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The role of the silicatein-α interactor silintaphin-1 in biomimetic biomineralization ☆

Biosilicification in sponges is initiated by formation of proteinaceous filaments, predominantly consisting of silicateins. Silicateins enzymatically catalyze condensation of silica nanospheres, resulting in symmetric skeletal elements (spicules). In order to create tailored biosilica structures in biomimetic approaches it is mandatory to elucidate proteins that are fundamental for the assembly of filaments. Silintaphin-1 is a core component of modularized filaments and also part of a spicule-enfolding layer. It bears no resemblance to other proteins, except for the presence of an interaction domain that is fundamental for its function as scaffold/template. In the presence of silicatein silintaphin-1 facilitates the formation of in vitro filaments. Also, it directs the assembly of gamma-Fe(2)O(3) nanoparticles and surface-immobilized silicatein to rod-like biocomposites, synthetic spicules. Thus, silintaphin-1 will contribute to biomimetic approaches that pursue a controlled formation of patterned biosilica-based materials. Its combination with gamma-Fe(2)O(3) nanoparticles and immobilized silicatein will furthermore inspire future biomedical applications and clinical diagnostics.

Morphogenetic Activity of Silica and Bio-silica on the Expression of Genes Controlling Biomineralization Using SaOS-2 Cells

In a previous study (Schröder et al., J Biomed Mater Res B Appl Biomater 75:387–392, 2005) we demonstrated that human SaOS-2 cells, when cultivated on bio-silica matrices, respond with an increased hydroxyapatite deposition. In the present contribution we investigate if silica-based components (Na-silicate, tetraethyl orthosilicate [TEOS], silica-nanoparticles) (1) change the extent of biomineralization in vitro (SaOS-2 cells) and (2) cause an alteration of the expression of the genes amelogenin, ameloblastin, and enamelin, which are characteristic for an early stage of osteogenesis. We demonstrate that the viability of SaOS-2 cells was not affected by the silica-based components. If Na-silicate or TEOS was added together with ß-glycerophosphate, an organic phosphate donor, a significant increase in biomineralization was measured. Finally, expression levels of the amelogenin, ameloblastin, and enamelin genes were determined in SaOS-2 cells during exposure to the silica-based components. After exposure for 2 days, expression levels of amelogenin and enamelin strongly increased in response to the silica-based components, while no significant change was seen for ameloblastin. In contrast, exposure of SaOS-2 cells to ß-glycerophosphate resulted in increased expression of all three genes. We conclude that the levels of the structural molecules of the enamel matrix, amelogenin and enamelin, increase in the presence of silica-based components and substantially contribute to the extent of hydroxyapatite crystallite formation. These results demonstrate that silica-based components augment hydroxyapatite deposition in vitro and suggest that enzymatically synthesized bio-silica (via silicatein) might be a promising route for tooth reconstruction in vivo.

Bioengineering of the silica-polymerizing enzyme silicatein-α for a targeted application to hydroxyapatite

Since its discovery, numerous biotechnological approaches have aimed to explore the silica-polymerizing catalytic activity of the enzyme silicatein. In vivo, silicatein catalyzes polymerization of amorphous silica nanospheres from soluble precursors. In vitro, it directs the formation of nanostructured biosilica. This is of interest for various applications that strive to benefit from both the advantages of the biological system (i.e., silica synthesis under physiological conditions) and the cell mineralization-stimulating effect of biosilica. However, so far immobilization of silicatein has been hampered by the complex multistep procedure required. In addition, the chemical surface modifications involved not only restrict the choice of carrier materials but also render application of silicatein to hydroxyapatite (HA) of mineralized tissue impossible. Here we describe the bioengineering of silicatein, adapted for application in the fields of bone regeneration, tissue engineering, and dental care. Inspired by Glu-rich sequences of mammalian proteins that confer binding affinity to HA, a novel protein-tag was developed, the Glu-tag. Following expression of Glu-tagged silicatein, the HA-binding capacity of the enzyme is demonstrated in combination with synthetic and dental HA. Furthermore, immobilized Glu-tagged silicatein catalyzes synthesis of biosilica coatings on both synthetic HA nanofibrils and dental HA. Hence, Glu-tagged silicatein reveals a considerable biomedical potential with regenerative and prophylactic implementations.

