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European Polymer Journal 49 (2013) 780–792

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European Polymer Journal
journal homepage: www.elsevier.com/locate/europolj

Feature Article

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

a r t i c l e

i n f o

Article history:
Received 10 October 2012
Received in revised form 12 December 2012
Accepted 15 December 2012
Available online 23 January 2013
Keywords:
Chitosan scaffolds
Biomaterials
Tissue engineering
Wound healing

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

Contents
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7.

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

⇑ Corresponding author. Tel.: +32 4 366 34 91; fax: +32 4 366 34 97.

1

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

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

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2). The deacetylation degree (DD) of chitosan. giving indication of the number of amino groups along the chains. The mushroom-extracted chitosan typically presents a narrower molecular mass distribution than the chitosan produced from seafood [12]. the current approach consists in analyzing the resulting product composition and properties rather than in targeting predefined characteristics by controlling the process of production. As such. Chitosan: structure and extraction Chitosan is a linear. and presents broad polydispersity.13]. if any . it can be easily derived from the partial deacetylation of a natural polymer: the chitin (Fig. but also UV-spectroscopy. biosourced components. Chitosan is also poorly soluble.6]. Chemical structure of chitosan. the physical properties of extracted chitosan being notably related to the growth substrate composition.4] (which corresponds to a deacetylation degree of 60). practical applications of chitin are extremely limited due to its poor solubility. from a functional point of view. Therefore. of limited supplies and with product variability which can lead to inconsistent physicochemical characteristics [10]. O HN OH 781 under severe alkaline conditions or by enzymatic hydrolysis in the presence of particular enzymes. the amino groups of the D-glucosamine residues might be protonated providing solubility in diluted acidic aqueous solutions (pH < 6) [14]. it is difficult to predict chitosan characteristics. and may also differ in terms of molecular mass. this is why producers mainly refer to samples viscosity rather than molecular mass. Consequently. Abundant naturally occurring polymers – as starch. 1. the two main sources of chitosan are crustaceans and fungal mycelia. while its molecular mass is typically deduced from viscosimetry or determined by size exclusion chromatography [2]. alginate. To be named ‘‘chitosan’’. 2. Moreover. the inherent characteristics of chitosan make it exploitable directly for itself. In this respect. cellulose and chitin [1] – represent attractive candidates as they could reduce the actual dependence on fossil fuels. depending on the source and preparation. Structure–properties relationship HO O O O HO OH HN O Fig. Chemical structure of chitin. Croisier. chitin is the second most abundant biopolymer [2] and is commonly found in invertebrates – as crustacean shells or insect cuticles – but also in some mushrooms envelopes. the animal source shows however some drawbacks as seasonal. green algae cell walls. Chitosan oligomers can be prepared by degradation of chitosan using specific enzyme [15] or reagent as hydrogen peroxide [16]. semi-crystalline polysaccharide composed of (1 ? 4)-2-acetamido-2-deoxy-b-D-glucan (N-acetyl D-glucosamine) and (1 ? 4)-2-amino-2-deoxyb-D-glucan (D-glucosamine) units [2]. Chitosan DD greatly varies between 60 and 100% while its molecular weight typically ranges from 300 to 1000 kDA [1]. O The presence of amino groups in the chitosan structure (Fig. Introduction Over the last years. At industrial scale. chitosan is a quite unique bio-based polymer: its intrinsic properties are so singular and valuable that chitosan possesses no actual petrochemical equivalent. The characterization of chitosan is thus quite of importance but it is not easygoing nor straightforward. 3. chitosan may be contaminated by organic and inorganic impurities. After cellulose. Jérôme / European Polymer Journal 49 (2013) 780–792 1. The deacetylation of chitin is conducted by chemical hydrolysis O HN OH HO O O O O HO OH NH2 Fig. colloidal titration and enzymatic degradation are reported in the literature for the determination of chitosan deacetylation degree [17]. except in acidic medium (as it will be discussed in the following paragraph) which makes analyzes difficult to perform. Indeed. DD and distribution of deacetylated groups [11. the deacetylated chitin should contain at least 60% of D-glucosamine residues [3. and consequently have a positive environmental impact. among of chitin deacetylase [5. The structure of this polymer is depicted in Fig. collagen. is calculated as the ratio of D-glucosamine to the sum of D-glucosamine and N-acetyl D-glucosamine. Being a natural-based compound. many attempts have been made to replace petrochemical products by renewable. Different methods including pH-potentiometric titration. the vegetable source is generally preferred to the crustacean one from an allergenic point of view – the produced chitosan being safer for biomedical and healthcare applications [12]. IR-spectroscopy. The most challenging part of this approach is to obtain bio-based materials with properties equivalent to those of fully synthetic products. In contrast. The mushroom source offers the advantage of a controlled production environment all year round that insures a better reproducibility of the resulting chitosan [11]. 1. 2. chitosan is not extensively present in the environment – however. gelatin. At the time of its production.F. 1H NMR spectroscopy. and yeasts [7–9]. and gives to this polymer many peculiar properties. 1) differentiates chitosan from chitin. C.

