Cu / Ta-Ti antibacterial composite coating for orthopedic implant

Abstract

The preparation method of the Cu / Ta-Ti antibacterial composite coating for the orthopedic implant comprises the following steps: providing a matrix for forming the orthopedic implant; cleaning the substrate; providing a composite deposition target material, wherein the composite deposition target material is formed by splicing a Cu target material, a Ta target material and a Ti target material; carrying out vacuum pre-sputtering treatment on the composite deposition target material; and the composite deposition target is sputtered and deposited on a base body to form a deposition coating, the mass content of Cu ranges from 0.30% to 2.00%, the mass content of Ta ranges from 0.10% to 0.17%, and the mass content of Ti ranges from 96.50% to 99.00%. The titanium-based antibacterial composite controllable sputtering coating disclosed by the invention is safely and reliably combined with a titanium alloy matrix, and the biocompatibility of the titanium-based antibacterial composite controllable sputtering coating is also considered on the basis of excellent antibacterial property.

Description

Technical Field

[0001] The present invention generally relates to an antimicrobial coating for a biological implant. Background Art

[0002] As the most common fixation device, external fixators are widely used in open fracture surgery. However, implants such as percutaneous needles and screws used in conjunction with external fixators are susceptible to infection. When the screws are implanted in the area around the fracture, the skin barrier is damaged and the soft tissue in this area is exposed to the external environment of the human body for a long time. At the same time, since the biologically inert surface of the screws is easy to adhere to bacteria such as Staphylococcus aureus and Staphylococcus epidermidis and form a biofilm, it is easy for the implant site to be infected after surgery, which reduces the stability of the external fixator. External fixation screws that are in direct contact with the human body are often made of titanium alloy or stainless steel. Among them, TC4 titanium alloy has been widely used in orthopedics in recent years due to its excellent corrosion resistance, elastic modulus close to human bone, and high fatigue resistance. However, since the screws made of TC4 alloy have a biologically inert surface and do not have antibacterial properties, the above-mentioned bacterial infection problem still exists after implantation in the human body.

[0003] At present, antibacterial coatings can be divided into inorganic antibacterial coatings and organic antibacterial coatings based on the type of antibacterial agents loaded on the coating. The former mainly releases metals, metal oxides or non-metallic elements, and kills bacteria through multiple mechanisms such as depolarization of bacterial cell membranes, compounding biomolecules, and generating ROS to induce oxidative stress. The latter exerts its antibacterial effect by releasing organic antibacterial agents such as antibiotics, chitosan and quaternary ammonium compounds.

[0004] CN106310371A discloses a bone implant coating having certain biocompatibility, antibacterial and corrosion resistance obtained by plasma chemical vapor deposition of a metal tantalum-copper coating (copper content in the coating is not more than 10%) on an implant substrate such as a titanium alloy. CN108904893B discloses a composite coating evaporated on a titanium or titanium alloy implant substrate, including first evaporating a tantalum-copper alloy antibacterial coating and then evaporating a tantalum or niobium biocompatible coating. The preparation process of this type of antibacterial coating has problems with the bonding strength with the titanium alloy substrate on the one hand, and also has problems with effective and reasonable control of the coating composition on the other hand. Summary of the invention

[0005] The purpose of the present invention is to provide a safer and more reliable antibacterial coating for implantation.

[0006] According to a first aspect of the present invention, there is provided a method for preparing an antibacterial composite coating for an orthopedic implant, comprising:

[0007] Providing a substrate for forming an orthopedic implant;

[0008] Cleaning the substrate;

[0009] Providing a composite deposition target material, which is composed of a Cu target material, a Ta target material and a Ti target material;

[0010] Vacuum pre-sputtering treatment of composite deposition targets; and

[0011] The composite deposition target material is sputtered and deposited on a substrate to form a deposition coating, wherein the mass content of Cu is 0.30% to 2.00%, the mass content of Ta is 0.10% to 0.17%, and the mass content of Ti is 96.50% to 99.00%.

