Biological and Medical Significance of Calcium Phosphate

Abstract

The inorganic part of hard tissues (bones and teeth) of mammals consists of calcium phosphate, mainly of apatitic structure. Similarly, most undesired calcifications (i.e. those appearing as a result of various diseases) of mammals also contain calcium phosphate. For example, atherosclerosis results in blood-vessel blockage caused by a solid composite of cholesterol with calcium phosphate. Dental caries result in a replacement of less soluble and hard apatite by more soluble and softer calcium hydrogenphosphates. Osteoporosis is a demineralization of bone. Therefore, from a chemical point of view, processes of normal (bone and teeth formation and growth) and pathological (atherosclerosis and dental calculus) calcifications are just an in vivo crystallization of calcium phosphate. Similarly, dental caries and osteoporosis can be considered to be in vivo dissolution of calcium phosphates. On the other hand, because of the chemical similarity with biological calcified tissues, all calcium phosphates are remarkably biocompatible. This property is widely used in medicine for biomaterials that are either entirely made of or coated with calcium phosphate. For example, self-setting bone cements made of calcium phosphates are helpful in bone repair and titanium substitutes covered with a surface layer of calcium phosphates are used for hip-joint endoprostheses and tooth substitutes, to facilitate the growth of bone and thereby raise the mechanical stability. Calcium phosphates have a great biological and medical significance and in this review we give an overview of the current knowledge in this subject.

Full text

1. Introduction
Calcium phosphates are the most important inorganic
constituents of biological hard tissues. In the form of
carbonated hydroxyapatite (HA), they are present in bone,
teeth, and tendons to give these organs stability, hardness, and
function. Calcium phosphate crystals are also found in ™dead∫
nature as mineral deposits of considerable size, having grown
over many years under sometimes extreme conditions of
pressure and temperature. In contrast, biologically formed
calcium phosphates are often nanocrystals that are precipi-
tated under mild conditions (ambient pressure, near room
temperature).
The biological formation of minerals by living organisms is
commonly called ™biomineralization∫.[1±9] Today more than
60 minerals are known that are used by organisms, for
example, for protection (shell), as tools (teeth), as gravity
sensors (octoconia or statoliths), or as a skeleton. In terms of
absolute quantity, calcium phosphates are minor compared to
calcium carbonate (CaCO3) and silicon dioxide (as silicic acid
SiO2¥nH2O), which both occur in huge amounts in marine
single-cell organisms. Another very important class of bio-
minerals are iron oxides that occur, for example, in snail teeth
or magnetotactic bacteria.[1] The presence of calcium phos-
phates in vertebrates (such as humans) makes them partic-
ularly important in biomedicine, as many diseases result from
irregularities in the skeletal system (i.e. in bone) or the dental
system (in teeth). It must also be stressed that, although the
presence of calcium phosphate in these hard tissues is crucial
for survival, there are occasions on which calcium phosphate
minerals crystallize in an irregular way in undesired regions.
These phenomena are called pathological crystallization or
ectopic mineralization, of which atherosclerosis, stone for-
mation, or dental calculus are prominent examples.
Herein, we give an overview of the occurrence, formation,
and significance of calcium phosphate minerals in living
organisms, with a special emphasis on current biomedical
questions.
Biological and Medical Significance of Calcium Phosphates
Sergey V. Dorozhkin and Matthias Epple*
Dedicated to Professor Sir John Meurig Thomas on the occasion of his 70th birthday
The inorganic part of hard tissues
(bones and teeth) of mammals consists
of calcium phosphate, mainly of apa-
titic structure. Similarly, most unde-
sired calcifications (i.e. those appear-
ing as a result of various diseases) of
mammals also contain calcium phos-
phate. For example, atherosclerosis
results in blood-vessel blockage caused
by a solid composite of cholesterol
with calcium phosphate. Dental caries
result in a replacement of less soluble
and hard apatite by more soluble and
softer calcium hydrogenphosphates.
Osteoporosis is a demineralization of
bone. Therefore, from a chemical point
of view, processes of normal (bone and
teeth formation and growth) and
pathological (atherosclerosis and den-
tal calculus) calcifications are just an
in vivo crystallization of calcium phos-
phate. Similarly, dental caries and
osteoporosis can be considered to be
in vivo dissolution of calcium phos-
phates. On the other hand, because of
the chemical similarity with biological
calcified tissues, all calcium phosphates
are remarkably biocompatible. This
property is widely used in medicine
for biomaterials that are either entirely
made of or coated with calcium phos-
phate. For example, self-setting bone
cements made of calcium phosphates
are helpful in bone repair and titanium
substitutes covered with a surface layer
of calcium phosphates are used for hip-
joint endoprostheses and tooth substi-
tutes, to facilitate the growth of bone
and thereby raise the mechanical sta-
bility. Calcium phosphates have a great
biological and medical significance and
in this review we give an overview of
the current knowledge in this subject.
Keywords: bioinorganic chemistry ¥
biomaterials ¥biomimetic synthesis ¥
biomineralization ¥materials science
[*] Prof. Dr. M. Epple, Dr. S. V. Dorozhkin
Solid-State Chemistry, Faculty of Chemistry
University of Bochum
Universit‰tsstrasse 150, 44780 Bochum (Germany)
Fax : ( 49)234-321-4558
E-mail: matthias.epple@ruhr-uni-bochum.de
REVIEWS
Angew. Chem. Int. Ed. 2002,41, 3130± 3146 ¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1433-7851/02/4117-3131 $ 20.00+.50/0 3131

REVIEWS M. Epple and S. V. Dorozhkin
2. Geological and Biological Occurrence
Calcium and phosphorus are widely distributed elements
on our planet. The surface layer of the Earth contains about
3.4 wt% of calcium and 0.10 wt % of phosphorus.[10] Combi-
nations of oxides of these two elements with or without
incorporation of water give different calcium phosphates.
Unless doped with a colored transition-metal ion (often the
case in nature), all calcium phosphates are white solids. Most
calcium phosphates are only sparingly soluble in water, and
some can be considered to be insoluble, but all dissolve in
acids. Ortho- (PO43), pyro- (P2O74), and poly- ((PO3)nn)
phosphates can be structurally distinguished. Although cal-
cium pyrophosphates occur in some pathological calcifica-
tions, only calcium orthophosphates will be considered here.
They are the major component of all human calcified tissues,
and natural calcium orthophosphates are the source for
phosphorus-containing fertilizers.[11±14]
Geologically, natural calcium orthophosphates are found in
different regions to fluoroapatite deposits, Ca10(PO4)6F2,or
phosphorites. Most geological environments contain calcium
phosphates, usually as accessory minerals (<5%). In some
sedimentary rocks (phosphorites) and rarely in igneous
segregations (fluoroapatite), the concentration is high enough
to permit an economic use. The largest world deposits of
natural phosphate rock are located in Morocco, Russia,
Kazakhstan, and the USA (Florida, Tennessee).[11±14] Most
natural calcium phosphates occur as small polycrystals. Larger
crystals usually have the crystal structure of apatites (hex-
agonal system, space group P63/m, or monoclinic system,
space group P21/b). None of these crystals are pure com-
pounds; they are always admixtures of other elements. For
example, calcium ions may be partially replaced by Sr, Ba,
Mg, K, Na, Fe; phosphate ions may be replaced by AsO43,
CO32, and VO43; hydroxide, chloride, bromide, carbonate,
and oxide ions may substitute fluoride ions in the crystal
lattice. Moreover, some ions in the crystal structure may be
missing, which leaves crystallographic defects. This leads to
the formation of nonstoichiometric compounds. Figure 1
shows polycrystalline and single-crystalline calcium phos-
phate minerals.
The major industrial application of calcium phosphate
minerals is in the production of agricultural fertilizers. Natural
calcium phosphates that are used for fertilizer production can
be of geological or of biological origin for example, guano
Figure 1. Polycrystalline (a) and single-crystalline (b) fluoroapatite (chem-
ical formula: Ca10(PO4)6F2) of geological origin. The single crystal has a
grey ± green color caused by incorporated transition metals.
