Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy.

Amelogenin proteins, the principal components of the developing dental enamel matrix, self-assemble to form nanosphere structures that are believed to function as structural components directly involved in the matrix mediated enamel biomineralization. The self-assembly behavior of a recombinant murine amelogenin (rM179) was investigated by atomic force microscopy (AFM) for further understanding the roles of amelogenin proteins in dental enamel biomineralization. Recombinant rM179 amelogenin was dissolved in a pH 7.4 Tris-HCl buffer at concentrations ranging from 12.5 to 300 microg/ml. The solutions were adsorbed on mica, fixed with Karnovsky fixative and rinsed thoroughly with water for atomic force microscopy (AFM). At low concentrations (12.5-50 microg/ml), nanospheres with diameters varying from 7 to 53 nm were identified while at concentrations ranging between 100-300 microg/ml the size distribution was significantly narrowed to be steadily between 10 and 25 nm in diameter. These nanospheres were observed to be the basic building blocks of both engineered rM179 gels and of the developing enamel extracellular matrix. The stable 15-20-nm nanosphere structures generated in the presence of high concentrations of amelogenins were postulated to be of great importance in facilitating the highly organized ultrastructural microenvironment required for the formation of initial enamel apatite crystallites.     

Control of Vertebrate Skeletal Mineralization by Polyphosphates

Background

Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO₃ ⁻)_n) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal Findings/Methodology

The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO₄ ³⁻) concentration while permitting the accumulation of a high total PO₄ ³⁻ concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO₄ ³⁻ and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4′,6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/Significance

We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.     

Effect of incorporation of nano bioactive silica into commercial Glass Ionomer Cement (GIC)

Four types of bioactive nano-silica were prepared by different methods, and were used to improve commercial dental Glass Ionomer Cement (GIC) bioactivity. The prepared powder samples were characterized by X-ray diffraction (XRD) to identify the formed phase; particle size and morphology were assessed by transmission electron microscope (TEM). The bioactivity of the prepared powder samples and its dental cement blends were applied in simulated body fluid (SBF). The change in surface morphology and composition after soaking in SBF after week at 37 °C were determined by scanning electron microscopy with energy dispersive spectroscopy (SEM with EDS) and Fourier transform infrared analyses (FTIR). Our results confirmed that the prepared silica powder exists in nano-scale. Precipitations of carbonate–apatite on the silica surface were observed by FT-IR spectroscopy and scanning electron microscopy. Silica dissolution and re-precipitation phenomena were also observed from SEM. The relationship between both phenomena during the in vitro test is discussed mainly in terms of structural and microstructural features of the silica. Combination of bioactive nano-silica with dental cement improves its bioactivity, which may be helpful to overcome marginal gap formation which is major disadvantage of the commercial dental cement.     

Long-lived radicals in irradiated apatites of biological interest: an e.s.r. study of apatite samples treated with 13CO2

Hydroxyapatite is used as a model for studying radical formation in the mineral compartment of irradiated calcified tissues. Treating this material with 13C-enriched CO2 confirms that radiogenic long-lived radicals correspond to carbon centred species. It is shown, however, that these radicals are not located on the carbonate ions which substitute either the phosphate or the hydroxyl groups. The conditions which allow the formation and the trapping of these radicals are investigated (role of humidity, CO2 and temperature) and this paramagnetic species is identified as CO-2 adsorbed at the surface of apatite crystallites.     

Diversity of mineral cell coverings and their formation processes: a review focused on the siliceous cell coverings

Mineral cell coverings are found in various protists. Some macroalgae accumulate calcium carbonate in the intercellular space, and some unicellular organisms use calcium carbonate or silica for the construction of loricas, scales, and frustules. Diatoms are representatives of those utilizing silica for the material of the cell covering called a frustule. The development of the frustule is initiated in a silica-deposition vesicle (SDV), which occurs just beneath the plasma membrane and, subsequently, the silicified cell covering expands its area, following the expansion of the SDV from valve face to valve mantle. Sequential valve development with whole valves is reviewed in several diatoms placed in different phylogenetic positions. Every diatom commences its valve formation from its pattern center and then develops by means of individual procedures. The results indicate that the valve development reflects the phylogeny of diatoms. In addition, recent progress in silica biomineralization is briefly reviewed, and the phylogeny of ability concerning siliceous cell covering formation is inferred. Electronic Publication     

