. . .tin .. . .. . .lvent sy. . .perties o. . .. . .. . .

Biotechnology Advances 31 (2013) 17761785

Contents lists available at ScienceDirect

Biotechnolog

j ourna l homepage: www.e lsev4.2. Cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17804.3. Bone and tendon tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1781

5. Chitin for stem cell technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17825.1. Applications of stem cells for regenerative medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17825.2. Chitin-based bers for the propagation and differentiation of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1782

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783 Corresponding author at: Institute of Bioengineering aWay, The Nanos, #04-01, 138669, Singapore. Tel.: +65 68

E-mail addresses: awan@ibn.a-star.edu.sg (A.C.A. Wan(B.C.U. Tai).

0734-9750/$ see front matter 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.biotechadv.2013.09.007. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779

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4. Chitin scaffolds for tissue engineering .

4.1. Modication of chitin scaffolds .1. Introduction . . . . . . . . . .2. Improving the processability of chi

2.1. Water-soluble chitin . . .2.2. Chitin derivatives . . . .2.3. Dissolution in alternative so2.4. -Chitin vs -chitin . . .

3. Physicochemical and biological pro3.1. Degradability of chitin . .3.2. Immunogenicity . . . . .3.3. Mechanical properties . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1776

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1778stems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779f chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1779ContentsResearch review paper

CHITIN A promising biomaterial for tissue engineering and stemcell technologies

Andrew C.A. Wan , Benjamin C.U. TaiInstitute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, 138669, Singapore

a b s t r a c ta r t i c l e i n f o

Article history:Received 2 July 2013Received in revised form 13 September 2013Accepted 23 September 2013Available online 29 September 2013

Keywords:ChitinStem cellTissue engineeringScaffolds

Chitin, after cellulose, is the second most abundant natural polymer. With a 200-year history of scienticresearch, 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 tothe perceived difculty in processing chitin. This reviewpresentsmethods to improve the processability of chitin,and goes on further to discuss the unique physicochemical and biological characteristics of chitin that favor it as abiomaterial for regenerative medicine applications. Examples of the latter are presented, with special attentionon the qualities of chitin that make it inherently suitable as scaffolds and matrices for tissue engineering, stemcell propagation and differentiation.

2013 Elsevier Inc. All rights reserved.ndNanotechnology, 31 Biopolis24 7134; fax: +65 6478 9082.), btai@ibn.a-star.edu.sg

ghts reserved.y Advances

i e r .com/ locate /b iotechadv1. Introduction

Chitin is the second most abundant polysaccharide in nature aftercellulose, found mainly as the exoskeleton material of arthropods andcrustaceans, and the cell wall of fungi (Buck and Obaidah, 1971). Itschemical structure is depicted in Fig. 1, in comparison to three closely

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1777A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785related biomolecules, chitosan, cellulose andmureic acid. The repeatingunit of chitin is an oxygen-containing hexose ring with an acetamidogroup at the second carbon position, strung together by -glycosidicbonds. The closest chemical entity to chitin, chitosan, is found naturallyas the main polysaccharide constituent of the cell wall for some fungalspecies (Bartnicki-Garcia, 1968). Chitin and chitosan are effectively thesame macromolecular entity, varying only in the fraction of acetylatedrepeating units, and possessing the chemical structure shown in Fig. 1.Here, x is the degree of acetylation, dened as themole ratio of acetylat-ed repeating units over that of the total repeating units. In fact, themainroute for chitosan production is via the deacetylation of chitin (Knorr,1991). However, deacetylation, in practice, does not proceed to comple-

