Ti6Al4V surface micro-arc zinc oxide modified coating as well as detection method and application thereof

Abstract

The invention relates to a Ti6Al4V surface micro-arc zinc oxide modified coating and a detection method and application thereof, and belongs to the technical field of material surface coating preparation. In order to solve the problems that an existing titanium oxide coating is poor in antibacterial performance and cannot be applied to a special oral environment, a zinc modified coating is prepared on the surface of Ti6Al4V through micro-arc oxidation, the zinc content of the zinc modified coating is 0-5.16%, a plurality of holes with the diameter being 1-2 micrometers are evenly distributed in the zinc modified coating, the contact angle of the zinc modified coating is 35-60 degrees, and the surface of the zinc modified coating is coated with the antibacterial coating. An oral microenvironment is simulated through antibacterial experiments and bacterium-cell co-culture, and it is verified that the antibacterial performance of the material is improved through introduction of zinc. In addition, the coating plays a role in promoting adhesion, proliferation and osteogenic differentiation capability of MC3T3-E1 cells in conventional and high-glucose states, and the coating is found to have better antibacterial property and hydrophilicity, and optimizes the osseointegration capability of the implant.

Description

Technical Field

[0001] The present invention relates to the technical field of material surface coating preparation, and in particular to a Ti6Al4V surface micro-arc zinc oxide modified coating and a detection method and application thereof. Background Art

[0002] Dental implants are increasingly becoming the preferred restoration option for edentulous patients due to their high comfort and safety against adjacent teeth. Ti6Al4V alloy is commonly used as an implant material in clinical practice. However, as a bioinert material, it poorly integrates with surrounding bone tissue. After implant placement, it easily forms a fibro-osseous bond with the surrounding fibrous tissue, resulting in poor stability and prone to implant loosening and loss. During implant surgery, the complex oral microbial environment and operating room environment can contribute to bacterial colonization on the implant surface. Similar to biofilm formation on natural teeth, bacterial colonization occurs within 30 minutes after implant surgery. The degree of bone integration is closely related to endogenous competition between bacteria and cells. Due to the poor antibacterial properties of Ti6Al4V alloy, once bacteria have a stronger competitive ability after implantation, they can proliferate and form a plaque biofilm that cannot be completely removed, even with antibiotic therapy, surgical irrigation, or debridement. Therefore, surface modification to repel or kill surrounding bacteria and enhance the adhesion and proliferation of surrounding osteoblasts is crucial for implant-bone integration. Under pathological conditions (such as diabetes), the unique blood environment in patients can also promote inflammation around implants, affecting their osseointegration ability. To address the challenges faced by Ti6Al4V alloy in medical applications and broaden its medical applications, surface modification by introducing various trace elements into implant materials to impart antibacterial properties and enhance osseointegration has become a current research hotspot.

[0003] Zinc is an essential trace element for the human body, playing a crucial role in bone formation, development, and mineralization. More importantly, zinc possesses superior antimicrobial properties, generating electrostatic forces through the interaction of positive and negative charges, forming strong ionic bonds with bacterial surfaces. Upon binding to bacteria, the permeability of the bacterial cell membrane increases, allowing zinc ions to more easily penetrate the interior of the bacteria, triggering additional toxic reactions. Zinc ions can also create pores on the bacterial surface, disrupting the integrity of the bacterial cell membrane and causing the cytoplasm and other components to leak out, leading to bacterial death.

[0004] At present, trace elements can be introduced into the surface of implants through acid etching-sandblasting, plasma spraying and micro-arc oxidation. Among them, micro-arc oxidation (MAO) technology has been widely used in implant surface coating modification due to its advantages such as simple preparation process, small footprint, high production efficiency and relatively environmental protection. At the same time, MAO can prepare a porous oxide ceramic film on the surface of the material to promote cell adhesion, proliferation and osteogenic differentiation. Studies have found that the in situ grown titanium oxide-based coating prepared by micro-arc oxidation is dense and uniform, and a variety of morphologies and structures are formed on the surface. In the early stage, a titanium oxide coating containing calcium and phosphorus elements was prepared by MAO, which was firmly bonded to the alloy substrate, formed 2-3μm pores on the surface, and was relatively evenly distributed. In vivo experiments showed that the experimental group had stronger bone bonding ability and good biological activity, but poor antibacterial properties and could not resist the potential infection risk after implant implantation. In addition, the microecological environment in the human oral cavity is complex. In clinical practice, the adhesion and growth of bacteria and cells often occur simultaneously. There has been no previous research on implants in such an environment. Moreover, for the special oral environment of some patients with hyperglycemia, high blood sugar can promote the production of reactive oxygen species. Excessive production of reactive oxygen species damages the adhesion ability of osteoblasts and blocks the BMP-2 signaling pathway, which will correspondingly weaken ALP activity, osteocalcin production and matrix mineralization ability, greatly affecting the placement of implants.

[0005] To this end, combined with the advantages of zinc for the human body, the feasibility of zinc-modified coating on the surface of Ti6Al4V in oral implant materials was studied through MAO technology, which provides more possibilities for expanding the application field of implants and has important influence and practical significance in clinical oral implant treatment. Summary of the Invention

[0006] In view of the defects existing in the above prior art, the present invention aims to provide a Ti6Al4V surface micro-arc zinc oxide modified coating and its detection method and application, so as to solve the problems existing in the background technology.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention discloses a Ti6Al4V surface micro-arc zinc oxide modified coating, wherein the zinc content of the zinc modified coating is 0-5.16%, a plurality of holes with a diameter of 1-2 μm are evenly distributed thereon, and the contact angle thereof is 35-60°.

[0009] As a further preferred solution of the above technical solution: the zinc content of the zinc-modified coating on the Ti6Al4V surface is 5.16%, the pore diameter is 1-1.5 μm, and the contact angle is 35°.

[0010] In a second aspect, the present invention further discloses a method for preparing a micro-arc zinc oxide modified coating on a Ti6Al4V surface, comprising the following steps:

[0011] S1: Processing and pretreatment of Ti6Al4V samples, and preparation of electrolyte;

[0012] S2: The pretreated Ti6Al4V sample was used as the anode and the stainless steel was used as the cathode, and placed in the electrolyte;

[0013] S3: Set the process parameters of micro-arc oxidation, then add a zinc acetate solution with a concentration of 8.5 g / L to the electrolyte to obtain a micro-arc zinc oxidation modified coating on the Ti6Al4V surface, and then disinfect and dry it.

[0014] A further preferred embodiment is that the preparation method further comprises a step of detecting the micro-arc zinc oxide modified coating on the Ti6Al4V surface, wherein the detection step comprises detecting cell adhesion, cell proliferation and cell osteogenic differentiation of the zinc modified coating under high sugar conditions.

[0015] A further preferred solution is that the process of cell adhesion detection is as follows:

[0016] (1) Ti6Al4V samples were placed in a 24-well plate, and 1 mL of high-glucose cultured MC3T3-E1 cell suspension was added to each well;

[0017] (2) Place the 24-well plate containing the MC3T3-E1 cell suspension in a cell culture incubator and culture for 1 to 24 hours;

[0018] (3) Remove the 24-well plate, wash the Ti6Al4V sample with PBS, and transfer it to a new 24-well plate. Fix, permeabilize, and stain the cells in sequence to observe cell adhesion.

[0019] A further preferred solution is that the process of cell proliferation detection is as follows:

[0020] (1) MC3T3-E1 cells were cultured in high glucose for 1 to 7 days;

[0021] (2) Place the Ti6Al4V sample in a 24-well plate, add 1 mL of cultured MC3T3-E1 cell suspension to each well, and culture at 37°C for 1 to 5 days;

[0022] (3) Discard the original culture medium and add 550 μL of a mixture of complete culture medium and CCK-8 to each well. Incubate for 2 h and use CCK-8 to detect cell proliferation.

[0023] A further preferred solution is that the process of detecting cell osteogenic differentiation is as follows:

[0024] (1) Ti6Al4V samples were placed in a 24-well plate, and 1 mL of high-glucose cultured MC3T3-E1 cell suspension was added to each well;

[0025] (2) When the cells grow to 70% to 80% fusion, replace the high-glucose mineralization induction medium and continue culturing. The high-glucose mineralization induction medium should be replaced every two days.

[0026] (3) On the 14th day, total cell protein was extracted and the expression of ALP, BMP-2 and OPN proteins was detected.

[0027] In a third aspect, the present invention also discloses an application of a Ti6Al4V surface micro-arc zinc oxide modified coating in oral implant materials.

[0028] Compared with the prior art, the present invention can produce the following beneficial effects:

[0029] 1. The present invention prepares a zinc-modified coating on the surface of Ti6AI4V by micro-arc oxidation, thereby changing the surface microstructure and phase composition of the material, so that the mass percentage of zinc element on the coating surface reaches 5.16%, the surface pore size is 1-1.5μm, and the contact angle is 35°, thereby enhancing the hydrophilicity of the material and better optimizing the implant-bone bonding ability.

[0030] 2. After zinc acetate is added to the micro-arc oxidation electrolyte in the present invention, anatase and rutile phases appear on the surface of the prepared zinc-modified coating. Compared with pure anatase, the rutile phase has better biological activity, can induce enhanced deposition of hydroxyapatite, and further promote bone bonding ability.

[0031] 3. The zinc-modified coating of the present invention has good antibacterial properties. On the one hand, zinc enhances the antibacterial ability by surface modification of Ti6AI4V. On the other hand, the rutile phase and anatase phase have certain antibacterial effects, which is consistent with the antibacterial experimental results, that is, the N-Zn group also exhibits certain antibacterial properties. However, the appearance of porous morphology on the coating surface is a double-edged sword for antibacterial properties. On the one hand, the porous morphology promotes the adhesion of bacteria. On the other hand, due to the increase in the surface area of the coating, more zinc combines with bacteria, enhancing the antibacterial properties.

[0032] 4. The present invention simulates the oral microenvironment through bacteria-cell co-culture and finds that the zinc-modified coating effectively inhibits bacterial activity in the competitive growth of bacteria-cells, promotes the adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells, and creates good conditions for improving bone bonding ability. It also promotes the adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells under high sugar conditions, providing a certain theoretical basis for broadening the adaptability of implants. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0034] Figure 1 This is a visual image of each group of samples after preparation of the present invention;

[0035] Figure 2 The surface morphology of each group of samples was observed using SEM in the present invention;

[0036] Figure 3 Elemental analysis diagram of the surface of the zinc-modified coating using EDS in the present invention; wherein A represents the EDS analysis diagram of each group, and B represents the histogram of the mass fraction and molecular weight fraction of zinc in each group;

[0037] Figure 4 This is the phase composition analysis diagram of each group of samples using XRD in the present invention;

[0038] Figure 5 Graph showing contact angle test results for each group of samples of the present invention;

[0039] Figure 6 This is a plate count chart of bacterial solutions of different concentrations according to the present invention;

[0040] Figure 7 This is a diagram showing the bacterial growth of each group of samples measured by the film sticking method of the present invention;

[0041] Figure 8 This is a graph showing bacterial activity detection of each group of samples using the Alma blue bacteria activity detection kit of the present invention;

[0042] Figure 9 This is a graph showing the results of the cytotoxicity test on MC3T3-E1 cells using the extracts of each group of samples in the present invention;

[0043] Figure 10 This is a diagram showing the adhesion and spreading of cells on the surface of each sample group under bacteria-cell co-culture in the present invention;

[0044] Figure 11 Figure 3 is a graph showing the live and dead status of cells on the surface of each sample group under bacteria-cell co-culture in the present invention; wherein A represents the fluorescent image of MC3T3-E1 cells co-cultured with bacteria on the sample surface, live cells (green), dead cells (red), B represents the number of live cells and dead cells, and C represents the ratio of dead cells to live cells.

[0045] Figure 12 The adhesion and spreading of each group of sample cells were stained by rhodamine-phalloidin for 1 hour;

[0046] Figure 13The adhesion and spreading of each group of sample cells were stained by rhodamine-phalloidin for 4 hours;

[0047] Figure 14 The adhesion and spreading of each group of sample cells were stained by rhodamine-phalloidin for 24 hours;

[0048] Figure 15 This is a graph showing the effects of each group of samples on the proliferation and migration of MC3T3-E1 cells detected by CCK-8 in the present invention;

[0049] Figure 16 This is the result of ALP staining of sample cells in each group observed under a stereo microscope (×25) after 7 days;

[0050] Figure 17 This is the result of ALP staining of sample cells in each group observed under a stereo microscope (×25) 14 days after the present invention;

[0051] Figure 18 The results of ALP activity determination of each group of sample cells at 7 and 14 days of mineralization induction in the present invention are as follows;

[0052] Figure 19 The effect of cell matrix mineralization on the cells of each sample group of the present invention after 14 days of cell mineralization induction; wherein A represents the results of Alizarin Red staining, and B represents the statistical analysis of the relative surface area of mineralized nodules of each sample group;

[0053] Figure 20 The effect of cell matrix mineralization on the cells of each sample group of the present invention after 21 days of cell mineralization induction; wherein A represents the results of Alizarin Red staining, and B represents the statistical analysis of the relative surface area of mineralized nodules of each sample group;

[0054] Figure 21 This is a semi-quantitative comparison of the extracellular matrix mineralization of each group of samples of the present invention;

[0055] Figure 22 The expression of osteogenic differentiation proteins (ALP, BMP-2, and OPN) was detected by Western Blot analysis in the present invention; wherein, A represents the measurement of ALP, BMP-2, and OPN by Western Blot analysis on the 7th day, and B represents the relative intensity of ALP, BMP-2, and OPN as a bar graph;

[0056] Figure 23 The present invention uses DAPI staining to observe the adhesion of cells in each group of samples under high glucose conditions;

[0057] Figure 24 The present invention uses CCK-8 method to observe the cell proliferation ability of each group of sample cells under high glucose conditions on the 1st, 3rd, 5th and 7th days;

[0058] Figure 25 The present invention detected the expression of ALP, BMP-2 and OPN proteins in each group of samples under high glucose conditions by Western Blot analysis; wherein, A represents the measurement of ALP, BMP-2 and OPN by Western Blot analysis on the 7th day, and B represents the relative intensity of ALP, BMP-2 and OPN shown in a bar graph. DETAILED DESCRIPTION

[0059] In order to make the purpose, 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 in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0060] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0061] Micro-arc oxidation related reagents: disodium ethylenediaminetetraacetic acid, hydrogen peroxide, calcium dihydrogen phosphate, sodium hydroxide, sodium silicate, zinc acetate, and calcium acetate were all obtained by domestic analysis and purification;

[0062] Cell counting kit (Cell Counting Kit-8, CCK-8) was purchased from Tokyo Tongren;

[0063] Sterile PBS solution was purchased from Meilun Biotechnology Co., Ltd.;

[0064] ALP test kit and ALP staining kit were purchased from Beyotime Biotechnology Research Institute.

[0065] Preparation of experimental reagents:

[0066] ① Preparation of LB culture medium: Add 1 L of distilled water to 40 g of culture medium, dissolve thoroughly, sterilize under high temperature and high pressure (121°C, 20 minutes), and store in a refrigerator at 4°C for later use.

[0067] ②) Preparation of LB agar medium: Add 1 L of distilled water to 32 g of the medium, dissolve thoroughly, and sterilize under high temperature and high pressure (121°C, 20 minutes) before use.

[0068] ③ Preparation of PBS: 2 L of double-distilled water and 1 bag of PBS powder. After fully dissolving, filter with a 0.22 μm filter, sterilize with high temperature and high pressure (121°C, 15 minutes), seal with sealing film, and store at 4°C for later use.

[0069] ④ Preparation of 10% FBS culture medium (complete culture medium): MEM-α culture medium (445 mL), FBS (50 mL), double antibody (5 mL) to obtain 500 mL of 10% FBS culture medium, filter with a 0.22 μm filter, mix well and store at 4°C.

[0070] ⑤ Preparation of high-glucose culture medium: Powdered glucose (2.63 g) + 45 mL 10% FBS MEM-α culture medium, filter to obtain a mother solution (330 mmol / L), and store at 4°C for later use. Dilute the mother solution to a glucose concentration of 33 mmol / L for cell culture.

[0071] ⑥ Preparation of mineralization induction solution: containing 10% FBS MEM-a culture medium + 10mmol / L β-glycerophosphate sodium + 10 -8 mol / L, dexamethasone + 50 μg / m ascorbic acid.

[0072] Reference Figure 1-Figure 25 The present invention provides a Ti6Al4V surface micro-arc zinc oxide modified coating, which is prepared by micro-arc oxidation (ie MAO):

[0073] The first step is to obtain Ti6Al4V and process it into Ti6Al4V samples, and pretreat the Ti6Al4V samples;

[0074] The Ti6Al4V in the present invention was purchased from Dongguan Guanyue Metal Co., Ltd. The chemical composition of Ti6Al4V is shown in Table 1.

[0075] Table 1 Chemical composition of Ti6Al4V

[0076] Material titanium aluminum vanadium iron carbon nitrogen hydrogen oxygen Ingredients (wt%) matrix 6.1 4.0 0.08 0.06 0.019 0.007 0.19

[0077] Ti6Al4V was processed into 1 cm × 1 cm × 1 mm alloy sheets. The surface oxide layer of the Ti6Al4V sample was removed by grinding with 200 mesh, 400 mesh, and 800 mesh sandpaper in sequence. The sample was then ultrasonically cleaned with acetone and anhydrous ethanol for 20 minutes, rinsed with deionized water, and dried for later use.

[0078] The second step is to prepare the electrolyte and prepare the micro-arc oxidation coating on the surface of the Ti6Al4V sample by micro-arc oxidation method, as follows:

[0079] The micro-arc oxidation equipment uses the MAO60-II equipment of the Special Ceramics Research Institute of Harbin Institute of Technology. The electrolytic cell is a 5L plastic barrel. The electrolyte consists of disodium ethylenediaminetetraacetic acid, calcium acetate, calcium dihydrogen phosphate, sodium silicate, sodium hydroxide and hydrogen peroxide. The concentrations of each component are 15g / L, 8.8g / L, 6.3g / L, 7.1g / L, 5g / L and 6mL / L, respectively. The pretreated Ti6Al4V sample is used as the anode and the stainless steel is used as the cathode. They are placed in the electrolyte. The process parameters of the MAO60-II equipment are set to a constant voltage of 400V, a frequency of 600Hz, a duty cycle of 8%, and a reaction time of 5 minutes.

[0080] The third step is to add a zinc acetate solution with a concentration of 0 to 10 g / L into the electrolyte to obtain a micro-arc zinc oxide modified coating on the surface of Ti6Al4V after the reaction, which is then disinfected and dried for later use.

