Recombinant collagen biomimetic artificial bone and preparation method and application thereof

Abstract

The present application relates to a kind of recombinant collagen biomimetic artificial bone and its preparation method and application, by crosslinking modification to recombinant collagen, increase its molecular weight and viscosity, make it form fiber network structure, then through soluble salt calcium chloride and sodium dihydrogen phosphate simulate the calcium-phosphorus ratio of hydroxyapatite, instead of insoluble hydroxyapatite, mineralization is carried out to recombinant collagen, the utilization rate of collagen is improved, the obtained biomimetic artificial bone can play better osteogenesis effect.The biomimetic artificial bone obtained in the present application has high biological safety, has high biomimetic characteristics, good biocompatibility and mechanical properties matched with human skeleton, can induce bone marrow mesenchymal stem cell osteogenic differentiation, has great potential in the field of bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a recombinant collagen biomimetic artificial bone, its preparation method, and its application. Background Technology

[0002] Bone defects are common orthopedic diseases caused by fractures, osteogenesis imperfecta, or osteoporosis. According to statistics, bone tissue is the second most needed tissue for transplantation worldwide, with approximately four million people requiring bone grafts or bone substitutes annually to treat bone defects. Bone diseases now account for half of chronic diseases in people over 50 years of age, making their treatment a significant clinical challenge. Autologous bone transplantation is an ideal treatment method, possessing osteoconduction and osteoinduction properties; however, it carries high risks, has limited bone donors, and is prone to severe pain and complications at the donor site post-surgery. Therefore, establishing effective bone regeneration treatment methods for precise treatment of bone diseases has significant clinical application value.

[0003] Bones are composed of a mesh-like framework of collagen fibers and nano-hydroxyapatite uniformly attached to the framework. The bone structure formed by the collagen and hydroxyapatite composite exhibits excellent microstructure and mechanical properties. Hydroxyapatite, as the main inorganic component of human bones, possesses good bioactivity and osteoconductivity, enabling it to form strong osseointegration with human bone tissue. Hydroxyapatite can be prepared by chemical deposition of calcium chloride and phosphate ions; this method is simple, safe, and produces no toxic substances. Bioceramic implants, represented by hydroxyapatite, exhibit good bioactivity and osseointegration capabilities. However, pure hydroxyapatite materials differ significantly from human bone in mechanical properties and microstructure, exhibiting lower mechanical strength, greater brittleness, and a degradation rate mismatch with autologous bone. Collagen is the main organic component of human bones; currently, most commercially available products are composites of animal-derived collagen and hydroxyapatite used for filling bone defects. Animal collagen is mainly derived from animal tissues such as pig skin, pig hooves, and bovine Achilles tendons. It carries risks of viral contamination, has low purity during preparation, and may lead to immune reactions and potential infectious diseases. Furthermore, animal collagen can only fully dissolve in acidic solutions to achieve its complexation effect with hydroxyapatite, which could significantly impact the protein activity and subsequent applications of the composite material.

[0004] Recombinant type I and type III humanized collagen are collagen proteins prepared using DNA recombination technology and microbial fermentation. Their amino acid sequences can be designed and improved according to needs. The gene sequence of recombinant humanized collagen is highly consistent with the human gene sequence, exhibiting good tissue compatibility. Literature (10.16190 / j.cnki.45-1211 / r.2018.03.011) reports the use of recombinant humanized collagen in the healing of deep burn wounds. Recombinant humanized collagen can reconnect broken collagen fibers caused by burns, synthesizing new type III humanized collagen, promoting wound healing, and reducing scar hyperplasia. This demonstrates that recombinant humanized collagen can be directly absorbed by the human body and participate in collagen construction, aiding in tissue repair and site filling.

