WO2017130029A1

WO2017130029A1 - Scratch resistance and corrosion behavior of nanotubular and nano-pitted anodic films on medical grade bulk titanium substrates

Info

Publication number: WO2017130029A1
Application number: PCT/IB2016/050464
Authority: WO
Inventors: Miklós WESZL; Imre Zoltán KIENTZL; Eszter BOGNÁR; Péter NAGY
Original assignee: Nanoti Limited
Priority date: 2016-01-29
Filing date: 2016-01-29
Publication date: 2017-08-03

Abstract

The present invention relates to durable, surface modified article used as medical device obtained by anodic oxidation and more particularly to a novel nanophase structure on the surface of said article and a method for producing the same.

Description

Scratch resistance and corrosion behavior of nanotubular and nano-pitted anodic films on medical grade bulk titanium substrates

Field of the invention

The present i nvention relates to durable, surface modified article used as medical device having non-phot ocatalytic antibacterial property obtained by anodic oxidation and more particularly to a novel nano-pitted nanophase film structure on the surface of said article, a method and an apparatusfor producing the same.

Background

Self-ordered, vertically oriented nanotubular titanium-dioxide (Ti0₂) arrays have proven ability to attenuate the attachment of biofilm forming pathogenic bacteria on the surface of medical grade titanium substrates, while mesenchymal stem cells and other stromal cells show improved viability on such surfaces ¹ . This phenomenon has made nanotubular Ti0₂ arrays promising candidates to enhance the biological performance of titanium bone substitutes, such as dental and orthopedic implants². The growing demand for a higher quality of life after joint and tooth replacement has become an essential requirement from the patients side, whereas the survival of those bone substitutes is compromised by the increasing incidence of implant-associated infections, recently³'⁴. The spread of antibiotic resistance among the biofilm forming bacteria exacerbates the problem that may reduce the success rate of implant revisions or even result in life-threatening complications⁵'⁶. Therefore, the development of alternative antibacterial strategies that do not contribute to the spreading antibiotic resistance, but rather prevent the occurrence of implant- associated infections has become an urgent issue not just for implant manufacturers but also for the world's health care system⁷.

It has been suggested that the anodic growth of nanotubular Ti0₂ arrays may offer a cost- effective and reliable method for the surface treatment of titanium implants so as to enhance their resistance against infections⁸'⁹. This idea is driven by the fact that through the precise control of the electrochemical process parameters homogenous nanotubular Ti0₂ arrays can be grown on titanium substrates, for instance on titanium foils¹⁰. The anodic growth of nanotubular Ti0₂ arrays can be a reliable method, provided that the process parameters are set in a suitable range that allows the production of uniform surfaces, which can be easily investigated in in vitro experimental settings, e.g. in biocompatibility and microbiology studies¹¹. The response of various mammal and bacterial cells was investigated on nanotubular Ti0₂ arrays and correlations have been demonstrated between the survival rate of the cells and the physical, chemical and physicochemical properties of the nanotubes 12 ' 13. These findings demonstrated the superiority of the anodic nanotubular Ti0₂ arrays over micro-rough implant surfaces that were created by etching, sandblasting or by the combinations thereof.

On the other hand, little is known about the mechanical resistance and the reproducibility of the anodic nanotubular Ti0₂ arrays on the surface of bulk titanium substrates in comparison to the conventional surface treatment methods, e.g. etching and sandblasting. Furthermore, there is limited information in the literature concerning the mechanical performance of the anodic nanotubular Ti0₂ arrays on the surface of 3 -dimensional implant geometries. As yet, the comparison of the mechanical integrity of the anodic nanotubular Ti0₂ arrays from practical point of view with other types of anodic films that exhibit different nanosurfaces has also been a lacking chapter in the art.

Anodization is a commonly used method for the formation of nanotubular films on titanium substrates¹⁴'¹⁵. However, the adhesion strengths of the nanotubular anodic films are usually poor that often result in the spontaneous pilling that of films that significantly limit their clinical applicability¹⁶'¹⁷'¹⁸. Various technologies have been developed to overcome the drawback but all of them have some significant weaknesses. For instance, Ti0₂ nanotubes have been anodized on laser- micromachined titanium substrate, but the improvement of adhesion was limited because of mechanical anchoring 17. Annealing treatment have also been applied to improve the interfacial structure and the adhesion between the nanotubular anodic film and Ti substrate, however the adhesion strength was strongly dependent on the film thickness and appeared relatively weak when it was thicker than 3.5 μ m¹⁹. Thermal treatment of the nanotubular anodic film in acetylene atmosphere enhanced its mechanical strength but the phase component of the film changed to

TiO_xC_z 20 that may alter both the mechanical and biological properties of the anodic film. Zhang and his co-workers developed an anodizing method that relies on the gradual sedimentation of the fluoride content of the electrolyte during the formation of nanotubular titanium oxide layer. Such way produced nanotubular oxide film showed significantly higher adhesion to the Ti substrate in scratch test compared to titanium oxide films that were growth in steady fluoride-containing electrolyte 21.

In spite of those scientific achievements under experimental conditions our own results showed that the reproduction of the nanotubular anodic films on 3 -dimensional implant surfaces is unreliable based on contact angle measurement and surface free energy calculation. The nanotubular anodic films that were produced in the same process showed higher wettability and surface free energy on the flat surface of grade 2 titanium discs than on the surface of dental screw implants. In contrast nano-pitted surfaces showed essentially similar wettability property and surface free energy on the surface of planar discs and dental implants (see Table 4 to 7 below). Furthermore, the wettability values showed remarkably lower standard deviation on the nano-pitted surfaces than on nanotublar surfaces both on discs and dental implants. These results suggests the better applicability of nano-pitted surfaces on implants than nanotubular surfaces because of the higher reproducibility in terms of physicochemical parameters, such as wettability and surface free energy.

Thus there is still room for improvements. Amongst others, there is a need for devices having improved antibacterial properties.

Therefore the object of the present invention is to solve the problems mentioned previously by providing a method and an apparatus for producing a durable nanophase structure by anodic oxidation on a surface of an article, belonging to e.g. medical implant, where the disadvantages related to fragmentation durability and reproducibility of vertically oriented nanotubes are overcome with a more durable structure, which is less likely to peel off and confer the article with preventive antibacterial effect while keeping the curative effect unchanged.

Summary of the invention

An aspect of the invention is providing a method for producing a nanophase structure on a surface of an object by anodic oxidation, the method comprising the steps of :

- electropolishing and chemical etching of an object;

- connecting the object to the anode connector;

- contacting the desired surface of an object used as anode with reaction media used as electrolyte in an electrochemical cell;

- providing a cathode and contacting with the electrolyte in an electrochemical cell;

- controlling the temperature and agitating the electrolyte;

- establishing a voltage between said anode object and said cathode for a predetermined period of time;

whereby forming nano-pitted structure

characterized in that the method is carried out firstly in a first reaction media and then in a second reaction media in separate and subsequent steps.

