2.213. Chitosan

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chitosan

Text of 2.213. Chitosan

  • 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

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    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