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

Keywords:Chitosan scaffoldsBiomaterialsTissue engineeringWound 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 fromthe presence of primary amines along the chitosan backbone. As a consequence, this poly-saccharide is a relevant candidate in the eld 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 lms and bers is dis-cussed, with a special focus on wound healing application.

2013 Elsevier Ltd.

Contents

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

5.1. Chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7835.1.1. 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7856. Chitosan 2D-scaffolds for wound dressing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

6.1. Chitosan films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7866.1.1. Improving properties of chitosan free-standing films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7866.1.2. Chitosan thin films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

6.2. Chitosan porous nanofiber membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7887. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

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

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: [email protected] (F. Croisier), [email protected] (C. Jrme).URL: http://www.cerm.ulg.ac.be (C. Jrme).

1 Tel.: +32 4 366 34 69; fax: +32 4 366 34 97.

European Polymer Journal 49 (2013) 780792

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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 reduce

the 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

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[79] . 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 of molecular 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 of chitosanusing specic enzyme [15] or reagent as hydrogenperoxide [16] .

At the time of its production, it is difcult to predictchitosan characteristics. Therefore, the current approachconsists in analyzing the resulting product compositionand properties rather than in targeting predened 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 difcultto 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. Structureproperties 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

O

OO

HO

NH 2

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.

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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 eld of cosmetics [2] .

Due to these amino groups, chitosan also efcientlycomplexes various species, such as metal ions, and there-fore is often used for the treatment of waste waters, puri-fying them by recovering heavymetals [2] . The complexingability of chitosan is further exploited for beverages (wine, juices . . .) clarication [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 lms, 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-specic reactions can beperformed on the alcohol groups, as etherication andesterication reactions [2] but remarkably, the aminogroup of D-glucosamine residues may be specically 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 eld of applications.

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] . Itcan 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 of chitosan 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 conrm this explanation[3234] .

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 of this 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 rst 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 of fundamental 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. Chitosanantimicrobialproperty 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 of the D-glucosamine residues can protonate in the presenceof proton ions that are released in the inammatory 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 identied, three of them possessing enzy-matic activity on chitosan [40] . The biodegradation of chitosan leads to the formation of non-toxic oligosaccha-rides of variable length. These oligosaccharides can 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 of N -acetyl D-glucosamine residues and the molecular massof chitosan [4244] .

To explain the relationship between chitosan biodegra-dation and DD, it is important to remember that chitosan isa semi-crystallinepolymer; 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 articial 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 modications 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 nally, 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 nely 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 waterwhile 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 [5254] .

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 modied 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. 4 a).

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 ne tuning of the swelling of the ob-tained hydrogel.


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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 broin) [5961] , anionic polysaccha-rides (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 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 of the 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

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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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 difcult 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. 4 b.

5.1.3. Chemically cross-linked chitosan hydrogelsThe formation of those hydrogels occurs via covalent

bonding between polymer chains ( Fig. 4 c). The obtainedhydrogels are much more stable than the previous physi-cally associated hydrogels since the gelation is irreversible.However, this approach requires the chemical modicationof 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 berstly modied 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(C

11H

14O

5, isolated from Genipa Americana in 1960 and

then 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 of chitosan that involves chemical modications of chitosanprimary amine might induce modications 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 uids (more than 20 times their dry weight),due to their micro-porosity. They typically offer good cellinteraction, still being soft and exible [80] .

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

These sponges are mainly obtained by freeze drying(lyophilization), a simple efcient process consisting infreezing a solution of chitosan followed by sublimation of the 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. ChitosanZnO composite spongesalso prepared by freeze-drying [80] showed good swelling,antibacterial and haemostatic activities, conrming theirpotential healing in wound dressing application.

Cross-linked chitosan sponges loaded with a model of antibiotic drug the noroxacin 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 (scCO 2) as a green medium to induceporosity to chitosan scaffolds [85] .

The scCO 2 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, specicrequirements have to be met to encounter efciency. The

100 m

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


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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 t quite well these requirements. Neverthe-less, as far as skin tissues are concerned, more 2D-shapedscaffolds, such as lms and porous membranes, are supple-mentary candidates, spreading the panel of structuresavailable to tackle the challenges addressed in skin repair.

6.1. Chitosan lms

Chitosan lms 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 lmsTo improve the properties of those lms, some physico-

chemical processes have been tested. For examples, (i)nitrogen or argon plasma [87] treatment of chitosan mem-branes increases the lm surface roughness and improvesthe cell adhesion and proliferation (especially for lmstreated 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(ethyleneglycol) [89] into the chitosan lms, their porosity can bearticially modied with macro and micro-pores (poressize ranging 0.5100 l m).

