Ag / ZnO-Ti antibacterial sputtering coating for medical implant

Abstract

The preparation method of the Ag / ZnO-Ti antibacterial sputtering coating for the medical implant comprises the step of forming a sputtering deposition coating on a titanium alloy substrate by using a composite deposition target material formed by splicing an Ag target material, a ZnO target material and a Ti target material. By reasonably controlling the content of Ag and Zn antibacterial agents in the coating, the implant antibacterial coating meeting the human body safety standard is obtained, the problem of postoperative complications of orthopedic implant infection is solved, and the antibacterial coating is particularly beneficial to inhibition of bacterial adhesion. In addition, Zn in the coating not only has antibacterial performance, but also can promote proliferation of osteoblasts and is beneficial to osseointegration.

Description

Technical Field

[0001] The present invention generally relates to an antimicrobial coating for medical implants. Background Art

[0002] Implants such as external fixation screws that come into direct contact with the human body are often made of titanium alloy or stainless steel. TC4 titanium alloy has been widely used in orthopedics in recent years due to its excellent corrosion resistance, elastic modulus close to that of human bone, and high fatigue resistance. To address the problem of bacterial infection after implantation into the human body, antimicrobial coatings such as inorganic antimicrobial coatings and organic antimicrobial coatings can be applied to the implants. The former primarily releases metals, metal oxides, or non-metallic elements, and kills bacteria through multiple mechanisms such as depolarization of bacterial cell membranes, complexing with biomolecules, and generating ROS to induce oxidative stress. The latter exerts its antimicrobial effect by releasing organic antimicrobial agents such as antibiotics, chitosan, and quaternary ammonium compounds.

[0003] In addition, CN106310371A discloses CVD deposition of a tantalum-copper metal coating on a titanium alloy implant substrate to produce a bone graft coating with certain biocompatibility, antibacterial, and corrosion resistance. CN108904893B discloses evaporation of a composite coating on a titanium or titanium alloy implant substrate, comprising first evaporating a tantalum-copper alloy antibacterial coating and then evaporating a tantalum or niobium biocompatible coating. Summary of the Invention

[0004] The purpose of the present invention is to provide a safer and more reliable antibacterial sputtering coating for medical implants.

[0005] According to a first aspect of the present invention, there is provided a method for preparing an antibacterial composite coating for a medical implant, comprising:

[0006] providing a substrate for forming an orthopedic implant;

[0007] Clean the substrate;

[0008] Providing a composite deposition target material, which is composed of Ag target material, ZnO target material and Ti target material;

[0009] Vacuum pre-sputtering treatment of composite deposition targets; and

[0010] The composite deposition target material is sputter-deposited on a substrate to form a deposition coating, wherein the mass content of Ag is 0.7% to 3.6%, the mass content of Zn is 0.2% to 0.35%, and the mass content of Ti is 94.8% to 98.5%.

[0011] According to the present invention, the composite deposition target is composed of sector-shaped Ag, ZnO, and Ti targets, assembled into a complete circular (cylindrical) target. The Ag target occupies 1% to 4% of the sector area, the ZnO target occupies 2%, and the Ti target occupies 94% to 97%. This composite circular target is very convenient for adjusting and controlling the composition of the sputtering deposition coating.

[0012] According to a preferred embodiment of the present invention, the Ag content of the deposited coating is 0.7% to 1.9%, the Zn content is 0.21% to 0.22%, and the Ti content is 97.2% to 98.4%. The properties of the deposited coating with this composition are optimized, achieving reliable bonding with the substrate while exhibiting not only improved biocompatibility but also superior antibacterial properties. When the composite deposition target material uses the aforementioned circular or cylindrical target material, the sector area occupied by the Ag target material is adjusted to 1% to 2%, the sector area occupied by the ZnO target material to 2%, and the sector area occupied by the Ti target material to 96% to 97%, thereby effectively achieving a deposited coating with the aforementioned corresponding composition.

