European Polymer Journal 49 (2013) 780–792

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European Polymer Journal

journal homepage: www.elsevier .com/locate /europol j

Feature Article

Chitosan-based biomaterials for tissue engineering

0014-3057 � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.eurpolymj.2012.12.009

⇑ 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.

Open access under CC BY-NC-ND license.

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:Received 10 October 2012Received in revised form 12 December 2012Accepted 15 December 2012Available online 23 January 2013

Keywords:Chitosan scaffoldsBiomaterialsTissue engineeringWound healing

Derived from chitin, chitosan is a unique biopolymer that exhibits outstanding properties,beside biocompatibility and biodegradability. Most of these peculiar properties arise fromthe presence of primary amines along the chitosan backbone. As a consequence, this poly-saccharide is a relevant candidate in the field of biomaterials, especially for tissue engi-neering. The current article highlights the preparation and properties of innovativechitosan-based biomaterials, with respect to their future applications. The use of chitosanin 3D-scaffolds – as gels and sponges – and in 2D-scaffolds – as films and fibers – is dis-cussed, with a special focus on wound healing application.

� 2013 Elsevier Ltd. Open access under CC BY-NC-ND license.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7812. Chitosan: structure and extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7813. Structure–properties relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7814. Remarkable properties of chitosan as biomaterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7825. Chitosan 3D-scaffolds for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

5.1. Chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

5.1.1. Physically associated chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7835.1.2. Chitosan cross-linked by coordination complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7855.1.3. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7866.1.2. Chitosan thin films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

6.2. Chitosan porous nanofiber membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

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1. Introduction

Over the last years, many attempts have been made toreplace petrochemical products by renewable, biosourcedcomponents. Abundant naturally occurring polymers – asstarch, collagen, gelatin, alginate, cellulose and chitin [1]– represent attractive candidates as they could reducethe actual dependence on fossil fuels, and consequentlyhave a positive environmental impact. The most challeng-ing part of this approach is to obtain bio-based materialswith 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 intrinsicproperties are so singular and valuable that chitosan pos-sesses no actual petrochemical equivalent. Consequently,the inherent characteristics of chitosan make it exploitabledirectly for itself.

2. Chitosan: structure and extraction

Chitosan is a linear, semi-crystalline polysaccharidecomposed 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 thispolymer 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 naturalpolymer: the chitin (Fig. 2). The deacetylation degree(DD) of chitosan, giving indication of the number of aminogroups 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 chitinshould contain at least 60% of D-glucosamine residues [3,4](which corresponds to a deacetylation degree of 60). Thedeacetylation of chitin is conducted by chemical hydrolysis

O

OO

HO

NH2

HO

OH

HN

O

O

OH

Fig. 1. Chemical structure of chitosan.

O

OO

HO

HN

HO

OH

HN

O

O

OH

O

Fig. 2. Chemical structure of chitin.

under severe alkaline conditions or by enzymatic hydroly-sis in the presence of particular enzymes, among of chitindeacetylase [5,6].

After cellulose, chitin is the second most abundant bio-polymer [2] and is commonly found in invertebrates – ascrustacean shells or insect cuticles – but also in somemushrooms envelopes, green algae cell walls, and yeasts[7–9]. At industrial scale, the two main sources of chitosanare crustaceans and fungal mycelia; the animal sourceshows however some drawbacks as seasonal, of limitedsupplies and with product variability which can lead toinconsistent physicochemical characteristics [10]. Themushroom source offers the advantage of a controlled pro-duction environment all year round that insures a betterreproducibility of the resulting chitosan [11], the physicalproperties of extracted chitosan being notably related tothe growth substrate composition. Moreover, the vegetablesource is generally preferred to the crustacean one from anallergenic point of view – the produced chitosan beingsafer for biomedical and healthcare applications [12]. Themushroom-extracted chitosan typically presents a nar-rower molecular mass distribution than the chitosan pro-duced from seafood [12], and may also differ in terms ofmolecular mass, DD and distribution of deacetylatedgroups [11,13]. Chitosan DD greatly varies between 60and 100% while its molecular weight typically ranges from300 to 1000 kDA [1], depending on the source and prepara-tion. Chitosan oligomers can be prepared by degradation ofchitosan using specific enzyme [15] or reagent as hydrogenperoxide [16].

At the time of its production, it is difficult to predictchitosan characteristics. Therefore, the current approachconsists in analyzing the resulting product compositionand 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 isnot easygoing nor straightforward. Being a natural-basedcompound, chitosan may be contaminated by organicand inorganic impurities, and presents broad polydisper-sity, this is why producers mainly refer to samples viscos-ity rather than molecular mass. Chitosan is also poorlysoluble, except in acidic medium (as it will be discussedin the following paragraph) which makes analyzes difficultto perform. Different methods including pH-potentiomet-ric titration, IR-spectroscopy, 1H NMR spectroscopy, butalso UV-spectroscopy, colloidal titration and enzymaticdegradation are reported in the literature for the determi-nation of chitosan deacetylation degree [17], while itsmolecular mass is typically deduced from viscosimetry ordetermined by size exclusion chromatography [2].

3. Structure–properties relationship

The presence of amino groups in the chitosan structure(Fig. 1) differentiates chitosan from chitin, and gives to thispolymer many peculiar properties. Indeed, the aminogroups of the D-glucosamine residues might be protonatedproviding solubility in diluted acidic aqueous solutions(pH < 6) [14]. In contrast, practical applications of chitinare extremely limited due to its poor solubility, if any

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[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 efficientlycomplexes 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 complexingability of chitosan is further exploited for beverages (wine,juices. . .) clarification [19].

Chitosan with protonated amino groups becomes apolycation that can subsequently form ionic complexeswith a wide variety of natural or synthetic anionicspecies [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 employedfor the preparation of multilayered films, using layer-by-layer deposition technique [22]. This particular case willbe detailed later on.

The amino and alcohol functions along chitosan chainsenable this polysaccharide to form stable covalent bon-dings with other species. Non-specific reactions can beperformed on the alcohol groups, as etherification andesterification reactions [2] but remarkably, the aminogroup of D-glucosamine residues may be specifically quat-ernized or reacted with aldehyde functions under mildconditions through reductive amination [2]. Various func-tionalizations can be introduced along chitosan backboneby this technique to further extend chitosan field ofapplications.

Besides, chitosan exhibits other remarkable intrinsicproperties: 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 itsdegradation being highly related to the molecular massof the polymer and its deacetylation degree – and hasproved to some extent biocompatibility with physiologicalmedium [29,30]. All these singular features make chitosanan outstanding candidate for biomedical applications.

4. Remarkable properties of chitosan as biomaterial

As already mentioned, several remarkable properties ofchitosan offered unique opportunities to the developmentof 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 alongD-glucosamine residues allows to elucidate most of chito-san properties. Indeed, the mucoadhesion of chitosan forexample, can be explained by the presence of negativelycharged residues (sialic acid) in the mucin – the glycopro-tein that composes the mucus. In acidic medium, chitosanamino groups are positively charged and can thus interactwith the mucin. This mucoadhesion is directly related tothe DD of chitosan: actually, if chitosan DD increases, thenumber of positive charges also increases, which leads toimproved mucoadhesive properties [31].

The haemostatic activity of chitosan can also be relatedto 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 activitythan chitosan, which tends to confirm this explanation[32–34].

Due to its positive charges, chitosan can also interactwith the negative part of cells membrane, which can leadto reorganization and an opening of the tight junction pro-teins, explaining the permeation enhancing property ofthis polysaccharide. As for mucoadhesion, if chitosan DDincreases, the permeation ability also increases [35].

