Flat Pearl Research Articles

Controlled in Vivo Nacre Formation in Flat Pearls with Hydroxyapatite Bioceramic Nuclei

We carried out in vivo experiments of cultured flat pearl production using disc-like hydroxyapatite (HAp) bioceramics as a nucleus, with and without electric polarization treatment, and evaluated the effects of electric polarization and the quality of HAp materials on flat pearl formation. The thickness of the lamellar structures of the nacreous aragonite layers increased when polarized HAp pellets, instead of nonpolarized HAp pellets, as the nucleus, were used as the nuclei, while the thickness of the columnar structures of the prismatic calcite layers decreased. These phenomena were observed remarkably on the negative potential surface of the polarized HAp pellet. The electric polarization treatment promoted the formation rate of the nacreous lamella structure. It was speculated that electric polarization may control the physiological actions of mantle tissue and regulate the formation of the prismatic calcite and nacreous aragonite layers. The polarization treatment leads to an interference color with ...

Mapping nanomechanical properties of freshly grown, native, interlamellar organic sheets on flat pearl nacre

We imaged surfaces of freshly grown flat pearl nacre (Haliotis tuberculata) in different stages of growth in seawater using an atomic force microscope (AFM). Characteristic mineral phases of nacre, such as aragonitic stacks of coins, as well as the associated organic sheets, could be detected. Apart from imaging, the acquisition of force volumes on freshly grown organic surface areas on flat pearl nacre was conducted with the AFM. The evaluation of the force volumes with the Hertz–Sneddon model resulted in Young’s moduli in the MPa range. The presented values are considerably smaller than values previously determined from macroscopic tensile tests. This might reflect the anisotropy of the organic nacre layers.

Correction: Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

In the Author Contributions statement, first author (LX) was incorrectly omitted from the list of those authors that Conceived and designed the experiments.

Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.

Reprint of: Growth of nacre in abalone: Seasonal and feeding effects

The processes of aggregation of mineral and organic materials to the growing surfaces in red abalone ( Haliotis rufescens) are analyzed. The flat pearl implantation method is used to observe the transient stages of calcium carbonate deposition, the structure of the organic interlayer, and the steady-state growth of aragonite tiles. The morphology of the organic interlayer is characterized by scanning electron microscopy. These results enable a realistic depiction of the formation of the terraced cones that comprise the principal biomineralization mechanism in this gastropod. In all cases, the growth initiated through spherulites, followed by tile formation. The transient stage with spherulitic formation was shorter at higher temperature; this is indicative of a greater activity of the animal at 21 °C. The growth rate in a normally fed gastropod was found to be higher compared with one provided with limited food. The effect of water temperature (seasonal) was also established, with growth proceeding faster in the summer (T ~ 21 °C) than in winter (15 °C). The structures of the organic interlayer and of the epithelium are revealed by scanning electron microscopy.

Growth of nacre in abalone: Seasonal and feeding effects

The processes of aggregation of mineral and organic materials to the growing surfaces in red abalone (Haliotis rufescens) are analyzed. The flat pearl implantation method is used to observe the transient stages of calcium carbonate deposition, the structure of the organic interlayer, and the steady-state growth of aragonite tiles. The morphology of the organic interlayer is characterized by scanning electron microscopy. These results enable a realistic depiction of the formation of the terraced cones that comprise the principal biomineralization mechanism in this gastropod. In all cases, the growth initiated through spherulites, followed by tile formation. The transient stage with spherulitic formation was shorter at higher temperature; this is indicative of a greater activity of the animal at 21°C. The growth rate in a normally fed gastropod was found to be higher compared with one provided with limited food. The effect of water temperature (seasonal) was also established, with growth proceeding faster in the summer (T ~21°C) than in winter (15°C). The structures of the organic interlayer and of the epithelium are revealed by scanning electron microscopy.

