An artificial bone material and a method for producing the same

Abstract

The application provides an artificial bone material and a preparation method thereof, wherein the material quality of the artificial bone material includes, with the total dry matter of the artificial bone material being 100%, 70%-90% of coral hydroxyapatite particles, 5%-30% of recombinant human collagen, and 0-7% of excipients. The artificial bone material provided by the application has high porosity and a longitudinal gradient structure, can simulate the characteristics of natural bone tissue, and has excellent biocompatibility, biodegradability, hydrophilicity, osteogenic performance and shape memory function.

Description

Technical Field

[0001] This invention belongs to the field of medical materials and relates to a method for preparing biomimetic materials, specifically an artificial bone material and its preparation method. Background Technology

[0002] Research on artificial bone biomaterials currently falls into several main categories: Polymer materials, such as polymethyl methacrylate (PMMA), i.e., bone cement, and polyethylene used in artificial joints. These materials have poor biocompatibility, with fibrous tissue acting as a barrier between them and bone tissue. Inorganic materials are the most widely used, primarily ceramic materials, which are categorized into bioinert, bioactive, and biodegradable materials. Bioinert materials include alumina ceramics; bioactive materials include glass ceramics, bioactive glass, and hydroxyapatite; and biodegradable ceramics are mainly β-tricalcium phosphate (β-TCP). Another type of biodegradable material, not belonging to ceramics, is natural coral. The main advantages of bioactive materials are good biocompatibility, the ability to chemically bond with bone tissue or degrade in vivo, and relatively high strength. The main disadvantage of ceramic materials is their high brittleness; their elastic modulus is difficult to match that of normal bone, which limits their clinical application to some extent.

[0003] Therefore, developing ideal bone graft substitutes has always been one of the important topics in the field of orthopedic surgery. Ideal bone graft substitutes should have the following characteristics: (1) osteoconductivity; (2) osteoinductive properties; (3) excellent hydrophilicity; (4) good biocompatibility; (5) biodegradability, with the in vivo degradation rate matching the rate of new bone regeneration; (6) suitable porosity and pore-connected structure; (7) good mechanical properties; (8) easy to operate during surgery; (9) easy to sterilize before use; and (10) able to be prepared into a certain size for easy filling, etc.

[0004] Hydroxyapatite (HAP) and collagen are the most abundant inorganic and organic components of natural bone, respectively, and both possess excellent biological properties. However, when used alone, they have different drawbacks, making it difficult to meet the performance requirements of biomaterials in tissue engineering. By combining collagen with hydroxyapatite, the binding properties of collagen can be effectively utilized to overcome some of the limitations of hydroxyapatite. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide an artificial bone material and its preparation method.

[0006] To achieve the above objectives, the present invention provides an artificial bone material, wherein, based on 100% of the total dry matter of the artificial bone material, its material comprises:

[0007] Coral hydroxyapatite particles 70%-90%,

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

[0009] Excipients: 0-7%.

[0010] According to a specific embodiment of the present invention, the artificial bone material is a solid porous material formed by the mutual adhesion of coral hydroxyapatite particles through recombinant human collagen.

[0011] According to a specific embodiment of the present invention, preferably, the porosity of the artificial bone material is 50%-99%, more preferably 80%-99%.

[0012] The recombinant human collagen described in this invention is the recombinant human collagen disclosed in CN108070032B [A purification method for recombinant human collagen].

[0013] According to a specific embodiment of the present invention, the recombinant human collagen has an amino acid sequence as shown in SEQ ID No:1.

[0014] According to a specific embodiment of the present invention, preferably, the artificial bone material comprises, based on 100% of the total dry matter, 70%-90% coral hydroxyapatite particles, 5%-23% recombinant human collagen, and 5-7% excipients.

[0015] 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%.

[0016] According to a specific embodiment of the present invention, preferably, the conversion rate of the coral hydroxyapatite particles is 5%-80%; more preferably, the conversion rate is 5%-30%.

[0017] According to a specific embodiment of the present invention, preferably, the excipient includes one or more of cross-linked porous starch, sodium carboxymethyl cellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methyl cellulose.

[0018] The recombinant human collagen of this invention has regular hydrophilic groups on the outside, exhibiting super-strong aggregation ability; and hydrophobic groups on the inside, forming a microscaffold. The recombinant human collagen of this invention possesses self-assembly capability, capable of self-assembly in low-oxygen or vacuum environments, and increasing temperature can promote self-assembly. This process avoids reagent residues caused by chemical cross-linking. The intelligent collagen hydrophilicity (structure) of this recombinant human collagen is as follows: Figure 1 As shown in the figure, the SEM image of the self-assembled structure of the recombinant human collagen is as follows. Figure 2 As shown.

[0019] According to a specific embodiment of the present invention, the coral hydroxyapatite particles are prepared by soaking coral stone in a cutting protective agent, crushing and granulating, and then subjecting it to hydrothermal exchange. The appearance of the coral stone described in this invention is shown in the image below. Figure 3 As shown, it has a three-dimensional network structure with interconnected pores. The microstructure of coral with different pore sizes is as follows: Figure 4 As shown, a represents a dense pore, b represents a medium pore, and c represents a large pore. Figure 4 The mesoporous coral shown in b is most similar to the structure of natural bone.

[0020] According to a specific embodiment of the present invention, the cutting protectant is a solution containing a polyol.

[0021] According to a specific embodiment of the present invention, preferably, the polyol is selected from one or more combinations of glycerol, ethylene glycol, sorbitol, and butylene glycol.

[0022] According to a specific embodiment of the present invention, the volume fraction of the polyol is ≥20% based on the total volume of the cutting protectant.

[0023] According to a specific embodiment of the present invention, the soaking time is ≥3h.

[0024] According to a specific embodiment of the present invention, the raw materials for the coral stone include natural coral and / or artificially cultured coral.

[0025] According to a specific embodiment of the present invention, preferably, the natural coral includes shore coral and / or horn-hole coral; more preferably, shore coral.

[0026] According to a specific embodiment of the present invention, the hydrothermal exchange step includes: impregnation with a saturated diammonium hydrogen phosphate solution, and reaction at 0.1-3 MPa and 150-220°C for 6-19 hours.

[0027] The method for preparing coral hydroxyapatite particles described in this invention further includes:

[0028] The coral hydroxyapatite particles are prepared by bleaching and cleaning the coral, followed by soaking in a cutting protective agent, crushing and granulating, and then undergoing hydrothermal exchange.

[0029] The coral hydroxyapatite particles obtained by the preparation method of this invention can form nanoflower-like hydroxyphosphorylated structures on their surface, resulting in "nanoflower"-shaped coral hydroxyapatite particles. Under a microscope, their surface exhibits a "nanoflower"-shaped structure, such as... Figure 5As shown. Soaking in a cutting protectant allows coral to retain its intact porous structure even when cut and polished into smaller particles, making it more similar to human cancellous bone. The "nanoflower"-shaped coral hydroxyapatite can also have its conversion rate controlled by adjusting temperature, time, and reagent dosage, thus preparing coral hydroxyapatite with different nanoflower ratios. When combined with recombinant human collagen, it can form biomimetic bone structures with different pore sizes, allowing for controllable degradation rates after implantation. The microstructures of "nanoflower"-shaped coral hydroxyapatite with different conversion rates are shown below. Figure 5 As shown, a represents no conversion, b represents slight conversion, and c represents complete conversion.

[0030] The nano-flower coral hydroxyapatite prepared by this invention, after being treated with a series of steps such as cutting protective agent, better protects the pore size and porosity, and greatly improves the yield.

[0031] According to a specific embodiment of the present invention, preferably, the artificial bone material has a bone tissue structure and exhibits excellent osteoconductivity and osteoinduction properties.

