UV-CURABLE, BIOCOMPATIBLE, SUPERAMPHIPHOBIC COATING

Technical Field

The present disclosure is related to medical device and biomaterial coatings.

The present disclosure is particularly related to a coating which has a superamphiphobic character providing both superoleophobicity and superhydrophobicity for coating medical device and biomaterial surfaces, is biocompatible as determined in vitro and in vivo, is curable with UV rays and has a unique chemical composition.

Prior Art

Medical device and biomaterial coatings are one of the most important application areas of the coating sector. Today, the coatings produced in current applications generally have a hydrophobic character. When the coating process for biomaterials is examined, it is seen that both superhydrophobicity and superoleophobicity are used as an important character. It is certain that the adhesion of non-polar body fluids and solutions containing protein on the biomaterial would have an adverse effect on the performance of surgical interventions and/or implants. In the current technique, similar hydrophobic structures have been investigated only as a chemical composition but they are not interpreted in the perspective as application. Therefore, studies are made by considering such structure as a compound, but not as a coating. Since they are investigated only in terms of the compound, there is no information on the metal coating performance of structures which can exhibit both superhydrophobic and superoleophobic character. The failure of current applications included in the superhydrophobic coating class to have biocompatibility character is one of the disadvantages. Selection of medical devices and biomaterials as the application area requires the coating to exhibit a biocompatible behavior.

In the literature, the following references have been found in the researches made on the subject matter.

In the patent application No. EP2900768B1 titled“Durable superhydrophobic coatings”, the invention is related to superhydrophobic coatings and particularly to superhydrophobic coatings containing particles which can attach covalently to various surfaces. The said invention is a superhydrophobic coating which contains a plurality of particles and a resin. Particles attach to the resin covalently and the resin does not fill up the pores of superhydrophobic particles, thus the three dimensional surface topology of superhydrophobic particles is maintained.

In the patent application No. TR 2004 00368 titled “Superhydrophobic polypropylene film or coating method”, the invention is related to a method for producing superhydrophobic film and coating from polypropylene plastic, and to a film and coating produced by using this method. The method applied to achieve the objectives of this invention essentially consists of the stages of dissolving isotactic polypropylene in a solvent at a certain temperature; pouring this solution onto the material to be coated in a certain temperature range and vaporizing the solution, and drying the coating completely. When the said patents are examined, it is seen that proposed coatings only provides the superhydrophobic character. For the coating of medical devices or biomaterials, no coating type could be found which provides both superhydrophobic and superoleophobic character at the same time, has a superamphiphobic character and is biocompatible as determined both in vitro and in vivo.

Consequently, it is required to engage into a new development in the related technical field due to above- mentioned problems and insufficiency of current solutions on the issue.

Aim of the Invention

The present disclosure is related to UV-curable, biocompatible, superamphiphobic coating which meet the aforementioned requirements, eliminate all disadvantages and bring some additional advantages, and synthesis method for such compounds.

The primary aim of the invention is to provide a coating which has a superamphiphobic character providing both superoleophobicity and superhydrophobicity for coating medical device and biomaterial surfaces, is biocompatible as determined in vitro and in vivo, is curable with UV rays and has a unique chemical composition.

An aim of the invention is to provide superhydrophobic and superoleophobic character to medical devices and biomaterials through its superamphiphobic character.

Another aim of the invention is to provide a coating which is curable with UV rays in a very short time for coating medical devices and biomaterials.

Another aim of the invention is to provide medical devices and biomaterials with biocompatible character thanks to its biocompatible character.

Another aim of the invention is to prevent adhesion of such biomaterials to be used inside the body as implants to the live tissue, therefore allow them to be placed and removed without causing any damage on the tissue, thus shorten the treatment and recovery period by means of its superamphiphobic character.

In order to achieve the aims described above, the invention is a UV-curable coating composition providing medical device or biomaterial surfaces with superamphiphobic and biocompatible character, wherein it contains,

• 25-80 % (w/w) of fluorous epoxy acrylate,

• 8-25 % (w/w) of hydrolyzed tetraethoxysilane or hydrolyzed triethoxy isobutylsilane,

• 10-60 % (w/w) of 1 ,6-hexanediol diacrylate,

• 1 -8 % (w/w) of 1 -hydroxycyclohexyl phenyl ketone or trimethylsulfonium hydroxide.

The invention is a method of coating with the mentioned coating composition wherein it consists of the following process steps:

a) mixing 25-80 % (w/w) of fluorous epoxy acrylate, 8-25 % (w/w) of hydrolyzed tetraethoxysilane or hydrolyzed triethoxy isobutylsilane, 10-60 % (w/w) of 1 ,6-hexanediol diacrylate, and 1 -8 % (w/w) of 1 -hydroxycyclohexyl phenyl ketone or trimethylsulfonium hydroxide until obtaining a homogeneous mixture, b) coating the medical device or biomaterial surface with the resulting mixture,

c) exposing the coated surface to UV light in order to cure the coating.

