Carrier composition for bone substitute materials

Abstract

The present invention relates to a carrier composition for particulate and granular bone substitute materials which is a hydrogel comprising a mixture of ethylene oxide (EO)-propylene oxide (PO) block copolymers and silica nanoparticles embedded therein. The present invention further relates to a bone substitute material containing osteoconductive and/or osteoinductive particles or granules in addition to the novel carrier composition. Processes for producing the novel carrier composition and the novel bone substitute material are likewise provided in the context of the invention.

Claims

1. A carrier composition for particulate and granular bone substitute materials, wherein the carrier composition is a hydrogel comprising: (a) an ethylene oxide (EO)-propylene oxide (PO) block copolymer or a mixture of ethylene oxide (EO)-propylene oxide (PO) block copolymers; and (b) silica nanoparticles with a size of between about 0.5 nm and about 10 nm.

2. The carrier composition according to claim 1, wherein the proportion of water in the hydrogel ranges from 60% to 90%.

3. The carrier composition according to claim 1, wherein the proportion of ethylene oxide (EO)-propylene oxide (PO) block copolymers in the hydrogel is between about 10% and 40% (w/w).

4. A carrier composition according to claim 1, wherein the proportion of silica nanoparticles is between about 2% and 12% (w/w).

5. A carrier composition according to claim 1, wherein the silica nanoparticles have a size between about 0.5 nm and 1.5 nm.

6. A carrier composition according to claim 1, wherein the silica nanoparticles form fractal aggregation clusters having an average size of less than 200 nm.

7. A carrier composition according to claim 1, wherein the ethylene oxide (EO)-propylene oxide (PO) block copolymers in the carrier composition have a molecular weight distribution between about 1,000 g/mol and 70,000 g/mol.

8. A carrier composition according to claim 1, wherein at least 30% (w/w) of the ethylene oxide (EO)-propylene oxide (PO) block copolymers in the carrier composition consist of a poloxamer.

9. A bone substitute material comprising: (a) a carrier composition according to claim 1; and (b) osteoconductive and/or osteoinductive particles or osteoconductive and/or osteoinductive granules.

10. A bone substitute material according to claim 9, wherein the osteoconductive or osteoinductive particles have a size between about 5 μm and 100 μm.

11. A bone substitute material according to claim 9, wherein the osteoconductive or osteoinductive particles are hollow spheres having an opening.

12. A bone substitute material according to claim 11, wherein the hollow spheres form clusters of a size between about 100 μm and 3,000 μm.

13. A bone substitute material according to claim 9, wherein the osteoconductive or osteoinductive particles or the osteoconductive and/or osteoinductive granules consist of hydroxyapatite crystallites which have the morphology of the biological hydroxyapatite of the bone and are coated with a matrix of silica xerogel.

14. A bone substitute material according to claim 9, wherein the osteoconductive and/or osteoinductive particles or the osteoconductive and/or osteoinductive granules are coated with a silica gel.

15. A process for the preparation of a bone substitute material comprising the steps of (a) providing a carrier composition according to claim 1; (b) optionally treating the carrier composition with gamma radiation; and (c) mixing the carrier composition with osteoconductive and/or osteoinductive particles or with osteoconductive and/or osteoinductive granules.

16. A carrier composition according to claim 8, wherein said poloxamer is poloxamer 407.

17. A carrier composition according to claim 8, wherein said poloxamer has an average molecular weight in the range of 9,800 to 14,600 g/mol.

18. A bone substitute material according to claim 14, wherein the silica concentration in the silica gel is between about 3% and 10%.

19. The process of claim 15, wherein the osteoconductive and/or osteoinductive particles or the osteoconductive and/or osteoinductive granules are coated with a silica hydrogel.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the molecular mass distribution of a SiO.sub.2-containing hydrogel based on Kolliphor P 407.

(2) FIG. 2 shows the molecular mass distribution of a SiO.sub.2-containing hydrogel based on Kolliphor P 407 after gamma irradiation.

(3) FIG. 3 shows the molecular mass distribution of a SiO.sub.2-containing hydrogel based on Kolliphor P 407 after storage of the hydrogel at 60° C. for 55 days.

(4) FIG. 4a shows the results of the viscosity measurement as a function of the shear rate. FIG. 4b shows the results of the measurement of the shear modulus as a function of frequency.

(5) FIG. 5 shows the formation of lamellar structures under the polarization microscope.

(6) FIG. 6 shows the application of the bone substitute material of the invention with an applicator.

(7) FIG. 7 shows the result of an examination of the bone substitute material of the invention after accelerated aging by means of reflected light microscopy.

(8) FIG. 8 shows the result of a HE staining 4 weeks after implantation of the bone substitute material of the invention into the hind leg of a rabbit.