In a preferred embodiment of the invention the object is bulk titanium.

In a preferred embodiment of the invention the first reaction media comprise electrolytes selected from the group consisting of aqueous hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, perchloric acid, ethanol, ethylene glycol and 2 -buthoxy ethanol, preferably hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, more preferably hydrogen fluoride solution.

In a preferred embodiment of the invention the second reaction media comprise electrolytes selected from the group consisting of aqueous hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, perchloric acid, ethanol, ethylene glycol and 2-buthoxyethanol, preferably aqueous hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, more preferably hydrochloric acid.

In a preferred embodiment the first reaction media comprise hydrogen fluoride solution and the second reaction media comprise hydrochloric acid.

In a preferred embodiment of the invention the concentration of the electrolyte in the first reaction media is between 0,001 to 10 wt%, preferably 0,01 to 1 wt%, more preferably 0,1 wt% and the temperature is between -40°C and +50°C, preferably about room temperature. Preferably said electrolyte is aqueous HF solution.

In a preferred embodiment of the invention the concentration of the electrolyte in the second reaction media is between 0,1M to 10M, preferably 1M and the temperature is between -40°C and +50°C, preferably about room temperature. Said electrolyte is preferably HC1 solution.

In a preferred embodiment of the invention the cathode is contiguous, perforated or mesh, said cathode is made of stainless steel, titanium or platinum, preferably titanium and of planar or circumferential shape. If the cathode is of circumferential shape, it is arranged circumferentially around the anode and anode object.

In a preferred embodiment of the invention the voltage between the anode object and the cathode is between IV to 60V, preferably 10V to 50V, more preferably 20V to 40V in the first reaction media.

In a preferred embodiment of the invention the voltage between the anode object and the cathode is between IV to 100V, preferably 10V to 50V, more preferably 10V to 25V in the second reaction media.

In a preferred embodiment of the invention the predetermined period of time of applying a voltage between said anode object and said cathode is between 150 and 450 sec, preferably between 150 and 300 sec, more preferably between about 150 sec and 250 sec in the first reaction media.

In a preferred embodiment of the invention the predetermined period of time of applying a voltage between said anode object and said cathode is between 50 and 200 sec, preferably between 50 and 150 sec, more preferably is between about 50 sec and 100 sec in the second reaction media.

In another preferred embodiment of the invention the electropolishing is carried out in the electrochemical cell.

In another preferred embodiment of the invention electropolishing is performed at 10 - 20 °C.

In another preferred embodiment of the invention electropolishing is performed in an electrolyte comprising perchloric acid, methanol, ethanol, ethylene glycol or combination thereof.

In a preferred embodiment of the invention the electrolyte is the combination of perchloric acid, methanol, and ethylene glycol and their concentration are preferably as follows: perchloric acid ranges from 1 v/v%, to 10 v/v%, methanol ranges from 40 v/v% to 50 v/v%, ethylene glycol ranges from 45 v/v% to 55 v/v%.

In a preferred embodiment of the invention the anode object is cleaned in absolute acetone and absolute ethanol for 5-5 minutes in ultrasonic cleaner.

In another preferred embodiment of the invention the chemical etching is performed in an aqueous electrolyte comprising HF, H3PO4, or combinations thereof. The duration of the chemical etching is between 0,1 to 10 minutes, preferably 1 to 5 minutes, more preferably 2 to 4 minutes.

In a preferred embodiment of the invention the electrolyte is a combination of HF and H3PO4, and the concentration of the hydrogen fluoride is 0.01 wt% to 10 wt%, preferably 0,1 to 1 wt%, the concentration of the phosphoric acid is 0,1 to 10 wt%, preferably 0,5 to 5 wt%, and the balance is distilled water.

In a preferred embodiment of the invention the anode object is cleaned after chemical etching in distilled water for 4 min in an ultrasonic bath and further cleaned in absolute acetone and absolute ethanol for 5-5 minutes in ultrasonic cleaner.

Another aspect of the invention is providing an article comprises a nano-pitted structure on a surface of the article wherein the nano-pits has a depth of 5 nm to 10 μηι, area of 0,01 to 0,9 μηι², the surface has a distilled water contact angle of 50° - 89° and diiodo- methane contact angle of 40° - 80°, and surface free energy of 15 - 55 Nm/m, preferably depth of 10 nm to 500 nm, area of 0,6 to 0,8 μηι², distilled water contact angle of 65° - 85° and diiodo- methane contact angle of 60° - 70°, and surface free energy of 25 - 45 Nm/m.

Another aspect of the invention is providing a method for treating a patient in need of replacing, supporting or enhancing a biological structure using a medical device comprising implanting an article selected from the group consisting of the article prepared in said apparatus, the article prepared by said method, and said article, according to standard orthopedic and dental procedures.

Brief description of the drawings

Figure 1A shows an embodiment of the apparatus according to the invention,

Figure IB shows another preferred embodiment of the apparatus according to the invention with cylindrical cathode mesh,

Figure 1C shows the cross section of an embodiment of the apparatus for bulk production wherein the tank incorporates more than one electrochemical cells

Figure 2A depict the anode connector with a sealing cap

Figure 2B shows the sealing cap 10 with a neck sealer

Figure 3A shows the object to be used as anode 11 with a socket 12 for releasable connection

Figure 3B shows anode connector 14 with sealing cap 9 attached to anode object 11

Figure 4A shows an anode object 11 sealer 13

Figure 4B shows the sealer 13 attached to the anode object 11

Figure 5A shows cell arrangement of immersed anode object 11

Figure 5B shows cell arrangement of partially immersed anode object 11

Figure 6 shows an overview about the sequences and control of the surface treatment process. Panel A shows the order of the individual surface treatment steps, while panel B shows the image of soft-LCD display with the controlled process parameters.

Figure 7 shows the scanning electron microscopic images of titanium discs that were subjected to anodization according to NT-1, NT-2, NT-3 and NT-4 process parameters.

Figure 8 shows the representative electron scanning microscopic images of nano- pitted anodic films that were created by two-stage anodizing on panel A and B.

Figure 9 shows the representative scanning electron microscopic images of the surface of NT, NP and EP titanium discs after scratch resistance test. Figure 10 shows the representative scanning electron microscopic images of NT-2 and NP anodic films before (A and C) and after corrosion test (B and D), respectively.

Figure 11 shows the concentration of Ti ions that were measured in element analysis (n=10).

Figure 12 demonstrates the biocompatibility of nano-pitted surface with human bone marrow derived mesenchymal stem cells (MSCs).

