Chitosan - A Versatile Semi-Synthetic Polymer in Biomedical

Progress in Polymer Science 36 (2011) 9811014
Contents lists available at ScienceDirect
Progress in Polymer Science

journal homepage: www.elsevier.com/locate/ppolysci
ChitosanA versatile semi-synthetic polymer in biomedical

applications
M. Dash a , F. Chiellini a , R.M. Ottenbrite b , E. Chiellini a,
a
Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab), UdR INSTM, Department of Chemistry and Industrial
Chemistry, University of Pisa, Pisa, Italy
b
Department of Chemistry, Virginia Commonwealth University, Richmond, VA, USA
a r t i c l e
i n f o
Article history:
Received 4 May 2010
Received in revised form 21 October 2010
Accepted 4 February 2011
Available online 22 February 2011
Keywords:
Chitosan
Tissue engineering
Drug delivery
Gene therapy
Bioimaging
a b s t r a c t
This review outlines the new developments on chitosan-based bioapplications. Over the
last decade, functional biomaterials research has developed new drug delivery systems
and improved scaffolds for regenerative medicine that is currently one of the most rapidly
growing elds in the life sciences. The aim is to restore or replace damaged body parts or
lost organs by transplanting supportive scaffolds with appropriate cells that in combination
with biomolecules generate new tissue. This is a highly interdisciplinary eld that encompasses polymer synthesis and modication, cell culturing, gene therapy, stem cell research,
therapeutic cloning and tissue engineering. In this regard, chitosan, as a biopolymer derived
macromolecular compound, has a major involvement. Chitosan is a polyelectrolyte with
reactive functional groups, gel-forming capability, high adsorption capacity and biodegradability. In addition, it is innately biocompatible and non-toxic to living tissues as well as
having antibacterial, antifungal and antitumor activity. These features highlight the suitability and extensive applications that chitosan has in medicine. Micro/nanoparticles and
hydrogels are widely used in the design of chitosan-based therapeuticsystems. The chemical structure and relevant biological properties of chitosan for regenerative medicine have
been summarized as well as the methods for the preparation of controlled drug release
devices and their applications.
2011 Elsevier Ltd. All rights reserved.
Abbreviations: AL, alginate; ASGPR, asialoglycoprotein receptor; RGD, arginineglycineaspartic acid; BAL, bioarticial liver; BMP, bone morphogenetic
protein; CP, calcium phosphate; CPC, calcium phosphate cement; CSF, colony-stimulating factor; DD, degree of deacetylation; DCs, dendritic cells; DTPA,
diethyl triamine penta acetic acid; EDC, 1-ethyl-3-[3-imethylaminopropyl]carbodiimide hydrochloride; EGFP, enhanced green uorescent protein; ECM,
extra cellular matrix; FGF-2, broblast growth factor-2; FRET, uorescence resonance energy transfer; FHF, fulminant hepatic failure; Gd, gadolinium;
GC, galactosylated chitosan; GDNF, glial cell line-derived nerve growth factor; GP, glycerophosphate; GAGs, glycosamine glycans; GM-CSF, granulocytemacrophage colony-stimulating factor; GTR, guided tissue regeneration; hGH, human growth hormone; hUCMSCs, human umbilical cord mesenchymal
stem cells; HA, hydroxyapetite; HEC, hydroxyethyl cellulose; IBL, implantable bioarticial liver; 131 I-NC, 131 I-norcholesterol; IL, interleukin; IPN, interpenetrating network; ILs, ionic liquids; LCST, lower critical solution temperature; MRI, magnetic resonance imaging; MSCs, mesenchymal stem cells; NHS,
N-hydroxysuccinimide; NCT, neutron-capture therapy; pDNA, plasmid DNA; PAA, poly(acrylic acid); PEC, polyelectrolyte complex; PEO, polyethylene oxide;
PEI, poly(ethylenimine); PVP, poly(vinyl pyrrolidine); PNIPAM, poly(N-isopropylacrylamide); PVA, poly vinyl alcohol; RES, reticuloendothelial system; RII,
retrograde intrabiliary infusion; RTILs, room temperature ionic liquids; RWM, round window membrane; SCs, Schwann cells; TPP, sodium tripolyphosphate;
SPIOs, super paramagnetic iron oxide; SBF, synthetic body uids; TCP, tricalcium phosphate; TGF-1, transforming growth factor 1; TEM, transmission
electron microscopy; TAA, triamcinolone acetonide; UV, ultra-violet; WSCLA, water-soluble chitosanlinoleic acid; XRD, X-ray diffraction.
Corresponding author. Tel.: +39 050 2210301/2/3; fax: +39 050 2210332.
E-mail address: emochie@dcci.unipi.it (E. Chiellini).
0079-6700/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2011.02.001
982
M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982
General aspects of chitosan-structural and functional features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
2.1.
Structure, source and physicochemical properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
2.2.
Structureproperty relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
2.3.
Biodegradability of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985
2.3.1.
Chitosan in-vitro biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985
2.3.2.
Chitosan in vivo biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985
2.4.
Biological properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985
2.5.
Chitosan toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
2.5.1.
In-vitro toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
2.5.2.
In vivo toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
Chitosan-based systems for biomedical applications types and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 987
3.1.
Chitosan micro/nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 987
3.1.1.
Emulsion cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 987
3.1.2.
Coacervation/precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 988
3.1.3.
Spray-drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 988
3.1.4.
Emulsion-droplet coalescence method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
3.1.5.
Ionic gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
3.1.6.
Reverse micellar method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
3.1.7.
Sieving method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
3.1.8.
Chitosan micro/nanoparticles drug loading and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991
3.2.
Chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991
3.2.1.
Physical association networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 992
3.2.2.
Cross-linked networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
3.2.3.
Chitosan hydrogels drug loading and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
Biomedicalpharmaceutical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
4.1.
Chitosan for tissue engineering applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
4.1.1.
Chitosan in bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
4.1.2.
Chitosan in cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
4.1.3.
Chitosan in liver tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 997
4.1.4.
Chitosan in nerve tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999
4.2.
Chitosan in wound-healing applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.3.
Chitosan in drug delivery applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.3.1.
Chitosan-based systems for the delivery of anti-cancer drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.3.2.
Chitosan-based systems for the delivery of proteins/peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.3.3.
Chitosan-based systems for the delivery of growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001
4.3.4.
Chitosan-based systems for the delivery of antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001
4.3.5.
Chitosan-based systems for the delivery of anti-infammatory drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002
4.3.6.
Chitosan-based systems for vaccines delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002
4.3.7.
Chitosan membranes in drug release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002
4.4.
Chitosan in gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
4.5.
Chitosan in bioimaging applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
4.6.
Chitosan in green chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006
1. Introduction
The history of chitosan dates back to the 19th century,
when Rouget [1] discussed the deacetylated forms of the
parent chitin natural polymer in 1859. During the past 20
years, a substantial amount of work has been reported
on chitosan and its potential use in various bioapplications. Chitosan is derived from naturally occurring sources,
which is the exoskeleton of insects, crustaceans and fungi
that has been shown to be biocompatible and biodegradable [2]. Chitosan polymers are semi-synthetically derived
aminopolysaccharides that have unique structures, multidimensional properties, highly sophisticated functionality
and a wide range of applications in biomedical and other
industrial areas [35]. They have become interesting not
only because they are made from an abundant renew-
able resource but because they are very compatible and

effective biomaterials that are used in many applications [68]. Chitosan is a linear copolymer of -(14)
linked 2-acetamido-2-deoxy--d-glucopyranose and 2amino-2-deoxy--d-glycopyranose (Fig. 1). It is obtained
by deacetylation of its parent polymer chitin, a polysaccharide widely distributed in nature (e.g. crustaceans, insects
and certain fungi) [9,10]. Due to chitins poor solubility
in aqueous solution and organic solvents, it does not nd
practical applications whereas chitosan as an articial variant of chitin is more suitable for useful bioapplications
[11]. The positive facets of excellent biocompatibility and
admirable biodegradability with ecological safety and low
toxicity with versatile biological activities such as antimicrobial activity and low immunogenicity have provided
ample opportunities for further development [1217].
983
Fig. 1. (a) Chitosan structure; (b) chemical structure of chitosan. Individual atoms are numbered. Dashed lines denote O3O5 hydrogen bonds. Two dihedral
angles (, ) dening the main chain conformation and one dihedral angle dening the O6 orientation are indicated [19]. Copyright 2009, Elsevier Ltd.
2. General aspects of chitosan-structural and

functional features
2.1. Structure, source and physicochemical properties of
chitosan
Chitosan molecule is a copolymer composed of Nacetyl-d-glucosamine and d-glucosamine units available in
different grades depending upon the degree of acetylated
moieties [18] (Fig. 1). It is a polycationic polymer that has
one amino group and two hydroxyl groups in the repeating
glucosidic residue [19] (Fig. 1). The carbohydrate backbone
is very similar to cellulose, which consists of -1,4-linked
d-glucosamine with a variable degree of N-acetylation,
except that the acetylamino group replaces the hydroxyl
group on the C2 position. Thus, chitosan is a copolymer consisting of N-acetyl-2-amino-2-deoxy-d-glucopyranose and
2-amino-2-deoxy-d-glucopyranose, where the two types
of repeating units are linked by (1 4)--glycosidic bonds
[20]. After renement, chitosan has a rigid crystalline structure through inter- and intra-molecular hydrogen bonding.
The source of chitosan is a naturally occuring polymer,
the chitin that is the second most abundant polysaccharide in nature, cellulose being the most abundant. Chitin
is found in the exoskeleton of crustacea, insects, and some
fungi. The main commercial sources of chitin are the shell
waste of shrimps, lobsters, krills and crabs. In the world
several millions tons of chitin are harvested annually and
hence this biopolymer represents a cheap and readily
available source [2022]. Chitosan is obtained by the thermochemical deacetylation of chitin in the presence of alkali
and naturally it occurs only in certain fungi (Mucoraceae)
[23]. Several alkaline methods have been proposed, most
of them involving the hydrolysis of the acetated position
using sodium or potassium hydroxide solutions as well
as a mixture of anhydrous hydrazine and hydrazine sulfate [24]. The treatment of chitin with an aqueous 4045%
(w/v) NaOH solution at 90120 C for 45 h results in
N-deacetylation of chitin. The conditions used for deacetylation determines the polymer molecular weight and the
degree of deacetylation (DD).
The active primary amino groups on the molecule being
reactive provide sites for a variety of side group attachment employing mild reaction conditions (Fig. 2). The facile
derivatization makes chitosan an ideal candidate for biofabrication [17]. In addition, the characteristic features of
chitosan such as being cationic, hemostatic and insoluble
at high pH, can be reversed by sulfating the amine which
makes the molecule anionic and water-soluble, with the
introduction of anticoagulant properties [25]. The attached
side groups on chitosan provide versatile materials with
specic functionality, alter biological properties or modify
physical properties.
2.2. Structureproperty relationship
Chitosans, with the main structural differences being
represented by the relative proportions of N-acetyl-dglucosamine and d-glucosamine residues, provide specic
structural changes. This difference in the structure gives
rise to several batches of chitosan that are distinguished
Fig. 2. Schematic illustration of chitosans versatility. At low pH (less than

about 6), chitosans amine groups are protonated conferring polycationic
behavior to chitosan. At higher pH (above about 6.5), chitosans amines
are deprontonated and reactive.
984
on the basis of their DD and molecular weight. DD and

molecular weight directly affect the chemical and biological properties of the polymer (Table 1). Commercial
chitosan as sold by SigmaAldrich is available in two grades
of high and low molecular weight. Low molecular weight
chitosan grade is characterized by molecular weight comprised between 20 kDa and 190 kDa with DD < 75%. The
high molecular weight chitosan grade is generally characterized by molecular weight comprised between 190 kDa
and 375 kDa with DD > 75%.
The parent chitin is insoluble in most organic solvents,
chitosan is readily soluble in dilute acidic solutions below
pH 6.0 due to the quaternisation of the amine groups that
have a pKa value of 6.3 making chitosan a water-soluble
cationic polyelectrolyte. The presence of the amino groups
indicates that pH substantially alters the charged state and
properties of chitosan [17]. At low pH, these amines get
protonated and become positively charged and that makes
chitosan a water-soluble cationic polyelectrolyte. On the
other hand, as the pH increases above 6, chitosans amines
become deprotonated and the polymer loses its charge
and becomes insoluble. The solubleinsoluble transition
occurs at its pKa value around pH between 6 and 6.5. As
the pKa value is highly dependent on the degree of Ndeacetylation, the solubility of chitosan is dependent on
the DD and the method of deacetylation used [26]. Apart
from the DD, the molecular weight is also an important
parameter that signicantly affects the solubility and other
properties [2731].
The choice of chitin and its isolation process are also factors that affect chitosan quality [32] in a signicant way.
There are three forms of chitin known , , . Depending on the origin of the polymer and its treatment during
the extraction process, chitosan shows crystallinity and
polymorphism. Crystallinity is maximum for chitin (i.e.
0% deacetylated) and fully deactylated chitosan (i.e. 100%
deacetylated) [33]. A linear unbranched structure and high
molecular chitosan is an excellent viscosity enhancing
agent in acidic environments and behaves as a pseudoplastic material demonstrating a decrease in viscosity with
increasing rates of shear. The viscosity of chitosan solution increases with an increase in the concentration of
chitosan, decrease in temperature and with increasing
DD. Viscosity also inuences biological properties such as
wound-healing properties and osteogenesis enhancement

as well as biodegradation by lysozyme [34].
The understanding and control of the degradation rate
of chitin and chitosan-based devices is of great interest
since degradation is essential in many small and large
molecule release applications and in functional tissue
regeneration applications. Degradation has been shown to
increase as DD decreases [3537]. It has also been observed
that the degradation kinetics seem to be inversely related
to the degree of crystallinity which is controlled mainly
by the DD. The distribution of acetyl groups also seem to
affect biodegradability, the arrangement of acetyl groups
and their homogeneous distribution (random rather than
block) results in very low rates of enzymatic degradation [25,38]. However, several studies reported that the
degradation rate is affected by the length of the chains
(Mw) as well [28,39,40]. Kofuji et al. investigated the enzymatic behaviors of various chitosans by observing changes
in the viscosity of chitosan solution in the presence of
lysozyme and found that chitosan with a low DD tend to
be degraded more rapidly [41]. Differences in degradation
due to variations in the distribution of acetamide groups in
the chitosan molecule have been observed [38,42]. Apparently, different deacetylation conditions can inuence the
viscosity of the chitosan solution by changing the inter- and
intramolecular repulsion forces [37]. Very fast degradation
rates of chitosan cause an accumulation of amino saccharide that can produce an inammatory response. While
the lower DD chitosans only induce an acute inammatory response; the higher DD produces a minimal response
due to the lower degradation rate. More specic information on the biodegradability of chitosan is provided
later in this review. The cytocompatibility of chitosan
has been observed in vitro with myocardial, endothelial
and epithellial cells, broblast, hepatocytes, condrocytes
and keratinocytes [29]. As the degree of deacetylation
(DD) of the polymer increases, the interactions between
chitosan and the cells increase due to the presence of
free amino groups, consequently, cell adhesion and proliferation, as well as cell type, depend on DD. Other
biological properties, such as analgesic, antitumor, haemostatic, hypocholesterolemic, antimicrobial, and antioxidant
properties are also affected by the physical properties of
chitosan [6,43,44]. The dependence of the various poly-
Table 1
Relationship between structural parameters and properties.
Property
Structural characteristicsa
References
Solubility
Crystallinity
Biodegradability
Viscosity
Biocompatibility
DD
DD
DD, Molecular weight
DD
DD
[17,2631]
[33]
[25,28,3542]
[41]
[29,45]
Biological
Mucoadhesion
Analgesic
Antimicrobial
Permeation enhancing effect
Antioxidant
Hemostatic