[0005] However, there are currently no reports disclosing the specific uses of recombinant type I and type III humanized collagen in the field of bone repair materials. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant collagen biomimetic artificial bone, its preparation method and application. The biomimetic artificial bone provided by the present invention broadens the application of collagen-based bone materials in bone repair, enabling it to be applied to bone defect repair in orthopedics, dentistry and other fields.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows: Technical Topic 1: This invention provides a recombinant collagen biomimetic artificial bone, the preparation method of which is as follows: S1. Cross-linking modification of recombinant collagen: Prepare a recombinant collagen solution, add NHS solution and EDC solution to it to obtain cross-linked recombinant collagen hydrogel, add an equal mass of purified water to it, homogenize it and then freeze-dry it under vacuum to obtain the recombinant collagen freeze-dried product. S2. Preparation of mineralized recombinant collagen: Add water to the freeze-dried product obtained in S1, stir and disperse after full swelling, add calcium chloride solution and sodium dihydrogen phosphate solution, and finally add sodium hydroxide solution to adjust the pH to 8~10. After obtaining a white suspension, let it stand to fully mineralize and obtain a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed into a dialysis bag for dialysis, the precipitate is collected by centrifugation, and the precipitate is placed in a mold for vacuum freeze-drying to obtain recombinant collagen biomimetic artificial bone; The collagen solution includes a recombinant type I humanized collagen solution or a mixed solution of recombinant type I and type III humanized collagen.

[0008] As a further embodiment of the present invention, the collagen in the collagen solution in S1 has a mass fraction of 5% to 15%.

[0009] As a further embodiment of the present invention, the proportion of recombinant type III humanized collagen protein in the recombinant type I and type III humanized collagen mixed solution in S1 does not exceed 1 / 9 of the total collagen protein mass.

[0010] As a further embodiment of the present invention, the concentration of the EDC solution in S1 is 0.5~2M; the concentration of the NHS solution is 0.5~2M; the ratio of the amount of EDC to collagen added is 0.0005~0.002mol:1g; and the molar ratio of EDC to NHS is 1:1~2.

[0011] As a further embodiment of the present invention, the vacuum freeze-drying procedure is as follows: pre-freezing at -30~-25℃ for 4~6 hours, then freeze-drying at -10~0℃ for 25~30 hours, and finally desorption drying at 20~25℃ for 8~10 hours; the vacuum degree during the vacuum freeze-drying process is 300~400μbar.

[0012] As a further embodiment of the present invention, the mass ratio of the freeze-dried product to water in S2 is 0.3~1:100.

[0013] As a further embodiment of the present invention, the concentration of the calcium chloride solution in S2 is 0.05~0.3M; the concentration of the sodium dihydrogen phosphate solution is 0.05~0.2M; the ratio of calcium ions to the lyophilized product obtained in S1 is 0.002~0.005mol:1g; and the molar ratio of phosphate ions to calcium ions is 3:5~6.

[0014] As a further embodiment of the present invention, the dialysis in S3 is as follows: the dialysis bag is placed in deionized water and dialyzed at low temperature at 2~6℃ for 3 days, with the water changed 3 times a day; the molecular weight cutoff of the dialysis bag is 1000~3000Da.

[0015] Technical Topic Two: This invention provides a method for preparing the recombinant collagen biomimetic artificial bone.

[0016] Technical Topic 3: This invention provides an application of the recombinant collagen biomimetic artificial bone in the preparation of bone defect filling and repair products, wherein the bone defects include various closed fractures of the limbs, vertebral bone defects, bone defects from maxillofacial surgery, bone defects from dental surgery, and / or skull defects.

[0017] The beneficial effects of adopting the above technical solution are as follows: 1. This invention provides a scheme for cross-linked modified recombinant collagen, which is prepared by chemical cross-linking recombinant humanized collagen to increase the mechanical strength of the biomimetic bone material and prolong the degradation time. The combined effect of multiple types of recombinant humanized collagen has better tissue support and repair capabilities, and can more significantly promote bone growth.

[0018] 2. This invention uses cross-linked modified recombinant collagen and hydroxyapatite mimicry for uniform co-deposition. The resulting material is similar in composition to natural bone (the ratio of inorganic to organic matter is approximately 65:35), which enhances the osteogenic differentiation capacity of cells and helps regenerate defective bone.

[0019] 3. This invention uses soluble calcium chloride / sodium dihydrogen phosphate instead of insoluble hydroxyapatite to mineralize recombinant collagen, thereby improving the utilization rate of collagen and achieving better osteogenic effects.

[0020] 4. The biomimetic artificial bone material obtained by this invention has good biocompatibility: The amino acid sequences of the two recombinant humanized collagen proteins used in the biomimetic artificial bone material obtained by this invention are consistent with the specific functional regions of the amino acid sequences of human collagen proteins, and the two collagen proteins have excellent cell adhesion effects. When applied to the human body, they avoid immune rejection and allergic reactions. At the same time, the raw materials used in the preparation process are all non-cytotoxic, have no viral risks or immunogenicity, have good biosafety, and can be widely used in the fields of medicine, medical devices and tissue engineering.

