A bioceramic nanostructured imitation tooth enamel with an amorphous-crystalline structure, its preparation method and application

Abstract

This invention relates to a dental enamel nanostructured amorphous-crystalline bioceramic, its preparation method, and its applications. The dental enamel nanostructured amorphous-crystalline bioceramic comprises: a three-dimensional continuous amorphous calcium phosphate matrix polymerized from calcium phosphate ion clusters (CPO), and inorganic micro / nanomaterials uniformly dispersed within the three-dimensional continuous amorphous calcium phosphate matrix; based on the total mass of the dental enamel nanostructured amorphous-crystalline bioceramic being 100%, the amorphous calcium phosphate matrix accounts for 20-80% of the mass, and the inorganic micro / nanomaterials account for 20-80% of the mass.

Description

Technical Field

[0001] This invention belongs to the field of bioceramic materials, specifically relating to a nano-amorphous-crystalline bioceramic with a tooth-like enamel structure, its preparation method, and its application. Background Technology

[0002] Bones are the main organs in living organisms, providing support and protection. They are a typical example of high-strength and high-toughness natural materials. Despite this, bone damage caused by accidents, diseases, and aging is very common. Repairing damaged bone tissue with artificial materials has always been a goal in clinical treatment. Bioceramics, with their excellent biocompatibility and bioactivity, are widely used in bone repair. Bioceramics are mostly prepared by high-temperature sintering, thus possessing high strength. However, their inherent brittleness and high stiffness still present challenges such as fragility, difficulty in processing, and poor bone mechanics compatibility, limiting their wider clinical application. Furthermore, the high-temperature sintering process affects the structure, physicochemical properties, and mechanical properties of bioceramic raw materials, reducing their bioactivity. Therefore, bioceramics prepared by traditional high-temperature sintering methods face the challenge of simultaneously achieving strength, toughness, and bioactivity. Developing new strategies for bioceramic preparation to produce bioceramics with excellent strength, toughness, and good bioactivity is of great significance for improving the clinical application of bioceramic materials and promoting new applications of bioceramics.

[0003] Improving the strength, toughness, and crack resistance of synthetic ceramic materials simultaneously is a challenging research area. However, there are inorganic materials in nature that can unify contradictory mechanical properties, and human tooth enamel is one of them. Tooth enamel is the hardest tissue in the human body, and its microstructure exhibits a unique "enamel prism" morphology. These prisms are arranged radially outward from the dentin-dentin junction and are composed of highly mineralized hydroxyapatite crystals. Between the crystals is a very thin layer of inorganic amorphous interphase. This "fiber-reinforced composite material"-like structure endows tooth enamel with extremely high hardness and wear resistance, while its multi-level ordered structure can also prevent crack propagation and dissipate chewing energy. The "amorphous-crystalline structure" in tooth enamel is its main component. This unique design, combining hard crystals with disordered amorphous materials, significantly improves the overall mechanical properties of tooth enamel through the synergistic effect of multiple core mechanisms. First, the highly oriented crystal array forms a rigid framework that resists external indentation and wear, providing high hardness and high elastic modulus. Second, the amorphous interphase layer fills the spaces between the crystals, and its interface with the crystals has extremely strong chemical bonds, effectively strengthening the connection between adjacent crystals, inhibiting interface slip and crack initiation. The amorphous layer can restrict the dislocation movement and deformation space of the internal crystals, allowing the material to withstand higher stresses. When crack propagation encounters the crystal / amorphous interface, the disordered structure of the amorphous phase can induce crack deflection, bridging, or absorption by the inorganic amorphous layer, thereby consuming a large amount of fracture energy, preventing the crack from penetrating further, and endowing the material with toughness. In summary, the amorphous-crystalline structure ingeniously reconciles the contradiction between high hardness and high toughness in the material, enabling tooth enamel to be both hard and wear-resistant while resisting catastrophic fracture. The nano-amorphous-crystalline structure of tooth enamel provides inspiration for the mechanical property design of bioceramics for artificial bone repair. Summary of the Invention

[0004] To address the problems of high brittleness, high modulus, and insufficient bone biomechanical compatibility in traditional high-temperature sintered bioceramics, this invention provides a method for preparing a tooth-inspired enamel nanocrystalline-amorphous bioceramic and its application in bone tissue repair. This tooth-inspired enamel nanocrystalline-amorphous bioceramic is prepared using a "hydroxyapatite nanowire assembly strategy based on calcium phosphate inorganic adhesive." It exhibits flexural strength approaching that of traditional high-temperature sintered hydroxyapatite ceramics, superior toughness and crack resistance, and a flexural modulus matching that of natural bone tissue. Furthermore, it possesses good biocompatibility and in vivo bone integration, showing broad prospects for clinical applications.

[0005] In a first aspect, the present invention provides a bioceramic with a nano-amorphous-crystalline structure resembling tooth enamel, the bioceramic comprising: a three-dimensional continuous amorphous calcium phosphate matrix polymerized from calcium phosphate ion clusters (CPO), and inorganic micro / nano materials uniformly dispersed within the three-dimensional continuous amorphous calcium phosphate matrix.

[0006] Preferably, based on the total mass of the dental enamel nano-amorphous-crystalline bioceramic structure being 100%, the mass percentage of the three-dimensional continuous amorphous calcium phosphate matrix is ​​20-80%, and the mass percentage of the inorganic micro-nano materials is 20-80%.

[0007] Preferably, the size of the calcium phosphate ion clusters is 8–10 nm; The inorganic micro / nano material is at least one of hydroxyapatite nanowires, hydroxyapatite nanoparticles, hydroxyapatite microrods, calcium silicate nanowires, alumina nanoparticles, magnesium oxide nanoparticles, and silicon nitride microparticles, preferably hydroxyapatite nanowires; more preferably, the hydroxyapatite nanowires have a diameter of 20–30 nm and a length of 80–100 μm.

[0008] Preferably, the dental enamel nano-amorphous-crystalline bioceramic is a plate-like ceramic with a dense internal structure; Preferably, the length of the dental enamel nano-amorphous-crystalline bioceramic is not less than 10 mm, the width is not less than 5 mm, and the thickness is not less than 0.5 mm.

[0009] Preferably, the flexural strength of the dental enamel-like nanocrystalline bioceramic is 41.4–117.8 MPa, the flexural modulus is 10.2–54.3 GPa, the compressive strength is 102.2–168.8 MPa, and the crack initiation fracture toughness is 3.82–4.52 MPa·m. 1 / 2 .

[0010] Secondly, the present invention provides a method for preparing the above-mentioned dental enamel nano-amorphous-crystalline bioceramic, comprising: mixing calcium phosphate ion clusters and inorganic micro / nanomaterials at a mass ratio, and stirring to obtain a mixed suspension; then, filtering the mixed suspension by vacuum filtration to obtain a calcium phosphate ion cluster-inorganic micro / nanomaterial composite gel; then, compacting the calcium phosphate ion cluster-inorganic micro / nanomaterial composite gel by a pressure-assisted densification process; finally, drying and densifying to obtain the dental enamel nano-amorphous-crystalline bioceramic.

[0011] Preferably, the calcium phosphate ion clusters are prepared by titration. The preparation method of the calcium phosphate ion clusters includes: dispersing 11.76 g CaCl2·2H2O in 1.6 L of anhydrous ethanol, sonicating for 30 min, adding 221.79 mL of triethylamine (TEA), and stirring for another 30 min to obtain a stable mixed solution. Then, slowly adding a pre-prepared phosphoric acid (4.8 mL) / anhydrous ethanol (80 mL) mixed solution, stirring for 12 h, to obtain a calcium phosphate ion cluster suspension. Washing the suspension three times with anhydrous ethanol to remove excess triethylamine, and finally dispersing it in anhydrous ethanol to obtain a calcium phosphate ion cluster suspension with a concentration of approximately 10 mg / mL. The calcium phosphate ion clusters are calcium phosphate inorganic glue, exhibiting polymerization characteristics similar to polymer materials. In the above titration synthesis process, discrete calcium phosphate ion clusters are stably present in anhydrous ethanol by the end-capping effect of small molecule triethylamine (TEA); in the subsequent pressure-assisted densification process, anhydrous ethanol and TEA volatilize, the end-capping effect is released, and the discrete calcium phosphate ion clusters form a continuous calcium phosphate material, i.e., calcium phosphate matrix, by linking and polymerizing with each other.