3132 Angew. Chem. Int. Ed. 2002,41, 3130 ± 3146
Matthias Epple studied chemistry at the Technical University of
Braunschweig and obtained his Ph.D. in 1992 (Prof. H. K.
Cammenga). In 1993, he held a postdoctoral position at the
University of Washington (Seattle, Prof. J. C. Berg). From 1994
to 1997 he worked on his Habilitation in the group of Prof.
Reller at the University of Hamburg, interrupted by research
stays at the Royal Institution in London with Prof. J. M.
Thomas. In 1998, he received the Heinz-Maier-Leibnitz prize
and a Heisenberg stipend from the DFG. Since 2000, he has
been Professor of Inorganic Chemistry at the University of
Bochum. His research interests include the reactivity of solids,
molecular crystals, synchrotron radiation, biomaterials, and
biomineralization. He has authored more than 90 publications
in these fields. He is also a member of the advisory board for synchrotron radiation at the German Electron Synchrotron
Facility (DESY, Hamburg) and a member of the board of the Ruhr Competence Center of Medical Technology.
Dr. Sergey V. Dorozhkin studied chemistry and chemical engineering at the Moscow Institute of Chemical Technology,
Moscow, USSR. Later he joined the Research Institute of Fertilizers (Moscow, USSR) where he worked on the dissolution
mechanism of natural fluoroapatite. He received his Ph.D. degree under the supervision of Prof. Igor V. Melikhov
(Chemistry Department of the M. V. Lomosonov Moscow State University, Russia) in 1992. Since that he has worked as a
post-doctoral researcher on calcium phosphates and biomaterials at the Universities of Strasburg and Nantes (both France),
as well as at the University of Aveiro (Portugal). From 2000 to 2002 he was a post-doctoral researcher on biomineralization
at the Department of Solid-State Chemistry of the University of Bochum (Germany).
S. V. DorozhkinM. Epple

REVIEWS
Biomineralization of Calcium Phosphates
(mineralized excrements of birds, accumulated over thou-
sands of years, e.g. in the South Sea at Nauru, Banaba, and
Makatea). On the 21 km2island of Nauru, about 2 million tons
of fertilizers are mined every year, which is leading to severe
ecological problems. The total capacity of industrial plants in
the world exceeds 25 million tons of phosphate fertilizers per
year (as P2O5).[12]
In biological systems, calcium orthophosphates occur as the
principal inorganic constituent of normal (bones, teeth, fish
enameloid, and some species of shells) and pathological
(dental and urinary calculus and stones, atherosclerotic
lesions) calcifications.[15±18] Structurally, they occur mainly in
the form of poorly crystallized nonstoichiometric sodium-,
magnesium-, and carbonate-containing HA (often called
™biological apatite∫ or dahllite). The main constituents of
human bones are calcium orthophosphates (50 ± 60 wt %),
collagen (30 ± 40 wt %), and water (10 wt %). In micro-
scopic studies of the interface between implanted calcium
phosphate biomaterials and the host bone, poorly crystallized
nonstoichiometric carbonated apatite similar to that of bone
apatite was found.[19±21] Detailed information on the chemical
composition of the most important human normal calcified
tissues is given in Table 1. Figure 2 shows a picture of a
calcined bone, that is, only the calcium phosphate skeleton,
after burning off all organic components.
As a variety of stoichiometric calcium phosphates is known,
abbreviations have traditionally been introduced to distin-
guish between the different compounds. Important parame-
ters are the molar Ca/P ratio and the solubility. Table 2
presents the known calcium phosphate phases. For the
chemically pure compounds, the Ca/P ratio can be between
0.5 ±2.0. In general, the lower this ratio, the more acidic and
soluble in water the calcium phosphate is (see ref. [22] for the
apparent solubility of these phases as a function of pH value
and calcium concentration). A brief description of all calcium
orthophosphates is given below. Table 3 contains their crys-
tallographic data.
Figure 2. Calcined porous bone (spongiosa) showing the high porosity and
the interconnecting network of pores (magnification : 20.4 ).
MCPM (monocalcium phosphate monohydrate, Ca(H2-
PO4)2¥H
2O) is the most acidic and water-soluble calcium
phosphate compound. It precipitates from highly acidic
solutions that are normally used in the industrial production
of phosphorus-containing fertilizer (™triple superphos-
phate∫).[12] At temperatures above 100 8C, it transforms into
MCPA (monocalcium phosphate anhydrate, Ca(H2PO4)2).
Because of its comparatively high acidity and solubility, MCPM
is never found in biological calcifications. However, MCPM is
used in some calcium phosphate cements in medicine.[23±27]
Other applications are as antacids, acidulents, and mineral
supplements for baking powders, foods, and beverages.[28]
MCPA is the anhydrous form of MCPM. It crystallizes
under similar conditions as MCPM but at temperatures above
1008C (e.g. from highly concentrated mother liquors in
fertilizer production). Like MCPM, MCPA never appears in
calcified tissues, and there is no current application in
medicine; it is mainly used as a fertilizer.[12, 28]
DCPD (dicalcium phosphate dihydrate, CaHPO4¥2H
2O;
the mineral brushite) can be easily crystallized from aqueous
Angew. Chem. Int. Ed. 2002,41, 3130 ± 3146 3133
Table 1. Comparative composition and structural parameters of inorganic phases of adult-human calcified tissues.[a][15, 21]
Composition Enamel Dentin Bone Hydroxyapatite (HA)
calcium [wt%][b] 36.5 35.1 34.8 39.6
phosphorus (as P) [wt %][b] 17.7 16.9 15.2 18.5
Ca/P (molar ratio)[b] 1.63 1.61 1.71 1.67
sodium [wt%][b] 0.5 0.6 0.9 ±
magnesium [wt%][b] 0.44 1.23 0.72 ±
potassium [wt%][b] 0.08 0.05 0.03 ±
carbonate (as CO32) [wt%][c] 3.5 5.6 7.4 ±
fluoride [wt %][b] 0.01 0.06 0.03 ±
chloride [wt%][b] 0.30 0.01 0.13 ±
pyrophosphate,(as P2O74) [wt%][c] 0.022 0.10 0.07 ±
total inorganic [wt %][c] 97 70 65 100
total organic [wt %][c] 1.5 20 25 ±
water [wt%][c] 1.5 10 10 ±
aaxis [ä][d] 9.441 9.421 9.41 9.430
caxis [ä][d] 6.880 6.887 6.89 6.891
crystallinity index, (HA 100) 70 ± 75 33 ± 37 33 ± 37 100
typical crystal sizes [nm][1, 105, 107] 100 50 50mm3525 45025 4 200 ± 600
ignition products (8008C) b-TCP HA b-TCP HA HA CaO HA
elasticity modulus (GPa)[261] 80 15 0.34 ± 13.8 10
compressive strength (MPa) 10 100 150 100
[a] Because of the considerable variation found in biological samples, typical values are given in these cases. [b] Ashed samples. [c] Unashed samples.
[d] Lattice parameters: 0.003 ä.

REVIEWS M. Epple and S. V. Dorozhkin
solutions. DCPD transforms into dicalcium phosphate anhy-
drate at temperatures above 808C. DCPD is of biological
importance because it is often found in pathological calcifi-
cations (dental calculi, crystalluria, chondrocalcinosis,[15±17]
and urinary stones[18] ). DCPD has been proposed as an
intermediate in both bone mineralization and dissolution of
enamel in acids (dental caries).[15±18] In surgery, DCPD is used
in calcium phosphate cements[27, 29±34] and, in dentistry, in
toothpaste together with fluoride-containing compounds (e.g.
