Bone oriented structure-imitating calcium pyrophosphate biological ceramic as well as preparation method and application thereof

Abstract

The invention relates to calcium pyrophosphate biological ceramic with a bone-imitating oriented structure as well as a preparation method and application of the calcium pyrophosphate biological ceramic. The bone-imitating oriented structure calcium pyrophosphate biological ceramic has a layered structure formed by alternately arranging calcium pyrophosphate crystal phase layers formed by dehydration condensation of brushite and amorphous carbon layers formed by carbonization of carboxymethyl chitosan. The bone-imitating oriented structure calcium pyrophosphate biological ceramic has significantly improved strength and toughness, also has good cytocompatibility, and is a potential load-bearing bone repair material. The bionic design based on the bone tissue orientation structure provides a new strategy for preparing high-strength and high-toughness bioactive materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials, and relates to a bone-like directional structure calcium pyrophosphate bioceramic and a preparation method and application thereof. BACKGROUND

[0002] The rapid development of artificial bone repair materials has played an important role in improving the health and quality of life of patients with orthopedic diseases. Among many artificial synthetic bone repair materials, calcium phosphate-based bioactive ceramics have attracted much attention due to their simple preparation, controllable composition and good osteogenic performance. As the tissue that mainly supports the human body, bone itself has the characteristics of high strength and high toughness. The compressive strength of human cortical bone can reach 100-230 MPa, the bending strength is 50-150 MPa, and the fracture toughness value is 2-12 MPa·m 1 / 2 Although calcium phosphate-based bioactive ceramics have good biological properties, the inherent brittleness of inorganic materials makes it difficult to match the toughness of bone tissue. This mechanical performance deficiency is a bottleneck problem that has long plagued calcium phosphate-based ceramics and even all bioceramics, limiting their more extensive clinical application.

[0003] Studies have shown that the excellent mechanical properties of many natural materials are closely related to their multi-level ordered structure from the nanoscale to the macroscopic scale and rich interface effects, and bone tissue is no exception. On the nanoscale, bone tissue is formed by periodic arrangement of hydroxyapatite nanocrystals along collagen fibers to form mineralized collagen fibers; the mineralized collagen fibers are further arranged to form bone units with concentric circular ordered layered structure. There is also a brittle interface between bone units with high mineralization and low collagen content. The ordered layered and interface structure of bone makes it possible to promote energy dissipation through various deformation and toughening methods such as crack bridging and crack deflection when cracks occur, thereby producing sufficient toughness to resist fractures. The ordered structure of bone tissue and its resulting toughening effect also provide a unique structural design strategy for the preparation of high-toughness bioceramics. SUMMARY

[0004] In view of the fact that calcium phosphate-based bioactive ceramics cannot overcome the problem of low toughness, inspired by the toughening effect of the ordered layered and interface structure of bone tissue, the present application provides a bone-like directional structure calcium pyrophosphate bioceramic and a preparation method and application thereof. Using two-dimensional sheet-like brushite as a template, which is safe, stable, and economically and easily available, and adding carboxymethyl chitosan with good biocompatibility as a binder, the brushite and carboxymethyl chitosan are induced to form an ordered structure alternately through directional assembly; during the subsequent vacuum hot-pressing sintering process, the brushite is converted into calcium pyrophosphate, and the carboxymethyl chitosan is carbonized and further used as a reverse template to induce the formation of an ordered structure and the grain-oriented arrangement of calcium pyrophosphate, thereby obtaining a bone-like structure calcium pyrophosphate bioceramic with high toughness and high strength.

[0005] In a first aspect, the present application provides a bone-like directional structure calcium pyrophosphate bioceramic, which has a layered structure formed by alternating arrangement of calcium pyrophosphate crystal phase layers formed by dehydration condensation of brushite and amorphous carbon layers formed by carbonization of carboxymethyl chitosan; the amorphous carbon layer is located between two layers of calcium pyrophosphate crystal phase layers and forms an interface structure between the strong and weak phases with the calcium pyrophosphate crystal phase layer. Among them, calcium pyrophosphate is a strong phase layer with more regular lattice structure and higher strength; amorphous carbon has poor crystallinity and is a weak phase layer. When damaged by external force, the crack will first expand along the amorphous carbon layer with weaker strength, and will not directly expand to the calcium pyrophosphate crystal phase. This interface structure between the strong and weak phases may be beneficial to increase the energy dissipation of the ceramic material when it breaks, thereby improving the mechanical properties of the material.

