This is the peer-reviewed version of the article
Uskoković, V. and Uskoković, D. P. (2011), Nanosized hydroxyapatite and other calcium phosphates:
Chemistry of formation and application as drug and gene delivery agents. J. Biomed. Mater. Res., 96B:
152–191.
which has been published in final form at http://dx.doi.org/10.1002/jbm.b.31746. This article may be used
for noncommercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
This work is licensed under
Creative Commons - Attribution-Noncommercial-NoDerivative Works 3.0 Serbia
Nanosized Hydroxyapatite and Other Calcium Phosphates:
Chemistry of Formation and Application as Drug and Gene Delivery Agents
Vuk Uskoković
1
, Dragan P. Uskoković
2
1
Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences,
University of California, 707 Parnassus Avenue, San Francisco, CA 94143, USA
2
Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Knez Mihailova 35/IV, 11000 Belgrade,
Serbia
Abstract The first part of this review looks at the fundamental properties of hydroxyapatite, the
basic mineral constituent of mammalian hard tissues, including the physicochemical features that
govern its formation by precipitation. A special emphasis is placed on the analysis of qualities of
different methods of synthesis and of the phase transformations intrinsic to the formation of
hydroxyapatite following precipitation from aqueous solutions. This serves as an introduction to
the second part and the main subject of this review, which relates to the discourse regarding the
prospects of fabrication of ultrafine, nanosized particles based on calcium phosphate carriers
with various therapeutic and/or diagnostic agents coated on and/or encapsulated within the
particles. It is said that the particles could be either surface-functionalized with amphiphiles,
peptides, proteins or nucleic acids or injected with therapeutic agents, magnetic ions or
fluorescent molecules. Depending on the additive, they could be subsequently used for a variety
of applications, including the controlled delivery and release of therapeutic agents
(extracellularly or intracellularly), magnetic resonance imaging and hyperthermia therapy, cell
separation, blood detoxification, peptide or oligonucleotide chromatography and ultrasensitive
detection of biomolecules, and in vivo and in vitro gene transfection. Calcium phosphate
nanoparticles as carriers of therapeutic agents that would enable a controlled drug release to treat
a given bone infection and at the same be resorbed in the body so as to regenerate hard tissue lost
to disease are emphasized hereby as one of the potentially attractive smart materials for the
modern medicine.
Keywords: Bone, Calcium phosphate, Drug Delivery, Hydroxyapatite, Precipitation, Soft
Chemistry, Theranostics
Introduction: Hydroxyapatite as the main constituent of bone
Hydroxyapatite (HAP) is to most people known as the mineral component of bone
1,2,3
.
Therein, stiff HAP crystals are responsible for imparting an appropriate compressive strength,
whereby collagen fibers, able to dissipate energy effectively, provide superior elastic properties,
thus ameliorating the brittleness of the sole HAP
4
. However, the exceptional stiffness and
strength of bone come not only from the synergetic combination of material properties of its
mineral and organic components, but from its hierarchical, superstructural organization as well
5
.
Bone is an organ that not only does provide a basic mechanical support to the body by
generating and transferring forces that are involved in locomotion, but it also has various other
functions
6
. For example, by storing minerals within, mostly calcium and phosphate, it presents
the main mineral reservoir for the body. Absorption and release of salts is the mechanism by
which bones buffer the blood and prevent excessive pH changes. Bones, such as skull or ribs,
also serve to physically protect vital internal organs, including brain, heart and lungs. Some
bones also act as fabrics for producing red and white blood cells. Bone has formed in co-
evolution with the surrounding tissues of organisms and the environment. In eukaryotic cells,
calcium ions play a plethora of functions, including that of a messenger in various signal
transduction pathways. Calcium-activated ATPase, Na
+
/Ca
2+
exchangers, calcium channels and
intracellular calcium binding proteins maintain a fine Ca
2+
homeostasis in cells such as
odontoblasts. On one hand, a controlled imbalance of internal calcium levels is a precursor for
the bone-building activity, which, unlike in the case of amelogenesis and dentinogenesis, always
proceeds through external accretion. On the other hand, increased amounts of intracellular Ca
2+
may result in uncontrolled secretion and internal precipitation. Namely, Ca
2+
is known to be a
part of harmful deposits in the body, such as atherosclerotic plaque, kidney stones and dental
calculi
7
. It also has a strong tendency to bond with many proteins, particularly phosphorylated
ones. In case of ATP, its aggregation may be induced thereby, resulting in the loss of biological
function. On the other hand, binding of Ca
2+
to phosphorylated proteins involved in
biomineralization, such as osteopontin in bone or dentin phosphophoryn, is a vital step in
formation of hard tissues of bone and dentin
8
.