NaF) for protection against caries.[35±38] Other applications are
in fertilizers,[12] glass production, calcium supplements in
foods, and mineral supplements in cereals.[28]
DCPA (dicalcium phosphate anhydrate, CaHPO4; the
mineral monetite) is the anhydrous form of DCPD. DCPA,
like DCPD, can be crystallized from aqueous solutions but at
1008C. Unlike DCPD, DCPA occurs in neither normal nor
pathological calcifications. It is used in calcium phosphate
cements,[33, 39±44] and other applications are as polishing agents,
sources of calcium and phosphate in nutritional supplements,
tabletting aids, and toothpaste components.[28]
OCP (octacalcium phosphate, Ca8(HPO4)2(PO4)4¥5H
2O) is
often found as an intermediate phase during the precipitation
of the thermodynamically more stable calcium phosphates
(e.g. HA, calcium-deficient HA (CDHA)) from aqueous
solutions. OCP consists of apatitic layers (with atomic
arrangements of calcium and phosphate ions similar to those
of HA) separated by hydrated layers (water molecules). OCP
is of great biological importance because it is one of the stable
components of human dental and urinary calculi.[45±47] It plays
an important role in the in vivo formation of apatitic
biominerals. A ™central OCP inclusion∫ (also known as
™central dark line∫) is seen by transmission electron micro-
scopy in many biological apatites and in some synthetically
precipitated HA (see below for a detailed discussion).[48±51]
Although OCP has not been observed in vascular calcifica-
tions, it has been strongly suggested as the precursor phase to
3134 Angew. Chem. Int. Ed. 2002,41, 3130 ± 3146
Table 2. Properties of the biologically relevant calcium orthophosphates.[a][103, 104]
Ca/P
ratio
Compound Formula Solubility at
258C, log(Ksp)
Solubility at
378C, log(Ksp)
pH stability
range in aqueous
solution at 258C
0.5 monocalcium phosphate monohydrate (MCPM) Ca(H2PO4)2¥H
2O 1.14 no data 0.0 ± 2.0
0.5 monocalcium phosphate anhydrate (MCPA) Ca(H2PO4)21.14 no data [d]
1.0 dicalcium phosphate dihydrate (DCPD, ™brushite∫) CaHPO4¥2H
2O 6.59 6.63 2.0 ± 6.0
1.0 dicalcium phosphate anhydrate (DCPA, ™monetite∫) CaHPO46.90 7.02 [d]
1.33 octacalcium phosphate (OCP) Ca8(HPO4)2(PO4)4¥5H
2O 96.6 95.9 5.5± 7.0
1.5 a-tricalcium phosphate (a-TCP) a-Ca3(PO4)225.5 25.5 [b]
1.5 b-tricalcium phosphate (b-TCP) b-Ca3(PO4)228.9 29.5 [b]
1.2 ± 2.2 amorphous calcium phosphate (ACP) Cax(PO4)y¥nH2O[c] [c] [e]
1.5 ± 1.67 calcium-deficient hydroxyapatite (CDHA) Ca10x(HPO4)x(PO4)6x(OH)2-x(0 <x<1) 85.1 85.1 6.5 ± 9.5
1.67 hydroxyapatite (HA) Ca10(PO4)6(OH)2116.8 117.2 9.5± 12
2.0 tetracalcium phosphate (TTCP) Ca4(PO4)2O 38 ± 44 37 ± 42 [b]
[a] The solubility is given as the logarithm of the ion product of the given formulae (excluding hydrate water) with concentrations in mol L1. [b] These compounds
cannot be precipitated from aqueous solutions. [c] Cannot be measured precisely. However, the following values were reported: 25.7 0.1 (pH 7.40), 29.9 0.1
(pH 6.00), 32.7 0.1 (pH 5.28).[78] [d] Stable at temperatures above 100 8C. [e] Always metastable. The composition of a precipitate depends on the solution
pH value and composition.
Table 3. Crystallographic data of calcium phosphates.[72, 73]
Compound Space group Unit cell parameters[a] Z[b] Density [g cm3]
MCPM triclinic P1
≈a5.6261(5), b11.889(2), c6.4731(8) 2 2.23
a98.633(6), b118.262(6), g83.344(6)
MCPA triclinic P1
≈a7.5577(5), b8.2531(6), c5.5504(3) 2 2.58
a109.87(1), b93.68(1), g109.15(1)
DCPD monoclinic Iaa5.812(2), b15.180(3), c6.239(2) 4 2.32
b116.42(3)
DCPA triclinic P1
≈a6.910(1), b6.627(2), c6.998(2) 4 2.89
a96.34(2), b103.82(2), g88.33(2)
OCP triclinic P1
≈a19.692(4), b9.523(2), c6.835(2) 1 2.61
a90.15(2), b92.54(2), g108.65(1)
a-TCP monoclinic P21/a a12.887(2), b27.280(4), c15.219(2) 24 2.86
b126.20(1)
b-TCP rhombohedral R3cH ab10.439(1), c37.375(6) 21[c] 3.07
g120
HA monoclinic P21/b a9.84214(8), b2a,c6.8814(7) 4 3.16
g120 (monoclinic)
or hexagonal P63/m ab9.4302(5), c6.8911(2) 2
g120 (hexagonal)
TTCP monoclinic P21a7.023(1), b11.986(4), c9.473(2) 4 3.05
b90.90(1)
[a] a,b,care given in ä and a,b,gin 8. [b] Number of formula units per unit cell. [c] Per hexagonal unit cell.

REVIEWS
Biomineralization of Calcium Phosphates
biological apatites found in natural and prosthetic heart
valves.[52, 53]
b-TCP (b-tricalcium phosphate) is the ™true calcium
orthophosphate∫ of the stoichiometric composition
Ca3(PO4)2. It cannot be precipitated from solution, but
may only be prepared by calcination, e.g. of CDHA (see
below), at temperatures above 800 8C [Eq. (1)]:
Ca9(HPO4)(PO4)5OH !3Ca3(PO4)2H2O (1)
At temperatures above 11258C, it transforms into the high-
temperature phase a-TCP. Being the stable phase at room
temperature, b-TCP is less soluble in water than a-TCP
(Table 2). Pure b-TCP never occurs in biological calcifica-
tions. Only the magnesium-containing form called ™whitlock-
ite∫ (chemical formula: b-(Ca,Mg)3(PO4)2) is found in dental
calculi and urinary stones,[15±18, 54] dental caries, salivary stones,
arthritic cartilage, as well as in some soft-tissue deposits.[15±18]
In biomedicine, b-TCP is used in calcium phosphate bone
cements.[23, 24, 55±58] In combination with HA, b-TCP is used as a
™biphasic calcium phosphate∫ (™BCP∫)[59±65] as a bone-sub-
stitution ceramic. Other applications include fertilizers,[12]
polishing and dental powders, porcelains, pottery, enamel,
and animal food supplements.[28]
a-TCP (a-tricalcium phosphate, a-Ca3(PO4)2) is a metasta-
ble phase at room temperature, prepared from b-TCP at
above 11258C. a-TCP is more reactive in aqueous systems
than b-TCP and can be hydrolyzed to a mixture of other
calcium phosphates. It never occurs in biological calcifications
and has a limited application in medicine in calcium
phosphate cements.[26, 31, 33, 34, 41±44, 66] a-TCP is also used as a
fertilizer.[28]
ACP (amorphous calcium phosphate) is often encountered
as a transient phase during the formation of calcium
phosphates in aqueous systems. Usually, ACP is the first
phase that is precipitated from a supersaturated solution
prepared by rapid mixing of solutions containing of calcium
cations and phosphate anions.[67±71] The chemical composition
of ACP strongly depends on the solution pH value and the
concentrations of calcium and phosphate ions in the mother
liquor. For example, ACP phases with Ca/P ratios in the range
of 1.18:1 (precipitated at solution pH 6.6) to 1.53:1 (precipi-
tated at solution pH 11.7)[72, 73] and even up to 2.5:1[15±17] have
been described.
The structure of ACP is still uncertain. IR spectra of ACP
show broad, featureless phosphate absorption bands. The
compounds are amorphous, according to X-ray diffraction
experiments. Electron microscopy of ACP usually reveals
spherical particles with typical diameters of 20 ± 200 nm.
