Silica as a morphogenetically active inorganic polymer.

At present the scaffolds used for bioprinting of cells do not elicit morphogenetic responses in the cells. In the present study we approached a solution by studying the effect of an inorganic silica supplement added to an Na-alginate matrix. Bone- and osteoblast-like SaOS-2 cells were embedded into this organic polymeric matrix which was additionally enriched with 400 μM prehydrolyzed TEOS [tetra-ethoxy-silane], a source of ortho-silicate. In this silica-based matrix the cells synthesized hydroxyapatite crystallites after exposure to a mineralization activation cocktail composed of β-glycerophosphate, ascorbic acid and dexamethasone. The degree of hydroxyapatite synthesis, determined by sta…

A cryptochrome-based photosensory system in the siliceous sponge Suberites domuncula (Demospongiae)

Based on the light-reactive behavior of siliceous sponges, their intriguing quartz glass-based spicular system and the existence of a light-generating luciferase [Muller WEG et al. (2009) Cell Mol Life Sci 66, 537–552], a protein potentially involved in light reception has been identified, cloned and recombinantly expressed from the demosponge Suberites domuncula. Its sequence displays two domains characteristic of cryptochrome, the N-terminal photolyase-related region and the C-terminal FAD-binding domain. The expression level of S. domuncula cryptochrome depends on animal’s exposure to light and is highest in tissue regions rich in siliceous spicules; in the dark, no cryptochrome transcri…

The Understanding of the Metazoan Skeletal System, Based on the Initial Discoveries with Siliceous and Calcareous Sponges

Initiated by studies on the mechanism of formation of the skeletons of the evolutionary oldest still extant multicellular animals, the sponges (phylum Porifera) have provided new insights into the mechanism of formation of the Ca-phosphate/hydroxyapatite skeleton of vertebrate bone. Studies on the formation of the biomineral skeleton of sponges revealed that both the formation of the inorganic siliceous skeletons (sponges of the class of Hexactinellida and Demospongiae) and of the calcareous skeletons (class of Calcarea) is mediated by enzymes (silicatein: polymerization of biosilica; and carbonic anhydrase: deposition of Ca-carbonate). Detailed studies of the initial mineralization steps i…

Crystalline nanorods as possible templates for the synthesis of amorphous biosilica during spicule formation in Demospongiae.

In tandem: High-resolution TEM shows that during the initial stages of demosponge spicule formation, a primordial crystalline structure is formed within the axial filament. The recently developed electron diffraction tomography technique (ADT) reveals that the nanorods have a layered structure that matches smectitic phyllosilicates. These intracellular nanorods have been considered as precursors of mature spicules. High-resolution microscopy shows that, during the initial stages of demosponge spicule formation, a primordial crystalline structure is formed within the axial filament. The recently developed electron diffraction tomography technique reveals that the nanorods have a layered stru…

Polyphosphate, the physiological metabolic fuel for corneal cells: a potential biomaterial for ocular surface repair

The regeneration of the epithelium, covering the avascular cornea, involves the processes of differentiation, proliferation and migration of cells originating from the corneal epithelial stem cells. We ask the question if these energy-consuming processes can be fueled by the physiological, inorganic polyphosphate (polyP), the main energy storage/donor molecule in the extracellular space. The ex vivo results reveal that addition of polyP, in the form of soluble Na-polyP, to the culture medium elicits a strong stimulatory effect on cell viability/growth and migration of corneal epithelial cells. Microscopic analyses of partially denuded cornea specimens show that in the presence of polyP, but…

Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions

Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditi…

Bifunctional dentifrice: Amorphous polyphosphate a regeneratively active sealant with potent anti- Streptococcus mutans activity

Abstract Objective In this study we demonstrate that inorganic polyphosphate (polyP) exhibits a dual protective effect on teeth: it elicits a strong antibacterial effect against the cariogenic bacterium Streptococcus mutans and, in form of amorphous calcium polyP microparticles (size of 100–400 nm), it efficiently reseals cracks/fissures in the tooth enamel and dentin. Methods Three different formulations of amorphous polyP microparticles (Ca-polyP, Zn-polyP and Sr-polyP) were prepared. Results Among the different polyP microparticles tested, the Ca-polyP microparticles, as a component of a newly developed formulation of a dentifrice, turned out to be most effective in inhibiting growth of …

Bio-Sintering/Bio-Fusion of Silica in Sponge Spicules

The synthesis of siliceous spicules in both demosponges and hexactinellids is enzymatically driven via silicatein. This enzyme exists both intra-spicularly and in the extra-spicular space. It catalyzes the formation of bio-silica constituting the silica lamellae that are formed during the appositional (layer-by-layer) growth of the spicules. The extent of (bio-silica forming) activity of silicatein from the demosponge Suberites domuncula measured in vitro reflects the amount of bio-silica required for the formation of spicules in vivo. It is shown that during growth and maturation of the spicules in demosponges a bio-fusion process occurs that results in an intra-spicular sintering of the s…

High biocompatibility and improved osteogenic potential of amorphous calcium carbonate/vaterite.

In human bone, amorphous calcium carbonate (ACC) is formed as a precursor of the crystalline carbonated apatite/hydroxyapatite (HA). Here we describe that the metastable ACC phase can be stabilized by inorganic polyphosphate (polyP) that is also used as a phosphate source for the non-enzymatic carbonate/phosphate exchange during HA formation. This polymer was found to suppress the transformation of ACC into crystalline CaCO3 at a percentage of 5% [w/w] ("CCP5") with respect to CaCO3 and almost completely at 10% [w/w] ("CCP10"). Both preparations (CaCO3/polyP) are amorphous, but also contain small amounts of vaterite, as revealed by XRD, FTIR and SEM analyses. They did not affect the growth/…

Biosilica-Based Strategies for Treatment of Osteoporosis and Other Bone Diseases

Osteoporosis is a common disease in later life, which has become a growing public health problem. This degenerative bone disease primarily affects postmenopausal women, but also men may suffer from reduced bone mineral density. The development of prophylactic treatments and medications of osteoporosis has become an urgent issue due to the increasing proportion of the elderly in the population. Apart from medical/hormonal treatments, current strategies for prophylaxis of osteoporosis are primarily based on calcium supplementation as a main constituent of bone hydroxyapatite mineral. Despite previous reports suggesting an essential role in skeletal growth and development, the significance of …

The Unique Invention of the Siliceous Sponges: Their Enzymatically Made Bio-Silica Skeleton

Sponges are sessile filter feeders that, among the metazoans, evolved first on Earth. In the two classes of the siliceous sponges (the Demospongiae and the Hexactinellida), the complex filigreed body is stabilized by an inorganic skeleton composed of amorphous silica providing them a distinct body shape and plan. It is proposed that the key innovation that allowed the earliest metazoans to form larger specimens was the enzyme silicatein. This enzyme is crucial for the formation of the siliceous skeleton. The first sponge fossils with body preservation were dated back prior to the “Precambrian-Cambrian” boundary [Vendian (610–545 Ma)/Ediacaran (542–580 Ma)]. A further molecule required for t…

Genetic, biological and structural hierarchies during sponge spicule formation: from soft sol–gels to solid 3D silica composite structures

Structural biomaterials are hierarchically organized and biofabricated. Although the structural complexity of most bioskeletons can be traced back from the millimeter-scale to the micrometer- or submicrometer-scale, the biological and/or genetic basis controlling the synthesis of these skeletons and their building blocks remained unknown. There is one distinguished example, the spicules of the siliceous sponges, for which the principle molecules and molecular-biological processes involved in their formation have been elucidated in the last few years. In this review, recent data on the different levels of molecular, biological and structural hierarchies controlling the synthesis of the pictu…

Hardening of bio-silica in sponge spicules involves an aging process after its enzymatic polycondensation: evidence for an aquaporin-mediated water absorption.

Abstract Background Spicules, the siliceous skeletal elements of the siliceous sponges, are synthesized enzymatically via silicatein. The product formed, bio-silica, constitutes their inorganic matrix. It remained unexplored which reactions are involved in molding of the amorphous bio-silica and formation of a solid and rigid biomaterial. Methods Cell and molecular biological techniques have been applied to analyze processes resulting in the hardening of the enzymatically synthesized bio-silica. The demosponge Suberites domuncula has been used for the studies. Results Cell aggregates (primmorphs) from the sponge S . domuncula , grown in the presence of Mn-sulfate, form spicules that compris…

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…

Metazoan Circadian Rhythm: Toward an Understanding of a Light-Based Zeitgeber in Sponges

In all eukaryotes, the 24-h periodicity in the environment contributed to the evolution of the molecular circadian clock. We studied some elements of a postulated circadian clock circuit in the lowest metazoans, the siliceous sponges. First, we identified in the demosponge Suberites domuncula the enzyme luciferase that generates photons. Then (most likely), the photons generated by luciferase are transmitted via the biosilica glass skeleton of the sponges and are finally harvested by cryptochrome in the same individual; hence, cryptochrome is acting as a photosensor. This information-transduction system, generation of light (luciferase), photon transmission (through the siliceous spicules),…

Inorganic Polyphosphates As Storage for and Generator of Metabolic Energy in the Extracellular Matrix.