as polymer crystallinity is inversely related to the biodegradation kinetic. The amino and alcohol functions along chitosan chains enable this polysaccharide to form stable covalent bondings with other species. which can lead to reorganization and an opening of the tight junction proteins. the permeation ability also increases [35]. Interestingly. These oligosaccharides can be incorporated in metabolic pathways or be further excreted [41]. to explain chitosan biodegradability. The second mechanism involves the binding of chitosan with the cell DNA (still via protonated amino groups). two main mechanisms have been reported in the literature to explain chitosan antibacterial and antifungal activities. the aqueous solubility of chitosan is pHdependent allowing processability under mild conditions [4]. mucoadhesive [25]. As a polyelectrolyte. The case of chitosan antimicrobial activity is slightly more complex. Indeed. which results in an increase of the biodegradation rate. the mucoadhesion of chitosan for example. if chitosan DD increases. Croisier. To explain the relationship between chitosan biodegradation and DD. The elucidation of their mechanism will lead to a better understanding of chitosan medical and pharmaceutical interest. The biodegradation of chitosan leads to the formation of non-toxic oligosaccharides of variable length. chitosan amino groups are positively charged and can thus interact with the mucin. and decreases for intermediate DD. Indeed. which would lead to the inhibition of the microbial RNA synthesis. if chitosan DD increases. 4. It can be biodegraded into non-toxic residues [27. Besides.36]. All these singular features make chitosan an outstanding candidate for biomedical applications. respectively). which tends to confirm this explanation [32–34]. positively charged chitosan can interact with negatively charged groups at the surface of cells. and mainly lysozyme [9. along with antifungal [9]. The degradation rate of chitosan is mainly related to its degree of deacetylation. analgesic [9] and haemostatic properties [26]. Indeed. Due to these amino groups. This particular case will be detailed later on. . it can be reasonably assumed that smaller chitosan chains will be more rapidly degraded into oligosaccharides than chitosan with higher molecular mass. This would prevent essential materials to enter the cells or/and lead to the leaking of fundamental solutes out of the cell. using layer-bylayer deposition technique [22]. but also to the distribution of N-acetyl D-glucosamine residues and the molecular mass of chitosan [42–44]. The haemostatic activity of chitosan can also be related to the presence of positive charges on chitosan backbone. and therefore is often used for the treatment of waste waters. which leads to improved mucoadhesive properties [31]. In the first proposed mechanism. which consequently contains breakable glycosidic bonds. crystallinity is indeed maximum for a DD equal to 0 or 100% (chitin or fully deacetylated chitosan. particularly in the field of cosmetics [2]. can be explained by the presence of negatively charged residues (sialic acid) in the mucin – the glycoprotein that composes the mucus. purifying them by recovering heavy metals [2]. naturally occurring polysaccharide [18]. such as lipids. but also a polysaccharide. as etherification and esterification reactions [2] but remarkably.21].20. the number of positive charges also increases. proteins. which opens prospects to a wide range of applications.782 F. it is important to remember that chitosan is a semi-crystalline polymer. its crystallinity also decreases.24]. The complexing ability of chitosan is further exploited for beverages (wine. chitosan can also interact with the negative part of cells membrane. Now.18. it is important to remember that chitosan is not only a polymer bearing amino groups. Non-specific reactions can be performed on the alcohol groups. when chitosan DD decreases (close to 60%).28] – the rate of its degradation being highly related to the molecular mass of the polymer and its deacetylation degree – and has proved to some extent biocompatibility with physiological medium [29. the amino group of D-glucosamine residues may be specifically quaternized or reacted with aldehyde functions under mild conditions through reductive amination [2]. . explaining the permeation enhancing property of this polysaccharide. Remarkable properties of chitosan as biomaterial As already mentioned. As a matter of fact. Till now. Besides.38. C.39]. Chitosan antimicrobial property might in fact result from a combination of both mechanisms [23. chitosan can notably be employed for the preparation of multilayered films. such as metal ions.30]. Various functionalizations can be introduced along chitosan backbone by this technique to further extend chitosan field of applications. and can thus interact with the positively charged chitosan. juices. alter its permeability. . Chitosan is actually degraded in vivo by several proteases. red blood cells membranes are negatively charged. eight human chitinases have been identified. Finally. The presence of the protonable amino group along D-glucosamine residues allows to elucidate most of chitosan properties.) clarification [19]. Chitosan with protonated amino groups becomes a polycation that can subsequently form ionic complexes with a wide variety of natural or synthetic anionic species [4]. Due to its positive charges. chitosan is the only positively charged. the amino groups of the D-glucosamine residues can protonate in the presence of proton ions that are released in the inflammatory area. and consequently the biodegradation rate. resulting in an analgesic effect [37]. DNA and some negatively charged synthetic polymers as poly(acrylic acid) [4. This mucoadhesion is directly related to the DD of chitosan: actually. Yet. Jérôme / European Polymer Journal 49 (2013) 780–792 [18]. and as a consequence. As for mucoadhesion. In acidic medium. three of them possessing enzymatic activity on chitosan [40]. several remarkable properties of chitosan offered unique opportunities to the development of biomedical applications. the distribution of acetyl residues along chitosan will also affect its crystallinity. chitosan also efficiently complexes various species. Besides. chitin shows less effective haemostatic activity than chitosan. chitosan exhibits other remarkable intrinsic properties: this polysaccharide exhibits antibacterial activity [23. The polycationic nature of chitosan also allows explaining chitosan analgesic effects.