[0081] In order to verify the effect of zinc content on the performance of zinc modified coating, the present invention sets up a control group and an experimental group. The control group is a polished Ti6Al4V sample (no-treatment control, NC group). The experimental group is divided into four groups according to the concentration of added zinc acetate, namely, no zinc group (N-Zn group), low zinc group (L-Zn group: 7 g / L), medium zinc group (M-Zn group: 8.5 g / L), and high zinc group (H-Zn group: 10 g / L). The prepared Ti6Al4V surface micro-arc zinc oxide modified coating is as follows Figure 1 As shown by Figure 1 It can be seen that after micro-arc oxidation, uniform coatings appeared on the surface of the materials of L-Zn, M-Zn, and H-Zn groups. With the increase of zinc ion concentration in the electrolyte, the coating color changed slightly, and uneven coating appeared on the surface of the H-Zn group.

[0082] The micro-arc zinc oxide modified coating on the Ti6Al4V surface was sprayed with gold, and the surface morphology of the material after micro-arc oxidation was observed using a scanning electron microscope (SEM). Figure 2 As shown, the surface of the NC group has no pore structure and is in a groove shape; the surfaces of the other four groups have pore structures, which are crater-like; the surface pore diameter of the N-Zn group is 2-3um; the surface pore diameter of the L-Zn group is 1-2mm; the surface pore diameter of the M-Zn group is 1-1.5um; as the zinc content increases, the micropore diameter becomes smaller and the micropore distribution becomes more uniform. When the zinc acetate concentration is 10g, the surface pore diameter in the H-Zn group is 1-2um but the coating is broken.

[0083] Energy Dispersive Spectroscopy (EDS) was used to analyze the surface zinc element distribution, atomic weight percentage, and mass percentage of the L-Zn group, M-Zn group, and H-Zn group. Figure 3 As shown in the figure, zinc element was successfully introduced into the surface of L-Zn, M-Zn and H-Zn groups, and the element distribution was relatively uniform. Compared with the other groups, the mass percentage and atomic weight percentage of zinc ions in M-Zn group were significantly higher than those in L-Zn and H-Zn groups (P<0.05).

[0084] Studies have shown that when the mass percentage of zinc on the coating surface is below 7%, it is non-toxic to cells and has antibacterial properties. When 8.5g / L zinc acetate is added to the electrolyte in the present invention, it is determined that the mass percentage of zinc on the surface of the zinc-modified coating reaches 5.16%. However, when the zinc acetate concentration in the electrolyte reaches 10g / L, the zinc content on the coating surface shows a downward trend. Therefore, zinc acetate with different concentration gradients is introduced based on the zinc acetate concentration of 8.5g / L. The results showed that the surface zinc content was: M-Zn group > H-Zn group > L-Zn group. The mass percentage and atomic weight percentage of zinc in the M-Zn group were significantly higher than those in the L-Zn and H-Zn groups; the mass percentage and atomic weight percentage of zinc in the H-Zn group were higher than those in the L-Zn group, indicating that the zinc content was related to the concentration of zinc acetate in the electrolyte, but the zinc content in the coating was not positively correlated with the concentration of zinc acetate in the electrolyte. This phenomenon was consistent with the surface morphology test results. The surface coating of the H-Zn group showed uneven distribution and fracture, which led to the obstruction of zinc introduction into the zinc-modified coating.

[0085] The phase analysis of Ti6Al4V surface coating was carried out by X-ray diffraction (XRD). Figure 4 As shown in the figure, the same waveform in the XRD pattern analysis represents the same crystal phase, and the height and number of the peaks represent the intensity of the crystallization. Figure 4 It can be seen that compared with the NC group, anatase phase (Rutile) and rutile phase (Anatase) appeared on the surface of the experimental group; currently, only rutile and anatase phases play a role in biomedical applications. After zinc acetate was added to the micro-arc oxidation electrolyte, anatase phase and rutile phase appeared on the surface of the experimental group. Compared with pure anatase, rutile phase has better biological activity, induces enhanced deposition of hydroxyapatite, and further promotes bone bonding ability.

[0086] The hydrophilicity of the micro-arc zinc oxide modified coating on the Ti6Al4V surface was tested using a contact angle meter. After a certain amount of distilled water was sucked up with a pipette, it was fixed vertically above the zinc modified coating. After pressing out the water, it was slowly dripped onto the surface of the zinc modified coating. The changes in the water droplets on the sample surface were recorded by software imaging equipment, and the average contact angle was calculated. The contact angle test results are shown in Figure 2. Figure 5 The results showed that the contact angles of the experimental groups were lower than those of the NC group. The M-Zn group had the smallest contact angle among the experimental groups, and the difference was statistically significant (P < 0.05).

[0087] Studies have found that a material surface contact angle between 35° and 80° is optimal for biomedical implants, and the smaller the contact angle, the higher the material's hydrophilicity. Under highly hydrophilic conditions, cell adhesion and proliferation are enhanced. However, when the contact angle is below 35°, it negatively impacts protein attachment and can easily lead to thrombosis. Studies have shown that implant coatings and surface modifications can improve hydrophilicity, thereby accelerating osseointegration. Results showed that the contact angle of the Ti6AI4V alloy was approximately 75°, while the contact angle of the coating surface after micro-arc oxidation ranged from 35° to 60°. The contact angles were in the order of M-Zn group > L-Zn group > H-Zn group > N-Zn group > NC group, indicating that the surface hydrophilicity of Ti6AI4V changed after micro-arc oxidation. Adjusting the zinc acetate concentration in the electrolyte microstructurally altered the pore size of the coating surface. As the pore size decreased, the contact angle decreased, and the hydrophilicity of the material surface increased.

[0088] The following is the antibacterial performance test of the micro-arc zinc oxide modified coating on the Ti6Al4V surface of the present invention:

[0089] Zinc ions have attracted widespread attention due to their simultaneous antibacterial properties and ability to promote bone differentiation. Zinc ions react with amino groups on the bacterial cell wall, destroying the structure of bacterial surface proteins and preventing the bacteria from proliferating. When bacteria necrotize and disintegrate, zinc ions will be released from the bacteria and continue to bind to free bacteria. After the implant is implanted, bacteria and host cells colonize on the surface of the implant in a competitive manner, and the results of the competition between bacteria and host cells will directly affect the implantation effect of the implant. In other words, if the implant material itself has antibacterial ability, it will weaken the competitiveness of bacteria in the early stage of implantation, and gain more colonization time for host cells (such as osteoblasts), which has an important impact on improving the success rate of implantation.

[0090] The E. coli international standard strain (8099) and S. aureus international standard strain (ATCC6538) used in the present invention were donated by Harbin Veterinary Research Institute.

