Calcium carbonate in biomineralisation and
biomimetic chemistry
F. C. Meldrum
Nature is adept at controlling mineralisation
processes to produce single crystals and
polycrystalline and amorphous structures with
remarkable morphologies and mechanical
properties. Calcium carbonate is one of the most
common biominerals, and as such has received
considerable attention, both in terms of research
into the processes used in vivo to control
calcification, and application of identified
strategies to control precipitation synthetically.
After providing an initial description of the
structure and properties of calcium carbonate, this
review will examine the mechanisms used by
organisms to influence calcium carbonate
nucleation and growth and will then demonstrate
how identified control strategies have been
applied to synthetic systems. The emphasis of the
review is on biomimetic engineering of calcium
carbonate which is considered in three principal
sections: control of crystal growth using soluble
additives, crystal growth in confinement and
calcium carbonate deposition on solid substrates.
IMR/385
The author is in the Department of Chemistry, Queen
Mary University of London, Mile End Road, London E1
4NS, UK (f.c.meldrum@qmul.ac.uk).
© 2003 IoM Communications Ltd and ASM International.
Published by Maney for the Institute of Materials, Minerals
and Mining and ASM International.
Introduction
The inorganic structures formed by organisms provide
a unique inspiration for materials design. Perhaps the
most immediately striking aspect of biominerals is
the range of truly exquisite and unique morphologies
observed, which are frequently entirely disparate from
their synthetic counterparts. Structurally, biominerals
are typically composite materials, being intimately
associated with organic macromolecules, and are
often hierarchically organised on a scale from ang-
stroms to millimetres. Nature necessarily achieves this
degree of control under ambient conditions,
employing specially designed macromolecules to
manipulate crystallisation processes. The organic
macromolecules are a vital component of the mineral,
being involved in nucleation and growth control, and
deŽ nition of mechanical properties. In consequence,
although precipitated under ambient conditions,
biominerals often exhibit superior mechanical prop-
erties, which is particularly important when they are
being exploited for skeletal roles. Clearly, nature has
evolved mechanisms to in uence crystal growth to
a degree unparalleled in any in vitro environment,
producing mineralised structures that are perfectly
optimised to their function within an organism.
DOI 10.1179/095066003225005836
The study of biomineralisation oVers valuable
insights into how mineralisation processes can be
controlled under ambient conditions to produce both
single crystal and polycrystalline structures, and has
prompted a range of studies using biomimetic routes
to control the synthesis of inorganic solids.1–7
Biomimetic strategies provide an attractive alternative
to high temperature syntheses, especially in appli-
cations where substrates cannot be exposed to high
temperatures.5 In comparison with vapour phase
methods of thin Ž lm formation, solution techniques
are cheaper, may oVer faster deposition rates, and
importantly, temperature sensitive materials can be
deposited.3 Given the demand for advanced materials
such as semiconductors, biomedical devices and
optical and electronic Ž lms which require control
over the size, orientation, phase and morphology of
the component crystals, a detailed study of biomimetic
processes will have important technological impact.6,8
Calcium carbonate is one of the most abundant
biominerals, and as such, has received considerable
interest from many branches of science. In particular,
the mechanical properties of these structures have
long fascinated, and frequently exceed all expectation
for such a traditionally poor engineering material as
calcium carbonate.9,10 These properties derive from
the composite character of CaCO biominerals, and
3
in particular the organisation of the components.10
Mollusk nacre provides an example of a polycrystal-
line calcium carbonate biomineral with excellent
mechanical properties. Despite having a very low
organic content (~1%), this material is superior to
most other composite ceramics in stiVness, strength
and toughness.11,12 and nacre is 3000 times more
resistant to fracture than a single crystal of pure
aragonite.13 These properties can be attributed to the
structural organisation of nacre, which comprises
layers of interlocking aragonite platelets separated by
a thin layer of organic material. When a crack propa-
gates through nacre it passes around the platelets by
a tortuous path,12,14 causing the plates to spring
apart, and extending the organic sheets.11,14 The
organic ‘adhesive’ between the plates appears to be
key in the fracture resistance of this material,15,16 and
elongates by pulling open folded domains and loops,
or breaking interchain bonds.16
The material presented in this review will examine
the mechanisms used by organisms to in uence cal-
cium carbonate nucleation and growth, and will then
demonstrate how identiŽ ed control strategies have
been applied to synthetic systems. The synthetic sys-
tems are subdivided into three principal sections:
control of crystal growth using soluble additives,
crystal growth in conŽ nement and calcium carbonate
deposition on solid substrates. This discussion is
preceded by an initial description of the structure and
properties of calcium carbonate. The subject of bio-
International Materials Reviews 2003 Vol. 48 No. 3 187
188 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry

mineralisation has been covered in detail in a number
of excellent reviews,2,17–25 and thus emphasis will be
placed on biomimetic strategies. That so many biomi-
metic studies have focused on control of calcium
carbonate growth can be attributed to the fact that
nature has already clearly demonstrated that it is
possible to control virtually any aspect of this mineral,
from morphology, texture and polymorph to orien-
tation and assembly. Calcium carbonate therefore
provides a ‘test case’ to demonstrate that the mechan-
isms used in biology can be applied in synthetic
systems to control crystal nucleation and growth.
Extension of these studies beyond calcium carbonate
is likely to lead to novel methods for the construction
of synthetic materials with optimised properties.
Calcium carbonate
Polymorphs of calcium carbonate
CaCO exists in a variety of polymorphic forms, of
3
which there are three anhydrous crystalline poly-
morphs, calcite, vaterite and aragonite, and two well
deŽ ned hydrated crystalline polymorphs, calcium car-
bonate hexahydrate (CaCO .6H O) and calcium car-
bonate monohydrate (CaCO 3 .H2 O). An amorphous
3 2 exists. Calcite and
form of calcium carbonate also
aragonite, which have similar thermodynamic stabil-
ities under standard conditions,17 are both common
in biological and geological samples (Table 1).
Vaterite is metastable with respect to calcite and
aragonite, and is extremely rare in nature, being
detected as a minor component of only a few bio-
mineralised structures, and not in geological samples.
There are few examples of the calcium carbonate
monohydrate in biology, and none of the hexahydrate,
although the latter has been found naturally26,27 in
sea water at temperatures from ­ 3 to 3°C. Current
available information suggests that amorphous cal-
cium carbonate (ACC) is also rare in nature. However,
this phase is easy to overlook when it is present in
combination with crystalline phases, and so it may
be more common than is currently believed.
Calcite is the most abundant carbonate mineral,
being somewhat more stable than aragonite under
ambient conditions.17,28 However, both forms are
common because the radius of the calcium ion is very
close to the limiting value for the transition from the
rhombohedral carbonate structure to that of the
orthorhombic aragonite structure.28,29 Traditionally,
the calcite structure is often considered by analogy to
NaCl, where the Na+ and Cl– ions are replaced by
Ca and CO groups respectively, and the unit cell is
distorted to3 accommodate the large planar CO2Õ
ions to give a face-centred rhombohedral cell.17,28 3
Calcite can be described by a number of unit cells,
including the cleavage cell (morphological rhombo-
hedron) which is commonly used in morphological
crystal studies and contains four CaCO units. The
3
smallest unit cell is the acute rhombohedron contain-
ing two CaCO units. For convenience, a hexagonal
unit cell which3 has the same height as the rhombo-
hedral cell, and contains six CaCO units is often
3 organisation
used (Fig. 1a). This cell clearly shows the
of the calcite structure, in which the Ca2+ and
CO2Õ ions are located in alternate layers, perpendicu-
3
lar to the c axis, and spaced at intervals of c/12. The
CO groups are identically oriented within a layer,
3
and the orientation reverses between adjacent layers.
Each Ca2+ ion has six immediate CO neighbours,
oriented such that one oxygen from each 3 forms the
immediate neighbours of calcium. Each Ca ion thus
occupies an octahedral environment of O atoms.
Aragonite is described by an orthorhombic unit
cell in which the metal ions exist in a pseudohexagonal
arrangement. In common with calcite, the Ca and
CO groups are organised in alternate layers perpen-
3 to the c axis, with the plane of the CO groups
dicular
3
lying perpendicular to the c axis (Fig. 1b).17,30 In
aragonite however, the CO layers are split into two
layers parallel to the a axis 3 such that they appear

Table 1 Occurrence of calcium carbonate in biology: adapted from Ref. 25

Mineral Formula Organism Location Function
Calcite CaCO3 Coccolithophores Cell wall scales Exoskeleton
Foraminifera Shell Exoskeleton
Mollusks Shell Exoskeleton
Crustaceans Crab cuticle Mechanical strength
Birds Eggshells Protection
Echinoderms Lens Optical imaging
Mammals Inner ear Gravity receptor
Mg–calcite (Mg,Ca)CO3 Octocorals Spicules Mechanical strength
Echinoderms Shell/spines Strength/protection
Aragonite CaCO3 Scleratinian corals Cell wall Exoskeleton
Mollusks Shell Exoskeleton
Gastropods Love dart Reproduction
Cephalopods Shell Buoyancy device
Fish Head Gravity receptor
Vaterite CaCO3 Gastropods Shell Exoskeleton
Ascidians Spicules Protection
Amorphous CaCO3 . nH2 O Crustaceans Crab cuticle Mechanical strength
Echinoids Larval spicules Precursor phase
Porifera Spicules Strength/protection
Ascidiacea Spicules Mechanical strength
Plants Leaves Calcium store

International Materials Reviews 2003 Vol. 48 No. 3

 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 189
1 Schematic diagrams of a hexagonal unit cell of calcite; b unit cell of aragonite, with additional CO23 Õ
groups outside cell shown to demonstrate coordination of Ca ion; and c subcell of vaterite, where
c¢ = c/2: reprinted from Ref. 17 with permission from Springer
corrugated. DiVerences also exists in the Ca coordi-
nation between calcite and aragonite. While each Ca
in the aragonite structure is also surrounded by six
CO groups, three CO groups are bonded via two
3 3
oxygens, while the remaining three are bonded by
one oxygen each, leading to nine immediate oxygen
neighbours. All of the Ca–O distances in aragonite
are substantially greater than in calcite, which at Ž rst
sight seems at odds with the greater density of aragon-
ite than calcite; more eYcient packing of the CO
3
groups occurs in aragonite.
As the third non-hydrated form of CaCO , vaterite
is described by a hexagonal unit cell (Fig.3 1c). The
Ca and CO groups are again organised in alternating
3 to the c axis, but in contrast to aragon-
layers parallel
ite and calcite, the plane of the CO groups is parallel
3
to the c axis.17,31 While rare in nature, vaterite is a
common synthetic product of solution precipitations
and high temperature preparations, being kinetically
favoured under certain conditions and subsequently
transforming to a more stable polymorph.31,32
The formation and stability of the CaCO poly-
3
morphs are highly sensitive to the experimental con-
ditions, such as temperature, supersaturation and the
presence of additives. There are three general ways of
precipitating calcium carbonate from aqueous solu-
tion. First, solutions containing the calcium and car-
bonate ions can be mixed. The second method
involves dissolution of calcium carbonate in water by
saturating the solution with carbon dioxide, and
reprecipitated on release of the carbon dioxide from
the solution. Third, exposure of a calcium ion solution
to ammonium carbonate results in calcium carbon-
ate precipitation. Calcite is typically precipitated at
room temperature,33 while aragonite is produced
as solution temperatures approach 100°C (Refs.
34–36). Syntheses of pure vaterite have also been
reported.37–39
Precipitation of these metastable phases is favoured
by increasing the rate of precipitation, lowering tem-
perature and by the addition of certain addi-
tives.26,40–42 Both the hydrated calcium carbonates,
CaCO .6H O and CaCO .H O, are unstable towards
3
conversion 2into the anhydrous
3 2 carbonates.17,26,40,42–44
The hexahydrate form can be prepared in the
absence of additives at low temperatures,26,41,45 and
is stabilised by additives such as polyphosphate
and Mg.26,40–42,46 In dilute solutions near 0°C,
CaCO .6H O is less soluble, and therefore more
stable 3than 2CaCO .H O.46 Control of temperature is
3 preparation
not as crucial for the 2 of calcium carbonate
monohydrate, which can be prepared at room temper-
ature in the presence of Mg.42 Monohydrate is also
formed on transformation of ACC in Mg solution.26,27
Amorphous calcium carbonate (ACC) is formed as
aggregates of spherical particles by homogeneous
precipitation under conditions of high supersat-
uration, and has been described26,27 as having com-
International Materials Reviews 2003 Vol. 48 No. 3
190 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
position CaCO .1·5H O. It is highly unstable, rapidly
transforming to3 the more
2 stable CaCO polymorphs
but transformation can be retarded 3 by low
temperatures.26,27,41,43,44,47
Influence of metal ions on calcium carbonate
precipitation
Precipitation of calcium carbonate in nature fre-
quently occurs in the presence of metal ions, which
can have signiŽ cant eVects on the polymorph and
habit of the crystals produced. Of the alkali metals,
Li is the only one to exert a signiŽ cant in uence on
calcite and aragonite growth.32 The alkali metal ions
occupy interstitial positions in the calcite structure,
and in the case of the small Li+ ion, large quantities
are incorporated within the calcite lattice, causing
distortion of the surface structure. Addition of Li+
ions therefore retards the growth of calcite, and the
transformation of aragonite and vaterite to cal-
cite.32,48 Lithium also aVects the habit of precipitated
calcium carbonate,49,50 resulting in calcite crystals
with well developed, basal (0001) faces. As the (0001)
face is homonuclear, and the carbonate ions are not
close packed, interstices large enough to accommo-
date the Li+ ions are present. Adsorption of Li+ ions
on this face may lead to its preferential stabilisation
by neutralisation of the surface dipole, without neces-
sitating surface reconstruction.49 Aragonite crystals
grown in the presence of Li+ ions were characterised
by well deŽ ned outgrowths, consisting of six prismatic
side faces, truncated by a (001) face.50
Many divalent metal ions can partially replace the
Ca2+ ions in calcite, and thus aVect CaCO precipi-
3
tation by a diVerent mechanism. The solid solution
of MgCO in calcite is one of the most important
carbonate 3 solid solutions in nature, and will be
considered separately (see following section).
Manganese rich calcite has been synthesised from
solution of up to 16 mol-%Mn, and natural samples
have been analysed as containing between 0 and
50 mol-%Mn. The substitution of Fe2+ for some Ca
is also quite common, and in natural samples,
5–10 mol-%Fe is known.29 Barium and strontium,
having a larger ionic radius than calcium, preferen-
tially incorporate into aragonite rather than calcite,
and suppress aragonite formation.32 Indeed, the most
important chemical substitution in aragonite bio-
minerals is Sr and with few exceptions, skeletal ara-
gonites have Sr/Ca ratios generally close to that of
sea water.30
Formation of Mg-calcites
The Mg2+ ion has a signiŽ cant eVect on CaCO
precipitation and, when present in suYcient concen-3
trations, generally results in the precipitation of ara-
gonite rather than the thermodynamically favoured
phase, calcite.17,51–56 The Mg2+ ion is more strongly
hydrated than Ca2+ and is strongly adsorbed on the
surface of the growing calcite crystal, poisoning active
growth sites.17,51,57 Dehydration of the Mg2+ ions
prior to incorporation in the calcite lattice creates a
barrier to the growth of calcite nuclei.17,58,59 This
eVect may be overcome by the initial formation of an
amorphous precursor phase,60 and may also provide
International Materials Reviews 2003 Vol. 48 No. 3
a mechanism for the occlusion of Mg in the product
calcite phase. Magnesian calcite is also more soluble
than pure calcite, which may lead to a decrease in
the thermodynamic driving force for crystallis-
ation.56,57 Adsorption of hydrated Mg2+ ions on
aragonite crystals does not pose a serious kinetic
obstacle to their growth since Mg2+ ions are not
incorporated into the aragonite lattice. Thus, when
the critical supersaturation for aragonite is exceeded,
aragonite rather than calcite is precipitated.17,51,56
Magnesian carbonates of geological origin display
a wide range of morphologies and compositions, as
dictated by factors such as the temperature, CO2Õ
concentration, the Mg/Ca ratio, the growth rate and 3
solution saturation.51,57,61–64 While conditions of low
Mg/Ca can only yield low Mg-calcites, elevated
Mg/Ca ratios can give calcites with highly variable
Mg contents. Magnesian calcite containing in excess
of 10 mol-%Mg is more soluble than aragonite, and
so is thermodynamically unstable with respect to this
phase.56 Syntheses leading to the precipitation of high
Mg-calcites often rely upon control of kinetics, such
as occurs during crystal growth in gels,57,65,66 or on
combination of high concentrations of CaCl /MgCl
solutions with sodium bicarbonate solution. The 2 latter2
reaction yields high Mg-calcites that are unstable
towards continued incubation in solution.67,68
Synthesis of Mg-calcites in the presence of organic
additives provides a biomimetic route towards stabil-
isation of high Mg-calcite and inhibition of aragon-
ite.55,60,65,69–71 Further discussion of the precipitation
of Mg-calcites in biology is given in the section
‘Magnesium incorporation’, below.
Atomistic simulations of calcite surfaces have
shown that kinetic eVects of surface-incorporated Mg
ions can lead to morphological changes in single
crystals of calcite.72 Mg2+ is preferentially adsorbed
on {100} faces, stabilising them relative to other faces.
Experimental observations on the growth of single
crystal magnesian calcites suggest that, in practice,
Mg2+ interacts speciŽ cally with the {011} faces of
calcite.73 These crystal faces exhibit alternating
anionic and cationic sites and the cations may be
suYciently far apart to facilitate adsorption of the
relatively large, hydrated Mg2+ ion. Magnesian cal-
cites elongated along the c axis and capped with
{104} faces have been crystallised from water–alcohol
mixtures,59,74 and on combination of CaCl /MgCl
solutions with sodium bicarbonate solution.67,71 2 2
Synthetic Mg-calcites with Mg contents in excess of
5–7% are invariably polycrystalline aggregates. If
both the growth and dissolution of the subcritical
nuclei are suYciently inhibited by adsorbed Mg2+
ions, then the critical size required for further growth
can only be reached by aggregation of subcritical
nuclei, and the supercritical nuclei become polycrys-
talline.75 Synthetic Mg-calcite polycrystalline aggre-
gates exhibit a far wider range of morphologies than
their single crystal counterparts, displaying forms
including peanuts, dumbbells, spheres, and oöid-like
structures.57,67,71,76,77
Calcium carbonate biomineralisation
Biomineralisation is very common in nature and over
60 diVerent biominerals are recognised. The calcium
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 191
based minerals represent approximately half of these,
and include both calcium carbonate and calcium
phosphate, which are important in skeleton forma-
tion. The predominance of Ca biominerals probably
re ects its abundance in the ocean, and the low
solubility products of many of its salts. It is also
widely used as a messenger in cells, which requires
eYcient removal to the cell exterior by ion pumps.18,24
Examples of calcium carbonate biominerals are given
in Table 1.
Biomineralisation has been divided into two funda-
mentally diVerent classes, based on the degree of
biological control.19,21,25 The precipitation of a min-
eral as a result of interactions between an organism
and the environment, when little control is exerted
over the mineralisation process, is termed ‘biologically
induced’ mineralisation. In ‘organic matrix mediated
mineralisation’ the organism directly controls miner-
alisation. Here, control over mineralisation relies
upon a number of key stages, some or all of which
may be involved in the precipitation of a given
mineral: conŽ ning a space, forming an organic matrix
framework, controlling ion input, constructing a
nucleation site, controlling crystal orientation and
growth and terminating crystal growth. The Ž rst
crucial step in control of mineralisation is the initial
isolation of space, which is usually achieved using
cellular membranes, vesicles or predeposited macro-
molecular matrix frameworks. Sophisticated control
can be achieved by further functionalisation of the
mineralisation environment using organic macro-
molecules. In this way, the number and orientation
of nucleating crystals can be dictated, as is necessary
to produce materials with superior properties.
Organic macromolecules
Organisms clearly cannot manipulate parameters
such as temperature or pressure, as is commonly done
synthetically to control crystal growth. Alternatively,
there is signiŽ cant experimental evidence that nature
uses organic macromolecules to produce minerals of
selected size, phase, morphology, texture and
assembly. At the most basic level, the isolated site at
which mineralisation occurs comprises a structured
organic matrix. This can vary from simple intercellu-
lar lipid vesicles, to more complex extracellular mac-
romolecular frameworks such as the organised
collagen Ž brils in bone. The three-dimensional shape
of the mineralisation site can play a key role in
determining the Ž nal morphology of the crystals
formed.
The majority of calcium carbonate biominerals also
contain organic macromolecules within the mineral
at levels of up to a few weight per cent. In vitro
growth of CaCO crystals in the presence of macro-
molecules isolated3 from calcium carbonate biominer-
als by gentle dissolution has demonstrated interaction
with speciŽ c crystal planes. This suggests that these
‘control’ macromolecules may be important in mor-
phological and textural control. Characterisation has
shown that the control macromolecules are usually
highly negatively charged and contain carboxyl, sul-
phate and phosphate groups. Aspartic acid and glu-
tamic acid are common, as are covalently bound
polysaccharides, which are generally sulphated and
carboxylated.78,79 Some common partial sequences of
amino acids have also been identiŽ ed, consisting of
runs of poly-Asp and alternating sequences with poly-
Asp at every other residue, commonly separated by
either glycine or serine.78 This sequence exhibits reg-
ular repeating negative charges which may bind Ca2+
ions and could be signiŽ cant in controlling crystal
growth. In addition, poly (aspartic acid) can adopt a
b-sheet structure on binding to Ca, which appears to
be important in enabling structured interaction with
crystal faces. IdentiŽ cation of common sequences
enables synthetic analogues of macromolecules active
in crystal growth control to be designed.
Adsorption of control macromolecules to the
framework matrix could provide a further route to
manipulation of crystal nucleation and growth.22
Oriented nucleation of calcium carbonate on a sub-
strate appears to depend upon the degree of organis-
ation of the nucleating organic matrix.8,80 The rate
of nucleation of calcium carbonate can also be
increased, and the morphology altered, by immobi-
lised control macromolecules, while the same proteins
acting from solution can inhibit crystal growth.79,81,82
The diVerence in behaviour between the immobilised
and free protein can be attributed to conformational
changes and rigidity on binding to a solid substrate.
Crystal nucleation
Selection of the number, orientation and structure of
crystals produced within a given environment occurs
at nucleation. Oriented nucleation must be deŽ ned
by an organised substrate, as simple concentration of
positive ions at a negatively charged substrate (an
ionotropic eVect) should be non-speciŽ c with respect
to crystal orientation and would be expected to yield
the most stable polymorph.22 In vitro experiment has
demonstrated that biological macromolecules are
active in controlling crystal orientation.83,84 The
aspartic acid rich proteins extracted from either the
calcitic or the aragonitic layer of a bivalve were
adsorbed on glass, producing a surface which
nucleated calcite crystals from the (001) face. The
crystals then continued to grow into solution to
develop regular, rhombohedral morphologies.83,84
The ability of the proteins to induce oriented
nucleation was attributed to their rigidity and adop-
tion of a b-sheet conformation after binding to the
substrate. In this conformation, the carboxylate
groups of the aspartic acid moieties emerge in a
planar array, which is available for binding to the
crystal.
Mollusk shell nacre has provided an excellent
system in which to study biological control over
nucleation, as there is a well deŽ ned relationship
between nascent crystals and the underlying organic
matrix (Fig. 2). Aragonitic nacre comprises layers of
tabular aragonite crystals which are separated by
interlamellar organic sheets, and are further delin-
eated laterally by vertical organic sheets, producing
stacks of polygon shaped crystals.17,85,86 In all mol-
lusks the c axes of the aragonite tablets are oriented
perpendicular to the shell surface, and the a and b
axes of the crystals within a stack are co-aligned.
International Materials Reviews 2003 Vol. 48 No. 3
192 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
2 Schematic diagram of relationship between
organic matrix and aragonite crystals in mollusk
shell nacre: reprinted from Ref. 128 with
permission from Oxford Univeristy Press
Separate stacks may be aligned or randomly oriented,
according to the species. The structure of the organic
matrix appears to be key in controlling the orientation
of the aragonite crystals, due to the well deŽ ned
spatial relationship between the matrix and inorganic
crystal. The organic matrix typically comprises thin
layers of b-chitin, sandwiched between layers of silk
Ž broin-like proteins in a b-sheet conformation, onto
which acidic macromolecules, which may also adopt
a b-sheet structure, are adsorbed.87 The chitin poly-
mers and the protein–polypeptide chains are ortho-
gonally aligned, in a plywood-like construction.
The aragonite crystals nucleate at speciŽ c sites on
the predeposited matrix such that the a and b axes
of the aragonite lattice are aligned with the directions
of the chitin Ž bres and the protein Ž bres respectively.
Binding of Ca2+ ions to the oriented proteins could
mimic the arrangement of ions in the ab face of the
nascent aragonite crystal, and thus cause the crystal
to nucleate with its c axis perpendicular to the nucleat-
ing surface. This structural relationship is valid even
in mollusks where the stacks of crystals are randomly
oriented, suggesting in this case that the matrix is
composed of a mosaic of relatively ordered areas.85
After nucleation, the aragonite crystals continue to
grow rapidly in the c direction, and initially exhibit
the typical needle-like morphology of aragonite.
When the crystals reach the surface of the next matrix
layer, their growth terminates in this direction, but
continues laterally.
It has been suggested that alignment within a stack
of aragonite crystals derives from mineral bridges
that pass through pores in the interlamellar organic
International Materials Reviews 2003 Vol. 48 No. 3
sheets, rather than from de novo nucleation of each
tablet on the organic sheet, although this opinion
remains controversial. Evidence for the mineral
bridges and pores in the organic matrix were obtained
from AFM study of the aragonitic nacreous layer of
the bivalve Atrina sp.,88 and of the ‘ at pearls’ pro-
duced on insertion of an abiotic substrate into the
gastropod Haliotis rufescens.89 The ‘ at pearls’ are
faithful biochemical, crystallographic and ultrastruc-
tural reproductions of the components of the native
shell and are deposited by an identical growth
sequence.90,91 The mineral bridges directly link the
crystals in a single stack such that each stack of
tabular crystals is continuous in structure and is
actually a single crystal.
Selection of polymorph at nucleation could be
achieved either by inhibiting the nucleation of a stable
form, or via preferred nucleation of a metastable
form.22 The former mechanism is achieved using
organic molecules, which bind to crystals of the stable,
but not the metastable form. This can delay nucleation
enough to allow crystallisation of the less stable form.
Given that the most frequently nucleated planes of
calcite and aragonite are the (001) faces, which are
virtually identical in the arrangement of Ca ions, it is
unlikely that calcite/aragonite polymorph selectivity
is determined by the structure of the nucleation site
alone. A cooperative mechanism may exist where the
substrate proteins are responsible for nucleation and
orientation, while an inhibitor of the stable poly-
morph selects the metastable polymorph.22
Polymorph selection
Many organisms are highly skilled in the selection of
calcium carbonate polymorph, and a number of
experiments have suggested that the organic control
macromolecules can select either for aragonite or
calcite. Mollusk shell, which comprises both calcitic
and aragonitic layers has again enabled calcite/
aragonite polymorphism to be studied. Calcium car-
bonate was grown on the nucleating protein sheets
isolated from a mollusk, in the presence of proteins
extracted from the aragonitic, or the calcitic layer of
the shell.92 In the absence of soluble proteins, rhombo-
hedral calcite crystals nucleated on the protein sheet,
whereas calcite crystals with spherulitic morphologies
were produced in the presence of the calcite derived
proteins. SigniŽ cantly, the aragonite derived proteins
induced the formation of aragonite crystals, which
had needle morphologies and were oriented in the
plane of the nucleating protein layer. When a mixture
of the aragonitic and calcitic proteins were used,
aragonite crystals were again precipitated on the
nucleating membrane, but were in the form of  at,
polycrystalline plates that were oriented on (001)
faces. Detailed examination showed that these plates
comprised oriented stacks of crystals, similar in form
to molluscan nacre.
Growth of calcite seed crystals in solutions of the
proteins yielded similar results.92 The aragonitic pro-
teins resulted in an overgrowth of aragonite needles
on the rhombohedral faces of the calcite crystals,
while the calcitic proteins caused calcite overgrowth.
Transition of layers from calcite to aragonite and
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 193

