Zirconia-based medical prosthesis material and preparation method and application thereof

Abstract

The application belongs to the technical field of medical prosthesis materials, and particularly relates to a zirconia-based medical prosthesis material and a preparation method and application thereof, the preparation method comprising the following steps: S1, pretreatment: firstly, a zirconia preform is subjected to one-time high-temperature sintering, then is subjected to polishing, polishing and one-time cleaning, and then is subjected to two-time high-temperature sintering and two-time cleaning; S2, preparation of a surface drug-loading structure: a honeycomb-shaped porous nanostructure for drug loading is prepared on the surface of the zirconia preform through phosphatization treatment; S3, loading of metal ions: the zirconia material is placed in a loading solution containing metal ions, and is reacted in a shaking table at 50-90 DEG C for 5-8 hours, and then is taken out after the reaction is completed, and is washed and dried to obtain the zirconia-based medical prosthesis material, the zirconia-based medical prosthesis material avoids the problems of poor wear resistance of PLGA, poor combination of a calcium phosphate coating and color of a dopamine coating, and realizes the slow release of metal ions, so that bone integration is more effectively promoted.

Description

Technical Field

[0001] This invention belongs to the field of medical prosthetic materials technology, and particularly relates to a zirconia-based medical prosthetic material, its preparation method and application. Background Technology

[0002] Compared to traditional titanium materials, zirconia exhibits unique advantages, but also faces some limitations. Firstly, zirconia possesses excellent biocompatibility, a characteristic that promotes good integration with surrounding tissues and reduces the likelihood of inflammation. Secondly, the natural bone color of zirconia gives zirconia-based medical prostheses a significant aesthetic advantage. Furthermore, zirconia also boasts excellent mechanical strength, corrosion resistance, and low magnetism, preventing artifacts in MRI scans. However, zirconia also has some drawbacks, with its bioinertness being a major reason for its limited adoption. Additionally, zirconia exhibits extremely high chemical stability, hardly reacting with bodily fluids or tissues, and is not easily corroded or dissolved. The high inertness of its surface and the lack of effective chemical functional groups make it difficult for bioactive substances to be stably fixed or adsorbed onto its surface, limiting its potential application in promoting bone repair and tissue engineering. Therefore, future research needs to focus on developing new surface modification technologies and material processing methods to improve the bioactivity of zirconia implants and promote better integration with bone tissue.

[0003] In the field of implant surface modification, polylactic acid-glycolic acid copolymer (PLGA), inorganic ion coatings such as calcium phosphate, and dopamine coatings are widely used to improve implant bioactivity and promote osseointegration by releasing metal ions. However, each of these methods has its limitations. For example, while PLGA has good biocompatibility and biodegradability, its wear resistance is poor, affecting its long-term stability; although calcium phosphate coatings can simulate the mineralization process of bone tissue, their bonding force with the implant substrate is insufficient, making them prone to detachment; and while dopamine coatings improve the bioactivity of the implant surface, their inherent color affects the aesthetics of the implant in the oral cavity. Summary of the Invention

[0004] This invention aims to provide a zirconia-based medical prosthesis material, its preparation method, and its application. By growing a stable and colorless metal ion coating in situ on the surface of the zirconia implant, the poor wear resistance of PLGA, the poor bonding of calcium phosphate coating, and the color problem of dopamine coating are avoided. At the same time, the sustained release of metal ions is achieved, thereby more effectively promoting osseointegration.

[0005] In view of this, the present invention provides a method for preparing a zirconia-based medical prosthesis material, comprising the following steps: S1, Pretreatment: First, the zirconia preform is subjected to high-temperature sintering, followed by grinding, polishing, and cleaning. Then, the zirconia preform is subjected to high-temperature sintering and cleaning again. S2, Preparation of surface drug-loaded structure: A honeycomb porous nanostructure for drug loading is prepared on the surface of the zirconia preform by phosphorylation treatment. S3, Loading of metal ions: The zirconia material treated in step S2 is placed in a loading solution containing metal ions and reacted in a shaker at 50~90℃ for 5~8h. After the reaction is completed, it is taken out, rinsed, and dried to obtain zirconia-based medical prosthesis material.

[0006] Furthermore, in step S1, the first high-temperature sintering process is as follows: first, the temperature is raised to 800-1000℃ at a rate of 5-8℃ / min and held for 40-60min, then the temperature is raised to 1350-1450℃ at a rate of 2-4℃ / min and held for 100-140min, and after the holding period, the temperature is cooled to room temperature with the furnace; the second high-temperature sintering process is as follows: first, the temperature is raised to 1450-1650℃ at a rate of 3-5℃ / min and held for 160-200min, and after the holding period, the temperature is cooled to room temperature with the furnace.

[0007] Furthermore, in step S1, the first cleaning process involves ultrasonic cleaning with anhydrous ethanol and ultrapure water in sequence; the second cleaning process involves ultrasonic cleaning with anhydrous ethanol and ultrapure water in sequence, and after the second cleaning, the zirconia preform is dried by nitrogen gas flow and stored in a vacuum tank for later use.

[0008] Furthermore, in step S2, the phosphorylation process is as follows: the pretreated zirconia preform is placed in a high-pressure reactor, and a phosphoric acid solution with a concentration of 2~3wt% is added. Then, the sealed high-pressure reactor is placed in a drying oven and kept at a constant temperature of 150~165℃ for 20~28h.

[0009] Furthermore, in step S3, the metal ion is selected from Ag. + Cu 2+ 、Sr 2+ One or more of them.

[0010] Furthermore, in step S3, the soluble nitrate or chloride of the corresponding metal ion is dissolved in deionized water to prepare the desired loading solution containing metal ions.

[0011] Furthermore, silver nitrate, copper nitrate, and strontium chloride were used to prepare solutions containing Ag. + Cu 2+ 、Sr 2+ The loading solution contains silver nitrate at a concentration of 10–50 μM, copper nitrate at a concentration of 100–400 mM, and strontium chloride at a concentration of 0.5–3 M.

[0012] Furthermore, in step S3, the rinsing and drying process is as follows: the prosthetic material is rinsed with deionized water, anhydrous alcohol and deionized water in sequence, and then the prosthetic material is placed in a vacuum oven to dry at 32~45℃ and stored in a vacuum container for later use.

[0013] In addition, the present invention also provides a zirconia-based medical prosthesis material, which is prepared by the above-described method for preparing zirconia-based medical prosthesis materials.

[0014] The present invention also provides the application of the above-mentioned zirconia-based medical prosthesis material in the preparation of dental prostheses and bone prostheses.

[0015] Compared with existing technologies, the zirconia-based medical prosthesis material, its preparation method, and its application described in this invention have the following advantages: 1. This invention first forms a honeycomb-like structure (ZrP) on the surface of zirconia material through phosphorylation treatment, and then loads active metal ions onto the ZrP surface through a hydrothermal method, endowing it with osteogenic differentiation regulation and antibacterial activity; 2. This invention, by growing a stable and colorless metal ion coating in situ on the surface of zirconia material, not only avoids the problems of poor wear resistance of PLGA, poor bonding of calcium phosphate coating, and color problem of dopamine coating, but also achieves sustained release of metal ions, thereby more effectively promoting osseointegration; 3. The colorless characteristic of the metal ion coating prepared by this invention makes the modified zirconia material more aesthetically pleasing in applications such as oral cavity, meeting the requirements for the appearance of prosthetic materials in clinical applications. Attached Figure Description

[0016] Figure 1 These are representative SEM images of the samples ZrO2, ZrP, Ag20@ZrP, Cu200@ZrP, and Sr2000@ZrP-E prepared in this invention; Figure 2 These are the surface elemental distribution analysis results of the samples prepared by this invention; where A represents the main elemental composition and distribution of each group of samples, and B represents the full XPS spectrum of each sample. Figure 3 These are peak fitting diagrams of Ag, Cu, and Sr in the sample prepared by this invention; wherein, C is the peak fitting diagram of Ag; D is the peak fitting diagram of Cu; and E is the peak fitting diagram of Sr. Figure 4 Ag in the sample prepared by this invention + Ca 2+ 、Sr 2+ Cumulative ion release curves at 0, 1, 3, 7, and 14 days; where A represents Ag. + Cumulative ion release curves at different immersion times; B represents Ca.2+ Cumulative ion release curves at different immersion times; C represents Sr 2+ Cumulative ion release curves at different immersion times; Figure 5 This is the test result of the surface water contact angle of the sample prepared by this invention; Figure 6 These are the cell viability test results for MC3T3-E1 cells; Figure 7 These are the results of MC3T3-E1 cell morphology and cell viability / death experiments; where A shows the cell morphology after culturing MC3T3-E1 cells on different sample surfaces; and B shows the results of the cell viability / death experiments. Figure 8 These are the results of the antibacterial rate test; where A is the CFU image of the bacterial plating plate; and B is the antibacterial rate calculated after colony counting. Figure 9 These are images of the inhibition zone and statistical results of the inhibition zone diameter measurement; where A is the inhibition zone image and B is the statistical result of the inhibition zone diameter measurement. Figure 10 These are scanning electron microscope (SEM) images of bacteria. Figure 11 The figures show the proliferation of samples from the Ag10@ZrP, Ag20@ZrP, and Ag40@ZrP groups over 12 hours; where A is the growth curve of Escherichia coli at OD600 nm over 10 hours; B is the growth curve of Staphylococcus aureus at OD600 nm over 10 hours; and C is the growth curve of Streptococcus mutans at OD600 nm over 10 hours. Figure 12 This is the live / dead staining of bacteria after co-incubation for 24 hours (green represents live bacteria, and red represents dead bacteria). Figure 13 These are the results of HUVEC cell viability assays; where A represents the HUVEC cell viability in each group of extracts after 3 and 5 days; B represents the fluorescence staining image of HUVEC cell morphology after 3 days of culture (green / blue: cytoskeleton / nucleus); and C represents the live / dead staining of HUVEC cells after 3 days (green / red: live cells / dead cells). Figure 14 The results are the in vitro angiogenesis performance evaluation results; where A is an angiogenesis image at 6 h; B is the migration image of HUVEC cells at 0 h and 24 h; and C is the expression of related genes of HUVECs after 7 d. Figure 15 These are the results of the MC3T3-E1 cell viability test; Figure 16Images show the morphology of MC3T3-E1 cells and the results of live / dead staining. Among them, A shows representative morphological images of MC3T3-E1 cells at 3 days and 5 days (green / blue: cytoskeleton / nucleus) and B shows the live / dead staining of MC3T3-E1 cells at 3 days (green / red: live cells / dead cells). Figure 17 These are ALP and ARS staining images of samples without EDTA chelating agent; Figure 18 These are the results of ALP activity and extracellular matrix mineralization detection in MC3T3-E1 cells; where A is an image of ALP activity and mineralization staining; B is the quantitative analysis result of ALP at 7 days; and C is the quantitative analysis result of mineralization at 14 days. Figure 19 The results show the expression of osteogenic-related genes in MC3T3-E1 cells after 14 days. Figure 20 This is a schematic diagram illustrating the antibacterial / osteogenic / angiogenic properties of zirconium oxide phosphorylation treatment and hydrothermal treatment loading of metal ions. Detailed Implementation

[0017] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application. In the description of this specification, the reference to terms such as "some embodiments," "examples," "specific examples," etc., means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Moreover, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in one or more embodiments or examples.

[0018] This invention provides a method for preparing a zirconia-based medical prosthesis material, comprising the following steps: S1, Pretreatment: First, the zirconia preform is sintered at high temperature, then polished and cleaned, and then sintered at high temperature and cleaned again. S2, Preparation of surface drug-loaded structure: A honeycomb porous nanostructure for drug loading was prepared on the surface of a zirconia preform by phosphorylation treatment; S3, Loading of metal ions: The zirconia material treated in step S2 is placed in a loading solution containing metal ions and reacted in a shaker at 50~90℃ for 5~8h. After the reaction is completed, it is taken out, rinsed and dried to obtain zirconia-based medical prosthesis material.

[0019] In the surface modification process of the aforementioned zirconia-based medical prosthesis material, the zirconia preform is first densified through a first high-temperature sintering process. This allows for full bonding between zirconia particles, reduces porosity, and forms a matrix with certain strength and structural stability, providing a basic structure for subsequent grinding, polishing, and secondary sintering. A second high-temperature sintering process further improves the density and structural uniformity of the zirconia preform, eliminating any micropores and structural defects that may exist after the first sintering. Simultaneously, it enhances the material's mechanical properties, such as hardness, strength, and toughness, to meet the requirements of medical prostheses used in the complex mechanical environment within the human body. After the second high-temperature sintering, the microstructure of the zirconia material is more complete, and the grain size is more uniform, resulting in superior wear resistance, corrosion resistance, and biocompatibility. This ensures that the prosthesis will not experience wear or corrosion due to insufficient material performance after long-term implantation, preventing problems such as inflammation and prosthesis failure. It also avoids the problem of excessive grain growth caused by prolonged temperature during a single sintering process, which can lead to structural inhomogeneity and difficulty in eliminating localized porosity, affecting the material's mechanical properties and subsequent processability. Meanwhile, good structural uniformity is also beneficial for the subsequent preparation of surface drug-loaded structures and loading of metal ions, ensuring that these functional structures can be uniformly and stably attached to the material surface.

[0020] Furthermore, the two high-temperature sintering processes endow the zirconia preform with high density and structural stability. During the subsequent phosphorylation treatment to prepare honeycomb porous nanostructures, the material does not deform or crack during the hydrothermal reaction due to its porous structure, ensuring the feasibility and structural integrity of the drug-loaded structure. Simultaneously, the more uniform and smoother surface of the material after the second sintering provides a good substrate for the uniform growth of porous nanostructures during the hydrothermal reaction. This facilitates the formation of a uniformly distributed and regularly morphologically regular honeycomb porous structure, ensuring the consistency of the drug-loading space and drug loading efficiency. It also facilitates the uniform adsorption and loading of metal ions within the pores, avoiding uneven metal ion loading caused by matrix structural defects.

[0021] More importantly, during the two high-temperature sintering processes mentioned above, the atomic bonds on the zirconium oxide surface undergo rearrangement, forming more unsaturated bonds, lattice defects, and active functional groups, such as hydroxyl groups and oxygen vacancies. These active sites provide stronger chemical bonding sites for metal ions. For example, metal ions can form coordination bonds with hydroxyl groups on the zirconium oxide surface or embed themselves into the crystal structure through lattice defects, thereby enhancing the bonding force between metal ions and the matrix and reducing the risk of their detachment from the bulk environment. After the two sintering processes, the surface activity and interfacial bonding force of the zirconium oxide surface are significantly improved. According to the principles of interfacial chemistry, the higher the surface energy, the stronger the interfacial bonding force between materials. Therefore, the interfacial bonding energy between metal ions and the zirconium oxide surface is increased, and the interaction between the two, such as electrostatic adsorption and chemical bonding, is stronger. This allows metal ions to remain stably attached even under physiological environments, such as the flushing of body fluids and the mechanical action of cells. Once the metal ions are firmly bonded to the zirconium oxide matrix, they will not be rapidly lost due to dissolution or metabolism in body fluids. They can continuously release metal ions for a longer period of time, effectively inhibiting the colonization and reproduction of bacteria around the prosthesis, reducing the risk of infection, or continuously stimulating the proliferation of vascular endothelial cells and the differentiation of osteoblasts. This maintains the long-term integration of the prosthesis with the host bone, improving the biomechanical stability and service life of the prosthesis.

[0022] Building upon this foundation, the present invention further incorporates a grinding and polishing process and a cleaning process between the first and second high-temperature sintering. This effectively removes roughness and residual impurities from the surface after the first sintering, resulting in a smoother and cleaner surface after the second sintering. This provides a more uniform reaction substrate for the subsequent phosphorylation treatment to prepare honeycomb porous nanostructures, ensuring the consistency of the morphology and distribution of the drug-loaded structure.

[0023] As a preferred example of the present invention, the first high-temperature sintering process is as follows: First, the temperature is raised to 800-1000°C at a rate of 5-8°C / min and held for 40-60 minutes. Then, the temperature is raised to 1350-1450°C at a rate of 2-4°C / min and held for 100-140 minutes. After the holding period, the temperature is cooled to room temperature with the furnace.

[0024] As a preferred example of the present invention, the temperature is first raised to 1450-1650°C at a rate of 3-5°C / min and held for 160-200min. After the holding period, the temperature is cooled to room temperature with the furnace.

[0025] In the two high-temperature sintering processes of this invention, the first high-temperature sintering process is mainly used to remove organic matter, such as binders and moisture, from the green body, allowing the zirconia particles to initially bond and obtain a green body with a certain strength and density, providing a structural basis for subsequent grinding and polishing. Its heating rate is moderately fast, thus balancing degreasing efficiency and green body integrity. Simultaneously, in the first holding stage, the holding time is extended to 40-60 minutes to allow for sufficient decomposition and removal of organic matter. Furthermore, in the second holding stage, the heating rate is slowed down to avoid excessive internal stress caused by rapid heating, and the final sintering temperature is limited to prevent excessive grain growth. In the second high-temperature sintering process, the heating rate is appropriately slowed down to ensure that the material's internal and external surfaces reach the sintering temperature simultaneously, avoiding the generation of new defects. The final sintering temperature is set higher than or equal to the final temperature of the first sintering to achieve further densification. At the same time, the holding time in the second high-temperature sintering is sufficiently long to provide ample time for grain growth and porosity elimination.

[0026] As a preferred example of the present invention, in step S1, the cleaning process is to perform ultrasonic cleaning with anhydrous ethanol and ultrapure water for 10-20 minutes in sequence.

[0027] As a preferred example of the present invention, in step S1, the secondary cleaning process involves ultrasonic cleaning with anhydrous ethanol and ultrapure water for 10-20 minutes in sequence, and after the secondary cleaning, the zirconia preform is dried by nitrogen gas flow and stored in a vacuum tank for later use.

