GB2532283A - Morphogenetically active calcium polyphosphate nanoparticles - Google Patents

Abstract

A method for the production of a solid, degradable polyphosphate material having calcium counterions comprises: (i) dissolving a polyphosphate salt in an aqueous solvent, such as water, and adjusting the pH value of the solution to alkaline values; (ii) adding a solution of a calcium salt solution to the polyphosphate salt solution, and adjusting the pH value to alkaline values, (iii) optionally, washing with a solvent, for example ethanol, and (iv) collecting the particles formed. The method in steps (i) to (iii) is performed at ambient temperature, and the material has a hardness similar to that of bone tissue, and is morphogenetically active. Preferably, the polyphosphate is sodium phosphate, and has a chain length in the range of 3 to 1000 phosphate units. The calcium salt solution may be calcium chloride, and the pH may be adjusted to 10. The amorphous nanoparticles made by the method may be used in bone regeneration and repair and in dentistry, or for drug delivery.

Description

MORPHOGENETICALLY ACTIVE CALCIUM POLYPHOSPHATE

NANOPARTICLES

This invention concerns a calcium polyphosphate material consisting of amorphous nanoparticles with a diameter of approximately 0.2 pm that displays a considerable hardness (elastic modulus) of about 1.3 GPa. The inventive non-crystalline and biodegradable material that is fabricated under mild conditions, at room temperature, is morphogenetically active and induces bone formation and the expression of the marker gene for osteoblast activity, alkaline phosphatase.

Background of Invention

Inorganic polyphosphate (polyP) is a nontoxic polymer existing in a wide range of organisms (Schroder HC, Muller WEG, eds (1999) Inorganic Polyphosphates -Biochemistry, Biology, Biotechnology. Prog Mol Subcell Biol 23:45-81; Kulaev IS, Vagabov V, Kulakovskaya T (2004) The Biochemistry of Inorganic Polyphosphates. New York: John Wiley & Sons Inc). It consists of usually linear molecules of tens to hundreds of phosphate units which are linked together via high energy phosphoanhydride bonds. Living organisms can produce this polymer metabolically at ambient temperatures, while the chemical synthesis of polyP requires high temperatures of several hundred degrees.

PolyP in bone Previous studies revealed that polyP molecules of different chain lengths accumulate especially in bone cells (Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Muller WEG, Schroder HC. Inorganic polyphosphate in human osteoblast-like cells. J Bone Mineral Res 1998;13:803-812; Schroder HC, Kurz L, Muller WEG, Lorenz B. Polyphosphate in bone. Biochemistry (Moscow) 2000;65:296-303). In addition, human osteoblast-like cells contain enzymes that hydrolyze polyP, e.g. the alkaline phosphatase (ALP) (Lorenz B, Schroder HC. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001;1547:254-261). PolyP is also found in platelets (Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey TH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA 2006;103:903-908), possibly playing a role in initiation of healing of bone fractures.

PolyP has an inductive effect on osteoblasts mainly as an anabolic polymer that stimulates differentiation of bone cells and mineralization (reviewed in: Wang XH, Schroder HC, Wiens M, Ushijima H, Muller WEG. Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation). Current Opinion Biotechnol 2012;23:570-578; Wang XH, Schroder HC and Muller WEG. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. Int Rev Cell Mol Biol 2014;313:27-77). In addition, polyP induces the ALP (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, Schloamacher U, Lieberwirth I, Glasser G, Wiens M and SchrOder HC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Can level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 2011;7:266h 2671).

Bone graft materials Bone graft materials must be biocompatible and comprise similar biomechanical properties like the physiological bone tissue. Favored are substitutes that are degradable and allow the surrounding tissue to migrate into at least the surface region of the non-self implant to strengthen the bridging of the biofabricated material to the surrounding tissue and to avoid inflammatory reactions.

Besides of cell-based bone graft substitutes ceramic-based materials have proven to be useful bone scaffold for implants. Among the calcium phosphate-based ceramics, the following materials are of particular relevance: Hydroxvapatite (HA). HA is produced at a high-temperature reaction and comprises a crystalline form of calcium phosphate (Noshi T, Yoshikawa T, Ikeuchi NI, Dohi Y, Ohgushi H, Horiuchi K, Sugimura M, Ichijima K, Yonemasu K. Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein. J Biomed Mater Res 2000;52:621-630). Since the composition of HA is Calo(PO4)6(OH)2 with a calcium-to-phosphate ratio of 1.67 this material is chemically very similar to the mineralized phase of physiological bone. Based on this property HA shows suitable biocompatibility (Nandi SK, Kundu B, Ghosh SK, De DK, Basu D. Efficacy of nanohydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat. J Vet Sci 2008;9:183-191).