[0032] According to a specific embodiment of the present invention, the artificial bone material may be in the form of blocks, flakes, granules or powder.

[0033] According to a specific embodiment of the present invention, the artificial bone material can be prepared in multiple forms in multiple molds before implantation into the patient. The artificial bone material of the present invention can be further subjected to cutting, crushing, sieving, and other steps, depending on the specific application, to obtain any desired form; it can also be further mixed with an aqueous liquid to obtain any desired material morphology.

[0034] In some embodiments, the artificial bone material is in powder form, with coral hydroxyapatite particles coated with collagen and / or excipients. When mixed with a liquid, it becomes viscous and can be used directly in powder form to fill bone defects; or an aqueous liquid can be added before filling to prepare a malleable paste; wherein the paste is injectable and used to fill bone defects.

[0035] According to a specific embodiment of the present invention, when the artificial bone material is in block form, its size range can 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, it can be the following sizes: 4mm×4mm×4mm, 6mm×6mm×6mm, 8mm×8mm×8mm, 10mm×10mm×10mm, 15mm×15mm×15mm, 20mm×20mm×20mm, 30mm×30mm×30mm, 65mm×65mm×65mm.

[0036] According to a specific embodiment of the present invention, when the artificial bone material is in sheet form, its size range can be (0.1-5)mm×(10-200)mm×(10-200)mm, (0.1-5)mm×(50-200)mm×(50-200)mm, or diameter (5-200)mm×height (1-5)mm; for example, it can be the following sizes: 0.5mm×10mm×10mm, 0.5mm×15mm×15mm, 0.5mm×20mm×20mm, 2mm×50mm×50mm, 5mm×100mm×100mm, etc.

[0037] According to a specific embodiment of the present invention, when the artificial bone material is in the form of granules, its particle size distribution range can be 0.18-2 mm; for example, it can be the following distribution ranges: 0.18 mm-0.25 mm, 0.25-0.3 mm, 0.3-0.5 mm, 0.5-1 mm, 1-2 mm.

[0038] On the other hand, the present invention also provides a method for preparing the above-mentioned artificial bone material, the method comprising:

[0039] The "nanoflower" coral hydroxyapatite particles were mixed with a recombinant human collagen solution and then freeze-dried to obtain a freeze-dried sample.

[0040] The artificial bone material is obtained by self-assembling and sterilizing the freeze-dried sample.

[0041] In the above preparation method, the recombinant human collagen solution contains 15%-50% by mass, preferably 20-50%, and more preferably 30%.

[0042] In the above preparation method, preferably, the solvent of the recombinant human collagen solution includes one or more of phosphate buffer, purified water, or water for injection.

[0043] In the above preparation method, preferably, the recombinant human collagen solution further includes an excipient. In this invention, the recombinant human collagen can form a collagen scaffold together with the excipient. In the above preparation method, the freeze-drying step includes:

[0044] (1) Quick freezing: The process parameters are to reach -80℃ to -60℃ within 30-240 minutes;

[0045] (2) Pre-freezing: The process parameters are to reach -50℃ to -30℃ within 60-240 min and continue for 120-600 min;

[0046] (3) Sublimation: Sublimate the pre-frozen product under a vacuum of 0.01-0.1 mbar, with the temperature reaching -50℃ to -5℃ within 10-60 min and maintained for 120-600 min.

[0047] (4) Desorption and drying: The sublimated product is desorbed and dried. The vacuum degree is set to 0.01-0.1 mbar, the temperature reaches 0-40℃ within 10-60 min, and it is maintained for 120-600 min.

[0048] In the preparation method of this invention, the specific process parameters for freeze drying are shown in Table 1.

[0049] Table 1

[0050]

[0051] In the above preparation method, the pore structure of the material can be controlled by controlling the freezing rate; the pore size is larger during slow freezing and smaller during rapid freezing.

[0052] The freeze-dried samples of the artificial bone material of the present invention can self-assemble under conditions of low oxygen content or no oxygen, including three cases: the first case is low-pressure self-assembly; the second case is nitrogen-filled vacuum self-assembly; and the third case is oxygen-free self-assembly.

[0053] In the above preparation method, the self-assembly step includes:

[0054] The freeze-dried sample is placed in a vacuum environment of -0.1 to -0.01 MPa or in a nitrogen atmosphere and heated to 100-300°C, then held for 0.5-8 hours; preferably, the heating temperature is 100-220°C and the vacuum degree of the nitrogen atmosphere is -0.01 to -100 kPa.

[0055] In the above preparation method, preferably, the self-assembly is carried out using an electric heating vacuum drying oven or a precision vacuum nitrogen-filled integrated drying oven.

[0056] In this invention, recombinant human collagen is prepared into a recombinant human collagen solution according to a specified ratio. After being freeze-dried and self-assembled using the methods of this invention to form a collagen scaffold, the porosity of the material is detected using the ethanol permeation method or the mercury porosimetry method. In this invention, the porosity of the collagen scaffold formed after freeze-drying and self-assembly of the recombinant human collagen solution is ≥85%.

[0057] In the above preparation method, preferably, the sterilization method is irradiation sterilization or ethylene oxide sterilization.

[0058] In the above preparation method, preferably, the irradiation sterilization method is cobalt-60 irradiation sterilization and / or electron beam irradiation sterilization, and the sterilization dose is 10-30 kGy.

[0059] In the above preparation method, preferably, the sterilization parameters for ethylene oxide sterilization are: sterilization temperature 40-60℃, sterilization humidity 40-60%, and ethylene oxide concentration 400-700 g / m³. 3 Vacuum degree -20-10KPa, sterilization time 6-12h.

[0060] In the above preparation method, preferably, the sterilization method is electron beam irradiation sterilization, and the sterilization dose is 10-25 kGy.

[0061] According to a specific embodiment of the present invention, the above preparation method includes the following steps:

[0062] The process includes: (1) preparing “nanoflower” coral hydroxyapatite by hydrothermal exchange; (2) preparing a recombinant human collagen solution; (3) dispersing “nanoflower” coral hydroxyapatite in the recombinant human collagen solution to obtain a suspension; (4) mixing the recombinant human collagen solution and the “nanoflower” coral hydroxyapatite suspension evenly, then quick-freezing and vacuum freeze-drying to obtain a freeze-dried sample; (5) self-assembling the freeze-dried sample under low oxygen content or anaerobic conditions; (6) pulverizing and sieving the self-assembled block or sheet sample to prepare granules; (7) finally sterilizing the block, sheet or granule material to obtain an artificial bone material with a three-dimensional network structure similar to natural bone tissue.

[0063] On the other hand, the present invention also provides the application of the above-mentioned artificial bone material in the preparation of products for bone repair.

[0064] According to a specific embodiment of the present invention, preferably, the bone repair includes filling and repairing bone defects.

[0065] In this invention, the "nanoflower"-shaped coral hydroxyapatite is prepared from natural coral through a series of processes including hydrothermal exchange, forming a unique "nanoflower" structure on the coral surface. The artificial bone material of this invention is prepared by mixing self-assembled recombinant human collagen with the "nanoflower"-shaped coral hydroxyapatite in a specific mass ratio, and then using freeze-drying and the material's bio-self-assembly capabilities to prepare a material with a cancellous bone-like structure and different pore sizes. This material should possess high porosity and a meridional gradient structure to mimic the characteristics of natural bone tissue.

[0066] The artificial bone material prepared in this invention possesses excellent biocompatibility, biodegradability, hydrophilicity, osteogenic properties, and shape memory function. The material softens rapidly upon contact with water, exhibiting elasticity and a certain degree of flexibility, allowing for arbitrary cutting to fit the shape of the defect area, making it suitable for filling bone defects at any location. Furthermore, because the artificial bone material of this invention uses recombinant human collagen, it eliminates the unavoidable viral risks associated with traditional animal collagen scaffold materials. No additives are used in the production process, significantly improving safety.

[0067] Specifically, the artificial bone material provided by this invention has the following advantages:

[0068] 1. Excellent biocompatibility: "Nanoflower" coral hydroxyapatite, or coral hydroxyapatite (HAP), exhibits excellent biocompatibility. Its composition and structure are similar to natural bone, and it does not cause systemic or local toxicity or immune rejection after implantation. The self-assembled recombinant human collagen and coral stone demonstrate excellent biocompatibility, and the preparation process primarily involves self-assembly under low or anaerobic conditions, leaving no chemical residues.

[0069] 2. Excellent Biodegradability: Self-assembled recombinant human collagen exhibits excellent biodegradability. The "nanoflower" coral hydroxyapatite, through its controlled conversion rate during preparation, ensures its degradation rate matches the growth and repair speed of bone tissue. The resulting artificial bone material degrades at the same rate as bone tissue ingrowth, achieving complete degradation and perfect fusion with bone tissue to form autologous bone. Its bone repair effect is second only to allogeneic bone, achieving perfect fusion with autologous bone within 3-6 months, without any foreign body sensation. The degradation rate matches the bone formation rate, and the implant feel and CT scans confirm that this material can perfectly fuse with autologous bone, ultimately growing into autologous bone. In comparison, bone materials prepared from nano-hydroxyapatite and bovine bone do not completely degrade. Bovine bone's bone repair effect is inferior to allogeneic bone or the artificial bone material of this invention. Furthermore, bovine bone has a slower degradation rate and longer bone absorption time, failing to fully fuse with autologous bone. Even 3-6 months after grafting, a gritty feeling remains upon re-implantation, making it a "semi-permanent" implant material. Long-term use may lead to local osteoporosis or displacement risks. Nano-hydroxyapatite has a better bone repair effect than bovine bone but not as good as homologous allogeneic bone and the artificial bone material of this invention. Moreover, the material is brittle and hard, difficult to degrade in the human body, and may remain in the human body for a long time.

[0070] 3. Excellent mechanical properties and shape memory function: The collagen network provides excellent mechanical support, giving the product moderate strength and good flexibility.

[0071] 4. Highly efficient cell adhesion and proliferation: The highly ordered network structure is conducive to cell adhesion, proliferation and migration.

[0072] 5. Excellent hydrophilicity: The porosity of this artificial bone material reaches 98.75% ± 0.56%, and it is fully rehydrated or re-blooded within 2-3 seconds. Good blood supply provides sufficient nutrients and oxygen for bone tissue regeneration, while also helping to remove metabolic waste, thus accelerating the bone healing process. Recent research shows that hydrophilic surfaces can accelerate osseointegration, shorten restoration waiting time, and significantly improve implant stability. For example, the Kewell superhydrophilic implant utilizes advanced surface treatment technology to provide extremely high hydrophilicity. This surface can accelerate bone tissue growth and reduce the occurrence of complications, which is particularly important for complex cases with poor periodontal condition or requiring full-mouth restoration. Furthermore, the overall stability of hydrophilic implants reaches its lowest value in the second week after implantation and achieves ideal osseointegration in the fourth to sixth week, indicating that hydrophilic surfaces can rapidly initiate the osseointegration process.

[0073] 6. Bionic structure: It has the radial gradient structure of natural bone tissue, which is closer to the structure of natural human bone tissue.

[0074] 7. Excellent clinical ease of use: Traditional tooth extraction socket filling materials, such as bone powder, require a covering membrane to prevent bone powder loss and promote its integration with bone tissue. However, with the artificial bone material of this invention, the extraction socket is mechanically sealed through its mesh structure, preventing infection and further tissue damage. No covering membrane is needed, and the operation time can be significantly shortened from over 20 minutes in traditional surgery to 2 minutes. Therefore, using artificial bone material not only simplifies the surgical procedure but also reduces the patient's treatment time and economic burden.

[0075] 8. Hemostatic and Healing-Promoting Effects: Due to its excellent adsorption properties, the artificial bone material of this invention can adsorb and activate platelets, promote blood clot formation, and form thrombi to exert a hemostatic effect. Simultaneously, the artificial bone material expands slightly after adsorbing blood, gently compressing the bone wall of the extraction socket to achieve a tight fit, thereby accelerating the healing and osteogenesis process. Furthermore, collagen, as a scaffold material, provides a site for cell attachment, promotes cell proliferation and differentiation, and thus promotes tissue remodeling and healing.

[0076] 9. Excellent osteogenic effect: The artificial bone material prepared by this invention is mainly composed of hydroxyapatite and collagen, which are the most important inorganic and organic components in natural bone. Using the artificial bone material prepared by this invention to fill tooth extraction sockets can prevent or reduce the degree of alveolar bone resorption, and also helps the gingival epithelium to creep and cover the socket, which is beneficial for restoring the bone height of the extraction socket. Attached Figure Description

[0077] Figure 1 This is a schematic diagram of the intelligent collagen hydrophilicity (structure) of recombinant human collagen.

[0078] Figure 2 This is a SEM image of the intelligent collagen self-assembly structure.

[0079] Figure 3 This is an image of the appearance of coral.

[0080] Figure 4 Microscopic structures of corals with different pore sizes.

[0081] Figure 5 Microstructure diagrams of hydroxyapatite from "nanoflower" corals with different conversion rates.

[0082] Figure 6 The images show the appearance and morphology of the artificial bone materials (blocks and sheets) obtained in the examples and comparative examples.

[0083] Figure 7 This is a morphological image of the artificial bone material (granular) in Example 2.

[0084] Figure 8 This is a microstructure diagram of the artificial bone material in Example 1.

[0085] Figure 9 The images show the rehydration state of the artificial bone materials in Examples 1 and 5.

[0086] Figure 10 The results are from the compression deformation test of the artificial bone material in Example 1.

[0087] Figure 11 The diagram illustrates the hydrophilic properties and shape memory function of the artificial bone materials obtained in the examples and comparative examples.

[0088] Figure 12 This is a diagram showing the cytotoxicity results of the artificial bone material in Example 1.

[0089] Figure 13 This is a diagram showing the cell migration results of the artificial bone material in Example 1.

[0090] Figure 14 This is a diagram showing the cell proliferation results of the artificial bone material in Example 1.

[0091] Figure 15 This is a diagram showing the cell adhesion results of the artificial bone material in Example 1.

[0092] Figure 16 This is a diagram showing the in vitro degradation results of the artificial bone material in Example 1.

[0093] Figure 17 This is an imaging image of bone repair at the rabbit femoral condyle defect site using the artificial bone material in Example 1.

[0094] Figure 18 This is an imaging image of bone repair at the rabbit femoral condyle defect site using the artificial bone material in Example 1.

[0095] Figure 19 This is an imaging image of bone repair in a clinical trial using artificial bone material for filling tooth extraction sockets, as shown in Example 1.

[0096] Figure 20 This is an imaging image of bone repair in a clinical trial using artificial bone material for filling tooth extraction sockets, as shown in Example 1. Detailed Implementation

[0097] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0098] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions are preferred embodiments for carrying out the invention; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of the invention. The scope of protection of this invention is determined by the appended claims.

[0099] The amino acid sequence of the recombinant human collagen used in the following examples is: GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGS PGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAG SPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP(SEQ ID No:1).

