Use of compositions in the manufacture of bone repair materials

Abstract

This invention provides the use of a composition in the manufacture of bone repair materials. [Solution] The present invention provides a method for using a composition in the manufacture of a bone repair material, wherein the total dry amount of the composition is 100%, and the composition comprises 70% to 90% coral hydroxyapatite particles, 5% to 30% recombinant human collagen, and 0% to 7% excipients, and the bone repair material is a simulated cancellous bone material with a porosity of 85% to 99%. [Effects] The composition provided by the present invention has a high porosity and radial gradient structure, can mimic the properties of natural bone tissue, and possesses excellent biocompatibility, biodegradability, hydrophilicity, bone formation performance, and shape memory function. It has excellent convenience for clinical use, hemostasis and healing promotion effects, and excellent bone formation effects, and has great potential in the manufacture of bone repair materials for filling and / or repairing bone defects.

Description

Technical Field

[0001] The present invention belongs to the field of medical materials, and specifically relates to use of a composition in the manufacture of a bone repair material. Background Art

[0002] In current research on artificial bone biomaterials, polymer materials such as polymethyl methacrylate (PMMA) are mainly used, namely bone cement, and high-molecular-weight polyethylene is used in artificial joints. Such materials have poor biocompatibility, and are separated from bone tissues by fibrous tissues. Inorganic materials are the most widely applied materials, are mainly ceramic materials, and are classified into bioinert, bioactive and degradable materials. Bioinert materials are, for example, alumina ceramics; bioactive materials include glass ceramics, bioactive glass, hydroxyapatite and the like; and degradable ceramics are mainly β-tricalcium phosphate (β-TCP). In addition, there are degradable materials not belonging to ceramics, which are natural corals. The main advantages of bioactive materials are good biocompatibility, capability of chemically bonding to bone tissues or being degraded in vivo, and high strength. The main drawbacks of ceramic materials are high brittleness, the elastic modulus thereof is difficult to match that of normal bone, and the clinical application thereof is limited to a certain extent.

[0003] Therefore, developing an ideal bone graft substitute has always been one of the important issues in the field of orthopedic surgery. An ideal bone graft substitute should have the following characteristics: (1) osteoconductivity, (2) osteoinductivity, (3) excellent hydrophilicity, (4) good biocompatibility, (5) degradability with an in vivo degradation rate matching the regeneration rate of new bone, (6) appropriate porosity and pore connectivity structure, (7) good mechanical performance, (8) ease of handling during surgery, (9) ease of disinfection before use, and (10) capability of being manufactured into a predetermined size and being easily filled.

[0004] Hydroxyapatite (HAP) and collagen (Col protein) are the most important inorganic and organic components in natural bone, and both possess excellent biological properties. However, when used individually, they have different drawbacks, making it difficult to meet the performance requirements of biomaterials as determined by tissue engineering. By compounding collagen with hydroxyapatite, the binding properties of collagen can be suitably utilized to overcome some of the limitations of hydroxyapatite. [Overview of the project]

[0005] To solve the above technical problems, the present invention aims to provide a composition for use in the manufacture of bone repair materials.

[0006] To achieve the above objective, the present invention relates to the use of a composition in the manufacture of a bone repair material,

[0007] Taking the total amount of dry material in the composition as 100%, the material of the composition is:

[0008] Coral hydroxyapatite particles 70%~90%

[0009] Recombinant human collagen 5%~30%, and

[0010] Use of compositions containing 0-7% excipients in the manufacture of bone repair materials.

[0011] The bone repair material is a simulated cancellous bone material with a porosity of 85% to 99%.

[0012] According to a specific embodiment of the present invention, preferably, the porosity of the simulated cancellous bone material is 95% to 99%.

[0013] According to specific embodiments of the present invention, preferably, the bone repair material includes a bone repair material used for filling and / or repairing bone defects.

[0014] According to specific embodiments of the present invention, preferably, the bone repair material includes bone repair materials used in oral surgery, orthopedic surgery, neurosurgery, and orthopedic surgery.

[0015] According to specific embodiments of the present invention, preferably, bone repair materials used in oral surgery include bone repair materials for filling tooth sockets after tooth extraction or root removal, alveolar ridge restoration, repair of alveolar bone defects due to periodontal disease, filling and repair of tooth and jaw bone defects or insufficient bone volume, and filling of bone defects in maxillofacial non-loading areas.

[0016] According to specific embodiments of the present invention, the bone repair material used in orthopedics preferably includes the repair of bone defects in unloaded areas, and more preferably includes bone repair materials for fracture-associated bone defects, bone failure or malunion and orthopedics, benign cystic bone lesions, spinal fusion surgery for lumbar instability or lumbar spinal stenosis, and bone grafting in arthrodesis.

[0017] According to specific embodiments of the present invention, preferably, bone repair materials used in neurosurgery include filling skull defects by craniotomy and perforation, filling skull defects by decompressive craniotomy, filling bone gaps by craniotomy and milling, repairing skull defects by intraoperative removal of skull with forceps, and bone repair materials in surgeries that are not contraindicated for other bone grafting in neurosurgery.

[0018] According to specific embodiments of the present invention, preferably, bone repair materials used in orthopedic surgery include bone repair materials used for filling and / or repairing bone defects.

[0019] According to a specific embodiment of the present invention, the composition is a solid porous material formed by coral hydroxyapatite particles adhering to each other with recombinant human collagen.

[0020] The recombinant human collagen described in this invention is CN108070032B This is recombinant human collagen as disclosed in (Method for Purifying Recombinant Human Collagen).

[0021] According to a specific embodiment of the present invention, said recombinant human collagen has the amino acid sequence set forth in SEQ ID No: 1.

[0022] According to a specific embodiment of the present invention, preferably, based on 100% of the total dry weight of said composition, the materials of said composition comprise 70% to 90% of coral hydroxyapatite particles, 5% to 23% of recombinant human collagen, and 5% to 7% of an excipient.

[0023] According to a specific embodiment of the present invention, said coral hydroxyapatite particles have a particle diameter ranging from 0.1 mm to 2 mm, a pore diameter ranging from 50 μm to 800 μm, and a porosity ranging from 50% to 90%.

[0024] According to a specific embodiment of the present invention, preferably, the conversion rate of said coral hydroxyapatite particles is 5% to 80%, and more preferably, the conversion rate is 5% to 30%.

[0025] According to a specific embodiment of the present invention, preferably, said excipient comprises one or a combination of two or more selected from the group consisting of crosslinked porous starch, sodium carboxymethyl cellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methyl cellulose.

[0026] The recombinant human collagen described in the present invention has regular hydrophilic groups on the exterior, strong aggregation ability, and hydrophobic groups on the interior, and forms a micro-stent. The recombinant human collagen described in the present invention has self-assembly ability, can perform self-assembly in a hypoxic or vacuum environment, and increasing the temperature can promote the self-assembly ability, and no reagent residue remains due to chemical crosslinking in this process. The hydrophilic structure of the intelligent recombinant human collagen of the present invention is shown in Figure 1, and the SEM image of the self-assembled structure of said recombinant human collagen is shown in Figure 2.

[0027] According to a specific embodiment of the present invention, the coral hydroxyapatite particles are produced by immersion of coral stone in a cutting protection agent, crushing granulation, and hydrothermal exchange. As shown in Figure 3, the appearance view of the coral stone according to the present invention has a three-dimensional network structure with interconnected pores. The microstructures of coral stones with different pore sizes are shown in Figure 4, wherein a indicates dense pores, b indicates mesopores, and c indicates macropores, and the mesoporous coral stone indicated by b in Figure 4 is most similar to the natural bone structure.

[0028] According to a specific embodiment of the present invention, the cutting protection agent is a solution containing a polyhydric alcohol.

[0029] According to a specific embodiment of the present invention, preferably, the polyhydric alcohol is one or a combination of two or more selected from glycerin, ethylene glycol, sorbitol, and butanediol.

[0030] According to a specific embodiment of the present invention, the volume fraction of the polyhydric alcohol relative to the total volume of the cutting protection agent is ≧20%.

[0031] According to a specific embodiment of the present invention, the immersion time is ≧3h.

[0032] According to a specific embodiment of the present invention, the raw material of the coral stone includes natural coral and / or artificially cultured coral.

[0033] According to a specific embodiment of the present invention, preferably, the natural coral includes Porites and / or Goniopora, and more preferably Porites.

[0034] According to a specific embodiment of the present invention, the hydrothermal exchange step comprises impregnation with a saturated diammonium hydrogen phosphate solution, and carrying out a reaction for 6 to 19 hours under the conditions of 0.1 to 3 MPa and 150 to 220°C.