13-Tricalcium phosphate ((3-TCP). Similar as HA, the chemical composition Ca3(PO4)2 and crystallinity of 13-TCP match the ones of the mineral phase of bone; in addition, this material is bioabsorbable and biocompatible (Daculsi G, LeGeros RZ, Heughebaert M, Barbieux I. Formation of carbonate apatite crystals after implantation of calcium phosphate ceramics. Calcif Tissue Int 1990;46:20-27). TCP implants have been successfully used as synthetic bone void fillers both in orthopedics and in dentistry (Shigaku S, Katsuyuki F. Beta-tricalcium phosphate as a bone graft substitute. Jikeikai Med J 2005;52:47-54).

Bioactive glass. Finally, bioactive glass ceramics ("bioglass") initially developed by Hench et al (Hench LL, Splinter RI, Allen WC, Greenlee TK. Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res Symp 1971;2:117-141; reviewed in: Chen Q, Roether JA, Boccaccini AR. Tissue engineering scaffolds from bioactive glass and composite materials. In: Ashammakhi N, Reis R, Chiellini F Topics in Tissue Engineering, Vol 4, pp. 1-27, 2008) is not only biocompatible but also osteoconductive and allows bone to bind without any intervening fibrous connective tissue interface (Zhang H, Ye XJ, Li IS. Preparation and biocompatibility evaluation of apatite/ wollastonite-derived porous bioactive glass ceramic scaffolds. Biomed Mater 2009;4: 45007). This mineralic glass is frequently used as filling material for bone defects either alone or in combination with autogeneic or allogenic cancellous bone graft (Dorea HC, McLaughlin RM, Cantwell HD, Read R, Armbrust L, Pool R, Roush JK, Boyle C. Evaluation of healing in feline femoral defects tilled with cancellous autograft, cancellous allograft or Bioglass. Vet Comp Orthop Traumatol 2005;18:157-168) or with morphogenetically active inorganic polymers, e.g. polyphosphate [polyP] (Wang XH, Tolba E, Schroder HC, Neufurth NI, Feng Q, Diehl-Seifert B, Muller WEG. Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting. PLoS ONE 2014;9:e112497).

Since all of these mineralic implant materials are fabricated at temperatures higher than 700°C and, in turn, are not, or only at a limited degree, osteoinductive like the bioglass, the inventors developed a new potential biomaterial likewise suitable to be used as bone implant.

The new material according to this invention is based on polyP. Previously polyP has been used, after calcinations, as potential scaffold for bone implants (Pilliar RM, Filiaggi MJ, Wells JD, Grynpas MD, Kandel RA. Porous calcium polyphosphate scaffolds for bone substitute applications -in vitro characterization. Biomaterials 2001;22:963-972; Qiu K, Wan CX, Zhao CS, Chen X, Tang CW, Chen YW. Fabrication and characterization of porous calcium polyphosphate scaffolds. J Materials Sci 2006;41:2429-2434; Ding YL, Chen YW, Qin YJ, Shi GQ, Yu XX, Wan CX. Effect of polymerization degree of calcium polyphosphate on its microstructure and in vitro degradation performance. J Mater Sci Mater Med 2008;19:1291-1295; Wang D, Wallace AF, De Yoreo JJ, Dove PM. Carboxylated molecules regulate magnesium content of amorphous calcium carbonates during calcification. Proc Natl Acad Sci USA 2009;106:21511-21516). In all those preparations, polyP had to be subjected to calcination with the result that the polyP chain might be degraded and the exact chain length is impossible to be determined. Furthermore those polyP scaffolds have not been described to act in an osteoinductive manner.

Summary of the invention

The material according to this invention is a hard amorphous polyP-based biomaterial that is produced at ambient conditions (i.e. at around 20°C ± 10°C) in the presence of a distinctly adjusted concentration of CaC12. The material obtained comprises a porous scaffold built of spherical, amorphous nanoparticles that are biodegradable and retain the morphogenetic activity of the inorganic polymer.

