Bone and Tooth
Mineralization:
Why Apatite?
Skull of a modern horse
Jill D. Pasteris1, Brigitte Wopenka1, and Eugenia Valsami-Jones2
DOI: 10.2113/ GSELEMENTS .4.2.97
T
The chemical–structural differences
between bone and tooth enamel
highlight the biological adaptability
of vertebrate animals, which is
mirrored by the crystal-chemical
versatility of the mineral apatite.
Bone consists of about 45–70 wt%
mineral, 10 wt% water, and the
remainder collagen plus a small
proportion of non-collagenous
KEYWORDS: apatite, bone, phosphate, biomineralization, biomaterials proteins (Rogers and Zioupos
1999; Skinner 2005; FIG. 1). The
mineral:collagen ratio not only
WHY APATITE?
differs among animals, among bones in the same animal,
Most mineralogists and geochemists are aware that the
and over time in the same animal, but it also exerts a major
mineral component of bones and teeth is a biologically procontrol on the material properties of bone, such as its
duced analog of hydroxylapatite, Ca5(PO4)3OH. But why is
toughness, ultimate strength, and stiffness. A higher mineral:
the biomineral not, for example, calcite? Clearly, the biocollagen ratio typically yields stronger, but more brittle,
mineral must possess the physical properties required for
bones (Rogers and Zioupos 1999; Currey 2004; Currey et al.
the functionality of the tissue, such as structural support
2004). For example, bone from the leg of a cow has a rela(bones) and mechanical grinding (teeth). Bone, however, is
tively high concentration of mineral (for support), whereas
also a chemical reservoir for phosphorus, which is a lifebone from the antler of a deer has a relatively high concenessential element. Phosphorus is present in a large array of
tration of collagen (for flexibility). Dentin has characteristics
biomolecules; for example, it occurs in DNA, RNA, collagen,
similar to those of bone, but enamel is strikingly different,
and other proteins. It is also a major component in, and
especially in its total lack of collagen and its 96 wt% mineral
indeed is essential to the formation of, ATP. [Note: All
content (FIG. 1). Thus, enamel is much more ceramic-like
words and acronyms in italics are defined in the accompaand brittle than dentin or bone.
nying glossary.] Apatite in bone stores 80 wt% of the body’s
phosphorus, as well as 99 wt% of its Ca and 50 wt% of its Bone is a composite material (FIG. 2A–H), whose two major
Mg (Skinner 2005; Glimcher 2006). But apatite is much components are microfibrils of collagen and crystallites of
more than a simple reservoir of elements: it can deliver bioapatite. These are well organized into arrays, even at the
those elements on demand.
nanometer scale (FIG. 2F). Note that the mineral:collagen
ratio of the entire bone is established at this nanoscale level.
These microfibril–bioapatite units, in turn, are bundled into
BIOAPATITE AND EXAMPLES
larger organized fibrils, which are grouped into even larger
OF BIOMINERALIZATION
mineralized fibers. In other words, bone, like many other
Biomineralized materials such as bones and teeth are combiomineralized materials, exhibits a hierarchical structure
posites of an inorganic (mineral) component, which is typthat is well organized at several spatial scales (Weiner and
ically nanocrystalline, and an organic component,
Wagner 1998; Glimcher 2006).
predominantly protein. The term “biomineral,” e.g. bioapatite, refers only to the inorganic component of the composite. In bone formation, during both primary development and
Among the remarkable aspects of bioapatite are the widely repair, collagen is laid down first and apatite mineralization
ranging properties and compositions it expresses in bone, follows. But the story does not end here. Bone mineralizadentin, enamel, and pathologic precipitates (“calcifications”); tion is a dynamic process. In the process of bone turnover
in altered form in fossilized teeth and bones; and in phos- (i.e. remodeling), which replaces essentially our entire
phorites, which are large sedimentary deposits dominated skeleton every 5–10 years (depending on age, diet, and
by phosphate materials, including fossil bones and teeth.
health), the collagen–mineral composite initially laid down
by osteoblast cells is constantly reworked, i.e. osteoclast cells
cause it to dissolve, so that osteoblasts subsequently can
deposit new bone material (FIGS. 2B, C). Moreover, bones that
1 Department of Earth and Planetary Sciences and the Center for
bear weight may increase in diameter through a feedback
Materials Innovation, Washington University, Campus Box 1169,
mechanism involving these same osteoclasts and osteoblasts.
St. Louis, MO 63130-4899, USA
Osteoclasts also can be deployed to release necessary calE-mail: [email protected]; [email protected]
cium or phosphate to the body fluid for use elsewhere
2 Department of Mineralogy, The Natural History Museum,
(Glimcher 2006; Boskey 2007).
Cromwell Road, London, SW7 5BD, UK
hrough evolution, vertebrates have “chosen” the calcium phosphate
mineral apatite to mineralize their teeth and bones. This article describes
the key characteristics of apatite in biological mineralization and explores
how the apatite structure allows biology to control mineral composition and
functionality. Through the synthesis and testing of calcium phosphates for biomaterials applications, we have gained further understanding of how sensitive
the chemical and physical properties of apatite are to its growth conditions.