The invention also includes medical devices or biomaterials coated with the mentioned method.

Structural and characteristic specifications as well as all advantages of the invention will be understood more clearly with the figures provided below and the detailed description written with reference to these figures, and thus, evaluation should be made by considering these figures and detailed description.

Figures to Help to Clarify the Invention

Figure 1 shows the angles of contact of unprocessed glass samples (a, b, c); coated glass samples (d, e, f); unprocessed CP-Ti samples (g, h, i); coated CP-Ti samples with water, ethylene glycol and hexadecane, respectively.

Figure 2 shows FTIR spectrums of the coating prepared for the method of curing with UV.

Figure 3 shows NMR result of the coating prepared for the method of curing with UV.

Figure 4 shows XPS spectrums of the coating prepared for the method of curing with UV.

Figure 5 shows 3D surface profiles of (a) unprocessed titanium, (b) superamphiphobic polymeric film.

Figure 6 shows (a) 2000X surface morphology of unprocessed CP-Ti, superamphiphobic polymeric structure; (b) 5000X surface morphology of unprocessed CP-Ti, superamphiphobic polymeric structure; (c) sectional SEM views of superamphiphobic polymeric film after UV coating.

Figure 7 is a graphic showing EDS analysis results of polymeric coated CP-Ti film.

Figure 8 shows optical light conductivity UV-vis spectrums of unprocessed and superhydrophobic polymeric coated glass material.

Figure 9 is a graphic showing OCP curve of unprocessed and polymeric coated CP-Ti material. Figure 10 is a graphic showing potentiodynamic polarization curve of unprocessed and superamphiphobic polymeric coated CP-Ti material.

Figure 1 1 shows SEM views of (a) unprocessed CP-Ti, (b) superamphiphobic polymeric coated CP- Ti material after corrosion.

Figure 12 shows graphics of (a) Nyquist curves (unprocessed and polymeric coating); (b) Bode curves (unprocessed and polymeric coating). Figure 13 shows graphics of (a) viability levels observed in 3T3 fibroblast cells at the end of 24 hours of incubation; (b) viability levels observed in 3T3 fibroblast cells at the end of 48 hours of incubation.

Figure 14 shows A- Unprocessed Glass group - Severe neutrophil leukocyte infiltration ( ^* ); B- Unprocessed Ti group - Severe fibroblast presence (arrow head); C- Polymeric coated Ti group - Medium level fibroblast presence (arrow head); D- Polymeric coated glass group - Medium level fibroblast presence (arrow head).

Figure 15 shows A- Unprocessed Glass group; B- Unprocessed Ti group - Low level; C- Polymeric coated Ti group - Low level; D- UV glass group - Low level, Collagen type I presence ( ^* ).

Figure 16 shows A- Unprocessed Glass group - Low level; B- Unprocessed Ti group - Medium level; C- Polymeric coated Ti group - Medium level; D-UV glass group - Medium level Collagen type III presence ( ^* ).

Detailed Description of the Invention

In this detailed description, UV-curable, biocompatible, superamphiphobic coating and its preferred embodiments are described just for a better understanding of the subject matter and in a way not to lead to a limiting effect.

The invention is related to a coating which has a superamphiphobic character providing both superoleophobicity and superhydrophobicity for coating medical device and biomaterial surfaces, is biocompatible as determined in vitro and in vivo, is curable with UV rays and has a unique chemical composition.

The term“superhydrophobic” is used herein for being water and dirt proof, removing water and dirt. It is characterized with the angle of contact of the water drop left on the surface with the surface. If the angle of contact measured with water is close to or more than 150 degrees, the surface shows a superhydrophobic character. The term“superoleophobic” is used for removing the oil on the surface, being oil proof. If the angle of contact is close to or more than 150 degrees as measured with ethylene glycol on the surfaces, it shows a superoleophobic character as defined in the literature. The term “superamphiphobic” means that the surface shows both superhydrophobic and superoleophobic character.

The coating of the invention shows superamphiphobic character. Thanks to this character of the coating, non-polar body fluids and protein-containing substances do not hold on the biomaterial or medical device coated with the coating of the invention. This situation prevents adhesion to instruments on which the coating of the invention is used. Therefore, if biomaterials and surgical procedures compatible with the coating are chosen, the treatment performance is increased positively and the treatment duration is shortened.

The studies made within the scope of the invention are not limited to chemical synthesis of the coating. In these studies, UV rays were used and coating was made on CP-Ti and borosilicate glass material which are currently preferred in biomedical field frequently. Afterwards, surface characteristics of the coated surface were determined. A type of coating has been obtained which can be applied on medical devices and biomaterials by means of all these processes.

