Silver free surface coating with palladium and gold

Abstract

An object is provided with a surface coating comprising particles of palladium and gold, where the coating is silver-free and neodymium-free. The total amount of the particles on the surface is in the range of 0.25 to 1.40 µg / cm², with gold constituting 45-60 wt% of the total amount. The particles and the outermost 10 µm of the object comprise less than 30 ppm silver. The mean particle diameter is between 20 and 80 nm, and particles with a diameter of 100 nm or more constitute at least 2% of the total number of particles. The coating provides both biocompatible and antimicrobial properties and is particularly advantageous for medical devices intended for contact with human or animal blood, as it counteracts blood clotting and thrombosis without the disadvantages associated with silver or neodymium.

Description

[0001] Silver free surface coating with palladium and gold

[0002] Field of the invention

[0003] The present invention relates to a surface coating comprising particles of palladium and gold, which coating is silver free and where neodymium is not added .

[0004] Background

[0005] Surfaces with antimicrobial and biocompatible properties are important within many applications . Examples of surfaces where such properties are of importance include surfaces intended to be in contact with a human or animal body including contact with the skin as well as body cavities and inside a body . Medical equipment , which is intended to be in contact with human or animal blood, should preferably have properties so that formation of blood clots and thrombosis is avoided .

[0006] US 6 , 224 , 983 discloses an article with an adhesive , antimicrobial and biocompatible coating comprising a layer of silver stabilised by exposure to one or more salts of one or more metals selected from the group consisting of platinum, palladium, rhodium, iridium, ruthenium and osmium . The thickness of the silver layer is in the range 2-2000A (A, Angstrom, Angstrom, 10-10m) and further disclosed ranges are 2-350A and 2-50A. There are also examples of a thickness of the silver layer of 50A, 350A, 500A, and 1200A. The substrate may be latex, polystyrene , polyester, polyvinylchloride , polyurethane, ABS polymers , polycarbonate , polyamide , polytetrafluoroethylene , polyimide or synthetic rubber . WO2007 / 117191 , W02007 / 117213 and W02007 / 117214 disclose a substrate having an electron donating surface, characterized in that there are metal particles on said surface, the metal particles comprise palladium and at least one metal selected from the group consisting of gold, ruthenium, rhodium, osmium, iridium, and platinum and wherein the amount of the metal particles is from about 0.001 to about 8 pg / cm2.

[0007] WO 2007 / 142579 discloses a polymer matrix, characterized in that it comprises a. an electron donating constituent and b. metal particles comprising at least one metal selected from the group consisting of palladium, gold, ruthenium, rhodium, osmium, iridium, and platinum.

[0008] WO 2019 / 206950 discloses a method for decreasing leakage of matter from an object to a surrounding, said object being coated with a coating at least partially applied on the object, said coating comprising an at least partially covering layer comprising silver, said object optionally comprising area(s) without said layer, said coating comprising metal particles applied on the layer and optionally on areas without said layer, said metal particles comprising palladium and at least one metal selected from the group consisting of gold, ruthenium, rhodium, osmium, iridium, niobium, neodymium and platinum and wherein the amount of the metal particles is in the interval 0.01-8 pg / cm2. It is disclosed that blood clotting can be reduced if the surface is exposed to blood from a human or animal, when the metal particles comprise palladium and neodymium. Silver is always present in the coating. There are disclosed specific examples for instance with the following content: Example 7: Pd 0.5 pg / cm2Au 0.3 pg / cm2and silver.

[0009] Example 8: Pd 0.2 pg / cm2Au 0.2 pg / cm2and silver. Example 9: Pd 0.3 pg / cm2Au 0.3 pg / cm2and silver.

[0010] Example 10: Pd 0.3 pg / cm2Au 0.3 pg / cm2and silver. Example 11: Pd 0.2 pg / cm2Au 0.2 pg / cm2and silver. Example 12: Pd 0.4 pg / cm2Au 0.4 pg / cm2and silver. Example 13: Pd 0.3 pg / cm2Au 0.3 pg / cm2and silver. Example 14: Pd 0.5 pg / cm2Au 0.3 pg / cm2and silver.

[0011] Example 15: Pd 0.7 pg / cm2Au 0.05 pg / cm2and silver as well as Pd 0.5 pg / cm2Au 0.1 pg / cm2and silver.

[0012] WO 2023 / 198555 discloses an object, wherein there are particles on the surface of the object, wherein the amount of particles is in the interval 0.041 - 6 pg / cm2, wherein the particles comprise palladium in an amount corresponding to 0.02 - 2 pg / cm2, neodymium in an amount corresponding to 0.001 - 2 pg / cm2, and gold in an amount corresponding to 0.02 -2 pg / cm2, and wherein the particles comprise less than 30 ppm silver. There are disclosed specific examples including :

[0013] Example 1 including neodymium with Pd 0.6 pg / cm2Au 0.2 pg / cm2, and a control sample including silver and neodymium with Pd 0.5 pg / cm2Au 0.4 pg / cm2.

[0014] Example 2 including neodymium with Pd 1.1 pg / cm2Au 0.6 pg / cm2, and a control sample including silver and neodymium with Pd 0.7 pg / cm2Au 0.6 pg / cm2.

[0015] Example 4 including neodymium with Pd 0.6 pg / cm2Au 0.4 pg / cm2, and a control sample including silver and neodymium with Pd 0.8 pg / cm2Au 0.3 pg / cm2.

[0016] Although the amount of silver, which is released from coatings such as the coatings, described in WO 2019 / 206950 is minimal, the silver may anyway have disadvantages in some cases. When applied to certain materials the silver may cause discoloration in spite of being applied in relatively low amounts according to the state of the art such as in WO 2019 / 206950 . Further, the application of silver requires some steps during the manufacture . The si lver i s typical ly applied form baths , which have to be replaced regularly .

[0017] Although the addition of neodymium instead of silver work well such as the coatings described in WO 2023 / 198555 , the addition of neodymium has a drawback in that the bath for application of neodymium ages fast and has to be replaced regularly . The durability and shel f li fe of such a neodymium bath makes it di f ficult to use . It is thus cumbersome to apply neodymium since it requires an additional step and frequent change of the bath for application of neodymium .

[0018] US 11 , 406 , 743 discloses an obj ect at least partially coated with an outer layer, the outer layer comprising an electron donating material and metal particles , with the proviso that the outer layer does not comprise s ilver, characteri zed in that the metal particles comprise palladium and at least one metal selected from the group consisting of gold, ruthenium, rhodium, osmium, iridium, and platinum, wherein the amount of the metal particles is from about 0 . 001 to about 8 pg / cm2, and wherein the metal particles have an average si ze of from about 10 to about 10 , 000 A. No further details regarding the composition of the silver free surface are disclosed . All examples are including silver .

[0019] Summary

[0020] One obj ect of the present invention is to obviate at least some of the disadvantages in the prior art and provide an improved surface coating without silver and without neodymium . In a first aspect there is provided an object, wherein there are particles on the surface of the object, the particles comprise palladium and gold, in a total amount of 0.25 - 1.40 pg / cm2, with gold constituting 45-60 wt% of the total amount and the particles comprising less than 30 ppm silver, and the outermost 10 pm of the object also comprising less than 30 ppm silver, wherein the mean particle diameter as measured according to ISO 19749:2021 is in the interval 20 - 80 nm and wherein particles with a diameter of 100 nm or more as measured according to ISO 19749:2021 constitute 2% or more of the total number of particles.

[0021] Further embodiments of the present invention are defined in the appended dependent claims.

[0022] An advantage is that there is provided a surface coating without silver and without neodymium, which at the same time is both antimicrobial and decreases blood clotting, when in contact with human or animal blood. The blood compatibility is at least as good and in some aspects better than previous surfaces including silver.

[0023] As evidenced by the examples, an advantage is that the coating according to the invention achieves a high degree of blood compatibility, comparable to the established silver-containing version, without the need for silver. This is a significant benefit if avoiding silver is desirable due to cost, regulatory concerns, or other factors.

[0024] The no-silver coatings according to the invention demonstrate a low degree of thrombo-inf lammatory reactions. They effectively reduce the activation of the coagulation cascade (lower TAT) and, in certain formulations, reduce inflammatory responses (lower PMN elastase) compared to uncoated materials.

[0025] Brief description of the drawings

[0026] Aspects and embodiments will be described with reference to the following drawings in which:

[0027] Figure 1 shows a comparison of a surface coating comprising silver and a silver free coating. Top sample, an uncoated control sample, middle sample a coating including silver, bottom sample a coating according to the present invention. It is seen that the silver free coating is completely clear, while the coating comprising silver is slightly greyish. The uncoated sample is also clear. The details of the coatings are outlined in example 5c.

[0028] Figure 2 shows a SEM picture of a coated object according to the invention as detailed in example 6 E. The magnification is 150.000 times.

[0029] Figure 3 shows fibrin deposition on material after being in contact with blood from BAC-333, BAC-334, BAC-335, BAC-336 and BAC-337 and after staining using toluidine blue.

[0030] Figure 4 is a graph that shows depletion of PTL in different loops .

[0031] Figure 5 is a graph that shows Generation of TAT in different loops .

[0032] Figure 6 is a graph that shows PMN elastase release in different loops. Figure 7 is a graph that shows release of C3a in different loops .

[0033] Figure 8 is a graph that shows FPA release in different loops .

[0034] Figure 9 is a graph that shows FPB release in different loops .

[0035] Figure 10 is a graph that shows IL-l / p release in different loops .

[0036] Figure 11 is a graph that shows IL-6 release in different loops .

[0037] Figure 12 is a graph that shows IL-8 release in different loops .

[0038] Figure 13 is a graph that shows MCP-1 release in different loops .

[0039] Figure 14 is a graph that shows TNFa release in different loops .

[0040] Figure 15 is a graph that shows VEGF release in different loops .

[0041] Figures 16A and 16B show fibrin deposition on material after being in contact with blood from BAC-355, BAC-356 and BAC- 357 and after staining using toluidine blue.

[0042] Figure 17 is a graph that shows depletion of PTL in different loops . Figure 18 is a graph that shows generation of TAT in different loops.

[0043] Figure 19 is a graph that shows release of PMN elastase in different loops.

[0044] Figure 20 is a graph that shows generation of FPA in different loops.

[0045] Figure 21 shows SEM analysis of BG non-silver coated TiGr2 plate with 5% Au.

[0046] Figure 22 shows SEM analysis of BG non-silver coated TiGr2 plate with 7.5% Au.

[0047] Figure 23 shows SEM analysis of BG non-silver coated TiGr2 plate with 10% Au.

[0048] Figure 24 shows SEM analysis of BG non-silver coated TiGr2 plate with 12.5% Au.

[0049] Figure 25 shows SEM analysis of BG non-silver coated TiGr2 plate with 15% Au.

[0050] Figure 26 shows SEM analysis of BG non-silver coated TiGr2 plate with 30% Au.

[0051] Figure 27 shows SEM analysis of BG non-silver coated TiGr2 plate with 80% Au. Figure 28 shows the distribution of surface immobilized particles of BG non-silver coated TiGr2 plates with different Au concentrations based on the average diameter.

[0052] Figures 29A and 29B show fibrin deposition on material after being in contact with blood from BAC-338, BAC-339, BAC-340 and BAC-341 and after staining using toluidine blue.

[0053] Figure 30 is a graph that shows depletion of PTL in different loops .

[0054] Figure 31 is a graph that shows generation of TAT in different loops.

[0055] Figure 32 is a graph that shows PMN elastase release in the different loops.

[0056] Figure 33 is a graph that shows release of C3a in different loops .

[0057] Figure 34 is a graph that shows release of FPA in different loops .

[0058] Figure 35 is an illustration of turbidimetric clotting and lysis variables.

[0059] Figure 36 is a graph that shows normal clot activation of NP (right) and clot activation of NP exposed to uncoated TiGr2 plate (left) .

[0060] Figure 37 is a graph that shows normal lysis of NP (right) and lysis of NP exposed to uncoated TiGr2 plate (left) . Figure 38 is a graph that shows the time it takes for a clot to start forming .

[0061] Figure 39 is a graph that shows the rate for a clot to start forming .

[0062] Figure 40 is a graph that shows the time it takes for lysis to start .

[0063] Figure 41 is a graph that shows the rate for lysis to start .

[0064] Figure 42 is a graph that shows the time duration from initiation for 50% completion of a lysis process .

[0065] Figure 43 is a graph that shows the time duration from initiation to complete a lysis process .

[0066] Figure 44 is a graph that shows the rate for a lysis process .

[0067] Detailed description

[0068] Before the invention is di sclosed and described in detail , it is to be understood that this invention is not limited to particular configurations , process steps and materials disclosed herein as such configurations , process steps and materials may vary somewhat .

[0069] It must be noted that , as used in thi s speci fication and the appended claims , the singular forms "a" , "an" and "the" include plural referents unless the context clearly dictates otherwise . The following terms are used throughout the description and the claims .

[0070] "Amount" of particles or other material on a surface is herein often given as pg / cm2. This is a suitable way of expressing the amount since the applied layer is very thin . For calculating the amount , the coated area of the obj ect is measured and the amount per the coated area is calculated . The amount is calculated by weight .

[0071] "Antimicrobial" as used herein is the property of suppressing or eliminating microbial growth . Microbial growth includes but is not limited to bacterial growth .

[0072] "Annular crown formations" as used herein refers to a speci fic particle morphology observed via SEM, characteri zed by larger, ring-like or crown-like structures formed by agglomerates of smaller primary particles . These formations are representative of particles with a diameter of 100 nm or more and are associated with the beneficial blood compatibility properties of the invention .

[0073] "Biocompatible" as used herein is the ability of a material to perform with an appropriate host response in a speci f ic application .

[0074] "Neodymium free" as used herein means that the coating is practically free of neodymium, which is interpreted so that the particles in the coating comprise less than 30 ppm neodymium . "Obj ect" as used herein i s the substrate , which is treated and at least partially surface coated according to the present invention .

[0075] "Outermost 10 pm of the obj ect" as used herein refers to the volume of the obj ect material extending from its surface to a depth of 10 pm, measured in a direction perpendicular to the surface . The composition within this volume , for instance the concentration of silver, is calculated by weight before the application of any particles .