The growth of nacre in the abalone shell

The process of mineral formation following periods of growth interruption (growth bands) is described. Flat pearl implantation as well as a new trepanning method are used to observe the transitory phases of calcium carbonate which nucleate and grow during this process. An initial random nucleation of the aragonite polymorph is observed followed by a transition towards spherulitic growth. During this transition the animal forms the structure of the shell through both mechanical and chemical actions. About 6 weeks after implantation a steady-state growth of aragonite tiles begins after shorter and more irregular tiles cover the outer surface of the spherulites. The growth rate of aragonitic spherulite during this transition period was calculated to be approximately 0.5 μm per day. An organic scaffolding is observed during the steady-state growth of tiled aragonite. Observations of mineral growth following the deposition of these membranes confirm the presence of mineral bridges originating from subsurface tiles and extending through the organic matrix, confirming the growth model proposed by Schäffer et al. [Schäffer TE, Ionescu-Zanetti C, Proksch R, Fritz M, Walters, DA, Almqvist N, et al. Does abalone nacre form by heteroepitaxial nucleation or by growth through mineral bridges? Chem Mater 1997;9:1731–40]. Field emission scanning electron microscopy of fractured deproteinated nacre shows the presence of mineral bridges existing between individual layers of tiles. Transmission electron microscopy provides further evidence of mineral bridges.

Growth and structure in abalone shell

The growth and self-assembly of aragonitic calcium carbonate found in the shell of abalone (Haliotis) is described. This was accomplished through the close examination of laboratory-grown flat pearl samples and cross-sectional slices of the nacreous shell. Further understanding of the sequenced assembly has been obtained. It has been confirmed that the growth of the aragonite component of the composite occurs by the successive nucleation of aragonite crystals and their arrest by means of a protein-mediated mechanism; it takes place in the “Christmas-tree pattern” [Nature 49 (1994) 371]. It is shown that the protein layer is virtually absent where plates on a same plane abut (along lateral surfaces of tiles). This suggests a mechanism of c -axis aragonite growth arrest by the deposition of a protein layer of approximately 20–30 nm that is periodically activated and determines the thickness of the aragonite platelets, which are remarkably constant (0.5 μm). This platelet size was measured for animals with shell diameters of 10, 50, and 200 mm and was found to be constant. The overall growth process is expressed in terms of parameters incorporating the anisotropy of growth velocity in aragonite ( V c , the velocity along c axis, and V ab , the velocity in basal plane). Comparison of laboratory-raised and naturally-grown abalone indicates growth regulated by the level of proteinaceous saturation. Naturally-grown abalone exhibits mesolayers (growth bands) ∼0.3 mm apart; it is proposed that they result from seasonal interruptions in feeding patterns, creating thicker (∼10–20 μm) layers of protein. These mesolayers play a critical role in the mechanical properties, and are powerful crack deflectors. The viscoplastic deformation of the organic inter-tile layers is responsible for the significant improvement of tensile strength over the tensile strength of monolithic aragonite.

Tissue Regeneration in the Shell of the Giant Queen Conch, Strombus gigas

The crossed-lamellar microarchitecture (microstructure) of the shell of Strombus gigas, the giant Queen conch native to Caribbean habitats, is the most common of the several shell microarchitectures known in the mollusk family. We have studied tissue regeneration in juvenile S. gigas conchs and compared the microstructure in this regenerated tissue to the microstructure of wild S. gigas shells. The regenerated hard tissue was of two types: hard tissue grown during wound repair, and so-called “flat pearls” which are hard tissue grown on abiotic substrates inserted between the mantle and the outer covering. In both cases, the crossed-lamellar microstructure is observed after formation of a transition structure consisting of a large quantity of matrix and aggregates of aragonite crystallites.

Structural and Microstructural Characterization of the Growth Lines and Prismatic Microarchitecture in Red Abalone Shell and the Microstructures of Abalone “Flat Pearls”

The structure of the growth lines in the shell of the red abalone, Haliotis rufescens, has been characterized by X-ray diffraction analysis and by scanning and transmission electron microscopy. The growth lines consist of a blocklike microstructure and a spherulitic microstructure, separated by a green organic matrix interlayer. The minerals in both the blocklike and spherulitic structures have been determined to be aragonite, the same CaCO3 polymorph as in the nacreous microstructure in these shells. The spherulitic structure is composed of radially distributed elongated crystals, whereas the blocklike structure involves crystalline aggregates with irregular shape. The individual aggregates are approximately single crystal, with orientations identical to that of the adjacent stack of tablets in the nacreous structure. The interfaces defining the transition from nacreous to blocklike microstructures are abrupt; on the other hand, the transition from spherulitic to nacreous microstructures shows more irregularity because of the occasional intergrowth of elongated crystals into the nacreous region. The microstructures of “flat pearls”, produced by mineralization of an abiotic substrate (a glass cover slip) inserted between the mantle tissue and the growing edge of the shell in a live abalone, have also been studied. A thin calcitic CaCO3 layer is produced on the center of the glass substrate, which is soon covered by green organic matrix. This matrix extends beyond the calcitic region; that is, it can be secreted directly onto the glass. Mineralization of this green matrix layer involves the deposition of spherulitic aragonite, similar to that occurring in the native shell, which is then capped by nacreous aragonite. Thus, the microstructures within the flat pearls mimic very closely certain aspects of the microstructures within the growth lines of the native shell.

