WO2015001366A2

WO2015001366A2 - Method to produce a metal implant with the properties of antimicrobiality and biocompatibility and such a metal implant

Info

Publication number: WO2015001366A2
Application number: PCT/HU2014/000048
Authority: WO
Inventors: Varsányi Magdolna LAKATOSNÉ; Tamás POZMAN; Mónika FURKÓ
Original assignee: Bay Zoltán Közhasznú Nonprofit Kft
Priority date: 2013-05-27
Filing date: 2014-05-27
Publication date: 2015-01-08
Also published as: WO2015001366A3; HUP1300343A2

Abstract

The invention relates, on the one hand, to a method for producing a medical metallic implant with the properties of antimicrobiality and biocompatibility and, on the other hand, to the metallic implant itself. According to the method, an antimicrobial coating is applied onto a metallic substrate of said metallic implant, the method comprising the steps of arranging said substrate as cathode against a counter electrode in an electrolytic solution containing at least a salt of the metal to be deposited to form the coating, and depositing the coating from the electrolytic solution onto the substrate surface by electrolysis. The essence of the present method is that after an initial pre-treatment of the substrate surface, the electrolysis is performed directly onto the substrate, and said coating is formed as a non-continuous nanostructured coating by pulse current series electrochemical deposition during the electrolysis. The medical metallic implant according to the invention comprises a metallic substrate and an antimicrobial coating, wherein the antimicrobial coating is bound in the form of a plurality of nanostructured microclusters directly to a substrate surface subjected to pre-treatment, and said microclusters are located on said substrate so as to form local galvanic cells when get into contact with an electrolyte.

Description

METHOD TO PRODUCE A METAL IMPLANT WITH THE PROPERTIES OF ANTIMICROBIALITY AND BIOCOMPATIBILITY AND SUCH A METAL IMPLANT

The present invention relates to a method to produce a medical metallic implant with the properties of antimicrobiality and biocompatibility, as well as to a metallic implant produced by the method.

Implants are present in human body as passive surfaces. Their passive state makes the implants susceptible to the adhesion of bacteria which can cause infection of the implants. In such a case an antibiotic treatment of the infection is ineffective, because a biofilm protects the pathogenic microorganisms which therefore become resistant to an- tibiotics. Therefore biofilms being formed on the surface of implants present a major (clinical) problem.

It has long been known about silver that it has antibacterial activity, however, its significance in the modern era may increase due to the wide-spreading of multiresistant bacteria. Before the widespread application of antibiotics, i.e. until about the first half of the 20^th century, medicaments containing colloidal silver were used in the field of therapy for its bactericidal effect or, in case of burn injuries, silver compounds to locally disinfect water. For the aforementioned reasons, due to their antibacterial and biocompatible properties, silver nanostructures formed on metallic implants with suitable parameters can be used advantageously for the prevention of infections by bacteria residing in hospital and clinical buildings and being resistant to effective antibiotic treatments.

Researchers from Miinster have deposited a 10-15 μηη thick silver layer galvanically onto a gold layer of 0.2 pm in thickness formed previously by vacuum vapor deposition onto metal prostheses. They have investigated the antimicrobial effectiveness and the possible side effects of silver-coated titanium megaendoprostheses by in vivo an- imal experiments. The results showed that silver-coated implants had an infection rate (by Staphylococcus aureus bacterium) of 7%, while for non-coated implants this was 47%. The results proved that the novel silver-coated Mutars prostheses in the referenced work reduced the infection rate without toxic side effects (see Gosheger, G., Hardes, J., Ahrens, H., Streitburger, A., Buerger, H., Erren, M., Gunsel, A., Kemper, F.H., Winkelmann, W., von Eiff, C, 2004. Silver-coated megaendoprostheses in a rabbit model - an analysis of the infection rate and toxicological side effect, Biomaterials 25 (24) pp. 5547-5556).

In order to found a base for the future clinical use of silver-coated implants, Secinti et al. (2008) investigated in vivo the antibacterial effect of silver-coated spinal implants under the effect of anodic polarization. They showed that anodic activation (1 μΑ for 1 hour) of the silver coating causes the bacterial growth to stop within the bacterial culture for at least three weeks and this effect remains even if frequent rinsing is applied and the electric current is switched off (see Secinti, K. D., Ayten, M., Kahilogullari, G., Kaygusuz, G., Ugur, H. C, Attar, A., 2008. Antibacterial effects of electrically activated vertebral implants. Journal of Clinical Neuroscience Ij^ pp. 434-439).

After the animal tests, researchers from Miinster have implanted silver-coated implants into 13 male and 7 female patients. The same method has been used for producing the silver coating as earlier in their animal tests. In the tests repeated every three months, they were searching for the signs of local or systemic argyria, difficulties of wound healing and symptoms of neurological damage. The silver concentration of blood and urine has been measured before and after the surgery. Liver function has been examined. None of the patients developed neurological damage which could be brought into connection with the surgery. None of the patients showed allergic reaction to the silver that coated the prosthesis. During the 19-month span of the test, the silver-coated megaendoprostheses in the 20 patients caused neither local nor systemic side effects (see Hardes, J., Ahrens, H., Gebert, C, Streitbuerger, A., Buerger, H., Erren, M., Gunsel, A., Wedemeyer, C, Saxler, G., Winkelmann, W., Gosheger, G., 2007. Lack of toxicological side-effects in silver-coated megaprostheses in humans. Biomaterials 28, pp. 2869-2875).