[0076] "Particle" as used herein is a small locali zed discrete obj ect which can be described by physical or chemical properties . Since metal particles according to the invention are on a surface and since they also can be formed by treating the substrate in a solution of metal ions , they may also be referred to as deposits of metal . All such deposits are referred to as particles , since they do not form a covering layer but are instead local i zed discrete obj ects on the substrate surface . The word particles is suitable with regard to the appearance of the coating in an SEM picture such as in Fig . 2 , where the particles of the coating can be seen as small dots which look like particles .

[0077] "Silver free" as used herein means that the coating is practically free of silver, which is interpreted so that the particles in the coating comprise less than 30 ppm si lver . A silver content of less than 30 ppm is considered as 30 ppm .

[0078] " Thrombo-inf lammatory reactions" as used herein refers to the cascade of biological responses initiated upon contact of a material with blood, involving both the coagulation cascade ( thrombosis ) and the activation of inflammatory pathways . In the context of this invention, a reduction in thrombo-inf lammatory reactions is evidenced by, for example , a lower generation of Thrombin-Antithrombin ( TAT ) complex and a lower release of PMN elastase compared to an uncoated control surface .

[0079] According to the present invention, a surface coating is applied on an obj ect to give it desired properties . The particles on the surface of the obj ect can be viewed as a surface coating . The obj ect can be made of a wide range of materials .

[0080] It has unexpectedly been found that by selecting certain amounts of palladium and gold and by having a certain ratio of palladium to gold, it is possible to use particles of only palladium and gold as an antimicrobial coating . It has further been found that by selecting these amounts of palladium and gold, it is possible to obtain an improved blood compatibility .

[0081] In the prior art , palladium and gold have been used together with silver and / or neodymium . It has been discovered that for combinations including silver and / or neodymium the amount of and relation between palladium and gold are not highly important .

[0082] After extens ive research, it has been discovered that when using only palladium and gold the amount of palladium and gold as well as the relation between the amounts of palladium and gold are much more critical . It has surprisingly been found that i f the amounts of and the relation between palladium and gold are kept within certain defined boundaries it is possible to use only palladium and gold and still obtain excellent antimicrobial properties as well as excellent blood compatibility.

[0083] In the first aspect there is provided an object, wherein there are particles on the surface of the object, the particles comprise palladium and gold, in a total amount of 0.25 - 1.40 pg / cm2, with gold constituting 45-60 wt% of the total amount and the particles comprising less than 30 ppm silver, and the outermost 10 pm of the object also comprising less than 30 ppm silver, wherein the mean particle diameter as measured according to ISO 19749:2021 is in the interval 20 - 80 nm and wherein particles with a diameter of 100 nm or more as measured according to ISO 19749:2021 constitute 2% or more of the total number of particles.

[0084] It has been found that neither silver nor neodymium has to be added if palladium and gold is used and if the amount of palladium and gold is kept within certain boundaries and if the amount of gold is 45-60 wt% of the total amount of palladium and gold. If the amounts of gold and palladium are kept within the boundaries then an antimicrobial and biocompatible surface coating is obtained. The absence of silver and neodymium is advantageous.

[0085] It has further been found that for the compositions where the beneficial effects occur, the number of particles with a bit larger diameter is significantly higher. This is observed in example 10, where annular crown formations are seen and which is represented in the diameter distributions as a higher fraction of larger particles with a diameter of 100 nm and more. This occurs for the samples "7.5%", "10%", " 12 . 5%" and " 15%" in example 10 . These are the gold amounts where the beneficial properties of the coating occur .

[0086] The obj ect is at least partially coated with metal particles on its surface . The metal particles are deposited on the surface of the obj ect . The word particles is used even i f the metal on the surface can be deposited from a solution comprising metal ions . Thus , also metal deposits on the surface is included in the word particles . It should be noted that the metal does not form a completely covering coating on the surface . Instead, it is a plurality of deposits or particles on the surface . The particles or deposits comprise gold and palladium in a mixture . The metal particles can both be deposited from metal ions in solution but can also be deposited from a suspension of metal particles in a liquid .

[0087] The obj ect is at least partially coated with particles so that there may be areas on the obj ect without particles . In one embodiment , the entire obj ect is coated with particles . In one embodiment , a part of the obj ect, which is dipped in a depositing solution is coated . An area, which is not coated with particles , is characteri zed in that the distance to the nearest particle on the surface is more than 1 mm . For a coating the particles on the surface are so abundant that the distance between the particles is much less than 1 mm, the distance between particles on the surface is several order of magnitude less than 1 mm . Areas without the coating can then by identi fied as having no particles . In order to determine an exact boundary where the coating ends , the criterion of 1 mm to the nearest particle can be used . Thi s criterion makes it easy to distinguish coated and non-coated areas since the distance between particles is very much lower than 1 mm in coated areas and since there are virtually no particles in non-coated areas . Non-coated areas have typically not been dipped or have been masked in some way .

[0088] The particles always comprise palladium and gold . The amounts of the di f ferent metals in the particles are calculated based on the weight of metal per area of the obj ect . The total amount of the particles is also calculated based on the weight of the particles per area of the obj ect . The total amount of the particles may comprise additional additives in addition to the two compulsory metals . I t should however be noted that the amount of impurities in general should be kept as low as possible since the coating is intended for medical products and the ef fect of additional impurities may be di f ficult to determine . As a precaution, it is advisable to keep the amount of impurities as low as practically possible in order to avoid unexpected side ef fects , which may otherwise occur from additional impurities .

[0089] The total amount of gold is in the range 45- 60 wt% of the total amount of palladium and gold . This is calculated by weight so that the weight of gold in the particles is divided by the total weight of gold and palladium . This is the total amount of gold in the particles in the finished coating . This value should not be mixed with some of the percentages in the examples , since the examples use percentages of a gold compound and a palladium compound which are used to prepare a solution comprising gold ions and palladium ions , which was used for instance as described in examples 5a and 5b . The surface coating is free of silver, which is interpreted so that the particles in the coating comprise less than 30 ppm silver . Any avoidable silver should be added neither to the particles nor to the surface of the obj ect . Even though no s ilver is added to the coating, the other metals in the surface coating may comprise small amounts of various impurities including silver . Thus , when the other metals are added it cannot be ruled out that there are some small amounts of silver as an impurity . Very small amounts of silver may be unavoidable because of impurities in the metals . For instance , there may be a small amount of silver as an impurity in the gold . A small amount of silver is a common impurity in gold . Thus , a very small amount of silver may be di f ficult to avoid even i f silver is not deliberately added . The amount of silver should be kept as low as practically possible . Since the amount of silver in commercially available high purity grades of the metals palladium and gold is in the order o f 10-30 ppm, it may be di f ficult to reach lower amounts of si lver in a commercial scale . It is thus motivated to refer to the surface as silver- free when the amount of silver in the particles is less than 30 ppm . By using higher quality grades of metals , it is possible to reach a silver amount in the particles of less than 20 ppm and even les s than 10 ppm . It may even be possible to reach a silver amount of less than 5 ppm .

[0090] No neodymium is deliberately added to the surface , which is an advantage since the additional step of neodymium would require and additional bath, which has the tendency to age fast and would require frequent changes with associated additional costs . Neodymium is typical ly not present as an impurity in commercially available grades of the metals palladium and gold, which are used in the present invention . Nevertheless, it cannot be ruled out that there may be traces of neodymium in the metals added to the surface coating. In one embodiment, the amount of neodymium in the particles and in the outermost 10 pm of the object is less than 30 ppm calculated by weight. In one embodiment, the particles comprising less than 30 ppm neodymium, and the outermost 10 pm of the object also comprising less than 30 ppm neodymium.

[0091] The object onto which the particles are deposited should also not comprise silver, at least it should comprise as little silver as possible, and otherwise the idea of a silver free coating would not be effective. The outermost 10 pm of the object comprises less than 30 ppm silver. The distance from the surface is measured in a direction perpendicular to the surface and 10 pm into the object. In this part of the object the amount of silver is calculated by weight. In one embodiment the outermost 10 pm of the object comprises less than 30 ppm neodymium.

[0092] In order to measure the amount in the outermost 10 pm of the object, the distance from the surface is measured in a direction perpendicular to the surface and 10 pm into the object. Within this volume the weight of the amount of for instance silver is calculated and then the total weight of the material in this volume is calculated and then the amount of for instance silver is calculated. The amount in the outermost 10 pm is measured or calculated before application of the particles. The same applies to both silver and neodymium.

[0093] In one embodiment, the object before application of the particles does not comprise more than 30 ppm silver. This embodiment takes into account the entire uncoated object, which should not comprise more than 30 ppm silver by weight.

[0094] The object onto which the particles are deposited does not comprise a coating comprising silver.

[0095] In one embodiment, the entire object including the particles, i.e. including the coating comprises less than 30 ppm silver, preferably less than 20 ppm silver, more preferably less than 10 ppm silver. In one embodiment, the object including the particles on the surface of the object comprises less than 30 ppm silver.

[0096] The object is also free of neodymium. The same limits and limitations as for silver can be applied also for neodymium.

[0097] The amount of silver and neodymium is calculated by weight in ppm (parts per million, 10-6) . 1 ppm as used herein corresponds to 0.0001 wt%.

[0098] In one embodiment, the object comprises at least one metal. Since the coating is to be silver-free, the object to be coated should also comprise as little silver as possible and thus the object cannot be made of silver. In line with the above description, a silver amount of maximum 30 ppm silver in the object is a suitable limit. Lower limits such as 20 ppm or 10 ppm can also be applied. For metals the total amount of particles comprising gold and palladium on the surface is in the interval 0.8 - 1.4 pg / cm2. This is inter alia because of the electrochemical exchange with metal substrates making a higher amount of particles suitable for metal substrates. In one embodiment, the at least one metal is selected from the group consisting of iron, titanium, cobalt, nickel, chromium, and mixtures thereof.

[0099] In one embodiment, the at least one metal is selected from the group consisting of steel, stainless steel, and nitinol.

[0100] In one embodiment the at least one metal is selected from the group consisting of medical grade titanium, medical grade stainless steel, and medical grade nitinol. The wording medical grade indicates that the metal or alloy is intended for medical use such as for implants or for being in contact with a human or animal body. Examples of medical grade stainless steel include but are not limited to SAE 316, SAE 440, SAE 420, and 316L produced according to ASTM F138 / F139. The medical grade also indicates that the metal alloy does not comprise metals or other additives, which are unsuitable and / or toxic to use in contact with a human body.

[0101] In one embodiment, the object comprises at least one ceramic material. A ceramic material is a material made by firing an inorganic, non-metallic material.

[0102] In one embodiment, the object comprises at least one selected from the group consisting of silicon nitride, and zirconium dioxide.

[0103] In one embodiment, the object comprises at least one selected from the group consisting of apatite, and hydroxyapatite .

[0104] In one embodiment, the object comprises at least one polymer. For polymers the total amount of particles comprising gold and palladium on the surface is in the interval 0.25 - 0.8 pg / cm2. This is inter alia because polymers are non-conducting. The exception is conducting polymers, where a higher amount of particles should be applied .

[0105] In one embodiment, the object comprises at least one polymer composite. A polymer composite comprises short or continuous fibres bound together by a matrix of organic polymers. In a polymer composite, a polymer is combined with various continuous and non-contiguous reinforcements / fibres , typically added to the polymer to improve the material performance. Also for polymer composites the total amount of particles comprising gold and palladium on the surface is in the interval 0.25 - 0.8 pg / cm2, unless the polymer composite is conducting, where a higher amount of particles should be applied.

[0106] In one embodiment the polymer is selected from the group consisting of latex, vinyl, polymers comprising vinyl groups, polyurethane urea, silicone, polyvinylchloride, polypropylene, styrene, polyurethane, polyester, copolymerisates of ethylene vinyl acetate, polytetrafluoroethylene (PTFE) , polyether ether ketone (PEEK) , polystyrene, polycarbonate, polyethylene, polyacrylate, polymethacrylate, acrylonitrile butadiene styrene (ABS) , polyamide, polyimide, and mixtures thereof. These polymers can be a part of a polymer composite or form an object.

[0107] In one embodiment, the object comprises at least one textile material. A textile is a material made of interlacing fibres. Both woven and non-woven textiles are encompassed within the term. Materials typically used for wound dressings are encompassed within the term textile. Medical textiles are encompassed, and are all types of textiles intended for medical use.

[0108] It must be noted that the particles in one embodiment are essentially homogenously composed of gold and palladium, i.e. they have essentially the same composition of metals throughout the particle.

[0109] The amount of palladium + gold on the surface is in the range 0.25 - 1.40 pg / cm2. Impurities in the particles will lead to a slightly higher amount of particles on the surface since any impurities are in addition to the amount of palladium and gold. In general, impurities should be avoided, but there may typically be small amounts of unavoidable impurities in the palladium and gold. The amount of such impurities are typically so low that the total amount of particles practically still is in the range of about 0.25 - 1.40 pg / cm2.

[0110] In one embodiment, the particles are separated particles, not in contact with each other, i.e. sparsely distributed particles, which are not in contact with each other. In an alternative embodiment, parts of the particles are in contact with each other to form agglomerates of particles. In such agglomerates, a number of particles are in contact to form an agglomerate. Nevertheless, the surface of the object is still accessible to an aqueous solution so that the coating with particles is percolated and permeable to aqueous solutions. The coating is never a completely covering and non-permeable layer of metal on the surface. Combinations of separated particles and agglomerates of particles are also encompassed.

[0111] In one embodiment, the particles have a size in the interval 5-500 nm. In one embodiment, the particles have a size in the interval 10-500 nm. The particle size is measured according to ISO 19749:2021 by scanning electron microscopy. A skilled person is aware that there are other methods of characterizing a surface such as an electron probe microanalyzer (EPMA) with WDX detectors and calculating a particle size from simulations of the measured data. However, the particle sizes as defined in the description and in the claims are as defined in ISO 19749:2021. In one embodiment, the particles have a size in the interval 10- 200 nm. In one embodiment, the particles have a size in the interval 5-200 nm. In one embodiment, the particles have a size in the interval 10-60 nm. A person skilled in the art realises that the particle size can be in different intervals from about 5 to about 500 nm. Examples of such intervals include but are not limited to 5-100 nm, 10-400 nm, 10-300 nm, 10-100 nm, 10-80 nm, 10-70 nm, 10-60 nm, 15- 150 nm, 15-100 nm, and 15-60 nm.