Hard Tissue Regeneration InStrombus Gigas, the Giant Queen Conch

AbstractHard tissue regeneration (i.e. shell repair) is an important biomineralization process in mollusks. Rapid regeneration is important in avoiding loss of fluids and preventing attacks by predators. We have studied such tissue regeneration in the Queen conch (Strombus Gigas) by inserting an abiotic glass cover slide between the mantle tissue and the shell. The glass cover slides were removed after mineralization periods extending from 6 hours to 4 days, and the deposited materials on the glass substrates (“flat pearls”) analyzed by X-ray diffraction, and scanning and transmission electron microscopy.Although the CaCO3in native Queen conch shell is exclusively aragonite, calcite was detected in the regenerated materials grown on glass substrates. Calcite formation occurred only during the very early stage of mineralization and the initial minerals formed were soon overgrown by aragonite. The initial aragonite overgrowth had a spherulitic morphology, and was thus relatively poorly oriented; after this spherulitic transient, the microstructure was recognizable as the crossed-lamellar structure of the natural shell.Thus, shell regeneration on abiotic substrates differs from shell formation during growth of conchs only in the very early stage. Once the crossed-lamellar microstructure has formed, further hard tissue development is identical to that occurring during natural shell growth.

First steps in harnessing the potential of biomineralization as a route to new high-performance composite materialsPaper presented at Sympos. Synergistic Synthesis of Inorganic Materials, March 1996, Schloß Ringberg, Germany.

The underlying molecular mechanisms that control biomineralization have long been thought to offer the potential for new routes to synthesis of high-performance nanocomposite materials, yet these mechanisms have until recently remained elusive. The biological mineralization of composites such as the molluscan shell generally has been thought to be directed by preformed organic arrays of proteins or other biopolymers [1]. A less explored structure-directing factor is the role of cooperative interactions between water-soluble protein molecules and the inorganic phase during crystal nucleation and growth [ Biomineralization, Chemical and Biochemical Perspectives VCH, New York, 1989, p. 133]. These interactions can control phase, morphology and growth dynamics of crystals on a time domain basis allowing the organism to rapidly introduce major structural changes in growing biomineralized composites over spatial scales ranging from angstroms to microns [ Mat. Res. Soc. Symp. Proc., 1993, 292, 59.]. We have purified and characterized the nucleating protein sheet and polyanionic proteins from the mineralized microlaminate composites of the abalone shell and flat pearl and resolved their roles controlling biomineralization. The protein sheet directs nucleation of oriented calcite to form a “primer”, while two distinct populations of soluble polyanionic proteins (at least one of which becomes occluded within the growing crystals) subsequently determine crystal phase, morphology and growth dynamics of the growing crystals. These polyanionic proteins allow us, in vitro, to abruptly and sequentially switch crystallographic phase from calcite to aragonite and vice-versa, in stereospecific directions, producing multiphase composites with micron-scale phase domains.

Does Abalone Nacre Form by Heteroepitaxial Nucleation or by Growth through Mineral Bridges?

We present experimental support for a model of abalone nacre growth that is based on mineral bridges between successive aragonite tablets rather than on heteroepitaxial nucleation. Interlamellar sheets of organic polymers delineate the aragonite tablets but allow the tablets to grow mineral bridges through pores in the sheets. Atomic force microscope images of interlamellar organic sheets from flat pearls made by Haliotis rufescens (red abalone; marine gastropod mollusk) reveal a fibrous core and holes of 5−50 nm in diameter. Scanning ion conductance microscopy shows that these holes are actually pores through the interlamellar sheets. With the help of statistical analysis we can associate the pore-to-pore spacings in the interlamellar sheets with the observed offsets of successive nacre tablets. These results, supplemented by AFM, SEM, and TEM images, support and extend the model of biofabrication of gastropod nacre which is based on mineral bridges between the aragonite tablets.

