involving small-size polyanions but
also polyelectrolytes appears straightforward (Fig. 4a).
Positively charged chitosan will interact with negatively charged molecules, such as phosphates, sulfates
and citrates ions [57,58], to form hydrogels. Varying concentration and size of the anionic species as well as the
numbers of D-glucosamine units vs. N-acetyl-D-glucosamine units allows ne tuning of the swelling of the obtained hydrogel.

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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
collagen) and anionic polysaccharides
(hyaluronic acid)).

Via coordination bonds with metal ions

(as Pd(II), Pt (II) and Mo(VI)).

Via amide or ester bonds formation

(with glyoxal, glutaraldehyde and
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 crosslinking (a), coordination complex cross-linking (b) and covalent cross-linking (c).

Larger negatively charged molecules, natural and synthetic polyanions, can also give gels with chitosan. In the
natural polyanion category, proteins (as gelatin, collagen,
keratin, albumin and broin) [5961], anionic polysaccharides (as hyaluronic acid, alginate, pectin, heparin, xanthan,
dextran sulfate, chodroitin sulfate, fucoidan) [6265],
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 stability of the formed hydrogels. Proper adjustment of the pH of
the medium is thus important.

It should be noted that electrostatic interactions can occur with other secondary interactions, as for example
hydrogen bonding [68,69]. However, the interactions between 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 additives, 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

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tissue used as mold or template, perfectly adjusting the

scaffold to the defect. However, the mechanical resistance/strength of the aforementioned hydrogels is quite
limited and uncontrolled dissolution of the gels can occur
[74]. Besides, it is difcult 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 suited for biomedical use. This gelation is depicted in Fig. 4b.

100 m
Fig. 5. SEM image of a chitosan sponge prepared by freeze-drying.

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 physically associated hydrogels since the gelation is irreversible.
However, this approach requires the chemical modication
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 chitosan 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
rstly modied by activated functional groups before
inducing cross-linking by photo-induced reaction or enzymatic 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 aforementioned cross-linkers may impart toxicity to the formed
hydrogels, which is a major concern given the future application 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 established [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 molecules to the moles of polymer repeating units [79].
It is important to note that chemical cross-linking of
chitosan that involves chemical modications of chitosan
primary amine might induce modications of chitosan
properties. However, these groups being more reactive

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 uids (more than 20 times their dry weight),
due to their micro-porosity. They typically offer good cell
interaction, still being soft and exible [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 nd
application in bone tissue engineering, as a lling material
[81].
These sponges are mainly obtained by freeze drying
(lyophilization), a simple efcient 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. ChitosanZnO composite sponges
also prepared by freeze-drying [80] showed good swelling,
antibacterial and haemostatic activities, conrming their
potential healing in wound dressing application.
Cross-linked chitosan sponges loaded with a model of
antibiotic drug the noroxacin were prepared by solvent 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 supercritical 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, specic
requirements have to be met to encounter efciency. The

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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 t quite well these requirements. Nevertheless, as far as skin tissues are concerned, more 2D-shaped
scaffolds, such as lms and porous membranes, are supplementary candidates, spreading the panel of structures
available to tackle the challenges addressed in skin repair.
6.1. Chitosan lms
Chitosan lms can be easily prepared by wet casting
from chitosan salt solutions, followed by drying (typically
using oven or infrared (IR) drying [86]). For example, HemCon bandage is an engineered chitosan acetate preparation designed as a haemostatic dressing [80].
6.1.1. Improving properties of chitosan free-standing lms
To improve the properties of those lms, some physicochemical processes have been tested. For examples, (i)
nitrogen or argon plasma [87] treatment of chitosan membranes increases the lm surface roughness and improves
the cell adhesion and proliferation (especially for lms
treated with nitrogen plasma at 20 W during 20 min), (ii)
ozone or UV irradiation promotes surface modication of
chitosan lms leading to depolymerization of chitosan
[88], (iii) by introducing silica particles or poly(ethylene
glycol) [89] into the chitosan lms, their porosity can be
articially modied with macro and micro-pores (pores
size ranging 0.5100 lm).
As far as mechanical properties are concerned, blending
and chemical modications of chitosan have proven efciency. To improve the lms 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 lm, the decrease of the modulus was 56%, while
the increase of the strain at break was 125%, in comparison
to pure chitosan lm) [90]. Amidation of the chitosan lms
(up to 50%) can occur with thermal treatment; it leads to
signicant strengthening of the lms and to a decrease of
the lm solubility in aqueous media [91]. Chitosan lms
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 obtained with a dialdehyde starch content of 5%, with a tensile strength of 113.1 MPa and an elongation at break of
27%) [92]. via Michael addition reaction, chitosan/polyethylene glycol diacrylate (PEGDA) blended lms were developed [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 values of tensile strength and modulus were obtained at a ratio of CS/PEGDA equal to 70/30).
Other noteworthy developments are also the preparation of: (i) phosphorylated chitosan lms (prepared from
the reaction of orthophosphoric acid and urea in DMF),
which present higher ionic conductivity than normal chitosan lms (conductivity of pure chitosan lm being equal to
8.3  105 S cm1, and reaching 1.2  103 S cm1 for a