[0112] Examples of objects comprising a substrate according to the present invention include but are not limited to medical devices, medical instruments, and medical disposable articles. In one embodiment, the object is selected from medical devices, medical instruments, and medical disposable articles. A medical device is a device, intended for medical use. A medical instrument is an instrument, intended for medical use. A medical disposable article is a disposable article, intended for medical use. Such objects are suitable to coat since it is an advantage that they are both antimicrobial and biocompatible.

[0113] In one embodiment, the object is an object intended to be in contact with human or animal blood. This use is particularly beneficial since the formation of blood clots is reduced compared to other materials. The risk for thrombosis is reduced. Objects intended to be inside a human or animal body or in contact with human or animal blood are suitable to coat according to the present invention. As an example, objects in devices intended to be in contact with human or animal blood are coated. Such objects include machines and devices where human or animal blood is processed and returned to a human or animal. For such procedures, the risk of thrombosis is reduced.

[0114] In one embodiment the object is at least one selected from the group consisting of a catheter, a central venous catheter, a peripheral venous catheter, a urinary catheter, a Foley catheter, an intermittent catheter, an implant, a dental implant, a dental abutment, a dental aligner, a dental prosthesis device, a bone replacing implant, an orthopaedic implant, a tissue replacing implant, a stent, a biliary stent, a tracheal stent, a peripheral stent, a glove, a pacemaker, a rupture net, a surgical instrument, a blood bag, an artificial heart valve, a vascular port, a haemodialysis equipment, a peritoneal dialysis equipment, a plasmapheresis device, an ecmo machine, a cardiopulmonary bypass device, an inhalation drug delivery device, a vascular graft, an arterial vascular graft, a venous vascular graft, a cardiac assist device, a wound dressing, a medical textile, an ECG electrode, an orthopaedic device, an intraocular lens, a suture, a needle, a staple, a mesh, a drug delivery device, an endotracheal tube, a shunt, a drain, a suction device, a hearing aid device, an urethral medical device, and an artificial blood vessel. All such objects are suitable to coat since it is an advantage that they are both antimicrobial and biocompatible. Further it is an advantage that they are free of silver and neodymium.

[0115] According to the invention, catheters can be coated. Examples of catheters include but are not limited to central venous catheters, peripheral venous catheters, urinary catheters, Foley catheters, and intermittent catheters.

[0116] Objects intended for dental use can be coated according to the invention. Examples of such objects include but are not limited to dental implants, dental abutments, dental aligners, and dental prosthesis devices.

[0117] According to the invention, implants can be coated. Implants for both dental applications as well as all other applications can be coated. Examples of implants include but are not limited to bone replacing implants, orthopaedic implants, and tissue replacing implants.

[0118] Stents can be coated according to the invention. Examples of stents include but are not limited to biliary stents, tracheal stents, and peripheral stents.

[0119] Objects intended to be in contact with human or animal blood during at least a part of their intended use can be coated according to the invention. Such objects include parts of devices and equipment, which are intended to be exposed to blood. Examples of such objects include but are not limited to haemodialysis equipment, peritoneal dialysis equipment, plasmapheresis devices, ecmo (Extracorporeal membrane oxygenation) machines, and cardiopulmonary bypass devices. For such objects, it is understood that the parts intended to come into contact with the blood are suitable to be coated .

[0120] Objects intended to be in contact with human or animal blood are suitable to coat and further examples include but are not limited to vascular grafts, arterial vascular grafts, venous vascular grafts, artificial blood vessels, artificial heart valves, blood bags, and vascular ports.

[0121] Also further objects are suitable to coat according to the invention and such objects include but are not limited to gloves, cardiac assist devices, pacemakers, rupture nets, surgical instruments, inhalation drug delivery devices, wound dressings, ECG electrodes, orthopaedic devices, intraocular lenses, sutures, needles, staples, meshes, drug delivery devices, endotracheal tubes, shunts, drains, suction devices, hearing aid devices, and urethral medical devices .

[0122] There is provided use of the coating, where the a coated object in contact with human or animal blood for preventing thrombosis. The coating is both antimicrobial and prevents thrombosis. Blood clots may form in contact with foreign uncoated objects such as tubes, pumps and so on and this is counteracted by using the present coating.

[0123] The invention is an improvement / derivative of the materials defined for instance in US patent 5,320,908. Differences include but are not limited to that the layer of silver in US patent 5,320,908 is excluded. In the present invention there are instead particles comprising gold and palladium. The amount of metals in the particles including the ratio between the metals are also different compared to the metals in the layer in US 5, 320, 908. In summary, it is an improvement of the materials described in US 5,320,908.

[0124] The applied amount of the metals is expressed in pg / cm2. This is calculated as the weight of the particles in relation to the coated area of the object. It is the weight of the metal in relation to the coated area of the object.

[0125] Now there is described one embodiment of the present invention for preparation of the coating. In one embodiment, the method includes the following steps:

[0126] 1. rinsing (optional)

[0127] 2. activation

[0128] 3. rinsing (optional)

[0129] 4. deposition of particles

[0130] 5. rinsing (optional)

[0131] 6. drying (optional)

[0132] Although the initial rinsing is optional, it is recommended.

[0133] The activation is made in an aqueous solution of a stannous salt containing 0.0005 to 30 g / 1 of stannous ions. The pH is 1 to 4 and adjusted by hydrochloric and / or sulphuric acid. The treatment time is 2-60 minutes at room temperature. After the pre-treatment, the surface is rinsed in demineralised water, but not dried.

[0134] In addition to the above activation, a treatment can be carried out before the activation in the stannous salt. Such an additional treatment is in one embodiment selected from the group consisting of treatment in alkali solution followed by neutralization in an acid solution, treatment in an NaOH solution followed by neutralization in HC1, treatment in an alkali solution upon heating to less than 90°C, treatment in an alcohol, and treatment in isopropanol.

[0135] Some polymeric objects are known to be difficult to coat in general such as for instance polytetrafluoroethylene (PTFE) . For such difficult objects comprising for instance polytetrafluoroethylene (PTFE) , polyether ether ketone (PEEK) , polypropylene, and hydroxyapatite, an alternative pre-treatment can be used to improve the adhesion to the object. In one embodiment, a pre-treatment is performed before the coating. A plasticizer based on an aliphatic polyisocyanate is dissolved in a solvent. Suitable solvents include but are not limited to n-butyl acetate, isopropanol, and xylene. The dissolved plasticizer is applied to the object to be coated and then dried. The concentration of plasticizer is adapted so that the dried layer of plasticizer is only a few molecules thick in one embodiment. For such a thin coating, there are no essential changes in most of the physical properties of the object. When the surface has been cured, the coating can proceed. By using this pre-treatment good adhesion is obtained for difficult objects comprising polytetrafluoroethylene (PTFE) , polyether ether ketone (PEEK) with carbon composite filler, nonwoven materials based on polypropylene, and hydroxyapatite. After the pre-treatment, the object is rinsed in demineralized water in one embodiment.

[0136] In one embodiment, colloidal suspensions of metals are used to obtain particles on the surface. The particles comprise a mixture of metals so that the desired composition is reached, i . e . all of the particles comprise the desired composition of metals . The particles are deposited from a suspension of the desired particles . The composition of the particles in the suspension is adj usted according to the desired amounts of the metals to be in the particles . The obj ect is dipped in the suspension of particles for a period from about a few seconds to about a few minutes or longer .

[0137] The substrate is treated with the suspension for a period from about a few seconds to about a few minutes or longer . After the treatment , the substrate is rinsed in a solvent or water such as demineralised water and left to dry in room temperature .

[0138] The particles can also be made from a solution comprising metal ions . The presence of palladium together with the activation with stannous ions gives an autocatalytic reaction so that metal ions of palladium, and gold are reduced to elemental metal in particles on the surface . The amount of gold ions and palladium ions in the solution is not the only factor which determines the amount of deposited particles on the surface . The type of substrate surface is also important . For instance on a metal substrate the amount of deposited particles tends to be higher compared to a non- metal substrate . For a metal substrate the type of metal and other factors determines the deposited amount of particles . Thus a measurement of the actual deposited amount should be made for each type of substrate and deposition solution to ensure the correct applied amount .

[0139] As evidenced by the appended experimental data both known coating (with silver ) and no-silver BG coatings , i . e . coatings according to the invention consistently demonstrate a higher degree of blood compatibility compared to uncoated material s . Thi s i s most evident in the signi ficantly lower generation of Thrombin-Antithrombin ( TAT ) complex, a primary marker for blood coagulation activation (Examples 9 , 11 ) .

[0140] It should be noted that the no-silver BG coating, i . e . the coating according to the invention provides a level of blood compatibility that is simi lar to the traditional silver- containing BG coating according to the prior art . The conclusion of Example 9 is that the no silver coating, ( i . e . according to the invention) shows similar blood compatibility when compared to the BG coating with silver . Thus it can be concluded that when carefully selecting the amounts of gold and palladium, then it is surpris ingly possible to exclude the silver and obtain a surface coating which has similar blood compatibility and antimicrobial properties , and in addition without the disadvantages of silver, such as risk for discoloration . This is a signi ficant benef it i f avoiding silver is desirable due to cost , regulatory concerns , or other factors .

[0141] In particular it should be noted that formulations of the no-silver coating according to the invention showed superior performance over the coatings according to the prior art . Speci fically, the no-silver coatings on silicone with 11 % and 16% compositions resulted in a lower release of PMN elastase ( an inflammatory marker ) and a lower generation of TAT complex compared to the uncoated strips . Their performance was comparable to or better than the standard silver-containing BG coating in these aspects (Example 11 ) . This makes these speci fic no-silver formulations a better choice in applications where minimi zing both clotting and inflammation is a priority .

[0142] The no-si lver BG coating on titanium surfaces was shown to delay the time it takes for a blood clot to start forming when compared to an uncoated titanium surface (Example 12 ) .

[0143] The no-silver coating according to the invention is better in any scenario where the performance of the silver- containing BG coating according to the prior art is desired, but the inclusion of silver itsel f is not is not desired . Since it provides similar blood compatibi lity it serves as an ef fective replacement .

[0144] Other features of the invention and their associated advantages will be evident to a person skilled in the art upon reading the description and the examples . It is understood that the disclosed embodiments can be freely combined with all other embodiments as long as it is not clearly contradictory .

[0145] It is to be understood that this invention is not limited to the particular embodiments shown here . The following examples are provided for illustrative purposes .

[0146] In the following, each of the described methods , apparatuses , examples and aspects , which do not ful ly correspond to the invention as defined in the claims is thus not according to the invention and is , as well as the whole following description, present for illustration purposes only or to highlight speci fic aspects or features of the claims Examples

[0147] Example 1 , comparative, not according to the invention

[0148] A sheet of medical grade silcone was etched in a solution containing ethanol and sodium hydroxide for 6 minutes and then neutralized in 10 wt% Hydrochloric acid and subsequently rinsed in deionized water. This was followed by an immersion in a water based solution containing 0.02 g / 1 Tin ( I I ) chloride for 5 minutes. After rinsing, it was dipped in a noble metal solution containing 0.009 g / 1 palladium ions and 0.004 g / 1 gold ions.

[0149] Analyzing the amount of noble metals showed a palladium concentration of 0.13 pg / cm2and 0.06 pg / cm2of gold. This means 32 wt% gold in the coating.

[0150] The Ahearn test is an in vitro test used to evaluate the adhesion of bacteria to a substrate compared to a control surface. The Ahearn test is described in D.G Ahearn et al, Current Microbiology, 2000:41, 120-125.

[0151] Measuring the bacterial adhesion in the Ahearn test showed a reduction of 36 % compared to functioning samples defined as those with a bacterial adhesion reduction of above 80%. The functioning samples are samples, which fall within the claims .

[0152] Example 2, comparative not according to the invention

[0153] 3 cm long cylinders with a diameter of 3 mm of the titanium alloy TiGr5 were degreased and then treated in a solution containing 0.22 g / 1 of Tin ( I I ) chloride for 5 minutes. After rinsing it was immersed in a solution containing 0.01 g / 1 palladium ions and 0.003 g / 1 gold ions. After drying the samples were analyzed with an amount of 0.13 pg / cm2palladium and 0.06 pg / cm2gold. The gold is only 32 wt% of the total noble metal content and the Ahearn test showed only 48 % bacterial growth reduction, whereas samples falling within the claims have over 80 % reduction.

[0154] Example 3, comparative not according to the invention

[0155] Rods with a diameter of 3 mm of the titanium alloy TiGr5 were degreased and then treated in a solution containing 0.08 g / 1 of Tin ( I I ) chloride for 10 minutes. After rinsing it was immersed in a solution containing 0.018 g / 1 palladium ions and 0.003 g / 1 gold ions. After drying the samples were analyzed with an amount of 0.52 pg / cm2palladium and 0.25 pg / cm2gold. The gold is only 32 wt% of the total noble metal content .

[0156] A Chandler loop test was carried out and it showed as much blood clotting as the control sample. This means that the surface coating did not have any noticeable effect on the blood clotting. There was no change in TAT value compared to the control and measurements of the inflammatory response were not possible.

[0157] Example 4, comparative , not according to the invention

[0158] Rods with a diameter of 3 mm of the titanium alloy TiGr5 were degreased and then treated in a solution containing 0.12 g / 1 of Tin ( I I ) chloride for 5 minutes. After rinsing it was immersed in a solution containing 0.012 g / 1 palladium ions and 0.015 g / 1 gold ions. After drying the samples were analyzed with an amount of 0.30 pg / cm2palladium and 0.78 pg / cm2gold. The gold is 72 wt% of the total noble metal content . A Chandler loop test was carried out and it showed as much blood clotting as the control sample. This means that the surface coating did not have any noticeable effect on the blood clotting. There was no change in TAT value compared to the control and measurements of the inflammatory response were not possible.