Fig. 1. Chemical structure of (A) chitin, when x N 0.5 or chitosan when x b 0.5; (B) cellu(D) schematic diagram of O-GlcNAcylation. The N-acetylglucosamine (GlcNAc) moiety is ltion, which implies that the chitosan obtained from commercial sourcesis chitin with a very low degree of acetylation (typically, x = 0.1 to0.25). 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 polysaccharideas 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 betweenchitin and chitosan by referring to the literature, based on the re-ported degree of acetylation. It is to be emphasized however, thatsuch a distinction is articial, and made merely for comparative pur-poses. In reality, chitin and chitosan are two points on a continuumof materials that share the same basic structure and differ only intheir 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, agreat disparitywould be observed. Using the keywords chitin or chitosanand tissue engineering while carefully observing the abovementionedcriteria on the denition of either polysaccharide based on degree ofacetylation, 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 chitinand chitosan. Chitosan's popularity as a biomaterial may derive fromthe fact that it possesses abundant amino groups. At sufciently lowpH, these become charged, allowing complexation with DNA that hasbecome 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 havebeen imbued with chitosan-DNA nanoparticle complexes where thetransfected genes may express bioactive proteins that facilitate tissueregeneration (Peng et al., 2009; Zhang et al., 2009). Polyionic complexesof chitosan with polyanions such as alginic acid (Tai et al., 2010a; Yimet al., 2007), heparin (Yim et al., 2007) and chondroitin sulfate (Chenet al., 2007) have been investigated for the fabrication of tissue scaffolds.Chitosan's abundant amino groups also provide the functionality for

; and (C) murein, an analogous carbohydrate to chitin found in the cell wall of bacteria;d via serine or threonine residues of the protein.crosslinking or conjugation of biologically active ligands, for example,via carbodiimide chemistry (Rafat et al., 2008). However, the primaryreason for chitosan's vastly higher usagemay be its ease of solubilization,making it amenable for processing into various forms. The amino groupof 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 homogenousfunctionalization reactions (Rafat et al., 2008) or even applied directlyin solution (Campos et al., 2006) or gel (Alemdaroglu et al., 2006) forms.

Chitin, on the other hand, is relatively difcult to process, and due toits insolubility in aqueous solution, has become less accessible tobiological laboratories. While the earlier work on chitin had establishedits solubility in strong acids such as dichloroacetic acid and trichloroace-tic acid (Austin, 1975a,b), and more recently, highly polar uorinatedsolvents such as hexauoroisopropyl alcohol (Capozza, 1976), these so-lution systems are unattractive due to their corrosive nature, toxicityand/or degradative effect on chitin polymer. There are, however, waysto make chitin more amenable to processing. This review is dividedinto four parts. The rst (Section 2) summarizes approaches towardsimproving the processability of chitin, which have allowed for thesuccessful employment of chitin in the area of stem cells and tissueengineering. In Section 3, the physicochemical and biological propertiesof chitin are discussed, where relevant, in comparison to the loweracetylated form (chitosan). Section 4 delves into the use of chitin as ascaffold for tissue engineering. In Section 5, we detail how chitin hasmore recently been applied for stem cell technologies, a developmentwhich relies on the advances described in the preceding sections.

2. Improving the processability of chitin

2.1. Water-soluble chitin

It was found that chitin,when deacetylated to a degree of acetylationof about 0.5 (Fig. 2A), becamewater-soluble (Sannan et al., 1976). Alter-natively, the samewater solubility could be imparted by acetylating halfof the amino groups available in chitosan (Kubota et al., 2000). Thiswater-solubility is postulated to result from the loss of crystallinityand intra-chain hydrogen-bonding with deacetylation, leading toa crystal structure approaching that of the more water-swellable-chitin (covered in Section 2.4) (Cho et al., 2000). For the purpose ofthis review, the water-soluble form of chitin/chitosan with degree ofdeacetylation 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 molecularweight can be obtained, which may confer an advantage in terms ofmechanical properties. In contrast to regular chitosan, which dissolvesin 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 severalapplications to that effect have been investigated (Leong et al., 2013;Lu et al., 2012b; Wan et al., 2004; Yim et al., 2006). Two points shouldbe noted when applying water-soluble chitin for stem cell or tissueengineering applications. Firstly, chitin and chitosan are degradable bylysozyme and chitinase to different extents depending on the respectivedegree of deacetylation (Sashiwa et al., 1990; Tomihata and Ikada,1997; Zhang and Neau, 2001). Thus, the in vivo degradation prole ofhighly acetylated chitin would differ signicantly from that of water-