Figure 13 shows a SEM image of amorphous surface.

Figure 14 shows a SEM image of irregular grain structure on the surface of an article.

Figure 15 shows another SEM image of irregular grain structure on the surface.

Detailed description of the invention

Herein is provided an electrochemical cell arrangement and a producing method to achieve a more durable nanosurface. The description also provides an article and its use in the field of medical prosthesis. The described materials, methods and examples are illustrative only and not intended to be limiting. The embodiments are disclosed by making references to drawings.

Figure 1A shows a preferred embodiment of the apparatus according to the invention. The apparatus comprises a tank 1, a cathode 2 body, an anode connector 14 and a power supply 15, wherein said tank 1 posses several joints 40, 41, 42 for a cooler, mixer, solvent exchanger respectively. Said cathode 2 may form a vessel 3 to be filled with an electrolyte solution and said vessel may also possess joints 40, 41 and 42. The cathode 2 body, the electrolyte, the anode connector 14 and the object 11 to be used as anode defines an electrochemical cell 16.

The curative effect of a nanosurface originates from anodic oxidation, thus the apparatus operates in electrolytes of organic and aqueous type, in particular in fluoride containing aqueous solution such as HF and NH₄F, perchloric acid. When the electrolyte contains fluoride ions the tank 1 incorporates the electrochemical cell 16 according to the invention is preferably made of plastic or any other material inert in the reaction of fluoride.

The tank 1 is adapted to operate in controlled temperature and, therefore, in a preferred embodiment it has a regulatory device for temperature control and a cooler (not shown). The cooler includes a heat exchanger connected to the source of the cryogenic liquid and the heat produced by the reaction is transferred to said cryogenic liquid from the electrolyte by circulating the cryogenic liquid inside the cooling system. In one embodiment of the invention the apparatus is a two electrode configuration electrochemical cell 16, wherein the reaction of interest is performed on the surface of the anode object 11.

The cathode 2 body used is inert and formed as a shape circumferentially arranged around the anode connector, preferably has a shape of a cylinder or a cone. Therefore, the cathode 2 has a vessel 3 and axis A of the circumferential vessel 3 could be defined.

In another preferred embodiment, the cathode is planar - in the form of a sheet (not shown) -, and a cell arrangement containing a planar cathode is readily understandable for a skilled person. The inert cathode 2 body used is selected from the group consisting of stainless steel, titanium and platinum. The body of the cathode 2 is discrete (continuous) or non-continuous e.g. perforated or mesh. In this case, optimal distance between the anode and the cathode can be determined by proper tests.

In a preferred embodiment the circumferentially arranged cathode 2 is discrete (continuous), has a closed bottom and said cathode 2 has the ability of retaining the reaction media. In this embodiment the cathode 2 forms a closed vessel 3, additional tank 1 is not necessary. In another preferred embodiment the circumferentially arranged cathode 2 is non- continuous thus the agitation of the bulk solvent is more uniform even in an apparatus for mass production wherein several electrochemical cell 16 is included. The internal surface of the cathode 2 body can be the negative of the geometry of the anode object 11 or it can exhibit any specific geometry.

Figure IB shows a preferred embodiment of the apparatus according to the invention wherein the cathode 2 body is a cylindrical cathode mesh. This preferable arrangement facilitates the uniform agitation of the bulk electrolyte by e.g. magnetic stirring.

Figure 1C shows the tank 1 capable of incorporating one or more electrochemical cell 16 for bulk production. The cathode 2 are shaped as a negative of the anode object 11, and separated as well insulated from each other by cathode housing 17. An object 11 is mounted to anode connector 14 for illustrative purpose, and separated from the cathode 2 by insulating ring 6.

The intensive stirring of the electrolyte can be performed using magnetic stirring, or ultrasound energy could be applied to mix the electrolyte or the electrolyte is moved intensely by an auxiliary pump through joint 41.

This invention has been carried out using a direct current - adjustable power supply 15 which connects to the electrodes through copper wires via ports 5 and any means necessary. The voltage applied can vary between IV and 300V depends on the anode. The object 11 used as anode is assembled to the anode connector 14 through releasable connections 7 (see Fig. 2A, 2B) such as screw or bolted connections, spline joints, clamp joints, keys and pins. In a preferred embodiment the joint between an object 11 to be treated and the anode connector 14 is also an electrical contact. Figure 2A and 2B depict embodiments of anode connectors 14. The anode connector 14 comprises a sealing cap which allows certain area of the surface to be excluded from the electrochemical process by covering said area and thereby insulating it. Fig 2A depicts the anode connector 14 with a sealing cap 9 to be abutted to a neck portion of the anode object 11 excludes the top of the anode object from anodic oxidation. Fig 2B shows the sealing cap 9 with neck sealer 10 mounted anode connector 14 wherein the neck portion of the anode object is also excluded from anodic process beside the top of the object. Figure 3A shows the object 11 to be used as anode with a socket 12 for releasable connection. Figure 3B shows anode connector 14 with mounted anode object 11 that has a sealing cap 9 enables the anodic oxidation of the whole lateral surface. Figure 3C depicts an anode connector 14 with mounted anode object 11 that seals the anode neck by sealing cap 9 with neck sealer 10 allowing the surface treatment only underneath. Fig 4A shows an anode object 11 sealer 13 having an internal space which is formed as a negative of the anode object 11. The mounted anode object 11 sealer 13 enables exposing a band located anywhere on the surface of the anode to the electrochemical process. Fig 4B shows the anode object 11 sealer 13 mounted to the anode object 11.

The anode connector 14 is assembled to a rotary axle 8. The axle 8 is rotatable around a rotational axis, has adjustable height position relative to the surface of the electrolyte and has adjustable angle relative to the axis A of the cathode 2 body. In an embodiment, the circular motion of the axle 8 around a rotational axis is provided by a conventional electric motor and a linear actuator creates motion in a straight line. The axle 8 could be moved along a skirt of a cone having an axis aligned with the axis of the cathode 2 body. In a preferred embodiment the axle 8 is moved along said skirt and its trajectory follows a helical path meanwhile the immersion of the anode object 11 is increasing. The movement of the axle 8 is controlled by an auxiliary instrument comprising a drive mechanism as well known by a skilled person. In a preferred embodiment the anode connector 14 is separated from the cathode 2 by an insulator ring 6 (see Fig. 1), which drives the anode connector 14 into the right position within the cathode 2 body. The aligned arrangement of axis A and axle 8 is also can be carried out by using the insulator ring 6. An object of the present invention is to describe a method for producing more durable, nano-pitted surface on an article. The anodic oxidation method comprises:

- an object is connected to the anode connector;

- controlling the agitation and the temperature of the electrolyte;

- establishing a voltage between said anode object and said cathode.

