GB2543529A

GB2543529A - Osteogenic scaffold based on amorphous calcium-carbonate-polyphosphate

Info

Publication number: GB2543529A
Application number: GB1518575.4A
Authority: GB
Inventors: Ernst Ludwig Georg Muller Werner
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-10-20
Filing date: 2015-10-20
Publication date: 2017-04-26
Also published as: GB201518575D0

Abstract

The present invention relates to a method for the preparation of biologically active amorphous calcium carbonate (ACC)-polyphosphate microparticles. The method comprises preparing an aqueous solution of a polyphosphate salt (polyP) in about 0.1M sodium hydroxide; adding about 0.5mol/L of sodium carbonate; diluting the resulting solution with about 1.5 volumes of deionized water; and mixing the solution with the same volume of an aqueous solution containing calcium chloride such that an about equimolar concentration ratio between calcium ions and carbonate ions results. The mixture is then washed with a lower alkyl ketone at about room temperature, followed by filtering and drying of a precipitate as formed. Preferably the polyphosphate salt is sodium polyphosphate (Na-polyP). Also claimed is an implant material produced by the method, preferably an artificial bone implant. The polyphosphate is thought to block the transformation of the metastable ACC into the most stable crystalline calcium carbonate polymorph. This stabilized ACC composition is claimed to be used as a food or dietary supplement, for use in the treatment of calcium deficiency and for use in the prophylaxis and/or therapy of osteoporosis.

Description

OSTEOGENIC SCAFFOLD BASED ON AMORPHOUS CALCIUM-CARBONATE-

POLYPHOSPHATE

This invention concerns a novel osteogenic material prepared from amorphous calcium carbonate (ACC) and inorganic polyphosphate (polyP). PolyP blocks the transformation of the metastable ACC into the most stable crystalline calcium carbonate polymorph, calcite. The inventive material containing polyP-stabilized ACC and small amounts of vaterite has superior properties compared to a calcite. It exhibits osteogenic activity, in contrast to the biologically inert calcium carbonate polymorph, and induces the expression of marker genes related to bone formation, encoding for alkaline phosphatase and bone morphogenic protein-2. The material turned out not only to be biocompatible but also to support the regeneration of the implant region in animal experiments and has the potential to be used as an osteogenic scaffold for bone implants.

Background of Invention

Currently available bone implant scaffold materials do not sufficiently satisfy the demand to such materials to promote a fast regeneration of the damaged bone tissue. Ideally such materials should be based on the principles underlying of the natural process of bone formation.

The basic building blocks of bone comprise, besides of collagen and water, carbonated apatite [Ca5(PC>4,C03)3(0H)], as well as hydroxyapatite. The crystalline minerals are likely to be formed from amorphous calcium phosphate (ACP) (Wang Y, Von Euw S, Fernandes FM, Cassaignon S, Selmane M, Laurent G, Pehau-Arnaudet G, Coelho C, Bonhomme-Coury L, Giraud-Guille MM, Babonneau F, Azais T, Nassif N (2013) Water-mediated structuring of bone apatite. Nat Mater 12:1144-1153).

Recent evidences suggest that amorphous calcium carbonate (ACC) acts as bioseed for the formation of ACP and carbonated apatite, a material that is formed by carbonic anhydrase(s) (CA), very likely by the soluble CA-II isoform and/or the cell-membrane-associated CA-IX (Wang XH, Schroder HC, Schlossmacher U, Neufurth M, Feng Q, Diehl-Seifert B, Muller WEG (2014) Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate. Calcif Tissue Int 94:495-509; Muller WEG, Schroder HC, Tolba E, Diehl-Seifert B, Wang XH (in press) Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions. FEBS J, in press, doi: 10.1111/febs. 13552). ACC is the least stable polymorph of calcium carbonate, which exists both in amorphous and crystalline phases; among the three major crystalline polymorphs, vaterite, aragonite, and calcite, the metastable vaterite is the thermodynamically least stable form of crystalline CaCC>3 (reviewed in: Meldrum FC, Colfen H (2008) Controlling mineral morphologies and structures in biological and synthetic systems. Chem Rev 108:4332-4432).

Accordingly, bone hydroxyapatite (HA) formation can be subdivided into the following three mechanically distinct phases: 1. Enzymatic formation of ACC bioseeds via carbonic anhydrase(s); 2. Non-enzymatic exchange of carbonate ions by phosphate under formation of ACP; and 3. Transition of ACP to the crystalline phase carbonated apatite/HA.

Inorganic polyphosphate (polyP) which is present in considerable amounts in the blood and in larger extent in blood platelets has been implicated as a phosphate source for the formation of the bone calcium phosphate deposits. From this polymer ortho-phosphate is enzymatically removed via the alkaline phosphatase (ALP) which might serve as donor for bone mineralization. The present state-of-the-art in enzyme-mediated bone formation and the role of polyP has been described in:

Wang XH, Schroder HC, Muller WEG (2015) Polyphosphate as a metabolic fuel in Metazoa: A foundational breakthrough invention for biomedical applications. Biotechnol J. doi: 10.1002/biot.201500168; and

Muller WEG, Neufurth M, Huang J, Wang K, Feng Q, Schroder HC, Diehl-Seifert B, Munoz-Espi R, Wang XH (2015) Non-enzymatic transformation of amorphous CaCC>3 into calcium phosphate mineral after exposure to sodium phosphate in vitro: Implications for in vivo hydroxyapatite bone formation. ChemBioChem 16:1323-1332

In recent years bioinspired as well as biomimetic approaches have been undertaken to develop functional materials capable of promoting bone tissue regeneration. Since collagen and HA are dominant in bone, biomaterials containing chemical-inducers of any of these materials or both have been extensively explored in bone tissue engineering with the hope to accelerate bone regeneration (reviewed in: Bose S, Roy M, Bandyopadhyay A (2012) Recent advances in bone tissue engineering scaffolds. Trends Biotechnol 30:546-554). Calcium phosphate salts in general and HA in particular have been found to be superior as regenerative materials than their non-mineralized counterparts (reviewed in: Guzman R, Nardecchia S, Gutierrez MC, Ferrer ML, Ramos V, del Monte F, Abarrategi A, Lopez-Lacomba JL (2014) Chitosan scaffolds containing calcium phosphate salts and rhBMP-2: in vitro and in vivo testing for bone tissue regeneration. PLoS One 9(2):e87149).

