GB2552649A

GB2552649A - Amorphous strontium polyphosphate microparticles for treatment of osteoporosis and inducing bone cell mineralization

Info

Publication number: GB2552649A
Application number: GB1612848.0A
Authority: GB
Inventors: Ernst Ludwig Georg Muller Werner
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-07-25
Filing date: 2016-07-25
Publication date: 2018-02-07
Also published as: GB201612848D0; WO2018019605A1

Abstract

A method for the preparation of morphogenetically active amorphous strontium-polyphosphate nanoparticles or microparticles ("Sr-a-polyP-MP") comprises: (a) dissolving sodium polyphosphate in an aqueous medium, such as distilled water, and adjustment to about pH10 with a basic aqueous solution, such as sodium hydroxide; (b) dissolving of a strontium salt in a suitable aqueous medium, such as aqueous ethanol; (c) slow (e.g. dropwise) addition of the polyphosphate solution to the strontium salt solution, keeping the pH at about 10 with a basic aqueous solution, such as sodium hydroxide, (d) forming particles, preferably by stirring of the suspension overnight at room temperature; and (e) collecting the particles, preferably by filtration, and washing with an aqueous medium, e.g. aqueous ethanol, and drying. The strontium polyphosphate particles may be encapsulated into acid-resistant capsules for oral administration. The microparticles or nanoparticles may be embedded into microspheres of poly(D,L)-lactide-co-glycolide (PLGA). The strontium nanoparticles or microparticles may be used in a method of preventing or treating bone-related diseases such as osteoporosis, and also in bone repair.

Description

(54) Title of the Invention: Amorphous strontium polyphosphate microparticles for treatment of osteoporosis and inducing bone cell mineralization

Abstract Title: Method for the preparation of amorphous strontium polyphosphate nanoparticles or microparticles (57) A method for the preparation of morphogenetically active amorphous strontium-polyphosphate nanoparticles or microparticles (Sr-a-polyP-MP) comprises: (a) dissolving sodium polyphosphate in an aqueous medium, such as distilled water, and adjustment to about pH 10 with a basic aqueous solution, such as sodium hydroxide; (b) dissolving of a strontium salt in a suitable aqueous medium, such as aqueous ethanol; (c) slow (e.g. dropwise) addition of the polyphosphate solution to the strontium salt solution, keeping the pH at about 10 with a basic aqueous solution, such as sodium hydroxide, (d) forming particles, preferably by stirring of the suspension overnight at room temperature; and (e) collecting the particles, preferably by filtration, and washing with an aqueous medium, e.g. aqueous ethanol, and drying. The strontium polyphosphate particles may be encapsulated into acid-resistant capsules for oral administration. The microparticles or nanoparticles may be embedded into microspheres of poly(D,L)-lactide-co-glycolide (PLGA). The strontium nanoparticles or microparticles may be used in a method of preventing or treating bone-related diseases such as osteoporosis, and also in bone repair.

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Intellectual

Property

Office

Application No. GB1612848.0

RTM

Date :18 January 2017

The following terms are registered trade marks and should be read as such wherever they occur in this document:

Nalgene

Cole-Parmer

Waring

Protelos

Sigma

Hitachi

Edax

Roche

Zeiss

Elsevier

Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo

- 1 AMORPHOUS STRONTIUM POLYPHOSPHATE MICROPARTICLES FOR TREATMENT OF OSTEOPOROSIS AND INDUCING BONE CELL MINERALIZATION

This invention concerns a novel preparation of amorphous strontium-polyphosphate microparticles (“Sr-a-polyP-MP”) that can be used for treatment of osteoporosis after oral administration and as a regeneratively active implant material for bone repair. The inventive particles are morphogenetically active. They induce a multifold higher expression of alkaline phosphatase and bone morphogenetic protein 2 than amorphous calcium-polyphosphate microparticles (“Ca-a-polyP-MP”), but, unexpectedly, the expression of sclerostin, an inhibitor of bone cell differentiation and mineralization is, if at all, only slightly affected, in contrast to “Ca-a-polyP-MP” that strongly increases the expression of this protein. As a result, the inventive particles show a significantly higher stimulatory effect on the growth of human mesenchymal stem cells (MSC) and on mineralization of osteoblast-like SaOS-2 cells compared to “Ca-a-polyP-MP” and the strontium salt. The superior properties of “Sr-a-polyPMP” compared to “Ca-a-polyP-MP” were confirmed in animal studies which revealed an increased healing/mineralization of bone defects even after short implantation periods. Consequently, the inventive microparticles (“Sr-a-polyP-MP”) are potentially applicable both in therapy of osteoporotic patients and bone repair.

Background of Invention

Osteoporosis has become a growing public health problem worldwide. This disease is characterized by a progressive bone mass reduction and micro-architectural deterioration, caused by an imbalance between osteoclasts (bone resorption) and osteoblasts (bone formation), leading to an increased fracture risk. Treatments of osteoporosis currently rely mainly on the use of agents that inhibit bone resorption, such as bisphosphonates or, more recently, RANKL inhibitors (denosumab, a monoclonal antibody mimicking OPG activity). In view of the reported side-effects of these medications there is a strong need to develop new strategies for treatment/prophylaxis of osteoporosis.

-2A promising element in treatment of skeletal disorders is strontium (Sr). Strontium has been reported to increase bone density in osteoporotic patients (Neuprez A, et al. (2008) Strontium ranelate: the first agent of a new therapeutic class in osteoporosis. Adv Ther 25:1235-1256). After long-term exposure, it is incorporated into bones and teeth; no adverse effects are seen at Sr doses of up to 80 mg/kg/day, if administered orally in rodents (Wiley J (2009) Strontium and its inorganic compounds. Weinheim: Wiley).

A strontium containing drug already on the market is strontium ranelate, a molecule containing two strontium atoms and ranelic acid, that dissociates in the gastro-intestinal tract. Strontium ranelate shows both anabolic and anti-catabolic properties (activation of osteoblasts and inhibition of osteoclasts) (Meunier PJ, et al. (2004) The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis. N Engl J Med 350:459-468). However, this drug has been repeatedly reported potentially to cause adverse effects, resulting in an increased cardiovascular risk (Reginster JY, et al. (2015) The position of strontium ranelate in today's management of osteoporosis. Osteoporos Int 26:1667-1671). Data on the possible role of the chelating activity of ranelic acid in these side-effects are not available.