Thus, in a first aspect thereof, this invention concerns a new morphogenetically active material consisting of calcium polyphosphate (polyP) nanoparticles that are (7) amorphous and (ii) display an unusual hardness not found for calcium polyphosphate materials prepared by state-of-the-art methods. The inventors developed a controlled and slow fabrication process that is performed at room temperature and unexpectedly resulted in the formation of a material showing these properties. The polyP material formed in the presence of CaCl2 at a stoichiometric ratio of around 1 or 2 (phosphate to calcium) is an amorphous powder that is composed of nanospheres with a diameter of approximately 0.2 p.m. The inventive material is degradable, in contrast to the Ca-polyP salt prepared by conventional methods which resists hydrolytic cleavage by phosphatases present in medium over longer time periods in cell culture experiments.

The following patent applications on polyP are deemed relevant; GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Milner WEG, Schroder HC, Wang XH; GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders. Inventors: Muller WEG, Schroder HC, Wang XH.

Detailed description of the invention

The inventors describe a novel polyphosphate (polyP) material that is characterized by the following properties: a) The material is amorphous (non-crystalline); b) The material has an unusual hardness (e.g., the elastic modulus of the Ca-polyP2 biopolymer according to this invention amounts to approximately 1.3 GPa (in the context of the present invention, "hard" or "hardness" means a value of approximately 1.3 GPa, such as between 0.8 and 1.8 GPa, preferably between 1.0 and 1.6 GPa), close to value measured for trabecular tissue surrounding human bone [6.9 GPa]); and preferably, c) The material consist of nanoparticles with a diameter of about 0.2 um (Ca-polyP2 particles according to this invention). -4 -

Advantageously, the inventive material can be prepared under mild conditions, such as ambient conditions, and particularly at room temperature.

Hardness can be measured using the Brinell and/or Vickers hardness test method, as known to the person of skill.

It was surprisingly found, that the inventive material is morphogenetically active, it induces bone alkaline phosphatase activity and new bone formation (hydroxyapatite synthesis); and that the material is biodegradable (e.g. by polyphosphatases, such as bone alkaline phosphatase).

This inventive material, whose properties make it superior compared to conventional polyphosphate preparations for a use, for example, in bone regeneration and replacement materials (e.g. GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue [Inventors: Muller WEG, Schroder MC, Wang XH]; GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders [Inventors: Muller WEG, Schroder HC, Wang XH]), can be prepared according to the following method, according to this invention: a) Dissolution of a polyP salt in water and adjustment of the pH to alkaline values, b) (slow) Addition of a solution of a calcium salt to the polyP salt (with Na+) solution, and adjustment of the pH to alkaline value, and c) Collection and drying of the particles thus formed, optionally after washing with ethanol.

This procedure is performed at room temperature.

As a preferred example, the method can be carried out using sodium polyphosphate (NapolyP) as a polyP salt (chain length: 50 phosphate units) in solution and calcium chloride as a calcium salt in solution as follows.

a) Dissolution of 10 g of Na-polyP in 500 ml of distilled water and adjustment of the pH to 10 with 1 M NaOH (room temperature), b) Slow, dropwise (1 ml/min), addition of a solution of 14 g of calcium chloride (Ca-polyP1) or 28 g of calcium chloride (Ca-polyP2) in 250 ml salt to the Na-polyP solution and adjustment of the pH to 10 (room temperature); c) Stirring of the thus formed suspension for about 4 h, and d) Collection of the particles formed, and optionally washing them with ethanol while filtering (0.45 pm filter; e.g., Nalgene Rapid-Flow), and drying at 60°C.

The Ca-polyP material obtained with 14 g of CaCl2 was named Ca-polyPl (stoichiometric ratio phosphate: calcium of 1); and the Ca-polyP material obtained with 28 g of CaCI1 was named "Ca-polyP2" (stoichiometric ratio phosphate: calcium of 1:2).

The nanoparticles consisting of the inventive Ca-polyP material are characterized by a high hardness (about 1.3 GPa; Ca-polyP2) compared to larger particles that are produced at a different phosphate to calcium ratio in solution or at harsh reaction conditions, e.g. in acid-flux of phosphoric acid with Ca(OH)) at high (250°C) temperature (Jackson LE, Kariuki BM, Smith ME, Barralet JE, Wright AJ. Synthesis and structure of a calcium polyphosphate with a unique criss-cross arrangement of helical phosphate chains. Chem Mater 2005;17:4642-4646), or the described calcium-polyphosphate complex (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, Schlo3macher U, Lieberwirth I, Glasser G, Wiens NI, Schroder HC. Inorganic -5 -polymeric phosphate/polyphosphate is an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 2011;7:26612671).