[0028] As a preferred example of the present invention, the phosphorylation process in step S2 is as follows: The pretreated zirconia preform was placed in a high-pressure reactor and a 2-3 wt% phosphoric acid solution was added. The sealed high-pressure reactor was then placed in a drying oven and allowed to stand at a constant temperature of 150-165℃ for 20-28 hours.

[0029] During the phosphorylation treatment, the amount of phosphoric acid solution added is ≥5 mL / cm², based on the surface area of ​​the zirconia preform. 2 .

[0030] More preferably, in step S2, the amount of phosphoric acid solution added is 10~30 mL / cm², based on the surface area of ​​the zirconia preform. 2 .

[0031] It should be noted that when the shape of the zirconia preform is special and the above-mentioned amount of phosphoric acid solution cannot completely cover the surface of the zirconia preform, the amount of phosphoric acid solution added can be appropriately increased to ensure that the phosphoric acid solution can completely cover the surface of the zirconia preform.

[0032] Phosphorylation treatment can form a honeycomb porous nanostructure for drug loading on the surface of zirconia preforms. Compared to smooth surfaces, the specific surface area of ​​the honeycomb porous structure is significantly increased, and the pore walls will form a large number of hydroxyl groups due to acid etching. These hydroxyl groups can react with metal ions, such as Ag. + Cu 2+ 、Sr 2+ Coordination bonds or electrostatic interactions are formed, allowing metal ions to be firmly adsorbed within the pores, preventing them from falling off during subsequent use. Simultaneously, the mild hydrothermal reaction employed in this invention does not damage the dense structure of the zirconia matrix; the porous layer is tightly bonded to the matrix. Even after subsequent loading of metal ions or drugs, the structure can still withstand the mechanical environment in vivo, such as chewing and movement, ensuring long-term stable function.

[0033] As a preferred example of the present invention, in step S3, the metal ion is selected from Ag. + Cu 2+ 、Sr 2+ One or more of them.

[0034] As a preferred example of the present invention, in step S3, a soluble nitrate or chloride of the corresponding metal ion is dissolved in deionized water to prepare the desired loading solution containing metal ions.

[0035] As a preferred example of the present invention, silver nitrate, copper nitrate, and strontium chloride are used to prepare solutions containing Ag. + Cu 2+ 、Sr 2+ The loading solution contains silver nitrate at a concentration of 10–50 μM, copper nitrate at a concentration of 100–400 mM, and strontium chloride at a concentration of 0.5–3 M.

[0036] As a preferred example of the present invention, in step S3, Sr is loaded onto the surface of the zirconium oxide material. 2+ During ionization, the phosphorylated prosthetic material can first be placed in a 0.3~0.7M EDTA solution and reacted in a shaker at 30~45℃ for 2.5~3.5h. After ultrasonic cleaning, it can be placed in a strontium chloride solution and reacted in a shaker at 70~90℃ for 20~50min. Afterwards, the obtained prosthetic material is ultrasonically washed with ultrapure water and then dried in a drying oven for later use.

[0037] As a preferred example of the present invention, in step S3, the amount of loading liquid added is preferably sufficient to completely submerge the prosthetic material.

[0038] As a preferred example of the present invention, in step S3, the amount of loading liquid added is ≥10 mL / cm², based on the surface area of ​​the zirconia preform. 2 .

[0039] As a preferred example of the present invention, in step S3, the molar ratio of EDTA to strontium chloride is 1:2.

[0040] As a preferred example of the present invention, in step S3, the rinsing and drying process is as follows: the prosthesis material is rinsed with deionized water, anhydrous alcohol and deionized water in sequence to remove free ions, and then the prosthesis material is placed in a vacuum oven to dry at 32~45°C and stored in a vacuum tank for later use.

[0041] Preferably, the zirconia preform of the present invention is prepared using yttrium-stabilized zirconia.

[0042] In this invention, a honeycomb-like structure (ZrP) is first formed on the surface of zirconia material through phosphorylation treatment. Then, active metal ions are loaded onto the ZrP surface via a hydrothermal method, endowing it with osteogenic differentiation-regulating and antibacterial activities. By growing a stable, colorless metal ion coating in situ on the surface of the zirconia material, not only are the poor wear resistance issues of PLGA, the poor bonding issues of calcium phosphate coatings, and the color issues of dopamine coatings avoided, but the sustained release of metal ions is also achieved, thereby more effectively promoting osseointegration. Furthermore, the colorless nature of the metal ion coating makes the modified zirconia material more aesthetically pleasing in applications such as the oral cavity, meeting the requirements for the appearance of prosthetic materials in clinical applications.

[0043] The following specific examples illustrate the zirconia-based medical prosthetic material and its preparation method according to the present invention: Example 1: Commercial yttrium stabilized zirconia blocks were cut into sheet samples measuring 10 mm × 10 mm × 1.8 mm and subjected to a first high-temperature sintering process. The first high-temperature sintering process was as follows: first, the temperature was increased to 900℃ at a rate of 6℃ / min and held for 50 min; then, the temperature was increased to 1400℃ at a rate of 3℃ / min and held for 120 min; after the holding period, the temperature was cooled to room temperature with the furnace. The zirconia sheets were then polished with 400 grit, 800 grit, 1000 grit, and 2000 grit sandpaper, respectively. The polished zirconia sheets were then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 15 min to remove surface organic contaminants and impurities. A second high-temperature sintering process was then performed in a high-temperature crystallization furnace. The second high-temperature sintering process was as follows: first, the temperature was increased to 1600℃ at a rate of 4℃ / min and held for 180 min; after the holding period, the temperature was cooled to room temperature with the furnace, yielding 8... Samples measuring 8 mm × 1.5 mm, conforming to the dimensions of the bottom of a 48-well plate, were ultrasonically cleaned again for 15 min each with anhydrous ethanol and ultrapure water to remove surface organic contaminants and impurities. Finally, they were dried with nitrogen and stored in a vacuum chamber for later use. The cleaned and dried zirconium sheets were placed in a 50 mL high-pressure reactor, and 25 mL of a 2.5 wt% phosphoric acid solution was added. The reactor was then reacted at 160 °C for 24 h in a drying oven to obtain phosphorylated zirconium sheets (denoted as ZrP).

[0044] Example 2: Take the phosphorylated zirconia sheet prepared in Example 1 above, and prepare silver nitrate solutions of different concentrations. Use a pipette to repeatedly blow and stir to fully disperse and dissolve the silver nitrate solution. Then, transfer the phosphorylated zirconia sheet and 30 mL of silver nitrate solution to a tetrafluoroethylene-lined reactor and react at 60°C in a shaker at 100 rpm for 6 hours. After the reaction is completed, take out the sample, rinse it with deionized water, anhydrous alcohol and deionized water in sequence to remove free ions, dry it in a vacuum oven at 37°C, and store it in a vacuum container for later use. Name the samples according to the concentration of silver nitrate solution used for impregnation, and label them as Ag10@ZrP group (10 μM silver nitrate solution), Ag20@ZrP group (20 μM silver nitrate solution), and Ag40@ZrP group (40 μM silver nitrate solution).

[0045] Example 3: The phosphorylated zirconia sheet prepared in Example 1 was taken, and then 30 mL of silver nitrate and copper nitrate solution was applied to the zirconia sheet and subjected to the same hydrothermal treatment as in Example 2 to obtain a sample. The obtained sample was ultrasonically washed with ultrapure water for 30 seconds and then dried in a drying oven. The obtained samples were labeled according to different salt solution concentrations as: Cu100@ZrP group (100 mM copper nitrate solution), Cu200@ZrP group (200 mM copper nitrate solution), and Cu400@ZrP group (400 mM copper nitrate solution).

[0046] Example 4: The phosphorylated zirconia sheet prepared in Example 1 was taken, and then the phosphorylated sample was reacted with 0.5M EDTA solution in a shaker at 100 rpm at 37°C for 3 h. After ultrasonic cleaning for 30 s, it was reacted with 30 mL of strontium chloride solutions of different concentrations in a shaker at 80°C at 100 rpm for 30 min. The resulting sample was ultrasonically washed with ultrapure water and dried in a drying oven. The molar ratio of EDTA to strontium chloride was 1:2. The obtained samples were labeled according to different salt solution concentrations: Sr1000@ZrP-E group (1M strontium chloride solution), Sr2000@ZrP-E group (2M strontium chloride solution).

[0047] Experimental Example 1 1.1 Ag loading on zirconia surface + Ca 2+ 、Sr 2+ Morphological evaluation of ionic honeycomb structures Figure 1Representative SEM images of samples ZrO2, ZrP, Ag20@ZrP, Cu200@ZrP, and Sr2000@ZrP-E are presented. The SEM images show that the ZrP group samples exhibit numerous honeycomb-like pores of varying sizes on their surface, with a relatively large pore size range of approximately 50–500 nm. The honeycomb structure on the Ag20@ZrP group sample surface changes, with dot-like substances surrounding the pores, and the ridged protrusions showing a tendency to thicken. The Cu200@ZrP group sample surface shows no significant difference compared to the ZrP group. The Sr2000@ZrP-E group sample surface appears to be covered by other substances, and the honeycomb pores are filled.

[0048] 1.2 Ag loading on zirconia surface + Ca 2+ 、Sr 2+ Chemical composition and chemical structure analysis of ionic honeycomb structures Figure 2 The surface elemental distribution analysis results of the samples prepared in this invention are shown below, where the main elemental composition and distribution of each group of samples are as follows: Figure 2 As shown in Figure A, the main constituent elements of the Ag20@ZrP, Cu200@ZrP, and Sr2000@ZrP-E groups are Zr, P, and O. Among them, Ag element was observed to be uniformly distributed on the surface of the Ag20@ZrP group, with a content of about 6.2%; Cu element was uniformly distributed on the sample surface of the Cu200@ZrP group, with a content of about 2.8%; and Sr element was uniformly distributed on the surface of the Sr2000@ZrP-E group, with a content of about 1.5%.

[0049] The full XPS spectra of each sample are as follows: Figure 2 In Figure B, the Ag20@ZrP group exhibited abnormal fluctuations in the 380eV-365eV range, detecting a strong Ag3d signal; the Cu200@ZrP group exhibited abnormal fluctuations in the 970eV-930eV range, detecting a strong Cu2p signal; and the Sr2000@ZrP-E group exhibited abnormal fluctuations in the 142eV-128eV range, detecting a strong Sr3d signal.

[0050] Figure 3 This is a peak fitting diagram of Ag, Cu, and Sr in the sample prepared by this invention. Figure 3 Figures C, D, and E show the peak fitting diagrams for Ag, Cu, and Sr, respectively. The Ag 3d peaks appear at binding energies of approximately 368.3 eV and 374.4 eV, representing Ag3d5 / 2 and Ag3d3 / 2 signals, respectively, and the presence of Ag2O is analyzed. The Cu 2p peaks appear at binding energies of approximately 955.1 eV and 952.8 eV, representing Cu2O signals. 2+ 2p signal, with a small amount of Cu +The presence of 2p indicates the presence of Cu metal oxides. The Sr 3d peaks appear at binding energies of approximately 133.6 eV and 135.6 eV, representing Sr 3d 3 / 2 and Sr 3d 5 / 2 signals, respectively, indicating the presence of Sr in oxide and ionic forms.

[0051] 1.3 Analysis of Ion Release Behavior One sample each of Ag20@ZrP, Cu200@ZrP, and Sr2000@ZrP-E was selected and immersed in 5 mL of PBS solution. The samples were removed at 1, 3, 7, and 14 days and placed in fresh PBS solution. The cumulative release of metal ions from the sample coatings was measured using ICP-OES. The results are detailed below. Figure 4 .

[0052] from Figure 4 As shown in Figures A, B, and C, Ag in Ag20@ZrP exhibits a high release rate on day 7, gradually slowing down thereafter, and showing a tendency to continue releasing even after day 14, before reaching its maximum release amount. A similar trend is observed in the Cu200@ZrP group. The Sr2000@ZrP-E group, however, exhibits explosive release, with the highest release amount and rate on day 1, followed by a slowdown and almost no release after day 1. Based on the cumulative release curves, Ag... 2+ The cumulative release concentration on day 14 was approximately 1.2 ppm; Cu 2+ The cumulative release concentration on day 14 was approximately 0.3 ppm; Sr 2+ The cumulative release concentration on day 14 was approximately 2.0 ppm.

[0053] 1.4 Water contact angle measurement results The smaller the water contact angle of a material, the greater the free energy of the material surface and the stronger its hydrophilicity. Figure 5 The results characterize the water contact angles of five material groups: ZrO2, ZrP, Ag20@ZrP, Cu200@ZrP, and Sr2000@ZrP-E. The average water contact angles for each group are approximately 61.6±3.4°, 34.1±2.8°, 32.0±4.4°, 41.2±4.7°, and 28.3±1.8°, respectively. This indicates a significant difference compared to zirconium oxide; the contact angles of all groups are smaller than those of the first group, ZrO2, indicating a significant improvement in hydrophilicity. However, there is no significant difference in hydrophilicity among the three experimental groups, and it is similar to that of the ZrP group. These results confirm that with the incorporation of metal elements, the water contact angles of each group do not decrease significantly relative to ZrP, and the hydrophilicity does not increase significantly.

[0054] 1.5 Analysis Conclusion: The above detection and analysis revealed that: (1) SEM analysis results show that after Ag + (Ag20@ZrP) and Cu 2+ The surface structure of the (Cu200@ZrP) doped sample did not show significant changes compared to the untreated phosphorylated sample (ZrP). This phenomenon may be attributed to the ion exchange characteristics of the honeycomb-like structure unique to the zirconium phosphate (ZrP) surface, which promotes metal ion doping without significantly altering the surface morphology. However, the Sr-doped sample... 2+ The (Sr2000@ZrP-E) doped sample exhibits different properties, with its surface pores appearing to be filled. This is likely due to the stable binding of EDTA to the honeycomb structure and its interaction with Sr. 2+ This chelation process leads to the filling of surface pores. The material's high surface hydrophilicity may facilitate protein adsorption and cell adhesion, thereby promoting bone tissue integration.

[0055] (2) The water contact angle test results further confirmed that the four samples ZrP, Ag20@ZrP, Cu200@ZrP and Sr2000@ZrP-E all showed high hydrophilicity, which helps cell adhesion and protein adsorption, providing an important physical basis for bone tissue engineering applications.

[0056] (3) Figure 4 Ag was displayed + The release rate gradually increased with soaking time, stabilizing after day 14. According to the World Health Organization (WHO) and other authoritative organizations, the release rate of Ag is 0.5–1.0 ppm. + The dilution concentration was sufficient to kill most bacteria and viruses. The Ag obtained in this study... + The release concentration reached 1.2 ppm, which is in line with international standards, and persisted for 14 days, indicating that Ag... + The release rate reached the expected concentration, exhibiting sustained release characteristics, indicating its potential application effects. In this invention, the Cu200@ZrP sample showed Cu... 2+ The rapid release followed by a gradual decrease in the release rate, culminating in a cumulative release concentration of 0.3 ppm by 14 days, consistent with previous studies on Cu. 2+ The concentrations that promote the proliferation, angiogenesis, and migration of human umbilical vein endothelial cells are similar. Further research indicates that appropriate concentrations of Sr... 2+ It is beneficial to cell proliferation, but concentrations exceeding 1400 ng / mL may have an adverse effect on osteoblast proliferation. In this invention, ICP measurements of the Sr2000@ZrP-E group showed that on day 1, Sr... 2+The release rate was highest, with a cumulative release of approximately 2 mg / L by day 21. Although this exceeded the maximum concentration for promoting cell proliferation, considering the body's fluid circulation and various metabolic reactions, it could relatively reduce Sr. 2+ Sr accumulates in the body, and in in vitro experiments, regular changes of the experimental fluid can reduce its accumulation. 2+ Accumulation in the experimental environment, thus Sr 2+ It may still have a sustained positive effect on osteoblasts.

[0057] Experimental Example 2: Ag loaded onto a honeycomb-like structure on a zirconia surface + Evaluation of post-biocompatibility and antibacterial properties 2.1 In vitro cell compatibility assessment of materials 2.1.1 MC3T3-E1 cell culture The time-dependent osteogenic differentiation of mouse embryonic osteoblast precursor cells (MC3T3-E1) is consistent with in vivo osteogenic differentiation, making it a classic in vitro osteogenic model. This experiment used MC3T3-E1 to investigate the effect of surface-modified zirconia implants on osteogenic development. The culture medium used for MC3T3-E1 was α-MEM containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin mixture. Cells were cultured in a 5% CO2 incubator. When the cells reached 80-90% confluence, the original culture medium was discarded, and the cells were washed twice with sterile PBS. 2 mL of preheated trypsin (37°C) was added, and the cells were digested in an incubator. Under a microscope, when the cells showed a shrunken, rounded shape, the trypsin was removed, 3 mL of complete culture medium was added to stop digestion, and the cells were detached from the culture flask using a Pasteur pipette. Fresh culture medium and an appropriate amount (approximately 1 / 3) of cell suspension were added to the culture flask for continued culture, or the cell suspension was added to well plates for subsequent experiments as required. Replace the culture medium in the culture flasks and well plates with the appropriate medium every 2-3 days until the cells reach confluence or the required time for further processing.

[0058] 2.1.2 MC3T3-E1 cell viability assay CCK-8 can be used for simple and accurate cell proliferation and toxicity analysis. Pre-culture the culture plates in an incubator for a period of time (37 ℃, 5% CO2), then seed two 48-well plates with a cell count of 8 × 10⁶ cells / well. 3Three replicates were performed per well to prepare for cell viability assays on days 3 and 5. On day 3, at the specified detection time point, the original culture medium was discarded, and 200 μL of serum-free culture medium containing 10% CCK-8 solution was added and incubated at 37 ℃ for 2 h. The official instructions recommend incubating the culture plate in the incubator for 1-4 h. Because the degree of color development varies depending on the cell type, if the color development is insufficient, the incubation time can be appropriately extended; in this cell assay, the incubation time is 2 h. The absorbance of the solution at 450 nm was then measured using a microplate reader. The procedure for day 5 was the same.