The case of chitosan antimicrobial activity is slightlymore complex; two main mechanisms have been reportedin the literature to explain chitosan antibacterial andantifungal activities. In the first proposed mechanism,positively charged chitosan can interact with negativelycharged groups at the surface of cells, and as a conse-quence, alter its permeability. This would prevent essentialmaterials to enter the cells or/and lead to the leaking offundamental solutes out of the cell. The second mechanisminvolves the binding of chitosan with the cell DNA (still viaprotonated amino groups), which would lead to the inhibi-tion of the microbial RNA synthesis. Chitosan antimicrobialproperty might in fact result from a combination of bothmechanisms [23,36].

The polycationic nature of chitosan also allows explain-ing chitosan analgesic effects. Indeed, the amino groups ofthe D-glucosamine residues can protonate in the presenceof 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, whichconsequently contains breakable glycosidic bonds. Chito-san is actually degraded in vivo by several proteases, andmainly 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 ofchitosan leads to the formation of non-toxic oligosaccha-rides of variable length. These oligosaccharides can beincorporated in metabolic pathways or be further excreted[41]. The degradation rate of chitosan is mainly related toits degree of deacetylation, but also to the distribution ofN-acetyl D-glucosamine residues and the molecular massof chitosan [42–44].

To explain the relationship between chitosan biodegra-dation and DD, it is important to remember that chitosan isa semi-crystalline polymer; crystallinity is indeed maximumfor a DD equal to 0 or 100% (chitin or fully deacetylatedchitosan, respectively), and decreases for intermediateDD. Yet, as polymer crystallinity is inversely related tothe biodegradation kinetic, when chitosan DD decreases(close to 60%), its crystallinity also decreases, which resultsin an increase of the biodegradation rate. Besides, the dis-tribution of acetyl residues along chitosan will also affectits crystallinity, and consequently the biodegradation rate.Finally, it can be reasonably assumed that smaller chitosanchains will be more rapidly degraded into oligosaccharidesthan chitosan with higher molecular mass.

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Considering all the aforementioned properties, it is notsurprising that chitosan was, is and will be tested in manybiomedical and pharmaceutical applications, notably forsutures, dental and bone implants and as artificial skin[2]. Within the framework of these tests, chitosan hasshown biocompatibility [9,45] and was notably approvedby the Food and Drug Administration (FDA) for use inwound dressings [46].

However, the compatibility of chitosan with physiolog-ical medium depends on the preparation method (residualproteins could indeed cause allergic reactions) and on theDD – biocompatibility increases with DD increase. Chito-san actually proved to be more cytocompatible in vitrothan chitin. Indeed, while the number of positive chargesincreases, 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 potentialapplications of chitosan as a biomaterial, and more pre-cisely on its processing methods for uses as biomedical3D-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 theelaboration of tissue engineering scaffolds also include thatthe scaffold should not induce acute or chronic response,should be biodegradable so that the cured tissue will beable to replace the biomaterial, possess surface propertiesthat will promote cell attachment, differentiation and pro-liferation, have suitable mechanical properties for handlingand 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 formissing or damaged tissue and organ [48], and allow cellattachment and proliferation [51] provided that 3D-scaf-folds might be produced. Such development relies thuson robust processing methods for chitosan making ableto adjust the scaffold properties at best to the diseased tis-sue requirements. Therefore, methodologies have beendeveloped to finely shape chitosan hydrogels and foams(or sponges) as pertinent 3D-scaffolds applicable for tissueengineering. These will be reviewed in the followingsections.

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 liquidphase. In hydrogels, the liquid phase is water (and some-times adjuvants). The solid phase ensures the consistency

of the gel, making it able to soak up/absorb large quantitiesof water while remaining insoluble in the liquid phase [12].Hydrogels are interesting biomaterials as their high watercontent makes them compatible with a majority of livingtissues. Moreover, they are soft and bendable, which min-imizes the damage to the surrounding tissue during andafter implantation in the patient [38]. The mechanicalproperties of hydrogels tend to mimic those of the softbody-tissues, which allows the gels to insure both func-tional and morphological characteristics of the tissue tobe repaired [38]. This is why hydrogels are often used asbiomedical scaffolds for tissue replacements, but also forother biomedical applications such as drug and growth fac-tor delivery [52–54].

Three main types of chitosan hydrogels have beendeveloped, presenting either reversible or irreversible gela-tion. Chitosan can indeed be either physically associated,coordinated with metal ions or irreversibly/chemicallycross-linked into hydrogels [38].

5.1.1. Physically associated chitosan hydrogelsThe formation of ‘‘physical’’ hydrogels is based on the

reversible interactions that can occur between polymerchains. Those interactions have a non-covalent nature,such as electrostatic interactions, hydrophobic interactionsor hydrogen bondings [55,56].

Those interactions can be dependent of various param-eters as pH, concentration, temperature. . ., which makethem not very stable, exhibiting reversible gelation. Theswelling of those hydrogels can be tuned by adjusting thenature and the quantities of each component, in order toincrease or decrease the number of interactions. As a rule,fewer interactions will lead to a softer gel while a highernumber of interactions will give a tighter and stiffer gel.

It is remarkable to observe that chitosan is able to forma gel, all by itself, without the need of any additive. Theprocess is based on the neutralization of chitosan aminogroups and thus the inhibition of the repulsion betweenchitosan chains. The formation of the hydrogel then occursvia 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-sensitivechitosan hydrogels can be obtained by blending chitosanwith 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 onthe temperature to form stable hydrogels [73].

Thanks to the polycationic nature of chitosan in acidicconditions, formation of chitosan hydrogels through elec-trostatic interactions involving small-size polyanions butalso polyelectrolytes appears straightforward (Fig. 4a).

Positively charged chitosan will interact with nega-tively charged molecules, such as phosphates, sulfatesand citrates ions [57,58], to form hydrogels. Varying con-centration and size of the anionic species as well as thenumbers of D-glucosamine units vs. N-acetyl-D-glucosa-mine units allows fine tuning of the swelling of the ob-tained hydrogel.

H bondsHydrophobic interactionsCrystallites

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…)).

Also via H bonds, hydrophobic

interactions and crystallization

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…).

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).

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Larger negatively charged molecules, natural and syn-thetic polyanions, can also give gels with chitosan. In thenatural 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 formhydrogels [67]. The charge density of chitosan and of thepolyanion will play a major role in the swelling and stabil-ity of the formed hydrogels. Proper adjustment of the pH ofthe medium is thus important.

It should be noted that electrostatic interactions can oc-cur with other secondary interactions, as for examplehydrogen bonding [68,69]. However, the interactions be-tween charged chitosan and anions or polyanions arestronger than these secondary bonding. The preparationof ionic chitosan hydrogels avoids the use of catalyst ortoxic reacting agent, which is a prerequisite for biomedicalapplications.

The easiness of gelation without the need of toxic addi-tives, in conditions compatible with the human body led tothe development of injectable solutions that exhibit asol/gel transition upon injection in the body, and offerthe unique opportunity to shape the gel within the diseased

100 µm

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

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tissue used as mold or template, perfectly adjusting thescaffold to the defect. However, the mechanical resis-tance/strength of the aforementioned hydrogels is quitelimited and uncontrolled dissolution of the gels can occur[74]. Besides, it is difficult to accurately control the sizeof the hydrogel pores. Therefore, irreversible chemicalcross-linking of the hydrogels have been investigated.

