Papers by Keyword: Biomineralization

Abstract: Underwater attachment is a significant challenge, for which we have no good general solutions in our technology. Yet, a number of biological organisms have evolved solutions to this problem. One intriguing approach to underwater attachment is that of the marine bivalve mussel Anomia simplex that uses a biomineralized byssus to permanently anchor itself to substrates. The byssus has a highly complex hierarchical structure and contains over 90 wt% CaCO₃. The byssus features a complex set of porosities, presumed to be highly important for the function of the attachment system. The pore space is the main focus of the present work. We characterize the three dimensional distribution of pore spaces in the byssus using micro-computed tomography (µCT) through a combination of in house mCT and high resolution synchrotron µCT. The pore structures are observed to fall into distinct categories in various parts of the byssus. We discuss the branching of one set of pores that reach the byssus substrate interface in particular. They form a network reaching the byssus surface that we now visualize in three dimensions.

Abstract: The teeth of sea urchins are highly complex composite structures, composed predominantly of high magnesium calcite, and of a minor heterogeneous assemblage of organic macromolecules that are occluded within the mineral. The organic matrix fulfils important functions in mineralization, in addition to giving the mineral phase peculiar mechanical properties, different from that of purely inorganic calcite. Nevertheless, the composition and function of individual components of the organic matrix still remains largely unknown. Up to now, the detailed protein repertoire of teeth from a single sea urchin species (Strongylocentrotus purpuratus, order Camarodonta) was investigated. In this study, we characterized for the first time the teeth skeletal matrix of another sea urchin, Arbacia lixula (order Arbacioida). The acetic acid soluble and acetic acid insoluble matrices, namely ASM and AIM respectively, were extracted and characterized with different biochemical methods including mono-dimensional SDS-PAGE, FT-IR spectroscopy, HPAE-PAD for monosaccharide analysis, and finally, proteomics. In spite of the paucity of peptide data, several of them displayed a high abundance of hydrophobic residues, i.e., alanine, glycine and valine, and of the apolar proline. We assert that the alanine- and proline-rich domains are important features of some of the matrix proteins associated to the teeth of sea urchins. None of the known skeletal matrix proteins from S. purpuratus teeth were identified in the organic matrix of A. lixula teeth. This might suggest major differences in teeth matrix protein repertoires of these two species belonging to orders that diverged in the Mesozoic times.

Abstract: From a biomineralization point of view, the protist world is far less investigated than its metazoan counterpart. However, eukaryotic single-celled organisms offer a very unique access to discover biomineralization mechanisms in vivo. With respect to intracellular mechanisms involved in ion enrichment, mineral transport or vesicle formation ciliates represent a good model system. One important group of protists, the ciliates, is very common and numerous studies have been performed on their ecology, cell biology, morphology or genetics. Ciliates are also known for their formation of diverse mineralized intracellular and extracellular structures. However, only limited numbers of detailed studies on the kind of minerals, their properties or their formation mechanisms have been reported so far. This article reviews older and more recent literature on biomineralization in ciliates.

Abstract: The contribution of microorganisms, particularly bacteria, in carbonate mineral formation, the main natural processes controlling CO₂ level in the atmosphere, has played an important role since the Archean Eon. In this study we review our recent experimental work on CaCO₃ precipitation induced by two anoxygenic phototrophic bacteria (APB), Rhodovulum steppense A-20s^T and Rhodovulum sp. S-17-65, and by cyanobacteria Gloeocapsa sp. f-6gl. These bacteria are representatives of two important groups of photosynthetic organisms present at the Earth surface both in the past and at the present times. The mechanisms of organomineralization deriving from APB and cyanobacteria activities are drastically different and relate to the main physical and chemical processes controlling CaCO₃ precipitation from aqueous solution, essentially local supersaturation with respect to carbonates induced by photosynthesis outside the living cells and Ca adsorption onto cell surface and the associated extracellular polymeric substances (EPS).The APB can physiologically control their surface potential to electrostatically attract nutrients at alkaline pH, while rejecting Ca ions to prevent Ca adsorption and subsequent CaCO₃ precipitation in the vicinity of cell surface and thus, cell incrustation. In contrast to other previously-investigated calcifying bacteria, no cellular protection mechanism against Ca²⁺ adsorption and subsequent carbonate precipitation has been evidenced for cyanobacteria Gloeocapsa sp. f-6gl. This is most likely linked to the peculiar cellular organization of this species that involves several cells clustered in one single capsule. In this regard, colony-forming, EPS-rich, capsular cyanobacteria may be among the most efficient calcifying microorganisms all along the Earth history and can be of interest for various technological applications involving carbonation.

Abstract: Denaturing 1D electrophoresis on acrylamide gels, also referred as SDS-PAGE, is a classical technique for fractionating and visualizing the macromolecular constituents of matrices associated to calcified tissues. This technique has been widely used in association with the subsequent silver nitrate staining. But because matrices associated to calcified tissues are very often glycosylated and constituted of numerous polydisperse macromolecules, the obtained pattern is frequently ‘smeary’ and discrete bands, when present on the gel, are often blurred and thickened. In this paper, we present a simple protocol that can circumvent this drawback and ‘clean’ the gels. In short, after the classical migration step of the matrix macromolecules, the gel is electro-blotted on a PVDF membrane, similarly to a Western blot, but for a shorter time (partial transfer, i.e., one hour or less). It is subsequently stained with silver nitrate. The likely effect of the transfer is to partly remove polydisperse macromolecules and to ‘sharpen’ the discrete bands. We think that this extra-step may improve in several cases the gel pictures, particularly when they are blurred. We illustrate this phenomenon with two examples taken from brachiopod and mollusc shell matrices.

