Bone Bio-Mineralization: in Depth Analysis of
Hydroxylapatite Crystallization Through
Experiments and Simulations
Barbara Pavan, Dan Zhou, Brandon Whitman, Marco Fornari and Mary
Tecklenburg
Science of Advanced Materials Program, Departments of Chemistry and Physics
Central Michigan University, Mt. Pleasant MI USA.
Calcium phosphate is the dominant solid mineral phase within skeletal and dental
tissue of vertebrates. It has a similar composition and structure to the apatite group
minerals, with general formula Ca10(PO4)6(OH,Br,F,Cl)2. The structural properties of
bones, such as density and strength, are related to the microstructure and morphology
of the apatitic material, which differs from the synthetic, highly crystallized and
geological one. The specific properties of biological apatites reflect their physiological
functions, both as a mechanical support and as an ion reservoir [1].
A detailed knowledge of the crystallization process of HAp is important not only to
understand the bone formation but also because apatitic materials are widely used in
bone and teeth implants [2].
The bio-mineralization of bone tissue is a complex process to study since it
involves biological and organic components, and the bones undergo continuous
remodeling and turnover. Bio-mineralization has been proposed to occur through a
progression of mineral phases and Octacalcium phosphate (OCP - structural formula
Ca8(HPO4)2(PO4)4·5H2O) has been suggested as a precursor to biological apatites. In
vitro studies proved the conversion of amorphous calcium phosphate (ACP) to
hydroxylapatite (HAp) [3]. While OCP, as an unstable intermediate, has been detected
by Raman micro-spectroscopy in biological samples [4], the evolution from
amorphous calcium phosphate to apatite, through octacalcium phosphate or OCP-like
intermediates, is not yet broadly accepted in the literature and the exact kinetics of the
mineralization process is still unknown.
The aim of our work was to elucidate the crystallization process of HAp under
physiological conditions (pH 7.4, 20-37°C), by Raman micro-spectroscopy. The shift
in the ν1PO4 vibration band was used to monitor the reaction evolution over time
(Figure 1). Calcium phosphate precipitation begins immediately after mixing but
remains in the amorphous state (ν1PO4 950 – 953 cm-1) for a time that is temperature
dependent and then very quickly transforms to apatite (ν1PO4 958 – 960 cm-1). The
samples were also characterized by XRD and SEM to determine their crystal phase
and morphology.
To provide a more accurate interpretation of the vibrational spectra obtained
experimentally, fluorapatite (FAp) and HAp were simulated by ab initio calculations.
The unit cells were built starting from available crystallographic data. A plane waves
basis set was used as implemented in the Quantum-ESPRESSO package [5].
After proper relaxation of the crystal lattices, the Raman vibration frequencies were
computed and compared to our experimental results.
FIGURE 1. (a) ν1PO4 bands for amorphous calcium phosphate to hydroxylapatite conversion,
shifting from 951 to 960 cm-1. The curves from left to right correspond to 10, 75, 89 and 180 minutes.
(b) ν1PO4 frequencies as function of reaction time at 20°C, pH 7.4. The frequency shift was found to follow a sigmoidal behavior.
Our study provided a further insight into the crystallization of hydroxylapatite
under physiological conditions. The crystallization process was confirmed to occur
through a progression of phases, from amorphous to a partially crystalline apatitic
product and the crystallization kinetics as function of temperature showed Arrhenius
behavior. The combination of experimental and theoretical results was demonstrated
to be a valuable tool to better understand how Raman frequencies of different
chemical groups are affected by their surrounding environment.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grant #AR047969-05A2.
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