The nucleation and growth of calcium phosphate by amelogenin

Abstract

The nucleation processes involved in calcium phosphate formation in tooth enamel are not well understood but are believed to involve proteins in the extracellular matrix. The ability of one enamel protein, amelogenin, to promote the nucleation and growth of calcium phosphate was studied in an in vitro system involving metastable supersaturated solutions. It was found that recombinant amelogenin (rM179 and rp(H)M180) promoted the nucleation of calcium phosphate compared to solutions without protein. The amount of calcium phosphate increased with increasing supersaturation of the solutions and increasing protein concentrations up to 6.5 μg/mL. At higher protein concentrations, the amount of calcium phosphate decreased. The kinetics of nucleation was studied in situ and in real time using a quartz crystal microbalance (QCM) and showed that the protein reduced the induction time for nucleation compared to solutions without protein. This work shows a nucleation role for amelogenin in vitro which may be promoted by the association of amelogenin into nanosphere templates, exposing charged functionality at the surface.

Fig. 1

Change in (a) calcium concentration and (b) particle density for nucleation from 2.2 mM CaCl₂ · 2H₂O solutions as a function of protein concentration after 24 h.

Fig. 2

FESEM images of calcium phosphate formed from 2.2 mM CaCl₂ · 2H₂O solutions after 24 h containing (a) no amelogenin, (b) 0.065 μg/mL rp(H)M180, (c) 0.65 μg/mL rp(H)M180, (d) 6.5 μg/mL rp(H)M180, (e) 65 μg/mL rp(H)M180, and (f) 6.5 μg/mL rM179 (parts (d) and (f) and their insets show the similarity in size, density, and morphology of the rp(H)M180 and rM179 grown under identical conditions). Controls showed essentially no calcium phosphate growth, consistent with ICP and pH measurements. The maximum nucleation occurred at 6.5 μg/mL, yielding the highest nucleation density and smallest particle size.

Fig. 3

Glancing angle X-ray diffraction spectra of calcium phosphate formed from 2.2 mM CaCl₂ · 2H₂O solutions, 6.5 μg/mL amelogenin solutions after 40 h.

Fig. 4

Kinetics of deposition of calcium phosphate from 2.2 mM CaCl₂ · 2H₂O solutions containing 6.5 and 65 μg/mL amelogenin using a quartz crystal microbalance.

Fig. 5

Schematic of a possible mechanism for nucleation: the association of the amelogenin monomers into a nanosphere (arrow indicating the C-terminal domain (red)), calcium and phosphate ion binding at the interface, and amino acid sequences of the C-terminal domain of amelogenin (red: acidic amino acids, blue: basic amino acids).