CN117802432A - Antibacterial bone-promoting titanium alloy with gradient nano structure and preparation method and application thereof - Google Patents
Antibacterial bone-promoting titanium alloy with gradient nano structure and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a gradient nano-structure antibacterial bone-promoting titanium alloy, which is prepared by carrying out surface mechanical rolling technology treatment on micro-alloyed copper or silver-titanium alloy, forming gradient nano-crystal layers with the grain size from 40nm to 100nm from top to bottom on the surface of the titanium alloy through different times of cyclic rolling, wherein the content of copper or silver in the micro-alloyed copper or silver-titanium alloy is lower than 1%, and the gradient nano-crystal layers are composed of Ti with the morphology of equiaxial crystals and the grain size of 40nm to 100nm 2 Cu phase or Ti 2 Ag phase composition, thereby imparting antibacterial properties and biological phase to the microalloyed titanium alloyThe compatibility, can promote cell proliferation, has no cytotoxicity, and has good function of inducing osteoblast differentiation. The obtained gradient nanocrystalline layer has stable structure and durable antibacterial performance, has antibacterial performance to staphylococcus aureus reaching more than 95%, and is favorable for popularization of biomedical copper/silver titanium alloy in orthopaedics materials in clinical medicine.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to preparation and application of a gradient nano-structure antibacterial bone titanium alloy.
Background
With the rapid development of the medical instrument industry, the demand of people for tissue repair substitute materials such as bone brackets, dental implants, artificial joints and the like is increasing, and titanium and the alloy thereof have the advantages of good biocompatibility, corrosion resistance, elastic modulus similar to that of bone tissues and the like, and have been widely applied to the materials. However, titanium itself does not have antibacterial properties, and may cause infection during use, so that it is required to provide titanium alloy with certain antibacterial properties. The existing antibacterial material is designed to mostly select Cu ions and Ag ions with good antibacterial performance, and the Cu ions or the Ag ions dissolved out from the surface of the material obtain good antibacterial performance. Therefore, the development of novel Cu-or Ag-containing titanium alloy materials is an option.
In practical applications, degradation of titanium and its alloys always starts from the surface. The corrosion resistance, wear resistance and biocompatibility of titanium and its alloys are significantly affected by the surface properties. At present, the research on antibacterial titanium alloy mainly focuses on adding a small amount of silver or copper alloying elements into a titanium matrix through powder metallurgy or smelting and subsequent heat treatment, and the antibacterial mechanism is generally attributed to dissolution of functional ions, so that a large amount of Cu ions or Ag ions are required to be dissolved out while good antibacterial performance is obtained. Current designs of antimicrobial titanium alloys therefore often require the addition of at least greater than 1wt.% elemental silver or greater than 3wt.% elemental copper.
However, long-term high concentration of functional ion release leads to cytotoxicity, allergy and immunityToxicity, and the like. In addition, the high mass fraction copper/silver ratio in titanium alloys also results in reduced plasticity and corrosion resistance of the matrix. Therefore, the design of the copper/silver-titanium alloy should control the content of copper/silver alloy element preferentially, and secondly, if the surface of the copper/silver-titanium alloy can inhibit bacterial adhesion and induce cell differentiation first, and form biological seal rapidly, the osseointegration efficiency can be effectively improved. Thus improving the biological activity of the alloy surface, reducing and controlling Cu 2+ Or Ag + The dissolution concentration is the current focus of research on copper/silver titanium alloys.
The surface modification technology of the dental implant widely used in clinic at present mainly comprises the following chemical method: acid etching, alkali heat treatment, and the like. The purpose of acid washing is to remove oxide layers or impurities from the surface of the material, and tiny corrosion pits are formed on the surface of titanium after acid treatment. The treatment can increase the roughness of the surface of the implant, and researches show that the rough surface can increase the bioadhesion more than the smooth surface, so that the biocompatibility is enhanced, but free hydrogen is easy to generate after acid treatment, so that the hydrogen embrittlement of the implant can be caused, the performance of the implant is influenced, and the human health is endangered.