DD
DD
DD
[39,4649]
[5053]
[54,55]
[45,5660]
[6163]
[6468]
Directly propotional to property; inversely propotional to property.
mer properties on the structural parameters are listed in

Table 1.
2.3. Biodegradability of chitosan
Biodegradation plays a major role in the metabolic
fate of chitosan in the body and is important to respect
all polymers used in drug delivery systems and scaffolds
in tissue engineering. In case of systemic absorption of
hydrophilic polymers, such as chitosan. For example, a
suitable molecular weight is required for renal clearance
from 30,000 to 40,000, depending on the polymer used. If
the administered polymers size is larger than this, then
the polymer must undergo degradation. Both chemical or
enzymatic biodegradation would provide fragments suitable for renal clearance. Chemical degradation is referred
to acid catalyzed degradation such as in the stomach.
Enzymatically, chitosan can be degraded by enzymes able
to hydrolyse glucosamineglucosamine, glucosamineNacetyl-glucosamine and N-acetyl-glucosamineN-acetylglucosamine linkages [69]. Even though depolymerisation
through oxidationreduction reaction [70] and free radical
degradation [71] of chitosan have been reported these are
unlikely to play a signicant role in the in vivo degradation.
Chitosan is known to be degraded in vertebrates predominantly by lysozyme and by certain bacterial enzymes
in the colon [72]. Eight human chitinases (in the glycoside hydrolase 18 families) have so far been identied,
three of which have shown enzymatic activity [73]. A variety of microorganisms synthesises and/or degrades chitin,
the biological precursor of chitosan. Both rate and extent
of chitosan biodegradability in living organisms are DD
dependent, with a decrease in the degradation rate with
increasing DD [74,75]. Basically, given adequate time and
appropriate conditions, chitosans, in most cases would
degrade sufciently to be excreted [69].
2.3.1. Chitosan in-vitro biodegradation
Viscometry and/or gel permeation chromatography are
the commonly used assays to determine the decrease in
molecular weight [76]. Following an in vitro incubation
using lysozyme at pH 5.5 in a phosphate buffer at 37 C
for 4 h, 50% acetylated chitosan lost 66% in viscosity [76].
This degradation is DD dependent with the degradation
of the higher acetylated chitosan behaving more like chitosan [77,78]. In addition, a range of proteases was found
to degrade chitosan lms, with leucine amino-peptidase
being the most effective with 38% over 30 days [65].
Pectinase isozyme from Aspergilus niger has also indicated
chitosan digestion at low pH releasing lower molecular
weight fragments [79,80]. From the digestion of chitosan
with rat cecal and colonic bacterial enzymes, Zhang and
Neau observed that extracellular enzymes were responsible for degradation that was related to both DD and
molecular weight [78]. McConnell et al. used human faecal preparations and studied the degradation of chitosan
lms, cross-linked by glutaraldehyde and tripolyphosphate
with interesting results [81]. The rate of porcine pancreatic
enzymes to degrade chitosan lms degradation was inuenced cross-linker; for example, glutaraldehyde degraded
more readily than tripolyphosphate.
985
2.3.2. Chitosan in vivo biodegradation

Very little has been reported on the in vivo degradation of chitosan. The mechanism of degradation is currently
unclear, especially after intravenous injection. However,
studies do indicate that distribution, degradation and elimination processes are strongly dependent on molecular
weight. The liver and kidney were found to be the possible sites of degradation inferred due to the localization
of chitosan. In rabbits, when injected intravenously, chitosan oligosaccharides enhanced lysozyme activity in the
blood [82]. Oral administration of chitosan has shown some
degradation in the gastrointestinal tract. The digestion of
chitosan, occurring predominantly in the gut, was found to
be species dependent with hens and broilers being more
efcient digesters (6798% degradation after oral ingestion) than rabbits (3983% degradation) [83]. In another
study, of subcutaneous implantation of chitosan, a proposed skin substitute such as glutaraldehyde cross-linked
chitosan/collagen was found to be relatively stable over
time compared to collagen alone when implanted subcutaneously in rabbits [84].
2.4. Biological properties of chitosan

Chitosan has proved to be a safe excipient in drug formulations over the last decades [85]. It has attracted attention
as an excellent mucoadhesive in its swollen state and a natural bioadhesive polymer that can adhere to hard and soft
tissues. Good adhesion was found in epithelial tissues and
in the mucus coat present on the surface of the tissues.
A number of colloidal delivery systems based on chitosan
have been presented in the literature for the mucosal delivery of polar drugs, peptides, proteins, vaccines and DNA.
Clinical tests carried out using chitosan-based biomaterials
do not report any inammatory or allergic reactions following implantation, injection, topical application or ingestion
in the human body [29]. The in vitro and in vivo cytocompatibility of chitosan lms with keratinocytes and broblasts
has demonstrated that DD has no signicant inuence. The
chitosan lms with a low DD are very good biomaterials for
supercial wound-healing [29]. Once placed on the wound,
they adhere to broblasts and favor the proliferation of
keratinocytes and thereby epidermal regeneration.
One of the upcoming areas of study related to chitosan
is its biodistribution, especially using methods other than
intravenous administration. The distribution of chitosan
formulates in the body is related to all aspects of the chitosan formulation from the molecular weight and DD to
the size of the delivery vehicle. For instance, in case of a
nanoparticulate formulations, the kinetics and biodistribution will initially be controlled by the size and charge of
the nanoparticles and not by chitosan structural features.
However, after particle decomposition to chitosan and free
drug, inside the cells or target tissues, free chitosan will distribute in the body and eliminate accordingly. Elimination
processes may be preceded by biodegradation. To understand chitosan biodistribution the kinetics of its labeled
(radio or uorescent) modications should be followed,
assuming that the label is neither labile nor affecting the
physicochemical properties of the chitosan [86].
986
Banerjee et al. investigated the distribution of intravenously injected 99mTc labeled nanoparticles (<100 nm)
in Swiss albino mice. Radio-label stability of the nanoparticles was tested and 80% of the radioactivity was associated
with the particles after 3 h. Nanoparticles were administered in mice and an apparent scavenging played of
the reticuloendothelial system (RES) was suggested as
radioactivity decreased in organs of this system but
remained stable in the blood after 1 h [87]. Unfortunately,
the nanoparticles were not sufciently stable to look at
long-term distributions. In a later study, by the same
author sodium borohydride was used in the preparation
conditions. The modied method prevented aggregation,
improved the kinetics of the nanoparticles and resulted in
both less blood clearance and liver accumulation i.e. avoidance of the RES. The radioactivity in blood was present for
up to 10% even after 2 h [88].
In most of the studies liver was found to be a signicant site of accumulation due to the action of Kepfer cells;
this could be due to this organ being a primary site of
metabolism as seen with radio-labeled dextran [89].
A suggestion was made by Kean and Thanou who proposed that a potential method to study native chitosan
without signicant modication would be to use 14 C as a
label e.g. in the food source for the animal/fungi producing the chitin so that the saccharide backbone is labeled, as
detection of native chitosan is appearing to be a challenge
[69].
To study the intraperitoneal administration of chitosan,
FITC-labeled chitosan (50% DD, 100 kDa) was prepared by
FITC coupling. It was observed that this labeled chitosan
was completely absorbed form the peritoneal cavity (no
evidence in abdominal uid after 14 h). FITC-chitosan was
found to be predominantly localised in the kidney at 1 h in
a mouse model. There was a rapid renal excretion rate (25%
at 1 h, 100% in 14 h) with evidence of degradation due to a
decrease in the molecular weight [76].
Some studies suggest that chitosan binds fat and
reduces cholesterol but the mechanism, is still somewhat questionable [43,90]. Apart from the effect that
chitosan may have on bile salts and gastrointestinal
milieu, the uptake of chitosan into the bloodstream is
generally not investigated in oral administration studies.
Molecular weight has been a parameter that largely inuences chitosans systemic absorption and distribution from
this route of delivery. It has been seen in some cases
that oligomers showed some absorption whereas larger
molecular weight chitosans were excreted without being
absorbed. This effect was investigated using FITC-labeled
chitosans with 3.8 kDa (88.4% DD) which was shown to
result into the greatest plasma concentration after oral
administration compared to 230 kDa (84.9% DD) for which
nearly no uptake was detected. In one of the studies aimed
at investigating plasma concentration after oral administration, the increasing molecular weight was seen to
decrease the plasma concentration [91].
Although intracellular uptake and distribution of native
chitosan have not been investigated, but chitosan/DNA
complexes have been studied in vitro [9294]. Chitosan
polyplex uptake at 37 C was 3-fold higher than at 4 C
which could be due to increased interaction and not an
ATP dependent endocytotic mechanism [92]. The authors

suggested nuclear localization and they also stated little
dissociation of the DNA from the chitosan. In a more extensive study, Leong et al. stained lysosomes and found some
co-localization with chitosan DNA nanoparticles. However,
the majority of the polyplexes were found in the cytosol
[93]. A complex of doxorubicin with chitosan was studied;
where is was observed that complexes enter cells through
an endocytotic mechanisms which was not further elucidated [95].
2.5. Chitosan toxicity
Chitosan is considered as being a non-toxic, biologically compatible polymer [96]. It is approved for dietary
applications in Japan, Italy and Finland [97] and it has been
approved by the FDA for use in wound dressings [98]. However, certain modications implemented on chitosan could
make it more or less toxic and any residual reactants should
be removed carefully.
2.5.1. In-vitro toxicity
In a series of studies, Schipper et al. observed the effects
of chitosan samples characterized by different molecular
weight and DD on CaCo-2 cells, HT29-H and in situ rat
jejunum. Toxicity was found to be dependent on DD and
molecular weight. At high DD the toxicity is related to the
molecular weight and the concentration, at lower DD toxicity is less pronounced and less related to the molecular
weight. Nevertheless, most of the chitosans tested did not
increase dehydrogenase activity signicantly in the concentration range tested (1500 g/ml) on Caco-2 cells. The
in situ rat jejunum study showed no increase in LDH activity
with any of the chitosan samples tested (50 g/ml) [45,99].
Red blood cell haemolysis assay is a study that reveals
safety of materials. No haemolysis was observed (<10%)
over 1 h and 5 h with chitosans of <5 kDa, 510 kDa and
>10 kDa at concentrations of up to 5 mg/ml [100]. Furthermore, no red blood cell lysis was observed with paclitaxel
chitosan micelles at 0.025 mg/ml [101].
Interestingly, chitosan and its derivatives seem to be
toxic to several bacteria [102], fungi [82] and parasites
[103]. This pathogen related toxicity is an effect which
could be benecial in the control of infectious disease. Bacterial inhibition took place in acidic solutions pH 55.3, and
a 87 kDa 92% DD chitosan was more effective than a 532 kDa
73% DD chitosan against both Pseudomonas aeruginosa and
Staphylococcus aureus. Antimycotic effect against Candida
albicans and Aspergillus niger was observed in a lipid emulsion of the same chitosans [102]. However, none of these
studies proposed a mechanism of action for the observed
inhibitory effect.
2.5.2. In vivo toxicity
In a study by Hirano et al. which was relatively
long (65 days), no detrimental effect on body weight
was found when chitosan oligosaccharides were injected
(7.18.6 mg/kg over 5 days). An increase in lysozyme activity was apparent on the rst day post injections [104].
Rao et al. stated no signicant toxic effects of chitosan
in acute toxicity tests in mice, no eye or skin irritation in
rabbits and guinea pigs respectively. He also concluded in

the same study that chitosan was not pyrogenic. However,
no concentration or DD of the chitosan used was noted
[65]. No detrimental effects were also noted by Richardson et al., even though there was mention of dose effect
in his work [100]. The LD50 of paclitaxel chitosan micelles
in mice was 72.2 mg/kg, no anaphylaxis was observed in
guinea pigs and no intravenous irritation was observed
histopathologically in rabbits at 6 mg/kg [101]. In a study on
fat chelation, 4.5 g/day chitosan (molecular weight and DD
not specied) in humans was not reported not to be toxic,
although no signicant reduction in fat was found [105].
Arai et al. found that chitosan has an LD50 comparable to
sucrose of >16 g/kg in oral administration to mice [106]. No
oral toxicity was found in mice treated with 100 mg/kg chitosan nanoparticles (80 kDa, 80% DD) [107]. Exposure of rat
nasal mucosa to chitosan solutions at 0.5% (w/v) over 1 h
caused no signicant changes in mucosal cell morphology
compared to control [108]. From most studies reported it
appears that chitosan shows minimal toxic effects and this
approves its adoption as a safe material in drug delivery.
3. Chitosan-based systems for biomedical
applications types and methods
Regenerative medicine offers the opportunity to drastically improve our ability to ght disease and to repair
damaged organs of the human body. It combines the
knowledge and skills of several disciplines towards the aim
of addressing impaired function in the body. It is important to note that the goal is not just to replace what is
malfunctioning, but to provide the elements required for
in vivo repair, to devise replacements that seamlessly interact with the living body and to stimulate the bodys intrinsic
capacities to regenerate [109].
3.1. Chitosan micro/nanoparticles
Chitosan offers several advantages, and these include
its ability to control the release of active agents and avoid
the use of hazardous organic solvents while fabricating
particles since it is soluble in aqueous acidic solution. In
view of the above-mentioned properties, chitosan is extensively used in developing drug delivery systems [110117].
Specically, chitosan has been used in the preparation
of mucoadhesive formulations [46,108,118,119], improv-
987
Table 2
Chitosan-based drug delivery systems prepared by different methods.
Type of system
Method of preparation
Tablets
Capsules
Microspheres
Matrix coating
Capsule shell
Emulsion cross-linking
Coacervation/precipitation
Spray-drying
Ionic gelation
Sieving method
Emulsion-droplet coalescence
Solution casting
Cross-linking
Nanoparticles
Beads
Films
Gel
ing the dissolution rate of the poorly soluble drugs

