-
2.213. Chitosanto, Porto, Portugal
2. 22. 22. 22. 32. 32.2 32.2 42. 52.2 52.2 52.2 52.2 62.2 62.2
62.2 62.213.1.5.8. Wound-healing properties 2262.213.1.5.9.
Bone-healing properties 226
to temperatures that cause the formation of ice crystals,
resulting from the association of a protein andwhich are removed by
sublimation under vacuum,
producing a porous structure.
glycosaminoglycans.2.213.1.6. Chitosan Functionalization
2272.213.2. Processing 2272.213.2.1. Films and Porous Scaffolds
(Freeze-Drying and Freeze-Gelling) 2272.213.2.2. Nanofibers
2272.213.2.3. Polyelectrolyte Complexes 2282.213.2.4. Micro- and
Nanoparticles 2292.213.2.5. Cross-linking 2292.213.3. Biomedical
Applications 2292.213.3.1. Wound Management 2292.213.3.2. Tissue
Repair and Regeneration 2302.213.3.3. Delivery of Therapeutic
Agents 2322.213.3.4. Other Applications 2342.213.4. Future
Prospects 235References 235
GlossaryCoacervation The process that results in the aggregation
of
molecules or colloidal particles under the action of
electrostatic attractive forces.
Degree of acetylation (DA) Molar fraction of N-acetylated
units in chitin/chitosan.
Electrospinning Technique used to produce
nanofibers, based on the application of a sufficiently high
voltage between a needle and a metallic collector, resulting
in a very thin jet of fluid which is projected against a
collector.
Endotoxin A toxin of internal origin. Endotoxins
should be absent from chitosan used for biomedical
applications.
Freeze-drying (of chitosan) Polymer solutions are frozen
Freeze-gelling (of chitosan) A method alternative to
freeze-drying to produce 3D-scaffolds. The method
is based on freezing and subsequent extraction of the
solution-rich phase by a nonsolvent for the polymer, while
the polymer-rich phase is gelled under the action of a
neutralizing agent.
Glycosaminoglycans A gel-forming repeating disaccharide
units of the extracellular matrix.
Neuroma A growth or tumor of nerve tissue.
Polycation A macromolecule with many positively charged
groups.
Polyelectrolyte complexes Self-assembled structures
formed by reacting two oppositely charged polyelectrolytes
in an aqueous solution.
Proteoglycans A constituent of the extracellular matrix 2011
Elsevier Ltd. All rights reserved.
213.1. Sources, Analysis, and Properties 22213.1.1. Chemical
Structure 22213.1.2. Solution Properties 22213.1.3. Chitosan
Preparation: Chitin Isolation and N-deacetylation 22213.1.4.
Chitosan Characterization 2213.1.4.1. Degree of acetylation
2213.1.4.2. Molecular weight 22213.1.5. General Aspects of
Biological Behavior 2213.1.5.1. Biocompatibility 2213.1.5.2.
Cytocompatibility 2213.1.5.3. Bacteriostatic and fungostatic
properties 2213.1.5.4. Enzymatic degradation 2213.1.5.5.
Immunoadjuvancy 2213.1.5.6. Hemostatic and blood clotting
properties 22M A Barbosa, A P Pego, and I F Amaral, Universidade do
Por
13.1.5.7. Cell-binding properties 22221
-
AbbreviationsA Absorbance
DA Degree of acetylation
EC Endothelial cells
ECM Extracellular matrix
FN Fibronectin
FT-IR Fourier transform infrared spectroscopy
GAG Glycosaminoglycan
H&E Hematoxylin and Eosin
lobster, and shrimp shells, adjacent sheets have opposite
direc-
tions, and thus it has an antiparallel chain arrangement. In
b- h he squidge
it
sh
ad
cosamine units by acid, the electrostatic repulsions between
NH3 groups lead to the destruction of interchain attractive
interactions, such as hydrogen bonds and hydrophobic inter-
t pH lower
a
-
-
h
-
s
e
e
f
e
c
s
f
FiD-b-
222 Materials of Biological Originnus Loligo, adjacent sheets
have the same direction, and thus
has a parallel chain arrangement. In g-chitin, every thirdeet
has the opposite direction to the previous two sheets. In
dition to intrasheet interchain hydrogen bonds, a-chitin
also
OO
NHRHO
OO
HO
O
NHRCH2OH
CH2OH
gure 1 Chemical structure of chitosan, a linear copolymer
ofglucosamine (RH) and N-acetyl D-glucosamine (R COCH3) in a(14)
linkage. Glucosamine is the predominant repeating unit.chitin, w
ich is the form occurring in the pen of tthan its pKa, which may
range from 6.5 to 7, chitosan ispolycation and at pH 4.0 and below,
it is completely proto
nated.6 Chitosan solubility depends on chitosan charge den
sity, which is tightly connected with structural parameters
suc
as DA, chain length, and distribution of N-acetylated glucos
amine units, as well as on environmental parameters, such a
pH, ionic strength, and dielectric constant of the media.7
Th
solubility range increases on increasing the DA, due to th
increase of the steric hindrance related to the increase o
the number of the acetyl groups, together with the increas
of the intrinsic pKa. According to Sorlier et al.,6 the
intrinsi
pKa of chitosan increases from 6.46 to 6.8 as the DA
increase
from 5% to 35%, respectively, revealing an increase oactions,
and consequently to chitosan solubility. AHA Hyaluronic acid
HLC Human-like collagen
IVD Intervertebral disc
LbL Layer-by-layer
Mn Number average molecular weight
2.213.1. Sources, Analysis, and Properties
2.213.1.1. Chemical Structure
Chitosan is a linear copolymer of D-glucosamine and
N-acetyl-
D-glucosamine in a b-(14) linkage, in which glucosamine isthe
predominant repeating unit (Figure 1). Chitosan itself may
be found in the mycelia of certain fungi in association with
other polysaccharides, but is mostly obtained by
deacetylation
of chitin. Chitin is the second most abundant polysaccharide
in nature after cellulose, occurring in the cell walls of
certain
fungi1 and yeasts, in plants as the equivalent to cellulose,
and
in many invertebrate groups such as mollusks and arthropods
as the fibrous support of the inorganic mineral phase of
their
exoskeleton, as an alternative to collagen.1 Chitin is a
high
molecular weight crystalline polysaccharide, which is
theoreti-
cally comprised entirely of N-acetylated D-glucosamine
units.
Naturally occurring chitin, however, is mostly present as a
copolymer, containing different proportions ofN-glucosamine
units, dependent on the source.2 In chitin, the chains are
arranged in sheets or stacks, the chains of each sheet
having
the same direction and being bonded through intrasheet
hydrogen bonds between two adjacent chains. Naturally occur-
ring chitins are found in three polymorphic forms, a-, b-,
andg-chitin, which differ in the arrangement of chains within
thecrystalline regions. In a-chitin, which is the one found in
crab,MSC Mesenchymal stem cells
Mw Weight average molecular weight
NMR Nuclear magnetic resonance
PDGF Platelet-derived growth factor
PECs Polyelectrolyte complexes
PEO Poly(ethylene oxide)
PLGA Poly(lactic-co-glycolic acid)
PLLA Poly(L-lactic acid)
SEC Size exclusion chromatography
SEM Scanning electron microscopy
TCP Tricalcium phosphate
TGF-b1 Transforming growth factor beta 1
g-PGA Gamma-poly(glutamic acid)
presents hydrogen bonds between adjacent chains. These
inter-
sheet bondings are responsible for the lack of swelling in
water
of a-chitin, whereas b-chitin swells readily in water and
formshydrates.2 Chitosan is also crystalline, but as compared
to
chitin, presents a longer distance between adjacent chains
belonging to the same sheet, due to the removal of
theN-acetyl
groups during the conversion from chitin to chitosan, which
hold together adjacent chains through C(2)NHOC(7)hydrogen
bonds.2 Instant differentiation between chitin and
chitosan can be made based on their solubility. While chitin
is
soluble in N,N-dimethylacetamide (DMAc) in the presence of
510% (w/v) lithium chloride and insoluble in dilute acid
solutions, the reverse is true for chitosan.2,3 In
chitin/chitosan
terminology, the molar fraction of N-acetylated units is
termed
the degree of acetylation (DA), expressed in percentage,
or fraction of N-acetylated units (FA).4,5 Since a DA around
or
lower than 50% is usually required for chitosan solubility
in
dilute acidic solutions, the term chitosan is applied both
to
fully-deacetylated chitin and partially deacetylated chitin
with
DAs 50%.