chitosan lm containing 87.31 mg/m2 of phosphorus

NB: conductivity measurements were realized after
hydratation of the lms) [94], (ii) chitosan blends with
minocycline hydrochloride, to prepare lms that accelerate
the healing of burns (reduction of the wound size) [95], (iii)
chitosan/poly(vinyl alcohol)/alginate lms by casting/
solvent evaporation method, in order to create a moist
environment within the framework of wound healing
[96], and (iv) non-adherent lm of b-glucan and chitosan
[97,98].
Heterogeneous chemical modication of the chitosan
lm can tune the surface properties of the lm; for instance, when a stearoyl group was attached to the chitosan
lms, they became more hydrophobic (contact angle of
pure chitosan lm being 89 6, while 101 4 was found
for N-stearoylchitosan lm) and promoted proteins
adsorption. When chitosan lms were reacted with succinic anhydride or phthalic anhydride, it resulted in more
hydrophilic lms (contact angles of 56 2 and 51 7
were found for N-succinylchitosan and N-phthalylchitosan
lms, respectively), which promoted lysozyme adsorption
[99]. Interestingly, a thermo-sensitive lm was prepared
by combining thiolated chitosan, poly(N-isopropyl acrylamide) and ciprooxacin, so that by decreasing the temperature, the lm can easily be removed from wound
[100].
Finally, incorporation of inorganic particles in these
lms allows synergistic effects of both materials leading
to high performance healing. Ag nanoparticles possess
identied antimicrobial activity, notably used in silverbased wound dressings [101]. Those Ag particles can be
added to chitosan lm, to further avoid microorganism
proliferation [102105]. Polyphosphate procoagulant can
also be incorporated to the chitosan/Ag lms [24]. Beside
Ag, zinc oxide (ZnO) nanoparticles are non-toxic and present antibacterial and good photocatalytic activities [106].
Like the Ag nanoparticles, they can be incorporated in
chitosan lms. Complex lms containing chitosan, Ag
nanoparticles and ZnO particles were prepared [107]. They
possess pronounced antibacterial activity (especially the
lm containing 0.1 wt.% Ag and 10 wt.% ZnO), thus promising application in wound dressing eld.
6.1.2. Chitosan thin lms
The properties of the wound dressing surface in contact
to the diseased body are particularly of importance to provide efcient healing. Multilayer coatings and nanostructured thin lms have thus also been investigated in order
to tune the surface properties of various medical devices.
Two methods to design chitosan thin lms in order to
modify the surface of a performed substrate will be shortly
described: LangmuirBlodgett (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 lm thickness, composition, morphology, and even
roughness [18].
To form a Langmuir [108] monolayer at the airwater
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 evaporated, the hydrophilic part of the amphiphile is anchored

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Fig. 6. Schematic representation of Langmuir lm (a) (left) and LangmuirBlodgett process (b) (right).