[113,120,121], drug targeting [122,123] and enhancement
of peptide absorption [108,118,124]. Different types of
chitosan-based drug delivery systems are summarized in
Table 2. In this section we have addressed the trends in the
area of micro/nanoparticulate chitosan-based drug delivery systems. Literature form past decade has been reviewed
and results are evaluated.
Different methods have been employed to prepare chitosan particulate systems. Selection of any of the methods
should take into consideration factors such as particle size
requirement, thermal and chemical stability of the active
agents, reproducibility of the release kinetic proles, stability of the nal product, residual toxicity associated with
the nal products, the nature of the active molecule as well
as the type of the delivery device [125].
3.1.1. Emulsion cross-linking
This method exploits the reactive functional amine
group of chitosan to cross-link with the available reactive groups of the cross-linking agent. In this method, a
water-in-oil (w/o) emulsion is prepared by emulsifying the
chitosan aqueous solution in the oil phase. A suitable surfactant is used to stabilize the aqueous droplets. Thereafter
stable emulsion is cross-linked by using an appropriate
cross-linking agent to harden the droplets. Microspheres
are ltered and washed repeatedly with alcohol and then
dried [126]. This method, is helpful in controlling the size
of the particles by controlling the size of aqueous droplets.
However, the particle size of nal product is dependent
on the extent of cross-linking agent used while hardening
Fig. 3. Schematic representation of preparation of chitosan particulate systems by emulsion cross-linking method.
988
Fig. 4. Schematic representation of preparation of chitosan particulate systems by coacervation/precipitation method.
along with the speed of stirring. This method is schematically represented in Fig. 3. The emulsion cross-linking
method involves a few drawbacks. Besides being tedious
it uses harsh cross-linking agents, which might possibly
induce chemical reactions with the active agent. Moreover,
complete removal of the unreacted cross-linking agent may
be a challenge.
Sankar et al. used this method to prepare the chitosanbased pentazocine microspheres for intranasal delivery.
Formulation parameters such as drug loading, polymer
concentration, stirring speed during cross-linking and oil
phase were modied to develop microspheres having good
in vivo performance. In vivo studies indicated a signicantly
improved bioavailability of pentazocine. In-vitro release
kinetic models indicated that these systems followed the
diffusion controlled release kinetics [127].
3.1.2. Coacervation/precipitation
The physicochemical property of chitosan is utilized in
this method since it is insoluble in alkaline pH medium,
but precipitates/coacervates when it comes in contact
with alkaline solution. Chitosan solution is blown into
an alkali solution like sodium hydroxide, NaOHmethanol
or ethanediamine using a compressed air nozzle to form
coacervate droplets. Separation and purication of particles are performed by ltration/centrifugation followed by
successive washing with hot and cold water. The method
is schematically represented in Fig. 4. Variation in compressed air pressure or spray-nozzle diameter can be done
to control the size of the particles. The drug release can be
controlled by using appropriate cross-linking agent.
This technique has been used to prepare chitosanDNA
nanoparticles [128]. Processing parameters such as concentrations of DNA, chitosan, sodium sulfate, temperature,
pH of the buffer and molecular weights of chitosan and DNA
have been investigated. The particle size was successfully
optimized to 100250 nm with a narrow distribution by
keeping the amino to phosphate group ratio between 3 and
8 and chitosan concentration of 100 g/ml. Surface charge
of these particles was slightly positive with a zeta potential of 112118 mV at pH lower than 6.0, and became nearly
neutral at pH 7.2. Results indicated that the nanoparticles
could partially protect the encapsulated plasmid DNA from
nuclease degradation.
3.1.3. Spray-drying
Spray-drying is a popular method to produce powders,
granules or agglomerates from the mixture of drug and
excipient solutions as well as suspensions. The method is
based on drying of atomized droplets in a stream of hot
air. Briey, chitosan is dissolved in aqueous acetic acid
solution, drug is then dissolved or dispersed in the solution followed by the addition of a suitable cross-linking
agent (Fig. 5). This solution or dispersion is then atomized
in a stream of hot air that leads to the formation of small
droplets, from which solvent evaporates instantaneously
leading to the formation of free owing particles [129].
Various process parameters should be controlled to get the
desired size of particles such as the size of nozzle, spray
ow rate, atomization pressure, inlet air temperature and
Fig. 5. Schematic representation of preparation of chitosan particulate

systems by spray-drying method.
989

systems by ionic gelation method.

systems by emulsion-droplet coalescence method.
extent of cross-linking. This method is however more commonly used for the preparation of microparticles than for
nanoparticles.
Huang et al. prepared betamethasone disodium phosphate chitosan microspheres by this method using type-A
gelatin and ethylene oxidepropylene oxide block copolymer poloxamer as modiers. Investigation of the surface
morphology and surface charges of the prepared microspheres were done which indicated that shape, size and
surface morphology of the microspheres were signicantly
inuenced by gelatin concentration. A good drug stability
(less 1% hydrolysis product), high entrapment efciency
(95%) and positive surface charge (37.5 mV) was observed.
In-vitro drug release from the microspheres was related to
gelatin content. The gelatin/chitosan ratio of 0.40.6 (w/w)
showed a fairly prolonged release up to 12 h [130].
3.1.4. Emulsion-droplet coalescence method
This emulsion-droplet coalescence method, developed
by Tokumitsu et al., which combines the principles of both
emulsion cross-linking and precipitation [131]. Instead of
cross-linking the stable droplets, precipitation is induced
by allowing coalescence of chitosan droplets with NaOH
droplets. Two separate emulsions are prepared, one containing aqueous solution of chitosan along with drug is
produced in liquid parafn oil, another containing chitosan
aqueous solution of NaOH is produced in the same manner. The emulsions are mixed under high-speed stirring,
droplets of each emulsion collide at random and coalesce, forming small sized particles that precipitate (Fig.
6). The particle size increased with the decrease in DD
of chitosan which in turn decreased the drug content.
Completely deacetylated chitosan produced particle size
of 452 nm with 45% drug loading. The efciency of this
method depends on the electrostatic interactions with the
amino groups of chitosan, which could not have occurred if
a cross-linking agent was used that blocked the free amino
groups of chitosan. Thus, it was possible to achieve higher

gadopentetic acid loading by using the emulsion-droplet
coalescence method that did not involve the use of any
cross-linking agent.
3.1.5. Ionic gelation
For the most part, complexation to prepare chitosan
microspheres has attracted much attention since the process is very simple and mild [132,133]. The reversible
physical cross-linking by electrostatic interaction, instead
of chemical cross-linking, decreases the potential toxicity impact of reagents and other undesirable effects.
For example, the polyanion, tripolyphosphate (TPP) is
a polyanion, which interacts electrostatically with the
cationic chitosan [114,134]. After Bodmeier et al. [135]
reported the preparation of TPPchitosan complex by
dropping chitosan droplets into a TPP solution, many
researchers have explored its potential pharmaceutical
usage [117,136140]. For ionic gelation, chitosan is dissolved in aqueous acidic solution which quaternizes the
chitosan amino groups making it soluble; this solution is
then added dropwise under constant stirring to polyanionic TPP solution. The complexation between oppositely
charged species, causes the chitosan to undergo ionic gelation and precipitate as spherical particles (Fig. 7).
Various formulations of chitosan nanoparticles produced by the ionic gelation of TPP and chitosan were
studied by Xu and Du [141]. The spherical shaped particles, 20 nm and 200 nm in size, were observed by TEM. The
factors that affected the release of bovine serum albumin
(BSA) as a model protein have been studied which include
molecular weight, DD and concentrations of chitosan and
BSA, as well as the presence of polyethylene glycol (PEG)
in the encapsulation medium.
3.1.6. Reverse micellar method
Reverse micelles are thermodynamically stable liquid
mixtures of water, oil and surfactant. These homogeneous and isotropic structured on microscopic scale
into aqueous and oil microdomains are separated by a
surfactant-rich lm. The dynamic behavior of these reverse
micelle systems provides very important characteristics.
990
Fig. 8. Schematic representation of preparation of chitosan particulate systems by reverse micellar method.
The nanoparticles prepared by conventional emulsion

polymerization methods are usually large (>200 nm), with
a broad size range. Ultrane polymeric nanoparticles with
narrow size distribution could be achieved by using reverse
micellar medium [142]. Due to the Brownian motion of
the micellar droplets, they undergo continuous coalescence followed by re-separation on a time scale that
varies between millisecond and microsecond [143]. A rapid
dynamic equilibrium maintains the size, polydispersity
and thermodynamic stability of these droplets. To prepare
reverse micelles, the surfactant is dissolved in an organic
solvent followed by the addition of chitosan and drug under
constant vortexing. To the transparent obtained solution, a
cross-linking agent is added with constant stirring, continued overnight. The maximum amount of drug that can be
dissolved in reverse micelles varies from drug to drug and

has to be determined by gradually increasing the amount
of drug until the clear microemulsion is transformed into
a translucent solution. This method is schematically represented in Fig. 8. Chitosan nanoparticles encapsulating
doxorubicindextran conjugate was prepared by reverse
micellar method [144]. The surfactant sodium bis(2-ethyl
hexyl) sulfosuccinate (AOT), was dissolved in n-hexane.
3.1.7. Sieving method
A simple, novel method to produce chitosan microparticles has been developed by Agnihotri and Aminabhavi
[145]. This method, is devoid of tedious procedures and
can be scaled up easily, microparticles are prepared by
cross-linking a 4% acetic acid chitosan solution to form
Fig. 9. Schematic representation of preparation of chitosan particulate systems by seiving method.
991
glassy hydrogels that are passed through a sieve (Fig. 9).

Clozapine (C18 H19 ClN4 ) was incorporated into chitosan gel
before cross-linking with 99% efciency. Irregular shaped
microparticles, 540700 m, were formed on seiving and
in vivo studies indicated a slow release of clozapine.
a sphere, the release mode is determined using equations

developed by Guy et al. [150].
The most commonly used equation for diffusion controlled matrix system is an empirical equation used by
Ritger and Peppas [151], in which the early time release
data can be tted to obtain the diffusion parameters,
3.1.8. Chitosan micro/nanoparticles drug loading and

release
Usually, drug loading in micro/nanoparticulate systems is achieved using one of the two following methods;
(a) incorporating the drug during the preparation of the
particles and (b) after the formation of the particles by
incubation the drug with them. In both systems, the
drug is physically embedded into the matrix as well as
adsorbed onto the surface. Both water-soluble and waterinsoluble drugs can be loaded by these techniques. The
water-soluble drugs are generally incorporated by mixing
with an aqueous chitosan solution to form a homogeneous
mixture, followed by particles production as described.
Water-insoluble drugs and drugs that precipitate in acidic
solutions are usually loaded by incubation which involves
soaking the pre-formed particles in a saturated solution
of drug [146]. Chitosan microspheres were loaded using
two different methods by Hejazi and Amiji [147]. In the
rst method, tetracycline was mixed with chitosan solution
and then simultaneously cross-linked and precipitated the
particles. In the second method, pre-formed microspheres
were incubated with the drug for 48 h. The accumulated
amount of tetracycline released and its stability was examined in different pH media at 37 C. When drug was added
to chitosan solution before cross-linking and precipitation, only 8% (w/w) was optimally incorporated into the
microsphere. But when the drug was incubated with the
pre-formed microspheres, 69% (w/w) could be loaded.
About 30% of tetracycline in solution or when released from
the microspheres was found to degrade at pH 1.2 in 12 h
[147].
The drug release from chitosan particulate systems usually follows three different, mechanisms: (a) release from
the surface of particles, (b) diffusion through the swollen
rubbery matrix and (c) release due to surface erosion.
In most cases, drug release follows more than one type
of mechanism. In case of release from the particle surface, adsorbed drug dissolves on contact with the release
medium; similarly, the drug entrapped onto the surface
layer of particles also follows this mechanism but perhaps
a little slower. These types of drug release lead to a burst
effect which is encountered in all delivery systems whereby
drug onto the surface is rapidly taken up followed by a
slower diffusion release from the inner matrix of the particle. Diffusion controlled drug release occurs in three steps; (a)
water penetrates into the particulate system, (b) the glassy
matrix becomes rubbery and swells, and (c) the drug diffuses from the swollen matrix. Hence, this type of release is
initially slow but becomes faster as the drug dissolves and
the matrix swells.
The Higuchi equation is one of the traditional methods
which was used to describe the release of a solute from a
at surface [148], but not from a sphere [149]. A good t
indicates that the release rate is dependant on the rate of
diffusion through the matrix. To describe the diffusion from
Mt
= kt n
M
(1)
where, Mt /M is the fractional drug released at time t, k is