2.213.1.2. Solution Properties
Chitosan is neither soluble in water nor in organic
solvents.
However, after protonation of amine functionalities from
glu-
-
Chitosan 223ried out at as low temperature as possible, under
inert atmo-
sphere, such as nitrogen or argon, or in the presence of
oxygen
scavengers or reducing agents, such as NaBH4. As chitin is
not
soluble in such systems, deacetylation occurs under
heteroge-
neous conditions. During deacetylation of a-chitin, NaOH
actsinitially on the amorphous regions of the polymer, and only
afterwards on the crystalline regions. Heterogeneous
deacetyla-
tion leads therefore to a block distribution of acetylated
units,
rather than a random distribution of the same. As a result,
the
characteristic infrared (IR) bands attributed to
crystallinizationcationicity of amine functionalities on increasing
the DA. As a
result, chitosans with DAs in the range of 4555% are water-
soluble, providing that the N-acetylated units are randomly
distributed. In the presence of high ionic strengths,
solubility
is reduced. The high concentration of protons leads to the
screening of the electrostatic interactions occurring
between
polymeric chains, with subsequent establishment of chain
interactions and polymer precipitation. As a result,
chitosan
is not soluble in strong acids such as hydrochloric acid
solu-
tions with molarities higher than 0.1M.7
2.213.1.3. Chitosan Preparation: Chitin Isolation
andN-deacetylation
Commercially available chitin is most commonly prepared
from the exoskeletons of crab, shrimp, and prawn, obtained
as waste from the seafood processing industry. In these,
chitin
is tightly associated with proteins, inorganic material
(mainly
CaCO3), pigments, and lipids. Deproteinization and deminer-
alization are generally carried out by treatment with 12M
NaOH at 70 C or higher temperature, and 1.25M HCl atroom
temperature, respectively, deproteinization being usually
done prior to demineralization. Both treatments may lead to
the cleavage of chitin polymeric chains. In this sense, a
number
of alternative methods have been proposed in order to mini-
mize the hydrolysis of glycosidic linkages during chitin
extrac-
tion, including the use of proteolytic enzymes to remove
protein and EDTA to remove mineral. Finally, the pigments
present in the exoskeletons of crustaceans can be extracted
with
ethanol, acetone, or oxidizing agents such as KMnO4.2 The
preparation of squid chitin, although similar, occurs under
milder conditions, as b-chitin is composed exclusively of
chitinand proteins, with only traces of metal salts.8
Deacetylation may be carried out under acid or basic
conditions, but basic conditions are preferred, due to the
sus-
ceptibility of chitin glycosidic linkages to acid hydrolysis.
The
deacetylation of a-chitin is usually carried out using
strongaqueous bases at 90150 C for a few hours, to produce
chit-osan with a DA between 5% and 30%.2,4,5,9 High reaction
temperatures reduce the time required for deacetylation, but
result in increased hydrolysis of polymeric chains.
Deacetyla-
tion of chitin proceeds rapidly in 50% (w/v) aqueous NaOH at
100 C during the first hour of alkali treatment, but extensionof
the reaction time results rather in chain hydrolysis than in
significant deacetylation.2 To obtain chitosans with low DAs
(
-
including elemental analysis, colloid titration with a
polya-
nion, dye adsorption, and spectroscopies such as ultraviolet
(UV), IR, and liquid/solid state nuclear magnetic resonance
(NMR). The advantages and drawbacks of each technique
have been discussed.2,4,5 Among these, high-resolution
proton
NMR (1H NMR) is considered the most accurate technique for
the determination of the DA of chitosan. 1H NMR is usually
performed in D2O containing DCl, the DA value being deter-
mined from the relative integrals of acetyl (N-acetyl and
AcOH) and combined H2H6 protons.16,17 For chitosans
with high acetyl contents, the use of solid state 13C CP/MAS
NMR is preferred to 1H NMR, since complete dissolution of
the sample prior to analysis is required for 1H NMR. Because
of its simplicity associated with accuracy, IR spectroscopy
is the most frequently used technique. The use of FT-IR
spec-
troscopy for the determination of the DA is based on the
variation of an absorbance band characteristic of N-acetyl
groups, as a function of an internal reference band. The DA
value can be extrapolated from a calibration curve estab-
lished using an absolute technique, such as NMR. A number
of different methods have been proposed, differing in terms
of the analytical and reference IR bands used, as well as in
terms of the baselines used for the determination of the
corre-
spondent absorbance values.18 One of the most frequently
employed method is the one described by Baxter et al.19
It uses the amide I band at 1655 cm1 as the analytical band
This relation was validated by dye adsorption, for DAs
comprised between 0 and 55%. Later on, Brugnerotto et al.20
proposed the use of the amide III band (CN stretching cou-
pled with NH in plane deformation) at 1320 cm1 as theanalytical
band and the band at 1420 cm1 as the internalreference band. The
authors analyzed chitin/chitosan samples
covering the entire range of DAs, and used samples from
different sources. A very good linear correlation between
the
ratio of absorbance bands (A1320/A1420) and the experimental
values obtained from NMR was found in all the range of DA
values, which could be expressed by the following relation:
A1320=A1420 0:3822 0:03133DA % Brugnerotto et al. found superior
agreement between the
experimental and estimated DA values using this ratio of
absor-
bance bands than those involving the band at 3450 cm1
(Figure 2).
2.213.1.4.2. Molecular weightDepending on its source and
preparation procedure, chitosan
molecular weight may range from 300 to over than 1000 kD,
squid pen chitosans presenting usually higher molecular
weight, as compared to chitosans obtained from the exoskele-
tons of crustaceans.21 Several techniques can be used to
esti-
mate the average molecular weight of chitosan. Among them
are capillary viscometry, ultracentrifugation, and size
exclusion
chromatography (SEC) coupled with light scattering. Whatever
l.
um
pos
224 Materials of Biological Origin(the baseline is the one
originally proposed for the A1655/A2867ratio) and the hydroxyl band
at 3450 cm1 as the internalreference band. The sample has,
therefore, to be perfectly dry
and the IR spectra immediately recorded. The DA is deter-
mined as follows:
DA % A1655=A3450 115
20003000400020
25
30
35
40
45
50
55
60
65
70
75
%T
3431
Baxter et al. Brugnerotto et a
Waven
Figure 2 Squid pen chitosan infrared spectra, showing the
baselines proA1320/A1420 and A1655/A3450 ratio, respectively.the
technique used, the tendency of the polymer chains to
aggregate in solution constitutes a problem.2,22 SEC is the
most direct one, providing, in a single measurement, the
weight-average molecular weight and the number-average
molecular weight. From these two values, the polydispersity
40010001500
1657
14211317
ber (cm1)
ed by Brugnerotto et al.20 and Baxter et al.19 for the
determination of the
-
Chitosan 225tion of neutrophils, which are cells usually
associated with
acute inflammation, no evidence of other signs associated
with an inflammatory response, such as erytema and edema,
were found. A very low incidence of chitosan-specific
immune reactions was observed and, with time, collagen
deposition within and surrounding the implant, with capsule
formation, was found. However, the capsule was always
highly cellular and its thickness decreased over time.