[0012] Preferably, when the inorganic micro / nanomaterial is hydroxyapatite nanowires, the hydroxyapatite nanowires are prepared using a calcium oleate precursor solvothermal method. The preparation method includes: mixing 210 mL of oleic acid, 120 mL of methanol, and 270 mL of deionized water while continuously stirring; adding sodium hydroxide aqueous solution (300 mL, 0.07 g / mL), calcium chloride aqueous solution (240 mL, 0.0275 g / mL), and sodium dihydrogen phosphate dihydrate aqueous solution (360 mL, 0.052 g / mL) sequentially at 25–35 minutes intervals; then transferring the mixed solution to a high-pressure reactor and reacting at 180–190 °C for 24–26 hours. After the reaction is completed and the temperature drops to room temperature, the white precipitate of hydroxyapatite nanowires at the bottom of the reactor is collected, washed three times alternately with anhydrous ethanol and deionized water, vacuum filtered, and dried to obtain hydroxyapatite nanowires.

[0013] Preferably, the mass ratio of the calcium phosphate ion clusters to the hydroxyapatite nanowires is 2:1 to 1:3, and more preferably 1:1.5 to 1:2.5.

[0014] Preferably, the stirring time is 24–48 h, more preferably 32–40 h.

[0015] Preferably, the parameters of the pressure-assisted densification process include: pressure of 100–900 MPa, preferably 350–450 MPa; temperature of 25–350 °C, preferably 90–110 °C; and time of 1 min–24 h, preferably 3.5–4.5 h.

[0016] Thirdly, the present invention provides an application of the above-mentioned dental enamel nano-amorphous-crystalline structure bioceramic in the preparation of bone repair materials. Beneficial effects

[0017] (1) This invention proposes a new strategy for the preparation of bioceramics based on the "assembly of hydroxyapatite nanowires based on calcium phosphate inorganic glue", and prepares a bioceramic with a nano-amorphous-crystalline structure similar to tooth enamel (CPO / HAP composite ceramic). This bioceramic has excellent mechanical properties, good bone mechanical matching, biocompatibility and bone integration, which enables it to have better integration with bone tissue after implantation to enhance the bone repair effect. (2) The bioceramic of the present invention, which combines excellent mechanical properties and good biological functions, is a composite of flexible hydroxyapatite nanowires with a polymerizable calcium phosphate ion cluster (i.e., CPO inorganic glue). Based on the polymerization characteristics of calcium phosphate inorganic glue, high-performance dental enamel nano-amorphous-crystalline structure CPO / HAP composite ceramics are prepared by combining "self-polymerization of CPO inorganic glue - fusion of CPO / HAP interface" under certain temperature and pressure conditions. (3) The preparation method of the bioceramic nano-amorphous-crystalline structure of dental enamel provided by the present invention is simple, low in energy consumption and low in cost, and can be manufactured on a large scale; (4) This invention is the first to prepare a bioceramic nano-amorphous-crystalline structure with excellent strength, toughness and crack resistance, bone mechanical matching, biocompatibility and bone integration, which promotes the development of bioceramic science and its clinical application. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the preparation process of the CPO / HAP composite ceramic in Example 1; Figure 2 This is the synthesis and characterization of CPO ion clusters in Example 1; where (a) is TEM images of CPO ion clusters at low and high concentrations, (b) is particle size analysis of CPO ion clusters, and (c) is FTIR analysis spectrum of CPO ion clusters. Figure 3 The polymerization characteristics of CPO ion clusters in Example 1 are shown; (a) is a photograph of CPO clusters and CPO gel, (b) is the injection and polymerization of CPO gel, (c) is a photograph of the CPO gel polymerization process, (d) is a SEM image of CPO bulk material, and (e) is an XRD pattern of CPO bulk material. Figure 4The preparation process of CPO / HAP composite ceramic in Example 1 is shown; where (a) is a photograph of the preparation process of CPO / HAP composite ceramic, and (b) is a photograph of large-size and irregularly shaped CPO / HAP composite ceramic. Figure 5 The mass ratio of CPO ion clusters to HAP nanowires was optimized; where (a) is the bending strength of CPO / HAP composite ceramics under different component mass ratios, (b) is the bending modulus of CPO / HAP composite ceramics under different component mass ratios, and (c) is the bending stress-strain curve of CPO / HAP composite ceramics under different component mass ratios. Figure 6 The bending properties of CPO / HAP composite ceramics under different pressing times are shown in Figure 1. (a) represents the bending strength of CPO / HAP composite ceramics under different pressing times, (b) represents the bending modulus of CPO / HAP composite ceramics under different pressing times, and (c) represents the bending stress-strain curves of CPO / HAP composite ceramics under different pressing times. Figure 7 The images show cross-sectional SEM images of CPO / HAP composite ceramics prepared by pressing for 5 min, (b) cross-sectional SEM images of CPO / HAP composite ceramics prepared by pressing for 1 h, (c) cross-sectional SEM images of CPO / HAP composite ceramics prepared by pressing for 4 h, and (d) cross-sectional SEM images of CPO / HAP composite ceramics prepared by pressing for 24 h. Figure 8 TEM images of CPO / HAP composite ceramics at different pressing times; where (a) is a cross-sectional TEM image of CPO / HAP composite ceramics prepared by pressing for 5 min at different magnifications, and (b) is a cross-sectional TEM image of CPO / HAP composite ceramics prepared by pressing for 24 h at different magnifications. Figure 9 SEM images of the bending properties and cross sections of CPO / HAP composite ceramics under different preparation pressures are shown. Among them, (a) is the bending strength of CPO / HAP composite ceramics under different pressure conditions, (b) is the bending modulus of CPO / HAP composite ceramics under different pressure conditions, (c) is the bending stress-strain curve of CPO / HAP composite ceramics under different pressure conditions, and (d) is the cross section SEM image of CPO / HAP composite ceramics prepared under different pressure conditions. Figure 10The images show the flexural properties and cross-sectional SEM images of CPO / HAP composite ceramics prepared at different temperatures. Among them, (a) shows the flexural strength of CPO / HAP composite ceramics under different temperature conditions, (b) shows the flexural modulus of CPO / HAP composite ceramics under different temperature conditions, (c) shows the flexural stress-strain curves of CPO / HAP composite ceramics under different temperature conditions, and (d) shows the cross-sectional SEM images of CPO / HAP composite ceramics prepared under different temperature conditions. Figure 11 The in-situ temperature-varying XRD patterns of CPO bulk material and CPO / HAP composite ceramic are shown below; where (a) is the in-situ temperature-varying XRD pattern of CPO bulk material and (b) is the in-situ temperature-varying XRD pattern of CPO / HAP composite ceramic. Figure 12 Optical photographs and surface and cross-sectional SEM images of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics are shown. Among them, (a) is an optical photograph, surface SEM image, and cross-sectional SEM image of HAP ceramics; (b) is an optical photograph, surface SEM image, and cross-sectional SEM image of PVA / HAP composites; (c) is an optical photograph, surface SEM image, and cross-sectional SEM image of S-HAP ceramics; and (d) is an optical photograph, surface SEM image, and cross-sectional SEM image of CPO / HAP composite ceramics. Figure 13 The images shown are TEM and selected area electron diffraction images of the CPO / HAP composite ceramic in Example 1. Figure 14 The images are TEM images of different materials; (a) is a TEM image of HAP ceramic at different magnifications, (b) is a TEM image of PVA / HAP composite material at different magnifications, and (c) is a TEM image of S-HAP ceramic at different magnifications. Figure 15 SEM images and elemental distributions of composite ceramics assembled from different inorganic micro / nano materials based on CPO inorganic adhesive; Figure 16The bending and compressive properties of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics are compared. Among them, (a) shows the bending strength of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; (b) shows the bending modulus of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; (c) shows the bending stress-strain curves of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; and (d) shows the compressive strength of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics. Figure 17 Fracture toughness tests and crack propagation path SEM images of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics are presented. Among them, (a) is a schematic diagram of fracture toughness test by the single-sided notched beam method, (b) shows the fracture toughness of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics, and (c) shows the crack propagation path SEM images of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics. Figure 18 Hydrophilicity and hydrophobicity and wet structural stability of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics were analyzed; (a) hydrophilicity and hydrophobicity analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics, and (b) structural stability analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics in water. Figure 19 Wet mechanical stability analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics; Figure 20 The wet mechanical properties of CPO / HAP composite ceramics and related photographs are shown; (a) shows the changes in bending properties under different immersion times in water, and (b) shows the optical photographs of CPO / HAP composite ceramics after immersion in water for 30 days. Figure 21 The images show surface and cross-sectional SEM images of CPO / HAP composite ceramics after immersion in water for 30 days; where (a) is a surface SEM image of CPO / HAP composite ceramics after immersion in water for 30 days, and (b) is a cross-sectional SEM image of CPO / HAP composite ceramics after immersion in water for 30 days. Figure 22The in vitro biocompatibility of S-HAP ceramics and CPO / HAP composite ceramics was evaluated. Among them, (a) is the immunofluorescence staining image of cells after co-culturing with ceramics for different time periods, (b) is the cell proliferation image of cells co-cultured with ceramics, (c) is the immunofluorescence staining image of intracellular BSP protein, and (d) is the quantitative statistical graph of intracellular BSP protein immunofluorescence intensity. Figure 23 The in vivo biocompatibility of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics was evaluated. Among them, (a) is a schematic diagram of the rat subcutaneous model creation process, and (b) is a tissue section H&E staining image 14 days after the material was implanted subcutaneously in rats. Figure 24 Micro-CT scan images related to in vivo bone integration analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics; where (a) shows the modeling steps of the rabbit femoral condyle defect model, and (b) shows the micro-CT analysis images of the blank group and PVA / HAP composites, S-HAP ceramics and CPO / HAP composite ceramics after 8 weeks of implantation. Figure 25 Van-Gieson stained images of tissue sections related to in vivo bone integration analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics. Detailed Implementation

[0019] The present invention is further illustrated by the embodiments described below. It should be understood that the embodiments described below are for illustrative purposes only and are not intended to limit the present invention.