The application of ACC as a potential regeneration-inducing/supporting material has been hampered by the fact that ACC, as such, is not stable. Stabilization of ACC in vivo is regulated by specialized proteins, often in combination with Mg2+, while under in vitro conditions non-biogenic additives, like soluble polycarboxylates, again Mg2+, triphosphate, or polyphosphonate species freeze ACC to a relative stable phase (see: Kellermeier M, Melero-Garcia E, Glaab F, Klein R, Drechsler M, Rachel R, Garcia-Ruiz JM, Kunz W (2010) Stabilization of amorphous calcium carbonate in inorganic silica-rich environments. J Am Chem Soc 132:17859-17866).

In contrast, vaterite is stable enough to allow dissociation and in turn might act as a potential ion buffering system for bone regeneration and by that could modify transformation processes from CaCC>3 to HA (Schroder R, Pohlit H, Schuler T, Panthofer M, Unger RE, Frey H, Tremel W (2015) Transformation of vaterite nanoparticles to hydroxycarbonate apatite in a hydrogel scaffold: Relevance to bone formation. J Mater Chem B 3:7079-7089).

Here the inventor describes that polyP can stabilize the ACC phase. In the inventive procedure, at a level of 5% [w/w], polyP considerably suppresses the transformation of ACC to crystalline CaCC>3 and at a percentage of 10% [w/w] the polymer almost completely blocks this process. This finding was unexpected. Previously it has been reported that soluble Na-polyP, spiked with defined molar ratios of Ca2+, can be processed to solid nanoscaled nano-/microparticles that remain amorphous (Muller WEG, Tolba E, Schroder HC, Wang S, Glafter G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166; Muller WEG, Tolba E, Schroder HC, Diehl-Seifert B and Wang XH (2015) Retinol encapsulated into amorphous Ca2+ polyphosphate nanospheres acts synergistically in MC3T3-E1 cells. Eur J Pharm Biopharm 93:214-223). It could not be expected that Ca-polyP can act as a stabilizer for metastable ACC; see also: GB1420363.2. Morphogenetically active calcium polyphosphate nanoparticles. Inventor: Muller WEG; and GB1502116.5. Synergistically acting amorphous calcium-polyphosphate nanospheres containing encapsulated retinol for therapeutic applications. Inventor: Muller WEG.

PolyP acts as a morphogenetically active inorganic molecule on bone cells and induces their mineralization, as previously disclosed by the inventor (see above [GB1420363.2 and GB1502116.5]; as well as: GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders Inventors: Muller WEG, Schroder HC, Wang XH). In the present application, the inventor additionally shows that CaCC>3, containing 5 or 10% [w/w] of polyP, comprises osteogenic potential in SaOS-2 cells as well as in human mesenchymal stem cells (MSC) by inducing ALP and bone morphogenic protein 2 (BMP2) gene expression. Even more surprising and unexpectedly, ACC enhanced the stimulatory effect of polyP on BMP2 expression in a “synergistic” way. Moreover, the inventor demonstrates that the inventive ACC/polyP hybrid material is biocompatible and supports regeneration in vivo, making it to a promising scaffold material for bone replacement/implants.

Summary of the invention

In human bone, ACC is formed as a precursor of the crystalline carbonated apatite/HA. The inventor surprisingly found that the metastable ACC phase can be stabilized by polyP. This polymer is used as a phosphate source for the non-enzymatic carbonate/phosphate exchange. The inventor demonstrates that polyP suppresses the transformation of ACC into crystalline CaCC>3 at a percentage of 5% [w/w] (termed “CCP5") with respect to CaCC>3 and almost completely at 10% [w/w] (termed “CCP10"). They show that both preparations are amorphous, but also contain small amounts of vaterite, as revealed by XRD, FTIR and SEM analyses.

The inventive ACC/polyP particles, for example “CCP5” and “CCP10”, do not affect the growth/viability of bone-forming SaOS-2 cells. In addition, using cell culture and Ca2+ release experiments the inventor shows that the CaCCh particles formed in the presence of polyP are less stable and become disintegrated compared with particles in the absence of the polymer.

The inventor demonstrates that the ACC/polyP particles according to this invention exhibit osteogenic activity, in contrast to calcite. They induce the expression of the gene encoding for alkaline phosphatase in SaOS-2 cells as well as in human mesenchymal stem cells (MSC), as well as the expression of bone morphogenic protein 2 gene.

Furthermore, the inventor demonstrates, in in vivo studies in rats, using PLGA microspheres containing the inventive ACC/polyP material and inserted in the muscles of the back of the animals, that the encapsulated ACC/polyP particles are not only biocompatible but also support the regeneration of the implant region.

It is surprising that ACC containing small amounts of vaterite has osteogenic potential and superior properties compared to a biologically inert calcite. Based on these properties the inventive material represents a promising scaffold material for bone implants.

The following patent applications on polyP are deemed relevant: GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Muller 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. WO 2012/010520. Hydroxyapatite-binding nano- and microparticles for caries prophylaxis and reduction of dental hypersensitivity. Inventors: Muller WEG, Wiens M. GB 1420363.2. Morphogenetically active calcium polyphosphate nanoparticles. Inventor: Muller WEG. GB1502116.5. Synergistically acting amorphous calcium-polyphosphate nanospheres containing encapsulated retinol for therapeutic applications. Inventor: Muller WEG. GB1510772.5. Method for preparation of teeth coatings with morphogenetic activity. Inventor: Muller WEG. GB1513011.5. Method for coating of titanium alloy with morphogenetically active Ca-polyphosphate microparticles. Inventor: Muller WEG. GB1515515.3. Amorphous inorganic polyphosphate-calcium-phosphate particles inducing bone formation. Inventor: Muller WEG.