[0021] 5. The biomimetic artificial bone material obtained by this invention has highly biomimetic characteristics: The biomimetic artificial bone material obtained by this invention has mechanical properties, porosity and suitable in vitro degradation rate that match human bones, and can induce osteogenic differentiation ability of bone marrow mesenchymal stem cells, which has great potential in the field of bone defect repair materials. Attached Figure Description

[0022] Figure 1 This is a flowchart of the preparation process of Embodiment 1 of the present invention; Figure 2 This is a picture of the lyophilized recombinant collagen biomimetic artificial bone RCAB(I / III) obtained in Example 1 of this invention; Figure 3 This is a SEM image of the recombinant collagen biomimetic artificial bone RCAB(I / III) sample obtained in Example 1 of this invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0024] The recombinant type III humanized collagen of this invention is prepared using the method disclosed in Chinese Invention Patent 202011366731.X, entitled "A recombinant human type III collagen and its preparation method and uses".

[0025] The preparation method of some recombinant type I collagen in this embodiment is as follows: (1) Gene design: Based on the sequence characteristics of human type I collagen, a collagen sequence was designed, the amino acid sequence of which is shown in SEQ ID NO.1, consisting of a tandem repeat of amino acids 671-750 of the α1 chain, repeated 4 times. The N-terminus begins with methionine to reduce the protein degradation rate; this is followed by a His-Tag of 6 histidine residues for easy identification and purification.

[0026] The encoding nucleic acid sequence was reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host *E. coli*. After the above optimization, the corresponding nucleic acid fragment encoding recombinant type I humanized collagen was obtained, and the nucleotide sequence corresponding to the above amino acid sequence is shown in SEQ ID NO.2.

[0027] (2) Gene synthesis: Based on the nucleic acid sequence shown above, Genscript Biotech Inc. synthesized a nucleic acid fragment encoding a recombinant human type I collagen fragment.

[0028] (3) Construction of expression vector pET30a(+)-HC90: The obtained nucleic acid fragment SEQ ID NO.2 was ligated to the pET30a(+) plasmid through the multiple cloning sites NdeI and XhoI to obtain the recombinant plasmid pET30a(+)-HC90.

[0029] (4) Construction of expression strain BL21(DE3) / pET30a(+)-HC90: The expression strain was constructed according to the method described in Molecular Cloning: A Laboratory Manual (3rd Edition) (by J. Sambrook et al.), and the expression strain BL21(DE3) / pET30a(+)-HC90 was obtained.

[0030] (5) Induction of expression of strain BL21(DE3) / pET30a(+)-HC90: Single colonies of the expression strain BL21(DE3) / pET30a(+)-HC90 were cultured overnight at 37°C and 200 rpm in LB broth containing 50 μg / mL Kan to activate the seed culture. These were then inoculated at a rate of 3% into a 5L fermenter containing 3L of CM medium. Fermentation was controlled at 37°C, 30% dissolved oxygen, and pH 7.0. When the OD600 reached 60, IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.5 mM was added to induce expression. The culture was continued for 12 hours, and the cells were collected by centrifugation.

[0031] (6) The product is obtained by separation and purification.

[0032] Example 1 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0033] Example 2 S1. Recombinant collagen cross-linking modification: Dissolve 10g of recombinant type I humanized collagen in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution and stir thoroughly for about 25min to form a hydrogel. Continue to cross-link at 4℃ for 24h to obtain CMRC(I) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I) freeze-dried product.

[0034] S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I) lyophilized product in 100ml of water, allow to swell fully at 4℃ for 24h, stir and disperse, add 17.9ml of 0.1M calcium chloride solution, stir thoroughly for 2h; then add 10.74ml of 0.1M sodium dihydrogen phosphate solution, stir thoroughly for 4h, add 0.1M sodium hydroxide to adjust the pH of the solution to 9; place at 4℃ for 24h to allow CMRC(I) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture; S3. Molding: The precipitate is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 5°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The precipitate is then freeze-dried, molded, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the finished product of recombinant collagen biomimetic artificial bone RCAB(I).

