This work was conducted on Pinctada maxima nacre (mother of pearl) in order to understand its multiscale ordering and the role of the organic matrix in its structure. Intermittent-contact atomic force microscopy with phase detection imaging reveals a nanostructure within the tablet. A continuous organic framework divides each tablet into nanograins. Their shape is supposed to be flat with a mean extension of 45nm. TEM performed in the darkfield mode evidences that at least part of the intracrystalline matrix is crystallized and responds like a 'single crystal'. The tablet is a 'hybrid composite'. The organic matrix is continuous. The mineral phase is thus finely divided still behaving as a single crystal. It is proposed that each tablet results from the coherent aggregation of nanograins keeping strictly the same crystallographic orientation thanks to a hetero-epitaxy mechanism. Finally, high-resolution TEM performed on bridges from one tablet to the next, in the overlying row, did not permit to evidence a mineral lattice but crystallized organic bridges. The same organic bridges were evidenced by SEM in the interlaminar sequence.
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.
Nacre organic matrix has been conventionally classified as both Ôwater-solubleÕ and Ôwater-insolubleÕ, based on its solubility in aqueous solutions after decalcification with acid or EDTA. Some characteristics (aspartic acid-rich, silkfibroin-like content) were specifically attributed to either one or the other. The comparative study on the technique of extraction (extraction with water alone vs. demineralization with EDTA) presented here, seems to reveal that this generally accepted classification may need to be reconsidered. Actually, the nondecalcified soluble organic matrix, extracted in ultra-pure water, displays many of the characteristics of what until now has been called Ôinsoluble matrixÕ. We present the results obtained on this extract and on a conventional EDTA-soluble matrix, with various characterization methods: fractionation by size-exclusion and anion-exchange HPLC, amino acid analysis, glycosaminoglycan and calcium quantification, SDS/PAGE and FTIR spectroscopy. We propose that the model for the interlamellar matrix sheets of nacre given by Nakahara Keywords: nacre; undecalcified soluble matrix; EDTAsoluble matrix; hydrophobicity; silk-fibroin-like-proteins.In the biomineralization field, the mollusk shell is one of the best studied of all calcium carbonate biominerals. Particular attention has been given to the organic matrix [1][2][3][4][5]. The latter is thought to promote the nucleation of the mineral component, to direct the crystal growth and to act as glue, preventing fracture of the shell [6][7][8][9]. The main biopolymers present in the organic matrix are essentially proteins, either glycosylated or not, acidic polysaccharides and chitin. In nacre, they represent 1-5% (w/w) of the structure.From the earliest experiments, it was believed that the biochemical properties of matrix constituents depend of the use of a decalcification procedure for removing the mineral component, which is strongly associated with the organic matrix [1,3]. Therefore, all investigations up until now used either EDTA, acetic acid or hydrochloric acid for this demineralization step and, subsequently, two fractions of the organic matrix were separated, based on their solubility in aqueous solutions. Accordingly, a designation of matrix into two classes, the soluble matrix and the insoluble matrix, has evolved from this extraction [10][11][12][13][14]. This paper presents for the first time the results of a comparative study on the organic matrix extracted from the nacreous layer of the shell from the pearl oyster Pinctada maxima by two very different methods. The first is a nondecalcifying technique obtained by an extraction in ultra-pure water. This unconventional approach arises from previous in vivo and in vitro experiments where we showed that biochemical signals from nacre chips were able to diffuse in the surrounding media and to induce new bone formation [15][16][17][18][19][20][21][22]. In an attempt to identify these signal molecules, we have previously perfected this original method of extraction of the ...
Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction patterns were used to analyze the mineral structure and organic matrix composition and thermal behavior of the internal nacreous layer (mother of pearl or nacre) of the shell of the giant oyster Pinctada maxima. Nacre is a natural biomaterial with osteogenic properties. The mineral of nacre is calcium carbonate crystallized as aragonite and it is highly crystallized. The FT-IR spectra showed amide, amine, and carboxylic acid groups in the organic matrix of the whole (organic and mineral) nacreous layer, with the HCO(-)(3) groups possibly at the organic-mineral interface. The insoluble organic matrix remaining after decalcification contained amide, amine, and carboxylic groups. The heated aragonite mineral structure of nacre underwent two transformations (X-ray diffraction), aragonite to calcite at 300-400 degrees C, and calcite to calcium oxide (CaO) at 500-600 degrees C. The organic matrix of nacre was destroyed around 550-600 degrees C, the same temperature as the calcite to CaO transformation, revealing the great thermal stability of the organic matrix and the organic-mineral bonding. This could be an useful feature for the in vivo use of this natural biomaterial as an implant.
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