U.S. patent application No. 2010/0316686 discusses the coating of surfaces of medical devices and implants by an antibacterial silver layer. At first, an oxide layer is formed by plasma electrolytic oxidation on the surface of the medical devices and implants made of metals/metal alloys and then silver islands are formed on said surface using silver colloid. The Ag/Ti0₂ coating shows excellent antibacterial, adhesion and bio- compatibility properties. In the plasma electrolytic oxidation process the base metal is connected as anode into the electrochemical cell, where an oxide layer is formed on the surface due to the electric current. The structure of this oxide layer is very porous. The silver nanoparticles are applied onto the surface in the oxidation process. The average size of the silver colloid is 50 nm, but the most preferred size is under 30 nm.

In additional investigations, this method has also been used to coat medical devices and implants with apatite, copper and zinc, which also showed antibacterial activity. In the case of hidroxyapatite, even the conditions of osteoconduction have been improved.

U.S. Patent No. 4,291 ,125 is directed to the destruction of plant and animal bacteria and plant viruses by silver ions generated by electric current. Production of silver ions is carried out by the very slow anodic dissolution, corrosion of silver wires. An electric cir- cuit is formed using an external power source, wherein a silver wire works as anode and another, non-corroding metal functions as cathode. In a suitable electrolyte, due to the electric current, silver ions are released from the silver wire and provide then an antibacterial effect. The silver wires are positioned very close to the infected area and by polarizing the anode, within the range of 5 mm, with a direct current in the order of magnitude of microampere, bacteria on the infected area got destroyed.

In the method according to U.S. Patent No. 6,365,220, a layer composed of a nobler metal is formed between the silver layer and the substrate. This method facilitates the release of silver ions, because micro galvanic cells form, in which silver functions as an- ode (dissolves), while the nobler metal works a cathode. The deposition of the silver layer is carried out by different, mostly physical processes and a suitable texture of the surface is mainly achieved by various after-treatments (etching, ultrasound, micro-abrasion).

In U.S. Patent No. 5,681 , 575A an antimicrobial and biocompatible silver layer is formed on the surface of medical instruments by vapor deposition technique. By this method, an atomic disorder is achieved on the surface of the substrate that provides a sufficient and continuous metal-ion release, thus providing the sufficient antimicrobial effect.

According to U.S. Patent No. 4,615,705 the antimicrobial effect of surgical implants, especially orthopedical endoprosthetic implants and sutures is achieved by forming a biodegradable metallic silver coating. Under in vivo circumstances silver ions are released from the silver coating at a maintainable rate in an amount that provides sufficient antimicrobial effect, but does not cause adverse side effects in the surrounding connective tissues. Antibacterial silver is embedded either in the inert implant material as a composite or a 35 nm thick silver layer is formed on the surface of the implant by a sputter coating technique, said layer covers the surface partially or entirely. In order to achieve the sufficient effect, the metallic silver layer has to be activated; this is carried out by abrasion, heating to above 180°C or using hydrogen peroxide. Furthermore, the structural material of the implant has to be bioinert in order to properly integrate into the body despite its release of silver ions. The implant can be made of any bioinert/biocompatible material; for this purpose, in particular, Ti and Ti alloys, CoCrMo, ceramics and non-toxic synthetic plastics can be equally used.

Published European patent application no. EP-2,229,962 A1 discloses the production of a silver-coated metallic implant with the substrate being mainly stainless steel. The partial, i.e. non-continuous silver-coating of the substrate surface is formed by galvanic deposition with masking said surface in patches of desired extent and shape or via immersing the substrate partially into an electrolyte.

The disadvantage of the prior art methods is that to provide a sufficient and constant rate of dissolution of the silver layer requires either (i) an additional layer deposited between the substrate and the silver layer, or (ii) a periodically repeated anodic oxidation of the implant by an external power source. In the former case it has to be taken into account, that the antimicrobial efficiency of the electrochemically deposited silver layer might not be significant, moreover, it can even lose its antimicrobial effect over time if no activation facilitating the release of silver ions has been carried out during pre-treatment of the silver-coated implants. Starting the dissolution of silver by activation, an external power source or an interposed layer of nobler metal can be achieved by alternative methods. It has been found in our investigations that local galvanic cells are created as a consequence of the microclustered arrangement of the nanostructured silver, wherein the anode of said galvanic cells is formed of silver. When body-fluids analogous to electrolytes con- taining NaCI contact simultaneously a substrate metal (i.e. the material of the implant) having an electrode potential more positive than that of silver and the antibacterial silver layer, a galvanic cell forms, in which silver gets oxidized, i.e. dissolves spontaneously at a given rate. Techniques used to create silver microclusters have to provide a large extent of disorder and a large specific area. These techniques usually involve the use of a pulsed laser and, therefore, production on the industrial scale is not solved by means of the given technology, i.e. due to the less economical production method, the cost of the obtained product is necessarily high.

As mentioned previously, a disadvantage of the electrochemical processes discussed above is that dissolution of the silver layer is achieved by either applying an inter- posed golden layer or through the periodically repeated anodic dissolution of the implant induced by an external power source.

In one aspect of the present invention, an electrochemical process to eliminate the aforementioned problems of economy is provided, wherein the rate of anodic dissolution of silver possessing antibacterial properties can be finely tuned by the parameters of the electrochemical process.