Aside from its mineral and protein components, bone is also populated by cells,
macromolecules and blood vessels. Three types of cells are involved in maintaining a healthy
bone structure: a) osteocytes involved in signal transduction of mechanical stimuli; b)
osteoblasts, which are derived from mesenchymal stem cells and secrete collagenous proteins,
thereby building the bone material; and c) osteoclasts, which are derived from hematopoietic
marrow cells and secrete acids and proteases, thereby degrading the mineralized tissue. RANKL,
a protein molecule bound to the osteoblast surface and serving to activate osteoclasts has been
intensively investigated because of its role in facilitating an optimal communication between
these two types of cells
9
. Overexpression of RANKL has been linked to a variety of degenerative
bone diseases, including rheumatoid arthritis and osteoporosis
10
. Through the cooperative action
of osteoblasts and osteoclasts, bone is constantly being remodeled in response to the
physiological requirements. Julius Wolff was the first to propose that bone remodels itself when
force is exerted on it by the mechanism according to which the internal architecture of the
trabecular bone first undergoes adaptive changes when placed under load, followed by secondary
changes to the external cortical portion of the bone
11
. An evidence of an impressive remodeling
capacity of bone has come from the observed modifications in bone shape and density in
astronauts subjected to microgravity conditions for prolonged periods of time
12
. Bone is, for
example, often regarded as a living mineral due to its continual growth and dissolution,
formation and degradation, renewal and remodeling, taking place during the organism’s lifetime.
Fig.1. Bone (left) is a complex, hierarchically structured biological material in which the building components are
precisely arranged at scales spanning half a dozen of orders of magnitude. The image on the left shows sketches of
the structural elements of cortical/compact bone (which comprises the harder, outer layer of the cross-section of
bone, surrounding the softer trabecular/spongy/cancellous bone) at different scales. The image on the upper right
side shows the nanostructure of mineralized collagen fibers in bone, whereby the image below displays the fine
structure of dental enamel, the hardest substance in the body. Within the former structure, HAP particles are
incorporated within the organic matrix, whereas the latter structure is composed of an almost pure mineral with
elongated HAP nanofibers connected into bundles and forming equally uniaxially directed enamel rods. Reprinted
with permission from Refs.13, 14, and 15.
Many mysteries, such as the role of “sacrificial” bonds that break under stress, but only to
be reformed at a later time, imparting durability and resilience to hard tissues, still surround the
superior functioning of this basic material of Nature
16
. Many new functions of bone have also
been gradually revealed over time. It has now been established that bone does not only act as a
reservoir for minerals, primarily calcium and phosphate, which circulate through blood in
supersaturated concentrations, but also stores growth factors, fatty acids, heavy metals and other
toxic elements, and is involved in buffering the blood by controlled release of alkaline salts. Be
that as it may, bone presents a connective tissue and a failure of the ability to stay “connected”
and properly transmit stress throughout the body has an implication of slowly bringing about
dysfunction of many other, close or distant segments of skeleton and the body. In view of that,
finding the ways to heal the impaired bone tissue in timely manner can be regarded as one of the
most important tasks that medicine can contribute to. In view of that, bone research has a special
meaning in the world of medicine. However, understanding bone is a challenging task for the
modern scientists, especially because it requires an atypical inter-disciplinary element in one’s
approach, explained by the fact that bone stands at the interface between many separate fields of
science. The more one focuses one’s attention on finer levels of organization, the more of the
biological approaches cede their place to fundamental physicochemical methods of probing bone
structure and properties.
