Mechanical properties of nacre and highly mineralized bone

We compared the mechanical properties of 'ordinary' bovine bone, the highly mineralized bone of the rostrum of the whale Mesoplodon densirostris, and mother of pearl (nacre) of the pearl oyster Pinctada margaritifera. The rostrum and the nacre are similar in having very little organic material. However, the rostral bone is much weaker and more brittle than nacre, which in these properties is close to ordinary bone. The ability of nacre to outperform rostral bone is the result of its extremely well-ordered microstructure, with organic material forming a nearly continuous jacket round all the tiny aragonite plates, a design well adapted to produce toughness. In contrast, in the rostrum the organic material, mainly collagen, is poorly organized and discontinuous, allowing the mineral to join up to form, in effect, a brittle stony material.     

Proteomics Analysis of the Nacre Soluble and Insoluble Proteins from the Oyster Pinctada margaritifera

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.     

Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.     

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus

In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.     

A scanning electron microscopic study of the inorganic and organic matrices comprising the mature shell of Amblema, a fresh-water mollusc

The scanning electron microscope has been used to describe the morphology of the mature shell in a fresh-water bivalve. The structure of the organic and inorganic components within the nacre, the myostracum, and the prismatic layer is described. A transitional or intermediate zone, interposed between the prismatic layer and the nacre, was identified. In demineralized samples, the organic component of the nacre was found to consist of parallel matricial sheets interconnected by irregular transverse bridges. The structure of the mineral component of the nacre was found to vary with the method of specimen preparation. With polished-etched samples, brick-like units were seen. When shells were simply broken and fixed in osmium, the layers of nacreous material consisted of fusing rhomboidal crystals of aragonite which demonstrated subconchoidal fractures. On the inner surface of the shell, the rhomboidal crystals showed an apparent spiral growth pattern. The myostracum was characterized by regions of modified nacreous structure consisting of enlarged aragonite crystals with a pyramidal morphology. The peripheral aspect of the muscle scars was characterized by rhomboidal crystals, the latter fusing to form the typical nacreous laminae. The uniqueness of the anterior adductor scar is exemplified by the presence of pores, each pore walled by pyramidal units, for the insertion of adductor fibres. In most regions of the shell, the prismatic layer consisted of one prism unit thickness with a height of approximately 225–250 μm. However, in two specialized regions of the shell, this layer was seen to consist of multiple layers of stacked prisms. The organic matrices of the prismatic layer are arranged in a honeycomb-like arrangement and packed with mineralized spherical subunits.     

Organization pattern of nacre in Pteriidae (Bivalvia: Mollusca) explained by crystal competition

Bivalve nacre is a brick-wall-patterned biocomposite of aragonite platelets surrounded by organic matter. SEM-electron back scatter diffraction analysis of nacre of the bivalve family Pteriidae reveals that early aragonite crystals grow with their c-axes oriented perpendicular to the growth surface but have their a- and b-axes disoriented. With the accumulation of successive lamellae, crystals progressively orient themselves with their b-axes mutually parallel and towards the growth direction. We propose that progressive orientation is a result of competition between nacre crystals at the growth front of lamellae, which favours selection of crystals whose fastest growth axis (b-axis) is oriented parallel to the direction of propagation of the lamella. A theoretical model has been developed, which simulates competition of rhombic plates at the lamellar growth front as well as epitaxial growth of crystals onto those of the preceding lamella. The model predicts that disordered nacre progressively produces bivalve-like oriented nacre. As growth fronts become diffuse (as is the common case in bivalves) it takes longer for nacre to become organized. Formation of microdomains of nacre platelets with different orientations is also reproduced. In conclusion, not only the organic matrix component, but also the mineral phase plays an active role in organizing the final microstructure.     

Identification of low molecular weight molecules as new components of the nacre organic matrix

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da–700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.     

Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals

We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre.     

The SEM and TEM study on the laminated structure of individual aragonitic nacre tablet in freshwater bivalve H. cumingii Lea shell

Nacre (mother-of-pearl), one of the natural composite materials, is renowned for its excellent mechanical properties and becomes a model for study on the biominerals. In the present study on bivalve H. cumingii Lea, the forming nacre tablet was observed with SEM to show laminated character on the lateral growing surfaces. Correspondingly, HRTEM showed dense crystal defects on (0 0 1) plane of the aragonite nacre tablet which might be caused by the adsorption of organic macromolecules on the plane. The correlation of the laminated growth mode and crystal defects on (0 0 1) plane was discussed. These findings could enhance our understanding to the formation mechanism of the nacre tablet as well as the superior mechanical properties of the nacre.     