by complexation or crosslinking. Such a process would be unnecessaryfor chitosan, which is insoluble at physiological pH. Here, methodsthat 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 polyanionicmolecules, although the relatively lower charge density (in comparisonto chitosan) would affect its complexation properties. Composite lmsof 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 bothhydroxyls can be used for functionalization; the primary hydroxyl onC6 is generally more reactive, as it is the less sterically hindered(Fig. 1). Chitosan, in comparison, possesses proportionally moreamino groups on C2 that could be employed for functionalization. Anobvious method to enhance the water-solubility of chitin would be tofunctionalize itwithhydrophilic groups or those containingmoieties ca-pable of hydrogen bonding. Mainly towards that end, carboxymethyl,hydroxypropyl, glycol, phosphoryl and sulfuryl derivatives of chitinhave been synthesized (Hirano, 1988; Nishi et al., 1986; Nishimuraet al., 1984; Park and Park, 2001; Tokura et al., 1994; Trujillo, 1968)(Fig. 2B). As for water-soluble chitin, a crosslinking strategy would beimportant to render these derivatives suitable for applications that re-quire a solid form of the material, for example, as tissue engineeringscaffolds. It is pertinent to note however, that these water soluble chitinderivatives may possessmarkedly different characteristics as comparedto their parent material, e.g. enzymatic degradation properties, and

1778 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785soluble chitin, which contains half the number of acetyl groups permole repeating unit. Second, the application of water-soluble chitin asa scaffold in an aqueous environment requires its prior insolubilizationFig. 2. Approaches towards improvshould therefore be separately investigated for the specic applicationsconcerned (Kaifu and Komai, 1982; Kumirska et al., 2010; Kurita et al.,2003; Saiki et al., 1990; Tong et al., 2011).ing the processability of chitin.

1779A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 177617852.3. Dissolution in alternative solvent systems

While chitin is insoluble in pure organic solvents such as N-methylpyrrolidone or dimethylacetamide, the addition of a small percentageof salt such as lithium chloride enhances its solubility dramatically(Austin, 1988; Austin et al., 1981). The observation can be explainedby invoking coordination of the salt with the acetylcarbonyl group ofchitin to form a soluble complex (Fig. 2C). Solutions of chitin in organicsolvents have been precipitated into bers (Zhong et al., 2010), cast intolms (Yusof et al., 2003) and used to form composites with inorganicmaterials such as hydroxyapatite (Wan et al., 1997). One concern inusing such processing routes for chitin would be the presence of resid-ual organic solvent in the nal biomaterial product. Thus, the absenceof solvent impurities should be ascertained by a stringent qualitycheck prior to the application.

Recently, chitin solutions in ionic liquids have also been obtained,which were processed into highly porous structures by supercriticaluid drying (Silva et al., 2011), ormixedwith cellulose to form compositegels and lms (Takegawa et al., 2010). Alcoholic calcium chloride solventsystems have also been employed to process chitin into scaffolds by ly-ophilization (Kumar et al., 2011;Maeda et al., 2008). As ameans to furtherimprove 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 interms of their polymer chain structure and crystallinity (Jang et al.,2004). Most chitins, such as insect and crustacean chitins, are -chitinin the native state, while the rarer -chitin allomorph occurs in squidpen and somediatoms.-Chitin exhibits a two-chain, antiparallel struc-ture, while -chitin has a one-chain unit cell with a parallel-chain struc-ture, with intramolecular hydrogen-bonding (Fig. 2D) (Blackwell, 1969;Minke and Blackwell, 1978; Rudall and Kenchington, 1973). Theweakerhydrogen-bonding from the parallel-chain structure of -chitinmay ac-count for its higher chemical reactivity (Atkins, 1985; Kurita et al.,2005). In addition, -chitin has the unique feature of incorporatingsmall molecules, including water, into its crystal lattice to form crystal-line complexes (crystallosolvates) (Li et al., 1999; Saito et al., 1997). Forthese reasons, -chitin is more easily hydrated and soluble in water,thus improving its processability. Nevertheless, a CaCl26H2O/methanolsolvent system has also been used to dissolve-chitin, allowing it to beprocessed into sponge forms for tissue engineering applications (Kumaret 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 followingsections are devoted to answering the question of whether chitinwould 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 polymericscaffolds would be of primary concern. The ideal situation would be agradual but complete scaffold degradation concomitant to tissueremodeling, leaving no foreign material that could elicit an adversehost 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 bethe enzyme mainly responsible for the degradation of chitin (Tomihataand Ikada, 1997). Chitin's susceptibility to lysozyme is a function of itsdegree of deacetylation Aiba reported a general increase in the initial