Detailed description of the invention

At physiological conditions, the turnover of HA is, if at all, very low, while the transition from ACC to calcium phosphate runs readily. The metastable ACC undergoes transformation to vaterite, and/or aragonite and calcite, unless this reactions chain is not blocked by inorganic or organic molecules (reviewed in: Muller WEG, Neufurth M, Huang J, Wang K, Feng Q, Schroder HC, Diehl-Seifert B, Munoz-Espi R, Wang XH (2015) Non-enzymatic transformation of amorphous CaCC>3 into calcium phosphate mineral after exposure to sodium phosphate in vitro: Implications for in vivo hydroxyapatite bone formation. ChemBioChem 16:1323-1332).

The inventor succeeded to fabricate an ACC polymorph that contains a small amount of vaterite. They added the Na+ salt of the anionic polymer polyP to the precursors of CaCC>3 (CaCb and NaiCCh) during the synthesis of ACC (Figure 1). Surprisingly, the inventor found that polyP prevented, at a final concentration of 10%, the transformation process of ACC to its crystalline polymorphs vaterite, aragonite and calcite almost totally.

Both the CaCC>3 solids and the polyP physiological metabolite, tested separately, have osteogenic potential and could serve as constituents of bioactive bone grafts. In turn, the scaffold developed in the present study exploits not only the morphogenetic potential of polyP but also utilizes the property of this polymer to freeze the CaCCE solids at the ACC stage. This material is superior to calcite with respect to the osteogenic activity; it strongly induces the expression of the gene encoding for ALP, a known marker for bone formation via stimulation of osteoblasts. This result has been obtained from studies with bone-like SaOS-2 cells and also with MSC.

Moreover, the inventor demonstrated that ACC/polyP strongly upregulates the expression of BMP2, an inducer of bone formation by osteoblasts. Even more important: They surprisingly found that ACC increases the induction of BMP2 expression by polyP in a “synergistic” way, resulting in a faster rise of the BMP2 transcript levels. It can be expected that this effect of the inventive ACC/polyP microparticles will result in a faster healing of bone defects.

The ACC/polyP material is not only biocompatible but also supports the cellular regeneration of the impaired implant region. To assess the biocompatibility of the ACC/polyP material in vivo, the inventor encapsulated the inventive material into PLGA microspheres. In parallel, control spheres remained without ACC/polyP. The pearls/microspheres were inserted in the muscles of the back of rats. After an observation period of 2, 4, and 8 weeks tissue samples were taken from the rats and inspected microscopically after slicing and staining with Mayer's hematoxylin. The inspections show that in the animals with the microspheres containing the ACC/polyP material, an advanced repopulation of the implant region with cells became evident after 4 weeks and 8 weeks, resp. In contrast, the microspheres lacking ACC/polyP were devoid of any cells. These results were supported by measurements of the hardness (median RedYM stiffness) of tissue samples of the implant region, which revealed a significant increase by 1.8-fold compared to control after a period of 8 weeks (81% of the value measured in muscle samples before implantation).

The preferred method for the preparation of the inventive ACC/polyp material developed by the inventor comprises the following steps.

a) Preparation of a solution containing a polyP salt in, for example, one liter of 0.1 M NaOH b) Addition of 0.5 mol/L ofNa2CC>3 to this solution c) Dilution of the resulting solution with the 1.5 volume of deionized water d) Rapid mixing of this solution with the same volume of an aqueous solution containing 0.5 mol/L of CaCl2-2H20 (resulting in an equimolar concentration ratio between calcium ions and carbonate ions); and e) Filtration and drying of the precipitate after washing with acetone at room temperature “About” shall mean +/- 10% of the value as indicated.

The preferred concentration of the polyP salt in the 0.1 M NaOH solution used for the preparation of the inventive ACC/polyP microparticles is in the range of 0.001 mol/L to 1.0 mol/L, preferably in the range of 0.01 mol/L to 0.1 mol/L (based on phosphate units).

Optimal results were achieved, if the concentration of the polyP salt in the 0.1 M NaOH solution used for the preparation of the inventive ACC/polyP microparticles is 0.025 mol/L or, even better, 0.05 mol/L (based on phosphate). The resulting preparations are termed “CCP5” and“CCP10”, respectively. The polyP salt is preferably Na-polyP.

The chain length of the polyP can be in the range of 3 to about 1000 phosphate units. Optimal results are achieved with polyP molecules with an average chain length of approximately 10 to about 100 phosphate units, and within this range optimally about 40 phosphate units. A further aspect of this invention concerns the finding that the inventive ACC/polyP particles exhibit osteogenic activity by inducing the expression of the genes encoding for ALP and for BMP2 in bone-forming SaOS-2 cells, as quantified by qRT-PCR.

The expression ALP gene is a reliable marker for differentiation and proliferation (Prins HJ, Braat AK, Gawlitta D, Dhert WJ, Egan DA, Tijssen-Slump E, Yuan H, Coffer PJ, Rozemuller H, Martens AC (2014) In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells. Stem Cell Res 12:428-440). In addition, ALP provides the ortho-phosphate to the osteoblasts for the formation of the bone HA biomineral (Golub EE, Boesze-Battaglia K (2007) The role of alkaline phosphatase in mineralization. CurrOpin Orthop 18:444-448). BMP2 is a potent inducer of differentiation of osteoblasts and plays an important role in the development of bone and cartilage (Chen D, Zhao M, Mundy GR (2004) Bone morphogenetic proteins. Growth Factors 22:233-241).

The ACC/polyP particles are biodegradable and display superior morphogenetic activity, compared to calcite which is rapidly formed from ACC in the absence of polyP.