Inorganic polyphosphates (polyP) consist of linear chains of orthophosphate residues, linked by high-energy phosphoanhydride bonds. They are evolutionarily old biopolymers that are present from bacteria to man. No other molecule concentrates as much (bio)chemically usable energy as polyP. However, the function and metabolism of this long-neglected polymer are scarcely known, especially in higher eukaryotes. In recent years, interest in polyP experienced a renaissance, beginning with the discovery of polyP as phosphate source in bone mineralization. Later, two discoveries placed polyP into the focus of regenerative medicine applications. First, polyP shows morphogenetic activity, i.e., induc…

Silicateins - A Novel Paradigm in Bioinorganic Chemistry: Enzymatic Synthesis of Inorganic Polymeric Silica

The inorganic matrix of the siliceous skeletal elements of sponges, that is, spicules, is formed of amorphous biosilica. Until a decade ago, it remained unclear how the hard biosilica monoliths of the spicules are formed in sponges that live in a silica-poor (<50 mu m) aquatic environment. The following two discoveries caused a paradigm shift and allowed an elucidation of the processes underlying spicule formation; first the discovery that in the spicules only one major protein, silicatein, exists and second, that this protein displays a bio-catalytical, enzymatic function. These findings caused a paradigm shift, since silicatein is the first enzyme that catalyzes the formation of an inorga…

Amorphous polyphosphate, a smart bioinspired nano-/bio-material for bone and cartilage regeneration: towards a new paradigm in tissue engineering

Recent developments in the field of biomaterials for tissue engineering open up new opportunities for regenerative therapy and prevention of progression of osteo-articular damage/impairment. A key advancement was the discovery of the regenerative activity of a group of physiologically occurring high-energy polymers, inorganic polyphosphates (polyP). These bio-polymers, in suitable bioinspired formulations, turned out to be capable of inducing proliferation and differentiation of mesenchymal stem cells into osteogenic or chondrogenic lineages through differential gene expression (morphogenetic activity). Unprecedented is the property of these biopolymers to deliver high-energy phosphate in t…

The sponge silicatein-interacting protein silintaphin-2 blocks calcite formation of calcareous sponge spicules at the vaterite stage

Ca-carbonate, the inorganic matrix of the spicules from the calcareous sponges, is formed as the result of an enzyme-catalyzed reaction with the carbonic anhydrase [CA] as a decisive component. The growth and the morphology of the spicules are genetically controlled, and are taxon-specific. In the present study it is shown that the silicatein-interacting protein silintaphin-2 is present at the surface of the siliceous spicules of the demosponge Suberites domuncula and prevents the association of calcareous crystals synthesized in vitro to these skeletal elements. Silintaphin-2 comprises a Ca2+-binding domain that is formed by a 22 amino acid-long peptide, N-DDDSQGEIQSDMAEEEDDDNVD-C. This ve…

Fabrication of amorphous strontium polyphosphate microparticles that induce mineralization of bone cells in vitro and in vivo.

Abstract Here we describe the fabrication process of amorphous strontium-polyphosphate microparticles (“Sr-a-polyP-MP”). The effects of these particles on growth and gene expression were investigated with SaOS-2 cells as well as with human mesenchymal stem cells (MSC) and compared with those particles prepared of amorphous calcium-polyphosphate (“Ca-a-polyP-MP”) and of strontium salt. The results revealed a markedly higher stimulation of growth of MSC by “Sr-a-polyP-MP” compared to “Ca-a-polyP-MP” and a significant increase in mineralization of SaOS-2 cells, as well as an enhanced upregulation of the expression of the genes encoding for alkaline phosphatase and the bone morphogenetic protei…

The Deep-Sea Natural Products, Biogenic Polyphosphate (Bio-PolyP) and Biogenic Silica (Bio-Silica), as Biomimetic Scaffolds for Bone Tissue Engineering: Fabrication of a Morphogenetically-Active Polymer

Bone defects in human, caused by fractures/nonunions or trauma, gain increasing impact and have become a medical challenge in the present-day aging population. Frequently, those fractures require surgical intervention which ideally relies on autografts or suboptimally on allografts. Therefore, it is pressing and likewise challenging to develop bone substitution materials to heal bone defects. During the differentiation of osteoblasts from their mesenchymal progenitor/stem cells and of osteoclasts from their hemopoietic precursor cells, a lineage-specific release of growth factors and a trans-lineage homeostatic cross-talk via signaling molecules take place. Hence, the major hurdle is to fab…

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 …

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 …

Chapter 3 Giant Siliceous Spicules From the Deep‐sea Glass Sponge Monorhaphis chuni

Only 13 years after realizing, during a repair of a telegraph cable pulled out from the deep sea, that the depth of the ocean is plentifully populated with a highly diverse fauna and flora, the Challenger expedition (1873-1876) treasured up a rich collection of vitreous sponges (Hexactinellida). They had been described by Schulze and represent the phylogenetically oldest class of siliceous sponges (phylum Porifera); they are eye-catching because of their distinct body plan, which relies on a filigree skeleton. It is constructed by an array of morphologically determined elements, the spicules. Soon after, during the German Deep Sea Expedition "Valdivia" (1898-1899), Schulze could describe th…

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…

Amorphous polyphosphate/amorphous calcium carbonate implant material with enhanced bone healing efficacy in a critical-size defect in rats

In this study the effect of amorphous calcium carbonate (ACC) microparticles and amorphous calcium polyphosphate (polyP) microparticles (termed aCa-polyP-MP) on bone mineral forming cells/tissue was investigated in vitro and in vivo. The ACC particles (termed ACC-P10-MP) were prepared in the presence of Na-polyP. Only the combinations of polyP and ACC microparticles enhanced the proliferation rate of human mesenchymal stem cells (MSCs). Gene expression studies revealed that ACC causes an upregulation of the expression of the cell membrane-associated carbonic anhydrase IX (CA IX; formation of ACC), while the transcript level of the alkaline phosphatase (ALP; liberation of orthophosphate from…

Morphogenetically-Active Barrier Membrane for Guided Bone Regeneration, Based on Amorphous Polyphosphate

We describe a novel regeneratively-active barrier membrane which consists of a durable electrospun poly(ε-caprolactone) (PCL) net covered with a morphogenetically-active biohybrid material composed of collagen and inorganic polyphosphate (polyP). The patch-like fibrous collagen structures are decorated with small amorphous polyP nanoparticles (50 nm) formed by precipitation of this energy-rich and enzyme-degradable (alkaline phosphatase) polymer in the presence of calcium ions. The fabricated PCL-polyP/collagen hybrid mats are characterized by advantageous biomechanical properties, such as enhanced flexibility and stretchability with almost unaltered tensile strength of the PCL net. The pol…

Front Cover Advanced Materials 3/2012

Enzyme-accelerated and structure-guided crystallization of calcium carbonate: Role of the carbonic anhydrase in the homologous system

Abstract The calcareous spicules from sponges, e.g. from Sycon raphanus, are composed of almost pure calcium carbonate. In order to elucidate the formation of those structural skeletal elements, the function of the enzyme carbonic anhydrase (CA), isolated from this species, during the in vitro calcium carbonate-based spicule formation, was investigated. It is shown that the recombinant sponge CA substantially accelerates calcium carbonate formation in the in vitro diffusion assay. A stoichiometric calculation revealed that the turnover rate of the sponge CA during the calcification process amounts to 25 CO2 s−1 × molecule CA−1. During this enzymatically driven process, initially pat-like pa…

Evagination of Cells Controls Bio-Silica Formation and Maturation during Spicule Formation in Sponges

The enzymatic-silicatein mediated formation of the skeletal elements, the spicules of siliceous sponges starts intracellularly and is completed extracellularly. With Suberites domuncula we show that the axial growth of the spicules proceeds in three phases: (I) formation of an axial canal; (II) evagination of a cell process into the axial canal, and (III) assembly of the axial filament composed of silicatein. During these phases the core part of the spicule is synthesized. Silicatein and its substrate silicate are stored in silicasomes, found both inside and outside of the cellular extension within the axial canal, as well as all around the spicule. The membranes of the silicasomes are inte…

Acquisition of Structure-guiding and Structure-forming Properties during Maturation from the Pro-silicatein to the Silicatein Form

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and res…

A bio-imitating approach to fabricate an artificial matrix for cartilage tissue engineering using magnesium-polyphosphate and hyaluronic acid

Here we describe an artificial cartilage-like material based on a hyaluronic acid-Mg/Ca-polyphosphate paste (HA-aMg/Ca-polyP-p) that is fabricated from a water-soluble Na-salt of energy-rich inorganic polyphosphate (polyP) and soluble hyaluronic acid in the presence of water-insoluble CaCO3. The resulting material, after conversion of Na-polyP into the less soluble Mg/Ca-salt consisting of amorphous Mg/Ca-polyP microparticles, was found to mimic the physiological cartilage tissue and to bind Ca2+ ions present in the synovial fluid. After the Mg2+/Ca2+ exchange and water extrusion, the polyP becomes more stable, but is still susceptible to hydrolytic cleavage by the alkaline phosphatase (ALP…

Enhancement of Wound Healing in Normal and Diabetic Mice by Topical Application of Amorphous Polyphosphate. Superior Effect of a Host⁻Guest Composite Material Composed of Collagen (Host) and Polyphosphate (Guest).

The effect of polyphosphate (polyP) microparticles on wound healing was tested both in vitro and in a mice model in vivo. Two approaches were used: pure salts of polyphosphate, fabricated as amorphous microparticles (MPs, consisting of calcium and magnesium salts of polyP, “Ca–polyp-MPs” and “Mg–polyp-MPs”), and host–guest composite particles, prepared from amorphous collagen (host) and polyphosphate (guest), termed “col/polyp-MPs”. Animal experiments with polyP on healing of excisional wounds were performed using both normal mice and diabetic mice. After a healing period of 7 days “Ca–polyp-MP” significantly improved re-epithelialization in normal mice from 31% (control) to 72% (polyP micr…

Differential Expression of the Demosponge (Suberites domuncula) Carotenoid Oxygenases in Response to Light: Protection Mechanism Against the Self-Produced Toxic Protein (Suberitine)

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7–8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the spo…

Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation.

Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3 (-) ) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, …

Biosilica

Biomineralization, biosilicification in particular (i.e. the formation of biogenic silica, SiO(2)), 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 demosponges, the involvement of further molecules in silica metabolism and their potential applications in nano-biotechnology and bio-medicine. While most forms of multicellular life have developed a calcium-based skel…

Retinol encapsulated into amorphous Ca2+ polyphosphate nanospheres acts synergistically in MC3T3-E1 cells

Both the quality and quantity of collagen, the major structural component of the skin, decrease in aging skin. We succeeded to encapsulate retinol into amorphous inorganic polyphosphate (polyP) nanoparticles together with calcium ions ("aCa-polyP-NP"), under formation of amorphous Ca-polyP/retinol nanospheres ("retinol/aCa-polyP-NS"). The globular nanospheres are not cytotoxic, show an almost uniform size of ≈ 45 nm and have a retinol content of around 25%. Both components of those nanospheres, retinol and the aCa-polyP-NP, if administered together, caused a strong increase in proliferation of mouse calvaria MC3T3 cells. The expressions of collagen types I, II and III genes, but not the exp…

Biomimetic transformation of polyphosphate microparticles during restoration of damaged teeth.