[0035] Example 3 S1. Cross-linking modification of recombinant collagen: Dissolve 9g of recombinant collagen (8g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 9ml of 1M NHS solution, and stir thoroughly. Then add 9ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0036] Example 4 S1. Recombinant collagen cross-linking modification: Dissolve 5g of recombinant collagen (4.5g of recombinant type I humanized collagen + 0.5g of recombinant type III humanized collagen) in 100ml of water, add 5ml of 1M NHS solution, and stir thoroughly. Then add 5ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0037] Example 5 S1. Cross-linking modification of recombinant collagen: 15g of recombinant collagen (13.5g of recombinant type I humanized collagen + 1.5g of recombinant type III humanized collagen) was dissolved in 100ml of water, and 15ml of 1M NHS solution was added and stirred thoroughly. Then, 15ml of 1M EDC solution was added and stirred thoroughly for about 15min to form a hydrogel. The hydrogel was allowed to stand at 4℃ for 24h to cross-link, and the cross-linked recombinant collagen CMRC(I / III) hydrogel was obtained. The hydrogel was washed with water 5 times and centrifuged to remove the washing water. An equal mass of purified water was added to the obtained CMRC(I / III) hydrogel and homogenized in a homogenizer to form a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum of 350μbar and pre-frozen at -25℃ for 5h, then freeze-dried at -5℃ for 30h, and finally desorbed and dried at 25℃ for 10h to obtain the CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0038] Example 6 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 5ml of 1M NHS solution, and stir thoroughly. Then add 5ml of 1M EDC solution and stir thoroughly for about 45min to form a hydrogel. Continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0039] Example 7 S1. Recombinant collagen cross-linking modification: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 20ml of 1M NHS solution, and stir thoroughly. Then add 20ml of 1M EDC solution, stir thoroughly for about 8min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0040] Example 8 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.3g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 10.74ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 6.44ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0041] Example 9 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 1g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 35.8ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 21.48ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0042] Example 10 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 10ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 6ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0043] Example 11 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell completely at 4℃ for 24h, then stir and disperse. Add 25ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 15ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0044] Example 12 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 8.95ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0045] Comparative Example 1 S1. Recombinant collagen cross-linking modification: Dissolve 10g of recombinant type III humanized collagen in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 30min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 was placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate was collected by centrifugation. The precipitate was placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it was freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product was freeze-dried, packaged, and sterilized by cobalt-60 irradiation with a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB(III) product.

[0046] Comparative Example 2 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) was dissolved in 100ml of water and stirred until dissolved. Then, 17.9ml of 0.1M calcium chloride solution was added and stirred thoroughly for 2 hours. Then, 10.74ml of 0.1M sodium dihydrogen phosphate solution was added and stirred thoroughly for 4 hours. 0.1M sodium hydroxide was added to adjust the pH of the solution to 9. The solution was placed at 4℃ for 24 hours to allow RC(I / III) to fully mineralize and precipitate. The solution was then placed in a dialysis bag with a molecular weight cutoff of 1000Da and placed in deionized water. Dialysis was performed at 5℃ for 3 days, with the water changed 3 times a day. The precipitate was collected by centrifugation. The solution was placed in a custom mold and placed in a freeze dryer with a vacuum of 350μbar. After pre-freezing and drying at -25℃ for 5 hours, the solution was freeze-dried at -5℃ for 30 hours and finally desorbed and dried at 25℃ for 10 hours to form the final freeze-dried product.

[0047] The resulting freeze-dried product has an uneven appearance. The upper layer is loose, spongy recombinant collagen, while the lower layer is relatively hard hydroxyapatite mimic particles. Therefore, the recombinant collagen without cross-linking modification cannot achieve uniform co-deposition of recombinant collagen and hydroxyapatite mimic, and thus cannot produce artificial bone that meets the performance requirements of osteogenic materials.