5.1.2. Chitosan cross-linked by coordination complexCoordinate-covalent bonds can also occur with chitosan

via metal ions as Pt (II), Pd (II), and Mo (VI) [68,69] leadingto 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 hydrogelsThe formation of those hydrogels occurs via covalent

bonding between polymer chains (Fig. 4c). The obtainedhydrogels are much more stable than the previous physi-cally associated hydrogels since the gelation is irreversible.However, this approach requires the chemical modificationof the primary structure of chitosan, which could alter itsinitial properties, particularly if amino groups are involvedin the reaction. In addition, the involved reaction might bea source of contamination by toxic residual reactants orcatalyst traces.

Both the amino and the hydroxyl groups of chitosan canform a variety of linkages, as amide and ester bonding, butalso Schiff base formation [55,75], which can lead to chito-san hydrogels formation. Small multifunctional moleculesor polymeric cross-linker can both be used to react withchitosan and induce cross-linking. Chitosan can also befirstly modified by activated functional groups beforeinducing cross-linking by photo-induced reaction or enzy-matic catalyzed reactions [38].

A straightforward way to obtain irreversible chitosanhydrogels is the use of dialdehyde cross-linkers, such asglyoxal and glutaraldehyde, which fastly react with theamino groups of the chitosan D-glucosamine units and, toa lesser extent, with the hydroxyl groups of chitosan[62]. Tripolyphosphate, ethylene glycol, diglycidyl etherand diisocyanate can also play the role of cross-linker toobtain chitosan hydrogels. However, all the aforemen-tioned cross-linkers may impart toxicity to the formedhydrogels, 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 andthen from Gardenia jasminoides Ellis) has been used asan alternative cross-linking agent [62]. This cross-linkerdegrades more slowly than other cross-linkers (such asglutaraldehyde) and its toxicity has not yet been estab-lished [62,77]. Genipin can react with primary amine andthus cross-link polymers containing such amino groups[77,78], like proteins and chitosan.

The properties of the resulting hydrogels will rely onthe 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 ofchitosan that involves chemical modifications of chitosanprimary amine might induce modifications of chitosanproperties. However, these groups being more reactive

than the hydroxyl groups, they are mostly used for thispurpose since the cross-linking degree might consumeonly few of the amino groups initially present on chitosan.Chitosan with high DD is then of interest as startingmaterial.

5.2. Chitosan sponges

Sponges are nothing else than foams with an openporosity. These solid structures are able to absorb highamount of fluids (more than 20 times their dry weight),due to their micro-porosity. They typically offer good cellinteraction, still being soft and flexible [80].

Chitosan sponges are mainly used as wound healingmaterials, as they can soak up the wound exudates, whilehelping the tissue regeneration. Chitosan sponges also findapplication in bone tissue engineering, as a filling material[81].

These sponges are mainly obtained by freeze drying(lyophilization), a simple efficient process consisting infreezing a solution of chitosan followed by sublimation ofthe solvent under reduced pressure (a SEM image of achitosan sponge prepared by this technique is presentedin Fig. 5). For example, chitosan [54], chitosan/tricalciumphosphate (TCP) [81,82], and chitosan/collagen sponges[83] were prepared by this technique and used as scaffoldsfor bone regeneration. Chitosan–ZnO composite spongesalso prepared by freeze-drying [80] showed good swelling,antibacterial and haemostatic activities, confirming theirpotential healing in wound dressing application.

Cross-linked chitosan sponges loaded with a model ofantibiotic drug – the norfloxacin – were prepared by sol-vent evaporation technique [84]. Fibrillar structure wasobtained and those sponges showed promising use forwound dressing application.

Recently, efforts were dedicated to the use of supercrit-ical carbon dioxide (scCO2) as a green medium to induceporosity to chitosan scaffolds [85].

The scCO2 method allows the preparation of porouschitosan scaffolds, suitable for cell cultures directly upondepressurization.

6. Chitosan 2D-scaffolds for wound dressing

In the particular case of wound dressing, specificrequirements 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, protectthe wound against microbial organisms and absorb woundexudates [80]. As mentioned above, chitosan sponges havebeen found to fit quite well these requirements. Neverthe-less, as far as skin tissues are concerned, more 2D-shapedscaffolds, such as films and porous membranes, are supple-mentary candidates, spreading the panel of structuresavailable to tackle the challenges addressed in skin repair.

6.1. Chitosan films

Chitosan films can be easily prepared by wet castingfrom chitosan salt solutions, followed by drying (typicallyusing oven or infrared (IR) drying [86]). For example, Hem-Con� bandage is an engineered chitosan acetate prepara-tion designed as a haemostatic dressing [80].

6.1.1. Improving properties of chitosan free-standing filmsTo 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 improvesthe cell adhesion and proliferation (especially for filmstreated with nitrogen plasma at 20 W during 20 min), (ii)ozone or UV irradiation promotes surface modification ofchitosan films leading to depolymerization of chitosan[88], (iii) by introducing silica particles or poly(ethyleneglycol) [89] into the chitosan films, their porosity can beartificially modified with macro and micro-pores (poressize ranging 0.5–100 lm).

As far as mechanical properties are concerned, blendingand chemical modifications of chitosan have proven effi-ciency. To improve the films ductility, chitosan can beblended (or copolymerized) with poly(ethylene glycol)(PEG), which results in a decrease of the modulus withan increase of the strain at break (for 50/50 chitosan/PEOblended film, the decrease of the modulus was 56%, whilethe increase of the strain at break was 125%, in comparisonto pure chitosan film) [90]. Amidation of the chitosan films(up to 50%) can occur with thermal treatment; it leads tosignificant strengthening of the films and to a decrease ofthe film solubility in aqueous media [91]. Chitosan filmsprepared in presence of dialdehyde starch, that plays therole of cross-linking agent, show improved mechanicalproperties 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 of27%) [92]. via Michael addition reaction, chitosan/polyeth-ylene glycol diacrylate (PEGDA) blended films were devel-oped [93], showing enhanced swelling (the maximumswelling was observed at a ratio of CS/PEGDA equal to40/60) 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 fromthe 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 to8.3 � 10�5 S cm�1, and reaching 1.2 � 10�3 S cm�1 for a

chitosan film containing 87.31 mg/m2 of phosphorus –NB: conductivity measurements were realized afterhydratation of the films) [94], (ii) chitosan blends withminocycline hydrochloride, to prepare films that acceleratethe 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 moistenvironment within the framework of wound healing[96], and (iv) non-adherent film of b-glucan and chitosan[97,98].

Heterogeneous chemical modification of the chitosanfilm can tune the surface properties of the film; for in-stance, when a stearoyl group was attached to the chitosanfilms, they became more hydrophobic (contact angle ofpure chitosan film being 89 ± 6�, while 101 ± 4� was foundfor N-stearoylchitosan film) and promoted proteinsadsorption. When chitosan films were reacted with succi-nic anhydride or phthalic anhydride, it resulted in morehydrophilic films (contact angles of 56 ± 2� and 51 ± 7�were found for N-succinylchitosan and N-phthalylchitosanfilms, respectively), which promoted lysozyme adsorption[99]. Interestingly, a thermo-sensitive film was preparedby 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 thesefilms allows synergistic effects of both materials leadingto high performance healing. Ag nanoparticles possessidentified antimicrobial activity, notably used in silver-based wound dressings [101]. Those Ag particles can beadded to chitosan film, to further avoid microorganismproliferation [102–105]. Polyphosphate procoagulant canalso be incorporated to the chitosan/Ag films [24]. BesideAg, zinc oxide (ZnO) nanoparticles are non-toxic and pres-ent antibacterial and good photocatalytic activities [106].Like the Ag nanoparticles, they can be incorporated inchitosan films. Complex films containing chitosan, Agnanoparticles and ZnO particles were prepared [107]. Theypossess pronounced antibacterial activity (especially thefilm containing 0.1 wt.% Ag and 10 wt.% ZnO), thus promis-ing application in wound dressing field.