[0059] 2.1.3 Morphology of MC3T3-E1 cells After complete sterilization, the samples were placed in 48-well plates, with MC3T3-E1 cells at a rate of 4 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well on the sample surface and incubated at 37 ℃ under 5% CO2 conditions, with the culture medium changed every 2 days. After 3 days, the culture medium was discarded, and the cells were washed 2-3 times with PBS solution. 200 μL of 4% tissue cell fixative was added to each well and incubated for 40 min. The fixative was then discarded, and the cells were washed 2-3 times with PBS solution. 200 μL of 0.1% Triton X-100 was added to each well and incubated for 10 min to increase cell membrane permeability. After PBS washing, the cells were incubated with FITC-labeled phalloidin (100 nM) and DAPI (10 μg / mL) in the dark for 60 min and 10 min, respectively, to stain the cytoskeleton and nucleus. Finally, five fields of view were randomly selected using an upright fluorescence microscope to capture 20x fluorescence morphology images of the cells, where green fluorescence represents the cytoskeleton and blue fluorescence represents the nucleus.

[0060] 2.1.4 MC3T3-E1 cell live / dead staining assay After removing the old culture medium from MC3T3-E1 cells, wash 2-3 times with sterile PBS, digest with trypsin for 2 min, neutralize with fresh complete culture medium, pipette and collect the cells, centrifuge at 1000 rpm for 5 min, resuspend the cells, and then centrifuge at 1×10⁻⁶ cells / min. 4 Cells were seeded at a density of [number] cells / well into 48-well plates and co-cultured with sterilized samples. The plates were incubated at 37°C in a 5% CO2 incubator, with the conditioned medium changed every other day. After 5 days of culture, the medium was aspirated, and the cells were gently washed twice with sterile PBS. 200 μL of cell live / dead cell staining working solution was added to each well (prepared in advance according to the manufacturer's instructions). The cells were incubated at 37°C for 10 min, the working solution was aspirated, and the cells were washed twice with PBS. Cells were then observed using an inverted fluorescence microscope. The staining process and fluorescence imaging were performed in the dark.

[0061] 2.2 Evaluation of the in vitro antibacterial properties of the material 2.2.1 Bacterial Culture 2.2.1.1 Bacterial cryopreservation Under aseptic conditions, add 500 μL of bacterial suspension and 500 μL of 50% glycerol solution to each of the sterilized 2 mL cryovials, mix gently, and the glycerol-freezing bacteria can be stored directly at -80°C. Sometimes, in order to reduce the death of bacteria due to ice crystal formation, liquid nitrogen is used for quick freezing before storing in a -80°C freezer.

[0062] 2.2.1.2 Bacterial resuscitation Prepare a suitable culture medium for the growth of the strain, restore the strain from its storage state to room temperature, select robust colonies from the culture medium, and inoculate a portion of these colonies into a new culture medium. Repeat this step 2-3 times to obtain well-grown colonies.

[0063] 2.2.1.3 Preparation of bacterial culture medium and agar plates Dissolve 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride in 1 L of ultrapure water. Adjust the pH to neutral with sodium hydroxide and autoclave to prepare Luria-Bertani medium (LB medium). LB medium is suitable for culturing aerobic bacteria such as Staphylococcus aureus and Escherichia coli. BHI medium is suitable for culturing facultative anaerobic bacteria such as Streptococcus mutans. Brain-Heart Broth (BHI) medium preparation method: tryptone, ox heart extract glucose, sodium chloride, and disodium hydrogen phosphate. Usage: Weigh 37 g of this product, add 1 L of distilled or deionized water, stir and heat to boiling until completely dissolved, dispense into test tubes, and sterilize at 121℃ for 15 min. The agar plate is prepared using LB medium or BHI medium, with an additional 18 g of agar powder added. After autoclaving at 121 °C for 30 min, the plate is poured out after the temperature drops to 60 °C. Once the agar plate has solidified, the edges of the petri dish are sealed and stored in a low-temperature refrigerator for later use.

[0064] 2.2.2 Antibacterial rate experiment Take fresh test bacteria (24 h old) and dilute with PBS to approximately 1×10⁻⁶. 5CFU / mL is prepared for use. Test samples and control samples are sterilized in prepared 75% alcohol. Take a sterile plate, use sterile forceps to place the test sample into the plate, and add 0.1 mL of the test bacterial suspension and 0.9 mL of LB liquid medium to each sample, starting the timer immediately. After 24 hours of contact between the test bacteria and the sample, add 100 μL of the co-culture solution to a 0.9 mL PBS tube and mix well. Determine the number of viable bacteria using the viable bacteria counting method, inoculating two plates for each sample. If the number of colonies growing on the plate is high, a 10-fold serial dilution can be performed before viable bacteria counting. The antibacterial rate is determined by imaging and counting CFU.

[0065] 2.2.3 Antibacterial ring test The size of the inhibition zone represents the antibacterial activity of a drug or material. Within a certain drug concentration range, the higher the drug concentration, the larger the diameter of the inhibition zone. Gradient concentration tests were conducted on the bacterial count in the test plates to determine the optimal concentration (10). 6 The optimal viable cell concentration is determined by the following steps: First, pour approximately 20 mL of culture medium into a sterilized agar plate, allow it to solidify horizontally, and then inoculate with 0.1 mL of 10... 7 Prepare the bacterial suspension by spreading it evenly. Sterilize the sample by inverting it onto an agar plate and marking it. Then, invert the plate and incubate it in a bacterial incubator for 24 hours. Take a picture the next day and measure the size of the inhibition zone with calipers.

[0066] 2.2.4 Bacterial Morphology For SEM observation, after culturing *S. aureus*, *E. coli*, and *S. mutans* for 24 h, the samples were gently washed three times with PBS, fixed overnight in 4% formaldehyde at 4 °C, washed 2–3 times with PBS, and dehydrated for 20 min at each concentration using a gradient of ethanol (30%, 50%, 70%, 90%, and 100%). Before SEM observation, the samples were sputtered onto a platinum target for 60 s.

[0067] 2.2.5 Turbidimetric method This method is used to help estimate the concentration of bacteria or other cells, or "cells per volume," in a liquid sample. It is commonly used in ELISA readers to estimate the concentration of bacteria or other cells in liquids because the 600 nm wavelength hardly damages or hinders their growth. (The last sentence appears to be incomplete and possibly refers to a different method.) 6 The bacteria were inoculated into a suitable liquid culture medium and cultured at a suitable temperature. Samples were taken at 0, 2, 4, 6, 8, and 10 h to measure the number of bacteria. The growth curves of the three bacteria were plotted with OD value on the ordinate and growth time on the abscissa.

[0068] 2.2.6 Staining of live and dead bacteria Bacterial live-dead staining techniques typically use fluorescent dyes or staining agents that interact with specific structures or compounds within bacterial cells to stain them. A common method is to use the fluorescent dyes SYTO-9 and PI for staining live and dead bacteria. SYTO-9 is a green fluorescent nucleic acid dye that can penetrate cell membranes and can label all bacteria in a colony—both with intact and damaged membranes. PI can only penetrate damaged membranes, but PI insertion causes a decrease in SYTO-9 staining fluorescence. Therefore, by using a moderate staining mixture of appropriate ratios of SYTO-9 and PI, bacteria with intact membrane structures will fluoresce green, while bacteria with damaged membrane structures will fluoresce red. The bacteria were incubated with different materials at 37°C for 24 h. After gentle pipetting, the bacterial solution was aspirated into 1.5 mL sterile EP tubes, centrifuged at 4000 rpm for 7 min, and the supernatant was removed. The live / dead staining solution was prepared using the LIVE / DEADBac-Ligh kit. 200 μL of staining solution was added to each EP tube, and the solution was pipetted evenly. The tubes were stained at room temperature in the dark for 20 min, then centrifuged. The tubes were then rinsed three times with deionized water to remove any residual staining agent. Finally, the bacterial solution was dropped onto a glass slide, covered with a coverslip, and observed and photographed using an inverted fluorescence microscope.

[0069] 2.3 Experimental Results 2.3.1 Results of in vitro cell compatibility assessment of the material 2.3.1.1 MC3T3-E1 cell viability MC3T3-E1 cell viability test results are as follows Figure 6 As shown. After 3 days of cultivation, Ag-doped cells can be observed. + The Ag10@ZrP and Ag20@ZrP groups with lower concentrations showed higher cell activity, while the Ag40@ZrP group with higher concentrations showed lower cell activity. Similarly, after 5 days of culture, all groups showed the same trend, with the test values ​​of each group increasing uniformly. The Ag40@ZrP group with higher concentrations showed a decreasing trend compared to other experimental groups, indicating reduced cell activity (P<0.05).