[0100] The recombinant human collagen lyophilized powder used in the following examples was obtained through large-scale fermentation and purification by high expression of novel recombinant human collagen in Pichia pastoris using optimized amino acid sequence design. Specifically, the hydrophilic Gly-XY repeat sequence is the smallest repeating unit of human type I collagen. Targeted arrangement and combination were used to design novel collagen nucleotide sequences. Then, the expression vector pPIC9K of Pichia pastoris was expressed in the host bacterium GS115 of Pichia pastoris via electroporation. After screening with antibiotic G418, the expression of high-copy strains was amplified through fermentation. High-purity recombinant human collagen was obtained by purification by ultrafiltration and ion exchange chromatography. The "nanoflower" coral hydroxyapatite used in the following examples was prepared by soaking coral stone in a 30% propylene glycol solution for 16 hours, crushing and granulating it to obtain a coral sample, and then subjecting the coral sample to hydrothermal exchange. The hydrothermal exchange step included: impregnating the coral sample with a saturated diammonium hydrogen phosphate solution and reacting it at 0.1-3 MPa and 150-220°C for 6-19 hours.

[0101] Example 1

[0102] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0103] Weigh 10g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 20g of phosphate buffer (pH=6.6), mix well, and prepare a 30g recombinant human collagen solution; weigh 70g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 15%, add it to the above recombinant human collagen solution, mix well, and obtain a 100g suspension; add... After the mold with a height of 15 mm was placed in a freezer at -80℃ for 60 min, it was then freeze-dried under vacuum to obtain 80 samples. The samples were then placed in a nitrogen-filled vacuum oven with a vacuum degree of -10 kPa and self-assembled at 100℃ for 6 h. After the self-assembly was completed, the samples were placed in aluminum foil bags, sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the artificial bone material. The porosity of the material was 98.17%.

[0104] The freeze-drying process is as follows:

[0105] During the pre-freezing stage, the temperature reaches -50℃ within 120 minutes and is maintained for 180 minutes.

[0106] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0107] The sublimated product was subjected to analytical drying under a vacuum of 0.1 mbar for 40 min, with the temperature reaching 25 °C and maintained for 240 min.

[0108] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in 'a'; the cross-sectional view is as follows. Figure 6 As shown in b, the "nanoflower" coral hydroxyapatite is visibly and uniformly distributed within the collagen scaffold; the scanning electron microscope image is shown below. Figure 8 As shown in a, b, and c, this indicates that the freeze-dried material possesses a three-dimensional structure, and the "nanoflower"-shaped coral hydroxyapatite is encapsulated by collagen and uniformly distributed within the collagen scaffold. This artificial bone material exhibits rapid rehydration, remains intact and does not easily disintegrate, and expands slightly (e.g., Figure 9 As shown in a), it has good toughness (such as...). Figure 10 (as shown) and hydrophilic properties and shape memory function (such as) Figure 11 (as shown in a) It also has good cell compatibility (e.g., Figure 12 , Figure 13 , Figure 14 , Figure 15 (as shown) and biodegradability (such as) Figure 16 (As shown).

[0109] Experiments were conducted using this artificial bone material to repair defects in the lateral femoral condyle of rabbits. The results showed that the material has a good bone repair effect (e.g., Figure 17 and Figure 18 As shown in experimental group 1), this artificial bone material was also used in a clinical trial to fill extraction sockets. The results showed that the material has a good bone repair effect (e.g., Figure 19 (As shown).

[0110] Example 2

[0111] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0112] Weigh 20g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 25g of phosphate buffer (pH=6.6), mix well, and prepare a 45g recombinant human collagen solution; weigh 55g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 20%, add it to the above recombinant human collagen solution, mix well, and obtain a 100g suspension; add... After forming a mold with a height of 15 mm, the sample was quick-frozen at -80℃ for 100 min, followed by vacuum freeze-drying to obtain 80 block samples. These samples were then self-assembled at 150℃ for 8 h in an electric vacuum drying oven with a vacuum level of -0.095 MPa. The block samples were then pulverized using a pulverizer (JC-FW-100) and sieved through a 10-65 mesh screen. The sieved samples were then placed in vials, sealed in aluminum foil bags, and sterilized by electron beam irradiation at a dose of 25 kGy to obtain the artificial bone material, which has a porosity of 98.62%.

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

[0114] During the pre-freezing stage, the temperature reaches -45℃ within 100 minutes and is maintained for 200 minutes;

[0115] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0116] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar, with the temperature reaching 27°C within 50 min and maintained for 250 min.

[0117] The appearance of the artificial bone material (granular) is shown in the image below. Figure 7 As shown, ae represent samples with particle size distributions of <0.25mm, 0.25-0.3mm, 0.3-0.5mm, 0.5-1mm, and 1-2mm, respectively.

[0118] Example 3

[0119] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0120] Weigh 15g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 45g of water for injection, suspend evenly, and prepare a 60g recombinant human collagen solution; weigh 40g of "nanoflower" coral hydroxyapatite with a particle size range of 0.5mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 22%, add it to the above recombinant human collagen solution, suspend evenly, and obtain a 100g suspension; add... After forming a mold with a height of 15mm, the sample was quick-frozen at -80℃ for 120 minutes, followed by vacuum freeze-drying to obtain 80 block samples. These samples were then self-assembled at 170℃ for 7 hours in an electrically heated vacuum drying oven with a vacuum level of -0.095MPa. The self-assembled samples were then sealed in double-layer blister packs and sterilized with ethylene oxide at the following parameters: sterilization temperature 55℃, sterilization humidity 50%, and ethylene oxide concentration 630g / m³. 3 The artificial bone material was obtained by sterilizing at a vacuum degree of -15 kPa for 10 hours, and the porosity of the material was 96.58%.

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

[0122] During the pre-freezing stage, the temperature reaches -50℃ within 140 minutes and is maintained for 190 minutes.

[0123] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0124] The sublimated product was subjected to analytical drying under a vacuum of 0.06 mbar, with the temperature reaching 20°C within 30 minutes and maintained for 250 minutes.

[0125] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in c; a clinical trial was conducted using this artificial bone material to fill extraction sockets, and the results showed that the material has a good bone repair effect (e.g., Figure 20 (As shown).

[0126] Example 4

[0127] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0128] Weigh 10g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 40g of water for injection, and suspend evenly to prepare 50g of recombinant human collagen solution; weigh 50g of "nanoflower" coral hydroxyapatite with a particle size range of 0.5mm-1.25mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 18%, and add it to the above recombinant human collagen solution and suspend evenly to obtain 100g of suspension; add... After the mold with a height of 15 mm was placed in a freezer at -80℃ for 180 min, it was then freeze-dried under vacuum to obtain 80 block samples. The samples were then self-assembled at 180℃ for 7 h in a nitrogen-filled vacuum oven with a vacuum degree of -15 kPa. After the self-assembly was completed, the samples were sealed in aluminum foil bags and then sterilized by cobalt-60 irradiation with an irradiation dose of 25 kGy to obtain the artificial bone material. The porosity of the material was 96.23%.

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

[0130] During the pre-freezing stage, the temperature was set to -50°C within 160 minutes and maintained for 220 minutes.

[0131] The pre-frozen product was sublimated under a vacuum of 0.05 mbar, with the temperature reaching -10°C within 50 min and maintained for 450 min.

[0132] The sublimated product was subjected to analytical drying under a vacuum of 0.07 mbar, with the temperature reaching 30°C within 40 min and maintained for 220 min.

[0133] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in d.