However, it is likely that ACP has an apatitic short-range
structure, but with a crystal size so small that it appears
amorphous in X-ray diffraction experiments (no coherent
X-ray scattering). This is supported by X-ray absorption
spectroscopic data (EXAFS; extended X-ray absorption fine
structure) on biogenic and synthetic samples.[74±77] On the
other hand, it was proposed that the basic structural unit of
ACP is a 9.5 ä diameter, roughly spherical cluster of ions with
the composition Ca9(PO4)6.[72, 73] These clusters were found
experimentally as seed nuclei during the crystallization of
HA, and a model was developed to describe the crystalliza-
tion of HA as a stepwise assembly of these units.[78] Bio-
logically, ACP (often containing magnesium, carbonate, and
pyrophosphate) is found in soft-tissue pathological calcifica-
tions (e.g. heart-valve calcifications of uremic patients).[15±18]
In medicine, ACP is sometimes used in calcium phosphate
cements.[31±33] Bioactive composites of ACP with polymers
have properties suitable for use in dentistry[79±82] and sur-
gery.[83±86]
CDHA (calcium-deficient hydroxyapatite) can be easily
prepared by the simultaneous addition of calcium- and
phosphate-containing solutions into boiling water, followed
by boiling the suspension for several hours. During this time,
initially precipitated OCP or ACP (this depends on the
solution pH value) are transformed into CDHA. On heating
above 700 8C, dry CDHA with Ca/P 1.5:1 will convert into b-
TCP and that with 1.5:1 <Ca/P <1.67:1 will convert into a
mixture of HA and b-TCP (the above-mentioned biphasic
calcium phosphate, BCP).[59±65]
Because of its nonstoichiometric character, CDHA always
contains other ions. The extent depends on the counterions of
the chemicals used for preparation (e.g. Na,Cl
). There have
been no direct determinations of the structures of CDHA and
the unit cell parameters are uncertain. As a first approxima-
tion, CDHA may be considered as HA with some ions
missing.[87] According to the chemical formula of CDHA
(Table 2), there are vacant calcium ion sites (mainly Ca2
sites,[88, 89] see HA below) and hydroxide ion sites in the crystal
structure of this compound. However, little is known about
the vacancies of phosphate ions: in CDHA, part of the
phosphate ions is either protonated or substituted by other
ions (e.g. carbonate).
Unsubstituted CDHA (i.e. containing calcium, phosphate,
hydrogenphosphate, and hydroxide ions only) does not exist
in biological systems; it occurs only with ionic substitutions:
Na,K
,Mg
2,Sr
2for Ca2; carbonate for phosphate;
fluoride, chloride, and carbonate for hydroxide, and some
water, form the so-called ™biological apatite∫ or dahllite–the
main inorganic component of animal and human normal and
pathological calcifications.[15, 16] Therefore, CDHA is a very
promising compound for the manufacture of artificial bone
substitutes.
HA (hydroxyapatite, Ca10(PO4)6(OH)2) is the most stable
and least soluble of all calcium orthophosphates (Table 2).
Pure HA crystallizes in the monoclinic space group P21/b.
However, at temperatures above 250 8C, there is a monoclinic
to hexagonal phase transition in HA[72, 73] (space group
P63/m).[90, 91] Some impurities, like partial substitution of
hydroxide by fluoride or chloride ions, stabilize the hexagonal
structure of HA at ambient temperature. For this reason, the
very rare single crystals of natural HA always exhibit a
hexagonal space group.
HA can be prepared in aqueous solutions by mixing exactly
stoichiometric quantities of calcium- and phosphate-contain-
ing solutions at pH >9, followed by boiling for several days
under a CO2-free atmosphere, filtration, and drying. Micro-
crystalline samples of HA can also be prepared by solid-state
reactions of other calcium phosphates (e.g. MCPM, DCPA,
DCPD, OCP) with CaO, Ca(OH)2, or CaCO3at temperatures
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REVIEWS M. Epple and S. V. Dorozhkin
above 12008C, in an atmosphere of equal volumes of water
and nitrogen. Single crystals of HA can be prepared by
hydrothermal synthesis.[72, 73] A water-free synthesis can be
performed in ethanol from Ca(OEt)2and H3PO4.[92, 93]
Pure HA never occurs in biological systems. However,
becuase of the chemical similarities to bone and teeth mineral
(Table 1), HA is widely used as a coating for orthopedic (e.g.
hip-joint prosthesis) and dental implants (reviewed in
refs. [94, 95]), and a calcium phosphate cement with HA has
also been developed.[29] Because of the great similarity to
bone mineral, HA is also used in liquid chromatography of
proteins and other biological compounds.[96±101]
TTCP (tetracalcium phosphate Ca4(PO4)2O) is the most
basic calcium orthophosphate. However, its solubility in
water is higher than that of HA (Table 2). TTCP cannot be
precipitated from aqueous solutions, and thus can only be
prepared by a solid-state reaction above 1300 8C, for
example, by heating homogenized, equimolar quantities of
DCPA and CaCO3in dry air, or in a stream of dry nitrogen
[Eq. (2)]:[72, 73]
2CaHPO42 CaCO3!Ca4(PO4)O 2CO2H2O (2)
TTCP is not very stable in aqueous solutions; it slowly
hydrolyses to HA and calcium hydroxide.[72, 73] Consequently,
TTCP is never found in biological calcifications. In medicine,
TTCP is widely used for the preparation of various self-setting
calcium phosphate cements.[27, 29±31, 39, 41, 102±104]
3. Biomineralization and Biological Hard Tissues
Biological mineralization (biomineralization) is the process
of in vivo formation of inorganic minerals. As shown in
Table 1 and discussed above, in the human body all normal
and most pathological calcifications consist of calcium phos-
phates. Other minerals such as calcium carbonate (found in
mollusk shells, algae, fish, ascidians, and plants), calcium
oxalate (present in plants), CaSO4(jellyfish), SrSO4(single-
celled sea organisms of the genus acantharia), and BaSO4
(algae), silicon dioxide (marine algae and plants), and iron
oxide (in bacteria, limpets, chitons, or mollusk teeth) are also
found in biological systems,[1, 4, 5] but that is another story.
Only the chemical and structural peculiarities of calcified
tissues consisting of calcium phosphates will be discussed
here.
According to Weiner and Wagner, ™the term bone refers to
a family of materials, all of which are built up of mineralized
collagen fibrils∫.[105, 106] This family of materials also includes
dentin (the material that constitutes the interior of a tooth),
cementum (the thin layer between the root of a tooth and the
jaw), and mineralized tendons.[105, 107] Let us start with the
™real∫ bones.
3.1. Bone
Bone is the major calcification present in a human body.[1]
It serves as structural (mechanical) support for the body
and as the major reservoir of calcium and phosphate ions
necessary for a wide variety of metabolic functions. From
the chemical point of view, bone is a composite material
(Table 1) of calcium phosphate and collagen. The physio-
logical fluids present in bone act as plasticizers. Porosity is
an important property of bone, as it allows the body fluids
and cells to access the various regions of the osseous
tissue while also influencing the mechanical anisotro-
py.[1, 5, 15±17, 19±21, 105, 108±112]
Usually bone is composed of a relatively dense outer
layer (Corticalis; the cortical or compact bone) surround-
ing a less dense, porous tissue (Spongiosa; cancellous
bone), which is filled with a gel-like tissue known as bone
marrow (Figure 3). Bone is a highly complex material
that exhibits a strongly hierarchical structure on different
length scales (see refs. [1, 5, 105, 108 ± 112] for detailed dis-
cussions).
Figure 3. A noncalcined cancellous bone (femoral head) showing the
transition from a more compact outer layer (corticalis) to a more porous
interior (spongiosa).
