PROCESS FOR THE PREPARATION OF A TOPOGRAPHY FOR IMPROVED PROTEIN ADHERENCE ON A BODY MADE OF TITANIUM OR A TITANIUM ALLOY

Abstract

A process for the preparation of a topography for improved protein adherence on a body made of titanium or a titanium alloy wherein the process includes the subsequent steps of pre-treating the surface including etching the surface with an etching solution containing a mineral acid, and forming on the pre-treated surface obtained under titanate-including sub-microscopic structures by treating the pre-treated surface with an aqueous solution containing an oxidative agent, the sub-microscopic structures extending in at least two dimensions to 1 m at most.

Claims

1. Process for the preparation of a topography for improved protein adherence on a body made of titanium or a titanium alloy, the process comprising the subsequent steps of a) etching the surface with an etching solution comprising a mineral acid, and b) forming on the etched surface obtained under a) titanate-comprising sub-microscopic structures by treating the etched surface with an aqueous solution comprising an oxidative agent, the sub-microscopic structures extending in at least two dimensions to 1 m at most.

2. Process according to claim 1, wherein by etching the surface according to step a) a microscopic topographical formation is formed, on which in step b) a layer comprising or essentially consisting of the sub-microscopic structures is formed, the layer forming a sub-microscopic topographical formation.

3. Process according to claim 2, wherein the microscopic topographical formation is defined by at least one of the following surface parameters: i) Sa being the arithmetic mean deviation of the surface in three dimensions and being in the range from 0.1 m to 2.0 m; ii) St being the maximum peak to valley height of the profile in three dimensions and being in the range from 1.0 m to 20.0 m, and/or iii) Ssk being the skewness of the profile in three dimensions and being in the range from 0.6 to 1.0.

4. Process according to claim 2, wherein by the sub-microscopic topographical formation formed in step b), at least one surface parameter defining the microscopic topographical formation formed in step a) and being selected from the group consisting of Sa, St and Ssk is changed by 50% at most.

5. Process according to claim 1, wherein the sub-microscopic structures extend to a length of more than 50 nm.

6. Process according to claim 1, wherein at least some of the sub-microscopic structures have the shape of a lamella, sheet and/or wire.

7. Process according to claim 6, wherein the thickness of the lamella, sheet and/or wire is in the range from 1 to 50 nm, and the lamella, sheet and/or wire extend in direction perpendicular to the direction of thickness from 50 to 1000 nm.

8. Process according to claim 1, wherein the treatment under step b) is performed under elevated temperatures.

9. Process according to claim 1, wherein the oxidative agent is an inorganic hydroxide.

10. Process according to claim 1, wherein the oxidative agent is a hydroxide of an alkali metal and/or a hydroxide of an alkaline earth metal.

11. Process according to claim 1, wherein the concentration of the oxidative agent is in the range from 0.1 to 5 M.

12. Process according to claim 1, wherein the treatment in step b) is carried out for a duration in the range from 0.25 hours to 12 hours.

13. Process according to claim 1, wherein in step b) the etched surface obtained in step a) is treated with an aqueous solution of sodium hydroxide and/or potassium hydroxide such that sodium titanate-comprising sub-microscopic structures or potassium titanate-comprising sub-microscopic structures are formed, respectively.

14. Process according to claim 1, wherein the body is a dental implant or a dental implant abutment and the topography is provided on at least a portion of the surface of the body that in use is intended to be in contact with hard tissue or soft tissue, respectively.

15. Body obtainable by the process according to claim 1.

16. Body according to claim 15, the surface of which being defined by a microscopic topographical formation and a sub-microscopic topographical formation formed on the microscopic topographical formation, said sub-microscopic topographical formation comprising or essentially consisting of a layer of titanate-comprising sub-microscopic structures extending in at least two dimensions to 1 m at most.