The inventor demonstrated that, using an ACC formulation with 10% [w/w] polyP (“CCP10”), the release of Ca2+, and simultaneously of CC^2-, is fast during the first 48 h of incubation, allowing the release of the biologically active anions CO3 and PO4 from the scaffold. The ortho-phosphate will be enzymatically liberated from polyP, as previously demonstrated by the inventor (Muller 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 level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). In _ turn, the CO3 as well as the HCO3' anions induce the mineralization process onto bone- forming cells, very likely via modulating the efficiency of the HCOf/Cl" anion exchanger, inserted into the plasma membrane not only of osteoclasts but also of osteoblasts. A further aspect of the inventive method is the application of this method for the fabrication of biologically active implant materials. Another aspect of the inventive method is the application of this method for preparation of artificial bone implants that stimulate osteoblast cell activity. Furthermore, another aspect of the invention described herein is an implant prepared by application of the inventive method.

The inventive method to stabilize the metastable ACC with polyP also allows the application of ACC/polyP particles as a dietary supplement. As demonstrated by the inventors, e g. in Figure 8, these particles, e.g. “CC10” release calcium over prolonged incubation periods, in contrast to the crystalline calcite polymorph.

Therefore, the ACC/polyP particles according to this invention can also be used as a dietary supplement for treatment of calcium deficiency.

Accordingly, another aspect of this invention is the use of the stabilized ACC (ACC/polyP) as a dietary supplement for prophylaxis/therapy of osteoporosis.

Calcium plays an important role in many biological processes, for example in intracellular signalling, muscle contraction, neuronal transmission, and vasoconstriction/vasodilatation. ACC stabilized by polyP can serve as an easily available food supplement for calcium for prophylaxis/therapy of many pathological conditions, associated with disturbances of calcium metabolism. _

Based on recent findings on the CaCC>3 nature of the bioseeds, the anion exchange of CO3 by P043' and the supply of ortho-phosphate from polyP the following series of mechanistically distinct processes can be described during bone formation (Figure 2). In the first phase during bone mineral deposition, like in the endochondral ossification, the cartilage in the metaphysis comprising the growth center between the epiphysis and the diaphysis of the long bone, calcifies. It is likely that this process of calcification is enzymatically driven by CA-II and/or CA-IX. Secondly, platelets that accumulate besides of the osteoblasts both in regions of bone formation and also at bone fracture sites release polyP into the extracellular space where the polymer undergoes ALP-mediated exohydrolysis under the release of orthophosphate. Thirdly, the available phosphate units, formed in a spatial vicinity to the bioseed synthesis, serve as the source for the formation of ACP.

As sketched in this scheme (Figure 2) the inventive material is a promising biocompatible and osteogenic scaffold that provides both the substrate for the bioseed development (CaCC>3 [CO3 ]) and for the subsequent transformation to the calcium phosphate (polyP [PO4 ]).

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 scheme of the preparation of calcite and CaCC>3 supplemented with polyP. The inserts show the SEM images of the respective product.

Figure 2 shows a scheme of the process of endochondral ossification and the proposed phases of bone mineral deposition. After penetration of blood vessels the hyaline cartilage at the primary ossification centers in the diaphysis starts to calcify. The formation of spongy bone at the secondary ossification centers in the epiphyses starts later. Two regions of hyaline cartilage remain, the articular cartilage at the surface of the epiphysis and the epiphyseal plate (growth region) between the epiphysis and diaphysis. The mineral deposition in the growth region is subdivided into phase I: Amorphous calcium carbonate (ACC) bioseeds are formed mediated by the membrane-associated CA-IX; phase II: PolyP released from platelets undergoes ALP-mediated hydrolysis under formation of ortho-phosphate for the carbonate-phosphate transfer reaction; and phase III: The phosphate units are used for the (carbonated) calcium phosphate formation.

Figure 3 shows the FTIR spectra of calcite as well as “CCP5” (0.05 g of Na-polyP/assay) and “CCP10” (0.1 g of Na-polyP). The major distinguishing vibration regions/signals for calcite versus ACC, the vibration range for O-H (around 3250 cm"1) and the asymmetric V2 line for CO3 at 725 cm'1 are circled.

Figure 4 shows the XRD pattern obtained from (A) calcite and (B) the two CaCC>3 preparations, containing two different concentrations of polyP, “CCP5” or “CCP10”. The characteristic signals are highlighted and marked with the respective Miller indices, given in parentheses. Please note the different scale of the ordinate captions between (A) and (B).

Figure 5 shows the morphology of the solids formed from CaCl2»2H20 and Na2C03; SEM analysis. (A and B) In the absence of polyP calcite crystals are formed. This morphology is changed after addition of polyP during the precipitation process. (C and D) In the presence of 5% polyP, the “CCP5” particles show a spherical appearance. (E and F) At 10% polyP, “CCP10”, the solids show a platelet-like shape, which corresponds to vaterite crystals (Vat).

Figure 6 shows the cell viability/growth of SaOS-2 cells after cultivation for 2 d and 3 d, respectively, in the absence of any CaCCb solids (control; open bars) or after exposure to 50 pg/mL of “CCP5” (left hatched bars), “CCP10” (right hatched bars) or calcite (filled bars). After terminating the cultivation, the assays were subjected to the MTT assay and the absorbance at 650 nm was determined. Data represent means ± SD of ten independent experiments.

Figure 7 shows the growth pattern of SaOS-2 cells in the presence of 50 pg/mL of “CCP10” (A and B) or calcite (C and D) after a 3 d incubation period. The cells were identified by phase contrast/Nomarski optics. The CaCC>3 particles in the assays became visible in the phase contrast images and are marked (> <).

Figure 8 shows the release of Ca2+ from the CaCC>3 particles. “CCP10” or calcite was incubated in Tris-HCl buffer (pH 7.4) for various time periods and the supernatant was analyzed for Ca2+ concentration. The results are means from 6 parallel experiments; * P < 0.01.

Figure 9 shows the steady-state expression levels of the ALP gene both in (A) SaOS-2 cells and in (B) MSCs. The cells remained without any CaCC>3 solids (control), or were exposed to 50 pg/mL of “CCP5” (left hatched bars), “CCP10” (right hatched bars), or calcite (filled bars). After the 3 d pre-incubation period in the absence of MAC the cells were continued to be incubated in the absence of MAC (minus MAC) or were exposed to MAC (plus MAC). After the 7 d incubation the cells were harvested, their RNA extracted and subjected to qRT-PCR analyses. The expression values are given as ratios to the reference gene GAPDH. The results are means from 5 parallel experiments; * P < 0.01; the values are computed against the expression measured in cells during seeding.