In light of the aforementioned, a further object of the present invention is to provide metallic implants suitable for being implanted into human or non-human animal bodies, which implants also have antimicrobial properties besides their biocompatibilty and thus can advantageously be used to prevent or reduce nosocomial infections possibly occur- ring after the implantation. A yet further object of the invention is to elaborate a method by which said implants can be produced at low costs, efficiently and in a relatively simple manner.

It is a further object of the invention to elaborate a method by means of which silver can be deposited electrochemically directly onto a metallic substrate (the metallic im- plant) and due to the suitable pre-treatment of said metallic implant and the suitably selected deposition parameters of pulse-current electrolysis, the dissolution of silver takes place spontaneously (by galvanic corrosion). Thus, the electrochemical process of the present invention can be performed in a single step.

The object aimed at the elaboration of an electrochemical process according to the invention has been achieved by the method according to claim 1. Further preferred exemplary embodiments of the electrochemical process according to the invention for producing an antimicrobial and biocompatible medical metallic implant are set forth in claims 2 to 9. The object aimed at providing a metallic implant according to the invention has been achieved by providing a metallic implant according to claim lOwith the antimicrobial prop- erty and biocompatibility. Possible further preferred exemplary embodiments of said metallic implant are set forth in claims 11 to 16.

In the process, the preferably surface treated substrate is placed into an electrolytic solution comprising one or more salts of the metal to be deposited and additives, and then silver is deposited onto said substrate against a suitably chosen counter electrode by a series of cathodic current pulses of preset current density, pulse- and relaxation time, wherein the deposited islanded (i.e. localised) silver has got a morphology and grain size that maintain a continuous and controlled rate of silver dissolution, as well as antibacterial properties (when said implant is implanted into a human or non-human animal body). Due to the antibacterial and biocompatible properties of the nanostructured silver formed on metallic implants with suitable parameters, it can be advantageously used in accordance with claim 17 to prevent infections caused by pathogens present in hospital and clinical buildings and resistant (multiresistant) to effective antibiotic therapies.

To maintain a long term spontaneous silver dissolution and the antimicrobial property, the silver islands, agglomerates formed by the electrochemical process according to the invention have nanocrystalline structure.

In what follows, two exemplary embodiments of the method according to invention will be disclosed to form silver films with antibacterial property and islanded nanostructure. The bases thereof are non-stationary and stationary electrochemical processes. Firstly, a method for depositing silver islands onto a titanium alloy by a pulse-current electrochemi- cal process is discussed. Secondly, a method for producing implants with the CoCrMo substrate coated with silver in islands by direct current electrolysis is disclosed.

Metallic implants made of titanium alloy or based on CoCrMo coated with silver in islands/islands provide a new perspective in orthopedic practice for the prevention of in- fections caused by bacteria on the knee- and hip prostheses.

It is known, that to form nanostructured metal and alloy films, pulse-current electrolysis is used more and more often and also on the industrial scale among other options. The main advantages of this techniques are its low cost, high productivity and that it can easily be automated. In this case the quality and morphology of the deposited layer are also affected significantly by the pulse parameters, besides the composition and the temperature of the electrolyte.

The core feature of the present invention is that a method for producing TiAI6V4 and CoCrMo based implants coated with a nanostructured silver layer in a non-continuous manner, in the form of tiny spots/islands is provided, wherein a properly surface-treated (grit-blasted) implant with a silver anode are placed into an electrolytic solution comprising a silver complex salt and other additives, and then said silver islands, agglomerates of desired structure and of antimicrobial and cytoconduction properties are formed by applying current pulses and currentless pulse periods according to a predetermined sequence. Silver agglomerates created in this way on the surface of the substrate that forms the im- plant have to dissolve at a minimal rate when implanted into a human or non-human animal body. Moreover, the substrate has to possess sufficient corrosion stability as well.

In what follows, the invention is described in detail with reference to the accompanying drawings, wherein

Figure 1 illustrates an exemplary embodiment of a series of cathodic current pulses used in a preferred embodiment of the method according to the invention to deposit nanostructured silver (Ag) onto an implant with metallic surface - the amplitude l_p of the individual pulses in the series of current pulses shown in Figure 1 and the current v. also shown in Figure 1 satisfy the equation of Ι_3ν.=γ Ιρ. wherein parameter γ is de- fined by the equation of *°^{n + t}°ff , wherein to_n is the length of the time period of the individual pulses and \_off stands for the length of the periods between said individual pulses forming said series of current pulses, i.e. γ can be interpreted as a duty factor;

Figure 2 shows the current-efficiency of nanosilver deposition as a function of the average current density for different implant base metals (TiAI6V4 [from now on: Ti]; stainless steel [from now on: steel] and CoCrMo) having a given base metal area (A = 2.3 cm²) treated by grit blasting and for different electrochemical process parameters (in this case: = 5 ms, t^ = 15 ms, l_p = 10; 20; 40; 60; 80 mAcm^"2; layer thickness of Ag: δ = 10 μΐη);