rate of chitin degradation by lysozyme with increasing degree ofacetylation. However, the studies also indicated that the pattern of acet-ylation of repeating units was important, and that sequences of morethan three N-acetyl D-glucosamine residues were required for digestionby lysozyme (Aiba, 1992). Similarly, using compressionmolded chitosanbulkmaterials, it was found that the initial degradation rate, equilibriumwater absorption, and swelling degree increased with increasing degreeof acetylation (Ren et al., 2005). In addition, chitinases have also been de-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 tobe absorbed within 12 weeks post-surgery (Funakoshi et al., 2006). Satoet al. (2000) reported that chitin implants exhibited aggressive tissueingrowth in a rabbit Achilles tendon defect model compared to poorertissue ingrowth for the slower degrading polylactic acid implants, indi-cating the importance of biomaterial degradation properties in tissueregeneration.

The suitability of any polymer as a temporary scaffold for tissuerepair also hinges on the products of its degradation. Here, chitin andchitosan exhibit a signicant difference as their breakdown productsare oligosaccharides and repeating units constituting primarily ofN-acetylglucosamine and glucosamine respectively. These shorter resi-dues of chitin and chitosan possess not only some similar propertieswith their parent polymers, but also special physiological or functionalproperties, especially in the immuno-modulatory aspect (Zhang et al.,2010). N-acetylglucosamine is a molecule with a substantial biologicalrole. Among other activities, it acts as the carbohydrate recognitionmolecule for uptake of pathogens by macrophages and dendritic cellsvia lectin-binding (Akira et al., 2006; Kumagai et al., 2008; Kumaret al., 2009). Post-translational modication of proteins withN-acetylglucosamine is also a cell-signaling mechanism that controlsvarious aspects of cell function (Yang et al., 2001) (Fig. 1D). BothN-acetylglucosamine and glucosamine are building blocks of glycos-aminoglycans which form a major macromolecular component of theextracellular matrix, and are particularly abundant in cartilaginoustissues (Safronova et al., 1991). Not surprisingly, both have beenresearched and used for treatment of skeletal diseases such as osteo-arthritis (Shikhman et al., 2001, 2009) and growth plate defects (Liet 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 degradationin vivo, as these are associated with signicant biological activity.

3.2. Immunogenicity

Another important aspect to be considered in the application ofchitin and chitosan in tissue engineering would be their effect on theimmune response. For applications in gene delivery, chitosan isfrequently cited to possess low immunogenicity as an advantage, andat the same time, the ability to act as an immunoadjuvant. The latterseemingly contradictory properties can be reconciled by the fact thatchitin/chitosan have been shown to depress adaptive type-2 allergic im-mune response, while stimulating macrophages to produce cytokinesand other compounds that confer non-specic host resistance againstbacterial and viral infections (Muzzarelli, 2010).

A major mechanism by which chitin stimulates macrophages is byinteracting with different cell surface receptors such as macrophagemannose 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 tovary correspondingly.