17. A method comprising using the body according to claim 16 as a dental implant or a dental implant abutment.

Description

[0099] The SEM images of the samples and the EDX spectra of the RXD SLActive NaOH sample are given in the attached figures, whereby

[0100] FIG. 1 relates to a SEM image of the surface of sample RXD SLActive NaOH in a magnification of about 1000, the scale corresponding to 10 micrometer being given in the bottom left corner of the image;

[0101] FIG. 2 relates to a SEM image of the surface of sample RXD SLActive in a magnification of about 1000, the scale corresponding to 10 micrometer being given in the bottom left corner of the image;

[0102] FIG. 3 relates to a SEM image of the surface of sample RXD SLActive NaOH in a magnification of about 20000, the scale corresponding to 200 nanometer being given in the bottom left corner of the image;

[0103] FIG. 4 relates to a SEM image of the surface of sample RXD SLActive in a magnification of about 20000, the scale corresponding to 200 nanometer being given in the bottom left corner of the image;

[0104] FIG. 5 relates to a SEM image of the surface of sample RXD SLA NaOH (storage in NaCl) in a magnification of about 50000, the scale corresponding to 200 nanometer being given in the bottom left corner of the image;

[0105] FIG. 6 relates to a SEM image of the surface of sample RXD SLA NaOH (storage in air) in a magnification of about 50000, the scale corresponding to 200 nanometer being given in the bottom left corner of the image; and

[0106] FIG. 7 relates to the EDX spectrum obtained on a 200 image of RD SLActive NaOH.

[0107] As can be seen from FIGS. 1 to 4, the RXD SLActive and RXD SLActive NaOH samples exhibit the macroscopic and microscopic topographical formation obtained by the SLA treatment, namely by sandblasting and etching the samples in a boiling mixture of HCl and H.sub.2SO.sub.4.

[0108] However, there are distinct differences in the appearance of a sub-microscopic topographical formation between sample RXD SLActive and RXD SLActive NaOH. Specifically, sub-microscopic structures in the form of lamellae or wires entangled with each other and, thus, forming a network are visible for sample RXD SLActive NaOH at the respective magnification shown in FIG. 3, whereas such sub-microscopic structures are not visible for sample RXD SLActive shown in FIG. 4. Further experiments have shown that the sub-microscopic topographical formation is maintained after storing the RXD SLActive NaOH samples in NaCl (FIG. 5) or in air (FIG. 6). In particular, FIG. 6 shows that the RXD SLA NaOH sample, which has been stored for 8 months in air, exhibits a very similar sub-microscopic topographical formation as the one of the RXD SLActive NaOH sample shown in FIG. 3.

[0109] As further shown by the EDX spectrum according to FIG. 7, the RXD SLActive NaOH sample has a relatively thick oxide layer thickness indicated by the pronounced signal for oxygen. Also, the RXD SLActive NaOH sample shows a pronounced signal for sodium, both signals being indicative of sodium-titanate comprising sub-microscopic structures. As a result of i.a. the increased oxide-layer thickness, the SEM image of the RXD SLActive NaOH sample shown in FIG. 1 is blurred.

[0110] Further experiments have shown that the oxide layer thickness is in the range from 500 nm to 800 nm and that sodium is observed to a depth of about 800 nm.

[0111] 1.2.3. Roughness Parameter Determination

[0112] Roughness images were acquired using a confocal microscope (psurf explorer, NanoFocus AG, Oberhausen, Germany) equipped with a 20 lens. Three measurements were performed on each sample disc and three discs were measured for each type of surface. The roughness parameters were calculated using the WinSAM software mentioned above. The whole roughness image with a size of 798 m (micrometer)798 m (micrometer) was used for the calculation of the 3D roughness parameters.

[0113] The values of the microscopic topographical formation (roughness) were determined using a moving average Gaussian filter with a cut-off wavelength of 30 m (x=31 m, y=30 m, 2019 image points). Then, the roughness parameters were calculated by means of a KFL analysis with limits from the amplitude density.

[0114] Specifically, S.sub.a (the arithmetic mean deviation of the surface in three dimension), S.sub.t (the maximum peak to valley height of the profile in three dimensions) and S.sub.sk (the skewness) were determined in analogy to EN ISO 4287 relating to the respective parameters R.sub.a, R.sub.t and R.sub.sk in two dimensions. For the parameters in three dimensions, it is further referred to ISO 25178, in which the symbol S.sub.z is used for the maximum peak to valley height of the profile (instead of the symbol S.sub.t used in the context of the present invention).

