The Microstructure,Proteomics and Crystallization of the Limpet Teeth

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase‐Rabs, and clathrin, are identified by proteomics. A goethite‐binding experiment coupled with proteomics and RNA‐seq highlights six chitin‐binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.     

Correlative transmission and scanning electron microscopy of the initial mineralization of healing alveolar bone in rats

Primary mineralization in healing sockets after extraction of molar teeth was studied in rats. The observations obtained by scanning electron microscopy were correlated by transmission electron microscopy. The process is characterized by abundance of extracellular matrix vesicles distributed between the forming cells and the calcifying fronts. The occurrence of osmiophilic material and solitary hydroxyapatitte crystals within the vesicles was followed by accumulation of hydroxyapatite crystals, disappearance of the vesicular membrane and formation of calcospherites that conglomerate into calcified fronts. The process described here in bone healing is essentially similar to primary mineralization in other normal and pathological calcified tissues.     

Matrix proteins of the teeth of the sea urchin Lytechinus variegatus

The teeth of the sea urchin Lytechinus variegatus grow continuously. The mineral phase, a high magnesium calcite, grows into single crystals within numerous compartments bounded by an organic matrix deposited by the odontoblasts. Electron microscopic examination of glutaraldehyde-fixed Ethylene Diamine Tetra acetic acid (EDTA) demineralized teeth shows the compartment walls to be organized from multiple layers of cell membrane which might contain cytoplasmic protein inclusions. Proteins extracted during demineralization of unfixed teeth were examined by gel electrophoresis, high performance liquid chromatography, and amino acid analysis. The tooth proteins were acidic, they contained phosphoserine, and they were rich in aspartic acid. By contrast, the proteins of similarly extracted mineralized Aristotle's lantern skeletal elements were nonphosphorylated and were rich in glutamic acid. Vertebrate tooth and bone matrix proteins show similar differences. Surprisingly, an antibody to the principle rat incisor phosphoprotein showed a significant cross-reactivity with the urchin tooth protein, by dot-blot and enzyme-linked immunosorbent assay procedures. Thus, the urchin tooth proteins contain epitope regions similar to those which are phenotypic markers of vertebrate odontoblasts. Whether this is an expression of convergent or divergent evolutionary processes, it is likely that the matrix proteins play a similar role in matrix mineralization. The sea urchin tooth may thus be an excellent model for the study of odontoblast-mediated mineral-matrix relationships.     

Self-assembly and mineralization of genetically modifiable biological nanofibers driven by β-structure formation

Bioinspired mineralization is an innovative approach to the fabrication of bone biomaterials mimicking the natural bone. Bone mineral hydroxylapatite (HAP) is preferentially oriented with c-axis parallel to collagen fibers in natural bone. However, such orientation control is not easy to achieve in artificial bone biomaterials. To overcome the lack of such orientation control, we fabricated a phage-HAP composite by genetically engineering M13 phage, a nontoxic bionanofiber, with two HAP-nucleating peptides derived from one of the noncollagenous proteins, Dentin Matrix Protein-1 (DMP1). The phage is a biological nanofiber that can be mass produced by infecting bacteria and is nontoxic to human beings. The resultant HAP-nucleating phages are able to self-assemble into bundles by forming β-structure between the peptides displayed on their side walls. The β-structure further promotes the oriented nucleation and growth of HAP crystals within the nanofibrous phage bundles with their c-axis preferentially parallel to the bundles. We proposed that the preferred orientation resulted from the stereochemical matching between the negatively charged amino acid residues within the β-structure and the positively charged calcium ions on the (001) plane of HAP crystals. The self-assembly and mineralization driven by the β-structure formation represent a new route for fabricating mineralized fibers that can serve as building blocks in forming bone repair biomaterials and mimic the basic structure of natural bones.     

Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals

Mollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have a strong influence on the growth of CaCO(3), whereas the role of the insoluble matrix on mineralization is unclear. Therefore, we mineralized the EDTA (ethylenediaminetetraacetic acid) insoluble organic matrix of abalone nacre with a modified double-diffusion set-up, where the diffusing solutions were constantly renewed. Control experiments were performed with cellulose and chitosan foils. The mineralized matrices/foils were analyzed with SEM. We show that the insoluble matrix of abalone nacre induces the growth of flat and roughly polygonal CaCO(3) crystals. In some of the experiments with the insoluble matrix, the growth of three-dimensional parallel sheets of densely packed platelets inside the insoluble matrix was observed. XRD on these samples revealed that they consist of oriented aragonite.     

