BONE REGENERATION MATERIAL

Abstract

The present invention relates to a bone regeneration material comprising: a solid first phase of hydroxyapatite of natural origin which is macroporous, having pores of diameters greater than or equal to 50 μm, this phase of hydroxyapatite is a crystalline solid phase of hydroxyapatite, whose crystals have a size comprised between 20 and 120 nm, and this solid phase of hydroxyapatite has a specific surface area comprised between 8 and 20 m.sup.2/g, the method of production and a method of bone repair based on this bone regeneration material.

Claims

1. A bone regeneration material consisting essentially of a solid phase of hydroxyapatite of natural origin which is macroporous having pores of diameters greater than or equal to 50 μm, wherein said phase of hydroxyapatite is a crystalline solid phase of hydroxyapatite, wherein the crystals have a size comprised between 20 and 120 nm, and said solid phase of hydroxyapatite has a specific surface area comprised between 8 and 20 m.sup.2/g.

2. The bone regeneration material according to claim 1, wherein the crystals of the solid phase of hydroxyapatite have a size comprised between 30 and 120 nm, preferably comprised between 40 and 100 nm, preferably between 45 and 80 nm or 50 and 80 nm, more preferably between 50 and 60 nm.

3. The bone regeneration material according to claim 1, wherein the specific surface area is comprised between 10 and 20 m.sup.2/g, preferably between 10 and 18 m.sup.2/g, more preferably between 12 and 16 m.sup.2/g.

4. The bone regeneration material according to claim 1, having a porosity comprised between 70 and 85%, preferably between 75 and 85%, preferably between 80 and 85%.

5. The bone regeneration material according to claim 1, having a granulometric distribution d.sub.50 comprised between 500 and 800 μm, preferably between 550 and 780 μm.

6. The bone regeneration material according to claim 1, having a granulometric distribution d.sub.90 comprised between 850 and 1250 μm, preferably between 850 and 1100 μm, more preferably between 850 and 1000 μm.

7. The bone regeneration material according to claim 1, being enriched by a second solid phase being a synthetic solid phase of calcium phosphate, said second phase having a Ca/P molar ratio of between 0.2 and 2, preferably of between 0.3 and 1.8, preferably of between 0.5 and 1.65, said bone regeneration material having a defined weight ratio between the first solid phase of hydroxyapatite of natural origin and said second synthetic solid phase of calcium phosphate of between 99/1 and 70/30, wherein said second synthetic solid phase of calcium phosphate having a Ks solubility product greater than that of said first phase of hydroxyapatite of natural origin.

8. The bone regeneration material of claim 1, wherein the first phase of hydroxyapatite of natural origin is hydroxyapatite obtained from a bone material of natural origin, in particular from a bone material of animal origin.

9. The bone regeneration material of claim 1, wherein the hydroxyapatite of natural origin which is macroporous of said first phase is a hydroxyapatite of natural origin which is microporous and at least partially sintered.

10. The bone regeneration material of claim 1, further comprising at least one therapeutic agent selected from the group constituted of antibiotics, antivirals, anti-inflammatories, hormones such as steroids, growth factors such as BMPs (Bone Morphogenetic Proteins), anti-rejection agents, stem cells, and the mixtures thereof.

11. The bone regeneration material of claim 1 intended to be used as an implant or prothesis for a bone formation, a bone regeneration or for a bone correction in a mammal, preferably in a human.

12. The bone regeneration material of claim 1 being sterile.

13. A method to produce a bone regeneration material comprising the following steps: contacting a bone material of natural origin comprising hydroxyapatite and organic substances with an extraction aqueous solution at a temperature between 150° C. and 300° C. and at a pressure comprised between 1500 kPa and 3500 kPa so as to obtain a first liquid phase comprising said organic substances and potential impurities extracted from the said bone material, and a second phase of solid hydroxyapatite, separating the said liquid phase from the said solid hydroxyapatite phase, sintering the said separated solid hydroxyapatite phase at a temperature comprised between 800° C. and 1200° C., optionally rinsing the said sintered hydroxyapatite, wherein the said sintered hydroxyapatite phase forms the said bone regeneration material.

14. The method of claim 13, being for the production of a bone regeneration material consisting essentially of a solid phase of hydroxyapatite of natural origin which is macroporous having pores of diameters greater than or equal to 50 μm, wherein said phase of hydroxyapatite is a crystalline solid phase of hydroxyapatite, wherein the crystals have a size comprised between 20 and 120 nm, and said solid phase of hydroxyapatite has a specific surface area comprised between 8 and 20 m.sup.2/g.

