Amorphous Inorganic Polyphosphate-Calcium-Phosphate And Carbonate Particles As Morphogenetically Active Coatings and Scaffolds
20190083679 · 2019-03-21
Inventors
Cpc classification
A61L2430/02
HUMAN NECESSITIES
A61L2300/112
HUMAN NECESSITIES
A61L2300/412
HUMAN NECESSITIES
International classification
Abstract
This invention concerns a method for the production of amorphous, nano- or microparticular materials based on inorganic polyphosphate (polyP) and calcium phosphate or calcium carbonate that show osteogenic activity. In one aspect of the invention, the inventor shows that amorphous calcium polyphosphate (Ca-polyP) microparticles can be used for biological functionalization of titanium alloy surfaces. The inventive method allows the fabrication of a durable and stable, almost homogeneous and morphogenetically active Ca-polyP layer on titanium oxidized Ti-6Al-4V scaffolds that supports the growth and enhances the functional activity of bone cells, in contrast to biologically inert non-modified titanium surfaces. A preferred aspect relates to the formation of amorphous calcium phosphate (CaP) particles in the presence of low concentrations of sodium polyP. This material causes a strong upregulation of the expression of proteins involved in bone formation. A further aspect of the invention concerns a material containing polyP-stabilized ACC and small amounts of vaterite that exhibits osteogenic activity and supports the regeneration of the implant region in animal experiments. The amorphous materials according to this invention have the potential to be used for bone implants.
Claims
1. A method selected from the group consisting of: A) a method for the production of biologically active coatings of titanium alloys, comprising the following steps: a) preparing Ca-polyP microparticles by mixing an aqueous solution of Na-polyP with an aqueous solution of calcium chloride dihydrate (CaCl.sub.2.2H.sub.2O) for several hours at an elevated temperature, under formation of a colloidal suspension; b) coupling said Ca-polyP microparticle colloidal suspension to a titanium alloy scaffold using a silane coupling agent; and c) adjusting the pH value of the suspension of b) to a slightly alkaline value to allow binding of polyP to the silane-functionalized metal scaffold via Ca.sup.2+ ionic bond formation; B) a method for the preparation of biologically active amorphous polyphosphate-substituted calcium phosphate particles (aCaP-polyP) comprising the following steps: a) adding an aqueous solution of a polyphosphate salt to an aqueous solution of a phosphate source; b) adding the resulting solution to a dissolved calcium salt; c) adjusting the pH to an alkaline value; and d) collecting, washing, and drying the resulting precipitate formed; and C) a method for the preparation of biologically active amorphous calcium carbonate (ACC)-polyphosphate microparticles, comprising the following steps: a) preparing an aqueous solution of a polyphosphate salt in about 0.1 M sodium hydroxide; b) adding about 0.5 mol/L of sodium carbonate to said solution; c) diluting the resulting solution with about 1.5 volumes of deionized water; d) mixing 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 at about room temperature; and f) filtering and drying a precipitate as formed.
2-3. (canceled)
4. The method according to claim 1, wherein, in the method of part A), said titanium alloy is Ti-6Al-4V.
5. The method according to claim 1, wherein, in the method of part A), said silane coupling agent is (3-aminopropyl)trimethoxysilane [APTMS].
6. The method according to claim 1, wherein, in the method of part C) 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, based on phosphate.
7. The method according to claim 6, 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.
8. The method according to claim 1, wherein the polyphosphate salt is sodium polyphosphate.
9. The method according to claim 1, wherein the chain length of the polyphosphate is about 3 to about 1000 phosphate units.
10. The method according to claim 2, wherein, in the method of part B), the amount of the polyphosphate salt is higher than 5 wt. % referred to the calcium phosphate preparation.
11. The method according to claim 2, wherein, in the method of part B), the calcium salt is calcium chloride (CaCl.sub.2) and the phosphate source is ammonium phosphate dibasic [(NH.sub.4).sub.2HPO.sub.4)].
12. The method according to claim 1, wherein, in the method of part A), the calcium polyphosphate microparticles are characterized by a stoichiometric ratio between 0.1 to 1 and 50 to 1 of phosphate to calcium.
13. The method according to claim 12, wherein the calcium polyphosphate microparticles are characterized by a stoichiometric ratio of 7 to 1 of phosphate to calcium.