Fig. 7. Schematic representation of LBL process.

to the aqueous subphase, while the hydrophobic part is directed toward air [18] (as depicted in Fig. 6a). The LangmuirBlodgett (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 lms can
be obtained.
Chitosan being poorly soluble in organic solvents,
chemical modication is required for this process to be
performed. Chitosan modied with long alkyl chains attached to the primary hydroxyl and amino groups, was
the rst example of LB chitosan lms formation [18,110],
this derivatization making the resulting chitosan soluble
in chloroform. Derivatives of chitosan pentamers were
used to produce LB lms [111,112], while other amphiphilic chitosans, namely, N,N-dialkyl-chitosans, were also
reported [113]. All these lms nd application mainly in
drug release and drug delivery systems, since the amphiphilic lms are able to entrap various drugs with efcacy.
To facilitate the optical characterization of the LB lms,
amphiphilic chitosans bearing an alkyl group combined

with a cinnamoyl chromophore were synthesized [114].

Mixed lms of cholesterol and chloroform-soluble O,O-dipalmitoyl chitosan are an example of two-component LB
lms [115].
On the other hand, the layer-by-layer (LBL) deposition
technique is based on the alternated adsorption of materials 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 molecules can be used, including synthetic and natural polyelectrolytes [111], nanotubes [116] and biomolecules
[117]. Such polyelectrolyte multilayered lms nd many
applications in the eld of sensors [118], lters, 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 chitosan-based lms are involved in many applications as sensors, 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, enzymes, antigens, nucleic acids, carbon nanotubes, phthalo-

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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 lms
are particularly interesting for their anticoagulation properties [18].
To improve mechanical properties of chitosan LBL lms,
which might be especially important when deposited in
soft substrates, chitosan can be combined in the lm with
clays such as Na-montmorillonite [120], with carbon nanotubes [121], or with cellulose nanowhiskers [122].
LBL lms entirely made of polysaccharides, such as
chitosan and hyaluronic acid [123], and of chitosan and
alginate [119] offer great prospect for wound dressing purpose, as they have shown abilities to accelerate the healing.
6.2. Chitosan porous nanober membranes
There are several ways to produce chitosan bers; the
rst 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 ber
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 distinguished as a versatile technique for the preparation of
polymer bers from a few nanometers to microns in diameter, 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 bers formation. Fig. 8
shows a schematic representation of this technique. The
mats of bers produced by this method possess high
porosity, high specic surface area, and are able to mimic
the natural extracellular matrix, which makes these mats
great candidates for biomaterial applications. Chitosan
(nano)bers nd 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,

during ESP, repulsive forces between charged species

within polymer backbone arise as a consequence of the
application of an important electric eld. This phenomenon
restricts the formation of continuous bers and often leads
to the creation of particles [133,134].
A few examples of pure electrospun chitosan bers are
reported; chitosan (nano)bers were successfully produced from triuoroacetic 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 molecules and facilitate the ESP process. In addition, the use
of DCM allows improving the homogeneity of the produced
bers. Another group used TFA in conjugation with glutaraldehyde, to produce cross-linked chitosan bers [138]. Finally, the use of concentrated aqueous acetic acid was
considered [139,140].
To facilitate the electrospinning of chitosan, some
authors report on the use of modied chitosan such as
hexanoyl chitosan [141] and PEGylated chitosan [142].
Nevertheless, the easiest way to produce chitosan (nano)bers is the electrospinning of chitosan as a blend with another polymer. Many examples of such chitosan blend
bers produced by ESP are reported in the literature.
The electrospinning of chitosan in presence of poly(ethylene oxide) (PEO) [143147] and ultrahigh-molecularweight poly(ethylene oxide) (UHMWPEO) [148] are
commonly reported. A SEM image of chitosan/PEO nanobers
is presented in Fig. 9. Poly(vinyl alcohol) (PVA) [149151]
can also be used to form chitosan (nano)bers. The uses
of poly(ethylene terephtalate (PET) [152], poly(vinyl
pyrrolidone) (PVP) [153] and poly(lactic acid) (PLA) [154]
were also reported.
Chitosan/PEO nanobers prepared by ESP exhibit cellular biocompatibility [133] [147]. The structure of the ber
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 bers ranging from less than an hundred nanometers to a few tens micrometers [148], while adding PVP allows decreasing the diameter of chitosan-based bers
[153]. These blends thus offer great prospects for designing
tissue engineering scaffolds [133]. The joint use of PVA and
chitosan allows the preparation of bers without beaded

10 m
Fig. 8. Schematic representation of the electrospinning set-up.

