Shell-derived biphase calcium phosphate composite bone repair material as well as preparation method and application thereof

Abstract

The invention discloses a shell-derived biphase calcium phosphate composite bone repair material as well as a preparation method and application thereof, and belongs to the field of biomedical materials. According to the preparation method, the shell-source calcium source and the calcium pyrophosphate are mixed, the biphase calcium phosphate (BCP) containing HAP and beta-TCP in different proportions is prepared through wet ball milling and solid-phase reaction, the reaction conditions are controlled in a simple mode, and the proportion of HAP to beta-TCP in the BCP is controllable. The biphase calcium phosphate prepared by the method is further compounded with sodium alginate, and the biphase calcium phosphate-sodium alginate composite scaffold is prepared by freeze drying and Cacrosslinking. The composite scaffold has a suitable porous structure, can exert in-vitro osteogenic induction activity and an in-vivo bone defect repair function, can significantly induce MC3T3-E1 cell alkaline phosphatase activity and induce calcium deposition, has osteogenic induction activity, can induce bone defect repair at an in-vivo rat skull defect part, and has a good application prospect. Excellent bone tissue engineering application prospects are shown.

Description

TECHNICAL FIELD

[0001] The present application relates to a bone repair material, in particular to a shell-derived biphasic calcium phosphate composite bone repair material and its solid-phase preparation method and application, belonging to the field of biomedical materials. BACKGROUND

[0002] Bone defect repair field mainly uses bone grafting technology for treatment. In clinical practice, autologous bone grafting is considered as the gold standard and has achieved significant efficacy, but still faces problems such as lack of donor source and complications at the bone harvesting site. The application of allogeneic demineralized bone is also limited, and its immunogenicity risk, unstable osteoinductive activity, high long-term absorption rate, and perioperative infection risks restrict its clinical promotion. Hydroxyapatite (HAP) has attracted attention due to its excellent biocompatibility, rapid bone conduction characteristics and direct bone integration ability. However, chemically synthesized HAP materials have defects such as high crystallinity, disordered spatial structure, slow in vivo degradation, and lack of osteoinductive activity, which limit their clinical application. β-tricalcium phosphate (β-TCP) is one of the most attractive bone graft substitutes in recent years, and has more advantages than HAP in terms of biodegradation performance and has properties such as bone induction and bone conduction. β-TCP can be degraded and absorbed by osteoclasts, achieving rapid bone conduction. However, its rapid degradation rate not only affects the quality of bone bonding, but also is not conducive to the ordered attachment of biological tissues on the surface of the material, thereby hindering the osteogenesis process. The mechanical strength of β-TCP also cannot be compared with that of living tissues, and it is easy to break brittlely. Based on the complementary characteristics of the above materials, biphasic calcium phosphate (BCP) has attracted widespread attention in the field of bone repair. Biphasic calcium phosphate is a widely used synthetic ceramic. This composite material is made by scientifically proportioning HAP and β-TCP, which not only maintains good bone conduction, but also significantly optimizes the in vivo degradation kinetics and osteoinductive properties of the material. Clinical studies have confirmed that BCP has unique advantages in promoting the integration of new bone tissue and the coordination of material degradation rate, and has been recognized as the most clinically applicable artificial bone substitute material, and has been widely recognized in the field of bone tissue engineering scaffolds.

[0003] The preparation methods of the reported biphasic calcium phosphate include: (1) physical compounding method: this process realizes phase compounding by mechanically mixing HAP and β-TCP powders, although the operation is simple and the compounding ratio is controllable, but the particle size distribution, crystallinity and dispersity of the raw materials directly affect the performance of the composite material. Since the HAP and β-TCP raw materials are synthesized by liquid phase method, the preparation process has problems such as long process flow, wastewater / gas emission, etc. (2) liquid phase method: this process obtains BCP powder through ammonia liquid phase reaction. This process consumes a large amount of ammonia water to adjust pH, and needs repeated washing to remove byproducts, which has wastewater treatment pressure. At the same time, the difference in solubility characteristics of β-TCP and HAP at different pH values leads to the difficulty in accurately controlling the phase ratio of the product (Zou Ping et al., Preparation and properties of β-TCP / HAP biphasic calcium phosphate composite powder, Journal of Sichuan University (Natural Science Edition), 1996, 33(2): 150-154). (3) sol-gel method: this process prepares a mixed sol with a certain molar ratio of calcium and phosphorus compounds, and then obtains a composite material after drying and sintering. The process is accompanied by air pollution caused by nitrogen dioxide, and the preparation of sol and solvent recovery are complex, which restricts the large-scale production (CN106242552A). (4) chelation calcination method: using calcium nitrate / phosphoric acid trimethyl ester as raw material, citric acid chelation and low-temperature calcination are used to prepare nano-powder. This method involves sol-gel transformation and calcination, which has high process complexity, and the cost of ester raw materials is high, which has organic waste gas emission problem (CN1557774A). (5) solid phase reaction method: using inorganic calcium source (such as calcium carbonate, calcium oxalate, etc.) and phosphorus source as raw materials, through wet ball milling, drying, and high-temperature calcination, HAP and β-TCP are formed, and the control of calcination temperature / time affects the grain size and phase ratio. However, the calcium source used in this process is inorganic calcium source, and it needs to control the reaction water vapor environment by adding deionized water, which is more complex in technology, and depends on peristaltic pump and other equipment (CN107176832A).

[0004] In addition, the biphasic calcium phosphate prepared in the prior art is usually micron-sized and has uneven particle size distribution and low crystallinity, and the HAP / β-TCP ratio cannot be accurately controlled, so it cannot meet the needs of different bone repair speeds and degradation rates. SUMMARY

[0005] In view of the problems of the existing biphasic calcium phosphate (BCP) preparation process, such as dependence on synthetic chemicals, complex process, serious environmental pollution, and insufficient biological activity of the product, the first object of the present application is to provide a preparation method of biphasic calcium phosphate with natural shell powder as calcium source, green and environmentally friendly, low cost, and osteogenic activity.

[0006] The second object of the present application is to provide a biphasic calcium phosphate with controllable HAP and β-TCP ratio prepared by the above method, which has nano-sized particles, uniform particle size distribution, high crystallinity, and excellent osteogenic induction activity.

[0007] The third object of the present application is to provide a composite bone repair material composed of the biphasic calcium phosphate prepared by the above method and natural polysaccharide sodium alginate, which constructs a three-dimensional scaffold with organic-inorganic biphasic structure simulating natural bone tissue, and realizes the repair of bone defects and the significant promotion of cell osteogenic differentiation.

[0008] The present application mixes the calcium source from shells with calcium pyrophosphate (Ca2P2O7) to prepare biphasic calcium phosphate (BCP) containing different proportions of HAP and β-TCP through wet ball milling and solid phase reaction. This preparation method controls the reaction conditions in a simple way, realizes the controllable proportion of HAP and β-TCP in BCP, and realizes the high-value utilization of aquatic waste shells. At the same time, the use of nitrate, ester or ammonia in the traditional chemical synthesis process is avoided, which has the characteristics of low cost, high performance and green environmental protection, and provides a breakthrough solution for the preparation of BCP.

[0009] The specific technical solutions of the present application are as follows:

[0010] A biphasic calcium phosphate from shells, characterized in that it comprises hydroxyapatite and β-tricalcium phosphate, the mass ratio of the hydroxyapatite and the β-tricalcium phosphate being 25:75-95:5; the biphasic calcium phosphate has uniform particle size distribution, the average particle size being between 100-600 nm, and the crystallinity being 65-100%.

[0011] A preparation method of biphasic calcium phosphate from shells, characterized in that it comprises the following steps:

[0012] (1) soaking clean scallop shells and other shells in 1-10% NaOH solution overnight to remove most of the organic matter, washing with water, drying, and crushing to obtain nacre layer powder;

[0013] (2) mixing the nacre layer powder with calcium pyrophosphate at a molar ratio of 4:3, adding water in a ball mill, and wet milling at a speed of 25-100 rps;

[0014] (3) drying the ground slurry and calcining at a temperature of 900-1100 ℃ to obtain biphasic calcium phosphate.

[0015] The wet milling time is 30-60 min.

[0016] The slurry is dried in a 100 ℃ vacuum drying oven for 24 h.

[0017] The high-temperature calcination time is 1-5 h.

[0018] The present application realizes the controllable proportion of hydroxyapatite and beta-tricalcium phosphate (beta-TCP) in the biphasic calcium phosphate between 25:75 and 95:5 by controlling the wet grinding time, the calcination temperature and the calcination time, thereby preparing the biphasic calcium phosphate containing different proportions of hydroxyapatite and beta-tricalcium phosphate.

[0019] The present application further utilizes the biphasic calcium phosphate prepared by the above method to be compounded with sodium alginate, and utilizes the freeze drying and Ca2+ crosslinking to prepare a biphasic calcium phosphate-sodium alginate composite scaffold, the composite scaffold has a suitable porous structure, can play the in-vitro osteogenic induction activity and the in-vivo bone defect repair function, can significantly induce the alkaline phosphatase activity of MC3T3-E1 cells, induce the calcium deposition, has the osteogenic induction activity, and can induce the repair of the bone defect in the in-vivo rat skull defect part.

[0020] (1) uniformly dispersing the biphasic calcium phosphate in water to form a uniform dispersion liquid; (2) adding sodium alginate to continue stirring to form a uniform colloidal suspension; (3) pouring the suspension into a mold to prepare the composite scaffold through freeze drying; (4) placing the freeze-dried composite scaffold in a 10% CaCl2 solution for crosslinking for 1 h; (5) washing away the excess Ca 2+ to obtain the biphasic calcium phosphate-sodium alginate composite scaffold.

[0021] The mass percentage of the biphasic calcium phosphate in the biphasic calcium phosphate / sodium alginate composite scaffold is 1-5%.

[0022] The pore size of the biphasic calcium phosphate-sodium alginate composite scaffold prepared by the above preparation method is 90-120 mu m.

[0023] The application of the biphasic calcium phosphate-sodium alginate composite scaffold prepared by the above preparation method as a bone repair material.

[0024] Compared with the prior art, the present application has the following significant innovations and technical advantages:

[0025] (1) Green raw materials and resource utilization

[0026] The present application first uses the aquatic processing waste, shell powder, as the main calcium source of BCP, avoids using chemical synthetic calcium salt (such as calcium carbonate, calcium oxide, etc.), not only reduces the raw material cost, but also effectively utilizes the renewable resources, and responds to the environmental protection requirements of sustainable development.

[0027] (2) Natural impurity ion function mechanism

[0028] The trace elements of strontium (Sr2+) and magnesium (Mg2+) naturally contained in scallop shells can partially enter the HAP / β-TCP crystal lattice in a high-temperature solid-phase reaction, adjust the nucleation and crystal growth process, and endow the final product with a more optimal crystal structure and biological function, so that the induced osteogenic activity can be enhanced without the need for external doping agents.

[0029] (3) Green solid-phase synthesis path, no pollution additives

[0030] The present application adopts pure water as a wet grinding medium, realizes reaction process control through ball milling, vacuum drying and atmospheric high-temperature calcination in three steps, and does not need chemical additives such as alcohol solvents, ammonia water and chelating agents, thereby avoiding the problems of organic waste gas or nitrogen-containing wastewater discharge in common processes.

[0031] (4) Precise regulation mechanism of HAP / β-TCP ratio

[0032] By systemically regulating the wet grinding time, calcination temperature and holding time, the HAP ratio in BCP can be controllably changed from 25% to 95%, and the nano-scale composite ceramic powder with uniform particle size distribution and high crystallinity is formed, thereby meeting the requirements of different bone repair speeds and degradation rates.

[0033] (5) Biomimetic composite scaffold construction and significantly enhanced osteogenic performance

[0034] The present application further mixes the prepared BCP powder with sodium alginate, and forms a porous composite scaffold through freeze-drying and Ca2+ crosslinking, which has the characteristics of a natural bone tissue structure. The scaffold can induce the differentiation of MC3T3-E1 cells, promote calcium deposition in vitro, and significantly promote new bone formation in a rat skull defect model, thereby showing excellent application prospects in bone tissue engineering. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The figure is an XRD spectrum of the composition analysis of biphasic calcium phosphate of different samples.

[0036] Figure 2 The figure is an SEM spectrum of the surface morphology of different samples.

[0037] Figure 3 The figure is a particle size distribution graph of biphasic calcium phosphate of different samples.

[0038] Figure 4 The figure is an SEM spectrum of the appearance and internal cross-section structure of biphasic calcium phosphate-sodium alginate composite scaffolds with different compositions.

[0039] Figure 5 The figure is an ALP staining observation graph of MC3T3-E1 cells cultured in different composite scaffold leaching liquids for 7 and 14 days.

[0040] Figure 6Figure 1 is a graph showing ALP activity of MC3T3-E1 cells cultured in different composite scaffold leaching liquor for 7 and 14 days.

[0041] Figure 7 Figure 2 is a graph showing alizarin red staining observation of MC3T3-E1 cells cultured in different composite scaffold leaching liquor for 14 and 21 days.

[0042] Figure 8 Figure 3 is a graph showing alizarin red quantitative determination of MC3T3-E1 cells cultured in different composite scaffold leaching liquor for 14 and 21 days.

[0043] Figure 9 Figure 4 is a Micro-CT three-dimensional graph of rat skull defect repair by different biphasic calcium phosphate-sodium alginate composite scaffolds.

[0044] Figure 10 Figure 5 is a cross-sectional graph of rat skull defect repair by different biphasic calcium phosphate-sodium alginate composite scaffolds. DETAILED DESCRIPTION

[0045] The present application will be further described in detail below with reference to the accompanying drawings and specific examples.

[0046] Example 1

[0047] (1) Fresh scallop shells were washed, the surface mud was removed, and the scallop shells were soaked in 10% NaOH overnight to remove most of the organic impurities. The excess NaOH on the surface of the scallop shells was washed off with tap water, and the scallop shells were naturally dried. The scallop shells were then crushed and sieved to obtain nacre powder.

[0048] (2) 9 g of nacre powder was mixed with calcium pyrophosphate at a molar ratio of 4:3, 6 ml of water was added to the ball mill, and wet grinding was carried out at a speed of 25 rps for 30 min.

[0049] (3) The ground slurry was placed in a 100°C vacuum drying oven and dried for 24 h. The dried mixture was calcined in a muffle furnace (calcined at 900°C for 1 h) to obtain biphasic calcium phosphate, labeled as H25, with a crystallinity of about 65%.

[0050] Example 2

[0051] (1) The same as step (1) of Example 1;

[0052] (2) The nacre powder was mixed with calcium pyrophosphate at a molar ratio of 4:3, 6 ml of water was added to the ball mill, and wet grinding was carried out at a speed of 25 rps for 60 min.

[0053] (3) The ground slurry was placed in a 100 °C vacuum drying oven for 24 h. The dried mixture was subjected to high-temperature calcination (calcination at 1000 °C for 1 h) in a muffle furnace to obtain the biphasic calcium phosphate, labeled as H50, with a crystallinity of about 75%.

[0054] Example 3:

[0055] (1) The same as step (1) of Example 1;

[0056] (2) The pearl layer powder and calcium pyrophosphate were mixed in a molar ratio of 4:3, 6 ml of water was added in a ball mill, and wet grinding was carried out at a speed of 25 rps for 60 min.

[0057] (3) The ground slurry was placed in a 100 °C vacuum drying oven for 24 h. The dried mixture was subjected to high-temperature calcination (calcination at 1000 °C for 1 h) in a muffle furnace to obtain the biphasic calcium phosphate, labeled as H50, with a crystallinity of about 75%.

[0058] Example 4:

[0059] (1) The same as step (1) of Example 1;

[0060] (2) The pearl layer powder and calcium pyrophosphate were mixed in a molar ratio of 4:3, 6 ml of water was added in a ball mill, and wet grinding was carried out at a speed of 25 rps for 60 min.

[0061] (3) The ground slurry was placed in a 100 °C vacuum drying oven for 24 h. The dried mixture was subjected to high-temperature calcination (calcination at 1000 °C for 1 h) in a muffle furnace to obtain the biphasic calcium phosphate, labeled as H50, with a crystallinity of about 75%.

[0062] Example 5:

[0063] (1) The same as step (1) of Example 1;

[0064] (2) The pearl layer powder and calcium pyrophosphate were mixed in a molar ratio of 4:3, 6 ml of water was added in a ball mill, and wet grinding was carried out at a speed of 25 rps for 60 min.

[0065] (3) The ground slurry was placed in a 100 °C vacuum drying oven for 24 h. The dried mixture was subjected to high-temperature calcination (calcination at 1000 °C for 1 h) in a muffle furnace to obtain the biphasic calcium phosphate, labeled as H50, with a crystallinity of about 75%.

[0066] Structure characterization of biphasic calcium phosphate:

[0067] The phase composition of the biphasic calcium phosphate prepared in Examples 1-5 was analyzed by X-ray diffraction (XRD): Cu target Kα ray (λ = 0.154 nm) was used, tube voltage was 40 kV, tube current was 40 mA, and the scanning range of wide-angle X-ray diffraction was 10-80° with a step of 4°. The particle size and particle size distribution of the biphasic calcium phosphate were analyzed by a particle size analyzer: the obtained biphasic calcium phosphate was uniformly dispersed in water to prepare a suspension, and the suspension was placed in the particle size analyzer to determine the particle size.

[0068] The XRD patterns of the biphasic calcium phosphate prepared in Examples 1-5 are shown in Figure 1 The XRD patterns of the above biphasic calcium phosphate samples were compared with the HAP standard pattern (ICDD NO. 09-0432) and the β-TCP (ICDD NO. 09-0169) standard pattern. By analyzing and calculating the components of the biphasic calcium phosphate samples by High Score Plus software, it can be concluded that the composition of H25 biphasic calcium phosphate is (25% HAP + 75% β-TCP), the composition of H50 biphasic calcium phosphate is (50% HAP + 50% β-TCP), the composition of H60 biphasic calcium phosphate is (60% HAP + 40% β-TCP), the composition of H70 biphasic calcium phosphate is (70% HAP + 30% β-TCP), and the composition of H95 biphasic calcium phosphate is (95% HAP + 5% β-TCP). The results prove that the solid-phase reaction between Ca2P2O7 and CaCO3 is affected by the wet milling time, calcination temperature and calcination time. With the extension of the wet milling time, the increase of the calcination temperature and the extension of the calcination time, the composition of the product is more converted into HAP.

[0069] The SEM morphology detection results of the biphasic calcium phosphate prepared in Examples 1-5 are shown in Figure 2 It can be seen from the figure that the surface of H25 has only a small amount of crystalline structure generated. The surfaces of H50, H60 and H70 have a large number of dense crystalline particles; the crystalline particles on the surface of H95 have been fused. The results prove that within the calcination temperature range of 900-1100 ℃, with the increase of the calcination temperature and the extension of the calcination time, the composition of the product is more converted into HAP.

[0070] The particle size distribution of the biphasic calcium phosphate prepared in Examples 1-5 is shown in Figure 3As shown in the figure, the particle sizes of the biphasic calcium phosphate powders all exhibit a normal distribution. The particle sizes of H25 and uncalcined nacre powder are similar, with smaller particles ranging from 100 to 200 nm. The particle sizes of H50 and H70 samples increased slightly, primarily around 300 nm. The H95 group had the largest particle size, primarily concentrated between 400 and 800 nm, with an average particle size of approximately 600 nm. These results indicate that the particle size of the resulting mixed powders gradually increases with increasing calcination temperature and time. All biphasic calcium phosphate particles are nanoscale in size. Cells have a stronger ability to absorb nanoscale BCP particles than micron-sized particles. Nanoscale biomaterial particles have a larger specific surface area, which facilitates cell adhesion, migration, proliferation, and differentiation, and enhances interactions between the material and cells.

[0071] The strontium (Sr²⁺) and magnesium (Mg²⁺) contents in nacre powder and some biphasic calcium phosphate samples are shown in Table 1. The trace elements strontium (Sr²⁺) and magnesium (Mg²⁺) naturally present in nacre powder can impart a superior crystal structure and osteoinductive activity to the final product.

[0072] Table 1. Strontium (Sr²⁺) and magnesium (Mg²⁺) contents in nacre powder and some biphasic calcium phosphate samples

[0073]

[0074] Example 6: Preparation of biphasic calcium phosphate-sodium alginate composite scaffold

[0075] (1) The biphasic calcium phosphate samples H25, H70, and pearl powder prepared in the above examples were uniformly dispersed in water and ultrasonically dispersed for 10 min to form a uniform dispersion; (2) Sodium alginate was added and magnetic stirring was continued to form a uniform colloidal suspension; (3) The suspension was poured into a mold and freeze-dried to obtain a composite scaffold; (4) The freeze-dried scaffold was placed in a 10% CaCl2 solution for cross-linking for 1 h; (5) The excess CaCl2 on the surface was washed off. 2+ , the obtained composite scaffolds were named NS (nacre powder-sodium alginate), H25S (H25-sodium alginate), and H70S (H70-sodium alginate).

[0076] The morphology of the composite scaffold was analyzed by scanning electron microscopy. Figure 4 As shown. Figure 4 As can be seen in the figure, the composite scaffold material has a sponge-like structure, and its shape can be plastic depending on the mold used for preparation. Scanning electron microscopy images show that the interior of the composite scaffold is a porous structure with interconnected pores, with an internal pore diameter of approximately 90-120 μm.

[0077] Example 7: Osteoinductive activity of biphasic calcium phosphate-sodium alginate composite scaffolds

[0078] MC3T3-E1 cells were seeded in 12-well plates at a cell density of 4 x 10 4 After 24 h of adherent culture, the experimental groups were replaced with the composite scaffold material extraction medium, and the blank group was replaced with the induction differentiation medium. The cells were fixed with 4% paraformaldehyde and subjected to ALP staining. The protein concentration was detected by spectrophotometry, and the ALP activity was quantitatively detected.

[0079] MC3T3-E1 cells were cultured for 14 and 21 days according to the above experimental method, and the in vitro biological mineralization activity of the cells was determined. After the cells were fixed with 4% paraformaldehyde, alizarin red staining solution was added, and the mixture was incubated at 37°C for 30 min. After multiple rinsing with distilled water until clear, the mixture was observed and photographed under an inverted microscope. 500 μL of 10% cetylpyridinium chloride was added to each well, and the mixture was incubated at 37°C for 1 h to dissolve the calcified nodules. 200 μL was transferred to a 96-well plate, and the absorbance value was measured at 540 nm.

[0080] The results of the alkaline phosphatase staining are shown in FIG. 5. After 7 days of culture, the cells grew well, and alkaline phosphatase was produced in the cells and the intercellular space, but there was no significant difference between the experimental groups and the blank group. After 14 days of culture, the alkaline phosphatase staining area in the experimental groups increased significantly, indicating that the ALP content in the cells increased significantly, and there was a significant difference compared with the blank group. The results of the ALP activity quantitative analysis are shown in FIG. 6. After 7 days of continuous culture of the cells with the composite scaffold material extraction liquid, the ALP activity of the H70S group was higher than that of the other experimental groups. After 14 days of continuous culture, the ALP activity of the cells in all groups increased significantly, and the ALP activity of the cells in the composite scaffold material extraction liquid group was significantly higher than that in the blank control group, and H70S > H25S > NS > blank. Figure 6 The results of the alizarin red staining are shown in FIG. 7. It can be seen that after 14 days, there was no significant mineralization in the experimental groups and the blank group. After 21 days, the cells in the composite scaffold material group showed significant mineralization nodules, which had a significant difference compared with the blank group. The biological mineralization level of the cells was quantitatively analyzed (FIG. 8).

[0081] Figure 7 The results of the alizarin red staining are shown in FIG. 7. It can be seen that after 14 days, there was no significant mineralization in the experimental groups and the blank group. After 21 days, the cells in the composite scaffold material group showed significant mineralization nodules, which had a significant difference compared with the blank group. The biological mineralization level of the cells was quantitatively analyzed (FIG. 8). Figure 8 ​It was found that the cell biomineralization level in the composite scaffold material group was significantly higher than that in the blank control group at 21 d. Mineralized nodules are a marker of cell osteogenic differentiation. The experimental results show that the composite scaffold material can induce calcium deposition and has good ability to induce biomineralization, and the H70S group has the most significant effect. The research results show that the biphasic calcium phosphate / sodium alginate composite scaffold has good potential for inducing osteogenic differentiation of MC3T3-E1 cells.

[0082] Example 8: Study on the bone defect repair function of the composite scaffold material

[0083] SD male rat skull defect models (5 mm in diameter) were prepared. The blank control group was not implanted with experimental materials. The experimental groups were implanted with NS, H25S and H70S materials in the skull defect area, sutured the subcutaneous soft tissue and skin, and iodophor debridement. The skull defect parts of the rats were taken for Micro-CT scanning at 1, 2 and 3 months after the operation, and three-dimensional images of the bone tissue were constructed to analyze the bone defect repair.

[0084] Figure 9 and Figure 10 are the Micro-CT three-dimensional reconstruction results and cross-sectional images of the rat skull, respectively. It can be seen that at 1 month after the operation, a small amount of new bone tissue was generated in the NS and H25S groups, and the amount of new bone tissue in the H70S group was significantly more than that in the other groups, among which the marginal osteogenesis was most active, and irregular new bone formation was visible at the edge of the bone defect. At 2 months after the operation, the amount of new bone in the H70S group was significantly more than that in the other groups. At 3 months after the operation, on the basis of marginal osteogenesis, point-like new bone generation appeared in the middle of the defect site in the H70S group, indicating that the composite scaffold can induce cell migration to the defect site, provide adhesion sites for osteoblasts, and be conducive to the calcification deposition of new bone. In the NS and H25S groups, the amount of new bone was relatively small. The animal experiment results show that the composite scaffold in the H70S group can better induce the generation of new bone and has a significant function of inducing bone regeneration, which promotes the repair of rat skull defects.

[0085] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, for those skilled in the art, the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A shell-derived biphasic calcium phosphate, characterized in that: The biphasic calcium phosphate comprises hydroxyapatite and β-tricalcium phosphate, wherein the mass ratio of the hydroxyapatite to β-tricalcium phosphate is 25:75 to 95:5; the particle size distribution of the biphasic calcium phosphate is uniform, the average particle size is between 100-600 nm, and the crystallinity is 65-100%.

2. A method for preparing biphasic calcium phosphate derived from shells, characterized in that: The following steps are involved: (1) Soak cleaned scallop shells or other shells in 1-10% NaOH solution overnight to remove most of the organic matter, and then wash, dry, and crush to obtain nacre powder; (2) Mix nacre powder and calcium pyrophosphate at a molar ratio of 4:3, add water in a ball mill, and wet grind at a speed of 25-100 rps; (3) The ground slurry is dried and then calcined at a temperature of 900-1100 °C to obtain biphasic calcium phosphate.

3. The method for preparing biphasic calcium phosphate according to claim 2, wherein: The wet grinding time in step (2) is 30-60 min.

4. The method for preparing biphasic calcium phosphate according to claim 2, wherein: The slurry in step (3) is dried in a vacuum drying oven at 100°C for 24 hours.

5. The method for preparing biphasic calcium phosphate according to claim 2, wherein: The high-temperature calcination time in step (3) is 1-5 h.

6. A method for preparing a biphasic calcium phosphate-sodium alginate composite scaffold, characterized in that: The following steps are involved: (1) Dispersing biphasic calcium phosphate uniformly in water to form a uniform dispersion; (2) Add sodium alginate and continue stirring to form a uniform colloidal suspension; (3) Pour the suspension into a mold and freeze-dry to obtain a composite scaffold; (4) Place the freeze-dried composite scaffold in a 10% CaCl2 solution for cross-linking for 1 h; (5) Rinse off excess Ca on the surface 2+ , and a biphasic calcium phosphate-sodium alginate composite scaffold was obtained.

7. The method for preparing the biphasic calcium phosphate-sodium alginate composite scaffold according to claim 6, characterized in that: The pore size of the composite scaffold is 90 μm to 120 μm.

8. The biphasic calcium phosphate-sodium alginate composite scaffold according to claim 6, characterized in that: The mass percentage of biphasic calcium phosphate in the composite scaffold is 1-5%.

9. Use of the biphasic calcium phosphate-sodium alginate composite scaffold prepared according to the preparation method of claim 6 as a bone repair material.

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