[0013] According to the present invention, the substrate is made of a titanium alloy, such as TC4 titanium alloy. Because the present invention utilizes a Ti-based target, a robust and reliable Ti-based coating can be deposited by sputtering on the titanium alloy substrate. The sputtered coating self-consistently fuses with the substrate, minimizing the risk of coating detachment.

[0014] According to another aspect of the present invention, a medical implant is provided, which has a substrate made of a titanium alloy, wherein the substrate is formed with an antibacterial composite coating obtained according to the above preparation method.

[0015] The titanium-based antibacterial composite controllable sputtering coating of the present invention is safely and reliably combined with the titanium alloy substrate, and has excellent antibacterial properties while also taking into account its biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a process flow chart of the coating sputtering deposition process used in the present invention;

[0017] Figure 2 This is a schematic diagram of the splicing of composite deposition targets used in the present invention;

[0018] Figure 3 SEM characterization images of the coating samples and blank samples prepared in the present invention;

[0019] Figure 4 This is a graph showing the silver ion release curve of the coating sample prepared in the present invention;

[0020] Figure 5 This is a graph showing the zinc ion release curve of the coating sample prepared in the present invention;

[0021] Figure 6 is the absorbance OD value of each group of samples at different time points;

[0022] Figure 7 This is a photo of the plate colony of the test sample;

[0023] Figure 8 The antibacterial rate of the coating sample prepared by the present invention is shown. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific examples.

[0025] Coating preparation

[0026] The corresponding coatings were prepared using a JGP450 high vacuum magnetron sputtering thin film deposition system produced by Shenyang Scientific Instrument Co., Ltd., Chinese Academy of Sciences. Figure 1 As shown and further described below.

[0027] First, a 10 mm diameter, 1 mm thick TC4 titanium alloy sheet was used as a substrate and ultrasonically cleaned in acetone, anhydrous ethanol, and deionized water for 20 min each, and then dried for later use.

[0028] Then, the Ag target, ZnO target and Ti target made into corresponding fan-shaped ones are spliced ​​together to form a complete circular composite deposition target to be used for sputtering, such as Figure 2 As shown;

[0029] When the vacuum degree in the deposition chamber drops to 6×10 -4 When the pressure is below 8 Pa, Ar gas is introduced into the chamber, and then pre-sputtering is performed for 12 minutes to remove oxides and other impurities on the surface of the sputtering target. The gas pressure used in the pre-sputtering is 8 Pa.

[0030] Next, the coating was deposited on the substrate at a deposition power of 35 W, a working pressure of 0.75 Pa, an argon flow rate of 30 sccm, and a rotational speed of 5 rpm. After the deposition was completed, the sputtered coating sample was removed and sealed for storage.

[0031] Sputtering target and coating composition

[0032] Figure 2 The composite deposition target shown is composed of Ag target, ZnO target and Ti target with the following sector ratios (or volume ratios):

[0033] Target 1: 97% Ti; 2% ZnO; 1% Ag;

[0034] Target 2: 96% Ti; 2% ZnO; 2% Ag;

[0035] Target 3: 95% Ti; 2% ZnO; 3% Ag;

[0036] Target 4: 94% Ti; 2% ZnO; 4% Ag.

[0037] Target materials 1-4 were used respectively to form corresponding sputtered coating samples 1-4 on corresponding substrates, and then their coating compositions were tested in turn (analyzed using a LabRAM HR Evolution laser Raman spectrometer from HORIBA FRANCE, France). The test results are shown in Table 1 below as the EPMA characterization results of the coatings, where coating sample 1 corresponds to target material 1, coating sample 2 corresponds to target material 2, coating sample 3 corresponds to target material 3, and coating sample 4 corresponds to target material 4.

