BIOACTIVE MATERIAL

Abstract

The present invention relates to a bioactive material and to a method of producing a bioactive material which is suitable for use as an implant or for use as a bone substitute for repairing bone.

Claims

1. A material suitable as an implant comprising a titanium or titanium alloy substrate comprising a first surface and a primary layer on the first surface, the primary layer comprising a plurality of micron scale structures, wherein the plurality of micron scale structures collectively comprise a second surface, and a surface layer on the second surface, the surface layer comprising alkali titanates, wherein the thickness of the surface layer is between 100 and 500 nm, wherein the surface layer comprises a plurality of nanoscale structures, and wherein the plurality of nanoscale structures collectively comprise a third surface.

2. The material of claim 1, wherein the alkali titanates comprise sodium titanate.

3. The material of claim 1, wherein the alkali titanates comprise discrete elements or fibrils comprising a width in the range of 2 to 20 nm and a length in the range of 200 nm to 300 nm.

4. The material of claim 1, wherein the thickness of the surface layer is between 100 and 500 nm.

5. The material of claim 1, wherein the primary layer further comprises hydroxyapatite.

6. The material of claim 1, wherein the primary layer further comprises titanium oxide.

7. The material of claim 1, wherein the first surface has a first surface area, the second surface has a second surface area, and the third surface has a third surface area, wherein the third surface area is greater than the second surface area, and wherein the second surface area is greater than the first surface area.

8. The material of claim 7, wherein the third surface area is between 1000 and 50000 times greater than the first surface area.

9. The material of claim 1, wherein the third surface comprises a reflectance to visible light in the range of 1% to 20%.

10. The material of claim 9, wherein the third surface comprises a reflectance to visible light in the range of 6% to 10%.

11. The material of claim 1, wherein the primary layer further comprises alumina in a concentration greater a concentration of alumina in the substrate.

12. The material of claim 11, wherein the surface layer of the primary layer comprises alumina in a concentration lesser than a concentration of alumina in a subsurface of the primary layer.

13. The material of claim 1, wherein the primary layer is formed by a process comprising soaking at least a portion of the substrate in an alkaline solution comprising a concentration of 2 to 6 molar at a temperature of 50 C. to 70 C., for 1 to 24 hours.

14. A material suitable as an implant comprising a titanium or titanium alloy substrate comprising a first surface and a primary layer on the first surface, the primary layer comprising a plurality of micron scale structures, wherein the plurality of micron scale structures collectively comprise a second surface; a surface layer on the second surface, wherein the surface layer comprises alkali titanates, wherein the alkali titanates comprise a plurality of nanoscale structures, and wherein the plurality of nanoscale structures collectively comprise a third surface; and alumina in a concentration greater than a concentration of alumina in the titanium or titanium alloy substrate.

15. The material of claim 14, wherein the surface layer of the primary layer comprises alumina in a concentration lesser than a concentration of alumina in a subsurface of the primary layer.

16. The material of claim 14, wherein the alkali titanates comprise discrete elements or fibrils comprising a width in the range of 2 to 20 nm and a length in the range of 200 nm to 300 nm.

17. The material of claim 14, wherein the primary layer further comprises hydroxyapatite.

18. The material of claim 14, wherein the primary layer further comprises titanium oxide.

19. The material of claim 14, wherein the first surface has a first surface area, the second surface has a second surface area, and the third surface has a third surface area, wherein the third surface area is between 1000 and 50000 times greater than the first surface area.

20. The material of claim 14, wherein the third surface comprises a reflectance to visible light in the range of 1% to 20%.

Description

[0031] Embodiments of the invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0032] FIG. 1 is a scanning electron micrograph (SEM) of a titanium alloy surface;

[0033] FIG. 2 is an SEM of the titanium alloy surface of FIG. 1 after grit blasting with alumina particles;

[0034] FIG. 3 is an SEM of a titanium alloy porous beaded surface;

[0035] FIG. 4 is an SEM of a titanium alloy sintered bead foam surface;

[0036] FIG. 5a is an SEM of titanium alloy surface after grit blasting with alumina particles;

[0037] FIGS. 5b-5g are SEMs of samples of the titanium alloy surface of FIG. 5a after soaking in a 2M (2 molar), 3M, 4M, 6M, 8M and 10M solution respectively of sodium hydroxide solution at 60 C. for 2 hours;