[0020] First, the present invention provides a dental enamel nano-amorphous-crystalline structure bioceramic, which comprises: a three-dimensional continuous amorphous calcium phosphate matrix polymerized from calcium phosphate ion clusters, and hydroxyapatite nanowires uniformly dispersed within the three-dimensional continuous amorphous calcium phosphate matrix.

[0021] In some embodiments, based on the total mass of the dental enamel nano-amorphous-crystalline bioceramic as 100%, the mass percentage of the amorphous calcium phosphate matrix is ​​20-80%, and the mass percentage of the uniformly dispersed hydroxyapatite nanowires is 20-80%. The dental enamel nano-amorphous-crystalline bioceramic provided by this invention is mainly composed of bioactive hydroxyapatite nanowires. The calcium phosphate ion clusters act as an inorganic glue to assemble the hydroxyapatite nanowires. This bioceramic preparation method avoids the high-temperature process of traditional high-temperature sintering, greatly reducing energy consumption. Simultaneously, the unsintered hydroxyapatite nanowires can maintain their initial high aspect ratio morphology, flexibility, and bioactivity. The prepared bioceramic also benefits from the biomimetic structure design, achieving excellent comprehensive mechanical properties that are more compatible with the mechanical properties of natural bone tissue.

[0022] The dental enamel nano-amorphous-crystalline bioceramic provided by this invention is a plate-like ceramic with a dense internal structure. Preferably, the shape of the dental enamel nano-amorphous-crystalline bioceramic is not limited to plate shape, but can also be cylindrical, curved, tubular, or other irregular shapes. Its size can be adjusted as needed. Preferably, the length of the dental enamel nano-amorphous-crystalline bioceramic is not less than 10 mm, the width is not less than 5 mm, and the thickness is not less than 0.5 mm.

[0023] In this invention, the dental enamel-inspired nanocrystalline-amorphous bioceramic is composed of an amorphous calcium phosphate matrix and hydroxyapatite nanowires uniformly dispersed within the matrix, forming a nanocrystalline-amorphous interface. Based on the synergistic strengthening and toughening effect of the nanocrystalline-amorphous interface structure and the flexible hydroxyapatite nanowires, the biomimetic ceramic possesses high strength, toughness, and crack resistance. Specifically, its strengthening and toughening mechanism is as follows: during material failure, as the calcium phosphate matrix fractures, the hydroxyapatite nanowires act as a bridge, gradually elongating, pulling out, and breaking as the crack propagates; simultaneously, when the crack propagates and encounters the amorphous-crystal interface, the disordered structure of the amorphous phase can induce crack deflection, bridging, or absorption by the inorganic amorphous layer. All of these processes consume a large amount of fracture energy, preventing further crack propagation, thereby endowing the material with strength and toughness. The bioceramic nanostructured amorphous-crystalline structure of dental enamel provided by this invention exhibits a flexural strength of 41.4–117.8 MPa, a flexural modulus of 10.2–54.3 GPa, a compressive strength of 102.2–168.8 MPa, and a crack initiation fracture toughness of 3.82–4.52 MPa·m. 1 / 2 .

[0024] The following exemplarily illustrates the preparation method of the dental enamel nano-amorphous-crystalline structure bioceramic provided by the present invention (wherein, the inorganic micro-nano material is hydroxyapatite nanowires).

[0025] Synthesis of calcium phosphate ion clusters. Calcium phosphate ion clusters were synthesized by titration: Triethylamine, a capping agent, was added to a calcium chloride dihydrate / anhydrous ethanol solution, followed by the slow addition of phosphoric acid / anhydrous ethanol solution to form calcium phosphate ion clusters. As an example, 11.76 g of CaCl2·2H2O was dispersed in 1.6 L of anhydrous ethanol, sonicated for 30 min, and then 221.79 mL of TEA was added. Stirring was continued for another 30 min to obtain a stable mixed solution. Subsequently, a pre-prepared phosphoric acid (4.8 mL) / anhydrous ethanol (80 mL) mixed solution was slowly added dropwise. After stirring for 12 h, a calcium phosphate ion cluster suspension was obtained. The suspension was washed three times with anhydrous ethanol to remove excess small-molecule TEA. Finally, the cleaned calcium phosphate ion clusters were dispersed in anhydrous ethanol (concentration approximately 10 mg / mL).

[0026] Synthesis of hydroxyapatite nanowires. HAP nanowires were synthesized using a conventional calcium oleate precursor solvothermal method: sodium hydroxide, calcium chloride, and sodium dihydrogen phosphate dihydrate were added sequentially to a mixed solution of deionized water, oleic acid, and methanol, followed by a hydrothermal reaction to obtain HAP nanowires. As an example: 270 mL of deionized water and 210 mL of oleic acid (C...) were mixed... 18 H 34 O2) and 120 mL of methanol (CH4O) were mixed under mechanical stirring. Every 30 minutes, sodium hydroxide aqueous solution (21 g / 300 mL), anhydrous calcium chloride aqueous solution (6.66 g / 240 mL), and sodium dihydrogen phosphate dihydrate aqueous solution (18.72 g / 360 mL) were slowly added to the mixed solution in sequence. Then, the reaction system was transferred to a polytetrafluoroethylene reaction vessel and kept at 180 °C for 24 h. After the temperature dropped to room temperature, the white precipitate HAP nanowires at the bottom of the reaction vessel was collected. The precipitate was washed three times alternately with anhydrous ethanol and deionized water. After cleaning, the mixture was filtered to obtain HAP nanowire filter cake, which was dried in a 60 °C oven for subsequent research.

[0027] Preparation of CPO / HAP composite ceramics. A certain volume of CPO ion cluster suspension was measured according to a certain mass ratio, and a certain mass of HAP nanowires was added to it. After stirring, a uniformly dispersed CPO / HAP mixed suspension was obtained. A certain amount of the mixed suspension was measured and filtered using a vacuum filter to obtain a CPO / HAP composite gel. Finally, the CPO / HAP composite gel was placed in a mold and pressed and compacted under certain pressure and temperature conditions. After holding the pressure for a certain time, and then drying and densifying, a dental enamel nano-amorphous-crystalline structure bioceramic was obtained. In the pressure-assisted densification stage of the CPO / HAP composite ceramic preparation process, the CPO ion clusters were regarded as an inorganic glue. The originally discrete CPO ion clusters gradually formed a large-size three-dimensional continuous matrix network through self-polymerization, and at the same time, a bonding interface was gradually formed with the HAP nanowires dispersed in the matrix, finally preparing a dental enamel nano-amorphous-crystalline structure bioceramic.

[0028] In some embodiments, the mass ratio of CPO ion clusters to HAP nanowires can be 2:1 to 1:3. Within this range, CPO / HAP composite ceramics can be prepared by the above preparation method, with a preferred mass ratio of 1:2. CPO / HAP composite ceramics are essentially a composite material, where CPO ion clusters polymerize to form a three-dimensional continuous CPO matrix, while HAP nanowires are a fibrous material that further reinforces the CPO matrix to obtain high-performance CPO / HAP composite ceramics. The mass ratio of the two has a decisive influence on the mechanical properties of the composite ceramic; both excessively high and excessively low mass ratios of CPO ion clusters to HAP nanowires will lead to a decrease in the mechanical properties of the CPO / HAP composite ceramic. If the CPO ion cluster content is too high, the CPO / HAP composite gel will be difficult to press into CPO / HAP composite ceramics due to its excessive fluidity. This is because the CPO gel obtained by vacuum filtration of CPO ion clusters has good fluidity, making it difficult to obtain dense CPO material by the "vacuum filtration-pressure assisted densification" method alone. The addition of HAP nanowires can make the CPO / HAP composite gel compressible, so the CPO content cannot be too high. When the CPO content is too low, the matrix component will be too small to completely encapsulate the HAP nanowires, making it difficult to fully build the bonding interface between the CPO matrix and HAP nanowires inside the ceramic, thus leading to a decrease in the mechanical properties of the CPO / HAP composite ceramic.

[0029] In some embodiments, the mixing and stirring time is 24 to 48 hours, preferably 36 hours. The more fully and uniformly the CPO ion clusters and HAP nanowires are mixed, the better it is for the construction of the interface between the two. If the stirring time is too short, the HAP nanowires cannot be well dispersed due to their own aggregation problem, making it more difficult to obtain excellent mechanical properties.

[0030] In some embodiments, the pressure of the pressure-assisted densification process can be 100–900 MPa, preferably 400 MPa. The preparation process of CPO / HAP ceramics is based on the polymerization of CPO ion clusters. Different pressures affect the CPO / HAP polymerization process, ultimately affecting the degree of polymerization of CPO ion clusters and the interfacial bonding strength between the CPO matrix and HAP nanowires. If the pressure is too low, the distance between CPO ion clusters is too large, making it difficult to link and polymerize with surrounding ion clusters, resulting in insufficient polymerization of CPO ion clusters, leading to a loose overall structure and poor mechanical properties of CPO / HAP. Increasing the pressure can improve the degree of polymerization of CPO ion clusters and the density of CPO / HAP composite ceramics. However, the plasticity of inorganic materials has an upper limit and they cannot be compressed indefinitely. Therefore, excessively high pressures are not suitable for the preparation of CPO / HAP composite ceramics. Ultimately, 400 MPa is preferred as the preparation pressure.

[0031] In some embodiments, the temperature of the pressure-assisted densification process can be 25–350 °C, preferably 100 °C. CPO ion clusters rely on the volatilization of anhydrous ethanol and TEA, and this volatilization process depends on the preparation temperature. When the temperature is too low, the volatilization rate of TEA is slow, thus requiring a longer preparation time and reducing material preparation efficiency. Conversely, when the temperature is too high, anhydrous ethanol and TEA may carbonize before complete volatilization due to the high temperature, resulting in residual impurities inside the CPO / HAP composite ceramic. Therefore, 100 °C is preferred as the optimal temperature, slightly higher than the boiling point of TEA (89.6 °C), allowing it to gradually volatilize without carbonizing during the preparation of the CPO / HAP composite ceramic.

[0032] In some embodiments, the holding time of the pressure-assisted densification process can be from 1 min to 24 h, preferably 4 h. The holding time directly determines the degree of polymerization of the CPO / HAP composite ceramic. If the holding time is too short, the CPO ion clusters will not be sufficiently developed, which can easily lead to a loose ceramic structure and poor mechanical properties. Extending the holding time can better ensure the full polymerization of the CPO ion clusters and the construction of the interface between the CPO matrix and the HAP nanowires, thereby improving the density and mechanical properties of the CPO / HAP composite ceramic.

[0033] As an example: Measure 20 mL of a CPO suspension with a concentration of 10 mg / mL, and follow M... CPO :M HAP400 mg of hydroxyapatite nanowires were added to the mixture at a mass ratio of 1:2. After vigorous stirring for 36 h, a uniformly dispersed CPO / HAP mixed suspension was obtained. The mixed suspension was then filtered using a vacuum filter to obtain a CPO / HAP composite gel. Finally, the composite gel was placed in a mold and compacted at 400 MPa and 100 ℃ for 4 h. After drying and densification, a nano-amorphous-crystalline bioceramic with a tooth-like enamel structure was obtained.

[0034] This invention proposes a "hydroxyapatite nanowire assembly strategy based on calcium phosphate inorganic adhesive" to prepare bioceramics based on hydroxyapatite nanowires. Utilizing the polymerization characteristics of CPO ion clusters, they are uniformly mixed with HAP nanowires and vacuum filtered to obtain a CPO / HAP composite gel. Subsequently, the composite gel is compacted using pressure-assisted densification. During pressure-assisted densification, the CPO ion clusters gradually polymerize with the volatilization of TEA molecules to form a three-dimensional continuous amorphous calcium phosphate matrix, simultaneously forming a stable bonding interface with the HAP nanowires dispersed in the calcium phosphate matrix. After drying and densification, the CPO / HAP composite gel achieves the preparation of a dental enamel-like nanocrystalline-amorphous bioceramic structure. This invention uses hydroxyapatite nanowires, a biomaterial with high aspect ratio, good flexibility, and good biocompatibility, as assembly units, and CPO ion clusters, a biomaterial with polymerization properties and good biocompatibility, as inorganic glue. It proposes a method of "hydroxyapatite nanowire assembly based on calcium phosphate inorganic glue" to prepare bioceramics with a nano-amorphous-crystalline structure similar to tooth enamel. This constructs a nano-amorphous-crystalline structure similar to tooth enamel, which is expected to give it excellent strength, toughness, and crack resistance.

[0035] Compared to traditional hydroxyapatite ceramics prepared by high-temperature sintering, the bioceramic prepared in this invention exhibits similar flexural strength and a lower flexural modulus, closely resembling the mechanical properties of human bone tissue. This allows for better matching with bone tissue, overcoming the problem of traditional bioceramics being difficult to match with bone tissue due to their high brittleness and modulus. Furthermore, compared to traditional bioceramics, thanks to its biomimetic nanocrystalline-amorphous structure, it possesses superior toughness and crack resistance. In addition, due to the high aspect ratio and good flexibility of the hydroxyapatite nanowires, they play a bridging role during the fracture process of the CPO / HAP composite ceramic, effectively increasing energy dissipation and resisting crack propagation, thereby improving the material's toughness.

[0036] The dental enamel nano-amorphous-crystalline bioceramic prepared by the above method has excellent comprehensive mechanical properties, including strength, toughness, crack resistance, good biocompatibility and osseointegration. It can be used to prepare bone repair materials and is expected to improve its in vivo osseointegration ability to further enhance the bone repair effect.

[0037] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention fall within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below. Example 1

[0038] (1) Synthesis of calcium phosphate ion clusters. 11.76 g CaCl2·2H2O was dispersed in 1.6 L of anhydrous ethanol and sonicated for 30 min. Then, 221.79 mL of TEA was added and stirred for another 30 min to obtain a stable mixed solution. Subsequently, a pre-prepared phosphoric acid (4.8 mL) / anhydrous ethanol (80 mL) mixed solution was slowly added dropwise and stirred for 12 h to obtain a calcium phosphate ion cluster suspension. The suspension was washed three times with anhydrous ethanol to remove excess small molecule TEA. Finally, the cleaned calcium phosphate ion clusters were dispersed in anhydrous ethanol (concentration of approximately 10 mg / mL). (2) Synthesis of hydroxyapatite nanowires. 270 mL of deionized water, 210 mL of oleic acid and 120 mL of methanol were mixed under mechanical stirring. Every 30 minutes, sodium hydroxide aqueous solution (21 g / 300 mL), anhydrous calcium chloride aqueous solution (6.66 g / 240 mL) and sodium dihydrogen phosphate dihydrate aqueous solution (18.72 g / 360 mL) were slowly added to the mixed solution in sequence. Then, the reaction system was transferred to four 500 mL polytetrafluoroethylene hydrothermal reactors and placed in an oven at 180 °C for 24 h. After the temperature dropped to room temperature, the white precipitate HAP nanowires were collected. The nanowires were washed three times alternately with anhydrous ethanol and deionized water. After cleaning, the nanowires were filtered to obtain HAP nanowire filter cake, which was then dried in an oven at 60 °C. (3) Preparation of CPO / HAP composite ceramics. Measure 20 mL of a CPO suspension with a concentration of 10 mg / mL, and follow M... CPO :M HAP 400 mg of hydroxyapatite nanowires were added to the mixture at a mass ratio of 1:2. After vigorous stirring for 36 h, a uniformly dispersed CPO / HAP mixed suspension was obtained. The mixed suspension was then filtered using a vacuum filter to obtain a CPO / HAP composite gel. Finally, the composite gel was placed in a mold and compacted at 400 MPa and 100 ℃ for 4 h. After drying and densification, a nano-amorphous-crystalline bioceramic with a tooth-like enamel structure was obtained. Example 2

[0039] In this Example 2, the preparation process of the dental enamel nano-amorphous-crystalline bioceramic structure is the same as in Example 1, except that in step (3), the mass ratio of CPO ion clusters to HAP nanowires M CPO :M HAP =2:1. Example 3

[0040] In this Example 3, the preparation process of the dental enamel nano-amorphous-crystalline bioceramic structure is the same as in Example 1, except that in step (3), the mass ratio of CPO ion clusters to HAP nanowires M CPO :M HAP =1:1. Example 4

[0041] In this Example 4, the preparation process of the dental enamel nano-amorphous-crystalline bioceramic structure is the same as in Example 1, except that in step (3), the mass ratio of CPO ion clusters to HAP nanowires M CPO :M HAP =1:3. Example 5

[0042] In this Example 5, the preparation process of the bioceramic nano-amorphous-crystalline structure of the dental enamel is the same as that in Example 1, except that the pressing pressure is 200 MPa in step (3). Example 6

[0043] In this Example 6, the preparation process of the imitation tooth enamel nano-amorphous-crystalline bioceramic is the same as that in Example 1, except that the pressing pressure is 600 MPa in step (3). Example 7

[0044] In this embodiment 7, the preparation process of the imitation tooth enamel nano-amorphous-crystalline structure bioceramic is the same as that in embodiment 1, except that in step (3), the pressing pressure is 800 MPa. Example 8

[0045] In this Example 8, the preparation process of the imitation tooth enamel nano-amorphous-crystalline structure bioceramic is the same as that in Example 1, except that the pressing temperature is 25℃ in step (3). Example 9

[0046] In this Example 9, the preparation process of the imitation tooth enamel nano-amorphous-crystalline structure bioceramic is the same as that in Example 1, except that the pressing temperature is 200℃ in step (3). Example 10

[0047] In this Example 10, the preparation process of the imitation tooth enamel nano-amorphous-crystalline bioceramic is the same as that in Example 1, except that the pressing temperature is 300 ℃ in step (3). Example 11

[0048] In this embodiment 11, the preparation process of the imitation tooth enamel nano-amorphous-crystalline structure bioceramic is the same as that in embodiment 1, except that the pressure holding time in step (3) is 1 h. Example 12

[0049] In this Example 12, the preparation process of the bioceramic nano-amorphous-crystalline structure of the dental enamel is the same as that in Example 1, except that the pressure holding time in step (3) is 2 h. Example 13

[0050] In this embodiment 13, the preparation process of the imitation tooth enamel nano-amorphous-crystalline structure bioceramic is the same as that in embodiment 1, except that the pressure holding time in step (3) is 24 h. Comparative Example 1

[0051] HAP ceramics were prepared as a control using the pressure densification process of HAP nanowires. HAP nanowires were dispersed in anhydrous ethanol, filtered to obtain a filter cake, placed in a mold, and pressed at 400 MPa and 100 ℃ for 4 h to obtain HAP ceramics. Comparative Example 2

[0052] As a control, an organic-inorganic composite strategy was used to prepare PVA / HAP composite materials. A 5% PVA aqueous solution was prepared. A certain amount of PVA solution was measured, and HAP nanowires were added to it at a mass ratio of 1:2 (PVA to HAP nanowires). After vigorous stirring for 36 h, the mixed suspension was frozen at -80 ℃ and dried in a freeze dryer for 48 h to obtain PVA / HAP composite aerogel. Subsequently, a certain amount of PVA / HAP aerogel was weighed and placed in a mold, and pressed at 400 MPa and 25 ℃ for 1 h to obtain the PVA / HAP composite material. Comparative Example 3

[0053] S-HAP ceramics prepared by conventional high-temperature sintering method were used as a control. 300 mg of HAP nanoparticles were weighed and placed into a mold, and held under pressure of 200 MPa for 5 min to obtain a HAP green body. The green body was then placed in a high-temperature sintering furnace for sintering. The sintering process was carried out according to the following heating curves: 1) heating from room temperature to 160 ℃ at a heating rate of 2.7 ℃ / min; 2) heating from 160 ℃ to 600 ℃ at a heating rate of 4 ℃ / min; 3) heating from 600 ℃ to 1100 ℃ / min at a heating rate of 5 ℃ / min; 4) heating from 1100 ℃ to 1300 ℃ at a heating rate of 6 ℃ / min; 5) holding at 1300 ℃ for 2 h and then cooling to room temperature at a rate of 1 ℃ / min to obtain S-HAP sintered ceramic.

[0054] Figure 1 This is a schematic diagram of the preparation process of the CPO / HAP composite ceramic in Example 1. CPO ion clusters and HAP nanowires were mixed uniformly in a beaker by physical stirring to obtain a CPO / HAO mixed suspension. After vacuum filtration, a CPO / HAP composite gel was obtained. Subsequently, the gel was compacted under certain temperature and pressure conditions using a pressure-assisted densification method. After drying and densification, the CPO / HAP composite ceramic was prepared.

[0055] Figure 2 This document describes the synthesis and characterization of CPO ion clusters in Example 1. (a) shows TEM images of the CPO ion clusters at low and high concentrations; (b) shows the particle size analysis of the CPO ion clusters; and (c) shows the FTIR spectrum of the CPO ion clusters. As shown in the figures, CPO ion clusters were synthesized using a titration method. The CPO clusters exhibited a fine nanofragment morphology with a size of approximately 8–10 nm. At low concentrations, the CPO oligomers were dispersed. Fourier transform infrared spectroscopy confirmed the stabilizing effect of triethylamine (TEA) on CPO. The Fourier transform infrared spectra of CPO and TEA / ethanol showed that the characteristic peak of the CN stretching vibration of TEA in ethanol appeared at 1197.70 cm⁻¹. -1 However, in CPO solution, this characteristic peak shifts to 1228.08 cm⁻¹. -1 This indicates an interaction between TEA and CPO. When the CPO concentration is high, they aggregate together, and as anhydrous ethanol and small TEA molecules volatilize, the stabilizing effect of TEA is removed, and the CPO ion clusters link and polymerize to form a continuous calcium phosphate material.

[0056] Figure 3The figure shows the polymerization characteristics of CPO ion clusters in Example 1. (a) is a photograph of the CPO clusters and CPO gel; (b) shows the injection and polymerization of the CPO gel; (c) is a photograph of the CPO gel polymerization process; (d) is a SEM image of the CPO bulk material; and (e) is an XRD pattern of the CPO bulk material. As shown in the figure, CPO, under the stabilizing effect of TEA, can be dispersed in anhydrous ethanol for a long time (60 days) and maintain its initial properties. Furthermore, after high-speed centrifugation, CPO gel can be obtained, containing anhydrous ethanol and TEA. Using an injection needle, the CPO gel is extruded to obtain strip-shaped gels. As the anhydrous ethanol and TEA evaporate, the stabilizing effect is eliminated, and CPO polymerizes to obtain a continuous calcium phosphate material with certain mechanical properties. Figure (c) shows the natural air-drying process of the CPO gel at room temperature. The results show that as the liquid in the gel evaporates, the polymerization process begins. During this process, the volume of the CPO gel slowly decreases, while it gradually becomes denser, ultimately yielding a continuous calcium phosphate material. The calcium phosphate bulk material obtained after centrifugal drying consists of micron-sized particles with a relatively loose internal structure. XRD patterns show that it is an amorphous phase.

[0057] Figure 4 The figure shows the preparation process of CPO / HAP composite ceramics in Example 1; where (a) is a photograph of the CPO / HAP composite ceramic preparation process, and (b) is a photograph of large-size and irregularly shaped CPO / HAP composite ceramics. As shown in the figure, after uniformly mixing CPO oligomers and HAP nanowires, CPO / HAP composite gels are obtained by vacuum filtration. The gels are then placed in a mold and densified under certain temperature and pressure conditions. During this process, CPO gradually polymerizes and bonds the HAP nanowires together, thus preparing CPO / HAP ceramics. The bioceramic preparation method based on the inorganic adhesive micro / nanomaterial assembly strategy is simple to operate and can prepare macroscopically large-size (6 cm × 6 cm) CPO / HAP composite ceramics. Furthermore, the preparation of ceramic materials with complex shapes is usually complex, with difficult and costly subsequent processing. Unlike traditional ceramic preparation methods, the inorganic adhesive micro / nanomaterial assembly method, thanks to the fluidity of the CPO / HAP composite gel, allows for the preparation of CPO / HAP composite ceramics with different structures using molds of different shapes, providing convenience for the preparation of irregularly shaped and complex ceramic materials.

[0058] Figure 5The mass ratio of CPO ion clusters to HAP nanowires was optimized. (a) shows the flexural strength of the CPO / HAP composite ceramic under different component mass ratios; (b) shows the flexural modulus of the CPO / HAP composite ceramic under different component mass ratios; and (c) shows the flexural stress-strain curves of the CPO / HAP composite ceramic under different component mass ratios. CPO / HAP composite ceramic is essentially an inorganic-reinforced ceramic matrix composite material. The proportion of HAP nanowires has a significant impact on the mechanical properties of the CPO / HAP composite ceramic. This invention sets different mass ratios of CPO ion clusters to HAP nanowires: 2:1, 1:1, 1:2, and 1:3. The flexural strength results show that as the amount of HAP nanowires added increases, the flexural strength of the CPO / HAP composite ceramic first increases and then decreases, reaching its maximum at a mass ratio of 1:2. The trends of its flexural modulus and flexural strength are consistent.

[0059] Figure 6 The bending properties of CPO / HAP composite ceramics under different pressing times are shown in Figure 1. (a) represents the bending strength of CPO / HAP composite ceramics under different pressing times, (b) represents the bending modulus of CPO / HAP composite ceramics under different pressing times, and (c) represents the bending stress-strain curves of CPO / HAP composite ceramics under different pressing times. The principle of CPO ion cluster polymerization is the volatilization of the end-capping agent TEA. Therefore, the degree of TEA volatilization directly affects the polymerization effect of CPO ion clusters. The volatilization of TEA depends on the time and temperature. TEA is a small molecule that is easy to volatilize, and its boiling point is 89.6 ℃. Therefore, the time was first screened at a temperature of 100 ℃, and four time periods were set: 1 h, 2 h, 4 h and 24 h. The results showed that as the pressing time of CPO / HAP increased, the bending strength of the obtained CPO / HAP composite ceramic gradually increased and tended to stabilize at a holding time of 4 h. Therefore, it can be concluded that, based on the CPO content used in this study, TEA can be completely volatilized within 4 h to achieve the maximum degree of CPO polymerization. However, when the holding time is short, TEA is not completely volatilized, which will lead to some CPO not being polymerized, thus failing to obtain the best bending resistance.

[0060] Figure 7The images show cross-sectional SEM images of CPO / HAP composite ceramics prepared by pressing for different times. (a) is the cross-sectional SEM image of the CPO / HAP composite ceramic prepared by pressing for 5 min, (b) is the cross-sectional SEM image of the CPO / HAP composite ceramic prepared by pressing for 1 h, (c) is the cross-sectional SEM image of the CPO / HAP composite ceramic prepared by pressing for 4 h, and (d) is the cross-sectional SEM image of the CPO / HAP composite ceramic prepared by pressing for 24 h. When the polymerization time is only 5 min, the internal structure of the ceramic is loose, and the CPO particles themselves have a low degree of polymerization. It is also evident that there is almost no interfacial bonding between the CPO particles and the HAP nanowires. When the time is extended to 1 h, it can be observed from the image that the internal structure density of the ceramic is improved, and a large amount of CPO polymerizes into continuous calcium phosphate material. However, the bonding interface between CPO and HAP nanowires is still relatively rare, and the mechanical load-bearing capacity mainly comes from the mechanical properties of CPO itself. When the polymerization time was further increased to 4 h and 24 h, the density of the ceramic was significantly improved. At 4 h, CPO was observed to be attached to the surface of HAP nanowires and form a relatively tight interface structure with them. In the ceramic polymerized for 24 h, a more obvious CPO / HAP nanowire interface bonding structure was observed inside, which is similar to the sintering neck that appears in the traditional high-temperature sintering process.

[0061] Figure 8 TEM images of CPO / HAP composite ceramics with different pressing times are shown. When the polymerization time is 5 min, interfacial bonding can also be formed between CPO and HAP nanowires. However, due to the short pressing time, a large number of pores appear inside the ceramic, resulting in a loose overall structure and insufficient mechanical properties. The density of CPO / HAP composite ceramics obtained after 24 h of polymerization is improved, no large-sized pores are observed between CPO and HAP nanowires, and the degree of interfacial bonding between the two is improved.

[0062] Figure 9The images show the flexural properties and cross-sectional SEM images of CPO / HAP composite ceramics under different preparation pressures. (a) shows the flexural strength of the CPO / HAP composite ceramics under different pressures; (b) shows the flexural modulus of the CPO / HAP composite ceramics under different pressures; (c) shows the flexural stress-strain curves of the CPO / HAP composite ceramics under different pressures; and (d) shows the cross-sectional SEM images of the CPO / HAP composite ceramics prepared under different pressures. This invention sets four pressures: 200 MPa, 400 MPa, 600 MPa, and 800 MPa. Adjusting the pressure value changes the state of the CPO / HAP composite ceramics during the polymerization process, from polymerization within a loose space at lower pressures to polymerization within a dense space at higher pressures, thus obtaining CPO / HAP composite ceramic materials with drastically different densities. The fracture mode of the material has gradually transitioned from CPO matrix fracture to HAP nanowire pull-out and HAP nanowire fracture. The preparation of CPO / HAP composite ceramics can be achieved by controlling the pressure value within a large range. In this invention, 400 MPa is preferred as the main preparation parameter for subsequent material preparation and research.

[0063] Figure 10 The images show the flexural properties and cross-sectional SEM images of CPO / HAP composite ceramics prepared at different temperatures. (a) shows the flexural strength of CPO / HAP composite ceramics under different temperature conditions, (b) shows the flexural modulus of CPO / HAP composite ceramics under different temperature conditions, (c) shows the flexural stress-strain curves of CPO / HAP composite ceramics under different temperature conditions, and (d) shows the cross-sectional SEM images of CPO / HAP composite ceramics prepared under different temperature conditions. This invention sets four temperatures: 25℃, 100℃, 200℃, and 300℃. When the preparation temperature is 25℃, the flexural strength of the CPO / HAP composite ceramic is relatively low. As the temperature increases to 100℃~300℃, the flexural strength of the CPO / HAP composite ceramic increases and then tends to stabilize, not increasing further with increasing temperature. However, the flexural modulus of the ceramic increases with increasing temperature. Further analysis of the structure of the ceramic prepared at different temperatures reveals that the fracture mode changes from CPO matrix fracture to HAP nanowire pull-out as the temperature increases. The particle size of CPO in the CPO / HAP composite ceramic shows a trend of increasing with temperature. This indicates that temperature changes the density of CPO by regulating the particle size, but does not affect the interfacial bonding strength of CPO / HAP.

[0064] Figure 11The images show the in-situ temperature-varying XRD patterns of bulk CPO and CPO / HAP composite ceramics. (a) shows the in-situ temperature-varying XRD pattern of bulk CPO, and (b) shows the in-situ temperature-varying XRD pattern of the CPO / HAP composite ceramic. Pure bulk CPO remains amorphous at room temperature and 100 °C. When the temperature rises to 200 °C, some calcium phosphate diffraction peaks are detected, indicating that CPO undergoes a phase transition at higher temperatures. The in-situ XRD patterns of the CPO / HAP ceramic show that the main phase of the CPO / HAP composite ceramic is hydroxyapatite at several temperature conditions. This suggests that CPO undergoes a partial phase transition and crystallizes into calcium phosphate when the temperature rises to a certain value, thus leading to an increase in the flexural modulus of the CPO / HAP composite ceramic. To ensure sufficient flexural strength and a nano-amorphous-crystalline structure of the CPO / HAP composite ceramic, and to minimize the energy consumption in ceramic preparation, 100 °C was ultimately selected as the optimal preparation temperature for the CPO / HAP ceramic.

[0065] Figure 12 Optical photographs and surface and cross-sectional SEM images of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics are shown. Among them, (a) is an optical photograph, surface SEM image, and cross-sectional SEM image of HAP ceramics; (b) is an optical photograph, surface SEM image, and cross-sectional SEM image of PVA / HAP composites; (c) is an optical photograph, surface SEM image, and cross-sectional SEM image of S-HAP ceramics; and (d) is an optical photograph, surface SEM image, and cross-sectional SEM image of CPO / HAP composite ceramics. HAP ceramics are directly pressed from HAP nanowires. SEM images of the surface show a disordered distribution of HAP nanowires and a loose internal structure. PVA / HAP composites are pressed from PVA / HAP composite aerogels. The HAP nanowires are encapsulated by PVA polymers, and the surface does not show a clear nanowire morphology. The cross-section exhibits large pores and tensile fracture characteristics of the PVA polymer. High-temperature sintered S-HAP ceramics are slowly sintered through multiple heating stages, resulting in a very dense microstructure with almost no obvious pores on the surface or inside. Unlike HAP ceramics, CPO / HAP composite ceramics, assembled using CPO inorganic adhesive, have pores between HAP nanowires filled by the CPO inorganic adhesive. The surface shows CPO covering the HAP nanowires, and the internal structure is denser than that of HAP ceramics and PVA / HAP composites. The CPO matrix tightly binds the HAP nanowires, thus causing them to be elongated, pulled out, and destroyed during ceramic fracture.

[0066] Figure 13The images show TEM and selected area electron diffraction (SED) images of the CPO / HAP composite ceramic in Example 1. The CPO / HAP composite ceramic contains both a CPO phase and a HAP nanowire phase. CPO forms a thick, continuous bonding layer between larger bundles of HAP fibers, bonding them together, and also fills the gaps between the HAP nanowires in smaller sizes, thus forming the CPO / HAP composite ceramic. SED images of CPO / HAP show it to be a polycrystalline ceramic material. Analysis of the interface structure between the CPO inorganic adhesive and the HAP nanowires reveals that the HAP nanowires are firmly bonded by the amorphous calcium phosphate CPO material. The interface forms a structure similar to the natural amorphous-crystalline nanostructure of tooth enamel, further confirming that CPO can undergo interfacial fusion with HAP nanowires during its own polymerization process, thereby completing the HAP nanowire assembly process based on CPO inorganic adhesive.

[0067] Figure 14 TEM images of HAP ceramics, PVA / HAP composites, and S-HAP ceramics are shown. (a) is a TEM image of HAP ceramics, (b) is a TEM image of PVA / HAP composites, and (c) is a TEM image of S-HAP ceramics. In HAP ceramics, nanowires are assembled through mechanical interlocking, resulting in a small number of pores between the nanowires. The TEM image of the PVA / HAP composite is similar to that of the CPO / HAP composite ceramic. PVA acts as a bonding layer, binding the upper and lower HAP fiber bundles together. However, the bonding between the HAP nanowires is not very dense, and pores of varying sizes still exist. This indicates that the wettability of PVA organic molecules to the nanowires is insufficient. Compared to nano-CPO ion clusters, PVA solution is more difficult to fill the pores within the HAP fiber bundles. Interface structure analysis results show that PVA can also form an adhesive interface with HAP nanowires. S-HAP ceramics are made by high-temperature sintering of HAP nanoparticles (20 nm in diameter). During the sintering process, the HAP nanoparticles coarsen and increase in size to more than 100 nm, and the particles are tightly fused together to form a distinct grain boundary structure.

[0068] In this invention, hydroxyapatite nanowires can also be replaced with various inorganic micro- and nanomaterials with different morphologies, sizes, and compositions, such as hydroxyapatite particles (HAP pa25icle), hydroxyapatite rods (HAP rod), calcium silicate (Ca2SiO3), aluminum oxide (Al2O3), magnesium oxide (MgO), and silicon nitride (Si3N4). Figure 15SEM images and elemental distributions of inorganic micro / nanomaterial composite ceramics assembled using CPO inorganic adhesive were obtained. CPO inorganic adhesive was used to assemble various inorganic micro / nanomaterials with different morphologies, sizes, and compositions, including hydroxyapatite particles, hydroxyapatite rods, calcium silicate (Ca2SiO3), alumina (Al2O3), magnesium oxide (MgO), and silicon nitride (Si3N4). All of these methods successfully produced dense CPO inorganic adhesive-based ceramic materials, demonstrating the flexibility and versatility of the CPO inorganic adhesive-based assembly strategy. A variety of CPO-based composite ceramics can be prepared through a simple mixing-filtration-pressure-assisted densification process.

[0069] Figure 16 The flexural and compressive properties of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics are compared. Among them, (a) is the flexural strength of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; (b) is the flexural modulus of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; (c) is the flexural stress-strain curve of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; and (d) is the compressive strength of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics. The CPO / HAP composite ceramics prepared by the HAP nanowire assembly strategy based on CPO inorganic adhesive exhibit bending and compressive properties comparable to those of traditional high-temperature sintered HAP ceramics, while also possessing a lower flexural modulus. As a bone repair material, it can enhance its mechanical compatibility with natural bone tissue. This low-temperature ceramic preparation strategy, which does not require high temperatures, not only significantly reduces energy consumption but also achieves superior comprehensive mechanical properties, fully demonstrating the feasibility, scientific validity, and application potential of this strategy.

[0070] Figure 17Fracture toughness tests and SEM images of crack propagation paths for HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics are shown. (a) is a schematic diagram of fracture toughness testing using the single-sided notched beam method; (b) shows the fracture toughness of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics; and (c) shows the SEM image of the crack propagation path. Cracks in HAP ceramics, PVA / HAP composites, and CPO / HAP composite ceramics exhibit a certain curved shape during propagation, while cracks in S-HAP ceramics penetrate the material directly via transgranular fracture paths, indicating that S-HAP has high brittleness and is difficult to resist crack tip propagation. The crack propagation mode of CPO / HAP composite ceramics combines a tortuous propagation path with the pull-out effect of HAP nanowires at the nanoscale. During the failure process of CPO / HAP ceramics, the cracks propagate and bifurcate in multiple directions, with the propagation path being the most tortuous. At the same time, as the cracks propagate forward, the HAP nanowires play a bridging and toughening role, effectively resisting further crack propagation, thus achieving superior comprehensive fracture toughness compared to traditional high-temperature sintered ceramics.

[0071] Figure 18 Hydrophilicity / phobicity and wet structural stability of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics were analyzed; (a) shows the hydrophilicity / phobicity analysis, and (b) shows the structural stability analysis in water. Due to the good interfacial adhesion of CPO inorganic adhesive, the CPO / HAP composite ceramics maintained good structural stability after ultrasonication in water for 30 min.

[0072] Figure 19 Wet mechanical stability analysis was performed on HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics. HAP ceramics, unable to resist water wetting, suffered structural damage and failed to maintain their initial mechanical properties. PVA / HAP composites exhibited significant changes in mechanical properties due to the swelling of PVA polymers in water, demonstrating the flexibility of polymer materials. S-HAP and CPO / HAP maintained their initial macroscopic support properties even after immersion in water for 1 hour, indicating that CPO inorganic adhesive can maintain good bonding function under wet conditions for a short period.

[0073] Figure 20The wet mechanical properties of CPO / HAP composite ceramics and related photographs are shown. (a) shows the change in flexural properties under different immersion times in water, and (b) is an optical photograph of the CPO / HAP composite ceramics after immersion in water for 30 days. With prolonged immersion time, the flexural strength of the CPO / HAP composite ceramics gradually decreases, dropping to about 75% of its initial strength after one day of immersion, half of its initial strength after seven days, and only about 30% of its initial strength after 30 days. This indicates that the adhesive function of CPO is gradually weakened under prolonged wet conditions, leading to the breakdown of the interface structure between CPO and HAP nanowires and a decline in the mechanical properties of the CPO / HAP composite ceramics.

[0074] Figure 21 The images show SEM images of the surface and cross-section of the CPO / HAP composite ceramic after immersion in water for 30 days. (a, b) are the surface (a) and cross-section (b) SEM images of the CPO / HAP composite ceramic after immersion in water for 30 days. Numerous micron-sized flake-like ceramic sheets precipitate on the ceramic surface. CPO inorganic adhesive is still observable in the internal structure, but the interface between the CPO inorganic adhesive and the HAP nanowires is disrupted, resulting in large interlayer pores between the HAP nanowires.

[0075] In vitro biocompatibility evaluation of CPO / HAP composite ceramics: The in vitro biocompatibility of S-HAP sintered ceramics and CPO / HAP composite ceramics was evaluated using cell adhesion and proliferation assays. S-HAP and CPO / HAP ceramic samples (5 mm × 5 mm × 1 mm) were prepared and sterilized at high temperature using a 1×10⁻⁶ cell adhesion and proliferation assay. 4 Cells were seeded at a density of cells / well on 48-well plates and cultured at 37 °C and 5% CO2 for 1, 4, and 7 days, respectively. After fixation with 4% paraformaldehyde for 24 hours, the cytoskeleton and nuclei were stained with phalloidin-FITC and DAPI, respectively, for 2 hours and 10 minutes, respectively. Cell adhesion was then observed using a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany). To determine cell proliferation, cells were seeded at a density of 1 × 10⁻⁶ cells / well. 4 Cells were seeded onto scaffolds in 48-well plates at a cell density of 1 × 10⁻⁶ cells / well and cultured in an incubator (37 °C, 5% CO₂) for 1, 4, and 7 days. CCK-8 assays were performed, and cell proliferation activity was measured at 450 nm using a microplate reader. For osteogenic differentiation protein staining analysis, BMSCs were seeded at a density of 1 × 10⁻⁶ cells / well. 4Cells were seeded at a density of [number] cells / well on ceramic scaffolds in 48-well plates and cultured for 7 days at 37 °C in a 5% CO2 incubator. Cells were fixed with 4% paraformaldehyde for 24 hours, followed by permeabilization with 0.1% Triton-X100 for 5 minutes and blocking with 5% bovine serum albumin for 1 hour. Primary antibody was then diluted 1:500 with PBS and incubated with the cells overnight at 4 °C. The next day, the cells were washed with PBS buffer, and secondary antibody was diluted 1:1000 with PBS and incubated with the cells at room temperature for 2 hours. The cytoskeleton and nuclei were then stained with phalloidin-FITC and DAPI, respectively. Fluorescence images were observed and acquired using CLSM, and quantitative analysis of fluorescence intensity was performed using ImageJ software.

[0076] Figure 22 This study evaluates the in vitro biocompatibility of S-HAP ceramics and CPO / HAP composite ceramics. (a) shows immunofluorescence staining of cells after co-culturing with the ceramics for different time periods; (b) shows cell proliferation after co-culturing with the ceramics; (c) shows immunofluorescence staining of intracellular BSP protein; and (d) shows the quantitative statistical graph of intracellular BSP protein immunofluorescence intensity. The figures show that the CPO / HAP composite ceramics support cell adhesion, proliferation, and expression of osteogenic-associated protein BSP, indicating good cell compatibility.

[0077] In vivo biocompatibility evaluation of CPO / HAP composite ceramics: A rat subcutaneous implantation model was established to study the in vivo biocompatibility of CPO / HAP bioceramics. The specific steps were as follows: Twelve 8-week-old male SD rats were randomly divided into HAP, PVA / HAP, S-HAP, and CPO / HAP groups. Rats were anesthetized by abdominal injection of anesthesia. Hair on the rat's back was removed and the area was disinfected with iodine. A 1 cm incision was then made in the skin on the rat's back using scissors. The incision was bluntly expanded on both sides, and a bioceramic sheet (5 mm × 5 mm × 1 mm) was implanted subcutaneously. On day 14 post-implantation, rats were euthanized by an overdose of anesthesia. Samples of the implanted material and surrounding tissue were collected. Samples were fixed with 4% paraformaldehyde solution for 24 h, and then decalcified with 10% EDTA decalcification solution for 20 days. After washing with PBS, the samples were sequentially dehydrated by incubating in 10 wt% sucrose PBS solution and 30 wt% sucrose PBS solution for 4 hours each. Then, the samples were embedded using cryoembedding medium, frozen and sectioned into 20 μm thick sections, and then hematoxylin-eosin (H&E) staining was performed to assess the in vivo inflammatory response. After staining, the stained tissue sections were photographed using an optical microscope.

[0078] Figure 23This study evaluates the in vivo biocompatibility of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics. (a) shows a schematic diagram of the rat subcutaneous model establishment process, and (b) shows H&E staining of tissue sections 14 days after subcutaneous implantation of the materials in rats. As shown in the figures, no significant inflammatory cell aggregation or infiltration was observed in the CPO / HAP composite ceramics 14 days after subcutaneous implantation in rats, indicating good in vivo biocompatibility.

[0079] In vivo bone integration evaluation of CPO / HAP composite ceramics: The in vivo osseointegration properties of CPO / HAP composite ceramics were investigated. PVA / HAP composites, S-HAP sintered ceramics, and CPO / HAP composite ceramics (6 mm in diameter and 6 mm in height) were prepared. Sixteen New Zealand white rabbits (weighing 3-3.5 kg) were randomly divided into four groups: Blank group (n=4), PVA / HAP group (n=4), S-HAP group (n=4), and CPO / HAP group (n=4). A rabbit bilateral femoral condyle injury model was established, and materials were implanted for 8 weeks of repair. The specific procedures were as follows: Rabbits were anesthetized by intramuscular injection of anesthetic. After preparing the surgical area and disinfecting with iodine solution, the skin at the surgical site was incised with a scalpel. After separating the fascia, a cylindrical defect with a diameter of 6 mm was created medially from the lateral femoral plateau. The depth of the defect was determined by slight bleeding within the defect. After implanting the material, the skin was sutured to complete the surgery. Eight weeks after scaffold implantation, rabbits were euthanized, and the femur was removed and fixed in 4% paraformaldehyde for 3 days for in vivo osseointegration evaluation. First, micro-CT scans were used to analyze the distribution of bone tissue around the scaffold and the integration of the scaffold edges with bone tissue. Subsequently, the samples underwent gradient dehydration and tissue was embedded in polymethyl methacrylate (PMMA). The embedded tissue blocks were used for hard tissue sections, and Van-Gieson staining of bone tissue was performed to further analyze the in vivo osseointegration of the CPO / HAP composite ceramic.

[0080] Figure 24Micro-CT scan images related to in vivo bone integration analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics are shown. (a) shows the modeling steps of the rabbit femoral condyle defect model, and (b) shows the micro-CT analysis images of the blank group and the PVA / HAP composite, S-HAP ceramic, and CPO / HAP composite ceramics after 8 weeks of implantation. The figures show that the Blank group rabbits had less regenerated bone and more obvious defects. The PVA / HAP group also showed a large defect area, but it was smaller than that of the Blank group. S-HAP and CPO / HAP are solid ceramic materials, and both integrated tightly with the surrounding bone tissue. It can also be seen that the CPO / HAP material has a higher bone content around it, which may be due to the continuous stimulation of bone tissue regeneration and maturation by the bioactive ions released by the material.

[0081] Figure 25 Van-Gieson stained images of tissue sections related to the in vivo osteointegration analysis of HAP ceramics, PVA / HAP composites, S-HAP ceramics, and CPO / HAP composite ceramics. PVA / HAP materials exhibited slight deformation due to in vivo mechanical instability, and some of its edges integrated with regenerated bone tissue. S-HAP and CPO / HAP both showed excellent osteointegration, with tight integration with regenerated bone tissue throughout their entire circumference. Furthermore, the bone tissue content around CPO / HAP was higher than that of S-HAP, an advantage attributed to the stimulating effect of slowly released bioactive ions.

[0082] Table 1 lists the performance parameters of the bioceramics and composite materials prepared in Examples 1-13 and Comparative Examples 1-3 of this invention.

[0083] Table 1: .

[0084] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A bioceramic with a nanocrystalline-amorphous structure resembling tooth enamel, characterized in that, The dental enamel nano-amorphous-crystalline bioceramic comprises: a three-dimensional continuous amorphous calcium phosphate matrix polymerized from calcium phosphate ion clusters (CPO), and inorganic micro- and nanomaterials uniformly dispersed within the three-dimensional continuous amorphous calcium phosphate matrix. Based on the total mass of the aforementioned dental enamel nano-amorphous-crystalline bioceramic as 100%, the mass percentage of the amorphous calcium phosphate matrix is ​​20-80%, and the mass percentage of the inorganic micro-nano materials is 20-80%.

2. The bioceramic with a nanocrystalline-amorphous structure resembling tooth enamel according to claim 1, characterized in that, The size of the calcium phosphate ion clusters is 8–10 nm.

3. The bioceramic nanostructured amorphous-crystalline structure of dental enamel according to claim 1 or 2, characterized in that, The inorganic micro / nano material is at least one of hydroxyapatite nanowires, hydroxyapatite nanoparticles, hydroxyapatite microrods, calcium silicate nanowires, alumina nanoparticles, magnesium oxide nanoparticles, and silicon nitride microparticles, preferably hydroxyapatite nanowires; more preferably, the hydroxyapatite nanowires have a diameter of 20–30 nm and a length of 80–100 μm.

4. The bioceramic nanostructured amorphous-crystalline structure of dental enamel according to any one of claims 1-3, characterized in that, The dental enamel nano-amorphous-crystalline bioceramic is a dense, sheet-like ceramic. Preferably, the dental enamel nano-amorphous-crystalline bioceramic has a length of not less than 10 mm, a width of not less than 5 mm, and a thickness of not less than 0.5 mm.

5. The bioceramic nanostructured amorphous-crystalline structure of dental enamel according to any one of claims 1-4, characterized in that, The described dental enamel nanocrystalline-amorphous bioceramic exhibits a flexural strength of 41.4–117.8 MPa, a flexural modulus of 10.2–54.3 GPa, a compressive strength of 102.2–168.8 MPa, and a crack initiation fracture toughness of 3.82–4.52 MPa·m. 1 / 2 .

6. A method for preparing a bioceramic nanostructured amorphous-crystalline structure imitating tooth enamel according to any one of claims 1-5, characterized in that, include: Calcium phosphate ion clusters and inorganic micro / nanomaterials were mixed at a certain mass ratio and stirred to obtain a mixed suspension. Then, the mixed suspension was filtered by vacuum filtration to obtain a calcium phosphate ion cluster-inorganic micro / nanomaterial composite gel. Next, the calcium phosphate ion cluster-inorganic micro / nanomaterial composite gel was compacted by a pressure-assisted densification process. Finally, after drying and densification, the dental enamel nano-amorphous-crystalline structure bioceramic was obtained.

7. The preparation method according to claim 6, characterized in that, The calcium phosphate ion clusters were prepared by titration; when the inorganic micro / nano material was hydroxyapatite nanowires, the hydroxyapatite nanowires were prepared by a solvothermal method using calcium oleate precursor.

8. The preparation method according to claim 6 or 7, characterized in that, The mass ratio of the calcium phosphate ion clusters to the hydroxyapatite nanowires is 2:1 to 1:3, preferably 1:1.5 to 1:2.

5.

9. The preparation method according to any one of claims 6-8, characterized in that, The stirring time is 24–48 h, preferably 32–40 h; The parameters of the pressure-assisted densification process include: pressure of 100–900 MPa, preferably 350–450 MPa; temperature of 25–350 °C, preferably 90–110 °C; and time of 1 min–24 h, preferably 3.5–4.5 h.

10. The application of a nano-amorphous-crystalline bioceramic structure based on any one of claims 1-5 in the preparation of bone repair materials.

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