14. The method according to claim 1, wherein, in the method of part B), the amount of the calcium salt and the amount of the reagent serving as phosphate source is calculated in order to obtain the Ca/P molar ratio for the calcium phosphate of 10:6.
15. The method according to claim 1, wherein, in the method of part A), the average size of the calcium polyphosphate microparticles is about 0.1 to about 30 m.
16. The method according to claim 1, wherein, in the method of part B), the average size of the polyphosphate-substituted calcium phosphate particles (aCaP-polyP) is in the range of about 20 to about 300 nm.
17. The method according to claim 1, further comprising, in the method of part A), the step of producing biologically active titanium alloy implants.
18. The method according to claim 1, further comprising the step of producing a biologically active implant material.
19. The method according to claim 18, further comprising the step of including at least one gallium salt into said implant.
20. The method according to claim 18, wherein said biologically active implant material is an artificial bone implant.
21. An implant prepared by the method according to claim 18.
22. Use, as an implant, of the coating as produced according to the method of part A) of claim 1.
23. The method according to claim 18, wherein the biologically active implant material is an artificial bone implant.
24. A stabilized amorphous calcium carbonate (ACC) composition produced by the method of part C) according to claim 1.
25. A method for providing a dietary supplement, treating calcium deficiency, and/or preventing or treating osteoporosis, wherein said method comprises administering a stabilized ACC composition according to claim 24.
26-27. (canceled)
Description
[0097] 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.
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
[0105]
[0106]
[0107]
[0108]
[0109]
[0110]
[0111]
[0112]
[0113]
[0114]
[0115]
[0116]
[0117]
[0118]
EXAMPLES
[0119] In the following examples, the inventive method is 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 about 20 to 100 units.
Titanium-Ca-polyP (TiCa-polyP) Discs
[0120] Titanium alloy (Ti-6Al-4V) disks were etched to allow cross-linking with the silane coupling agent APTMS (
[0121] A comparison between the titanium alloy discs and the TiCa-polyP discs (light microscopic images) revealed that, in contrast to the dark gray surface color of the titanium alloy discs, the TiCa-polyP discs have a shiny silver-white appearance. After the coating of the surfaces of the discs with polyP they lose their high roughness. While the untreated discs have an average roughness of 5.5 m with a maximum of 7.02 m (
[0122] Element-specific analyse of the surfaces of the titanium discs was performed by EDX spectroscopy (
Durability of the Ca-polyP Coat
[0123] The surface coat of the polyP was measured by the determination of Ca.sup.2+ release from the coated discs in SBF (lacking Ca.sup.2+ as component), as described under Methods. In parallel assays, the release of Ca.sup.2+ from TiCa-polyP discs as well as from untreated titanium discs (control) was measured. As an additional control one TiCa-polyP disc each was inserted in the SBF incubation medium supplemented with 1 g/ml of ALP; all samples were incubated at 37 C. At time zero in all three assays the Ca.sup.2+ concentration was <3 g/ml. After one d in the incubation medium the amount of Ca.sup.2+ was determined as follows: TiCa-polyP discs: <3 g/ml (<3 g/ml [control]; 123 g/ml [TiCa-polyP discs+ALP]); 5 parallel assays were performed. The Ca.sup.2+ release increased slightly in assays containing the TiCa-polyP discs after a 3 d incubation period, in contrast to the assays of TiCa-polyP discs together with ALP. The following values are measured: 5.20.8 g/ml [TiCa-polyP discs] (<3 g/ml [control]; 86.93.2 g/ml [TiCa-polyP discs+ALP]). After 12 d in the incubation assay the values are as follows: 9.71.2 g/ml [TiCa-polyP discs]; <3 g/ml [control]; 153.117.1 g/ml [TiCa-polyP discs+ALP].
Growth of SaOS-2 Cells on the Titanium Discs
[0124] The overall growth rate of the bone-like SaOS-2 cells was determined by the XTT assay as described under Methods. The cells were seeded at a density of 310.sup.4 cells/well (2 ml assays) for all three parallel series of experiments; assays without titanium discs, titanium alloy discs, TiCa-polyP discs (
[0125] The property of the discs, coated with polyP, being a very suitable matrix for SaOS-2 cells to grow onto, was also underscored by staining the surface of the discs. Titanium alloy discs, not coated with polyP were incubated for 3 d with SaOS-2 cells; after that period no cells could be visualized onto the discs. In contrast, if polyP-coated TiCa-polyP discs are incubated in the presence of SaOS-2 cells for the same period of time an almost homogenous mono-cell layer is observed. A closer inspection at higher magnification revealed that the cells show the property of cell spreading, a characteristic sign for vital survival and growth of cells.