Figure 10 shows the steady-state expression levels of the BMP2 gene both in SaOS-2 cells in the presence of “CCP10” and polyP (Ca complex). The cells remained without any additive (control), or were exposed to 50 pg/ml of “CCP10” (right hatched bars), 5 pg/ml of polyP (Ca2+ complex; 50 μΜ phosphate units; cross hatched bars), or 50 pg/ml of calcite (filled bars). After the 3 d pre-incubation period in the absence of MAC the cells were continued to be incubated in the presence of MAC for up to 7 days, and the expression BMP2 was analyzed by qRT-PCR. The expression values are given as ratios to the reference gene GAPDH. The results are means from 5 parallel experiments; * P < 0.01; the values are computed against the expression measured in cells during seeding (day 0); P < 0.01 (only for “CCP10”); the values are computed against the expression measured in cells with polyP (Ca2+ complex) at the respective incubation periods.

Figure 11 shows the morphology of the microspheres; (A) control spheres “cont-mic” and (B) polyP loaded spheres, “polyP-mic”.

Figure 12 shows the implantation of the microspheres (A and B) into muscle of the back of a test animal. (C to H) Cytochemical analysis of the regions around the microspheres after a period of 2 weeks (C, D), 4 weeks (E, F) and 8 weeks (G, H) of transplantation. The animals received either “cont-mic” microspheres (C, E, G) or microspheres, filled with “CCP10”; staining of the slices was performed with hematoxylin. Microspheres (mic) and muscle areas are marked (m).

Examples

In the following examples, the inventive method described only for polyP molecules with a chain length of 40 phosphate units. Similar results can be obtained by using polyP molecules with lower and higher chain lengths, such as between 100 to 20 units.

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 Ca-carbonate microparticles

Ca-carbonate (CaCCL) is prepared by direct precipitation in aqueous solutions (at room temperature), using CaCl2*2H20 solution and Na2C03 solution at equimolar concentration ratio between Ca2+ and CO32' through rapid mixing; scheme in Figure 1.

To study the effect of polyP on precipitated CaCC>3 the solution of 20 ml of 0.1 M NaOH is supplemented with 0.05 g or 0.1 g of Na-polyP to which 1.05 g of Na2CC>3 is added; subsequently this solution is diluted with 30 mL of deionized water. Then 50 mL water, containing 1.47 g CaCl2*2H20, is added. By this, 5% [w/w] (addition of 0.05 g Na-polyP) and 10% [w/w] (0.1 g Na-polyP) of polyP, respectively, is added to the CaCCL precipitation assay. The suspensions obtained are filtrated, washed with acetone and dried at room temperature. The samples are termed “CCP5” (0.05 g Na-polyP per CaCCL precipitation assay) or “CCP10” (0.1 g). X-rav diffraction analyses X-ray diffraction (XRD) experiments can be performed as described by (Raynaud S, Champion E, Bernache-Assollant D, Thomas P (2002) Calcium phosphate apatites with variable Ca/P atomic ratio I. Synthesis, characterisation and thermal stability of powders. Biomaterials 23:1065-1072).

Fourier transformed infrared spectroscopy

Fourier transformed infrared spectroscopic (FTIR) analyses can be performed, for example, with micro-milled (agate mortar and pestle) mineral powder in an ATR (attenuated total reflectance)-FTIR spectroscope/Varian 660-IR spectrometer (Agilent), fitted with a Golden Gate ATR unit (Specac).

Scanning electron microscopic studies

Scanning electron microscopic (SEM) analyses can be performed, for example, with an SU 8000 instrument (Hitachi High-Technologies Europe), at low voltage (1 kV).

Release of Ca from the CaCCL particles

In separate assays 100 pg/ml of either cal cite or “CCP10” are added into an Eppendorf tube containing 1 mL of 1 M Tris-HCl (pH 7.4). After incubating at room temperature for 2 h, 2 d, 3 d and 8 d samples of 100 μΐ are taken, centrifuged and the supernatant analyzed for Ca2+ concentration. The determination can be performed, for example, with the photometric test kit (e.g., Millipore/Merck Chemicals; article no. 100858 “Calcium Cell Test”). The blank values are subtracted from the test assays.

Cultivation of SaOS-2 cells

The human osteogenic sarcoma cells SaOS-2 are cultured in McCoy’s medium (Biochrom-Seromed), supplemented with 2 mM L-glutamine and enriched with 15% heat-inactivated fetal calf serum (FCS). Where indicated, the cultures are first incubated for a period of 3 d in the absence the mineralization-activating cocktail (MAC). Then the cultures are continued to be incubated for additional 7 d in the absence or presence of the MAC, comprising 5 mM β-glycerophosphate, 50 mM ascorbic acid and 10 nM dexamethasone to induce biomineralization.

Cell viability assay

SaOS-2 cells are seeded into the 6-well plates and cultured for 3 d in McCoy’s medium/15% FCS. Where indicated under Examples, the cultures are supplemented with 30 pg (in 3 mL) of the respective CaCC>3 preparation, as described and the cell viability was determined with 3-[4,5-dimethyl thiazole-2-yl]-2,5-diphenyl tetrazolium (MTT; #M2128, Sigma) (Wang XH, Schroder HC, Schlossmacher U, Neufurth M, Feng Q, Diehl-Seifert B and Muller WEG (2014) Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate. Calcif Tissue Int 94:495-509).

Human mesenchymal stem cells

The expression of ALP is determined, in parallel to the one in SaOS-2 cells, with human mesenchymal stem cells (MSC). The cells are isolated and cultivated using established methods (Wang XH, Schroder HC, Grebenjuk V, Diehl-Seifert B, Mailander V, Steffen R, SchloBmacher U, Muller WEG (2014) The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for differentiation of human multipotent stromal cells: Potential application in 3D printing and distraction osteogenesis. Marine Drugs 12, 1131-1147).