6.1.2. Chitosan thin filmsThe 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 orderto tune the surface properties of various medical devices.Two methods to design chitosan thin films in order tomodify the surface of a performed substrate will be shortlydescribed: Langmuir–Blodgett (LB), and the layer-by-layer(LBL) deposition techniques. These two processes are ofimportance since both of them allow to control parameterssuch as film thickness, composition, morphology, and evenroughness [18].

To form a Langmuir [108] monolayer at the air–waterinterface, a solution in an organic volatile solvent of anamphiphilic water-insoluble (macro)molecule is spreadon the surface of water. When the organic solvent is evap-orated, the hydrophilic part of the amphiphile is anchored

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

Fig. 7. Schematic representation of LBL process.

F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792 787

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 transferringthis Langmuir monolayer onto a solid support. This canbe 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 canbe obtained.

Chitosan being poorly soluble in organic solvents,chemical modification is required for this process to beperformed. Chitosan modified with long alkyl chains at-tached to the primary hydroxyl and amino groups, wasthe first example of LB chitosan films formation [18,110],this derivatization making the resulting chitosan solublein chloroform. Derivatives of chitosan pentamers wereused to produce LB films [111,112], while other amphi-philic chitosans, namely, N,N-dialkyl-chitosans, were alsoreported [113]. All these films find application mainly indrug 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 LBfilms [115].

On the other hand, the layer-by-layer (LBL) depositiontechnique is based on the alternated adsorption of materi-als bearing complementary charged or functional groups[22,112], in aqueous medium. A schematic representationof 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 manyapplications in the field of sensors [118], filters, but alsoas biomaterial for tissue engineering [119].

The polycationic nature of chitosan makes this polymerwell-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, boneimplants and wound dressings [18].

Chitosan can be LBL co-deposited with several specieson 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 ina chitosan matrix that helps maintaining the biomoleculeactivity. For coronary stents, chitosan/heparin LBL filmsare particularly interesting for their anticoagulation prop-erties [18].

To improve mechanical properties of chitosan LBL films,which might be especially important when deposited insoft substrates, chitosan can be combined in the film withclays such as Na-montmorillonite [120], with carbon nano-tubes [121], or with cellulose nanowhiskers [122].

LBL films entirely made of polysaccharides, such aschitosan and hyaluronic acid [123], and of chitosan andalginate [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; thefirst example was reported as early as 1926 [124,125].Fibers were notably produced using dry [126] and wetspinning [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 fiberproperties, blends of chitosan with other polymers werealso 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 ofpolymer fibers from a few nanometers to microns in diam-eter, depending on the processing conditions. ESP uses ahigh voltage to create an electrically charged jet of polymersolution or melt which can lead to fibers formation. Fig. 8shows a schematic representation of this technique. Themats of fibers produced by this method possess highporosity, high specific surface area, and are able to mimicthe natural extracellular matrix, which makes these matsgreat candidates for biomaterial applications. Chitosan(nano)fibers find many applications for the preparation ofwound dressing and for tissue engineering [133].

However, as chitosan is a polyelectrolyte in acidicmedium, its electrospinning turns out to be tricky. Indeed,

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

during ESP, repulsive forces between charged specieswithin polymer backbone arise as a consequence of theapplication of an important electric field. This phenomenonrestricts the formation of continuous fibers and often leadsto the creation of particles [133,134].

A few examples of pure electrospun chitosan fibers arereported; chitosan (nano)fibers were successfully pro-duced from trifluoroacetic acid (TFA) and dichloromethane(DCM) mixtures [135,136]. It was suggested [135] that theamino 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 useof DCM allows improving the homogeneity of the producedfibers. 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 wasconsidered [139,140].

To facilitate the electrospinning of chitosan, someauthors report on the use of modified chitosan such ashexanoyl 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 blendfibers 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] arecommonly reported. A SEM image of chitosan/PEO nanofibersis presented in Fig. 9. Poly(vinyl alcohol) (PVA) [149–151]can also be used to form chitosan (nano)fibers. The usesof poly(ethylene terephtalate (PET) [152], poly(vinylpyrrolidone) (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 fibermats was found to promote the attachment of human cells,while preserving their morphology and viability [147]. Theaddition of UHMWPEO to chitosan ESP solution allowsforming 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 designingtissue engineering scaffolds [133]. The joint use of PVA andchitosan allows the preparation of fibers without beaded

10 µm

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

Table 1Main 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-ated (reversible)

Soft flexible non-toxic Not stable (uncontrolleddissolution may occur)

Tissue replacements/engineeringdrug/growth factor delivery

Low mechanical resistancePore size difficult to control

� Chemically cross-linked(irreversible)

Soft flexible stablecontrolled pore size

May be toxic

Crosslinking may affectchitosan 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 theconstruction of multilayers

Coatings for a variety of scaffoldswound dressings skin substitutes

� Thin (LBL) Material coatingMultilayerconstruction

Many steps

Porous Membranes (2D) Nanofibers High porosity Coatings for a variety of scaffoldswound dressings skin substitutesMimic skin

extracellular matrixESP of pure chitosan difficult

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defects; chitosan/PVA nanofibers are notably used for avariety 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 ESPprocess is actually a way to improve the properties of theresulting fibers such as the mechanical properties, the bio-compatibility, and the antibacterial behavior [133]. Elec-trospun chitosan-based nanofiber mats appear thus todayas an emerging biomaterial in wound dressing applicationssustained by the apparition of products such as Chitoflex�

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 highlyvaluable physico-chemical properties but also particularinteractions with proteins, cells and living organisms. Inaddition, this polycation (upon protonation in acidic condi-tions) shows improved solubility in aqueous media whichallows 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 easilymade available with good control on their composition,function and morphology. The main advantages and disad-vantages of the chitosan biomaterials presented in thisarticle are pointed out in Table 1, with respect to their bio-medical applications. Therefore, this polymer is veryattractive for biomedical uses, notably in tissue engineer-ing. The wide panel of chitosan properties and processedmaterials gives to this biosourced polymer a quite promis-ing future as biomaterial as demonstrated by emergingproducts on the market notably in the wound dressingfield.

Acknowledgements

F.C. is grateful to the Belgian ‘‘Fonds pour la Formation àla Recherche dans l’Industrie et dans l’Agriculture’’ (FRIA)for financial support. The present work has been supportedby 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.

References

[1] Liu X, Ma L, Mao Z, Gao C. Chitosan-based biomaterials for tissuerepair and regeneration. Chitosan for biomaterials II. In: JayakumarR, Prabaharan M, Muzzarelli RAA, editors. Advances in polymerscience. Heidelberg: Springer Berlin; 2011. p. 81–127.

[2] Rinaudo M. Chitin and chitosan: properties and applications. ProgPolym Sci 2006;31:603–32.

[3] Acosta N, Jimenez C, Borau V, Heras A. Extraction andcharacterization of chitin from crustaceans. Biomass Bioenergy1993;5:145–53.

[4] Madihally SV, Matthew HWT. Porous chitosan scaffolds for tissueengineering. Biomaterials 1999;20:1133–42.

[5] Jayakumar R, Menon D, Manzoor K, Nair SV, Tamura H. Biomedicalapplications of chitin and chitosan based nanomaterials. A shortreview. Carbohydr Polym 2010;82:227–32.