Disclosure of Invention
In view of the above problems, the invention develops a novel surface modification method for microalloyed copper/silver titanium alloy, and the microalloyed antibacterial titanium alloy forms a gradient nanostructure by Surface Mechanical Rolling (SMRT). The formed gradient nano-structure surface layer can improve the quantity of new bone on the surface of the copper/silver-titanium alloy, improve the bone-joining efficiency and separate out Ti 2 Cu/Ti 2 The Ag phase has long-term and stable antibacterial activity.
The invention aims to provide a preparation method and application of a gradient nano-structure antibacterial bone titanium alloy, namely, the surface of the titanium alloy added with trace copper/silver is formed into gradient nano-structures with different depths through surface mechanical rolling, the method is simple to operate, and even if the titanium alloy is exposed in air or is always soaked in simulated body fluid, the antibacterial property of the titanium alloy to staphylococcus aureus is still more than 90%, the stability of a gradient nano-crystal layer is proved, and the titanium alloy has no cytotoxicity, and can promote cell proliferation and induce osteoblast differentiation.
In order to solve the technical problems, the invention provides a gradient nano-structure antibacterial bone titanium alloy, which is formed by a gradient nano-crystal layer with the grain size gradually ranging from 40nm to 100nm from top to bottom, wherein the gradient nano-crystal layer is formed by Ti with the morphology of equiaxed crystals and the grain size ranging from 40nm to 100nm, and the gradient nano-crystal layer is formed on the surface of micro-alloyed copper or silver titanium alloy 2 Cu phase or Ti 2 The Ag phase is formed, and the thickness of the gradient nanocrystalline layer is 100-1000 mu m.
Furthermore, the gradient nano-structure antibacterial effect bone titanium alloy provided by the invention comprises 0.1-1% of copper or silver in weight percentage.
Meanwhile, the invention also provides a preparation method of the gradient nano-structure antibacterial bone titanium alloy, which mainly comprises the following steps: removing a surface oxide layer of the microalloyed copper or silver-titanium alloy; then the micro-alloyed copper or silver-titanium alloy upper surface is circularly rolled for 2 to 20 times with single pressing quantity of 5 mu m to 30 mu m, ti is separated out on the surface after the circularly rolled 2 Cu phase or Ti 2 Ag phase, thereby forming the gradient nanocrystalline layer.
In the preparation method, the number of circulating rolling is preferably 5, and the single pressing amount is 10 mu m.
And sequentially ultrasonically cleaning the titanium alloy after the cyclic rolling in dichloromethane and absolute ethyl alcohol, and drying. The time for ultrasonic cleaning of the titanium alloy in dichloromethane is 20min. The time for ultrasonic cleaning of the titanium alloy in absolute ethyl alcohol is 5min.
The gradient nano-structure antibacterial bone titanium alloy disclosed by the invention has the antibacterial property of more than 90% on staphylococcus aureus even if being exposed in air or being soaked in simulated body fluid all the time, and has the antibacterial property and biocompatibility for staphylococcus aureus, more than 95% on staphylococcus aureus through antibacterial and biocompatibility tests, no cytotoxicity, and the function of promoting cell proliferation and osteoblast differentiation, can be used as an orthopedic material in clinical medicine, and is favorable for popularization of biomedical copper/silver titanium alloy in the orthopedic material in clinical medicine.
Compared with the prior art, the invention has the beneficial effects that:
(1) The method reduces the weight fraction of the antibacterial metal to 1% or below, fully reduces cytotoxicity caused by heavy metal ions, and simultaneously ensures that the titanium alloy has strong (more than 90%) antibacterial performance.
(2) The titanium alloy obtained by the method is nanocrystalline with equiaxial size, the nanocrystalline is favorable for biological adhesion, has no cytotoxicity, can promote cell proliferation and has the function of inducing osteoblast differentiation, and inflammation is reduced.
(3) The surface mechanical rolling technology is simple to operate, the modified titanium alloy presents a gradient nano structure, and the gradient nano crystal layer can promote precipitation of Ti in the titanium matrix 2 Cu/Ti 2 The Ag phase has obviously improved antibacterial effect, and is favorable for popularization of biomedical copper/silver-titanium alloy in orthopaedics materials in clinical medicine.
Drawings
FIG. 1 is a TEM image of the surface of the micro-alloyed copper-titanium alloy of example 1 after mechanical rolling treatment, showing the top-layer image.
Fig. 2 is a photograph of a coating count of staphylococcus aureus after mechanically rolling the micro-alloyed copper/silver alloy on the surface of example 1 and comparative sample 1 or 2, wherein the weight percentage of copper/silver is not more than 1%. The control group Ti recipe was consistent with copper/silver, with GNS Ti-Cu, GNS Ti-Ag being the samples obtained in example 1, ti-Cu being the comparative sample 1, and Ti-Ag being the comparative sample 2.
FIG. 3 shows the cell proliferation of the micro-alloyed copper/silver alloy of example 1 and comparative sample 1 on day 3 after the mechanical rolling treatment, wherein GNS Ti-Cu and GNS Ti-Ag are the samples obtained in example 1, ti-Cu is the sample obtained in comparative sample 1, and Ti-Ag is the sample obtained in comparative sample 2.
FIG. 4 is a graph of CCK-8 test results on day 3 after mechanical rolling treatment of the surface of the microalloyed copper/silver alloy of example 1 and comparative sample 1 or 2. Wherein, the A diagram is microalloyed Cu-containing titanium alloy, and the B diagram is microalloyed Ag-containing titanium alloy. Wherein GNS Ti-Cu and GNS Ti-Ag are the samples obtained in example 1, ti-Cu in the A diagram is obtained as a comparative sample 1, and Ti-Ag in the B diagram is obtained as a comparative sample 2.
FIG. 5 is a graph of alkaline phosphatase (ALP) results at day 7 after mechanical rolling treatment of the surface of the microalloyed copper/silver alloy of example 1 and comparative sample 1 or 2. Wherein, A is a microalloyed Cu-containing titanium alloy and B is a microalloyed Ag-containing titanium alloy. Wherein GNS Ti-Cu and GNS Ti-Ag are the samples obtained in example 1, ti-Cu in the A diagram is obtained as a comparative sample 1, and Ti-Ag in the B diagram is obtained as a comparative sample 2.
FIG. 6 is a graph showing the results of alizarin red staining mineralized nodules on day 28 after mechanical rolling treatment of the surface of the micro-alloyed copper/silver alloy of example 1 and comparative sample 1 or 2. Wherein, the A diagram is microalloyed Cu-containing titanium alloy, and the B diagram is microalloyed Ag-containing titanium alloy. Wherein GNS Ti-Cu and GNS Ti-Ag are the samples obtained in example 1, ti-Cu in the A diagram is obtained as a comparative sample 1, and Ti-Ag in the B diagram is obtained as a comparative sample 2.
