Heinz-Christoph Schröder

Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro

Inorganic polymeric phosphate is a physiological polymer that accumulates in bone cells. In the present study osteoblast-like SaOS-2 cells were exposed to this polymer, complexed in a 2:1 stoichiometric ratio with Ca(2+), polyP (Ca(2+) salt). At a concentration of 100 μM, polyP (Ca(2+) salt) caused a strong increase in the activity of the alkaline phosphatase and also an induction of the steady-state expression of the gene encoding this enzyme. Comparative experiments showed that polyP (Ca(2+) salt) can efficiently replace β-glycerophosphate in the in vitro hydroxyapatite (HA) biomineralization assay. In the presence of polyP (Ca(2+) salt) the cells extensively form HA crystallites, which remain intimately associated with or covered by the plasma membrane. Only the tips of the crystallites are directly exposed to the extracellular space. Element mapping by scanning electron microscopy/energy-dispersive X-ray spectroscopy coupled to a silicon drift detector supported the finding that organic material was dispersed within the crystallites. Finally, polyP (Ca(2+) salt) was found to cause an increase in the intracellular Ca(2+) level, while polyP, as well as inorganic phosphate (P(i)) or Ca(2+) alone, had no effect at the concentrations used. These findings are compatible with the assumption that polyP (Ca(2+) salt) is locally, on the surface of the SaOS-2 cells, hydrolyzed to P(i) and Ca(2+). We conclude that the inorganic polymer polyP (Ca(2+) salt) in concert with a second inorganic, and physiologically occurring, polymer, biosilica, activates osteoblasts and impairs the maturation of osteoclasts.

Silicate modulates the cross‐talk between osteoblasts (SaOS‐2) and osteoclasts (RAW 264.7 cells): Inhibition of osteoclast growth and differentiation

It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS‐2 sarcoma cells in vitro. In contrast, silicate does not affect the steady‐state gene expression level of the osteoclastogenic ligand receptor activator of NF‐κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS‐2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10–100 µM. Applying a co‐cultivation assay system, using SaOS‐2 cells and RAW 264.7 cells, it is shown that in the presence of 10 µM silicate the number of RAW 264.7 cells in general, and the number of TRAP+ RAW 264.7 cells in particular markedly decreases. The SaOS‐2 cells retain their capacity of differential gene expression of OPG and RANKL in favor of OPG after exposure to silicate. It is concluded that after exposure of the cells to silicate a factor(s) is released from SaOS‐2 cells that causes a significant inhibition of osteoclastogenesis of RAW 264.7 cells. It is assumed that it is an increased secretion of the cytokine OPG that is primarily involved in the reduction of the osteoclastogenesis of the RAW 264.7 cells. It is proposed that silicate might have the potential to stimulate osteogenesis in vivo and perhaps to ameliorate osteoporotic disorders. J. Cell. Biochem. 113: 3197–3206, 2012.

Bio-vaterite formation by glycoproteins from freshwater pearls

A 48 kDa acidic and putative calcium-binding glycoprotein was isolated from pearls of the freshwater mussel Hyriopsis cumingii. This protein was compared with a related 46 kDa polypeptide, obtained from the nacreous shell of the same species. Separation by two-dimensional gel electrophoresis revealed that the difference in molecular weight is due to the higher extent of glycosylation of the 48 kDa protein existing in pearls. Evidence is presented that the sugar moieties of the protein contribute to crystal growth, starting with the nucleation step. In in vitro precipitation experiments, the 48 kDa glycoprotein of pearls directed the formation of round-shaped vaterite crystals while the 46 kDa glycoprotein of shells promoted formation of small irregular calcite particles. Furthermore, both proteins, 48 kDa/46 kDa, comprised carbonic anhydrase activity that has been implicated in CaCO(3) formation. Thus, a function of the isolated glycoproteins in biomineralization is proposed together with the mechanism by which they can stabilize different calcium carbonate polymorphs.

Sponge Biosilica Formation Involves Syneresis Following Polycondensation in vivo

Syneresis is a process observed during the maturation/aging of silica gels obtained by sol–gel synthesis that results in shrinkage and expulsion of water due to a rearrangement and increase in the number of bridging siloxane bonds. Here we describe how the process of biosilica deposition during spicule (“biosilica” skeleton of the siliceous sponges) formation involves a phase of syneresis that occurs after the enzyme‐mediated polycondensation reaction. Primmorphs from the demosponge Suberites domuncula were used to study syneresis and the inhibition of this mechanism. We showed by scanning electron microscopy that spicules added to primmorphs that have been incubated with manganese sulfate fuse together through the deposition of silica spheres and bridges. Energy‐dispersive X‐ray mapping of the newly formed deposits showed high silicon and oxygen content. These biosilica deposits contain a comparably higher percentage of water than mature/aged spicules. Quantitative real‐time polymerase chain reaction analyses revealed that the addition of silicate to primmorph cultures resulted in a marked upregulation of the expression of the aquaporin gene and of the genes encoding the silica anabolic enzyme silicatein‐α and the silica catabolic enzyme silicase. On the other hand, addition of manganese sulfate, either alone or together with silicate, caused a strong reduction in the level of aquaporin transcripts, although this metal ion did not essentially affect the silicate‐induced increase in silicatein‐α and silicase gene expression. We conclude that the secondary silica deposits formed on spicules under physiological conditions in the presence of silicate fuse together and subsequently undergo syneresis, which is facilitated by the removal of water through aquaporin channels. In growing spicules, these processes of biosilica formation and syneresis in the lamellar monolithic structures precede the final step of “biosintering” during which the massive biosilica rods of the spicules are formed.