Microscopically, the constituent building blocks of bone are
mineralized collagen fibrils of 80 ± 100 nm thickness and a
length of a few to tens of microns (Figure 4). These fibrils are
composites of biological apatite (i.e. CDHA with ionic
substitutions) and molecules of type I collagen. The crystals
of biological apatite in bone are always plateletlike (elongated
along the crystallographic caxis) and very thin; 2 ± 4 nm (in
other words, just a few unit cells thick!–see Table 1). The
crystals insert themselves in a parallel fashion into the
collagen fibrils, while the latter are formed by self-assembly
of collagen triple helices.[105] Recently, this lowest level of
hierarchical organization of bone has been successfully
simulated by HA precipitation on amphiphilic peptide nano-
fibers.[113] However, the interface between collagen and
crystals of biological apatite is still poorly understood. It is
not known why the crystals of biological apatite are platelet-
shaped.[1, 5, 105, 108±112]
In general, a sequence of temporal events can be recog-
nized during bone formation. The first stage involves the
synthesis and extracellular assembly of the collagen I matrix
framework of fibrils, followed by its mineralization. The
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REVIEWS
Biomineralization of Calcium Phosphates
Figure 4. Schematic drawing of the mineralized collagen fibrils that are the
basic constituents of bone. Platelet-shaped nanocrystals of CDHA are
incorporated in a parallel way between collagen molecules, with the
crystallographic caxis parallel to the fiber axis.
crystals of biological apatite grow with a specific crystalline
orientation–the caxes of the crystals are roughly parallel to
the long axes of the collagen fibrils within which they are
deposited.[5, 105, 107] The same is true for dentin and enam-
el,[114, 115] as well as for more primitive living organisms. For
example, in the shell of the mollusk Lingula unguis which
consists of CDHA, the crystal caxes are oriented parallel to
the b-chitin fibrils.[116] Therefore, the orientation of CDHA
crystals parallel to the long axes of an organic framework
could be a general feature of the calcium phosphate bio-
mineralization process.
Unlike other mineralized tissues, bone continuously under-
goes a so-called ™remodeling∫ process as it is resorbed by
specialized cells called osteoclasts and formed by another type
of cells called osteoblasts in a delicate equilibrium. Osteopo-
rosis is the condition in which bone resorption dominates, and
in osteopetrosis, the reverse process is dominant. That is why
mature bone consists of a very complex assembly of bone
™patches∫, each of which has a slightly different structure and
a different age.[1, 5, 105, 107±112]
There is no general agreement on the chemical mechanism
of bone formation. It is clear that the inorganic part of bone
consists of biological apatite, that is, CDHA in which some
ions have been replaced but (surprisingly!) without detect-
able amounts of hydroxide ions.[117±119] However, various
in vitro experiments on the precipitation of CDHA and HA
revealed that none of these compounds directly precipitates
from supersaturated aqueous solutions containing calcium
and phosphate ions: some intermediate phases (so-called
™precursors∫) are always involved.[15±17, 48±53, 67±71] Three com-
pounds (DCPD, ACP, and OCP) are possible precursors to
CDHA and HA precipitation in vitro. Therefore, the same
compounds are suggested as the precursors to in vivo bone
formation. Evidently, the precursor phase of bone is of a
transient nature, which complicates its detection, especially
in vivo. In 1966, Brown et al. suggested that OCP is the
original precipitate on which biological apatite nucleates
in the following step.[120] This idea was extended in
their further investigations.[121±124] By use of high-resolution
transmission electron microscopy, this hypothesis was sup-
ported: computer-simulated lattice images of the ™central
dark line∫ in mineralized tissues revealed that it consisted of
OCP.[48±50]
Simultaneously with Brown, the research group led by
Posner proposed that ACP is the initially precipitated phase of
bone formation in vivo.[125±127] This conclusion was drawn from
the following facts:
*When calcium orthophosphates are prepared by rapid
precipitation from aqueous solutions containing calcium
cations and phosphate anions at pH >8.5 in vitro, the
initial solid phase that appears is amorphous.
*Mature bone mineral is a mixture of ACP and poorly
crystallized CDHA.
*Early bone mineral has a lower crystallinity than mature
bone,[125±133] which suggests that after being formed the
crystals of bone mineral undergo some transformations
during maturation.
For obvious reasons, there is only indirect evidence for the
in vivo crystal growth of bone mineral. Studies of animal
bones of different ages showed that the X-ray diffraction
peaks become sharper with increasing age, that is, the
crystallinity and/or the domain size increase. This change
occurs anisotropically, that is, it is more pronounced in the
crystallographic aaxis [(310) reflections] than the caxis
[(002) reflections].[134, 135] In addition to this, other changes,
such as an increase of calcium content and a decrease of
HPO42occur in bone mineral with age.[136, 137] Both crystal
size and carbonate content increase during aging in rats and
cows.[137] From a chemical point of view, these changes
indicate a slow transformation of a poorly crystallized CDHA
into a better crystallized HA.
There is a current debate on the question of whether bone
formation is an active or a passive process. As an ™active
process∫, one describes the assembly of calcium phosphate
nanocrystals within a spatially confined compartment of an
osteoblast, that is, within a matrix vesicle. These structures
have been found by transmission electron microscopy for
bone and tooth formation.[138±140] The term ™passive process∫
comes from the observation that blood serum is supersatu-
rated with respect to calcium phosphate precipitation,[141]
therefore mineralization should occur spontaneously at a
suitable nucleus (i.e. on a collagen fibril). The collagen fibrils
have a specific structure with a periodicity of 67 nm and 35±
40 nm gaps or holes between the ends of the collagen
molecules, where bone mineral is incorporated in the
mineralized fibril. A nucleation within these holes would
lead to discrete crystals with a size related to the nucleating
cavity in the collagen fibril. It was proposed that the
temporary absence of specific inhibitors leads to precipitation
and thereby regulates this physicochemical bone forma-
tion.[142±144] The question of whether cells do actively form
and deposit bone mineral or whether a systemic regulation of
inhibitors controls bone formation is still open.[145] The truth
probably lies somewhere in between, that is, calcium phos-
phate nanocrystals are formed within cells from a super-
saturated medium and excreted near the collagen fibers where
they are finally deposited.
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REVIEWS M. Epple and S. V. Dorozhkin
3.2. Teeth
Teeth are the second major normal calcification present
in mammals.[1] The structure of teeth is even more com-
plicated than that of bone (Figure 5). For example, unlike
bone, teeth consist of at least two different biominerals:
enamel (outside) and dentin (interior). As shown in
Table 1, dentin and bone have many similarities, and in
most aspects they can be regarded as being essentially the
same material.[1, 72, 73, 105, 107±112, 136] Therefore, most statements
made above for bone are also valid for dentin.
Figure 5. Schematic picture of a tooth and its local chemical composition.
Tooth enamel contains crystals of biological apatite that are
much larger than those of bone and dentin (Table 1). In
addition, its organic phase does not contain collagen. At the
interface between enamel and dentin, there is an ™enameloid∫
phase; a hard tissue that contains enamel-like crystals of
biological apatite and collagen fibrils.[1]
Enamel and enameloid consist of biological apatite crystals
that are remarkably different from the other mineralized
tissues in humans and vertebrates. In enamel, needlelike
crystal rods are tens of microns long (up to 100 mm) but
sometimes only 50 nm wide,[146±150] which is much larger than
the mineral crystals of dentin and bone (Table 1), but
nevertheless consist of carbonated CDHA.[151±153] On the
surface, there is also some fluoride content in place of
hydroxide ions[154] although the overall content of fluoride
ions in enamel is small (about 0.01 wt %;[16] see also Table 1).
Note that fluoroapatite is not found in enamel.[1]
The enamel crystals are generally organized into parallel
arrays under strict biological control. This structure can be
deduced from the observation that, at every stage, the parallel
arrays are well-ordered and that the crystal rods all have a
remarkably uniform cross section (Figure 6).[146±148] The first
detectable crystals in enamel formation are flat, thin rib-
bons,[146±148] that were reported to be OCP,[109, 155±157] b-
(Ca,Mg)3(PO4)2,[156] or DCPD.[117, 119] During maturation of
the enamel, the mineral content increases from initially
45 wt% to 98 ±99 wt %,[117] accompanied by widening and
thickening of the crystal rods.[117, 119, 158, 159] Simultaneously, the
Figure 6. Scanning electron micrograph of the forming enamel on a
continuously growing rat incisor, which shows ordered rods of calcium
phosphates. Scale bar: 10 mm (taken from ref. [1] with permission).