[0159] Example 5a

[0160] A percutaneous dressing consisting of a polyurethane foam was coated with gold and palladium. It was rinsed and then immersed in a water based solution containing 0.1 g / 1 Tin ( I I ) chloride for 5 minutes. After rinsing, it was dipped in a noble metal solution containing 0.12 g / 1 palladium ions and 0.055 g / 1 gold ions. Analyzing the amount of noble metals showed a palladium concentration of 0.3 pg / cm2and 0.3 pg / cm2of gold. (50 wt% gold)

[0161] Measuring the bacterial adhesion in the Ahearn test showed a reduction of 95 %.

[0162] Exampl e 5b

[0163] Two different titanium rods made of the alloy TiGr5 were coated. It was rinsed and then immersed in a water based solution containing 0.1 g / 1 Tin ( I I ) chloride for 5 minutes. After rinsing, it was dipped in a noble metal solution containing 0.12 g / 1 palladium ions and 0.055 g / 1 gold ions. Analyzing the amount of noble metals showed a palladium concentration of 0.55 pg / cm2and 0.5 pg / cm2of gold. (48 wt% gold)

[0164] Measuring the bacterial adhesion in the Ahearn test showed a reduction of 97 % and 99 respectively for the two different s amp les. Example 5c

[0165] Medical grade silicone samples were coated with a) palladium, gold and silver as well as b) only palladium and gold. An uncoated sample was included as well. The concentrations of the metals were as follows: a) palladium 0.3 pg / cm2, gold 0.1 pg / cm2, silver 1.2 pg / cm2. b) Palladium 0.3 pg / cm2, gold 0.3 pg / cm2. (50 wt% gold) c) Uncoated.

[0166] Pictures of the three different samples are shown in fig 1. Sample c) (uncoated) is the top sample in fig 1. Sample a) (including silver, i.e. not according to the invention) is the middle sample in fig 1. Sample b) (according to the invention) is the bottom sample in fig 1. It can be seen that the coating comprising silver has a slight grey tone, whereas the uncoated sample and the sample according to the present invention does not show a greyish tone.

[0167] Exampl e 6

[0168] Five different materials were investigated. All samples were made and analyzed in triplicate.

[0169] A) Polyurethane

[0170] A CVC-catheter made of polyurethane was as a pretreatment immersed in a solution containing a mixture of potassium permanganate and sodium hydroxide at a temperature of 50 degrees Celsius for 15 minutes. After rinsing, the surface had got a brownish color which consisted of manganous oxide. The catheter was then dipped in an aqueous solution containing 0.02 g / 1 stannous ions for 8 minutes so that the tin reacted with the oxygen in the manganous oxide. After rinsing, the catheter was treated for 10 minutes in a water- based solution containing 0.018 g / 1 palladium ions and 0.08 g / 1 gold ions. The sample was rinsed in deionized water and after that dried. A part of the catheter was used for analysis of the content of the noble metals. The results were 0.26 pg / cm2of palladium and 0.25 pg / cm2of gold.

[0171] B) Silicone

[0172] A sheet of medical grade silicone (PE-4062 from Sterne sas, France) was pretreated for 10 minutes in a solution containing water, ethanol and sodium hydroxide. After neutralizing in 10 % hydrochloric acid the sample was rinsed in deionized water and then immersed in an acid solution containing tin ( I I ) chloride . The amount of tin in the solution was 0.32 g / 1. After leaving the sample in the solution for 5 minutes it was then rinsed in deionized water and then dipped in a solution containing both palladium ions and gold ions. The amount of palladium ions was 0.018 g / 1 and for gold ions 0.008 g / 1.

[0173] A piece of the sheet was used for determining the content of the noble metals. The analysis showed a content of palladium of 0.15 pg / cm2and gold of 0.14 pg / cm2.

[0174] The remaining part of the sample was used for blood evaluation in the Chandler loop test.

[0175] C) Nitinol

[0176] A tubing of nitinol used for producing bare metal stents was used for coating with a mixture of gold and palladium. The sample was first cleaned with a common alkaline degreasing agent, rinsed in deionized water and after drying immersed in an etching solution consisting of a mixture of 0.2 % hydrofluoric acid and 2 % nitric acid for 5 minutes at room temperature. After a new rinsing the sample was dipped for 4 minutes in a solution containing an acid solution of tin ( I I ) chloride where the concentration of tin ions was 0.23 g / 1. After a new rinsing in deionized water the sample was immersed in an aqueous solution containing 0.032 g / 1 palladium and 0.12 g / 1 gold. After drying apart of the nitinol was used for analyzing. The content of palladium was 0.59 pg / cm2and for gold 0.65 pg / cm2.

[0177] The remaining part of the tubing was used for testing with blood according to the Chandler loop test and testing for various inflammatory markers could be done.

[0178] D) 316L Stainless steel

[0179] A part of a surgical instrument made of 316 L stainless steel medical grade was used for coating with the noble metals palladium and gold. As a first step, the sample was degreased in a degreasing solution from Turtle® and then rinsed in deionized water. The next step was a pretreatment in an etching solution for 6 minutes. The etching solution consisted of 0.3 % hydrofluoric acid and 1.5 % nitric acid. After a thorough rinsing the sample was placed for 8 minutes in a water based solution containing tin (IT) -ions. The concentration was 0.24 g / 1 of the ions. After a new rinsing, the sample was immersed in a gold / palladium solution containing 0.025 g / 1 palladium and 0.010 g / 1 gold. After rinsing and drying a part of the sample was used for analysis of the noble metals. The composition of the coating was 0.68 pg / cm2of palladium and 0.65 pg / cm2of gold.

[0180] The remaining part of the sample was used for blood analysis.

[0181] E) Titanium alloy A trauma implant material of the in the implant business common titanium alloy TiGr5 (T16A14V) was used for coating with gold and palladium. The titanium alloy TiGr5 (ASTM Grade 5) is an alpha-beta titanium alloy with a high specific strength and excellent corrosion resistance. The samples consisted of cylindrical rods with a diameter of 3 mm. First, the samples were degreased in a 5 % alkaline solution (Ikaclean®) at 40 °C for 5 minutes and later neutralized in 2 wt% hydrochloric acid followed by rinsing in deionized water. After that, an immersion in a sensitizing solution followed. The content in the solution consisted of 0.22 g / 1 tin ( I I ) chloride at a pH of 2.4 for 6 minutes. After a new rinsing, the samples were placed in a water based solution containing 0.018 g / 1 palladium and 0.008 g / 1 gold. After rinsing and drying a sample was analyzed and the result was 0.37 pg / cm2palladium and 0.53 pg / cm2gold. A SEM picture of this sample is shown in Fig. 2 in 150.000 times magnification. The particles of the coating can be seen as tiny white dots.

[0182] Some samples were used for testing with Chandler Loop.

[0183] For all samples A-E also three control samples including silver were manufactured. The amount of silver was in the interval 0.8 - 1.2 pg / cm2.

[0184] All samples were prepared and analyzed in triplicate and a mean and standard deviation was calculated using all measurements .

[0185] A summary of the different samples are provided in the following table:

[0186] All samples were contacted with blood at 37 °C in a Chandler loop apparatus. For all measurements there were i) an uncoated control sample, (control) , ii) the coating as described above (BG-Ag) , iii) the same coating as ii) but including silver (BG) . There were also a blood control with the first blood from the donor and which blood was not contacted with any samples to establish a zero level. Further, a loop control was made in only the Chandler loop to check the influence of the loop and any spontaneous activation of blood clotting.

[0187] For all samples, the following measurements were made. The measurements were repeated for all of the three samples of each material.

[0188] The units in the above table is used for all values.

[0189] The different analyses were carried out as follows:

[0190] Platelets (PTL) : Analysis carried out at Danderyd hospital, Karolinska Universitet Laboratoriet , registered with the id: Studie S-2181 Ytbehandling av CVK. The laboratory has been accredited by Swedac Acreditering . Accreditation nr: 1886

[0191] PMN : A kit from abeam for Human PMN elastese ELISA, Ref. AB19553-1001 Human PMN Elastase ELISA Kit is a Sandwich

[0192] (quantitative) ELISA kit for the measurement of Human PMN Elastase .

[0193] TAT: a kit from Siemens Healthinner. Enzygnos®TAT micro Ref. OWMG15. For quantitative determination of thrombin / antithrombin III complex (TAT) in human plasma.

[0194] FPA: from Cloud-Clone Corp. ELISA kit for FPA: Ref. CEB307HU. An ELISA Kit for Fibrinopept ide A (FPA) .

[0195] IL-1, IL-6, IL-8, and TNF-a: a kit from MSD: V-PLEX

[0196] Proinf lammatory Panel 1 Human Kit. Ref. A151A1H-1. MCP-1: a kit from MSD: Chemokine panel 1 Gen B kit : Ref. K15705.

[0197] VEGF: a kit from MSD: Human Angiogenesis: Ref. A151A6H-1 for Human VEGF.

[0198] C3a: a kit MicroVue Complement. MicroVue C3a plus EIA Ref A032. MicroVue C3a Plus Enzyme Immunoassay is for the measurement of C3a in plasma and serum.

[0199] The instructions provided by the manufacturers of the kits were followed.

[0200] In the below table the results from the measurements on the different substrates are summarized. The result is given as a mean of three different measurements, where the average ± one standard deviation is given.

[0201]

[0202] The number of signi ficant figures in the table does not necessarily correspond to the measurement accuracy in the experiments .

[0203] As a brief summary, it can be concluded that within the calculated standard deviations only a few properties are better without silver . Within the calculated standard deviations it can be concluded that the coating without silver is definitely not worse than the coating including silver . I f disregarding the calculated standard deviations it can be seen a trend that some properties are improved without silver, although it is not statistically signi ficant .

[0204] The coating without silver has a number of other advantages , such as no discoloration, and it has been shown that the coating without silver according to the present invention can be used instead of the coating including silver with respect to blood compatibility as well as antimicrobial properties .

[0205] Exampl e 7

[0206] When biomaterials come into contact with biological fluids such as blood, an immediate and spontaneous adsorption of proteins around the foreign material occurs. This reaction is responsible for a cascade of reactions that leads to acceptance or rejection by the body. The adsorption process is complex and dynamic that depends on many parameters such as the physical and chemical properties of the material including hydrophilic / hydrophobic condition, surface charge and roughness. The adsorption of proteins is also dependent on other types of parameters such as chemical composition of the proteins, protein complexity and concentration. In addition to these, the pH of the medium and the presence of ionic strength, the temperature and the flows to which the biomaterial is subjected must also be taken into account. Furthermore, the reversibility or irreversibility of protein adsorption, Vroman effect, multilayer adsorption, energetics and many other parameters have to be taken into account too.

[0207] There are a number of technologies to study protein adsorption such as radiolabeling, spectroscopy , quartz crystal microbalance (QCM) , atomic force microscopy (ARM) , surface plasmon resonance (SPR) and fourier transform infrared spectroscopy (FTIR) . All these methods are unable to give us a discriminatory identification of the protein composition of complex samples such as blood proteins. On the other hand, mass spectroscopy (MS) is selective and sensitive enough for the determination of large amounts of peptides and proteins. It is necessary to desorb the proteins from the exposed surface in order to carry out the analysis.

[0208] In order to perform this analysis on silicone materials with the present coating, an analysis protocol known as on-surface enzymatic digestion (oSED) was used, in which the proteins adsorbed on the coated material were reduced, alkylated and enzymatically cleaved. These peptides were desorbed and monitored using high- performance liquid chromatography technique with tandem mass spectrometry (LC-MS / MS) . The results can be interpreted as the peptide sequences that were involved in the protein adsorption process.

[0209] Samples were made on silicone following the method of example 6 B. The following samples were made with the following amounts of metals on the surface:

[0210] BG coating: Ag: 0.8 pg / cm2, Pd: 0.15 pg / cm2, Au 0.14 pg / cm2.

[0211] BG coating without silver: Pd: 0.15 pg / cm2, Au 0.14 pg / cm2. (48 wt% gold)

[0212] Briefly, BG coating and BG coating without silver (BG - Ag) silicone samples were incubated with anticoagulated EDTA blood at 37°C. Afterwards, the bound proteins were oSEDed with trypsin and analyzed using LG- Orbitrap MS / MS at the MS Facility. A high number of proteins were found and identified against human (Homo sapiens) proteome. The peptide sequences related to fibrinogen is shown in Table 1. Fibrinogen is a fibrous glycoprotein present in human blood plasma that essential for haemostasis, wound healing, inflammation, angiogenesis, and other biological functions. Once the coagulation cascade is activated and the thrombin enzyme is formed, this macromolecule is converted to fibrin that forms an insoluble gel to prevents blood loss. This gel is a viscoelastic polymer characterized by its rigidity and flexibility properties that determine how the clot can respond to blood flow. Fibrinogen has integrin receptors specific for platelets, endothelial cells, leukocytes and other cell types, hence playing a crucial role in platelet aggregation and amplification of clot formation as well as in the inflammation reaction.

[0213] Fibrinogen molecule is composed of three different chains with different functions known as Alpha, Beta and Gamma chains . In the Gamma chain, integrin receptors for platelets are specifically found between amino acid sequences 19-38 ( FLSSTCVAYVATRDNCCILD) responsible for platelet aggregation, in which it binds specifically to glycoprotein lllb / llla (GPllb / llla) also known as CD41 . The GPllla receptor is found between amino acids 50-84 (IADFLSTYQTKVDKDLQSLEDILHQVENKTSEVK) whilst the platelet activation CD61-P and the region between amino acids 423 to 437 (GQQHHLGGAKQVRPE) for platelets aggregation . In this region there is also found the receptor for Staphylococci sp which are significant for bacterial clumping reaction. The region between amino acids 387 and 442 (NGYDNGI IWATWKTRW) has affinity for leukocytes ICAM bind receptor (CDllb / CD18, Mac-1, CR3) , which is important for the inflammatory response. Interpretation :

[0214] Once the proteins are adsorbed on the surfaces , they undergo a conformational change and can be spontaneous ly activated, inhibited or denaturatated . Several studies demonstrated that the abovementioned fibrinogen sequences lose their biological functions when blocked .