[0006] Preferably, in the bone-like directional structure calcium pyrophosphate bioceramic, the mass fraction of calcium pyrophosphate is 98.88% to 99.82%, and the mass fraction of amorphous carbon is 0.18% to 1.12%.

[0007] Preferably, the thickness of the calcium pyrophosphate crystal phase layer is 3 to 5 μm; and the thickness of the amorphous carbon layer is <100 nm.

[0008] Preferably, the flexural strength of the bone-like directional structure calcium pyrophosphate bioceramic is 84.9 to 94.1 MPa, and the fracture toughness is 1.08 to 1.6 MPa·m 1 / 2 .

[0009] In a second aspect, the present application provides a preparation method of a bone-like directional structure calcium pyrophosphate bioceramic, which comprises the following steps: (1) preparing a brushite / carboxymethyl chitosan composite ceramic body by filtration assisted directional assembly, taking two-dimensional sheet-shaped brushite powder as a template and carboxymethyl chitosan as a binder; (2) hot-pressing sintering the brushite / carboxymethyl chitosan composite ceramic body to obtain the bone-like directional structure calcium pyrophosphate bioceramic.

[0010] Preferably, the two-dimensional sheet-shaped brushite powder is prepared by a chemical precipitation method; the chemical precipitation method comprises: dissolving potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4) and anhydrous calcium chloride (CaCl2) in deionized water, magnetically stirring under ice water bath conditions to react, after the reaction is completed, separating the precipitate from the mixed solution by filtration, and drying and grinding to obtain the two-dimensional sheet-shaped brushite powder.

[0011] Preferably, the molar ratio of the potassium dihydrogen phosphate, the disodium hydrogen phosphate and the anhydrous calcium chloride is (1 to 3):(6 to 8):(8 to 10), and is preferably 2:7:9.

[0012] Preferably, the temperature of the ice-water bath is 0–5 °C; and the magnetic stirring time is 1–2 hours.

[0013] Preferably, the mass ratio of the two-dimensional flaky phosphogypsum powder to carboxymethyl chitosan is (90-95):(5-10).

[0014] Preferably, the parameters of the hot pressing sintering include: sintering temperature of 900-1100 ℃, sintering pressure of 10-30 MPa, sintering time of 1-4 hours, and sintering atmosphere of vacuum atmosphere.

[0015] Thirdly, the present invention provides an application of the above-mentioned bone-inspired directional calcium pyrophosphate bioceramic in the preparation of bone defect repair materials. Beneficial effects

[0016] (1) The bone-like calcium pyrophosphate bioactive ceramic prepared in this invention has a long-range ordered microstructure with alternating parallel lamellar bone layers; and it has a unique interfacial bonding structure formed by an amorphous carbon layer and a calcium pyrophosphate crystalline phase layer, which is beneficial to increasing energy dissipation during fracture. The flexural strength of the bone-like calcium pyrophosphate bioceramic is 84.9–94.1 MPa, and the fracture toughness is 1.08–1.6 MPa·m. 1 / 2 The mechanical properties of the bone-like ceramics can basically meet the requirements of load-bearing bones. After co-culturing bone marrow mesenchymal stem cells and endothelial cells for 24 hours, the ceramics did not cause significant cell death, indicating that the ceramics have no obvious cytotoxicity. Furthermore, the ceramics effectively supported cell adhesion on their surface, exhibited good cytoskeleton spreading, and promoted cell migration. (2) The bone-inspired calcium pyrophosphate ceramic prepared by this invention has significantly improved strength and toughness, and also has good cell compatibility, making it a potential load-bearing bone repair material. This biomimetic design based on the directional structure of bone tissue provides a new strategy for preparing high-strength, high-toughness bioactive materials. Attached Figure Description