[0035] The method for producing coral hydroxyapatite particles described in the present invention further includes:

[0036] The process includes bleaching and washing the coral, followed by immersion in a cutting protective agent, crushing and granulation, and hydrothermal exchange to produce the coral hydroxyapatite particles.

[0037] The coral hydroxyapatite particles obtained by the manufacturing method of the present invention have a nano-flower-like hydroxyl group phosphorylation structure formed on their surface, resulting in "nano-flower" coral hydroxyapatite particles, whose surface exhibits a "nano-flower" structure under a microscope, as shown in Figure 5. When the coral is cut into small particle sizes and polished by immersion in a cutting protective agent, it can still maintain a complete porous structure, which is closer to the spongy bone of the human body. The conversion rate of the "nano-flower" coral hydroxyapatite can also be controlled by controlling the temperature, time, and amount of reagent added, thereby producing coral hydroxyapatite with different proportions of nano-flowers, forming a bionic bone structure with a different pore size than recombinant human collagen, and controlling the rate of degradation when implanted in the body. The microstructures of "nano-flower" coral hydroxyapatite with different conversion rates are shown in Figure 5, where a represents unconverted, b represents mildly converted, and c represents completely converted.

[0038] The "nano-flower" coral hydroxyapatite produced by this invention is treated in a series of processes, including the use of cutting protective agents, to better protect the pore size and void ratio, thereby significantly improving yield.

[0039] According to specific embodiments of the present invention, preferably, the composition has a bone tissue structure and exhibits excellent bone conduction and bone induction performance.

[0040] According to specific embodiments of the present invention, the form of the composition includes lumps, sheets, particles, or powders.

[0041] According to a specific embodiment of the present invention, when the composition is in the form of lumps, sheets, or parts, the porosity is 85% to 99%, preferably 90% to 99%.

[0042] According to a specific embodiment of the present invention, when the composition is in the form of a block, its dimensional range may be (1~10) mm × (1~10) mm × (1~10) mm, (10~100) mm × (10~100) mm × (10~100) mm, diameter (2~100) mm × height (5~100) mm, small end diameter (2~100) mm × large end diameter (2~100) mm × height (1~100) mm, for example 4 mm × 4 mm × 4 mm, 6 mm × 6 mm × 6 mm, 8 mm × 8 mm × 8 mm, 10 mm × 10 mm × 10 mm, 15 mm × 15 mm × 15 mm, 20 mm × 20 mm × 20 mm, 30 mm × Dimensions such as 30mm x 30mm, 65mm x 65mm x 65mm, φ5mm x height 8mm, φ5mm x height 12mm, φ5mm x height 15mm, φ8mm x height 8mm, φ8mm x height 12mm, φ8mm x height 15mm, φ10mm x height 8mm, φ10mm x height 12mm, φ10mm x height 15mm, φ5~8mm x height 10mm, φ6~10mm x height 10mm, φ7~12mm x height 10mm, φ8~15mm x height 10mm, φ5~8mm x height 15mm, φ6~10mm x height 15mm, φ7~12mm x height 15mm, and φ8~15mm x height 15mm are also acceptable.

[0043] According to a specific embodiment of the present invention, when the composition is in the form of a sheet, its dimensional range may be (0.1~5) mm × (10~200) mm × (10~200) mm, (0.1~5) mm × (50~200) mm × (50~200) mm, diameter (5~200) mm × height (1~5) mm, for example, 0.5 mm × 10 mm × 10 mm, 0.5 mm × 15 mm × 15 mm, 0.5 mm × 20 mm × 20 mm, 2 mm × 50 mm × 50 mm, 5 mm × 100 mm × 100 mm, φ8 mm × height 2 mm, φ8 mm × height 3 mm, φ10 mm × height 2 mm, φ10 mm × height 3 mm, φ20 mm × height 5 mm, φ25 mm × height 5 mm, φ50 mm × height 5 mm, φ100 mm × height 5 mm.

[0044] According to a specific embodiment of the present invention, when the composition is in particulate form, the particle size distribution range may be 0.18 to 2 mm, or for example, a distribution range such as 0.18 mm to 0.25 mm, 0.25 to 0.3 mm, 0.3 to 0.5 mm, 0.5 to 1 mm, or 1 to 2 mm.

[0045] According to a specific embodiment of the present invention, the composition can be manufactured in multiple forms in (multiple) molds before being implanted into a patient.

[0046] Depending on the specific application, the composition of the present invention can be further subjected to steps such as cutting, shredding, crushing, and sieving to obtain the bone repair material, or an aqueous liquid can be added to obtain the bone repair material, the aqueous liquid including blood, water for injection, phosphate buffer, purified water, etc.

[0047] In some embodiments, the composition is in powder form, with the surface of coral hydroxyapatite particles coated with collagen and / or excipients, and when mixed with a liquid, it becomes viscous and can be used in powder form to fill bone defects, or an aqueous liquid can be added before filling to form a moldable paste, thereby obtaining the bone repair material, in which case the paste is injectable and used to fill bone defects.

[0048] According to a specific embodiment of the present invention, the bone repair material is a bone tissue filling material and / or a bone tissue stent material.

[0049] On the other hand, the present invention further provides a method for producing the above-described composition. The production method is

[0050] Nano-flower-shaped coral hydroxyapatite particles and recombinant human collagen solution are uniformly mixed and freeze-dried to obtain a freeze-dried sample.

[0051] The freeze-dried sample is self-assembled and sterilized to obtain the composition.

[0052] This includes the following.

[0053] In the manufacturing method described above, the mass fraction of recombinant human collagen in the recombinant human collagen solution is 15% to 50%, preferably 20% to 50%, and more preferably 30%.

[0054] In the manufacturing method described above, preferably, the solvent for the recombinant human collagen solution includes one or more of the following: phosphate buffer, purified water, or water for injection.

[0055] In the above-described manufacturing method, preferably, the recombinant human collagen solution further comprises an excipient. In the present invention, the recombinant human collagen can form a collagen stent together with the excipient. In the above-described manufacturing method, the freeze-drying step is

[0056] Rapid freezing: Process parameters include reaching a temperature of -80°C to -60°C within 30-240 minutes.

[0057] Pre-freezing: As a process parameter, the temperature should be raised to -50°C to -30°C within 60 to 240 minutes and maintained for 120 to 600 minutes.

[0058] Sublimation: Sublimate the pre-frozen product, set the vacuum to 0.01~0.1 mbar, and within 10~60 min, raise the temperature to -50°C~-5°C and maintain it for 120~600 min.

[0059] Vacuum drying: The sublimation product is vacuum dried by setting the vacuum level to 0.01-0.1 mbar, raising the temperature to 0-40°C within 10-60 minutes, and maintaining this temperature for 120-600 minutes.

[0060] This includes the following.

[0061] Table 1 shows the specific process parameters for freeze-drying in the manufacturing method of the present invention.

[0062] [Table 1]

[0063] In the manufacturing method described above, the pore size structure of the material can be controlled by controlling the freezing rate; the pore size becomes larger when frozen slowly and smaller when frozen rapidly.

[0064] Freeze-dried samples of the compositions of the present invention can self-assemble under low oxygen content or oxygen-free conditions, and include three conditions: a first condition of low-pressure self-assembly, a second condition of nitrogen-filled vacuum self-assembly, and a third condition of oxygen-free self-assembly.

[0065] In the manufacturing method described above, the self-assembly step is:

[0066] The freeze-dried sample is placed in a vacuum of -0.1 to -0.01 MPa or a nitrogen atmosphere, heated to 100 to 300°C, and then held for 0.5 to 8 hours, preferably with a heating temperature of 100 to 220°C and a vacuum of -0.01 to -100 kPa in the nitrogen gas environment.

[0067] In the manufacturing method described above, the self-assembly is preferably carried out using an electric vacuum drying box or a precision vacuum nitrogen-filled integrated oven.

[0068] In this invention, recombinant human collagen is added to a recombinant human collagen solution in an appropriate proportion, and a collagen stent is formed by the freeze-drying and self-assembly process of this invention. The porosity of the material is then measured by ethanol infiltration or mercury intrusion. In this invention, the porosity of the collagen stent formed after freeze-drying and self-assembly of the recombinant human collagen solution is ≥85%.

[0069] In the manufacturing method described above, the sterilization method is preferably radiation sterilization or ethylene oxide sterilization.

[0070] In the manufacturing method described above, preferably, the radiation sterilization method is cobalt-60 irradiation sterilization and / or electron beam irradiation sterilization, and the sterilization dose is 10 to 30 kGy.

[0071] In the manufacturing method described above, preferably, the sterilization parameters for ethylene oxide sterilization are: sterilization temperature 40-60°C, sterilization humidity 40-60%, and ethylene oxide concentration 400-700 g / m³. 3 The vacuum level is -20 to 10 kPa, and the sterilization time is 6 to 12 hours.

