The invention relates to the application of a combination of at least one inorganic polyphosphate (polyP) and/or a salt of said polyP and calcium [polyP (Ca2+ salt)] and bicarbonate/calcium-carbonate, optionally together with at least one carbonic anhydrase activatora carbonic anhydrase (CA) activator or a material comprising these combinations for the treatment of bone defects, such as, for example, for a prophylaxis and treatment of osteoporosis and other bone diseases. The combinations, e.g. of a CA activator and polyP or polyP (Ca ²⁺ complex) can be used both as a drug or food supplement, and as a material to be injected into bone tissue.

Background of Invention

There is an increasing need for new materials and pharmaceuticals for the treatment of bone fractures. The number of bone injuries increases worldwide, both as a result of traffic accidents and as a result of the aging population. For example, the number of fractures caused by osteoporosis has doubled in the last decade. As a consequence of the reduced density of bone mineral in osteoporosis, the risk of bone fractures increases. Hip fractures and vertebral fractures often occur in osteoporotic patients. The costs caused by osteoporosis and other bone diseases, as well as the costs for treatment of bone defects, are tremendous and will continue to increase in future worldwide.

Bone formation

Bone consists of an organic matrix and a mineral phase. The dominant component of the inorganic phase is calcium phosphates, more specifically calcium hydroxyapatite [Ca ₅ (P0 ₄ )3(OH); abbreviation: HA]. In addition, the bone mineral contains carbonate. In the apatite crystal lattices the carbonate exists as C0 ₃ ^2~ ions that substitute for the P0 ₄ ^3~ ions and/or the OH ^" ions (for a review, see: Posner AS (1969) Crystal chemistry of bone mineral. Physiol Rev 49:760-792).

The formation of bone tissue depends on the functional interaction of HA-forming osteoblasts and HA-dissolving osteoclasts. The maturation of osteoblasts and osteoclasts from their precursor cells and the cross talk between these cells are controlled by a series of cytokines, among them bone morphogenetic protein 2 (BMP-2), receptor activator of the NF-kB ligand (RANKL) and osteoprotegerin (OPG). RANKL stimulates osteoclast precursor cells to differentiate into mature osteoclasts, while the maturation of osteoblasts from their precursor cells is promoted by BMP-2. The stimulatory effect of RANKL is abolished by OPG.

Several mechanisms have been proposed to explain early bone mineral formation. It is assumed that the early mineralization process starts intracellularly, in matrix vesicles, under formation of poorly crystalline carbonated apatite deposits (Boonrungsiman S, et al. (2012) The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc Natl Acad Sci USA 109: 14170-14175). The crystallites then migrate to the extracellular space. Evidence has been presented showing that the intracellularly formed amorphous Ca- phosphate particles are formed from polyphosphate (polyP) after hydro lytic degradation of this polymer (Omelon S, et al. (2009) Control of vertebrate skeletal mineralization by polyphosphates. PLoS One 4:e5634).

Recent results revealed that CaC0 ₃ deposits function as bio-seeds for Ca-phosphate precipitation onto bone forming cells (Miiller WEG, Schroder HC, Schlossmacher U, Grebenjuk VA, Ushijima H and Wang XH (2013) Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation. Biomaterials 34:8671-8680).

Furthermore, it was found that carbonic anhydrases are involved in CaC0 ₃ deposition in vitro (Miiller WEG, Schroder HC, Schlossmacher U, Neufurth M, Geurtsen W, Korzhev M and Wang XH (2013) The enzyme carbonic anhydrase as an integral component of biogenic Ca- carbonate formation in sponge spicules. FEBS Open Bio 3:357-362) and likely also in vivo (Miiller WEG, Schlossmacher U, Schroder HC, Lieberwirth I, Glasser G, Korzhev M, Neufurth M and Wang XH (2013) Enzyme-accelerated and structure-guided crystallization of Ca-carbonate: role of the carbonic anhydrase in the homologous system. Acta Biomater 10.1016/j.actbio.2013.08.025).

Carbonic anhydrases (CAs) have been identified not only in bone-forming osteoblasts, but also in bone-resorbing osteoclasts. The CAs catalyze the rapid interconversion of carbon dioxide and water to bicarbonate (HC0 ₃ ) and H ⁺ . The most abundant isoforms of the mammalian CAs are the cytosolic CA II (-75%) and the cytosolic CA I (-20%). One considerable fraction of the CAs is associated with the cell membrane.

At present, only little information is available about the potential therapeutic effect of CA inhibitors on bone anabolism (for a review see: Supuran CT, Scozzafava A (2000) Carbonic anhydrase inhibitors and their therapeutic potential. Expert Opinion on Therapeutic Patents 10: 575-600).

Only a few CA activators have been identified, but none of them had been tested for their potential in the treatment of bone disorders (for a review see: Pastorekova S, Parkkila S, Pastorek J, Supuran CT (2004) Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem 19: 199-229; Supuran CT (2008) Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 7: 168-181 ; and Supuran CT (2008) Carbonic anhydrases - an overview. Curr Pharm Des 14:603-614).

Scozzafava and Supuran (in: Scozzafava A, Supuran CT. Carbonic anhydrase activators. Part 24. High affinity isozymes I, II and IV activators, derivatives of 4-(4- chlorophenylsulfonylureido-amino acyl)ethyl-lH-imidazole. Eur J Pharm Sci. 2000 Mar; 10(1):29-41) describe a series of compounds with the general formula cpu-AA-Hst (cpu, 4- C1C(6)H(4)S0(2)NHC0) as efficient activators of three CA isozymes. Best activity was detected against hCA I and bCA IV, for which some of the new compounds showed affinities in the 1-10 nM range (h, human; b, bovine isozymes). hCA II, on the other hand, was less prone to activation by the new derivatives, which possessed affinities around 20-50 nM for this isozyme.