Quantitative real-time polymerase chain reaction: ALP expression

The SaOS-2 or MSCs cells are pre-cultivated for 3 d in medium/serum. Then the cultures are split and incubated either in the absence of any CaCC>3 (control) or with 50 pg/mL of “CCP5”, “CCP10” or calcite and the cultivation is continued for an additional 7 d in the absence or presence of MAC. Subsequently, the cells are harvested, RNA extracted and subjected for quantitative real-time RT [reverse transcriptionj-polymerase chain reaction (qRT-PCR). The following primer pairs are used: ALP [alkaline phosphatase; NM 000478.4] Fwd: 5 '-TGC AGTACGAGCTGAACAGGAACA-3' [ntmi to ntii64] and Rev: 5'-TCCACCAAATGTGAAGACGTGGGA-3' [ntnis to nti395; product size of 278 bp], BMP2 [bone morphogenic protein 2; NM 001200.2] Fwd: 5’-ACCCTTTGTACGTGGACTTC-3’ [nti68i to ntnoo] and Rev: 5’-GTGGAGTTCAGATGATCAGC-3’ [ntnss to ntiso4, 124 bp], and as reference GAPDH [glyceraldehyde 3-phosphate dehydrogenase; NM 002046.3] Fwd: 5'-CCGTCTAGAAAAACCTGCC-3' [nt929 to nt947] and Rev: 5'- GCCAAATTCGTTGTCATACC-3' [ntn45 to ntn26; 199 bp].

Preparation of PLGA-based microspheres

The microspheres, used for the animal experiments are produced as described in details (Wang SF, Wang XH, Draenert FG, Albert 0, Schroder HC, Mailander V, Mitov G and Muller WEG (2014) Bioactive and biodegradable silica biomaterial for bone regeneration. Bone:67:292-304). The microspheres lacking CCP10 are fabricated with 4 ml of a PLGA/dichloromethane solution (volume ratio 1:5); they are termed “cont-mic” (PLGA: poly(D,L-lactide-co-glycolide); lactide:glycolide [75:25]; mol.wt. 66,000-107,000). For the fabrication of microspheres containing CaC03/polyP, “CCP10” microspheres (“polyP-mic”) are added to the PLGA/dichloromethane mixture at a concentration of 20%. The viscous reaction mixture is pressed through a syringe with an aperture of 0.8 mm. By this approach, microspheres with an average diameter of ~ 820 pm are obtained.

The content of polyP in the microspheres is determined as described (Mahadevaiah MS, Kumar Y, Abdul-Galil MS, Suresha MS, Sathish MA, Nagendrappa G (2007) A simple spectrophotometric determination of phosphate in sugarcane juices, water and detergent samples. E-Joumal of Chemistry 4:467-473).

Determination of the mechanical properties

The mechanical properties of the microspheres and of the muscle tissue of the implant region (regenerating zone) can be determined, for example, with a nanoindenter, equipped with a cantilever that has been fused to the top of a ferruled optical fiber (Wang SF, Wang XH, Draenert FG, Albert O, Schroder HC, Mailander V, Mitov G, Muller WEG (2014) Bioactive and biodegradable silica biomaterial for bone regeneration. Bone 67:292-304). Using this technique the reduced Young’s modulus (RedYM) is quantified.

Compatibility studies in vivo

In the experiments described under Examples, Wistar rats of (male) genders, weighting between 240 g and 290 g (age: two months) are used; 3 animals from each group are used. Diet and water are provided ad libitum during the total experimental period. Prior to surgery the animals are treated with Ciprofloxacins 10 ml/kg of body weight for antibiotic prophylaxis. Then the animals are narcotized with chlorpromazine/Ketamin via intramuscular injection. Following routine disinfection incisions of -1 cm are made in the right and left half, perpendicularly to the vertebral axis at the upper limbs level. Following skin incision, the muscle is incised and dissected to accomodate the microspheres. The microspheres (-20 mg in a volume of 100 pL) are introduced into the muscle and stabilized there in the deeper layer (Vidya S., Parameswaran A., Sugumaran VG (1994) Comparative evaluation of tissue. Compatibility of three root canal. Sealants in Rattus norwegicus: A Histopathological study. Endodontology 6: 7-17). After a period of 2, 4, or 8 weeks the animals are sacrificed and the specimens with the surrounding tissue are dissected and sliced. The samples are inspected macroscopically for inflammation, infection and discoloration.

The samples are fixed in formalin, sliced, stained with Mayer's hematoxylin and then analyzed by optical microscopy (e.g., with an Olympus AHBT3 microscope).

Statistical analysis

After finding that the values follow a standard normal Gaussian distribution, the results can be statistically evaluated using paired Student’s /-test.

Effect of polvP on cal cite formation: FTIR and XRD spectra

For all CaCOs solids the following FTIR signals were recorded: Vi (symmetric stretching) at -1080 cm-1; V2 (out of-plane bending) at -870 cm-1; v3 (doubly degenerate planar asymmetric stretching) at -1400 cm-1 and V4 (doubly degenerate planar bending) at 700 cm-1. The published IR data (Rodriguez-Bianco JD, Shaw S and Benning LG (2011) The kinetics and mechanisms of amorphous calcium carbonate (ACC) crystallization to calcite, via vaterite.

Nanoscale 3:265-271) which were obtained with FTIR/KBr pellets, include peaks located at around 1400 cm-1 (v3), 876 cm-1 (V2), and 714 cm-1 (V4) for calcite and 1090 cm-1 (vi), 870 cm-1 (V2), and 745 cm'1 (V4) for vaterite (Figure 3). Our samples prepared in the absence of polyP are characterized as follows. For calcite the typical vibration bands 1391, 872 and 712 cm'1 were recorded, while the samples prepared in presence of polyP showed the adsorption peaks at 1398, 869 and 742 cm'1 for “CCP5” polyP as well as the bands at 1398, 869 and 741cm'1 for “CCP10” proving the formation of vaterite. It is apparent that the strength of the signal for vaterite around 741 cm'1 decreases at higher content of polyP in the fabricated CaC03 solids, “CCP10” versus “CCP5”. This is indicative for the formation of ACC. Beside of the CO3 absorption peaks, the peaks from 1200 cm to 950 cm correspond to the absorption peaks of phosphate in polyP.

