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INTRODUCTION

BIOLOGICAL COMPOSITE MATERIALS such as bones,teeth, diatoms, and sea shells are distinguished from

synthetic composites by their structural and organiza-tional complexity, and by the processes whereby they aregenerated.1,2 In biological composites the mineral pre-cipitates in situ within swollen polymer matrices. Cellsexert control over the crystallization process by regula-tion of the concentration of solutes, pH, and redox po-tentials in the extracellular space.3 The controlled envi-ronment is relatively large (in the range of micrometers).Metabolic control of the ion flow between the cell andthe matrix is an important kinetic aspect, through which“inorganic mediation” of crystal growth is achieved. Su-persaturation, pH, and redox potential are all presumablyoptimized beforehand. The more delicate, specific as-pects of the control over formation of biocomposites arethe most intriguing, and involve mediation by biologicalmacromolecules. These include the ability of macromol-ecules to specifically induce nucleation of one polymorphover the other in an oriented manner, and to form regu-

lar arrays of crystals and macromolecules, which arespecifically tailored to fulfill their function.4

Biological composite materials are remarkable strongand tough, even though they are usually made predomi-nantly of calcium carbonate or phosphate. Synthetic ma-terials containing such minerals show inferior mechani-cal properties. This feature prevents their use fortechnological applications.

Organisms, however, utilize them to form tough anddurable structures.5 The inorganic parts of skeletons aremade of highly strong materials that are manipulated byan array of strategies. These include the formation of nat-ural composite materials in diverse “fiber matrix”arrangements. The control over crystallite size and ori-entation is probably an additional strengthening mecha-nism. In mollusks, where many crystals are embedded inan organic matrix, more than 40 types of ultrastructuralarrangement are known.6 They reflect, besides the evo-lutionary history of the taxons, their adaptation to the me-chanical requirements of each organism. The variation inbiological mineralization is enormous and, hence, manyalternative solutions to similar problems have been found.

TISSUE ENGINEERINGVolume 10, Number 1/2, 2004© Mary Ann Liebert, Inc.

Review

Chitin Mineralization

GIUSEPPE FALINI, Ph.D and SIMONA FERMANI, Ph.D

ABSTRACT

The polysaccharide chitin is found in nature as a major component of the organic fraction of sev-eral biocomposites in which an organic matrix is associated with an inorganic fraction. The rela-tionship between the mineral phase and the organic phase implies a high level of molecular recog-nition. Chitin in mineralized biological systems is present in different polymorphs and has a crucialrole in the hierarchical control of the biomineralization processes; the nacre of the mollusk shell isa representative example. Biologically inspired synthesis has been used for the production of min-eral–chitin composites. Their actual and future applications move from the medical field as bonerepair (chitin–calcium phosphate composites) to the industrial field as catalyst (silica chitin struc-ture replica).

Department of Chemistry G. Ciamician, Alma Mater Studiorum Università di Bologna, Bologna, Italy.

CHITIN IN MINERALIZED BIOLOGICALSYSTEMS

Chitin, the second most common portion of biomass,is produced by a variety of animals, insects, and fungi.It is a linear polysaccharide containing chains of b(1-4)2-acetamido-2-deoxy-D-glucopyranose residues7–9 (Fig. 1).

In the crustacean exoskeleton the framework macro-molecule is a-chitin10 and in the mollusk shell it is b-chitin.4

In the nacre mollusk shell, the mineral phase forms in-side a preformed organic matrix. X-ray and transmissionelectron microscopy (TEM) studies of the matrix in themineralized tissue showed that it is composed of thin lay-ers of b-chitin sandwiched between two thicker layers ofsilklike proteins, onto which acidic macromolecules richin aspartic acid are adsorbed.11,12 The fiber axis of thechitin and the silk proteins are perpendicular to each otherand aligned with the a and b axes of the mineral phase,which is the aragonite polymorph of calcium carbon-ate.13,14 Cryo-TEM studies of the matrix of bivalve At-rina rigida embedded in vitrified ice have shown that theintralamellar sheets are composed mainly of highly or-dered and aligned b-chitin fibrils. The silk, which is quan-titatively an important component of the matrix, couldnot be imaged between the sheets. Organic material was,however, observed between the sheets. It was postulatedthat this was the location of the silk. Because this mate-rial reveals no regular structure it has been suggested thatbefore mineralization the silk forms a hydrated gel.15