E-mail: [email protected]
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The spatial and chemical mechanisms that guide bioapatite
to precipitate in regular patterns and with fixed crystallite
size on the collagen fibrils of bone (FIGS. 2E, F) are not fully
understood. Precipitation apparently is initiated biochemically by the removal of a ubiquitous nucleation inhibitor
and the concurrent increase in concentration of apatitic
components, especially phosphorus. In other words,
although a specific organism produces an individualized
biological–biochemical environment for mineral formation, that crystalline solid ultimately is stabilized according
to the principles of inorganic chemistry; element concentrations must equal or exceed saturation, and nucleation
must occur either spontaneously (homogeneously) or
through templating (heterogeneously), which is the primary nucleation mechanism in biomineralization (Mann
2001). As well as can be determined in bone’s intimately
intergrown nanocomposite, apatite crystallites nucleate on
and grow within the collagen network in two environments
of different size (FIG. 2F), i.e. in larger “holes” between the
terminations of end-to-end-aligned collagen microfibrils
and in smaller “pores” between side-by-side collagen
microfibrils. The c-axes of the apatite crystallites lie parallel
to the long axis of the microfibril (FIG. 2G). The crystallites
are plate-like rather than prismatic, which is the typical
shape of geologically formed crystals (LeGeros and LeGeros
1984; Elliott 2002; Glimcher 2006; Boskey 2007). The
nature of the binding between the mineral and the collagen
has yet to be determined, but this affinity must be strong,
as demonstrated by the remarkable flexibility and strength
of bones (Currey 2004; Currey et al. 2004) and the difficulty
in separating the two components physically or chemically.
Tooth mineralization is accomplished through a totally different set of cells than those forming bone. Tooth-forming
cells are specialized into different types, the most common
of which produce dentin (odontoblasts) or enamel
(ameloblasts). Like bone, dentin comprises collagen and
bioapatite nanocrystals, but dentin typically does not
undergo remodeling. In enamel formation, the ameloblasts
first secrete proteins that become mineralized (as in bone)
but later help remove those proteins from the maturing
enamel. Enamel ultimately contains only 1–2 wt% organic
compounds, and its crystallites are about 10 times wider
and up to 1000 times longer than bone and dentin crystallites (LeGeros and LeGeros 1984; Mann 2001; Skinner 2005;
Glimcher 2006; also FIG. 1).
The major components of apatite-mineralized tissue
(bone, dentin, enamel) are mineral, collagen (organic),
and water. The mineral component dominates the bone by weight but
occurs in subequal concentrations with collagen by volume (data for A
and B from Skinner 2005; data for C from LeGeros and LeGeros 1984).
FIGURE 1
Seen One Bioapatite, NOT Seen Them All
There is an old joke about how some people use duct tape
to activate or repair any conceivable device. Bioapatite does
duct tape one better—it changes its properties to suit the
functional demand of the device. For enamel, which is not
subject to remodeling but must resist abrasion and acid
attack, there is one composition of apatite. For bone crystallites, in contrast, which must bind to the surface of collagen
and dissolve under controlled conditions to be repaired or
to release Ca or P, there is a different composition of apatite.
the biological task at hand, and why/how can this differ
with, for example, species or age? What was the optimization
process that produced the specific ranges of coordinated
parameter values (e.g. as carbonate concentration increases,
hydroxyl concentration and size decrease) that now are
expressed in bone, dentin, and enamel apatite?
Mineralogists, crystallographers, geochemists, paleontologists,
medical (especially orthopedic) and dental researchers, and
biomaterials engineers all have chipped away at certain
aspects of the above questions. Their focal point is apatite,
whose structure is sufficiently pliable to accommodate
almost half the periodic table through elemental substitution (Hughes and Rakovan 2002; Pan and Fleet 2002). There
are some absolute rules and some incidental constraints on
the mineral-properties optimization scheme of bioapatite,
however. Apatite has atomic sites of several different shapes
and sizes, in which a wide range of ions can substitute for
each other (FIG. 2H). Like every mineral, however, apatite is
subject to the requirement of charge balance, which limits
the list of elements that could substitute or calls for coordinated substitutions. That list is also constrained by chemical
availability, with the body’s extracellular fluid providing
FIGURES 1A and B are reminders of how the proportions of
mineral differ among the three biomineralized tissues.
Biominerals themselves differ among the tissues (FIG. 1C), as
indicated by the contrasting size, hydroxyl concentration,
and carbonate concentration among crystallites of human
bone, dentin, and enamel. Reflection on these mineralogical variations brings up such questions as: What are the specific relations among the measurable physicochemical
parameters of apatites and their functional properties, e.g.
hardness, solubility, surface reactivity? How does an organism
create the biochemical environment necessary to stabilize
the nucleation and precipitation of an apatite with the size,
shape, composition, and solubility required to accomplish
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radiating channels that provide nutrients and presumably biochemical
signals to the specialized bone cells. The circles indicate the stepwise
infilling of new bone by osteoblasts. The black elliptical spots are lacunae, which house osteocytes. Photomicrograph of a single osteon in a
thin-section of bison jaw bone, taken in transmitted light. (D) One collagen fiber, created by the bundling of hundreds of fibrils, forms the
structural framework of bone. Evenly spaced, dark, spiraling bands represent periodic gaps (i.e. “holes” seen in F) that occur between the ends
of collagen fibrils laid down in overlapping arrays. (E) The smallest unit
of the organic component in bone is the triple-helix collagen molecule.