Studies on formation and resorption of fish scales

Summary Scale formation in Cyprinodon variegatus was found to be initiated at about 26 to 30 days after hatching. Ultrastructural investigation revealed that within 4 to 6 h in the first-formed scales the marginal cells begin to flatten and differentiate into osteogenic cells, which later change to osteoblasts and fibroblasts. These cells are separated from the surrounding epithelial cells by a basal lamina. The osteoid is formed by the marginal and osteogenic cells; the osseous layer by the osteoblasts; and the fibrillary plate by the fibroblasts.The osteoid is formed within 2 to 3 h after the initiation of the scale, and within 20 to 24 h the osseous layer is formed. Hydroxyapatite crystals are deposited in the matrix of the osseous layer without apparent association with collagen fibers. No matrix vesicles or dense bodies are evident at the sites of calcification. The fibrillary plate arises 18 to 20 h after the initiation of the scale. It is also partially calcified, but not before the third week of scale formation. The crystals develop almost exclusively between the collagen fibers at the extreme edge of the calcifying front, but solid calcification of the fibers results with further growth of the crystals. The fibroblasts appear to participate in calcification of the fibrillary plate.Contribution No. 332, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.     

Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study

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.     

Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia cuprina

The gut of most insects is lined with a peritrophic matrix that facilitates the digestive process and protects insects from invasion by micro-organisms and parasites. It is widely accepted that the matrix is composed of chitin, proteins and proteoglycans. Here we critically re-examine the chitin content of the typical type 2 peritrophic matrix from the larvae of the fly Lucilia cuprina using a range of techniques. Many of the histochemical and biochemical techniques indicate the presence of chitin, although they are often adversely influenced by the presence of highly glycosylated proteins, a principal component of the matrix. The alkali-stable fraction, which is used as an indicator of the maximum chitin content in a biological sample, is only 7.2% of the weight of the matrix. Larvae fed on the potent chitin synthase inhibitor polyoxin D or the chitin-binding agent Calcofluor White, showed strong concentration-dependent inhibition of larval weight and survival but no discernible effects on the matrix structure. A bacterial endochitinase fed to larvae had no effect on larval growth and no observable effect in vitro on the structure of isolated peritrophic matrix. RT–PCR did not detect a chitin synthase mRNA in cardia, the tissue from which PM originates. It is concluded that chitin is a minor structural component of the type 2 peritrophic matrix of this insect.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo,Determined by Immunolabelling

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.     

CRYSTAL GROWTH IN RAT ENAMEL

Observations have been made, using electron microscopy and x-ray diffraction, on the changes in crystal size and shape which occur in developing rodent enamel during mineralization. Small enamel pieces isolated from ground sections of rat molars and incisors were either embedded in methacrylate and sectioned with a diamond knife for electron microscopy, or they were mounted intact on glass fibers in a Debye-Sherrer type powder camera for x-ray diffraction. By either approach it was found that the apatite crystals were very long in the c axis direction from the beginning of enamel mineralization. Morphologically, the early crystals took the shape of extremely thin, long plates arranged in such a manner that there seemed to be little room for any further length-wise growth. It was demonstrated clearly, on the other hand, that the crystals increased in both thickness and width with advancing mineralization. As a result, the thin crystal plates gradually developed into hexagonal rods, which in the most mature enamel examined measured 500 to 600 A in width and 250 to 300 A in thickness.     

Ultrastructural studies of initial stages of mineralization of long bones and vertebrae in human fetuses

We studied 27 embryos of 5-12 weeks gestational age where pregnancy was interrupted due to paramedical reasons, in order to find the developmental stages at which matrix vesicles appear in cartilage, and whether they are involved in the mineralization process. Specimens of long bones, lumbar and thoracic vertebral column were prepared for light, transmission and scanning electron microscopic studies. In the cartilaginous models of long bones, matrix vesicles were found amongst maturing and hypertrophic chondrocytes already by the 6th week after fertilization. By that stage, bone rudiments consisted of only cartilage that was not yet mineralized. In the vertebral column matrix, vesicles were found in the vertebral bodies amongst maturing and hypertrophic chondrocytes at the beginning of the 8th week. At that stage, although hypertrophy of chondrocytes was observed, mineralization was still absent. No matrix vesicles were found in the perichondrium, investing mesenchyme and intervertebral discs. Mineralization of cartilage in long bone rudiments started in the form of hydroxyapatite crystals within or around the matrix vesicles at 7 weeks of age and in the vertebral column at 11 weeks. As mineralization progressed, more hydroxyapatite crystals were observed around the matrix vesicles, forming typical calcospherites . Mineralization then progressed in the form described in other animals.     