One of the proteins that is affected by strontium is sclerostin, an osteocyte-specific protein that acts as a negative regulator of bone formation through inhibiting the Wnt signaling pathway (Rybchyn MS, et al. (2011) An Akt-dependent increase in canonical Wnt signaling and a decrease in sclerostin protein levels are involved in strontium ranelate-induced osteogenic effects in human osteoblasts. J Biol Chem 286:23771-23779). The canonical Wnt signaling pathway is an important regulatory pathway during the osteogenic differentiation of mesenchymal stem cells. The activation of this pathway requires a member of the frizzled family and one of two transmembrane proteins, LRP-5 and LRP-6. This receptor system is blocked by sclerostin, the SOST gene protein product, via binding to LRP5/6 (Li X, et al. (2005) Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem 280:19883-19887).

Previously, the inventor found that inorganic polyphosphate (polyP) causes both in osteoblastlike SaOS-2 cells and in human mesenchymal stem cells (MSC) an increased mineral deposition (reviewed in: Wang XH, et al. (2014) Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine.

- 3 Int Rev Cell Mol Biol 313:27-77). PolyP increases the expression of the bone differentiation marker proteins alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2) (Muller WEG, et al. (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; Wang XH, et al. (2013) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Engin Regen Med 7:767-776). This anabolic stimulatory pathway includes the activation of the BMP-2 - RUNX2 pathway (Wang XH, et al. (2014) Isoquercitrin and polyphosphate coenhance mineralization of human osteoblast-like SaOS-2 cells via separate activation of two RUNX2 cofactors AFT6 and Etsl. Biochemical Pharmacol 89:413-421).

Recently the technology for the fabrication of a morphogenetically active amorphous polyP bone implant material has been introduced. This material is obtained by co-precipitating of Na-polyP with CaCh in the presence of poly(ethylene glycol) (PEG); under those conditions amorphous calcium polyP microparticles (“Ca-a-polyP MP”) with a size range of 150 to 250 nm are formed (Muller WEG, et al. (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Lett 148:163-166). These microparticles turned out to be biologically active in both MSC and SaOS-2 cells. They elicit the expression of ALP and BMP-2 (Wang XH, et al. (2016) Polyphosphate as a metabolic fuel in Metazoa: A foundational breakthrough invention for biomedical applications. Biotechnol. J 11:11-30).

The state-of-the-art of polyP has been described in, for example, Muller WEG, et al. (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci 15:1182-1197.

Summary of the invention

Here the inventor produced amorphous microparticles from SrCh and polyP (“Sr-a-polyPMP”) and studied their effect on gene expression of the genes that encode for proteins involved in the anabolic pathway of mineralization, such as, for example, ALP and BMP-2, and the SOST gene, that encodes the anti-anabolically acting sclerostin product. The inventor demonstrates that “Sr-a-polyP-MP” causes a significant increase in the expression of the ALP and BMP-2 gene, compared to “Ca-a-polyP-MP” and Sr ions.

-4Surprising and unexpectedly is the finding of the inventor that - in contrast to the expression of ALP and BMP-2 - “Sr-a-polyP-MP” markedly reduces the upregulation of the expression of sclerostin if compared to “Ca-a-polyP-MP”.

Therefore, the strontium salt of polyP, produced as amorphous microparticles, adds a new, unexpected biological activity to the functions of the “Ca-a-polyP MP” as identified previously.

The property of the “Sr-a-polyP-MP” to elicit in bone forming cells, for example SaOS-2 cells, an increased expression of ALP and BMP-2, while the steady-state expression of sclerostin, an inhibitor of mineralization and bone cell differentiation, is only slightly affected provides these particles with a multifold higher anabolic activity on bone formation compared to “Ca-a-polyP-MP”.

The data underlying this invention qualifies the inventive material “Sr-a-polyP-MP” for use/application a) in the treatment of osteoporotic patients after oral administration; and b) as an implant material for healing of bone defects, including fractures caused by osteoporosis.

Furthermore, these particles are non-toxic and by far less expensive to produce than, e.g., the anti-osteoporotic compound Sr-ranelate. A summary of the present invention is shown in Fig.

1. It is outlined that the low expression of sclerostin (SOST gene), a negative regulator of the Wnt pathway that acts via interference with the FRP5/6 co-receptor, is not changed by “Sr-apolyP-MP”. As a result, an increase in mineralization of bone-forming cells occurs, via the Wnt-FRP5/6 pathway.

The following patent applications on polyP are assumed relevant: GB 1420363.2. Morphogenetically active calcium polyphosphate nanoparticles. Inventor: Muller WEG; GB 1502116.5. Synergistically acting amorphous calcium-polyphosphate nanospheres containing encapsulated retinol for therapeutic applications. Inventor: Muller WEG; 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, and PCT/EP2011/062159. Hydroxyapatite-binding nano- and

- 5 microparticles for caries prophylaxis and reduction of dental hypersensitivity. Inventors: Muller WEG, Wiens M.

Detailed description of the invention

This invention concerns the preparation of novel amorphous strontium-polyphosphate nanoparticles or microparticles (“Sr-a-polyP-MP”) which are morphogenetically active and can be used for oral administration in patients with osteoporosis and as a regeneratively active implant material for bone repair.

Previously, the inventor described a technology to fabricate morphogenetically active amorphous calcium-polyphosphate nanoparticles or microparticles (“Ca-a-polyP-MP”) (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166). The properties of these microparticles that have been disclosed in patent application GB 1420363.2. They are superior compared to conventional polyP preparations for application as a bone regeneration material (e.g., GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue [Inventors: Muller WEG, Schroder HC, Wang XH]; and GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders [Inventors: Muller WEG, Schroder HC, Wang XH]).

These “Ca-a-polyP-MP” are (i) amorphous and (ii) biologically active (Muller WEG, Tolba E, Schroder HC, Wang XH (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci 15:1182-1197).

Now the inventor succeeded to develop a method for the preparation of morphogenetically active “Sr-a-polyP-MP” that are superior to “Ca-a-polyP-MP”. The inventive method comprises or preferably consists of the following steps:

a) Dissolution of sodium polyphosphate (Na-polyP) in aqueous medium (e.g. distilled water) and adjustment to about pH 10.0 with basic solution, e.g. sodium hydroxide (NaOH) solution;

b) Dissolution of a strontium salt, preferably strontium chloride (SrCk), in a suitable solvent, e.g. aqueous ethanol;

c) Slow (e.g. dropwise) addition of the Na-polyP solution to the strontium salt solution and keeping the pH at about 10 with NaOH solution;

- 6d) Formation of the particles, preferably by stirring of the suspension overnight at room temperature; and

e) Collection of the particles, preferably by filtration, washing with suitable solvent, e.g. aqueous ethanol, and drying.

The chain length of the polyP can be 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.

In the context of the present invention, the term “about” shall mean +/- 10 percent of a given value.