[0012] According to the present invention, the composite deposition target is composed of a Cu target, a Ta target and a Ti target, each of which is a fan-shaped target, and is spliced ​​into a complete circular target, wherein the Cu target occupies 1% to 4% of the fan area, the Ta target occupies 6% of the fan area, and the Ti target occupies 90% to 93% of the fan area. The composite circular target is very convenient for adjusting and controlling the composition of the sputtering deposition coating.

[0013] According to the preferred embodiment of the present invention, the mass content of Cu in the deposited coating is 1.4% to 1.9%, the mass content of Ta is 0.14% to 0.16%, and the mass content of Ti is 97.00% to 97.60%. The characteristics of the deposited coating of this composition are optimized. On the basis of reliable bonding with the substrate, it not only has better biocompatibility, but also has better antibacterial properties (up to Class I antibacterial properties). In the case where the composite deposition target adopts the above-mentioned circular target, the sector area occupied by the Cu target is adjusted to 3% to 4%, the sector area occupied by the Ta target is 6%, and the sector area occupied by the Ti target is 90% to 91%, so that the deposition coating with the above-mentioned corresponding composition can be conveniently and reliably realized.

[0014] According to the present invention, the substrate is made of a titanium alloy, such as TC4 titanium alloy. Since the present invention uses a Ti-based target material, a strong and reliable Ti-based deposited coating can be sputtered on the titanium alloy substrate. The sputtered coating is self-consistently fused with the substrate, thereby eliminating the problem of coating shedding to the greatest extent.

[0015] According to another aspect of the present invention, an orthopedic implant is provided, which has a substrate made of titanium alloy, wherein the substrate is formed with an antibacterial composite coating obtained according to the above preparation method.

[0016] 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

[0017] Figure 1 The process flow chart of the coating sputtering deposition adopted by the present invention;

[0018] Figure 2 A schematic diagram of the splicing of the composite deposition target material used in the present invention;

[0019] Figure 3 The SEM characterization images of the coating samples and blank samples prepared in the present invention;

[0020] Figure 4 The copper ion release curve of the coating sample prepared by the present invention;

[0021] Figure 5 A tantalum ion release curve diagram of the coating sample prepared in the present invention;

[0022] Figure 6 is the absorbance OD value of each group of samples at different time points;

[0023] Figure 7 This is a photo of the plate colonies of the test sample;

[0024] Figure 8 The antibacterial rate of the coating sample prepared by the present invention is shown. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific examples.

[0026] Coating preparation

[0027] 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.

[0028] Firstly, a TC4 titanium alloy sheet with a diameter of 10 mm and a thickness of 1 mm 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;

[0029] Then, the Cu target, Ta target and Ti target which are made into corresponding fan-shaped ones are spliced ​​into a complete circular composite deposition target to be used for sputtering, such as Figure 2 As shown;

[0030] 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.

[0031] Next, the coating was deposited on the substrate with a deposition power of 40 W, a working pressure of 0.75 Pa, an argon flow rate of 30 sccm, and a rotation rate of 5 r / min. After the deposition was completed, the sputtered coating sample was taken out and sealed for storage.

[0032] Sputtering coating composition

[0033] Figure 2 The composite deposition target shown is composed of Cu target, Ta target and Ti target with the following sector ratios (or volume ratios):

[0034] Target 1: 93% Ti; 6% Ta; 1% Cu;

[0035] Target 2: 92% Ti; 6% Ta; 2% Cu;

[0036] Target 3: 91% Ti; 6% Ta; 3% Cu;

[0037] Target 4: 90% Ti; 6% Ta; 4% Cu.

[0038] 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 by LabRAM HR Evolution laser Raman spectrometer produced by HORIBAFRANCE, France). The test results are the coating EPMA characterization results shown in Table 1, wherein 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.

