Medical implant with lithium disilicate coating and preparation method thereof

Abstract

The invention belongs to the technical field of artificial prosthesis materials, and particularly relates to a medical implant with a lithium disilicate coating and a preparation method thereof, and the preparation method comprises the following steps: (1) placing a prefabricated matrix in a high-temperature environment for staged sintering, and then carrying out surface polishing, cleaning and drying treatment; (2) mixing lithium ethoxide and tetraethoxysilane at room temperature, and reacting for 2-4 hours while stirring to obtain lithium disilicate sol; the preparation method comprises the following steps: preparing a prefabricated substrate, soaking the prefabricated substrate in lithium disilicate sol for 3-7 minutes, performing sol permeation for 3-10 minutes under the negative pressure of more than 30 mm Hg, adding an ethanol water solution to initiate gelation reaction, taking out the prefabricated substrate after the reaction is completed, drying and sintering to obtain the medical implant with the lithium disilicate coating. According to the method, the uniform and continuous lithium disilicate coating which is not easy to fall off can be formed on the surface of the prefabricated matrix of the implant, so that the surface performance of the implant is improved, and the osteogenesis promoting performance and the angiogenesis promoting performance of the implant are optimized.

Description

Technical Field

[0001] This invention belongs to the field of artificial prosthetic materials technology, and particularly relates to a medical implant with a lithium disilicate coating and its preparation method. Background Technology

[0002] Currently, zirconia (ZrO2) medical implants, with their translucent aesthetic appearance, excellent corrosion resistance, low plaque adhesion rate, and significant biocompatibility advantages, hold promise as a next-generation alternative to medical implant materials. However, zirconia implants exhibit high chemical inertness and extremely low hydroxyl density, preventing them from actively binding specifically to biomolecules. They can only provide physical anchoring points through surface morphology, attracting osteoblasts to passively adhere. Therefore, their osseointegration process primarily relies on the host's own bone repair capacity, making it difficult for osteoblasts to migrate from surrounding bone tissue to the implant surface, resulting in low proliferation and differentiation efficiency. Furthermore, bone matrix can only deposit in a shallow layer on the implant surface, failing to fill the tiny interfacial gaps, leading to a low bone-implant contact rate and poor bone performance, necessitating surface modification for optimization. In addition, the surface of zirconia implants is unlikely to release active ions or regulate cytokines, causing angiogenesis to rely entirely on the host's own repair response after surgical trauma, such as inflammatory cells in the wound area secreting vascular endothelial growth factor to induce slow ingrowth of surrounding blood vessels. This process is characterized by slow angiogenesis rate and low vascular density, making it difficult to quickly provide sufficient nutrition to osteoblasts, resulting in a long osseointegration cycle. This is especially true in areas with poor blood supply, such as the maxilla, where osseointegration is prone to delays or even failure. Therefore, it is necessary to improve the angiogenesis properties of zirconia implants by surface modification to enhance early stability and improve long-term clinical success rates.

[0003] To enhance the bioactivity and therapeutic potential of ZrO2 implants, surface modification is a promising approach. Currently, various physical, chemical, and electrochemical surface treatment strategies, such as laser treatment, acid etching, anodizing, and sandblasting, have been developed and applied to enhance the performance of implants by controlling their microstructure and chemical composition. However, these methods all have limitations: physical manipulation of surface morphology offers limited controllability, making nanoscale precision customization difficult; chemical treatments may damage the material's inherent properties; and the long-term stability of electrochemical treatments remains to be tested. Based on these limitations of surface treatment technologies, there is an urgent need to find a more feasible novel functionalization strategy.

[0004] Currently, coating technology, with its excellent operability, surface controllability, and bioactivity, has gradually become a research hotspot for the surface modification of ZrO2 implants. In recent years, silicon, lithium, and their compounds have received considerable attention in the biomedical field, particularly in dental and orthopedic applications. For example, Li2Si2O5 microcrystalline glass, due to its good mechanical properties and excellent translucency, has been widely used in oral restoration. Currently, the preparation of Li2Si2O5 coatings mainly includes melting methods, sol-gel methods, hydrothermal methods, solid-phase reaction methods, and chemical vapor deposition methods to meet different production application needs. Among them, the sol-gel method is a commonly used wet chemical synthesis method for preparing inorganic materials, ceramic materials, and thin films. The specific steps involve dissolving lithium and silicon source precursors in a suitable solvent to form a sol solution, followed by gelation and drying. The dried gel is then subjected to high-temperature calcination to remove residual organic matter and promote the crystallization of lithium disilicate. However, the process of preparing lithium disilicate coatings on the surface of medical zirconia implants using the sol-gel method presents the following challenges: First, after high-temperature sintering, the pre-fabricated zirconia substrate has an extremely low surface hydroxyl density and exists in a stable Zr-OH form, making it difficult to undergo condensation reactions with the silanol groups in the sol. Only weak van der Waals forces or hydrogen bonds can form between the coating and the substrate, resulting in generally low interfacial bonding strength. Furthermore, commercially available pre-fabricated zirconia implants often have complex morphologies such as threaded or conical shapes, with irregular areas on the surface including threaded grooves and inverted conical structures. This easily leads to uneven coating thickness during sol coating. Additionally, substances remaining on the pre-fabricated substrate surface can hinder sol wetting during coating preparation, causing defects such as missed coating and pinholes. Moreover, there is a difference in the coefficient of thermal expansion between the pre-fabricated substrate material and lithium disilicate. During the sol-gel sintering stage, the interfacial tensile stress generated by the difference in thermal shrinkage accumulates linearly with increasing coating thickness, leading to coating cracking and detachment. In summary, the combined effect of these factors makes the lithium disilicate coating loaded on the implant surface prone to peeling off during implantation surgery or postoperative chewing.

[0005] Second, the sol system has poor compatibility with the pre-fabricated substrate, and the pre-fabricated substrate cannot participate in the network construction of the sol, which makes the sol system prone to repulsion from the substrate. For example, the inertness of the zirconium oxide substrate surface will exacerbate the phase separation of the sol on its surface, causing the silicon and lithium components in the sol to preferentially self-aggregate and form independent microparticles rather than a uniformly spread liquid film. Ultimately, it is difficult to form a uniform coating, resulting in uneven coating thickness and composition.

[0006] Third, during the sintering process, the sintering process lacks compatibility with both the coating and the precast substrate, resulting in post-treatment processes such as sol-gel drying and sintering failing to simultaneously ensure the coating performance and the stability of the precast substrate. For example, during sintering, the coating needs to be sintered at 700-800℃ to achieve lithium disilicate crystallization, forming a β phase with biological activity, and the temperature needs to be increased slowly to avoid cracking. However, for the precast substrate, although zirconium oxide is heat-resistant, prolonged exposure (>2 hours) to a high-temperature environment above 700℃ will cause the surface tetragonal phase (t-ZrO2) to transform into a monoclinic phase (m-ZrO2), leading to volume expansion of the precast substrate surface and tearing of the formed coating.

[0007] In view of this, those skilled in the art urgently need to propose a method to optimize the overall performance of zirconia implants by surface modification. Summary of the Invention

[0008] The present invention aims to provide a uniform, continuous, and non-detachable film layer that can be formed on the surface of a prefabricated substrate of an implant to improve the surface properties of the implant, optimize its osteogenic and angiogenic properties, thereby enhancing the early stability of the implant and improving the long-term clinical success rate.