[0038] FIG. 6a-6c are SEMs of an alumina grit blast titanium alloy surface, a titanium porous beaded surface, and a titanium sintered bead foam surface respectively, soaked in a 4M sodium hydroxide solution at 60 C. for 2 hours;

[0039] FIGS. 7 and 8 are magnified views of the titanium alloy surfaces FIG. 6b and FIG. 6c respectively;

[0040] FIG. 9 is an SEM of a Porous Beaded Titanium surface prior to forming the primary layer;

[0041] FIG. 10 is an SEM of the porous beaded titanium surface of FIG. 9, the primary layer having been formed by soaking in a 4M sodium hydroxide solution at 60 C. for 2 hours in a sonicating water bath;

[0042] FIG. 11a is an SEM of the Porous Beaded Titanium surface of FIG. 9 soaked in a 2M solution of sodium hydroxide at 60 C. for 10 minutes;

[0043] FIG. 11b is a magnified SEM of the Porous Beaded Titanium surface of FIG. 11a, more clearly showing the early formation of the nanostructured primary layer comprising nano-sized fibrils having a size in the region of 1-20 nanometers;

[0044] FIG. 11e is an SEM of the Porous Beaded Titanium surface of FIG. 11a soaked in a 2 Molar solution of sodium hydroxide at 60 C. for a further 15 minutes clearly showing the development of the nanostructured primary layer;

[0045] FIG. 11d is an SEM of a different portion of the Porous Beaded Titanium surface of FIG. 11a clearly showing the irregular nature of the formation of the primary layer;

[0046] FIG. 12 is an SEM of a commercially pure titanium surface after grit blasting with alumina particles with subsequent soaking in a 4M solution of sodium hydroxide solution at 60 C. for 2 hours;

[0047] FIGS. 13a-13c are SEMs of increasing magnification of areas of the surface of TiAlNb alloy after grit blasting with alumina particles but prior to soaking in sodium hydroxide, the SEM employing a 2 kv beam to analyse the upper structure of the primary layer created;

[0048] FIGS. 14a-14c are SEMs of the same areas of the surface of the TiAlNb alloy of FIGS. 13a-13c after soaking in 4M sodium hydroxide solution at 60 C. for 2 hours, the SEM employing a 2 kv beam to analyse the upper structure of the completed primary layer;

[0049] FIGS. 15a and 15b are SEMs of the same areas of the surface of the TiAlNb alloy of FIGS. 14b and 14c respectively, the SEM employing a 15 kv beam to analyse the substructure of the completed primary layer;

[0050] FIG. 16 is a graph showing the percentage reflectance from the surface of the primary layer for different substrates; and

[0051] FIGS. 17a and 17b are pictorial views of samples of the material according to the present invention showing the primary layer prior to treatment with sodium hydroxide and post treatment with sodium hydroxide respectively.

[0052] FIG. 18 shows grit blasted titanium coupons p-NPP data normalised to DNA (Pico Green) with error bars of standard deviation. This data is from Table 2.

[0053] FIG. 19 shows porous beaded titanium coupons p-NPP data normalised to DNA (Pico Green) with error bars of standard deviation. This data is from Table 3.

[0054] FIG. 20 shows polished titanium coupons p-NPP data normalised to DNA (Pico Green) with error bars of standard deviation. This data is from Table 4.

[0055] Sample titanium alloy plates of various dimensions having surface areas ranging from approximately 40 mm.sup.2 to 100 mm.sup.2 were washed cleaned and dried to form sample substrates. The prepared or sample substrate surfaces are approximately smooth. This can be seen most clearly from FIG. 1 which is a view of the substrate surface taken by a ultra-high resolution scanning electron microscope.

[0056] A FEI Nova 200 NanoSEM ultra-high resolution Scanning Electron Microscope with a stated resolution of 1.8 nm at 3 kV and 1 nm at 15 kV using immersion optics was used to characterise primary layers formed on the titanium alloy substrate. The views or micrographs of the primary layer show detail on the nanoscale. However, it will be appreciated that other suitable methods and equipment may also be used to explore the surface detail of the primary layer.