[0038] Table 1: Coating composition (mass percentage; excluding oxygen)

[0039] Coating samples Ag content Zn content Ti content 1 0.77 0.21 98.41 2 1.85 0.22 97.18 3 2.94 0.32 95.79 4 3.63 0.35 94.81

[0040] Coating sample performance test

[0041] The four coating samples obtained in Table 1 and the blank sample of TC4 titanium alloy sheet were subjected to SEM characterization using a Zeiss Sigma-300 scanning electron microscope produced by Carl Zeiss, Germany. Figure 3 , where (a), (b), (c), and (d) correspond to coating samples 1-4 in Table 1, and (e) corresponds to the blank sample. Figure 3 As shown, the surface of the (polished) blank sample is very smooth and free of obvious defects. Typical nanoparticles formed on the surfaces of the other four samples. These particles are mostly spherical, well-spaced, and very evenly distributed, essentially covering the entire substrate surface. The nanoparticles on the surface of coating sample 1 are the smallest, with an average particle size of only 26 nm and the largest spacing. The nanoparticles on the surface of coating sample 2 increase in both size and number, but their shapes are the most irregular, with a large number of striped and square nanoparticles. On the surface of coating sample 3, two sizes of nanoparticles are observed: the larger spherical nanoparticles are approximately 73 nm in size and spaced farther apart. Compared to coating sample 3, the large nanoparticles on the surface of coating sample 4 decrease slightly in size but increase in number, becoming more densely packed. Smaller particles, approximately 24 nm in size, can be seen interspersed between the large particles, exhibiting the roughest surface morphology of the four samples. The higher number of nanoparticles on the surfaces of coating samples 3 and 4 may be attributed to the target composition providing more nucleation sites on the coating surface, thus facilitating the formation and rapid growth of nanoparticles.

[0042] Coating sample Ag 2+ 、Zn 2+ Release Test

[0043] The Agilent 720ES inductively coupled plasma mass spectrometer produced by Agilent Technologies, USA, was used to measure the corresponding ion concentrations released by the above coating samples 1-4 at different time points. Before the experiment, the above samples 1-4 were placed in a 20ml test tube and 10ml of phosphate buffer was added. Then, the samples were sealed and placed in a constant temperature incubator at 37°C. At the points of 12, 24, 36, 48 and 72 hours, 1ml of PBS solution in the test tube was taken each time to detect the Ag content. 2+ At the same time, at 12, 24, 36 and 48 hours, 1 mL of PBS solution was taken from another tube of the same group of test tubes to detect the Zn 2+ The concentration of PBS solution was added to the test tube at the same time. The above samples were repeated 3 times. The ion release test results are shown in Figure 4 and Figure 5 ,in Figure 4 Ag 2+ A graph showing the concentration of versus time; Figure 5 Zn 2+ The concentration of is plotted against time. Figure 4 and Figure 5 It can be seen that coating samples 1-4 can continuously release Ag in water environment. 2+ and Zn 2+ .pass Figure 4 The release curves show that the Ag of coating samples 1-4 in the first 36 hours + The release curve showed a rapid upward trend, indicating that the Ag in the coating was quickly released into the PBS solution. + The release rate of Ag in PBS solution began to slow down significantly. + The concentration tended to be stable and remained at a relatively high level without a significant decrease until 72 hours. + The gradient change of the release curve is basically consistent with the Ag content in the corresponding coating. The Ag content of the 4-Ag / Zn group samples at each time point is + The concentration was basically the highest, reaching 734.26μg / L at 72h. Figure 5 The release curve shows that compared with Ag + , Zn content of each sample 2+ The release rate increased steadily until it reached its highest point at 48h, but the overall Zn 2+ The concentration is much lower than that of Ag + At 48h, the Zn in sample groups 1, 2, 3 and 4 2+The concentrations were 27.4μg / L, 49.78μg / L, 51.3μg / L and 69.36μg / L respectively.