Another enzyme, the bone-specific alkaline phosphatase (ALP), plays a prominent role in bone phosphate metabolism. This enzyme (i) generates the inorganic phosphate (Pi) which is required for HA formation and (ii) hydrolyzes inorganic pyrophosphate (PPi) which acts as a mineralization inhibitor. The bone ALP is one potential target of bisphosphonates which are used in treatment of osteoporosis and act as inhibitors of that enzyme.

Polyphosphates

Inorganic polyphosphates (PolyP) are nontoxic multifunctional molecules, consisting of linear polymers of tens to hundreds of phosphate units which are linked together via high-energy phosphoanhydride bonds.

Various functions of polyP have been proposed. PolyP has been proposed to act, among others, as a storage substance of energy, as chelator for metal cations, as donor for sugar and adenylate kinase, and as an inducer of apoptosis. The function of polyP in bone mineralization has been proposed by the inventors and confirmed by other groups more than 15 year ago.

PolyP is synthesized from ATP by polyP kinases. The degradation of polyP is catalyzed by several endo- and exopolyphosphatases (for a review, see: Schroder HC, Miiller WEG (eds) (1999) Inorganic Polyphosphates. Biochemistry. Biology. Biotechnology. Vol 23, Springer Verlag, Berlin Heidelberg; Kulaev IS, Vagabov VM, Kulakovskaya TV (2004) The Biochemistry of Inorganic Polyphosphates. Chichester, England: John Wiley & Sons Ltd, pp 1-277; Rao N, Gomez-Garcia MR, Kornberg A (2009) Inorganic polyphosphate: Essential for growth and survival. Annu Rev Biochem 78:605-647; Akiyama M, Crooke E, Kornberg A (1992) The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein. J Biol Chem 267:22556-22561; Lorenz B, Schroder HC (2001) Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 1547:254-261; Lorenz B, Miiller WEG, Kulaev IS, Schroder HC (1994) Purification and characterization of an exopolyphosphatase activity from Saccharomyces cerevisiae. J Biol Chem 269:22198-22204; and Lorenz B, Munkner J, Oliveira MP, Kuusksalu A, Leitao JM, Miiller WEG, Schroder HC (1997) Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis. Biochim Biophys Acta 1335:51-60.

Several methods for the analysis of polyP, including quantification and determination of chain length, have been developed that facilitate the experimental investigation of this polymer and its (enzymatic) formation/degradation (for a review, see: Lorenz B, Schroder HC (1999) Methods for investigation of inorganic polyphosphates and polyphosphate-metabolizing enzymes. In: Inorganic Polyphosphates - Biochemistry, Biology, Biotechnology (Schroder HC, Miiller WEG, eds). Prog Mol Subcell Biol 23:217-239 ; and Lorenz B, Munkner J, Oliveira MP, Leitao JM, Miiller WEG, Schroder HC (1997) A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2. Anal Biochem 246: 176-184).

PolyP in bone

Previous studies have revealed that polyP molecules of different chain lengths accumulate especially in bone cells. In addition, human osteob last-like cells contain enzymes that hydrolyze polyP. Based on these results, further studies focused on the effect of polyP on ALP present in bone forming cells. It has been proposed that polyP, after hydrolysis to Pi by phosphatases, is involved in the control of mineralization processes during vertebrate skeleton formation. The degradation of polyP by various exo- and endopolyphosphatases as well as ALP has been demonstrated. The release of Ca ²⁺ during the enzymatic hydrolysis of polyP has been proposed to be utilized as a Ca ²⁺ source for HA formation (for a review, see: Schroder HC, Kurz L, Miiller WEG, Lorenz B (2000) Polyphosphate in bone. Biochemistry (Moscow) 65:296-303; Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Miiller WEG, Schroder HC (1998) Inorganic polyphosphate in human osteoblast-like cells. J Bone Mineral Res 13:803-812; Kornberg A (1999) Inorganic polyphosphate: a molecule of many functions. In: Schroder HC, Miiller WEG (eds) Inorganic polyphosphates: biochemistry, biology, biotechnology. Prog Molec Subcell Biol 23. SpringenBerlin, pp 1-26; Omelon S, et al. (2009) Control of vertebrate skeletal mineralization by polyphosphates. PLoS ONE 4:e5634; Omelon SJ, Grynpas MD (2008) Relationships between polyphosphate chemistry, biochemistry and apatite biomineralization. Chem Rev 108:4694-4715; and Schroder HC, Lorenz B, Kurz L, Miiller WEG (1999) Inorganic polyP in eukaryotes: enzymes, metabolism and function. In: Schroder HC, Miiller WEG, eds, Inorganic Polyphosphates - Biochemistry, Biology, Biotechnology. Prog Mol Subcell Biol 23 :45-81).

The present state of knowledge regarding the effect of polyP on bone-forming and bone- resorbing cells can be summarized as follows.

PolyP, as Ca ²⁺ salt, is a strong inducer of HA formation in SaOS-2 cells (Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, SchloBmacher U, Wiens M, Miiller WEG (2012a) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Eng Regen Med, in press, doi: 10.1002/term.l465).

PolyP, as Ca ²⁺ salt, causes an induction of the expression of the BMP-2 gene in SaOS-2 cells (Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, SchloBmacher U, Wiens M, Miiller WEG (2012) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Eng Regen Med; doi: 10.1002/term. l465).

PolyP, as Ca ²⁺ salt, impairs osteoclastogenesis; it inhibits the progression of RAW 264.7 cells into functional osteoclasts (Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, SchloBmacher U, Wiens M, Miiller WEG (2012) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Eng Regen Med; doi: 10.1002/term. l465).

PolyP, as Ca ²⁺ salt, inhibits the phosphorylation/activation of ΙκΒα kinase in osteoclast-like RAW 264.7 cells, which mediates the activation of NF-κΒ during RANKL-induced differentiation of (pre)osteoclasts (Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, SchloBmacher U, Wiens M, Miiller WEG (2012) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Eng Regen Med; doi: 10.1002/term. l465).

PolyP, as Ca ²⁺ salt, is a potent activator of the ALP and also induces the expression of the gene encoding the bone-specific, the tissue-nonspecific ALP (TNAP) (Miiller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca ²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

PolyP, as Ca ²⁺ salt, causes an increase in the intracellular Ca ²⁺ level of SaOS-2 cells (Miiller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca ²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). PolyP is deposited in vesicles as insoluble Ca ²⁺ -polyP salt.