[0091] The two bacteria were inoculated into LB medium respectively and cultured at 37℃ for 24 hours before use. The revived bacteria were transferred for two generations. The strains were inoculated into LB liquid medium with an inoculation loop and cultured at 37℃ for 24 hours. The concentration of the bacterial solution was adjusted to 1×10 5 cfu / mL, 1×10 6 cfu / mL, 1×10 7 cfu / mL, 1×10 8 cfu / mL, 100 μL of bacterial suspension was taken from each group for plate coating, the experiment was repeated 3 times, and the appropriate bacterial suspension concentration was selected for subsequent experiments based on the experimental results.

[0092] like Figure 6 As shown, the concentration is 1x10 8 cf / mL, 1x10 7 cfi / mL, the plate surface is completely covered with bacteria, and the concentration is 1x10 6 cf / mL, the colonies are uniform and independent; the concentration is 1x10 5 cfu / mL, the number of bacteria on the plate surface is too small, which is not conducive to subsequent experiments. Therefore, 1x10 6 cfu / mL bacterial solution concentration for subsequent experiments.

[0093] After resuscitation, the bacteria were transferred for two generations, and the strain was inoculated into LB liquid medium with an inoculation loop and cultured at 37°C for 24 hours. The concentration of the bacterial solution was adjusted to 1×10 6 cfu / mL. The experiment was divided into 5 groups, with 6 samples in each group, and placed in a 24-well plate. Take 20μL of bacterial suspension and drip it on the surface of the specimen. Cover it with a 0.8cm×0.8cm sterile PE film to spread the bacterial liquid but not overflow. After constant temperature incubation at 37℃ for 24 hours, take out the specimen and elute it in 20mLPBS. Take 100μL of the eluate and drip it on the LB solid culture medium plate and spread it evenly. After constant temperature incubation at 37℃ for 24 hours, count the plates and calculate the average colony count of each group. The experiment was repeated 3 times. The bacterial growth of each group is shown as follows. Figure 7 The colony count results are shown in Table 2, and the antibacterial rate calculation formula is as follows:

[0094] Antibacterial rate = [(number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group] × 100%

[0095] Table 2 Antibacterial effect of different densities of Staphylococcus aureus and Escherichia coli on zinc modified coatings for 24 h

[0096]

[0097] Bacterial activity was further analyzed. The experiment was divided into 5 groups, with 8 samples in each group placed in a 24-well plate. 60 μL of bacterial suspension (1×106 cfu / mL) was pipetted and inoculated on the surface of each group of samples. The samples were cultured at 37°C for 3, 6, 12, and 24 hours. The following experiment was repeated at each time point. 0.5 mL of 10% Alamar blue solution was added to each sample and cultured for another 2 hours. 100 μL of the solution was pipetted and added to a 96-well plate with 3 replicates. The fluorescence intensity at 560 nm excitation and 590 nm emission was measured using a microplate reader. The proliferation rate was calculated using the following formula:

[0098] Proliferation rate = [(control group - experimental group) / control group] × 100%

[0099] The results are as follows Figure 8 As shown in the results, compared with the NC group, the bacterial activity of each experimental group was significantly reduced (P<0.05). Compared with the H-Zn group, the bacterial activity of the N-Zn group and the L-Zn group was relatively higher (P<0.05). There was no significant difference between the M-Zn group and the H-Zn group (P>0.05).

[0100] Cytotoxicity test of micro-arc zinc oxide modified coating on Ti6Al4V surface:

[0101] Because zinc has a certain degree of cytotoxicity, low-dose zinc ions can enhance cell number and metabolic activity, but high-dose zinc ion release can adversely affect cell proliferation. Therefore, it is essential to test the cytotoxic effects of the zinc-modified coatings produced by micro-arc oxidation of T6AI4V. For solid materials (such as metals or alloys), cytotoxicity can be tested using the extract obtained after immersing the material in culture medium at a ratio of 3 cm²:1 mL for three days according to ISO 10993-5.

[0102] The experiment was divided into five groups. The high-temperature and high-pressure samples were placed in sterile centrifuge tubes. According to the ISO10993-5 standard, 9 samples and 6 mL of complete culture medium were placed in each centrifuge tube. The tubes were sealed with sealing film and placed in an incubator for 3 days. The sample extracts were obtained and transferred to 15 mL sterile centrifuge tubes. The extracts were stored in a 4°C refrigerator. MC3T3-E1 cells were revived, digested, resuspended, and the cells were counted. Based on the seeding of 2 × 103 cells per well, the required cell resuspension volume per well was calculated. The cells were seeded into 96-well plates and incubated at 37°C for 24 hours. The extracts stored at 4°C were removed in advance and cooled to room temperature. The extracts of the experimental group and the NC group were added to the 96-well plates, 0.2 mL per well, and cultured for 1 day and 3 days respectively. The original culture medium was discarded, and complete culture medium and CCK-8 were mixed at a ratio of 10:1. 110 μL was added to each well and incubated for 2 hours. The absorbance value was detected at a wavelength of 450 nm using a microplate reader.

[0103] The results of the cytotoxicity test on MC3T3-E1 cells using the extract are as follows: Figure 9 As shown in the results, after 1 and 3 days of culture, there was no significant difference in cell proliferation compared with the NC group (P>0.05), indicating that the zinc-modified coating of the present invention has no cytotoxicity.

[0104] Due to the complex environment of the oral microecology, during the implant placement process, bacteria in the oral cavity, such as the cheek, tongue, periodontal pocket, and restoration, can shift, then colonize and attach around the implant. Studies have found that within 30 minutes of implant placement, bacteria from other parts of the mouth can infiltrate around the implant through the edge of the healing abutment, further colonize and grow on the surface, and eventually form a biofilm. In addition, even after high temperature and high pressure, the implant surface still carries trace amounts of bacterial microorganisms, and the bacteria present in the operating room air environment may cause infection at the surgical site.

[0105] In clinical practice, bacterial adhesion and cell growth often occur simultaneously. To investigate the growth of MC3T3-E1 cells in the presence of bacteria, we co-cultured MC3T3-E1 cells with E. coli and S. aureus. The experiment was divided into five groups, with six samples from each group placed in a 24-well plate. 60 μL of a mixed bacterial suspension at a concentration of 1.0 × 10⁶ cfu / mL was dripped onto the sample surface and incubated in a 37°C incubator for 90 minutes. Cell morphology and viability were observed.

[0106] (1) Observe cell morphology:

[0107] The 24-well plate was removed and washed with PBS to remove unadhered bacteria. Then, 1 mL of the cell suspension (2 × 104 cells / mL) was inoculated per well and cultured in a cell incubator for an additional 24 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes, then stained for the cytoskeleton with rhodamine-labeled phalloidin for 20 minutes. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 30 seconds. After washing three times with PBS, the cells were photographed under a fluorescence microscope.

[0108] like Figure 10 As shown in the data, the number of cell adhesion in the NC and N-Zn groups was small, the cells were wrinkled, and the cell structure was incomplete; the introduction of zinc-modified coating was conducive to cell adhesion and spreading, among which the fluorescence intensity of the surface of the M-Zn and H-Zn groups was higher, the cytoskeleton was clearly visible, the pseudopodia were well extended, and the cell spreading was more uniform. Within 24 hours, the number of cell adhesion was significantly increased compared with the NC, N-Zn, and L-Zn groups.

[0109] (2) Cell live / death status:

[0110] The specimens were washed with PBS to remove non-adherent bacteria, and then 1 mL of cell suspension (5 × 104 cells / mL) was inoculated into each well. After continued culture in a cell culture incubator for 24 hours, the specimens were washed three times with PBS. Calcein-AM, Calcein-PI, and 1 × Assay Buffer were mixed at a ratio of 1:3:1000 according to the instructions of the cell viability staining kit. 500 μL was added to each well and incubated in the dark for 30 minutes. Cell viability was observed using a fluorescence microscope.

[0111] The number of live cells in each experimental group was significantly higher than that in the NC group, and the number of dead cells in each experimental group was significantly lower than that in the NC group (P<0.05). In order to exclude the influence of the number of cell adhesion on the cell survival, the number of dead cells / live cells (%) on the surface of the material was statistically analyzed. Figure 11 The results showed that the dead cell / live cell ratio (%) of each experimental group was significantly lower than that of the NC group, and the difference was statistically significant. Compared with the experimental groups, the dead cell / live cell ratio (%) of the M-Zn group was significantly lower than that of the N-Zn, L-Zn, and H-Zn groups, and the difference was statistically significant (P<0.05).

[0112] Combined with the physical properties and antibacterial test results of the above zinc-modified coatings, the surface coating of the H-Zn group broke, and compared with the M-Zn group, it did not show specific antibacterial properties. For subsequent cell studies, the N-Zn, L-Zn, and M-Zn groups will be selected as experimental groups.

[0113] Cell adhesion, proliferation, and osteogenic differentiation:

[0114] (1) Cell adhesion and spreading

[0115] Three samples were taken from each group and placed in a 24-well plate. 1 mL of cell suspension (2×104 cells / mL) was added to each well and the cells were cultured in a cell culture incubator for 1, 4, and 24 hours. The 24-well plate was removed and each sample was washed three times with PBS. The samples were transferred to a new 24-well plate and 0.5 mL of 4% paraformaldehyde was added to each well to fix the cells for 15 minutes. The samples were washed three times with PBS and 0.5 mL of 0.1% (v / v) Triton X-100 was added to permeabilize the cells at room temperature for 5 minutes. The samples were washed three times with PBS and 0.5 mL of rhodamine-labeled phalloidin was added to the surface to stain the cells at room temperature. After being kept away from light for 30 minutes, the samples were washed three times with PBS and 1 mL of LDAPI dye was added to each well to stain the cell nuclei at room temperature. After being kept away from light for 30 seconds, the samples were washed three times with PBS and the cell adhesion and spreading were observed using an inverted fluorescence microscope.

[0116] The adhesion and spreading of cells were observed by rhodamine-phalloidin. Figure 12-14 As shown, compared with the NC group, the number of cell adhesions in each experimental group increased significantly, and the extension of cell pseudopodia was obvious over time.

[0117] Cells were seeded and stained 1 hour later. Figure 12 As shown in the figure, there were fewer adherent cells in the NC and N-Zn groups, the cell morphology was oval, and the spreading was poor. The number of adherent cells in the L-Zn and M-Zn groups was significantly increased compared with the NC and N-Zn groups, and the cells spread better, with stronger initial adhesion ability.

[0118] Cells were seeded and stained 4 hours later. Figure 13 As shown, the number of cell adhesions in the N-Zn, L-Zn, and M-Zn groups was significantly greater than that in the NC group, and the cell spreading area was larger and the pseudopodia extended significantly; the number of cell adhesions in the NC group increased and the initial adhesion began.

[0119] Cells were seeded and stained 24 hours later. Figure 14 As shown in the figure, the spreading of cells on the material surface increased. Compared with the NC group, the experimental group had more cell adhesion and a larger spreading area. The M-Zn group had better adhesion ability than the N-Zn and L-Zn groups, indicating that different concentrations of zinc-modified coatings Ti6AI4V promoted the adhesion of MC3T3-E1 cells.

[0120] (2) Cell proliferation

[0121] Cell proliferation is one of the physiological activities of cells and is the basis for cells to carry out life activities and self-renewal. The enhancement of cell proliferation ability is conducive to cell differentiation.

[0122] Cell proliferation was also detected using the CCK-8 assay. MC3T3-E1 preosteoblasts were revived, digested, and resuspended before cell counting. Three samples from each group were placed in a 24-well plate. 1 mL of cell suspension (2 × 104 cells / mL) was added to each well and cultured at 37°C for 1, 3, and 5 days. The original culture medium was discarded, and 550 μL of complete culture medium mixed with CCK-8 at a ratio of 10:1 was added to each well. The cells were incubated for 2 hours, and absorbance was measured at 450 nm using a microplate reader.

[0123] like Figure 15 As shown in the figure, at each observation time point (1, 3, and 5 days), the OD values of cells in the NC group and the experimental group increased significantly over time. On the first day, there was no significant difference in cell proliferation ability between the experimental group and the NC group, and there was no difference between the experimental groups. On the third and fifth days, cell proliferation in the NC group was significantly lower than that in the other experimental groups, suggesting that micro-arc oxidation surface treatment can promote the proliferation of MC3T3-E1 cells. Compared with the experimental groups, the M-Zn group had a significantly higher ability to promote cell proliferation than the N-Zn group and the L-Zn group (P < 0.05), indicating that cell proliferation ability is related to zinc content.

[0124] (3) Cell differentiation into osteoblasts

[0125] ALP is an early marker of osteoblast differentiation and maturation. Quantitative detection of ALP can reflect the differentiation level of osteoblasts. The higher its activity, the more obvious the differentiation of pre-osteoblasts into mature osteoblasts.