[0215] The sequences o f the peptides isolated from the surface of the BG coatings show that they had been adsorbed in such a way that their elution required a strong digestion and desorption proces s . The sequences involved in protein adsorption have lost their biological capacity, thus preventing both platelets and leukocytes from being recruited to the surface of the coated material . Consequently , this decreases the ampli fication of the potential activation of coagulation cascade , as well as reducing the poss ibil ity of the activation of the inflammatory reaction by the leukocyte recruitment . Most importantly, the presence of conformational change in the receptor for Staphyl ococci sp could lead to the poss ibil ity of less aggregation of staphyl ococci and therefore reducing the formation of a biof ilm and infection .

[0216] The sequences related to platelet adhesion and aggregation, namely DNCCILD, IADFLSTYQTK and GQQHHLGGAK, are al so neutral i zed due to the conformational change on the surface o f BG coated material s . Meanwhile , the si licone control material (without coating) presents only the sequence IADFLSTYQTK involved in adsorption whi le the others remain exposed to attract platelets and this i s one of the reasons why fibrin deposition and clot formation are observed on the uncoated surface . Thi s indicates that the sequences are unique characteristics of the peptides on the surface of materials with BG coating as well as BG-Ag coating. A conclusion is that the exact binding and mechanisms of action are slightly different between the BG and BG-Ag surface coatings.

[0217] Table 1. This table shows the different peptide sequences that were isolated from the control silicone, BG-coated silicone and BG-Ag-coated silicone material after exposure to blood.

[0218] Exampl e 8

[0219] 1. Purpose and scope

[0220] The purpose of this example is to evaluate the effect of blood compatibility to uncoated and Bactiguard (BG) coated silicone strips, using Chandler loop model (WI-02916) . The Chandler loop model has been used for evaluation of standard blood compatibility markers like platelet consumption, thrombin antithrombin ( TAT ) formation, PMN elastase release , C3a release , f ibrinopeptide A ( FPA) and f ibrinopeptide B ( FPB ) release using ELISA kits , fibrin deposition on the surface and inflammatory markers and vascular endothelial growth factor (VEGF) using Mesoscale MS Di .

[0221] There are three di f ferent kinds of coatings in this coating evaluation, one normal BG according to the prior art , one inventive BG coating with no silver ( -Ag) and one comparative BG coating with no silver but with neodymium ( - Ag + Nd) . The BG coating with no si lver ( -Ag) is according to the invention, whereas the others are comparative .

[0222] 2. Introducti on

[0223] The Chandler loop model is an ex-vi vo research and development tool to evaluate how the natural defense mechanism of the body is influenced when biomaterials are in contact with blood . The model determines platelet depletion from the blood as a consequence of their activation and adsorption to a surface , fibrin deposition on a surface , activation of the coagulation mechanism, activation of the complement system and degree of hemolysis . In the model , blood is drawn from healthy human donors and un- f ractioned heparin is added . The blood is exposed to the selected test material and controls in a PVC loop . One loop without test material ( control ) is included into each experimental set for control of the blood reaction with the PVC material . The loop is circulated on a vertical plate rotating at 10 rpm, which is submerged in a water bath at 37 ° C . The blood i s analyzed after 1 hour for the number of platelets, compared with the initial number in order to determine relative platelet depletion. The rest of the blood is then centrifuged, and the plasma is analyzed for hemolysis degree and blood markers. The test material is rinsed and photographed to perform visual inspection of fibrin deposits. For more details and information read The Chandler loop model, WI-02916.

[0224] 3. Material and methods

[0225] 3.1 Test material

[0226] Uncoated silicone samples and different batches of three different kinds of BG coated silicone strips. The BG coated silicone strips according to the invention were made according to Example 6B . The other samples including silver and including neodymium were made similarly but with the addition of the different metals.

[0227] Table 1. Materials used in the study

[0228] 3.2 Experimental setup

[0229] The present investigation included different blood donors identified as BAG-333, BAG-334, BAG- 335, BAG-336 and BAG- 337. Blood collection and handling was performed according to the Chandler loop model, WI-02916.

[0230] Table 2. Placement of the silicone strips in the loops, identical for all blood donors

[0231] 4. Results and discussion

[0232] 4.1 Baseline for measured blood parameters before experimental phase

[0233] The baseline parameters for platelets, TAT, FP A and FPB were normal levels in blood and demonstrate that the blood withdrawal did not activate the blood coagulation, see Table 3.

[0234] Table 3. Baseline levels of platelets, TAT-complex , PMN- elastase , C3a, FPA and FPB in the donor blood before experiment phase.

[0235] * the values are excluded because they are too low to calculate a reasonable mean value, with a large standard deviation , and are therefore considered outliers .

[0236] 4.2 Visual fibrin deposition

[0237] Figure 3 show fibrin deposition on the material after being in contact with blood from BAC-

[0238] 333, BAC-334, BAC-335, BAC-336 and BAC-337 and after staining using toluidine blue.

[0239] The visual control of the fibrin deposition is subjective and not quantitative and is only used as an indication. There is no visual difference in the fibrin deposition between uncoated and all three different BG coated silicone strips .

[0240] 4.3 Platelet depletion

[0241] The platelets (PTL) remaining in blood plasma after Chandlers loop experiment is presented in figure 4 and the table below.

[0242] Table 5. Percentage of remaining platelets, mean and standard deviation from each sample and experiment BAC-333, BAC-334, BAC-335, BAC-336 and BAC-337.

[0243] The presented data show no difference in PTL consumption between uncoated and all three different BG coated silicone strips .

[0244] 4.4 Thrombin antithrombin (TAT) complex

[0245] The TAT generation in blood plasma after Chandlers loop experiment is presented in figure 5 and the table below.

[0246] In figure 5, *=0.01-0.05 **=0.001-0.01 ***=0.0001-0.001.

[0247] Table 6. TAT complex formation in plasma, mean and standard deviation from each sample and experiment BAC-333 , BAC-334 , BAC-335, BAC-336 and BAC-337.

[0248] Uncoated silicone strips show the highest generation of TAT .

[0249] There is a lower amount of TAT generation when in contact with all three di f ferent BG coated silicone strips , especially for the BG -Ag +Nd coated silicone strips . The loop control shows that there is no additional activation of TAT generation .

[0250] 4 . 5 PMN elastase The PMN elastase release in blood plasma after Chandlers loop experiment is presented in figure 6 and the table below .

[0251] Table 7 . PMN el astase rel ease in pl asma , mean and standard deviati on from each sampl e and experiment BAC-333 , BAC-334 , BAC-335, BAC-336 and BAC-337.

[0252] * the values are excluded because they are too high to calculate a reasonable mean value, with a large standard deviation, and are therefore considered outliers. Due to the high standard deviation in the present evaluation, it is di f ficult to draw conclusion, even when looking at the loop control .

[0253] 4 . 6 C3a The C3a in blood plasma after Chandlers loop experiment is presented in figure 7 and the table below . Table 8. C3a release In plasma, mean and standard deviation from each sample and experiment BAC-333 , BAC-334 , BAC-335 , BAC-336 and BAC-337.

[0254] * the values are excluded because they are too high to calculate a reasonable mean value, with a large standard deviation, and are therefore considered outliers.

[0255] The presented data show no difference in C3a release between uncoated and all three different BG coated silicone strips. The loop control shows that there is no additional release of C3a.

[0256] 4.7 Fibrinopeptide A

[0257] FPA release in the different loops is shown in figure 8.

[0258] Table 9. FPA release in plasma, mean and standard deviationfrom each sample and experiment BAC-333 , BAC-334 , BAC-335, BAC-336 and BAC-337.

[0259] The presented data show no difference in FPA release between uncoated and all three different BG coated silicone strips. The loop control shows that there is no additional release of FPA. 4.8 Fibrinopeptide B

[0260] FPB release in the different loops is shown in figure 9.

[0261] Table 10. FPB release in plasma, mean and standard deviation from each sample and experiment BAC-333 , BAC-334 , BAC-335rBAC-336 and BAC-337.

[0262] The presented data show no difference in FPB release between uncoated and all three different BG coated silicone strips. However, there is a trend of higher release of the BG-Ag+Nd coated silicone strips. The loop control shows that there is no additional release of FPB.

[0263] 4.9 Interleukin Ip

[0264] IL-l / p release in the different loops is shown in figure 10.

[0265] There is no difference between uncoated and all three different BG coated silicone strips in the release ofIL-lp. However, there is a trend of a lower amount of IL-lp release when in contact with all three different BG coated silicone strips, especially for the BG-Ag coated silicone strips. The loop control shows that there is no additional activation of IL-lp release.

[0266] 4.10 Interleukin 6

[0267] IL-6 release in the different loops is shown in figure 11. The graph shows no difference between uncoated silicone strips and all three different BG coated silicone strips. However, there is a trend of lower release of IL-6 for the three different BG coated silicone strips. The loop control shows that there is no additional release of silicone strips .

[0268] 4.11 Interleukin 8

[0269] IL-8 release in the different loops is shown in figure 12.

[0270] The graph shows no difference in the IL-8 release. However, there is a trend of lower release of all three different BG coated silicone strips.

[0271] 4.12 MCP-1

[0272] MCP-1 release in the different loops is shown in figure 13.

[0273] The presented data shows no difference in MCP-1 release between uncoated and all three different BG coated silicone strips. The loop control shows that there is no additional release of MCP-1 release.

[0274] 4.13 TNFa

[0275] TNFa release in the different loops is shown in figure 14.

[0276] The graph shows no difference between uncoated and all three different BG coated silicone strips in the release of TNFa. However, there is a trend of lower release when in contact with BG-Ag coated silicone strips. The loop control shows that there is no additional release of TNFa.

[0277] 4.14 VEGF

[0278] VEGF release in the different loops is shown in figure 15. The presented data show no difference in VEGF release between uncoated and all three different BG coated silicone strips. The loop control shows that there is no additional release of VEGF release.

[0279] 5. Summary and conclusion

[0280] 5.1 Summary

[0281] ♦♦♦ There is no visual difference in the fibrin deposition between uncoated and all three different BG coated silicone strips .

[0282] ♦♦♦ The generation of TAT complex show a difference when comparing the uncoated and all three different BG coated silicone strips.

[0283] ♦♦♦ The release of IL-lp, IL-6, and IL-8 shows no difference but trends of lower release of these chemokines when in contact with all three different BG coated silicone strips compared to the uncoated silicone strips were observed.

[0284] ♦♦♦ The release of TNFa shows no difference but trends of lower release when in contact with the BG -Ag coated silicone strips compared to the uncoated silicone strips was observed .

[0285] ♦♦♦ The PTL consumption and the release of PMN elastase, C3a, FPA, FPB, MCP-1 and VEGF show no difference when comparing the uncoated and all three different BG coated silicone strips .

[0286] ♦♦♦ BG-Ag+Nd coated silicone strips show a trend of higher release of FPB. However, FPA does not show the same trend. Higher release of FPB could potentially be caused by the background noise as FPB releases after the release of FPA. 5 . 2 Conclusion

[0287] It seems that the BG coated silicone strips show a low degree of thrombo-inf lammatory reactions . The results presented in this technical report provide more emphasi zed explanation of the mode of action of the BG coated silicone strips .

[0288] Exampl e 9

[0289] 1 . Purpose and scope

[0290] The purpose of this example is to evaluate the ef fect of blood compatibility of coated TiGr5 and silicone with di f ferent Au concentrations in the coating solution using Chandler loop model (WI- 02916 ) .

[0291] The Chandler loop model has been used for evaluation of standard blood compatibility markers like platelet consumption, thrombin antithrombin ( TAT ) formation, PMN elastase release .

[0292] 2. Introducti on

[0293] The Chandler loop model is an ex-vi vo research and development tool to evaluate how the natural defense mechanism of the body is influenced when biomaterials are in contact with blood . The model determines platelet depletion from the blood as a consequence of their activation and adsorption to a surface , fibrin deposition on a surface , activation of the coagulation mechanism, activation of the complement system and degree of hemolysis . In the model , blood is drawn from healthy human donors and un- f ractioned heparin is added . Blood is exposed to the selected test material and controls in a PVC loop . One loop without test material ( control ) is included in each experimental set for control of the blood reaction with the PVC material . The loop is circulated on a vertical plate rotating at 10 rpm, which is submerged in a water bath at 37°C. The blood is analyzed after 1 hour for the number of platelets, compared with the initial number in order to determine relative platelet depletion. The rest of the blood is then centrifuged, and the plasma is analyzed for hemolysis degree and blood markers. The test material is rinsed and photographed to perform visual inspection of fibrin deposits. For more details and information read The Chandler loop model, WI-02916.

[0294] 3. Material and methods

[0295] 3.1. Test material

[0296] Both uncoated and BG coated TiGr5 and silicone were used. The TiGr5 samples were coated as in Example 6E and the silicone samples were coated as in Example 6B . The samples according to the invention are denoted BG(-Ag) and the comparative samples with silver are denoted as BG.

[0297] Table 1 Materials used in the study

[0298] 3.2. Experimental setup

[0299] The present investigation included different blood donors identified as BAC-355, BAC-356 and BAC-357. Blood collection and handling was performed according to the Chandler loop model, WI-02916.

[0300] The samples denoted BG(-Ag) were according to the invention. Table 2. Placement of the CVC samples in the loops, identical for all blood donors

[0301] 4. Results and discussion

[0302] 4.1. Baseline for measured blood parameters before experimental phase

[0303] The baseline parameters for platelets, and TAT were normal levels in blood and demonstrate that the blood withdrawal did not activate the blood coagulation, see Table 3.

[0304] Table 3. Baseline levels of platelets and TAT-complex, PMN- elastase and FPA in the donor blood before experiment phase.

[0305] 4.2. Visual fibrin deposition

[0306] Figures 16A and 16B show fibrin depositions on the CVC after being in contact with blood from BAC-355, BAC-356 and BAC- 357. In figures 16A and 16B, * Uncoated sample not available due to scarcity of sample.

[0307] The visual control of the fibrin deposition is subjective and not quantitative and is only used as an indication. There is a small visual difference in the fibrin deposition between uncoated and BG coated TiGr5 and silicone samples.