The method is carried out in two different stages each of which utilizes a different reaction media.

In some embodiments, the anode object used is selected from the group consisting of silicon, titania, zirconia, cobalt-chromium, alumina, silica, barium aluminate, barium titanate, iron oxide, and zinc oxide, nitinol, elastinite, tantalum, elgiloy, phynox, Ti6A14V, CoCr, TiC, TiN, L605, 316, MP35N, MP20N, stainless steel alloy, 316L stainless steel alloy, 304 stainless steel alloy, or combinations thereof.

In a preferred embodiment the anode object used is a titanium based bulk material. This can be pure titanium metal, a titanium alloy or a titanium coating on a carrier. In a preferred embodiment the anode object can be used as medical device manufactured to replace, support or enhance a biological structure and it can have any suitable shape or size. For example the medical device may be surface modified implant such as bone screws, cylinder implants, shaped bone prosthetics and all other types of implants for use at or near bone. In a preferred embodiment the medical device made of said titanium based material can be a stent selected from the group comprising cardiac stents, drug eluting stents, coronary stents, uretric stents and biliary stents.

In a preferred embodiment pure titanium (Grade 2) used as anode object.

Although the method disclosed herein is to be applied to articles e.g. implants or stents, the method elaborated in details comprises titanium disc as anode object 11. In the sense of the anodic oxidation process the disc used as anode object 11 is analogous to any medical devices that could be used as implants or stents. The titanium discs (Grade 2) have the following parameter: 14 mm diameter; 2 mm thickness.

Anodic oxidation is preceded by electropolishing. Electropolishing can be performed in the apparatus applied for the anodic oxidation process. The titanium disc can be connected to the apparatus and used as the anode object. The cathode body 2 and titanium samples are connected to a power supply 15 through copper wires. The titanium disc is mounted to the anode connector 14, positioned right in the center of the cylindrical cathode body 2 and immersed into electrolyte. In such a configuration the planes of the anode and the axis A of the cathode are parallel and the distance is equal between the planes (Figure 5A). Alternatively, planar cathode can be also used. The electropolishing of titanium discs is performed in the compound of perchloric acid, methanol, ethanol and ethylene glycol and at 10 - 20 °C. Various time can be applied during electropolishing in the range of 0,1 to 10 min in order to obtain smooth surface without micropits and grooves. In a preferred embodiment of the invention the electrolyte is the combination of perchloric acid, methanol, and ethylene glycol and their concentration are preferably as follows: perchloric acid ranges from 1 v/v%, to 10 v/v%„ methanol ranges from 40 v/v% to 50 v/v%, ethylene glycol ranges from 45 v/v% to 55 v/v%.. In a preferred embodiment of the invention the anode object is cleaned in absolute acetone and absolute ethanol for 5-5 minutes in ultrasonic cleaner.

Anodic oxidation is also preceded by chemical/acid etching. Chemical etching is the most commonly used treatment method on implant materials that yields micro-sized titanium-dioxide structures on the surface. Chemical etching initiates the formation of hydroxide islands on the surface that catalyze nanopore formation. The proper surface roughness facilitates further treatment procedures. In a preferred embodiment acid etching is performed in the time range of 0,1 to 10 minutes, preferably 1 to 5 minutes, more preferably 2 to 4 minutes at ambient temperature. The electrolyte is a combination of HF and H3PO4, and the concentration of the hydrogen fluoride is 0.01 wt% to 10 wt%, preferably 0,1 to 1 wt%, the concentration of the phosphoric acid is 0,1 to 10 wt%.. preferably 0,5 to 5 wt%, and the balance is distilled water. In the most preferred embodiment chemical etching of the electropolished anode object is carried out in the compound of about 0.1 wt% HF, about 1 wt% H3PO4 and distilled water in an ultrasonic bath for about 3 min at room temperature. After etching the anode object were rinsed in distilled water for 4 min in an ultrasonic bath in order to remove residual acid molecules from the surface. After rinsing the workpieces were further cleaned in absolute acetone and absolute ethanol for 5-5 minutes in ultrasonic cleaner.

The first stage of the anodic oxidation can be performed in 0,001 to 10 wt%, preferably 0,01 to 1 wt%, more preferably 0,1 wt% HF at the temperature range between 5 and 25 °C. Various time and voltage can be applied in the range between 150 and 450 sec, preferably between 150 and 300 sec, more preferably is between about 150 sec and 250 sec , and IV to 60V, preferably 10V to 50V, more preferably 20V to 40V V, respectively. In a preferred embodiment the anode object is contacted with the electrolyte and immersed completely in that. In another preferred embodiment the anodic oxidation is performed by the titanium disc laid off onto the meniscus of the electrolyte as anode object and positioned into the centre of the cathode body. This means that the anode object 11 is immersed only partially.

The second stage of the anodic oxidation can be performed in 0,1M to 10M, preferably 1M HC1 at the temperature range between 5 and 25 °C. Various time and voltage can be applied in the range between 50 and 200 sec, preferably between 50 and 150 sec, more preferably is between about 50 sec and 100 sec, and IV to 100V, preferably 10V to 50V, more preferably 10V to 25V, respectively. The anode object can be immersed completely or partially into the reaction media.

After said method carried out nano-pitted surface structure can be observed on the surface of Grade 2 and Grade 5 titanium discs.

The nano-pitted surface is characterized in that the depth of pits is 5 nm to 10 μηι, the area of pits is 0,01 to 0,9 μηι², the contact angle is 50° - 89° (distilled water) and 40° - 80° (diiodo-methane), and the surface free energy is 15 - 55 Nm/m. Preferred nano-pitted surfaces has depth of 10 nm to 500 nm, area of 0,6 to 0,8 μηι², contact angle of 65° - 85° (distilled water) and 60° - 70° (diiodo-methane), and surface free energy of 25 - 45 Nm/m. Conic section method was used to determine the contact angles of the drops, while surface free energy, polar part and disperse part were calculated. The area of pits was determined by quantitative analyses of 2-dimensional SEM images with image processing software according to known methods. The surface roughness and surface profile was measured by confocal microscopy. The structure is shown on Figure 8 A and B.

Nano-pitted structure does not imply nanotubes. Nanotube (Figure 7) can be characterized by a tubular structure that is perpendicular to the plane of the bulk material. The tubular structure can be further characterized by inner and outer walls, both having substantially circular shape, which are not applicable for nano-pitted structure.

