Sophie Berland

Interface between bone and nacre implants in sheep.

We have investigated the interface between bone and chronic implants of nacre in sheep. There was no foreign body reaction over the period of 10 months and the implants were not broken down. Light microscopy indicated activity within an osteoprogenitor cellular layer lining the implant, resulting in a complete sequence of new bone formation. Nacre appeared to bind directly to newly formed bone without any intervening fibrous tissue. Scanning electron microscopy and energy dispersive photon X-microanalysis showed calcium and phosphate ions lining the nacre within the osteoprogenitor tissue. These studies show a dynamic activity of the bone/nacre interface, leading to continuity between the nacre and the bone.

Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies

There is frequently a loss of vertebral bone due to disease or aging. Nacre (mother of pearl from the oyster Pinctada maxima) stimulates bone cell differentiation and bone formation in vitro and in vivo. Experimental bone defects were prepared in the vertebrae of sheep and used to test the suitability of nacre as an injectable osteogenic biomaterial for treating vertebral bone loss. Twenty-one cavities were prepared in the first four upper lumbar vertebrae of 11 sheep and filled with nacre powder. The lumbar vertebrae were removed after 1 to 12 weeks, embedded undecalcified in methacrylate, and processed for histological studies. The nacre slowly dissolved and the experimental cavities contained a large active cell population. By 12 weeks, the experimental cavity was occupied by newly matured bone trabeculae in contact with or adjacent to the dissolving nacre. The functional new bone trabeculae were covered with osteoid lined with osteoblasts, indicating continuing bone formation. The in vitro study on rat bone marrow explants cultured with a water-soluble extract of the nacre organic matrix also resulted in the stimulation of osteogenic bone marrow cells with enhanced alkaline phosphatase activity. Thus, both the in vivo and in vitro findings suggest that nacre contains one or more signal molecules capable of activating osteogenic bone marrow cells.

Reconstruction of human maxillary defects with nacre powder: histological evidence for bone regeneration

The defective areas in the premolar-molar region of maxillary alveolar bone of eight patients were reconstructed using powdered nacre from the giant oyster Pinctada maxima. Histological, microradiographic and polarized light studies of drill biopsies taken 6 months postoperatively showed that nacre was tightly bound to newly-formed bone. The nacre was gradually and centripetally biodissolved and replaced with immature and then mature lamellar bone. These results are in agreement with our previous experimental in vitro data indicating that nacre has good osteogenic properties.

Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells and osteoblasts.

In vivo and in vitro studies provide strong evidence of the osteogenic activity of nacre obtained from Pinctada maxima. The in vitro studies indicate that diffusible factors from nacre are involved in cell stimulation. The water-soluble matrix (WSM) was extracted from nacre by a non-decalcifying process, and four fractions (SE(1)-SE(4)) were separated by SE-HPLC. Those fractions were tested in vitro on MRC5 fibroblasts. Alkaline phosphatase (ALP) activity was measured as a marker of osteoblastic differentiation. The anti-apoptotic protein Bcl-2 was also immunodetected in cultured osteoblasts from rat calvaria. WSM and fraction SE(4) increased ALP activity. BMP-2 had the same effect on the cells as WSM and SE(4). WSM greatly increased the amount of Bcl-2 in the cytoplasm and nucleus of osteoblasts. These in vitro studies support our initial hypothesis that nacre organic matrix (WSM) of a bivalve mollusk contains signal-molecules that can stimulate the osteogenic pathway in mammalian cells that are targets for bone induction.

Bone reactions to nacre injected percutaneously into the vertebrae of sheep.

We have studied the osteogenic effects of nacre (mother of pearl) placed in experimental cavities prepared in the lumbar vertebrae of sheep. Some of cavities were filled with nacre, some with PMMA, and some were left empty. The vertebrae were removed 1, 8, 12 weeks after surgery, and assessed histologically and morphometrically. The nacre particles in the bone cavity and the surrounding intertrabecular spaces gradually dissolved beginning at 8 weeks after surgery. There were layers of newly formed bone, both woven and lamellar, in various stages of maturation in contact with or adjacent to the dissolving nacre. Quantitative assessment of the activation of bone formation adjacent to the cavities filled with nacre indicated significant activation of bone formation, which continued until week 12. There was also increased mineralization of the host bone at this time. There was no new bone formation in the empty cavities, or in those filled with PMMA. PMMA also caused necrosis of surrounding bone cells with a change in bone architecture and significant reductions in bone formation and mineralization. This study demonstrates that nacre stimulates bone-forming cells in vertebrae and appears to result in new bone formation.

Zona localization of shell matrix proteins in mantle of Haliotis tuberculata (Mollusca, Gastropoda).

Organic matrix from molluscan shells has the potential to regulate calcium carbonate deposition and crystallization. Control of crystal growth thus seems to depend on control of matrix protein secretion or activation processes in the mantle cells, about which little is known. Biomineralization is a highly orchestrated biological process. The aim of this work was to provide information about the source of shell matrix macromolecule production, within the external epithelium of the mantle. An in vivo approach was chosen to describe the histologic changes in the outer epithelium and in blood sinus distribution, associated with mantle cells implicated in shell matrix production. Our results characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis.

Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata

An integrated study of shell formation was initiated covering the entire life cycle of the marine gastropod Haliotis tuberculata. Shell microstructure, chemistry and mineralogy were investigated by polarized microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and infra-red (IR) spectroscopy. SEM images of trochophore and veliger larvae showed the different stages of shell growth from the initial shell field to the late calcified protoconch. Cross-sections revealed the microstructural arrangement of biominerals, showing the progressive mineralization of the organic protoconch prior to metamorphosis. To gain more information on mineralogical composition, EDX analyses and IR spectroscopy were performed along the development stages. The results demonstrated that early protoconch was mostly composed of amorphous calcium carbonate, while veliger stages showed a gradually crystallization under the form of aragonite. Post-metamorphic shell contained two distinct parts, the original protoconch supporting the new juvenile shell characterized by a marked sculptural pattern. The shells from post-larval and juvenile abalones were essentially made of aragonite.

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.

Coupling Proteomics and Transcriptomics for the Identification of Novel and Variant Forms of Mollusk Shell Proteins: A Study with P. margaritifera

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell‐forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine‐rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra‐ and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase‐like protein previously identified in Crassostrea gigas, two chitin‐binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif‐forming sequences, such as chitin‐binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.

The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (p<0.001) by 100 μg proteins/ml extract and 20% (p<0.001) by 50 μg proteins/ml extract, but a low concentration of extract decreased the ALP activity by 8%. Cells treated with a high aspartic acid content fraction of the extract had increased ALP activity (23%, p<0.0001). Nacre extract and the fraction have no effect on the proliferation of mature osteoblasts. Immunoreactive Bcl-2 was overproduced in the cytoplasm and nuclei of osteoblasts at all stages of culture. Bcl-2 was found over the whole chromatin in quiescent and mitotic cells at the end of mitosis in the two nuclei in one cell, before cytodieresis. Bcl-2 was also found over chromosomes. Thus, nacre extract stimulates Bcl-2 production in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration.