CN111876707B

CN111876707B - Modification method of titanium alloy with bone regeneration promoting and antibacterial functions and modified titanium alloy

Info

Publication number: CN111876707B
Application number: CN202010772457.XA
Authority: CN
Inventors: 黄润; 黄雷; 黄明策; 杜超
Original assignee: Anhui University of Science and Technology
Current assignee: Anhui University of Science and Technology
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2021-11-19
Anticipated expiration: 2040-08-04
Also published as: CN111876707A

Abstract

The invention discloses a method for modifying a titanium alloy with bone regeneration promoting and antibacterial functions, which relates to the technical field of material surface modification and comprises the following steps: (1) carrying out solution treatment on the Ti-Nb-Zr-Sn-Mo titanium alloy at 750 ℃ for 1h, cooling in air, then placing at 550 ℃ for aging treatment for 6h, and then fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine for grinding treatment; (2) and (2) treating the nanocrystalline sample in the step (1) in an ion implanter, and performing ion implantation under a vacuum condition by using Mg and Ag target materials as ion sources. The invention has the beneficial effects that: according to the invention, magnesium and silver ions are introduced into the surface of the nanocrystalline Ti-Nb-Zr-Sn-Mo titanium alloy by adopting an ion injection method, so that a surface layer with double functions of promoting new bone formation and high-efficiency antibiosis is prepared on the surface of the titanium alloy.

Description

Modification method of titanium alloy with bone regeneration promoting and antibacterial functions and modified titanium alloy

Technical Field

The invention relates to the technical field of material surface modification, in particular to a modification method of a titanium alloy with double functions of promoting bone regeneration and resisting bacteria and a modified titanium alloy.

Background

Because of good physical, chemical and mechanical properties, titanium metal is the preferred material for repairing and replacing hard tissues.

Surface mechanical polishing (SMAT) is an emerging surface treatment method, and the working diagram is shown in FIG. 1. The method is a surface treatment method for obtaining a structure with a nanocrystalline surface and a gradient structure with gradually increased grain size along the depth direction by making the surface of a metal material into a nano-grade through strong plastic deformation. In the surface mechanical grinding treatment process, steel balls (or other materials such as glass balls or ceramic balls) with smooth surfaces act on the surface of a sample in different directions, and a stress field is formed near the surface of the sample, so that the surface grain size of the treated sample is continuously refined to a nanometer scale.

Ion implantation is a physical process in which gas or elemental vapor is ionized to form ions, which are accelerated by a high-voltage electric field to strike the ions at high speed onto the surface of a solid. After the high-speed ions are injected into the metal, the ions elastically or inelastically collide with atoms, electrons and the like on the surface of the metal, so that the kinetic energy of the ions is gradually transferred to recoil atoms or electrons to complete energy transfer and deposition, as shown in fig. 2. The ion implantation modification method has the following characteristics: I) the ion implantation is a non-thermal equilibrium process, and in principle, any element on the periodic table can be implanted into the surface of any base material without metallurgical limitation; II) the variety, energy and dosage of the implanted elements can be selected, the surface prepared by the method is not limited by classical thermodynamic parameters and an equilibrium phase diagram, the implanted surface layer is often in a metastable state, and new phases or compounds which are difficult to obtain by a conventional method, such as supersaturated solid solutions, amorphous phases and the like are obtained; III) the ion injection layer has no obvious interface relative to the base material, the surface has no cracking or peeling problem, and the problem of the firm film-base combination of other biomaterial surface modification methods is avoided; IV) the ion implantation process is easy to control and has good repeatability, and the concentration, distribution and depth of the implanted elements can be regulated and controlled by process parameters; v) ion implantation is generally carried out under normal temperature and vacuum, the shape of a workpiece is not changed, oxidation is avoided, the original size precision and surface roughness of the surface of a sample can be maintained, and the method is particularly suitable for the final process of high-precision parts.

For example, patent application with publication number CN109468605A discloses a method for modifying the surface of a titanium alloy and a modified titanium alloy, which comprises the steps of mechanically treating the surface of the titanium alloy, injecting Co ions into the titanium alloy by using a Co target as an ion source in an ion injection manner, and modifying the titanium alloy, wherein the modified Ti-Nb-Zr-Sn titanium alloy can significantly promote differentiation of stem cells towards angiogenesis.

However, there are two major problems with titanium-based implants in clinical applications: 1) titanium-based metals have a poor ability to induce new bone formation and often fail to form an effective repair when faced with bone defects. 2) The titanium-based alloy has no antibacterial property, when clinical operation is carried out, bacteria easily form a layer of biomembrane on the surface of the titanium-based alloy, and the biomembrane can protect the bacteria in the titanium-based alloy from being attacked by the immune system of a host, so that bacterial plaque is formed on the surface of the implant, and the infection and necrosis of surrounding tissues are caused.

Disclosure of Invention

The technical problems to be solved by the invention are that the titanium alloy in the prior art has weak capacity in inducing new bone formation, has no antibacterial property, often cannot form effective repair when being used for treating bone defect, and tissues around the implanted titanium alloy are susceptible to necrosis, so that the modification method of the titanium alloy with the bone regeneration promoting and antibacterial functions is provided.

The invention solves the technical problems through the following technical means:

a method for modifying a titanium alloy with double functions of promoting bone regeneration and resisting bacteria comprises the following steps:

(1) preparing a nanocrystalline sample: carrying out solution treatment on the Ti-Nb-Zr-Sn-Mo titanium alloy at 730-780 ℃ for 0.75-3h, cooling in air, then placing the Ti-Nb-Zr-Sn-Mo titanium alloy at 550 ℃ for aging treatment for 5-8h, and then fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine for grinding treatment;

(2) and (2) treating the nanocrystalline sample in the step (1) in an ion implanter, and performing ion implantation under a vacuum condition by using Mg and Ag target materials as ion sources.

Has the advantages that: according to the invention, magnesium (Mg) and silver (Ag) ions are simultaneously introduced into the surface of the nanocrystalline Ti-Nb-Zr-Sn-Mo titanium alloy by adopting an ion injection method, so that a surface layer with good dual functions of promoting new bone formation and high-efficiency antibiosis is prepared on the surface of the Ti-Nb-Zr-Sn-Mo titanium alloy.

The invention adopts the aging temperature of 550 ℃ to obtain a matrix with a needle-like alpha phase embedded in a beta phase, and then the biocompatibility of the matrix can be improved only by obtaining the nanocrystalline on the surface layer of the matrix through SMAT treatment.

Preferably, the Ti-Nb-Zr-Sn-Mo titanium alloy in the step (1) is a circular plate with the diameter of 100mm and the thickness of 5 mm.

Preferably, in the step (1), GCr15 steel balls are adopted to impact the Ti-Nb-Zr-Sn-Mo titanium alloy.

Preferably, the diameter of the GCr15 steel ball is 3 mm.

Preferably, the vibration frequency of the grinding treatment in the step (1) is 2000 Hz.

Preferably, the impact time in step (1) is at least 30 min.

Has the advantages that: the Ti-Nb-Zr-Sn-Mo titanium alloy is subjected to solid solution for 1h at 750 ℃, then is cooled in air, is subjected to aging for 6h at 550 ℃, and then is subjected to SMAT treatment, wherein alpha phase is continuously converted into beta phase along with the prolonging of SMAT treatment time, and simultaneously, the diffraction peak is wider and wider, and the surface layer is converted into single beta-phase nanocrystalline at 30 min.

If the skin layer is treated with SMAT for 15min under the above conditions, the needle-like alpha phase still exists on the skin layer, and no beta-phase nanocrystal is formed, so the SMAT treatment time is required to be 30min or more, but the skin layer can be nanocrystallized in 30 min.

Preferably, the vacuum degree in the vacuum chamber of the ion implanter in the step (2) is 4 × 10^-4Pa and the accelerating voltage is 50 kV.

Preferably, the implantation doses of the Mg and Ag ions are 1 × 10 respectively¹⁷ions/cm²。

Has the advantages that: when the implantation dose is less than 1 × 10¹⁷ions/cm²When the antibacterial agent is used, the antibacterial activity to bacteria adhered to the surface is low, and when the injection dosage is more than 1 x 10¹⁷ions/cm²Is 2X 10¹⁷ions/cm²In the process, the bombardment time of the implanted ions is long, so that the nanocrystalline obtained on the surface layer is damaged into amorphous, and the modified titanium alloy also generates certain cytotoxicity to stem cells.

The technical problems to be solved by the invention are that the titanium alloy in the prior art has weak capability in inducing new bone formation, has no antibacterial property, often cannot form effective repair when facing bone defect, and tissues around the implanted titanium alloy are susceptible to necrosis, so that the titanium alloy with the double functions of promoting bone regeneration and resisting bacteria is provided.

the titanium alloy prepared by the modification method has the double functions of promoting bone regeneration and resisting bacteria.

Has the advantages that: the modified titanium alloy has a surface layer with good double functions of promoting new bone formation and high-efficiency antibiosis.

The invention has the advantages that: according to the invention, magnesium (Mg) and silver (Ag) ions are simultaneously introduced into the surface of the nanocrystalline Ti-Nb-Zr-Sn-Mo titanium alloy by adopting an ion injection method, and a surface layer with good double functions of promoting new bone formation and high efficiency and antibiosis is prepared on the surface of two kinds of Ti-Nb-Zr-Sn-Mo titanium alloys by virtue of good bone regeneration promoting capability of the Mg ions and outstanding antibacterial function of the Ag ions. Compared with a titanium alloy sample injected with coarse crystal double ions (Mg and Ag), the surface of the nanocrystalline titanium alloy sample has more crystal boundaries and can provide more diffusion channels for ion injection, so that the nanocrystalline titanium alloy sample has higher concentration of injected ions under the same ion injection process and shows stronger osteoinduction and antibacterial dual biological functions.

Compared with a coarse crystal-MgAg co-injection sample, the stem cells are more fully developed on the surface of the nanocrystalline-MgAg co-injection sample, which shows that the stem cells have a better stem cell adhesion environment. The expression condition of a representative osteogenesis related gene-RUNX 2 transcription factor shows that compared with a macrocrystalline-MgAg co-injection sample, the nanocrystalline-MgAg co-injection sample can up-regulate the gene expression thereof, and shows better capability of promoting the differentiation of stem cells from bone; and the staphylococcus aureus shows less bacteria on the surface of the nanocrystalline-MgAg co-injection sample, which shows that the nanocrystalline-MgAg co-injection sample also has better antibacterial capability. The results jointly show that the difunctional biomedical surface with good bone regeneration promoting and antibacterial functions can be prepared on the surface of the Ti-Nb-Zr-Sn-Mo titanium alloy by a method of injecting Mg and Ag ions in advance and then simultaneously into the surface of the Ti-Nb-Zr-Sn-Mo titanium alloy, and the service life of the titanium-based implant in clinic is prolonged.

Drawings

FIG. 1 is a schematic view of a surface mechanical polishing process;

FIG. 2 illustrates various processes initiated after ion implantation into a metal surface;

FIG. 3 is a phase composition XRD diffraction pattern of the surface of the nanocrystalline samples of comparative example 11, comparative example 2 and comparative example 3 according to the present invention;

FIG. 4 is a transmission electron micrograph of a sample of comparative example 3 of the present invention;

FIG. 5 is a transmission electron micrograph of a sample in example 1 of the present invention;

FIG. 6 is a transmission electron micrograph of a sample of comparative example 7 of the present invention;

FIG. 7 is a transmission electron micrograph of a sample of comparative example 6 of the present invention;

FIG. 8 is a transmission electron micrograph of a sample of comparative example 11 of the present invention;

FIG. 9 is a transmission electron micrograph of a sample of comparative example 4 of the present invention; wherein a is a transmission electron microscope bright field image, b is corresponding diffraction, and c is a high resolution image;

FIG. 10 is a graph showing the distribution of Mg ion concentration in the surface layer with depth at different grain sizes after ion co-implantation in example 1 and comparative example 1;

FIG. 11 is a graph showing the distribution of Ag ion concentration in the surface layer with depth at different grain sizes after co-ion implantation in example 1 and comparative example 1;

FIG. 12 is an AFM image of the surfaces of comparative examples 11, 7, 8, 1, 12, 1 according to the present invention;

FIG. 13 is a graph showing the results of hydrophilicity measurements of the surfaces of comparative examples 11, 7, 8, 1, 12, 1 according to the present invention;

FIG. 14 is a graph showing the results of measurement of Ag ions precipitated from the surface of a sample into a cell culture solution in example 1, comparative example 1 and comparative example 7 of the present invention;

FIG. 15 is a graph showing the results of measurement of Mg ions injected in example 1, comparative example 1 and comparative example 8 of the present invention precipitated from the sample surface into the cell culture solution;

FIG. 16 is a spreading pattern diagram of rabbit mesenchymal stem cells of the present invention after cultured on the surface of the sample of comparative example 1 for 5 hours;

FIG. 17 is a spreading pattern of rabbit mesenchymal stem cells of the present invention after cultured on the surface of the sample of example 1 for 5 hours;

FIG. 18 shows the expression of intracellular RUNX2 gene after rabbit mesenchymal stem cells are injected on the surface at different grain sizes and cultured for different times;

FIG. 19 shows the expression of the intracellular BSP gene of rabbit mesenchymal stem cells of the present invention after injected on the surface at different grain sizes and cultured for different times;

FIG. 20 shows the intracellular OCN gene expression of rabbit mesenchymal stem cells of the present invention after injected on the surface at different grain sizes and cultured for different times;

FIG. 21 shows the Col-I gene expression of rabbit mesenchymal stem cells of the present invention after being injected into the surface at different grain sizes and cultured for different times;

FIG. 22 is a graph showing the result of ALP activity measurement in rabbit mesenchymal stem cells injected on the surface at different grain sizes and cultured for different times;

FIG. 23 is a graph showing the result of measuring the content of OPN protein in rabbit mesenchymal stem cells injected on the surface of different crystal grain sizes and cultured for different time periods;

FIG. 24 is a graph showing the results of the measurement of the content of OCN in rabbit mesenchymal stem cells after the rabbit mesenchymal stem cells are injected into the surface at different grain sizes and cultured for different times;

FIG. 25 is a diagram showing the results of measuring the Col-I protein content in rabbit mesenchymal stem cells after the rabbit mesenchymal stem cells are injected into the surface at different grain sizes and cultured for different times;

FIG. 26 shows the results of the measurement of collagen secretion from cells on the surface of a sample according to the present invention;

FIG. 27 shows the results of the cell matrix mineralization detection on the surface of a sample according to the present invention;

FIG. 28 is a colony morphology of Staphylococcus aureus of the present invention cultured on the surface of the sample of comparative example 1 for 4 hours;

FIG. 29 is a colony morphology of Staphylococcus aureus of the present invention after 4 hours of incubation on the surface of the sample of example 1;

FIG. 30 is a colony morphology of Staphylococcus aureus of the present invention cultured on the surface of the sample of comparative example 11 for 4 hours;

FIG. 31 is a graph showing the results of counting the number of viable cells cultured on the surface of a sample by the agar plate method.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.

Example 1

The modification method of the titanium alloy with the double functions of promoting bone regeneration and resisting bacteria comprises the following steps:

(1) nanocrystalline sample (NG) preparation: carrying out solution treatment on a Ti-Nb-Zr-Sn-Mo titanium alloy circular plate with the diameter of 100mm and the thickness of 5mm at 750 ℃ for 1h, cooling the titanium alloy circular plate in air for 2h, then placing the titanium alloy circular plate at 550 ℃ for aging treatment for 6h, then fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine, starting the machine, and allowing a GCr15 steel ball with the diameter of 3mm to impact the titanium alloy circular plate for 30min under the condition that the vibration frequency is 2000 HZ; the titanium alloy in this example is a TLM titanium alloy: ti-25Nb-3Mo-2Sn-3Zr (wt%);

(2) the titanium alloy circular plate after surface mechanical grinding treatment is placed in an ion implanter for treatment, Mg and Ag target materials are taken as ion sources, ion implantation is carried out under the vacuum condition, and the treatment process comprises the following steps: the vacuum degree in the vacuum chamber is 4X 10 during ion implantation^-4Pa, acceleration voltage of 50kV, and implantation doses of 1 × 10¹⁷ions/cm²And the modified sample is named as NG-MgAg or nanocrystalline-MgAg co-injection.

Example 2

(1) nanocrystalline sample (NG) preparation: carrying out solution treatment on a Ti-Nb-Zr-Sn-Mo titanium alloy circular plate with the diameter of 100mm and the thickness of 5mm at 730 ℃ for 3h, cooling the titanium alloy circular plate in air for 2h, then placing the titanium alloy circular plate at 550 ℃ for aging treatment for 5h, then fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine, starting the machine, and allowing a GCr15 steel ball with the diameter of 3mm to impact the titanium alloy circular plate for 30min under the condition that the vibration frequency is 2000 HZ; the titanium alloy in this example is a TLM titanium alloy: ti-25Nb-3Mo-2Sn-3Zr (wt%);

Example 3

(1) nanocrystalline sample (NG) preparation: carrying out solution treatment on a Ti-Nb-Zr-Sn-Mo titanium alloy circular plate with the diameter of 100mm and the thickness of 5mm at 780 ℃ for 3h, cooling the titanium alloy circular plate in air for 2h, then placing the titanium alloy circular plate at 550 ℃ for aging treatment for 8h, then fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine, starting the machine, and allowing a GCr15 steel ball with the diameter of 3mm to impact the titanium alloy circular plate for 30min under the condition that the vibration frequency is 2000 HZ; the titanium alloy in this example is a TLM titanium alloy: ti-25Nb-3Mo-2Sn-3Zr (wt%);

Comparative example 1

(1) Preparation of coarse-grained sample (CG): carrying out solution treatment on a Ti-Nb-Zr-Sn-Mo titanium alloy circular plate with the diameter of 100mm and the thickness of 5mm at 750 ℃ for 1h, taking out the titanium alloy after reaching the time, fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine, starting the machine, and allowing a GCr15 steel ball with the diameter of 3mm to impact the titanium alloy circular plate for 30min under the condition that the vibration frequency is 2000 HZ;

(2) placing the titanium alloy circular plate subjected to the surface mechanical grinding treatment in the step (1) into an ion implanter for treatment, and performing ion implantation under a vacuum condition by taking Mg and Ag targets as ion sources, wherein the treatment process comprises the following steps: the vacuum degree in the vacuum chamber is 4X 10 during ion implantation^-4Pa, acceleration voltage of 50kV, and implantation doses of 1 × 10¹⁷ions/cm²The modified sample is named CG-MgAg or coarse crystal-MgAg co-injection.

Comparative example 2

This comparative example differs from example 1 in that: in the step (1), a GCr15 steel ball with the diameter of 3mm is allowed to impact a titanium alloy circular plate for 5min under the condition that the vibration frequency is 2000 Hz.

Comparative example 3

This comparative example differs from example 1 in that: in the step (1), a GCr15 steel ball with the diameter of 3mm is allowed to impact a titanium alloy circular plate for 15min under the condition that the vibration frequency is 2000 Hz.

Comparative example 4

This comparative example differs from example 1 in that: the implantation doses are each 2X 10¹⁷ions/cm²。

Comparative example 5

This comparative example differs from example 1 in that: the implantation doses are each 5 × 10¹⁷ions/cm²。

Comparative example 6

This comparative example differs from example 1 in that: the implantation doses are each 1 × 10¹⁶ions/cm²。

Comparative example 7

This comparative example differs from example 1 in that: only Ag ions were implanted and the modified sample was named NG-Ag.

Comparative example 8

This comparative example differs from example 1 in that: only Mg ions were implanted and the modified sample was named NG-Mg.

Comparative example 9

The present comparative example differs from comparative example 1 in that: only Ag ions are injected, and the modified sample is named as CG-Ag.

Comparative example 10

The present comparative example differs from comparative example 1 in that: only Mg ions were implanted and the modified sample was named CG-Mg.

Comparative example 11

This comparative example differs from example 1 in that: only nanocrystalline samples were prepared, named NG.

Comparative example 12

The present comparative example differs from comparative example 1 in that: only a coarse-grained sample was prepared and designated CG.

Example 4

The performance of the modified titanium alloy in example 1 and comparative examples 1-10 was tested.

Transmission electron microscope analysis:

the microstructure morphology of the sample was analyzed by a JEM-2100F Transmission Electron Microscope (TEM) manufactured by JEOL corporation, and the operating voltage of the electron microscope was 200 kV. The TEM flakes were prepared as follows: grinding single side of each sample to 30-50 μm, punching into phi 3mm shape with sample punching device, thinning with MTP-TA type double-spraying electrolyte instrument with 5% HClO + 95% CH₃Adding nitrogen into OH solution, cooling to freeze the solution, thinning the solution with the working voltage of 10V, and thinning the solution by double spraying until the sample is perforated.

(II) phase detection:

the phase before and after the surface mechanical grinding treatment was identified by X' Pert PRO multifunctional X-ray diffractometer (XRD for short) from Panalytal, Netherlands. During testing, CuK alpha, an X-ray source and a graphite monochromatic tube are adopted, the tube pressure is 40kV, the tube flow is 40mA, the continuous scanning mode is adopted, the step length is 0.02 degrees, each step is 0.5s, and the scanning range of the diffraction angle is 30-80 degrees.

(III) analysis of the concentration of implanted ions:

stripping the surface of the ion-implanted sample by using an ULVACPHI-700 Auger electron spectrometer produced in Japan to analyze the distribution of the implanted Mg and Ag elements on the surface of the implanted sample along the depth, wherein the accelerating voltage of the incident argon ion beam is 4kV, and the stripping speed is 16 nm.min^-1。

(IV) surface roughness and hydrophilicity experiments:

the surface morphology of the sample was observed and the roughness of the sample surface was measured by an atomic force microscope (SPM-9500J 3 model, manufactured by Japan) using Si₃N₄Probe having cantilever spring constant of 60mN · m^-1The imaging of the atomic force microscope adopts a non-contact mode, the test result is displayed on a computer in a three-dimensional shape, and then a photo is taken.

Three representative indexes Ra, Rq and Rz are selected to evaluate the roughness of the surface of the sample, each sample is provided with three parallel samples, different areas of the surface of each sample are tested for three times, and the average value is taken as a corresponding index test value.

Wherein Ra: the arithmetic mean of the profiles refers to the arithmetic mean of the profile offsets within the sampling length; rq: a geometric mean of the profile, which refers to the n-th root of the product of n values of a set of statistical data; rz: 10 point micro-unevennesses means the sum of the mean of the 5 largest profile peaks high and the mean of the 5 largest profile valleys deep within the sampling length.

The hydrophilic properties of the coating surface were tested using a DSA30 model wetting angle tester from KRUSS. The liquid for testing is secondary deionized water, a video real-time tracking mode is used in the testing process, and the image of the third frame after the liquid drops are dripped is uniformly taken as data for analyzing the wetting angle. The hydrophilic property of the material surface is reflected by measuring the size of the obtained contact angle.

(V) ion elution experiment:

after soaking the sample for different time, taking the sample out of the soaking solution, collecting the soaking solution, centrifuging at 1000rpm for 15min, taking the supernatant, and carrying out concentration test on Mg and Ag ions in the solution by using ICP-AES.

(VI) cell experiment:

(1) obtaining and culturing stem cells: selecting 2-month-old New Zealand rabbits, taking tibial periosteum and femoral periosteum under aseptic condition, digesting with 0.25% pancreatin and 0.1% collagenase I for 30min and 2h respectively, centrifuging, collecting cells, culturing in a 5% culture box at 37 deg.C, wherein the culture medium is DMEM containing 20% fetal calf serum, 10-7 dexamethasone, 50mg/L vitamin C, penicillin 100U/ml and streptomycin 100 μ g/ml. The primary cells were passaged to 100ml flasks and when 80% of the cells fused, passaged at a ratio of 1: 4.

(2) Adhesion proliferation assay:

when the stem cells reach 80% fusion, digesting with 500 mu L of 0.25% pancreatin containing 0.02% ethylenediaminetetraacetic acid, adding a complete culture medium containing serum to stop digestion when the cells become round and begin to be separated from the wall of a culture flask, centrifuging for 5min at 1000prm, discarding the supernatant, adding the complete culture medium to suspend the cells, and counting by a blood counting cell counting plate under an inverted microscope; the total volume was 1mL, containing 8X 10⁴Injecting a cell culture medium of each osteoblast into a 24-pore plate containing an experimental sample, and placing the 24-pore plate in a cell culture box; after 1, 5, 24, 72 and 168h of incubation, the culture broth in the 24-well plate was aspirated, gently rinsed 3 times with PBS (to remove cells that did not adhere to the surface of the material), and the sample was transferred to a new 24-well plate; adding 300 μ L of pancreatin per well for digestion for 3-5min, and adding 700 μ L of cell culture medium to stop digestion; blowing to obtain single cell suspension, and counting.

After the cells were cultured on the surface of the sample for 5 hours, the morphology was observed by a field emission scanning electron microscope. The specific method comprises the following steps: the total volume was 500. mu.L, containing 8X 10⁴The cell culture medium of each osteoblast was injected into a 24-well plate containing the test sample, and cultured in a cell incubator. By the time of the target, the 24-well plate was removed, the medium in the well was aspirated, gently rinsed 3 times with PBS, and the experimental sample was transferred to a new 24-well plate. Adding 300 μ L of 2.5% glutaraldehyde into each well, and fixing at 4 deg.C for 1 h; after the glutaraldehyde is discarded, the reaction mixture is cooled to 30%, 50% or 70% at room temperatureDehydration is carried out for 10min by gradient alcohol with the concentration of 90 percent, 95 percent and 100 percent respectively; after dehydration, the cells are put into a vacuum drying oven overnight, and the shapes of the cells are observed by a field emission scanning electron microscope after gold spraying on the surfaces of the cells.

(3) Intracellular gene expression experiments:

in order to detect the influence of the sample surface on the expression of the osteogenesis related genes of the stem cells, real-time quantitative PCR (qRT-PCR) is used for detecting the mRNA level expression of the osteogenesis related genes after the stem cells are cultured on the sample surface for different times. The experimental procedure was as follows:

(a) extraction of sample surface stem cell total RNA: 4 samples of each were placed in 24-well plates and 1mL of a solution containing 8X 10 was added to each well⁴The culture medium of each cell is placed in an incubator at 37 ℃ and 5% CO₂And 3, 7 and 14d under saturated humidity conditions. After the target time is reached, removing the cell culture solution, rinsing with PBS for 3 times, adding 1mL of TRIzol to dissolve cells on 4 parallel samples, and transferring the cell lysate to a l.5mL centrifuge tube; adding 0.2mL of chloroform, mixing by inversion, standing at room temperature for 1min, at 4 deg.C and 12000 r.min^-1Centrifuging for 15min under the condition; adding colorless water phase liquid into another 1.5mL centrifuge tube, adding 0.5mL isopropanol, standing at room temperature for l 0min, at 4 deg.C and 12000r min^-1Centrifuging for 10min under the condition, and then discarding the liquid; adding 75% ethanol lmL, heating at 4 deg.C and 7500r min^-1Centrifuging for 5min under the condition, and then discarding the liquid; adding 0.02mL of 0.1% DEPC water to dissolve RNA, quantitatively analyzing the nucleic acid concentration of the total RNA extract of the cells by using a spectrophotometer, and adjusting to make the total RNA concentration uniform;

(b) reverse transcription of RNA into cDNA: taking 4 mu L of template RNA from each group of samples, adding 1 mu L of oligo (dT) and DEPC water respectively, mixing uniformly, reacting at 70 ℃ for 10min, and rapidly performing ice bath for 2 min; adding 6 mu L of template obtained by reaction into 0.25 mu L of 5 XM-MLV Buffer 2 mu L, dNTP 0.5.5 mu L, RNA enzyme inhibitor 0.25 mu L, M-MLV enzyme 0.25 mu L and 1 mu L of DEPC water, uniformly mixing, reacting the mixed system at 42 ℃ for 60min, and then reacting at 70 ℃ for 15 min; placing each group of cDNA obtained by reverse transcription on ice for cooling, and placing at-20 ℃ for storage for later use;

(c) real-time quantitative PCR reaction: primer and probe sequence design is shown in Table 1. Diluting the cDNA obtained by reverse transcription in the step (2) by 5 times by using double distilled water, sucking 2 mu L, transferring the cDNA into a centrifuge tube special for Real-Time quantitative PCR, and uniformly mixing the cDNA with 10 mu L of Real-Time PCR (polymerase chain reaction) medium Mix, 7 mu L of double distilled water and 0.5 mu L of each of an upstream primer and a downstream primer to form a 20 mu L reaction system. The reaction System carries out PCR reaction on a Bio-Rad iQ5 Real-time PCR instrument, uses IQ5 SYBR Green I Supermix PCR System to carry out Real-time fluorescent quantitative reaction, and the reaction process is as follows: first, pre-denaturation is carried out at 95 ℃ for 30s, and then target gene amplification reaction is carried out at 60 ℃ for 10s and at 40 ℃ for 20s, and amplification cycle is carried out for 40 times.

This experiment detects 6 genes associated with cell osteogenesis: the method comprises the following steps of taking osteogenesis related gene transcription factors (Runx2, BSP, collagen type I (Col-I), alkaline phosphatase (ALP), Osteopontin (OPN) and Osteocalcin (OCN) as housekeeping genes, taking GAPDH as housekeeping genes, setting 3 times of repetition for each group of samples, and dividing the Ct value of a target gene of each test group by the Ct value of the housekeeping genes by adopting a drawn gradient dilution DNA standard curve to process experimental data so as to eliminate system errors and ensure consistency in result analysis.

Table 1 shows the sequences of upstream and downstream primers used for real-time quantitative PCR of each gene

(4) And (3) detecting the intracellular alkaline phosphatase and the specific protein:

after the stem cells are cultured on the surfaces of the three samples, the activity of intracellular alkaline phosphatase (ALP) and the content of intracellular specific protein are detected by an enzyme-linked immunoassay method. The total volume was 500. mu.L, containing 8X 10⁴Injecting the cell culture medium of each osteoblast into a 24-well plate containing an experimental sample, culturing for 3, 7 and 14 days, and then gently rinsing with PBS for three times; treating the test sample with 200 μ L of 0.1% Triton X-100 per well, repeatedly freezing and thawing for 5 cycles to lyse cells, and shaking for 5 min; 4 ℃ at 1000r min^-1Centrifuging for 10min, collecting supernatant, and storing at-80 deg.C. Respectively using corresponding human enzyme-linked immunoassay kit (R)&D, USA), the specific assay procedures were referred to kit instructions. This experiment examined the intracellular ALP activity of osteoblasts toAnd the concentration of three intracellular specific proteins, type I collagen (Col-I), Osteopontin (OPN), and Osteocalcin (OCN).

(5) And (3) detecting collagen secretion of cells on the surface of the sample:

the collagen secretion of stem cells cultured on the surface of a sample for different time is detected by a sirius red staining method, and the specific experimental steps are as follows: cells were cultured at 8X 10⁴Inoculating to 24-well plate containing test sample, culturing for 3, 7 and 14 days, rinsing with PBS for 3 times, and fixing with 4% paraformaldehyde at room temperature for 30 min; after transferring the sample to a new hole, continuously rinsing the sample with PBS for three times, sucking and removing rinsing liquid, and dyeing collagen secreted by stem cells on the experimental sample with 0.1% sirius red solution dissolved in saturated picric acid, wherein the dyeing process lasts for 18 hours at room temperature; after the target time was reached, the test specimens were repeatedly rinsed with 0.1M acetic acid until no red color had precipitated. For quantitative comparison, after staining the experimental samples of

cell cultures

3, 7 and 14d, 500. mu.L of elution solution (prepared from 0.2M sodium hydroxide and methanol at a ratio of 1: 1) was added to each well, the dye on the surface of the experimental sample was eluted by shaking for 15min, and the absorbance at 540nm was measured in a spectrophotometer.

(6) And (3) mineralization detection of cell matrixes on the surface of the sample:

after the stem cells are cultured on the surface of the sample, the mineralization level of the extracellular matrix of the stem cells is detected by using an alizarin red staining method. Cells were cultured at 8X 10⁴The cells/well were inoculated in 24-well plates containing test samples, and after 3, 7 and 14d incubation, they were rinsed three times with PBS and fixed with 75% alcohol at room temperature for 1h, and then the cells on the surface of the three samples were stained with 40mM alizarin Red (pH4.2) at room temperature for 10 min. After dyeing, the sample was repeatedly rinsed with distilled water until no further decolorization occurred. To quantitatively analyze the mineralization of the extracellular matrix, after staining samples of

cell cultures

3, 7 and 14d, 10% cetylpyridinium chloride (solvent 10mM sodium phosphate (pH 7)) solution was added to each well, shaken for 15min, the dye on the surface of the sample was eluted, and the absorbance at 620nm was measured in a spectrophotometer.

(7) And (3) antibacterial property experiment:

gold is mixed withThe staphylococcus aureus ATCC25923 was co-cultured with the sample for 4h to examine the adhesion of bacteria on the surface. The bacteria were formulated to a concentration of 10⁷cell·mL^-1The sample was then steam-sterilized at 120 ℃ and 0.1MPa for 40min and placed in a 24-well plate with the treatment side facing upward. Subsequently, two bacterial suspensions each having a volume of 1mL were injected into a 24-well plate containing the test sample, and incubated at 37 ℃ in an incubator for 4 hours. After the target time is reached, the sample is taken to a new hole, the surface of the sample is gently rinsed by PBS to remove the non-adhered bacteria, and 2.5% glutaraldehyde is used for fixation for 1h at 4 ℃; after the glutaraldehyde is absorbed and removed, respectively dehydrating the solution for 10min at room temperature in gradient alcohols of 30%, 50%, 70%, 90%, 95% and 100%; and (4) placing the dehydrated bacteria in a vacuum drying oven overnight, spraying gold on the surface of the bacteria, and observing the shape of the bacteria by using a field emission scanning electron microscope.

Statistical analysis: four replicates were used for each set of experiments and the data are expressed as mean ± standard deviation and analyzed using SPSS 14.0 software. Two-way anova and SNK test compare differences between groups. p <0.01 is considered to be a very large statistical difference.

And (3) measuring results:

(1) fig. 3 is a phase composition XRD diffractogram of the surface of the nanocrystalline sample in comparative example 11, comparative example 2 and comparative example 3, and it can be seen that as the SMAT treatment time is prolonged, the α phase is continuously transformed into the β phase, and the diffraction peak is wider and wider, and at 30min, the surface layer is transformed into single β phase nanocrystalline. Fig. 4 is a transmission electron micrograph of the sample in comparative example 3, and if only SMAT treatment is performed for 15min under the above-described conditions, the surface layer still has a needle-striped α phase and no nanocrystals of the β phase are formed, so the SMAT treatment time must be 30min or more, but the surface layer nanocrystallization can be achieved already at 30 min.

(2) Fig. 5 to 8 are transmission electron micrographs of the samples of example 1, comparative example 7, comparative example 8, and comparative example 11, respectively, and it can be seen that the size of the β -Ti nanocrystal particles (about 40 nm) was not changed by ion implantation, that the implanted magnesium ions were present as magnesium oxide of about 15nm, and that the implanted silver ions were present as elemental silver particles of about 7nm on the surface layer.

(3) FIG. 9 is a transmission electron microscope of comparative example 4Figure, when the implantation dose is increased to 2 x 10¹⁷ions/cm²In the meantime, a in fig. 9 is a transmission electron microscope bright field image, the bright field has no contrast and shows an amorphous characteristic, b in fig. 9 is corresponding diffraction, the diffraction shows halo and shows an amorphous characteristic, c in fig. 9 is a high resolution image, it can be seen that atoms are disordered and show an amorphous characteristic, and the surface layer is damaged to be amorphous due to the long bombardment time of the implanted ions.

(4) Fig. 10 and 11 are depth distribution curves of injected magnesium ions and silver ions on the surface of the sample, respectively, the Ag concentration peak values of the NG-MgAg and CG-MgAg samples are 9.1% and 5.0%, respectively, and the Mg concentration peak values of the NG-MgAg and CG-MgAg samples are 30.0% and 19.8%, respectively, and it can be seen that the nanocrystalline series surface energy is higher in the amount of the injected ions.

(5) Fig. 12 is an AFM image of the surfaces of the samples of comparative example 11, comparative example 7, comparative example 8, example 1, comparative example 12 and comparative example 1, and it can be seen from the images that the surface roughness of these samples is similar, and the surface roughness of the samples is not significantly changed by the nanocrystalline sample, the macrocrystalline sample and the subsequent ion implantation.

(6) Fig. 13 shows the hydrophilicity of the surfaces of the samples of comparative example 11, comparative example 7, comparative example 8, example 1, comparative example 12, and comparative example 1, and it can be seen that the hydrophilicity of the nanocrystalline sample is superior to that of the macrocrystalline sample, the magnesium ion implantation can increase the hydrophilicity, and the silver ion implantation decreases the hydrophilicity.