Examples

Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the invention. However the scope of this invention is not to be in any way limited by the examples set forth herein. MATERIALS AND METHODS

Surface treatment process

Various electrochemical treatment parameters were applied to Grade 2 titanium (Createch, France) discs (h = 2 mm, 014 mm) and screw dental implants (Sanatmetal, Hungary) so as to create homogenous anodic films that exhibit either nanotubular (NT) or nano-pitted (NP) Ti0₂ features on the surface. Table 1 gives an overview on the sequence of the surface treatment steps and the range of parameters that were systematically optimized. The discs were subjected to a three-sequence surface treatment process. In the first step, the machining marks were removed by electrochemical polishing. In the second step, the polished discs were subjected to acid etching in order to initiate the formation of hydroxide islands on the surface that catalyze nanopore formation 22. In the third step, nanotubular and nano-pitted anodic films were grown on the surface of the discs (Figure 6A). In the following the final parameter sets and experimental set-ups were further detailed that had been applied for the production of the test samples.

Electrochemical polishing

The electrochemical polishing was carried out in a two-electrode setup (anode-cathode distance was 5 mm) by using a DC power source (Elektro-Automatik, EA-PS 8080-40) applying 30V for 35 sec in a steady electrolyte flow with 0.11/min velocity using a thermoplastic mag drive centrifugal pump (HTM6 PP, GemmeCotti), while the temperature of the electrolyte was kept at 15 °C. The cathode is planar. As electrolyte, the compound of CH₃OH, C₂H₄(OH)₂, HC10₄ (Molar Chemicals) was used (Table 2, 3). The composition of the electrolyte is 390 ml CH₃OH, 350 ml C₂H₄(OH)₂ , and 60 ml HCI0₄.

Chemical etching

The chemical etching of the electropolished workpieces was carried out in the compound of 0.1 wt% HF, 1 wt% H₃P0₄ and distilled water (Molar Chemicals) in an ultrasonic bath for 3 min at room temperature. After etching the workpieces were rinsed in distilled water for 4 min in an ultrasonic bath in order to remove residual acid molecules from the surface. After rinsing the workpieces were further cleaned in absolute acetone and absolute ethanol for 5-5 minutes in ultrasonic cleaner (Table 2, 3).

Anodization The anodization of the electrochemically polished and etched workpieces was carried out in a two-electrode electrochemical reactor using a continuous direct power supply (Elektro-Automatik, EA-PS 8360-15 2U). Planar cathode was used in this experiment. The anodizing parameters of NT samples are given explicitly in Table 2. The anodizing parameters of NP samples are 20V voltage, duration is 180 sec and the electrolyte is 0.1 wt% HF (first stage, first reaction media). The anode objects (NP and NT samples) were cleaned in an ultrasonic cleaner.

The parameters of the second stage anodization are as follows: duration of 60 sec, voltage of 14 V, and the second reaction media is 1M HC1 (Table 3).

Example 1

If the anodization time is less than 150 sec in the first reaction media and less than 50 sec in the second reaction media than amorphous features growth on the surface of the titanium substrate that is shown on Figure 13. Similar irregular surface structures appear, if the substrate is not subjected to etching in the compound of HF and H₃P0₄ before anodizing.

Example 2

If the titanium substrate is not subjected to electrochemical polishing and acid etching according to the abovementioned protocol and subjected to anodizing in HF electrolyte applying 20V then irregular grain sructures develop on the surface as it is shown on Figure 14.

Example 3

If the titanium substrate is not subjected to electrochemical polishing and acid etching according to the abovementioned protocol and subjected to anodizing in HF electrolyte applying 60V instead of 20V similar irregular grain structures develop on the surface as it is shown on Figure 15. The findings of Example 2 and example 3 suggest that the preparation of the titanium substrate by etching and/or electrochemical polishing is necessary to grow homogeneous, self-ordered nano-pitted anodic film.

Example 4

Nano-pitted structure is developed on titanium substrate (anode object) subjected to the method according to Table 3. The respective SEM image is shown on Figure 8.

Comparative examples

Experimental design One optimal NT forming and one optimal NP forming protocol were selected for further mechanical and microscopic investigations. The mechanical characteristics of the NT and the NP anodic films were assessed from practical point of view first on the surface of discs. Then the selected types of the films were further assessed on the surface of dental implants. The anodic films were subjected to scratch resistance and corrosion tests on the surface of discs, while they were investigated in screwing tests on the surface of dental implants. Concerning discs, the NT and NP surfaces were compared to electrochemically polished (EP) and a sandblasted/acid etched (SBAE) reference surfaces (Table 8). The sandblasting of the discs was performed by KLS Martin GmbH (Freiburg, Germany), while the acid etching was carried out in a subsequent step according our own protocol (Table 8). Concerning implants, the NT and NP surfaces were compared to each other in screwing tests without external reference surface. In the following the investigation methods will be detailed.

Microscopy

Stereomicroscopic (Olympus SZX16, Pennsylvania, United States) and scanning electron microscopic (Philips XL 30, Zagreb, Croatia) images were taken of the titanium discs and dental screw implants in order to investigate the anodic films.

Scratch resistance test

The mechanical integrity and faiure mode of NP and NT anodic films were investigated on the surface of discs in scratch resistance test. A tensile testing machine (INSTRON 5965 (5 kN) with a high-performance pneumatic wedge grip with 2 kN lateral force capacity) was used to perform the tests by making 5 scratches on the surface of the titanium discs. A custom-made martensitic stainless steel stylus was produced by the 90° bending of a commercially available tweezers (VetusTweezers). The quantitative set of the normal load was not possible in this setting; however, the achievement of identical normal loads was attempted by the fixation of the disc and stylus in the same position relative to each other in all measurements through the lateral adjustment of the lower and the upper wedge grips of the tensile testing machine. The stylus had a tapered head with 10 μ m radius. The displacement rate of the stylus was 100 μ m/sec. During the measurements the lateral load-displacement diagram was recorded by BlueHill 3 software (Materials Testing Software, Instron, Norwood, MA, USA) (data not shown). As reference, electrochemically polished discs were prepared as it was described above. Screwing test

The mechanical integrity and failure mode of NP and NT anodic films were investigated on the surface of dental implants in screwing test. Solid rigid polyurethane foam blocks (Sawbones®) were used to simulate trabecular and cortical bone density types. According to the instructions of the implant manufacturer cylindrical holes were drilled in advance into the foam blocks in order to reduce the mechanical stress that occurs during the driving of the screw. Scanning electron microscopic (SEM) and stereomicroscopic images were taken from the surface of the implants before and after the screwing test.

