A method for imparting antimicrobial properties to metals, metallic alloys, ceramics, glass, glass-ceramic, and composite substrates

Abstract

The present invention concerns a method for imparting antimicrobial properties to substrates.

Description

[0001] “A method for imparting antimicrobial properties to metals, metallic alloys, ceramics, glass, glass-ceramic, and composite substrates”

[0002] DESCRIPTION

[0003] Biomedical materials have developed a wide range of applications in biomedicine. They are used in skin tissue engineering, cardiovascular devices, drug delivery, and orthopedics. Because each of these applications requires customized material properties and good biocompatibility, there are several types of biomaterials, which can be categorized into four classes: metals, polymers, ceramics, and composites (Figure 1 ). Among them, the most common biomaterial class for implant applications is metals.

[0004] Metals

[0005] Because of their excellent thermal and electrical conductivity as well as mechanical properties, biomaterials made from metals are widely implemented in biomedicine. Because electrons in metals are independent, thermal energy and electric charges can be transferred quickly. These free electrons provide a strong bonding force, holding positively charged metal ions together. Because of the fundamental nondirectional nature of metallic bonds, metal ions can be displaced without breaking the crystal structure. This allows solid material to plastically deform. In addition, some metals have good corrosion resistance.

[0006] Because of these capabilities, several metals are applied in orthopedics, replacing hard tissues such as hips and knees, or are used in fracture-healing purposes for bones, such as spinal fixation devices, and bone screws and plates. The first attempt to implement metallic materials as medical implants was witnessed in the 19th century when the Industrial Revolution resulted in an expansion of applications from the metal industry. They were used as implant materials to provide the fixation of fractured long bones. However, implants made from silver, gold, or iron did not provide efficacious outcomes. In the 1860s, Lister introduced the aseptic surgical technique, which allowed to implementation of metallic materials as medical implants. After that, metallic materials became major materials in orthopedics. Furthermore, the application of metal alloys improved their outcomes thanks to enhanced mechanical and tribological properties, which led to better biocompatibility for implants. The first metallic implant for the human body was Sherman Vanadium steel; however, it was found to be toxic in the long run because of its rapid corrosion. Alternatively, in 1920s, 18Cr-8Ni (wt%) stainless steel was developed as an alloy with better corrosion resistance. Later, by adding molybdenum, 316 stainless steel was obtained, providing improved corrosion resistance. Then, in 1940, titanium and its alloys were studied as implant materials for orthopedic applications. The reason behind that was the successful implementation of these materials in aircraft applications thanks to their superior corrosion resistance in salty seawater.

[0007] Meanwhile, in the 1950s, the amount of carbon in 316 stainless steel was decreased from 0.08wt% to 0.03 wt% to obtain increased corrosion resistance as well as weldability. This introduced a new 316L stainless steel grade. By containing Cr, the formation of rust in the human body was prevented. In addition, the low carbon percentage in the compound increases corrosion resistance, preventing the formation of chromium carbides. However, the risk of stress corrosion can lead to unexpected cracking failures in the implant. Thus, this material is used only as a temporary implant in some cases. In comparison, CoCr alloys have higher corrosion resistance. This is thanks to the formation of the Cr2Os oxide layer in the environment of the human body. However, the toxic effects are caused by releasing Ni, Cr, and Co, leading to several diseases. As an alternative, Ti alloys have gained enormous demand thanks to several advantages in terms of mechanical properties (high specific strength), good corrosion resistance from the formed oxide layer, and excellent biocompatibility. The density of Ti alloy is the lowest among the three mentioned alternatives that are closer to bone. In terms of corrosion resistance, the unique behavior of Ti is the rapid formation of an oxide layer. It can be useful in cases where a layer is to be broken (pitting corrosion). The release of toxic elements is prevented from the immediate reformation of a new layer. Thus, Ti and its alloys were found to be better options for implant material in orthopedic applications.

[0008] Currently, there are a variety of metals that can be manufactured, but only a few of them can be applied as a biomedical material, because of their biocompatibility requirements. According to the mentioned criteria, there are different types of metals, and their alloys have been used as implant materials for bone replacement. Nowadays, biocompatible metallic materials are separated into four groups. They are cobalt-based alloys, stainless steel metals, titanium-based alloys, and miscellaneous metals. The first three groups are frequently used as biomedical materials.

[0009] Stainless Steel

[0010] Stainless steel is a type of metal alloy based on iron. It has a high amount of chromium (1 1 %— 30 wt%) and a varying percentage of nickel. In recent years, AISI 316L stainless steel grades have been the main grades used in biomedicine, among other types of stainless steel. It is composed of a high amount of chromium (16-18%) and nickel (10-14%) and a low amount of carbon (below 0.03%). Moreover, some additional elements are added, such as molybdenum (2-3%) and manganese (2%), as well as a tiny amount of sulfur, phosphorous, silicon, and nitrogen. This grade has good ductility, fatigue properties and work hardenability. In addition, the attractive side of the material is its low cost, production ease and availability. Despite its good biocompatibility, stainless steel alloys lack blood compatibility, bioactivity, and osteoconductivity.

[0011] According to their chemical composition, these metals are divided into two groups: the chromium type and the chromium-nickel type. Furthermore, based on molecular microstructure, they are classified into four groups: martensite, austenite, ferrite, and duplex (mix of ferrite and austenite). The first three groups are used in medical applications. Ferritic stainless steels are used in medical devices. Meanwhile, martensite stainless steels have appropriate hardness characteristics reaching 97HRB, which makes them suitable to be used in surgical and dental instruments. Austenitic stainless steels find their applications in non-implantable devices, which require high corrosion resistance and strength. In addition to that, most of the implant materials made from stainless steel are austenitic.

[0012] CoCr-Based Alloys

[0013] Alloys based on cobalt were initially used in aircraft applications. Throughout the past century, their applications were developed in biomedical applications. Compared with stainless steel alloys, cobalt-based alloys offer better corrosion resistance, less wear, and less fatigue. CoCrMo alloys are currently the most common materials used in implants for hard- tissue replacement with high loads. Because of their high fatigue resistance, these materials can be used in permanent applications, which can last for more than 20 years, showing their high long-term biocompatibility. Nevertheless, there are still several issues that needs further investigation. Cobalt-based alloys suffer several failures in terms of fretting, corrosion fatigue, wearing and stress-shielding effects, causing aseptic loosening and releasing of particles such as Co, Ni and Co ions, which leads to biological toxicity.

[0014] In addition, the high elastic modulus of these alloys is another disadvantage. Currently, wrought CoNiCrMo and cast CoCrMo alloys are the main cobalt-chromium type of alloys used in biomedical applications as replacements for knee joints and hip stems. The high ultimate tensile strength (655-1 172 MPa) and fatigue strength (up to 107 cycles) of wrought alloys allow them to be used in implants for long-term applications, upwards of 20 years. Moreover, a small amount of Cu is added to obtain better antibacterial and mechanical performance.

