Biomedical Engineering Reference
In-Depth Information
4.4
Controlling Nucleation and Aggregation of HAP or OCP
Mouse amelogenin (composed of 179 amino acids), which is involved in enamel
formation, was expressed in
Escherichia coli
using a recombinant DNA technique.
When recombinant mouse amelogenin (rM179) was mixed in a calcium phosphate
solution, which is supersaturated with respect to HAP and OCP, OCP formation was
accelerated [
48
]. Nucleation of crystals occurs when the thermodynamic driving
force of the crystal phase overcomes the negative effect of the surface free energy of
small particles. If a part of the atomic arrangement of rM179 is complementary
to the OCP crystal faces, the binding of rM179 to calcium phosphate particles
reduces the surface free energy of the particles, which induces heterogeneous
nucleation of the OCP. This heterogeneous nucleation apparently accelerates OCP
precipitation. The amount of OCP precipitated depends on the rM179 concentration.
The maximum amount of OCP was precipitated at an rM179 concentration of
6.5
g/mL, and OCP deposition decreased as the rM179 concentration was
increased. This was explained as follows: an increased amount of rM179 results
in more binding of calcium and phosphate ions to the protein, which reduces the
degree of solution supersaturation.
Moreover, rM179 not only accelerates nucleation but also regulates the assembly
of HAP crystals. To investigate the function of the charged C-terminal domain of
amelogenin, Beniash et al. generated rM166, which lacks the C-terminal domain
of rM179. Crystallization experiments were performed in the presence of these
proteins, and the properties of the resultant crystals were compared [
49
]. The HAP
crystals that formed in the presence of rM179 were bundle-like aggregates aligned
with the
c
-axis and were organized in a manner similar to the HAP in enamel. Those
that formed in the presence of rM166 assembled into aggregates with a random
orientation. This indicates that the C-terminal domain of amelogenin is essential for
ordered assembly of HAP crystals. When pre-aggregated amelogenin was added to
supersaturated solution, The HAP crystals aggregated with a random orientation,
and aggregates aligned along the
c
-axis were not obtained. This suggests that the
self-assembly of amelogenin and the organization of HAP crystals proceed in a
cooperative manner.
Wang et al. expressed full-length recombinant porcine amelogenin (rP172) in
E. coli
and investigated the effect of the protein on crystallization and crystalline
calcium phosphate aggregation. Spherical aggregates of OCP crystals were formed
in the absence of rP172, and HAP crystal aggregates aligned along the
c
-axis formed
in its presence. The induction time for nucleation decreased as the concentration of
amelogenin was increased (measured in the concentration range of 0.5-5.0
g/mL;
maximum acceleration took place at 5
g/mL).
In addition to the decreased induction time for nucleation, HAP and not OCP was
obtained, suggesting the presence of structural matching at the interface between
atomic arrangements of the amelogenin surface and HAP crystal faces ([
50
]; A role
of amelogenin proposed by Wang et al. was also described in Sect. 5.4.4 in Chap. 5).
This suggestion is based on the theory that the nucleation energy barrier decreases