Abstract Objective In the present study, we investigated the fusion process between amorphous microparticles of the calcium salt of the physiological polymer comprising orthophosphate units, of inorganic polyphosphate (polyP), and enamel. Methods This polymer was incorporated as an ingredient into toothpaste and the fusion process was studied by electron microscopy and by synchrotron-based X-ray tomography microscopy (SRXTM) techniques. Results The data showed that toothpaste, supplemented with the amorphous Ca-polyP microparticles (aCa-polyP-MP), not only reseals tooth defects on enamel, like carious lesions, and dentin, including exposed dentinal tubules, but also has the potential to ind…

Porous titania surfaces on titanium with hierarchical macro- and mesoporosities for enhancing cell adhesion, proliferation and mineralization

Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5 μm. An additional mesoporous titania top layer following the contour of the macropores, of 100-200 nm thickness and with a pore diameter of 10nm, was formed by using the evaporation-induced self-assembly (EISA) method with titanium (IV) tetraethoxide as the precursor. A coherent laminar titania surface layer was thus obtained, creating a hierarchical macro- and mesoporous surface that was characterized by high-resolution electron microscopy. The interfacial bonding between the surface layers and the titanium matrix was characterized by the scratch test that con…

Cover Picture: NanoSIMS: Insights into the Organization of the Proteinaceous Scaffold within Hexactinellid Sponge Spicules (ChemBioChem 8/2010)

Biologically-induced transition of bio-silica sol to mesoscopic gelatinous flocs: a biomimetic approach to a controlled fabrication of bio-silica structures

Siliceous sponges form their skeletal elements, the spicules, enzymatically via the sponge-specific enzyme silicatein. The enzymatic product of silicatein in vitro is a bio-silica polymer that is not processed/hardened by phase separation. In the present study we applied a two-phase system to investigate the transition of bio-silica, formed by silicatein, from the lucid state to the opaque/turbid state. We report for the first time that the polyether polyethylene glycol [PEG] causes a rapid transition of the lucid bio-silica to the opaque/turbid state. For the experiments the recombinant silicatein from the demosponge Suberites domuncula had been used. This process is rapid (1 h) and procee…

Polyphosphate as a metabolic fuel in Metazoa: A foundational breakthrough invention for biomedical applications

In animals, energy-rich molecules like ATP are generated in the intracellular compartment from metabolites, e.g. glucose, taken up by the cells. Recent results revealed that inorganic polyphosphates (polyP) can provide an extracellular system for energy transport and delivery. These polymers of multiple phosphate units, linked by high-energy phosphoanhydride bonds, use blood platelets as transport vehicles to reach their target cells. In this review it is outlined how polyP affects cell metabolism. It is discussed that polyP influences cell activity in a dual way: (i) as a metabolic fuel transferring metabolic energy through the extracellular space; and (ii) as a signaling molecule that amp…

Whole-Ocean Changes in Silica and Ge/Si Ratios During the Last Deglacial Deduced From Long-Lived Giant Glass Sponges

Morphology of Sponge Spicules: Silicatein a Structural Protein for Bio-Silica Formation

Most forms of multicellular life have developed a calcium-based skeleton, while only a few specialized organisms complement their body plan with silica, such as sponges (phylum Porifera). However, the way in which sponges synthesize their silica is exceptional. They use an enzyme, silicatein, for the polymerization/polycondensation of silica, and thereby form their highly resistant and stabile massive siliceous skeletal elements (spicules). 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. This peculiar phenomenon has…

Fabrication of a new physiological macroporous hybrid biomaterial/bioscaffold material based on polyphosphate and collagen by freeze-extraction

We describe the fabrication of a new scaffold, an inorganic–organic hybrid biomaterial, consisting of the physiological polymers: the inorganic polymer polyphosphate (polyP), as well as the organic macromolecules collagen and chondroitin sulfate. The polyP polymer is composed of multiple phosphate orthophosphate units linked together by high-energy phosphoanhydride bonds. Chondroitin sulfate has been included due to its hydrogel-forming properties. In the presence of Ca2+ ions, the randomly coiled polyP reorganizes together with collagen and chondroitin sulfate to linear molecules which undergo hardening. This scaffold is deposited as amorphous Ca–polyP nanoparticles (size ≈20–40 nm large) …

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

Abstract 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-κB 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 e…

Bacterial sensors based on biosilica immobilization for label-free OWLS detection

In the last years, a new group of enzymes, the so-called silicateins, have been identified and characterized, which form the axial filaments of the spicules of the siliceous sponges, consisting of not only amorphous silica among others. These enzymes are able to catalyze the polycondensation and deposition of silica at mild conditions. Silicateins can be expressed in Escherichia coli. The recombinant proteins are expressed on the surface of the cell wall and are able to catalyze the formation of a polysilicate net around the bacterial cells providing the possibility for further attachment to the surface of SiO2 containing sensor chips. With this mild immobilization process it is now possibl…

Polyphosphate Reverses the Toxicity of the Quasi-Enzyme Bleomycin on Alveolar Endothelial Lung Cells In Vitro

Simple Summary Bleomycin (BLM) is a medication introduced used to treat various types of cancer, including testicular cancer, ovarian cancer, and Hodgkin’s disease. Its most serious side effect is pulmonary fibrosis and impaired lung function. Using A549 human lung cells it is shown that, in parallel to an increased cell toxicity and DNA damage, BLM causes a marked enlargement of the cell nucleus. This effect is abolished by inorganic polyphosphate (polyP), if this physiological polymer is administered together with BLM. The detoxification of BLM is–most likely–caused by the upregulation of the gene encoding the BLM hydrolase which inactivates BLM in vitro and in vivo. This study contribute…

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…

The silicatein propeptide acts as inhibitor/modulator of self-organization during spicule axial filament formation.

Silicateins are crucial enzymes that are involved in formation of the inorganic biosilica scaffold of the spicular skeleton of siliceous sponges. We show that silicatein acquires its structure-guiding and enzymatically active state by processing of silicatein from pro-silicatein to the mature enzyme. A recombinant propeptide (PROP) of silicatein from the siliceous demosponge Suberites domuncula was prepared, and antibodies were raised against the peptide. In sponge tissue, these antibodies reacted with both surface structures and the central region of the spicules. Using phage display expression, spicule-binding 12-mer peptides were identified that are rich in histidine residues. In the pre…

Sponges (Porifera) as living metazoan witnesses from the Neoproterozoic: biomineralization and the concept of their evolutionary success

Terra Nova, 22, 1–11, 2010 Abstract The emergence of the Metazoa can be dated back to the Neoproterozoic Era which comprises the Cryogenian Period during which two major glaciations occurred, the Sturtian and the Varanger-Marinoan. At that time, the phylum Porifera (sponges) evolved as the first animals and developed a hard skeleton. The two classes of siliceous sponges, the Hexactinellida and the Demospongiae, are already provided with the major genetic repertoire and gene regulatory networks that also exist in modern multicellular animals. Besides these metazoan innovations, the siliceous sponges display one autapomorphic character, silicatein, an enzyme which mediates bio-silica formatio…

The inorganic polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations

Graphical abstract The inorganic physiological polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations. This discovery proposes polyphosphate as a new member of the host's antiviral innate immune defense.

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…

Silicatein: Nanobiotechnological and Biomedical Applications

Silica-based materials are used in many high-tech products including microelectronics, optoelectronics, and catalysts. Siliceous sponges (Demospongiae and Hexactinellida) are unique in their ability to synthesize silica enzymatically. We have cloned the silica-forming enzymes, silicateins, from both demosponges (marine and freshwater sponges) and hexactinellid sponges. The recombinant enzymes allow the synthesis of silica under environmentally benign ambient conditions, while the technical (chemical) production of silica commonly requires high temperatures and pressures, and extremes of pH. Silicateins can be used for the fabrication of highly-ordered inorganic–organic composite materials w…

Hierarchical architecture of sponge spicules: biocatalytic and structure-directing activity of silicatein proteins as model for bioinspired applications

Since the first description of the silicateins, a group of enzymes that mediate the formation of the amorphous, hydrated biosilica of the skeleton of the siliceous sponges, much progress has been achieved in the understanding of this biomineralization process. These discoveries include, beside the proof of the enzymatic nature of the sponge biosilica formation, the dual property of the enzyme, to act both as a structure-forming and structure-guiding protein, and the demonstration that the initial product of silicatein is a soft, gel-like material that has to undergo a maturation process during which it achieves its favorable physical-chemical properties allowing the development of various t…

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…

Bioinspired Fabrication of Bio-Silica-Based Bone-Substitution Materials

The gold standard for bone reconstruction is the use of autogeneic grafts from various donor regions, since they possess osteoinductive as well as osteoconductive potential. Only a few synthetic materials possess/display properties that allow optimal bone reconstitution. Previously, we showed that the natural product, bio-silica, comprises osteoinductive, and probably also osteoconductive activity. Bio-silica is formed enzymatically via silicatein; this enzyme has been isolated from siliceous sponges and has also been cloned and prepared recombinantly. In the present study, silicatein was encapsulated together with its substrate, sodium metasilicate, in poly(D,L-lactide)/poly(vinyl pyrrolid…

Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship.

Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI's of…

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 …

Uptake of polyphosphate microparticles in vitro (SaOS-2 and HUVEC cells) followed by an increase of the intracellular ATP pool size

Recently two approaches were reported that addressed a vitally important problem in regenerative medicine, i. e. the successful treatment of wounds even under diabetic conditions. Accordingly, these studies with diabetic rabbits [Sarojini et al. PLoS One 2017, 12(4):e0174899] and diabetic mice [Müller et al. Polymers 2017, 9, 300] identified a novel (potential) target for the acceleration of wound healing in diabetes. Both studies propose a raise of the intracellular metabolic energy status via exogenous administration either of ATP, encapsulated into lipid vesicles, or of polyphosphate (polyP) micro-/nanoparticles. Recently this physiological polymer, polyP, was found to release metabolic …

The biomaterial polyphosphate blocks stoichiometric binding of the SARS-CoV-2 S-protein to the cellular ACE2 receptor

The effect of the polyanionic polymer of inorganic polyphosphate (polyP) involved in innate immunity on the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the cellular ACE2 receptor was studied. The RBD surface comprises a basic amino acid stretch of four arginine residues which interact with the physiological polyP (polyP40) and polyP3. Subsequently, the interaction of RBD with ACE2 is sensitively inhibited. After the chemical modification of arginine, an increased inhibition by polyP, at a 1 : 1 molar ratio (polyP : RBP), is measured already at 0.1 μg mL−1. Heparin was ineffective. The results suggest a potential therapeutic benefit of polyP against SARS-C…

In Situ Polyphosphate Nanoparticle Formation in Hybrid Poly(vinyl alcohol)/Karaya Gum Hydrogels: A Porous Scaffold Inducing Infiltration of Mesenchymal Stem Cells

Abstract The preparation and characterization of a porous hybrid cryogel based on the two organic polymers, poly(vinyl alcohol) (PVA) and karaya gum (KG), into which polyphosphate (polyP) nanoparticles have been incorporated, are described. The PVA/KG cryogel is prepared by intermolecular cross‐linking of PVA via freeze‐thawing and Ca2+‐mediated ionic gelation of KG to form stable salt bridges. The incorporation of polyP as amorphous nanoparticles with Ca2+ ions (Ca‐polyP‐NP) is achieved using an in situ approach. The polyP constituent does not significantly affect the viscoelastic properties of the PVA/KG cryogel that are comparable to natural soft tissue. The exposure of the Ca‐polyP‐NP w…

Contribution of aerobic anoxygenic phototrophic bacteria to total organic carbon pool in aquatic system of subtropical karst catchments, Southwest China: evidence from hydrochemical and microbiological study

Aerobic anoxygenic phototrophic bacteria may play a particular role in carbon cycling of aquatic systems. However, little is known about the interaction between aerobic anoxygenic phototrophic bacteria and hydrochemistry in groundwater-surface water exchange systems of subtropical karst catchments. We carried out a detailed study on the abundance of aerobic anoxygenic phototrophic bacteria and bacterioplankton, hydrochemistry and taxonomy of bacterioplankton in the Maocun watershed, Southwest China, an area with karst geological background. Our results revealed that bacteria are the important contributors to total organic carbon source/sequestration in the groundwater-surface water of this …

Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges

Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro , change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin …

Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation.

Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethason…

Enzymatically Synthesized Biosilica

Structural biomaterials are hierarchically organized and biofabricated. Biosilica represents the main mineral component of the sponge skeletal elements, the spicules. We summarize recent data on the different levels of molecular, biological, and structural hierarchies controlling the synthesis of the picturesquely and intricately architectured spicules/skeletons.

Role of ATP during the initiation of microvascularization: acceleration of an autocrine sensing mechanism facilitating chemotaxis by inorganic polyphosphate

The in vitro tube formation assay with human umbilical vein endothelial cells (HUVEC) was applied to identify the extra- and intracellular sources of metabolic energy/ATP required for cell migration during the initial stage of microvascularization. Extracellularly, the physiological energy-rich polymer, inorganic polyphosphate (polyP), applied as biomimetic amorphous calcium polyP microparticles (Ca-polyP-MP), is functioning as a substrate for ATP generation most likely via the combined action of the alkaline phosphatase (ALP) and the adenylate kinase (AK). The linear Ca-polyP-MP with a size of 40 phosphate units, close to the polyP in the acidocalcisomes in the blood platelets, were found …

Amorphous Ca2+ polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells

ABSTRACT Polyphosphate (polyP) is a physiologically occurring polyanion that is synthesized especially in bone-forming osteoblast cells and blood platelets. We used amorphous polyP nanoparticles, complexed with Ca2+, that have a globular size of ∼100 nm. Because polyP comprises inorganic orthophosphate units that are linked together through high-energy phosphoanhydride bonds, we questioned whether the observed morphogenetic effect, elicited by polyP, is correlated with the energy-generating machinery within the cells. We show that exposure of SaOS-2 osteoblast-like cells to polyP results in a strong accumulation of mitochondria and a parallel translocation of the polyP-degrading enzyme alka…

Involvement of aquaporin channels in water extrusion from biosilica during maturation of sponge siliceous spicules.

Aquaporins are a family of small, pore-forming, integral cell membrane proteins. This ancient protein family functions as water channels and is found in all kingdoms (including archaea, eubacteria, fungi, plants, and animals). We discovered that in sponges aquaporin plays a novel role during the maturation of spicules, their skeletal elements. Spicules are synthesized enzymatically via silicatein following a polycondensation reaction. During this process, a 1:1 stoichiometric release of water per one Si-O-Si bond formed is produced. The product of silicatein, biosilica, is a fluffy, soft material that must be hardened in order to function as a solid rod. Using the model of the demosponge sp…

Silica-protein composite layers of the giant basal spicules from Monorhaphis: Basis for their mechanical stability

The hexactinellid sponge Monorhaphis chuni possesses with its giant basal spicules the largest biosilica structure on Earth. The approximately 8.5-mm-thick spicules are composed of up to 800 lamellae. By application of high-resolution electron microscopy (HR-SEM), it is shown that within the siliceous lamellae a proteinaceous scaffold exists which is composed of one protein of a size of 27 kDa. Analyses with Fourier transform infrared (FT-IR) emission and energy-dispersive X-ray (EDX) spectroscopy support this localization of the protein. No evidence for the presence of protein on the surfaces of the lamellae could be obtained. Heating the giant basal spicule to 600 °C destroys and eliminat…

Isoquercitrin and polyphosphate co-enhance mineralization of human osteoblast-like SaOS-2 cells via separate activation of two RUNX2 cofactors AFT6 and Ets1.

Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3-O-glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like SaOS-2 cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of SaOS-2 cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3μM), if given simultaneously with polyP (as Ca(2+) salt; at 3, 10, 30 and 100μM) amplifies the mineralization-enhanci…

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: Biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth o…

Amorphous polyphosphate–hydroxyapatite: A morphogenetically active substrate for bone-related SaOS-2 cells in vitro

There is increasing evidence that inorganic calcium-polyphosphates (polyP) are involved in human bone hydroxyapatite (HA) formation. Here we investigated the morphology of the particles, containing calcium phosphate (CaP) with different concentrations of various Na-polyP concentrations, as well as their effects in cell culture. We used both SaOS-2 cells and human mesenchymal stem cells. The polymeric phosphate readily binds calcium ions under formation of insoluble precipitates. We found that addition of low concentrations of polyP (10wt.%, referred to the CaP deposits) results in an increased size of the HA crystals. Surprisingly, at higher polyP concentrations (10wt.%) the formation of cr…

Biogenic Inorganic Polysilicates (Biosilica): Formation and Biomedical Applications

The siliceous sponges, the demosponges and hexactinellid glass sponges, are unique in their ability to form biosilica structures with complex architectures through an enzyme-catalyzed mechanism. The biosilica skeleton of these sponges with its hierarchically structure and exceptional opto-mechanical properties has turned out to be an excellent model for the design of biomimetic nanomaterials with novel property combinations. In addition, biosilica shows morphogenetic activity that offers novel applications in the field of bone tissue engineering and repair. In recent years, much progress has been achieved towards the understanding of the principal enzymes, the silicateins that form the spon…

Biosilica-based immobilization strategy for label-free OWLS sensors

Abstract In the last years, a new group of enzymes, so-called silicateins, have been identified and characterized, which form the axial filaments of the spicules of the siliceous sponges, consisting of amorphous silica. Silicateins are able to catalyze the polycondensation and deposition of silica at mild conditions (low temperature and physiological pH). By means of these enzymes it is possible for the first time to produce silica nanostructures biocatalytically, which opens new ways for construction of biosensors. The cDNAs encoding the responsible enzymes have been isolated and the proteins can be produced in a recombinant way. Here we demonstrate the silicatein-mediated biosilica format…

Inorganic Polymers: Morphogenic Inorganic Biopolymers for Rapid Prototyping Chain

In recent years, considerable progress has been achieved towards the development of customized scaffold materials, in particular for bone tissue engineering and repair, by the introduction of rapid prototyping or solid freeform fabrication techniques. These new fabrication techniques allow to overcome many problems associated with conventional bone implants, such as inadequate external morphology and internal architecture, porosity and interconnectivity, and low reproducibility. However, the applicability of these new techniques is still hampered by the fact that high processing temperature or a postsintering is often required to increase the mechanical stability of the generated scaffold, …

Amplified morphogenetic and bone forming activity of amorphous versus crystalline calcium phosphate/polyphosphate.

Amorphous Ca-phosphate (ACP) particles stabilized by inorganic polyphosphate (polyP) were prepared by co-precipitation of calcium and phosphate in the presence of polyP (15% [w/w]). These hybrid nanoparticles showed no signs of crystallinity according to X-ray diffraction analysis, in contrast to the particles obtained at a lower (5% [w/w]) polyP concentration or to hydroxyapatite. The ACP/15% polyP particles proved to be a suitable matrix for cell growth and attachment and showed pronounced osteoblastic and vasculogenic activity in vitro. They strongly stimulated mineralization of the human osteosarcoma cell line SaOS-2, as well as cell migration/microvascularization, as demonstrated in th…

Marine biominerals: perspectives and challenges for polymetallic nodules and crusts.

Deep sea minerals in polymetallic nodules, crusts and hydrothermal vents are not only formed by mineralization but also by biologically driven processes involving microorganisms (biomineralization). Within the nodules, free-living and biofilm-forming bacteria provide the matrix for manganese deposition, and in cobalt-rich crusts, coccolithophores represent the dominant organisms that act as bio-seeds for an initial manganese deposition. These (bio)minerals are economically important: manganese is an important alloying component and cobalt forms part of special steels in addition to being used, along with other rare metals, in plasma screens, hard-disk magnets and hybrid car motors. Recent p…

Biocalcite, a multifunctional inorganic polymer: Building block for calcareous sponge spicules and bioseed for the synthesis of calcium phosphate-based bone

Calcium carbonate is the material that builds up the spicules of the calcareous sponges. Recent results revealed that the calcium carbonate/biocalcite-based spicular skeleton of these animals is formed through an enzymatic mechanism, such as the skeleton of the siliceous sponges, evolutionarily the oldest animals that consist of biosilica. The enzyme that mediates the calcium carbonate deposition has been identified as a carbonic anhydrase (CA) and has been cloned from the calcareous sponge species Sycon raphanus. Calcium carbonate deposits are also found in vertebrate bones besides the main constituent, calcium phosphate/hydroxyapatite (HA). Evidence has been presented that during the init…

Optical properties of in-vitro biomineralised silica

Silicon is the second most common element on the Earth's crust and its oxide (SiO(2)) the most abundant mineral. Silica and silicates are widely used in medicine and industry as well as in micro- and nano-optics and electronics. However, the fabrication of glass fibres and components requires high temperature and non-physiological conditions, in contrast to biosilica structures in animals and plants. Here, we show for the first time the use of recombinant silicatein-α, the most abundant subunit of sponge proteins catalyzing biosilicification reactions, to direct the formation of optical waveguides in-vitro through soft microlithography. The artificial biosilica fibres mimic the natural spon…

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…

Spatial and temporal dynamics of bacterioplankton community composition in a subtropical dammed karst river of southwestern China.