[0126] As a further support of the conclusion that SaOS-2 cells are growing readily onto TiCa-polyP discs the areas, covered with cells, were analyzed for the distribution of elements carbon (C), titanium (Ti) and phosphorus (P). The semiquantitative determinations of the elements were performed by SEM-based EDX mappings. The localization of the cells was obtained by recording the back-scattered electrons. Within the regions where the cells grow a high accumulation for the element C is measured, while titanium and polyP are highlighted outside of the cell areas, at the surrounding surface of the discs onto which the cells grow.
Expression of Carbonic Anhydrase IX and Alkaline Phosphatase
[0127] As a marker for the functional activity of the SaOS-2 cells, growing onto titanium discs, the expression of the two genes encoding for the enzymes carbonic anhydrase IX (CA IX) and alkaline phosphatase (ALP) was determined by quantitative qRT-PCR. The studies for the steady-state level of transcripts of CA IX in SaOS-2 cells growing for 3 d in the absence of the MAC showed for the cultures which contained titanium alloy discs a significant decrease of the expression levels from 0.310.03 (time at seeding) to 0.120.01, while the levels in the cells cultured in the absence of discs or the presence of the TiCa-polyP discs increased from 0.270.02 and 0.250.03 to 0.380.04 and 0.400.05, respectively (
[0128] In parallel, the expression of the gene for the enzyme ALP was determined, likewise by qRT-PCR. Again the data (
[0129] In a further set of experiments, the assays were performed in the presence of 100 M gallium nitrate (see Table 1). The cells were cultivated either without any titanium discs, or either onto titanium alloy discs or on TiCa-polyP discs, as described above, first in the absence of the MAC for 3 d and then in medium supplemented with the MAC for additional 5 d. The results revealed that in the absence of discs, the steady-state level of CA IX transcripts in SaOS-2 cells growing for 3 d in the absence of the MAC and subsequently for 5 d in the presence of the MAC increased from 0.240.05 to 0.890.11 (Table 1), compared to 0.270.02 to 0.810.08 in the absence of gallium nitrate (see also
[0130] Similar results were obtained, if the effect of gallium nitrate on the steady-state levels of ALP transcripts in the absence of discs and in the presence of titanium alloy discs or of TiCa-polyP discs were determined. In the absence of the titanium discs, the addition of the gallium salt had only a small effect on the ALP transcript levels, compared to the assay without this additive, while in the presence of the TiCa-polyP discs, again a strong synergistic effect on the Ca-polyP-caused increase of the ALP transcript levels was observed (increase from 0.0270.003 to 0.1150.007), if compared with the assay without this supplement (increase from 0.0300.003 to 0.0740.007; see also
TABLE-US-00001 TABLE 1 Effect of gallium on the expression of the genes encoding for CA IX and for ALP. without Ga with Ga MAC +MAC MAC +MAC Incubation Gene 0 d 5 d 0 d 5 d Without CA IX 0.27 0.02 0.81 0.08 0.24 0.05 0.89 0.11 discs Titanium CA IX 0.31 0.03 nd 0.28 0.05 nd alloy discs TiCapolyP CA IX 0.25 0.03 0.59 0.06 0.27 0.04 0.96 0.15 discs Without ALP 0.029 0.004 0.097 0.007 0.027 0.004 0.111 0.010 discs Titanium ALP 0.028 0.003 nd 0.030 0.002 nd alloy discs TiCapolyP ALP 0.030 0.003 0.074 0.007 0.027 0.003 0.115 0.007 discs The experiment was performed as described in the legend to FIG. 8, in the absence or in the presence of 100 M gallium nitrate in the assay mixture. The expression values were normalized to the expression of GAPDH. The cells were cultivated either without any titanium discs, or either onto titanium alloy discs or on TiCapolyP discs. The cultures were incubated at first in the absence of the MAC for 3 d and then transferred to medium, supplemented with the MAC, and the incubation was continued for additional 3 or 5 d. nd, not detectable.
