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Research review paper

CHITIN A promising biomaterial for tissue engineering and stem


cell technologies
Andrew C.A. Wan , Benjamin C.U. Tai
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, 138669, Singapore
a b s t r a c t a r t i c l e i n f o
Article history:
Received 2 July 2013
Received in revised form 13 September 2013
Accepted 23 September 2013
Available online 29 September 2013
Keywords:
Chitin
Stem cell
Tissue engineering
Scaffolds
Chitin, after cellulose, is the second most abundant natural polymer. With a 200-year history of scientic
research, chitin is beginning to see fruitful application in the elds of stem cell and tissue engineering. To date,
however, research in chitin as a biomaterial appears to lag far behind that of its close relative, chitosan, due to
the perceived difculty inprocessing chitin. This reviewpresents methods to improve the processability of chitin,
and goes on further to discuss the unique physicochemical and biological characteristics of chitin that favor it as a
biomaterial for regenerative medicine applications. Examples of the latter are presented, with special attention
on the qualities of chitin that make it inherently suitable as scaffolds and matrices for tissue engineering, stem
cell propagation and differentiation.
2013 Elsevier Inc. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1776
2. Improving the processability of chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1778
2.1. Water-soluble chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1778
2.2. Chitin derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1778
2.3. Dissolution in alternative solvent systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779
2.4. -Chitin vs -chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779
3. Physicochemical and biological properties of chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779
3.1. Degradability of chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779
3.2. Immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779
3.3. Mechanical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779
4. Chitin scaffolds for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1780
4.1. Modication of chitin scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1780
4.2. Cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1780
4.3. Bone and tendon tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1781
5. Chitin for stem cell technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1782
5.1. Applications of stem cells for regenerative medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1782
5.2. Chitin-based bers for the propagation and differentiation of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1782
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783
1. Introduction
Chitin is the second most abundant polysaccharide in nature after
cellulose, found mainly as the exoskeleton material of arthropods and
crustaceans, and the cell wall of fungi (Buck and Obaidah, 1971). Its
chemical structure is depicted in Fig. 1, in comparison to three closely
Biotechnology Advances 31 (2013) 17761785
Corresponding author at: Institute of Bioengineering and Nanotechnology, 31 Biopolis
Way, The Nanos, #04-01, 138669, Singapore. Tel.: +65 6824 7134; fax: +65 6478 9082.
E-mail addresses: awan@ibn.a-star.edu.sg (A.C.A. Wan), btai@ibn.a-star.edu.sg
(B.C.U. Tai).
0734-9750/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.09.007
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related biomolecules, chitosan, cellulose and mureic acid. The repeating
unit of chitin is an oxygen-containing hexose ring with an acetamido
group at the second carbon position, strung together by -glycosidic
bonds. The closest chemical entity to chitin, chitosan, is found naturally
as the main polysaccharide constituent of the cell wall for some fungal
species (Bartnicki-Garcia, 1968). Chitin and chitosan are effectively the
same macromolecular entity, varying only in the fraction of acetylated
repeating units, and possessing the chemical structure shown in Fig. 1.
Here, x is the degree of acetylation, dened as the mole ratio of acetylat-
ed repeating units over that of the total repeating units. In fact, the main
route for chitosan production is via the deacetylation of chitin (Knorr,
1991). However, deacetylation, in practice, does not proceed to comple-
tion, which implies that the chitosan obtained fromcommercial sources
is chitin with a very low degree of acetylation (typically, x = 0.1 to
0.25). Similarly, chitin derived from natural sources has been re-
ported to contain a certain percentage of anhydroglucosamine re-
peating units, i.e. not being completely acetylated (Rudall, 1963).
For the purpose of this review, we would refer to the polysaccharide
as chitin ((1 4)-2-acetamido-2-deoxy--D-glucan) when x N 0.5,
and refer to it as chitosan ((1 4)-2-amino-2-deoxy--D-glucan)
when x b 0.5. This would enable us to make comparisons between
chitin and chitosan by referring to the literature, based on the re-
ported degree of acetylation. It is to be emphasized however, that
such a distinction is articial, and made merely for comparative pur-
poses. In reality, chitin and chitosan are two points on a continuum
of materials that share the same basic structure and differ only in
their degree of acetylation.
If one were to carry out a literature survey on the volume of past re-
search done on chitosan and chitin in the area of tissue engineering, a
great disparity would be observed. Using the keywords chitinor chitosan
and tissue engineering while carefully observing the abovementioned
criteria on the denition of either polysaccharide based on degree of
acetylation, publications on chitosan biomaterials on the Medline data-
base number about 892 in the period from 2002 to 2012. In contrast,
the same search on chitin biomaterials generates only 165 publications.