[0126] For ALP staining and activity assays, six samples from each group were placed in a 24-well plate, and 1 mL of cell suspension (2 × 104 cells / mL) was added to each well. After two days of cell culture, the mineralization-inducing medium was replaced and cultured. ALP staining and ALP activity assays were performed on days 7 and 14. The experimental procedures were performed according to the instructions for the Beyotime ALP staining kit and the ALP assay kit, respectively.

[0127] The ALP staining results of each group of cells at different time points were observed under a stereo microscope (x25). Figure 16-17 As shown, the results showed that compared with the control group, the number of stained cell clusters in the different zinc intervention groups was significantly increased and the color was darker, with concentration-dependence and time-dependence characteristics.

[0128] The results of ALP activity determination at 7 and 14 days after mineralization induction in each group were as follows: Figure 18As shown in the data, the ALP activity of the experimental group was significantly increased compared with that of the NC group (P<0.05). Comparison among the groups showed that the ALP activity in the M-Zn and L-Zn groups was higher than that in the N-Zn group, which was statistically significant (P<0.05). In addition, the ALP activity was positively correlated with the zinc content on the coating surface, that is, the M-Zn group > the L-Zn group.

[0129] During induction of osteoblastic differentiation in MC3T3-E1 cells in vitro, individual cells first grow and stack into clusters. Subsequently, mineralized nodules form as calcium and phosphate accumulate. The formation of mineralized nodules is a key indicator of late osteoblastic differentiation. Alizarin red chelates with calcium ions produced by MC3T3-E1 cells to form a complex, producing orange-red deposits (i.e., calcium nodules) that visually reflect the mineralization of the cells.

[0130] Three samples from each group were placed in a 24-well plate, and 1 mL of cell suspension (2 × 104 cells / mL) was added to each well. After 2 days of cell culture, the cells were replaced with mineralization medium and stained with Alizarin Red on days 14 and 21. The solution was discarded, the cells were washed three times with phosphate buffer, and fixed with paraformaldehyde for 15 minutes. The solution was discarded, and the cells were washed three times with phosphate buffer for 5-10 minutes each time. 0.1% Alizarin Red stain was added to the surface of each sample, fully submerged, and incubated at room temperature for 30 minutes. The stain was aspirated, and the cells were washed three times with phosphate buffer. The cells were observed and photographed under a stereomicroscope. For semi-quantitative analysis of extracellular matrix mineralization, 10% cetylpyridinium chloride was prepared, and 500 μL of eluent was added to the surface of each sample. After thorough shaking, the OD value was measured at a wavelength of 620 nm.

[0131] BMP-2 is an important extracellular signaling molecule that promotes endochondral bone formation and induces osteoblast differentiation. OPN is a marker protein for late bone formation, playing a role in both bone matrix formation and absorption and is associated with the initiation of mineralization.

[0132] Three samples from each group were placed in a 24-well plate, and 1 mL of cell suspension (2×104 cells / mL) was added to each well. After culturing the cells for 2 days, the mineralization induction medium was replaced and cultured. The medium was replaced every two days, and total cell protein was extracted on the 14th day.

[0133] The results of Alizarin red staining of cells in each group after 14 days of mineralization induction are shown in the figure below. Figure 19 As shown in (A), under a stereo microscope (x25), the number of mineralized nodules in the experimental group was significantly increased compared with the NC group. Figure 20(A) The number of surface mineralized nodules in the NC and N-Zn groups did not increase significantly, while the number of mineralized nodules in the L-Zn and M-Zn groups was relatively large. The relative surface area of mineralized nodules in each group was statistically analyzed. Figure 19 (B) and 20(B) show that the relative surface area of mineralized nodules in the experimental group was significantly greater than that in the NC group, and the difference was statistically significant (P<0.05).

[0134] To semi-quantitatively detect the mineralization of the extracellular matrix, 10% cetylpyridinium chloride was prepared and 500 μl of the eluent was added to the surface of each sample. After thorough shaking, the OD value was measured at a wavelength of 620 nm. The results showed that the extracellular matrix mineralization ability of the L-Zn group and the M-Zn group was significantly higher than that of the NC group and the N-Zn group, and the difference was statistically significant (P<0.05). The ability of the M-Zn group to promote extracellular matrix mineralization was higher than that of the L-Zn group ( Figure 21 ).

[0135] After 14 days of cell mineralization induction in each group, the expression of osteogenic differentiation proteins (ALP, BMP-2 and OPN) was detected by Western Blot, with GAPDH as the internal reference. Figure 22 As shown in the data, compared with the NC group, the experimental groups upregulated the expression of ALP, BMP-2 and OPN proteins, which was statistically significant (P<0.05). Compared with the experimental groups, the M-Zn group promoted the expression of osteogenic differentiation proteins more significantly, and the difference was statistically significant (P<0.05), indicating that the increase of zinc content on the surface of the material can promote the osteogenic differentiation ability of MC3T3-E1 in a dose-dependent manner.

[0136] Hyperglycemia impairs osteoblast adhesion, proliferation, and differentiation, causing an imbalance between osteoblasts and osteoclasts and affecting bone tissue metabolism. High glucose levels can also alter the biomineralization process, inhibiting osteoblast mineralization and the expression of osteogenic markers (ALP, Runx2, BMP-2, and osteocalcin) and promoting the expression of adipogenic markers. Hyperglycemia also leads to the accumulation of advanced glycation end products (AGEs), which can inhibit osteoblast proliferation and promote osteoclast-related bone resorption, leading to an overall deterioration in bone quality. High glucose levels promote the production of reactive oxygen species (ROS). Excessive ROS production impairs osteoblast adhesion and blocks the BMP-2 signaling pathway, subsequently reducing ALP activity, osteocalcin production, and matrix mineralization.

[0137] Based on the above experimental results, a suitable zinc-modified coating on the Ti6Al4V surface was screened for subsequent research under high-glucose conditions. The following experiments were divided into three groups: NC group, N-Zn group, and M-Zn group. The specific experimental process was similar to the above method, in which the final glucose concentration was 33 mmol / L.

[0138] (1) Cell adhesion:

[0139] Three samples from each of the NC, N-Zn, and M-Zn groups were placed in a 24-well plate. 1 mL (5 × 104 cells / mL) of MC3T3-E1 cell suspension cultured in high glucose was added to each well. The plates were then cultured in a cell culture incubator for 24 hours, and cell adhesion was observed.

[0140] like Figure 23 As shown in the figure, the cell adhesion was observed by DAPI staining. Compared with the NC group, the number of cells adhered in each experimental group was significantly increased (P<0.05). Compared with the N-Zn group, the M-Zn group significantly improved the cell adhesion ability.