15. The method according to claim 13, wherein the extraction aqueous solution is at a temperature between 170° C. and 280° C., preferably between 190° C. and 260° C. or 210° C. and 240° C., more preferably between 220° C. and 230° C.

16. The method according to claim 13, wherein the extraction aqueous solution is at a pressure between 2000 and 3500 kPa, preferably between 2500 and 3500 kPa, more preferably between 3200 and 3500 kPa.

17. The method according to claim 13, wherein the sintering step is performed for a period between 40 minutes and 4 hours, preferably between 1 and 3 hours, more preferably between 1 and 2 hours or between 1 hour and 90 minutes.

18. The method according to claim 13, wherein the sintering step is performed at a temperature between 800° C. and 1150° C., preferably between 800° C. and 1100° C. or between 800° C. and 1050° C., more preferably between 800° C. and 850° C. or between 810° C. and 830° C.

19. The method of claim 13, further comprising a sieving step, after the separation step and before the sintering step of the said solid hydroxyapatite phase, preferably the said sieving step comprises at least a first sieving on a 1 mm-mesh and a second sieving on a 0.25 mm mesh.

20. The method of claim 19, wherein metallic balls are added during the sieving step and are moved on the mesh so as to improve the said sieving step.

21. The method of claim 19 further comprising, after the sieving step and before the sintering step of the said solid hydroxyapatite phase, a step of treating the solid hydroxyapatite by peroxides such as hydrogen peroxide, optionally following by a drying step of the said hydroxyapatite.

22. The method of claim 13, further comprising the step of enriching the bone regeneration material in calcium and phosphorous by at least one first and at least one second separate soaking succeeding one another in any order, said at least one first soaking taking place in a first solution comprising calcium and said at least one second soaking taking place in a second solution comprising phosphorus, wherein preferably the calcium concentration of the said first solution is of at least 1 M, and/or preferably the phosphorous concentration in the said second solution is of at least 0.4 M, and preferably wherein the said first solution comprises Ca(NO.sub.3).sub.2.4H.sub.2O, CaCl.sub.2.2H.sub.2O, CaSO.sub.4.2H.sub.2O, CaCO.sub.3 or a mixture thereof, so as to allow the said calcium concentration of at least 1M, and preferably wherein the said second solution comprises Na.sub.3PO.sub.4, Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4.H.sub.2O, K.sub.3PO.sub.4, K.sub.2HPO.sub.4, KH.sub.2PO.sub.4, K.sub.2HPO.sub.4, (NH.sub.4).sub.3PO.sub.4, (NH.sub.4).sub.2HPO.sub.4, NH.sub.4H.sub.2PO.sub.4, or a mixture thereof, so as to allow the said phosphorous concentration of at least 0.5 M.

23. The method of claim 13, further comprising the step of sterilization of the bone regeneration material or of the enriched bone regeneration material, preferably a sterilization upon ionizing radiation.

24. A method of repair a bone defect in a patient comprising the steps of: measuring the size of the defect to correct; placing a synthetic bone regeneration device arranged to increase bone regeneration in the bone defect, being obtained by 3D printing and comprising at least one shell made of a porous matrix and presenting pores having a size comprised between 50 and 1000 μm, and at least a holder shaft bound on this porous matrix and on the bone surface of this bone defect, this device being arranged so as to encompass an empty zone, which is arranged to house a bone volume to regenerate, filling the said empty zone formed by the said device by the bone regeneration obtainable by the method of claim 13.

25. The method of claim 24 for a bone subject to heavy mechanical constraints, such as a bone of the masticatory apparatus.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0101] FIGS. 1a, 1b and 1c illustrate respectively the development of the calcium (Ca) concentration over time for a solid phase of hydroxyapatite of natural bovine origin, non-enriched, immersed in an HBSS medium, the development of the phosphorus (P) concentration over time for a solid phase of hydroxyapatite of natural bovine origin, non-enriched, immersed in an HBSS medium, and the weight increase over time for a solid phase of hydroxyapatite of natural bovine origin, non-enriched, immersed in an HBSS medium.