One may attribute this to the difference in functionality between chitin
and chitosan. Chitosan's popularity as a biomaterial may derive from
the fact that it possesses abundant amino groups. At sufciently low
pH, these become charged, allowing complexation with DNA that has
become the basis of its application in gene delivery (Cui and Mumper,
2001; Ercelen et al., 2006; Fang et al., 2001; Germershaus et al., 2008;
Hejazi and Amiji, 2003; Jayakumar et al., 2010a; Katas and Alpar, 2006;
Tahara et al., 2008). In the area of tissue engineering, scaffolds have
been imbued with chitosan-DNA nanoparticle complexes where the
transfected genes may express bioactive proteins that facilitate tissue
regeneration (Peng et al., 2009; Zhang et al., 2009). Polyionic complexes
of chitosan with polyanions such as alginic acid (Tai et al., 2010a; Yim
et al., 2007), heparin (Yim et al., 2007) and chondroitin sulfate (Chen
et al., 2007) have been investigated for the fabrication of tissue scaffolds.
Chitosan's abundant amino groups also provide the functionality for
crosslinking or conjugation of biologically active ligands, for example,
via carbodiimide chemistry (Rafat et al., 2008). However, the primary
reason for chitosan's vastly higher usage may be its ease of solubilization,
making it amenable for processing into various forms. The amino group
of chitosan is protonated in dilute weak acids such as acetic acid,
resulting in the formation of a soluble polybaseacid complex. As such,
chitosan can easily be cast into membranes, formed into bers or cap-
sules by precipitation in a coagulating bath, subject to homogenous
functionalization reactions (Rafat et al., 2008) or even applied directly
in solution (Campos et al., 2006) or gel (Alemdaroglu et al., 2006) forms.
Chitin, on the other hand, is relatively difcult to process, and due to
its insolubility in aqueous solution, has become less accessible to
biological laboratories. While the earlier work on chitin had established
its solubility instrong acids suchas dichloroacetic acid and trichloroace-
tic acid (Austin, 1975a,b), and more recently, highly polar uorinated
solvents such as hexauoroisopropyl alcohol (Capozza, 1976), these so-
lution systems are unattractive due to their corrosive nature, toxicity
and/or degradative effect on chitin polymer. There are, however, ways
to make chitin more amenable to processing. This review is divided
into four parts. The rst (Section 2) summarizes approaches towards
improving the processability of chitin, which have allowed for the
successful employment of chitin in the area of stem cells and tissue
engineering. In Section 3, the physicochemical and biological properties
of chitin are discussed, where relevant, in comparison to the lower
acetylated form (chitosan). Section 4 delves into the use of chitin as a
scaffold for tissue engineering. In Section 5, we detail how chitin has
more recently been applied for stem cell technologies, a development
which relies on the advances described in the preceding sections.
Fig. 1. Chemical structure of (A) chitin, when x N 0.5 or chitosan when x b 0.5; (B) cellulose; and (C) murein, an analogous carbohydrate to chitin found in the cell wall of bacteria;
(D) schematic diagram of O-GlcNAcylation. The N-acetylglucosamine (GlcNAc) moiety is linked via serine or threonine residues of the protein.
1777 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785
2. Improving the processability of chitin
2.1. Water-soluble chitin
It was foundthat chitin, whendeacetylated to a degree of acetylation
of about 0.5 (Fig. 2A), became water-soluble (Sannan et al., 1976). Alter-
natively, the same water solubility could be imparted by acetylating half
of the amino groups available in chitosan (Kubota et al., 2000). This
water-solubility is postulated to result from the loss of crystallinity
and intra-chain hydrogen-bonding with deacetylation, leading to
a crystal structure approaching that of the more water-swellable
-chitin (covered in Section 2.4) (Cho et al., 2000). For the purpose of
this review, the water-soluble form of chitin/chitosan with degree of
deacetylation of approx. 0.5 would be referred to as water-soluble chi-
tin. Compared to water-soluble chitosan (degree of acetylation b0.5),
which is commonly obtained by the depolymerization of chitosan
(Qin et al., 2002), water-soluble chitin of relatively high molecular
weight can be obtained, which may confer an advantage in terms of
mechanical properties. In contrast to regular chitosan, which dissolves
in weak acids and requires a pH of b~5 to remain in solution, water-
soluble chitin is soluble at neutral or near-neutral pHs, i.e. under condi-
tions that preserve the viability of living cells. Cells can thus be encapsu-
lated in water-soluble chitin-based hydrogels or bers, and several
applications to that effect have been investigated (Leong et al., 2013;
Lu et al., 2012b; Wan et al., 2004; Yim et al., 2006). Two points should
be noted when applying water-soluble chitin for stem cell or tissue
engineering applications. Firstly, chitin and chitosan are degradable by
lysozyme and chitinase to different extents depending onthe respective
degree of deacetylation (Sashiwa et al., 1990; Tomihata and Ikada,
1997; Zhang and Neau, 2001). Thus, the in vivo degradation prole of
highly acetylated chitin would differ signicantly from that of water-
soluble chitin, which contains half the number of acetyl groups per
mole repeating unit. Second, the application of water-soluble chitin as
a scaffold in an aqueous environment requires its prior insolubilization
by complexation or crosslinking. Such a process would be unnecessary
for chitosan, which is insoluble at physiological pH. Here, methods
that have been fruitfully applied to the crosslinking of chitosan (e.g.
genipin (Mi et al., 2002)) would directly apply to water-soluble chitin.