[0057] A surface of a prepared substrate sample was blasted with abrasive alumina particles otherwise known as alumina grit-blast. The process of alumina grit-blasting roughens the surface of the substrate creating or partially forming a primary layer which has a greater surface area than the surface area of the prepared substrate prior to grit-blasting. This can be seen most clearly in FIGS. 2 and 5a. The primary layer was completed by soaking the substrate with the partially formed primary layer in a 4 molar solution of sodium hydroxide at 60 C. for two hours. FIG. 5b is a view of the completed primary layer which clearly shows the development of strut-like formations, fibres or fibrils of sodium titanate having dimensions on the nanoscale. The diameter or width of these fibrils fall within the range of between 1 and 20 nanometers. Approximately 80% of the fibrils have been measured as having a diameter in the range of 5 to 12 nanometers. The length of the fibrils are between 200 and 300 nanometers.

[0058] Five further samples of the prepared substrate were alumina grit blasted and soaked in sodium hydroxide solutions of concentration 3 molar, 4 molar, 6 molar, 8 molar and 10 molar respectively at 60 C. for two hours and FIGS. 5c to 5g are views of the completed primary layer formed in each case. As can be seen from FIGS. 5c to 5g, the best etching, texturing or nanostructure formed or greatest density of fibril formation was observed where the substrate was soaked in 4 molar solution of sodium hydroxide. The greater the density of fibril formation, the greater the surface area of the primary layer. Treatment with higher concentrations of sodium hydroxide was less effective and lead to re-dissolution of the nanostructure of the primary layer resulting in a smoother surface and thus a reduced surface area.

[0059] FIGS. 6a to 6c are views of a primary layer formed according to the present invention wherein the starting substrate and thus the initial topography is different in each case. FIG. 6a is a view of a primary layer which has been formed on a surface of a solid titanium alloy substrate which has been blasted with abrasive alumina particles and subsequently chemically treated by soaking in a 4 molar concentrated solution of sodium hydroxide at 60 C. for 2 hours. FIG. 6b and FIG. 6c are views of the primary layer which have been formed on a surface of a porous beaded titanium substrate and a titanium foam substrate respectively, which have been chemically treated in the same way as the solid titanium alloy substrate.

[0060] The porous beaded titanium substrate and titanium foam substrate were not subjected to any physical treatment such as in the case of the solid titanium alloy substrate. It was found that the greater the surface area of the starting substrate, the greater the surface area of the primary layer formed. As can be quite clearly seen from FIGS. 6a to 6c the fibrils formed in the case of the titanium foam substrate, which had the greatest starting substrate surface area, were the most fine, and thus fibril formation density was greatest presenting the highest primary layer surface area. FIG. 9 is an SEM of a portion of a surface of a porous beaded titanium alloy prior to completing the primary layer. FIGS. 11a to 11d illustrate the development of the primary layer over time when soaked in a 2 molar solution of sodium hydroxide at 60 C.

[0061] FIGS. 13a-13c are SEMs of increasing magnification (200, 500 and 1200 times magnified respectively) of areas of the surface of TiAlNb alloy after grit blasting with alumina particles but prior to soaking in sodium hydroxide; the SEM employing a 2 kv beam to analyse the upper structure of the completed primary layer. FIGS. 14a-14c are SEMs of the same areas and at the same magnifications of the surface of the TiAlNb alloy of FIGS. 13a-13c respectively after soaking in 4 molar sodium hydroxide solution at 60 C. for 2 hours; the SEM employing a 2 kv beam to analyse the upper structure of the primary layer thus formed. FIGS. 15a and 15b are SEMs of same areas and at the same magnifications of the surface of the TiAlNb alloy of FIGS. 13b and 13c respectively; the SEM employing a 15 kv beam to analyse the substructure of the primary layer formed.

[0062] The surfaces of the primary layers were analysed by scanning electron microscopy (SEM) before and after soaking the substrate in sodium hydroxide solution to analyse surface topography and alumina content throughout the primary layer. This technique can be carried out at different voltages which enables the surface and subsurface of the primary layer to be analysed; the greater the voltage the deeper the penetration of the beam. Titanium alloy has a higher average atomic number than alumina. The higher the average atomic number of the material being analysed using SEM, the greater will be the electron backscatter and thus the brighter will be the SEM image.

