Recombinant collagen bionic artificial bone as well as preparation method and application thereof

Abstract

The invention relates to a recombinant collagen bionic artificial bone and a preparation method and application thereof.The recombinant collagen is subjected to cross-linking modification, the molecular weight and viscosity of the recombinant collagen are increased, a fiber network structure is formed, then the calcium-phosphorus ratio of hydroxyapatite is simulated through soluble salts of calcium chloride and sodium dihydrogen phosphate, and the recombinant collagen bionic artificial bone is obtained. The biomimetic artificial bone can replace insoluble hydroxyapatite to mineralize the recombinant collagen, so that the utilization rate of the collagen is improved, and the obtained biomimetic artificial bone can exert a better osteogenesis effect. The obtained bionic artificial bone is high in biological safety, has high bionic characteristics, good biocompatibility and mechanical properties matched with human bones, can induce bone marrow mesenchymal stem cell osteogenic differentiation, and has great potential in the field of bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular, relates to a recombinant collagen bionic artificial bone and a preparation method and application thereof. Background Art

[0002] Bone defects are common orthopedic diseases caused by fractures, osteogenesis imperfecta or osteoporosis. According to relevant statistics, bone tissue is the second largest tissue in the world that needs to be transplanted. About four million people need bone grafts or bone substitute materials to treat bone defects each year. Bone diseases now account for half of chronic diseases in people over 50 years old, and their treatment has become a huge clinical challenge. Autologous bone transplantation is an ideal treatment method with the characteristics of bone conduction and bone induction, but it has high risks, few bone sources, and is prone to severe pain and complications at the donor site after surgery. Therefore, it is of great clinical significance to establish an effective bone regeneration treatment method and accurately treat bone diseases.

[0003] Bones are composed of a mesh scaffold formed by collagen fibers and nano-hydroxyapatite uniformly attached to the scaffold. The bone structure formed by the composite of collagen and hydroxyapatite has good microscopic morphology and mechanical properties. As the main inorganic component of human bones, hydroxyapatite has good biological activity and osteoconductivity, and can form a firm bone integration with human bone tissue. Hydroxyapatite can be prepared by chemical deposition of calcium chloride and phosphate ions, which is simple to operate, highly safe, and does not produce toxic substances. Bioceramic material implants represented by hydroxyapatite have good biological activity and bone integration ability, but pure hydroxyapatite materials have significant differences in mechanical properties and microstructure from human bones, poor mechanical strength, large brittleness, and degradation rate that does not match autologous bone. Collagen is the main organic component of human bones. Currently, most of the products on the market are composites of animal-derived collagen and hydroxyapatite to fill bone defects. Animal collagen is mainly derived from animal tissues such as pig skin, pig trotters and cattle Achilles tendons, which have potential risks of viruses, low purity, immune response and potential infectious diseases. Animal collagen can only be completely dissolved in an acidic solution to achieve a composite effect with hydroxyapatite, which is likely to affect the protein activity and later application in the composite material.

[0004] Recombinant humanized type I and type III collagen are collagens prepared by DNA recombination technology and microbial fermentation, and their amino acid sequences can be designed and improved according to needs. The gene sequence of recombinant humanized collagen is highly consistent with that of humans, and has good tissue compatibility. A document (10.16190 / j.cnki.45-1211 / r.2018.03.011) reported that recombinant humanized collagen was used to heal the wounds of patients with deep burns. Recombinant humanized collagen can reconnect the broken collagen fibers caused by burns, synthesize new type III humanized collagen, promote wound healing, and reduce scar hyperplasia, proving that recombinant humanized collagen can be directly absorbed by the human body and participate in the construction of collagen, helping with tissue repair and site filling.

[0005] However, there are currently no reports disclosing the specific uses of recombinant humanized type I and type III 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 bionic artificial bone and its preparation method and application. The bionic artificial bone provided by the present invention broadens the application of collagen-based bone materials in bone repair, so that it can be used in bone defect repair in orthopedics and dentistry.