The new, hard polyP material according to this invention is biodegradable and displays superior morphogenetic activity, compared to the Ca-polyP salts prepared by conventional techniques.

In addition, the inventive hard Ca-polyP nanoparticles are prone to cellular uptake (even observed for larger, 600 nm particles; Zhao Y, Sun X, Zhang G, Trewyn BG, Slowing II, Lin VS. Interaction of mesoporous silica nanoparticles with human red blood cell membranes: size and surface effects. ACS Nano 2011;5:1366-1375) and subsequent metabolization, a property that is not possible for the free polyanionic polyP polymer.

The chain lengths of the polyP molecules can be in the range 3 to up to 1000 phosphate units. Optimal results are achieved with polyP molecules with an average chain length of approximately 200 to 20, and optimally about 50 phosphate units.

A further aspect of the invention concerns the material as obtained by one of the methods described above.

Further, the inventors demonstrate that the inventive method can be used for the preparation of hard amorphous and morphogenetically active polyP nanoparticles.

A further aspect of the invention concerns a material comprising the nanoparticles as obtained by one of the methods described above.

The technology according to this invention can be used for the fabrication of nanoparticles or for a material containing such nanoparticles to be used, preferably, in bone regeneration and repair and in dentistry.

A further aspect of the invention concerns the application of the inventive nanoparticles in drug delivery, again, preferably, in bone regeneration and repair and in dentistry, in analogy to, for example, systems described in Kwon et al. and Yang et al. (Kwon S, Singh RK, Perez RA, Abou Neel EA, Kim EIW, Chrzanowski W. Silica-based mesoporous nanoparticles for controlled drug delivery. J Tissue Eng. 2013 Sep 3; 4:2041731413503357. eCollection 2013. Yang P, Gai S, Lin J. Functionalized mesoporous silica materials for controlled drug delivery. Chem Soc Rev. 2012 May 7;41(9):3679-98).

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 listing, Figure 1 shows the FTIR spectra of Na-polyP, Ca-polyP1 and Ca-polyP2; (A) wavenumbers 4000 to 600 cm-1 and (B) wavenumbers 1400 and 600 cm-1 Figure 2 shows the SEM micrographs taken from the following polyP powders: (A and B) Na-polyP, (C and D) Ca-polyP1 and (E and F) Ca-polyP2. -6 -

Figure 3 shows the biological properties of the Ca-polyP material, developed here. (A) SaOS2 cells remained without phosphate material (none), or were incubated with 10 ttg/mL of NapolyP, Ca-polyP1, Ca-polyP2, HA, or 0-TCP for 7 d in the presence of the activation cocktail OC. Then the coverslips with the cells were stained with Alizarin Red S. (B) Expression level of ALP in SaOS-2 cells, in response to exposure to phosphate material. After an 1 or 7 d incubation period the cells were harvested and RNA was extracted and subjected to qRT-PCR analysis. The steady-state-levels of the ALI' transcripts were measured and normalized to the expression of GA1'011. Data are expressed as mean values ± SD for four independent experiments; each experiment was carried out in duplicate. Differences between the groups were evaluated using unpaired f-test. *p < 0.05. (C) Degradation of polyP in vitro, using the 16.5% polycrylamide/7 NI urea gel electrophoresis technique. The cells were incubated with 50 ttg/m1 of solid Ca-polyP2 either in the presence of SaOS-2 cells and medium/serum or PBS [phosphate buffered saline]; after an incubation period of 1 or 7 d, samples were taken for chain length determination. Synthetic polyP markers with an average chain length of 80, 40 and 3 units were run in parallel.

Examples

In the following examples, the inventive method described only for polyP molecules with a chain length of 50 phosphate units. Similar results can be obtained by using polyP molecules with lower and higher chain lengths, such as between 100 to 20 units.