[0015] Titanium and Titanium-Based Alloys

[0016] Titanium is so far one of the most preferred metal materials for orthopedic applications thanks to its inert behavior and high corrosion resistance. Compared with other metals, it has better direct bonding between the surface of the implant and the bone. Its compatibility was revealed by observing the calcification, osteoblast activity, the formation of calcium phosphate in reproduced body fluid, and the development of hard tissue on the implant surface of animals with implanted titanium.

[0017] It was perceived that implanting titanium in bone allows it to increase early contact between implant and bone, resulting in better bone-bonding strength. The surface morphology of an implant is also a vital factor because the compatibility of hard tissue depends on the adhesion and proliferation of its osteogenic cells.

[0018] It was reported that the molecular structure of Ti and that of its alloys have a significant effect on biocompatibility. Its general molecular structure is hexagonal and close-packed, which is called the a-phase. However, it also can be transformed into a body-centered cubic molecular structure called the p-phase. These phase transformations can be manipulated by alloying them with specific elements. These additives are called a (Al, O, C) and (Mo, Nb, Ta) stabilizers. By manipulating the amount of the composition of both stabilizers, Ti alloys can be divided into three main groups: a- and near-a-type (the implementation of these materials is suitable for a high-temperature environment, despite that, the lower capacity of mechanical and the fatigue strength of these materials make them less desirable for bone replacement implants), a+p-type (the main advantage of these materials is their high strength, but conversely, a+p alloys lead to bone resorption as well as the loosening of the implant), and p-type alloys (they have significantly lower elastic modulus with a high density).

[0019] Ceramic

[0020] Another material group that was developed for use in biomedicine is ceramic biomaterials. Initially, their application in medicine was limited because the materials are brittle and their tensile strength is low. However, in this century, these materials have developed an application for the replacement of body parts, especially bones.

[0021] The combination of high compressive strength and inertness within the body environment favors implementing the material. Bioceramics found their place in biomedicine thanks to the studies of Larry Hench. Because metallic and polymeric implant materials were bioinert, by discovering materials that can generate bonding with bone, the first bioactive glass was invented in 1969. A degradable glass, currently termed 45S5, was able to generate bonding with bone strong enough that to remove the implant, bone breaking was required. After this invention, biomedicine found a new biomaterial group of bioactive ceramics materials, which include ceramic, glass, and glass-ceramic materials.

[0022] Currently, one of the major applications of bioceramic materials in biomedicine is bone tissue engineering because of their ability to facilitate the growth of bone tissues at contact zones. Because of that, ceramics implant materials are produced, providing a three-dimensional (3D) porous environment called a scaffold. This allows for the transfer of body cells, providing cell interactions while preventing toxicity and degradation at a certain rate.

[0023] Composite

[0024] Biomaterials that consist of two or more phases or materials larger than molecular scales are called composite biomaterials. By combining different materials into composites, several characteristics of materials, such as elastic modulus, can be significantly changed compared with homogeneous materials. As examples of composite materials, bone, skin, and wood can be classified as composite materials. In detail, bone consists of carbonated apatite and collagen, where the first item provides reinforcement and stiffness and the second provides toughness and flexibility. Composite materials have vast potential in biomedical applications thanks to the possibility of obtaining strong, lightweight, and stiff materials. However, all components of a composite implant are required to be biocompatible, and the degradation of interfaces between components needs to be avoided. Currently, composite biomaterials are used mostly in dental and orthopedic applications.

[0025] These biomedical alloys possess excellent corrosion resistance and mechanical properties, which makes them excellent materials for future biomedical uses. However, there are still problems on their surfaces during service. Developing surface treatment methods has attracted increasing attention. Many efforts have proved that physical surface modifications are effective and stable ways to enhance surface bio-functionality. Common physical surface modifications are thermal spraying, glow discharge plasma, ion implantation, ultrasonic nanocrystal surface modification, and physical vapor deposition. Despite the wide use of biomaterials in clinical environments, the biocompatibility of the materials is still far from ideal and a variety of adverse reactions such as inflammation, fibrosis, infection, and thrombosis, may be triggered.

[0026] Blood is often the first body fluid that comes into contact with blood-contacting devices. Blood-material interactions trigger a complex series of events including protein adsorption, platelet adhesion and activation, coagulation, and thrombosis. Rapid adsorption of plasma proteins is the first event occurring on the biomaterial surface during blood / material interactions and leads to activated, adsorbed proteins that can catalyze, mediate, or moderate the subsequent biological responses to biomaterials. Surface- induced thrombosis is the main problem impeding the development of long-term bloodcontacting devices. Thrombus formation on device surfaces is partially due to platelet- mediated reactions and partially due to coagulation of blood plasma. Biological responses are a complex process governed by many factors, but it is widely accepted that the surface properties of a biomaterial dictate the biological response. Surface properties such as chemistry, topography, surface free energy, elasticity, and charge may moderate protein and cell interactions, and ultimately the host response. For example, platelet adhesion and activation on biomaterial surfaces are influenced by surface properties such as energy, charge, and composition. However, platelet adhesion and activation are largely mediated by proteins such as fibrinogen. As plasma proteins can rapidly adsorb onto the material surface to form a “conditioning film” following blood contact, this adsorbed protein layer may minimize the direct effect of biomaterial surface properties on cell responses, and the surface-biology interactions are mediated by proteins.

[0027] The main process of physical surface modifications is applying coatings on the substrates. Thus, investigating novel coating materials will be helpful in the future. Moreover, reasonably combining these physical surface modifications can have them support each other and overcome their disadvantages. In summary, more efforts are needed to develop physical surface modifications on biomedical alloys for medical applications.

[0028] Brief description of the figures

[0029] Figure 1 - Most common biomaterial class for biomedical applications.

[0030] Figure 2 - Surgical steel (SS), NiTinol (NiT), chromium / cobalt (CC), titanium (Ti), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=l 0 for each type of treatment), underwent a Thrombin Generation Assay Test (Haemoscan, Groningen, Netherlands).

[0031] Figure 3 - Surgical steel (SS), chromium / cobalt (CC), NiTinol (NiT), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=10 for each type of treatment), underwent a bacterial adhesiveness evaluation assessed forS. aureus and E. coli. Treatment condition (A).

[0032] Figure 4 - Surgical steel (SS), chromium / cobalt (CC), NiTinol (NiT), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=10 for each type of treatment), underwent a bacterial adhesiveness evaluation assessed forS. aureus and E. coli. Treatment condition (B).

[0033] Figure 5 - Surgical steel (SS), chromium / cobalt (CC), NiTinol (NiT), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=l 0 for each type of treatment), underwent a bacterial adhesiveness evaluation assessed forS. aureus and E. coli. Treatment condition (C). Figure 6 - Surgical steel before (NT), and after (T) treatment with a solution based on caffeic acid (n=5 for each type of treatment), underwent a biochemical assay to evaluate the presence of sessile (surface-adhered) bacteria.

[0034] Figure 7-SEM evaluation of original and treated Surgical steel sample (Magnification 1.500X).

[0035] Figure 8- SEM evaluation of original and treated Surgical steel sample (Magnification 3.000X).

[0036] Figure 9 - Percentage of protein adsorption of surgical steel (SS), chromium / cobalt (CC), NiTinol (NiT), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=10 for each type of treatment). Treatment condition (A).

[0037] Figure 10 - Percentage of protein adsorption of surgical steel (SS), chromium / cobalt (CC), NiTinol (NiT), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=10 for each type of treatment). Treatment condition (B).

[0038] Figure 1 1 - Percentage of protein adsorption of surgical steel (SS), chromium / cobalt (CC), NiTinol (NiT), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=10 for each type of treatment). Treatment condition (C).

[0039] Figure 12 - SEM evaluation of CC sample.

[0040] Figure 13 - SEM evaluation of NiT sample.

[0041] Figure 14 - SEM evaluation of T sample.

[0042] Figure 15 - Fluorescence evaluation of G sample.

[0043] Object of the invention

[0044] In a first object, the present invention discloses a method for imparting antimicrobial properties to a substrate represented by metals, metallic alloys, ceramics, glass, glassceramic, and composite substrates.

[0045] In an embodiment, said substrate is represented by: stainless steel alloys, nitinol, cobalt-based alloys, titanium alloys, ceramic, glass, and glass / ceramic.

[0046] In an embodiment, the method of the invention provides to said substrate one or more properties selected from the group comprising: inhibition of the surface adhesiveness to circulating proteins and inhibition of microbial adhesion.

[0047] In particular, the method of the invention provides one or more of: inhibition of adhesiveness to circulating proteins, thrombin generation, inhibition of bacterial adhesion. In particular, the method of the invention provides resistance to tissue bacterial adhesion.

[0048] In a second object, the present invention discloses a substrate obtained according to the method described and a medical device comprising such a substrate.

[0049] In a particular embodiment, said medical device is selected from the group comprising: implants, such as arthroplasty, hip joints, craniofacial, maxillofacial, external and internal prostheses, surgical instrumental applications, and healthcare goods. The most prevalent biomedical application is currently for hip and knee replacements, with shoulder and elbow joint implants following closely. Metals, ceramics, and alloys have also seen frequent utilization in the spinal area forspinal correction parts, spinal fixation devices, spinal fusion cages, and, in recent years, replacements of spinal disks. Rib cages, finger and toe implants, as well as tibial nails employed in the reinforcement of lower leg fractures, are also made of metal and alloys. Fixation and reconstructive devices that support broken bones, such as bone plates, mesh, pins, screws, and rods made of metals, ceramics, and alloys, are frequently used. Neurosurgical and cranioplasty applications of metals, ceramics, and alloys include cranial plates, mesh, and acrylic. Owing to the inherent properties of metals, ceramics, and alloys, such as corrosive resistance, low weight, and toughness, their alloys are used extensively for the fabrication of temporary or long-term external devices and fixations, including artificial limbs and orthopedic calipers. Metals, ceramics, and alloys are used to secure false ears and eyes, providing fixation for interorbital fractures. The aural applications of metals, ceramics, and alloys include bone conduction hearing aids anchored with devices made of metals, ceramics, and alloys that are connected to the middle ear. The carrier structure for replacement heart valves, coronary angioplasty catheters, defibrillators, intravascular stents, pacemaker cases, and vascular access ports is also made of metal and alloys. Infusion pumps utilize metal and alloys that flex when the applied electrical current enables the creation of a heating and cooling cycle that changes the shape of the chamber. Urethral strictures are treated with urethral stents made from metals and alloys.

[0050] An extensive range of surgical instruments, such as dental drills, forceps, and laser electrodes, often contains metals, ceramics, and alloys due to their antibacterial properties, corrosion resistance, compatibility with radiation, durability, and lightweight nature. Their low weight reduces the onset of fatigue for surgeons wielding the instrument for extended periods. For microsurgical operations, such as ocular surgery, metal and alloy surgical instruments are usually anodized to produce a non-reflecting surface essential for such operations. The utilization of metals, ceramics, and alloys for the fabrication of healthcare goods includes external prostheses and wheelchairs, particularly those used for sporting purposes.

[0051] Detailed description of the invention

[0052] According to the first object of the invention it is disclosed a method for imparting antimicrobial properties to a substrate represented by metals, metallic alloys, ceramics, glass, glass-ceramic, and composite substrates.

[0053] In an embodiment, said substrate may be represented by stainless steel alloys, nitinol, cobalt-based alloys, titanium alloys, ceramic, glass, and glass / ceramic.

[0054] According to the present invention, the following substrates are not included: polyurethane, polyesters, polyamides, polyethylene, silicones, PEEK, polyacrylates, acrylic hydrogels, Teflon, polysiloxane, fluorinated polymers.

[0055] In particular, stainless steel alloys include, for instance, the families: Austenitic, Ferritic, Martensitic, Duplex, and Precipitation hardening.

[0056] In particular, Austenitic stainless steel includes the sub-groups, 200 series and 300 series: type 200 series are chromium-manganese-nickel alloys; type 201 is hardenable through cold working; type 202 is general-purpose stainless steel; type 300 series are chromium-nickel alloys that achieve their austenitic microstructure almost exclusively by nickel alloying; type 304, also known as 18 / 8 and 18 / 10 for its composition of 18% chromium and 8% or 10% nickel, respectively; type 316 and its low-carbon versions such as 316L or304L.

[0057] In particular, Ferritic stainless steels include type 430, 409, and 409 Cb, 439 and 441 , 430 and 440C).

[0058] In particular, Martensitic stainless steels include Fe-Cr-C grades (these were the first grades used and are still widely used in engineering and wear-resistant applications), Fe-Cr- Ni-C grades (they offer higher toughness and higher corrosion resistance such as Grade EN 1.4303, casting grade CA6NM), Precipitation hardening grades such as Grade EN 1.4542 (also known as 17-4 PH), Creep-resisting grades.

[0059] In particular, there are three types of Precipitation hardening stainless steels: (1 ) Martensitic 17-4 PH (AISI 630 EN 1 .4542) contains about 17% Cr, 4% Ni, 4% Cu, and 0.3% Nb, (2) Semi-austenitic 17-7 PH (AISI 631 EN 1 .4568) contains about 17% Cr, 7.2% Ni, and 1 .2% Al, and Austenitic A286(ASTM 660 EN 1 .4980) contains about Cr 15%, Ni 25%, Ti 2.1 %, Mo 1 .2%, V 1 .3%, and B 0.005%.

[0060] In particular, the following designations are used to describe stainless steel finishes by ASTM A480 / A480M-18 (DIN): No. 0 (Hot-rolled, annealed, thicker plates), No. 1 (I D, Hot- rolled, annealed and passivated), No. 2D (cold rolled, annealed, pickled and passivated), No. 2B (same as above with additional pass-through highly polished rollers), No. 2BA (2R, bright annealed, BA or 2R same as above then bright annealed under oxygen-free atmospheric condition), No. 3 (G-2G, coarse abrasive finish applied mechanically), No. 4 (1 J-2J, brushed finish), No. 5 (satin finish), No. 6 ( 1 K-2K, matte finish), No. 7 (1 P-2P, reflective finish), No. 8 (mirror finish), No. 9 (bead blast finish), and No. 10 (heat colored finish).