The process is based on the neutralization of chitosan amino groups and thus the inhibition of the repulsion between chitosan chains. Chitosan can indeed be either physically associated. and a liquid phase. it is not surprising that chitosan was.. while the number of positive charges increases. Jérôme / European Polymer Journal 49 (2013) 780–792 Considering all the aforementioned properties. and 2D-scaffolds especially for wound healing purposes. be manufactured into variety of shapes [49. According to literature. some chemical modifications of chitosan structure could induce toxicity [38]. which make them not very stable. scaffold morphology and porosity. should be biodegradable so that the cured tissue will be able to replace the biomaterial. temperature. Three main types of chitosan hydrogels have been developed. typically representing less than 10% of the total volume of the gel. Thanks to the polycationic nature of chitosan in acidic conditions. mechanical properties. However. The formation of the hydrogel then occurs via hydrogen bonds. as poly(vinyl alcohol) (PVA) [55. notably for sutures. but also for other biomedical applications such as drug and growth factor delivery [52–54]. presenting either reversible or irreversible gelation. 3. making it able to soak up/absorb large quantities of water while remaining insoluble in the liquid phase [12]. as glycerol phosphate disodium salt [72]. C. possess surface properties that will promote cell attachment.1. Chitosan structure can also be modified to form physical hydrogels: chitosan-g-poly(ethylene glycol) (PEG) graft copolymer is able of self-organization depending on the temperature to form stable hydrogels [73]. which tends to improve biocompatibility [47]. Chitosan hydrogels Gels are composed of a solid phase.1. N-acetyl-D-glucosamine units allows fine tuning of the swelling of the obtained hydrogel. as well as healing and tissue replacement capacity [48]. the compatibility of chitosan with physiological medium depends on the preparation method (residual proteins could indeed cause allergic reactions) and on the DD – biocompatibility increases with DD increase. chitosan has shown biocompatibility [9. 5. These will be reviewed in the following sections. Croisier. as illustrated in Fig. and more precisely on its processing methods for uses as biomedical 3D-scaffolds for tissue engineering. The solid phase ensures the consistency 783 of the gel. the interaction between cells and chitosan increases as well. and finally.58].1. chitosan hydrogels can be formed by blending chitosan with other water-soluble non-ionic polymers. such as electrostatic interactions. differentiation and proliferation. the liquid phase is water (and sometimes adjuvants). Positively charged chitosan will interact with negatively charged molecules. In hydrogels. formation of chitosan hydrogels through electrostatic interactions involving small-size polyanions but also polyelectrolytes appears straightforward (Fig. Thermo-sensitive chitosan hydrogels can be obtained by blending chitosan with polyol salts. chitosan appears thus as a relevant candidate for the preparation of such biomaterials. fewer interactions will lead to a softer gel while a higher number of interactions will give a tighter and stiffer gel. Therefore. the following will focus on the potential applications of chitosan as a biomaterial. Indeed. Further on. is and will be tested in many biomedical and pharmaceutical applications. As a rule. without the need of any additive. all by itself. they are soft and bendable. which minimizes the damage to the surrounding tissue during and after implantation in the patient [38].45] and was notably approved by the Food and Drug Administration (FDA) for use in wound dressings [46]. Those interactions can be dependent of various parameters as pH.71]. . in order to increase or decrease the number of interactions. The mechanical properties of hydrogels tend to mimic those of the soft body-tissues.F. Besides. hydrophobic interactions or hydrogen bondings [55. Chitosan 3D-scaffolds for tissue engineering During the fabrication of implantable scaffolds particular attention should be paid to body compatibility. which allows the gels to insure both functional and morphological characteristics of the tissue to be repaired [38]. Varying concentration and size of the anionic species as well as the numbers of D-glucosamine units vs.56].70]. 4a).50]. to form hydrogels. Physically associated chitosan hydrogels The formation of ‘‘physical’’ hydrogels is based on the reversible interactions that can occur between polymer chains. have suitable mechanical properties for handling and to mimic the damaged tissue. and allow cell attachment and proliferation [51] provided that 3D-scaffolds might be produced. the main requirements for the elaboration of tissue engineering scaffolds also include that the scaffold should not induce acute or chronic response. 5. This is why hydrogels are often used as biomedical scaffolds for tissue replacements. exhibiting reversible gelation. Moreover. It is remarkable to observe that chitosan is able to form a gel. methodologies have been developed to finely shape chitosan hydrogels and foams (or sponges) as pertinent 3D-scaffolds applicable for tissue engineering. coordinated with metal ions or irreversibly/chemically cross-linked into hydrogels [38]. sulfates and citrates ions [57. . Such development relies thus on robust processing methods for chitosan making able to adjust the scaffold properties at best to the diseased tissue requirements. Due to its aforementioned remarkable properties. . The swelling of those hydrogels can be tuned by adjusting the nature and the quantities of each component. concentration. Chitosan actually proved to be more cytocompatible in vitro than chitin. such as phosphates. hydrophobic interactions and chitosan crystallites [38. 5. Hydrogels are interesting biomaterials as their high water content makes them compatible with a majority of living tissues. dental and bone implants and as artificial skin [2]. After describing chitosan main properties in the previous paragraphs. which could substitute for missing or damaged tissue and organ [48]. Those interactions have a non-covalent nature. Within the framework of these tests.