(9) FIG. 9 shows the result of one of the histomorphometric evaluation after implantation of the bone substitute material of the invention into the hind leg of a rabbit.

(10) FIG. 10 shows the use of microparticles in the form of hollow spheres with an opening and a diameter of about 40 μm in the bone substitute material of the invention.

(11) FIG. 11 schematically shows the coating of clusters of microparticles with pure silica hydrogel before embedding them in the poloxamer-silica hydrogel.

(12) FIG. 12a shows the application of the shear [“shear stress”] as a function of the shear rate [“shear rate”] for different compositions of the carrier material: A 19.6% Kolliphor P 407, 0% SiO.sub.2; B 19.6% Kolliphor P 407, 4.8% SiO.sub.2; C 36.0% Kolliphor P 407, 0% SiO.sub.2; D 36.0% Kolliphor P 407, 3.8% SiO.sub.2.

(13) FIG. 12b shows the complex shear modulus (storage modulus [“storage modulus] G′; loss modulus [gloss modulus”] G″) for different carrier material compositions: A 36.0% Kolliphor P 407, 0% SiO.sub.2; B 36.0% Kolliphor P 407, 5.0% SiO.sub.2.

(14) FIG. 12c shows the complex shear modulus (storage modulus G′; loss modulus G″) for different carrier material compositions: A 19.4% Kolliphor P 407, 0% SiO.sub.2; B 19.4% Kolliphor P 407, 4.8% SiO.sub.2.

(15) FIG. 12d shows the complex shear modulus (storage modulus G′; loss modulus G″) for different carrier material compositions: A 16.4% Kolliphor P 407, 0% SiO.sub.2; B 16.4% Kolliphor P 407, 5.0% SiO.sub.2; C 16.4% Kolliphor P 407, 7.4% SiO.sub.2.

EXAMPLES

(16) The following examples illustrate the effectiveness as well as the advantages of the carrier composition according to the invention and the bone substitute material formulated from it.

Example 1: Production of Hydrogels with and without SiO.SUB.2

(17) For comparison purposes, SiO.sub.2-free and SiO2-containing hydrogels were produced on the basis of Kolliphor P 407. For the production of the SiO.sub.2-free hydrogels, 23.5 g Kolliphor P 407 from BASF were mixed with 76.5 g water. For the hydrogels containing SiO.sub.2, a sol was prepared by ion exchange with a SiO.sub.2 concentration of 4% and 6%, respectively. Concentrated sodium water glass solution from Merk (specification: Na.sub.2O: 7.5-8.5%; SiO.sub.2: 25.5-28.5%) was used and diluted with ultrapure water. A Lewatit MonoPlus SP 112Na+ column was used as an ion exchanger. The soles had a pH value of 2.7 and were cooled down to 5° C. In each 76.5 g of the sol 23.5 g of Kolliphor P 407 were stirred in. The resulting hydrogels contain polymers with the molecular mass distribution shown in FIG. 1 (molecular mass distribution A). The molecular mass distribution can be determined by chromatography. The analysis led to the following peaks:

(18) Peak 1: Position: 5,550 g/mol, proportion: 17.8%

(19) Peak 2: Position: 11,000 g/mol, proportion: 8.2%

(20) Peak 3: Position: 13,470 g/mol, proportion: 73.1%

(21) Peak 4: Position: 25,500 g/mol, proportion: 0.8%

(22) The peaks at 5,550 g/mol and 11,000 g/mol represent fragments of Kolliphor 407. The peak at 25,500 g/mol results from the cross-linking of two chains.

(23) Some of the prepared samples were treated with gamma radiation (17.5 to 30 kGray, radiation source: cobalt 60, maximum activity 111 PBq). The gamma radiation leads to a cross-linking of the polymer chains. At the same time, chains are also broken. The result is a polymer with a broad molecular mass distribution.

(24) These hydrogels contain polymers with the molecular mass distribution shown in FIG. 2 (molecular mass distribution B). The analysis led to the following peaks:

(25) Peak 1: Position: 5,400 g/mol, proportion: 8.1%

(26) Peak 2: Position: 11,000 g/mol, proportion: 4.0%

(27) Peak 3: Position: 13,400 g/mol, proportion: 37.0%

(28) Peak 4: Position: 17,000 g/mol, proportion: 2.7%

(29) Peak 5: Position: 25,500 g/mol, proportion: 4.2%

(30) Peak 6: Position: 35,000 g/mol, proportion: 0.2%

(31) After irradiation, the proportion of the continuous mass distribution was 43.8%. The original Kolliphor 407 only has a proportion of 37%. Molecules with a continuous size distribution of up to approx. 70,000 g/mol have the largest proportion of 43.8%.