3.3. Mechanical properties

Native -chitin exhibits a crystalline structure with both inter-

molecular and intramolecular hydrogen-bonding, by virtue of the

1780 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785N-acetylglucosamine groups being distributed along the polymer back-bone (Ogawa et al., 2011). By X-ray diffraction, the elastic moduli of thecrystalline regions of chitin and chitosan have been determined to be41 GPa and 65 GPa respectively (Nishino et al., 1999), although thechitin modulus was measured to be 59.3 GPa in a later work (Ogawaet al., 2011). The actual mechanical properties of chitin and chitosanasmaterials, however, dependon the specic processed formof thema-terial. For example, chitin from various sources were found to exhibitelastic moduli ranging between 1.1 to 2.9 GPa by tensile testing, in thedry state, and 0.3 to 0.6 GPa in thewet state (Hepburn et al., 1975). Chi-tin sutures aged in ambient atmosphere for various time periods of upto two weeks exhibited elastic moduli ranging between 2.8 and5.5 MPa (Notin et al., 2006), while hydrated chitosan scaffolds exhibit-ed elastic moduli ranging from 0.1 MPa to 7 MPa, depending on theporosity of the sample involved (Suh andMatthew, 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 towater molecules, resulting in a strong afnity for water. Water bindingin 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 chitinand chitosan have been used to modulate mechanical properties forsome applications (Khor and Lim, 2003; Kuo and Lin, 2006; Kuo andTsai, 2010). Due to its favorable mechanical properties, chitin hasbeen 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 thevarious means by which it could be processed into useful biomaterialforms, the present section delves into its use as a material for tissueengineering. Tissue engineering has its roots in the experiments ofLanger and Vacanti in the late 80s (Vacanti et al., 1988), leading to theconceptualization of a new eld (Langer and Vacanti, 1993). The basicpremise of tissue engineering lies in the use of a biomaterial scaffoldto dene the shape and structure of cell growth. As discussed earlierin 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 beenapplied in the form of hydrogels, brous scaffolds or porous sponges,within which the appropriate cell types are seeded for in vitro orin vivo culture and evaluation.

4.1. Modication of chitin scaffolds

In applying chitin as scaffolds for tissue engineering, the need mayarise tomodify it, tomodulate its physical or biochemical characteristicsfor interaction with the biological environment. Modication can becarried out by simply blending chitin with one or more other polymers,or could be effected by chemically conjugating smaller molecules suchas sugars or peptides to the chitin scaffold. For example, hybrid nano-bers of chitin with silk-broin and poly(glycolic acid) have beenprepared by electrospinning (Park et al., 2006a,b). These scaffolds sup-ported the attachment and spreading of human keratinocytes and bro-blasts (Noh et al., 2006; Yoo et al., 2008). Hybrid microspheres of chitinand 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 drugdelivery 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, fusionproteins of RGD and a human chitin-binding domain have been

designed with the intention of mediating cell-adhesion on reacetylatedchitosan (Carvalho et al., 2008). This approach, however, negativelyaffected anchorage of the cells to the surface, thus inhibiting cell adhe-sion and proliferation. A more successful approach towards promotingcell 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 ofchitin-based scaffolds has mainly relied on chemically modifyingthe 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-activatedscaffolds could then be conjugated to the desired protein or peptideusing the appropriate chemistry. An alternative route involves thecrosslinking of proteins onto chitin-based scaffolds using genipin orcarbodiimide/N-hydroxy succinimide (Kuo and Chung, 2012).

Chitin-based polyelectrolyte complex bers can be modied withproteins 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 theiractivity after being released from the bers. In the case of protein deliv-ery, incorporation of heparin as an additional polyanion component ledto a longer period of sustained growth factor release from the bers, at-tributed to growth factor-binding by heparin. In vivo studies indicatedgood biocompatibility and blood compatibility of chitin-alginate bers,which could again be improved by incorporating a small amount ofheparin during ber formation (Yim et al., 2007).

Chitin scaffolds have been applied chiey to the regeneration ofcartilage, bone and tendon tissue, as presented in Sections 4.2 and 4.3.

4.2. Cartilage tissue engineering

Chitinmay be inherently suitable as a biomaterial to support cartilageregeneration, as its repeatingunit, N-acetylglucosamine (Fig. 1), is a build-ing block of glycosaminoglycans such as keratin sulfate and hyaluronate,which formamajor 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 wassubsequently removed (Kuo and Tsai, 2011), to serve as scaffolds for theculture of chondrocytes. An interesting alternative process involves thederivation 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 whichthese scaffolds could potentially be derived from are typically chitin,and not chitosan-based. These chitin scaffolds supported deposition of aproteoglycan-rich extracellular matrix by chondrocytes when implantedsubcutaneously in mice. A chitinchitosanpolyethylene oxide scaffoldwas found to support adhesion and proliferation of bovine kneechondrocytes and the deposition of their extracellular matrix over4 weeks in vitro, when surface modied with elastin, poly(L-lysine) orpolyethyleneimine (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 moresuitable than chitosan as a matrix for cartilage tissue regeneration. Theability of scaffolds composed of different chitinchitosan ratios tosupport chondrogenesis was investigated in two separate studies (Kuoand Lin, 2006; Suzuki et al., 2008). While the studies differed in the iso-form of chitin ( vs ), degree of acetylation of chitin and processingconditions thatwere used, both pointed to higher deposition of cartilag-inous extracellular matrix and quality of cartilage regeneration with ahigher percentage of chitin in mixed chitinchitosan scaffolds.