Abstract: The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called “calcifying matrix” is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, the molecular basis that dictates nacre formation remains largely uncharacterized.Recent expressed sequence tag (EST) investigations of the freshwater mussels (Elliptio complanata and Villosa leinosa) provide an opportunity to further characterize the proteins in the bivalve shell by a proteomic approach. In this study, we have identified a total of 15 proteins from their nacre insoluble matrices. Few of these proteins, such as Pif, MSI60, Nacrein-like, Shematrin, Kunitz-like containing, Papilin-like, LamG containing, Chitin-binding containing, M-rich and Q-rich proteins, appear to be analogs, if not true homologs, of proteins previously described from the pearl oyster or the edible mussel nacre matrices. This work constitutes a comprehensive EST-based nacre proteomic study of non-pteriomorphid bivalves that concretely gives us the opportunity to describe the molecular basis of deeply conserved nacre biomineralization toolkit within nacreous shell bearing bivalves.

Abstract: Proteomics is an efficient high throughput technique developed to identify proteins from a crude extract using sequence homology. Advances in Next Generation Sequencing (NGS) have led to increase knowledge of several non-model species. In the field of calcium carbonate biomineralization, the paucity of available sequences (such as the ones of mollusc shells) is still a bottleneck in most proteomic studies. Indeed, this technique needs proteins databases to find homology. The aim of this study was to perform different data mining approaches in order to identify novel shell proteins. To this end, we disposed of several publicly non-model molluscs databases. Previously identified molluscan shell matrix sequences were used as keyword to query annotated databases. BLAST tools and KASS program (KEGG Automatic Annotation Server) were developed to analyse other non-annotated databases. Our results suggest that the efficiency of these methods depends on the quality of the shared data. Finally, an in-house shell matrix protein database has been generated.

Abstract: Gold nanoparticles (GNPs) have shown great promises in biomedical applications due to their distinct dimensions, non-toxicity, regulated drug release capability and adjustable surface functionalities. This study illustrates a green method for GNPs synthesis from electronic scrap material (ESM) using Cupriavidus metallidurans and Delftia acidovorans. Leaching of pre-treated electronic scrap materials at 0.5 % (w/v) pulp density using spent medium genetically from modified Chromobacterium violaceum pBAD strain recovered 37.8% of gold. The recovered gold in solution was converted in GNPs using C. metallidurans and D. acidovorans. The synthesized GNPs were mostly spherical in shape and crystalline in nature.

Abstract: Hydroxyapatite (HA) is a well known bioactive material in the application of coated orthopaedic and dental implants. Recently, biomimetic technique has been explored to deposit a stable carbonated HA on a metal surface, mimicking the properties of natural bone. The aim of this study is to surface characterize the biomimetic hydroxyapatite (HA) and metallized silver (Ag) functionalized on a polydopamine film grafted titanium alloy (Ti6Al4V). The Ti6Al4V disks were grafted with the polydopamine film to provide catechol/quinone groups for chemical binding process. The grafted surfaces were metallized with Ag in silver nitrate solution. The metallized surfaces were then grafted with the second layer of polydopamine film and further biomineralized with HA in 1.5 simulated body fluid (SBF) solution for 3 and 7 days. The chemical compositions and chemical functionalities of those functionalized surfaces were characterized by XPS, FTIR and EDS. The morphologies of the surfaces were viewed under SEM. Finally, the wettability properties of the surfaces were investigated by water contact angle analysis. The XPS results showed that the polydopamine films were grafted on the Ti6Al4V surfaces. The polydopamine films became the chemical binding medium for functionalization of Ag and HA as the existence of both elements were clarified in XPS and EDS data. The appearance of HA functional groups (phosphate groups) were only noticed on FTIR spectrum when the biomimetic process was performed at 7 days. The formation of biomimetic HA has produced a hydrophilic surface with an appearance of hemispherical lath-like HA structure which is crucial for osseointegration and bone growth stability.

Abstract: Transient or metastable phases offer an intermediate phase with additional flexibility for creating the end product. The processing pathway remains unknown when the final product is viewed. Biological processes frequently employ the amorphous phase as the transition phase. This is shown in mineralized tissues: invertebrates, pathological calcified deposits and murine fetal teeth. After a fast transition from an amorphous calcium phosphate to crystalline material, smaller changes occur over time. For an appreciation of the transition state, crystallinity is defined and measurement methods outlined. Biotechnology using transition material states offers fast, low temperature access to nanosized high temperature phases. Alfa tricalcium phosphate and apatite is made by a phase transition, but peroxyapatite requires long-range diffusion within the same structure. Tetracalcium phosphate is also possible, but this requires multiple transition states and phase decomposition. The pathway via the amorphous state offers an alternative route to biologically important materials.