(7) Fig. 14 shows the case where the injected Ag ions were eluted from the sample surface into the cell culture solution, and fig. 15 shows the case where the injected Mg ions were eluted from the sample surface into the PBS solution, and it can be seen that the ion elution was higher in the nanocrystalline sample than in the macrocrystalline sample.

(8) Fig. 16 shows the spreading form of rabbit mesenchymal stem cells after being cultured on the surface of the macrocrystalline-MgAg co-injected sample for 5 hours, and fig. 17 shows the spreading form of rabbit mesenchymal stem cells after being cultured on the surface of the nanocrystalline-MgAg co-injected sample for 5 hours, so that, compared with the macrocrystalline-MgAg co-injected sample, the stem cells are more fully developed on the surface of the nanocrystalline-MgAg co-injected sample, which indicates that the rabbit mesenchymal stem cells have a better stem cell adhesion environment.

(9) Fig. 18 shows the expression of intracellular RUNX2 gene after rabbit mesenchymal stem cells are injected into the surface at different grain sizes and cultured for different times, and fig. 19-21 show the expression of intracellular BSP gene, OCN gene and Col-I gene after rabbit mesenchymal stem cells are injected into the surface at different grain sizes and cultured for different times.

The intracellular ALP activity, OPN, OCN and Col-I contents are shown in FIGS. 22-25, respectively, and the intracellular ALP activity and OPN, OCN and Col-I contents with the culture time have a trend similar to that of the corresponding gene expression in surface cells.

(10) Fig. 26 is a detection result of collagen secretion of cells on the surface of a sample, and fig. 27 is a detection result of mineralization of cell matrixes on the surface of the sample, which shows that the collagen secretion and the cell matrix mineralization of a nanocrystal-MgAg co-injected sample are obviously higher than those of a macrocrystalline-MgAg co-injected sample, and the surface of the nanocrystal-MgAg co-injected sample can promote the mineralization of stem cells in the osteogenic direction faster, so that the formation of bone matrixes is facilitated, and the formation of new bones is promoted earlier.

(11) Fig. 28 is a colony morphology of staphylococcus aureus cultured on the surface of a macrocrystalline-MgAg co-injected sample for 4 hours, fig. 29 is a colony morphology of staphylococcus aureus cultured on the surface of a nanocrystalline-MgAg co-injected sample for 4 hours, and fig. 30 is a colony morphology of staphylococcus aureus cultured on the surface of an NG sample for 4 hours, which shows that the double effects of osteogenic differentiation promotion and bacteriostasis are more remarkably shown on the surface of the nanocrystalline-MgAg sample. Fig. 31 shows the number of viable cells counted by culturing the cells adhered to the surface of the sample by the agar plate method, and it can be seen that the sterilization effect (antimicrobial rate 91.2%) of the mixed nanocrystal injection is better than the sterilization effect (antimicrobial rate 74.5%) of the mixed crude injection.

(12) The sample surface in comparative example 5 was not only effective in killing adherent bacteria, but also was somewhat cytotoxic to stem cells.

(13) The sample of comparative example 6 weakly induced differentiation of stem cells toward osteogenic direction, but showed low antibacterial activity against surface-adhered bacteria.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A method for modifying titanium alloy with double functions of promoting bone regeneration and resisting bacteria is characterized in that: the method comprises the following steps:

(1) preparing a nanocrystalline sample: carrying out solution treatment on the Ti-Nb-Zr-Sn-Mo titanium alloy at 750 ℃ for 1h, cooling in air, then placing at 550 ℃ for aging treatment for 6h, then fixing the treated Ti-Nb-Zr-Sn-Mo titanium alloy in a treatment cavity of a surface mechanical grinding treatment machine for grinding treatment, and impacting the Ti-Nb-Zr-Sn-Mo titanium alloy for at least 30min by adopting GCr15 steel balls;

(2) processing the nanocrystalline sample in the step (1) in an ion implanter, and performing ion implantation under a vacuum condition by taking Mg and Ag target materials as ion sources, wherein the implantation doses of the Mg and Ag ions are respectively 1 × 10¹⁷ions/cm²。

2. The method for modifying the titanium alloy with the double functions of promoting bone regeneration and resisting bacteria according to claim 1, wherein the method comprises the following steps: the Ti-Nb-Zr-Sn-Mo titanium alloy in the step (1) is a circular plate with the diameter of 100mm and the thickness of 5 mm.

3. The method for modifying the titanium alloy with the double functions of promoting bone regeneration and resisting bacteria according to claim 1, wherein the method comprises the following steps: the diameter of the GCr15 steel ball is 3 mm.

4. The method for modifying the titanium alloy with the double functions of promoting bone regeneration and resisting bacteria according to claim 1, wherein the method comprises the following steps: the vibration frequency of the grinding treatment in the step (1) is 2000 Hz.

5. The method for modifying the titanium alloy with the double functions of promoting bone regeneration and resisting bacteria according to claim 1, wherein the method comprises the following steps: the vacuum degree in the vacuum cavity of the ion implanter in the step (2) is 4 multiplied by 10^-4Pa and the accelerating voltage is 50 kV.

6. A titanium alloy with the functions of promoting bone regeneration and resisting bacteria, which is prepared by the modification method of any one of claims 1 to 5.