Thus, polyP as a Ca ²⁺ salt, displays a dual effect on bone-forming osteoblasts and bone- resorbing osteoclasts: (i) it promotes - through upregulation of BMP-2 expression - the maturation of osteoblasts, resulting in an enhanced mineralization (HA deposition); (ii) it inhibits the differentiation of osteoclast precursors to functionally active osteoclasts through interfering with the NF-κΒ signaling pathway. In addition, polyP (Ca ²⁺ salt) stimulates bone ALP.

The following patent publications are related to the analysis of polyp; DE19703025C2, and DE 4309248 Al .

US 2011/0104278 Al describes a solidifiable bone precursor composition which can be used to produce a mineralized substitute bone tissue material and the method of making the substitute bone tissue material. The substitute bone tissue material includes a collagen matrix comprising pores, and an apatitic mineral within said pores.

EP1935414 describes intraneuronal carbonic anhydrase activator compounds for improving attentive cognition.

The above publications do not describe any (synergistic) effect of combinations of polyP (Ca ²⁺ salt) with bicarbonate (HC0 ₃ ^~ ), and with activators of the CA, on HA formation, which would be of great advantage for therapy and prophylaxis of osteoporosis and related diseases, and for treatment of bone defects.

Summary of the invention

The object of the invention is solved according to the appended claims. In a first aspect thereof, the problem is solved by a composition comprising a combination of at least one inorganic polyphosphate (polyP) and/or a salt of said at least one polyP and calcium [polyP (Ca2+ salt)], and bicarbonate/calcium-carbonate, optionally together with at least one carbonic anhydrase activator.

The invention is based on the discovery of the inventors that (i) the CA-driven CaC0 ₃ deposition can be stimulated by CA activators both in vitro and in vivo and (ii) the stimulatory effect of polyP on bone formation (HA synthesis) by osteoblasts is enhanced in a synergistic manner by CA activators. The inventors demonstrate that sponge extracts, more specifically extracts from the marine demosponge Suberites domuncula are able to enhance the deposition of CaC0 ₃ mediated by the bone-specific CA. One component, quinolinic acid (QA), isolated from those extracts, is part of the inventive material, described herein. Unexpectedly, the inventors found that the effect of polyP or polyP (Ca ²⁺ salt) on mineralization / HA formation of bone- forming osteoblasts can be amplified by bicarbonate and, synergistically, by addition of sponge extracts, or components thereof, like QA, that act as activators of CA reaction. This finding was unexpected since monomeric phosphate formed from polyP by degradation / hydrolysis by bone-specific ALP and providing the substrate for HA deposition, inhibits the CA involved in early bone mineral formation. Therefore, compositions comprising combinations of CA activators, such as sponge extracts and QA, and polyP or polyP (Ca ²⁺ salt) represent a novel, inventive material that can be used as a constituent of a scaffold material, material for injection, drug or food supplement for prophylaxis or therapy of osteoporosis and other bone diseases or treatment of bone fractures or defects. The inventors demonstrate that this material is superior in activation of new bone formation / HA formation than polyP or polyP (Ca ²⁺ salt) present in conventional materials for bone tissue engineering and repair or prophylaxis / therapy of osteoporotic disorders.

Thus, the material, drug or food supplement ("composition") according to this invention, can be combined with other materials enhancing bone formation, like bio silica, which can be produced by silicatein, which turned out to be morphogenetically active. The state-of-the-art of biosilica and silicatein has been described in, for example, Schroder HC, Wang XH, Tremel W, Ushijima H, Miiller WEG (2008) Bio fabrication of biosilica-glass by living organisms. Nat Prod Rep 25:455-474; Wang XH, SchloBmacher U, Wiens M, Batel R, Schroder HC, Miiller WEG (2012) Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber-like spicules. FEBS J 279: 1721-1736; and Miiller WEG, Schroder HC, Burghard Z, Pisignano D, Wang XH (2013) Silicateins - a novel paradigm in bioinorganic chemistry: enzymatic synthesis of inorganic polymeric silica. Chem Eur J 19:5790-5804.

The following patents or patent applications relate to biosilica, which may be used in combination with CA activators and polyP or polyP (Ca ²⁺ salt), and its application in biomedicine and dentistry, nevertheless, these patents/patent applications do not include the approach described in the present patent application: European patent EP 1320624, German patent DE10352433, United States patent US6670438 , European patent EP1546319, German patent DE10246186, EP1740707, European patent EP1682658, patent application EP09005849.6, DE102004021229.5, EP2007007363, patent application PCT/US2009/005302, and patent application EP 10167744.1.

The material, drug or food supplement according to this invention, can also be modified by replacing the polyP component with non-hydrolyzable polyP analogs such as the non- hydrolyzable pyrophosphate analogs bisphosphonates which contain a P-C-P bond instead a hydro lyzable P-O-P bond that cannot be cleaved by ALP.

A schematic presentation of the biological processes of early bone formation and of the targets of the inventive material, drug or food supplement, or its components, is shown in Figure 1. In this scheme, the polyP (Ca ²⁺ salt) storing cell (upper bone cell) is depicted separately from the biomineralizing cell (lower bone cell), for reasons of simplicity. PolyP is stored as polyP (Ca ²⁺ salt) in vesicles that are sequestered into the extracellular space where polyP induces the expression of ALP. The ALP in turn hydrolyzes the polyP (Ca ²⁺ salt) under formation of monomeric inorganic phosphate (Pi) and Ca ²⁺ that serve as building blocks for Ca-phosphate / HA formation. The mineralizing cell (lower bone cell) is synthesizing CaC0 ₃ bio-seeds in close association of the cell membrane, either extracellularly or intracellularly under the control of the CA. This enzyme catalyzes the rate-limiting step to the HC0 ₃ . The exchange of HC0 ₃ between the extracellular and intracellular compartments is mediated by transporters and exchangers, e.g. NBC or AE. After formation of the CaC0 ₃ bio-seeds the activity of the CA is downregulated by Pi. In the subsequent step the HC0 ₃ bio-seeds are partially transformed to Ca-carbonated-phosphate followed by the deposition of Ca- phosphate. The activity of the two regulator enzymes, ALP and CA, is under feed-back control. The ALP is activated by polyP, while the CA is inhibited by Pi. The latter enzyme is stimulated by the sponge extract or QA, being components of the inventive material.

