Pyrophosphate type material, process for preparing such a material and use for bone repair

Abstract

A material, especially a glassy material of pyrophosphate type, corresponding to the general formula (I): {[(M.sup.2+).sub.1x(R.sup.+).sub.2x].sub.2[(P.sub.2O.sub.7.sup.4).sub.1y(PO.sub.4.sup.3).sub.4y/3]} n(H.sub.2O) in which x and y are positive rational numbers each between 0 and 0.8, and n is such that the weight percentage of water in the material is greater than 0 and less than or equal to 95. M.sup.2+ represents a bivalent ion of a metal chosen from calcium, magnesium, strontium, copper, zinc, cobalt, manganese and nickel, or any mixture of such bivalent ions. R.sup.+ represents a monovalent ion of a metal selected from potassium, lithium, sodium, and silver, or any mixture of such monovalent ions. This material in particular can be used in manufacturing of bone replacements or prosthesis coatings.

Claims

1. A material comprising a compound of general formula (I):
{[(M.sup.2+).sub.1x(R.sup.+).sub.2x].sub.2[(P.sub.2O.sub.7.sup.4).sub.1y(PO.sub.4.sup.3).sub.4y/3]}n(H.sub.2O)(I) wherein:
0<x0.8,
0y0.8, n is a positive rational number such that a percentage by weight of water in the material is greater than 0 and less than or equal to 95, M.sup.2+ represents a divalent ion of a calcium, and R.sup.+ represents a monovalent ion of a sodium.

2. The material as claimed in claim 1, wherein y in the general formula (I) is such that: 0y0.5.

3. The material as claimed in claim 1, wherein n in the general formula (I) is such that the percentage by weight of water in the material is greater than or equal to 5 and less than or equal to 95.

4. The material as claimed in claim 1, wherein n in the general formula (I) is such that the percentage by weight of water in the material is greater than 0 and less than or equal to 20, and wherein the material is an amorphous material.

5. The material as claimed in claim 4, wherein the material is porous.

6. The material as claimed in claim 1, wherein n in the general formula (I) is such that the percentage by weight of water in the material is greater than 20 and less than or equal to 95, and wherein the material is a gel.

7. The material as claimed in claim 1, wherein a R.sup.+/P molar ratio is less than or equal to 0.3.

8. The material as claimed in claim 1, wherein a R.sup.+/P molar ratio is greater than 0.3.

9. The material as claimed in claim 1, doped with a percentage by weight of between 0 and 15%, limits included, of an element selected from the group consisting of copper, iron, chromium, manganese, zinc, lanthanum, lithium, cerium, praseodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, erbium, thulium, neodymium and ytterbium, and of any mixture of said elements.

10. The material as claimed in claim 1, in a form of monoliths, nanoparticles, microparticles, a thin layer with a thickness of less than 10 m, a thick layer with a thickness of greater than 10 m or fibers.

11. A method of manufacturing bone substitutes, said method comprising a step of manufacturing bone substitutes from the material as claimed in claim 1.

12. A method of manufacturing prostheses coatings, said method comprising a step of manufacturing prostheses coatings, from the material as claimed in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The characteristics and advantages of the invention will become clearly apparent in the light of the examples below, provided simply by way of illustration and without being limiting of the invention, with the support of FIGS. 1-11, in which:

(2) FIG. 1 shows a photograph, with a magnification of 3.2, of a vitreous material in accordance with the invention with the composition
{[(Ca.sup.2+).sub.0.84(K.sup.+).sub.0.33].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}.0.43H.sub.2O;

(3) FIG. 2 shows the thermogravimetric analysis curves of vitreous materials in accordance with the invention, the Material A being a glass with the composition {[(Ca.sup.2+).sub.0.84(K.sup.+).sub.0.33].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}. 0.43H.sub.2O, and the Material B for its part being a glass-ceramic with the composition {[(Ca.sup.2+).sub.0.80(K.sup.+).sub.0.40].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}.0.3H.sub.2O;

(4) FIG. 3 shows X-ray diffractograms obtained for the vitreous materials Material A and Material B of FIG. 2;