Carbonate facies‐specific stable isotope data record climate,hydrology, and microbial communities in Great Salt Lake,UT

Organic and inorganic stable isotopes of lacustrine carbonate sediments are commonly used in reconstructions of ancient terrestrial ecosystems and environments. Microbial activity and local hydrological inputs can alter porewater chemistry (e.g., pH, alkalinity) and isotopic composition (e.g., δ¹⁸O_water, δ¹³C_DIC), which in turn has the potential to impact the stable isotopic compositions recorded and preserved in lithified carbonate. The fingerprint these syngenetic processes have on lacustrine carbonate facies is yet unknown, however, and thus, reconstructions based on stable isotopes may misinterpret diagenetic records as broader climate signals. Here, we characterize geochemical and stable isotopic variability of carbonate minerals, organic matter, and water within one modern lake that has known microbial influences (e.g., microbial mats and microbialite carbonate) and combine these data with the context provided by 16S rRNA amplicon sequencing community profiles. Specifically, we measure oxygen, carbon, and clumped isotopic compositions of carbonate sediments (δ¹⁸O_carb, δ¹³C_carb, ?₄₇), as well as carbon isotopic compositions of bulk organic matter (δ¹³C_org) and dissolved inorganic carbon (DIC; δ¹³C_DIC) of lake and porewater in Great Salt Lake, Utah from five sites and three seasons. We find that facies equivalent to ooid grainstones provide time‐averaged records of lake chemistry that reflect minimal alteration by microbial activity, whereas microbialite, intraclasts, and carbonate mud show greater alteration by local microbial influence and hydrology. Further, we find at least one occurrence of ?₄₇ isotopic disequilibrium likely driven by local microbial metabolism during authigenic carbonate precipitation. The remainder of the carbonate materials (primarily ooids, grain coatings, mud, and intraclasts) yield clumped isotope temperatures (T(?₄₇)), δ¹⁸O_carb, and calculated δ¹⁸O_water in isotopic equilibrium with ambient water and temperature at the time and site of carbonate precipitation. Our findings suggest that it is possible and necessary to leverage diverse carbonate facies across one sedimentary horizon to reconstruct regional hydroclimate and evaporation–precipitation balance, as well as identify microbially mediated carbonate formation.     

Prescribing diatom morphology: toward genetic engineering of biological nanomaterials

The formation of inorganic materials with complex form is a widespread biological phenomenon (biomineralization). Among the most spectacular examples of biomineralization is the production by diatoms (a group of eukaryotic microalgae) of intricately nanopatterned to micropatterned cell walls made of silica (SiO2). Understanding the molecular mechanisms of diatom silica biomineralization is not only a fundamental biological problem, but also of great interest in materials engineering, as the biological self-assembly of three-dimensional (3D) inorganic nanomaterials has no man-made analog. Recently, insight into the molecular mechanism of diatom silica formation has been obtained by structural and functional analysis of biomolecules that are involved in this process. Furthermore, the rapid development of diatom molecular genetics has provided new tools for investigating the silica forming machinery of diatoms and for manipulating silica biogenesis. This has opened the door for the production, through genetic engineering, of unique 3D nanomaterials with designed structures and functionalities.     

Isotopic signatures (δO and δC) of bivalve shells from cold seeps and hydrothermal vents

The oxygen and carbon isotopic compositions of 108 modern shells of various bivalve species collected from cold seeps and hydrothermal vents were investigated in order to evaluate whether these parameters can provide information on environmental geochemical variability as well as on bivalve species and on the type of symbiotic bacteria present in their gills. The results show that the carbonate of bivalve shells from hydrothermal vents is characterized by abnormal positive δ¹³C values due to kinetic isotope effects, whereas the carbonate of bivalve shells from cold seeps exhibits positive as well as negative δ¹³C values suggesting that oxidized methane emitted by the seeping fluids may be incorporated in the shell. Comparison of the δ¹⁸O and δ¹³C values of bivalve shells hosting different chemosymbiotic bacteria suggests that each type of symbiosis is associated with a specific environment and bivalve species, indicating that there is a strong physiological/metabolic control on the incorporation of stable isotopes during the biomineralization process.     

Biomineralization by photosynthetic organisms: Evidence of coevolution of the organisms and their environment?