[0048] Comparative Example 3 S1. Cross-linking modification of recombinant collagen: 10g of recombinant collagen (8.75g of recombinant type I humanized collagen + 1.25g of recombinant type III humanized collagen) was dissolved in 100ml of water, and 10ml of 1M NHS solution was added and stirred thoroughly. Then, 10ml of 1M EDC solution was added and stirred thoroughly for about 15min to form a hydrogel. The hydrogel was allowed to stand at 4℃ for 24h to cross-link, and the cross-linked recombinant collagen CMRC(I / III) hydrogel was obtained. The hydrogel was washed with water 5 times and centrifuged to remove the washing water. An equal mass of purified water was added to the obtained CMRC(I / III) hydrogel and homogenized in a homogenizer to form a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum of 350μbar and pre-frozen at -25℃ for 5h, then freeze-dried at -5℃ for 30h, and finally desorbed and dried at 25℃ for 10h to obtain the CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0049] Comparative Example 4 S1. Recombinant collagen cross-linking modification: 20g of recombinant collagen (18g of recombinant type I humanized collagen + 2g of recombinant type III humanized collagen) was dissolved in 100ml of water, and 20ml of 1M NHS solution was added and stirred. Then, 20ml of 1M EDC solution was added and stirred thoroughly for about 10min to form a hydrogel. The hydrogel was allowed to stand at 4℃ for 24h to cross-link, and the cross-linked recombinant collagen CMRC(I / III) hydrogel was obtained. The hydrogel was washed with water 5 times and centrifuged to remove the washing water. An equal mass of purified water was added to the obtained CMRC(I / III) hydrogel and homogenized in a homogenizer to form a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum of 350μbar and pre-freezed at -25℃ for 5h, then freeze-dried at -5℃ for 30h, and finally desorbed and dried at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product.

[0050] S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, stir at 4℃ for 24h, then add 17.9ml of 0.1M calcium chloride solution and stir for 2h; add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir for 4h; finally add 0.1M sodium hydroxide to adjust the pH of the solution to 9; place at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, thus obtaining a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0051] The comparative sample had a high collagen content. Due to the high collagen concentration, it was difficult to stir the solution thoroughly, resulting in the generation of many bubbles in the solution during the preparation process. Consequently, the lyophilized product exhibited uneven cross-linking and swelling.

[0052] Comparative Example 5 S1. Recombinant collagen cross-linking modification: 2.5g of recombinant collagen (2.25g of recombinant type I humanized collagen + 0.25g of recombinant type III humanized collagen) was dissolved in 100ml of water, and 2.5ml of 1M NHS solution was added and stirred thoroughly. Then, 2.5ml of 1M EDC solution was added, and stirring was performed, but no hydrogel could be formed. Cross-linking was continued at 4℃ for 24h, but cross-linked recombinant collagen CMRC(I / III) hydrogel could not be obtained. The concentration of recombinant collagen in this comparative example was too low to allow the cross-linking reaction to occur.

[0053] Comparative Example 6 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 2.5ml of 1M NHS solution, and stir thoroughly; then add 2.5ml of 1M EDC solution, stir thoroughly for about 2h to form a hydrogel, and continue to stand at 4℃ for cross-linking for 24h to obtain cross-linked modified recombinant collagen CMRC(I / III) hydrogel.

[0054] In this comparative example, when the crosslinking agent was removed by washing with water, the viscous colloid dissolved in the water again, and the crosslinked sample was unstable, making it impossible to obtain the crosslinked modified recombinant collagen CMRC(I / III).

[0055] Comparative Example 7 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 5ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 3ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place the solution at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0056] Comparative Example 8 S1. Cross-linking modification of recombinant collagen: Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water, add 10ml of 1M NHS solution, and stir thoroughly. Then add 10ml of 1M EDC solution, stir thoroughly for about 15min to form a hydrogel, and continue to cross-link at 4℃ for 24h to obtain cross-linked recombinant collagen CMRC(I / III) hydrogel. Wash with water 5 times and centrifuge to remove the washing water. Add an equal mass of purified water to the obtained CMRC(I / III) hydrogel, place it in a homogenizer to disperse it into a viscous colloidal solution, place it in a freeze dryer with a vacuum degree of 350μbar, pre-freeze dry at -25℃ for 5h, freeze dry at -5℃ for 30h, and finally desorb and dry at 25℃ for 10h to obtain CMRC(I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5g of CMRC(I / III) lyophilized product in 100ml of water, allow it to swell fully at 4℃ for 24h, then stir and disperse. Add 30ml of 0.1M calcium chloride solution and stir thoroughly for 2h. Add 18ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h. Finally, add 0.1M sodium hydroxide to adjust the pH of the solution to 9. Place at 4℃ for 24h to allow CMRC(I / III) to fully mineralize and deposit, resulting in a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed in a dialysis bag with a molecular weight cutoff of 1000 Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water changed 3 times a day. The precipitate is collected by centrifugation. The precipitate is placed in a custom mold and put into a freeze dryer with a vacuum degree of 350 μbar. After pre-freezing and drying at -25°C for 5 hours, it is freeze-dried at -5°C for 30 hours, and finally desorbed and dried at 25°C for 10 hours. The product is freeze-dried, packaged, and sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the recombinant collagen biomimetic artificial bone RCAB (I / III) finished product.