Similarly, the availability of amino groups on water-soluble chitin al-
lows it to undergo polyelectrolyte complexation with other polyanionic
molecules, although the relatively lower charge density (in comparison
to chitosan) would affect its complexation properties. Composite lms
of water-soluble chitin and other polysaccharides, such as amylose,
have also been fabricated and studied for their antibacterial activity
(Suzuki et al., 2005).
2.2. Chitin derivatives
Chitin contains two hydroxyl groups per repeating unit, and both
hydroxyls can be used for functionalization; the primary hydroxyl on
C6 is generally more reactive, as it is the less sterically hindered
(Fig. 1). Chitosan, in comparison, possesses proportionally more
amino groups on C2 that could be employed for functionalization. An
obvious method to enhance the water-solubility of chitin would be to
functionalize it withhydrophilic groups or those containing moieties ca-
pable of hydrogen bonding. Mainly towards that end, carboxymethyl,
hydroxypropyl, glycol, phosphoryl and sulfuryl derivatives of chitin
have been synthesized (Hirano, 1988; Nishi et al., 1986; Nishimura
et al., 1984; Park and Park, 2001; Tokura et al., 1994; Trujillo, 1968)
(Fig. 2B). As for water-soluble chitin, a crosslinking strategy would be
important to render these derivatives suitable for applications that re-
quire a solid form of the material, for example, as tissue engineering
scaffolds. It is pertinent to note however, that these water soluble chitin
derivatives may possess markedly different characteristics as compared
to their parent material, e.g. enzymatic degradation properties, and
should therefore be separately investigated for the specic applications
concerned (Kaifu and Komai, 1982; Kumirska et al., 2010; Kurita et al.,
2003; Saiki et al., 1990; Tong et al., 2011).
Fig. 2. Approaches towards improving the processability of chitin.
1778 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785
2.3. Dissolution in alternative solvent systems
While chitin is insoluble in pure organic solvents such as N-methyl
pyrrolidone or dimethylacetamide, the addition of a small percentage
of salt such as lithium chloride enhances its solubility dramatically
(Austin, 1988; Austin et al., 1981). The observation can be explained
by invoking coordination of the salt with the acetylcarbonyl group of
chitin to form a soluble complex (Fig. 2C). Solutions of chitin in organic
solvents have been precipitated into bers (Zhong et al., 2010), cast into
lms (Yusof et al., 2003) and used to form composites with inorganic
materials such as hydroxyapatite (Wan et al., 1997). One concern in
using such processing routes for chitin would be the presence of resid-
ual organic solvent in the nal biomaterial product. Thus, the absence
of solvent impurities should be ascertained by a stringent quality
check prior to the application.
Recently, chitin solutions in ionic liquids have also been obtained,
which were processed into highly porous structures by supercritical
uid drying (Silva et al., 2011), or mixed with cellulose to formcomposite
gels and lms (Takegawa et al., 2010). Alcoholic calciumchloride solvent
systems have also been employed to process chitin into scaffolds by ly-
ophilization(Kumar et al., 2011; Maeda et al., 2008). As a means tofurther
improve its solubility, chitin is sometimes subject to depolymerization
(Kubota et al., 2000).
2.4. -Chitin vs -chitin
Chitin exists as different allomorphic forms in nature, which vary in
terms of their polymer chain structure and crystallinity (Jang et al.,
2004). Most chitins, such as insect and crustacean chitins, are -chitin
in the native state, while the rarer -chitin allomorph occurs in squid
penand some diatoms. -Chitinexhibits a two-chain, antiparallel struc-
ture, while -chitin has a one-chain unit cell with a parallel-chain struc-
ture, withintramolecular hydrogen-bonding (Fig. 2D) (Blackwell, 1969;
Minke andBlackwell, 1978; Rudall and Kenchington, 1973). The weaker
hydrogen-bonding fromthe parallel-chain structure of -chitin may ac-
count for its higher chemical reactivity (Atkins, 1985; Kurita et al.,
2005). In addition, -chitin has the unique feature of incorporating
small molecules, including water, into its crystal lattice to form crystal-
line complexes (crystallosolvates) (Li et al., 1999; Saito et al., 1997). For
these reasons, -chitin is more easily hydrated and soluble in water,
thus improving its processability. Nevertheless, a CaCl
2
6H
2
O/methanol
solvent systemhas also been used to dissolve -chitin, allowing it to be
processed into sponge forms for tissue engineering applications (Kumar
et al., 2011).
3. Physicochemical and biological properties of chitin
In Section 2, we have addressed the processability of chitin, includ-
ing its ability to be functionalized and form complexes. The following
sections are devoted to answering the question of whether chitin
would command the same attention as chitosan, based on its (chitin)
inherent physicochemical and biological properties.
3.1. Degradability of chitin
For tissue engineering applications, the degradability of polymeric
scaffolds would be of primary concern. The ideal situation would be a
gradual but complete scaffold degradation concomitant to tissue
remodeling, leaving no foreign material that could elicit an adverse
host tissue response in the long term. Chitin is susceptible to lysozyme,
which is primarily a muramidase with a weaker chitinase activity.