The coating of the invention and the chemical content produced newly and used in the coating have a completely unique value. Therefore, the coating of the invention shows biocompatibility thanks to its different chemical content. The presence of this character has been proven in vivo and in vitro. It outperforms current polymeric coatings with this aspect.

The following Table 1 shows chemical composition of the coating of the invention and their component ratios by weight.

Table 1. Chemical composition of the coating of the invention

The coating of the invention contains 25-80 % (w/w) of reactive resin, 8-25 % (w/w) of rigidity agent, I Q- 60 % (w/w) of reactive solvent (cross-linker), and 1 -8 % (w/w) of photo-initiator.

Mentioned reactive resin herein is fluorous epoxy acrylate; mentioned rigidity agent is tetraethoxysilane hydrolyzed triethoxy isobutylsilane; mentioned reactive solvent (cross-linker agent) is 1 ,6-hexanediol diacrylate; mentioned photo-initiator is 1 -hydroxycyclohexyl phenyl ketone or trimethylsulfonium hydroxide.

A preferred embodiment of the invention prepared in the studies made within the scope of the invention contains 54.7 % (w/w) of fluorous epoxy acrylate, 19.5 % (w/w) of hydrolyzed TEOS (tetraethoxysilane), 23.4 % (w/w) of 1 ,6-hexanediol diacrylate, 2.4 % (w/w) of Irgacure184 ® (1 -hydroxycyclohexyl phenyl ketone).

Fluorous epoxy acrylate is the reactive resin component providing basic characteristics of the coating that is subject of the invention. The fluorine component which is included by the fluorous epoxy acrylate enhances amphiphobic character of the film. To prepare the fluorous epoxy acrylate; 27-30 % (v/v) of 1 H,1 H,2H,2H-perfluorohexane-1 -ol or 1 H,1 H-perfluoroheptane-1 -ol) and 53-55 % (v/v) of hexamethylene diisocyanate or 1 ,4-diisoscyanatebutane are mixed in a magnetic stirrer for about 3-4 hours under nitrogen gas at a temperature of 60-70 O. The resu lting mixture is added 15-1 7 % (v/v) of N-N dimethylformamide and stirring is continued until the mixture gets clear. The clarified mixture is added epoxy acrylate in a way to obtain a total solid content of 85 % (w/v), and stirred in magnetic stirrer for I Q- 12 hours without heating. At the end of this duration, fluorination is completed and fluorous epoxy acrylate is obtained.

In the studies made within the scope of the invention, 1 .71 ml 1 H,1 H,2H,2H- perfluorohexane-1 -ol and 3.24 ml Hexamethylene diisocyanate were stirred under nitrogen gas at 60 T) for 3 hours. Afterwards, N - N dimethylformamide was added to this mixture until it becomes clear. Then, epoxy acrylate was added to the mixture in a way to obtain a total solid content of 85 % (w/v). The mixture was stirred without heating for 12 hours and fluorination was completed, and fluorous epoxy acrylate was obtained.

Hydrolyzed tetraethoxysilane or hydrolyzed triethoxy isobutylsilane form the Silicon component in the coating film of the invention, and increase the rigidity of the coating film.

For the hydrolysis of the rigidity agent, 55-57 % (v/v) of TEOS (tetraethoxysilane) or Triethoxy isobutylsilane, 12-14 % (v/v) of water and 1 -3 % (w/v) of P-toluenesulfonic acid (catalyst) are added in 28- 30 % (v/v) of ethanol or methanol and this mixture is stirred in a magnetic stirrer for 10-12 hours at a temperature of 20-25‘C. At the end of this duratio n, hydrolysis is completed and hydrolyzed rigidity agent is obtained.

In the studies made within the scope of the invention, for the hydrolysis of TEOS, 3.28 ml TEOS (tetraethoxysilane), 0.77 g water and 0.1 g P-toluenesulfonic acid (catalyst) were mixed in 1 .69 ml ethanol at 25‘C. The mixture prepared was hydrolyzed by st irring for 12 hours at room temperature.

1-6 hexanediol diacrylate is the reactive solvent component which increases the cross linking rate of the coating film of the invention, and adjusts such physical characteristics of the film as rigidity, durability and resistance to chemical substances.

1-hydroxycyclohexyl phenyl ketone or trimethylsulfonium hydroxide is a photo-initiator which initiates polymerization process of the coating of the invention under UV light at a suitable wavelength and energy.

In order to obtain the coating solution; 25-80 % (w/w) of fluorous epoxy acrylate, 8-25 % (w/w) of hydrolyzed tetraethoxysilane or hydrolyzed triethoxy isobutylsilane, 10-60 % (w/w) of 1 ,6-hexanediol diacrylate, and 1 -8 % (w/w) of 1 -hydroxycyclohexyl phenyl ketone or trimethylsulfonium hydroxide are mixed until a homogeneous mixture is obtained.