[0007] To solve the above problems, the technical solution adopted by the present invention is: Technical topic 1: The present invention provides a recombinant collagen bionic artificial bone, and the preparation method is as follows: S1. Cross-linking modification of recombinant collagen: prepare a recombinant collagen solution, add NHS solution and EDC solution thereto to obtain a cross-linked recombinant collagen hydrogel, add an equal amount of purified water thereto, break up with a homogenizer, and then freeze-dry in a vacuum to obtain a recombinant collagen lyophilized product; S2. Preparation of mineralized recombinant collagen: add water to the lyophilized product obtained in S1, stir and disperse after sufficient swelling, add calcium chloride solution and sodium dihydrogen phosphate solution, and finally add sodium hydroxide solution to adjust the pH to 8-10, obtain a white suspension, and then let it stand for full mineralization to obtain a mineralized recombinant collagen mixture; S3, molding: putting the mixed solution obtained in S2 into a dialysis bag, dialyzing, collecting the precipitate by centrifugation, placing the precipitate in a mold and vacuum freeze-drying to obtain a recombinant collagen bionic artificial bone; The collagen solution includes a recombinant type I humanized collagen solution or a recombinant type I and type III humanized collagen mixed solution.

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

[0009] As a further embodiment of the present invention, in the mixed solution of recombinant humanized type I and type III collagen described in S1, the mass of recombinant humanized type III collagen accounts for no more than 1 / 9 of the total mass of collagen.

[0010] As a further embodiment of the present invention, S1 the concentration of the EDC solution is 0.5~2M; the concentration of the NHS solution is 0.5~2M; the ratio of the added amount of EDC to collagen is 0.0005~0.002mol:1g; 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: pre-freezing temperature -30~-25°C freeze-drying for 4~6 hours, then freeze-drying at -10~0°C for 25~30 hours, and finally desorption drying at 20~25°C 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 the added amount 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: placing the dialysis bag in deionized water, low-temperature dialysis at 2-6°C for 3 days, and changing the water 3 times a day; the molecular weight cutoff of the dialysis bag is 1000-3000Da.

[0015] Technical theme 2: The invention provides a method for preparing the recombinant collagen bionic artificial bone.

[0016] Technical Theme 3: The present invention provides an application of the recombinant collagen bionic artificial bone in the preparation of bone defect filling and repair products, wherein the bone defects include various closed fracture bone defects of limbs, spinal bone defects, bone defects of maxillofacial surgery, bone defects of dental surgery and / or skull defects.

[0017] The beneficial effects of adopting the above technical solution are: 1. The present invention provides a scheme of using cross-linked modified recombinant collagen, in which recombinant humanized collagen is prepared into cross-linked modified recombinant collagen by chemical cross-linking, thereby increasing the mechanical strength of the later bionic bone material and prolonging the degradation time. The composite effect of various types of recombinant humanized collagen has more excellent tissue support and repair capabilities, and can more significantly exert the osteogenic effect.

[0018] 2. The present invention uses cross-linked modified recombinant collagen and hydroxyapatite mimics to be evenly distributed and co-deposited. The resulting material is close to the composition of natural bone (the ratio of inorganic matter to organic matter is about 65:35), which improves the osteogenic differentiation ability of cells and helps regenerate defective bone.

[0019] 3. The present invention uses soluble salt calcium chloride / sodium dihydrogen phosphate instead of insoluble hydroxyapatite to carry out mineralization reaction on recombinant collagen, thereby improving the utilization rate of collagen and exerting a better osteogenesis effect.

[0020] 4. The bionic artificial bone material obtained by the present invention has good biocompatibility: the amino acid sequence of the two recombinant humanized collagens used in the bionic artificial bone material obtained by the present invention is consistent with the specific functional region of the collagen amino acid sequence of the human body, and the two collagens have excellent cell adhesion effect. Applying them to the human body avoids the production of immune rejection and allergic reactions; at the same time, the raw materials used in the preparation process of the material are non-cytotoxic, free of any viral risks and immunogenicity, have good biosafety, and can be widely used in the fields of medicine, medical devices and tissue engineering.

[0021] 5. The bionic artificial bone material obtained by the present invention has a high degree of bionic properties: the bionic artificial bone material obtained by the present invention has mechanical properties, porosity and suitable in vitro degradation rate matching those of human bones, can induce the osteogenic differentiation ability of bone marrow mesenchymal stem cells, and has great potential in the field of bone defect material repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flow chart of the preparation process of Example 1 of the present invention; Figure 2 This is a physical picture of the lyophilized product of the recombinant collagen bionic artificial bone RCAB (I / III) obtained in Example 1 of the present invention; Figure 3 This is a SEM image of the recombinant collagen bionic artificial bone RCAB (I / III) sample obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0024] The recombinant humanized type III collagen of the present invention is prepared by the method disclosed in Chinese invention patent 202011366731.X, entitled “A recombinant human type III collagen, its preparation method and use”.

