Artificial bone material and method for manufacturing the same

Abstract

Hydroxyapatite (HAP) and collagen (Col protein) are the most important inorganic and organic components of natural bone, and both possess good biological properties. However, when used individually, they have different defects, making it difficult to meet the performance requirements of biomaterials through tissue engineering. [Solution] The present invention provides an artificial bone material, wherein the total amount of the dry material of the artificial bone material is 100%, and the material comprises 70% to 90% coral hydroxyapatite particles, 5% to 30% recombinant human collagen, and 0% to 7% excipients, as well as a method for producing the same. The artificial bone material provided by the present invention has a high porosity and radial gradient structure, can mimic the characteristics of natural bone tissue, and has excellent biocompatibility, biodegradability, hydrophilicity, bone formation performance, and shape memory function.

Description

Technical Field

[0001] The present invention belongs to the field of medical materials and relates to a method for manufacturing a bioactive material, specifically an artificial bone material and a method for manufacturing the same.

Background Art

[0002] Regarding the research of artificial bone biomaterials, currently, there are mainly polymer materials such as polymethyl methacrylate (PMMA), that is, bone cement, and the polymer polyethylene used in artificial joints. Such materials have poor biocompatibility and are separated from bone tissue by fibrous tissue. Inorganic materials are the most widely applied materials, mainly ceramic materials, which are classified into bioinert, bioactive, and degradable materials. Bioinert materials are, for example, alumina ceramics, bioactive materials include glass ceramics, bioactive glass, hydroxyapatite, etc., and degradable ceramics are mainly β-tricalcium phosphate (β-TCP). There are also degradable materials that do not belong to ceramics, such as natural coral. The main advantages of bioactive materials are good biocompatibility, the ability to chemically bond with bone tissue or be decomposed in the body, and high strength. The main disadvantages of ceramic materials are high brittleness, their elastic modulus is difficult to match that of normal bone, and their clinical application 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 a matching in vivo degradation rate and new bone regeneration rate, (6) appropriate porosity and pore connection structure, (7) good mechanical properties, (8) ease of handling during surgery, (9) ease of disinfection before use, and (10) can be manufactured in a predetermined size and is easy to fill.

[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 Initiative]

[0005] To solve the above technical problems, the present invention aims to provide an artificial bone material and a method for manufacturing the same.

[0006] To achieve the above objective, the present invention provides an artificial bone material, wherein the total amount of the dry material of the artificial bone material is 100%, and its material is

[0007] Coral hydroxyapatite particles 70%~90%

[0008] Recombinant human collagen 5%~30%, and

[0009] Excipient 0-7%

[0010] We provide artificial bone materials that include [the specified component].

[0011] According to a specific embodiment of the present invention, the artificial bone material is a solid porous material formed by coral hydroxyapatite particles adhering to each other with recombinant human collagen,

[0012] According to a specific embodiment of the present invention, the porosity of the artificial bone material is preferably 50% to 99%, and more preferably 80% to 99%.

[0013] The recombinant human collagen described in this invention is the recombinant human collagen disclosed in CN108070032B, "Method for Purifying Recombinant Human Collagen."

[0014] According to a specific embodiment of the present invention, the recombinant human collagen has the amino acid sequence shown in SEQ ID No:1.

[0015] In a specific embodiment of the present invention, preferably, with the total dry amount of the artificial bone material being 100%, the material comprises 70% to 90% coral hydroxyapatite particles, 5% to 23% recombinant human collagen, and 5% to 7% excipients.

[0016] According to a specific embodiment of the present invention, 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%.

[0017] According to specific embodiments of the present invention, the conversion rate of the coral hydroxyapatite particles is preferably 5% to 80%, and more preferably 5% to 30%.

[0018] According to specific embodiments of the present invention, preferably, the excipient comprises one or more combinations of crosslinked porous starch, sodium carboxymethylcellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methylcellulose.

[0019] The recombinant human collagen described in the present invention has regular hydrophilic groups on the outside, strong aggregation ability, and hydrophobic groups on the inside, forming microstents. The recombinant human collagen described in the present invention has self-assembly ability and can self-assemble in low-oxygen or vacuum environments, and its self-assembly ability can be promoted by raising the temperature, and in this process there is no reagent residue due to chemical crosslinking. The hydrophilic (structure) of the recombinant human collagen intelligent collagen is shown in Figure 1, and the SEM of the recombinant human collagen self-assembly structure is shown in Figure 2.