Corrosion test

The corrosion test of the NT-2 and NP anodic films on the surface of discs was performed in 10 parallel measurements by the static immersion method in accord with ISO 10271:2011 "Corrosion test methods for metallic materials". In the first step, the surface of the discs was cleaned ultrasonically in ethanol for 5 minutes then dried with compressed air. The individual specimens were investigated in 10 separate containers that were filled with the aqueous solution of 0.1 mol/1 sodium chloride and 0,1 mol/1 lactic acid, resulting in a pH value of 2.29. The containers were sealed and kept at 37°C for 7 days. The pH of the residual and reference solutions was recorded after the 7 days incubation period by a pH meter (Voltcraft PHT-02 ATC). The calculated disc area was 3.96 cm , which was related to the electrochemically polished surface and did not implicate the surface augmentation owing to nano-, and micropores. As reference, sandblasted/etched (SBAE) and electrochemically polished (EP) discs were used.

Element analysis

An inductively coupled plasma optical emission spectrometer (ICP-OES, Plasmalab) with 40-channel analyzer (Labtest) was applied to measure the concentration of the dissolved titanium ions in the aqueous chloride medium that was used for corrosion testing. After the 7 days incubation period samples were taken from the supernatant and analyzed. Three (3) samples were taken from each container for spectrophotometric analysis in three consecutive measurements. The means of the measured concentrations were calculated along with standard deviations. As reference, the titanium ion content of a blank solution was determined in three measurements and the mean (0.93 mg/1) was calculated, which was deducted from the means of the test solutions.

Statistical analysis One-way ANOVA analysis was performed (Tukey's post hoc test) in order to investigate the difference in the concentrations of dissolved Ti ions that were measured in element analysis concerning the NT and NP anodic films and the reference surfaces. A p value < 0.05 was considered significant.

In vitro bacterial adhesion test

Anodized and control samples were γ-sterilized (25 kGy) by an accredited service provider before in vitro microbiology tests. The samples were stored in ABS rack that assured the mechanical protection and prevented the contamination of the samples during the transportation and storage. The surfaces were tested for bacterial adhesion and biofilm formation of Streptococcus sanguinis. As reference surfaces electrochemically polished and sandblasted/etched (provided by KLS Martin) titanium samples were analyzed, as well. The γ-sterilized samples were coated with human saliva for 4 h at 37 °C and then inoculated with 2.5 x 106 cells/mL of S. sanguinis. After 24 h static cultivation at 37 °C the attached cells were detected using fluorescence in situ hybridization (FISH). The biofilm covered surface [%] was calculated using the software Fiji (ImageJ, Particle Analysis).

Experimental groups in bacterial adhesion tests

Nano-pitted anodic film was tested against four types of nanoporous anodic films (TOA_l, TOA_2, TOA_3, TOA_4). The TO A abbreviation referrers to the type of anodization in terms of applied process parameters. As reference sandblasted/etched (provided by the company of KLS Martin) and electrochemically polished surfaces were used.

Contact angle measurement

Distilled water and diiodo -methane were used as test fluids for contact angle measurements on the surface of Grade 2 titanium discs and screw implants using drop shape analyzer (Kriiss, DSA25). The measurement was performed on the surfaces that had been produced 3 months before the experiment and were stored in non-sealing containers under ambient atmosphere. The surfaces were not subjected to UV irradiation or any other manipulation before contact angle measurement. The measurement started after 3 sec of dropping. Conic section method was used to determine the contact angles of the drops, while surface free energy, polar part and disperse part were calculated.

RESULTS Homogenous nanotubular arrays appeared on the surface of discs that were anodized according to NT-1, NT-2 and NT-3 parameters, whereas the nanotubes slightly deformed when NT- 4 parameters were applied (Figure 7). The reproducibility of the various NT anodic films showed considerable variance that often detrimentally affected of the surface quality, which was manifested either in spontaneous peeling or in the emergence of crevices (data not shown). The growth of nanotubular anodic film according to NT-2 process parameters showed the highest reliability compared to other NT anodizing parameters, thus the NT-2 surface was selected for further investigations as a representative nanotubular anodic film (Table 8). On the other hand, the growth of NP anodic film through two-stage anodization showed high reliability that allowed reproducing essentially similar NP surfaces (Figure 8).

Significant difference was revealed in the mechanical integrity of NT-2 and NP anodic films in the screwing test. The NT-2 films exfoliated from the surface of the screw dental implants when they were introduced either into trabecular or cortical bone density foam blocks. In contrast, the NP anodic film remained intact on the surface of the dental implants even in the cortical bone density foam block (data not shown).

The scratch resistance tests revealed the tendency of NT anodic films to flake off when ~ 2N lateral force was applied to the surfaces of the discs. Contrarily, the NP anodic films did not exfoliate when the same lateral force was applied to them but moderate sideward and terminal material extrusion appeared on the track of the stylus (Figure 9).

The corrosion resistance of NT-2 anodic films was significantly lower (concentration of dissolved Ti ions: 23.89 ± 6.7 mg/ml; p* < 0.001) than that of NP anodic films (concentration of dissolved Ti ions: 1.11 ± 0.77 mg/ml) and reference surfaces. After corrosion test slight recesses appeared on the surface of NT-2 that disintegrated the vertical homogeneity of the nanotubular arrays (Figure 10). On the other hand, the corrosion behavior of NP anodic films was essentially similar to that of the sandblasted/acid etched Ti0₂ surfaces that exhibit micro-rough features.

Interestingly, there was not credible difference between the concentrations of dissolved titanium ions concerning the electrochemically polished (concentration of dissolved Ti ions: 0.343 ± 0.009 mg/ml) and sandblasted/acid etched surfaces (concentration of dissolved Ti ions: 0.589 ± 0.463 mg/ml), and the NP anodic films (Figure 11).

Results of the bacterial adhesion test

The results of the first experiment are shown on the bar diagram below (darker bars). Sandblasted/etched samples were most covered with bacteria (49 ± 5 %). The electrochemically polished reference surface showed more biofilm coverage (25 ± 1 %) than the anodized surfaces (TOA_l : 11 ± 6%; TOA_2: 10 ± 2 %; TOA_3 : 19 ± 1 %; TOA_4: 17 ± 7%). The results of the second experiment (brighter bar) show no difference between electropolished and anodized surfaces (electropolished: 15 ± 1 %; nano-pitted anodic film: 17 ± 3 %; TOA_2: 17 ± 1 %; TOA_3 : 16 ± 3 %; TOA 4: 16 ± 1 %). In The summary, the nano-pitted anodic film attenuated the attachment of S. sanguinis as well as nanoporous anodic film compared to sandblasted/etched surface.

eSeetropo!isfi-ed nsno-pitieiJ TOA.,1 TO A 2 TOA.,3 TOA., samJtilfisle<i%tctse-l

Analysis of bacterial adhesion and biofilm formation of Streptococcus sanguinis (biofilm covered surface [%]) on titanium discs. So far, two rounds of bacterial adhesion tests were performed. The nano-pitted surfaces were tested only in the second round of experiments, while TOA_l were excluded from the second round that explains why single bars belong to those two experimental groups.