The above result was confirmed with XRD in which the diffraction peaks of the sample prepared in absence of polyP, at approximately 23°, 30°, 36° and 40°, is given; those signals correspond to calcite. In contrast, the samples prepared in the presence of polyP (“CCP5") showed peaks at approximately 24°, 27°, 32° and 44°, which also reflect the existence of vaterite. Furthermore, these data prove that the CaCC>3 solids, prepared in the absence of polyP were pure calcite (Figure 4A), while the “CCP5" samples were composed of vaterite in association with ACC, as can be deduced from the low intensities of the signals and also the broadening of the diffraction peaks for sample "CCP5" (Figure 4B). In consequence, the increase of the amount of polyP, as in “CCP10", decreases the rate of transformation of ACC to vaterite. This is evident from the XRD pattern of "CCP10" sample which exhibits the amorphous nature of the sample, but also containing small amounts of vaterite.

Morphology of the solids formed

The solids formed by precipitation from CaCl2*2H20 and Na2CC>3 were studied by SEM. The photomicrographs of the particles, formed in the absence of polyP, show the typical crystalline calcite, the rhombohedral crystals surrounded by {104} faces; Figure 5A and B. The size of the particles varies between 5.3 to 8.9±2.4 pm. In contrast, those solids formed from CaCl2*2H20 and Na2CC>3 in the presence of polyP show a different morphology. At the lower polyP concentration, the “CCP5” particles show a spherical appearance with an average size of the spherical crystals of 9.4±3.7 pm (Figure 5C and D); we attribute these particles to vaterite. They are surrounded by very abundantly accumulating small nanoparticles with a size range of 100 to 200 nm, which we assigned as ACC. Increasing the polyP, as in “CCP10”, the globular particles disappear and are replaced by penta/hexagonal flake shaped particles, 5-10 pm sized vaterite (Figure 5E and F).

Effect of CaCCF samples on cell growth/viability

The cell growth/viability of SaOS-2 cells after exposure to the CaCC>3 preparations was determined by applying of the MTT assay (see above). The CaCC>3 samples were added at a concentration of 50 pg/mL to the cells. In parallel, a control assay lacking any CaCC>3 solids was performed (Figure 6). The results revealed that the increase in cell growth/viability from 0.70±0.11 at time 0 to approximately 1.1 absorbance units after 2 d and 2.35 units after 3 days changes only non-significantly among the control assays and the three CaCC>3 series (“CCP5”, “CCP10” or calcite).

Stability of the CaCCF solids in the culture medium

SaOS-2 cells grow in an adherent manner (Pautke C, Schieker M, Tischer T, Kolk A, Neth P, Mutschler W, Milz S (2004) Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res 24:3743-3748). If the cultures are exposed to either calcite or “CCP5” solids the growth behavior onto the surfaces of the culture dishes is similar in assays containing either “CCP10” (Figure 7A and B) or calcite (Figure 7C and D). After 3 d the cells grow almost to confluency. However, it is remarkable that the number of mineral particles, floating in the culture medium, after this period of time, is strongly reduced in the assays containing “CCP10”, compared to those seen in calcite assays. This observation can be taken as an indication that the “CCP10” particles undergo dissolution during the 5 d incubation period. This finding is supported by the determination revealing that after 3 d incubation period in simulated body fluids (Oyane A, Kim HM, Furuya T, Kokubo T, Miyazaki T, Nakamura T (2003) Preparation and assessment of revised simulated body fluids. J Biomed Mater Res A 65:188-195) the amount of calcite particles decreases only by 5-10%, while only 35% of the “CCP10” particles can be recovered, as measured on the basis of sedimentable carbonate (data not shown).

Release of Ca2+ from the CaCCE particles

In separate assays either calcite or “CCP10” was added into an 1 mL assay buffered with 1 M Tris-HCl (pH 7.4). While almost no Ca2+ is released from the calcite sample, already 6.8±1.1 pg/ml (68% of the total Ca2+ in the reaction mixture) was released from the “CCP10” after a period of 48 hr; this extent increases further during the total 192 hr of incubation (Figure 8).

Expression of ALP in SaOS-2 cells as well as in MSCs

The morphogenetic activity of the CaCCb samples towards SaOS-2 cells as well as the MSCs was determined in the absence and presence of MAC. Using SaOS-2 cells it was determined that in the absence of MAC the expression ratio between the ALP and the reference gene expression (GAPDH) significantly increases from 0.31±0.9 to ~0.6. Within the sets of experiments without the MAC no significant differences are measured, irrespectively of the absence (control) or presence of the CaCC>3 samples in the assays (Figure 9A). However, if the expression ratio (ALP.GAPDH) is determined in MAC activated cells then a significant increase of the ratio to 0.87±0.12 (in the control), to 1.74±0.23 (“CCP5”) or to 1.86±0.29 (“CCP10”) is measured. In contrast, no response of the cells in assays with calcite is measured (0.14±0.05). A similar expression pattern of the ALP, if correlated to the reference GAPDH gene expression, is found if MSCs are used for the experiments. Again, in the presence of the MAC a significant increase of the expression ratio is seen assays in the absence of any CaCC>3 solid, as well as in the presence of both “CCP5” and “CCP10”. No inducing effect is determined in cells exposed to calcite (Figure 9B).