[0009] In view of this, the present invention provides a method for preparing a medical implant with a lithium disilicate coating, comprising the steps of: (1) Pre-treatment of the surface of the precast substrate: The precast substrate is placed in a high-temperature environment for staged sintering, followed by surface polishing, cleaning and drying. The phased sintering process is as follows: First, the temperature is increased to 800-1000℃ at a heating rate of 3-8℃ / min and held for 20-40min. Then, the temperature is increased to 1300-1700℃ at a heating rate of 2-5℃ / min and held for 100-150min before entering the cooling process. During the cooling process, the precast substrate is first cooled to 700-400℃ with the furnace, then cooled to 250-350℃ after the furnace is opened. Finally, the precast substrate is taken out and allowed to cool naturally to room temperature. (2) Preparation of coating by negative pressure infiltration combined with sol-gel method: Lithium ethanol and tetraethyl orthosilicate are used as lithium and silicon sources for reaction. Lithium ethanol and tetraethyl orthosilicate with a molar ratio of 1~1.15:1 are mixed at room temperature and reacted under stirring for 2~4h to obtain lithium disilicate sol. Then, the pre-prepared substrate after step (1) is immersed in lithium disilicate sol for 3~7min and then infiltrated with sol under a negative pressure of more than 30mmHg for 3~10min. After that, an aqueous ethanol solution is added to initiate the gelation reaction. After the gelation reaction is completed, the pre-prepared substrate is taken out and dried and sintered to obtain a medical implant with lithium disilicate coating.

[0010] Furthermore, the material of the prefabricated substrate is zirconium oxide.

[0011] Furthermore, the pre-prepared substrate after step (1) is first subjected to surface phosphorylation treatment, and then a lithium disilicate coating is prepared in the manner described in step (2).

[0012] Furthermore, the surface phosphorylation treatment process is as follows: First, the pre-prepared matrix is ​​immersed in a phosphoric acid solution in a high-pressure reactor. Then, the high-pressure reactor is sealed and placed in a vacuum drying oven at 140~180℃ for 20~30 hours. The concentration of the phosphoric acid solution is 2~3wt%.

[0013] Preferably, the staged sintering process in step (1) is as follows: First, the temperature is increased to 900℃ at a rate of 5℃ / min and held for 30 min. Then, the temperature is increased to 1500℃ at a rate of 3℃ / min and held for 120 min before entering the cooling process. During the cooling process, the precast substrate is first cooled to 500℃ with the furnace, then the furnace is partially opened to cool down to 300℃. Finally, the precast substrate is removed and allowed to cool naturally to room temperature.

[0014] Furthermore, the surface polishing, cleaning, and drying process of the prefabricated substrate in step (1) is as follows: Gradual polishing was performed using silicon carbide sandpaper ranging from 400 to 2000 grit. Then, ultrasonic cleaning was performed in acetone, ethanol solution, and ultrapure water, respectively. Finally, place it in a vacuum drying oven to dry and store it.

[0015] Furthermore, the sintering procedure in step (2) is as follows: First, heat the temperature to 500-700℃ at a rate of 1.5-2.5℃ / min and hold for 1.5-2.5 hours. Then, heat the temperature to 900-1000℃ at a rate of 1.3-2.5℃ / min and hold for 1.8-2.3 hours. Finally, cool the temperature to room temperature at a rate of 3-7℃ / min.

[0016] Preferably, the sintering procedure in step (2) is as follows: First, the temperature is increased to 600℃ at a rate of 2℃ / min and held for 2 hours. Then, the temperature is increased to 950℃ at a rate of 2℃ / min and held for 2 hours. Finally, the temperature is decreased to room temperature at a rate of 5℃ / min.

[0017] In addition, the present invention also provides a medical implant with a lithium disilicate coating, which is prepared according to the above-described preparation method.

[0018] In addition, the present invention also provides the application of the above-mentioned medical implant with lithium disilicate coating in the preparation of dental prostheses and bone prostheses.

[0019] Compared with existing technologies, the medical implant with lithium disilicate coating and its preparation method described in this invention have the following advantages: 1. This invention optimizes the surface structure and hydroxyl distribution of the pre-fabricated zirconia substrate through a staged sintering pretreatment, improving surface activity and enabling better condensation reactions with the silanol groups in the sol, thereby enhancing the adhesion between the coating and the substrate. Furthermore, this invention utilizes negative pressure infiltration combined with a sol-gel method to allow the sol to penetrate more fully into the micropores of the substrate surface, further improving interfacial bonding strength and reducing the risk of coating peeling during use.

[0020] 2. The negative pressure permeation process used in this invention helps the sol to spread evenly and continuously on the surface of the pre-prepared substrate, overcoming the problem of phase separation of the sol on the surface of the zirconium oxide substrate in the traditional sol-gel method. This allows the silicon-lithium components to be evenly and continuously distributed, forming a lithium disilicate coating with uniform thickness and composition, thus improving the effect of implant surface modification.

[0021] 3. The lithium disilicate coating sintering process in this invention has been optimized. It adopts a step-by-step heating and holding method, which can ensure that the lithium disilicate coating crystallizes at a suitable temperature to form a biologically active β phase, and can also avoid the transformation of the surface tetragonal phase to monoclinic phase caused by the pre-fabricated substrate being in a high-temperature environment for a long time. This prevents the coating from tearing due to the volume expansion of the substrate surface, effectively balancing the coating performance and the stability of the pre-fabricated substrate.

[0022] 4. The lithium disilicate coating prepared by this invention has a honeycomb microstructure, high surface roughness, and large specific surface area, which can enhance cell adhesion and adsorption capacity for bioactive molecules, and is not easy to fall off. 5. The Li in the lithium disilicate coating prepared by this invention + The release behavior exhibits a two-stage characteristic: rapid release in the early stage, which may promote early cellular response; and slow release in the later stage, which helps maintain long-term biological effects. 6. The in vitro experiments in this invention verified that the Li2Si2O5 coating has good cell compatibility and confirmed that the material is non-cytotoxic; at the same time, it verified that the medical implant loaded with Li2Si2O5 coating has excellent bone-promoting and angiogenesis-enhancing functions. Attached Figure Description

[0023] Figure 1 These are the morphological observation results of the four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention. Figure 2 These are the water contact angle detection images and WCA quantitative detection results of the four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention. Figure 3 These are the XPS full spectrum and XRD spectrum of the four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention. Figure 4 The Li content of the Zr@LS sample prepared in Example 3 of this invention + Cumulative release curve; Figure 5 The results of MC3T3-E1 cell proliferation and morphology detection on the surfaces of four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention are shown. Figure 6 The results of ALP and mineralization assays of MC3T3-E1 cells from four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention are shown. Figure 7 The results of HUVEC cell viability and cell scratch detection for the four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention; Figure 8 The results of HUVEC cell tubule formation assays for the four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention; Figure 9 The results of the bone-bonded tissue section analysis of the three groups of samples (Zr, ZrP, and ZrP@LS) prepared in Example 3 of this invention are shown. Figure 10 The results of angiogenic tissue section analysis of the three groups of samples (Zr, ZrP, and ZrP@LS) prepared in Example 3 of this invention are shown. Detailed Implementation

[0024] The technical solutions in the embodiments of this application will be clearly described below with reference to specific examples. 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.