Synthesis of the neurotoxin quinolinic acid in apoptotic tissue from Suberites domuncula: cell biological, molecular biological, and chemical analyses.

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. Using the sponge Suberites domuncula we document for the first time that in its apoptotic tissue a toxic compound is produced that very likely controls the elimination of the dying tissue. Apoptosis was induced by exposing the sponges to 2,2?-dipyridyl or by maintaining them under nonaeration conditions. After that treatment at least one eukaryotic epibiont (Bittium sp.) could be found grazing on apoptotic tissue. Cell proliferation assays demonstrated that aqueous extracts from unaffected sponge tissue displayed no cytotoxicity. However, addition of an extract from apoptotic tissue to neuronal cells from rat brain exerted strong toxicity. The underlying compound was identified as quinolinic acid; quantitative determination showed that quinolinic acid is present only in apoptotic tissue (4.8 mg/g dry wet weight). The complementary DNA encoding the key enzyme of the quinolinic acid pathway, 3-hydroxyanthranilate 3,4-dioxygenase, was cloned and characterized. The expression of this gene is up-regulated in apoptotic tissue. These data suggest that a complex molecular network controls apoptotic elimination of sponge tissue, which results in the synthesis of the bioactive compound quinolinic acid that controls the elimination of the tissue, perhaps via differential effects on grazing epibionts.

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were assessed. Finally, the cultivation of osteoblastic SaOS-2 cells on the surface-functionalized CHS-g-PCL was followed by analyses of cell viability, mineral deposition and alkaline phosphatase activity. These characterizations revealed a composite that combines the versatile properties of CHS-g-PCL with the osteogenic activity of the silicatein/biosilica coating and, hence, represents an innovative alternative to conventionally used CHS/PCL composites for biomedical applications, where stable bone-material interfaces are required.

Silicatein conjugation inside nanoconfined geometries through immobilized NTA-Ni(II) chelates.

The chemical modification and bioconjugation processes inside confined geometries by His-tagged silicatein promote sensitive changes in the polarity and surface charge density that mainly contribute to the ionic current rectification properties of the single conical nanopores.

Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution.

Chemical mimicry: hierarchical 1D TiO2@ZrO2 core-shell structures reminiscent of sponge spicules by the synergistic effect of silicatein-α and silintaphin-1.

In nature, mineralization of hard tissues occurs due to the synergistic effect of components present in the organic matrix of these tissues, with templating and catalytic effects. In Suberites domuncula, a well-studied example of the class of demosponges, silica formation is mediated and templated by an axial proteinaceous filament with silicatein-α, one of the main components. But so far, the effect of other organic constituents from the proteinaceous filament on the catalytic effect of silicatein-α has not been studied in detail. Here we describe the synthesis of core-shell TiO(2)@SiO(2) and TiO(2)@ZrO(2) nanofibers via grafting of silicatein-α onto a TiO(2) nanowire backbone followed by a coassembly of silintaphin-1 through its specifically interacting domains. We show for the first time a linker-free, one-step funtionalization of metal oxides with silicatein-α using glutamate tag. In the presence of silintaphin-1 silicatein-α facilitates the formation of a dense layer of SiO(2) or ZrO(2) on the TiO(2)@protein backbone template. The immobilization of silicatein-α onto TiO(2) probes was characterized by atomic force microscopy (AFM), optical light microscopy, and high-resolution transmission electron microscopy (HRTEM). The coassembly of silicatein-α and silintaphin-1 may contribute to biomimetic approaches that pursue a controlled formation of patterned biosilica-based biomaterials.

Growth of fibrous aggregates of silica nanoparticles: Fibre growth by mimicking the biogenic silica patterning processes

We describe the self-assembly of discrete SiO2 nanofibers via grafting of silicatein side chains to a polymer backbone. The covalent binding of silicatein to the backbone of the polymer is based on the affinity of the nitrilotriacetic acid (NTA) side chain, which serves as a ligand for the immobilization of His-tagged silicatein. The surface charge and the bulkiness of the protein moieties prevent the entropically favoured coil formation of the polymer and force it to adopt an open chain structure after hydrolysis of the silica precursors. The probes were characterized by scanning force microscopy (SFM) and optical light microscopy. Surface complexation of the resulting silica nanoparticles by the polymer-bound silicatein were analysed using high resolution tranmission electron microscopy (HRTEM).