[0017] Figure 1 The image shows the characterization of the two-dimensional permeable phosphatite powder prepared in Example 1 of this invention; wherein, A is the XRD pattern of the permeable phosphatite powder; B and C are the microstructures of the permeable phosphatite powder; Figure 2 The microstructures of calcium pyrophosphate bioceramics with different components prepared in Examples 1-2 and Comparative Examples 1-2 of this invention are shown. Figure 3 The elemental spectra of calcium pyrophosphate bioceramics with different components prepared in Examples 1-2 and Comparative Examples 1-2 of this invention are shown below. Figure 4The XRD patterns of different components of calcium pyrophosphate bioceramics prepared in Examples 1-2 and Comparative Example 2 of this invention are shown. Figure 5 Characterization of the interface structure of the bone-inspired ordered calcium pyrophosphate ceramic (CPP) prepared in Example 1 of the present invention; wherein, A is a TEM image at the interface between the CPP layer and the amorphous carbon layer; B is the elemental distribution at the interface between the CPP layer and the amorphous carbon layer; C is a TEM image of the crack propagation in the amorphous carbon layer in 95CPP ceramic; Figure 6 The mechanical properties of the bone-inspired ordered structure calcium pyrophosphate ceramics prepared in this invention are shown below; wherein, A represents the flexural strength of the calcium pyrophosphate ceramics prepared in Examples 1-2 and Comparative Examples 1-2; B represents the typical stress-strain curves of the flexural tests in Examples 1-2 and Comparative Examples 1-2; C represents the fracture toughness of the calcium pyrophosphate ceramics prepared in Examples 1-2; and D represents the elastic modulus of the calcium pyrophosphate ceramics prepared in Examples 1-2. Figure 7 The in vitro bioactive ion release performance of the bone-inspired ordered structure calcium pyrophosphate ceramics prepared in Examples 1-2 and Comparative Examples 1-2 of this invention is shown; wherein, a is the concentration of calcium ions released by the calcium pyrophosphate ceramics; and b is the concentration of phosphorus ions released by the calcium pyrophosphate ceramics. Figure 8 The in vitro biological properties of the bone-inspired ordered structure calcium pyrophosphate ceramics prepared in Examples 1-2 of this invention are shown; where a is the cytotoxicity experiment (live / dead staining); b is cell adhesion; and c is cell migration. Detailed Implementation

[0018] To more clearly illustrate the technical solution, features, and practical effects of the present invention, a detailed description is provided below with reference to specific embodiments. It should be understood that the modification method of the present invention is not limited to the described embodiments. Equivalent substitutions or modifications made by those skilled in the art based on the teachings of the present invention, without departing from the spirit of the invention, are also within the scope of protection of the claims of the present invention. In each embodiment, unless otherwise specified, the percentage content refers to mass percentage content.

[0019] Calcium-phosphate-based bioceramics (such as hydroxyapatite, tricalcium phosphate, and calcium pyrophosphate) are widely used in bone repair due to their ease of preparation, controllable composition, and good osteogenic properties. Human bone tissue is a typical high-strength and high-toughness natural material. However, the inherent brittleness of inorganic materials makes it difficult for traditional calcium-phosphate-based bioceramics to match the toughness of bone tissue, limiting their wider clinical application. Studies have shown that the excellent toughness of bone tissue is closely related to its multi-level ordered structure. Inspired by the ordered layered and interfacial structure of bone tissue, this invention provides a bone-mimicking oriented calcium pyrophosphate bioceramic. This bioceramic has a layered structure formed by alternating layers of calcium pyrophosphate crystalline phase formed by the dehydration condensation of calcium phosphate and amorphous carbon layers formed by the carbonization of carboxymethyl chitosan. The amorphous carbon layer is located between the two calcium pyrophosphate crystalline phase layers and forms a strong-weak interfacial structure with the calcium pyrophosphate crystalline phase layers. The thickness of the calcium pyrophosphate crystalline phase layer is 3–5 μm; the thickness of the amorphous carbon layer is <100 nm.

[0020] Furthermore, this invention also provides a method for preparing bone-inspired oriented calcium pyrophosphate bioceramics. Based on a combined strategy of template-assisted reverse-induced assembly and hot-pressing sintering, a tougher, bone-inspired ordered calcium pyrophosphate bioactive ceramic is prepared. In this invention, a calcium phosphate template is first used to promote the oriented assembly of carboxymethyl chitosan. Then, the calcium phosphate is converted into calcium pyrophosphate through vacuum hot-pressing sintering. Simultaneously, the carbonized carboxymethyl chitosan reverse-induced the oriented arrangement of calcium pyrophosphate ceramic grains, forming an ordered micro / nano structure, resulting in the final calcium pyrophosphate bioactive ceramic. Specifically, calcium phosphate (CaHPO4·2H2O) undergoes proton transfer during dehydration to generate calcium hydrogen pyrophosphate (CaH2P2O7), which is then calcined at high temperature to generate the final calcium pyrophosphate (Ca2P2O7). The carboxylated chitosan groups are eliminated during high-temperature calcination in a vacuum atmosphere, leaving only the unreacted carbon skeleton to form an amorphous carbon layer.