Detailed Description
The invention provides a gradient nano-structure antibacterial bone titanium alloy, which is characterized in that a gradient nano-crystal layer with the thickness of 100-1000 mu m is formed on the surface by circularly rolling the surface of micro-alloyed copper or silver titanium alloy for a plurality of times, and the gradient nano-crystal layer is formed by Ti precipitated on the surface of the micro-alloyed copper or silver titanium alloy 2 Cu phase or Ti 2 Ag phase composition, said Ti 2 Cu phase or Ti 2 The morphology of the Ag phase is equiaxed crystal and nanocrystalline with the grain size of 40 nm-100 nm, and the Ag phase is distributed according to the gradient that the grain size gradually transits from 40nm to 100nm from top to bottom. In the invention, the weight percentage of copper or silver in the microalloyed copper or silver-titanium alloy is only 0.1-1%. The invention relates to a preparation method of a gradient nano-structure antibacterial bone-promoting titanium alloy, which mainly comprises the following steps: removing a surface oxide layer of the microalloyed copper or silver-titanium alloy; then, the single pressing amount is 5-30 μm for 2-20 times from top to bottom, and a gradient nanocrystalline layer structure with the grain size gradually transiting from 40nm to 100nm from top to bottom is formed. Thereby more easily precipitating Ti on the surface 2 Cu/Ti 2 Ag phase, the titanium alloy after the cyclic rolling is firstly ultrasonically cleaned in dichloromethane for 20min,then ultrasonic cleaning is carried out in absolute ethyl alcohol for 5min, and drying is carried out.
The technical scheme of the invention is further specifically described below with reference to the accompanying drawings and specific embodiments. The following examples are in no way limiting of the invention and the invention is not limited to copper/silver titanium alloys.
Example 1
1. The preparation of the bone titanium alloy is facilitated by the antibacterial gradient nano structure, which comprises the following steps:
step 1) pretreatment: and fixing the microalloyed copper-titanium alloy with copper content of 1wt% on a milling machine, and milling the surface oxide layer by using a cutter to make the surface smooth and clean.
Step 2) surface mechanical rolling treatment: 5 times of circulating rolling with each pressing amount of 10 mu m are carried out on the surface of the copper-titanium alloy from top to bottom, so that the structure of the gradient nanocrystalline layer with the thickness of 200 mu m is obtained on the surface of the micro-alloyed copper-titanium alloy, and the grain size is gradually increased from 40nm (namely the smallest grain size of the cross section) to 100nm (namely the largest grain size of the cross section) from top to bottom.
Step 3) treating the surface of the sample after the cyclic rolling: and ultrasonically cleaning the mechanically rolled sample with dichloromethane and absolute ethyl alcohol for 5min and 10min to remove greasy dirt and impurities on the surface.
In order to evaluate the microscopic morphology of the gradient nanocrystalline layer obtained in example 1, the cross-sectional outermost layer region of the microalloyed copper-containing titanium alloy after the cyclic rolling treatment was tested by a TEM electron microscope, and as shown in fig. 1, the outermost layer thereof was seen to be nanocrystalline with equiaxial, and was composed of white shiny α -Ti phase and black shiny β -Ti phase, and the thickness of the obtained nanocrystalline layer was 200 μm, and the grain size was gradually increased, the grain size of the outermost layer was 40nm, and the grain size was gradually increased to 100nm with the increase of depth, so that it was confirmed from the figure that the structure formed by the method was a gradient nanostructure.
2. Antibacterial and biocompatibility testing
1) Evaluation of antibacterial experiment
In order to evaluate the antibacterial performance of the gradient nanocrystalline layer obtained by the preparation method of the present invention, the gradient nanocrystalline layer obtained in example 1 was subjected to antibacterial experimental evaluation.
Cutting the surface-mechanically rolled titanium alloy of example 1 to a diameter of Φ15X2mm 3 Ultraviolet irradiation is carried out after standard original tablets are subjected to sterilization for 1h; respectively placing the sterilized pure Ti samples of the samples and the control group of the invention and the copper-titanium alloy samples and the silver-titanium alloy samples with untreated surfaces into 12 pore plates, respectively dripping 100 mu L of bacterial liquid on the surfaces of all the samples, and culturing in an incubator at 37 ℃ for 24 hours; and placing the sample which is cultured for 24 hours with the bacterial liquid into a centrifuge tube containing 2mL of sterile PBS solution, and uniformly eluting bacteria on the surface by ultrasonic vibration for 3 min.