[0115] Table 2 presents the mean values of the microroughness values of the two samples. The table shows that at least the values of S.sub.a and S.sub.t of both RXD SLActive and RXD SLActive NaOH lie within the same range typically observed for SLA/SLActive implants. Similar values are obtained for RXD SLActive NaOH samples after storing in 0.9% NaCl solution or after storing in air for 1 month. Specifically, the S.sub.a, S.sub.t and S.sub.sk values of RD SLActive NaOH deviate from the respective values of the RXD SLActive samples by less than 25%.

TABLE-US-00002 TABLE 2 Values of the microscopic topographical formation Sa Std Sa St Std St Std [m] [m] [m] [m] Ssk Ssk RXD 1.183 0.037 7.91 0.31 0.215 0.041 SLActive NaOH RXD 0.970 0.046 6.38 0.26 0.192 0.051 SLActive

[0116] 1.2.4. Protein Adsorption Measurements

[0117] Albumin (from bovine serum (BSA), Alexa Fluor 647 conjugate, Invitrogen, USA), fibrinogen (from human plasma; HPF, Alexa Fluor 546 conjugate, Invitrogen, USA) and fibronectin (Rhodamine Fibronectin from bovine plasma; BSF, Cytoskeleton, Inc., USA) were used as model proteins to study their adsorption (or adherence) behaviour on the different surfaces by means of fluorescence microscopy using a fluorescence scanner.

[0118] Stock solutions of 0.5 mg/ml albumin and 0.5 mg/ml fibrinogen have been made according to the product manuals. For storage these stock solutions were divided into 0.5 ml aliquots and frozen at 20 C. Fibronectin solutions were made directly from the 20 g vials, without making a stock solution.

[0119] All protein-adsorption solutions were made with HEPES 2 buffer prepared with 10 mM 4-(2-hydroxylethyl)-piperazine-1-ethanesulfonic acid (HEPES) and 150 mM NaCl with pH 7.4. Prior to use the HEPES 2 buffer was filtered (Whatman FP 30/0.2 CA-S, size 0.2 m, maximum pressure 7 bar).

[0120] For low protein concentration experiments, the protein solution consisted of filtered HEPES 2 and fluorescently labelled protein of defined concentration (see Table 3 below). For high concentration experiments, unlabelled protein was added additionally to simulate the real protein concentration in human blood (Table 3). To enhance the solubility of unlabelled proteins, HEPES 2 was heated to 37 C. (water bath, INCO 2/108, Memmert GmbH&Co, Germany) prior to the preparation of the solution. The different proteins were tested separately; therefore, the prepared protein solutions always contained only one type of protein. It is assumed that the labelled proteins behave like the unlabelled ones.

[0121] To reduce a possible uncertainty in the results due to the instability of the fluorescence marker, the protein solution was freshly prepared right before the adsorption experiments.

[0122] The method applied was based on the application of fluorescently labelled proteins and intensity measurements as well as comparison of fluorescence scanning images.

[0123] For the albumin and fibrinogen experiments, the samples were generally immersed into 2 ml of protein solution for 10 minutes. The adsorption process was carried out in 24-well plates. Experiments with fibronectin were carried out in 96-well plates and 0.3 ml protein solution but also with an adsorption time of 10 minutes. All adsorption experiments were performed at room temperature.

[0124] Proteins not adsorbed onto the surface were removed by submerging the samples in 2 ml of pure HEPES 2 for 10 seconds. Next, they were pivoted in 5 ml of HEPES 2 for 5 seconds followed by a rinsing step with the same 5 ml of HEPES 2. Additionally, the samples were rinsed with ultrapure water for 3 seconds, dried in a stream of nitrogen (at a pressure of about 1 bar) and stored at room temperature in a 24-well plate. To avoid bleaching of the fluorescent label of the adsorbed proteins, the well plates were covered with aluminium foil.