[0072] In the manufacturing method described above, preferably, the sterilization method is electron beam irradiation sterilization, and the sterilization dose is 10 to 25 kGy.

[0073] According to a specific embodiment of the present invention, the above-described manufacturing method includes the following steps.

[0074] (1) Prepare a recombinant human collagen solution. (2) Disperse the nano-flowered coral hydroxyapatite in the recombinant human collagen solution to obtain a suspension. (4) After uniformly mixing the recombinant human collagen solution and the nano-flowered coral hydroxyapatite suspension, rapidly freeze and then vacuum freeze-dry to obtain a freeze-dried sample. (5) Self-assemble the freeze-dried sample under low oxygen content or oxygen-free conditions. (6) The self-assembled lump or sheet-like sample may be crushed and sieved to produce particulate matter. (7) Finally, sterilize the lump, sheet-like, or particulate material to obtain a composition with a three-dimensional network structure similar to natural bone tissue.

[0075] In this invention, "nano-flower" coral hydroxyapatite is produced from natural coral through a series of processes including hydrothermal exchange, and a unique "nano-flower" structure is formed on the surface of the coral rock. The production of the composition of this invention involves mixing self-assembling recombinant human collagen and "nano-flower" coral hydroxyapatite in a predetermined mass ratio, and producing a material with a simulated cancellous bone structure having different pore sizes through freeze-drying and the material's biological self-assembly ability. This material has a high porosity and a radial gradient structure in order to simulate the characteristics of natural bone tissue.

[0076] The composition produced by this invention possesses excellent biocompatibility, biodegradability, hydrophilicity, bone formation performance, and shape memory function. The material softens rapidly upon contact with water, is highly elastic and somewhat flexible, can be cut arbitrarily according to the shape of the defect area, and is suitable for filling bone defects in any location. Furthermore, the composition of this invention uses recombinant human collagen to avoid the potential problem of viruses unavoidable in conventional animal collagen stent materials, and no additives are added during the production process, thus significantly improving safety of use.

[0077] Specifically, the compositions provided by the present invention have the following advantages.

[0078] Excellent Biocompatibility: Coral hydroxyapatite (HAP), which is a "nano-flower" coral hydroxyapatite, exhibits excellent biocompatibility. Its composition and structure are similar to natural bone, and it does not cause systemic or local toxic reactions after implantation in the body, nor does it cause immune rejection. Self-assembling recombinant human collagen and coralite also exhibit excellent biocompatibility, and since the manufacturing process mainly involves self-assembly under low-oxygen or oxygen-free conditions, there are no residual chemical reagents.

[0079] Excellent Biodegradability: In the compositions provided by the present invention, the self-assembling recombinant human collagen exhibits excellent biodegradability, and the conversion rate of the "nano-flower" coral hydroxyapatite is controlled by the manufacturing process so that its degradation rate matches the growth and repair rate of bone tissue. The degradation rate of the manufactured simulated cancellous bone material matches the growth rate of bone tissue, and it can decompose completely and fuse perfectly with bone tissue to form autologous bone. The bone repair effect is second only to allogeneic bone, and it can fuse perfectly with autologous bone in 3 to 6 months, without any foreign body sensation, with a degradation rate that matches the bone growth rate. From the palpable feel of the implant and CT scans, it can be seen that this material can fuse perfectly with autologous bone and ultimately grow into autologous bone. Relatively speaking, bone materials made from nanohydroxyapatite and bovine bone do not decompose completely, the bone repair effect of bovine bone is inferior to that of allogeneic bone or the simulated cancellous bone material of the present invention, and the rate of decomposition of bovine bone is slow, the bone resorption time is long, and it cannot completely fuse with the autologous bone. When re-implanting after 3-6 months of bone transplantation, it still feels grainy, and it is a "semi-permanent" transplant material, and long-term use may cause a risk of localized osteoporosis or displacement. The bone repair effect of nanohydroxyapatite is superior to that of bovine bone, but it is inferior to that of allogeneic bone and the simulated cancellous bone material of the present invention, and its material is hard, difficult to decompose in the human body, and may remain in the human body for a long period of time.

[0080] Excellent mechanical performance and shape memory function: The collagen network provides good mechanical support, giving the product appropriate strength and good flexibility.

[0081] Efficient cell adhesion and proliferation capacity: The highly ordered reticular structure is advantageous for cell adhesion, proliferation, and migration.

[0082] Excellent hydrophilicity: The porosity of this composition can reach 98.75% ± 0.56%, and it is completely absorbed in 2-3 seconds when mixed with water or blood, exhibiting excellent hydrophilicity. Good blood transport provides sufficient nutrients and oxygen for bone tissue regeneration and contributes to the removal of metabolic waste, thereby accelerating the bone healing process. Recent research shows that hydrophilic surfaces can accelerate bone integration, shorten the waiting time for restoration, and significantly improve implant stability. For example, Cowell superhydrophilic implants employ advanced surface treatment technology to provide extremely high hydrophilicity. Such surfaces can accelerate bone tissue growth and reduce the occurrence of complications, which is especially important for complex cases with poor periodontal conditions or those requiring full-mouth restoration. Furthermore, hydrophilic implants show a minimum overall stability at 2 weeks post-implantation and can reach ideal osseointegration at 4-6 weeks, indicating that hydrophilic surfaces can rapidly initiate the osseointegration process.

[0083] Bionic structure: It has a radial gradient structure similar to that of natural bone tissue, making it closer to the structure of natural bone tissue in the human body.

[0084] Superior clinical convenience: Traditional tooth socket filling materials, such as bone powder, require a cover film to prevent bone powder leakage and promote bonding with bone tissue. However, after using the composition of the present invention, its mesh structure mechanically seals the tooth socket, preventing infection and further tissue damage, eliminating the need for a cover film and significantly reducing surgical time, from more than 20 minutes in traditional surgery to just 2 minutes. Therefore, bone repair materials manufactured using the composition of the present invention not only simplify the surgical process but also reduce the patient's treatment time and financial burden.

[0085] Hemostatic and Healing-Promoting Effects: Due to its excellent adsorption properties, the composition of the present invention can adsorb and activate platelets, promote the formation of blood clots, and exert a hemostatic effect by forming thrombi. Furthermore, after adsorbing blood, the composition expands slightly in volume, gently compressing and adhering to the bone wall of the tooth extraction socket, thereby accelerating the healing and bone formation process. In addition, collagen, as a stent material, provides a site for cell adhesion and promotes cell proliferation and differentiation, thereby promoting tissue reformation and healing.

[0086] 9. Excellent bone formation effect: The main components of the composition produced in this invention are hydroxyapatite and collagen, which are the most important inorganic and organic components in natural bone. Using the composition produced in this invention to seal tooth extraction sockets can prevent or reduce alveolar bone resorption, contribute to the creeping coverage of gingival epithelium, and is advantageous for restoring bone height at the extraction site. [Brief explanation of the drawing]

[0087] [Figure 1] This is a schematic diagram of the hydrophilic (structure) intelligent collagen of recombinant human collagen. [Figure 2] This is an SEM image of the self-assembly structure of intelligent collagen. [Figure 3] This is an external view of a coral rock. [Figure 4] These are microstructure diagrams of coralite with different pore sizes. [Figure 5] This is a microstructure diagram of "nano-flower" coral hydroxyapatite with different conversion rates. [Figure 6] These are diagrams showing the external appearance of the compositions (in block form and sheet form) obtained in the examples and comparative examples. [Figure 7] This is a diagram showing the external appearance of the composition (particulate) in Example 2. [Figure 8] This is a microstructure diagram of the composition in Example 1. [Figure 9] Water mixing phase diagrams of the compositions in Example 1 and Example 5. [Figure 10] This shows the results of the compression deformation experiment of the composition in Example 1. [Figure 11] This figure shows the hydrophilic properties and shape memory functions of the compositions obtained in the examples and comparative examples. [Figure 12] This is a diagram showing the results of the cytotoxicity of the composition in Example 1. [Figure 13] This is a diagram showing the results of cell migration of the composition in Example 1. [Figure 14] This is a diagram showing the results of cell proliferation with the composition in Example 1. [Figure 15] This is a diagram showing the results of cell adhesion of the composition in Example 1. [Figure 16] This is a diagram showing the results of the extracorporeal degradation of the composition in Example 1. [Figure 17] This is an imaging diagram of bone repair at a femoral condyle defect site in a rabbit, showing the composition used in Example 1. [Figure 18] This is an imaging diagram of bone repair at a femoral condyle defect site in a rabbit, showing the composition used in Example 1. [Figure 19] This is an imaging diagram of bone repair in which the composition from Example 1 was filled into a tooth extraction socket in a clinical trial. [Figure 20] This is an imaging diagram of bone repair in which the composition from Example 3 was filled into a tooth extraction socket in a clinical trial. [Modes for carrying out the invention]

[0088] The following describes in detail the proposed technical aspects of the present invention in order to provide a clearer understanding of its constituent elements, objectives, and beneficial effects, but this does not limit the scope of the invention's applicability.