The preferred composition of the Sr-polyP nanoparticles or microparticles used in the inventive method is a stoichiometric ratio of between 0.1 to 3 (strontium to phosphate), preferably between 0.3 and 1.0, and most preferred by a stoichiometric ratio of 0.4 to 0.5.

Surprisingly, the Sr-polyP microparticles are biologically active although their diameter (0.8 and 0.9 pm) is outside the range allowing receptor-mediated endocytosis (around 50 nm).

A further aspect of the inventive method concerns the encapsulation of the inventive Sr-polyP nanoparticles or microparticles into acid-resistant capsules, or tablets for oral administration.

These acid-resistant capsules can consist, for example, of hydroxypropyl methyl cellulose (HPMC), but also of any other material that prevents the degradation of the polyP component of the inventive Sr-polyP nanoparticles or microparticles during stomach passage.

The inventive Sr-polyP nanoparticles or microparticles encapsulated into acid-resistant capsules for oral administration can be applied for treatment or prevention of osteoporosis.

A further aspect of the inventive method concerns the packaging of the Sr-polyP nanoparticles or microparticles into microspheres.

These microspheres can be produced from the Sr-polyP nanoparticles or microparticles and poly(D,L-lactide-c0-glycoiide (PLGA).

-Ί The inventive method can be applied for the fabrication of a regeneratively active implant material for bone and cartilage.

The inventive “Sr-a-polyP-MP” cause a significantly higher stimulation of growth of human mesenchymal stem cells (MSC) compared to “Ca-a-polyP-MP” and the strontium salt.

In addition, the inventive “Sr-a-polyP-MP” cause a significantly higher increase in mineralization of bone-forming cells, e.g. SaOS-2 cells, compared to “Ca-a-polyP-MP” and the strontium salt.

Moreover, the inventive “Sr-a-polyP-MP” upregulate the expression of the genes encoding for ALP and BMP-2 to a significantly higher extent in bone-forming cells, e.g. SaOS-2, compared to “Ca-a-polyP-MP” and the strontium salt.

By contrast, the inventive “Sr-a-polyP-MP” have the unique property to change only slightly the expression of the osteocyte-specific sclerostin. Sclerostin is a negative regulator of the canonical Wnt signaling pathway and an inhibitor of bone cell differentiation and mineralization that is expressed at later stages during MSC differentiation, as well as in SaOS2. This effect is opposite the effect of “Ca-a-polyP-MP” which strongly increase the expression of SOST (sclerostin) gene.

The beneficial properties of the inventive material (“Sr-a-polyP-MP”) can be demonstrated in animal experiments after entrapment into poly(D,L-lactide-co-glycolide (PLGA) microspheres. Implantation of microspheres containing the “Sr-a-polyP-MP” into critical-size calvarial defects in rats revealed that the amorphous Sr-polyP-containing microspheres cause an increased healing/mineralization of the bone defect even after short implantation periods if compared to control microspheres containing β-tri-calcium phosphate or “Ca-a-polyP-MP”.

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,

-8Figure 1 shows a scheme summarizing the invention. It is proposed that “Sr-a-polyP-MP” increases the differentiation and mineralization processes in bone/SaOS-2 cells via the Wnt/LRP5/6 pathway (left panel). Sclerostin, a proposed inhibitor of the Wnt pathway, acts via interference with the LRP5/6 co-receptor and is assumed to be internalized and degraded together with the LRP5/6 co-receptor (middle). The “Sr-a-polyP-MP” particles significantly upregulate the expression of BMP-2 and ALP (not shown) very likely via activation of the Runx2 transcription factor, and apparently do not interfere with the Wnt pathway and do not affect the expression of the SOST gene, encoding for sclerostin (right). These properties qualify the inventive “Sr-a-polyP-MP” particles as a novel agent for treatment or prophylaxis of osteoporosis as well as a regeneratively active material for bone repair.

Figure 2 shows the morphology of the polyP nanoparticles/microparticles; SEM. (A) Image from amorphous “Ca-a-polyP-MP” and (B and C) images from “Sr-a-polyP-MP”.

Figure 3 shows the FTIR spectra from “Sr-a-polyP-MP” and Na-polyP (starting material for the preparation of “Sr-a-polyP-MP”) within the range of wavenumbers 2000 to 300 cm'¹.

Figure 4 shows the EDX spectra for (A) “Ca-a-polyP-MP” and (B) “Sr-a-polyP-MP”. The respective signals for the different atoms are marked.

Figure 5 shows the effect of the different concentrations of microparticles, either “Ca-apolyP-MP” (hatched to the right) or “Sr-a-polyP-MP” (cross hatched), Ca²⁺-complexed NapolyP (hatched to the left), or Sr (as SrCf · 6H₂O; open bars) on viability/growth of MSC. The number of viable cells was determined by the XTT assay (A450 values). The absorbance level at time zero for the controls (without polyP or Sr ) is given as a bar, white hatched to the right on black; the value measured at the end of the 3 d incubation period is in black. Ten parallel assays have been performed and the mean values (± SD) have been determined for significance between the assays marked with rectangular brackets (**/?<0.001).

Figure 6 shows the quantitative determination of the mineralization potency of SaOS-2 cells, based on alizarin red S (AR) staining. Cells were grown in medium/FCS in the absence or presence of the activation cocktail (- MAC / + MAC) for 5 d without polyP or with either “Ca-a-polyP-MP” or “Sr-a-polyP-MP”, at the concentrations indicated. Then the cells were collected and their extract was stained with alizarin red S; the quantitative determination was

-9performed as described in “Methods”; the extent of biomineralization is correlated with the cell number (as measured by the DNA content). Values represent the means (± SD) from 10 separate experiments each (*/?<0.05; **/?<0.001).

Figure 7 shows the effect of “Ca-a-polyP-MP”, “Sr-a-polyP-MP” and Sr²⁺ ions (from SrCfi) on the expression of the genes, encoding for (A) ALP and (B) BMP-2, as well as (C) for the SOST gene, encoding sclerostin in SaOS-2 cells. The cells were incubated in the presence of MAC for 3 d. Then the RNA from the samples was isolated and the respective gene expressions were quantified by qRT-PCR and correlated to the expression of the GAPDH house-keeping gene. The cultures were incubated in the absence of the compounds or in the presence of (3 to 100 pg/mL) “Ca-a-polyP-MP”, “Sr-a-polyP-MP” or Sr²⁺ ions (as SrCfi). The results are means calculated from 5 parallel experiments. The values are computed against the expression measured in the controls of the respective incubation time point (marked with §§). In addition, the significances of the difference of the expression levels between the cultures incubated with “Sr-a-polyP-MP” and the respective cell assays exposed to “Ca-a-polyP-MP” or Sr ions (**/?<0.01) (as in A and B) are given. In C, the significance between the exposure assays with “Ca-a-polyP-MP” are compared to those exposed to “Sr-apolyP-MP” and marked just as before (**/?<0.01).