Biomineralization is widespread among photosynthetic organisms in the ocean, in inland waters and on land. The most quantitatively important biogeochemical role of land plants today in biomineralization is silica deposition in vascular plants, especially grasses. Terrestrial plants also increase the rate of weathering, providing the soluble substrates for biomineralization on land and in water bodies, a role that has had global biogeochemical impacts since the Devonian. The dominant photosynthetic biomineralizers in today's ocean are diatoms and radiolarians depositing silica and coccolithophores and foraminifera depositing calcium carbonate. Abiotic precipitation of silica from supersaturated seawater in the Precambrian preceded intracellular silicification dominated by sponges, then radiolarians and finally diatoms, with successive declines in the silicic acid concentration in the surface ocean, resulting in some decreases in the extent of silicification and, probably, increases in the silicic acid affinity of the active influx mechanisms. Calcium and bicarbonate concentrations in the surface ocean have generally been supersaturating with respect to the three common calcium carbonate biominerals through geological time, allowing external calcification as well as calcification in compartments within cells or organisms. The forms of calcium carbonate in biominerals, and presumably the evolution of the organisms that produce them, have been influenced by abiotic variations in calcium and magnesium concentrations in seawater, and calcium carbonate deposition has probably also been influenced by carbon dioxide concentration whose variations are in part biologically determined. Overall, there has been less biological feedback on the availability of substrates for calcification than is the case for silicification.     

Osteodentine and vascular osteodentine of Anarhichas lupus (L.)

Summary TEM, SEM and X-ray diffraction analysis demonstrate the heterogeneity of the dentinal tissue of Anarhichas lupus, a vascular osteodentine. The disordered aspect of collagen fibres, incompletely mineralized (the periodical striation being still visible), explains the scattered distribution of crystallites since they are responsible for their arrangement. The low degree of mineralization revealed by the visible collagen striation is confirmed by X-ray diffraction analysis (the crystallinity of vascular osteodentine being much lower than that of the peripheral dental tissue) as well as by high resolution TEM, since no lattice planes could be observed. Osteodentine, supporting bone and proper bone have in common a mineral phase, more or less organized, different from the apatite system.The authors thank Mireille Cottrel-Gengoux for her technical assistance     

Calcium isotope fractionation by intracellular amorphous calcium carbonate (ACC) forming cyanobacteria

The formation of intracellular amorphous calcium carbonate (ACC) by various cyanobacteria is a widespread biomineralization process, yet its mechanism and importance in past and modern environments remain to be fully comprehended. This study explores whether calcium (Ca) isotope fractionation, linked to ACC-forming cyanobacteria, can serve as a reliable tracer for detecting these microorganisms in modern and ancient settings. Accordingly, we measured stable Ca isotope fractionation during Ca uptake by the intracellular ACC-forming cyanobacterium Cyanothece sp. PCC 7425. Our results show that Cyanothece sp. PCC 7425 cells are enriched in lighter Ca isotopes relative to the solution. This finding is consistent with the kinetic isotope effects observed in the Ca isotope fractionation during biogenic carbonate formation by marine calcifying organisms. The Ca isotope composition of Cyanothece sp. PCC 7425 was accurately modeled using a Rayleigh fractionation model, resulting in a Ca isotope fractionation factor (Δ⁴⁴Ca) equal to −0.72 ± 0.05‰. Numerical modeling suggests that Ca uptake by these cyanobacteria is primarily unidirectional, with minimal back reaction observed over the duration of the experiment. Finally, we compared our Δ⁴⁴Ca values with those of other biotic and abiotic carbonates, revealing similarities with organisms that form biogenic calcite. These similarities raise questions about the effectiveness of using the Ca isotope fractionation factor as a univocal tracer of ACC-forming cyanobacteria in the environment. We propose that the use of Δ⁴⁴Ca in combination with other proposed tracers of ACC-forming cyanobacteria such as Ba and Sr isotope fractionation factors and/or elevated Ba/Ca and Sr/Ca ratios may provide a more reliable approach.     

How corals made rocks through the ages

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef‐building corals retain information about the marine environment in their skeletons, which is an organic–inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue–skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification.     