The possible mechanism of the interaction of QA, being part of the inventive material, with the Zn atom at the catalytic site of the CA is shown in Figure 2. QA with its N-heteroatom in the pyridine backbone, as well as the dicarboxylic acid side chains are proposed to interact with the Zn-containing CA as follows. The zinc prosthetic group in the CA is coordinated in three positions by histidine side-chains, by H1S94, H1S96, and Hisng. The fourth coordination position at Zn is occupied by one water molecule, and causes there a polarization of the hydrogen-oxygen bond, resulting in an increased negativity of the oxygen and a weakening of the bond to the Zn atom. A fourth histidine, His ₆ 4, is placed close to the substrate of water and accepts a proton from the water molecule through a general acid-base catalysis under formation of a hydroxide bound to the zinc. The hydrogen bridge bond that can be postulated between the N-heteroatom of QA and the water no is assumed to facilitate the rate-limiting proton transfer step. The dicarboxylic acid side chains of QA could interact, via their conjugated side chains with the enzyme-bound Zn ion.

A further aspect of this invention concerns a combination of a CA activator and polyP or polyP (Ca ²⁺ complex) with silicic acid or polysilicic acid, as well as their salts, or enzymatically synthesized forms (biosilica). In earlier studies, the inventors have demonstrated that in osteoblasts biosilica not only induces HA formation (EP 2005004738; US 11579020) but also stimulates OPG synthesis (EP10167744.1), thus counteracting the function of RANKL by increasing the OPG : RANKL ratio that is crucial for preventing the development of osteoporosis.

This invention concerns a material, drug or food supplement, wherein the CA activator is a sponge extract or a component thereof.

The sponge extract, being part of the inventive material, drug or food supplement can be an extract from the demosponge Suberites domuncula.

The component of the sponge extract, being part of the inventive material, drug or food supplement, can be a component of the kynurenine pathway.

The component of the sponge extract, being part of the inventive material, drug or food supplement, can be QA.

A further aspect of this invention is a material, drug or food supplement, consisting of a combination of a CA activator and polyP or polyP (Ca ²⁺ salt) whereby the chain length of the polyP molecules or of the polyP molecules of the polyP (Ca ²⁺ salt) can be in the range between 2 to 1000 phosphate units, but also chain length between 10 to 100 phosphate units, or more narrow ranges of chain lengths are possible for specific applications.

A further aspect of this invention is a material, drug or food supplement, consisting of a combination of a CA activator and polyP or polyP (Ca ²⁺ salt), wherein the phosphate units of the polyP molecules are linked by non- hydro lyzable bonds, or being a bisphosphonate.

A further aspect of this invention is a material, drug or food supplement, consisting of a combination of a CA activator and polyP or polyP (Ca ²⁺ salt), and monomeric silicic acid (orthosilicic acid) or polymeric silicic acid (silica).

Moreover, polymeric silicic acid can be added that has been formed by an enzyme or protein involved in biosilica (amorphous, hydrated silicon oxide) metabolism, such as silicatein or silicatein fusion proteins or combinations thereof.

A further aspect of this invention is a material, drug or food supplement, consisting of a combination of a CA activator and polyP or polyP (Ca ²⁺ salt), wherein said material, drug or food supplement is supplemented with calcium carbonate, a CA polypeptide A, a CA fusion protein or combinations thereof.

The CA polypeptide or the CA fusion protein can be produced using a prokaryotic or eukaryotic expression system, or synthetically.

A further aspect of this invention is method of producing the composition according to the invention, comprising the steps of identifying an activator of a carbonic anhydrase by contacting a carbonic anhydrase polypeptide or a carbonic anhydrase fusion protein that is produced synthetically or produced using a prokaryotic or eukaryotic expression system with a sample comprising a candidate substance, and identifying a substance that activates said carbonic anhydrase, and combining said activator as identified.

Preferably, any sample can be used for the identification (screening), as long as it potentially contains a prospective activator of the CA.

Assays for screening can be found, for example, in Bertucci A, Zoccola D, Tambutte S, Vullo D, Supuran CT. Carbonic anhydrase activators. The first activation study of a coral secretory isoform with amino acids and amines. Bioorg Med Chem. 2010 Mar 15;18(6):2300-3. doi: 10.1016/j.bmc.2010.01.059. Epub 2010 Feb 4, or Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C. T. Biochemistry 1997, 36, 10384.

Preferred is the method of producing the composition according to the invention, wherein said sample is derived from a sponge extract, and preferably is a sample comprising a component of the kynurenine pathway and/or wherein said activator is quinolinic acid.

In addition, it is possible to encapsulate combinations of a CA activator and polyP or polyP (Ca ²⁺ salt) or combinations thereof with calcium carbonate or monomeric or polymeric silicic acid or one or more of the components (enzymes, proteins, and substrates) involved in their formation, in an organic polymer, consisting of shellac, alginate, or poly(lactic acid), or poly(lactide/glycolide), or polycaprolactone, or collagen, or silk fibroin, or starch, or chitosan, or poly(3-hydroxybutyrate), or poly(D,L-lactide)/polyvinyl pyrrolidone-based microspheres.

Based on its properties, the material according to this invention can be used as a food supplement for prophylaxis or therapy of osteoporosis or other bone disorders.

Furthermore, the material according to this invention can be used for the formulation of an orally or parenterally-administrable drug for therapy or prophylaxis of osteoporosis or other bone disorders.

This material can be implanted for treatment of bone defects.

