Noninvasive Microcomputerized X-ray Tomography Visualization of Mineralization Directed by Sea Urchin- and Nacre-Specific Proteins

The biomineralization process offers novel principles for crystal engineering and solid-state chemistry, but to achieve this we must first understand how organisms such as the mollusk and sea urchin craft skeletal elements such as the shell and embryonic spicule, respectively. In vitro studies of mollusk- and sea urchin-associated proteins reveal that these proteins form hydrogel particles that control the nucleation process, assemble mineral nanoparticles, and modify the surfaces and interiors of existing crystals. However, visualization of these processes is hampered by destructive and invasive methods such as those used for conventional scanning electron microscopy/transmission electron microscopy. In this report we detail the novel use of microcomputerized X-ray tomography (μCT) imaging to nondestructively investigate the in vitro calcium carbonate mineralization process in the presence of a recombinant sea urchin spicule matrix protein, rSpSM50, and a recombinant mollusk shell nacre protein, rPif97. Relative to the protein-free control scenario, both proteins generate calcite crystals that are consistent with the results obtained from previous studies, but with μCT we discovered new features; each protein generates a different number of mineral deposits, exhibits unique domains or motifs, and creates a stratification of mineral phases into layers. These results coincide with the known function of these proteins in vitro and in situ, and provide new information regarding biomineralization protein hydrogels and how they influence nucleation and crystal growth. We foresee that μCT imaging could, for appropriately sized systems, offer a bridge between in vitro experiments and in situ investigations of time-resolved nucleation and crystal growth phenomena in organisms, tissues, and synthetic materials.