[0125] The experimental conditions are given in Table 3 below:

TABLE-US-00003 TABLE 3 Experimental conditions of protein adsorption experiments Protein Protein Concentration solution Time Samples low protein concentration Albumin 3 g/mL 2 mL 10 min 6 Fibrinogen 7 g/mL 2 mL 10 min 6 Fibronectin 3 g/mL 0.3 mL 10 min 6 high protein concentration Albumin 3 g/mL + 10 mg/mL* 2 mL 10 min 6 Fibrinogen 7 g/mL + 1 mg/mL* 2 mL 10 min 6 Fibronectin 3 g/mL + 0.2 mg/mL* 0.3 mL 10 min 6 *= unlabelled protein

[0126] The amount of protein attached to the surface was measured qualitatively using a microarray fluorescence scanner (Axon Genepix 4200A, Molecular Devices, USA). For intensity measurements, the resolution was set to 100 m/pixel and only one scan per line was performed. For imaging the resolution was set to 5 m/pixel and three scans per line were performed. To read out the albumin adsorption a laser with a wavelength of 635 nm was used whereas the scanning of fibrinogen and fibronectin adsorbed surfaces was performed using a 532 nm laser. The best focus position was determined separately for every sample.

[0127] The photo-multiplier tube (PMT) of the fluorescence scanner was specified to be linear between a gain of 350 to 600. For that reason, all scans were performed in this PMT range. The gain was adapted for each combination of surface, protein and concentration in order to stay in the grey-scale limits of the fluorescence signal of a sample. All gains chosen are listed in Table 4.

TABLE-US-00004 TABLE 4 Gains chosen for the RXD SLActive NaOH sample to measure the protein adsorption RXD SLActive NaOH low protein concentration Albumin 400 Fibrinogen 350 Fibronectin 350 high protein concentration Albumin 550 Fibrinogen 350 Fibronectin 450

[0128] To evaluate the homogeneity of the protein adsorption, high-resolution images were compared with each other by visual examination.

[0129] The fluorescence intensity data acquired by fluorescence scanning are given in the attached

[0130] FIG. 8 showing a diagram relating to the fluorescence intensities measured for albumin, fibrinogen and fibronectin on RXD SLActive NaOH surfaces at the low protein concentration defined above; and

[0131] FIG. 9 showing a diagram relating to the fluorescence intensities measured for albumin, fibrinogen and fibronectin on RXD SLActive NaOH surfaces at the high protein concentration defined above.

[0132] All values presented in FIGS. 8 and 9 are normalized to the respective intensities measured for a titanium body treated according to the SLActive protocol (Ti SLActive). The error bars indicate the standard deviation.

[0133] According to FIGS. 8 and 9, an extremely high adsorption of the proteins fibrinogen and fibronectin was determined for the RXD SLActive NaOH sample. Whereas at low protein concentration, the improvement in fibrinogen adsorption was more than three times higher than for the Ti SLActive reference, the factor in adsorption improvement was more than 57 at high protein concentration.

[0134] Thus, the sample according to the present invention exhibits a highly improved adsorption of the proteins fibrinogen and fibronectin, to both of which an important role in mediating blood coagulation and cell attachment and, thus, tissue interaction with the body is attributed. The adsorption of these proteins is also highly specific, given that the adsorption of non-specific albumin is far lower than for fibrinogen and fibronectin, both at low and high protein concentration.

[0135] 1.2.5. Assessment of Fibrin Network Formation after Whole Human Blood Incubation

[0136] RXD SLA NaOH samples were incubated with whole human blood and analysed for fibrin network formation by SEM and CLSM (confocal laser scanning microscopy) imaging.

[0137] Specifically, whole human blood obtained from healthy volunteers was partially heparinized directly with 3 IU/ml sodium heparin (final concentration 0.5 IU heparin/ml blood) and used for the experiments within 1 hour after withdrawal.

[0138] Samples were placed into a sample holder and freshly withdrawn blood was added until all samples were covered with a 4 mm thick layer of blood. To prevent further contact with air, the sample holder was closed with a lid and sealed with parafilm before incubation on a tumbling shaker at 10 rpm at room temperature.

[0139] The incubation time was determined for each experiment individually. For this, whole blood was spiked with labeled fibrinogen (Alexa488), which allows live monitoring of the blood coagulation on the samples using the fluorescence microscope. As reference, the samples RXD SLA (and RXD SLActive) were used and two time points were chosen (t1: thin, t2: thick fibrin network present on the reference sample).