Figure 8 shows the implant material. (A and B) Morphology of the “Sr-a-polyP-MS” microspheres, used for the fabrication of the (C) implant discs; optical images.

Figure 9 shows the histological sections through rat calvariae. (A) Untreated sample; the mineralized cells/tissues are stained in green, while the marrow and soft tissues are in red. (B to I) Calvariae with critical-size defects treated with discs, prepared from (D and G) “β-TCPMS”, (Β, E, H) “Ca-a-polyP-MS” and (C, F, I) “Sr-a-polyP-MS”. After 4 wk (upper row), 8 wk (middle), and 12 wk (lower), tissue samples are removed, sectioned and stained with Masson’s trichrome. The regenerative zones (reg) are marked; the microspheres are abbreviated with “mic”. The defect margin (double-headed line) indicates the border between the regenerative zones and the “old bone”. Some osteocytes (oc) / primary ossification centers (poc) are marked.

Figure 10 shows the determination of the stiffness of the tissue around the implanted microspheres (“Ca-a-polyP-MS” and “Sr-a-polyP-MS”). The determinations were performed

- 10between two microspheres which were separated by 80 to 150 pm. The stiffness/RedYM of the regenerating/mineralizing tissue between spheres is given in MPa. For a comparison the stiffness for the calvarial bone is given. The significance (box plot characteristics) are given by comparing the control implants (“β-TCP-MS”) with the implants, prepared from “Ca-apolyP-MS” and “Sr-a-polyP-MS”. The horizontal bars within the box indicate the median value. **/><0.001; * /><0.05.

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 10 to 100 units.

Methods

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).

Production of Sr-polyP microparticles

The amorphous Sr-polyphosphate nanoparticles/microparticles, Sr-polyP microparticles (“Sra-polyP-MP”), are prepared, basically following the described procedure (Muller WEG, et al. (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166). In brief, 1 g Na-polyP dissolved in 50 mL distilled water is adjusted to pH 10.0. In parallel, 5.16 g of strontium chloride hexahydrate is dissolved in 50 mL of 70% [v/v] ethanol. Subsequently, the polyP solution is added dropwise to the strontium solution, keeping the pH at 10 with sodium hydroxide solution. This suspension is left for overnight under stirring (at room temperature); during this time the particles are formed. They can be collected by filtration through, for example, a Nalgene Filter Unit (pore size 0.45 pm; ColeParmer) and washed three times with 70% [v/v] ethanol. During this step the non-bound Sr²⁺is separated from the particles. The material is dried at 60°C to obtain the Sr-polyP microparticles. Then, the particles are grinded in a Waring blender and sieved through 100 pm mesh.

- 11 In the Examples, the resulting atomic ratio of Sr to P has been determined to be 0.44 and the one for Na to P 0.18, using EDX and inductively coupled plasma mass spectrometry. The particles are termed “Sr-a-polyP-MP”.

X-ray diffraction analysis and Fourier transform infrared spectroscopic analysis can be applied to verify the amorphous state of polyP.

In the Examples, for comparison reasons Ca-polyP microparticles (“Ca-a-polyP-MP”) have been prepared as described (Muller WEG, et al. (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166).

Where indicated in the Examples, Na-polyP has been added together with CaCh in a stoichiometric ratio of 2 moles of polyP (referred to the monomer unit of polyP): 1 mole of

CaCh [designated “Na-polyP/Ca ”]. The addition of CaCh compensates any chelating

2₊ activity of polyP to Ca .

Encapsulation of the amorphous Sr-polyP microparticles into acid-resistant capsules The microparticles “Sr-a-polyP-MP” were packaged into acid-resistant vegetable empty capsules (size: 0; product no.: LK-MSR; info@neue-lebensqualitaet.com) suitable for stomach passage. These capsules, consisting of hydroxypropyl methyl cellulose (HPMC) with a volume of 0.68 ml, were filled with 150 mg each of the microparticles using a capsule filling device (aponorm; WEPA, Hillscheid; Germany).

Determination of the strontium content within the acid-resistant capsules filled with amorphous Sr-polyP microspheres

The strontium content in the microparticles “Sr-a-polyP-MP” was determined to be 45% (based on EDX analysis). This means that 1 g “Sr-a-polyP-MP” contains 450 mg of strontium. From this value, it can be calculated that, after loading with 150 mg “Sr-a-polyP-MP”, one acid-resistant capsule contains 67.5 mg of strontium.

For comparison: The daily dose of strontium ranelate used as a commercial drug for treatment of osteoporosis (PROTELOS, Servier, France), a granulate that must be suspended in water, is 2 g. The molar mass of strontium ranelate (CnTy+OgSS^) amounts to 513.49 g mol’¹. Based on the atomic weight of strontium (87.62) and the formula of strontium

- 12ranelate (CnHilSriOgSS^) it can be calculated that a dose of 2 g strontium ranelate contains

680 mg strontium. This corresponds to about 1.5 g of “Sr-a-polyP-MP”.

Oral uptake and urinary excretion of strontium present in the Sr-polyP microparticles in vivo

The capsules (3 capsules thrice a day, containing in total 607.5 mg of “Sr-a-polyP-MS”) were administered to a volunteer (male; age 64, body weight 79 kg) at day 1. The urine of this volunteer was collected over the following 5 days. Aliquots of the samples were stored at 20°C until analysis. In the Examples, the concentrations of strontium in the samples were measured using ICP-AES (Inductively Coupled Plasma Atomic Emission Spectrometry).

Preparation of the PLGA-based microspheres

Microspheres can be produced as described (Wang SF, et al. (2014) Bioactive and biodegradable silica biomaterial for bone regeneration. Bone 67:292-304; Tolba E., et al. (2016) High biocompatibility and improved osteogenic potential of amorphous calcium carbonate/vaterite. J. Mat. Chem B 4:376-386). The active components, the microparticles, can be embedded into poly(D,L-lactide-co-glycolide (PEGA); mol. wt. 66,000-107,000; e.g., Pl 941 Sigma.

In the Examples, three different microsphere samples have been prepared.