(5) FIG. 4 shows scanning electron microscopy photographs obtained for the vitreous materials Material A and Material B of FIG. 2;

(6) FIG. 5 shows a scanning electron microscopy photograph obtained for the vitreous material Material B of FIG. 2, with a greater magnification (magnification of 500 times);

(7) FIG. 6 shows .sup.31P NMR spectra obtained for the vitreous materials Material A and Material B of FIG. 2;

(8) FIG. 7 shows Raman microspectroscopy spectra obtained for the vitreous materials Material A and Material B of FIG. 2;

(9) FIG. 8 shows graphs illustrating, for Material A of FIG. 2, the change in the concentrations of calcium and of phosphorus as a function of the duration of immersion of a sample of this material in simulated body fluid (SBF) at 37 C.;

(10) FIG. 9 shows a graph illustrating the cell viability and the metabolic activity of mesenchymal stem cells isolated from human bone marrow stroma, after culturing in a medium which has been brought into contact beforehand with Material A of FIG. 2 for 24 h (1), 48 h (2) and 72 h (3);

(11) FIG. 10 shows a scanning electron microscopy photograph obtained for a vitreous material in accordance with the invention with the composition
{[(Ca.sup.2+).sub.0.84(Na.sup.+).sub.0.33].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}.0.43H.sub.2O (Material C); and

(12) FIG. 11 shows an X-ray diffractogram obtained for the vitreous material of FIG. 10.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1Preparation of Vitreous Materials in Accordance with the Invention

(13) Vitreous materials in accordance with the invention, respectively called Material A and Material B, are prepared, which materials have the following compositions:

(14) Material A:
{[(Ca.sup.2+).sub.0.84(K.sup.+).sub.0.33].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}.0.43H.sub.2O
Material B:
{[(Ca.sup.2+).sub.0.80(K.sup.+).sub.0.40].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}.0.37H.sub.2O.
Material A is a glass, whereas Material B is a glass-ceramic.

(15) These materials are each prepared by a process in accordance with the present invention, in the following way.

(16) Step 1

(17) A solution (1) of calcium precursor is prepared by dissolving CaCl.sub.2 in 20 ml of distilled water, in a proportion of 0.4 g for Material A and 1.6 g for Material B.

(18) A solution (2) of pyrophosphate precursor is prepared by dissolving 5.5 g of K.sub.4P.sub.2O.sub.7 in 200 ml of distilled water.

(19) Step 2

(20) The solution (1) is added dropwise to the solution (2) over a period of time of 5 min.

(21) Step 3

(22) The resulting solution is washed with demineralized water and subjected to centrifugation for 5 min at 7500 revolutions per minute, in order to obtain a hydrated gel in accordance with the invention.

(23) Step 4

(24) This gel is poured into a container and then treated at a temperature of 70 C. for 48 h, so as to obtain, respectively, the vitreous materials Material A and Material B.

Example 2Characterization of the Materials

(25) For each of the Materials A and B obtained in example 1, the following characterization experiments were carried out.

(26) Photography

(27) A photograph of the Material A obtained is shown in FIG. 1.

(28) It is observed therein that this material exists in the form of transparent millimetric pieces, which are characteristic of a glass.

(29) Analysis by ICP-AES Spectrometry (Inductively Coupled Plasma-Atomic Energy Spectroscopy)

(30) For the implementation of this technique, 100 mg of sample, mixed with 500 mg of lithium metaborate LiBO.sub.2, were heated at 1100 C. The resulting solid was dissolved in 200 ml of nitric acid at 1 mol/l. The solution was nebulized in the spectrometer (Ultima Horiba device). This technique made it possible to determine the calcium, phosphorus and potassium concentrations of each of the Materials A and B, and thus to confirm their composition and their chemical formulae.

(31) The Ca, P and K concentrations thus determined are shown in table 1 below.

(32) TABLE-US-00001 TABLE 1 Ca, K and P concentrations for Materials A and B in accordance with the invention, determined by ICP-AES Material A B [Ca] (mol/l) 0.61 0.54 [K] (mol/l) 0.16 0.27 [P] (mol/l) 0.66 0.63
Thermogravimetric Analysis

(33) This analysis was carried out using a Setaram Instrumentation Setsys Evolution device. The measurements were carried out from 25 C. to 900 C., with a gradient of 5 C./min.