This material can also be used for percutaneous injection into fractured bone tissue or for prophylactic stabilization of vertebral bodies in osteoporotic patients or patients with other bone disorders.

The experiments underlying this invention have been performed with SaOS-2 cells as a model system for osteoblasts, having the capacity of HA formation. The results are described in the following. The CA is the prime enzyme involved in CaC0 ₃ deposition. Previous results of the inventors revealed that HC0 ₃ causes an induction of CA in SaOS-2 cells and a subsequent increase in mineralization (Miiller WEG, Schroder HC, Schlossmacher U, Grebenjuk VA, Ushijima H and Wang XH (2013) Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation. Biomaterials 34:8671-8680).

The inventors screened for CA activators and used sponges as a starting biological material. The inventors screened organic extracts from sponges, and among them, the ethyl-acetate extract from the demosponge Suberites domuncula [SubDo-extr] showed a distinct activation of the CA enzyme during the in vitro CaC0 ₃ formation.

The inventors demonstrate that QA causes a significant upregulation of the bio mineralization process by SaOS-2 cells in vitro.

QA is a bioactive compound from S. domuncula (Schroder HC, Sudek S, De Caro S, De Rosa S, Perovic S, Steffen R, Miiller IM, Miiller WEG (2002) Synthesis of the neurotoxin quinolinic acid in apoptotic tissue from Suberites domuncula: Cell biological, molecular biological and chemical analyses. Mar Biotechnol 4:546-558).

Studies using scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed that the element carbon (a component of Ca-carbonate deposits) is accumulated in the mineral nodules on the surface of SaOS-2 cells, both if incubated with sponge extract or with QA.

The inventors could demonstrate for the first time that an enhancement - in a synergistic manner - of the effect of polyP (Ca ²⁺ salt) on mineralization by bicarbonate (HC0 ₃ ) and extracts or compounds that increase CA-mediated mineralization occurs. This finding is remarkable since polyP causes this effect in the presence of Pi, present in the culture medium. This finding implies that polyP, as such, causes an enhancing effect on mineral deposit formation in the polymeric state. The explanation for this amplifying effect of polyP might be a local extrusion and sequestration of Ca ²⁺ -polyP from cells adjacent to the bone-forming sites of the osteoblasts. The ALP activity is spatially localized on the apical region of the (secretory) cell membranes. As schematically outlined in Figure 1 the extracellularly localized ALP, induced by polyP, in a positive auto-circle increasingly degrades Ca ²⁺ -polyP to Ca ²⁺ and Pi, perhaps allowing Ca-carbonated-phosphate to form onto the CaC0 ₃ bio-seeds. It should be noted that Pi enzymatically released from polyP negatively controls back the formation of CaC0 ₃ by inhibiting the CA (Miiller WEG, Schroder HC, Schlossmacher U, Grebenjuk VA, Ushijima H and Wang XH (2013) Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation. Biomaterials 34:8671-8680).

The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures,

Figure 1 shows a schematic representation of the early stages of the enzymatic synthesis of the bio-seeds during bone mineralization. Two osteoblasts are depicted, one of them is involved in polyP formation/release. After enzymatic formation and its compartmentalization into vesicles, the charges of polyP are neutralized by cations, e.g. Ca ²⁺ . The vesicles are extruded and polyP (Ca ²⁺ salt) is discharged. Driven by the membrane-bound CA, HC0 ₃ and subsequently CaC0 ₃ deposits are formed that act as bio-seeds for carbonate phosphate and HA. It is outlined that polyP activates the ALP, and in turn the released ortho-phosphate inhibits the CA. The CA on the other side is activated by HC0 ₃ , and also by SubDo-extr and QA.

Figure 2 shows a scheme of the proposed interaction of QA (in red) with the active center of the human CA. Three His residues are interacting with the central Zn ²⁺ ion. The three hydrogen bond network, formed by Wat-150, Wat-129 and Wat-130, is indicated. It is proposed that QA interacts with its N-heteroatom to Wat-130 and its one carboxylic acid group to Zn ²⁺ .

Figure 3 shows the formation of CaC0 ₃ in the ammonium carbonate diffusion assay in the absence (A) or presence of 35 W-A units CA per 500 μΐ, crystallization assay (B). The reaction assays remained either free of additional compound(s) (--■--) or were supplemented with 5 μg/500 μΐ. of SubDo-extr (· · ·) or with 10 μΜ QA (·- Α-·). At the indicated time points samples were taken and the free Ca ²⁺ concentration was determined. The decrease in the concentration of the free Ca ²⁺ indicates the increase in deposited CaC0 ₃ . Samples of six parallel determinations were quantitated; the means ± S.D. are given. *p < 0.05.

Figure 4 shows the formation of calcitic crystals in the carbonate diffusion assay found in the presence of 35 W-A units CA/500 μΐ, (B, D, F and H) if compared with assays without the enzyme (A, C, E and G). Two different morphologies of the calcitic crystals can be distinguished, the round- shaped vaterite deposits (d) and the rhombohedral prisms of calcite (p). Light microscopical images.

Figure 5 shows the results of crystallization studies in the carbonate diffusion assay that was performed again in the absence (A, C, E and G) or presence of 35 W-A units/500 μΐ, of CA (B, D, F and H). All assays contained, in addition, 5 μg/500 μΐ SubDo-extr. Some vaterite deposits (d) and rhombohedral prisms of calcite (p) are marked.

Figure 6 shows the results of the in vitro crystallization assay in the absence (A, C, E and G) or presence of 35 W-A units/500 μΐ, of CA (B, D, F and H); the assays have been supplemented with 10 μΜ QA. Vaterite deposits (d), rhombohedral prisms of calcite (p).