Angio-
genic activity associated with the external implant surface
was
observed. Overall, chitosan with DA 8% was found to have a
high degree of biocompatibility. Recently, we assessed the
inflammatory response to chitosan porous scaffolds with
two different DAs (DA 4% and DA 15%) using a subcutane-
ous air-pouch model of inflammation.28 Implantation of
chitosan scaffolds with DA 15% induced a higher recruit-
ment of neutrophils and increased adhesion of inflammatory
cells during the early phase of implantation, while DA 4%
merely caused a slight increase in the number of leukocytes
present in the inflammatory exudates. With time, chitosanindex
Mw/Mn can be easily determined. Still, this technique
requires previous calibration of the SEC system with narrow
polydispersity standards of knownmolecular weight. The asso-
ciation of a light-scattering detector with SEC provides
infor-
mation on the absolute molecular weight as well as on
molecular size parameters, such as the radius of gyration
Rg.
The analysis is usually performed using CH3COOH/
CH3COONa buffers at pH near 4.5 as mobile phase, salt
being added to screen electrostatic repulsion between proto-
nated amine groups in chitosan.23 For the analysis, the
refrac-
tive index increment value (dn/dC) of chitosan in the
CH3COONa/CH3COOH system used is required. The dn/dC
values for chitosan may be found in literature, including
some
that are dependent on the DA.24 In this case, previous
charac-
terization of the DA may be required. Alternatively and if
possible, the dn/dC value can be measured using a
differential
refractometer. SEC coupled with light scattering is also the
technique described in ASTM guidelines for the determination
of chitosan molecular weight.
2.213.1.5. General Aspects of Biological Behavior
2.213.1.5.1. BiocompatibilityOne of the major issues that have
to be addressed while
envisaging biomedical applications of a biomaterial is bio-
compatibility. The nontoxicity of chitosan films was
initially
showed by Rao and Sharma25 using standard in vivo toxico-
logical tests to evaluate chitosan safety and haemostatic
potential. The biocompatibility of films with different DAs
was examined by Tomihata and Ikada,26 using a rat subcuta-
neous implant model. While chitosan films with DA 31%
induced a relatively severe inflammatory reaction, with
almost complete resorption after 4weeks of implantation,
films with lower DAs led to a lower inflammatory reaction
and degraded at a slower rate. Films with DAs 16% showeda very
mild tissue reaction. The biocompatibility of chitosan
porous scaffolds (DA 8%) was addressed by VandeVord
et al.,27 using a mouse intraperitoneal and subcutaneous
implant model. Histological assessment revealed an early
migration of neutrophils into the implantation area, which
resolved over implantation time. Besides this early
accumula-2.213.1.5.3. Bacteriostatic and fungostatic
propertiesChitosan exhibits an intrinsic antibacterial activity,
inhibiting
bacteria and fungi growth. As an example, in Staphylococcus
aureus cultures, chitosan treatment promotes structural
changes in the so-called membranewall complex leading to
the impairment of surface cell structures and to
bacterial2.213.1.5.2. CytocompatibilityA wide number of cells have
been successfully cultured on 2D
and 3D chitosan matrices envisaging cell-based regenerative
therapies, among them keratinocytes,29 chondrocytes,30,31
osteoblasts,3234 hepatocytes,35 and Schwann cells.36 The DA
was found to be an important parameter affecting cell adhe-
sion, lower DAs favoring cell adhesion. This effect was
reported
for a number of anchorage-dependent cells, such as keratino-
cytes,29 fibroblasts,29,37 dorsal root ganglion neurons,38,39
and
Schwann cells.40 In our lab, we investigated the DA effect
on
the behavior of osteogenic cells on chitosan films and
porous
matrices, using DAs in the range of 449%.32,33,41,42 These
studies revealed a similar tendency to increased cell
adhesion
on decreasing the DA, and showed that differences in the DA
as
small as 9% can be critical in terms of osteoblastic response
to
chitosan.41,42 For instance, in the case of rat bone marrow
stromal cells, cell adhesion, cytoskeleton organization,
prolif-
eration, and osteogenic differentiation were only observed
on
chitosan with DA 4%, while the same were hampered on
chitosans with higher DAs (13%). We hypothesize that theDA could
influence cell adhesion and osteoblast differentia-
tion by influencing the adsorbed layer of adhesion proteins.
For that, we performed protein adsorption studies
using125I-fibronectin. In line with the higher cell adhesion
levels
found, chitosan with DA 4% showed the highest fibronectin
(FN) adsorption both from a single FN protein solution and
from diluted serum.32,33 From these results we may speculate
that protonated amine groups from glucosamine units in chit-
osan may modulate cell adhesion to chitosan by promoting
the adsorption of cell adhesive proteins such as FN.
To improve cell behavior on chitosan matrices, several
attempts have been made, including physiadsorption of adhe-
sive proteins43 and covalent binding of cell adhesion pep-
tides.44,45 For instance, endothelial cell (EC) adhesion to
porous chitosan matrices is significantly enhanced upon
previ-
ous incubation of chitosan matrices in an FN solution.46
Inter-
estingly, we found that EC adhesion to FN-coated chitosan
matrices is also dependent on the DA. While cell adhesion
was impaired on DA 15%, ECs were able to adhere, spread,
and colonize chitosan matrices with DA 4%. Later on, protein
adsorption studies on scaffolds with DA 4% revealed a higher
number of exposed FN cell-binding domains, as well as
greater
ability to adsorb FN and to retain and exchange adsorbed FN
in
the presence of competitive proteins, in agreement with the
higher cell numbers found.scaffolds with DA 15% induced the
formation of a thicker
collagenous capsule and a high infiltration of inflammatory
cells within the scaffold. Since inflammation and healing
are
interrelated, these results showed the importance of the
care-
ful selection of the DA while developing chitosan porous
implants for tissue repair and regeneration.
-
47
226 Materials of Biological Origindeath. The biological
mechanisms underlying this property
remain poorly understood. Bacterial growth inhibition is
believed to be related to chitosan ability to establish
electro-
static interactions between chitosan cationic amino groups
and
anions, such as N-acetylmuramic acid, sialic acid, and
neura-
minic acid, present on the bacterial cell wall. In addition
to
electrostatic interactions, hydrophobic interactions
resultant
from the presence of N-acetylated residues in chitosan are
also thought to contribute to chitosan bacteriostatic
properties,
highly acetylated chitosans being reported to be excellent
floc-
culants of Escherichia coli suspensions.48
2.213.1.5.4. Enzymatic degradationIn nature, chitosan can be
hydrolysed by chitinases, chitosanases,
and lysozymes, as well as by nonspecific hydrolases, such as
a-amilases and lipases. In addition, like all polysaccharides,
chit-osan is vulnerable to acid hydrolysis and to
oxidativereductive
depolymerization reactions.49,50 In vivo, the human enzymes
involved in chitosan hydrolysis are only partially known.
In human serum partially, N-acetylated chitosans are
mainly depolymerized by lysozyme.50 Lysozyme is normally
present in the human serum, saliva, and other fluids, hydro-
lyzing preferentially the b-(14) glicosidic linkages
betweenN-acetylglucosamine (NAGA) and N-acetylmuramic acid
residues that occur in the cell walls of bacteria.
Therefore,
in addition to its natural substrate, lysozyme can hydrolyse
partially N-acetylated chitosans. The active site of
lysozyme
binds six sugar rings, and three
consecutiveN-acetyl-D-glucos-
amine residues are required for lysozyme catalytic
activity.51
As a consequence, the susceptibility of chitosan to lysozyme
depolymerization in vitro depends not only on the DA but
also on the distribution of N-acetylated units along
chitosan
chains. The initial degradation rate increases with the DA,
and this increase is more pronounced for N-acetylated chit-
osans prepared under homogeneous conditions than for
chitosans obtained by heterogeneous deacetylation.50,52 The
same trend was observed in vivo upon subcutaneous implan-
tation of chitosan films in a rat animal model.26 Chitosans
with low DAs may last several months in vivo.27
Crystallinity
is another parameter significantly influencing chitosan sus-
ceptibility to lysozyme hydrolysis, by reducing lysozyme
accessibility to the substrate. For instance, the low
degrada-
tion rates reported for chitosans with very low DAs have
been
frequently associated with the high levels of crystallinity
and
inter-molecular bindings present in these chitosans.26,27
Finally, chitosan solubility may eventually shade the effect
of lysozyme on chitosan degradation. This is the case of the
accelerated mass loss observed for chitosans with DAs close
to 50%, attributed to the enhanced solubility of chitosan
mole-
cules at physiologic pH.38,39
The enzymatic hydrolysis of chitosan in wound-healing
process was addressed by Muzarelli.49 Upon hydrolysis of
chitosan by lysozyme, the oligomers released activate macro-
phages, inducing the production of diffusible molecules,
such as interferon, tumor necrosis factor-a, and
interleukin-1.Activated macrophages secrete more lysozyme as well
as
N-acetyl-b-D-glucosaminidase and chitinase, which
furthercatalyze the depolymerization of chitosan into monomers.