[0061] In particular, cobalt-based alloys include Co-28Cr-6Mo, which is cast (ASTM F75), forged (ASTM F799), or wrought (ASTM Fl 537); Co-20Cr-l 5W-10NL which is wrought (ASTM F90); and Co-35Ni-20Cr-10Mo, which is wrought (ASTM F562) or forged (ASTM 961 ), CoCrMo (CCM Plus, BS 7252), L605 (AMS 5537, and AMS 5796), and MP35N (ASTM F562, AMS 5758, AMS 5844, AMS 5845).

[0062] In particular Titanium alloys include (1 ) alpha alloys which contain neutral alloying elements (such as tin) and / oralpha stabilizers (such as aluminum or oxygen) only (examples include Ti-5AI-2Sn-ELI, Ti-8AI-l Mo-l V), (2) near-alpha alloys contain small amount of ductile beta-phase. Besides alpha-phase stabilizers, near-alpha alloys are alloyed with 1-2% of beta-phase stabilizers such as molybdenum, silicon, or vanadium (examples include Ti-6AI- 2Sn-4Zr-2Mo, Ti-5AI-5Sn-2Zr-2Mo, IMI 685, Ti 1 100), (3) alpha and beta alloys, which are metastable and generally include some combination of both alpha and beta stabilizers (examples include TI-6AI-4V, Ti-6AI-4V-ELI, Ti-6AI-6V-2Sn, Ti-6AI-7Nb, and Ti62A), (4) beta and near beta alloys, which are metastable and which contain sufficient beta stabilizers (such as molybdenum, silicon and vanadium) to allow them to maintain the beta phase when quenched, and which can also be solution treated and aged to improve strength (examples include Ti-10V-2Fe-3AI, Ti-29Nb-13Ta-4.6Zr, TI-13V-1 1 Cr-3AI, Ti-8Mo-8V-2Fe-3AI, Beta C, Ti-15-3).

[0063] In particular titanium alloys include all the following treatments: Grade 1 , Grade 2, Grade 2H, Grade 3, Grades 1 -4 considered commercially pure or CP, Grade 5 also known as TI6AI4V, TI-6AI-4V or Ti 6-4, Grade 6 also known as Ti-5AI-2.5Sn, Grade 7, Grade 7H, Grade 9, Grade 1 1 , Grade 12, Grades 13, 14, and 15, Grade 16, Grade 16H, Grade 17, Grade 18, Grade 19, Grade 20, Grade 21 , Grade 23 also known as TI-6AI-4V-ELI or TAV-ELI, Grade 24, Grade 25, Grades 26, 26H, and 27, Grade 28, Grade 29, Grades 30 and 31 , Grade 32, Grades 33 and 34, Grade 35, Grade 36, 37 and 38.

[0064] For the purposes of the present invention, surgical steel (AC), NiTinol (NiT), chromium / cobalt (CC), titanium (Ti) and aluminum (Al) are intended as metals or metal alloys.

[0065] In particular, ceramics include bioceramics, glidcop, and cermet.

[0066] In particular bioceramics include alumina, zirconia, calcium phosphate, glass ceramics, and pyrolytic carbons. In particular, bioceramics can be grouped, based on their biocompatibility, in biopassive (bioinert), bio-active, or resorbable ceramics.

[0067] In particular, ceramics include the LAS system: U2OxAI2O3xnSiC>2, a mix of lithium, silicon, and aluminum oxides with additional components, e.g., glass-phase-forming agents such as Na2O, K2O, and CaO and refining agents. As nucleation agents most commonly zirconium (IV) oxide in combination with titanium (IV) oxide is used.

[0068] In particular, Glidcop includes copper-based metal matrix composite (MMC) alloys mixed primarily with small amounts of aluminum oxide ceramic particles.

[0069] In particular, cermet includes oxide-based cermets, carbide-based cermets, boride cermets, and MAX-phases.

[0070] According to the present invention, the method disclosed may comprise a preliminary step of pre-treatment of said substrate.

[0071] In particular, said pre-treatment comprises anodic oxidation, alkaline or acidic treatment, hydrogen peroxide incubation, electrospinning, polishing, grinding, sandblasting, microarc oxidation, electrical discharge machining (micro and powdermixing), and laser etching.

[0072] In particular, the technology can be applied to diamond-like carbon or Ti nitride- coated substrates.

[0073] Specifically, the substrate can be pre-treated with methods including physical vapor deposition and sputtering (including the addition of TiO2, Y2O3, AI2O3, polyethylene, chitosan, antibiotics, metallic ions, yttrium stabilized zirconia, NisAI, carbon nanotubes, hydroxyapatite, 3-aminorpopyltriethoxysilane (APTES), dopamines, poly-caprolactone, nanodiamond coatings, and gelatine).

[0074] In a preferred condition, said pre-treatment comprises the incubation of the substrate in a pre-treatment solution of alcohol.

[0075] In particular, the C1 -C4 alcohol is selected in the group comprising: methanol, ethanol, isopropanol, heptanol, ethylene glycol, octanol, or butanol and is preferably represented by ethanol.

[0076] More in particular said alcohol has a concentration of about 10-100% (v / v) and preferably of 100% (v / v).

[0077] In an embodiment, the incubation of the pre-treatment is continued for a period of time from 2 minutes to 24 hours.

[0078] In a preferred embodiment, the pre-treatment incubation is performed for about 10 minutes. According to a preferred embodiment of the invention, before said pre-treatment step, the pre-treatment solution is maintained at a temperature of about -25°C to -15°C.

[0079] In a preferred embodiment, the pre-treatment solution is maintained at a temperature of about -20°C.

[0080] In a preferred embodiment, the pre-treatment solution is maintained at the disclosed temperature for a period of time from about 10 minutes to 5 hours.

[0081] For the purposes of the present invention, the method of the invention comprises a step of contacting said substrate with a treatment solution based on caffeic acid.

[0082] In particular, said treatment solution based on caffeic acid has a concentration of caffeic acid of between 0.1 -10 mg / ml.

[0083] In a preferred embodiment, said treatment solution has a concentration of caffeic acid of about 2-4 mg / ml.

[0084] The treatment solution of the invention is prepared by dissolving caffeic acid in a percentage of the final volume between 1% - 99% (v / v) in alcohol, preferably 10%.

[0085] In particular, for the preparation of the treatment solution, the caffeic acid is dissolved in a C1-C4 alcohol.

[0086] In a preferred embodiment, the Cl -C4 alcohol is selected from the group comprising: methanol, ethanol, isopropanol, heptanol, ethylene glycol, octanol, or butanol.

[0087] The pH of the treatment solution is then adjusted to a range between 2.5 and 9.0 and preferably between pH 5.5 and pH 8.0.

[0088] According to an embodiment of the present invention, the treatment solution comprises a second component.

[0089] For the purposes of the present invention, said second component is selected from the group comprising polyphenols and their salts or esters, phenolic compounds and their salts and derivatives, antibiotics or antimicrobial agents, methylated phenols, fatty acids and their esters and metal-based solutions.