Five collagen molecules are bundled side by side in a staggered array,
forming a microfibril (F). Microfibrils, in turn, are bundled into fibrils (E).
(F) Enlargement of collagen microfibrils. Note that apatite crystallites
(not to scale) form in voids of two sizes and shapes, i.e. holes (or gaps)
between opposing ends of fibers and pores (or channels) created along
the lengths of adjacent microfibrils. Each microfibril is ~300 nm long
and ~4 nm thick. (G) Individual platelet of bioapatite. Unlike hydroxylapatite or fluorapatite, which crystallize into elongated prisms, carbonated apatite forms platelets. Bioapatite platelets are only about 2–3 unit
cells thick. (H) View of the atomic structure of fluorapatite (as a standin for compositionally more-complex, less-symmetric bioapatite),
viewed down the c-axis. For clarity, only the first couple of layers of
atoms are shown, with PO4 groups indicated by tetrahedra. Yellow =
calcium atoms; red = oxygen; dark blue = phosphate tetrahedra; light
blue = hydroxyl in channel sites.
Sketches of the hierarchical levels of typical cortical bone,
e.g. in a femur. A wedge (B) of bone can be dissected into
incrementally finer and finer well-organized structures, culminating with
the individual collagen molecule (E, top) and the unit cell of the mineral
(H). (A) Longitudinal section of long bone showing the open network
of bone struts in cancellous bone (ends) and dense cortical bone (shaft).
(B) Enlargement of a cross-sectional slice of cortical bone. Most of the
volume of mature cortical bone consists of cylindrical osteons (circular or
oval in cross-section), indicating secondary dissolution and reprecipitation of bone. Photomicrograph in transmitted light of a thin-section of
cortical bone in a bison jaw, showing numerous osteons. (C) Enlargement of one osteon, consisting of a central vascular cavity (i.e. blood
vessel), concentric circles, and thin radiating lines. The latter are narrow,
FIGURE 2
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the reservoir of the elements available for incorporation into
bioapatite. Despite these constraints, biological apatites
incorporate many elements, some of them at the ppm level
(LeGeros and LeGeros 1984; Elliott 2002; Skinner 2005).
tion is observed between the concentrations of carbonate
and hydroxyl in bioapatites, because the substitution of X
moles of phosphate by carbonate is charge-balanced
through X moles of vacancies in both the calcium and
hydroxyl sites (formula above, from Cazalbou et al. 2004).
Both Raman and infrared spectroscopy of a variety of bioapatites (FIGS. 3 and 4) confirm the depletion of OH- as carbonate concentration increases; this is especially clear in
bone apatite.
There are several ways to represent the range of compositional variability in bioapatite and the interdependence of
the substitutions. Skinner (2005) represented the overall
range of bioapatite chemistry as (Ca,Na,Mg,K,Sr,Pb,...)10
(PO4,CO3,SO4,...)6(OH,F,Cl,CO3)2, whereas Cazalbou et al.
(2004) use Ca8.3 1.7(PO4)4.3(HPO4 and CO3)1.7(OH and
0.5CO3)0.3 1.7 to indicate the extremely important role of
structural carbonate in bone apatite (where is a vacancy,
i.e. unfilled site, in the structure). The multiple substitutions
shown above also explain why/how typical bioapatites
have lower Ca:P atomic ratios than the 1.67 value for stoichiometric hydroxylapatite. Studies also have shown that
the carbonate concentration in apatite correlates positively
with its solubility (Ito et al. 1997; Elliott 2002). Thus, the
contrasting carbonate concentrations in bone and enamel
bioapatite coincide with the very different solubility
requirements of bone (resorbable) and enamel (chemically
resistant).
The incorporation of about 6 wt% CO3 in bone apatite and
about 3.5 wt% in enamel (FIG. 1C) highlights the interconnection between composition and structure in the mineral
and between the combined structure–composition and its
physical and chemical properties. The above formulas show
that the trigonal planar CO32- anion can fit into two structural sites, i.e. it can replace either a tetrahedral PO43- group
or an OH- in the large channel site. Clearly, both of these
substitutions flaunt Pauling’s rules; neither the charge of
the proposed substituent nor its size and shape match those
of the typical occupant. Yet, the substitution occurs
(LeGeros and LeGeros 1984; Elliott 2002; Glimcher 2006).
