Ute Schloßmacher

The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells

Earlier studies have demonstrated that biosilica, synthesized by the enzyme silicatein, induces hydroxyapatite formation in osteoblast-like SaOS-2 cells. Here we study the effect of biosilica on the expressions of osteoprotegerin [OPG] and the receptor activator for NF-kappaB ligand [RANKL] in the SaOS-2 cell model. We show that during growth of SaOS-2 cells on biosiliceous matrices hydroxyapatite formation is induced, while syntheses of cartilaginous proteoglycans and sulfated glycosaminoglycans are down-regulated. Furthermore, quantitative real-time RT-PCR analysis revealed a strong time-depended increase in expression of OPG in biosilica exposed SaOS-2 cells while the steady-state expression level of RANKL remained unchanged. These results have been corroborated on the protein level by ELISA assays. Therefore, we propose that biosilica stimulated OPG synthesis in osteoblast-like cells counteracts those pathways that control RANKL expression and function (e.g. maturation of pre-osteoclasts and activation of osteoclasts). Hence, the data obtained in the present study reveal the considerable biomedical potential of biosilica for treatment and prophylaxis of osteoporotic disorders.

Sponge spicules as blueprints for the biofabrication of inorganic–organic composites and biomaterials

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e., biosilicification) hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results.

Poly(silicate)-metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (silica polymerase and silica esterase)

Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica‐filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization/polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester‐like bond in bis(p‐aminophenoxy)‐dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E‐64. We consider that the dual function of silicatein (silica polymerase and silica esterase) will be useful for the rational synthesis of structured new silica biomaterials.

Enzymatic production of biosilica glass using enzymes from sponges: basic aspects and application in nanobiotechnology (material sciences and medicine)

Biomineralization, biosilicification in particular (i.e. the formation of biogenic silica, SiO2), has become an exciting source of inspiration for the development of novel bionic approaches following “nature as model”. Siliceous sponges are unique among silica forming organisms in their ability to catalyze silica formation using a specific enzyme termed silicatein. In this study, we review the present state of knowledge on silicatein-mediated “biosilica” formation in marine sponges, the involvement of further molecules in silica metabolism and their potential application in nanobiotechnology and medicine.

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.

Silicatein expression in the hexactinellid Crateromorpha meyeri: the lead marker gene restricted to siliceous sponges

The siliceous spicules of sponges (Porifera) are synthesized by the enzyme silicatein. This protein and its gene have been identified so far in the Demospongiae, e.g., Tethya aurantium and Suberites domuncula. In the Hexactinellida, the second class of siliceous sponges, the mechanism of synthesis of the largest bio-silica structures on Earth remains obscure. Here, we describe the morphology of the spicules (diactines and stauractines) of the hexactinellid Crateromorpha meyeri. These spicules are composed of silica lamellae concentrically arranged around a central axial canal and contain proteinaceous sheaths (within the siliceous mantel) and proteinaceous axial filaments (within the axial canal). The major protein in the spicules is a 24-kDa protein that strongly reacts with anti-silicatein antibodies in Western blots. Its cDNA has been successfully cloned; the deduced hexactinellid silicatein comprises, in addition to the characteristic catalytic triad amino acids Ser-His-Asn and the “conventional” serine cluster, a “hexactinellid C. meyeri-specific” Ser cluster. We show that anti-silicatein antibodies react specifically with the proteinaceous matrix of the C. meyeri spicules. The characterization of silicatein at the genetic level should contribute to an understanding of the molecular/biochemical mechanism of spiculogenesis in Hexactinellida. These data also indicate that silicatein is an autapomorphic molecule common to both classes of siliceous sponges.