Figure 7 shows the effect of polyP (Ca ²⁺ salt) on the extent of mineralization (HA formation) onto SaOS-2 cells in assays, in the absence (A) or presence of HC0 ₃ (B); Alizarin Red S spectrophotometric assay. As indicated, the experiments were performed in the absence (- MAC; open bars/left hatched in black) or presence (+ MAC; grey-colored bars/right hatched in white) of mineralization activation cocktail (MAC), composed of 5 mM β- glycerophosphate, 50 mM ascorbic acid and 10 nM dexamethasone. As marked, polyP (Ca ²⁺ salt) was added in the absence of MAC (- MAC + polyP; left hatched in black) or the presence of MAC (+ MAC + polyP; right hatched in white). Standard errors of the means are shown (n = 6); *p < 0.05. The results show, in the presence of a phosphate source (MAC), a clear synergistic effect of polyP and bicarbonate (HC0 ₃ ), the product of the CA reaction that was found to be activated by the sponge extract (SubDo-extr) or QA (see Figure 3).

Figure 8 shows the stimulatory effect on HA formation of co-incubation of SaOS-2 cells with MAC and polyP (Ca ²⁺ salt), if administered together with 10 μΜ QA (stippled bars) or of 5 μg SubDo-extr per assay (filled bars). The control values without QA or SubDo-extr are given in comparison (right hatched in white). Means (n = 6) together with *p < 0.05 are given. The results show that the effect of polyP and bicarbonate (HC0 ₃ ), the product of the CA reaction, in the presence of a phosphate source (MAC), is significantly amplified after addition of the sponge extract (SubDo-extr) or QA that activate the CA-driven mineralization reaction (see Figure 3).

Figure 9 shows the Alizarin Red staining staining of the cells, incubated either in the absence (- MAC) or presence of MAC (+ MAC) in dependence of the S. domuncula compounds. The assays remained in the absence of QA (- QA) or the presence of QA (+ QA) and were incubated without the sponge compounds (- QA/- SubDo-extr) or with the compound(s) (+ QA/+ SubDo-extr). The incubation was terminated after 5 d and the overslips were stained with 10% Alizarin Red S.

Figure 10 shows the results of semiquantitative determinations of the accumulation calcium [Ca], phosphorous [P] and carbon [C] around growing nodules (no), which have been distinguished by secondary electron imaging. Element mapping was performed by SEM- based EDX mapping. (A) Secondary electron image. Mapping for Ca (B), P (C), and C (D). The intensity scale for the pseudocolor documentation is given below the images; dark: low intensities; red/white high intensities. The SaOS-2 cells were incubated in the presence of HC0 ₃ and MAC for 5 d.

Figure 11 shows the results of mapping studies for the elements Ca, P and C around growing nodules (no) onto SaOS-2 cells. The cells were grown for 5 d in the presence of HCO3 and MAC and, in addition, of 10 μΜ QA. (A) Secondary electron image; mapping for Ca (B), P (C), and C (D). It is obvious that the areas of increase of impulses for C (D) extend the areas of increased signals for Ca (B) and P (C).

Examples

Effect of sponge extracts and QA on CA-mediated CaCQ ₃ deposit formation

Under the conditions used described under "Methods" 20% of the soluble CaCl ₂ salt was converted to insoluble CaC0 ₃ after an incubation period of 240 min by using the ammonium carbonate diffusion assay, in the absence of CA (Figure 3A). Addition of 5 μg/assay [500 μί] of SubDo-extr or of 10 μΜ QA does not significantly change the extent of CaC0 ₃ formation; in the presence of the SubDo-extr the amount of CaC0 ₃ formation was 32.5±4.9% and in the presence of QA 28.4±3.7% after the 240 min incubation period.

In the presence of CA the rate of CaC0 ₃ formation increases significantly to 34.2=1=5.1% during 240 min and again significantly to 66.9±7.5% in the presence of QA and 84.9±8.3% in the presence of SubDo-extr (Figure 3B).

Conclusion:

The CA-driven CaC0 ₃ formation is significantly amplified after addition of the sponge extract or QA.

Rate of the CaCQ ₃ crystal formation in dependence on the presence of CA, SubDo-extr and QA

No crystals are seen at the beginning of the reaction in the diffusion/desiccator assays (Figure 4A and B). In the absence of CA only very scarcely, 42±3 μιη (n=10) large crystals are seen after 30 min (Figure 4C); in contrast, in the presence of CA 105±16 μηι sized crystals are regularly seen (Figure 4D). After an incubation period of 60 min the size of the crystals increases to 122±25 μιη in both assays (Figure 4E and F), again with a much higher abundance in the assays with the CA. After a further, extended incubation for 180 min (Figure 4G and H) the crystals, especially in the assays containing CA, further increase in size. Especially in the assays containing the CA two forms of calcitic crystals are see, first the round-shaped deposits and second the rhombohedral prisms. The round-shaped deposits consists of the metastable vaterite with the characteristic peaks of the FT-IR spectra at 875 cm ^"1 and 744 cm ^"1 , while the prisms consist of calcite with the FT-IR peaks at 873 cm ^"1 and 71 1 cm ^"1 .

In the presence of the SubDo-extr, 5 μg · 500 μί ^"1 , the abundance of the calcitic crystals increases significantly in the assays. While at time zero again no crystals are seen (Figure 5 A and B), the number of the crystals increases gradually during the incubation from 30 min (Figure 5C and D), over 60 min (Figure 5E and F), to 180 min (Figure 5G and H). Significantly higher is the number of crystals in the CA containing assays (Figure 5D, F and H), compared to those lacking the enzyme (Figure 5C, E and G).

A similar effect on the number of crystals formed can be seen if the crystallization studies are performed with 10 μΜ QA. Again the prevalence of the distinctly formed crystals increases during the 180 min incubation period (Figure 6) especially in the assays containing the CA (Figure 6D, F and H), compared to the assays lacking CA (Figure 6C, E and G). However, in addition to the crystals also small-sized deposits are seen together with the crystals that accumulate on the aqueous/air interphase

Conclusion:

The CA-driven formation of CaC0 ₃ crystals is significantly amplified after addition of the sponge extract or QA.