Research on bone apatite and its synthetic analogs indicates
that, in precipitation at body temperature, most of the CO3
is accommodated in the PO4 site and relatively little in the
channel site (Elliott 2002). Nevertheless, an inverse correla-
Structurally, the CO32- substitution also has important ramifications. The high degree of misfit of the CO32- ion in the
occupied sites creates strain in the structure. This strain is
reflected in peak-broadening in X-ray diffractograms and
Raman and infrared spectra (FIG. 3 AND 4; LeGeros and
LeGeros 1984; Elliott 2002; Pasteris et al. 2004; Wopenka
and Pasteris 2005; Skinner 2005; Glimcher 2006) and also
may account for the very small grain size of carbonated
apatite synthesized at room/body temperature (LeGeros and
LeGeros 1984). It is possible that the high concentration of
CO3 in bone apatite may place an upper boundary on crystallite size, i.e. that the strain accrued during growth may
create an insurmountable energy barrier once a certain crystallite size is reached.
The size of the individual types of bioapatite crystallites
actually is extremely important (FIG. 1C). The nanometer
size of the crystallites provides very high ratios of surface
area to volume, imparting high dissolution rates and surface activities, especially for the smallest crystallites. Eppell
et al. (2001) used atomic force microscopy to measure the
crystallites of mature cow bone. The widths (27–172 nm)
averaged 64 nm, and the lengths (43–226 nm) averaged
90 nm. Amazingly, 98% of the crystallites were less than 2
nm thick, i.e. only about 2 apatite unit cells (FIG. 2G).
Because of the extremely small crystallite size of bone mineral, a huge proportion of the atoms are either directly on
Infrared spectra from microgram amounts of powdered
materials, acquired through attenuated total reflectance
(ATR) using a diamond prism. (A) Synthetic hydroxylapatite, where
excellent crystallinity is reflected in the narrowness of the P–O stretching band at about 1020 cm-1. (B) In the rostrum, no bands for collagen
are detected at about 2900 cm-1, confirming its hypermineralized
nature. The apatite is carbonated, as indicated by the starred bands and
the consequent broadening of the P–O bands. (C) The spectrum for
geologic calcite confirms positions of the bands caused by C–O vibrations. (D) Turkey bone shows bands for collagen (compare with E) as
well as bioapatite. The presence of collagen in typical bone (D) severely
compromises detection of the most intense carbonate band at ~1415
cm-1. The broadening of apatite’s main P–O band at about 1020 cm-1
causes overlap with the C–O band at ~870 cm-1, complicating the
quantification of carbonate in bone apatite.
FIGURE 4
Raman microprobe spectra taken from individual 2-micrometer spots on cross-sections of bone, dentin, and
enamel in the jawbone and teeth of a young, domesticated bison.
Raman spectra indicate structure–composition units, such as phosphate,
but do not provide elemental compositional analyses. Peaks for the individual mineral (apatite, labeled A) and organic components (mostly collagen, labeled C) can be resolved. Most peaks below 1100 Δcm-1 represent vibrations of bonded atoms in the apatite, whereas most peaks
above 1100 Δcm-1 come from collagen. In the apatite, enhanced intensity of the peak at about 1070 Δcm-1 (starred) indicates greater incorporation of carbonate.
FIGURE 3
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the surface or bonded to an atom on the surface. The bulk
properties of such small entities are thus dominated by surface phenomena (Cazalbou et al. 2004; Wopenka and
Pasteris 2005; Glimcher 2006; Boskey 2007).
In part to recognize the large specific surface areas of bone
crystallites and in part to explain various spectroscopic phenomena that had been observed, some medical researchers
introduced the term “labile” for components intimately
associated with bioapatite but not structurally incorporated,
such as CO32- and HPO42- (Cazalbou et al. 2004; Glimcher
2006). From a geochemical perspective, such associations
could be viewed as adsorption, i.e. a surface property. Thus,
the mineral chemistry controls the size of the crystallites
and their surface properties, both of which must have an
influence on how strongly the crystals adhere to the collagen
microfibrils.
Why do we not know more about bone apatite? The small
size is the main explanation. If one combines the nanoscale
of the crystallites with the intimacy of their intergrowth
with collagen, one can see how bone apatite continues to
present an analytical challenge. Even for instruments with
spatial resolution on the order of tens of nanometers, there
are limitations posed by hydrated biological tissue and
organic components. Complete physical separation of bone
apatite from collagen is not possible. Chemical isolation of
bone apatite is possible through the use of very strong oxidizing agents, such as sodium hypochlorite, to destroy and
dissolve the surrounding tissue, but there are doubts that
the bioapatite crystallites remain unchanged by such a
treatment. Transmission electron microscopy and atomic
force microscopy have been applied to image and measure
physically ground bone fragments (Weiner and Wagner
1998; Elliott 2002; Glimcher 2006), but such sample handling raises concerns about induced comminution and strain
in the crystallites.
Several types of spectroscopy, including Raman and
infrared, have been used on whole bone, because the
organic and inorganic components produce spectrally separated or separable signatures (FIGS. 3 and 4). However,
these vibrational spectroscopic techniques yield combined
chemical–structural information, rather than explicit
chemical or crystallographic data. Another approach to
understanding bone apatite is through analogous materials
that are much easier to analyze, i.e. either synthetic compounds or biological materials almost devoid of organic
components. The hypermineralized bone-like rostrum and
bulla of whales permit bulk and microbeam analyses of
essentially pure bioapatite (FIG. 4; Rogers and Zioupos
1999). However, such unusual forms of bioapatite are recognizably different from typical bone apatite, e.g. in the former’s elevated carbonate concentration. Likewise, tooth
enamel is ideal for analysis, because it has much larger crystallites than bone and almost no organic “contamination.”