[0102] FIGS. 2a, 2b and 2c illustrate respectively the development of the calcium (Ca) concentration over time for a material immersed in an HBSS medium and comprising a solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 90/10), the development of the phosphorus (P) concentration over time for a material immersed in an HBSS medium and comprising a solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 90/10) and the weight increase for a material immersed in an HBSS medium and comprising a solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 90/10).

[0103] FIGS. 3a, 3b and 3c illustrate respectively the development of the calcium (Ca) concentration over time for a material immersed in an HBSS medium and comprising a solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 80/20), the development of the phosphorus (P) concentration over time for a material immersed in an HBSS medium and comprising a solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 80/20) and the weight increase for a material immersed in an HBSS medium and comprising a solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 80/20).

[0104] FIG. 4 illustrates an image obtained by scanning electronic microscopy of the structure of a bone regeneration material according to the prior art, sintered at a temperature above 1200° C.

[0105] FIGS. 5a and 5b illustrates an image obtained by scanning electronic microscopy of the structure of a bone regeneration material sintered at a temperature of 820° C. at a 5000 and 10000 magnification.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Examples

[0106] Comparison of Calcium and Phosphorus Salting-Outs in the Form of Free Ions Over Time for Different Bone Regeneration Materials—Solubility Tests

[0107] Comparative tests have been carried out in order to determine the calcium and phosphorus quantities (concentrations) salted-out over time (solubility tests) at the start of different bone regeneration materials, this in an medium reproducing the in vivo implantation conditions.

1. Methodology

[0108] To do this, the calcium and phosphorus ratios and quantities (concentrations) salted-out have been measured in a medium simulating the in vivo implantation conditions (human blood plasma), i.e. in an HBSS medium (Hank's balanced salt medium) having a pH of 7 and of which the composition is outlined in table 1 below:

TABLE-US-00001 TABLE 1 Concentrations (mM) Na.sup.+ 142.8 K.sup.+ 5.8 Mg.sup.2+ 0.9 Ca.sup.2+ 1.3 Cl.sup.− 146.8 HCO.sup.3− 4.2 HPO.sub.4.sup.2− 0.3 H.sub.2PO.sup.4− 0.4 SO.sub.4.sup.2− 0.4 Glucose 5.6

[0109] The following bone regeneration materials have been tested: [0110] Material 1: 0.5 g of solid phase of hydroxyapatite of natural bovine origin, non-enriched, having pores of a size of between 50 and 100 μm; [0111] Material 2: 0.5 g of solid phase of hydroxyapatite of natural bovine origin having pores of a size of between 50 and 100 μm and enriched by a synthetic solid phase of calcium phosphate according to a ratio by weight of solid phase of hydroxyapatite/synthetic solid phase of calcium phosphate of 90/10, the phase of calcium phosphate having a Ca/P molar ratio of less than 1.67; and [0112] Material 3: 0.5 g of solid phase of hydroxyapatite of natural bovine origin having pores of a size of between 50 and 100 μm and enriched by a synthetic solid phase of calcium phosphate according to a ratio by weight of solid phase of hydroxyapatite/synthetic solid phase of calcium phosphate of 80/20, the phase of calcium phosphate having a Ca/P molar ratio of less than 1.67.

[0113] The solid phase of hydroxyapatite of natural bovine origin is obtained according to the method described in document WO2015/049336 and is more specifically composed of hydroxyapatite particles having a size of between 0.25 mm and 1 mm.

[0114] The materials comprising a first solid phase of hydroxyapatite of natural origin and a second synthetic solid phase of calcium phosphate have been obtained by successive and alternate soakings for a duration of 5 minutes of the first solid phase of hydroxyapatite of natural origin (hydroxyapatite particles) in separate baths of Ca(NO.sub.3).sub.2.4H.sub.2O (1M, pH=10) and of NaH.sub.2PO.sub.4.H.sub.2O (0.5M, pH=10). The first bath makes it possible to enrich the hydroxyapatite calcium (Ca.sup.2+) particles of natural origin, the second phosphorus (PO.sub.4.sup.3−) bath. Following 4 successive soakings (2 soakings in each bath) to obtain a ratio by weight of solid phase of hydroxyapatite/synthetic solid phase of calcium phosphate of 90/10 or following 6 successive soakings (3 soakings in each bath) to obtain a ratio by weight of solid phase of hydroxyapatite/synthetic solid phase of calcium phosphate of 80/20, the hydroxyapatite particles of natural origin enriched in calcium and in phosphorus have been rinsed in order to remove the excess calcium and phosphorus then dried at a temperature of 100° C. for 6 hours.