Aggregation of some water-soluble derivatives of chitin in aqueous solutions: Role of the degree of acetylation and effect of hydrogen bond breaker

By dynamic light scattering in combination with fluorescence spectroscopy and TEM it was shown that aggregation in aqueous solutions is inherent not only to chitosan, but also to two other water-soluble derivatives of chitin: O-carboxymethylchitin and di-N,N-carboxymethylchitosan. Aggregation is observed even for the samples without N-acetyl-d-glucosamine units, which remain upon incomplete chemical modification of chitin, indicating that specific interactions between residual chitin repeat units cannot be the main reason for the aggregation. At the same time, 7 M urea weakens the aggregation, thus testifying that hydrogen bonding and/or hydrophobic interactions are partially responsible for this phenomenon. The incomplete disruption of aggregates in 7 M urea may arise from crystallization of junction zones between different macromolecules, which makes some hydrogen bonds inaccessible for urea or too stable for breaking by this agent.     

A transmission electron microscope study using vitrified ice sections of predentin: structural changes in the dentin collagenous matrix prior to mineralization

The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize.     

Study on reaction mechanism of bioleaching of nickel and cobalt from lateritic chromite overburdens

Depletion of high-grade ores and presence of significant quantities of metals in low-grade oxide ores has enforced to utilize the overburdens (COB) and wastes (low-grade ores) generated during mining operations. The impact of ore mineralogy and mineral–microbe interaction during bioleaching could not be ignored. Seeking to the need, a systematic study was performed to establish the reaction mechanism involved for recovery of nickel and cobalt from chromite overburden (COB), Sukinda, Orissa using pure culture of Aspergillus niger. Mineralogical analysis reveals a complete conversion of goethite into hematite phase leading to exposure of nickel particles into the micro-pores and cracks developed in the matrix which was initially found to be intertwined in the goethite lattice. As a result, it became more susceptible to attack by the fungal bio acids which in turn accelerate the dissolution rate. Organic acids like oxalic and citric acids were detected in the culture filtrate using HPLC. TEM analysis of the leached samples shows that nickel dissolute into the solution leaving a porous space in the matrix of the hematite by forming nickel oxalate or nickel citrate. Kinetics of the nickel bioleaching was studied to support the mechanism of the reaction. It was observed that the initial rate of reaction follows the chemical control dissolution reaction where as the later part fits to shrinking core model. 18% of nickel and 37.8% of cobalt was recovered from pre-treated COB at 2.5% pulp-density with 10% (v/v) fungal inoculum at 30 °C within 25 days in shake flask while 32.5% of nickel and 86% of cobalt was recovered in bioreactor.     

Structural Organisation of the Cusps of the Radular Teeth of the Chiton Plaxiphora albida

Abstract The structure, morphology and organisation of the cusps of the major lateral radula teeth of the chiton Plaxiphora albida have been examined using light, transmission and scanning electron microscopy, together with energy dispersive X-ray analysis and Mössbauer spectroscopy. In this chiton species, both the anterior and posterior surfaces of the major lateral teeth are composed of magnetite, which is indicated to be non-stoichiometric and associated with some maghemite, together with small amounts of phosphorus and silicon. This outer layer surrounds an inner core region of the tooth, which only reaches the surface through a small window zone on the anterior surface and which contains large amounts of iron and phosphorus presumably in the form of iron(III) phosphate. The organic matrix, on which the teeth are constructed, consists of a zone of densely packed fine fibres at the surface of the tooth, underlain by larger fibres which become sparser deeper into the cusp. The core region is characterized by the presence of densely packed short fibres. In contrast to the situation found in most other species of chiton, large fibres of the organic matrix extend throughout the region of magnetite mineralization, leading to the suggestion that the matrix exerts more control over the mineralization of magnetite than has previously been thought.     

Biomineralization of calcium carbonate in the cell wall of Lithothamnion crispatum (Hapalidiales,Rhodophyta): correlation between the organic matrix and the mineral phase

Over the past few decades, progress has been made toward understanding the mechanisms of coralline algae mineralization. However, the relationship between the mineral phase and the organic matrix in coralline algae has not yet been thoroughly examined. The aim of this study was to describe the cell wall ultrastructure of Lithothamnion crispatum, a cosmopolitan rhodolith‐forming coralline algal species collected near Salvador (Brazil), and examine the relationship between the organic matrix and the nucleation and growth/shape modulation of calcium carbonate crystals. A nanostructured pattern was observed in L. crispatum along the cell walls. At the nanoscale, the crystals from L. crispatum consisted of several single crystallites assembled and associated with organic material. The crystallites in the bulk of the cell wall had a high level of spatial organization. However, the crystals displayed cleavages in the (104) faces after ultrathin sectioning with a microtome. This organism is an important model for biomineralization studies as the crystallographic data do not fit in any of the general biomineralization processes described for other organisms. Biomineralization in L. crispatum is dependent on both the soluble and the insoluble organic matrix, which are involved in the control of mineral formation and organizational patterns through an organic matrix‐mediated process. This knowledge concerning the mineral composition and organizational patterns of crystals within the cell walls should be taken into account in future studies of changing ocean conditions as they represent important factors influencing the physico‐chemical interactions between rhodoliths and the environment in coralline reefs.     

Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone

High-voltage (1.0 MV) electron microscopy and stereomicroscopy, electron probe microanalysis, electron diffraction and three-dimensional computer reconstruction, have been used to examine the spatial relationship between the inorganic crystals of calcium phosphate and the collagen fibrils of pickerel and herring bone. High-voltage stereo electron-micrographs were obtained of cross-sections of the cylinder-shaped intramuscular bones in uncalcified regions, in regions where only one or only several crystals had been deposited in some of the fibrils, and in successive sections containing progressively more mineral crystals until the stage of full mineralization was reached. High-resolution electron probe microanalysis confirmed that the electron-dense particles contained calcium and phosphorus. In the earliest stages of mineralization and progressing throughout the mineralization process, the crystals are located only within the collagen fibrils; crystals are not observed free in the extracellular spaces between collagen fibrils. The progressive increase in the mass of mineral deposited in the bone tissue with time occurs, essentially, completely within the collagen fibrils including the stage of full mineralization. At this stage, cross-sectional profiles of collagen fibrils are completely obliterated by mineral. A small number of crystals that are located on or close to the surface of the fibrils appear to extend a very short distance into the spaces between the fibrils. These ultrastructural observations of the very onset of calcification in which nucleation of the calcium phosphate crystals is clearly shown to begin within specific volumes of collagen fibrils, and of the subsequent temporal and spatial sequences of this phenomenon, which shows that calcification continues wholly within the collagen fibrils until maximum calcification is achieved, add important information on the basic physical chemical mechanism of the calcification and the structural elements that are involved. The spatial and temporal independence of the sites where mineralization is initiated establishes that such ultrastructural locations within individual collagen fibrils represent independent, physical chemical nucleation loci. The findings are totally inconsistent with the proposal that crystals must first be deposited in matrix vesicles, or other components such as mitochondria, and subsequently released and propagated in the interfibrillar space, until they eventually reach and impregnate the hole zone regions of the collagen fibrils. Three-dimensional computer reconstruction of serial transverse and longitudinal sections demonstrates periodic swellings along the collagen fibrils, corresponding to the hole zone region of their axial period as mineralization proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Skeletal formation in the modern but ultraconservative chaetetid spongeSpirastrella (Acanthochaetetes) wellsi (demospongiae,porifera)

Summary The modern hadromerid coralline spongeSpirastrella (Acanthochaetetes) wellsi exhibits a unique secondary high-Mg calcite (>19 mol % MgCO₃) basal skeleton. The basal skeleton is constructed of bundles of elongated crystals more or less tangentially orientated. The initial formation of these crystals is controlled by soluble highly acidic aspartic and glutamic-rich (40%) macromolecules. The skeletal mineralization occurs in four different loci: in the top of the calicles, at the tabulae, on collagenous anchor fibres, and within closed spaces between the tabulae. The clicle walls are formed on the uppermost top of the basal skeleton as a continuous process. Based on long term stainings with Ca²⁺-chelating fluorochroms (calcein, chlorotetracyclines) the growth rate of this sponge is extremely low with ca. 50–100μm/a. The skeletal formation takes places outside the sponge, within a narrow zone (300–500 nm) between the basopinacoderm and the mature basal skeleton. The sponge produces thread-like folded templates (‘spaghetti fibres’) of 0,5–2 μm size, the shape controlling insoluble organic matrix. These templates become mineralized in a first step as MgCO₃, then are stretched. A soluble organic matrix is also secreted, and remains are included inside the mineralized skeleton. This organic matrix consists of in a complex mixture containing small very acidic proteins (5, 13, 31 KD; 40% Asp and Glu and therefore most probably Ca²⁺-binding) and high molecular weight glycoproteins among several other organic compounds. The mature crystals are high-Mg calcites. During calcification large cells with large reserve granules (LCG) are always present in a tight connection with the basopinacoderm. These cells form also the collagenous anchor fibres. Primary tabulae are formed by a non-collagenous organic sheet. Calcification happens only when LCG cells are enriched on the organic sheet. Randomly oriented high-Mg calcite crystals are growing on the collagenous anchor fibres. The same type of the mineralization is observed within the spaces of the tabulae. This particular case of mineralization is controlled by decaying sponge tissue (ammonification). The δ¹³C values are in equilibrium with the ambient sea water and vary between +3.2 and +2.8 ‰. The mode of mineralization of the basal skeleton can be described as biologically induced resp. matrix mediated.