The shell of the pink shrimp Pandalus borealis has amatrix of a-chitin. Amorphous calcium carbonate is as-sociated with the chitin. Interestingly, during continuedfrozen storage the amorphous phase crystallizes in theform of the two polymorphs calcite and vaterite. Thechitin is an integral part of the crystallized regions andtherefore it is suggested to play an important role in cal-cium carbonate deposition.16

The internal shell of the cuttlefish (Sepia officinalis),the familiar cuttlebone, is a highly organized calcium car-

FALINI AND FERMANI

bonate biomineral with elaborate macroporous architec-ture. The structure consists of sheets of aragonitic cal-cium carbonate separated by pillars in a manner similarto an honeycomb.17,18 The pillars are sigmoidal in cross-section. This provides maximum resistance to crushingwith minimum mass of material. The complex inorganicstructure is deposited on a preformed b-chitin template,which is revealed when the inorganic component is re-moved by dissolution in acid.

The shell of Lingula unguis is composed of apatite andan organic matrix. The apatite crystals precipitate in theorganic scaffold, forming consecutive mineralized andorganic layers to produce a laminated structure with im-proved mechanical properties. The organic matrix is con-stituted mainly of b-chitin. The degree of orientation ofapatite showed correlation to that of b-chitin, the fiberaxis of b-chitin being parallel to the c axis of apatite.Moreover, it was suggested that a close relationship ofunit cell dimensions of chitin and apatite indicates thatthe fibrous structure of the organic matrix assists the ori-entation of the apatite crystals.19

CHITIN–CALCIUM CARBONATECOMPOSITES

Chitin is a support on which calcite may nucleate andgrow. The crystallization was studied at constant solu-tion supersaturation and thermodynamic data were cal-culated.20 Chitin had been used as an organic matrix toform organic/inorganic composites and has biological rel-evance for controlling CaCO3 crystallization in molluskshells. An artificial assembly of b-chitin and silk fibroin,together with acidic macromolecules extracted from dif-ferent mollusk shells, was designed to match roughly thebiological composite.21 This assembly was shown to beable to control calcium carbonate polymorphism. Thesame mollusk shell acidic macromolecules that exclu-sively induce calcite when adsorbed on plastic substrateswere shown to retain the polymorphism specificity in the

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FIG. 1. Schematic representation of the b(1-4)2-acetamido-2-deoxy-D-glucopyranose residue, which constitutes the biopoly-mer chitin. The molecule is shown along two directions: (A) view normal to the molecular axis; (B) view along the molecularaxis.

appropriately assembled artificial microenvironment.Once adsorbed on this scaffold, the macromolecules ex-tracted from aragonitic mollusk shell layers induced ara-gonite formation, whereas those extracted from calciticlayers induced calcite formation, with total fidelity. Allthe components of the assembly have a specific role andonly their ordered and controlled association leads to thecontrol of polymorphism. Chitin, purified from squid pen(Loligo sp.), has a porous framework, which facilitatesthe diffusion of macromolecules and ions into the struc-ture. Fluorescence light microscopy has demonstratedthat silk fibroin is intimately associated with the chitinframework. One particular fraction of the assemblage ofthe mollusk shell macromolecules extracted from anaragonitic shell layer is able to induce specifically ara-gonite crystal formation in vitro. The use of syntheticpeptides containing leucine residues and aspartic or glu-tamic residues as acidic macromolecules analog showedthat only aspartic–leucine peptide is capable of specifi-cally inducing aragonite formation.15 The mineral de-posited inside the matrix and formed a material in whichthe organic and inorganic phases possess some featuresof composite materials at the nanometer scale. This is thescale at which the chitin fibers are intergrown intimatelywith the crystallites. Interestingly, in the artificial as-sembly the single crystalline domain within the poly-crystalline spherulites of calcite preserves the size andmorphology typical of the mineral (Fig. 2).

Belcher et al. have studied a different in vitro system,using as a nucleating matrix the so-called green layersheet isolated from abalone shells.22 This nucleatingsheet is formed by an internal core of b-chitin sandwichedbetween two layers of different fibrous proteins, one ofwhich is rich in tyrosine residues (G. Falini, unpublisheddata). Aragonite or calcite crystallization has been ob-served when proteins extracted from aragonitic or cal-citic layers of the abalone shell were, respectively, addedto the green layer.

