SOLID SUSPENSION

Abstract

A solid suspension for use in bone regeneration and/or the repair of bone defects, comprising a source of at least one group II metal cation and a source of zinc cations, wherein the source of zinc cations comprises zinc oxide and wherein where there is only one group II metal cation this is strontium. A bone graft comprising a solid suspension, a method of preparation of a solid suspension and a use of a solid suspension in bone regeneration and/or in the repair of bone defects.

Claims

1. A solid suspension for use in bone regeneration and/or the repair of bone defects, comprising a source of at least one group II metal cation and a source of zinc cations, wherein the source of zinc cations comprises zinc oxide and wherein where there is only one group II metal cation this is strontium.

2. A solid suspension according to claim 1, wherein the group II metal cations are selected from calcium, strontium, magnesium and combinations thereof.

3. A solid suspension according to claim 2, wherein the group II metal cations are selected from calcium, strontium and combinations thereof.

4. A solid suspension according to claim 2 or claim 3, wherein the calcium cations are provided as a calcium compound selected from calcium sulfate anhydrite (CaSO.sub.4), calcium sulfate hemihydrate (CaSO.sub.4.0.5H.sub.2O), calcium sulfate dihydrate (CaSO.sub.4.2H.sub.2O), monocalcium phosphate (Ca(H.sub.2PO.sub.4).sub.2), dicalcium phosphate anhydrous (CaHPO.sub.4), tricalcium phosphate (Ca.sub.3(PO.sub.4).sub.2), tetracalcium phosphate (Ca.sub.4(PO.sub.4).sub.2O), octacalcium phosphate (Ca.sub.8H.sub.2(PO.sub.4).sub.6.5H.sub.2O), hydroxyapatite (Ca.sub.5(PO.sub.4).sub.3(OH)), calcium carbonate (CaCO.sub.3), calcium oxide (CaO) and calcium silicate (CaSiO.sub.3) or combinations thereof.

5. A solid suspension according to claim 4, wherein the calcium compound comprises a phosphate.

6. A solid suspension according to claim 5, wherein the calcium compound comprises hydroxyapatite.

7. A solid suspension according to any preceding claim, wherein the strontium cations are provided as a strontium compound selected from strontium sulfate (SrSO.sub.4), strontium phosphate (Sr.sub.3(PO.sub.4).sub.2), strontium carbonate (SrCO.sub.3), strontium oxide (SrO), strontium peroxide (SrO.sub.2), strontium phosphide (Sr.sub.3P.sub.2), strontium sulfide (SrS), strontium chloride (SrCl.sub.2), strontium-substituted hydroxyapatite (Sr.sub.5(PO.sub.4).sub.3(OH)) and strontium ranelate (C.sub.12H.sub.6N.sub.2O.sub.8SSr.sub.2) or combinations thereof.

8. A solid suspension according to claim 7, wherein the strontium compound comprises strontium-substituted hydroxyapatite.

9. A solid suspension according to any of claims 4 to 8, wherein the hydroxyapatite comprises nano-hydroxyapatite.

10. A solid suspension according to any of claims 4 to 9, comprising a ratio of calcium:strontium cations in the range 0:100-99:1.

11. A solid suspension according to any preceding claim, wherein the zinc oxide is of mean particle size in the range 0.01 μm-100 μm.

12. A solid suspension according to any preceding claim, further comprising a liquid component comprising water.

13. A solid suspension according to any preceding claim, comprising in the range 20-60 wt % solid.

14. A solid suspension according to any preceding claim, comprising zinc oxide in the range 0.25-5.0 wt % of the solid suspension.

15. A solid suspension according to any preceding claim, wherein the solid suspension is at least partially bioresorbable.

16. A solid suspension according to any preceding claim, for use in bone regeneration and/or in the repair of bone defects.

17. A solid suspension according to any preceding claim, for use in the treatment of bone defects arising from osteoporosis, wherein the solid suspension is to be administered to the bone defect site by injection.

18. A solid suspension according to any of claims 1 to 17, for use in the treatment of bone defects arising from osteoarthritis, wherein the solid suspension is to be administered to the bone defect site by injection.

19. A bone graft, comprising the solid suspension of any preceding claim.

20. A method of preparing a solid suspension according to any of claims 1 to 18, comprising: mixing at least one group II metal cation with zinc oxide, wherein where there is only one group II metal cation this is strontium.

21. A method of forming a bone graft comprising delivering a solid suspension of any of claims 1 to 18 to an implantation location.

22. A method according to claim 21, wherein the implantation location is in the human or animal body.

23. A method according to claim 21 or claim 22, wherein the implantation location is orthopaedic, spinal or dental.

24. A use of the solid suspension of any of claims 1 to 18 in bone regeneration and/or in the repair of bone defects.

25. A use according to claim 24, in orthopaedic or spinal bone regeneration.

26. A use according to claim 24, in the treatment of bone defects arising from osteoporosis or osteoarthritis.

27. A use according to claim 24, wherein the bone regeneration is cosmetic.

28. A use according to claim 27, wherein the cosmetic bone regeneration is dental.

Description

[0031] In order that the invention may be more readily understood, it will be described further with reference to the figures and to the specific examples hereinafter.