[0025] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0026] This invention provides a method for preparing a medical implant with a lithium disilicate coating, comprising the following steps: (1) Pre-treatment of the surface of the precast substrate: The precast substrate is placed in a high-temperature environment for staged sintering, followed by surface polishing, cleaning and drying. The phased sintering process is as follows: First, the temperature is increased to 800-1000℃ at a heating rate of 3-8℃ / min and held for 20-40min. Then, the temperature is increased to 1300-1700℃ at a heating rate of 2-5℃ / min and held for 100-150min before entering the cooling process. During the cooling process, the precast substrate is first cooled to 700-400℃ with the furnace, then cooled to 250-350℃ after the furnace is opened. Finally, the precast substrate is taken out and allowed to cool naturally to room temperature. (2) Preparation of coating by negative pressure infiltration combined with sol-gel method: Lithium ethanol and tetraethyl orthosilicate are used as lithium and silicon sources for reaction. Lithium ethanol and tetraethyl orthosilicate with a molar ratio of 1~1.15:1 are mixed at room temperature and reacted under stirring for 2~4h to obtain lithium disilicate sol. Then, the pre-prepared substrate after step (1) is immersed in lithium disilicate sol for 3~7min and then infiltrated with sol under a negative pressure of more than 30mmHg for 3~10min. After that, an aqueous ethanol solution is added to initiate the gelation reaction. After the gelation reaction is completed, the pre-prepared substrate is taken out and dried and sintered to obtain a medical implant with lithium disilicate coating.

[0027] In this invention, a lithium disilicate coating was successfully prepared on the surface of zirconium-based materials by combining negative pressure infiltration technology with the sol-gel method. Through systematic research on the characterization, osteogenic induction, and angiogenic properties of the coating, it was found that the zirconium-based materials loaded with this coating exhibited significant improvements in both osteogenic and angiogenic properties. This research provides new research ideas and theoretical basis for the application of lithium disilicate coatings in the field of implants.

[0028] Preferably, the material of the prefabricated substrate is zirconium oxide.

[0029] Furthermore, in this invention, the pre-prepared substrate after step (1) can be first subjected to surface phosphorylation treatment, and then a lithium disilicate coating can be prepared in the manner described in step (2).

[0030] As some examples of the present invention, the surface phosphorylation treatment process is as follows: First, the pre-prepared matrix is ​​immersed in a phosphoric acid solution in a high-pressure reactor. Then, the high-pressure reactor is sealed and placed in a vacuum drying oven at 140~180℃ for 20~30 h. The concentration of the phosphoric acid solution is 2~3wt%.

[0031] Preferably, the staged sintering process in step (1) is as follows: First, the temperature is increased to 900℃ at a rate of 5℃ / min and held for 30 min. Then, the temperature is increased to 1500℃ at a rate of 3℃ / min and held for 120 min before entering the cooling process. During the cooling process, the precast substrate is first cooled to 500℃ with the furnace, then the furnace is partially opened to cool down to 300℃. Finally, the precast substrate is removed and allowed to cool naturally to room temperature.

[0032] Furthermore, the surface polishing, cleaning, and drying process of the prefabricated substrate in step (1) is as follows: Gradual polishing was performed using silicon carbide sandpaper ranging from 400 to 2000 grit. Then, ultrasonic cleaning was performed in acetone, ethanol solution, and ultrapure water, respectively. Finally, place it in a vacuum drying oven to dry and store it.

[0033] Furthermore, the sintering procedure in step (2) is as follows: First, heat the temperature to 500-700℃ at a rate of 1.5-2.5℃ / min and hold for 1.5-2.5 hours. Then, heat the temperature to 900-1000℃ at a rate of 1.3-2.5℃ / min and hold for 1.8-2.3 hours. Finally, cool the temperature to room temperature at a rate of 3-7℃ / min.

[0034] Preferably, the sintering procedure in step (2) is as follows: first, the temperature is raised to 600°C at a heating rate of 2°C / min and held for 2 hours; then, the temperature is raised to 950°C at a heating rate of 2°C / min and held for 2 hours; finally, the temperature is lowered to room temperature at a rate of 5°C / min.

[0035] In addition, the present invention also provides a medical implant with a lithium disilicate coating, which is prepared according to the above-described preparation method.

[0036] Based on this, the present invention also provides the application of the above-mentioned medical implant in the field of preparing dental prostheses and bone prostheses.

[0037] The following specific embodiments illustrate the medical implant with a lithium disilicate coating and its preparation method according to the present invention: Example 1 Preparation of medical implants with lithium disilicate coating: (1) Pretreatment of the surface of the precast substrate: Zirconia bioceramic blocks are used as raw materials. After drying in a vacuum drying oven, they are transferred to a zirconia sintering furnace for staged sintering. The sintering procedure is as follows: first, the temperature is raised to 800℃ at a heating rate of 3℃ / min and held for 40 min, then the temperature is raised to 1700℃ at a heating rate of 2℃ / min and held for 100 min before entering the cooling procedure. After the temperature in the furnace drops to 400℃, the furnace is partially opened and the temperature is lowered to 250℃. Finally, the precast substrate is taken out and allowed to cool naturally to room temperature.

[0038] Subsequently, the ZrO2 prefabricated substrate underwent surface polishing and cleaning: it was sequentially polished using 400-mesh, 600-mesh, 1000-mesh, and 2000-mesh silicon carbide sandpaper, then ultrasonically cleaned for 8 minutes each in acetone, 75% ethanol solution, and ultrapure water, and finally dried in a vacuum drying oven for storage.

[0039] (2) Preparation of coating by negative pressure infiltration combined with sol-gel method: using tetraethyl orthosilicate (TEOS) (C8H) 20 Using TEOS (2 mol / L) and lithium ethoxide (LiOEt, 0.82 mol / L) as the silicon and lithium sources for the reaction, according to the molecular formula of Li2Si2O5, TEOS and lithium ethoxide in a molar ratio of 1:1.15 were mixed at room temperature (25°C), and the mixture was stirred at 300 r / min for 4 h on a magnetic stirrer. After completion, the Tyndall effect of the solution was checked to ensure that the Li2Si2O5 sol was prepared completely. Next, the samples were placed in a 24-well plate and immersed in 400 μL of lithium disilicate sol at room temperature and pressure for 7 min. Then, the sol was permeated for 3 min under a vacuum pressure of 50 mmHg. Immediately afterward, 200 μL of an ethanol-water aqueous solution (2:1 volume ratio) was added to each well to initiate a gelation reaction. After 50 s, the samples were removed and placed in a new well plate, then dried at 60 °C for 12 h. Following these steps, the dried samples were heat-treated as follows: sintering at 500 °C for 2.5 h (heating rate 1.5 °C / min) → sintering at 900 °C for 2.3 h (heating rate 2.5 °C / min) → cooling to room temperature (cooling rate 7 °C / min).

[0040] Example 2 Preparation of medical implants with lithium disilicate coating: (1) Pretreatment of the surface of the precast substrate: Zirconia bioceramic blocks are used as raw materials. After drying in a vacuum drying oven, they are transferred to a zirconia sintering furnace for staged sintering. The sintering procedure is as follows: first, the temperature is raised to 1000℃ at a heating rate of 8℃ / min and held for 20 min, then the temperature is raised to 1300℃ at a heating rate of 5℃ / min and held for 150 min before entering the cooling procedure. After the temperature inside the furnace drops to 700℃, the furnace is partially opened and the temperature is lowered to 350℃. Finally, the precast substrate is taken out and allowed to cool naturally to room temperature. Subsequently, the ZrO2 prefabricated substrate underwent surface polishing and cleaning: it was sequentially polished using 400-grit, 600-grit, 1000-grit, and 2000-grit silicon carbide sandpaper, followed by ultrasonic cleaning for 6 minutes each in acetone, 75% ethanol solution, and ultrapure water. Finally, it was dried in a vacuum drying oven and stored.

[0041] (2) Phosphorylation treatment of pre-prepared substrate surface: The pretreated ZrO2 sample was immersed in a high-pressure reactor lined with a polytetrafluoroethylene container with a phosphoric acid solution diluted to 2wt%. Then, the high-pressure reactor was sealed and placed in a vacuum drying oven at 180℃ for 20 h. After the reaction was completed, the sample was taken out for cleaning and drying.