These become available for further incorporation into
hyalur-
onate, keratan sulfate, and chondroitin sulfate.2.213.1.5.5.
ImmunoadjuvancyChitosan is chemotactic for neutrophils, which has
been
attributed to specific interactions of chitosan or its
oligosac-
charides with neutrophils receptors, such as selectins.27 In
addition to being chemotactic to neutrophils, chitosan shows
a biological aptitude for activating macrophages for
tumorici-
dal activity and for production of interleukin-1,53 as well
as
nitric oxide.54 The immunoadjuvant properties of chitosan
have been attributed to the NAGA units, rather than to
the glucosamine units. Macrophage activation appears to be
dependent on binding of NAGA to specific cell membrane
receptors, namely involved in the binding of mannose- and
NAGA-glycoproteins.54
2.213.1.5.6. Hemostatic and blood clotting propertiesChitosan is
a powerful hemostatic agent25 that induces blood
clotting, even in the presence of extensive anticoagulation
ther-
apy.55 Blood clotting was suggested to be related to the
possible
formation of polyelectrolyte complexes (PECs), involving
chit-
osan amino functionalities and negatively charged acidic
groups
present at the surface of erytrocytes. Benesch and
Tengvall56
suggested that the procoagulant activity of chitosan could
be
related to the ability of chitosan to bind fibrinogen. In
contrast,
chitin displays anticoagulant properties, increasing upon
O-sulfation,57 which is attributed to its similarity to heparin,
a
naturally occurring sulfated glycosaminoglycan (GAG) used as
anticoagulant agent in clinic.
2.213.1.5.7. Cell-binding propertiesChitosan is able to
agglutinate a variety of mammalian cells in
suspension. Cell adhesion to chitosan is attributed to
nonspe-
cific electrostatic interactions occurring directly between
pro-
tonated amine groups from glucosamine units and negatively
charged carboxylate and sulfate groups found in cell surface
proteoglycans.29,58 Close to the physiologic pH, the
majority
of chitosan ammonium groups are dissociated and subse-
quently uncharged. Still, the presence of a small amount of
nondissociated ammonium groups in chitosan chains is suffi-
cient to provide enough cationic sites and allow the
establish-
ment of electrostatic interactions.30
2.213.1.5.8. Wound-healing propertiesChitosan has been found to
accelerate dermal wound healing
and inhibit fibroplasia, showing a biological aptitude to
stim-
ulate cell proliferation and the deposition of an orderly
organized connective tissue.59 This behavior has been
related
to chitosan ability to activate macrophages for cytokine
production (transforming growth factor beta 1 (TGF-b1)and
platelet-derived growth factor (PDGF)), upon hydrolysis
by lysozyme.60,61 Moreover, Howling et al.62 suggested
that chitosan may interact with growth factors present in
serum, potentiating their effect, as chitosan has a
stimulatory
effect on dermal fibroblast proliferation when added to
serum-
containing culture medium, highly deacetylated chitosans
producing a stronger mitogenic response, as compared to sam-
ples with lower levels of deacetylation.
2.213.1.5.9. Bone-healing propertiesThe osteogenic potential of
chitosan was first reported by
Borah et al.63 in 1992. In this study, chitosan was used to
-
Chitosan 227In the control group, no sign of ostegenesis or
reparative
process was observed and bone marrow was rich in adipo-
cytes. The modified chitosan was reported to have a stimula-
tory effect on bone formation. Although several works have
demonstrated the osteoconductive properties of chitosan, it
was often combined with therapeutic molecules, growth fac-
tors, or calcium phosphates. The bone regenerative
properties
of unmodified chitosan are reported in a study of Park et
al.66
In this study, unmodified and PDGF-releasing chitosan
sponges were applied to rat calvarial defects. Both matrices
led to a significant increase in new bone formation, as com-
pared to untreated defects, which became completely filled
with fibrous connective tissue. In addition, a marked
increase
of bone formation and mineralization was observed in the
presence of PDGF, as expected. The subsequent studies of
Lee et al.67 supported these results. The effect of the DA
on osteogenesis was addressed by Hidaka et al.,68 who
implanted subperiosteally over the calvaria of rats mem-
branes prepared from hydroxyapatite and chitosan. DAs of
0, 6, 20, 30, and 35% were used. The authors reported for
DAs 20% a marked inflammatory reaction, followedby accumulation
of osteocalcin positive cells at the site of
implantation, while for lower DAs a mild inflammation
with minimal osteogenesis was observed.
2.213.1.6. Chitosan Functionalization
Chitosan has both reactive amino or amido groups at C(2)
positions, as well as primary and secondary hydroxyl groups
at C(6) and C(3) positions which can be used to prepare
derivatives with well-defined structures and biological pro-
perties under mild reaction conditions. Two modification
reactions were already addressed in this chapter, namely
dea-
cetylation and N-acetylation. Other chemical modifications
often explored to prepare versatile precursors and chitosan
derivatives for biomedical applications include other acyla-
tion reactions, N-phthaloylation, Schiffs base formation,
N,O-carboxymethylation, N-carboxyalkylation, and graft
copolymerization.2,9 Graft copolymerization, in particular,
has been extensively used to introduce side chains onto
chit-
osan, namely to obtain tailored hybrid materials composed
of natural polysaccharides and synthetic polymers.treat bone
defects, made in the endochondral long bones of
the rabbit. Chitosan reportedly stimulated osteogenesis with
closure of the critical size bone defects, as compared to
controls, in 812 weeks. As time progressed, the possible
oste-
ogenic, osteoconducting, and osteoinducting properties
became the subject of investigation. The most significant
works demonstrating the osteoconductive properties of chito-
san are possibly those carried out by Muzarelli et al.64,65 In
the
first work, Muzzarelli et al.65 treated bone defects made in
the tibiae of rabbits with freeze-dried methyl pyrrolidinone
chitosan. The experimental sites showed signs of neoformed
bone tissue, as opposed to controls, originating from the
pre-
existing bone, as well as from the periosteum. Subsequently,
Muzzarelli et al. prepared a modified chitosan (DA 8%)
carrying imidazole groups, to treat bone defects made in the
femoral condyle of sheep. Within 40 days after surgery,
the neoformed tissue occluded the surgical hole and assumed
a trabecular structure in the peripheral area of the
lesion.2.213.2. Processing
Chitosan is extremely versatile in terms of processing.
Films,
micro- and nanoparticles, porous scaffolds, micro- and nano-
fibers, and meshes can be produced with chitosan for a wide
range of applications.
2.213.2.1. Films and Porous Scaffolds (Freeze-Drying
andFreeze-Gelling)
Taking advantage of the solubility of chitosan in mildly
acidic
conditions, films can be produced by casting the resulting
gel
on a flat surface and then allowing the solvent to
evaporate.
Neutralization with an alkaline solution, washing, and
drying
are often employed to stabilize the films. These can then be
used as substrates for cell culture experiments.