[0020] According to a specific embodiment of the present invention, the coral hydroxyapatite particles are produced by immersion with a cutting protector for coral, crushing and granulation, and hydrothermal exchange. As shown in FIG. 3, the external view of the coral described in the present invention has a three-dimensional network structure in which each pore communicates. The microstructure of corals with different pore diameters is shown in FIG. 4, where a represents dense pores, b represents mesopores, c represents macropores, and the coral with mesopores shown by b in FIG. 4 is most similar to the natural bone structure.

[0021] According to a specific embodiment of the present invention, the cutting protector is a solution containing a polyhydric alcohol.

[0022] 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.

[0023] According to a specific embodiment of the present invention, the volume fraction of the polyhydric alcohol is ≧20% with respect to the total volume of the cutting protector.

[0024] According to a specific embodiment of the present invention, the immersion time is ≧3 h.

[0025] According to a specific embodiment of the present invention, the raw material of the coral includes natural coral and / or artificially cultured coral.

[0026] According to a specific embodiment of the present invention, preferably, the natural coral includes Porites and / or Goniopora, and more preferably Porites.

[0027] According to a specific embodiment of the present invention, the hydrothermal exchange step includes permeating with a saturated solution of diammonium hydrogen phosphate and performing a reaction at 0.1 to 3 MPa and 150 to 220 °C for 6 to 19 h.

[0028] The method for producing coral hydroxyapatite particles described in the present invention further includes:

[0029] 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.

[0030] 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.

[0031] 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.

[0032] According to specific embodiments of the present invention, preferably, the artificial bone material has a bone tissue structure and possesses excellent bone conduction and bone induction properties.

[0033] According to a specific embodiment of the present invention, the form of the artificial bone material includes lumps, sheets, particles, or powder.

[0034] According to a specific embodiment of the present invention, the artificial bone material can be manufactured in multiple forms using (multiple) molds before being implanted in a patient. Depending on the specific application, the artificial bone material of the present invention can be further subjected to steps such as cutting, crushing, and sieving to obtain any desired form, and an aqueous liquid can also be added to obtain any desired material form.

[0035] In some embodiments, the artificial bone material 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 prepare a moldable paste, in which case the paste is injectable and used to fill bone defects.

[0036] According to a specific embodiment of the present invention, when the artificial bone material 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.

[0037] According to a specific embodiment of the present invention, when the artificial bone material 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.

[0038] According to a specific embodiment of the present invention, when the artificial bone material 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.

[0039] On the other hand, the present invention further provides a method for manufacturing the artificial bone material described above. The manufacturing method is

[0040] Nano-flower-shaped coral hydroxyapatite particles and recombinant human collagen solution are uniformly mixed and freeze-dried to obtain a freeze-dried sample.

[0041] The freeze-dried sample is self-assembled and sterilized to obtain the artificial bone material.

[0042] This includes the following.

[0043] 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%.

[0044] In the manufacturing method described above, preferably, the solvent for the recombinant human collagen solution includes one or more combinations of phosphate buffer, purified water, or water for injection.

[0045] 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

[0046] (1) Rapid freezing: Process parameters include reaching a temperature of -80°C to -60°C within 30 to 240 minutes.

[0047] (2) Pre-freezing: Process parameters include raising the temperature to -50°C to -30°C within 60 to 240 minutes and maintaining it for 120 to 600 minutes.

[0048] (3) Sublimation: Sublimate the pre-frozen product, set the vacuum to 0.01~0.1mbar, and within 10~60min, raise the temperature to -50℃~-5℃ and maintain it for 120~600min.

[0049] (4) Vacuum drying: The sublimation product is vacuum dried, with the vacuum set to 0.01-0.1 mbar, the temperature raised to 0-40°C within 10-60 minutes, and maintained for 120-600 minutes.

[0050] This includes the following.

[0051] Table 1 shows the specific process parameters for freeze-drying in the manufacturing method of the present invention.

[0052] [Table 1]

[0053] 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.