A new printable and durable N,O-carboxymethyl chitosan–Ca2+–polyphosphate complex with morphogenetic activity

Biomimetic materials have been gaining increasing importance in tissue engineering since they may provide regenerative alternatives to the use of autologous tissues for transplantation. In the present study, we applied for bioprinting of a functionalized three-dimensional template, N,O-carboxymethyl chitosan (N,O-CMC), mimicking the physiological extracellular matrix. This polymer, widely used in tissue engineering, has been provided with functional activity by integration of polyphosphate (polyP), an osteogenically acting natural polymer. The two polymers, N,O-CMC and polyP, are linked together via Ca2+ bridges. This N,O-CMC + polyP material was proven to be printable and durable. The N,O-CMC + polyP printed layers and tissue units retain their properties to induce SaOS-2 bone-like cells to biomineralization. Subsequent in vivo experiments revealed a strong regeneration-inducing activity of the material in the rat calvarial defect model. In turn, N,O-CMC + polyP represents a promising hybrid material useful as a potential custom-designed scaffold for alternative tissue-engineering solutions.

Isolation of the silicatein-α interactor silintaphin-2 by a novel solid-phase pull-down assay.

The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, of the growing spicule was discovered. In this study, a new interactor has been identified by both a conventional yeast two-hybrid library screening and a newly established pull-down assay. For the latter approach, silicatein-α has been bioengineered to carry a Glu tag, which confers binding affinity to hydroxyapatite. After immobilization on a solid-phase matrix (hydroxyapatite), the Glu-tagged silicatein was used as bait for the identification of interactors. Both approaches revealed a 15 kDa polypeptide, and its identity was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization of silintaphin-2 and silicatein-α within the axial filament and on the spicule surface was shown by immunohistological analyses. Subsequent autoradiography demonstrated the Ca(2+) binding affinity of this silicatein interactor. These findings indicate that both proteins operate in concert during spiculogenesis. Besides binding of calcium, silintaphin-2 shares several structural features with certain acidic, secreted extracellular matrix proteins that facilitate tissue mineralization in Metazoa. Hence, silintaphin-2 might mediate signal transduction during spiculogenesis or may play a more direct role during biosilica formation, in concert with silicatein.

Biosilica-loaded poly(ϵ-caprolactone) nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast-related SaOS-2 cells

Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell-cell, and cell-substrate contact formation of the matrix-embedded cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(ϵ-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related SaOS-2 cells in vitro. Analysis of the microstructure of the 300-700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the cells are embedded in a softer matrix.

Development of a morphogenetically active scaffold for three‐dimensional growth of bone cells: biosilica–alginate hydrogel for SaOS‐2 cell cultivation

Polymeric silica is formed from ortho‐silicate during a sol–gel formation process, while biosilica is the product of an enzymatically driven bio‐polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP‐2) and osteoprotegerin in osteoblast‐related SaOS‐2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three‐dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS‐2 cells were embedded into a Na‐alginate‐based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS‐2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 μm ortho‐silicate; (c) the hydrogel supplemented with 400 μm ortho‐silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho‐silicate and BSA. The SaOS‐2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica‐containing hydrogels. After an incubation period of 2 weeks, cells present in silica‐containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP‐2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na‐alginate‐based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing. Copyright

Bacteria survival and growth in multi-layered silica thin films

A layer-by-layer approach was used to build up silica thin films (<500 nm) compatible with Escherichia coli bacteria immobilization and spatially confined growth.

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.

Haloperoxidase Mimicry by CeO2−x Nanorods Combats Biofouling

CeO2-x nanorods are functional mimics of natural haloperoxidases. They catalyze the oxidative bromination of phenol red to bromophenol blue and of natural signaling molecules involved in bacterial quorum sensing. Laboratory and field tests with paint formulations containing 2 wt% of CeO2-x nanorods show a reduction in biofouling comparable to Cu2 O, the most typical biocidal pigment.