Lysozyme is found ubiquitously in humans where it is thought to be
the enzyme mainly responsible for the degradation of chitin (Tomihata
and Ikada, 1997). Chitin's susceptibility to lysozyme is a function of its
degree of deacetylation Aiba reported a general increase in the initial
rate of chitin degradation by lysozyme with increasing degree of
acetylation. However, the studies also indicated that the pattern of acet-
ylation of repeating units was important, and that sequences of more
than three N-acetyl D-glucosamine residues were required for digestion
by lysozyme (Aiba, 1992). Similarly, using compression molded chitosan
bulk materials, it was found that the initial degradation rate, equilibrium
water absorption, and swelling degree increased with increasing degree
of acetylation(Renet al., 2005). Inaddition, chitinases have also beende-
tected in human body uids (Guan et al., 2009; Paoletti et al., 2007;
Tjoelker et al., 2000), which may also contribute towards chitin degrad-
ability in vivo. In a rabbit model, implanted chitin fabric was reported to
be absorbed within 12 weeks post-surgery (Funakoshi et al., 2006). Sato
et al. (2000) reported that chitin implants exhibited aggressive tissue
ingrowth in a rabbit Achilles tendon defect model compared to poorer
tissue ingrowth for the slower degrading polylactic acid implants, indi-
cating the importance of biomaterial degradation properties in tissue
regeneration.
The suitability of any polymer as a temporary scaffold for tissue
repair also hinges on the products of its degradation. Here, chitin and
chitosan exhibit a signicant difference as their breakdown products
are oligosaccharides and repeating units constituting primarily of
N-acetylglucosamine and glucosamine respectively. These shorter resi-
dues of chitin and chitosan possess not only some similar properties
with their parent polymers, but also special physiological or functional
properties, especially in the immuno-modulatory aspect (Zhang et al.,
2010). N-acetylglucosamine is a molecule with a substantial biological
role. Among other activities, it acts as the carbohydrate recognition
molecule for uptake of pathogens by macrophages and dendritic cells
via lectin-binding (Akira et al., 2006; Kumagai et al., 2008; Kumar
et al., 2009). Post-translational modication of proteins with
N-acetylglucosamine is also a cell-signaling mechanism that controls
various aspects of cell function (Yang et al., 2001) (Fig. 1D). Both
N-acetylglucosamine and glucosamine are building blocks of glycos-
aminoglycans which form a major macromolecular component of the
extracellular matrix, and are particularly abundant in cartilaginous
tissues (Safronova et al., 1991). Not surprisingly, both have been
researched and used for treatment of skeletal diseases such as osteo-
arthritis (Shikhman et al., 2001, 2009) and growth plate defects (Li
et al., 2004). While there appears to be much potential for the applica-
tion of both chitin and chitosan as scaffolds for tissue repair, special at-
tention should be paid towards the products of their degradation
in vivo, as these are associated with signicant biological activity.
3.2. Immunogenicity
Another important aspect to be considered in the application of
chitin and chitosan in tissue engineering would be their effect on the
immune response. For applications in gene delivery, chitosan is
frequently cited to possess low immunogenicity as an advantage, and
at the same time, the ability to act as an immunoadjuvant. The latter
seemingly contradictory properties can be reconciled by the fact that
chitin/chitosan have been shown to depress adaptive type-2 allergic im-
mune response, while stimulating macrophages to produce cytokines
and other compounds that confer non-specic host resistance against
bacterial and viral infections (Muzzarelli, 2010).
A major mechanism by which chitin stimulates macrophages is by
interacting with different cell surface receptors such as macrophage
mannose receptor, toll-like receptor-2 (TLR-2) and C-type lectin recep-
tor (Lee, 2009). As chitin and chitosan show differential binding afni-
ties to the various receptors (Kristiansen et al., 1999; Sashiwa et al.,
2000), their immunoadjuvant activities would also be expected to
vary correspondingly.
3.3. Mechanical properties
Native -chitin exhibits a crystalline structure with both inter-
molecular and intramolecular hydrogen-bonding, by virtue of the
1779 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785
N-acetylglucosamine groups being distributed along the polymer back-
bone (Ogawa et al., 2011). By X-ray diffraction, the elastic moduli of the
crystalline regions of chitin and chitosan have been determined to be
41 GPa and 65 GPa respectively (Nishino et al., 1999), although the
chitin modulus was measured to be 59.3 GPa in a later work (Ogawa
et al., 2011). The actual mechanical properties of chitin and chitosan
as materials, however, dependonthe specic processed formof the ma-
terial. For example, chitin from various sources were found to exhibit
elastic moduli ranging between 1.1 to 2.9 GPa by tensile testing, in the
dry state, and 0.3 to 0.6 GPa in the wet state (Hepburn et al., 1975). Chi-
tin sutures aged in ambient atmosphere for various time periods of up
to two weeks exhibited elastic moduli ranging between 2.8 and
5.5 MPa (Notin et al., 2006), while hydrated chitosan scaffolds exhibit-
ed elastic moduli ranging from 0.1 MPa to 7 MPa, depending on the
porosity of the sample involved (Suh and Matthew, 2000). These differ-
ences can be accounted for by the difference in crystallinity of the sam-
ples, and the role of water as a plasticizer. Like other polysaccharides,
chitin possesses abundant hydroxyl groups that can hydrogen bond to
water molecules, resulting in a strong afnity for water. Water binding
in turn leads to a lowering of the glass transition temperature, hence,
the polymer becomes more rubbery (lower modulus) at room temper-
ature. To exploit their apparent complementarity, mixtures of chitin
and chitosan have been used to modulate mechanical properties for
some applications (Khor and Lim, 2003; Kuo and Lin, 2006; Kuo and
Tsai, 2010). Due to its favorable mechanical properties, chitin has
been researched for applications that require good integrity and physi-
cal strength, such as surgical sutures, medical textiles and bone substi-
tute materials.