In the studies made within the scope of the invention; in order to prepare the coating solution, 3.36 g of the fluorous epoxy acrylate obtained, 1 .44 g of 1 -6 hexanediol diacrylate, 1 .2 g of hydrolyzed TEOS, 0.144 g of Irgacure 184 (1 -hydroxycyclohexyl phenyl ketone) were added and the mixture was stirred in a magnetic stirrer until it becomes homogeneous.

In the studies made within the scope of the invention, the coating solution prepared was applied on CP-Ti (commercially pure titanium) and glass (borosilicate) samples. The coating solution prepared was applied on surfaces to be coated homogeneously by using immersion or spin coating methods. Surfaces coated with the composition were exposed to UV light for 150-3600 seconds, preferably for 180 seconds, in a system equipped with a 350 W mercury lamp with a wavelength of 365 nm, in order to cure the coating. During the curing with UV light source, the samples dipped up in the coating solution were placed about 20 cm away from the light source. Surfaces coated and cured with UV were subjected to heat treatment for post-curing for 1 hour in an oven at 90-100 TT After the post-curing process, coated surfaces were put to rest in a fume hood at room temperature for 10-12 hours.

In the studies made within the scope of the invention, the following analyses were made on CP-Ti and glass samples in order to identify properties of the coating film obtained with the method of curing with UV.

Angle of contact and surface energy measurements:

Superamphiphobic character of the polymeric film formed on the surface was identified with the angle of contact of fluids formed on the surface. Superhydrophobicity of the surface was identified with water, superoleophobicity was identified with ethylene glycol; and the surface energy was measured with OWRK/Fowkes method by identifying the angle of contact of three fluids (water, hexane and ethylene glycol). Results of angles of contact on the obtained surface are shown in the Chart 1 , and their views are shown in Figure 1 .

Figure 1 shows the angles of contact of unprocessed glass samples (a, b, c); coated glass samples (d, e, f); unprocessed CP-Ti samples (g, h, i); coated CP-Ti samples with water, ethylene glycol and hexadecane, respectively. Static angle of contact measurements were taken from“Attension Theta Lite tensiometer” device.

When the angle of contact of water on the obtained coating was measured on borosilicate based materials, the lowest angle of contact was measured as 148 degrees, and the highest angle of contact was measured as 150 degrees, and it was observed that the material had a superhydrophobic character as a result of the coating obtained.

Chart 1. Angle of contact and surface energy values measured with water, ethylene glycol and hexane for UV-cured samples

Characterization of chemical composition:

FTIR, XPS and NMR analyses were made for chemical characterization of the coating of the invention.

Figure 2 shows FTIR spectrums of the coating composition prepared for the method of curing with UV. FTIR analysis was made with Thermo Scientific (Nicolet 6700) FT-IR spectrometer. Figure 2 shows FTIR spectrums of the coating solution prepared for the method of curing with UV. The lack of spectrum peak at 1730-2800 crrr ¹ shows that the first stage of the solution has been synthesized with the loss of the peak of isocyanate groups(-NCO). Examining FTIR Spectrum; it was observed that the tensile peak of NH groups were at 3336 crrr ¹ , tensile peak of asymmetric and symmetric CH groups was at 2930 to 2858 crrr ¹ , tensile peak of the ester group ( C=0) and amide group (-NHC=0) from carboxyl groups was between 1600-1750 crrr ¹ , tensile peaks of C=C, CH2 (bending), CO, C-C groups were at 1507, 1437, 1407 and 1385 cm-1 , respectively, tensile peak of C-N groups was at 1235 crrr ¹ , tensile peaks of CF2 and CF3CF2 groups were at 1 183-1 134 and 658 crrr ¹ , tensile peaks of Si-O-C and Si-O-Si were at 1089 and 1062 crrr ¹ ’de, tensile peaks of S1CH2 groups were at 864 and 830 crrr ¹ ’de and bend peaks of SiO groups were between 400 to 500 cm- ¹ .

Figure 3 shows NMR result of the coating composition prepared for the method of curing with UV. It was measured by using Nuclear Magnetic Resonance (NMR) Spectroscopy.

H-NMR Spectrum was performed particularly to observe the addition of flour and silicon to the structure. When the H-NMR values were examined, S1-CH2 was observed at d = 0.2-0.8 ppm for shifting values of protons in the structure. The presence of CH-(CF3)2 structure was also confirmed with proton shifts determined at 5=3.1 -4.2 ppm. These values are also compatible with H-NMR chemical shift charts and literature values. From these results, it was seen that silicon and fluorine were added to the structure successfully.

Figure 4 shows XPS spectrums of the coating composition prepared for the method of curing with UV. Specs-Flex was taken from X-Ray Photoelectron Spectroscopy (XPS) device.