Diluting the bacterial solution to 1X 10 with sterile PBS 6 CFU/mL, and 50 μl of diluted bacterial liquid was plated and cultured for 24 hours, and the antibacterial ratio was calculated according to the following formula: antibacterial ratio (%) = [ (number of colonies on surface of control sample-number of colonies on surface of copper/silver-titanium alloy after cyclic rolling)/number of colonies on surface of control sample]×100％。
The gradient nanocrystalline layer prepared on the surface of the microalloyed copper-titanium alloy in the embodiment 1 has good antibacterial performance, and as shown in figure 2, the antibacterial rate to staphylococcus aureus is over 95 percent.
2) Cell proliferation and toxicity evaluation:
in order to evaluate the osteogenic properties of the gradient nanocrystalline layer prepared according to the invention, it was subjected to cell proliferation (FDA staining) and toxicity evaluation (CCK-8), alkaline phosphatase Assay (ALP) and (alizarin red) mineralization nodule assay, respectively.
The titanium alloy subjected to the surface mechanical rolling treatment in example 1 was co-cultured with mouse osteoblast MC3T3 at an inoculation density of 1X 10 4 cells/cm 2 Density standard, after co-cultivation for 3 days, the supernatant was aspirated and discarded;
washing with sterile PBS for 2-3 times to remove non-viable cells, adding 200 μL diacetic acid Fluorescein (FDA) in dark place, staining for 2min, and observing cell proliferation with an inverted fluorescence microscope.
The titanium alloy subjected to the surface mechanical rolling treatment in example 1 was put into a 24-well plate and protected from light200. Mu.L of CCK-8 mixed solution was added to the surface of the titanium alloy in a 24-well plate under the same conditions, and the mixture was reacted in a constant temperature incubator at 37℃for 2 hours. 100. Mu.L of the mixed solution was pipetted into a 96-well plate and the absorbance (OD) values of osteoblasts at a wavelength of 450nm on the samples were measured using a multifunctional microplate reader. The relative proliferation rates of cells (relative growth rate, RGR) were calculated according to International Standard ISO 10993-5 biological assessment of medical instruments-in vitro cytotoxicity test): RGR= (OD Material group /OD Control group )×100％。
The FDA detection and CCK-8 detection are important marks for judging whether cells proliferate and have no toxicity, the FDA result is shown in figure 3, the gradient nanocrystalline layer obtained after surface mechanical rolling treatment can be seen, the number and spreading area of the cells are increased, the gradient nanostructure is proved to promote cell proliferation, the CCK-8 result is shown in figure 4, the absorbance of the copper/silver titanium alloy after surface mechanical rolling treatment is obviously enhanced, and the copper/silver titanium alloy is proved to have no cytotoxicity.
3) Alkaline phosphatase Assay (ALP)
Placing the titanium alloy subjected to the surface mechanical rolling treatment in the embodiment 1 into a 24-hole plate, sucking out the complete culture medium in the material, adding 300 mu L of pancreatin for digestion, centrifuging, and removing the supernatant; PBS buffer was added to 10mL and centrifuged at 1500rpm for 10min. Removing supernatant, adding 100 μl of cell lysate solution, repeatedly freezing and thawing for 5-10 times, placing the repeatedly frozen and thawing solution into a centrifuge, centrifuging at 3000rpm for 15min, and collecting supernatant. The alkaline phosphatase content of the sample was determined according to the alkaline phosphatase kit instructions.
Alkaline phosphatase is an important marker for osteoblast differentiation, and the osteoblast properties of the cells are described from another aspect, and as a result, as shown in FIG. 5, a gradient nanocrystalline layer formed after surface mechanical rolling treatment can be seen, and the absorbance increases, thus proving to have the function of inducing osteoblast differentiation.