FTIR analyses The two phosphate materials, Ca-polyP1 and Ca-polyP2, were characterized by FTIR and compared with the spectrum obtained with Na-polyP (Fig. 1). The complete spectra between the wavenumbers 4000 and 600 cm1 are shown in Fig. 1A, while segments between 1400 and 600 cm-I are given in Fig. 1B. The band near 1250 cm-I is assigned to the asymmetric stretching mode of the two non-bridging oxygen atoms bonded to phosphorus atoms in the P02 metaphosphate units, vas(P02)-. The weak band at 1190 cm-1 is the P02 symmetric stretching mode vs(P02)-.The absorption bands close to 1083 and 999 cm-1 are assigned to the asymmetric and symmetric stretching modes of chain-terminating P03 groups [vas(P03)2 and vs(P03)21. The absorption band near 864 cm-I is attributed to the asymmetric stretching modes of the P-O-P linkages, vas (P-O-P) and the partially split band centered around 763 cm-1 is due to the symmetric stretching modes of these linkages, vs (P-O-P).

The comparison of the spectra of Na-polyP and Ca-polyP shows that the polyP features are seen in the 1300-1000 cm-1 region. There, most of the chemical characteristics of polyP chains are found. In all three samples a similar pattern is seen, reflecting that the polyP chain backbones are not broken down during the reaction with the Ca2+ ions. However, the peaks are shifted in the Ca-polyP1 and Ca-polyP2 samples if compared the Na-polyP spectrum. This shifting is also seen for other bands in the Ca-polyP spectra; they even increase by increasing the Cat-content in the polyP sample (from Ca-polyP1 to Ca-polyP2). Moreover, it has been reported that the adsorptions bands near 1100-1000 cm-1 are attributed to the ionic stretching mode of the P-0-group, the shifting as well as broadening of this peak of the Ca-polyP samples is attributed to the formation of P-O-N1-(±) (where NI is Ca2-). In conclusion, the 1R spectra confirm the interaction between Ca2+ and polyP and the formation of Ca-PolyP.

XRD analyses were performed with both Ca-polyP1 and Ca-polyP2; the patterns showed no sign of crystallinity, like Na-polyP (Fig. 1C). -7 -

Morphology of polyP samples The three samples, Na-polyP, Ca-polyP1 and Ca-polyP2, were analyzed by SEM (Fig. 2). The Na-polyP particles, of a non-regular shapes, often show a tapered morphology (Fig. 2A and B). The sizes of the particles vary between 1 and 300 um with an average size of =---1 00 pm. Likewise non-regular shapes show the Ca-polyPl particles (Fig. 2C and D). They are smaller than the Na-polyP particles with an average diameter of =4 um. Even smaller are the CapolyP2 particles with an average diameter of =02 pm (Fig. 2 E and F).

PolyP-induced mineralization of SaOS-2 cells The cells were incubated with 10 ug/mL of Na-polyP, Ca-polyP1, Ca-polyP2, HA, or f3-TCP in the presence of the activation cocktail OC for 7 d. Then the coverslips onto which the cells had been cultivated were removed and stained with Alizarin Red S, as described under Methods. Eye-inspection revealed that the intensity of the color reaction, which reflects the extent of minerals being present in the samples, is highest for Ca-polyP1 and for Ca-polyP2. The degree of color reaction is lower for Na-polyP, 13-TCP and HA; the intensities of those samples are only slightly higher, compared to the control (Fig. 3A).

Expression level of ALP Since the Alizarin Red S color reaction might not be sensitive enough due to cross-reactivity with exogenously added grains, the steady-state-expression level of ALP in SaOS-2 cells was quantified by qRT-PCR. The inventors determined previously that the expression level of this enzyme is a reliable marker for the polyP-induced activation of bone cells (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, Schlol3macher U, Lieberwirth I, Glasser G, Wiens M and Schroder HC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca--level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011j;7:2661-2671). Therefore, the inventors subjected the cells, after incubation with the different phosphate samples and in the presence of the OC activation cocktail to qRT-PCR analysis. The data revealed that at day 1 the expression level of ALP is statistically not different between the different polyphosphate samples used. However, after an incubation period of 7 d the steady-state-expression levels of ALP in the cells exposed to Ca-polyP1 and Ca-polyP2 are significantly higher 0.10 expression units compared to the one of the reference GAPDH) than those measured for Na-polyP, fl-TCP or HA (cc. 0.055 expression units). The expression levels of the latter three assays are not significantly higher than the one seen in control assays (no phosphate sample added); Fig. 3B.

Hardness of the Ca-polyP2 material Determination of the mechanical properties (elastic modulus) of the Ca-polyP2 biopolymer was performed with a ferrule-top cantilever and found to be of 1.3 GPa, close to values measured for trabecular tissue that is surrounding human bone with 6.9 GPa (Zysset PK, Guo XE, Hoffler CE, Moore KE, Goldstein SA. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. J Biomech 1999; 321005-1012).