[0025] The preparation method of some recombinant type I collagen in this example is as follows: (1) Genetic design: According to the sequence characteristics of human type I collagen, the collagen sequence was designed, and its amino acid sequence is shown in SEQ ID NO.1, which is a tandem repeat of amino acids 671-750 of the α1 chain, with a repeat number of 4. Its N-terminus starts with methionine to reduce the degradation rate of the protein, followed by a His-Tag of 6 histidines for easy identification and purification.

[0026] The coding nucleic acid sequence was reversely designed using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for host Escherichia coli expression. 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 Corporation 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 connected 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 construction of the expression strain was based on the method described in Molecular Cloning Experiment Manual (3rd Edition) (J. Sambrook et al.), and the expression strain BL21(DE3) / pET30a(+)-HC90 was obtained.

[0030] (5) Induced expression of expression strain BL21(DE3) / pET30a(+)-HC90: A single colony of the expression strain BL21(DE3) / pET30a(+)-HC90 was cultured in LB liquid medium containing 50μg / mL Kan at 37°C and 200r / min overnight to become an activated seed, and then inoculated into a 5L fermenter containing 3L CM medium at a 3% inoculum. The fermentation process was controlled at 37°C, 30% dissolved oxygen, and pH = 7.0. When OD600 reached 60, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.5mM to induce expression, and the culture was continued for 12 hours, and the bacteria were collected by centrifugation.

[0031] (6) Separate and purify to obtain.

[0032] Example 1 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 17.9 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 10.74 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0033] Example 2 S1. Cross-linking modification of recombinant collagen: Dissolve 10 g of recombinant type I humanized collagen in 100 ml of water, add 10 ml of 1 M NHS solution, and stir well to mix; then add 10 ml of 1 M EDC solution, stir well for about 25 min to form a hydrogel, and continue to stand at 4°C for cross-linking for 24 h to obtain CMRC (I) hydrogel, wash with water 5 times, and remove the washing water by centrifugation; add an equal amount of purified water to the obtained CMRC (I) hydrogel, put it into a homogenizer to break it up into a viscous colloidal solution, put it into a freeze dryer with a vacuum degree of 350 μbar, pre-freeze-dry at -25°C for 5 h, freeze-dry at -5°C for 30 h, and finally desorb and dry at 25°C for 10 h to obtain a CMRC (I) lyophilized product.

[0034] S2. Preparation of mineralized recombinant collagen: 0.5 g of CMRC (I) freeze-dried product was dissolved in 100 ml of water, fully swelled at 4°C for 24 hours, stirred to break up, and 17.9 ml of 0.1 M calcium chloride solution was added, and fully stirred for 2 hours; then 10.74 ml of 0.1 M sodium dihydrogen phosphate solution was added, and fully stirred for 4 hours, and 0.1 M sodium hydroxide was added to adjust the solution pH to 9; the solution was placed at 4°C for 24 hours to allow CMRC (I) to fully mineralize and precipitate, thereby obtaining a mineralized recombinant collagen mixture; S3, molding: put it into a dialysis bag with a molecular weight cutoff of 1000Da, place it in deionized water, and dialyze it at a low temperature of 5°C for 3 days, changing the water 3 times a day, and collect the precipitate by centrifugation; put the obtained precipitate into a customized mold, put it into a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dry it at a temperature of -25°C for 5h, freeze-dry it at -5°C for 30h, and finally desorb and dry it at 25°C for 10h, freeze-dry it into shape, package it, and sterilize it by irradiation with cobalt 60 at a dose of 25kGy to obtain the finished product of recombinant collagen bionic artificial bone RCAB (I).