[6] Venkatesan J, Kim S-K. Chitosan composites for bone tissueengineering – an overview. Mar Drugs 2010;8:2252–66.

[7] Rane KD, Hoover DG. Production of chitosan by fungi. FoodBiotechnol 1993;7:11–33.

[8] Aranaz I, Harris R, Heras A. Chitosan amphiphilic derivatives.Chemistry and applications. Curr Org Chem 2010;14:308–30.

[9] Aranaz I, Mengibar M, Harris R, Panos I, Miralles B, Acosta N, et al.Functional characterization of chitin and chitosan. Curr Chem Biol2009;3:203–30.

[10] Kannan M, Nesakumari M, Rajarathinam K, Ranjit SAJA. Productionand characterization of mushroom chitosan under solid-statefermentation conditions. Adv Biol Res 2010;4:10–3.

[11] Wu T, Zivanovic S, Draughon FA, Sams CE. Chitin and chitosanvalue-added products from mushroom waste. J Agricult Food Chem2004;52:7905–10.

[12] Riva R, Ragelle H, des Rieux A, Duhem N, Jérôme C, Préat V. Chitosanand chitosan derivatives in drug delivery and tissue engineering.Chitosan for biomaterials II. In: Jayakumar R, Prabaharan M,Muzzarelli RAA, editors. Advances in polymer science. Heidelberg:Springer Berlin; 2011. p. 19–44.

790 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792

[13] Teng WL, Khor E, Tan TK, Lim LY, Tan SC. Concurrent production ofchitin from shrimp shells and fungi. Carbohydr Res2001;332:305–16.

[14] Leedy M, Martin H, Norowski P, Jennings J, Haggard W, BumgardnerJ. Use of chitosan as a bioactive implant coating for bone-implantapplications. Chitosan for biomaterials II. In: Jayakumar R,Prabaharan M, Muzzarelli RAA, editors. Advances in polymerscience. Heidelberg: Springer Berlin; 2011. p. 129–65.

[15] Nanba T, Izume M. Cosmetics containing chitosan oligosaccharides.1987; JP 62273905 A: 12p.

[16] Zhang Y, Zhang X-W, Li Y-M. Study on the preparation of chitosanoligomer through the chitosan degraded by hydrogen peroxide.Anhui Nongye Kexue 2011;39:13060–2.

[17] Balazs N, Sipos P. Limitations of pH-potentiometric titration for thedetermination of the degree of deacetylation of chitosan. CarbohydrRes 2006;342:124–30.

[18] Pavinatto FJ, Caseli L, Oliveira Jr ON. Chitosan in nanostructuredthin films. Biomacromolecules 2010;11:1897–908.

[19] Craveiro AA, Craveiro AC, Pontes DF, Brasil IM, Cesar LT, Cabral MdFet al. Use of chitosan in clarification of beverages. 2012; BR2010001795 A2: 11p.

[20] Takahashi T, Takayama K, Machida Y, Nagai T. Characteristics ofpolyion complexes of chitosan with sodium alginate and sodiumpolyacrylate. Int J Pharm 1990;61:35–41.

[21] Kim T-H, Jiang H-L, Jere D, Park I-K, Cho M-H, Nah J-W, et al.Chemical modification of chitosan as a gene carrier in vitro andin vivo. Prog Polym Sci 2007;32:726–53.

[22] Decher G. Fuzzy nanoassemblies: toward layered polymericmulticomposites. Science 1997;277:1232–7.

[23] Sudarshan NR, Hoover DG, Knorr D. Antibacterial action of chitosan.Food Biotechnol 1992;6:257–72.

[24] Ong S-Y, Wu J, Moochhala SM, Tan M-H, Lu J. Development of achitosan-based wound dressing with improved hemostatic andantimicrobial properties. Biomaterials 2008;29:4323–32.

[25] Lehr CM, Bouwstra JA, Schacht EH, Junginger HE. In vitro evaluationof mucoadhesive properties of chitosan and some other naturalpolymers. Int J Pharm 1992;78:43–8.

[26] Yang J, Tian F, Wang Z, Wang Q, Zeng Y-J, Chen S-Q. Effect ofchitosan molecular weight and deacetylation degree on hemostasis.J Biomed Mater Res, Part B 2007;84B:131–7.

[27] Bagheri-Khoulenjani S, Taghizadeh SM, Mirzadeh H. Aninvestigation on the short-term biodegradability of chitosan withvarious molecular weights and degrees of deacetylation. CarbohydrPolym 2009;78:773–8.

[28] Vaarum KM, Myhr MM, Hjerde RJN, Smidsroed O. In vitrodegradation rates of partially N-acetylated chitosans in humanserum. Carbohydr Res 1997;299:99–101.

[29] VandeVord PJ, Matthew HWT, DeSilva SP, Mayton L, Wu B, WooleyPH. Evaluation of the biocompatibility of a chitosan scaffold inmice. J Biomed Mater Res 2002;59:585–90.

[30] Sashiwa H, Aiba S. Chemically modified chitin and chitosan asbiomaterials. Prog Polym Sci 2004;29:887–908.

[31] He P, Davis SS, Illum L. In vitro evaluation of the mucoadhesiveproperties of chitosan microspheres. Int J Pharm 1998;166:75–88.

[32] Park P-J, Je J-Y, Jung W-K, Ahn C-B, Kim S-K. Anticoagulant activityof heterochitosans and their oligosaccharide sulfates. Eur Food ResTechnol 2004;219:529–33.

[33] Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies onits safety and hemostatic potential. J Biomed Mater Res 1997;34:21–8.

[34] Klokkevold PR, Fukayama H, Sung EC, Bertolami CN. The effect ofchitosan (poly-N-acetyl glucosamine) on lingual hemostasis inheparinized rabbits. J Oral Maxillofac Surg 1999;57:49–52.

[35] Smith J, Wood E, Dornish M. Effect of chitosan on Epithelial CellTight Junctions. Pharm Res 2004;21:43–9.

[36] Chung Y-C, Chen C-Y. Antibacterial characteristics and activity ofacid-soluble chitosan. Bioresour Technol 2008;99:2806–14.

[37] Okamoto Y, Kawakami K, Miyatake K, Morimoto M, Shigemasa Y,Minami S. Analgesic effects of chitin and chitosan. CarbohydrPolym 2002;49:249–52.

[38] Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosan – a versatilesemi-synthetic polymer in biomedical applications. Prog Polym Sci2011;36:981–1014.

[39] Kean T, Thanou M. Chitin and chitosan: sources, production andmedical applications. In: Williams PA, editor. Renewable resourcesfor functional polymers and biomaterials. Polysaccharides, proteinsand polyesters. 1st ed. London: Royal Society of, Chemistry; 2011.p. 292–318.

[40] Funkhouser JD, Aronson Jr NN. Chitinase family GH18: evolutionaryinsights from the genomic history of a diverse protein family. BMCEvol Biol. 2007; 7:96:16 pp.

[41] Pangburn SH, Trescony PV, Heller J. Lysozyme degradation ofpartially deacetylated chitin, its films and hydrogels. Biomaterials1982;3:105–8.

[42] Aiba S. Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans. Int J Biol Macromol 1992;14:225–8.

[43] Tomihata K, Ikada Y. In vitro and in vivo degradation of films inchitin and its deacetylated derivatives. Biomaterials1997;18:567–75.

[44] Zhang H, Neau SH. In vitro degradation of chitosan by a commercialenzyme preparation: effect of molecular weight and degree ofdeacetylation. Biomaterials 2001;22:1653–8.