[0070] 3.1.2 Results of MC3T3-E1 cell morphology and cell viability assays After culturing MC3T3-E1 cells on different sample surfaces for 3 and 5 days, the cell morphology was as follows: Figure 7 As shown in Figure A, green and blue represent the cytoskeleton and nucleus, respectively; cells on the Ag40@ZrP sample surface showed varying degrees of atrophy. However, some cells on the ZrO2, ZrP, Ag10@ZrP, and Ag20@ZrP surfaces exhibited larger cell spreading areas. Cell density and viability were similar in the 3-day and 5-day cell morphology images, indicating that these coatings promoted cell adhesion. Figure 7 Analysis B revealed that on day 5, no significant dead cells were observed in MC3T3-E1 cells from the ZrO2, ZrP, Ag10@ZrP, and Ag20@ZrP groups. However, some red cells were observed in Ag40@ZrP cells, and the cell count was reduced. Green and red represent live and dead cells, respectively. In conclusion, Ag10@ZrP and Ag20@ZrP exhibited good biocompatibility, while Ag40@ZrP showed cytotoxicity.

[0071] 2.3.2 Results of in vitro antibacterial performance evaluation of the material 2.3.2.1 Antibacterial rate For detailed results of the antibacterial rate test, please refer to [link / reference]. Figure 8 In the figure, A represents the CFU image of the bacterial plating plate, and B represents the inhibition rate calculated after colony counting, with a confidence coefficient set at 95%. Agar plate analysis showed that the Ag20@ZrP and Ag40@ZrP groups had significantly fewer Staphylococcus aureus, Escherichia coli, and Streptococcus mutans samples than the other groups. The plating count images and related statistical results of each group of materials after 24 h of culture in Staphylococcus aureus, Escherichia coli, and Streptococcus mutans are shown.

[0072] In addition, such as Figure 8 As shown in Figure A, the bacterial colonies counted by centrifugation and plating of the Ag20@ZrP and Ag40@ZrP groups were significantly lower than those in the ZrO2 and ZrP groups, indicating that the number of the three types of bacteria adhering to the surface was the lowest, followed by Ag10@ZrP. Both ZrO2 and ZrP showed a relatively high number of bacteria adhering to their surfaces. Further calculations and analysis (see Figure A for details) are needed. Figure 8 In group B), the inhibition rates of Ag10@ZrP, Ag20@ZrP, and Ag40@ZrP against Escherichia coli, Staphylococcus aureus, and Streptococcus mutans were approximately 21%, 72%, 98%, 19%, 70%, 97%, 85%, 92%, and 90%, respectively (P<0.05). This demonstrates that the two higher concentration groups exhibited significant antibacterial effects.

[0073] 2.3.2.2 Antibacterial ring Figure 9 Images of inhibition zones and statistical results of inhibition zone diameter measurements are shown. In image A, the inhibition zone image is shown, and in image B, the inhibition zone diameter measurement results are shown. In image A, the group at the top is ZrO2, and the groups from left to right are ZrO2, ZrP, Ag10@ZrP, Ag20@ZrP, and Ag40@ZrP. Figure 9In the figure, B indicates that the inhibition zone diameters for Escherichia coli are approximately 0 mm, 0 mm, 2-2.5 mm, 3-3.5 mm, and 3.5-4 mm, respectively; for Staphylococcus aureus, the inhibition zone diameters are approximately 0 mm, 0 mm, 1-1.5 mm, 1.5-2 mm, and 2.5-3 mm, respectively; and for Streptococcus mutans, the inhibition zone diameters are approximately 0 mm, 0 mm, 2.5-3 mm, 3-3.5 mm, and 4-5 mm, respectively.

[0074] 2.3.2.3 Bacterial Morphology Previous experiments have shown that Ag10@ZrP has insufficient antibacterial properties and Ag40@ZrP has poor biocompatibility. Therefore, ZrO2, ZrP, and Ag20@ZrP are the experimental groups for SEM bacterial morphology. Figure 10 These are scanning electron microscope (SEM) images of bacteria, from... Figure 10 It is evident that the ZrO2 and ZrP groups exhibited a greater adhesion of Staphylococcus aureus, Escherichia coli, and Streptococcus mutans, with intact bacterial morphology, indicating a lack of antibacterial properties in these two groups. On the surface of the Ag20@ZrP group samples, the amount of bacterial adhesion was significantly reduced, and the bacteria shrank or cracked, showing significant morphological changes.

[0075] 2.3.2.4 Turbidimetric method The proliferation of Ag10@ZrP, Ag20@ZrP, and Ag40@ZrP samples within 12 h is as follows: Figure 11 As shown in Figures A, B, and C, A represents the growth curve of *Escherichia coli* at OD 600 nm within 10 hours; B represents the growth curve of *Staphylococcus aureus* at OD 600 nm within 10 hours; and C represents the growth curve of *Streptococcus mutans* at OD 600 nm within 10 hours.

[0076] Compared with the ZrO2 and ZrP groups, in the first 3 hours, there were no significant changes in the values ​​of Escherichia coli and Staphylococcus aureus in any group, while the Ag20@ZrP and Ag40@ZrP groups of Streptococcus mutans showed a slight increase, and the values ​​of ZrO2, ZrP, and Ag10@ZrP showed a larger increase. At 6 hours, the OD values ​​of the ZrO2, ZrP, and Ag10@ZrP groups of Escherichia coli and Staphylococcus aureus increased to 0.2, while the Ag20@ZrP and Ag40@ZrP groups remained at 0.03. In the Streptococcus mutans group, there was no significant difference in the ZrO2, ZrP, and Ag10@ZrP groups, while the value of the Ag20@ZrP group reached 0.07, and the Ag40@ZrP group still showed no significant increase. At 9 and 12 hours, significant differences were observed among the groups. The values ​​of ZrO2, ZrP, and Ag10@ZrP showed a linear increase, while Ag20@ZrP remained at a moderate level, falling between the other groups and Ag40@ZrP. The Ag40@ZrP group maintained a stable reading at OD 600 nm, approximately 0.03 for Escherichia coli and Staphylococcus aureus, and approximately 0.05 for Streptococcus mutans. Therefore, it can be concluded that Ag40@ZrP exhibited the most significant antibacterial activity, Ag20@ZrP had a moderate antibacterial effect, and Ag10@ZrP showed almost no antibacterial effect, similar to the control group.

[0077] 2.3.2.5 Staining of live and dead bacteria like Figure 12 As shown, after culturing samples with Escherichia coli, Staphylococcus aureus, and Streptococcus mutans for 24 h, inverted fluorescence microscopy revealed that the ZrO2 and ZrP groups had more green live bacteria and fewer red dead bacteria. The Ag10@ZrP group had a majority of live and dead bacteria, while the Ag20@ZrP and Ag40@ZrP groups showed a significant majority of red dead bacteria and very few live bacteria.

[0078] Experimental Example 3: Cu loaded onto a honeycomb-like structure on a zirconia surface 2+ Post-angiogenic performance evaluation 3.1 Experimental Methods 3.1.1 Preparation of extracts and in vitro biocompatibility assessment of materials 3.1.1.1 Preparation of Extract Extracts for each group of samples were prepared according to ISO 10993-12 standard. First, the samples were sterilized by immersion in 75% alcohol for 30 min, followed by infusion into DMEM serum-free medium at a depth of 1.25 cm⁻¹. 2 The sample was added in a volume of / mL to the well plate containing the sample, and then incubated in a 37℃ constant temperature incubator under 5% CO2 conditions for 72 h.

[0079] 3.1.1.2 HUVEC cell culture Cu 2+It plays an important role in the proliferation and differentiation of HUVECs and can promote angiogenesis. The culture and passage methods are the same as for osteoblasts.

[0080] 3.1.1.3 HUVEC cell viability HUVEC cells were planted at 8 × 10⁸ cells per well. 3 HUVEC cells were seeded per well in 48-well plates using a 10% FBS extract. Cell viability was assessed on the surface of each sample after 3 and 5 days of culture to evaluate the cell compatibility of the material. The specific procedure was the same as that for MC3T3-E cell viability testing.

[0081] 3.1.1.4 HUVEC cell morphology and live / dead staining experiments HUVEC cells were pre-seeded at a rate of 4 × 10⁶ cells per well. 3 HUVEC cells were seeded per well in 48-well plates using a medium containing 10% FBS. After 3 days, HUVEC cells on the surface of each group of zirconia cells were fluorescently stained and photographed under an inverted fluorescence microscope. The specific procedures were the same as those for morphology and viability assays of MC3T3-E cells.

[0082] 3.1.2 In vitro angiogenesis performance evaluation 3.1.2.1 HUVEC cell migration assay Cell migration assays were performed using a scratch assay. HUVEC cells were cultured at a concentration of 2 × 10⁻⁶ cells / year. 4 One day in advance, cells were seeded into 24-well plates at a density of [number] cells / well, in DMEM medium containing 10% FBS. Using a sterile 10 μL pipette tip, a cell-free area of ​​approximately uniform width, resembling a cross, was drawn at the bottom of the plate. The medium was discarded, and floating cells were washed twice with sterile PBS. Extraction medium still containing 1% FBS was added, and the cells were cultured further. Images of four fields of view around the cross were taken at 0 and 24 h using an inverted phase-contrast microscope. The final results were compared between the microscopic images and the newly covered area of ​​the blank area with the control group. The experiment was repeated three times.