[0308] 4.3. Platelet depletion

[0309] The platelets (PTL) remaining in blood plasma after

[0310] Chandlers loop experiment is presented in figure 17 and the table below.

[0311] Figure 17 shows the depletion of PTL in the different loops.

[0312] (A) TiGr5 (B) Silicone strips.

[0313] Table 5. Percentage of remaining platelets, mean and standard deviation from each sample and experiment BAC-355, BAC-356 and BAC-357.

[0314] ★Uncoated sample not available due to scarcity of sample.

[0315] The presented data shows no difference in PTL consumption between uncoated and BG coated TiGr5 and silicone, for all different coatings. 4.4. (TAT) complex

[0316] The TAT generation in blood plasma after Chandlers loop experiment is presented in figure 18 and the table below.

[0317] In figure 18, generation of TAT in the different loops is shown, wherein (A) TiGr5 (B) Silicone strips. The samples denoted No-Ag are according to the invention. *0.01-0.05

[0318] Table 6. TAT complex formation in plasma, mean and standard deviation from each sample and experiment BAC-355 , BAC-356 and BAC-357.

[0319] *Uncoated sample not available due to scarcity of sample.

[0320] Uncoated TiGr5 and silicone show the highest generation of TAT compared to their respective samples. TiGr5 samples generates TAT more than 5 times higher as compared to silicone samples. The loop control shows that there is no additional activation of TAT generation.

[0321] 4.5. PMN Elastase

[0322] The PMN elastase release in blood plasma after Chandlers loop experiment is presented in figure 19 and the table below . Table 7. PMN elastase release in plasma, mean and standard deviation from each sample and experiment BAC-355 , BAC-356 and BAC-357.

[0323] Uncoated sample not available due to scarcity of sample.

[0324] **The absorbance was higher than the highest standard thus not available

[0325] Silicone samples exhibit higher PMN elastase generation, even higher than the highest standard that was tested. BG coated TiGr5 shows higher PMN elastase generation but no conclusion can be drawn due to high standard deviation within the respective group. The loop control shows that there is no additional release of PMN elastase.

[0326] 4.6. Fibrinopeptide A

[0327] Generation of FPA in the different loops is shown in figure 20. (A) TiGr5 FPA of uncoated TiGr5 was higher than the highest standard [lOOpg / mL] thus not available (B) Silicone.

[0328] Table 8. FPA generation in plasma, mean and standard deviation from each sample and experiment BAC-355 , BAC-356 and BAC-357.

[0329] * Uncoa ted sample not available due to scarci ty of sample .

[0330] * *The absorbance was higher than the highest s tandard thus not available

[0331] Higher FPA generation was observed for both uncoated TiGr5 and silicone when compared to their respective BG coated samples . Within the TiGr5 group, standard deviation was too high to draw any conclusion . Meanwhile , within the silicone group, although the uncoated silicone was higher than BG coated silicone , no di f ference was observed .

[0332] 5. Summary and concl usi on

[0333] 5 . 1 Summary

[0334] There is a small visual di fference in the fibrin deposition between uncoated and BG coated TiGr5 and silicone samples ♦♦♦ There is no di f ference in PTL consumption between uncoated and BG coated TiGr5 and silicone , for all di f ferent coatings .

[0335] ♦♦♦ Uncoated TiGr5 and silicone show the highest generation of TAT compared to their respective samples . TiGr5 samples generates TAT more than 5 times higher as compared to silicone samples .

[0336] ♦♦♦ Silicone samples exhibit higher PMN elastase generation, even higher than the highest standard that was tested . BG coated TiGr5 shows a trend of higher PMN elastase generation . ♦♦♦ Higher FPA generation was observed for both uncoated TiGr5 and silicone when compared to their respective BG coated s amp les .

[0337] 5 . 2 . Conclusion

[0338] Both BG coated TiGr5 and silicone show high degree of blood compatibility in terms of activation of coagulation ( TAT and FPA) . No si lver coating, ( i . e . according to the invention) shows similar blood compatibility when compared to the BG coating with silver .

[0339] Exampl e 10

[0340] 1 . Purpose and scope

[0341] The main obj ective of this example is to investigate the structure of the Bactiguard (BG) non silver coating on TiGr2 plates with di f ferent gold (Au ) concentrations in the mixture of gold / palladium bath in the production process . This material was used in the development of the new concentrate , where silver was eliminated as a component metal of the BG coating . Bactiguard (BG) non silver coating is according to the invention .

[0342] 2. Background

[0343] The resolution capacity of the human eye is limited, being able to distinguish a distance of only 0 . 2 mm separation but using lenses it can reach at least 0 . 1 mm . Due to this limitation, the microscope was developed as an ef fective tool for studying and characteri zing materials . Two fundamental factors for the development of the microscope are the number of lenses used, and the wavelength of the light source used . This is why microscopes are divided into optical microscopy ( OM) with a visible light source and electron microscopy (EM) that uses a beam of accelerated and focused electrons. Although investigation of material surfaces can be performed with both, EM is preferred because the accelerated electrons focused on the surface provide better image resolution with a fine and very small scale.

[0344] There are two main types of electron microscopy (EM) : scanning electron microscopy (SEM) and transmission electron microscopy (TEM) . In the present report, we used SEM because it can detect the electrons emitted from the surface providing information on the shape, height, size and distribution of the immobilized particles as well as the morphology and composition of the BG coating producing black and white dimensional and 3D images for easy interpretation.

[0345] From preliminary experiments (REP-20503) , high degree of blood compatibility was observed from the gold / palladium compound between 11-16% for silicone strips. Therefore, it is necessary to investigate whether there is a relationship in the size of the surface immobilized particles if the concentration of the gold is varied in the coating process of BG non-silver technology.

[0346] 4. Material and procedure

[0347] 4.1. Test materials

[0348] The TiGr2 plates used in the present investigation consist of pure titanium in which the surface has a natural layer of titanium dioxide. The coating is non-silver coating with different Au concentrations in the mixture of the gold / palladium bath in the production process. The coating procedure of Example 6E was used, but with different concentrations of gold and palladium. The percentages for Au referred in this example (5 wt%, 7.5 wt%, 10 wt%, 12.5 wt% , 15 wt% , 30 wt% , and 80 wt% ) refer to the percentage of the gold compound which is used to make the solution of gold ions and palladium ions . The selection of the gold compound and palladium compound is not critical as long as there are gold and palladium ions in the solution . The amount of gold in the particles of the finished coating is in the claimed interval 45- 60 wt% for the percentages 7 . 5 wt% , 10 wt% , 12 . 5 wt% , and 15 wt% . The percentages in this example re fers to percentage of the gold compound which is used to make the solution of metal ions . The method outlined in Example 6E was used for the coating .

[0349] Table 1. Test materials Material identification

[0350] RND25-004-01 Uncoated TiGr2 plate

[0351] RND25-004-02 TiGr2 plate coated Au 5%

[0352] RND25-004-03 TiGr2 plate coated Au 7.5%

[0353] RND25-004-04 TiGr2 plate coated Au 10%

[0354] RND25-004-05 TiGr2 plate coated Au 12.5%

[0355] RND25-004-06 TiGr2 plate coated Au 15%

[0356] RND25-004-07 TiGr2 plate coated Au 30%

[0357] RND25-004-08 TiGr2 plate coated Au 80%

[0358] 4 . 2 . Experimental procedure

[0359] SEM pictures of both uncoated and BG coated TiGr2 plates were taken using Zeiss Ultra 55 GEMINI , a high-resolution field emission SEM at SWERIM Kista . No sputtering was performed on the test materials . Evaluation of SEM pictures was conducted in accordance with the working instructions described in Example 14 .

[0360] 5. Resul ts

[0361] Figure 21 summari zes the SEM evaluation for BG non-silver coated TiGr2 plates with 5% Au . The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 29.6 nm, which has a density of 47.9 particles per square micrometer. The majority of particles are observed in the 20 - 40 nm range. However, an equal and low distribution is also observed between the diameters 50 - 80 nm. The generated plot of the surface shows that the immobilized particles do not have a great height.

[0362] Figure 22 shows SEM analysis of BG non-silver coated TiGr2 plate with 7.5% Au.

[0363] The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 50.2 nm, which has a density of 48.3 particles per square micrometer. The majority of particles are observed in the 10 - 20 nm range. However, an equal and low distribution is also observed between the diameters 30 - 50 nm and between 70 - 110 nm. The surface plot generation shows a different structure for the immobilized particles with a larger diameter. These large particles present a formation in the form of an annular crown. The small diameter particles show the same trend as the previous figure Au 5%.

[0364] Figure 23 summarizes the SEM evaluation for BG non-silver coated TiGr2 plates with 10% Au.

[0365] The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 36.9 nm, which has a density of 33.0 particles per square micrometer. The majority of particles are observed in the 10 - 20 nm range. However, an equal and low distribution is also observed between the diameters 30 - 100 nm. The surface plot generation shows a different structure for the immobilized particles with a larger diameter. These large particles present a formation in the form of an annular crown. The small diameter particles show the same trend as the previous figure Au 5%.

[0366] Figure 24 summarizes the SEM evaluation for BG non-silver coated TiGr2 plates with 12.5% Au.

[0367] The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 33.2 nm, which has a density of 50.7 particles per square micrometer. The majority of particles are observed in the 10 - 20 nm range. However, an equal and low distribution is also observed between the diameters 30 - 100 nm. The surface plot generation shows a different structure for the immobilized particles with a larger diameter. These large particles present a formation in the form of an annular crown .

[0368] Figure 25 summarizes the SEM evaluation for BG non-silver coated TiGr2 plates with 15% Au.

[0369] The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 37.1 nm, which has a density of 25.7 particles per square micrometer. The majority of particles are observed in the 10 - 20 nm range. However, an equal and low distribution is also observed between the diameters 40 - 90 nm. The surface plot generation shows a different structure for the immobilized particles with a larger diameter. These large particles present a formation in the form of an annular crown although not as defined as in the previous photographs. The small diameter particles show the same trend as the previous figure Au 5%.

[0370] Figure 26 summarizes the SEM evaluation for BG non-silver coated TiGr2 plates with 30% Au.

[0371] The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 31.0 nm, which has a density of 19.9 particles per square micrometer. The majority of particles are observed in the 10 to- 20 nm range. However, an equal and low distribution is also observed between the diameters 40

[0372] 90 nm. The surface plot generation shows a different structure for the immobilized particles with a larger or middle diameter. The small diameter particles show the same trend as the previous figure Au 5%.

[0373] Figure 27 summarizes the SEM evaluation for BG non-silver coated TiGr2 plates with 80% Au.

[0374] The SEM image shows a distribution of immobilized particles of different diameters. The average diameter of these particles is 18.3 nm, which has a density of 189.6 particles per square micrometer. The majority of particles are observed in the 10 nm. A decrease in particle diameter is observed from 20 nm, which declines to 60 nm with minimum values up to 110 nm. The surface plot generation shows a different structure for the immobilized particles with a larger or middle diameter. The small diameter particles show the same distribution trend as the previous figure Au 5% but the shape is sharper. Figure 28 summarizes the distribution of surface immobilized particles of BG no-silver coated materials with different Au concentrations based on the average diameter. The average diameter of surface immobilized particles is similar for the coating with different Au concentration except 7.5% and 80%. However, since the standard deviation is very high, no definitive conclusion can be made.

[0375] 6. Summary and conclusion

[0376] 6.1. Summary

[0377] ♦♦♦ In the present investigation, TiGr2 plates coated with BG non-silver technology show different distribution patterns in the diameter of the surface immobilized particles.

[0378] ♦♦♦ Predominantly, the diameter of the surface immobilized particles is between 10 and 30nm.

[0379] ♦♦♦ For smaller particles as shown in Au 5% and 80%, the geometric characteristic formation of the particles is triangular and pyramidal. Meanwhile, the particles of Au 7.5% to 30% are more complex, in which sharper edges with more pronounced walls and typical multiple-twin crown were observed .

[0380] ♦♦♦ For the samples according to the invention (7.5%, 10%, 12.5%, and 15% Au) the analysis showed that 2% or more of the number of the particles had a diameter of 100 nm or more .

[0381] ♦♦♦ Three different density per area were observed, namely Au 5% to 12.5% (45-50) , Au 15% and 30% (20-25) and Au 80% (> 180) . 6.2. Conclusion

[0382] The topographic formation of the surface immobilized particles of BG non-silver coating with different Au concentration could have an effect on the degree of biological compatibility of the coated material. Particularly, the protein adsorption, activation and inhibition to the crown of the coating and its biological effects .

[0383] Exampl e 11

[0384] 1. Purpose and scope

[0385] The purpose of this example is to evaluate the effect of blood compatibility on uncoated and Bactiguard (BG) coated silicone strips, using Chandler loop model (WI-02916) . The Chandler loop model has been used for evaluation of standard blood compatibility markers like platelet consumption, thrombin antithrombin (TAT) formation, PMN elastase release and f ibrinopeptide A (FPA) release using ELISA kits, fibrin deposition on the surface.

[0386] There are four different kinds of BG coatings in this coating evaluation, one normal BG, three BG coatings with no silver (-Ag) 6 wt%, 11 wt% and 16 wt%. The BG coatings (11 wt% and 16 wt%) with no silver are according to the invention.

[0387] 2. Introduction

[0388] The Chandler loop model is an ex-vivo research and development tool to evaluate how the natural defense mechanism of the body is influenced when biomaterials are in contact with blood. The model determines platelet depletion from the blood as a consequence of their activation and adsorption to a surface, fibrin deposition on a surface, activation of the coagulation mechanism, activation of the complement system and degree of hemolysis. In the model, blood is drawn from healthy human donors and un-f ractioned heparin is added. The blood is exposed to the selected test material and controls in a PVC loop. One loop without test material (control) is included into each experimental set for control of the blood reaction with the PVC material. The loop is circulated on a vertical plate rotating at 10 rpm, which is submerged in a water bath at 37°C. The blood is analyzed after 1 hour for the number of platelets, compared with the initial number in order to determine relative platelet depletion. The rest of the blood is then centrifuged, and the plasma is analyzed for hemolysis degree and blood markers. The test material is rinsed and photographed to perform visual inspection of fibrin deposits. For more details and information read The Chandler loop model, WI-02916.

[0389] 3. Material and methods

[0390] 3.1 Test material

[0391] Uncoated and four different kinds of BG coated silicone strips. The method described in example 6B was used for the coating albeit with different amounts of metal. The percentages 6 wt%, 11 wt% and 16 wt% refer to percentages of Au-compound used. The percentages 11 wt% and 16 wt% of Au compound give values within 45 - 60 wt% Au in the finished particles, whereas 6 wt% gives values slightly below the interval 45 - 60 wt% Au in the finished particles.

[0392] Table 1. Materials used in the study

[0393] 3.2 Experimental setup

[0394] The present investigation included different blood donors identified as BAC-338, BAC-339, BAC-340 and BAC-341. Blood collection and handling was performed according to the Chandler loop model, WI-02916.

[0395] Table 2. Placement of the silicone strips in the loops, identical for all blood donors.

[0396] 4. Results and discussion

[0397] 4.1 Baseline for measured blood parameters before experimental phase

[0398] The baseline parameters for platelets, TAT, PMN elastase, C3a and FP A were normal levels in blood and demonstrate that the blood withdrawal did not activate the blood coagulation, see Table 3.

[0399] Table 3. Baseline levels of platelets, TAT-complex , P MN- elastase , C3a and FP A in the donors ' blood before experiment phase.

[0400] * The value is excluded because it is higher than the highest standard and therefore considered as outlier .

[0401] 4.2 Visual fibrin deposition

[0402] Figures 29A and 29B show fibrin depositions on the silicone strips, after being in contact with blood from BAC-338, BAC- 339, BAC-340 and BAC-341.

[0403] In figures 29A and 29B, * represents Not available due to clotting of the blood.

[0404] The visual control of the fibrin deposition is subjective and not quantitative and is only used as an indication. There is no visual difference in the fibrin deposition between uncoated and all four different BG coated silicone strips. Notably, clotting happened to the uncoated silicone strip when exposed to blood from BAC-341.

[0405] 4.3 Platelet depletion

[0406] The platelets (PTL) remaining in blood plasma after Chandler loop experiment is presented in figure 30 and the table below .

[0407] Table 5. Percentage of remaining platelets, mean and standard deviation from each sample and experiment BAC-338 , BAC-339, BAC-340 and BAC-341.

[0408] *Not available as the blood clotted during the experiment.

[0409] **Not available as the data was not generated by the Kemlab at Danderyd hospi tai . The presented data show no difference in PTL consumption between uncoated and all four different BG coated silicone strips .

[0410] 4.4 Thrombin antithrombin (TAT) complex

[0411] The TAT generation in blood plasma after Chandler loop experiment is presented in Figure 31 and the table below. In figure 31, *=0.01-0.05

[0412] Table 6. TAT complex formation in plasma, mean and standard deviation from each sample and experiment BAC-338rBAC-339 , BAC-340 and BAC-341. ★Not available due to clotting of the blood during the experiment.

[0413] Uncoated silicone strips show the highest generation of TAT. There is a lower amount of TAT generation when in contact with all four different BG coated silicone strips, especially for the normal BG, BG -Ag 11 % and 16% coated silicone strips. The loop control shows that there is no additional activation of TAT generation.

[0414] 4.5 PMN Elastase

[0415] The PMN elastase release in blood plasma after Chandler loop experiment is presented in figure 32 and the table below. In figure 32, *=0.01-0.05

[0416] Table 7. PMN elastase release in plasma, mean and standard deviation from each sample and experiment BAC-338 , BAC-339 , BAC-340 and BAC-341.

[0417] BG -Ag 11 % and 16% coated silicone strips have lower release of PMN elastase compared to the uncoated silicone strips. However, due to the high standard deviation even for loop control, it is difficult to draw conclusions. 4.6 C3a

[0418] The C3a in blood plasma after Chandler loop experiment is presented in figure 33 and the table below.

[0419] Table 8. C3a release in plasma mean and standard deviation . from each sample and experiment BAC-338, BAC-339, BAC-340 and BAC-341.

[0420] ★Not available due to clotting of the blood.

[0421] The presented data show no difference in C3a release between uncoated and all four different BG coated silicone strips.

[0422] 4.7 Fibrinopeptide A (FPA)

[0423] The FPA in blood plasma after Chandler loop experiment is presented in figure 34 and the table below.

[0424] Table 9. FPA release in plasma, mean and standard deviation from each sample and experiment BAC-338 BAC-339 , BAC-340 and BAC-341.

[0425] *Not available due to clotting of the blood.

[0426] 5. Summary and conclusion

[0427] 5.1 Summary

[0428] ♦♦♦ There is no visual difference in the fibrin deposition between uncoated and all four different BG coated silicone strips, except for BAG-341.

[0429] ♦♦♦ There is no difference in platelet consumption, C3a and FP A generation between uncoated and all four different BG coated silicone strips.

[0430] ♦♦♦ There is a lower amount of TAT generation when in contact with all four different BG coated silicone strips, especially for the normal BG, BG -Ag 11 wt% and 16 wt% coated silicone strips. It is important to have low TAT generation for a biocompatible object when the object comes into contact with blood — especially in scenarios where triggering the coagulation cascade would be harmful or undesirable .

[0431] ♦♦♦ There is a lower amount of PMN elastase release when in contact with BG -Ag 11 wt% and 16 wt% coated silicone strips. The good values for the 11 wt% and 16 wt% is consistent with giving a gold content in the interval 45-60 wt% in the finished particles, i.e according to the invention.

[0432] 5.2 Conclusion

[0433] All BG coated silicone strips show a low degree of thrombo- inflammatory reactions. Notably, BG -Ag coated silicone strips have significantly lower TAT complex and PMN elastase as compared to the uncoated silicone strips . Therefore , these coatings demonstrate good compatibility with blood .

[0434] Exampl e 12

[0435] 1 . Purpose and scope

[0436] The aim of thi s example was to evaluate the clot formation and lysis between uncoated and Bactiguard (BG) no silver coated titanium grade 2 ( TiGr2 ) plate (non-anodi zed) with di f ferent Au concentration . Bactiguard (BG) no silver was according to the invention .

[0437] Although there are a lot o f factors that contribute to the activation of the thromboinf lammatory reaction, many of them are associated with the di f ferent components of the biomaterials . These contribute to undesired reactions when the biomaterials are exposed to blood or tissue that is constantly irrigated by blood, such as af fecting the bone structure , inducing collateral consequences such as worsening infections and poor osseointegration, and lastly as a fatal result , loss of the implant .

[0438] Both clot formation and fibrinolysis are part of the dynamic processes that regulate the thrombo-inf lammatory reaction and are directly related to the risk of bio-incompatibility of the material . This process is reflected in the structure and function of fibrin and its subsequent degradation . Collet and colleagues ( 2003 ) evaluated the structure / function of the fibrin clot in relation to biomaterials have shown that dense structures , with greater rigidity, lower permeability and less lysis of the clot are observed in materials that accelerate thrombin formation . It is important to be able to evaluate and identi fy the factors and how the BG coating and its di f ferent prototypes can influence the formation of the fibrin structure and fibrinolysis after been in contact with normal plasma (NP) . This can indicate the risk and bene fit of these prototypes and their potential applications .

[0439] One of the analyses that are commonly used to evaluate clot formation, its morphology and the degree of lys is in NP is based on turbidimetric monitoring of fibrin formation and its dissolution . Thi s method is known as the Turbidimetric Clotting Assay and Turbidimetric Lysis Assay .

[0440] Clotting and fibrinolysis are two biological defense phenomena which is initiated simultaneously in vi vo . Fibrinolysis can also occur without any individuali zed lys is front , known as intrinsic fibrinolysis .

[0441] Coagulation originates from two activation pathways that ended up forming fibrin that protects us from blood loss . The biological mechanism of tissue origin induces the generation of thrombin in an accelerated manner, which leads to a rapid trans formation of fibrinogen to fibrin . The other mechanism is related to the intrinsic activation of coagulation . This mechanism is oriented by the activation of the contact mechanism, mainly by the adsorption and activation of factor XI I which stimulates the cascade of enzymatic reactions .

[0442] Fibrinogen and fibrin are essential for hemostasis and are maj or factors in thrombosis , wound healing, inflammation, angiogenesis , and several other biological functions . Fibrinogen is a soluble macromolecule that is converted into fibrin (clot or insoluble gel) by the action of the serine protease thrombin. This is activated by a series of enzymatic reactions that can be triggered by an injury to the vascular wall, blood cells or foreign surface. The mechanical stability of the clot is important for hemostasis and promote tissue wound healing. Fibrin clots are dissolved by the fibrinolytic system, acting in a series of positive and negative feedback enzymatic reactions.

[0443] 1.1. Clot and lysis principal analysis of generated data

[0444] Figure 35 is an illustration of turbidimetric clotting and lysis variables. Turbidimetric clotting assay: lag time (Lagc) ; maximum absorbance (MaxAbsc) ; clot rate (CRc) . B Turbidimetric lysis assay: lag time (LagL) ; maximum absorbance (MaxAbsL) ; clot rate (CRL) ; lysis times Lys5Oto, Lys50MA, LysT; lysis rate (LR) ; area under curve (AUG) .

[0445] Lag time (Lagc) represents the time at which sufficient f ibrin-f ibres have been formed to enable lateral aggregation and coagulation. The monitoring process was taken as the time point at which an exponential increase in absorbance occurred .

[0446] Maximum absorbance of coagulation (MaxAbsc) occurs when all the fibrinogen is converted to fibrin by activation by thrombin. The observations were taken as the absorbance at which 3 consecutive readings were identical corrected for the Lagcabsorbance. The crude rate of clot formation (CRc) was derived from time and absorbance values for Lagcand MaxAbsc. For the turbidimetric lysis assay, in addition to the lag time (LagL) , maximum absorbance (MaxAbsL; the highest absorbance value adjusted for LagL) , and crude rate of clot formation (CRL) .

[0447] 2. Definitions and acronyms

[0448] Term Explanation

[0449] TiGr2 Titanium grade 2 plate material

[0450] Human plasma The secondary product after blood centrifugation

[0451] Normal plasma Human plasma pooled from different donors tPA Tissues plasminogen activator

[0452] CaC12 Calcium chloride

[0453] 4. Experimental material and method

[0454] The TiGr2 plates used in the present investigation consist of pure titanium with surface that has a natural layer of titanium dioxide. The material was coated with no silver coating with different Au concentration. For the coating, the method outlined in Example 6E was used but with different gold concentrations. The samples with percentages 7.5 wt%, 10 wt%, 12.5 wt%, and 15 wt% gave gold concentrations in the interval 45-60 wt% in the finished particles. Normal plasma (NP) was used and obtained according to WI-02916

[0455] The Chandler Loop

[0456] Model, Form 4 Protocol for preparation of pool plasma.

[0457] Table 2 Material information

[0458] 4.1. Method

[0459] 1. TiGr2 plates (1.5x0.3 cm) were incubated in 250 pL NP in a 96 well plate for 20 minutes.

[0460] 2. Another 96 well plate was prepared by adding 2 pL of 1 M CaCb to the well for clot analysis. 6 pL of 100 pg / mL tissue plasminogen activator (tPA) was added to the wells for lysis analysis, see table 3.

[0461] 3. TiGr2 plates were taken out and 100 pL of the NP was transferred to the prepared wells for turbidimetric assays (clot and lysis analysis) .

[0462] Table 3. The 96-well plate layout

[0463] 4. Plates were shaken and read at 340 nm every 12 sec for 1 hour using TECAN Sunrise, Magellan V.6.3 (2006) .

[0464] 5. A customised software was commissioned to analyse the large amount of raw data generated. 5. Results

[0465] The results are shown in the tables and figures 36 to 44 .

[0466] Table 4.

[0467] Figures 36 and 37 show examples of graphs generated from the software from one experimental with NP and uncoated control TiGr2 plate.

[0468] Figure 36 represents normal clot activation of NP (right) and clot activation of NP exposed to uncoated TiGr2 plate (left) .

[0469] Figure 37represents normal lysis of NP (right) and lysis of NP exposed to uncoated TiGr2 plate (left) .

[0470] Figures 38 and 39 show clot analysis from NP, uncoated control, seven different Au concentration BG coatings TiGr2 plate .

[0471] Figure 38 shows the time that takes for the clot to start forming, where NP, uncoated control and seven different Au concentration BG coatings TiGr2 plate was exposed.

[0472] Figure 39 shows the rate for the clot to start forming, where NP, uncoated control and seven different Au concentration BG coatings TiGr2 plate was exposed.

[0473] The uncoated TiGr2 plate showed shorter clotting time compared to NP and all different BG coated TiGr2 plate. The coated TiGr2 plates showed similar CRccompared to the uncoated TiGr2 plate and NP .

[0474] Figures 40 to 44 show lysis analysis from NP, uncoated control , seven di f ferent Au concentration BG coatings TiGr2 plate .

[0475] Figure 40 shows the time that takes for the lysis to start , where NP, uncoated control and seven di f ferent Au concentration BG coatings TiGr2 plate was exposed .

[0476] Figure 41 shows the rate for the lysi s to start , where NP, uncoated control and seven di f ferent Au concentration BG coatings TiGr2 plate was exposed .

[0477] Figure 42 shows the time duration from initiation for 50% completion of the lysis process , where NP, uncoated control and seven di f ferent Au concentration BG coating TiGr2 late was exposed .

[0478] Figure 43 shows the time duration from initiation to complete the lysi s process , where NP, uncoated control and seven di fferent Au concentration BG coatings TiGr2 late was exposed .

[0479] Figure 44 shows the rate for the lysis process , derived from MaxAbsLand the point at which absorbance values returned to baseline , where NP, uncoated control and seven di f ferent Au concentration BG coatings TiGr2 late was exposed .