Effect of polyP and CaCQ ₃ on mineralization of SaOS-2 cells

As expected, the addition of MAC to the SaOS-2 cells causes an increase in mineralization, which becomes significant after an incubation period of 3 d. The extent of the MAC-caused increase in mineral deposition is lower in the absence of HC0 ₃ in the assays (Figure 7A), compared with the increase that is measured in the presence of HC0 ₃ (Figure 7B). Especially at day 5 of incubation, in the presence of HC0 ₃ , the increase is high with 5.3-fold, compared to the 1.3-fold increase in the absence of HC0 ₃ . If polyP (Ca ²⁺ salt) is added to the SaOS-2 cultures the basis level (absence of MAC) is not markedly different in the two series of experiments, without or with HC0 ₃ (Figure 7A; Figure 7B). In contrast, if polyP (Ca ²⁺ salt) is added to the cultures, the MAC-caused stimulation, e.g. at day 5, in the presence of polyP (Ca ²⁺ salt) is significantly higher in the presence of HC0 ₃ ([3.6-fold] Figure 7B), if compared to the experiments in the absence of HC0 ₃ ([1.5-fold] Figure 7A).

Conclusion:

In the presence of a phosphate source (MAC), there is a clear synergistic effect on osteoblast cell mineralization (HA formation) of polyP and bicarbonate (HC0 ₃ ), the product of the CA reaction that was found to be activated by the sponge extract or QA (see Figure 3). Effect of SubDo-extr and QA on biomineralization onto SaOS-2 cells

Both the SubDo-extr and QA did not significantly change the low extent, ~ 0.1 nmol Alizarin Red S staining per 1 μg DNA at day 1 and 3, or ~ 0.1 nmol^g, in the absence of MAC, or in medium lacking HCO3 . However, in the presence of MAC and polyP (Ca ²⁺ salt) the two natural components, SubDo-extr and QA, significantly increased the extent of mineral deposition onto SaOS-2 (Figure 8). This increase is especially pronounced at day 3; at this time point the extent of biomineralization, after addition of 5 μg SubDo-extr per assay, or of 10 μΜ QA, is amplified to 2.3-fold, or 2.1-fold, respectively. At day 5 of mineralization, the increase is still significant, but less pronounced.

Figure 9 shows the Alizarin Red S staining of the cells that have been incubated in the absence or presence of MAC and the S. domuncula compounds (QA or sponge extract).

Conclusion:

The effect of polyP and bicarbonate (HCO3 ), the product of the CA reaction on osteoblast cell mineralization (HA formation) in the presence of a phosphate source (MAC) is significantly amplified after addition of the sponge extract or QA that activate the CA-driven mineralization reaction (see Figure 3).

Carbon-rich deposits formed onto SaOS-2 cells after HCO exposure: element mapping

The cells were incubated in the presence of HCO ₃ and the MAC for 5 d and then - after fixation - subjected to element mapping, using two-dimensional element mapping in the SEM/EDX set-up adjustment (Figure 10). As shown in Figure 10A the nodules are highlighting at the secondary electron image setting. Around those nodules strong increases of the mapping impulses for calcium [Ca] (Figure 10B), phosphorous [P] (Figure IOC) and also carbon [C] (Figure 10D) are evident. The signals for Ca, P, and C are matching the area of the nodules.

The same series of experiments have been performed for SaOS-2 cells that had been grown in the presence of HCO ₃ and MAC, again for 5 d, followed by element mapping (Figure 11). If to this experimental set-up 10 μΜ QA is added as well. The growing nodules can be distinguished by secondary electron imaging (Figure 11 A). Within the areas of nodules a strong increase of the impulses for Ca (Figure 11B), P (Figure 11C) and especially C (Figure 11D) is seen. It should be particularly mentioned that the regions with increased C signals expand those regions highlighting for the nodules (secondary electron imaging) and Ca or P.

Conclusion:

The mineral deposits formed in the presence of a phosphate source (MAC) and HCO ₃ , the product of the CA reaction, contain, in addition to calcium and phosphorus (components of calcium phosphate / HA), calcium and carbon (components of calcium carbonate, formed during the early mineralization reaction).

Methods

Cultivation of SaOS-2 cells SaOS-2 cells (human osteogenic sarcoma cells) were cultured in McCoy's medium (Biochrom-Seromed), lacking Na-bicarbonate but containing 2 mM L-glutamine and 1 mM CaCl ₂ ). The medium was supplemented with 10% heat-inactivated fetal calf serum (FCS) and 100 units/mL penicillin/ 100 μg/mL streptomycin. Where indicated 20 mM Na-bicarbonate (NaHC0 ₃ ) was added as well. The medium/serum was buffered with 25 mM HEPES (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid) to a pH of 7.4. The cells were incubated in 25 cm ² flasks or in six-well plates (surface area 9.46 cm ² ; Orange Scientifique) in a humidified incubator at 37°C. Routinely, 3 · 10 ⁵ cells/well were added (total volume 3 mL). Where indicated the cultures were supplemented with the mineralization activation cocktail (MAC), composed of 5 mM β-glycerophosphate, 50 mM ascorbic acid and 10 nM dexamethasone. The mineralization activation cocktail was added 3 days after starting the experiments. Medium was changed every 3 days and new MAC was added. Where indicated with the respective assay either S. domuncula extract [SubDo-extr] or QA was added at the respective concentration.

Mineralization by SaOS-2 cells in vitro

The extent of mineralization in assays with SaOS-2 cells was determined quantitatively using the Alizarin Red S spectrophotometric assay (Gregory CA, Gunn WG, Peister A, Prockop DJ (2004) An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 329:77-84) The amount of bound Alizarin Red S is given in nmoles. Values were normalized to total DNA in the samples.

In one series of experiments the cultures, growing into culture wells were stained directly on the coverslips with 10% Alizarin Red S, after fixation with ethanol (Schroder HC, Borejko A, Krasko A, Reiber A, Schwertner H, Miiller WEG (2005) Mineralization of SaOS-2 cells on enzymatically (Silicatein) modified bioactive osteoblast-stimulating surfaces. J Biomed Mat Res Part B - Appl Biomater 75B:387-392).