Abstract River damming influences the hydro‐physicochemical variations in karst water; however, such disruption in bacterioplankton communities has seldom been studied. Here, three sampling sites (city‐river section, reservoir area, and outflow area) of the Ca2+–Mg2+–HCO 3 −–SO 4 2− water type in the dammed Liu River were selected to investigate the bacterioplankton community composition as identified by high‐throughput 16S rRNA gene sequencing. In the dammed Liu River, thermal regimes have been altered, which has resulted in considerable spatial‐temporal differences in total dissolved solids (TDSs), oxidation‐reduction potential (Eh), dissolved oxygen (DO), and pH and in a different microe…

Fractal-related assembly of the axial filament in the demosponge Suberites domuncula: relevance to biomineralization and the formation of biogenic silica,

Abstract The siliceous spicules of sponges (Porifera) show great variations of sizes, shapes and forms; they constitute the chief supporting framework of these animals; these skeletal elements are synthesized enzymatically by silicatein. Each sponge species synthesizes at least two silicateins, which are termed − α and − β . In the present study, using the demosponge Suberites domuncula , we studied if the silicateins of the axial filament contribute to the shape formation of the spicules. For these experiments native silicateins have been isolated by a new Tris/glycerol extraction procedure. Silicateins isolated by this procedure are monomeric (24 kDa), but readily form dimers through non-…

Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligop…

Two-Armed Activation of Bone Mineral Deposition by the Flavones Baicalin and Baicalein, Encapsulated in Polyphosphate Microparticles

In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to…

New Target Sites for Treatment of Osteoporosis

In the last few years, much progress has been achieved in the discovery of new drug target sites for treatment of osteoporotic disorders, one of the main challenging diseases with a large burden for the public health systems. Among these new agents promoting bone formation, shifting the impaired equilibrium between bone anabolism and bone catabolism in the direction of bone synthesis are inorganic polymers, in particular inorganic polyphosphates that show strong stimulatory effects on the expression of bone anabolic marker proteins and hydroxyapatite formation. The bone-forming activity of these polymers can even be enhanced by combination with certain small molecules like quercetin, or if …

Biocalcite and Carbonic Acid Activators

Based on evolution of biomineralizing systems and energetic considerations, there is now compelling evidence that enzymes play a driving role in the formation of the inorganic skeletons from the simplest animals, the sponges, up to humans. Focusing on skeletons based on calcium minerals, the principle enzymes involved are the carbonic anhydrase (formation of the calcium carbonate-based skeletons of many invertebrates like the calcareous sponges, as well as deposition of the calcium carbonate bioseeds during human bone formation) and the alkaline phosphatase (providing the phosphate for bone calcium phosphate-hydroxyapatite formation). These two enzymes, both being involved in human bone for…

Acousto-optic cavity coupling in 2D phoxonic crystal with combined convex and concave holes

International audience; A two-dimensional cross-like phoxonic crystal (PxC) model is proposed, which exhibits simultaneously large complete photonic crystal (PtC) and phononic crystal (PnC) bandgaps. The most salient trait of the structure is the wide range of geometrical parameters compatible with large complete bandgaps. After geometrical optimization, photonic and phononic bandgaps with gap-to-midgap ratios of 11.5% and 90.7% are obtained, respectively. These values are close to the best topology-optimized reported values but are obtained with simple shapes compatible with nanoscale fabrication technology. These characteristics make the convex–concave topology a promising candidate for P…

Rebalancing β-Amyloid-Induced Decrease of ATP Level by Amorphous Nano/Micro Polyphosphate: Suppression of the Neurotoxic Effect of Amyloid β-Protein Fragment 25-35

Morbus Alzheimer neuropathology is characterized by an impaired energy homeostasis of brain tissue. We present an approach towards a potential therapy of Alzheimer disease based on the high-energy polymer inorganic polyphosphate (polyP), which physiologically occurs both in the extracellular and in the intracellular space. Rat pheochromocytoma (PC) 12 cells, as well as rat primary cortical neurons were exposed to the Alzheimer peptide Aβ25-35. They were incubated in vitro with polyphosphate (polyP); ortho-phosphate was used as a control. The polymer remained as Na+ salt; or complexed in a stoichiometric ratio to Ca2+ (Na-polyP[Ca2+]); or was processed as amorphous Ca-polyP microparticles (C…

An unexpected biomaterial against SARS-CoV-2: Bio-polyphosphate blocks binding of the viral spike to the cell receptor.

Graphical abstract

Engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells.

Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially i…

Bioactive and biodegradable silica biomaterial for bone regeneration.

Biosilica, a biocompatible, natural inorganic polymer that is formed by an enzymatic, silicatein-mediated reaction in siliceous sponges to build up their inorganic skeleton, has been shown to be morphogenetically active and to induce mineralization of human osteoblast-like cells (SaOS-2) in vitro. In the present study, we prepared beads (microspheres) by encapsulation of β-tricalcium phosphate [β-TCP], either alone (control) or supplemented with silica or silicatein, into the biodegradable copolymer poly(d,l-lactide-co-glycolide) [PLGA]. Under the conditions used, ≈5% β-TCP, ≈9% silica, and 0.32μg/mg of silicatein were entrapped into the PLGA microspheres (diameter≈800μm). Determination of …

Potentiation of the cytotoxic activity of copper by polyphosphate on biofilm-producing bacteria: A bioinspired approach

Adhesion and accumulation of organic molecules represent an ecologically and economically massive problem. Adhesion of organic molecules is followed by microorganisms, unicellular organisms and plants together with their secreted soluble and structure-associated byproducts, which damage unprotected surfaces of submerged marine structures, including ship hulls and heat exchangers of power plants. This is termed biofouling. The search for less toxic anti-biofilm strategies has intensified since the ban of efficient and cost-effective anti-fouling paints, enriched with the organotin compound tributyltin, not least because of our finding of the ubiquitous toxic/pro-apoptotic effects displayed b…

Biologization of Allogeneic Bone Grafts with Polyphosphate: A Route to a Biomimetic Periosteum

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni.

SUMMARYSilicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e.g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial f…

Biomimetic Alginate/Gelatin Cross-Linked Hydrogels Supplemented with Polyphosphate for Wound Healing Applications

In the present study, the fabrication of a biomimetic wound dressing that mimics the extracellular matrix, consisting of a hydrogel matrix composed of non-oxidized and periodate-oxidized marine alginate, was prepared to which gelatin was bound via Schiff base formation. Into this alginate/oxidized-alginate-gelatin hydrogel, polyP was stably but reversibly integrated by ionic cross-linking with Zn2+ ions. Thereby, a soft hybrid material is obtained, consisting of a more rigid alginate scaffold and porous structures formed by the oxidized-alginate-gelatin hydrogel with ionically cross-linked polyP. Two forms of the Zn-polyP-containing matrices were obtained based on the property of polyP to f…

ChemInform Abstract: Genetic, Biological and Structural Hierarchies During Sponge Spicule Formation: from Soft Sol-Gels to Solid 3D Silica Composite Structures

Bioorganic/inorganic hybrid composition of sponge spicules: matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis.

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3 m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 mu m), (ii) the bulky axial cylinder (radius: <75 mu m), and (iii) the central axial canal (diameter: <2 mu m) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated …

Utilization of metabolic energy in treatment of ocular surface disorders: polyphosphate as an energy source for corneal epithelial cell proliferation.

Impaired regeneration of the corneal epithelium, as found in many ocular surface diseases, is a major clinical problem in ophthalmology. We hypothesized that corneal epithelial regeneration can be promoted by the physiological, energy-delivering as well as “morphogenetically active” polymer, inorganic polyphosphate (polyP). Corneal limbal explants (diameter, 4 mm) were cultivated on collagen-coated well plates in the absence or presence of polyP (chain length, ∼40 Pi units; 50 μg ml−1) or human platelet lysate (hp-lysate; 5% v/v). Cell outgrowth and differentiation were analyzed after staining with DRAQ5 (nuclei) and rhodamine phalloidin (cytoskeleton), as well as by environmental scanning …

A Novel Biomimetic Approach to Repair Enamel Cracks/Carious Damages and to Reseal Dentinal Tubules by Amorphous Polyphosphate.

Based on natural principles, we developed a novel toothpaste, containing morphogenetically active amorphous calcium polyphosphate (polyP) microparticles which are enriched with retinyl acetate (“a-polyP/RA-MP”). The spherical microparticles (average size, 550 ± 120 nm), prepared by co-precipitating soluble Na-polyP with calcium chloride and supplemented with retinyl acetate, were incorporated into a base toothpaste at a final concentration of 1% or 10%. The “a-polyP/RA-MP” ingredient significantly enhanced the stimulatory effect of the toothpaste on the growth of human mesenchymal stem cells (MSC). This increase was paralleled by an upregulation of the MSC marker genes for osteoblast differ…

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP.

Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular …

Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro

Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca2+ complex)] on the osteoblast-like SaOS-2 cells and the osteoclast-like RAW 264.7 cells was determined. Both polymers are non-toxic for these cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca2+ complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca2+ complex) triggered in SaOS-2 cell…

Interaction of the retinoic acid signaling pathway with spicule formation in the marine sponge Suberites domuncula through activation of bone morphogenetic protein-1

Abstract Background The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin. Scope of review Molecular cloning of the cDNAs (carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM. Major conclusions We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13 kDa calcium-b…

NanoSIMS: insights into the organization of the proteinaceous scaffold within Hexactinellid sponge spicules.