a constant characteristic of the drugpolymer interaction
and n is an empirical parameter characterizing the release
mechanism.
The drug transport can be classied as Fickian behavior
(n = 0.5), Case II transport (n = 1), non-Fickian or anomalous
behavior (0.5 < n < 1) and super Case II (n > 1), based on the
diffusion exponent [152].
The dynamic swelling data for chitosan microparticles was analysed by Agnihotri and Aminabhavi using
equation (1) to predict drug release from microparticles
cross-linked with 5.0, 7.5 and 10.0 104 ml of glutaraldehyde/mg of chitosan [145]. It was found that the swelling
of chitosan microparticles decreased as the cross-linking
increased. And the values of n < 0.5 were due to the irregular shaped particles and that n decreased systematically
with increasing cross-linking.
A good correlation for the cumulative drug released
vs. the square root of time was obtained by Jameela et
al. [153]; this indicated that drug release from the microsphere matrix is diffusion controlled and obeys the Higuchi
equation [148]. In addition, the smaller sized microspheres
released drugs faster than the large microspheres due to
their relative greater surface area and that the duffusion
path length was shorter from the smaller particles into the
dissolution medium.
As stated earlier, the release of a specic drug from
chitosan-based particulate systems depends upon several
factors, such as the cross-linking, morphology, size and
density of the particles used. Other considerations involve
the physicochemical properties of the drug as well as the
presence of adjuvants. In-vitro release also depends on
pH, polarity and presence of enzymes in the dissolution
media.
3.2. Chitosan hydrogels
Hydrogels are composed of three-dimensional polymer
networks that can absorb large quantities of water. Consequently, they are soft, pliable, wet materials with a wide
range of potential biomedical applications. Hydrogels are
widely used in bioapplications and play a crucial role in
current strategies to remedy malfunctions in and injuries
to living systems. The high water content of hydrogels renders them compatible with most living tissue and their
viscoelastic nature minimizes damage to the surrounding tissue when implanted in the host. In addition, their
mechanical properties parallel those of soft tissue, making
them particularly appealing with the host tissues, assisting
and improving the healing process, and mimicking functional and morphological characteristics of organ tissue.
Chitosan hydrogels can be prepared with a variety of ways.
992
actions including hydrogen bonding between chitosans

hydroxyl groups and the ionic molecules, or interactions
between deacetylated chitosan chains after neutralization
of their cationic charge [159,162]. These interactions often
enhance certain physical properties of the hydrogel, which
can be modulated to express unique material properties,
such as pH sensitivity.
Fig. 10. Schematic representation of chitosan hydrogel networks derived

by physical associations: networks formed with ionic molecules, polyelectrolyte polymer and neutral polymers [161]. Copyright 2009, Elsevier
Ltd.
In each process, chitosan is either physically associated or

chemically cross-linked networks to form hydrogel.
3.2.1. Physical association networks
To construct a stable hydrogel, the chitosan polymer
network must satisfy two conditions: (a) interchain interactions must be strong enough to form semi-permanent
junction points in the molecular network and (b) the network must be capable of exchanging water molecules with
the hydrated polymer network. The chitosan hydrogels
that meet these basic requirements may be prepared by
non-covalent strategies that take advantage on electrostatic, hydrophobic, and hydrogen bonding forces between
polymer chains [154,155]. The schemes of four major
physical interactions ionic, polyelectrolyte, interpolymer
complex, and hydrophobic associations that lead to the
gelation of a chitosan solution are in Fig. 10. Since all of
these are purely physical interactions, gel formation can be
reversed. Swelling behavior can be tuned by adjusting the
concentration and nature of the component(s) used during
the fabrication process. However, the number of applications of chitosan hydrogels is limited due to the weak
mechanical strength and uncontrolled dissolution [156].
3.2.1.1. Ionic complexes. Ionic complexes are readily
formed by ionic interactions between the cationic chitosan and negatively charged molecules, such as sulfates,
citrates, and phosphates ions [157,158] and anionic metals
Pt(II), Pd(II), and Mo(VI) [159,160]. These interactions can
be manipulated to form hydrogels with varying structural
properties that depend upon the charge density and
size of the anionic agents, as well as chitosan DD and
concentration [161].
Anions and small molecules bind chitosan via its
protonated amino groups, whereas metal ions form
coordinatecovalent bonds with the polymer instead of
electrostatic interactions [159,160]. Ionic complexation is
usually accompanied by other secondary interchain inter-
3.2.1.2. Polyelectrolyte complexes (PEC). Chitosan electrostatic interactions with polyelectrolytes, are different from
the ionic complexes in that they are larger molecules. The
interactions between the chitosan and polyelectrolytes are
stronger than other secondary binding interactions like
hydrogen bonding. This type of complexes avoids the use
of organic precursors, catalysts, or reactive agents, alleviating the concern about toxicity or reactions with a
therapeutic payload. Since, PEC only consist of chitosan
and the polyelectrolyte, their complexation is generally
straightforward and reversible [163]. Chitosan-based PEC
networks have been produced with DNA, anionic polysaccharides such as alginate, glucosamineglycans, chondroitin
sulfate, hyaluronic acid, heparin, carboxymethyl cellulose,
pectin, dextran sulfate, xanthan [164166], proteins such
as gelatin, albumin, broin, keratin, and collagen [167169]
and synthetic anionic polymers such as (polyacrylic acid)
[170]. The stability of these complexes is dependent on
charge density, solvent, ionic strength, pH, and temperature [171]. The charge of the anionic molecule under
physiological conditions is an important factor for PEC to
occur; since the pH of the hydrogel environment modies
the ionic interactions, in vivo environment must be considered when using PEC for hydrogels.
3.2.1.3. Physical mixtures and secondary bonding. In addition, hydrogels are formed by polymer blends between
chitosan and other water-soluble nonionic polymers, such
as poly(vinly alcohol) (PVA). After lyophilization or a series
of freezethaw cycles, these polymer mixtures form junction points in the form of crystallites and interpoymer
complexation [154,172]. The chainchain interactions perform as cross-linking sites of the hydrogel formation. In
the case of chitosanPVA polymer blends, increasing the
chitosan content negatively affects the formation of PVA
crystallites, leading to the formation of poorer hydrogel
structures. Chitosan is also capable of forming hydrogels by
itself without the use of any polymer or complexing agent.
A chitosan hydrogel using a hydro-alcohol gel formation
process based on the neutralization of chitosans amino
groups using a sodium hydroxide solution was prepared
by Ladet et al. [173]. This process inhibited ionic repulsion
between the polymer chains that allowed the formation
of hydrogen bonds, hydrophobic interactions, and chitosan
crystallites to form the necessary cross-links for hydrogel
formation (Fig. 11). An interrupted gelation method led to
multilayered onion-like hydrogels (Fig. 11); this unique
feature is being explored to encapsulate drugs to co-deliver
multiple therapeutics and for pulse-like delivery of a given
payload [174,175].
3.2.1.4. Thermoreversible hydrogels and hydrophobic associations. Thermoreversible hydrogels and hydrophobic
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Fig. 11. (a) Multi-membrane biomaterial with onion-like structure based on chitosan hydrogel. (b) Schematic diagram of the multi-membrane onionlike structures. (c) Variation of hydrogel shrinkage during neutralization as a function of the concentration of sodium hydroxide (the initial polymer
concentration in the non-neutralized alcohol gel is constant and close to 4.5 wt.% in each case). (d) Evolution of the chitosan mass fraction in the gel (WCH)
as a function of the NaOH neutralization [173]. Copyright 2008, Nature Publishing Group.
association form an interesting class of hydrogels that are

transient gels or in liquid states depending upon the environmental temperature. These hydrophobic and secondary
bonding interactions develop junctions between the polymer chains that form semi-rigid gels from a owable liquid
solution. When temperature of the system is below the
lower critical solution temperature (LCST), the material
undergoes a hydrophilichydrophobic transition. Thus, a
polymer solution that has a low viscosity at room temperature, can form a gel above the LCST; this is very signicant
as these materials can be injected into the body as a liquid below LCST and then it forms a gel in situ at body
temperature which is above the LCST. This carrier matrix
application is being used in a wide range of biomedical
and pharmaceutical applications [176,177]. The injectable
gelling systems can be introduced into the body without
the need for invasive surgery and deliver the bioactive
agents to the defect site without signicant side effects.
The hydrogels prepared by aggregation of chitosan-based
copolymers or by neutralization with polyol salts show
promising thermoreversible gelation properties in aqueous
media [178182]. The latter strategy uses the temperaturesensitivity of glycerol phosphate disodium salt (GP) and
chitosan physical mixture. The phosphates of the GP salt are
believed to neutralize the ammonium groups of chitosan,
thus increasing the hydrophobic and hydrogen bonding
between the chitosan chains at elevated temperatures
[182].
3.2.2. Cross-linked networks

Although physically bonded hydrogels have the advantage of gel formation without the use of cross-linking
moieties, they have certain limitations. For example, it is
difcult to accurately control the physical gel pore size,
chemical functionalizations, dissolution and degradation.
Consequently they often provide inconsistent in vivo performance. Better chitosan hydrogel mechanical properties
can be produced using irreversible networks. To form
the hydrogel, the polymer chains are covalently bonded
together by small cross-linker molecules, secondary
polymerizations, or irradiation. Covalently cross-linked
hydrogels are also obtained by attaching photo-reactive
or enzyme-sensitive molecules on the chitosan, followed
by their subsequent exposure to UV or sensitive enzymes,
respectively. The properties of cross-linked hydrogels
depend mainly on their cross-linking density and the ratio
cross-linker molecules to the moles of polymer repeating
units [183]. The different method to make irreversible chitosan hydrogels is described below.
3.2.2.1. Chemical cross-linking. Chemical cross-linking is
the most straightforward method to produce permanent
hydrogel networks using covalent bonding between the
polymer chains. The available NH2 and OH groups on
chitosan are active sites capable of forming a number
of linkage, including amide and ester bonding as well
as Schiff base formation [162,184,185]. These networks
994
can be formed by using small molecule cross-linkers,

polymerpolymer reactions between activated functional
groups, photosensitive agents and enzyme-catalyzed reactions.
The new cross-linking agent, genipin (C11 H14 O5 ), is a
naturally derived chemical that can bind biological tissues
and biopolymers through covalent coupling [185187].
Genipin is particularly effective for cross-linking polymers
containing amino groups. In addition it is less toxic and
degrades slower than agents like glutraldehyde [188,189].
Genipin also provides extended drug release by chitosan
hydrogels cross-linked in situ [179,190]. Although, genipin
has good biocompatibility, it is susceptible to interact with
the encapsulated drugs; this can be a problem for gelation
in the presence of a therapeutic agent [161].
A thermo-sensitive, chitosanpluronic hydrogel was
also produced using ultraviolet (UV) photo-cross-linking
[191]. The UV exposure cross-links chitosan and pluronic
groups functionalized with photosensitive acrylate groups.
These systems form physical networks at temperatures above the LCST. Thermo-sensitive hydrogels
have been used for sustained release of encapsulated
human growth hormone (hGH) and plasmid DNA and
exhibit potential application for different types of drugs
[191,192].
3.2.2.2. Interpenetrating networks (IPN). Entangled polymer networks within the cross-linked networks can be
strengthened by interlacing them with secondary polymers. In this case, a cross-linked chitosan network is
allowed to swell in an aqueous solution of monomers.
When these monomers are polymerized, a physically
entangled polymer mesh is formed called an interpenetrating (IPN) network. A semi-IPN involves one
cross-linked polymer network with another polymer
in the linear states. There are several chitosan-based
semi-IPNs reported in literature prepared with polyether
[193,194], silk [195], polyethylene oxide (PEO) [196], and
polyvinylpyrrolidine (PVP) [197] and full-IPNs (prepared
with poly(N-isopropylacrylamide) (PNIPAM) [198]).
This technique has the advantage of specically selecting polymers that can complement the deciencies of one
another. Based on the target application, cross-linking density, hydrogel porosity, and gel stiffness can be adjusted
in IPN-based hydrogels; however it is more difcult to
encapsulate a wide variety of therapeutic agents, especially sensitive biomolecules. IPN preparations generally
involve the use of toxic agents to initiate or catalyze
the polymerization or the cross-linking; their complete
removal is very difcult, making the clinical application a
concern.
3.2.3. Chitosan hydrogels drug loading and release
The drug loading in a hydrogel depends upon the physical and chemical properties of the gel as well as from the
structural features of the therapeutic agent. Three major
approaches to drug loading can be summarized as: diffusion, entrapment, and tethering [199203]; each method
has advantages and disadvantages. The easiest drug loading
method is to place the fully formed hydrogel into a medium
saturated with the therapeutic agent [204,205]. The drug
slowly diffuses into the gel depending upon the porosity of the hydrogel, the size of the drug and the chemical
properties of each, such as hydrophobicity/hydrophilicity.
When placed in vivo, the drug diffuses out of the hydrogel
into the neighboring tissue. This technique has proved to
be effective in loading small molecules, but larger therapeutics peptides and proteins, in particular, do not readily
able to migrate through the small pores of the hydrogel
[161]. However, this process is also very time consuming
and, therefore, it is not recommended for manufacturing
purposes. Therefore, in the case of large sized drugs and
bioligands, it is preferable to have the drug entrapped
during the gelation process. Usually, the drug is mixed
in a polymer solution, and a cross-linking or complexation agent is added. The chemistry of the drug molecule
must be considered to prevent unwanted cross-linking or
deactivation of the therapeutic agent during gelation. Free
movement of the therapeutic agent out of the hydrogel
network is takes place in both diffusion and entrapment
systems. This process often leads to an initial burst release
after implantation of the hydrogel in vivo due to the
concentration gradient formed between the gel and the
surrounding environment.
To reduce the loss of the therapeutic reserve and the
risk of toxic exposure, drugs usually covalently or physically are linked to the polymer chains prior to gelation.
This technique is termed as tethering which limits tissue
exposure to the agent only when the hydrogel breaks down
or the molecular tether is broken [184]. Drug loading can
become complicated by molecules that have the opposite
salvation features or similar charge as the continuous polymer matrix [206]. An alternative in such cases is to form
a complex with amphiphilic additives before the hydrogel and drug are blended in solution [207,208]. This has
been accomplished by binding paclitaxel (C47 H51 NO14 ) to
albumin (Abraxane) or by mixing it in an aqueous citric acid/glyceryl monooleate solution prior to hydrogel
loading [208]. Therapeutic agents can also been loaded
into small secondary release vehicles, such as microparticles, microgels, liposomes, and micelles, prior to hydrogel
encapsulation [209,210].
The release of a therapeutic from a hydrogel can
follow one of three different modes: diffusion, chemical/environmental stimulation, or enzyme-specic stimulation [211]. Diffusion is regulated by the movement of the
drug through the polymer matrix or by bulk erosion of the
hydrogel carrier as it breaks down in vivo. Environmentally responsive hydrogels swell in response to external
conditions, such as pH and temperature that effectively
open the pores to enhance diffusion of the entrapped
therapeutic under predetermined conditions [161]. This
type of controlled release is used to limit drug release
outside of the effective range of the diseased tissue. The
release of a drug payload can also be triggered by local
enzymatic action. These biochemically responses occur
by tethering drugs to the hydrogel via labile domains
that are susceptible to enzymes or using polymers that
are targeted by enzymes [212]. This method though not
widely used offers selective, sustained release mechanisms
is beginning to receive attention from chitosan hydrogel
engineers.
4. Biomedicalpharmaceutical applications
4.1. Chitosan for tissue engineering applications
Tissue engineering is a highly interdisciplinary eld
that combines the principles and methods of life sciences
and engineering to utilize structural and functional relationships in normal and pathological tissue to develop
biological substitutes to restore, maintain, or improve
biofunction [213]. It involves the in vitro seeding and proliferation of relevant cells in a scaffold support. Since it
is biodegradable, non-toxic and can be formulated in a
variety of forms including powders, gels and lms for applications, it has a wide range of potential applications in
tissue engineering. In addition to being implied in the formulation of controlled delivery systems a wide range of
chitosan modications can be made to improve the cell
seedings. Many tissue analogs including cartilage, bone,
liver, and nerve have been prepared using this engineering
technology.
4.1.1. Chitosan in bone tissue engineering
Chitosan has been extensively used in bone tissue engineering, since it was shown to promotes cell growth and
mineral rich matrix deposition by osteoblasts cells in culture [214]. The biocompatibility of chitosan minimizes
additional local inammation, and it can be molded into
porous structures to allow osteoconduction [215]. Several studies have focused on chitosancalcium phosphate
(CP) composites for this purpose [216218] with a 3D
macroporous CP bioceramic embedded with porous chitosan sponges being developed by Zhang and Zhang [219].
This nested chitosan sponge enhances the mechanical
strength of the ceramic phase through matrix reinforcement and preserves the osteoblast phenotype [220]. The
macroporous chitosan scaffolds incorporating hydroxyapatite (HA) or CP glass had an interconnected porosity of
approximately 100 m and are used for clinical applications [221]. Hu et al. prepared a chitosanHA multilayer
nanocomposite with high strength and bending modulus
rendering the material suitable for possible application for
internal xation of long bone fractures [222]. A series of
chitosan- tricalcium phosphate (TCP) composite scaffolds
were developed for bone tissue engineering using freezedrying process which provide macroporous composite
scaffolds with different pore structures. Compressive properties were improved, especially compressive modulus
from 4 MPa to 11 MPa. The biocompatibility, evaluated subcutaneously on rabbits indicated that these scaffolds can
be utilized in non-loading bone regeneration [223]. Chitosan is also used as an adjuvant with bone cements to
increase their injectability while keeping the chemicophysical properties suitable for surgical use with respect to
setting time and mechanical properties [224]. The choice of
chitosan for this purpose is based on the property that chitosan solutions tend to gel in response to a pH change from
slightly acidic to physiological environment. It is important
to note that, the chitosanCP composites address the need
to develop bone llers that set in response to physiological
conditions, but not while mixing the components in vitro
[221].
995
Xu et al. studied the feasibility of creating macropores