- Figure 3 illustrates the grain size distribution of Ag layers deposited by the method according to the invention onto Ti [Figures 3A and 3B] and steel [Figure 3C] base metals by indicating the average grain size d_averag_e for the following electrochemical process parameters - Figure 3A: l_p = 100 mAcm^"2, to_n = 1 ms, t_off = 10 ms, d_average = 68 nm; Figure 3B: l_p = 10 mAcm^"2, t_on = 5 ms, t_off = 5 ms, d_average = 59 nm; Figure 3C: l_p = 100 mAcm^"2, ton = ms, to_ff = 9 ms, d_average = 160 nm;

Figures 4A and 4B are scanning electron microscope (SEM) images taken of the silver layers deposited by the method according to the invention onto Ti and CoCrMo, respectively, base metals (here = 5 ms, t_off = 15 ms, l_p = 40 mAcm^"2; layer thickness of Ag: δ = 5 pm)

- Figure 5 illustrates the results of corrosion potential measurements on different metals against a saturated calomel electrode as a function of time, performed in 0.9 wt% NaCI solution at room temperature and atmospheric pressure;

Figure 6 shows the surface of a TiAI6V4 alloy coated with an Ag layer [top line, plot (a)] using the method according to the invention and the Ti base metal surface that pops up in spots (see the areas encircled) after the corrosion of the alloy in an NaCI solution (taking place for 1 month) [top line, plot (b)], as well as the EDS spectra [bottom line, plots (a) and (b)] of said alloy at the two instants (i.e. right after the deposition and 1 month later);

Figure 7 illustrates the results of the EIS measurements performed for TiAI6V4 [plot a] and CoCrMo [plot b] implant base metal discs (electro)coated with silver in islands by the method according to the invention after storing said discs in an isotonic salt solution for 5 days;

Figure 8 shows the results of the EIS measurements carried out for non-coated base metals surface-treated by grit blasting and for Ag islands/agglomerates (elec- tro)deposited by the method according to the invention onto implant base metals

TiAI6V4 and CoCrMo after immersing said samples into an isotonic salt solution for 48 hours (the electrochemical deposition parameters in this case were: 5ms/15ms/4mAcm^"2 (for TiAI6V4) and 1 mAcm^"2 (for CoCrMo));

Figure 9 illustrates an equivalent circuit (the so-called Randies circuit) used for the evaluation of impedance plots composing part of the measurement results; Figure 10 shows the change of the calculated silver dissolution (inversely proportional to the charge transfer resistance R,) over time in the case of Ag/TiAI6V4 samples (left side plot) and Ag/CoCrMo samples (right side plot);

Figure 1 1 shows the change of the corrosion potential E(V) over time against a satu- rated calomel electrode, measured in NaCI solution for different metals coated with a non-continuous Ag layer according to the invention (i.e. coated with silver in grains/islands);

Figure 12 illustrates the antibacterial effect of TiAI6V4 alloys coated with nanosilver by the method according to the invention performed with different electrochemical process parameters (see Table 1 );

Figure 13 shows the optical and SEM images, as well as the EDX spectrum of a TiAI6V4 implant coated with nanosilver islands by the electrochemical process according to the invention;

Figure 14 illustrates the results of surface SEM inspections performed after a rapid electrochemical nanosilver deposition according to the invention onto TiAI6V4 [Figures A, B] and CoCrMo [Figure C] substrates with different electrochemical deposition parameters (A: to_n = 5 ms, t_off = 15 ms, l_p = 40 mAcm^"2, Q = 100 mCcm^"2; B: to_n = 5 ms, toff = 15 ms, l_p = 4 mAcm^"2, Q = 50 mCcm^"2; C; l_p = 1 mAcm^"2, Q = 50 mCcm^"2);

Figure 15 summarizes the results of cell viability and biocompatibility investigations carried out for different implant base metals (TiAI6V4 and CoCrMo) provided with a non-continuous silver layer after 14 days of culturing, with the indication of the average fluorescence values obtained from resazurin assay; number of samples: n=6 discs (**p<0.01 ; * p<0.05); and

Figure 16 shows the cell viability data measured after 14 days of culturing on TiAI6V4 and CoCrMo implant base metal discs not coated with silver and coated with silver using the indicated parameters; number of samples: n=6 discs (*p<0.05).

The present invention is based on the recognition that the rate of silver dissolution is determined collectively by the morphology, the grain size, the silver deposition in islands, as well as the titanium, CoCrMo and stainless steel alloys (i.e. the alloys serving as the base metal of the implants) that are suitably surface treated, that is, exhibit a sufficiently, preferably physically, in particular by grit blasting roughened surface. According to the investigations carried out, the extent of surface roughness is preferably at most 30 μπι, more preferably at most 24 μηι, wherein the "extent of surface roughness" means the height difference between the highest and the deepest point of the surface treated sur- face. The composition of the silver bath used for the electrochemical deposition performed by cathodic current pulses/direct current according to the invention is the following: potassium silver cyanide (54%) 56 gdm^"3

potassium cyanide (free of Na) 154 gdm^"3

Grundzusatz ELFIT 73 (base additive) 20 mldm^"3

Grundzusatz ELFIT 73 (light additive) 2.5 mldm^"3

T /°C/ 15-25

pH 10 .

In what follows, two preferable exemplary variants of the electrochemical deposi- tion process according to the invention are disclosed.