[0042] (3) Preparation of coating by negative pressure infiltration combined with sol-gel method: using tetraethyl orthosilicate (TEOS) (C8H) 20 Using TEOS (2 mol / L) and lithium ethoxide (LiOEt, LiOC2H5, 0.82 mol / L) as the silicon and lithium sources for the reaction, according to the molecular formula of Li2Si2O5, TEOS and lithium ethoxide were mixed at a molar ratio of 1:1.05 at room temperature (25℃), and the mixture was stirred at 300 r / min for 2 h on a magnetic stirrer. After completion, the Tyndall effect of the solution was checked to ensure that the Li2Si2O5 sol was prepared completely. Next, the samples were placed in a 24-well plate and immersed in 400 μL of lithium disilicate sol at room temperature and pressure for 3 min. Then, the sol was permeated for 10 min under a vacuum pressure of 40 mmHg. Immediately afterward, 200 μL of an ethanol-water aqueous solution (2:1 volume ratio) was added to each well to initiate a gelation reaction. After 50 s, the samples were removed and placed in a new well plate, then dried at 60 °C for 12 h. Following these steps, the dried samples were heat-treated as follows: sintering at 700 °C for 1.5 h (heating rate 2.5 °C / min) → sintering at 1000 °C for 1.8 h (heating rate 1.3 °C / min) → cooling to room temperature (cooling rate 3 °C / min).

[0043] Example 3 Preparation of experimental samples for the Zr group, ZrP group, Zr@LS group, and ZrP@LS group: (1) Grouping of experimental samples: According to the experimental design, the samples were divided into the following 4 groups: 1) Zr group: Zirconia (ZrO2) sheets and ZrO2 cylinders that have been cut, sintered and polished; 2) ZrP group: Based on the Zr group, ZrO2 sheets and ZrO2 cylinders are phosphorylated. 3) Zr@LS group: Based on the Zr group, lithium ethanol and tetraethyl orthosilicate were used as precursors, and ZrO2 sheets and ZrO2 cylinders with lithium disilicate coating were prepared on the material surface by negative pressure infiltration combined with sol-gel method; 4) ZrP@LS group: Based on the ZrP group, ZrO2 sheets and ZrO2 cylinders with Li2Si2O5 coating were prepared on the surface.

[0044] (2) Zirconia surface pretreatment: Zirconia bioceramic blocks were used as raw materials, and two different specifications of samples were prepared by diamond wire cutting machine: 10 mm*10 mm ZrO2 sheets with a thickness of 1.5 mm and ZrO2 cylinders with a diameter of 5 mm and a length of 10 mm. After drying in a vacuum drying oven, all samples were transferred to a zirconia sintering furnace for high-temperature crystallization in standard sintering mode. The sintering procedure was as follows: first, the temperature was raised to 900℃ at a heating rate of 5℃ / min and held for 30 min, then the temperature was raised to 1500℃ at a heating rate of 3℃ / min and held for 120 min before entering the cooling procedure. After the temperature in the furnace dropped to 500℃, the furnace was partially opened and the temperature was lowered to 300℃. Finally, the temperature was allowed to drop naturally to room temperature. Subsequently, the ZrO2 samples underwent surface polishing and cleaning: they were sequentially polished using 400-mesh, 600-mesh, 1000-mesh, and 2000-mesh silicon carbide sandpaper, followed by ultrasonic cleaning in acetone, 75% ethanol solution, and ultrapure water for 5 minutes each. Finally, they were dried in a vacuum drying oven and stored in a dry place.

[0045] (3) Phosphorylation treatment of zirconia surface: The pretreated ZrO2 sample was immersed in a high-pressure reactor lined with a polytetrafluoroethylene container with a phosphoric acid solution diluted to 2.4 wt% and placed in a vacuum drying oven at 160 ℃ for 24 h. After the reaction was completed, the sample was taken out for cleaning and drying.

[0046] (4) Preparation of coating by negative pressure infiltration combined with sol-gel method: using tetraethyl orthosilicate (TEOS) (C8H) 20Using TEOS (2 mol / L) and lithium ethoxide (LiOEt, 0.82 mol / L) as the silicon and lithium sources, respectively, according to the molecular formula of Li₂Si₂O₅, the TEOS and lithium ethoxide in a molar ratio of 1:1 were mixed at room temperature (25°C), and the mixture was stirred at 300 r / min for 3 h on a magnetic stirrer. After completion, the Tyndall effect of the solution was checked to ensure that the Li₂Si₂O₅ sol was prepared completely. The following is the reaction equation of the solvent: ① Hydrolysis reaction of TEOS: Si(OC2H5)4 + 4H2O → Si(OH)4 + 4C2H5OH; ② Condensation reaction of lithium ethoxide and silanol: 2LiOEt + 2Si(OH)4 → Li2Si2O5 + 3H2O + 2C2H5OH; ③ Overall reaction equation: 2 Si(OC2H5)4 + 2LiOEt + 2H2O → Li2Si2O5 + 10C2H5OH; Next, the samples were placed in a 24-well plate and immersed in 400 μL of lithium disilicate sol at room temperature and pressure for 5 min. Then, the sol was permeated in a vacuum pot under a negative pressure of 50 mmHg for 5 min. Immediately afterwards, 200 μL of an ethanol-water aqueous solution with a volume ratio of 2:1 was added to each well to initiate a gelation reaction. After 50 s, the samples were removed and placed in a new well plate, and dried at 60 °C for 12 h. After completing the above steps, the dried samples were heat-treated as follows: sintering at 600 °C for 2 h (heating rate of 2 °C / min) → sintering at 950 °C for 2 h (heating rate of 2 °C / min) → cooling to room temperature (cooling rate of 5 °C / min).

[0047] Experimental Example 1: Sample Performance Characterization (1) Morphological analysis Using a blue plastic backing as the shooting background, four groups of ZrO2 samples (Zr, ZrP, Zr@LS, and ZrP@LS) were photographed and recorded. The macroscopic morphology observation results are as follows: Figure 1 As shown in Figure A; subsequently, the surfaces of the four groups of samples were pretreated with gold sputtering using an ion sputtering instrument, and their surface morphology was observed under SEM. The obtained SEM images are shown below. Figure 1 As shown in B.

[0048] according to Figure 1It can be seen that the Li2Si2O5 coating prepared by phosphorylation treatment and sol-gel method showed no significant color change compared with the pure ZrO2 group, with only a slight reduction in light reflection. This indicates that the ZrO2 surface treatment method of this invention does not alter the aesthetic effect of the material itself. Furthermore, according to the surface SEM images of the four groups of samples, except for the ZrO2 group, all other samples exhibited a rough, porous surface structure. The ZrP group showed a multi-layered, uniformly sized honeycomb porous surface with pore diameters of approximately 300-500 nm, which differs from the ultrathin hexagonal α-ZrP layered nanostructures described in conventional studies. The Li2Si2O5 coating prepared by negative pressure infiltration + sol-gel method exhibited a continuous multi-layered porous network structure. Under the microscope, the Zr@LS group showed a relatively loose yet dense porous structure with numerous fibrous connectives on the surface, exhibiting highly porous characteristics overall. The ZrP@LS group had larger pores, obvious surface particles, and a strong three-dimensional effect between the particles, showing obvious particle aggregation. This structure can increase the surface roughness of the material, increase the specific surface area, and provide excellent catalytic and adsorption properties.

[0049] (2) Contact angle measurement The contact angle is an important parameter for measuring the wettability of a material surface. It refers to the contact angle between a droplet (usually water) and a solid surface, and its magnitude is commonly used to measure the hydrophilicity or hydrophobicity of a material. Four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) were each configured with three parallel controls. The ultrapure water droplet volume was set to 2 μL / drop. After the ultrapure water droplet stabilized on the sample surface for 5 seconds, images of the ultrapure water contacting the sample surface were recorded using a high-speed camera, and then analyzed using accompanying software. The water contact angle detection images and WCA quantitative detection results for the four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) are shown below. Figure 2 As shown: Among them, Figure 2 Image A shows the water contact angle images of four groups of samples, and image B shows the quantitative detection results of WCA.