Enamel yields better-resolved XRD patterns and vibrational
spectra than bone apatite (FIG. 3; LeGeros and LeGeros
1984; Rogers and Zioupos 1999), but its composition is distinctly different.
The Role of Biological Fluids
Body fluids are naturally saturated with respect to hydroxylapatite (FIG. 5). It is only the action of biochemical
inhibitors of nucleation and/or crystallization that prevents
us from being mineralized all over. Pathological calcifications
occur when this inhibitory process goes wrong (LeGeros
and LeGeros 1984; Glimcher 2006). There are both intracellular and extracellular fluids in the body, and these have
different compositions. Bioapatite precipitates from extracellular fluid, whose composition and pH, therefore, must
ELEMENTS
Log–log plot of mineral solubility as a function of pH,
indicating the degree of saturation of body fluid and
saliva with respect to hydroxylapatite (OHAp) and the significantly lesssoluble fluorapatite (FAp) (calculated with MINEQL+v4.5 at 37°C,
assuming 1 g/L apatite and a dilute solution). Blood and saliva have very
similar calcium concentrations, but blood has a much more limited pH
range than saliva (the most common range is shown as a solid line).
Clearly, blood and saliva must be supersaturated with respect to OHAp
and FAp. Incorporation of carbonate makes bioapatite more soluble (i.e.
the saturation curve is raised) than OHAp and FAp. Nucleation and crystallization inhibitors in saliva and blood prevent constant, ubiquitous
apatite precipitation in the body.
FIGURE 5
be compatible with the apatite composition that characterizes
healthy bones and teeth. It is interesting that extracellular
fluid resembles seawater: it is dominated by Na and Cl, with
P and Ca in much lower concentration; its pH is about 7;
and it is oxidized (Skinner 2005). The significant substitution of Na and Mg in the apatite of typical cortical bone and
hypermineralized bone, like that in the rostrum and bulla,
partly arises from the high abundance of those ions in
extracellular fluid.
The neutral pH of the fluid is especially important because
a number of calcium phosphate phases are stable at body
temperature, but at somewhat different pH. In the pure
CaO–P2O5–H2O system, there is a very small compositional
region and pH range (within a couple of tenths of pH 7)
within which hydroxylapatite is the single stable phase; the
constraints on bioapatite stability are similar (LeGeros and
LeGeros 1984; Skinner 2005). Indeed, this is one reason that
urine must be acidic. If urine becomes more alkaline than
about pH 5 or 6, insoluble calcium phosphates can precipitate and form “stones.” The body must control the carbonate and bicarbonate concentration of bone-forming fluid to
assure both the proper solution pH and the desired carbonate
concentration within the precipitated bioapatite. Two
corollaries of this fluid-mediated precipitation are: (1) trace
(even toxic) elements that are ingested, e.g. Pb and Sr, can
be incorporated into bone mineral as it precipitates, and (2)
the fluid that constantly bathes the bone must be in equilibrium with bioapatite, or the bone mineral would dissolve.
Analogously, saliva must be saturated with respect to the
bioapatite of tooth enamel, or teeth would dissolve (FIG. 5).
Problems can arise with both the inorganic and the organic
components of the bone-forming fluid, as well as with the
specialized bone cells. Bone diseases, such as osteoporosis,
osteopetrosis, and osteogenesis imperfecta, are reminders
of the complex, sensitive interrelations between the biological and chemical processes of biomineralization. Some
bone diseases reflect an imbalance in the resorption and
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reprecipitation cycles. Whereas the approach to treating
osteoporosis is to focus on maintaining or increasing bone
density (i.e. concentration of mineral), this must not be
done at the expense of bone or mineral quality. Which mineral qualities? At least one previous attempt by physicians to
enhance bone mass failed because of unintended effects on
mineral quality. Osteoporotic women were given fluoride supplements, which resulted in an increase in their bone density, but also in an increase in the incidence of bone
fractures. Excessive incorporation of fluoride into bone
apatite apparently disrupts mineral–collagen interactions
and diminishes composite material strength. Dental fluoridation, on the other hand, is clearly advantageous (LeGeros
and LeGeros 1984; Elliott 2002). It enhances resistance to
acid attack by stabilizing fluorapatite on the tooth surfaces.
Fluorapatite is significantly less soluble than hydroxylapatite (FIG. 5). However, excessive fluoride in the tooth
results in discoloration and lesions in the enamel.
BIOMATERIALS: ENHANCED
UNDERSTANDING THROUGH IMITATION
The first evidence of biomineralization on Earth dates back
more than half a billion years. Organisms have had a great
deal of time to exploit the feedback between composition
and structure in apatite, on the one hand, and benefit from
its biological functionality, on the other. Biomaterials scientists, in contrast, have had only a few decades of experience
in trying to emulate biological materials. They have a long
way to go before they can “outdo” Nature.