[0035] Example 3 S1. Cross-linking modification of recombinant collagen: 9 g of recombinant collagen (8 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 9 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then, 9 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the solution was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and the solution was pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 17.9 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 10.74 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0036] Example 4 S1. Cross-linking modification of recombinant collagen: 5 g of recombinant collagen (4.5 g of recombinant type I humanized collagen + 0.5 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 5 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then, 5 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the mixture into a viscous colloidal solution. The mixture was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 17.9 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 10.74 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

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

[0038] Example 6 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 5 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then, 5 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 45 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the solution was put into a homogenizer to be broken up into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 17.9 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 10.74 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0039] Example 7 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 20 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 20 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 8 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the solution was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 17.9 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 10.74 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0040] Example 8 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: 0.3 g of CMRC (I / III) freeze-dried product was dissolved in 100 ml of water, fully swollen at 4°C for 24 hours, then stirred and dispersed, 10.74 ml of 0.1 M calcium chloride solution was added, and fully stirred for 2 hours; 6.44 ml of 0.1 M sodium dihydrogen phosphate solution was added, and fully stirred for 4 hours; finally, 0.1 M sodium hydroxide was added to adjust the pH value of the solution to 9; the solution was placed at 4°C for 24 hours to allow CMRC (I / III) to be fully mineralized and deposited, thereby obtaining a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0041] Example 9 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 1g of CMRC (I / III) freeze-dried product in 100ml of water, fully swell at 4°C for 24h, stir and disperse, add 35.8ml of 0.1M calcium chloride solution, and fully stir for 2h; add 21.48ml of 0.1M sodium dihydrogen phosphate solution, and fully stir for 4h; finally, add 0.1M sodium hydroxide and adjust the pH value of the solution to 9; place at 4°C for 24h to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0042] Example 10 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 10 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 6 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0043] Embodiment 11 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, incubate at 4°C for 24 hours to fully swell, stir and disperse, add 25 ml of 0.1 M calcium chloride solution, and stir for 2 hours; add 15 ml of 0.1 M sodium dihydrogen phosphate solution, and stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0044] Example 12 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: 0.5 g of CMRC (I / III) freeze-dried product was dissolved in 100 ml of water, fully swollen at 4°C for 24 hours, then stirred and dispersed, 17.9 ml of 0.1 M calcium chloride solution was added, and fully stirred for 2 hours; 8.95 ml of 0.1 M sodium dihydrogen phosphate solution was added, and fully stirred for 4 hours; finally, 0.1 M sodium hydroxide was added to adjust the pH value of the solution to 9; the solution was placed at 4°C for 24 hours to allow CMRC (I / III) to be fully mineralized and deposited, thereby obtaining a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0045] Comparative Example 1 S1. Cross-linking modification of recombinant collagen: dissolve 10g of recombinant type III humanized collagen in 100ml of water, add 10ml of 1M NHS solution, and stir well to mix; then add 10ml of 1M EDC solution, stir well for about 30min to form a hydrogel, and continue to stand at 4°C for cross-linking for 24h to obtain a cross-linked recombinant collagen CMRC (III) hydrogel, wash it with water 5 times, and remove the washing water by centrifugation; add an equal amount of purified water to the obtained CMRC (III) hydrogel, put it into a homogenizer to break it into a viscous colloidal solution, put it into a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dry it at a temperature of -25°C for 5h, freeze-dry it at -5°C for 30h, and finally desorb and dry it at 25°C for 10h to obtain a CMRC (III) freeze-dried product; S2. Preparation of mineralized recombinant collagen: 0.5 g of CMRC (III) freeze-dried product was dissolved in 100 ml of water, fully swollen at 4°C for 24 hours, then stirred and dispersed, 17.9 ml of 0.1 M calcium chloride solution was added, and fully stirred for 2 hours; 10.74 ml of 0.1 M sodium dihydrogen phosphate solution was added, and fully stirred for 4 hours; finally, 0.1 M sodium hydroxide was added to adjust the pH value of the solution to 9; the solution was placed at 4°C for 24 hours to allow CMRC (III) to be fully mineralized and deposited, thereby obtaining a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (III) finished product.

[0046] Comparative Example 2 Dissolve 10g of recombinant collagen (9g of recombinant type I humanized collagen + 1g of recombinant type III humanized collagen) in 100ml of water and stir to dissolve; then add 17.9ml of 0.1M calcium chloride solution and stir thoroughly for 2h; then add 10.74ml of 0.1M sodium dihydrogen phosphate solution and stir thoroughly for 4h, add 0.1M sodium hydroxide and adjust the pH value of the solution to 9; place it at 4℃ for 24h to allow RC(I / III) to fully mineralize and precipitate, then put it into a dialysis bag with a molecular weight cutoff of 1000Da, place it in deionized water, and dialyze it at 5℃ for 3 days, changing the water 3 times a day, and collect the precipitate by centrifugation; place it in a customized mold and put it into a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dry it at -25℃ for 5h, freeze-dry it at -5℃ for 30h, and finally desorb and dry it at 25℃ for 10h to freeze-dry it into shape.