(32) Another part of the prepared samples was stored at elevated temperature for a longer period of time. After storage for 55 days at 60° C., the hydrogels contained polymers with the molecular mass distribution shown in FIG. 3 (molecular mass distribution C). The analysis led to the following peaks:

(33) Peak 1: Position: 5,350 g/mol, proportion: 10.6%

(34) Peak 2: Position: 8,200 g/mol, proportion: 13.7%

(35) Peak 3: Position: 13,470 g/mol, proportion: 23.6%

(36) Peak 4: Position: 17,700 g/mol, proportion: 1.9733%

(37) Peak 5: Position: 24,700 g/mol, proportion: 1.5018%.

(38) Peak 6: Position: 35,000 g/mol, proportion: 0.0%

(39) It can be seen that also in this case molecules with a size distribution ranging from about 1,000 g/mol to about 70,000 g/mol show the largest proportion of 48.7%.

(40) For all samples the viscosity was measured as a function of the shear rate (StrainSweep Test, oscillation rheometer ARES—T.A. Instruments). The results are shown in FIG. 4a. It can be seen that the viscosity of both the SiO.sub.2-free hydrogels and the SiO.sub.2-containing hydrogels increases with the broadening of the molecular mass distribution. The samples with the molecular mass distribution A are not optically active, they show no contrast in the polarization microscope. This means that the polymers form micelles. The samples with the molecular mass distribution B, on the other hand, are optically active. They show a contrast in the polarizing microscope. This shows that the samples also contain so-called lamellar structures in addition to micelles. At high concentrations, some surfactants form lamellar structures in which the water is located in the polar intermediate layers of the associations. This optical anisotropy changes the plane of oscillation of the linearly polarized light so that characteristic light-dark appearances can be seen under the polarization microscope. FIG. 5 shows a typical example that documents the emergence of lamellar structures. Furthermore, FIG. 4a shows that the viscosity strongly increases with increasing SiO.sub.2 content in the hydrogel. At a shear rate of 50 1/s, the viscosity increases 10-fold with the addition of 4.5% SiO.sub.2. This is of decisive importance for the applicability of the gels as carriers for bone substitute materials.

(41) In addition, the shear modulus was measured as a function of frequency. This measurement provides information about the vibration behaviour of viscoelastic materials under oscillating shear stress and allows conclusions to be drawn about the interaction of the molecules in the system. FIG. 4b shows the storage portion of the shear modulus as a function of frequency for different hydrogels. A polymer with the molecular mass distribution A was selected here. On the one hand, the effect can be seen that the storage portion of the shear modulus increased with increasing polymer concentration. On the other hand, the storage portion of the shear modulus increased strongly with increasing SiO.sub.2. For the example with 25% polymer content, the storage portion increased by 10 times if 4.5% silica nanoparticles were present in the gel. This shows the interaction between the polymer chains and the silica nanoparticles which is important for the application of the gels.

(42) FIG. 12a shows the shear as a function of the shear rate for different compositions of the carrier material. The shear measurements were performed at 20° C. Curve A corresponds to the carrier material with 19.6% Kolliphor without silica nanoparticles. The curve corresponds to that of a liquid, since the gel formation only begins at about 25° C. at this proportion of the Kolliphor. The curves B, C and D show a typical course for hydrogels. A flow limit is visible (shear at which the material begins to flow). Curve B shows that the addition of 4.8% SiO.sub.2 converts the liquid into a gel. Curve C corresponds to the carrier material with 36.0% Kolliphor without silica nanoparticles. A gel is formed here at 20° C. by the formation of micelles. If 3.8% SiO.sub.2 are added to this sample, much higher shear is required to make the material flow. Gel formation here is based on the interaction of the polymers with the silica nanoparticles.

(43) This effect is also documented by the measurements of the complex shear modulus as a function of shear, which are shown in FIGS. 12b, 12c and 12d. The measurements were carried out at 20° C. If the storage portion G′ in the curve is larger than the loss portion G″, the material is a gel. If the two curves intersect, the material begins to flow. If the loss portion G″ in the curve is greater than the storage portion G′, the behaviour indicates a liquid.

(44) FIG. 12b shows the behaviour of a carrier material with 36.0% Kolliphor. Without silica nanoparticles (A), the material forms a gel at 20° C. which shows transition to liquid at a shear of approx. 500 Pa. If 5% SiO.sub.2 are added to the carrier material (B), the material remains a gel in the entire measuring range. The curves of G′ and G″ hardly approach each other. For the application this means that the carrier material with silica nanoparticles is much more stable and ensures improved handling.

