Nacre ultrastructureAmorphous precursors, aggregation and crystallization

Abstract

Abstract Biominerals are organo-mineral composites synthetized by organisms. A composite is defined as the material that results from the combination of two or more different and structurally complementary materials. A composite has certain structural or functional properties, which are not present in the individual components. These emergent properties appear from the interaction of their individual constituents, which normally involves an organic matrix and a reinforcing material, organized in a hierarchical manner. Vertebrate bone, echinoderm spines and molluscs shells are some examples of composite biomaterials. The field of biomineralization aims to understand the formation, structure and properties of the minerals deposited by biological systems. Molluscs are excellent biocomposite constructors. Making use of a reduced number of elements (shells are mainly composed of calcium carbonate, in the form of calcite or aragonite, and 1-5.5% of organic material) they can produce more than a dozen microstructures. Crystallites and the organic matter organize into particular patterns termed microstructures, characterized by recurrent spatial and crystallographic relationships. Frequently, shells are composed by layers with different microstructures, and even different mineralogies. For this to occur, organisms exert an exquisite multi-level control over the transport, aggregation and deposition of the mineral. The formation processes of the different microstructures are of great interest, in part due to their applicability in the development of biomimetic materials. One of the hot topics today is the formation of crystalline materials through amorphous phases. Its discovery attracted great scientific interest, since the classical mineralization models developed in the last century described the crystal growth through the addition of ions, but not by aggregation of higher-order particles. The present PhD work addresses the issue of the amorphous precursors in one of the best studied mollusc microstructures, the nacre. The presence of amorphous calcium carbonate (ACC) in the forming tablets has been recently demonstrated. Our approach was to make a high resolution study encompassing two main lines of evidence: imaging and elemental analysis. We have studied three stages of the nacre formation process using a gastropod species as model system. Gastropod peculiarity is the presence of a dense organic membrane (the surface membrane), which covers the mineralization compartment. Within such compartment, the organic membranes (interlamellar membranes) are arranged first and the mineral forms between them. The gastropod nacre tablets stack forming towers. This PhD study is divided in three sections: - Determination of the transport mechanism of the ACC: vesicles function and surface membrane dynamics. - Characterization of the ACC in the forming tablets (distribution and relationships with the crystalline phase). - Study of the morphology in the mature tablet (nanogranularity). For the first section we have used transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). We have imaged and analysed the vesicles segregated by the mantle cells and the surface membrane. The results show that some of these vesicles transport calcium-rich material attached to their inner surface; the membranes of these vesicles also contain calcium. The surface membrane and the interlamellar membranes show certain amount of calcium. The surface membrane undergoes a structure modification process, from a fuzzy aspect looking towards the mantle cells, to a defined porous structure at the mineralization compartment side. When the interlamellar membranes detach from the surface membrane the porous structure is evident. For the second section, about the transformation and crystallization of ACC, the amorphous phase has been imaged by means of TEM/STEM. The forming tablets are composed by an aggregation of globular particles (20-50 nm) that present a crystalline core (~ 30 nm average size) embedded in amorphous matrix (5-10 nm thickness). This amorphous layer presents a mixture of organic material and ACC, both identified by EELS. The crystalline nanodomains present complex, pseudodendritic forms, which might be connected in the third dimension. The third section of this PhD study is dedicated to the study of the lineations in the surface of the nacre tablets. It was carried out by scanning electron microscopy (SEM), electron backscattered diffraction (EBSD) and atomic force microscopy (AFM). Images clearly show that lineations are the result of the aggregation of calcium carbonate nanoglobules. EBSD maps show that the lineations are aligned with the a-axis of the tablets. Our model proposes that this morphology results from the differential absorption of macromolecules along the aragonite crystallographic axes. Finally, it is interesting to note that the results of the two latter sections match well, reinforcing our hypothesis that the nanogranular pattern characteristic of biominerals might not represent the aggregation unit sizes as was implied by some authors, but are the result of the subsequent crystallization process.