[0063] Alumina has an average atomic number less than titanium alloy and thus an SEM image of titanium alloy with alumina present is darker than titanium alloy without alumina. It is quite clear when comparing FIG. 1, which shows a titanium alloy substrate prior to alumina grit blasting, with FIGS. 13a to 13c, for example, which show the titanium alloy substrate post alumina grit blasting, that quite a substantial amount of alumina becomes embedded in the surface of the substrate forming the primary layer. FIGS. 14a to 14c represent the primary layer of FIGS. 13a to 13c which have been completed by chemical treatment with sodium hydroxide as described above. As can be seen, the SEM images of the primary layer illustrated in FIGS. 14a to 14c are brighter than the SEM images of the primary layer illustrated in FIGS. 13a to 13c because the sodium titanate layer formed masks the alumina particles present in the upper surface of the primary layer. Sodium titanate has an average atomic number higher than that of alumina and thus the SEM image of the completed primary layer will appear brighter than the primary layer created by alumina grit blasting and prior to treatment with sodium hydroxide.

[0064] The higher voltage SEM images illustrate the composition of the subsurface of the primary layer which is clearly darker and thus higher in alumina content than the upper surface regions. However, it is only critical to mask the alumina particles in the upper surface of the primary layer which forms the implant-to-bone interface and thus is in direct contact with the bone, as contamination of the subsurface of the primary layer with abrasive particles has little affect on the bond formed between the bone and implant.

[0065] Analysis of the reflectance of various substrates prior to and after treatment with 4 molar sodium hydroxide was also undertaken. As can be seen quite clearly from table I below, the greater the surface area of the primary layer, the less visible light is reflected. The titanium foam substrate which produced the primary layer having the greatest surface area reflected only between 5 and 10% of the visible light. All the primary layers completed were black in colour when viewed by the naked eye.

TABLE-US-00001 TABLE 1 4M NaOH 4M NaOH Control Control treated treated 4M NaOH Wavelength Grit-Blast Porous Control Grit-Blast Porous treated (nm) Ti6Al4V Beaded Ti Foam Ti Ti6Al4V Beaded Ti Foam Ti 400 22.83 17.99 17.61 12.71 4.78 5.56 410 23.23 18.32 17.86 12.61 4.94 5.71 420 23.6 18.58 18.05 12.53 5.06 5.82 430 23.95 18.75 18.19 12.47 5.12 5.88 440 24.26 18.92 18.3 12.47 5.18 5.96 450 24.55 19.17 18.44 12.54 5.31 6.1 460 24.82 19.42 18.6 12.67 5.45 6.25 470 25.11 19.6 18.8 12.88 5.59 6.39 480 25.39 19.79 19.03 13.14 5.72 6.53 490 25.64 19.99 19.31 13.41 5.84 6.63 500 25.92 20.28 19.61 13.74 5.98 6.76 510 26.3 20.75 19.92 14.18 6.2 7 520 26.67 21.19 20.21 14.63 6.4 7.23 530 26.89 21.4 20.42 14.99 6.51 7.37 540 27.07 21.54 20.61 15.33 6.6 7.49 550 27.76 21.72 20.8 15.7 6.7 7.61 560 27.44 21.89 20.97 16.06 6.8 7.74 570 27.6 22.02 21.09 16.41 6.91 7.88 580 27.74 22.15 21.19 16.72 7 8.01 590 27.87 22.32 21.27 16.95 7.02 8.1 600 27.99 22.51 21.35 17.15 7.04 8.19 610 28.12 22.68 21.48 17.36 7.15 8.31 620 28.28 22.85 21.67 17.6 7.3 8.46 630 28.52 23.06 21.95 17.95 7.54 8.63 640 28.78 23.27 22.23 18.29 7.74 8.8 650 28.9 23.4 22.34 18.42 7.73 8.92 660 28.96 23.51 22.38 18.45 7.62 9 670 29.04 23.65 22.41 18.46 7.55 9.06 680 29.14 23.79 22.46 18.47 7.52 9.11 690 29.31 23.88 22.59 18.48 7.55 9.18 700 29.52 23.94 22.78 18.49 7.63 9.27

[0066] Sample titanium materials, titanium alloy coupons, having different pre-treatments (grit blasted, polished, porous beaded) were compared for osteogenic activity on the surface after being chemically treated with an alkaline solution, compared to each other type and of pre-treatment and to not being chemically treated.