(45) FIG. 12c shows this effect for smaller Kolliphor concentrations (19.6%). Without silica nanoparticles there is no gel formation at 20° C. However, the addition of silica nanoparticles leads to gel formation. FIG. 12c documents the dependence of this effect on the SiO.sub.2 concentration. The starting point is a carrier material with 16.4% Kolliphor, which does not form a gel at 20° C. (A). By adding 5.0% SiO.sub.2, the material becomes a gel which shows transition to liquid at a shear rate of approx. 500 Pa (B). With 7% SiO.sub.2 a gel is formed which proves to be stable in the entire measuring range (C). These results show that the rheological properties of the composition can be modulated by changing the ratio of Kolliphor, silica nanoparticles and water. This makes it possible to optimize the carrier material specifically for different applications.

Example 2: Embedding of Porous Bone Substitute Materials

(46) Osteoinductive granules of hydroxyapatite (HA) in the form of fir cones were used (Nanobone, Artoss GmbH, Rostock, Germany). These were on average 3 mm long and had a diameter between 0.5 and 1.0 mm. The HA showed a crystallographic morphology similar to that of biological HA. This HA was embedded in a highly porous matrix of silica xerogel. The porosity of the granules was about 50%, the specific surface area was about 200 m.sup.2/g, and the pore size distribution showed a maximum at 4 nm.

(47) The granules were impregnated in a mass ratio of 1:1 with a pure silica sol with a SiO.sub.2 concentration of 6% and a pH value of 7.0. In contact with the solid, the silica sol gels. Granules are produced which are filled with a silica gel and are coated with same.

(48) To produce the poloxamer-silica hydrogel, 35 g Kolliphor P 407 (BASF) were stirred in 65 g silica sol with a SiO.sub.2 content of 6%. The sol was previously cooled to 1° C. Cross-linking is achieved by gamma irradiation in the range of 17.5 to 30 kGrey. This polymer-silica hydrogel was mixed with the coated granules in a mass ratio of 1:1. The resulting pasty bone substitute material is very easy to shape and can be inserted into bone defects with an applicator. FIG. 6 shows the use of the bone substitute material with an applicator.

(49) The stability of the coating of the granules with pure silica hydrogel was controlled by subjecting the material to accelerated ageing for 1 year according to ASTM F 1980-07. After removing the poloxamer-silica hydrogel by rinsing with water, granules coated with pure silica hydrogel could be seen under the microscope. FIG. 7 shows the analysis of the granules using reflected light microscopy.

Example 3: Functionality in Animal Experiments

(50) The experiments were carried out with female rabbits (New Zealand White, 3-4 kg, Charles River, Sulzfeld, Germany). The bone substitute material produced according to Example 2 was implanted bilaterally into the hind legs. The cut through the cutis and subcutis has a length of approx. 2.5 cm. The musculature was also severed in a small area in order to keep the injuries as small as possible, and then the periosteum was carefully detached from the bone at the defect site to be placed. A cylindrical defect (5 mm in diameter and 10 mm in length) was then inserted into each of the lateral condyles of the femora. A standard drill (Ø 4.5 mm) was used for this purpose. During defect settlement, the area was rinsed with 0.9% NaCl solution to prevent necrosis of bone tissue due to heat exposure.

(51) Anaesthesia was administered subcutaneously to the neck fold by injection of 10% ketamine (30-60 mg/kg body weight) and 2% xylazine (5 mg/kg body weight). After 10 min, 0.3 ml atropine (0.5 mg/ml) was administered. In addition, novamine sulfone (500 mg/ml) was injected as an analgesic and penicillin G (intramuscular 150,000 i.U.) as an antibiotic. Local anaesthesia was performed with 2 ml xylocitin-loc (2%/ml). After implantation, the wound area was rinsed with gentamicin (80 mg/2 ml, 1:5 dilution with NaCl). The wound closure (point seam) was made with vicryl suture material.

(52) After trial periods of 4, 8 and 12 weeks, the corresponding trial groups were removed from the trial. The euthanasia was performed on the anaesthetised animal (10% ketamine and 2% xylazine, subcutaneously) using Release® (300 mg/ml corresponding to: 1 ml/kg body weight) intravenously. Histological sections were made for the evaluation. The defect regions were explanted, decalcified and embedded in paraffin. A hematoxylin and eosin stain was applied.

(53) Result: After 4 weeks neither the polymer-silica hydrogel nor the pure silica hydrogel was detectable. A complete resorption occurred. Changes in the temporal sequence to granules embedded in the patient's blood were not detectable during defect healing. FIG. 8 shows a histological image (HE staining) 4 weeks after the procedure. New bone formation and resorption of the granules is not influenced by the original embedding in the two hydrogels. The results of the histomorphometric evaluation of the animal experiments are shown in FIG. 9. A defect healing is documented.