XRD Analyses
[0131] The phase identification of the HA as well as the polyP-HA particles was performed by applying the powder X-ray diffraction (XRD) method (
FTIR Spectral Analysis
[0132] All the spectra for CaP recorded here, like pure HA, as well as HA(2.5)polyP and HA(5)polyP, showed the typical HA bandings (
[0133] For comparison, the spectrum of polyP is also included in the CaP tracings (
TEM and Particle Size Distribution Results
[0134] The morphologies of the CaP samples were analyzed by TEM. The HA sample showed needle-like nano-crystals with an average length of 398 nm and a width of 144 nm (
Effect of CaP Samples and of Ca-polyP Nanoparticles on Cell Growth
[0135] The cell viability and growth of SaOS-2 cells onto the CaP samples were tested by applying the MTT assay. Those samples were added at a concentration of 100 g/mL to the cells. In parallel, an incubation was performed with 10 g/mL of Ca-polyP nanoparticles, aCa-polyP-NP, a sample which has been proven to increase the growth rate of the cells and to cause an increased gene expression of ALP and COL-I.
[0136] The results revealed that, after an incubation period of 2 d no significant differences in the growth of the cells on the different substrates are seen. However, after an incubation period of 3 d a significant increase in the growth of the SaOS-2 cells in the presence of aCa-polyP-NP (from 1.740.19 [day 2] to 2.450.20 absorbance units) is measured. The increase of the growth onto the different CaP samples is lower and significant for HA (from 1.540.18 to 1.930.21) and for aCaP(10)polyP (from 1.540.18 to 2.310.25).
SEM Analyses
[0137] Cells were cultivated onto either pure HA or onto aCaP(10)polyP discs for 3 d. Then the samples were fixed with paraformaldehyde and inspected by SEM. It is seen that in both assays the cells firmly attach to the substrate both for the HA and the aCaP(10)polyP cultures. At higher magnification the property of the cells for spreading becomes obvious.
Gene Expression Propensity of SaOS-2 Cells on CaP
[0138] The bone-related SaOS-2 cells were cultivated initially for 3 d and then transferred into new medium, lacking or supplemented with MAC and containing also the CaP samples (100 g/mL) or the polyP nanoparticles (10 g/mL). Then the incubation was continued for 7 d prior to qRT-PCR analyses to determine the steady-state level of transcripts for COL-I or ALP (
[0139] The determinations revealed that the expression of COL-I at the time of seeding the cells is low with 0.260.07 expression units, related to the expression of GAPDH. In the absence of MAC and the 7 d presence of the CaP samples or the polyP nanoparticles the expression level significantly increased after incubation with HA(2.5)polyP (to 0.350.05), HA(5)polyP (to 0.430.05), as well as aCaP(10)polyP (to 0.520.06), and, as expected, also for polyP aCa-polyP-NP (to 0.630.07);
[0140] A comparable inducing expression pattern is recorded for the ALP gene, if correlated to the reference gene GAPDH. Again, in the absence of MAC the ALP expression level is lower compared to the values measured for cells incubated for 7 d in the presence of MAC (
Effect of polyP on Calcite Formation: FTIR and XRD Spectra
[0141] For all CaCO.sub.3 solids the following FTIR signals were recorded: .sub.1 (symmetric stretching) at 1080 cm.sup.1; .sub.2 (out of-plane bending) at 870 cm.sup.1; .sub.3 (doubly degenerate planar asymmetric stretching) at 1400 cm.sup.1 and .sub.4 (doubly degenerate planar bending) at 700 cm.sup.1. The published IR data (Rodriguez-Blanco J D, Shaw S and Benning L G (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.sup.1 (3), 876 cm.sup.1 (.sub.2), and 714 cm.sup.1 (.sub.4) for calcite and 1090 cm.sup.1 (.sub.1), 870 cm.sup.1 (.sub.2), and 745 cm.sup.1 (.sub.4) for vaterite (
[0142] 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 CaCO.sub.3 solids, prepared in the absence of polyP were pure calcite (
Morphology of the Solids Formed
[0143] The solids formed by precipitation from CaCl.sub.2.2H.sub.2O and Na.sub.2CO.sub.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;
Effect of CaCO.SUB.3 .Samples on Cell Growth/Viability
[0144] The cell growth/viability of SaOS-2 cells after exposure to the CaCO.sub.3 preparations was determined by applying of the MTT assay (see above). The CaCO.sub.3 samples were added at a concentration of 50 g/mL to the cells. In parallel, a control assay lacking any CaCO.sub.3 solids was performed. The results revealed that the increase in cell growth/viability from 0.700.11 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 CaCO.sub.3 series (CCP5, CCP10 or calcite).