Percentage ratios of elements contained in the structure were revealed when the results of X-Ray Photoelectron Spectroscopy (XPS) analysis were evaluated. As a result of the XPS analysis made, peak values of carbon, oxygen, fluorine and silicon elements were identified. Accordingly, percentage amounts of silicon which increases the rigidity of the coating material obtained and fluorine which gives superhydrophobicity in the composition were revealed. Accordingly, it was determined that the structure comprises approximately 69% of carbon, 16% of oxygen, 1 1 % fluorine and 5% silicon. Thus, the presence of silicon and fluorine in the structure has been confirmed.

Roughness of surface:

Roughness of unprocessed CP-Ti and superamphiphobic coated surface was measured with a 3D profilometer. Figure 5 shows 3D surface profiles of unprocessed titanium and (b) superamphiphobic polymeric film-coated titanium surface. Roughness of surface was measured by using a Bruker Contour GT-K profilometer device. When the said surface profiles are examined, it is observed that the roughness of surface increased after the coating.

The most critical parameters affecting the angle of contact on superamphiphobic coatings are the surface energy and surface roughness. Roughness of the coating film causes the drop formed on the surface stay spherical form instead of spreading. It was observed from the SEM views obtained (Figure 6) that microscopic pores were formed on the surface after the coating. Therefore, roughness was increased on these surfaces and thereby, the angle of contact was increased and this led to an increase both in superhydrophobicity and in superoleophobicity. SEM and EDS Analysis of CP-Ti Samples after UV-Coating:

Figure 6 shows (a) 2000X surface morphology of unprocessed CP-Ti, superamphiphobic polymeric structure; (b) 5000X surface morphology of unprocessed CP-Ti, superamphiphobic polymeric structure; (c) sectional SEM views of superamphiphobic polymeric film after UV coating. SEM images was obtained from SEM FEI Quanta 250 device.

The roughness of surface was increased in order to enhance superhydrophobic and superoleophobic character of surfaces. There are micro grooves inside the surface whose surface roughness is increased, and entrained air in mentioned grooves. When a fluid contacts the surface, the fluid cannot enter in due to surface tension. Since the surface area contacting with the fluid is small, the affinity between the fluid and the solid surface decreases. Therefore, micro grooves on the surface also shows the superhydrophobicity on the surface. From the image obtained, it is observed that the gaps between particles are quite intense compared with the unprocessed sample. When the cross-section images are examined, it is seen that a continuous and significant layer is formed on the surface. The diffusion layer is seen under the compound layer. It was observed that the thickness of the layer formed on the surface after the coating is between 3- 4 pm.

Figure 7 shows EDS analysis results of the film obtained after the coating. Elemental composition of the film was made in EDS, FEI Quanta 250 device. EDS results shows that C, O, F, Si and N, which were expected to form on the film, formed on the coating layer. According to the results shown in Chart 2, polymeric coating components were determined as titanium (56.27%), oxygen (23.31 %), carbon (1 1 .16%), fluorine (3.7%) and silicon (3.29%), nitrogen (2.28%).

Chart 2. EDS analysis results of polymeric coated CP-Ti film

Element % Weight ratio % Atomic weight

C 1 1 .16 23.29

N 2.28 4.08

O 23.31 36.52

F 3.7 2.33

Si 3.29 4.34

Ti 56.27 29.45

Micro-rigidity analysis after UV-Coating:

While the rigidity of unprocessed CP-Ti sample was 140 HVo.i , it was observed that the rigidity value increased by approximately 2.5 times after polymeric coating as seen in the Chart 3.

Chart 3. Micro-rigidity analysis results of CP-Ti surface

Applied Surface Treatment Surface rigidity HV ₀ .i

UV-cured CP-Ti 380

Unprocessed CP-Ti 140 Optical Analysis:

Figure 8 shows UV-vis spectrums of thin film formed on glass plates with the method technique of curing with UV rays. They were taken with a Shimadzu brand UV-3600 model UV-VIS-NIR Spectrometer. The mean permeability of superhydrophobic glass material is about 95% in the rage of 562-940 nm. UV-VIS-

5 NIR spectrum of the thin film was formed on glass plate by means of the method of curing with UV.

Electrochemical Analyses:

Electrochemical development was analyzed under conditions simulating biological interaction of samples with human body. For this purpose, measurements were made on unprocessed CP-Ti and coated CP-Ti0 samples with SBF (simulated body fluid) at 37 Ό.

Figure 9 shows the OCP curve of unprocessed and polymeric CP-Ti material. OCP measurements were made on Series G750TM Potentiostat/Galvanostat/ZRA device from GAMRY firm. OCP analysis is important to determine when the system has become stabilized, and when the shifts between different situations such as passive and active behavior will occur. Figure 9 shows OCP curves of unprocessed5 and UV-coated CP-Ti samples by keeping them under open grid conditions for 7200 seconds in SBF (simulated body fluid) in order to identify balance potentials of samples. Open circuit potential (OCP) values are more positive means the material is more resistant to corrosion. It is observed from the graphic that the OCP curve obtained from polymeric coated sample with superhydrophobic and superoleophobic character has a superior character compared to unprocessed sample.