To perform the first variant of the method (i.e. coating a TiAI6V4 alloy with silver in islands), the average current density when applying a coating onto the titanium alloy in aqueous solution by cathodic current pulses was chosen to be 1-10 mAcm^"2. The pulse length of the cathodic current pulses is preferably 5 ms, while the length of time periods without current is preferably 5-15 ms. The current intensity of the pulse is chosen to be 4- 40 mA. The concentration of silver in the electrolytic solution is 34.94 gdm^"3, which was kept at a constant value during the electrochemical deposition by dissolving the silver anode. The electrolysis was carried out substantially at room temperature.

In the second variant of the method, silver agglomerates/islands were deposited onto a CoCrMo alloy from an electrolytic bath with a composition used in the first variant of the method. A coating with antibacterial and biocompatible properties was formed by pulsed current and direct current electrolysis using a direct current density of 1 mAcm^"2 with a charge input of 50 mCcm^"2.

During the investigations it has been concluded that to form Ag is- lands/agglomerates that are suitable from the point of view of the inventive solutions, a charge input of preferably at least about 25 mCcm^"2 and at most about 100 mCcm^"2 is required, while a charge input of 50 mCcm^"2 seems to be the optimal choice.

In vitro investigation of silver-coated implants obtained by the method according to the invention

(1) Investigation of silver dissolution

The susceptibility to spontaneous dissolution, corrosion of silver deposited onto the two different metal alloys in islands has been investigated in physiological salt solution, in 0.9 wt% NaCI solution in vitro by the EIS technique (electrochemical impedance spectroscopy) over the 100 kHz - 10 mHz frequency domain. The open circuit potential (ocp) has been measured for 1 hour before the measurements and every EIS measurement was carried out along with potential control, wherein the potential compared to a reference electrode was equal to the open circuit potential. As reference electrode Ag/AgCI has been used, while as counter electrode a Pt mesh with a high surface area was used. The evaluation of the impedance spectra and the dissolution of silver were performed on the basis of a suitably chosen electric circuit model. Water, oxygen, diffusion of the electrolyte and the galvanic microcells formed over time have all a crucial role in the degradation and corrosion of said silver islands.

Figure 7 shows the EIS plots taken after 5 days of immersing the two samples having been deposited by applying two different current densities, but equal charge inputs. Of the two samples prepared by deposition onto the TiAI6V4 alloy by the pulsed technique, the layer deposited at the smaller current pulses dissolves slower. The same tendency can be observed in the case of Ag/CoCrMo samples. In the Nyquist plot the high frequency part relates to the properties of the coating, while the low frequency part relates to the processes taking place on the electrode surface. The course of change of the EIS spectra may be clearly interpreted by the corrosion and dissolution of the coating, that provides the antibacterial activity of the samples. The deposition was performed with the parameters optimized earlier that correspond to average current densities of 1 and 10 mAcm^"2.

Figure 8 shows further comparative EIS investigations, wherein the electrochemical behavior of samples, i.e. of silver islands electrodeposited by cathodic current pulses according to the inventive method has been compared with the behavior of the base metal and a silver plate. The obtained graphs clearly show that the pure Ag plate has the greatest corrosion (the measured resistance values are lower). Contrary to this, the corrosion resistances of pure base metals are high, and they actually do not change over immersion time. In this case the Nyquist impedance spectra do not bend at the low frequency portions. Base metals behave as dissipative capacitors (the value of the exponent calculated from the Bode plots is n=0.80-0.88). EIS measurements show, that the susceptibility to dissolution of the samples prepared by the method according to the present invention falls between that of the base metal and that of the pure metallic silver plate, thereby providing the desired antibacterial property, but avoiding the risk of metallosis.

The goodness factor of the most simple equivalent circuit, the Randies circuit according to Figure 9 has been proven to be sufficient for the characterization of the boundary surface processes taking place on the galvanic granular silver and on the silver plate, as well as on the substrates, and also for the interpretation of the impedance data. Due to a surface pre-treatment, the base metal surface is rough, therefore the capacitor is substituted by a constant phase element (CPE). (Z_CPE = [Ο0^ω)^η]^"1 where Q is the non-dissipative capacity if the exponent n has a value of 1 ). The elements (R1Q1 ) of the model connected in parallel describe the charge transfer process occurring at the base metal/silver layer boundary surface; here Ri represents the polarization resistance of the Ag/base metal boundary surface, represents the capacity of the silver layer at the metal/silver boundary surface, and R_s is the resistance of the solution. The change in the calculated silver dissolution over time (which is inversely proportional to the R charge transfer resistance) is illustrated in Figure 10.

The charge transfer resistance shows a decreasing tendency over time, and after a certain time it does not change significantly. This change is more dominant in the case of the TiAI6V4 base metal, while for the CoCrMo base metal its fluctuation is larger. The decrease in the resistance corresponds to the increase in the corrosion rate according to the relation of 1/R ~ i_corr..

The silver-coated surface structure which is in contact with the electrolyte has the characteristics that for a galvanic corrosion effect to set in, one of the electrodes, the anode has to be discretely dispersed on the surface regions e.g. in the form of microclusters, and the distance between two adjacent electrodes has to be appropriate, because the potential difference generates a field strength sufficient for the continuous maintenance of a controlled silver dissolution at the desired rate and for the production of the antibacterial activity only within a suitably selected anode-cathode distance. The polarity of the contact corrosion microcells has been investigated by long term measurement of electrode potential in a NaCI solution for different contacting pairs. The results of the electrode potential measurements are shown in Figure 7.