[0050] Depend on Figure 2 It can be seen that the WCA value of phosphorylated ZrO2 sheets is significantly lower than that of the Zr group, while the WCA values ​​of the latter two groups treated with Li2Si2O5 coating are not significantly different from those of the control group. In other words, phosphorylation treatment can increase the hydrophilicity of the material, while the hydrophilicity of the coated material is not significantly different from that of the pretreated ZrO2 sheets.

[0051] (3) X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) detection In this study, XPS was first used to characterize the chemical composition and relative content of each element in the Li2Si2O5 coating, and then XRD was used to determine the crystal structure of the coating on the surface of each sample.

[0052] Figure 3 The XPS full spectrum and XRD spectrum of four groups of samples are shown below. Figure 3 In the figure, A is the XPS full spectrum of the four groups of samples, B is the XRD spectrum of the four groups of samples, and Table 1 below shows the percentage of each element content on the surface of the four groups of samples: Zr, ZrP, Zr@LS, and ZrP@LS.

[0053] Table 1. XPS Element Ratio The test results revealed that Li failed to appear in the XPS spectra of all samples due to its weak signal and high reactivity, which makes it prone to volatilization or deep reaction in a vacuum environment. P, Li, and Si were all undetectable in the Zr group. However, in the phosphorylated ZrP and ZrP@LS groups, P content was 18.56 at% and 7.21 at%, respectively; Si content was detected on the surfaces of the Zr@LS and ZrP@LS samples with Li₂Si₂O₅ coatings, at 26.49 at% and 19.5 at%, respectively. Correspondingly, outside the Zr group, the Zr content on the surfaces of the ZrP, Zr@LS, and ZrP@LS groups gradually decreased until it became undetectable.

[0054] The XRD results show that the main diffraction peaks of the Zr group appear at 2θ≈30°, 35°, and 50°, which is typical of the tetragonal crystal structure of ZrO2, indicating that the ZrO2 matrix has strong crystallinity and no interference from other phases. Compared with ZrO2, the ZrP group shows characteristic peaks of a monoclinic phase at 20-30°, indicating that the zirconium phosphate coating may be a thin coating with low crystallinity. The diffraction peaks at 2θ≈30°, 35°, and 50° are not very obvious, proving that the newly formed zirconium phosphate covers the ZrO2 surface, and the crystal structure of the ZrO2 substrate has been slightly affected. In addition to the diffraction peaks of zirconium oxide and zirconium phosphate, the Zr@LS and ZrP@LS groups also show new diffraction peaks around 20-25°, indicating that a new crystalline phase has formed on the material surface and that the coating has a certain degree of crystallinity.

[0055] Overall, XPS and XRD analyses indicate the successful formation of zirconium phosphate and Li₂Si₂O₅ coatings on the ZrO₂ surface. XPS analysis confirmed the elemental composition and chemical state of the coatings, demonstrating uniform loading of the Li₂Si₂O₅ coating on both Zr and ZrP surfaces. XRD analysis revealed the crystallinity of the coatings and their minimal impact on the crystal structure of the ZrO₂ substrate.

[0056] (4) Detection of Li by inductively coupled plasma optical emission spectrometry (ICP / OES) + concentration In this invention, the cumulative release of Li+ from the sample coating was determined using ICP-OES to reveal the coating's sustained-release function. The cumulative Li+ release of Zr@LS over 0, 1, 4, 10, 14, and 21 days was statistically analyzed. + The cumulative Li+ release curve of the Zr@LS sample was obtained, as shown in the figure below. Figure 4 As shown: From Li + The release curve shows that Li in the Zr@LS group + Rapid release occurs on day 1 and continues until approximately day 10. After 10 days, the release rate in each group stabilizes and begins to slow down.

[0057] (5) Statistical analysis In this invention, all experiments were repeated at least three times, and the data were analyzed using Graphpad Prism with one-way ANOVA and Students't test. Statistical results are presented as mean ± standard deviation (Mean ± SD). The confidence coefficient was set at 95% (*p < 0.05).

[0058] Based on the above test results, it can be concluded that in this invention: 1) Li2Si2O5 coatings were successfully prepared on the surfaces of two different zirconium oxide materials using negative pressure infiltration combined with sol-gel technology. Under electron microscopy, the coatings exhibited a continuous multilayer porous network structure, which increased the surface roughness and specific surface area of ​​the substrate.

[0059] 2) The Li2Si2O5 coating does not significantly increase the hydrophilicity of the substrate, but its hydrophilicity is basically the same as that of ZrO2.

[0060] 3) Li in the Li2Si2O5 coating + It is released rapidly in the initial stage, and then shows a slow release trend in the later stage.

[0061] Experimental Example 2: Evaluation of the in vitro osteogenic and angiogenic properties of lithium disilicate coatings (I) Experimental Methods: Cell Culture and Passaging: MC3T3-E1 is an osteogenic progenitor cell line derived from mouse embryos. Due to its stable osteogenic capacity, well-defined differentiation process, and good experimental reproducibility, it is widely used in in vitro osteogenic studies, especially for evaluating the osteogenic properties of biomaterials. Human umbilical vein endothelial cells (HUVECs), extracted from primary cells of the umbilical vein endothelium of healthy newborns, are cell models used to study whether biomaterials affect endothelial cell adhesion, growth, and function. In this experiment, the MC3T3-E1 cell line and the HUVEC cell line were used for in vitro osteogenic and angiogenic performance testing, respectively. MC3T3-E1 cells were cultured in α-MEM medium (containing a mixture of 10% FBS and 1% antibiotics), while HUVECs were cultured in high-glucose modified Eagle medium (DMEM) complete medium (containing a mixture of 10% FBS and 1% antibiotics), where the antibiotic mixture contained penicillin (100 IU / mL) and streptomycin (100 μg / mL). Subculturing should be performed after cell confluence reaches 80%-90%: Discard the original culture medium in the culture flask, rinse 2-3 times with sterile phosphate-buffered saline (PBS), add 1 mL of trypsin containing EDTA and incubate in a constant temperature incubator (37℃, 5% CO2) for 2 minutes. When the cells appear as slightly shiny, wrinkled, rounded cells under an inverted microscope, add 3 mL of culture medium to stop the digestion. Gently pipette to detach the cells, collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in an appropriate amount of culture medium. Subculture at a 1:3 ratio. Change the medium every 2 days until the next cell passage or cryopreserve the cells.