[0134] Example 5

[0135] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0136] Weigh 10g of recombinant human collagen lyophilized powder (recombinant human collagen is the protein with the sequence listed in SEQ ID NO: 10g). A protein with the amino acid sequence IDNo:1 was added to 40g of purified water and mixed evenly to prepare a 50g recombinant human collagen solution. 50g of "nanoflower" coral hydroxyapatite with a particle size range of 0.5mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 15% was added to the above recombinant human collagen solution and mixed evenly to obtain a 100g suspension. This suspension was then placed in a 1cm×1cm×1cm mold and quick-frozen at -80℃ for 180min, followed by vacuum freeze-drying to obtain 80 block samples. These samples were then self-assembled at 180℃ for 6h in an electric vacuum drying oven with a vacuum level of -0.095MPa. After self-assembly, the samples were sealed in aluminum foil bags and sterilized by electron beam irradiation at a dose of 25kGy to obtain the artificial bone material, which has a porosity of 96.06%.

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

[0138] During the pre-freezing stage, the temperature reaches -50℃ within 200 minutes and is maintained for 200 minutes.

[0139] The pre-frozen product was sublimated under a vacuum of 0.1 mbar, with the temperature reaching -10°C within 60 min and maintained for 500 min.

[0140] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar for 50 min, with the temperature reaching 25 °C and maintained for 200 min.

[0141] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in 'e', ​​this artificial bone material remains intact and does not easily disintegrate after rehydration (e.g., Figure 9 As shown in b), it has good toughness, hydrophilicity, and shape memory function (e.g., Figure 11 (as shown in b).

[0142] Example 6

[0143] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0144] Weigh 12g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 40g of purified water, and suspend evenly to prepare a 52g recombinant human collagen solution; weigh 48g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 15%, and add it to the above recombinant human collagen solution, suspend evenly to obtain a 100g suspension; add... The 5mm high mold was placed in a -80℃ freezer for 180 minutes and then vacuum freeze-dried to obtain 26 sheet samples. The samples were then self-assembled at 220℃ for 6 hours in an electric vacuum drying oven with a vacuum degree of -0.095MPa. After self-assembly, the samples were sealed in aluminum foil bags and then sterilized by electron beam irradiation with an irradiation dose of 25kGy to obtain the artificial bone material with a porosity of 97.79%.

[0145] The freeze-drying process is as follows:

[0146] During the pre-freezing stage, the temperature reaches -50℃ within 200 minutes and is maintained for 200 minutes.

[0147] The pre-frozen product was sublimated under a vacuum of 0.1 mbar, with the temperature reaching -10°C within 60 min and maintained for 500 min.

[0148] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar for 50 min, with the temperature reaching 25 °C and maintained for 200 min.

[0149] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in f in the figure.

[0150] Example 7

[0151] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0152] Three 15g portions of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing) were weighed separately, and 40g of purified water was added. The mixture was then thoroughly mixed to prepare three 55g portions of recombinant human collagen solution. Three 45g portions of "nanoflower" coral hydroxyapatite with three conversion rates (10%, 55%, and 80%) were weighed separately. These particles had a particle size range of 0.5mm-1mm, a pore size of 100μm-800μm, and a porosity of 50%-80%. These were added to the above recombinant human collagen solution and thoroughly mixed to obtain three 100g portions of suspension. These suspensions were then added to… After the mold with a height of 15 mm is placed in a freezer at -80℃ for 200 min, it is then freeze-dried under vacuum. The dried sample is placed in an electric vacuum drying oven with a vacuum degree of -0.095 MPa at 180℃ for 6 h to allow it to self-assemble. The self-assembled sample is then placed in an aluminum foil bag, sealed, and sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the artificial bone material.

[0153] The freeze-drying process is as follows:

[0154] During the pre-freezing stage, the temperature reaches -50℃ within 200 minutes and is maintained for 200 minutes.

[0155] The pre-frozen product was sublimated under a vacuum of 0.1 mbar, with the temperature reaching -10°C within 60 min and maintained for 500 min.

[0156] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar for 50 min, with the temperature reaching 25 °C and maintained for 200 min.

[0157] The pore structure of the artificial bone material is shown in Table 2 below. As can be seen from the table, the higher the conversion rate of the "nanoflower" coral hydroxyapatite, the larger its pore size and the greater its porosity. Therefore, the artificial bone material prepared in this way also has a greater porosity.

[0158] Table 2

[0159]

[0160] Example 8

[0161] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0162] Weigh 5g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No: 1 in the sequence listing), add 20g of phosphate buffer (pH=6.6), mix well, and prepare 25g of recombinant human collagen solution; weigh 5g of cross-linked porous starch and add it to the prepared recombinant human collagen solution and mix well; then weigh 70g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 15%, add it to the above mixed solution and mix well to obtain 100g of viscous solution; add... After the mold with a height of 15 mm was placed in a freezer at -80℃ for 200 min, it was then freeze-dried under vacuum to obtain 80 samples. The samples were then placed in a nitrogen-filled vacuum oven with a vacuum degree of -10 kPa and self-assembled at 100℃ for 6 h. After the self-assembly was completed, the samples were placed in aluminum foil bags, sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the artificial bone material. The porosity of the material was 97.43%.

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

[0164] During the pre-freezing stage, the temperature reaches -50℃ within 120 minutes and is maintained for 180 minutes.

[0165] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0166] The sublimated product was subjected to analytical drying under a vacuum of 0.1 mbar for 40 min, with the temperature reaching 25 °C and maintained for 240 min.

[0167] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in g in the figure.

[0168] Example 9

[0169] This embodiment provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped coral hydroxyapatite, which is prepared by the following steps:

[0170] Weigh 5g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No: 1 in the sequence listing), add 20g of phosphate buffer (pH=6.6), mix well, and prepare 25g of recombinant human collagen solution; weigh 5g of sodium carboxymethyl cellulose and add it to the prepared recombinant human collagen solution and mix well; then weigh 70g of "nanoflower" coral hydroxyapatite with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 10%, add it to the above mixed solution and mix well to obtain 100g of viscous solution; add... After the mold with a height of 15 mm was placed in a freezer at -80℃ for 200 min, it was then freeze-dried under vacuum to obtain 80 samples. The samples were then placed in a nitrogen-filled vacuum oven with a vacuum degree of -10 kPa and self-assembled at 100℃ for 6 h. After the self-assembly was completed, the samples were placed in aluminum foil bags, sealed, and then sterilized by electron beam irradiation with an irradiation dose of 25 kGy to obtain the artificial bone material. The porosity of the material was 98.83%.

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

[0172] During the pre-freezing stage, the temperature reaches -50℃ within 120 minutes and is maintained for 180 minutes.

[0173] The pre-frozen product was sublimated under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0174] The sublimated product was subjected to analytical drying under a vacuum of 0.1 mbar for 40 min, with the temperature reaching 25 °C and maintained for 240 min.

[0175] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in h.

[0176] Comparative Example 1

[0177] This comparative example provides an artificial bone material prepared by the following steps:

[0178] Weigh 12g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 40g of purified water, suspend evenly, and prepare a 52g recombinant human collagen solution; weigh 48g of nano-hydroxyapatite, a nanoscale material with no pore structure (manufacturer: Zhejiang Aipuri Nanomaterials Co., Ltd., particle size: 20nm, purity: 99%), add it to the above recombinant human collagen solution, suspend evenly, and obtain a 100g suspension; add... The 15mm high mold was placed in a -80℃ freezer for 180 minutes and then vacuum freeze-dried to obtain 80 block samples. The samples were then self-assembled at 180℃ for 6 hours in an electric vacuum drying oven with a vacuum degree of -0.095MPa to obtain the artificial bone material.

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

[0180] During the pre-freezing stage, the temperature reaches -50℃ within 200 minutes and is maintained for 200 minutes.

[0181] The pre-frozen product was sublimated under a vacuum of 0.1 mbar, with the temperature reaching -10°C within 60 min and maintained for 500 min.

[0182] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar for 50 min, with the temperature reaching 25 °C and maintained for 200 min.