4. Chitin scaffolds for tissue engineering
Having reviewed the physicochemical properties of chitin and the
various means by which it could be processed into useful biomaterial
forms, the present section delves into its use as a material for tissue
engineering. Tissue engineering has its roots in the experiments of
Langer and Vacanti in the late 80s (Vacanti et al., 1988), leading to the
conceptualization of a new eld (Langer and Vacanti, 1993). The basic
premise of tissue engineering lies in the use of a biomaterial scaffold
to dene the shape and structure of cell growth. As discussed earlier
in Sections 3.13.3 respectively, chitin possesses the requisite proper-
ties to act as a scaffold for tissue engineering, with respect to its degrad-
ability, immunogenicity and mechanical strength. Chitin has been
applied in the form of hydrogels, brous scaffolds or porous sponges,
within which the appropriate cell types are seeded for in vitro or
in vivo culture and evaluation.
4.1. Modication of chitin scaffolds
In applying chitin as scaffolds for tissue engineering, the need may
arise to modify it, to modulate its physical or biochemical characteristics
for interaction with the biological environment. Modication can be
carried out by simply blending chitin with one or more other polymers,
or could be effected by chemically conjugating smaller molecules such
as sugars or peptides to the chitin scaffold. For example, hybrid nano-
bers of chitin with silk-broin and poly(glycolic acid) have been
prepared by electrospinning (Park et al., 2006a,b). These scaffolds sup-
ported the attachment andspreading of human keratinocytes and bro-
blasts (Noh et al., 2006; Yoo et al., 2008). Hybrid microspheres of chitin
and poly-leucine have also been synthesized via an interfacial polymer-
ization process based on the ring-opening polymerization of an alpha-
amino acid N-carboxyanhydride (NCA), with potential utility for drug
delivery and/or tissue engineering (Wang et al., 2008) (Fig. 3A). Modi-
cation of chitin surfaces to make it amenable to cell attachment is im-
portant for its application as a tissue scaffold. Towards that end, fusion
proteins of RGD and a human chitin-binding domain have been
designed with the intention of mediating cell-adhesion on reacetylated
chitosan (Carvalho et al., 2008). This approach, however, negatively
affected anchorage of the cells to the surface, thus inhibiting cell adhe-
sion and proliferation. A more successful approach towards promoting
cell adhesion appeared to be the conjugation of wheat germ agglutinin,
but this was applied to chitosan, and not to chitin membranes
(Wang et al., 2003). Our own approach towards the modication of
chitin-based scaffolds has mainly relied on chemically modifying
the polyanion (e.g. by biotinylation or thiol-activation), to be subse-
quently complexed with water soluble chitin or chitosan (Tai et al.,
2010b; Wan et al., 2004). The resulting biotinylated or thiol-activated
scaffolds could then be conjugated to the desired protein or peptide
using the appropriate chemistry. An alternative route involves the
crosslinking of proteins onto chitin-based scaffolds using genipin or
carbodiimide/N-hydroxy succinimide (Kuo and Chung, 2012).
Chitin-based polyelectrolyte complex bers can be modied with
proteins and genes, to be delivered in a spatially and temporally con-
trolled manner (Liao et al., 2005; Lim et al., 2006) (Fig. 4A). These stud-
ies demonstrated that the genes and growth factors retained their
activity after being released fromthe bers. In the case of protein deliv-
ery, incorporation of heparin as an additional polyanion component led
to a longer period of sustained growth factor release fromthe bers, at-
tributed to growth factor-binding by heparin. In vivo studies indicated
good biocompatibility and blood compatibility of chitin-alginate bers,
which could again be improved by incorporating a small amount of
heparin during ber formation (Yim et al., 2007).
Chitin scaffolds have been applied chiey to the regeneration of
cartilage, bone and tendon tissue, as presented in Sections 4.2 and 4.3.