The giant basal spicules (GBS) from Monorhaphis chuni (Porifera [sponges], Hexactinellida) represent the largest biosilica structures on Earth and can reach lengths of 300 cm (diameter of 1.1 cm). The amorphous silica of the inorganic matrix is formed enzymatically by silicatein. During this process, the enzyme remains trapped inside the lamellar-organized spicules. In order to localize the organic silicatein scaffold, the inside of a lamella has been analyzed by nano-secondary ion mass spectrometry (NanoSIMS). It is shown that the GBSs are composed of around 245 concentrically arranged individual siliceous lamellae. These surround an internal siliceous axial cylinder. The lamellae adjacent…

Axial growth of hexactinellid spicules: Formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size …

Morphogenetic (Mucin Expression) as Well as Potential Anti-Corona Viral Activity of the Marine Secondary Metabolite Polyphosphate on A549 Cells

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phospha…

Transformation of Amorphous Polyphosphate Nanoparticles into Coacervate Complexes: An Approach for the Encapsulation of Mesenchymal Stem Cells.

Inorganic polyphosphate [polyP] has proven to be a promising physiological biopolymer for potential use in regenerative medicine because of its morphogenetic activity and function as an extracellular energy-donating system. Amorphous Ca2+ -polyP nanoparticles [Ca-polyP-NPs] are characterized by a high zeta potential with -34 mV (at pH 7.4). This should contribute to the stability of suspensions of the spherical nanoparticles (radius 94 nm), but make them less biocompatible. The zeta potential decreases to near zero after exposure of the Ca-polyP-NPs to protein/peptide-containing serum or medium plus serum. Electron microscopy analysis reveals that the particles rapidly change into a coacerv…

Collagen-inducing biologization of prosthetic material for hernia repair: Polypropylene meshes coated with polyP/collagen

Prostethic mesh material such as nonabsorbable polypropylene used in open and laparoscopic hernia repair are characterized by controllable mechanical properties but may elicit undesirable physiological reactions due to the nonphysiological inert polymer material. We succeeded in developing a biocompatible coating for these meshes, based on a physiological inorganic polymer, polyphosphate (polyP) that is morphogenetically active and used as a metabolic energy source, and a collagen matrix. The polyP/collagen hydrogel material was prepared by a freeze-extraction method, with amorphous Ca-polyP microparticles. Electron microscopy (SEM and REM) studies revealed that the polyP/collagen coats are…

Polyphosphate: A Morphogenetically Active Implant Material Serving as Metabolic Fuel for Bone Regeneration

The initial mineralization centers during human bone formation onto osteoblasts are composed of CaCO3 . Those bioseeds are enzymatically formed via carbonic anhydrase(s) in close association with the cell surface of the osteoblasts. Subsequently, the bicarbonate/carbonate anions are exchanged non-enzymatically by inorganic phosphate [Pi ]. One source for the supply of Pi is polyphosphate [polyP] which is a physiological polymer, formed in the osteoblasts as well as in the platelets. The energy-rich acid anhydride bonds within the polyP chain are cleaved by phosphatase(s); during this reaction free-energy might be released that could be re-used, as metabolic fuel, for the maintenance of the …

Amorphous polyphosphate nanoparticles: application of the morphogenetically active inorganic polymer for personalized tissue regeneration

The enzyme carbonic anhydrase as an integral component of biogenic Ca-carbonate formation in sponge spicules

The inorganic scaffold of the spicules, the skeletal elements of the calcareous sponges, is formed of calcium carbonate (CaCO3). The growth of the approximately 300-μm large spicules, such as those of the calcareous sponge Sycon raphanus used in the present study, is a rapid process with a rate of about 65 μm/h. The formation of CaCO3 is predominantly carried out by the enzyme carbonic anhydrase (CA). The enzyme from the sponge S. raphanus was isolated and prepared by recombination. The CA-driven deposition of CaCO3 crystallites is dependent on temperature (optimal at 52 °C), the pH value of the reaction assay (7.5/8.0), and the substrate concentration (CO2 and Ca2+). During the initial pha…

Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-α gene

Bioencapsulation is an intriguing way to immobilize biological materials, including cells, in silica, metal-oxides or hybrid sol-gel polymers. Until now only the sol-gel precursor technology was utilized to immobilize bacteria or yeast cells in silica. With the discovery of silicatein, an enzyme from demosponges that catalyzes the formation of poly(silicate), it became possible to synthesize poly(silicate) under physiological (ambient) conditions. Here we show that Escherichia coli can be transformed with the silicatein gene, its expression level in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG) can be efficiently intensified by co-incubation with silicic acid. This effect co…

Enzymatically Synthesized Inorganic Polymers as Morphogenetically Active Bone Scaffolds

In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of …

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…

Triple-target stimuli-responsive anti-COVID-19 face mask with physiological virus-inactivating agents

Conventional face masks to prevent SARS-CoV-2 transmission are mostly based on a passive filtration principle. Ideally, anti-COVID-19 masks should protect the carrier not only by size exclusion of virus aerosol particles, but also be able to capture and destroy or inactivate the virus. Here we present the proof-of-concept of a filter mat for such a mask, which actively attracts aerosol droplets and kills the virus. The electrospun mats are made of polycaprolactone (PCL) a hydrophilic, functionalizable and biodegradable polyester, into which inorganic polyphosphate (polyP) a physiological biocompatible, biodegradable and antivirally active polymer (chain length, ∼40 Pi units) has been integr…

Enzyme-based biosilica and biocalcite: biomaterials for the future in regenerative medicine

The oldest animals on Earth, sponges, form both the calcareous and the siliceous matrices of their spicules enzymatically. Until recently, it has been neglected that enzymes play crucial roles during formation of these biominerals. This paradigm shift occurred after the discovery that the enzyme silicatein, which catalyzes the polycondensation of silica, and the enzyme carbonic anhydrase (CA), which catalyzes the formation of bicarbonate (HCO3(-)/CaCO3), produce solid amorphous bioglass or biocalcite. This suggests that in mammals, biosilica and biocalcite can act anabolically during hydroxyapatite (HA) synthesis and bone formation. Biosilica and biocalcite are thus promising candidates for…

Concept of Biogenic Ferromanganese Crust Formation: Coccoliths as Bio-seeds in Crusts from Central Atlantic Ocean (Senghor Seamount/Cape Verde)

At depths of 2,000 to 3,000 m, seamounts from the Cape Verde archipelago (Central Atlantic Ocean) are largely covered with ferromanganese crusts. Here we studied 60 to 150 mm thick crusts from the Senghor Seamount (depth: 2257.4 m). The crusts have a non lamellated texture and are covered with spherical nodules. The chemical composition shows a dominance of MnO2 (26.1%) and Fe2O3 (38.8%) with considerable amounts of Co (0.74%) and TiO2 (2.1%). Analysis by scanning electron probe microanalyzer (EPMA) revealed a well defined compositional zonation of micro-layers; the distribution pattern of Mn does not match that of Fe. Analysis by high resolution scanning electron microscopy (SEM) revealed…

Amorphous, Smart, and Bioinspired Polyphosphate Nano/Microparticles: A Biomaterial for Regeneration and Repair of Osteo-Articular Impairments In-Situ

Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form of amorphous Ca-polyP nano/microparticles, polyP retains its function to act as both an intra- and extracellular metabolic fuel and a stimulus eliciting morphogenetic signals. The method for synthesis of the nano/microparticles with the polyanionic polyP also allowed the fabrication of hybrid particles with the bisphosphonate zoledronic acid, a drug used in therapy of bone metastases in cancer patients. The r…

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 o…

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…

Flashing light signaling circuit in sponges: Endogenous light generation after tissue ablation in Suberites domuncula

The skeleton of siliceous sponges (phylum Porifera: classes Demospongiae and Hexactinellida), composed of tightly interacting spicules that assemble to a genetically fixed scaffold, is formed of bio-silica. This inorganic framework with the quality of quartz glass has been shown to operate as light waveguide in vitro and very likely has a similar function in vivo. Furthermore, the molecular toolkit for endogenous light generation (luciferase) and light/photon harvesting (cryptochrome) has been identified in the demosponge Suberites domuncula. These three components of a light signaling system, spicules—luciferase—cryptochrome, are concentrated in the surface layers (cortex) of the poriferan…

Inorganic Polyphosphates: Biologically Active Biopolymers for Biomedical Applications

Inorganic polyphosphate (polyP) is a widely occurring but only rarely investigated biopolymer which exists in both prokaryotic and eukaryotic organisms. Only in the last few years, this polymer has been identified to cause morphogenetic activity on cells involved in human bone formation. The calcium complex of polyP was found to display a dual effect on bone-forming osteoblasts and bone-resorbing osteoclasts. Exposure of these cells to polyP (Ca2+ complex) elicits the expression of cytokines that promote the mineralization process by osteoblasts and suppress the differentiation of osteoclast precursor cells to the functionally active mature osteoclasts dissolving bone minerals. The effect o…

Self-healing, an intrinsic property of biomineralization processes

The sponge siliceous spicules are formed enzymatically via silicatein, in contrast to other siliceous biominerals. Originally, silicatein had been described as a major structural protein of the spicules that has the property to allow a specific deposition of silica onto their surface. More recently, it had been unequivocally demonstrated that silicatein displays a genuine enzyme activity, initiating and maintaining silica biopolycondensation at low precursor concentrations (<2 mM). Even more, as silicatein becomes embedded into the biosilica polymer, formed by the enzyme, it retains its functionality to enable a controlled biosilica deposition. The protection of silicatein through the biosi…

Self-Healing Properties of Bioinspired Amorphous CaCO3/Polyphosphate-Supplemented Cement

There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% (w/w) Na&ndash

Flashing light in sponges through their siliceous fiber network: A new strategy of “neuronal transmission” in animals

Sponges (phylum Porifera) represent a successful animal taxon that evolved prior to the Ediacaran-Cambrian boundary (542 million years ago). They have developed an almost complete array of cell- and tissue-based interaction systems necessary for the establishment of a functional, multicellular body. However, a network of neurons, one cell/tissue-communication system is missing in sponges. This fact is puzzling and enigmatic, because these animals possess receptors known to be involved in the nervous system in evolutionary younger animal phyla. As an example, the metabotropic glutamate/GABA-like receptor has been identified and cloned by us. Recently, we have identified a novel light transmi…

Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP …

3D printing of hybrid biomaterials for bone tissue engineering: Calcium-polyphosphate microparticles encapsulated by polycaprolactone.