in calcium phosphate cement (CPC) using chitosan and/or
absorbable mesh. This injectable, bioabsorbable composite
material formed interconnected macropores (osteoconductive) and provided strength to the implant during
tissue regeneration [225]. Zhao et al. used phase separation
technique to fabricate biomimetic HA/chitosangelatin
network composites in the form of 3D-porous scaffolds
that improved adhesion, proliferation and expression of
rat calvaria osteoblasts on these highly porous scaffolds [226]. Kim et al. showed the application of this
property through composites of chitosan with poly methylmethacrylate (PMMA). This specially developed composite
material exhibited lower exothermic curing temperatures and possessed higher interconnected porosity with
a pore size suitable for osteoconduction with better
anchorage to the surrounding bone. It was observed that
the pore size of this composite material increased with
time due to biodegradation of the chitosan [227]. Chitosan is also used to modify the surface properties of
prosthetic materials for the attachment of osteoblasts
[228,229].
Recently, Zhao et al., using a scaffold with CPC and
chitosan bers, harvested human umbilical cord mesenchymal stem cells (hUCMSCs) without an invasive
procedure that is commonly required when studying bone
marrow mesenchymal stem cells (MSCs). The objectives
were to develop CPC scaffolds with improved resistance
to fatigue and fracture, for the delivery of hUCMSC aimed
at bone tissue engineering. In fast fractures, CPC with
15% chitosan and 20% polyglactin [(C2 H2 O2 )m (C3 H4 O2 )n ]
bers (CPCchitosanber scaffold) had exural strength
of 26 MPa, while CPC control was 10 MPa. The hUCMSCs showed excellent viability when seeded in CPC and
CPCchitosanber scaffolds, the live cell retention was
9699%. The cell density was about 300 cells/mm2 on day
1; and with proliferation 700 cells/mm2 at day 4 (Fig. 12).
Wst-1 assay showed that the stronger CPCchitosanber
scaffold had hUCMSC viability that matched the CPC control
(p > 0.1). This study indicated that chitosan and polyglactin
bers substantially increased the fatigue resistance of CPC,
and that hUCMSCs had excellent proliferation and viability
on the scaffolds [230].
A robotic desktop rapid prototyping (RP) system to
fabricate scaffolds for tissue engineering applications
was designed by Ang et al. The set-up consisted of a
computer-guided desktop robot and a one-component
pneumatic dispenser. The dispensing material, chitosan
and chitosanhydroxyapatite (HA) dissolved in acetic acid,
was forced out through a small Teon lined nozzle into a
dispensing sodium hydroxideethanol medium. Layer-bylayer, the chitosan was fabricated with a pre-programmed
lay-down pattern. The attachment between layers allowed
the chitosan matrix to form interconnected channeled
architectures. The in vitro osteoblast cell culture studies revealed the biocompatibility of the scaffolds with
cells exhibiting healthy morphology and avid proliferation throughout the culture period. The rapid prototyping
robotic dispensing (RPBOD) system is capable of fabricating
three-dimensional (3D) scaffolds with regular and reproducible macropore architecture [231].
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Fig. 12. (1) hUCMSCs were cultured on CPC control and CPCchitosanber for 1, 4, and 8 days: (A) percent of live cells, and (B) live cell attachment
(mean sd; n = 5). PLive reached 9699%, not different from each other (p > 0.1). CAttach was less than 300 cells/mm2 at day 1; it more than doubled to
700 cells/mm2 at day 4, due to hUCMSC proliferation. In (B), dissimilar letters indicate values that are signicantly different (p < 0.05). (2) SEM of hUCMSC
attachment on: (A) CPC control, and (B) CPCchitosanber scaffold. Cells are designated as C, which anchored to CPC in (A), and to the bers in the scaffold
in (B). Cells developed long, cytoplasmic extensions E, shown in (C) at a higher magnication, attaching rmly to the ber in the CPCchitosanber
scaffold [230]. Copyright 2010, Elsevier Ltd.
4.1.2. Chitosan in cartilage tissue engineering

The choice of biomaterial is very critical for the success
of tissue engineering approaches, especially in cartilage
repair [232]. The ideal cell-carrier substance should mimic
the natural environment in the articular cartilage matrix.
The cartilage-specic extracellular matrix (ECM) components such as type II collagen and GAGs, play a critical
role in regulating expression of the chondrocytic phenotype and in supporting chondrogenesis in vitro and in vivo
[233,234].
Three-dimensional (3D) scaffolds are especially important for fabricating articular cartilage. Ideal scaffolds are
designed to be biocompatible, bioabsorbable and exhibit
predictable porosity and degradation rate. They provide
a framework that facilitates new tissue in growth; moreover and mechanical characteristics that match those of the
native tissue; this increases the chances that the reparative

process will be compatible with the hosts tissue physiology
[235,236]. Chitosan was chosen as a scaffolding material in
articular cartilage engineering due to its structural similarity with various GAGs found in articular cartilage [25,237].
This is of importance since GAGs are considered to play
a pivotal role in modulating chondrocytes morphology,
differentiation, and function [238]. An alginatechitosan
hybrid based on polymer bers, that increased cell attachment and proliferation in vitro compared to alginate was
reported by Iwasaki et al. [239]. These hybrid polymer
bers showed increased tensile strength, implying a possible use in developing a 3D load-bearing scaffold for
cartilage regeneration. Chondrocytes cultured on chitosan
substrates in vitro maintained a round morphology and
cell-specic ECM [237,240]. Chitosan modied PLLA sub-
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Fig. 13. (a) Accumulation of matrix in the tissue-engineered cartilage. Histology and immunohistochemistry of chondrocytes cultured in chitosan hydrogels
3 weeks. The results showed that the chondrocytes in the chitosan hydrogels accumulated pericellular sulfated GAG-containing matrix. A, H.E. staining;
B, type II collagen immunohistochemical staining; C, toluidine blue staining; D, safranin O staining. Star: chitosan hydrogel; arrowhead: cell nucleus;
arrow: matrix of the chondrocytes. Bar = 100 m. (b) Gross observation of the articular cartilage repair at 24 weeks post-operation. A, the defect part of
the cartilage in the experimental group was covered by consistently smooth, glistening white hyaline tissue nearly indistinguishable from the surrounding
normal cartilage. No clear signs of margin with normal cartilage could be spotted on the surface of the regenerated areas; B, the defects in control group 1
were partially repaired with ber-like tissue, leaving a small depression in the defect areas; C, the defects in control group 2 detected a thin and irregular
surface tissue, with obvious defects and cracks surrounding the normal cartilage. Arrow: the defect; Bar = 0.5 cm [252]. Copyright 2010, Elsevier Ltd.(For
interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)
strate showed increased cell adhesion, proliferation and

biosynthetic activity [241]. Chondrocyte adhesion, proliferation, and the synthesis of aggrecan and type II collagen
were signicantly higher on the hybrid ber than on
chitosan [242]. Similarly, chitosanalginatehyaluronan
complexes increase the cellular adhesiveness with or without covalent attachment with arginineglycineaspartic
acid (RGD) containing protein. When chondrocytes seeded
scaffolds were implanted into rabbit knee cartilage defects,
partial repair was observed after 1 month both in presence
or absence of RGD indicating potential of this composite
material for cartilage regeneration [243].
Chitosan-based scaffolds deliver growth factors in a
controlled fashion to promote the in-growth and biosynthetic ability of chondrocytes. Lee et al. [244] reported
porous collagen/chitosan/GAG scaffolds loaded with transforming growth factor (TGF)-1. This scaffold exhibited
controlled release of TGF-1 and promoted cartilage regeneration. The addition of chitosan to the collagen scaffold
improved the mechanical properties [244] and the stability
of the collagen network by inhibiting the action of collagenases [245]. Kim et al. used a porous freeze-dried chitosan
scaffold incorporating TGF-1-containing microspheres,
for the treatment of cartilage defects [246]. The TGF-1 was
released in a sustained fashion, and promoted chondrocyte proliferation and matrix formation. In a similar trial, Lu
et al. studied the effect of intraarticular injection of chitosan
on regeneration of articular cartilage. An increase in epiphysis cartilage in the tibial and femoral joints was seen with
an activation of chondrocytes proliferation. Similarly, an
intra-articular brous tissue was observed for the 6 weeks
of the experiment, together with residual injected chitosan
[247].
A noteworthy accomplishment was achieved by Hoemann et al. who showed that microfractured ovine
defects are repaired with more hyaline cartilage when
the defect is treated with in situ-solidied implants of
chitosanGP mixed with autologous whole blood, compared to microfracture alone in an ovine model at 6 months
[248]. Since bleeding has been identied as an initiating event in post-surgical repair, it was hypothesized that
microfracture-based repair could be improved by stabilizing the clot formed in the lesion with chitosan that is
thrombogenic and actively stimulates the wound-healing
process. These chitosanGP/blood clots are adhesive and
contract much less than whole blood clots, thereby maintaining a voluminous scaffold [248]. ChitosanGP/blood
implants were applied to marrow-stimulated chondral
defects in rabbit cartilage repair models [249], where they
induced greater ll of chondral defects with repair of tissue
compared to marrow-stimulation alone [248]. In addition,
a more cellular and hyaline repair cartilage was produced
with a porous subchondral bone structure [248251].
A chitosan hydrogel in the form of a scaffold was
prepared for chondrocyte cells to reconstruct tissueengineered cartilage and repair articular cartilage defects
in the sheep model was reported by Hao et al. [252]. In this
study, temperature-responsive chitosan hydrogels were
prepared by combining chitosan, GP and hydroxyethyl
cellulose (HEC). The in vitro tissue-engineered cartilage
reconstructions were made by mixing sheep chondrocytes
with a chitosan hydrogel. Cell survival and matrix accumulation analysis were done after 3 weeks in culture (Fig. 13).
For in vivo repair, reconstructions cultured for 1 day and
transplanted to the freshly prepared defects of the articular cartilage of sheep. The cultured chondrocytes survived
and retained their ability to secrete. Thus in vivo transplantation repaired cartilage defects completely within 24
weeks (Fig. 13). This study showcased the success of a new
technique in its ability to repair articular cartilage defects.
4.1.3. Chitosan in liver tissue engineering
Due to the insufcient donor organs for orthotopic
liver transplantation the need for new therapies for
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Fig. 14. (A) SEM images of hepatocytes cultured within fructose-modied chitosan scaffold: (a) low-magnication view showing the higher density of
hepatocytes and cell aggregates; (b) high-magnication of the microvilli on cell surface and the cell morphology. (B) Urea synthesis of hepatocytes cultured
within different scaffolds. (Each time point represents the mean standard deviation of the mean of three experiments) [260]. Copyright 2003, Elsevier
Ltd.
acute and chronic liver disease is critical [253]. Currently,

bioarticial liver (BAL) is a promising application of tissue engineering for the treatment of fulminant hepatic
failure (FHF). An important issue for BAL devices is the
proper choice of cell sources, such as primary hepatocytes, hepatic cell lines, and liver stem cells [254]. The
primary hepatocyte of these cells represent the most
direct approach to BAL devices however hepatocytes
are anchor dependent cells and are highly sensitive to
the ECM milieu for the maintenance of their viability
and differentiation functions [255257]. The structural
similarity of chitosan to GAGs and since GAGs are components of the liver ECM, chitosan was selected as a scaffold
material for hepatocytes culture [258261]. Wang et al.
prepared chitosan/collagen matrix (CCM) by cross-linking
agent
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride (EDC) in N-hydroxysuccinimide (NHS)
buffer system [261]. The cross-linked matrix had moderate mechanical strength, good hepatocyte compatibility
as well as excellent blood compatibility. However,
implantable bioarticial liver (IBL) has severe complexities, unlike those for bioarticial skin, bone and cartilage.
Since thrombus formation can lead to occlusion and
decrease membrane efciency, special designs of the
complex architecture, as well as the anti-thrombogenic
extracellular component, were necessary to develop this
blood-contact device [254]. A superior blood compatible
chitosan/collagen/heparin matrix in implantable bioarticial liver (IBL) applications was, subsequently, developed
by Wang et al. [262].
Multivalent galactose residues to bind to the asialoglycoprotein receptor (ASGPR) expressed on the surface
of hepatocytes is another strategy for liver tissue engineering [263,264]. Typical cellmatrix interactions are
mediated by an adhesion receptor like integrin which
specically binds RGD sequence [265]. The ASGPR was the
rst reported mammalian lectin, or carbohydrate-binding
protein discovered [266,267]. The hepatic ASGPR is a classical system for studying receptor-mediated endocytosis.
Chung et al. reported the potential ability to improve hepa-
999
Fig. 15. Optical microscope longitudinal view of (a) a chitin hydrogel tube and (b) a chitin gel tube reinforced with a PLGA coils embedded in the wall
[280]. Copyright 2005, Elsevier Ltd.
tocyte attachment to alginate (AL)/galactosylated chitosan