[0057] Example 1: In vitro cytotoxicity study (1) Preparation of recombinant collagen biomimetic artificial bone extract: Weigh the lyophilized recombinant collagen biomimetic artificial bone and extract it using serum-containing 1640 cell culture medium at a ratio of 0.1 g / ml. The extracted sample was extracted at 37℃±1℃ for 24±2 h. The extract was used as the test solution.

[0058] (2) The concentration is 1×10 5 L929 cell suspension of 100 μl / well was added to a 96-well plate and cultured in a 5% CO2 incubator at 37°C for 24 h. The culture medium was then aspirated and replaced with culture medium containing recombinant collagen biomimetic bone extract from Example 1, and cultured for another 24 h. The 96-well plate was then removed, and CCK8 was added to each well at 10 μl / well. The plate was then incubated for another 2 h, and the absorbance at 450 nm was measured using a microplate reader to calculate the cell viability.

[0059] (3) The results of the cytotoxicity test showed that the cell survival rate of the RCAB(I / III) extract prepared in Example 1 was not significantly different from that of the normal culture group, with a survival rate of 103%, indicating that the biomimetic artificial bone has no potential cytotoxicity and good biocompatibility.

[0060] Example 2: Porosity Investigation (1) The porosity of the recombinant collagen biomimetic artificial bone materials obtained in Examples 1-12 and Comparative Examples 1, 3, 4, 7, and 8 was detected by liquid displacement method in this invention. The initial mass of the biomimetic artificial bone was weighed and recorded as W1; the biomimetic artificial bone was immersed in anhydrous ethanol and weighed after 48 hours and recorded as W2; the total volume of the biomimetic artificial bone sample and the absorbed ethanol was measured and recorded as V. The porosity of the material (%) = (W2-W1) / ρV*100.

[0061] (2) The porosity results of recombinant collagen biomimetic artificial bone are shown in Table 1: The porosity of the recombinant collagen biomimetic artificial bone material prepared by the methods in Examples 1 to 12 is between 60% and 90%, which is close to the porosity between the trabeculae of normal human cancellous bone, meets the requirements of cancellous bone, and has high biomimetic performance.

[0062] Example 3: Mechanical Performance Examination (1) The compressive strength and elastic modulus of the biomimetic artificial bone materials prepared in Examples 1-12 and Comparative Examples 1, 3, 4, 7 and 8 were tested using a universal electronic material testing machine. The crosshead loading speed of the testing machine was 0.5 mm / min, and the sample was a circular piece with a size of φ8 mm × 10 mm.

[0063] (2) According to literature reports, the compressive strength of normal human cortical bone is 130-180 MPa, and the elastic modulus is 17-20 GPa. This invention studied the mechanical properties of the artificial bones prepared in the examples and comparative examples, and the results are shown in Table 1. The compressive strength of the biomimetic artificial bone materials prepared in Examples 1-12 is all above 145 MPa, which is higher than the compressive strength of normal human cortical bone. The elastic modulus is basically 15 GPa, close to the elastic modulus of normal human cortical bone, and can meet the mechanical requirements of artificial bone repair materials.

[0064] Example 4: In vitro degradation rate investigation (1) Immerse the bionic artificial bone in 10 mL of PBS solution. After 48 h, weigh the bionic artificial bone material and record the initial mass as W1. Shake in a constant temperature shaker at 37℃ (100 rpm). Change the PBS solution every 3 days. At weeks 1, 2, 3 and 4 of degradation, take out the bionic artificial bone material, freeze-dry it for 12 h, and accurately weigh the degraded bionic artificial bone material as W2. Calculate the degradation rate of the bionic artificial bone material at different time points. Material degradation rate (%) = (W1-W2) / W1*100.