Fluoride and carbonate incorporation into hydroxyapatite under condition of cyclic pH variation

Fluoride and carbonate ions, which are present in the inorganic phase of bone, enamel, and dentine, are known to play an important and opposite role in the process of recrystallization. We have investigated the incorporation of fluoride and carbonate ions into hydroxyapatite structure under conditions of cyclic pH fluctuation and the effect of these incorporations on the conversion of hydroxyapatite into β-tricalcium phosphate after heat treatment. Fluoro-hydroxyapatite has been obtained as unique crystalline phase up to 20 fluoride at. %. The degree of substitution of fluoride for hydroxyl does not depend on the extent of carbonate incorporated into the apatite structure. On the other hand, the carbonate incorporation into the apatite structure seems to be hindered by the contemporary presence of fluoride. Both fluoride and carbonate exhibit a stabilizing effect on the apatite structure, as far as its conversion into β-tricalcium phosphate is concerned. The order of efficiency in stabilizing the apatite structure is F⁻ > F⁻ + CO₃²⁻ > C0₃²⁻.     

Influence of microgravity on crystal formation in biomineralization.

Biomineralized tissues are widespread in animals. They are essential elements in skeletons and in statocysts. The function of both can only be understood with respect to gravitational force, which has always been present. Therefore, it is not astonishing to identify microgravity as a factor influencing biomineralization, normally resulting in the reduction of biomineralized materials. All known biominerals are composite materials, in which the organic matrix and the inorganic materials, organized in crystals, interact. If, during remodeling and turnover processes under microgravity, a defective organization of these crystals occurs, a reduction in biomineralized materials could be the result. To understand the influence of microgravity on the formation of biocrystals, we studied the shell-building process of the snail Biomphalaria glabrata as a model system. We show that, under microgravity (space shuttle flights STS-89 and STS-90), shell material is built in a regular way in both adult snails and snail embryos during the beginning of shell development. Microgravity does not influence crystal formation. Because gravity has constantly influenced evolution, the organization of biominerals with densities near 3 must have gained independence from gravitational forces, possibly early in evolution.     

Mineralization of pristine chitosan film through biomimetic process

The biomineralization of pristine chitosan film without any prior surface treatment was evaluated by immersing the film in simulated body fluid (SBF) at 37 °C for 3 weeks. The film was prepared by solvent casting method using chitosan of known degree of deacetylation (DD). The formation of the hydroxyapatite (HA) phase on the film surface after immersion was studied periodically by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), energy dispersive X-ray analysis (EDX) and scanning electron microscopy (SEM) methods. The electron micrographs showed the morphology of the deposited apatite as small globules appearing uniformly throughout the films surfaces. The Ca/P ratio of the apatite was found to increase with increase in immersion time and approaching towards the stoichiometric value of the HA phase. The mineralized chitosan film could be of promising support to hard tissue regeneration.     

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.     

厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。     

Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

Plasticity of mandibular biomineralization in myostatin-deficient mice

Compared with the normal or wild-type condition, knockout mice lacking myostatin (Mstn), a negative regulator of skeletal muscle growth, develop significant increases in relative masticatory muscle mass as well as the ability to generate higher maximal muscle forces. Wild-type and myostatin-deficient mice were compared to assess the postweaning influence of elevated masticatory loads because of increased jaw-adductor muscle and bite forces on the biomineralization of mandibular cortical bone and dental tissues. Microcomputed tomography (microCT) was used to quantify bone density at a series of equidistant external and internal sites in coronal sections for two symphysis and two corpus locations. Discriminant function analyses and nonparametric ANOVAs were used to characterize variation in biomineralization within and between loading cohorts. Multivariate analyses indicated that 95% of the myostatin-deficient mice and 95% of the normal mice could be distinguished based on biomineralization values at both symphysis and corpus sections. At the corpus, ANOVAs suggest that between-group differences are due to the tendency for cortical bone mineralization to be higher in myostatin-deficient mice, coupled with higher levels of dental biomineralization in normal mice. At the symphysis, ANOVAs indicate that between-group differences are related to significantly elevated bone-density levels along the articular surface and external cortical bone in the knockout mice. Both patterns, especially those for the symphysis, appear because of the postweaning effects of increased masticatory stresses in the knockout mice versus normal mice. The greater number of symphyseal differences suggest that bone along this jaw joint may be characterized by elevated plasticity. Significant differences in bone-density levels between normal and myostatin-deficient mice, coupled with the multivariate differences in patterns of plasticity between the corpus and symphysis, underscore the need for a comprehensive analysis of the plasticity of masticatory tissues vis-à-vis altered mechanical loads.