back to calcite again was also demonstrated on

addition of the appropriate proteins. The results
therefore demonstrated that while the nucleating pro-
tein sheet controlled the orientation of the calcite
primer layer, and the insoluble proteins of the matrix
deŽ ne the plate thicknesses, the soluble proteins are
then active in controlling further aspects of shell
growth such as crystal polymorph and morphology.
It has also been suggested that the insoluble organic
matrix may play a role in polymorph control. Calcium
carbonate was deposited on a substrate constructed
from b-chitin, silk Ž broin and soluble macromolecules
obtained either from the calcitic or aragonitic layers
of shells, an organisation which mimics that present
in the mollusk.93,94 Crystallisation on partial or com-
plete substrate assemblies resulted in the formation
of CaCO spherulites on the surface and inside the
3
chitin scaVold. The complete matrix assembly induced
calcite growth inside the chitin when calcitic macro-
molecules were used, and aragonite when the macro-
molecules had been extracted from an aragonite
layer.93 The ability of the macromolecules extracted
from the calcitic or aragonitic shell layers to induce
the speciŽ c nucleation of calcite and aragonite crystals
respectively was only achieved with the complete
substrate assembly.
Polymorph selectivity in this system appeared to
rely on both specialised macromolecules and a con-
trolled microenvironment.94 The b-chitin framework
has a porous layered structure that would allow
diVusion of ions and macromolecules into the struc-
ture, while the silk is essential for the adsorption of
the macromolecules. The macromolecules which pro-
moted calcite nucleation are strongly polyanionic and
more strongly acidic than aragonite inducing ones.
They may have provided a strong binding site for
Ca2+ ions, creating a high local supersaturation. In
contrast, the macromolecules that promote aragonite 3 Micrographs of a spine and b skeletal plate of
nucleation are unique to the aragonite layer. The sea urchin Heliocidaris erythrograma
structural features of macromolecules active in ara-
gonite selection were investigated by fractionating the
macromolecules extracted from the aragonite nacre- morphologies, the spines and skeletal plates of sea
ous layer of a mollusk and adsorbing them to the urchins are single crystals of calcite.
chitin–Ž broin matrix.94 One fraction which comprised Biominerals with such complex morphologies
6 mol-% of the total amino acids was unique in cannot possibly form through the action of additives
supporting massive crystallisation of needle shaped alone.25 As emphasised above, biologically controlled
aragonite crystals within the chitin layer. This fraction mineralisation takes place within localised volumes,
was absent in the proteins extracted from the calcite whose structure and microenvironment exerts an
layer. Aragonite crystals of similar morphology were external control over crystal morphology. Biological
produced when a protein of sequence poly (Asp-Leu) control over crystal growth is time dependent, as
was adsorbed on the matrix. demonstrated by the evolution of morphologies with
time. For example, the Ž rst deposit in the formation
of a sea urchin larval spicule is a regular rhombo-
Crystal growth hedron of calcite, which clearly forms with minimal
Following nucleation, the growth of crystal nuclei external control.95 As growth continues, the organism
must be controlled to produce desired shapes, sizes actively modulates the crystal morphology to generate
and textures. Every crystal has a characteristic morph- a single crystal with tri-radiate structure. Morphology
ology, which is governed by the crystal structure and may be determined by controlled ion  ux through
symmetry, and the conditions in which the crystal ion pumps and channels into the mineralisation site.
grows. Organisms override this basic growth form Change in the activity or positioning of ion pumps
using a combination of mechanisms to produce crys- and channels during crystal development may lead
tals whose overall morphologies often bear no relation to crystal growth in preferred directions. Alternatively,
to the symmetry of the crystal lattice. An excellent growth may be promoted in speciŽ c locations by
example is provided by the skeletal elements of sea introduction of storage granules of enriched ions or
urchins (Fig. 3). Despite their intricate sponge-like amorphous material into the mineralisation site. The
International Materials Reviews 2003 Vol. 48 No. 3
194 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
physical form of the compartment in which mineralis-
ation occurs is also of fundamental importance in
control of crystal morphology. Many biominerals,
such as the calcite scales produced by coccoliths,96 or
sea urchin larval spicules,97 form within vesicles with
deŽ ned shapes and grow until they impinge upon the
vesicle, which then eVectively acts as a mould. The
size and shape of the vesicle may be altered during
the crystal growth process.
There is considerable experimental evidence that
the soluble macromolecules occluded within calcium
carbonate biominerals may also play a role in control
of crystal morphology. Indeed, calcium carbonate
precipitated in vitro in the presence of isolated
macromolecules has shown modiŽ ed crystal
morphologies,98,99 and speciŽ c crystallographic inter-
actions.73,100–104 These molecules may possess struc-
tural motifs that match the atomic arrangement on
one set of crystal planes, causing the additive to
interact with and stabilise these faces. The occluded
macromolecules are unique to a particular biomineral.
Further, speciŽ c moieties present on extracted pro-
teins interact diVerently with growing calcite crys-
tals.104 Following adsorption to speciŽ c crystal faces,
the macromolecules become overgrown and are
occluded within the crystals, as demonstrated by
calcite crystals grown in the presence of  uorescence
labelled sea urchin macromolecules.102
Much of the validity of these experiments depends
upon the extracted macromolecules retaining the
same conformation as they exhibit in the in vivo
situation. In order to preserve the in vivo protein
conformation as far as possible, biological calcite
crystals have been immersed in CaCl solution, which
2
results in local dissolution of the crystal surface and
release of macromolecules. Slow diVusion of CO into
the solution then results in epitaxial overgrowth 2 of
synthetic calcite on the biogenic crystals.103,105 In all
cases the overgrowth crystals displayed new, well
deŽ ned faces which demonstrates speciŽ c interaction
of the released macromolecules with certain crystal
faces. The crystals deposited on sea urchin elements
showed the same set of faces as produced with the
macromolecules extracted by full dissolution of the
calcium carbonate biomineral. As the majority of
polyelectrolytes would be anticipated to interact non-
speciŽ cally (and thus produce particles with rounded
surfaces), this suggests that these biological macro-
molecules have evolved to bind to speciŽ c faces.
The role of biological growth additives in con-
trolling crystal morphology has also been demon-
strated by incubating pieces of molluscan, aragonitic
nacre in a continuous  ow of sea water in which the
carbonate ion concentration had been enhanced by a
factor of two.106 This solution falls within the pH and
ion concentration range of mollusk extrapallial  uid.
Aragonite growth occurred on the nacre surface as a
continuation of the existing structure and took the
form of needle-like extensions of the (001) surface
rather than the characteristic layered tablets. This
suggests that once the organism has controlled the
nucleation of aragonite, it could continue to grow
inorganically from the extrapallial  uid. The role of
the soluble macromolecules may then be in morpho-
logical control.
International Materials Reviews 2003 Vol. 48 No. 3
Direct evidence of the interaction of biological
macromolecules with the faces of growing crystals
has been obtained using AFM. Immersion of a geo-
logical calcite crystal in a saturated calcium carbonate
solution, in the presence of intracrystalline proteins
extracted from calcitic and aragonitic layers of the
red abalone Haliotis rufescens, resulted in changes in
the structure of the (10.4) faces.107 In the absence of
added proteins, the calcite (104) face showed straight
step edges with some kinks, and sharp corners
between diVerent step edges. After brief exposure to
solutions containing calcite derived proteins,107 the
corners between step edges had become rounded, and
the step edges appeared more convoluted and kinked.
The proteins agglomerated at the step edges and
appeared to attach more strongly to step edges than
to the terraces. The aragonitic proteins had markedly
diVerent eVects on crystal growth, causing step edges
to become highly convoluted, terraces to become
extremely rough, and ultimately resulted in the cess-
ation of crystal growth.108 Examination of the atomic
structure of the calcite {104} face also demonstrated
a change in the lattice structure consistent with over-
growth of aragonite from a (001) plane.
SpeciŽ c interaction between proteins rich in
aspartic acid and calcite have also been predicted by
energy optimisation calculations.109 Studies using
AFM showed that polyaspartate containing up to 20
residues bound to the calcite (0001) surface as parallel
arrays, whereas polyanionic protein extracted from
oyster shell, (which has domains of Asp ) bound as
n
globular molecules on this surface. In contrast, oyster
shell proteins appeared to bind with a speciŽ c orien-
tation, along (11Â 0) terraces parallel to the c axis.
Molecular simulations on the interactions of poly-
aspartate in the b-pleated sheet conformation and
calcite crystal faces demonstrated that binding to the
(0001) plane was strongest. Binding to (11Â 0) prism
faces is also energetically favoured, and is selective
for orientations parallel and perpendicular to the c
axis. Binding to the (110) faces is signiŽ cantly weaker
and orientation non-speciŽ c, due to the diVerent
orientation of the carbonate ions in this face. Binding
to (104) faces is weak, which is consistent with the
common exhibition of unmodiŽ ed (104) cleavage
planes.
A recent paper has suggested that control of crystal
morphologies with organic molecules relies upon
binding of organic molecules to the surface step edges
rather than single crystal faces.110 The Ž nal crystal
morphology is therefore the product of both stereo-
chemical recognition and the eVects of binding on the
interfacial energies of the growing crystal. An AFM
study of the growth of the calcite (10.4) face in the
presence of the chiral molecules D-aspartic acid and
L-aspartic acid showed that interaction with the
crystal steps was asymmetric. Modelling of the inter-
action of these molecules with the step edges suggested
that D-Asp binds to the (014Â ) riser such that one of
its negatively charged carboxyl groups completes the
coordination of calcium ions, whereas the positively
charged (NH+ ) group remains in registry with the
3 the surface. At the step, the remaining
positive ions at
carboxyl group can bind to the two adjacent {10.4}
surfaces. In contrast, the carboxyl group of D-Asp
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 195
does not closely match the carbonate groups on the
(11 4 ) riser. The speciŽ c amino acid enantiomers there-
fore bind to the step edges that oVer the best geometric
and chemical Ž t. This changes the step edge free
energies, which in turn results in macroscopic crystal
shape modiŽ cations.
Modulation of crystal texture
Beyond control of morphology, organisms have also
developed strategies to produce single crystals with
mechanical properties far superior to their synthetic
counterparts, a characteristic that is essential if the
crystals are to be used for mechanical support and
protection. An example is provided by the skeletal
elements of echinoderms. Despite being single crystals
of calcite, these fracture with diYculty to give con-
choidal fracture surfaces (Fig. 4), and have strength/
weight ratios exceeding those of many man made
construction materials such as brick and con-
crete.111,112 These properties occur because sea urchin
calcite occludes ~0·1 wt-% organic macromolecules
and is eVectively a composite material.9,23,113–115
These macromolecules interfere with crack propa-
gation along the cleavage planes, and reinforce the
crystal against fracture. While it is often stated that
the fenestrated structure interferes with crack propa-
gation, this is actually incorrect for a brittle material
such as calcite.9 Measurement of the Young’s modulus
of simple (non-fenestrated ) sea urchin larval spicules
gave a value four times lower than inorganic calcite,
in a direction parallel to the length (and the c axis),
demonstrating that they are unusually resistant
to bending.114 The spicules are also stronger than
inorganic calcite and have a higher resistance
to fracture.115
The ability of occluded organic molecules to
enhance the fracture properties of single crystals has
been demonstrated through microindentation experi-
ments.23 Similarly, the fracture properties of crystals
grown in the presence of macromolecules extracted
from a sea urchin and from the calcitic prismatic
layer of a mollusk were modiŽ ed as compared with
control crystals grown in the absence of protein.102
The control crystals fractured along {104} planes,
producing smooth surfaces and well deŽ ned steps.
Crystals with occluded mollusk proteins were brittle
and tended to disintegrate readily, with broken sur-
faces showing a series of {104} steps. In contrast,
crystals containing sea urchin proteins cleaved much
less readily, producing conchoidal fracture surfaces.
Detailed studies on the incorporation of macro-
molecules within biological single crystals have been
carried out using high resolution synchrotron X-ray
diVraction (XRD).101,105,116–119 While deŽ nition of a
single crystal suggests that it is defect free, this is
clearly never valid. Real crystals contain defects that
are described in terms of the coherence length (the
size of perfect crystalline domains) and the domain
spread (the misalignment of the crystalline blocks).
Defects in biogenic crystals are principally caused by
occlusion of macromolecules which are too large to
be incorporated into the perfect lattice of a single
crystal. Measurement of the coherence length and
domain spread along diVerent crystallographic direc-
4 Micrograph of fracture surface of skeletal
plate of sea urchin Heliocidaris erythrograma
showing conchoidal fracture surfaces
tions therefore provides information on the location
of the macromolecules.
Textural measurements of biogenic calcites showed
that the texture of each crystal is an intrinsic property
that varies between taxonomic groups.118 A series of
single crystal, calcite sponge spicules, which varied
according to morphology, were studied to determine
the relationship between the crystallographic axes
and overall morphology.101,117 Measurement of
coherence lengths along diVerent crystallographic
directions enabled the average shape of domains to
be determined, and revealed a distinct correlation
with the overall morphology of the spicules.
Generally, longer coherence lengths were present in
crystallographic directions close to the morphological
axes of the spicules. However, this was only true for
spicules containing occluded macromolecules and no
correlation was detected for pure calcite spicules.101
Some of the spicules studied exhibited morpho-
logies inconsistent with the symmetry of the calcite
lattice.117 In these spicules, the measured coherence
lengths of symmetry related faces were not ident-
ical.101 This suggests that control beyond speciŽ c
protein–crystal interactions must be operative, cre-
ating an anisotropic environment for crystal
growth.105 This could be achieved through oriented
nucleation of a crystal from a speciŽ c crystallographic
plane, such that it grew in one direction only.
Macromolecules could then be adsorbed on speciŽ c
planes that are exposed during growth. This two-
stage mechanism was supported by  uorescence label-
ling experiments, which revealed that deposition of
curved monaxon spicules occurs from one spicule
extremity. Growth then proceeds along one direction
towards the sponge external surface.105
The distribution of defects was also investigated in
a series of synthetic calcite crystals grown in the
presence of macromolecules extracted from selected
biogenic calcite crystals,116,119 including young and
mature sea urchin spines.116 Macromolecules
extracted from the young and mature spines had
identical eVects on calcite crystal morphologies,
inducing the formation of well developed, stepped
International Materials Reviews 2003 Vol. 48 No. 3
196 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
{10l} (l # ­ 1·5) faces, in addition to {104} faces. An
XRD study of these crystals showed that coherence
lengths were smaller in the [100] as compared with
the [110] direction, which was consistent with prefe-
rential adsorption of proteins on the {100} as com-
pared with {110} planes, as had been suggested by
the morphological studies. Comparison of these data
with measurements made on sea urchin spines surpris-
ingly demonstrated that a higher degree of molecular
recognition occurs between the macromolecules and
growing crystals for the synthetic calcite crystals than
for the immature spines. These results suggest that
cellular activity acts to override the tendency of the
macromolecules to be directed to speciŽ c planes
only.116
Amorphous calcium carbonate
Amorphous calcium carbonate (ACC) is highly
unstable and rapidly transforms into a crystalline
phase. In contrast, biogenic ACC can be stable for
long periods of time, demonstrating that organisms
deliberately inhibit crystallisation. The beneŽ ts that
organisms obtain from ACC remain a matter of
speculation.21,22 Study of the moult cycle of a terres-
trial isopod has demonstrated that ACC can act as a
temporary storage site.120 During the biphasic moult
cycle of this animal, about 40% of the cuticular
calcium is stored in the form of amorphous calcium
deposits, before being rapidly incorporated into the
new soft cuticle. Storage of calcium in the form of
ACC facilitates its rapid mobilisation during
regrowth. Similarly, amorphous 25 nm Ca-polyanion
particles have been observed as intermediate struc-
tures during coccolith formation in the coccolitho-
phorid alga Pleurochrysis carterae.121
It has also been suggested that ACC may possess
advantageous mechanical properties, since amorph-
ous materials have no intrinsic fracture planes,22 that
it could provide an organism with buoyancy, or that
it may provide a mechanism for more rapid skeleton
formation.122 Amorphous materials may also be
important in morphological control as they have no
preferred morphology, and could potentially be
moulded into more intricate structures than crystal-
line materials.21 Investigation of the structures of
some ACC biominerals using EXAFS has shown that
biogenic ACCs can be structurally distinct.123,124
Biogenic ACC may potentially contain some short
range order which could be important in deŽ ning the
mechanism of subsequent crystallisation and poly-
morph selection.123
Stabilisation of ACC again involves specialised
macromolecules. This has been demonstrated by
extraction of glycoproteins from ACC contained in
spicules from the sponge Clathrina and from the
ascidian Pyura pachydermatina.125 The Clathrina spic-
ules are composed of a crystalline calcite core, a thick
layer of ACC, and a thin outer coating of calcite,
while the Pyura spicules only comprise ACC. The
proteins contained within the ACC of both spicules
were quite distinct from those within the calcite
component of the Clathrina spicule. In vitro precipi-
tation of CaCO in the presence of the macro-
3
molecules extracted from the ACC phases of the
International Materials Reviews 2003 Vol. 48 No. 3
spicules resulted in ACC that was stable to transform-
ation in the dry state in excess of three months. In
contrast, the proteins extracted from the calcitic por-
tion of the Clathrina spicules induced the formation
of calcite crystals with well formed (001) faces.
Organisms can also control the transformation of
ACC into calcite. Mineralisation in sea urchin larvae
represents the Ž rst example in biology of ACC acting
as a transient precursor to single crystal calcite.122,126
Crystal growth in sea urchins is intracellular, and
occurs within a membrane delineated compartment.
Following formation of an oriented calcite crystal
within this compartment, growth continues by
addition of ACC, and the mineral deposit gradually
transforms into a single crystal of calcite. During the
Ž rst stages of spicule growth, ACC is present in
relatively large amounts, as compared with the crys-
talline component. The proportion of ACC decreases
on aging of the spicule. The transformation process
of the biogenic ACC is much slower than for synthetic
ACC but is also signiŽ cantly faster than the crystallis-
ation of dried spicules isolated from the larvae. This
suggests that crystallisation in vivo must be mediated
by the organism.
SpeciŽ c macromolecules appear to be involved
in the stabilisation of ACC in sea urchins.127
Fractionation of the organic matrix extracted from
spines of the sea urchin Paracentrotus lividus
was carried out to produce O-glycoprotein and
N-glycoprotein fractions, fractions containing ter-
minal sialic acids, and two organic matrix proteins
(SM30 and SM50). Labelling experiments showed
that these components were not homogeneously
distributed throughout the spine stereom. The
N-glycoproteins were preferentially located in the
inner core and were postulated to be involved in
the stabilisation of an ACC phase.
Magnesium incorporation
An intriguing aspect of biological calcites is that they
often contain Mg as a solid solution within the calcite
lattice. Levels of a few per cent to around 20% are
common,128–133 and values of 44% have been
observed in sea urchin teeth.17 Single crystals of
Mg-calcite are also common, which directly contrasts
with room temperature laboratory syntheses of high
Mg-calcites which produce crystals that are almost
without exception polycrystalline. Both the role of
Mg incorporation in biological calcite, and the
mechanisms by which organisms control the pre-
cipitation of single crystals of Mg-calcite are
unclear.59,60,70,73,74,85
Polycrystalline high Mg calcites can be precipitated
from aqueous solution in the presence of organic
additives that chelate the Mg2+ ions.55,60,65,69–71
CaCO has been grown in agar gels containing cal-
3
cium-binding polysaccharides isolated from cal-
careous algae, which resulted in an enhanced ratio of
magnesian calcite to aragonite and magnesian calcites
containing up to 7·5%MgCO .65 Similarly, mag-
3
nesian calcite was almost the sole phase precipitated
from solution in the presence of macromolecules
extracted from the alga Corallina elongata.60 The
calcites contained 11–14%Mg and exhibited either
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 197
spherical or dumbbell morphologies, although a
smaller number of elongated particles containing up
to 21 mol-%Mg were also observed. The Mg-calcite
phases formed via an amorphous phase. Magnesium
in solution increases the supersaturation prior to
crystallisation, which in turn results in the rapid
precipitation of the amorphous phase. The amorph-
ous phase was signiŽ cantly stabilised in the presence
of the organic macromolecules.
It has been suggested that Mg incorporation may
be relevant to morphological control.60,71,74 Indeed,
Mg2+ ions interact speciŽ cally with the {011} faces
of calcite and can cause elongation along the c
axis.59,73,74 Stabilisation of an amorphous phase as a
precursor to the crystalline phase could provide a
mechanism for enabling a high concentration of Mg
to enter the calcite lattice, and could additionally
provide a route to morphology control. In the biologi-
cal situation, Mg2+ ions may be delivered to a
growing crystal in a partially dehydrated state due to
complexation with organic macromolecules, which
may promote incorporation within the calcite struc-
ture.73 Indeed, precipitation of magnesium calcium
carbonate from water–alcohol solutions in which the
Mg2+ ions are less hydrated resulted in Mg-calcites
with Mg contents up to 14 mol%.59
Soluble additives
Calcium carbonate is common and of low toxicity
and as such is used for a wide range of purposes
including neutralising agents, Ž llers and cements.
Given the frequency of adventitious calcium carbon-
ate precipitation, it is also important in Ž elds such as
geology, the chemical industry and water treatment.
Traditional routes to crystal growth control rely upon
manipulation of such variables as supersaturation,
reactant  owrates, temperature and pressure. An
alternative to these techniques gains inspiration from
biological systems, and uses organic molecules as
additives to control crystallisation and produce pow-
ders with homogenous particle sizes and shapes. Due
to the relevance of calcium carbonate precipitation
to many industries, there is clearly a wealth of litera-
ture regarding the application of additives to growth
control. The description of calcium carbonate control
using soluble additives given in the present review is
therefore not intended to be exhaustive, and will focus
on biomimetic approaches.
The principal drive of the work described in this
section is in understanding how control over calcium
carbonate polymorph and morphology by soluble
additives can be rationalised in terms of the structure
of the additives. This is described for a range of
molecules varying from relatively simple species such
as a, v-dicarboxylates, to more complex molecules
such as block copolymers, which can exert signiŽ cant
control over polymorph and morphology according
to the functional groups present and the size of the
blocks. Of note also is the design of peptides in which
not only the primary, but also the secondary structure,
is designed to interact with speciŽ c crystal faces.
Calcification in presence of functionalised and
non-functionalised a, x-dicarboxylates
Calcite was precipitated in the presence of a series of
functionalised and non-functionalised a, v-dicarb-
oxylates (of generic structure HO C–(CH ) –CO H )
and the in uence of subtle changes 2 2 n additive
in the 2
structure was studied.134 Of the non-functionalised
dicarboxylate molecules, malonic acid (n=1) was the
most eVective in inducing a morphological change.
At a Ca/additive ratio of ~3, crystals were elongated
parallel to the c axis, were capped with rhombohedral
{104} end faces, and displayed curved {11Â 0} faces
approximately parallel to the axis of elongation.
Similar morphologies were observed with the other
non-functionalised dicarboxylate molecules, but this
eVect was reduced with increase in the chain length.
The morphological changes were considered to
re ect the interaction of the organic additive with
crystal faces. A high degree of molecular recognition
between the crystal face and additive results in the
appearance of a set of symmetry related faces. The
crystal faces of calcite lying parallel to the c axis are
characterised by carbonate groups oriented perpen-
dicular to the face. As the additives must interact
with the crystal via bidentate binding of the carb-
oxylate groups, these crystal faces may be stabilised
via stereoselective adsorption of the acids. That
increase in the chain length reduced the eVect of the
additives suggests that cooperative binding of the
carboxylate groups may occur in the case of the short
chain additives, while the carboxylate groups behave
independently in the longer chain additives.
Functionalisation of the lower acid chains had a
marked eVect on crystal morphology. Crystals grown
in the presence of aspartate (a-aminosuccinate)
exhibited well deŽ ned {11 0} prismatic faces at
Ca/additive=17, whereas c-carboxyglutamate had a
pronounced eVect at ratios as high as 85. The
increased eVect of these modiŽ ed additives can be
attributed to the cooperative eVect of the functional
groups present.
Peptide design to control calcite morphology
A number of a-helical peptides were synthesised with
well deŽ ned structures designed to interact with
speciŽ c calcite faces.135 While some information has
been obtained on the amino acid sequence of isolated
intracrystalline proteins, these proteins are typically
diYcult to characterise and little information is avail-
able on their secondary structures. Synthetic peptides
with well characterised secondary structures therefore
enable the role of the secondary structure in protein–
crystal binding to be investigated. a-helical proteins
were selected as they can be synthesised with well
deŽ ned secondary structures and can be active in
controlling biological crystal growth.
Peptides were synthesised containing an array of
aspartate residues that were postulated to interact
with the {11Â 0} face of calcite, and were added to
growing rhombohedral calcite seed crystals. The helix
content of a synthesised calcium binding peptide
designated CBP1 was 89% at 3°C and 42% at 25°C.
At 3°C, CBP1 produced crystals elongated along the
[001] direction, with rhombohedral {104} caps. At
25°C epitaxial crystal growth occurred on the six
{104} faces to give protrusions that tapered towards
the end, producing ‘studded’ crystals. The rhomboid
surfaces were frequently truncated at the acute cor-
International Materials Reviews 2003 Vol. 48 No. 3
198 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry

5 Schematic diagram of poly(propylene imine)

dendrimer modified with long chain alkyl
chains: reprinted from Ref. 137 with permission
from the Royal Society of Chemistry

ners, resulting in an hexagonal shape, which suggested

that growth occurred along an axis with hexagonal
symmetry. This idea was conŽ rmed by the obser-
vation of CBP1 labelled with  uorescein on the newly
formed non-rhombohedral surfaces only. That CBP1
interacts diVerently with the growing crystals at 3
and 25°C can be attributed to the loss in structural
order at room temperature, and consequent non-
speciŽ c binding to calcite. Indeed, other synthesised
peptides of low helical content also interacted to
produce studded growth.

Calcification in association with dendrimers 6 Electron micrographs of calcium carbonate

Dendrimers represent a relatively new class of organic
molecules, and when applied as additives, can be
eVective in controlling both the morphology and
phase of calcium carbonate.136,137 SigniŽ cantly, selec-
ted dendrimers are also active in stabilising amorph-
ous calcium carbonate (ACC) and can support the
coexistence of both ACC and calcite. These properties
are characteristic of biological systems, in which the
highly unstable ACC phase is stabilised by specialised
organic macromolecules (see above).
Precipitation of CaCO in the presence of poly (ami-
3
doamine) (PAMAM) dendrimers (generation G=1·5)
bearing external carboxylate groups was initiated by
simultaneous injection of CaCl and (NH ) CO solu-
2 42 3
tions into the dendrimer solution until a sudden
increase in turbidity was observed.136 Stirring was
then continued for a further 4 days under nitrogen.
Under these conditions, 1–2 mm spherical vaterite
particles, with a dendrimer content of 33 wt-% were
produced. Notably, when the Na salt of PAMAM
was employed, only ACC was detected after 4 days.
As an elegant route to systematically changing the
surface chemistry of dendrimer molecules, poly (prop-
ylene imine) dendrimers were modiŽ ed with long
International Materials Reviews 2003 Vol. 48 No. 3
particles produced in presence of octadecyl-
amine/modified poly(propylene imine) den-
drimers after a 4 and b 14 days: reprinted from
Ref. 137 with permission from the Royal Society
of Chemistry
aliphatic chains (Fig. 5).137,138 Intercalation of surfac-
tants into the exposed long chains on the dendrimer
then generates a new class of amphiphile whose size,
shape, exposed surface groups, and surface charge
density can be readily modiŽ ed according to the
structure of the selected surfactant. The dendrimer/
surfactant species formed aggregates, which varied in
size and structure according to the surfactant used.
While octadecylamine (ODA)/dendrimer aggregates
were polyhedral in shape and 250 nm in diameter,
cetylammonium bromide (CTAB)/dendrimer aggre-
gates were 20 nm in size and less rigid in structure.
Sodium dodecylsulphate (SDS)/dendrimer aggregates
were 140 nm in size, and exhibited an approximately
spherical morphology.138
Within 15 min of introduction into a saturated
calcium carbonate solution, dendrimers functionalised
by intercalated ODA had supported the growth of
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 199

7 a crystalline aggregate containing helical protrusion, resulting from addition of 10 mg mLÕ 1 poly(a,L-
aspartate); b helical particle etched with 0·2% acetic acid for 10 min, neutralised with 0·1M NaOH, and
rinsed with distilled water: reprinted from Ref. 139, ©1998, with permission from Elsevier Science