Chitin fibers have been coated with a film of calciumcarbonate in the presence of poly(acrylic acid) (pAA),poly(L-aspartate) (pD), and poly(L-glutamate) (pE).23

These polymers have been widely used as soluble addi-tives for the crystallization of CaCO3, because their pres-ence influences the morphology and the crystallinity of theinorganic compounds.24 Calcite and vaterite crystals formfrom supersaturated calcium bicarbonate solution in thepresence of pAA. The polymorph of CaCO3 switches fromcalcite to vaterite with the increase in the concentration ofpAA. Only vaterite was observed in the presence of pD orpE. The presence of polyelectrolytes is an essential re-quirement for mineral deposition. In fact, no mineral filmformation has been observed in the presence of monomericcompounds such as amino acids and propionic acid.23

Composites of b-chitin with calcium carbonate poly-morphs were prepared by precipitation of the mineral into

CHITIN MINERALIZATION

a chitin scaffold, obtained from squid pen (Loligo sp.),by means of a double diffusion system. The three mainpolymorphs of calcium carbonate (aragonite, calcite, andvaterite) were observed. Their location within the matrixdepends on the kind of polymorph (Fig. 3). The super-saturation inside the compartmentalized space in thechitin governs the location and polymorphism of the crys-tals.25

Open-pore chitin matrices can be produced using gelscast from chitin solutions loaded with calcium carbonatecrystals. The CaCO3–chitin gels were treated with acidicsolution to remove the mineral and to obtain highlyporous matrices with good water vapor permeability, wa-ter uptake profile, and enhanced mechanical properties.26

CHITIN–CALCIUM PHOSPHATECOMPOSITES

Chitin has been combined advantageously with hy-droxylapatite (HA) to produce new composite materials.

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FIG. 2. Synthetic composite materials produced in vitro con-taining (a) calcite and (b) aragonite crystals in a matrix. Thematrix is composed of b-chitin and silk fibroin, as well as sol-uble macromolecules extracted from the calcitic (a) and thearagonitic (b) layers of the shell of Atrina serrata. (Picturestaken by the author at the Weizmann Institute of Science, Is-rael.) Scale bar: 1 mm.

The role of chitin as the organic component able to re-inforce the mineral phase is analogous to collagen, whichis the main organic component of bone. This property, inaddition to the inherent biodegradability, biocompatibil-ity, and good mechanical properties of chitin, suggestspotential use of chitin–HA composites as a hard tissuesubstitute. These composites are potentially useful as abone material substitute because they allow the intro-duction of osteoconductive HA into regions of bone loss,with the chitin matrix acting as a binder to prevent post-operative migration of the HA particles.

Often soluble chitosan, obtained by means of partialdeacetylation of chitin, is used to prepare composites. Ho-

FALINI AND FERMANI

mogeneous chitosan–HA composite nanostructures havebeen prepared by a coprecipitation method. The com-posites form elliptic aggregations with a width of about50 nm and a length of about 230 nm, corresponding ap-proximately to those of a chitosan molecule. The sizes ofthe constituent HA crystallites were 30 nm in length and10 nm in width, with their c axis oriented in the same di-rection of the chitin molecular axis.27

The anionic chitin derivatives sodium carboxymethylchitin and phosphoryl chitin have strong influence on thecrystallization of calcium phosphate from supersaturatedsolutions. They inhibit the growth of HA and retard therate of calcium phosphate precipitation. These chitin de-rivatives are incorporated into the precipitate and influ-ence both the phase and morphology of the calcium phos-phate formed.28 Chitin–HA composite has been formedin situ onto porous chitin scaffolds. These scaffolds havebeen prepared from chitin solutions and subsequent

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FIG. 3. Calcium carbonate chitin scaffold mineralized for atime of 24 h, using a double-diffusion system: on one side of thematrix is the calcium reservoir and on the other side is the car-bonate solution reservoir.26 (a) Calcium side; (b) cross-section;(c) carbonate side. Rhombohedral calcite crystals are present onthe scaffold surfaces. Scale bars: (a) 50 mm; (b and c) 100 mm.

FIG. 4. Calcium phosphate chitin scaffold mineralized for atime for 24 h, using a double-diffusion system. The layeredstructure of the matrix is well evident and the mineral crystalsare intimately associated with the organic phase. (Pictures atdifferent magnifications; scale bars, 50 mm.)

carboxymethylation or phosphorylation. N,N-Dicarboxy-methyl chitosan mixed with calcium acetate and di-sodium hydrogen phosphate yields solutions from whichan amorphouse material containing an inorganic compo-nent is isolated. This compound was used for the treat-ment of bone lesions in experimental surgery and in den-tistry.29