[0062] (II) In vitro assessment of osteogenic properties: (1) Cell viability and proliferation assay Four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) were sterilized and dried in a clean bench using ultraviolet light and 75% ethanol solution, and then placed in sterile 24-well plates. Five parallel controls were set up for each group, and 2 × 10⁶ samples were inoculated per well. 4One MC3T3-E1 cell was placed on the sterilized sample surface in a 24-well plate, and α-MEM complete medium was added. The plate was then incubated in a constant temperature incubator (37 ℃, 5% CO2), with the medium changed every two days. Cell viability and proliferation were assessed on days 1, 3, and 7. The CCK-8 assay kit was used for detection. The medium was aspirated from the wells in a clean bench, and the cells were gently rinsed twice with PBS. 200 mL of CCK-8 solution (α-MEM medium containing 10% CCK-8 reagent) was added to each well, and the plate was incubated for 1 hour. When the solution turned orange, 100 μL of solution was transferred from each well to a 96-well plate, and the absorbance was read at 450 nm using a microplate reader. (All steps involving CCK-8 reagent were performed in a dark environment.) (2) Cell morphology record Four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) were sterilized and dried before being placed in sterile 24-well plates, with four replicates for each group. Two × 10⁶ samples were inoculated into each well. 4 MC3T3-E1 cells were placed on the sample surface and incubated in a constant temperature incubator. The culture medium was changed regularly, and the morphology of MC3T3-E1 cells on the material surface was detected on days 1 and 7. The specific steps were as follows: the culture medium in the well plate was discarded, and the cells were gently washed 2-3 times with PBS solution (avoiding direct rinsing of the material surface). The cells were then fixed with 200 μL of 4% paraformaldehyde per well for 40 minutes. After discarding the paraformaldehyde solution, the cells were washed 2-3 times with PBS solution, and reacted with 200 μL / well of 0.1% Triton X-100 for 10 minutes to create pores in the cell membrane. After washing with PBS again, the samples were immersed in FITC-labeled phalloidin staining solution (100 nM) and 4',6-diamidinyl-2-phenylindole (DAPI) staining solution (10 μg / mL) for 40 minutes and 10 minutes respectively to stain the cytoskeleton and nucleus. After removing the staining solution, the cells were rinsed with PBS solution. After completing the above steps, use an inverted fluorescence microscope to randomly select different fields of view to photograph the fluorescence morphology of the cells. The cytoskeleton is represented by the green fluorescent area, while the cell nucleus is represented by the blue fluorescent area. (All steps involving fluorescent dyes must be performed in a dark environment.) (3) Assessment of alkaline phosphatase (ALP / AKP) activity in cells The four groups of samples were sterilized and dried, with four replicates for each group. Two × 10⁶ samples were inoculated per well. 4 One MC3T3-E1 sample was placed on the surface of a sterilized sample in a 24-well plate and incubated in a constant temperature incubator. The culture medium was changed periodically. After incubation for 7 days, the culture medium was aspirated, and the sample was washed with PBS. Alkaline phosphatase (ALP) staining reagent was added and stained for 4 hours in the dark at room temperature. The sample was washed again and photographed and recorded using a stereomicroscope.

[0063] Next, ALP activity was detected using an ALP kit. Cells washed with PBS were lysed with 1% Triton X-100 solution. After preparing the reaction solution according to the instructions, the reaction solution was added to the lysis buffer all at once, and a standard curve was prepared simultaneously. After the reaction was terminated, the absorbance was measured at a wavelength of 520 nm. At the same time, the absorbance was measured at a wavelength of 562 nm using a BCA kit. Finally, the relative alkaline phosphatase activity values ​​of each group were calculated using a formula.

[0064] (4) Extracellular matrix mineralization assay After sterilization and drying, the samples were placed in sterile 24-well plates, with 4 replicates per group. 2 × 10⁶ samples were inoculated per well. 4 MC3T3-E1 cells were placed on sterilized sample surfaces and incubated in an incubator, with the culture medium changed every 48 hours. After 14 and 21 days of culture, the original culture medium was removed, and the cells were gently washed with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed twice with ultrapure water, and then stained with 200 μL of Alizarin Red S solution in each well at room temperature in the dark for about 30 minutes. Residual staining was then washed away with ultrapure water. The formation of mineralized nodules on the sample surface was observed and recorded using a stereomicroscope.

[0065] Subsequently, 500 μL of pre-prepared 10% cetylpyridine chloride solution was added to each well. The plate was then fixed on a shaker and shaken until the mineralized nodules on the sample surface were completely dissolved (generally requiring 30 min). After mixing with a pipette, 150 μL was aspirated from each well into a 96-well plate, and the absorbance value was measured at 540 nm using a microplate reader to obtain the quantitative results of extracellular matrix mineralization assay.

[0066] (III) Evaluation of in vitro angiogenesis performance (1) Preparation of sample extract First, the samples were placed in a clean bench and disinfected by immersion in 75% alcohol for 30 minutes, followed by UV irradiation and drying. Then, they were placed in a 24-well plate, with each well spaced 1.25 cm apart. 2 Add serum-free high-glucose DMEM at a volume of / mL, incubate in a constant temperature incubator for 24 hours, and then collect the extract for later use.

[0067] (2) Cell scratch test First, the sterilized cell scratching insert (Ibidi, Germany) was placed into the 24-well plate using sterile forceps. Then, the centrifuged and resuspended HUVEC cells were sputtered at 5 × 10⁻⁶. 4Cells were added to each well of the insert at a density of 70 μL of cell suspension and incubated at 37 ℃ with 5% CO2 for 24 hours. After confirming confluence under a microscope, the insert was removed using sterile forceps in a clean bench. The exfoliated cells were gently washed away with PBS solution, and sample extract containing 1% serum was added for further incubation. Cell migration was observed and photographed at time points 0, 3, and 8 using an inverted phase-contrast microscope. Cell migration ability was quantitatively analyzed using ImageJ software. Specifically, the central blank area at 0 h was used as the baseline (S0), and the remaining blank areas at 3 h and 8 h (S3 and S8) were measured. The migration rate at 3 h was calculated as (S0-S3) / S0 × 100%, and the cell migration rate at 8 h was calculated in the same way.

[0068] (3) Tube formation experiment Two hours beforehand, thaw the dispensed matrix gel at 4°C. Pre-cool the 24-well plate and pipette tips at -20°C. In a clean bench, slowly pipette the pre-thawed matrix gel onto the bottom of the pre-cooled 24-well plate using a pre-cooled pipette tip, spreading it evenly in a circular motion with 1 mL of pipette tip (avoiding the gel from touching the sidewalls of the wells). Gently shake to remove air bubbles and incubate overnight at 4°C to allow the matrix gel to spread evenly. Place the plate in an incubator for 30 minutes to allow the matrix gel to solidify. Incubate HUVEC cells at 1.5 × 10⁻⁶ cells / well. 5 Cells / well were seeded at a density of [number] cells / well on a matrix gel, and after incubation for 4 hours with sample extract containing 10% serum, the cells were observed and recorded using an inverted microscope. Finally, the tube images were analyzed using AngloTool, and the results were processed using GraphPad.

[0069] (4) Statistical analysis: In this invention, all data were analyzed using Graphpad Prism with one-way ANOVA and Students't test. Statistical results are presented as mean ± standard deviation (Mean ± SD). Confidence coefficients were set at 95% (*p < 0.05).

[0070] (iv) Experimental results: (1) Results of MC3T3-E1 cell proliferation and morphology Figure 5 The above describes the detection results of MC3T3-E1 cell proliferation and morphology on the surfaces of four groups of samples (Zr, ZrP, Zr@LS, and ZrP@LS) prepared in Example 3 of this invention. Figure 5 The middle image shows representative images of MC3T3-E1 cell morphology at 1 day and 7 days (green / blue: cytoskeleton / nucleus). Figure 5In the middle B, the results represent the quantitative results of cell viability. *p<0.05, **p<0.01.

[0071] Quantitative cell viability analysis revealed the survival of MC3T3-E1 cells in four sample environments. The bar chart shows that on day 1, the osteoblast viability of the two groups coated with lithium disilicate was slightly lower than that of the control group, and the difference was statistically significant. However, on days 3 and 7, the cell viability of all treatment groups was slightly lower than that of the Zr group, but the difference was not statistically significant. This indicates that phosphorylation and the lithium disilicate coating have good biocompatibility.

[0072] Simultaneously, we performed cell morphology staining experiments on samples inoculated with MC3T3-E1 cells for 1 day and 7 days to visually understand the impact of each group of samples on cell proliferation. Cell morphology staining images are shown below. Figure 5 Image A shows the effect of surface treatment on cell spreading, with the cell nucleus and cell membrane stained blue and green fluorescence, respectively. From the images on day 1, we can see that the number of cells in the three treated groups was higher than in the control group, with the ZrP@LS group showing the best cell spreading. Images on day 7 show that the number of cells in all groups increased significantly, with overlapping pseudopodia and cells stacked in multiple layers.