To qualify the point that a higher degree of acetylation is not alwaysbetter for tissue regeneration, chitosan has been demonstrated to farebetter than chitin for wound healing in rats (Minagawa et al., 2007).Although chitin and chitosan themselves, their oligomers, as well asmonomers were found to enhance wound healing, the wound break

strength and collagenase activity of the chitosan group was found to

1781A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785be 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), wasused to support the formation of a surface cartilage layer by culture ofprimary rabbit knee chondrocytes for 4 weeks (Abe et al., 2004). Sucha conguration was aimed at producing an implant that could bepress-t into articular cartilage defectswithout covering the periosteumor suturing the implant. At the end of the culture period, the cell layer atthe surface of the sponge was lled with chondrocytes and abundantextracellular matrix, of a composition and structure that resembledarticular cartilage.

4.3. Bone and tendon tissue engineering

Early investigations on the use of chitin for bone tissue engineeringfocused on its ability to chemically interact and form composites withapatite, the mineral phase of bone. In a series of papers from the 90s,Khor and co-workers demonstrated how apatite could be depositedonto chitin scaffolds, co-precipitated with aqueous chitin solutions orblended and cast with chitin using an organic solvent system (Wanet al., 1996; Wan et al., 1998a; Wan et al., 1998b). These studies revealedfavorable mechanical properties of the chitin-apatite composites and theinherent ability of particular chitin derivatives to nucleate calciumphosphate formation. More recent follow-up work by the same lab-oratory established that a chitinhydroxyapatite composite loaded

Fig. 3. Various chitin forms for tissue engineering. (A) Cross-sectional SEM of a chitin-gpolyp(Reprinted with permission from Kuo and Chung, 2012. 2012, Elsevier.) (B) The three-dimenarea, which enables considerable liquid absorption to take place by capillary attraction. (Reprinchondrocyte-chitin sponge composite at week 4. The arrow indicates a cartilage-like layer for2004. ;2004, Mary Ann Liebert.) (D) Scanning electronmicrograph of nonwoven chitin fabric (ment of rotator cuff tendon defects. (Reprinted with permission from Funakoshi et al., 2006. with mesenchymal stem cell-induced osteoblasts, when implantedinto bone defects of rabbit femur, was able to support bone regenera-tion (Ge et al., 2004). Labelling studies showed that the osteoblastsnot only proliferated, but also recruited the ingrowth of surrounding tis-sue. Other work on chitin-based composites for bone tissue engineeringhas since followed, although these lacked in vivo demonstration. Com-posite scaffolds of chitinwith nano hydroxyapatite and nano titania, ob-tained by dispersing the particles in a chitin hydrogel cast from a CaCl2/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 byemulsication of a chitin solution.

The benets of chitin for use in bone regeneration extend to morethan just being a matrix of a composite material. Chitin hexamershave 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 thebone. These effects are mediated by the production of cytokines andgrowth factors by broblasts that come into contact with the chitinmaterial, for example, interleukin-8, which in turn induces the migra-tion of broblasts and vascular endothelial cells (Mori et al., 1997).

eptide microsphere. The inset shows a close-up of a broken sphere, scale bar = 100 m.sional chitinous matrix isolated from Aplysina cauliformis possesses large internal surfacetedwith permission from Ehrlich et al., 2010. 2010, Elsevier.) (C) Photograph of culturedmed on the top surface of the -chitin sponge. (Reprintedwith permission fromAbe et al.,original magnication 3000)whichwas investigated as an acellular matrix for the treat-2006, Elsevier.)