[0065] (2) The results of the in vitro degradation rate are shown in Table 1: As the in vitro degradation time increased, the mass of the biomimetic artificial bone material gradually decreased. After 4 weeks, the degradation rates of Examples 1-12 were basically between 10% and 15%, which matched the rate of new bone formation. This indicates that the recombinant collagen biomimetic artificial bone samples in the Example group had a suitable degradation rate. The materials in the comparative group degraded too quickly or too slowly, which was not conducive to the regeneration of autologous bone. The biomimetic artificial bone in the Example group can continuously and slowly degrade in vitro. In the early stage, it can provide good support in the bone defect area, and in the later stage, it does not hinder the regeneration of new bone, providing a new solution for clinical bone defect repair.

[0066] Case Study 5: Effect of Inducing Osteogenic Differentiation of Stem Cells This invention uses ALP (alkaline phosphatase) activity assay and ALP mRNA expression assay to detect the effect of the biomimetic artificial bone materials obtained in Examples 1, 2, 1, 3 and 8 on the differentiation capacity of bone marrow mesenchymal stem cells.

[0067] (1) ALP activity assay: BMSCs cells were inoculated at 1×10 5Cells were seeded at a density of [insert density here] onto biomimetic artificial bone material. After incubation for 7 days, total cell protein and ALP activity were measured using a BCA kit and an ALP kit. Following the kit instructions, ALP activity was normalized to the total protein level. The seeded biomimetic artificial bone material was digested with trypsin for 3-4 minutes, and fetal bovine serum was added to terminate the digestion. Cells adhering to the recombinant collagen biomimetic artificial bone material were gently pipetted to completely detach them. The resulting liquid was transferred to EP tubes and centrifuged at 600 rpm for 8 minutes to collect the cells. The cells were washed with PBS to remove trypsin. The cells were resuspended in PBS, and cell lysis buffer was added to completely lyse the cells. The supernatant was centrifuged at 4000 rpm for 5 minutes to remove all insoluble debris. 50 μL of the sample was added to a 96-well plate, along with 50 μL of matrix solution. The plate was incubated at 37°C for 10 minutes, and then 100 μL of stop solution was added. The OD value of each well was measured at 405 nm.

[0068] (2) ALP mRNA expression level detection method: BMSCs cells were injected with 1×10 5 Cells were seeded at varying densities onto recombinant collagen-based biomimetic bone materials. After 7 days of incubation, the differences in ALP mRNA expression levels among cells seeded with different biomimetic bone materials were detected by qRT-PCR. The control group consisted of BMSCs cultured in osteogenic induction medium; the experimental group consisted of BMSCs cultured in osteogenic induction medium containing the biomimetic bone materials from Examples 1, 2, 1, 3, and 8. The ALP primer sequences are shown in SEQ ID NO. 3; the internal control β-action primer sequence is shown in SEQ ID NO. 4.

[0069] (3) The quantitative analysis results are shown in Table 1: The ALP activity of BMSCs cells cultured on each group of materials increased with time; on the 7th day, the ALP expression activity of the recombinant collagen biomimetic artificial bone material group prepared in the example was significantly higher than that of the comparative group, among which the expression level of the Example 1 group was the best, indicating that it has the highest osteogenic induction performance and good biocompatibility.

[0070] (4) The results of ALP mRNA expression detection are shown in Table 1: Compared with the blank control group, the expression level of ALP mRNA in the example group was significantly increased, which was significantly higher than that in the control group. Among them, the expression level in the example 1 group was the highest. This result also shows that RCAB(I / III) material can induce osteogenic differentiation of bone marrow mesenchymal stem cells and significantly promote the repair performance of osteogenic defects.

[0071] Table 1 Comparison of the performance of biomimetic artificial bone in each experimental group In summary, the recombinant collagen biomimetic artificial bone material prepared by this invention has a similar structure and chemical composition to natural bone tissue, and its porosity and mechanical properties meet the requirements for bone repair materials. Furthermore, the biomimetic artificial bone material obtained by this invention exhibits good in vitro degradation rate and excellent osteogenic differentiation induction ability, effectively promoting the healing of bone defects. This biomimetic bone repair scaffold material retains the bioactivity of recombinant collagen to the greatest extent, demonstrating good biosafety and efficacy.