[0083] 3.1.2.2 Angiogenesis Experiment 3.1.2.2.1 Subpackaging of the matrix adhesive: ① One day in advance, place the unopened, frozen matrix gel in a foam box with crushed ice and thaw it overnight at 4°C. ② One hour in advance, pre-cool the consumables used for dispensing the matrix gel (such as pipette tips and 1.5 mL EP tubes) in a -20°C environment. ③ Before dispensing, place the thawed matrix gel and pre-cooled consumables in a clean bench, and then place them on an ice box and ice plate that have been disinfected with alcohol. Ensure that the entire dispensing process is performed on ice. ④ Open the matrix gel packaging, being careful to hold the top of the packaging to avoid warming the gel at the bottom due to body heat. Dispense the required amount of matrix gel into EP tubes for each experiment, and quickly freeze them at -20°C for storage, avoiding repeated freeze-thaw cycles.

[0084] 3.1.2.2.2 Applying adhesive: ① The steps for melting the matrix gel overnight and pre-cooling consumables (including 96-well plates) are the same as above. ② Place an appropriate amount of melted matrix gel and pre-cooled consumables on an ice box sprayed with alcohol inside a clean bench. When mixing the diluted matrix gel by pipetting, be careful to avoid blowing in air bubbles. If air bubbles have formed, you can briefly centrifuge at a low speed. ③ Add 50 μL of matrix gel to each well of the pre-cooled 96-well plate, making sure the pipette tip is inserted directly to the bottom to avoid the matrix gel sticking to the side wall. Reverse pipetting can be used to avoid air bubbles in the matrix gel. ④ After adding the matrix gel, gently tap the 96-well plate to make the liquid surface even and level, and incubate in a 37°C cell culture incubator for 30 min to 1 h.

[0085] 3.1.2.2.3 Cell preparation: ① While waiting for the matrix gel to solidify, prepare the HUVEC cells. Digest the cells using trypsin digestion solution, centrifuge at 1000 rpm for 5 min, resuspend the cells, and count them. ② The cell quantity per well should be determined in advance according to needs. In this experiment, approximately 5.0 × 10⁶ cells per well of a 96-well plate was used. 4 ③ After the matrix gel solidifies, gently drop the prepared cell suspension onto each well of the matrix gel, being careful not to apply too much pressure and damage the smooth surface of the matrix gel. Note that HUVEC cells tend to settle easily; therefore, thoroughly agitate the cells before each addition to avoid significant differences in cell count between wells. ④ Place the 96-well plate in a cell culture incubator for angiogenesis experiments, and take photos after 2-4 hours.

[0086] 3.1.2.3 Expression of angiogenesis-related genes Gene expression levels were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).

[0087] HUVEC at 1×10 4Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in DMEM medium containing 10% FBS. After 7 days of culture, total RNA was extracted from the cells. The total RNA was obtained using an RNA extraction kit, and the RNA concentration was determined using a micro-spectrophotometer. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. Finally, the gene expression of VEGF, FGFR, and TIE was quantified using a quantitative real-time polymerase chain reaction (qPCR) analyzer with a dye-based real-time fluorescence quantitative PCR kit.

[0088] 3.2 Experimental Results 3.2.1 Results of in vitro biocompatibility assessment of materials 3.2.1.1 HUVEC cell viability HUVEC cell viability assay results are as follows: Figure 13 As shown, A represents the HUVEC cell viability in the extracts after 3 and 5 days; B represents the fluorescence staining image of HUVEC cell morphology after 3 days of culture (green / blue: cytoskeleton / nucleus); C represents the live / dead staining of HUVEC cells after 3 days (green / red: live / dead cells). After 3 days of culture, Cu-doped cells can be observed. 2+ The lower concentration Cu100@ZrP and Cu200@ZrP groups showed higher cell activity, while the higher concentration Cu400@ZrP group showed lower cell activity. Similarly, after 5 days of culture, all groups showed the same trend, with the test values ​​of each group increasing uniformly. Likewise, the higher concentration Cu400@ZrP group showed a decreasing trend compared to other experimental groups, indicating lower cell activity.

[0089] 3.2.1.2 Results of HUVEC cell morphology and live / dead staining assays To further characterize the state of cells after co-culturing with sample extract, the cells were stained with fluorescence and observed. The results are as follows: Figure 13 As shown in Figure B, after 3 days of co-culturing cells with the sample, the cell morphology did not change significantly. The Cu100@ZrP and Cu200@ZrP groups still exhibited extended structures with a large cell spreading area, while the Cu400@ZrP group showed a decrease in cell number and varying degrees of cell shrinkage in the image field of view. Figure 13 As shown in Figure C, no obvious dead cells were observed in the ZrO2 group, ZrP group, Cu100@ZrP, and Cu200@ZrP. Some red dead cells were observed in Cu400@ZrP, and the number of cells was significantly reduced. Therefore, Cu400@ZrP was not used as the subsequent experimental group.

[0090] 3.2.2 Results of in vitro angiogenesis performance evaluation 3.2.2.1 HUVEC cell migration results Cell migration is an important step in angiogenesis. This experiment uses a scratch assay to verify the effect of copper-doped zirconia implants on cell migration. Figure 14 The results are the in vitro angiogenesis performance evaluation results. Among them, A is the angiogenesis image at 6 h; B is the migration image of HUVEC cells at 0 h and 24 h; and C is the related gene expression of HUVECs after 7 d.

[0091] Figure 14 Image B shows images of cells around the scratch at 0 h and 24 h. After 24 h, cells migrated into the cell-free zone within the scratch. The migration areas, arranged from largest to smallest, were: Cu200@ZrP group, Cu100@ZrP group, ZrP group, and ZrO2 group. The edges of the cell-free zones in the ZrO2 and ZrP groups remained relatively clear. The Cu200@ZrP group showed the most significant migration trend towards the central cell-free zone, with the narrowest cell-free zone. The data indicate that the Cu100@ZrP and Cu200@ZrP groups were more conducive to endothelial cell migration than the ZrO2 group.

[0092] 3.2.2.2 Results of angiogenesis experiment Angiogenesis assays are a method for rapidly measuring angiogenesis capacity in vitro. Endothelial cells connect to form lines, and the nodes at their ends represent typical early stages of angiogenesis. In the middle and later stages, the vascular branches formed by endothelial cells cross-link to form a vascular network structure; the points where these branches cross-link are called junctions. Figure 14 As shown in A, after incubation in the extracts of each group for 4 h, obvious endothelial cell nodes and tubular branching junctions were observed in the Cu100@ZrP and Cu200@ZrP groups, and the cell junctions initially showed tubular structures. In the ZrO2 and ZrP groups, only simple short lines and circular structures formed by cell junctions were observed.

[0093] 3.2.2.3 PCR Experiment Results The expression levels of VEGF, FGFR, and TIE angiogenesis-related genes are as follows: Figure 14 As shown in Figure C, VRGF expression was highest in the Cu200@ZrP group after 7 days of culture, while VEG expression levels were significantly reduced in the ZrO2 and ZrP groups. The Cu200@ZrP group exhibited extremely high TIE expression after 7 days of culture. In addition, the Cu100@ZrP and Cu200@ZrP groups showed a stable increase in FGFR expression levels compared to the ZrO2 and ZrP groups.

[0094] Experimental Example 4: Sr loaded onto a honeycomb-like structure on a zirconia surface 2+ Post-osteogenic performance assessment 4.1 Experimental Methods 4.1.1 In vitro biocompatibility assessment of materials 4.1.1.1 MC3T3-E cell viability With 1×10 per hole 4 MC3T3-E cells were seeded onto the surfaces of each group of materials at a density of 10% FBS and cultured in α-MEM medium containing a mixture of 10% FBS and 1% antibiotics. Cell viability of MC3T3 cells on the zirconia surfaces of each group was assessed after 3 and 5 days of culture to evaluate the cell compatibility of the materials. Other procedures were the same as above.

[0095] 4.1.1.2 Morphology and live / dead staining experiments of MC3T3-E1 cells MC3T3-E cells were pre-seeded at a rate of 1 × 10⁶ cells per well. 4 HUVEC cells were seeded per well in 48-well plates using a medium containing 10% FBS. After 3 days, HUVEC cells on the surface of each group of zirconia were fluorescently stained and photographed under an inverted fluorescence microscope. The specific procedure was the same as above.

[0096] 4.1.2 In vitro osteogenic performance evaluation of materials 4.1.2.1 Assessment of alkaline phosphatase activity in cells MC3T3-E1 cells were also used at a rate of 1×10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per group onto the surface of each zirconia sample and cultured in α-MEM medium containing 10% FBS and 1% antibiotics for 24 h. ALP activity was assessed after 7 days of culture. Lysis buffer was added to the surface of the material until complete coverage, and the mixture was incubated at 4 °C for 30 min to lyse the cells and release intracellular proteins into the lysate. Lysates containing intracellular proteins were collected from each group and centrifuged at 12,000 rpm to obtain the supernatant. The alkaline phosphatase in the supernatant reacted with the substrate in the kit at 37 °C for 15 min to produce p-nitrophenol. A stop solution was then added, and the absorbance was measured using a microplate reader. A standard curve was then plotted to calculate the ALP activity data for each sample. The total intracellular protein concentration was determined using a BCA kit, and the AKP activity of each group was standardized. Finally, the relative AKP activity and total ALP values ​​for each group were calculated using a formula.