Abstract Here we describe the formulation of a morphogenetically active bio-ink consisting of amorphous microparticles (MP) prepared from Ca 2+ and the physiological inorganic polymer, polyphosphate (polyP). Those MP had been fortified by mixing with poly-e-caprolactone (PCL) to allow 3D-bioprinting. The resulting granular PCL/Ca-polyP-MP hybrid material, liquefied by short-time heating to 100 °C, was used for the 3D-printing of tissue-like scaffolds formed by strands with a thickness of 400 µm and a stacked architecture leaving ≈0.5 mm 2 -sized open holes enabling cell migration. The printed composite scaffold turned out to combine suitable biomechanical properties (Young’s modulus of 1.60…

Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni☆

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processe…

Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were pr…

ChemInform Abstract: Biofabrication of Biosilica-Glass by Living Organisms

Back Cover: Macromol. Biosci. 9/2015

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-…

3D bioprinting of tissue units with mesenchymal stem cells, retaining their proliferative and differentiating potential, in polyphosphate-containing bio-ink

Abstract The three-dimensional (3D)-printing processes reach increasing recognition as important fabrication techniques to meet the growing demands in tissue engineering. However, it is imperative to fabricate 3D tissue units, which contain cells that have the property to be regeneratively active. In most bio-inks, a metabolic energy-providing component is missing. Here a formulation of a bio-ink is described, which is enriched with polyphosphate (polyP), a metabolic energy providing physiological polymer. The bio-ink composed of a scaffold (N,O-carboxymethyl chitosan), a hydrogel (alginate) and a cell adhesion matrix (gelatin) as well as polyP substantially increases the viability and the …

Circumferential spicule growth by pericellular silica deposition in the hexactinellid sponge Monorhaphis chuni.

SUMMARY The giant basal spicule of the hexactinellid sponge Monorhaphis chuni represents the longest natural siliceous structure on Earth. This spicule is composed of concentrically arranged lamellae that are approximately 10 μm thick. In the present study, we investigated the formation of outer lamellae on a cellular level using microscopic and spectroscopic techniques. It is shown that the formation of an outermost lamella begins with the association of cell clusters with the surface of the thickening and/or growing spicule. The cells release silica for controlled formation of a lamella. The pericellular (silica) material fuses to a delimited and textured layer of silica with depressions …

Polyphosphate as a Bioactive and Biodegradable Implant Material: Induction of Bone Regeneration in Rats

Inorganic polyphosphate (polyP) is a naturally occurring polymer that is bioresorbable and anabolically active on bone forming cells in vitro. In order to demonstrate if polyP also shows morphogenetic activity in vivo, animal studies are performed applying the rat calvarial defect model. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres with a narrow size distribution (≈820 μm) are prepared, containing either encapsulated polyP or β-tricalcium phosphate (β-TCP), used as a reference material. Discs are prepared from the microspheres and inserted into 10 mm large defects created in the calvaria of rats. Both the formation of COL-I and the expression of ALP is upregulated, as well as the exte…

The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl 3 , as an inducer of hydroxyapatite formation in vitro

Inorganic polyphosphate (polyP) is a physiological polymer composed of tens to hundreds of phosphate units linked together via phosphoanhydride bonds. Here we compared the biological activity of polyP (chain length of 40 phosphate units), complexed with Gd(3+) (polyP·Gd), with the one caused by polyP (as calcium salt) and by GdCl3 alone, regarding their potencies to induce hydroxyapatite (HA) formation in SaOS-2 cells in vitro. The three compounds, GdCl3, polyP and polyP·Gd were found to be non-toxic at concentrations up to at least 30μM. Selecting a low, 5μM, concentration it was found that polyP·Gd significantly induced HA formation, as determined by Alizarin Red S staining and by quantit…

A Milestone in Biomineralization: From a Structural Assessment to a Genetic Concept of Mineral Formation. Or How the Enzyme Silicatein Forms a Solid Silica Rod

Biologizing titanium alloy implant material with morphogenetically active polyphosphate

As a further step towards a new generation of bone implant materials, we developed a procedure for biological functionalization of titanium alloy surfaces with inorganic calcium polyphosphate (Ca-polyP). This polymer has been demonstrated to exhibit morphogenetic activity. The coating of titanium oxidized Ti-6Al-4V scaffolds with biologically active amorphous Ca-polyP microparticles is formed by Ca2+ ion bridges to the silane coupling agent APTMS. This surface is durable and stable as an almost homogeneous Ca-polyP layer onto the metal. The homogenously coated Ca-polyP titanium scaffold was found to be biologically active and supported the growth and functional activity of bone cell-related…

A biomimetic approach to ameliorate dental hypersensitivity by amorphous polyphosphate microparticles.

Abstract Objective Dental hypersensitivity has become one of the most common and most costly diseases in the world, even though those maladies are very rarely life threatening. Using amorphous microparticles, fabricated from the natural polymer (polyphosphate), we intend to reseal the dentinal tubules exposed and reduce by that the hypersensitivity. Methods Amorphous microparticles (termed aCa-polyP-MP) were prepared from Na-polyphosphate (polyP) and CaCl 2 , then incubated with human teeth. The potential of the microparticles to plug the dentinal tubules was determined by microscopic and spectroscopic techniques. Results We demonstrate that, in contrast to polyP, the aCa-polyP-MP efficient…

Electrospun bioactive mats enriched with Ca-polyphosphate/retinol nanospheres as potential wound dressing

Background While electrospun materials have been frequently used in tissue engineering no wound dressings exist that significantly improved wound healing effectively. Methods We succeeded to fabricate three-dimensional (3D) electrospun poly(D,l-lactide) (PLA) fiber mats into which nanospheres, formed from amorphous calcium polyphosphate (polyP) nanoparticles (NP) and encapsulated retinol (“retinol/aCa-polyP-NS” nanospheres [NS]), had been incorporated. Results Experiments with MC3T3-E1 cells revealed that co-incubation of the cells with Ca-polyP together with retinol (or incubation with retinol/aCa-polyP-NS) resulted in a significant synergistic effect on cell growth compared with particle-…

Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation)

Bio-silica represents the main mineral component of the sponge skeletal elements (siliceous spicules), while bio-polyphosphate (bio-polyP), a multifunctional polymer existing in microorganisms and animals acts, among others, as reinforcement for pores in cell membranes. These natural inorganic bio-polymers, which can be readily prepared, either by recombinant enzymes (bio-silica and bio-polyP) or chemically (polyP), are promising materials/substances for the amelioration and/or treatment of human bone diseases and dysfunctions. It has been demonstrated that bio-silica causes in vitro a differential effect on the expression of the genes OPG and RANKL, encoding two mediators that control the …

Heparin-based ELISA reduces background reactivity in virus-like particle-based papillomavirus serology.

The interaction between human papillomavirus (HPV) particles and cell surface heparan sulfate requires intact conformation of the HPV particles. Type-specific HPV serology is currently based on virus-like particles (VLPs) with intact conformation. Presence of incorrectly folded VLPs in VLP preparations is recognized as an important cause of cross-reactivity in HPV serology. Heparin-coated microtitre plates were evaluated for capturing conformationally correct VLPs and improving the type specificity of HPV serology. Hybrid VLPs between HPV16 and HPV11, which had been found to have significant reactivity with children's sera and a batch of HPV18 VLPs that had failed the quality control becaus…

&lt;p&gt;Cuprous oxide nanoparticles reduces hypertrophic scarring by inducing fibroblast apoptosis&lt;/p&gt;

Background Less apoptosis and excessive growth of fibroblasts contribute to the progression of hypertrophic scar formation. Cuprous oxide nanoparticles (CONPs) could have not only inhibited tumor by inducing apoptosis and inhibiting proliferation of tumor cells, but also promoted wound healing. The objective of this study was to further explore the therapeutic effects of CONPs on hypertrophic scar formation in vivo and in vitro. Methods In vivo, a rabbit ear scar model was established on New Zealand albino rabbits. Six full-thickness and circular wounds (10 mm diameter) were made to each ear. Following complete re-epithelization observed on postoperative day 14, an intralesional injection o…

Inducible ASABF-Type Antimicrobial Peptide from the Sponge Suberites domuncula: Microbicidal and Hemolytic Activity in Vitro and Toxic Effect on Molluscs in Vivo

Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demospongeSuberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSαβ structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs ; it is distantly related to the defensins. The recom…

Molecular Biomineralization: Toward an Understanding of the Biogenic Origin of Polymetallic Nodules, Seamount Crusts, and Hydrothermal Vents

Polymetallic nodules and crusts, hydrothermal vents from the Deep Sea are economically interesting, since they contain alloying components, e.g., manganese or cobalt, that are used in the production of special steels; in addition, they contain rare metals applied for plasma screens, for magnets in hard disks, or in hybrid car motors. While hydrothermal vents can regenerate in weeks, polymetallic nodules and seamount crusts grow slowly. Even though the geochemical basis for the growth of the nodules and crusts has been well studied, the contribution of microorganisms to the formation of these minerals remained obscure. Recent HR-SEM (high-resolution scanning electron microscopy) analyses of …

The Marine Sponge-Derived Inorganic Polymers, Biosilica and Polyphosphate, as Morphogenetically Active Matrices/Scaffolds for the Differentiation of Human Multipotent Stromal Cells: Potential Application in 3D Printing and Distraction Osteogenesis

The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to &gt;100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by …

Restoration of Impaired Metabolic Energy Balance (ATP Pool) and Tube Formation Potential of Endothelial Cells under “high glucose”, Diabetic Conditions by the Bioinorganic Polymer Polyphosphate

Micro-vascularization is a fast, energy-dependent process that is compromised by elevated glucose concentrations such as in diabetes mellitus disease. Here, we studied the effect of the physiological bioinorganic polymer, polyphosphate (polyP), on the reduced ATP content and impaired function of endothelial cells cultivated under "high glucose" (35 mM diabetes mellitus conditions) concentrations. This high-energy biopolymer has been shown to provide a source of metabolic energy, stored in its phosphoanhydride bonds. We show that exposure of human umbilical vein endothelial cells (HUVEC cells) to "high glucose" levels results in reduced cell viability, increased apoptotic cell death, and a d…

Heparan sulfate proteoglycans interact exclusively with conformationally intact HPV L1 assemblies: basis for a virus-like particle ELISA.