[0054] The freeze-dried sample of the artificial bone material of the present invention can undergo self-assembly under low oxygen content or oxygen-free conditions, and includes 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.

[0055] In the manufacturing method described above, the self-assembly step is:

[0056] 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.

[0057] 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.

[0058] 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%.

[0059] In the manufacturing method described above, the sterilization method is preferably radiation sterilization or ethylene oxide sterilization.

[0060] 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.

[0061] 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.

[0062] In the manufacturing method described above, preferably, the sterilization method is electron beam irradiation sterilization, and the sterilization dose is 10 to 25 kGy.

[0063] According to a specific embodiment of the present invention, the above-described manufacturing method includes the following steps.

[0064] (1) "Nano-flower" coral hydroxyapatite is produced by hydrothermal exchange. (2) A recombinant human collagen solution is prepared. (3) The "nano-flower" coral hydroxyapatite is dispersed in the recombinant human collagen solution to obtain a suspension. (4) The recombinant human collagen solution and the "nano-flower" coral hydroxyapatite suspension are uniformly mixed, then rapidly frozen and further vacuum freeze-dried to obtain a freeze-dried sample. (5) The freeze-dried sample is self-assembled 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 material. (7) Finally, the lump, sheet-like, or particulate material is sterilized to obtain an artificial bone material with a three-dimensional network structure similar to natural bone tissue.

[0065] On the other hand, the present invention also provides the use of the above-mentioned artificial bone material in the manufacture of bone repair products.

[0066] According to specific embodiments of the present invention, the bone repair preferably includes filling and repairing a bone defect.

[0067] 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 artificial bone material 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 spongy bone structure having different pore sizes through freeze-drying and the material's biological self-assembly ability. This material should have a high porosity and a radial gradient structure in order to simulate the characteristics of natural bone tissue.

[0068] The artificial bone material manufactured according to the present invention possesses excellent biocompatibility, biodegradability, hydrophilicity, bone-forming properties, 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 artificial bone material of the present invention uses recombinant human collagen to avoid the potential problem of viruses unavoidable in traditional animal collagen stent materials, and no additives are added during the production process, thus significantly improving safety of use.

[0069] Specifically, the artificial bone material provided by the present invention has the following advantages.

[0070] 1. Excellent Biocompatibility: Coral hydroxyapatite (HAP), which is "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 coral rock 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.

[0071] 2. Excellent Biodegradability: Self-assembling recombinant human collagen exhibits excellent biodegradability, and the conversion rate of "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 artificial bone material matches the growth rate of bone tissue, allowing it to decompose completely and fuse perfectly with bone tissue to form autologous bone. The bone repair effect is second only to allogeneic bone, allowing for perfect fusion with autologous bone in 3-6 months, without any foreign body sensation, and the degradation rate matches the bone growth rate. From the palpable feel of the implant and CT scans, it can be seen that this material fuses perfectly with autologous bone and can 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 artificial 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-implantation is performed 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 artificial 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.

[0072] 3. Excellent mechanical performance and shape memory function: The collagen network provides good mechanical support, giving the product appropriate strength and good flexibility.

[0073] 4. Efficient cell adhesion and proliferation capacity: The highly ordered reticular structure is advantageous for cell adhesion, proliferation, and migration.

[0074] 5. Excellent Hydrophilicity: The porosity of this artificial bone material can reach 98.75% ± 0.56%, and it is completely absorbed in 2-3 seconds when mixed with water or blood. 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.

[0075] 6. 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.

[0076] 7. Excellent Clinical Convenience: Traditional tooth extraction 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 artificial bone material of the present invention, its mesh structure mechanically seals the tooth extraction 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, using the artificial bone material not only simplifies the surgical process but also reduces the patient's treatment time and financial burden.

[0077] 8. Hemostatic and Healing-Promoting Effects: Due to its excellent adsorption properties, the artificial bone material 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 artificial bone material 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.