[0044] In vitro cytotoxicity test of coating samples

[0045] Experimental materials: The cells used in this experiment are rat bone marrow mesenchymal stem cells (rBMSCs) (Sybio (Shanghai) Biotechnology Co., Ltd., Catalog No.: RAT-iCell-s018). Other major materials used include: iCell primary mesenchymal stem cell culture system (Sybio (Shanghai) Biotechnology Co., Ltd., Catalog No.: PriMed-iCell-012); CCK-8 kit (Invigentech, USA, Catalog No.: IV08-100); phosphate buffer solution (PBS) (Hyclone, USA); trypsin (Guangzhou Shuopu Biotechnology Co., Ltd., Catalog No.: BL512A); 96-well plate (Slip (Shanghai) Medical Equipment Co., Ltd.); anhydrous ethanol (Shanghai Shengxi Chemical Co., Ltd.); anhydrous formaldehyde (Shanghai Shengxi Chemical Co., Ltd.).

[0046] Experimental equipment: The main equipment used in this experiment are: CO2 constant temperature incubator (Binder, Germany, model CB260); centrifuge (Eppendorf, Germany, model 5810R); pipette (Eppendorf, Germany, model: 3120000240); ultra-clean bench (BioX Technology Co., Ltd., USA, model 2284); microplate reader (Berteng Instrument Co., Ltd., USA, model ELX800); UV spectrophotometer (Beckman Coulter, USA)

[0047] Cytotoxicity assay: CCK-8 assay was used to detect cytotoxicity

[0048] The CCK-8 assay is a widely used method for measuring cell viability and proliferation in vitro. It relies on the reduction of a tetrazolium salt (WST-8) by cellular dehydrogenases to form a water-soluble formazan dye. Its absorbance (OD) is then quantitatively measured using a UV spectrophotometer. The OD value reflects cell viability.

[0049] The experiment used rBMSCs cells in the logarithmic growth phase and the cells were plated at a rate of 1.5×10 4Cells were seeded at a density of 100 μL into 96-well plates containing the corresponding samples and then incubated in a constant temperature incubator at 5% CO₂ and 37°C for 1, 3, and 5 days. At each incubation time point, the culture medium was removed, each well was washed three times with PBS, and 1000 μL of culture medium containing 10% CCK-8 was added. The wells were then incubated again in a constant temperature incubator at 5% CO₂ and 37°C for 2 hours. After the final incubation, 800 μL of supernatant was transferred from each well to a new 24-well plate, and the absorbance at 450 nm was measured using a microplate reader.

[0050] The relative growth rate (RGR) of cells was calculated using the following formula for the final absorbance OD value of each sample group:

[0051]

[0052] In formula (1), RGRx represents the relative cell viability of the sample group numbered x, and Ox is its corresponding OD value. O1 is the OD value of the blank group TC4 titanium alloy sheet, O b is the background OD value, that is, the OD value of the multi-well plate itself without culture medium and cells.

[0053] Figure 6 The OD values ​​of samples cultured with rat bone marrow mesenchymal stem cells (rBMSCs) at different time points are shown. Specifically, the OD values ​​of the background multiwell plate (left 1 in each group), the blank sample (TC4 titanium alloy sheet; left 2 in each group), and coated samples 1-4 (left 3-6 in each group, respectively) at 24, 72, and 120 hours are shown in Table 2. The specific values ​​and their corresponding relative toxicity levels (RGRs) are shown. As can be seen, there were no significant differences in the OD values ​​between coated samples 1-4 at these time points (P>0.05), nor were there significant differences in the OD values ​​between coated samples 1-4 and the uncoated blank sample (P>0.05). The RGRs of coated samples 1 and 2 were both above 75%, corresponding to only Grade I cytotoxicity, thus meeting safety toxicity standards. The RGR of coated sample 3 in the first 72 hours indicated a potential safety risk to humans, with a Grade II toxicity rating, but it reached Grade I safety standards after 120 hours. The OD values ​​of coating sample 4 were the lowest at 24h, 72h and 120h, among which the RGR at 24h was only 33.51%, and the toxicity level reached Level III, indicating that the coating poses an extremely high safety risk to biological cells and does not meet human safety standards.