The results of the measurements show that a certain incubation time has to pass until the start of spontaneous silver dissolution. After the incubation time, the potential values on the base metal alloys coated with silver in islands are always more positive than the potentials of pure silver, which means that silver in the silver/TiAI6V4 and sil- ver/CoCrMo galvanic cells is going to work as anode. This provides a continuous silver dissolution, i.e. an antibacterial activity of the samples. In line with the above discussion, the potentials that forms on the silver-coated samples after the incubation time are in the form of the mixed potentials of silver and the base metal.

(2) Microbiological investigations

Microbiological investigations have been carried out to determine if the novel im- plants coated with silver in islands according to the invention have bactericide effect and how the Ag-grains deposited onto the surface of the discs modify the biocompatibility of said discs, what effect they have on the growth and the viability of bone cells.

Investigation of antibacterial and antifungal effects

Pathogenic Gram positive and Gram negative strains of bacteria and fungi responsible for common infections have been selected for the investigations.

Metal discs to be investigated with a diameter of 19 mm and a thickness of 2 mm to be used as the base material for the implants have been placed in the sterile Petri dishes a to f (with a diameter of 90 mm) shown in Figure 12, and then 20 ml of Mueller-Hinton (BioMerieux) agar have been layered onto each of them. Table 1 below lists the electrochemical process parameters used to form the Ag layer on the discs.

Table 1. The electrochemical process parameters used to prepare nanosilver- coated TiAI6V4 alloys.

Metal discs layered with the agar have been left to rest at room temperature for 3 days after jellification of the agar. A suspension with the turbidity of 0.05 McF has been prepared from the microbes shown in Table 2 below, and then 10-10 μΙ of this suspension has been layered onto the solid culture medium containing the discs directly above each of the discs. As self-control, the same amount has been dropped on and spread over an area of the same size within the region of the culture medium not containing disc. The inoculated culture media have been incubated for 24 hours in a thermostat at 37°C with normal and increased (5%) C0₂ concentration atmosphere. To measure bacterial growth, after the incubation the colonies developing on the sites of incubation have been collected by a sterile loop and washed into 1 ml physiological salt solution. During this, colonies have been collected from areas above the different discs and from areas not containing discs at all. Densities of the obtained suspensions have been read at the wavelengths of 620 and 450 nm by an ELISA reader (manufacturer: Thermo Fisher Scientific Oy, Vantaa, Finland; further details: User Manual Rev. 3.4; October 2008, catalogue no. 1507300). To express the percentile extent of growth, the absorbance value obtained for the area above the discs has been divided by the absorbance value obtained for the area containing no discs at all. The measurements have been performed three times, each time carrying out three-three parallel measurements for a given strain of bacterium or fungi. The significance of the difference between the results has been determined by a post hoc test fol- lowing a variance analysis by means of the Statistica 8 software. The data obtained have been summarized in Table 2, wherein the reference numbers of the microbes as used in Figure 12 are indicated next to the names (in parentheses) of said microbes.

Table 2. Measurement results of the antimicrobial investigations.

Based on the investigations covering nine bacterial strains and one fungal species, the TiAI6V4 discs coated with Ag grains according to the invention show considerable bacterial growth impairing effect against the bacterial strains of exceptional importance in nosocomial infections (S. aureus, E. coli, K. pneumonie, P. aeruginosa); this effect is significant compared to that of the areas without discs and the non-treated TiAI6V4 discs.

Biocompatibility investigations

Prior to the experiments, the TiAI6V4 discs were placed one by one in bags made of heat-resistant foil, said bags were then sealed and subsequently sterilized in an autoclave.

For the investigation, an MC3T3-E1 (CRL-2593) mice preosteoblast cell line was used. For culturing the cells, cc-MEM (Minimum Essential Medium Eagle, Alpha Modifica- tion, Sigma) culturing liquid complemented with 10% serum (FBS, Gibco-lnvitrogen), Na- pyruvate, L-glutamine and penicillin/streptomycin solution was used. Cells have been cul- tured on the surface of different metal discs for 14 days and then cell viability was determined with resazurin. For culturing, the discs was placed into a culturing plate with 12 holes and then 1 ml cell suspension (25000 cells/ml) was measured onto each disc. The culturing liquid was changed in every 2-3 days.

At the end of the culturing period, the discs intended for resazurin assay were washed in 1.5 ml PBS (phosphate-buffer saline), then were placed into a new culturing plate with 12 holes, wherein 1.5 ml culturing liquid with resazurin (1350 μΙ culturing liquid and 150 μΙ resazurin) was applied to the cells. After 5 hours of incubation (37°C, 5% C0₂) a sample of 100 ml was pipetted from each hole onto a plate with 96 holes. Then fluores- cence was measured by a Wallac Victor fluorescent plate reader. The significance of the difference between the results obtained from cell counting and cell viability measurements was determined by a post hoc test following variance analysis using Statistica 8 software.

Cell viability investigations on TiAI6V4 and CoCrMo discs with Ag grains

Cell viability measurements have been used to compare the biocompatibility of the surfaces of TiAI6V4 and CoCrMo alloy discs according to the invention coated with Ag grains. The silver-coated discs had a diameter of 19 mm and a thickness of 1-2 mm. Cell viability data are summarized in Figure 15.

According to the measurements performed, the viability of cells was significantly high on the CoCrMo discs having surfaces with Ag grains electrodeposited by direct current (1 mA/cm²) according to the invention. High flourescence values and good cell viability values have been obtained for the TiAI6V4 and CoCrMo alloy discs having surfaces with Ag grains electrodeposited by pulsed current (4 mA/cm²) according to the invention as well.