1. Spheres that contain β-tri-calcium phosphate (#49963 Sigma; β-TCP). They are fabricated from 60 mg of β-TCP and 600 mg of PLGA. PLGA is dissoluted with 3 mL of DCM (dichloromethane); then 0.8 mL of water is added and the mixture is intensely blended (2 min) to reach a homogeneous emulsion. The resulting viscous mixture is pressed through a syringe needle (opening 0.8 mm) into 300 mL of 1% poly(vinyl alcohol) (87-90% hydrolyzed; mol. wt. 30,000-70,000; e,g., P8136 Sigma) using a the syringe pump with a speed of 12 mL/h. After stirring for 4 h the microspheres are collected, washed five times with water and collected onto filter paper. Then the sample (termed “β-TCP-MS”) is freeze dried; these microspheres are used as a control.

2. The amorphous Ca-polyP microparticles, “Ca-a-polyP-MP”, are packed into PLGA in the same way by replacing the 60 mg of β-TCP with 60 mg of the Ca-polyP microparticles (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166); they are designated “Ca-a-polyP-MS”.

- 13 3. The Sr-polyP microparticles “Sr-a-polyP-MP” are similarly embedded into PLGA and labeled “Sr-a-polyP-MS”.

The ~ 830 to 840 pm large microspheres (40 mg) were pressed into discs of a diameter of about 8 mm and then inserted into the bone defect of the opened rat calvariae. Prior to use, the microparticles/microspheres/discs are treated by ultraviolet light.

The polyP content in the microspheres can be determined after dissolving the microspheres in dichloromethane and a treatment with 1 M sulfuric acid, to hydrolyze polyP to orthophosphate, with ammonium molybdate. β-TCP and Sr can be quantitated on the basis of calcium/strontium using the ICP-AES (Inductively Coupled Plasma Atomic Emission Spectrometer.

Electron microscopy and energy dispersive X-ray spectroscopy

The scanning electron microscopic (SEM) analyses can be performed, for example, with a HITACHI SU 8000 electron microscope and the EDX spectroscopy with an EDAX Genesis EDX System attached to a scanning electron microscope operating at 10 kV with a collection time of 30-45 s.

Cell culture experiments

The human mesenchymal stem cells (MSC) used in the Examples are from normal (nondiabetic) adult human bone marrow of normal volunteers; they have been purchased from Lonza Cologne (Cologne; Germany). Incubation can be performed as described (Wang XH, et al. (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. Mar Drugs 12:11311147). The cells are maintained in 75 cm flasks and cultivated in α-MEM, supplemented with 20% FCS (fetal calf serum) and 0.5 mg mL'¹ of gentamycin, 100 units mL'¹ penicillin, 100 mg mL'¹ of streptomycin and 1 mM pyruvate. Incubation is performed in a humidified incubator at 37°C. In the Examples, the assays for cell viability and gene expression studies were started with an inoculum of 1·10⁴ cells per well (48 well plates) in a total volume of 0.5 mL. The cultures were first incubated for 4 d in the absence of the mineralization-activating cocktail (MAC).

- 14SaOS-2 cells (human osteogenic sarcoma cells) can be cultured in McCoy’s medium (containing 1 mM CaCh) with 5% heat-inactivated fetal calf serum (FCS), 2 mM Lglutamine, and gentamicin (50 mg/mL) in 25 cm flasks or in six-well plates (surface area

9.46 cm ; e.g. from Orange Scientifique) in a humidified incubator at 37°C.

Where indicated in the Examples, the mineralization activating cocktail (MAC), comprising 50 mM ascorbic acid and 10 nM dexamethasone has been added to the cultures to induce biomineralization. The third component usually used in the MAC, β-glycerophosphate, has been omitted since polyP has been shown to be sufficient as a phosphate supply (Muller WEG, et al. (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

Strontium ions were added as strontium chloride hexahydrate to the cultures, as indicated.

Mineralization by SaOS-2 cells in vitro

SaOS-2 cells can be used and incubated on plastic coverslips (e.g., Nunc), placed into 24-well plates (14.5 mm well diameter) in McCoy’s medium and 10% FCS. The cells are used either in the absence or presence of MAC (see above). After an initial incubation periods of 1 d the samples are processed for 5 d, either in the absence or presence of MAC, and subsequently the intensity of alizarin red S staining is quantitatively assessed by application of a spectrophotometric assay (Gregory CA, et al. (2004) An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 329:77-84). In turn, the cells are transferred into acetic acid, followed by mechanical scraping off and centrifugation. Then the supernatant obtained is neutralized with acid and the optical density is read at 405 nm. In order to correlate with the cell number, the amount of bound alizarin red S is given in moles, which are determined after setting up a calibration curve and the values are normalized to total DNA, using the PicoGreen method (Schroder HC, et al. (2005) Mineralization of SaOS-2 cells on enzymatically (Silicatein) modified bioactive osteoblast-stimulating surfaces. J Biomed Mat Res Part B - Applied Biomaterials 75B:387-392); calf thymus DNA is used as a standard.

Cell proliferation/cell viability assays

- 15 Quantifying cell growth/metabolic activity can be performed, for example, by a colorimetric method based on the tetrazolium salt XTT (e.g., Cell Proliferation Kit II; Roche), as described in (Mori K, et al. (2007) Receptor activator of nuclear factor-kappaB ligand (RANKL) directly modulates the gene expression profile of RANK-positive Saos-2 human osteosarcoma cells. Oncol Rep 18:1365-1371). The absorbance is determined at 450 nm and subtracted by the background values (500 nm). Routinely the viable cells are determined after 72 h.

Gene expression studies: qRT-PCR

The technique of quantitative real-time reverse transcription polymerase chain reaction (qRTPCR) is applied to quantitate the effect of the two polyP particle samples “Ca-a-polyP-MP” and “Sr-a-polyP-MP” on the steady-state expression in SaOS-2 cells, in the presence of the MAC. In the Examples, the following three genes for the human SaOS-2 cells have been selected, and primers have been designed against them. First, the ALP (alkaline phosphatase) NM 000478.4) Fwd: 5'-TGCAGTACGAGCTGAACAGGAACA-3' (SEQ ID NO. 1) (ntn₄i to ntn₆₄) and Rev: 5'-TCCACCAAATGTGAAGACGTGGGA-3' (SEQ ID NO. 2) (ntuis to nti3₉₅; 278 bp), second BMP-2 (bone morphogenetic protein-2; NM_001200.2) Fwd: 5'ACCCTTTGTACGTGGACTTC-3' (SEQ ID NO. 3) (ntiesi to ntroo); and Rev: 5'GTGGAGTTCAGATGATCAGC-3' (SEQ ID NO. 4) (nti₈₀₄ to nti₇₈₅; 124 bp), and third the sclerostin (SOST) NM 025237) Fwd: 5'-AAGCTATGCTGCTTCCCAGCC-3' (SEQ ID NO. 5) (ntisio to nti₅₃o) and Rev: 5'-ATTTCTATCCCTCCCACCACCCTC-3' (SEQ ID NO. 6) (ntli63₈ to nti6i5; 129 bp). As a reference gene, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) NM_002046.3) Fwd: 5'-CCGTCTAGAAAAACCTGCC-3' (SEQ ID NO. 7) (nt₉29 to nt₉₄₇); and Rev: 5 '-GCC AAATTCGTTGTC AT ACC-3' (SEQ ID NO. 8) (ntn₄₅ to ntn26; 217 bp) is used. The cells are extracted for RNA using the TRIzol reagent and then subjected to qRT-PCR. The fluorescence data are computed at the 80°C step. The quantitative real-time PCR experiments can be performed, for example, in an iCycler (Bio-Rad); the mean C_t values and efficiencies are calculated with the iCycler software; the estimated PCR efficiencies range between 93% and 103%.