In this article, we demonstrate that interaction of human papillomavirus-like particles (HPV-VLPs) with the putative glucosaminoglycan binding receptor is strictly dependent on conformational integrity. Such conformations are present on VLPs and capsomeres but not on monomers of the major capsid protein, L1, confirming reports that capsomeres can induce virus-neutralizing antibodies. Furthermore, we show the suitability of this specific interaction for development of VLP-based enzyme-linked immunosorbent assays (ELISAs), using heparin for indirect coupling of VLPs to microtiter plates, which may add an intrinsic quality control. This avoids presentation of linear, often highly cross-reactiv…

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 r…

A physiologically active interpenetrating collagen network that supports growth and migration of epidermal keratinocytes: zinc-polyP nanoparticles integrated into compressed collagen.

The distinguished property of the physiological polymer, inorganic polyphosphate (polyP), is to act as a bio-intelligent material which releases stimulus-dependent metabolic energy to accelerate wound healing. This characteristic is based on the bio-imitating feature of polyP to be converted, upon exposure to peptide-containing body fluids, from stable amorphous nanoparticles to a physiologically active and energy-delivering coacervate phase. This property of polyP has been utilized to fabricate a wound mat consisting of compressed collagen supplemented with amorphous polyP particles, formed from the inorganic polyanion with an over-stoichiometric ratio of zinc ions. The proliferation and t…

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…

Silicatein: from chemical through enzymatic silica formation, to synthesis of biomimetic nanomaterials

Silicateins are the enzymes that had been identified in sponges, then sequenced and expressed. They are not only the enzymes facilitating biosilica synthesis but also function as structure-guiding and structure-forming proteins. The three minireviews highlight the principles of silicatein-mediated biosilica formation and outline the bionic strategies which might be used for the design and fabrication of novel materials.

Electrospinning of Bioactive Wound-Healing Nets

The availability of appropriate dressings for treatment of wounds, in particular chronic wounds, is a task that still awaits better solutions than provided by currently applied materials. The method of electrospinning enables the fabrication of novel materials for wound dressings due to the high surface area and porosity of the electrospun meshes and the possibility to include bioactive ingredients. Recent results show that the incorporation of biologically active inorganic polyphosphate microparticles and microspheres and synergistically acting retinoids into electrospun polymer fibers yields biocompatible and antibacterial mats for potential dressings with improved wound-healing propertie…

Molecular Evolution of Defense Pathways in Sponges: Self–Self-recognition and Fight against the Nonself

This article provides an overview of the immune system in poriferans at a tissue, cellular, and molecular level. Despite their simple organization, sponges have developed molecular mechanisms to recognize and resist foreign organisms. They are able to distinguish among food organisms, pathogens, and sponge-associated organisms. Although they lack specialized immune cells, sponges display molecular precursors, which are similar to molecular mediators involved in innate and adaptive immune systems, present in more evolutionarily advanced taxa, as outlined in succeeding articles in the Phylogeny section.

The unique skeleton of siliceous sponges (Porifera; Hexactinellida and Demospongiae) that evolved first from the Urmetazoa during the Proterozoic: a review

Abstract. Sponges (phylum Porifera) had been considered as an enigmatic phylum, prior to the analysis of their genetic repertoire/tool kit. Already with the isolation of the first adhesion molecule, galectin, it became clear that the sequences of the sponge cell surface receptors and those of the molecules forming the intracellular signal transduction pathways, triggered by them, share high similarity to those identified in other metazoan phyla. These studies demonstrated that all metazoan phyla, including the Porifera, originate from one common ancestor, the Urmetazoa. The sponges evolved during a time prior to the Ediacaran-Cambrian boundary (542 million years ago (myr)). They appeared du…

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 roughn…

Biofabrication of biosilica-glass by living organisms

Biosilicification is an evolutionarily old and widespread type of biomineralization both in unicellular and multicellular organisms, including sponges, diatoms, radiolarians, choanoflagellates, and higher plants. In the last few years combined efforts in molecular biology, cell biology, and inorganic and analytical chemistry have allowed the first insight into the molecular mechanisms by which these organisms form an astonishing variety of siliceous structures that cannot be achieved by chemical methods. Here we report about the present stage of knowledge on structure, biochemical composition, and mechanisms of biosilica formation, focusing our attention particularly on sponges because of t…

New biomaterials: Biofabrication of biosilica-glass by sponges

Influence of Altered Microbes on Soil Organic Carbon Availability in Karst Agricultural Soils Contaminated by Pb-Zn Tailings.

Soil organic carbon (SOC) availability is determined via a complex bio-mediated process, and Pb-Zn tailings are toxic to the soil microbes that are involved in this process. Here, Pb-Zn-tailings- contaminated karst soils with different levels (paddy field &gt; corn field &gt; citrus field &gt; control group) were collected to explore the intrinsic relationship between Pb-Zn tailings and microbes due to the limited microbial abundance in these soils. The SOC concentration in the paddy fields is the highest. However, based on the soil microbial diversity and sole-carbon-source utilization profiles, the rate of SOC availability, McIntosh index, Shannon-Wiener diversity index, Simpson’s diversi…

The therapeutic potential of inorganic polyphosphate: A versatile physiological polymer to control coronavirus disease (COVID-19).

Rationale: The pandemic caused by the novel coronavirus SARS-CoV-2 is advancing rapidly. In particular, the number of severe courses of the disease is still dramatically high. An efficient drug therapy that helps to improve significantly the fatal combination of damages in the airway epithelia, in the extensive pulmonary microvascularization and finally multiorgan failure, is missing. The physiological, inorganic polymer, polyphosphate (polyP) is a molecule which could prevent the initial phase of the virus life cycle, the attachment of the virus to the target cells, and improve the epithelial integrity as well as the mucus barrier. Results: Surprisingly, polyP matches perfectly with the ca…

Transformation of Construction Cement to a Self-Healing Hybrid Binder

A new biomimetic strategy to im prove the self-healing properties of Portland cement is presented that is based on the application of the biogenic inorganic polymer polyphosphate (polyP), which is used as a cement admixture. The data show that synthetic linear polyp, with an average chain length of 40, as well as natural long-chain polyP isolated from soil bacteria, has the ability to support self-healing of this construction material. Furthermore, polyP, used as a water-soluble Na-salt, is subject to Na+/Ca2+ exchange by the Ca2+ from the cement, resulting in the formation of a water-rich coacervate when added to the cement surface, especially to the surface of bacteria-containing cement/c…

ChemInform Abstract: Silicateins - A Novel Paradigm in Bioinorganic Chemistry: Enzymatic Synthesis of Inorganic Polymeric Silica

Amorphous Polyphosphate and Ca‐Carbonate Nanoparticles Improve the Self‐Healing Properties of both Technical and Medical Cements

Cement is used both as a construction material and for medical applications. Previously, it has been shown that the physiological polymer inorganic polyphosphate (polyP) is morphogenetically active in regeneration of skin, bone, and cartilage. The present study investigates the question if this polymer is also a suitable additive to improve the self-healing capacity not only of construction cement but also of inorganic bone void fillers. For the application in the cement, two different polyP-based amorphous nanoparticles (NP) are prepared, amorphous Ca-polyP NP and amorphous Ca-carbonate (ACC) NP. The particles are integrated into poly(methyl methacrylate) in a concentration ratio of 1:10. …

Biomimetic routes to micro/nanofabrication

Abstract The discovery of inorganic polyphosphate (polyP), an evolutionarily ancient biopolymer, and of its function in energy storage/supply and induction of cell differentiation through specific gene expression (“morphogenetic activity”) has opened new opportunities in regenerative medicine, including therapy of bone, cartilage, and vascular diseases. Inspired by nature, various procedures have been developed for the preparation of amorphous nano and microparticles of polyP with different counterions, which mimic the physiological polyP found in blood platelets/acidocalcisomes, as well as for the preparation of morphogenetically active polyP coacervate complexes. This chapter summarizes t…

Manganese/polymetallic nodules: Micro-structural characterization of exolithobiontic- and endolithobiontic microbial biofilms by scanning electron microscopy

Polymetallic/ferromanganese nodules (Mn-nodules) provide a rich source for manganese. It is not yet known if the nodules have a biogenic or an abiogenic origin. Here we applied the technique of high-resolution scanning electron microscopy, in combination with energy dispersive X-ray spectroscopical (EDX) analysis, to trace the existence of microbial biofilms. Two spatially separated assemblies exist, the exolithobiontic- and endolithobiontic colonizations. The exolithobiontic colonization is seen in the micro-canals, which traverse the outer surface layer of the nodules and are formed by elongated filamentous organisms, which show no signs of mineralization. In the center of the nodules thr…

Evidence for a biogenic, microorganismal origin of rock varnish from the Gangdese Belt of Tibet

In the present study we examined material from the Ashikule Basin of Tibet. Chemical analyses were performed by use of energy dispersive X-ray spectroscopy and electron probe microanalysis to clarify whether the varnish layers that had developed on the surface of the rhyolite are indeed composed of varnish bodies and silica glaze. Electron microscopic analyses revealed that the surface of the varnish is covered both by filamentous hyphae bacterial and cocci-shaped forms. Within the varnish mineral layer in those samples two forms of bacteria-like microorganisms exist; cocci as tightly packed bacterial aggregates [within varnish bodies], and bacillus-like microorganisms [within the varnish m…

Biosilica aging: From enzyme-driven gelation via syneresis to chemical/biochemical hardening

Abstract Background The distinguished property of the siliceous sponge spicules is their enzyme (silicatein)-catalyzed biosilica formation. The enzymatically formed, non-structured biosilica product undergoes a molding, syneresis, and hardening process to form the species-specifically shaped, hard structured skeletal spicules. Besides of silicatein, a silicatein-associated protein, silintaphin-2, is assumed to be involved in the process of biosilica formation in vivo. Methods Biosilica has been synthesized enzymatically and determined quantitatively. In addition, the subsequent hardening/aging steps have been followed by spectroscopic and electron microscopic analyses. Results The young spi…

Cryptochrome in Sponges: A Key Molecule Linking Photoreception with Phototransduction

Sponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now…