The charge density of chitosan and of the polyanion will play a major role in the swelling and stability of the formed hydrogels. sulfates and citrates) or polyanions (as proteins (gelatin and collagen…) and anionic polysaccharides (hyaluronic acid…)). prepared in presence of additives. natural and synthetic polyanions. fucoidan) [62–65]. alginate. the interactions between charged chitosan and anions or polyanions are stronger than these secondary bonding. It should be noted that electrostatic interactions can occur with other secondary interactions. hydrophobic interactions and crystallization Fig. carboxymethyl cellulose and glycosaminoglycans [62. coordination complex cross-linking (b) and covalent cross-linking (c). However. anionic polysaccharides (as hyaluronic acid. Via coordination bonds with metal ions (as Pd(II).784 F. Via amide or ester bonds formation (with glyoxal. C. which is a prerequisite for biomedical applications. Non-covalent cross-linking (a) Coordination complex cross-linking (b) Covalent cross-linking (c) Via electrostatic interactions with anions (as phosphates. Schematic representation of three types of chitosan hydrogels. Larger negatively charged molecules. The easiness of gelation without the need of toxic additives. proteins (as gelatin. In the natural polyanion category. pectin. 4. dextran sulfate. without any additives. Also via H bonds. can also give gels with chitosan. 3. Gels can be formed through non-covalent crosslinking (a). Croisier. Schematic representation of physical chitosan hydrogel. keratin. albumin and fibroin) [59–61]. xanthan. heparin. Proper adjustment of the pH of the medium is thus important.69]. Jérôme / European Polymer Journal 49 (2013) 780–792 H bonds Hydrophobic interactions Crystallites Fig. collagen. and offer the unique opportunity to shape the gel within the diseased . Pt (II) and Mo(VI)). The preparation of ionic chitosan hydrogels avoids the use of catalyst or toxic reacting agent.66] have been reported for such purpose. as for example hydrogen bonding [68. in conditions compatible with the human body led to the development of injectable solutions that exhibit a sol/gel transition upon injection in the body. chodroitin sulfate. glutaraldehyde and Genipin…). Synthetic polyanions – as poly(acrylic acid) (PAA) – were also used to form hydrogels [67].