[0072] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant collagen biomimetic artificial bone, characterized in that, The preparation method is as follows: S1. Cross-linking modification of recombinant collagen: Prepare a recombinant collagen solution, add NHS solution and EDC solution to it to obtain cross-linked recombinant collagen hydrogel, add an equal mass of purified water to it, homogenize it and then freeze-dry it under vacuum to obtain the recombinant collagen freeze-dried product. S2. Preparation of mineralized recombinant collagen: Add water to the freeze-dried product obtained in S1, stir and disperse after full swelling, add calcium chloride solution and sodium dihydrogen phosphate solution, and finally add sodium hydroxide solution to adjust the pH to 8~10. After obtaining a white suspension, let it stand to fully mineralize and obtain a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed into a dialysis bag for dialysis, the precipitate is collected by centrifugation, and the precipitate is placed in a mold for vacuum freeze-drying to obtain recombinant collagen biomimetic artificial bone; The recombinant collagen solution is a mixture of recombinant type I and type III humanized collagen; the protein mass ratio of recombinant type I and type III humanized collagen is 9:

1. The molar ratio of phosphate ions to calcium ions is 3:5~6, and the ratio of calcium ions to the amount of lyophilized product obtained from S1 is 0.002~0.005 mol:1g. The ratio of EDC to recombinant collagen added is 0.0005~0.002 mol: 1 g; the molar ratio of EDC to NHS is 1:1~2.

2. The recombinant collagen biomimetic artificial bone according to claim 1, characterized in that, The recombinant collagen solution described in S1 contains 5% to 15% by mass of recombinant collagen.

3. The recombinant collagen biomimetic artificial bone according to claim 1, characterized in that, The concentration of the EDC solution in S1 is 0.5~2M; the concentration of the NHS solution is 0.5~2M; and the ratio of the amount of EDC to the amount of recombinant collagen added is 0.0005~0.002mol:1g.

4. The recombinant collagen biomimetic artificial bone according to claim 1, characterized in that, The vacuum freeze-drying procedure is as follows: pre-freezing at -30~-25℃ for 4~6 hours, then freeze-drying at -10~0℃ for 25~30 hours, and finally desorption drying at 20~25℃ for 8~10 hours; the vacuum degree during the vacuum freeze-drying process is 300~400μbar.

5. The recombinant collagen biomimetic artificial bone according to claim 1, characterized in that, The mass ratio of the freeze-dried product to water in S2 is 0.3~1:

100.

6. The recombinant collagen biomimetic artificial bone according to claim 1, characterized in that, The concentration of the calcium chloride solution in S2 is 0.05~0.3M; the concentration of the sodium dihydrogen phosphate solution is 0.05~0.2M.

7. The recombinant collagen biomimetic artificial bone according to claim 1, characterized in that, The dialysis described in S3 is as follows: the dialysis bag is placed in deionized water and dialyzed at a low temperature of 2~6℃ for 3 days, with the water changed 3 times a day; the molecular weight cutoff of the dialysis bag is 1000~3000 Da.

8. A method for preparing recombinant collagen biomimetic artificial bone according to any one of claims 1 to 7, characterized in that, The steps include the following: S1. Cross-linking modification of recombinant collagen: Prepare a recombinant collagen solution, add NHS solution and EDC solution to it to obtain cross-linked recombinant collagen hydrogel, add an equal mass of purified water to it, homogenize it and then freeze-dry it under vacuum to obtain the recombinant collagen freeze-dried product. S2. Preparation of mineralized recombinant collagen: Add water to the freeze-dried product obtained in S1, stir and disperse after full swelling, add calcium chloride solution and sodium dihydrogen phosphate solution, and finally add sodium hydroxide solution to adjust the pH to 8~10. After obtaining a white suspension, let it stand to fully mineralize and obtain a mineralized recombinant collagen mixture. S3, Molding: The mixture obtained in S2 is placed into a dialysis bag for dialysis, the precipitate is collected by centrifugation, and the precipitate is placed in a mold for vacuum freeze-drying to obtain recombinant collagen biomimetic artificial bone; The recombinant collagen solution is a mixture of recombinant type I and type III humanized collagen; the protein mass ratio of recombinant type I and type III humanized collagen is 9:

1. The molar ratio of phosphate ions to calcium ions is 3:5~6, and the ratio of calcium ions to the amount of lyophilized product obtained from S1 is 0.002~0.005 mol:1g. The ratio of EDC to recombinant collagen added is 0.0005~0.002 mol: 1 g; the molar ratio of EDC to NHS is 1:1~2.

9. The application of the recombinant collagen biomimetic artificial bone as described in any one of claims 1 to 7 in the preparation of bone defect filling and repair products.

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