[0021] The following exemplarily illustrates the preparation method of the bone-inspired oriented calcium pyrophosphate bioceramic provided by the present invention.

[0022] Two-dimensional permeable calcium phosphate powder was synthesized by chemical precipitation. Potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), and anhydrous calcium chloride (CaCl2) were dissolved in deionized water and reacted under magnetic stirring in an ice-water bath (reaction temperature 0–5 °C). After the reaction was completed, the mixed solution was filtered to separate the precipitate, and then dried and ground to obtain the two-dimensional flaky permeable calcium phosphate powder. As an example, 0.012 mol potassium dihydrogen phosphate and 0.042 mol disodium hydrogen phosphate were dissolved in 1.4 L of deionized water, and 0.054 mol anhydrous calcium chloride was dissolved in 0.4 L of deionized water. The solutions were placed in a constant-temperature magnetically stirred water bath and stirred for 0.5 hours, with the reaction temperature controlled by an ice-water bath. The 0.4 L calcium chloride solution was slowly added to a 1.4 L phosphate mixture and magnetically stirred for 1.5 hours under ice-water bath conditions (reaction temperature 1°C). After stirring, the solution was filtered to separate the precipitate, which was then placed in a fume hood to air dry naturally. The dried precipitate was then ground in a mortar and pestle to obtain phosphogypsum powder.

[0023] In an optional embodiment, the molar ratio of potassium dihydrogen phosphate, disodium hydrogen phosphate and anhydrous calcium chloride is (1-3):(6-8):(8-10), preferably 2:7:9.

[0024] Prepare a 10 mg / mL carboxylated chitosan solution by mixing the carboxylated chitosan solution and deionized water at a volume ratio of 1:20, and then add 100 mg / mL calcium chloride solution to allow the carboxylated chitosan to precipitate completely and obtain a suspension.

[0025] Add calcium phosphate powder to the obtained carboxylated chitosan suspension, stir for 1-2 hours, and then filter to obtain a green body. Place the green body in a fume hood to dry completely.

[0026] In an optional embodiment, the mass ratio of the permeapatite powder to carboxymethyl chitosan is (90-95):(5-10). If the mass ratio of permeapatite powder to carboxymethyl chitosan is too small, i.e., the content of carboxylated chitosan is too large, the bonding between the amorphous carbon layer and the calcium pyrophosphate layer is poor, resulting in poor mechanical properties; if the mass ratio of permeapatite powder to carboxymethyl chitosan is too large, i.e., the content of carboxylated chitosan is too small, the reverse template induction effect during the hot pressing sintering process is poor, it is difficult to form a layered structure of alternating calcium pyrophosphate and amorphous carbon, and the thickness of the calcium pyrophosphate layer varies greatly.

[0027] The dried green body is placed in a hot-pressing sintering furnace for sintering. The specific hot-pressing sintering process is as follows: Under a vacuum atmosphere, the furnace temperature is raised from room temperature to the highest hot-pressing sintering temperature, and during this process, the pressure is gradually increased to the required highest hot-pressing sintering pressure; after maintaining the temperature and pressure for a certain period of time, the process ends, and the body is allowed to cool naturally to room temperature to obtain the final bone-like ordered structure calcium pyrophosphate bioceramic.

[0028] In an optional embodiment, the parameters of the hot pressing sintering include: sintering temperature of 900-1100 ℃, sintering pressure of 10-30 MPa, sintering time of 1-4 hours, and sintering atmosphere of vacuum atmosphere.