a simple efficient process consisting in freezing a solution of chitosan followed by sublimation of the solvent under reduced pressure (a SEM image of a chitosan sponge prepared by this technique is presented in Fig. ethylene glycol. Recently. the mechanical resistance/strength of the aforementioned hydrogels is quite limited and uncontrolled dissolution of the gels can occur [74]. which fastly react with the amino groups of the chitosan D-glucosamine units and. perfectly adjusting the scaffold to the defect. Croisier. they are mostly used for this purpose since the cross-linking degree might consume only few of the amino groups initially present on chitosan.2. 4b. confirming their potential healing in wound dressing application. the genipin (C11H14O5. such as glyoxal and glutaraldehyde. Chitosan–ZnO composite sponges also prepared by freeze-drying [80] showed good swelling. but also Schiff base formation [55. The properties of the resulting hydrogels will rely on the cross-linking density and the ratio of cross-linker molecules to the moles of polymer repeating units [79]. Therefore. but less suited for biomedical use. However.78]. 4c). These solid structures are able to absorb high amount of fluids (more than 20 times their dry weight). due to their micro-porosity.F. However. as a filling material [81]. antibacterial and haemostatic activities. while helping the tissue regeneration. isolated from Genipa Americana in 1960 and then from Gardenia jasminoides Ellis) has been used as an alternative cross-linking agent [62]. The . which is a major concern given the future application of the hydrogels as biomaterials [76]. These sponges are mainly obtained by freeze drying (lyophilization). Chemically cross-linked chitosan hydrogels The formation of those hydrogels occurs via covalent bonding between polymer chains (Fig. diglycidyl ether and diisocyanate can also play the role of cross-linker to obtain chitosan hydrogels. irreversible chemical cross-linking of the hydrogels have been investigated. which could alter its initial properties. C. Chitosan sponges are mainly used as wound healing materials. Tripolyphosphate. SEM image of a chitosan sponge prepared by freeze-drying. Chitosan cross-linked by coordination complex Coordinate-covalent bonds can also occur with chitosan via metal ions as Pt (II). For example. Chitosan sponges also find application in bone tissue engineering.75]. 5. to a lesser extent. Chitosan sponges Sponges are nothing else than foams with an open porosity. This cross-linker degrades more slowly than other cross-linkers (such as glutaraldehyde) and its toxicity has not yet been established [62. it is difficult to accurately control the size of the hydrogel pores. This gelation is depicted in Fig.82]. Chitosan with high DD is then of interest as starting material.69] leading to the formation of another type of hydrogels. and chitosan/collagen sponges [83] were prepared by this technique and used as scaffolds for bone regeneration. Genipin can react with primary amine and thus cross-link polymers containing such amino groups [77. In addition. 5). as amide and ester bonding. The scCO2 method allows the preparation of porous chitosan scaffolds. as they can soak up the wound exudates. still being soft and flexible [80]. 6. Lately. Pd (II). A straightforward way to obtain irreversible chitosan hydrogels is the use of dialdehyde cross-linkers. They typically offer good cell interaction. 5. Both the amino and the hydroxyl groups of chitosan can form a variety of linkages. Chitosan 2D-scaffolds for wound dressing In the particular case of wound dressing. these groups being more reactive than the hydroxyl groups. Jérôme / European Polymer Journal 49 (2013) 780–792 785 tissue used as mold or template.1. like proteins and chitosan. Fibrillar structure was obtained and those sponges showed promising use for wound dressing application. particularly if amino groups are involved in the reaction.77]. this approach requires the chemical modification of the primary structure of chitosan. efforts were dedicated to the use of supercritical carbon dioxide (scCO2) as a green medium to induce porosity to chitosan scaffolds [85]. Besides. chitosan [54]. It is important to note that chemical cross-linking of chitosan that involves chemical modifications of chitosan primary amine might induce modifications of chitosan properties. specific requirements have to be met to encounter efficiency. chitosan/tricalcium phosphate (TCP) [81. and Mo (VI) [68. However. Small multifunctional molecules or polymeric cross-linker can both be used to react with chitosan and induce cross-linking. 5.1. suitable for cell cultures directly upon depressurization.2. Chitosan can also be firstly modified by activated functional groups before inducing cross-linking by photo-induced reaction or enzymatic catalyzed reactions [38]. The obtained hydrogels are much more stable than the previous physically associated hydrogels since the gelation is irreversible. Cross-linked chitosan sponges loaded with a model of antibiotic drug – the norfloxacin – were prepared by solvent evaporation technique [84]. the involved reaction might be a source of contamination by toxic residual reactants or catalyst traces. However. 100 µm Fig.3. all the aforementioned cross-linkers may impart toxicity to the formed hydrogels. a natural derived cross-linker. 5. with the hydroxyl groups of chitosan [62]. which can lead to chitosan hydrogels formation.