Why develop synthetic substitutes for bone? Most bonegraft procedures currently performed are “natural bone
transplants.” However, there are major disadvantages in
such procedures: autografts (own bone) cause further
trauma to the patient, and allografts (donor bone) are limited in supply and could be unsafe. Thus, there is much
incentive to optimize bone-replacement biomaterials
(Williams 1990).
There are some essential qualities that all bone replacement
materials must have: they must be non-toxic, non-carcinogenic, non-allergenic, and non-thrombogenic (Sammons et
al. 2004), and they must be generally biocompatible. Other
desirable qualities for such materials include bioactivity,
osteoconductivity, and osteoinductivity (Urist et al. 1967;
LeGeros 2002; Sammons et al. 2004; Matsumoto et al. 2007).
Most importantly, however, the mechanical properties
must be adequate, with appropriate strength and flexibility
or (inversely) stiffness, i.e. Young’s modulus (FIG. 6). Bone
owes both its strength and elasticity to its biocomposite
nature, as reflected in the Young’s modulus of cortical bone,
which falls between those of its components, hydroxylapatite and collagen (FIG. 6A). Compared to other materials
used for bone replacements (e.g. metals, polymers, glasses,
natural materials such as coral), synthetic calcium phosphates, which in many cases are apatite analogs, have the
advantage that they most closely approximate the composition and properties of bone and therefore are intrinsically
biocompatible.
The development of an apatitic bone substitute, however, is
no trivial task. Current technologies cannot yet reproduce
the multistage biosynthesis, the hierarchical structure, and
the mechanical properties of bone. Most composites of
polymers and hydroxylapatite are too flexible (FIG. 6A).
Various methods have been used to synthesize hydroxylapatites as biomaterials, including high-temperature vapor
deposition, hydrothermal methods, hydrolysis of other calcium phosphates, and sol–gel methods, as reviewed by Gross
A
B
(A) Log–log plot of Young’s modulus (degree of stiffness)
versus density for natural cancellous and cortical bone,
several other natural materials, and synthetic materials and composites
that can be used as bone and tooth replacements. PLA = polylactide
(polymer), HDPE = high-density polyethylene (figure modified from
Gross and Berndt 2002). (B) Young’s modulus (linear scale) plotted
against the weight % mineral in totally dried samples of cortical bone
from animals. All labeled samples are of femur bone, except where
noted. A strong positive correlation exists between percent mineralization and degree of stiffness. Note the extremely low degree of mineralization of the deer antler, which is much more flexible than the deer
femur (in green). The significant increase in mineralization from the 5year-old to the 35-year-old human femur (in red) parallels a notable
increase in stiffness. Also note the differences among the dentin, rib,
bulla, and rostrum of whales (in blue). The hypermineralized whale bulla
and rostrum are exceptionally stiff, but not as much as expected from
projecting the trend established by the other data (derived from Rogers
and Zioupos 1999; Currey 2004; Currey et al. 2004).
FIGURE 6
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and Berndt (2002). The most common synthesis method is
precipitation through drop-wise addition of calcium salt
and phosphate salt solutions into a thermostated and pHbuffered solution. Biologically important properties of the
precipitated apatites, such as solubility, crystallinity, and
stoichiometry, can be manipulated by changing the synthesis
conditions or by subsequent aging. Properties can also be
manipulated by the introduction of foreign ions, such as
fluoride, carbonate, and organic species.
In the future, biomaterial production will be aided by a better
understanding of (1) how specialized bone cells respond to
the structural and chemical properties of calcium phosphates
and (2) the effects of natural composition variability on
bone properties. Importantly, even the least soluble apatite
substitutes are remodeled by the host tissue when
implanted into bone (Benhayoune et al. 2000). If desired,
faster resorption can be achieved by (partial) introduction
of more-soluble calcium phosphates. Recent advances in
nature-inspired approaches, i.e. biomimetic tissue engineering,
replicate the structure and function of natural biocomposites. Porous and biodegradable scaffold materials are developed
to act as temporary 3-D templates, which, when combined
with bioactive materials, eventually gain full biological
functionality and allow self-repair through the formation of
new tissue (Boccaccini and Blaker 2005; Jones et al. 2007).
If imitation is the highest form of flattery, then apatite is
among the most highly acclaimed minerals. The synthesis
and testing of calcium phosphates for biomaterials applications have revealed how minor differences in the growth
environment (e.g. temperature, pH, precursor solutions),
minor-element chemistry, and degree of crystallinity of
apatite can affect its solubility, adhesion to a substrate, and
biological compatibility.