[0090] In a preferred embodiment, said second component is selected from the group comprising polyphenols and their salts or esters, phenolic compounds and their salts and derivatives,

[0091] In particular, said polyphenols are selected from the group comprising: resveratrol, aloin, cyanarin, epigallocatechin, tannic acid, chlorogenic acid, hydroxy tyrosol, rosmarinic acid, narigenin, gallic acid, hesperidin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictyol, theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid, baicalein, 5,7-dihydroxy-4- phenyl coumarin, rutin hydrate, 5,8-dihydroxy-l,4-naphthoquinone, 2,3-dichloro-5,8- dihydroxy-1 ,4-naphthoquinone, ethyl-3,4-dihydroxy-cinnamate, butyl gallate, 4-hydroxyl-4- biphenyl-carboxylic acid, oleuropein, garlic acid, magnolol, curcumin, ethyl-3,5-dihydroxy- benzoate.

[0092] In particular, said phenolic compounds are selected from the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavonoids, flavones, chaicones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acids, phenylpropanoids.

[0093] Derivatives of such phenolic compounds, represented by salts and esters, are also included.

[0094] In particular, said antibiotics or antimicrobial agents are selected from the group comprising: penicillins, aminoglycosides, carbapenems, glycopeptides, and lipoglycopeptides such as vancomycin, monobactams aztreonam, oxazolidinones such as linezolid and tedizolid, rifamycins, streptogramins such as quinupristin and dalfopristin, cephalosporins, tetracyclines, macrolides, fluoroquinolones, sulfonamides.

[0095] In particular, said methylated phenols are selected from the group comprising: a- tocopherol, pi-tocopherol, y-tocopherol, 8-tocopherol and tocotrienols.

[0096] In particular, said metal-based solution is selected from the group comprising: acetates, sulfates, phosphates, chlorides, nitrites, nitrates, or carbonates.

[0097] Metal can be selected from the group comprising: iron, silver, gold, zinc, copper, barium, magnesium, and aluminum.

[0098] For instance, there can be used barium carbonate, iron (II) chloride, iron (III) chloride, iron (II) nitrate, iron (III) nitrate, aluminum chloride, calcium chloride, calcium carbonate, calcium nitrate, copper sulfate, and copper nitrate.

[0099] For the purposes of the present invention, said second component has a concentration of about 0.1 -20 mg / ml.

[0100] According to this embodiment, the treatment solution of the invention is prepared by admixing a solution of caffeic acid with a solution of the second component.

[0101] In particular, the solution of caffeic acid is preferably represented by an alcoholic solution of caffeic acid.

[0102] In a preferred embodiment, the solution of caffeic acid is prepared by dissolving caffeic acid in a percentage of the final volume between 1 % - 99% (v / v) in alcohol, preferably 10%.

[0103] In particular, for the preparation of the treatment solution, the caffeic acid is dissolved in a C1-C4 alcohol. In a preferred embodiment, the C1 -C4 alcohol is selected in the group comprising: methanol, ethanol, isopropanol, heptanol, ethylene glycol, octanol, or butanol.

[0104] In a preferred embodiment, the solution of the second component is prepared by dissolving the second component in an aqueous buffer.

[0105] For the purposes of the present invention, a suitable buffer can be selected from the group comprising: PBS (phosphate buffer), bicarbonate buffer, Dulbecco's Phosphate Buffered Saline, TBE (tris / borate / EDTA buffer), TE (Tris / EDTA) buffer, Tris-buffered saline (TBS), SSC (sodium chloride / sodium citrate), and SSPE (sodium chloride / sodium phosphate / EDTA).

[0106] Finally, the two solutions are mixed and the pH is adjusted in a range between 2.5 and 9.0 and preferably between pH 5.5 and pH 8.0.

[0107] For the purposes of the present invention, the method of the present invention comprises at least one treatment cycle, which comprises the steps wherein: i) said substrate is incubated in said treatment solution and then ii) said incubated substrate is washed.

[0108] In particular, said step i) of incubation is performed for a period of time from about 1 minute to 8 days and preferably for about 60 minutes.

[0109] In particular, said step ii) of washing is performed for a period of time from about 2 to 240 minutes and preferably for about 15 minutes.

[0110] For the purposes of the present invention, the treatment step i) comprises at least one cycle performed at pH 5.5.

[0111] In an embodiment of the present invention, the treatment step i) comprises from 1 to 5 treatment cycles performed at pH 5.5.

[0112] Preferably, the treatment step i) comprises 2 treatment cycles performed at pH 5.5.

[0113] For the purposes of the present invention, the treatment step i) is performed in the dark.

[0114] As per step ii), the washing is performed with a washing solution represented by a buffer solution.

[0115] In particular, the buffer solution is selected from the group comprising: PBS (phosphate buffer), bicarbonate buffer, Dulbecco's Phosphate Buffered Saline, TBE (tris / borate / EDTA buffer), TE (Tris / EDTA) buffer, Tris-buffered saline (TBS), SSC (sodium chloride / sodium citrate), and SSPE (sodium chloride / sodium phosphate / EDTA)

[0116] In particular, the treatment step i) can be conducted completely or at least partially under irradiation with ultraviolet (UV) light.

[0117] For the purposes of the present invention, after the treatment step, it is further performed a drying step. In particular, said drying step is performed at a temperature of about 10-85°C.

[0118] In particular, said drying step is performed for a period of time from about 1 minute to 5 hours and preferably for about 30 minutes.

[0119] According to the present invention, the method disclosed above provides antimicrobial properties to the treated synthetic substrate.

[0120] In particular, said antimicrobial properties are versus Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coll, Proteus mirabilis, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica typhimurium, Streptococcus viridans, nontuberculous mycobacteria such as Mycobacterium chelonae, yeast such as Candida albicans and fungus such as Aspergillus brasiliensis.

[0121] In a preferred embodiment, said antimicrobial properties are versus Staphylococcus aureus, and Escherichia coli.

[0122] In addition, the method of the invention provides one or more of the following properties: inhibition of the surface adhesiveness to circulating proteins and inhibition of microbial adhesion.

[0123] In particular, the method of the invention provides one or more of: inhibition of adhesiveness to circulating proteins, thrombin generation, inhibition of bacteria adhesion.

[0124] In particular, the method of the invention provides resistance to tissue bacterial adhesion.

[0125] In particular, said properties of resistance to tissue microbial adhesion are versus Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica typhimurium, Streptococcus viridans, nontuberculous mycobacteria such as Mycobacterium chelonae, yeast such as Candida albicans and fungus such as Aspergillus brasiliensis.

[0126] In a preferred embodiment, said properties of resistance to tissue bacterial adhesion are versus Staphylococcus aureus, and Escherichia coli.

[0127] According to a second object of the invention, it is disclosed a synthetic substrate obtained according to the method of the invention.

[0128] Furthermore, it is disclosed a medical device comprising such a substrate.

