Cell migration, DNA fragmentation and antibacterial properties of novel silver doped calcium polyphosphate nanoparticles.

To combat infections, silver was used extensively in biomedical field but there was a need for a capping agent to eliminate its cytotoxic effects. In this study, polymeric calcium polyphosphate was doped by silver with three concentrations 1, 3 or 5 mol.% and were characterized by TEM, XRD, FTIR, TGA. Moreover, cytotoxicity, antibacterial, cell migration and DNA fragmentation assays were done to assure its safety. The results showed that the increase in silver percentage caused an increase in particle size. XRD showed the silver peaks, which indicated that it is present in its metallic form. The TGA showed that thermal stability was increased by increasing silver content. The antibacterial tests showed that the prepared nanoparticles have an antibacterial activity against tested pathogens. In addition, the cytotoxicity results showed that the samples exhibited non-cytotoxic behavior even with the highest doping concentration (5% Ag-CaPp). The cell migration assay showed that the increase in the silver concentration enhances cell migration up to 3% Ag-CaPp. The DNA fragmentation test revealed that all the prepared nanoparticles caused no fragmentation. From the results we can deduce that 3% Ag-CaPp was the optimum silver doped calcium polyphosphate concentration that could be used safely for medical applications.

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 cationic groove on the RBD. Subsequent binding studies disclosed that polyP, with a physiological chain length of 40 phosphate residues, abolishes the binding propensity of the RBD to the ACE2 receptor. In addition to this first mode of action of polyP, this polymer causes in epithelial cells an increased gene expression of the major mucins in the airways, of MUC5AC and MUC1, as well as a subsequent glycoprotein production. MUC5AC forms a gel-like mucus layer trapping inhaled particles which are then transported out of the airways, while MUC1 constitutes the periciliary liquid layer and supports ciliary beating. As a third mode of action, polyP undergoes enzymatic hydrolysis of the anhydride bonds in the airway system by alkaline phosphatase, releasing metabolic energy. Conclusions: This review summarizes the state of the art of the biotherapeutic potential of the polymer polyP and the findings from basic research and outlines future biomedical applications.

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 form, at neutral pH, a coacervate-the physiologically active form of the polymer. At alkaline conditions (pH 10), it will form nanoparticles, acting as a depot that is converted at pH 7 into the coacervate phase. Both polyP-containing hydrogels were biologically active and significantly enhanced cell growth/viability and attachment/spreading of human epidermal keratinocytes compared to control hydrogels without any adverse effect on reconstructed human epidermis samples in an in vitro skin irritation test system. From these data, we conclude that polyP-containing alginate/oxidized-alginate-gelatin hydrogels may provide a suitable regeneratively active matrix for wound healing for potential in vivo applications.

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 the migration of human skin keratinocytes in those matrices were investigated. If the cells were embedded into the mat they respond with a significantly higher motility when zinc-polyP particles are present. Interestingly, only keratinocytes that were grown in a polyP environment developed well-structured microvilli, reflecting an increased biological activity. The data show that Zn-polyP particles incorporated into wound mats are a potent cell growth and cell migration-stimulating inorganic bio-material.

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-polyP. Embedding these particles in the hydrated cement resulted in the formation of calcite crystals after a hardening time of 10 days, which were not seen in controls, indicating that the particles dissolve and then transform into calcite. While there was no significant repair in the controls without ACC, almost complete closure of the cracks was observed after a 10 days healing period in the ACC-supplemented samples. Nanoindentation measurements on the self-healed crack surfaces showed a similar or slightly higher elasticity at a lower hardness compared to non-cracked surfaces. Our results demonstrate that bioinspired approaches, like the use of polyP-stabilized ACC shown here, can significantly improve the repair capacity of Portland cement.

Biomimetic transformation of polyphosphate microparticles during restoration of damaged teeth.

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 induce re-mineralization in the enamel and dentin regions. The formation of a regeneration mineralic zone on the tooth surface induced by aCa-polyP-MP was enhanced upon exposure to artificial saliva, as demonstrated by SRXTM. Energy dispersive X-ray analysis revealed an increase in the calcium/phosphorus atomic ratio of the enamel deposits to values characteristic for the particles during the treatment with polyP applied in the toothpaste, indicating a fusion of the particles with the tooth mineral. SIGNIFICANCE: Our results suggest that toothpaste enriched with aCa-polyP-MP is a promising biomimetic material for accelerating enamel and dentin restoration.