ACC. After 4 days, the solution comprised large ACC
particles of diameter " 10 mm (Fig. 6a), together with
calcium carbonate particles with rhombohedral mor-
phologies, formed by growth of calcite over a spherical
ACC nucleus (Fig. 6b). Continued aging increased the
proportion of rhombohedral particles. Formation and
stabilisation of ACC was attributed to the low charge
density, and high rigidity of these aggregates, which
limits the nucleation and growth of the crystalline
polymorphs. Binding of the aggregates to the ACC
surface, and possible incorporation within the ACC
particles, may also have limited transformation into
crystalline polymorphs.137,138 In contrast, dendrimers
modiŽ ed by CTAB did not support ACC precipi-
tation, producing instead calcite crystals covered with
vaterite discs. Loss of the vaterite phase occurred on
continued incubation of the solution. The positive
charge of the CTAB modiŽ ed dendrimer aggregates
will cause adsorption of carbonate ions, which
is believed to promote vaterite formation.137,138
Addition of the SDS/dendrimer aggregates to the
CaCO solution resulted in 1–20 mm spheres, which
3 the type of particles produced. Mosaic Ž lms were
principally comprised organic material. Over 4 days,
precipitation of calcite crystals incorporating the
organic spheres occurred. The SDS/dendrimer species
strongly bind Ca2+ ions which results in their agglom-
eration, and consequent depletion of free Ca2+ ions
in solution. Precipitation of CaCO in the solution is
3 Ž lms also contained a proportion of calcium carbon-
therefore retarded.138
Calcium carbonate growth in solutions of
poly(aspartate)
Poly(aspartate) is considered to provide a structural
analogue to many biological control macromolecules,
which contain repeated runs of aspartic acid. The
interaction of low concentrations of the charged
polypeptides poly (a,L-aspartate) and poly (a,b-D,L-
aspartate) with growing calcium carbonate crystals
in solution was shown to produce some remark-
able particle morphologies.139 Additive levels of
5–30 mg mLÕ 1 induced the formation of aggregates
of rounded particles, which frequently grew quite
remarkable helical protrusions (Fig. 7a). The helices
and supporting crystal aggregates were vaterite, and
both right and left handed helices were formed, even
in the presence of achiral poly (a,b-D,L-aspartate).
Acid etching of the rounded particles and helices
revealed spherulitic textures and the presence of a
‘membrane’ which appeared not simply to be a pre-
cipitated form of calcium poly (aspartate) (Fig. 7b).
Hollow helices that may have represented an inter-
mediate growth form were occasionally observed, and
appeared to have grown inward from the membrane
template rather than from a central core.
At additive concentrations of 10–30 mg mLÕ 1, a
highly birefringent inorganic Ž lm initially formed in
streaks and patches on the glass slide at the base of
the crystallisation dish. The Ž lms were either mosaic
or spherulitic in structure, and appeared to control
composed of interconnected single crystals, princi-
pally of rhombohedral calcite, while Ž lms with spheru-
litic texture had vaterite lobes and helices associated
with them, suggesting that they may have developed
into the membrane surrounding the particles. Some
ate hexahydrate, which may have been stabilised by
the polymers, and could have formed as a precursor
to the more stable non-hydrated phases.
This work was extended to form calcium carbonate
Ž lms by a polymer induced liquid precursor (PILP)
process.140 DiVusion of ammonium carbonate vapour
into a CaCl solution containing poly-a,L-aspartate
2
or poly-a,b,D,L-aspartate resulted in the phase sep-
aration of 2–5 mm amorphous droplets (the so called
International Materials Reviews 2003 Vol. 48 No. 3
200 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
PILP phase) from the solution, and accumulation of
these particles on the cover slide placed in contact
with the upper surface of the solution. The droplets
then coalesce to form a continuous isotropic Ž lm.
With time, crystal tablets nucleate and grow within
this amorphous Ž lm, and ultimately spread laterally
to form a continuous crystalline Ž lm. These Ž lms are
composed of either single crystal areas of calcite, or
spherulitic patches of vaterite. In addition to the
crystalline thin Ž lms, other crystal products including
aggregates of distorted calcite crystals and the helical
protrusions of vaterite (described in detail in Ref. 139)
were often observed. Notably, addition of magnesium
at a ratio Mg/Ca=5 promotes the PILP process
such that only thin Ž lms are produced, without associ-
ated crystal aggregates. The role of poly (aspartate) in
the PILP mechanism is envisaged as sequestering and
concentration of the ionic species, while delaying the
nucleation and growth of crystalline phases long
enough to generate a metastable solution. The soluble
acidic macromolecules associated with the vast
majority of minerals are considered to behave simi-
larly during biomineralisation processes.
Poly(aspartate) has also been demonstrated to be
active in modifying the morphology of vaterite
particles.50 Precipitation of calcium carbonate in the
presence of a linear poly (a,b-aspartate), resulted in
vaterite particles, which formed as 50–500 mm aggre-
gates of rounded, disc shaped units with unusual
helicoid outgrowths. The outgrowths appeared to
comprise either a right or left handed stack of disc
shaped vaterite crystals aligned along their c axes. At
early stages of growth, a surface membrane was
observed on the aggregates, which was considered to
have been a CaCO –peptide gel generated on adsorp-
3
tion of the poly (aspartate) on the growing crystals.
Control of calcium carbonate precipitation
using block copolymers
Recent work has demonstrated that double hydro-
philic block copolymers are very eVective in con-
trolling the morphologies of calcium carbonate
particles.141–145 These polymers comprise one block
that interacts strongly with the mineral surface and a
second block which interacts only weakly with the
mineral surface. The structure of the polymer can be
carefully controlled to independently vary the prop-
erties of the two functional blocks, and thus to control
the overall in uence of the polymer on particle
morphology.
The Ž rst experiments investigating the in uence of
block copolymers on calcium carbonate precipitation
utilised anionically polymerised poly (ethylene oxide)-
block-poly (methacrylic acid) (i.e. PEO-b-PMAA)
polymers.145 Investigation into the behaviour of the
separate polymer blocks showed that the PEO homo-
polymer had no eVect on CaCO precipitation,
3
whereas the PMAA homopolymer suppressed crystal-
lisation, leading to the precipitation of irregularly
shaped calcite particles of diameters less than 2 mm.
The PEO-b-PMAA polymers also induced calcite
precipitation, but produced markedly diVerent crystal
morphologies. The crystals were elongated along the
c axis, were capped by well deŽ ned {10.4} faces, and
International Materials Reviews 2003 Vol. 48 No. 3
showed very rough surfaces parallel to the long axis,
suggesting that the polymers interacted speciŽ cally
with crystal faces lying parallel to the c axis.
A series of detailed studies have been carried out
using polymers constructed from a poly (ethylene
glycol ) (PEG) block, and a selected hydrophilic
block.141–144 The in uence of experimental variables
such as pH, polymer concentration, ionic strength,
and the ratio of polymer functional groups to the
mineral was determined for calcium carbonate pre-
cipitation in the presence of poly (ethylene glycol)-
block-poly (methacrylic acid) (PEG-b-PMAA).142
Control experiments performed using PEG and
PMAA homopolymers produced rhombohedral and
rounded particles (comprising aggregates of 40 nm
crystals) respectively, indicating that PEG has no
in uence on crystal morphology, while the carboxylic
groups of PMAA bind strongly, but non-speciŽ cally
to the mineral surface. The PEG-b-PMMA caused
dramatic changes in particle morphology. At a poly-
mer concentration of 0·2 g LÕ 1, a CaCO concen-
tration of 8 mmol LÕ 1 and pH 10, twinned 3 calcite
crystals with a ‘peanut’ morphology formed. At pH 9
the particles assumed rodlike morphologies, while
increase in pH to 10·5 and 11 resulted in a mixture
of ellipsoidal particles and irregular starlike aggre-
gates. Reduction in the polymer concentration to
0·1 g LÕ 1 at pH 10 produced rodlike or branched
particles with rugged and faceted surfaces. Further
reduction in the polymer concentration to 0·05 g LÕ 1
resulted in ineVective control over polymorph and
morphology.
Increase in the polymer concentration beyond
0·2 g LÕ 1 at 8 mmol LÕ 1 CaCO and pH 10 produced
3
calcite particles with dumbbell morphologies, while
increase to 1 g LÕ 1 resulted in particles where the
dumbbell heads had grown back over to give almost
spherical morphologies. The Ž rst species to form
during the precipitation of these particles was a
polymer stabilised amorphous intermediate. With
time, a number of large spherical particles were
produced, suggesting that while nucleation of calcite
from the amorphous precursor is fairly slow, sub-
sequent growth is rapid. Continued aging resulted in
an increase in the number of particles and precipi-
tation of smaller peanuts and dumbbells. The calcium
carbonate concentration also aVected the morphology
of the precipitated particles. At pH 10 and 0·2 g LÕ 1
polymer, a 4 mmol LÕ 1 CaCO concentration
3
produced peanut shaped calcite particles, while
16 mmol LÕ 1 resulted in a mixture of spherical vater-
ite particles and poorly deŽ ned calcite particles.
The in uence of the functional groups on the
hydrophilic block of the polymer was investigated
using the copolymers listed in Table 2.141,143 Calcium
carbonate was prepared in the presence of a range of
double hydrophilic block copolymers using a double
jet reactor,143 and the crystals produced were incu-
bated in the solution for 12 months prior to isolation.
The particles produced in the presence of PEG poly-
mers containing only one terminal functional unit
(COOH, Asp, EDTA) were calcite, and were typically
large crystals of ill deŽ ned morphology or aggregates.
The only exception were the bis-phosphonated groups
which produced smaller crystals, with roughly spheri-
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 201
cal morphologies and consisting of calcite and vater-
ite. The most eVective crystal growth inhibitors were
the bis-phosphonate, followed by the EDTA moieties,
while the COOH group oVered the weakest eVect.
In contrast, all of the block copolymers had a
speciŽ c eVect on crystal growth, and produced
particles with well deŽ ned morphologies. All particles
were in the micrometre size range, and many exhibited
spherical morphologies. The PMMA, and PMAA-
Asp block copolymers induced the formation of
spherical calcite particles; monophosphonation of the
COOH groups of PEG-b-PMAA lead to very well
deŽ ned spherical particles that were 75% vaterite and
25% calcite and comprised aggregated nanocrystal-
lites. Spherical vaterite/calcite particles with well
deŽ ned sizes and smooth surfaces were also obtained
in the presence of CH O-PEG-b-pAsp. The CH O-
PEG-b-PEIPA polymer 3 was unique in producing 3 the ends. A phosphonated polymer, PEG-b-PEI-
pure vaterite in the form of hollow spheres, which
were stable in solution for over a year.
The block length of the polymers had a marked
eVect on calcium carbonate precipitation. Strong
binding did not necessarily correlate with superior
control over crystal growth, as demonstrated by the
EDTA functionalised polymer. Whereas no control
over CaCO precipitation is achieved when only one
EDTA group 3 is attached to the PEG chain, attach-
ment of an EDTA trimer enables partial control.
Substitution of a PEIPA block (eVectively a poly-
EDTA block) yielded hollow vaterite spheres.
Investigation of the crystallisation inhibition eYciency
for these polymers demonstrates that the polymers
with one EDTA group and the PEIPA block show
low inhibition eYciency (with the former being slightly
poorer than the latter), whereas the EDTA trimer
polymer is markedly more eYcient.143
ModiŽ cation of hydrophobic copolymers with a
controlled number of hydrophobic groups can have
a dramatic eVect on their ability to control crystal
growth.146 Based on this rationale, a series of hydro-
phobically modiŽ ed double hydrophilic block copoly-
mers was synthesised by chemical modiŽ cation
of poly (ethylene glycol )-block-poly (ethyleneimine),
Table 2 Copolymers used in control of calcium
carbonate precipitation, as described in
Ref. 141
CH3 O-PEG-COOH CH3 O-PEG-EDTA3
CH3 O-PEG-EDTA CH3 O-PEG-b-PEIPA
PEG-b-PMAA PEG-b-PMAA-Asp
PEG-b-PMAA-PO3 H2 CH3 O-PEG-Asp
CH3 O-PEG-b-pAsp CH3 O-PEG-CP2 O7 HNa4
Abbreviation Sample
PEG poly(ethylene glycol)
EDTA ethylenediaminetetraacetic acid
PEIPA poly(ethyl-enimine)-poly(acetic acid)
pAsp poly(aspartic acid)
PMAA poly(methacrylic acid)
PMAA-pAsp poly(methacrylic acid) modified with
aspartic acid, statisatically at every
second group
PMAA-PO3 H2 monophosphonated poly(methacrylic
acid)
CP2 O7 HNa4 sodium salt of a bisphosphonated
COOH
(PEG-b-PEI), and functionalisation with –CO H,
–SO H, –PO H and –SH groups.144 All the polymers 2
3 3 2
investigated in uenced particle morphology, and typi-
cally produced spherical particles comprising aggre-
gated nanoparticles. The lengths of both the PEI
block and the hydrophobic group deŽ ned how eVec-
tive the polymers were in stabilising the CaCO
particles against aggregation. A number of polymers3
produced alternative particle morphologies. A poly-
mer with a long PEI block, modiŽ ed with COOH
groups, and a rather long hydrophobic group (PEG-
b-PEI-COC H (CH COOH) ) produced a range
17 35 2 n
of morphologies, according to the reaction time. At
early times, the particles were predominantly rodlike
(calcite) or spherical (vaterite). With time, the vaterite
particles dissolved and were replaced with the
rodlike calcite particles, which become overgrown at
COC H (CH CH PO H ) , induced the formation
11 23 2 2 3 2n
of hollow vaterite spheres. The spheres were initially
solid, and dissolved from the inside to yield the hollow
structure. An analogous polymer with smaller PEI
block and thus fewer phosphonate groups produced
similar spheres, but the dissolution process was con-
siderably slower. Hexagonal vaterite particles formed
in the presence of the sulphonated polymers.
Control of calcium carbonate precipitation
using homopolymers
Calcium carbonate precipitation in the presence of a
series of well deŽ ned water soluble polymers, based
upon functionalised norbornene monomers, gener-
ated using ROMP living polymerisations was investi-
gated.147 Whereas many of the polymers investigated
were inactive, polymers derived from the exo,endo-
monomer of 5,6-methylcarboxylates of norborene did
in uence calcium carbonate precipitation. At a Ca2+ /
polymer carboxyl group ratio of 10 : 1, calcite crystals
were precipitated, and the nucleation density increased
on increasing the polymer number average molecular
weight. All the crystals were of identical morphologies
and were modiŽ ed on one crystal face only. The other
structural isomers of this monomer did not display
the same degree of crystal growth control.
Control of calcium carbonate polymorphs
using ‘latent inductor’
It has been suggested that organisms may control
crystal growth by interaction with the mineralisation
process at speciŽ c stages of crystallisation.148 The
potential of this route was investigated by selective
interaction of organic additives with calcium carbon-
ate at diVerent stages of the crystallisation process.
CaCO was precipitated by injection of CaCl and
3 2
(NH ) CO solutions into a solution of sodium acry-
4 2 3
late. Subsequent addition of a radical initiator to the
solution at speciŽ c times resulted in the in situ forma-
tion of poly (acrylate). While sodium acrylate has no
in uence over CaCO precipitation, poly (acrylate)
strongly interacts with 3growing crystals. Initiator was
added after periods of 1, 3 and 20 min, and resulted
in the precipitation of aragonite, a mixture of vaterite
and calcite, and calcite respectively. Poly(acrylate)
may interact with the developing nuclei in solution,
International Materials Reviews 2003 Vol. 48 No. 3
202 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
and therefore dictate the polymorph formed, accord-
ing to the structure of the nuclei present at the time
at which the active additive was generated.
Crystallisation of calcium carbonate in
presence of colloidal additives
Precipitation of calcium carbonate from a viscous
solution containing hydroxyethyl cellulose (HEC) in
a counter-diVusion set-up led to the precipitation of
2–3 cm shell-like structures.149 The apparatus com-
prised one beaker containing a solution of CaCl and
2
HEC, placed in a second larger beaker Ž lled with
Na CO and HEC solution. DiVusion occurred
2
through 3a liquid bridge to precipitate calcium carbon-
ate at a number of sites within the apparatus. In the
presence of HEC, calcium carbonate precipitated as
a 60 mm thick, shell shaped mass attached to the
outer wall of the inner beaker. The shell comprised
closely packed crystals, the inner side being vaterite
and the outer side being principally vaterite, together
with small quantities of calcite and aragonite. The
crystals took the form of Ž nger shaped aggregates
that were oriented in the direction of the shell’s
growth. In turn, each of these aggregates was com-
prised of hexagonal platelets aligned along their c
axes and whose thickness increased with increasing
HEC concentration. The colloidal species HEC and
potato starch were also investigated as growth addi-
tives.150 Calcite was produced in all experiments,
precipitating as unmodiŽ ed rhombohedra in the pres-
ence of HEC, but as thin, rectangular plates in the
presence of potato starch.
Crystal growth in confinement
Controlled crystal growth in biological systems takes
place in localised spaces, which provides the organism
with the opportunity to in uence the environment in
which mineralisation occurs. The mineralisation
environment can be of deŽ ned geometry, and in
addition to further functionalisation using organic
macromolecules, the organism can manipulate such
variables as the rate and location of ion  ow into the
space. There are clearly many factors that contribute
to control over crystallisation in conŽ nement in vivo,
and mimicking these in synthetic models is extremely
challenging. For example, although vesicles can read-
ily be prepared in the laboratory, and have been used
to localise crystal growth, they provide a poor model
of biological crystallisation as ions are not continu-
ously pumped across the membrane to the site of
crystal growth.
This section describes a number of diverse systems
in which crystal growth occurs in conŽ nement. The
use of gels to support crystallisation is well known,
and has resulted in some truly remarkable morpho-
logies. Precipitation of calcium carbonate in oriented
and non-oriented collagenous matrices has clearly
demonstrated that the microenvironment in which
crystallisation occurs can deŽ ne polymorph and
orientation of the crystals formed. Surfactant systems
have provided a versatile means of controlling miner-
alisation, producing products ranging from nano-
particles in reverse micelles, to particles with ‘micro-
sponge’ textures on evaporation of microemulsions.
International Materials Reviews 2003 Vol. 48 No. 3
Finally, growth of calcium carbonate with rigid poly-
mer membranes has produced crystals with morpho-
logies dictated by the polymer template.
Calcium carbonate growth in gels
The method of crystal growth in gels dates back to
as early as the nineteenth century with the experi-
ments carried out by Liesengang, and the observation
of the bands of crystals known as ‘Liesengang’s
rings’.151 Experimentally straightforward, the tech-
nique typically leads to the formation of large, high
quality single crystals. In modern experiments a range
of gels are used, including silica and gelatin, the
structure of which comprises a three-dimensional
network. Silica gels are reported to support the
growth of superior quality crystals and are generally
preferred.151 The rate of crystal growth can be con-
trolled according to the density of the gel, and the
concentration of the reactant solutions.
Two principal methods have been employed to
precipitate calcium carbonate in silica gels. In the
Ž rst method, the gel contains the carbonate ions. An
aqueous solution of CaCl (or equivalent) is placed
on top of the set gel, and 2 diVusion is allowed.
Alternatively, a pure silica gel can be prepared in a
U tube and the reagent solutions added to each arm.
The reagents diVuse into the gel, and calcium carbon-
ate precipitates where they meet. Unlike the majority
of other gel grown crystals, calcite does not displace
the gel during growth, but incorporates it within the
lattice, producing turbid crystals.151,152 Acid dissolu-
tion of these crystals leaves a SiO matrix that main-
2 crystal.152 XRD
tains the form of the original calcite
studies suggest that the calcite lattice remains unper-
turbed, even though up to 1·7%SiO has been
recorded within calcite crystals.152 2
Calcium carbonate
The polymorph and morphologies of calcium carbon-
ate precipitated in silica gels depends upon the pH
and the relative and absolute reagent concentrations
of the reactant ions.66,152 One study reported that
calcite was the sole phase produced at low and high
pH values, while clusters and spherulites of aragonite
and vaterite were also precipitated66 at pH values
between 8 and 10. The quantity of aragonite precipi-
tated also depended on the relative reagent concen-
trations. The morphology of the calcite crystals was
pH dependent, and at pH 7–9·5, calcite rhombohedra
formed. Polycrystalline aggregates with a feathery
structure were produced at pH 9·0–10·5, whereas
increase in pH to above 10·5 resulted in spicular or
spherulitic particles.
Calcite aggregates with unique, non-crystallo-
graphic morphologies have been produced in silica
gels.153–157 Experiments were carried by counter-
diVusion of Na CO and CaCl solutions into the gel
2 3 The Ž rst2 precipitates observed
in a U shaped cassette.
were constructed from sheaf-of-wheat aggregates and
fell into two structural types: displaying, or not dis-
playing, a clear banding structure. The rhythmic
structure of the banded precipitates derived from
alternating bands of low and high densities of particles
(Fig. 8).153–155 Each band was itself formed by a radial
aggregation of microsheaves, each sheaf comprising
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 203
8 Electron micrograph of structure of sheaf-of-
wheat aggregates, formed in silica gels:
reprinted from Ref. 153, ©1986, with permission
from Elsevier Science
radial Ž bres emanating from a growth centre. The
low and high density bands corresponded to the
centre point and expanded fan ends of the sheaves
respectively. The non-banded sheaf of wheat aggre-
gates were composed of Ž bres arranged radially from
the centre of the sheaf.
In both aggregate types, the individual Ž bres form-
ing the sheaves-of-wheat appeared to comprise
rhombohedral subunits aligned along their c axes.
Indeed, in the growth front adjacent to a growing
aggregate, individual oriented rhombohedra were
observed without physical contact between them. This
suggests that a connection exists between the existing
aggregate and new crystals, possibly in the form of a
silicate derived matrix.155,157 The membrane may act
as a substrate for nucleation, could support the epitax-
ial growth of further CaCO and is likely to be
3
involved in controlling the arrangement of crystallites
within the aggregate.157 The importance of a sili-
caceous membrane in aggregate formation was further
supported by a kinetic study of aggregate formation.
Examination of the rate of growth of aggregate struc-
tures formed at the gel/glass wall interface suggested
that growth was not exclusively diVusion controlled,
further implicating a silica based matrix in growth
control.154
Calcite crystals with unusual rounded morpho-
logies have also been precipitated in silica gels, by
double diVusion of CaCl and K C O .156 While the
major product was CaC O 2 , CaCO2 2was
4 also precipi-
2 4 3 playing steep, striated {02.1} faces and the tips of
tated due to CO2Õ ions as a signiŽ cant impurity in
3 displayed both singular and non-
the gel. The crystals
singular curved faces and contained 0·7%Si, which
was evenly distributed throughout the particles. The
formation of curved faces was attributed to the growth
kinetics, as crystallisation in very low concentration
gradients can produce crystals with curved faces.156
Calcium carbonate in presence of Mg2 +
The Mg2+ ion is an important impurity (see
‘Formation of Mg-calcites’, above), and as such a
number of studies have addressed the eVect of Mg2+
on calcium carbonate precipitation in gels.57,66,158,159
Mg2+ ions favoured the formation of aragonite in
silica,66 and agar gels.158 Silica gels were used as a
medium in which to precipitate magnesium calcite
crystals over a range of Mg/Ca ratios.159 Both calcite
and aragonite were formed in mixed Ca–Mg solu-
tions, with aragonite predominating when the Mg/Ca
ratio exceeded 2. The calcite crystals occluded Mg
within the crystal lattice, with the percentage of Mg
increasing as a function of the Mg/Ca ratio up to
approximately Mg/Ca=5, where crystals containing
as much as 14–16%Mg were recorded. No inform-
ation was given on whether the crystals produced
were single crystal or polycrystalline aggregates. In
some experiments, aragonite overgrowth of initial
calcite nuclei was observed. As crystallisation of cal-
cium carbonate proceeds in the gel, greater depletion
of Ca2+ than Mg2+ ions occurs, increasing the local
Mg/Ca ratio, hence favouring aragonite over calcite
precipitation.
A detailed study on the crystallisation of calcium
carbonate in the presence of Mg2+ in silica gels was
carried out in which nucleation behaviour was studied
by measuring the location of the Ž rst precipitate, the
nucleation density and the time taken for the Ž rst
crystals to be observed under magniŽ cation (the
experimental waiting period ).57 The pH and concen-
tration of reagents at any point in the gel varied
according to the rate of diVusion and were determined
by analysing slices of the gel at diVerent times. On the
basis of this information, the supersaturation of the
solution with respect to the diVerent calcium carbon-
ate phases was calculated at the point of nucleation.
In the absence of Mg, the Ž rst crystal phase precipi-
tated was always calcite of rhombohedral habit. With
growth, these crystals exhibited dendritic, and sub-
sequently hopper-like morphologies. As crystallis-
ation proceeded, spherulites of aragonite precipitated
in the region of the gel closer to the Na CO reservoir.
Addition of Mg to the system increased 2 3 both the
waiting time for nucleation, and the supersaturation
at the position of nucleation, although no change was
noted in the position in the column of the Ž rst crystals
in the gel, nor in the sequence of crystallisation of the
phases. The Mg had a signiŽ cant in uence on calcite
crystal morphologies. Crystals were precipitated in a
strict morphological sequence. This began with
200 mm spheres with very rough surfaces, and con-
tinued with dumbbell-like forms, with early crystals
displaying smooth surfaces and rounded morpho-
logies, and later members displaying bundle or sheaf-
like morphologies with some well deŽ ned crystal
faces. The sequence ended with single crystals dis-
crystals were capped by {10.4} faces. The calcite
crystals contained occluded Mg. The single crystal
species had average compositions of 1·5 mol-%
MgCO while the spherical end members of the series
contained3 up to 5 mol-%. Microprobe analysis of the
particles demonstrated the existence of Mg zoning,
and the highest Mg concentrations were recorded in
the spherical particles which contained 15 mol-
%MgCO at the centre, decreasing to 3 mol-% at
the edges.3
The morphological evolution of calcite crystals
precipitated in the presence of Mg relates to changes
International Materials Reviews 2003 Vol. 48 No. 3
204 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
in the growth mechanisms. Mg poisons the active
growth sites on calcite nuclei, an eVect which is
overridden at high supersaturation levels where crys-
tal surfaces become atomically rough. In the presence
of Mg, initial nucleation occurs at very high levels of
supersaturation and crystals show very rough sur-
faces. Under these conditions of rapid growth less
distinction between Ca2+ and Mg2+ is made, and a
higher percentage of MgCO is incorporated within
3
the calcite lattice. Complex morphological forms
therefore correlate with high MgCO incorporation.
Continued crystal growth occurs at 3 progressively
lower supersaturation values, which is re ected in the
sequence of morphologies from complex to regular
crystalline forms, and in decreased MgCO
3
incorporation.
Calcium carbonate in presence of Mg2 + and
organic additives
High Mg-calcites have been precipitated in agar gels
in the presence of Mg and organic additives.65,160 The
Mg-calcite was precipitated in agar gels in the pres-
ence of carboxylic acids: acrylic, maleic, tartaric,
malonic, malic, succinic and citric acid.160 In the
absence of the carboxylic acids, the product was
almost exclusively aragonite. Addition of the car-
boxylic acids induced Mg-calcite precipitation, and
at suYciently high concentrations no aragonite was
precipitated. The calcite crystals contained from 1·5
to 7·0%MgCO , depending on the reagent concen-
3 tricarboxylic acids were the most
trations used. The
eVective, and the monocarboxylic acids were least
eVective, in stabilising calcite with respect to aragon-
ite. The organic additives also aVected the morph-
ology of the Mg-calcite crystals. The tricarboxylic
acids produced crystals which resembled those of the
control Mg-calcite crystals, showing elongation and
evidence of deŽ ned crystal faces. Calcite particles
precipitated with the less eVective acids were of ill
deŽ ned shape, and in the presence of acrylic acid,
clearly aggregates.
The in uence of three acid polysaccharides, isolated
from calcareous marine algae, on Mg-calcite precipi-
tation was also studied.65 One polysaccharide was
extracted from the cell walls of an alga which produces
Mg-calcite scales containing 21 mol-%MgCO . The
other two were isolated from whole cells of an 3 alga
which produces calcite scales containing 3 mol-%
MgCO . An increase in the concentration of any of
3
the added polysaccharides resulted in an increased
induction time, and a higher ratio of calcite to aragon-
ite. Magnesian carbonates containing up to
7·5%MgCO were recorded, and showed a range of
3 Calcium carbonate precipitation
morphologies ranging from rhombohedral to ovoid.
The ovoid particles were clearly aggregates rather
than single crystals. In the absence of the polysaccha-
rides only aragonite precipitated. No correlation
between the MgCO content of the alga scales and
the MgCO content3 of the synthetic calcite crystals
3 organised structures.55,161–164 Experiments were car-
produced in the presence of the extracted polysaccha-
rides was observed.
Calcium carbonate in presence of divalent metal
ions
Calcium carbonate has also been precipitated in gels
in the presence of a range of metal ions.66,158 The
International Materials Reviews 2003 Vol. 48 No. 3
9 Ranges of concentration under which calcium
carbonate polymorphs formed in undeformed
(undef), and uniaxially deformed (def)
collagenous matrices, containing entrapped
poly(aspartic acid), (Poly(asp)) or poly(glutamic
acid) (Poly(glu)): solid lines represent non-
oriented crystals, unfilled lines oriented
crystals; reprinted from Ref. 161, ©2000, with
permission from Elsevier Science
additives Ni2+ and Sr2+ favoured the formation of
Sr-aragonite and Ni-aragonite particles which con-
tained optically dense cores. The core of the
Sr-aragonite particles was a prismatic SrCO crystal,
which would have provided a nucleus for the 3epitaxial
overgrowth of aragonite. The cores of the
Ni-aragonite particles were Ni rich, but could not be
classiŽ ed as a unique crystalline phase. Addition of
Ba2+ induced the formation of vaterite and calcite
crystals with modiŽ ed morphologies and isolated
BaCO spherulites.66 That isolated BaCO particles
3
were observed suggests that BaCO does not 3 support
3
overgrowth of calcium carbonate.
An alternative study investigated the eVect of the
divalent cations Fe2+ , Ni2+ , Co2+ , Zn2+ and Cd2+
on calcium carbonate precipitation in agar gels.158
All these ions, with the exception of Cd2+ , promoted
aragonite formation. At lower additive concentrations
vaterite and calcite were also co-precipitated, with
the metal ions being occluded within the calcite lattice.
The aragonite particles were spherulitic in morph-
ology, whereas calcite crystals were distorted rhom-
bohedra. In the case of Cd2+ , CdCO precipitated
prior to aragonite, removing Cd2+ ions3 from the gel;
only calcite formed. In the presence of the impurity
ions, aragonite was stabilised with respect to the
metal ion doped calcite.
Calcium carbonate precipitation in
collagenous matrices
The eVect on calcium carbonate precipitation of the
speciŽ c microenvironment in which the crystals form
was investigated in a system comprising the poly-
electrolytes poly-L-aspartate (poly-Asp) and poly-L-
glutamate (poly-Glu), adsorbed on collagen gels with
ried out with crosslinked and uncrosslinked gelatin
Ž lms, and with uniaxially deformed (crosslinked )
Ž lms, all of which contained adsorbed polypeptides
and Ca2+ ions.55,164 The CaCO was precipitated by
diVusion of (NH ) CO vapour3into CaCl solutions
42 3 2
of the gelatin Ž lms and crystals were produced on the
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 205
10 Images of crystals grown within uniaxially deformed collagenous matrices containing poly(Asp):
a aragonite aggregates grown in matrices containing 0·5 mg poly-Asp/g gelatin; b vaterite aggregates
grown in matrices containing 10 mg poly-Asp/g gelatin; reprinted from Ref. 161, ©2000, with
permission from Elsevier Science
surface and/or inside the Ž lms. The degree of deforma-
tion of the gelatin Ž lms, and the type and concen-
tration of the polypeptide were important factors in
deŽ ning polymorph selectivity and crystal orientation.
This information is summarised in Fig. 9,161 although
the data given in this table somewhat contradict
information given in an earlier reference.162 Shorter
induction times and higher nucleation densities
were recorded with increasing polyelectrolyte
concentration.
Unstretched gelatin Ž lms containing poly-Asp or
poly-Glu supported the growth of non-oriented crys-
tals, with the one exception of Ž lms containing con-
centrations of poly-Asp less than 0·5 mg/g gelatin.
Here, calcite crystals were precipitated on the surface
of the Ž lm, and many were oriented with the c axis
perpendicular to the Ž lm surface. They also exhibited
rough faces, approximately parallel to the c axis, due
to speciŽ c interaction with small quantities of poly-
Asp released from the Ž lm163,164 The unique action
of poly-Asp in in uencing oriented calcite nucleation
was possibly due to its adopting a b-sheet confor-
mation on absorption to the collagen matrix.161 In
contrast, poly-Glu adopts a random conformation
under the experimental conditions used.161 The sole
phase deposited at polypeptide concentrations
<300 mg/g gelatin was calcite. Increase in the concen-
tration resulted Ž rst in the precipitation of non-
oriented aragonite, and then non-oriented vaterite
aggregates.55,161,162 The concentration range under
which aragonite formed without co-precipitation of
vaterite was signiŽ cantly smaller in the presence of
poly-Glu as compared with poly-Asp.
Substitution of stretched collagen Ž lms resulted in
a signiŽ cant increase in the degree of crystal orien-
tation, and a change in the polypeptide concentration
ranges under which speciŽ c polymorphs were precipi-
tated (Fig. 9). Oriented crystals of all three poly-
morphs were obtained in the presence of poly-Asp,
whereas only oriented aragonite and vaterite were
obtained with poly-Glu.161 Poly-Glu was markedly
less selective of polymorph type than poly-Asp. The
oriented aragonite crystals produced within the Ž lms
were rods aligned parallel to the direction of elong-
ation, with the c axis perpendicular to the rod length
(Fig. 10a). This suggested that their morphologies
were dictated by the initial shape of the sites in which
they grew. The oriented vaterite crystals precipitated
within cavities of the stretched Ž lms such that the c
axis was parallel to the collagen Ž bre axis (Fig. 10b).
The orientation was again attributed to the b-pleated-
sheet structure of poly-Asp, which may have inter-
acted most favourably with the (100) plane of vaterite,
which is the most positively charged crystal face
(Fig. 11).
The sequential precipitation of calcite, aragonite
and Ž nally vaterite with increasing concentration of
polypeptide suggests kinetic control of polymorphism.
Higher polypeptide concentrations increase Ca2+ ion
diVusion into the Ž lms, which in turn could have
produced high local supersaturations exceeding the
thermodynamic solubility product. Vaterite, as the
least stable phase with highest solubility, would then
be predicted to precipitate Ž rst. Polymorphic control
depended on the degree of uniaxial deformation.
Under deformation, the gelatin layers reorganise into
bundles parallel to the direction of elongation which
contain oriented segments of collagen molecules.
Stretching reduces the average cavity size and hence
increases supersaturation.163
Mg-calcite precipitation
Calcium carbonate was also precipitated in
crosslinked gelatin Ž lms containing poly-Asp, from
CaCl –MgCl solutions.55 In the absence of poly-
2 2
Asp, magnesian calcite crystals containing up to
12 mol-%MgCO were precipitated on the surface of
3
unstretched gelatin Ž lms. At Mg/Ca=1 the initial
calcite precipitates were prisms elongated along the c
axis and capped with rhombohedral {104} faces.
Continued growth produced aggregates with an angu-
lar spread of the hexagonal axis of a few degrees, and
Ž nally particles with a globular shape. The gelatin
matrix therefore regulated the precipitation of high
Mg-calcite, possibly by reducing the hydration sphere
of Mg on nucleation.
In the presence of poly-Asp and at Mg/Ca<2·5,
magnesian calcites with a MgCO content of
3
~12 mol-% were the principal product formed on
the unstretched Ž lms. Poly-Asp induced morphologi-
cal changes in the magnesian calcite crystals. At
Mg/Ca=1, the rhombohedral faces exhibited
rounded edges which disappeared completely at a
poly-Asp concentration of 0·5 mg/g gelatin.
Magnesian calcite aggregates were produced at
Mg/Ca=2·5 which consisted of a smaller hemisphere
International Materials Reviews 2003 Vol. 48 No. 3
206 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
11 Schematic diagram of relationship between vaterite crystals and entrapped poly-Asp in uniaxially
deformed collagenous matrices: reprinted from Ref. 162 with permission from Wiley-VCH
grown within the Ž lm and joined to a larger hemi-
sphere grown towards the solution. With poly-Asp at
0·5–500 mg/g gelatin, the smaller hemisphere had a
central core surrounded by concentric layers of radi-
ally oriented crystals. Concentrations of poly-Asp of
0·5–1·0 mg/g gelatin induced a change from calcite to
aragonite, independent of the Mg content in solution.
The aragonite crystals nucleated within the gelatin
Ž lms and were organised into globular aggregates.
Uniaxial deformation of the gelatin Ž lms resulted
in distinct changes in magnesian calcite precipitation.
Even at concentrations of poly-Asp<0·5 mg/g gela-
tin, the inorganic phase was principally precipitated
within the Ž lm. Aragonite was precipitated at mark-
edly lower poly-Asp concentrations (0·5–500 mg/g
gelatin, independent of the Mg concentration) than
in the unstretched Ž lms. The aragonite aggregates
appeared as  at lozenges with contours and lengths
depending on the elongation of the Ž lms and on the
Mg concentration. Increased Mg in solution induced
formation of more rounded lozenges which were
shaped by the bundles of collagen molecules.
Calcium carbonate precipitation in micelles
and microemulsions
Formation of calcium carbonate particles in water-
in-oil microemulsions
Water-in-oil (W/O) microemulsions were prepared by
solubilising Ca(OH) solution in the cores of reverse
2 nonionic surfactant hexaethy-
micelles formed by the
leneglycol dodecyl ether (DP-6) in cyclohexane.165
Precipitation of calcium carbonate was initiated by
bubbling carbon dioxide through the solution. The
method produced a uniform population of 5·4 nm
diameter particles, which were reported to be stable.
A similar method was used to precipitate calcium
carbonate in reverse micelles of calcium 1,2-bis-
(2-ethylhexylcarbonyl)-1-ethane sulphonate (CaOT)
in cyclohexane.166 Water was added to the micelles
over a range deŽ ned by R=[H O]/[CaOT].
Particles of CaCO of diameter 48–63 nm 2 were pro-
3
duced up to R =10, and converged to between 110
and 130 nm above R =10. The large increase in
International Materials Reviews 2003 Vol. 48 No. 3
particle size at R=10 was attributed to structural
changes in the water core which occur on formation
of microemulsions at R =10. Below R =10, the water
molecules interacted strongly with the surfactant mol-
ecules, whereas the water structure approached that
of bulk water above R =10. Particle diameters were
always considerably larger than the size of the micelle
water core, and there were a factor of 108 times more
micelles than CaCO particles. The particles therefore
3
clearly formed via fusion of the micelles.
Calcium carbonate was also precipitated in cetyl-
trimethylammonium bromide (CTAB)/n-butanol/
hexadecane/water microemulsions,167 by mixing sep-
arate solutions containing dissolved ammonium
carbonate and calcium chloride. Four solution com-
positions were investigated and in all cases, calcite
was precipitated. The morphologies of the particles,
however, depended on the region of the phase diagram
selected, and varied from intergrown rhombohedra,
to isolated rhombohedra, monodisperse spheres and
irregular rhombohedral particles.
Calcium carbonate particles with ‘microsponge’
structure
Precipitation of calcium carbonate in surfactant sys-
tems has produced some truly remarkable morpho-
logies, as exempliŽ ed by the structure of particles
produced by evaporation of W/O microemulsions.168
Spherical vaterite particles with sponge-like micro-
structures and diameters of 4–16 mm were synthesised
via passive evaporation of SDS (sodium dodecyl
sulphate)/octane/supersaturated calcium bicarbonate
solution micelles (Fig. 12). The particles exhibited an
irregular arrangement of hemispherical surface
depressions and pores, 0·3–5 mm in size. Many were
highly porous with a perforated outer shell and a
partially hollow centre, while others were more highly
mineralised, with one large hole or indentation.
Prior to mineralisation, the microemulsion solution
contained micrometre scale water droplets whose size
depended on the solution composition. In the initial
stages of growth, mineral aggregates formed along
the surface of water droplets to generate perforated
hollow shells with smooth surfaces. The presence and
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 207
12 Scanning electron image of vaterite particles
with microsponge texture, produced from
70·8 : 0·7 : 3·5 : 25 (wt-%) octane–dodecanol–
SDS–CaHCO3 microemulsion: reprinted from
Ref. 168 with permission from Wiley-VCH
stability of the water droplets was essential for the
formation of the patterned spheres. Crystallisation in
the gel phase yielded only irregular vaterite particles,
while addition of a co-surfactant to stabilise the
microemulsions produced particles with superior pat-
terning. A growth mechanism was proposed in which
bubbles of CO released from the saturated bicarbon-
2 acetone.171 The separated emulsion phase was Ž nally
ate solution form at the oil/water interface of the
aqueous droplets, where they remain due to their low
diVusion rate through the oil phase. Calcium carbon-
ate then precipitates around the bubbles, generating
the observed surface patterns. Subsequent growth
proceeds inwards as the droplets progressively
decrease in volume. The preferential stabilisation of
vaterite over the more stable polymorph calcite was
attributed to interaction of the surfactant sulphate
headgroups with the Ca2+ ions.
Hollow porous shells of calcium carbonate from
self-organising media
Aragonite with an intergrown, ‘skeletal’ structure
was precipitated from bicontinuous microemulsions
of DDAB (didodecyldimethylammonium bromide),
tetradecane and hexadecane, and a saturated calcium–
magnesium bicarbonate solution.169 Magnesium was
essential for aragonite formation, and disorganised
aggregates of calcite crystals were produced in its
absence. Films of aragonite were deposited on solid
substrates by placing a drop of the microemulsion
solution on a substrate, immersing it in hot chloro-
form and Ž nally evaporating any residual solvent.
They exhibited a honeycomb microstructure of irregu-
lar shaped columnar pores with interconnecting crys-
talline walls often at ~120° to each other. The pore
size depended on the composition of the micro-
emulsions, with larger pores being produced with
increasing oil content. Hollow shells of skeletal ara-
gonite were formed using a similar strategy involving
dipping polystyrene spheres into the microemulsion
solution, washing in hexane and Ž nally dissolving
away the spheres.
The intergrown aragonite structures appeared to
form during the solvent extraction step, as simple
storage of the microemulsion solution did not lead to
calciŽ cation. Precipitation of calcium carbonate in
this system is likely to have been signiŽ cantly retarded
due to the low diVusion rate of CO through the
2
viscous oil. Solvent extraction may have induced
phase separation, producing oil droplets in the aque-
ous solution, and allowing rapid mineralisation.
Complete removal of the oil then leaves an inorganic
replica of the self-assembled foam. Although the over-
all structures of the bicontinuous microemulsions and
the continuous mineral network are similar, the length
scales are totally incommensurate which suggests that
phase separation and reorganisation of the micro-
emulsions structure must occur during crystallisation.
Calcium carbonate particles produced in
pseudovesicular double emulsion
A double water-in-oil-in-water (W/O/W) emulsion
system, with a pseudo-vesicular structure, was used
as an environment for the preparation of calcium
carbonate particles.170,171 A W/O emulsion, contain-
ing an aqueous solution of Na CO , NaOH and
NaNO was prepared, and was poured 2 3 into an exter-
3
nal aqueous phase of Ca(NO ) solution. The
32
resulting mixture was stirred to produce a W/O/W
emulsion, to extract the Ca2+ ions into the internal
aqueous phase and to allow precipitation to occur.
On termination of this stage, the W/O emulsion was
washed and was demulsiŽ ed either by adding etha-
nol,170 using a freeze–thaw process,170 or adding
scrubbed with ethanol or acetone, and water and the
CaCO particles were isolated.
The 3phase and morphology of the calcium carbon-
ate particles formed depended on the composition of
the internal aqueous phase, the demulsiŽ cation
method, and the surfactant used. Using sorbitan
sesquioleate (Span 83) as the surfactant and pH<11,
aggregates of vaterite particles were produced.170 Use
of ethanol in the demulsiŽ cation process induced
immediate separation of the vaterite particles, produc-
ing dense, random aggregates. In contrast, use of the
freeze–thaw demulsiŽ cation method produced hollow
vaterite spheres of narrow size distribution. These
structures were considered to form by nucleation of
vaterite crystals at the oil/internal phase interface,
and their subsequent aggregation during the freeze–
thaw cycle. At pH values of 13 and above, micrometre
sized calcite rhombohedra were precipitated in
addition to vaterite. The results demonstrated that
spatial control of supersaturation, rather than tem-
plating by an organised organic structure, was respon-
sible for controlling mineralisation. A brief report was
also made using Span 20 as the surfactant, which
yielded calcite rhombohedra.171
Crystal growth in polymer templates
Control of calcium carbonate morphology by
transformation of amorphous precursor in
constrained volume
It has been suggested that amorphous calcium car-
bonate may play an important role in morphological
control of calcium carbonate (see above). This premise
has been examined by investigating the morphological
changes of ACC particles to calcite within a restricted
volume.172 SpeciŽ cally, calcium carbonate was pre-
International Materials Reviews 2003 Vol. 48 No. 3
208 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
13 Calcium carbonate crystals grown within pores of track etch membranes: a at 4°C after 24 h; b at 4°C
after 15 min; c showing crystalline faces characteristic of calcite; d at room temperature after 24 h
cipitated in the 3 mm diameter, cylindrical pores of
track etch membranes by counter-diVusion of CaCl
and Na CO solutions into the membrane pores.2
2 3
Experiments were carried out at room temperature
and at low temperature (4–6°C). The intramembrane
particles produced at low temperature exhibited rod-
like morphologies and dimensions of 3×10 mm
(Fig. 13a), which had clearly been dictated by the
geometry of the membrane channels.
Examination of these particles at early stages of
growth demonstrated that they formed via an ACC
precursor (Fig. 13b). Spherical particles of diameter
0·5 mm, which are characteristic of ACC, initially
coated the walls of the pores, before Ž lling in the
entire volume and generating the Ž nal cylindrical
form. Morphological evidence suggested that the
mature cylinders were calcite (Fig. 13c). Repetition of
the experiments at room temperature produced crys-
tals of irregular morphologies (Fig. 13d ). The results
therefore suggest that transformation of ACC within
a constrained volume can produce calcite crystals of
morphology imposed by the environment.
Crystal growth in bicontinuous polymer template
Single crystals of calcite with complex form were
produced in the absence of additives, by precipitation
in a polymer membrane with sponge-like structure.173
The membrane was templated by a sea urchin plate
International Materials Reviews 2003 Vol. 48 No. 3
and was produced by dipping a plate in the polymer
monomer solution, curing the polymer, and Ž nally
dissolving away the calcium carbonate to generate
the polymer replica. As the porous and inorganic
fractions of the sea urchin plate occupy equal volumes,
and have identical morphologies, the polymer mem-
brane produced had an identical structure to the
original calcium carbonate plate (Fig. 14a). Calcium
carbonate was precipitated in the membrane by plac-
ing it between two half U tube arms, and Ž lling them
with CaCl and Na CO solutions respectively, in
2 2 3
the concentration range 0·001–0·800 mol LÕ 1. The
particles produced were isolated from the membrane
by heating to burn oV the polymer.
Evidence of templating of the particle morphology
by the membrane was observed for all solution con-
centrations used, but was far superior at low concen-
trations. At higher concentrations, the structure was
polycrystalline calcite and replication was poor, with
limited network formation being observed (Fig. 14b).
In marked contrast, perfect replication of the mem-
brane structure was achieved at low reagent concen-
trations (Fig. 14c). Under these conditions, large
single crystals of calcite, whose morphologies were
identical to the structure of the polymer membrane
(and therefore sea urchin plates) were produced. This
category of particles showed both non-crystallo-
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 209

