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 i…

ATP distribution and localization of mitochondria in Suberites domuncula (Olivi 1792) tissue

SUMMARY The metabolic energy state of sponge tissue in vivo is largely unknown. Quantitative bioluminescence-based imaging was used to analyze the ATP distribution of Suberites domuncula (Olivi 1792) tissue, in relation to differences between the cortex and the medulla. This method provides a quantitative picture of the ATP distribution closely reflecting the in vivo situation. The obtained data suggest that the highest ATP content occurs around channels in the sponge medulla. HPLC reverse-phase C-18, used for measurement of ATP content, established a value of 1.62 μmol ATP g–1 dry mass in sponge medulla, as opposed to 0.04 μmol ATP g–1 dry mass in the cortex, thus indicating a specific and…

Bioengineering of the silica-polymerizing enzyme silicatein-alpha 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 modific…

Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component

Abstract Background Laccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates. Methods We describe the first poriferan laccase from the marine demosponge Suberites domuncula. Results This enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which con…

A new polyphosphate calcium material with morphogenetic activity

Abstract Polyphosphate [polyP] has been proven to elicit morphogenetic activity on bone cells. By applying mild reaction conditions, a Ca-polyP material that displays a hardness of ≈1.3 GPa has been fabricated. The Ca-polyP granules are prone to hydrolytic degradation during in vitro incubation of the cells, suggesting that this property is associated with the observed bioactivity.

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(e-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicate…

Dissection of the structure-forming activity from the structure-guiding activity of silicatein: a biomimetic molecular approach to print optical fibers

Silicateins, a group of proteins forming the proteinaceous axial filaments of the inorganic biosilica spicules of the siliceous sponges, are unique in their dual function to exhibit both structure-guiding (providing the structural platform for the biosilica product) and structure-forming activities (enzymatic function: biosilica synthesis from ortho-silicate). The primary translation product of the silicatein gene comprises a signal peptide, a pro-peptide and, separated by an autocatalytic cleavage site glutamine/aspartic acid [Q/D], the sequence of the mature silicatein protein. In order to dissect the biocatalytic, structure-forming activity of silicatein from its structure-guiding functi…

The role of the silicatein-alpha 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 sil…

Modular Small Diameter Vascular Grafts with Bioactive Functionalities.

We report the fabrication of a novel type of artificial small diameter blood vessels, termed biomimetic tissue-engineered blood vessels (bTEBV), with a modular composition. They are composed of a hydrogel scaffold consisting of two negatively charged natural polymers, alginate and a modified chitosan, N,O-carboxymethyl chitosan (N,O-CMC). Into this biologically inert scaffold two biofunctionally active biopolymers are embedded, inorganic polyphosphate (polyP) and silica, as well as gelatin which exposes the cell recognition signal, Arg-Gly-Asp (RGD). These materials can be hardened by exposure to Ca(2+) through formation of Ca(2+) bridges between the polyanions, alginate, N,O-CMC, and polyP…

Bacteria survival and growth in multi-layered silica thin films

International audience; The fields of application of sol–gel encapsulation technology for living cells have been greatly extended over the last few years. Photobioreactors,1 biofuel cells,2 bio-remediation materials,3 biosensors4 and biomedical devices5 are currently being developed taking advantage of the robustness of inorganic materials compared to their (bio)-organic counterparts. In many of these applications, the formation of stable thin films (<1 μm) would be highly desirable for integration, for instance, in bio-chips.6 However, several limitations exist when trying to transfer a protocol mainly developed from bulk hydrogels to thin films. Especially, the storage stability becomes m…

Silica-coated Au@ZnO Janus particles and their stability in epithelial cells

Multicomponent particles have emerged in recent years as new compartmentalized colloids with two sides of different chemistry or polarity that have opened up a wide field of unique applications in medicine, biochemistry, optics, physics and chemistry. A drawback of particles containing a ZnO hemisphere is their low stability in biological environment due to the amphoteric properties of Zn2+. Therefore we have synthesized monodisperse Au@ZnO Janus particles by seed-mediated nucleation and growth whose ZnO domain was coated selectively with a thin SiO2 layer as a protection from the surrounding environment that imparts stability in aqueous media while the Au domain remained untouched. The thi…

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 novel TiO2-assisted magnetic nanoparticle separator for treatment and inactivation of bacterial contaminants in aquatic systems

Ferromagnetic nanoparticles (Fe-nanoparticles) have been functionalized with recombinant poly- Glu [glutamic acid]-tagged silicatein, a biomineral-synthesizing enzyme from siliceous sponges that forms the inorganic silica skeleton of those animals. The biocatalytic activity of silicatein was used to form a titania (TiO2) shell around the iron nanoparticle core, using the water-soluble non- natural substrate titanium bis(ammonium lactato)-dihydroxide (TiBALDH). Thereby the diameter of the nanoparticles increases from 7 nm to ≈22 nm. This procedure also allows the layer-by-layer fabrication of titania/silica- Fe-nanoparticles. SEM/EDX analysis confirmed the presence of the Ti and Si signals i…

An approach to a biomimetic bone scaffold: increased expression of BMP-2 and of osteoprotegerin in SaOS-2 cells grown onto silica-biologized 3D printed scaffolds

Three-dimensional printed (3D printed) bone material is needed to close the shortage and to avoid the potential health risks associated with autografts and allografts, in the treatment of bone fractures/nonunions or bone trauma. Here we describe the fabrication of 3D printed scaffold, initially prepared form Ca-sulfate that has been impregnated/biologized with Ca-phosphate or with silica. The 3D printed grids had a size mesh of 200 μm; the chemical composition was determined by energy dispersive X-ray spectroscopy or conventional chemical analysis. Using human SaOS-2 cells (human osteogenic cells) it is shown that both the Ca-sulfate, and the Ca-phosphate or the silica impregnated Ca-sulfat…

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-…

Self‐Organized Arrays of SnO 2 Microplates with Photocatalytic and Antimicrobial Properties

Formation of a micropatterned titania photocatalyst by microcontact printed silicatein on gold surfaces

The enzyme silicatein has been bioengineered to carry a thiol-bearing Au-affinity tag (Cys-tag) for direct immobilization on gold carriers in shortest time without the need for prior surface functionalization. Through microcontact printing, defined silicatein micropatterns were created on gold surfaces, facilitating the subsequent enzymatically controlled synthesis of photocatalytically active TiO(2).

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…

Haloperoxidase Mimicry by CeO2−xNanorods 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.