[0183] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in i in the figure, the scanning electron microscope image is as follows. Figure 8 As shown in 'd', this indicates that after freeze-drying, nano-hydroxyapatite is encapsulated by collagen, forming a relatively dense three-dimensional structure. This artificial bone material was used in experiments to repair bone defects in the lateral femoral condyle of rabbits (e.g., Figure 18 As shown in Experiment 2), the results indicate that the bone repair effect of this material is worse than that of the blank control, but not as good as that of Example 1. The porous structure of the "nanoflower" hydroxyapatite of the present invention is closer to that of human cancellous bone. The three-dimensional spatial channels formed by its porous structure increase the interface between the material and the recipient tissue, which is conducive to accelerating the reaction process of interface bonding and providing space for bone-inducing substances in vivo. Secondly, the interconnected pores facilitate nutrient transfer and the exchange of fibrovascular tissue, which is more conducive to guiding the ingrowth of new bone. At the same time, the "nanoflower" hydroxyapatite has a suitable conversion rate and can be degraded in vivo along with bone repair, while nano hydroxyapatite degrades more slowly and requires a longer time to be completely absorbed and replaced by the body.

[0184] Comparative Example 2

[0185] This comparative example provides an artificial bone material prepared by the following steps:

[0186] Weigh 1g of collagen sponge (generic name: medical collagen sponge, trade name: Kejibang, manufacturer: Wuxi Bedy Biotechnology Co., Ltd.), cut it into small pieces, disperse the collagen sponge in 50ml of purified water, and then homogenize it using a homogenizer for 10 minutes to obtain collagen slurry; mix 8g of "nanoflower" coral hydroxyapatite particles with a particle size range of 0.25mm-1mm, a pore size of 100μm-800μm, a porosity of 50%-80%, and a conversion rate of 15% with the collagen slurry, and then use... A magnetic stirrer was used to mix the collagen, coral, and hydroxyapatite mixture for 20 minutes to obtain a collagen, coral, and hydroxyapatite slurry. The slurry was then transferred to a mold and compressed to remove water for 12 hours. After demolding, it was freeze-dried to obtain a shaped collagen, coral, and hydroxyapatite scaffold. The scaffold was then placed in an electric vacuum drying oven and heated to 60-180°C under a vacuum of -0.095 MPa for 2 hours for thermal cross-linking treatment to obtain the final collagen, coral, and hydroxyapatite composite scaffold with a porosity of 88.05%.

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

[0188] During the pre-freezing stage, the temperature reaches -50℃ within 200 minutes and is maintained for 200 minutes.

[0189] The pre-frozen product was sublimated under a vacuum of 0.1 mbar, with the temperature reaching -10°C within 60 min and maintained for 500 min.

[0190] The sublimated product was subjected to analytical drying under a vacuum of 0.08 mbar for 50 min, with the temperature reaching 25 °C and maintained for 200 min.

[0191] The appearance of the collagen-coral-hydroxyapatite composite scaffold is shown in the image below. Figure 6 As shown in j in the figure, the rehydration experiment is as follows: Figure 11 As shown in 'c', this indicates that the collagen-coral-hydroxyapatite composite scaffold is easily dispersed after rehydration and is fragile when pressed by hand. The strength and toughness of the sample are far inferior to those of the example, and the product of the example is more in line with clinical requirements.

[0192] Comparative Example 3

[0193] This comparative example provides an artificial bone material prepared by the following steps:

[0194] Weigh 10g of recombinant human collagen lyophilized powder (raw material from other companies on the market, white or off-white spongy solid, purity 99.9%), add 20g of phosphate buffer (pH=6.6), mix well to prepare 30g of recombinant human collagen solution; weigh 70g of "nanoflower" hydroxyphosphorylated coral stone with a particle size range of 0.25mm-1mm, pore size of 200μm-800μm, porosity of 50%-70%, and conversion rate of 15%, add to the above recombinant human collagen solution and mix well to obtain 100g of suspension; add... After the mold with a height of 15 mm was placed in a -80℃ freezer for 60 min, it was then vacuum freeze-dried to obtain 80 samples. The samples were then placed in a nitrogen-filled vacuum oven with a vacuum degree of -10 kPa and treated at 100℃ for 6 h to obtain the artificial bone material, which has a porosity of 78.55%.

[0195] The freeze-drying process is as follows:

[0196] (1) During the pre-freezing stage, the temperature reaches -50℃ within 120 minutes and is maintained for 180 minutes;

[0197] (2) Sublimate the pre-frozen product under a vacuum of 0.01 mbar, with the temperature reaching -10°C within 60 min and maintained for 600 min.

[0198] (3) The sublimated product was desorbed and dried. The vacuum degree was set to 0.1 mbar, the temperature reached 25℃ within 40 min, and was maintained for 240 min.

[0199] The appearance of the artificial bone material is shown in the image below. Figure 6 As shown in k, the appearance is slightly yellow, and the rehydration test is as follows. Figure 11 As shown in d, this indicates that the artificial bone material slightly dissolves after rehydration, and the particles are easily dispersed. The strength and toughness of the sample are far inferior to those of the example, and the product of the example is more in line with clinical use requirements.

[0200] Comparative Example 4

[0201] This comparative example provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped hydroxyphosphorylated coral stone, which is prepared by the following steps:

[0202] Weigh 6g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No:1 in the sequence listing), add 14g of purified water, suspend evenly, and prepare a 20g recombinant human collagen solution; weigh 80g of "nanoflower" hydroxyphosphorylated coral stone with a particle size range of 0.25mm-1mm, a pore size of 200μm-800μm, a porosity of 50%-70%, and a conversion rate of 15%, add it to the above recombinant human collagen solution, suspend evenly, and obtain a 100g suspension; add... The mold with a height of 15mm was placed in a -80℃ freezer for 180min and then vacuum freeze-dried to obtain 80 samples. The samples were then self-assembled at 160℃ for 6h in an electric vacuum drying oven with a vacuum degree of -0.095MPa to obtain the artificial bone material with a porosity of 71.52%.

[0203] The freeze-drying process is as follows:

[0204] (1) During the pre-freezing stage, the temperature reaches -50℃ within 200 minutes and is maintained for 200 minutes;

[0205] (2) Sublimate the pre-frozen product under a vacuum of 0.1 mbar, with the temperature reaching -10°C within 60 min and maintained for 500 min.

[0206] (3) The sublimated product was desorbed and dried. The vacuum degree was set to 0.08 mbar, the temperature reached 25℃ within 50 min, and was maintained for 200 min.

[0207] The artificial bone material contains 93% (dry weight) of nano-flower-shaped hydroxyphosphorylated coral stone. The sample appearance is shown in the image below. Figure 6 As shown in the image (l), the appearance is slightly yellow, and the rehydration test is as follows: Figure 11 As shown in 'e', ​​this indicates that the artificial bone material is relatively hard, slow to rehydrate, and the particles are easily dispersed after rehydration. The sample lacks toughness and is far inferior to the product of the example. The product of the example is more in line with the requirements for clinical use.

[0208] Comparative Example 5

[0209] This comparative example provides an artificial bone material containing self-assembled collagen and "nanoflower"-shaped hydroxyphosphorylated coral stone, which is prepared by the following steps:

[0210] Weigh 10g of recombinant human collagen lyophilized powder (recombinant human collagen is a protein with the amino acid sequence SEQ ID No: 1 in the sequence listing), add 20g of phosphate buffer (pH=6.6), mix well, and prepare a 30g recombinant human collagen solution; weigh 70g of "nanoflower" hydroxyphosphorylated coral stone with a particle size range of 0.25mm-1mm, a pore size of 200μm-800μm, a porosity of 50%-70%, and a conversion rate of 15%, add it to the above recombinant human collagen solution, mix well, and obtain a 100g suspension; add... After the mold with a height of 15 mm was placed in a -80℃ freezer for 60 min, it was then vacuum freeze-dried to obtain 80 samples. The samples were then placed in a nitrogen-filled vacuum oven with a vacuum degree of -10 kPa and self-assembled at 100℃ for 6 h to obtain the artificial bone material. The porosity of the material was 78.18%.