Ca/P ratio increases[158, 159] and the carbonate content de-
creases,[160±162] which finally results in the most highly miner-
alized and hardest skeletal tissue.
Enamel crystals show the (100) face at the sides and
presumably the (001) face at the ends,[163, 164] as usual for
HA. A ™central dark line∫ is observed by TEM in the centers
of enamel crystals (also observed in bone and dentin), which
consists of OCP.[48±51] As described above for bone, X-ray
diffraction shows that the crystals of ™younger∫ dentin are less
ordered than those of more mature dentin.[136] Therefore,
maturation of dentin is a slow transformation of a poorly
crystallized CDHA into a better crystallized HA.
The development of individual enamel and dentin crystals
was studied by high-resolution transmission electron micros-
copy.[165±167] Both processes appear to be roughly comparable
and were described in a four-step process. The first two steps
include the initial nucleation and formation of nanometer-
sized particles of CDHA. They are followed by formation of
ribbonlike crystals, which until recently was considered to be
the first step of biological crystal formation in the tooth.[165±167]
These complicated processes, starting with the heterogeneous
nucleation of inorganic calcium phosphate on an organic
extracellular matrix, are controlled in both tissues by the
organic matrix and are under cellular control (odontoblasts
and ameloblasts).[168] To complicate the process even further,
regular and discrete domains of various charges or charge
densities on the surface of CDHA crystals derived from the
maturation stage of enamel development were recently
discovered by a combination of atomic and chemical force
microscopy.[169] Organic molecules (e.g. amelogenin)[169] at
physiological solution pH values appear to bind on the
charged surface domains of CDHA.
On the other hand, dentin and enamel share a common
starting location : the dentin ± enamel junction.[170±172] The
steps of enamel crystal growth at the junction are a matter
of current debate. Some authors claim that the enamel crystals
grow epitaxially on the pre-existing dentin crystals, because of
a high continuity between enamel and dentin crystals.[173±175]
Others have shown that enamel crystals are formed at a given
distance from the dentin surface[155±157, 176] and could either
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REVIEWS
Biomineralization of Calcium Phosphates
reach dentin crystals by a subsequent growth[177] or remain
distant.[176, 178] Thus, both structure and formation of the teeth
appear to be more complicated than those of the bone.
A physicochemical mineralization occurs every day on our
teeth. Enamel is only formed during dentinogenesis in the jaw,
that is, it will never be repaired by cellular action. If it is
etched, for example, by acidic food or beverages, CDHA is
dissolved. Fortunately, the saliva in the mouth is supersatu-
rated with respect to CDHA deposition (as is the blood
serum), and after a while, the surface layer is restored again.
This process does not involve any biological action and
therefore can be classified as ™passive mineralization∫ (see
also the discussion above on bone formation). Replacement
of some hydroxide ions with fluoride ions (which leads to
fluorohydroxyapatite) lowers the solubility and therefore
improves the acid resistance.[154]
3.3. Cartilage
Cartilage is usually (but not exclusively) part of the
endoskeleton of animals[1, 179] and exists both in mineralized
and unmineralized forms. Only vertebrates develop mineral-
ized cartilage, in some cases in the central portions of the
vertebra and close to the surface of jaws. Except for
pathological cases, the mineralization of cartilage occurs in
two situations in the body: First, during bone formation in the
endochondral plate (in almost all vertebrates) and second, as
final mineralized product (only in sharks and certain other
fishes[180]).[1]
Mineralized cartilage consists of the unmineralized carti-
lage plus crystals of CDHA, as well as considerable amounts
of amino acids, phosphoserine, and other biological com-
pounds. The molecular organization of macromolecules of
cartilage and CDHA crystals is still not fully understood.
Mineralized cartilage and bone coexist in close proximity in
the endochondral plate during bone formation. They have
similar macromolecular constituents, and both contain
CDHA.[1] However, the shape of the CDHA crystals in
mineralized cartilage, in general, resembles that in enamel:
the crystals were found to be needlelike (CDHA crystals of
bone are platelike),[1, 105, 108±112] but much shorter (25 ±
75 nm[181] or 50 ± 160 nm[182] ) than those of enamel (up to
100 mm[146±150]). The average thickness of the CDHA crystals
in mineralized cartilage was reported as 5 ± 7.5 nm[181] and
1.8 nm.[182]
The process of cartilage mineralization has been well-
described elsewhere.[183±185] Before the crystal formation, the
organic matrix (consisting of proteoglycans, type II collagen
and water)[1] first takes up calcium and then phosphate.[185]
The first crystals of CDHA, those formed in cartilage, were
needlelike and located inside cellular matrix vesicles.[183, 184]
After growth within the vesicles, the crystals extend out of
these containers into the surrounding organic matrix. They
aggregate into clusters of randomly oriented crystals. In a
second step, these clusters further aggregate to form the
mature mineralized-cartilage structure with a random ar-
rangement of crystals.[181] Physicochemical investigations of
the crystals revealed their very poor crystallinity and the
presence of significant amounts of nonapatitic calcium
phosphates. The concentration of such nonapatitic phosphates
was found to increase during the early stages of cartilage
mineralization but then decreased as the mineral content
steadily rose, until full mineralization was achieved.[186]
Therefore, the CDHA crystals in the vesicles act as centers
of cartilage mineralization. However, a detailed understand-
ing of the mechanisms of crystal nucleation and growth in
these vesicles is not yet available.[1]
3.4. Shells
Rarely, calcium phosphates are encountered in mollusk
shells (that in most cases consist of calcium carbonate).[187, 188]
When biomineralization was ™invented∫ by nature about
570 million years ago, there were both mollusks with calcium
carbonate and calcium phosphate shells. Over time, the ones
with calcium phosphate shells mostly disappeared (so-called
™problematica∫), and today the overwhelming majority of
mollusk has shells of calcium carbonate.[1] Figure 7 shows
fossilized shells of the species Lingula that consist of calcium
phosphate (apatite).[116]
Figure 7. Fossilized shells of the brachiopod Lingula from the
Lower Triassic, consisting of calcium phosphate (taken from
http://inyo.topcities.com/ef/lingula.html with permission).
4. Pathological Crystallization of Calcium
Phosphates
Unwanted deposition of calcium phosphates in the body
can lead to severe diseases. Calcium phosphate depositions
are responsible, among other things, for urinary
stones,[15, 189, 190] atherosclerosis,[141, 191±193] dental calculus,[45, 46]
calcification of artificial heart valves,[194±198] and calcified
menisci (™chondrocalcinosis∫).[199, 200] Figure 8 shows an ex-
ample of atherosclerotic depositions of calcium phosphate
(together with cholesterol) that was isolated from arter-
ies.[193, 201] Blockage of arteries by such deposits is the major
cause of death in developed countries.
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REVIEWS M. Epple and S. V. Dorozhkin
Figure 8. Spherical calcium phosphate particles isolated from an athero-
sclerotic lesion. Scale bar : 4 mm (taken from ref. [193] with permission).