[0115] A total of 36 samples (of 0.5 g) have been tested simultaneously by immersing in 10 ml of HBSS medium at a temperature of 37° C. Calcium and phosphorus salting-out measurements in the form of free ions have been taken after 8 hours, 24 hours, 48 hours, 1 week, 2 weeks and 3 weeks on two samples for each material and by duration, these samples being rinsed and dried at a temperature of 100° C. after each of these durations.

[0116] The calcium and phosphorus quantities (concentrations) salted-out at the start of each sample in the HBSS medium have been measured according to the ICP-AES (Inductively Coupled Plasma—Atomic Emission Spectroscopy) technique, this following a removal of possible materials suspended in the HBSS medium by centrifugation. The weight of the materials have been defined by weighing (gravimetry) before and after immersion in the HBSS medium, this for each of the durations mentioned above.

2. Results

[0117] The results obtained are illustrated in FIGS. 1 to 3. For each of the materials (samples), the starting calcium and phosphorus concentrations are respectively 1.26 mmol/l and 0.78 mmol/l, which corresponds to the concentrations of these ions in the initial HBSS medium.

[0118] These results show that, when the material 1—solid phase of hydroxyapatite of natural bovine origin, non-enriched—is immersed in the HBSS medium, the calcium and phosphorus concentrations in the form of free ions decrease before being stabilised (FIGS. 1a and 1b). At the same time, an increase by weight of the material 1 is observed (FIG. 1c). For example, after 3 weeks, the calcium concentration in the medium is 0.06 mmol/l while the phosphorus concentration in the medium is 0.03 mmol/l for an average weight increase per sample of 5 mg.

[0119] For the material 2—solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid calcium phase (ratio 90/10)—the calcium and phosphorus concentrations start by decreasing with a quick stabilisation of the calcium concentration (0.6 mmol/l) while the phosphorus concentration progressively increases up to reaching a final value of 1.34 mmol/l after 3 weeks of immersion (FIGS. 2a and 2b), this concentration being greater than the initial phosphorus concentration in the HBSS medium. The weights of the samples of the material 2 have first barely increased before decreasing down to a loss by weight of 3.5 mg (FIG. 2c).

[0120] Concerning the material 3—solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 80/20)—the calcium and phosphorus concentrations increase over time while the weights of the samples decreases (FIGS. 3a to 3c). The final calcium and phosphorus concentrations, after 3 weeks of immersion in the HBSS medium, are respectively 3.69 mmol/l and 2.78 mmol/l for a loss by weight of 12.5 mg, these concentrations being greater than those initially measured in the HBSS medium.

3. Conclusion

[0121] The results obtained with the material 1—solid phase of hydroxyapatite of natural bovine origin, non-enriched—highlight the capacity of hydroxyapatite to fix the calcium and the phosphorus in the form of free ions present in the HBSS medium to form an apatite layer on the surface thereof. Already after 8 hours of immersion, most of the calcium and phosphorus free ions are absorbed on the surface of the samples. These results correspond to the gravimetric recordings which indicate that the samples are heavier after 3 weeks of immersion. These results therefore indicate that the material 1 (non-enriched hydroxyapatite) fixes the calcium and the phosphorus in the form of free ions, and that the dissolution of hydroxyapatite in the HBSS medium is marginal.

[0122] These results show, for the material 2—solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 90/10)—that two phenomena take place concurrently since the calcium and phosphorus concentrations first both decrease, which is again linked to the capacity that hydroxyapatite has to fix these ions. However, the phosphorus concentration in the medium then increases and even exceeds the concentration of this compound in the initial HBSS medium, while the calcium concentration remains less than that initially measured in the HBSS medium by however being greater than that measured when the material 1 is immersed in this same medium. At the same time, after 3 weeks, the gravimetric results show that the samples have lost weight. All of these results for the material 2 indicate that this material is dissolved in the HBSS medium, which can only be attributed to the enriching phase (synthetic solid phase of calcium phosphate), since the results obtained with the material 1 show that the dissolution of hydroxyapatite under the same conditions is totally marginal.