[0067] The alkaline solution was a 4 molar solution of sodium hydroxide for 2 hours at 60 (as described here before).

[0068] The pre-treatments of the titanium alloy coupons were polishing the surface, grit blasting and porous beading as known in the art.

[0069] After the chemical treatment the titanium alloy coupons were inserted into individual silicone tubes so that any fluid placed on to the coupon remained on the test surface. Coupons were then sterilised.

[0070] Human mesenchymal stem cells were resurrected and passaged in suitable medium and incubated overnight. After incubation the medium was replaced with an osteogenic medium containing R-Glycerophosphate and this was changed twice a week.

[0071] Live/dead staining on the cells was performed on all surface types at all time points.

[0072] The samples were subject to cell lysis and P-nitrophenol alkaline phosphatase p-NPP assay analysis to indicated osteogenic activity of the cells and thus, bone formation.

[0073] The results are as shown in Table 2 for grit blasted pre-treated coupons alkaline solution treated compared to not being subject to alkaline solution.

TABLE-US-00002 TABLE 2 GB GB GB GB Non- GB Non- GB Non- GB Non- GB treated Alkali treated Alkali treated Alkali treated Alkali Grit (Day (Day (Day (Day (Day (Day (Day (Day blasted 3) 3) 7) 7) 14) 14) 21) 21) p-NPP Rep 1 25.493 38.760 79.569 155.813 165.721 211.735 149.917 226.772 Rep 2 21.630 37.081 63.447 142.378 184.362 263.529 228.443 380.482 Rep 3 19.783 36.745 95.859 129.446 235.583 306.969 243.480 327.018 Pico Rep 1 6.702 5.965 5.227 7.439 11.863 7.439 10.388 9.651 Green Rep 2 6.702 6.702 6.702 5.227 10.388 5.965 8.176 8.176 Rep 3 6.702 7.439 6.702 5.965 9.651 7.439 10.388 8.176 Normal- Rep 1 3.804 6.498 15.222 20.945 13.970 28.463 14.432 23.498 isation Rep 2 3.228 5.533 9.467 27.237 17.748 44.183 27.940 46.535 Rep 3 2.952 4.939 14.304 21.703 24.411 41.265 23.439 39.996 Mean 3.328 5.657 12.998 23.295 18.709 37.970 21.937 36.676 Standard 0.435 0.787 3.092 3.435 5.286 8.362 6.878 11.872 deviation

[0074] Table 3 shows pre-treated porous beaded coupons with and without alkaline solution treatment.

TABLE-US-00003 TABLE 3 Porous Porous Porous Porous Non- Porous Alkali + Non- Porous Alkali + treated Alkali heat treated Alkali Heat Porous (Day (Day (Day (Day (Day (Day beaded 3) 3) 3) 7) 7) 7) p-NPP Rep 1 74.363 125.584 150.271 76.404 295.273 198.369 Rep 2 75.874 161.187 187.553 153.259 322.005 250.163 Rep 3 94.851 151.278 174.622 139.893 231.784 265.200 Pico Rep 1 14.074 5.965 8.914 10.388 8.914 6.702 Green Rep 2 8.914 8.176 8.914 11.125 9.651 6.702 Rep 3 8.176 8.176 8.176 9.651 7.439 6.702 Normal- Rep 1 5.284 21.055 16.859 7.355 33.126 29.600 isation Rep 2 8.512 19.714 21.041 13.776 33.366 37.328 Rep 3 11.601 18.502 21.357 14.495 31.158 39.572 Mean 8.466 19.757 19.752 11.875 32.550 35.500 Standard 3.159 1.277 2.511 3.931 1.212 5.231 deviation Porous Porous Porous Porous Non- Porous Alkali + Non- Porous Alkali + treated Alkali Heat treated Alkali Heat Porous (Day (Day (Day (Day (Day (Day beaded 14) 14) 14) 21) 21) 21) p-NPP Rep 1 612.718 1137.336 734.683 636.108 744.708 945.199 Rep 2 617.730 1132.324 709.622 843.282 968.589 1035.420 Rep 3 729.671 1052.127 846.624 764.757 1100.579 1017.041 Pico Rep 1 11.125 8.176 8.176 10.388 6.702 8.914 Green Rep 2 10.388 8.914 8.176 11.125 8.914 9.651 Rep 3 11.125 8.176 9.651 10.388 8.176 9.651 Normal- Rep 1 55.074 139.102 89.855 61.235 111.121 106.041 isation Rep 2 59.466 127.034 86.790 75.799 108.665 107.289 Rep 3 65.587 128.681 87.726 73.619 134.606 105.384 Mean 60.042 131.606 88.124 70.218 118.131 106.238 Standard 5.280 6.544 1.571 7.855 14.321 0.967 deviation