0 Figure 10 shows potentiodynamic polarization curves of (a) unprocessed and (b) superamphiphobic polymeric coated CP-Ti samples, and Chart 4 shows the results. Potentiodynamic polarization measurements were made on Series G750TM Potentiostat/Galvanostat/ZRA device from GAMRY firm. After the polymeric coating, the corrosion potential of polymeric coated sample changed positively compared to the unprocessed sample, and the anodic part of the curve reached a lower current density.5 Examining the results obtained, after the corrosion applied in SBF electrolyte, corrosion current density of polymeric coated sample decreased to unprocessed sample. The corrosion potential (Ecorr) value of unprocessed CP-Ti is -335 mV/Ag/AgCI, and its corrosion current density (lcorr) value is 1 .43e-4 mA/cm ² . The corrosion current value of polymeric coated CP-Ti is 1 .05e-5 mA/cm ² . Polymeric coating on metal surfaces acts as a barrier, and thus, provides an anodic protection for the metal.

0

Chart 4. Corrosion test results of unprocessed and coated and UV-cured CP-Ti samples.

Material Ecorr lcorr lcorr,a lcorr,c E _pit (mV) E _paS s pa pa Rp (kD Corrosi

(mV) (mA/cm (mV) (mV/d (mV/ cm ² ) on rate

² ) ec) dec) (mpv)

Unproces 4335 1 .43 e-4 1 .68e-4 0.62e-4 434.2 Ϊ53 328.6 29?7 84.59 0.254 sed

Polymeric -180 1 .05 e-5 1 .28e-5 0.82e-4 - 682 452.3 232.1 6351 .47 0.0033 coated Figure 1 1 shows SEM views of (a) unprocessed CP-Ti, (b) superamphiphobic polymeric coated CP-Ti material after corrosion. SEM images was obtained from SEM, FEI Quanta 250 device. Examining SEM images after corrosion, it is seen that the corrosion damage forming on the surface of unprocessed CP-Ti sample is a damage of well-type. It is observed from these images that, on UV-cured polymeric film, the wells disappear compared to the unprocessed sample, and that corrosion occurs only locally.

Electrochemical impedance spectroscopy (EIS) analyses:

Figure 12 shows Nyquist and Bode Curves of the coating film (taken from Series G750TM Potentiostat/Galvanostat/ZRA device from GAMRY), and Chart 5 shows EIS results. In the range of 10 ² - 10 ⁵ Hz, the phase angle of unprocessed sample is unstable. As for the polymeric coated sample, these values are quite stable. The highest phase angle for polymeric coated sample in this region is -44°. T his shows that a protective and preventive passive film layer forms on the coated sample. Furthermore, impedance values of the coated sample are higher than the values of unprocessed sample. The passive film on the coated sample causes to obtain higher impedance data.

Chart 5. EIS results of unprocessed and polymeric coated CP-Ti in SBF

Biocompatibility and Cytotoxicity test results:

For the evaluation of cytotoxicity levels of new materials, in vitro tests are used firstly. Cytotoxic researches have been made by using MTT and LDH tests. The findings obtained were compared to control+ and control-, and % cell viability values were calculated. Control ⁺ refers to environment prepared to add cytotoxic substance triton-x to the environment, and Control refers to environment prepared to compare cell viability without any addition to the environment. In this study, it was determined as a result of preferred in vitro cytotoxicity analyses (MTT and LDH oscillation tests) that cytotoxicity levels of all tested materials are below 30%. According to biocompatibility levels of the materials used in the study; viability values in control-, control+, polymeric coated metal, polymeric coated glass, unprocessed glass, unprocessed metal and mouse fibroblast cells treated for 24 hours were identified as 100, 18.3, 97.2, 79.2, 96.1 , 93.7, and 88.2%, respectively, in MTT test, and 100, 28.4, 98.7, 98.9, 99.6, 97.5, and 96.5% in LDH test.

Figure 13 shows cytotoxicity test results with graphics of (a) viability levels observed in 3T3 fibroblast cells at the end of 24 hours of incubation; (b) viability levels observed in 3T3 fibroblast cells at the end of 48 hours of incubation. Viability levels in mouse fibroblast cells treated for 24 hours were read at 450 nm wavelengths in plate spectrometer according to MTT and LDFI tests and absorbance values were obtained, and % viability and cytotoxicity values were calculated with formulas and finally, graphics were created in Excel format.