[0078] 9. Excellent bone formation effect: The main components of the artificial bone material manufactured in this invention are hydroxyapatite and collagen, which are the most important inorganic and organic components in natural bone. Using the artificial bone material manufactured in this invention to fill 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]

[0079] [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 artificial bone materials (lump-like and sheet-like) obtained in the examples and comparative examples. [Figure 7] This is a diagram showing the external appearance of the artificial bone material (particulate) in Example 2. [Figure 8] This is a microstructure diagram of the artificial bone material in Example 1. [Figure 9]Diagrams showing the water mixing state of the artificial bone material in Examples 1 and 5. [Figure 10] This shows the results of the compression deformation experiment on the artificial bone material in Example 1. [Figure 11] This figure shows the hydrophilic properties and shape memory functions of the artificial bone materials obtained in the examples and comparative examples. [Figure 12] This figure shows the results of the cytotoxicity of the artificial bone material in Example 1. [Figure 13] This is a diagram showing the results of cell migration in the artificial bone material in Example 1. [Figure 14] This figure shows the results of cell proliferation in the artificial bone material in Example 1. [Figure 15] This is a diagram showing the results of cell adhesion of the artificial bone material in Example 1. [Figure 16] This is a diagram showing the results of extracorporeal decomposition of the artificial bone material in Example 1. [Figure 17] This is an imaging diagram of bone repair of a condylar defect in the femoral bone of a rabbit using artificial bone material in Example 1. [Figure 18] This is an imaging diagram of bone repair of a condylar defect in the femoral bone of a rabbit using artificial bone material in Example 1. [Figure 19] This is an imaging diagram of bone repair in which the artificial bone material from Example 1 was used to fill an extracted tooth socket in a clinical trial. [Figure 20] This is an imaging diagram of bone repair in which the artificial bone material used in Example 3 was filled into a tooth extraction socket in a clinical trial. [Modes for carrying out the invention]

[0080] 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.

[0081] 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.

[0082] The amino acid sequence of recombinant human collagen used in the following examples is:

[0083] GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPG KPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSN GPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP (SEQ ID No: 1).

[0084] 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. 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.

[0085] Example 1

[0086] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0087] 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 artificial bone material, which has a porosity of 98.17%.

[0088] The freeze-drying process is as follows:

[0089] During the pre-freezing stage, the temperature should reach -50°C within 120 minutes and be maintained for 180 minutes.

[0090] 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;

[0091] 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.

[0092] As shown in Figure 6a for the external view of the artificial bone material and Figure 6b for the cross-sectional view, 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 has a three-dimensional structure, and the nano-flowered coral hydroxyapatite is encapsulated by collagen and uniformly distributed within the collagen stent. The artificial bone material 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).

[0093] Experiments using the artificial bone material to repair lateral femoral condyle defects in rabbits showed that the material has a good bone repair effect (as shown in experimental group 1 in Figures 17 and 18). Furthermore, clinical trials using the artificial bone material to fill tooth extraction sockets showed that the material has a good bone repair effect (as shown in Figure 19).

[0094] Example 2

[0095] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0096] 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 min, and then vacuum freeze-dried to produce 80 chunk 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 using 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 artificial bone material, which has a porosity of 98.62%.

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

[0098] During the pre-freezing stage, the temperature should reach -45°C within 100 minutes and be maintained for 200 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 0.08 mbar, the temperature is raised to 27°C within 50 minutes, and maintained for 250 minutes.

[0101] As shown in Figure 7, the external appearance of the artificial bone material (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.

[0102] Example 3

[0103] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0104] 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 sterilization process was performed under a vacuum of -15 kPa and a sterilization time of 10 hours to obtain the artificial bone material, which has a porosity of 96.58%.

[0105] The freeze-drying process is as follows:

[0106] During the pre-freezing stage, the temperature should reach -50°C within 140 minutes and be maintained for 190 minutes.

[0107] 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;

[0108] 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.

[0109] As shown in Figure 6c, the external appearance of the artificial bone material was shown, and when a clinical trial of tooth extraction socket filling was conducted using the artificial bone material, it was shown that the material has a good bone regeneration effect (as shown in Figure 20).

[0110] Example 4

[0111] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0112] 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, thereby preparing 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, yielding 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 samples. The artificial bone material was obtained by self-assembling the sample 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 artificial bone material, which has a porosity of 96.23%.

[0113] The freeze-drying process is as follows:

[0114] During the pre-freezing stage, the temperature should reach -50°C within 160 minutes and be maintained for 220 minutes.

[0115] 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;

[0116] 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.