4) Mineralized nodule detection (alizarin red)
Placing the titanium alloy subjected to the surface mechanical rolling treatment in the embodiment 1 into a 24-hole plate, adding paraformaldehyde solution into the hole plate to immerse a sample, sucking out after 10min, and cleaning with PBS for 2-3 times;
adding 400uL of alizarin red, placing in an incubator at 37 ℃ for dyeing for 30min, and washing with PBS for 2-3 times until the PBS is colorless;
after each well was dissolved by adding chlorohexadecyl oxapyridine, 200. Mu.L of the lysate was added to a 96-well plate, and the absorbance (OD) value of osteoblast cells at 560nm on the sample was measured using a multifunctional microplate reader.
The alizarin red dye can identify calcium salt, the calcium salt is a mark of osteoblast differentiation and osteogenic potential, the result is shown in figure 6, the gradient nanocrystalline layer obtained after surface mechanical rolling treatment can be seen, the absorbance is increased, and the osteogenic potential is proved.
In conclusion, through antibacterial and biocompatibility tests, it can be judged that the gradient nanocrystalline layer formed in the invention has antibacterial capability and osteogenic potential, and the application direction of the gradient nanocrystalline layer can be used as a bone substitute in clinical medicine to reduce inflammation.
Examples 2 to 10
The gradient nanostructure antimicrobial contributes to the preparation of bone titanium alloys, which were prepared in essentially the same manner as in example 1, except that: in the step 1), the adopted microalloyed copper-titanium alloy or silver-titanium alloy contains different copper or silver; in the step 2), the number of cyclic rolling and the single pressing amount are different, and the thickness and the grain size of the finally obtained gradient nanocrystalline layer are different, as shown in table 1.
TABLE 1
Examples 1-10 are copper/silver-titanium alloys with a mass fraction of 0.1-1% of the cyclic rolling treatment, and as can be seen from fig. 2, when the mass fraction of the copper/silver-titanium alloy is 1%, the antibacterial effect on staphylococcus aureus is most obviously improved and is higher than 95%, and the antibacterial rate of the rest of the microalloyed copper/silver-titanium alloys after treatment is gradually improved. The method is fully proved to be suitable for copper/silver-titanium alloy.
Examples 11 to 15
The gradient nano-structure antibacterial effect is achieved in the preparation process of the bone titanium alloy, wherein the preparation process is the same as that of the example 1, and microalloyed copper titanium alloy or silver titanium alloy with copper content or silver content of 1 weight percent is adopted, and the preparation process is different from the example 1 only in that: in the step 2), the number of cyclic rolling and the single pressing amount are different, and the thickness and the grain size of the finally obtained gradient nanocrystalline layer are different, as shown in table 2.
TABLE 2
In examples 11-15, the mass fraction of the microalloyed copper/silver was 1%, and the surface treatment process was different, and the antibacterial effect and bone property improvement were not obvious with the increase of the thickness of the nanolayer and the increase of the grain size, and the important regulation and control mechanism of the antibacterial property and bone property was provided by the nanocrystals during the process, and the regulation and control function was gradually reduced with the gradual increase of the size.
Comparative example 1
The preparation of the bone titanium alloy is promoted by the antibacterial effect of the gradient nano structure, and the process is as follows:
step 1) pretreatment: fixing copper-titanium alloy with copper content of 0.5wt% prepared by smelting or powder metallurgy on a milling machine, and milling the surface oxide layer by a turning tool to make the surface smooth and clean.
Step 2) surface mechanical rolling treatment and step 3) the treatment of the surface of the sample after cyclic rolling was the same as steps 2) and 3) of example 1, and finally comparative sample 1 was obtained.
Comparative example 2
The preparation of the bone copper/silver titanium alloy is promoted by the antibiosis, the micro-alloyed silver titanium alloy with silver content of 0.5 weight percent is fixed on a milling machine, and a cutter is used for milling the surface oxide layer of the micro-alloyed silver titanium alloy, so that the surface is smooth and clean, and a comparison sample 2 is obtained.