And just as usual, as scientific attention is focused to ever smaller physical details, the
things do not get simplified, but become ever more complex instead
17
. Hence, the structural
arrangement of nanosized HAP crystals within the collagen matrix is still a subject of scrutiny. It
is still not resolved whether mineralization first occurs within the 40 nm wide gap or the 27 nm
wide overlap region of collagen fibers. Contrary to earlier assumptions, a recent cryo-TEM study
has shown that upon mineralization of collagen the mineral first fills the overlap region and only
then it proceeds to incorporate itself within the gap region
18
. Two forms of HAP crystals in
dentin and bone can also be distinguished based on where they are found: extrafibrillar and
intrafibrillar
19
. The former are larger nanocrystals (plate-shaped with 2 – 3 nm in thickness and a
few tens of nanometers in length and width) existing in-between individual fibrils (that is,
bundles of collagen fibers having ~ 1.5 – 3.5 nm in diameter, and are essentially individual
elongated, triple helix molecules), whereas the latter are found to figure as links between
individual fibers along their long axis. However, the role of each has not been discerned yet
20
.
Also, just as the structural water has been added to the basic structural diagrams of
proteins in recent years
21
, its role for the proper mechanical performance of hard tissues has been
increasingly pointed out by researchers
22
. Collagenous tissues, such as bone or dentin, which
contain approximately 70 wt% apatite, 20 wt% collagen and 10 wt% water (with only about 3 %
of noncollagenous proteins, including some polysaccharides as well), are nowadays known to
partly owe their mechanical properties to the structural water
23,24
. Hydrated dentin is, for
example, shown to dramatically degrade in toughness following its dehydration
25,26
. Also, in
case of collagen molecules wherein backbone hydrogen bonding between polypeptide chains in
its triple-stranded structure does not present the major stabilizing force, unlike in the case of α–
helices and β–sheets, additional enthalpic contributions are known to come from water molecules
that form a “scaffold” around the surface of the triple helix, implying that water plays an intimate
role in stabilizing this protein
27
. This observation coincides with the recently observed 10-fold
drop in tensile properties of single fibrils of collagen following desorption of the bound water in
vacuum, even though the strength of the molecule was the same in water and air
28
.
Hence, even though the fascinating properties of bone are products of precise and
intricate arrangement of its building blocks on many different levels – from nanometer to
millimeter scales (Fig.1) – the complexity of each one of these building blocks is equally
complex as to deserve paying sole research attention thereto. In fact, the complexity of this
material has ever since puzzled scientists involved in bone research; hence, the name of this
compound, apatite, derives from Greek απαταο, which means “to deceive”. The respective
mineral was, however, named so because it had easily been mistaken for other, more precious
minerals
29
; yet, a drop of lime juice was sufficient to dissolve it. Unlike some other similarly
complex materials, such as doped manganites which exhibit an enormous set of electric and
magnetic behaviors depending on the structural arrangement of the constitutive ions
30,31
, in case
of HAP the main emphasis is on the breadth of possible mechanical properties depending on
different phase arrangements and the structure and morphology of the compound. Another
remarkable feature of this material is a considerably low crystal growth rate even under relatively
high supersaturations. The reason for this is thought to lie in the complex growth mechanism that
involves precipitation of amorphous, ~ 1 nm sized solid units called Posner’s clusters in the first
stage of the process, and their aggregation and ionic rearrangement followed by an increased
compactness and crystallinity in the second stage
32
. Owing to the fact that this mechanism
resembles the one of the growth of protein and viral crystals that involves chirality selection and
orientation arrangements, the low crystal growth rate of HAP is often compared to that of these
biological compounds
33
. Low crystallinity of particles precipitated under physiological
conditions and stoichiometric sensitivity to mildest changes in synthesis conditions are additional