Stability of the CaCO.SUB.3 .Solids in the Culture Medium
[0145] SaOS-2 cells grow in an adherent manner (Pautke C, et al (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 (
Release of Ca.sup.2+ from the CaCO.sub.3 Particles
[0146] 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 Ca.sup.2+ is released from the calcite sample, already 6.81.1 g/ml (68% of the total Ca.sup.2+ 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 (
Expression of ALP in SaOS-2 Cells as Well as in MSCs
[0147] The morphogenetic activity of the CaCO.sub.3 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 0.310.9 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 CaCO.sub.3 samples in the assays (
[0148] 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 CaCO.sub.3 solid, as well as in the presence of both CCP5 and CCP10. No inducing effect is determined in cells exposed to calcite (
Expression of BMP2 in SaOS-2 Cells
[0149] The expression level of BMP2 in response to CCP10 and polyP (Ca.sup.2+ 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 g/ml), polyP (Ca.sup.2+ complex; 5 g/ml; corresponding to 50 M with respect to phosphate) or calcite (50 g/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
Microspheres, Used for the Animal Studies
[0150] The control spheres, the cont-mic had a size of (845 m [82060 m]; n=50), while those containing polyP were insignificantly slightly smaller (838 m [81665 m]);
Compatibility Studies in Rats
[0151] 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. 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 and the polyP-mic series. After 2 weeks the regions, where the microspheres had been placed into the muscle, a few cells are scattered within the microsphere areas. However, after a 4 and 8 weeks 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 an accumulation of the cells within the spheres are evident. After 8 weeks the spheres are almost filled with infiltrating cells.
[0152] Determinations of the hardness of the implant region after 8 weeks revealed a significant increase of the median RedYM stiffness of 33.137.97 kPa for the cont-mic and 60.1112.13 kPa for the polyP-mic microspheres. The muscles of the back of rats before implantation have a median RedYM stiffness of 74.4014.33 kPa.
Methods
Polyphosphate
[0153] 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).
TiCa-polyP Discs
[0154] Titanium alloy (Ti-6Al-4V) disks (15 mm in diameter and 2 mm in thickness, can be obtained, for example, from Nobel Biocare. Prior to use they are polished with emery paper (silicon carbide; Matador) followed by ultrasonic cleaning in distilled water, and subsequently washing in acetone (10 min) and in 40% ethyl alcohol solution (15 min), and finally rinsing in distilled water for 20 min. The samples are dried at 50 C. for 24 h. Then titanium alloy discs are incubated in 20 mL of 5 M HCl at room temperature for 6 h. After gentle washing in distilled water the discs were dried at room temperature and the treated disc samples were overlayed with 10 ml Ca-polyP nanoparticle suspension in the presence of the silane coupling agent (3-aminopropyl)trimethoxysilane [APTMS] (e.g., from Sigma-Aldrich).
[0155] Ca-polyP microparticles are prepared by mixing of 0.5 g of Na-polyP with ATPMS solution (1 wt %) in 100 ml water; then 0.1 g Ca.sup.2+-chloride dihydrate (CaCl.sub.2.2H.sub.2O) was added. The titanium disks were incubated in the above suspension for 3 h at a 90 C.; under those conditions a colloidal suspension was initially formed. The pH of the environment was adjusted to 8.0 to allow binding of polyP to the silane-etched titanium discs via Ca.sup.2+ ionic bonds/bridging. The samples remained in this suspension for 1 d. The influence of two different ATMPS concentrations (1 mg/assay and 2 mg/assay, respectively) on the morphology of the coat formed onto the titanium surface was studied. Finally, the specimens, titanium-Ca-polyP (TiCa-polyP)discs, were removed and dried at 100 C. (see
[0156] In the experiments described under Examples, if not mentioned otherwise, discs prepared with the higher proportion of APTMS and then with Ca-polyP have been used.
Synthesis of HA and polyP-Hydroxyapatite
[0157] Hydroxyapatite (HA) nanoparticles can be synthesized by a wet chemical precipitation method from calcium chloride (CaCl.sub.2) as Ca.sup.2+ source, and ammonium phosphate dibasic ((NH.sub.4).sub.2HPO.sub.4) as phosphate source. To precipitate stoichiometric HA (Ca.sub.10(PO.sub.4).sub.6(OH).sub.2; Ca/P ratio of 1.667), 100 mL of 0.3 M aqueous solution of (NH.sub.4).sub.2HPO.sub.4 is dropwise added to 100 mL 0.5 M aqueous solution of CaCl.sub.2. The amount of reagents is calculated in order to obtain the Ca/P molar ratio for HA of 10:6. The pH of the reaction is maintained at 10 with the addition of sodium hydroxide solution.