[0029] The bone-inspired directional calcium pyrophosphate bioceramic prepared by this invention has a flexural strength of 84.9–94.1 MPa and a fracture toughness of 1.08–1.6 MPa·m. 1 / 2 This material can basically meet the strength and toughness requirements for load-bearing bone repair. The bone-mimetic calcium pyrophosphate bioactive ceramic prepared by this invention has good biocompatibility and bioactivity, and can continuously release calcium and phosphorus bioactive ions. It can well support the adhesion and proliferation of mesenchymal stem cells on its surface and promote cell migration, making it a potential bone repair material.

[0030] 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 are 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

[0031] The preparation method of the two-dimensional plaque-like calcium pyrophosphate bioceramic (95CPP) with an initial mass fraction of 95% in Example 1 includes the following steps: (1) Dissolve 0.012 mol potassium dihydrogen phosphate and 0.042 mol disodium hydrogen phosphate in 1.4 L of deionized water, and dissolve 0.054 mol anhydrous calcium chloride in 0.4 L of deionized water. Place them in a constant temperature magnetic stirring water bath and stir for 0.5 hours. Control the reaction temperature by using an ice-water bath. Slowly add the 0.4 L calcium chloride solution prepared above to the 1.4 L phosphate mixed solution and stir magnetically for 1.5 hours under ice-water bath conditions (1 °C). After stirring, separate the precipitate by vacuum filtration and place the precipitate in a fume hood to air dry naturally. Grind the air-dried precipitate with a mortar and pestle to obtain two-dimensional flaky calcium phosphate powder. (2) Take 30 mL of carboxylated chitosan solution (10 mg / mL) and add it to 600 mL of deionized water. Then, dissolve 5 g of calcium chloride in 50 mL of deionized water and add the mixture to the carboxylated chitosan solution to allow the carboxylated chitosan to precipitate completely. Subsequently, add 5.7 g of calcium phosphate powder (the mass ratio of carboxylated chitosan to calcium phosphate is 5:95), stir for 2 hours, and then filter to obtain the green body. Place the green body in a fume hood to dry completely. (3) The blank is placed in a hot pressing sintering furnace for hot pressing sintering; the parameters of the hot pressing sintering include: under vacuum atmosphere, the temperature is raised from room temperature to 1000℃ at a heating rate of 10℃ / min; then the temperature is raised to 1100℃ at a heating rate of 5℃ / min, and the pressure is gradually increased to 30 MPa during this process. The temperature and pressure are maintained for 1 hour and then the temperature is naturally cooled to room temperature to obtain the final bone-like ordered structure calcium pyrophosphate bioceramic. Example 2

[0032] The preparation process of the bone-like calcium pyrophosphate bioceramic (85CPP) with an initial mass fraction of 90% of two-dimensional permeable phosphatite in Example 2 is the same as that in Example 1, except that in step (2), the mass ratio of carboxylated chitosan to permeable phosphatite is 10:90. Comparative Example 1

[0033] The preparation process of the bone-like calcium pyrophosphate bioceramic (85CPP) with an initial mass fraction of 85% of two-dimensional permeable phosphatite in Comparative Example 1 is the same as that in Example 1, except that in step (2), the mass ratio of carboxylated chitosan to permeable phosphatite is 15:85. Comparative Example 2

[0034] The preparation process of the bone-like calcium pyrophosphate bioceramic (97.5CPP) with an initial mass fraction of 97.5% of two-dimensional permeable phosphatite in Comparative Example 2 is the same as that in Example 1, except that in step (2), the mass ratio of carboxylated chitosan to permeable phosphatite is 2.5:97.5.

[0035] Figure 1 This is a characterization diagram of the two-dimensional permealumina powder prepared in Example 1 of the present invention. As can be seen from the figure, the permealumina powder has a typical two-dimensional lamellar structure with a size of approximately 5 μm.

[0036] Figure 2The microstructures of different components of calcium pyrophosphate bioceramics prepared in Examples 1-2 and Comparative Examples 1-2 of this invention are shown in the figures. As can be seen from the figures, the synthesized bone-like calcium pyrophosphate bioceramics all possess a long-range ordered layered microstructure, similar to the alternating parallel bone lamellae in bone tissue. The calcium pyrophosphate layer thickness of 85CPP, 90CPP, and 95CPP is relatively uniform, approximately 3–5 μm. For the 97.5CPP bioceramic, due to its initially low content of carboxylated chitosan, the reverse template induction effect during hot pressing sintering is poor, resulting in a large difference in interlayer thickness. The 85CPP ceramic, due to the addition of more carboxylated chitosan to the green body, although successfully constructing a long-range ordered bone-like lamellae structure, exhibits poor interfacial bonding between the calcium pyrophosphate layer and the carbonized organic layer.