As many body fluids (blood, saliva) are supersaturated with
respect to HA precipitation,[141] we may conclude that
calcification is thermodynamically feasible but kinetically
hindered in most parts of the body. Therefore, suitable
inhibitory mechanisms must be at work to prevent an
unwanted mineralization in the body. The mechanisms of this
inhibition are a topic of current research in molecular
medicine, as it can be concluded that disruptions of this
inhibition are probably the cause of pathological calcifica-
tions. In addition, the fine-tuned equilibrium of bone resorp-
tion and formation may be based on such processes. For
instance, in mice in which the genes that are responsible for
the production of the specific blood proteins (fetuine,[142, 144]
matrix Gla protein[143, 202] ) were knocked out, uncontrolled
calcification in the arteries occurs. Obviously, these proteins
serve as inhibitors of calcium phosphate precipitation by
suitable complexation of the dissolved ions or by effectively
preventing formed nuclei from further growth by preferential
adsorption.[203±205]
On the other hand, some mechanisms have been identified
that enhance crystallization.[141] Currently discussed, especial-
ly for the case of atherosclerosis, are:
*the heterogeneous nucleation of calcium phosphates on the
membranes of dead cells that contain phospholipids
(phosphate groups act as nucleators),[192, 195]
*nucleation by antibodies that are specific for cholester-
ol,[205, 206] and
*cellular action of osteoblast-like cells (so-called pericytes)
within arteries that form bonelike tissue.[207]
For the case of atherosclerosis, obviously a number of
effects are responsible for the pathological calcification ; these
range from purely physicochemical effects (supersatura-
tion)[141] over biologically induced nucleation to the bio-
logically controlled deposition of calcium phosphates by
specialized cells.[141]
Similar effects exist during the calcification of artificial
heart valves. The replacement of heart valves by implants of
either biological (porcine heart valves) or synthetic origin is
now a common procedure in cardiosurgery (about 150 000 are
implanted every year worldwide).[196] However, the implanted
devices tend to calcify after implantation (in some cases even
after a few months), that is, they become stiff because of
deposition of calcium phosphate. The origin of this behavior is
not yet clear but, at least with heart valves of biological origin,
a nucleation by membranes of dead cells (phospholipids)
appears likely.[194±198]
5. Calcium Phosphates as Biomaterials
The treatment of injuries or diseases often requires surgical
action. For the past 50 years, biomaterials have increasingly
been applied to improve surgical procedures or to restore lost
body functions. Bone fractures are usually treated with
metallic wires, nails, screws, and plates, joints are replaced
by artificial endoprostheses (hip or knee), and lost teeth are
replaced by metallic implants in the jaw, to name a few
examples. As soon as foreign materials come into internal
contact with the body, the question of biocompatibility
becomes paramount, as any adverse effect (namely toxicity,
allergy, inflammation, corrosion, and mechanical failure)
must be strictly avoided. The search for optimally designed
biomaterials is still ongoing as a joint effort of physicians,
engineers, chemists, and physicists.[15, 194, 208±213]
Calcium phosphates generally have an excellent biocom-
patibility, that is, they are well-accepted by the body and
integrate well, for example, into bone upon implantation. This
is because of their almost ubiquitous presence in the body in
either the dissolved or solid form. Consequently, they have
found important applications as biomaterials, particularly for
hard-tissue regeneration.[21, 47, 66, 214±221]
In the bulk form, calcium phosphates are used as artificial
bone-substitution material for surgical treatment of bone
defects by orthopedic surgeons and maxillofacial sur-
geons.[15, 16, 219, 222] A bone defect that is caused, for example,
by tumor extraction, complicated fracture, or inflammation
must be filled with a suitable material to permit growth of new
bone into this defect. Otherwise, ingrowth of fibrous tissue
would prevent bone formation within the defect. Because the
ideal substitute (the ™golden standard∫), a patient×s own
spongious bone from the Iliac crest (hip) is usually not
available in sufficient quantities, and as materials of biological
origin are critically discussed because of possible infections or
immune reactions, the need for a fully synthetic material is
evident. Today, many different calcium phosphate ceramics
are on the market for the treatment of bone defects (see, for
example, refs. [15, 16, 219, 222] for overviews).
Chemically, synthetic bone-substitution materials are usu-
ally based on HA, b-TCP, or BCP (i.e. a composite of HA and
b-TCP).[15, 16, 219, 222, 223] The requirements for an ideal substi-
tute are usually:
*a porosity with a pore diameter of some 100 mm size (to
permit ingrowth of bone cells; see Figures 2 and 3),
*a biodegradation rate comparable to the formation of bone
tissue (i.e. between a few months and about two years), and
*a sufficient mechanical stability.[15, 16, 219, 222]
HA is more stable than a- and b-TCP under physio-
logical conditions, as it has a lower solubility and slower
resorption kinetics.[15, 16, 219, 222] Implants of calcined HA of
high crystallinity are present in a defect even years after
implantation in a virtually unchanged form, therefore b-
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Biomineralization of Calcium Phosphates
TCP[218] or BCP[21, 61, 63, 64] ceramics are favored today. An ideal
material should be degraded inside the defect simultaneously
with the formation of a new bone, that is, the full restoration
of the defect with biological material is desired. Figure 9
shows three examples of calcium phosphate-based bone-
substitution materials of different origins. Implant porosity is
a very important property to allow cell invasion and bone
ingrowth.
Figure 9. Examples of porous calcium phosphate-based bone-substitution
materials: a) Cerabone (hydroxyapatite) from spongious calcined bovine
bone (about 3 11cm
3); b) Algipore (hydroxyapatite) from hydro-
thermal processing of calcium carbonate-containing algae with ammonium
phosphate. Scale bar: 100 mm; c) Cerasorb (synthetic phase-pure b-TCP)
with CNC (computer numerical control)-drilled holes (about 1 1
2cm
3).
A new concept in the treatment of bone defects was
introduced with bone cements based on calcium phosphates,
which harden inside the defect. Although different formula-
tions are on the market (see the discussion of the different
calcium phosphates above), they usually consist of solid
calcium phosphates that are mixed with a solution to induce
the precipitation of a CDHA-like phase [Eq. (3), not stoichio-
metrically balanced]:[66, 102±104, 211, 212]
Ca(H2PO4)¥2H
2O (s) b-Ca3(PO4)2(s) CaCO3(s) Na2HPO4(aq) !
Ca8.8(HPO4)0.7(PO4)4.5(CO3)0.7(OH)1.3 (s) (3)
The advantage of this procedure is that the cement adapts
better to the defect geometry than ceramic materials that are
implanted as solids. The structure and composition of the
hardened calcium phosphate is close to that of bone mineral;
therefore, a facilitated resorption is observed.[66]
Calcium phosphate coatings on metals are often applied in
medicine. Metallic implants are encountered in endoprosthe-
ses (total hip-joint replacements) and artificial tooth sockets.
The requirement for mechanical stability necessitates the use
of a metallic body for such devices. As metals usually do not
undergo bone bonding, that is, they do not form a mechan-
ically stable bond between implant and bone tissue, ways have
been sought to improve the mechanical contact at the
interface.[194, 208, 224] One possibility is to coat the metal with
calcium phosphate ceramics; these increase the roughness of
the bone surface and thereby facilitate bone bonding, and may
therefore serve as a ™glue∫ between the metal and bone
(Figure 10).
Figure 10. Calcium phosphates in hip endoprostheses: a ceramic ball joint
(Al2O3), a calcium-phosphate coated endoprosthesis (™cementless endo-
prosthesis∫) and an uncoated endoprosthesis that must be fixed in place
with PMMA bone cement.