[0047] The appearance of the freeze-dried product is uneven, with the upper layer being loose sponge-like recombinant collagen and the lower layer being harder hydroxyapatite mimic particles. Therefore, recombinant collagen that has not been cross-linked cannot achieve uniform co-deposition of recombinant collagen and hydroxyapatite mimics, and artificial bone that meets the required performance of osteogenic materials cannot be obtained.

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

[0049] Comparative Example 4 S1. Cross-linking modification of recombinant collagen: 20 g of recombinant collagen (18 g of recombinant type I humanized collagen + 2 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 20 ml of 1 M NHS solution was added and stirred; then 20 ml of 1 M EDC solution was added and stirred for about 10 min to form a hydrogel, which was then allowed to stand at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water 5 times and centrifuged to remove the washing water; an equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the solution was placed in a homogenizer to break up into a viscous colloidal solution, which was placed in a freeze dryer with a vacuum degree of 350 μbar, pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain CMRC (I / III) freeze-dried solution.

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

[0051] The collagen content in this comparative example is relatively high. Due to the high concentration of collagen, it is difficult to fully stir, resulting in more bubbles in the solution during the preparation process, and the freeze-dried product has uneven cross-linking and swelling.

[0052] Comparative Example 5 S1. Cross-linking modification of recombinant collagen: 2.5g recombinant collagen (2.25g recombinant type I humanized collagen + 0.25g recombinant type III humanized collagen) was dissolved in 100ml water, 2.5ml 1M NHS solution was added, and the mixture was stirred thoroughly; then 2.5ml 1M EDC solution was added, and the mixture was stirred but no hydrogel was formed. The cross-linking was continued at 4°C for 24h, but no cross-linked recombinant collagen CMRC (I / III) hydrogel was obtained. The concentration of recombinant collagen in this comparative example was too low, and no cross-linking reaction could 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 well to mix; then add 2.5ml of 1M EDC solution, stir well for about 2h to form a hydrogel, and continue to cross-link at 4°C for 24h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel.

[0054] When the comparative example was washed with water to remove the cross-linking agent, the viscous colloid dissolved in the water again, the cross-linked sample was unstable, and the cross-linked modified recombinant collagen CMRC (I / III) could not be obtained.

[0055] Comparative Example 7 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 5 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 3 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0056] Comparative Example 8 S1. Cross-linking modification of recombinant collagen: 10 g of recombinant collagen (9 g of recombinant type I humanized collagen + 1 g of recombinant type III humanized collagen) was dissolved in 100 ml of water, and 10 ml of 1 M NHS solution was added, and the mixture was stirred thoroughly. Then 10 ml of 1 M EDC solution was added, and the mixture was stirred thoroughly for about 15 min to form a hydrogel. The mixture was allowed to stand for cross-linking at 4°C for 24 h to obtain a cross-linked recombinant collagen CMRC (I / III) hydrogel, which was washed with water for 5 times and centrifuged to remove the washing water. An equal amount of purified water was added to the obtained CMRC (I / III) hydrogel, and the mixture was placed in a homogenizer to break up the solution into a viscous colloidal solution. The solution was placed in a freeze dryer with a vacuum degree of 350 μbar, and pre-lyophilized at -25°C for 5 h, freeze-dried at -5°C for 30 h, and finally dried at 25°C for 10 h to obtain a CMRC (I / III) freeze-dried product. S2. Preparation of mineralized recombinant collagen: Dissolve 0.5 g of CMRC (I / III) freeze-dried product in 100 ml of water, fully swell at 4°C for 24 hours, stir to disperse, add 30 ml of 0.1 M calcium chloride solution, and fully stir for 2 hours; add 18 ml of 0.1 M sodium dihydrogen phosphate solution, and fully stir for 4 hours; finally, add 0.1 M sodium hydroxide to adjust the pH value of the solution to 9; place at 4°C for 24 hours to allow CMRC (I / III) to fully mineralize and precipitate, and obtain a mineralized recombinant collagen mixture; S3, molding: the mixed solution obtained in S2 is loaded into a dialysis bag with a molecular weight cutoff of 1000Da, placed in deionized water, and dialyzed at 4°C for 3 days, with the water replaced 3 times a day, and the precipitate is collected by centrifugation; the obtained precipitate is placed in a customized mold, and placed in a freeze dryer with a vacuum degree of 350μbar, pre-freeze-dried at -25°C for 5h, freeze-dried at -5°C for 30h, and finally desorbed at 25°C for 10h, freeze-dried, packaged, and sterilized by cobalt 60 irradiation with a dose of 25kGy to obtain the recombinant collagen bionic artificial bone RCAB (I / III) finished product.