[0117] The external view of the artificial bone material is shown in Figure 6d.

[0118] Example 5

[0119] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0120] 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 artificial bone material is obtained by self-assembling it at 180°C for 6 hours in an electric vacuum drying box set to a vacuum of -0.095 MPa, sealing the self-assembling sample in an aluminum foil bag, and then sterilizing it with electron beam irradiation at an irradiation dose of 25 kGy, resulting in a porosity of 96.06%.

[0121] The freeze-drying process is as follows:

[0122] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0123] 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;

[0124] 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.

[0125] As shown in Figure 6e, the appearance of the artificial bone material is complete and does not easily scatter after mixing with water (as shown in Figure 9b), and it has good toughness, hydrophilicity, and shape memory function (as shown in Figure 11b).

[0126] Example 6

[0127] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0128] 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 artificial bone material is obtained by self-assembling it at 220°C for 6 hours in an electric vacuum drying box set to a vacuum of -0.095 MPa, sealing the self-assembling sample in an aluminum foil bag, and then sterilizing it with electron beam irradiation at an irradiation dose of 25 kGy, resulting in a porosity of 97.79%.

[0129] The freeze-drying process is as follows:

[0130] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0131] 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;

[0132] 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.

[0133] The external view of the artificial bone material is shown in Figure 6f.

[0134] Example 7

[0135] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0136] 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 placed in 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 artificial bone material.

[0137] The freeze-drying process is as follows:

[0138] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0139] 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;

[0140] 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.

[0141] As shown in Table 2 below, the pore size structure of the artificial bone material is such that the higher the conversion rate of "nano-flower" coral hydroxyapatite, the larger the pore size and the larger the porosity, which in turn increases the porosity of the manufactured artificial bone material.

[0142] [Table 2]

[0143] Example 8

[0144] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0145] 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 artificial bone material, which has a porosity of 97.43%.

[0146] The freeze-drying process is as follows:

[0147] During the pre-freezing stage, the temperature should reach -50°C within 120 minutes and be maintained for 180 minutes.

[0148] 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;

[0149] 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.

[0150] The external appearance of the artificial bone material is shown in Figure 6g.

[0151] Example 9

[0152] This embodiment provides an artificial bone material having self-assembling collagen and "nano-flower" coral hydroxyapatite, which is manufactured by the following steps.

[0153] 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 artificial bone material, which has a porosity of 98.83%.

[0154] The freeze-drying process is as follows:

[0155] During the pre-freezing stage, the temperature should reach -50°C within 120 minutes and be maintained for 180 minutes.

[0156] 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;

[0157] 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.

[0158] The external view of the artificial bone material is shown in Figure 6h.

[0159] Comparative Example 1

[0160] This comparative example provides an artificial bone material, which is manufactured by the following steps.

[0161] 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 mins, and then vacuum freeze-dried to produce 80 chunk-like 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 artificial bone material.

[0162] The freeze-drying process is as follows:

[0163] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0164] 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;

[0165] 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.

[0166] As shown in Figure 6i (visual view) and Figure 8d (scanning electron microscope image) of the artificial bone material, it was shown that after freeze-drying, the nanohydroxyapatite was encased in collagen, forming a relatively dense three-dimensional structure. When this artificial bone material was used in a rabbit femoral lateral condyle defect bone repair experiment (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 "nanoflower" 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" hydroxyphosphorylated 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.

[0167] Comparative Example 2

[0168] This comparative example provides an artificial bone material, which is manufactured by the following steps.

[0169] 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%.

[0170] The freeze-drying process is as follows:

[0171] During the pre-freezing stage, the temperature should reach -50°C within 200 minutes and be maintained for 200 minutes;

[0172] 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;

[0173] 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.

[0174] 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.

[0175] Comparative Example 3

[0176] This comparative example provides an artificial bone material, which is manufactured by the following steps.

[0177] 10 g of recombinant human collagen freeze-dried powder (a commercially available raw material from another company, white or nearly white sponge-like solid, 99.9% purity) was weighed, and 20 g of phosphate buffer (pH=6.6) was added to uniformly suspend it, preparing 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, uniformly suspending 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 sample was 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 artificial bone material, the porosity of which was 78.55%.

[0178] The freeze-drying process is as follows:

[0179] (1) During the pre-freezing stage, the temperature should be raised to -50°C within 120 minutes and maintained for 180 minutes;

[0180] (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;

[0181] (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.

[0182] As shown in Figure 6k, the appearance of the artificial bone material is slightly yellowish. As shown in Figure 11d, the water mixing experiment showed that the artificial bone material dissolved 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. This indicates that the product of the examples is better suited to meet the requirements of clinical use.

[0183] Comparative Example 4

[0184] This comparative example provides an artificial bone material having self-assembled collagen and "nano-flower" hydroxyphosphorylated coralite, which is manufactured by the following steps.

[0185] 6 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 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 artificial bone material, which has a porosity of 71.52%.

[0186] The freeze-drying process is as follows:

[0187] (1) In the pre-freezing stage, the temperature should be raised to -50°C within 200 minutes and maintained for 200 minutes;

[0188] (2) 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;

[0189] (3) 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.

[0190] The proportion of "nano-flower" hydroxyphosphorylated coralite in the artificial bone material 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 artificial bone material 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 better met the requirements for clinical use.

[0191] Comparative Example 5

[0192] This comparative example provides an artificial bone material having self-assembled collagen and "nano-flower" hydroxyphosphorylated coralite, which is manufactured by the following steps.

[0193] 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 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 sample was 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 artificial bone material, which has a porosity of 78.18%.

[0194] The freeze-drying process is as follows:

[0195] (1) During the pre-freezing stage, the temperature should reach -50°C within 60 minutes and be maintained for 180 minutes;

[0196] (2) 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;

[0197] (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.

[0198] 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 artificial bone material 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 water mixing of the artificial bone material was slow, the particles scattered easily after water mixing, the sample lacked toughness and was far inferior to the example. This demonstrated that the product of the example is better able to meet the requirements for clinical use.

[0199] The product obtained in Example 1 was used as a representative example for relevant performance measurements, which are as follows:

[0200] 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.

[0201] 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.

[0202] 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 3, 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.

[0203] [Table 3]

[0204] Cytotoxicity measurement: A cytotoxicity test was performed on the artificial bone material obtained in Example 1 according to GB / T 16886.5-2017 Biological evaluation of medical devices, Part 5: Extracorporeal cytotoxicity testing, MTT method (two parallel experiments were performed, with both Sample 1 and Sample 2 being the products obtained in Example 1). The specific procedure is as follows.

[0205] Using a high-sugar DMEM culture medium as the extrusion medium, the artificial bone material was first completely swollen, and then extruded at a ratio of 0.1 g / ml. The extrusion temperature was 37°C and the extrusion time was 72 hours. 1.2 × 10⁶ L929 cells in the logarithmic growth phase were extracted. 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).

[0206]

number

[0207] 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.

[0208] The experimental results are shown in Table 4 and Figure 12.

[0209] [Table 4]

[0210] As can be seen from Table 4, the artificial bone material of the present invention has good biocompatibility and is non-cytotoxic.

[0211] Cell migration:

[0212] Leaching: 4g of artificial bone material was mixed with 20mL of physiological saline solution and leached in a pressure steam sterilizer at 121°C for 1 hour.

[0213] 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.

[0214] 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).

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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 artificial bone material had a significant effect in promoting osteoblast migration.

[0220] Cell proliferation:

[0221] 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.

[0222] Addition of cell suspension: After immersing the artificial bone material in normal culture medium for 2 hours, the medium was discarded, and 200 μL of cell suspension was gradually added 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.

[0223] 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).

[0224] Measurement: After the culture period, the culture medium was removed, washed 2-3 times with PBS, and the cells were digested with 0.25% trypsin to perform cell counting. The average value of the results was then taken. As shown in Figure 14, the experimental results demonstrated that the artificial bone material had a significant effect in promoting osteoblast proliferation.

[0225] Cell adhesion:

[0226] Cell culture: Using 2nd-3rd generation MC3T3-E1 cells, after the degree of cell fusion reaches 80%, the cells are digested and collected, counted on a cell counter, and the cell inoculation density is set to 2 × 10⁻⁶. 7 The concentration was adjusted to 1 / mL.