Figure 12 demonstrates the biocompatibility of nano-pitted surface with human bone marrow derived mesenchymal stem cells (MSCs).. As reference electrochemically polished, sandblasted/etched and nanoporous Ti02 surfaces were used. Panel A shows electrochemically polished surface with MSCs that exhibit healthy (plane) morphology. Panel B shows nanoporous surface, panel C shows nano-pitted surface and panel D shows sandblasted/etched surface with healthy MSCs.

DISCUSSION

Our results show that the growth of homogenous, self-ordered, vertically oriented nanotubular Ti0₂ anodic films on the surface of bulk titanium substrates is particularly sensitive to the anodizing process parameters that detrimentally affects their reproducibility. The low reproducibility of the nanotubular anodic films was manifested in their weak mechanical integrity that resulted either in spontaneous peeling or in exfoliation when moderate lateral force was applied to the surfaces. Contrarily, the NP anodic film was highly reproducible in a two-stage anodizing process. Both in its mechanical integrity and corrosion resistance the NP anodic film outperformed the NT-2 film. Our findings suggest that the applicability of nanotubular anodic films on the surface of titanium bone substitutes may be limited because of some mechanical considerations. On the other hand, it is worth to consider the applicability of nano-pitted surfaces over nanotubular anodic films for the purpose of the enhancement of bone substitutes, such as dental implants.

The mechanical integrity of the surface of a titanium bone substitute is essential so as to achieve its intended biological performance 23. However, it is very difficult to grow a nanotubular anodic film that shows homogenous microscopic appearance and mechanical integrity even through the systematic optimization of the anodizing parameters 24. Among the various anodizing parameters the temperature was found to be the most critical in our setup that affected the practical adhesion of the nanotubular anodic films to the bulk titanium substrates. Interestingly, the local temperature had to be kept between 5-8°C in the close proximity of the anodic workpiece when NT-2 surfaces were grown, otherwise spontaneous peeling of the anodic films occurred. However, in spite of the deliberate control of the process parameters of anodizing the NT-2 anodic film easily exfoliated even when moderate forces were applied to the surface. In this study we did not investigate the underlying reason of the low mechanical resistance of nanotubular anodic films but it has been reported by other authors that the detachment of such anodic films may be associated with the formation of a titanium fluoride layer between the oxide film and the metal substrate. According to Habasaki and his co-workers the development of the titanium fluoride layer might be the result of the fast inward migration of fluoride ions during anodic film growth under high electric field 25. Our findings might be supported by the hypothesis of Habasaki, if we consider the anodizing parameters under the NP and NT-2 films were grown. In the first sequence of the anodic growth of NP films considerably lower fluoride ion concentration and field force was applied for shorter time then in the case of the NT-2 films. The lower exposure to fluoride ions may reduced the rate of the development of the titanium fluoride layer between the NP anodic film and the titanium substrate, which may prevented the detachment of the NP films.

On the other hand, the development of a titanium-fluoride layer between the anodic film and the titanium substrate does not explain the significant differences in the corrosion behavior of the NP and NT-2 films. Presumably, the difference in the electric properties of the NP and NT-2 anodic films is responsible for the different corrosion resistance, however this hypothesis should be confirmed in further experiments in the future. The intense Ti ion dissolution and the microscopic appearance of NT-2 anodic films after the corrosion test suggested that crevice corrosion occurred. From practical point-of-view, the intense corrosion would result in the corrosion fatigue, whereas the exfoliation of the anodic film may reduce the biological performance of an implant.

The high sensitivity of the surface quality of the nanotubular anodic films to the variance of the anodizing parameters may extremely increase the complexity of the process validation, which may cause undue costs for an implant manufacturer. The validation of critical processes in the production line is a general requirement for the European and American implant manufacturers and the enforcement of this requirement has become more imperative, recently²⁶. However, our findings suggest that the reproducibility and the mechanical integrity of the nanotubular anodic films on 3- dimensional implant geometries is low, if the conventional anodizing parameters are applied, e.g. continuous direct current is applied in aqueous fluoride containing media for minutes or hours. On the other hand, our results do not imply any manner the non-existence of a parameter set that is suitable to create nanotubular anodic film on the surface of bulk titanium substrates. Nevertheless, the improvement of anodizing processes may lead to new nanosurfaces with physical characteristics that are more suitable to enhance the biological performance of titanium bone substitutes than the currently known nanotubular anodic films. Our novel two-sequence anodizing process supports this hypothesis, given that it is a robust method that yields reproducible nano-pitted anodic films with good mechanical integrity and corrosion resistance.

TABLES

Electrochemical polishing

Anode-cathode distance: 5 - 65 mm

Anode-cathode configuration

Cathode geometry: planar, cylindrical mesh

316L stainless steel, grade 2 titanium, aluminum,

Material of the cathode

magnesium

Struers All solution, and

Electrolytes*

CH₃OH + C₂H₄(OH)₂ + HCIO₄

Voltage 10V - 80V

Temperature - 40°C - (+ 40°C)

Stirring: 100 - 1000 rpm, or

Agitation

Laminar flow: 0.1 - 0.51 min

Time 10 sec - 1200 sec

Etching HC1, H₃PO₄, (COOH)₂x2H₂0 + H₂0₂,

Etchants*

HF + H₃PO₄ + dH₂0

Temperature 20°C - 60°C

Agitation Ultrasonic

Time 30 sec - 1200 sec

Anodization

Anode-cathode distance: 3 - 65 mm

Anode-cathode configuration

Cathode geometry: planar, cylindrical mesh

Material of the cathode 316L stainless steel, grade 2 titanium

HF, HC1, H₃PO₄,

Electrolyte* NH₄F + H₂0 + C₂H₄(OH)₂,

NH₄F + H₂0 + C₃H₈0₂

Voltage 20V - 100V

Temperature - 40°C - (+ 50°C)

Stirring 100 - 1000 rpm, laminar flow 0.1 -

Agitation

0.51 min, no agitation

Time 200 sec - 3600 sec

Table 1

Table 2 Raw material Grade 2 titanium discs (14x2 mm)

Cleaning Absolute acetone (5 min) and absolute ethanol (5 min) in an ultrasonic cleaner

Electropolishing

Voltage 30V

Time 35 sec

Electrolyte Solution: 390 ml CH₃OH + 350 ml

C₂H₄(OH)₂ + 60 ml HCI0₄

Chemical etching

Ethcing compound 0.1 wt% HF + 1 wt% H3PO4 + _dH₂0

Ecthing time 3 minutes in an ultrasonic bath

Cleaning Distilled water (4 min) in an ultrasonic cleaner

Anodization 1.