14 Electron micrographs of: a polymer membrane produced as template of sea urchin skeletal plate;
b polycrystalline calcium carbonate precipitated in membrane from 0·400 mol LÕ 1 reagents;
c templated single crystal of calcite precipitated from 0·020 mol LÕ 1 solutions; d templated single
crystal of calcite isolated after 5 min reaction time

graphic curved surfaces, as well as the planar faces
characteristic of crystalline materials.
Isolation of the single crystals after only 5 min
reaction time yielded particles 50–100 mm in size.
Longer incubation times increased the number of
particles present in a membrane but did not signiŽ -
cantly increase the size of individual particles. In
contrast to the entirely smooth surfaces of the mature
particles, the surfaces of the 5 min particles showed
many well deŽ ned crystal faces (Fig. 14d). The single
crystals must derive from an isolated nucleation site
and then grow by extension through the porous
network. Continued growth then occurs at the particle
surface until the crystal impinges on the polymer
matrix, which limits further growth. Crystals of this
complexity are characteristic of biologically con-
trolled mineralisation. However, the results demon-
strate that single crystals with intricate structures can
be produced synthetically by precipitation in a suit-
able constrained volume.
Crystal growth on solid films
Organisms are adept at controlling mineralisation
such that crystal nucleation and growth occurs at
speciŽ c locations (see ‘Crystal nucleation’, above).
Further, oriented arrays of particles can be produced
using ordered organic matrices as growth templates,
as is beautifully demonstrated by mollusk nacre.
Many organic substrates have been employed syn-
thetically to control calcium carbonate nucleation,
and experiments have met with considerable success.
Both naturally derived and synthetic polymer Ž lms
have supported the deposition of CaCO , and thin
Ž lms of calcite have been produced on 3addition of
suitable soluble additives to the growth medium. A
novel approach has been used in which a polymer
was imprinted with existing calcite crystals. The
resulting polymer was active in selecting the calcite
polymorph and producing oriented crystals.
Langmuir monolayers have provided a valuable
system in which to study the relationship between an
organised organic surface and a nucleated crystal.
Early experiments employed monolayers constructed
from simple long chain alkyl compounds, which were
active in selecting polymorph and the crystal orien-
tation according to the headgroup expressed. A higher
degree of control has been achieved using monolayers
with superior organisation. The application of self-
assembled monolayers (SAMs) to control crystal
International Materials Reviews 2003 Vol. 48 No. 3
210 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry

nucleation and growth has demonstrated an impress-

ive degree of control: SAMs have a number of advan-
tages over Langmuir monolayers in that they are
easily prepared with a high degree of molecular order
and they can be patterned. These patterned substrates
have been used to direct the formation of two-
dimensional patterned arrays of oriented crystals.

