The in vitro osteoclastic degradation of nacre.

Osteoclast activity was studied on nacre, the mother of pearl (MOP) in order to assess the plasticity of bone resorbing cells and their capacity to adapt to a biomineralized material with a different organic and mineral composition from that of its natural substrate, bone. Pure MOP, a natural biomineralized CaCO(3) material, was obtained from Pinctada oyster shell. When implanted in the living system, nacre has proven to be a sustainable bone grafting material although a limited surface degradation process. Osteoclast stem cells and mature osteoclasts were cultured on MOP substrate and osteoclast precursor cells were shown to differentiate into osteoclasts capable of resorbing nacre substrate. However, analysis of the organization of the cytoskeleton showed that both a sealing zone and a podosome structure were observed on the nacre substrate. Moreover, MOP resorption efficiency was consistently found to be lower than that of bone and appeared to be a limited process.

Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.

Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95-99% calcium carbonate and 1-5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.

Role of near-infrared fluorescence imaging in the resection of metastatic lymph nodes in an optimized orthotopic animal model of HNSCC.

OBJECTIVES: To study the role of near-infrared fluorescence imaging in the detection and resection of metastatic cervical lymph nodes in head and neck cancer. MATERIALS AND METHODS: CAL33 head and neck cancer cells of human origin were implanted in the oral cavity of nude mice. The mice were followed up after tumor resection to detect the development of lymph node metastases. A specific fluorescent tracer for αvß3 integrin expressed by CAL33 cells was injected intravenously in the surviving mice between the second and the fourth month following tumor resection. A near-infrared fluorescence-imaging camera was used to detect tracer uptake in metastatic cervical lymph nodes, to guide of lymph-node resection for histological analysis. RESULTS: Lymph node metastases were observed in 42.8% of surviving mice between the second and the fourth month following orthotopic tumor resection. Near-infrared fluorescence imaging provided real-time intraoperative detection of clinical and subclinical lymph node metastases. These results were confirmed histologically. CONCLUSION: Near infrared fluorescence imaging provides real-time contrast between normal and malignant tissue, allowing intraoperative detection of metastatic lymph nodes. This preclinical stage is essential before testing the technique in humans.

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.

Characterization and quantification of chitosan extracted from nacre of the abalone Haliotis tuberculata and the oyster Pinctada maxima.

This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (eta) of extraction was calculated (eta = 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (eta = 0.053%) was isolated rather than chitosan.

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.

Role of water-soluble matrix fraction, extracted from the nacre of Pinctada maxima, in the regulation of cell activity in abalone mantle cell culture (Haliotis tuberculata).

In mollusks, the mantle is responsible for the secretion of an organic matrix that mineralizes to form the shell. A model of mantle cell culture has been established from the nacreous gastropod Haliotis tuberculata. First, viability of cells, quantified by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) reduction assay, was monitored in order to determine a cell density and a time-culturing period in order to investigate biomineralization processes in vitro. During the first 11 days of culture, an increase of MTT response demonstrated an activation of cultured cells mitochondrial activity as confirmed by the total protein content assay. The effect of a water-soluble extract from the organic matrix of Pinctada maxima (WSM) was tested on this cell culture system for 11 days-period exposure. WSM reduced the global viability of mantle cells in a dose-dependent way which corresponded to a cell death. Alkaline phosphatase activity normalized to total protein content increased in the presence of WSM. This increase may be due to an activation of cells and a selection of one (or a few) cell type(s). Further investigations will help us to determine this selectivity issue.

Partial purification of parathyrin from the corpuscles of Stannius (PCS) of the eel (Anguilla anguilla, L.).

It has been previously shown that the eel corpuscles of Stannius (CS) synthesize and secrete a substance (PCS) which is functionally and immunologically related to the mammalian parathyrin family. Purification of PCS, including anion-exchange chromatography, ODS C-18 reverse-phase HPLC, and affinity chromatography, showed that a biologically active peak, eluted in 32% acetonitrile, contains a 32- to 34-kDa protein which is 600-fold more potent than the crude extract is a test involving the hypocalcemic response in the CS-deprived eel. Specific immunoprecipitation of protein encoded by mRNA extracted from eel CS indicates that a 45-kDa precursor is involved in PCS synthesis. The hypothetical significance of a "large" parathyrin-like molecule in fish is suggested in relation to what is known about mammalian parathyrin gene expression.