Silintaphin-1-interaction with silicatein during structure-guiding bio-silica formation

Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin‐1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal‐like structures; and finally (c) assembly of fractal‐like structures to form filaments. The present study was aimed at exploring the effect of self‐assembled complexes of silicatein and silintaphin‐1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin‐1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein‐α reaggregates to randomly organized structures, coincubation of silicatein‐α and silintaphin‐1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal‐like patterned self‐assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein‐α strongly increased by 5.3‐fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein‐α‐mediated biosilicification depends on the concomitant presence of silicatein‐α and silintaphin‐1. Accordingly, silintaphin‐1 might not only enhance the enzymatic activity of silicatein‐α, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer.

Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber‐like spicules

Biomineralization processes are characterized by controlled deposition of inorganic polymers/minerals mediated by functional groups linked to organic templates. One metazoan taxon, the siliceous sponges, has utilized these principles and even gained the ability to form these polymers/minerals by an enzymatic mechanism using silicateins. Silicateins are the dominant protein species present in the axial canal of the skeletal elements of the siliceous sponges, the spicules, where they form the axial filament. Silicateins also represent a major part of the organic components of the silica lamellae, which are cylindrically arranged around the axial canal. With the demosponge Suberites domuncula as a model, quantitative enzymatic studies revealed that both the native and the recombinant enzyme display in vitro the same biosilica‐forming activity as the enzyme involved in spicule formation in vivo. Monomeric silicatein molecules assemble into filaments via fractal intermediates, which are stabilized by the silicatein‐interacting protein silintaphin‐1. Besides the silicateins, a silica‐degrading enzyme silicase acting as a catabolic enzyme has been identified. Growth of spicules proceeds in vivo in two directions: first, by axial growth, a process that is controlled by evagination of cell protrusions and mediated by the axial filament‐associated silicateins; and second, by appositional growth, which is driven by the extraspicular silicateins, a process that provides the spicules with their final size and morphology. This radial layer‐by‐layer accretion is directed by organic cylinders that are formed around the growing spicule and consist of galectin and silicatein. The cellular interplay that controls the morphogenetic processes during spiculogenesis is outlined.

Silicatein-Mediated Polycondensation of Orthosilicic Acid: Modeling of a Catalytic Mechanism Involving Ring Formation

The sponge protein silicatein is the first enzyme that has been described to form an inorganic polymer (silica) from a monomeric precursor (tetraethoxysilane or orthosilicic acid). The models proposed for silicatein-mediated silica formation are mainly based on the use of synthetic substrates (hydrolytic cleavage of tetraethoxysilane to silanol compounds) or only consider the formation of less reactive silicic acid dimers (disilicic acid). Here we propose a new model for the catalytic mechanism of silicatein that leads to the formation of reactive, cyclic silicic acid species (trisiloxane rings and higher-membered siloxane rings) which easily promote the silica polycondensation reaction.

Alginate/silica composite hydrogel as a potential morphogenetically active scaffold for three-dimensional tissue engineering

Pursuing our aim to develop a biomimetic synthetic scaffold suitable for tissue engineering, we embedded bone cells, osteoblast-related SaOS-2 cells and osteoclast-like RAW 264.7 cells, into beads, formed of a Na-alginate-based or a silica-containing Na-alginate-based hydrogel matrix. The beads were incubated either separately (only one cell line in a culture dish) or co-incubated (SaOS-2-containing beads and RAW 264.7 beads). The alginate and alginate/silica hydrogel matrices were found not to impair the viability of the encapsulated cells. In these matrices the SaOS-2 cells retain their capacity to synthesize hydroxyapatite crystallites. The mechanical properties, including surface roughness and hardness, of the hydrogel were determined. If silica is included in the hydrogel matrix, the encapsulated SaOS-2 cells were found to increasingly express the gene encoding for osteoprotegerin in co-cultivation experiments with RAW 264.7 cell beads, suggesting that under the applied conditions the differentiation capacity of the RAW 264.7 cells is impaired. In continuation it was found that under these conditions (SaOS-2 cells cultured together with RAW 264.7 cells) the RAW 264.7 cells show a reduced capacity to express the gene for tartrate-resistant acid phosphatase. It is concluded that the applied bead-encapsulation of bone cells is a useful technique to produce bioactive programmable hydrogels.