Animal studies

In the Examples, the studies have been performed with male Sprague-Dawley rats, 6 weeks old (190+30 g). The animals have been purchased from the Animal Resources Centre (Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing; China). Permissions for the implementation of the animal experiments have been obtained by the ethics committee

- 16at the Dongzhimen Hospital at the Beijing University of Chinese Medicine (No. 5a Haiyuncang Road, Dongcheng District, Beijing 100700; Beijing Committee of Science and Technology). The certificate number for the approval is 2012-0001_g/02. After the operation the animals were checked for health daily; heart rate, blood pressure and body temperature were monitored daily. None of the experimental animals showed signs of pain, suffering or distress and no unexpected surgical events or deaths occurred. Prior to surgery the animals were anaesthesized with medetomidine, midazolam and fentanyl.

A 3 cm midline incision was set over the calvaria; after removal of the periosteum a 10 mm craniotomy size defect was drilled. This dimension is 2 mm larger than the critical size defect of 8 mm to ensure that the defect does not completely heal over the natural lifetime of the animal. Furthermore, smaller discs (diameter of 8 mm) were inserted as implants to allow the fibrin clot to develop. After closing the skin with 4-0 silk sutures, the rats were given an intramuscular injection of an analgesic (0.05 mg kg'¹ buprenorphine).

After a healing period of 4, 8 or 12 weeks (wk) the animals were sacrificed with inhaled CO₂and the calvariae were removed for histological analysis. Three groups of rats were arranged at random (4 specimens in each group per time point) and implanted with one disc composed of microspheres each; in group 1 (controls) “P-TCP-MS”, group 2 “Ca-a-polyP-MS and group 3 “Sr-a-polyP-MSP

Histology

The removed calvarial specimens are fixed with 10% formaldehyde (overnight) and then decalcified with 0.5% formaldehyde, supplemented with 10% EDTA (pH 7.4). After dehydration in a graded alcohol series the specimens are embedded in paraffin and 5 pm thick sections are prepared to comprise a plane of analysis through the defect. The sections are stained with Masson's trichrome to discriminate between mineralized collagen and bone areas highlighted in green or blue-green and the muscle fibers that light up in red. The samples are inspected randomly using, for example, a Zeiss Axiophot microscope.

Determination of the mechanical properties (hardness)

For the quantification of the local mechanical properties of the regenerating bone tissue and of the microspheres, for example, the Opticsll Piuma Nanoindenter can be used, equipped with a cantilever-based optomechanical indentation probe (Opticsll, Amsterdam). By this method

- 17the reduced Young’s modulus (RedYM) can be quantified. For each sample ten independent measurements are performed at ten different sites.

Statistical analysis

After finding that the respective values follow a standard normal Gaussian distribution and that the variances of the respective groups are equal, the results are statistically evaluated using the independent two-sample Student’s Z-test. The Box plot analysis is applied for the quantification of the hardness results of the regenerated implants. For the indent determinations within the region of trabecular bone tissue, or in the implant regions, 30 individual measurements each, from 4 separate animals are performed. The hardness of the newly formed bone tissue around the microspheres is likewise measured. From the 2 mm thick slices (4 slices from each group) 40 individual measurements are performed. Both the lower and the upper limits (whiskers) are set to 2.5th and 97.5th percentile.

The Sr-polyP microparticles

The nanoparticles/microparticles, Sr-polyP microparticles “Sr-a-polyP-MP”, were prepared, by co-precipitation of an aqueous Na-polyP solution and an ethanolic SrCl₂ solution. The resulting particles had a size varying between 120 and 800 nm (average 340±165 nm); Fig. 2B and C. In comparison, the particles of the Ca-polyP microparticles “Ca-a-polyP-MP” are smaller and measured in the average 170±65 nm (Fig. 2A). The atomic ratio between Sr and P was found to 0.44±0.6. The material had an amorphous state as determined by X-ray diffraction analysis (data not shown) and Fourier transform infrared spectroscopic analysis.

The FTIR spectra of both the “Sr-a-polyP-MP” and the starting material Na-polyP are shown in Fig. 3. The spectra are very related and show only some shifts, which can referred to 741 cm'¹ (“Sr-a-polyP-MP”) versus 750 cm'¹ (Na-polyP), ascribed to a symmetric stretching of PO-P; 876 cm'¹ versus 867 cm'¹ to asymmetric stretching of P-O-P, 1089 cm'¹ and 995/986 cm' ¹ for the asymmetric and symmetric stretching of P-O-P. The broad signals at 1250 - 1285 cm' ¹ are ascribed to the asymmetric stretching reflecting P=O bonds. Moreover, the bands near 3428 cm'¹ are ascribed to stretching vibrations of hydroxyl groups (not shown in Fig. 3), whereas the band at 1638 cm^-1 is due to the deformation of water molecules absorbed through the “Sr-a-polyP-MP” particle surface.

- 18 The EDX spectra demonstrate that the “Sr-a-polyP-MP” are primarily composed of Sr, O and

P and only of minute amounts of Na (Fig. 4B). In comparison, the “Ca-a-polyP-MP” particles contain mainly Ca, O and P and only small amount of Na, originating from Na-polyP (Fig.

4A).

Uptake of strontium after oral administration of the amorphous Sr-polyP microparticles: in vivo study

The polyP component of the Sr-polyP microparticles is stable in neutral or alkaline solution (minimum of the hydrolysis pH 9; Corbridge DEC (1990) Phosphorus: An Outline of its Chemistry, Biochemistry, and Technology, 4^th edn, Elsevier, Amsterdam), but hydrolyzed at low pH (Farrokhpay S, et al. (2012) Stability of sodium polyphosphate dispersants in mineral processing applications. Miner Engineer 39:39-44; Rashchi F, Finch JA (2000) Polyphosphates: a review. Min Engineer 13:1019-1035). In order to allow the oral administration and stomach passage of the particles without degradation, they had to be packed into acid-resistant capsules.