[0057] Investigation Example 1 In vitro cytotoxicity investigation (1) Preparation of recombinant collagen bionic artificial bone extract: Weigh the recombinant collagen bionic artificial bone freeze-dried product, and extract it with 1640 cell culture medium containing serum at a ratio of 0.1g / ml. Extract the sample at 37℃±1℃ for 24±2h. Take the extract as the test solution.

[0058] (2) The concentration is 1×10 5 A L929 cell suspension of 100 μl / well was added to a 96-well plate, and after culturing in a 5% CO2, 37°C incubator for 24 hours, the culture medium was aspirated and replaced with a culture medium containing the recombinant collagen bionic artificial bone extract of Example 1 and continued to be cultured for 24 hours; the 96-well plate was taken out, CCK8 was added at 10 μl / well, and the plate was placed in an incubator and continued to be cultured for 2 hours. The absorbance at 450 nm was measured on a microplate reader to calculate the cell survival rate.

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

[0060] Investigation Example 2: Porosity Investigation (1) The present invention uses a liquid replacement method to detect the porosity of the recombinant collagen bionic artificial bone materials obtained in Examples 1 to 12 and Comparative Examples 1, 3, 4, 7, and 8. The initial mass of the bionic artificial bone is weighed, recorded as W1; the bionic artificial bone is soaked in anhydrous ethanol and weighed after 48 hours, recorded as W2; the total volume of the bionic artificial bone sample and the absorbed ethanol is measured, recorded as V. Material porosity (%) = (W2-W1) / ρV*100.

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

[0062] Investigation Example 3 Mechanical Properties Investigation (1) The compressive strength and elastic modulus of the bionic artificial bone materials prepared in Examples 1 to 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 disc with a size of φ8 mm×10 mm.

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

[0064] Investigation Example 4 Investigation of in vitro degradation rate (1) Soak the bionic artificial bone in 10 mL of PBS solution, weigh the bionic artificial bone material after 48 hours, and record the initial mass as W1; shake in a 37°C constant temperature shaker (100 rpm), and replace the PBS solution every 3 days. Take out the bionic artificial bone material at the 1st, 2nd, 3rd and 4th week of degradation, freeze-dry it for 12 hours, and accurately weigh the mass of the bionic artificial bone material after degradation 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 in vitro degradation rate investigation are shown in Table 1: As the in vitro degradation time increases, the mass of the bionic artificial bone material gradually decreases. After 4 weeks, the degradation rates of Examples 1 to 12 were basically between 10% and 15%, which matched the rate of new bone formation, indicating that the recombinant collagen bionic artificial bone samples in the Example group had a more suitable degradation rate. The degradation of the materials in the Control Group was too fast or too slow, which was not conducive to the regeneration of autologous bone. The bionic artificial bone in the Example group can be continuously and slowly degraded in vitro, which can provide good support in the bone defect area in the early stage and does not hinder the regeneration of new bone in the later stage, providing a new solution for clinical bone defect repair.

[0066] Investigation Example 5 Investigation of the effect of inducing osteogenic differentiation of stem cells The present invention detects the effects of the bionic artificial bone materials obtained in Example 1, Example 2, Comparative Example 1, Comparative Example 3 and Comparative Example 8 on the differentiation ability of bone marrow mesenchymal stem cells through an ALP (alkaline phosphatase) activity detection test and an ALP mRNA expression detection test.

[0067] (1) ALP activity detection method: BMSCs cells were cultured at 1×10 5The cells were inoculated on the biomimetic artificial bone material at a density of 100 μg / mL. After incubation for 7 days, the total cell protein and ALP activity were determined using the BCA kit and ALP kit. According to the kit instructions, the activity of ALP was normalized to the total protein level. The biomimetic artificial bone material inoculated with cells was digested with trypsin for 3-4 minutes, and fetal bovine serum was added to terminate the digestion. The cells adhering to the recombinant collagen biomimetic artificial bone material were blown slowly with a pipette to completely remove the cells. The resulting liquid was transferred to an EP tube and centrifuged at 600 r / min for 8 minutes to collect the cells; the cells were washed with PBS to remove the trypsin; the cells were resuspended with PBS, and cell lysis buffer was added to completely lyse the cells; the supernatant was centrifuged at 4000 rpm / min for 5 minutes to remove all insoluble fragments; 50ul of the sample was added to a 96-well plate, 50μl of the matrix solution was added, incubated at 37°C for 10 minutes, and 100μl of the stop solution was added. The OD value of each well was measured at 405nm.