[0211] The freeze-drying process is as follows:

[0212] (1) During the pre-freezing stage, the temperature reaches -50℃ within 60 minutes and is maintained for 180 minutes;

[0213] (2) Sublimate the pre-frozen product under a vacuum of 0.01 mbar for 300 min, and maintain the temperature at -10°C for 600 min.

[0214] (3) The sublimated product was desorbed and dried. The vacuum degree was set to 0.1 mbar, the temperature reached 25℃ within 40 min, and was maintained for 240 min.

[0215] This comparative example primarily slows down the heating rate in the freeze-drying process. The appearance of the artificial bone material sample is shown in the image below. Figure 6 As shown in m, a slow heating rate will lead to uneven temperature distribution inside the product, causing structural collapse and reduced porosity. The rehydration experiment is as follows: Figure 11 As shown in f, this indicates that the artificial bone material rehydrates slowly and the particles easily disperse after rehydration. The sample lacks toughness and is far inferior to the product of the example. The product of the example is more in line with clinical requirements.

[0216] The relevant performance tests were conducted using the product obtained in Example 1 as an example, as detailed below:

[0217] Appearance: When observed with the naked eye under fluorescent light, it appears yellow or pale yellow, and the color is uniform. Figure 6 As shown in a and b in the figure.

[0218] Compression deformation determination: The test was conducted according to ASTM F1566-15, "Standard Test Method: Compression Test of Medical Sponge". After rehydration, the material was placed on the compression fixture platform of an electronic universal testing machine. The test speed was set to 10 mm / min to simulate the force rate of the material in actual application. The electronic universal testing machine was started to begin the compression test. The test process was monitored in real time, and the deformation during compression was recorded. The results are as follows: Figure 10 As shown, this indicates that the cancellous bone material has good toughness and mechanical strength, and will not be easily crushed or crumbled.

[0219] Swelling determination: The actual size V0 of each group of samples was measured using vernier calipers, and then their initial weight (w0) was measured and recorded. After immersing the samples in distilled water for 30 seconds, the surface moisture was absorbed with filter paper, and the samples were weighed (w). The size V after water absorption and swelling was measured using vernier calipers. Swelling rate (%) = ((w-wo) / wo) × 100%, and the volume ratio before and after swelling = V / V0. The results are shown in Table 3, indicating that the material has good water absorption and the swelling volume is within a certain range, and it will not compress surrounding tissues during clinical use.

[0220] Table 3. Results of swelling test

[0221] Swelling rate / % (n = 10) 48.22±6.33 Volume ratio before and after swelling (n=10) 1.48±0.06

[0222] Cytotoxicity assay: The artificial bone material obtained in Example 1 was subjected to cytotoxicity test according to GB / T 16886.5-2017 Biological evaluation of medical devices Part 5: In vitro cytotoxicity test MTT method (two parallel experiments were conducted, and sample 1 and sample 2 were both products obtained in Example 1). The specific operation was as follows: the artificial bone material was first thoroughly swollen with high glucose DMEM culture medium, and then extracted at a ratio of 0.1 g / ml. The extraction temperature was 37℃ and the extraction time was 72 h. L929 cells in the logarithmic growth phase were seeded into 96-well plates at 1.2 × 105 cells / mL, 100 μL / well, and cultured for 24 h. When the cell seeding rate in the 96-well plate reached 40%–60%, the following were administered: ① Sample group: 100 μL of culture medium containing different concentrations of the original extract (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 / zeroing group (BC): No cells were added, only 100 μL of culture medium was added; ④ Blank control group (SC): Cells were added, only 100 μL of culture medium was added; Cultured for 24 h. The medium was discarded and replaced with 150 μL / well of culture medium containing MTT (0.5 mg / mL), and cultured for another 4 h. The medium was discarded again, and 150 μL / well of DMSO was added. After thorough shaking and color development, the OD was measured using a microplate reader. 490 Calculate the relative cell viability according to formula (1):

[0223]

[0224] Criteria for determining cytotoxicity: If the relative cell viability is greater than 70%, it is considered that there is no cytotoxic reaction; otherwise, it is considered that there is potential cytotoxicity.

[0225] The experimental results are shown in Table 4 and Figure 12 As shown.

[0226] Table 4. Cytotoxicity test results of artificial bone material-L929

[0227] Grouping SC 3.13％ 6.25％ 12.50％ 25.00％ 50％ 100％ PC mean 0.955 1.189 1.099 1.07 1.038 1.012 0.856 0.452 Relative survival rate % 100.00％ 124.51％ 115.12％ 112.05％ 108.73％ 106.01％ 89.70％ 47.35％ SD 0.07 0.03 0.03 0.02 0.01 0.02 0.04 0.08 P / 0.01 0.03 0.05 0.11 0.24 0.11 0

[0228] As shown in Table 4, the artificial bone material of the present invention has good biocompatibility and no cytotoxicity.

[0229] Cell migration: Extraction: Add 4 grams of artificial bone material to 20 ml of physiological saline and extract at 121°C for 1 hour in a pressure steam sterilizer.

[0230] Coating: Add 2 mL of the sample extract to a 6-well plate, incubate at 37°C in a 5% CO2 incubator for 2 h, discard the excess extract in the wells, add 2 mL of 1% BSA-PBS solution, incubate at 37°C in a 5% CO2 incubator for 1 h, discard the liquid in the wells, wash 3 times with PBS, discard the liquid in the wells, seal with sealing film and store at 4°C for later use.

[0231] Inoculation: Inoculate cells into 6-well plates at a seeding density of 1.4E5 cells / well and incubate overnight (24h) in an incubator (37°C, 5% CO2).

[0232] Scratching: According to the experimental design, cells were divided into groups, with 3 replicates per group. Cells were incubated in an incubator (37℃, 5% CO2) for 24 hours. When the cell deposition rate in the 6-well plates reached over 90%, scratching was performed. Two vertical scratches were made in the 6-well plate using a 10µL pipette tip, with the vertical scratch serving as the baseline (the pipette tip perpendicular to the edge of the ruler). The spacing between the scratches was maintained at 2cm. After making the vertical scratches, horizontal scratches were made near the centerline of the 6-well plate, perpendicular to the baseline. When scratching, the pressure should be as even as possible to ensure a consistent scratch width.

[0233] Use a 10μL pipette tip to make two vertical lines in the six-well plate as a baseline (the pipette tip is perpendicular to the edge of the ruler), with the distance between the lines kept at 2cm.

[0234] After making the vertical marks, make horizontal marks near the center axis of the six-hole plate, perpendicular to the baseline. When making the marks with the gun tip, try to apply even pressure and keep the width of the marks consistent.

[0235] Washing: After scratching, gently wash the cells with 1 mL of PBS solution in each well, repeating 3 times to remove cells detached due to the scratching. After washing, add 2 mL of culture medium (serum-free) to each well and incubate at 37°C in a 5% CO2 incubator.

[0236] Photography: Photos were taken under a 4x microscope 0 h after the scratch; 24 h after the scratch, the area was washed once with PBS and photographed under a 4x microscope; if no significant migration was observed, photos were taken under a 4x microscope 48 h after the scratch, after washing once with PBS. The scratch area between the two baselines and the two intersections of the transverse scratch was used as the observation area, observed from left to right. Nine typical areas were photographed at 0 h, and nine consecutive photos were taken at 24 h and 48 h (exfoliated cells were washed away with PBS before photographing at 24 h and 48 h). Experimental results are as follows: Figure 13 As shown, this artificial bone material has a significant effect on promoting osteoblast migration.

[0237] Cell proliferation: Cell culture: Digest and collect cells, count them with a cell counting chamber, and adjust the cell seeding density to 10,000 cells / mL.

[0238] Adding cell suspension: After immersing the artificial bone material in ordinary culture medium for 2 hours, discard the culture medium and slowly add 200 μL of cell suspension from the top of the sterile sample (two parallel experiments were performed; sample 1 (2#-1) and sample 2 (2#-2) were both products obtained in Example 1) until the cell suspension was completely absorbed into the sample. Place the culture dish containing the sample into a cell culture incubator.

[0239] Incubation: After 6 hours, slowly add a small amount of culture medium around the sample until it is completely submerged. After 16 hours, slowly add 1-2 ml of culture medium into the culture dish. Incubate for 1 day, 3 days, 5 days, 7 days, and 9 days respectively (change the culture medium every other day).

[0240] Detection: After culture, remove the culture medium, wash 2-3 times with PBS, digest cells with 0.25% trypsin, and count the cells. The average value of the results is taken. Experimental results are as follows: Figure 14 As shown, this artificial bone material has a significant effect on promoting osteoblast proliferation.

[0241] Cell adhesion: Cell culture: Use 2-3 passages of MC3T3-E1 cells. When the cell confluence reaches 80%, digest and collect the cells. After counting with a cell counting chamber, adjust the cell seeding density to 2 × 10⁻⁶. 7 per mL.

[0242] Add cell suspension: After immersing the artificial bone material in ordinary culture medium for 2 hours, discard the culture medium and add 500 μL of cell suspension (4 × 10⁻⁶ cells / mL). 6Add the cells slowly in three portions from the center of the sterile sample until the cell suspension is completely absorbed into the sample gaps. During the operation, care should be taken to avoid the cell suspension falling into the well plate.

[0243] Incubation and culture: After 6 hours, slowly add a small amount of culture medium around the sample until it covers the sample. After 16 hours, slowly add 1-2 ml of culture medium into the culture dish and incubate for 2 hours and 4 hours respectively.

[0244] Detection: After culture, two osteoblast / artificial bone material composite culture specimens were randomly selected, along with two uninoculated blank artificial bone material specimens. The culture medium was removed, and the specimens were gently washed 2-3 times with PBS. They were then fixed with 3% glutaraldehyde for 30 minutes, followed by washing 2-3 times with PBS. The specimens were then dehydrated stepwise with ethanol at concentrations of 30%, 50%, 70%, 90%, and 100% (each concentration for 2 minutes of soaking and washing), vacuum dried, sputter-coated with gold, and the cell adhesion morphology on the material surface was observed using a scanning electron microscope at different time points. Experimental results are as follows: Figure 15 As shown, osteoblasts can adhere to this artificial bone material and form a lamellar structure, which is beneficial to the bone repair process.

[0245] In vitro degradation assay: The sample was cut into four uniform pieces. Then, the four pieces of sample were mixed with prepared, filtered, sterilized 0.01 mol PBS buffer (pH = 7.4) according to m... 本产品 V PBS Add 1 g of PBS to a sterile centrifuge tube at a ratio of 1 g / 200 ml. Gently agitate to ensure the material is fully in contact with the PBS solution. Simulate degradation in a 37°C incubator or water bath. Collect residual samples at 1, 2, 4, 9, 14, 17, 20, and 30 days, and calculate the degradation rate using the constant weight method. Results are as follows: Figure 16 As shown, this indicates that the cancellous bone material has good degradation properties.

[0246] In summary, the above are merely preferred embodiments of the present invention and are not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An artificial bone material, wherein, Based on the total dry matter content of the artificial bone material as 100%, its material composition includes: Coral hydroxyapatite particles 70%-90%, Recombinant human collagen 5%-30%, and Excipients 0-7%; The artificial bone material is a solid porous material formed by coral hydroxyapatite particles being bonded together by recombinant human collagen. The porosity of the artificial bone material is 80%-99%; The recombinant human collagen has the amino acid sequence shown in SEQ ID No:

1.

2. The artificial bone material according to claim 1, wherein, 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%.

3. The artificial bone material according to claim 1, wherein, The conversion rate of the coral hydroxyapatite particles is 5%-80%.

4. The artificial bone material according to claim 1, wherein, The conversion rate of the coral hydroxyapatite particles is 5%-30%.

5. The artificial bone material according to claim 1, wherein, The excipients include one or more of cross-linked porous starch, sodium carboxymethyl cellulose, chitosan, carboxymethyl chitosan, and hydroxypropyl methyl cellulose.

6. The artificial bone material according to claim 1, wherein, The coral hydroxyapatite particles are prepared by soaking coral stone in a cutting protective agent, crushing and granulating it, and then subjecting it to hydrothermal exchange. The cutting protectant is a solution containing polyols.

7. The artificial bone material according to claim 6, wherein, The polyol is selected from one or more of glycerol, ethylene glycol, sorbitol, and butylene glycol.

8. The artificial bone material according to claim 6, wherein, The mass fraction of the polyol is ≥20% based on the total mass of the cutting protectant.

9. The artificial bone material according to claim 6, wherein, The soaking time is ≥3 h.

10. The artificial bone material according to claim 6, wherein, The raw materials for the coral stone include natural coral and / or cultured coral.

11. The artificial bone material according to claim 10, wherein, The natural corals include shore corals and / or horn-hole corals.

12. The artificial bone material according to claim 11, wherein, The natural coral in question is a shore coral.

13. The artificial bone material according to claim 6, wherein, The hydrothermal exchange steps include: impregnation with a saturated diammonium hydrogen phosphate solution, and reaction at 0.1-3 MPa and 150-220°C for 6-19 h.

14. The artificial bone material according to claim 1, wherein it is in granular, block, flake, or powder form.

15. A method for preparing an artificial bone material according to any one of claims 1-14, wherein, The preparation method includes: Coral hydroxyapatite particles were mixed with recombinant human collagen solution and then freeze-dried to obtain freeze-dried samples. The artificial bone material is obtained by self-assembling and sterilizing the freeze-dried sample.

16. The preparation method according to claim 15, wherein, The recombinant human collagen solution contains 15%-50% recombinant human collagen by mass.

17. The preparation method according to claim 15, wherein, The solvent for the recombinant human collagen solution includes one or more of phosphate buffer, purified water, or water for injection.

18. The preparation method according to claim 15, wherein, The freeze-drying step includes: (1) Quick freezing: The process parameters are to reach a temperature of -80℃ to -60℃ within 30-240 min; (2) Pre-freezing: The process parameters are to reach -50℃ to -30℃ within 60-240 min and continue for 120-600 min; (3) Sublimation: Sublimate the pre-frozen product under a vacuum of 0.01-0.1 mbar, with the temperature reaching -50℃ to -5℃ within 10-60 min and continuing for 120-600 min. (4) Desorption and drying: The sublimated product is desorbed and dried. The vacuum degree is set to 0.01-0.1 mbar, the temperature reaches 0-40℃ within 10-60 min, and it is maintained for 120-600 min.

19. The preparation method according to claim 15, wherein, The self-assembly steps include: The freeze-dried sample was placed in a vacuum environment of -0.1 to -0.01 MPa or in a nitrogen atmosphere and heated to 100-300°C, and then held for 0.5-8 h.

20. The preparation method according to claim 19, wherein, The temperature for the heating is 100-220℃.

21. The preparation method according to claim 19, wherein, The self-assembly is carried out using an electric heating vacuum drying oven or a precision vacuum nitrogen-filled integrated drying oven.

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