[45] Thanou M, Verhoef JC, Junginger HE. Oral drug absorptionenhancement by chitosan and its derivatives. Adv Drug DeliveryRev 2001;52:117–26.

[46] Wedmore I, McManus JG, Pusateri AE, Holcomb JB. A special reporton the chitosan-based hemostatic dressing: experience in currentcombat operations. J Trauma 2006;60:655–8.

[47] Chatelet C, Damour O, Domard A. Influence of the degree ofacetylation on some biological properties of chitosan films.Biomaterials 2000;22:261–8.

[48] Dutta P, Rinki K, Dutta J. Chitosan: a promising biomaterial fortissue engineering scaffolds. Chitosan for biomaterials II. In:Jayakumar R, Prabaharan M, Muzzarelli RAA, editors. Advances inpolymer science. Heidelberg: Springer Berlin; 2011. p. 45–79.

[49] Olteanu CE, Enescu D. Chitosan involved tissue engineering andregenerative medicine. Roum Biotechnol Lett 2007;12:3217–33.

[50] Zhu X, Chian KS, Chan-Park MBE, Lee ST. Effect of argon-plasmatreatment on proliferation of human-skin-derived fibroblast onchitosan membrane in vitro. J Biomed Mater Res, Part A2005;73A:264–74.

[51] Jagur-Grodzinski J. Biomedical applications of polymers 2001–2002. e-Polym. 2003; Paper No. 12.

[52] Muzzarelli RAA, Muzzarelli C. Chitosan chemistry: relevance to thebiomedical sciences. Adv Polym Sci 2005;186:151–209.

[53] Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffolddesign variables and applications. Biomaterials 2003;24:4337–51.

[54] Seol Y-J, Lee J-Y, Park Y-J, Lee Y-M, Ku Y, Rhyu I-C, et al. Chitosansponges as tissue engineering scaffolds for bone formation.Biotechnol Lett 2004;26:1037–41.

[55] Berger J, Reist M, Mayer JM, Felt O, Gurny R. Structure andinteractions in chitosan hydrogels formed by complexation oraggregation for biomedical applications. Eur J Pharm Biopharm2004;57:35–52.

[56] Boucard N, Viton C, Domard A. New aspects of the formation ofphysical hydrogels of chitosan in a hydroalcoholic medium.Biomacromolecules 2005;6:3227–37.

[57] Shu XZ, Zhu KJ. Controlled drug release properties of ionicallycross-linked chitosan beads: the influence of anion structure. Int JPharm 2002;233:217–25.

[58] Shen EC, Wang C, Fu E, Chiang CY, Chen TT, Nieh S. Tetracyclinerelease from tripolyphosphate–chitosan cross-linked sponge: apreliminary in vitro study. J Periodontal Res 2008;43:642–8.

[59] Yin YJ, Yao KD, Cheng GX, Ma JB. Properties of polyelectrolytecomplex films of chitosan and gelatin. Polym Int 1999;48:429–32.

[60] Mao JS, Cui YL, Wang XH, Sun Y, Yin YJ, Zhao HM, et al. Apreliminary study on chitosan and gelatin polyelectrolyte complexcytocompatibility by cell cycle and apoptosis analysis. Biomaterials2004;25:3973–81.

[61] Jiang T, Zhang Z, Zhou Y, Liu Y, Wang Z, Tong H, et al. Surfacefunctionalization of titanium with chitosan/gelatin viaelectrophoretic deposition: characterization and cell behavior.Biomacromolecules 2010;11:1254–60.

[62] Djerassi C, Gray JD, Kinci FA. Naturally occurring oxygenheterocycles. IX. Isolation and characterization of genipin. J OrgChem 1960;25:2174–7 [Francesko A, Tzanov T. Chitin, chitosan andderivatives for wound healing and tissue engineering. Adv BiochemEng/Biotechnol 2011; 125: 1–27].

[63] Sezer AD, Cevher E, Hatipoglu F, Ogurtan Z, Bas AL, Akbuga J.Preparation of fucoidan–chitosan hydrogel and its application asburn healing accelerator on rabbits. Biol Pharm Bull2008;31:2326–33.

[64] Boddohi S, Moore N, Johnson PA, Kipper MJ. Polysaccharide-basedpolyelectrolyte complex nanoparticles from chitosan, heparin, andhyaluronan. Biomacromolecules 2009;10:1402–9.

[65] Il’ina AV, Varlamov VP. Chitosan-based polyelectrolyte complexes:a review. Appl Biochem Microbiol 2005;41:5–11.

F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792 791

[66] Denuziere A, Ferrier D, Damour O, Domard A. Chitosan–chondroitin sulfate and chitosan–hyaluronate polyelectrolytecomplexes: biological properties. Biomaterials 1998;19:1275–85.

[67] de Oliveira HCL, Fonseca JLC, Pereira MR. Chitosan-poly(acrylicacid) polyelectrolyte complex membranes: preparation,characterization and permeability studies. J Biomater Sci PolymEd 2008;19:143–60.

[68] Brack HP, Tirmizi SA, Risen Jr WM. A spectroscopic and viscometricstudy of the metal ion-induced gelation of the biopolymer chitosan.Polymer 1997;38:2351–62.

[69] Dambies L, Vincent T, Domard A, Guibal E. Preparation of chitosangel beads by ionotropic molybdate gelation. Biomacromolecules2001;2:1198–205.

[70] Ladet S, David L, Domard A. Multi-membrane hydrogels. Nature2008;452:76–9.

[71] Cascone MG, Maltinti S, Barbani N, Laus M. Effect of chitosan anddextran on the properties of poly(vinyl alcohol) hydrogels. J MaterSci: Mater Med 1999;10:431–5.

[72] Chenite A, Chaput C, Wang D, Combes C, Buschmann MD,Hoemann CD, et al. Novel injectable neutral solutions ofchitosan form biodegradable gels in situ. Biomaterials2000;21:2155–61.

[73] Bhattarai N, Ramay HR, Gunn J, Matsen FA, Zhang M. PEG-graftedchitosan as an injectable thermosensitive hydrogel for sustainedprotein release. J Controlled Release 2005;103:609–24.

[74] Peppas N, editor. Hydrogels in medicine and pharmacy, vol. 1.Fundamentals: CRC Press; 1986.

[75] Hennink WE, van NCF. Novel crosslinking methods to designhydrogels. Adv Drug Delivery Rev 2002;54:13–36.

[76] Leung H-W. Ecotoxicology of glutaraldehyde: review ofenvironmental fate and effects studies. Ecotoxicol Environ Saf2001;49:26–39.

[77] Sung HW, Huang RN, Huang LL, Tsai CC. In vitro evaluation ofcytotoxicity of a naturally occurring cross-linking reagent forbiological tissue fixation. J Biomater Sci Polym Ed 1999;10:63–78.

[78] Sung H-W, Liang IL, Chen C-N, Huang R-N, Liang H-F. Stability of abiological tissue fixed with a naturally occurring crosslinking agent(genipin). J Biomed Mater Res 2001;55:538–46.

[79] Park K, Shalaby WSE, Park H. Biodegradable hydrogels for drugdelivery. Technomic; 1993.

[80] Jayakumar R, Prabaharan M, Sudheesh KPT, Nair SV, Tamura H.Biomaterials based on chitin and chitosan in wound dressingapplications. Biotechnol Adv 2011;29:322–37.

[81] Costa-Pinto AR, Reis RL, Neves NM. Scaffolds based bone tissueengineering: the role of chitosan. Tissue Eng Part B2011;17:331–47.