Fig. 9. SEM image of electrospun chitosan/PEO blended bers.

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10

F. Croisier, C. Jrme / European Polymer Journal xxx (2013) xxxxxx

Table 1
Main advantages, disadvantages and applications of the chitosan biomaterials presented.
Biomaterial type
Hydrogels (3D)

Specications

Main advantage(s)

Main disadvantage(s)

Main biomedical application(s)

 Physically associated (reversible)

Soft exible non-toxic

Tissue replacements/engineering
drug/growth factor delivery

 Chemically crosslinked
(irreversible)

Soft exible stable

controlled pore size

Not stable (uncontrolled

dissolution may occur)
Low mechanical resistance
Pore size difcult to control
May be toxic

Crosslinking may affect

chitosan intrinsic properties
Sponges (3D)
Films (2D)

Porous Membranes (2D)

 Free-standing

High porosity
Soft

May shrivel
Low porosity

Tissue engineering (lling material)

Wound dressings skin substitutes

 Thin (LB)

Material coating

Coatings for a variety of scaffolds

wound dressings skin substitutes

 Thin (LBL)

Material coating
Multilayer
construction

Laborious for the

construction of multilayers
Many steps

Nanobers

High porosity
Mimic skin
extracellular matrix

defects; chitosan/PVA nanobers are notably used for a

variety of biomedical applications, as they also proved biocompatible [149151]. Chitosan/PET nanobers present,
besides biocompatibility, enhanced antibacterial properties, in comparison to pure PET bers or chitin/PET bers
[148].
The addition of another polymer to facilitate the ESP
process is actually a way to improve the properties of the
resulting bers such as the mechanical properties, the biocompatibility, and the antibacterial behavior [133]. Electrospun chitosan-based nanober mats appear thus today
as an emerging biomaterial in wound dressing applications
sustained by the apparition of products such as Chitoex
on the market.
7. Conclusions
Chitosan is a quite unique biosourced polymer characterized by primary amines along the backbone. Such structure 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 conditions) shows improved solubility in aqueous media which
allows the processing of this material in very mild conditions in a wide variety of shapes. Soft gels and thin lms,
so as stronger sponges and nanober mats are thus easily
made available with good control on their composition,
function and morphology. The main advantages and disadvantages of the chitosan biomaterials presented in this
article are pointed out in Table 1, with respect to their biomedical applications. Therefore, this polymer is very
attractive for biomedical uses, notably in tissue engineering. The wide panel of chitosan properties and processed
materials gives to this biosourced polymer a quite promising future as biomaterial as demonstrated by emerging
products on the market notably in the wound dressing
eld.

ESP of pure chitosan difcult

Coatings for a variety of scaffolds

wound dressings skin substitutes

Acknowledgements
F.C. is grateful to the Belgian Fonds pour la Formation
la Recherche dans lIndustrie et dans lAgriculture (FRIA)
for nancial support. The present work has been supported
by the Science Policy Ofce of the Belgian Federal Government (IAP 7/05). CERM thanks the Walloon Region for supporting researches on chitosan in the frame of CHITOPOL,
TARGETUM and GOCELL projects.
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Florence Croisier received her masters degree

in chemical sciences from the University of
Lige (ULg, Belgium) in 2007. She is currently
nishing her Ph.D. under the supervision of
Professor Christine Jrme in the Center for
Education and Research on Macromolecules
(ULg, Belgium). Her research focuses on the
preparation of chitosan-based nanobers
with multilayered structure, using a combination of electrospinning and layer-by-layer
deposition techniques.

Christine Jrme completed her PhD on conjugated polymers for aqueous waste treatments 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 Scientic Research in the
group of Professor R. Jrme, 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 biomaterials.

Please cite this article in press as: Croisier F, Jrme C. Chitosan-based biomaterials for tissue engineering. Eur Polym J (2013), http://
dx.doi.org/10.1016/j.eurpolymj.2012.12.009