REFERENCES
Benhayoune H, Jallot E, Laquerriere P,
Balossier G, Bonhomme P, Frayssinet P
(2000) Integration of dense HA rods into
cortical bone. Biomaterials 21: 235-242
Boccaccini AR, Blaker JJ (2005) Bioactive
composite materials for tissue engineering
scaffolds. Expert Review of Medical
Devices 2: 303-317
Boskey A (2007) Mineralization of bones
and teeth. Elements 3: 385-391
Cazalbou S, Combes C, Eichert D, Rey C
(2004) Adaptive physico-chemistry of
bio-related calcium phosphates. Journal
of Materials Chemistry 14: 2148-2153
Currey JD (2004) Tensile yield in compact
bone is determined by strain, post-yield
behaviour by mineral content. Journal of
Biomechanics 37: 549-556
Currey JD, Brear K, Zioupos P (2004) Notch
sensitivity of mammalian mineralized
tissues in impact. Proceedings of the
Royal Society of London B217: 517-522
Elliott JC (2002) Calcium phosphate
biominerals. In: Kohn MJ, Rakovan J,
Hughes JM (eds) Phosphates: Geochemical,
Geobiological, and Material Importance.
Reviews in Mineralogy & Geochemistry
48, pp 427-454
ELEMENTS
CONCLUSIONS
Why is apatite an ideal choice as a skeletal-framework mineral? Calcium and phosphorus are two biologically abundant elements, and phosphorus is an element shared by the
collagen that directs biomineral precipitation. Apatite is a
“sparingly soluble salt,” making it a safe reservoir for Ca
and P for use in other biological functions. Apatite’s properties can be tailored to some extent through its ability to
accept a wide range of chemical substitutions, each of
which affects its chemical and physical properties. Most
important is apatite’s extensive carbonate substitution,
which controls lattice strain, solubility, the nature of substitution, and perhaps apatite’s maximum crystal size. Even
after an organism’s death, its bioapatite remains reactive, as
demonstrated by the complex process of fossilization (Kohn
et al. 1999) and by the ability of bone apatite to remediate
metal-contaminated aqueous systems (Manning 2008 this
issue). Not only is it challenging to fully characterize bioapatite, but it is difficult to synthesize material that faithfully replicates its properties through harnessing the
feedback between apatite’s composition and structure, i.e.
to imitate what biological systems do routinely. Apatite is
where mineralogy meets geochemistry and biology.
ACKNOWLEDGMENTS
We thank numerous colleagues and students who have
worked with us over the years on apatite and bone. JDP and
BW thank Shuyi Man for the drawings used in Figure 2,
John Freeman for IR spectra of apatite, Chaoxian Zhou for
Raman spectra of bison tooth and bone, Daniel Giammar
for calculating the solubility of apatite, and Stephanie
Novak for preparing the thin section of the bison jaw. We
thank Matthew Silva and Eric Oelkers for commenting on
an earlier version of the manuscript. JDP and BW gratefully
acknowledge partial funding from Washington University’s
Center for Materials Innovation. EVJ acknowledges support
from the UK Natural Environment Research Council
(NER/D/S/2003/00678). Eppell SJ, Tong W, Katz JL, Kuhn L,
Glimcher MJ (2001) Shape and size of
isolated bone mineralites measured using
atomic force microscopy. Journal of
Orthopaedic Research 19: 1027-1034
Glimcher MJ (2006) Bone: Nature of the
calcium phosphate crystals and cellular,
structural, and physical chemical
mechanisms in their formation. In: Sahai
N, Schoonen MAA (eds) Medical
Mineralogy and Geochemistry. Reviews
in Mineralogy & Geochemistry 64, pp
223-282
Gross KA, Berndt CC (2002), Biomedical
application of apatites. In: Kohn MJ,
Rakovan J, Hughes JM (eds) Phosphates:
Geochemical, Geobiological, and Material
Importance. Reviews in Mineralogy &
Geochemistry 48, pp 631-672
Hughes JM, Rakovan J (2002) The crystal
structure of apatite, Ca5(PO4)3(F,OH,Cl).
In: Kohn MJ, Rakovan J, Hughes JM (eds)
Phosphates: Geochemical, Geobiological,
and Material Importance. Reviews in
Mineralogy & Geochemistry 48, pp 1-12
Ito A, Maekawa K, Tsutsumi S, Ikazaki F,
Tateishi T (1997) Solubility product of
OH-carbonated hydroxylapatite. Journal of
Biomedical Materials Research 36: 522-528
103
Jones JR, Gentleman E, Polak J (2007)
Bioactive glass scaffolds for bone
regeneration. Elements 3: 393-399
Kohn MJ, Schoeninger MJ, Barker WW
(1999) Altered states: effects of diagenesis
on fossil tooth chemistry. Geochimica et
Cosmochimica Acta 63: 2737-2747
LeGeros RZ (2002) Properties of osteoconductive biomaterials: Calcium phosphates.
Clinical Orthopaedics and Related Research
395: 81-98
LeGeros RZ, LeGeros JP (1984) Phosphate
minerals in human tissue. In: Nriagu JO,
Moore PB (eds) Phosphate Minerals.
