Proteomic and Transcriptomic Analyses in the Slipper Snail Crepidula fornicata Uncover Shell Matrix Genes Expressed During Adult and Larval Biomineralization.

The gastropod shell is a composite composed of minerals and shell matrix proteins (SMPs). SMPs have been identified by proteomics in many molluscs, but few have been studied in detail. Open questions include (1) what gene regulatory networks regulate SMP expression, (2) what roles individual SMPs play in biomineralization, and (3) how the complement of SMPs changes over development. These questions are best addressed in a species in which gene perturbation studies are available; one such species is the slipper snail, Crepidula fornicata. Here, SEM and pXRD analysis demonstrated that the adult shell of C. fornicata exhibits crossed lamellar microstructure and is composed of aragonite. Using high-throughput proteomics we identified 185 SMPs occluded within the adult shell. Over half of the proteins in the shell proteome have known biomineralization domains, while at least 10% have no homologs in public databases. Differential gene expression analysis identified 20 SMP genes that are up-regulated in the shell-producing mantle tissue. Over half of these 20 SMPs are expressed during development with two, CfSMP1 and CfSMP2, expressed exclusively in the shell gland. Together, the description of the shell microstructure and a list of SMPs now sets the stage for studying the consequences of SMP gene knockdowns in molluscs.

A method for mapping submicron-scale crystallographic order/disorder applied to human tooth enamel.

Tooth enamel, the outermost layer of human teeth, is a complex, hierarchically structured biocomposite. The details of this structure are important in multiple human health contexts, from understanding the progression of dental caries (tooth decay) to understanding the process of amelogenesis and related developmental defects. Enamel is composed primarily of long, nanoscale crystallites of hydroxyapatite that are bundled by the thousands to form micron-scale rods. Studies with transmission electron microscopy show the relationships between small groups of crystallites and X-ray diffraction characterize averages over many rods, but the direct measurement of variations in local crystallographic structure across and between enamel rods has been missing. Here, we describe a synchrotron X-ray-based experimental approach and a novel analysis method developed to address this gap in knowledge. A ~500-nm-wide beam of monochromatic X-rays in conjunction with a sample section only 1 µm in thickness enables 2D diffraction patterns to be collected from small well-separated volumes within the enamel microstructure but still probes enough crystallites (~300 per pattern) to extract population-level statistics on crystallographic features like lattice parameter, crystallite size, and orientation distributions. Furthermore, the development of a quantitative metric to characterize relative order and disorder based on the azimuthal autocorrelation of diffracted intensity enables these crystallographic measurements to be correlated with their location within the enamel microstructure (e.g., between rod and interrod regions). These methods represent a step forward in the characterization of human enamel and will elucidate the variation of the crystallographic structure across and between enamel rods for the first time.

A contribution to the characterization of the silicate-water interface - Part II: Atom Probe Tomography of tricalcium silicate.

This work explores the possibility to investigate the nanoscale cement-water interface by means of atom-probe tomography (APT). For this purpose, the main compound of Ordinary Portland Cement, tricalcium silicate, and its hydration product calcium-silicate-hydrate have been analyzed by APT. Of special interest was the surface of anhydrous and hydrated tricalcium silicate. The results show, that a nanoscale characterization of tricalcium silicate with APT is possible by carefully controlling the various measurement parameters. Furthermore, our results indicate, that the conditions during focused ion beam sample preparation, especially the high vacuum and energy input, are potentially harmful to calcium-silicate-hydrate. Future developments in cryo sample preparation will greatly enhance the applicability of APT on cement and its hydration products.

A contribution to the characterization of the silicate-water interface - Part I: Implication of a new polished sample hydration technique.

The analysis of the atomic composition of the interface between tricalcium silicate (C3S), the main compound of Ordinary Portland Cement, and surrounding solution is still a challenging task. At the same time, that knowledge is of profound importance for describing the basic processes during hydration. By means of Scanning Electron Microscopy (SEM) and Atom Probe Tomography (APT) we combine modern techniques in order to shed light on this topic in the present study. The results of these methods are compared with conduction calorimetry as a standard technique to study the hydration kinetics of cement. The tests were carried out on powders as well as on polished C3S samples. Results indicate that the progress of hydration is strongly increased when the C3S is used in the form of polished specimen. First C-S-H phases are detected in the powder 2.2 h after contact with water, on the polished section after 5 min. Besides SEM, the formation of C-S-H phases can be detected by APT, leading to an advantageous atomic resolution compared to EDX analysis. We propose that the use of APT will lead to deeper insights on the hydration progress and on the composition of the sensitive C-S-H phases based on these first results.

Characterization of enamel caries lesions in rat molars using synchrotron X-ray microtomography.

Dental caries is a ubiquitous infectious disease with a nearly 100% lifetime prevalence. Rodent caries models are widely used to investigate the etiology, progression and potential prevention or treatment of the disease. To explore the suitability of these models for deeper investigations of intact surface zones during enamel caries, the structures of early-stage carious lesions in rats were characterized and compared with previous reports on white spot enamel lesions in humans. Synchrotron X-ray microcomputed tomography non-destructively mapped demineralization in carious rat molar specimens across a range of caries severity, identifying 52 lesions across the 30 teeth imaged. Of these lesions, 13 were shown to have intact surface zones. Depth profiles of fractional mineral density were qualitatively similar to lesions in human teeth. However, the thickness of the surface zone in the rat model ranges from 10 to 58 µm, and is therefore significantly thinner than in human enamel. These results indicate that a fraction of lesions in rat caries possess an intact surface zone and are qualitatively similar to human lesions at the micrometer scale. This suggests that rat caries models may be a suitable analog through which to investigate the structure of surface zone enamel and its role during dental caries.