In vivo tests:

In vivo material and method: At first, the hair was removed on the area from under the knee up to the thigh of each anesthetized animal, and the application area was sterilized by dying the extremities with liquid povidone iodine (Poviodeks, Kim-Pa, Istanbul, Turkey). After the completion of anesthesia protocol, an incision of about 2 cm width was made on the anterior face of the femur on the rear left legs of each subject. After subcutaneous fascia is retracted, musculus biceps femoris muscle (thigh rear site muscle) was revealed. After a pocket was created with a width in which the implant can fit in the muscle fascia, the implants were placed. After the muscle fascia was stitched, subcutaneous fascia and connective tissue were closed with number 2/0 polyglactin 910 (Ethicon, Vicryl, US). Finally, the incision line was closed with 2/0 silk yarn, and protected under a bandage. Wounds and bandages of subjects were checked daily in the post-operative period. On the 14th post-operative day, after all rats were euthanized in a C02 unit, the implant site was dissected and samples were collected.

Histopathologic Examinations: Necropsy examination was made on the rats after the euthanasia, and implants were removed. Surrounding tissues contacting the implant were taken into a 10% buffered formalin solution. Collected samples were then in embed paraffin blocks after immersed routine alcohol- xylol series. 5 pm sections taken from the blocks were examined with hematoxylene-eosine. The evaluation was made according to the modification of the method used by Lehle et al. (2004). For this purpose, activities such as the neutrophil leukocyte, fibroblast presence, and fibrous capsule formation were evaluated as none (0), low (1 ), medium (2) and severe (3).

Immunohistochemical examinations: After the paraffin on 5 pm sections taken to polylysine slides was removed letting them in the oven for 1 hour, the preparations were passed through xylol and alcohol series. After keeping them in distilled water for 5 minutes, the sections were kept in 3% FI2O2 for 10 min. to ensure endogenous peroxidase inactivation, and then washed with PBS 2 times. They were treated with antigen retrieval solution for 2 x 5 min. at 500 Watt and washed with PBS 3 times in order to reveal the antigen in tissues. In order to prevent non-specific bonding, tissues were contoured with a PAP pen and protein block was dropped on them, and after a waiting duration of 10 min., they were washed with PBS 1 time. Afterwards, tissues washed with PBS were left to incubation for 30 min. at a dilution ratio of 1/100 at room temperature with Anti-Collagen I antibody (dilution ratio: 1/2000, Abeam, Catalogue no. ab90395) and Anti-Collagen III antibody (dilution ratio: 1/2000, Abeam, Catalogue no. ab6310) prime antibodies. After the incubation, biotinylated secondary and HRP conjugate were dropped on tissues washed 3 times with PBS respectively, and tissues were left to rest for 15 min. On the tissues washed 4 times with PBS after HRP conjugate, 3,3 diaminobenzidine (DAB) was dropped chromogenously at a rate of 1 ml of DAB to 30 pi of Substrate. After the sections kept in chromogen for about 30 seconds were washed with distilled water, contrast dyeing was made with Mayer’s hematoxylene. After entellan was dropped on the sections which had been passed through alcohol-xylol series again, the sections were examined under light microscope.

Statistical analysis: SPSS program ver. 16.0 was used in the statistical evaluation. In the examination of data regarding neutrophil leukocyte, fibroblast activity and fibrous capsule formation obtained semi- quantitatively in histopathological examination and of the data of collagen type I and collagen type III obtained in immunohistochemical examinations, the difference between groups was identified with Kruskal Wallis test, and the groups creating the difference were identified with Mann Whitney U test. p<0.05 value was considered statistically significant.

In vivo results:

Histopathologic Examinations: Chart 6 shows neutrophil leukocyte, fibroblast activity and fibrous capsule formation of unprocessed glass, Polymeric coated Ti and Polymeric coated glass samples. Statistical difference was identified between groups (p < 0.05). It was determined that the neutrophil leukocyte presence was strongest in the unprocessed glass group, and the weakest in polymeric coated Ti group.

Chart 6. In vivo results

Unprocessed Unprocessed Ti Polymeric Polymeric

Glass coated Ti coated glass

Neutrophil Sample No ^* Sample No ^** Sample No ^** Sample No ^**

Leucocyte

1 . Severe (3) 1 . Medium (2) 1 . Medium (2) 1 . Medium (2)

2. Severe (3) 2. Severe (3) 2. Medium (2) 2. Medium (2)

3. Severe (3) 3. Medium (2) 3. Medium (2) 3. Medium (2)

4. Severe (3) 4. Medium (2) 4. Medium (2) 4. Medium (2)

5. Severe (3) 5. Severe (3) 5. Medium (2) 5. Medium (2)

6. Severe (3) 6. Severe (3) 6. Medium (2) 6. Medium (2)

Fibroblast Sample No ^* Sample No ^** Sample No ^*** Sample No ^***

1 . None (0) 1 . Severe (3) 1 . Medium (2) 1 . Medium (2)

2. Low (1 ) 2. Severe (3) 2. Medium (2) 2. Medium (2)

3. None (0) 3. Severe (3) 3. Medium (2) 3. Severe (3) 4. Low (1 ) 4. Medium (2) 4. Medium (2) 4. Medium (2)

5. Low (1 ) 5. Severe (3) 5. Medium (2) 5. Medium (2)

6. Low (1 ) 6. Severe (3) 6. Medium (2) 6. Medium (2)

Fibrous capsule Sample No ^* Sample No ^** Sample No ^*** Sample No ^*** formation