[0097] 4.1.2.2 Extracellular matrix mineralization (ARS) assay The seeding density and culture conditions of MC3T3-E1 cells were the same as before, with 6 replicate wells per group. After 14 days of culture, alizarin red staining and quantitative analysis were used to assess the mineralization of the extracellular matrix. First, cells were fixed with 4% tissue fixative at room temperature for 30 min, washed 3-5 times with ultrapure water, and then 500 μL of alizarin red staining solution was added to each well for 40 min of alizarin red staining. The reaction was then terminated by washing with ultrapure water until the wash solution was colorless. Images of alizarin red-stained mineralized nodules were observed and photographed using a stereomicroscope. Quantitative analysis of the degree of mineralization involved completely dissolving the surface mineralized nodules in 10 wt% hexadecylpyridine chloride solution and measuring the absorbance at 540 nm using a microplate reader.

[0098] 4.1.2.3 Expression of Osteogenesis-Related Genes After sterilization, the prepared samples were placed in 48-well plates, with MC3T3-E1 cells at a rate of 1 × 10⁶ cells per well. 4 Cells were inoculated at a density of [number] cells onto the sample surface and incubated at 37°C for 14 days, with the culture medium changed every two days. After 14 days, intracellular RNA was extracted using a total RNA extraction kit. The obtained RNA was reverse transcribed into cDNA using a PrimeScript RT kit, and finally, cDNA was amplified using a real-time quantitative PCR instrument. Target genes included osteogenic family transcription factor (RUNX2), osteocalcin (OCN), and osteopontin (OPN). Collagen (COL) and glycerol triphosphate dehydrogenase (GAPDH) were used as internal control genes. Other procedures were the same as above.

[0099] 4.2 Experimental Results 4.2.1 Results of in vitro biocompatibility assessment of materials 4.2.1.1 Results of MC3T3-E1 cell viability MC3T3-E1 cell viability test results are as follows Figure 15 As shown. After 3 days of culture, there was no significant difference in cell viability between Sr1000@ZrP-E, Sr2000@ZrP-E and the control group. Similarly, after 5 days of culture, all groups showed the same trend, with the test values ​​of each group increasing uniformly and without statistical difference (P<0.05).

[0100] 4.2.1.2 Morphology and live / dead staining results of MC3T3-E1 cells Figure 16 These are images of MC3T3-E1 cell morphology and live / dead staining results. A shows representative images of MC3T3-E1 cells at 3 days and 5 days (green / blue: cytoskeleton / nucleus), and B shows live / dead staining of MC3T3-E1 cells at 3 days (green / red: live / dead cells).

[0101] The cell morphology of MC3T3-E1 cells after culturing on different sample surfaces for 3 and 5 days is as follows: Figure 16 As shown in A and B. Under a 20x microscope, the Sr1000@ZrP-E and Sr2000@ZrP-E coatings showed the largest number of MC3T3-E1 cells adhering to them, and the cells exhibited good spreading on the coating surface, with no obvious dead cells.

[0102] 4.2.2 Results of in vitro osteogenic performance evaluation of materials 4.2.2.1 Results of ALP activity and extracellular matrix mineralization of MC3T3-E1 The short-term osteogenic capacity of MC3T3-E1 cells can be assessed by detecting alkaline phosphatase levels, while the late-stage extracellular matrix mineralization capacity can be detected by alizarin red staining. Figure 17 The images show ALP and ARS staining of samples without EDTA chelating agent. The matrix mineralization images of the S1000@ZrP and S2000@ZrP samples, which were simply immersed in strontium chloride solution without chelating agent, are deep purple and no red mineralized nodules are produced, indicating that they do not promote osteogenic differentiation of MC3T3-E1 cells.

[0103] Figure 18 The results show the ALP activity and extracellular matrix mineralization of MC3T3-E1 cells. A represents the staining images of ALP activity (4 and 7 days) and mineralization (14 days); B represents the quantitative analysis results of ALP at 7 days; and C represents the quantitative analysis results of mineralization at 14 days. The confidence coefficient was set at 95%.

[0104] according to Figure 18 ALP staining in samples A and B showed increased color intensity in S1000@ZrP-E and Sr2000@ZrP-E. ALP quantification analysis also showed an increasing trend compared to the control group. Therefore, S1000@ZrP-E and Sr2000@ZrP-E have the ability to promote short-term osteoblast formation, with Sr2000@ZrP-E showing a more significant effect. Similarly... Figure 18 Images and quantitative analysis of alizarin red stained mineralized nodules in groups A and C showed that at 14 days, the Sr2000@ZrP-E group exhibited the highest mineralization level among all groups, followed by the S1000@ZrP-E group, whose mineralization level was slightly higher than that of the ZrO2 and ZrP groups.

[0105] 4.2.2.2 PCR test results Figure 19 These are the results of osteogenic-related gene expression in MC3T3-E1 cells after 14 days. Figure 19The expression levels of osteogenic-related genes (RUNX2, OPN, OCN, COL) were displayed. RUNX2 expression was highest in the Sr2000@ZRP-E group after 14 days of culture, while RUNX2 expression levels decreased significantly in the ZrO2 and ZrP groups. In the Sr2000@ZrP-E group, OCN, OPN, and COL showed a stable increase in expression levels after 14 days of culture.

[0106] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing a zirconia-based medical prosthetic material, characterized in that, Including the following steps: S1, Pretreatment: First, the zirconia preform is sintered at high temperature, then polished and cleaned, and then sintered at high temperature and cleaned again. S2, Preparation of surface drug-loaded structure: A honeycomb porous nanostructure for drug loading was prepared on the surface of a zirconia preform by phosphorylation treatment; S3, loading of metal ions: The zirconia material treated in step S2 is placed in a loading solution containing metal ions and reacted in a shaker at 50~90℃ for 5~8h. After the reaction is completed, it is taken out, rinsed and dried to obtain zirconia-based medical prosthesis material. In step S1: the secondary high-temperature sintering process is as follows: first, the temperature is raised to 1450~1650℃ at a rate of 3~5℃ / min, and held for 160~200min. After the holding period, the temperature is cooled to room temperature with the furnace. In step S2, the phosphorylation process is as follows: the pretreated zirconia preform is placed in a high-pressure reactor and a phosphoric acid solution with a concentration of 2~3wt% is added. Then, the sealed high-pressure reactor is placed in a drying oven and kept at a constant temperature of 150~165℃ for 20~28h. In step S3, the soluble nitrate or chloride of the corresponding metal ion is dissolved in deionized water to prepare the required loading solution containing metal ions.

2. The method for preparing zirconia-based medical prosthesis material according to claim 1, characterized in that, In step S1: The high-temperature sintering process is as follows: First, the temperature is raised to 800-1000℃ at a rate of 5-8℃ / min and held for 40-60min. Then, the temperature is raised to 1350-1450℃ at a rate of 2-4℃ / min and held for 100-140min. After the holding period, the temperature is cooled to room temperature with the furnace.

3. The method for preparing zirconia-based medical prosthesis material according to claim 1 or 2, characterized in that, In step S1: The cleaning process described above involves ultrasonic cleaning with anhydrous ethanol and ultrapure water in sequence. The secondary cleaning process involves ultrasonic cleaning with anhydrous ethanol and ultrapure water in sequence, followed by drying of the zirconia preform with nitrogen gas flow after the secondary cleaning and storing it in a vacuum tank for later use.

4. The method for preparing the zirconia-based medical prosthesis material according to claim 1, characterized in that, In step S3, the metal ion is selected from Ag. + Cu 2+ 、Sr 2+ One or more of them.

5. The method for preparing zirconia-based medical prosthetic material according to claim 1, characterized in that, Silver nitrate, copper nitrate, and strontium chloride were used to prepare solutions containing Ag. + Cu 2+ 、Sr 2+ The loading solution contains silver nitrate at a concentration of 10–50 μM, copper nitrate at a concentration of 100–400 mM, and strontium chloride at a concentration of 0.5–3 M.

6. The method for preparing the zirconia-based medical prosthesis material according to claim 1, characterized in that, In step S3, the rinsing and drying process is as follows: The prosthetic material was rinsed with deionized water, anhydrous alcohol and deionized water in sequence. Then the prosthetic material was placed in a vacuum oven and dried at 32~45℃ and stored in a vacuum container for later use.

7. A zirconia-based medical prosthetic material, characterized in that, It is prepared using the method for preparing zirconia-based medical prosthesis materials as described in any one of claims 1 to 6.

8. The application of the zirconia-based medical prosthesis material according to claim 7 in the preparation of dental prostheses and bone prostheses.

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