Figure 16 shows the average of the fluorescence values obtained by a resazurin measurement performed for CoCrMo and Ti alloys with and without silver coating. After the measurement, the normalized values have been plotted, i.e. values obtained at various instants have been divided at every single time of measurement by the average obtained for the CoCrMo discs, thus values belonging to different measurements, times of measurement can be compared.

According to the thus obtained results, the TiAI6V4 discs having surfaces with Ag grains electrodeposited by pulsed current (4 mA/cm²) were the most advantageous for bone cells growth. It has also been shown that the deposition of Ag did not modify the biocompatibility of TiAI6V4 discs. In this case, for the CoCrMo discs having surfaces with Ag grains electrodeposited by direct current (1 mA/cm²), lower cell viability values but within the error limit have been obtained and the CoCrMo discs without Ag were also less advantageous for bone cells growth.

Biocompatibility measurements (cell counting and especially resazurin measurement) showed that the Ag deposition according to the invention has adverse effect on nei- ther the biocompatibility of the discs nor the viability of the cells being adhered thereon.

The most advantageous results were obtained for discs of TiAI6V4 coated with silver by pulsed current (4 mA/cm²) and for discs of CoCrMo coated with silver in islands by direct current (1 mA/cm²).

In summary:

By applying the two exemplary variants of the inventive method discussed previously, TiAI6V4 and CoCrMo implants coated with nanosilver can be produced that provide long term controlled silver dissolution, sufficient antibacterial property and biocompatibility. The advantage of the method according to the invention is that a silver coating with the desired structure can be formed at room temperature without modifying the composition of the electrolytic solution used for depositing conventional metallic silver coatings, and merely by varying the pulse parameters used for the pulse current series electrochemical deposition and a suitable pre-treatment of the substrate surface of the metallic implant.

TiAI6V4, CoCrMo and stainless steel are widely used as orthopedic prostheses. This is due to the excellent mechanical properties, corrosion resistance and biocompatibil- ity of said alloys. However the adhesion of bacteria and biofilm formation on said metallic implants can cause various infections. Removal of the biofilm and the bacteria from the surface of the metallic implant is not possible and systemic antibiotic treatment is not effective either. The problem is present and intensive research is going on to solve it. Our research towards the inventive solution was motivated partially by the reduction and the prevention of infections caused by implants. According to some literature data, infections are reduced on silver-coated medical devices and on implants coated by different physical processes and silver implants activated electrically. Considering the resistance of bacteria against antibiotics, it is thought that the implant coated with silver by a specific electrochemical process according to the present invention, i.e. pulse current series electro- chemical deposition, may provide an effective means to prevent infections caused by bacteria.

Figures 1-16 illustrate the electrochemical deposition of silver onto suitably pre- treated TiAI6V4 and CoCrMo alloys. In fact, the electrolysis has been carried out by square wave current pulses from a cyanide-containing electrolytic solution. Table 1 sum- marizes the typical parameters of the electrochemical process. The average current den- sity was 5 or 10 mA/cm². Before deposition, the oxide layer present on the surface of the substrate has been removed by sand-blasting. The change of current-efficiency has been investigated as a function of the average current density. The efficiency increases to 99% as a function of current density, then is barely changes over the average current density.

Considering that only Ag⁺ ions provide biocompatibility of the silver-coated implant, the silver layer on the surface has to dissolve spontaneously and continuously in such a way that the silver-coated titanium be antibacterial but do not cause argyria. The dissolution of silver has been tracked by different investigations. At first, the long-term, 1 month or longer, corrosion potential measurements carried out on silver-coated implants and dif- ferent substrates were preferred. Corrosion potentials of the substrates have always been more positive than that of silver or AgTi coatings. In this case a bimetallic galvanic corrosion takes place on the silver-coated metallic implants. Silver works as anode in the galvanic corrosion cell. Based on the measured potentials, it may be assumed that anodic- cathodic regions are formed in the chloride solution between the base metal and the coat- ing. The cathode process is the reduction process of the solved oxygen taking place on the free, not silver-coated portions of the titanium surface. The SEM images of Figure 6 clearly show the dissolution of silver. According to the results of EDX analysis, on silver- coated titanium surfaces that has not yet contacted with the chloride solution only silver can be identified, while on the surface of an AgTi sample stored for 1 month in a chloride solution, element analysis also showed traces of titanium. The thickness of the silver layer was 10 pm in these experiments. This is clearly visible in the cross-section image taken of the microstructure of the coating and from the results of the EDX element analysis. The rate of silver dissolution has been systematically investigated by in vitro experiments in a model solution (NaCI solution) using electrochemical impedance spectroscopy. The EIS measurements (see Figure 7) have been carried out on the corrosion potential, in the 100 kHz-10 mHz frequency domain. Impedance measurements were repeated several times as a function of time in order to gain an insight into the process of dissolution and degradation of the silver coating. The spectra have been analyzed on the basis of Nyquist and Bode plots. Results of impedance measurements showed that the corrosion rate of the samples increases significantly right after said samples have been immersed into the solution. However, as immersion time increases, the increasing tendency of dissolution gets flattened and reaches a nearly stationary value. The rate of dissolution can be increased by increasing the temperature of the solution. The rate of dissolution can also be increased by shifting the electrode potential into the anodic direction. As expected, mixing also increases the dissolution rate and the corrosion rate of the silver coating. The imped- ance response is nearly capacitive in the first two days of the immersion. Over time, as the silver-coated and non-coated parts contact the chloride electrolytic solution, the conditions for the galvanic corrosion set in and the rate of silver dissolution increases. This leads to a reduction in the diameter of the Nyquist plot, which indicates definite electrode reaction and a charge transfer process. Electrochemical properties of the silver layer have been investigated by polarization measurements and also Tafel plots, as well as polarization plots of titanium alloys coated with silver and without silver have been recorded. The corrosion current densities determined from these plots confirm the results of a solution analysis not discussed here.