[0037] Figure 3 The figures show the elemental spectra of calcium pyrophosphate bioceramics with different compositions prepared in Examples 1-2 and Comparative Examples 1-2 of this invention. As can be seen from the figures, the calcium pyrophosphate bioceramics prepared in Examples 1-4 mainly contain calcium (Ca), phosphorus (P), and oxygen (O) elements, and the atomic ratios of Ca / P / O are close to 2:2:7, which is the same as the atomic ratios of each element in calcium pyrophosphate (Ca2P2O7).

[0038] Figure 4 The XRD patterns of different components of calcium pyrophosphate bioceramics prepared in Examples 1-2 and Comparative Example 2 of this invention are shown. The results show that the bioceramics prepared in Examples 1 and 3-4 are composed of calcium pyrophosphate. Figures 2-4 Experimental results show that it is entirely feasible to construct the ordered structure of bone-mimicking calcium pyrophosphate bioceramics by combining template-assisted reverse induced assembly and hot pressing sintering.

[0039] Figure 5 The figure shows the characterization of the interface structure of the bone-inspired ordered calcium pyrophosphate ceramic (CPP) prepared in Example 1 of this invention. As can be seen from the figure, the carbonized organic layer in the middle of the bone-inspired calcium pyrophosphate bioceramic interface structure is an amorphous phase, while the two sides are composed of calcium pyrophosphate crystalline phases, and the two are well bonded together, forming a strong-weak phase interfacial bond. Furthermore, when subjected to external force, the crack first propagates along the amorphous carbon layer of the CPP ceramic, rather than directly extending to the calcium pyrophosphate crystalline phase. This strong-weak phase interfacial structure may help increase energy dissipation during ceramic material fracture, thereby improving the mechanical properties of the material.

[0040] Figure 6The mechanical properties of the bone-inspired ordered calcium pyrophosphate ceramics prepared in this invention are shown in the figure. As can be seen from the figure, the flexural strength of the materials was determined using a three-point bending test. The flexural strengths of 90CPP, 95CPP, and 97.5CPP reached 94.1 MPa, 84.9 MPa, and 81.3 MPa, respectively, all reaching the flexural strength of cortical bone (50–150 MPa). For the 85CPP ceramic, due to its poor interfacial bonding, interlayer cracking is very easy, resulting in a lower flexural strength, below 20 MPa. The fracture toughness of the 90CPP and 95CPP ceramics was determined using the single-sided notched beam method, reaching 1.08 MPa·m, respectively. 1 / 2 and 1.6 MPa·m 1 / 2 Among them, the fracture toughness of 95CPP ceramic is close to the lower limit of fracture toughness of cortical bone (2 MPa·m). 1 / 2 ).

[0041] Figure 7 This study investigates the in vitro bioactive ion release performance of the bone-inspired ordered-structure calcium pyrophosphate ceramics prepared in Examples 1-2 and Comparative Examples 1-2 of this invention. CPP bone-inspired ceramics with different component contents were immersed in Tris-HCl solution (pH=7.4) and placed in a constant-temperature shaker at 37 ℃. The weight loss and ion release of each material were measured at different time points (3, 7, 14, and 21 days). The weight change of the CPP bone-inspired ceramics with different components was relatively small after immersion for 21 days, indicating slow degradation. The concentrations of calcium and phosphorus active ions in the immersion solution were determined using ICP. The results showed that the rate of calcium and phosphorus ion release from the bone-inspired ceramics decreased with increasing calcium pyrophosphate content. 85% CPP bone-inspired ceramics exhibited the highest calcium and phosphorus ion release rate, while 97.5% CPP bone-inspired ceramics released fewer active ions. This may be because with increasing CPP content, the strong-weak interface in the microstructure formed after hot pressing and sintering decreases, making the bone-inspired ceramics more dense and thus exhibiting a lower degradation and ion release rate.