[0089] In the specification and claims, terms are used to refer to specific components. Those skilled in the art should understand that the same component may be referred to by different terms. In this specification and claims, the distinction between components is based on their functional differences, not on differences in terminology. For example, the terms "includes" or "inclusive" as used in the specification and claims are open terms and should be interpreted as "includes but not limited to." The subsequent descriptions in the specification are preferred embodiments for carrying out the invention, but these descriptions are intended to convey the general principles of the specification and do not limit the scope of the invention. The scope of protection of the invention is as defined in the appended claims.

[0090] The amino acid sequence of recombinant human collagen used in the following examples is:

[0091] GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPG KPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSN GPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP (SEQ ID No: 1).

[0092] The recombinant human collagen freeze-dried powder used in the following examples was obtained by optimizing the amino acid sequence design, highly expressing novel recombinant human collagen in Pichia yeast, and then large-scale fermentation and purification. Specifically, the hydrophilic Gly-XY repeat sequence is the smallest repeat unit of human type I collagen, and a novel collagen nucleotide sequence was designed using target sequences and combinations. Subsequently, the Pichia yeast expression vector pPIC9K was expressed in the Pichia yeast host bacterium GS115 by electrotransformation. After screening with the antibiotic G418, the expression of high-copy strains was expanded through the fermentation process, and high-purity recombinant human collagen was obtained by ultrafiltration and ion-exchange chromatography.

[0093] The "nano-flower" coral hydroxyapatite used in the following examples is produced by obtaining a coral sample from coral rock through 16 hours of immersion in propylene glycol at a concentration of 30% as a cutting protective agent, followed by crushing and granulation, and then further by hydrothermal exchange of the coral sample. The hydrothermal exchange step involves permeating the coral sample with a saturated solution of diammonium hydrogen phosphate and reacting it for 6 to 19 hours under conditions of 0.1 to 3 MPa and 150 to 220°C.

[0094] Example 1

[0095] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0096] 10 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, 20 g of phosphate buffer (pH=6.6) was added, and the mixture was homogenized to prepare 30 g of recombinant human collagen solution. 70 g of "nano-flower" coral hydroxyapatite, with a particle size range of 0.25 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 15%, was weighed and added to the above recombinant human collagen solution, and homogenized to obtain 100 g of suspension. After adding to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 60 mins, and then vacuum freeze-dried to produce 80 samples. The samples were placed in a nitrogen-filled vacuum oven set to a vacuum of -10 kPa and self-assembled at 100°C for 6 hours. After the self-assembly of the sample is completed, it is placed in an aluminum foil bag and sealed, then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 98.17%.

[0097] The freeze-drying process is as follows:

[0098] During the pre-freezing stage, the temperature should reach -50°C within 120 minutes and be maintained for 180 minutes.

[0099] Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 600 minutes;

[0100] The sublimated product is vacuum-dried, the vacuum is set to 0.1 mbar, the temperature is raised to 25°C within 40 minutes, and maintained for 240 minutes.

[0101] As shown in Figure 6a for the external view and Figure 6b for the cross-sectional view of the composition, the nano-flowered coral hydroxyapatite was observed to be uniformly distributed within the collagen stent with the naked eye. As shown in Figures 8a, b, and c for scanning electron microscope images, it was shown that after freeze-drying, it had a three-dimensional structure, and the nano-flowered coral hydroxyapatite was encapsulated by collagen and uniformly distributed within the collagen stent. The composition has a fast water mixing rate, is complete and does not scatter easily, expands slightly (as shown in Figure 9a), has good toughness (as shown in Figure 10), hydrophilicity and shape memory function (as shown in Figure 11a), as well as good cytocompatibility (as shown in Figures 12, 13, 14, and 15) and biodegradability (as shown in Figure 16). (1) An experiment was conducted to repair a lateral femoral condyle defect in rabbits using the composition.

[0102] in particular,

[0103] Test animals: Healthy male New Zealand white rabbits were selected, aged 6-8 months and weighing 2.0-2.5 kg.

[0104] Anesthesia and surgical preparation: All animals were fasted for 24 hours prior to surgery. Anesthesia was administered by intravenously injecting 2.5% pentobarbital sodium into the edge of the rabbit's ear at a rate of 1 ml / kg. After anesthesia, the rabbits' hind leg knee joints were slightly flexed, the skin was tightened, and hair was removed.

[0105] Construction of a lateral femoral condyle defect model: A bone defect measuring 6 mm in diameter and 5 mm in depth was created by intermittently drilling vertically into the lateral aspect of the distal end of the femur of rabbits using a 6 mm diameter skull drill. This process was performed by dripping in physiological saline.

[0106] Material transplantation: The composition from Example 1 (φ10mm, height 15mm) was trimmed to an appropriate size to obtain bone repair material. The experimental groups were divided according to Table 2 and transplanted, with each of the two groups sharing one rabbit, and each of the two groups having five rabbits at each time point, for a total of three time points (30 days, 60 days, and 90 days).

[0107] The control group underwent transplantation using a similar product, a bone graft material (Geistlich Bio-Oss Collagen).

[0108] No blank control group was used.

[0109] [Table 2]

[0110] After closing the defect, the lower limbs were not immobilized, and the experimental rabbits were allowed to move freely. Postoperatively, 400,000 U / d of penicillin was administered intramuscularly for three consecutive days, and the rabbits were then kept in separate cages and raised individually.

[0111] Experimental period:

[0112] The animals were sacrificed on the 30th, 60th, and 90th days, respectively.

[0113] General observations:

[0114] Postoperatively, the animals' diet, activity level, and the condition of the skin and soft tissues around the incision were observed, and the healing of the incision and symptoms of inflammation and infection were checked. The surface morphology of the specimen and the presence or absence of malformations were also observed.

[0115] X-ray examination:

[0116] Anterior and lateral X-ray examinations of the distal ends of both femurs were performed on days 30, 60, and 90, respectively, under imaging conditions of 70KV, 80mA, and 32ms, to observe the repair of bone defects and the degradation of bone material.

[0117] MicroCT scan:

[0118] The animals were sacrificed on days 30, 60, and 90, fixed in 10% formaldehyde for one week, then washed and subjected to MicroCT scans.

[0119] MicroCT was used to reconstruct images in three dimensions, and qualitative analysis was performed to observe the growth status of newly formed bone and changes in the bone defect area.

[0120] As a result, the material was shown to have a good bone repair effect, and the rate of bone repair was superior to that of similar bone graft materials (Geistlich Bio-Oss Collagen) (as shown in Figures 17 and 18). a) A clinical trial was conducted using the composition to fill tooth extraction sockets.

[0121] in particular,

[0122] Patient selection and preoperative preparation:

[0123] Appropriate patients were selected for the tooth extraction socket filling experiment, and surgical contraindications such as diabetes and osteoporosis were eliminated. Patients are required to sign informed consent.

[0124] Minimally invasive tooth extraction:

[0125] To minimize damage to the alveolar bone and soft tissues, minimally invasive tooth extraction was performed under local anesthesia. At least three sides of the bone wall were ensured to remain in the extraction socket.

[0126] Selection and application of bone materials

[0127] Using the composition from Example 1 (φ10 mm, height 15 mm), a bone repair material was obtained by trimming it to an appropriate size. Taking advantage of the rapid blood-forming properties of this material, it was directly placed into the tooth extraction socket without prior mixing with water, and the wound was directly sutured closed without covering it with a collagen membrane.

[0128] Postoperative care:

[0129] Postoperatively, the healing of soft and hard tissues was observed, and imaging tests (e.g., cone-beam CT or X-ray) were performed 3 to 6 months postoperatively to evaluate changes in bone mass.

[0130] Experimental results and analysis:

[0131] Experimental results (as shown in Figure 19) demonstrated that the material could perfectly repair an L-shaped bone defect of approximately 11 mm, without the need for membrane covering during surgery or fixation with metal fixatives. Bone formation occurred within 3 months, tooth transplantation was possible within 5 months, complete fusion with the autogenous bone was achieved, there was no foreign body, no bone depression, and the height and width of the alveolar ridge were maintained.

[0132] Example 2

[0133] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0134] 20 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, 25 g of phosphate buffer (pH=6.6) was added, and the mixture was homogenized to prepare 45 g of recombinant human collagen solution. 55 g of "nano-flower" coral hydroxyapatite, with a particle size range of 0.25 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 20%, was weighed and added to the above recombinant human collagen solution, and the mixture was homogenized to obtain 100 g of suspension. After adding to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 100 mins, and then vacuum freeze-dried to produce 80 chunk-like samples. After self-assembly at 150°C for 8 hours in an electric vacuum drying chamber set to a vacuum of -0.095 MPa, the lump sample is pulverized with a pulverizer (JC-FW-100). After pulverization, the sample is sieved through a 10-mesh to 65-mesh screen. The sieved sample is placed in a vial, then sealed in an aluminum foil bag, and finally sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 98.62%.