The microparticles were filled into commercially available acid-resistant vegetable empty capsules (consisting of hydroxypropyl methyl cellulose; capsule volume, 0.68 ml) suitable for stomach passage, using a capsule filling device. A single dose of 607 mg of “Sr-a-polyP-MP” (present in 3x3 capsules) was administered to a healthy volunteer (male; age 64, body weight 79 kg). This dose (607 mg) is close to the daily dose of 2 g strontium ranelate (containing 680 mg of strontium; see “Methods”). The total urine of this volunteer was collected during the subsequent 5 days after administration. Aliquots of the urine samples were stored at -20°C. The concentrations of strontium in the samples were determined by ICP-AES before and after “Sr-a-polyP-MP” administration.

The total volume of the urine collected until day 5 was 6.08 L. The concentration of strontium in urine before the administration of the single dose was determined to be 130 pg/L of urine. The determination of strontium after the administration of the Sr-polyP capsules revealed that until day 5, a total amount of 40.2 mg of strontium has been excreted via the urine. After subtraction of the amount of “endogenous” strontium present in this volume (0.79 mg) the amount of “additional”, administered strontium excreted until day 5 amounts of 39.8 mg.

- 19This result indicates that a considerable portion of the strontium present in the “Sr-a-polyPMP” capsules has been taken up after stomach passage. Based on the cumulative urinary excretion and the administered dose of 607 mg of strontium, 6.6% of the administered dose was cleared into the urine until day 5.

Effect of the different states of polyP on growth of human MSC

In the absence of any polyP the growth/metabolic activity of the MSC increases from 0.31±0.05 absorbance units (AU at 450 nm) at time zero (seeding) to 0.82±0.11 after a 3 d incubation period (Fig. 5). The ion Sr (added as SrCf · 6H₂O) and the Na-polyP, complexed with Ca , did not alter the metabolic activity within the concentration range 3 and 100 pg/mL. The “Ca-a-polyP-MP” particles displayed only at the concentrations of 10 and 30 pg/mL a relatively small (1.25-fold and 1.41-fold, respectively), but significant, increase in AU450 nm· This is in contrast to the effect which is displayed by “Sr-a-polyP-MP”; with those particles the metabolic activity is significantly higher, both with respect to the controls (absence of any polyP) and “Ca-a-polyP-MP” and reaches between 10 and 30 pg/mL an increased absorbance level (with respect to the controls) of 1.6- to 1.8-fold; Fig. 5.

PolyP-induced mineralization in SaOS-2 cells

SaOS-2 cells were incubated in medium/FCS for 1 d and subsequently for additional 5 d in the absence or presence of the MAC and with polyP particles within the concentration range of 0 to 100 pg/mL. The results (Fig. 6) revealed that in the absence of MAC only a slight increase in mineralization in the cell assays occurred, in the presence of the particles which is hardly specific at ~ 30 pg/mL. A different situation was found when the cells were incubated with the microparticles. The “Ca-a-polyP-MP” caused, in the presence of the MAC, within the concentration range of 3 to 100 pg/mL a significant increase in the mineralization, which reaches a value of 3.8-fold (with respect to the corresponding absorbance in the assays without the MAC). Even stronger is the effect of “Sr-a-polyP-MP” which causes even at the low concentration of 3 pg/mL a significant higher value for the mineralization, if compared with the activated cultures treated with “Ca-a-polyP-MP”. Within the complete concentration range (3 to 100 pg/mL) a significant larger mineralization is measured for the MAC-activated cells in the presence of the “Sr-a-polyP-MP”, if compared with the corresponding “Ca-apolyP-MP” assays. The maximum of activation is seen at the concentration of 100 pg/mL “Sr-a-polyP-MP” with a 5.5-fold increase (with MAC), with respect to the Alizarin Redpositive reaction measured in the absence of MAC.

-20'Λ j

Differential effects of “Ca-a-polyP-MP”, “Sr-a-polyP-MP” and Sr on gene expression

2d^- 2d^-

The mineralization studies revealed that polyP, in dependence on its salt (Ca / Sr ) state and Sr differentially affects the gene expression of the ALP, BMP-2 (two marker genes of osteoblast differentiation and mineralization [anabolic effect on bone formation]) and sclerostin (“target” gene [anti-anabolic effector]).

The steady-state-expression of ALP is readily and significantly upregulated at all concentrations tested between 3 and 100 pg/mL for the two polyP particles, the “Ca-a-polyPMP” as well as the “Sr-a-polyP-MP” (Fig. 7A). However, while the Ca -salt particles caused at the concentration of 30 pg/mL, a 2-fold upregulation (with respect to the controls that were incubated neither with the particles nor with Sr ), the maximal value, the “Sr-a-polyP-MP” particles were significantly effective already at 3 pg/mL, the maximum value of the increased expression was measured at 30 pg/mL. Interestingly, the “Sr-a-polyP-MP” elicited - at all concentrations tested - a significantly higher transcript level for ALP compared to “Ca-apolyP-MP” and Sr²⁺.

A similar gene induction pattern is seen for the response of SaOS-2 cells towards polyP and Sr²⁺ with respect to BMP-2 (Fig. 7B). Again, both particles, “Ca-a-polyP-MP” and “Sr-apolyP-MP”, significantly induce the expression of BMP-2 within the concentration range of 3 to 100 pg/mL. Again the inducing potency was higher for “Sr-a-polyP-MP” with respect to the potency of “Ca-a-polyP-MP”. Maximum values of induction are seen within the range of 10 and 30 pg/mL with 1.9-fold for “Ca-a-polyP-MP” and 2.9-fold for “Sr-a-polyP-MP”, respectively. Also in this test system Sr did not cause a BMP-2 gene induction.

In contrast to the induction pattern seen for the anabolic proteins of bone mineralization, the gene encoding for the catabolic protein acting on bone mineral formation, sclerostin was only slightly (1.3- to 1.6-fold, with respect to the controls) induced by “Sr-a-polyP-MP”, while “Ca-a-polyP-MP” caused a marked upregulation with 2.4-fold of SOST (Fig. 7C). Surprisingly, Sr²⁺ did not cause any significant change of SOST expression in this in vitro cell system.