[0039] Table 1: Coating composition (mass percentage; excluding oxygen)

[0040] Coating samples Cu content Ta content Ti content 1 0.45 0.11 98.69 2 0.79 0.12 98.27 3 1.44 0.14 97.55 4 1.87 0.16 97.01

[0041] Coating sample performance test

[0042] The four coating samples obtained in Table 1 and the blank sample of TC4 titanium alloy sheet were subjected to SEM characterization. The equipment used was a Zeiss Sigma-300 scanning electron microscope produced by Carl Zeiss of Germany. The characterization results are shown in Figure 3 , where (a), (b), (c), and (d) correspond to coating samples 1-4 in Table 1, respectively, and (e) corresponds to the blank sample. Figure 3As shown in the figure, the surface of the blank sample is very flat and has no obvious morphology. Irregularly shaped nanoparticles were observed on the surfaces of the other four samples. The spacing between the particles was relatively stable and evenly distributed on the entire Cu / Ta-Ti coating surface. More nanoparticles were formed on the surfaces of coating samples 3 and 4, which may be attributed to the fact that the target material of this composition can provide more nucleation sites on the coating surface, thus facilitating the formation and rapid growth of nanoparticles.

[0043] Coating sample Cu 2+ 、 5+ Release Test

[0044] The Agilent 720ES inductively coupled plasma mass spectrometer produced by Agilent Technologies, Inc., USA, was used to measure the concentration of corresponding ions released by the above coating samples 1-4 at different time points. Before the experiment, the above samples 1-4 were placed in a test tube with a volume of 20 ml and 10 ml of phosphate buffer was added, and then sealed and placed in a constant temperature incubator at 37°C. At the nodes of 12, 24, 36, 48 and 72 hours, 1 ml of PBS solution in the test tube was taken each time to detect the Cu 2+ At the same time, at 12, 24, 36 and 48 hours, 1 mL of PBS solution was taken from another tube in the same group to detect the Ta content. 5+ The concentration of PBS solution was added to the test tube at the same time. The above samples were repeated 3 times for each group. The ion release test results are shown in Figure 4 and Figure 5 ,in Figure 4 Cu 2+ A graph showing the concentration of versus time; Figure 5 For her 5+ The concentration of is plotted against time. Figure 4 and Figure 5 It can be seen that coating samples 1-4 can continuously release the Cu 2+ and Ta 5+ .pass Figure 4 The release curves show that the Cu released by coating samples 1-4 at various time points 2+ The concentration has a relatively obvious gradient, which corresponds to the Cu content of each group measured by electron probe microanalysis (EPMA); within 12 hours, Cu 2+ The concentration of Cu detected in each group of samples increased rapidly at 72h. 2+ The concentrations all reached the highest level, which were 164.82μg / L, 242.29μg / L, 423.38μg / L and 589.7μg / L respectively. Figure 5 The release curves show that the Ta released by coating samples 1-4 5+The concentration showed a rapid increase in the first 48 hours and reached the maximum at 48 hours. 5+ The concentrations were 18.22μg / L, 22.18μg / L, 30.82μg / L and 42.06μg / L respectively.

[0045] In vitro cytotoxicity test of coating samples

[0046] Experimental materials: The cells used in this experiment are rat bone marrow mesenchymal stem cells (rBMSCs) (Sybikang (Shanghai) Biotechnology Co., Ltd., Catalog No.: RAT-iCell-s018). Other main materials used are: iCell primary mesenchymal stem cell culture system (Sybikang (Shanghai) Biotechnology Co., Ltd., Catalog No.: PriMed-iCell-012); CCK-8 kit (Invigentech, USA, Catalog No.: IV08-100); phosphate buffer solution (PBS) (Hyclone, USA); pancreatic enzyme (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.).

[0047] Experimental equipment: The equipment used in this experiment mainly includes: CO2 constant temperature incubator (Binder, Germany, model CB260); centrifuge (Eppendorf, Germany, model 5810R); pipette (Eppendorf, Germany, model: 3120000240); clean bench (BioX Technology Co., Ltd., USA, model 2284); microplate reader (Berteng Instrument Co., Ltd., USA, model ELX800); UV spectrophotometer (BeckmenCoulter, USA)

[0048] Cytotoxicity assay: CCK-8 assay was used to detect cytotoxicity

[0049] The CCK-8 method is a widely used method for detecting cell viability and proliferation in vitro. It is based on the reduction of tetrazolium salt (WST-8) by cell dehydrogenase to form a water-soluble formazan dye, and then its absorbance OD value is quantitatively measured using a UV spectrophotometer. The size of the OD value can reflect the activity of the cells.