[0135] The freeze-drying process is as follows:

[0136] During the pre-freezing stage, the temperature should reach -45°C within 100 minutes and be maintained for 200 minutes.

[0137] Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 600 minutes;

[0138] The sublimated product is vacuum-dried, the vacuum is set to 0.08 mbar, the temperature is raised to 27°C within 50 minutes, and maintained for 250 minutes.

[0139] As shown in Figure 7, the appearance of the composition (particulate) is shown in a to e, which represent samples with particle size distribution ranges of <0.25 mm, 0.25 to 0.3 mm, 0.3 to 0.5 mm, 0.5 to 1 mm, and 1 to 2 mm, respectively.

[0140] Example 3

[0141] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0142] 15 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, 45 g of sterile water for injection was added, and the mixture was homogenized to prepare 60 g of recombinant human collagen solution. 40 g of "nano-flower" coral hydroxyapatite, with a particle size range of 0.5 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 22%, was weighed and added to the above recombinant human collagen solution, and the mixture was homogenized to obtain 100 g of suspension. After adding to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 120 mins, and then vacuum freeze-dried to produce 80 chunk samples. The samples underwent self-assembly for 7 hours at 170°C in an electrically heated vacuum drying chamber set to a vacuum of -0.095 MPa. After self-assembly, the samples were placed in a two-layer blister case, sealed, and then sterilized with ethylene oxide. The sterilization parameters were: sterilization temperature 55°C, sterilization humidity 50%, and ethylene oxide concentration 630 g / m³. 3 The vacuum level was -15 kPa and the sterilization time was 10 hours to obtain the above composition, which had a porosity of 96.58%.

[0143] The freeze-drying process is as follows:

[0144] During the pre-freezing stage, the temperature should reach -50°C within 140 minutes and be maintained for 190 minutes.

[0145] Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 600 minutes;

[0146] The sublimated product is vacuum-dried, the vacuum is set to 0.06 mbar, the temperature is raised to 20°C within 30 minutes, and then maintained for 250 minutes.

[0147] The external view of the composition is shown in Figure 6c.

[0148] A clinical trial experiment involving filling tooth extraction sockets was conducted using the composition in question.

[0149] in particular,

[0150] Patient selection and preoperative preparation:

[0151] Appropriate patients were selected for the tooth extraction socket filling experiment, and surgical contraindications such as diabetes and osteoporosis were eliminated. Patients are required to sign informed consent.

[0152] Minimally invasive tooth extraction:

[0153] To minimize damage to the alveolar bone and soft tissues, minimally invasive tooth extraction was performed under local anesthesia. At least three sides of the bone wall were ensured to remain in the extraction socket.

[0154] Selection and application of bone materials

[0155] The composition from Example 3 (φ10 mm, height 15 mm) was trimmed to an appropriate size to obtain a bone repair material. Taking advantage of the rapid blood-forming properties of this material, it was directly placed into the tooth extraction socket without prior mixing with water, and the wound was directly sutured closed without covering it with a collagen membrane.

[0156] Postoperative care:

[0157] Postoperatively, the healing of soft and hard tissues was observed, and imaging tests (e.g., cone-beam CT or X-ray) were performed 3 to 6 months postoperatively to evaluate changes in bone mass.

[0158] Experimental results and analysis:

[0159] Experimental results (as shown in Figure 20) demonstrated that the material could perfectly repair bone defects in 14 weeks, without the need for membrane covering during surgery, without the need for metal fixation, and without preserving the height and width of the alveolar ridge. Examinations showed that bone formation occurred in 3 months, and the patient underwent tooth transplant surgery in the second week.

[0160] Example 4

[0161] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0162] 10 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 40 g of sterile water for injection was added to uniformly suspend it to prepare 50 g of recombinant human collagen solution. 50 g of "nano-flower" coral hydroxyapatite, with a particle size range of 0.5 mm to 1.25 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 18%, was weighed and added to the above recombinant human collagen solution to uniformly suspend it to obtain 100 g of suspension. After adding it to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 180 mins, and then vacuum freeze-dried to produce 80 chunk-shaped samples. The self-assembly process was carried out at 180°C for 7 hours in a nitrogen-filled vacuum oven set to a vacuum of -15 kPa. After the self-assembly was completed, the sample was placed in an aluminum foil bag and sealed, and then sterilized by cobalt-60 irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 96.23%.

[0163] The freeze-drying process is as follows:

[0164] During the pre-freezing stage, the temperature should reach -50°C within 160 minutes and be maintained for 220 minutes.

[0165] Sublimate the pre-frozen product, set the vacuum to 0.05 mbar, and within 50 minutes, raise the temperature to -10°C and maintain it for 450 minutes;

[0166] The sublimated product is vacuum-dried, the vacuum is set to 0.07 mbar, the temperature is raised to 30°C within 40 minutes, and maintained for 220 minutes.

[0167] The external view of the composition is shown in Figure 6d.

[0168] Example 5

[0169] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0170] 10 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 40 g of purified water was added to uniformly suspend it to prepare 50 g of recombinant human collagen solution. 50 g of "nano-flower" coral hydroxyapatite, which has a particle size range of 0.5 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 15%, was weighed and added to the above recombinant human collagen solution to uniformly suspend it to obtain 100 g of suspension. After adding it to a 1 cm × 1 cm × 1 cm mold, it was rapidly frozen in a -80°C refrigerator for 180 mins, and then vacuum freeze-dried to produce 80 chunk-shaped samples. The sample was subjected to self-assembly at 180°C for 6 hours in an electric vacuum drying box set to a vacuum of -0.095 MPa. After the self-assembly was completed, the sample was placed in an aluminum foil bag and sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 96.06%.

[0171] The freeze-drying process is as follows:

[0172] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0173] Sublimate the pre-frozen product, set the vacuum to 0.1 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 500 minutes;

[0174] The sublimated product is vacuum-dried, the vacuum is set to 0.08 mbar, the temperature is raised to 25°C within 50 minutes, and then maintained for 200 minutes.

[0175] As shown in Figure 6e, the composition is complete and does not easily scatter after mixing with water (as shown in Figure 9b), and has good toughness, hydrophilicity, and shape memory function (as shown in Figure 11b).

[0176] Example 6

[0177] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0178] 12 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 40 g of purified water was added to uniformly suspend it, preparing 52 g of recombinant human collagen solution. 48 g of "nano-flower" coral hydroxyapatite, with a particle size range of 0.25 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 15%, was weighed and added to the above recombinant human collagen solution, uniformly suspending it to obtain 100 g of suspension. After adding it to a mold with a diameter of 30 mm and a height of 5 mm, it was rapidly frozen in a refrigerator at -80°C for 180 min, and then vacuum freeze-dried to produce 26 sheet-like samples. The self-assembly process was carried out at 220°C for 6 hours in an electric vacuum drying box set to a vacuum of -0.095 MPa. After the self-assembly was completed, the sample was placed in an aluminum foil bag and sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 97.79%.

[0179] The freeze-drying process is as follows:

[0180] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0181] Sublimate the pre-frozen product, set the vacuum to 0.1 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 500 minutes;

[0182] The sublimated product is vacuum-dried, the vacuum is set to 0.08 mbar, the temperature is raised to 25°C within 50 minutes, and then maintained for 200 minutes.

[0183] The external view of the composition is shown in Figure 6f.

[0184] Example 7

[0185] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0186] Three sets of 15g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing) were weighed, and 40g of purified water was added to uniformly suspend them, thereby preparing 55g of three sets of recombinant human collagen solutions. Three sets of 45g of "nano-flower" coral hydroxyapatite with three different conversion rates (10%, 55%, and 80%) were weighed, with particle size ranges of 0.5mm to 1mm, pore size of 100μm to 800μm, and porosity of 50% to 80%. These were each added to the above recombinant human collagen solutions and uniformly suspended, yielding 100g of three sets of suspensions. These were added to a mold with a diameter of 10 mm and a height of 15 mm, then rapidly frozen in a -80°C refrigerator for 200 minutes, followed by vacuum freeze-drying. The dried samples were placed in an electric vacuum drying box set to a vacuum of -0.095 MPa and self-assembled at 180°C for 6 hours. After the self-assembly was complete, the samples were placed in an aluminum foil bag and sealed, then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition.