[0054] Table 2: Absorbance values ​​and RGR of test samples

[0055]

[0056] In vitro antibacterial activity test of coating samples

[0057] Experimental materials: The strain used in this experiment is Staphylococcus aureus (Sigma-Aldrich, USA, No.: ATCC6538). Other main materials include tryptone soy agar plates (Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.); tryptone soy culture medium (Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.); PBS buffer (Hyclone, USA); anhydrous ethanol (Shanghai Shengxi Chemical Co., Ltd.); coating rod (Changde Beekman Biotechnology Co., Ltd.); culture dish (Sigma-Aldrich, USA, specification 100mm×20mm).

[0058] Experimental equipment: constant temperature incubator (Liebherr, Germany); high temperature and high pressure steam sterilizer (Sanyo, Japan, model MLS-3030CH); pipette (Eppendorf, Germany, model: 3120000240); electronic analytical balance (Mettler Toledo, Switzerland, model AL104); ultraviolet spectrophotometer (Beckman Coulter, USA); centrifuge (Eppendorf, Germany, model 5810R).

[0059] Preparation of culture medium and bacterial suspension: Preparation of TSA liquid culture medium: Use an electronic analytical balance to weigh 3g of tryptone soy culture medium solid powder and add it to a clean and sterile flask, and add 100mL of distilled water to the flask to achieve the required TSA liquid culture medium concentration. Gently shake the flask or bottle to completely dissolve the powder. Then place the flask in a high-temperature and high-pressure steam sterilizer and autoclave at 121°C for 15-20 minutes to sterilize the TSA liquid culture medium. Finally, place the TSA liquid culture medium in an incubator and store it at 4°C for later use. Preparation of bacterial suspension: Culture Staphylococcus aureus on a tryptone soy agar plate to obtain its isolated single colony, and then place the individual colonies in a test tube containing 3mL of TSA liquid culture medium and place it in a constant temperature oscillator and shake and culture at 37°C for 24 hours. The concentration of Staphylococcus aureus in the test tube is adjusted to 10 by using an ultraviolet spectrophotometer to measure the optical density of the bacterial culture at a specific wavelength and comparing it with a bacterial turbidimetric tube. 7 CFU / mL was used to achieve the bacterial concentration required for the experiment. To prevent contamination, the bacterial suspension was stored in a sterile environment until use.

[0060] Co-culture-plate coating method for antimicrobial performance testing: Before the experiment, the coated samples were disinfected. A cotton ball was soaked in anhydrous ethanol for approximately 10 seconds. The sample surface was then wiped with an alcohol-soaked cotton ball to sterilize and remove any debris and other impurities. After wiping, the sample was rinsed three times with sterile distilled water and air-dried in a laminar flow hood.

[0061] After disinfection is completed, the bacterial suspension for plate coating is prepared by co-cultivation. First, take out the prepared sample piece and place it in a 24-well plate. Use a pipette to drop 500 μL of bacterial suspension into the corresponding wells in turn. Then put the 24-well plate containing the sample and bacterial suspension into a constant temperature incubator and co-cultivate for 24 hours in a dark anaerobic environment at 37 ° C. After the incubation is completed, take out the 24-well plate from the incubator, shake it slightly to separate the bacterial suspension on the surface of the sample and make it fall off, then remove the co-cultivated sample in the well and collect the bacterial suspension in the well. Transfer the latter to a sterile container and dilute it with PBS buffer before preparing for plate coating.

[0062] Once all co-cultured bacterial suspensions are obtained, pipette 50 μL of each suspension onto separate tryptone soy agar plates. Use a spreading stick to evenly spread the suspension across the plates. Each plate is then numbered and placed in a 37°C incubator. After 24 hours, remove the plates and count the colonies.

[0063] In order to quantitatively compare and evaluate the antibacterial effects of each group of samples, this experiment used the inhibition rate to calculate the antibacterial performance of the samples. The calculation formula is as follows:

[0064]

[0065] Rx represents the inhibition rate (%) of the sample numbered x, C1 represents the number of colonies on the surface of the agar plate of the blank group (cfu / plate), and Cx represents the number of colonies on the surface of the agar plate of the xth group (cfu / plate).