Mineral proximity influences mechanical response of proteins in biological mineral-protein hybrid systems

The organic phase of nacre, which is composed primarily of proteins, has an extremely high elastic modulus as compared to that of bulk proteins, and also undergoes large deformation before failure. One reason for this unusually high modulus could be the mineral-organic interactions. In this work, we elucidate the specific role of mineral proximity on the structural response of proteins in biological structural composites such as nacre through molecular modeling. The "glycine-serine" domain of a nacre protein Lustrin A has been used as a model system. It is found that the amount of work needed to unfold is significantly higher when the GS domain is pulled in the proximity of aragonite. These results indicate that the proximity of aragonite has a significant effect on the unfolding mechanisms of proteins when pulled. These results will provide very useful information in designing synthetic biocomposites, as well as further our understanding of mechanical response in structural composites in nature.     

珍珠颜色和贝壳珍珠层颜色研究进展

颜色及其色度均一性是衡量珍珠价值的重要指标之一。珍珠颜色及贝壳珍珠层颜色的研究涉及多个学科领域,研究表明,珍珠的颜色与制片蚌外套膜对应的珍珠层颜色相一致,而蚌的珍珠层颜色主要由遗传因素决定。现有的研究资料对珍珠层颜色形成的机理虽然还不能给出一个系统、合理的诠释,但金属元素、卟啉、类胡萝卜素和物理结构等因素可能和珍珠层颜色形成密切相关,珍珠层中含有少量以蛋白质为主的有机基质,这些蛋白调控珍珠层的结构和颜色的形成,可能是解释珍珠层颜色形成机理的关键。本文对珍珠颜色和贝壳珍珠层颜色研究进展进行系统综述,探讨珍珠颜色的影响因素及相互关联,旨在为进一步研究珍珠和贝壳珍珠层颜色提供借鉴与思路。     

Flexible nacre in the nautiloid Isorthoceras, with remarks on the evolution of cephalopod nacre

Secondarily phosphatized shells of Isorthoceras sociale (Hall) from the Upper Ordovician Maquokcta shale, Iowa, U.S.A., often have their original ultrastructurc preserved. Scanning Electron Microscope study reveals that the nacreous layer is similar to that in the shells of Recent Nautilus , except that the nacreous tablets usually have a much larger diameter, and that numerous vertical interspaces arc left between adjacent stacks of tablets. Because of the latter condition, the nacre in A sociale is porous, whereas it is compact in Nautilus. The pores in the nacre were probably filled with a non-mincralizcd organic matrix. The high porosity and high content of organic matrix made the nacreous layer of I. sociale much more flexible and considerably less strong than that in Nautilus. As a consequence, the shells of I. sociale , and probably also of other orthoconic cephalopods, were more flexible and could not withstand such high hydrostatic pressures as the shells of Nautilus. During evolution, the porosity of the nacreous layer became completely reduced. As a result, the mechanical properties of the nacre changed towards a lower flexibility and a higher tensile strength. Cephalopoda, Nautiloidea, nacre, ultrastructurc, phosphatization, Upper Ordovician.     

Heterogeneity of Proteinase Inhibitors in the Water-Soluble Organic Matrix from the Oyster Nacre

We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.     

Derivation of the Stress-Strain Behavior of the constituents of Bio-Inspired Layered TiO₂/PE-Nanocomposites by Inverse Modeling Based on FE-Simulations of Nanoindentation Test

Owing to the apparent simple morphology and peculiar properties, nacre, an iridescent layer, coating of the inner part of mollusk shells, has attracted considerable attention of biologists, material scientists and engineers. The basic structural motif in nacre is the assembly of oriented plate-like aragonite crystals with a ’brick’ (CaCO₃ crystals) and ’mortar’ (macromolecular components like proteins) organization. Many scientific researchers recognize that such structures are associated with the excellent mechanical properties of nacre and biomimetic strategies have been proposed to produce new layered nanocomposites. During the past years, increasing efforts have been devoted towards exploiting nacre’s structural design principle in the synthesis of novel nanocomposites. However, the direct transfer of nacre’s architecture to an artificial inorganic material has not been achieved yet. In the present contribution we report on laminated architecture, composed of the inorganic oxide (TiO₂) and organic polyelectrolyte (PE) layers which fulfill this task.
To get a better insight and understanding concerning the mechanical behaviour of bio-inspired layered materials consisting of oxide ceramics and organic layers, the elastic-plastic properties of titanium dioxide and organic polyelectrolyte phase are determined via FE-modelling of the nanoindentation process. With the use of inverse modeling and based on numerical models which are applied on the microscopic scale, the material properties of the constituents are derived.     

Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters

Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO₃ and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.     