[0187] The freeze-drying process is as follows:

[0188] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0189] Sublimate the pre-frozen product, set the vacuum to 0.1 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 500 minutes;

[0190] The sublimated product is vacuum-dried, the vacuum is set to 0.08 mbar, the temperature is raised to 25°C within 50 minutes, and then maintained for 200 minutes.

[0191] As shown in Table 3, the pore size structure of the composition is such that the higher the conversion rate of "nano-flower" coral hydroxyapatite, the larger the pore size and the greater the porosity, which in turn increases the porosity of the manufactured composition.

[0192] [Table 3]

[0193] Example 8

[0194] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0195] 5 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 20 g of phosphate buffer (pH=6.6) was added to uniformly suspend it, thereby preparing 25 g of recombinant human collagen solution. 5 g of cross-linked porous starch was weighed and added to the above prepared recombinant human collagen solution and mixed uniformly. Furthermore, 70 g of "nano-flower" coral hydroxyapatite, which has a particle size range of 0.25 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 15%, was weighed and added to the above mixed solution and mixed uniformly to obtain 100 g of viscous solution. After adding it to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 200 min, and then vacuum freeze-dried to produce 80 samples. The sample is placed in a nitrogen-filled vacuum oven set to a vacuum of -10 kPa and self-assembled at 100°C for 6 hours. After the self-assembly is complete, the sample is placed in an aluminum foil bag and sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 97.43%.

[0196] The freeze-drying process is as follows:

[0197] During the pre-freezing stage, the temperature should reach -50°C within 120 minutes and be maintained for 180 minutes.

[0198] Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 600 minutes;

[0199] The sublimated product is vacuum-dried, the vacuum is set to 0.1 mbar, the temperature is raised to 25°C within 40 minutes, and maintained for 240 minutes.

[0200] The external view of the composition is shown in Figure 6g.

[0201] Example 9

[0202] This embodiment provides a composition having self-assembled collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0203] 5 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 20 g of phosphate buffer (pH=6.6) was added to uniformly suspend it, thereby preparing 25 g of recombinant human collagen solution. 5 g of carboxymethylcellulose sodium was weighed and added to the prepared recombinant human collagen solution, which was then uniformly mixed. Furthermore, 70 g of "nano-flower" coral hydroxyapatite, which has a particle size range of 0.25 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 10%, was weighed and added to the above mixed solution, which was then uniformly mixed to obtain 100 g of viscous solution. After adding it to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 200 mins, and then vacuum freeze-dried to produce 80 samples. The sample is placed in a nitrogen-filled vacuum oven set to a vacuum of -10 kPa and self-assembled at 100°C for 6 hours. After the self-assembly is complete, the sample is placed in an aluminum foil bag and sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the composition, which has a porosity of 98.83%.

[0204] The freeze-drying process is as follows:

[0205] During the pre-freezing stage, the temperature should reach -50°C within 120 minutes and be maintained for 180 minutes.

[0206] Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 600 minutes;

[0207] The sublimated product is vacuum-dried, the vacuum is set to 0.1 mbar, the temperature is raised to 25°C within 40 minutes, and maintained for 240 minutes.

[0208] The external view of the composition is shown in Figure 6h.

[0209] Comparative Example 1

[0210] This comparative example provides a composition which is manufactured by the following steps.

[0211] 12 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein with amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 40 g of purified water was added to uniformly suspend it, preparing 52 g of recombinant human collagen solution. 48 g of nanohydroxyapatite, a nanoscale material without a pore structure, was weighed (manufacturer: Zhejiang Aipurui Nano New Materials Co., Ltd., particle size: 20 nm, purity: 99%). It was added to the above recombinant human collagen solution and uniformly suspended to obtain 100 g of suspension. After adding a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 180 min, and then vacuum freeze-dried to produce 80 chunky samples. Self-assembly was performed at 180°C for 6 hours in an electric vacuum drying chamber set to a vacuum of -0.095 MPa to obtain the above composition.

[0212] The freeze-drying process is as follows:

[0213] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0214] Sublimate the pre-frozen product, set the vacuum to 0.1 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 500 minutes;

[0215] The sublimated product is vacuum-dried, the vacuum is set to 0.08 mbar, the temperature is raised to 25°C within 50 minutes, and then maintained for 200 minutes.

[0216] As shown in Figure 6i (visual view) and Figure 8d (scanning electron microscope image) of the composition, it was demonstrated that after freeze-drying, the nanohydroxyapatite was encased in collagen, forming a relatively dense three-dimensional structure. When a bone repair material was manufactured using this composition and subjected to bone repair experiments on lateral femoral condyle defects in rabbits (as shown in experimental group 2 of Figure 18), it was shown that the bone repair effect of the material was superior to that of the blank control, but not as effective as that of Example 1. The porous structure of the "nano-flower" coral hydroxyapatite of the present invention is closer to that of human cancellous bone, and the three-dimensional spatial conduits formed by this porous structure increase the interface between the material and the transplanted tissue, which is advantageous for accelerating the reaction process of interfacial bonding. Furthermore, it provides space for in-vivo bone-inducing substances, and the communication of pores is advantageous for nutrient transport and the exchange of fibrous vascular tissue, which is advantageous for inducing the growth of new bone. Furthermore, while "nano-flower" hydroxyphosphoric coralite has an appropriate conversion rate and can be broken down in the body during bone repair, the breakdown of nanohydroxyapatite is slow, and it takes a longer time for it to be completely absorbed and replaced by the body.

[0217] Comparative Example 2

[0218] This comparative example provides a composition which is manufactured by the following steps.

[0219] 1 g of collagen sponge (general name: medical collagen sponge, trade name: Kejibang, manufacturer: Wuxi Beidi Biotechnology Co., Ltd.) was weighed and sheared. The collagen sponge was then dispersed in 50 ml of purified water and homogenized using a homogenizer for 10 minutes to obtain a collagen slurry. 8 g of "nano-flower" coral hydroxyapatite particles, with a particle size range of 0.25 mm to 1 mm, a pore size of 100 μm to 800 μm, a porosity of 50% to 80%, and a conversion rate of 15%, were mixed with the collagen slurry. The mixture was stirred using a magnetic stirrer for 20 minutes to obtain a collagen coral hydroxyapatite mixed slurry. The obtained collagen coral hydroxyapatite mixed slurry was transferred to a mold and compressed for 12 hours to dehydrate it. After demolding, it was freeze-dried to obtain a molded collagen coral hydroxyapatite stent. The above-mentioned collagen coral hydroxyapatite stent was placed in an electric vacuum drying box and subjected to thermal crosslinking treatment by heating at 60-180°C under a vacuum of -0.095 MPa for 2 hours to obtain the final collagen coral hydroxyapatite composite stent, the porosity of which is 88.05%.

[0220] The freeze-drying process is as follows:

[0221] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0222] Sublimate the pre-frozen product, set the vacuum to 0.1 mbar, and within 60 minutes, raise the temperature to -10°C and maintain it for 500 minutes;

[0223] The sublimated product is vacuum-dried, the vacuum is set to 0.08 mbar, the temperature is raised to 25°C within 50 minutes, and then maintained for 200 minutes.

[0224] The external appearance of the collagen coral hydroxyapatite composite stent is shown in Figure 6j, and as shown in Figure 11c, the water mixing experiment revealed that the collagen coral hydroxyapatite composite stent was prone to scattering after water mixing, easily broken when pressed by hand, and the strength and toughness of the sample were far inferior to that of the example, indicating that the product of the example is better suited to meet the requirements of clinical use.

[0225] Comparative Example 3

[0226] This comparative example provides a composition which is manufactured by the following steps.

[0227] 10 g of recombinant human collagen freeze-dried powder (a commercially available raw material from another company, white or nearly white sponge-like solid, purity 99.9%) was weighed, and 20 g of phosphate buffer (pH=6.6) was added to uniformly suspend it to prepare 30 g of recombinant human collagen solution. 70 g of "nano-flower" hydroxyphosphorylated coral stone, with a particle size range of 0.25 mm to 1 mm, a pore size of 200 μm to 800 μm, a porosity of 50% to 70%, and a conversion rate of 15%, was weighed and added to the above recombinant human collagen solution to uniformly suspend it to obtain 100 g of suspension. After adding it to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 60 mins, and then vacuum freeze-dried to produce 80 samples. The samples were placed in a nitrogen-filled vacuum oven set to a vacuum of -10 kPa and treated at 100°C for 6 hours to obtain the above composition, the porosity of the material being 78.55%.

[0228] The freeze-drying process is as follows: (1) During the pre-freezing stage, the temperature should be raised to -50°C within 120 minutes and maintained for 180 minutes; (2) Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 60 minutes raise the temperature to -10°C and maintain it for 600 minutes; (3) The sublimated product is vacuum dried, the vacuum is set to 0.1 mbar, the temperature is raised to 25°C within 40 minutes and maintained for 240 minutes.