Referring to fig. 2, it is apparent that the antibacterial effect of the Ti-Cu of comparative sample 1 and the Ti-Ag of comparative sample 2 is not remarkable because the surface is peeled off only by the mechanical friction of the turning tool rotating at high speed, the grains are not refined, and the antibacterial effect is not remarkable, since only the surface scale is removed, and the antibacterial performance is not different from that of the copper/silver alloy produced by powder metallurgy or smelting.
As can be seen from examples 1-15 and comparative samples 1-2 and FIGS. 1-6, the number of cyclic rolling and the single pressing amount have an effect on the antibacterial performance of the copper/silver alloy, and the formed gradient nanocrystalline layer refines the grain size to the nano-scale, thereby making Ti easier 2 Cu/Ti 2 Ag is compatible and easy to release, so the antibacterial mechanism of the invention is as follows: micro-alloyed copper/silver-titanium alloy is processed by cyclic rolling from top to bottom to form a gradient nanocrystalline layer structure on the surface of the micro-alloyed copper/silver-titanium alloy, so that nanocrystalline Ti is easier to make 2 Cu/Ti 2 The Ag phase is easier to release, and then contacts with bacteria to destroy bacterial cell membranes and membrane proteins, so that the sterilizing effect is realized. In addition, the proliferation and differentiation of osteoblasts can be promoted by both high surface energy and hydrophilic property caused by high-density crystal defects on the surface of the nanocrystals. Has great application potential in clinical medicine.
Although the invention has been described above with reference to the accompanying drawings, the invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many changes may be made by those skilled in the art without departing from the spirit of the invention, which are all within the protection of the invention.
Claims (9)
1. A gradient nanostructured antimicrobial osteo-titanium alloy, characterized by: the gradient nanocrystalline layer is formed by a gradient nanocrystalline layer which is formed on the surface of microalloyed copper or silver-titanium alloy and has the grain size from 40nm to 100nm gradually, and the gradient nanocrystalline layer is formed by Ti with the morphology of equiaxed crystals and the grain size from 40nm to 100nm 2 Cu phase or Ti 2 Ag phase, the thickness of the gradient nanocrystalline layer is 100-1000μm。
2. The gradient nanostructured antimicrobial osteogenic titanium alloy of claim 1, wherein the weight percent of copper or silver in the microalloyed copper or silver titanium alloy is between 0.1 and 1%.
3. A method of preparing a gradient nanostructured antimicrobial osteo-titanium alloy according to claim 1, wherein the micro-alloyed copper or silver-titanium alloy is subjected to surface oxide layer removal; then the micro-alloyed copper or silver-titanium alloy upper surface is circularly rolled for 2 to 20 times with single pressing quantity of 5 mu m to 30 mu m, ti is separated out on the surface after the circularly rolled 2 Cu phase or Ti 2 Ag phase, thereby forming the gradient nanocrystalline layer.
4. The method of claim 3, wherein the number of circulating rolls is 5 and the single pressing amount is 10. Mu.m.
5. The method according to claim 3, wherein the titanium alloy after the cyclic rolling is sequentially ultrasonically cleaned in dichloromethane and absolute ethanol and dried.
6. The method of claim 4, wherein the titanium alloy is ultrasonically cleaned in methylene chloride for 20 minutes.
7. The method of manufacturing according to claim 4, wherein: the time for ultrasonic cleaning of the titanium alloy in absolute ethyl alcohol is 5min.
8. An application of a gradient nano-structure antibacterial bone titanium alloy is characterized in that: use of the titanium alloy according to claim 1 or 2 obtained by the preparation method according to any one of claims 3 to 7 in orthopedic materials in clinical medicine.
9. The application of the gradient nano-structure antibacterial bone titanium alloy according to claim 8, which is characterized in that the gradient nano-structure antibacterial bone titanium alloy has stable structure and durable antibacterial performance, and the antibacterial rate of the gradient nano-structure antibacterial bone titanium alloy to staphylococcus aureus reaches more than 95%; the titanium alloy has no cytotoxicity, can promote cell proliferation, and has the function of inducing osteoblast differentiation.
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