Porous scaffolds can be prepared by freeze-drying, also
known as lyophilization. Acidic chitosan solutions are
frozen
to temperatures of 20 to 80 C, which causes formation ofice
crystals. The rate and temperature of freezing influence the
size of the ice crystals and consequently the size of the
pores
formed during the sublimation phase. The latter is carried
out
in a vacuum at low temperature, in order to avoid melting of
the chitosan-rich phase. After elimination of the ice
crystals,
drying can be continued to eliminate unfrozen water mole-
cules. Pores with dimensions in the range 40250mm and
porosities of 80% can be obtained. By applying a temperature
gradient along a certain direction, preferential orientation
of
the pores can be obtained to produce structures with aligned
porosity. Figure 3 presents laser scanning confocal and
scan-
ning electron microscopy (SEM) images of chitosan sponges
with two DAs (4% and 30%), which have been colonized
by MG-63 cells.42 It shows that a better spreading is
reached
with the lower DA.
In some cases, the freeze-drying method can damage the
pore walls during evaporation of the solvent. Also,
formation
of skin can also occur. To avoid these problems, the method
of
freeze-gelling has been proposed for producing porous struc-
tures of poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic
acid)
(PLGA), chitosan, and alginate.69 The method is based on the
extraction of the solution-rich phase by a nonsolvent for
the
polymer. Exemplifying for the case of chitosan, the
procedure
is the following. An acetic acid solution of chitosan is
frozen
to 20 C, which causes the formation of ice crystals. To formthe
pores, the ice crystals are removed by immersing the frozen
chitosan solution in an NaOH/ethanol solution at 20
C.Neutralization of the acid by the sodium hydroxide causes
the gelation of chitosan, whereas the ethanol dissolves the
ice
crystals. Subsequent evaporation of the ethanol at room tem-
perature produces the pores. The method has also been
applied
to produce composite scaffolds of chitosan and gamma-poly
(glutamic acid) (g-PGA) for the delivery of rhBMP-2.70
Con-centrations of chitosan and g-PGA were 4% and 1%,
respec-tively, in a 0.2M acetic acid solution. Freezing was
done
at 80 C and gelation was achieved by immersing the
frozenchitosan/g-PGA in a 3-M NaOH/ethanol solution at 20 C.
2.213.2.2. Nanofibers
Nanofibers of chitosan can be produced by electrospinning.
This technique has become extremely useful for the
-
Dimagidiud. M
228 Materials of Biological Originpreparation of nanofibers for
tissue engineering, due to the
wide range of polymers that can be processed to obtain nano-
fibers with diameters in the range 20400nm. Briefly, the
application of a sufficiently high voltage between a needle
and a metallic collector overcomes the surface tension
holding
a drop of liquid at the tip of the needle, resulting in a very
thin
jet of fluid being projected against the collector. The solvent
is
evaporated during the trajectory between needle and
collector,
resulting in a nonwoven structure. One of problems with
electrospun meshes is the slow cell infiltration due to the
high packing density of the nanofibers. To circumvent this
problem fast degrading nanofibers can be combined with
slow degrading fibers. This strategy has been developed
by,71
who have co-electrospun poly(epsilon-caprolactone) with
poly(ethylene oxide) (PEO) from two separate spinnerets.
The former polymer is slowly degradable, whereas the second
one is water-soluble, acting as a sacrificial component of
the
scaffold. Seeding these scaffolds with mesenchymal stem
cells
(MSCs) has show that cell infiltration improved with the
frac-
tion of sacrificial polymer. High porosities (of the order of
95%
to 97%) can be obtained also by coating microfibers with
Figure 3 Fluorescence microscopy and scanning electron
microscopicDAs. Red lines: chitosan walls of the pores; nuclei were
stained with prop(green). Adapted from Amaral, I. F; Sampaio, P.;
Barbosa, M. A. J. BiomeDA 30%nanofibers, as demonstrated by.72
Scaffolds made from poly-
caprolactone nanofibers electrospun onto polylactic acid
microfibers supported infiltration of human chondrocytes,
with higher porosities favoring cell infiltration.
2.213.2.3. Polyelectrolyte Complexes
The self-assembly of polymer chains due to electrostatic
inter-
actions is the basis for the formation of PECs. PECs are
formed
by reacting two oppositely charged polyelectrolytes in an
aque-
ous solution. PECs are generally biocompatible networks and
can be easily produced in the lab. PECs between chitosan
and many polyanions have been described. Polysaccharides
such as alginate, chondroitin sulfate, dextran sulfate and
gel-
lum gum form PECs with chitosan. Similarly, polyamino
acids (e.g., poly-L-Lysine and poly(aspartic acid)),
proteins
(e.g., collagen), and glycosoaminoglycans (e.g., hyaluronic
acid, HA) also bind electrostatically to chitosan, via
anionic
carboxylic or sulfate groups. The cationic amino groups
ofchitosan are responsible for the formation of electrostatic
bonds with the anionic groups of the other polyelectrolyte.
The formation of PEC hydrogels requires that the two
polymers are oppositely charged. Since the pKa of chitosan
is in the vicinity of 6.5, the other polymer must have a
lower pKa for electrostatic aggregation to occur. If
attraction
is too strong, a precipitate, and not a hydrogel, will form.
By modifying the ionic strength of the aqueous solution,
for example, by addition of salts such as NaCl, the electro-
static interactions can be modulated so that a homogeneous
hydrogel is formed. Functionalization of chitosan with posi-
tively charged (e.g., glycolchitosan73), or negatively
charged
(e.g., sulfate32,33) groups expands the range of PECs
that can be produced. Since no cross-linkers are required to
form the hydrogel, these materials usually have excellent
cytocompatibilty.
Using the principles that govern PEC formation, a film can
be produced using the layer-by-layer (LbL) method, by alter-
nate immersion of a substrate in solutions of positively and
negatively charged polyelectrolytes. LbL films are usually
very
thin (typically
-
form the chitosan/DNA complex nanoparticles. Chitosan
anteed during washing, which has limited the use of this
has been reported to have no such toxic effects.
Cross-linking
2.213.3. Biomedical Applications
Because of its well-known biocompability, chitosan is being
widely explored for biomedical and pharmaceutical applica-
tions. In the following sections the main areas of
application
and research will be discussed.
2.213.3.1. Wound Management
Chitosan is referenced in the wound management field for its
hemostatic properties85 Furthermore, the biological
properties
including bacteriostatic and fungistatic86 properties are
partic-
ularly useful for wound treatment. It also affects
inflammatory
cell function that helps in faster wound healing, and has an
aptitude to stimulate cell proliferation and
histoarchitectural
tissue organization.87
Chitosan has been explored as a topical hemostatic agent in
a variety of forms,88 reaching the market in recent years.
Agents
being commercialized include: (1) a dressing, which works by
becoming extremely adherent when in contact with blood,
sealing the wound and controlling bleeding (HemConMedical
Strain (%)
Chitosan 229cross-linker. Genipin, which is a natural product
used in tradi-
tional Chinese medicine and extracted from gardenia
fruit,nanoparticles with various ratios of chitosan to plasmid
OP-1
were used to transfect chondrocytes.81 Nanoparticles with a
chitosan/plasmid weight ratio of 10:1 entered the cells and
resulted in the expression of the plasmid, which maintained
its structural integrity. The size of the plasmid did,
however,
affect the efficiency of transfection to the cells.
2.213.2.5. Cross-linking
Like in other polymers, cross-linking of chitosan molecules
is
often used to increase the mechanical and chemical stability
of
chitosan. Cross-linking may involve covalent binding of
chit-
osan molecules to chitosan molecules, or to other polymer
chains. A hybrid polymer network is formed in this case.
The covalent bonds are formed between the two different
molecules, but they may also be established between mole-
cules of the same polymer. Another possibility is to add to
the
chitosan solution a nonreacting polymer before
cross-linking.