The antibacterial activity and the fungicide effect of silver-coated titanium alloy discs have been investigated against three common bacterium strains and one fungus. Silver-coated samples were placed in Petri dishes after sterilization and culturing solution was poured onto the samples. This resulted in a 2-3 mm thick layer on the silver-coated samples. Said samples were left to rest for two days at 35°C after inoculating bacteria into the culture medium. If silver-coated samples prevent bacterial growth/reproduction, a clean area free of bacterial colonies has to remain on top of the samples.

Biocompatible properties of the samples have always been kept in mind throughout the experiments. Because the initial experiments based on depositing silver in a layer of 10 pm in thickness by pulse current electrolysis showed that electrochemically coated samples have an adverse effect on the growth of bone cells, in further experiments non- continuous silver coatings, i.e. silver islands were formed electrochemically on the surfaces of implants. In this case, certain parts of the base metal were free of coating which allowed the initial adhesion and a subsequent growth/reproduction of bone cells on the implant. Biocompatibility assaying of said samples clearly proved that a non-continuous (is- landed) silver layer formed on the surface of the metallic implants by means of an electrochemical process does not weaken biocompatibility of the implant.

Claims

1. A method to produce a medical metallic implant with the properties of antimicrobiality and biocompatibility, wherein an antimicrobial coating is applied onto a metallic substrate of said metallic implant, comprising the steps of

- arranging said substrate as cathode against a counter electrode in an electrolytic solution containing at least a salt of the metal to be deposited to form the coating, and

- depositing the coating from the electrolytic solution onto the substrate surface by electrolysis, characterized in that

after an initial pre-treatment of the substrate surface, the electrolysis is performed directly onto the substrate, and said coating is formed as a non-continuous nanostructured coating by pulse current series electrochemical deposition during the electrolysis.

2. The method according to claim 1 , characterized in that said surface pre- treatment is carried out by a grit blasting directed onto the surface of the substrate.

3. The method according to claim 1 or 2, characterized in that relaxation periods having a length that is at least as long as the length of the current pulses are scheduled into between the current pulses applied in the pulse current series electrochemical deposition.

4. The method according to any of claims 1 to 3, characterized in that as the salt for the metal to be deposited to form said coating a silver complex salt is used.

5. The method according to any of claims 1 to 4, characterized in that the substance of the substrate is chosen amongst biocompatible materials, and preferably from the group of titanium and its alloys, platinum, cobalt/chromium alloys, as well as stainless steel.

6. The method according to any of claims 1 to 5, characterized in that current pulses having a length of 5 ms and a current intensity of 4-40 mA are applied in the pulse current series electrochemical deposition.

7. The method according to any of claims 1 to 6, characterized in that relaxation periods with a length falling between 5 ms and 15 ms are applied in the pulse current series electrochemical deposition.

8. The method according to any of claims 1 to 7, characterized in that the average current density in the pulse current series electrochemical deposition is maintained between 1 mAcm ² and 100 mAcm^"2.

9. The method according to any of claims 1 to 8, characterized in that said non- continuous nanostructured coating is provided in the form of microclusters containing silver.

10. A medical metallic implant comprising a metallic substrate and an antimicrobial coating, characterized in that the antimicrobial coating is bound in the form of a plurality of nanostructured microclusters directly to a substrate surface subjected to pre-treatment, and said microclusters are located on said substrate so as to form local galvanic cells when get into contact with an electrolyte.

11. The metallic implant according to claim 10, characterized in that the substance of said antimicrobial coating is electronegative compared to the substance of said substrate.

12. The metallic implant according to claim 10 or 1 1 , characterized in that the mean grain size of said microclusters is at most 150 nm, preferably is at most 80 nm, most preferably is 30 nm.

13. The metallic implant according to any of claims 10 to 12, characterized in that the substrate is made of a biocompatible material, the substance of the substrate is chosen preferably from the group of titanium and its alloys, platinum, cobalt/chromium alloys, as well as stainless steel.

14. The metallic implant according to claim 13, characterized in that said substrate is made of TiAI6V4 or CoCrMo alloy or stainless steel.

15. The metallic implant according to any of claims 10 to 14, characterized in that said antimicrobial coating contains silver, preferably in the form of nanosilver.

16. The metallic implant according to claim 14 or 15, characterized in that said microclusters are present on the substrate surface in a spatial distribution ensuring a con- tinuous silver dissolution at a controlled rate when said microclusters get into contact with the electrolyte.

17. Use of a medical metallic implant produced by any of claims 1 to 9 or in accordance with any of claims 10 to 6 when implanted into a human or a non-human animal body to prevent microbial infections.