Marthe Rousseau

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.

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone.

Dynamics of sheet nacre formation in bivalves.

Formation of nacre (mother-of-pearl) is a biomineralization process of fundamental scientific as well as industrial importance. However, the dynamics of the formation process is still not understood. Here, we use scanning electron microscopy and high spatial resolution ion microprobe depth-profiling to image the full three-dimensional distribution of organic materials around individual tablets in the top-most layer of forming nacre in bivalves. Nacre formation proceeds by lateral, symmetric growth of individual tablets mediated by a growth-ring rich in organics, in which aragonite crystallizes from amorphous precursors. The pivotal role in nacre formation played by the growth-ring structure documented in this study adds further complexity to a highly dynamical biomineralization process.

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.

Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates

Calcitonin gene related peptide (CGRP) related molecules were purified from sardine hydrolysates prepared using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography and HPLC performed purification of these molecules. The purified molecules were characterised using specific CGRP radioimmunoassays and radioreceptoraasays. From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules, the molecular weight determined by mass spectrophotometry was 6000 daltons. The biological activity of these molecules was analysed using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. The purified molecules induced an inhibition of the CGRP stimulated adenylate cyclase activity, this effect was specific as no such effect was observed on the glucagon stimulated adenylate cyclase activity measured in the same rat liver membrane preparation. These results suggest that the purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. These new antagonists may be of particular importance in various aspects of CGRP action in vertebrates.

Nacre biocrystal thermal behaviour

The thermal behaviour of Pinctada margaritifera nacre was studied at different temperatures by means of thermal gravimetric, thermo-mechanical and Rock-Eval analyses. From the mechanical point of view nacre exhibited a complete reversible behaviour up to 230 °C. The bio-aragonite allotrope was seen to be as stable as the abiotic aragonite up to 470–500 °C. It was also evidenced that the organic phase was keeping cracking oxygen functions at temperatures as high as 650 °C. Nacre thermal behaviour could be described following four distinctive stages and discussed in comparison with previous data obtained in oxidative conditions.

CGRP stimulates gill carbonic anhydrase activity in molluscs via a common CT/CGRP receptor.

The physiological significance of calcitonin gene-related peptide (CGRP) during biomineralization was investigated by assessing the effect of human CGRP on the carbonic anhydrase activity in gill membranes of the pearl oyster, Pinctada margaritifera. Salmon CT and human CGRP were able to induce a 150% increase of the basal activity. No additive effect was observed suggesting that both activities are mediated by the same receptor. The CGRP-stimulated effect was specific as demonstrated by the inhibition produced by the CGRP antagonist, hCGRP8-37. So, CGRP by its specific action on gill carbonic anhydrase controls the calcification process, an ancient role both in invertebrates and non-mammalian vertebrates.

Multi-Scale Structure and Growth of Nacre: A New Model for Bioceramics

Nacre is the internal lustrous ‘mother of pearl’ layer of many molluscan shells. The structure is a brick and mortar arrangement: the bricks are fla t po ygonal crystals of aragonite and the mortar consists of organic compounds.The biological mineralization of composites such as the molluscan shell has generally been thought to be directed by preformed ganic arrays of proteins or others biopolymers.The possibility that the organic matrix behaves as a template for crystal formation by heteroepitaxial growth, is examined. The structure and g rowth of nacre of Pinctada margaritifera and Pinctada maxima are studied at different scales.We propose a new model in nacre growth and maturation. Understanding the molecular mechanisms that reg ulate biomineralization may thus provide practical routes to the synthesis of new high-performance composite m aterials. Nacreous shell growth was studied by electron microscopy after fi xation of immature nacre. It shows that the first step in crystal formation takes place in a biofilm, as evidenced by Laser Raman Spectroscopy.This biofilm is composed of organic matrix and cal cium carbonate in the aragonite crystal form. Crystalin nuclei grow in the biofilm as an aligned sequence, starting with a row of intitial nucleation centres, gradually increasing concentric ally in size and fusing together to form polygons in the next highest nacreous level. In Pinctada biomineralization takes place in a biopolymer that has the ability to initiate nucleation and to control orientation in the aragonite crystal form and growth. Al so is stressed the existence of several levels or steps of mineralization, simultaneously, as a ke y mechanism for the thickening of the shell. This natural bioceramic is studied with the aim of using it as a biomater ial in bone repair. Introduction The active role of proteins in biomineralization is fundamental [1-3]. It represents a source of inspiration for future nanotechnology with a bottom-up approach. The brick and mor tar ordering of nacre has already inspired the toughening of ceramic materials by co-processing rigid ceramic as silicon carbide and supple interlayers as boron nitride [4]. The interdigitating brickwork array of tablets, specific, in bivalve s (“sheet nacre”) is not the only interesting aspect of nacre structure. The bio-crystal itse f is a composite. It has not only the mineral structure of aragonite but possesses intracristalline or ganic material [5]. Among others Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 1009-1012 doi:10.4028/www.scientific.net/KEM.254-256.1009

Multi-Scale Structure of the Pinctada Mother of Pearl: Demonstration of a Continuous and Oriented Organic Framework in a Natural Ceramic

Sheet nacre is a promising natural bioceramic, which consists on the internal lustrous “mother of pearl” layer of many molluscan shells, e.g. Pinctada, our model. The aim of this work is to study the structure of the flat polygonal tablets of nacre, in order to understand the multi-scale organization of this composite material and the role of the organic template during the growth of the biocrystal. We studied the organic matrix, in situ with techniques such as darkfield transmission electronic microscopy (TEM) on small cross-sections of nacre of Pinctada maxima, or intermittent-contact atomic force microscopy coupled with phase imaging on samples of nacre of Pinctada margaritifera polished parallel to the surface of the tablets. In this study, we demonstrate the continuity of the organic framework and the crystallographic orientation in the biocrystal at 2 relevant levels : nano- and micro-scale.

Low Molecular Weight Molecules of Oyster Nacre Induce Mineralization of the MC3T3-E1 Cells

The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurence of clusters of spherical particles ressembling to hydroxyapatite. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair.