Use of a composition for the preparation of a bone repair material

Abstract

The application provides application of a composition in preparation of a bone repair material, wherein the composition comprises, based on the total dry matter of the composition: 70-90% of coral hydroxyapatite particles, 5-30% of recombinant human collagen, and 0-7% of excipients, and the bone repair material is a cancellous bone-like material with a porosity of 85-99%. The composition has high porosity and a longitudinal gradient structure, can simulate the characteristics of natural bone tissue, has excellent biological compatibility, biodegradability, hydrophilicity, osteogenic performance and shape memory function, has excellent clinical use convenience, hemostatic and healing promotion effects, excellent osteogenesis, and has a wide application prospect in preparation of the bone repair material for filling and / or repairing bone defects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical materials, and particularly relates to application of a composition in preparation of a bone repair material. BACKGROUND

[0002] For the research of artificial bone biomaterials, there are mainly the following types: high polymer materials, such as polymethyl methacrylate (PMMA), i.e. bone cement, and high polymer polyethylene used for artificial joints, which have poor biocompatibility and are separated from bone tissue by fibrous tissue. Inorganic materials are the most commonly used materials, mainly ceramic materials, which are divided into biologically inert, bioactive and degradable materials. Biologically inert materials are, for example, alumina ceramics, bioactive materials are, for example, glass ceramics, bioactive glass, hydroxyapatite, etc., and the degradable ceramic is mainly beta-tricalcium phosphate (beta-TCP). In addition, there is another type of degradable material that is not a ceramic, which is natural coral. The main advantages of bioactive materials are good biocompatibility, chemical combination with bone tissue or degradation in the body, and high strength. The main disadvantage of ceramic materials is brittleness, and their elastic modulus is difficult to match that of normal bone, which limits their application in clinical practice to a certain extent.

[0003] Therefore, developing an ideal bone graft substitute has been one of the important topics in the field of bone surgery. The ideal bone graft substitute should have the following characteristics: (1) bone conduction; (2) bone induction; (3) excellent hydrophilicity; (4) good biocompatibility; (5) degradability, the in vivo degradation rate matches the new bone regeneration rate; (6) appropriate porosity and pore connection structure; (7) good mechanical properties; (8) easy to operate during surgery; (9) easy to sterilize before use; (10) can be prepared into a certain size for filling, etc.

[0004] Hydroxyapatite (HAP) and collagen (Col protein) are the main inorganic and organic components in natural bone, and both have good biological properties. However, when used alone, they have different defects and cannot meet the requirements of tissue engineering for the performance of biomaterials. By compounding collagen and hydroxyapatite, the adhesion of collagen is better utilized to overcome some limitations of hydroxyapatite. SUMMARY

[0005] To solve the above technical problems, the purpose of the present application is to provide application of a composition in preparation of a bone repair material.

[0006] In order to achieve the above purpose, the present application provides application of a composition in preparation of a bone repair material, wherein the material quality of the composition includes, based on the total dry matter of the composition being 100%:

[0007] coral hydroxyapatite particles 70-90%,

[0008] recombinant human collagen 5-30%, and

[0009] excipients 0-7%.

[0010] The bone repair material is a cancellous bone-like material, and the porosity is 85-99%.

[0011] According to a specific embodiment of the present application, preferably, the porosity of the cancellous bone-like material is 95-99%.

[0012] According to a specific embodiment of the present application, preferably, the bone repair material comprises a bone repair material used in filling and / or repairing of bone defects.

[0013] According to a specific embodiment of the present application, preferably, the bone repair material comprises a bone repair material used in oral surgery, orthopedics, neurosurgery, plastic surgery.

[0014] According to a specific embodiment of the present application, preferably, the bone repair material used in oral surgery comprises a bone repair material used in filling of tooth extraction socket after tooth extraction or extraction of residual root, restoration of alveolar ridge, repair of alveolar bone defects caused by periodontal disease, filling and repair of dental and jaw bone defects or insufficient bone mass, and filling of non-bearing parts of maxillofacial bone defects.

[0015] According to a specific embodiment of the present application, preferably, the bone repair material used in orthopedics comprises a bone repair material used in repair of non-bearing parts of bone defects; more preferably, a bone repair material used in bone grafting in fracture with bone defects, nonunion or malunion and orthopedics, benign cystic lesions of bone, spinal fusion caused by lumbar instability or lumbar spinal stenosis, and arthrodesis.

[0016] According to a specific embodiment of the present application, preferably, the bone repair material used in neurosurgery comprises a bone repair material used in filling of skull defects caused by craniotomy drilling, filling of skull defects caused by decompressive craniectomy, filling of bone suture caused by craniotomy milling, repair of skull defects caused by intraoperative bone resection, and bone repair material used in other types of neurosurgery without bone grafting contraindications.

[0017] According to a specific embodiment of the present application, preferably, the bone repair material used in plastic surgery comprises a bone repair material used in filling and / or repair of bone defects.

[0018] According to a specific embodiment of the present application, the composition is a solid porous material formed by mutual adhesion of coral hydroxyapatite particles with recombinant human collagen.

[0019] The recombinant human collagen in the present application is the recombinant human collagen disclosed in CN108070032B

A purification method of recombinant human collagen

[0020] According to a specific embodiment of the present application, the recombinant human collagen has an amino acid sequence as shown in SEQ ID No: 1.

[0021] According to a specific embodiment of the present application, preferably, the material comprises, based on the total dry matter of the composition: 70-90% of coral hydroxyapatite particles, 5-23% of recombinant human collagen, and 5-7% of excipients.

[0022] According to a specific embodiment of the present application, the particle size of the coral hydroxyapatite particles ranges from 0.1 mm to 2 mm, the pore size ranges from 50 μm to 800 μm, and the porosity ranges from 50% to 90%.

[0023] According to a specific embodiment of the present application, preferably, the conversion rate of the coral hydroxyapatite particles is 5-80%; preferably, the conversion rate is 5-30%.

[0024] According to a specific embodiment of the present application, preferably, the excipients include one or more than two combinations of cross-linked porous starch, sodium carboxymethyl cellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methyl cellulose.

[0025] The recombinant human collagen has regular hydrophilic groups on the outside, super strong aggregation ability; and has hydrophobic groups on the inside, forming a micro-scaffold. The recombinant human collagen has the ability of self-assembly, and can be self-assembled in a low-oxygen or vacuum environment. The self-assembly ability can be promoted by increasing the temperature, and the process does not cause reagent residues caused by chemical cross-linking. The smart collagen hydrophilicity (structure) of the recombinant human collagen is as shown in Figure 1 , and the SEM of the self-assembly structure of the recombinant human collagen is as shown in Figure 2 .

[0026] According to a specific embodiment of the present application, the coral hydroxyapatite particles are prepared by soaking, crushing and granulating coral stone with a cutting protective agent, and water heat exchange. The appearance of the coral stone in the present application is as shown in Figure 3 , and has a three-dimensional network structure with interconnected pores. The microstructure of the coral stone with different pore sizes is as shown in Figure 4 , wherein a represents dense pores, b represents mesopores, and c represents macropores, Figure 4 The mesoporous coral stone represented by b is most similar to the structure of natural bone.

[0027] According to a specific embodiment of the present application, the cutting protective agent is a solution containing a polyol.

[0028] According to the specific embodiment of the present application, preferably, the polyol is selected from one or a combination of two or more of glycerol, ethylene glycol, sorbitol, and butanediol.

[0029] According to the specific embodiment of the present application, the volume fraction of the polyol is ≥20% based on the total volume of the cutting protective agent.

[0030] According to the specific embodiment of the present application, the soaking time is ≥3h.

[0031] According to the specific embodiment of the present application, the raw material of the coral stone comprises natural coral and / or artificially cultured coral.

[0032] According to the specific embodiment of the present application, preferably, the natural coral comprises Porites and / or Montastrea; more preferably, Porites.

[0033] According to the specific embodiment of the present application, the step of hydrothermal exchange comprises: soaking in a saturated solution of diammonium hydrogen phosphate, and reacting at 0.1-3Mpa and 150-220℃ for 6-19h.

[0034] The method for preparing the coral hydroxyapatite particles in the present application further comprises:

[0035] The coral is bleached and cleaned, and then soaked in a cutting protective agent, crushed and granulated, and subjected to hydrothermal exchange to prepare the coral hydroxyapatite particles.

[0036] The coral hydroxyapatite particles prepared by the method in the present application can form a nano-flower-like hydroxyapatite structure on the surface, obtaining "nano-flower" coral hydroxyapatite particles, the surface of which presents a "nano-flower" structure under a microscope, as shown in Figure 5 The soaking in a cutting protective agent can make the coral retain the complete porous structure when cut and polished into smaller particle sizes, and be closer to human cancellous bone. The "nano-flower" coral hydroxyapatite can also be controlled in conversion rate by controlling the temperature, time, and amount of reagent, so as to prepare coral hydroxyapatite with different nano-flower proportions, and form a biomimetic bone structure with different pore diameters with recombinant human collagen, so that the degradation rate of the implant in the body is controllable. The microstructures of the "nano-flower" coral hydroxyapatite with different conversion rates are shown in Figure 5 , wherein a represents unconverted, b represents slightly converted, and c represents completely converted.

[0037] The "nano-flower" coral hydroxyapatite prepared in the present application is better protected in pore size and porosity after a series of steps such as cutting protective agent, greatly improving the yield.

[0038] According to a specific embodiment of the present application, preferably, the composition has a bone tissue structure, and has excellent bone conduction and bone induction properties.

[0039] According to a specific embodiment of the present application, the form of the composition includes a block, a sheet, a granule, or a powder.

[0040] According to a specific embodiment of the present application, when the form of the composition includes a block, a sheet, or a granule, the porosity is 85-99%; preferably, 90-99%.

[0041] According to a specific embodiment of the present application, when the form of the composition is a block, the size thereof can range from (1-10) mm x (1-10) mm x (1-10) mm, (10-100) mm x (10-100) mm x (10-100) mm, diameter (2-100) mm x height (5-100) mm, small head diameter (2-100) mm x large head diameter (2-100) mm x height (1-100) mm; for example, it can be the following sizes: 4 mm x 4 mm x 4 mm, 6 mm x 6 mm x 6 mm, 8 mm x 8 mm x 8 mm, 10 mm x 10 mm x 10 mm, 15 mm x 15 mm x 15 mm, 20 mm x 20 mm x 20 mm, 30 mm x 30 mm x 30 mm, 65 mm x 65 mm x 65 mm, φ 5 mm x height 8 mm, φ 5 mm x height 12 mm, φ 5 mm x height 15 mm, φ 8 mm x height 8 mm, φ 8 mm x height 12 mm, φ 8 mm x height 15 mm, φ 10 mm x height 8 mm, φ 10 mm x height 12 mm, φ 10 mm x height 15 mm, φ 5-8 mm x height 10 mm, φ 6-10 mm x height 10 mm, φ 7-12 mm x height 10 mm, φ 8-15 mm x height 10 mm, φ 5-8 mm x height 15 mm, φ 6-10 mm x height 15 mm, φ 7-12 mm x height 15 mm, φ 8-15 mm x height 15 mm.

[0042] According to a specific embodiment of the present application, when the composition is in the form of a sheet, the size thereof can range from (0.1-5) mm x (10-200) mm x (10-200) mm, (0.1-5) mm x (50-200) mm x (50-200) mm, diameter (5-200) mm x height (1-5) mm; for example, it can have the following dimensions: 0.5 mm x 10 mm x 10 mm, 0.5 mm x 15 mm x 15 mm, 0.5 mm x 20 mm x 20 mm, 2 mm x 50 mm x 50 mm, 5 mm x 100 mm x 100 mm, φ 8 mm x height 2 mm, φ 8 mm x height 3 mm, φ 10 mm x height 2 mm, φ 10 mm x height 3 mm, φ 20 mm x height 5 mm, φ 25 mm x height 5 mm, φ 50 mm x height 5 mm, φ 100 mm x height 5 mm.

[0043] According to a specific embodiment of the present application, when the composition is in the form of a granule, the particle size distribution thereof can range from 0.18-2 mm; for example, it can have the following distribution range: 0.18 mm-0.25 mm, 0.25-0.3 mm, 0.3-0.5 mm, 0.5-1 mm, 1-2 mm.

[0044] According to a specific embodiment of the present application, the composition can be prepared in various forms in a mold before being implanted into a patient.

[0045] The composition of the present application can be further subjected to steps such as cutting, trimming, crushing, and sieving, to obtain the bone repair material; and / or can be further added with an aqueous liquid to obtain the bone repair material; the aqueous liquid includes blood, water for injection, phosphate buffer, purified water, and the like.

[0046] In some embodiments, the composition is in the form of a powder, and the surface of the coral hydroxyapatite granule is wrapped with collagen and / or excipient; when mixed with a liquid, it can generate viscosity, and can be directly used in the form of a powder to fill a bone defect site; or, before filling, it can be added with an aqueous liquid to become a paste that can be shaped, to obtain the bone repair material, wherein the paste is injectable and used to fill a bone defect site.

[0047] According to a specific embodiment of the present application, the bone repair material is a filling material for bone tissue and / or a scaffold material for bone tissue.

[0048] In another aspect, the present application further provides a preparation method of the above-mentioned composition, which comprises:

[0049] The "nanoflower" coral hydroxyapatite granule is mixed with a solution of recombinant human collagen, and freeze-dried to obtain a freeze-dried sample.

[0050] The composition is obtained by self-assembly and sterilization of the freeze-dried sample.

[0051] In the above preparation method, the mass fraction of the recombinant human collagen in the recombinant human collagen solution is 15%-50%, preferably 20-50%, and more preferably 30%.

[0052] In the above preparation method, preferably, the solvent of the recombinant human collagen solution comprises one or a combination of more than two of a phosphate buffer, purified water or water for injection.

[0053] In the above preparation method, preferably, the recombinant human collagen solution further comprises an excipient. In the present application, the recombinant human collagen can form a collagen scaffold together with the excipient. In the above preparation method, the step of freeze-drying comprises:

[0054] (1) Quick freezing: the process parameters are that the temperature reaches -80℃ to -60℃ within 30-240 min;

[0055] (2) Pre-freezing: the process parameters are that the temperature reaches -50℃ to -30℃ within 60-240 min, and the temperature is maintained for 120-600 min;

[0056] (3) Sublimation: the pre-frozen product is subjected to sublimation, the vacuum degree is set to 0.01-0.1 mbar, the temperature reaches -50℃ to -5℃ within 10-60 min, and the temperature is maintained for 120-600 min;

[0057] (4) Desorption drying: the sublimated product is subjected to desorption drying, the vacuum degree is set to 0.01-0.1 mbar, the temperature reaches 0-40℃ within 10-60 min, and the temperature is maintained for 120-600 min.

[0058] In the preparation method of the present application, the specific process parameters of freeze-drying are shown in Table 1.

[0059] Table 1

[0060]

[0061] In the above preparation method, the pore size structure of the material can be controlled by controlling the freezing rate. When slow freezing, the pore size is relatively large, and when quick freezing, the pore size is relatively small.

[0062] The freeze-dried sample of the composition of the present application can be self-assembled under conditions of low oxygen content or no oxygen, including three cases: the first case is low-pressure self-assembly; the second case is nitrogen-filled vacuum self-assembly; and the third case is oxygen-free self-assembly.

[0063] In the above preparation method, the step of self-assembly comprises:

[0064] The freeze-dried sample is heated to 100-300℃ and kept for 0.5-8h in a vacuum degree of -0.1 to -0.01 MPa or a nitrogen environment; preferably, the heating temperature is 100-220℃, and the vacuum degree of the nitrogen environment is -0.01 to -100 KPa.

[0065] In the above preparation method, preferably, the self-assembly is performed by using an electric heating vacuum drying oven or a precision vacuum nitrogen-filling integrated oven.

[0066] In the present application, the recombinant human collagen is proportionally prepared into a recombinant human collagen solution, and the collagen scaffold is formed after the freeze-drying and self-assembly of the recombinant human collagen solution. The porosity of the collagen scaffold is detected by using an ethanol infiltration method or a mercury intrusion method. In the present application, the porosity of the collagen scaffold formed after the freeze-drying and self-assembly of the recombinant human collagen solution is ≥85%.

[0067] In the above preparation method, preferably, the sterilization method is radiation sterilization or ethylene oxide sterilization.

[0068] In the above preparation method, preferably, the radiation sterilization method is 60Co radiation sterilization and / or electron beam radiation sterilization, and the sterilization dose is 10-30 kGy.

[0069] In the above preparation method, preferably, the sterilization parameters of the ethylene oxide sterilization are: a sterilization temperature of 40-60℃, a sterilization humidity of 40-60%, an ethylene oxide concentration of 400-700 g / m 3 , a vacuum degree of -20-10 KPa, and a sterilization time of 6-12h.

[0070] In the above preparation method, preferably, the sterilization method is electron beam radiation sterilization, and the sterilization dose is 10-25 kGy.

[0071] According to a specific embodiment of the present application, the above preparation method comprises the following steps: (1) preparing "nanoflower" coral hydroxyapatite by water heat exchange; (2) preparing a recombinant human collagen solution; (3) dispersing the "nanoflower" coral hydroxyapatite in the recombinant human collagen solution to obtain a suspension; (4) uniformly mixing the recombinant human collagen solution and the "nanoflower" coral hydroxyapatite suspension, then rapidly freezing and vacuum freeze-drying to obtain a freeze-dried sample; (5) self-assembling the freeze-dried sample under a condition of low oxygen content or no oxygen; (6) crushing and sieving the self-assembled blocky, flaky sample to prepare a granular sample; and (7) finally sterilizing the blocky, flaky or granular sample to obtain a composition with a three-dimensional network structure similar to natural bone tissue.

[0072] The "nano-flower" coral hydroxyapatite in the present application is prepared from natural coral through a series of processes such as hydrothermal exchange, and forms a unique "nano-flower" structure on the surface of the coral stone. The preparation of the composition of the present application is mixing self-assembled recombinant human collagen and "nano-flower" coral hydroxyapatite at a certain mass ratio, and preparing a material with different pore sizes of the simulated cancellous bone structure through freeze-drying and the biological self-assembly ability of the material. The material has high porosity and longitudinal gradient structure to simulate the characteristics of natural bone tissue.

[0073] The composition prepared in the present application has excellent biocompatibility, biodegradability, hydrophilicity, osteogenic performance and shape memory function. The material is soft and elastic when it meets water, and has certain flexibility, and can be cut according to the shape of the defect area, suitable for filling bone defects at any site. At the same time, the composition of the present application uses recombinant human collagen protein, which eliminates the virus hidden danger that cannot be avoided in traditional animal collagen protein scaffold materials, and does not add any auxiliary agent in the production process, so the safety is greatly improved.

[0074] Specifically, the composition provided by the present application has the following advantages:

[0075] 1. Good biocompatibility: "Nano-flower" coral hydroxyapatite, i.e. coral hydroxyapatite (HAP), has good biocompatibility. Its composition and structure are similar to natural bone, and it will not produce systemic or local toxicity reaction after being implanted into the body, and also has no immune rejection reaction. Self-assembled recombinant human collagen and coral stone have good biocompatibility, and the preparation process is mainly self-assembly under the condition of low oxygen content or no oxygen, and there is no residue of any chemical reagent.

[0076] 2. Good biodegradability: The composition provided by the present application, wherein the self-assembled recombinant human collagen has good biodegradability; the "nanoflower" hydroxyapatite coral controls its conversion rate through the preparation process, so that its degradation rate matches the growth and repair speed of bone tissue, and the degradation rate of the prepared cancellous bone material is consistent with the growth rate of bone tissue, which can completely degrade and perfectly fuse with bone tissue to form autologous bone. The bone repair effect is only second to allogeneic bone, and can perfectly fuse with autologous bone in 3-6 months without foreign body sensation. The degradation rate matches the bone formation rate, and the CT image shows that the material can perfectly fuse with autologous bone and finally grow into autologous bone. In comparison, the bone material prepared from nanohydroxyapatite and bovine bone cannot completely degrade, the bone repair effect of bovine bone is not as good as allogeneic bone or the cancellous bone material of the present application, and the degradation rate of bovine bone is slower, the bone absorption time is longer, and it cannot completely fuse with autologous bone. After 3-6 months of bone implantation, there is still a sand-like feeling when replanting, which is a "semi-permanent" implant material, and long-term use may cause local osteoporosis or displacement risk. The bone repair effect of nanohydroxyapatite is better than that of bovine bone but not as good as allogeneic bone and the cancellous bone material of the present application, and the material is brittle and hard, which is difficult to degrade in the human body and may exist in the human body for a long time.

[0077] 3. Excellent mechanical properties and shape memory function: The collagen network provides good mechanical support, making the product have moderate strength and good flexibility.

[0078] 4. High efficiency of cell adhesion and proliferation: The highly ordered network structure is conducive to cell adhesion, proliferation and migration.

[0079] 5. Excellent hydrophilicity: The porosity of the composition can reach 98.75%±0.56%, and it can be filled up in 2-3 seconds when rehydrated or rebleeding, which has excellent hydrophilicity. Good blood supply can provide sufficient nutrients and oxygen for bone tissue regeneration, and help to remove metabolic waste, thereby accelerating the bone healing process. Recent studies have shown that a hydrophilic surface can accelerate bone integration, shorten the repair waiting time, and significantly improve the stability of the implant. For example, the Covail super-hydrophilic implant uses advanced surface treatment technology to provide extremely high hydrophilicity. This surface can accelerate bone tissue growth and reduce the incidence of complications, which is particularly important for complex cases with poor periodontal conditions or full-mouth restoration. In addition, the total stability of the hydrophilic implant decreases to the lowest value at the second week after implantation, and reaches the ideal bone integration at the fourth to sixth week, which indicates that the hydrophilic surface can quickly start the bone integration process.

[0080] 6. Biomimetic structure: It has a radial gradient structure similar to that of natural bone tissue, which is closer to the structure of human natural bone tissue.

[0081] 7. Excellent clinical use convenience: Traditional tooth socket filling materials such as bone powder require a cover film to prevent the loss of bone powder and promote its combination with bone tissue. However, after using the composition of the present application, the tooth socket is mechanically closed by its network structure to prevent infection and further tissue damage, without the need for a cover film, and the operation time can be greatly shortened from more than 20 minutes in traditional surgery to 2 minutes. Therefore, the bone repair material prepared using the composition of the present application not only simplifies the surgical procedure, but also shortens the treatment time and economic burden of the patient.

[0082] 8. Hemostatic and healing-promoting effects: The composition of the present application can adsorb and activate platelets due to its good adsorption, promote the formation of blood clots, and form thrombus to play a hemostatic role. At the same time, the volume of the composition after adsorbing blood expands slightly, slightly pressing the bone wall of the tooth socket to achieve close fitting, thereby accelerating healing and osteogenesis. In addition, collagen as a scaffold material provides a place for cell attachment, promotes cell proliferation and differentiation, and thus promotes tissue remodeling and healing.

[0083] 9. Excellent osteogenesis: The composition prepared by the present application is mainly composed of hydroxyapatite and collagen, which are the main inorganic and organic components in natural bone. Using the composition prepared by the present application to fill the tooth socket can prevent or reduce the degree of alveolar bone resorption, and is helpful for the creeping coverage of the gingival epithelium, which is beneficial to the recovery of the bone height of the tooth extraction wound. BRIEF DESCRIPTION OF DRAWINGS

[0084] Figure 1 Intelligent collagen hydrophilicity (structure) schematic diagram of recombinant human collagen.

[0085] Figure 2 SEM image of self-assembly structure of intelligent collagen.

[0086] Figure 3 Coral stone appearance diagram.

[0087] Figure 4 Microstructure diagram of coral stone with different pore sizes.

[0088] Figure 5 Microstructure diagram of "nanoflower" transformed coral hydroxyapatite with different conversion rates.

[0089] Figure 6 Appearance diagram of the composition (blocky, flaky) obtained in the examples and comparative examples.

[0090] Figure 7 Appearance diagram of the composition (granular) in Example 2.

[0091] Figure 8 Microstructure diagram of the composition in Example 1.

[0092] Figure 9 Figure 1 is a graph showing the rehydration profile of the composition in Example 1 and Example 5.

[0093] Figure 10 Figure 2 is a graph showing the compression set test results of the composition in Example 1.

[0094] Figure 11 Figure 3 is a graph showing the hydrophilic properties and shape memory function of the composition obtained in Example 1 and Comparative Example.

[0095] Figure 12 Figure 4 is a graph showing the cytotoxicity results of the composition in Example 1.

[0096] Figure 13 Figure 5 is a graph showing the cell migration results of the composition in Example 1.

[0097] Figure 14 Figure 6 is a graph showing the cell proliferation results of the composition in Example 1.

[0098] Figure 15 Figure 7 is a graph showing the cell adhesion results of the composition in Example 1.

[0099] Figure 16 Figure 8 is a graph showing the in vitro degradation results of the composition in Example 1.

[0100] Figure 17 Figure 9 is a graph showing the bone repair radiographic images of the composition in Example 1 for the femoral condyle defect of a rabbit.

[0101] Figure 18 Figure 10 is a graph showing the bone repair radiographic images of the composition in Example 1 for the femoral condyle defect of a rabbit.

[0102] Figure 19 Figure 11 is a graph showing the bone repair radiographic images of the composition in Example 1 for tooth extraction socket filling in a clinical trial.

[0103] Figure 20 Figure 12 is a graph showing the bone repair radiographic images of the composition in Example 1 for tooth extraction socket filling in a clinical trial. DETAILED DESCRIPTION

[0104] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application.

[0105] It should be noted that some terms are used in the description and claims herein throughout. It is to be understood that a person skilled in the art can use different names to refer to the same component. The description and claims herein do not take the difference in names as a way to distinguish components, but take the difference in functions of components as the criterion for distinction. "Include" or "comprise" mentioned throughout the description and claims is an open term, which should be interpreted as "including but not limited to". The subsequent description in the description is a preferred embodiment for implementing the present application, but the description is for the purpose of the general principles of the description, and is not intended to limit the scope of the present application. The scope of protection of the present application is defined by the appended claims.

[0106] The amino acid sequence of the recombinant human collagen used in the following examples is: GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP (SEQ ID No: 1).

[0107] The recombinant human collagen lyophilized powder used in the following examples is designed by optimizing the amino acid sequence, expressed by Pichia pastoris, and obtained by large-scale fermentation and purification. Specifically, the hydrophilic Gly-X-Y repeat sequence is the smallest repeat unit of human type I collagen, which is targeted and combined to design a new collagen nucleotide sequence, and then the expression vector pPIC9K of Pichia pastoris is expressed in Pichia pastoris host bacteria GS115 by electroporation. After screening with antibiotic G418, the expression of high copy strains is expanded by fermentation process, and high purity recombinant human collagen is obtained by ultrafiltration and ion exchange chromatography purification.

[0108] The "nanoflower" coral hydroxyapatite used in the following examples was prepared by soaking coral stone in a 30% propylene glycol solution for 16 hours, crushing and granulating it to obtain a coral sample, and then subjecting the coral sample to hydrothermal exchange. The hydrothermal exchange step included: impregnating the coral sample with a saturated diammonium hydrogen phosphate solution and reacting it at 0.1-3 MPa and 150-220°C for 6-19 hours.

[0109] Example 1

[0110] This embodiment provides a composition containing self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0111] Weigh 10g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 20g of phosphate buffer (pH=6.6), mix well, and prepare a 30g recombinant human collagen solution; weigh 70g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 15%, add it to the above recombinant human collagen solution, mix well, and obtain a 100g suspension; add... After the mold with a height of 15 mm was placed in a freezer at -80℃ for 60 min, it was then freeze-dried under vacuum to obtain 80 samples. The samples were then placed in a nitrogen-filled vacuum oven with a vacuum degree of -10 kPa and self-assembled at 100℃ for 6 h. After the self-assembly was completed, the samples were placed in aluminum foil bags, sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition with a porosity of 98.17%.

[0112] The freeze-drying process is as follows:

[0113] During the pre-freezing stage, the temperature reaches -50℃ within 120 minutes and is maintained for 180 minutes.

[0114] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0115] The sublimated product was subjected to analytical drying under a vacuum of 0.1 mbar for 40 min, with the temperature reaching 25 °C and maintained for 240 min.

[0116] The appearance of the composition is shown in the figure below. Figure 6 As shown in 'a'; the cross-sectional view is as follows. Figure 6 As shown in b, the "nanoflower" coral hydroxyapatite is visibly and uniformly distributed within the collagen scaffold; the scanning electron microscope image is shown below.Figure 8 As shown in a, b, and c, this indicates that the freeze-dried material possesses a three-dimensional structure, and the "nanoflower"-shaped coral hydroxyapatite is encapsulated by collagen and uniformly distributed within the collagen scaffold. This composition exhibits rapid rehydration, remains intact and does not easily disperse, and expands slightly (as shown in the image). Figure 9 As shown in a), it has good toughness (such as...). Figure 10 (as shown) and hydrophilic properties and shape memory function (such as) Figure 11 (as shown in a) It also has good cell compatibility (e.g., Figure 12 , Figure 13 , Figure 14 , Figure 15 (as shown) and biodegradability (such as) Figure 16 (As shown).

[0117] (1) The composition was used to conduct a bone repair experiment on the lateral femoral condyle defect in rabbits.

[0118] Specifically:

[0119] Experimental animals: Healthy male New Zealand white rabbits, aged 6 to 8 months and weighing 2.0 to 2.5 kg, were selected.

[0120] Anesthesia and surgical preparation: All animals were fasted and deprived of water for 24 hours before surgery. Rabbits were anesthetized via intravenous injection of 2.5% sodium pentobarbital at a dose of 1 ml / kg. After successful anesthesia, the rabbit's hind legs were slightly flexed at the knee joint, the skin was stretched taut, and the hair was removed.

[0121] Establishment of a femoral lateral condyle defect model: A 6mm diameter cranial drill was used to intermittently drill vertically into the distal lateral femur of rabbits, creating a bone defect with a diameter of 6mm and a depth of 5mm. This process was performed under saline infusion.

[0122] Material implantation: using the composition of Example 1 ( The bone repair material was trimmed to the appropriate size (15mm in height) to obtain the bone repair material. The experimental groups were implanted according to the grouping in Table 2. Each pair of groups shared one rabbit, and each pair of groups had 5 rabbits at each time point, for a total of three time points (30 days, 60 days, and 90 days).

[0123] The control group received bone grafts made from a similar product (Geistlich Bio-Oss Collagen).

[0124] The blank control group did not use any materials.

[0125] Table 2

[0126]

[0127] After the defect is closed, the lower limbs are not fixed, and the experimental rabbits are allowed to move freely. After the operation, 400,000 U of penicillin is injected intramuscularly per day for 3 consecutive days, and the animals are fed separately in cages.

[0128] Experimental period:

[0129] The animals are sacrificed at 30 days, 60 days, and 90 days, respectively.

[0130] General observation:

[0131] The diet, activity, and skin and soft tissue around the incision of the animals after the operation are observed, and the healing and inflammatory symptoms of the incision are observed. The surface morphology of the specimen is observed, and whether there is deformity or not is observed.

[0132] X-ray examination:

[0133] At 30 days, 60 days, and 90 days, respectively, the distal end of the bilateral femur is subjected to X-ray examination, and the conditions for shooting are 70KV, 80mA, and 32ms. The repair of the bone defect and the degradation of the material are observed.

[0134] MicroCT examination:

[0135] At 30 days, 60 days, and 90 days, respectively, the animals are sacrificed, fixed with 10% formaldehyde for 1 week, washed, and subjected to MicroCT examination.

[0136] Through the three-dimensional reconstruction image of MicroCT, the growth of the new bone and the changes in the bone defect area are qualitatively analyzed and observed;

[0137] The results show that the material has good bone repair effect, and the bone repair rate is better than that of the same product bone filling material (Geistlich Bio-Oss Collagen) (as shown in Figure 17 、 Figure 18 ).

[0138] (2) The composition is used for clinical trial tooth extraction socket filling experiment, specifically:

[0139] Patient selection and preoperative preparation:

[0140] Suitable patients are selected for tooth extraction socket filling experiment, and surgical contraindications such as diabetes and osteoporosis are excluded. The patient needs to sign an informed consent form.

[0141] Minimally invasive tooth extraction:

[0142] Minimally invasive tooth extraction is performed under local anesthesia, and the damage to the alveolar bone and soft tissue is minimized. It is ensured that at least three bone walls of the tooth extraction socket are retained.

[0143] Selection and application of bone material:

[0144] The composition of Example 1 is used Trim the material to the appropriate size (15mm in height) to obtain bone repair material. Utilize the rapid hemolysis property of this material to directly fill the extraction socket without prior rehydration and without covering it with a collagen membrane. Then, suture the wound directly to close it.

[0145] Postoperative care:

[0146] Postoperatively, observe the healing of soft and hard tissues and perform imaging examinations (such as cone-beam CT or X-ray) 3-6 months after surgery to assess changes in bone mass.

[0147] Experimental Results and Analysis:

[0148] Experimental results show (e.g.) Figure 19 As shown in the image, this material can perfectly repair an L-shaped bone defect of approximately 11mm. No covering membrane or metal fixation is required during surgery. Bone formation occurs in 3 months, and dental implantation can be performed in 5 months. It completely fuses with the patient's own bone, resulting in no foreign bodies or bone collapse, and maintains the height and width of the alveolar ridge.

[0149] Example 2

[0150] This embodiment provides a composition containing self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0151] Weigh 20g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 25g of phosphate buffer (pH=6.6), mix well, and prepare a 45g recombinant human collagen solution; weigh 55g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 20%, add it to the above recombinant human collagen solution, mix well, and obtain a 100g suspension; add... After forming a mold with a height of 15 mm, the sample was quick-frozen at -80℃ for 100 min, followed by vacuum freeze-drying to obtain 80 block samples. These samples were then self-assembled at 150℃ for 8 h in an electric vacuum drying oven with a vacuum level of -0.095 MPa. The block samples were then pulverized using a pulverizer (JC-FW-100) and sieved through a 10-65 mesh screen. The sieved samples were then placed in vials, sealed in aluminum foil bags, and sterilized by electron beam irradiation at a dose of 25 kGy to obtain the composition with a porosity of 98.62%.

[0152] The freeze-drying process is as follows:

[0153] During the pre-freezing stage, the temperature reaches -45℃ within 100 minutes and is maintained for 200 minutes;

[0154] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0155] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar, with the temperature reaching 27°C within 50 min and maintained for 250 min.

[0156] The appearance of the composition (granular) is shown in the image below. Figure 7 As shown, ae represent samples with particle size distributions of <0.25mm, 0.25-0.3mm, 0.3-0.5mm, 0.5-1mm, and 1-2mm, respectively.

[0157] Example 3

[0158] This embodiment provides a composition containing self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0159] Weigh 15g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 45g of water for injection, suspend evenly, and prepare a 60g recombinant human collagen solution; weigh 40g of "nanoflower" coral hydroxyapatite with a particle size range of 0.5mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 22%, add it to the above recombinant human collagen solution, suspend evenly, and obtain a 100g suspension; add... After forming a mold with a height of 15mm, the sample was quick-frozen at -80℃ for 120 minutes, followed by vacuum freeze-drying to obtain 80 block samples. These samples were then self-assembled at 170℃ for 7 hours in an electrically heated vacuum drying oven with a vacuum level of -0.095MPa. The self-assembled samples were then sealed in double-layer blister packs and sterilized with ethylene oxide at the following parameters: sterilization temperature 55℃, sterilization humidity 50%, and ethylene oxide concentration 630g / m³. 3 The composition was obtained by sterilizing at a vacuum of -15 kPa for 10 hours, with a porosity of 96.58%.

[0160] The freeze-drying process is as follows:

[0161] During the pre-freezing stage, the temperature was set to -50°C within 140 minutes and maintained for 190 minutes.

[0162] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0163] The sublimated product was subjected to analytical drying under a vacuum of 0.06 mbar, with the temperature reaching 20°C within 30 minutes and maintained for 250 minutes.

[0164] The appearance of the composition is shown in the figure below. Figure 6 As shown in c in the figure.

[0165] The composition was used in a clinical trial for filling extraction sockets, specifically as follows:

[0166] Patient selection and preoperative preparation: Select suitable patients for tooth extraction socket filling tests and rule out contraindications such as diabetes and osteoporosis. Patients must sign an informed consent form.

[0167] Minimally invasive tooth extraction: Performed under local anesthesia, this minimally invasive procedure minimizes damage to the alveolar bone and soft tissues. Ensure that at least three sides of the extraction socket are preserved.

[0168] Selection and application of bone materials: using the composition of Example 3 ( Trim the material to the appropriate size (15mm in height) to obtain bone repair material. Utilizing the rapid hemolysis properties of this material, it can be directly inserted into the extraction socket without prior rehydration, and the wound can be closed directly by suturing without covering it with a collagen membrane.

[0169] Postoperative management: Postoperatively observe the healing of soft and hard tissues, and perform imaging examinations (such as cone-beam CT or X-ray) 3-6 months after surgery to assess changes in bone mass.

[0170] Experimental Results and Analysis: The experimental results show (e.g.) Figure 20 As shown in the image, this material can perfectly repair bone defects in 14 weeks. No covering membrane or metal fixation is needed during surgery, and it can maintain the height and width of the alveolar ridge. After examination, bone formation was observed in 3 months, and the patient underwent dental implant surgery in the second week.

[0171] Example 4

[0172] This embodiment provides a composition containing self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0173] Weigh 10g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 40g of water for injection, suspend evenly, and prepare 50g of recombinant human collagen solution; weigh 50g of "nanoflower" coral hydroxyapatite with a particle size range of 0.5mm-1.25mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 18%, add it to the above recombinant human collagen solution, suspend evenly, and obtain 100g of suspension; add... The mold with a height of 15 mm was placed in a -80℃ refrigerator for quick freezing for 180 min, and then vacuum freeze-drying was performed; 80 block-shaped samples were prepared; nitrogen-filling vacuum drying at 180℃ was performed in a nitrogen-filling vacuum oven with a vacuum degree of -15 KPa for 7 h; the sample after self-assembly was sealed in an aluminum foil bag and subjected to cobalt 60 irradiation sterilization, and the irradiation dose was 25 kGy, thereby obtaining the composition, and the porosity was 96.23%;

[0174] The freeze-drying process was as follows:

[0175] In the pre-freezing stage, the temperature reached -50℃ within 160 min and lasted for 220 min;

[0176] Sublimation was performed on the pre-frozen product, the vacuum degree was set to 0.05 mbar, the temperature reached -10℃ within 50 min and lasted for 450 min;

[0177] Desiccation was performed on the sublimated product, the vacuum degree was set to 0.07 mbar, the temperature reached 30℃ within 40 min and lasted for 220 min.

[0178] The appearance diagram of the composition is shown in FIG. d. Figure 6

[0179] Example 5

[0180] The composition provided in this embodiment is a composition of self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0181] 10 g of lyophilized powder of recombinant human collagen (the recombinant human collagen is a protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing) was weighed, 40 g of purified water was added, and the mixture was uniformly suspended to prepare 50 g of a recombinant human collagen solution; 50 g of "nanoflower" coral hydroxyapatite with a particle size range of 0.5 mm-1 mm, a pore size of 100 μm-800 μm, a porosity of 50%-80%, and a conversion rate of 15% was added to the above-mentioned recombinant human collagen solution and uniformly suspended to obtain 100 g of a suspension; a 1 cm×1 cm×1 cm mold was placed in a -80℃ refrigerator for quick freezing for 180 min, and then vacuum freeze-drying was performed; 80 block-shaped samples were prepared; self-assembly was performed at 180℃ in an electric vacuum drying oven with a vacuum degree of -0.095 MPa for 6 h; the sample after self-assembly was sealed in an aluminum foil bag and subjected to electron beam irradiation sterilization, and the irradiation dose was 25 kGy, thereby obtaining the composition, and the porosity was 96.06%;

[0182] The freeze-drying process was as follows:

[0183] ​Pre-freezing stage, temperature reached -50℃ within 200 min, and lasted for 200 min;

[0184] Sublimation to the product after pre-freezing, vacuum degree was set to 0.1 mbar, temperature reached -10℃ within 60 min, and lasted for 500 min;

[0185] Analytical drying to the product after sublimation, vacuum degree was set to 0.08 mbar, temperature reached 25℃ within 50 min, and lasted for 200 min.

[0186] The appearance diagram of the composition is shown as e in Figure 6 , the composition is not easy to scatter after rehydration (as shown in b in Figure 9 ), has good toughness and hydrophilic performance and shape memory function (as shown in b in Figure 11 ).

[0187] Example 6

[0188] The embodiment provides a composition of self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0189] 12 g of lyophilized powder of recombinant human collagen (the recombinant human collagen is a protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing) is weighed, 40 g of purified water is added, and the suspension is uniformly mixed to prepare 52 g of a recombinant human collagen solution; 48 g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25 mm-1 mm, a pore size of 100 μm-800 μm, a porosity of 50%-80%, and a conversion rate of 15% is added to the above-mentioned recombinant human collagen solution and uniformly mixed to obtain 100 g of a suspension; and The height of the mold is 5 mm, and the mold is placed in a-80℃ refrigerator for quick freezing for 180 min, and then vacuum freeze-drying is performed; 26 sheet-shaped samples are prepared; self-assembly is performed at 220℃ in an electric heating vacuum drying box with a vacuum degree of-0.095 MPa for 6 h; the sample after self-assembly is sealed in an aluminum foil bag and subjected to electron beam irradiation sterilization, and the irradiation dose is 25 kGy, thereby obtaining the composition, and the porosity is 97.79%;

[0190] The freeze-drying process is as follows:

[0191] Pre-freezing stage, temperature reached -50℃ within 200 min, and lasted for 200 min;

[0192] Sublimation to the product after pre-freezing, vacuum degree was set to 0.1 mbar, temperature reached -10℃ within 60 min, and lasted for 500 min;

[0193] The product after sublimation is desolvated, the vacuum degree is set to 0.08 mbar, the temperature reaches 25°C within 50 min, and the temperature is maintained for 200 min.

[0194] The appearance of the composition is shown in FIG. f of Figure 6

[0195] Example 7

[0196] The present example provides a composition with self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0197] Three portions of 15 g of lyophilized powder of recombinant human collagen (the recombinant human collagen is a protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing) are weighed, 40 g of purified water is added, and the mixture is uniformly suspended to prepare three portions of 55 g of recombinant human collagen solution; three kinds of "nanoflower" coral hydroxyapatite with conversion rates of 10%, 55%, and 80% are weighed, 45 g each, the particle size range is 0.5 mm-1 mm, the pore size is 100 μm-800 μm, and the porosity is 50%-80%, which are added to the above-mentioned recombinant human collagen solution and uniformly suspended to obtain three portions of 100 g of suspension; the suspension is added to a mold with a height of 15 mm, and then placed in a-80°C refrigerator for quick freezing for 200 min, followed by vacuum freeze drying; the dried sample is placed in an electric vacuum drying oven with a vacuum degree of-0.095 MPa, and self-assembled at 180°C for 6 h; the sample after self-assembly is sealed in an aluminum foil bag and subjected to electron beam irradiation sterilization, and the irradiation dose is 25 kGy, thereby obtaining the composition;

[0198] The freeze-drying process is as follows:

[0199] In the pre-freezing stage, the temperature reaches-50°C within 200 min, and the temperature is maintained for 200 min;

[0200] The product after pre-freezing is sublimated, the vacuum degree is set to 0.1 mbar, the temperature reaches-10°C within 60 min, and the temperature is maintained for 500 min;

[0201] The product after sublimation is desolvated, the vacuum degree is set to 0.08 mbar, the temperature reaches 25°C within 50 min, and the temperature is maintained for 200 min.

[0202] The pore size structure of the composition is shown in Table 3, and it can be seen from Table 3 that the higher the conversion rate of the "nanoflower" coral hydroxyapatite, the larger the pore size and the porosity, and the larger the porosity of the composition prepared thereby.

[0203] Table 3

[0204] ​​

[0205] Example 8

[0206] The present example provides a composition having self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0207] Weigh 5 g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence of SEQ ID No: 1 in the sequence listing), add 20 g of phosphate buffer (pH = 6.6), mix uniformly, and prepare 25 g of recombinant human collagen solution; Weigh 5 g of cross-linked porous starch into the above prepared recombinant human collagen solution and mix uniformly, then weigh 70 g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25 mm-1 mm, a pore size of 100 μm-800 μm, a porosity of 50%-80%, and a conversion rate of 15%, and mix them into the above solution uniformly to obtain 100 g of viscous solution; After adding a mold with a height of 15 mm, place it in a -80℃ refrigerator for quick freezing for 200 min, then vacuum freeze dry to prepare 80 samples; Place the samples in a nitrogen-filled vacuum oven with a vacuum degree of -10 KPa at 100℃ for self-assembly for 6 h; After the self-assembly is completed, the samples are sealed in aluminum foil bags and subjected to electron beam irradiation sterilization with an irradiation dose of 25 kGy, and the composition is obtained, and the porosity is 97.43%;

[0208] The freeze-drying process is as follows:

[0209] In the pre-freezing stage, the temperature reaches -50℃ within 120 min and lasts for 180 min;

[0210] Sublimate the product after pre-freezing, set the vacuum degree to 0.01 mbar, the temperature reaches -10℃ within 60 min, and lasts for 600 min;

[0211] Resolve the product after sublimation, set the vacuum degree to 0.1 mbar, the temperature reaches 25℃ within 40 min, and lasts for 240 min.

[0212] The appearance of the composition is shown in Figure 6 .

[0213] Example 9

[0214] The present example provides a composition having self-assembled collagen and "nanoflower" coral hydroxyapatite, which is prepared by the following steps:

[0215] Weigh 5 g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing), add 20 g of phosphate buffer (pH = 6.6), mix uniformly, and prepare 25 g of recombinant human collagen solution; weigh 5 g of sodium carboxymethyl cellulose and add it to the above prepared recombinant human collagen solution and mix uniformly, then weigh 70 g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25 mm-1 mm, a pore size of 100 μm-800 μm, a porosity of 50%-80%, and a conversion rate of 10%, and add it to the mixed solution and mix uniformly to obtain 100 g of a viscous solution; add a mold with a height of 15 mm into a -80℃ refrigerator for quick freezing for 200 min, and then vacuum freeze-drying to prepare 80 samples; place the samples into a nitrogen-filled vacuum oven with a vacuum degree of -10 KPa at 100℃ for self-assembly for 6 h; seal the samples after self-assembly in an aluminum foil bag and perform electron beam irradiation sterilization with an irradiation dose of 25 kGy to obtain the composition, and the porosity is 98.83%;

[0216] The freeze-drying process is as follows:

[0217] In the pre-freezing stage, the temperature reaches -50℃ within 120 min and lasts for 180 min;

[0218] Sublimation is performed on the pre-frozen product, the vacuum degree is set to 0.01 mbar, the temperature reaches -10℃ within 60 min and lasts for 600 min;

[0219] Analytical drying is performed on the sublimated product, the vacuum degree is set to 0.1 mbar, the temperature reaches 25℃ within 40 min and lasts for 240 min.

[0220] The appearance diagram of the composition is shown in FIG. h. Figure 6

[0221] Comparative Example 1

[0222] This comparative example provides a composition prepared by the following steps:

[0223] Weigh 12 g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing), add 40 g of purified water, mix uniformly, and prepare 52 g of recombinant human collagen solution; weigh 48 g of nanometer hydroxyapatite, which is a nanometer material without pore size structure. (Supplier: Zhejiang Aipu Rui New Material Co., Ltd., particle size: 20 nm, purity: 99%) and add it to the above recombinant human collagen solution to mix uniformly to obtain 100 g of a suspension; add ​The mold with a height of 15 mm was put into a -80℃ refrigerator for quick freezing for 180 min, and then vacuum freeze-drying was performed; 80 block-shaped samples were prepared; and self-assembly was performed in an electric heating vacuum drying box with a vacuum degree of -0.095 MPa at 180℃ for 6 h, to obtain the composition.

[0224] The freeze-drying process is as follows:

[0225] In the pre-freezing stage, the temperature reached -50℃ within 200 min and was maintained for 200 min;

[0226] Sublimation was performed on the pre-frozen product, the vacuum degree was set to 0.1 mbar, the temperature reached -10℃ within 60 min and was maintained for 500 min;

[0227] Analytical drying was performed on the sublimated product, the vacuum degree was set to 0.08 mbar, the temperature reached 25℃ within 50 min and was maintained for 200 min.

[0228] The appearance of the composition is shown in i of Figure 6 , the scanning electron microscope image is shown in d of Figure 8 , which indicates that the freeze-dried nano-hydroxyapatite is wrapped by collagen to form a relatively dense three-dimensional structure. The composition is used to prepare a bone repair material for rabbit femoral condyle defect bone repair experiments (as shown in experimental group 2 of Figure 18 ), and the results show that the bone repair effect of the material is better than that of the blank control, but not as good as that of Example 1. The porous structure of the “nano-flower”ized coral hydroxyapatite of the application is closer to human cancellous bone, the three-dimensional space channels formed by the porous structure increase the interface between the material and the implanted tissue, which is beneficial to accelerate the reaction process of interface combination, and provides space for bone inducting substances in the body. In addition, the pores are connected, which is beneficial to the mutual transmission of nutrients and fibrovascular tissue, and is more conducive to guiding the growth of new bone. At the same time, the “nano-flower”ized hydroxyapatite coral has a suitable conversion rate, which can be degraded in the body along with the repair of the bone, while the nano-hydroxyapatite degrades slowly and needs a longer time to be completely absorbed and replaced by the body.

[0229] Comparative Example 2

[0230] This comparative example provides a composition prepared by the following steps:

[0231] 1 g of collagen sponge (common name: medical collagen sponge, trade name: Kebang, manufacturer: Wuxi Bedi Biotechnology Co., Ltd.) was weighed, cut into pieces, and then dispersed in 50 ml of purified water. The collagen slurry was obtained by homogenizing the mixture for 10 minutes. 8 g of "nanoflower" coral hydroxyapatite particles with a particle size range of 0.25 mm-1 mm, a pore size of 100 μm-800 μm, a porosity of 50%-80%, and a conversion rate of 15% were mixed with the collagen slurry. The mixture was stirred for 20 minutes using a magnetic stirrer to obtain a collagen coral hydroxyapatite mixed slurry. The collagen coral hydroxyapatite mixed slurry was transferred to a mold and compressed to remove water for 12 hours. After demolding, the material was freeze-dried to obtain a shaped collagen coral hydroxyapatite scaffold. The collagen coral hydroxyapatite scaffold was placed in an electric vacuum drying oven and heated to 60-180°C under a vacuum of -0.095 MPa for 2 hours to obtain a final collagen coral hydroxyapatite composite scaffold. The porosity of the material was 88.05%.

[0232] The freeze-drying process was as follows:

[0233] In the pre-freezing stage, the temperature reached -50°C within 200 min and was maintained for 200 min.

[0234] The product after pre-freezing was subjected to sublimation under a vacuum of 0.1 mbar. The temperature reached -10°C within 60 min and was maintained for 500 min.

[0235] The product after sublimation was subjected to desorption drying under a vacuum of 0.08 mbar. The temperature reached 25°C within 50 min and was maintained for 200 min.

[0236] The appearance of the collagen coral hydroxyapatite composite scaffold is shown in FIG. j, and the rehydration experiment is shown in FIG. c. The collagen coral hydroxyapatite composite scaffold was easy to disperse after rehydration, and was easy to break by hand. The strength and toughness of the sample were much worse than those of the product of the example, and the product of the example was more suitable for clinical use. Figure 6 Figure 11 Comparative Example 3

[0237] The comparative example provided a composition prepared by the following steps:

[0238] The comparative example provided a composition prepared by the following steps:

[0239] ​Weigh 10 g of recombinant human collagen lyophilized powder (raw material of other companies on the market, white or white-like sponge solid, purity 99.9%), add 20 g of phosphate buffer (pH = 6.6), mix uniformly, and prepare 30 g of recombinant human collagen solution; Weigh 70 g of "nanoflower" hydroxyapatite coral stone with a particle size range of 0.25 mm-1 mm, a pore size of 200 μm-800 μm, a porosity of 50%-70%, and a conversion rate of 15%, and add it to the above-mentioned recombinant human collagen solution to mix uniformly, to obtain 100 g of suspension; Add The mold with a height of 15 mm is placed in a-80℃ refrigerator for quick freezing for 60 min, and then vacuum freeze-drying is performed to prepare 80 samples; The samples are placed in a nitrogen-filled vacuum oven with a vacuum degree of-10 KPa and treated at 100℃ for 6 h to obtain the composition. The porosity of the material is 78.55%;

[0240] The freeze-drying process is as follows:

[0241] (1) Pre-freezing stage, the temperature reaches-50℃ within 120 min, and lasts for 180 min;

[0242] (2) Sublimation of the pre-frozen product, the vacuum degree is set to 0.01 mbar, the temperature reaches-10℃ within 60 min, and lasts for 600 min;

[0243] (3) Analytical drying of the sublimated product, the vacuum degree is set to 0.1 mbar, the temperature reaches 25℃ within 40 min, and lasts for 240 min.

[0244] The appearance diagram of the composition is shown as k in Figure 6 The appearance is slightly yellow, the rehydration experiment is shown as d in Figure 11 , which indicates that the composition is slightly dissolved after rehydration, the particles are easy to disperse, and the strength and toughness of the sample are far inferior to those of the examples. The product of the examples can better meet the requirements of clinical use.

[0245] Comparative Example 4

[0246] This comparative example provides a composition with self-assembled collagen and "nanoflower" hydroxyapatite coral stone, which is prepared by the following steps:

[0247] Weigh 6 g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence of SEQ ID No: 1 in the sequence listing), add 14 g of purified water, mix uniformly, and prepare 20 g of recombinant human collagen solution; weigh 80 g of "nanoflower" hydroxylated coral stone with a particle size range of 0.25 mm-1 mm, a pore size of 200 μm-800 μm, a porosity of 50%-70%, and a conversion rate of 15%, and add it to the above-mentioned recombinant human collagen solution to mix uniformly, obtaining 100 g of suspension; add The height 15 mm mold is placed in a-80℃ refrigerator for quick freezing for 180 min, and then vacuum freeze-drying is performed; 80 samples are prepared; self-assembly is performed at 160℃ in an electric vacuum drying oven with a vacuum degree of-0.095 MPa for 6 h, and the composition is obtained; the porosity of the material is 71.52%;

[0248] The freeze-drying process is as follows:

[0249] (1) The pre-freezing stage is that the temperature reaches-50℃ within 200 min and lasts for 200 min;

[0250] (2) The product after pre-freezing is subjected to sublimation, the vacuum degree is set to 0.1 mbar, the temperature reaches-10℃ within 60 min, and lasts for 500 min;

[0251] (3) The product after sublimation is subjected to desorption drying, the vacuum degree is set to 0.08 mbar, the temperature reaches 25℃ within 50 min, and lasts for 200 min.

[0252] The "nanoflower" hydroxylated coral stone in the composition accounts for 93% (dry weight), and the sample appearance diagram is shown as l in Figure 6 , the appearance is slightly yellow, the rehydration experiment is shown as e in Figure 11 , which indicates that the composition is relatively hard, rehydrates slowly, and the particles are easy to disperse after rehydration, the sample has no toughness, and is far inferior to the example, and the product of the example can better meet the clinical use requirements.

[0253] Comparative Example 5

[0254] This comparative example provides a composition with self-assembled collagen and "nanoflower" hydroxylated coral stone, which is prepared by the following steps:

[0255] Weigh 10 g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing), add 20 g of phosphate buffer (pH = 6.6), mix uniformly, and prepare 30 g of recombinant human collagen solution; weigh 70 g of "nanoflower" hydroxylated coral stone with a particle size range of 0.25 mm-1 mm, a pore size of 200 μm-800 μm, a porosity of 50%-70%, and a conversion rate of 15%, and add it to the above-mentioned recombinant human collagen solution to mix uniformly, to obtain 100 g of suspension; add a mold with a height of 15 mm, and then place it in a -80℃ refrigerator for quick freezing for 60 min, and then vacuum freeze-dry to prepare 80 samples; place the samples in a nitrogen-filled vacuum oven with a vacuum degree of -10 KPa at 100℃ for self-assembly for 6 h to obtain the composition, and the porosity of the material is 78.18%;

[0256] The freeze-drying process is as follows:

[0257] (1) pre-freezing stage, the temperature reaches -50℃ within 60 min, and lasts for 180 min;

[0258] (2) sublimation of the pre-frozen product, the vacuum degree is set to 0.01 mbar, the temperature reaches -10℃ within 300 min, and lasts for 600 min;

[0259] (3) desorption drying of the sublimated product, the vacuum degree is set to 0.1 mbar, the temperature reaches 25℃ within 40 min, and lasts for 240 min.

[0260] This comparative example mainly slows down the temperature rising rate in the freeze-drying process, and the appearance of the composition sample is shown as m in Figure 6 , and a too slow temperature rising rate will cause uneven internal temperature distribution of the product, resulting in sample structure collapse and porosity reduction, and the rehydration experiment is shown as f in Figure 11 , indicating that the composition rehydrates slowly and the particles are easy to disperse after rehydration, and the sample has no toughness, which is far inferior to the example, and the product of the example can better meet the clinical use requirements.

[0261] The product obtained in Example 1 is taken as a representative to perform related performance detection, and the details are as follows:

[0262] Appearance: observed under daylight lamp with naked eyes, the appearance is yellow or light yellow, and the color is uniform, as shown as a, b in Figure 6 .

[0263] Compression deformation test: according to ASTM F1566-15 "Standard Test Method: Compression Test of Medical Sponges", the material was placed on the compression fixture table of the electronic universal testing machine after being rehydrated; the test speed was set to 10 mm / min to simulate the stress speed of the material in actual application; the electronic universal testing machine was started to begin the compression test process; the test process was monitored in real time, and the deformation during compression was recorded. The results, as shown in Table 1, indicate that the cancellous bone material has good toughness and mechanical strength and will not be easily crushed or crumbled. Figure 10

[0264] Swelling test: the actual size V0 of each group of samples was measured using a vernier caliper, and then the initial weight (w0) was measured, and the data was recorded. After immersing the sample in distilled water for 30 s, the surface water was absorbed with filter paper, weighed (w), and the size V after water absorption was measured using a vernier caliper. Swelling rate (%) = ((w-wo) / wo) x 100%, volume ratio before and after swelling = V / V0. The results, as shown in Table 4, indicate that the material has good water absorption and the swelling volume is within a certain range, and will not compress the surrounding tissue during clinical use.

[0265] Table 4 Swelling test results

[0266] Swelling rate / % (n = 10) 48.22±6.33 Volume ratio before and after swelling (n = 10) 1.48±0.06

[0267] Cytotoxicity test: according to GB / T 16886.5-2017 Medical Devices-Biological Evaluation-Part 5: In Vitro Cytotoxicity Test-MTT Method, the composition obtained in Example 1 was subjected to a cytotoxicity test (two parallel experiments were performed, and sample 1 and sample 2 were both products obtained in Example 1). The specific operation was as follows: high-sugar DMEM culture solution was used as the extraction medium, the composition was completely swelled, and then extracted at a ratio of 0.1 g / ml, the extraction temperature was 37℃, and the extraction time was 72 h; L929 cells in the logarithmic growth phase were inoculated into a 96-well plate at a concentration of 1.2 x 105 / mL, 100 μL / well, and cultured for 24 h. When the cell plating rate in the 96-well plate reached 40%-60%, the drug was given, ① sample group: 100 μL of culture solution containing different extraction sample dilution concentrations (100%, 50%, 25%, 12.5%, 6.25%, 3.13%) was added to each well; ② positive control group (PC): 100 uL of culture solution containing 5% DMSO was added; ③ blank zero group (BC): no cells were added, only 100 μL of culture solution was added; ④ blank control group (SC): containing cells, only 100 μL of culture solution was added; cultured for 24 h. Discard the liquid and replace it with MTT (0.5 mg / mL) culture solution, 150 μL / well, continue to culture for 4 h. Discard the liquid, add 150 μL / well of DMSO, shake well, develop color, and then use an enzyme-labeled instrument to detect OD 490 , and calculate the relative cell viability according to formula (1): 490 , and calculate the relative cell viability according to formula (1):

[0268]

[0269] Cytotoxicity determination criteria: if the relative viability of cells is greater than 70%, it is considered that there is no cytotoxic reaction; otherwise, it is considered that there is potential cytotoxicity.

[0270] The experimental results are shown in Table 5 and Figure 12 .

[0271] Table 5 Composition-L929 cytotoxicity test results

[0272] Grouping SC 3.13％ 6.25％ 12.50％ 25.00％ 50％ 100％ PC mean 0.955 1.189 1.099 1.07 1.038 1.012 0.856 0.452 Relative survival rate % 100.00％ 124.51％ 115.12％ 112.05％ 108.73％ 106.01％ 89.70％ 47.35％ SD 0.07 0.03 0.03 0.02 0.01 0.02 0.04 0.08 P / 0.01 0.03 0.05 0.11 0.24 0.11 0

[0273] As can be seen from Table 5, the composition of the application has good biological compatibility and no cytotoxicity.

[0274] Cell migration: 4 grams of the composition were added to 20 milliliters of normal saline and placed in a pressure steam sterilizer at 121°C for 1 hour.

[0275] Coating: 2 mL of the sample leaching solution to be tested was added to a 6-well plate, incubated at 37°C in a 5% carbon dioxide incubator for 2 hours, the excess leaching solution in the wells was discarded, 2 mL of 1% BSA-PBS solution was added, incubated at 37°C in a 5% carbon dioxide incubator for 1 hour, the liquid in the wells was discarded, washed with PBS for 3 times, the liquid in the wells was discarded, sealed with sealing film and placed at 4°C for standby.

[0276] Inoculation: the cells were inoculated into the 6-well plate at a seeding density of 1.4E5 per well, and incubated in an incubator (37°C, 5% CO2) overnight (24 hours).

[0277] Scratching: according to the experimental design, grouping was carried out, and 3 replicate wells were set in each group. The incubator (37°C, 5% CO2) was continued to be cultured for 24 hours. When the plating rate of the cells in the 6-well plate reached more than 90%, scratching was carried out. Two vertical scratches were made in the 6-well plate with a 10 uL gun head, and the scratches were used as the baseline (the gun head was perpendicular to the edge of the ruler), and the distance between the scratches was kept at 2 cm. After the vertical scratches were completed, horizontal scratches were made perpendicular to the baseline near the axis of the 6-well plate. When the scratches were made with the gun head, the force was uniform as much as possible, and the width of the scratches was kept consistent as much as possible.

[0278] Two vertical scratches were made in the 6-well plate with a 10 uL gun head as the baseline (the gun head was perpendicular to the edge of the ruler), and the distance between the scratches was kept at 2 cm.

[0279] After the vertical scratches were completed, horizontal scratches were made perpendicular to the baseline near the axis of the 6-well plate. When the scratches were made with the gun head, the force was uniform as much as possible, and the width of the scratches was kept consistent as much as possible.

[0280] Washing: After the scratch, each well was gently washed with 1 mL of PBS solution, and the washing was repeated 3 times to remove the cells that were detached due to the scratch. After the washing, 2 mL of culture medium (without serum) was added to each well, and the cells were cultured at 37°C in a 5% CO2 incubator.

[0281] Photographing: The photographing was performed at 0 h after the scratch under a 4-fold microscope, and the photographing was performed under a 4-fold microscope after the scratch for 24 h after the washing with PBS once. If there was no obvious migration, the photographing was performed under a 4-fold microscope after the scratch for 48 h after the washing with PBS once. The observation was performed from left to right with the observation area being the scratch between the two intersections of the two baselines and the horizontal scratch. Nine photographs were taken at a typical area at 0 h, and nine photographs were taken continuously at 24 h and 48 h (the detached cells were removed by washing with PBS before the photographing at 24 h and 48 h). The experimental results are shown in FIG. 2, which indicates that the composition has a remarkable effect of promoting the migration of osteoblasts. Figure 13

[0282] Cell proliferation: Culturing cells: After the cells were digested and collected, the cell count was counted, and the planting density of the cells was adjusted to 10,000 cells / mL.

[0283] Adding cell suspension: After the composition was soaked in the general culture medium for 2 h, the culture medium was discarded, and 200 μL of cell suspension was slowly added from the top of the sterile sample (two parallel experiments were performed, sample 1 (2#-1) and sample 2 (2#-2) were both the product obtained in Example 1), so that the cell suspension was completely absorbed in the sample. The culture dish with the sample was placed in the cell culture box.

[0284] Culturing: After 6 hours, a small amount of culture medium was slowly added around the sample until the sample was covered, and after 16 hours, 1-2 ml of culture medium was slowly added to the culture dish. The culturing was performed for 1 day, 3 days, 5 days, 7 days, and 9 days (the culture medium was changed every other day).

[0285] Detection: After the culturing was completed, the culture medium was removed, the cells were washed with PBS for 2-3 times, the cells were digested with 0.25% trypsin for cell counting, and the results were averaged. The experimental results are shown in FIG. 3, which indicates that the composition has a remarkable effect of promoting the proliferation of osteoblasts. Figure 14

[0286] Cell adhesion: Culturing cells: 2-3 generation MC3T3-E1 cells were used, and when the cell confluence reached 80%, the cells were digested and collected, the cell count was counted, and the planting density of the cells was adjusted to 2 x 10 7

[0287] Adding cell suspension: After the composition was soaked in the general culture medium for 2 h, the culture medium was discarded, and 500 μL of cell suspension (4 x 10 6 ​​​Add the cells slowly in three portions from the center of the sterile sample until the cell suspension is completely absorbed into the sample gaps. During the operation, care should be taken to avoid the cell suspension falling into the well plate.

[0288] Incubation and culture: After 6 hours, slowly add a small amount of culture medium around the sample until it covers the sample. After 16 hours, slowly add 1-2 ml of culture medium into the culture dish and incubate for 2 hours and 4 hours respectively.

[0289] Detection: After culture, two osteoblast / composite culture specimens were randomly selected, along with two uninoculated blank specimens. The culture medium was removed, and the specimens were gently washed 2-3 times with PBS. They were then fixed with 3% glutaraldehyde for 30 minutes, followed by washing with PBS 2-3 times. The specimens were then dehydrated stepwise with ethanol at concentrations of 30%, 50%, 70%, 90%, and 100% (each concentration for 2 minutes of immersion and washing), vacuum dried, sputter-coated with gold, and the cell adhesion morphology on the material surface was observed using a scanning electron microscope at different time points. Experimental results are as follows: Figure 15 As shown, osteoblasts can adhere to the composition and form a lamellar structure, which is beneficial to the bone repair process.

[0290] In vitro degradation assay: The sample was cut into four uniform pieces. Then, the four pieces of sample were mixed with prepared, filtered, sterilized 0.01 mol PBS buffer (pH = 7.4) according to m... 本产品 V PBS Add 1 g of PBS to a sterile centrifuge tube at a ratio of 1 g / 200 ml. Gently agitate to ensure the material is fully in contact with the PBS solution. Simulate degradation in a 37°C incubator or water bath. Collect residual samples at 1, 2, 4, 9, 14, 17, 20, and 30 days, and calculate the degradation rate using the constant weight method. Results are as follows: Figure 16 As shown, this indicates that the cancellous bone material has good degradation properties.

[0291] In summary, the above are merely preferred embodiments of the present invention and are not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Use of a composition in the manufacture of a bone repair material, wherein, The material includes, based on the total dry matter of the composition as 100%: 70%-90% of coral hydroxyapatite particles, 5%-30% of recombinant human collagen, and 0-7% of excipients; The bone repair material is cancellous bone-like material, and the porosity is 85%-99%. The recombinant human collagen has an amino acid sequence as shown in SEQ ID No:

1.

2. The use according to claim 1, wherein, The bone repair material includes a bone repair material used in filling and / or repairing of bone defects.

3. The use according to claim 1, wherein, The bone repair material includes a bone repair material used in oral surgery, orthopedics, neurosurgery, and plastic surgery.

4. The use according to claim 1, wherein, The bone repair material used in oral surgery includes a bone repair material used in filling of tooth extraction socket after tooth extraction or extraction of residual root, recovery of alveolar ridge, repair of alveolar bone defects caused by periodontal disease, filling and repair of dental and jaw bone defects or insufficient bone mass, and filling of non-bearing parts of maxillofacial bone defects.

5. The use according to claim 1, wherein, The bone repair material used in orthopedics includes a bone repair material used in repair of non-bearing part bone defects.

6. The use according to claim 1, wherein, The bone repair material used in orthopedics includes a bone repair material used in bone grafting in fracture with bone defect, nonunion or malunion, orthopedics, benign cystic lesion of bone, spinal fusion caused by lumbar instability or lumbar spinal stenosis, and joint fusion.

7. The use according to claim 1, wherein, The bone repair material used in neurosurgery includes a bone repair material used in filling of skull defects caused by craniotomy drilling, filling of skull defects caused by decompression with bone flap, filling of bone suture caused by craniotomy milling, and repair of skull defects caused by intraoperative bone removal.

8. The use according to claim 1, wherein, The bone repair material used in plastic surgery includes a bone repair material used in filling and / or repair of bone defects.

9. The use according to claim 1, wherein, The composition is a solid porous material formed by mutual adhesion of coral hydroxyapatite particles and recombinant human collagen.

10. The use according to claim 1, wherein, The coral hydroxyapatite particles have a particle size range of 0.1 mm-2 mm, a pore size of 50 μm-800 μm, and a porosity of 50%-90%.

11. Use according to claim 1, wherein, The conversion rate of the coral hydroxyapatite particles is 5%-80%.

12. The use according to claim 1, wherein, The conversion rate of the coral hydroxyapatite particles is 5%-30%.

13. The use according to claim 1, wherein, The excipients include one or more than two combinations of cross-linked porous starch, sodium carboxymethyl cellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methyl cellulose.

14. The use according to claim 1, wherein, The coral hydroxyapatite particles are prepared by soaking, crushing, and granulating coral stone with a cutting protection agent and water heat exchange. The cutting protection agent is a solution containing a polyol.

15. Use according to claim 14, wherein, The polyol is selected from one or more than two combinations of glycerol, ethylene glycol, sorbitol, and butanediol.

16. The use according to claim 14, wherein, The mass fraction of the polyol is ≥20% based on the total mass of the cutting protection agent.

17. The use according to claim 14, wherein, The soaking time is ≥3 h.

18. The use according to claim 14, wherein, The raw material of the coral stone includes natural coral and / or artificially cultured coral.

19. The use according to claim 18, wherein, The natural coral includes Porites and / or Montastrea.

20. The use according to claim 18, wherein, The natural coral is Porites.

21. The use of claim 14, wherein, The water heat exchange includes soaking in a saturated diammonium hydrogen phosphate solution, reacting at 0.1-3 MPa and 150-220 °C for 6-19 h.

22. The use of claim 1, wherein, The composition is in the form of particles, blocks, tablets, or powder. ​

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