(34) The thermogravimetric analysis made it possible to confirm the amount of water in Materials A and B and thus their chemical formulae.

(35) From the thermogravimetric analysis curves shown in FIG. 2, it was possible to establish a water loss of 14.3% for Material A and of 12.3% for Material B.

(36) X-Ray Diffraction

(37) This analysis was carried out using an Inel CPS 120 instrument, at a wavelength of cobalt (K1)=1.78897 , from 3 to 110 with a step of 0.02.

(38) The diffractograms obtained have made it possible to demonstrate the amorphous nature of Material A, having a K/P atomic ratio of less than 0.3, as shown in FIG. 3. In this figure, a halo characteristic of a vitreous material is observed for Material A. For Material B, having a K/P atomic ratio of greater than 0.3, it is indeed observed that the X-ray diffractogram still consists of a halo characteristic of a vitreous material but that peaks have appeared, corresponding to a crystalline phase (Ca.sub.10K.sub.4(P.sub.2O.sub.7).sub.6.9H.sub.2O). This combination is typical of a glass-ceramic, formed of a crystalline phase distributed in a vitreous matrix.

(39) Scanning Electron Microscopy

(40) This analysis was carried out using a Leo 435 VP scanning microscope with an acceleration voltage of 15 kV.

(41) The photographs obtained, for Material A and Material B, are shown in FIG. 4. They demonstrate a morphology typical of a glass for Material A and crystalline entities in a vitreous matrix for Material B, which is characteristic of a glass-ceramic. These results thus confirm those obtained by X-ray diffraction.

(42) Furthermore, a photograph obtained for Material B with a greater magnification is presented in FIG. 5. This photograph clearly shows the porosity of this material.

(43) Solid-State Nuclear Magnetic Resonance (NMR)

(44) This analysis was carried out using a 600 MHz NMR spectrometer, 20 kHz MAS, 3.2 mm MAS probe, 0 C. reg .sup.31P one pulse; DS=4, NS=4, 45 pulse, recycling time 90 s.

(45) The spectra obtained for each of Material A and Material B are shown in FIG. 6.

(46) It is observed therein that the phosphorus contained in the materials is present therein in two forms: pyrophosphate (major) and orthophosphate (minor), whether this be for the glass material (Material A) or for the glass-ceramic material (Material B). A quantitative evaluation of the two forms of phosphates present has been carried out. The results are shown in FIG. 6 (orthophosphate/pyrophosphate distribution: 15%/85%).

(47) Raman Microspectroscopy

(48) This analysis was carried out using a LabRam HR800 confocal microspectrometer (Horiba Jobin Yvon), AR-diode laser/wavelength =532 nm.

(49) The spectra obtained are shown in FIG. 7. They confirm, in particular by the presence of the band at 740 cm.sup.1, which is a characteristic of the P-O-P vibration, the presence in the materials of phosphorus in the pyrophosphate entity form, this being the case both for Material A and for Material B.

(50) Furthermore, Material A exhibits bands characteristic of orthophosphates, in particular the band at 963 cm.sup.1, which are more intense than for Material B, which is consistent with the quantitative analyses obtained by solid-state NMR, shown in FIG. 6 (orthophosphate/pyrophosphate distribution 15%/85%).

Example 3Study of the Dissolution in a Solution Simulating Blood Plasma

(51) The kinetics of dissolution of Material A were studied in a solution simulating blood plasma (SBF, simulated body fluid). SBF has an ion composition similar to that of human blood plasma (Kokubo et al., 1990, J. Biomed. Mater. Res., 24, 721-734).

(52) The dissolution test was carried out by immersion of samples of Material A in the solution of simulated body fluid SBF. The principle of the use of this solution is to demonstrate the bioactivity of the material, characterized by its dissolution in the solution, followed by the formation of a precipitate, such as hydroxyapatite, at the surface of the material. The operating protocol is as described in the standard ISO 23317:2007.