Degradation of polyP in vitro In one series of experiments, the SaOS-2 cells were incubated with 50 pg/m1 of solid CapolyP2 and incubated in the standard assay for 1 d or 7 d either in the presence of SaOS-2 cells and medium/serum or in PBS. Then samples were taken and assayed for the chain length of polyP (Lorenz B, Schroder I-1C. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001;1547:254-261). The gels were stained with o-toluidine blue. The results (Fig. 3C) show that Ca-polyP2, added to the assays, does not undergo hydrolytic degradation during the 7 d long incubation period if dissolved in -8 -PBS. In contrast the average chain length of Ca-polyP2 drops from an average chain length of 40 to around 3 Pi units if the polymer is incubated in the assays which contained SaOS-2 cells and medium/serum. This result indicates that Ca-polyP2 is prone to hydrolysis by ALP (exopolyphosphatase), but also to other polyP hydrolyzing enzymes, endophosphatases, that exist in serum.

Methods Polyphosphate The sodium polyphosphate (Na-polyP of an average chain of 40 phosphate units) used in the Examples has been obtained from Chemische Fabrik Budenheim (Budenheim; Germany).

Preparation of calcium polyphosphate nanospheres Ten g of Na-polyP are dissolved in 500 ml of distilled water and the pH is adjusted to 10 with 1 M NaOH. A solution of 14 g of calcium chloride dihydrate or 28 g of CaC12 in 250 ml is added slowly, dropwise (1 ml/min) to the Na-polyP solution, adjusting steadily the pH to 10 at room temperature. The suspension formed is stirred for 4 hr. Then the particles formed are collected by washing twice with ethanol while filtering through a 0.45 p.m filter (e.g., Nalgene Rapid-Flow). Then the particles are dried at 60°C. The Ca-polyP material obtained by addition of 14 g of CaC12 is named "Ca-polyP1", and the one with 28 g of CaC12 is termed "Ca-polyP2".

Chemical characterization by FTIR Fourier transformed infrared (FTIR) spectroscopy in the attenuated total reflectance (ATR) mode is applied, using, for example, the Varian 660-IR spectrometer with Golden Gate ATR auxiliary (Agilent). Spectra between the wavenumbers 4000 and 600 cm-1 are recorded.

Scanning electron microscopy For the scanning electron microscopic (SEM) analyses, for example, a HITACHI SU 8000 can be employed at low voltage (<1 kV; analysis of near-surface organic surfaces).

XRD analyses X-ray diffraction (XRD) is performed using established procedures (Fischer V, Lieberwirth I, Jakob G, Landfester K, Mulioz-Espi R. Metal oxide/polymer hybrid nanoparticles with versatile functionality prepared by controlled surface crystallization. Adv Funct Mat 2013;23:451-466).

Cells and cell culture conditions Human osteogenic sarcoma cells, SaOS-2 cells, are used for the experiments and cultivated in McCoy's medium with fetal calf serum [FCS] (Wiens M, Wang XH, Schlof3macher U, Lieberwirth I, Glasser G, Ushijima H, Schroder HC, Muller WEG. Osteogenic potential of bio-silica on human osteoblast-like (SaOS-2) cells. Calcif Tissue Intern 2010;87:513-524). Cultivation of the cells is performed in 24-well plates; 3X104 cells are seeded per well. After an initial incubation period of 3 d, the cultures are supplemented either with 10 mg/mL of solid Na-polyP, supplemented with CaC12 in a 2:1 stoichiometric ratio, in order to compensate for the chelating activity of polyP as described (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Cat-1 level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011; 7;2661-26719), or with the two polyP samples, prepared here, with Ca-polyP1 or with CapolyP2. In comparison also nano-hydroxyapatite (HA; for example: 677418 Sigma-Aldrich) or p-TCP (for example: 13204 Sigma-Aldrich) had been included in the test series. As controls none of those polymers is added. Then the cells are continued to be incubated in the presence of the osteogenic cocktail [OC], containing 10 nM dexamethasone, 5 mM 13-glycerophosphate and 50 mM ascorbic acid.