The chains of this polymer become entangled in the structure
of the cross-linked chitosan, contributing to its physical
rein-
forcement. These are called semi-interpenetrating networks
(semi-IPN). The reinforcing polymer can be subsequently
cross-linked to form a full-IPN. The most common cross-
linkers that have been used are dialdehydes, and
particularly
glutaraldhyde.82 The aldehyde group forms imine bonds with
the amino groups of chitosan and the reaction can be carried
out in aqueous medium, without the need for other molecules
to initiate the reaction. One of the disadvantages of the
method is the toxicity of glutaraldhyde. Removal of residual
glutaraldehyde molecules from the hydrogel cannot be
guar-bilayers and using higher concentrations of cross-linker,
the
effect on adhesion was more pronounced.
2.213.2.4. Micro- and Nanoparticles
Micro- and nanoparticles of chitosan can be prepared by
vari-
ous methods. The most common method consists in ionotro-
pic gelation of chitosan molecules by anions, such as the
polyanion tripolyphosphate (TPP)77 and sulfate.78 The
experi-
mental procedure involves drop-wise addition of the anion to
a chitosan solution under agitation. Agitation can be per-
formed mechanically, ultrasonically, or by a combination of
both. The rate of agitation controls the size of the
particles.
Chitosan nanoparticles prepared by gelation with TPP were
uptaken by A549 cells in percentages that depended on the
molecular weight (Mw) and DA.79 Uptake decreased by 26%
when the Mw decreased from 213 000 to 10 000 and by 41%
when the DA increased from 12% to 54%. The nanoparticles
cytotoxicity was lower for the higher DA.
Nanoparticles have been produced for incorporating
DNA, proteins and therapeutic agents. A complex coacervation
method has been often used for incorporating DNA. In com-
plex coacervation, separation of two oppositely charged col-
loids occurs. Normally, a chitosan solution (e.g., in sodium
acetate buffer) and a DNA solution (e.g., in sodium sulfate)
are
preheated to 50 C and then quickly mixed and vortexed to80of
chitosan/PEO blends with genipin resulted in more stable
and elastic films.83 Figure 4 shows the stressstrain curves
of chitosan/PEO obtained with different concentrations of
genipin. 0.1% genipin resulted in films that exhibited a
strain
to fracture of 90%. Genipin has also been used to stabilize
polylectrolyte multilayers of chitosan/hyaluronan and chito-
san/alginate.76 Cell adhesion was markedly influenced by
cross-linking. Water-soluble chitosan chlorides (low and
high
molecular weights) and chitosan glutamates (low and
high molecular weights) were cross-linked using various con-
centrations (5% to 20%) of genipin, to encapsulate cells
removed from bovine intervertebral discs.84 A cell viability
of
95% was obtained with the high molecular weight chitosan
glutamate cross-linked with 5% genipin.Figure 4 Stressstrain
curves of chitosan/poly(ethylene oxide)(PEO) films cross-linked
with genipin. The PEO used (LPEO) had aMw 600. Reproduced from Jin,
J.; Song, M.; Hourston, D. J.Biomacromolecules 2004, 5(1), 162168,
with permission from Elsevier.0
7
14
21
28
35
0.01% 0.1%
0.8%
5% water
0%
CSR/LPEO50
Str
ess
(MP
a)
0 20 40 60 80 100
0.5%
-
well as bone fillers, such as cements, adhesives for tissue
repair,
230 Materials of Biological Originand scaffolds for tissue
engineering. Among the latter, skin,
bone, cartilage, and nervous tissues are those where more
investigation is taking place, involving the use of chitosan
as
a scaffolding material or as an extracellular matrix (ECM)
analog.9496 The popularity of chitosan for tissue repair and
regeneration is owing to the fact that it can be readily
processed
into a variety of forms that include fibers, films, sponges,
or
hydrogels. This provides the possibility to mimic the shape
of
target tissue or interfaces. Moreover, the similarity of its
chem-
ical structure to some polysaccharide constituents of the
ECM
and the possibility to chemically modify it to impart
desired
functionalities add to the great potential of chitosan as a
bio-
material for tissue repair and regeneration.
In orthopedics, chitosan has been often used in combina-
tion with ceramics, such as hydroxiapatite and other
calcium-
containing ceramics to produce composites. These have been
investigated as bone fillers, and many were found suitable
as
bone-filling materials.97,98 The advantage of this approach is
to
develop bone substitute materials, which combine the biode-
gradability, strength, and flexibility of chitosan with the
osteo-
genic potential and hardness of the mineral filler. In
addition,
the chitosan matrix acts as a binder, preventing
postoperative
migration of the mineral phase.99 Chitosan has also been
explored as an adjuvant additive, to render calcium
phosphate
cements injectable and to enable their use in minimally
inva-
sive surgical procedures.100 Other bone cements combining
chitosan, hydroxiapatite, and poly(methyl methacrylate)
wereTechnologies, Inc.); (2) high surface area flakes which,
when
in contact with blood, swell, gel, and stick together to make
a
gel-like clot (being commercialized by MedTrade Products
Ltd.); and (3) a chitosan-coated nonwoven pad used in the
management of bleeding wounds (Abbot Vascular, USA). The
mechanisms underlying the action of chitosan are not com-
pletely understood, but it has been suggested to involve
vasoconstriction, and the rapid mobilization of red blood
cells, clotting factors and platelets to the site of the injury
as a
result of the positive charge on the chitosan molecule.89
This
action is reported to occur even in patients on
anticoagulants.
Because of its high absorption capacity of fluids, wound-
healing properties, adhesiveness, antibacterial activity,
and
film forming properties, chitosan has been explored for burn
and wound dressings.90 The modification of chitosan proper-
ties, either at the structural level or by association with
other
materials, has been explored60,61 to potentially improve its
biological performance. Despite these efforts, the
commercial
exploration of a wound dressing material based on chitosan
is
yet in its infancy.
Although the apparent potential of chitin and chitosan deri-
vatives in the preparation of sutures have long been
recognized,
there is still no commercial production of chitin-based
absorb-
able suture materials because of insufficient elasticity of
chitin
threads and certain limitations of their processability into
the
fiber form.91,92 Strategies to overcome some of these
hurdles
have been thoroughly reviewed by Pillai and coworkers.93
2.213.3.2. Tissue Repair and Regeneration
Internal medical applications for tissue repair and
regeneration
include orthopedic implants, such as bone pins and plates,
asalso developed, and reported to provide porous spaces for
osteoconduction, after chitosan degradation.101 Finally, the
association of growth factors with chitosan-based bone
fillers
has also been investigated, as a combinative strategy of
con-
trolled local drug delivery and bone regenerative therapy.49
Besides being used as a filler material, chitosan has also
been
employed as a coating material, in order to improve titanium
and hydroxyapatite implants osteointegration.102104 Envisa-
ging to improve the biocompatibility of electrolytically
depos-
ited apatite coatings on Ti alloys, Wang et al.105 prepared
a
hybrid calcium phosphate/chitosan coating. When compared
to apatite coatings, this hybrid coating revealed to be more
cytocompatible towards bone marrow stromal cells.
There are a wide variety of adhesives available for use in
surgery, ranging from cyanoacrylates to fibrin-based
mixtures.
Chitosan use in the design of new tissue adhesives was moti-
vated by the fact that it can bind to collagen due to
hydrogen
bonding and polyanionicpolycationic interactions. It is said
to overcome some of the limitations of currently available
materials. It is not blood-derived, and in light activated
appli-
cations can be used without significant temperature raise.
Lauto and coworkers106,107 have developed flexible and
insol-
uble strips of chitosan-based adhesives that incorporate
indo-
cyanin green dye, for use as a bandage to fix rectangular
sections of sheep intestine using a diode laser. The
chitosan
bandage bonded effectively to tissue without sutures and
pre-
served the ECM structure avoiding irreversible thermal dena-
turation of imbedded bioactive proteins. A
photo-cross-linked
chitosan hydrogel showed strong sealing ability when tested
in
a rabbit animal model of a punctured carotid artery and
lung,
stopping the bleeding and air leakage, respectively.108 The
sealing ability of the chitosan hydrogel was found to be
similar
or even stronger than that of fibrin.