[0042] Figure 8The in vitro biological properties of the bone-inspired ordered structure calcium pyrophosphate ceramics prepared in Examples 1-2 of this invention were investigated. The cytotoxicity of the materials was explored using live / dead staining. Cells were stained with Ethidium homodimer-1 and Calcein AM, and cell viability was observed using confocal laser scanning microscopy. The results showed that after co-culturing cells (blank group) with 90CPP and 95CPP bone-inspired ceramics for 24 hours, mesenchymal stem cells and endothelial cells were in good condition, and no large number of dead cells were observed, indicating that neither type of bone-inspired ceramic had significant cytotoxicity. Cell adhesion experiments further observed the state of cells on the material surface. Cell nuclei and cytoskeleton were stained with DAPI and FITC, respectively, and confocal laser scanning microscopy revealed that both types of bone-inspired ceramics could effectively support the adhesion of mesenchymal stem cells to their surfaces, and the cytoskeleton spread well. A cell migration scratch assay was used to evaluate the effect of the materials on cell migration. In this assay, scratches were made on a cell monolayer to artificially remove the central portion of cells, and the cells were then cultured again. The migration of peripheral cells to the central scratched area was observed. The results showed that after 16 hours of culture, the cells in the blank group did not show a significant tendency to migrate towards the center; while in the 90CPP and 95CPP bone-mimetic ceramic groups, cells could be clearly observed migrating towards the center of the scratch. This phenomenon may be due to the effect of the calcium and phosphorus bioactive ions slowly released by the bone-mimetic ceramic.

Claims

1. A bone-inspired oriented calcium pyrophosphate bioceramic, characterized in that, The bone-inspired oriented calcium pyrophosphate bioceramic has a layered structure formed by alternating layers of calcium pyrophosphate crystalline phase formed by the dehydration condensation of phosphogypsum and amorphous carbon layers formed by the carbonization of carboxymethyl chitosan.

2. The bone-inspired oriented calcium pyrophosphate bioceramic according to claim 1, characterized in that, In the bone-inspired oriented calcium pyrophosphate bioceramic, the mass percentage of calcium pyrophosphate is 98.88%–99.82%, and the mass percentage of amorphous carbon is 0.18%–1.12%.

3. The bone-inspired oriented calcium pyrophosphate bioceramic according to claim 1 or 2, characterized in that, The thickness of the calcium pyrophosphate crystalline phase layer is 3–5 μm; the thickness of the amorphous carbon layer is <100 nm.

4. The bone-inspired oriented calcium pyrophosphate bioceramic according to any one of claims 1-3, characterized in that, The bone-inspired oriented calcium pyrophosphate bioceramic exhibits a flexural strength of 84.9–94.1 MPa and a fracture toughness of 1.08–1.6 MPa·m. 1 / 2 .

5. A method for preparing a bone-inspired oriented calcium pyrophosphate bioceramic according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Using two-dimensional sheet-like permeable calcium phosphate powder as a template and carboxymethyl chitosan as a binder, permeable calcium phosphate / carboxymethyl chitosan composite ceramic green body was prepared by directional assembly assisted by filtration. (2) The calcium pyrophosphate bioceramic with bone-like directional structure is obtained by hot pressing and sintering the calcium pyrophosphate composite green body.

6. The preparation method according to claim 5, characterized in that, The two-dimensional flaky phosphatite powder is synthesized by chemical precipitation. The chemical precipitation method includes: dissolving potassium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous calcium chloride in deionized water, reacting them under magnetic stirring in an ice-water bath, separating the precipitate by filtration after the reaction, and then drying and grinding to obtain the two-dimensional flaky phosphatite powder. Preferably, the temperature of the ice-water bath is 0-5 °C, and the magnetic stirring time is 1-2 hours.

7. The preparation method according to claim 5 or 6, characterized in that, The molar ratio of potassium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous calcium chloride is (1-3):(6-8):(8-10), preferably 2:7:

9.

8. The preparation method according to any one of claims 5-7, characterized in that, The mass ratio of the two-dimensional flaky phosphogypsum powder to carboxymethyl chitosan is (90-95):(5-10).

9. The preparation method according to any one of claims 5-8, characterized in that, The parameters for hot pressing sintering include: sintering temperature of 900–1100 °C, sintering pressure of 10–30 MPa, sintering time of 1–4 hours, and sintering atmosphere of vacuum.

10. The application of a bone-inspired oriented calcium pyrophosphate bioceramic according to any one of claims 1-4 in the preparation of bone defect repair materials.

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