Tooth enamel proteins enamelin and amelogenin cooperate to regulate the growth morphology of octacalcium phosphate crystals

Abstract

To examine the hypothetical cooperative role of enamelin and amelogenin in controlling the growth morphology of enamel crystals in the post-secretory stage, we applied a cation selective membrane system for the growth of octacalcium phosphate (OCP) in the truncated recombinant porcine amelogenin (rP148) with and without the 32kDa enamelin fragment. Enamelin alone inhibited the growth in the c-axis direction more than rP148, yielding OCP crystals with the smallest aspect ratio of all conditions tested. When enamelin was added to the amelogenin "gel-like matrix", the inhibitory action of the protein mixture on the growth of OCP in the c-axis direction was diminished, while that in the b-axis direction was increased. As a result, the length to width ratio (aspect ratio) of OCP crystal was markedly increased. Addition of enamelin to amelogenin enhanced the potential of amelogenin to stabilize the amorphous calcium phosphate (ACP) transient phase. The ratio of enamelin and amelogenin was crucial for stabilization of ACP and the growth of OCP crystals with larger aspect ratio. The cooperative regulatory action of enamelin and amelogenin was attained, presumably, through co-assembling of enamelin and amelogenin. These results have important implications in understanding the growth mechanism of enamel crystals with large aspect ratio.

Figure 1

X-ray diffraction patterns of the products grown in (a) 10% rP148, (b) 4μg/ml enamelin in 10% rP148 (rP148+4En), (c) 10μg/ml enamelin in 10% rP148 (rP148+10En), (d) 40μg/ml enamelin in 10% rP148 (rP148+40En) and (e) 40μg/ml enamelin (40En).

Figure 2

SEM images of small particles adhere to crystals grown in (a)10μg/ml enamelin in 10% rP148 (rP148+10En) and (b) 40μg/ml enamelin in 10% rP148 (rP148+40En).

Figure. 3

(a) Length to width (L/W) ratio and (b) width to thickness (W/T) ratio of crystals grown in 10% rP148, 4μg/ml enamelin in 10% rP148 (rP148+4En), 10μg/ml enamelin in 10% rP148 (rP148+10En), 40μg/ml enamelin in 10% rP148 (rP148+40En) and 40μg/ml enamelin (40En).

Figure 4

SEM images of the products grown in amelogenin with increasing concentration of the 32kDa enamelin (a) 10% rP148, (b) 40μg/ml enamelin (40En), (c) 10μg/ml enamelin in 10% rP148 (rP148+10En) and (d) 40μg/ml enamelin in 10% rP148 (rP148+40En). In (d2), it is important to note that the small particles adhered to crystals. (a1-d1 are at a magnification of 3K and a2-d2 are at a magnification of 10K).

Figure 5

SEM images and EDXA mapping of the pseudo-spherical particles observed in the rP148+40En sample. The brightness and contrast of the EDXA maps were altered for clarity. (a) A low resolution SEM image of the area that was mapped. Scale bar 500nm. (b) The EDXA map of calcium (Ca-K_α x-ray radiation at 3.691 keV). (c) The EDXA map of phosphorus (P-K_α x-ray radiation at 2.013 keV). It is important to note that the sample was coated in gold and some of the phosphorus signal may have come from Au-M_α x-ray radiation at 2.120 keV, although the Au-M_α and P-K_α spectral windows do not overlap. (d) The EDXA spectrum of the particles. The calcium K_α and K_β peaks are clearly visible, but the phosphorus K_α peak only appears as a shoulder on the side of a large gold M_α peak.