[0480] The uncoated showed shorter lysis time compared to NP and all di f ferent BG coated TiGr2 plate . The coated plates showed slower lysis CRLcompared to the uncoated and NP . Lys5Oto of BG coated plates are slightly faster than uncoated and NP . The duration from initiation to completion of lysis process of different BG coated plates are between the uncoated and NP . The rate of lysis process of BG coated plates is similar to the uncoated and NP except Au 12.5% coated TiGr2 plate.

[0481] Table 7. Summary of all mean values of relevant analysis parameters of all three experiments with TiGr2 plates.

[0482] 6. Conclusions

[0483] All BG coated TiGr2 plates with different Au concentration show slower clot formation (Lagc) in comparison to the uncoated TiGr2 plate showing a good affinity of the coating with the coagulation activation system. Other parameters were similar between uncoated and BG coated TiGr2 plates.

[0484] Exampl e 13

[0485] In order to further illustrate the measurements using scanning electron microscopy according to ISO 19749:2021 the following working instructions are given. The working instructions were followed when analyzing the material.

[0486] Surface characterization is necessary for a better understanding of the physical and chemical properties. It is an important step in designing and optimizing material performance. The physicochemical properties of the materials can be characterized using various methods.

[0487] One of these is the scanning electron microscopy (SEM) , which evaluates the morphology, porosity and size of the immobilized structures present on the surface. When a sample is radiated with an electron beam, "elastic scattering" results from the atomic nucleus or the outer shell electrons with similar energy. The incident electrons that are elastically scattered at an angle larger than 90° are called backscattered electrons (BSEs) . BSE signals provide both compositional and topographic information of the sample. When the incident electrons and the electrons and atoms of the specimen interact, "inelastic scattering" occurs. During inelastic interactions, the accelerated electrons transfer considerable energy to the specimen atoms, causing the excitation of the sample electrons and leading to the generation of secondary electrons (SEs) defined with energies less than 50eV. SEs are the most extensively used signals in SEM, as the emitted electrons have low energy; they can only escape from a region within a few nanometers of the sample, providing accurate topographic information with good resolution. SEM contains an electron gun that accelerates the electrons through 0.1-30 keV. Electromagnetic lenses and apertures are used to focus the electrons on the beam and demagnify them to the size of the electron source (~50m for a tungsten filament) before the electron beam hits the sample to the required size (1-100 nm) . The analysis is performed in a high-vacuum environment to avoid electron scattering. SEM is a highly versatile tool for the structural characteri zation of biobased materials . These materials are , in general , composed of long chains of the repeating units of low-atomic- number elements like carbon, hydrogen, nitrogen, and oxygen . These lighter elements have scarcer interactions with the accelerated electrons , which yield poor contrast ; therefore, regularly coating or staining the polymers is necessary to study the sample . Most polymer samples are often sputter-coated with metals like gold to achieve optimum imaging with SEM .

[0488] SEM pictures must be taken with a minimum defined scale resolution of 100K . I f multiple images are to be evaluated, it is neces sary to keep the parameters constant throughout the evaluation so that the results can be compared . The sharpest images with a defined focus should be obtained . Three pictures should be taken at di f ferent representative locations of a sample . The pictures will then be analysed using Image J .

[0489] The image analysis tool , ImageJ, must be installed on the computer to evaluate the SEM pictures taken at SWERIM . The software is available for free at https : / / imagej . net / ij / download . html .

[0490] Pre-analysi s parameters

[0491] The SEM picture can be opened by either dragging the picture to the panel or opening directly from the software by clicking File > Open image . Di f ferent analyses can be performed on SEM pictures , but several parameters must be set beforehand, namely the scale , image type and threshold . Scale

[0492] Each SEM picture has its own scale bar that serves to provide a visual indication of distance on the picture. To obtain a unified analysis, the software's scale must be calibrated according to the scale bar. This can be achieved by following step :

[0493] 1. Select straight or continues line on the control panel and place it over the scale bar of the SEM picture

[0494] 2. Click Analyse > Set scale

[0495] 3. Enter the known distance (e.g. : 200) and the unit of length (e.g. : nm)

[0496] 4. Click OK

[0497] 5. Close the window

[0498] Image type

[0499] The SEM picture must be converted into bitmap. This can be done by clicking Image > Type> 8-bit.

[0500] Threshold

[0501] To adjust the threshold, click Image > Adjust > > Threshold. A new pop-up window will appear. Choose Default and B&W as shown on Figure 4. DO NOT reset the range. Instead, click on the black and white image generated by ImageJ and move the black slider (slider on the top) until the point where the curve begins (which can be between 100 to 120) . Then, move the white slider (slider at the bottom) below 253 to the point where the details of the letters on the SEM picture are removed. Apply and close the pop-up window.

[0502] Before analyzing the particles, go to the Analyze command bar and scroll to Set measurements. There, select Area> Mean Gray Values> Feret's Diameter. With the table generated during the analysis, you can use the area to obtain the equivalent diameter according to equation below or, if you prefer, you can use the Feret diameter for the following evaluations .

[0503] Analysis of SEM picture

[0504] Surface immobilized particles

[0505] There are two ways to analyse the surface immobilized particles, either analyse the entire area of the SEM picture or several representative areas (preferably 5 areas) . The procedure for both ways is the same as follows ( :

[0506] 1. Select the rectangle area selection tool on the control panel

[0507] 2. Mark the chosen area of analysis (either the entire picture or representative area as abovementioned)

[0508] 3. Click Analyse > Analysis of particles

[0509] 4. Set size between 10 to 40000

[0510] 5. Set circularity between 0.00 to 1.00

[0511] 6. Select show outlines

[0512] 7. Tick Display results (the measurements for each particle will be displayed) and Clear results (any previous measurements listed in the result table will be cleared)

[0513] 8. Click OK

[0514] Two new windows will be generated by ImageJ, namely the binary image and the results table. Save the binary image by clicking File > Save as > choose Tiff image. It is essential to save the binary image as it serves as the internal control during the data processing step. Save the results table by clicking File > Save as > choose excel or csv format.

[0515] Plot profile analysis

[0516] Plot profile analysis helps to identify the structure of the different types of particles by displaying a two-dimensional graph of the intensities of pixels along a line selection. The procedure to perform plot profile analysis is as follows :

[0517] 1. Reload the SEM picture

[0518] 2. Adjust the scale as described in 4.3

[0519] 3. Choose the straight-line selection tool 0 on the control panel

[0520] 4. Select one or more particle (s) by dragging the line completely across the particle

[0521] 5. Click Analyse> Plot profile

[0522] 6. Save the plot profile by clicking File > Save as

[0523] 7. The diameter and height of the particle (s) can be measured by the measure function in Analyse

[0524] Several measurements will appear in the generated result table. There are two relevant measurements, namely angle and length. The angle indicates the degrees in which the measurement was taken, both in diameter (-2° to +2°) and length (85° - 95°) . Length is the most important measurement that corresponds to the diameter and height of the particle (s) . Depending on the type of coating, several peaks might appear instead of a single peak. In this case, measure all the peaks and count the average as the peak for the particle .

[0525] Save all the measurement by clicking File> Save as.

[0526] Surface plot analysis

[0527] Surface plot displays a three-dimensional graph of the intensities of the pixels in a grayscale or pseudo colour image. The procedure to perform plot profile analysis is as follows :

[0528] 1. Reload the same SEM picture as the particle size analysis 2. Reset all the pre-analysis parameters

[0529] 3. Select the rectangle area selection tool on the control panel

[0530] 4. Mark the desired area of analysis

[0531] 5. Click Analyse> Surface plot

[0532] 6. Set Polygon multiplier at I 00%

[0533] 7. Select Shade, Draw Axis and Smooth

[0534] 8. Click OK

[0535] ImageJ will generate a 3-D surface plot of the immobilized particle (s) along with the measurements of the analysed particle ( s ) .

[0536] Analysis of the result

[0537] Particle measurement

[0538] There are two methods for evaluating the diameter of particles immobilized on a surface, namely using the Feret diameter or the equivalent diameter.

[0539] • The Feret diameter measures the distance between two parallel lines tangent to the outermost edges of a particle in a specific direction.

[0540] • The equivalent diameter assumes that particles are spherical and that the projected area corresponds to a real, measurable surface.

[0541] If the particles are not spherical, the Feret diameter provides a more accurate representation of their shape and size. However, the equivalent diameter is a simplified metric commonly used in physical and chemical calculations.

[0542] In the case of the BG coating, the immobilized particles are mostly circular, making the use of the equivalent diameter more objective and representative. It exhibits less variability, yields statistically robust results , and allows for comparisons with previous measurements .

[0543] Additionally, it is less dependent on orientation, since the Feret diameter can vary depending on the angle of measurement .

[0544] To further analyse the particle measurement obtained from Image J, open the raw data and proceed with following steps :

[0545] 1 . Open the excel file that contains the particle measurements

[0546] 2 . Convert the column using text to column command

[0547] 3 . Select the column ' Area ' and convert it from text to numeric format

[0548] 4 . Move the numeric format column to a new excel sheet

[0549] 5 . Calculate the particle diameter based on the area using the following formula :

[0550] .

[0551] Equivalent diameter

[0552] 6 . Determine the particle distribution range using COUNTI F function in excel as following table :

[0553] Table 2 : Excel formula for particle distribution range

[0554] Diameter and height ratio measurement

[0555] To further analyse the plot profile measurement obtained from ImageJ, open the raw data and proceed with following steps :

[0556] 1. Open the excel file that contains the plot profile measurement

[0557] 2. Convert the csv file to excel file 3. Adjust the height measurements according to the numbering order from the ImageJ generated results. If multiple particles were measured, calculate the average.

[0558] 4. Create a bar chart from the data

Claims

Claims1. An object, wherein there are particles on the surface of the object, the particles comprising palladium and gold, in a total amount of 0.25 - 1.40 pg / cm2, with gold constituting 45-60 wt% of the total amount and the particles comprising less than 30 ppm silver, and the outermost 10 pm of the object also comprising less than 30 ppm silver, wherein the mean particle diameter as measured according to ISO 19749:2021 is in the interval 20 - 80 nm and wherein particles with a diameter of 100 nm or more as measured according to ISO 19749:2021 constitute 2% or more of the total number of particles.

2. The object according to claim 1, wherein the object including the particles on the surface of the object comprises less than 30 ppm silver.

3. The object according to any one of claims 1-2, wherein the particles comprise less than 20 ppm silver.

4. The object according to any one of claims 1-3, wherein the particles comprise less than 10 ppm silver.

5. The object according to any one of claims 1-4, wherein the object is selected from the group consisting of medical devices, medical instruments, and medical disposable articles .

6. The object according to any one of claims 1-5, wherein the object is any object intended to be in contact with human or animal blood.

7. The object according to any one of claims 1-6, wherein the object comprises at least one metal and wherein the total amount of particles comprising palladium and gold on the surface is in the interval 0.8 - 1.40 pg / cm2.

8. The object according to claim 7, wherein the at least one metal is selected from the group consisting of iron, titanium, cobalt, nickel, chromium, and mixtures thereof.

9. The object according to claim 7, wherein the at least one metal is selected from the group consisting of steel, stainless steel, and nitinol.

10. The object according to claim 7, wherein the at least one metal is selected from the group consisting of medical grade titanium, medical grade stainless steel, and medical grade nitinol.

11. The object according to any one of claims 1-10, wherein the object comprises at least one ceramic material.

12. The object according to any one of claims 1-11, wherein the object comprises at least one selected from the group consisting of silicon nitride, and zirconium dioxide.

13. The object according to any one of claims 1-12, wherein the object comprises at least one selected from the group consisting of apatite, and hydroxyapatite.

14. The object according to any one of claims 1-13, wherein the object comprises at least one polymer and wherein the total amount of particles comprising palladium and gold on the surface is in the interval 0.25 - 0.8 pg / cm2.

15. The object according to any one of claims 1-14, wherein the object comprises at least one polymer composite and wherein the total amount of particles comprising palladium and gold on the surface is in the interval 0.25 - 0.8 pg / cm2.

16. The object according to anyone of claims 14-15, wherein the polymer is selected from the group consisting of latex, vinyl, polymers comprising vinyl groups, polyurethane urea, silicone, polyvinylchloride, polypropylene, styrene, polyurethane, polyester, copolymerisates of ethylene vinyl acetate, polytetrafluoroethylene (PTFE) , polyether ether ketone (PEEK) , polystyrene, polycarbonate, polyethylene, polyacrylate, polymethacrylate, acrylonitrile butadiene styrene (ABS) , polyamide, polyimide, and mixtures thereof.

17. The object according to any one of claims 1-16, wherein the object comprises at least one textile material.

18. The object according to any one of claims 1-17, wherein the particles are separated particles, not in contact with each other.

19. The object according to any one of claims 1-17, wherein at least a part of the particles are in contact with other particles to form agglomerates of particles.

20. The object according to any one of claims 1-19, wherein the particles have a size in the interval 10-500 nm as measured according to ISO 19749:2021.

21. The object according to any one of claims 1-20, wherein the object is at least one selected from the group consisting of a catheter, a central venous catheter, a peripheral venous catheter, a urinary catheter, a Foley catheter, an intermittent catheter, an implant, a dental implant, a dental abutment, a dental aligner, a dental prosthesis device, a bone replacing implant, an orthopaedic implant, a tissue replacing implant, a stent, a biliary stent, a tracheal stent, a peripheral stent, a glove, a pacemaker, a rupture net, a surgical instrument, a blood bag, an artificial heart valve, a vascular port, a haemodialysis equipment, a peritoneal dialysis equipment, a plasmapheresis device, an ecmo machine, a cardiopulmonary bypass device, an inhalation drug delivery device, a vascular graft, an arterial vascular graft, a venous vascular graft, a cardiac assist device, a wound dressing, a medical textile, an ECG electrode, an orthopaedic device, an intraocular lens, a suture, a needle, a staple, a mesh, a drug delivery device, an endotracheal tube, a shunt, a drain, a suction device, a hearing aid device, an urethral medical device, and an artificial blood vessel.

22. The object according to any one of claims 1-21 for use in contact with human or animal blood for preventing thrombosis .

23. The object according to any one of claims 1-22, wherein the particles comprising less than 30 ppm neodymium, and the outermost 10 pm of the object also comprising less than 30 ppm neodymium.

Images