[0141] (2) Cell proliferation:

[0142] Three samples from each of the NC, N-Zn, and M-Zn groups were placed in a 24-well plate. MC3T3-E1 cells were cultured in high glucose for 1, 3, 5, and 7 days. Cell proliferation was detected by CCK-8 assay. The results are shown in Figure 3. Figure 24 As shown in the figure, at each observation time point (1, 3, 5, and 7 days), the cells in the NC group and the experimental group showed a proliferative state, and the OD values of each group increased significantly with the extension of time.

[0143] On day 1, the cell proliferation ability of the experimental groups was significantly enhanced compared with the NC group, but there was no statistical difference between the experimental groups. On days 3, 5, and 7, the cell proliferation ability of the NC group was significantly lower than that of the experimental group. Compared with the experimental groups, the M-Zn group had a significantly higher ability to promote cell proliferation than the N-Zn group (P < 0.05).

[0144] (3) Western Blot detection of ALP, BMP-2 and OPN protein expression:

[0145] Three samples from each of the NC, N-Zn, and M-Zn groups were placed in a 24-well plate, and 1 mL of high-glucose culture cell suspension (5 × 10 4 cells / mL), and when the cells grew to 70%-80% fusion, they were cultured in high-glucose mineralization induction medium. The culture medium was changed every two days, and total cell protein was extracted on the 14th day for Western Blot analysis.

[0146] like Figure 25As shown, cells in each group were cultured with high glucose for 14 days and mineralization was induced. Western Blot analysis was used to detect the expression of ALP, BMP-2, and OPN proteins. GAPDH was used as an internal control. Compared with the NC group, the N-Zn and M-Zn groups upregulated the expression of ALP, BMP-2, and OPN proteins, with statistical significance (P<0.05). Compared with the N-Zn group, the M-Zn group significantly upregulated the expression of ALP, BMP-2, and OPN proteins (P<0.05).

[0147] This study simulated a high-glucose state by adjusting the glucose concentration in the culture medium. The researchers then observed cell adhesion on the zinc-modified coating to determine whether the coating retained the biological properties observed at normal glucose concentrations under high-glucose conditions. The results showed that, under high-glucose conditions, the number of cells adhering to the zinc-modified coating increased after micro-arc oxidation compared to the NC coating, indicating that the introduction of zinc further enhanced cell adhesion.

[0148] These results indicate that the micro-arc oxidation coating exhibited the same biological properties under high glucose conditions as under normal glucose concentrations, promoting MC3T3-E1 cell adhesion and proliferation. Furthermore, the introduction of the zinc coating further enhanced cell adhesion and proliferation compared to micro-arc oxidation alone. Furthermore, the trends in cell mineralization under high glucose concentrations were similar to those under normal glucose concentrations. Compared to the NC group, both the N-Zn and M-Zn groups upregulated the expression of ALP, BMP-2, and OPN proteins, with the M-Zn group showing a more significant upregulation of these proteins compared to the N-Zn group.

[0149] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A Ti6Al4V surface micro-arc zinc oxide modified coating, characterized in that: The zinc content of the zinc modified coating is 0-5.16%, a plurality of holes with a diameter of 1-2 μm are evenly distributed on the coating, and the contact angle is 35-60 degrees.

2. The Ti6Al4V surface micro-arc zinc oxide modified coating according to claim 1, characterized in that: The zinc content of the zinc-modified coating on the Ti6Al4V surface is 5.16%, the pore diameter is 1 to 1.5 μm, and the contact angle is 35°.

3. The method for preparing a Ti6Al4V surface micro-arc zinc oxide modified coating according to any one of claims 1-2, characterized in that: The following steps are involved: S1: Processing and pretreatment of Ti6Al4V samples, and preparation of electrolyte; S2: The pretreated Ti6Al4V sample was used as the anode and the stainless steel was used as the cathode, and placed in the electrolyte; S3: Set the process parameters of micro-arc oxidation, then add a zinc acetate solution with a concentration of 8.5 g / L to the electrolyte to obtain a micro-arc zinc oxidation modified coating on the Ti6Al4V surface, and then disinfect and dry it.

4. The method for preparing a Ti6Al4V surface micro-arc zinc oxide modified coating according to claim 3, characterized in that: The method also includes a detection step for the micro-arc zinc oxide modified coating on the Ti6Al4V surface.

5. The method for preparing a Ti6Al4V surface micro-arc zinc oxide modified coating according to claim 4, characterized in that: The detection steps include cell adhesion detection, cell proliferation detection and cell osteogenic differentiation detection of the zinc-modified coating under high sugar conditions.

6. The method for preparing a Ti6Al4V surface zinc modified coating based on micro-arc oxidation according to claim 5, characterized in that: The process of cell adhesion assay is as follows: (1) Ti6Al4V samples were placed in a 24-well plate, and 1 mL of high-glucose cultured MC3T3-E1 cell suspension was added to each well; (2) Place the 24-well plate containing the MC3T3-E1 cell suspension in a cell culture incubator and culture for 1 to 24 hours; (3) Remove the 24-well plate, wash the Ti6Al4V sample with PBS, and transfer it to a new 24-well plate. Fix, permeabilize, and stain the cells in sequence to observe cell adhesion.

7. The method for preparing a Ti6Al4V surface zinc modified coating based on micro-arc oxidation according to claim 5, characterized in that: The process of cell proliferation assay is as follows: (1) MC3T3-E1 cells were cultured in high glucose for 1 to 7 days; (2) Place the Ti6Al4V sample in a 24-well plate, add 1 mL of cultured MC3T3-E1 cell suspension to each well, and culture at 37°C for 1 to 5 days; (3) Discard the original culture medium and add 550 μL of a mixture of complete culture medium and CCK-8 to each well. Incubate for 2 h and use CCK-8 to detect cell proliferation.

8. The method for preparing a Ti6Al4V surface zinc modified coating based on micro-arc oxidation according to claim 5, characterized in that: The process of cell osteogenic differentiation detection is as follows: (1) Ti6Al4V samples were placed in a 24-well plate, and 1 mL of high-glucose cultured MC3T3-E1 cell suspension was added to each well; (2) When the cells grow to 70% to 80% fusion, replace the high-glucose mineralization induction medium and continue culturing. The high-glucose mineralization induction medium should be replaced every two days. (3) On the 14th day, total cell protein was extracted and the expression of ALP, BMP-2 and OPN proteins was detected.

9. Use of a Ti6Al4V surface micro-arc zinc oxide modified coating according to any one of claims 1-2 in oral implant materials.

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