Ultrastructure and mineral distribution in the tergite cuticle of the beach isopod Tylos europaeus Arcangeli, 1938

The crustacean cuticle is a hierarchically organised material composed of an organic matrix and mineral. It is subdivided into skeletal elements whose physical properties are adapted to their function and the eco-physiological strains of the animal. Using a variety of ultrastructural and analytical techniques we studied the organisation of the tergite cuticle of the sand burrowing beach isopod Tylos europaeus. The surface of the tergites bear epicuticular scales, sensilla and micro-tubercles. A distal layer of the exocuticle is characterised by a low density of organic fibres and the presence of magnesium-calcite. Surprisingly, the mineral forms regions containing polyhedral structures alternating with smooth areas. Between sub-domains within the distal exocuticle calcite varies in its crystallographic orientation. Proximal layers of the exocuticle and the endocuticle are devoid of calcite and the mineral occurs in the form of amorphous calcium carbonate (ACC). Using thin sections of mineralised cuticle we describe for the first time that ACC forms tubes around single protein-chitin fibrils.     

不同pH值对三角帆蚌珍珠质分泌的影响

运用多种组织化学方法和透射电镜技术,研究了５种ｐＨ水环境（ｐＨ５、６、７、８、９）对三角帆砷外套膜珍珠质分泌的影响机制,结果表明,在中性水环境中,贝体能积极地从外界水环境中吸收钙,并能旺盛地合成和分泌贝壳珍珠层及珍珠有机基质前体物质,持续的酸性水环境导致贝体的钙严重丢失,并引起珍珠质分泌细胞对有机基质前体物质的合成和分泌能力减弱,持续的碱性水环境虽能导致贝体对钙的积累,但珍珠质分泌细胞合成和分泌珍     

Interfacial mechanical behaviour of protein–mineral nanocomposites: A molecular dynamics investigation

Biological composite materials, such as bone, tooth and nacre, are comprised of a mixture of nano-sized hard components (e.g. mineral platelets) and soft components (e.g. protein molecules). Their mechanical behaviour greatly depends on the protein–mineral interfaces. This paper investigates the effects of mineral surface nanostructures on the interfacial interaction and mechanical behaviour of protein–mineral nanocomposites. Interfacial shear between osteopontin (OPN) and hydroxyapatite (HA) mineral layers with surface nanostructures is investigated using the atomistic molecular dynamics (MD) simulations. The results indicate that the OPN residues can be attached to HA surfaces but the surface nanostructures greatly affect the interfacial interaction and mechanical behaviour. The HA layers with a higher number of nano-sized grooves (defects) increase the surface roughness but reduce the pulling force and energy dissipation.     

Binding of adenine and adenine-related compounds to the clay montmorillonite and the mineral hydroxylapatite

The first living things may have consisted of no more than RNA or RNA-like molecules bound to the surfaces of mineral particles. A key aspect of this theory is that these mineral particles have binding sites for RNA and its prebiotic precursors. The object of this study is to explore the binding properties of two of the best studied minerals, montmorillonite and hydroxylapatite, for possible precursors of RNA. The list of compounds investigated includes purines, pyrimidines, nucleosides, nucleotides, nucleotide coenzymes, diaminomaleonitrile and aminoimidazole carbox-amide. Affinities for hydroxylapatite are dominated by ionic interactions between negatively charged small molecules and positively charged sites in the mineral. Binding to montmorillonite presents a more complex picture. These clay particles have a high affinity for organic ring structures which is augmented if they are positively charged. This binding probably takes place on the negatively charged faces of these sheet-like clay particles. Additional binding sites on the edges of of these sheets have a moderate affinity for negatively charged molecules.Small molecules that bind to these minerals sometimes bind independently to sites on the minerals and sometimes bind cooperatively with favorable interactions between the bound molecules.     

On the fracture mechanisms of nacre: Effects of structural orientation

The nacre of mollusk shells is distinguished by an exceptional mechanical efficiency which is derived essentially from its lamellar structure and frequently acts as a source of inspiration for the development of biomimetic materials. The structure and mechanical properties of nacre have been intensively investigated with a special focus on its toughening strategies; nevertheless, the fracture mechanisms, more specifically the critical stress/strain conditions for the failure of nacre, and the effects of structural orientation and hydration state remain largely unexplored. Here uniaxial compression tests were performed on nacre of both dry and hydrated states with different off-axis angles, i.e., the inclination of loading axis with respect to the lamellar structure, ranging from 0° to 90°. The mechanical properties and fracture characteristics of nacre and their dependences on the structural orientation and hydration state were elucidated in terms of mechanics behind failure. Quantitative relationships were established between the mechanical properties and off-axis angle based on different failure criteria. The competition between the fracture modes of fragmentation and shearing was quantified by comparing their respective driving force and resistance on the interfacial plane. This study may aid the understanding on the mechanical behavior of nacre and nacre-inspired synthetic materials and promote a better replication of the underlying design principles of nacre in man-made systems.