Authors: Alexandre Parker, Françoise Immel, Nathalie Guichard, Cédric Broussard, Frédéric Marin
Abstract: Mollusc shells are organic-inorganic composites that are often preserved in the fossil record. However, the way the organic fraction, also called shell matrix, gets fossilized remains an unsolved question, in spite of several old and more recent studies. In the present paper, we have tried to mimic a diagenetic process by constantly heating for ten days at 100°C fresh nacre powder samples of the Polynesian pearl oyster Pinctada margaritifera. Each day, aliquots of nacre powder were sampled and the matrix was subsequently extracted. It was further analysed by direct weigh quantification, by immunological techniques and by proteomics. Our preliminary data suggest that nacre proteins, when heated at 100°C in dry condition, degrade rather slowly. We evidenced a differential degradation pattern of the soluble and insoluble fractions, and showed that some nacre proteins of the insoluble fraction are stable after ten days of heating. Factors that influence the diagenetic stability of some shell proteins are discussed.
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Authors: Antonio Osuna-Mascaro, Teresa Cruz-Bustos, Benjamin Marie, Antonio G. Checa, Frédéric Marin
Abstract: It is known for a long time that calcified tissues secreted by aquatic or terrestrial invertebrates – such as mollusc shells – have the ability to concentrate large amounts of pollutants, in particular heavy metals. In the present paper, we have found an extremely rapid and easy procedure to qualitatively detect the putative presence of heavy metals in shells, without having to use sophisticated techniques such as Wavelength Dispersive Spectroscopy, atomic adsorption spectroscopy or ICP-MS. Our method rests on the capacity of the silver enhancement chemicals that are traditionally used in immunogold localization experiments to increase the size of heavy metal nanoparticles, whatever the chemical element. It goes as follows: freshly broken pieces of shells that are suspected to contain traces of heavy metals are simply incubated 15 minutes in few drops of a silver enhancement solution (British Biocell International), and, after short rinsing and drying, the shell fragments are directly observed with a tabletop Scanning Electron Microscope, under back scattered electron (BSE) mode without any further preparation. Heavy metals nanoparticles are detected as bright spots. Our method is extremely fast (about half an hour in total), and may be used as a quick check for pre-selecting series of calcified samples prior to the quantitative analysis of their heavy metal content.
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Authors: Frédéric Marin, Nathalie Le Roy, Benjamin Marie, Paula Ramos-Silva, Stephan Wolf, Sana Benhamada, Nathalie Guichard, Françoise Immel
Abstract: In Nature, calcium carbonate biomineralizations are the most abundant mineralized structures of biological origin. Because many exhibit remarkable characteristics, several attempts have been made to use them as substitution materials for bone reconstruction or as models for generating biomimetic composites that exhibit tailored properties. CaCO3 biomineralizations contain small amounts of amalgamate of proteins and polysaccharides that are secreted during the calcification process. They contribute to control the morphology of the crystallites and to spatially organize them in well-defined microstructures. These macromolecules, collectively defined as the skeletal matrix, have been the focus of a large number of studies aiming at synthesizing in vitro biomimetic materials, according to a bottom-up approach. However, recent proteomic investigations performed on the organic matrices associated to mollusc shells or to coral skeletons have quashed our hopes to generate, with only few macromolecular ingredients, biomimetic materials with properties approaching to those of natural biominerals. As a mean value, each matrix comprises a minimum of few tens of different proteins that seem to be strictly associated to calcium carbonate biominerals. Among the proteins that are currently detected, one finds RLCDs-containing proteins (Repetitive-Low-Complexity Domains), enzymes, proteins with protease inhibitors domains and at last, proteins that contains typical ECM (ExtraCellular Matrix) domains. Today, we still do not understand how the skeletal matrix works, and unveiling its complex functioning is one of the challenges for the coming decade, both from fundamental and applied viewpoints. Is it realistic to attempt generating abiotically, in a test tube at room temperature, biomimetic composites that mimic natural biomineralizations in their properties If so, and by supposing that we know the individual functions of all the components of the matrix, is there a minimal number of proteins required for producing in vitro calcium carbonate biomaterials that approximate natural biominerals These issues are of importance for the future research directions in biomaterials science.
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Authors: Faik Nuzhet Oktar, Patricia Valério, G. Göller, Simeon Agathopoulos, Alfredo Goes, M. Fatima Leite
Abstract: The in vitro biocompatibility of aragonite material obtained from inner and out layers of
four different molluscs was tested. After grinding and sieving, the obtained fine powders were put in contact with primary osteoblasts derived from rat calvariae. The viability of the cells increased at about 10% in the presence of powders derived from Vennus Gallina outer layer and from Pecten Jacobaeus inner layer. In the case of the presence of the other 6 tested powders, there was no statistical difference in cells’ viability. With regard to alkaline phosphatase production, all the tested
powders induced a decrease of the production of this enzyme by osteoblasts. There was no evidence of any alterations in collagen production.
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Authors: Daniel Chateigner, M. Morales, E.M. Harper
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