[0129] In a particular embodiment, said medical device is selected from the group comprising: implants, such as arthroplasty, hip joints, craniofacial, maxillofacial, external and internal prostheses, surgical instrumental applications, and healthcare goods. The most prevalent biomedical application is currently for hip and knee replacements, with shoulder and elbow joint implants following closely. Metals, ceramics, and alloys have also seen frequent utilization in the spinal area forspinal correction parts, spinal fixation devices, spinal fusion cages, and in recent years, replacements of spinal disks. Rib cages, finger and toe implants, as well as tibial nails employed in the reinforcement of lower leg fractures, are also made of metal and alloys. Fixation and reconstructive devices that support broken bones, such as bone plates, mesh, pins, screws, and rods made of metals, ceramics, and alloys, are frequently used. Neurosurgical and cranioplasty applications of metals, ceramics, and alloys include cranial plates, mesh, and acrylic. Owing to the inherent properties of metals, ceramics, and alloys, such as corrosive resistance, low weight, and toughness, its alloys are used extensively for the fabrication of temporary or long-term external devices and fixations, including artificial limbs and orthopedic calipers. Metals, ceramics, and alloys are used to secure false ears and eyes, providing fixation for interorbital fractures. The aural applications of metals, ceramics, and alloys include bone conduction hearing aids anchored with devices made of metals, ceramics, and alloys that are connected to the middle ear. The carrier structure for replacement heart valves, coronary angioplasty catheters, defibrillators, intravascular stents, pacemaker cases, and vascular access ports are also made of metal and alloys. Infusion pumps utilize metal and alloys that flex when the applied electrical current enables the creation of a heating and cooling cycle that changes the shape of the chamber. Urethral strictures are treated with urethral stents made from metals and alloys.

[0130] An extensive range of surgical instruments, such as dental drills, forceps, and laser electrodes, often contain metals, ceramics, and alloys due to their antibacterial properties, corrosion resistance, compatibility with radiation, durability, and lightweight nature. Their low weight reduces the onset of fatigue for surgeons wielding the instrument for extended periods. For microsurgical operations, such as ocular surgery, metals and alloys surgical instruments are usually anodized to produce a non-reflecting surface essential for such operations. The utilization of metals, ceramics, and alloys for the fabrication of healthcare goods includes external prostheses and wheelchairs, particularly those used for sporting purposes.

[0131] The present invention will be further disclosed by the following experimental section.

[0132] Experimental section

[0133] Alloys

[0134] Starting from sheets measuring approximately 10cm X 10cm, 50 samples measuring 0.5cm x 0.5cm of the following metal alloys were obtained: AISI 316 surgical steel (AC), NiTinol (NiT), chromium / cobalt (CC), titanium (Ti) and aluminum (Al). Samples were incubated for 10 minutes in a 100% (v / v) isopropanol solution. Before use, the alcoholic solution has been placed at -20°C for a time interval ranging from 10 minutes to 5 hours. Subsequently, each type of alloy was divided into 3 groups, and each group was subjected to 3 different treatment variants as described below:

[0135] Variant 1 (VI) (A) caffeic acid at a concentration of 2 mg / ml and tannic acid at a concentration of 4 mg / ml was chosen. The caffeic acid was dissolved in 10% of the final volume in isopropanol. The second polyphenol was dissolved in 90% of the final volume in PBS (phosphate buffer). Finally, the two solutions are mixed and the pH is adjusted in a range between pH 5.5 and pH 8.0.

[0136] The alloy samples are then incubated under moderate but constant agitation in these caffeic acid-based solutions for 120 minutes, followed by a wash in phosphate buffer lasting 30 minutes. Incubation and washing have been performed in the dark.

[0137] After the treatment cycles, the samples were placed in a stove at 37°C for a time of 30 minutes.

[0138] The same treatment variant was performed using other polyphenol mixtures, including resveratrol, epigallocatechin, chlorogenic acid, rosmarinic acid, narigenin, hesperetin, gallic acid, cynarin, and aloin. In particular, (B) caffeic acid at a concentration of 10 mg / ml and rosmarinic acid at a concentration of 10 mg / ml, and (C) caffeic acid at a concentration of 1 mg / ml, and hesperetin at a concentration of 15 mg / ml. All other combinations listed above were tested at concentrations ranging from 0.1 to 20 mg / ml.

[0139] Variant 2 (V2): (A) caffeic acid at a concentration of 10 mg / ml and tannic acid at a concentration of 4 mg / ml was chosen. The caffeic acid was dissolved in 10% of the final volume in isopropanol. The second polyphenol was dissolved in 90% of the final volume in PBS (phosphate buffer). Finally, the two solutions are mixed and the pH is adjusted in a range between pH 5.5 and pH 8.0.

[0140] The alloy samples are then incubated under moderate but constant agitation in these caffeic acid-based solutions for 60 minutes in the dark. A second step of incubation was performed for another 60 minutes under UV irradiation, followed by a wash in phosphate buffer lasting 30 minutes. The first incubation step and washing have been performed in the dark.

[0141] After the treatment cycles, the samples were placed in a stove at 37°C for a time of

[0142] 30 minutes.

[0143] The same treatment variant was conducted using other polyphenol mixtures, including resveratrol, epigallocatechin, chlorogenic acid, rosmarinic acid, narigenin, hesperetin, gallic acid, cynarin, and aloin. In particular, (B) caffeic acid at a concentration of 4 mg / ml and rosmarinic acid at a concentration of 1 mg / ml, and (C) caffeic acid at a concentration of 10 mg / ml and hesperetin at a concentration of 20 mg / ml. All other combinations listed above were tested at concentrations ranging from 0.1 to 20 mg / ml.

[0144] Variant 3 (V3) (A) caffeic acid at a concentration of 5 mg / ml and tannic acid at a concentration of 4 mg / ml was chosen. The caffeic acid was dissolved in 10% of the final volume in isopropanol. The second polyphenol was dissolved in 90% of the final volume in PBS (phosphate buffer). Finally, the two solutions are mixed and the pH is adjusted in a range between pH 5.5 and pH 8.0.

[0145] The alloy samples are then incubated under moderate but constant agitation in these caffeic acid-based solutions for 120 minutes under UV irradiation, followed by a wash in phosphate buffer lasting 30 minutes. The washing step has been performed in the dark.

[0146] After the treatment cycles, the samples were placed in a stove at 37°C for a time of 30 minutes.

[0147] The same treatment variant was conducted using other polyphenol mixtures, including resveratrol, epigallocatechin, chlorogenic acid, rosmarinic acid, narigenin, hesperetin, gallic acid, cynarin, and aloin. In particular, (B) caffeic acid at a concentration of 4 mg / ml and rosmarinic acid at a concentration of 1 mg / ml and (C) caffeic acid at a concentration of 0.5 mg / ml, and hesperetin at a concentration of 18 mg / ml. All other combinations listed above were tested at concentrations ranging from 0.1 to 20 mg / ml.

[0148] Treated and untreated samples for each type of alloy were evaluated for effect on microbial and bacterial adhesion, inhibition of protein adsorption as well as thrombus formation, and surface evaluation using scanning electron microscopy (SEM).