[0229] As shown in Figure 6k, the appearance of the composition is slightly yellowish, and as shown in Figure 11d, the water mixing experiment showed that the composition dissolved only slightly after mixing with water, the particles scattered easily, and the strength and toughness of the sample were far inferior to those of the examples, indicating that the products of the examples are better suited to clinical use.

[0230] Comparative Example 4

[0231] This comparative example provides a composition having self-assembled collagen and "nano-flower" hydroxyphosphorylated coralite, which is manufactured by the following steps.

[0232] 6 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein having the amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 14 g of purified water was added to uniformly suspend it to prepare 20 g of recombinant human collagen solution. 80 g of "nano-flower" hydroxyphosphorylated coral stone, with a particle size range of 0.25 mm to 1 mm, a pore size of 200 μm to 800 μm, a porosity of 50% to 70%, and a conversion rate of 15%, was weighed and added to the above recombinant human collagen solution to uniformly suspend it to obtain 100 g of suspension. After adding it to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 180 mins, and then vacuum freeze-dried to produce 80 samples. Self-assembly was performed at 160°C for 6 hours in an electric vacuum drying box set to a vacuum of -0.095 MPa to obtain the above composition, the porosity of the material being 71.52%.

[0233] The freeze-drying process is as follows: (4) During the pre-freezing stage, the temperature should be raised to -50°C within 200 minutes and maintained for 200 minutes; (5) Sublimate the pre-frozen product, set the vacuum to 0.1 mbar, and within 60 minutes raise the temperature to -10°C and maintain it for 500 minutes; (6) The sublimated product is vacuum dried, the vacuum is set to 0.08 mbar, the temperature is raised to 25°C within 50 minutes and maintained for 200 minutes.

[0234] The proportion of "nano-flower" hydroxyphosphorylated coralite in the composition was 93% (dry weight). As shown in Figure 6l, the appearance of the sample was slightly yellowish. As shown in Figure 11e, the water mixing experiment revealed that the composition was relatively hard, mixed slowly, and the particles scattered easily after mixing. The sample lacked toughness and was far inferior to the example, indicating that the product of the example was better suited to meet the requirements for clinical use.

[0235] Comparative Example 5

[0236] This comparative example provides a composition having self-assembled collagen and "nano-flower" hydroxyphosphorylated coralite, which is manufactured by the following steps.

[0237] 10 g of recombinant human collagen freeze-dried powder (recombinant human collagen is a protein having the amino acid sequence SEQ ID No:1 in the sequence listing) was weighed, and 20 g of phosphate buffer (pH=6.6) was added to uniformly suspend it to prepare 30 g of recombinant human collagen solution. 70 g of "nano-flower" hydroxyphosphorylated coral stone, with a particle size range of 0.25 mm to 1 mm, a pore size of 200 μm to 800 μm, a porosity of 50% to 70%, and a conversion rate of 15%, was weighed and added to the above recombinant human collagen solution to uniformly suspend it to obtain 100 g of suspension. After adding it to a mold with a diameter of 10 mm and a height of 15 mm, it was rapidly frozen in a refrigerator at -80°C for 60 mins, and then vacuum freeze-dried to produce 80 samples. The samples were placed in a nitrogen-filled vacuum oven set to a vacuum of -10 kPa and self-assembled at 100°C for 6 hours to obtain the above composition, the porosity of the material being 78.18%.

[0238] The freeze-drying process is as follows: (7) During the pre-freezing stage, the temperature should be raised to -50°C within 60 minutes and maintained for 180 minutes; (8) Sublimate the pre-frozen product, set the vacuum to 0.01 mbar, and within 300 minutes raise the temperature to -10°C and maintain it for 600 minutes; (9) The sublimated product is vacuum dried, the vacuum is set to 0.1 mbar, the temperature is raised to 25°C within 40 minutes and maintained for 240 minutes.

[0239] In this comparative example, the heating rate in the freeze-drying process was mainly slowed down. As shown in Figure 6m of the appearance of the composition sample, if the heating rate is too slow, the temperature distribution inside the product becomes uneven, causing the sample structure to collapse and the porosity to decrease. As shown in Figure 11f of the water mixing experiment, the composition mixes slowly with water, the particles tend to scatter after mixing, the sample lacks toughness and is far inferior to the example. This shows that the product of the example is better able to meet the requirements of clinical use.

[0240] The product obtained in Example 1 was used as a representative example for relevant performance measurements, which are as follows:

[0241] Appearance: As shown in Figures 6a and 6b, when observed with the naked eye under fluorescent light, the appearance was yellow or pale yellow, and the color was uniform.

[0242] Compression deformation measurement: The test was conducted according to ASTM F1566-15 "Standard test method: Compression test of medical sponge." After mixing the material with water, it was placed on the compression jig of an electronic universal testing machine, and the test speed was set to 10 mm / min to simulate the load rate of the material in actual application. The electronic universal testing machine was started and the compression test process was initiated. The test process was monitored in real time, and the deformation status during the compression process was recorded. As a result, as shown in Figure 10, the cancellous bone material was shown to have good toughness and mechanical strength and not easily crushed or shed debris.

[0243] Swelling Measurement: The actual dimensions V0 of each sample were measured using calipers, and then their initial weight (w0) was weighed and the data recorded. After immersing the samples in distilled water for 30 seconds, they were removed, the moisture on the sample surface was absorbed with filter paper, the weight (w) was weighed, and the dimensions V after water absorption and expansion were measured using calipers. The swelling rate (%) = ((w-wo) / wo) × 100%, and the volume ratio before and after swelling = V / V0. As shown in Table 4, the results indicate that the material has good water absorption, its swollen volume is within the specified range, and it does not compress surrounding tissues when used clinically.

[0244] [Table 4]

[0245] Cytotoxicity Measurement: A cytotoxicity test was performed on the composition obtained in Example 1 according to GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests MTT method (two parallel experiments were performed, with both Sample 1 and Sample 2 being the product obtained in Example 1). The specific procedure was as follows: A high-sugar DMEM culture medium was used as the leachate medium. First, the composition was completely swollen, and then leached at a ratio of 0.1 g / ml. The leachate temperature was 37°C, and the leachate time was 72 h. 1.2 × 10⁶ L929 cells in the logarithmic growth phase were used. 5 Cells / mL were inoculated into 96-well plates and cultured at 100 μL / well for 24 hours. When the plate development rate of cells in the 96-well plates reached 40% to 60%, the following were administered: 1. Sample group: 100 μL of culture medium containing samples at different leaching solution dilution concentrations (100%, 50%, 25%, 12.5%, 6.25%, 3.13%) was added to each well; 2. Positive control group (PC): 100 μL of culture medium containing 5% DMSO was added; 3. Blank zero adjustment group (BC): No cells were added, only 100 μL of culture medium was added; 4. Blank control group (SC): Cells were included, only 100 μL of culture medium was added, and the cells were cultured for 24 hours. The liquid was discarded and replaced with culture medium containing MTT (0.5 mg / mL), and cultured continued at 150 μL / well for 4 hours. Discard the liquid, add DMSO at 150 μL / well, shake well, allow to develop color, then use a microplate reader to obtain OD (Optical Color Dioxide).490 The values ​​were measured, and the relative cell activity was calculated according to equation (1).

number

[0246] Cytotoxicity assessment criteria: If the relative cell activity is greater than 70%, it is considered that there is no cytotoxic reaction, or conversely, that there is potential cytotoxicity.

[0247] The experimental results are shown in Table 5 and Figure 12.

[0248] [Table 5]

[0249] As can be seen from Table 5, the compositions of the present invention have good biocompatibility and are non-cytotoxic.

[0250] Cell migration:

[0251] Infusion: 4g of the composition was mixed with 20mL of physiological saline solution and infused in a pressure steam sterilizer at 121°C for 1 hour.

[0252] Coating: Add 2 mL of sample leachate to a 6-well plate, incubate for 2 hours at 37°C in a 5% carbon dioxide incubator, discard excess leachate from the wells, add 2 mL of 1% BSA-PBS solution, incubate for 1 hour at 37°C in a 5% carbon dioxide incubator, discard the liquid from the wells, wash three times with PBS, discard the liquid from the wells, seal with a sealing film, and leave at 4°C to prepare for use.

[0253] Inoculation: Cells were inoculated into 6-well plates at an inoculation density of 1.4E5 cells / well and incubated overnight (24 hours) in an incubator (37°C, 5% CO2).