Expression of BMP2 in SaOS-2 cells

The expression level of BMP2 in response to “CCP10” and polyP (Ca complex) was determined by qRT-PCR analysis. SaOS-2 cells were incubated in mineralization medium (McCoy’s medium/MAC) for up to 7 days. “CCP10” (50 pg/ml), polyP (Ca2+ complex; 5 pg/ml; corresponding to 50 μΜ with respect to phosphate) or calcite (50 pg/ml) were added to the cultures at the beginning of the experiments. After termination RNA was extracted from the cultures and subjected to qRT-PCR. The expression of the housekeeping gene GAPDH was used as reference. As shown in Figure 10 the expression levels of BMP2 significantly increased 3 to 7 days after addition of “CCP10” or polyP (Ca2+ complex). However, the increase in BMBP2 expression was much faster for “CCP10” compared with polyP (Ca complex). Maximum levels of BMB2 gene expression were already achieved after an incubation period of 3 days for “CC10P”, while the expression of this gene induced by polyP (Ca complex) reached maximum levels (and similar levels compared with “CCP10”) only after a longer, 5 day incubation period. At day 3 the expression level of BMP2 in response to “CCP10” was significantly (about 2-fold) higher compared with polyP (Ca2+ complex), indicating a “synergistic” effect of both components. After 7 days, the expression levels decreased for both “CCP10” and polyP (Ca2+ complex) but remained still significant. Calcite that is formed from metastable ACC in the absence of polyP did not show any stimulatory effect on BMP2 gene expression.

Microspheres, used for the animal studies

The control spheres, the “cont-mic” had a size of (-845 μιη [820±60 μηι]; n=50), while those containing polyP were insignificantly slightly smaller (-838 μιη [816±65 μιη]); Figure 11A and B. The texture of the microspheres surfaces was porous and had pores of 25-30 nm (not shown here). The content of polyP in the “polyP-mic” was 7.26±0.92%. The hardness of the particles was determined for both the “cont-mic” and the “polyP-mic”; the median RedYM stiffness of 26.99±6.22 kPa for the “cont-mic” and 23.96±23.96 kPa for the “polyP-mic” microspheres.

Compatibility studies in rats

The microsphere samples (20 mg), both “cont-mic” and “polyP-mic” were inserted in the muscles of the back of rats, as described under “Materials and Methods” (Figure 12A and B). After 2, 4, or 8 weeks tissue samples with the microspheres were removed, sliced and stained with hematoxylin solution. In none of the excised specimens any sign for a histopathological alteration could be seen in all of the three sacrificed laboratory animals per group both for the “cont-mic” (Figure 12C, E and G) and the “polyP-mic” series (Figure 12D, F and H). Typical images for the sample sections, stained with hematoxylin are shown. It is evident that after 2 weeks the regions, where the microspheres had been placed into the muscle, a few cells are scattered within the microsphere areas (Figure 12C and D). However, after a 4 (Figure 12E) and 8 weeks (Figure 12G) stay of the “cont-mic” microspheres in the muscle area they appear to be empty or close to be cell-free. In contrast, within the “polyP-mic” microspheres already after 4 weeks (Figure 12F) an accumulation of the cells within the spheres are evident. After 8 weeks the spheres are almost filled with infiltrating cells (Figure 12H).

Determinations of the hardness of the implant region after 8 weeks revealed a significant increase of the median RedYM stiffness of 33.13±7.97 kPa for the “cont-mic” and 60.11±12.13 kPa for the “polyP-mic” microspheres. The muscles of the back of rats before implantation have a median RedYM stiffness of 74.40±14.33 kPa. SEQUENCE LISTING <110> Muller, Werner E.G.

<120> OSTEOGENIC SCAFFOLD BASED ON AMORPHOUS CALCIUM-CARBONATE-POLYPHOSPHATE

<130> M3 3110GB <160> 6 <170> Patentln version 3.5 <210> 1 <211> 24

<212> DNA <213> Homo sapiens <400> 1 tgcagtacga gctgaacagg aaca 24 <210> 2 <211> 24

<212> DNA <213> Homo sapiens <400> 2 tccaccaaat gtgaagacgt ggga 24 <210> 3 <211> 20

<212> DNA <213> Homo sapiens <400> 3 accctttgta cgtggacttc 20 <210> 4 <211> 20

<212> DNA <213> Homo sapiens <400> 4 gtggagttca gatgatcagc 20 <210> 5 <211> 19

<212> DNA <213> Homo sapiens <400> 5 ccgtctagaa aaacctgcc 19 <210> 6 <211> 20

<212> DNA <213> Homo sapiens < 4 0 0> 6 gccaaattcg ttgtcatacc 20 - 1 -

Claims

1. Method for the preparation of biologically active amorphous calcium carbonate (ACC)-polyphosphate microparticles, comprising the following steps: a) Preparing of an aqueous solution of a polyphosphate salt in about 0.1 M sodium hydroxide, b) Adding of about 0.5 mol/L of sodium carbonate to said solution, c) Diluting of the resulting solution with about 1.5 volumes of deionized water, d) Mixing of said solution with the same volume of an aqueous solution containing calcium chloride, so that an about equimolar concentration ratio between calcium ions and carbonate ions results, e) Washing with a lower alkyl ketone, such as acetone, at about room temperature, and f) Filtering and drying of a precipitate as formed.

2. The method according to claim 1, wherein the concentration of the polyphosphate salt in step a) is in the range of about 0.001 mol/L to about 1.0 mol/L, preferably in the range of about 0.01 mol/L to about 0.1 mol/L, based on phosphate.

3. The method according to claim 2, wherein the concentration of the polyphosphate salt in step a) is about 0.025 mol/L or about 0.05 mol/L, based on phosphate.

4. The method according to any of claims 1 to 3, wherein said polyphosphate salt is sodium polyphosphate.

5. The method according to any one of claims 1 to 4, wherein the chain length of said 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 40 phosphate units.

6. The method according to any one of claims 1 to 5, further comprising the step of producing a biologically active implant material.

7. The method according to claim 6, wherein said biologically active implant material is an artificial bone implant.

9. An implant material produced by the method according to any one of claims 1 to 7.

10. A stabilized ACC composition produced by the method according to any one of claims 1 to 5.

11. Use of the stabilized ACC composition according to claim 10 as a food or dietary supplement.

12. The stabilized ACC composition according to claim 10 for use in the treatment of calcium deficiency.

13. The stabilized ACC composition according to claim 10 for use in the prophylaxis and/or therapy of osteoporosis.