[0227] Addition of cell suspension: After immersing the artificial bone material in normal culture medium for 2 hours, discard the medium and add 500 μL (4 × 10) cell suspension. 6 The individual cells should be added gradually in three separate steps, starting from the middle of the sterile sample, until the entire cell suspension is absorbed into the sample's voids. It is important to avoid letting the cell suspension fall into the well plate during the process.

[0228] Incubation and culturing: 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, and the samples were incubated for 2 hours and 4 hours, respectively.

[0229] Measurement: After the culture period, two osteoblast / artificial bone material composite culture samples were randomly selected, and two blank artificial bone material samples that had not been inoculated with cells were also selected. The culture medium was removed, the samples were gently shaken and washed 2-3 times with PBS, fixed with 3% glutaraldehyde for 30 minutes, washed 2-3 more times with PBS, dehydrated stepwise with ethanol in concentration gradients of 30%, 50%, 70%, 90%, and 100% (immersion washing for 2 minutes at each concentration), vacuum dried, and gold plated. The cell adhesion morphology on the material surface at different time points was then observed using a scanning electron microscope. As shown in Figure 15, the experimental results demonstrated that osteoblasts could adhere to the artificial bone material and form lamellar structures, which is advantageous in the bone repair process.

[0230] In vitro degradation analysis: After uniformly cutting the sample into four parts, the four samples were mixed with the prepared, filtration-treated 0.01 mol PBS buffer (pH=7.4) and 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.

[0231] 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. An artificial bone material, wherein the total amount of the dry material of the artificial bone material is 100%, and the material is Coral hydroxyapatite particles 70% to 90% Recombinant human collagen 5% to 30%, and Contains 0-7% excipients. The aforementioned artificial bone material is a solid porous material formed by the adhesion of coral hydroxyapatite particles to each other by recombinant human collagen. The porosity of the artificial bone material is 80% to 99%. The recombinant human collagen has the amino acid sequence shown in SEQ ID No: 1, and the coral hydroxyapatite particles are "nano-flower" coral hydroxyapatite, which 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, crushing and granulation, and then further exchanging the hydrothermal energy of the coral sample. The aforementioned hydrothermal exchange step includes 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. The aforementioned 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%. An artificial bone material in which the conversion rate of the coral hydroxyapatite particles is 10% to 80%.

2. The artificial bone material according to claim 1, wherein the conversion rate of the coral hydroxyapatite particles is 10% to 30%.

3. The artificial bone material according to claim 1, wherein the excipient comprises one or more combinations of cross-linked porous starch, sodium carboxymethylcellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methylcellulose.

4. The artificial bone material according to claim 1, which is in the form of particulate matter, lumps, sheets, or powder.

5. Coral hydroxyapatite particles and recombinant human collagen solution are uniformly mixed and freeze-dried to obtain a freeze-dried sample. A method for producing an artificial bone material according to any one of claims 1 to 4, comprising: placing the freeze-dried sample under a vacuum of -0.1 to -0.01 MPa or a nitrogen atmosphere, raising the temperature to 100 to 300°C, holding for 0.5 to 8 hours, and sterilizing it to obtain the artificial bone material.

6. The manufacturing method according to claim 5, wherein the mass fraction of recombinant human collagen in the recombinant human collagen solution is 15% to 50%.

7. The manufacturing method according to claim 5, wherein the solvent for the recombinant human collagen solution includes one or more combinations of phosphate buffer, purified water, or water for injection.

8. The freeze-drying step described above is (1) Rapid freezing: Process parameters include raising the temperature to -80°C to -60°C in 30 to 240 minutes. (2) Pre-freezing: Process parameters include raising the temperature to -50°C to -30°C for 60 to 240 mins and maintaining it for 120 to 600 mins. (3) Sublimation: Sublimate the pre-frozen product, set the vacuum level to 0.01 to 0.1 mbar, raise the temperature to -50°C to -5°C within 10 to 60 minutes, and maintain it for 120 to 600 minutes. (4) Vacuum drying: The manufacturing method according to claim 5, comprising vacuum drying the product after sublimation, setting the vacuum level to 0.01 to 0.1 mbar, raising the temperature to 0 to 40°C within 10 to 60 mins, and maintaining it for 120 to 600 mins.

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