(GC) scaffolds for short-term culture [268] while Seo et al.,
showed enhanced hepatocyte functions in AL/GC scaffolds
[269]. The study was done for long periods and hepatocytes
cultured enhanced function through its spheroid formation
in co-culture condition with broblast cells.
Concurrently, fructose known as a specic ligand of
ASGPR in hepatocyte, was conjugated, by Li et al., on porous
chitosan scaffolds by forming Schiffs bases. The chitosan
surface modied with fructose induced the formation of
cellular aggregates and enhanced liver specic metabolic
activities and cell density to a satisfactory level (Fig. 14)
[259,260].
Chitosan micro/nanobers can be fabricated by chemical and electrospinning techniques, however, Lee et al.
developed a microuidic-based pure chitosan microbers
for liver tissue engineering applications without the use of
any chemical additives [270]. To evaluate the capability of
the microbers, hepatoma HepG2 cells were seeded onto
the chitosan microbers. The HepG2 cells self-aggregated
forming spheroids that had a higher liver function that was
conrmed by albumin secretion and urea synthesis. This
method represents a potentially useful tool for liver tissue
engineering applications.
4.1.4. Chitosan in nerve tissue engineering
Once the nervous system is impaired, its recovery is difcult and malfunctions in other parts of the body generally
occurs [271]. Nerve injuries complicate successful rehabilitation since mature neurons like many other cells in the
body do not undergo cell division. The goal to repair nerve
lesions is to direct the regenerating nerve bers into the
proper endoneurial tubes. The current strategies can be
classied into two categories: (a) bridging, using grafting
and tubulization techniques and (b) end-to-end suturing
of the nerve stumps. The former technique seems to be
more effective, as it avoids tension across the repair site
[272]. A wide variety of biocompatible, non-degradable
and degradable materials have been suggested for the production of articial tubes for nerve repair. However, the
articial tubes have been suggested for the production
of articial tubes for nerve repair. However, the articial tubes lack sufcient internal surface area for nerve
bers and Schwann cells (SCs) to cohere [254]. For articial tubes to bridge large defects in nerve repair the
biodegradable matrix should provide a cellular, and molecular framework for SCs and neurite migration across the
nerve gap. Chitosan is suitable for nerve regeneration based
on its biocompatibility and biodegradability. Haipeng et al.
reported that neurons cultured on the chitosan membrane
can grow well and that chitosan tube can promote repair
of the peripheral nervous system [273]. Yuan et al. found
that chitosan bers supported the adhesion, migration
and proliferation of SCs, which provide a similar guide
for regenerating axons to Bngner bands in the nervous
system [274]. A novel biomaterial for nerve regeneration
through immobilization of laminin peptide in molecularly aligned chitosan by covalent bonding was designed
by Matsuda et al. [275]. Progestrone delivered from chitosan prostheses were reported by, Chavez-Delgado et al.,
to provide better facial nerve regenerative response of
the rabbits than chitosan prostheses without progesterone
[276]. An improved attachment, differentiation and growth
on the chitosan/poly(l-lysine) composite materials when
compared to cells cultured on chitosan membranes was
found by Mingyu et al. The improved nerve cell afnity on the chitosan/poly(l-lysine) composite materials
was attributed to the increased hydrophilicity by the
hydroxyl groups and the positive surface charge of chitosan
[277].
Gelatin added by Cheng et al. provided a soft and elastic
complex that has good nerve cell afnity. The composite
lm exhibited a lower modulus with a higher percentage of
elongation at break compared to chitosan lms. In addition,
PC12 cells cultured on the composite lms differentiated
more rapidly and with longer neurites than on chitosan
lms [278,279]. Using mold casting techniques Frier et al.
developed chitin hydrogel tubes by using no toxic crosslinking agent (Fig. 15). Both chitin and chitosan indicated
nerve cell adhesion and neurite outgrowth, indicating the
potential of these materials for scaffolds in neural tissue
engineering [280].
A new method was developed by Yeh et al. for
culturing in chitosan microbers scaffolds; a chitosan
laminar ow by sheath force in a microuidic chip was
used to produce sodium tripolyphosphate (TPP) chitosan
1000
microbers via the ionic cross-linking reaction. A polymethyl-methacrylate (PMMA) microuidic chip with a 45
cross-junction microchannel was fabricated using a CO2
laser machine to generate the chitosan microbers. By
changing the ratios of the core and sheath ow rate, different sizes of chitosan microbers were produced. The
microbers were then coated with collagen and Schwann
cells and broblast cells were cultured on the chitosan
microbers. Both cell types adhered to the surface of the
chitosan microbers and began to proliferate after 24 h.
After 72 h, the Schwann cells had proliferated linearly while
the broblast cells covered the surface of the chitosan
microbers. The chitosan microbers provide very good
scaffolds for many tissue engineering applications with the
advantages of ease of fabrication, simplicity and cost effectiveness [281].
4.2. Chitosan in wound-healing applications
In wound-healing, an ideal dressing should protect the
wound from bacterial infection as well as promote healing [282]. Chitosan-based materials, produced in varying
formulations, have been used in a number of woundhealing applications. Chitosan induces wound-healing on
its own and produces less scarring [283285]. It seems to
enhance vascularization and the supply of chito-oligomers
at the lesion site, which have been implicated in better
collagen bril incorporation into the extracellular matrix
[65,286]. While different material dressings have been
used to enhance endothelial cell proliferation, the delivery
of growth factors involved in the wound-healing process can improve that process [287]. In addition to the
reparative nature of the chitosan hydrogels they can also
deliver a therapeutic payload to the local wound, for example, broblast growth factor-2 (FGF-2) which stimulates
angiogenesis by activating capillary endothelial cells and
broblasts [288,289]. To sustain FGF-2 residence at the
wound site, FGF-2 was incorporated into a high molecular
weight chitosan hydrogel, formed by UV-initiated crosslinking [290].
A chitosan hydrogel scaffold impregnated with -FGFloaded microspheres were developed by Park et al. that
accelerates wound closure in the treatment of chronic
ulcers [288]. Films of chitosan, in combination [291] were
found to promote accelerated healing of incisional wounds
in a rat model [292]. The wounds closed within 14 days
and mature epidermal architecture observed histologically
with keratinized surface of normal thickness and a subsided inammation in the dermis.
4.3. Chitosan in drug delivery applications
Drug delivery has been a very active area, especially for
chitosan as a carrier for various active agents [293295].
Chitosan has been effectively used in drug delivery as a
hydrogel system, drug conjugate, biodegradable release
system, and PEC for many components. Chitosan-based
systems are used for the delivery of proteins/peptides,
growth factors, anti-inammatory drugs, antibiotics, as
well as, in gene therapy and bioimaging applications.
4.3.1. Chitosan-based systems for the delivery of

anti-cancer drugs
Cisplatin loaded chitosan microspheres were prepared
using a w/o emulsion system; the incorporation efciency was 30% [296]. The type of oil used was found
to affect release properties of cisplatin, and the initial
burst effect. Pharmacokinetics, targeting, embolization
effects and alteration of liver function using cisplatin chitosan microspheres were evaluated after hepatic arterial
embolization in dogs. A remarkable decrease in the number of arterioles in liver, necrosis of nodules and hepatic cell
degeneration in the embolized region. A chitosan-based
hydrogel with 131 I-norcholesterol (131 I-NC) was tested in a
breast cancer xenograft mouse model by Azab et al. This
hydrogel, cross-linked with glutaraldehyde, reduced the
progression of the tumor and [297,298] prevented 69% of
tumor recurrence and metastatic spreading. Most importantly, there was little or no systemic distribution of the
radioisotope after hydrogel implantation. Recently, paclitaxel was effectively delivered from salicylic acid-grafted
chitosan oligosaccharide nanoparticle by Wei et al. [299].
proteins/peptides
Chitosan particles or polyelectrolytes complexes have
been studied for nasal delivery of therapeutic proteins have
been done [300304]. It was found that insulin-loaded
chitosan nanoparticles enhanced nasal absorption of proteins to a greater extent than relevant chitosan solutions
[300,301,304]. Insulin-loaded nanoparticles were made by
spray-drying a mannitol/lactose solution to yield 13 m
microparticle powders for alveolar deposition [305307].
The nanoparticles have a good loading capacity (6580%)
and a fast release of insulin [307]. An inhalable chitosanbased powder formulation of salmon calcitonin containing
mannitol (as a cryoprotecting agent) was prepared by Yang
et al. using spray-drying. The dissolution rate of the protein
decreased when formulated with chitosan, which might be
due to irreversible complex formation between the (aggregated) protein and chitosan during the drying process
[308]. In a recent study, Ma and Liu prepared proteinloaded chitosan microspheres by a modied ionotropic
gelation method combined with a high voltage electrostatic
eld [309]. The morphology, particle size, encapsulation
efciency and in vitro release behavior of the prepared
microspheres were investigated. Microspheres with good
dispersity and spherical shape were obtained from a mixture of TPP and ethanol was applied as the coagulation
solution. The encapsulation efciency depended on the
ratio of BSA to chitosan with 35% of the BSA being released
from the microspheres cured in 3% coagulation solution,
and >50% of the BSA was released from the microspheres
cured in 1% coagulation solution. The combination of an
ionotropic gelation method with a high voltage electrostatic eld seems to be an effective method to fabricate
chitosan microspheres for sustained delivery of protein.
Hu et al. polymerized acrylic acid with chitosan to form
nanoparticles; the lower molecular weight, the better the
yield (70%) [310]. The release of silk peptide occurred
as an initial burst followed by prolonged release up to 10
days. These nanospheres were suggested for pH dependent
1001
Fig. 16. (A) TEM images of chitosanTPP nanocomplexes formed by ionically cross-linking in (a) adipic acid medium and (b) acetic acid medium. (B) TEM
photos of (a) chitosanTPP nanoparticles and (b) chitosanTPP nanobers after soaking in SBF at 37.0 0.5 C for 7 days [311]. Copyright 2009, Elsevier Ltd.
drug release applications in the gastric cavity. Chitosanbased nanocomplexes were prepared by Zeng et al. by
ionically cross-linking with TPP in different acidic media
under mild conditions. The self-assembly and ionic interactions of chitosan and TPP were affected by reaction media;
chitosan-based nanobers could be obtained in adipic acid
medium while nanoparticles were formed in acetic acid
(Fig. 16A). The in vitro drug release of BSA indicated that
chitosan-based nanobers and nanoparticles have similar
prolonged release prole. The bioinspired mineralization
of both chitosan-based nanobers and nanoparticles was
performed by soaking them in synthetic body uids (SBF).
Transmission electron microscopy (TEM) (Fig. 16B) and
X-ray diffraction (XRD) data indicate that chitosan-based
nanobers induced nanohydroxyapatite formation better than chitosan-based nanoparticles. These biomimetic
chitosan-based systems have a controlled release capacity
of bioactive factors that may be of use in bone tissue engineering to enhance the bioactivity and bone inductivity
[311].
4.3.3. Chitosan-based systems for the delivery of growth
factors
Chitosan hydrogels coupled with bone morphogenetic
protein (BMP)-7 have shown the ability to enhance lesion
repair [312]. Chondroitin sulfate, a GAG molecule found
in cartilage, was immobilized in chitosan hydrogels to
enhance cartilage formation [25]. A platelet derived growth
factor has also been loaded into chitosan gels to enhance
osteoinduction as the hydrogel degraded at the defect
site [313,314] while chitosanalginate hydrogels loaded
with BMP-2 and MSCs were shown to induce subcutaneous bone formation [315]. Chitosanlaminin nerve
guides loaded with glial cell line-derived nerve growth
factor (GDNF) enhanced both the functional and sensory
nerve recovery by releasing GDNF in the early stages of

implantation [316]. Growth factors that have short therapeutic half-lives, such as endothelial growth factor, require
frequent administration to maintain an effective concentration [161]. Chitosanalbumin hydrogel microspheres
have shown continuous release for over 3 weeks after subcutaneous implantation in rats, indicating possible success
for in vivo applications [317].
antibiotics
The gastric residence time of tetracycline loaded chitosan microspheres (prepared by ionic cross-linking and
precipitation method) was examined following oral administration in gerbils by Hejazi and Amiji [318]. The gastric
retention was determined by administering radioiodinated
[125 I] chitosan microsphere in nonacid-suppressed and
acid-suppressed states and then measuring the buildup
of radioactivity in specic tissues and uids. The tetracycline concentration prole in the stomach following
administration of microsphere formulation was similar to
that of aqueous solution. The chitosan microspheres did
not prolong the residence time in the fasted gerbil stomach. Magnetic chitosan nanoparticles, as multifunctional
nanocarriers, were loaded with bleomycin and proved to
be a very effective as targeting system by Kavaz et al. [319].
In another study, the use of chitosan drug delivery to the
inner ear across the round window membrane (RWM)
was examined by Saber et al. [294]. Three structurally
different chitosans loaded with a tracer drug, neomycin
(C23 H46 N6 O13 ), were injected into the middle ear cavity of
albino guinea pigs (n = 35). After 7 days, the hearing organ
was examined for hair cell loss and the RWM evaluated
in term of thickness. All chitosan formulations successfully released the loaded neomycin which diffused across
1002
the RWM, and exerted a concentration dependent ototoxic