After a 7 d incubation period the cells are either stained with Alizarin Red S to assess the extent of mineralization (Schroder HC, Borejko A, Krasko A, Reiber A, Schwertner H, Muller WEG. Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bioactive osteoblast-stimulating surfaces. J Biomed Mater Res B Appl Biomater 2005; 75:387-392) or subjected to qRT-PCR [quantitative real-time RT-PCR] analysis (Wiens M, Wang XH, Schloemacher U, Lieberwirth I, Glasser G. Ushijima H, Schroder HC, Muller WEG. Osteogenic potential of bio-silica on human osteoblast-like (SaOS-2) cells. Calcif Tissue Intern 2010;87:513-524).

Transcripts expression For qRT-PCR reactions the primer pair Fwd: 5'-TGCAGTACGAGCTGAACAGGAACA-3' (SEQ ID NO: 1) [ntii4i to nti ii,4] and Rev: 5'-TCCACCAAATGTGAAGACGTGGGA-3' (SEQ ID NO: 2) [nti418 to nti395; PCR product length 278 bp] can be used for the quantification of the alkaline phosphatase [ALP] transcripts (accession number NM 000478.4). The expression level of the ALI' can be normalized to the one of the reference gene GAPIEI (glyceralclehyde 3-phosphate dehydr*ogenase; NM 002046.3) using the primer pair Fwd: 5'-CCGTCTAGAAAAACCTGCC-3' (SEQ ID NO: 3) [ntios to ntii63] and Rev: 5'-GCCAAATTCGTTGTCATACC-3' (SEQ ID NO: 4) [ntio5u to ntinm; 215 bp].

Determination of the hardness The hardness of the polyphosphate material Ca-polyP2 can be determined, for example, by a ferruled optical fiber-based nanoindenter as described (Chavan D, Andres D, Iannuzzi D. Ferrule-top atomic force microscope. II. Imaging in tapping mode and at low temperature. Rev Sci Instrum 2011; 82:046107. doi: 10.1063/1.3579496).

PolyP degradation in vitro In one series of experiments the SaOS-2 cells are incubated with 50 pg/ml of solid Ca-polyP or Ca-polyP2 and incubated in the standard assay for 4 d. Then samples (50 pi) are taken and assayed for the chain length of polyP (for example, see: Lorenz B, Schroder HC. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001;1547:254-261). The gels are stained with o-toluidine blue.

Statistical analysis The results can be statistically evaluated using the paired Student's t-test.

Claims (10)

1. -10 -CLAIMS1. Method for the production of a solid, degradable amorphous polyphosphate material having calcium counterions, comprising the steps of i) dissolving of a polyphosphate salt in an aqueous solvent, such as water, and adjusting the pH value of the solution to alkaline values, ii) adding a solution of a calcium salt solution to said polyphosphate salt solution, and adjusting the pH value to alkaline values, iii) optionally, washing with a solvent, for example ethanol, and iv) collecting of the particles formed, wherein said method in steps i) to iii) is performed at ambient temperature, and wherein said material hasa hardness similar to that of bone tissue, and is morphogenetically active.
2. 2. The method according to claim 1, wherein the said polyphosphate salt is sodium polyphosphate.
3. 3. The method according to claim 1 or 2, wherein the chain length of the polyphosphate is in the range of about 3 to about 1000 phosphate units, preferably in the range of about 10 to about 100 phosphate units, and most preferred about 50 phosphate units.
4. 4. The method according to any of claims 1 to 3, wherein the calcium salt is calcium chloride.
5. 5. The method according to any of claims 1 to 4, wherein the pH is adjusted to 10.
6. 6. The method according to any of claims 1 to 5, wherein the calcium polyphosphate material is formed from sodium polyphosphate in the presence of calcium chloride at a stoichiometric ratio of 0.1 to 10 (phosphate to calcium), preferably of 1 to 2.
7. 7. The method according to any of claims 1 to 6, wherein the calcium polyphosphate material is obtained by addition of a solution containing 14 g/L of calcium chloride or 28 g/L of calcium chloride to a solution containing 10 g/L of sodium polyphosphate.
8. 8. The method according to any of claims 1 to 7, further comprising the step of producing hard amorphous and morphogenetically active polyphosphate nanoparticles, and/or a material containing such nanoparticles.
9. 9. The nanoparticles or the material containing such nanoparticles produced according to claim 8 for use in bone regeneration and repair and in dentistry.
10. 10. The nanoparticles or the material containing such nanoparticles produced according to claim 8 for use in in drug delivery.