such as films and porous membranes. respectively). keep moist environment. Like the Ag nanoparticles. that plays the role of cross-linking agent. via Michael addition reaction. When chitosan films were reacted with succinic anhydride or phthalic anhydride. and reaching 1.1. blending and chemical modifications of chitosan have proven efficiency. Chitosan films prepared in presence of dialdehyde starch. and (iv) non-adherent film of b-glucan and chitosan [97. which promoted lysozyme adsorption [99]. Other noteworthy developments are also the preparation of: (i) phosphorylated chitosan films (prepared from the reaction of orthophosphoric acid and urea in DMF). Improving properties of chitosan free-standing films To improve the properties of those films. Heterogeneous chemical modification of the chitosan film can tune the surface properties of the film. the decrease of the modulus was 56%. a solution in an organic volatile solvent of an amphiphilic water-insoluble (macro)molecule is spread on the surface of water. are supplementary candidates. These two processes are of importance since both of them allow to control parameters such as film thickness. (iii) chitosan/poly(vinyl alcohol)/alginate films by casting/ solvent evaporation method. For examples. They possess pronounced antibacterial activity (especially the film containing 0. Interestingly. 6. which results in a decrease of the modulus with an increase of the strain at break (for 50/50 chitosan/PEO blended film. allow gas exchanges. to further avoid microorganism proliferation [102–105]. (ii) chitosan blends with minocycline hydrochloride. HemConÒ bandage is an engineered chitosan acetate preparation designed as a haemostatic dressing [80]. To improve the films ductility. Polyphosphate procoagulant can also be incorporated to the chitosan/Ag films [24]. As far as mechanical properties are concerned.2. Ag nanoparticles possess identified antimicrobial activity.1 MPa and an elongation at break of 27%) [92].786 F. As mentioned above. chitosan sponges have been found to fit quite well these requirements. composition. (iii) by introducing silica particles or poly(ethylene glycol) [89] into the chitosan films. chitosan/polyethylene glycol diacrylate (PEGDA) blended films were developed [93]. as far as skin tissues are concerned. Nevertheless. it leads to significant strengthening of the films and to a decrease of the film solubility in aqueous media [91]. and the layer-by-layer (LBL) deposition techniques. show improved mechanical properties and better water-swelling (best values were obtained with a dialdehyde starch content of 5%.1 wt. notably used in silverbased wound dressings [101]. in order to create a moist environment within the framework of wound healing [96]. Those Ag particles can be added to chitosan film. the film can easily be removed from wound [100]. for instance. morphology. poly(N-isopropyl acrylamide) and ciprofloxacin. more 2D-shaped scaffolds. it resulted in more hydrophilic films (contact angles of 56 ± 2° and 51 ± 7° were found for N-succinylchitosan and N-phthalylchitosan films. (ii) ozone or UV irradiation promotes surface modification of chitosan films leading to depolymerization of chitosan [88]. Chitosan films Chitosan films can be easily prepared by wet casting from chitosan salt solutions. Chitosan thin films The properties of the wound dressing surface in contact to the diseased body are particularly of importance to provide efficient healing.2  103 S cm1 for a chitosan film containing 87. while the increase of the strain at break was 125%.1. (i) nitrogen or argon plasma [87] treatment of chitosan membranes increases the film surface roughness and improves the cell adhesion and proliferation (especially for films treated with nitrogen plasma at 20 W during 20 min). Beside Ag. their porosity can be artificially modified with macro and micro-pores (pores size ranging 0. Two methods to design chitosan thin films in order to modify the surface of a performed substrate will be shortly described: Langmuir–Blodgett (LB). when a stearoyl group was attached to the chitosan films. so that by decreasing the temperature. while 101 ± 4° was found for N-stearoylchitosan film) and promoted proteins adsorption. protect the wound against microbial organisms and absorb wound exudates [80]. and even roughness [18].31 mg/m2 of phosphorus – NB: conductivity measurements were realized after hydratation of the films) [94]. a thermo-sensitive film was prepared by combining thiolated chitosan. chitosan can be blended (or copolymerized) with poly(ethylene glycol) (PEG). 6. in comparison to pure chitosan film) [90]. showing enhanced swelling (the maximum swelling was observed at a ratio of CS/PEGDA equal to 40/60) and good mechanical properties (the maximum values of tensile strength and modulus were obtained at a ratio of CS/PEGDA equal to 70/30). which present higher ionic conductivity than normal chitosan films (conductivity of pure chitosan film being equal to 8. Croisier. followed by drying (typically using oven or infrared (IR) drying [86]). Amidation of the chitosan films (up to 50%) can occur with thermal treatment.1. thus promising application in wound dressing field. they became more hydrophobic (contact angle of pure chitosan film being 89 ± 6°. Complex films containing chitosan. with a tensile strength of 113.3  105 S cm1. Multilayer coatings and nanostructured thin films have thus also been investigated in order to tune the surface properties of various medical devices. 6.1.98]. incorporation of inorganic particles in these films allows synergistic effects of both materials leading to high performance healing. For example. zinc oxide (ZnO) nanoparticles are non-toxic and present antibacterial and good photocatalytic activities [106]. they can be incorporated in chitosan films. Jérôme / European Polymer Journal 49 (2013) 780–792 wound dressing should be non-toxic and non-allergenic. the hydrophilic part of the amphiphile is anchored . to prepare films that accelerate the healing of burns (reduction of the wound size) [95]. Finally. To form a Langmuir [108] monolayer at the air–water interface.% Ag and 10 wt.% ZnO). some physicochemical processes have been tested. C. Ag nanoparticles and ZnO particles were prepared [107].5–100 lm). spreading the panel of structures available to tackle the challenges addressed in skin repair. When the organic solvent is evaporated.