[82] Lee Y-M, Park Y-J, Lee S-J, Ku Y, Han S-B, Choi S-M, et al. Tissueengineered bone formation using chitosan/tricalcium phosphatesponges. J Periodontol 2000;71:410–7.

[83] Arpornmaeklong P, Pripatnanont P, Suwatwirote N. Properties ofchitosan–collagen sponges and osteogenic differentiation of rat-bone-marrow stromal cells. Int J Oral Maxillofac Surg2008;37:357–66.

[84] Denkbas EB, Ozturk E, Ozdemir N, Kececi K, Agalar C. Norfloxacin-loaded chitosan sponges as wound dressing material. J BiomaterAppl 2004;18:291–303.

[85] Rinki K, Tripathi S, Dutta PK, Dutta J, Hunt AJ, Macquarrie DJ, et al.Direct chitosan scaffold formation via chitin whiskers by asupercritical carbon dioxide method: a green approach. J MaterChem 2009;19:8651–5.

[86] Khan TA, Peh KK, Ch’ng HS. Mechanical, bioadhesive strength andbiological evaluations of chitosan films for wound dressing. J PharmPharm Sci 2000;3:303–11.

[87] Silva SS, Luna SM, Gomes ME, Benesch J, Pashkuleva I, Mano JF, et al.Plasma surface modification of chitosan membranes:characterization and preliminary cell response studies. MacromolBiosci 2008;8:568–76.

[88] Matienzo LJ, Winnacker SK. Dry processes for surface modificationof a biopolymer: chitosan. Macromol Mater Eng 2002;287:871–80.

[89] Clasen C, Wilhelms T, Kulicke WM. Formation and characterizationof chitosan membranes. Biomacromolecules 2006;7:3210–22.

[90] Kolhe P, Kannan RM. Improvement in ductility of chitosan throughblending and copolymerization with PEG: FTIR investigation ofmolecular interactions. Biomacromolecules 2003;4:173–80.

[91] Zotkin MA, Vikhoreva GA, Kechek’yan AS. Thermal modification ofchitosan films in the form of salts with various acids. VysokomolSoedin Ser A Ser B 2004;46:359–63.

[92] Tang R, Du Y, Fan L. Dialdehyde starch-crosslinked chitosan filmsand their antimicrobial effects. J Polym Sci Part B: Polym Phys2003;41:993–7.

[93] Zhang X, Yang D, Nie J. Chitosan/polyethylene glycol diacrylatefilms as potential wound dressing material. Int J Biol Macromol2008;43:456–62.

[94] Wan Y, Creber KAM, Peppley B, Bui VT. Synthesis, characterizationand ionic conductive properties of phosphorylated chitosanmembranes. Macromol Chem Phys 2003;204:850–8.

[95] Aoyagi S, Onishi H, Machida Y. Novel chitosan wound dressingloaded with minocycline for the treatment of severe burn wounds.Int J Pharm 2007;330:138–45.

[96] Pei HN, Chen XG, Li Y, Zhou HY. Characterization and ornidazolerelease in vitro of a novel composite film prepared with chitosan/poly(vinyl alcohol)/alginate. J Biomed Mater Res Part A2008;85A:566–72.

[97] Kofuji K, Huang Y, Tsubaki K, Kokido F, Nishikawa K, Isobe T, et al.Preparation and evaluation of a novel wound dressing sheetcomprised of b-glucan–chitosan complex. React Funct Polym2010;70:784–9.

[98] Xu H, Ma L, Shi H, Gao C, Han C. Chitosan-hyaluronic acid hybridfilm as a novel wound dressing: in vitro and in vivo studies. PolymAdv Technol 2007;18:869–75.

[99] Tangpasuthadol V, Pongchaisirikul N, Hoven VP. Surfacemodification of chitosan films. Effects of hydrophobicity onprotein adsorption. Carbohydr Res 2003;338:937–42.

[100] Radhakumary C, Antonty M, Sreenivasan K. Drug loadedthermoresponsive and cytocompatible chitosan based hydrogel asa potential wound dressing. Carbohydr Polym 2011;83:705–13.

[101] Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation ofantimicrobials. Biotechnol Adv 2009;27:76–83.

[102] Lu S, Gao W, Gu HY. Construction, application and biosafety ofsilver nanocrystalline chitosan wound dressing. Burns2008;34:623–8.

[103] Mi F-L, Wu Y-B, Shyu S-S, Chao A-C, Lai J-Y, Su C-C. Asymmetricchitosan membranes prepared by dry/wet phase separation: a newtype of wound dressing for controlled antibacterial release. JMembr Sci 2003;212:237–54.

[104] Thomas V, Yallapu MM, Sreedhar B, Bajpai SK. Fabrication,characterization of chitosan/nanosilver film and its potentialantibacterial application. J Biomater Sci Polym Ed 2009;20:2129–44.

[105] Vimala K, Mohan YM, Sivudu KS, Varaprasad K, Ravindra S, ReddyNN, et al. Fabrication of porous chitosan films impregnated withsilver nanoparticles: a facile approach for superior antibacterialapplication. Colloids Surf B 2010;76:248–58.

[106] Cohen ML. The theory of real materials. Ann Rev Mater Sci2000;30:1–26.

[107] Li L-H, Deng J-C, Deng H-R, Liu Z-L, Li X-L. Preparation,characterization and antimicrobial activities of chitosan/Ag/ZnOblend films. Chem Eng J 2010;160:378–82.

[108] Langmuir I. Constitution and fundamental properties of solids andliquids. II. Liquids. J Am Chem Soc 1917;39:1848–906.

[109] Blodgett KB. Films built by depositing successive unimolecularlayers on a solid surface. J Am Chem Soc 1935;57:1007–22.

[110] Nishimura S, Miura Y, Ren L, Sato M, Yamagishi A, Nishi N, et al. Anefficient method for the syntheses of novel amphiphilicpolysaccharides by regio- and thermoselective modifications ofchitosan. Chem Lett 1993:1623–6.

[111] Fendler JH. Self-assembled nanostructured materials. Chem Mater1996;8:1616–24.

[112] Hammond PT. Form and function in multilayer assembly: newapplications at the nanoscale. Adv Mater 2004;16:1271–93.

[113] Li M, Su S, Xin M, Liao Y. Relationship between N,N-dialkyl chitosanmonolayer and corresponding vesicle. J Colloid Interface Sci2007;311:285–8.

[114] Wu Y, Hisada K, Maeda S, Sasaki T, Sakurai K. Fabrication andstructural characterization of the Langmuir–Blodgett films from anew chitosan derivative containing cinnamate chromophores.Carbohydr Polym 2007;68:766–72.

[115] Tong Y, Wang S, Xu J, Chua B, He C. Synthesis of O,O0-dipalmitoylchitosan and its amphiphilic properties and capability ofcholesterol absorption. Carbohydr Polym 2005;60:229–33.

[116] Kim SN, Rusling JF, Papadimitrakopoulos F. Carbon nanotubes forelectronic and electrochemical detection of biomolecules. AdvMater 2007;19:3214–28.

[117] He J-A, Samuelson L, Li L, Kumar J, Tripathy SK. Orientedbacteriorhodopsin/polycation multilayers by electrostatic layer-by-layer assembly. Langmuir 1998;14:1674–9.

792 F. Croisier, C. Jérôme / European Polymer Journal 49 (2013) 780–792

[118] Lee S-H, Kumar J, Tripathy SK. Thin film optical sensors employingpolyelectrolyte assembly. Langmuir 2000;16:10482–9.

[119] Wittmer CR, Phelps JA, Lepus CM, Saltzman WM, Harding MJ, VanTPR. Multilayer nanofilms as substrates for hepatocellularapplications. Biomaterials 2008;29:4082–90.