[0254] Scratching: Based on the experimental design, cells were divided into groups, and each group had three double wells. Cells were cultured in an incubator (37°C, 5% CO2) for 24 hours. Scratching was performed when the plate expansion rate of cells in the 6-well plate reached 90% or more. Using a 10 μL pipette tip, two lines were drawn longitudinally on the 6-well plate, with the longitudinal marks serving as the baseline (the pipette tip perpendicular to the edge of the ruler), and the distance between marks was maintained at 2 cm. After drawing the longitudinal marks, the pipette tip was positioned perpendicular to the baseline and scratched horizontally near the central axis of the 6-well plate. When scratching with the pipette tip, the force was applied as uniformly as possible to ensure that the width of the scratches was as uniform as possible.

[0255] Using a 10 μL pipette tip, two longitudinal marks were drawn along the longitudinal direction on a 6-well plate to serve as baselines (the pipette tip was perpendicular to the edge of the ruler), and the distance between the marks was maintained at 2 cm.

[0256] After drawing vertical marks, hold the pipette tip perpendicular to the baseline and scratch horizontally near the central axis of the 6-well plate. When scratching, apply as even pressure as possible to make the scratch width as uniform as possible.

[0257] Washing: After scratching was complete, 1 mL of PBS solution was added to each well to gently wash the cells, and this process was repeated three times to wash away any cells that had been removed by scratching. After washing, 2 mL of culture medium (serum-free) was added to each well, and the cells were cultured at 37°C in a 5% CO2 incubator.

[0258] Photography: Images were taken with a 4x magnifying mirror at 0 hours after scratching. After 24 hours, the area was washed once with PBS and photographed with a 4x magnifying mirror. If no obvious migration was observed, the area was washed once with PBS and photographed with a 4x magnifying mirror at 48 hours after scratching. The scratch area between the two baselines and the two intersections of the transverse marks was used as the observation area, and observations were made from left to right. Nine images were taken at 0 hours from a typical area, and nine consecutive images were taken at 24 hours and 48 hours (shed cells were washed off with PBS before taking images at 24 hours and 48 hours). As shown in Figure 13, the experimental results demonstrated that the composition has a significant effect in promoting osteoblast migration.

[0259] Cell proliferation:

[0260] Cell culture: Cells were digested and collected, counted on a cell counter, and then the cell inoculation density was adjusted to 10,000 cells / mL.

[0261] Addition of cell suspension: After immersing the composition in normal culture medium for 2 hours, discard the medium and gradually add 200 μL of cell suspension to the tip of the sterile sample (two parallel experiments were performed, and both Sample 1 (2#-1) and Sample 2 (2#-2) were products obtained in Example 1) until all of the cell suspension was absorbed into the sample. The culture dish containing the sample was placed in an incubator.

[0262] Culture: After 6 hours, a small amount of culture medium was gradually added around the sample until it exceeded the sample size. After 16 hours, 1-2 ml of culture medium was gradually added to the culture dish. Each sample was cultured for 1, 3, 5, 7, and 9 days (the culture medium was changed every other day).

[0263] Measurement: After culturing, the culture medium was removed, washed 2-3 times with PBS, and the cells were digested with 0.25% trypsin for cell counting. The average value was then taken. As shown in Figure 14, the experimental results demonstrated that the composition had a significant effect in promoting osteoblast proliferation.

[0264] Cell adhesion:

[0265] Cell culture: MC3T3-E1 at passage 2 to 3 is used. After the cell confluence reaches 80%, the cells are digested and collected, counted with a cell counting plate, and the cell seeding density is adjusted to 2×10 7 cells / mL.

[0266] Addition of cell suspension: After the composition is immersed in a conventional medium for 2 h, the medium is discarded. 500 μL of cell suspension (containing 4×10 6 cells) is gradually added in three portions from the middle position of the sterile sample, until all the cell suspension is absorbed into the voids inside the sample. It is necessary to avoid the cell suspension from dripping into the well plate during the operation.

[0267] Incubation and culture: After 6 hours, a small amount of medium is gradually added around the sample until the liquid level exceeds the sample. After 16 hours, 1 to 2 mL of medium is gradually added into the culture dish, followed by culture for 2 hours and 4 hours respectively.

[0268] Measurement: After the culture is completed, two osteoblast / composition composite culture samples are randomly taken, and two blank composition samples without inoculated cells are additionally taken. The medium is removed, the samples are washed by gentle shaking with PBS for 2 to 3 times, fixed with 3% glutaraldehyde for 30 minutes, washed with PBS for another 2 to 3 times, dehydrated stepwise with ethanol at concentration gradients of 30%, 50%, 70%, 90% and 100% (immersed and washed for 2 minutes at each concentration), vacuum dried and gold-plated, then the cell adhesion morphology on the material surface at different time points is observed by scanning electron microscope. As shown in Figure 15, the experimental results show that osteoblasts can adhere to the composition and form lamellar structures, which is beneficial to the process of bone repair.

[0269] In vitro degradation measurement: After the sample is evenly cut into four pieces, the four samples and the prepared 0.01 mol PBS buffer solution (pH=7.4) that has been filtered and sterilized are mixed at a ratio of m 本製品 :V PBSThe material was added to a sterile centrifuge tube at a ratio of 1g / 200ml, gently shaken to ensure thorough contact with the PBS solution, and then decomposition was simulated in a 37°C constant temperature incubator or water bath. Residual samples were taken out on days 1, 2, 4, 9, 14, 17, 20, and 30, and the decomposition rate was calculated using the constant gravimetric method. As shown in Figure 16, the cancellous bone material demonstrated good decomposition performance.

[0270] As described above, these are merely preferred embodiments of the present invention and do not limit the invention to other forms. Those skilled in the art can use the technical content disclosed above to make equivalent changes and modifications to achieve equivalent effects. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical substance of the invention without departing from the technical concept of the present invention remain within the scope of protection of the technical concept of the present invention.

Claims

1. The use of a composition in the manufacture of bone repair materials, With the total amount of dry material in the above composition being 100%, the material of the above composition is: Coral hydroxyapatite particles 70% to 90% Recombinant human collagen 5% to 30%, and Contains 0-7% excipients. The bone repair material is a simulated cancellous bone material with a porosity of 85% to 99%. The recombinant human collagen is used in the production of a bone repair material, having the amino acid sequence shown in SEQ ID No:

1.

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

3. The use according to claim 1, wherein the bone repair material includes bone repair materials used in oral surgery, orthopedic surgery, neurosurgery, and orthopedic surgery.

4. The use of bone repair materials in oral surgery includes filling extraction sockets after tooth extraction or root removal, restoring alveolar ridges, repairing alveolar bone defects due to periodontal disease, filling and repairing tooth and jawbone defects or insufficient bone volume, and filling bone defects in maxillofacial non-loading areas, as described in claim 1.

5. The use of bone repair material used in orthopedics includes the repair of bone defects in non-weight-bearing areas, as described in claim 1.

6. The use of bone repair materials used in orthopedics includes bone repair materials in bone grafting for bone defects associated with fractures, inability to heal or malunion of bones, orthopedics, benign cystic lesions of bone, spinal fusion surgery for lumbar spinal instability or lumbar spinal stenosis, and arthrodesis surgery, as described in claim 1.

7. The use of bone repair materials in neurosurgery, as described in claim 1, includes filling skull defects by craniotomy and perforation, filling skull defects by decompressive craniotomy, filling bone gaps by craniotomy and milling, and repairing skull defects by intraoperative removal of skull with forceps.

8. The use of bone repair materials used in orthopedic surgery, including bone repair materials in filling and / or repairing bone defects, as described in claim 1.

9. The use according to claim 1, wherein the composition is a solid porous material formed by coral hydroxyapatite particles adhering to each other with recombinant human collagen.

10. The coral hydroxyapatite particles have a particle size range of 0.1 mm to 2 mm, a pore size of 50 μm to 800 μm, and a porosity of 50% to 90%, as described in claim 1.

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

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

13. The use according to claim 1, wherein the excipient comprises one or more combinations of crosslinked porous starch, sodium carboxymethylcellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methylcellulose.

14. The aforementioned coral hydroxyapatite particles were produced by immersion in a cutting protective agent of coral rock, crushing and granulation, and hydrothermal exchange. The use according to claim 1, wherein the cutting protective agent is a solution containing a polyhydric alcohol.

15. The use according to claim 14, wherein the polyhydric alcohol is one or more selected from glycerin, ethylene glycol, sorbitol, and butanediol.

16. The use according to claim 14, wherein the mass fraction of the polyhydric alcohol is ≥ 20% of the total mass of the cutting protective agent.

17. The use according to claim 14, wherein the immersion time is ≥ 3 hours.

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

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

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

21. The use according to claim 14, wherein the step of hydrothermal exchange includes permeation with a saturated solution of diammonium hydrogen phosphate and carrying out the reaction for 6 to 19 hours under conditions of 0.1 to 3 MPa and 150 to 220°C.

22. The use according to claim 1, wherein the composition is in particulate, lumpy, sheet-like, or powder form.

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