[0050] The experiment used rBMSCs cells in the logarithmic growth phase. The cells were cultured at a rate of 1.5×10 4The cells were inoculated at a density of 100 μL into a 96-well plate containing the corresponding samples, and then placed in a constant temperature incubator for 1 / 3 / 5 days at 5% CO2 and 37°C. The culture medium was removed at each time point after the culture was completed, each well was washed 3 times with PBS solution, and 1000 μL of culture medium containing 10% CCK-8 was added, and then placed in a constant temperature incubator at 5% CO2 and 37°C for 2 hours. After the final culture was completed, 800 μL of supernatant was taken from each well into a new 24-well plate, and the absorbance value at 450 nm was detected using an ELISA reader.

[0051] The relative growth rate (RGR) of cells was calculated using the following formula for the final absorbance OD value of each group of samples:

[0052]

[0053] 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 It is the background OD value, that is, the OD value of the multi-well plate itself without adding culture medium and cells.

[0054] Figure 6 The absorbance OD values ​​of samples measured by rat bone marrow mesenchymal stem cells (rBMSCs) at different time points include the cell absorbance OD values ​​of the background multi-well plate (left 1 of each group), blank sample (TC4 titanium alloy sheet; left 2 of each group), and coating samples 1-4 (corresponding to left 3-6 of each group) at 24h, 72h and 120h respectively; Table 2 gives the specific values ​​and their corresponding RGR. It can be seen that there is no statistically significant difference in the OD values ​​of coating samples 1-4 at the above time points (P>0.05), and there is no significant difference in the OD values ​​between coating samples 1-4 and uncoated blank samples (P>0.05). The RGR of coating samples 1-4 at all time points is higher than 75%, reaching the lowest level I cytotoxicity, so they are all biosafe materials. With the increase of time, the RGR of coating samples 3 and 4 showed a significant increase, indicating that the coating has excellent biocompatibility and can promote the proliferation of rBMSCs cells.

[0055] Table 2: Absorbance values ​​and RGR of test samples

[0056]

[0057] In vitro antibacterial activity test of coating samples

[0058] Experimental materials: The strain used in this experiment is Staphylococcus aureus (Sigma-Aldrich, USA, No.: ATCC6538). Other main materials include tryptone soy agar plate (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).

[0059] 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 (MettlerToledo, Switzerland, model AL104); ultraviolet spectrophotometer (Beckman Coulter, USA); centrifuge (Eppendorf, Germany, model 5810R).

[0060] Preparation of culture medium and bacterial suspension: Preparation of TSA liquid culture medium: Weigh 3g of solid powder of tryptone soy culture medium with an electronic analytical balance and add it to a clean and sterile flask, and add 100mL of distilled water to the flask to achieve the required concentration of TSA liquid culture medium. Gently shake the flask or bottle to completely dissolve the powder. Then put the flask into a high-temperature and high-pressure steam sterilizer and sterilize the TSA liquid culture medium by autoclaving at 121°C for 15-20 minutes. 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 colony in a test tube containing 3mL of TSA liquid culture medium, and place it in a constant temperature oscillator and shake it at 37°C for 24 hours. The concentration of Staphylococcus aureus in the test tube is adjusted to 10 by measuring the optical density of the bacterial culture at a specific wavelength using an ultraviolet spectrophotometer and comparing it with a bacterial turbidimetric tube. 7 CFU / mL to achieve the bacterial concentration required for the experiment. To prevent contamination, the bacterial suspension was stored in a sterile environment for later use.