[0075] Table 4 shows polished coupons with and without alkaline solution treatment.

TABLE-US-00004 TABLE 4 Polished Polished Polished Polished Non- Polished Alkali + Non- Polished Alkali + treated Alkali heat treated Alkali heat (Day (Day (Day (Day (Day (Day Polished 3) 3) 3) 7) 7) 7) p-NPP Rep 1 20.287 37.920 38.088 136.668 186.209 87.630 Rep 2 24.149 34.729 31.203 94.683 152.958 118.866 Rep 3 20.119 36.913 31.203 67.477 148.927 132.133 Pico Rep 1 8.914 5.227 6.702 5.965 5.965 5.227 Green Rep 2 6.702 5.227 9.651 7.439 5.227 5.227 Rep 3 12.600 4.490 5.965 22.921 5.227 5.227 Normal- Rep 1 2.276 7.254 5.683 22.913 31.220 16.764 isation Rep 2 3.603 6.644 3.233 12.728 29.261 22.740 Rep 3 1.597 8.221 5.231 2.944 28.490 25.278 Mean 2.492 7.373 4.716 17.821 29.657 21.594 Standard 1.021 0.795 1.304 7.202 1.407 4.371 deviation Polished Polished Polished Polished Non- Polished Alkali + Non- Polished Alkali + treated Alkali Heat treated Alkali Heat (Day (Day (Day (Day (Day (Day Polished 14) 14) 14) 21) 21) 21) p-NPP Rep 1 206.723 527.509 158.271 288.590 15.488 20.500 Rep 2 265.200 470.703 357.091 352.079 908.442 484.069 Rep 3 196.699 554.241 731.342 7.903 527.509 388.836 Pico Rep 1 5.965 8.176 5.965 8.914 0.671 0.067 Green Rep 2 6.702 5.227 5.227 8.176 8.176 9.651 Rep 3 7.439 5.965 7.439 0.067 7.439 6.702 Normal- Rep 1 34.659 64.517 26.535 32.377 23.091 308.186 isation Rep 2 39.572 90.048 68.313 43.061 111.107 50.159 Rep 3 26.441 92.923 98.311 118.804 70.911 58.020 Mean 33.557 82.496 64.387 37.719 91.009 54.089 Standard 6.634 15.636 36.049 7.555 28.423 5.559 deviation

[0076] The results show that there is more Osteogenic activity where the coupons have been chemically treated e.g. with an alkaline solution compared to not being chemically treated.

[0077] It will be appreciated that the primary layer may include various bio-active materials including antimicrobials. It will also be appreciated that the primary layer may be further treated to impart anti-biofouling, cytogenic, catalytic, osteogenic or electrochemical properties to the implant.

[0078] It will be further appreciated that the substrate may comprise other metals or alloys instead of titanium, for example, nitinol or zirconium.

[0079] It is envisaged that the material formed maybe subjected to further physical treatment steps to improve or enhance the surface characteristics of the primary surface layer. For example, on completion of the primary layer, the material can be rinsed in water or phosphate buffered-saline solution to remove the alkali. After drying the material, it can be heated to a target temperature of between 300-600 C. The target temperature can be reached by raising the temperature of the material by 5 C. per minute. The target temperature, once reached can be maintained for at least one hour.

[0080] It will be appreciated that the invention is not limited to the embodiments hereinbefore described but may be varied in construction and detail within the scope of the appended claims.