Two methods of bone coating are currently applied :
Application of molten calcium phosphate by high-temper-
ature plasma spraying and precipitation from a supersatu-
rated calcium phosphate solution. The first ap-
proach[94, 95, 216, 224] is very rough from a chemical point of
view. Solid calcium phosphate is injected into a plasma flame
and directed towards an implant that is appropriately rotated
to achieve a uniform coating. This extremely fast quenching
leads to the formation of a mixture of calcium phosphates on
the implant surface. Metal and calcium phosphate are strongly
joined after this procedure.[94, 95, 216, 224]
The second approach involves dipping metallic implants
into supersaturated calcium phosphate solutions. This method
was strongly promoted by the work of Kokubo and co-
workers and van Blitterswijk and co-workers, who showed
that after appropriate surface etching, a stable interface
evolves between metal and ceramic.[225±230] The method also
permits coating of internal surfaces (difficult with plasma
spraying) and the incorporation of biologically active sub-
stances, for example, proteins or antibiotics into the coat-
ing.[231] A special case is surface coating with a biomimetic
defect apatite by dipping into simulated body fluid (SBF), a
solution that contains the inorganic ions of human blood
plasma in almost natural concentrations.[225±228, 232±235] Fig-
ure 10 shows both a calcium phosphate-coated and an un-
coated hip endoprosthesis. The latter has to be fixed in the
femur bone by a suitable bone cement based on poly(me-
Angew. Chem. Int. Ed. 2002,41, 3130 ± 3146 3141

REVIEWS M. Epple and S. V. Dorozhkin
thylmethacrylate) (PMMA). Note that this polymer is not
biodegradable and remains in the operation site.[224, 236]
The same principles are valid for tooth implant systems that
are fixed into the jawbone, onto which artificial teeth are
attached. In general, the mechanical contact between implant
and bone is crucial, as considerable forces have to be
withstood. Coating of such dental implants with calcium
phosphates (usually by plasma spraying) leads to better and
faster bone attachment. Figure 11 shows such a plasma-spray-
coated tooth implant in low and high magnification. Finally,
Figure 12 shows the surface of a nickel-titanium shape-
memory alloy (NiTi, ™Nitinol∫) that was coated with calcium
phosphate from solution to improve its biocompatibility.[237]
Figure 11. Dental implants (by Friadent) coated with calcium phosphate
by a plasma-spray process. a: 10, b: 1000. Note the irregular, rough
structure of the deposited calcium phosphate at the higher magnification.
Scale bars 1 mm and 10 mm, respectively.
Figure 12. The surface of a nickel ± titanium shape-memory alloy (™Niti-
nol∫) that was coated with a calcium phosphate layer by dipping into an
supersaturated calcium phosphate solution. The front part shows the
etched metal surface from which the calcium phosphate layer has been
mechanically removed. Scale bar : 2 mm.
6. Biomimetic Crystallization of Calcium
Phosphates
Nature×s ability to assemble inorganic compounds into the
biological structures (shells, spicules, teeth, bone, skeletons) is
still not reproducible by synthetic procedures. Because of its
potential benefits for materials science, research groups
around the world are increasingly addressing the question of
biomineralization. When considering calcium phosphates, the
demand of clinical medicine to design biocompatible implants
and to treat diseases related to crystallization phenomena
adds a strong practical impetus to understanding these
processes. The fundamentals of biomineralization have been
reviewed extensively.[1, 3±7, 238±241] We will limit ourselves to
considerations of biologically inspired crystallization of
calcium phosphates and present a few examples that demon-
strate the current possibilities.
An approach to the preparation of biomimetic bone-
substitution materials was made by Pompe et al., who crystal-
lized HA on collagen to obtain a bonelike composite.[242]
Although the ultrastructure of bone could not be realized,
such collagen ± HA tapes are currently under investigation for
clinical use. Note that the final step to make bone out of
artificial implants is up to the body×s own remodeling
function. Ozin et al. precipitated HA in the presence of
surfactants, to obtain a biomimetic lamellar product.[243] Stupp
et al. have prepared so-called ™organoapatites∫ with a bone-
like crystallinity by precipitation of calcium phosphate in the
presence of organic polyelectrolytes.[214, 217, 244, 245] Kokubo and
co-workers and van Blitterswijk and co-workers were suc-
cessful in coating different substrates with a bonelike apatite
layer (see refs. [229, 234] and those given above on coated
metal prostheses). We have recently prepared bulk samples of
bonelike apatite and composites of it with biodegradable
polymers.[84±86, 246]
Nancollas and co-workers invented the ™Constant-Compo-
sition Technique∫ to monitor and control the external
conditions (mainly solution pH value and concentrations of
participating ions) during a crystallization experiment.[22, 247]
Generally, during precipitation of calcium phosphates from a
neutral solution, the pH value decreases because of the
release of protons that were formerly bound to hydrogen
phosphate or dihydrogen phosphate [Eq. (4)].
5Ca2(aq) 3 HPO42(aq) 5H2O (l) !
Ca5(PO4)3OH (s) 4H3O(aq) (4)
One of the main differences between chemical and bio-
logical crystallization is the rate of precipitation. Usually in
chemistry, precipitation occurs fast whereas in biology the
crystals need days, weeks, or months to grow. A suitable
simulation of this process, especially in the presence of
(bio)organic additives, must therefore slow down the crystal-
lization. This can be achieved by separating the two compo-
nents with a suitable membrane or medium that acts as a
diffusion barrier (a double-diffusion technique). If this
medium itself contains some biomimetic functional groups,
it can have a templating influence on the growing crystals.
Work along this line has been carried out by Iijima et al.
3142 Angew. Chem. Int. Ed. 2002,41, 3130 ± 3146

REVIEWS
Biomineralization of Calcium Phosphates
(collagen matrix from bovine achilles tendon,[248] and mem-
branes in the presence of bovine[249] and murine[250] ameloge-
nins), Kniep and co-workers (matrix of denaturated colla-
gen),[154, 251, 252] Epple and co-workers (matrix of microporous
polyglycolide),[253±256] Falini et al. (matrix of collagen),[257] and
Stupp and co-workers (carbon-coated TEM grid).[113] Work on
the crystallization from SBF under static and dynamic
conditions to yield bonelike apatite was also reported recently
by Vallet-Regi and co-workers[258, 259] and by Epple and co-
workers.[256, 260]
Interactions between collagen and growing fluoroapatite
crystals are responsible for a fractal growth of fluoro-
apatite into dumbbell shapes that finally close to give
spheres.[154, 251, 252] Figure 13 shows this special morphology.
Figure 13. A biomimetically grown aggregate of fluoroapatite that was
crystallized in a gelatin matrix. The crystal shape can be explained and
simulated by a fractal growth mechanism. Scale bar: 10 mm (taken from
ref. [252] with permission).
By combining the constant-composition technique with the
double-diffusion setup, we were able to identify different
crystal morphologies of fluoroapatite as functions of overall
concentration (i.e. supersaturation), pH value, and fluoride
ion concentration.[254, 255] Figure 14 shows a uniform crystal
population that was prepared by this method.
Figure 14. Hexagonal fluoroapatite crystals that were grown by a double-
diffusion technique under controlled conditions (pH 7.4, 378C, constant ion
concentrations, 7 days). Note the well-shaped crystals and their uniform
size and morphology. Scale bar : 10 mm.
7. Summary and Outlook
Although it may appear surprising to the nonspecialist,
there are still many open questions within the area of calcium
phosphate chemistry. The basic questions concerning crystal-
lography, thermodynamics, and phase relationships have been
answered. Nevertheless, when it comes to the biological
formation of calcium phosphates, issues including rate of
crystallization, control of morphology, incorporation of for-
eign ions, and interaction with biomolecules remain hot topics
that are not well understood even today. A better under-
standing of structure, formation, and dissolution of such
biominerals will lead to improved biomaterials that can
substitute bone and teeth. This knowledge will also help to
counter widespread pathological calcifications such as athe-
rosclerosis, stone formation, or dental calculus. Further
progress of unforeseeable impact will come from modern
genetics, where gene structures are currently related to hard-
tissue formation.
We thank Alexander Becker, Dr. Jongsik Choi, Elena
Dorozhkina, Dr. Bernd Hasse (now at DESY), Dr. Fabian
Peters (now at Curasan), Carsten Schiller, Dr. Karsten Schwarz
(now at Tutogen), Dr. Michael Siedler, and Drazen Tadic for
their research contributions during the past years. We also
thank Dr. Jˆrg Arnoldi (Mathys), Dr. Philip Cantzler (Fria-
dent), Dr. Peter Seidel (Coripharm), and Prof. Gerd Willmann
(Ceramtec) for providing material. We are also grateful to the
Deutsche Forschungsgemeinschaft (DFG), to the Fonds der
Chemischen Industrie, to the Deutscher Akademischer Aus-
tauschdienst (DAAD), and to HASYLAB at DESY (Ham-
burg) for generous support of our work during the past years.
Received: December 3, 2001 [A 505]
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