[0066] Figure 7 Table 3 shows the colony count results of the test samples. Figure 8 The graph shows the inhibition rates of coating samples 1-4 against Staphylococcus aureus.

[0067] Table 3

[0068]

[0069] As shown above, compared to the unmodified blank TC4 sample, all four experimental groups coated with Ag-ZnO / Ti composite coatings demonstrated significant antibacterial efficacy against Staphylococcus aureus, killing a significant portion of the bacteria with average inhibition rates exceeding 90%. Despite having the lowest Ag content of 0.77 wt.%, the 1-Ag / Zn experimental group still achieved an inhibition rate exceeding 97.7%, while the 4-Ag / Zn experimental group exhibited virtually no bacterial colonies on the surface. Overall, the four experimental groups demonstrated significantly improved antibacterial performance with increasing Ag content in the coatings, with inhibition rates of approximately 99.03%, 99.62%, and 99.98% for the 2-Ag / Zn, 3-Ag / Zn, and 4-Ag / Zn groups, respectively. Overall, the 2-Ag / Zn coating was the best performing antibacterial coating, achieving an antibacterial rate of 99.62%. Its cytotoxicity also met safety standards for biomaterials, demonstrating excellent biocompatibility.

[0070] In summary, increasing the proportion of Ag target material significantly increases the number of nanoparticles on the coating surface and increases the roughness of the coating. In addition, the Ag-ZnO / Ti coating transforms the surface of the TC4 material into a hydrophobic surface.

[0071] In summary, this invention uses radio frequency magnetron sputtering technology to prepare an Ag-ZnO / Ti composite antimicrobial coating co-doped with Ag and Zn on the surface of TC4 material. By rationally controlling the content of the Ag and Zn antimicrobial agents in the coating, an implant antimicrobial coating that meets human safety standards is obtained, addressing the postoperative complications of orthopedic implant infection and being particularly beneficial in inhibiting bacterial adhesion. Furthermore, the Zn in the coating not only has antimicrobial properties but also promotes osteoblast proliferation, facilitating osseointegration.

Claims

1. A method for preparing an antibacterial composite coating for medical implants, comprising: providing a substrate for forming an orthopedic implant; Clean the substrate; Providing a composite deposition target material, which is composed of Ag target material, ZnO target material and Ti target material; Vacuum pre-sputtering treatment of composite deposition targets; and The composite deposition target material is sputter-deposited on a substrate to form a deposition coating, wherein the mass content of Ag is 0.7% to 3.6%, the mass content of Zn is 0.2% to 0.35%, and the mass content of Ti is 94.8% to 98.5%.

2. The preparation method according to claim 1, wherein the composite deposition target is composed of a sector-shaped Ag target, a ZnO target and a Ti target, each of which is spliced ​​into a complete circular target, wherein the Ag target occupies 1% to 4% of the sector area, the ZnO target occupies 2% of the sector area, and the Ti target occupies 94% to 97% of the sector area.

3. The preparation method according to claim 1, wherein the mass content of Ag in the deposited coating is 0.7% to 1.9%, the mass content of Zn is 0.21% to 0.22%, and the mass content of Ti is 97.2% to 98.4%.

4. The preparation method according to claim 3, wherein the composite deposition target is composed of a sector-shaped Ag target, a ZnO target, and a Ti target, each of which is spliced ​​into a complete circular target, wherein the Ag target occupies 1% to 2% of the sector area, the ZnO target occupies 2% of the sector area, and the Ti target occupies 96% to 97% of the sector area. The preparation method according to claim 1 , wherein the substrate is made of titanium alloy.

6. A medical implant comprising a substrate made of a titanium alloy, wherein the substrate is provided with an antibacterial composite coating obtained by the preparation method according to any one of claims 1 to 4.

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