[0032] FIG. 1 shows the particle size distribution of zinc oxide used in the test examples;

[0033] FIG. 2 is a graph illustrating the biocompatibility of the pastes/suspensions tested. Calcium hydroxyapatite pastes containing 0, 1, 2 or 3 wt % zinc oxide, no paste and strontium-substituted hydroxyapatite paste were tested;

[0034] FIG. 3 is a graph illustrating the antibacterial effect of increasing concentration of zinc oxide in a hydroxyapatite paste. Shown are the number of viable S. aureus after 24 h. Error bars±S.E.M. Significance: *p<0.05;

[0035] FIG. 4 is a graph illustrating the reduction in the number of viable S. aureus after 24 h at different concentrations of zinc oxide in hydroxyapatite, based on the data of FIG. 3. Error bars±S.E.M; and

[0036] FIG. 5 is a graph illustrating the antibacterial effect of each paste of hydroxyapatite, hydroxyapatite+2 wt % zinc oxide, strontium-substituted hydroxyapatite, and strontium-substituted hydroxyapatite+2 wt % zinc oxide. Shown are the number of viable S. aureus attached to HA and SrHA pastes containing 2 wt % ZnO after 24 h. Error bars±S.E.M. Significance: *p<0.05.

[0037] FIG. 6 is haematoxylin and eosin stained histology sections of defect sites from rabbit femoral condyle implantation of 100 wt. % strontium-substituted hydroxyapatite+2 wt. % zinc oxide paste, which show the paste in contact with bone (i) 6 weeks, (ii) 12 weeks, and (iii) 19 weeks after implantation. In FIG. 6, P represents paste, B represents bone, F represents fibrous tissue, and the scale bar represents 200 μm.

[0038] FIG. 7 is an X-ray diffraction pattern of a composition containing 30 wt. % strontium-substituted hydroxyapatite+4 wt. % zinc oxide, wherein “∇” represents 35 wt % strontium-substituted hydroxyapatite (ICDD PDF 04-016-3586), and “.circle-solid.” represents zinc oxide (ICDD PDF 5-664).

[0039] FIG. 8 is an X-ray diffraction pattern of a composition containing 100 wt. % strontium-substituted hydroxyapatite+2 wt. % zinc oxide, wherein “.Math.” represents 100 wt % strontium-substituted hydroxyapatite (ICDD PDF 33-1348), and “.circle-solid.” represents zinc oxide (ICDD PDF 5-664).

EXAMPLES

[0040] Paste Manufacture

[0041] Pastes were prepared by mixing crystalline zinc oxide powder with hydroxyapatite (HA) slurries. Depending on the example, the hydroxyapatite slurries may be calcium, strontium or combinations thereof in the cationic ratios discussed. A portion of the water content was then evaporated in a drying oven resulting in a paste consistency. The amount of zinc oxide was calculated as a percentage of the final HA paste (HA solids+water) with the residual solid content of the HA paste being in the region of 38-40 wt %.

[0042] Test Methods

[0043] Antibacterial Evaluation

[0044] The ability of the pastes to prevent bacterial colonisation on the paste surface was assessed using a biofilm initialisation model. The pastes were tested for antibacterial activity against a clinical isolate of Staphylococcus aureus (S235). The attachment and survival of bacteria to the surface of the pastes was assessed after a 24 h incubation.

[0045] 10 mm (±1 mm) lengths of sterile pastes extruded from a standard luer lock syringe were placed into sterile 1 ml Eppendorfs, with 3 samples prepared for each paste. 500 μL of sterile PBS (phosphate buffered saline) was added to each Eppendorf. A bacterial suspension of OD 0.05 was prepared in PBS and 500 μL of bacterial suspension was added to the Eppendorf tubes containing paste (n=3). The tubes were incubated at 37° C. for 20 h. The PBS was removed from all tubes and the paste lengths were washed twice with 1 ml PBS. To assess the number of viable bacteria attached to the paste after the overnight incubation the pastes were suspended and serial dilutions were plated on agar plates to allow for colonies to be counted. In detail, the washed pastes were suspended in 1 ml PBS using a vortex. Two serial dilutions were then performed per sample in PBS and the following dilutions were plated up by placing 10 μL sample onto BHI (brain heart infusion) agar plates: 1 in 10.sup.0, 10.sup.1, 10.sup.2, 10.sup.3 and 10.sup.4. The plates were incubated overnight at 37° C. and the colonies were counted at an appropriate dilution where single colonies could easily be identified.

[0046] Statistical analysis was carried out on the log numbers from the experiment using SPSS software. The statistical analysis firstly involved a one way ANOVA test. The post hoc test was then selected based on the homogeneity of the variance between the experimental groups. Levene's test was used to determine the homogeneity of the variances. If the variances were not significantly different from each other, Tukey's multiple comparisons test was used. If the variances were significantly different from each other, Games-Howell comparison was applied.