effect on the cochlear hair cells. Chitosans had no harmful
effect on the cochlear hair cells and were determined to be
safe and effective carriers for inner ear therapy.
anti-infammatory drugs
The loading and release study of triamcinolone
acetonide (TAA) [C24 H31 FO6 ], a drug used to reduce
inammation in the treatment of mouth ulcers, in
chitosanpoly(acrylic acid) (PAA) membrane was investigated by Ahn et al. [320]. The TAA in this membrane
was found to be effective as a transmucosal drug delivery system that responded to pH changes. Indomethacin
(C19 H16 ClNO4 ) loaded chitosan microspheres were prepared as polyelectrolyte complexation of TPP and chitosan
by Shiraishi et al. [117]. After oral administration of
chitosan gel beads to beagle dogs, the plasma exhibited a sustained release pattern for indomethacin. A
signicant correlation was observed between the molecular weight of chitosan and dissolution rate constant
or the mean absorption time and the area under the
plasma concentrationtime curve. Another study reported,
the preparation of indomethacin loaded chitosan microspheres using only aqueous solvents [321]. The inuence
of formulation variables on indomethacin content in the
microspheres and time for release of indomethacin from
the microspheres was assessed. Huang et al. observed that
betamethasone disodium phosphate (C22 H28 FNa2 O8 P)
loaded microspheres had good drug stability (<1% hydrolysis product), high entrapment efciency (95%) and positive
surface charge (37.5 mV) [322]. The results also indicated that yield and size of particles was increased with
increasing betamethasone amount but both zeta potential and density of the particles decreased with increasing
betamethasone loaded amount. The in vitro release had a
dose-dependent burst, followed by a slower release that
was drug loading between 5% and 30% (w/w) [323].
A novel inorganicorganic pH-sensitive membrane
based on an interpenetrating network utilizing inorganic
silicate and organic chitosan was proposed by Park et al.
[324]. Percolation of lidocaine-HCl (C14 H22 N2 O), sodium
salicylate (C7 H5 NaO3 ) and 4-acetomidophenol (C8 H9 NO2 )
into this membrane was studied and found to be sensitive
to the external pH as well as the ionic interactions of drugs
with chitosan. The membrane was also sensitive to other
stimuli such as temperature and light that could serve as
alternate routes for drug loading. In a similar approach, the
in situ light initiated polymerization of acrylic acid in the
presence of chitosan was used to derive a novel mucoadhesive membrane [320]. The interactions between the two
polymers were determined to be based on hydrogen bonding and the strong adhesive property of this membrane
rendered it suitable for transmucosal drug delivery applications.
4.3.6. Chitosan-based systems for vaccines delivery
Chitosan-based powders and micro/nanoparticles for
parenteral and mucosal delivery of antigen vaccines have
been studied [325344]. Immunizations with various
antigens co-administered with chitosan produced both
systemic and local immune responses. In a phase I clinical study, intranasal immunization with inuenza vaccine
formulated with soluble chitosan glutamate showed positive effects [345]. After intraperitoneal administration in
mice and guinea pigs, the adjuvant activity of chitosan
and its precursor chitin, were investigated in terms of
induction of cytokines, long-lasting circulating antibodies
and cell-based immunity against bacterial alpha-amylase
and Escherichia coli infection [346,347]. In another study,
chitosan induced cytokines, interleukin (IL)-1 and colonystimulating factor (CSF) in macrophages in vitro [348].
Zaharoff et al. found that chitosan dissolved in buffer at pH
6.2 enhanced the immunoadjuvant properties of cytokines
such as granulocyte-macrophage colony-stimulating factor (GM-CSF), when co-administered subcutaneously.
Chitosan seems to slow the dissemination of GM-CSF at
the site of injection which prolongs its exposure and
enhances the immunoadjuvant properties of GM-CSF. After
a single subcutaneous injection of GM-CSF/chitosan solution, the cytokine expanded lymph nodes were about
5-fold greater than GM-CSF injected four times alone.
The chitosan may also be enhancing the antigen capability to present dendritic cells (DCs) and induce greater
allogeneic T-cell proliferation [349]. It was found that
soluble chitosan substantially increased antigen-specic
antibody titers and antigen-specic CD4+ proliferation
upon subcutenous administration of an aqueous solution of -galactosidase, as a mode antigen and chitosan.
The authors suggested that the ability of soluble chitosan to enhance humoral and cell-mediated immunity
is related to its physicochemical characteristics that
increases the retention of formulations at the injection
sites and to induce transient cellular expansion in draining lymph nodes [350]. Ghendon et al. demontrated that
intramuscular administration of soluble chitosan with
monovalent and trivalent split inactivated inuenza vaccine gave strong humoral and cell-immunity responses
against different variants of A- and B-type human inuenza
viruses [351,352]. The soluble chitosan mixed with inactivated inuenza vaccine increased cytotoxic activity of
the splenic NK T-lymphocytes and enhanced the proliferative activity of mononuclear lymphocytes in the spleen.
Moreover, it was shown that the number of CD3, C3/NK
and CD25 T-cells also increased. The chitosan may be
activating cell immunity because of its proliferation activity, initiated through the receptor complex TCRCD3 as
well as activation signals linked with lectin receptors
[352].
4.3.7. Chitosan membranes in drug release
The interaction of oxidized glucose dialdehyde with chitosan is another method of generating chitosan membranes
[353]. N-alkyl groups of varying chain lengths were used to
modify the hydrophobicity of these chitosan membranes;
the longer the alkyl chain, the more hydrophobic the chitosan membrane which affected the drug release rate. The
permeation and diffusion of B2 vitamin as the drug model
decreased with increasing pH 8; above pH 8 the hydrophobicity of the chitosan increased. In-vitro studies showed no
toxic effects for this membrane system. Chitosangelatin
lms entrapped danshen, an herbal extract, for delivery in
the abdominal cavity without the need for cross-linking

[354].
Recently asymmetric chitosan membranes were developed for the guided tissue regeneration (GTR) by using
the two-step phase separation [355]. The liquidliquid
demixing by non-solvent induction formed bicontinuous
strucutures and the pore size ranged from 0.5 to 2 m. The
membrane showed good biocompatibility, tissue integration, cell occlusivity and osteoconduction, for 3 months.
The chitosan membrane prevented bacterial proliferation,
that was signicantly superior to current commercial GTR
products for now. The asymmetric structure would be
appropriate for the release of complex drugs with different
effective time periods. These asymmetric structures appear
to be capable of releasing complex drugs with different
effective time periods.
4.4. Chitosan in gene therapy
Many nucleic acid delivery vehicles have been investigated due to the low transfection efciency of naked
nucleic acid injection in vitro and in vivo. Chitosan-based
gene delivery systems have been proven to be effective
for non-viral gene therapy [125]. The chitosanDNA complexes are very easy to synthesise and are more effective
compared to the commonly used polygalactosamineDNA
complexes, however, their use is limited because of the
lower transfection efciency [356].
Various factors affecting chitosan delivery of nucleic
acids were studied by Mao et al. [357] and Sato et al. [358].
The molecular weight of chitosan, the charge ratio between
the luciferase plasmid to chitosan and the pH of the culture media were found to be the factors related to the
in vitro transfection efciency. Chitosan enhanced adenovirus infectivity to mammalian cells in gene therapy [359]
and low concentration and low molecular weight chitosans
were better in enhancing adenovirus activity [360]. The stability of the DNAchitosan complexes depends on several
factors such as the chitosan chain length and the amount
of chitosan. Increasing the chitosan molecular weight and
chitosan concentration yielded more stable complexes,
indicating that varying the chitosan chain length may
provide a tool for controlling the ability of the polyplex
to deliver therapeutic gene vectors to cells [361,362].
Spherical chitosan/DNA nanoparticles with an average
38 nm diameter were prepared by using a simple osmosisbased process [363]. About 30% DNA was incorporated
with prolonged release time. By varying the solvent/nonsolvent couple, temperature and membrane cut-off, several
nanostructured systems of different size and shape were
obtained and used for several biomedical and biotechnological applications. Using a novel technique, Dai et al.
studied the gene delivery by chitosanDNA nanoparticles
through retrograde intrabiliary infusion (RII) and examined
the efcacy of liver specic targeting [364]. The transfection efciency of chitosanDNA nanoparticles, compared
with poly(ethylenimine) (PEI)DNA nanoparticles, was
evaluated in Wistar rats by infusing into the common bile
duct, portal vein, or the tail vein. Luciferase expression
was not detected when chitosanDNA nanoparticles were
administrated through the portal vein or tail vein, however,
1003
the rats that received chitosanDNA nanoparticles had 500

times higher luciferase expression in the liver 3 days after
RII; and transgene expression levels decreased gradually
over 14 days. Luciferase expression in the kidney, lung,
spleen, and heart was negligible compared with that in the
liver. RII of chitosanDNA nanoparticles did not have any
signicant toxicity or damage to the liver and biliary tree.
Luciferase expression by RII of PEIDNA nanoparticles was
17-fold lower than that of chitosanDNA nanoparticles on
day 3, but then it increased slightly over time. These results
suggest that gene delivery by chitosanDNA nanoparticles
through RII is a possible routine to achieve liver-targeted
gene delivery; both gene carrier characteristics and mode
of administration signicantly inuence gene delivery efciency.
In an attempt, to track the efciency of DNA delivery, Lee et al. employed uorescence resonance energy
transfer (FRET) to monitor the molecular dissociation of
a chitosan/DNA complex with chitosan characterized by
different molecular weights [365]. Plasmid DNA and chitosan were labeled with Quantum Dots and Texas Red,
respectively, and confocal microscopy and uorescence
spectroscopy were used to monitor the dissociation of
the complexes. As the chitosan molecular weight in the
chitosan/DNA complex increased the Texas Red-labeled
chitosan gradually lost FRET-induced uorescence light.
This observation was also observed when HEK293 cells
were incubated with chitosan/DNA complex. This indicated
that the dissociation of the chitosan/DNA complex was
greater with the higher molecular weight chitosan/DNA
complex. Fluorescence spectroscopy analysis conrmed
that the molecular dissociation of the chitosan/DNA complex at pH 7.4 and 5.0 and conrmed that the dissociation
occurred in acidic environments. Therefore, it appears that
the high molecular weight chitosan/DNA complexes dissociate better in lysosomes than the low molecular weight
complexes. Furthermore, the high molecular weight
chitosan/DNA complex showed superior transfection efciency in relation to the low molecular weight complex.
It was concluded that the dissociation of the chitosan/DNA
complex is a critical event in obtaining the high transfection
efciency of the gene carrier/DNA complex [365].
Chitosan/pluronic hydrogels as injectable systems for
gene therapy to enhance local transgene expression at
the site of injection were prepared by Lee, Kim and
Yoo. Transfection studies employing HEK293 cells showed
that released fractions from chitosan/pluronic hydrogels
showed better transfection efciency than those from
pluronic hydrogels [192].
In another study, Khatri et al. investigated the preparation and in vivo efcacy of plasmid DNA(pDNA) loaded
chitosan nanoparticles for nasal mucosal immunization
against hepatitis B. Chitosan/pDNA nanoparticles were prepared using a complex coacervation process [366]. The
chitosan nanoparticles produced humoral (both systemic
and mucosal) and cellular immune responses upon nasal
administration. This study signied the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for
effective immunization through non-invasive nasal route.
Even though, the conventional high molecular chitosans
have a few drawbacks such as aggregation, low solubility
1004
Fig. 17. MR images of the central region of mouse liver before (A) and after (BC) injection of SPION-loaded WSCLA nanoparticles. Images were obtained
at (B) 30 min and (C) 1 h after injection of the nanoparticles. L = left [369]. Copyright 2009, Elsevier Ltd.
at neutral pH, and high viscosity at concentrations used for

in vivo delivery and a slow onset of action [31], the non-viral
gene delivery systems based on chitosan are still regarded
as one of the most efcient system for DNA vaccine delivery.
To develop chitosan nanoparticles for siRNA delivery, Katas and Alpar, prepared chitosan nanoparticles by
two methods of ionic cross-linking, simple complexation
and ionic gelatin using TPP. In-vitro studies using two
types of cells lines, CHO K1 and HEK 293, revealed that
the preparation method of siRNA association to the chitosan plays an important role on the silencing effect.
ChitosanTPP nanoparticles with entrapped siRNA had
better vectors than chitosansiRNA complexes; this may
be due to their high binding capacity and loading efciency the chitosanTPP nanoparticles appear to have good
potential as viable vector candidates with safer and costeffective siRNA delivery [367]. Exploring the efciency of
chitosan/siRNA nanoparticles as a therapeutic agent, Liu
et al. reported that the physicochemical properties and
in vitro gene silencing of chitosan/siRNA nanoparticles are
strongly dependent on chitosan molecular weight and DD
[368]. Chitosan with high molecular weight and high level
of DD resulted in the formation of discrete stable nanoparticles of 200 nm in size. Chitosan/siRNA formulations (N/P:
50) prepared with low molecular weight chitosan (10 kDa)
showed almost no knockdown of endogenous enhanced
green uorescent protein (EGFP) in H1299 human lung
carcinoma cells, whereas those prepared from higher
molecular weight (65170 kDa) and DD (80%) showed
greater gene silencing ranging between 45% and 65%.
The highest gene silencing efciency (80%) was achieved
using chitosan/siRNA nanoparticles at N/P 150 using higher
molecular weight (114 kDa and 170 kDa) and DD (84%)
that correlated with formation of stable nanoparticles of
200 nm. From their studies it can be concluded that there
is still scope for improvement and for the optimization
of gene silencing using chitosan/siRNA nanoparticles and
certain improvements in the polymeric properties would
make signicant differences.
4.5. Chitosan in bioimaging applications
Chitosan appears to be an exemplary polymer in
biological applications owing to its biocompatible prop-
erties. In this context, its use in bioimaging applications