1782 A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785Chitin appears to be an idealmaterial for the regenerationof ligamentsand tendons, as illustrated by the efcacy of a chitin-coated polyestergraft as a scaffold for anterior cruciate ligament reconstruction (Kawaiet al., 2010). Here, the chitin coating enhanced bone formation in the 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 bywhich the fabric could helpdirect and improve tissueregeneration (Funakoshi et al., 2006) (Fig. 3D). Firstly, it was observedthat the collagen bers were deposited in the fabric and were regularlyoriented, which was attributed to chitin's ability to moderate the stressesat the bone-tendon interface. Chitin could thus be used to facilitate thedeposition, alignment and maturation of extracellular matrix. Secondly,the biodegradability of chitin fabric could be easily tuned by changingthe 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 morecomprehensive introduction on these different stem cell types and their

Fig. 4. Chitin-basedpolyelectrolyte complexbers for 3D cell culture and tissue engineering. (A)PEIDNA nanoparticles containing GFP-encoding plasmid. At 12 days postseeding, large numbe2006. 2006, Nature Publishing Group). (B) A human embryonic stem cell line (BG01V/hOG) enLu et al., 2012b. 2012, Elsevier.) (C) Confocal microscopy image of three cell types within a th(NIH/3T3), greenHepG2 cells. (Reprintedwith permission fromWan et al., 2012. 2012 Johnone component with, and the other without collagen. Cell elongation and spreading are obser2012. 2012 JohnWiley and Sons.)utility in regenerative medicine, the reader is referred to two excellentreviews (Sylvester and Longaker, 2004; Wu and Hochedlinger, 2011).Biomaterials, such as chitin, can help to realize the vast potential ofstem cells in regenerative medicine in one of two ways.

Firstly, large numbers of stem cells are required for therapeuticapplications. For instance, the number of insulin-secreting beta cellsrequired for therapy of Diabetes Type I has been estimated at610 108 cells per patient, while tissue engineering of the bladderwould require approximately 1.5 108 cells (Mason and Dunnill,2009). Here, biomaterials may play a role in providing substrates thatsupport stem cell self-renewal, while maintaining stem cell pluri- ormultipotency. The second potential contribution of biomaterials tostem cell therapy would be the provision of a matrix permissive tostem cell differentiation, or in a more active manner, one bearingligands and soluble factors that direct differentiation of stem cells intothe 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 ofpluripotent 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

Transgene expression of human dermalbroblasts seeded onbrous scaffolds loadedwithrs of broblasts were actively expressing GFP. (Reprinted with permission from Lim et al.,capsulated and propagated in 3Dmicrobrous scaffolds. (Reprintedwith permission fromree-component ber: red human umbilical vein endothelial cells, blue broblast cellsWiley and Sons.) (D)Humanmesenchymal stem cells cultured in a two-component ber,ved for cells in the collagen-containing half. (Reprinted with permission from Wan et al.,

1783A.C.A. Wan, B.C.U. Tai / Biotechnology Advances 31 (2013) 17761785spooled around planar supports or entangled to form constructs forlong term cell culture. Water-soluble chitin is uniquely suitable forcell 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 supportedthe self-renewal of two embryonic and two induced pluripotentstem cell lines for at least ten passages in culture, while maintaininga stable cell karyotype. The success of chitin in this application can beattributed to the provision of a non cell-adhesive, three-dimensionalhydrogel microenvironment within which the stem cells proliferat-ed in the form of aggregates. The high enzymatic susceptibility of chi-tin to chitinase afforded another advantage to the platform, as it allowedconvenient dissociation of the ber and release 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 vitrostem cell expansion. The major protein factors responsible for self-renewal of pluripotent stem cells, Oct4 and Sox2, were found to bemod-ied with N-acetylglucosamine, the repeating unit of chitin (Jang et al.,2012). The same modication was also found to induce the expressionof other pluripotent genes. In the paper itself, it was suggested thatthis proffered an explanation for the positive effect of glucosamineand N-acetylglucosamine in culture media on embryonic stem cellmaintenance. While the chitin ber matrix used to encapsulate thepluripotent stem cells in our experimentswas polymeric in nature, deg-radation of chitin that occurs under culture conditions, especially whenin contact with cells, may generate N-acetylglucosamine. Furthermore,during the process of decapsulation, when chitin is depolymerized toN-acetylglucosamine oligomers and its repeating units by the additionof chitinase, a high N-acetylglucosamine concentration in the mediamay help to preserve pluripotency.