Table 1 provides examples of works being developed in the
field of tissue engineering using chitosan as a
scaffold/matrix
material.
Many studies have been reported on the use of chitosan as a
scaffold in skin tissue engineering due to its many
advantages
for wound healing, such as hemostasis, accelerating tissue
regeneration, and stimulating the fibroblast synthesis of
colla-
gen.121 The use of chitosan for the preparation of skin
substi-
tutes was successfully explored in a blend with bovine
collagen
types I and III and GAGs, where fibroblast and keratinocytes
have been cocultured. The blend-based porous substrate acts
as
a scaffold for fibroblasts, thereby producing a living
dermal
equivalent, which once epithelialized results in
reconstructed
skin.122 The use of this skin substitute has been proven to
lead not only to the reconstruction of surface epithelia for
the treatment of pathological conditions of skin, but also
as
a testing platform for pharmatoxicologic studies, serving as
a
validated alternative to animal tests.
In articular cartilage tissue engineering, the ideal
scaffold
should mimic the natural environment in the articular
cartilage
matrix, which is highly hydrated and rich in ECM components,
such as type II collagen and GAGs. These are known to play a
key role in the expression of the chondrocytic phenotype
and in supporting chondrogenesis in vitro as well as in
vivo.
Chitosan has been found particularly interesting for hyaline
cartilage tissue engineering, due to its structural similarity
with
HA, a GAG abundant in the ECM of cartilage.123 The
-
Table 1 Current applications of chitosan scaffolds in tissue
engineering
Application Scaffold DA (%) Scaffold properties Performed
biological studies References
Skin Asymmetric genepin-cross-linked chitosan membranecontaining
collagen Inanospheres
15 Asymmetricmembrane(porosity 4258%)
In vitro static primary culture of rat skinfibrobasts
In vivo evaluation of skin regeneration in arat animal model
109
Cartilage Chitosan/b-chitin scaffolds 2 Porous (pore
size50100mm)
Rabbit chondrocytes (isolated fromarticular cartilage) culture
and ECMproduction assessment
110
Cartilage Temperature-responsivechitosan hydrogel
(withb-sodiumglycerophosphate andhydroxyethyl cellulose)
14 Hydrogel In vitro cultures with adult sheep
articularcondrocytes
In vivo implantation in articular cartilagedefects in the sheep
model
111
Nucleuspulposus ofintervertebraldiscs (IVD)
Chitosan/glycerophosphategels
Chitosan/glycerophosphate/hydroxyethyl cellulose
gelsChitosan/genipin gels
Chitosan:5% and21%Glutamatesalt ofchitosan:15%
Hydrogel In vitro primary cultures of peripheralannulus fibrosus
and the centralnucleus pulposus cells isolated fromIVDs from the
tails of bovine steers
112
Bone Chitosan/TCP scaffolds n.m. Porous (pore size100mm)
Fetal rat calvarial osteoblastic cellsproliferation, viability,
anddifferentiation
113
Bone Chitosan/nonsinteredhydroxylapatite particles
12 BilayeredPorous layer (poresize 100400mm;porosity 30%)
Cytotoxicity of extracts (fibroblasts)Osteogenic and
chondrogenicdifferentiation studies withmesenchymal stem cell (from
humanadipose tissue)
114
Bone Chitosan and PDGF-BBloaded chitosan scaffolds
n.m. Porous (pore size100mm)
Cytotoxicity tests: rat calvarialosteoblastic cells adhesion
andproliferationBone regeneration ability in a ratcalvarial
defect
66
Bone Chitosan and chitosan/chondroitin
sulfatescaffoldsChitosan/PLLA scaffoldsChitosan coated
PLLAscaffolds(all loaded or not withPDGF-BB)
30 Porous (pore size100150mm)Porous (pore size150200mm)Porous
(pore size100150mm)
Rat calvarial osteoblastic cells adhesionand proliferationBone
regeneration ability in a ratcalvarial defect
67
Bone Chitosan/gelatin andchitosan/gelatin/b-TCPmacroporous
scaffolds
10 Porous (pore size322mm)Porous (pore size185420mm)
Biocompatibility evaluation(subcutaneous implantation in a
rabbitmodel)
115
Bone Chitosan scaffolds andchitosan scaffoldsmodified with RGDS
orRGES peptides
15 n. m. Rat osteoblast-like cells (ROS 17/2.8)adhesion,
proliferation, anddifferentiation
45
Nerve Chitosan conduits containingoriented filaments
ofpolyglycolic acid
7.7 Nonporous chitosanconduit containingoriented filamentsof
polyglycolic acid
Bridging sciatic nerve across a 30-mmdefect in Beagle dogs
116
Nerve Chitosan 8 Nonporous conduit Implantation of
extramedullary conduitsseeded with neural stem and progenitorcells
derived from transgenic greenfluorescent protein rats after
spinalcord transection
117
Ligament Chitosan-based hyaluronanhybrid polymer fibers
19 Fibers Rabbit fibroblast adhesion andproliferation; ECM
productionassessment
118
(Continued)
Chitosan 231
-
fold
s wrag160 nm and chitosan films
/chitffol 9rosi
232 Materials of Biological Originpossibility of using chitosan
in the hydrogel form is an added
value in such application. In this form it may closely match
the
natural mechanical properties of hyaline cartilage. Further-
more, its application can be performed by aminimally
invasive
method. A number of in situ gelling chitosan-based hydrogels
are currently under investigation for this purpose.124,125
In recent years, considerable attention has been given to
the
application of chitosan-based materials in the field of
orthope-
dic tissue engineering. Interesting characteristics that
render
chitosan suitable for this purpose are its
biocompatibility/bio-
degradability, structural similarity to ECMGAGs, intrinsic
anti-
bacterial nature, ability to bind anionic molecules such as
growth factors, GAGs, and DNA, as well as its ability to be
molded into porous structures suitable for cell ingrowth and
osteoconduction.126 In this sense, a wide number of support
matrices in the form of injectable gels or porous scaffolds
have
been developed for bone tissue-engineering applications.
Most
often, chitosan is used in combination with a variety of
materi-
als, such as ceramics, PLLA, gelatin, GAGs, as well as
growth
factors, in an attempt to improve its mechanical properties,
osteoconduction, and ability to induce bone regeneration
(see
Table 1). Binding with cell adhesive motifs has also been
explored, in order to promote cell adhesion.
Although chitosan has been used as a scaffold for articular
cartilage and bone formation by direct differentiation of
mesen-
chymal cells into chondrocytes and osteoblast, respectively,
only
recently it has been used as a template for endochondral
ossifi-
cation.31,127 The endochondral ossification pathway involves
an
intermediate cartilage stage and is responsible for the
formation
of long bones, vertebra, and the cranial base during
develop-
ment. Oliveira et al. have subcutaneously implanted a
transient
cartilage scaffold based on chitosan and a permanent
cartilage
scaffold in nude mice (see Figure 5). Only in the former,
the
Table 1 (Continued)
Application Scaffold DA (%) Scaf
Liver Galactosylated chitosanelectrospun nanofibers
15 Fiberaveof
Vascular Human-like collagen (HLC,produced by recombinantE.
coli)/chitosan tubularscaffolds
2025 HLCsca46po
n. m. not mentioned.depositionof ectopic bonewas detected, as
early as 1month after
implantation. After 3months, bone trabeculae and bone mar-
row cavities were formed inside the scaffolds. The bone
depos-
itedwas similar to the boneof themice vertebra. Interestingly,
no
bone formation was observed in control implants, for the
time
span of the study.