is also gaining rapid attention. The incorporation of
imaging agents is enabling its use for bioimaging, for
example, the incorporation of imaging agents such as
Fe3 O4 for Magnetic Resonance Imaging (MRI) into the
self-assembled nanoparticles could enhance hepatocytetargeted imaging [369] and the particle could serve as
MR molecular imaging agent. Several inorganic materials
including metals are being incorporated into the chitosan
composite preparations and their combined characteristics are proving benecial for biomedical applications [19].
Chitosan polyion complex composites can be prepared
by interactions of chitosan with natural and synthetic
polyanion molecules [370]. PAA (Carbopol), an anionic
synthetic polymer having mucoadhesive properties, is
extensively used with chitosan to form polymer composites, which have longer circulation times in vivo, resulting
in higher bioavailability of incorporated therapeutic agents
[243,370372]. These composite systems are being widely
investigated by incorporating contrast agents for imaging purposes. Preparing uorescent chitosan quantum dot
composites enables the combination of targeted drug and
gene delivery with optical imaging [373,374]. Lee et al.
have developed novel self-assembling nanoparticles composed of amphipathic water-soluble chitosanlinoleic acid
(WSCLA) conjugates for encapsulation of super paramagnetic iron oxide (SPIOs) as a contrast agent to target
hepatocytes [369]. The WSCLA conjugates self-assembled
into coreshell structures in aqueous solution. Since its
incorporation in nanoparticles, its potential for in vivo
molecular imaging applications has increased tremendously (Fig. 17).
Chitosan-based gadolinium (Gd)-nanoparticles were
prepared by incorporating Gd complexed with diethyl triamine penta acetic acid (DTPA) using the emulsion-droplet
coalescence technique [6]. Their release properties and
their ability for long-term retention of Gd-DTPA in the
tumor indicated that these Gd nanoparticles might be useful as an intratracheal injectable device for gadolinium
neutron-capture therapy (Gd-NCT) [131]. The Gd loading
depended on the deacetylation of the chitosan used. The
highest Gd load was achieved with 100% deacetylated chitosan in 15% Gd-DTPA aqueous solution, and the particle
size was 452 nm, whereas chitosan with lower deacetylation level produced much larger particles with decreased
Gd-DTPA content. After an in vivo intratumoral injection

the Gd-DTPA-chitosan nanoparticles displayed prolonged
retention in the tumor tissue [375,376]. Kumar et al. also
described the chemistry and preparations of Holmium-166
and Samarium-153 chitosan complexes, that are mainly
suited for radiopharmaceutical applications [6].
4.6. Chitosan in green chemistry
Ionic liquids (ILs), have recently received much attention as green solvents as a result of the development of
green chemistry and the requirement for environmental
protection [377]. The acceptance of ILs has come as a revolution that has excited both the academia and the chemical
industries. The terms room temperature ionic liquid (RTIL),
nonaqueous ionic liquid, molten salt, liquid organic salt and
fused salt have all been used to describe ILs (salts in the
molten state) [378]. The ILs have a unique physicochemical
properties that are very useful when conventional organic
solvents are not sufciently effective or not applicable
[379].
Based on the combination of their unique properties,
ionic liquids have a number of advantages for synthesis and extractions. These liquids have negligible vapor
pressure, a liquid range of up to more than 400 K and densities greater than water. Ionic liquids are miscible with
substances within a wide range of polarities, as well as,
simultaneously dissolve organic and inorganic substances.
In many cases, some processes would be impossible with
conventional solvents because of their limited liquid range
or miscibility. Even greater potential is the use of RTILs
for chemical synthesis because the charged nature of these
solvents can inuence the synthesis itself. The most novel
and striking feature of RTILs as solvents is the possibility to design one with specialized properties needed
for a specic application, known as designer solvents
[380]. Ionic liquids have been applied as alternative solvents in many catalytic organic transformations [378,381].
They efciently dissolve biological macromolecules that
are linked by intermolecular hydrogen bonds, such as carbohydrates, cellulose, wool keratin and silk broin [382]
ILs are also being used to dissolve chitosan for waste water
treatment, cosmetics, heavy metal chelation, heterogeneous catalysts.
Chitosan has one amino group and two hydroxyl groups
in the repeating hexosaminide residue (Fig. 1). During the
chemical modication of any biopolymer, it is essential to
form a stable homogeneous solution in order to improve
the efciency of modication. However, the strong interand intramolecular hydrogen bonding between the chitin
and chitosan chains decreases their solubility in many
organic solvents. Therefore, dilute aqueous solutions of
organic and mineral acids solutions are used to dissolve
chitosan. However, these solvents are corrosive and require
an alkaline solution treatment process to remove the acid
after the process. Furthermore, in certain applications like
transition metal sorbents [383] and drug carriers [384], the
polyelectrolyte solutions formed have limited application
since bioactive agents may be affected by the presence of
acetic acid used for dissolving chitosan. Consequently, a
new processing strategy for developing potential applica-
1005
tions of these biorenewable resources is desirable [377].

Xie et al., utilized 1-butyl-3-methyl-imidazolium chloride as a solvent for chitin and chitosan and used them
as substitutes for amino-functionalized synthetic polymers for capturing and releasing CO2 . To have efcient
CO2 recovery methods from industrial waste gases is
very important to both reutilization of the CO2 as a carbon resource as well as environmental issues related to
greenhouse effects. Traditionally, the most commonly used
process for CO2 recovery is chemically reversible CO2 xation with amines at room temperature to form ammonium
carbamates; the CO2 is then released from the ammonium
carbamates by heating [385]. Amino-functionalized synthetic polymers and ionic liquids have also been developed
to xate CO2 based on this principle [386]. Chitosan is a natural polyamine, and hence, could be a suitable alternative
amino-functionalized polymer for CO2 xation. Although
the ionic liquids do not completely disrupt the crystalline
domains of chitosan and only a partially dissolved solution
is obtained, it apparently does not affect their utilization.
Both the chitin/IL and chitosan/IL were effective for the
reversible xation of carbon dioxide. This environmentally
friendly process has the potential not only for recovering
CO2 from industrial exhaust but also for CO2 sensing [377].
A novel polymer/RTIL composite material based on
chitosan and 1-butyl-3-methyl-imidazolium tetrauoroborate (BMIM.BF4) was developed by Lu et al. The composite
system could be readily used as an immobilization matrix
to entrap proteins and enzymes. Hemoglobin (Hb) was
chosen as a model protein to investigate the composite
system. The bioactive composite lm-modied glassy carbon (GC) electrode was prepared by direct electron transfer
between the protein and the GC electrode. The lm electrode exhibited dramatically enhanced bioelectrocatalytic
activity towards oxygen and trichloroacetic acid along
with good stability in solution. Two well-dened quasireversible redox peaks for hemoglobin were observed at the
composite lm-modied GC electrode. Thermogravimetric
analysis (TGA) indicates that the chitosanBMIM.BF4Hb
composite had higher thermal stability than chitosanHb
itself. These unique composite material could provide a
good electrochemical sensing platform for redox proteins
and enzymes, and thus have potential applications in direct
electrochemistry, biosensors, and biocatalysis by direct
electron transfer [387].
A sensitive glucose biosensor, using an electrodepositing chitosanionic liquidglucose oxidase biocomposite
onto nano-gold electrode, was fabricated by Zeng et al. A
nano-gold electrode was constructed by electrochemically
depositing gold nanoparticles onto a at gold electrode
surface followed by immersing into a bath containing pbenzoquinone (BQ), chitosan, glucose oxidase (GOD) and IL
for electrodeposition of enzymatic electrode. This biosensor exhibits a fast amperometric response (<5 s) to glucose
with a high current sensitivity, that is 2.8 times better than a
biosensor prepared by electrodepositing chitosanILGOD
biocomposite on a at gold electrode. The detection limit
for glucose was 20-fold better compared to the biosensor
prepared on the at gold electrode. The biosensor has high
reproducibility, long-time storage stability and satisfactory
anti-interference ability [388].
1006
Chitosan/cellulose composites, as biodegradable

biosorbents, were prepared based on ionic liquids; no
cross-linking agent and adsorption for heavy metal ions
by Sun et al. The freeze-dried chitosan/cellulose biosorbent was indicated to possess higher adsorption capacity
together with better stability. The interaction of the two
components and the resulting materials adsorption capacity to Ni(II) were conrmed by IR and XPS. In addition,
other heavy metal ions, were effectively adsorbed with
the following capacity Cu(II) > Zn(II) > Cr(VI) > Ni(II) > Pb(II)
[389].
An ingenious approach for the fabrication of a promising
glucose sensor, GOx/C60-Fc-CS-IL, that exploits the synergistic benets of fullerene (C60), ferrocene (Fc), chitosan
and ionic liquid (IL) for glucose oxidase (GOx), was developed by Zhilei et al. The electrocatalytic activity of C60
and Fc remarkably improved the electron relays which
activated the oxidation of the glucose and accelerated the
electrochemical reaction while the chitosanIL network
provided a favorable microenvironment that maintained
the bioactivity of GOx. The biosensor exhibited a very high
sensitivity, low detection limit, fast response time, wide
calibration range and excellent long-term stability up to
30 weeks [390].
Although chitosan is relatively non-toxic, biocompatible material, care must be taken to ensure that it is pure
and free from contaminations, such as protein, metal or
other contaminants that could potentially cause many
deleterious effects both in cross-linking approaches and
in dosage forms. After each chemical reaction, care must
be taken to thoroughly remove the unreacted reagents,
particularity those that are cytotoxic, to prevent confusing
results.
5. Conclusions
Regenerative medicine has witnessed a signicant
progress with the development of modern science and
technology. Chitosan, with its exiciting properties, is one
of the most promising bio-based polymers for drug delivery, tissue engineering, gene therapy and theranostics. It
is almost the only cationic polysaccharide in nature with
such great innate medical potential. The various preparation techniques presented herein can be helpful in deciding
the context of using chitosan in selectively capturing a therapeutic payload and control release in a target site as well
as for tissue engineering purposes. It has been documented
that chitosan particle/hydrogels can be used as carriers for
encapsulation and controlled release. There is a window
for the preparative conditions to systematically manipulate
the incorporation process and to control the basic properties, such as size and surface charge density, to develop a
successful system.
Chitosan has been shown to improve the dissolution
rate of poorly soluble drugs, and thus, can be exploited
for bioavailability enhancement of drugs and their delivery. Various therapeutic agents, such as anticancer,
anti-inammatory, antibiotics, antithrombotic, steroids,
proteins, amino acids, antidiabetic and diuretics have been
incorporated in chitosan-based systems to achieve controlled release. A new application related to chitosan is
based on the fact that chitosan particles provide an excellent template for bioimaging.
Chitosans tissue engineering potential as a biomaterial to generate structures with predictable pore sizes and
degradation rates makes it particularly suitable for bone
and cartilage regeneration. However, efforts to improve the
mechanical properties of chitosan-based composite biomaterials are essential for this type of application. Another
very signicant chitosan property is its capability to bind
anionic molecules, such as growth factors, glucosamine
glycans and DNA. The ability to link chitosan to DNA
molecules as a substrate for gene activated matrices makes
this material a good candidate in gene therapy applications.
In fact, the combination of chitosans good biocompatibility, intrinsic antibacterial activity, ability to bind growth
factors and to be processed into a variety forms, makes
it an appropriate candidate as scaffold material for tissue
engineering. Chitosan is also contributing to green chemistry by being actively used with liquid solvents. The chief
application of this combination is in waste water treatment,
heavy metal chelation, and biosensing. Although some of
the parameters, such as molecular weight and viscosity
have to be considered in order to use chitosan at its full
potential, the benets are signicant enough to make the
effort highly rewarding.
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Progress in Polymer Science 36 (2011) 9811014

Contents lists available at ScienceDirect

Progress in Polymer Science

ChitosanA versatile semi-synthetic polymer in biomedical

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

able resource but because they are very compatible and

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

2. General aspects of chitosan-structural and

Fig. 2. Schematic illustration of chitosans versatility. At low pH (less than

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

on the basis of their DD and molecular weight. DD and

wound-healing properties and osteogenesis enhancement

DD, Molecular weight

Directly propotional to property; inversely propotional to property.

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

mer properties on the structural parameters are listed in

2.3.2. Chitosan in vivo biodegradation

2.4. Biological properties of chitosan

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

ATP dependent endocytotic mechanism [92]. The authors

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

rabbits and guinea pigs respectively. He also concluded in

ing the dissolution rate of the poorly soluble drugs

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

Fig. 4. Schematic representation of preparation of chitosan particulate systems by coacervation/precipitation method.

Fig. 5. Schematic representation of preparation of chitosan particulate

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

Fig. 7. Schematic representation of preparation of chitosan particulate

Fig. 6. Schematic representation of preparation of chitosan particulate

groups of chitosan. Thus, it was possible to achieve higher

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

The nanoparticles prepared by conventional emulsion

dissolved in reverse micelles varies from drug to drug and

Fig. 9. Schematic representation of preparation of chitosan particulate systems by seiving method.

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glassy hydrogels that are passed through a sieve (Fig. 9).

a sphere, the release mode is determined using equations

3.1.8. Chitosan micro/nanoparticles drug loading and

where, Mt /M is the fractional drug released at time t, k is

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actions including hydrogen bonding between chitosans

Fig. 10. Schematic representation of chitosan hydrogel networks derived

In each process, chitosan is either physically associated or

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association form an interesting class of hydrogels that are

3.2.2. Cross-linked networks

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can be formed by using small molecule cross-linkers,

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Xu et al. studied the feasibility of creating macropores

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4.1.2. Chitosan in cartilage tissue engineering

native tissue; this increases the chances that the reparative

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strate showed increased cell adhesion, proliferation and

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acute and chronic liver disease is critical [253]. Currently,

M. Dash et al. / Progress in Polymer Science 36 (2011) 9811014

tocyte attachment to alginate (AL)/galactosylated chitosan

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4.3.1. Chitosan-based systems for the delivery of

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nerve recovery by releasing GDNF in the early stages of