[0061] Co-culture-plate coating method to test antibacterial properties: Before the experiment, the coating samples were first disinfected. Soak the cotton ball in anhydrous ethanol for about 10 seconds, and then use the alcohol cotton ball to wipe the sample surface for sterilization, while removing possible debris and other impurities. After wiping, rinse the sample with sterile distilled water for 3 times, and place the sample in a sterile environment of a laminar flow hood to air dry for later use.

[0062] After disinfection, prepare the bacterial suspension for plate coating by co-cultivation. First, take out the prepared sample piece and put it into the 24-well plate, and use a pipette to drop 500μL of bacterial suspension into the corresponding wells in turn. Then put the 24-well plate with 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 cultivation is completed, take out the 24-well plate from the incubator, and separate the bacterial suspension on the surface of the sample by slight vibration to 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 to prepare for plate coating.

[0063] After obtaining the bacterial suspensions of all samples co-cultured, use a pipette to take 50 μL of each group of bacterial suspensions and add them to different tryptone soy agar plates, and evenly spread them on the surface of these plates with a coating rod. Then, number each plate in sequence and put it in a constant temperature incubator at 37°C after completion. After 24 hours, take out the agar plate and count the number of colonies on its surface.

[0064] 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 properties of the samples. The calculation formula is as follows:

[0065]

[0066] Rx represents the antibacterial 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). After obtaining the antibacterial rate Rx, the antibacterial properties of the sample groups are graded with reference to the classification standards for the antibacterial properties of coatings in the national standard "HG / T3950-2007": when Rx≥99, the sample has strong antibacterial properties, which is Class I; when 90≤Rx≤99, the sample has antibacterial properties, which is Class II.

[0067] Figure 7 Table 3 shows the plate colony count results of the test samples, where (a), (b), (c), and (d) correspond to coating samples 1-4, respectively, and (e) corresponds to the blank group. Figure 8 The antibacterial rates of coating samples 1-4 against Staphylococcus aureus are shown.

[0068] Table 3

[0069]

[0070] As can be seen from the above, the number of colonies on the surface of coating samples 1-4 showed a gradual decreasing trend as a whole, among which the number of colonies on the surface of coating sample 3 has been significantly reduced, and the antibacterial rate has reached 98.03%, with level II antibacterial property. The number of colonies on the surface of coating sample 4 is the least, and the antibacterial rate is about 99.29%, with the highest level I antibacterial property among the four groups, showing excellent antibacterial effect.

Claims

1. A method for preparing an antibacterial composite coating for orthopedic implants, comprising: Providing a substrate for forming an orthopedic implant; Cleaning the substrate; Providing a composite deposition target material, which is composed of a Cu target material, a Ta target material and a Ti target material; Vacuum pre-sputtering treatment of composite deposition targets; and The composite deposition target material is sputtered and deposited on a substrate to form a deposition coating, wherein the mass content of Cu is 0.30% to 2.00%, the mass content of Ta is 0.10% to 0.17%, and the mass content of Ti is 96.50% to 99.00%.

2. The preparation method according to claim 1, wherein the composite deposition target is composed of a Cu target, a Ta target and a Ti target, each of which is fan-shaped, spliced ​​into a complete circular target, wherein the Cu target occupies 1% to 4% of the fan area, the Ta target occupies 6% of the fan area, and the Ti target occupies 90% to 93% of the fan area.

3. The preparation method according to claim 1, wherein the Cu mass content in the deposited coating is 1.4% to 1.9%, the Ta mass content is 0.14% to 0.16%, and the Ti mass content is 97.00% to 97.60%.

4. The preparation method according to claim 3, wherein the composite deposition target is composed of a Cu target, a Ta target and a Ti target, each of which is fan-shaped, spliced ​​into a complete circular target, wherein the Cu target occupies 3% to 4% of the fan area, the Ta target occupies 6% of the fan area, and the Ti target occupies 90% to 91% of the fan area. The preparation method according to claim 1 , wherein the substrate is made of titanium alloy.

6. An orthopedic implant having a substrate made of a titanium alloy, wherein the substrate is formed with an antibacterial composite coating obtained according to the preparation method of any one of claims 1 to 4.

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