Fish Otolith as a Model for in Vitro Mineralization Studies

Otoliths are biominerals that grow in the endolymphatic fluid of the balance organs of vertebrates, and are composed of calcium carbonates with low amounts of organic matter [1]. In fish they are composed mainly of aragonite, and participate also in the transduction mechanism of hearing [2]. The microstructure of otoliths give information about their own process of growth, as it is possible to observe the growth lines (alternate pattern of more and less mineralized regions), in histological sections. The objective of this study was to elucidate the spatial organization of the components of otoliths from the sacullus (sagitta) of corvine fish (Micropogonias furnieri) aiming to understand how the arrangement between mineral and organic parts was correlated to the whole structure and its process of growth. We have also isolated the soluble matrix from sagitta by dialysis using EDTA (Ethylene-diamine-tetraacetic acid), and performed in vitro mineralization of calcium carbonates in the presence or absence of these molecules to access their possible role in the determination of the polymorphs produced. The sagitta were easily extracted. Sections of circa 200 μm thick were obtained with a low speed diamond wheel saw (South Bay Technology Inc.), and were further ground and polished. Whole otoliths partially demineralized in a solution containing EDTA and glutaraldehyde as fixative, were embedded in epoxi resin from where semi-thin sections were obtained. Inner otolithic surfaces exposed by sectioning with a diamond knife, were gold sputtered (BAL-TEC) and analysed by scanning electron microscopy (SEM; Jeol 5310 operated at 15 kV). Histological sections of embedded demineralized otoliths, stained with toluidine blue, showed a high concentration of growth lines (Fig. 1, and insert). Sectioned surfaces showed the very compact organization of mineral and organic parts (Fig. 2). When these sections were briefly etched with EDTA (10% in water), the highly mineralized and more organic regions became clearly distinguished (Fig. 3). In these sections we also observed discontinuities between crystallites that correspond to the short connections linking growth lines seen in figure 1 (Fig. 3). Polished sections of otoliths similar to the previous one, partially demineralized in EDTA, critical point dried (Balzers), gold sputtered, and observed by SEM, showed that the non-soluble organic matrix is composed mainly of parallel organic sheets (Fig. 4). Polished sections of the whole otolith parallel to and near the macula region, slightly etched with EDTA, showed that crystallites were organized in clusters (Fig. 5) and were oriented perpendicularly to the growth surfaces (Fig. 5, insert). In vitro mineralization of calcium carbonates in the presence of the soluble matrix from sagitta, showed that these molecules could modulate the polymorphs produced (Fig. 6), when compared with the control experiment (insert in Fig. 6). It was concluded that sagitta presented insoluble organic matrix distributed as approximately parallel sheets, with short connections oriented perpendicularly, and that the soluble matrix was capable to modulate the in vitro mineralization of calcium carbonate polymorphs. We suggest that the demineralized organic matrix of fish otolith should be used as scaffold for in vitro mineralization assays in the presence/ absence of soluble matrix molecules.