This can be done by immersing (or emerging) a solid support in (or from) the aqueous subphase to recover the monolayer (Fig.N-dialkyl-chitosans. nucleic acids. By repeating the process. Chitosan modified with long alkyl chains attached to the primary hydroxyl and amino groups. while other amphiphilic chitosans. Jérôme / European Polymer Journal 49 (2013) 780–792 787 Fig. For sensing applications. On the other hand. Chitosan can be LBL co-deposited with several species on metallic devices [18]. Chitosan being poorly soluble in organic solvents. Schematic representation of Langmuir film (a) (left) and Langmuir–Blodgett process (b) (right). in aqueous medium. 6a). bone implants and wound dressings [18]. Mixed films of cholesterol and chloroform-soluble O. filters. Fig. amphiphilic chitosans bearing an alkyl group combined with a cinnamoyl chromophore were synthesized [114]. 7.112]. this derivatization making the resulting chitosan soluble in chloroform. chemical modification is required for this process to be performed. carbon nanotubes.112]. Derivatives of chitosan pentamers were used to produce LB films [111. to the aqueous subphase. phthalo- . Croisier. A large number of molecules can be used. but also as biomaterial for tissue engineering [119]. 7. 6b). was the first example of LB chitosan films formation [18. haemostatic devices.F. Such polyelectrolyte multilayered films find many applications in the field of sensors [118]. C. including synthetic and natural polyelectrolytes [111]. namely. drug delivery systems. enzymes. LBL chitosan-based films are involved in many applications as sensors.110]. while the hydrophobic part is directed toward air [18] (as depicted in Fig. were also reported [113]. A schematic representation of LBL process is presented in Fig. Schematic representation of LBL process. The polycationic nature of chitosan makes this polymer well-suited for LBL processes.O-dipalmitoyl chitosan are an example of two-component LB films [115]. since the amphiphilic films are able to entrap various drugs with efficacy. All these films find application mainly in drug release and drug delivery systems. nanotubes [116] and biomolecules [117]. multilayered films can be obtained. To facilitate the optical characterization of the LB films. 6. the layer-by-layer (LBL) deposition technique is based on the alternated adsorption of materials bearing complementary charged or functional groups [22. As a consequence. N. antigens. The Langmuir–Blodgett (LB) process [109] consists in transferring this Langmuir monolayer onto a solid support.