Voltage 20V

Time 180 sec

Electrolyte 0.1 wt% HF

Cleaning Distilled water (10 min) in an ultrasonic cleaner

Anodization II.

Voltage 14V

Time 60 sec

Electrolyte 1M HCI

Cleaning Distilled water (10 min) in an ultrasonic cleaner

Table 3

NANOTUBULAR ANODIC FILM ON PLANAR DISCS

Water (n=10) diiodo-methane (n=10) middle edges middle edges

Representative ! f ?

⁹ images/

parameters OMOi u 'fifiill" ^

left angle (°) 42, 13±15 38, 1 l±15 38, 10±17 30, 16±15 right angle (°) 41,51±15 38,38±14 38,96±15 31,81±14 average angle 41,82±15,48 38,24±14,83 38,53±16,24 30,99±15, 13 edges 66,59±14,78

SFE (mN/m)

middle 62,84±17,54 disperse edges 43,81±6,41

(mN/m) middle 40,34±7,99

edges 22,78±8,36 polar (mN/m)

middle 22,50±9,55

Table 4

Table 5

Table 6 NANO-PITTED ANODIC FILM ON IMPLANT

Water (n=6) diiodo-methane (n=6)

Representative

images/

parameters

eft angle (°) 75,43±7,50 64,22±5,05 right angle (°) 76,29±6,95 68,79±6, 19 average angle 75,86±7, 14 66,50±5,27

SFE (mN/m) 34,35±6,98

disperse (mN/m) 24,85±2,99

polar (mN/m) 9,50±3,99

Table 7

Table 8 REFERENCES

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Claims

1. A method for producing a nanophase structure on a surface of an object (11) by anodic oxidation, the method comprising the steps of:

- electropolishing and chemical etching of an object (11);

- connecting the object to the anode connector;

- contacting the desired surface of an object (11) used as anode with reaction media used as electrolyte in an electrochemical cell (16);

- providing a cathode (2) arranged circumferentially around the anode and contacting with the electrolyte in an electrochemical cell (16);

- controlling the temperature and agitating the electrolyte;

- establishing a voltage between said anode object (11) and said cathode (2) for a predetermined period of time; whereby forming nano-pitted structure

characterized in that the method is carried out firstly in a first reaction media and then in a second reaction media in separate, subsequent steps.

2. The method according to claim 1 wherein the anode object is bulk titanium.

3. The method according to claim 1 or 2 wherein the first reaction media comprise electrolytes selected from the group consisting of aqueous hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, perchloric acid, ethanol, ethylene glycol and 2-buthoxyethanol, preferably hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, more preferably hydrogen fluoride solution; and the second reaction media comprise electrolytes selected from the group consisting of aqueous hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, perchloric acid, ethanol, ethylene glycol and 2-buthoxyethanol, preferably aqueous hydrogen fluoride solution, ammonium fluoride solution, phosphoric acid, hydrochloric acid, more preferably hydrochloric acid.

4. The method according to claim 3 wherein the first reaction media comprise hydrogen fluoride solution and the second reaction media comprise hydrochloric acid.

5. The method according to any of claim 1 to 4, wherein the HF concentration of the first reaction media comprising HF electrolyte is between 0,001 to 10 wt%, preferably 0,01 to 1 wt%, more preferably 0,1 wt% and the temperature is between -40°C and +50°C, preferably about room temperature.

6. The method according to any of claim 1 to 5, wherein the HC1 concentration of the second reaction media comprising HC1 electrolyte is between 0,1M to 10M, preferably 1M and the temperature is between -40°C and +50°C, preferably about room temperature.

7. The method according to any of claim 1 to 6, wherein the cathode (2) is continuous, perforated or mesh, said cathode is made of stainless steel, titanium or platinum.

8. The method according to any of claim 1 to 7, wherein the voltage between said anode object (11) and said cathode (2) is between IV to 60V, preferably 10V to 50V, more preferably 20V to 40V in the first reaction media.

9. The method according to any of claim 1 to 8, wherein the voltage between said anode object (11) and said cathode (2) is between IV to 100V, preferably 10V to 50V, more preferably 10V to 25V in the second reaction media.

10. The method according to any of claim 1 to 9, wherein the predetermined period of time of applying a voltage between said anode object and said cathode is between 150 and 450 sec, preferably between 150 and 300 sec, more preferably between about 150 sec and 250 sec in the first reaction media.

11. The method according to any of claim 1 to 10, wherein the predetermined period of time of applying a voltage between said anode object and said cathode is between 50 and 200 sec, preferably between 50 and 150 sec, more preferably is between about 50 sec and 100 sec in the second reaction media.

12. The method according to any of claim 1 to 11, wherein the electropolishing is performed at 10 - 20 °C in an electrolyte comprising perchloric acid, methanol, ethanol, ethylene glycol or combination thereof.

13. The method according to claim 12, wherein the electrolyte is the combination of perchloric acid, methanol, and ethylene glycol and their concentration are preferably as follows: perchloric acid ranges from 1 v/v%, to 10 v/v%, methanol ranges from 40 v/v% to 50 v/v%, ethylene glycol ranges from 45 v/v% to 55 v/v%.

14. The method according to any of claim 1 to 13, wherein the chemical etching is performed in an aqueous electrolyte comprising HF, H₃P0₄, or combinations thereof, and the duration of the chemical etching is between 0,1 to 10 minutes, preferably 1 to 5 minutes, more preferably 2 to 4 minutes.

15. The method according to any of claim 14, wherein the aqueous electrolyte is a combination of HF and H₃PO₄, and the concentration of the hydrogen fluoride is 0.01 wt% to 10 wt%, preferably 0,1 to 1 wt%, the concentration of the phosphoric acid is 0, 1 to 10 wt%, preferably 0,5 to 5 wt%,

16. An article comprising a nano -pitted structure on a surface of the article wherein the nano-pits has a depth of 5 nm to 10 μιη, area of 0,01 to 0,9 μπι², and the surface has a distilled water contact angle of 50° - 89° and diiodo-methane contact angle of 40° - 80°, and surface free energy of 15 - 55 Nm/m.

17. Article according to claim 16, wherein the nano-pits has a depth of 10 nm to 500 nm, area of 0,6 to 0,8 μπι², and the surface has a distilled water contact angle of 65° - 85° and diiodo-methane contact angle of 60° - 70°, and surface free energy of 25 - 45 Nm/m.

18. Article obtainable by the method according to any of claim 1 to 15.

19. A method for treating a patient in need of replacing, supporting or enhancing a biological structure using a medical device comprising implanting an article selected from the group consisting of the article prepared by the method according to any of claim 1 to 15, and the article according to claim 17 or 18, according to standard orthopedic and dental procedures.