[0140] After incubation, blood was removed and the samples were washed three times by adding pre-warmed PBS into the sample holder, then incubated on a tumbling shaker at 10 rpm for 1 minute for each washing step. Thereafter, the samples were transferred into a new 96-well plate for further treatment.

[0141] For SEM imaging, samples were fixed in modified Karnovsky solution for 1 h at room temperature (RT) and then washed twice in PBS. Thereafter, the samples were dehydrated by immersing the samples in solutions of a gradient series of ethanol (50, 70, 80, 90 and 100%), followed by incubation in hexamethyldisilazane (HMDS) for 30 min. Finally, samples were placed into a new 96-well plate and dried overnight at RT. On the next day samples were sputter-coated with gold/palladium (high vacuum coater Leica EM ACE 600, Switzerland). SEM imaging was performed using a Hitachi S-4800 (Hitachi High-Technologies, Canada) at an accelerating voltage of 2 kV and 10 A current flows.

[0142] For CLSM analysis, samples were incubated for 30 min in PBS with 5% goat serum and 1% FCS before staining platelets with Alexa546-labeled phalloidin for 1 h at RT. The platelets and the fibrin network (visible due to spiking of the blood with Alexa488-labeled fibrinogen) were imaged with the CLSM (10, 40 magnification). Depending on the coverage and thickness of the fibrin network seen by SEM imaging, only one time point (t1 or t2) was imaged. On samples showing complete coverage with fibrin on the surface, the thickness of the fibrin network was measured from z-stack images. To assess the thickness of the fibrin network, 4 z-stack images of 2 samples (2 images per sample with 4 to 6 measurements per image) were analysed to measure the distance from the sample surface to the top surface of the fibrin network using the measure function of the Zeiss ZEN software. CLSM analysis was performed with three independent experiments.

[0143] The semi-quantitative analysis of the SEM imaging revealed a trend for higher fibrin network thickness and larger sample coverage of the RXD SLA NaOH sample in comparison to the RXD SLA sample.

[0144] This is evidenced by the following figures, of which

[0145] FIG. 10 relates to a SEM image of the surface of sample RXD SLA NaOH after 14 minutes of incubation in a magnification of about 800, the scale corresponding to 50 micrometer being given in the bottom right corner of the image; and

[0146] FIG. 11 relates to a SEM image of the surface of sample RXD SLA after 14 minutes of incubation in a magnification of about 800, the scale corresponding to 50 micrometer being given in the bottom right corner of the image.

[0147] Evaluating the presence, distribution and thickness of the fibrin network, the semi-quantitative analysis of the SEM images is summarized in Table 5 showing for four different experiments the value of a qualitative ranking from 0 to 4 taken in each case for two samples and for two different incubation periods given in the table, the ranking starting from patches of blood cells with only few visible fibrin fibers (0) to thick fibrin networks completely covering the sample surface (4).

TABLE-US-00005 TABLE 5 Semi-quantitative analysis of the SEM images for four different experiments performed on RXD SLA NaOH and RXD SLA samples Incubation period Experiment (min) RXD SLA NaOH RXD SLA 1 10 2.5 2.5 1 1.5 15 4 4 0 1 2 12 0 4 0 0 15 4 4 2 2 3 14 4 3 0 0 17 4 4 0 1.5 4 15 4 4 0 0 18 4 4 0 2

[0148] The thickness of fibrin formed on samples incubated with whole blood (partially heparinized 0.5 IU/ml) for 17 minutes has been assessed as described above, the results of which are given in Table 6.

TABLE-US-00006 TABLE 6 Analysis of fibrin layer thickness of RXD SLA NaOH and RXD SLA samples, the layer thickness was measured, otherwise, no measurement was done (n.m.) Sample fibrin layer thickness [m] RXD SLA NaOH homogenous 11.02 RXD SLA none n.m.

[0149] Thus, in contrast to the RXD SLA samples showing no fibrin network formation, a relatively high fibrin thickness was determined on the samples according to the present invention.