Calcium carbonate precipitation on polymer

films
Thin film deposition on chitin/chitosan substrates
A common mechanism of growth control in biological
systems employs the combined eVects of an insoluble
organic matrix, which deŽ nes the nucleation event,
and soluble macromolecules that control crystal
growth (see ‘Organic macromolecules’ and ‘Crystal
nucleation’, above). These conditions have been mim-
icked in a number of systems. Thin Ž lms of calcium
carbonate have been deposited on insoluble matrices
of chitin174–176 and chitosan,175–180 in the presence of
a range of soluble additives. The soluble additive has
a dual role in promoting thin Ž lm formation.
Adsorption of the additive on the substrate generates
a surface that concentrates Ca2+ ions and induces
crystal nucleation, while in solution the additive inter-
acts with the growing nuclei, inhibiting growth into
the solution.
The Ž rst demonstration that thin Ž lms of calcium
carbonate could be prepared using the combined
action of an insoluble matrix and soluble additives
was made by depositing CaCO on spin coated 15 Scanning electron images of calcium
chitosan substrates in the presence 3 of poly (acrylic carbonate thin films grown on chitosan
acid) (PAA) as an additive.177 In the presence of PAA, matrices in the presence of 4·4 wt-ppm poly-
spherical calcite crystals were initially precipitated Asp in a absence and b presence of MgCl2
(Mg:Ca = 6): reprinted from Ref. 178 with
homogeneously over the Ž lm. Continued crystallis-
permission from the Royal Society of
ation produced a thin, continuous Ž lm of calcite over Chemistry
the entire chitosan substrate that was preferentially
oriented with the {110} plane parallel to the substrate.
strate. Rhombohedral calcite crystals were deposited
In the absence of PAA, only isolated calcite crystals
on all substrates in the absence of PAA. On addition
were produced. The orientational control of the chito-
san Ž lm/PAA system may have derived from the close of PAA, substrates CE, CH, CHS and CH derivatised
all supported thin Ž lm formation, whereas the deriv-
geometric match of Ca–Ca distance in the {110}
atised CE and CH polymer matrices did not. The
calcite plane with the carboxylate group separation
in PAA. activity of the substrates depended on adsorption of
PAA to the polymer matrix; no PAA adsorbed to the
Deposition of calcium carbonate on spin coated
derivatised CE or CH substrates.
chitosan Ž lms was also investigated with a wider
range of soluble additives.180 Thin Ž lms of calcite Aragonite thin Ž lms were produced on chitosan
substrates by addition of magnesium ions to the
were deposited on chitosan substrates in the presence
supersaturated calcium carbonate solution.178 Poly-
of PAA (Fig. 15a) and poly (glutamic acid) (poly-Glu),
while poly (allylamine) completely inhibited crystal Asp was used to induce thin Ž lm formation, producing
circular thin Ž lms of aragonite that grew radially
growth in both the presence and absence of the
from a central point (Fig. 15b). Repeated cycles of
substrate. Spherical calcite particles were produced in
bulk solution and on the substrate in the presence of spin coating and thin Ž lm growth produced layered
inorganic/organic composites (Fig. 16). This tech-
DNA, whereas rhombohedral calcite crystals precipi-
nique was also used to generate layered calcite/
tated on the chitosan Ž lms in the absence of additives.
The in uence of the substrate on calcium carbonate polymer structures.175 Calcite thin Ž lms were
deposited on chitin and chitosan substrates, by precip-
deposition in the presence of PAA was investigated
itating CaCO in the presence of PAA. Spin coating
by precipitating CaCO on cellulose (CE), chitin (CH) 3
3
and chitosan (CHS) substrates, and derivatives of of a second polysaccharide layer on the surface of the
CaCO thin Ž lm then provided a suitable surface for
these substrates.176 Cellulose has only OH functional 3
the growth of a second layer of CaCO .
groups, while chitin and chitosan have OH and NH 3
groups. In the derivatised CE and CH Ž lms, all Crystallisation on biologically derived fibres
proton donating groups were protected, whereas only In related work, calcium carbonate has also been
NH groups remained on the derivatised CHS sub- precipitated on a range of biologically derived Ž bres,
International Materials Reviews 2003 Vol. 48 No. 3
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 211
16 Scanning electron image of double layer
aragonite/chitosan composite structure
formed by deposition of aragonite on chitosan
matrix in the presence of 4·4 wt-ppm poly-Asp
and MgCl2 (Mg:Ca = 6), by alternate spin
coating and crystallisation: reprinted from
Ref. 178 with permission from the Royal
Society of Chemistry
which have determined the polymorph precipitated,
and controlled the kinetics of precipitation. Calcium
carbonate deposition was carried out under con-
ditions where the solution supersaturation was main-
tained at a constant value during crystallisation.
Fibrin Ž bres supported the formation of spherical
vaterite crystals,181 whereas rhombohedral calcite
crystals deposited on the chitin,174 and cellulose
Ž bres.182 Crystallisation in the presence of elastin
resulted in nucleation and growth of calcite crystals
with intergrown, rhombohedral morphologies on the
elastin Ž bres.183 The nucleating capability, and poly-
morph selectivity of the Ž bres depended on the ability
of the biopolymer to bind Ca2+ ions at the functional
groups on the polymer surface, and the stereochemis-
try of these groups. Thin Ž lms of calcium carbonate
have also been deposited on chitin Ž bres in the
presence of PAA.184 The chitin Ž bres therefore support
identical CaCO growth in the presence and absence
of PAA as do the 3 spin coated chitin Ž lms (see preced-
ing section).
Calcium carbonate deposition on spin coated
polymer substrates
Calcium carbonate was grown in the presence of
powdered poly (vinyl chloride-co-vinyl actetate-co-
maleic acid), consisting of 83, 13 and 1% of the
respective monomers, using a constant composition
method.185 Spherulitic vaterite particles nucleated on
the polymer particles, and subsequently transformed
into calcite. In contrast, a vinyl chloride–vinyl acetate
copolymer, and a carboxylated vinyl chloride–vinyl
acetate copolymer containing 1·8% ionisable groups
failed to nucleate calcium carbonate. A fully soluble
styrene–maleic anhydride copolymer with 50% maleic
anhydride groups acted as a Ca2+ sequestering agent,
but did not support calcium carbonate growth.
Selectivity of the crystal polymorph was attributed to
the kinetics of growth of the supercritical nuclei on
the substrates, rather than geometrical eVects.
Continuous thin Ž lms of calcite were produced on
spin cast poly (ethylenimine) Ž lms, in the presence of
generation G=3·5 poly (amidoamine) (PAMAM)
dendrimers.186 In contrast, rhombohedral calcite crys-
tals were deposited on the substrate in the presence
of a G =1·5 PAMAM dendrimer, and also in control
experiments in the absence of dendrimer additives.
Interaction of the poly (ethylenimine) Ž lm with the
PAMAM dendrimers may have produced an insol-
uble Ž lm which supported calcite nucleation. The
PAMAM in solution then inhibited crystal growth
into the solution, resulting in the formation of a thin
Ž lm of calcite on the substrate. That the G=1·5
dendrimer failed to support thin Ž lm formation can
be attributed to its weaker complexation with Ca2+
ions.
Deposition on sulphonated polystyrene films
The soluble control macromolecules used by organ-
isms to directly control crystal growth often contain
ordered aspartic acid domains, together with disor-
ganised covalently bound polysaccharides, which are
generally sulphated and carboxylated (see ‘Organic
macromolecules’, above). The sulphate groups may
be active in concentrating calcium ions, while the
b-sheet structured carboxylate domains provide a
nucleation site. The cooperative action of sulphate
and carboxylate groups was investigated in a model
system by generating substrates through adsorption
of poly (aspartic acid) (poly-Asp), poly (glutamic acid)
(poly-Glu), poly (acrylic acid) (PAA) and proteins
extracted from the calcitic prismatic layer of the
mollusk Mytilus californianus, on sulphonated poly-
styrene Ž lms.187 The nucleation density of calcite
crystals on the sulphonated polystyrene Ž lms in the
absence of adsorbed polymer progressively increased
on Ž lms sulphonated for up to 8 h, and then decreased
with longer treatment, probably due to a loss in
rigidity of the Ž lm surface. The percentage of crystals
nucleating speciŽ cally on the (001) plane of calcite
also peaked on the 8 h Ž lms, reaching 60–65%. The
large number of oriented crystals observed under
these conditions probably derived from non-speciŽ c
electrostatic eVects.
Films prepared by adsorption of poly-Asp on sul-
phonated polystyrene Ž lms were considerably less
eVective in nucleating and orienting crystals, as com-
pared with the polystyrene Ž lms alone. In contrast,
sulphonated Ž lms formed by adsorption of poly-Asp
in the presence of calcium associated over twice as
much Ca and exhibited a three-fold increase in the
number of (001) oriented crystals (25%), as compared
with identical Ž lms without poly-Asp. Neither poly-
Glu nor PAA induced oriented nucleation, suggesting
that the ordered b-sheet conformation of poly-Asp
was required for oriented nucleation. The aspartic
acid rich proteins isolated from mollusk shells were
also capable of inducing (001) oriented calcite. A
single glycoprotein containing both sulphate and
carboxylic groups was isolated from the protein
assembly. This protein was eVective in inducing a
twofold increase in (001) oriented crystals. Blockage
of the carboxylate groups suppressed oriented
nucleation, demonstrating that both the sulphonate
and carboxylate ligands are required to induce ori-
ented calcite nucleation.
International Materials Reviews 2003 Vol. 48 No. 3
212 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
17 Schematic diagrams of a formation of imprint of rhombohedral calcite crystal; b nucleation and
growth of large single crystal of calcite from imprinted site: reprinted from Ref. 191 with permission
from Wiley-VCH
Calcite nucleation on poly(organosiloxane)
membranes
The majority of studies focusing on control of calciŽ -
cation at surfaces have employed organic substrates.
Experiments using poly (organosiloxane) membranes
demonstrated that inorganic materials may also
be active in controlling crystal nucleation and
growth.188,189 Unfunctionalised poly (dimethylsilox-
ane) (PDMS) membranes, prepared by crosslinking
Me SiO[MeSi (H )O] SiMe with an a,v-dihydroxy-
poly3 (dimethylsiloxane)x in the3 presence of tetraethoxy-
silane and a suitable catalyst (dibutyltindilaurate,
dibutyltindiactetate, dibutyltindimethoxide or zinc
octadecanoate) were mounted on glass slides which
were then immersed in saturated solutions of calcium
hydrogencarbonate. At low dibutyltindilaurate cata-
lyst concentrations only calcite was deposited, while
higher concentrations resulted in calcium silicate
overgrowth of the calcite. The calcium silicate mater-
ial was amorphous and contained 5–10% calcite.
Membranes prepared using dibutyltindiactetate, di-
butyltindimethoxide or zinc octadecanoate catalysts
supported calcite deposition without calcium silicate
overgrowth. The unique action of the dibutyltindilaur-
ate catalyst in promoting calcium silicate formation
was attributed either to fragmentation of the SiO
4/2
and R SiO centres, or the heterosiloxane linkages
2 2/2
due to free lauric acid liberated from the catalyst.
Curing of the dibutyltindilaurate membranes at
80–90°C reduced the rate of mineral deposition, and
completely inhibited deposition after 3–4 days’ curing.
Heating was likely to have caused rearrangement of
Si–O–Sn linkages to more stable Si–O–Si and
Sn–O–Sn combinations. Acid treatment of uncured
dibutyltindilaurate Ž lms enhanced solid formation,
while washing of the cured Ž lms with 2M HCl re-
activated them. Membranes containing functionalised
side chains were also investigated, and again sup-
ported the growth of calcite with a calcium silicate
overgrowth. The yields of CaCO were signiŽ cantly
3
higher than on the unfunctionalised membranes, with
ester, ether and  uoroaromatic groups being most
eVective. Glass slides coated with similarly func-
tionalised silanes were inactive towards mineral
deposition.
International Materials Reviews 2003 Vol. 48 No. 3
Directed nucleation of calcium carbonate at
imprinted polymer surfaces
A novel templating route in which synthetic polymers
were imprinted with the motif of crystal surfaces was
used to direct the growth of calcium carbonate on
polymer substrates.190,191 Molecular imprinting of
synthetic polymers is a technique that creates speciŽ c
recognition sites for target molecules to be formed in
a polymer substrate.192,193 Typically, functional and
cross-linking monomers are co-polymerised in the
presence of the imprint molecule. The functional
monomers initially form a complex with the imprint
molecule, and following polymerisation, their func-
tional groups are held in a position by the highly
cross-linked polymer structure. Subsequent removal
of the imprint molecule reveals binding sites that are
complementary in size and shape to the original
template molecule (Fig. 17).
Imprinted polymers have been prepared from
6-methacrylamidohexanoic acid as the functional
monomer and divinylbenzene as the crosslinking
agent, using calcite or aragonite crystals as tem-
plates.190 Calcite crystals with contrasting rhombo-
hedral, lozenge and dumbbell morphologies were also
used.191 In all cases, removal of the template crystals
left voids in the polymer that were consistent in
morphology with the original crystals. Calcium car-
bonate was then regrown on the polymer template
from supersaturated solutions of calcium carbonate.
Imprints formed by rhombohedral calcite crystals
promoted the regrowth of single crystals of calcite
from a {10.4} face with identical rhombohedral mor-
phologies, but which were signiŽ cantly larger than
their template counterparts. The structural inform-
ation contained in the single {10.4} face of calcite in
contact with the polymer during templating was
therefore successfully translated into the organisation
of carboxylate groups on the polymer surface. In
contrast, polymers imprinted with lozenge and dumb-
bell shaped calcite crystals promoted the growth of
aggregates of calcite crystals in the imprints. The
overall shapes of the clusters bore some resemblance
to the morphologies of the template crystals. The
template crystals were probably polycrystalline rather
than single crystal in character, resulting in random
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 213
18 a type I and b type II vaterite crystals
orientation of nucleating crystals within the polymer
void.
The polymers templated with rhombohedral calcite
crystals were also active in controlling polymorph.
Indeed, at higher temperatures, which favour aragon-
ite precipitation, calcite crystals still precipitated at
the imprinted areas, while aragonite was produced in
the bulk solution. The aragonite imprinted polymers
failed to select for the aragonite polymorph, and a
mixture of calcite and aragonite crystal were produced
in the voids.
Langmuir monolayers
Long chain aliphatic monolayers
The Ž rst experiments in which calcium carbonate was
precipitated in association with Langmuir monolayers
demonstrated that both the orientation and the phase
of the crystals could be determined by the surfactant
structure and the subphase concentration.194–201
Monolayers of octadecylamine (ODA),197–200 stearic
acid,197–200 eicosyl sulphate,201 or eicosyl phosphon-
ate,201 were spread on supersaturated solutions of
calcium bicarbonate, and calcium carbonate precipi-
tated at the monolayer on passive loss of CO from
the solution. 2 trast with that of the ODA and stearic acid mono-
Crystallisation under ODA monolayers from
subphases of concentration 4·5 <[Ca]<9·0 mmol
LÕ 1 produced oriented vaterite crystals in two forms,
termed type I and type II),197–200 (Fig. 18). Type I
crystals developed from (0001) oriented hexagonal
discs to produce mature crystals with  oret morpho-
logies. Type II crystals nucleated from the (11.0) face.
Early crystals were disc-like, whereas mature crystals
had a complex shape and exhibited twofold symmetry.
Crystal nucleation and growth under stearic acid
monolayers, unlike the ODA system, was dependent
on the subphase Ca concentration.196–200 At [Ca]=
9 mmol LÕ 1, oriented calcite crystals were preferen-
tially nucleated in two morphological forms, termed
type I and type II. Both crystal types were plate-like
at early growth stages, and nucleated from a (11Â .0)
plane. Realignment during growth resulted in the
capped rhombohedral plates of the type I crystals
and a related triangular morphology in the case
of the type II crystals. When the concentration
of subphase calcium was reduced to [Ca2+ ]=
4·5 mmol LÕ 1, only oriented type I vaterite crystals
were observed.
The in uence of the monolayer on calcium carbon-
ate precipitation was considered on the basis of ion
binding, and on geometric and stereochemical com-
plementarity between the monolayer and the nucleat-
ing crystal face. That vaterite was precipitated under
both positively and negatively charged monolayers,
whereas calcite was only produced under a negatively
charged surface, suggested that Ca binding was
required for the crystallisation of calcite but not of
vaterite. In addition, high Ca concentrations resulted
in precipitation of the thermodynamically favoured
phase, calcite, under stearic acid monolayers. On
reduction of the Ca concentration, the activation
energy for calcite nucleation increases until it becomes
comparable with that of the metastable vaterite phase,
and vaterite is precipitated.
Stereochemical and geometric matching occurs
between the stearic acid monolayer and the (11Â .0)
face of calcite. In terms of geometric complementarity,
the headgroup spacings in the monolayer closely
approach the carbonate–carbonate spacing in the
(11Â .0) face of calcite. Stereochemical matching also
occurs in that the orientation of the carboxylate
groups in the monolayer mimics the rows of perpen-
dicular carbonate ions in the (11Â .0) calcite face. In
the case of vaterite nucleation under stearic acid
monolayers, no geometric match exists and oriented
crystal growth is directed by stereochemical matching
between the carbonate groups in the (0001) and (11.0)
crystal faces and the carboxylate headgroups in the
monolayer. With the ODA monolayers, no Ca2+
binding takes place at the monolayer and only vaterite
crystals were produced. The amine headgroups in the
ODA monolayers have no stereochemical equivalent
in the vaterite crystal. Oriented nucleation may have
been due to electrostatic binding of HCOÕ ions
3
orthogonal to the NH+ headgroups, which could
3
provide an indirect means of stereochemical
recognition.
The headgroups of n-eicosyl sulphate and n-eicosyl
phosphonate exhibit trigonal symmetries, which con-
layers. Immature crystals grown under the sulphate
monolayers were pseudohexagonal in form, nucleated
from a (0001) face, and developed to a trigonal
pyramidal morphology201 (Fig. 19). Monolayers of
International Materials Reviews 2003 Vol. 48 No. 3
214 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
19 Calcite crystal nucleated under n-eicosyl
sulphate monolayer, and schematic diagram
showing orientation of crystal
the corresponding phosphonate surfactant yielded
similar results, although a reduced nucleation density
was obtained. An approximate geometric matching
exists between the hexagonal array of Ca2+ ions in
the (0001) calcite face, and the pseudohexagonal
packing of the headgroups in the sulphate and phos-
phonate monolayers. However, the stereochemical
recognition between the monolayer and nucleating
crystal is probably the overriding factor determining
the orientation. The monolayer headgroups have trig-
onal symmetry, which mimics that of the planar
carbonate ions in the (0001) face of calcite.
The in uence on CaCO growth of soluble addi-
3 investigated. Aragonite
tives in the subphase was also
was precipitated under monolayers of n-eicosanoic
acid, n-eicosyl sulphate and n-eicosyl phosphonate by
addition of Mg2+ to the subphase.201 n-eicosanoic
acid monolayers supported the growth of intergrown
bundles of acicular crystals, oriented with the [100]
axis perpendicular to the monolayer. In contrast,
crystals precipitated under the sulphate and phos-
phonate monolayers were discrete, pseudohexagonal
in morphology, and were oriented with the [001]
axis perpendicular to the monolayer. As the arrange-
ment of ions in the (001) plane of aragonite is very
similar to that in the (0001) face of calcite, rationalis-
ation of the nucleation plane is stereochemistry based
as described for (0001) calcite nucleation under sul-
phate monolayers. Addition of Li+ ions to the sub-
phase produced thin triangular tablets of calcite in
International Materials Reviews 2003 Vol. 48 No. 3
association with the n-eicosyl sulphate monolayers,
due to inhibition of crystal growth along the [001]
axis.195 Use of malonic acid as a growth additive
modiŽ ed the morphology of calcite crystals precipitat-
ing under carboxylate monolayers by stabilising
{11Â .0} faces.195
Influence of monolayer structure on mineralisation
Brewster angle microscopy (BAM) provides an
opportunity to visualise both the surfactant mono-
layer and the growing crystals, and was used to study
CaCO precipitation under monolayers of diacetylene
3 by glycine (Gly-DA).202 Crystals grown
modiŽ ed
under Gly-DA monolayers at surface pressure p=
0–5 mN mÕ 1, where the monolayer spontaneously
organised into smectic domains, were predominantly
calcite. At p=20 mN mÕ 1, the monolayer adopted a
pseudo-focal-conic texture,203 and the crystals precipi-
tated were solely vaterite. Notably, the distance
between the foci of the monolayer pseudo-focal-conic
textures almost exactly equalled the separation of the
vaterite crystals grown under the monolayer, suggest-
ing a direct spatial correlation between the monolayer
template and growing crystals. The vaterite crystals
were likely to have grown under the most densely
packed domains since higher surface pressures pro-
moted vaterite growth. The monolayer packing den-
sity therefore appears to be important to polymorph
selectivity, whereas modulations in the monolayer
textures may deŽ ne the location of the precipitated
crystals.
Total alignment of calcite at acidic polydiacetylene
films
Total alignment of calcite crystals was achieved with
respect to polymeric Langmuir–Schäfer Ž lms of
10,12-pentacosadiynoic acid (p-PDA).204,205 Films of
PDA prepared as Langmuir monolayers were ultra-
violet polymerised and were subsequently horizon-
tally transferred to hydrophobised solid supports (a
Langmuir–Schäfer Ž lm) such that the carboxylic head
groups were exposed. Calcium carbonate crystallis-
ation was initiated by placing a drop of supersaturated
CaCO solution on the polymer Ž lm.
3
Calcite crystals that were either rounded or triangu-
lar in morphology were deposited on the Ž lms and
were co-aligned within single domains of the PDA
Ž lms. The rounded morphology represented a further
growth stage of the triangular crystals. The crystals
nucleated from a (01.2) plane and AFM demonstrated
that the a axis was oriented parallel to the polymer
backbone direction. Both the crystal and the p-PDA
polymer have a periodicity of ~5 A Ã along this direc-
tion such that the carboxylate groups along the
polymer background Ž t closely to the calcite crystal
structure (Fig. 20). The stereochemistry of the carb-
oxylate groups was also signiŽ cant in the selection of
the (01.2) crystal face. The (01.2) face comprises
alternating planes of Ca2+ ions and CO2Õ ions that
3
are tilted 28° to the normal of the (01.2) plane. This
orientation is closely matched by the 30° tilt of the
side chains and carboxylate groups on the polymer.
The stereochemical match thus uniquely selects for
the (01.2) plane, rather than, for example, the (0001)
face of calcite, which oVers a similar geometric, but
poor stereochemical match.
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 215
20 Schematic diagram showing potential
geometric relationship between (012) face of
calcite and p-PDA template. Left diagram is
view along calcite a axis, down polymer
backbone direction, and edge-on (012) basal
plane: alternating calcium (grey circles; dark in
front, lighter toward back) and tilted carbonate
layers characteristic of (012) plane are depicted;
calcite unit cell is shown as dashed, truncated
rectangle; partial structure of the p-PDA film is
shown: polymer backbone axis is symbolised
as continuous ‘w’; tilt angle of the polymer
alkyl chains and headgroups (o) emulate
carbonate stereochemistry and are implicated
in alignment of crystal about backbone axis.
On right is schematic diagram of single calcite
crystal with respect to polymer backbone
direction, indicating the dihedral angles: (014Â )
face corresponds to that viewed edge-on in
right diagram; reprinted with permission from
Ref. 204, ©1995 American Association for the
Advancement of Science
Investigation of conformational changes in the
polymer Ž lm at the onset of crystal growth demon-
strated that cooperativity at the organic/inorganic
interface contributed to the total orientational control
of the calcite crystals. The p-PDA Ž lms deposited on
the substrate were blue in colour, but turned red
subsequent to the onset of calcite growth, due to
changes in the orientation of side groups. FTIR of
the Ž lms in situ during crystallisation also showed
that rotations of side groups occurred at the onset of
crystallisation. This process is likely to correspond to
the movement of carboxylate groups into the optimal
position for achieving a stereochemical match.
Porphyrin amphiphiles as templates for calcium
carbonate nucleation
Oriented thin Ž lms of calcite were grown under
monolayers of amphiphilic tricarboxyphenylporphy-
rin iron(III) m-oxo dimers (Fig. 21), in the presence
of the soluble additive poly (acrylic acid) (PAA).206
The results obtained were independent of the degree
of monolayer compression, suggesting spontaneous
condensation of the porphyrin units. In the absence
of PAA, the principal product was rhombohedral
calcite crystals, many of which displayed pronounced
rectangular cavities on at least one of the distal {104}
faces.207 The crystals were oriented such that they
were suspended from the monolayer by one corner,
corresponding to a (001) face. Selection of the (001)
21 Amphiphilic tricarboxyphenylporphyrin iron(III)
l-oxo dimers used to generate monolayer:
reprinted with permission from Ref. 206, ©1998
American Chemical Society
face was attributed to the epitaxial match between
à Ca–Ca ion separation in the (001) calcite face
4·99 A
and the 5 A Ã intercarboxylate distance in the mono-
layer dimer unit.
In the presence of 20–30 ppm PAA, mineralisation
occurred more slowly to produce a uniform, thin
calcium carbonate Ž lm, several square centimetres in
area.206 The Ž lm surface in contact with the mono-
layer was  at and featureless, whereas the side directed
into the solution was rough, comprising intergrown
particles. Structurally, the Ž lm contained both calcite
and amorphous calcium carbonate (ACC), and the
extent of crystallisation was inhomogeneous across
the Ž lm. The crystalline areas of the Ž lm were (001)
oriented, as was observed in the absence of PAA.
Repetition of the experiment at low temperature
resulted in Ž lms composed entirely of ACC, and
demonstrated that the calcite Ž lms formed via an
ACC precursor. Continued maintenance of the Ž lms
on the subphase resulted in slow transformation
to calcite.
Hydrogen bonded Langmuir monolayers
Monolayers with well deŽ ned structures can be gener-
ated through linkage of the monolayer components
by hydrogen bonding. These monolayers exhibit
superior control over crystal orientation and
polymorph.208,209 This has been well demonstrated
by H bonded monolayers prepared by spreading
a monolayer of the amphiphile N,Nê -diocta-
decyltriazine-2,4,6-triamine (1) on a saturated calcium
hydrogen carbonate solution, to which the second
component, a cyanuric acid derivative (2), was
added.208 Compound (2) possessed a tailgroup R,
which was directed into the solution and was termin-
ated by either alcohol, carboxy, amino or phosphate
groups. The two monolayer components self-organ-
ised via hydrogen bonding such that the spatial
separation of like components along the ribbon was
à , whereas the ribbons were separated by 4·5 A
9·5 A Ã
(Fig. 22).
Monolayers incorporating carboxy terminated (2)
supported the growth of calcite rhombohedra from
International Materials Reviews 2003 Vol. 48 No. 3
216 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
22 Representation of malanine–cyanuric acid
monolayer normal (top) and parallel (bottom)
to air/water interface: R =CH2 CH2 CO2 H,
CH2 CH2 CH2 OPO3 H2 , CH2 CH2 CH2 NH2 ; reprinted
from Ref. 208 with permission from Wiley-VCH
the {01.2} crystal face. In contrast, the phosphate
modiŽ cation of (2) induced the formation of pyrami-
dal calcite crystals, nucleated from a triangular (001)
face and the side faces formed by three {10.4} planes.
The orientational control of both of these monolayers
was attributed to a stereochemical correspondence
between the monolayer and the crystal nucleation
face. The carbonate groups in the {01.2} face of calcite
possess a close geometric match with the carboxyl
groups in the monolayer template. Unique selection
of the {01.2} face over others which provide a similar
lattice match derives from the orientation of the
carboxyl groups in the {01.2} plane, which provides
a good match with the monolayer. In the case of the
phosphate terminated (2) monolayer, a close lattice
matching occurs between the positions of the phos-
phate groups and the planar carbonate ions in the
unique (001) calcite face, which selects for nucleation
from the (001) face. Monolayers containing amine
terminated (2) favoured the growth of vaterite rather
than calcite. Morphological analysis revealed two
populations of crystals, which were aligned with either
their [001] or # 11.0$ axes perpendicular to the
monolayer. The proportion of the # 11.0$ oriented
crystals was signiŽ cantly higher than [001] oriented
crystals. Hydroxyl terminated monolayers exerted no
in uence on crystal growth.
Further demonstration of the degree of control
achievable using highly ordered H bonded mono-
layers was achieved using monolayers constructed
from amide containing phospholipids, identiŽ ed as
(3), (4) and (5) in Fig. 23.209 Monolayers of (3) in
either a liquid or solid phase supported the oriented
nucleation of prismatic calcite crystals from the (10.0)
face. Selection of the (10.0) was attributed to the
monolayer structure. Molecules (3) self-assemble in a
linear array with an intermolecular distance of
approximately 5 A Ã due to H bonds between the amide
moieties. A further intramolecular bond between one
of the P–O–H groups and an ether oxygen results in
a molecular conformation in which the phosphate
group is bent back, leaving only two phosphate
International Materials Reviews 2003 Vol. 48 No. 3
23 Molecules 1, 2 and 3, used to form monolayers
at the air/water interface: from Ref. 209
oxygen atoms in contact with the solution. This motif
closely matches the orientation and spacing of the
carbonate ions along the b axis in the (10.0) face
of calcite.
The proposed templating mechanism was veriŽ ed
using monolayers of related compounds (4) and (5).
Molecule (4) carries a single methyl substitution on
the phosphate group, and thus cannot form an intra-
molecular bond with the phenoxy group. Monolayers
of (4) supported the growth of CaCO crystals
3
nucleated from the (10.0) face under the solid state
monolayer only. Compression presumably resulted in
a structural reorganisation of molecules (4), such that
they presented a headgroup orientation similar to (3).
Molecule (5) possesses an OH group in place of the
phosphate group, and monolayers of (5) failed to act
as templates for CaCO growth.
3
Monolayers of 5-hexadecyloxyisophthalic acid
(C ISA), which are ordered due to hydrogen bonds
16
between adjacent carboxylic acid groups and water,
supported the precipitation of aragonite.210 The crys-
tals nucleated as hexagonal tablets from the ac crystal
face and developed to form bundles of needles that
had grown radially outwards from a central crystal.
It was postulated that the C ISA molecules organ-
16
ised on the water surface such that the carboxyl
groups were periodically spaced at 4·4 and 5·5 A Ã
along the a and c directions respectively. The Ca2+
ions then bound either as double chelate complexes
with carboxylate groups on an individual molecule,
or in a bridging mode between carboxylate groups of
diVerent molecules. Both models give a close spatial
and geometrical correspondence between the array of
carboxylate groups in the C ISA template and the
16
Ca2+ ions in the ac face of aragonite. A further
possibility was that the bidentate motif of the meta-
disposed carboxylates complemented the stereochem-
ical arrangement of the carbonate groups in the
ac face.
Self-assembled monolayers
Silane monolayers on silicon substrates
A series of terminally functionalised organosilane
monolayers on silicon wafer substrates exerted some
control over the orientation, size and nucleation
density of calcium carbonate crystals.211 The mono-
layers were formed either by direct self-assembly
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 217
of the selected silane on the Si substrate, or
through chemical modiŽ cation of monolayers of
(3-aminopropyl )triethoxysilane (APS) (Table 3). All
aminosilane surfaces tended to support the formation
of {104} oriented rhombohedral crystals. The APS-
DCDA monolayers always produced the highest den-
sity of crystals, although the APS-NTA surfaces were
also eVective. Approximately the same number of
crystals deposited on all other monolayers. The calcite
crystals on the APS-DCDA monolayers were prefer-
entially nucleated from the (001) plane, with signiŽ -
cant numbers also nucleating from (101), (10 10) and
(018) planes. On the APS-NTA surfaces, the (119)
and (110) nucleation faces were most frequent, with
(116), (211), (113) and (018) orientations also being
favoured. The APS-SA and APS-POCl3 monolayers
enhanced (001) and (012) orientations.
The selection of certain crystal faces was interpreted
in terms of enhanced Ca2+ binding at the monolayer/
solution interface (an ‘ionotropic’ eVect). Nucleation
from (001), (018) and (10 10) faces on APS-DCDA
and APS-Acids monolayers therefore indicates that
crystal faces close to the (001) are stabilised by the
ionotropic eVect. The data for the APS-NTA system
suggest that a speciŽ c interaction occurs between the
nucleating calcite plane and the monolayer terminal
groups. Of the common nucleation planes observed,
many (the (001), (113), (116) and (211)) contain
carbonate ions that are oriented at equivalent distance
and angles to the plane. This speciŽ c angle may
provide pairs of carboxylate sites that are oriented
such that they mirror the pair of carboxyl groups
expressed by the NTA moiety.
Alkanethiol monolayers on gold and silver
A series of SAMs of v-terminated alkanethiols on
gold and silver thin Ž lms evaporated on to glass
substrates were investigated, and were demonstrated
to be highly eVective in controlling the orientation
of deposited calcite crystals, with almost 100%
orientation being achieved in some systems.212
Monolayers of HS(CH ) CO H, HS(CH ) OH,
HS(CH ) SO H, HS(CH 2 15 ) PO 2 H , HS(CH
2 11 )
2 11 3 2 11 3 2 2 11
N(CH ) Cl, and HS (CH ) CH were investigated.
3 3 2 15 3
The COÕ , SOÕ and OH terminated SAMs induced
2 3 growth of calcite and were active
highly oriented
in promoting crystal nucleation. The PO2Õ mono-
layers induced limited crystallographic 3speciŽ city
in that crystals nucleated from a variety of planes,
but all were at an identical angle to the c axis.
The N(CH )+ and CH terminated monolayers
33 3 2 15
Table 3 Silane self-assembled monolayers used to control calcium carbonate
nucleation and growth: information summarised from Ref. 210
Silane
(3-aminopropyl)triethoxysilane
(4-aminobutyl)triethoxysilane
[(N-(6-aminohexyl)amino)propyl]trimethoxysilane
[3-(N-(2-aminoethyl)amino)propyl]trimethoxysilane
Trimethoxysilylpropyldiethylenetriamine
(((((aminoethyl)amino)methyl)phenyl)ethyl)trimethoxysilane
APS-Nitrilotriacetic acid coupled
APS-Docosanedioic acid coupled
APS+Amidophosphonation
APS+Succinamidation
inhibited crystallisation. In comparison, the bare Au
and Ag substrates both supported the growth of a
mixture of randomly oriented calcite (~85%) and
vaterite (~15%) crystals.
The crystals produced in the CO (Au) system were
predominantly (015) oriented, in the 2 OH(Au) system
(104) oriented, and in the SO (Au) system (10 12)
3
oriented. The PO monolayers induced crystal growth
3
from planes oriented at 24° with respect to the c axis.
The crystals were largely randomly oriented in the
remaining Au systems. The crystals grown on Ag
substrates predominantly nucleated on the following
planes; CO (Ag) on (102), OH(Ag) on (103), and
SO (Ag) on2 (107). The PO monolayers induced
3 nucleation from faces at
crystal 3 a given angle to the
c axis, in this case at ~40° . Again, the remaining
systems were largely randomly oriented.
The molecular organisation of the monolayer was
of primary importance to the determination of crystal
orientation. Indeed, a smaller proportion of oriented
crystals were observed on CO H (Au) SAMs contain-
ing defects. The importance of2the organisation of the
SAM in the presence of counterions prior to crystal
growth was demonstrated by growing crystals on the
SAMs by mixing 0·1 mol LÕ 1 solutions of NaHCO
and CaCl in diVerent orders. For SAMs of COÕ 3
(Au), COÕ 2 (Ag), SOÕ (Au) and SOÕ (Ag), highly 2
2 3 3
oriented nucleation was only observed when the SAM
was exposed to CaCl solution Ž rst. No preferred
orientation occurred when 2 the Ž lms were Ž rst exposed
to the NaHCO solution. For SAMs of OH (Au) and
OH(Ag), the degree3 of orientation was signiŽ cantly
reduced after initial exposure to the NaHCO
solution. 3
The results demonstrate that the calcite nucleation
face is determined by the structure, chemical character
and coordination number of the monolayer head-
group. The basic lattice structure of the SAMs is the
same for a given metal and no lattice match occurs
between the metal lattice and the calcite nucleating
plane. In all cases of oriented nucleation, the Au and
Ag substrates induced nucleation from faces diVering
in orientation by 15–20° , which is identical to the
diVerence in the tilt angle of the thiol molecules on
Au and Ag. The COÕ , OHÕ and SOÕ SAMs therefore
appear to selectively2 nucleate calcite3 from one plane
only. In the case of the POÕ headgroup, the mono-
3 the c axis makes with
layer selects only the angle that
the substrate.
In an alternative experiment, CaCO was grown
on HS(CH ) CO H SAMs on 200 nm3 evaporated
2
Abbreviation
APS
ABS
AHAP
EDA
DETA
PEDA
APS-NTA
APS-DCDA
APS-POCl3
APS-SA
International Materials Reviews 2003 Vol. 48 No. 3
218 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry

gold Ž lms on mica.213 The mica substrate was heated

to 200°C during evaporation, and was annealed for
1 h, resulting in signiŽ cant growth of the Au single
crystal {111} domains to 250×250 mm in size.
Analysis of the resultant Au Ž lms demonstrated that
the polycrystalline domains were co-aligned with
respect to the single crystal mica substrate. Growth
of calcite on HS(CH ) CO H SAMs on these sub-
2 15
strates resulted in arrays 2of crystals which were
aligned over areas exceeding several square milli-
metres, and which were oriented either on a {102}
face (90% of crystals) or {015} face (10% of crystals).
That the predominant nucleation face was {102} as
opposed to the {105} recorded in the aforementioned
studies,212 was attributed to diVerences in the solu-
tion pH.
Previous investigations into calcium carbonate
deposition on v-functionalised alkyl thiol monolayers
on Au achieved a signiŽ cantly lower level of orien- 24 ‘Spherule’ formed by CaCO3 precipitation on
tational control.214–217 It is possible that the experi- alkylthiol coated gold particles
mental procedure used yielded less ordered
monolayers, a factor demonstrated to be important
non-polar monolayers, the longer chains tended to
in determining the ability of the SAM to control
promote oriented nucleation. Short chain thiols are
crystal orientation. The SAMs used in these studies
poorly ordered and would be anticipated to be less
are given in Table 4, and vary according to the
eVective in promoting oriented nucleation.
chain length and terminal functional group. The
highest nucleation densities were recorded on Calcium carbonate was also precipitated on SAMs
of anthracene-2-carboxylic acid (ANTH) on gold.
HS(CH ) CO H and HS (CH ) SO H SAMs,
2 10 2 2 10 3 ANTH monolayers exhibit a centred rectangular lat-
which are polar surfaces, and on the naked gold
substrate. The other SAMs were approximately 2–3 tice on Au(111) which was considered to mimic the
structure of, and thus template for, orthorhombic
times less eVective, with the exception of
aragonite.214 Results were compared with mercap-
HS(CH ) CH and glass, which were less eVective
again. 2 15 3 tohexadecane (CH (CH ) SH ) (MHD) monolayers,
3 on 2Au(111)
15
which self assemble with an hexagonal
At 22°C the predominant polymorphs precipitated
lattice. At 22°C, the proportions of crystals precipitat-
were calcite and vaterite. The vaterite crystals
nucleated either from (001) or (010) planes, whereas ing on the ANTH and MHD monolayers were
approximately 40 : 45 : 15 calcite–vaterite–aragonite
calcite crystals on some monolayers displayed evi-
and 45 : 55 calcite–vaterite respectively. At 45°C, 90%
dence of orientation, with crystals nucleating from
the (001) face. Aragonite was only observed on the of the crystals deposited on the ANTH SAMs were
aragonite, whereas 90% of the crystals precipitating
HS(CH ) SO H as ~20% of the total mass precipi-
23 3 on the MHD monolayer were calcite. The calcite
tated. At 45°C, all three polymorphs were observed
on the majority of the substrates, although no crystals precipitated on the MHD substrate were
preferentially oriented with the c axis perpendicular
aragonite precipitated on the HS (CH ) OH,
2 11 to the substrate, whereas the calcite crystals on the
HS(CH ) CH or HS(CH ) CH SAMs or on the
2 10 3 2 15 3
naked gold substrate. Generally, aragonite appeared ANTH SAMs were non-oriented.
to be induced by rough, polar surfaces. Comparing Crystallisation on SAM coated gold particles
the eVect of diVerent chain lengths in both polar and Monodisperse populations of nanosized gold
particles, coated with mercaptophenol, were used as
nuclei on which to grow calcium carbonate.218,219 At
Table 4 x-terminated alkane thiols used to pH 12, and all conditions of temperature and colloid
construct self-assembled monolayers, to concentration, calcite precipitated on the gold col-
control calcium carbonate nucleation and loids, forming ‘spherules’. At 22°C and a seed concen-
growth: information summarised from tration of 10 mg/100 mL solution, 5–10 mm spherules
Refs. 214, 215
were produced which comprised radially organised
Abbreviation Formula rhombohedral calcite crystals, organised such that
their c axes were approximately perpendicular to the
C11H HS(CH2 )10 CH3
C16H HS(CH2 )15 CH3
particle surface (Fig. 24). ModiŽ cation of the reaction
C11OH HS(CH2 )11 OH conditions to 4°C and a seed concentration of
C12SH HS(CH2 )12 SH 10 mg/100 mL solution resulted in spherules with
C16SH HS(CH2 )16 SH smoother surfaces, formed from needle-like crystals
C10COOH HS(CH2 )10 CO2 H radiating outwards from the centre of the spherule.
C15COOH HS(CH2 )15 CO2 H Each of the CaCO coated spherules was likely to
C3SO3H HS(CH2 )3 SO3 HNa 3
have contained a number of gold particles. In com-
C10SO3H HS(CH2 )10 SO3 HNa parison, only calcite crystals with poorly deŽ ned
C3PO4H2 HS(CH2 )3 PO4 H2 morphologies and rounded surfaces were deposited
International Materials Reviews 2003 Vol. 48 No. 3
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 219
25 Ordered two-dimensional arrays of single
calcite crystals deposited on patterned
SAMs consisting of stamped regions
of HS(CH 2 )15 CO2 H, and remaining areas
functionalised with HS(CH 2 )15 CH3 : a SAMs
formed on Au: nucleating plane (015), crystal
size d= 35 mm, spacing p=100 mm, [Ca2 + ]=
10 mmol LÕ 1 ; b SAMs formed on Ag:
nucleating plane (012), d= 15 mm, p=100 mm,
[Ca2+ ]= 10 mmol LÕ 1 ; reprinted from Ref. 222
with permission of the Nature Publishing
Group
at 22°C and pH 12 on equivalent SAMs formed on
planar gold substrates.
Patterned crystallisation
Patterning of SAMs enables periodic arrays of discrete
crystals with controlled densities, location, size and
orientation to be produced.220–222 The SAMs were
patterned using a microcontact printing technique to
produce areas with diVerent nucleating abilities.
Hydrophilic thiols (either HS (CH ) CO H,
HS(CH ) OH or HS (CH ) SO H ) were 2located 15 2 on
2 11 2 11 3
the metal substrate by microcontact printing of a
square array of circular domains, and the substrate
was then washed with an ethanolic solution of
HS(CH ) CH to passivate the uncoated areas.222
2 15 and
The location 3 density of growing crystals, the
number of crystals nucleating within a given region
and the orientation of the crystals could all be deŽ ned
according to the density and sizes of features on the
stamp, the concentration of the crystallising solution
and the terminal groups of the SAM (Fig. 25).
Control over patterned crystal growth was investi-
gated in more detail using a stamped pattern of
HS(CH ) CO H, and HS(CH ) CH functionalis-
2 15 2 2 15 3
ing the remaining areas.221 Localised crystallisation
occurs in this system due to diVusion limited
nucleation.221,222 Crystals nucleate Ž rst on the areas
of hydrophilic, carboxyl terminated thiols, and trans-
port of ions to these growing crystals reduces the
concentration of calcium and carbonate ions in the
adjacent hydrophobic areas (the methyl terminated
thiols), and prevents non-speciŽ c nucleation within a
characteristic distance of the interface. Variation of
the size and periodicity of patterns and the concen-
tration of the crystallising solution, deŽ nes the mass
transport to diVerent regions of the substrate and
oVers control over crystal patterning.
Stamps bearing circular relief structures, in a range
of circle diameter d and periodicity p were used.221,222
For large values of p, crystallisation occurred in a
‘halo’ pattern, and no nucleation was observed within
a certain distance of the more active region.221 The
most ordered crystal growth was observed when the
carboxyl terminated thiols were stamped in features
smaller than the depletion zone. These conditions
induced the highest densities of identically sized crys-
tals on the carboxyl regions and the number of
crystals produced depended linearly on the area.
Crystal growth was entirely inhibited on the methyl
regions.221 The solution concentration also aVected
the size of the halo pattern, the induction time,
nucleation density and the sizes of the crystals. The
edge resolution of the crystal patterns was substan-
tially increased by increasing the concentration of the
crystallisation solution, enabling structures with an
edge resolution of <50 nm to be produced.221
As an alternative to microcontact printing, pat-
terned SAMs were prepared using substrates gener-
ated from mixed metals.220 This technique enables
SAMs containing sub-50 nm patterns to be created,
as compared with the 100–200 nm patterns possible
using microcontact printing. The mixed metal sub-
strates were prepared by initial evaporation of either
Ag or Au, followed by application of a second metal
(Ag, Au) using a stencil or a photoresist mask. The
pattern in the substrate is transferred to the SAM as
the interface between adjacent metal domains pro-
duces disorder in the monolayer, which is translated
into the growing crystals. On these substrates SAMs
of HS(CH ) CO H were formed that supported the
2 15 2
formation of patterned arrays of calcite crystals. The
disordered regions in the patterned SAMs acted as
the initial nucleation sites for the calcite crystals. The
 ux of ions created towards the growing crystals
generates a depletion region where the concentration
of ions is below saturation.
The pattern of crystals can be controlled according
to the size of the metal domains in the substrate, and
the concentration of the crystallisation solution. This
was demonstrated by investigating crystal growth on
Ag/Au substrates, patterned using the stencil method,
under a range of solution concentrations (Fig. 26).
For C <10 mmol LÕ 1 (the local eVective concen-
trationeffin solution), which is below C (the saturation
concentration) for both substrates,satcrystal growth
occurred at the metal domain boundaries only. At
International Materials Reviews 2003 Vol. 48 No. 3
220 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry

26 a–d Patterned calcite formation on SAMs of HS(CH2 )15 CO2 H supported on substrates that were
micropatterned with Au and Ag using stencil mask(TEM grid): sequence of metal deposition and the
concentration range are indicated (see text); e non-patterned nucleation of oriented calcite crystals
on SAMs of HS(CH2 )15 CO2 H supported on gold substrates, micropatterned with gold islands; f complex
ornament of crystals formed on mixed metal substrate further patterned using microcontact printing:
reprinted with permission from Ref. 220, ©2000 American Chemical Society