When the extent of nerve damage prohibits the direct
approximation of the two nerve stumps in peripheral nerve
repair, autologous nerve grafting is considered the surgical
treatment of choice. Artificial nerve guides, where a tube
bridges the nerve gap, offer a promising alternative,
preventing
the sacrifice of a donor nerve and possible neuroma
formation
at the donor site. Furthermore, the use of synthetic nerveguides
reduces operation time and prevents the mismatch
between the damaged nerve and the graft. Peripheral nerve
correction performed with chitosan-based nerve conduits has
been explored with success.116 Chitosan-based conduits pres-
ent a number of advantages over other proposed guides,
namely, the readiness of tuning their physical
properties38,39
and surface chemistry by changing the DA of chitosan, which
in turn influences cell adhesion behavior of the key player
cells
in the nerve regeneration process.40 The surface
modification
of chitosan scaffolds with cell adhesion proteins or motifs,
such as the pentapeptides YIGSR and IKVAV, which bind to
neural cell membrane receptors, has also been investigated
either by physiadsorption128 or chemical grafting.129131 The
use of chitosan-based conduits for spinal cord injury
treatment
is also under study with the first in vivo studies showing
promising results.117,132,133 The incorporation into the
scaf-
folds of proteins that can enhance nerve regeneration is
being
assessed as well.134 Moreover, the possibility of culturing
neu-
ral stem cells in these constructs also opens new avenues
for
addressing nerve lesion treatments.135
A transversal problem in the field of tissue engineering has
been the vascularization of the tissue-engineered constructs
postimplantation. The insufficient supply of oxygen and
nutri-
ents to the inner part of the cellmatrix constructs in the
initial
phases after implantation136 was found to be contributing to
the limited success of many of the proposed tissue
regeneration
strategies. To overcome this unsolved issue, a number of
stra-
tegies are being developed to create a vascular network able
to
deliver oxygen and nutrients throughout an implanted cell
matrix construct. One of the promising approaches has been
the precolonization of the scaffolds with vascular cell
types,
such as ECs or endothelial progenitor cells.137,138 We have
shown that the endothelization of chitosan scaffolds was
Hepatocytes function assessmentosan tubulard (pore sizemm;ty
85%)
In vitro human venous fibroblast cultureIn vivo biocompatability
assessment inrabbits livers
120properties Performed biological studies References
ith ane diameter
Attachment and culture of primary rathepatocytes on the fibrous
scaffolds
119found to be possible when these were precoated with the
ECM protein FN.46 However, as observed for other cell types,
the DA was found to be a key parameter modulating EC adhe-
sion to FN-coated chitosan scaffolds (see Figure 6) by
influen-
cing the adsorbed protein layer. The selection of suitable
DAs will therefore be highly important for the design of new
vascularization strategies.
2.213.3.3. Delivery of Therapeutic Agents
The use of chitosan in the pharmaceutical industry spans
from
its application as an excipient139 to its application as a
vehicle
for the controlled delivery of therapeutic agents.
-
(q)(p)
1st month 2nd month 3rd month 4th month
(a)
(e)
(i)
(j)
(m) (n)
(f) (g) (h)
5th month
ExperimentalVertebraControl
(b) (c) (d)
50mm
200mm200mm200mm200mm
50mm
2 mm
50mm50mm
200mm
50mm
50mm 50mm 50mm
50mm50mm
200mm 200mm(l)
(o)
(r)
(k)
Figure 5 Histological analysis of chondrocyte/chitosan scaffolds
implanted in the subcutaneous region of nude mice. Axial paraffin
sections were cutthrough the body of the mouse (I, 5month time
point) to include the experimental and control scaffolds. Sections
were stained with H&E andphotographed. Details of bone forming
in experimental scaffolds during the first 4months of implantation
are shown in images (a) through (d). Images(e), (f), (g), and (h)
correspond to high magnifications of (a), (b), (c), and (d),
respectively, showing the beginning of vascular invasion (arrow
heads).The control scaffolds do not undergo visible changes during
the first 5months of implantation, and therefore only the 5th month
is included in thisfigure. A complete section is shown after
5months of implantation in image (i). Control scaffold is on the
left and experimental is seen on the right side ofthe image.
Details of control scaffold (j), vertebral body (k), and
experimental scaffold (l) can be observed. Higher magnifications of
control (m, p),mouse vertebra (n, q), and experimental scaffold (o,
r) show tissue details. Deep in the control sample (m), only
cartilage tissue can be observed.Vascular invasion of cartilage
(arrow head in o) and bone deposition (arrow in o) can be observed
in the experimental sample. Detail of vertebra (n, q)shows the
presence of bone (arrow) and blood vessels (arrow head). Note bone
formation deep in the experimental scaffold as well as the presence
ofblood vessels (r). Adapted from Oliveira, S. M.; Mijares, D. Q.;
Turner, G.; Amaral, I. F.; Barbosa, M. A.; Teixeira, C. C. Tissue
Eng. 2009, 15(3): 635643,with permission from Liebert.
Chitosan 233
-
234 Materials of Biological Origin72h
DA 4%
(a) (d)
4h
300mmTaking advantage of the fact that positively charged
chitosan
can bind to negatively charged lipids and bile salt
components,
interfering with the lipid absorption process in the gut,
chito-
san has been introduced in the market as a weight and
choles-
terol control agent. However, the efficacy of its use has
been
questioned in the field.140,141 Furthermore, chitosan has
been used for the production of controlled release implant
systems for delivery of hormones, proteins, and other
peptides
over extended periods of time.142144 The mucoadhesive prop-
erties and transmucosal absorption-promoting characteristics
of chitosan have been exploited especially for nasal145,146
and
oral delivery147151 of polar drugs to include peptides,
proteins,
and nucleic acids and for vaccine development.132,133,152
The immune adjuvant behavior of chitosan153 has also been
drawing the attention of the field to its application in
vaccine
development154156 and cancer treatment.157159
Additionally, its cationic nature and high charge density in
slightly acidic conditions allow chitosan to form PECs
nucleic
(b)
(c)
(e)
(f)
144
300mm
300mm
Figure 6 Confocal laser scanning microscopy of human pulmonary
microvascaffolds previously incubated in a 40mgml1 fibronectin
solution. Cells werImages were collected after 4, 72, and 144 h
after cell seeding. HPMEC-ST1.6the scaffolds (a) to (f). Cell
spreading as well as the cellular layer covering themagnification
images (d) to (f). In these, the 3D scaffold is shown in blue
duchitosan scaffolds with DA 15%, very few cells were found after
72 and 144 hfor which the same bar corresponds to 75 mm. Adapted
from Amaral, I. F.; Upermission from Elseiver.(g) 300mm75mmDA
15%acids both DNA and RNA.94,95,160,161 Although chitosan is
able to condense nucleic acids and protect them from
nuclease
degradation, its main advantage over other nonviral vectors
relies on its low cytotoxicty and biodegradability. However,
chitosans poor solubility under physiological conditions and
low transfection efficacy has impeded its use as a nucleic
acid
carrier. To overcome such limitations, a number of strategies
for
chitosan modification have been proposed by our group and
others. These include the increase of chitosan overall
positive
charge by quaternization,162 modification of its buffering
capac-
ity163 and functionalization of chitosan with cell-binding
mole-
cules for receptor-mediated cell internalization.164
2.213.3.4. Other Applications
Other potential applications of chitosan include cosmetics,
contact lenses, dialysis membranes, and coatings of the
inner
lumen of blood-contacting polymeric tubes, onto which an
(h)
(i) 300mm
300mm
75mm
74.14mm
scular endothelial cells (HPMEC-ST1.6R cell line) grown on
chitosane labeled with calcein AM resulting in green fluorescence
of viable cells.R cells seeded on scaffolds with DA 4% were able to
spread and colonizepore walls after 144 h of cell culture are shown
in more detail in the highere to chitosan autofluorescence upon
excitation by the 405 nm laser. Onof cell culture (h) and (i).
Scale bar is 300 mm except for images (d) to (f),nger, R. E.;
Fuchs, S. et al. Biomaterials 2009, 30(29), 54655475, with
-
tested in depth.
16. Fernandez-Megia, E.; Novoa-Carballal, R.; Quinoa, E.;
Riguera, R. Carbohydr.Polym. 2005, 61(2), 155161.
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