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.

Chitosan 23517. Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26(1), 8794.18. Shigemasa, Y.; Matsuura, H.; Sashiwa, H.; Saimoto, H. Int. J. Biol. Macromol.

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