[In vitro translation of RNA extracted from corpuscules of Stannius of the eel (Anguilla anguilla); identification of the precursor of parathyrin in the corpuscules of Stannius (PCS)]. / Traduction in vitro des ARN extraits des corpuscules de Stannius de l'Anguille (Anguilla anguilla); identification du précurseur de la parathyrine des corpuscules de Stannius (PCS).

Corpuscles of Stannius (CS) of Teleosts secrete a PTH like hormone (Parathyrin of CS: PCS) which is involved in calcium metabolism by acting mainly at the gill level. Translated products encoded by mRNA extracted from Eel Corpuscles of Stannius and among these, translated products immunoprecipitated by anti b-PTH immunserum were compared to the products synthetized during incubation of the glands with labelled methionine. The analysis was performed by polyacrylamide gel electrophoresis and fluorography. Biosynthesis of the PCS involves a mRNA encoding for a 45 kD precursor which is immunoprecipitated by anti b-PTH immunserum and totally displaced from antibody, either by 1-34 h-PTH or by purified PCS.

[Effect of salmon calcitonin on intestinal absorption of calcium in the eel (Anguilla anguilla L.)]. / Effet de la calcitonine de Saumon sur l'absorption intestinale du calcium chez l'Anguille (Anguilla anguilla L.).

Salmon calcitonin (SCT) acted, in eel, on Ca intestinal absorption by modulating Ca flux lumen-plasma (phi Calp) intensity. This response was dependent upon the basal value of the intestinal Ca absorption; a linear relation between the basal phi Calp and its variation after SCT was shown. After Stannius corpuscles removal the lower phi Calp was stimulated by SCT in all cases.

[Are Stannius corpuscles the parathyroid glands of teleost fishes? Ultrastructural, cytologic and immunocytochemical arguments]. / Les corpuscules de Stannius sont-ils les glandes parathyroïdes des poissons téléostéens? Arguments ultrastructuraux, cytologiques et immunocytochimiques.

Parathyrin of Stannius corpuscles (PCS), glands which are restricted to Holostei and Teleostei, is closely related to mammalian parathyrin (PTH) secreted by the parathyroids [( 6] to [9]). In unstimulated and stimulated CS we have shown the same structure and cellular types that are described in mammalian parathyroids. We observed three types of cells; the two sorts of cells already described [14] present such a difference of density (Fig. 1) that type I may be compared to chief dark cells and type II to chief light cells. The difference between these two forms was particularly marked in activated CS; a similar observation has been reported concerning PT [15]. Furthermore, we have detected a third type of cell present either singly in unactivated CS or in small groups between chief cells in activated CS. They showed all the characteristics of oxyphil cells [17]; they present an extremely dense cytoplasm with numerous mitochondria and a typical stellate form with cytoplasmic processes extending between chief cells (Figs. 2, 3). In CS of untreated eels, we have shown by means of indirect immunocytology (using an immunserum raised against the active fragment 1-34 bPTH) that the immunostained reaction product was limited to dilated cisternae and ribosomes of the rough endoplasmic reticulum and to most of the granules in all the chief cells (Figs. 4, 5). No immunoreaction was observed in Golgi area. Oxyphil cells did not present an immune localization of PCS. CS are structurally and cytologically similar to mammalian PT; furthermore their chief cells synthesize, stock and secrete a substance immunologically similar to mammalian PTH; the exact function of oxyphil cells has to be demonstrated.

Responses of the ultimobranchial body in eels (Anguilla anguilla L.) maintained in sea water and experimentally matured, to injections of synthetic salmon calcitonin.