4.2. Cartilage tissue engineering
Chitin may be inherently suitable as a biomaterial to support cartilage
regeneration, as its repeating unit, N-acetylglucosamine (Fig. 1), is a build-
ing block of glycosaminoglycans such as keratin sulfate and hyaluronate,
whichforma major component of articular cartilage. Sponge forms of chi-
tin have been obtained by lyophilizing chitin solutions (Maeda et al.,
2008) or inltrating chitin gel into a colloidal crystal template that was
subsequently removed (Kuo and Tsai, 2011), to serve as scaffolds for the
culture of chondrocytes. An interesting alternative process involves the
derivation of three dimensional chitin scaffolds from natural sponges
(Ehrlich et al., 2010) (Fig. 3B). Here, chitin may possess a unique advan-
tage over chitosan, as sponges and other aquatic organisms from which
these scaffolds could potentially be derived from are typically chitin,
and not chitosan-based. These chitin scaffolds supported deposition of a
proteoglycan-rich extracellular matrix by chondrocytes when implanted
subcutaneously in mice. A chitinchitosanpolyethylene oxide scaffold
was found to support adhesion and proliferation of bovine knee
chondrocytes and the deposition of their extracellular matrix over
4 weeks in vitro, when surface modied with elastin, poly(L-lysine) or
polyethyleneimine (Kuo and Chung, 2012; Kuo and Ku, 2009). The chon-
drogenesis promoting effect appeared to be greater for the case of elastin,
when compared to poly(L-lysine).
Collective evidence from the literature suggests that chitin is more
suitable than chitosan as a matrix for cartilage tissue regeneration. The
ability of scaffolds composed of different chitinchitosan ratios to
support chondrogenesis was investigated in two separate studies (Kuo
and Lin, 2006; Suzuki et al., 2008). While the studies differed in the iso-
form of chitin ( vs ), degree of acetylation of chitin and processing
conditions that were used, bothpointed to higher deposition of cartilag-
inous extracellular matrix and quality of cartilage regeneration with a
higher percentage of chitin in mixed chitinchitosan scaffolds.
To qualify the point that a higher degree of acetylation is not always
better for tissue regeneration, chitosan has been demonstrated to fare
better than chitin for wound healing in rats (Minagawa et al., 2007).
Although chitin and chitosan themselves, their oligomers, as well as
monomers were found to enhance wound healing, the wound break
strength and collagenase activity of the chitosan group was found to
1780 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785
be higher than that of the chitin group. In fact, Howling et al. (2001)
found that a lower degree of acetylation stimulated broblast prolifera-
tion but had an inhibitory effect on human keratinocytes.
A -chitin scaffold, molded into the shape of a pillar (Fig. 3C), was
used to support the formation of a surface cartilage layer by culture of
primary rabbit knee chondrocytes for 4 weeks (Abe et al., 2004). Such
a conguration was aimed at producing an implant that could be
press-t into articular cartilage defects without covering the periosteum
or suturing the implant. At the end of the culture period, the cell layer at
the surface of the sponge was lled with chondrocytes and abundant
extracellular matrix, of a composition and structure that resembled
articular cartilage.
4.3. Bone and tendon tissue engineering
Early investigations on the use of chitin for bone tissue engineering
focused on its ability to chemically interact and form composites with
apatite, the mineral phase of bone. In a series of papers from the 90s,
Khor and co-workers demonstrated how apatite could be deposited
onto chitin scaffolds, co-precipitated with aqueous chitin solutions or
blended and cast with chitin using an organic solvent system (Wan
et al., 1996; Wan et al., 1998a; Wan et al., 1998b). These studies revealed
favorable mechanical properties of the chitin-apatite composites and the
inherent ability of particular chitin derivatives to nucleate calcium
phosphate formation. More recent follow-up work by the same lab-
oratory established that a chitinhydroxyapatite composite loaded
with mesenchymal stem cell-induced osteoblasts, when implanted
into bone defects of rabbit femur, was able to support bone regenera-
tion (Ge et al., 2004). Labelling studies showed that the osteoblasts
not only proliferated, but alsorecruitedthe ingrowthof surrounding tis-
sue. Other work on chitin-based composites for bone tissue engineering
has since followed, although these lacked in vivo demonstration. Com-
posite scaffolds of chitinwithnano hydroxyapatite and nano titania, ob-
tained by dispersing the particles in a chitin hydrogel cast froma CaCl
2
/
methanol solvent system and freeze-drying the mixture, supported ap-
atite deposition and adhesion of a variety of cell lines (Jayakumar et al.,
2010b; Kumar et al., 2011). In a more indirect use of chitin for bone tis-
sue engineering, Peng et al. (2010) made hydroxyapatite spherules by
emulsication of a chitin solution.
The benets of chitin for use in bone regeneration extend to more
than just being a matrix of a composite material. Chitin hexamers
have been shown to promote osteogenesis in mesenchymal stem cells,
and their effect is greater than that of chitosan hexamers (Lieder et al.,
2012). There have also been abundant reports of chitin's ability to accel-
erate wound healing (Muzzarelli, 2009), and the same biological activ-
ity of enhancing cell migration (Okamoto et al., 1993; Su et al., 1999)
and forming granulation tissue with angiogenesis (Okamoto et al.,
1997, 2002) likely extends to other vascularized tissue, including the
bone. These effects are mediated by the production of cytokines and
growth factors by broblasts that come into contact with the chitin
material, for example, interleukin-8, which in turn induces the migra-
tion of broblasts and vascular endothelial cells (Mori et al., 1997).
Fig. 3. Various chitin forms for tissue engineering. (A) Cross-sectional SEM of a chitin-gpolypeptide microsphere. The inset shows a close-up of a broken sphere, scale bar = 100 m.
(Reprinted with permission from Kuo and Chung, 2012. 2012, Elsevier.) (B) The three-dimensional chitinous matrix isolated from Aplysina cauliformis possesses large internal surface
area, which enables considerable liquid absorption to take place by capillary attraction. (Reprinted with permission fromEhrlich et al., 2010. 2010, Elsevier.) (C) Photograph of cultured
chondrocyte-chitin sponge composite at week 4. The arrowindicates a cartilage-like layer formed on the top surface of the -chitin sponge. (Reprinted with permission fromAbe et al.,
2004. ;2004, Mary Ann Liebert.) (D) Scanning electron micrograph of nonwoven chitin fabric (original magnication 3000) which was investigated as an acellular matrix for the treat-
ment of rotator cuff tendon defects. (Reprinted with permission from Funakoshi et al., 2006. 2006, Elsevier.)
1781 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785
Chitinappears tobe anideal material for the regenerationof ligaments
and tendons, as illustrated by the efcacy of a chitin-coated polyester
graft as a scaffold for anterior cruciate ligament reconstruction (Kawai
et al., 2010). Here, the chitincoating enhancedbone formationinthe fem-
oral bone tunnel and soft tissue formation in the articular cavity, thus in-
creasing the attachment strength of the graft to the bone. Funakoshi et al.
employed a chitin fabric in the repair of rotator cuff tendon defects, sug-
gesting twoways by whichthe fabric couldhelpdirect andimprove tissue
regeneration (Funakoshi et al., 2006) (Fig. 3D). Firstly, it was observed
that the collagen bers were deposited in the fabric and were regularly
oriented, which was attributed to chitin's ability to moderate the stresses
at the bone-tendon interface. Chitin could thus be used to facilitate the
deposition, alignment and maturation of extracellular matrix. Secondly,
the biodegradability of chitin fabric could be easily tuned by changing
the ber diameter and fabric density.
5. Chitin for stem cell technologies
5.1. Applications of stem cells for regenerative medicine
Stem cells can be classied broadly into embryonic stem cells, post-
natal or adult stem cells and induced pluripotent stem cells. For a more
comprehensive introduction on these different stemcell types and their
utility in regenerative medicine, the reader is referred to two excellent
reviews (Sylvester and Longaker, 2004; Wu and Hochedlinger, 2011).
Biomaterials, such as chitin, can help to realize the vast potential of
stem cells in regenerative medicine in one of two ways.
Firstly, large numbers of stem cells are required for therapeutic
applications. For instance, the number of insulin-secreting beta cells
required for therapy of Diabetes Type I has been estimated at
610 10
8
cells per patient, while tissue engineering of the bladder
would require approximately 1.5 10
8
cells (Mason and Dunnill,
2009). Here, biomaterials may play a role in providing substrates that
support stem cell self-renewal, while maintaining stem cell pluri- or
multipotency. The second potential contribution of biomaterials to
stem cell therapy would be the provision of a matrix permissive to
stem cell differentiation, or in a more active manner, one bearing
ligands and soluble factors that direct differentiation of stem cells into
the desired lineages and cell types.
5.2. Chitin-based bers for the propagation and differentiation of stem cells
We have recently applied chitin towards the in vitro expansion of
pluripotent stem cells (Lu et al., 2012b) (Fig. 4B). Water-soluble chi-
tin was rst used to encapsulate cells in bers by a process of inter-
facial complexation with alginic acid. These bers could be either
Fig. 4. Chitin-basedpolyelectrolyte complex bers for 3Dcell culture and tissue engineering. (A) Transgene expressionof human dermal broblasts seeded onbrous scaffolds loadedwith
PEIDNA nanoparticles containing GFP-encoding plasmid. At 12 days postseeding, large numbers of broblasts were actively expressing GFP. (Reprinted with permission from Limet al.,
2006. 2006, Nature Publishing Group). (B) A human embryonic stemcell line (BG01V/hOG) encapsulated and propagated in 3Dmicrobrous scaffolds. (Reprinted with permission from
Lu et al., 2012b. 2012, Elsevier.) (C) Confocal microscopy image of three cell types within a three-component ber: red human umbilical vein endothelial cells, blue broblast cells
(NIH/3T3), green HepG2 cells. (Reprinted with permission fromWan et al., 2012. 2012 John Wiley and Sons.) (D) Human mesenchymal stemcells cultured ina two-component ber,
one component with, and the other without collagen. Cell elongation and spreading are observed for cells in the collagen-containing half. (Reprinted with permission from Wan et al.,
2012. 2012 John Wiley and Sons.)
