Christian Milet

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.

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.

Effects of calcitonin and ultimobranchialectomy (UBX) on calcium and bone metabolism in the eel,Anguilla anguilla L.

Prolonged administration of synthetic salmon calcitonin (SCT) to immature female silver eels, maintained in sea water, provoked a decrease of the serum calcium concentration and an increase of both the osteoblastic apposition and of the degree of mineralization of the intercellular matrix in the vertebral bone. The osteoclastic resorption and osteocytic osteolysis were not significantly affected, however the osteoclastic index was reduced. The ultimobranchial body, site of CT secretion, was cauterized in immature female silver eels mainained in Ca++ rich tap water. This operation resulted in a rise in serum calcium levels with a maximal response after two weeks. After UBX, the vertebral bone osteoblastic apposition stopped completely but the osteoclastic resorption was not modified. The degree of osteocytic osteolysis did not vary. We also observed a significant decrease in the degree of mineralization of the bone organic matrix. The observations made after UBX confirm those obtained after exogenous CT treatment SCT administered preventively to immature female eels (maintained in sea water), before experimental maturation, inhibited, at least partially, the acute osteoclastic resorption and completely inhibited the bone demineralization induced by carp pituitary extract. The increase of osteocytic osteolysis, usually observed, did not appear.

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.

Soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone Haliotis tuberculata:: extraction and partial analysis of nacre proteins

Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and beta-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.

Evolution of Nacre: Biochemistry and Proteomics of the Shell Organic Matrix of the Cephalopod Nautilus macromphalus

Matrix evolutions: We have biochemically characterized the nacre matrix of the cephalopod Nautilus macromphalus, in part by a proteomic approach applied to the acetic acid‐soluble and ‐insoluble shell matrices, as well as to spots obtained after 2D gel electrophoresis. Strikingly, most of the obtained partial sequences are entirely new, whereas a few correspond only partly with bivalvian nacre proteins. Our findings shed new light on the macroevolution of nacre matrix proteins.

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.

Immunocytochemical detection in eel corpuscles of Stannius of a mammalian parathyroid-like hormone.

Stannius corpuscles of the eel synthesize and secrete a mammalian parathyroid-like hormone called parathyrin of CS (PCS). PCS has been localized in the cytoplasm of all cells in the corpuscles, detection being by indirect immunofluorescence with an antiserum anti-1-84 bovine hormone (PTH). The specificity of the reaction was demonstrated by inhibition of the fluorescent staining with 1-84 bovine PTH and the active fragment 1-34 of human PTH. Variations of the cellular localization of the PCS or a complete depletion of the hormonal content, in all cells, were observed in eels made hypercalcemic by Ca overloading. The secretory activity of the two types of CS cells may be regulated by the plasma Ca2+ concentration.

Gill calcium fluxes in the eel, Anguilla angvilla (L.). effects of stannius corpuscles and ultimobranchial body

Abstract The effects of Stannius corpuscles (SC) or ultimobranchial body (UB) removal and SC extracts or calcitonin (CT) perfusion on gill calcium fluxes were described in eel Anguilla anguilla (L.). The perfusion of isolated gill arches made possible the measurements of bidirectional gill calcium fluxes. 1. 1. SC removal (SCX) enhanced gill calcium influx and decreased outflux. Saline extract of SC (SCSE) elicited a transient influx increase (not significant) and an outflux increase (81%). Methylene chloride extract of SC (SCOE) decreased influx (35%) and promoted a strong outflux increase (240%). 2. 2. Calcium influx of ultimobranchialectomized eels (UBX) was lower than of controls while outflux was unchanged. CT perfusion brought about a decrease in influx (39%) and a marked increase in ouflux (47%) in sham-operated eels, while the influx decrease and the outflux increase were respectively 26 and 80% in UBX. Interrelationships between the effect of UB and SC hormones on gills and serum calcium levels are discussed with regards to the general regulation of calcium metabolism.

Nacre Calcification in the Freshwater Mussel Unio pictorum: Carbonic Anhydrase Activity and Purification of a 95 kDa Calcium-Binding Glycoprotein

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid‐soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium‐binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre‐soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of ∼4, or ∼7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.