[0149] Glass

[0150] Samples measuring 4cm x 2cm of bio-glass for medical application were incubated for 10 minutes in a 100% (v / v) isopropanol solution. Before use, the alcoholic solution has been placed at -20°C for a time interval ranging from 10 minutes to 5 hours. Subsequently, samples were incubated in a solution of caffeic acid at a concentration of 2 mg / ml and tannic acid at a concentration of 4 mg / ml. The caffeic acid was dissolved in 10% of the final volume in isopropanol. The second polyphenol was dissolved in 90% of the final volume in PBS (phosphate buffer). Finally, the two solutions are mixed and the pH is adjusted in a range between pH 5.5 and pH 8.0.

[0151] The glass samples are then incubated under moderate but constant agitation in these caffeic acid-based solutions for 120 minutes, followed by a wash in phosphate buffer lasting 30 minutes. Incubation and washing have been performed in the dark. After the treatment cycles, the samples were placed in a stove at 37°C for a time of 30 minutes.

[0152] The same treatment variant was conducted using other polyphenol mixtures, including resveratrol, epigallocatechin, chlorogenic acid, rosmarinic acid, narigenin, hesperetin, gallic acid, cynarin, and aloin. In particular, (B) caffeic acid at a concentration of 4 mg / ml and rosmarinic acid at a concentration of 4 mg / ml and (C) caffeic acid at a concentration of 5 mg / ml, and hesperetin at a concentration of 6 mg / ml. All other combinations listed above were tested at concentrations ranging from 0.1 to 20mg / ml.

[0153] Thrombin generation assay test (TGA)

[0154] Samples of AISI 316 surgical steel (SS), NiTinol (NiT), chromium / cobalt (CC), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=10 for each type of treatment), underwent a Thrombin Generation Assay Test (Haemoscan, Groningen, Netherlands). Thrombin is a key enzyme of the coagulation cascade. Its measurement gives direct information about the thrombogenicity of a biomaterial (i.e. its ability to form blood clots). In normal plasma, thrombin is captured into the fibrin meshwork and is rapidly inactivated by antithrombin III or other antiproteases. The short half-life of thrombin hampers its accurate enzymatic determination. The Thrombin Generation Assay is based on a special plasma product that enables the determination of thrombin activity in an incubation medium after this has been exposed to a biomaterial. This method is suited to evaluate the haemocompatibility of biomaterials and medical devices according to the international standard ISO 10993- 4:2002. Specimens were processed by following the instructions provided by the manufacturer. Briefly, samples were incubated in modified human plasma (plasma was provided by the manufacturer) with subsequent withdrawals at different time points. The thrombin concentration of the samples was determined from a calibration curve of optical density at 405 nm. The thrombin generation curve for each specimen was constructed by plotting the thrombin concentration versus the time points at which the samples were taken. The curve is used to determine the speed of thrombin generation, expressed as per cm2of a sample. Reference materials were provided by the manufacturer, in particular: low- density Polyethylene (LDPE, low propensity to thrombin generation) and Medical steel (MS, high propensity to thrombin generation). The results shown in Figure 2 demonstrate as the treatment with the caffeic acid-based solution can significantly reduce the thrombotic propensity in all the treated materials, in particular: SS -84.8%, NiT -63%, CC -72.8%, Al -63%, T

[0155] -65.7%, and G -68%.

[0156] Resistance to Tissue Bacterial Adhesion The anti-adhesive bacterial activity was evaluated regarding the Staphylococcus aureus (S. aureus), and Escherichia coll (E. coli). The bacteria were grown overnight in Tryptic Soy Broth (TSB) at 37°C. The total bacterial load was assessed by 10-factor serial dilutions in TSB (10-1 to 10-7), sown in Petri dishes with appropriate selective medium, and kept in an overnight incubator. Following incubation, the CFU was counted to determine the effective concentration of the microorganism. Furthermore, the optical density at 600 nm was determined from each tiled dilution to verify the linearity between the latter and the effective microbial load of the broth.

[0157] Samples of surgical steel AISI 316 (SS), Nitinol ( NiT), cobalt / chromium (CC), aluminium (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n=l 0 for each type of treatment) as from treatment variant V3 were prepared to obtain the same effective surface for bacterial adhesion. To eliminate any bacterial load before the adhesiveness test, samples were washed with PBS and incubated overnight at RT in PBS, supplemented with gentamicin (300 g / mL) under moderate but constant agitation. Following overnight incubation, the samples were washed extensively in PBS to remove any remaining antibiotics that could skew the test results. Subsequently, the treated and untreated samples were exposed singularly to S. aureus, E. coli, and P. mirabilis bacterial suspensions (bacterial load 1 >< 107CFU / mL) for 90 min at RT under moderate but constant agitation.

[0158] Subsequently, the samples were subjected to three moderate vortexing passages to facilitate the detachment of the loosely bound bacteria and serial dilutions of the washing were plated in Petri dishes containing the appropriate selective growth media. Finally, after 24 hr of incubation at 37°C, the CFU was counted for each type of sample.

[0159] Considering the number of colonies found in the untreated samples as 100%, the percentage of adhesion inhibition was calculated for every single sample treated with the caffeic acid-based solution.

[0160] As shown in Figure 3-5, the caffeic acid-based solution of the invention proved to be effective in inhibiting the surface adhesion of both considered bacteria.

[0161] Confirmation of the mechanism of action of technology

[0162] To investigate the mechanism of action and confirm the anti-adhesive effect against microorganisms, specific experiments were carried out. Two sets of AISI 316 Surgical Steel discs were prepared (1 .5cm 0). The samples, both untreated and treated with the coating technology, were inoculated with a known concentration of a bacterial mixture (bacterial load 1 >< 108CFU / mL) in a maintenance medium. Following a 24-hour incubation at 37°C, the samples were gently washed to remove weakly adhered bacteria. One set was then used for a biochemical assay aimed at detecting the presence of sessile (metabolically active) bacteria, while the second set was analyzed using Scanning Electron Microscopy (SEM) to observe bacterial adhesion at the surface level.

[0163] Biochemical assay for the detection of metabolically active bacteria The test is based on the principle that, in the sessile state, most bacteria contain high levels of peroxidases. These enzymes catalyze the conversion of 3, 5,3', 5'- tetramethylbenzidine (TMB), initially colorless, into a visible oxidized blue form in the presence of H2O2. Surgical Steel discs, both untreated and treated with the coating technology, were incubated for 25 minutes with TMB, to which H2O2was directly added. The incubation was then extended for an additional 10 minutes. The reaction mixture was subsequently analyzed using a plate reader to acquire an absorbance spectrum ranging from 300 nm to 700 nm.

[0164] Concerning the absorbance spectra graphs (Figure 6), the data include a curve corresponding to a Positive Control (metabolically active bacteria adhered to the surface) and a curve representing a Negative Control (bacteria in suspension in their planktonic form).

[0165] Treated samples are represented by a dashed line, while untreated (original) samples are shown with a dotted line. The untreated samples exhibit activity curves comparable to that of the positive control, indicating the presence of metabolically active sessile bacteria. In contrast, the treated samples show a curve that closely overlaps with the Negative Control, suggesting that the microorganisms remained in their planktonic state and did not adhere to the surface.

[0166] Scanning Electron Microscopy observation (SEM)

[0167] SEM observation of the surface of the Surgical Steel discs revealed the presence of bacterial colonies exclusively on the original (untreated) samples, as shown in Figures 7 and 8. All treated samples showed no evidence of bacterial adhesion.