(53) More specifically, for each sample, 100 mg of Material A were immersed at 37 C. in 50 ml of SBF. At regular time intervals, a sample was withdrawn and analyzed by ICP-AES, after filtration and ten-fold dilution, for its concentration of calcium and phosphorus.

(54) The curves illustrating the change in these concentrations as a function of the duration of immersion in SBF are shown in FIG. 8.

(55) A very slight decrease in the calcium concentration and a very slight increase in the phosphorus concentration are observed therein after 7 days of immersion. The amplitude of these variations is only a tenth of a ppm.

(56) In comparison, for a conventional bioactive glass provided by the prior art SiO.sub.2CaOP.sub.2O.sub.5 synthesized by the sol-gel route (Sepulveda et al., 2002, Journal of Biomedical Materials, 61, 301-311), after immersion of 0.5 g of material in 45 ml of SBF, a strong decrease in the phosphorus concentration and a high increase in the calcium concentration (with amplitudes of several hundred ppm) are observed in only a few hours. These variations reflect a very high dissolution of the material and possibly a precipitation of phases.

Example 4Cytotoxicity Test

(57) Material A in accordance with the invention, in the powder form, was sterilized by gamma rays, at a dose of 25 KGy, and then subjected to an indirect cytotoxicity test with mesenchymal stem cells, isolated from human bone marrow stroma (HBMSCs).

(58) A culture medium solution was brought together with the material (in a proportion of 100 mg material/ml of medium) for 24 h. After 24 h, a first sample of 100 ml of this solution was withdrawn, supplemented with 10% v/v of fetal calf serum and tested on an HBMSC culture for 24 h (extract (1)).

(59) At the same time, immediately after withdrawal of the first sample, an equivalent volume of fresh medium was added to the solution in order to keep the 100 mg of material/ml of medium ratio constant. After an additional 24 h, a second sample of 100 ml of this solution was withdrawn, supplemented with 10% v/v of fetal calf serum and tested on an HBMSC culture for 24 h (extract (2)).

(60) At the same time, immediately after the withdrawal of a second sample, an equivalent volume of fresh medium was added in order to keep the 100 mg of material/ml of medium ratio constant. After an additional 24 h, a third sample of 100 ml of this solution was withdrawn, supplemented with 10% v/v of fetal calf serum and tested on an HBMSC culture for 24 h (extract (3)).

(61) For each of the extracts, the cell viability and the metabolic activity of the cells were evaluated after culturing for 24 h, respectively using the test with neutral red (NR) and the test with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Parish et al., 1983, Journal of Immunological Methods, 58, 225-37).

(62) In accordance with the standard AFNOR 6NFEN30993, it was considered for this experiment that a product is regarded as cytotoxic when the percentage of mortality is greater than 25% (that is to say that the cell viability is less than 75%).

(63) The results obtained are shown in FIG. 9.

(64) They demonstrate that the cells are viable and active whatever the extract used. A significant difference is observed relating to the cell viability (NR) between the first extract and the following two. The degree of cell viability of the first extract, although lower, remains very high, greater than 95%. The living cells are in addition active, no significant variation being revealed by the test with MTT.

(65) These cell tests demonstrate the noncytotoxicity of Material A in accordance with the invention.

Example 5Preparation of a Vitreous Material in Accordance with the Invention

(66) The glass in accordance with the invention, called Material C, is prepared, which material has the following composition:
{[(Ca.sup.2+).sub.0.84(Na.sup.+).sub.0.33].sub.2[(P.sub.2O.sub.7.sup.4).sub.0.80(PO.sub.4.sup.3).sub.0.27]}.0.43H.sub.2O.

(67) This material is prepared according to the process described in Example 1 above for the preparation of Material A, except that the pyrophosphate precursor used is Na.sub.2P.sub.2O.sub.7.

(68) This material is subjected to analysis by scanning electron microscopy, as described in Example 2 above. The photograph obtained is shown in FIG. 10. A morphology typical of a glass is observed therein.

(69) An analysis by X-ray diffraction, carried out in accordance with the conditions described in Example 2 above, confirms this observation. The diffractogram obtained, presented in FIG. 11, indeed shows a diffuse halo characteristic of a vitreous material.