Springer-Verlag, New York, pp 351-395
Mann S (2001) Biomineralization:
Principles and Concepts in Bioinorganic
Materials Chemistry. Oxford University
Press, Oxford, 198 pp
Manning DAC (2008) Phosphate minerals,
environmental pollution and sustainable
agriculture. Elements 4: 105-108
Matsumoto T, Okazaki M, Nakahira A,
Sasaki J, Egusa H, Sohmura T (2007)
Modification of apatite materials for bone
tissue engineering and drug delivery
carriers. Current Medicinal Chemistry
14: 2726-2733
A PRIL 2008
Pan Y, Fleet ME (2002) Compositions of
the apatite-group minerals: Substitution
mechanisms and controlling factors. In:
Kohn MJ, Rakovan J, Hughes JM (eds)
Phosphates: Geochemical, Geobiological,
and Material Importance. Reviews in
Mineralogy & Geochemistry 48, pp 13-50
Pasteris JD, Wopenka B, Freeman JJ, Rogers
K, Valsami-Jones E, van der Houwen
JAM, Silva MJ (2004) Lack of OH in
nanocrystalline apatite as a function of
degree of atomic order: implications for
bone and biomaterials. Biomaterials 25:
229-238
Rogers KD, Zioupos P (1999) The bone
tissue of the rostrum of a Mesoplodon
densirostris whale: A mammalian
biomineral demonstrating extreme
texture. Journal of Materials Science
Letters 18: 651-654
Sammons RL, Marquis PM, Macaskie LE,
Yong P, Basner C (2004) Developments
in the use of calcium phosphates as
biomaterials. In: Valsami-Jones E (ed)
Phosphorus in Environmental Technology:
Principles and Applications. IWA
Publishing, London, pp 582-609
Skinner HCW (2005) Biominerals.
Mineralogical Magazine 69: 621-641
Weiner S, Wagner HD (1998) The material
bone: Structure-mechanical function
relations. Annual Review of Materials
Science 28: 271-298
Williams D (ed) (1990) Concise Encyclopaedia
of Medical & Dental Materials. Pergamon
Press, Oxford, 412 pp
Wopenka B, Pasteris JD (2005) A mineralogical perspective on the apatite in bone.
Materials Science and Engineering
C25: 131-143 Urist MR, Silverman MF, Buring K, Dubek
FL, Rosenburg JM (1967) The bone
induction principle. Clinical
Orthopaedics 53: 243-283
GLOSSARY
Apatite – General name for a group of mineral species with the
same structure but a range of compositions, including
Ca5(PO4,CO3)3(OH,F,Cl)
Lacunae – Small voids that develop in the lamellae of osteons and
house osteocytes and their elongated projections; one osteocyte
per lacuna
ATP – Adenosine triphosphate, the energy currency of life. By losing
the endmost of its three phosphate groups, ATP releases energy,
which organisms use to carry out all biological functions.
Osseous – Of the bone, bony
Bioactivity – In the context of this paper, the ability to nucleate
bioapatite in vitro or in vivo
Biocompatibility – The ability of a material or device to provide
appropriate functionality in the host in a specific application,
e.g. not toxic
Biomaterials – Synthetic substitute materials for biological applications
Biomimetic – Mimicking a biological material; application of natural biological methods or systems to understand or design a (synthetic) material or process
Biomineralized materials – Natural biological composites consisting
of biologically precipitated mineral and an organic component,
typically a protein; examples: bones, teeth, shells
Bulla – A bony structure that houses the inner ear of the whale; it
has a much greater degree of mineralization than typical bone,
i.e. it is hypermineralized.
Cancellous bone (also known as spongy or trabecular bone) – Osseous
tissue of high porosity and surface area, but low density and
strength, normally found in the ends of long bones
Carbonated apatite – A descriptive term, rather than a formal mineral
species; the formal species most similar to bone apatite is
carbonate-hydroxylapatite.
Ceramic – A synthetic, inorganic, non-metallic material hardened
by heat
Osteoblasts – Cells that synthesize type-I collagen matrix of new
bone, which subsequently becomes mineralized; they reside
only on the surface of bone tissue and eventually may become
osteocytes.
Osteoclasts – Large, multinucleated, mobile cells that resorb bone
mineral and collagen; using their ruffled borders, they seal off a
volume of bone and then secrete collagenase and acid into the
defined microenvironment.
Osteoconductivity – The ability to bond with bone and support
bone growth
Osteocytes – Those osteoblasts that eventually become surrounded
by bone and incorporated into lamellae of the osteon; they
reside in lacunae.
Osteoinductivity – The ability to allow cellular functions that lead to
osteoblast cell formation and growth of new bone in non-osseous
sites
Osteons – Concentrically layered, cylindrical structures (seen as circles
in thin section) of the cortical bone, which surround a central
vascular (Haversian) canal (FIG. 2C). They are caused by the
remodeling of the bone, i.e. dissolution and new precipitation
of collagen and bioapatite.
Rostrum – The projecting snout of a “beaked” whale; it is almost
totally mineralized (i.e. hypermineralized) bone and it shows
extensive osteon development.
Young’s modulus – A measure of “stiffness,” i.e. resistance of a
material to elongation and deformation
Cortical bone (also known as dense or compact bone) – Dense
osseous tissue that comprises the shafts of long bones and the
hard outer layer of most bones
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