The effect of a synthetic salmon calcitonin (SCT) treatment on ultimobranchial body (UB) activity in eels (Anguilla anguilla L.) maintained in seawater and submitted to experimental maturation, has been studied histologically. 2. The activity of the glands of a control group of eels maintained in sea water was taken as a reference. 3. The UB parenchyma showed a marked atrophy in the fish treated with SCT alone and serum calcium decreased significantly in this group. 4. Immature female silver eels receiving carp pituitary extract (CPE 1 mg/100 g body wt. per injections) until complete maturation presented high hypercalcemia associated with cellular hypertrophy and hyperplasia in the UB. 5. SCT treatment did not prevent the hypercalcemia provoked by CPE injections. UB activity was strongly increased in this case. 6. These data indicate that the activity of the UB in eels varies with both physiological and experimental hypercalcemia, and responds to SCT injections.

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 maintained 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 ostelysis, usually observed, did not appear.

[Morphological, cytological, and biochemical changes of Anguilla anguilla L. gills deprived of Stannius corpuscles (S. C.) (author's transl)]. / Modifications morphologiques, cytologiques et biochimiques de la branchie d'Anguilla anguilla L. après ablation des corpuscules de Stannius.

In the silver female eel (Anguilla anguilla L.) maintained in fresh water, surgical removal of Stannius corpuscles (SC) resulted in: a) A significant increase of wet weight of gill cells and the dry weight of gill filaments expressed as function of body weight. b) A proliferation and a hypertrophy of chloride cells shown by a significant rise of cell volume expressed as a precentage of the epithelial volume. c) An increase of the calcium (Ca) binding protein activity in the total branchial mucosa. These modifications are not observed when eels are maintained in Ca-free water. The results are discussed in relation to the Ca fluxes across gills in eels deprived of SC and gill morphological changes during sea water adaptation.

Comparative effects of nacre water-soluble matrix and dexamethasone on the alkaline phosphatase activity of MRC-5 fibroblasts.

In this study we demonstrate, for the first time, that dexamethasone and BMP-2 stimulated alkaline phosphatase (ALP) activity in MRC-5 fibroblasts, a cell line derived from human fetal lung. Previously we reported that the water-soluble matrix (WSM) of nacre obtained from the inner shell layer of the oyster Pinctada maxima, promoted an increase in ALP activity that was dose-dependent. In this work, we show that the effect of WSM is also time-dependent. As a comparison, the effect of WSM was also tested in bone marrow stromal cells because marrow and other bone surface-derived osteoblast stem cells have the inherent direct potential for osteogenesis. WSM promotes cell proliferation and ALP activity when tested with bone marrow cells in concentrations between 135 and 540 microg protein/mL. The effect of WSM on ALP activity of bone marrow stromal cells is similar to that obtained by dexamethasone. These results imply that MRC-5 fibroblasts respond to differentiating factors that promote osteoblastic phenotype in bone-derived cell cultures.

[Structural similarities between the hypocalcemia hormone of the corpuscles of Stannius of the eel (Anguilla anguilla L.) and the mammalian parathyroid hormone]. / Similitudes structurales entre l'hormone hypocalcemiante des corpuscules de Stannius (PCS) de l'Anguille (Anguilla anguilla L.).

Extracts of the corpuscules of Stannius enhance Eel gill calcium efflux and increase bone resorption in the Rat. Thus the hormone is hypocalcaemic in the Eel although hypercalcaemic in the Rat. We show here that a 15 min. pulse perfusion of a synthetic fragment of human PTH 1-34 had a similar effect on gill calcium fluxes to that of CS extract. Further, we found that Eel plasma contains a substance reacting with five different antibodies against bovine PTH. The concentration of this plasma material is enhanced seven-fold by parenteral calcium and rendered extremely low or absent by CS removal. A convenient name for this major calcium-regulating hormone is parathyrine of the corpuscles of Stannius (PCS) since this reflects both its glandular source and chemical structure.

[Comparative effects of an extract of Anguilla Stannius corpuscles and of an active fragment of human parathyroid hormone (1-34 hPTH) on an anuran batracian, Xenopus laevis]. / Effets comparés de l'extrait de corpuscules de Stannius d'Anguille et du fragment actif de la parathyrine humaine (1-34 hPTH) chez un batracien anoure, Xenopus laevis.