As far as mechanical properties are concerned, blendingand chemical modications of chitosan have proven ef-ciency. To improve the lms 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 lm, the decrease of the modulus was 56%, whilethe increase of the strain at break was 125%, in comparisonto pure chitosan lm) [90] . Amidation of the chitosan lms(up to 50%) can occur with thermal treatment; it leads tosignicant strengthening of the lms and to a decrease of the lm solubility in aqueous media [91] . Chitosan lmsprepared 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 of 27%) [92] . via Michael addition reaction, chitosan/polyeth-ylene glycol diacrylate (PEGDA) blended lms 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 lms (prepared fromthe reaction of orthophosphoric acid and urea in DMF),which present higher ionic conductivity than normal chito-san lms (conductivity of pure chitosan lm being equal to8.3 10 5 S cm 1 , and reaching 1.2 10 3 S cm 1 for a

chitosan lm containing 87.31 mg/m 2 of phosphorus NB: conductivity measurements were realized afterhydratation of the lms) [94] , (ii) chitosan blends withminocycline hydrochloride, to prepare lms that acceleratethehealing 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 moistenvironment within the framework of wound healing[96] , and (iv) non-adherent lm of b-glucan and chitosan[97,98] .

Heterogeneous chemical modication of the chitosanlm can tune the surface properties of the lm; for in-stance, when a stearoyl group was attached to the chitosanlms, they became more hydrophobic (contact angle of pure chitosan lm being 89 6 , while 101 4 was foundfor N-stearoylchitosan lm) and promoted proteinsadsorption. When chitosan lms were reacted with succi-nic anhydride or phthalic anhydride, it resulted in morehydrophilic lms (contact angles of 56 2 and 51 7were found for N-succinylchitosan and N-phthalylchitosanlms, respectively), which promoted lysozyme adsorption[99] . Interestingly, a thermo-sensitive lm was preparedby combining thiolated chitosan, poly( N -isopropyl acryl-amide) and ciprooxacin, so that by decreasing the tem-perature, the lm can easily be removed from wound[100] .

Finally, incorporation of inorganic particles in theselms allows synergistic effects of both materials leadingto high performance healing. Ag nanoparticles possessidentied antimicrobial activity, notably used in silver-based wound dressings [101] . Those Ag particles can beadded to chitosan lm, to further avoid microorganismproliferation [102105] . Polyphosphate procoagulant canalso be incorporated to the chitosan/Ag lms [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 lms. Complex lms containing chitosan, Agnanoparticles and ZnO particles were prepared [107] . Theypossess pronounced antibacterial activity (especially thelm containing 0.1 wt.% Ag and 10 wt.% ZnO), thus promis-ing application in wound dressing eld.

6.1.2. Chitosan thin lmsThe properties of the wound dressing surface in contact

to the diseased body are particularly of importance to pro-vide efcient healing. Multilayer coatings and nanostruc-tured thin lms have thus also been investigated in orderto tune the surface properties of various medical devices.Two methods to design chitosan thin lms in order tomodify the surface of a performed substrate will be shortlydescribed: LangmuirBlodgett (LB), and the layer-by-layer(LBL) deposition techniques. These two processes are of importance since both of them allow to control parameterssuch as lm thickness, composition, morphology, and evenroughness [18] .

To form a Langmuir [108] monolayer at the airwaterinterface, 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


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to the aqueous subphase, while the hydrophobic part is di-rected toward air [18] (as depicted in Fig. 6 a). The Lang-muirBlodgett (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. 6 b). By repeating the process, multilayered lms canbe obtained.

Chitosan being poorly soluble in organic solvents,chemical modication is required for this process to beperformed. Chitosan modied with long alkyl chains at-tached to the primary hydroxyl and amino groups, wasthe rst example of LB chitosan lms formation [18,110] ,this derivatization making the resulting chitosan solublein chloroform. Derivatives of chitosan pentamers wereused to produce LB lms [111,112] , while other amphi-philic chitosans, namely, N,N-dialkyl-chitosans, were alsoreported [113] . All these lms nd application mainly indrug release and drug delivery systems, since the amphi-philic 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-di-palmitoyl chitosan are an example of two-component LBlms [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 lms nd manyapplications in the eld of sensors [118] , lters, 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 lms 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-

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

Fig. 7. Schematic representation of LBL process.


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cyanines, and porphyrins can be trapped by this process ina chitosan matrix that helps maintaining the biomoleculeactivity. For coronary stents, chitosan/heparin LBL lmsare particularly interesting for their anticoagulation prop-erties [18] .

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

LBL lms 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 nanober membranes

There are several ways to produce chitosan bers; therst 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 berproperties, 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 of polymer bers 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 polymer

solution or melt which can lead to bers formation. Fig. 8shows a schematic representation of this technique. Themats of bers produced by this method possess highporosity, high specic surface area, and are able to mimicthe natural extracellular matrix, which makes these matsgreat 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 acidicmedium, its electrospinning turns out to be tricky. Indeed,

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

A few examples of pure electrospun chitosan bers arereported; chitosan (nano)bers were successfully pro-duced from triuoroacetic 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 producedbers. Another group used TFA in conjugation with glutar-aldehyde, to produce cross-linked chitosan bers [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 modied chitosan such ashexanoyl chitosan [141] and PEGylated chitosan [142] .Nevertheless, the easiest way to produce chitosan (nano)-bers is the electrospinning of chitosan as a blend with an-other polymer. Many examples of such chitosan blendbers produced by ESP are reported in the literature.

The electrospinning of chitosan in presence of poly(eth-ylene oxide) (PEO) [143147] and ultrahigh-molecular-weight poly(ethylene oxide) (UHMWPEO) [148] arecommonly reported. A SEMimage of chitosan/PEO nanobersis presented in Fig. 9 . Poly(vinyl alcohol) (PVA) [149151]can also be used to form chitosan (nano)bers. The usesof poly(ethylene terephtalate (PET) [152] , poly(vinylpyrrolidone) (PVP) [153] and poly(lactic acid) (PLA) [154]were also reported.

Chitosan/PEO nanobers prepared by ESP exhibit cellu-lar biocompatibility [133] [147] . The structure of the bermats was found to promote the attachment of human cells,while preserving their morphology and viability [147] . Theaddition of UHMWPEO to chitosan ESP solution allowsforming bers 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 bers[153] . These blends thus offer great prospects for designingtissue engineering scaffolds [133] . The joint use of PVA andchitosan allows the preparation of bers without beaded

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

10 m

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


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defects; chitosan/PVA nanobers are notably used for avariety of biomedical applications, as they also proved bio-compatible [149151] . Chitosan/PET nanobers present,besides biocompatibility, enhanced antibacterial proper-ties, in comparison to pure PET bers or chitin/PET bers[148] .

The addition of another polymer to facilitate the ESPprocess is actually a way to improve the properties of theresulting bers such as the mechanical properties, the bio-compatibility, and the antibacterial behavior [133] . Elec-trospun chitosan-based nanober mats appear thus todayas an emerging biomaterial in wound dressing applicationssustained by the apparition of products such as Chitoexon 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 lms,so as stronger sponges and nanober 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 dressingeld.

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 supportedby the Science Policy Ofce of the Belgian Federal Govern-ment (IAP 7/05). CERM thanks the Walloon Region for sup-porting researches on chitosan in the frame of CHITOPOL,TARGETUM and GOCELL projects.

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Table 1

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

Biomaterial type Specications Main advantage(s) Main disadvantage(s) Main biomedical application(s)

Hydrogels (3D) Physically associ-ated (reversible)

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

Tissue replacements/engineeringdrug/growth factor delivery

Low mechanical resistancePore size difcult to control

Chemically cross-

linked(irreversible)

Soft exible stable

controlled pore size

May be toxic

Crosslinking may affectchitosan intrinsic properties

Sponges (3D) High porosity May shrivel Tissue engineering (lling 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) Nanobers High porosity Coatings for a variety of scaffoldswound dressings skin substitutesMimic skin

extracellular matrixESP of pure chitosan difcult


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Florence Croisier received her masters degreein chemical sciences from the University of Lige (ULg, Belgium) in 2007. She is currentlynishing her Ph.D. under the supervision of Professor Christine Jrme in the Center forEducation and Research on Macromolecules(ULg, Belgium). Her research focuses on thepreparation of chitosan-based nanoberswith multilayered structure, using a combi-nation of electrospinning and layer-by-layerdeposition techniques.

Christine Jrme 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 of the 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 Scientic Research in the

group of Professor R. Jrme, where she became Professor in 2006 anddirector of the Center for Education and Research on Macromolecules in2007. Today full Professor, her research interests include biosourcedpolymers, functional macromolecular systems and advanced biomateri-als.


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