Magnesium-aspartate-based crystallization switch inspired from shell molt of crustacean

Abstract

Many animals such as crustacean periodically undergo cyclic molt of the exoskeleton. During this process, amorphous calcium mineral phases are biologically stabilized by magnesium and are reserved for the subsequent rapid formation of new shell tissue. However, it is a mystery how living organisms can regulate the transition of the precursor phases precisely. We reveal that the shell mineralization from the magnesium stabilized precursors is associated with the presence of Asp-rich proteins. It is suggested that a cooperative effect of magnesium and Asp-rich compound can result into a crystallization switch in biomineralization. Our in vitro experiments confirm that magnesium increases the lifetime of amorphous calcium carbonate and calcium phosphate in solution so that the crystallization can be temporarily switched off. Although Asp monomer alone inhibits the crystallization of pure amorphous calcium minerals, it actually reduces the stability of the magnesium-stabilized precursors to switch on the transformation from the amorphous to crystallized phases. These modification effects on crystallization kinetics can be understood by an Asp-enhanced magnesium desolvation model. The interesting magnesium-Asp-based switch is a biologically inspired lesson from nature, which can be developed into an advanced strategy to control material fabrications.

Fig. 1.

Morphology, phase, and composition of cuticles at different molt stages. (A) Photographs of Armadillidium vulgare in the different molt states (–5). (B) SEM images and SAED patterns of the cross-sections of exocuticle layer shown in A. (C and D) XRD patterns and FT-IR spectra, respectively. (E) Organic contents of the cuticles during the molt process.

Fig. 2.

Structure, element, and phase distribution in an inter-molt cuticle. (A) Distribution of Mg, Ca, and P elements in an inter-molt cuticle by electron probe microanalysis corresponding to the cuticle in B. (B) SEM image of sagittally cleaved fully mineralized cuticle of Armadillidium vulgare. The cross-section of cuticle showed the thin epicuticle (ep), calcified exocuticle (ex), and endocuticle (en), and membranous layers (ml). One through three indicate the positions where Raman spectra were collected. (C) Raman spectra recorded at corresponding sites indicated in B. A spectrum of sternal ACC is used as the reference. Curve 1: exocuticle contained well-crystalline calcite and minor ACC; Curve 2: boundary between exocuticle and endocuticle composed of ACC and minor calcite; Curve 3: endocuticle composed of ACC phase without any detectable calcite. (D) A magnified image of spectra in C.

Fig. 3.

Kinetics of phase transformation from ACC to calcite. (A) Kinetic plot of phase transformation in the absence and presence of magnesium and Asp. (B) FT-IR spectra of slightly crystallized samples extracted at different timescales under different conditions. The inset images are in situ phase evolution investigated by polarizing microscopy in the solvent. (Scale bar, 100 μm.) The calcite and ACC phases are expressed as the bright and dark on the image, respectively. The bright densities can reflect the crystallization degrees during the transition visually.

Fig. 4.

Kinetics of phase transformation of ACP at different conditions. (A) Kinetic plot of phase transformation from ACP to HAP under different conditions. Splitting function (SF) of calcium phosphate is used as the crystallization indicator due to the characteristic splitting of the phosphate anti-symmetric bending mode (SI Text and Fig. S5B). (B) FT-IR spectra of slightly crystallized calcium phosphates extracted at different timescales under different conditions. Pure ACP is used as the reference.