Parathyrin of the corpuscles of Stannius (PCS), glands which are restricted to Holostei and Teleostei, resembles mammalian parathyroid hormone secreted by the parathyroids. These glands made their first phylogenic appearance with Amphibians, whereas the Stannius corpuscles disappeared. We have compared, in the clawed toad, Xenopus laevis, the effects of injections of extracts of corpuscles of Stannius and of the active synthetic fragment of parathyrin (1-34 hPTH). Both hormones elicited hypocalcemia and hypophosphatemia within a 4 days treatment. These results are interpreted according to the hypothesis that in the most primitive Amphibians there are receptors leading to hypocalcemic effect co-existing with the appearance of "new" glands which secrete a hypercalcemic hormone in mammals.

Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts.

A new approach to the isolation of the water-soluble factors from nacre without any demineralization is described and examined their effect on fibroblast cells in culture. The soluble matrix in pure water from the nacre of Pinctada maxima was analysed by size-exclusion HPLC. Four fractions (SE1-SE4) of the water-soluble matrix (WSM) were further analysed by anion-exchange HPLC. The amino acid composition of the WSM showed that it is mainly composed of glycine and alanine. SE1 and SE4 had different amino acid compositions from the whole WSM. The WSM and SE4 tested on a culture of human foetus lung tissue fibroblasts increased the alkaline phosphatase (ALP) activity. SE1 caused a decrease in ALP activity. Our results support the hypothesis that WSM promotes the differentiation of cells in vitro.

Calcium-dependent metabolism of 25-hydroxycholecalciferol in silver eel tissues.

We investigated the in vitro metabolism of [26,27-3H]-25-(OH)D3 in different eel tissues. After incubation with [3H]-25-(OH)D3, tissues were extracted with methanol-chloroform and chromatographed on Sephadex LH 20 columns. Two derivatives less polar than 25-(OH)D3 were detected, the first one being sensitive to KOH treatment. Three peaks more polar than 25-(OH)D3 were also found: peak I migrated close to the 24,25-(OH)2D3 area and was quantitatively the most important, but the presence of 24,25-(OH)2D3 could not be demonstrated; peak II migrated in the 1,25-(OH)2D3 region; and peak III had an elution position twice that of peak II. After 6-h incubation of tissues isolated from control eels, peak I was found in all tissues including intestine and gills. It was highest in pituitary gland and brain and lowest in ovaries and muscle. It was not significantly modified 20 days after ablation of the corpuscules of Stannius. In contrast, in vivo daily calcium chloride injection was followed 24 hr later by a significant increase in the [3H]-25-(OH)D3 conversion into peak I in gills, intestine, and the spinal cord and by an inhibition of this conversion in pituitary gland, skin, and muscle. The inhibition was found in all tissues after five daily calcium injections. Calcium injection had no effect on the in vitro metabolite synthesis by the corpuscules of Stannius. These results suggest that vitamin D is not metabolized in the same way in eel as in mammals and that this metabolism could in part be calcium dependent.

[Immunocytochemical detection in Stannius corpuscles of the eel (Anguilla anguilla L.) of a hormone similar to the mammalian parathyroid hormone]. / Détection immunocytochimique dans les corpuscules de Stannius de l'anguille (Anguilla anguilla L.) d'une hormone proche de l'hormone parathyroïdienne mammalienne.

In eels the parathyrin of the corpuscles of Stannius (PCS), mammalian parathyroid-like hormone, has been localized in the cytoplasm of all the cells in the corpuscles. This detection was done by indirect immunofluorescence with an antiserum anti 1-84 bovine parathormone. The specificity of the reaction was demonstrated by inhibition of the coloration obtained with the 1-84 bovine parathormone and the active fragment 1-34 of human parathormone. Variations of the cellular localization of the PCS or a complete depletion of the hormonal content were observed in eels made hypercalcaemic by calcium overloading.