[0158] In order to prepare polyP-substituted HA nanoparticles of various polyP content, the starting components (CaCl.sub.2 and (NH.sub.4).sub.2HPO.sub.4) are additionally supplemented with 2.5, 5 or 10 wt. % of Na-polyP (referred to HA, or the respective CaP preparation) as follows. The respective amount of Na-polyP, 0.12 g [HA(2.5)polyP], 0.25 g [HA(5)polyP] or 0.50 g [aCaP(10)polyP], is added to the aqueous solution of (NH.sub.4).sub.2HPO.sub.4; then this solution is added to the dissolved CaCl.sub.2. The pH is kept at 10. The resulting precipitates are left at room temperature for 24 h. Then the precipitates are filtered, washed 3-times with distilled water before being dried in a hot air oven at 60 C. for 24 h. The final powders are termed HA, HA(2.5)polyP, HA(5)polyP and aCaP(10)polyP.
Fabrication of the polyP Nanoparticles
[0159] For comparative functional/biological studies amorphous Ca-polyP nanoparticles can be prepared as described (Mller W E G, Tolba E, Schrder H C, Diehl-Seifert B and Wang X H. Retinol encapsulated into amorphous Ca.sup.2+ polyphosphate nanospheres acts synergistically in MC3T3-E1 cells. Eur J Pharm Biopharm 2015; 93:214-223). In brief, 2.8 g of CaCl.sub.2 in 30 mL distilled water are added dropwise to 1 g of Na-polyP, dissolved in 50 mL distilled water at a pH of 10.0. The amorphous Ca-polyP nanoparticles formed are washed in water and then dried at 50 C.; the preparation is termed aCa-polyP-NP. The average diameter of the spherical particles is 9628 nm and they have an amorphous state (Mller W E G, et al. A new polyphosphate calcium material with morphogenetic activity. Materials Letters 2015c; 148:163-166).
Preparation of Ca-Carbonate Microparticles
[0160] Ca-carbonate (CaCO.sub.3) is prepared by direct precipitation in aqueous solutions (at room temperature), using CaCl.sub.2.2H.sub.2O solution and Na.sub.2CO.sub.3 solution at equimolar concentration ratio between Ca.sup.2+ and CO.sub.3.sup.2 through rapid mixing; for a scheme, see
[0161] To study the effect of polyP on precipitated CaCO.sub.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 Na.sub.2CO.sub.3 is added; subsequently this solution is diluted with 30 mL of deionized water. Then 50 mL water, containing 1.47 g CaCl.sub.2.2H.sub.2O, 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 CaCO.sub.3 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 CaCO.sub.3 precipitation assay) or CCP10 (0.1 g).
Durability of the Ca-polyP Coat
[0162] The stability and the durability of the Ca-polyP coat around the titanium discs can be quantified, for example, by determination of the Ca.sup.2+ release from the discs. The control discs, as well as the TiCa-polyP discs are submersed in simulated body fluid (SBF) but omitting Ca.sup.2+ as component; the pH is adjusted to 8.0. The assay volume is 1 ml and incubation is performed at 37 C. The Ca.sup.2+ concentration is determined by applying the complexometric titration method; the reagent Eriochrome Black T is used (e.g., from Sigma-Aldrich). In the experiments described under Examples, the surface thickness of the polyP coat on one plane of the discs has been determined microscopically to be 5 m. In turn, the total amount of Ca-polyP (density of 3 g/ml) on one plane of the discs had a value of 2.4 mg.
[0163] Where indicated under Examples, 5 g of alkaline phosphatase (ALP) from bovine intestinal mucosa (e.g. from Sigma; 6,500 DEA units/mg protein) was added to the reaction mixture.
Microscopic Analysis
[0164] The light microscopic inspection of the discs can be performed, for example, with a VHX-600 Digital Microscope from KEYENCE, equipped either with a VH-Z25 zoom lens (25 to 175 magnification) or a VH-Z-100 long-distance high-performance zoom lens (up to 1000 magnification). The surface roughness can be measured, for example, by using the KEYENCE VK-analyser software. For the scanning electron microscopic (SEM) analyses, for example, a HITACHI SU 8000 electron microscope (Hitachi High-Technologies Europe GmbH, Krefeld, Germany) can be employed.