[0047] Biocompatibility of Pastes

[0048] The biocompatibility of the pastes was investigated by seeding 50,000 MG63 cells per well with 1 mL basal media (made up of the following v/v %: α-MEM supplemented with 10% foetal calf serum, 1% l-alanyl-l-glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids) in a 24 well plate. The cells were incubated at 37° C., 5% CO.sub.2 for 24 h after which the media was removed and 0.9 mL media was added. Permeable Millicell® hanging inserts (0.4 μm pore size, Merck Millipore) were then placed in each well and 0.2 mL complete media was added inside each insert. 0.1 mL paste was then added to each insert in triplicate and the plates were incubated at 37° C., 5% CO.sub.2 for an additional 24 h. After the incubation the inserts were removed and the cells were imaged using light microscopy. The media was removed from the cells and 0.5 mL of a 10% (v/v) PrestoBlue® solution in complete media was added to each well. PrestoBlue® solution was also added in triplicate to empty wells as a control to subtract from the fluorescence values obtained. The samples were incubated at 37° C., 5% CO.sub.2 until appropriate colour change was observed. At each time point 0.2 mL solution was placed into a 96 well plate. The fluorescence of the solutions was measured using a plate reader with an excitation wavelength of 535 nm and an emission wavelength of 590 nm. The same statistical approach was used as described above for the antibacterial testing.

[0049] Test Results

[0050] Particle Size Distribution of Zinc Oxide

[0051] The particle size distribution of the crystalline zinc oxide used in these tests was as shown in Tables 1 and 2 below. FIG. 1 illustrates this in graphical form.

TABLE-US-00001 TABLE 1 Particle size distribution Under 0.4 μm 0.6 μm 0.8 μm 1 μm 3 μm % 5-15 20-40 45-60 60-80 100

TABLE-US-00002 TABLE 2 Particle size distribution D10 D50 D90 μm 0.30-0.45 0.70-0.85 1.20-1.70

[0052] Biocompatibility of Pastes

[0053] FIG. 2 shows the excellent biocompatibility of the suspensions. Specifically it shows good cell viability (MG63 cells) with calcium hydroxyapatite pastes containing 0, 1, 2 and 3 wt % zinc oxide, with strontium-substituted hydroxyapatite paste and in the absence of paste. It is clear that cell viability is not significantly affected by the presence of the suspensions.

[0054] Antibacterial Activity of Zinc Oxide

[0055] FIGS. 3 and 4 show the antibacterial activity of hydroxyapatite pastes containing zinc oxide in a range of concentrations. It can be seen that the presence of zinc oxide enhances the antibacterial properties of the hydroxyapatite, this enhancement becoming significant when the zinc oxide is present at a level above 0.25 wt % of the solid.

[0056] Antibacterial Activity of Zinc Oxide Combined with Strontium

[0057] The antibacterial activity of the following four compositions was measured and compared: [0058] 1. hydroxyapatite, [0059] 2. hydroxyapatite+2 wt % of the solid suspension zinc oxide, [0060] 3. strontium-substituted hydroxyapatite, and [0061] 4. strontium-substituted hydroxyapatite+2 wt % of the solid suspension zinc oxide

[0062] As can be seen from FIG. 5, and discussed above, the addition of zinc oxide alone enhances the antibacterial properties of hydroxyapatite. FIG. 5 also shows that the presence of strontium enhances the antibacterial properties of hydroxyapatite. Unexpectedly, however, where both zinc oxide and strontium are present a synergistic effect is observed. Specifically, it has been shown that the combination of the zinc and strontium has a greater antibacterial effect in terms of the reduction of bacterial numbers than would be expected from an additive effect of each of the zinc and strontium compounds alone (i.e. the number of viable bacteria attached to the SrHA+2% ZnO is significantly lower than the number of viable bacteria attached to SrHA and HA+2 wt % ZnO pastes). Therefore, the zinc oxide and strontium appear to be working together, in synergy, to provide an antibacterial effect.

[0063] It would be appreciated that the methods of the invention are capable of being implemented in a variety of ways, only a few of which have been illustrated and described above.

[0064] In Vivo Bone Regeneration

[0065] FIG. 6 shows haematoxylin and eosin stained histology sections of defect sites from rabbit femoral condyle implantation of 100 wt. % strontium-substituted hydroxyapatite+2 wt. % zinc oxide paste, which show the paste in contact with bone (i) 6 weeks, (ii) 12 weeks, and (iii) 19 weeks after implantation. These results show in vivo bone regeneration.

[0066] Crystallinity

[0067] FIG. 7 shows the X-ray diffraction pattern of a composition containing 30 wt. % strontium-substituted hydroxyapatite+4 wt. % zinc oxide. The peaks are indicative of the presence of a mixture of strontium-substituted hydroxyapatite and crystalline zinc oxide.

[0068] FIG. 8 shows the X-ray diffraction pattern of a composition containing 100 wt. % strontium-substituted hydroxyapatite+2 wt. % zinc oxide. As with FIG. 7, the peaks are indicative of the presence of a mixture of strontium-substituted hydroxyapatite and crystalline zinc oxide.