1 . None (0) 1 . None (0) 1 . Medium (2) 1 . Medium (2)

1 . None (0) 2. Low (1 ) 2. Medium (2) 2. Medium (2)

1 . None (0) 3. Low (1 ) 3. Medium (2) 3. Medium (2) 1 . None (0) 4. Low (1 ) 4. Medium (2) 4. Medium (2) 1 . None (0) 5. Low (1 ) 5. Medium (2) 5. Medium (2) 1 . None (0) 6. Low (1 ) 6. Medium (2) 6. Medium (2)

Figure 14 shows A- unprocessed glass group - severe neutrophil leukocyte infiltration ( ^* ); B- unprocessed Ti group - severe fibroblast presence (arrow head); C- polymeric coated Ti group - medium level fibroblast presence (arrow head); D- polymeric coated glass group - medium level fibroblast presence (arrow head). images were taken from DP72 model Olympus BX52 model light microscope with camera.

It was determined that the fibroblast activity was strongest in the unprocessed Ti group, and the weakest in polymeric coated Ti group. Whereas 4 of the samples in the unprocessed glass group showed fibroblast activity, 2 of them did not show any fibroblast activity. In the unprocessed Ti group, 4 samples showed severe level, and 1 sample showed medium level fibroblast activity. Fibroblast activity was determined at medium level in all the samples in the polymeric coated Ti group, at medium level in 4 samples in the polymeric coated glass group, and at severe level in 1 sample. It was determined that the unprocessed glass group and polymeric coated Ti group were different than other groups, and the Unprocessed Ti group was different than all the other groups. No statistically significant difference was identified between polymeric coated Ti and polymeric coated glass groups (p<0.05).

Immunohistochemical analysis results: Chart 7 shows collagen type I and collagen type III density of Unprocessed glass, Polymeric coated Ti and Polymeric coated glass samples. Collagen type I in fibrous tissue was the highest in polymeric coated Ti.

Chart 7.lmmunohistochemical analysis results

Statistical difference was identified between groups in terms of collagen type III. Collagen type III in fibrous tissue was the highest in polymeric coated Ti. It was determined that 3 of 4 samples in this group had a severe level of collagen type III. A medium level of collagen type III was observed in unprocessed Ti, polymeric coated Ti, polymeric coated glass groups (p > 0.05).

In figure 15, collagen type I presence is obtained low in A- Unprocessed Glass group and B- Unprocessed Ti group; low in C- Polymeric coated Ti group and low in D- UV glass group ( ^* ). images were taken from DP72 model Olympus BX52 model light microscope with camera.

Figure 16 shows that the level of Collagen type III presence is low in in A- Unprocessed Glass group, medium in B- Unprocessed Ti group; medium in C- Polymeric coated Ti group and medium in D- UV glass group ( ^* ). images were taken from DP72 model Olympus BX52 model light microscope with camera.

Collagen type I in fibrous tissue was the highest in polymeric coated Ti. It was determined that 5 of 6 samples in this group had a medium level of collagen type I. A low level of collagen type I was observed in unprocessed Ti, polymeric coated Ti, polymeric coated glass groups. It was determined that 3 of 6 samples in unprocessed glass group had a low level of collagen type I, whereas no collagen saving was determined in the other 3 samples (Figure 15).

Statistical difference was identified between groups in terms of collagen type III. Collagen type III in fibrous tissue was the highest in polymeric coated Ti. It was determined that 5 of 6 samples in this group had a severe level of collagen type III. A medium level of collagen type III was observed in unprocessed Ti, polymeric coated Ti, polymeric coated glass groups. It was determined that 3 of 6 samples in unprocessed glass group had a low level of collagen type III, whereas no collagen savingwas determined in the remaining samples (p < 0.05) (Figure 16).

As a result of the analyses made, it is determined that the coating of the invention shows superhydrophobicity and superoleophobicity at the same time, in other words it is superamphiphobic. Moreover, it is an innovative property that the coating is obtained in a very short time with UV rays. Biocompatibility properties of the coating film obtained were determined both in vitro and in vitro envoriment. In applications in which a short-term implant treatment is required and the implant is not permanent, an instrument having such a surface characteristic (superamphiphobic) will be separated from the tissue easily. Adhesion during the removal of the implant is at a minimum level compared to uncoated implants. Since adhesion is at a minimum level between the tissue and implant surface, it is not possible for the live tissue to adhere to the implant surface and thereby, surface characteristics of the tissue is protected substantially, and the operation is completed with minimum subcutaneous damage. Damaged site which can heal in a short period will be exposed to less force and less incisions in the second operation (in which the implant is removed). Therefore, the total healing period is shortened considerably.