[0123] The results obtained with the material 3—solid phase of hydroxyapatite of natural bovine origin enriched by a synthetic solid phase of calcium phosphate (ratio 80/20)—demonstrate that no decrease in calcium and phosphorus concentrations takes place, that on the contrary, since the calcium and phosphorus concentrations increase over time at the same time with a loss of weight of the samples. From these observations, it can be concluded that these increases of calcium and phosphorus concentrations are due to a dissolution of the second phase of the material 3 in the HBSS medium since the results obtained with the material 1 show that the dissolution of hydroxyapatite under the same conditions is totally marginal. Moreover, it can be observed that the material 3 does not fix the calcium and the phosphorus in the form of free ions salted-out or only marginally, which ensures that the latter remain in solution.

[0124] All of these results also show that the enriching (synthetic solid phase of calcium phosphate) is more soluble than hydroxyapatite and that it is able to salt-out the calcium and the phosphorus in the form of free ions (Ca.sup.2+ and PO.sub.4.sup.3−) in the surrounding medium (HBSS imitating blood plasma).

Example 2. Bone Regeneration Material at Controlled Sintering Temperatures

[0125] A bone regeneration material has been obtained according to the present invention and consisting essentially of a hydroxyapatite solid phase. In the present example, the material has been sterilized.

[0126] Several analyses have been performed; among them the composition of the solid phase, the crystal size, the volumic porosity, the particle size, the specific surface; this has been done for three samples of the bone regeneration material.

[0127] The three samples have all 100% of hydroxyapatite, meeting the requirement of consisting essentially of hydroxyapatite.

[0128] The sample 1 has a crystal size of 54.6 nm; sample 2 of 54.2 nm and sample 3 of 54.4 nm. The median size of the crystals of the bone regeneration material according to the present invention is thus of 54.4 nm.

[0129] The volumic porosity of sample 1 is of 82.3%; sample 2: 82.1% and sample 3: 82.5%. The mean volumic porosity is thus of 82.3%.

Sample 1 has a granulometric distribution d.sub.10 of 381 μm, d.sub.50 of 553 μm and d.sub.90 of 877 μm.
Sample 2 has a granulometric distribution d.sub.10 of 452 μm, d.sub.50 of 782 μm and d.sub.90 of 1243 μm.
Sample 3 has a granulometric distribution d.sub.10 of 474 μm, d.sub.50 of 756 μm and d.sub.90 of 1115 μm.
Hence the mean granulometric distribution d.sub.10 is thus of 436 μm, the d.sub.50 is of 697 μm and the d.sub.90 is of 1079 μm.

[0130] Moreover, the bone regeneration material according to the present invention has been obtained after a sintering step at 820° C. for a duration between 45 and 60 min. (plateau), thereby allowing a more rigid and resistant bone regeneration material, having a rough surface topography, improving the bone regeneration potential.

[0131] Indeed, as shown in the FIGS. 5A and 5B, the present bone regeneration material presents substantially spherical-like or ball-like elements made of hydroxyapatite, from a size between 150 and 350 nm, forming together a rough surface. This contrasts with the material shown at FIG. 4, which has been sintered at a temperature above 1200° C., which presents a smooth surface hampering the bone regeneration potential.

Example 3—Preparation of the Bone Regeneration Material According to the Present Invention

[0132] A batch of bone regeneration material has been prepared in obtaining bovine bones, then in cutting them so as to form a bone material.

[0133] The bone regeneration material comprising hydroxyapatite and organic substances has been put in contact with an aqueous extraction solution under supercritical conditions, for instance between 220° C. and 230° C. and at a pressure comprised between 3200 kPa and 3500 kPa so as to obtain a first liquid phase with the organic substances, and a second solid phase of hydroxyapatite; these two phases are then separated.

[0134] The separated solid hydroxyapatite phase has been weakened by the supercritical extraction process, and is placed on a first sieve of 1 mm for a first sieving step, then on a second sieve of 0.25 mm for a second sieving step. Metallic balls have been added to help the sieving step. If needed, the sieving steps can be repeated, for instance 2 or three times.

[0135] The hydroxyapatite is then sintered at 820° C. (plateau sub-step) for 45 to 60 minutes. This forms the bone regeneration material according to the present invention, which is more rigid and more resistant and with a rough surface topography, thereby improving the bone regeneration potential.

[0136] This sintered hydroxyapatite phase can be then rinsed and/or enriched in calcium and phosphorus by distinct immersion steps, then sterilized, preferably by ionising radiation.

[0137] It is well understood that the present invention is, in no manner, limited to the embodiments described above, and that modifications can be contributed to it without moving away from the scope of the appended claims.