Microspheres used as implant material

-21 Into PLGA fabricated microspheres with a size of -830 to 840 pm the active ingredients βTCP, amorphous Ca-polyP nanoparticles/microparticles or amorphous Sr-a-polyP particles were embedded and used for the animal experiments; they are termed “β-TCP-MS”, “Ca-apolyP-MS” and “Sr-a-polyP-MS”, respectively. The newly described microspheres “Sr-apolyP-MS” (Fig. 8A) fall in a size range of 842±40 pm (n = 25) and comprise, like the other microspheres, a porous texture on their surfaces (Fig. 8B). The hardness of the particles, the (median) RedYM, has a value of 28.19±7.90 MPa. The active ingredients in the PLGA-based microsphere were determined to be for β-TCP (in the “β-TCP-MS”) 7.93±0.38% and the polyP content in both the “Ca-a-polyP-MS” and the “Sr-a-polyP-MS” was 7.14±0.85%. The Sr content in the particles was determined to be 1.74±0.26%.

Healing/bone regeneration after rat critical size calvarial defect

Implants, 8 mm discs were inserted into the 10 mm large calvarial defects over the dural and brain tissue, as described under “Methods”. Three groups of animals were arranged and received the following disc implants: Group 1 “β-TCP-MS” as control, group 2 “Ca-a-polyPMS” and group 3 “Sr-a-polyP-MS”. After a period of 4, 8 or 12 wk the animals were sacrificed and subjected to histological analysis.

As shown in Fig. 9A the untreated calvarial sample shows an almost uniform tissue, stained in green for osteoid/bone cells/tissue, which is interrupted by the marrow and soft tissues that are stained in red. The microspheres within the discs “β-TCP-MS” remained at 4 wk empty from any invading cells/tissue (not shown). Also during the following 4 wk (total 8 wk; Fig. 9D) and 8 wk (12 wk; Fig. 9G) the microspheres are only occasionally filled with cell patches. Very different from the “β-TCP-MS” controls are the histological sections thought the regenerating calvarial defects, implanted with the polyP-containing microparticles, “Ca-apolyP-MS” and “Sr-a-polyP-MS”. Already after 4 wk of regeneration time at the “Ca-apolyP-MS” healing defects most of the microspheres are invaded by cells/tissues, which are strained in green and by that reflecting bone-like areas (Fig. 9B). During the following 8 wk in regeneration the invaded areas within the microspheres increase in size and (Fig. 9E and H) and show occasionally primary ossification centers, as marked in Fig. 9H. Even more accelerated are the healing processes around the “Sr-a-polyP-MS” implants. In this series of regeneration studies the microspheres are almost homogeneously filled with cells/tissue after 4 wk and 8 wk, which display mineralizing phenotype, stained in green, (Fig. 9C, F and I) and also osteocyte clusters.

-22Determination of the regeneration/mineralization within the implant regions by nanoindentation

The hardness, based on the determination of the reduced Young's modulus (RedYM) was applied to assess more quantitatively the degree of mineralization within the disc implants zones, applying the recently developed load-displacement measurements/nanoindentation procedure. Those areas within the excised/removed implant region were selected in which the -840 pm microspheres are separated from each other by at least 80 to 150 pm. The determinations were performed after a 12 wk healing period (Fig. 10). The data for the RedYM revealed for the surrounding trabecular bone tissue a hardness of 3.05 MPa, a value that is almost reached by the samples from calvarial defects that had been implanted by discs prepared from “Sr-a-polyP-MS” with 2.93±0.38 MPa. The modulus of the implants with the “β-TCP-MS” controls amounted to 0.84±0.13 MPa. Significantly higher (/»<0.001) are the RedYM for the two polyP-containing microspheres. Also a significant difference (p<0.05) is seen for the RedYM values of “Sr-a-polyP-MS” towards the “Ca-a-polyP-MS” discs (Fig. 10).

	SEQUENCE LISTING
<110>	Muller, Werner E.G.
<120>	AMORPHOUS STRONTIUM POLYPHOSPHATE MICROPARTICLES FOR TREATMENT OF OSTEOPOROSIS AND INDUCING BONE CELL MINERALIZATION
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Claims

1. Method for the preparation of morphogenetically active amorphous strontiumpolyphosphate nanoparticles or microparticles, comprising the following steps:

a) Dissolving of sodium polyphosphate in an aqueous medium, e.g. distilled water, and adjustment to about pH 10 with a basic aqueous solution, e.g. sodium hydroxide solution;

b) Dissolving of a strontium salt in a suitable aqueous medium, e.g. aqueous ethanol;

c) Slow (e.g. dropwise) addition of said polyphosphate solution to said strontium salt solution and keeping the pH at about 10 with a basic aqueous solution, e.g. sodium hydroxide solution;

d) Forming of particles, preferably by stirring of the suspension overnight at room temperature; and

e) Collecting of the suitably produced particles, preferably by filtration, and washing with a suitable aqueous medium, e.g. aqueous ethanol, and drying.

2. The method according to claim 1, wherein said strontium salt is strontium chloride.

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 40 phosphate units.

4. The method according to any one of claims 1 to 3, wherein the strontium polyphosphate nanoparticles or microparticles are characterized by a stoichiometric ratio between 0.1 to 3 (strontium to phosphate), preferably between 0.3 and 1.0, and most preferred by a stoichiometric ratio of 0.4 to 0.5.

5. The method according to any one of claims 1 to 4, wherein the average size of the strontium polyphosphate nanoparticles or microparticles is in the range of about 0.1 to about 10 pm.

6. The method according to any one of claims 1 to 5, wherein the average size of the strontium polyphosphate microparticles is between 0.8 and 0.9 pm.

7. The method according to any one of claims 1 to 6, wherein the strontium polyphosphate nanoparticles or microparticles are encapsulated into acid-resistant capsules suitable for oral administration.

8. The method according to claim 7, wherein said acid-resistant capsules, or tablets consist of hydroxypropyl methyl cellulose (HPMC).

9. The method according to any one of claims 1 to 6, wherein the strontium polyphosphate nanoparticles or microparticles are embedded into microspheres.

10. The method according to any one of claims 1 to 6 or 9, wherein the microspheres are produced from strontium polyphosphate nanoparticles or microparticles and poly(D,L-lactideco-glycolide (PLGA).

11. A method for producing a regenerative active implant material for bone and cartilage, comprising the method according to any of claims 1 to 6 and claims 9 and 10, and further formulating said nanoparticles or microparticles into an implant material.

12. Morphogenetically active amorphous strontium-polyphosphate nanoparticles or microparticles produced according to any one of claims 1 to 10 for use in the prevention or treatment of bone-related diseases, such as osteoporosis.

13. A method for preventing or treating of bone-related diseases, such as osteoporosis, comprising administering to a patient in need thereof strontium-polyphosphate nanoparticles or microparticles produced according to any one of claims 1 to 10 and/or a regenerative active implant material produced according to claim 11.

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Application No: GB1612848.0 Examiner: Gareth Prothero