1782 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785
spooled around planar supports or entangled to form constructs for
long term cell culture. Water-soluble chitin is uniquely suitable for
cell encapsulation. The polymer is of adequate positive charge densi-
ty (sufcient amino groups) for complexation with the polyanion,
and yet not too deacetylated to the extent of requiring an acid sol-
vent. Polyelectrolyte complex bers of chitin and alginate supported
the self-renewal of two embryonic and two induced pluripotent
stem cell lines for at least ten passages in culture, while maintaining
a stable cell karyotype. The success of chitin in this application can be
attributed to the provision of a non cell-adhesive, three-dimensional
hydrogel microenvironment within which the stem cells proliferat-
ed in the formof aggregates. The high enzymatic susceptibility of chi-
tin to chitinase afforded another advantage to the platform, as it allowed
convenient dissociation of the ber andrelease of cell aggregates for fur-
ther application. A recently published paper suggests an intriguing, sec-
ond explanation for the effectiveness of chitin as a matrix for in vitro
stem cell expansion. The major protein factors responsible for self-
renewal of pluripotent stemcells, Oct4 and Sox2, were foundto be mod-
ied with N-acetylglucosamine, the repeating unit of chitin (Jang et al.,
2012). The same modication was also found to induce the expression
of other pluripotent genes. In the paper itself, it was suggested that
this proffered an explanation for the positive effect of glucosamine
and N-acetylglucosamine in culture media on embryonic stem cell
maintenance. While the chitin ber matrix used to encapsulate the
pluripotent stemcells in our experiments was polymeric in nature, deg-
radation of chitin that occurs under culture conditions, especially when
in contact with cells, may generate N-acetylglucosamine. Furthermore,
during the process of decapsulation, when chitin is depolymerized to
N-acetylglucosamine oligomers and its repeating units by the addition
of chitinase, a high N-acetylglucosamine concentration in the media
may help to preserve pluripotency.
Besides the expansion of stem cells, chitin-based microbrous
scaffolds can be used just as effectively for differentiation of stem cells,
when the latter are provided with the appropriate signals. Again, the
use of water-soluble chitin is advantageous as it allows encapsulation
of cells at near-neutral pH conditions, and maintains a matrix which is
both permissible and non-detrimental to soluble differentiation factors.
In our laboratory, we have developed a neuronal differentiation proto-
col where bers encapsulating pluripotent stem cells are rst trans-
ferred from stem cell expansion medium to neural induction medium,
whereby the cells are differentiated to neural progenitors. Subsequent-
ly, a second transfer of the cell-ber construct to neural maturation me-
dium leads to a high yield of terminally differentiated neurons (N90%)
(Lu et al., 2012a). In a similar way, mesenchymal stem cells seeded or
encapsulated in water-soluble chitin-alginate brous scaffolds have
been differentiated into chondrogenic and osteogenic lineages by
immersion in the respective differentiation media (Yim et al., 2006).
In these experiments, better cell proliferation and differentiation was
observed for cell-encapsulated, as compared to cell-seeded scaffolds.
The advantages of a chitin matrix in supporting stem cell proliferation
and differentiation extends to its performance in vivo, where chitin
has been shown to be effective as a carrier material for mesenchymal
stem cells in the treatment of large physeal defects (Li et al., 2004).
The polyelectrolyte complex ber route towards stemcell differenti-
ation, utilizing water-soluble chitin, holds additional promise the
mild aqueous based process supports the incorporation of ECMcompo-
nents such as collagen and heparin, which could be used to further
modulate cell differentiation (Liao et al., 2005; Wan et al., 2004).
While earlier constructs took the form of non-woven matrices (Wan
et al., 2006), the importance of well-dened domains for culture of dif-
ferent cell types and ECMled us to develop a process to formmulticom-
ponent bers composed of water-soluble chitin and alginate (Limet al.,
2013; Wan et al., 2012). The ability to spatially pattern biological
components (cells and ECM) in three dimensions and recapitulate the
interactions that occur between them is deemed important to repro-
duce the complexity of human tissue and organ systems (Fig. 4C, D).
6. Conclusion
At the outset, this reviewhas endeavored to provide an explanation
for the disparity in the application of chitin and chitosanas biomaterials.
It appears that the lesser use of chitin as comparedto chitosanis not due
to inherently less favorable biomaterial characteristics, but rather, stems
fromthe perception that it is more difcult to process. In the rst part of
the review, various methods that have been employed to improve the
processability of chitin have been presented. Where the issues of pro-
cessing have been overcome, the literature has in fact demonstrated
the vast potential of chitin as a biomaterial, specically for tissue engi-
neering and stem cell applications. Chitin's biomedical potential stems,
not only from its easy availability as a waste product of the seafood in-
dustry, but also from its inherent material and chemical properties
such as degradability, mechanical strength and biological activity. To re-
alize its full potential though, due attention should be given to several
aspects of its utilization. Firstly, as emphasized in this review, the per-
formance of chitin in specic applications depends greatly on its degree
of acetylation, and the latter should therefore be characterized precisely
in any investigation. The same applies to the molecular weight distribu-
tion of chitin used in the experiment, as it affects the mechanical and
biological properties of the material. Last but not the least, prior knowl-
edge of chitin biochemistry and biological activity would be helpful in
evaluating its suitability for particular cell or tissue types. The design
of chitin and other biomaterials should ideally keep abreast with
developments in the biology of stem cells and tissue regeneration, so
that they could be applied in the most fruitful and effective way.
Having encapsulated the state-of-art in the application of chitin in
the tissue engineering and stem cell elds, it is hoped that this review
will encourage more researchers to employ chitin as part of the growing
biopolymer arsenal for regenerative medicine.
Acknowledgment
This work is funded by the Institute of Bioengineering and Nanotech-
nology (Biomedical ResearchCouncil, Agency for Science, Technology and
Research, Singapore).
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