Besides the expansion of stem cells, chitin-based microbrousscaffolds can be used just as effectively for differentiation of stem cells,when the latter are provided with the appropriate signals. Again, theuse of water-soluble chitin is advantageous as it allows encapsulationof cells at near-neutral pH conditions, and maintains a matrix which isboth 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 neuralmaturationme-dium leads to a high yield of terminally differentiated neurons (N90%)(Lu et al., 2012a). In a similar way, mesenchymal stem cells seeded orencapsulated in water-soluble chitin-alginate brous scaffolds havebeen differentiated into chondrogenic and osteogenic lineages byimmersion in the respective differentiation media (Yim et al., 2006).In these experiments, better cell proliferation and differentiation wasobserved for cell-encapsulated, as compared to cell-seeded scaffolds.The advantages of a chitin matrix in supporting stem cell proliferationand differentiation extends to its performance in vivo, where chitinhas been shown to be effective as a carrier material for mesenchymalstem cells in the treatment of large physeal defects (Li et al., 2004).

The polyelectrolyte complex ber route towards stem cell differenti-ation, utilizing water-soluble chitin, holds additional promise themild aqueous based process supports the incorporation of ECM compo-nents such as collagen and heparin, which could be used to furthermodulate cell differentiation (Liao et al., 2005; Wan et al., 2004).While earlier constructs took the form of non-woven matrices (Wanet al., 2006), the importance of well-dened domains for culture of dif-ferent cell types and ECM led us to develop a process to formmulticom-ponent bers composed of water-soluble chitin and alginate (Lim et al.,2013; Wan et al., 2012). The ability to spatially pattern biologicalcomponents (cells and ECM) in three dimensions and recapitulate theinteractions 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 review has endeavored to provide an explanationfor the disparity in the application of chitin and chitosan as biomaterials.It appears that the lesser use of chitin as compared to chitosan is not dueto inherently less favorable biomaterial characteristics, but rather, stemsfrom the perception that it is more difcult to process. In the rst part ofthe review, various methods that have been employed to improve theprocessability of chitin have been presented. Where the issues of pro-cessing have been overcome, the literature has in fact demonstratedthe 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 propertiessuch as degradability, mechanical strength and biological activity. To re-alize its full potential though, due attention should be given to severalaspects of its utilization. Firstly, as emphasized in this review, the per-formance of chitin in specic applications depends greatly on its degreeof acetylation, and the latter should therefore be characterized preciselyin any investigation. The same applies to themolecular weight distribu-tion of chitin used in the experiment, as it affects the mechanical andbiological properties of thematerial. Last but not the least, prior knowl-edge of chitin biochemistry and biological activity would be helpful inevaluating its suitability for particular cell or tissue types. The designof chitin and other biomaterials should ideally keep abreast withdevelopments in the biology of stem cells and tissue regeneration, sothat they could be applied in the most fruitful and effective way.

Having encapsulated the state-of-art in the application of chitin inthe tissue engineering and stem cell elds, it is hoped that this reviewwill encouragemore researchers to employ chitin as part of the growingbiopolymer arsenal for regenerative medicine.

Acknowledgment

This work is funded by the Institute of Bioengineering and Nanotech-nology (Biomedical Research Council, Agency for Science, Technology andResearch, Singapore).

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CHITIN A promising biomaterial for tissue engineering and stem cell technologies1. Introduction2. Improving the processability of chitin2.1. Water-soluble chitin2.2. Chitin derivatives2.3. Dissolution in alternative solvent systems2.4. -Chitin vs -chitin

3. Physicochemical and biological properties of chitin3.1. Degradability of chitin3.2. Immunogenicity3.3. Mechanical properties

4. Chitin scaffolds for tissue engineering4.1. Modification of chitin scaffolds4.2. Cartilage tissue engineering4.3. Bone and tendon tissue engineering

5. Chitin for stem cell technologies5.1. Applications of stem cells for regenerative medicine5.2. Chitin-based fibers for the propagation and differentiation of stem cells

6. ConclusionAcknowledgmentReferences