while adding PVP allows decreasing the diameter of chitosan-based fibers [153]. 6. and are able to mimic the natural extracellular matrix. The addition of UHMWPEO to chitosan ESP solution allows forming fibers ranging from less than an hundred nanometers to a few tens micrometers [148]. the first example was reported as early as 1926 [124. chitosan (nano)fibers were successfully produced from trifluoroacetic acid (TFA) and dichloromethane (DCM) mixtures [135. to produce cross-linked chitosan fibers [138].140]. which makes these mats great candidates for biomaterial applications. and porphyrins can be trapped by this process in a chitosan matrix that helps maintaining the biomolecule activity. This phenomenon restricts the formation of continuous fibers and often leads to the creation of particles [133. Another group used TFA in conjugation with glutaraldehyde. A SEM image of chitosan/PEO nanofibers is presented in Fig. as they have shown abilities to accelerate the healing. SEM image of electrospun chitosan/PEO blended fibers.788 F. C. The joint use of PVA and chitosan allows the preparation of fibers without beaded 10 µm Fig. A few examples of pure electrospun chitosan fibers are reported. Jérôme / European Polymer Journal 49 (2013) 780–792 cyanines. The electrospinning of chitosan in presence of poly(ethylene oxide) (PEO) [143–147] and ultrahigh-molecularweight poly(ethylene oxide) (UHMWPEO) [148] are commonly reported.125]. The structure of the fiber mats was found to promote the attachment of human cells. Chitosan/PEO nanofibers prepared by ESP exhibit cellular biocompatibility [133] [147]. Finally. blends of chitosan with other polymers were also considered: sodium alginate [129]. ESP uses a high voltage to create an electrically charged jet of polymer solution or melt which can lead to fibers formation. as chitosan is a polyelectrolyte in acidic medium. its electrospinning turns out to be tricky. poly(vinyl pyrrolidone) (PVP) [153] and poly(lactic acid) (PLA) [154] were also reported.136]. Lithium chloride/ N. To improve mechanical properties of chitosan LBL films. poly(acrylic acid) [131] were thereby employed. which destroy the interactions between chitosan molecules and facilitate the ESP process. chitosan/heparin LBL films are particularly interesting for their anticoagulation properties [18]. high specific surface area. Croisier. Chitosan porous nanofiber membranes There are several ways to produce chitosan fibers. For coronary stents. and of chitosan and alginate [119] offer great prospect for wound dressing purpose. 8 shows a schematic representation of this technique. tropocollagen [130]. the easiest way to produce chitosan (nano)fibers is the electrospinning of chitosan as a blend with another polymer. the use of concentrated aqueous acetic acid was considered [139. 9. with carbon nanotubes [121]. In recent years. Fibers were notably produced using dry [126] and wet spinning [127] from acetic acid solution. LBL films entirely made of polysaccharides. Indeed. the use of DCM allows improving the homogeneity of the produced fibers. In addition.2. repulsive forces between charged species within polymer backbone arise as a consequence of the application of an important electric field. Schematic representation of the electrospinning set-up. 8. sodium hyaluronate. The uses of poly(ethylene terephtalate (PET) [152]. during ESP. Poly(vinyl alcohol) (PVA) [149–151] can also be used to form chitosan (nano)fibers. . Fig.N-dimethylacetamide was also used as a solvent [128]. The mats of fibers produced by this method possess high porosity. sodium chondroitin sulfate [124]. Chitosan (nano)fibers find many applications for the preparation of wound dressing and for tissue engineering [133]. It was suggested [135] that the amino groups of the chitosan form salts with TFA [137]. which might be especially important when deposited in soft substrates. To facilitate the electrospinning of chitosan. electrospinning (ESP) [132] has distinguished as a versatile technique for the preparation of polymer fibers from a few nanometers to microns in diameter. depending on the processing conditions. such as chitosan and hyaluronic acid [123]. Fig.134]. In order to decrease production cost and to improve fiber properties. Nevertheless. or with cellulose nanowhiskers [122]. while preserving their morphology and viability [147]. cellulose. sodium heparin. some authors report on the use of modified chitosan such as hexanoyl chitosan [141] and PEGylated chitosan [142]. Many examples of such chitosan blend fibers produced by ESP are reported in the literature. chitosan can be combined in the film with clays such as Na-montmorillonite [120]. However. 9. These blends thus offer great prospects for designing tissue engineering scaffolds [133].

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CERM thanks the Walloon Region for supporting researches on chitosan in the frame of CHITOPOL. so as stronger sponges and nanofiber mats are thus easily made available with good control on their composition. The addition of another polymer to facilitate the ESP process is actually a way to improve the properties of the resulting fibers such as the mechanical properties. Ma L. Biomaterial type Hydrogels (3D) Specifications Main advantage(s) Main disadvantage(s) Main biomedical application(s)  Physically associated (reversible) Soft flexible non-toxic Tissue replacements/engineering drug/growth factor delivery  Chemically crosslinked (irreversible) Soft flexible stable controlled pore size Not stable (uncontrolled dissolution may occur) Low mechanical resistance Pore size difficult to control May be toxic Crosslinking may affect chitosan intrinsic properties Sponges (3D) Films (2D) Porous Membranes (2D)  Free-standing High porosity Soft May shrivel Low porosity Tissue engineering (filling material) Wound dressings skin substitutes  Thin (LB) Material coating Coatings for a variety of scaffolds wound dressings skin substitutes  Thin (LBL) Material coating Multilayer construction Laborious for the construction of multilayers Many steps Nanofibers High porosity Mimic skin extracellular matrix defects. Extraction and characterization of chitin from crustaceans. 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