concentrations of 20–40 mmol LÕ 1, which corres-
ponds to conditions where C (Ag)>C
C (Au)<C , crystal growth was eff restrictedsatto the
eff sat
Ag regions only. At concentrations of >50 mmol LÕ 1,
where C (Ag) and C (Au)>C , crystal growth
occurred effover the whole
eff patterned
sat surface. When
domains of the same metal were created, no patterning
of crystals was observed. For patterned substrates
produced using the photoresist technique, the inter-
domain regions were suYciently disordered to create
patterned calcite arrays even when a single metal was
used. Combination of the mixed metal substrate and
microcontact printing of hydrophobic thiols was also
used to produce patterned crystalline arrays of more
sophisticated design.220
Deposition on CaCO 3 on surfaces functionalised by
gold colloids
Localisation of gold colloids on surfaces provides a
route for the controlled modiŽ cation of a  at gold
surface, varying aspects such as the roughness and
hydrophilicity of the surface. This was investigated
by adsorbing gold colloids to planar gold substrates,
and determining the in uence of the modiŽ ed sub-
strate on the nucleation and growth of calcium car-
bonate.223 Immersion of SAMs of HS(CH ) SH on
gold substrates into a solution of uncoated 2 12 gold
colloids resulted in adsorption of the particles onto
the substrate. The colloid modiŽ ed substrates sup-
ported the nucleation and growth of 80 : 20 aragonite–
calcite, whereas 50 : 50 calcite–vaterite deposited on
International Materials Reviews 2003 Vol. 48 No. 3
the clean gold surface at signiŽ cantly lower crystal
and densities.
Summary
While biomineralisation was historically a subject
studied by biologists, the last 10–15 years have seen
an explosion of interest in the topic, from researchers
with backgrounds ranging from physics, to chem-
istry and materials science. This has principally
derived from the recognition that biological systems
operate under ambient conditions to achieve un-
parallelled morphological control and superior mech-
anical properties. To be able to emulate such
processes in synthetic crystal growth experiments,
which under traditional methods often rely upon
high temperatures and pressures and non-aqueous
conditions, oVers clear advantages. For example,
moderate conditions oVer the potential for greater
structural control of the Ž nal product, are signiŽ -
cantly less expensive and enable the use of polymer
substrates.
In the absence of the extreme conditions available
to synthetic crystal growth experiments, organic mol-
ecules ( both as soluble organic additives and insoluble
organic frameworks) provide the fundamental mech-
anism of control over mineralisation by organisms.
As demonstrated in this review, this route has also
provided the principal basis for biologically inspired
crystal growth experiments. In terms of soluble
organic additives, superior control of particle size,
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 221
structure, polymorph and morphology has been
achieved by tailoring the structure of block copoly-
mers.141–145 Selected functionalised dendrimers have
also exhibited remarkable control over calcium car-
bonate polymorph, providing the Ž rst demonstration
of co-stabilisation of amorphous and crystalline
phases.136,137
Calcium carbonate deposition has also been carried
out on a range of organic substrates. There now exist
many examples of control over crystal orientation
and polymorph by organised organic substrates.
Precise control over orientation has been achieved
using highly ordered Langmuir monolayers, in which
the monolayer structure is locked-in using intermolec-
ular bonding,206–209 or polymerisation.204,205 Self-
assembled monolayers (SAMs) of functionalised thiols
on suitable metal substrates are also highly eVective
in determining crystal orientation, with almost 100%
orientation being achieved in some systems.212,213
SAMs are also especially valuable in that they can be
used to direct the formation of arrays of crystals,
patterned in the plane of the monolayer.220–222 Of
particular interest is also the combined eVect of
soluble organic additives and an organised insoluble
organic matrix, which has led to the deposition of
uniform, oriented calcite thin Ž lms under Langmuir
monolayers of porphyrin amphiphiles.206
An immediate diVerence between crystal growth in
biological and synthetic systems is that it occurs
within restricted volumes, rather than in bulk solu-
tion. This has inevitable eVects on crystallisation, as
demonstrated by polymorph variation on precipi-
tation within aligned and non-aligned precipitation
in collagenous matrices in the presence and absence
of additives.55,161–164 Micelles and microemulsions
oVer a constrained crystallisation volume and have
exhibited control over morphology and polymorph.
This is exempliŽ ed by the sponge-like, polycrystalline
spherical vaterite particles produced on evaporation
of water-in-oil microemulsions.168 The pores of track
etch membranes have been used to demonstrate that
crystallisation of amorphous calcium carbonate
within small volumes may be important in morpho-
logical control of the Ž nal single crystal product.172
Further, precipitation of calcium carbonate within
rigid moulds has shown that it is possible to produce
single crystals of calcite with complex, sponge-like
morphologies in the absence of soluble additives, by
external imposition of morphology only.173
The research described in this review demonstrates
that insight into the mechanisms controlling mineral-
isation in biological systems has led to signiŽ cant
advances in the control of crystal size, morphology,
polymorph and orientation in synthetic systems.
Continued research into biomineralisation processes,
such as further elucidation of key amino acid
sequences in proteins which determine polymorph
selectivity, or understanding of the role that amorph-
ous precursor phases play in determining the form of
the product crystal, will undoubtedly provide further
inspiration for routes to synthetic crystal growth
control. In addition, while calcium carbonate has
provided the focus for many biologically inspired
crystal growth experiments, it will be interesting to
investigate to what degree the same principles can be
applied to the control of the structure and properties
of a wider range of minerals, and ultimately whether
they can be integrated into the preparation of func-
tional materials.
Acknowledgements
I am most grateful to those authors who provided
me with copies of their Ž gures, which has signiŽ cantly
contributed to this article.
References
1. s. mann: Ang. Chem. Int. Ed., 2000, 39, 3392–3406.
2. s. mann, d. d. archibald, j. m. didymus, t. douglas, b. r.
heywood, f. c. meldrum and n. j. reeves: Science, 1993, 261,
1286–1292.
3. p. calvert and p. rieke: Chem. Mater., 1996, 8, 1715–1727.
4. s. i. stupp and p. v. braun: Science, 1997, 277, 1242–1248.
5. i. a. aksay, m. trau, s. manne, i. honma, n. yao, l. zhou,
p. fenter, p. m. eisenberger and s. m. gruner: Science, 1996,
273, 892–898.
6. b. c. bunker, p. c. rieke, b. j. tarasevich, a. a. campbell,
g. e. fryxell, g. l. graff, l. song, j. liu, j. w. virden and
g. l. mcvay: Science, 1994, 264, 48–55.
7. s. mann: Nature, 1993, 365, 499–505.
8. a. a. campbell, Curr. Op. Coll. Int. Sci., 1999, 4, 40–45.
9. j. d. currey: J. Exp. Biol., 1999, 202, 3285–3294.
10. m. f. ashby, l. j. gibson, u. wegst and r. olive: Proc. R. Soc.
L ond. A, 1995, 450, 123–140.
11. a. p. jackson, j. f. v. vincent and r. m. turner: Proc. R.
Soc. L ond. B, 1988, 234, 415–440.
12. a. p. jackson, j. f. v. vincent and r. m. turner: J. Mater.
Sci., 1990, 25, 3173–3178.
13. j. d. currey: Proc. R. Soc. L ond. B, 1977, 196, 443–463.
14. r. z. wang, h. b. wen, f. z. cui, h. b. zhang and h. d. li:
J. Mater. Sci., 1995, 30, 2299–2304.
15. j. d. currey, p. zioupos, p. davies and a. casinos: Proc. R.
Soc. L ond. B, 2001, 268, 107–111.
16. b. l. smith, t. e. schäffer, m. viani, j. b. thompson, n. a.
frederick, j. kindt, a. belcher, g. d. stucky, d. e. morse
and p. k. hansma: Nature, 1999, 399, 761–763.
17. f. lippmann: ‘Sedimentary carbonate minerals’; 1973, Berlin,
Springer-Verlag.
18. s. mann: J. Chem. Soc. Dalton T rans., 1993, 1–9.
19. m. a. crenshaw: in ‘Skeletal biomineralization: patterns,
processes and evolutionary trends’, Vol. 1, (ed. J. G. Carter),
1–9; 1991, New York, Van Nostrand Reinhold.
20. s. mann: J. Inorg. Biochem., 1986, 28, 363–371.
21. s. mann: Struct. Bond., 1983, 54, 125–174.
22. s. weiner and l. addadi: J. Mater. Chem., 1997, 7, 689–702.
23. s. weiner, l. addadi and h. d. wagner: Mater. Sci. Eng. C,
2000, 11, 1–8.
24. l. addadi and s. weiner: Angew. Chem. Int. Ed., 1992,
31, 153–169.
25. s. mann: ‘Biomineralization: principles and concepts in
bioinorganic materials chemistry’; 2001, Oxford, Oxford
University Press.
26. y. kojima, a. kawanobe, t. yasue and y. arai: J. Ceram. Soc.
Jpn Int. Edn, 1994, 102, 1131–1138.
27. y. kojima, a. kawanobe, t. yasue and y. arai: J. Ceram. Soc.
Jpn, 1994, 102, 1128–1136.
28. r. j. reeder: in ‘Carbonates: mineralogy and chemistry’,
Vol. 11, (ed. R. J. Reeder), 1–47; 1983, Washington, DC,
Mineralogical Society of America.
29. w. a. deer, r. a. howie and j. zussman, ‘Non-silicates’; 1974,
London, Longman.
30. j. a. speer: in ‘Carbonates: mineralogy and chemistry’, Vol.
11, (ed. R. J. Reeder), 145–190; 1983, Washington, DC,
Mineralogical Society of America.
31. w. d. carson: in ‘Carbonates: mineralogy and chemistry’,
Vol. 11, (ed. R. J. Reeder), 191–225; 1983, Washington, DC,
Mineralogical Society of America.
32. k. sawada: in ‘Crystallization processes’, Vol. 3, (ed.
H. Ohtaki), 39–68; 1999, New York, John Wiley and Sons.
33. d. söhnel and j. w. mullin: J. Cryst. Growth, 1982, 60,
239–250.
International Materials Reviews 2003 Vol. 48 No. 3
222 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
34. y. kitano, k. park and d. w. hood: J. Geophys. Res., 1962,
67, 4873–4874.
35. l. wang, i. sondi and e. matijevic: J. Coll. Int. Sci., 1999,
218, 545–553.
36. d. chakrabarty and s. mahapatra: J. Mater. Chem., 1999, 9,
2953–2957.
37. l. dupont, f. portemer and m. figlarz: J. Mater. Chem.,
1997, 7, 797–800.
38. a. j. easton and d. claugher: Mineral. Mag., 1986, 50,
332–336.
39. h. j. meyer: Z. Kristal., 1965, 121, 220–242.
40. r. brooks, l. m. clark and e. f. thurston: Philos. T rans.
Roy. Soc., 1951, A243, 145–167.
41. j. r. clarkson, t. j. price and c. j. adams: J. Chem. Soc.
Faraday T rans., 1992, 88, 243–249.
42. k. kamiya, s. sakka and k. terada: Mater. Res. Bull., 1977,
12, 1095–1102.
43. n. koga, y. nakagoe and h. tanaka: T hermochim. Acta.,
1998, 318, 239–244.
44. l. brecevic and a. e. nielsen: J. Cryst. Growth, 1989, 98,
504–510.
45. l. brecevic and a. e. nielsen: Acta Chimica Scand., 1993,
47, 668–673.
46. b. dickens and w. e. brown: Inorg. Chem., 1970, 9, 480–486.
47. y. kojima, a. kawanobe, t. yasue and y. arai: J. Ceram. Soc.
Jpn, 1993, 101, 1145–1152.
48. t. ogino, t. suzuki and k. sawada: J. Cryst. Growth, 1990,
100, 159–167.
49. s. rajam and s. mann: J. Chem. Soc. Chem. Commun., 1990,
1789–1791.
50. s. d. sims, j. m. didymus and s. mann: J. Chem. Soc. Chem.
Commun., 1995, 1031–1032.
51. a. mucci and j. w. morse: Geochim. Cosmochim. Acta, 1983,
47, 217–233.
52. a. katz: Geochim. Cosmochim. Acta, 1973, 37, 1563–1586.
53. m. m. reddy and g. h. nancollas: J. Cryst. Growth, 1976,
35, 33–38.
54. m. m. reddy and k. k. wang: J Cryst. Growth, 1980, 50,
470–480.
55. g. falini, s. fermani, m. gazzano and a. ripamonti: Chem.
Eur. J., 1997, 3, 1807–1814.
56. r. a. berner: Geochim. Cosmochim. Acta, 1975, 39, 489–504.
57. l. ferna’ndez-di’az, a. putnis, m. prieto and c. v. putnis:
J. Sedimen. Res., 1996, 66, 482–491.
58. g. h. nancollas and k. sawada: J. Petrol. T echnol., 1982,
34, 645–652.
59. g. falini, m. gazzano and a. ripamonti: Chem. Commun.,
1996, 1037–1038.
60. s. raz, s. weiner and l. addadi: Adv. Mater., 2000, 12, 38–42.
61. f. t. mackenzie, w. d. bischoff, f. c. bishop, m. loijens,
j. schoonmaker and r. wollast: in ‘Reviews in mineralogy:
carbonates: mineralogy and chemistry’, Vol. 11, (ed.
R. J. Reeder), 97–144; 1983, Washington, DC, Mineralogical
Society of America.
62. r. k. given and b. h. wilkinson: J. Sedim. Petrol., 1985,
55, 109–119.
63. l. a. gonzalez, s. j. carpenter and k. c. lohmann: J. Sedim.
Petrol., 1992, 62, 382–399.
64. r. d. cody: J. Sedim. Petrol., 1991, 61, 704–718.
65. n. wada, m. okazaki and s. tachikawa: J. Cryst. Growth,
1993, 132, 115–121.
66. j. w. mccauley and r. roy: Am. Min., 1974, 59, 947–963.
67. d. m. devery and a. j. ehlmann: Am. Min., 1981, 66, 592–595.
68. e. d. glover and r. f. sippel: Geochim. Cosmochim. Acta.,
1967, 31, 603–613.
69. y. kitano and n. kanamori: Geochim. J., 1966, 1, 1–10.
70. g. falini, m. gazzano and a. ripamonti: J. Cryst. Growth,
1994, 137, 577–584.
71. f. c. meldrum and s. hyde: J. Cryst. Growth, 2001, 231,
544–558.
72. j. o. titiloye, s. c. parker and s. mann: J. Cryst. Growth,
1993, 131, 533–545.
73. s. albeck, j. aizenberg, l. addadi and s. weiner: J. Am.
Chem. Soc., 1993, 115, 11691–11697.
74. g. falini, s. fermani, m. gazzano and a. ripamonti: J. Mater.
Chem., 1998, 8, 1061–1065.
75. m. prieto, a. putnis, l. ferna’ndez-di’az and s. lo’pez-andre’s:
J. Cryst. Growth, 1994, 142, 225–235.
76. s. l. tracy, c. j. p. françois and h. m. jennings: J. Cryst.
Growth, 1998, 193, 374–381.
International Materials Reviews 2003 Vol. 48 No. 3
77. j. ferguson, b. bubela and p. j. davies: Chem. Geol., 1978,
22, 285–308.
78. s. weiner and l. hood: Science, 1975, 190, 987–989.
79. a. p. wheeler, k. c. low and c. s. sikes: in ‘Surface reactive
peptides and polymers’, Vol. 444, (ed. C. S. Sikes and A. P.
Wheeler), 72–84; 1991, Washington, DC, ACS.
80. g. k. hunter: Curr. Op. Solid State and Int. Sci., 1996,
1, 430–435.
81. a. p. wheeler, j. w. george and c. a. evans: Science, 1981,
212, 1397–1398.
82. a. p. wheeler and c. s. sikes: Am. Zool., 1984, 24, 933–944.
83. l. addadi and s. weiner: Proc. Natl Acad. Sci., 1985, 82,
4110–4114.
84. l. addadi and s. weiner: Mol. Cryst. L iq. Cryst., 1986,
134, 305–322.
85. s. weiner and w. traub: Phil. T rans. R. Soc. L ond. B, 1984,
304, 425–434.
86. s. weiner and w. traub: FEBS L ett., 1980, 111, 311–316.
87. d. worms and s. weiner: J. Exp. Zool., 1986, 237, 11–20.
88. s. manne, c. m. zaremba, r. giles, l. huggins, d. a. walters,
a. belcher, d. e. morse, g. d. stucky, j. m. didymus, s. mann
and p. k. hansma: Proc. R. Soc. L ond. B, 1994, 256, 17–23.
89. t. e. schäffer, c. ionescu-zanetti, r. proksch, m. fritz,
d. a. walters, n. almqvist, c. m. zaremba, a. m. belcher,
b. l. smith, g. d. stucky, d. e. morse and p. k. hansma:
Chem. Mater., 1997, 9, 1731–1740.
90. m. fritz, a. m. belcher, m. radmacher, d. a. walters, p. k.
hansma, g. d. stucky, d. e. morse and s. mann: Nature, 1994,
371, 49–51.
91. c. m. zaremba, a. m. belcher, m. fritz, y. li, s. mann, p. k.
hansma, d. e. morse, j. s. speck and g. d. stucky: Chem.
Mater., 1996, 8, 679–690.
92. a. m. belcher, x. h. wu, r. j. christensen, p. k. hansma,
g. d. stucky and d. e. morse: Nature, 1996, 381, 56–58.
93. g. falini, s. albeck, s. weiner and l. addadi: Science, 1996,
271, 67–69.
94. y. levi, s. albeck, a. brack, s. weiner and l. addadi: Chem.
Eur. J., 1998, 4, 389–396.
95. k. okazaki and s. inoue: Dev. Growth DiVer., 1976, 18,
413–434.
96. j. r. young, s. a. davis, p. r. brown and s. mann: J. Struct.
Biol., 1999, 126, 195–215.
97. p. dubois and c.-p. chen: in ‘Echinoderm studies’, (ed.
M. Jangoux and J. M. Lawrence); 1989, Rotterdam/
BrookŽ eld, A. A. Balkema.
98. j. m. didymus, p. oliver, s. mann, a. l. devries, p. v. hauschka
and p. westbroek: J. Chem. Soc. Faraday T rans., 1993, 89,
2891–2000.
99. k. m. mcgrath: Adv. Mater., 2001, 13, 989–992.
100. s. albeck, l. addadi and s. weiner: Connec. T issue Res.,
1996, 35, 365–370.
101. j. aizenberg, j. hanson, m. ilan, l. leiserowtiz, t. f. koetzle,
l. addadi and s. weiner: FASEB J., 1995, 9, 262–268.
102. a. berman, l. addadi and s. weiner: Nature, 1988, 331,
546–548.
103. j. aizenberg, s. albeck, s. weiner and l. addadi: J. Cryst.
Growth, 1994, 142, 156–164.
104. a. albeck, s. weiner and l. addadi: Chem. Eur. J., 1996,
2, 278–284.
105. j. aizenberg, m. ilan, s. weiner and l. addadi: Connec. T iss.
Res., 1996, 34, 225–261.
106. r. giles, s. manne, s. mann, d. e. morse, g. d. stucky and
p. k. hansma: Biol. Bull., 1995, 188, 8–15.
107. d. a. walters, b. l. smith, a. m. belcher, g. t. paloczi, g. d.
stucky, d. e. morse and p. k. hansma: Biophys. J., 1997, 72,
1425–1433.
108. j. b. thompson, g. t. paloczi, j. h. kindt, m. michenfelder,
b. l. smith, g. stucky, d. e. morse and p. k. hansma:
Biophys. J., 2000, 79, 3307–3312.
109. a. wierzbicki, c. s. sikes, j. d. madura and b. drake: Calcif.
T issue Int., 1994, 54, 133–141.
110. c. a. orme, a. noy, a. wierzbicki, m. t. mcbride, m. grantham,
h. h. teng, p. m. dove and j. j. deyoreo: Nature, 2001,
411, 775–779.
111. j. weber, r. greer, b. voight, e. white and r. roy:
J. Ultrastruc. Res., 1969, 26, 355–366.
112. j. d. currey: J. Mar. Biol. Assoc. UK, 1975, 55, 419–424.
113. a. burkhardt, w. hansmann, k. märkel and h.-j. niemann:
Zoomorph., 1983, 102, 189–203.
 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry 223
114. r. b. emlet: Biol. Bull., 1982, 163, 264–275.
115. a. b. smith: in ‘Skeletal biomineralization: patterns, processes
and evolutionary trends’, Vol. 1, (ed. J. G. Carter), 413–443;
1991, New York, Van Nostrand Reinhold.
116. j. aizenberg, j. hanson, t. f. koetzle, s. weiner and
l. addadi: J. Am. Chem. Soc., 1997, 119, 881–886.
117. j. aizenberg, j. hanson, t. f. koetzle, l. leiserowtiz,
s. weiner and l. addadi: Chem. Eur. J., 1995, 1, 414–422.
118. a. berman, j. hanson, l. leiserowtiz, t. f. koetzle, s. weiner
and l. addadi: Science, 1993, 259, 776–779.
119. a. berman, j. hanson, l. leiserowtiz, t. f. koetzle, s. weiner
and l. addadi: J. Phys. Chem., 1993, 97, 5162–5170.
120. a. ziegler: J. Struct. Biol., 1994, 112, 110–116.
121. m. e. marsh: Protoplasma, 1994, 177, 108–122.
122. e. beniash, j. aizenberg, l. addadi and s. weiner: Proc. R.
Soc. L ond. B, 1997, 264, 461–465.
123. y. levi-kalisman, s. raz, s. weiner, l. addadi and i. sagi:
J. Chem. Soc. Dalton T rans., 2000, 3977–3982.
124. m. g. taylor, k. simkiss, g. n. greaves, m. okazaki and
s. mann: Proc. R. Soc. L ond. B, 1993, 252, 75–80.
125. j. aizenberg, g. lambert, l. addadi and s. weiner: Adv.
Mater., 1996, 8, 222–226.
126. e. beniash, l. addadi and s. weiner: J. Struct. Biol., 1999,
125, 50–62.
127. l. ameye, g. d. becker, c. killian, f. wilt, r. kemps,
s. kuypers and p. dubois: J. Struct. Biol., 2001, 134, 56–66.
128. h. a. lowenstam and s. weiner: ‘On biomineralization’; 1989,
Oxford, Oxford University Press.
129. j. r. goldsmith, d. l. graf and o. i. joensuu: Geochim.
Cosmochim. Acta., 1995, 7, 212–230.
130. k. e. chave: J. Geol., 1954, 62, 266–283.
131. k. e. chave: J. Geol., 1954, 62, 587–599.
132. j. n. weber: J. Geol., 1973, 81, 543–556.
133. w. bischoff, f. t. mackenzie and f. c. bishop: Geochim.
Cosmochim. Acta., 1987, 51, 1413–1423.
134. s. mann, j. m. didymus, n. p. sanderson, b. r. heywood and
e. j. a. samper: J. Chem. Soc. Faraday T rans., 1990, 86,
1873–1880.
135. d. b. deoliveira and r. a. laursen: J. Am. Chem. Soc., 1997,
119, 10627–10631.
136. k. naka, y. tanaka, y. chujo and y. ito: Chem. Commun.,
1999, 1931–1932.
137. j. j. j. m. donners, b. r. heywood, e. w. meijer, r. j. m.
nolte, c. roman, a. p. h. j. schenning and n. a. j. m.
sommerdijk: Chem. Commun., 2000, 1937–1938.
138. j. j. j. m. donners, b. r. heywood, e. w. meijer, r. j. m. nolte
and n. a. j. m. sommerdijk: Chem. Eur. J., 2002, 8, 2561–2567.
139. l. a. gower and d. a. tirrell: J. Cryst. Growth, 1998,
191, 153–160.
140. l. b. gower and d. j. odom: J. Cryst. Growth, 2000, 210,
719–734.
141. h. cölfen and m. antionetti: L angmuir, 1998, 14, 582–589.
142. h. cölfen and l. qi: Chem. Eur. J., 2001, 7, 106–116.
143. m. sedla’k, m. antionetti and h. cölfen: Macromol. Chem.
Phys., 1998, 199, 247–254.
144. m. sedla’k and h. cölfen: Macromol. Chem. Phys., 2001,
202, 587–597.
145. j. m. marentette, j. norwig, e. stöckelmann, w. h. meyer
and g. wegner: Adv. Mater., 1997, 9, 647–651.
146. m. antionetti, m. breulmann, c. g. göltner, h. cölfen,
k. k. wong, d. walsh and s. mann: Chem. Eur. J., 1998, 4,
2493–2500.
147. w. j. feast, f. cacialli, r. daik, r. h. friend, e. herzog,
b. r. heywood, l. hobson, j. l. megson and d. snowden:
Macromol. Symp., 1999, 143, 81–93.
148. k. naka, d. k. keum, y. tanaka and y. chujo: Chem. Commun.,
2000, 1537–1538.
149. l. pach, z. hrabe, s. komarneni and r. roy: J. Mater. Res.,
1990, 5, 2928–2932.
150. l. pach, s. duncan, r. roy and s. komarneni: J. Mater. Sci.,
1996, 31, 6565–6569.
151. h. k. henisch: ‘Crystals in gels and Liesegang rings’; 1988,
Cambridge, Cambridge University Press.
152. h. j. nickl and h. k. henisch: J. Electrochem. Soc., 1969, 116,
1258–1260.
153. s. dominguez-bella and j. m. garcia-ruiz: J. Cryst. Growth,
1986, 79, 236–240.
154. s. dominguez-bella and j. m. garcia-ruiz: J. Mater. Sci.,
1987, 22, 3095–3102.
155. j. m. garcia-ruiz: J. Cryst. Growth, 1985, 73, 251–262.
156. m. prieto, j. m. garcia-ruiz and j. l. amoro’s: J. Cryst.
Growth, 1981, 52, 864–867.
157. j. m. garzia–ruiz and j. l. amoro’s: Bull. Mineral., 1981,
104, 107–113.
158. n. wada, k. yamashita and t. umegaki: J. Cryst. Growth,
1995, 148, 297–304.
159. b. d. ricketts: J. Sedimentary Petrol., 1980, 50, 963–969.
160. n. wada, k. yamashita and t. umegaki: J. Coll. Int. Sci., 1999,
212, 357–364.
161. g. falini: Int. J. Inorg. Mat., 2000, 2, 455–461.
162. g. falini, s. fermani, m. gazzano and a. ripamonti: Chem.
Eur. J., 1998, 4, 1048–1052.
163. g. falini, s. fermani, m. gazzano and a. ripamonti: J. Chem.
Soc. Dalton T rans., 2000, 3983–3987.
164. g. falini, m. gazzano and a. ripamonti: Adv. Mater., 1994,
6, 46–48.
165. k. kandori, k. kon-no and a. kitahara: J. Coll. Int. Sci.,
1987, 115, 579–582.
166. k. kandori, k. kon-no and a. kitahara: J. Coll. Int. Sci.,
1988, 122, 78–82.
167. m. l. rock, l. j. tranchitella and r. s. pilato: Colloid
Polym. Sci., 1997, 275, 893–896.
168. d. walsh, b. lebeau and s. mann: Adv. Mater., 1999, 11,
324–328.
169. d. walsh and s. mann: Nature, 1995, 377, 320–323.
170. t. hirai, s. hariguchi, i. komasawa and r. j. davey: L angmuir,
1997, 13, 6650–6653.
171. r. j. davey and t. hirai: J. Cryst. Growth, 1997, 171, 318–320.
172. e. loste and f. c. meldrum: Chem. Commun., 2001, 10,
901–902.
173. r. j. park and f. c. meldrum: Adv. Mater., 2002, 14,
1167–1169.
174. f. manoli, s. koutsopoulos and e. dalas: J. Cryst. Growth,
1997, 182, 116–124.
175. t. kato, t. suzuki and t. irie: Chem. L ett., 2000, 186–187.
176. n. hosoda and t. kato: Chem. Mater., 2001, 13, 688–693.
177. s. zhang and k. e. gonsalves: J. Appl. Polym. Sci., 1995,
56, 687–695.
178. a. sugawara and t. kato: Chem. Commun., 2000, 487–488.
179. t. kato: Adv. Mater., 2000, 12, 1543–1546.
180. t. kato, t. suzuki, t. amamiya and t. irie: Supramol. Sci,
1998, 5, 411–415.
181. j. kanakis and e. dalas: J. Cryst. Growth, 2000, 219, 277–282.
182. e. dalas, p. g. klepetsanis and p. g. koutsoukos: J. Coll. Int.
Sci., 2000, 224, 56–62.
183. f. manoli and e. dalas: J. Cryst. Growth, 1999, 204, 369–375.
184. t. kato and t. amamiya: Chem. L ett., 1999, 199–200.
185. e. dalas, p. klepetsanis and p. g. koutsoukos: L angmuir,
1999, 15, 8322–8327.
186. y. tanaka, t. nemoto, k. naka and y. chujo: Polymer Bull.,
2000, 45, 447–450.
187. l. addadi, j. moradian, e. shay, n. g. maroudis and s. weiner:
Proc. Natl Acad. Sci., 1987, 84, 2732–2736.
188. b. j. brisdon, b. r. heywood, a. g. w. hodson, s. mann and
k. k. w. wong: Adv. Mater., 1993, 5, 49–51.
189. k. k. w. wong, b. j. brisdon, b. r. heywood, a. g. w. hodson
and s. mann: J. Mater. Chem., 1994, 4, 1387–1392.
190. s. m. d’souza, c. alexander, s. w. carr, a. m. waller, m. j.
whitcome and e. n. vulfson: Nature, 1999, 398, 312–316.
191. s. m. d’souza, c. alexander, m. j. whitcome, a. m. waller
and e. n. vulfson: Polymer Int., 2001, 50, 429–432.
192. e. yilmaz, k. haupt and k. mosbach: Angew. Chem. Int. Ed.,
2000, 39, 2115–2118.
193. k. haupt and k. mosbach: Chem. Rev., 2000, 100, 2495–2504.
194. b. r. heywood and s. mann: Adv. Mater., 1994, 6, 9–20.
195. b. r. heywood: in ‘Biomimetic materials chemistry’, (ed.
S. Mann), 143–173; 1996, New York, VCH.
196. s. mann, b. r. heywood, s. rajam and d. birchall: Nature,
1988, 334, 692–695.
197. s. mann, b. r. heywood, s. rajam, j. b. a. walker, r. j. davey
and d. birchall: Adv. Mater., 1990, 2, 257–261.
198. s. mann, b. r. heywood, s. rajam and j. b. a. walker: J. Phys.
D: Appl. Phys., 1991, 24, 154–164.
199. s. rajam, b. r. heywood, j. b. a. walker, s. mann, r. j. davey
and j. d. birchall: J. Chem. Soc. Faraday T rans., 1991,
87, 727–734.
200. b. r. heywood, s. rajam and s. mann: J. Chem. Soc. Faraday
T rans., 1991, 87, 735–743.
International Materials Reviews 2003 Vol. 48 No. 3
224 Meldrum Calcium carbonate in biomineralisation and biomimetic chemistry
201. b. r. heywood and s. mann: Chem. Mater., 1994, 6, 311–318.
202. a. l. litvin, l. a. samuelson, d. h. charych, w. spevak and
d. l. kaplan: J. Phys. Chem., 1995, 99, 12065–12068.
203. a. l. litvin, l. a. samuelson, d. h. charynch, w. spevak and
d. l. kaplan: J. Phys. Chem., 1995, 99, 492–495.
204. a. berman, d. j. ahn, a. lio, m. salmeron, a. reichert and
d. charych: Science, 1995, 269, 515–518.
205. a. berman and d. charych: J. Cryst. Growth, 1999, 198/199,
796–801.
206. g. xu, n. yao, i. a. aksay and j. t. groves: J. Am. Chem. Soc.,
1998, 120, 11977–11985.
207. j. lahiri, g. xu, d. m. dabbs, n. yao, i. a. aksay and j. t.
groves: J. Am. Chem. Soc., 1997, 119, 5449–5450.
208. s. champ, j. a. dickinson, p. s. fallon, b. r. heywood and
m. mascal: Angew. Chem. Int. Ed., 2000, 39, 2716–2719.
209. p. j. j. a. buijnsters, j. j. j. m. donners, s. j. hill, b. r.
heywood, r. j. m. nolte, b. zwanenburg and n. a. j. m.
sommerdijk: L angmuir, 2001, 17, 3623–3628.
210. a. l. litvin, s. valiyaveettil, d. l. kaplan and s. mann: Adv.
Mater., 1997, 9, 124–127.
211. d. d. archibald, s. b. qadri and b. p. gaber: L angmuir, 1996,
12, 538–546.
International Materials Reviews 2003 Vol. 48 No. 3
212. j. aizenberg, a. j. black and g. m. whitesides: J. Am. Chem.
Soc., 1999, 121, 4500–4509.
213. a. m. travaille, j. j. j. m. donners, j. w. gerritsen, n. a. j. m.
sommerdijk, r. j. m. nolte and h. van-kempen: Adv. Mater.,
2002, 14, 492–495.
214. j. küther, g. nelles, r. seshadri, m. schaub, h. j. butt and
w. tremel: Chem. Eur. J., 1998, 4, 1834–1842.
215. j. küther and w. tremel: T hin Solid Films, 1998, 327–329,
554–558.
216. j. küther, r. seshadri, w. knoll and w. tremel: J. Mater.
Chem., 1998, 8, 641–650.
217. j. küther and w. tremel: Chem. Commun., 1997, 2029–2030.
218. j. küther, r. seshadri, g. nelles, w. assenmacher, h. j.
butt, w. mader and w. tremel: Chem. Mater., 1999, 11,
1317–1325.
219. j. küther, r. seshadri and w. tremel, Angew. Chem. Int. Ed.,
1998, 37, 3044–3047.
220. j. aizenberg: J. Chem. Soc. Dalton T rans., 2000, 3963–3968.
221. j. aizenberg: J. Cryst. Growth, 2000, 211, 143–148.
222. j. aizenberg, a. j. black and g. m. whitesides: Nature, 1999,
398, 495–498.
223. j. küther, r. seshadri, g. nelles, h. j. butt, w. knoll and
w. tremel: Adv. Mater., 1998, 10, 401–404.