Electron Microscopy and Energy Dispersive X-Ray Spectroscopy
[0165] For the transmission electron microscopic (TEM) analyses, for example, the TemCam-F416 (4K4K) CCD camera (TVIPS), operated on a Tecnai 12 transmission electron microscope (FEI) at an accelerating voltage of 120 kV, can be used. The equipment is connected with a particle size analyzer (ImageJ); in the experiments, described under Examples, 25-50 crystals/spheres have been evaluated.
[0166] Scanning electron microscopic (SEM) analyses can be performed, for example, with an SU 8000 instrument (Hitachi High-Technologies Europe), at low voltage (1 kV). For the studies described under Examples, the cells were grown in the 6-well plates onto CaP preparations that had been pressed to 1 mm thick discs, with a diameter of 34 mm, for 3 d. The cells, growing on the CaP substrates are fixed with 4% paraformaldehyde.
[0167] Energy dispersive X-ray (EDX) spectroscopy can be performed, for example, with an EDAX Genesis EDX System attached to a scanning electron microscope (Nova 600 Nanolab; FEI) operating at 10 kV with a collection time of 30-45 s. Areas of approximately 5 m.sup.2 are analyzed.
[0168] EDX mapping can be performed, for example, with the Hitachi SU 8000 microscope, carried out at low voltage (<1 kV, analysis of near-surface organic surfaces). The SEM is coupled with an XFlash 5010 detector, an X-ray detector that allows the simultaneous EDX-based elemental analyses. This combination of devices is used for higher-voltage (10 kV) analysis, during which the XFlash 5010 detector is used for element mapping of the surfaces of the deposits. The HyperMap database is used for interpretation.
X-Ray Diffraction Analyses
[0169] The X-ray diffraction (XRD) experiments can be performed as described (Raynaud S, et al. Calcium phosphate apatites with variable Ca/P atomic ratio I. Synthesis, characterisation and thermal stability of powders. Biomaterials 2002; 23:1065-1072). The patterns of dried powders can be registered, for example, on a Philips PW 1820 diffractometer with Cu.sub.K radiation (=1.5418 , 40 kV, 30 mA) in the range 2=5-65 (2=0.02, t=5 s). The HA crystals can be identified as described (Lee D S H, Pai Y, Chang S. Effect of thermal treatment of the hydroxyapatite powders on the micropore and microstructure of porous biphasic calcium phosphate composite granules. J Biomat Nanobiotechnol 2013; 4: 114-118).
Fourier Transformed Infrared Spectroscopy
[0170] The Fourier transformed infrared (FTIR) spectroscopic analyses can be performed by using micro-milled (agate mortar and pestle) mineral powder, for example, in an ATR-FTIR spectroscope/Varian 660-IR spectrometer (Agilent), equipped with a Golden Gate ATR unit (Specac). Each spectrum shown under Examples represents the average of 100 scans with a spectral resolution of 4 cm.sup.1 (typically 550-1800 cm.sup.1). Baseline correction, smoothing, and analysis of the spectra can be achieved, for example, with the Varian 660-IR software package 5.2.0 (Agilent). Graphical display and annotation of the spectra can be performed, for example, with Origin Pro (version 8.5.1; OriginLab).
Release of Ca.sup.2+ from the CaCO.sub.3 Particles
[0171] In separate assays 100 g/ml of either calcite 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 l are taken, centrifuged and the supernatant analyzed for Ca.sup.2+ 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
[0172] Bone cell like SaOS-2 cells (human osteogenic sarcoma cells) are cultured in McCoy's medium (Biochrom-Seromed), supplemented with 2 mM L-glutamine, 10% or 15% heat-inactivated fetal calf serum (FCS), and 100 units/ml penicillin and 100 g/ml streptomycin. The cells are incubated in 25-cm.sup.2 flasks or in 6-well plates (surface area 9.46 cm.sup.2; e.g. from Orange Scientifique) in a humidified incubator at 37 C. Routinely, the cultures are started with 310.sup.4 or 110.sup.4 cells/well in a total volume of 3 ml. Where indicated, the cultures are first incubated for a period of 3 d in the absence the mineralization-activating cocktail (MAC), comprising 5 mM -glycerophosphate, 50 mM ascorbic acid and 10 nM dexamethasone. Then the cultures are continued to be incubated for up to 7 d in the absence or presence of the MAC. The HA/polyP mineral samples (100 g/mL [HA, CaP] or 10 g/mL [aCa-polyP-NP]), are added to each well at the beginning of the experiments. Every third day the culture medium is replaced by fresh medium/serum and, where indicated, also with MAC. For the studies with the discs, 24-well plates (e.g., from Corning; diameter of each well 15.6 mm) are used into which the 15 mm large discs are inserted. The assays are performed with a total volume of 2 ml of cells/medium/FCS.