[0168] Given that the biochemical assay confirmed the absence of metabolically active (sessile) bacteria and scanning electron microscopy revealed no bacterial presence on the surface, it is reasonable to hypothesize that the mechanism of action of the applied technology does not involve direct bactericidal activity. Instead, the results suggest a surface-masking effect that prevents bacterial adhesion and subsequent colonization of the material. This interpretation supports the notion that the technology acts by modifying the surface properties in a way that renders it unfavorable for microbial attachment, rather than exerting a cytotoxic effect on the microorganisms themselves.

[0169] Inhibition of surface adhesiveness to serum proteins

[0170] Samples of AISI 316 surgical steel (SS), NiTinol (NiT), chromium / cobalt (CC), titanium (T), aluminum (Al), and bio-glass (G) before (NT), and after (T) treatment with a solution based on caffeic acid (n= 10 for each type of treatment), were incubated for 24 hours at 37°C in phosphate buffer containing 50 g / ml of bovine serum albumin (BSA, 66 kDa) or bovine thyroglobulin (BT, 670 kDa), with moderate but constant stirring. Subsequently, all samples were subjected to 3 washes in phosphate buffer for 3 minutes each to remove any protein residues not firmly bound to the surface. The protein adhered to the surface was measured and, considering the quantity of protein quantified on the untreated samples as a value of 100, the percentage of reduction in the variously treated samples was derived. As reported in Figure 9-1 1 , the treatment with the caffeic acid-based solution of the invention can ensure a significant reduction of protein adhesion for both BSA and BT.

[0171] Scanning electron microscopy evaluation (SEM)

[0172] The treated and untreated specimens were subjected to scanning electron microscopy (SEM) for surface evaluation.

[0173] The cobalt / chromium (CC) samples were subjected to mechanical insult (scratching) to remove part of the coating and better appreciate the surface difference between coated (treated) and uncoated areas (treated and scratched surface). In Figure 12, it is possible to appreciate the difference in color between the coated area and the uncoated area.

[0174] The nitinol (NiT) samples were subjected to mechanical insult (scratching) to remove part of the coating and better appreciate the surface difference between coated and uncoated areas. In Figure 13, it is possible to appreciate the sail-like presence of the polyphenolic coating, especially in the interface area of the scratch (green and orange boxes). It was also possible to measure the thickness of the coating.

[0175] The titanium (T) samples were analyzed, treated and untreated separately. In Figure 14, it is possible to appreciate that, at low magnifications, there are no differences on the surface of the sample. With greater magnification, however, the presence of the polyphenolic coating is evident, which tends to form a texture similar to that of cobblestones.

[0176] Fluorescence microscopy evaluation

[0177] The bio-glass (G) samples were irradiated with light at a specific wavelength to exploit the fluorescent properties of polyphenols. In Figure 15, it is possible to appreciate how the treated sample appears to emit blue, whereas the untreated one does not emit any signal.

Claims

CLAIMS1. A method for imparting antimicrobial properties to a substrate represented by metals, metallic alloys, ceramics, glass, glass-ceramic, and composite substrates, comprising the step of contacting said substrate with a treatment solution based on caffeic acid.

2. The method for imparting antimicrobial properties to a substrate according to the preceding claim, which comprises the steps of: i) incubating said substrate in said treatment solution based on caffeic acid and then ii) washing said incubated substrate.

3. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said step i) of incubation is performed for a period of time from about 1 minute to 8 days and preferably of about 60 minutes.

4. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein, said step ii) of washing is performed for a period of time from about 2 to 240 minutes and preferably of about 15 minutes.

5. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment step i) comprises at least one cycle performed at pH 5.5.

6. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment step i) is performed in the dark or at least partially under irradiation with ultraviolet (UV) light.

7. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said washing step ii) is performed with a washing solution represented by a buffer solution.

8. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said substrate is selected from the group comprising: stainless steel alloys, cobalt-based alloys, titanium alloys, ceramic, glass, and glass / ceramic.

9. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said substrate is not represented by polyurethane, polyesters, polyamides, polyethylene, silicones, PEEK, polyacrylates, acrylic hydrogels, Teflon, polysiloxane, fluorinated polymers.

10. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, which is one or more of the following: a method for the inhibition of the surface adhesiveness to circulating proteins, a method for the inhibition ofmicrobial adhesion, a method for the inhibition of thrombin generation, a method to increase the resistance to tissue bacterial adhesion.

11. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, which further comprises a pre-treatment step of any one of: anodic oxidation, alkaline or acidic treatment, hydrogen peroxide incubation, electrospinning, polishing, grinding, sandblasting, microarc oxidation, electrical discharge machining (micro and powder-mixing), and laser etching; or of any one of: the substrate can be pre-treated with methods including physical vapor deposition and sputtering (including the addition of TiC>2, Y2O3, AI2O3, polyethylene, chitosan, antibiotics, metallic ions, yttrium stabilized zirconia, NisAI, carbon nanotubes, hydroxyapatite, 3- aminorpopyltriethoxysilane (APTES), dopamines, poly-caprolactone, nanodiamond coatings, and gelatine), incubation in a solution of alcohol.

12. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment solution based on caffeic acid has a concentration of caffeic acid of between 0.1 -10 mg / ml.

13. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment solution has a concentration of caffeic acid of about 2-4 mg / ml.

14. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment solution is an alcoholic solution.

15. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment solution has a pH in a range between 2.5 and 9.0 and preferably of between pH 5.5 and pH 8.0.

16. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said treatment solution further comprises a second component.

17. The method for imparting antimicrobial properties to a substrate according to the preceding claim, wherein said second component is selected from the group comprising polyphenols and their salts or ester, phenolic compounds and their salts and derivatives, antibiotics or antimicrobial agents, methylated phenols, fatty acids and their esters and metal-based solutions.

18. The method for imparting antimicrobial properties to a substrate according to the preceding claim, wherein said second component is selected from the group comprising polyphenols and their salts or esters, phenolic compounds and their salts and derivatives.

19. The method for imparting antimicrobial properties to a substrate according to claim 16, wherein said second component has a concentration of about 0.1 -20 mg / ml.

20. The method for imparting antimicrobial properties to a substrate according to the preceding claim 10, wherein said properties of resistance to tissue microbial adhesion are versus microbes selected from the group comprising: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coll, Proteus mirabilis, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica typhimurium, Streptococcus viridans, nontuberculous mycobacteria such as Mycobacterium chelonae, yeast such as Candida albicans and fungus such as Aspergillus brasiliensis, and preferably are versus Staphylococcus aureus, and Escherichia coli.

21. The method for imparting antimicrobial properties to a substrate according to any one of the preceding claims, wherein said properties of resistance to tissue bacterial adhesion are versus Staphylococcus aureus and Escherichia coli.

22. A synthetic substrate obtained according to the method for imparting antimicrobial properties to a substrate according to any one of the preceding claims.

23. A synthetic substrate according to the preceding claim, which is a medical device or a part of a medical device.

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