Wind Erosion Control with Crop Residues in the Sahel

AbstractMulching for wind erosion control in Sahelian farming systems is limited by low biomass production and use of crop residues for other purposes. The aim of this study was to test the effectiveness in soil protection created by two low amounts of crop residues. A field experiment was conducted in southwest Niger, on a Psammentic Paleustalf (sandy, siliceous, isohyperthermic) during the early rainy seasons of 1994 and 1995. Particle mass transport was quantified in two plots of 55 by 70 m. During the first storms of both seasons, the plots were without a mulch cover. Afterward, one plot was covered with flat pearl millet [Pennisetum glaucum (L.) R. Br.] stalks. The application rates were 1500 and 1000 kg ha‐1 during the first and second seasons, respectively. To quantify the mulch effect, mass transport rate differences between the two bare plots were quantified with a multiple linear regression model (R2 = 0.89), using wind speed (7.4–12.3 m s‐1), wind direction, and storm duration (464–3835 s). Total mass transport rates were reduced from 365.2 to 132.9 g m‐1 s‐1 (63.6%) with 1500 kg ha‐1, and from 325.1 to 188.0 g m‐1 s‐1 (42.2%) with 1000 kg ha‐1 of crop residues. Soil protection tended to decrease with increasing wind speed. Linear regression indicated that the reduction in mass transport becomes zero at wind speeds of 11.1 and 16.0 m s‐1 for the 1000 and 1500 kg ha‐1 covers, respectively. The 1000 kg ha‐1 cover even enhanced sediment transport by 6.5% during one storm with a wind speed of 11.3 m s‐1. The 1500 kg ha‐1 mulch cover reduced sediment transport from 49.7 to 80.2% during five storms with wind speeds varying from 8.3 to 10.6 m s‐1, and is therefore recommended as the better application rate for wind erosion control in the Sahel.

Critical Transitions in the Biofabrication of Abalone Shells and Flat Pearls

Analyses of biopolymer/calcium carbonate composites grown on inorganic abiotic substrates implanted between the shell and the shell-secreting epithelium of live red abalones (Haliotis rufescens) provide detailed spatial and temporal data on the in vivo assembly process that generates the shell. X-ray diffraction and scanning electron microscopy analyses of the growth of these flat pearl composites reveal that biomineralization is initiated by the deposition of an organic sheet on the implanted substrate, followed by the growth of a calcite layer with preferred {10.4} orientation and, finally, by the growth of nacreous aragonite. The calcite layer is structurally similar to the green organic/calcite heterolayer of native shell nacre. It comprises 0.2−2.0-μm-diameter elongated crystallites of typical geological habits in various aggregate arrangements. The shell also contains an external layer of (00.1)-oriented prismatic calcite, which is deposited on one edge of a flat pearl and has a morphology similar to that of the {10.4}-oriented calcite layer. The transition from {10.4}-oriented calcite to aragonite in both the shell and the flat pearl is abrupt. In vitro calcium carbonate growth experiments reveal that a similar calcite-to-aragonite transition is induced by the addition of soluble proteins isolated from the aragonitic nacre. The growth of flat pearls is highly sensitive to physical and chemical properties of the abiotic substrate. Either roughened or hydrophobic substrates result in abnormal arrangements of the basal calcite layer, which are corrected for by a reinitiation of the biomineralization process, beginning with the deposition of an organic sheet. Insertion of flat pearls as substrates, however, results in continued nacre growth without the deposition of an organic sheet and a calcite layer.

Flat pearls from biofabrication of organized composites on inorganic substrates

THE study of biomineralization is inspiring new approaches to the controlled fabrication of synthetic materials such as nanoparticles, polymer–mineral composites and templated crystals1–3. Although this biomimetic approach is gaining momentum, the biological mechanisms involved in biomineralization remain relatively unexplored. One major reason for this is the difficulty of analysing biomineralization processes in their native dynamic state. Here we demonstrate that a highly organized composite material—a 'flat pearl'—can be biofabricated on disks of glass, mica and MoS2 inserted between the mantle and shell of Haliotis rufescens (red abalone). We show that the construction of this material is spatially and temporally regulated and proceeds through a developmental sequence that closely resembles that at the growth front of the natural shell. Recognition of the implanted inorganic surfaces by mantle cells apparently governs a switch, perhaps genetically controlled, from aragonite to calcite biomineralization. Once a partially oriented calcite—protein primer layer has been deposited, there is a switch back to the nucleation and assembly of columnar stacks of highly ordered aragonitic nacre. Thus the presence of an inorganic surface between the mantle and shell of the organism triggers a change in the nature of the mineral phase deposited.