[0073] (2) ALP and mineralization results in MC3T3-E1 cells For detailed results of ALP and mineralization assays in MC3T3-E1 cells, please refer to [link / reference]. Figure 6 ,exist Figure 6 In the figure, Figure A shows ALP staining images of different groups of samples (7 d), Figure B shows representative alizarin red staining images of different groups of samples (14 d and 21 d), and Figure C shows the quantitative results of ALP activity (7 d) and (D) mineralization (14 d and 21 d).

[0074] ALP activity assays are often used to assess the osteogenic differentiation potential of cells, especially in the early stages of osteoogenesis; while Alizarin Red staining is a method used to identify the mineralization capacity of cultured cells in vitro, i.e., to determine the mineralization capacity of the extracellular matrix. Figure 6 As shown in Figure A, under a stereomicroscope, the ALP staining results in the non-control group were better than those in the control group, but the staining results in the ZrP, Zr@LS, and ZrP@LS groups appeared to be similar. Figure 6 The results showed that the ALP activity in the ZrP@LS group was significantly higher than that in the Zr group after 7 days, with a statistically significant difference.

[0075] Regarding mineralization, the results of qualitative and quantitative analysis ( Figure 6Both B and D in the data corroborate that the ZrP@LS group showed the best results. Furthermore, in the alizarin red staining images, the differences in mineralized nodule staining among the Zr, ZrP, and Zr@LS groups were not significant, with the latter two groups showing only slightly better mineralization than the Zr group. Quantitative results showed that at 14 days, the Zr@LS group had better mineralization than the Zr group, and by day 21, the ZrP group also showed significantly better mineralization than the control group.

[0076] (3) HUVEC cell viability and cell scratch results Figure 7 The results show the HUVEC cell viability and cell scratch assay results. A represents the migration images of HUVECs at 0 h, 3 h, and 8 h; B represents the quantitative results of HUVEC cell viability; and C represents the quantitative analysis of cell migration area after culturing in different extracts for 3 h and 8 h.

[0077] First, HUVEC cells were incubated with the sample extract for 1 day, 4 days, and 7 days to test cell viability. The results are shown below. Figure 7 In section B, no statistically significant difference was found in the proliferation of HUVEC cells among the four groups of samples. Quantitative statistics... Figure 7 Figure A shows the effect of the extracts from the four groups of samples on HUVEC cell migration in the cell scratch assay. Figure 7 As shown, after 3 hours of incubation, the cell migration rate of the ZrP, Zr@LS, and ZrP@LS groups was faster than that of the Zr group, especially the ZrP@LS group. After 8 hours, the cell migration rate of the ZrP@LS group was still far ahead, and the scratch wound was basically healed. The ZrP and Zr@LS groups were also superior to the Zr group.

[0078] Through further statistical analysis of the scratch test, we... Figure 7 The percentage of cell area for each group relative to 0 hours was obtained in C, and it can be seen that the quantitative results are basically consistent with the trend of the scratch image.

[0079] (4) Results of HUVEC cell tubule formation Figure 8 The results of the HUVEC cell tubule formation assay are shown. In the figure, A is a tubule formation image at 4 h; B and C are the quantitative analysis of the number of tubule formation nodes and the percentage of blood vessel area, respectively.

[0080] The tubule formation assay is a classic method for assessing in vitro angiogenesis capacity. The number of nodes, i.e., the number of branching points, reflects the complexity of the vascular network; the number of branches reflects the degree of differentiation of the vascular network. More nodes and branches indicate more active angiogenesis and a more mature vascular network. For example... Figure 8As shown in Figure A, the Zr group has fewer nodes and relatively less blood vessel area, while the other three groups, especially ZrP@LS, have more obvious tubular structures, which, as seen in the figure, already show initial tubular formation. Quantitative results of the number of tubular formation nodes and the percentage of blood vessel area confirm that the ZrP@LS group has the best angiogenesis function.

[0081] We conducted a series of experiments using HUVEC cells. Based on the cell scratch assay and tubule formation assay, it is clear that ZrP, Zr@LS, and ZrP@LS all have good angiogenesis-inducing abilities. In the cell migration assay, HUVEC cells migrated faster in the first 3 hours than in hours 3-8. This may be due to insufficient energy required for cell culture in the later stages and the increased susceptibility of HUVEC cells to Li₂ in the later stages. + Si 4+ Excessive consumption is related to a decrease in cell migration ability in the later stages. The effect of the material on endothelial cells can be explained by the following three points: ①Li + It can significantly promote angiogenesis by activating signaling pathways such as Wnt / β-catenin and PI3K / Akt, upregulating VEGF expression, and promoting endothelial cell proliferation; ②Si 4+ By stimulating the expression of angiogenic factors such as VEGF, endothelial cell proliferation, migration, and lumen formation are promoted, thus significantly promoting angiogenesis. ③ Although there is no literature confirming that phosphate ions directly affect angiogenesis, they can indirectly promote angiogenesis through energy metabolism, signal transduction, bone formation, cell proliferation and differentiation, matrix mineralization, and inflammatory responses. These points well explain the results of the cell migration experiments in each group. The angiogenesis experiment showed a trend consistent with the cell scratch assay. In summary, zirconium phosphate and lithium disilicate coatings, through the release of ions from their surface coatings, stimulate early migration and angiogenesis of HUVEC cells.

[0082] In summary, among the four groups of samples prepared by this invention: 1) all four groups of samples showed good cell compatibility; 2) the ZrP, Zr@LS, and ZrP@LS groups had good osteogenic properties, with the ZrP@LS group showing the best effect; 3) the ZrP, Zr@LS, and ZrP@LS groups significantly improved the migration ability of endothelial cells and induced in vitro angiogenesis, with the ZrP@LS group showing the best experimental effect.

[0083] Experimental Example 3: Evaluation of the in vivo osteogenic and angiogenic properties of lithium disilicate coatings (a) Laboratory animals Twelve female New Zealand white rabbits, 6 months old and weighing 3-3.5 kg, were provided by the Wenzhou Institute of the Chinese Academy of Sciences (CAS), with ethics review approval number WIUCAS24052702. All procedures performed on the animals during the experiment complied with animal ethics requirements.

[0084] (II) Experimental Methods (1) Implant preparation Because the promoting effect of Zr@LS on osteogenic and angiogenesis was less significant than that of ZrP@LS in in vitro experiments, only three groups were set up for in vivo experiments: Zr, ZrP, and ZrP@LS. The preparation method was the same as that for the zirconium sheets of the Zr, ZrP, and ZrP@LS groups.

[0085] (2) Animal model preparation Rabbits were randomly divided into three groups: Zr, ZrP, and ZrP@LS. All rabbits underwent general anesthesia before surgery, with local anesthesia administered to the femoral surgical area to further reduce the anesthetic effect. Hair was shaved from both femurs, and the femoral area was disinfected with povidone-iodine to keep the surgical area clean.

[0086] Surgical steps: 1) Incision Design and Exposure: A longitudinal incision of approximately 15 mm was made lateral to the patellar ligament in both hind legs of all rabbits to expose the lateral femoral condyle. 2) Implantation Site Preparation and Implant Placement: A cylindrical bone defect with a diameter of 4 mm and a depth of 8 mm was created near the femoral epiphysis using a 4 mm drill bit. After thoroughly rinsing the fractured bone fragments with saline solution, a pre-prepared and sterilized zirconium screw was implanted. 3) Suturing and Postoperative Care: The surgical wound was sutured in layers. Penicillin was administered for 3 days postoperatively to prevent infection. 4) Tissue Collection: Rabbits were euthanized 6 weeks post-implantation, and the femur was harvested for histological staining experiments.