The in situ precipitation strategies are similar to thoseemployed in naturally occurring biocomposites. Com-posites of b-chitin from Loligo squid pens with octacal-cium phosphate or HA have been prepared by precipita-tion of the mineral into the chitin scaffold by means ofa double diffusion system. The octacalcium phosphatecrystals form with the usual form of [001] blades insidechitin layers preferentially oriented with the [100] facesparallel to the surface of the squid pen (Fig. 4). Thesecrystals are more stable to the hydrolysis to HA with re-spect to those precipitated in solution. In these in vitroexperiments the compartmentalized space in the chitingoverns the orientation of the crystals, even if epitaxialfactors might play a role in the nucleation processes.30

Different results are obtained when suspensions in-stead of solutions are used. Chitin strips, sheet, films,plates, and membranes modified with the bioactive com-ponent, HA, were synthesized. HA was dispersed at var-ious percentages in a chitin solution of 5% LiCl in N,N-dimethylacetamide. The composite was obtained bycontrolled drying of the gel. Scanning electron mi-croscopy examination shows a homogeneous dispersionof HA filler in the chitin matrix, which meets require-ments of a uniform composite.31

Composites of HA and a network formed by cross-linking of chitosan and gelatin with glutaraldehyde wereproduced. The presence of HA did not retard the forma-tion of the chitosan–gelatin network and the polymer ma-trix did not influence he crystallinity of HA.32

It has been shown by mechanical testing that the vis-coelastic properties of the chitin are retained even with ahigh loading (50% by mass) of HA. This indicates thatthe composite has good fracture toughness and impact re-sistance. A composite that contained 50% by mass offiller exhibited a tensile strength of approximately 29MPa and an elastic modulus of 2.4 GPa in the dry form.This strength is expected to diminish in an aqueous en-vironment but not to a detrimental level.31

CHITIN–SILICA COMPOSITES

Chitin fiber structures have been used as replicas toobtain silica macroporous and microporous materialswith potential catalytic applications.

Aquagels, in which the solid phase consists of bothchitosan and silica, have been prepared by using an acidicsolution of chitosan to catalyze the hydrolysis and con-

CHITIN MINERALIZATION

densation of tetraethylorthosilicate. By standard dryingprocesses the corresponding aerogels have been obtained.The amount of chitosan in the gel plays a role in theshrinkage of the aerogel during drying. Pyrolysis undernitrogen produces a darkened aerogel due to the thermaldecomposition of the chitosan; however, the aerogel re-tains its monolithic form. Biocompatibility screening ofthis material shows a high value of hemolysis, but a lowvalue for cytotoxicity.33

A hydrothermal technique was used to solidify cal-cium silicate hydrate (xonotlite) powders at low tem-perature and low pressure. Chitosan was used to rein-force the mechanical strength of the calcium silicatecompacts. The low-temperature solidification methodis expected to be useful in the development of an en-vironment-friendly processing route for making artifi-cial wood.34

Cuttlebone b-chitin has been used as a highly orga-nized template with macroscopic porosity to prepareanalogous silica polysaccharides replicas with a three-di-mensional interconnecting box structure. The results in-dicate that the fidelity of the replication and the inorganiccontent can be controlled by the degree of silicate su-persaturation and the extent of deacetylation of the or-ganic matrix.35

CONCLUSIONS

Chitin and chitosan are widespread low-cost biopoly-mers with numerous potential applications in materialsscience as well as in medicine. Their structural organi-zation favors interaction with minerals of biological rel-evance such as calcium phosphate and carbonate. So faronly a few mechanisms of inorganic–organic phase in-teraction have been proposed19,30; this area of researchrepresents a new and stimulating frontier for the designof chitin-based materials.

ACKNOWLEDGMENTS

We thank Prof. A. Ripamonti and Dr. M. Gazzano forcriticism and suggestions. Financial support from theConsiglio Nazionale delle Ricerche, the Ministero dell’Università e della Ricerca Scientifica, and the Universityof Bologna (Funds for Selected Research Topics) is grate-fully acknowledged.

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18. Seizen, R. Cuttlebone: The buoyant skeleton. Sea Front.32, 115, 1986.

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22. Belcher, A.M., Wu, X.H., Christensen, R.J., Hansma, P.K.,Stucky, G.D., and Morse, D.E. Control of crystal phaseswitching and orientation by soluble mollusk shell proteins.Nature 381, 56, 1996.

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24. Mann, S., and Ozin, G.A. Synthesis of inorganic materialswith complex form. Nature 382, 313, 1996.

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Address reprint requests to:Giuseppe Falini, Ph.D.

Dipartimento di Chimica G. CiamicianAlma Mater Studiorum Università di Bologna

Via Selmi 2I-40126 Bologna, Italy

E-mail: falini@ciam.unibo.it

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