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 ortho-phosphate used for bone hydroxyapatite formation. Here we show that the flavone-loaded Ca-polyP microparticles are readily taken up by phOSB, resulting in the accumulation of polyP around the nuclei and the formation of intracellular vesicles containing the ALP. In addition, we demonstrate that baicalin/baicalein causes a rise of the intracellular calcium [Ca[Formula: see text]]i a level which markedly is augmented after encapsulation into Ca-polyP, through activation of the phospholipase C. Moreover, both flavones, either alone or associated with Ca-polyP microparticles, upregulate the expression of the osteoblast calcium efflux channel, the plasma membrane Ca[Formula: see text]-ATPase (PMCA), while the expression of ALP, which promotes bone mineralization, is induced by Ca-polyP and by the flavones only if present in the Ca-polyP-microparticle-associated form. As a result, the extent of bone mineralization is markedly enhanced. Based on the two-armed activating function, new applications of baicalin/baicalein as a component of nutriceuticals for osteoporosis prevention or bone implants can be envisaged.

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 formation, open novel not yet exploited targets for pharmacological intervention of human bone diseases like osteoporosis, using compounds that act as activators of these enzymes. This chapter focuses on carbonic anhydrases of biomedical interest and the search for potential activators of these enzymes, was well as the interplay between carbonic anhydrase-mediated calcium carbonate bioseed synthesis and metabolism of energy-rich inorganic polyphosphates. Beyond that, the combination of the two metabolic products, calcium carbonate and calcium-polyphosphate, if applied in an amorphous form, turned out to provide the basis for a new generation of scaffold materials for bone tissue engineering and repair that are, for the first time, morphogenetically active.

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 differentiation, collagen type I and alkaline phosphatase. In addition, polyP, applied as Zn-polyP microparticles ("Zn-a-polyP-MP"), showed a distinct inhibitory effect on growth of Streptococcus mutans, in contrast to a toothpaste containing the broad-spectrum antibiotic triclosan that only marginally inhibits this cariogenic bacterium. Moreover, we demonstrate that the "a-polyP/RA-MP"-containing toothpaste efficiently repairs cracks/fissures in the enamel and dental regions and reseals dentinal tubules, already after a five-day treatment (brushing) of teeth as examined by SEM (scanning electron microscopy) and semi-quantitative EDX (energy-dispersive X-ray spectroscopy). The occlusion of the dentin cracks by the microparticles turned out to be stable and resistant against short-time high power sonication. Our results demonstrate that the novel toothpaste prepared here, containing amorphous polyP microparticles enriched with retinyl acetate, is particularly suitable for prevention/repair of (cariogenic) damages of tooth enamel/dentin and for treatment of dental hypersensitivity. While the polyP microparticles function as a sealant for dentinal damages and inducer of remineralization processes, the retinyl acetate acts as a regenerative stimulus for collagen gene expression in cells of the surrounding tissue, the periodontium.

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

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

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-free polyP complexed with Ca2+ or amorphous Ca-polyP NPs and retinol alone. Incubation of the cells in the presence of the retinol/aCa-polyP NSs also caused a significant increase of the expression levels of the genes encoding for the fatty acid binding protein 4 (FABP4), as well as of the genes encoding for leptin and the leptin receptor. In contrast, the single components, soluble Na-polyP, complexed to Ca2+, or retinol-free aCa-polyP NPs, and retinol, had no significant effect on the expression of these genes. CONCLUSIONS: These results indicate that the PLA fibers, supplemented with aCa-polyP-NP or retinol/aCa-polyP-NS, elicit morphogenetic activity, suggesting that these fiber mats, along with the antibacterial effect of polyP, have a beneficial potential as wound dressings combining antimicrobial and regenerative (wound healing) properties. GENERAL SIGNIFICANCE: The PLA-based fiber mats, containing retinol and polyP nanoparticles, provide promising bioactive meshes that are urgently needed as dressings for chronic wounds.

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 • Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP • Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP • Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.

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 ß-tricalcium phosphate [ß-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% ß-TCP, ≈9% silica, and 0.32µg/mg of silicatein were entrapped into the PLGA microspheres (diameter≈800µm). Determination of the biocompatibility of the ß-TCP microspheres, supplemented with silica or silicatein, revealed no toxicity in the MTT based cell viability assay using SaOS-2 cells. The adherence of SaOS-2 cells to the surface of silica-containing microspheres was higher than for microspheres, containing only ß-TCP. In addition, the silica-containing ß-TCP microspheres and even more pronounced, a 1:1 mixture of microspheres containing ß-TCP and silica, and ß-TCP and silicatein, were found to strongly enhance the mineral deposition by SaOS-2 cells. Using these microspheres, first animal experiments with silica/biosilica were performed in female, adult New Zealand White rabbits to study the effect of the inorganic polymer on bone regeneration in vivo. The microspheres were implanted into 5mm thick holes, drilled into the femur of the animals, applying a bilateral comparison study design (3 test groups with 4-8 animals each). The control implant on one of the two hind legs contained microspheres with only ß-TCP, while the test implant on the corresponding leg consisted either of microspheres containing ß-TCP and silica, or a 1:1 mixture of microspheres, supplemented with ß-TCP and silica, and ß-TCP and silicatein. The results revealed that tissue/bone sections of silica containing implants and implants, composed of a 1:1 mixture of silica-containing microspheres and silicatein-containing microspheres, show an enhanced regeneration of bone tissue around the microspheres, compared to the control implants containing only ß-TCP. The formation of new bone induced by the microspheres is also evident from measurements of the stiffness/reduced Young's modulus of the regenerated bone tissue. The reduced Young's modulus of the regenerating bone tissue around the implants was markedly higher for the silica-containing microspheres (1.1MPa), and even more for the 1:1 mixture of the silica- and silicatein-containing microspheres (1.4MPa), compared to the ß-TCP microsphere controls (0.4MPa). We propose that based on their morphogenetic activity on bone-forming cells in vitro and the results of the animal experiments presented here, silica/biosilica-based scaffolds are promising materials for bone repair/regeneration.