[0173] In a further series of experiments, shown under Examples, the assays have been performed in the presence of 100 M gallium nitrate.
Cell Proliferation/Cell Viability Assays
[0174] Cell proliferation/growth can be determined, for example, by the colorimetric method, based on the tetrazolium salt XTT, e.g., Cell Proliferation Kit II (Roche), or 3-[4,5-dimethyl thiazole-2-yl]-2,5-diphenyl tetrazolium (MTT; #M2128, Sigma) (Wang X H, et al. (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
[0175] 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 X H, 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. Marine Drugs 12, 1131-1147).
Reverse Transcription-Quantitative Real-Time PCR Analyses
[0176] The quantitative real-time RT [reverse transcription]-PCR (qRT-PCR) technique is applied to determine the effect of the discs on the expression levels of the following genes in SaOS-2 cells. In brief, RNA was extracted from the cells and the PCR reaction is performed using the following primer pairs: carbonic anhydrase IX (CA IX; NM_001216 human) Fwd: 5-ACATATCTGCACTCCTGCCCTC-3 [nt.sub.977 to nt.sub.998] (SEQ ID NO. 1) and Rev: 5-GCTTAGCACTCAGCATCACTGTC-3 [nt.sub.1105 to nt.sub.1083] (SEQ ID NO. 2), alkaline phosphatase (ALP; NM_000478.4) Fwd: 5-TGCAGTACGAGCTGAACAGGAACA-3 [nt.sub.1141 to nt.sub.1164] (SEQ ID NO. 3) and Rev: 5-TCCACCAAATGTGAAGACGTGGGA-3 [nt.sub.1418 to nt.sub.1395] (SEQ ID NO. 4), type I collagen (Col I; NM_000088.3) Fwd: 5-GACTGCCAAAGAAGCCTTGCC-3 [nt.sub.5073 to nt.sub.5093] (SEQ ID NO: 5) and Rev: 5-TTCCTGACTCTCCTCCGAACCC-3 [nt.sub.51196 to nt.sub.5175] (SEQ ID NO: 6), and BMP2 (bone morphogenic protein 2; NM_001200.2) Fwd: 5-ACCCTTTGTACGTGGACTTC-3 [nt.sub.1681 to nt.sub.1700] (SEQ ID NO: 7) and Rev: 5-GTGGAGTTCAGATGATCAGC-3 [nt.sub.1785 to nt.sub.1804] (SEQ ID NO: 8). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference (NM_002046.5) Fwd: 5-CCGTCTAGAAAAACCTGCC-3 [nt.sub.929 to nt.sub.947] (SEQ ID NO. 9) and Rev: 5-GCCAAATTCGTTGTCATACC-3 [nt.sub.1145 to nt.sub.1126] (SEQ ID NO. 10). The PCR reactions can be performed, for example, in an iCycler (Bio-Rad), applying the respective iCycler software. After determinations of the C.sub.t values the expression of the respective transcripts are calculated.
Preparation of PLGA-Based Microspheres
[0177] The microspheres, used for the animal experiments are produced as described in details (Wang S F, et al. (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 CaCO.sub.3/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 m are obtained.
[0178] The content of polyP in the microspheres is determined as described (Mahadevaiah M S, et al. (2007) A simple spectrophotometric determination of phosphate in sugarcane juices, water and detergent samples. E-Journal of Chemistry 4:467-473).
Determination of the Mechanical Properties
[0179] 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 S F, et al. (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
[0180] 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 accommodate the microspheres. The microspheres (20 mg in a volume of 100 L) are introduced into the muscle and stabilized there in the deeper layer (Vidya S., Parameswaran A., Sugumaran V G (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
[0181] The results are statistically evaluated using paired Student's t-test.