[0068] (2) ALP mRNA expression detection method: BMSCs cells were cultured at 1×10 5 The cells were inoculated at a density of 100 μg / cm2 on the recombinant collagen bionic artificial bone material. After incubation for 7 days, the difference in ALP mRNA expression of the cells inoculated with each bionic artificial bone material was detected by qRT-PCR. The control group was BMSCs cells cultured in osteogenic induction medium; the experimental group was BMSCs cells cultured in osteogenic induction medium containing bionic artificial bone materials of Example 1, Example 2, Comparative Example 1, Comparative Example 3 and Comparative Example 8. The ALP primer sequence is shown in SEQ ID NO.3; the internal reference β-action primer sequence is shown in SEQ ID NO.4.

[0069] (3) The results of quantitative analysis 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 bionic artificial bone material group prepared in the example was significantly higher than that of the control 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 are shown in Table 1: Compared with the blank control group, the expression of ALP mRNA in the example group was significantly increased, which was significantly higher than that in the comparative example group. The expression level in Example 1 group was the highest. This result also shows that the RCAB (I / III) material can induce osteogenic differentiation of bone marrow mesenchymal stem cells and promote osteogenic defect repair.

[0071] Table 1 Comparison of bionic artificial bone performance in each experimental group In summary, the recombinant collagen bionic artificial bone material prepared by the present invention is similar to the structure and chemical composition of natural bone tissue, and its porosity and mechanical performance parameters meet the requirements of bone repair materials; and the bionic artificial bone material obtained by the present invention has a good in vitro degradation rate and a good ability to induce osteogenic differentiation, effectively promoting the healing of bone defect tissue. The bionic bone repair scaffold material retains the biological activity of recombinant collagen to the greatest extent, and has good biological safety and effectiveness.

[0072] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; 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 bionic 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 thereto to obtain a cross-linked recombinant collagen hydrogel, add an equal amount of purified water thereto, break up with a homogenizer, and then freeze-dry in a vacuum to obtain a recombinant collagen lyophilized product; S2. Preparation of mineralized recombinant collagen: add water to the lyophilized product obtained in S1, stir and disperse after sufficient swelling, add calcium chloride solution and sodium dihydrogen phosphate solution, and finally add sodium hydroxide solution to adjust the pH to 8-10, obtain a white suspension, and then let it stand for full mineralization to obtain a mineralized recombinant collagen mixture; S3, molding: putting the mixed solution obtained in S2 into a dialysis bag, dialyzing, collecting the precipitate by centrifugation, placing the precipitate in a mold and vacuum freeze-drying to obtain a recombinant collagen bionic 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.

2. The recombinant collagen bionic artificial bone according to claim 1, characterized in that: The mass fraction of collagen in the collagen solution described in S1 is 5%~15%.

3. The recombinant collagen bionic artificial bone according to claim 1, characterized in that: In the mixed solution of recombinant humanized type III collagen and recombinant type III collagen described in S1, the mass of recombinant type III humanized collagen does not exceed 1 / 9 of the total mass of collagen.

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

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

6. The recombinant collagen bionic 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.

7. The recombinant collagen bionic 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; the ratio of the added amount of calcium ions to the lyophilized product obtained in S1 is 0.002~0.005mol:1g; the molar ratio of phosphate ions to calcium ions is 3:5~6.

8. The recombinant collagen bionic artificial bone according to claim 1, characterized in that: The dialysis described in S3 is as follows: placing the dialysis bag in deionized water, low-temperature dialysis at 2-6°C for 3 days, and changing the water 3 times a day; the molecular weight cutoff of the dialysis bag is 1000-3000Da.

9. A method for preparing the recombinant collagen bionic artificial bone according to any one of claims 1 to 8.

10. Use of the recombinant collagen bionic artificial bone according to any one of claims 1 to 8 in the preparation of bone defect filling and repair products.

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