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 >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 exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca²âº salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also morphogenetic potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are morphogenetically active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.

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 ß-glycerophosphate, ascorbic acid and dexamethasone. The degree of hydroxyapatite synthesis, determined by staining the cells with the OsteoImage dye, strongly increased after exposure of the cells to silica. In a previous study we reported that ortho-silicate induces the expression of the gene encoding BMP-2 [bone morphogenetic protein-2]. Now we asked the question whether, in the presence of the mineralization activation cocktail, silica induces differentially the fibrillar proteins type I collagen [COLI] and type V collagen [COLV], as well as the non-collagenous proteins alkaline phosphatase [ALP], osteopontin [OPN], osteonectin [ON], osteocalcin [OC], and bone sialoprotein II [BSP]. Those expression values were correlated with the transcript levels of RUNX2 [Runt-related transcription factor 2]. The data show that the steady-state transcript level of RUNX2 remained unchanged in the presence of silica, while this inorganic polymer caused an elevated BMP-2 transcript level, and simultaneously also a significant upregulation of the COLI, COLV, OPN and ON genes. In contrast, the level of expression of OC and BSP remained unchanged in the presence of silica. It is concluded that silica causes its morphogenetic effect with respect to some bone-specific genes, COLI, COLV, OPN and ON, in a RUNX2-independent way.

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, as well as a post-processing, i.e., surface modification/functionalization to enhance the biocompatibility of the scaffold or to bind some bioactive component. A solution might be provided by the introduction of novel inorganic biopolymers, biosilica and polyphosphate, which resist harsh conditions applied in the RP chain and are morphogenetically active and do not need supplementation by growth factors/cytokines to stimulate the growth and the differentiation of bone-forming cells.

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 oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2)) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+)-depletion condition (1 mM CaCl(2)). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast).

Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea).

The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules) are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract.

The largest Bio-Silica Structure on Earth: The Giant Basal Spicule from the Deep-Sea Glass Sponge Monorhaphis chuni.

The depth of the ocean is plentifully populated with a highly diverse fauna and flora, from where the Challenger expedition (1873-1876) treasured up a rich collection of vitreous sponges [Hexactinellida]. They have 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. Later, during the German Deep Sea Expedition "Valdivia" (1898-1899), Schulze could describe the largest siliceous hexactinellid sponge on Earth, the up to 3 m high Monorhaphis chuni, which develops the equally largest bio-silica structures, the giant basal spicules (3 m × 10 mm). With such spicules as a model, basic knowledge on the morphology, formation, and development of the skeletal elements could be elaborated. Spicules are formed by a proteinaceous scaffold which mediates the formation of siliceous lamellae in which the proteins are encased. Up to eight hundred 5 to 10 µm thick lamellae can be concentrically arranged around an axial canal. The silica matrix is composed of almost pure silicon and oxygen, providing it with unusual optophysical properties that are superior to those of man-made waveguides. Experiments indicated that the spicules function in vivo as a nonocular photoreception system. In addition, the spicules have exceptional mechanical properties, combining mechanical stability with strength and stiffness. Like demosponges the hexactinellids synthesize their silica enzymatically, via the enzyme silicatein. All these basic insights will surely contribute also to a further applied utilization and exploration of bio-silica in material/medical science.

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 trace element silicon in human bone formation has attracted major scientific interest only rather recently. The interest in silicon has been further increased by the latest discoveries in the field of biosilicification, the formation of the inorganic silica skeleton of the oldest still extant animals on Earth, the sponges, which revealed new insights in the biological function of this element. Sponges make use of silicon to build up their inorganic skeleton which consists of biogenously formed polymeric silica (biosilica). The formation of biosilica is mediated by specific enzymes, silicateins, which have been isolated, characterized, and expressed in a recombinant way. Epidemiological studies revealed that dietary silicon reduces the risk of osteoporosis and other bone diseases. Recent results allowed for the first time to understand the molecular mechanism underlying the protective effect of silicic acid/biosilica against osteoporosis. Biosilica was shown to modulate the ratio of expression of two cytokines involved in bone formation-RANKL and osteoprotegerin. Hence, biosilica has been proposed to have a potential in prophylaxis and therapy of osteoporosis and related bone diseases.