[0087] (3) Histological analysis Femoral bone samples containing implants were collected and fixed in 4% paraformaldehyde for 48 hours. Subsequently, the samples were decalcified in 12% EDTA solution for 45 days, with the decalcification solution changed every three days. After decalcification, the implants were gently separated from the bone tissue, and the bone samples underwent graded ethanol dehydration (70%, 80%, 90%, 100%), paraffin embedding, and section preparation. The sections were then stained with hematoxylin and eosin (HE), Masson's trichrome stain, and vWF and CD34 immunohistochemical staining for analysis. Finally, the stained sections were scanned and recorded using a slide scanning imaging system for quantitative evaluation.

[0088] (4) Statistical analysis: In this study, all data were analyzed using Graphpad Prism one-way ANOVA and the Students't test. All results are expressed as mean ± standard deviation (Mean ± SD). The confidence coefficient was set at 95% (*p < 0.05).

[0089] (III) Experimental Results (1) Analysis of osteosynthesis tissue sections Figure 9 The results of bone-bonded tissue section analysis of the three groups of samples (Zr, ZrP, and ZrP@LS) prepared in Example 3 of this invention are shown below. Figure 9 Image A shows HE and Masson staining images of the bone tissue surrounding the implant 6 weeks after implantation. Images B and C show the quantitative results of HE and Masson staining. *p<0.05, **p<0.01.

[0090] Figure 9 The "A" in the figure indicates that HE and Masson staining reflect early new bone formation and osseointegration at the bone interface around the implant. In Masson staining, the blue area represents immature trabeculae, and the red area represents mature trabeculae. At different magnifications, it can be observed that the blue area in the tissue surrounding the Zr implant is significantly less than in the other two groups, while the blue area in the ZrP@LS group is the thickest and largest. HE staining also shows less osteoid tissue around the Zr implant, while osteoid tissue is more prominent in the other two groups. This indicates that at 6 weeks post-operation, the ZrP and ZrP@LS groups show better new bone formation, especially the latter. Figure 9 B and C in the chart illustrate this point more intuitively through a bar chart.

[0091] (2) Analysis of angiogenic tissue sections Figure 10 The results of angiogenic tissue section analysis of three groups of samples: Zr, ZrP, and ZrP@LS are shown. Figure 10 Image A shows the immunohistochemical staining results of vWF and CD34 in the peri-implant tissue 6 weeks after implantation. Figure 10 B and C represent the quantitative results of immunohistochemical staining for vWF and CD34, respectively. *p<0.05, **p<0.01.

[0092] To verify the in vivo angiogenesis effect of the samples, we performed vWF and CD34 immunohistochemical staining on tissue sections from three groups of samples. Figure 10 As shown in Figure A, the yellow or brown areas in the image are positive regions. We can see that the positive area is largest in the ZrP@LS group. Furthermore, continuous vascular structures are observed in both the ZrP and ZrP@LS groups, indicating a good induction of angiogenesis in surrounding tissues after implantation. The ZrP@LS group implant exhibits the best pro-angiogenic properties. This is also clearly demonstrated by quantitative calculations of the immunohistochemical results.

[0093] In summary, the ZrP@LS group samples provided by this invention can promote new bone formation around the implant and improve osseointegration; at the same time, the ZrP@LS group samples have the function of inducing angiogenesis around the implant, which can promote tissue repair.

[0094] 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 producing a medical implant having a coating of lithium disilicate, characterized in that The method comprises the steps of: (1) pre-treating the surface of the preform: placing the preform in a high-temperature environment for staged sintering, and then performing surface polishing, cleaning and drying treatment; wherein the staged sintering procedure is: first, heating to 800-1000℃ at a heating rate of 3-8℃ / min and maintaining for 20-40min, and then heating to 1300-1700℃ at a heating rate of 2-5℃ / min and maintaining for 100-150min before entering the cooling procedure; during the cooling process, the preform is first cooled to 700-400℃ with the furnace, and then cooled to 250-350℃ with the furnace opened, and finally the preform is taken out and naturally cooled to room temperature; (2) preparing the coating by negative pressure infiltration combined with sol-gel method: taking lithium ethoxide and tetraethyl orthosilicate as the lithium source and silicon source for the reaction, mixing lithium ethoxide and tetraethyl orthosilicate with a molar ratio of 1-1.15:1 at room temperature, and then reacting for 2-4h under stirring to obtain lithium disilicate sol; then, the preform pre-treated in step (1) is soaked in the lithium disilicate sol for 3-7min, and then sol infiltration is performed in a negative pressure of 30mmHg or above for 3-10min, and then an ethanol aqueous solution is added to initiate the gelation reaction; after the gelation reaction is completed, the preform is taken out, dried and sintered to obtain a medical implant body with a lithium disilicate coating.

2. The method of producing a medical implant body having a coating of lithium disilicate according to claim 1, characterized in that The material of the preform is zirconia.

3. Process for the production of a medical implant with a coating of lithium disilicate according to claim 1 or 2, characterized in that The preform pre-treated in step (1) is first subjected to surface phosphating treatment, and then the lithium disilicate coating is prepared according to the method in step (2).

4. The method of producing a medical implant body having a coating of lithium disilicate according to claim 3, characterized in that The surface phosphating treatment process is as follows: First, immerse the preform in a phosphoric acid solution in a high-pressure reaction kettle, then seal the high-pressure reaction kettle and place it in a vacuum drying oven at 140-180℃ for 20-30h, wherein the concentration of the phosphoric acid solution is 2-3wt%.

5. The method of producing a medical implant body having a coating of lithium disilicate according to claim 1, characterized in that The staged sintering procedure in step (1) is as follows: First, heat to 900℃ at a heating rate of 5℃ / min and maintain for 30min, and then heat to 1500℃ at a heating rate of 3℃ / min and maintain for 120min before entering the cooling procedure; during the cooling process, the preform is first cooled to 500℃ with the furnace, and then cooled to 300℃ with the furnace half-opened, and finally the preform is taken out and naturally cooled to room temperature.

6. The method of producing a medical implant body having a coating of lithium disilicate according to claim 1, characterized in that The surface polishing, cleaning and drying treatment process of the preform in step (1) is as follows: Gradient polishing is performed using 400-2000 mesh silicon carbide sandpaper in sequence; Then, ultrasonic cleaning is performed in acetone, ethanol solution and ultrapure water, respectively; Finally, dry in a vacuum drying oven and store dry.

7. The method of producing a medical implant body having a coating of lithium disilicate according to claim 1, characterized in that The sintering procedure in step (2) is: First, heat to 500-700℃ at a heating rate of 1.5-2.5℃ / min and maintain for 1.5-2.5h, and then heat to 900-1000℃ at a heating rate of 1.3-2.5℃ / min and maintain for 1.8-2.3h, and finally cool to room temperature at a rate of 3-7℃ / min.

8. The method of producing a medical implant body with a coating of lithium disilicate according to claim 7, characterized in that The sintering procedure in step (2) is: First, the temperature is raised to 600°C at a rate of 2°C / min, and then held for 2 h. Next, the temperature is raised to 950°C at a rate of 2°C / min, and then held for 2 h. Finally, the temperature is lowered to room temperature at a rate of 5°C / min.

9. A medical implant having a coating of lithium disilicate, characterized in that The medical implant is prepared according to the preparation method of any one of claims 1-8.

10. Use of the medical implant with lithium disilicate coating according to claim 9 in the field of the preparation of dental restorations and bone restorations.

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