[120] Darder M, Colilla M, Ruiz-Hitzky E. Biopolymer-claynanocomposites based on chitosan intercalated inmontmorillonite. Chem Mater 2003;15:3774–80.

[121] Wang S-F, Shen L, Zhang W-D, Tong Y-J. Preparation andmechanical properties of chitosan/carbon nanotubes composites.Biomacromolecules 2005;6:3067–72.

[122] Liu Y, Hu N. Enhanced DNA oxidative damage by acrylonitrile andprotecting DNA from damage with an outer catalase layer: anelectrochemical study. Electroanalysis 2008;20:2671–6.

[123] Salomaki M, Kankare J. Influence of synthetic polyelectrolytes onthe growth and properties of Hyaluronan–Chitosan multilayers.Biomacromolecules 2009;10:294–301.

[124] Pillai CKS, Paul W, Sharma CP. Chitin and chitosan polymers:chemistry, solubility and fiber formation. Prog Polym Sci2009;34:641–78.

[125] Kunike G. Chitin and chitosan. J Soc Dyers Colorists 1926;42:24.[126] Rigby GW. Process for the preparation of films and filaments and

products thereof. 1936; US 2040880: p. 4.[127] East GC, Qin Y. Wet spinning of chitosan and the acetylation of

chitosan fibers. J Appl Polym Sci 1993;50:1773–9.[128] Agboh OC, Qin Y. Chitin and chitosan fibers. Polym Adv Technol

1997;8:355–65.[129] Tamura H, Tsuruta Y, Tokura S. Preparation of chitosan-coated

alginate filament. Mater Sci Eng C 2002;C20:143–7.[130] Hirano S, Zhang M, Nakagawa M, Miyata T. Wet spun chitosan–

collagen fibers, their chemical N-modifications, and bloodcompatibility. Biomaterials 2000;21:997–1003.

[131] Ohkawa K, Ando M, Shirakabe Y, Takahashi Y, Yamada M, Shirai H,et al. Preparing chitosan-poly(acrylic acid) composite fibers by self-assembly at an aqueous solution interface. Text Res J2002;72:120–4.

[132] Formhals A. Artificial threads and method of producing same.1940; US 2187306. p. 4.

[133] Jayakumar R, Prabaharan M, Nair SV, Tamura H. Novel chitin andchitosan nanofibers in biomedical applications. Biotechnol Adv2010;28:142–50.

[134] Min B-M, Lee SW, Lim JN, You Y, Lee TS, Kang PH, et al. Chitin andchitosan nanofibers: electrospinning of chitin and deacetylation ofchitin nanofibers. Polymer 2004;45:7137–42.

[135] Ohkawa K, Cha D, Kim H, Nishida A, Yamamoto H. Electrospinningof chitosan. Macromol Rapid Commun 2004;25:1600–5.

[136] Sangsanoh P, Supaphol P. Stability improvement of electrospunchitosan nanofibrous membranes in neutral or weak basic aqueoussolutions. Biomacromolecules 2006;7:2710–4.

[137] Hasegawa M, Isogai A, Onabe F, Usuda M, Atalla RH.Characterization of cellulose-Chitosan blend films. J Appl PolymSci 1992;45:1873–9.

[138] Schiffman JD, Schauer CL. Cross-linking chitosan nanofibers.Biomacromolecules 2007;8:594–601.

[139] Geng X, Kwon O-H, Jang J. Electrospinning of chitosan dissolved inconcentrated acetic acid solution. Biomaterials 2005;26:5427–32.

[140] Vrieze S, Westbroek P, Camp T, Langenhove L. Electrospinning ofchitosan nanofibrous structures: feasibility study. J Mater Sci2007;42:8029–34.

[141] Neamnark A, Rujiravanit R, Supaphol P. Electrospinning ofhexanoyl chitosan. Carbohydr Polym 2006;66:298–305.

[142] Du J, Hsieh Y-L. PEGylation of chitosan for improved solubility andfiber formation via electrospinning. Cellulose 2007;14:543–52.

[143] Spasova M, Manolova N, Paneva D, Rashkov I. Preparation ofchitosan-containing nanofibers by electrospinning of chitosan/poly(ethylene oxide) blend solutions. e-Polym; 2004 [No pp.given].

[144] Duan B, Dong C, Yuan X, Yao K. Electrospinning of chitosansolutions in acetic acid with poly(ethylene oxide). J Biomater SciPolym Ed 2004;15:797–811.

[145] Matsumoto H, Yako H, Minagawa M, Tanioka A. Characterizationof chitosan nanofiber fabric by electrospray deposition:electrokinetic and adsorption behavior. J Colloid Interface Sci2007;310:678–81.

[146] Ojha SS, Stevens DR, Hoffman TJ, Stano K, Klossner R, Scott MC,et al. Fabrication and characterization of electrospun chitosannanofibers formed via templating with polyethylene oxide.Biomacromolecules 2008;9:2523–9.

[147] Bhattarai N, Edmondson D, Veiseh O, Matsen FA, Zhang M.Electrospun chitosan-based nanofibers and their cellularcompatibility. Biomaterials 2005;26:6176–84.

[148] Zhang YZ, Su B, Ramakrishna S, Lim CT. Chitosan nanofibers froman easily electrospinnable UHMWPEO-doped chitosan solutionsystem. Biomacromolecules 2008;9:136–41.

[149] Huang X-J, Ge D, Xu Z-K. Preparation and characterization of stablechitosan nanofibrous membrane for lipase immobilization. EurPolym J 2007;43:3710–8.

[150] Duan B, Yuan X, Zhu Y, Zhang Y, Li X, Zhang Y, et al. A nanofibrouscomposite membrane of PLGA-chitosan/PVA prepared byelectrospinning. Eur Polym J 2006;42:2013–22.

[151] Li L, Hsieh Y-L. Chitosan bicomponent nanofibers and nanoporousfibers. Carbohydr Res 2006;341:374–81.

[152] Jung K-H, Huh M-W, Meng W, Yuan J, Hyun SH, Bae J-S, et al.Preparation and antibacterial activity of PET/chitosan nanofibrousmats using an electrospinning technique. J Appl Polym Sci2007;105:2816–23.

[153] Ignatova M, Manolova N, Rashkov I. Novel antibacterial fibers ofquaternized chitosan and poly(vinyl pyrrolidone) prepared byelectrospinning. Eur Polym J 2007;43:1112–22.

[154] Peesan M, Rujiravanit R, Supaphol P. Electrospinning of hexanoylchitosan/polylactide blends. J Biomater Sci Polym Ed2006;17:547–65.

Florence Croisier received her master’s degreein chemical sciences from the University ofLiège (ULg, Belgium) in 2007. She is currentlyfinishing her Ph.D. under the supervision ofProfessor Christine Jérôme in the Center forEducation and Research on Macromolecules(ULg, Belgium). Her research focuses on thepreparation of chitosan-based nanofiberswith multilayered structure, using a combi-nation of electrospinning and layer-by-layerdeposition 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-dosctoralresearcher on developing the electrograftingprocess of acrylates. In 2000, she joined theUniversity of Ulm in Germany as a recipient ofthe Humboldt scholarship and studied thesynthesis of functional magnetic nanohybrids.She returned to the University of Liege in2001 as Research Associate of the NationalFoundation of the Scientific Research in the

group of Professor R. Jérôme, where she became Professor in 2006 anddirector of the Center for Education and Research on Macromolecules in2007. Today full Professor, her research interests include biosourced

polymers, functional macromolecular systems and advanced biomateri-als.