Incubation of the SaOS-2 cells with polyP

SaOS-2 cells were incubated, under otherwise identical conditions, with polyP. As source, Na-polyP (average chain of approximately 40 phosphate units), obtained from Chemische Fabrik Budenheim (Budenheim; Germany) was used for the studies. To compensate for any effect, caused by the chelating activity to Ca ²⁺ after hydrolysis of polyP to monomeric phosphate (or pyrophosphate) by phosphatases in vitro, polyP was mixed together with CaCl ₂ in a stoichiometric ratio of 2: 1 (polyP:CaCl ₂ ), as described (Miiller WEG, Wang XH, Diehl- Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M and Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca ²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 7:2661-2671). The salt, designated as "polyP (Ca ²⁺ salt)", was added at a concentration of 50 μΜ to the assays.

CA-mediated CaCQ ₃ precipitation assay

For the CaC0 ₃ precipitation assay the ammonium carbonate diffusion method has been used, as described (Miiller WEG, Schlossmacher U, Schroder HC, Lieberwirth I, Glasser G, Korzhev M, Neufurth M and Wang XH (2013) Enzyme-accelerated and structure-guided crystallization of Ca-carbonate: role of the carbonic anhydrase in the homologous system. Acta Biomater; 10.1016/j.actbio.2013.08.025). In a 3.4 1 large desiccator C0 ₂ vapor was generated from a 1 M NH ₄ HCO3 solution which had be placed into the lower compartment of the desiccator. C0 ₂ diffused into the upper compartment which contained in eight-well square chamber slides 50 mM CaCl ₂ in 30 mM Tris-HCl (pH 8.3). The slides with an edge length of 10 mm (Lab-Tek II slides #154534) came from Thermo Scientific). The surface area of the 500 assay was 0.7 cm ² . Where indicated the recombinant human CA2 enzyme, expressed in Escherichia coli (C6624; Sigma), with a specific activity of ~ 5,000 units/mg (Magid E (1968) The dehydration kinetics of human erythrocytic carbonic anhydrases B and C. Biochim Biophys Acta 151 :236-244; Carter ND, Chegwidden WR, Hewett-Emmett D, Jeffery S, Shiels A, Tashian RE (1984) Novel inhibition of carbonic anhydrase isozymes I, II and III by carbamoyl phosphate. FEBS Lett 165: 197-200) was added at a concentration of 35 W-A units (10 μg)/500 of CaCl ₂ to the assays. Prior to the addition of the Tris/CaCl ₂ reaction vessels, the desiccator was pre-equilibrated with C0 ₂ . The formation of CaC0 ₃ was quantitatively determined on the basis of the consumption of free Ca ²⁺ by using the EDTA titration procedure described (Slowinski EJ, Wolsey WC, Masterton WL (2009) Chemical Principles in the Laboratory. 9th ed, Belmont, CA:Brooks/Cole; Miiller WEG, Schlossmacher U, Schroder HC, Lieberwirth I, Glasser G, Korzhev M, Neufurth M and Wang XH (2013) Enzyme-accelerated and structure-guided crystallization of Ca-carbonate: role of the carbonic anhydrase in the homologous system. Acta Biomater; 10.1016/j.actbio.2013.08.025). The crystals formed were visualized using a light digital microscope.

Preparation of S. domuncula extract

Freshly collected sponge specimens, dredged 5 km offshore from Rovinj (Adriatic Sea; Croatia), were cleaned and cut into small pieces. The material (1 kg) was homogenized in a Waring Blender in the presence of 100 mL of ethanol; then the slurry was extracted ethyl acetate (3 x 250 mL) at room temperature. The combined ethyl acetate extracts were washed with water (3 x 200 mL), dried over Mg ₂ S0 ₄ , filtered and evaporated to dryness yielding ~ 4 g of dark tar-like residue. This material, termed SubDo-extr, was dissolved in dimethyl sulfoxide (DMSO) and subjected to testing in the cell culture. Control cultures received the carrier solvent alone (1.0% DMSO).

Extraction and identification of quinolinic acid

QA was extracted as described (Schroder HC, Sudek S, De Caro S, De Rosa S, Perovic S, Steffen R, Miiller IM, Miiller WEG (2002) Synthesis of the neurotoxin quinolinic acid in apoptotic tissue from Suberites domuncula: Cell biological, molecular biological and chemical analyses. Mar Biotechnol 4:546-558). The extract was shaken in a separation funnel with chloroform to eliminate nonpolar components. The residue was used for the determination of QA by high-performance liquid chromatography (HPLC). The eluate was monitored with an UV detection at 268 nm. At this wavelength the molar extinction coefficient (ε) is 4000 as M ^"1 cm ^"1 (Reinhard JF, Erickson JB, Flanagan EM (1994) Quinolinic acid in neurological disease: Opportunities for novel drug discovery. Adv Pharmacol 30:85-127).

Scanning electron microscopy and energy-dispersive X-ray analysis

The scanning electron microscopic (SEM) analysis was performed with a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH) microscope, employed at low voltage (<1 kV; analysis of near-surface organic surfaces). The SEM microscope was coupled to an XFlash 5010 detector, an X-ray detector that allows simultaneous energy-dispersive X-ray (EDX)- based elemental analyses. Likewise, the same combination of devices was used for higher voltage (10 kV) analysis during which the XFlash 5010 detector was used for element mapping of the surfaces of the deposits. The HyperMap database was used for interpretation, as described (Salge T, Terborg R (2009) EDS microanalysis with the silicon drift detector (CDD): innovative analysis options for mineralogical and material science application. Anadolu Univ J Sci Technol 10:45-55).

Additional methods

The results were statistically evaluated (Sachs L (1984) Angewandte Statistik. Berlin: Springer, p 242). DNA content was determined by application of the PicoGreen method as described (Schroder HC, Borejko A, Krasko A, Reiber A, Schwertner H, Miiller WEG (2005) Mineralization of SaOS-2 cells on enzymatically (Silicatein) modified bioactive osteoblast- stimulating surfaces. J Biomed Mat Res Part B - Appl Biomater 75B:387-392) using calf thymus DNA as a standard.